FOLR1 binding agents, conjugates thereof and methods of using the same

Abstract

To provide FOLR1 antibodies, antigen-binding portions thereof, other binding agents and FOLR1 conjugates thereof for use in treatment of cancer.SOLUTION: Provided is a binding agent that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the VH region comprising complementarity determining regions HCDR1, HCDR2, and HCDR3 arranged in a heavy chain variable region framework region, the VL region comprising LCDR1, LCDR, and LCDR3 arranged in a light chain variable region framework region, and the VH and VL CDRs having amino acid sequences selected from a set of amino acid sequences shown in the group consisting of specific sequences.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Sequence Listing Description The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the Sequence Listing is named 760270_404WO_SEQUENCE_LISTING.txt. The text file is 35.8 KB, was created on April 5, 2022, and has been submitted electronically via EFS-Web. [Background technology]

[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 5-methyltetrahydrofolate, the physiological folate, into the interior of cells. FOLR1 is overexpressed in the majority of ovarian cancers, as well as many uterine, endometrial, pancreatic, renal, lung, and breast cancers, whereas FOLR1 expression in normal tissues is restricted to the apical membrane of renal proximal tubules, lung alveolar pneumocytes, bladder, testis, choroid plexus, and thyroid epithelial cells (Weitman SD et al., Cancer Res 52:3396-3401 (1992); Antony AC, Annu Rev Nutr 16:501-521 (1996); Kalli KR 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 with FOLR1 antibodies and FOLR1 antibody-drug conjugates have met with limited success. The present invention addresses this and other needs. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Weitman SD et al., Cancer Res 52:3396-3401 (1992) [Non-patent document 2] Antony AC, Annu Rev Nutr 16:501~521(1996) [Non-patent document 3] Kalli KR et al. Gynecol Oncol 108:619~626 (2008) Summary of the Invention [Means for solving the problem]

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

[0006] In some embodiments, a binding agent is provided 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 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, and the VH and VL CDRs have amino acid sequences selected from the set of amino acid sequences set forth in the group 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, respectively; 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 regions are human framework regions.

[0007] In some embodiments, the VH and VL regions have amino acid sequences selected from pairs of amino acid sequences set forth in the group consisting of 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, respectively; and SEQ ID NO:23 and SEQ ID NO:24, respectively, wherein the heavy chain framework regions and light chain framework regions are optionally modified with substitutions, deletions, or insertions of 1 to 8 amino acids within the framework regions.

[0008] In some embodiments, the VH region and VL region have amino acid sequences selected from pairs of amino acid sequences set forth in the group consisting of 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, respectively; and SEQ ID NO:23 and SEQ ID NO:24, respectively.

[0009] In some embodiments, the VH and VL regions have amino acid sequences selected from pairs of amino acid sequences set forth in the group consisting of 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, respectively; and SEQ ID NO:21 and SEQ ID NO:22, respectively.

[0010] In some embodiments, the VH and VL regions have amino acid sequences selected from pairs of amino acid sequences set forth in the group consisting of SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; and SEQ ID NO:21 and SEQ ID NO:22, respectively. In some embodiments, the VH and VL regions have amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively. In some embodiments, the VH and VL regions have amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively. In some embodiments, the VH and VL regions have amino acid sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively.

[0011] In some embodiments, the binding agent is an antibody or antigen-binding portion thereof. In some embodiments, the binding agent is a monoclonal antibody, Fab, Fab', F(ab'), 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 an 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 an IgG4 constant region. In some embodiments, the heavy chain constant region further comprises an amino acid modification that reduces binding affinity to at least human Fc gamma RIII. 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, pharmaceutical compositions are provided comprising any of the binding agents described herein and a pharmaceutically acceptable carrier. In some embodiments, nucleic acids encoding any of the binding agents described herein are provided. In some embodiments, vectors are provided comprising any of the nucleic acids encoding any of the binding agents described herein. In some embodiments, cell lines are provided 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 comprising any of the binding agents described herein, at least one linker attached to the binding agent, and at least one drug attached to each linker. In some embodiments, each drug is selected from a cytotoxic agent, an immunomodulator, a nucleic acid, a growth inhibitory agent, a PROTAC, a toxin, and a radioisotope. In some embodiments, each linker is attached to the binding agent via an interchain disulfide residue, a lysine residue, an engineered cysteine ​​residue, a glycan, a modified glycan, the N-terminal residue of the binding agent, or a polyhistidine peptide attached to the binding agent. In some embodiments, 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.

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

[0016] In some embodiments, the linker comprises mc-VC-PAB, CL2, CL2A, or (succinimide-3-yl-N)-(CH2)nC(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)- (wherein n=1-5). 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 (succinimide-3-yl-N)-(CH2)nC(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)-. The conjugate of claim 43, wherein the linker is attached 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 TRL7 agonist, TLR8 agonist, STING agonist or RIG-I agonist.In some embodiments, the immunomodulator is a TLR7 agonist.In some embodiments, the TLR7 agonist is imidazoquinoline, imidazoquinoline amine, thiazoquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazol-2-amine, tetrahydropyridopyrimidine, heteroaromatic azido-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-benzimidazol-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 (succinimide-3-yl-N)-(CH)C(=O)-Gly-Gly-Phe-Gly-NH-CH-O-CH-(C=O)- (wherein n=1 to 5).

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

[0019] In some embodiments, a method of treating a FOLR1+ cancer is provided, comprising administering a therapeutically effective amount of any of the binding agents described herein, any of the conjugates described herein, or any of the pharmaceutical compositions of the binding agents or conjugates described herein to a subject in need thereof. 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 carcinoma. 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 an antibody that specifically binds 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 includes administering to a subject any of the conjugates described herein or any of the pharmaceutical compositions described herein. In some embodiments, the binding agent, conjugate, or pharmaceutical composition is administered intravenously. In some embodiments, the binding agent, 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 subject's outcome is improved. In some embodiments, the improved outcome is an objective response selected from stable disease, partial response, or complete response. In some embodiments, the improved outcome is a reduction in tumor burden. In some embodiments, the improved outcome is progression-free survival or disease-free survival.

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

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

[0025] [Figure 1] FIG. 1 shows a comparison of anti-FOLR1 antibody binding to Hela cells. [Figure 2] FIG. 1 shows a comparison of the binding ability of anti-FOLR1 antibodies to RPTEC / TERT1 cells. [Figure 3] FIG. 1 shows dose-dependent binding of anti-FOLR1 antibodies to Hela cells. [Figure 4] FIG. 1 shows dose-dependent binding of anti-FOLR1 antibodies to RPTEC / TERT1 cells. [Figure 5] FIG. 1 shows the internalization of anti-FOLR1 antibodies into Hela cells. [Figure 6] FIG. 1 shows the internalization of anti-FOLR1 antibodies into RPTEC / TERT1 cells. [Figure 7] FIG. 1 shows a comparison of anti-FOLR-1 conjugates binding to the target FOLR1 protein. [Figure 8] FIG. 1 shows a comparison of anti-FOLR-1 conjugates binding to the target FLOR1 protein. [Figure 9] FIG. 1 shows a comparison of anti-FOLR-1 conjugate binding to Hela cells. [Figure 10] FIG. 1 shows a comparison of an-huFOLR-1 conjugate binding to IGROV-1, OVCAR3, and OV90 cells. [Figure 11] FIG. 1 shows a comparison of internalization of anti-FOLR-1 conjugates into Hela cells. [Figure 12] FIG. 1 shows a comparison of internalization of anti-FOLR-1 conjugates into OVCAR-3 cells. [Figure 13] FIG. 1 shows a comparison of internalization of anti-FOLR-1 conjugates into OV90 cells. [Figure 14] FIG. 1 shows a comparison of internalization of anti-FOLR-1 conjugates into IGROV-1 cells. [Figure 15] FIG. 1 shows a comparison of the cytotoxicity of anti-huFOLR-1 conjugates against Hela cells. [Figure 16] FIG. 1 shows a comparison of the cytotoxicity of anti-huFOLR-1 conjugates against OV90 cells. [Figure 17] FIG. 1 shows a comparison of the cytotoxicity of anti-huFOLR-1 conjugates against OVCAR-3 cells. [Figure 18] FIG. 1 shows a comparison of the cytotoxicity of anti-huFOLR-1 conjugates against IGROV-1 cells. [Figure 19] FIG. 1 shows the pharmacokinetics of anti-FOLR-1 conjugates. [Figure 20] FIG. 1 shows the effect of anti-FOLR-1 conjugates on body weight. [Figure 21] FIG. 1 shows F131 binding assay to JEG-3 by FACS. [Figure 22] FIG. 1 shows F131 binding assay to PC-3 by FACS. [Figure 23] FIG. 1 shows F131 internalization in tumor cell lines. [Figure 24] FIG. 1 shows the in vivo efficacy of F131 conjugates in CDX against OVCAR-3. [Figure 25] FIG. 1 shows the in vivo efficacy of F131 conjugate in CDX against HCC827. [Figure 26]FIG. 1 shows the in vivo efficacy of F131 conjugates in CDX versus H441. [Figure 27] FIG. 1 shows the in vivo efficacy of F131 conjugates in CDX against OVCAR-3. [Figure 28] FIG. 1 shows the in vivo efficacy of F131 conjugates in CDX against KB. [Figure 29] FIG. 1 shows the in vivo efficacy of F131 conjugate in CDX against HCC827. [Figure 30] FIG. 1 shows the in vivo efficacy of F131 conjugates in CDX versus H441. [Figure 31] FIG. 1 shows the in vivo efficacy of F131 conjugates in CDX against OV90. [Figure 32] FIG. 1 shows the in vivo efficacy of F131 conjugates in CDX against OVCAR-3. [Figure 33] FIG. 1 shows the in vivo efficacy of F131 conjugates in CDX against KB. [Figure 34] FIG. 1 shows PK studies of F131 and conjugates in a rat model. [Figure 35] FIG. 1 shows PK studies of F131 and conjugates in a rat model. [Figure 36] FIG. 1 shows the tolerability of F131-deruxtecan in a pilot cynomolgus monkey toxicity study. [Figure 37] FIG. 1 shows the tolerability of F131-deruxtecan in a pilot cynomolgus monkey toxicity study. [Figure 38] FIG. 1 shows F131-deruxtecan PK in a pilot cynomolgus monkey toxicity study. DETAILED DESCRIPTION OF THE INVENTION

[0026] definition For convenience, certain terms in the specification, examples, and claims are defined here. Unless otherwise specified or implied from context, the following terms and phrases have the meanings provided below. The definitions are provided to aid in the description of particular 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" shall be construed to mean "one," "at least one," or "one or more." Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0028] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprise," "comprising," and the like are to be construed in an inclusive sense, rather than an exclusive or exhaustive sense; that is, in the sense of "including, but not limited to."

[0029] The terms "reduced," "lowering," "reduced," "reduction," "reduction," and "inhibit" are all used generally herein to mean a decrease by a statistically significant amount compared to a reference.

[0030] The terms "increased," "increase," or "enhance," or "activate" are all used herein to generally mean an increase by a statically significant amount compared to a reference.

[0031] As used herein, the terms "protein" and "polypeptide" are used interchangeably 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., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, the use of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein to refer 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 to 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 conventionally bound by an immunoglobulin VH / VL pair, such as the antibodies, antigen-binding portions thereof, and other binding agents described herein. Epitopes can be formed on polypeptides of contiguous or noncontiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least three, more usually at least five, about nine, or about eight to ten amino acids in a unique spatial arrangement. An epitope defines the minimal binding site of an antibody, its antigen-binding portion, and other binding agent and thus represents the target of specificity of the antibody, its antigen-binding portion, or other immunoglobulin-based binding agent. In the case of single-domain antibodies, an epitope represents the structural unit bound by the variable domain alone.

[0034] As used herein, "specifically binds" means that a binding agent (e.g., an antibody or antigen-binding portion thereof) described herein specifically binds to an antigen of interest. -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 -12Specific binding refers to the ability to bind to a target, such as human FOLR1, with a KD of M or less. Specific binding can be influenced, for example, by the affinity and avidity of the antibody, antigen-binding portion, or other binding agent and the concentration of the target polypeptide. Those skilled in the art can determine appropriate conditions under which the antibodies, antigen-binding portions, and other binding agents described herein selectively bind to FOLR1 using any appropriate method, such as titrating the binding agent in an appropriate cell binding assay. A binding agent that specifically binds to FOLR1 is not displaced by a non-similar competitor. In certain embodiments, a 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 antibody or antigen-binding portion thereof or other binding agent described herein is administered intravenously. -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 -12 M or less dissociation constant (KD or K D In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a FOLR1 polypeptide at about 10 -5 M~10 -6 In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a FOLR1 polypeptide with a dissociation constant (KD) of about 10 M. -6 M~10 -7 In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a FOLR1 polypeptide with a dissociation constant (KD) of about 10 M. -7 M~10 -8In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a FOLR1 polypeptide with a dissociation constant (KD) of about 10 M. -8 M~10 -9 In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a FOLR1 polypeptide with a dissociation constant (KD) of about 10 M. -9 M~10 -10 In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a FOLR1 polypeptide with a dissociation constant (KD) of about 10 M. -10 M~10 -11 In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a FOLR1 polypeptide with a dissociation constant (KD) of about 10 M. -11 M~10 -12 In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a FOLR1 polypeptide with a dissociation constant (KD) of 10 M. -12 It specifically binds to the FOLR1 polypeptide with a dissociation constant (KD) of less than M.

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

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

[0038] Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean + / - 1%.

[0039] The term "statistically significant" or "significantly" refers to statistical significance, generally meaning a difference of 2 standard deviations (2SD) above or below a reference value.

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

[0041] Provided herein are FOLR1-binding antibodies (also referred to as FOLR1 antibodies) and antigen-binding portions thereof, as well as other binding agents, that specifically bind to human FOLR1. Also provided herein are conjugates of FOLR1 antibodies and antigen-binding portions and other binding agents (also referred to as FOLR1 conjugates) linked to drugs, such as cytotoxic agents or immunomodulatory agents. 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, wherein the VH and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NOs: 1 and 2, respectively; SEQ ID NOs: 3 and 4, respectively; SEQ ID NOs: 5 and 6, respectively; SEQ ID NOs: 7 and 8, respectively; SEQ ID NOs: 9 and 10, respectively; SEQ ID NOs: 11 and 12, respectively; SEQ ID NOs: 13 and 14, respectively; SEQ ID NOs: 15 and 16, respectively; SEQ ID NOs: 17 and 18, respectively; SEQ ID NOs: 19 and 20, respectively; SEQ ID NOs: 21 and 22, respectively; and SEQ ID NOs: 23 and 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, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NOs: 1 and 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, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NOs: 3 and 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, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NOs: 5 and 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, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NOs: 7 and 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, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NOs: 9 and 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, wherein the VH region and VL region have the amino acid sequences set forth in 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, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NOs: 13 and 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, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NOs: 15 and 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, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NOs: 17 and 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, wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NOs: 19 and 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, wherein the VH and VL regions 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, wherein the VH and VL regions 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, wherein the VH region and VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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, respectively; 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. and SEQ ID NO:23 and SEQ ID NO:24, respectively; wherein the heavy chain variable framework region and the light chain variable framework region are optionally modified with substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.The phrase "unmodified CDRs of the heavy or light chain variable region" 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 the 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 with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, 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 the 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 with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, 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 the amino acid sequences set forth in a pair of amino acid sequences selected from 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 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, 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 the amino acid sequences set forth in a pair of amino acid sequences selected from 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 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[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 the amino acid sequences set forth in a pair of amino acid sequences selected from 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 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, 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 the amino acid sequences set forth in a pair of amino acid sequences selected from 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 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[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, wherein the VH region and the VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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 regions, 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 the amino acid sequences set forth in a pair of amino acid sequences selected from 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[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 have the amino acid sequences set forth in a pair of amino acid sequences selected from 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 regions, 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 the amino acid sequences set forth in a pair of amino acid sequences selected from 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[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 VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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 regions, 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 VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[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 VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, 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 VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[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 VL region have the amino acid sequences set forth 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 with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, 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 VL region have the amino acid sequences set forth 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 with substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[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 VL region have the amino acid sequences set forth 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 with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, 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 VL region have the amino acid sequences set forth 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 with substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[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 VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, 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 VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[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 VL region have the amino acid sequences set forth in 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 with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, 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 VL region have the amino acid sequences set forth in 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 with substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[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 VL region have the amino acid sequences set forth in 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 with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, 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 VL region have the amino acid sequences set forth in 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 with substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0056] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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, respectively; and SEQ ID NO:23 and SEQ ID NO:24, respectively. 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 VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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, respectively; and SEQ ID NO:23 and SEQ ID NO:24, respectively; and the binding agent specifically binds to FOLR1 with a higher binding affinity (lower Kd) than 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 VL region have the amino acid sequence set forth in a pair of amino acid sequences selected from 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, respectively; and SEQ ID NO:23 and SEQ ID NO:24, respectively; and the heavy chain variable framework region and 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 regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified. and SEQ ID NO:23 and SEQ ID NO:24, respectively; wherein the heavy chain variable framework region and the light chain variable framework region are optionally modified with substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified.As described herein, the binding agent comprises a FOLR1 antibody or antigen-binding portion thereof, and can include other peptides or polypeptides covalently bound to the FOLR1 antibody or antigen-binding portion thereof. In any of these embodiments, the binding agent specifically binds to FOLR1.

