FOLR1 binder, its conjugate, and method of using the same

Abstract

The present invention provides FOLR1 antibodies, antigen-binding portions thereof, other binding agents and FOLR1 conjugates thereof for use in the treatment of cancer.

Description

Technical Field

[0001] Description of Sequence Listing The sequence listing related to this application is provided in text format instead of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 760270_404WO_SEQUENCE_LISTING.txt. The text file is 35.8 KB, was created on April 5, 2022, and was electronically submitted via EFS-Web.

Background Art

[0002] Folate receptor 1 (FOLR1), also known as folate receptor alpha or folate-binding protein, is an N-glycosylated protein expressed on the plasma membrane of cells. FOLR1 has a high affinity for folic acid and several reduced folate derivatives. FOLR1 mediates the delivery of the physiological folate 5-methyltetrahydrofolate into cells. FOLR1 is overexpressed in most ovarian cancers, as well as many uterine, endometrial, pancreatic, kidney, lung, and breast cancers, while FOLR1 expression in normal tissues is restricted to the apical membranes of renal proximal tubules, alveolar lung cells of the lung, bladder, testis, choroid plexus, and epithelial cells of the thyroid (Weitman S D et al., Cancer Res 52:3396-3401 (1992); Antony A C, Annu Rev Nutr 16:501-521 (1996); Kalli K R et al. Gynecol Oncol 108:619-626 (2008)). This expression pattern of FOLR1 makes it a desirable target for FOLR1-directed cancer therapy.

[0003] Although FOLR1 is present in various cancer types, clinical trials using FOLR1 antibodies and FOLR1 antibody-drug conjugates have had limited success. The present invention addresses this need and other needs.

Prior Art Documents

Non-Patent Documents

[0004] [Non-Patent Document 1] Weitman S D et al., Cancer Res 52:3396 - 3401(1992) [Non-Patent Document 2] Antony A C, Annu Rev Nutr 16:501 - 521(1996) [Non-Patent Document 3] Kalli K R et al., Gynecol Oncol 108:619 - 626(2008) [Summary of the Invention] [Means for Solving the Problems]

[0005] This specification provides FOLR1 antibodies, antigen-binding portions and other binding agents, and conjugates of such antibodies, antigen-binding portions and other binding agents. Methods of using FOLR1 antibodies, antigen-binding portions and other binding agents and their conjugates for treating cancer and other diseases are also provided. The invention disclosed herein is based in part on FOLR1 antibodies, antigen-binding portions and other binding agents and their conjugates that specifically bind to FOLR1 and exhibit improved properties. FOLR1 is an important and advantageous therapeutic target for the treatment of certain cancers. FOLR1 antibodies, antigen-binding portions, other binding agents and their conjugates provide compositions and methods based on the use of such antibodies, antigen-binding portions and related binding agents, and their conjugates in the treatment of FOLR1+ cancers and other diseases.

[0006] In some embodiments, a binder is provided that includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region includes complementarity determining regions HCDR1, HCDR2, and HCDR3 disposed in a heavy chain variable region framework region, the VL region includes LCDR1, LCDR, and LCDR3 disposed in a light chain variable region framework region, and the VH and VL CDRs have amino acid sequences selected from the set of amino acid sequences consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; and SEQ ID NO: 31, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively. In some embodiments, the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively. In some embodiments, the framework region is a human framework region.

[0007] In some embodiments, the VH region and the VL region have amino acid sequences selected from the pair of amino acid sequences consisting of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively, and the heavy chain framework region and the light chain framework region are optionally modified by substitution, deletion, or insertion of 1 to 8 amino acids within the framework region.

[0008] In some embodiments, the VH region and the VL region each have an amino acid sequence selected from the pair of amino acid sequences shown in the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

[0009] In some embodiments, the VH region and the VL region each have an amino acid sequence selected from the pair of amino acid sequences shown in the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; and SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

[0010] In some embodiments, the VH region and the VL region each have an amino acid sequence selected from the pair of amino acid sequences shown in the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 7 and SEQ ID NO: 8; and SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In some embodiments, the VH region and the VL region each have the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. In some embodiments, the VH region and the VL region each have the amino acid sequence shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In some embodiments, the VH region and the VL region each have the amino acid sequence shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

[0011] In some embodiments, the binding agent is an antibody or an antigen-binding portion thereof. In some embodiments, the binding agent is a monoclonal antibody, Fab, Fab’, F(ab’)₂, Fv, scFv, single-domain antibody, diabody, bispecific antibody, or multispecific antibody. In some embodiments, the heavy-chain variable region further comprises a heavy-chain constant region. In some embodiments, the heavy-chain constant region is of the IgG isotype. In some embodiments, the heavy-chain constant region is the IgG1 constant region. In some embodiments, the IgG1 constant region has the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the heavy-chain constant region is the IgG4 constant region. In some embodiments, the heavy-chain constant region further comprises an amino acid modification that reduces at least the binding affinity for human FcγRIII. In some embodiments, the light-chain variable region further comprises a light-chain constant region. In some embodiments, the light-chain constant region is of the kappa isotype. In some embodiments, the light-chain constant region has the amino acid sequence set forth in SEQ ID NO: 40.

[0012] In some embodiments, the binding agent is monospecific. In some embodiments, the binding agent is bivalent. In some embodiments, the binding agent is bispecific.

[0013] In some embodiments, there is provided a pharmaceutical composition comprising any of the binding agents described herein and a pharmaceutically acceptable carrier. In some embodiments, there is provided a nucleic acid encoding any of the binding agents described herein. In some embodiments, there is provided a vector comprising any of the nucleic acids encoding any of the binding agents described herein. In some embodiments, there is provided a cell line comprising any of the vectors encoding any of the binding agents described herein or any of the nucleic acids encoding any of the binding agents described herein.

[0014] In some embodiments, a conjugate is provided that includes any of the binders described herein, at least one linker bound to the binder, and at least one drug bound to each linker. In some embodiments, each drug is selected from a cytotoxin, an immunomodulator, a nucleic acid, a growth inhibitor, a PROTAC, a toxin, and a radioisotope. In some embodiments, each linker is bound to the binder via an interchain disulfide residue, a lysine residue, an engineered cysteine residue, a glycan, a modified glycan, an N-terminal residue of the binder, or a polyhistidine peptide bound to the binder. In some embodiments, the average drug load of the conjugate is from about 1 to about 8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, from about 3 to about 5, from about 6 to about 8, or from about 8 to about 16.

[0015] In some embodiments of the conjugate, the drug is a cytotoxin. In some embodiments, the cytotoxin is selected from the group consisting of auristatin, maytansinoid, camptothecin, duocarmycin, or calicheamicin. In some embodiments, the cytotoxin is auristatin. In some embodiments, the cytotoxin is MMAE or MMAF. In some embodiments, the cytotoxin is camptothecin. In some embodiments, the cytotoxin is exatecan. In some embodiments, the cytotoxin is SN-38. In some embodiments, the cytotoxin is calicheamicin. In some embodiments, the cytotoxin is a maytansinoid. In some embodiments, the maytansinoid is maytansine, maytansinol, or a maytansine analog of DM1, DM3, and DM4, or ansamitocin-2.

[0016] In some embodiments, the linker comprises mc-VC-PAB, CL2, CL2A or (succinimid-3-yl-N)-(CH2)n-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)-, where n = 1 to 5. In some embodiments, the linker comprises mc-VC-PAB. In some embodiments, the linker comprises CL2A. In some embodiments, the linker comprises CL2. In some embodiments, the linker comprises (succinimid-3-yl-N)-(CH2)n-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)-. The conjugate according to claim 43, wherein the linker is bound to at least one molecule of exatecan. In some embodiments,

[0017] In some embodiments, the drug is an immunomodulator. In some embodiments, the immunomodulator is selected from the group consisting of a TLR7 agonist, a TLR8 agonist, a STING agonist, or a RIG-I agonist. In some embodiments, the immunomodulator is a TLR7 agonist. In some embodiments, the TLR7 agonist is imidazoquinoline, imidazoquinoline amine, thiazoloquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazole-2-amine, tetrahydropyridopyrimidine, heteroarothiadiazide-2,2-dioxide, benzonaphthyridine, guanosine analog, adenosine analog, thymidine homopolymer, ssRNA, CpG-A, polyG10, and polyG3. In some embodiments, the immunomodulator is a TLR8 agonist. In some embodiments, the TLR8 agonist is selected from imidazoquinoline, thiazoloquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazole-2-amine, tetrahydropyridopyrimidine or ssRNA. In some embodiments, the immunomodulator is a STING agonist. In some embodiments, the immunomodulator is a RIG-I agonist. In some embodiments, the RIG-I agonist is selected from KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400 and KIN2000. In some embodiments, the linker is selected from the group consisting of mc-VC-PAB, CL2, CL2A and (succinimid-3-yl-N)-(CH2)n-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)-(where n = 1 to 5).

[0018] In some embodiments, there is provided a pharmaceutical composition comprising any of the conjugates described herein and a pharmaceutically acceptable carrier.

[0019] In some embodiments, provided is a method of treating FOLR1+ cancer, comprising administering to a subject in need thereof a therapeutically effective amount of any of the binders described herein, any of the conjugates described herein, or any of the pharmaceutical compositions of the binders or conjugates described herein. In some embodiments, the FOLR1+ cancer is a solid tumor. In some embodiments, the FOLR1+ cancer is selected from lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, pancreatic cancer, and renal cell cancer. In some embodiments,

[0020] In some embodiments, the method further comprises administering an immunotherapy to the subject. In some embodiments, the immunotherapy comprises a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is selected from antibodies that specifically bind to human PD-1, human PD-L1, or human CTLA4. In some embodiments, the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab. In some embodiments, the method further comprises administering a chemotherapy to the subject.

[0021] In some embodiments, the method comprises administering to the subject any of the conjugates described herein or any of the pharmaceutical compositions described herein. In some embodiments, the binder, conjugate, or pharmaceutical composition is administered intravenously. In some embodiments, the binder, conjugate, or pharmaceutical composition is administered at a dose of about 0.1 mg / kg to about 12 mg / kg.

[0022] In some embodiments of the method, the treatment outcome of the subject is improved. In some embodiments, the improved treatment outcome is an objective response selected from stable disease, partial response, or complete response. In some embodiments, the improved treatment outcome is a reduction in tumor volume. In some embodiments, the improved treatment outcome is progression-free survival or disease-free survival.

[0023] In some embodiments, provided is the use of any of the binders described herein or any of the pharmaceutical compositions of the binders described herein for treating a subject with FOLR1+ cancer. In some embodiments, provided is the use of any of the conjugates described herein or any of the pharmaceutical compositions described herein for treating a subject with FOLR1+ cancer.

[0024] These and other aspects of the invention can be more fully understood by reference to the following detailed description, non-limiting examples of specific embodiments, and the accompanying drawings.

Brief Description of the Drawings

[0025]

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Mode for Carrying Out the Invention

[0026] Definition For the sake of simplicity, certain terms used in this specification, the examples and the claims are defined herein. Unless otherwise specified or implied from the context, the following terms and phrases have the meanings provided below. The definitions are provided to assist in the description of specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0027] As used herein, unless otherwise indicated, the terms "a" and "an" are to be construed as meaning "one," "at least one," or "one or more." Unless the context specifically requires otherwise, singular terms used herein shall include the plural, and plural terms shall include the singular.

[0028] Throughout the specification and claims, unless the context clearly dictates otherwise, words such as "comprise," "comprising," etc. are to be construed in an inclusive sense, i.e., in the sense of "including, but not limited to"; and not in an exclusive or exhaustive sense.

[0029] The terms "decreased," "reduced," "lowered," "reduction," "decrease," and "inhibited" are all generally used herein to mean a statistically significant decrease in amount as compared to a reference.

[0030] The terms "increased," "increase," "enhanced," or "activated" are all generally used herein to mean a statically significant increase in amount as compared to a reference.

[0031] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues connected to each other by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" also refer to polymers of amino acids, including modified amino acids (e.g., phosphorylation, glycosylation, glycosylation, etc.) and amino acid analogs, regardless of their size or function. The terms "protein" and "polypeptide" are often used with respect to relatively large polypeptides, whereas the term "peptide" is often used with respect to small polypeptides, but the use of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to encoded gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0032] FOLR1, or folate receptor alpha, is a cell surface protein that binds folate and reduced folate derivatives and mediates the delivery of 5-methyltetrahydrofolate and folate analogs into cells. It is also known as FR-alpha, adult folate binding protein, FBP, folate receptor 1, folate receptor-adult, KB cell FBP, and ovarian tumor-associated antigen MOv18. Human FOLR1 polypeptides include, but are not limited to, those having the amino acid sequence shown in UniProt identifier P15328-1; this sequence is incorporated herein by reference.

[0033] As used herein, "epitope" refers to amino acids that are customarily bound by an immunoglobulin VH / VL pair such as an antibody, an antigen-binding portion thereof, and other binding agents described herein. An epitope can be formed on a polypeptide of contiguous or non-contiguous amino acids juxtaposed by the tertiary folding of a protein. An epitope formed from contiguous amino acids is typically retained upon exposure to a denaturing solvent, whereas an epitope formed by tertiary folding is typically lost upon treatment with a denaturing solvent. An epitope typically contains at least 3, more usually at least 5, about 9, or about 8 - 10 amino acids in a unique spatial arrangement. An epitope defines the minimal binding site of an antibody, an antigen-binding portion thereof, and other binding agents, and thus represents the target of the specificity of an antibody, an antigen-binding portion thereof, or other immunoglobulin-based binding agent. In the case of a single-domain antibody, an epitope represents the structural unit to which the variable domain binds alone.

[0034] As used herein, "specifically binds" means that a binding agent (e.g., an antibody or an antigen-binding portion thereof) described herein binds to a target molecule with a dissociation constant (Kd) of 10 -5 M (10000 nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12Refers to the ability to bind to a target, such as human FOLR1, with a KD of M or less. Specific binding can be affected, for example, by the affinity and binding activity of an antibody, antigen-binding portion or other binding agent, as well as the concentration of the target polypeptide. One of ordinary skill in the art can determine the appropriate conditions for the antibodies, antigen-binding portions and other binding agents described herein to selectively bind to FOLR1 using any appropriate method, such as titration of the binding agent in an appropriate cell binding assay. A binding agent that specifically binds to FOLR1 cannot be replaced by a non-analogous competitor. In certain embodiments, an FOLR1 antibody or antigen-binding portion thereof or other binding agent is said to specifically bind to FOLR1 if it preferentially recognizes its target antigen FOLR1 in a complex mixture of proteins and / or macromolecules.

[0035] In some embodiments, the FOLR1 antibodies or antigen-binding portions thereof or other binding agents described herein have a dissociation constant (KD or K -5 of 10 M or less, for example 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or less, and specifically bind to the FOLR1 polypeptide. In some embodiments, the FOLR1 antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to the FOLR1 polypeptide with a dissociation constant (KD) of about 10 D M to 10 -5 M. In some embodiments, the FOLR1 antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to the FOLR1 polypeptide with a dissociation constant (KD) of about 10 -6 M to 10 -6 M. In some embodiments, the FOLR1 antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to the FOLR1 polypeptide with a dissociation constant (KD) of about 10 -7 M to 10 -7 M to 10 -8It binds specifically to the FOLR1 polypeptide with a dissociation constant (KD) of M. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein has a dissociation constant (KD) of about 10 -8 M to 10 -9 It binds specifically to the FOLR1 polypeptide with a dissociation constant (KD) of M. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein has a dissociation constant (KD) of about 10 -9 M to 10 -10 It binds specifically to the FOLR1 polypeptide with a dissociation constant (KD) of M. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein has a dissociation constant (KD) of about 10 -10 M to 10 -11 It binds specifically to the FOLR1 polypeptide with a dissociation constant (KD) of M. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein has a dissociation constant (KD) of about 10 -11 M to 10 -12 It binds specifically to the FOLR1 polypeptide with a dissociation constant (KD) of M. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein has a dissociation constant (KD) of less than 10 -12 M and binds specifically to the FOLR1 polypeptide.

[0036] As used herein, the term "consisting essentially of" refers to the elements necessary for a given embodiment. This term allows for the presence of elements that do not substantially affect the basic and novel or functional characteristics of that embodiment.

[0037] As used herein, the term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any element not recited in the description of that embodiment.

[0038] Except where otherwise indicated or unless otherwise specified, all numbers expressing amounts of ingredients or reaction conditions used in this specification are to be understood as being modified in all instances by the term "about." The term "about," when used in connection with percentages, can mean + / - 1%.

[0039] The terms "statistically significant" or "significantly" refer to statistical significance and generally mean a 2 standard deviation (2SD) difference above or below a reference value.

[0040] Other terms are defined herein within the scope of the description of the various aspects of the invention.

[0041] Provided herein are FOLR1-binding antibodies (also referred to as FOLR1 antibodies) that specifically bind to human FOLR1, antigen-binding portions thereof, and other binding agents. Also provided herein are conjugates of FOLR1 antibodies, antigen-binding portions, and other binding agents conjugated to a drug such as a cytotoxic agent or an immunomodulatory agent (also referred to as FOLR1 conjugates). In some embodiments, the FOLR1 antibodies, antigen-binding portions, other binding agents, and conjugates specifically bind to and reduce the number of FOLR1+ cells in a subject. In some embodiments, the FOLR1 antibodies, antigen-binding portions, other binding agents, and / or conjugates specifically bind to and reduce the number of FOLR1+ cancer cells in a subject. In some embodiments, the FOLR1 antibodies, antigen-binding portions, other binding agents, and / or conjugates specifically bind to and reduce the number of FOLR1+ cells associated with a disease or condition in a subject.

[0042] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have amino acid sequences shown by pairs of amino acid sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have amino acid sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have amino acid sequences shown by SEQ ID NO: 3 and SEQ ID NO: 4, respectively. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have amino acid sequences shown by SEQ ID NO: 5 and SEQ ID NO: 6, respectively. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have amino acid sequences shown by SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have amino acid sequences shown by SEQ ID NO: 9 and SEQ ID NO: 10, respectively. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have amino acid sequences shown by SEQ ID NO: 11 and SEQ ID NO: 12, respectively.In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

[0043] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have the amino acid sequences shown in pairs of amino acid sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region and VL region have the amino acid sequences shown in pairs of amino acid sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.The phrase "the CDRs of the heavy chain variable region or the light chain variable region are unmodified" refers to VH and VL CDRs that have no amino acid substitutions, deletions, or insertions.

[0044] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0045] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by a pair of amino acid sequences each selected from SEQ ID NO: 3 and SEQ ID NO: 4; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by a pair of amino acid sequences each selected from SEQ ID NO: 3 and SEQ ID NO: 4; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0046] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by a pair of amino acid sequences each selected from SEQ ID NO: 5 and SEQ ID NO: 6; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by a pair of amino acid sequences each selected from SEQ ID NO: 5 and SEQ ID NO: 6; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0047] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the VH region and the VL region each having an amino acid sequence shown in a pair of amino acid sequences selected from SEQ ID NO: 7 and SEQ ID NO: 8; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the VH region and the VL region each having an amino acid sequence shown in a pair of amino acid sequences selected from SEQ ID NO: 7 and SEQ ID NO: 8; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion or insertion of 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0048] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region each have an amino acid sequence shown in a pair of amino acid sequences selected from SEQ ID NO: 9 and SEQ ID NO: 10; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region each have an amino acid sequence shown in a pair of amino acid sequences selected from SEQ ID NO: 9 and SEQ ID NO: 10; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0049] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region each have an amino acid sequence shown in a pair of amino acid sequences selected from SEQ ID NO: 11 and SEQ ID NO: 12; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region each have an amino acid sequence shown in a pair of amino acid sequences selected from SEQ ID NO: 11 and SEQ ID NO: 12; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0050] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region each have an amino acid sequence shown in a pair of amino acid sequences selected from SEQ ID NO: 13 and SEQ ID NO: 14; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region each have an amino acid sequence shown in a pair of amino acid sequences selected from SEQ ID NO: 13 and SEQ ID NO: 14; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0051] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown in a pair of amino acid sequences selected from SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown in a pair of amino acid sequences selected from SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0052] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown in a pair of amino acid sequences selected from SEQ ID NO: 17 and SEQ ID NO: 18, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown in a pair of amino acid sequences selected from SEQ ID NO: 17 and SEQ ID NO: 18, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0053] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region each have an amino acid sequence shown in a pair of amino acid sequences selected from SEQ ID NO: 19 and SEQ ID NO: 20; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region each have an amino acid sequence shown in a pair of amino acid sequences selected from SEQ ID NO: 19 and SEQ ID NO: 20; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0054] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by a pair of amino acid sequences selected from SEQ ID NO: 21 and SEQ ID NO: 22, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by a pair of amino acid sequences selected from SEQ ID NO: 21 and SEQ ID NO: 22, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion or insertion of 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0055] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by a pair of amino acid sequences selected from SEQ ID NO: 23 and SEQ ID NO: 24, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by a pair of amino acid sequences selected from SEQ ID NO: 23 and SEQ ID NO: 24, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion or insertion of 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0056] In some embodiments, a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown in pairs of amino acid sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively; a binder that specifically binds to FOLR1 is provided herein. In some embodiments, the binder comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown in pairs of amino acid sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and the binder specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107.In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown in pairs of amino acid sequences selected from, respectively, SEQ ID NO: 1 and SEQ ID NO: 2; respectively, SEQ ID NO: 3 and SEQ ID NO: 4; respectively, SEQ ID NO: 5 and SEQ ID NO: 6; respectively, SEQ ID NO: 7 and SEQ ID NO: 8; respectively, SEQ ID NO: 9 and SEQ ID NO: 10; respectively, SEQ ID NO: 11 and SEQ ID NO: 12; respectively, SEQ ID NO: 13 and SEQ ID NO: 14; respectively, SEQ ID NO: 15 and SEQ ID NO: 16; respectively, SEQ ID NO: 17 and SEQ ID NO: 18; respectively, SEQ ID NO: 19 and SEQ ID NO: 20; respectively, SEQ ID NO: 21 and SEQ ID NO: 22; and respectively, SEQ ID NO: 23 and SEQ ID NO: 24; and the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown in pairs of amino acid sequences selected from, respectively, SEQ ID NO: 1 and SEQ ID NO: 2; respectively, SEQ ID NO: 3 and SEQ ID NO: 4; respectively, SEQ ID NO: 5 and SEQ ID NO: 6; respectively, SEQ ID NO: 7 and SEQ ID NO: 8; respectively, SEQ ID NO: 9 and SEQ ID NO: 10; respectively, SEQ ID NO: 11 and SEQ ID NO: 12; respectively, SEQ ID NO: 13 and SEQ ID NO: 14; respectively, SEQ ID NO: 15 and SEQ ID NO: 16; respectively, SEQ ID NO: 17 and SEQ ID NO: 18; respectively, SEQ ID NO: 19 and SEQ ID NO: 20; respectively, SEQ ID NO: 21 and SEQ ID NO: 22; and respectively, SEQ ID NO: 23 and SEQ ID NO: 24; and the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.As described herein, the binder may comprise an FOLR1 antibody or an antigen-binding portion thereof, and may further comprise another peptide or polypeptide covalently bound to the FOLR1 antibody or an antigen-binding portion thereof. In any of these embodiments, the binder specifically binds to FOLR1.

