Anti-folate receptor alpha antibodies and methods of use

Abstract

Antibody constructs that bind to human folate receptor alpha (FRalpha or FOLR1) and antibody-drug conjugates (ADCs) comprising anti-FRalpha 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 targeting human folate receptor alpha (hFRα). Background Art

[0002] Folate receptor α (FRα) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein encoded by FOLR1 and is a member of the high-affinity FR family, which also includes FRβ (FOLR2), FRγ (FOLR3) and FRδ (FOLR4). FRα has been identified as a highly relevant target for cancer therapy because it is overexpressed in a variety of cancers, including ovarian cancer, triple-negative breast cancer (TNBC), endometrial cancer, mesothelioma, and lung cancer, with minimal expression in non-malignant tissues.

[0003] Several clinical studies involving FRα-targeted agents in cancer treatment are currently underway, 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 potentially relevant to the present disclosure. It is not necessarily intended to, nor should it be construed as, an admission that any of the foregoing information constitutes prior art to the claimed invention. Summary of the invention

[0005] Anti-FRα antibodies and methods of use are described herein. One aspect of the present disclosure relates to an antibody construct comprising an antigen binding domain that specifically binds to human folate receptor α (hFRα), wherein the antibody construct competes for binding to hFRα with a reference antibody that specifically binds to an epitope within hFRα comprising 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 α (hFRα), wherein the antigen binding domain comprises a heavy chain CDR amino acid sequence (HCDR1, HCDR2 and HCDR3) and a light chain CDR amino acid sequence (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDR amino acid sequence comprises the sequence shown in SEQ ID NOs: 3, 4 and 5, and the light chain CDR amino acid sequence comprises the sequence shown 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, wherein each of the antigen binding domains specifically binds to human folate receptor alpha (hFRα) and comprises:

[0009] (a) the VL amino acid sequence as shown in SEQ ID NO:39 and the VH amino acid sequence as shown in SEQ ID NO:19; or

[0010] (b) the VL amino acid sequence as shown in SEQ ID NO: 124 and the VH amino acid sequence as shown in SEQ ID NO: 91; or

[0011] (c) the VL amino acid sequence as shown in SEQ ID NO: 64 and

[0012] (i) the VH amino acid sequence as shown in SEQ ID NO: 50, or

[0013] (ii) the VH amino acid sequence as shown in SEQ ID NO: 54, or

[0014] (iii) the VH amino acid sequence as shown in SEQ ID NO: 57, or

[0015] (iv) the VH amino acid sequence as shown in SEQ ID NO: 61, or

[0016] (v) the VH amino acid sequence as shown in SEQ ID NO: 76, or

[0017] (vi) the VH amino acid sequence as shown in SEQ ID NO: 79, or

[0018] (vii) the VH amino acid sequence as shown in SEQ ID NO: 82, or

[0019] (viii) the VH amino acid sequence as shown in SEQ ID NO: 85, or

[0020] (ix) the VH amino acid sequence as shown in SEQ ID NO: 88, or

[0021] (x) the VH amino acid sequence as shown in SEQ ID NO: 106; or

[0022] (d) the VL amino acid sequence as shown in SEQ ID NO: 130 and

[0023] (i) the VH amino acid sequence as shown in SEQ ID NO: 99, or

[0024] (ii) the VH amino acid sequence as shown in SEQ ID NO: 106, or

[0025] (iii) the VH amino acid sequence as shown in SEQ ID NO: 113, or

[0026] (iv) the VH amino acid sequence as shown in SEQ ID NO: 116, or

[0027] (v) the VH amino acid sequence as shown in SEQ ID NO: 133, or

[0028] (vi) the VH amino acid sequence as shown in SEQ ID NO: 136; or

[0029] (e) the VL amino acid sequence as shown in SEQ ID NO: 119 and

[0030] (i) the VH amino acid sequence as shown in SEQ ID NO: 106, or

[0031] (ii) the VH amino acid sequence as shown in SEQ ID NO:116.

[0032] Another aspect of the present disclosure relates to a polynucleotide or a set of polynucleotides encoding an anti-FRα antibody construct as described herein.

[0033] Another aspect of the present 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 present disclosure relates to a host cell comprising the expression vector or a set of expression vectors.

[0034] Another aspect of the present disclosure relates to an antibody-drug conjugate comprising an anti-FRα antibody construct as described herein conjugated to one or more drug moieties.

[0035] Another aspect of the present disclosure relates to antibody-drug conjugates having the general formula I:

[0036] A-(L-(D) m ) n (I) In which:

[0037] A is an anti-FRα antibody construct as described herein;

[0038] L is the connector;

[0039] D is the drug part;

[0040] m is between 1 and about 8, and

[0041] n is 1 and about 12.

[0042] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an anti-FRα antibody construct as described herein or an antibody-drug conjugate as described herein and a pharmaceutically acceptable carrier or diluent.

[0043] Another aspect of the present disclosure relates to an anti-FRα antibody construct as described herein or an antibody-drug conjugate as described herein for use in therapy, such as for use in the treatment of cancer.

[0044] Another aspect of the present disclosure relates to the use of an anti-FRα antibody construct as described herein or an antibody-drug conjugate as described herein in the manufacture of a medicament for treating cancer.

[0045] Another aspect of the present disclosure relates to a method of inhibiting the growth of FRα-positive tumor cells, comprising contacting the cells with an anti-FRα antibody construct as described herein or an antibody-drug conjugate as described herein.

[0046] Another aspect of the present disclosure relates to a method of treating a subject having cancer, comprising administering to the subject an effective amount of an anti-FRα antibody construct as described herein or an antibody-drug conjugate as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1A shows the sequence of the rabbit heavy chain variable domain CDRs of the chimeric antibody v23924 grafted onto a human VH framework (IGHV3-23*01) (SEQ ID NO: 155), and Figure 1B The sequence of the rabbit light chain variable domain CDRs of the chimeric antibody v23924 grafted onto a human VL framework (IGKVI-39*01) (SEQ ID NO: 156) is shown. The CDRs are designated according to the AbM definition and are marked in bold italic font.

[0048] Figure 2 A- Figure 2D shows the profiles of purified parental chimeric variant v23924 and purified representative humanized variant v30384 as analyzed by electrophoresis and UPLC-SEC. Figure 2 A and Figure 2 C shows the electrophoretic profiles of the parental chimeric variant v23924 (2A) and the purified representative humanized variant v30384 (2C) after preparative SEC purification (post-prep-SEC) or after protein A purification (post-pA) under non-reducing (NR) and reducing (R) conditions, Figure 2 B and Figure 2 D shows the UPLC-SEC profiles of the parental chimeric variant v23924 after preparative SEC purification (2B) and a representative humanized variant v30384 purified after Protein A purification (2D).

[0049] Figure 3 A and Figure 3 B depicts biolayer interferometry (BLI) sensorgrams of the parental chimeric variant v23924 (3A) and the purified representative humanized variant v30384 (3B).

[0050] Figure 4 A- Figure 4 D depicts the complete LC / MS spectra of representative humanized variants v30384 (4A, expanded view of the main peak in 4B) and v31422 (4C, expanded view of the main peak in 4D).

[0051] Figure 5 A and Figure 5 B shows the receptor-mediated internalization capacity of chimeric antibody v23924, representative humanized variant v30384, and FRα-targeting antibodies Sumituximab and Fatuzumab 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.

[0052] Figure 6 A and Figure 6 B shows the receptor-mediated internalization capacity of chimeric anti-FRα antibody v23924, representative humanized variant v30384, and FRα-targeting antibodies Sumituximab and Fatuzumab 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.

[0053] Figure 7 Shown is 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.

[0054] Figure 8 A and Figure 8 B shows summary (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 complex.

[0055] Fig. 9 A- Fig. 9 C shows the amide deuteration level of peptide 119-126 (WEDCRTSY) (SEQ ID NO: 152) after 1 hour hydrogen / deuterium exchange mass spectrometry (HDX-MS): hFOLR1 (9A) versus hFOLR1-v23924 complex (9B), and the difference graph (9C).

[0056] Fig.10 A and Fig.10 B shows the receptor-mediated internalization capacity of the parental humanized antibody variant v30384 and the representative affinity matured variant v35356 in the FRα expressing cell lines IGROV-1 (10A) and JEG-3 (10B) as determined by flow cytometry after 5 and 24 hour incubation periods. Palivizumab was included as a non-targeting control.

[0057] Fig.11 A- Fig.11 D shows the intracellular payload delivery capacity of representative ADCs in cell lines JEG-3 (11A), Caov-3 (11B), H2110 (11C) and HEC-1-A (11D) assessed by mass spectrometry. The ADCs are humanized antibody variant v30384 and affinity matured variant v35356, each conjugated to drug-linker DL1.

[0058] Fig.12 A- Fig.12 H shows 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 (somitoxin Fab with HetFc) conjugated to drug-linker DL1 or DL6.

[0059] Fig.13In vivo antitumor activity of ADCs comprising chimeric antibody v23924 or humanized variants v30384 or v30399, each conjugated to drug-linker DL1, administered at 4 mg / kg or 9 mg / kg in the OVCAR3 ovarian cancer model expressing FRα at medium / high levels is shown.

[0060] Fig.14 A- Fig.14 E shows the in vivo antitumor activity of ADCs containing humanized variant v30384 conjugated to drug-linkers DL1 or DL8 administered at the indicated doses as assessed in a xenograft model: H2110 CDX (14A), SKOV3 CDX (14B and 14C), IGROV-1 CDX (14D), and LXFA737 PDX (14E). Control ADCs were v17717 (somitoxin Fab with HetFc) conjugated to drug-linker DL6; v17716 (somitoxin Fab with HomoFc) conjugated to drug-linker DL6, and for H2110 CDX, v31629 (fatuzumab) conjugated to drug-linker DL4.

[0061] Fig.15 A- Fig.15 D shows the in vivo antitumor activity of ADCs comprising the humanized variant v30384 conjugated to the drug-linker DL5 administered at the indicated doses in xenograft models: OV90 (15A), H2110 (15B), and OVCAR-3 (15C and 15D).

[0062] Fig.16 A- Fig.16 H shows the results of pharmacokinetic analysis, indicating the concentration of serum IgG or ADC over time in sera taken from animals treated with ADCs comprising 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).

[0063] Fig.17 A table showing the CDR sequences of representative anti-FRα antibodies as defined by the IMGT, Chothia, Kabat, Contact and AbM definitions is presented.

[0064] Fig.18 A table showing the VH and VL sequences of representative anti-FRα antibodies is presented.

[0065] Fig.19 The cell growth inhibition (cytotoxicity) ability of ADCs comprising humanized antibody v30384 conjugated to drug-linker DL1 and ADCs 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) is shown.

[0066] Fig. 20 A- Fig. 20 D shows the penetration of anti-FRα humanized antibody variant v36675 in JEG-3 cell spheroids compared to sometuximab and negative control palivizumab at 4 hours (20A), 24 hours (20B), 48 hours (20C), and 96 hours (20D).

[0067] Fig.21 A and Fig.21 B shows fixed cell confirmation screening images of 20 μg / mL of anti-FRα humanized antibody variant v36675 (21A) and 1 μg / mL of control antibody (rituximab biosimilar) (21B) using Retrogenix cell microarray technology to screen for specific off-target binding interactions.

[0068] Fig. 22 Shown is competition binding between chimeric anti-FRα antibody v23294 and anti-FRα antibodies sometuximab and fatuzumab evaluated in H2110 cells.

[0069] Fig.23 Shown is the receptor-mediated internalization capacity of the humanized variant v30384 compared to the biparatopic anti-FRα antibody B5327A (v36264) and the anti-FRα antibody Sumetuximab (v17716) in the FRα expressing cell line IGROV-1 as determined by flow cytometry after a 5 h incubation period.

[0070] Fig.24 Shown is the penetration of the anti-FRα humanized antibody variant v36675 in JEG-3 cell spheroids at 96 hours compared to the biparatopic anti-FRα antibody B5327A (v36264) and the anti-FRα antibody Sumetuximab (v17716). DETAILED DESCRIPTION

[0071] The present disclosure relates to antibody constructs that bind to human folate receptor alpha (FRα; also referred to herein as FOLR1), but do not show significant binding to folate receptor beta (FOLR2), gamma (FOLR3), or delta (FOLR4). In certain embodiments, the anti-FRα antibody constructs of the present disclosure are also capable of binding to cynomolgus monkey FRα.

[0072] The present disclosure also relates to antibody-drug conjugates (ADCs) comprising an anti-FRα antibody construct as 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 agents or diagnostic agents. Certain aspects of the present disclosure relate to methods and uses of anti-FRα antibody constructs and ADCs, for example, in the treatment of cancer. Some aspects relate to diagnostic methods and uses of anti-FRα antibody constructs and ADCs, for example, in the diagnosis or analysis of cancer.

[0073] definition

[0074] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0075] As used herein, the term "about" refers to a variation of approximately + / - 10% from a given value. It should be understood that such variations are always included in any given value provided herein, whether or not specifically mentioned.

[0076] When used in conjunction with the term "comprising" in this document, the use of the word "a / kind" may mean "one / kind", but it also has the meaning of "one / kind or more / kinds", "at least one / kind" and "one / kind or more than one / kind".

[0077] As used herein, the terms "comprising," "having," "including," and "containing," and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unlisted elements and / or method steps. When used in conjunction with a composition, use, or method herein, the term "consisting essentially of means that additional elements and / or method steps may be present, but these additions do not substantially affect the manner in which the enumerated composition, method, or use functions. The term "consisting of" does not include the presence of additional elements and / or method steps when used in combination with a composition, use, or method herein. A composition, use, or method described herein as comprising certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments, and consist of those elements and / or steps in other embodiments, whether or not these embodiments are specifically mentioned.

[0078] "Complementarity determining region" or "CDR" is an amino acid sequence that contributes to antigen binding specificity and affinity. "Framework" region (FR) can help maintain the correct conformation of CDR to promote the binding between antigen binding region and antigen. From N-terminus to C-terminus, the light chain variable region (VL) and heavy chain variable region (VH) of an antibody generally include domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three heavy chain CDRs are referred to as HCDR1, HCDR2 and HCDR3 in this article, and the three light chain CDRs are referred to as LCDR1, LCDR2 and LCDR3. CDR provides most of the contact residues for the binding of an antibody to an antigen or epitope. Generally, three heavy chain CDRs and three light chain CDRs are required to bind antigen. However, in some cases, even a single variable domain can also confer antigen binding specificity. In addition, as known in the art, in some cases, antigen binding can also occur by a combination of at least one or more CDRs (e.g., HCDR3) selected from VH and / or VL domains.

[0079] Many different definitions of CDR sequences are commonly used, 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 IMGT, AbM (University of Bath) and Contact (MacCallum et al., 1996, J Mol Biol, 262 (5): 732-745). For example, the definitions of CDRs according to Kabat, Chothia, IMGT, AbM and Contact are provided in Table 1 below. Therefore, it will be apparent to one skilled in the art that the exact numbering and placement of CDRs may differ based on the numbering system employed. However, it should be understood that the disclosure of VH herein includes disclosure of the associated (intrinsic) heavy chain CDRs (HCDRs) as defined by any known numbering system. Similarly, disclosure of a VL herein includes disclosure of the associated (intrinsic) light chain CDRs (LCDRs) as defined by any known numbering system.

[0080] Table 1: Common CDR definitions 1

[0081]

[0082] 1Either the Kabat or Chothia numbering system may be used for all defined HCDR2, HCDR3 and light chain CDRs except Contact which uses Chothia numbering.

[0083] 2 Kabat numbering is used. The positions in the Kabat numbering scheme that delineate the ends of the Chothia and IMGT CDR-H1 loops vary depending on the length of the loop, as Kabat places insertions outside of those CDR definitions at positions 35A and 35B. However, the IMGT and Chothia CDR-H1 loops can be clearly defined using Chothia numbering. CDR-H1 definitions using Chothia numbering: Kabat H31-H35, Chothia H26-H32, AbM H26-H35, IMGT H26-H33, Contact H30-H35.

[0084] In the context of two or more polynucleotides or polypeptide sequences, the term "identical" refers to two or more identical sequences or subsequences. When sequences are compared and aligned to obtain the maximum consistency measured as one of the conventional sequence comparison algorithms known to those of ordinary skill in the art or by manual alignment and visual inspection on a comparison window or in a specified region, if the sequence has a certain percentage of identical amino acid residues or nucleotides (e.g., about 80%, about 85%, about 90%, about 95% or about 98% identity in a specified region), the sequence is "substantially identical". For sequence comparison, a test sequence is usually compared with a specified reference sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0085] A "comparison window" refers to a segment of a sequence comprising contiguous amino acid or nucleotide positions, which can be, for example, about 10 to 600 contiguous amino acid or nucleotide positions, or about 10 to about 200, or about 10 to about 150 contiguous amino acid or nucleotide positions, over which a test sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are known to those of ordinary skill in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c; the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; the similarity search method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444; or by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining percentage of sequence identity are 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 analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI).

[0086] As used herein, the term "subject" refers to an animal, in some embodiments a mammal, that is the subject of treatment, observation, or experiment. The animal may be a human, a non-human primate, a companion animal (e.g., a dog, a cat, etc.), a farm animal (e.g., a cow, a sheep, a pig, a horse, etc.), or a laboratory animal (e.g., a rat, a mouse, a guinea pig, a non-human primate, etc.). In certain embodiments, the subject is a human.

[0087] It is contemplated that any embodiment discussed herein can be implemented by any method, use, or composition disclosed herein, and vice versa.

[0088] Certain features, structures, and / or characteristics described in connection with one embodiment disclosed herein may be combined with features, structures, and / or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more further embodiments.

[0089] It should also be understood that the positive recitation of a feature in one embodiment is a basis for excluding that feature in another embodiment. For example, where 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 the shortened list may form an alternative embodiment, regardless of whether such an alternative embodiment is specifically mentioned.

[0090] Anti-FRα antibody constructs

[0091] The present disclosure relates to antibody constructs that specifically bind to human FRα (hFRα). In this context, the term "antibody construct" refers to a polypeptide or a group of polypeptides comprising one or more antigen binding domains, wherein each of the one or more antigen binding domains specifically binds to an epitope or antigen. When the antibody construct comprises two or more antigen binding domains, each antigen binding domain 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 may also include a scaffold, and the one or more antigen binding domains may be fused or covalently linked to the scaffold, optionally connected via a linker, as described herein.

[0092] According to the present disclosure, the anti-FRα antibody construct comprises at least one antigen binding domain that specifically binds to hFRα. "Specific binding" to hFRα means that the antibody construct binds to hFRα but does not exhibit significant binding to any of the human folate receptors β (FOLR2), γ (FOLR3), or δ (FOLR4). In certain embodiments, the anti-FRα antibody construct of the present disclosure is capable of binding to FRα from one or more non-human species. In certain embodiments, the anti-FRα antibody construct of the present disclosure is capable of binding to cynomolgus monkey FRα.

[0093] 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, those 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 (SEQ ID NO: 2; NCBI reference sequence: XP_005579002.2) is also provided in Table 2.

[0094] Table 2: Human and cynomolgus monkey FRα protein sequences

[0095]

[0096]

[0097] Specific binding of the antigen binding domain to the target antigen or epitope can be measured, for example, by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (using, for example, 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, for example, less than about 10% of the binding to hFRα as measured by ELISA or flow cytometry to a non-target protein (such as FOLR2, FOLR3, or FOLR4). In certain embodiments, specific binding of an antibody construct to FRα can be measured by a dissociation constant (K D ) < 1 μΜ, e.g. < 500nM, < 250nM, < 100nM, < 50nM or < In certain embodiments, the specific binding of an antibody construct to a particular antigen or epitope can be measured by the dissociation constant (K D ) is 10 -6 M or smaller, e.g. 10 -7 M or less or 10 -8 In some embodiments, the specific binding of an antibody construct to a particular antigen or epitope can be measured by the dissociation constant (K D) between 10 -6 M and 10 -9 M, for example, between 10 -7 M and 10 -9 M is defined between.

[0098] In certain embodiments, the anti-FRα antibody constructs of the present disclosure show higher internalization into FRα-expressing cells than the reference antibodies somituximab (huMov19 or huFR107) and faetuzumab (MORAb-003).

[0099] Antibody internalization can be measured using methods known in the art, for example, by direct internalization according to the protocol detailed in Schmidt, M. et al., 2008, Cancer Immunol. Immunother., 57:1879-1890, or using commercially available fluorescent dyes such as pHAb dye (Promega Corporation, Madison, WI), pHrodo iFL and Deep Red dye (ThermoFisher Scientific Corporation, Waltham, MA) and Fabfluor-pH antibody labeling reagent (Sartorius AG, Germany), and analytical techniques such as microscopy, FACS, high-content imaging or other plate-based assays.

[0100] In certain embodiments, when the amount of anti-FRα antibody construct internalized into FRα-expressing cells under the same test conditions is at least 1.2 times the amount of reference antibody internalized into the same FRα-expressing cells, the anti-FRα antibody construct is considered to exhibit higher internalization into FRα-expressing cells than the corresponding reference antibody (somituximab or fatuzumab). In certain embodiments, the amount of internalized antibody is determined using appropriate fluorescent dyes and high-content imaging. In some embodiments, the amount of internalized antibody in cells expressing FRα at high levels is determined. In some embodiments, the amount of internalized antibody in IGROV-1 cells or cells expressing FRα at a level similar to that of IGROV-1 cells is determined. 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.

[0101] In certain embodiments, when the amount of anti-FRα antibody construct internalized into FRα expressing cells under the same test conditions is at least 1.3 times, at least 1.4 times, at least 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times or 2.0 times the amount of reference antibody internalized into the same FRα expressing cells, the anti-FRα antibody construct is considered to be higher than the corresponding reference antibody (somituximab or fatuzumab) in internalization into FRα expressing cells. In certain embodiments, the amount of internalized antibody is determined using appropriate fluorescent dyes and high-content imaging. In some embodiments, the amount of internalized antibody in cells expressing FRα at high levels is determined. In some embodiments, the amount of internalized antibody in IGROV-1 cells or cells expressing FRα at a level similar to that of IGROV-1 cells is determined. 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.

[0102] Antigen binding domain

[0103] The anti-FRα antibody constructs disclosed herein comprise 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 (sdAb).

[0104] A "Fab fragment" contains the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, as well as the variable domains of the light and heavy chains (VL and VH, respectively). A Fab' fragment differs from a Fab fragment in that it has several amino acid residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. A Fab fragment can also be a single-chain Fab molecule, i.e., a Fab molecule in which a Fab light chain and a 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 can be connected to the N-terminus of a Fab heavy chain in a single-chain Fab molecule.

[0105] "scFv" comprises the heavy chain variable domain (VH) and light chain variable domain (VL) of an antibody in a single polypeptide chain. ScFv may optionally further comprise a polypeptide linker between the VH and VL domains so that the scFv can form a structure required for antigen binding. For example, scFv may include a VL connected to the N-terminus of VH from the C-terminus via a polypeptide linker. Alternatively, scFv may comprise a VH connected to the N-terminus of VL via its C-terminus via a polypeptide linker (see the review in Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0106] The "sdAb" format refers to a single immunoglobulin domain. sdAb can be, for example, of camel origin. Camel antibodies lack light chains, and their antigen binding sites consist of a single domain called "VHH". sdAb contains three CDR / hypervariable loops CDR1, CDR2, and CDR3 that form the antigen binding site. sdAb is quite stable and easy to express, for example, expressed as a fusion with an antibody Fc chain (see, e.g., Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77 (1): 13-22).