[0057] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. 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 VL region have the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; and the binding agent specifically binds to FOLR1 with a higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0058] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. 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 VL region have the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; and the binding agent specifically binds to FOLR1 with a higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0059] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. 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 VL region have the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the binding agent specifically binds to FOLR1 with a higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0060] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. 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 VL region have the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the binding agent specifically binds to FOLR1 with a higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0061] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. 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 VL region have the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; the binding agent specifically binds to FOLR1 with a higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0062] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. 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 VL region have the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and the binding agent specifically binds to FOLR1 with a higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0063] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein 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 binding agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0064] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein 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 binding agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; and the binding agent specifically binds to FOLR1 with a higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0065] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein 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 binding agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively; and the binding agent specifically binds to FOLR1 with a higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0066] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein 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 binding agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively; and the binding agent specifically binds to FOLR1 with a higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0067] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein 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 binding agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; the binding agent specifically binds to FOLR1 with a higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0068] In some embodiments, provided herein is a binding agent that specifically binds to FOLR1, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and VL region have the amino acid sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. 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 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 higher binding affinity (lower Kd) than antibody FR107. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0069] In some embodiments, an antibody or antigen-binding portion is provided 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 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, and the VH and VL CDRs have the amino acid sequences set forth in a set 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, respectively; 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. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0070] In some embodiments, an antibody or antigen-binding portion is provided 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 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, 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 and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0071] In some embodiments, an antibody or antigen-binding portion is provided 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 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, 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 and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0072] In some embodiments, a binding agent is provided 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 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, and the VH and VL CDRs have the amino acid sequences set forth in a set 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, respectively; 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. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0073] In some embodiments, a binding agent is provided 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 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, 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 and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0074] In some embodiments, a binding agent is provided 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 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, 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 and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

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

[0076] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds to an antigen, e.g., human FOLR1. The term generally refers to antibodies that are composed of two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions, including full-length antibodies (having heavy 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 a CL domain. The VH and VL regions are further divided into hypervariable regions called complementarity-determining regions (CDRs), and may be interspersed with conserved regions called framework regions (FRs). Thus, each VH and VL region consists of three CDRs and four FRs arranged in the following order from N-terminus to 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 a portion of the FOLR1 antibody described herein that has the VH and VL sequences or CDRs of the FOLR1 antibody and specifically binds to FOLR1. Examples of antigen-binding portions include Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, single-domain antibodies (also called VHHs, VNARs, sdAbs, or nanobodies), or diabodies (see, e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 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 composed of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked together within the hinge region via a disulfide bridge; and (iii) an Fv fragment, in each case composed of the VL and VH domains of a FOLR1 antibody. The two domains of the Fv fragment, i.e., the VL and VH, are encoded by separate coding regions, but can be further linked to each other using a synthetic linker, such as a poly-G4S amino acid sequence ("(G4S)n" disclosed as SEQ ID NO: 38) (where n = 1 to 5), allowing the preparation of a single protein chain in which the VL and VH domains are linked 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.Diabodies are bivalent, bispecific antibodies in which a VH domain and a VL domain are expressed on a single polypeptide chain, but a linker connecting the VH domain and the VL domain is used that is too short to allow the two domains to bind on the same chain, thereby allowing the VH domain and the VL domain to pair with complementary domains (VL and VH, respectively) on different chains to form two antigen-binding sites (e.g., Holliger, R et al. (1993) Proc. Natl. Acad. Sci. USA 90:64446448; Poljak, RJ 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 an antibody heavy chain derived from a camelid (e.g., a nanobody or VHH moiety). Additionally, 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., DABs or VHHs) are known in the art, as disclosed, for example, in 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 novel epitopes inaccessible to conventional VH-VL pairs (see, e.g., Muyldermans et al., 2001). Alpaca serum IgG contains approximately 50% camelid heavy chain-only IgG antibodies (HCAbs) (see, e.g., Maass et al., 2007). Alpacas can be immunized with antigens, and VHHs that bind to and neutralize the target antigen can be isolated (see, e.g., Maass et al., 2007). PCR primers that amplify alpaca VHH coding sequences have been identified and can be used to construct alpaca VHH phage display libraries, which can be used to isolate antibody fragments by standard biopanning techniques well known in the art (see, e.g., 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, the 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] VH and VL domain modifications With respect to VH and VL amino acid sequences, one of skill in the art will recognize that individual substitutions, deletions, or additions (insertions) to the nucleic acid encoding VH or VL, or amino acids in a polypeptide, that alter a single amino acid or a small percentage of amino acids in the encoded sequence, are "conservatively modified variants," in which the alteration results in the substitution of an amino acid with a chemically similar amino acid (conservative amino acid substitution), and the altered polypeptide retains the ability to specifically bind to FOLR1.

[0083] In some embodiments, conservatively modified variants of a FOLR1 antibody, or antigen-binding portion thereof, can have modifications in the framework regions (FRs) (i.e., outside the CDRs). For example, a conservatively modified variant of a FOLR1 antibody has the amino acid sequences of the VH and VL CDRs (set forth in the set of amino acid sequences (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; 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) with at least one conservative amino acid substitution in the framework region. In some embodiments, the VH and VL amino acid sequences collectively have no more than 8, 6, 4, 2, or 1 conservative amino acid substitutions in the FRs 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 compared to the amino acid sequences of the unmodified VH and VL regions. In a further aspect of any of these embodiments, conservatively modified variants of FOLR1 antibodies, antigen-binding portions thereof, or other binding agents exhibit specific binding to FOLR1.

[0084] For conservative amino acid substitution, a given amino acid can be replaced with a residue having similar physicochemical properties, for example, an aliphatic residue can be replaced with another aliphatic residue (e.g., Ile, Val, Leu, or Ala for each other), or a polar residue can be replaced with another polar residue (e.g., Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative amino acid substitutions, such as the substitution of entire regions with similar hydrophobic properties, are well known. Polypeptides 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, for example, antigen binding activity and specificity, is maintained.

[0085] In some embodiments, a FOLR1 antibody or antigen-binding portion thereof or other binding agent can be further optimized to reduce potential immunogenicity or optimize other functional properties while maintaining functional activity, e.g., for treatment in humans. 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 and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from 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, respectively; 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 regions, 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 or other binding agent 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 a pair of amino acid sequences selected from 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, respectively; 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0086] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0087] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0088] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0089] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0090] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0091] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein are binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0092] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy chain variable region or light chain variable region are unmodified. In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0093] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or light chain variable region are unmodified.

[0094] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or light chain variable region are unmodified.

[0095] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0096] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or light chain variable region are unmodified.

[0097] In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified. In some embodiments, provided herein is a FOLR1 antibody, or antigen-binding portion thereof, or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and 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 substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0098] In any of these embodiments, the functional activity of a FOLR1-binding antibody or antigen-binding portion thereof or other binding agent includes specific binding to FOLR1. Additional functional activities include depletion of FOLR1+ cells (e.g., cancer cells). Furthermore, a FOLR1 antibody or antigen-binding portion thereof or other binding agent having functional activity refers to a polypeptide that exhibits an activity similar to or better than the activity of a reference antibody or antigen-binding portion thereof described herein (e.g., a reference FOLR1-binding antibody or antigen-binding portion thereof, or a variant thereof, described herein, comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 36 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 37), regardless of whether or not it is dose-dependent. If a dose-dependence exists, it need not be identical to the dose-dependence of the reference antibody or antigen-binding portion thereof, but rather is substantially similar to or better than the dose-dependence of 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] For conservative substitutions, amino acids can be grouped according to the similarity of their side chain properties (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, for conservative substitutions, naturally occurring residues can be grouped based on shared 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 that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging members of one of these classes for another.

[0101] Particular 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, conservatively modified variants of a FOLR1 antibody or antigen-binding portion thereof are 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 a reference VH or VL sequence, with the VH and VL CDRs unmodified. The degree of homology (percent identity) between a reference sequence and a modified sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly 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 no more than 8, 6, 4, 2, or 1 conservative amino acid substitutions in the framework regions 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 no more than 8, 6, 4, 1, or 2, or 1 conservative amino acid substitutions in the framework regions 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 no more than 8, 6, 4, 2, or 1 amino acid substitutions, deletions, or insertions in the framework regions compared to the amino acid sequences of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences have no more than 8, 6, 4, 1, or 2, or 1 conservative amino acid substitutions in the framework regions 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 no more than 8, 6, 4, 2, or 1 amino acid substitutions, deletions, or insertions compared to the amino acid sequences of the unmodified VH and VL regions.

[0104] Modification of a 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 oligonucleotides containing the desired mutated sequence flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes a variant with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide modified nucleotide sequences with specific codons altered according to the desired substitution, deletion, or insertion. Techniques for making 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. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entireties.

[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 human heavy chain constant regions and human light chain constant regions are known in the art. The constant region can be of any suitable type selected from the immunoglobulin classes, 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 comprising constant regions from more than one isotype. 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 set forth in SEQ ID NO: 39. In some embodiments, the FOLR1 antibody light chain is of the kappa isotype and has the amino acid sequence set forth in SEQ ID NO: 40.

[0107] Furthermore, a 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 binding of the antibody or antigen-binding portion to one or more other proteins or peptides. Related to such binding agents are, for example, the use of streptavidin core regions to prepare tetrameric scFv molecules (Kipriyanov, SM et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidinyl peptides, such as hexahistidinyl tags ("hexahistidinyl tag" disclosed as SEQ ID NO: 41), to generate bivalent and biotinylated scFv molecules (Kipriyanov, SM et al. (1994) Mol. Immunol. 31:1047-1058).

[0108] Fc domain modifications to alter effector function In some embodiments, the Fc region or Fc domain of a FOLR1 antibody or antigen-binding portion thereof or other binding agent 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 at least a 1000-fold decrease in binding affinity (e.g., an increase in Kd) for an Fc gamma receptor. 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 exhibits weak or no binding to any of the Fc gamma receptors. In some embodiments, the Fc null domain or region exhibits at least a 1000-fold reduction in binding affinity (i.e., an increased Kd) to the Fc gamma receptor.

[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 cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). In some embodiments, the Fc domain exhibits reduced ADCC, ADCP, or CDC activity compared to a wild-type Fc domain. In some embodiments, the Fc domain exhibits reduced ADCC, ADCP, and CDC compared to a wild-type Fc domain. In some embodiments, the Fc domain exhibits substantially no effector function (i.e., the ability to stimulate or effect ADCC, ADCP, or CDC). As used herein, "substantially no effector function" refers to at least a 1000-fold reduction in effector function activity compared to a 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 at least a 10-fold, at least a 20-fold, at least a 30-fold, at least a 50-fold, at least a 100-fold, or at least a 500-fold decrease in the ADCC activity of the Fc domain.

[0111] In some embodiments, the Fc domain has reduced or no CDC activity. As used herein, reduced or no CDC activity refers to at least a 10-fold, at least a 20-fold, at least a 30-fold, at least a 50-fold, at least a 100-fold, or at least a 500-fold decrease in the CDC activity of the Fc domain.

[0112] In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted ADCC and / or CDC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks Fc gamma receptor binding (and therefore likely lacks ADCC activity). NK cells, the primary cells for mediating ADCC, express only Fc gamma RIII, whereas 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 to assess ADCC activity of a molecule of interest are described in U.S. Pat. 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. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods 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)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).

[0113] C1q binding assays can also be performed to confirm that antibodies or Fc domains or regions cannot bind to C1q and therefore lack or have reduced CDC activity.See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402.To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)).

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

[0115] ADCP binding assays can also be performed to confirm that an antibody or Fc domain or region lacks or has reduced ADCP activity. See, e.g., U.S. Patent Application Publication Nos. 20190079077 and 20190048078 and references therein.

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

[0117] In some embodiments, the FOLR1 antibody or antigen-binding portion thereof or other binding agent comprises an Fc domain or region having one or more amino acid substitutions that reduce Fc gamma R binding, e.g., 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 EU numbering. In some embodiments, the Fc domain comprises D265A and / or P329G in the Fc region derived from a human IgG1 Fc region according to Kabat EU numbering. In some embodiments, the substitutions are L234A, L235A, and P329G in the Fc region derived from a human IgG1 Fc region according to Kabat EU numbering (LALA-PG) (see, e.g., WO 2012 / 130831). In some embodiments, the substitutions are L234A, L235A and D265A of the Fc region from a human IgG1 Fc region according to EU numbering of Kabat (LALA-DA).

[0118] In some embodiments, modifications to the Fc region are made that result in altered (i.e., decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, 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, their antigen-binding portions, 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, their antigen-binding portions, and other binding agents. This allows for the production of FOLR1 antibodies and a range of FOLR1 antigen-binding portions and other binding agents (including fusion proteins) in a selected host species. Production of FOLR1 antibodies, their antigen-binding portions, and other binding agents in bacteria, yeast, transgenic animals, and chicken eggs is also an alternative to cell-based production systems. The main advantage of transgenic animals is the potential high yield from renewable resources.

[0120] In some embodiments, a nucleic acid encodes a 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, a nucleic acid encodes a 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, a nucleic acid encodes a FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a nucleic acid encodes a FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, a nucleic acid encodes a FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, a nucleic acid encodes a FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, a nucleic acid encodes a FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, a nucleic acid encodes a FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the nucleic acid encodes a FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the nucleic acid encodes a FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the nucleic acid encodes a FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the nucleic acid encodes a FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the nucleic acid encodes a FOLR1 VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the nucleic acid encodes a 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 VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 1 and 2. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 3 and 4. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 5 and 6. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 7 and 8. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 9 and 10. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 11 and 12. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 13 and 14. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 15 and 16. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 17 and 18. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 19 and 20. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 21 and 22. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs: 23 and 24.

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

[0124] Nucleic acid molecules encoding the amino acid sequence of a FOLR1 antibody, its antigen-binding portion, or other binding agent can be prepared by various methods known in the art. These methods include, but are not limited to, the preparation of synthetic nucleotide sequences encoding a FOLR1 antibody, antigen-binding portion, or other binding agent. Furthermore, oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding a FOLR1 antibody or antigen-binding portion, or other binding agent. Nucleic acid sequences encoding at least the FOLR1 antibody, its antigen-binding portion, binding agent, or polypeptide 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 suitable sticky ends, filling in sticky ends as necessary, alkaline phosphatase treatment to avoid undesired ligation, and ligation with an appropriate ligase, 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] A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide when it contains a nucleotide sequence containing transcriptional and translational regulatory information, and such a sequence is "operably linked" to a nucleotide sequence encoding the polypeptide. An operable linkage is one in which the regulatory DNA sequence and the DNA sequence desired to be expressed (e.g., a FOLR1 antibody or antigen-binding portion thereof or other binding agent) are connected in a manner that allows for gene expression of recoverable amounts of the polypeptide or antigen-binding portion. The exact nature of the regulatory regions required for gene expression can vary from organism to organism, as is well known in the art. See, e.g., Sambrook et al., 1989; Ausubel et al., 1987-1993.

