Antibodies specific for the folate receptor alpha

Abstract

Antibodies specific for folate receptor alpha (also known as folate receptor 1 or FOLR1) and their uses for therapeutic and diagnostic purposes. Also provided herein are chimeric antigen receptors (CARs) comprising an extracellular antigen-binding fragment that binds to FOLR1 and immune cells expressing the same.

Description

[Technical field]

[0001] 2. Background of the Invention Folate receptor alpha, also known as folate receptor 1 (FOLR1), belongs to the folate receptor family, whose members have high binding affinity to folic acid and / or its derivatives (e.g., 5-methyltetrahydrofolate).FOLR1 has been reported to be overexpressed in a number of epithelial-derived tumors, including ovarian, breast, renal, lung, colorectal, and brain.Therefore, this receptor can be a target for the treatment of such epithelial-derived tumors. Therefore, it is of great importance to develop effective FOLR1 antagonists, such as anti-FOLR1 antibodies, for use in both cancer treatment and diagnosis. Summary of the Invention

[0002] SUMMARY OF THE PRESENT APPLICATION The present disclosure is based, at least in part, on the development of a number of antibodies specific for FOLR1. Such antibodies have high binding affinity to the target FOLR1 antigen and / or FOLR1 + It showed high inhibitory activity against the cells. Accordingly, one aspect of the disclosure features an isolated antibody that binds to FOLR1 (anti-FOLR1 antibody), where the antibody binds to the same epitope of human FOLR1 as a reference antibody, which is FOLR1-Ab1, FOLR1-Ab4, FOLR1-Ab14, FOLR1-Ab20, or FOLR1-Ab23, the structural characteristics of each of which are provided herein.

[0003] In some embodiments, the anti-FOLR1 antibodies described herein are (a)X 1 YTFTX 2 YX 3 Heavy chain complementarity determining region 1 (HC CDR1), represented as 1 is G or I, and X 2 is D or S, and X 3 is W, N, or S; (b)INX 1 X 2 X 3 X 4 X 5 X 6 Heavy chain complementarity determining region 2 (HC CDR2), represented as: 1 is P or T, and X 2 is N, Y, or E, and X 3 is N, D, or T, and X 4 is G or S, and X 5 is G or E, and X 6 is T or P; and (c)ARX 1 X 2 X 3 YX 4 X 5 X 6 X 7 X 8 X 9 X 10 Heavy chain complementarity determining region 3 (HC CDR3), represented as: 1 is S, K or M, and X 2 is G or P, and X 3 is G or Y, and X 4 is G or absent, X 5 is P or absent, and X 6 is A, R, or K, and X 7 is W, Y or I, and X 8 is F or M, and X 9 is D or A, and X 10 is a heavy chain variable region (V H ).

[0004] Such anti-FOLR1 antibodies include Hwherein HC CDR1, HC CDR2, and HC CDR3 are collectively at least 85% (e.g., at least 90%, at least 95%, at least 98% or more) identical to the HC CDR1, HC CDR2, and HC CDR3 of the reference antibody. In some cases, the antibody may comprise a V that contains the same HC CDR1, HC CDR2, and HC CDR3 as one of the reference antibodies noted above. H In other embodiments, the anti-FOLR1 antibodies described herein may comprise a V sequence comprising HC CDR1, HC CDR2, and HC CDR3 that collectively comprise up to 5, 4, 3, 2, or 1 mutations relative to the HC CDR1, HC CDR2, and HC CDR3 of a reference antibody. H may include.

[0005] Alternatively or additionally, the anti-FOLR1 antibodies described herein may be characterized as having: (a) light chain complementarity determining region 1 (LC CDR1), denoted as ESVDNYGISF or QSLLYSSSQKNY; (b)X 1 Light chain complementarity determining region 2 (LC CDR2), denoted as AS, where X 1 is V, A, or W; and (c)QQX 1 X 2 X 3 X 4 PX 5 Light chain complementarity determining region 3 (LC CDR3), denoted as T, where X 1 is Y or S, and X 2 is Y or K, and X 3 is E or S, and X 4 is Y or V, and X 5 is a light chain variable region (V) that includes one or more of W, Y, or absent. L ).

[0006] Such antibodies are Lwherein the LC CDR1, LC CDR2, and LC CDR3 are collectively at least 85% (e.g., at least 90%, at least 95%, at least 98% or more) identical to the LC CDR1, LC CDR2, and LC CDR3 of the reference antibody. In some cases, the antibody may comprise the same LC CDR1, LC CDR2, and LC CDR3 as one of the reference antibodies noted above. In other embodiments, the anti-FOLR1 antibodies described herein may comprise an LC CDR1, LC CDR2, and LC CDR3 that collectively comprise up to 5, 4, 3, 2, or 1 mutation relative to the LC CDR1, LC CDR2, and LC CDR3 of the reference antibody.

[0007] In some examples, the anti-FOLR1 antibodies described herein contain the same heavy and / or light chain CDRs as one of the reference antibodies noted above. In some cases, such anti-FOLR1 antibodies contain the same V and / or V CDRs as the reference antibody. H and / or V L may include. Any of the anti-FOLR1 antibodies described herein can specifically bind to human FOLR1. In some cases, the anti-FOLR1 antibody can cross-react with human FOLR1 and non-human FOLR1, such as rodent FOLR1 or primate FOLR1. The antibody can be a human antibody or a humanized antibody. In some cases, it can be a chimeric antibody. In some embodiments, the anti-FOLR1 antibody can be a full-length antibody (e.g., an IgG molecule) or an antigen-binding fragment thereof. Alternatively, it can be a single-chain antibody.

[0008] In another aspect, the present disclosure features a nucleic acid or a set of nucleic acids (e.g., two nucleic acids) that collectively encode any of the anti-FOLR1 antibodies described herein, and a vector or a set of vectors (e.g., two vectors) that includes a nucleic acid(s) encoding an anti-FOLR1 antibody. In some cases, the vector or set of vectors can be an expression vector(s). Also provided herein is a host cell that includes a nucleic acid(s) or a vector(s). Furthermore, the present disclosure provides a method for producing an anti-FOLR1 antibody described herein, the method comprising culturing a host cell that includes a vector or a set of vectors that includes an antibody coding sequence, where the coding sequence is operably linked to a suitable promoter, and collecting the antibody so produced, for example, from the host cell or culture medium. In addition, the disclosure provides antibody-drug conjugates (ADCs) comprising any of the anti-FOLR1 antibodies described herein and at least one therapeutic agent covalently conjugated to the antibody. In some examples, the therapeutic agent can be a cytotoxic agent, such as monomethylauristatin E.

[0009] In some embodiments, the antibody and the therapeutic agent may be conjugated through a linker. In some instances, the linker may be a cleavable linker, such as a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker. In some cases, the linker may be a protease-sensitive linker that may include a peptide having 2-5 amino acids. The peptide may include naturally occurring amino acid residues, non-naturally occurring amino acid residues, or a combination thereof. In one example, the peptide may include valine-citrulline. In another example, the linker may be a non-cleavable linker. Such a non-cleavable linker may include an optionally substituted alkane or thioether.

[0010] In some embodiments, the linker may comprise a functional group that forms a covalent bond between the antibody and the linker. Exemplary functional groups include, but are not limited to, maleimide groups, iodoacetamide groups, vinylsulfone groups, acrylate groups, acrylamide groups, acrylonitrile groups, and methacrylate groups. In one example, the linker is represented by Formula I: [ka] During the ceremony, R1 may further include a molecular spacer represented by: optionally substituted C1-6 alkyl, optionally substituted phenyl, optionally substituted C2-6 alkylene, optionally substituted C2-6 alkenylene, optionally substituted C2-6 alkynylene, or optionally substituted triazole; and X is O, S, or N.

[0011] Further, the present disclosure provides a chimeric antigen receptor (CAR) that may include (i) an extracellular domain that includes an antigen-binding fragment that binds to FOLR1, (ii) a transmembrane domain, and (iii) one or more intracellular stimulatory domains. The antigen-binding fragment may bind to the same epitope of human FOLR1 as any of the reference antibodies described herein. In some examples, the antigen-binding fragment may include the same HC CDRs and / or LC CDRs of any of the reference antibodies. Such antigen-binding fragments may include the same V CDRs as the reference antibodies. H and V L In some examples, the antigen-binding fragment may be a single chain antibody (scFv).

[0012] In any of the CARs described herein, the transmembrane domain may comprise a transmembrane domain derived from CD28 or CD8. Alternatively or in addition, the one or more intracellular stimulatory domains may comprise a signaling domain from CD3zeta, and optionally a costimulatory signaling domain that may be from 4-1BB, CD7, CD27, CD28, CD40, OX40, ICOS, GITR, HVEM, TIM1, or LFA-1. The nucleic acid encoding any of the CARs described herein, the vector comprising it, and the host cell expressing the CAR are also within the scope of the present disclosure. In some examples, the host cell expressing the CAR is an immune cell, such as a T cell.