[0057] In some embodiments, provided herein is a binder that comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and that specifically binds to FOLR1. In some embodiments, the binder comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; and the binder specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binder that comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the heavy-chain variable framework region and the light-chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy-chain variable region or the light-chain variable region are not modified. In some embodiments, provided herein is a binder that comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the heavy-chain variable framework region and the light-chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy-chain variable region or the light-chain variable region are not modified.

[0058] In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and specifically bind to FOLR1. In some embodiments, the binder comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; and the binder specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1-8, 1-6, 1-4 or 1-2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1-8, 1-6, 1-4 or 1-2 amino acid substitutions, deletions or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0059] In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively, and specifically bind to FOLR1. In some embodiments, the binder comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; and the binder specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1-8, 1-6, 1-4, or 1-2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0060] In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and which specifically binds to FOLR1. In some embodiments, the binder comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; and the binder specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0061] In some embodiments, provided herein is a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and specifically bind to FOLR1. In some embodiments, the binding agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; and the binding agent specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0062] In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the binder specifically binds to FOLR1. In some embodiments, the binder comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and the binder specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0063] In some embodiments, provided herein is a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and specifically bind to FOLR1. In some embodiments, the binding agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; and the binding agent specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1-8, 1-6, 1-4, or 1-2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0064] In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and specifically bind to FOLR1. In some embodiments, the binder comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; and the binder specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0065] In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and the binder specifically binds to FOLR1. In some embodiments, the binder comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively; and the binder specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0066] In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and specifically bind to FOLR1. In some embodiments, the binder comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively; and the binder specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1-8, 1-6, 1-4 or 1-2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1-8, 1-6, 1-4 or 1-2 amino acid substitutions, deletions or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0067] In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and specifically bind to FOLR1. In some embodiments, the binder comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; and the binder specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0068] In some embodiments, provided herein is a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively, and specifically bind to FOLR1. In some embodiments, the binding agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and the binding agent specifically binds to FOLR1 with a binding affinity (lower Kd) higher than that of antibody FR107. In some embodiments, provided herein is a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0069] In some embodiments, an antibody or antigen-binding portion thereof that includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region includes complementarity-determining regions HCDR1, HCDR2, and HCDR3 disposed in a heavy-chain variable-region framework region, the VL region includes LCDR1, LCDR, and LCDR3 disposed in a light-chain variable-region framework region, and the VH and VL CDRs have amino acid sequences set forth in (i) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; and (ii) a set of amino acid sequences each selected from SEQ ID NO: 31, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively, is provided. In some embodiments, each VH region and VL region includes a humanized framework region. In some embodiments, each VH region and VL region includes a human framework region.

[0070] In some embodiments, an antibody or antigen-binding portion thereof that includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region includes complementarity-determining regions HCDR1, HCDR2, and HCDR3 disposed in a heavy-chain variable-region framework region, the VL region includes LCDR1, LCDR, and LCDR3 disposed in a light-chain variable-region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively, is provided. In some embodiments, each VH region and VL region includes a humanized framework region. In some embodiments, each VH region and VL region includes a human framework region.

[0071] In some embodiments, an antibody or antigen-binding portion thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region is provided, wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 disposed in a heavy chain variable region framework region, the VL region comprises LCDR1, LCDR, and LCDR3 disposed in a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO: 31, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively. In some embodiments, each VH region and VL region comprises a humanized framework region. In some embodiments, each VH region and VL region comprises a human framework region.

[0072] In some embodiments, a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region is provided, wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 disposed in a heavy chain variable region framework region, the VL region comprises LCDR1, LCDR, and LCDR3 disposed in a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in (i) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; and (ii) a set of amino acid sequences selected from SEQ ID NO: 31, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively. In some embodiments, each VH region and VL region comprises a humanized framework region. In some embodiments, each VH region and VL region comprises a human framework region.

[0073] In some embodiments, a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region is provided, wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 disposed in a heavy chain variable region framework region, the VL region comprises LCDR1, LCDR, and LCDR3 disposed in a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively. In some embodiments, each VH region and VL region comprises a humanized framework region. In some embodiments, each VH region and VL region comprises a human framework region.

[0074] In some embodiments, a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region is provided, wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 disposed in a heavy chain variable region framework region, the VL region comprises LCDR1, LCDR, and LCDR3 disposed in a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO: 31, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively. In some embodiments, each VH region and VL region comprises a humanized framework region. In some embodiments, each VH region and VL region comprises a human framework region.

[0075] In some embodiments, the compositions and methods described herein relate to the reduction of FOLR1+ cells in a subject (e.g., a decrease in the number of FOLR1+ cells in a cancer or tumor) by an in vivo FOLR1 antibody, an antigen-binding portion thereof, another binder, or a conjugate thereof. In some embodiments, the compositions and methods described herein relate to the treatment of FOLR1+ cancer in a subject by administering an FOLR1 antibody, an antigen-binding portion thereof, another binder, or a conjugate thereof. In some embodiments, the compositions and methods described herein relate to a decrease in the number of FOLR1+ cells in a subject by administering an FOLR1 antibody, an antigen-binding portion thereof, another binder, or a conjugate thereof.

[0076] As used herein, the term "antibody" refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that specifically binds to an antigen, e.g., human FOLR1. This term generally refers to an antibody composed of two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions, including full-length antibodies (having heavy chain constant regions and light chain constant regions).

[0077] Each heavy chain is composed of a variable region (abbreviated as VH) and a constant region. The heavy chain constant region may include three domains CH1, CH2, and CH3, and optionally a fourth domain CH4. Each light chain is composed of a variable region (abbreviated as VL) and a constant region. The light chain constant region is the CL domain. The VH region and the VL region are further divided into hypervariable regions called complementarity-determining regions (CDRs), and conserved regions called framework regions (FRs) may be interspersed. Thus, each VH region and VL region consists of three CDRs and four FRs arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. This structure is well known to those skilled in the art.

[0078] As used herein, the "antigen-binding portion" of a FOLR1 antibody refers to the portion of the FOLR1 antibody described herein that has the VH and VL sequences of the FOLR1 antibody or the CDRs of the FOLR1 antibody and specifically binds to FOLR1. Examples of antigen-binding portions include Fab, Fab’, F(ab’)2, Fv, scFv, disulfide-bonded Fv, single-domain antibodies (also referred to as VHH, VNAR, sdAb, or nanobodies) or diabodies (see, e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference). As used herein, the terms Fab, F(ab’)2, and Fv refer to (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab’)2 fragment, i.e., a bivalent fragment containing two Fab fragments linked to each other within the hinge region via a disulfide bridge; and (iii) in each case, the Fv fragment of the FOLR1 antibody, consisting of the VL and VH domains. The two domains of the Fv fragment, i.e., VL and VH, are encoded by separate coding regions, but these can be further linked to each other using a synthetic linker, such as the polyG4S amino acid sequence (the "(G4S)n" disclosed as SEQ ID NO: 38, where n = 1-5), to form a single protein chain to which the VL and VH regions bind to form a monovalent molecule (known as a single-chain Fv or scFv). The term "antigen-binding portion" of an antibody is also intended to include such single-chain antibodies. Other forms of single-chain antibodies, such as "diabodies", are also included herein.The diabody has VH and VL domains expressed on a single polypeptide chain, but uses a linker that connects the VH and VL domains, which are too short for the two domains to be able to bind on the same chain, thereby pairing the VH and VL domains with complementary domains on different chains (VL and VH, respectively) to form two antigen-binding sites, a bivalent bispecific antibody (e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J. et al. (1994) Structure 2: 1121-1123).

[0079] A single-domain antibody is an antibody moiety consisting of a single monomeric variable antibody domain. Single-domain antibodies can be derived from the variable domain of the heavy chain of antibodies from camelids (e.g., nanobodies or VHH moieties). Furthermore, the term single-domain antibody includes autonomous human heavy chain variable domains (aVH) or VNAR moieties derived from sharks (see, e.g., Hasler et al., Mol. Immunol. 75: 28-37, 2016).

[0080] Techniques for generating single-domain antibodies (e.g., DAB or VHH) are known in the art, as disclosed, for example, by Cossins et al. (2006, Prot Express Purif 51:253-259) and Li et al. (Immunol. Lett. 188:89-95, 2017). Single-domain antibodies can be obtained, for example, from camels, alpacas or llamas by standard immunization techniques. (See, for example, Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; and Maass et al., J Immunol Methods 324:13-25, 2007). VHH can have strong antigen-binding ability and can interact with novel epitopes inaccessible to conventional VH-VL pairs (see, for example, Muyldermans et al., 2001). Alpaca serum IgG contains approximately 50% IgG antibodies (HCAb) consisting only of camelid heavy chains (see, for example, Maass et al., 2007). Alpacas can be immunized with an antigen, and VHHs that bind to and neutralize the target antigen can be isolated (see, for example, Maass et al., 2007). PCR primers for amplifying alpaca VHH coding sequences have been identified and can be used to construct an alpaca VHH phage display library, which can be used for antibody fragment isolation by standard biopanning techniques well known in the art (see, for example, Maass et al., 2007).

[0081] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof is part of a bispecific or multispecific binding agent. Bispecific and multispecific antibodies include scFv1-scFv2, scFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb and scFv-HSA-scFv. In some embodiments, IgG-scFv is IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG or IgG-2scFv. See, for example, Brinkmann and Kontermann, MAbs 9(2):182-212 (2017); Wang et al., Antibodies, 2019, 8, 43; Dong et al., 2011, MAbs 3:273-88; Natsume et al., J. Biochem. 140(3):359-368, 2006; Cheal et al., Mol. Cancer Ther. 13(7):1803-1812, 2014; and Bates and Power, Antibodies, 2019, 8, 28.

[0082] Modifications of the VH and VL regions Regarding the VH amino acid sequence and the VL amino acid sequence, one of ordinary skill in the art will recognize that individual substitutions, deletions or additions (insertions) to the nucleic acid encoding an amino acid in VH or VL, or in a polypeptide, that change a single amino acid or a small percentage of the amino acids in the encoded sequence result in an amino acid substitution with a chemically similar amino acid (conservative amino acid substitution) and that the modified polypeptide is a "conservative modification variant" that retains the ability to specifically bind to FOLR1.

[0083] In some embodiments, a conservative modified variant of a FOLR1 antibody or an antigen-binding portion thereof can have modifications in the framework regions (FRs) (i.e., other than the CDRs). For example, a conservative modified variant of a FOLR1 antibody has the amino acid sequences of the VH and VL CDRs ((i) each set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; and (ii) each set forth in SEQ ID NO: 31, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35), and has at least one conservative amino acid substitution in the framework region. In some embodiments, the VH and VL amino acid sequences have collectively 8 or 6 or 4 or 2 or 1 or fewer conservative amino acid substitutions in the FRs as compared to the amino acid sequences of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences have 8 to 1, 6 to 1, 4 to 1, or 2 to 1 conservative amino acid substitutions in the FRs as compared to the amino acid sequences of the unmodified VH and VL regions. In a further aspect of any of these embodiments, the conservative modified variant of the FOLR1 antibody, its antigen-binding portion, or other binder exhibits specific binding to FOLR1.

[0084] For conservative amino acid substitutions, a given amino acid can be replaced by a residue having similar physicochemical properties. For example, one aliphatic residue can be substituted for another (e.g., Ile, Val, Leu, or Ala for each other), or one polar residue can be substituted for another (e.g., between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative amino acid substitutions, such as substitutions of entire regions having similar hydrophobic properties, are well known. A polypeptide containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity of the native or reference polypeptide, i.e., activity against FOLR1, such as antigen-binding activity and specificity, is retained.

[0085] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent can be further optimized, for example, for treatment in humans, to reduce potential immunogenicity while maintaining functional activity or to optimize other functional properties. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown in pairs of amino acid sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown as pairs of amino acid sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion or insertion of 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0086] In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0087] In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0088] In some embodiments, provided herein is an anti-FOLR1 antibody or an antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is an anti-FOLR1 antibody or an antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0089] In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0090] In some embodiments, provided herein is an FOLR1 antibody or an antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is an FOLR1 antibody or an antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0091] In some embodiments, provided herein is an FOLR1 antibody or an antigen-binding portion or other binder thereof that includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the heavy-chain variable framework region and the light-chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy-chain variable region or the light-chain variable region are not modified. In some embodiments, provided herein is a binder that includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the heavy-chain variable framework region and the light-chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy-chain variable region or the light-chain variable region are not modified.

[0092] In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0093] In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0094] In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively; the heavy-chain variable framework region and the light-chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy-chain variable region or the light-chain variable region are not modified. In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively; the heavy-chain variable framework region and the light-chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy-chain variable region or the light-chain variable region are not modified.

[0095] In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is an anti-FOLR1 antibody or antigen-binding portion or other binder thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0096] In some embodiments, provided herein is an FOLR1 antibody or antigen-binding portion or other binder thereof that includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified. In some embodiments, provided herein is an FOLR1 antibody or antigen-binding portion or other binder thereof that includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; the heavy chain variable framework region and the light chain variable framework region are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified.

[0097] In some embodiments, provided herein is an FOLR1 antibody or an antigen-binding portion or other binder thereof that includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively; the heavy-chain variable framework region and the light-chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy-chain variable region or the light-chain variable region are not modified. In some embodiments, provided herein is an FOLR1 antibody or an antigen-binding portion or other binder thereof that includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively; the heavy-chain variable framework region and the light-chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy-chain variable region or the light-chain variable region are not modified.

[0098] In any of these embodiments, the functional activity of the FOLR1-binding antibody or antigen-binding portion thereof or other binding agent includes specific binding to FOLR1. Further functional activities include depletion of FOLR1+ cells (e.g., cancer cells). Further, an FOLR1 antibody or antigen-binding portion thereof or other binding agent having functional activity, regardless of the presence or absence of dose-dependence, when measured in a specific assay such as a biological assay, is a polypeptide that exhibits activity similar to or better than that of a reference antibody or antigen-binding portion thereof described herein (e.g., a reference FOLR1-binding antibody or antigen-binding portion thereof or variant thereof having (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 36 and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 37 as described herein). If dose-dependence is present, it need not be identical to that of the reference antibody or antigen-binding portion thereof, rather it is substantially similar to or better than the dose-dependence at a given activity compared to the reference antibody or antigen-binding portion thereof described herein (i.e., the candidate polypeptide exhibits greater activity compared to the reference antibody).

[0099] In the case of conservative substitutions, amino acids can be grouped according to the similarity of the properties of their side chains (A.L. Lehninger, Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).

[0100] Alternatively, in the case of conservative substitutions, naturally occurring residues can be grouped based on common side-chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging one member of these classes or another class.

[0101] Certain conservative substitutions include, for example,; Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0102] In some embodiments, a conservatively modified variant of the FOLR1 antibody or antigen-binding portion thereof is preferably 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 more identical to the reference VH or VL sequence, and the VH and VL CDRs are not modified. The degree of homology (percent identity) between the reference and modified sequences can be determined, for example, by comparing the two sequences using freely available computer programs generally employed for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0103] In some embodiments, the VH and VL amino acid sequences collectively have 8 or 6 or 4 or 2 or 1 or fewer conservative amino acid substitutions in the framework region as compared to the amino acid sequences of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences collectively have 8 to 1, or 6 to 1, or 4 to 1, or 2 to 1 conservative amino acid substitutions in the framework region as compared to the amino acid sequences of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences collectively have 8 or 6 or 4 or 2 or 1 or fewer amino acid substitutions, deletions or insertions in the framework region as compared to the amino acid sequences of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences have 8 to 1, 6 to 1, 4 to 1, or 2 to 1 conservative amino acid substitutions in the framework region as compared to the amino acid sequences of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences collectively have 8 or 6 or 4 or 2 or 1 or fewer amino acid substitutions, deletions or insertions as compared to the amino acid sequences of the unmodified VH and VL regions.

[0104] Modification of the native (or reference) amino acid sequence can be achieved by any of several techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing an oligonucleotide containing the desired mutant sequence adjacent to a restriction site that allows ligation to a fragment of the native sequence. After ligation, the resulting reconstructed sequence encodes a variant having the desired amino acid insertion, substitution, or deletion. Alternatively, an oligonucleotide-directed site-specific mutagenesis procedure can be employed to provide a modified nucleotide sequence having a specific codon modified according to the desired substitution, deletion, or insertion. Techniques for performing such modifications are very well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 and 4,737,462, which are hereby incorporated by reference in their entirety.

[0105] constant region In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent has a fully human constant region. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent has a fully humanized constant region. In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent has a non-human constant region. The immunoglobulin constant region refers to a heavy chain constant region or a light chain constant region. The amino acid sequences of the human heavy chain constant region and the human light chain constant region are known in the art. The constant region can be of any suitable type that can be selected from the classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM. Some immunoglobulin classes can be further divided into isotypes, such as IgG1, IgG2, IgG3, IgG4, or IgA1, and IgA2. The heavy chain constant regions (Fc) corresponding to different classes of immunoglobulins can be α, δ, ε, γ, and μ, respectively. The light chain can be either kappa (or κ) or lambda (or λ).

[0106] In some embodiments, the constant region can have an IgG1 isotype. In some embodiments, the constant region can have an IgG2 isotype. In some embodiments, the constant region can have an IgG3 isotype. In some embodiments, the constant region can have an IgG4 isotype. In some embodiments, the Fc domain can have a hybrid isotype that includes constant regions from two or more isotypes. In some embodiments, the immunoglobulin constant region can be an IgG1 or IgG4 constant region. In some embodiments, the FOLR1 antibody heavy chain is of the IgG1 isotype and has the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the FOLR1 antibody light chain is of the kappa isotype and has the amino acid sequence shown in SEQ ID NO: 40.

[0107] Furthermore, the FOLR1 antibody or antigen-binding portion thereof or other binding agent can be part of a larger binding agent formed by covalent or non-covalent attachment of the antibody or antigen-binding portion to one or more other proteins or peptides. Examples of such binding agents include the use of streptavidin core regions for preparing tetrameric scFv molecules (Kipriyanov, S.M. et al. (1995), Human Antibodies and Hybridomas 6:93-101), as well as the use of cysteine residues, marker peptides, and C-terminal polyhistidinyl peptides, such as the hexahistidinyl tag (the "hexahistidinyl tag" disclosed as SEQ ID NO: 41), for generating divalent and biotinylated scFv molecules (Kipriyanov, S.M. et al. (1994) Mol. Immunol. 31:1047-1058).

[0108] Fc domain modification for modifying effector function In some embodiments, the Fc region or Fc domain of an anti-FOLR1 antibody or its antigen-binding portion or other binder does not substantially bind to at least one Fc receptor selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). In some embodiments, the Fc region or domain does not substantially bind to any of the Fc receptors selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). As used herein, "does not substantially bind" refers to weak or no binding to one or more selected Fc gamma receptors. In some embodiments, "does not substantially bind" refers to a decrease in binding affinity (e.g., an increase in Kd) for the Fc gamma receptor by at least one thousand-fold. In some embodiments, the Fc domain or region is Fc null. As used herein, "Fc null" refers to an Fc region or Fc domain that shows weak or no binding to any of the Fc gamma receptors. In some embodiments, the Fc null domain or region shows a decrease in binding affinity (i.e., an increase in Kd) for the Fc gamma receptor by at least one thousand-fold.

[0109] In some embodiments, the Fc domain has reduced or substantially no effector function activity. As used herein, "effector function activity" refers to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). In some embodiments, the Fc domain exhibits reduced ADCC, ADCP or CDC activity as compared to the wild-type Fc domain. In some embodiments, the Fc domain exhibits a reduction in ADCC, ADCP and CDC as compared to the wild-type Fc domain. In some embodiments, the Fc domain substantially does not exhibit effector function (i.e., the ability to stimulate or effect ADCC, ADCP or CDC). As used herein, "substantially no effector function" refers to a reduction in effector function activity of at least one thousandth-fold as compared to the wild-type or reference Fc domain.

[0110] In some embodiments, the Fc domain has reduced or no ADCC activity. As used herein, reduced or no ADCC activity refers to a reduction in ADCC activity of the Fc domain of at least one tenth-fold, at least one twentieth-fold, at least one thirtieth-fold, at least one fiftieth-fold, at least one hundredth-fold or at least one five-hundredth-fold.

[0111] In some embodiments, the Fc domain has reduced or no CDC activity. As used herein, reduced or no CDC activity refers to a reduction in CDC activity of the Fc domain of at least one tenth-fold, at least one twentieth-fold, at least one thirtieth-fold, at least one fiftieth-fold, at least one hundredth-fold or at least one five-hundredth-fold.

[0112] In vitro and / or in vivo cytotoxicity assays can be performed to confirm a decrease / depletion of ADCC and / or CDC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks Fc gamma receptor binding (and thus presumably lacks ADCC activity). NK cells, which are the major cells mediating ADCC, express only Fc gamma RIII, while monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, a non-radioactive assay method may be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and CytoTox 96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Nat’l Acad. Sci. USA 95:652-656 (1998).

[0113] A C1q binding assay can also be performed to confirm that the antibody or Fc domain or region is unable to bind to C1q and thus lacks CDC activity or has reduced CDC activity. See, for example, the C1q and C3c binding ELISAs of WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)).

[0114] In some embodiments, the Fc domain has reduced or no ADCP activity. As used herein, having reduced or no ADCP activity refers to a reduction in the ADCP activity of the Fc domain by at least 1 / 10-fold, at least 1 / 20-fold, at least 1 / 30-fold, at least 1 / 50-fold, at least 1 / 100-fold or at least 1 / 500-fold.

[0115] An ADCP binding assay can also be performed to confirm that the antibody or Fc domain or region lacks ADCP activity or has reduced ADCP activity. See, for example, US 2019 / 0079077, US 2019 / 0048078 and the references disclosed therein.

[0116] FOLR1 antibodies or antigen-binding portions or other binders thereof with reduced effector function activity include those having one or more substitutions of Fc region residues such as 238, 265, 269, 270, 297, 327, and 329 according to Kabat's EU numbering (see, for example, U.S. Patent No. 6,737,056). Such Fc variants include Fc variants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine according to Kabat's EU numbering (see U.S. Patent No. 7,332,581). Certain antibody variants with reduced binding to FcR are also known. (See, for example, U.S. Patent No. 6,737,056; International Publication No. 2004 / 056312 pamphlet, and Shields et al., J. Biol. Chem. 9(2):6591 - 6604 (2001)). FOLR1 antibodies or antigen-binding portions or other binders thereof containing such amino acid modifications and having reduced binding to FcR can be prepared.

[0117] In some embodiments, the FOLR1 antibody or antigen-binding portion or other binder thereof comprises an Fc domain or region having one or more amino acid substitutions that reduce Fc gamma R binding, such as substitutions at positions 234 and 235 (EU numbering of residues) in the Fc region. In some embodiments, the substitutions are L234A and L235A (LALA) according to Kabat's EU numbering. In some embodiments, the Fc domain comprises D265A and / or P329G in an Fc region derived from the human IgG1 Fc region according to Kabat's EU numbering. In some embodiments, the substitutions are L234A, L235A, and P329G (LALA - PG) in an Fc region derived from the human IgG1 Fc region according to Kabat's EU numbering (see, for example, International Publication No. 2012 / 130831 pamphlet). In some embodiments, the substitutions are L234A, L235A, and D265A (LALA - DA) in an Fc region derived from the human IgG1 Fc region according to Kabat's EU numbering.