[0107] In those embodiments where the anti-FRα antibody construct comprises two or more antigen binding domains, each additional antigen binding domain can independently be an immunoglobulin-based domain (such as an antigen-binding antibody fragment) or a non-immunoglobulin-based domain (such as a non-immunoglobulin-based antibody mimetic), or other polypeptides or small molecules that are capable of specifically binding to their targets (e.g., natural or engineered ligands). Non-immunoglobulin-based antibody mimetic forms include, for example, anticalins, fynomers, affimers, alphabodies, DARPins, and avimers.

[0108] The present disclosure describes herein the identification of an antibody that specifically binds hFRα (variant v23924), as well as representative humanized versions of the antibody (variants v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, and v31426) and representative affinity matured versions of the antibody (variants v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, and v36675) (see Examples and Sequence Listing). Figure 7 Epitope mapping of the hFRα sequence shown (SEQ ID NO: 15) determined that the epitope within the hFRα protein to which variant v23924 binds comprises amino acid residues E120, D121, R123, T124, S125 and Y126 of SEQ ID NO: 15 (see Example 13).

[0109] In certain embodiments, the at least one antigen binding domain that binds hFRα comprised by the anti-FRα antibody constructs of the present disclosure binds to an epitope of amino acid residues E120, D121, R123, T124, S125, and Y126 within the hFRα protein comprising SEQ ID NO: 15. In some embodiments, the hFRα epitope bound by the anti-FRα antibody construct is a non-linear (or discontinuous) epitope comprising amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15. In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain that competes for binding to hFRα with an antibody that binds to an epitope within the hFRα protein comprising amino acid residues E120, D121, R123, T124, S125, and Y126. In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain that competes for binding to hFRα with antibody v23924 described herein.

[0110] Competition assays known in the art can be used to determine whether an antibody construct competes with an antibody that binds to an epitope comprising amino acid residues E120, D121, R123, T124, S125 and Y126 within the hFRα protein or with antibody v23924 for binding to hFRα. For example, an antibody that binds to an epitope comprising amino acid residues E120, D121, R123, T124, S125 and Y126 within the hFRα protein or antibody v23924 (reference antibody) is first allowed to bind to hFRα under saturation conditions, and then the ability of the test antibody construct to bind to hFRα is measured. If the test antibody construct is able to bind to hFRα simultaneously with the reference antibody, it is considered that the test antibody construct binds to a different epitope than the reference antibody. Conversely, if the test antibody construct cannot bind to hFRα simultaneously with the reference antibody, it is considered that the test antibody construct binds to an epitope that is identical to the epitope bound to the reference antibody, an overlapping epitope, or an epitope that is closely adjacent to the epitope bound to the reference antibody. Competition assays can also be run in which the order of binding of the reference and test antibodies is reversed, ie, the test antibody is first allowed to bind hFRα under saturating conditions and the ability of the reference antibody construct to bind hFRα is then measured.

[0111] Such competition 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 the binding of the reference antibody to its epitope in a competition assay by 50% or more.

[0112] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise at least one antigen binding domain that specifically binds to hFRα, wherein 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 the antibody are shown in Fig.17 Analysis of CDR sequences from parental and affinity matured anti-FRα antibodies identified the minimum amino acid sequence present in each CDR as defined by any of the IMGT, Chothia, Kabat, Contact or AbM numbering systems. These amino acid sequences are represented by the minimum consensus CDR sequences provided in Table 3. An expanded version of these CDR consensus sequences based on the CDR sequences defined by the AbM numbering system is shown in Table 4.

[0113] Table 3: Minimal CDR consensus sequences of anti-FRα antibodies

[0114]

[0115] Table 4: CDR consensus sequences of anti-FRα antibodies based on the AbM numbering system

[0116]

[0117] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 3, 4, and 5, and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 6, 7, and 8.

[0118] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

[0119] (i) the HCDR1 amino acid sequence as shown in SEQ ID NO: 3; the HCDR2 amino acid sequence as shown in SEQ ID NO: 4; and the HCDR3 amino acid sequence as shown in SEQ ID NO: 5, wherein X 2 is L and X 3 A, or X 2 is H and X 3 is P, and

[0120] (ii) the LCDR1 amino acid sequence as shown in SEQ ID NO: 6, wherein X 4 is G and X 5 D, or X 4 is W and X 5 is Y; the LCDR2 amino acid sequence as shown in SEQ ID NO: 7; and the LCDR3 amino acid sequence as shown in SEQ ID NO: 8, wherein X 6 For S, X 7 N, X 8 V and X 9 D, or X 6 W, X 7 H, X 8 I and X 9 For L.

[0121] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 9, 10, and 11, and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 12, 13, and 14.

[0122] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

[0123] (i) the HCDR1 amino acid sequence as shown in SEQ ID NO: 9; the HCDR2 amino acid sequence as shown in SEQ ID NO: 10, wherein X 11 is S or A and X 12 V, or X 11 is S and X 12 L; and the HCDR3 amino acid sequence as shown in SEQ ID NO: 11, wherein X 13 is L and X 14 A, or X 13 is H and X 14 is P, and

[0124] (ii) the LCDR1 amino acid sequence as shown in SEQ ID NO: 12, wherein X 15 R or Q, X 16 is G and X 17 D, or X 15 R, X 16 is W and X 17 is Y; the LCDR2 amino acid sequence as shown in SEQ ID NO: 13; and the LCDR3 amino acid sequence as shown in SEQ ID NO: 14, wherein X 18 For S, X 19 N, X 20 V and X 21 D, or X 18 W, X 19 H, X 20 I and X 21 For L.

[0125] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

[0126] (i) a HCDR1 amino acid sequence selected from the group consisting of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v3 a HCDR1 amino acid sequence selected from 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 a HCDR3 amino acid sequence selected from variants v23924, v30618, v30384, v30389, v3 the HCDR3 amino acid sequence of any one of v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, and

[0127] (ii) a LCDR1 amino acid sequence selected from the group consisting of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35355 a LCDR1 amino acid sequence selected from variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v31427, v31428, v31429, v3142A, v3142B, v3142C, v3142D, v3142E, v3142F, v3142A, v3142A, v3142A, v3142B, v3142C, v3142D, v3142E, v3142A, v3142A, v3142A 6, a LCDR2 amino acid sequence of any one of v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675; and a LCDR3 amino acid sequence selected from variants v23924, v30618, v30384, v30389, v35391, v35410, v35411, v35413, v35414, v35415 the LCDR3 amino acid sequence of any one of v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675,

[0128] wherein the CDR amino acid sequences are as defined in any of the IMGT, Chothia, Kabat, Contact or AbM numbering systems (see Fig.17 ).

[0129] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) selected from variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35357, v35358, v35359, v35361, v35362, v35363, v35364, v35365, v35366, v35367, v35368, v35369, v35370, v35371, v35372, v35373, v35374, v35375 The heavy chain CDR amino acid sequence (HCDR1, HCDR2 and HCDR3) of any one of v6167, v36168 or v36675, and the light chain CDR amino acid sequence is selected from variants v23924, v30618, v30384, v30389, v30394, v33967, v33968, v33969, v34081, v34083, v34084, v34085, v34086, v34087, v34088, v34089, v34083, v34087, v34088, v34089, v34081 The light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) of any one of: V399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675.

[0130] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, as defined by any of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems. The heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) and light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) of any one of v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675.

[0131] 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 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 the CDR sequences of the VL domain of any 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. Fig.18 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.

[0132] 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.

[0133] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a VH amino acid sequence and a VL amino acid sequence selected from the VH and VL amino acid 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.

[0134] Those skilled in the art will appreciate that a limited number of amino acid substitutions can be introduced into the CDR sequences or VH or VL sequences of a known antibody without the antibody losing its ability 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 binding activity of the resulting variants tested by standard techniques. Thus, in certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a variant v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35355 A set of CDRs of any one of v56, v35358, v36167, v36168 or v36675 has a set of CDRs (i.e., heavy chain HCDR1, HCDR2 and HCDR3 and light chain LCDR1, LCDR2 and LCDR3) with 90% or more, 95% or more, 98% or more, 99% or more or 100% sequence identity, wherein the % sequence identity is calculated in all six CDRs, and wherein the antigen binding domain retains the ability to bind to hFRα.

[0135] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen binding domain comprising a variant of a 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 to 10 amino acid substitutions between the set of CDRs (i.e., the CDRs can be modified by up to 10 amino acid substitutions, wherein any combination of the 6 CDRs are modified), and wherein the antigen binding domain retains the ability to bind to hFRα. In some embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v353 The invention relates to a variant of a set of CDR sequences of any one of v50, v35354, v35356, v35358, v36167, v36168 or v36675, wherein the variant comprises 1 to 7 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 4 amino acid substitutions, 1 to 3 amino acid substitutions, 1 to 2 amino acid substitutions or 1 amino acid substitution in the set of CDRs, and wherein the antigen binding domain retains the ability to bind to hFRα.

[0136] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a polypeptide that is identical to variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, The VH sequence of any one of v35354, v35356, v35358, v36167, v36168 or v36675 has 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, wherein the antigen binding domain retains the ability to bind to hFRα. In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a polypeptide that is identical to variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, The VL sequence of any one of v35354, v35356, v35358, v36167, v36168 or v36675 has 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, wherein the antigen binding domain retains the ability to bind to hFRα.

[0137] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

[0138] (i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28, 31, 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 27, 29, 32, 51, 58, 100, 101, 102, 103, 109, 137, 138 or 139; and a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25, 30, 107, 108 or 110, and

[0139] (ii) a LCDR1 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 40, 43, 45, 65, 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 42, 47, 120 or 121.

[0140] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a CDR sequence of a VH domain having a sequence as shown 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, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a CDR sequence of a VL domain having a sequence as shown in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

[0141] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

[0142] (a) a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 43 or 45; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; and a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 27, 29 or 32; and a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30;

[0143] (b) a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; and a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 51; and a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30;

[0144] (c) a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; and

[0145] (i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 51; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

[0146] (ii) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 58; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

[0147] (iii) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 24, 100, 101, 102 or 103; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

[0148] (d) a LCDR1 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 120 or 121; and

[0149] (i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 24, 100, 101, 102 or 103; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

[0150] (ii) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; or

[0151] (iii) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

[0152] (iv) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; or

[0153] (v) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

[0154] (vi) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 137, 138 or 139; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; or

[0155] (e) a LCDR1 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 120 or 121; and

[0156] (i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; or

[0157] (ii) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; and a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110.

[0158] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain having:

[0159] (a) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 27, 29 or 32; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 43 or 45; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or

[0160] (b) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 51; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or

[0161] (c) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 58; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or

[0162] (d) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 51; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or

[0163] (e) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 24, 100, 101, 102 or 103; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

[0164] (f) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or

[0165] (g) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

[0166] (h) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

[0167] (i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

[0168] (j) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

[0169] (k) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

[0170] (l) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or

[0171] (m) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 137, 138 or 139; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121.

[0172] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a VH amino acid sequence selected from the group consisting of the VH amino acid sequence shown 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, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a VL amino acid sequence selected from the group consisting of the VL amino acid sequence shown in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

[0173] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising a VH amino acid sequence selected from the VH amino acid sequence shown 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 sequence shown in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

[0174] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising:

[0175] (a) the VL amino acid sequence as shown in SEQ ID NO:39 and the VH amino acid sequence as shown in SEQ ID NO:19; or

[0176] (b) the VL amino acid sequence as shown in SEQ ID NO: 124 and the VH amino acid sequence as shown in SEQ ID NO: 91; or

[0177] (c) the VL amino acid sequence as shown in SEQ ID NO: 64 and

[0178] (i) the VH amino acid sequence as shown in SEQ ID NO: 50, or

[0179] (ii) the VH amino acid sequence as shown in SEQ ID NO: 54, or

[0180] (iii) the VH amino acid sequence as shown in SEQ ID NO: 57, or

[0181] (iv) the VH amino acid sequence as shown in SEQ ID NO: 61, or

[0182] (v) the VH amino acid sequence as shown in SEQ ID NO: 76, or

[0183] (vi) the VH amino acid sequence as shown in SEQ ID NO: 79, or

[0184] (vii) the VH amino acid sequence as shown in SEQ ID NO: 82, or

[0185] (viii) the VH amino acid sequence as shown in SEQ ID NO: 85, or

[0186] (ix) the VH amino acid sequence as shown in SEQ ID NO: 88, or

[0187] (x) the VH amino acid sequence as shown in SEQ ID NO: 106; or

[0188] (d) the VL amino acid sequence as shown in SEQ ID NO: 130 and

[0189] (i) the VH amino acid sequence as shown in SEQ ID NO: 99, or

[0190] (ii) the VH amino acid sequence as shown in SEQ ID NO: 106, or

[0191] (iii) the VH amino acid sequence as shown in SEQ ID NO: 113, or

[0192] (iv) the VH amino acid sequence as shown in SEQ ID NO: 116, or

[0193] (v) the VH amino acid sequence as shown in SEQ ID NO: 133, or

[0194] (vi) the VH amino acid sequence as shown in SEQ ID NO: 136; or

[0195] (e) the VL amino acid sequence as shown in SEQ ID NO: 119 and

[0196] (i) the VH amino acid sequence as shown in SEQ ID NO: 106, or

[0197] (ii) the VH amino acid sequence as shown in SEQ ID NO:116.

[0198] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise an antigen binding domain comprising:

[0199] (i) the VH amino acid sequence shown in SEQ ID NO: 19 and the VL amino acid sequence shown in SEQ ID NO: 39, or

[0200] (ii) the VH amino acid sequence shown in SEQ ID NO:50 and the VL amino acid sequence shown in SEQ ID NO:64, or

[0201] (iii) the VH amino acid sequence shown in SEQ ID NO:54 and the VL amino acid sequence shown in SEQ ID NO:64, or

[0202] (iv) the VH amino acid sequence shown in SEQ ID NO:57 and the VL amino acid sequence shown in SEQ ID NO:64, or

[0203] (v) the VH amino acid sequence shown in SEQ ID NO:61 and the VL amino acid sequence shown in SEQ ID NO:64, or

[0204] (vi) the VH amino acid sequence shown in SEQ ID NO:76 and the VL amino acid sequence shown in SEQ ID NO:64, or

[0205] (vii) the VH amino acid sequence shown in SEQ ID NO:79 and the VL amino acid sequence shown in SEQ ID NO:64, or

[0206] (viii) the VH amino acid sequence shown in SEQ ID NO: 82 and the VL amino acid sequence shown in SEQ ID NO: 64, or

[0207] (ix) the VH amino acid sequence shown in SEQ ID NO: 85 and the VL amino acid sequence shown in SEQ ID NO: 64, or

[0208] (x) the VH amino acid sequence shown in SEQ ID NO: 88 and the VL amino acid sequence shown in SEQ ID NO: 64, or

[0209] (xi) the VH amino acid sequence shown in SEQ ID NO:91 and the VL amino acid sequence shown in SEQ ID NO:124, or

[0210] (xii) the VH amino acid sequence shown in SEQ ID NO:99 and the VL amino acid sequence shown in SEQ ID NO:130, or

[0211] (xiii) the VH amino acid sequence shown in SEQ ID NO: 106 and the VL amino acid sequence shown in SEQ ID NO: 64, or

[0212] (xiv) the VH amino acid sequence shown in SEQ ID NO: 106 and the VL amino acid sequence shown in SEQ ID NO: 119, or

[0213] (xv) the VH amino acid sequence shown in SEQ ID NO: 106 and the VL amino acid sequence shown in SEQ ID NO: 130, or

[0214] (xvi) the VH amino acid sequence shown in SEQ ID NO: 113 and the VL amino acid sequence shown in SEQ ID NO: 130, or

[0215] (xvii) the VH amino acid sequence shown in SEQ ID NO: 116 and the VL amino acid sequence shown in SEQ ID NO: 119, or

[0216] (xviii) the VH amino acid sequence shown in SEQ ID NO: 116 and the VL amino acid sequence shown in SEQ ID NO: 130, or

[0217] (xix) the VH amino acid sequence shown in SEQ ID NO: 133 and the VL amino acid sequence shown in SEQ ID NO: 130, or

[0218] (xx) the VH amino acid sequence shown in SEQ ID NO:136 and the VL amino acid sequence shown in SEQ ID NO:130.

[0219] Format

[0220] The anti-FRα antibody constructs of the present disclosure may have various formats. The minimum component of the anti-FRα antibody construct is an antigen binding domain that binds hFRα. The anti-FRα antibody construct may also optionally include 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 within hFRα, may bind to different epitopes within hFRα, or may bind to different antigens. Therefore, the anti-FRα antibody construct may be, for example, monospecific, biparatopic, bispecific, or multispecific.

[0221] In certain embodiments, the anti-FRα antibody construct comprises at least one antigen binding domain that binds hFRα and a scaffold, wherein the antigen binding domain is operably linked to the scaffold. As used herein, the term "operably linked" 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.

[0222] In certain embodiments, the anti-FRα antibody construct comprises two antigen binding domains that are optionally operably linked 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 domains can each be independently operably linked to the scaffold or the first antigen binding domain, or when there are more than two antigen binding domains, operably linked to another antigen binding domain.

[0223] Anti-FRα antibody constructs lacking a scaffold may include a single antigen binding domain in an appropriate format, such as an sdAb, or they may include two or more antigen binding domains optionally operably connected by one or more joints. In such anti-FRα antibody constructs, the antigen binding domain may be in the format of scFv, Fab, sdAb, or a combination thereof. For example, using scFv as an antigen binding domain, a format such as a tandem scFv ((scFv)2 or taFv) may be constructed, wherein the scFvs are connected together by a flexible joint. ScFv can also be used to construct a double antibody format, which includes two scFvs connected by a short joint (usually about 5 amino acids in length). The restricted joint length causes the scFv to dimerize in a head-to-tail manner. In any of the aforementioned forms, the scFv can be further stabilized by including an interdomain disulfide bond. For example, a disulfide bond can be introduced between VL and VH by introducing an additional cysteine ​​residue in each chain (e.g., at position 44 of VH and position 100 of VL) (see, e.g., Fitzgerald et al., 1997, Protein Engineering, 10: 1221-1225), or a disulfide bond can be introduced between the two VHs to provide a construct with a DART format (see, e.g., Johnson et al., 2010, J Mol. Biol., 399: 436-449).

[0224] Similarly, in some embodiments, a format comprising two sdAbs (such as VH or VHH) linked together by a suitable linker may be employed. Other examples of anti-FRα antibody construct formats lacking a scaffold include those based on Fab fragments, such as Fab2 and F(ab')2 formats, in which the Fab fragments are linked by a linker or an IgG hinge region.

[0225] Combinations of antigen binding domains of varying sizes can also be employed to generate alternative scaffold-free formats. For example, a scFv or sdAb can be fused to the C-terminus of one or both of the light and heavy chains of a Fab fragment, thereby generating a bivalent (Fab-scFv / sdAb) construct.

[0226] In certain embodiments, the anti-FRα antibody construct may be an antibody format based on an immunoglobulin (Ig). 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 specified immunoglobulin isotype, it means that the anti-FRα antibody construct comprises all or part of the constant region of the specified 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, wherein the scaffold comprises an Fc region from a given isotype and optionally an Ig hinge region from the same or different isotypes. It should be understood that in some embodiments, the anti-FRα antibody construct may also comprise a hybrid of an isotype and / or subclass. It should also be understood that the Fc region and / or hinge region may be optionally modified to confer one or more desired functional properties known in the art.

[0227] In some embodiments, the anti-FRα antibody construct can be derived from two or more immunoglobulins from different species, for example, the anti-FRα antibody construct can be a chimeric antibody or a humanized antibody. The terms "chimeric antibody" and "humanized antibody" generally refer to antibodies that combine immunoglobulin regions or domains from more than one species.

[0228] "Chimeric antibodies" typically comprise at least one variable domain from a non-human antibody, such as a rabbit or rodent (e.g., murine) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not have the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81: 6851-55 and U.S. Pat. No. 4,816,567.

[0229] "Humanized antibodies" are a class of chimeric antibodies that contain minimal sequences derived from non-human antibodies. Typically, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable regions (CDRs) of the recipient are replaced with residues from hypervariable regions (CDRs) of non-human species (donor antibodies) that have the desired specificity and affinity for the target antigen, such as mice, rats, rabbits, or non-human primates. This technique for creating humanized antibodies is often referred to as "CDR transplantation."

[0230] In some cases, additional modifications may be made to humanized antibodies to further improve antibody performance. For example, the framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues, or humanized antibodies may include residues not found in recipient antibodies or donor antibodies. In general, the variable domains in humanized antibodies will include all or nearly all hypervariable regions from non-human immunoglobulins and all or nearly all FRs from human immunoglobulin sequences. Humanized antibodies are described in more detail in, for example, 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.

[0231] Many methods are known in the art for selecting the most appropriate human framework for transplanting non-human CDR therein. Early methods use a limited subset of fully characterized human antibodies, independent of the sequence identity of the non-human antibodies providing CDR ("fixed framework" method). The most recent method has adopted a variable region with high amino acid sequence identity to the variable region of the non-human antibodies providing CDR ("homologous matching" or "best fit" method). An alternative method is to select a fragment of framework sequence from each light chain or heavy chain variable region from several different human antibodies. In some cases, CDR transplantation may cause the affinity of the transplanted molecule to its target antigen to be partially or completely lost. In such cases, affinity can be restored by backmutating some of the residues in some human sources to corresponding non-human residues. Methods for preparing humanized antibodies by these methods 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).

[0232] Alternatively, or in addition to these traditional methods, newer technology can be used to further reduce the immunogenicity of the humanized antibody of CDR transplantation.For example, a framework based on human germline sequence or consensus sequence can be used as a receptor human framework rather than a human framework with somatic mutations.Another technology intended to reduce the potential immunogenicity of non-human CDR is to transplant specificity determining residues (SDR) only.In this method, only the minimum CDR residues (" SDR ") required for antigen binding activity are transplanted into the human germline framework.This method improves the "humanity" (i.e., similarity to human germline sequence) of humanized antibodies, and therefore can contribute to reducing the immunogenicity risk of variable regions. These techniques have been 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).

[0233] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise humanized antibody sequences, such as one or more humanized variable domains. In some embodiments, the anti-FRα antibody constructs may 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 Examples and Sequence Listing).

[0234] branch

[0235] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise one or more antigen binding domains operably linked to a scaffold. The antigen binding domain 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, heterodimerized peptides (such as leucine zippers, "zipper" peptides derived from Jun and Fos forming heterodimers, IgG CH1 and CL domains, or barnase-barstar toxins), cytokines, chemokines, or growth factors. Other examples include DOCK-AND-LOCK based on the DOCK-AND-LOCK developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. TM (DNL TM ) technology (see, e.g., Chang et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0236] The scaffold can be a peptide, polypeptide, polymer, nanoparticle or other chemical entity. When the scaffold is a polypeptide, each antigen binding domain of the anti-FRα antibody construct can be connected to the N-terminus or C-terminus of the polypeptide scaffold. Anti-FRα antibody constructs comprising polypeptide scaffolds are also contemplated in certain embodiments, wherein one or more antigen binding domains are connected to regions other than the N-terminus or C-terminus, for example, via amino acid side chains with or without a linker.

[0237] In embodiments where the anti-FRα antibody construct comprises a scaffold that is a peptide or polypeptide, the antigen binding domain can be attached to the scaffold by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen binding domain is attached to the scaffold by genetic fusion. In some embodiments, where the scaffold is a polymer or nanoparticle, the antigen binding domain can be attached to the scaffold by chemical conjugation.

[0238] Many protein domains are known in the art that contain selective pairing of two different polypeptides and can be used to form a scaffold. Examples are leucine zipper domains that are selectively paired together, such as Fos and Jun (Kostelny et al., J Immunol, 148: 1547-53 (1992); Wranik et al., J. Biol. Chem., 287: 43331-43339 (2012)). Other selectively paired molecular pairs include, for example, barnase-barstar pairs (Deyev et al., Nat Biotechnol, 21: 1486-1492 (2003)), DNA strand pairs (Chaudri et al., FEBS Letters, 450 (1–2): 23-26 (1999)) and split fluorescent protein pairs (International Patent Application Publication No. WO 2011 / 135040).