[0013] In another aspect, the disclosure provides a pharmaceutical composition comprising (i) a host cell expressing one or more of the anti-FOLR1 antibodies described herein, a nucleic acid or set of nucleic acids encoding same, an antibody-drug conjugate described herein, or a CAR construct described herein, and (ii) a pharma- ceutical composition comprising a pharma-ceutical acceptable carrier. Moreover, the present disclosure provides + The present disclosure also features a method for reducing the number of cells, comprising administering an effective amount of any of the pharmaceutical compositions described herein to a subject in need thereof.In some embodiments, the subject may be a human patient with cancer or suspected of having cancer, for example, the cancer is epithelial cancer.Also within the scope of the present disclosure are the pharmaceutical compositions described herein for use in treating any of the target diseases described herein (e.g., cancers such as epithelial cancers) or for use in manufacturing medicaments for treating target diseases.

[0014] In addition, the present disclosure provides + The present invention features a method for detecting the presence of i.FOLR1 cells, the method comprising: + contacting a sample suspected of having cells with any of the anti-FOLR1 antibodies described herein that are conjugated to a labeling agent; and ii. detecting FOLR1 in the sample. +The method includes detecting the presence of a cell based on binding of an antibody to a cell in the sample. In some cases, the sample is derived from a human patient at risk for or suspected of having a cancer, such as an epithelial cancer. The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims. [Brief description of the drawings]

[0015] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] 1A-1E include diagrams showing that a number of anti-FOLR1 antibodies, including FOLR1-Ab1, FOLR1-Ab4, FOLR1-Ab14, FOLR1-Ab20, and FOLR1-Ab23, bound to cells expressing surface FOLR1. [Figure 1B] 1A-1E include diagrams showing that a number of anti-FOLR1 antibodies, including FOLR1-Ab1, FOLR1-Ab4, FOLR1-Ab14, FOLR1-Ab20, and FOLR1-Ab23, bound to cells expressing surface FOLR1. [Figure 1C] 1A-1E include diagrams showing that a number of anti-FOLR1 antibodies, including FOLR1-Ab1, FOLR1-Ab4, FOLR1-Ab14, FOLR1-Ab20, and FOLR1-Ab23, bound to cells expressing surface FOLR1. [Figure 1D] 1A-1E include diagrams showing that a number of anti-FOLR1 antibodies, including FOLR1-Ab1, FOLR1-Ab4, FOLR1-Ab14, FOLR1-Ab20, and FOLR1-Ab23, bound to cells expressing surface FOLR1. [Figure 1E] 1A-1E include diagrams showing that a number of anti-FOLR1 antibodies, including FOLR1-Ab1, FOLR1-Ab4, FOLR1-Ab14, FOLR1-Ab20, and FOLR1-Ab23, bound to cells expressing surface FOLR1.

[0016] [Figure 2A-B] 2A-2D include diagrams showing the inhibitory effects of exemplary anti-FOLR1 antibodies shown on FOLR1+ SK-OV-3 cells. [Figure 2C-D] 2A-2D include diagrams showing the inhibitory effects of exemplary anti-FOLR1 antibodies shown on FOLR1+ SK-OV-3 cells.

[0017] Detailed Description of the Invention High binding affinity to the target FOLR1 antigen and / or FOLR1 + Disclosed herein are a number of anti-FOLR1 antibodies that exhibit excellent characteristics, including high inhibitory activity against cells. Accordingly, provided herein are antibodies capable of binding to FOLR1, nucleic acids encoding same, antibody-drug conjugates (ADCs) and chimeric antigen receptors (CARs) comprising anti-FOLR1 antibodies, and their uses for both therapeutic and diagnostic purposes. Also provided herein are kits for the therapeutic and / or diagnostic use of the antibodies and / or ADCs and CARs comprising same, as well as methods for producing anti-FOLR1 antibodies.

[0018] Antibody that binds to FOLR1 The present disclosure provides an antibody that binds to folate receptor alpha, also known as folate receptor 1 (FOLR1). As a member of the folate receptor family, FOLR1 has high binding affinity to folate and its derivatives. In humans, FOLR1 is encoded by the FOLR1 gene. FOLR1 has been found to be overexpressed in various epithelial tumors (e.g., ovarian cancer). Thus, this receptor can serve as a target and / or biomarker for the treatment and diagnosis of target cancer. Consequently, the anti-FOLR1 antibodies disclosed herein can be used in treating and / or diagnosing the target cancers described herein, either by themselves or conjugated to other moieties, for example, conjugated to therapeutic agents to form antibody-drug conjugates, or by becoming the extracellular antigen-binding domain in chimeric antigen receptors.

[0019] Antibodies (used interchangeably in the plural) are immunoglobulin molecules capable of specifically binding to targets such as carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact (i.e., full-length) polyclonal or monoclonal antibodies, as well as antigen-binding fragments thereof (Fab, Fab', F(ab')2, Fv, etc.), single chains (scFv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, bispecific antibodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified conformation of an immunoglobulin molecule that comprises an antigen recognition site of the required specificity (including glycosylation variants of antibodies), amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0020] A typical antibody molecule consists of a heavy chain variable region (V H ) and the light chain variable region (V L ), which are mostly involved in antigen binding. H and V L The regions can be further subdivided into regions of high variability, also known as "complementarity determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs").H and V L is typically composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of framework regions and CDRs can be precisely identified using methodologies known in the art (e.g., by the Kabat definition, the Chothia definition, the AbM definition, and / or the Contact definition), all of which are well known in the art. See, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.

[0021] In some embodiments, the anti-FOLR1 antibodies described herein can bind to and inhibit the activity of FOLR1 by at least 50% (e.g., 60%, 70%, 80%, 90%, or 95% or more). The apparent inhibition constant (Ki) provides a measure of the potency of the inhibitor. app or K i,app ) is related to the concentration of inhibitor required to reduce enzyme activity and is independent of enzyme concentration. The inhibitory activity of the anti-FOLR1 antibodies described herein can be determined by routine methods known in the art.

[0022] Antibody Ki, app The value can be determined by measuring the inhibitory effect of various concentrations of antibody on the extent of the reaction (e.g., enzyme activity). Fitting the change in the pseudo-first-order rate constant (v) as a function of inhibitor concentration to a modified Morrison equation (Equation 1) provides an estimate of the apparent Ki value. For competitive inhibitors, the Ki app is K i, app It can be obtained from the y-intercept extracted from linear regression analysis of a plot of versus substrate concentration.

number

[0023] In some embodiments, the anti-FOLR1 antibodies described herein have a Ki of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 pM or less for a FOLR1 antigen or antigenic epitope thereof. app In some embodiments, the anti-FOLR1 antibody may have a lower Ki value for a first target compared to a second target. app It may have Ki app The difference (e.g., for specificity or other comparisons) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 It could be double.

[0024] The antibodies described herein may be murine, rat, human, or of any other origin (including chimeric or humanized antibodies). Such antibodies are not naturally occurring, i.e., are not produced in an animal without the action of a human (e.g., immunizing such an animal with a desired antigen or a fragment thereof). Any of the antibodies described herein can be either monoclonal or polyclonal. A "monoclonal antibody" refers to a homogeneous antibody population, whereas a "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the source of the antibody or the method by which it is made.

[0025] In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibody refers to a form of non-human (e.g., murine) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or antigen-binding fragment thereof that contains minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient complementarity determining region (CDR) are replaced by residues from the CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit that has the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the imported CDR or framework sequence, but are included to further refine and optimize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of a human immunoglobulin consensus sequence. A humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an altered Fc region as described in WO99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, and / or six) that are altered with respect to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. A humanized antibody may also be affinity matured.

[0026] In another example, the antibody described herein may be a chimeric antibody that may include heavy and light chain constant regions from a human antibody. Chimeric antibody refers to an antibody that has a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable region of an antibody from one species of mammal (e.g., non-human mammals such as mice, rabbits, and rats), while the constant portion is homologous to the sequence of an antibody from another mammal, such as humans. In some embodiments, amino acid modifications may be made in the variable and / or constant regions.

[0027] In some embodiments, the anti-FOLR1 antibodies described herein specifically bind to the corresponding target antigen or epitope thereof. An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, with a longer duration, and / or with greater affinity to a particular target antigen than it does to alternative targets. An antibody "specifically binds" to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to a FOLR1 antigen or an antigenic epitope therein is an antibody that binds to this target antigen with greater affinity, avidity, more readily, and / or with greater duration than it binds to other antigens or other epitopes of the same antigen. By this definition, it is also understood that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. In some examples, an antibody that "specifically binds" to a target antigen or its epitope may not bind to other antigens or other epitopes of the same antigen. In some embodiments, the anti-FOLR1 antibody described herein specifically binds to human FOLR1. In some examples, its binding activity to non-human FOLR1 antigens is undetectable or extremely low by conventional assays, and therefore will not have significant biological significance as known to those skilled in the art. In other examples, the anti-FOLR1 antibody described herein may cross-react with FOLR1 from different species, for example, between human FOLR1 and non-human FOLR1 (e.g., FOLR1 from experimental animals such as non-human primates, mice, or rats).