[0126] Therefore, the expression of the FOLR1 antibody or its antigen-binding portion described herein can be carried out in either prokaryotic or eukaryotic cells. Suitable hosts include yeast, insect, fungal, avian, and mammalian cells in vivo or in situ, or bacterial or eukaryotic hosts, including 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. Furthermore, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved, for example, by using the yeast ubiquitin hydrolase system. The fusion protein thus produced can be processed in vivo or purified and processed in vitro to allow the synthesis of the FOLR1 antibody or its antigen-binding portion or other binding agent described herein having a specified amino-terminal sequence. Furthermore, problems associated with retaining the methionine residue from the start codon 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 binding agents, can be produced using any of a range of yeast gene expression systems incorporating promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

[0127] Production of FOLR1 antibodies or antigen-binding portions thereof or other binding agents in insects can be accomplished, for example, by infecting an insect host with a baculovirus engineered to express the polypeptide 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 capable of autonomous replication in the recipient host cell. Any of a wide variety of vectors can be employed for this purpose, which are known and available to those skilled 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 that do not contain 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 host cells of different species.

[0129] Exemplary prokaryotic vectors known in the art include plasmids, such as those that can replicate in E. coli. Other gene expression elements useful for expressing DNA encoding a FOLR1 antibody or its antigen-binding portion 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)), Rous sarcoma virus LTR (Gorman et al., 79 PNAS 6777 (1982)), and 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 (Okayama 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 enhancer, the mouse immunoglobulin heavy chain promoter enhancer, the SV40 late region mRNA splicing sequence, the rabbit S globin intervening sequence, the immunoglobulin and rabbit S globin polyadenylation sites, and the SV40 polyadenylation element as expression elements, as described by Liu et al., infra, and Weidle et al., 51 Gene 21 (1987).

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

[0131] In some embodiments, for the expression of the DNA coding region in rodent cells, the transcription promoter can be a viral LTR sequence, and the transcription promoter enhancer can be either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, as well as polyadenylation and transcription termination regions. In other embodiments, DNA sequences encoding other proteins are 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.Then, recipient cells capable of expressing FOLR1 variable region or its antigen-binding portion or other binding agent are transfected with the nucleotide encoding FOLR1 antibody or antibody polypeptide or its antigen-binding portion or other binding agent alone, or co-transfected with the polynucleotide encoding VH and VL chain coding region or other binding agent.The transfected recipient cells are cultured under conditions that allow the expression of the incorporated coding region, and the expressed antibody chain or intact antibody or antigen-binding portion or other binding agent is recovered from the culture.

[0133] In some embodiments, nucleic acids containing coding regions encoding a FOLR1 antibody or antigen-binding portion thereof or other binding agent are assembled into separate expression vectors that are then used to cotransfect recipient host cells. Each vector can contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, with one selectable gene designed for selection in a bacterial system and a second selectable gene designed for selection in a eukaryotic system, with each vector carrying a set of coding regions. This strategy results in a vector that first directs the production of a nucleotide sequence in a bacterial system and allows for amplification. The DNA vector thus produced and amplified in the bacterial host is then used to cotransfect eukaryotic cells, allowing for the selection of cotransfected cells carrying the desired transfected nucleic acid (e.g., containing the heavy and light chains of a FOLR1 antibody). Non-limiting examples of selectable genes for use in bacterial systems are genes that confer resistance to ampicillin and genes that confer resistance to chloramphenicol. Selectable genes for use in eukaryotic transfectants include the xanthine guanine phosphoribosyltransferase gene (called gpt) and the phosphotransferase gene from Tn5 (called neo). Alternatively, fused 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 antigen-binding portion thereof 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 the transfected immunoglobulin genes and have mechanisms for immunoglobulin glycosylation. For example, in some embodiments, the recipient cells are recombinant Ig-producing myeloma cells SP2 / 0. SP2 / 0 cells produce only the immunoglobulins encoded by the transfected genes. Myeloma cells can be grown in culture or in the peritoneal cavity of mice, and secreted immunoglobulins can be obtained from the ascites fluid.

[0135] An expression vector encoding a FOLR1 antibody or antigen-binding portion thereof 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. (Johnston et al., 240 Science 1538 (1988), known to those skilled in the art.)

[0136] Yeast offers certain advantages over bacteria for the production of immunoglobulin heavy and light chains. Yeast performs post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies exist that utilize strong promoter sequences and high-copy-number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes leader sequences in cloned mammalian gene products and secretes polypeptides bearing leader sequences (i.e., prepolypeptides). See, e.g., Hitzman et al., 11th ed. Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpelier, 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 incorporating promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium are available. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, the promoter and terminator signals 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 the optimal expression plasmid for immunoglobulin expression in yeast. See, for example, II DNA Cloning 45 (Glover, ed., IRL Press, 1985) and 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 antigen-binding portions thereof and other binding agents described herein. E. coli K12 strains such as E. coli W3110, Bacillus species, enterobacteria such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species can be used. Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in conjunction with these bacterial hosts. The vectors contain replication sites and specific genes that can provide phenotypic selection in transformed cells. Several approaches can be used 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 and 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 antigen-binding portions thereof or other binding agents.

[0140] In addition to the cells of lymphoid origin described above, mammalian cells that can be useful as hosts for producing antibody proteins include cells of fibroblast origin, 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 make desired post-translational modifications to the heavy and / or light chains. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

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

[0142] In some embodiments, the antibody or antigen-binding portion thereof or other binding agent is produced in a cell-free system. Non-limiting exemplary 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] Many 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, VH and VL chains, and optionally associated constant regions, can be coexpressed in the same cell to achieve intracellular association and linkage of the VH and VL chains into a complete tetrameric H2L2 antibody or its antigen-binding portion. Coexpression can be achieved by using either the same or different plasmids in the same host. Nucleic acids encoding VH and VL chains or their antigen-binding portions or other binding agents can be placed on the same plasmid, which is then transfected into cells, thereby directly selecting for cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, e.g., the VL chain, and the resulting cell line subsequently transfected with a VH chain plasmid containing a second selection marker. Cell lines producing antibodies, antigen-binding portions thereof, via either route can be transfected with plasmids encoding additional copies of the peptide, VH, VL, or VH+VL chain, along with additional selectable markers, to generate cell lines with enhanced properties, such as higher production of assembled FOLR1 antibodies, or antigen-binding portions thereof, or other binding agents, or increased stability of the transfected cell line.

[0144] Furthermore, plants have emerged as a convenient, safe, and economical alternative expression system for recombinant antibody production based on the large-scale culture of microorganisms or animal cells. FOLR1-binding antibodies or their antigen-binding portions or other binding agents can be expressed in plant cell cultures or conventionally grown plants. Expression in plants can be systemic, restricted to intracellular plastids, or restricted 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. Several plant-derived antibodies have reached advanced stages of development, including clinical trials (see, for example, Biolex, NC).

[0145] In the case of intact antibodies, the variable regions (VH and VL regions) of the FOLR1 antibody are typically linked to at least a portion of an immunoglobulin constant region (Fc) or domain, typically at least a portion 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 (WO 87 / 02671). 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. Pat. No. 4,946,778; 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), which are incorporated herein by reference in their entirety) can be adapted to produce single-chain antibodies that specifically bind to FOLR1. Single-chain antibodies are formed by linking the heavy and light chain variable regions of the Fv region via an amino acid bridge, resulting in 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 incorporated herein by reference in its entirety).

[0147] In some embodiments, the antigen-binding moiety or other binding agent comprises one or more scFvs. An scFv can be a fusion protein of the variable regions of an antibody's heavy chain (VH) and light chain (VL), connected by a short linker peptide, e.g., 10 to about 25 amino acids. The linker is typically glycine-rich for flexibility and serine- or threonine-rich for solubility, and can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. The protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. scFv antibodies are described, for example, in Houston, J.S., Methods in Enzymol 203 (1991) 46-96. Methods for producing scFv molecules and designing suitable 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, which is an antigen-binding portion consisting of a single monomeric variable antibody domain. Single-domain antibodies can be derived from the variable domain of an antibody heavy chain from a camelid (e.g., a nanobody or VHH portion). Furthermore, single-domain antibodies can be autonomous 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 generating single-domain antibodies (DABs or VHHs) are known in the art, as disclosed, for example, in 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 ability and can interact with epitopes inaccessible to conventional VH-VL pairs (see, e.g., Muyldermans et al., 2001). Alpaca serum IgG contains approximately 50% camelid heavy chain-only IgG antibodies (HCAbs) (see, e.g., Maass et al., 2007). Alpacas can be immunized with antigens, and VHHs that bind to and neutralize the target antigen can be isolated (see, e.g., Maass et al., 2007). PCR primers that amplify alpaca VHH coding sequences have been identified and can be used to construct alpaca VHH phage display libraries, which can be used to isolate antibody fragments by standard biopanning techniques well known in the art (see, e.g., Maass et al., 2007).

[0150] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with 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 "knobs-in-holes" technology (see, e.g., U.S. Pat. No. 5,731,168; Carter (2001), J Immunol Methods 248, 7-15). Multispecific antibodies can also be produced by electrostatic steering to generate antibody Fc-heterodimeric molecules. steering effect (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or antigen-binding portions thereof (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); Antibody portions can also be produced using "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and by the use of single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by the preparation of trispecific antibodies as described, for example, in Tutt et al., J. Immunol. 147:60 (1991).

[0151] Engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies," can also be binders (see, eg, US Patent Application Publication No. 2006 / 0025576).

[0152] The binding agent (e.g., antibody or antigen-binding portion) herein also includes a "dual-acting FAb" or "DAB" that contains antigen-binding sites that bind to 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., WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, and WO 2013 / 026833).

[0153] In some embodiments, the binding agent comprises 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 contained in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. Therefore, in order to improve the yield and purity of bispecific molecules in recombinant production, it is advantageous to introduce modifications into the Fc domain of the binding agent that promote the association of the desired polypeptides.

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

[0155] In some embodiments, the binding agent 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, the single polypeptide chain can comprise two single-chain Fv (scFv) moieties, each having a variable heavy (VH) and variable light (VL) domain separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. The single polypeptide further comprises a polypeptide spacer sequence between the two scFvs. Each scFv recognizes a different epitope, and these epitopes can be specific for different proteins, so that both proteins are bound by the BiTE.

[0156] Because it 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 1 691 833) may be required to separate the monomeric bispecific T cell engager from other multimeric species that may have biological activity 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 with 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 bispecific antibody binding agent is composed of a single polypeptide chain comprising two single-chain FV moieties (scFVs) fused to each other by a peptide linker.

[0157] In some embodiments, the binding agent 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 for 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 dual variable domain antibodies (DVD-Ig) have been described by Wu et al., 2007, Nat Biotechnol 25:1290-97; Hasler et al., Mol. Immunol. 75:28-37, 2016, and in WO 08 / 024188 and WO 07 / 024715. Triomabs have been described by Chelius et al., MAbs 2(3):309-319, 2010. 2-in-1 IgGs have been described by Kontermann et al., Drug Discovery Today 20(7):838-847, 2015. Tanden antibodies or TandAbs have been described by Kontermann et al., supra. ScFv-HSA-scFv antibodies have also been described by Kontermann et al. (ibid.).

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

[0160] Antibody-drug conjugates In some embodiments, the FOLR1 antibody, antigen-binding portion, or other binding agent described herein is part of a FOLR1 antibody-drug conjugate (also referred to as a FOLR1 conjugate or FOLR1 ADC). In some embodiments, the FOLR1 antibody, antigen-binding portion, or other binding agent 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 immunomodulator, a nucleic acid (including siRNA), a growth inhibitory agent, 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 these cells.

[0161] cytotoxic agents 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, dolastatins, tubulysins, colchicine, vinca alkaloids, taxanes, cryptophycins, maytansinoids, hemiasterins, and other tubulin disrupting agents. Auristatins are derivatives 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 U.S. Patent Application Publication Nos. 2021 / 0008099, 2017 / 0121282, 2013 / 0309192, and 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 U.S. Patent Application Publication No. 2001 / 0018422). Additional dolastatin derivatives contemplated for use herein are disclosed in U.S. Patent No. 9,345,785, which is incorporated herein by reference.

[0163] Tubulysins include, but are not limited to, tubulysin D, tubulysin M, tubuphenylalanine, and tubutyrosine. WO 2017 / 096311 and WO 2016 / 040684 describe tubulysin analogs, including tubulysin M.

[0164] Colchicines include, but are 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, maytansine analogs DM1, DM3 and DM4, or ansamitocin-2. Exemplary maytansinoid drug moieties include those with modified aromatic rings such as C-19-dechloro (U.S. Pat. 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. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), + / -dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acyl chloride), as well as those with modifications at other positions.

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

[0170] Hemiasterins include, but are not limited to, hemiasterin and HTI-286.

[0171] Other tubulin disrupting agents include taccalonolide A, taccalonolide B, taccalonolide AF, taccalonolide AJ, taccalonolide Al-epoxide, discodermolide, epothilone A, epothilone B, and laulimalide.

[0172] In some embodiments, the cytotoxic agent may be a topoisomerase inhibitor, such as camptothecin. Exemplary camptothecins include, for example, camptothecin, irinotecan (also known as 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 International Publication No. WO 1996 / 021666, International Publication No. WO 00 / 08033, U.S. Patent Application Publication No. 2016 / 0229862, and International Publication No. WO 2020 / 156189.

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

[0174] immunomodulators 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, thiazoquinolines, aminoquinolines, aminoquinazolines, pyrido[3,2-d]pyrimidine-2,4-diamines, pyrimidine-2,4-diamines, 2-aminoimidazoles, 1-alkyl-1H-benzimidazol-2-amines, tetrahydropyridopyrimidines, heteroaromatic azides-2,2-dioxides, benzonaphthyridines, guanosine analogs, adenosine analogs, thymidine homopolymers, ssRNA, CpG-A, polyG10, and polyG3. In some embodiments, the TLR7 agonist is selected from imidazoquinolines, imidazoquinoline amines, thiazoquinolines, aminoquinolines, aminoquinazolines, pyrido[3,2-d]pyrimidine-2,4-diamines, pyrimidine-2,4-diamines, 2-aminoimidazoles, 1-alkyl-1H-benzimidazol-2-amines, tetrahydropyridopyrimidines, heteroaromatic azides-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, as well as compounds disclosed in U.S. Patent Application Publication No. 20160168164 (Janssen), U.S. Patent Application Publication No. 20150299194 (Roche), U.S. Patent Application Publication No. 20110098248 (Gilead Sciences), U.S. Patent Application Publication No. 20100143301 (Gilead Sciences), and U.S. Patent Application Publication No. 20150299194 (Roche). Sciences) and compounds disclosed in 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-benzimidazol-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-benzimidazol-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, and VTX-1463.

[0177] In some embodiments, the TLR8 agonist can be any of the compounds described in WO2018 / 170179, WO2020 / 056198, and WO2020056194.

[0178] Other TLR7 and TLR8 agonists are described, for example, in WO 2016142250, WO 2017046112, WO 2007024612, WO 2011022508, WO 2011022509, WO 2012045090, WO 2012097173, WO 2012097177, WO 2017079283, U.S. Patent Application Publication No. 20160008374, U.S. Patent Application Publication No. 20160194350, U.S. Patent Application Publication No. 20160289229, U.S. Patent No. 6,043,238, U.S. Patent Application Publication No. 20180086755 (Gilead), WO 2017216054 (Roche), WO 2017190669 (Shanghai De Novo Pharmatech), WO 2017202704 (Roche), WO 2017202703 (Roche), WO 20170071944 (Gilead), U.S. Patent Application Publication No. 20140045849 (Janssen), U.S. Patent Application Publication No. 20140073642 (Janssen), WO 2014056953 (Janssen), WO 2014076221 (Janssen), WO 2014128189 (Janssen), U.S. Patent Application Publication No. 20140350031 (Janssen), WO 2014023813 (Janssen), U.S. Patent Application Publication No. 20080234251 (Array Biopharma), U.S. Patent Application Publication No. 20080306050 (Array Biopharma), U.S. Patent Application Publication No. 20100029585 (Ventirx Pharma), U.S. Patent Application Publication No. 20110092485 (Ventirx Pharma), U.S. Patent Application Publication No. 20110118235 (Ventirx Pharma), U.S. Patent Application Publication No. 20120082658 (VentirxNo. 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), WO 2018198091 (Novartis AG), and U.S. Patent Application Publication No. 20170131421 (Novartis AG).

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

[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 an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0182] radioactive isotope In some embodiments, the drug is a radioactive atom.Various radioisotopes can be used to prepare radioconjugate.Examples include I131, I125, Y90, Re186, Re188, Sm153, Bi213, P32, Pb212 and the radioisotope of lutetium (for example, Lu177).