[0239] 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., Müller 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).

[0240] For example, it has been demonstrated that fusing an antigen-binding moiety such as scFv, diabody or single-chain diabody to albumin can improve the serum half-life of the antigen-binding moiety (Müller et al., supra). The antigen-binding moiety may be fused to the N-terminus and / or C-terminus of albumin, optionally via a linker.

[0241] Albumin derivatives in the form of heteromultimers have been described, which comprise two transporter polypeptides obtained by fragmenting albumin, such that the transporter polypeptides self-assemble to form a natural albumin-like structure (see International Patent Application Publication Nos. WO 2012 / 116453 and WO 2014 / 012082). Due to the fragmentation of albumin, the heteromultimer comprises four termini and can therefore optionally be fused to up to four different antigen-binding moieties via a linker.

[0242] 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 (e.g., an IgG Fc region).

[0243] Fc region

[0244] As used herein, the term "Fc region," "Fc" or "Fc domain" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also known 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).

[0245] 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 consist of two Fc polypeptides, or alternatively, the Fc region may consist of a single polypeptide.

[0246] In the case of dimeric Fc, "Fc polypeptide" refers to one of the two polypeptides forming the dimeric Fc domain, i.e., a polypeptide comprising one or more C-terminal constant regions of immunoglobulin heavy chains capable of stable self-association. When referring to polypeptides forming the dimeric Fc region, the terms "first Fc polypeptide" and "second Fc polypeptide" can be used interchangeably, provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0247] The Fc region may comprise a CH3 domain or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, the Fc polypeptide 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.

[0248] 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.

[0249] In certain embodiments, the anti-FRα antibody construct may comprise a scaffold based on an IgG Fc region, which is a homodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence and optionally a CH2 sequence, and wherein the amino acid sequence of the first and second Fc polypeptides are identical.

[0250] In certain embodiments, the anti-FRα antibody construct may include a scaffold based on an IgG Fc region, the IgG Fc region being a heterodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence and optionally a CH2 sequence, and wherein the amino acid sequences of the first and second Fc polypeptides are different. In some embodiments, the anti-FRα antibody construct may include a scaffold based on an Fc region, the Fc region comprising two CH3 sequences, wherein at least one CH3 sequence comprises one or more amino acid modifications. In some embodiments, the anti-FRα antibody construct may include a scaffold based on an Fc region, the Fc region comprising two CH3 sequences and two CH2 sequences, wherein at least one CH2 sequence comprises one or more amino acid modifications.

[0251] In some embodiments, the anti-FRα antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, wherein the modified CH3 domain is an asymmetrically modified CH3 domain containing one or more asymmetric amino acid modifications. As used herein, "asymmetric amino acid modification" refers to a modification, such as a substitution or insertion, in which the amino acid at a specific position on the first CH3 or CH2 sequence is different from the amino acid at the same position on the 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 corresponding amino acid position on each sequence, or different modifications of the two amino acids at the same corresponding 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.

[0252] In some embodiments, the anti-FRα antibody construct may comprise a heterodimeric Fc containing a modified CH3 domain, wherein the modified CH3 domain comprises one or more amino acid modifications that promote heterodimeric Fc formation over homodimeric Fc formation. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0253] Amino acid modifications that can be made to the CH3 domain of Fc to promote the formation of heterodimeric Fc are known in the art and include, for example, those described in International Publication No. WO 96 / 027011 ("knob-mortise"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic manipulation"), Davis et al., 2010, Prot Eng Des Sel, 23(4): 195-202 (chain 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 methods of combining positive and negative design strategies to generate stable asymmetric modified Fc regions, such as those described in International Publication Nos. WO 2012 / 058768 and WO 2013 / 063702. In certain embodiments, an anti-FRα antibody construct may comprise a modified Fc region based scaffold as described in International Publication Nos. WO 2012 / 058768 or WO 2013 / 063702.

[0254] Table 5 provides the amino acid sequence of a human IgG1 Fc sequence (SEQ ID NO: 16), which corresponds to amino acids 231 to 447 of a full-length human IgG1 heavy chain. The CH3 sequence comprises amino acids 341-447 of a full-length human IgG1 heavy chain. Also shown in Table 5 are CH3 domain amino acid modifications that promote heterodimeric Fc formation, as described in International Patent Application Publication Nos. WO 2012 / 058768 and WO 2013 / 063702.

[0255] In certain embodiments, the anti-FRα antibody construct may comprise a heterodimeric Fc scaffold with a modified CH3 domain comprising modifications of any of Variant 1, Variant 2, Variant 3, Variant 4, or Variant 5, as shown in Table 5.

[0256] Table 5: Human IgG1 Fc sequences that promote heterodimer formation 1 and CH3 domain amino acid modifications

[0257]

[0258] 1 Sequence from position 231 to 447 (EU numbering)

[0259] In some embodiments, the anti-FRα antibody construct may comprise a scaffold based on an Fc region comprising two CH3 sequences and two CH2 sequences, wherein at least one CH2 sequence comprises one or more amino acid modifications. Modifications in the CH2 domain may affect the binding of Fc receptors (FcRs) to Fc, such as receptors of the FcγRI, FcγRII, and FcγRIII subclasses.

[0260] In some embodiments, the anti-FRα antibody construct comprises an IgG Fc based scaffold with a modified CH2 domain, wherein the modification of the CH2 domain results in altered binding to one or more of the FcγRI, FcγRII, and FcγRIII receptors.

[0261] A variety of amino acid modifications that selectively change the affinity of Fc for different Fcγ receptors to the CH2 domain are known in the art. Amino acid modifications that lead to increased binding and amino acid modifications that lead to reduced binding can each be used for certain indications. For example, increasing the binding affinity of Fc to FcγRIIIa (an activating receptor) can cause an increase in antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn causes an increase in the lysis of target cells. Reducing the binding to FcγRIIb (an inhibitory receptor) may also be beneficial in some cases. In certain indications, it may be necessary to reduce or eliminate ADCC and complement-mediated cytotoxicity (CDC). In such cases, a modified CH2 domain ("knockout" variant) comprising an amino acid modification that leads to an increase in binding to FcγRIIb or reduces or eliminates the binding of the Fc region to all Fcγ receptors may be useful.

[0262] Examples of amino acid modifications to the CH2 domain that alter the binding of Fcγ receptors to Fc include, but are not limited to, the following: 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., 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 the binding of FcγRIIb to Fc are described in International Publication No. WO 2021 / 232162. Additional modifications that affect the binding of Fc to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, October 2012, page 283).

[0263] In certain embodiments, the anti-FRα antibody construct comprises an IgG Fc based scaffold with a modified CH2 domain comprising one or more amino acid modifications that result in reduced or eliminated binding of the Fc region to all Fcγ receptors (ie, a "knockout" variant).

[0264] Various publications describe strategies that have been used to engineer antibodies to produce "knockout" variants (see, e.g., 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, pp. 225-249). These strategies include reducing effector function by glycosylation modification, using IgG2 / IgG4 scaffolds, or introducing mutations in the hinge or CH2 domains of Fc (see also U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO 2006 / 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).

[0265] Examples of mutations that can be introduced into the hinge or CH2 domain to generate "knockout" variants include amino acid modifications L234A / L235A and L234A / L235A / D265S.

[0266] In certain embodiments, the anti-FRα antibody constructs described herein may comprise an IgG Fc-based scaffold in which the native glycosylation has been modified. As is known in the art, the sugar groups of Fc can 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 the production of aglycosylated Fc lacking all effector functions (Bolt et al., 1993, Eur. J. Immunol., 23: 403-411; Tao & Morrison, 1989, J. Immunol., 143: 2595-2601).

[0267] In contrast, 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 low-fucose antibodies can be produced, for example, in knockout Chinese hamster ovary (CHO) cells that lack fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622); in a variant CHO cell line Lec 13 with reduced ability to attach fucose to N297-linked carbohydrates (International Publication No. WO 03 / 035835), or other cells that produce afucosylated 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). Additionally, International Publication No. WO 2009 / 135181 describes the addition of a fucose analog to the culture medium during antibody production to inhibit the incorporation of fucose into carbohydrates on the antibody.

[0268] Other methods for producing antibodies containing little or no fucose at the Fc glycosylation site (N297) are well known in the art. For example, Technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12): 1607-1618 and US Pat. No. 8,409,572).

[0269] Other glycosylation variants include those with bisected oligosaccharide variants, for example, wherein the biantennary oligosaccharide connected to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc) variants. Such glycosylation variants can have reduced fucosylation and / or improved ADCC function (see, e.g., International Publication No. WO 2003 / 011878, U.S. Patent No. 6,602,684 and U.S. Patent Application Publication No. US 2005 / 0123546). Useful glycosylation variants also include those variants having at least one galactose residue in the oligosaccharide connected to the Fc region, which can have improved CDC function (see, e.g., International Publication No. WO 1997 / 030087, WO 1998 / 58964 and WO 1999 / 22764).

[0270] In certain embodiments, the anti-FRα antibody construct has the format of a full-size antibody (FSA). In some embodiments, the anti-FRα antibody construct has an IgG FSA format, such as 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 and comprises H1, H2, L1, and L2 sequences, wherein H1 and H2 have the same amino acid sequence, and L1 and L2 have the same amino acid sequence. In some embodiments, the anti-FRα antibody construct is a monospecific FSA with 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 the same amino acid sequence. In some embodiments, the anti-FRα antibody construct is a bispecific or biparatopic FSA with 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.

[0271] In certain embodiments, the anti-FRα antibody construct is a FSA having a set of H1, H2, L1, and L2 sequences comprising the H1, H2, L1, and L2 amino acid sequences as shown in Tables A and B for any 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 in certain cell lines or from certain expression vectors can result in the inclusion of a C-terminal lysine residue on one or both heavy chains. 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 comprising the H1, H2, L1, and L2 amino acid sequences as shown in Tables A and B for any 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 comprise a C-terminal lysine (see, e.g., SEQ ID NO:157).

[0272] Preparation of anti-FRα antibody constructs

[0273] 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,” 2nd edition, Greenfield, ed., Cold Spring Harbor Laboratory Press, New York, 2014).

[0274] Typically, in order to recombinantly produce an antibody construct, a polynucleotide or a set of polynucleotides encoding an anti-FRα antibody construct is produced and inserted into one or more vectors for further cloning and / or expression in a host cell. Polynucleotides encoding anti-FRα antibody constructs can be produced by standard methods known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 and updates, and "Antibodies: A Laboratory Manual," 2nd edition, Greenfield ed., Cold Spring Harbor Laboratory Press, New York, 2014). As will be appreciated by those skilled in the art, the number of polynucleotides required to express an anti-FRα antibody construct will depend on the format of the construct, including whether the antibody construct contains a scaffold. For example, when the anti-FRα antibody construct is in the mAb format with a homodimer Fc, two polynucleotides encoding each polypeptide chain will be required, and when the anti-FRα antibody construct is in the mAb format with a heterodimer Fc, three polynucleotides encoding each polypeptide chain will be required. When multiple polynucleotides are desired, they can be incorporated into one vector or more than one vector.

[0275] Typically, for expression, a polynucleotide or a group of polynucleotides is incorporated into one or more expression vectors together with one or more regulatory elements, such as transcription elements, which are required for the effective transcription of the polynucleotides. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. It will be appreciated by those skilled in the art that the selection of regulatory elements depends on the host cell selected for expressing the antibody construct, and such regulatory elements may be derived from a variety of sources, including bacteria, fungi, viruses, mammals, or insect genes. The expression vector may optionally further contain a heterologous nucleic acid sequence, which 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 integration vector.

[0276] Suitable host cells for cloning or expressing anti-FRα antibody constructs include various prokaryotic or eukaryotic cells 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, Escherichia coli, Aeromonas salmonicida, or Bacillus subtilis cells.

[0277] In certain embodiments, anti-FRα antibody constructs can be produced in bacteria, particularly when glycosylation and Fc effector functions are not desired, as described, for example, in U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; and in Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, BKC Lo, ed., Humana Press, Totowa, NJ, 2003.

[0278] In certain embodiments, eukaryotic microorganisms such as filamentous fungi or yeast may be suitable expression host cells, particularly fungi and yeast strains whose glycosylation pathways have been "humanized" to result 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).

[0279] Suitable host cells for expressing glycosylated anti-FRα antibody constructs are generally eukaryotic cells. For example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 describe PLANTIBODIES for producing antigen-binding constructs in transgenic plants. TM Mammalian cell lines adapted to growth in suspension are particularly useful for expressing antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (COS-7), 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 liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells (see, e.g., Mather et al., 1982, Annals NY Acad Sci, 383:44-68), MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR -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 producing antibody constructs are reviewed in Yazaki and Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKCLo ed., Humana Press, Totowa, NJ, 2003).

[0280] In certain embodiments, the host cell can be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell can be a mammalian HEK293T, CHO, HeLa, NS0 or COS cell line, or a cell line derived from any of these cell lines. In some embodiments, the host cell can be a stable cell line that allows mature glycosylation of the antibody construct.

[0281] Host cells containing expression vectors encoding anti-FRα antibody constructs can be cultured using conventional methods to produce anti-FRα antibody constructs. Alternatively, in some embodiments, host cells containing expression vectors encoding anti-FRα antibody constructs can be used therapeutically or prophylactically to deliver anti-FRα antibody constructs to subjects, or polynucleotides or expression vectors can be administered ex vivo to cells from subjects and then the cells are returned to the subject's body.

[0282] Typically, the anti-FRα antibody construct is purified after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3rd Edition, Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques, including ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, fractionated chromatography or gel filtration and reverse phase chromatography, performed at atmospheric pressure or high pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoresis, immunoassays, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration are also useful. As is well known in the art, a variety of natural proteins bind to Fc and antibodies, and these proteins can be used to purify certain antibody constructs. For example, bacterial proteins A and G bind to the Fc region. Similarly, the bacterial protein L binds to the Fab region of some antibodies. Purification can also be performed by specific fusion partners. For example, if a GST fusion is used, glutathione resin can be used to purify the antibody, and if a His tag is used, Ni can be used. +2The antibody is purified by affinity chromatography, or if a Flag tag is used, the antibody can be purified using an immobilized anti-Flag antibody. The degree of purification necessary will vary depending on the anti-FRα antibody construct used. In some cases, purification may not be necessary.

[0283] 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 (such as a natural cell, or a host cell in the case of a recombinantly produced construct). 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.

[0284] Certain embodiments of the present disclosure relate to methods for preparing an anti-FRα antibody construct, the methods comprising culturing a host cell into which has been introduced one or more polynucleotides encoding the anti-FRα antibody construct or one or more expression vectors encoding the anti-FRα antibody construct under conditions suitable for expression of the anti-FRα antibody construct, and optionally recovering the anti-FRα antibody construct from the host cell (or from the host cell culture medium).

[0285] Post-translational modification

[0286] In certain embodiments, the anti-FRα antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications may occur in vivo, or in vitro after the anti-FRα antibody construct is isolated from a host cell.

[0287] Post-translational modifications include various modifications as known in the art (see, e.g., Proteins-Structure and Molecular Properties, 2nd Edition, TECreighton, WH Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, BC Johnson, ed., Academic Press, New York, pp. 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 those embodiments where the anti-FRα antibody construct comprises one or more post-translational modifications, the construct may comprise the same type of modification at one or several sites, or it may comprise different modifications at different sites.

[0288] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage, or specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, or NaBH4.

[0289] Other examples of post-translational modifications include, for example, the addition or removal of N-linked or O-linked carbohydrate chains, chemical modifications of N-linked or O-linked carbohydrate chains, treatment of the N-terminus or C-terminus, attachment of chemical moieties to the amino acid backbone, and addition or deletion of N-terminal methionine residues produced by prokaryotic host cell expression. Post-translational modifications may also include modification with a detectable label (such as an enzyme label, a fluorescent label, a luminescent label, an isotope label, or an affinity label) to allow detection and separation of proteins. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, and phycoerythrin. Examples of luminescent materials include luminol and bioluminescent materials such as luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

[0290] Additional examples of post-translational modifications include acetylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or a nucleotide derivative, covalent attachment of a lipid or a 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.

[0291] Polynucleotides, vectors and host cells

[0292] Certain embodiments of the present disclosure relate to an isolated polynucleotide or a set of polynucleotides encoding an anti-FRα antibody construct described herein. A polynucleotide in this context may encode all or part of an anti-FRα antibody construct.

[0293] The terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or their analogs) of any length. 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.

[0294] A polynucleotide that "encodes" a given polypeptide is one that is transcribed (in the case of DNA) or translated (in the case of mRNA) into the 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' (carboxyl) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0295] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding the anti-FRα antibody constructs described herein. The one or more polynucleotides may be contained in a single vector, or in more than one vector. In some embodiments, the polynucleotides are contained in a multicistronic vector.

[0296] Certain embodiments of the present disclosure relate to host cells comprising polynucleotides encoding anti-FRα antibody constructs described herein or one or more vectors comprising the polynucleotides. In some embodiments, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0297] Antibody-drug conjugates

[0298] Certain embodiments of the present disclosure relate to antibody-drug conjugates (ADCs) comprising an anti-FRα antibody construct as described herein conjugated to one or more drug moieties, such as a cytotoxin or an immunomodulator.

[0299] Typically, in an ADC, the anti-FRα antibody construct is conjugated to a drug moiety via a linker, which may be a cleavable linker or a non-cleavable linker. The anti-FRα antibody construct may be conjugated to a single drug molecule, or it 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 to antibody ratio (DAR). In certain embodiments, in the ADC of the present disclosure, the DAR is in the range of about 1 to about 12, or about 2 to about 12, or about 2 to about 8.

[0300] In certain embodiments, the ADC comprising the anti-FRα antibody construct has the general formula I:

[0301] A-(L-(D) m ) n (I)

[0302] wherein A is an anti-FRα antibody construct as described herein; L is a linker; D is a drug moiety; m is between 1 and about 8, and n is between 1 and about 12.

[0303] In certain embodiments of Formula I, m is between 1 and 6. In some embodiments, m is 1 or 2. In some embodiments, n is between about 1 and about 8, such as between about 2 and about 8.

[0304] Various compounds known to be useful as cytotoxic or immunomodulatory ADC payloads can be used as drug moieties in ADCs comprising the anti-FRα antibody constructs. Examples include, but are not limited to, maytansinoids and maytansinoid analogs, benzodiazepines, and pyrrolobenzodiazepine Duocarmycins (such as CC-1065) and their analogs, calicheamicin and calicheamicin analogs, auristatins and auristatin analogs, hemiasterlin and hemiasterlin analogs, tubulysin and tubulysin analogs, amatoxin and amatoxin analogs, camptothecin and camptothecin analogs, eribulin, TLR agonists (such as TLR7 and / or TLR8 agonists) or STING agonists.

[0305] In certain embodiments, the drug moiety comprised by the ADC of the present disclosure is an auristatin or an auristatin analog, hemistatin or a hemistatin analog, camptothecin or a camptothecin analog, or eribulin.

[0306] Typically, in the ADC of the present disclosure, the drug moiety is connected to the anti-FRα antibody construct via a linker. A linker is a bifunctional or multifunctional moiety capable of connecting one or more drug molecules to the antibody construct. In some embodiments, the linker may be bifunctional (or monovalent) such that it connects a single drug molecule to a single site on the antibody construct. In some embodiments, the linker may be multifunctional (or multivalent) such that it connects more than one drug molecule to a single site on the antibody construct. In some embodiments, a multifunctional linker may also be used to connect one drug molecule to more than one site on the antibody construct.

[0307] Attachment of the linker to the anti-FRα antibody construct can be achieved in a variety of ways, such as through surface lysines, reductive coupling with oxidized carbohydrates, or through cysteine ​​residues released by reduction of interchain disulfide bonds. Alternatively, the linker can be attached to the anti-FRα antibody construct by modifying the antibody construct to include an additional cysteine ​​residue (see, e.g., U.S. Pat. Nos. 7,521,541; 8,455,622 and 9,000,130) or providing a non-natural amino acid with a reactive handle, 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., 2014, Bioconj. Chem., 25: 351-361). Another option is to use GlycoConnect TM Technology (Synaffix BV, Nijmegen, Netherlands) involves enzymatic remodeling of antibody glycans to allow attachment of linkers via metal-free click chemistry (see, eg, European Patent No. EP 2 911 699).

[0308] The linker generally includes a functional group capable of reacting with one or more target groups on the antigen-binding construct, and one or more functional groups capable of reacting with the target group on the drug moiety. Suitable functional groups are known in the art and include those described in, for example, Bioconjugate Techniques (GT Hermanson, 2013, Academic Press). Non-limiting examples of the functional group reacting with free cysteine ​​or thiol include maleimide, haloacetamide, haloacetyl, activated ester (such as succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester), anhydride, acyl chloride, sulfonyl chloride, isocyanate and isothiocyanate. In this case, "self-stabilizing" maleimide can also be used, such as Lyon et al., 2014, Nat.Biotechnol., 32: those described in 1059-1062. Non-limiting examples of functional groups for reacting with surface lysine and amines include activated esters (such as N-hydroxysuccinamide (NHS) esters and sulfo-NHS esters), imidoesters (such as Traut's reagent), isothiocyanates, aldehydes, and anhydrides (such as diethylenetriaminepentaacetic anhydride (DTPA)). Other examples include the use of succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolephosphonium hexafluorophosphate (PyBOP) to convert carboxylic acids into activated esters, which can then react with amines. Non-limiting examples of functional groups capable of reacting with electrophilic groups (such as aldehyde or keto carbonyl) on antibody constructs or drug moieties include hydrazides, oximes, amino groups, hydrazines, thiosemicarbazones, carboxylic acid hydrazides, and aromatic hydrazides.

[0309] In certain embodiments, a linker including a functional group that allows for bridging of two interchain cysteines on the antibody binding construct may be used, such as ThioBridge TM 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 linker (Lee et al., 2016, Chem. Sci., 7: 799-802) or dibromopyridazinedione-based linker (Maruani et al., 2015, Nat. Commun., 6: 6645).

[0310] A variety of linkers for attaching drugs to antibodies are known in the art, including hydrazone-, disulfide- and peptide-based linkers. Linkers can be cleavable or non-cleavable. Cleavable linkers are generally susceptible to cleavage under intracellular conditions, such as by lysosomal processes. Examples include protease-sensitive, acid-sensitive or reduction-sensitive linkers. In contrast, non-cleavable linkers rely on degradation of the antibody in the cell, which generally results in release of the amino acid-linker-drug moiety.

[0311] Examples of cleavable linkers that can be used in certain embodiments are peptide-containing linkers that can be cleaved by intracellular proteases such as lysosomal proteases or endosomal proteases. Examples include dipeptide-containing linkers, such as 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, )Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys or Met-(D)Lys; tripeptide-containing linkers, such as those containing the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe-Lys or (D)Ala-Phe-Lys, and tetrapeptide-containing linkers, such as those containing the tetrapeptides Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly or Ala-Leu-Ala-Leu.

[0312] Additional useful cleavable linkers include linkers containing disulfide bonds and linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers. Examples of linkers containing disulfide bonds include, but are not limited to, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB). Linkers containing disulfide bonds may optionally include additional groups to provide steric hindrance near the disulfide bonds to improve the extracellular stability of the linker, for example, including geminal dimethyl groups. Linkers containing combinations of these functionalities may also be useful, for example, linkers containing both hydrazones and disulfide bonds are known in the art.