[0118] In some embodiments, for example, modifications of the Fc region are made that result in a modification (i.e., a decrease in either) of C1q binding and / or complement-dependent cytotoxicity (CDC), as described in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642 pamphlet, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0119] Methods of making antibodies, antigen-binding portions, and other binding agents In various embodiments, FOLR1 antibodies, antigen-binding portions thereof, and other binding agents can be produced in human, mouse, or other animal-derived cell lines. Recombinant DNA expression can be used to produce FOLR1 antibodies, antigen-binding portions thereof, and other binding agents. This enables the production of FOLR1 antibodies as well as a range of FOLR1 antigen-binding portions and other binding agents (including fusion proteins) in a selected host species. Production of FOLR1 antibodies, antigen-binding portions thereof, and other binding agents in bacteria, yeast, transgenic animals, and chicken eggs are also alternative methods to cell-based production systems. The main advantage of transgenic animals is the potential high yield from renewable resources.

[0120] In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23. In some embodiments, the nucleic acid encodes an FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the nucleic acid encodes an FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 23.

[0121] In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the nucleic acid encodes a FOLR1 VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 24.

[0122] In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 1 and 2. In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 3 and 4. In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 5 and 6. In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 7 and 8. In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 9 and 10. In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 11 and 12. In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 13 and 14. In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 15 and 16. In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 17 and 18. In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 19 and 20. In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 21 and 22. In some embodiments, the nucleic acid encodes a VH polypeptide and a VL polypeptide having the amino acid sequences set forth in SEQ ID NOs: 23 and 24.

[0123] As used herein, the terms "nucleic acid", "nucleic acid sequence", "polynucleotide sequence", or "nucleotide" refer to polymeric molecules incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be a single strand of a denatured double-stranded DNA. In some embodiments, the nucleic acid can be cDNA, e.g., a nucleic acid lacking introns.

[0124] Nucleic acid molecules encoding the amino acid sequences of FOLR1 antibodies, antigen-binding portions thereof, and other binding agents can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the preparation of synthetic nucleotide sequences encoding FOLR1 antibodies, antigen-binding portions, or other binding agents. In addition, oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding FOLR1 antibodies or antigen-binding portions and other binding agents. At least the nucleic acid sequences encoding the FOLR1 antibodies, antigen-binding portions, binding agents, or polypeptides thereof described herein can be recombined with vector DNA according to conventional techniques such as, for example, blunt or sticky ends for ligation, restriction enzyme digestion to provide appropriate sticky ends, filling in of sticky ends if necessary, alkaline phosphatase treatment to avoid unwanted ligation, and ligation with appropriate ligases, or other techniques known in the art. Techniques for such manipulations are disclosed, for example, by Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons), 1987-1993, and can be used to construct nucleic acid sequences and vectors encoding FOLR1 antibodies or antigen-binding portions thereof, or VH or VL polypeptides thereof, or other binding agents.

[0125] Nucleic acid molecules such as DNA contain nucleotide sequences that contain transcriptional and translational regulatory information, and such sequences are said to be "able to express" a polypeptide when they are "operably linked" to a nucleotide sequence encoding a polypeptide. An operable linkage is a linkage in which a regulatory DNA sequence and a DNA sequence that is required to be expressed (e.g., a FOLR1 antibody or an antigen-binding portion thereof or other binding agent) are connected such that gene expression of a recoverable amount of the polypeptide or antigen-binding portion is enabled. The exact nature of the regulatory regions required for gene expression can vary from organism to organism, as is well known in the relevant art. See, for example, Sambrook et al., 1989; Ausubel et al., 1987-1993.

[0126] Accordingly, expression of the FOLR1 antibodies or antigen-binding portions thereof described herein can be carried out in either prokaryotic or eukaryotic cells. Suitable hosts include bacteria or eukaryotic hosts, including yeast, insects, fungi, avian, and mammalian cells in vivo or in situ, or host cells of mammalian, insect, avian, or yeast origin. Mammalian cells or tissues can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, although any other mammalian cells can be used. Further, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved, for example, through use of the yeast ubiquitin hydrolase system. The fusion proteins thus produced can be processed in vivo or purified and processed in vitro to enable synthesis of the FOLR1 antibodies or antigen-binding portions thereof or other binders described herein having the designated amino-terminal sequences. Further, problems associated with retention of the start codon-derived methionine residue in direct yeast (or bacterial) expression can be avoided (see, e.g., Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989)). Recombinant FOLR1 antibodies or antigen-binding portions thereof or other binders can be obtained using any of a series of yeast gene expression systems incorporating promoter elements and terminator elements from highly expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast are grown in glucose-rich media. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, the promoter signal and terminator signal of the phosphoglycerate kinase gene can be utilized.

[0127] Production of FOLR1 antibodies or antigen-binding portions thereof or other binders in insects can be achieved, for example, by infecting insect hosts with baculoviruses engineered to express polypeptides by methods known to those of skill in the art. See Ausubel et al., 1987 - 1993.

[0128] In some embodiments, the introduced nucleic acid sequence (encoding the FOLR1 antibody or antigen-binding portion thereof or other binding agent or polypeptide thereof) is incorporated into a plasmid or viral vector that can autonomously replicate in the recipient host cell. Any of a variety of vectors can be employed for this purpose, and these are known and available to those of skill in the art. See, for example, Ausubel et al., 1987 - 1993. Important factors in selecting a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from recipient cells not containing the vector; the copy number of the vector desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between different species of host cells.

[0129] Exemplary prokaryotic vectors known in the art include plasmids such as plasmids that can replicate in Escherichia coli (E. coli). Other gene expression elements useful for the expression of DNA encoding an FOLR1 antibody or antigen-binding portion thereof or other binding agent include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), the Rous sarcoma virus LTR (Gorman et al., 79 PNAS 6777 (1982)), and the Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b) splice regions and polyadenylation sites, such as those derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites, such as those in SV40 (Okayama et al., 1983). DNA genes encoding immunoglobulins can be expressed using the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancer, SV40 late region mRNA splicing, rabbit S globin intervening sequences, immunoglobulin and rabbit S globin polyadenylation sites, and the SV40 polyadenylation element as expression elements, as described by the following Liu et al., and Weidle et al., 51 Gene 21 (1987).

[0130] In the case of immunoglobulins encoding nucleotide sequences, the transcription promoter can be, for example, human cytomegalovirus, and the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulins.

[0131] In some embodiments, for the expression of a DNA coding region in a rodent cell, the transcription promoter can be a viral LTR sequence, the transcription promoter enhancer can be either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, and can be a polyadenylation and transcription termination region. In other embodiments, a DNA sequence encoding another protein is combined with the above expression elements to achieve protein expression in mammalian cells.

[0132] Each coding region or gene fusion is assembled or inserted into an expression vector. A recipient cell capable of expressing the FOLR1 variable region or its antigen-binding portion or other binder is then transfected alone with a nucleotide encoding an FOLR1 antibody or antibody polypeptide or its antigen-binding portion or other binder, or co-transfected with polynucleotides encoding VH and VL chain coding regions or other binders. The transfected recipient cells are cultured under conditions that allow expression of the incorporated coding region, and the expressed antibody chain or intact antibody or antigen-binding portion or other binder is recovered from the culture.

[0133] In some embodiments, a nucleic acid containing a coding region encoding an FOLR1 antibody or an antigen-binding portion thereof or other binding agent is assembled into a separate expression vector that is then used to co-transfect recipient host cells. Each vector can contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, with the first selectable gene designed for selection in a bacterial system and the second selectable gene designed for selection in a eukaryotic system, and each vector having a set of coding regions. This strategy results in vectors that first direct the production of nucleotide sequences in a bacterial system and allow amplification. The DNA vectors thus produced and amplified in a bacterial host are then used to co-transfect eukaryotic cells, enabling selection of co-transfected cells having the desired transfected nucleic acid (e.g., containing the heavy and light chains of an FOLR1 antibody). Non-limiting examples of selectable genes for use in a bacterial system are genes conferring resistance to ampicillin and genes conferring resistance to chloramphenicol. Selectable genes for use in eukaryotic transfectants include the xanthine-guanine phosphoribosyl transferase gene (referred to as gpt) and the phosphotransferase gene derived from Tn5 (referred to as neo). Alternatively, the fusion nucleotide sequences encoding the VH and VL chains can be assembled on the same expression vector.

[0134] For transfection of the expression vector and production of the FOLR1 antibody or its antigen-binding portion or other binding agent, the recipient cell line can be a Chinese hamster ovary cell line (e.g., DG44) or a myeloma cell. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by transfected immunoglobulin genes and have mechanisms for glycosylation of immunoglobulins. For example, in some embodiments, the recipient cell is the recombinant Ig-producing myeloma cell SP2 / 0. SP2 / 0 cells produce only the immunoglobulin encoded by the transfected gene. Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse, and the secreted immunoglobulin can be obtained from ascites fluid.

[0135] The expression vector encoding the FOLR1 antibody or its antigen-binding portion or other binding agent can be introduced into a suitable host cell by any of a variety of suitable means, including biochemical means such as transformation, transfection, protoplast fusion, calcium phosphate precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and particle bombardment. See Johnston et al., 240 Science 1538 (1988), which is known to those skilled in the art.

[0136] Yeast offers certain advantages over bacteria for production of immunoglobulin heavy and light chains. Yeast performs post-translational peptide modifications including glycosylation. There are several recombinant DNA strategies that utilize strong promoter sequences and high copy number plasmids that can be used to produce the desired protein in yeast. Yeast recognizes the leader sequences of cloned mammalian gene products and secretes polypeptides having the leader sequence (i.e., pre-polypeptides). See, for example, Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpellier, France, 1982).

[0137] Yeast gene expression systems can be routinely evaluated for the production, secretion, and stability levels of antibodies, as well as assembled FOLR1 antibodies and their antigen-binding portions and other binding agents. Various yeast gene expression systems can be utilized that incorporate promoter elements and termination elements from highly expressed genes encoding glycolytic enzymes that are produced in large quantities when yeast are grown in glucose-rich media. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter signal and terminator signal of the phosphoglycerate kinase (PGK) gene can be utilized. Another example is the translation elongation factor 1 alpha promoter, such as that derived from Chinese hamster cells. Several approaches can be taken to evaluate optimal expression plasmids for the expression of immunoglobulins in yeast. See II DNA Cloning 45, (ed. Glover, IRL Press, 1985) and, for example, U.S. Patent Application Publication No. 2006 / 0270045.

[0138] Bacterial strains can also be used as hosts for the production of the antibody molecules or their antigen-binding portions and other binding agents described herein. E. coli K12 strains such as E. coli W3110, enterobacteria such as Bacillus species, Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species can be used. Plasmid vectors containing replicons and control sequences derived from species compatible with the host cell are used in connection with these bacterial hosts. The vector has a replication site and specific genes that can provide phenotypic selection in the transformed cells. Several approaches can be taken to evaluate expression plasmids for the production of FOLR1 antibodies and their antigen-binding portions and other binding agents in bacteria (see Glover, 1985; Ausubel, 1987, 1993; Sambrook, 1989; Colligan, 1992-1996).

[0139] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin molecules, including removal of leader peptides, folding and assembly of VH and VL chains, glycosylation of antibody molecules, and secretion of functional antibodies and / or their antigen-binding portions or other binding agents.

[0140] In addition to the above lymphoid-derived cells, mammalian cells that can be useful as hosts for the production of antibody proteins include fibroblast-derived cells such as Vero cells or CHO-K1 cells. Exemplary eukaryotic cells that can be used to express immunoglobulin polypeptides include, but are not limited to, COS cells including COS7 cells; 293 cells including 293-6E cells; CHO cells including CHO-S and DG44 cells; PERC6™ cells (Crucell); and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform desired post-translational modifications on the heavy and / or light chains. For example, in some embodiments, CHO cells produce polypeptides with a higher level of sialylation than the same polypeptide produced in 293 cells.

[0141] In some embodiments, one or more FOLR1 antibodies or their antigen-binding portions or other binding agents can be produced in vivo in an animal engineered or transfected with one or more nucleic acid molecules encoding the polypeptide according to any suitable method.

[0142] In some embodiments, an antibody or its antigen-binding portion or other binding agent is produced in a cell-free system. Exemplary non-limiting cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); and Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0143] A number of vector systems are available for the expression of VH and VL chains in mammalian cells (see Glover, 1985). Intact antibodies can be obtained according to various approaches. As discussed above, it is possible to co-express the VH and VL chains, and optionally the associated constant regions, in the same cell to achieve intracellular association and ligation of the VH and VL chains into a complete tetrameric H2L2 antibody or its antigen-binding portion. Co-expression can be carried out by using either the same or different plasmids in the same host. Nucleic acids encoding the VH and VL chains or their antigen-binding portion or other binding agents can be placed on the same plasmid, which is then transfected into cells, thereby directly selecting cells that express both chains. Alternatively, cells can first be transfected with a plasmid encoding one chain, e.g., the VL chain, and subsequently transfected with a VH chain plasmid containing a second selectable marker. Cell lines producing antibodies or their antigen-binding portions can be transfected via either route with a plasmid encoding additional copies of a peptide, VH, VL, or VH+VL chains, together with an additional selectable marker, to generate cell lines with enhanced properties such as higher production of the assembled FOLR1 antibody or its antigen-binding portion or other binding agent or enhanced stability of the transfected cell line.

[0144] Furthermore, plants are emerging as a convenient, safe and economic alternative expression system for the production of recombinant antibodies based on large-scale culture of microorganisms or animal cells. FOLR1-binding antibodies or antigen-binding portions thereof or other binding agents can be expressed in plant cell cultures or in plants grown conventionally. Expression in plants may be systemic, limited to intracellular plastids, or limited to seeds (endosperm). See, for example, U.S. Patent Application Publication No. 2003 / 0167531; U.S. Patent No. 6,080,560; U.S. Patent No. 6,512,162; and International Publication No. 0129242 pamphlet. Some plant-derived antibodies have reached advanced development stages, including clinical trials (see, for example, Biolex, N.C.).

[0145] In the case of intact antibodies, the variable regions (VH region and VL region) of the FOLR1 antibody are typically linked to at least a part of an immunoglobulin constant region (Fc) or domain, typically at least a part of a human immunoglobulin constant region or domain. Human constant region DNA sequences can be isolated from various human cells such as immortalized B cells according to well-known procedures (International Publication No. 87 / 02671 pamphlet). The FOLR1-binding antibody can contain both a light chain constant region and a heavy chain constant region. The heavy chain constant region can include a CH1 region, a hinge region, a CH2 region, a CH3 region, and optionally a CH4 region. In some embodiments, the CH2 domain can be deleted or omitted.

[0146] Techniques described for the production of single-chain antibodies (see, e.g., U.S. Patent No. 4,946,778, which is hereby incorporated by reference in its entirety; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single-chain antibodies that specifically bind to FOLR1. Single-chain antibodies are formed by linking the heavy-chain variable region and the light-chain variable region of the Fv region via an amino acid bridge to yield a single-chain polypeptide. Techniques for the assembly of functional Fv portions in E. coli can also be used (see, e.g., Skerra et al., Science 242:1038-1041 (1988), which is hereby incorporated by reference in its entirety).

[0147] In some embodiments, the antigen-binding portion or other binding agent comprises one or more scFvs. An scFv can be, for example, a fusion protein of the variable regions of the heavy-chain (VH) and light-chain (VL) variable regions of an antibody connected by a short linker peptide of about 10 to about 25 amino acids. The linker is typically rich in glycine for flexibility and rich in serine or threonine for solubility and can connect the N-terminus of VH to the C-terminus of VL or vice versa. This protein retains the specificity of the original antibody despite removal of the constant regions and introduction of the linker. scFv antibodies are described, for example, in Houston, J.S., Methods in Enzymol 203 (1991) 46-96. Methods for making scFv molecules and for designing appropriate peptide linkers are described, for example, in U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; Raag and Whitlow, FASEB 9:73-80 (1995) and Bird and Walker, TIBTECH, 9:132-137 (1991). ScFv-Fc is described by Sokolowska-Wedzina et al., Mol. Cancer Res. 15(8):1040-1050, 2017.

[0148] In some embodiments, the antigen-binding portion or other binding agent is a single-domain antibody that is an antigen-binding portion consisting of a single monomeric variable antibody domain. Single-domain antibodies can be derived from the variable domains of heavy chains of antibodies from camelids (e.g., nanobodies or VHH portions). Additionally, single-domain antibodies can be autologous human heavy chain variable domains (aVH) or VNAR portions derived from sharks (see, e.g., Hasler et al., Mol. Immunol. 75:28-37, 2016).

[0149] Techniques for making single-domain antibodies (DABs or VHHs) are known in the art, as disclosed, for example, by Cossins et al. (2006, Prot Express Purif 51:253-259) and Li et al. (Immunol. Lett. 188:89-95, 2017). Single-domain antibodies can be obtained, for example, from camels, alpacas, or llamas by standard immunization techniques. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; and Maass et al., J Immunol Methods 324:13-25, 2007). VHHs can have strong antigen-binding capabilities and can interact with epitopes inaccessible to conventional VH-VL pairs (see, e.g., Muyldermans et al., 2001). Alpaca serum IgG contains approximately 50% IgG antibodies of only camelid heavy chains (HCAb) (see, e.g., Maass et al., 2007). Alpacas can be immunized with an antigen, and VHHs that bind to and neutralize the target antigen can be isolated (see, e.g., Maass et al., 2007). PCR primers for amplifying alpaca VHH coding sequences have been identified and can be used to construct alpaca VHH phage display libraries, which can be used for antibody fragment isolation by standard biopanning techniques well known in the art (see, e.g., Maass et al., 2007).

[0150] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-chain / light-chain pairs having different specificities (see, e.g., Milstein and Cuello, Nature 305:537 (1983); WO 93 / 08829; and Traunecker et al., EMBO J. 10:3655 (1991)), and the "knobs-into-holes" technique (see, e.g., U.S. Patent No. 5,731,168; Carter (2001), J Immunol Methods 248, 7-15). Multispecific antibodies can also be made by engineering the electrostatic steering effect for making antibody Fc-heterodimer molecules (see, e.g., WO 2009 / 089004); cross-linking of two or more antibodies or antigen-binding portions thereof (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); use of leucine zippers for making bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); use of the "diabody" technique for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and use of single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and can also be made, for example, by the preparation of trispecific antibodies as described in Tutt et al. J. Immunol. 147:60 (1991).

[0151] Engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies," can also serve as binding agents (see, e.g., US 2006 / 0025576 A1).

[0152] The binding agents (e.g., antibodies or antigen-binding portions) herein also include “dual action Fabs” or “DAFs” that include antigen-binding sites that bind two different antigens (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820 and Bostrom et al., 2009, Science 323:1610-14). “Crossmab” antibodies are also included herein (see, e.g., International Publication No. WO 2009 / 080251, International Publication No. WO 2009 / 080252, International Publication No. WO 2009 / 080253, International Publication No. WO 2009 / 080254, and International Publication No. WO 2013 / 026833).

[0153] In some embodiments, the binding agent includes different antigen-binding sites fused to one or the other of the two subunits of the Fc domain; thus, the two subunits of the Fc domain can be included in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. Thus, it is advantageous to introduce modifications that promote the association of the desired polypeptides into the Fc domain of the binding agent to improve the yield and purity of the bispecific molecule in recombinant production.

[0154] Generally, this method involves substituting one or more amino acid residues at the interface of the two Fc domains with charged amino acid residues such that homodimer formation is electrostatically disfavored, but heterodimerization is electrostatically favored.

[0155] In some embodiments, the binder is a "bispecific T cell engager" or BiTE (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain can comprise two single-chain Fv (scFv) moieties having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of sufficient length to permit intramolecular association between the two domains. This single polypeptide further comprises a polypeptide spacer sequence between the two scFvs. Each scFv recognizes a different epitope, and since these epitopes can be specific for different proteins, both proteins are bound by the BiTE.

[0156] Since this is a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic expression system known in the art, such as a CHO cell line. However, specific purification techniques (see, e.g., EP 1691833) may be required to separate the monomeric bispecific T cell engager from other multimeric species that may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography and the polypeptide is eluted with a gradient of imidazole concentration. This eluate is further purified using anion exchange chromatography and the polypeptide is eluted using a gradient of sodium chloride concentration. Finally, this eluate is subjected to size exclusion chromatography to separate the monomer from the multimeric species. In some embodiments, the binder, which is a bispecific antibody, is composed of a single polypeptide chain comprising two single-chain FV moieties (scFV) fused to each other by a peptide linker.

[0157] In some embodiments, the binder is multispecific, for example an IgG-scFV. IgG-scFv formats include IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG and IgG-2scFv. These and other bispecific antibody formats and methods of making them are described, for example, in Brinkmann and Kontermann, MAbs 9(2):182-212 (2017); Wang et al., Antibodies, 2019, 8, 43; Dong et al., 2011, MAbs 3:273-88; Natsume et al., J. Biochem. 140(3):359-368, 2006; Cheal et al., Mol. Cancer Ther. 13(7):1803-1812, 2014; and Bates and Power, Antibodies, 2019, 8, 28.

[0158] IgG-like bispecific variable domain antibodies (DVD-Ig) are described by Wu et al., 2007, Nat Biotechnol 25:1290-97; Hasler et al., Mol. Immunol. 75:28-37, 2016, and in International Publication No. 08 / 024188 pamphlet and International Publication No. 07 / 024715 pamphlet. Triomab is described by Chelius et al., MAbs 2(3):309-319, 2010. 2-in-1-IgG is described by Kontermann et al., Drug Discovery Today 20(7):838-847, 2015. Tandem antibody or TandAb is described by Kontermann et al., ibid. ScFv-HSA-scFv antibodies are also described by Kontermann et al. (ibid.).

[0159] Intact (e.g., whole) antibodies, their dimers, individual light and heavy chains, or their antigen-binding portions, as well as other binders, can be recovered and purified by known techniques, such as immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination thereof. Generally, see Scopes, Protein Purification (Springer-Verlag, New York, 1982). Substantially pure FOLR1-binding antibodies or their antigen-binding portions or other binders with at least about 90% - 95% homogeneity are advantageous, and in particular for pharmaceutical use, those having a homogeneity of 98% - 99% or more are advantageous. Once partially or to the desired homogeneity purified, the intact FOLR1 antibody or its antigen-binding portion or other binder can be used therapeutically or in developing and performing assay procedures, immunofluorescent staining, etc. Generally, see Vols. I&II Immunol. Meth. (eds. Lefkovits & Pernis, Acad. Press, New York, 1979 and 1981).

[0160] Antibody-drug conjugate In some embodiments, the FOLR1 antibodies, antigen-binding portions or other binders described herein are part of an antibody-drug conjugate (also referred to as a FOLR1 conjugate or FOLR1 ADC). In some embodiments, the FOLR1 antibody, antigen-binding portion or other binder is attached to at least one linker, and at least one drug is attached to each linker. As used herein, in the context of a conjugate, the term "drug" refers to a cytotoxic agent (such as a chemotherapeutic agent or drug), an immunomodulatory agent, a nucleic acid (including siRNA), a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), a radioisotope, a PROTAC, and other compounds that are active against target cells when delivered to those cells.

[0161] Cytotoxic agent In some embodiments, the FOLR1 conjugate comprises at least one drug that is a cytotoxic agent. A "cytotoxic agent" refers to an agent that has a cytotoxic effect on cells. A "cytotoxic effect" refers to the depletion, elimination, and / or killing of target cells. Cytotoxic agents include, for example, tubulin-disrupting agents, topoisomerase inhibitors, DNA minor groove binders, and DNA alkylating agents.