[0183] PROTAC In some embodiments, the drug is a proteolysis-directing 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 FOLR1 conjugates typically include at least one linker, with each linker having at least one drug attached thereto. Typically, the conjugate includes a linker between the FOLR1 antibody (or antigen-binding portion thereof or other binding agent) and the drug. In various embodiments, the linker is 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 sulfide bond (e.g., Jain et al., Pharm. Res. 32:3526-3540 (2015); Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. 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 photodegradable 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 cleavage of the linker releases the drug from the antibody (or antigen-binding portion thereof or other binding agent) and / or the linker in the intracellular environment. For example, in some embodiments, the linker can be cleaved by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, 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, a lysosomal or endosomal protease (see, e.g., WO 2004 / 010957, U.S. Patent Application Publication No. 20150297748, U.S. Patent Application Publication No. 2008 / 0166363, U.S. Patent Application Publication No. 20120328564, and U.S. Patent Application Publication No. 20200347075). Typically, the peptidyl linker is at least one amino acid long or at least two amino acids long. Intracellular cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug within target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). The most typical peptidyl linker is 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 tissues, 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 U.S. Pat. No. 6,214,345. In specific embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. 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., U.S. Patent Application Publication No. 2015 / 0297748).One advantage of using intracellular proteolytic release of drugs is that the drug is typically attenuated when conjugated, and the serum stability of the conjugates 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 a metabolic process or reaction within a cell on an antibody-drug conjugate by which the covalent bond, e.g., linker, between the drug (e.g., cytotoxic agent) and the antibody is broken, yielding free drug or other metabolic product of the conjugate dissociated from the antibody within the cell. 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, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used. (See, for example, U.S. Pat. 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 below pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker, such as a thioether attached to the drug via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929).

[0188] In some embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). For example, a variety of disulfide linkers are known, including those 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.S. Press, 1987); see also U.S. Pat. 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, e.g., 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 about 20% or less, typically about 15% or less, more typically about 10% or less, and even more typically about 5%, about 3%, or about 1% or less of the linkers in a sample of an antibody-drug conjugate (ADC) are cleaved when the ADC is present in an extracellular environment (e.g., plasma). Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating both (a) the ADC ("ADC sample") and (b) an equimolar amount of 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 to that present in the control sample, as measured, for example, by high-performance liquid chromatography.

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

[0192] Various linkers that can be used in the compositions and methods of the present invention are described in WO2004010957. In some embodiments, the protease-cleavable linker comprises a thiol-reactive spacer and a dipeptide. In some embodiments, the 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 and WO 2016 / 040684).

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

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

[0196] In other embodiments, conjugates of FOLR1 antibodies (or antigen-binding moieties or other binding agents) and drugs may be made using a variety of 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-azido 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 conjugation of radionucleotides to antibodies, antigen-binding portions thereof, or other binding agents are described, for example, in WO 94 / 11026.

[0197] Conjugates of FOLR1 antibodies (or antigen-binding portions or other binding agents) include, but are not limited to, 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, which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Illinois, USA), and such conjugates prepared using cross-linkers including SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0198] In some embodiments, the linker can be attached to the end of the amino acid sequence of an antibody, antigen-binding moiety, or other binding agent, or to the side chain of a modified antibody, antigen-binding moiety, or other binding agent, such as lysine, serine, threonine, cysteine, tyrosine, aspartic acid, non-natural amino acid residue, glutamine, or glutamic acid residue. The bond between an antibody, antigen-binding moiety, or other binding agent and a linker or drug can be via any of several bonds, including, but not limited to, an amide bond, an ester bond, an ether bond, a carbon-nitrogen bond, a carbon-carbon single bond, a double bond, or a triple bond, a disulfide bond, or a thioether bond. Functional groups that can form such bonds include, for example, amino groups, carboxyl groups, aldehyde groups, azide groups, alkyne and alkene groups, ketones, carbonates, and carbonyl functional groups linked to leaving groups such as cyano groups, succinimidyl groups, and hydroxyl groups.

[0199] In some embodiments, the linker is attached to the antibody, antigen-binding moiety, or other binding agent at an interchain disulfide. In some embodiments, the linker is attached to the antibody, antigen-binding moiety, or other binding agent at a hinge cysteine ​​residue. In some embodiments, the linker is attached to the antibody, antigen-binding moiety, or other binding agent at an engineered cysteine ​​residue. In some embodiments, the linker is attached to the antibody, antigen-binding moiety, or other binding agent at a lysine residue. In some embodiments, the linker is attached to the antibody, antigen-binding moiety, or other binding agent at an engineered glutamine residue. In some embodiments, the linker is attached to the antibody, antigen-binding moiety, or other binding agent at an unnatural amino acid engineered into the heavy chain.

[0200] In some embodiments, the linker is attached to the antibody, antigen-binding moiety, or other binding agent via a sulfhydryl group. In some embodiments, the linker is attached to the antibody, antigen-binding moiety, or other binding agent via a primary amine. In some embodiments, the linker is attached via a bond created between an unnatural amino acid on the antibody, antigen-binding moiety, 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 attached to the antibody, antigen-binding moiety, or other binding agent via a sortase A linker, which can be generated by a sortase A enzyme fusing 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 drug such as a tubulin disrupting agent, e.g., an auristatin, is attached to the linker by its C-terminal carboxyl group, which forms an amide bond with the linker (e.g., a linker unit (LU) described in U.S. Pat. No. 9,463,252, incorporated herein by reference). In some embodiments, the linker comprises at least one amino acid.

[0203] In some embodiments, the linker also comprises a stretcher unit and / or an amino acid unit. Exemplary stretcher units and amino acid units are described in U.S. Patent Nos. 9,345,785 and 9,078,931, each of which is incorporated herein by reference.

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

[0205] In some embodiments, the FOLR1 conjugate has the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein mAb is a FOLR1 antibody, antigen-binding portion thereof, or other binding agent; S is a sulfur atom of the antibody, antigen-binding portion, or other binding agent; A is a Stretcher unit; and p is from about 3 to about 5, or from about 3 to about 8.

[0206] Drug loading is represented by p, the average number of drug molecules (e.g., cytotoxic agents) per antibody (or antigen-binding moiety or other binding agent) in the conjugate. For example, if p is about 4, then the average drug loading, considering all of the antibodies (or antigen-binding moieties or other binding agents) present in the composition, is about 4. In some embodiments, p is in the range of about 3 to about 5, about 3.6 to about 4.4, or about 3.8 to about 4.2. In some embodiments, p can be about 3, about 4, or about 5. In some embodiments, p is in the range of about 6 to about 8, more preferably 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 moiety or other binding agent) in a preparation can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of antibody-drug conjugates with respect to p can also be determined. In some instances, separation, purification, and characterization of homogeneous antibody-drug conjugates with a constant value of p from antibody-drug conjugates with other drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0208] In some embodiments, a Stretcher unit can link an antibody (or antigen-binding portion or other binding agent) to an amino acid or peptide (e.g., a valine-citrulline peptide) via a sulfhydryl group on the antibody (or antigen-binding portion or other binding agent). Sulfhydryl groups can be generated, for example, by reduction of the interchain disulfide bond of a FOLR1 antibody (or antigen-binding portion or other binding agent). For example, a Stretcher unit can be linked to an antibody (or antigen-binding portion or other binding agent) via a sulfur atom generated from reduction of the interchain disulfide bond of the antibody (or antigen-binding portion or other binding agent). In some embodiments, a Stretcher unit is linked to an antibody (or antigen-binding portion or other binding agent) solely via a sulfur atom generated from reduction of the interchain disulfide bond of the antibody. In some embodiments, sulfhydryl groups can be generated by reaction of amino groups on lysine moieties of a FOLR1 antibody (or antigen-binding portion or other binding agent) with 2-iminothiolane (Traut's reagent) or other sulfhydryl-generating reagents. In some embodiments, the FOLR1 antibody (or antigen-binding portion or other binding agent) 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 binding agent) is engineered to have an additional cysteine, such as an additional sulfhydryl group, e.g., an engineered cysteine.

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

[0210] Representative Stretcher units are set forth within the brackets of formulas IIa and IIb in US Pat. No. 9,211,319, incorporated herein 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-aminobenzyloxycarbonyl or PAB spacer.

[0212] The abbreviation "PAB" stands for self-immolating spacer: [ka] Refers to...

[0213] The abbreviation "MC" stands for stretcher maleimidocaproyl: [ka] Refers to...

[0214] In other exemplary embodiments, the conjugate has the following general formula: Ab-[L3]-[L2]-[L1] m -AA n -drugs, where Ab is a FOLR1 antibody (or antigen-binding moiety or other binding agent); the drug can be, for example, a cytotoxic agent such as a tubulin-disrupting agent or a topoisomerase inhibitor; L3 is a component of a linker comprising an antibody coupling moiety (such as a stretcher unit) and one or more acetylene (or azide) groups; L2 comprises an optional PEG (polyethylene glycol) azide (or acetylene) at one end that is complementary to the acetylene (or azide) moiety of L3 and a reactive group such as a carboxylic acid or hydroxyl group at the other end; L1 comprises a foldable unit (e.g., a self-immolative group), or a peptidase-cleavable moiety or acid-cleavable moiety optionally attached to the foldable unit; 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. Such linkers can be assembled via click chemistry (see, e.g., U.S. Pat. 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 known as CPT-11), belotecan, topotecan, 10-hydroxy-CPT, exatecan, DXd, and / or SN-38. Representative structures are shown below. [ka]

[0216] Conjugated Ab-[L3]-[L2]-[L1] m -AA n -Referring to the drug, in some embodiments, m is 0. Conjugate Formula Ab-[L3]-[L2]-[L1] m -AA nIn some embodiments, L2 is absent, referring to the drug. In such embodiments, an 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, Fmoc, or monomethoxytrityl (MMT) derivative, which is deprotected after forming an ester bond with the hydroxyl group of the cytotoxic agent. Since "monomethoxytrityl (MMT)" is removable by mild acid treatment, such as dichloroacetic acid, which does not cleave the BOC group, selective removal of the amine protecting group can be achieved in the presence of a BOC protecting group at the hydroxyl position of a cytotoxic agent containing an additional hydroxyl group by using MMT as a protecting group for the amino group of the amino acid or polypeptide involved in ester formation. After the amino group of the amino acid or polypeptide that forms the ester bond with the hydroxyl of the drug is unmasked, the amino group is reacted with the activated form of the COOH group of the PEG moiety of L2 (if present) under standard amide-forming conditions. In preferred embodiments, L3 comprises a thiol-reactive group that links to a thiol group of an antibody (or antigen-binding portion or other binding agent). The thiol-reactive group is optionally maleimide or vinyl sulfone, or bromoacetamide, or iodoacetamide, that links to a thiol group of the antibody. In some embodiments, the reagent having a thiol-reactive group is generated from, for example, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or succinimidyl-(epsilon-maleimido)caproate, and the thiol-reactive group is a maleimide group.

[0217] In another embodiment, m is 0 and AA comprises a peptide moiety, preferably a di-, tri-, or tetrapeptide, that is cleavable by an intracellular peptidase, such as cathepsin B. 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, e.g., WO 2014 / 057687).

[0218] In some embodiments, L1 is comprised 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), with the benzyl alcohol moiety directly linked to the hydroxyl group of a drug, such as a chloroformate form of a cytotoxic agent. 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 attached to the N-terminus of the amino acid or peptide linked at the hydroxyl group of the cytotoxic agent through the activated form of p-amidobenzyl alcohol, i.e., PABOCOPNP, in which PNP is p-nitrophenyl. In some embodiments, the linker comprises a thiol-reactive group that links to a thiol group of an antibody (or antigen-binding moiety or other binding agent). The thiol-reactive group is optionally maleimide or vinyl sulfone, or bromoacetamide, or iodoacetamide, that links to a thiol group of an antibody (or antigen-binding moiety or other binding agent). In a preferred embodiment, the component having a thiol-reactive group is generated from, for example, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or succinimidyl-(epsilon-maleimido)caproate, where the thiol-reactive group is a maleimide group.

[0219] In some embodiments, when the drug is a 20-hydroxyl-bearing camptothecin or an analog or derivative thereof, L1 is comprised of an intracellularly cleavable peptide, such as a cathepsin B-cleavable peptide, connected at the C-terminus of the peptide to a foldable linker, p-aminobenzyl alcohol (or p-amino-benzyloxycarbonyl), with the benzyl alcohol moiety directly attached 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 attached to the N-terminus of the amino acid or polypeptide linked at the 20-position of CPT through an activated 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 couples to a thiol group on an antibody (or antigen-binding moiety or other binding agent). The thiol-reactive group is optionally maleimide or vinyl sulfone, or bromoacetamide, or iodoacetamide, which is linked to a thiol group on an antibody (or antigen-binding portion or other binding agent). In a preferred embodiment, the component bearing the thiol-reactive group is generated from, for example, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or succinimidyl-(epsilon-maleimido)caproate, and the thiol-reactive group is a maleimide group.

[0220] In some embodiments, the L2 component of the conjugate is present and contains a polyethylene glycol (PEG) spacer, which can be up to about MW 5000 in size; in preferred embodiments, the PEG is a defined PEG having 1-12 or 1-30 repeating monomer units. In some embodiments, the PEG is a defined PEG having 1-12 repeating monomer units. Introduction of PEG can involve the use of commercially available heterobifunctional PEG derivatives. Heterobifunctional PEGs typically contain azide or acetylene groups. An example of a heterobifunctional defined PEG containing 8 repeating monomer units where "NHS" is succinimidyl is shown below in the following formula: [ka]

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

[0222] A representative conjugate is shown below in which the drug is a cytotoxic agent such as SN-38 (a CPT analog) prepared using a maleimide-containing SN-38-linker derivative, with the linkage to the antibody (referred to as MAb) represented as a succinimide: Here, m=0, the 20-O-AA ester attached to SN-38 is a glycinate; azide-acetylene coupling of L2 and L3 results in a triazole moiety as shown. [ka]

[0223] Another representative conjugate prepared using a maleimide-containing SN-38-linker derivative, with the antibody (MAb) linkage represented as the succinimide, is shown below, where n=0 in general formula 2; "L1" contains the cathepsin B-cleavable dipeptide Phe-Lys linked to a foldable p-aminobenzyl alcohol moiety, the latter being linked to SN-38 as a carbonate bond at position 20; and azide-acetylene coupling joining the "L2" and "L3" moieties results in a triazole moiety as shown. [ka]

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

[0225] In another representative example, "L1" contains a single amino acid attached to a foldable p-aminobenzyl alcohol moiety, where the p-aminobenzyl alcohol is substituted or unsubstituted (R). The general conjugate formula is Ab-[L3]-[L2]-[L1]m-AAn-drug, where m = 1 and n = 0, and the drug is exemplified by SN-38. The structure is depicted below (referred to as MAb-CLX-SN-38). The single amino acid 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 hydrogen or an alkyl group selected from C1-C10 alkyl groups. [ka]

[0226] An embodiment of mAb-CLX-SN-38 (above) in which the single amino acid AA is L-lysine, R=H, and the drug is a cytotoxic agent exemplified by SN-38 (referred to as mAb-CL2A-SN-38) is shown below: [ka]

[0227] In other embodiments, the drug is a cytotoxic agent attached to a linker comprising a Stretcher unit (Z) attached to an amino acid unit (AA) attached to a Spacer unit (Y), the Stretcher unit being attached to an antibody (or antigen-binding portion thereof or other binding agent, referred to as an Ab or MAb), and the Spacer unit being attached to an amino group of the cytotoxic agent. Such linkers have the following formula: Ab-Z-AA-Y-cytotoxic agent wherein Z is -(succinimide-3-yl-N)-(CH) 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 -C(=O)--, n 2 represents an integer from 2 to 8, and n 3 represents an integer from 1 to 8, and n 4 represents an integer of 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: [ka] (having It has.

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

[0229] In some embodiments, the cytotoxic agent 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-cytotoxic agent has the following structure: [ka] and the released cytotoxic agent is DXd (see US Pat. No. 9,808,537).