[0313] Another example of a cleavable linker is a linker comprising β-glucuronide, which can be cleaved by β-glucuronidase, an enzyme present in lysosomes and tumor stroma (see, e.g., De Graaf et al., 2002, Curr. Pharm. Des., 8: 1391-1403).

[0314] The cleavable linker may optionally further comprise one or more additional functional groups, such as a self-immolative / self-immolative group, a stretch or a hydrophilic moiety.

[0315] Self-decomposition and self-eliminating groups that can be used for joints include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, methylated ethylenediamine (MED) and hemiacetal groups. Other examples of self-decomposition groups include, but are not limited to, aromatic compounds similar to PABC or PABE groups in electronic properties, such as heterocyclic derivatives, such as 2-aminoimidazole-5-methanol derivatives, as described in U.S. Patent No. 7,375,078. Other examples include groups that undergo cyclization when the amide bond is hydrolyzed, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, ChemistryBiology, 2: 223-227) and 2-aminophenylpropionic acid amides (Amsberry et al., 1990, J.Org.Chem., 55: 5867-5877). Self-decomposition / self-eliminating groups, alone or in combination, are generally included in peptide-based joints, but may also be included in other types of joints. In some embodiments, the linker can include one or more self-immolative / self-degrading groups, such as a PABC group, a PABE group, or a combination of a PABC or PABE group and a MED.

[0316] Extensions that can be used in the linker of ADC include, for example, alkylene and extensions based on aliphatic acids, diacids, amines or diamines, such as diglycolates, malonates, caproates and caproamides. Other extensions include, for example, glycine-based extensions and polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG) extensions. PEG and mPEG extensions also act as hydrophilic moieties and can be particularly useful with hydrophobic drugs, but their use in linkers with other drugs is also contemplated in some embodiments.

[0317] In certain embodiments, the extension segment may have one of the following structures:

[0318]

[0319] in:

[0320] R is H or C1-C6 alkyl;

[0321] t is an integer between 2 and 10, and

[0322] u is an integer between 1 and 10.

[0323] In some embodiments, in the ADC of Formula I, Linker L is a cleavable linker having Formula II:

[0324]

[0325] in:

[0326] Z is a linker group that connects the linker to the target group on the anti-FRα antibody construct A;

[0327] Str is the extension segment;

[0328] AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] r Formation of protease cleavage sites;

[0329] X is a self-decomposing group;

[0330] q is 0 or 1;

[0331] r is 1, 2, or 3;

[0332] s is 0, 1, or 2;

[0333] # is the connection point with the anti-FRα antibody construct A, and

[0334] % is the point of attachment to the drug moiety D.

[0335] In some embodiments, in the linker of formula (II):

[0336] Z is Where # is the point of connection to A and * is the point of connection to the rest of the linker.

[0337] In some embodiments, in the linker of formula (II), Str is selected from:

[0338]

[0339] in:

[0340] R is H or C1-C6 alkyl;

[0341] t is an integer between 2 and 10, and

[0342] u is an integer between 1 and 10.

[0343] In some embodiments, the ADC of Formula I may comprise a disulfide bond-containing linker. In some embodiments, in the ADC of Formula I, the linker L is a cleavable linker having Formula III:

[0344]

[0345] in:

[0346] Z is a linker group that connects the linker to the target group on the anti-FRα antibody construct A;

[0347] Q is -(CH2) p -or-(CH2CH2O) q -, wherein p and q are each independently an integer between 1 and 10;

[0348] Each R is independently H or C1-C6 alkyl;

[0349] n is 1, 2 or 3;

[0350] # is the connection point with the anti-FRα antibody construct A, and

[0351] % is the point of attachment to the drug moiety D.

[0352] In some embodiments, the ADC of Formula I may comprise a b-glucuronide-containing linker.

[0353] Various non-cleavable joints are known in the art for connecting drugs to antibodies, and can be used in ADCs of the present disclosure in certain embodiments. Examples of non-cleavable joints include having N-succinimidyl esters or N-sulfosuccinimidyl esters for reacting with antibodies, and joints for reacting with drugs based on maleimido or haloacetyl parts (or vice versa). Examples of such non-cleavable joints are based on sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylates (sulfo SMCC). Sulfo-SMCC conjugation usually occurs via maleimide groups reacted with sulfhydryl (thiol, -SH), and sulfo-NHS esters are reactive to primary amines. Other non-limiting examples of such linkers include those based on N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-aminohexanoate) ("long chain" SMCC or LC-SMCC), kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), gamma-maleimidobutyric acid N-succinimidyl ester (GMBS), epsilon-maleimidobutyric acid N-succinimidyl ester (EMB), and succinimidyl-succinimidyl ester (SMB), succinimidyl-succinimidyl ester (SMB), and succinimidyl-succinimidyl ester (SMB). Examples include imidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(αmaleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-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).

[0354] ADCs comprising anti-FRα antibody constructs as described herein can be prepared by one of several routes known in the art, using standard organic chemistry reactions, conditions and reagents (see, e.g., Bioconjugate Techniques (G T Hermanson, 2013, Academic Press). For example, conjugation can be achieved by: (1) reacting a functional group of the antibody construct with a divalent 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 the 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 the 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.

[0355] 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 and March, 2006, 6th edition, Wiley); Toki et al., 2002, J. Org. Chem., 67: 1866-1872; Frisch et al., 1997, Bioconj. Chem., 7: 180-186; Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press) and Antibody-Drug Conjugates: Methods in Molecular Biology (Ducry (ed.), 2013, Springer)). In addition, many preformed drug-linkers suitable for reaction with a selected antibody construct are also commercially available, e.g., drug-linkers comprising DM1, DM4, MMAE, MMAF, or duocarmycin SA are commercially available from Creative BioLabs (Shirley, NY). Various antibody-drug conjugation services are also 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).

[0356] Once prepared, the ADC can be purified by standard techniques such as chromatography (eg, HPLC, size exclusion, adsorption, ion exchange, and / or affinity capture), dialysis, and / or tangential flow filtration.

[0357] How to use

[0358] Certain aspects of the present disclosure relate to therapeutic or diagnostic uses of anti-FRα antibody constructs and ADCs. FRα is overexpressed in a variety of cancers, and therefore certain embodiments of the present disclosure relate to methods of using anti-FRα antibody constructs and ADCs in the treatment or diagnosis of FRα-positive cancers.

[0359] 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 construct and ADC can be used in methods of treating FRα-positive cancers or tumors in subjects.

[0360] 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 methods for treating FRα-positive cancers with anti-FRα antibody constructs or ADCs as 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 can be used to treat triple-negative breast cancer (TNBC).

[0361] Treatment of FRα-positive cancer may result in one or more of the following: relief 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, reduction in the rate of disease progression, improvement or palliation of the disease state, improved survival, increased progression-free survival, remission and / or improved prognosis.

[0362] In certain embodiments, when used to treat cancer, the anti-FRα antibody construct or ADC can be systemically administered 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 locally administered to the subject at the site to be treated.

[0363] It is expected that the anti-FRα antibody construct or ADC can be used alone or in combination with one or more known chemotherapeutic agents or immunotherapeutic agents commonly used to treat cancer. The combination of the anti-FRα antibody construct or ADC and standard chemotherapeutic agents or immunotherapeutic agents can be used to improve the efficacy of chemotherapeutic agents or immunotherapeutic agents, and thus can improve standard cancer therapy. This application may be important in the treatment of drug-resistant cancers that do not respond to standard treatment. When used together with one or more known chemotherapeutic agents or immunotherapeutic agents, the anti-FRα antibody construct or ADC can be administered before or after the administration of the chemotherapeutic agent or immunotherapeutic agent, or they can be administered simultaneously.

[0364] The dose of the anti-FRα antibody construct or ADC to be administered is not limited, but it will be a therapeutically effective amount. "Therapeutically effective amount" refers to the amount of the anti-FRα antibody construct or ADC described herein that is sufficient to achieve treatment for a specific indication when administered to a subject. For cancer treatment, a therapeutically effective amount of an anti-FRα antibody construct or ADC may, for example, have one or more of the following effects: reducing the number of cancer cells, reducing tumor size, inhibiting cancer cell infiltration into surrounding organs, inhibiting tumor metastasis, inhibiting tumor growth; increasing survival time and / or alleviating one or more symptoms associated with cancer to a certain extent. For cancer therapy, efficacy may alternatively be measured, for example, by assessing time to disease progression (TTP) and / or and / or determining a response rate (RR).

[0365] Certain embodiments relate to methods for detecting the presence of FRα in a biological sample (such as a sample containing cells or tissue) using an anti-FRα antibody construct described herein. In some embodiments, the biological sample can be taken from a patient, such as a patient known or suspected of having cancer. Some embodiments relate to methods for detecting the presence of FRα in a biological sample, comprising contacting the sample with an anti-FRα antibody construct described herein.

[0366] Certain embodiments relate to methods of diagnosing a disorder associated with increased FRα expression (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 the subject, or may be an in vitro method in which a sample taken from the subject is contacted with the anti-FRα antibody construct. For in vivo methods, administration may be systemic or local.

[0367] In methods of detecting the presence of FRα or diagnosing a disorder associated with increased expression of FRα, the anti-FRα antibody construct can be labeled with a detectable label known in the art, such as a fluorescent, luminescent, chromogenic, chemiluminescent, radioactive or enzymatic label.

[0368] Pharmaceutical composition

[0369] For therapeutic use, the anti-FRα antibody construct and ADC can be provided in the form of a pharmaceutical composition comprising the anti-FRα antibody construct or ADC and a pharmaceutically acceptable carrier or diluent. The composition can be prepared by known procedures using well-known and readily available ingredients.

[0370] The pharmaceutical composition can be formulated for administration to a subject, for example, parenteral, oral (including, for example, buccal or sublingual), topical, rectal or vaginal routes, or by inhalation or spraying. "Parenteral" administration can be subcutaneous injection or intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal or intrathecal injection or infusion. The pharmaceutical composition will generally be formulated into a form suitable for administration to a subject, such as a syrup, elixir, tablet, lozenge, pastille, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granules, emulsion, injection or solution. The pharmaceutical composition can be provided as a unit dose formulation.

[0371] In certain embodiments, pharmaceutical compositions comprising the anti-FRα antibody constructs or ADCs may be formulated for parenteral administration by infusion or in a unit dose injectable form (eg, as a lyophilized formulation or an aqueous solution).

[0372] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl paraben or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin 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 ions; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as polyethylene glycol (PEG).

[0373] In certain embodiments, the pharmaceutical composition comprising the anti-FRα antibody construct or ADC may be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions may be prepared using suitable dispersants 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 used include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile fixed oils may be used as solvents or suspension media. To this end, various mild fixed oils may be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used in injectable preparations. Adjuvants, such as local anesthetics, preservatives, and / or buffers may also be included in injectable solutions or suspensions.

[0374] In certain embodiments, the pharmaceutical composition comprising the anti-FRα antibody construct or ADC can be formulated for intravenous administration to a subject, such as a human. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and / or a local anesthetic, such as lidocaine, to relieve pain at the injection site. Typically, the components are provided separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or a sachet) indicating the amount of the active agent. When the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0375] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0376] Medicine prescription box

[0377] Certain embodiments relate to pharmaceutical kits comprising an anti-FRα antibody construct or ADC as described herein.

[0378] The kit will typically include a container containing the anti-FRα antibody construct or ADC and a label and / or package insert on or attached to the container. The label or package insert contains instructions typically included in the commercial packaging of the therapeutic product, providing information about the indications, usage, dosage, administration, contraindications and / or warnings for the use of such therapeutic products. The label or package insert may also include a notice in a form prescribed by a government agency regulating the manufacture, use or sale of drugs or biological products, which reflects the approval of the use or sale of the manufacturing agency 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 a vial with a stopper pierceable by a hypodermic needle.

[0379] In addition to the container containing the anti-FRα antibody construct or ADC, the kit may optionally include one or more additional containers containing other components of the kit, for example, a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution), other buffers or diluents.

[0380] Suitable containers include, for example, bottles, vials, syringes, and intravenous solution bags, etc. The container may be made of various materials such as glass or plastic. Where appropriate, one or more components of the kit may be lyophilized or provided in a dry form (such as a powder or granules), and the kit may additionally contain a suitable solvent for reconstituting the lyophilized or dried component.

[0381] The kit may also include other materials desirable from a commercial and user standpoint, such as filters, needles, and syringes.

[0382] The following examples are offered for illustrative purposes and are not intended to limit the scope of the present invention in any way.

[0383] Example

[0384] Overview

[0385] Bioassays: Expression levels of FRα in cell lines and CDX models were assessed in-house using a research-grade IHC assay and assigned relative expression levels (high / medium / low or strong / medium / weak). PDX models were similarly assessed using archived tumor samples.

[0386] Example 1: Preparation of anti-folate receptor α antibody

[0387] Antibodies that specifically bind to folate receptor alpha (FRα) were generated by immunizing rabbits with human FRα antigen, isolated and sequenced as described below.

[0388] 1.1 Immunity

[0389] Antibodies to FRα were generated in rabbits immunized with soluble HIS-labeled human folate receptor 1 antigen (FRα-HIS) (ACROBiosystems, Newark, DE; Catalog No. 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 3 sites along the rabbit's dorsal body (0.333 mL / site). These were followed by 4 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 for serological analysis before the fourth immunization.

[0390] 1.2 Selection of animals for harvest

[0391] Anti-human FRα antibody titers were determined by flow cytometry using CHO cells expressing human FRα. TM CHO cells were transiently transfected with a pTT5-based expression plasmid encoding human FRα (National Research Council of Canada) using the instructions of 2000 (Thermo Fisher Scientific Corp., Waltham, MA). Dilutions of immune rabbit serum starting at 1:400 and serially diluted 1:2 for 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 using 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 a fluorescent signal at least 2 times background.

[0392] 1.3 Recovery of B cells and discovery of anti-human FRα antibodies

[0393] Immunized rabbits with desired titers above 100,000 were sacrificed and spleens were harvested. Lymphoid cells were dissociated by trituration in FACS buffer (PBS, 2% v / v FBS) to release the cells from the tissue. The cells were pelleted and then washed with 5 mL of Pharm Lyse TM (Becton, Dickinson & Co., Franklin Lakes, NJ) for 1 minute to lyse red blood cells. Add an equal volume of FACS buffer to neutralize Pharm LyseTM The resulting lymphocyte samples were pelleted and resuspended in FACS buffer.

[0394] 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. TM IgG+B cells were sorted and counted on a ELISA kit (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 in 384-well plates at different densities from a single cell to 50 cells, 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.

[0395] Human FRα-specific monoclonal antibodies in the supernatant were screened 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 blocking buffer (2% skim milk, PBS) was added and the plates were incubated at room temperature for 1 hour. After incubation, the plates were washed and 12.5 μL / well of antibody-containing supernatant + 12.5 μL blocking buffer and positive and negative controls were added, and the plates were incubated at room temperature for 2 hours.

[0396] After incubation, the plates were washed, 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 plates were incubated for 1 hour at room temperature. After incubation, the plates were washed and 25 μL of tetramethylbenzidine (TMB) was added, and the plates were allowed to develop for approximately 10 minutes (until the negative control wells began to show signal). 25 μL of stop solution (1N HCL) was then added to each well and the plates were incubated at room temperature for 1 hour. TM The plates were read on H1 microplates (BioTek Instruments, Winooski, VT) at a wavelength of 450 nm.

[0397] 1.4 Sequencing of Anti-human FRα Antibodies

[0398] 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 using SuperScript TM cDNA was transcribed from mRNA using PCR products (Thermo Fisher Scientific Corp., Waltham, MA) and oligo-dT20 (Integrated DNA Technologies, Inc., Coralville, IA). 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 using cDNA as a 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. Zero Blunt TM TOPO TM The PCR product was cloned into pCRTOPO4 using a PCR cloning kit (ThermoFisher Scientific Corp., Waltham, MA) and transformed into Cells (Lucigen Corporation, Middleton, WI). Antibiotic-resistant clones were subjected to Sanger sequencing and analyzed for unique antibody coding sequences.

[0399] 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). Unique heavy and light chain sequences from single-well samples were co-expressed in all possible combinations in HEK293-6E cells (National Research Council of Canada) to determine the correct heavy and light chain pairing. The generated antibodies were assayed for binding to antigens transiently expressed on HEK293 cells.

[0400] 1.5 Production of chimeric antibodies

[0401] The coding sequences of the antibody variable regions were cloned in-frame into the huIgG1 expression vector and the huCK expression vector (based on the pTT5 vector). The huIgG1 constant region starts at alanine Kabat-118, and the huCK constant region starts at arginine Kabat-108. The activity of the resulting recombinant chimeric antibodies was confirmed in a specific binding assay and found to be equivalent to the parental antibody.

[0402] Example 2: Humanization of anti-FRα antibody

[0403] One of the chimeric anti-human FRα (anti-hFRα) generated as described in Example 1, variant v23924, 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.

[0404] Table 2.1: CDR sequences of anti-hFRα antibody v23924

[0405]

[0406] Table 2.2: VH and VL sequences of anti-hFRα antibody v23924

[0407]

[0408] 2.1 Humanization

[0409] The rabbit VH and VL sequences of v23924 were aligned with the corresponding human germline sequences, and IGHV3-23*01 and IGKVI-39*01 were identified as the closest and most common human germline sequences. The CDR sequences defined according to AbM (see Table 2.1) were transplanted onto the framework of these selected human germline sequences, as shown in Figure 1. Back mutations to rabbit residues at positions in the resulting sequence that were judged to be important for maintaining binding affinity to the antigen hFRα were included to generate several humanized sequences, where the generated sequences were largely built on the previous sequence and where the first humanized sequence contained the minimum number of back mutations. No variant modified the CDRs of the parent antibody as defined by the AbM method.

[0410] The process is carried out in two cycles, wherein the first cycle ("cycle 1") produces six variable heavy chain humanized sequences and five variable light chain humanized sequences. The second cycle ("cycle 2") is expanded to another 5 variable heavy chain humanized sequences, in pursuit of a closer parent-like affinity of humanized antibodies to hFRα. Complete heavy chain sequences containing humanized heavy chain variable domains (VH) and hIgG1 heavy chain constant domains (CH1, hinge, CH2, CH3), and complete light chain sequences containing humanized light chain variable domains (VL) and human κ light chain constant domains (κCL) are assembled. Monoclonal antibody (mAb) variants are then assembled so that in cycle 1, each humanized heavy chain is paired with each humanized light chain to provide 30 humanized variants, and in cycle 2, additional humanized heavy chains are paired with selected humanized light chains to obtain another 15 humanized variants, with a total of 45 humanized variants to be evaluated using experimental methods.

[0411] 2.2 Generation of humanized antibodies

[0412] Each of the 45 humanized mAb constructs, as well as the parental v23924 mAb construct, was produced in a full-size antibody (FSA) format containing two identical full-length heavy chains (parental v23924 and 15 humanized variants), resulting in a homodimeric Fc region (HomoFc), or containing heterodimeric full-length heavy chains (30 humanized variants) in the CH3 region that contained complementary mutations to drive unique heavy chain pairing, resulting in a heterodimeric Fc region (HetFc). A version of v23924 containing HetFc instead of HomoFc was also produced (variant v30618). All constructs included two identical kappa light chains.

[0413] The two identical full-length heavy chains contained in the HomoFc region contain 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 (HetFc-A and HetFc-B) contained in the HetFc region contain the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01, and have the following mutations in the Fc region:

[0414] HetFc-A:T350V_L351Y_F405A_Y407V

[0415] HetFc-B:T350V_T366L_K392L_T394W

[0416] The sequences of HetFc-A (SEQ ID NO: 148) and HetFc-B (SEQ ID NO: 149) are provided in Table 2.3. For all constructs, the human kappa CL sequence of IGKC*01 (SEQ ID NO: 147; see Table 2.3) was used.

[0417] Table 2.3: HomoFc and HetFc sequences

[0418]

[0419] Each humanized VH domain sequence from cycle 1 was appended to the human CH1-hinge-CH2-CH3 (HetFc-A and HetFc-B) domain sequence of IGHG1*01 to provide twelve humanized full heavy chain sequences (six humanized VH x2). Rabbit VH and each of the five additional humanized VH domain sequences from cycle 2 were appended to the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 to provide the parent rabbit-human chimeric full heavy chain sequence and five additional humanized full heavy chain sequences. Each VL domain sequence was appended to the human κCL sequence of IGKC*01 to provide five humanized light chain sequences. All sequences were reverse translated into DNA, codon optimized for mammalian expression, and gene synthesized.

[0420] 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 terminating at residue G446 (EU numbering) of the CH3 domain were ligated into the pTT5 vector to generate a heavy chain expression vector. The light chain vector insert containing the same signal peptide was ligated into the pTT5 vector to generate a light chain expression vector. The resulting heavy and light chain expression vectors were sequenced to confirm the correct reading frame and sequence of the encoding DNA.

[0421] The heavy and light chains of each humanized antibody variant were expressed in 200 ml CHO-3E7 cell culture. Briefly, the cells were cultured at 37°C in FreeStyle 400 μL PBS supplemented with 4 mM glutamine (GE Life Sciences, Marlborough, MA) and 0.1% Pluronica F-68 (Gibco / Thermo Fisher Scientific, Waltham, MA). TMCHO-3E7 cells were cultured in F17 medium (ThermoFisher Scientific, Waltham, MA) at a density of 1.7-2 x 10 6 cells / ml, viability>95%. Use (Polyscience, Inc., Philadelphia, PA), a total of 200ug DNA (100ug antibody DNA and 100ug GFP / AKT / filler DNA) was used to transfect CHO-3E7 cells in a total volume of 200ml at a DNA:PEI ratio of 1:4 (w / w) + 1x antibiotic / antimycotic (GE Life Sciences, Marlborough, MA). Twenty-four hours after adding the DNA-PEI mixture, 0.5mM 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 another 6 days before harvesting.

[0422] In batch mode or using 1mL HiTrap TM MabSelect TM SuRe TM Protein A purification was performed using a Cytiva ® column (Cytiva, Marlborough, MA). In batch mode, the clarified supernatant samples were mixed with mAb Select SuRe TM The resin (GE Healthcare, Chicago, IL) was incubated in batches, cleaned in situ (CIP'd) with NaOH and balanced in Dulbecco's PBS (DPBS). The resin was poured into a CIP'd column and the column was washed with DPBS. In both purification modes, the protein was eluted with 100 mM sodium citrate buffer (pH 3.0). The eluted fraction was pH adjusted by adding 10% (v / v) 1M HEPES (pH about 10.6-10.7) to obtain a final pH of 6-7. The sample buffer was exchanged into DPBS. Protein was quantified based on the absorbance at 280 nm (A280 nm). After protein A purification, the parent 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 a DPBS mobile phase.

[0423] After purification, high-throughput protein expression assays and Caliper The purity of the samples was assessed by electrophoresis under non-reducing and reducing conditions using a GXII or GXII Touch HT (Perkin Elmer, Waltham, MA). The procedure was followed according to the User Guide, Version 2, with the following modifications. 2 μl or 5 μl of antibody sample (concentration range 5-2000 ng / μl) was added to individual wells in a 96-well plate (BioRad, Hercules, CA) along with 7 μl of HT protein expression sample buffer (Perkin Elmer, catalog number 760328). The antibody samples were then denatured at 70°C for 15 minutes. The instrument was operated using the HT Protein Express Chip (Perkin Elmer, Waltham, MA) and the Ab-200 assay setup.

[0424] The yields of the 45 humanized antibody variants and the parent chimeric antibody v23924 (after protein A purification) ranged from approximately 9-17 mg (or 45-85 mg / L). Figure 2 A and Figure 2 C shows the Caliper electrophoresis results of the parent chimeric antibody v23924 and the representative humanized variant v30384. Figure 2 As can be seen, for a representative humanized antibody sample, both non-reduced (NR) and reduced (R) Calipers reflect a single species corresponding to the full-size antibody and intact heavy and light chains. This is also the case for the other humanized variants.