[0162] Tubulin-disrupting agents include, for example, auristatins, dolastatin, tubulysin, colchicine, vinca alkaloids, taxanes, cryptophycins, maytansinoids, hemiasterlin, and other tubulin-disrupting agents. Auristatin is a derivative of the natural product dolastatin 10. Exemplary auristatins include MMAE (N-methylvaline-valine-dolaisoleucine-dolaproine-norephedrine), MMAF (N-methylvaline-valine-dolaisoleucine-dolaproine-phenylalanine), and AFP (see WO 2004 / 010957 and WO 2007 / 008603). Other auristatin-like compounds are disclosed, for example, in US 2021 / 0008099, US 2017 / 0121282, US 2013 / 0309192, and US 2013 / 0157960. Dolastatins include, for example, dolastatin 10 and dolastatin 15 (see, for example, Pettit et al., J. Am. Chem. Soc., 1987, 109, 6883-6885; Pettit et al., Anti-Cancer Drug Des., 1998, 13, 243-277; and US 2001 / 0018422). Further dolastatin derivatives contemplated for use herein are disclosed in US 9,345,785, which is incorporated herein by reference.

[0163] Tube lysins include, but are not limited to, tube lysin D, tube lysin M, tubuphenylalanine, and tubutyrosine. WO 2017 / 096311 pamphlet and WO 2016 / 040684 pamphlet describe tube lysin analogs including tube lysin M.

[0164] Colchicine includes, but is not limited to, colchicine and CA-4.

[0165] Vinca alkaloids include, but are not limited to, vinblastine (VBL), vinorelbine (VRL), vincristine (VCR), and vindesine (VOS).

[0166] Taxanes include, but are not limited to, paclitaxel and docetaxel.

[0167] Cryptophycins include, but are not limited to, cryptophycin-1 and cryptophycin-52.

[0168] Maytansinoids include, but are not limited to, maytansine, maytansinol, DM1, DM3 and DM4 maytansine analogs, or ansamitocin-2. Exemplary maytansinoid drug moieties include C-19-dechloro (U.S. Patent No. 4,256,746) (prepared by lithium aluminum hydride reduction of ansamitocin P2); C-20-hydroxy (or C-20-demethyl) + / - C-19-dechloro (U.S. Patent Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and those having modified aromatic rings such as C-20-demethoxy, C-20-acetyloxy (-OCOR), + / - dechloro (U.S. Patent No. 4,294,757) (prepared by acylation using acyl chloride), as well as those having modifications at other positions.

[0169] The maytansinoid drug moieties include those having modifications such as C-9-SH (U.S. Patent No. 4,424,219) (prepared by the reaction of maytansinol with H2S or P2S5); C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Patent No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Patent No. 4,450,254) (prepared from Nocardia); C-15-hydroxy / acyloxy (U.S. Patent No. 4,364,866) (prepared by the conversion of maytansinol by Streptomyces); C-15-methoxy (U.S. Patents 4,313,946 and 4,315,929) (isolated from Trewia nudiflora); C-18-N-demethyl (U.S. Patents No. 4,362,663 and 4,322,348) (prepared by the demethylation of maytansinol by Streptomyces); and 4,5-deoxy (U.S. Patent No. 4,371,533) (prepared by the titanium trichloride / LAH reduction of maytansinol), etc.

[0170] Hemiasterlin includes, but is not limited to, hemiasterlin and HTl-286.

[0171] Other tubulin-disrupting agents include, but are not limited to, tacrolonolide A, tacrolonolide B, tacrolonolide AF, tacrolonolide AJ, tacrolonolide Al-epoxide, discodermolide, epothilone A, epothilone B, and laulimalide.

[0172] In some embodiments, the cytotoxic agent can be a topoisomerase inhibitor such as camptothecin. Exemplary camptothecins include, for example, camptothecin, irinotecan (also called CPT-11), belotecan, (7-(2-(N-isopropylamino)ethyl)camptothecin), topotecan, 10-hydroxy-CPT, SN-38, exatecan, and exatecan analog DXd (see U.S. Patent Application Publication No. 20150297748). Other camptothecins are disclosed in WO 96 / 21666, WO 00 / 08033, U.S. Patent Application Publication No. 2016 / 0229862, and WO 2020 / 156189.

[0173] In some embodiments, the cytotoxic agent is a duocarmycin comprising synthetic analogs, KW-2189, and CBI-TMI.

[0174] Immunomodulatory agent In some embodiments, the drug is an immunomodulatory agent. The immunomodulatory agent can be, for example, a TLR7 and / or TLR8 agonist, a STING agonist, or a RIG-I agonist or other immunomodulatory agent.

[0175] In some embodiments, the drug is an immunomodulatory agent such as a TLR7 and / or TLR8 agonist. In some embodiments, the TLR7 agonist is selected from imidazoquinolines, imidazoquinoline amines, thiazoloquinolines, aminoquinolines, aminoquinazolines, pyrido[3,2-d]pyrimidine-2,4-diamines, pyrimidine-2,4-diamines, 2-aminoimidazoles, 1-alkyl-1H-benzimidazole-2-amines, tetrahydropyridopyrimidines, heteroarothiadiazide-2,2-dioxides, benzonaphthyridines, guanosine analogs, adenosine analogs, thymidine homopolymers, ssRNA, CpG-A, poly G10, and poly G3. In some embodiments, the TLR7 agonist is selected from imidazoquinolines, imidazoquinoline amines, thiazoloquinolines, aminoquinolines, aminoquinazolines, pyrido[3,2-d]pyrimidine-2,4-diamines, pyrimidine-2,4-diamines, 2-aminoimidazoles, 1-alkyl-1H-benzimidazole-2-amines, tetrahydropyridopyrimidines, heteroarothiadiazide-2,2-dioxides or benzonaphthyridines. In some embodiments, the TLR7 agonist is a non-naturally occurring compound. Examples of TLR7 modulators include GS-9620, GSK-2245035, imiquimod, resiquimod, DSR-6434, DSP-3025, IMO-4200, MCT-465, MEDI-9197, 3M-051, SB-9922, 3M-052, Limtop, TMX-30X, TMX-202, RG-7863, RG-7795, and the compounds disclosed in US Patent Application Publication No. 20160168164 (Janssen), US Patent Application Publication No. 20150299194 (Roche), US Patent Application Publication No. 20110098248 (Gilead Sciences), US Patent Application Publication No. 20100143301 (Gilead Sciences) and US Patent Application Publication No. 20090047249 (Gilead Sciences).

[0176] In some embodiments, the TLR8 agonist is selected from benzazepine, imidazoquinoline, thiazoloquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazole-2-amine, tetrahydropyridopyrimidine, or ssRNA. In some embodiments, the TLR8 agonist is selected from benzazepine, imidazoquinoline, thiazoloquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazole-2-amine, and tetrahydropyridopyrimidine. In some embodiments, the TLR8 agonist is a non-naturally occurring compound. Examples of TLR8 agonists include motolimod, resiquimod, 3M-051, 3M-052, MCT-465, IMO-4200, VTX-763, VTX-1463.

[0177] In some embodiments, the TLR8 agonist can be any of the compounds described in WO 2018 / 170179, WO 2020 / 056198, and WO 2020 / 056194.

[0178] Other TLR7 and TLR8 agonists are, for example, International Publication No. WO 2016 / 142250 pamphlet, International Publication No. WO 2017 / 046112 pamphlet, International Publication No. WO 2007 / 024612 pamphlet, International Publication No. WO 2011 / 022508 pamphlet, International Publication No. WO 2011 / 022509 pamphlet, International Publication No. WO 2012 / 045090 pamphlet, International Publication No. WO 2012 / 097173 pamphlet, International Publication No. WO 2012 / 097177 pamphlet, International Publication No. WO 2017 / 079283 pamphlet, US Patent Application Publication No. US 2016 / 0008374 specification, US Patent Application Publication No. US 2016 / 0194350 specification, US Patent Application Publication No. US 2016 / 0289229 specification, US Patent No. 6,043,238 specification, US Patent Application Publication No. US 2018 / 0086755 specification (Gilead), International Publication No. WO 2017 / 216054 pamphlet (Roche), International Publication No. WO 2017 / 190669 pamphlet (Shanghai De Novo Pharmatech), International Publication No. WO 2017 / 202704 pamphlet (Roche), International Publication No. WO 2017 / 202703 pamphlet (Roche), International Publication No. WO 2017 / 0071944 pamphlet (Gilead), US Patent Application Publication No. US 2014 / 0045849 specification (Janssen), US Patent Application Publication No. US 2014 / 0073642 specification (Janssen), International Publication No. WO 2014 / 056953 pamphlet (Janssen), International Publication No. WO 2014 / 076221 pamphlet (Janssen), International Publication No. WO 2014 / 128189 pamphlet (Janssen), US Patent Application Publication No. US 2014 / 0350031 specification (Janssen), International Publication No. WO 2014 / 023813 pamphlet (Janssen), US Patent Application Publication No. US 2008 / 0234251 specification (Array Biopharma), US Patent Application Publication No. US 2008 / 0306050 specification (Array Biopharma), US Patent Application Publication No. US 2010 / 0029585 specification (Ventirx Pharma), US Patent Application Publication No. US 2011 / 0092485 specification (Ventirx Pharma), US Patent Application Publication No. US 2011 / 0118235 specification (Ventirx Pharma), US Patent Application Publication No. US 2012 / 0082658 specification (VentirxDisclosed in U.S. Patent Application Publication No. 20120219615 (Ventirx Pharma), U.S. Patent Application Publication No. 20140066432 (Ventirx Pharma), U.S. Patent Application Publication No. 20140088085 (Ventirx Pharma), U.S. Patent Application Publication No. 20140275167 (Novira Therapeutics), and U.S. Patent Application Publication No. 20130251673 (Novira Therapeutics), International Publication No. 2018198091 Pamphlet (Novartis AG), and U.S. Patent Application Publication No. 20170131421 (Novartis AG).

[0179] In some embodiments, the immunomodulatory agent is a STING agonist. Examples of STING agonists include, for example, those disclosed in International Publication No. 2020059895 Pamphlet, International Publication No. 2015077354 Pamphlet, International Publication No. 2020227159 Pamphlet, International Publication No. 2020075790 Pamphlet, International Publication No. 2018200812 Pamphlet, and International Publication No. 2020074004 Pamphlet.

[0180] In some embodiments, the immunomodulatory agent is a RIG-I agonist. Examples of RIG-I agonists include KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400, and KIN2000.

[0181] toxin In some embodiments, the drug is, but not limited to, an enzymatically active toxin or a fragment thereof, including but not limited to diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crocin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene.

[0182] Radioisotope In some embodiments, the drug is a radioactive atom. Various radioisotopes are available for the production of radioactive conjugates. Examples include I131, I125, Y90, Re186, Re188, Sm153, Bi213, P32, Pb212, and radioactive isotopes of lutetium (e.g., Lu177).

[0183] PROTAC In some embodiments, the drug is a proteolysis-inducing chimeric molecule (PROTAC). PROTACs are described, for example, in U.S. Patent Application Publication Nos. 20210015942, 20210015929, 20200392131, 20200216507, 20200199247, and 20190175612, the disclosures of which are incorporated herein by reference.

[0184] Linker The FOLR1 conjugate typically includes at least one linker, and each linker has at least one drug attached thereto. Typically, the conjugate includes a linker between the FOLR1 antibody (or an antigen-binding portion or other binder thereof) and the drug. In various embodiments, the linker can be a protease-cleavable linker, an acid-cleavable linker, a disulfide linker, a sulfide-containing linker or a disulfide-containing linker having a dimethyl group adjacent to the disulfide bond (see, e.g., Jain et al., Pharm. Res. 32:3526-3540 (2015); Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020), a self-stabilizing linker (see, e.g., WO 2018 / 031690; WO 2015 / 095755 and Jain et al., Pharm. Res. 32:3526-3540 (2015)), a non-cleavable linker (see, e.g., WO 2007 / 008603), a photocleavable linker, and / or a hydrophilic linker (see, e.g., WO 2015 / 123679).

[0185] In some embodiments, the linker is a cleavable linker that can be cleaved under intracellular conditions such that upon cleavage of the linker, an antibody (or an antigen-binding portion or other binder thereof) and / or a drug is released from the linker in the intracellular environment. For example, in some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within lysosomes or endosomes or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to lysosomal or endosomal proteases (see, e.g., WO 2004 / 010957 pamphlet, US 2015 / 0297748 specification, US 2008 / 0166363 specification, US 2012 / 0328564 specification, and US 2020 / 0347075 specification). Typically, the peptidyl linker is at least 1 amino acid in length or at least 2 amino acids in length. Intracellular cleaving agents can include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to effect release of the active drug in target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typically, it is a peptidyl linker cleavable by an enzyme present in target antigen-expressing cells. For example, a peptidyl linker cleavable by the thiol-dependent protease cathepsin B, which is highly expressed in cancerous tissue, can be used (e.g., Phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO: 42)). Other such linkers are described, for example, in US Pat. No. 6,214,345. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., US Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a val-cit linker) or a Gly-Gly-Phe-Gly linker (SEQ ID NO: 43) (see, e.g., US 2015 / 0297748 specification).One advantage of using intracellular proteolytic release of a drug is that the drug is typically attenuated when conjugated and the serum stability of the conjugate is typically high. See also U.S. Patent No. 9,345,785.

[0186] As used herein, the terms "cleaved intracellularly" and "intracellular cleavage" refer to an intracellular metabolic process or reaction on an antibody-drug conjugate by which a covalent bond, e.g., a linker, between a drug (e.g., a cytotoxic agent) and an antibody is broken and a free drug or other metabolite of the conjugate dissociated from the antibody within the cell is obtained. Thus, the cleaved portion of the conjugate is an intracellular metabolite.

[0187] In some embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, the pH-sensitive linker is hydrolysable under acidic conditions. For example, acid-labile linkers hydrolysable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic acid amides, orthoesters, acetals, ketals, etc.) can be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; and 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions such as the pH in blood, but are unstable at a pH of about 5.5 or less than 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolysable linker is a thioether linker (e.g., a thioether bonded to a drug via an acylhydrazone linkage) (see, e.g., U.S. Patent No. 5,622,929).

[0188] In some embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). For example, various disulfide linkers are known that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB, and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel, ed., Oxford U.Press, 1987). See also U.S. Patent No. 4,880,935).

[0189] In some embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res., 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12). In some embodiments, the linker unit is not cleavable, such as a maleimidocaproyl linker, and the drug is released by antibody degradation (see U.S. Patent Application Publication No. 2005 / 0238649).

[0190] In some embodiments, the linker is substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment" in the context of a linker means that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, even more typically no more than about 5%, about 3%, or about 1% of the linker in a sample of an antibody-drug conjugate (ADC) is cleaved when the ADC is present in the extracellular environment (e.g., in plasma). Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating both (a) an ADC ("ADC sample") and (b) an equimolar amount of an unconjugated antibody or drug ("control sample") independently with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of unconjugated antibody or drug present in the ADC sample, as measured, for example, by high performance liquid chromatography, to that present in the control sample.

[0191] In some embodiments, the linker promotes endocytosis. In some embodiments, the linker promotes endocytosis when conjugated to a drug such as a cytotoxic agent (i.e., in the linker-drug environment of an ADC as described herein). In still other embodiments, the linker promotes endocytosis when conjugated to both a drug and an FOLR1 antibody (i.e., in the environment of an ADC as described herein).

[0192] The various linkers that can be used in the compositions and methods of the present invention are described in WO 2004 / 010957. In some embodiments, a protease-cleavable linker comprises a thiol-reactive spacer and a dipeptide. In some embodiments, a protease-cleavable linker consists of a thiol-reactive maleimidocaproyl spacer, a valine-citrulline dipeptide, and a p-aminobenzyloxycarbonyl spacer.

[0193] In some embodiments, the acid-cleavable linker is a hydrazine linker or a quaternary ammonium linker (see WO 2017 / 096311 pamphlet and WO 2016 / 040684 pamphlet).

[0194] In some embodiments, the linker is a self-stabilizing linker containing a maleimide group as described in US Patent No. 9,504,756.

[0195] In some embodiments, the linker is a hydrophilic linker such as, for example, the hydrophilic peptide of WO 2015 / 123679 pamphlet and the sugar alcohol polymer-based linker disclosed in WO 2013 / 012961 pamphlet and WO 2019 / 213046 pamphlet.

[0196] In other embodiments, conjugates of a FOLR1 antibody (or antigen-binding portion or other binder) and a drug can be made using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Chelating agents for the conjugation of radioactive nucleotides to an antibody, its antigen-binding portion or other binder are described, for example, in WO 94 / 11026 pamphlet.

[0197] Conjugates of the FOLR1 antibody (or antigen-binding portion or other binding agent) include, but are not limited to, such conjugates prepared using crosslinking agents including, for example, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Illinois, USA).

[0198] In some embodiments, the linker is attached to the end of the amino acid sequence of the antibody, antigen-binding portion or other binding agent, or can be attached to the side chain modification of the antibody, antigen-binding portion or other binding agent, such as the side chain of lysine, serine, threonine, cysteine, tyrosine, aspartic acid, unnatural amino acid residues, glutamine or glutamic acid residues. The bond between the antibody, antigen-binding portion or other binding agent and the linker or drug can be via some bonds, such as, but not limited to, amide bond, ester bond, ether bond, carbon-nitrogen bond, carbon-carbon single bond, double bond or triple bond, disulfide bond, or thioether bond. Functional groups capable of forming such bonds include, for example, amino groups, carboxyl groups, aldehyde groups, azide groups, alkyne and alkene groups, ketones, carbonates, carbonyl functional groups bonded to leaving groups such as cyano groups and succinimidyl groups and hydroxyl groups.

[0199] In some embodiments, the linker is linked to the antibody, antigen-binding portion, or other binding agent by an interchain disulfide. In some embodiments, the linker is connected to the antibody, antigen-binding portion, or other binding agent by a hinge cysteine residue. In some embodiments, the linker is linked to the antibody, antigen-binding portion, or other binding agent by an engineered cysteine residue. In some embodiments, the linker is connected to the antibody, antigen-binding portion, or other binding agent by a lysine residue. In some embodiments, the linker is connected to the antibody, antigen-binding portion, or other binding agent by an engineered glutamine residue. In some embodiments, the linker is connected to the antibody, antigen-binding portion, or other binding agent by an engineered non-natural amino acid in the heavy chain.

[0200] In some embodiments, the linker is linked to the antibody, antigen-binding portion, or other binding agent via a sulfhydryl group. In some embodiments, the linker is linked to the antibody, antigen-binding portion, or other binding agent via a primary amine. In some embodiments, the linker is linked via a bond created between unnatural amino acids on the antibody, antigen-binding portion, or other binding agent by reacting with an oxime bond formed by modifying a ketone group on the drug with an alkoxyamine.

[0201] In some embodiments, the linker is linked to the antibody, antigen-binding portion, or other binding agent via a sortase A linker. The sortase A linker can be generated by a sortase A enzyme that fuses an LPXTG recognition motif (SEQ ID NO: 44) to an N-terminal GGG motif to regenerate a native amide bond.

[0202] Exemplary linker-drug combinations In some embodiments, a tubulin-disrupting agent, such as a drug like auristatin, is linked to the linker by a C-terminal carboxyl group that forms an amide bond with the linker (e.g., linker unit (LU) as described in U.S. Patent No. 9,463,252, which is incorporated herein by reference). In some embodiments, the linker comprises at least one amino acid.

[0203] In some embodiments, the linker also includes a stretcher unit and / or an amino acid unit. Exemplary stretcher units and amino acid units are described in U.S. Patent No. 9,345,785 and U.S. Patent No. 9,078,931, which are hereby incorporated by reference in their entireties.

[0204] In some embodiments, the antibody-drug conjugate includes an anti-FOLR1 antibody covalently linked to MMAE through an mc-val-cit-PAB linker.

[0205] In some embodiments, the FOLR1 conjugate has the following formula: [Chemical formula] Or a pharmaceutically acceptable salt thereof (wherein mAb is an anti-FOLR1 antibody, an antigen-binding portion thereof, or another binder, S is a sulfur atom of the antibody, antigen-binding portion, or other binder, A is a stretcher unit, and p is from about 3 to about 5, or from about 3 to about 8).

[0206] The drug load is represented by p, which is the average number of drug molecules (e.g., a cytotoxic agent) per antibody (or antigen-binding portion or other binder) in the conjugate. For example, when p is about 4, the average drug load considering all of the antibodies (or antigen-binding portions or other binders) present in the composition is about 4. In some embodiments, p ranges from about 3 to about 5, from about 3.6 to about 4.4, or from about 3.8 to about 4.2. In some embodiments, p can be about 3, about 4, or about 5. In some embodiments, p ranges from about 6 to about 8, more preferably from about 7.5 to about 8.4. In some embodiments, p can be about 6, about 7, or about 8.

[0207] The average number of drugs per antibody (or antigen-binding portion or other binder) in the preparation can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the antibody-drug conjugate with respect to p can also be determined. In some examples, the separation, purification, and characterization of a homogeneous antibody-drug conjugate with a constant value of p from antibody-drug conjugates having other drug loads can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0208] In some embodiments, the stretcher unit can link an antibody (or antigen-binding portion or other binder) to an amino acid or peptide (e.g., valine-citrulline peptide) via a sulfhydryl group of the antibody (or antigen-binding portion or other binder). The sulfhydryl group can be generated, for example, by reduction of the interchain disulfide bond of a FOLR1 antibody (or antigen-binding portion or other binder). For example, the stretcher unit can be linked to the antibody (or antigen-binding portion or other binder) via a sulfur atom generated from reduction of the interchain disulfide bond of the antibody (or antigen-binding portion or other binder). In some embodiments, the stretcher unit is linked to the antibody (or antigen-binding portion or other binder) only via a sulfur atom generated from reduction of the interchain disulfide bond of the antibody. In some embodiments, the sulfhydryl group can be generated by reaction of an amino group of the lysine portion of a FOLR1 antibody (or antigen-binding portion or other binder) with 2-iminothiolane (Traut's reagent) or other sulfhydryl-generating reagent. In some embodiments, the FOLR1 antibody (or antigen-binding portion or other binder) is a recombinant antibody and is engineered to have one or more lysines. In some embodiments, the recombinant FOLR1 antibody (or antigen-binding portion or other binder) is engineered to have additional sulfhydryl groups, such as additional cysteines, for example engineered cysteines.

[0209] The synthesis and structure of MMAE are described in U.S. Patent No. 6,884,869, which is hereby incorporated by reference in its entirety for all purposes. The synthesis and structure of exemplary linker units and methods of making antibody-drug conjugates are described, for example, in U.S. Patent Application Publication Nos. 2006 / 0074008 and 2009 / 0010945, respectively, each of which is hereby incorporated by reference in its entirety.

[0210] Exemplary linker units are described within the square brackets of Formulas Illa and Illb of U.S. Patent No. 9,211,319, which is hereby incorporated by reference.

[0211] In some embodiments, the FOLR1 conjugate comprises monomethyl auristatin E (MMAE) and a protease-cleavable linker. It is contemplated that the protease-cleavable linker comprises a thiol-reactive spacer and a dipeptide. In various embodiments, the protease-cleavable linker comprises a thiol-reactive maleimidocaproyl spacer, a valine-citrulline (val-cit) dipeptide, and a p-aminobenzyl oxycarbonyl or PAB spacer.