[0231] Conjugation of Drug-Linkers to Antibodies, Antibody Binding Moieties, and Other Binding Agents Techniques for attaching drugs to antibodies (or antigen-binding portions thereof or other binding agents) via linkers are well known in the art. See, e.g., Alley et al., Current Opinion in Chemical See Biology 2010 14:1-9; Senter, Cancer J., 2008, 14(3):154-169. In some embodiments, the linker is first attached to the drug (e.g., a cytotoxic agent), and then the drug-linker is attached to the antibody or antigen-binding portion thereof or other binding agent. In some embodiments, the linker is first attached to the antibody or antigen-binding portion thereof or other binding agent, and then the drug is attached to the linker. In the following discussion, the term drug-linker is used to illustrate the attachment of a linker or drug-linker to an antibody or antigen-binding portion thereof or other binding agent; one of skill in the art will recognize that the attachment method selected can be selected according to the linker and the cytotoxic agent or other drug. In some embodiments, the drug is attached to the antibody or antigen-binding portion thereof or other binding agent 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] In general, conjugates can be prepared by several routes employing organic chemical reactions, conditions, and reagents known to those skilled in the art, including: (1) reacting a nucleophilic group on an antibody (or antigen-binding portion thereof or other binding agent) with a bivalent linker reagent to form an antibody-linker intermediate via a covalent bond, followed by reaction with a drug (e.g., a cytotoxic agent); and (2) reacting a nucleophilic group on a drug (e.g., a cytotoxic agent) with a bivalent linker reagent to form a drug-linker via a covalent bond, followed by reaction with a nucleophilic group on an antibody or antigen-binding portion thereof or other binding agent. An exemplary method for preparing conjugates via the latter route is described in U.S. Pat. No. 7,498,298, which is expressly incorporated herein by reference.

[0233] Nucleophilic groups on antibodies, antigen-binding moieties, and other binding agents 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 on glycosylated antibodies. Amine, thiol, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups. Certain antibodies (and antigen-binding moieties or other binding agents) have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies (and antigen-binding moieties and other binding agents) can be made reactive for conjugation with linker reagents by treating them with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) so that the antibody is fully or partially reduced. Thus, each cysteine ​​bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies (and antigen-binding moieties and other binding agents) through modification of lysine residues, for example, by reacting the lysine residue with 2-iminothiolane (Traut's reagent) to convert the amine to a thiol. Reactive thiol groups can also be introduced into antibodies (and antigen-binding moieties and other binding agents) by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing antibodies, antigen-binding moieties, and other binding agents containing one or more non-naturally occurring cysteine ​​amino acid residues).

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

[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; and (iii) amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups, which can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including aldehyde, ketone, carboxyl, and maleimide groups.

[0236] Non-limiting exemplary cross-linking agents that can be used to prepare conjugates are described herein or known to those skilled in the art. Methods for using such cross-linking agents to link two moieties, including an antibody (or antigen-binding portion or other binding agent) and a chemical moiety, are known in the art. In some embodiments, a fusion protein comprising an antibody or antigen-binding portion and a drug can be produced, for example, by recombinant technology or peptide synthesis. A recombinant DNA molecule can include a region encoding an antibody (or its antigen-binding portion or other binding agent) and a region encoding the active portion of the conjugate (e.g., a cytotoxic moiety), either adjacent to each other or separated by a region encoding a linker that does not destroy the desired properties of the conjugate.

[0237] In some embodiments, the drug-linker is attached to an interchain cysteine ​​residue of an antibody (or antigen-binding portion thereof or other binding agent). See, for example, International Publication Nos. WO 2004 / 010957 and WO 2005 / 081711. In such embodiments, the linker typically contains a maleimide group for attachment to the cysteine ​​residue of the interchain disulfide. In some embodiments, the linker or drug-linker is attached to a cysteine ​​residue of an antibody or antigen-binding portion thereof, as described in U.S. Pat. Nos. 7,585,491 or 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 antigen-binding portion thereof or other binding agent), as described in WO 2005 / 037992 or WO 2010 / 141566. The drug loading of the resulting conjugate is typically in the range of 1-8.

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

[0240] In some embodiments, the drug-linker is attached to an engineered cysteine ​​residue in an Fc residue other than the interchain disulfide. In some embodiments, the drug-linker is attached to an engineered cysteine ​​residue in an Fc residue other than the interchain disulfide. In some embodiments, the drug-linker is attached to an engineered cysteine ​​residue in an Fc residue other than the interchain disulfide. In some embodiments, the drug-linker is attached to an engineered cysteine ​​residue in an Fc residue other than the interchain disulfide. , 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, and / or to an engineered cysteine ​​introduced into the light chain at positions 106, 108, 142 (light chain), 149 (light chain), and / or V205. 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 is according to 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 thereof or other binding agent), as described in WO2006 / 034488, WO2011 / 156328, and / or WO2016040856.

[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 moiety or a glycoengineered antibody (or other binding agent). For example, see International Publication No. WO2017 / 147542, International Publication No. WO2020123425, International Publication No. WO2014 / 072482; International Publication No. WO2014 / 065661, International Publication No. WO2015 / 057066 and International Publication No. WO2016 / 022027.

[0243] Pharmaceutical preparations Another aspect of the FOLR1 antibody and its antigen-binding portion or other binding agent, as well as conjugates of any of these, relates to a composition comprising an active ingredient (i.e., a FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein, or a conjugate thereof, or a nucleic acid encoding the antibody or antigen-binding portion thereof or other binding agent described herein). In some embodiments, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to an active agent in combination 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 the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0244] The preparation of pharmacological compositions containing active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on any particular formulation. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; however, solid forms suitable for rehydration or suspension in liquid before use can also be prepared. Preparations can also be emulsified or provided as liposomal compositions. The FOLR1 antibody or its antigen-binding portion or other binding agent or conjugate thereof can be mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient, in an amount appropriate for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Additionally, if desired, pharmaceutical compositions can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance or maintain the effectiveness of the active ingredient (e.g., the FOLR1 antibody or its antigen-binding portion or other binding agent or conjugate thereof). The pharmaceutical compositions described herein can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of a polypeptide) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, and the like. 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, and the like. Physiologically acceptable carriers are well known in the art. An exemplary liquid carrier is a sterile aqueous solution containing an active ingredient (e.g., a FOLR1 antibody and / or its antigen-binding portion, other binding agent, or conjugate thereof) and water, and may contain both buffers such as sodium phosphate at physiological pH values, saline, or phosphate-buffered saline. Furthermore, aqueous carriers can contain two or more buffer salts, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes.Liquid compositions can also contain liquid phases in addition to and other than water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent that will be effective in 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, a pharmaceutical composition comprising a FOLR1 antibody or antigen-binding portion thereof or other binding agent or conjugate thereof described herein, or a nucleic acid encoding a FOLR1 antibody or antigen-binding portion thereof or other binding agent described herein, may be a lyophilizate.

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

[0247] Cancer treatment In some embodiments, the FOLR1 antibodies or antigen-binding portions thereof, binding agents and conjugates described herein may be used in methods comprising administering a FOLR1 antibody or antigen-binding portion thereof or other binding agent or conjugate thereof described herein to a subject in need thereof, for example, a subject with cancer.

[0248] In some embodiments, a method is provided comprising administering a FOLR1 antibody or 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 VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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, respectively; and SEQ ID NO:23 and SEQ ID NO:24, respectively. In some embodiments, a method is provided that includes administering a FOLR1 antibody or 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 VL region have the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In some embodiments, a method is provided that includes administering a FOLR1 antibody or 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 VL region have the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. In some embodiments, a method is provided that includes administering a FOLR1 antibody or 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 VL region have the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. In some embodiments, a method is provided that includes administering a FOLR1 antibody or 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 VL region have the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.In some embodiments, a method is provided that includes administering a FOLR1 antibody or 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 VL region have the amino acid sequences set forth in SEQ ID NOs: 9 and 10, respectively. In some embodiments, a method is provided that includes administering a FOLR1 antibody or 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 VL region have the amino acid sequences set forth in SEQ ID NOs: 11 and 12, respectively. In some embodiments, a method is provided that includes administering a FOLR1 antibody or 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 VL region have the amino acid sequences set forth in SEQ ID NOs: 13 and 14, respectively. In some embodiments, a method is provided that includes administering a FOLR1 antibody, or 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 VL region have the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively. In some embodiments, a method is provided that includes administering a FOLR1 antibody, or 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 VL region have the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively. In some embodiments, a method is provided that includes administering a FOLR1 antibody, or 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 VL region have the amino acid sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.In some embodiments, methods are provided that include administering a FOLR1 antibody or 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 VL region have the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In some embodiments, methods are provided that include administering a FOLR1 antibody or 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 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 is provided that includes administering a FOLR1 antibody or 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 VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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, respectively; and SEQ ID NO:23 and SEQ ID NO:24, respectively; and wherein 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 regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified.In some embodiments, a method is provided that includes administering a FOLR1 antibody or 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 VL region have the amino acid sequences set forth in a pair of amino acid sequences selected from 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, respectively; and SEQ ID NO:23 and SEQ ID NO:24, respectively; and wherein the heavy chain variable framework region and the light chain variable framework region are optionally modified with substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids within the framework regions, and wherein the CDRs of the heavy chain variable region or the light chain variable region are unmodified.

[0250] In some embodiments, a method is provided comprising administering a FOLR1 antibody or 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 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, and the VH and VL CDRs have the amino acid sequences set forth in a set 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, respectively; 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. 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 is provided comprising administering a FOLR1 antibody or 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 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, and the VH and VL CDRs (i) 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 and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0252] In some embodiments, a method is provided comprising administering a FOLR1 antibody or 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 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, 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.

[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 with a FOLR1+ cancer or malignancy. In some embodiments, the method is for treating lung cancer in a subject. In some embodiments, the method is for treating non-small cell lung cancer in a subject. In some embodiments, the method is for treating breast cancer in a subject. In some embodiments, the method is for treating ovarian cancer in a subject. In some embodiments, the method is for treating cervical cancer in a subject. In some embodiments, the method is for treating endometrial cancer in a subject. In some embodiments, the method is for treating renal cell carcinoma in a subject. In some embodiments, the method is for treating uterine cancer in a subject. In some embodiments, the method is for treating pancreatic cancer in a subject.

[0254] The methods described herein include administering a therapeutically effective amount of a FOLR1-binding antibody or antigen-binding portion thereof, or other binding agent, or conjugate thereof, to a subject with a FOLR1+ cancer or malignant tumor. As used herein, the phrase "therapeutically effective amount," "effective amount," or "effective dose" refers 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 a cancer or malignant tumor, e.g., an amount that provides a statistically significant reduction in at least one symptom, sign, or marker of a tumor or malignant tumor. Determining a therapeutically effective amount is well within the capabilities of one skilled in the art. Generally, a therapeutically effective amount may vary depending on the subject's medical history, age, condition, sex, 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 proliferation of cells that interferes with the normal function of bodily organs and systems. Cancer or malignant tumors can be primary or metastatic, i.e., they have become invasive, seeding tumor growth in tissues distant from the original tumor site. A "tumor" refers to the uncontrolled proliferation of cells that interferes with the normal function of bodily organs and systems. A subject with cancer is one who has objectively measurable cancer cells present within the subject's body. This definition includes benign tumors and malignant cancers, as well as latent dormant tumors and micrometastases. Cancers that migrate from their original location and disseminate to other vital organs can ultimately lead to the subject's death through the functional decline of the affected organ. Hematologic malignancies (hematopoietic cancers), such as leukemia and lymphoma, can, for example, overwhelm a subject's normal hematopoietic compartment, thereby resulting in hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia) and ultimately death.

[0256] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancer include, but are not limited to, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer (e.g., triple-negative breast cancer), peritoneal cancer, cervical cancer; bile duct cancer, 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 gastric cancer), glioblastoma (GBM), liver Cancer, hepatoma, intraepithelial neoplasia, kidney or renal cancer (e.g., clear cell carcinoma), 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's lymphoma and non-Hodgkin's 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 Cancer of the salivary gland, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine or endometrial cancer, uterine serous carcinoma, urinary system cancer, vulvar cancer; and other carcinomas and sarcomas, as well as B-cell lymphomas (low-grade / follicular non-Hodgkin's 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 nucleus NHL, These include follicular NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (such as that 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 carcinoma. In some embodiments, the cancer or malignant tumor is FOLR1 positive (FOLR1+). As used herein, the term "FOLR1 positive" or "FOLR1+" is used to describe cancer cells, cancer cell clusters, tumor masses, or metastatic cells that express FOLR1 on the cell surface (membrane-bound FOLR1). 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 carcinoma.

[0258] It is contemplated that the methods herein reduce tumor size or tumor burden in a subject and / or reduce metastasis in a subject. In various embodiments, the subject's tumor size is reduced by about 25-50%, about 40-70%, or about 50-90% or more. In various embodiments, the method reduces tumor size by 10%, 20%, 30% or more. In various embodiments, the method reduces 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. Typically, an animal is a vertebrate, such as a primate, a rodent, a domestic animal, or a 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 and game animals include cattle, 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 trout, catfish, and salmon. In certain embodiments, the subject is a mammal, such as a primate, for example, a human. The terms "patient," "individual," and "subject" are used interchangeably herein.

[0260] Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects, for example, animal models of various cancers. Furthermore, 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 may be a subject who has been previously diagnosed with FOLR1+ cancer or who has been identified as having FOLR1+ cancer and in need of treatment, but who does not need to have already received treatment for FOLR1+ cancer. In some embodiments, the subject may also be a subject who has not previously been diagnosed with FOLR1+ cancer in need of treatment. In some embodiments, the subject may be a subject who exhibits one or more risk factors for a condition or one or more complications associated with FOLR1+ cancer, or a subject who does not exhibit risk factors. A subject "in need" of treatment for FOLR1+ cancer may particularly be a subject who has the condition or has been diagnosed with the condition. In other embodiments, a subject "at risk of developing" a condition refers to a subject who has been diagnosed as being at risk of developing the condition or at risk of re-developing cancer (e.g., FOLR1+ cancer).

[0262] As used herein, the terms "treat," "treatment," "treating," or "amelioration," when used in reference to a disease, disorder, or medical condition, refer to therapeutic treatment of a condition with the goal of reversing, alleviating, improving, inhibiting, slowing, or halting the progression or severity of the symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of the condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the condition is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation of symptoms or at least a slowing of progression or worsening that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, a reduction in FOLR1+ cancer cells in a subject, alleviation of one or more symptoms, a reduction in the degree of the defect, a stabilized (i.e., non-worsening) state of the cancer or malignant tumor, a delay or slowing of tumor growth and / or metastasis, and an increase in lifespan compared to that expected in the absence of treatment. As used herein, the term "administering" refers to providing 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, to a subject by a method or route that results in binding of the FOLR1-binding antibody or antigen-binding portion thereof or other binding agent or conjugate to FOLR1+ cancer cells or malignant cells. 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 by any suitable route that results in effective treatment in the subject.

[0263] The dosage range of a FOLR1-binding antibody or antigen-binding portion thereof, or binding agent or conjugate, depends on efficacy and includes an amount sufficient to produce the desired effect, such as slowing tumor growth or reducing tumor size. The dosage should not be so high as to cause unacceptable adverse side effects. Generally, dosages vary depending on the age, condition, and sex of the subject and can be determined by one of ordinary skill in the art. Dosages can also be adjusted by an individual physician in the event of any complications. In some embodiments, dosages range from 0.1 mg / kg to 10 mg / kg body weight. In some embodiments, dosages range from 0.5 mg / kg to 15 mg / kg body weight. In some embodiments, the dosage range is from 0.5 mg / kg to 5 mg / kg body weight. Alternatively, the dosage range can be metered to maintain serum levels between 1 μg / mL and 1000 μg / mL. When administered systemically, a subject can be administered 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 more.

[0264] The administration of the doses listed above can be repeated.In a preferred embodiment, the doses listed above are administered every week, every other week, every 3 weeks or every month for several weeks or months.The duration of treatment depends on the clinical progress of the patient and the responsiveness to treatment.

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

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

[0267] In some embodiments, the dose may be administered weekly. In some embodiments, the dose may be administered every other week. In some embodiments, the dose may be administered about every two weeks. In some embodiments, the dose may be administered about every three weeks. In some embodiments, the dose may be administered about every four weeks.