[0425] 2.3 Quality assessment of humanized antibodies

[0426] 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.

[0427] The samples were analyzed using a Waters Acquity UPLC system set at 30 °C with a photodiode array (PDA) detector. TMUPLC-SEC was performed on a Waters Acquity BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 μm particles) (Waters LTD, Mississauga, ON) on an H-Class Bio system. The mobile phase was Dulbecco's phosphate buffered saline (DPBS) (pH 7.4) containing 0.02% Tween 20, and the flow rate was 0.4 ml / min. The total run time for each injection was 7 minutes, 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. A Waters 3 software, using Apex Track TM Peak integration was performed and shoulder features were detected.

[0428] Figure 2 B and Figure 2 D shows the UPLC-SEC profiles of the parent chimeric antibody v23924 (after SEC purification) and the representative humanized antibody v30384 (after protein A purification), respectively. The UPLC-SEC profiles of the representative humanized antibody samples reflect the same high species homogeneity as the parent chimeric antibody samples. Samples from the remaining humanized antibody variants have similar characteristics as those shown in the representative humanized antibody samples.

[0429] Example 3: Binding of humanized antibodies to hFRα

[0430] 3.1 Evaluation of affinity of humanized antibodies for hFRα

[0431] To determine whether the humanization process affected the affinity of the humanized variants for their target, the ability of 45 purified humanized antibody variants to bind to the hFRα antigen was assessed by biolayer interferometry (BLI) as follows.

[0432] use The RED96 system (ForteBio, Fremont, CA) was used to assess hFRα antigen binding by cycling the following steps: loading the antibody (0.9 μg / mL) onto the anti-human IgG Fc capture (AHC) biosensor within 200 s; stabilizing the baseline for 60 s; and quantifying the hFRα antigen binding across the expected K. DAssociation with recombinant His-tagged human FRα (ACRO Biosystems, Newark, DE) at multiple relevant concentrations of 400-500 seconds; dissociation was recorded for 500-1000 seconds; regeneration was performed by cycling 3 times between 10 mM glycine pH 1.5 (15 seconds) and assay buffer (1 second) before proceeding to the next antibody. The assay buffer used was KB buffer (kinetic buffer, composed 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 cases 1% BSA. The experiment was performed at 30°C with a shaking speed of 1000 rpm.

[0433] Data analysis was performed using ForteBio 9.0 (ForteBio, Fremont CA). Reference-subtracted binding curves were globally fit to a 1:1 interaction model to generate binding kinetic parameters k on , k off and the dissociation constant K D .

[0434] Nine of the 45 humanized antibody variants were found to have K values ​​ranging from about 63 nM to 210 nM. D The K values ​​of the parental chimeric antibody (v23924) were determined. D The humanized antibody variants that exhibit binding to hFRα are characterized by a decrease in affinity of about 2-fold to about 8-fold compared to the parent chimeric antibody. As can be seen from Table 3.1, all successful humanized variants have the same variable light chain (4L) but differ in variable heavy chain composition. The light and heavy chain sequences of successful humanized variants are provided in Table 3.2. Figure 3 BLI sensorgrams of the parental chimeric antibody v23924 and the representative humanized antibody v30384 are shown.

[0435] Table 3.1: Pass Antigen binding assessment of humanized variants

[0436]

[0437]

[0438] 1 HetFc = heterodimeric Fc region; HomoFc = homodimeric Fc region (see Example 2)

[0439] 2 n=2

[0440] Table 3.2: Amino acid sequences of humanized VH and VL chains

[0441]

[0442] 3.2 Evaluation of the affinity of humanized antibodies for hFRα

[0443] The affinity of antigen binding of the parental and selected humanized variants in FSA format was assessed by surface plasmon resonance (SPR) as described below.

[0444] The SPR assays for determining the hFRα affinity and avidity of the parent chimeric antibody (v23924) and humanized variants were performed at 25°C on a Biacore TM The T200 SPR system was run with PBS-T (PBS + 0.05% (v / v) Tween 20) running buffer (with 0.5M EDTA stock solution added to reach a final concentration of 3.4mM). CM5 Series S sensor chip, Biacore TM 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).

[0445] Screening for variants binding to the hFRα antigen was performed in two steps: capture of hFRα onto the surface followed by injection of 3 to 5 concentrations of variants. The hFRα surface was prepared on a CM5 Series S sensor chip by standard amine coupling methods as described by the manufacturer (GE Healthcare LifeScience, Mississauga, ON, Canada). Biacore was used for resonance units (RU) ranging from 5 to 200 RU. TM T200 Immobilization Wizard and amine coupling method were used for immobilization of hFRα. Using multi-cycle kinetics, a two-fold dilution series of samples starting at 300nM with blank buffer was injected at 50uL / min for 180 seconds with a 600-second dissociation phase to generate a set of sensorgrams with buffer blank reference. The hFRα surface was regenerated in preparation for the next injection cycle by a single pulse of 10mM glycine / HCl (pH 1.5) at 30uL / min for 30 seconds. Biacore TM The blank-subtracted sensorgrams were analyzed using T200 Evaluation Software v3.0. The blank-subtracted sensorgrams were then fitted to a 1:1 Langmuir binding model.

[0446] The results are shown in Table 3.3. The parent antibody (v23924) and two humanized variants (v30384 and v30399) in the conventional bivalent antibody format (FSA) all exhibited avidity binding to the hFRα antigen. That is, the K obtained in FSA was significantly higher than that in the one-armed antibody (OAA) format at medium-low antigen density. D The values ​​were about 1 / 17 in the case of the parental sample and about 1 / 27-1 / 39 in the case of the humanized sample, which were further reduced to about 1 / 108 and about 1 / 116-1 / 181, respectively, at high antigen density.

[0447] Table 3.3: Affinity assessment of selected humanized variants by SPR

[0448]

[0449]

[0450] 1 OAA = one-armed antibody, FSA = full-sized antibody; OAA antibody samples are equivalent to the corresponding FSA samples and were produced in a similar manner as described in Example 2 for the FSA samples.

[0451] 2 n=2

[0452] Example 4: Purity of humanized anti-FRα antibody

[0453] Following protein A purification (Example 2) and native deglycosylation, the apparent purity of the humanized antibody variants from Example 3 was assessed using mass spectrometry.

[0454] Since the antibody variant samples only contained Fc N-linked glycans, the samples were treated with N-glycosidase F (PNGase-F) only. The purified samples were deglycosylated with PNGase F as follows: 0.1 U PNGase F / μg antibody in 50 mM Tris-HCl (pH 7.0), incubated overnight at 37°C, with a final protein concentration of 0.48 mg / mL. After deglycosylation, the samples were stored at 4°C before LC-MS analysis.

[0455] Use with LTQ-Orbitrap TMDeglycosylated protein samples were analyzed by intact LC-MS using an Agilent 1100 HPLC system (adjusted for optimal detection of larger proteins (>50 kDa)) coupled to an XL mass spectrometer (ThermoFisher, Waltham, MA) via an Ion Max electrospray source. Samples were injected onto a 2.1 x 30 mm Poros R2 reverse phase column (Applied Biosystems Corp., Waltham, MA) and resolved using a linear gradient of 0.1% formic acid in water / acetonitrile (degassed) consisting of increasing concentrations (20%-90%) of acetonitrile. The column was heated to 82.5°C and the solvent was heated to 80°C before the column 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 analysis of IgG samples using a deglycosylated IgG standard (Waters IgG standard) and a deglycosylated mAb standard mixture (25:75 half-size: full-size antibody). For each LC-MS analysis, mass spectra acquired across the antibody peak (typically 3.6-4.1 minutes) were summed and analyzed using MassLynx TM The entire multiply charged ion envelope (m / z 1,200-4,000) was deconvoluted into a molecular weight profile using the MaxEnt 1 module of the data analysis software (Waters, Milford, MA). The apparent amount of each antibody species in each sample was determined from the peak heights in the resulting molecular weight profile.

[0456] The results are shown in Table 4.1. Almost all humanized variants are highly pure, ranging from about 89% to 100% of the desired species, with only v30389 showing a lower purity (82.6%). The purity of the four variants v30384, v30389, v30394 and v30399 is slightly lower than that of the remaining variants because these variants contain heterodimeric CH3 regions that result in the presence of some half antibodies (see Example 2). Figure 4 Depicted are the LC / MS spectra of two representative humanized variants, v30384 and v31422. In the LC / MS spectra of all samples, a side peak of approximately +266 Da was observed, which may be an artifact of the analysis.

[0457] Table 4.1: Purity of humanized variants determined by LC / MS

[0458] Variants Required type% v30384 93.2 v30389 82.6 v30394 89.1 v30399 91.8 v31422 100 v31423 100 v31424 100 v31425 100 v31426 100

[0459] Example 5: Thermal stability of humanized anti-FRα antibodies

[0460] The thermal stability of the humanized antibody variants was assessed by differential scanning calorimetry (DSC) as described below.

[0461] 400 μ L purified samples mainly at 0.4 mg / mL concentration in PBS are used to perform DSC analysis using VP-capillary DSC (GE Healthcare, Chicago, IL). At the beginning of each DSC run, 5 buffer blank injections are performed to stabilize the baseline, and buffer injections are arranged before each sample injection for reference. Each sample is scanned from 20 ° C to 100 ° C at a rate of 60 ° C / h using low feedback, 8 second filters, 3 minutes of pre-scan thermostats and 70 psi nitrogen pressure. The resulting thermal spectra are referenced and analyzed using Origin 7 software (OriginLab Corporation, Northampton, MA) to determine the melting temperature (Tm) as a thermal stability indicator.

[0462] The Fab Tm values ​​determined for the humanized variants are shown in Table 5.1. All humanized variants exhibited increased thermal stability compared to the parent antibody v23924 (Fab Tm of about 72°C), with Fab Tm values ​​ranging from about 81-84°C. Among the humanized variants, v30394 showed the highest thermal stability.

[0463] Table 5.1: Thermal stability of humanized variants

[0464] Variants Fab Tm(℃) v23924 (parent) 72.1 v30384 81.7 v30389 80.7 v30394 84.4 v30399 83.4 v31422 82.6 v31423 83.0 v31424 81.3 v31425 82.4 v31426 81.9

[0465] Example 6: Determination of isoelectric point of humanized anti-FRα antibody

[0466] The isoelectric points of the humanized antibody variants were determined by capillary isoelectric focusing (cIEF) as described below.

[0467] cIEF was performed using a CE-UV Agilent 7100 capillary electrophoresis (CE) system. 5ug (or maximum 2.5uL) sample was applied to the capillary (ampholyte range of 3.0-10.0). A pI standard mixture, 4.1, 4.22, 5.5, 7.0 and 10.0 were used for system suitability tests, and 4.1 and 10.0 were used for sample analysis. For all CE runs, an Agilent 7100 CE system equipped with an external water bath set to 6°C, a detector filter assembly (280nm) and an external pressure of 9 bar was used. Neutral coated capillaries (fluorocarbons) were cut at a distance of 8.5cm and 24.5cm from the detection window at both ends, respectively, equipped with a green alignment interface and assembled into an Agilent capillary box. Once a day, capillaries were conditioned as follows: high pressure rinsed with 350mM acetic acid at 3.5 bar for 5 minutes, rinsed with water for 2 minutes and rinsed with cIEF gel for 5 minutes. Before each operation, the capillary was adjusted as follows: high pressure washing at 3.5 bar for 3 minutes with 4.3M urea solution and rinsed with water for 2 minutes. By applying 2 bar high pressure for 100 seconds, the inlet electrode and outlet electrode water soaking were then carried out to inject the sample. Focusing was performed for 10 minutes at 25kV using 200mM phosphoric acid as anolyte and 300mM NaOH as catholyte. Using chemical mobilization, the outlet bottle was replaced with 350mM acetic acid and 30kV was applied for 30 minutes. After each operation, high pressure washing was performed with water at 3.5 bar for 2 minutes. Manual integration of peak RT and electrophoretogram was obtained using Agilent OpenLAB Intelligent Reporting A.01.06.111 software. Raw data (signal and retention time) were exported to CVS files and the pI (based on internal pI standards) of main isoform pI, pI range and mass center were calculated in Microsoft Excel.

[0468] The results are shown in Table 6.1. The pi values ​​determined for the major isoforms of most humanized variants ranged from 7.78 to 7.97, with one variant having a pi of 8.25 (v31426), all falling within the typical range for therapeutic antibodies and relatively comparable to the pi value of the parental chimeric antibody v23924 (pi of 7.65).

[0469] Table 6.1: Isoelectric points of humanized variants

[0470] Variants pI (major isoform) v23924 (parent) 7.65 v30384 7.79 v30389 7.78 v30394 7.89 v30399 7.89 v31422 7.93 v31423 7.94 v31424 7.97 v31425 7.93 v31426 8.25

[0471] Example 7: Chromatographic analysis of anti-FRα antibodies

[0472] Parental and humanized variants were analyzed by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) as described below.

[0473] 7.1 HIC analysis

[0474] The hydrophilicity / hydrophobicity of the antibody was assessed by HIC as described in Antibody Drug Conjugates, Methods in Molecular Biology, 2013, Vol. 1045, pp. 275-284, edited by L. Ducry. The antibody was pre-equilibrated with 5 column volumes of buffer A (1.5 M (NH4)2SO4, 25 mM NaH2PO4, pH 6.95) at room temperature. Experiments were performed on an Agilent Infinity II 1290 HPLC column (2.5 μm, 4.6 x 35 mm, TOSOH Bioscience GmbH, Griesheim, Germany). Typically, 20 to 30 mg of sample at a concentration of 2 to 3 mg / mL was loaded into a column with 95% buffer A and 5% buffer B (75% 25 mM PO4 3- 25% isopropanol, pH 6.95) was loaded onto the column and run at 0.5 mL / min for 15 minutes using the gradient shown in Table 7.1. The HIC chromatogram was integrated using appropriate parameters that provided a complete baseline to baseline integration.

[0475] Table 7.1: HIC solvent gradient

[0476] Time (minutes) Buffer A% Buffer B% 0 95 5 0.1 95 5 5 80 20 9.5 65 35 11.5 50 50 12.5 5 95 13.5 5 95 12.6 95 5 15 95 5

[0477] 7.2 SEC Analysis

[0478] At room temperature, SEC was performed using an Advance Bio SEC column ( Analytical SEC was performed on an Agilent Infinity II 1260 HPLC (2.7 μm, 7.8 × 150 mm), and the column was balanced with a mobile phase A (150 mM Na2PO4, pH 6.95) of 5 column volumes. Typically, 20-30 mg samples with a concentration of 2-3 mg / mL were eluted with 1 mL / min isostatic pressure for 7 minutes and absorbance was monitored at A280. The chromatogram was integrated to provide a complete baseline-baseline integration of each peak, and the separation position between the partially separated peaks was reasonable. Based on the SEC spectrum of the control trastuzumab, the peak corresponding to the main component of IgG (approximately retention time 3.3 minutes) was reported as a monomer. Any peak appearing before 3.3 minutes was designated as a high molecular weight species (HMWS), and any peak appearing after 3.3 minutes was designated as a low molecular weight species (LMWS), excluding solvent peaks (over 5.2 minutes).

[0479] 7.3 Results

[0480] A summary of the HIC retention time (HIC-RT) and SEC monomer % of the parent and humanized variants is provided in Table 7.2. In summary, HIC and SEC show favorable biophysical behavior of all humanized variants. The parent chimeric antibody v23924 eluted at 6.5 minutes on the HIC gradient, while all humanized variants eluted between 6.0-6.7 minutes. The SEC profile shows >90% monomer for all humanized variants, with variant v30389 having the lowest monomer % (94%). All of these variants have <5% HMWS and <5% LMWS.

[0481] Table 7.2: HIC and SEC analysis of parental and humanized anti-FRα antibodies

[0482]

[0483] *No prior Prep-SEC

[0484] Example 8: Additional stability studies

[0485] To further investigate the stability of the humanized antibody variants, 40°C stability and acid stability studies were performed, wherein samples were characterized at specific time points by Caliper and UPLC-SEC as described in Example 2, and in the case of the 40°C stability studies additionally by cIEF and Octet antigen binding as described in Example 6 and Example 3, respectively. Trastuzumab was used as a control in these studies.

[0486] The selected humanized variants (v30389, v30394, v30399, v31423 and v31424) were subjected to a 40°C stability study for 14 days in PBS pH 7.4 buffer at a sample concentration of about 1 mg / ml. Samples were characterized at time points 0, 5, 7, 11 and 14 days. Acid stability studies were performed after the sample buffer was exchanged for an acetate buffer of pH 3.6 at 25°C for 1 hour at different sample concentrations, wherein the time points were characterized at 0, 15, 30 and 60 minutes. In addition, freeze-thaw (from -80°C to room temperature) was performed in PBS (pH 7.4) at a sample concentration of about 1 mg / ml, and freeze-thaw constituted three cycles of 30 minutes per cycle.

[0487] The results are shown in Table 8.1. No significant issues with the stability of the humanized variants were identified in the study. Freeze-thaw and acid stability studies did not reveal any changes in sample composition during the study as determined by Caliper and UPLC-SEC. The 40°C stability study showed minor changes in the Caliper and UPLC-SEC profiles as the study proceeded (specifically, some amounts of low molecular weight species appeared, ranging from about 10%-17%). No changes in antigen binding affinity were observed. cIEF revealed the expected small increase in more acidic species relative to the major isoform.

[0488] Table 8.1: Stability assessment of humanized variants at 40°C, acid stability, and freeze-thaw studies

[0489]

[0490] 1 Trastuzumab control

[0491] 2 Err = Error Baseline

[0492] Example 9: Functional Characterization of Anti-FRα Antibodies - FRα Specificity

[0493] The parental chimeric antibody v23924 was evaluated for binding cross-reactivity to FRα (FOLR1), FOLR2, FOLR3, and FOLR4 by flow cytometry and ELISA. FRα, FOLR2, and FOLR4 binding were evaluated by flow cytometry using HEK293 transfected cells. FOLR3 binding was evaluated by ELISA because FOLR3 is a soluble protein. A control anti-FOLR2 (mouse anti-human FOLR2; Nordic BioSite AB, Sweden; catalog number AFC-4544-2), anti-FOLR3 (mouse anti-human FOLR3; LS Bio, Seattle, WA; catalog number LS-C125621), and anti-FOLR4 (mouse anti-His DyLight TM 650; Novus Biologicals, Littleton, CO; catalog number NBP2-31055C) antibody was included in these experiments.

[0494] FRα, FOLR2, and FOLR4 bindingBriefly, HEK293-6e cells were transfected for approximately 24 hours to transiently express human FRα (Cat. No. 13420), FOLR2 (Cat. No. 13481), and FOLR4 (Cat. No. 13483) (all from GenScript Biotech, Piscataway, NJ) with 1 ug DNA per 1 million cells. After transfection, 50,000 cells were seeded in a V-bottom 96-well plate and incubated with 50 nM primary antibody for 45 minutes under standard culture conditions. After incubation, cells were washed and stained with anti-human IgG FcAF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) at room temperature (RT) for 45 minutes. After incubation and washing, cells were stained with BD LSRFortessa TM Fluorescence was detected by flow cytometry on a cell analyzer (BD Biosciences, Franklin Lake, NJ), and a minimum of 1,000 events were collected per well.

[0495] FOLR3 binding : ELISA 96-well plates were coated with commercially purified FOLR3 protein (R&D Systems, Inc., Minneapolis, MN; catalog number 5319-FR) at 37°C for 1 hour. The plates were blocked with 1% milk in PBS (pH 7.4) at room temperature for 1 hour. After blocking, primary antibodies were added at 7nM for 1 hour at room temperature. HRP-conjugated secondary antibodies (Jackson Immuno Research Labs, West Grove, PA; catalog number 109-035-098) were then added at 0.4μg / ml at room temperature for 1 hour. The plates were developed using tetramethylbenzidine (TMB) and the reaction was terminated using HCl. The plates were incubated with Synergy TM The absorbance was read at 450 nm using an H1 microplate reader (BioTek Instruments, Winooski, VT).

[0496] result

[0497] The results are shown in Table 9.1 and Table 9.2.

[0498] Anti-FRα, anti-FOLR2, anti-FOLR3, and anti-FOLR4 control antibodies showed expected binding to the corresponding target proteins by flow cytometry or ELISA. TMThe live single cell population was gated using BD Biosciences v8 software (BD Biosciences, Franklin Lake, NJ), and the AF647 GeoMean and % positive binding were determined for each antibody in that population. For ELISA, the % positive binding for anti-FOLR3 and v23924 antibodies was determined using raw absorbance values ​​compared to the negative control absorbance signal. v23924 showed expected binding to human FRα, while exhibiting no binding cross-reactivity to FOLR2, FOLR3, or FOLR4, indicating FRα specificity.

[0499] Table 9.1: Binding to FRα, FOLR2 and FOLR4 assessed by flow cytometry

[0500]

[0501]

[0502] Table 9.2: Binding to FOLR3 assessed by ELISA

[0503] Antibody Absorbance 450nm Combined % v23924 0.29 8.9 Anti-FRα control 0.24 7.4 Anti-FOLR3 control 3.27 100.0 Human IgG 0.15 4.5

[0504] Example 10: Functional Characterization of Anti-FRα Antibodies - Binding to Cynomolgus Monkey FRα

[0505] The parental chimeric antibody v23924 was evaluated for cross-reactivity with human and cynomolgus monkey FRα by flow cytometry using transfected CHO-S cells as described below. Palivizumab (anti-RSV) (v22277) was used as a negative control.

[0506] Briefly, CHO-S cells were transfected for approximately 24 hours to transiently express human or cynomolgus monkey FRα with 1 ug DNA per 1 million cells. After transfection, cells were seeded at 50,000 cells / well in a V-bottom 96-well plate 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 ImmunoResearch Labs, West Grove, PA; catalog number 109-605-098) at 4°C for 30 minutes. After incubation and washing, cells were stained with BD LSRFortessa TM Fluorescence was detected by flow cytometry on a cell analyzer (BD Biosciences, Franklin Lake, NJ).

[0507] The results are shown in Table 10.1. v23924 showed equivalent 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 for the control v22277.

[0508] Table 10.1: Binding to cynomolgus monkey FRα

[0509]

[0510]

[0511] Example 11: Functional Characterization of Anti-FRα Antibodies - Cell Binding

[0512] The on-cell binding abilities of the parental chimeric antibody v23924 and the representative humanized variant v30384 were assessed by flow cytometry on JEG-3 and HEC-1-A endogenous FRα expressing cell lines as described below.

[0513] 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 ImmunoResearch Labs, West Grove, PA; catalog number 109-605-098) for 30 minutes at 4°C. After incubation and washing, cells were stained with BD LSRFortessa TM Fluorescence was detected by flow cytometry on a cell analyzer (BD Biosciences, Franklin Lake, NJ), with a minimum of 1,000 events collected per well. The 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).

[0514] The results are shown in Table 11.1. Both the chimeric (v23924) and humanized (v30384) antibodies produced equivalent apparent Kd and Bmax values ​​in both JEG-3 and HEC-1-A cell lines (high and moderate endogenous FRα expression, respectively).

[0515] Table 11.1: Cell binding

[0516]

[0517] Example 12: Functional Characterization of Anti-FRα Antibodies - Internalization

[0518] The receptor-mediated internalization capacity of the parent chimeric antibody v23924 and the representative humanized variant v30384 in FRα-expressing cell lines (IGROV-1 and OVCAR-3) was determined by high-content imaging as described below. The antibodies targeting FRα, somituximab and fatuzumab, were used as positive controls, and palivizumab (anti-RSV) (v22277) was used as a negative control.