[0212] The abbreviation "PAB" refers to a self-immolative spacer:

Chem.

[0213] The abbreviation "MC" refers to a linker maleimidocaproyl:

Chem.

[0214] In other exemplary embodiments, the conjugate has the following general formula: Ab-[L3]-[L2]-[L1] m -AA n -drug, (Wherein, Ab is an anti-FOLR1 antibody (or antigen-binding portion or other binding agent); the drug can be a cytotoxic agent such as, for example, a tubulin-disrupting agent or a topoisomerase inhibitor; L3 is a component of an antibody coupling portion (such as a stretcher unit) and a linker containing one or more acetylene (or azide) groups; L2 contains at one end any PEG (polyethylene glycol) azide (or acetylene) complementary to the acetylene (or azide) portion of L3, and contains a reactive group such as a carboxylic acid or hydroxyl group at the other end; L1 contains a foldable unit (such as a self-immolative group), or a peptidase-cleavable portion optionally attached to the foldable unit, or an acid-cleavable portion; AA is an amino acid; m is an integer having a value of 0 or 1, and n is an integer having a value of 0, 1, 2, 3, or 4) has. Such linkers can be assembled via click chemistry (see, for example, U.S. Patent Nos. 7,591,944 and 7,999,083).

[0215] In some embodiments, the drug is camptothecin or a camptothecin (CPT) analog, such as irinotecan (also called CPT-11), belotecan, topotecan, 10-hydroxy-CPT, exatecan, DXd and / or SN-38. Representative structures are shown below.

Chemical formula

[0216] For the conjugate formula Ab-[L3]-[L2]-[L1] m -AA n -drug, in some embodiments, m is 0. The conjugate formula Ab-[L3]-[L2]-[L1] m -AA n- With reference to the drug, in some embodiments, L2 is absent. In such embodiments, the ester moiety is first formed between the carboxylic acid of an amino acid (AA) such as glycine, alanine, or sarcosine, or the carboxylic acid of a peptide such as glycylglycine, and the hydroxyl group of a drug such as a cytotoxic agent. In this example, the N-terminus of the amino acid or polypeptide can be protected as a Boc or Fmoc or monomethoxytrityl (MMT) derivative, which is deprotected after the formation of the ester bond with the hydroxyl group of the cytotoxic agent. Since "monomethoxytrityl (MMT)" can be removed by mild acid treatment such as dichloroacetic acid that does not cleave the BOC group, MMT can be used as a protecting group for the amino group of the amino acid or polypeptide involved in ester formation to achieve selective removal of the amine protecting group in the presence of the BOC protecting group at the hydroxyl position of the cytotoxic agent containing an additional hydroxyl group. After the amino group of the amino acid or polypeptide that forms an ester bond with the hydroxyl of the drug is de-masked, the amino group reacts with the activated form of the COOH group of the PEG moiety of L2 (if present) under standard amide formation conditions. In a preferred embodiment, L3 comprises a thiol-reactive group that links to the thiol group of an antibody (or antigen-binding portion or other binder). The thiol-reactive group is optionally a maleimide or vinyl sulfone that links to the thiol group of the antibody, or bromoacetamide, or iodoacetamide. In some embodiments, a reagent having a thiol-reactive group is generated, for example, from succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or succinimidyl-(epsilon-maleimid)caproate, and the thiol-reactive group is a maleimide group.

[0217] In another embodiment, m is 0 and AA contains a peptide moiety cleavable by an intracellular peptidase such as cathepsin B, preferably a di-, tri- or tetrapeptide. Examples of cathepsin B-cleavable peptides are Phe-Lys, Val-Cit (Dubowchick, 2002), Ala-Leu, Leu-Ala-Leu, Ala-Leu-Ala-Leu (SEQ ID NO: 45) (Trouet et al., 1982), and Gly-Gly-Phe-Gly (SEQ ID NO: 43) (see, for example, WO 2014 / 057687 pamphlet).

[0218] In some embodiments, L1 is composed of an intracellularly cleavable peptide such as a cathepsin B-cleavable peptide connected at the C-terminus of the peptide to a foldable unit such as p-aminobenzyl alcohol (or p-amino-benzyloxycarbonyl), and the benzyl alcohol moiety thereof is directly bound to the hydroxyl group of a drug such as a cytotoxic agent in chloroformate form. In this embodiment, n is 0. Alternatively, when "n" is non-zero, the benzyl alcohol moiety of the p-amidobenzyl alcohol (or p-amino-benzyloxycarbonyl) moiety is bound to the N-terminus of an amino acid or peptide linked by the hydroxyl group of the cytotoxic agent through the active form of p-amidobenzyl alcohol, i.e., PABOCOPNP where PNP is p-nitrophenyl. In some embodiments, the linker contains a thiol-reactive group that links to the thiol group of an antibody (or antigen-binding portion or other binder). The thiol-reactive group is optionally a maleimide or vinyl sulfone that links to the thiol group of an antibody (or antigen-binding portion or other binder), or bromoacetamide, or iodoacetamide. In a preferred embodiment, the component having the thiol-reactive group is generated from, for example, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or succinimidyl-(epsilon-maleimid)caproate, and the thiol-reactive group is a maleimide group.

[0219] In some embodiments, when the drug is camptothecin having 20-hydroxyl or an analog or derivative thereof, L1 is composed of an intracellular cleavable peptide such as a cathepsin B cleavable peptide connected to p-aminobenzyl alcohol (or p-amino-benzyloxycarbonyl), a linker foldable at the C-terminus of the peptide, and the benzyl alcohol moiety thereof is directly bound to CPT-20-O-chloroformate. In this embodiment, n is 0. Alternatively, when "n" is non-zero, the benzyl alcohol moiety of the p-amidobenzyl alcohol moiety is bound to the N-terminus of an amino acid or polypeptide that links at the 20-position of CPT through the active form of p-amidobenzyl alcohol, i.e., PABOCOPNP where PNP is p-nitrophenyl. In a preferred embodiment, the linker contains a thiol-reactive group that links to the thiol group of an antibody (or antigen-binding portion or other binder). The thiol-reactive group is optionally a maleimide or vinyl sulfone, or bromoacetamide, or iodoacetamide that links to the thiol group of an antibody (or antigen-binding portion or other binder). In a preferred embodiment, the component having the thiol-reactive group is generated, for example, from succinimidyl-4-(N maleimidomethyl) cyclohexane-1-carboxylate (SMCC) or succinimidyl-(epsilon-maleimide) caproate, and the thiol-reactive group is a maleimide group.

[0220] In some embodiments, an L2 component of the conjugate is present and contains a polyethylene glycol (PEG) spacer that can be up to about MW5000 in size, and in a preferred embodiment, the PEG is a defined PEG having (1 to 12 or 1 to 30) repeating monomer units. In some embodiments, the PEG is a defined PEG having 1 to 12 repeating monomer units. The introduction of PEG can involve the use of commercially available heterobifunctional PEG derivatives. Heterobifunctional PEGs typically contain an azide group or an acetylene group. An example of a heterobifunctional defined PEG containing 8 repeating monomer units where "NHS" is succinimidyl is shown below by the following formula:

Chem.

[0221] In some embodiments, L3 has a plurality of acetylene (or azide) groups in the range of 2 to 40, preferably 2 to 20, more preferably 2 to 5, and a single antibody binding moiety.

[0222] Representative conjugates are shown below, which are cytotoxic agents such as SN-38 (a CPT analog) prepared with a maleimide-containing SN-38-linker derivative and having a binding to an antibody (referred to as MAb) represented as succinimide. Here, m = 0, the 20-O-AA ester binding to SN-38 is glycinate; the azide-acetylene coupling bond of L2 and L3 results in a triazole moiety as shown.

Chem.

[0223] Another representative conjugate prepared with a maleimide-containing SN-38-linker derivative and having a binding to an antibody (MAb) represented as succinimide is shown below. Here, in General Formula 2, n = 0; "L1" contains a cathepsin B-cleavable dipeptide Phe-Lys bound to a foldable p-aminobenzyl alcohol moiety, and the latter is bound to SN-38 as a carbonate bond at the 20th position; the azide-acetylene coupling that binds the "L2" and "L3" moieties results in a triazole moiety as shown.

Chem.

[0224] Another representative SN-38 conjugate, mAb-CL2-SN-38, was prepared using a maleimide-containing SN-38-linker derivative having a bond with an antibody represented as succinimide and is shown below. Here, the 20-O-AA ester bound to SN-38 is glycinate that is bound to the L1 moiety via a p-aminobenzyl alcohol moiety and a cathepsin B-cleavable dipeptide; the latter is bound to "L2" via an amide bond, and the "L2" and "L3" moieties are coupled via azide-acetylene "click chemistry".

Chem.

[0225] In another representative example, "L1" contains a single amino acid bound to a foldable p-aminobenzyl alcohol moiety, the p-aminobenzyl alcohol is substituted or unsubstituted (R), in the general conjugate formula Ab-[L3]-[L2]-[L1]m-AAn-drug, m = 1 and n = 0, and the drug is exemplified by SN-38. The structure is represented below (referred to as MAb-CLX-SN-38). The single amino acid of AA can be selected from any one of the following L-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. The substituent R of the 4-aminobenzyl alcohol moiety is an alkyl group selected from hydrogen or a C1-C10 alkyl group.

Chem.

[0226] An embodiment of mAb-CLX-SN-38 (described above), called mAb-CL2A-SN-38, where the single amino acid AA is L-lysine, R = H, and the drug is a cytotoxic agent exemplified by SN-38, is shown below: [Chemical formula]

[0227] In other embodiments, the drug is a cytotoxic agent bound to a linker comprising a stretcher unit (Z) bound to an amino acid unit (AA) bound to a spacer unit (Y), wherein the stretcher unit is bound to an antibody (or an antigen-binding portion thereof or other binder, referred to as Ab or MAb), and the spacer unit is bound to the amino group of the cytotoxic agent. Such a linker has the following formula: Ab-Z-AA-Y-cytotoxic agent (wherein Z is -(succinimid-3-yl-N)-(CH2) n 2 -C(=O)--, --CH2--C(=O)--NH--(CH2)n 3 -C(=O)--, -C(=O)-cyc.Hex(1,4)-CH2--(N-ly-3-diminiccuS)-, or -C(=O)--(CH2)n 4 selected from, n 2 represents an integer from 2 to 8, n 3 represents an integer from 1 to 8, n 4 represents an integer from 1 to 8; cyc.Hex(1,4) represents a 1,4-cyclohexylene group; (N-ly-3-diminiccuS)- represents a structure represented by the following formula: [Chemical formula] (having) having.

[0228] In some embodiments, AA is a peptide of 2 to 7 amino acids. In some embodiments, the spacer unit Y is -NH-(CH2) b -(C=O)- or -NH-CH2-O-CH2-(C=O)-, and b is an integer from 1 to 5.

[0229] In some embodiments, the cytotoxin is exatecan. In some embodiments, the amino acid unit (AA) is -Gly-Gly-Phe-Gly-. In some embodiments, the spacer unit Y is -NH-CH2-O-CH2-(C=O)-.

[0230] In some embodiments, the linker-cytotoxin has the following structure:

Chemical formula

[0231] Binding of drug-linkers to antibodies, antibody binding portions, and other binders Techniques for conjugating a drug to an antibody (or antigen-binding portion thereof or other binder) via a linker are well known in the art. See, for example, Alley et al., Current Opinion in Chemical Biology 2010 14:1-9; Senter, Cancer J., 2008, 14(3):154-169. In some embodiments, the linker is first conjugated to a drug (e.g., a cytotoxin), and then the drug-linker is conjugated to an antibody or antigen-binding portion thereof or other binder. In some embodiments, the linker is first conjugated to an antibody or antigen-binding portion thereof or other binder, and then the drug is conjugated to the linker. In the following discussion, the term drug-linker is used to exemplify the conjugation of a linker or drug-linker to an antibody or antigen-binding portion thereof or other binder; one of ordinary skill in the art will recognize that the conjugation method selected can be chosen according to the linker and cytotoxin or other drug. In some embodiments, the drug is conjugated to an antibody or antigen-binding portion thereof or other binder via a linker in a manner that reduces the activity of the drug until it is released from the conjugate (e.g., by hydrolysis, proteolysis, or a cleaving agent).

[0232] Generally, conjugates can be prepared by several routes that employ organic chemical reactions, conditions, and reagents known to those of skill in the art, including (1) reacting a nucleophilic group of an antibody (or an antigen-binding portion or other binding agent thereof) with a bivalent linker reagent to form an antibody-linker intermediate via a covalent bond and subsequently reacting with a drug (e.g., a cytotoxic agent); and (2) reacting a nucleophilic group of a drug (e.g., a cytotoxic agent) with a bivalent linker reagent to form a drug-linker and subsequently reacting with a nucleophilic group of an antibody or an antigen-binding portion or other binding agent thereof. An exemplary method for preparing conjugates via the latter route is described in U.S. Patent No. 7,498,298, which is hereby expressly incorporated by reference.

[0233] Nucleophilic groups on antibodies, antigen-binding portions, and other binders include, but are not limited to, (i) N-terminal amine groups, (ii) side-chain amine groups such as lysine, (iii) side-chain thiol groups such as cysteine, and (iv) sugar hydroxyl or amino groups to which the antibody is glycosylated. Amine groups, thiol groups, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophilic groups on (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl halides and benzyl halides such as haloacetamides; and (iii) linker moieties and linker reagents containing aldehyde groups, ketone groups, carboxyl groups, and maleimide groups. Certain antibodies (and antigen-binding portions or other binders) have reducing interchain disulfides, i.e., cysteine bridges. Antibodies (and antigen-binding portions and other binders) can be made reactive with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol) or TCEP (tricarbonylethylphosphine) so that the antibody is fully or partially reduced. Thus, each cysteine bridge theoretically forms two reactive thiol nucleophiles. For example, additional nucleophilic groups can be introduced into the antibody (and antigen-binding portion and other binders) through modification of lysine residues by reacting the lysine residues with 2-iminothiolane (Traut reagent) to convert the amine to a thiol. Reactive thiol groups can also be introduced into the antibody (and antigen-binding portion and other binders) by introducing one, two, three, four, or more cysteine residues (e.g., by preparing antibodies, antigen-binding portions, and other binders that contain one or more non-natural cysteine amino acid residues).

[0234] Conjugates can also be generated by the reaction between an electrophilic group on an antibody (or its antigen-binding portion or other binding agent), such as an aldehyde group or a ketone carbonyl group, and a nucleophilic group on a linker reagent or a drug. Useful nucleophilic groups on linker reagents include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide. In one embodiment, the antibody (or its antigen-binding portion or other binding agent) is modified to introduce an electrophilic moiety capable of reacting with a nucleophilic substituent on a linker reagent or a drug. In another embodiment, the sugar of a glycosylated antibody is oxidized, for example, with a periodate oxidation reagent to form an aldehyde group or a ketone group capable of reacting with an amine group of a linker reagent or a drug moiety. The resulting imine Schiff base group can form a stable bond or can be reduced, for example, with a borohydride reagent to form a stable amine bond. In one embodiment, the reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium metaperiodate can result in a carbonyl (aldehyde and ketone) group in the antibody (or its antigen-binding portion or other binding agent) capable of reacting with a suitable group on a drug (see, e.g., Hermanson, Bioconjugate Techniques). In another embodiment, an antibody containing an N-terminal serine or threonine residue can react with sodium metaperiodate to generate an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. Patent No. 5,362,852). Such an aldehyde can be reacted with a cytotoxic agent or a linker.

[0235] Exemplary nucleophilic groups on drugs such as cytotoxic agents include, but are not limited to, (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl halides and benzyl halides such as haloacetamides; (iii) amine groups, thiol groups, hydroxyl groups, hydrazide groups, oxime groups, hydrazine groups, thiosemicarbazone groups, hydrazine carboxylate groups, and aryl hydrazide groups that can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents containing aldehyde groups, ketone groups, carboxyl groups, and maleimide groups.

[0236] Non-limiting exemplary crosslinking agents that can be used to prepare conjugates are described herein or are known to those of skill in the art. Methods of linking two moieties comprising an antibody (or antigen-binding portion or other binder) and a chemical moiety using such crosslinking agents are known in the art. In some embodiments, fusion proteins comprising an antibody or antigen-binding portion and a drug can be made, for example, by recombinant techniques or peptide synthesis. Recombinant DNA molecules can comprise a region encoding an antibody (or antigen-binding portion or other binder) and a region encoding the active portion of the conjugate (e.g., a cytotoxic portion), either adjacent to each other or separated by a region encoding a linker that does not disrupt the desired properties of the conjugate.

[0237] In some embodiments, the drug-linker is attached to an interchain cysteine residue of an antibody (or an antigen-binding portion or other binder thereof). See, for example, WO 2004 / 010957 and WO 2005 / 081711. In such embodiments, the linker typically comprises a maleimide group for attaching to the cysteine residue of an interchain disulfide. In some embodiments, the linker or drug-linker is attached to a cysteine residue of an antibody or an antigen-binding portion thereof as described in U.S. Pat. No. 7,585,491 or U.S. Pat. No. 8,080,250. The drug loading of the resulting conjugate is typically in the range of 1 to 8.

[0238] In some embodiments, the linker or drug-linker is attached to a lysine or cysteine residue of an antibody (or an antigen-binding portion or other binder thereof) as described in WO 2005 / 037992 or WO 2010 / 141566. The drug loading of the resulting conjugate is typically in the range of 1 to 8.

[0239] In some embodiments, engineered cysteine residues, polyhistidine sequences, glycoengineering tags, or transglutaminase recognition sequences can be used for site-specific attachment of a linker or drug-linker to an antibody or an antigen-binding portion or other binder thereof.

[0240] In some embodiments, the drug-linker is attached to a cysteine residue engineered with an Fc residue other than an interchain disulfide. In some embodiments, the drug-linker is attached to engineered cysteines introduced at positions 118, 221, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 275, 276, 278, 280, 281, 283, 285, 286, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 302, 305, 313, 318, 323, 324, 325, 327, 328, 329, 330, 331, 332, 333, 335, 336, 396, and / or 428 of IgG (typically IgG1) and / or positions 106, 108, 142 (light chain), 149 (light chain), and / or V205 of the light chain according to the Kabat EU numbering. An exemplary substitution for site-specific conjugation using an engineered cysteine is S239C (see, e.g., U.S. Patent Application Publication No. 20100158909; numbering of the Fc region follows the EU index).

[0241] In some embodiments, the linker or drug-linker is attached to one or more introduced cysteine residues of an antibody (or antigen-binding portion or other binder thereof) as described in WO 2006 / 034488, WO 2011 / 156328, and / or WO 2016 / 040856.

[0242] In some embodiments, exemplary substitutions for site-specific conjugation using bacterial transglutaminase are N297S or N297Q in the Fc region. In some embodiments, a linker or drug-linker is attached to the glycan or modified glycan of an antibody or antigen-binding portion or glycoengineered antibody (or other binder). See, for example, WO 2017 / 147542 pamphlet, WO 2020123425 pamphlet, WO 2014 / 072482 pamphlet; WO 2014 / / 065661 pamphlet, WO 2015 / 057066 pamphlet and WO 2016 / 022027 pamphlet.

[0243] Pharmaceutical formulation Other aspects of the FOLR1 antibody and its antigen-binding portion or other binder and conjugates of any of these relate to compositions comprising an active ingredient (i.e., an FOLR1 antibody or its antigen-binding portion or other binder or conjugate described herein, or a nucleic acid encoding an antibody or its antigen-binding portion or other binder described herein). In some embodiments, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to an active agent combined with a pharmaceutically acceptable carrier approved for use in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment, without undue toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0244] The preparation of a pharmacological composition containing an active ingredient dissolved or dispersed therein is well understood in the art and need not be limited to any particular formulation. Typically, such compositions are prepared as injectable either as a liquid solution or suspension; however, solid forms suitable for rehydration or suspension in a liquid prior to use can also be prepared. The preparation can also be emulsified or provided as a liposomal composition. The FOLR1 antibody or its antigen-binding portion or other binder or its conjugate can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient in an amount suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Further, if desired, the pharmaceutical composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, etc. that enhance or maintain the effectiveness of the active ingredient (e.g., FOLR1 antibody or its antigen-binding portion or other binder or its conjugate). The pharmaceutical compositions described herein can contain pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid (formed with the free amino groups of the polypeptide). Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions containing the active ingredient (e.g., FOLR1 antibody and / or its antigen-binding portion, other binder or its conjugate) and water, and can contain both a buffer such as sodium phosphate at physiological pH values, saline, or phosphate-buffered saline. Still further, the aqueous carrier can contain two or more buffer salts, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol and other solutes.The liquid composition can also contain, in addition to water and excluding water, a liquid phase. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of the active agent effective for treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0245] In some embodiments, the pharmaceutical composition comprising the FOLR1 antibody or antigen-binding portion thereof or other binder or conjugate thereof described herein, or the nucleic acid encoding the FOLR1 antibody or antigen-binding portion thereof or other binder described herein, can be a lyophilized product.

[0246] In some embodiments, a syringe containing a therapeutically effective amount of the FOLR1 antibody or antigen-binding portion thereof or other binder or conjugate thereof described herein, or a pharmaceutical composition, is provided.

[0247] Treatment of cancer In some embodiments, the FOLR1 antibody or antigen-binding portion thereof, binder, and conjugate described herein can be used in a method comprising administering the FOLR1 antibody or antigen-binding portion thereof or other binder or conjugate thereof described herein to a subject in need thereof, such as a subject having cancer.

[0248] In some embodiments, a method comprising the step of administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by pairs of amino acid sequences selected from, respectively, SEQ ID NO: 1 and SEQ ID NO: 2; respectively, SEQ ID NO: 3 and SEQ ID NO: 4; respectively, SEQ ID NO: 5 and SEQ ID NO: 6; respectively, SEQ ID NO: 7 and SEQ ID NO: 8; respectively, SEQ ID NO: 9 and SEQ ID NO: 10; respectively, SEQ ID NO: 11 and SEQ ID NO: 12; respectively, SEQ ID NO: 13 and SEQ ID NO: 14; respectively, SEQ ID NO: 15 and SEQ ID NO: 16; respectively, SEQ ID NO: 17 and SEQ ID NO: 18; respectively, SEQ ID NO: 19 and SEQ ID NO: 20; respectively, SEQ ID NO: 21 and SEQ ID NO: 22; and respectively, SEQ ID NO: 23 and SEQ ID NO: 24, is provided. In some embodiments, a method comprising the step of administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, is provided. In some embodiments, a method comprising the step of administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by SEQ ID NO: 3 and SEQ ID NO: 4, respectively, is provided. In some embodiments, a method comprising the step of administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by SEQ ID NO: 5 and SEQ ID NO: 6, respectively, is provided. In some embodiments, a method comprising the step of administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by SEQ ID NO: 7 and SEQ ID NO: 8, respectively, is provided.In some embodiments, provided is a method comprising the step of administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. In some embodiments, provided is a method comprising the step of administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In some embodiments, provided is a method comprising the step of administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In some embodiments, provided is a method comprising the step of administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively. In some embodiments, provided is a method comprising the step of administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively. In some embodiments, provided is a method comprising the step of administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.In some embodiments, provided is a method comprising administering an anti-FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In some embodiments, provided is a method comprising administering an anti-FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have the amino acid sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

[0249] In some embodiments, a method comprising administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown in pairs of amino acid sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and the heavy chain variable framework region and the light chain variable framework region are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 conservative amino acid substitutions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified, is provided.In some embodiments, a method comprising administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences shown by pairs of amino acid sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and the heavy chain variable framework region and the light chain variable framework region are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework region, and the CDRs of the heavy chain variable region or the light chain variable region are not modified, is provided.