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

[0269] Pharmaceutical compositions containing a FOLR1-binding antibody or antigen-binding portion thereof, or other FOLR1-binding agent or FOLR1 conjugate thereof can be administered in unit doses. The term "unit dose," when used in reference to pharmaceutical compositions, refers to physically discrete units suitable as unitary administration for a subject, each unit containing a predetermined amount of active material (e.g., a FOLR1-binding antibody or antigen-binding portion thereof, or other binding agent or conjugate thereof) calculated to produce a desired therapeutic effect, together with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0270] In some embodiments, FOLR1 binding antibody or its antigen-binding portion or other binding agent or its conjugate, or any of these pharmaceutical compositions, is administered together 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 malignant tumors.Examples of immunotherapy include, but are not limited to, antibodies such as checkpoint inhibitors.

[0271] In some embodiments, immunotherapy involves administration of a checkpoint inhibitor. In some embodiments, immune checkpoint inhibitors include agents that inhibit CTLA-4, PD-1, PD-L1, etc. Suitable anti-CTLA-4 inhibitors include, for example, ipilimumab, tremelimumab, antibodies disclosed in PCT Publication No. WO 2001 / 014424, antibodies disclosed in PCT Publication No. WO 2004 / 035607, antibodies disclosed in U.S. Patent Application Publication No. 2005 / 0201994, and antibodies disclosed in granted European Patent No. 1212422. 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 and WO 00 / 37504; 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 invention include those disclosed, for example, in WO 98 / 42752; U.S. Pat. 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), U.S. Pat. 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 incorporated herein 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 for improving the therapeutic outcome of a subject receiving immunotherapy is provided. The method generally includes the steps of administering an effective amount of immunotherapy to a subject with cancer; and administering to the subject a therapeutically effective amount of a FOLR1 antibody, antigen-binding portion, other binding agent, or conjugate thereof, or a pharmaceutical composition thereof, wherein the FOLR1 antibody, antigen-binding portion, other binding agent, or conjugate thereof specifically binds to FOLR1+ cancer cells; and improving the therapeutic outcome of the subject compared to administration of immunotherapy alone. In some embodiments, the FOLR1 antibody, antigen-binding portion, other binding agent, or conjugate thereof comprises any of the embodiments of the FOLR1 antibody, antigen-binding portion, other binding agent, or conjugate thereof described herein. In some embodiments, the binding agent is an antibody or antigen-binding portion thereof. In some embodiments, the binding agent is a monoclonal antibody, Fab, Fab', F(ab'), Fv, scFv, single-domain antibody, diabody, bispecific antibody, or multispecific antibody. In some embodiments, the binding agent is a FOLR1 monoclonal antibody, Fab, Fab', F(ab'), Fv, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody conjugate.

[0276] In some embodiments, the improved outcome is an objective response selected from stable disease, partial response, or complete response as determined by standard medical criteria for the cancer being treated. In some embodiments, the improved outcome is a reduction in tumor burden. In some embodiments, the improved 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. Heavy chain variable (VH) region and light chain variable (VL) region A binder comprising: The VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 arranged in heavy chain variable region framework regions, the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, and the VH and VL CDRs are 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; 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 A binding agent having an amino acid sequence selected from the set of amino acid sequences set forth in the group consisting of: 2. The VH region and the VL region are 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, respectively; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively and having an amino acid sequence selected from the pair of amino acid sequences shown in the group consisting of: 2. The binding agent of embodiment 1, wherein the heavy chain framework regions and the light chain framework regions are optionally modified by substitution, deletion or insertion of 1 to 8 amino acids within the framework regions. 3. The VH region and the VL region are 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, respectively; and SEQ ID NO: 23 and SEQ ID NO: 24, respectively 3. The binding agent of embodiment 1 or 2, having an amino acid sequence selected from the pair of amino acid sequences set forth in the group consisting of: 4. The VH region and the VL region are 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, respectively; and SEQ ID NO: 21 and SEQ ID NO: 22, respectively 4. The binding agent of any one of embodiments 1 to 3, having an amino acid sequence selected from the pair of amino acid sequences set forth in the group consisting of: 5. The VH region and the VL region are SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO: 7 and SEQ ID NO: 8, respectively; and SEQ ID NO: 21 and SEQ ID NO: 22, respectively 5. The binding agent of any one of embodiments 1 to 4, having an amino acid sequence selected from the pair of amino acid sequences set forth in the group consisting of: 6. The binding agent of embodiment 1, wherein the framework regions are human framework regions. 7. The binding agent of any one of embodiments 1 to 6, which is an antibody or an antigen-binding portion thereof. 8. The binding agent of any one of embodiments 1 to 7, which is a monoclonal antibody, Fab, Fab', F(ab'), Fv, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody. 9. The binding agent of any one of embodiments 1 to 8, wherein the heavy chain variable region further comprises a heavy chain constant region. 10. The binding agent of embodiment 7, wherein the heavy chain constant region is of the IgG isotype. 11. The binding agent of embodiment 10, wherein the heavy chain constant region is an IgG1 constant region. 12. The binding agent of embodiment 10, wherein the heavy chain constant region is an IgG4 constant region. 13. The binding agent of embodiment 11, wherein the IgG1 constant region has the amino acid sequence set forth in SEQ ID NO: 39. 14. The binding agent of any one of embodiments 1 to 13, wherein the light chain variable region further comprises a light chain constant region. 15. The binding agent of embodiment 14, wherein the light chain constant region is of the kappa isotype. 16. The binding agent of embodiment 15, wherein the light chain constant region has the amino acid sequence set forth in SEQ ID NO: 40. 17. The binding agent of any one of embodiments 9 to 16, wherein the heavy chain constant region further comprises an amino acid modification that reduces binding affinity to at least human Fc gamma RIII. 18. The binding agent of any one of embodiments 1 to 17, which is monospecific. 19. The binding agent of any one of embodiments 1 to 18, which is bivalent. 20. The binding agent of any one of embodiments 1 to 17, which is bispecific. 21. A pharmaceutical composition comprising the binding agent of any one of embodiments 1 to 20 and a pharmaceutically acceptable carrier. 22. A nucleic acid encoding a binding agent according to any one of embodiments 1 to 20. 23. A vector comprising the nucleic acid of embodiment 22. 24. A cell line comprising the vector of embodiment 22 or the nucleic acid of embodiment 21. 25. A binder according to any one of embodiments 1 to 20, at least one linker attached to the binding agent; at least one drug attached to each linker; A conjugate comprising: 26. The conjugate of embodiment 25, wherein each drug is selected from a cytotoxic agent, an immunomodulator, a nucleic acid, a growth inhibitory agent, a PROTAC, a toxin, and a radioisotope. 27. The conjugate of any one of embodiments 25 to 26, wherein each linker is attached to the binder via an interchain disulfide residue, a lysine residue, an engineered cysteine ​​residue, a glycan, a modified glycan, the N-terminal residue of the binder, or a polyhistidine peptide attached to the binder. 28. The conjugate of 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 of any one of embodiments 25 to 28, wherein the drug is a cytotoxic agent. 30. The conjugate of embodiment 29, wherein the cytotoxic agent is selected from the group consisting of an auristatin, a maytansinoid, a camptothecin, a duocarmycin, or a calicheamicin. 31. The conjugate of embodiment 30, wherein the cytotoxic agent is an auristatin. 32. The conjugate of embodiment 31, wherein the cytotoxic agent is MMAE or MMAF. 33. The conjugate of embodiment 30, wherein the cytotoxic agent is camptothecin. 34. The conjugate of embodiment 33, wherein the cytotoxic agent is exatecan. 35. The conjugate of embodiment 33, wherein the cytotoxic agent is SN-38. 36. The conjugate of embodiment 30, wherein the cytotoxic agent is calicheamicin. 37. The conjugate of embodiment 30, wherein the cytotoxic agent is a maytansinoid. 38. The conjugate of embodiment 37, wherein the maytansinoid is maytansine, maytansinol, or maytansine analogues of DM1, DM3 and DM4, or ansamitocin-2. 39. The conjugate of any one of embodiments 25 to 38, wherein the linker comprises mc-VC-PAB, CL2, CL2A, or (succinimide-3-yl-N)-(CH2)nC(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)- (wherein n=1 to 5). 40. The conjugate of embodiment 39, wherein the linker comprises mc-VC-PAB. 41. The conjugate of embodiment 39, wherein the linker comprises CL2A. 42. The conjugate according to embodiment 39, wherein the linker comprises CL2. 43. The conjugate of embodiment 39, wherein the linker comprises (succinimide-3-yl-N)—(CH2)nC(═O)-Gly-Gly-Phe-Gly-NH-CH2-O—CH2-(C═O)—. 44. The conjugate according to embodiment 43, wherein the linker is attached to at least one molecule of exatecan. 45. The conjugate of any one of embodiments 25 to 28, wherein the drug is an immunomodulator. 46. ​​The conjugate of embodiment 45, wherein the immunomodulatory agent is selected from the group consisting of a TRL7 agonist, a TLR8 agonist, a STING agonist, or a RIG-I agonist. 47. The conjugate of embodiment 46, wherein the immunomodulatory agent is a TLR7 agonist. 48. The conjugate of embodiment 47, wherein the TLR7 agonist is imidazoquinoline, imidazoquinoline amine, thiazoquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazol-2-amine, tetrahydropyridopyrimidine, heteroaromatic azide-2,2-dioxide, benzonaphthyridine, guanosine analogue, adenosine analogue, thymidine homopolymer, ssRNA, CpG-A, polyG10, and polyG3. 49. The conjugate of embodiment 46, wherein the immunomodulatory agent is a TLR8 agonist. 50. The conjugate of embodiment 49, wherein the TLR8 agonist is selected from imidazoquinolines, thiazoloquinolines, aminoquinolines, aminoquinazolines, pyrido[3,2-d]pyrimidine-2,4-diamines, pyrimidine-2,4-diamines, 2-aminoimidazoles, 1-alkyl-1H-benzimidazol-2-amines, tetrahydropyridopyrimidines or ssRNA. 51. The conjugate of embodiment 46, wherein the immunomodulatory agent is a STING agonist. 52. The conjugate of embodiment 46, wherein the immunomodulatory agent is a RIG-I agonist. 53. The conjugate of embodiment 52, wherein the RIG-I agonist is selected from KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400 and KIN2000. 54. The conjugate of any one of embodiments 45 to 53, wherein the linker is selected from the group consisting of mc-VC-PAB, CL2, CL2A and (succinimide-3-yl-N)-(CH2)nC(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)- (wherein n=1 to 5). 55. A pharmaceutical composition comprising a conjugate according to any one of embodiments 25 to 54 and a pharmaceutically acceptable carrier. 56. A method for treating FOLR1+ cancer, comprising administering a therapeutically effective amount of a binding agent described in any one of embodiments 1 to 20, a conjugate described in any one of embodiments 25 to 54, or a pharmaceutical composition described in embodiment 21 or 55 to a subject in need thereof. 57. The method of embodiment 56, wherein the FOLR1+ cancer is a solid tumor. 58. The method of 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 carcinoma. 59. The method of any one of embodiments 56 to 58, further comprising administering immunotherapy to the subject. 60. The method of embodiment 59, wherein the immunotherapy comprises a checkpoint inhibitor. 61. The method of embodiment 60, wherein the checkpoint inhibitor is selected from an antibody that specifically binds to human PD-1, human PD-L1, or human CTLA4. 62. The method of embodiment 61, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab. 63. The method of any one of embodiments 56 to 62, further comprising administering chemotherapy to the subject. 64. The method according to any one of embodiments 56 to 63, comprising administering a conjugate according to any one of embodiments 25 to 54 or a pharmaceutical composition according to embodiment 55. 65. The method of any one of embodiments 56 to 64, wherein the binding agent, conjugate or pharmaceutical composition is administered intravenously. 66. The method of embodiment 6, wherein the binding agent, conjugate or pharmaceutical composition is administered at a dose of about 0.1 mg / kg to about 12 mg / kg. 67. The method of any one of embodiments 56 to 66, wherein the subject's outcome is improved. 68. The method of embodiment 67, wherein the improved outcome is an objective response selected from stable disease, partial response, or complete response. 69. The method of embodiment 67, wherein the improved outcome is a reduction in tumor burden. 70. The method of embodiment 67, wherein the improved outcome is progression-free survival or disease-free survival. 71. Use of a binding agent according to any one of embodiments 1 to 20 or a pharmaceutical composition according to embodiment 21 for treating FOLR1+ cancer in a subject. 72. Use of a conjugate according to any one of embodiments 25 to 54 or a 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 disclosure to the precise form disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes, but those skilled in the art will recognize that 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 appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as appropriate, to adopt the compositions, functions, and concepts of the above references and applications to provide still further embodiments of the present disclosure. These and other changes can be made to the present disclosure in light of the detailed description.

[0279] Specific elements of any of the foregoing embodiments may be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages to fall within the scope of the present disclosure.

[0280] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications that might be used in connection with the present invention. These publications are provided solely 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 prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0281] Example Example 1: Generation of human antibodies against human FOLR1 Antibodies targeting human FOLR-1 were screened using a fully human antibody library, a semi-synthetic human antibody library in which Fabs are displayed on the surface of phage.

[0282] Standard protocols were followed for library panning. Specifically, PolySorp or MaxiSorp Nunc-Immuno Tubes (Nunc-MG Scientific) were coated with 0.5 ml of 6 μg / ml human FOLR1 (ACRO-FO1-H52H1) antigen (see panning summary, Table 1) and placed in the refrigerator overnight. The tubes were washed once with PBS, blocked with 1% BSA / PBS, and incubated at room temperature for 1 hour. The tubes were then incubated with the indicated amount of library phage sample (CFU, see panning summary, Table 1) for 1 hour at room temperature. The tubes were washed 10 times with PBST buffer. To elute bound phage, 0.5 ml of 100 mM TEA (triethylamine) was added and incubated at room temperature 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 logarithmic-phase E. coli TG1 (OD600 ~ 0.5), mixed thoroughly, and incubated at 37°C (water bath) for 30 min without shaking. Ten-fold dilutions of the culture were made in 2xTY medium, and 10 μl of each dilution was plated onto TYE / amp / glu plates and incubated overnight at 30°C. The following day, the number of colonies in each dilution was counted, and the CFU (colony-forming units) of the panning output were calculated. The remaining culture was centrifuged at 2,800 g for 15 min, resuspended in 0.5 ml of 2xTY medium, plated onto two 150 mm TYE / amp / glu plates, and incubated overnight at 30°C. The following 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. Glycerol stocks were made by mixing 1.5 ml of bacteria with 0.5 ml of 80% glycerol and the stocks were placed at -80°C.

[0283] To prepare phage particles for the next round of selection, the glycerol stock was inoculated into 40 ml of 2xTY / amp / glu medium, starting at 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 30 minutes of shaking at 37°C. The bacterial culture was centrifuged at 3,000 rpm for 20 minutes, and the supernatant was removed. The pellet was resuspended in 100 ml of 2xTY / amp / glu medium and then grown overnight at 30°C with shaking. The culture was harvested by centrifugation at 6,000 g for 30 minutes. Phage particles were precipitated by adding 1 / 5 volume of PEG solution to the supernatant, followed by incubation on ice for 1 hour and centrifugation at 4,000 g for 20 minutes at 4°C. The supernatant was completely discarded. 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. 100 ul of logarithmic-phase E. coli TG1 was diluted 10-fold with phage solution (2xTY, 10 -11 The titer of the phage preparation was determined by infecting 1000 cells / ml of phage cultures (reduced to 10 ...

[0284] A total of four rounds of panning were performed, with the concentration of PBS-Tween 20 in the washing buffer increasing gradually to 0.2%, 0.3%, and 0.4% in the second, third, and fourth rounds, respectively.

[0285] After four rounds of screening, the target-positive enrichment rate was 1.5 × 10 4The results were significantly different from the blank control, as shown in Table 1. Clones from two 96-well plates were picked for phage ELISA validation; clones with high binding affinity to FOLR-1 were selected for sequencing.

[0286] A total of 69 clones were sequenced, yielding 12 unique VH sequences. Analysis of these 12 VH sequences revealed two unique HCDR3 sets, as shown in Tables 2 and 4. For the clones with the 12 unique VH sequences, the VL sequences were then determined. As shown in Tables 3 and 4, two unique VL sequences were obtained using two groups of unique LCDR3s.