[0519] Briefly, the antibodies were fluorescently labeled by coupling with 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 5:1 molar excess at 4 ° C for 24 hours. Cells were seeded in 96-well plates and incubated overnight at 37 ° C in 5% CO2. The next day, the coupled antibodies were added to the cells and incubated at 37 ° C for 6-24 hours to allow internalization. After incubation, the cells were stained with dye cycle violet (Dye Cycle Violet) (ThermoFisher Scientific Corporation, Waltham, MA; Catalog No. V35003) for live cell identification and using CellInsight TM Internalized fluorescence in living cells was analyzed by high content imaging using the CX5 High Content Screening (HCS) platform (ThermoFisher Scientific Corporation, Waltham, MA). The fold-over fluorescence of the chimeric antibody v23924 was plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0520] result

[0521] The results are as follows Figure 5 A and Figure 5 B (IGROV-1 cells) and Figure 6 A and Figure 6B (OVCAR-3 cells). The chimeric antibody v23924 and the humanized variant v30384 showed equivalent internalization levels in both IGROV-1 cells (high FRα) and OVCAR-3 cells (medium FRα). In IGROV-1 and OVCAR cells, the chimeric antibody v23924 and the humanized variant v30384 showed increased internalization compared to the positive controls of sometuximab and fatuzumab at all tested concentrations (25 to 1 nM) and time points (6 hours and 24 hours). For example, after 6 hours of incubation in IGROV-1 cells, the humanized variant v30384 showed a 3.3-fold and 6.1-fold increase in internalization fluorescence compared to sometuximab and fatuzumab at 25 nM, respectively, and a 2.6- and 19.1-fold increase in internalization fluorescence compared to sometuximab and fatuzumab at 5 nM, respectively ( Figure 5 A). Similarly, after 24 h incubation in IGROV-1 cells, the humanized variant v30384 showed a 2.1-fold and 3.9-fold increase in internalized fluorescence at 25 nM compared to sometuximab and fatuzumab, respectively, and a 1.9-fold and 4.7-fold increase in internalized fluorescence at 5 nM compared to sometuximab and fatuzumab, respectively ( Figure 5 B).

[0522] Example 13: Epitope Mapping

[0523] 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.

[0524] 13.1 Sample preparation for HDX-MS

[0525] Lyophilized hFRα was purchased from ACROBiosystems (Newark, DE; catalog number: FO1-H5229) and dissolved to a concentration of 2.5 mg / ml. Antigen-antibody complexes were prepared by mixing hFRα with the parent chimeric antibody v23924 at a molar ratio of 2:1. All samples had a pH of 7.4 and were clear (no precipitation 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 to 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 out at 20 seconds, 7 minutes, 1 hour, and 4 hours, and immediately quenched by adding a 200 mM TCEP solution containing 4 M guanidine. These samples were flash frozen in liquid nitrogen and stored at -80°C. During LC-MS experiments, protein aliquots were rapidly thawed and kept on ice for 2 min of reduction followed by pepsin digestion at 0°C for 2 min.

[0526] 13.2 LC-MS and LC-MS / MS

[0527] In the LC-MS experiments, a 20 μL aliquot of each sample was immediately injected into 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 ThermoScientific Orbitrap Fusion TM Mass spectrometer connection. The column, accessories, syringes and solvent delivery lines were embedded in an ice bucket to minimize H / D back exchange. The syringes used for injection were cooled on ice. The mobile phases were 0.1% formic acid (A) and 100% acetonitrile / 0.1% formic acid (B), and the peptides were separated by a 13-minute gradient. MS survey scans were performed in the range of m / z 300-1600 with a mass resolution of 120,000 FWHM. The Orbitrap detector was calibrated to an error of <3 ppm by using a calibration mixture (Calmix; ThermoFisher Scientific Corporation, Waltham, MA). In the electron transfer dissociation (ETD) experiment, the fluoranthene radical anion was introduced into the ion trap within 50 milliseconds. Collision induced dissociation (CID) and ETD fragment ions were detected in the Orbitrap using a scan range of 150-2000 m / z.

[0528] For data analysis, Proteome Discoverer TM Bottom-up LC-MS / MS raw data were processed using the Xcalibur ELISA software suite (Thermo Fisher Scientific). The resulting peak list was submitted to the in-house Mascot 2.2 server and searched against the sequence of hFRα. The peptides thus identified were used for HDX data analysis. TM Software (ThermoFisher Scientific Corporation, Waltham, MA) was used to process the ETD data, and the resulting ETD peak list was searched against the sequence of hFRα using ProteinProspector (available online from the University of California, San Francisco website; http: / / prospector.ucsf.edu). The matched ions were also checked and confirmed by manual inspection. The mass shift of the peptides and the deuteration state of each amide were determined based on their center of mass 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). Deuterium incorporation percentage values ​​were obtained by comparing the number of deuteriums acquired with the total number of amide hydrogens contained in each peptide. Amide level deuteration information was calculated based on deuterium uptake of the ETD fragments.

[0529] 13.3 Results

[0530] Protein sequence coverage and peptide identification

[0531] The presence of protein disulfide bonds has a significant impact on the pepsin digestion pattern and efficiency in peptide-based HDX-MS analysis. Since 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 each optimized to 2 minutes, and these conditions were applied to both hFRα and the hFRα-v23924 complex. The peptides so identified covered 100% of the antigen sequence (see Figure 7 ).

[0532] Comparison of HDX at the peptide level

[0533] Deuterium incorporation levels of peptides from hFRα and v23924 complexes were plotted versus HDX time (20 seconds, 7 minutes, 1 hour, and 4 hours). The results are summarized and shown in Figure 8 Most peptides have the same deuterium uptake behavior before and after antibody binding ( Figure 8 A), indicating that the binding site (epitope) of the v23924 antibody is quite localized. Figure 8In the difference map shown in B, three peptides (Nos. 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.

[0534] Epitope determination at the amino acid level

[0535] Although the three epitope peptides differ in sequence, their HDX differences are identical ( Figure 8 ), and they all contain the sequence WEDCRTSY(119-126) (SEQ ID NO:152). This indicates that all epitope residues are included in the shortest peptide 119-126. Multiple differential peptides were observed in the same region, providing further confirmation that this region of the protein is the binding site for the v23924 antibody. To further localize the HDX differences and pinpoint the epitope to a single amino acid, MSMS was performed on peptide 119-126 using ETD. The 1 hour HDX time point was chosen as this produced the greatest differences. ETD fragments provide single residue resolution. The deuteration level of each amino acid between the hFRα and v23924 complexes was calculated and compared ( Fig. 9 ). Based on the results of the difference map, the epitope residues were determined to be E120, D121, R123, T124, S125 and Y126 of SEQ ID NO: 15 (ie, the epitope sequence is: EDRTSY; SEQ ID NO: 154).

[0536] Example 14: Affinity maturation of anti-FRα antibodies

[0537] Using HuTarg TM The humanized antibody v30384 (see Example 3) was affinity matured using the ELISA system (Innovative Targeting Solutions, Vancouver, BC, Canada). Genetic recombination was applied to the variable regions of the humanized variant v30384, and high affinity mutants were identified using next generation sequencing (NGS).

[0538] 14.1 Design of plasmid library

[0539] The CDR loops of the humanized variant v30384 variable domains are interspersed with RAG1 / 2 recombination signal sequences (RSS).These variable domains were synthesized at Integrated DNA Technologies, Inc. (Coralville, IA) and cloned into plasmid E951 (Innovative Targeting Solutions, Vancouver, BC, Canada).

[0540] 14.2 HuTarg TM Library generation

[0541] The following steps were performed according to the Innovative Targeting System protocol. TM The HuTarg protein was successfully reconstituted into the cells and induced RAG1 / 2 expression for 48 hours. The HuTarg protein was selected after staining with PE-conjugated goat anti-human κ light chain antibody (Bio-Rad Laboratories, Hercules, CA; Catalog No. 206009). TM cells for further study.

[0542] 14.3 FACS-based selection of affinity mutants

[0543] In BD FACSAria TM HuTarg was analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ). TM Cells were subjected to multiple rounds of FACS-based sorting, each using decreasing amounts of biotinylated soluble HIS-tagged FRα antigen (FRα-HIS; ACROBiosystems Newark, DE; Catalog No. FO1-H82E2) and detected with streptavidin conjugated to AlexaFluor-647 (Thermo Fisher Scientific Corp., Waltham, MA; Catalog No. S11223). HuTarg TM Cells were sorted directly into RNAzol RT (Sigma-Aldrich, St. Louis, MI; Catalog No. R4533) in preparation for next generation sequencing.

[0544] 14.4 Next-generation sequencing of affinity mutants

[0545] Total RNA was isolated from cells lysed in RNAzol following the manufacturer's instructions. TMRNA was digested with 5% paraformaldehyde (Thermo Fisher Scientific Corp., Waltham, MA; Catalog No. 11766051) and expressed using Superscript TM IV (Thermo Fisher Scientific Corp., Waltham, MA; Catalog No. 18090010) and gene-specific primers to transcribe cDNA. The VH and VK domains were targeted for PCR amplification and PCR amplification with Ultra TM Molecular barcoding was performed using the DNA library preparation kit (New England Biolabs, Ipswich, MA; catalog number E7370L). Samples were pooled and analyzed on an Illumina MiSeq TM The sequences were run on a sequencer using a 500-cycle kit using v2 chemistry (Illumina, San Diego, CA; catalog number MS-102-2003).Sequence analysis was performed to identify mutations within the VH and VK sequences that showed a high probability of conferring increased affinity.

[0546] 14.5 Recombinant Expression of Affinity Mutants

[0547] DNA sequences encoding mutant VH and VK domains were synthesized as "MiniGenes" (Integrated Technologies, Inc., Coralville, IA) and cloned into expression vectors to provide expression plasmids encoding complete human IgG1 heavy chains and human kappa light chains, respectively. The expression plasmids were matrixed to each other so that each heavy chain plasmid was paired with each light chain plasmid. The matrix was prepared in Expi293 TM Recombinantly expressed in ELISA cells (Thermo Fisher Scientific Corp., Waltham, MA; Catalog No. A14635) according to the manufacturer's instructions to generate 64 samples.

[0548] 14.6 Evaluation of affinity mutants

[0549] Protein G particles (Spherotech Inc., Lake Forest, IL) were coated with normalized concentrations of humanized variant v30384 or affinity matured antibodies. Soluble human FRα was diluted to the 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 Fcy (both from Jackson Laboratories, Bar Harbor, ME), respectively. TM Samples were analyzed by flow cytometry on a cytometer (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 the affinity grade humanized variant v30384 for the affinity matured antibody.

[0550] Single point affinity ranking was performed by measuring the ratio of antibody captured on the beads to the amount of antigen captured by the antibody. Using a human FRα concentration of 1.9 nM, variant v30384 binding was minimal (3x background), while most mutant variants showed higher binding ratios. Of the 64 mutant variants, 3 variants showed a 4-fold increase in binding ratio, and 10 variants showed equivalent binding ratios.

[0551] Example 15: Affinity assessment of affinity matured antibodies for FRα

[0552] The 10 affinity matured variants described in Example 14 were produced in full-size antibody (FSA) format at WuXi Biologics (Hong Kong) Limited via transient transfection and affinity capture purification in CHO-K1 cells, with subsequent polishing steps (if necessary) primarily involving preparative SEC or CEX chromatography, yielding greater than 97% sample purity by HPLC-SEC. The FSA format is similar to the parent humanized variant v30384, except that these variants contain HomoFc instead of HetFc. The FSA format was used as described in Example 3. The RED96 system characterizes the binding of 10 affinity matured variants to hFRα.

[0553] result

[0554] The results are shown in Table 15.1. Affinity maturation resulted in an affinity for hFRα significantly higher than that of the parent humanized antibody v30384 (K DThe humanized variants of 1.27E-07M were successful, with affinity improvements ranging from about 12 to 58 fold. The lowest K of 2.2E-09M was observed for variant v35348. D It was determined that the increase in affinity was mainly achieved by reducing the dissociation constant.

[0555] Table 15.1: Affinity evaluation of affinity matured variants

[0556]

[0557]

[0558] *n=2

[0559] Example 16: Chromatographic Analysis of Affinity Matured Antibodies

[0560] 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 the variable HIC-RT. The antibody monomer content was above 97% in all cases and was independent of HIC-RT.

[0561] Table 16.1: HIC and SEC analysis of affinity matured anti-FRα antibodies

[0562]

[0563] Example 17: Functional Characterization of Affinity Matured Antibodies - Cell Binding

[0564] The on-cell binding ability of the representative affinity matured variant v35356 was assessed by flow cytometry on IGROV-1 and JEG-3 endogenous FRα expressing cell lines as described in Example 11.

[0565] result

[0566] The results are shown in Table 17.1. Both the parental humanized variant v30384 and the affinity matured variant v35356 produced equivalent apparent Kd and Bmax values ​​in both IGROV-1 and JEG-3 cell lines (high and medium endogenous FRα expression, respectively).

[0567] Table 17.1: Cell Binding

[0568]

[0569] Example 18: Functional Characterization of Affinity Matured Antibodies - Internalization

[0570] The receptor-mediated internalization capacity of the parental humanized variant v30384 and the 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.

[0571] Briefly, the antibodies were fluorescently labeled by coupling with Fab-AF488 anti-human IgG Fc labeling reagent (Jackson ImmunoResearch Labs, West Grove, PA; catalog number 109-547-008) at a 1:1 molar ratio at 4°C for 24 hours. Cells were seeded in 48-well plates and incubated overnight at 37°C in 5% CO2. The next day, the coupled antibodies were added to the cells and incubated at 37°C for 24 hours to allow internalization. After incubation, the cells were dissociated, washed, and surface AF488 fluorescence was quenched using 100 nM anti-488 antibody at 4°C for 45 minutes. The cells were then incubated in a BD LSRFortessa TM All samples were analyzed by flow cytometry for quenched AF488 fluorescence (internalized fluorescence) on a cell analyzer (BD Biosciences, Franklin Lake, NJ), with a minimum of 1,000 events collected per well. The AF488 / FITC-AGeoMean in live cell populations was plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0572] result

[0573] The results are shown in Fig.10 The parental humanized variant v30384 and the affinity matured variant v35356, when administered at 20 nM, showed significant increases in IGROV-1 ( Fig.10 (A)) and JEG-3( Fig.10 (B) ) cells showed equivalent internalization.

[0574] Example 19: Preparation of Antibody-Drug Conjugates - Cysteine ​​Conjugation

[0575] Antibody-drug conjugates (ADCs) comprising chimeric parent antibody v23924, humanized antibody variants, or affinity matured antibody variants conjugated to a maleimide-containing drug linker were prepared at various drug to antibody ratios (DARs). Exemplary schemes are provided below. The drug-linkers employed are listed in Table 19.1. The ADCs prepared and the conjugation conditions employed are summarized in Table 19.2.

[0576] 19.1 Conjugation of v23924 (DAR 4) via partial reduction of interchain disulfide bonds

[0577] A solution (5.14 mL) of chimeric antibody v23924 (25 mg) was diluted to 3.57 mg / mL with 5 mM DTPA (diethylenetriaminepentaacetic acid) in PBS (pH 7.4), and 10 mM tris(2-carboxyethyl)phosphine (TCEP) (1.78 equiv., 31 μL) was added to the solution. After incubation for 2 hours in a 37°C water bath, drug-linker DL1 (10 equiv., 87 μL) in the form of a 20 mM DMSO stock solution was added. The conjugation reaction was thoroughly mixed by pipetting, and the reaction was allowed to proceed on ice for up to 1 hour, followed by quenching excess drug-linker with an aqueous stock solution of 20 mM N-acetylcysteine ​​(9 equiv., 78 μL) for 30 minutes, and then ADC was purified from the small molecule. v23924 was also conjugated to drug-linkers DL2 and DL3 with DAR 4 using the same conjugation procedure.

[0578] 19.2 After complete reduction of interchain disulfide bonds, conjugation with v23924 (DAR 10)

[0579] A solution (3.1 mL) of chimeric antibody v23924 (15 mg) was diluted to 3.57 mg / mL with 5 mM 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 hours and then eluted with 10 mL of 40 kD Zeba TM Excess TCEP was removed by spin desalting columns (Thermo Fisher Scientific, Waltham, MA). The fully reduced antibody was conjugated with a 20 mM DMSO stock solution of drug-linker DL1 (18 eq, 94 μL) on ice for up to 1 hour, followed by quenching excess drug-linker with an aqueous stock solution of 20 mM N-acetyl cysteine ​​(12 eq, 63 μL) for 30 minutes before purifying the ADC from the small molecule.

[0580] 19.3 Conjugation of v30384 (DAR 4) via partial reduction of interchain disulfide bonds

[0581] A solution (2.1 mL) of humanized antibody variant v30384 (10 mg) was diluted to 3.52 mg / mL with 5 mM 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 hours before conjugation to a 20 mM DMSO stock solution of drug-linker DL1 (10 eq., 34.7 μL) on ice for up to 1 hour, followed by quenching excess drug-linker with a 20 mM aqueous stock solution of N-acetyl cysteine ​​(9 eq., 31.2 μL) for 30 minutes before purifying the ADC from the small molecule.

[0582] 19.4 Complete reduction of interchain disulfide bonds followed by conjugation with v30384 (DAR 8)

[0583] A solution of humanized antibody variant v30384 (20 mg) (958.3 μL) was diluted to 5 mg / mL with 5 mM DTPA 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 and then eluted with 10 mL of 40 kD Zeba pre-equilibrated with 10 mM sodium acetate (pH 5.5). TM Excess TCEP was removed by spin desalting columns (ThermoFisher Scientific, Waltham, MA). The fully reduced antibody was conjugated with a 10 mM DMSO stock solution of drug-linker DL5 (15 eq., 208 μL) in the presence of 10% DMSO (vol / vol) at room temperature with continuous stirring in the dark for up to 2 hours, followed by quenching of excess drug-linker with an aqueous stock solution of 10 mM N-acetylcysteine ​​(20 eq., 274 μL) for 30 minutes before purifying the ADC from the small molecule.

[0584] Table 19.1: Drug-Linkers for Preparation of Cysteine-Conjugated ADCs

[0585] Drug-Linker <![CDATA[Connector 1 > Payload DL1 MTvc <![CDATA[Compound 1 1 > DL2 MTvc <![CDATA[Compound 2 1 > DL3 MCvc-PABC MMAE DL4 MCvc-PABC Eribulin DL5 MC-GGFG DxD DL7 MTv <![CDATA[Compound 2 1 >

[0586] 1 The structure shown below.

[0587] MTvc:

[0588]

[0589] MCvcPABC:

[0590]

[0591] MC-GGFG:

[0592]

[0593] MTvk:

[0594]

[0595] Compound 1:

[0596]

[0597] (See International Patent Application Publication No. WO 2016 / 041082) Compound 2:

[0598]

[0599] (See International Patent Application Publication No. WO 2014 / 144871)

[0600] Table 19.2: Summary of Cysteine ​​Conjugated ADC Preparations

[0601]

[0602]

[0603] 1 See Example 2

[0604] 2 Variant v36675 is identical to v30384 but contains HomoFc instead of HetFc

[0605] Example 20: Preparation of Antibody-Drug Conjugates - Lysine Conjugation

[0606] Antibody-drug conjugates (ADCs) comprising humanized antibody variant v30384 conjugated to drug-linkers DL6 or DL8 (see Table 20.1) with a target DAR of 3.3 or 4, respectively, were prepared as described below and summarized in Table 20.2.

[0607] DL6: A solution (207 μL) of humanized antibody v30384 (1 mg) was reacted with a 10 mM DMSO stock solution of drug-linker DL6 (14 equiv., 9.7 μL) in PBS pH 7.4. The conjugation reaction was mixed well by pipetting and allowed to proceed at room temperature for up to 17 hours.

[0608] DL8: A solution (5.9 mL) of humanized antibody v30384 (30 mg) was reacted with a 20 mM DMSO stock solution of drug-linker DL8 (9.5 equiv., 99 μL) in PBS pH 7.4. The conjugation reaction was mixed thoroughly by pipetting and allowed to proceed at room temperature for up to 18 hours.

[0609] Table 20.2: Drug-Linkers for Preparation of Lysine-Conjugated ADCs

[0610] Drug-Linker <![CDATA[Connector 1 > Payload DL6 sSPDB DM4 DL8 NHS-ADvc <![CDATA[Compound 1 2 >

[0611] 1 The structure shown below.

[0612] 2 See footnote to Table 19.1

[0613] sSPDB:

[0614]

[0615] NHS-ADvc:

[0616]

[0617] Table 20.2: Summary of Lysine Conjugated ADC Preparations

[0618]

[0619] *See Example 2

[0620] Example 21: Purification and characterization of ADC

[0621] The appropriate size 40 kD Zeba TM ADCs prepared as described in Example 19 and Example 20 were purified on a spin desalting column (Thermo Fisher Scientific, Waltham, MA). ADCs produced at >1 mg scale were sterile filtered (0.22 μm).

[0622] The purified ADC was stored at 4°C and analyzed for total protein content using absorbance at 280 nm or using bicinchoninic acid (BCA) assay with reference to a standard curve generated from trastuzumab 1 mg / mL. The ADC was also characterized by HPLC-HIC, SEC, CE-SDS, and RP-HPLC-MS as described below. The average DAR and DAR distribution of the ADC were obtained from the HIC and LC-MS data. The ToxinSensor TM Endotoxin levels were assessed using a single test kit (Genescript BioTech, Piscataway, NJ; Catalog No. L00450) with a threshold set at 0.5 EU / mg. Residual free drug and drug-linker levels (% FD) were assessed by RP-HPLC-MS and calculated based on the following equation with a threshold set at 1 mol % DAR:

[0623]

[0624] The biophysical properties of the ADCs measured are summarized in Table 21.4.

[0625] 21.1 Determination of DAR of Cysteine-Conjugated ADCs by HIC

[0626] The average DAR was estimated by HIC as described in Antibody Drug Conjugates, Methods in Molecular Biology, 2013, Vol. 1045, pp. 275-284. L. Ducry, ed., using 5 column volumes of buffer A (1.5 M (NH4)2SO4, 25 mM NaH2PO4, pH = 6.95) pre-equilibrated. Butyl-NPR column (2.5 μm, 4.6x35mm, TOSOH Bioscience GmbH, Griesheim, Germany) and at room temperature, on Agilent InfinityII 1290HPLC (Agilent Technologies, Santa Clara, CA) experiments were performed. Typically, 20-30 μg of sample at a concentration of 2-3 mg / mL was loaded onto a 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. The HIC chromatogram was integrated using appropriate parameters that provided a complete baseline-baseline integration of each peak, followed by integration of each peak that showed reasonable separation. As a reference, a non-conjugated naked antibody was run on the same gradient to obtain the HIC retention time of a substance with DAR=0.

[0627] Table 21.1: HIC gradient

[0628] Time (minutes) Buffer A% Buffer B% 0 95 5 0.1 95 5 5 80 20 9.5 65 35 11.5 50 50 12.5 5 95 13.5 5 95 12.6 95 5 15 95 5

[0629] 21.2 Determination of DAR of Lysine-Conjugated ADCs by RP-HPLC-MS

[0630] ADC samples were deglycosylated using Endo S at room temperature for 1 hour and injected onto an Agilent 1290 Infinity II LC (Agilent Technologies, Santa Clara, CA) coupled to an Agilent 6545 quadrupole time-of-flight (Q-TOF) mass spectrometer. 8uM, 50×2.1 mm) at a flow rate of 0.3 ml / min using the gradient shown in Table 21.2 to separate protein species. Buffer A: 0.1% formic acid (FA), 0.025% trifluoroacetic acid (TFA), and 10% isopropanol (IPA) in water. Buffer B: 0.1% FA and 10% IPA in acetonitrile (ACN).