[0250] In some embodiments, a method comprising administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 disposed in a heavy chain variable region framework region, the VL region comprises LCDR1, LCDR, and LCDR3 disposed in a light chain variable region framework region, and the VH and VL CDRs have amino acid sequences shown by sets of amino acid sequences selected from (i) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; and (ii) SEQ ID NO: 31, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively, is provided. In some embodiments, each VH region and VL region comprises a humanized framework region. In some embodiments, each VH region and VL region comprises a human framework region.

[0251] In some embodiments, a method comprising administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 disposed in a heavy chain variable region framework region, the VL region comprises LCDR1, LCDR and LCDR3 disposed in a light chain variable region framework region, and the VH and VL CDRs have amino acid sequences as set forth in (i) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively, is provided. In some embodiments, each VH region and VL region comprises a humanized framework region. In some embodiments, each VH region and VL region comprises a human framework region.

[0252] In some embodiments, a method comprising administering an FOLR1 antibody or an antigen-binding portion thereof or other binding agent or conjugate thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 disposed in a heavy chain variable region framework region, the VL region comprises LCDR1, LCDR and LCDR3 disposed in a light chain variable region framework region, and the VH and VL CDRs have amino acid sequences as set forth in SEQ ID NO: 31, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively, is provided. In some embodiments, each VH region and VL region comprises a humanized framework region. In some embodiments, each VH region and VL region comprises a human framework region.

[0253] In some embodiments, the subject is in need of treatment for cancer and / or malignancy. In some embodiments, the subject is in need of treatment for a FOLR1+ cancer or FOLR1+ malignancy such as, for example, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, pancreatic cancer, and renal cell cancer. In some embodiments, the method is for treating a subject having a FOLR1+ cancer or malignancy. In some embodiments, the method is for treating the subject's lung cancer. In some embodiments, the method is for treating the subject's non-small cell lung cancer. In some embodiments, the method is for treating the subject's breast cancer. In some embodiments, the method is for treating the subject's ovarian cancer. In some embodiments, the method is for treating the subject's cervical cancer. In some embodiments, the method is for treating the subject's endometrial cancer. In some embodiments, the method is for treating the subject's renal cell cancer. In some embodiments, the method is for treating the subject's uterine cancer. In some embodiments, the method is for treating the subject's pancreatic cancer.

[0254] The methods described herein include administering to a subject having a FOLR1+ cancer or malignancy a therapeutically effective amount of a FOLR1-binding antibody or antigen-binding portion thereof or other binding agent or conjugate thereof. As used herein, the phrases "therapeutically effective amount," "effective amount," or "effective dosage" refer to an amount of a FOLR1 antibody or antigen-binding portion thereof or other binding agent or conjugate described herein that provides a therapeutic benefit in the treatment, management, or prevention of recurrence of cancer or malignancy, e.g., an amount that provides a statistically significant decrease in at least one symptom, sign, or marker of the tumor or malignancy. Determination of a therapeutically effective amount is well within the ability of one of ordinary skill in the art. Generally, the therapeutically effective amount can vary depending on the subject's medical history, age, condition, gender, and the severity and type of the subject's medical condition, as well as the administration of other pharmaceutically active agents.

[0255] The terms "cancer" and "malignant tumor" refer to the uncontrolled growth of cells that interfere with the normal functions of the organs and systems of the body. A cancer or malignant tumor can be primary or metastatic, i.e., invasive enough to seed tumor growth in tissues distant from the original tumor site. A "tumor" refers to the uncontrolled growth of cells that interfere with the normal functions of the organs and systems of the body. A subject having cancer is a subject having objectively measurable cancer cells present in the body of the subject. This definition includes benign tumors and malignant cancers, as well as latent dormant tumors and micrometastases. Cancers that migrate from their original location and seed other vital organs can ultimately result in the death of the subject through the deterioration of the function of the affected organs. Hematological malignancies (hematopoietic cancers), such as leukemia and lymphoma, for example, can overcome the subject's normal hematopoietic compartment, thereby resulting in hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia), and ultimately cause death.

[0256] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include, but are not limited to, basal cell cancer, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer (e.g., triple negative breast cancer), peritoneal cancer, cervical cancer; cholangiocarcinoma, choriocarcinoma, chondrosarcoma, colon and rectal cancer (colorectal cancer), connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer and stomach cancer), glioblastoma (GBM), liver cancer, hepatoma, intraepithelial neoplasia, kidney cancer or renal cancer (e.g., clear cell cancer), laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lymphoma including Hodgkin lymphoma and non-Hodgkin lymphoma, melanoma, mesothelioma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, uterine serous cancer, urinary system cancer, vulvar cancer; as well as other carcinomas and sarcomas, as well as B cell lymphoma (including low grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade / follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatosis, edema (such as those associated with brain tumors), and Meigs syndrome.

[0257] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a solid tumor including, but not limited to, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, pancreatic cancer, and renal cell cancer. In some embodiments, the cancer or malignancy is FOLR1 positive (FOLR1+). As used herein, the terms "FOLR1 positive" or "FOLR1+" are used to describe cancer cells that express FOLR1 on the cell surface (membrane-bound FOLR1), clusters of cancer cells, tumor masses, or metastatic cells. Some non-limiting examples of FOLR1-positive cancers include, for example, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, pancreatic cancer, and renal cell cancer.

[0258] The methods herein are contemplated to reduce the tumor size or tumor burden of a subject and / or reduce metastases in a subject. In various embodiments, the tumor size of the subject is reduced by about 25-50%, about 40-70% or about 50-90% or more. In various embodiments, the method reduces the tumor size by 10%, 20%, 30% or more. In various embodiments, the method reduces the tumor size by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0259] As used herein, "subject" refers to a human or an animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic animals and game animals include cows, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, wolves, avian species such as chickens, emus, ostriches, and fish such as salmon, catfish and trout. In certain embodiments, the subject is a mammal such as a primate such as a human. The terms "patient", "individual" and "subject" are used interchangeably herein.

[0260] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Non-human mammals can be advantageously used, for example, as subjects for animal models of various cancers. Further, the methods described herein can be used to treat domestic animals and / or pets. The subject can be male or female. In certain embodiments, the subject is a human.

[0261] In some embodiments, the subject can be a subject who has been previously diagnosed with FOLR1+ cancer or has been identified as having FOLR1+ cancer and in need of treatment, but does not necessarily have already received treatment for FOLR1+ cancer. In some embodiments, the subject can also be a subject who has not been previously diagnosed as having FOLR1+ cancer in need of treatment. In some embodiments, the subject can be a subject who exhibits one or more risk factors for one or more conditions or complications associated with the condition or FOLR1+ cancer, or a subject who does not exhibit risk factors. A "subject in need of treatment" for FOLR1+ cancer can in particular be a subject who has the condition or has been diagnosed as having the condition. In other embodiments, a subject "at risk of developing" the condition refers to a subject who has been diagnosed as being at risk of developing the condition or at risk of developing cancer again (e.g., FOLR1+ cancer).

[0262] As used herein, the terms "treat," "treatment," "treating," or "ameliorate," when used with respect to a disease, disorder, or medical condition, refer to a therapeutic treatment of a condition aimed at reversing, alleviating, ameliorating, inhibiting, slowing down, or halting the progression or severity of a symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of a condition is reduced or halted. That is, "treatment" includes not only an improvement in symptoms or markers, but also halting or at least slowing the progression or worsening of a symptom that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, a decrease in FOLR1+ cancer cells in a subject, alleviation of one or more symptoms, a decrease in the degree of deficit, stabilization (i.e., not worsening) of a cancer or malignancy, delay or slowing of tumor growth and / or metastasis, and an increase in lifespan as compared to a lifespan expected in the absence of treatment. As used herein, the term "administering" refers to providing to a subject a FOLR1-binding antibody or antigen-binding portion thereof or other binding agent or conjugate described herein, or a nucleic acid encoding a FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein, by a method or route that results in binding to FOLR1+ cancer cells or malignant cells of the FOLR1-binding antibody or antigen-binding portion thereof or other binding agent or conjugate. Similarly, a pharmaceutical composition comprising a FOLR1-binding antibody or antigen-binding portion thereof or other binding agent or conjugate described herein, or a nucleic acid encoding a FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein, can be administered to a subject by any suitable route that results in an effective treatment in the subject.

[0263] The dosage range of the FOLR1-binding antibody or its antigen-binding portion or conjugate or agent is dependent on efficacy and includes an amount sufficient to produce the desired effect, such as slowing of tumor growth or reduction of tumor size. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary according to the age, condition and sex of the subject and can be determined by one of ordinary skill in the art. The dosage can also be adjusted by the individual physician in the event of any complications. In some embodiments, the dosage ranges from 0.1 mg / kg body weight to 10 mg / kg body weight. In some embodiments, the dosage ranges from 0.5 mg / kg body weight to 15 mg / kg body weight. In some embodiments, the dosage range is from 0.5 mg / kg body weight to 5 mg / kg body weight. Alternatively, the dosage range can be quantified to maintain a serum level between 1 ug / mL and 1000 ug / mL. In the case of systemic administration, a therapeutic amount, for example, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 12 mg / kg or an amount in excess thereof can be administered to the subject.

[0264] Administration of the dosages listed above can be repeated. In a preferred embodiment, the dosages listed above are administered weekly, bi-weekly, every three weeks or monthly over a period of several weeks or months. The duration of treatment depends on the clinical progression of the subject and responsiveness to treatment.

[0265] In some embodiments, the dosage can be from about 0.1 mg / kg to about 100 mg / kg. In some embodiments, the dosage can be from about 0.1 mg / kg to about 25 mg / kg. In some embodiments, the dosage can be from about 0.1 mg / kg to about 20 mg / kg. In some embodiments, the dosage can be from about 0.1 mg / kg to about 15 mg / kg. In some embodiments, the dosage can be from about 0.1 mg / kg to about 12 mg / kg. In some embodiments, the dosage can be from about 1 mg / kg to about 100 mg / kg. In some embodiments, the dosage can be from about 1 mg / kg to about 25 mg / kg. In some embodiments, the dosage can be from about 1 mg / kg to about 20 mg / kg. In some embodiments, the dosage can be from about 1 mg / kg to about 15 mg / kg. In some embodiments, the dosage can be from about 1 mg / kg to about 12 mg / kg. In some embodiments, the dosage can be from about 1 mg / kg to about 10 mg / kg.

[0266] In some embodiments, the dosage can be administered intravenously. In some embodiments, the intravenous administration can be an infusion administered over a period of about 10 minutes to about 4 hours. In some embodiments, the intravenous administration can be an infusion administered over a period of about 30 minutes to about 90 minutes.

[0267] In some embodiments, the dosage can be administered weekly. In some embodiments, the dosage can be administered bi - weekly. In some embodiments, the dosage can be administered every about 2 weeks. In some embodiments, the dosage can be administered every about 3 weeks. In some embodiments, the dosage can be administered every about 4 weeks.

[0268] In some embodiments, a total of about 2 to about 10 dosages are administered to the subject. In some embodiments, a total of 4 dosages are administered. In some embodiments, a total of 5 dosages are administered. In some embodiments, a total of 6 dosages are administered. In some embodiments, a total of 7 dosages are administered. In some embodiments, a total of 8 dosages are administered. In some embodiments, a total of 9 dosages are administered. In some embodiments, a total of 10 dosages are administered. In some embodiments, more than a total of 10 dosages are administered.

[0269] A pharmaceutical composition containing an anti-FOLR1 antibody or an antigen-binding portion thereof or another FOLR1-binding agent or a conjugate thereof can be administered in unit dosage. As used in connection with a pharmaceutical composition, the term "unit dosage" refers to a physically discrete unit suitable as a unit dose for administration to a subject, each unit containing a predetermined quantity of the active material (e.g., an anti-FOLR1 antibody or an antigen-binding portion thereof or another binding agent or a conjugate thereof) calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0270] In some embodiments, an anti-FOLR1 antibody or an antigen-binding portion thereof or another binding agent or a conjugate thereof, or a pharmaceutical composition thereof, is administered in combination with immunotherapy. As used herein, "immunotherapy" refers to a therapeutic strategy designed to induce or enhance the subject's own immune system to fight cancer or malignancy. Examples of immunotherapy include, but are not limited to, antibodies such as checkpoint inhibitors.

[0271] In some embodiments, the immunotherapy involves administration of a checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor includes an agent that inhibits CTLA-4, PD-1, PD-L1, etc. Suitable anti-CTLA-4 inhibitors include, for example, ipilimumab, tremelimumab, the antibodies disclosed in PCT Publication No. WO 2001 / 014424 pamphlet, the antibodies disclosed in PCT Publication No. WO 2004 / 035607 pamphlet, the antibodies disclosed in U.S. Patent Application Publication No. 2005 / 0201994 specification, and the antibodies disclosed in the granted European Patent No. 1212422 specification. Additional anti-CTLA-4 antibodies are described in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; PCT Publication Nos. WO 01 / 14424 pamphlet and WO 00 / 37504 pamphlet; and U.S. Patent Application Publication Nos. 2002 / 0039581 and 2002 / 086014. Other anti-CTLA-4 antibodies that can be used in the methods of the present invention include, for example, WO 98 / 42752 pamphlet; U.S. Patent Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camacho et al., J. Clin. Oncology, 22(145):Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res, 58:5301-5304 (1998), the antibodies disclosed in U.S. Patent Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281.

[0272] Suitable anti-PD-1 inhibitors include, for example, nivolumab, pembrolizumab, pidilizumab, MEDI0680, and combinations thereof. In other specific embodiments, anti-PD-L1 therapeutic agents include atezolizumab, BMS-936559, MEDI4736, MSB0010718C, and combinations thereof.

[0273] Suitable anti-PD-1 inhibitors include, for example, those described in Topalian et al., Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy, Cancer Cell 27:450-61 (April 13, 2015), which is hereby incorporated by reference in its entirety.

[0274] In some embodiments, the checkpoint inhibitor is ipilimumab (Yervoy), nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi).

[0275] In some embodiments, a method of improving the treatment outcome of a subject undergoing immunotherapy is provided. Generally, the method includes administering an effective amount of immunotherapy to a subject having cancer; and administering to the subject a therapeutically effective amount of an FOLR1 antibody, antigen-binding portion, other binder or conjugate thereof, or a pharmaceutical composition thereof, wherein the FOLR1 antibody, antigen-binding portion, other binder or conjugate thereof specifically binds to FOLR1+ cancer cells; and the treatment outcome of the subject is improved as compared to administration of immunotherapy alone. In some embodiments, the FOLR1 antibody, antigen-binding portion, other binder or conjugate thereof includes any of the embodiments of the FOLR1 antibody, antigen-binding portion, other binder or conjugate thereof described herein. In some embodiments, the binder is an antibody or an antigen-binding portion thereof. In some embodiments, the binder is a monoclonal antibody, Fab, Fab’, F(ab’), Fv, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody. In some embodiments, the binder is a conjugate of an FOLR1 monoclonal antibody, Fab, Fab’, F(ab’), Fv, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody.

[0276] In some embodiments, the improved treatment outcome is an objective response selected from stable, partial response or complete response, determined by standard medical criteria for the cancer being treated. In some embodiments, the improved treatment outcome is a reduction in tumor burden. In some embodiments, the improved treatment outcome is progression-free survival or disease-free survival.

[0277] The present invention is further illustrated by the following embodiments, which should not be construed as limiting. 1. A binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region wherein the binder The VH region includes complementarity-determining regions HCDR1, HCDR2, and HCDR3 arranged in the heavy-chain variable-region framework region, the VL region includes LCDR1, LCDR, and LCDR3 arranged in the light-chain variable-region framework region, and the VH and VL CDRs are respectively, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; and respectively, SEQ ID NO: 31, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 A binder having an amino acid sequence selected from the set of amino acid sequences shown in the group consisting of 2. The VH region and the VL region are respectively, SEQ ID NO: 1 and SEQ ID NO: 2; respectively, SEQ ID NO: 3 and SEQ ID NO: 4; respectively, SEQ ID NO: 5 and SEQ ID NO: 6; respectively, SEQ ID NO: 7 and SEQ ID NO: 8; respectively, SEQ ID NO: 9 and SEQ ID NO: 10; respectively, SEQ ID NO: 11 and SEQ ID NO: 12; respectively, SEQ ID NO: 13 and SEQ ID NO: 14; respectively, SEQ ID NO: 15 and SEQ ID NO: 16; respectively, SEQ ID NO: 17 and SEQ ID NO: 18; respectively, SEQ ID NO: 19 and SEQ ID NO: 20; respectively, SEQ ID NO: 21 and SEQ ID NO: 22; and respectively, SEQ ID NO: 23 and SEQ ID NO: 24 selected from the pair of amino acid sequences shown in the group consisting of, and The binder according to Embodiment 1, wherein the heavy-chain framework region and the light-chain framework region are optionally modified by substitution, deletion, or insertion of 1 to 8 amino acids within the framework region. 3. The VH region and the VL region are respectively, SEQ ID NO: 1 and SEQ ID NO: 2; respectively, SEQ ID NO: 3 and SEQ ID NO: 4; respectively, SEQ ID NO:5 and SEQ ID NO:6; respectively, SEQ ID NO:7 and SEQ ID NO:8; respectively, SEQ ID NO:9 and SEQ ID NO:10; respectively, SEQ ID NO:11 and SEQ ID NO:12; respectively, SEQ ID NO:13 and SEQ ID NO:14; respectively, SEQ ID NO:15 and SEQ ID NO:16; respectively, SEQ ID NO:17 and SEQ ID NO:18; respectively, SEQ ID NO:19 and SEQ ID NO:20; respectively, SEQ ID NO:21 and SEQ ID NO:22; and respectively, SEQ ID NO:23 and SEQ ID NO:24 The binder according to Embodiment 1 or 2, having an amino acid sequence selected from a pair of amino acid sequences shown in the group consisting of 4. The VH region and the VL region are respectively, SEQ ID NO:3 and SEQ ID NO:4; respectively, SEQ ID NO:7 and SEQ ID NO:8; respectively, SEQ ID NO:9 and SEQ ID NO:10; respectively, SEQ ID NO:11 and SEQ ID NO:12; respectively, SEQ ID NO:15 and SEQ ID NO:16; respectively, SEQ ID NO:17 and SEQ ID NO:18; respectively, SEQ ID NO:19 and SEQ ID NO:20; and respectively, SEQ ID NO:21 and SEQ ID NO:22 The binder according to any one of Embodiments 1 to 3, having an amino acid sequence selected from a pair of amino acid sequences shown in the group consisting of 5. The VH region and the VL region are respectively, SEQ ID NO:3 and SEQ ID NO:4; respectively, SEQ ID NO:7 and SEQ ID NO:8; and respectively, SEQ ID NO:21 and SEQ ID NO:22 The binder according to any one of Embodiments 1 to 4, having an amino acid sequence selected from a pair of amino acid sequences shown in the group consisting of 6. The binder according to embodiment 1, wherein the framework region is a human framework region. 7. The binder according to any one of embodiments 1 to 6, which is an antibody or an antigen-binding portion thereof. 8. The binder according to any one of embodiments 1 to 7, which is a monoclonal antibody, Fab, Fab’, F(ab’)₂, Fv, scFv, single-domain antibody, diabody, bispecific antibody, or multispecific antibody. 9. The binder according to any one of embodiments 1 to 8, wherein the heavy-chain variable region further comprises a heavy-chain constant region. 10. The binder according to embodiment 7, wherein the heavy-chain constant region is of the IgG isotype. 11. The binder according to embodiment 10, wherein the heavy-chain constant region is an IgG1 constant region. 12. The binder according to embodiment 10, wherein the heavy-chain constant region is an IgG4 constant region. 13. The binder according to embodiment 11, wherein the IgG1 constant region has the amino acid sequence shown in SEQ ID NO: 39. 14. The binder according to any one of embodiments 1 to 13, wherein the light-chain variable region further comprises a light-chain constant region. 15. The binder according to embodiment 14, wherein the light-chain constant region is of the kappa isotype. 16. The binder according to embodiment 15, wherein the light-chain constant region has the amino acid sequence shown in SEQ ID NO: 40. 17. The binder according to any one of embodiments 9 to 16, wherein the heavy-chain constant region further comprises an amino acid modification that reduces at least the binding affinity for human FcγRIII. 18. The binder according to any one of embodiments 1 to 17, which is monospecific. 19. The binder according to any one of embodiments 1 to 18, which is bivalent. 20. The binder according to any one of embodiments 1 to 17, which is bispecific. 21. A pharmaceutical composition comprising the binder according to any one of embodiments 1 to 20 and a pharmaceutically acceptable carrier. 22. A nucleic acid encoding the binder according to any one of embodiments 1 to 20. 23. A vector comprising the nucleic acid according to Embodiment 22. 24. A cell line comprising the vector according to Embodiment 22 or the nucleic acid according to Embodiment 21. 25. A conjugate comprising the binder according to any one of Embodiments 1 to 20, at least one linker bound to the binder, and at least one drug bound to each linker . 26. The conjugate according to Embodiment 25, wherein each drug is selected from a cytotoxin, an immunomodulator, a nucleic acid, a growth inhibitor, a PROTAC, a toxin, and a radioisotope. 27. The conjugate according to any one of Embodiments 25 to 26, wherein each linker is bound to the binder via an interchain disulfide residue, a lysine residue, an engineered cysteine residue, a glycan, a modified glycan, an N-terminal residue of the binder, or a polyhistidine peptide bound to the binder. 28. The conjugate according to any one of Embodiments 25 to 27, wherein the average drug loading of the conjugate is about 1 to about 8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 3 to about 5, about 6 to about 8, or about 8 to about 16. 29. The conjugate according to any one of Embodiments 25 to 28, wherein the drug is a cytotoxin. 30. The conjugate according to Embodiment 29, wherein the cytotoxin is selected from the group consisting of auristatin, maytansinoid, camptothecin, duocarmycin, and calicheamicin. 31. The conjugate according to Embodiment 30, wherein the cytotoxin is auristatin. 32. The conjugate according to Embodiment 31, wherein the cytotoxin is MMAE or MMAF. 33. The conjugate according to Embodiment 30, wherein the cytotoxin is camptothecin. 34. The conjugate according to Embodiment 33, wherein the cytotoxin is exatecan. 35. The conjugate according to Embodiment 33, wherein the cytotoxin is SN-38. 36. The conjugate according to embodiment 30, wherein the cytotoxic agent is calicheamicin. 37. The conjugate according to embodiment 30, wherein the cytotoxic agent is a maytansinoid. 38. The conjugate according to embodiment 37, wherein the maytansinoid is maytansine, maytansinol, or a maytansine analog of DM1, DM3, and DM4, or ansamitocin-2. 39. The conjugate according to any one of embodiments 25 to 38, wherein the linker comprises mc-VC-PAB, CL2, CL2A, or (succinimid-3-yl-N)-(CH2)n-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)-, where n = 1 to 5. 40. The conjugate according to embodiment 39, wherein the linker comprises mc-VC-PAB. 41. The conjugate according to embodiment 39, wherein the linker comprises CL2A. 42. The conjugate according to embodiment 39, wherein the linker comprises CL2. 43. The conjugate according to embodiment 39, wherein the linker comprises (succinimid-3-yl-N)-(CH2)n-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)-. 44. The conjugate according to embodiment 43, wherein the linker is bound to at least one molecule of exatecan. 45. The conjugate according to any one of embodiments 25 to 28, wherein the drug is an immunomodulatory agent. 46. The conjugate according to embodiment 45, wherein the immunomodulatory agent is selected from the group consisting of a TLR7 agonist, a TLR8 agonist, a STING agonist, or a RIG-I agonist. 47. The conjugate according to embodiment 46, wherein the immunomodulatory agent is a TLR7 agonist. 48. The conjugate according to embodiment 47, wherein the TLR7 agonist is imidazoquinoline, imidazoquinoline amine, thiazoloquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazole-2-amine, tetrahydropyridopyrimidine, heteroarothiadiazide-2,2-dioxide, benzonaphthyridine, guanosine analog, adenosine analog, thymidine homopolymer, ssRNA, CpG-A, poly G10, and poly G3. 49. The conjugate according to embodiment 46, wherein the immunomodulatory agent is a TLR8 agonist. 50. The conjugate according to embodiment 49, wherein the TLR8 agonist is selected from imidazoquinoline, thiazoloquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazole-2-amine, tetrahydropyridopyrimidine or ssRNA. 51. The conjugate according to embodiment 46, wherein the immunomodulatory agent is a STING agonist. 52. The conjugate according to embodiment 46, wherein the immunomodulatory agent is a RIG-I agonist. 53. The conjugate according to embodiment 52, wherein the RIG-I agonist is selected from KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400 and KIN2000. 54. The conjugate according to any one of embodiments 45 to 53, wherein the linker is selected from the group consisting of mc-VC-PAB, CL2, CL2A and (succinimid-3-yl-N)-(CH2)n-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)- (where n = 1 to 5). 55. A pharmaceutical composition comprising the conjugate according to any one of embodiments 25 to 54 and a pharmaceutically acceptable carrier. A method for treating FOLR1+ cancer, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a binder according to any one of embodiments 1 to 20, a conjugate according to any one of embodiments 25 to 54, or a pharmaceutical composition according to embodiment 21 or 55. 57. The method according to embodiment 56, wherein the FOLR1+ cancer is a solid tumor. 58. The method according to embodiment 57, wherein the FOLR1+ cancer is selected from lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, pancreatic cancer, and renal cell cancer. 59. The method according to any one of embodiments 56 to 58, further comprising the step of administering immunotherapy to the subject. 60. The method according to embodiment 59, wherein the immunotherapy comprises a checkpoint inhibitor. 61. The method according to embodiment 60, wherein the checkpoint inhibitor is selected from antibodies that specifically bind to human PD-1, human PD-L1, or human CTLA4. 62. The method according to embodiment 61, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab. 63. The method according to any one of embodiments 56 to 62, further comprising the step of administering chemotherapy to the subject. 64. The method according to any one of embodiments 56 to 63, comprising the step of administering a conjugate according to any one of embodiments 25 to 54 or a pharmaceutical composition according to embodiment 55. 65. The method according to any one of embodiments 56 to 64, wherein the binder, conjugate, or pharmaceutical composition is administered intravenously. 66. The method according to embodiment 6, wherein the binder, conjugate, or pharmaceutical composition is administered at a dose of about 0.1 mg / kg to about 12 mg / kg. 67. The method according to any one of embodiments 56 to 66, wherein the treatment outcome of the subject is improved. 68. The method according to embodiment 67, wherein the improved treatment outcome is an objective response selected from stable, partial response, or complete response. 69. The method according to embodiment 67, wherein the improved treatment outcome is a reduction in tumor mass. 70. The method according to embodiment 67, wherein the improved treatment outcome is progression-free survival or disease-free survival. 71. Use of the binder according to any one of embodiments 1 to 20 or the pharmaceutical composition according to embodiment 21 for treating FOLR1+ cancer in a subject. 72. Use of the conjugate according to any one of embodiments 25 to 54 or the pharmaceutical composition according to embodiment 55 for treating FOLR1+ cancer in a subject.