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

[0288] [Table 1]

[0289] [Table 2]

[0290] [Table 3]

[0291] [Table 4]

[0292] [Table 5]

[0293] Example 2: Validation of antibodies produced by HEK293 cells After obtaining the sequences of the antibody clones (as described above), further analysis was performed using the complete IgG molecules. First, full-length antibody molecules with IgG1 Fc were expressed in 48- or 96-well microplates, and the supernatants were collected for detection of expression levels 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 in vector PTT5. HEK293 cells were harvested and cultured at 1 × 10 6 The cells were adjusted to a cell density of 1 / ml and plated at 200 or 400 μL per well into 48- or 96-well cell culture plates 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 and mixed thoroughly. 2.5 μL of transfection reagent T1 (plasmid:T1 = 1:5) was diluted in 20 μL of OPTI medium, mixed thoroughly, and incubated at room temperature for 5 minutes. The transfection reagent T1 diluent was added to the DNA, mixed thoroughly, and incubated at room temperature for 30 minutes. Transfection complexes formed during incubation. The transfection complexes were added to the cells, mixed thoroughly, and incubated at 37°C in a 5% CO2 incubator for 48 hours. When transfecting 48-well plates, the amounts of plasmid and transfection reagent were doubled. Two days after transfection, supernatants were collected and antibody bioactivity was detected by ELISA or FACS.

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

[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-link to cynomolgus monkey FOLR1 protein was tested by standard ELISA. Briefly, His-tagged human FOLR1 protein (ACRO-FO1-H52H1) or cynomolgus monkey FOLR1 protein (ACRO, F01-C52H8) was diluted to 5 μg / ml in a carbonate coating solution at pH 9.6, and 100 μL of the antigen was coated onto each well of a 96-well microtiter plate overnight at 4°C. The liquid in the wells was discarded, and the wells were washed three times with PBST. The wells were then blocked with 4% nonfat dry milk-PBS (Sigma, D5652-1L) at 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 three times with PBS. Samples were added at 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 three times with PBST. HRP-goat anti-human IgG (Sigma, I18885-2ML) was added (1:5000 dilution, 100 μL / well), and the plate was incubated at 37°C for 1 hour. The liquid in the wells was then discarded and the wells were washed five times with PBST. TMB solution was added using 100 μL / well. 2M H2SO4 was added using 50 μL to each well to stop the reaction after 10-15 minutes. The A450 value was read using a microplate reader.

[0297] The expression levels of IgG in microtiter plates and binding to human FOLR1 protein are shown in Table 6. All antibodies except clone F50 had normal binding to human FOLR1 protein.

[0298] The results of anti-FOLR1 antibody cross-binding to cynomolgus monkey FOLR1 protein are shown in Table 7. All antibodies except clone F50 had good cross-binding to cynomolgus monkey FOLR1 protein.

[0299] 2.4 Antibody binding to tumor cell lines expressing high FOLR1. The binding activity of the antibody to 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, 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 collected at 1 × 10 6 Cells were added to a 1.5 ml centrifuge tube at 1500 rpm for 5 minutes, and the supernatant was discarded. All procedures were then performed in an ice bath. 100 μL of transfection supernatant was added to each 1.5 ml centrifuge tube. Blank cells, blank cells + secondary antibody, medium, and HEK293 supernatant were set up as controls. The reaction was performed in an ice bath for 1 hour. Cells were then pelleted and washed twice with PBS. The secondary antibody, goat anti-human IgG (PE, Abcam, ab98596), was diluted (1:200) and added using 100 μL per tube. The reaction was performed in the dark in an ice bath for 1 hour. Cells were pelleted again, washed twice with PBS, resuspended in 300 μL of PBS, and FL2 fluorescence readings were measured by cytometry. Results were analyzed using FlowJo™10 software.

[0300] The results of anti-FOLR1 antibody binding 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 eight clones were positive for Hela cell binding.

[0301] The results of anti-FOLR1 antibody binding 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 nine clones were positive for RPTEC / TERT1 cell binding.

[0302] [Table 6] TIFF2025160300000020.tif48160

[0303] [Table 7]

[0304] Example 3: Characterization of anti-human FOLR1 antibodies produced by HEK293 cell expression in shake flasks Anti-FOLR1 antibody binding was quantitatively tested by expressing it in suspension cells to obtain sufficient amounts of protein. Plasmids were transfected into suspension cells for expression. The supernatant was collected for antibody purification. Highly purified antibodies were used to quantitatively detect antibody binding and internalization 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 and transfected at 1 × 10 6 The cells were adjusted to a cell density of 1 / ml and cultured in 30 mL of medium in a 125 mL shake flask in a shaker at 37°C with 5% CO2 for later use. For transfection, 30 μg of plasmid was diluted in 1500 μL of KPM medium and mixed thoroughly. 150 μL of transfection reagent T1 (plasmid:T1 = 1:5) was diluted in 1500 μL of KPM medium, mixed thoroughly, and incubated at room temperature for 5 minutes. The transfection reagent T1 diluent was added to the DNA, mixed thoroughly, and incubated at room temperature for 30 minutes to form the transfection complex. The transfection complex was added to the cells, mixed thoroughly, and incubated at 37°C with 5% CO2 at 120 rpm for 48 hours. TN1 solution was added to a final concentration of 0.5% 24 hours later. Six days after transfection, the supernatant was collected and purified.

[0306] Antibody purification was performed using standard processes 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, at a flow rate of 1 ml / min. Fractions of approximately 1.5–3.5 ml were collected and neutralized by adding 10% volume of 1 M Tris-HCl, pH 9.0. The antibody sample was then dialyzed twice overnight against 1x PBS 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. Tests were performed as described above. The results are shown in Figures 3 and 4. All antibodies bind to Hela cells and RPTEC / TERT1 cells in a dose-dependent manner.

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

[0311] HeLa or RPTEC / TERT1 cells were seeded at 15,000 cells per well in 100 μL and cultured at 37°C in a 5% CO2 incubator for 20–24 hours. pHAb-labeled test antibodies were added to the wells at a concentration of 10 μg / ml. Plates were then read on a Thermo Varioscan Flash using an excitation wavelength of 520 nm and an absorption wavelength of 570 nm at 0, 1, 4, 6, and 23 hours, respectively.

[0312] The results are shown in Figures 5 and 6. All tested anti-FOLR1 antibodies showed a time-dependent increase in pHAb fluorescence in FOLR1-expressing HeLa and RPTEC / TERT1 cells. These results indicate that each antibody was internalized into HeLa and RPTEC / TERT1 cells, with antibodies F8 and F131 having the strongest internalization rates.

[0313] Example 4: Characterization of anti-FOLR-1 immunoconjugates Further characterization of the anti-FOLR-1 antibody as an immunoconjugate was performed.

[0314] 4.1 Expression of the 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 U.S. Pat. No. 8,557,966 (SEQ ID NOs: 36 and 37, respectively) and codon-optimized. Optimized cDNAs encoding huFR107 and antibodies F8, F26, and F131 were constructed in the vector pcDNA3.4. The plasmids were then transiently transfected into ExpiCHO-S cells in Erlenmeyer flasks using the standard ExpiFectamine CHO Transfection procedure (Gibco, A29129). The suspended transient transfections were incubated for 10 days, and then the clarified supernatant was purified by protein A column followed by SDS-PAGE as described above.

[0315] 4.2 Preparation of anti-FOLR-1 immunoconjugates The pH of the antibody solution was adjusted to within the range of pH 7.0-7.5 by adding 0.5 M disodium phosphate. The indicated amount of 0.5 M 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 min. DMSO was then added to achieve a 10% v / v concentration. 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 cysteine ​​thiol available. The conjugation reaction was left at RT for 30 min. NAC (N-acetyl-L-cysteine) stock solution was added to achieve a NAC / Mc-VC-PAB-MMAE molar ratio of 5. The quenched reaction was left at RT for 15 min. Purification was carried out on a PD10 column.

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

[0317] The hydrophobicity of the anti-FOLR-1 immunoconjugates was evaluated by hydrophobic interaction chromatography (HIC) on a TosoHaas TSK Gel Butyl-NPR column (4.6 mm i.d. × 3.5 cm, 2.5 μm particle size) using a Waters HPLC E2695&2489 system. Briefly, the HPLC system was operated at 25 °C with 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 phase was filtered through a 0.22 μm membrane filter (Millipore) and run at a flow rate of 0.5 mL for 30 min. The linear gradient parameters are shown in Table 10. The DAR (drug-antibody ratio) of the anti-FOLR-1 immunoconjugates was determined according to the HIC data and was in the range of 3–4 (data not shown).

[0318] [Table 9]

[0319] [Table 10]

[0320] Example 5: Binding Characterization of Immunoconjugates Comparison of anti-FOLR1 conjugate binding to FOLR1-his or FOLR1 high-expressing tumor cell lines was performed by standard ELISA or FACS.

[0321] 5.1 ELISA testing. Recombinant His-tagged FOLR1 was coated onto a 96-well microplate (Thermo, catalog number: 468667) in PBS overnight at 2 μg / ml, 100 μL / well. 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 of blocking buffer (2% BSA / TBST) per well. The plate was placed 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 in a 1:3 serial dilution. The plate was placed 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 1:20,000 in blocking buffer and added to the plate at 100 μL per 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 left in the dark for 3-10 minutes. 50 μL of stop solution (2 M H2SO4) was added, and the optical density at 450 nm and 630 nm was read. Data were analyzed using GraphPad Prism 5 software.

[0322] The ELISA results are shown in Figures 7 and 8. The data showed that the activity of the conjugates 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 testing. FOLR1-expressing Hela cells, OVCAR3 cells (ATCC® HTB-161™, provided by COBIOER), OV90 cells (ATCC® CRL-11732™, provided by COBIOER), and IGROV-1 cells (provided by COBIOER) were incubated with various concentrations of anti-FOLR-1 conjugates. Each antibody conjugate was incubated for 0.5 hours in 0.1 ml of FACS buffer (PBS supplemented with 0.1% BSA). The cells were then pelleted, washed, and incubated with 0.1 ml of PE-conjugated goat anti-human IgG antibody (Abcam, Ab98596) for 0.5 hours. The cells were pelleted again, washed with PBS, and resuspended in 100 μL of PBS. Samples were analyzed using a CytoFLEX (Beckman).

[0324] The results are shown in Figures 9 and 10. There was no significant difference in binding to the cell lines by the three antibody conjugates compared to the reference antibody conjugate. All three anti-FOLR1 conjugates had stronger binding to OVCAR3 than to IGROV-1 cells or 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 x 10 cells were isolated from tissue culture flasks by treatment with 0.25% trypsin / EDTA. 5Cells were harvested and then incubated with each immunoconjugate at 10 micrograms / ml in FACS buffer (1x PBS containing 0.1% BSA) at 4°C for 30 minutes. A 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 set time points (0, 4, and 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 surface levels of immunoconjugates or isotype controls in Hela and OVCAR3 cell lines maintained at 4°C over the course of 4 or 24 hours. Surface levels of immunoconjugates significantly decreased when cells were shifted to 37°C over the course of the assay. This finding suggests that there were no significant differences in internalization of 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 anti-FOLR1 immunoconjugates in the tumor cell line OV90. The results showed that the internalization of F8-ADC was better than that of other ADCs in the OV90 cell line. From the results shown in Figure 14, it was not possible to determine internalization in the IGROV-1 tumor cell line.

[0329] The internalization results of 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 into 96-well solid white flat-bottom plates at the indicated amounts (according to cell growth rate) before adding anti-FOLR1 conjugates. The next day, cells were exposed to conjugates ranging in drug concentration from 30 micrograms / ml to 0.37 micrograms / ml or 100 micrograms / ml to 0.015 micrograms / ml using a 1:3 serial dilution in duplicate wells. Plates were incubated at 37°C for 120 hours. 40 μL of CTG (Promega, G7572) was then added per well to the plates, and after a 5-minute incubation, the plates were read on an MD I3X reader. Growth inhibition was measured as percent proliferation 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 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 Penalty kick BALB / c normal mice were purchased from JOINN Laboratories (Suzhou) and used after one week of incarceration. Mice were group-housed in sterile cages and maintained under pathogen-free conditions. Laboratory environmental conditions were as follows: temperature 20–22°C, humidity 59–78%, and 12 h of artificial lighting. Mouse cages were polysulfone boxes measuring 325 mm × 210 mm × 180 mm and autoclaved before use. Up to five animals were housed in each cage, and the experimental number, experimental start time, project leader, experimental personnel, animal source, group, and animal number were indicated on the cage card. Experimental animals were ear-marked. Mice were fed an FR-2 diet and provided with tap water (autoclaved before use). Mice weighed approximately 20–22 g at the time of administration.

[0336] Four groups, each containing six mice, were treated intravenously (IV) with a single 3 mg / kg dose of F8, F26, F131, or FR107 immunoconjugates. Blood samples were collected 10 minutes, 4 hours, 1 day, 4 days, 7 days, 10 days, 14 days, and 21 days after immunoconjugate administration, followed by centrifugation (4°C, 10,000 x g, 3 minutes) to separate serum. The total antibody concentration of each conjugate in the serum was detected by ELISA and analyzed using Winnonlin 8.2 software.

[0337] Goat anti-human IgG Fc (Invitrogen, 31125) was coated at 2 micrograms / ml onto a 96-well microplate (Thermo, catalog number: 468667) in PBS overnight at 4°C 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 in 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 (2 M H2SO4) was added, and the optical density was read at 450 nm and 630 nm. Data were analyzed using GraphPad Prism 5 software.

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

[0339] 8.2 Safety effects in mice. The mice used in the safety study were as described above. Five groups of six mice each were treated intravenously (IV) with a single 30 mg / kg dose of F8, F26, F131, and FR107 immunoconjugates. Animals were checked daily for feeding, drinking, and activity, weight gain / loss (body weight was measured once every two days), eye / hair matting, and other abnormal effects, and deaths and observed clinical signs were recorded.

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

[0341] Example 9: Affinity data of F131 for FOLR family proteins tested by BLI Recombinant proteins consisting of the extracellular domains of FOLR family proteins linked to His tags were purchased (from ACRO Systems) or synthesized in-house. For binding studies via biolayer interferometry (BLI), F131 (16.67 nM) was immobilized on an anti-human IgG Fc biosensor chip (Fortebio). Binding assays using various concentrations of recombinant antigen protein in solution (from 500 nM to 7.8 nM) were performed using an Octet RED (Fortebio) detector. The association time was set to 180 s and the dissociation time was set to 300 s. Binding affinities were calculated using ForteBio Data Acquisition 6.3 software (ForteBio), and affinities were 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, but had low responses to human FOLR2 and no responses to human FOLR3, demonstrating its binding specificity (Table 13). F131 exhibited high binding affinity to human and cynomolgus monkey FOLR1, with equilibrium dissociation constants (KD) of 1.5 nM and 8.1 nM, respectively. F131 showed no cross-reactivity with rat FOLR1 and low cross-reactivity with 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 assessed by flow cytometry (Beckman, Cytoflex) using a cell line with high FOLR1 target expression (JEG-3) or a cell line with no FOLR1 target expression (PC-3). 3 × 10 cells per well were cultured. 5 Cells were seeded into 96-well plates and incubated with 100 μl of F131 in serial dilutions. After incubation at 4° C. for 30 minutes, the cells were washed twice with PBS and stained with 1:200 diluted PE-conjugated anti-human Fc in 100 μl of FACS buffer (1×PBS containing 1% BSA), followed by incubation 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 (FIG. 21), but not to the human FOLR1-negative cell line, PC-3 (FIG. 22).

[0345] Example 11: F131 internalization in tumor cell lines Internalization assay was performed over time. 5 Individual cells were incubated with 10 μg / ml F131 in FACS buffer (1×PBS containing 0.1% BSA) at 4°C for 30 minutes. Cells were washed at 4°C to remove unbound material and kept on ice or transferred to 37°C as needed. At progressive time points (0, 0.5, 1, 2, 3, and 4 hours), cells were stained with PE-conjugated anti-human Fc for 30 minutes at 4°C 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, 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 not in FOLR1-non-expressing cells (PC-3) (Figure 23).