[0631] Table 21.2: RP-HPLC-MS gradient

[0632] Time (minutes) Buffer A% Buffer B% 0 80 20 20 60 40 22 10 90 22.5 1 99 24 1 99

[0633] The MS source conditions are shown in Table 21.3 and the acquisition parameters are as follows:

[0634] Mode: MS; Mass range: 500 to 7000 m / z; Acquisition rate: 1 spectrum / s and 1000 ms / spectrum, 3354 transients / spectrum.

[0635] Table 21.3: MS source conditions

[0636] Gas temperature: 300℃ VCap: 5000V Dry gas: 13l / min Nozzle voltage: 2000V Atomizer: 45psig Fragmenter: 170V Sheath gas temperature: 400℃ Skimmer: 65V Sheath gas flow rate: 12l / min Oct RF Vpp:750V

[0637] Deconvolution and data analysis were performed using qualitative analysis using MassHunter software (Agilent Technologies, Santa Clara, CA). The deconvolution parameters were as follows:

[0638] Deconvolution algorithm: Maximum Entropy; Mass Range: 70000-160000; Mass Step: 1.0; Limited m / z Range Used: 1000-7000; Baseline Subtraction: 7.0; Adduction: Protons; Isotope Width: Automatic; Height Filter: Peak Signal-to-Noise Ratio >= 30.0; Maximum Number of Peaks: Limited by Height 100.

[0639] The average DAR was calculated from the deconvoluted spectra using the following equation:

[0640]

[0641] 21.3 SEC-HPLC Analysis of ADC

[0642] At room temperature, SEC was performed using an Advance Bio SEC column ( Analytical SEC was performed on an Agilent Infinity II 1260 HPLC (Agilent Technologies, Santa Clara, CA) with a column volume of 2.7 μm, 7.8×150 mm; Agilent Technologies), and the column was balanced with 5 column volumes of buffer (150 mM Na2PO4, pH 6.95). Typically, 20-30 μg of sample at a concentration of 2-3 mg / mL was eluted isostatically at 1 mL / min for 7 minutes and the absorbance was monitored at A280. The chromatogram was integrated to provide a complete baseline-baseline integration of each peak, and the separation positions between partially separated peaks were reasonable. Based on the SEC profile of the unmodified chimeric antibody v23924, the peak corresponding to the main component of IgG (approximately retention time 3.3 minutes) was reported as a monomer. Any peak appearing before 3.3 minutes was designated as a high molecular weight species (HMWS), and any peak appearing after 3.3 minutes was designated as a low molecular weight species (LMWS), excluding solvent peaks (over 5.2 minutes).

[0643] 21.4 CE-SDS Analysis of ADC

[0644] All samples were first diluted to 1 mg / mL and then assayed according to the manufacturer's protocol (Protein Express AssayLabChip TM ; PerkinElmer, Inc., Waltham, MA) in 96-well PCR plates. Briefly, 2 μg of ADC was mixed with 7 uL ProteinExpress buffer in the presence (reducing) or absence (non-reducing) of 400 mM dithiothreitol (DTT), followed by heat denaturation at 95°C for 5 minutes. The samples were then diluted in dH2O at a ratio of 1:2 before data acquisition. After each CE-SDS run, the samples were analyzed using LabChip TM The gels and corresponding electropherograms were analyzed by Reviewer (PerkinElmer, Inc., Waltham, MA).

[0645] Table 21.4: Biophysical properties of ADCs

[0646]

[0647]

[0648] 1 For details of drug-linkers (DL), see Example 19 and Example 20.

[0649] 2 ND = Not Determined

[0650] 3 Small scale preparation, %FD and endotoxin not determined

[0651] 4 Large-scale preparation

[0652] Example 22: In vitro cytotoxicity of ADC

[0653] The cell growth inhibitory (cytotoxic) ability of ADCs comprising the humanized variant v30384 or the affinity matured variant v35356, each conjugated to the drug-linker DL1, was determined in a panel of FRα expressing cell lines as described below (see Example 19).

[0654] Briefly, cells were seeded in 384-well plates at a density of 1,000 cells / well and treated with test article titrations generated in complete cell growth medium. Treated cells were incubated for 4 days under standard culture conditions (37°C / 5% CO2). Reagent (Promega Corporation, Madison, WI; Catalog No. G7570) was added to all wells and the cells were analyzed using Synergy TM The luminescence corresponding to the ATP present in each well was measured by an H1 microplate reader (BioTek Instruments, Winooski, VT). Based on blank wells (no test article added, only blank medium), % cytotoxicity values ​​were calculated using ATP measurement RLU values ​​(relative light units) and the test article concentrations were plotted using GraphPad Prism 9 software (GraphPad Software, San Diego, CA).

[0655] result

[0656] The results are shown in Table 22.1 and Fig.19 The ADC containing the humanized variant v30384 and the ADC containing the affinity matured variant v35356 showed equivalent in vitro cytotoxicity in FRα-expressing cells KB-Hela, IGROV-1, JEG-3, and SKOV-3. In the FRα-negative MDA-MB-468 cell line, none of the ADCs showed any nonspecific cytotoxicity.

[0657] Table 22.1: In vitro cytotoxicity

[0658]

[0659] Example 23: Release and quantification of intracellular payload

[0660] The intracellular payload delivery ability of representative ADCs in cell lines JEG-3 (high FRα), Caov-3 (medium FRα), HEC-1-A (medium FRα) and H2110 (medium / low FRα) was evaluated using mass spectrometry as described below. The ADCs tested comprised humanized variant v30384 or affinity matured variant v35356, each conjugated to drug-linker DL1 (see Example 19).

[0661] In brief, cells were seeded in 12-well plates at 80,000 cells / well and treated with various concentrations of ADC under standard culture conditions for 24 hours. After incubation, cells were washed with PBS (pH 7.4), collected, counted and frozen at -80 ° C. After thawing, cells were lysed using pure acetonitrile. The cell lysate supernatant was injected into an Agilent 1290 Infinity II LC (Agilent Technologies, Santa Clara, CA) coupled to an Agilent 6470 triple quadrupole (QQQ) mass spectrometer, and free payload (compound 1) in the cell lysate sample was quantified using a known concentration of free payload standard.

[0662] Results The results are shown in Fig.11 In the high FRα expressing cell line JEG-3, both ADCs showed equivalent intracellular payload (Compound 1) delivery. In the lower FRα expressing cell lines Caov-3, H2110 and HEC-1-A, the ADC containing the affinity matured variant v35356 showed higher payload delivery at 24 hours compared to the ADC containing the parental humanized variant v30384.

[0663] Example 24: In vivo efficacy study - chimeric anti-FRα antibody ADC

[0664] As described below, the in vivo anti-tumor activity of the parent chimeric antibody v23924 conjugated to the drug-linker DL1 or DL7 was evaluated in a variety of xenograft models expressing different levels of FRα. The activity of a control ADC containing the known FRα-targeting antibody Suometuximab was evaluated for comparison. The control ADCs were v17717 (Suometuximab Fab with HetFc) conjugated to the drug-linker DL1 and v17717 conjugated to the drug-linker DL6. For statistical analysis, a linear mixed effects model was used to fit the log-transformed tumor volume, followed by an F-test for the null hypothesis of equal mean growth rates, and post hoc pairwise comparisons.

[0665] The ADCs used in each xenograft study, xenograft models, doses, and study durations are summarized in Table 24.1. For each xenograft study, tumor volume and body weight of the animals were measured twice weekly.

[0666] Table 24.1: Study parameters

[0667]

[0668]

[0669] For the CTG-0848 PDX model, tumor fragments were implanted subcutaneously into female nude mice. When the average tumor volume reached approximately 100-250 mm 3 At day 0, animals were matched by tumor size and assigned to treatment groups (n=5 per group) and treated on day 0 with a single IV dose of ADC as shown in Table 24.1.

[0670] For the OV90 model, tumor cell suspension (0.1 ml 50% 1×10 7 cells) were implanted subcutaneously into female CB.17SCID mice. When the average tumor volume reached 100-150mm 3 Animals were divided into groups (n=9 per group) and treated on study day 1 with a single IV dose of ADC as shown in Table 24.1.

[0671] For the OVCAR3 model, tumor fragments (approximately 1 mm 3 ) were implanted subcutaneously into female CB.17SCID mice. When the average tumor volume reached about 100-150mm 3 Animals were divided into groups (n=8 per group) and treated on study day 1 with a single IV dose of ADC as shown in Table 24.1.

[0672] For the LXFA737 PDX model, tumor fragments (3-4 mm edge length) were implanted subcutaneously into female nude mice. When the average tumor volume reached approximately 80-200 mm 3 Animals were divided into groups (n=6 per group) and treated on study day 0 with a single IV dose of ADC as shown in Table 24.1.

[0673] For the JEG3 CDX model, tumor cell suspension (0.1 ml 50% 1×10 7 cells) were implanted subcutaneously into NOD / SCID mice. When the average tumor volume reached about 100 mm 3Animals were divided into groups (n=5 per group) and treated with a single IV dose of ADC on study day 0 as shown in Table 24.1.

[0674] For the HCC1954 CDX model, tumor cell suspension (0.1 ml 50% 5×10 6 When the average tumor volume reached about 150 mm 3 Animals were divided into groups (n=5 per group) and treated on study day 0 with a single IV dose of ADC as shown in Table 24.1.

[0675] For the SKOV3 CDX model, tumor cell suspension (0.1 ml 50% 1×10 7 When the average tumor volume reached about 170 mm 3 Animals were divided into groups (n=5 per group) and treated with a single IV dose of ADC on study day 0 as shown in Table 24.1.

[0676] For the KB CDX model, tumor cell suspension (3 × 10 in 0.1 ml PBS) was added. 6 When the average tumor volume reached about 100-150mm 3 Animals were divided into groups (n=5 per group) and treated on study day 1 with a single IV dose of ADC as shown in Table 24.1.

[0677] For all models except the HCC1954 and KB CDX models, serum was collected at multiple time points for pharmacokinetic analysis as described in Example 28.

[0678] result

[0679] The results are shown in Fig.12 middle.

[0680] In the CTG-0848 PDX model, v23924-DL1 caused sustained regression of tumor growth when administered at 5 mg / kg and 10 mg / kg ( Fig.12 A). In the OV90 model, v23924-DL1 caused moderate inhibition of tumor growth rate when administered at 9 mg / kg and 18 mg / kg ( Fig.12B), but this was not statistically significantly different from the 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 with significantly stronger inhibition of tumor growth rate than v17717-DL6 (p<0.001) ( Fig.12 C).

[0681] In the LXFA737 PDX model, v23924-DL1, v17717-DL6, and v17717-DL1 caused 198%, 144%, and 165% inhibition of tumor growth rate, respectively, when dosed at 5 mg / kg (each tumor growth rate was significantly different from control, p<0.001) ( Fig.12 D). In the JEG3 CDX model, v23924-DL1, v17717-DL6, and v17717-DL1 caused transient tumor regressions when administered at 3 mg / kg, with tumor growth rate inhibition of 132%, 148%, and 139%, respectively (each tumor growth rate was significantly different from control, p<0.001) ( Fig.12 E). In the HCC1954 CDX model, v23924-DL1, v17717-DL6, and v17717-DL1 caused transient tumor regression when administered at 10 mg / kg, with tumor growth rate inhibition of 287%, 278%, and 242%, respectively (each tumor growth rate was significantly different from the control, p<0.01) ( Fig.12 F).

[0682] In the SKOV3 CDX model, v23924-DL1, v17716-DL6, and v17717-DL1 caused transient tumor regressions when administered at 10 mg / kg, with tumor growth rate inhibition of 287%, 243%, and 278%, respectively (each tumor growth rate was significantly different from control, p<0.01) ( Fig.12 G). In the KB CDX model, when administered at 5 mg / kg, v23924-DL1, v23924-DL7, and v17717-DL6 caused 289%, 171%, and -1% inhibition of tumor growth rate, respectively, with both v23924 ADCs significantly superior to vehicle control and v17717-DL6 (p<0.01). When administered at 10 mg / kg, all ADCs caused sustained tumor regression in this model ( Fig.12 H).

[0683] Example 25: In vivo efficacy studies - chimeric and humanized anti-FRα antibody ADCs

[0684] As described below, chimeric antibody v23924 and humanized variants v30384 and v30399 (see Example 3) conjugated to drug-linker DL1 (see Example 19) were evaluated for in vivo antitumor activity in the OVCAR3 ovarian cancer model expressing medium / high levels of FRα. For statistical analysis, a linear mixed effects model was fitted to log-transformed tumor volumes, followed by an F-test for the null hypothesis of equal mean growth rates and post hoc pairwise comparisons.

[0685] Tumor fragments (about 1 mm 3 ) were implanted subcutaneously into female CB.17SCID mice. When the average tumor volume reached about 100-150mm 3 Animals were divided into groups (n=8 per group) at 40 days and treated with a single IV dose of 4 mg / kg or 9 mg / kg ADC on study day 1. Tumor volume and body weight were measured twice a week for a study duration of 60 days.

[0686] result

[0687] The results are shown in Fig.13 v23924-DL1 and humanized v30384-DL1 and v30399-DL1 caused similar tumor regressions followed by regrowth when administered at 4 mg / kg. All ADCs caused sustained tumor regressions when administered at 9 mg / kg.

[0688] Example 26: In vivo efficacy study - humanized anti-FRα antibody ADC #1

[0689] As described below, the in vivo anti-tumor activity of the humanized variant v30384 (see Example 3) conjugated to the drug-linker DL1 or DL8 (see Examples 19 and 20) was evaluated in multiple xenograft models expressing different levels of FRα. The activity of the control ADC containing the known FRα-targeting antibody Suometuximab was evaluated in the selected model for comparison. The control ADC was v17717 (Suometuximab Fab with HetFc) (v17717-DL6) conjugated to the drug-linker DL6 and v17716 (Suometuximab Fab with HomoFc) (v17716-DL6) conjugated to the drug-linker DL6. In the H2110CDX model, v31629 (fatuzumab) (v31629-DL4) conjugated to the drug-linker 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.

[0690] The ADCs used in each xenograft study, xenograft models, doses, and study durations are summarized in Table 26.1. For each xenograft study, tumor volume and body weight of the animals were measured twice weekly.

[0691] Table 26.1: Study parameters

[0692]

[0693] For the H2110 CDX model, tumor cell suspension (0.1 ml 50% 1×10 7 cells) were implanted subcutaneously into CB.17SCID mice. When the average tumor volume reached about 155 mm 3 Animals were divided into groups (n=8 per group) and treated with a single IV dose of ADC on study day 0 as shown in Table 26.1. Serum was collected at multiple time points for pharmacokinetic analysis as described in Example 28.

[0694] For the SKOV3 CDX model, tumor cell suspension (0.1 ml 50% 1×10 7 cells) were implanted subcutaneously into nude mice. When the average tumor volume reached about 175 mm 3 At 1:10, animals were divided into groups (n=9 per group for repeated dose groups and n=7 per group for single dose groups) and treated with ADC according to the dose levels and schedules shown in Table 26.1 starting on study day 0. Serum was collected at multiple time points for pharmacokinetic analysis as described in Example 28.

[0695] For the IGROV-1 model, tumor cell suspension (0.1 ml 50% 1×10 7 cells) were implanted subcutaneously into female nude mice. When the average tumor volume reached about 100-150 mm 3 At 4 hr, animals were divided into groups (n=12 per group) and treated with ADC according to the dose levels and schedules as shown in Table 26.1.

[0696] For the LXFA737 CDX model, tumor fragments (3-4 mm edge length) were implanted subcutaneously into nude mice. When the average tumor volume reached approximately 100 mm 3 Animals were divided into groups (n=10 per group) and treated on study day 0 with a single IV dose of ADC as shown in Table 26.1.

[0697] result

[0698] The results are shown in Fig.14 middle.

[0699] In the H2110 CDX model, both v30384-DL1 and v17716-DL6 caused small to moderate tumor growth inhibition in a superficial dose response when dosed at 1.25 mg / kg, 2.5 mg / kg, and 5 mg / kg, but these were not statistically significant ( Fig.14 A). v31629-DL4 dosed at 2.5 mg / kg caused a smaller inhibition of tumor growth rate comparable to v17716-DL6 and v30384-DL1.

[0700] In the SKOV3 CDX model, a single administration of 8 mg / kg of v30384-DL1, v30384-DL8, and v17716-DL6 caused significant inhibition of tumor growth rate by 203%, 178%, and 232%, respectively (p<0.01), with v17716-DL6 having a moderate but statistically significantly greater inhibition of tumor growth rate (p<0.01) ( Fig.14 Compared with single-dose administration, repeated dosing (v30384-DL1 every 10 days for a total of 4 doses; v17716-DL6 every 15 days for a total of 3 doses) had less effect on tumor growth ( Fig.14 C).

[0701] In the IGROV-1 model, when administered at 8 mg / kg, v30384-DL1 (once every 10 days for a total of 4 doses) and v17716-DL6 (once every 15 days for a total of 3 doses) caused significant inhibition of tumor growth compared to the control (p<0.001). When administered at 4 mg / kg, only v30384-DL1 (once every 10 days for a total of 4 doses) caused significant inhibition of tumor growth compared to the control (p<0.001). At the 4 mg / kg and 8 mg / kg dose levels, v30384-DL1 caused significantly stronger inhibition of tumor growth rate than v17716-DL6 (p<0.001 and p=0.014, respectively) ( Fig.14 D).

[0702] In the LXFA737 CDX model, v30384-DL8 and v17716-DL6 caused significant inhibition of tumor growth rate by 154% and 313%, respectively, when administered at 8 mg / kg (p<0.01). v17716-DL6 caused 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) ( Fig.14 E).

[0703] Example 27: In vivo efficacy study - humanized anti-FRα antibody ADC #2

[0704] As described below, the in vivo antitumor activity of the humanized variant v30384 (see Example 3) conjugated to the drug-linker DL5 (see Example 19) was evaluated in multiple xenograft models expressing different levels of FRα. For statistical analysis, a linear mixed effects model was fitted to the log-transformed tumor volumes, followed by an F-test of the null hypothesis of equal mean growth rates and post hoc pairwise comparisons.

[0705] The ADCs used in each xenograft study, xenograft models, doses, and study durations are summarized in Table 27.1. For each xenograft study, tumor volume and body weight of the animals were measured twice weekly.

[0706] Table 27.1: Study parameters

[0707] Xenograft Models Cancer Type FRα expression Dose level mg / kg Study duration OV90 Ovarian cancer middle 1、3、10 61 days H2110 Lung cancer Medium / Low 0.3、1、3、10 28 days OVCAR3 Ovarian cancer Medium / High 0.25、0.75、1.5、3 61 days OVCAR3 Ovarian cancer Medium / High 6 61 days

[0708] For the OV90 CDX model, tumor cell suspension (0.1 ml 50% 1×10 7 cells) were implanted subcutaneously into CB.17SCID mice. When the average tumor volume reached about 100-150mm 3 Animals were divided into groups (n=6 per group) and treated on study day 1 with a single IV dose of ADC as shown in Table 27.1.

[0709] For the H2110 CDX model, tumor cell suspension (0.1 ml 50% 1×10 7 cells) were implanted subcutaneously into CB.17SCID mice. When the average tumor volume reached about 150 mm 3 At 4 hrs, animals were divided into groups (n=5 per group) and treated with a single IV dose of ADC as shown in Table 27.1.

[0710] For the OVCAR3 CDX model, tumor fragments (approximately 1 mm 3 ) were implanted subcutaneously into CB.17SCID mice. When the average tumor volume reached about 100-150mm 3 Animals were divided into groups (n=5 per group) and treated on study day 1 with a single IV dose of ADC as shown in Table 27.1.

[0711] For each model, serum was collected at multiple time points for pharmacokinetic analysis as described in Example 28.

[0712] result

[0713] The results are shown in Fig.15 In all three models, v30384-DL5 produced a dose response at the doses tested.

[0714] In the OV90 CDX model, v30384-DL5 produced a dose response at 1 mg / kg, 3 mg / kg, and 10 mg / kg (inhibition of tumor growth rate at the 3 mg / kg and 10 mg / kg dose levels was significant (p<0.01) by 25%, 47%, and 185%, respectively) ( Fig.15 A). In the H2110 CDX model, v30384-DL5 produced a dose response at 0.3 mg / kg, 1 mg / kg, 3 mg / kg, and 10 mg / kg dose levels ( Fig.15 B). In the OVCAR3 CDX model, v30384-DL5 produced a dose response at 0.25 mg / kg, 0.75 mg / kg, 1.5 mg / kg, and 3 mg / kg dose levels, with the higher dose groups resulting in sustained regressions with regrowth approximately 5 weeks after dosing ( Fig.15 C). In another study in the OVCAR3 CDX model, v30384-DL5 administered at 6 mg / kg resulted in durable regressions ( Fig.15 D).

[0715] Example 28: Pharmacokinetic Study

[0716] From a subset of the xenograft studies described in Examples 24, 26, and 27, sera were collected as described and analyzed for the pharmacokinetics (PK) of the evaluated ADCs as described below. In summary, these studies in immunocompromised tumor-bearing mice demonstrate that v30384-based ADCs have favorable PK properties.

[0717] The test article (total antibody and / or intact ADC) concentration was measured from mouse serum by 384-well plate ELISA. To detect total IgG in serum from mice dosed with ADCs based on chimeric v23924, humanized v30384, and comparators v17717 and v17716, ELISA plates were coated with goat anti-human IgG Fc capture antibody (Jackson Immuno Research Laboratories, West Grove, PA). For ELISA of DL1 intact ADC, rabbit anti-compound 1 capture antibody was used. For ELISA of DL6 intact ADC, mouse anti-DM1 / 4 capture antibody (Levena Biopharma, San Diego, CA) was used. 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). The absorbance at 450 nm was measured after application of 3,3',5,5'-tetramethylbenzidine (TMB) and HCl quenching. The concentration was determined with reference to standards (GraphPad Prism Software, San Diego, CA).

[0718] The results are shown in Fig.16 Analysis of multiple studies demonstrated that v23924-DL1 and v17717-DL1 were equivalent in total IgG and ADC PK, with typical antibody-like prolonged exposure (see Fig.16 A- Fig.16 F). v17717-DL6 and v17716-DL6 ADC PK consistently demonstrated lower exposure and greater clearance compared to their corresponding total IgG PK.

[0719] v30384-DL5 demonstrated dose proportional PK across 1 mg / kg, 3 mg / kg, and 10 mg / kg in the OV90 model ( Fig.16 G). In the H2110 model, v30384-DL5 showed prolonged exposure at 10 mg / kg and greater clearance at lower doses ( Fig.16 H).

[0720] Example 29: Penetration of anti-FRα antibodies in multicellular tumor spheroids

[0721] The ability of anti-FRα antibodies to penetrate spheroids of FRα-expressing cell lines was evaluated according to the following method. Spheroids provide a three-dimensional organization of cells with different cell population stratification and the formation of different gradients from the outer to the inner regions. Cell signaling is more complex in spheroids than in two-dimensional cell cultures. Due to these characteristics, spheroids have the potential to reproduce drug resistance and metabolic adaptation.

[0722] The spheroid penetration capacity of humanized variant v36675 was compared with that of Sumetuximab (v17716) and the non-FRα-targeted control Palivizumab (anti-RSV) (v22277). Variant v36675 is identical to variant v30384 but contains HomoFc instead of HetFc. The cell line used was high FRα-expressing JEG-3 (placental choriocarcinoma).