[0278] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the exact forms disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes, but as will be recognized by those skilled in the art, various equivalent modifications are possible within the scope of the present disclosure. The teachings of the disclosure provided herein can be applied to other procedures or methods as needed. Combinations of the various embodiments described herein can provide further embodiments. Aspects of the present disclosure can be modified to adopt the compositions, functions, and concepts of the above references and applications to provide still further embodiments of the present disclosure as needed. In light of the detailed description, these and other changes can be made to the present disclosure.

[0279] Any specific element of the above embodiments can be combined with or replaced by an element of other embodiments. Further, although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments can also exhibit such advantages, and not all embodiments necessarily exhibit such advantages in order to fall within the scope of the present disclosure.

[0280] All patents and other publications specified are hereby expressly incorporated by reference herein for the purpose of, for example, describing and disclosing the methodologies described in such publications that may be used in connection with the present invention. These publications are provided only for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or content of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the date or content of these documents.

[0281] Examples Example 1: Preparation of a Human Antibody Against Human FOLR1 Antibodies targeting human FOLR-1 were screened using a fully human antibody library. This library is a semi-synthetic human antibody library in which Fab is displayed on the phage surface.

[0282] The panning of the library was carried out according to the standard protocol. Specifically, PolySorp or MaxiSorp Nunc-Immuno Tubes (Nunc-MG Scientific) were coated with 0.5 ml of 6 μg / ml (see panning summary, Table 1) of human FOLR1 (ACRO-FO1-H52H1) antigen and placed in the refrigerator overnight. The tubes were washed once with PBS, blocked with 1% BSA / PBS, and incubated at RT (room temperature) for 1 hour. The tubes were incubated with the indicated amount (CFU, see panning summary, Table 1) of the library phage sample at RT for 1 hour. The tubes were washed 10 times with PBST buffer. To elute the bound phage, 0.5 ml of 100 mM TEA (triethylamine) was added, incubated at RT for 2 minutes, the eluate was transferred to a new tube, and immediately neutralized by adding 0.25 ml of 1.0 M Tris-HCL, pH 8.0 while mixing. The eluate (0.75 ml) was added to 10 ml of log-phase E. coli TG1 (OD600 ~ 0.5), mixed well, and incubated at 37 °C (water bath) for 30 minutes without shaking. 10-fold dilutions of the culture were made in 2xTY medium, 10 μl of each dilution was plated on TYE / amp / glu plates, and incubated at 30 °C overnight. The next day, the number of colonies in each dilution was counted, and the CFU (colony-forming units) of the panning output was calculated. The remaining culture was centrifuged at 2,800 g for 15 minutes, resuspended in 0.5 ml of 2×TY medium, plated on two 150 mm TYE / amp / glu plates, and incubated at 30 °C overnight. The next day, 3 - 5 ml of 2xTY / amp / glu medium was added to each plate, and the bacteria were scraped off the plates with a cell spreader. A glycerol stock was made by mixing 1.5 ml of bacteria with 0.5 ml of 80% glycerol, and the stock was placed at -80 °C.

[0283] To prepare phage particles for the next selection round, a glycerol stock was inoculated into 40 ml of 2xTY / amp / glu medium starting from an OD600 of approximately 0.01 - 0.05. The culture was grown at 37°C with shaking (300 rpm) until the OD600 reached 0.4 - 0.6. The culture was infected by adding helper phage CM13 to the culture at a helper phage:bacteria ratio of 5 - 10:1. The culture was incubated at 37°C for 30 minutes with occasional mixing and standing in a water bath, followed by shaking at 37°C for 30 minutes. The bacterial culture was centrifuged at 3000 rpm for 20 minutes and the supernatant was removed. The pellet was resuspended in 100 mL of 2xTY / amp / kan and then grown with shaking overnight at 30°C. The culture was harvested by centrifugation at 6,000g for 30 minutes. 1 / 5 volume of PEG solution was added to the supernatant, followed by incubation on ice for 1 hour, and then phage particles were precipitated by centrifugation at 4,000g at 4°C for 20 minutes. The supernatant was discarded completely. The phage pellet was resuspended in 1 - 2 ml of cold PBS. Residual bacteria were removed by microcentrifugation at maximum speed for 5 minutes at 4°C. The phage thus prepared can be used immediately for selection or stored at -80°C in aliquots containing 10% glycerol. The titer of the phage preparation was determined by infecting 100 ul of log-phase E. coli TG1 with a 10-fold diluted phage solution (in 2xTY, 10 -11 down to). Selection was repeated for a total of 3 - 4 rounds starting from step 1.

[0284] A total of 4 pannings were performed. The concentration of the washing buffer PBS-Tween20 was gradually increased to 0.2%, 0.3% and 0.4% for the 2nd, 3rd and 4th washings respectively.

[0285] After 4 screenings, the target positive enrichment rate was 1.5×10 4It reached, and as shown in Table 1, there was a significant difference from the blank control. For phage ELISA verification, clones were collected from two 96-well plates; clones with high binding affinity to FOLR-1 were selected for sequencing.

[0286] A total of 69 clones were sequenced, and 12 unique VH sequences were obtained. When these 12 VH sequences were analyzed, as shown in Tables 2 and 4, they had two distinct sets of HCDR3. For the clones with 12 unique VH sequences, the VL sequences were then determined. As shown in Tables 3 and 4, using two groups of unique LCDR3, two unique VL sequences were obtained.

[0287] Further analysis of the clone sequences using the Kabat system for CDR regions showed that, as shown in Table 5, clones F1 / 8 / 9 / 26 / 48 / 50 / 100 / 112 / 123 / 131 / 138 had the same HCDR and LCDR, but different heavy-chain framework (HFR) and light-chain framework (LFR) sequences. Clone F40 had different HCDR and LCDR, and different HFR and LFR, as shown in Table 5.

[0288]

Table 1

[0289]

Table 2

[0290]

Table 3

[0291]

Table 4

[0292]

Table 5

[0293] Example 2: Verification of Antibodies Produced by HEK293 Cells (As described above), after obtaining the sequences of the antibody clones, further analysis was performed using the complete IgG molecule. First, the expression of the full-length antibody molecule by IgG1 Fc was carried out in 48-well or 96-well microplates, and the supernatant was collected for detection of the expression level and antigen or cell-binding ability.

[0294] 2.1 Antibody Expression in 48 or 96-Well Plates. cDNA sequences encoding the heavy and light chains of antibodies F1, F8, F26, F40, F48, F50, F100, F112, F123, F131, and F138 were constructed into vector PTT5. HEK293 cells were harvested, adjusted to a cell density of 1×10 6 / ml, and plated in 48 / 96-well cell culture plates at 200 or 400 μL / well in a 37°C incubator with 5% CO2 for later use. For transfection in 96-well plates, 0.5 μg of plasmid was diluted in 20 μL of OPTI medium, mixed well, and 2.5 μL of transfection reagent T1 (plasmid:T1 = 1:5) was diluted in 20 μL of OPTI medium, mixed well, and incubated at room temperature for 5 minutes. The transfection reagent T1 diluent was added to the DNA, mixed well, and incubated at room temperature for 30 minutes. A transfection complex was formed during the incubation. The transfection complex was added to the cells, mixed well, and incubated at 37°C in a 5% CO2 incubator for 48 hours. When transfecting in 48-well plates, the amounts of plasmid and transfection reagent were doubled. On the second day after transfection, the supernatant was collected to detect antibody bioactivity by ELISA or FACS.

[0295] 2.2 IgG Expression Level. Antibody expression levels in 96 wells were tested by standard ELISA. Briefly, anti-human IgG Fc antibody (Sigma, 18885-2ML) was diluted to 5 μg / ml with carbonate coating solution at pH 9.6, and 100 μL was coated onto each well of a 96-well microtiter plate at 4 °C overnight. The liquid in the wells was discarded, and the wells were washed 3 times with PBST, blocked with 4% non-fat dry milk-PBS (Sigma, D5652-1L), 300 μL / well, and incubated at 37 °C for 1 hour. The liquid in the wells was discarded, and then the wells were washed 3 times with PBS. Samples were added to the 96-well microtiter plate using 100 μL / well. PBS was added to the control group. The plate was incubated at 37 °C for 1 hour, then the liquid was discarded, and the wells were washed 3 times with PBST. HRP-goat anti-human IgG (Sigma, I18885-2ML) was added using 100 μL / well (1:5000 dilution), and the plate was incubated at 37 °C for 1 hour. Then the liquid in the plate was discarded, and the plate was washed 5 times with PBST. TMB solution was added using 100 μL / well. Then 2M H2SO4 was added to each well using 50 μL / well to stop the reaction after 10 - 15 minutes. The A450 value was read using a microplate reader. The results are shown in Table 6. All antibodies had normal expression except for clone F50.

[0296] 2.3 Antibodies that bind to human and cynomolgus monkey FOLR1 proteins. The ability of antibodies to bind to human FOLR1 protein or cross-react with cynomolgus monkey FOLR1 protein was tested by standard ELISA. Briefly, human FOLR1 protein with His tag (ACRO-FO1-H52H1) or cynomolgus monkey FOLR1 protein (ACRO, F01-C52H8) was diluted to 5 μg / ml with carbonate coating solution at pH 9.6, and 100 μL of the antigen was coated overnight at 4 °C in each well of a 96-well microtiter plate. The liquid in the wells was discarded, and the wells were washed 3 times with PBST. Then, the wells were blocked with 4% skim milk-PBS (Sigma, D5652-1L) using 300 μL / well, and the plate was incubated at 37 °C for 1 hour. The liquid in the wells was discarded, and the wells were washed 3 times with PBS. Samples were added using 100 μl / well; PBS was added to the control group. The plate was incubated at 37 °C for 1 hour. The liquid in the wells was discarded, and the wells were washed 3 times with PBST. HRP-goat anti-human IgG (Sigma, I18885-2ML) was added (1:5000 dilution, 100 μ / well), and the plate was incubated at 37 °C for 1 hour. Then, the liquid in the wells was discarded, and the wells were washed 5 times with PBST. TMB solution was added using 100 μL / well. 2M H2SO4 was added to each well using 50 μL to stop the reaction after 10 - 15 minutes. The A450 value was read using a microplate reader.

[0297] Table 6 shows the expression levels of IgG in the microtiter plate and the binding to human FOLR1 protein. All antibodies except clone F50 had normal binding to human FOLR1 protein.

[0298] Table 7 shows the results of anti-FOLR1 antibody cross-reactivity to cynomolgus monkey FOLR1 protein. All antibodies except clone F50 had good cross-reactivity to cynomolgus monkey FOLR1 protein.

[0299] 2.4 Antibody binding to tumor cell lines expressing high levels of FOLR1. The binding activity of antibodies against Hela cells (ATCC® CCL-2 provided by COBIOER) and RPTEC / TERT1 cells (ATCC® CRL-4031 provided by COBIOER) was tested by flow cytometry using the transfection supernatant. Briefly, the target cells were digested with 0.02% EDTA-2Na, centrifuged at 1500 rpm for 3 minutes, and resuspended in PBS. After counting, the cells were added to 1.5 ml centrifuge tubes at 1×10 6 cells / tube, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. Subsequently, all operations were performed in an ice bath. 100 μL of the transfection supernatant was added to each 1.5 ml centrifuge tube. Blank cells, blank cells + secondary antibody, medium, and HEK293 supernatant were set as controls. The reaction was carried out in an ice bath for 1 hour. Then, the cells were pelleted and washed twice with PBS. The secondary antibody, goat anti-human IgG (PE, abcam, ab98596), was diluted (1:200), and 100 μL per tube was used for addition. The reaction was carried out in the dark in an ice bath for 1 hour. The cells were pelleted again, washed twice with PBS, resuspended in 300 μL of PBS, and the FL2 fluorescence reading value was measured by cytometry. The results were analyzed by FlowJoTM 10 software.

[0300] The results of the binding of anti-FOLR1 antibody to Hela cells are shown in Figure 1. The results demonstrate that clone F50 was negative for Hela cell binding. Clones F40 and F138 were weakly positive for Hela cell binding. The remaining 8 clones were positive for Hela cell binding.

[0301] The results of the binding of anti-FOLR1 antibody to RPTEC / TERT1 cells are shown in Figure 2. The results demonstrate that clone F50 was negative for RPTEC / TERT1 cell binding. Clone F138 was weakly positive for RPTEC / TERT1 cell binding. The remaining 9 clones were positive for RPTEC / TERT1 cell binding.

[0302]

Table 6

[0303]

Table 7

[0304] Example 3: Characterization of anti-human FOLR1 antibody produced by HEK293 cell expression in a shaking flask The binding of the anti-FOLR1 antibody was quantitatively tested by expressing the anti-FOLR1 antibody in floating cells to obtain sufficient amounts of protein. Plasmids were transfected into floating cells for expression. Supernatants were collected for antibody purification. High-purity antibodies were used to quantitatively detect the binding and internalization of the antibody on tumor cells with high FOLR1 protein levels.

[0305] 3.1 Antibody expression and purification. Plasmids encoding antibodies F8, F26, F40, F48, F100, F112, F123 and F131 were transfected into HEK293 cells. Briefly, HEK293 cells were harvested, adjusted to a cell density of 1×10 6 / ml, and then cultured in 30 mL of medium in a 125 mL shaking flask in a 37 °C shaker with 5% CO2 for later use. For transfection, 30 μg of plasmid was diluted in 1500 μL of KPM medium, mixed well, 150 μL of transfection reagent T1 (plasmid:T1 = 1:5) was diluted in 1500 μL of KPM medium, mixed well, and incubated at room temperature for 5 minutes. The transfection reagent T1 diluent was added to the DNA, mixed well, and incubated at room temperature for 30 minutes to form a transfection complex. The transfection complex was added to the cells, mixed well, and incubated at 37 °C for 48 hours in a 5% CO2 shaker at 120 rpm. TN1 solution was added after 24 hours to a final concentration of 0.5%. On the 6th day after transfection, the supernatant was collected and purified.

[0306] Antibody purification was performed by a standard process using Protein A or Protein G. Briefly, each supernatant was filtered through a 0.22 μm filter membrane and loaded onto a column equilibrated with binding buffer (PB, pH 7.2). The column was washed with binding buffer until a stable baseline with no absorbance at 280 nm was obtained. The antibody was eluted with 0.1 M citrate buffer containing 0.15 M NaCl, pH 3.4, using a flow rate of 1 ml / min. Fractions of approximately 1.5 - 3.5 ml were collected and neutralized by the addition of 10% volume of 1 M Tris-HCl, pH 9.0. Subsequently, the antibody sample was dialyzed twice against 1×PBS overnight and sterilized by filtration through a 0.2 μm filter membrane. Purity was tested using 12% SDS-PAGE.

[0307] The expression levels and purification results are shown in Table 8. Antibodies F8, F26, and F131 had higher expression levels, and antibody F100 had the lowest expression level. All antibodies had high purity (data not shown).

[0308]

Table 8

[0309] 3.2 Antibody binding to tumor cell lines with high FOLR1 levels. Anti-FOLR1 antibody binding to Hela cells and RPTEC / TERT1 cells was tested by FACS. The test was performed as described above. The results are shown in Figures 3 and 4. All antibodies bound to Hela cells and RPTEC / TERT1 cells in a dose-dependent manner.

[0310] 3.3 Characterization of the internalization rate. Anti-FOLR1 antibodies F8, F26, F40, F48, F100, F112, F123 and F131 were tested for their ability to internalize into FOLR-1-expressing tumor cell lines Hela and RPTEC / TERT1 using a pHAb assay in which the antibodies were labeled with a pHAb fluorescent dye. Antibody labeling was performed according to the kit instructions. Specifically, 50 μL of magnetic beads were added to a 1.5 ml EP tube. The EP tube was placed on a magnetic stand for 10 seconds to remove the protective solution on the magnetic beads. Each tube of magnetic beads was washed with 250 μL of PB, and 100 μg of antibody was added to each tube of magnetic beads (buffer system: citrate / Tris-HCl sodium (pH 6.0)). The volume was made up to 1 ml with PB, the reaction solution was mixed, and rotated at room temperature for 1 hour. Then, the magnetic beads were washed with 250 μL of PB and equilibrated with 250 μL of NaHCO3. 100 μL of NaHCO3 and 1.2 μL of the prepared pHAb dye (prepared before use) were added to each tube, and the reaction was left in the dark for 1 hour. Each tube was washed twice with 250 μL of PB. 50 mM glycine was added to each tube using 100 μL at room temperature for 5 minutes, and then the labeled antibody was eluted. Then, 2M Tris buffer was added to the eluate for neutralization. The final labeled antibody was stored in the dark for later use.

[0311] Hela cells or RPTEC / TERT1 cells were seeded at 15,000 cells per well in 100 μL and cultured in a 5% CO2 incubator at 37 °C for 20 - 24 hours. The pHAb-labeled test antibody was added to the wells at a concentration of 10 μg / ml. Then, the plates were read using a Thermo VARIOSKAN FLASH at an excitation wavelength of 520 nm and an absorption wavelength of 570 nm at 0 hours, 1 hour, 4 hours, 6 hours, and 23 hours, respectively.

[0312] The results are shown in FIGS. 5 and 6. All anti-FOLR1 antibodies tested showed a time-dependent increase in pHAb fluorescence in FOLR1-expressing Hela cells and RPTEC / TERT1 cells. This result indicates that each antibody internalizes into Hela cells and RPTEC / TERT1 cells, and that antibodies F8 and F131 have the strongest internalization rates.

[0313] Example 4: Characterization of anti-FOLR-1 immunoconjugates Further characterization of anti-FOLR-1 antibodies as immunoconjugates was performed.

[0314] 4.1 Expression of reference antibody and antibodies F8, F26 and F131 The anti-FOLR-1 antibody mirvetuximab (huFR107) from ImmunoGen Inc. was used as a control. The amino acid sequences of the VH and VL regions of huFR107 were obtained from the specification of US Patent No. 8,557,966 (SEQ ID NOs: 36 and 37, respectively) and codon-optimized. Optimized cDNAs encoding huFR107 and encoding antibodies F8, F26 and F131 were constructed in the vector pcDNA3.4. The plasmid was then transiently transfected into ExpiCHO-S cells in a shake flask using the standard ExpiFectamine CHO Transfection procedure (Gibco, A29129). The suspended transient transfection was incubated for 10 days, then the clarified supernatant was purified by a protein A column and subsequently SDS-PAGE was performed as described above.

[0315] 4.2 Preparation of anti-FOLR-1 immunoconjugates By adding 0.5M disodium phosphate, the pH of the antibody solution was adjusted within the range of pH 7.0 - 7.5. The indicated amount of 0.5M EDTA was added to achieve a final EDTA concentration of 5 mM in the antibody solution. The indicated amount of 10 mM TCEP (tris(2-chloroethyl)phosphate) solution was added to achieve the desired TCEP / mAb molar ratio. The reduction reaction was left at RT for 90 minutes. DMSO was then added to achieve 10% v / v. The drug-linker mc-VC-PAB-MMAE was dissolved in DMSO to achieve a final concentration of 10 mM, and the indicated amount was added to the reaction solution at a 30 - 50% molar excess compared to the number of moles of available cysteine thiols. The conjugation reaction was left at RT for 30 minutes. NAC (N-acetyl-L-cysteine) stock solution was added to achieve an NAC / Mc-VC-PAB-MMAE molar ratio of 5. The quenched reaction was left at RT for 15 minutes. Purification was performed by a PD10 column.