[0346] Example 12: In vivo efficacy of F131 conjugates The antitumor activity of F131 in conjugates with various benchmark linker-drugs (Table 15) was evaluated in a cell line-derived xenograft (CDX) model. To prepare F131-soraftansine, a solution of sulfo-SPDB-DM4 (10 mg / mL in DMSO) was added to 2 mL of antibody solution (10 mg / mL in 50 mM phosphate buffer containing 5 mM EDTA pH 7.4) to achieve a sulfo-SPDB-DM4 to mAb molar ratio of 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-deruxtecan, 2 mL of antibody (10 mg / mL) in 50 mM sodium phosphate buffer (pH 6.9) containing 5 mM EDTA was added to 10 mM aqueous TCEP HCl (tris(2-carboxyethyl)phosphine HCl) solution, resulting in a TCEP to mAb molar ratio of 8.0. The reduction reaction was carried out at 25 °C for 2 hours. Deruxtecan was dissolved in DMSO at a concentration of 20 mg / mL and added to the reduced antibody at a molar ratio of 12 (deruxtecan / mAb). The coupling reaction was stirred at 25 °C for 8 hours. Excess deruxtecan 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 antibody (10 mg / mL) in 50 mM sodium phosphate buffer (pH = 6.9) containing 5 mM EDTA was added to 10 mM aqueous TCEP HCl (tris(2-carboxyethyl)phosphine HCl) solution, resulting in a TCEP to mAb molar ratio of 2.2. The reduction reaction was carried out at 25 °C for 2 h. 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 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. To characterize the target (FOLR1) copy number (binding site) 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. Then, the cells, setup beads, and calibration beads were labeled in parallel with a fluorescein-conjugated anti-mouse secondary antibody. Samples were analyzed by flow cytometry, and the copy number was calculated based on the calibration curve. For CDX testing using the F131 conjugate, an appropriate amount of cells suspended in either Matrigel / medium (1:1) or medium was subcutaneously injected into female BALB / c nude mice. On days 6–26 after tumor inoculation, tumors with an average size of 110–180 mm were obtained. 3 Mice were selected and assigned to treatment groups (n = 6-9 per group) using stratified randomization based on tumor volume. Treatment with intravenous injection of F131 conjugate or vehicle control began 1 day after randomization and was administered in either a single-dose (day 1, Figures 24, 25, 26, 32, 33) or multiple-dose (days 1, 4, 8, 11) model (Figures 27, 28, 29, 30, 31). Tumor size was measured twice weekly using standard methods. Animal weight was monitored as an indirect measure of toxicity. No morbidity or mortality was observed in any of the treatment groups during the treatment period. Compared to vehicle control, the F131 conjugate provided substantial tumor growth inhibition in all models tested.

[0347] [Table 15]

[0348] Example 13: PK study of F131 and conjugates in a rat model F131 and its conjugates were intravenously administered to male Sprague-Dawley rats at a single dose of 3 mg / kg (n=3 / group). Orbital blood samples were taken from each rat at various time points after administration. Total Ab concentrations (detecting F131 and its conjugates in plasma) were analyzed by ELISA kit (Genscript) and calculated using Winnonlin 8.2 software. F131-deruxtecan showed excellent PK in rats, indistinguishable from that of the parent mAb (Figure 34). F131-vedotin showed stable PK in rats, although its clearance appeared to be somewhat faster than that of the parent mAb (Figure 35).

[0349] Example 14: F131-deruxtecan PK and tolerability in a pilot cynomolgus monkey toxicity study F131-deruxtecan was administered intravenously to one male and one female cynomolgus monkey at a single dose of 60 mg / kg on Day 1. Clinical signs, body weight, food consumption, and clinical pathology were monitored throughout the study. Necropsy was scheduled for Day 22. Toxicokinetic samples were collected from each animal at 0, 24, 72, 120, 336, and 504 hours after the end of dosing. Total Ab concentrations, expressed as F131 and F131 conjugates in plasma, were analyzed by ELISA kit (Genscript) and calculated using Winnonlin 8.2 software. Both animals survived until the scheduled necropsy. Clinical findings, hematology, and clinical chemistry are shown in Table 16 and Figures 36 and 37. All changes showed a tendency toward recovery by Day 22. No toxicological abnormalities were observed in body weight, temperature, coagulation, urinalysis, or gross necropsy. F131-deruxtecan exhibited stable pharmacokinetic properties in cynomolgus monkey plasma (FIG. 38).

[0350] [Table 16]

[0351] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0352] Various publications, including patents, patent application publications, and scientific literature, are cited herein, the disclosures of which are incorporated by reference in their entireties for all purposes.

[0353] [Sequence table] SEQ ID NO: 1 F1 VH amino acid sequence EVQLLESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA VISYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR PRAYYGAYGSSFDY WGQGTQVTVSS SEQ ID NO: 2 F1 VL amino ac...

Claims

1. 1. A binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, The VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, and the VH and VL CDRs are a. 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; and b. 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 A binding agent having an amino acid sequence selected from the set of amino acid sequences set forth in the group consisting of:

2. the VH region and the VL region a. SEQ ID NO: 1 and SEQ ID NO: 2, respectively; b. SEQ ID NO: 3 and SEQ ID NO: 4, respectively; c. SEQ ID NO:5 and SEQ ID NO:6, respectively; d. SEQ ID NO: 7 and SEQ ID NO: 8, respectively; e. SEQ ID NO: 9 and SEQ ID NO: 10, respectively; f. SEQ ID NO:11 and SEQ ID NO:12, respectively; g. SEQ ID NO: 13 and SEQ ID NO: 14, respectively; h. SEQ ID NO: 15 and SEQ ID NO: 16, respectively; i. SEQ ID NO: 17 and SEQ ID NO: 18, respectively; j. SEQ ID NO: 19 and SEQ ID NO: 20, respectively; k. SEQ ID NO: 21 and SEQ ID NO: 22, respectively; and l. SEQ ID NO: 23 and SEQ ID NO: 24, respectively having an amino acid sequence selected from the set of amino acid sequence pairs shown in the group consisting of:

2. The binding agent of claim 1, wherein the heavy chain framework regions and the light chain framework regions are optionally modified by substitution, deletion or insertion of 1 to 8 amino acids within the framework regions.

3. the VH region and the VL region a. SEQ ID NO: 1 and SEQ ID NO: 2, respectively; b. SEQ ID NO: 3 and SEQ ID NO: 4, respectively; c. SEQ ID NO:5 and SEQ ID NO:6, respectively; d. SEQ ID NO: 7 and SEQ ID NO: 8, respectively; e. SEQ ID NO: 9 and SEQ ID NO: 10, respectively; f. SEQ ID NO:11 and SEQ ID NO:12, respectively; g. SEQ ID NO: 13 and SEQ ID NO: 14, respectively; h. SEQ ID NO: 15 and SEQ ID NO: 16, respectively; i. SEQ ID NO: 17 and SEQ ID NO: 18, respectively; j. SEQ ID NO: 19 and SEQ ID NO: 20, respectively; k. SEQ ID NO: 21 and SEQ ID NO: 22, respectively; and l. SEQ ID NO: 23 and SEQ ID NO: 24, respectively 3. The binding agent of claim 1, having an amino acid sequence selected from the set of amino acid sequence pairs shown in the group consisting of:

4. the VH region and the VL region a. SEQ ID NO: 3 and SEQ ID NO: 4, respectively; b. SEQ ID NO: 7 and SEQ ID NO: 8, respectively; c. SEQ ID NO: 9 and SEQ ID NO: 10, respectively; d. SEQ ID NO:11 and SEQ ID NO:12, respectively; e. SEQ ID NO: 15 and SEQ ID NO: 16, respectively; f. SEQ ID NO: 17 and SEQ ID NO: 18, respectively; g. SEQ ID NO: 19 and SEQ ID NO: 20, respectively; and h. SEQ ID NO: 21 and SEQ ID NO: 22, respectively 4. The binding agent according to any one of claims 1 to 3, having an amino acid sequence selected from the set of amino acid sequence pairs shown in the group consisting of:

5. the VH region and the VL region a. SEQ ID NO: 3 and SEQ ID NO: 4, respectively; b. SEQ ID NO: 7 and SEQ ID NO: 8, respectively; and c. SEQ ID NO: 21 and SEQ ID NO: 22, respectively 5. The binding agent of claim 1, having an amino acid sequence selected from the set of amino acid sequence pairs shown in the group consisting of:

6. 2. The binding agent of claim 1, wherein the framework regions are human framework regions.

7. 7. The binding agent of any one of claims 1 to 6, which is an antibody or an antigen-binding portion thereof.

8. 8. The binding agent of any one of claims 1 to 7, which is a monoclonal antibody, Fab, Fab', F(ab'), Fv, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody.

9. 9. The binding agent of any one of claims 1 to 8, wherein the heavy chain variable region further comprises a heavy chain constant region.

10. The binding agent of claim 7, wherein the heavy chain constant region is of the IgG isotype.

11. 11. The binding agent of claim 10, wherein the heavy chain constant region is an IgG1 constant region.

12. 11. The binding agent of claim 10, wherein the heavy chain constant region is an IgG4 constant region.

13. 12. The binding agent of claim 11, wherein the IgG1 constant region has the amino acid sequence set forth in SEQ ID NO:

39.

14. 14. The binding agent of any one of claims 1 to 13, wherein the light chain variable region further comprises a light chain constant region.

15. 15. The binding agent of claim 14, wherein the light chain constant region is of the kappa isotype.

16. 16. The binding agent of claim 15, wherein the light chain constant region has the amino acid sequence set forth in SEQ ID NO:

40.

17. 17. The binding agent of any one of claims 9 to 16, wherein the heavy chain constant region further comprises an amino acid modification that reduces binding affinity to at least human Fc gamma RIII.

18. 18. The binding agent of any one of claims 1 to 17, which is monospecific.

19. 19. The binding agent of any one of claims 1 to 18, which is bivalent.

20. 18. The binding agent of any one of claims 1 to 17, which is bispecific.

21. 21. A pharmaceutical composition comprising the binding agent of any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

22. 21. A nucleic acid encoding the binding agent of any one of claims 1 to 20.

23. 23. A vector comprising the nucleic acid of claim 22.

24. 23. A cell line comprising the vector of claim 22 or the nucleic acid of claim 21.

25. A binder according to any one of claims 1 to 20; at least one linker attached to the binding agent; at least one drug attached to each linker; A conjugate comprising:

26. 26. The conjugate of claim 25, wherein each drug is selected from a cytotoxic agent, an immunomodulatory agent, a nucleic acid, a growth inhibitory agent, a PROTAC, a toxin, and a radioisotope.

27. 27. The conjugate of any one of claims 25 to 26, wherein each linker is attached 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 attached to the binder.

28. 28. The conjugate of any one of claims 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. 29. The conjugate of any one of claims 25 to 28, wherein the drug is a cytotoxic agent.

30. 30. The conjugate of claim 29, wherein the cytotoxic agent is selected from the group consisting of an auristatin, a maytansinoid, a camptothecin, a duocarmycin, or a calicheamicin.

31. 31. The conjugate of claim 30, wherein the cytotoxic agent is an auristatin.

32. 32. The conjugate of claim 31, wherein the cytotoxic agent is MMAE or MMAF.

33. 31. The conjugate of claim 30, wherein the cytotoxic agent is camptothecin.

34. 34. The conjugate of claim 33, wherein the cytotoxic agent is exatecan.

35. 34. The conjugate of claim 33, wherein the cytotoxic agent is SN-38.

36. 31. The conjugate of claim 30, wherein the cytotoxic agent is calicheamicin.

37. 31. The conjugate of claim 30, wherein the cytotoxic agent is a maytansinoid.

38. 38. The conjugate of claim 37, wherein the maytansinoid is maytansine, maytansinol, or maytansine analogs of DM1, DM3 and DM4, or ansamitocin-2.

39. The linker is mc-VC-PAB, CL2, CL2A or (succinimide-3-yl-N)-(CH 2 ) n -C(=O)-Gly-Gly-Phe-Gly-NH-CH 2 -O-CH 2 39. The conjugate of any one of claims 25 to 38, comprising -(C=O)-, where n=1 to 5.

40. 40. The conjugate of claim 39, wherein the linker comprises mc-VC-PAB.

41. 40. The conjugate of claim 39, wherein the linker comprises CL2A.

42. 40. The conjugate of claim 39, wherein the linker comprises CL2.

43. The linker is (succinimide-3-yl-N)-(CH 2 ) n -C(=O)-Gly-Gly-Phe-Gly-NH-CH 2 -O-CH 2 40. The conjugate of claim 39, comprising -(C=O)-.

44. 44. The conjugate of claim 43, wherein the linker is attached to at least one molecule of exatecan.

45. 29. The conjugate of any one of claims 25 to 28, wherein the drug is an immunomodulatory agent.

46. 46. ​​The conjugate of claim 45, wherein the immunomodulatory agent is selected from the group consisting of a TRL7 agonist, a TLR8 agonist, a STING agonist, or a RIG-I agonist.

47. 47. The conjugate of claim 46, wherein the immunomodulatory agent is a TLR7 agonist.

48. 48. The conjugate of claim 47, wherein the TLR7 agonist is imidazoquinoline, imidazoquinoline amine, thiazoquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazol-2-amine, tetrahydropyridopyrimidine, heteroarothiadiazide-2,2-dioxide, benzonaphthyridine, guanosine analogue, adenosine analogue, thymidine homopolymer, ssRNA, CpG-A, polyG10, and polyG3.

49. 47. The conjugate of claim 46, wherein the immunomodulatory agent is a TLR8 agonist.

50. 50. The conjugate of claim 49, wherein the TLR8 agonist is selected from imidazoquinolines, thiazoloquinolines, aminoquinolines, aminoquinazolines, pyrido[3,2-d]pyrimidine-2,4-diamines, pyrimidine-2,4-diamines, 2-aminoimidazoles, 1-alkyl-1H-benzimidazol-2-amines, tetrahydropyridopyrimidines, or ssRNA.

51. 47. The conjugate of claim 46, wherein the immunomodulatory agent is a STING agonist.

52. 47. The conjugate of claim 46, wherein the immunomodulatory agent is a RIG-I agonist.

53. 53. The conjugate of claim 52, wherein the RIG-I agonist is selected from KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400 and KIN2000.

54. The linker is mc-VC-PAB, CL2, CL2A, and (succinimide-3-yl-N)-(CH 2 ) n -C(=O)-Gly-Gly-Phe-Gly-NH-CH 2 -O-CH 2 54. The conjugate of any one of claims 45 to 53, selected from the group consisting of -(C=O)-, where n=1 to 5.

55. 55. A pharmaceutical composition comprising the conjugate of any one of claims 25 to 54 and a pharmaceutically acceptable carrier.

56. A method for treating FOLR1+ cancer, comprising administering a therapeutically effective amount of a binding agent described in any one of claims 1 to 20, a conjugate described in any one of claims 25 to 54, or a pharmaceutical composition described in claim 21 or 55 to a subject in need thereof.

57. 57. The method of claim 56, wherein the FOLR1+ cancer is a solid tumor.

58. 58. The method of claim 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. 59. The method of any one of Claims 56 to 58, further comprising administering to the subject an immunotherapy.

60. 60. The method of claim 59, wherein the immunotherapy comprises a checkpoint inhibitor.

61. 61. The method of claim 60, wherein the checkpoint inhibitor is selected from an antibody that specifically binds to human PD-1, human PD-L1, or human CTLA4.

62. 62. The method of claim 61, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

63. 63. The method of any one of claims 56 to 62, further comprising administering chemotherapy to the subject.

64. 64. The method of any one of claims 56 to 63, comprising administering a conjugate of any one of claims 25 to 54 or a pharmaceutical composition of claim 55.

65. 65. The method of any one of claims 56 to 64, wherein the binding agent, conjugate or pharmaceutical composition is administered intravenously.

66. 7. The method of claim 6, wherein the binding agent, conjugate or pharmaceutical composition is administered at a dose of about 0.1 mg / kg to about 12 mg / kg.

67. 67. The method of any one of claims 56 to 66, wherein the subject's outcome is improved.

68. 68. The method of claim 67, wherein the improved outcome is an objective response selected from stable disease, partial response, or complete response.

69. 68. The method of claim 67, wherein the improved outcome is a reduction in tumor burden.

70. 68. The method of claim 67, wherein the improved outcome is progression-free survival or disease-free survival.

71. 22. Use of a binding agent according to any one of claims 1 to 20 or a pharmaceutical composition according to claim 21 for treating FOLR1+ cancer in a subject.

72. 56. Use of a conjugate according to any one of claims 25 to 54 or a pharmaceutical composition according to claim 55 for treating FOLR1+ cancer in a subject.

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