[0028] As used herein, the terms "folate receptor alpha", "folate receptor 1", or "FOLR1" refer to the folate receptor alpha of any suitable species, e.g., human, non-human mammals such as non-human primates, or rodents (e.g., mouse or rat). FOLR1 is a single-chain membrane protein capable of binding folic acid and its derivatives. The amino acid sequence of an exemplary human FOLR1 is provided below (see also GenBank Accession No. P15328): [ka] FOLR1 molecules from other species are well known in the art and their amino acid sequences can be retrieved from publicly available databases, such as GenBank.

[0029] In some embodiments, the anti-FOLR1 antibodies described herein have suitable binding affinity to a target antigen (e.g., human FOLR1) or an antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or K A Refers to. A is the dissociation constant (K D The anti-FOLR1 antibodies described herein have a binding affinity of at least 10 to a target antigen or antigenic epitope. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , or 10 -10 Binding affinity (K D The increased binding affinity may be expressed as a decreased K D A higher affinity binding of an antibody to a first antigen compared to a second antigen corresponds to a higher K A (or a number K D ) for binding to the first antigen A (or a smaller number K D) in such cases. In such cases, the antibody has specificity for a first antigen (e.g., a first protein in a first conformation or a mimetic thereof) compared to a second antigen (e.g., the same first protein in a second conformation or a mimetic thereof; or a second protein). In some embodiments, the anti-FOLR1 antibodies described herein have a higher binding affinity (higher K A or smaller K D The difference in binding affinity (e.g., for specificity or other comparison) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 In some embodiments, any of the anti-FOLR1 antibodies may be further affinity matured to increase the binding affinity of the antibody to a target antigen or antigenic epitope thereof.

[0030] Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (using, as an example, a fluorescence assay). Exemplary conditions for assessing binding affinity are HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([bound]) is generally related to the concentration of free target protein ([free]) by the following formula: [Bound]=[Free] / (Kd+[Free])

[0031] However, K A It is not necessary to perform an exact determination of K since, for example, it may be determined using methods such as ELISA or FACS analysis. AThis is because it may be sufficient to simply obtain a quantitative measure of affinity (such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measure of affinity, or to obtain an estimate of affinity, e.g., by activity in a functional assay, e.g., in an in vitro or in vivo assay) that is proportional to and can therefore be used for comparison.

[0032] In some embodiments, the anti-FOLR1 antibody described herein binds to the same epitope of FOLR1 antigen (e.g., human FOLR1) as one of the reference antibodies provided herein, or binds to FOLR1 antigen in competition with the reference antibody. The reference antibodies provided herein include FOLR1-Ab217, FOLR1-Ab218, FOLR1-Ab220, FOLR1-Ab221, and FOLR1-Ab222, the structural characteristics of each of which are provided herein. An antibody that binds to the same epitope as the reference antibody described herein may bind to exactly the same epitope as the reference antibody or to a substantially overlapping epitope (e.g., comprising less than three non-overlapping amino acid residues, less than two non-overlapping amino acid residues, or only one non-overlapping amino acid residue). Whether two antibodies compete with each other to bind to a cognate antigen can be determined by a competitive assay, which is well known in the art. Such antibodies can be identified, as known to those skilled in the art, by, for example, having substantially similar structural features (e.g., complementarity determining regions) and / or by assays known in the art. For example, a competition assay can be performed using one of the reference antibodies to determine whether a candidate antibody binds to the same epitope as the reference antibody or whether it competes with the reference antibody to bind to the FOLR1 antigen.

[0033] The anti-FOLR1 antibodies described herein comprise: (a) X 1 YTFTX 2 YX 3 Heavy chain complementarity determining region 1 (HC CDR1), represented as 1 is G or I, and X 2 is D or S, and X3 is W, N, or S; (b) INX 1 X 2 X 3 X 4 X 5 X 6 Heavy chain complementarity determining region 2 (HC CDR2), represented as: 1 is P or T, and X 2 is N, Y, or E, and X 3 is N, D, or T, and X 4 is G or S, and X 5 is G or E, and X 6 is T or P; (c) ARX 1 X 2 X 3 YX 4 X 5 X 6 X 7 X 8 X 9 X 10 Heavy chain complementarity determining region 3 (HC CDR3), represented as: 1 is S, K or M, and X 2 is G or P, and X 3 is G or Y, and X 4 is G or absent, X 5 is P or absent, and X 6 is A, R, or K, and X 7 is W, Y or I, and X 8 is F or M, and X 9 is D or A, and X 10 is Y or V, or (d) a heavy chain variable region (V H In some cases, the antibody may comprise a HC CDR1 of (a), a HC CDR2 of (b), and a HC CDR3 of (c).

[0034] In some embodiments, the HC CDR1 motif X 1 YTFTX 2 YX 3 is the position X 1 G, position X2 D and / or position X 3 Alternatively or additionally, the HC CDR2 motif INX may contain N. 1 X 2 X 3 X 4 X 5 X 6 is the position X 1 P, position X 2 and / or position X 3 N, position X 4 G, position X 5 G or S, and / or position X 6 Alternatively or additionally, the HC CDR3 motif ARX may include a T. 1 X 2 X 3 YX 4 X 5 X 6 X 7 X 8 X 9 X 10 is the position X 1 K at position X 2 P or G, position X 3 Y, position X 4 G, position X 5 P, position X 6 K or R, position X 7 Y, position X 8 F, position X 9 D and / or position X 10 may contain Y or V.

[0035] Table 1 provides the amino acid sequences (according to the IMGT definition) of the heavy chain CDRs of exemplary anti-FOLR1 antibodies. Antibodies having the same heavy chain CDR1, CDR2, and CDR3 regions as those exemplary anti-FOLR1 antibodies are also within the scope of this disclosure. [Table 1]

[0036] Alternatively or additionally, the anti-FOLR1 antibodies described herein may comprise (a) a light chain complementarity determining region 1 (LC CDR1) designated as ESVDNYGISF or QSLLYSSSQKNY; (b) X 1 Light chain complementarity determining region 2 (LC CDR2), denoted as AS, where X 1 is V, A, or W; (c) QQX 1 X 2 X 3 X 4 PX 5 X 6 Light chain complementarity determining region 3 (LC CDR3), represented as: 1 is Y or S, and X 2 is Y or K, and X 3 is E or S, and X 4 is Y or V, and X 5 is W, Y, or T, and X 6 is T or absent; or (d) a light chain variable region (V L ) containing a light chain variable domain (V L ).

[0037] In some examples, the LC CDR1 of the anti-FOLR1 antibody is ESVDNYGISF. In other examples, the LC CDR1 of the antibody is QSLLYSSSQKNY. Alternatively or additionally, the LC CDR2 motif X 1 AS is position X 1 Alternatively or additionally, the LC CDR3 motif QQX 1 X 2 X 3 X 4 PX 5 T is position X 1 S at position X 2 K at position X 3 E, and / or position X 4 and / or position X 5 does not contain any residues.

[0038] Table 2 provides the amino acid sequences of the light chain CDRs of exemplary anti-FOLR1 antibodies. Antibodies having the same light chain CDR1, CDR2, and CDR3 regions as those exemplary anti-FOLR1 antibodies are also within the scope of this disclosure. [Table 2]

[0039] The heavy and light chain CDRs of the reference antibodies provided herein are determined based on the IMGT approach, which is well known in the art. In some cases, the anti-FOLR1 antibodies disclosed herein may contain the same heavy and light chain CDRs of any of the reference antibodies disclosed herein. The same V H and / or V L Two antibodies that have CDRs mean that their CDRs are identical when determined by the same approach (e.g., those described herein and / or known in the art).

[0040] In some examples, the anti-FOLR1 antibodies disclosed herein have the same V H and / or V L The sequence may include (CDRs in bold): FOLR1-Ab1 : [ka] FOLR1-Ab4 [ka]

[0041] FOLR1-Ab14 [ka] FOLR1-Ab20 [ka] FOLR1-Ab23 [ka]

[0042] Also within the scope of this disclosure are functional variants of any of the reference anti-FOLR1 antibodies disclosed herein (e.g., those listed in Tables 1 and 2 above). Functional variants include up to five (e.g., 4, 3, 2, or 1) amino acid residue variations in one or more heavy and light chain CDR regions of the reference antibody and have substantially similar affinities (e.g., the same order of K D The functional variants can bind to the same epitope of the FOLR1 antigen with a CDR having a CDR value of 0.01 to 0.02. In some cases, each of the heavy and / or light chain CDRs of the functional variants contains no more than two amino acid residue variations relative to the corresponding CDR of the reference antibody. In some examples, each of the heavy and / or light chain CDRs of the functional variants contains only one amino acid residue variation relative to the corresponding CDR of the reference antibody.

[0043] In one example, the variation of amino acid residue is a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, such as those found in references that summarize such methods (e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York.). Conservative amino acid substitutions include those made between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0044] In some embodiments, the anti-FOLR1 antibody comprises heavy chain CDRs that are collectively at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs of the reference antibody, and / or light chain CDRs that are collectively at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the light chain CDRs of the reference antibody. In some embodiments, the anti-FOLR1 antibody comprises a heavy chain variable region (V) that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the heavy chain variable region of any reference antibody. H ), and / or a light chain variable region (V) that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the light chain variable region of the reference antibody. L ).