[0316] The purity of the anti-FOLR-1 immunoconjugate was evaluated by size exclusion chromatography (SEC) using a Waters HPLC E2695&2489 system with a TSK gel G3000SWXL, 7.8×300 mm column (Tosoh Bioscience). The operation was carried out at 25 °C using a mobile phase of 50 mM Na2PO4 (pH 6.7) and 10% IPA, and was run for 20 minutes at a flow rate of 0.8 mL / min. Referring to Table 9, all four ADCs had high purity.

[0317] The hydrophobicity of the anti-FOLR-1 immunoconjugate was evaluated by hydrophobic interaction chromatography (HIC) using a Waters HPLC E2695&2489 system and a TosoHaas TSK gel Butyl-NPR column (inner diameter 4.6 mm × 3.5 cm, particle size 2.5 μm). Briefly, the HPLC system was operated at 25 °C using mobile phase A: 50 mM Na2PO4 / 1.5 M (NH4)2SO4 pH 7.0 and mobile phase B: 50 mM Na2PO4 / 25% IPA, pH 7.0. The mobile phases were filtered through a 0.22 μm membrane filter (Millipore) and run at a flow rate of 0.5 mL for 30 minutes. The parameters of the linear gradient are shown in Table 10. When the DAR (drug-to-antibody ratio) of the anti-FOLR-1 immunoconjugate was determined according to the HIC data, it was in the range of 3 - 4 (data not shown).

[0318]

Table 9

[0319]

Table 10

[0320] Example 5: Evaluation of the Binding Characteristics of Immunoconjugates The binding of the anti-FOLR1 conjugate to FOLR1-his or FOLR1-highly expressing tumor cell lines was compared by standard ELISA or FACS.

[0321] 5.1 ELISA test. Recombinant His-tagged FOLR1 was coated overnight at 2 μg / ml, 100 μL / well on a 96-well microplate (Thermo, catalog number: 468667) in PBS. The coating solution was removed, and the plate was washed twice by filling the wells with 350 μL / well of TBST. The plate was blocked by adding 200 μL / well of blocking buffer (2% BSA / TBST). The plate was incubated at 37 °C for 2 hours. The plate was washed twice with 350 μL / well of TBST. Samples were added at a starting concentration of 10 μg / ml and titrated by 1:3 serial dilution. The plate was incubated at room temperature for 1 hour. The solution was removed, and the plate was washed twice with 350 μL / well of TBST. Goat anti-human IgG Fc HRP (abcam, ab98624) was diluted 1:20000 with blocking buffer and added to the plate at 100 μL / well. The plate was incubated at room temperature for 1 hour. The plate was washed four times with 350 μL / well of TBST. 100 μL of TMB (solution A: solution B, 1:1) solution was added to each well, and the reaction was placed in the dark for 3 - 10 minutes. 50 uL of stop solution (2M H2SO4) was added, and the optical density at 450 nm and 630 nm was read. The data was analyzed with GraphPad Prism 5 software.

[0322] The results of ELISA are shown in Figures 7 and 8. The data showed that the activity of the conjugate in binding to the target FOLR1 protein was not affected after conjugation, and there was no significant difference among the three ADCs in binding to the recombinant protein FOLR-1.

[0323] 5.2 FACS assay. FOLR1-expressing Hela cells, OVCAR3 cells (provided by COBIOER, ATCC® HTB-161™), OV90 cells (provided by COBIOER, ATCC® CRL-11732™) and IGROV-1 (provided by COBIOER) were incubated with anti-FOLR-1 conjugates at various concentrations. Each antibody conjugate was incubated for 0.5 h in 0.1 ml of FACS buffer (PBS supplemented with 0.1% BSA). The cells were then pelleted, washed and incubated for 0.5 h with 0.1 ml of PE-conjugated goat anti-human IgG antibody (Abcam, Ab98596). The cells were pelleted again, washed with PBS and resuspended in 100 μL of PBS. Samples were analyzed using CytoFLEX (Beckman).

[0324] The results are shown in FIGS. 9 and 10. There was no significant difference in the binding of the three antibody conjugates to the cell lines compared to the reference antibody conjugate. All three anti-FOLR1 conjugates had stronger binding to OVCAR3 than to binding to IGROV-1 cells or to the OV90 cell line.

[0325] Example 6: Internalization of anti-FOLR1 immunoconjugates Anti-FOLR1 conjugates (F8-ADC, F26-ADC, F131-ADC and control FR107-ADC) were tested for their ability to internalize into FOLR1-expressing Hela, OVCAR3, IGROV-1 and OV90 tumor cells using an immunofluorescence staining assay.

[0326] Specifically, 3×10 5Cells were harvested and then incubated with each immunoconjugate at 10 micrograms / ml in FACS buffer (1×PBS containing 0.1% BSA) for 30 minutes at 4°C. Human IgG1 isotype control was used as a negative control. Cells were washed to remove unbound material and incubated at 4°C or transferred to 37°C. At the set time points (0 hours, 4 hours, 24 hours), cells were stained with a PE-conjugated anti-human Fc antibody (Abcam, Ab98596) for 30 minutes at 4°C and analyzed by flow cytometry. The internalization ratio was determined by subtracting the MFI at 37°C from the MFI at 4°C and then compared to the MFI at 4°C.

[0327] Figures 11 and 12 show the changes in the surface levels of immunoconjugates or isotype controls in the Hela and OVCAR3 cell lines maintained at 4°C over a 4- or 24-hour period. When the cells were transferred to 37°C over the course of the assay, the surface levels of the immunoconjugates significantly decreased. This finding suggests that there was no significant difference in internalization among the three tested anti-FOLR1 immunoconjugates, as well as the reference antibody conjugate, in the two tumor cell lines.

[0328] Figure 13 shows the internalization results of the anti-FOLR1 immunoconjugate in the OV90 tumor cell line. The results showed that the internalization of F8-ADC was better than that of the other ADCs in the OV90 cell line. From the results shown in Figure 14, the internalization in the IGROV-1 tumor cell line could not be determined.

[0329] The internalization results of the anti-FOLR1 conjugates in Hela, OVCAR3, and OV90 are summarized in Table 11.

[0330]

Table 11

[0331] Example 7: In Vitro Cytotoxicity Assay The ability of the F8, F26, F131, and FR107 conjugates to inhibit cell proliferation was measured using an in vitro cytotoxicity assay. The following method was used.

[0332] Cells were harvested and seeded in 96-well solid white flat-bottom plates at the indicated amounts (depending on the cell proliferation rate) before adding the anti-FOLR1 conjugate. The next day, the cells were exposed to the conjugate in a drug range of 30 micrograms / ml to 0.37 micrograms / ml or 100 micrograms / ml to 0.015 micrograms / ml using 1:3 serial dilutions with replicate wells. The plates were incubated at 37 °C for 120 hours. Then, 40 μL of CTG (Promega, G7572) per well was added to the plates, and after a 5-minute incubation, the plates were read on an MD I3X reader. Growth inhibition was measured as the percentage of growth relative to untreated cells using Microsoft Excel and Prism software.

[0333] The results are shown in Figures 15 to 18 and Table 12. As shown in Figures 15 and 18, all three anti-FOLR1 conjugates (F8-ADC, F26-ADC, and F131-ADC) had slightly better cytotoxicity against Hela cells and IGROV-1 cells than the reference antibody conjugate (FR107-ADC). Furthermore, F131-ADC had somewhat better cell growth inhibitory activity against the IGROV-1 cell line than F8-ADC and F26-ADC.

[0334]

Table 12

[0335] Example 8: Pharmacokinetics (PK) and Safety of Anti-FOLR-1 Immunoconjugates in a Mouse Model 8.1 PK BALB / c normal mice were purchased from JOINN Laboratories (Suzhou) and used after one week of accommodation. The mice were housed in sterile cages in groups and maintained under pathogen-free conditions. In the laboratory, the environmental conditions were as follows: temperature 20°C - 22°C and humidity 59% - 78%, 12-hour artificial lighting. The mouse cages were polysulfone boxes with a specification of 325 mm × 210 mm × 180 mm and were used after autoclaving. A maximum of 5 animals were bred in each box, and the experiment number, experiment start time, project leader, experiment personnel, animal source, group, and animal number were indicated on the cage card. The experimental animals were ear-marked. The mice were fed with FR-2 diet and given tap water (used after autoclaving). The body weight of the mice was approximately 20 - 22 g at the time of administration.

[0336] Four groups, each containing 6 mice per group, were intravenously (IV) treated with a single dose of 3 mg / kg of F8, F26, F131, and FR107 immunoconjugates. Blood samples were collected 10 minutes, 4 hours, 1 day, 4 days, 7 days, 10 days, 14 days, and 21 days after the administration of the immunoconjugate and subsequently centrifuged (4°C, 10000×g, 3 minutes) to separate the serum. The total antibody concentration of each conjugate in the serum was detected by ELISA and analyzed by Winnonlin 8.2 software.

[0337] Goat anti-human IgG Fc (Invitrogen, 31125) was coated overnight at 4°C on a 96-well microplate (Thermo, catalog number: 468667) in PBS at 2 micrograms / ml using 100 μL / well. The next day, the solution was removed and the plate was washed twice with 350 μL / well of TBST. The plate was blocked by adding 200 μL / well of blocking buffer (3% BSA / TBST). The plate was incubated at 37°C for 2 hours and washed twice with 350 μL / well of TBST. A series of concentrations of standards and samples were added to each well and the plate was incubated at room temperature for 2 hours. The solution was removed and the wells were washed twice with 350 μL / well of TBST. Goat anti-human kappa light chain (HRP) (abcam, ab202549) was diluted with blocking buffer and added at 100 μL per well. The plate was incubated at room temperature for 1 hour. The plate was then washed four times with 350 μL / well of TBST. 100 μL of TMB (solution A: solution B, 1:1) solution was added to each well and the plate was placed in the dark for 3 - 10 minutes. 50 μL of stop solution (2M H2SO4) was added and the optical density at 450 nm and 630 nm was read. The data was analyzed with GraphPad Prism5 software.

[0338] The results are shown in Figure 19. FR107-ADC had higher serum clearance than F8-ADC and F131-ADC.

[0339] 8.2 Safety effects on mice. The mice used in the safety test were as described above. Five groups, each containing 6 mice, were treated intravenously (IV) with a single dose of 30 mg / kg of F8, F26, F131, and FR107 immunoconjugates. The animals were checked daily for food intake, water consumption and activity, weight gain / loss (weight was measured once every two days), eye / hair entanglement and other abnormal effects, and deaths and observed clinical signs were recorded.

[0340] The weight results are as shown in Figure 20. The data showed no significant increase or decrease in weight in the treated mice.

[0341] Example 9: Affinity Data of F131 for FOLR Family Proteins Tested by BLI A recombinant protein consisting of the extracellular domain of the FOLR family protein linked to a His tag was purchased (from ACRO systems) or synthesized in-house. For the binding assay via biolayer interferometry (BLI), F131 (16.67 nM) was immobilized on an anti-human IgG Fc biosensor chip (Fortebio). A binding assay using recombinant antigen proteins at various concentrations (from 500 nM to 7.8 nM) in solution was performed using Octet RED (Fortebio). The association time was set to 180 seconds and the dissociation time was set to 300 seconds. The binding affinity was calculated using ForteBio Data Acquisition 6.3 software (Fortebio), and the affinity was derived by fitting the kinetic data to a 1:1 Langmuir binding model using a global fitting algorithm. F131 showed high affinity for human FOLR1, had a low response to human FOLR2, and had no response to human FOLR3, demonstrating the binding specificity of F131 (Table 13). F131 showed high binding affinity for human and cynomolgus monkey FOLR1, and the equilibrium dissociation constants (KD) were 1.5 nM and 8.1 nM, respectively. F131 showed no cross-reactivity to rat FOLR1 and low cross-reactivity to mouse FOLR1 (KD = 2.9 μM).

[0342]

Table 13A

Table 13B

[0343]

Table 14A

Table 14B

[0344] Example 10: F131 Binding Assay Data The binding activity of F131 was evaluated by flow cytometry (Beckman, Cytoflex) using a cell line with high FOLR1 target expression (JEG-3) or a cell line without FOLR1 target expression (PC-3). 3×10 5 cells per well were seeded in a 96-well plate and incubated with 100 μl of F131 at serial dilutions. After incubation at 4°C for 30 minutes, the cells were washed twice with PBS and stained with a 1:200 dilution of PE-conjugated anti-human Fc in 100 μl of FACS buffer (1×PBS containing 1% BSA), and then incubated at 4°C for 30 minutes. The cells were then washed twice with PBS and analyzed by flow cytometry. F131 showed strong binding to the human FOLR1-positive cell line, JEG-3 (Figure 21), and no binding to the human FOLR1-negative cell line, PC-3 (Figure 22).

[0345] Example 11: F131 Internalization in Tumor Cell Lines The internalization assay was performed over time. 3×10 5 cells were incubated with 10 μg / ml of F131 in FACS buffer (1×PBS containing 0.1% BSA) at 4°C for 30 minutes. The cells were washed at 4°C to remove unbound material and kept on ice or transferred to 37°C as needed. At the indicated time points (0 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours), the cells were stained with PE-conjugated anti-human Fc at 4°C for 30 minutes and analyzed by flow cytometry. The internalization rate was calculated by subtracting the mean fluorescence intensity (MFI) of cell surface-bound antibody at 37°C at each time point from the MFI of cell surface-bound antibody at 4°C at time 0, and then dividing by the MFI of cell surface-bound antibody at 4°C at time 0. F131 showed rapid internalization in FOLR1-expressing cell lines (OVCAR-3, KB, JEG-3, NCI-H441, OV90), but no internalization in FOLR1-non-expressing cells (PC-3) (Figure 23).

[0346] Example 12: In Vivo Efficacy of the F131 Conjugate In a cell line-derived xenograft (CDX) model, the antitumor activity of F131 in conjugates with various benchmarking linker drugs (Table 15) was evaluated. To prepare F131-sorafenatin: A solution of sulfo-SPDB-DM4 (10 mg / mL in DMSO) was added to 2 mL of the antibody solution (10 mg / mL in 50 mM phosphate buffer containing 5 mM EDTA, pH 7.4), and the molar ratio of sulfo-SPDB-DM4 to mAb was set to 6.0. The reaction was carried out at 25 °C for 6 hours. Excess sulfo-SPDB-DM4 and its impurities were removed by ultrafiltration using 50 mM sodium phosphate buffer. The ADC was stored in 20 mM histidine buffer containing 6% sucrose and 0.02% (w / v) Tween 20 by UFDF. The purity by SEC-HPLC was 97.9%, and the DAR value was 3.5 based on LC-MS. To prepare F131-dextecan, 2 mL of the antibody (10 mg / mL) in 50 mM sodium phosphate buffer containing 5 mM EDTA (pH = 6.9) was added to 10 mM TCEP HCl aqueous solution (tris(2-carboxyethyl)phosphine HCl), and the molar ratio of TCEP to mAb was set to 8.0. The reduction reaction was carried out at 25 °C for 2 hours. Dextecan was dissolved in DMSO at a concentration of 20 mg / mL and added to the reduced antibody at a molar ratio of 12 (dextecan / mAb). The coupling reaction mixture was stirred at 25 °C for 8 hours. Excess dexteran and its impurities were removed by ultrafiltration using 50 mM sodium phosphate buffer. The ADC was stored in 20 mM histidine buffer containing 6% sucrose and 0.02% (w / v) Tween 20 by UFDF. The purity by SEC-HPLC was 97.5%, and the DAR value was 7.7 based on LC-MS. To prepare F131-vedotin, 2 mL of the antibody (10 mg / mL) in 50 mM sodium phosphate buffer containing 5 mM EDTA (pH = 6.9) was added to 10 mM TCEP HCl aqueous solution (tris(2-carboxyethyl)phosphine HCl), and the molar ratio of TCEP to mAb was set to 2.2. The reduction reaction was carried out at 25 °C for 2 hours. Vedotin was dissolved in DMSO at a concentration of 20 mg / mL and added to the reduced antibody at a molar ratio of 5.0 (vedotin / mAb).The coupling reaction product was stirred at 25 °C for 2 hours. Excess vedotin and its impurities were removed by ultrafiltration using 50 mM sodium phosphate buffer. The ADC was stored in 20 mM histidine buffer containing 6% sucrose and 0.02% (w / v) Tween 20 by UFDF. The purity by SEC-HPLC was 97.5% and the DAR value was 3.9 based on HIC-HPLC. For the characterization of the target (FOLR1) copy number (binding sites) per cell (Table 15): The assay was performed according to the instructions of the QIFIKIT (DAKO, K0078) assay kit. Briefly, cells were labeled with a primary mouse monoclonal antibody against human FOLR1. Subsequently, the cells, setup beads and calibration beads were labeled in parallel with a fluorescein-conjugated anti-mouse secondary antibody. The samples were analyzed by flow cytometry and the copy number was calculated based on the calibration curve. For the CDX test using the F131 conjugate:...

Claims

**Claim 1**: An antibody or antigen-binding portion thereof that binds to folate receptor alpha (FOLR1) and comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region are each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 15 and SEQ ID NO: 16; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 17 and SEQ ID NO: 18; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20; and each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22; and the antibody or antigen-binding portion thereof has an amino acid sequence selected from the pair of amino acid sequence sets shown in the group consisting of the antibody or antigen-binding portion thereof. **Claim 2** the VH region and the VL region are each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 15 and SEQ ID NO: 16; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 17 and SEQ ID NO: 18; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20; and each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22 and the antibody or antigen-binding portion thereof according to claim 1 has an amino acid sequence selected from the pair of amino acid sequence sets shown in the group consisting of **Claim 3** the VH region and the VL region are each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4; each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8; and each, amino acid sequences selected from the pair of amino acid sequence sets shown in the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22 and the antibody or antigen-binding portion thereof according to claim 1 has an amino acid sequence selected from the pair of amino acid sequence sets shown in the group consisting of **Claim 4**: An antibody or antigen-binding portion thereof that binds to FOLR1 and comprises a heavy chain variable (VH) region containing the amino acid sequence shown in SEQ ID NO: 21 and a light chain variable (VL) region containing the amino acid sequence shown in SEQ ID NO:

22. **Claim 5**: The antibody according to claim 1, further comprising a heavy chain constant region, wherein the heavy chain constant region is of the IgG isotype. **Claim 6**: The antibody according to claim 4, further comprising a heavy chain constant region, wherein the heavy chain constant region is of the IgG isotype. **Claim 7**: The antibody according to claim 1, further comprising a light chain constant region, wherein the light chain constant region is of the kappa isotype. **Claim 8**: The antibody according to claim 1, further comprising a light chain constant region, ​ ​ ​ The constant region of the light chain is of the kappa isotype, The antibody according to claim 4.

9. Further comprising a constant region of the light chain, The constant region of the light chain is of the kappa isotype, The antibody according to claim 6.

10. The antibody according to claim 5, wherein the constant region of the heavy chain comprises an amino acid modification that at least reduces the binding affinity for human Fc gamma RIII.

11. The antibody according to claim 6, wherein the constant region of the heavy chain comprises an amino acid modification that at least reduces the binding affinity for human Fc gamma RIII.

12. Further comprising a constant region of the light chain, The constant region of the light chain is of the kappa isotype, The antibody according to claim 11.

13. The antibody according to claim 1 or an antigen-binding portion thereof; At least one linker bound to the antibody or an antigen-binding portion thereof; and At least one drug bound to each linker A conjugate comprising.

14. The antibody according to claim 2 or an antigen-binding portion thereof; At least one linker bound to the antibody or an antigen-binding portion thereof; and At least one drug bound to each linker A conjugate comprising.

15. The antibody according to claim 3 or an antigen-binding portion thereof; At least one linker bound to the antibody or an antigen-binding portion thereof; and At least one drug bound to each linker A conjugate comprising.

16. The antibody according to claim 4 or an antigen-binding portion thereof; At least one linker bound to the antibody or an antigen-binding portion thereof; and At least one drug bound to each linker A conjugate comprising.

17. An antibody or an antigen-binding portion thereof that binds to FOLR1, comprising a heavy chain variable (VH) region containing the amino acid sequence shown in SEQ ID NO: 21 and a light chain variable (VL) region containing the amino acid sequence shown in SEQ ID NO: 22; At least one linker bound to the antibody or an antigen-binding portion thereof; and At least one drug bound to each linker A conjugate comprising.

18. The conjugate according to claim 13, wherein the average drug load of the conjugate is 1 to 8.

19. The conjugate according to claim 14, wherein the average drug load of the conjugate is 1 to 8.

20. The conjugate according to claim 15, wherein the average drug load of the conjugate is 1 to 8.

21. The conjugate according to claim 16, wherein the average drug loading of the conjugate is from 1 to 8.

22. The conjugate according to claim 17, wherein the average drug loading of the conjugate is from 1 to 8.

23. The conjugate according to claim 13, wherein the drug is a cytotoxic agent.

24. The conjugate according to claim 14, wherein the drug is a cytotoxic agent.

25. The conjugate according to claim 15, wherein the drug is a cytotoxic agent.

26. The conjugate according to claim 16, wherein the drug is a cytotoxic agent.

27. The conjugate according to claim 17, wherein the drug is a cytotoxic agent.

28. The conjugate according to claim 21, wherein the drug is a cytotoxic agent.

29. The conjugate according to claim 23, wherein the cytotoxic agent is selected from the group consisting of auristatin, maytansinoid, camptothecin, duocarmycin or calicheamicin.

30. The conjugate according to claim 24, wherein the cytotoxic agent is selected from the group consisting of auristatin, maytansinoid, camptothecin, duocarmycin or calicheamicin.

31. The conjugate according to claim 25, wherein the cytotoxic agent is selected from the group consisting of auristatin, maytansinoid, camptothecin, duocarmycin or calicheamicin.

32. The conjugate according to claim 26, wherein the cytotoxic agent is selected from the group consisting of auristatin, maytansinoid, camptothecin, duocarmycin or calicheamicin.

33. The conjugate according to claim 27, wherein the cytotoxic agent is selected from the group consisting of auristatin, maytansinoid, camptothecin, duocarmycin or calicheamicin.

34. The conjugate according to claim 28, wherein the cytotoxic agent is selected from the group consisting of auristatin, maytansinoid, camptothecin, duocarmycin or calicheamicin.

35. The conjugate according to claim 23, wherein the cytotoxic agent is exatecan.

36. The conjugate according to claim 24, wherein the cytotoxic agent is exatecan.

37. The conjugate according to claim 25, wherein the cytotoxic agent is exatecan.

38. The conjugate according to claim 26, wherein the cytotoxic agent is exatecan.

39. The conjugate according to claim 27, wherein the cytotoxic agent is exatecan.

40. The conjugate according to claim 28, wherein the cytotoxic agent is exatecan.

41. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 12 or an antigen-binding portion thereof and a pharmaceutically acceptable carrier.

42. A pharmaceutical composition comprising the conjugate according to any one of claims 13 to 40 and a pharmaceutically acceptable carrier.

43. The pharmaceutical composition according to claim 42 for treating FOLR1+ cancer.

44. The pharmaceutical composition according to claim 43, wherein the FOLR1+ cancer is selected from lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, pancreatic cancer, and renal cell cancer.

45. The pharmaceutical composition according to claim 43, wherein the FOLR1+ cancer is ovarian cancer.

46. A nucleic acid encoding the antibody according to any one of claims 1 to 12 or an antigen-binding portion thereof.

47. A cell line comprising the nucleic acid according to claim 46.

48. A vector comprising the nucleic acid according to claim 46.

49. A cell line comprising the vector according to claim 48.

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