[0723] The antibodies were fluorescently labeled by coupling with Fab fragment AF488 conjugate targeting anti-human IgG Fc (Jackson ImmunoResearch Labs, West Grove, PA; catalog number 109-547-008) at a 1:1 molar ratio in PBS (pH 7.4) (ThermoFisher Scientific, Waltham, MA; catalog number 10010-023) at 4°C for 24 hours.

[0724] Using TrypLE TM JEG-3 cells were detached from the culture vessel using the 1X expression enzyme (Thermo Fisher Scientific, Waltham, MA) and cultured using MX high-throughput automated cell counter (Nexcelom Bioscience LLC, Lawrence, MA) was used for counting. Cells were diluted in complete growth medium (minimal 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.

[0725] After spheroid formation, Fab-AF488 conjugate antibody was added to the spheroid at a final concentration of 25 nM and incubated for 4-96 hours under standard culture conditions. After incubation, excess antibody was removed by adding 100 μL of complete growth medium and removing 100 μL of medium from the wells, washing three times in total. Spheroids were treated with a solution of 1 μM Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA) and 100 nM anti-Alexa Fluor 488 antibody (Thermo Fisher Scientific, Waltham, MA; Catalog No. A-11094) and incubated for 2 hours at 37°C / 5% CO2.

[0726] Using an Operetta CLS with confocal acquisition and 10× magnification air objective TM Imaging was performed using the High Content Analysis System (Perkin Elmer, Waltham, MA). Z-stacks of 15 planes spaced 15 μm apart were acquired, and the slice with the largest diameter representing the central slice of the spheroid was selected for 2D analysis. Image analysis was performed using 4.5 software (PerkinElmer, Waltham, MA). In brief, spheroid identification was performed by applying a mask to one spheroid per well around Hoechst 33342-positive objects. The spheroid region was divided into subregions of concentric bands, each subregion accounting for 10% of the spheroid region. The average AF488 fluorescence within each subregion band was quantified, corrected by subtracting the internal 10% average AF488 fluorescence, and plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0727] result

[0728] The results are summarized in Tables 29.1 and Fig. 20 Humanized antibody variant v36675 showed greater penetration into JEG-3 spheroids than somituximab, as measured by AF488 intensity at each subregion band and by distance from the spheroid edge to where AF488 fluorescence was detectable at all time points assessed. The non-binding control palivizumab showed less AF488 signal throughout the spheroids compared to both anti-FRα antibodies at all time points assessed.

[0729]

[0730] Example 30: Specificity evaluation of anti-FRα antibodies

[0731] The following anti-FRα antibodies were screened for any specific off-target binding interactions using Retrogenix cellular microarray technology (Charles River Laboratories, Wilmington, MA): humanized variant v36675 and affinity matured variant v35356.

[0732] Retrogenix Cell Microarray technology identifies interactions with cell surface receptors and secreted proteins by screening cDNA clone libraries representing more than 6,300 human proteins for bound test ligands. These proteins include plasma membrane monomers, heterodimers (formed by co-expression of individual subunits), and secreted proteins (expressed with inert cell membrane tethers). Each cDNA is spotted in duplicate onto a specialized slide and covered with HEK293 cells. These cells become reverse transfected, generating cell clusters that each overexpress a different single protein (or heterodimeric complex).

[0733] The study was conducted at Charles River Laboratories and consisted of three phases: prescreening, library screening, and confirmation screening. Prescreening was first performed to determine the appropriate concentration of the test antibody 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, the test antibodies were screened as a library against fixed HEK293 cells expressing approximately 6,300 human proteins alone. In the confirmation screening phase, each library hit was re-expressed and re-tested using fixed and live HEK293 cells alone.

[0734] In all three stages, slides were spotted individually with expression vectors encoding ZsGreen1 (for assessment of transfection efficiency) and (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 in duplicate on multiple microarray slides ("slide sets"), where two replicate slides were screened for each slide set; or (3) in the confirmatory screening stage: protein hits identified in the library screen or control receptors (EGFR and CD20) arrayed in duplicate.

[0735] In the prescreening phase, 2, 5, or 20 μg / mL of each selected antibody, 1 μg / mL of a control antibody (a CD20-binding rituximab biosimilar), or PBS was added to the slides after cell fixation. In the library screening phase, a pool of two antibodies (20 μg / mL of variant v36675 and 5 μg / mL of variant v35356) was added to each slide after cell fixation. In the confirmatory screening phase, slides were treated with the individual antibodies without fixation (live cells; n = 1 slide per treatment) and after cell fixation (n = 2 slides per treatment): 20 μg / mL of variant v36675, 5 μg / mL of variant v35356, or 1 μg / mL of a control antibody (a rituximab biosimilar), or no test article.

[0736] use Binding was detected by 647-labeled anti-human IgG H+L (AF647 anti-hIgG H+L), followed by fluorescence imaging. TM Fluorescence images were analyzed and quantified using ImageQuant software (version 8.2; GE Healthcare, Chicago, IL). Protein hits were defined as duplicate spots showing increased signal compared to background levels and were quantified using the ImageQuant software. TM The gridded images on the software were identified by visual inspection. Hits were classified as strong, medium, weak or very weak based on the intensity of the replicate spots by two experienced scientists by visual inspection.

[0737] result

[0738] Most of the initial hits identified in the library screen (spot intensities ranged from very weak to strong) were confirmed as hits in the confirmation screen for variant v36675 and variant v35356. However, in addition to the hits reflecting the expected strong interaction with FRα, other hits were considered nonspecific because these hits 1) were also observed for the control antibody (rituximab biosimilar) because they included FcgR receptors (due to the signal generated by the Fc domain-mediated interaction) or 2) included various immunoglobulins recognized by the test antibody.

[0739] For the humanized variant v36675, no other interactions were identified (see Fig.21 ), indicating that variant v36675 has high specificity for its primary target FRα. The weak interaction observed for this variant with the heterodimer FCGR3A+CD247 was not observed in the control (compare Fig.21 A and Fig.21 B), considered not strong enough to qualify as a hit.

[0740] For affinity matured variant v35356, a weak intensity signal for protein COL6A2 isoform 2C2A was detected in the library screen. The "hits" were confirmed in the fixed cell screen but not in the live cell screen, indicating that the observed inconsistency may be caused by fixation artifacts.

[0741] Example 31: Competition Assay

[0742] Competition binding to the FRα target between the parental chimeric antibody v23924 and the anti-FRα antibodies sometuximab (v17716) and fatuzumab (v31629) was assessed by flow cytometry in the moderate FRα expressing tumor cell line H2110 as follows.

[0743] Briefly, antibodies were fluorescently labeled with Alexa Fluor 647 (AF647; ThermoFisher Scientific, Waltham, MA; Catalog No. A20006) according to the manufacturer's instructions prior to cell treatment. Tumor cells were seeded at 50,000 cells / well in a V-bottom 96-well plate 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, cells were stained with BD LSRFortessa TM Fluorescence was detected by flow cytometry on a cell analyzer (BD Biosciences, Franklin Lake, NJ), with 1,000 minimum events collected per well. Competitive binding % relative to untreated controls was calculated using the AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) in the live cell population, and data were plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0744] result

[0745] The results are as follows Fig. 22 As shown in Table 31.1. It was found that the chimeric antibody v23924 competed with fatuzumab for FRα binding in the H2110 cell line, producing nearly 100% competitive binding in both binding directions (primary antibody fatuzumab and fluorescently labeled secondary antibody variant v23924, and primary antibody variant v23924 and fluorescently labeled secondary antibody fatuzumab). It was found that the chimeric antibody v23924 did not compete with sometuximab for FRα binding in the H2110 cell line. These antibodies showed low levels of competition % in both binding directions. In contrast, fatuzumab showed competitive binding with sometuximab. Therefore, the chimeric antibody v23924 exhibited binding characteristics different from those of sometuximab and fatuzumab.

[0746] Table 31.1: Competition binding with facilitation and somituximab on H2110 cells

[0747]

[0748] Example 32: Additional functional characterization of anti-FRα antibodies

[0749] The functional activity of humanized antibody variants v30384 (HetFc) or v36675 (HomoFc) was evaluated by cell binding, antigen-mediated antibody internalization, antibody penetration into tumor cell spheroids, and 3D cell growth inhibition of spheroids in comparison with the biparatopic anti-FRα antibody B5327A (IMGN151; v36264) and the anti-FRα antibody sometuximab (v17716) using FRα-expressing tumor cell lines.

[0750] 32.1 Cell Binding

[0751] The ability of humanized antibody variants v30384 (HetFc), v36264 (B5327A) and v17716 (somituximab) to bind to FRα-expressing tumor cell lines was assessed by flow cytometry in the endogenous tumor cell line IGROV-1 (ovarian adenocarcinoma; FRα-high) expressing FRα following the same general procedure as described in Example 11.

[0752] The results are shown in Table 32.1. Humanized variants v30384 and Sumetuximab (v17716) exhibited similar binding abilities on cells, with equivalent Bmax values ​​(16,599 and 19,505 intensity geometric means, respectively) and apparent Kd values ​​(0.43 nM and 0.69 nM, respectively). The biparatopic antibody B5327A (v36264) exhibited an increased Bmax compared to the other two antibodies (26,267 intensity geometric means), as expected for a biparatopic antibody.

[0753] Table 32.1 Cellular binding of anti-FRα antibodies on IGROV-1 cell line

[0754] sample Bmax Kd(nM) Slope(h) v30384 16,599 0.43 1.2 v36264(B5327A) 26,267 0.70 1.4 v17716 (somituximab) 19,505 0.69 1.4

[0755] 32.2 Internalization

[0756] The receptor-mediated internalization capacity of the humanized antibody variants v30384 (HetFc), v36264 (B5327A) and v17716 (somitoximab) was assessed by uptake into the endogenous tumor cell line IGROV-1 (ovarian adenocarcinoma) expressing FRα by flow cytometry as follows.

[0757] The antibodies were fluorescently labeled by coupling with anti-human IgG Fc Fab fragment AF488 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-547-008) at a 1: 1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; Catalog No. 10010-023) at 4°C for 24 hours. Cells were seeded in 48-well plates at 50,000 cells / well and incubated overnight under standard culture conditions (37°C / 5% CO2) to allow attachment. The next day, the coupled antibodies were added to the cells at 25nM and incubated under standard culture conditions for 5 hours to allow internalization. After incubation, the cells were dissociated, washed, and the surface AF488 fluorescence was quenched at 100nM at 4°C for 30 minutes using an anti-AF488 antibody (Life Technologies, Carlsbad, CA; Catalog No. A-11094). In BD LSRFortessa TM The quenched AF488 fluorescence (internalized fluorescence) was detected by flow cytometry on a cell analyzer (BD Biosciences, Franklin Lake, NJ), and a minimum of 1,000 events were collected per well. TM AF488 / FITC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human Fab AF488 labeling) of live single-cell populations was calculated using GraphPad® version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0758] The results are shown in Fig.23 After incubation of IGROV-1 cells with 25 nM of the test antibodies for 5 h, the humanized antibody variant v30384 showed an internalization fluorescence equivalent to that of the biparatopic antibody v36264 (1.1-fold that of v30384), whereas sometuximab (v17716) showed a significantly lower degree of internalization fluorescence (0.5-fold that of v30384).

[0759] 32.3 Antibody Penetration into 3D Spheroids

[0760] The ability of humanized antibody variants v36675 (HomoFc), v36264 (B5327A) and v17716 (somitoxin) to penetrate FRα-expressing cell line spheroids was assessed by treating spheroids formed from the high FRα-expressing cell line JEG-3 (placental choriocarcinoma) with fluorescently labeled antibodies following the same general procedure described in Example 29.

[0761] The results are shown in Fig.24 After incubation of JEG-3 spheroids with 25 nM fluorescently labeled antibodies for 96 hours, humanized antibody variant v36675 showed greater fluorescence intensity from the second layer inward than biparatopic antibody B5327A (v36264) or Sumetuximab (v17716), indicating greater cellular uptake of v36675 in all layers and an overall greater distance of penetration into the spheroid. In contrast, on the outermost layer, biparatopic antibody B5327A (v36264) showed greater fluorescence intensity than v36675 or v17716, indicating accumulation of the antibody on the spheroid surface.

[0762] 32.4 3D Spheroid Growth Inhibition

[0763] The 3D cytotoxicity capabilities of humanized antibody variants v30384 (HetFc), v36264 (B5327A) and v17716 (somituximab), each conjugated to the drug-linker DL1, were evaluated in IGROV-1 (ovarian adenocarcinoma) and JEG-3 (placental choriocarcinoma) cell lines expressing FRα as follows.

[0764] Briefly, cells were seeded at 3,000 cells / well in Ultra-Low Attachment 384-well plates (Corning, New York, NY), centrifuged, and cultured for 3 days under standard culture conditions to allow for spheroid formation and growth. Spheroids were then treated with test article titrations generated in cell growth medium. Spheroids were incubated for 6 days under standard culture conditions. 3D reagent (Promega Corporation, Madison, WI) was added to all wells. The plates were incubated in the dark for 1 hour at room temperature and luminescence was quantified using a BioTek Cytation 5 Cell Imaging Multimode Reader (Agilent Technologies, Inc., Santa Clara, CA). Percent cytotoxicity values ​​were calculated based on blank wells (no test article added) and the test article concentrations were plotted using GraphPad Prism 9 software (GraphPad Software, San Diego, CA). EC was calculated by GraphPad Prism 9 based on nonlinear regression log (agonist) versus response, variable slope (four parameters) 50 value.

[0765] The results are shown in Table 32.2. Against IGROV-1 spheroids, all three antibody-drug conjugates (ADCs) showed equivalent cell growth inhibition ability. Against JEG-3 spheroids, v30384-DL1 was similar in potency to v17716-DL1 (EC 50 0.18 nM and 0.17 nM, respectively), while the biparatopic ADC, v36264-DL1, displayed slightly lower potency (0.35 nM).

[0766] Table 32.4 3D in vitro cell growth inhibition

[0767]

[0768] The disclosures of all patents, patent applications, publications, and database entries mentioned in this specification are hereby expressly incorporated by reference in their entirety to the same extent as if each individual patent, patent application, publication, and database entry was specifically and individually indicated to be incorporated by reference.

[0769] Modifications of the specific embodiments described herein which are apparent to those skilled in the art are intended to be within the scope of the following claims.

[0770] Sequence Listing

[0771] Table A: Clone numbers of variants

[0772]

[0773] Table B: Cloned sequences

[0774]

[0775]

[0776]

[0777]

[0778]

[0779]

[0780]

[0781]

[0782]

[0783]

[0784]

[0785]

[0786]

[0787]

[0788]

[0789]

[0790]

[0791]

[0792]

[0793]

[0794]

[0795]

[0796]

[0797]

[0798]

[0799]

[0800]

[0801]

[0802]

[0803]

[0804]

[0805]

[0806]

[0807]

[0808]

[0809]

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

Claims

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

15.

2. The antibody construct according to claim 1, wherein the epitope is a discontinuous epitope.

3. The antibody construct according to claim 1 or 2, wherein the reference antibody comprises a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 27, 29 or 32; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 43 or 45; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47.

4. An antibody construct comprising an antigen binding domain, wherein the antigen binding domain 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.

5. The antibody construct according to claim 4, wherein the epitope is a discontinuous epitope.

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

7. The antibody construct of claim 4 or 6, wherein the antigen binding domain comprises a CDR sequence of a VH domain having a sequence as shown in any one of SEQ ID NO: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133 or 136.

8. The antibody construct of any one of claims 4, 6 and 7, wherein the antigen binding domain comprises a CDR sequence of a VL domain having a sequence as shown in any one of SEQ ID NO: 39, 64, 119, 124 or 130.

9. The antibody construct according to claim 4 or 6, wherein the antigen binding domain comprises: (i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28, 31, 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 27, 29, 32, 51, 58, 100, 101, 102, 103, 109, 137, 138 or 139; and a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25, 30, 107, 108 or 110, and (ii) a LCDR1 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 40, 43, 45, 65, 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 42, 47, 120 or 121.

10. The antibody construct according to claim 4 or 6, wherein the antigen binding domain comprises: (a) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 27, 29 or 32; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 43 or 45; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or (b) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 51; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or (c) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 58; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or (d) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 51; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or (e) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 24, 100, 101, 102 or 103; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or (f) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 42 or 47; or (g) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or (h) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or (i) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or (j) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 40, 45 or 65; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or (k) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 107, 108 or 110; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or (l) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 20, 23, 26, 28 or 31; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 29, 32 or 109; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121; or (m) a HCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 92, 93, 94, 95 or 96; a HCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 21, 24, 137, 138 or 139; a HCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 22, 25 or 30; a LCDR1 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 125, 126 or 127; a LCDR2 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 41, 44 or 46; and a LCDR3 amino acid sequence selected from the group consisting of the amino acid sequences shown in any one of SEQ ID NOs: 120 or 121.

11. The antibody construct of claim 4 or 6, wherein the antigen binding domain comprises a VH amino acid sequence selected from the VH amino acid sequence shown in any one of SEQ ID NO: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133 or 136.

12. The antibody construct of any one of claims 4, 6 and 11, wherein the antigen binding domain comprises a VL amino acid sequence selected from the VL amino acid sequence shown in any one of SEQ ID NO: 39, 64, 119, 124 or 130.

13. An antibody construct according to claim 4 or 6, wherein the antigen binding domain comprises: (i) the VH amino acid sequence as shown in SEQ ID NO: 19 and the VL amino acid sequence as shown in SEQ ID NO: 39, or (ii) the VH amino acid sequence shown in SEQ ID NO:50 and the VL amino acid sequence shown in SEQ ID NO:64, or (iii) the VH amino acid sequence shown in SEQ ID NO:54 and the VL amino acid sequence shown in SEQ ID NO:64, or (iv) the VH amino acid sequence shown in SEQ ID NO:57 and the VL amino acid sequence shown in SEQ ID NO:64, or (v) the VH amino acid sequence shown in SEQ ID NO:61 and the VL amino acid sequence shown in SEQ ID NO:64, or (vi) the VH amino acid sequence shown in SEQ ID NO:76 and the VL amino acid sequence shown in SEQ ID NO:64, or (vii) the VH amino acid sequence shown in SEQ ID NO:79 and the VL amino acid sequence shown in SEQ ID NO:64, or (viii) the VH amino acid sequence shown in SEQ ID NO: 82 and the VL amino acid sequence shown in SEQ ID NO: 64, or (ix) the VH amino acid sequence shown in SEQ ID NO: 85 and the VL amino acid sequence shown in SEQ ID NO: 64, or (x) the VH amino acid sequence shown in SEQ ID NO: 88 and the VL amino acid sequence shown in SEQ ID NO: 64, or (xi) the VH amino acid sequence shown in SEQ ID NO:91 and the VL amino acid sequence shown in SEQ ID NO:124, or (xii) the VH amino acid sequence shown in SEQ ID NO:99 and the VL amino acid sequence shown in SEQ ID NO:130, or (xiii) the VH amino acid sequence shown in SEQ ID NO: 106 and the VL amino acid sequence shown in SEQ ID NO: 64, or (xiv) the VH amino acid sequence shown in SEQ ID NO: 106 and the VL amino acid sequence shown in SEQ ID NO: 119, or (xv) the VH amino acid sequence shown in SEQ ID NO: 106 and the VL amino acid sequence shown in SEQ ID NO: 130, or (xvi) the VH amino acid sequence shown in SEQ ID NO: 113 and the VL amino acid sequence shown in SEQ ID NO: 130, or (xvii) the VH amino acid sequence shown in SEQ ID NO: 116 and the VL amino acid sequence shown in SEQ ID NO: 119, or (xviii) the VH amino acid sequence shown in SEQ ID NO: 116 and the VL amino acid sequence shown in SEQ ID NO: 130, or (xix) the VH amino acid sequence shown in SEQ ID NO: 133 and the VL amino acid sequence shown in SEQ ID NO: 130, or (xx) the VH amino acid sequence shown in SEQ ID NO:136 and the VL amino acid sequence shown in SEQ ID NO:

130.

14. The antibody construct of any one of claims 1 to 13, further comprising a scaffold, wherein the antigen binding domain is operably linked to the scaffold.

15. The antibody construct of claim 14, wherein the scaffold comprises an IgG Fc region.

16. The antibody construct according to any one of claims 1 to 13, wherein the antibody construct further comprises a second antigen binding domain. The antibody construct of claim 16 , wherein the second antigen binding domain specifically binds hFRα. The antibody construct of claim 16 , wherein the second antigen binding domain binds an antigen other than hFRα.

19. The antibody construct according to any one of claims 16 to 18, further comprising one or more additional antigen binding domains.

20. The antibody construct of any one of claims 16 to 19, further comprising a scaffold, wherein at least one antigen binding domain is operably linked to the scaffold.

21. The antibody construct of claim 20, wherein the scaffold comprises an IgG Fc region.

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

116.

23. A polynucleotide or a group of polynucleotides encoding an antibody construct according to any one of claims 1 to 22.

24. An expression vector or a set of expression vectors comprising the polynucleotide or a set of polynucleotides according to claim 23.

25. A host cell comprising the expression vector or a group of expression vectors according to claim 24.

26. An antibody-drug conjugate comprising the antibody construct according to any one of claims 1 to 22 conjugated to one or more drug moieties.

27. The antibody-drug conjugate of claim 26, wherein the antibody conjugate is conjugated to 1 to about 8 drug moieties.

28. An antibody-drug conjugate having the general formula I: A-(L-(D) m ) n (I) in: A is an antibody construct according to any one of claims 1 to 22; L is the connector; D is the drug part; m is between 1 and about 8, and n is 1 and about 12.

29. The antibody-drug conjugate of claim 28, wherein m is 1 or 2.

30. The antibody-drug conjugate of claim 28 or 29, wherein n is between about 2 and about 8.

31. The antibody-drug conjugate of any one of claims 26 to 30, wherein the drug moiety is a maytansinoid, a maytansinoid analog, a benzodiazepine , pyrrolobenzodiazepine , duocarmycin, calicheamicin, a calicheamicin analog, auristatin, auristatin analog, hemiesterin, hemiesterin analog, tubulysin, tubulysin analog, amatoxin, amatoxin analog, camptothecin, camptothecin analog, eribulin, TLR agonist or STING agonist.

32. The antibody-drug conjugate of any one of claims 26 to 30, wherein the drug moiety is an auristatin, an auristatin analog, hemistatin, a hemistatin analog, camptothecin, a camptothecin analog, or eribulin.

33. A pharmaceutical composition comprising the antibody construct according to any one of claims 1 to 22 and a pharmaceutically acceptable carrier or diluent.

34. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 26 to 32 and a pharmaceutically acceptable carrier or diluent.

35. An antibody construct according to any one of claims 1 to 22 for use in therapy.

36. An antibody construct for use according to claim 35, wherein the therapy comprises the treatment of cancer.

37. The antibody-drug conjugate of any one of claims 26 to 32 for use in therapy.

38. The antibody-drug conjugate for use according to claim 37, wherein the therapy comprises the treatment of cancer.

39. Use of an antibody construct according to any one of claims 1 to 22 in the manufacture of a medicament for the treatment of cancer.

40. Use of an antibody-drug conjugate according to any one of claims 26 to 32 in the manufacture of a medicament for treating cancer.

41. A method of inhibiting the growth of FRα-positive tumor cells, comprising contacting the cells with an antibody construct according to any one of claims 1 to 22.

42. A method of inhibiting the growth of FRα-positive tumor cells, comprising contacting the cells with an antibody-drug conjugate according to any one of claims 26 to 32.

43. A method of treating a subject suffering from cancer, comprising administering to the subject an effective amount of an antibody construct according to any one of claims 1 to 22.

44. A method of treating a subject having cancer, comprising administering to the subject an effective amount of the antibody-drug conjugate of any one of claims 26 to 32.

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