[0045] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990 (modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0046] The present disclosure also provides germline variants of any of the reference anti-FOLR1 antibodies disclosed herein. A germline variant contains one or more mutations in the framework region relative to its parent antibody relative to the corresponding germline sequence. To generate a germline variant, the heavy or light chain variable region sequence or a portion thereof (e.g., framework sequence) of the parent antibody can be used as a query against an antibody germline sequence database (e.g., bioinfo.org.uk / abs / , www.vbase2.org, or imgt.org) to identify the corresponding germline sequence used by the parent antibody and the variation of amino acid residues in one or more framework regions between the germline sequence and the parent antibody. One or more amino acid substitutions can then be introduced into the parent antibody based on the germline sequence to produce a germline variant.

[0047] In some embodiments, the heavy chain of any of the anti-FOLR1 antibodies described herein may further comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, e.g., human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is derived from human IgG (gamma heavy chain). Optionally, the anti-FOLR1 antibody described herein may comprise a modified constant region. For example, it may comprise a modified constant region that is immunologically inert, e.g., does not cause complement-mediated lysis or does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity may be assessed using the methods disclosed in U.S. Pat. No. 5,500,362. In other embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT / GB99 / 01441; and / or UK Patent Application No. 9809951.8.

[0048] Any of the anti-FOLR1 antibodies described herein may further comprise a light chain comprising a light chain variable region and optionally a light chain constant region, which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. Antibody heavy and light chain constant regions are well known in the art, and are provided, for example, in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0049] Preparation of anti-FOLR1 antibody Antibodies capable of binding to FOLR1 as described herein can be produced by any method known in the art, see, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, antibodies specific to target FOLR1 antigen (e.g., human FOLR1) can be produced by conventional hybridoma technology. A full-length target antigen or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal to generate antibodies that bind to the antigen. The route and schedule of immunization of the host animal is generally consistent with established conventional techniques for stimulating and producing antibodies, as further described herein. General techniques for producing mouse, humanized, and human antibodies are known in the art and described herein. It is contemplated that any mammalian animal subject, including humans, or antibody-producing cells derived therefrom can be engineered to serve as the basis for the production of mammalian animal hybridoma cell lines, including humans. Typically, a host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and / or intradermally with an amount of immunogen, including those described herein.

[0050] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497, or as modified by Buck, DW, et al., In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those from Salk Institute, Cell Distribution Center, San Diego, Calif., USA, can be used for hybridization. In general, this technique involves fusing myeloma cells and lymphoid cells using a fusing agent such as polyethylene glycol or by electrical means well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parent cells. Any of the media described herein, with or without serum supplementation, can be used to culture hybridomas secreting monoclonal antibodies. As another alternative to cell fusion techniques, EBV-immortalized B cells can be used to produce the anti-FOLR1 monoclonal antibodies described herein. Hybridomas are expanded and subcloned as necessary, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

[0051] Hybridomas that can be used as a source of antibodies include all derivatives, progeny cells of parent hybridomas that produce monoclonal antibodies capable of blocking FOLR1 activity. Hybridomas producing such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture media or body fluids, if necessary, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesirable activity, if present, can be removed, for example, by running the preparation over an adsorbent made of immunogen attached to a solid phase, and eluting or releasing the desired antibody from the immunogen. Immunization of a host animal with a target antigen or fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (linkage via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl, or soybean trypsin inhibitor using R1N=C=NR (where R and R1 are different alkyl groups), can generate a population of antibodies (e.g., monoclonal antibodies).

[0052] If necessary, the antibody of interest (monoclonal or polyclonal) (e.g., produced by a hybridoma) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell, which can then be expanded and frozen for future use. In the alternative, the polynucleotide sequence can be used to "humanize" the antibody, or for genetic engineering to improve affinity (affinity maturation), or other characteristics of the antibody. For example, if the antibody is to be used in human clinical trials and treatments, the constant region can be engineered to be more similar to human constant regions to avoid immune responses. It may be desirable to genetically engineer the antibody sequence to obtain greater affinity for the target antigen and greater efficacy in inhibiting the activity of FOLR1. It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to an antibody and still maintain its binding specificity to the target antigen.

[0053] In other embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response can also be used to generate humanized or human antibodies. An example of such technology is the Xenomouse mouse from Amgen, Inc. (Fremont, Calif.). RTM and HuMAb-Mouse from Medarex, Inc. (Princeton, NJ). RTMand TC MouseTM. In another alternative, antibodies can be made recombinantly by phage display or yeast technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

[0054] In some embodiments, an antibody capable of binding to a FOLR1 antigen can be isolated from an antibody library, such as a phage display antibody library or a yeast display antibody library. In one example, the anti-FOLR1 antibody described herein can be isolated from a monoclonal antibody library, for example, according to the method disclosed in US2015 / 0153356, the relevant disclosure of which is incorporated herein by reference for the purposes or subject matter referred to herein.

[0055] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared via routine methods, for example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragment.

[0056] Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single chain antibodies, and bispecific antibodies, can be produced, for example, through conventional recombinant techniques. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into one or more expression vectors and then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. For example, see PCT Publication No. WO87 / 04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, genetically engineered antibodies such as "chimeric" or "hybrid" antibodies can be prepared that have the binding specificity of a target antigen.

[0057] Techniques developed for the production of "chimeric antibodies" are well known in the art. See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452. Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the V of the parent non-human antibody is H and V L The variable regions of the human VV are subjected to three-dimensional molecular modeling analysis according to methods known in the art. Framework amino acid residues predicted to be important for the formation of the correct CDR structure are then identified using the same molecular modeling analysis. In parallel, a human VVV having an amino acid sequence that is homologous to the amino acid sequence of the parent non-human antibody is generated. H and V L The chain is the parent V as a search query. H and V L The sequence is then used to identify the human V H and V L The acceptor gene is selected.

[0058] The CDR regions in the selected human acceptor gene may be replaced with CDR regions from a parent non-human antibody or a functional variant thereof. If necessary, residues in the framework regions of the parent chain that are predicted to be important for interactions with the CDR regions (see above) can be used to replace the corresponding residues in the human acceptor gene. Single chain antibodies can be prepared through recombinant technology by linking the nucleotide sequence encoding the heavy chain variable region with the nucleotide sequence encoding the light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, the techniques described for producing single chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce phage or yeast scFv libraries, and FOLR1 specific scFv clones can be identified from the libraries according to routine procedures. Positive clones can be subjected to further screening to identify clones that inhibit FOLR1 activity.

[0059] The antibodies obtained according to the methods known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope that an antigen binds to, i.e., "epitope mapping". There are many methods known in the art for mapping and characterizing the location of an epitope on a protein, including elucidating the crystal structure of an antibody-antigen complex, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In an additional example, epitope mapping can be used to determine the sequence that an antibody binds to. An epitope can be a linear epitope (i.e., contained in a single stretch of amino acids) or a conformational epitope formed by the three-dimensional interaction of amino acids that are not necessarily contained in a single stretch (linear sequence of primary structure). Peptides of various lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used in binding assays with antibodies. In another example, the epitope to which an antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to a gene fragment expression assay, an open reading frame encoding a target antigen is fragmented, either randomly or by specific gene construction, and the reactivity of the expressed fragments of the antigen with the antibody being tested is determined. The gene fragments can be produced, for example, by PCR, and then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. Binding of the antibody to the radiolabeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis.An epitope can also be identified by using a large library of random peptide sequences (phage libraries) displayed on the surface of phage particles. Alternatively, a defined library of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In an additional example, mutagenesis of antigen-binding domains, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues necessary, sufficient, and / or required for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen, where various fragments of the FOLR1 polypeptide are replaced (swapped) with sequences from closely related but antigenically distinct proteins. The importance of specific antigen fragments to antibody binding can be evaluated by assessing the binding of the antibody to mutant FOLR1.

[0060] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as the other antibody. Competition assays are well known to those of skill in the art. In some instances, anti-FOLR1 antibodies are prepared by recombinant techniques, as exemplified below. The nucleic acids encoding the heavy and light chains of the anti-FOLR1 antibody described herein can be cloned into one expression vector, and each nucleotide sequence is operably linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operably linked to a separate prompter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter, so that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the coding sequences of the heavy and light chains.

[0061] In some examples, the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cell that expresses it, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to allow the formation of the antibody. In general, the nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art.For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.Alternatively, synthetic nucleic acid linkers can be linked to the ends of the gene.These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector.The choice of expression vector / promoter will depend on the type of host cell for use in producing the antibody.

[0062] A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter. Regulatable promoters can also be used, including those that use the lac repressor from E. coli as a transcriptional modulator to regulate transcription from mammalian cell promoters with lac operators [Brown, M. et al., Cell, 49:603-612 (1987)], and those that use the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16 or p65 using astrodiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0063] Regulatable promoters with repressors in the operon can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from mammalian cell promoters with lac operators [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with a transcriptional activator (VP16) to create the tetR-mammalian cell transcriptional activator fusion protein tTa (tetR-VP16), which was combined with a minimal promoter with tetO from the human cytomegalovirus (hCMV) major immediate early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operator is correctly placed downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR), but not the tetR-mammalian cell transcription factor fusion derivative, can function alone as a powerful transmodulator to regulate gene expression in mammalian cells (Yao et al., Human Gene Therapy).One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins, which may be toxic to cells in some cases, to achieve its regulatable effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0064] In addition, the vectors may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene, for selection of stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; the SV40 polyoma origin of replication and ColE1 for correct episomal replication; an internal ribosome binding site (IRES), a versatile multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0065] Examples of polyadenylation signals useful in carrying out the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal. One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies can be introduced into a suitable host cell for producing the antibody. The host cells can be cultured under conditions suitable for expression of the antibody or any polypeptide chains thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or culture supernatant) via conventional methods, e.g., affinity purification. If necessary, the polypeptide chains of the antibody can be incubated for a suitable period of time under suitable conditions to allow production of the antibody.

[0066] In some embodiments, the method for preparing the antibody described herein includes a recombinant expression vector encoding both the heavy and light chains of the anti-FOLR1 antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cell) by conventional methods, e.g., calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions that allow the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cell can be incubated under suitable conditions that allow the formation of the antibody.

[0067] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-FOLR1 antibody and the other encoding the light chain of an anti-FOLR1 antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cell) by conventional methods, e.g., calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow the expression of the polypeptide chain of the antibody. When the two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or culture medium. If necessary, the polypeptide chain can be recovered from the host cell or culture medium and then incubated under suitable conditions that allow the formation of the antibody. When the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or the corresponding culture medium. The two polypeptide chains can then be incubated under suitable conditions for the formation of the antibody.

[0068] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G-bound matrix. Any of the nucleic acids encoding the heavy chain, light chain, or both of the anti-FOLR1 antibodies described herein, vectors containing same (e.g., expression vectors); and host cells containing the vectors are within the scope of this disclosure.

[0069] Antibody-drug conjugates The present disclosure also provides an antibody-drug conjugate comprising any of the anti-FOLR1 antibodies described herein covalently linked to a therapeutic agent. As used herein, the term "antibody-drug conjugate" or "ADC" refers to a conjugate in which an anti-FOLR1 antibody described herein and a therapeutic agent are covalently linked. In general, the antibody-drug conjugate may include an anti-FOLR1 antibody, a therapeutic agent, and optionally a linker between the antibody and the therapeutic agent. The ADS is a conjugate that binds the FOLR1 targeted by the antibody. + Cells, especially FOLR1 + The therapeutic effect can be increased by delivering the therapeutic agent to the cancer cells. The antibody-drug conjugate can be prepared by various methods for preparing antibody-drug conjugates known in the art.

[0070] The therapeutic agent of the ADC described herein can be a toxin, a chemotherapeutic agent, an antibiotic, an ADP-ribosyltransferase, a radioisotope, or a nuclease. In some cases, the therapeutic agent is a cytotoxic agent. Examples include, but are not limited to, anthracyclines, auristatins (e.g., auristatin E), camptothecins, combretastains, dolastatins, duocarmycins, enediynes, geldanamycins, indolino-benzodiazepine dimers, maytansines, puromycins, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, tubulysins, hemiasterins, spliceostatins, pladienolides, and calicheamicins.

[0071] In some embodiments, the anti-FOLR1 antibody and the therapeutic agent are connected via a linker. Such a linker may be a cleavable linker, for example, cleavable under certain pH conditions (pH-sensitive linker), cleavable by a protease (protease-sensitive linker), or cleavable in the presence of glutathione (glutathione-sensitive linker). In some examples, the linker includes a protease cleavage site, which may include 2-5 amino acid residues that are recognizable and / or cleavable by a suitable protease. Such peptides may include naturally occurring amino acid residues, non-naturally occurring amino acid residues, or combinations thereof. In one example, the peptide linker may be a dipeptide linker. Examples include a valine-citrulline (val-cit) linker, a phenylalanine-lysine (phe-lys) linker, or a maleimidocaproic-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker. Alternatively, the linker may be non-cleavable, for example a linker comprising an optionally substituted alkane or a thioether.

[0072] In some examples, the linker may include a functional group capable of forming a covalent bond with the antibody. Exemplary functional groups include, but are not limited to, maleimide, iodoacetamide, vinylsulfone, acrylate, acrylamide, acrylonitrile, or methacrylate groups. In some cases, the linker may include one or more reactive amines, including, but not limited to, acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC) or aminoPEG6-propionyl. For example, see WO2012 / 059882. Other exemplary linkers include sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). Sulfo-smcc conjugation occurs via a maleimide group that reacts with sulfhydryls (thiols, -SH), while the sulfo-NHS ester is reactive toward primary amines (found in lysine and the N-terminus of proteins or peptides).

[0073] In some instances, the linker is a molecular spacer, such as a linker represented by Formula I: [ka] In the formula, R 1 is an optionally substituted C 1~6 Alkyl (e.g., C 1~3 alkyl), optionally substituted phenyl, optionally substituted C 2~6 Alkylene, optionally substituted C 2~6 Alkenylene, optionally substituted C 2~6 and / or X may be O, S, or N.

[0074] Methods for conjugating cytotoxic agents or other therapeutic agents to antibodies are known in the art and have been described in various publications.For example, chemical modifications can be made in antibodies through lysine side chain amines or through cysteine ​​sulfhydryl groups that are activated by reducing interchain disulfide bonds for conjugation reactions to occur.See, for example, Tanaka et al., FEBS Letters 579:2092-2096, (2005), and Gentle et al., Bioconjug. Chem. 15:658-663, (2004).Reactive cysteine ​​residues engineered at specific sites of antibodies for specific drug conjugation with defined stoichiometry have also been described.See, for example, Junutula et al., Nature Biotechnology, 26:925-932, (2008). Conjugation using acyl donor glutamine-containing tags and / or endogenous glutamines made reactive by polypeptide engineering in the presence of transglutaminase and amines, e.g., cytotoxic agents modified with reactive amines, has also been described in WO2012 / 059882, Strop et al., Chem. Biol. 20(2):161-167 (2013), and Farias et al., Bioconjug. Chem. 25(2):245-250 (2014). The relevant disclosures of such publications are incorporated herein by reference for the purposes and subject matter referenced therein.

[0075] Chimeric antigen receptors (CARs) and immune cells expressing them The present disclosure also features chimeric antigen receptors that target FOLR1 and immune cells that express it. The chimeric antigen receptors (CARs) disclosed herein target FOLR1, such as epithelial-derived cancer cells. +FOLR1 is an artificial cell surface receptor that redirects the binding specificity of immune cells (e.g., T cells) expressing it to the cell, thereby eliminating the target disease cell, e.g., via the effector activity of the immune cell. CAR constructs often include at least an extracellular antigen binding domain fused to an intracellular signaling domain. Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010. The extracellular antigen binding domain, which may be a single-chain antibody fragment (scFv), is specific for the FOLR1 antigen, and the intracellular signaling domain may mediate cell signaling leading to the activation of the immune cell. Thus, immune cells expressing a CAR construct specific for FOLR1 can bind to disease cells (e.g., tumor cells) expressing FOLR1, resulting in the activation of the immune cell and the elimination of the disease cell.

[0076] Any of the anti-FOLR1 antibodies described herein can be used to produce a CAR construct, also described herein. For example, the V H and V L The domain can be fused to an intracellular signaling domain(s) to produce a CAR construct using conventional recombinant techniques. In some examples, the V of anti-FOLR1 H and V L The domains are connected via peptide linkers to form the scFv fragment.

[0077] The CAR construct disclosed herein may comprise one or more intracellular signaling domains. In some examples, the CAR comprises an intracellular signaling domain that comprises an immunoreceptor tyrosine-based activation motif (ITAM). Such an intracellular signaling domain may be derived from CD3zeta. In addition, the CAR construct may further comprise one or more costimulatory signaling domains, which may be derived from, for example, a costimulatory receptor from 4-1BB (CD137), CD7, CD27, CD28, CD40, OX40, ICOS, GITR, HVEM, TIM1, or LFA-1.

[0078] The CAR constructs disclosed herein may further comprise a transmembrane hinge domain that can be obtained from a suitable cell surface receptor, e.g., CD28 or CD8. Also provided are isolated nucleic acid molecules and vectors encoding any of the anti-FOLR1 CARs disclosed herein, and host cells, such as host immune cells (e.g., T cells and natural killer cells) that contain the nucleic acid molecules or vectors. Immune cells expressing anti-FOLR1 CARs that contain FOLR1-specific antibody binding fragments can be used to treat cancers that express FOLR1. Thus, the present invention provides a method for treating cancers that express FOLR1 by selecting a subject with a cancer that expresses FOLR1 and administering to the subject a therapeutically effective amount of immune cells that express a CAR that targets FOLR1. + Methods of treating a subject with cancer are also provided.

[0079] Pharmaceutical Compositions Anti-FOLR1 antibodies, encoding nucleic acids or nucleic acid sets, vectors containing them, or host cells containing vectors described herein, as well as ADCs containing anti-FOLR1 antibodies and / or immune cells expressing CARs targeting FOLR1, can be mixed with pharmaceutically acceptable carriers (excipients) to form pharmaceutical compositions for use in treating target diseases. "Acceptable" means that the carrier must be compatible with the active ingredients of the composition (and preferably be able to stabilize the active ingredients) and not harmful to the subject being treated. Pharmaceutically acceptable excipients (carriers) include buffers that are well known in the art. For example, see Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0080] The pharmaceutical compositions used in the method may contain pharma- ceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used and may include: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0081] In some instances, the pharmaceutical compositions described herein include liposomes containing the antibody (or encoding nucleic acid, or ADC), which can be prepared by methods known in the art, such as those described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to produce liposomes with the desired diameter.

[0082] The antibody, encoding nucleic acid(s), or ADC can also be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, for example, in microcapsules prepared by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively). Such techniques are known in the art, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0083] In other examples, the pharmaceutical compositions described herein can be formulated in sustained release format.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles (for example, films or microcapsules).Examples of sustained release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0084] Pharmaceutical compositions used for in vivo administration must be sterile. This can be easily accomplished, for example, by filtration through a sterile filtration membrane. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper that can be pierced by a hypodermic injection needle. The pharmaceutical compositions described herein may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0085] To prepare solid compositions such as tablets, the primary active ingredient may be mixed with conventional tablet ingredients such as pharmaceutical carriers, e.g., corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents (e.g., water) to form a solid preformulation composition containing a homogenous mixture of the compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. When these preformulation compositions are referred to as homogenous, it means that the active ingredient is evenly dispersed throughout the composition such that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the novel composition may be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action. For example, the tablet or pill may contain an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0086] Suitable surfactants include non-ionic agents such as polyoxyethylene sorbitan (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitan (e.g., Span™ 20, 40, 60, 80 or 85), among others. Compositions with surfactants conveniently contain between 0.05% and 5% surfactant, and may be between 0.1% and 2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma- ceutically acceptable vehicles, may be added as required.

[0087] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient can be dissolved in a premixed emulsion composition, or alternatively, it can be dissolved in an emulsion formed when mixed with oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and phospholipid (e.g., egg phospholipid, soybean phospholipid or soybean lecithin) and water. It will be understood that other ingredients, such as glycerol or glucose, can be added to adjust the osmotic pressure of the emulsion. Suitable emulsions typically contain up to 20% oil, for example between 5% and 20%.

[0088] The emulsion composition may be prepared by mixing the antibody with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water). Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably sterile pharma-ceutically acceptable solvents can be nebulized by using gas.Nebulized solutions can be directly breathed from the nebulizing device, or the nebulizing device can be attached to a face mask, tent or intermittent positive pressure breathing machine.Solution, suspension or powder compositions can be administered from a device that delivers the formulation in an appropriate manner, preferably orally or nasally.

[0089] Treatment and diagnostic methods Any of the anti-FOLR1 antibodies, encoding nucleic acids or nucleic acid sets, vectors containing the same, ADCs containing anti-FOLR1 antibodies, and immune cells (e.g., T cells or NK cells) expressing a CAR targeting FOLR1 described herein may be used to treat FOLR1. + FOLR1 in cancer cells + Useful for inhibiting and / or eliminating diseased cells, thereby preventing FOLR1 + This provides benefit in the treatment of diseases or disorders associated with diseased cells. To carry out the methods disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as, for example, intravenous administration, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation or topical route, as a bolus, or by continuous infusion over a period of time. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, the antibodies described herein can be aerosolized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized and milled powders.

[0090] The subject to be treated by the methods described herein may be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. Human subjects in need of treatment may be those that express FOLR1. + The target disease / disorder may be a human patient at risk of having or suspected of having a disease cell-associated target disease / disorder. +The diseased cells are cancer cells, such as epithelial cancer cells (i.e., derived from epithelial cells). Examples include, but are not limited to, ovarian cancer cells, breast cancer cells, renal cancer cells, lung cancer cells, colorectal cancer cells, and brain cancer cells. Subjects with target diseases or disorders can be identified by routine medical examinations, such as laboratory tests, organ function tests, CT scans, or ultrasound. Subjects suspected of having any of such target diseases / disorders can exhibit one or more symptoms of the disease / disorder. Subjects at risk for a disease / disorder can be subjects with one or more risk factors for the disease / disorder.

[0091] As used herein, "effective amount" refers to the amount of each active agent, alone or in combination with one or more other active agents, required to provide a therapeutic effect to a subject. In some embodiments, the therapeutic effect is reduced FOLR1 activity or FOLR1 activity. + The activity of cells. It will be clear to those skilled in the art how to determine whether the amount of antibody or other therapeutic agent (e.g., ADC or CAR-T cell) containing it achieves therapeutic effect. As recognized by those skilled in the art, effective amount varies depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex and weight, duration of treatment, nature of concomitant treatment (if any), specific route of administration, and similar factors within the knowledge and expertise of medical practitioners. These factors are well known to those skilled in the art and can be addressed only by routine experimentation. In general, it is preferable to use the maximum dose of each component or combination thereof, i.e., the highest safe dose according to sound medical judgment.

[0092] Empirical considerations such as half-life generally contribute to the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of treatment, and is generally, but not necessarily, based on the treatment and / or suppression and / or remission and / or delay of the target disease / disorder. Alternatively, a continuous sustained release formulation of the antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art. In one example, the dosage of the antibody described herein can be empirically determined in an individual who has received one or more administrations (single or multiple) of the antibody. The individual is given increasing dosages of the antagonist. Indicators of disease / disorder can be followed to assess the effectiveness of the antagonist.

[0093] In general, for administration of any of the anti-FOLR1 antibodies or ADCs comprising same described herein, the initial candidate dosage may be about 2 mg / kg. For purposes of this disclosure, a typical daily dosage may range anywhere from approximately 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg to 30 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or more, depending on the condition, treatment is sustained until a desired suppression of symptoms occurs or until a therapeutic level sufficient to alleviate the target disease or disorder, or a symptom thereof, is achieved. An exemplary dosing regimen includes administering an initial dose of about 2 mg / kg, followed by a weekly maintenance dose of the antibody of about 1 mg / kg, or followed by a maintenance dose of about 1 mg / kg every other week. However, other dosing regimens may be useful depending on the pattern of pharmacokinetic decay the practitioner wishes to achieve. For example, dosing 1 to 4 times per week is contemplated. In some embodiments, a dosage range of about 3 μg / mg to about 2 mg / kg (such as about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 2 mg / kg) may be used. In some embodiments, the dosage frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks, or once every month, every 2 months, or every 3 months, or once for a longer period. The progress of this treatment is easily monitored by conventional techniques and assays. The dosage regimen (including the antibody used) may vary over time.

[0094] For purposes of this disclosure, the appropriate dosage of the antibodies described herein will depend on the particular antibody(ies), antibodies, and / or non-antibody peptides (or compositions thereof) used, the type and severity of the disease / disorder, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the antagonist, and the discretion of the attending physician. Typically, the clinician will administer the antibody until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a reduction in thrombosis. Methods for determining whether a dosage has produced a desired result will be apparent to one of skill in the art. Administration of one or more antibodies may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to the skilled practitioner. Administration of the antibody may be essentially continuous over a preselected period of time, or may be a series of spaced doses, for example, either before, during, or after the onset of the target disease or disorder.

[0095] As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, with the intent to cure, heal, alleviate, mitigate, alter, cure, ameliorate, or improve or affect the disorder, a symptom of the disease, or a predisposition to a disease or disorder. Alleviating the target disease / disorder includes delaying the onset or progression of the disease, or reducing the severity of the disease. Alleviating the disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of the target disease or disorder means deferring, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the disease. This delay can be of various lengths of time, depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease, or delaying the onset of a disease, is a method that reduces the likelihood of developing one or more symptoms of the disease in a given time frame and / or reduces the severity of symptoms in a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to produce statistically significant results.

[0096] "Onset" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of the disease. Onset of a disease can be detected and evaluated using standard clinical techniques well known in the art. However, onset also refers to progression that may be undetectable. For the purposes of this disclosure, onset or progression refers to the biological course of a symptom. "Onset" includes onset, recurrence, and onset. As used herein, "onset" or "onset" of a target disease or disorder includes initial onset and / or recurrence. In some embodiments, the antibodies described herein are administered to a subject in need of treatment in an amount sufficient to inhibit the activity of one or both of the target antigens in vivo by at least 20% (e.g., by 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more). In other embodiments, the antibodies are administered in an amount effective to reduce the activity level of the target antigen by at least 20% (e.g., by 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more).

[0097] Depending on the type of disease or site of disease to be treated, the pharmaceutical composition can be administered to the subject using conventional methods known to those skilled in the art of medicine. The composition can also be administered by other conventional routes, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via an injectable depot route of administration, such as using a one-month, three-month, or six-month depot injectable or biodegradable material and method. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

[0098] Injectable compositions may contain various carriers, such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered by drip infusion, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. The physiologically acceptable excipient may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipient. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0099] In one embodiment, antibody is administered via site-specific or targeted local delivery technique.Examples of site-specific or targeted local delivery technique include various implantable depot sources of antibody, or local delivery catheters such as injection catheters, indwelling catheters, or needle catheters, synthetic grafts, foreign wraps, shunts and stents, or other implantable devices, site-specific carriers, direct injection, or direct application.See, for example, PCT Publication No. WO00 / 53211 and U.S. Patent No. 5,981,568. Targeted delivery of therapeutic compositions comprising antisense polynucleotides, expression vectors or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (JA Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

[0100] Therapeutic compositions containing polynucleotides (e.g., those encoding the antibodies described herein) are administered in the range of about 100 ng to about 200 mg for local administration in gene therapy protocols. In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg or more of DNA may also be used during gene therapy protocols. The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (see generally Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of coding sequences can be either constitutive or regulated.

[0101] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include recombinant retroviruses (see, e.g., PCT Publication Nos. WO90 / 07936; WO94 / 03622; WO93 / 25698; WO93 / 25234; WO93 / 11230; WO93 / 10218; WO91 / 02805; U.S. Patent Nos. 5,219,740 and 4,777,127; British Patent No. 2,200,651; and European Patent No. 0 345 242), alphavirus-based vectors (see, e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR-1251), and / or other vectors, including vectors that are suitable for use in the preparation of recombinant human encephalitis vectors. ATCC VR-532) and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94 / 12649, WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984 and WO95 / 00655). Administration of DNA linked to killed adenovirus, such as described in Curiel, Hum. Gene Ther. (1992) 3:147, may also be used.

[0102] Non-viral delivery vehicles and methods can also be used, including, but not limited to, polycationic condensed DNA linked or not linked to killed adenovirus alone (see, for example, Curiel, Hum. Gene Ther. (1992) 3:147); DNA linked to a ligand (see, for example, Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicle cells (see, for example, U.S. Pat. No. 5,814,482; PCT Publication Nos. WO95 / 07994; WO96 / 17072; WO95 / 30763; and WO97 / 42338) and neutralization of nucleic acid charge or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA introduction methods are described in PCT Publication No. WO90 / 11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publication Nos. WO95 / 13796; WO94 / 23697; WO91 / 14445; and European Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0103] The particular dosing regimen used in the methods described herein, i.e., dosage, timing, and repetition, will depend on the particular subject and the subject's medical history. When immune cells expressing a CAR targeting FOLR1 are used to treat a disease, patients receive a therapeutically effective dose of such immune cells, such as T lymphocytes or NK cells, at approximately 10 per kilogram of body weight. 5 ~10 10 Treatment can be by injecting in the range of 10 cells / Kg or more. Injections can be repeated as many times as the patient can tolerate until the desired response is achieved. The appropriate infusion volume and schedule will vary from patient to patient and can be determined by the physician treating the particular patient. Typically, infusions are administered in doses of approximately 10 6An initial dose of cells / Kg was injected, 10 8 The number of cells / Kg or more can be increased. IL-2 can be co-administered to expand the injected cells. The amount of IL-2 is approximately 1-5x10 per square meter of body surface area. 6 It can be an international unit.

[0104] In some embodiments, a combination of two or more antibodies, or an antibody and another suitable therapeutic agent, may be administered to a subject in need of treatment. The antibodies, ADCs and / or CAR-T cells comprising same may also be used in conjunction with other agents that serve to enhance and / or complement the effectiveness of the agents. The effectiveness of treatment of the target disease / disorder can be assessed by methods well known in the art. Any of the anti-FOLR1 antibodies described herein can be used to detect FOLR1 in a sample. + The diagnostic assays can be used to detect the presence or level of cells. Such diagnostic assays can be performed in vitro or in vivo.

[0105] For diagnostic applications, the anti-FOLR1 antibodies described herein can be conjugated with a detectable label (e.g., an imaging agent such as a contrast agent) for either in vivo or in vitro diagnostic purposes. As used herein, "conjugated" or "attached" means that two entities are associated, preferably with sufficient affinity such that the therapeutic / diagnostic benefit of the association between the two entities is realized. The association between the two entities can be direct or via a linker, such as a polymeric linker. Conjugation or attachment can include covalent or non-covalent binding, as well as other forms of association, such as entrapment (e.g., one entity on or within the other, or either or both entities on or within a third entity, such as a micelle).

[0106] In one example, the anti-FOLR1 antibodies described herein can be attached to a detectable label, which is a compound capable of directly or indirectly emitting a detectable signal, allowing the aptamer to be detected, measured, and / or qualified in vitro or in vivo. Examples of such "detectable labels" include, but are not limited to, fluorescent labels, chemiluminescent labels, colorimetric labels, enzyme markers, radioisotopes, and affinity tags such as biotin. Such labels can be directly or indirectly conjugated to the aptamer by conventional methods.

[0107] In some embodiments, the detectable label detects FOLR1 in vivo. + A suitable agent for imaging cells, which may be a radioactive molecule, a radiopharmaceutical, or an iron oxide particle. Suitable radioactive molecules for in vivo imaging include: 122 I, 123 I, 124 I, 125 I, 131 I, 18 F, 75 Br, 76 Br, 76 Br, 77 Br, 211 At, 225 Ac, 177 Lu, 153 Sm, 186 Re, 188 Re, 67 Cu, 213 Bi, 212 Bi, 212 Pb, and 67 Exemplary radiopharmaceuticals suitable for in vivo imaging include, but are not limited to, Ga. 111 Inoxyquinoline, 131 I sodium iodide, 99m Tc mebrophenin, and 99m Tc red blood cells, 123 I sodium iodide, 99m Tc Examethadime, 99m Tc macroaggregated albumin, 99m Tc medronate, 99mTc Meltiatide, 99m Tc oxidronate, 99m Tc pentetate, 99m Tc pertechnetate, 99m Tc sestamibi, 99m Tc sulfur colloid, 99m Reporting agents include Tc tetrofosmin, thallium-201, and xenon-133. Reporting agents also detect FOLR1 in tissue samples. + The dye may be, for example, a fluorophore, which is useful for detecting cell-mediated diseases.

[0108] To perform the in vitro diagnostic assay, anti-FOLR1 antibodies were used + The antibody can be contacted with a sample suspected to contain cells. The antibody and the sample can be incubated under suitable conditions for a suitable period of time to allow the antibody to bind to the FOLR1 antigen. Such interaction can then be detected by conventional methods, for example, ELISA or FACS. To perform diagnostic assay in vivo, a suitable amount of anti-FOLR1 antibody conjugated with a label can be administered to the subject that needs to be examined. The presence of the labeled antibody can be detected based on the signal emitted by the label by conventional methods.

[0109] Kits for Use in Treatment and Diagnostics The present disclosure also provides + Kits are provided for use in inhibiting and / or eliminating disease cells, thereby alleviating diseases / disorders associated with such disease cells. Such kits may include one or more containers containing anti-FOLR1 antibodies, ADCs containing same, or immune cells expressing CAR polypeptides targeting FOLR1, e.g., any of those described herein. In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administering an anti-FOLR1 antibody, ADC, or immune cell to treat, delay onset, or alleviate a target disease, such as those described herein. The kit may further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has a target disease. In yet other embodiments, the instructions include instructions for administering an antibody, ADC, or immune cell to an individual at risk for a target disease.

[0110] The instructions for use of anti-FOLR1 antibody, ADC comprising it, or immune cells expressing CAR targeting FOLR1 generally include information on dosage, dosing schedule, and route of administration for intended treatment. Containers can be unit dose, bulk package (e.g., multi-dose package) or sub-unit dose. The instructions provided in the kit of the present invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. The label or package insert shall state that the composition is an anti-FOLR1 + It is indicated for use in treating, delaying the onset of, and / or alleviating a cell-related disease or disorder. Instructions can be provided for carrying out any of the methods described herein.

[0111] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers) or injection devices, such as mini-pumps, are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an anti-FOLR1 antibody, an ADC containing it, or an immune cell expressing a CAR that targets FOLR1, such as those described herein.

[0112] The kit may optionally provide additional components such as buffers and interpretive information. Typically, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described above. FOLR1 in the sample + Also provided herein is a kit for use in detecting cells. Such a kit may include any of the anti-FOLR1 antibodies described herein. In some cases, the anti-FOLR1 antibody may be conjugated with a detectable label, such as those described herein. As used herein, "conjugated" or "attached" means that two entities are associated, preferably with sufficient affinity, such that the therapeutic / diagnostic benefit of the association between the two entities is realized. The association between the two entities may be direct or via a linker, such as a polymeric linker. Conjugation or attachment may include covalent or non-covalent binding, as well as other forms of association, such as entrapment (e.g., one entity on or within the other, or either or both entities on or within a third entity, such as a micelle). Alternatively, or in addition, the kit may include a secondary antibody capable of binding to the anti-FOLR1 antibody. + The method may further include instructions for using the anti-FOLR1 antibody to detect

[0113] Common techniques The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1989) Academic Press; Animal Cell Culture (RI Freshney, ed. 1987); Introuction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds.1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985≫; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984≫; Animal Cell Culture (R.I. Freshney, ed. (1986≫; Immobilized Cells and Enzymes (lRL Press, (1986≫; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.) and other sources.

[0114] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. Accordingly, the following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0115] example Example 1: Generation of anti-FOLR1 antibodies Reagents and general methods Hybridoma cell culture medium (PFHM-II Protein-Free Hybridoma Medium; #12040077) was purchased from Thermo Fisher. RNA was isolated using standard protocols and TRIzol Reagent from Thermo Fisher (#15596018). The resulting cDNA molecules were generated using a cDNA synthesis kit from Takara (PrimeScript II 1st strand cDNA synthesis kit; #6210A). Amplification of antibody V regions was performed using Premix Taq from Takara (#RR901A). Standard PCR primer sets (Ig-Primer Sets #TB326) were obtained from Novagen. Genes were cloned into pET28a (Novagen; #69864) using standard techniques including the use of EcoRI, HindIII, SalI, and T4 ligase (all from NEB). Some, but not all, oligonucleotide molecules were purified utilizing the QIAEX II Gel Extraction Kit (QIAgen #20021). SK-OV-3 and Daudi cell cultures were grown in 5% CO 2 The tumor cells were maintained in vitro as monolayer cultures at 37° C. in a 5% CO atmosphere. Tumor cells were passaged periodically as needed.

[0116] Screening an antibody library to discover anti-FOLR1 antibodies A large library of monoclonal antibodies (>100,000 total) was generated using a mixture of proteomic and peptide antigens as previously described in US2015 / 0153356. The library was partitioned into a series of high-density antibody arrays before screening against cancer tumor samples and the FDA Normal Tissue Panel.

[0117] We found that many of the antibodies isolated from the library, including FOLR1-Ab1, FOLR1-Ab4, FOLR1-Ab14, FOLR1-Ab20, and FOLR1-Ab23, differentially targeted the SK-OV-3 cell line. FOLR1 was identified as the target antigen via immunoprecipitation with the antibody followed by mass spectrometry. Subsequent knockdown of FOLR1 using standard antisense oligonucleotide techniques and overexpression of FOLR1 confirmed that FOLR1 was the target antigen bound by the antibodies.

[0118] Production of antibody clones Individual hybridoma clones were cultured in T25 flasks with 10 mL hybridoma cell culture medium (PFHM-II Protein-Free Hybridoma Medium). Cells were grown at 37°C until 80% confluent. Culture medium was then removed and cells were washed twice with 1xPBS. TRIzol reagent (1 mL volume) was added directly to the flask and cells were lysed by pipette mixing. Cell lysates were then harvested from the T25 flasks and total RNA was isolated using standard methods. RNA concentrations were then measured with a Nanodrop 2000 (Thermo Fisher). cDNA strands were then generated from the isolated RNA following the Takara PrimeScript II 1st strand cDNA synthesis kit protocol. Amplification of the hybridoma V region of the resulting cDNA was then performed following Novagen user protocol TB326. Primer pairs as shown in Table 3 below were used for amplification.

[0119] [Table 3-1] [Table 3-2]

[0120] The PCR products were checked on a 1% agarose gel. The positive PCR products were recovered using a QIAgen gel extraction kit and subsequently cloned into a pET28a vector using restriction enzymes (from NEB) corresponding to the primer sequences. The pET28a vectors with the PCR product inserts were transformed into DH5α bacterial cells and grown on ampicillin-positive agar plates. Each bacterial clone was sent for Sanger sequencing using MuIgGVH3'-2, MuIgkVL3'-1 or MuIglVL3'-1 primers. The obtained sequences were compared for consistency to identify the target VH3'-2, MuIgkVL3'-1 or MuIglVL3'-1 primers, respectively. H and V L The sequence was then confirmed. H and V L The sequences were analyzed in the IGMT database (http: / / www.imgt.org / ), and V H and V L The V regions, frame and CDR elements of the ribozyme were provided.

[0121] Example 2: Evaluation of anti-FOLR1 antibodies FOLR1-overexpressing SK-OV-3 cells (FOLR1 + SK-OV-3) and Daudi cells negative for FOLR1 expression (FOLR1 - The FOLR1 cells (Followers 1 and 2) were harvested using trypsin-EDTA partial digestion followed by centrifugation at 1000 rpm for 5 min. The cells were resuspended in cold PBS and aliquoted. Anti-FOLR1 antibody was diluted in PBS and used to detect FOLR1. + SK-OV-3 cells or FOLR1 -Daudi cells. The cell solutions were mixed and incubated at 4 °C in the dark, washed with PBS, and then a secondary antibody conjugate was added (for detection purposes). After incubation, the cells were washed with PBS, fixed with fixative, and then subjected to FACS analysis. As shown in Figures 1A-1E, these antibodies were able to bind to FOLR1 + These antibodies showed saturable binding to FOLR1. - No saturable binding to Daudi was observed.

[0122] The antibodies were tested in an antigen binding assay using an ELISA titration experiment. The antibodies were incubated with various concentrations of recombinant FOLR1 protein (rProtein). As shown in Table 4 below, all antibodies tested bound with affinities ranging from 0.39 to 12.5 nM.

[0123] SK-OV-3 cells were dosed with four anti-FOLR1 antibody clones. For the purposes of this experiment, dosing with IgG alone served as a control experiment. After dosing, cell viability was determined to evaluate the indirect cytotoxicity of all antibody clones. As shown in Table 4 below and Figures 2A-2D, in both cell lines, anti-FOLR1 antibodies reduced cell viability to 26-32% total viability, resulting in an IC 50 Values ​​ranged from 26 to 33.73 pM. The IgG control antibody did not result in significant loss of cell viability.

[0124] [Table 4]

[0125] Other Aspects All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to adapt the present invention to various applications and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the appended claims.

[0126] Equivalent While several inventive aspects have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or results and / or obtaining one or more of the advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive aspects described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application(s) for which the teachings of the invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive aspects described herein. Thus, the above embodiments are presented by way of example only, and it will be understood that, within the scope of the appended claims and equivalents thereto, the inventive aspects can be practiced otherwise than as specifically described and claimed. The inventive aspects of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more of such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0127] All definitions and those used herein should be understood to control for any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and in some cases may include the entire document. As used herein, and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless clearly indicated to the contrary.

[0128] In the present specification and claims, the phrase "and / or" as used herein should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related to the elements specifically identified or not. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising", may refer in one embodiment to A only (optionally including elements other than B); in another embodiment to B only (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements), etc.

[0129] In the present specification and claims, "or" as used herein shall be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive; that is, to include at least one, but also include a plurality, and optionally include additional unlisted items, of a number or list of elements. Only terms clearly indicated to the contrary, such as "only one" or "exactly one," or "consisting of" when used in the claims, shall refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall be interpreted to indicate exclusive alternatives (i.e., "one or the other, but not both") only when preceded by a term of exclusivity, such as "either," "one," "only one," or "exactly one." "Consisting essentially of" when used in the claims shall have its ordinary meaning as used in the field of patent law.

[0130] As used herein and in the claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, can optionally be present, whether or not related to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer, in one embodiment, to at least one (optionally including more than one) A (in which B is absent) (and optionally including elements other than B); in another embodiment, to at least one (optionally including more than one) B (in which A is absent) (and optionally including elements other than A); in yet another embodiment, to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements), etc. It is also to be understood that, unless expressly indicated to the contrary, in any method described herein that includes multiple steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

Claims

1. 1. An isolated antibody that binds to FOLR1, comprising a heavy chain variable region (VH) having a heavy chain complementarity determining region 1 (HC CDR1) of the amino acid sequence GYTFTSYW, a heavy chain complementarity determining region 2 (HC CDR2) of the amino acid sequence INPYDSET, and a heavy chain complementarity determining region 3 (HC CDR3) of the amino acid sequence ARSGGYAWFAY, and a light chain variable region (VL) having a light chain complementarity determining region 1 (LC CDR1) of the amino acid sequence QSLLYSSSQKNY, a light chain complementarity determining region 2 (LC CDR2) of the amino acid sequence WAS, and a light chain complementarity determining region 3 (LC CDR3) of the amino acid sequence QQYYSYPWT, wherein the antibody specifically binds to human FOLR1.

2. The antibody described in claim 1, which (a) cross-reacts with human FOLR1 and non-human FOLR1, and optionally the non-human FOLR1 is rodent FOLR1 or primate FOLR1; (b) is a full-length antibody or an antigen-binding fragment thereof, or a single-chain antibody; (c) is an IgG molecule; and / or (d) is a humanized antibody or a chimeric antibody.

3. A nucleic acid or set of nucleic acids encoding the antibody of claim 1 or 2; or a vector or set of vectors comprising said nucleic acid(s), optionally wherein the vector(s) is / are expression vector(s); or a host cell comprising said vector or set of vectors.

4. An antibody-drug conjugate (ADC), comprising: i. an antibody according to claim 1 or 2; and ii. at least one therapeutic agent, optionally a cytotoxic agent, e.g., monomethylauristatin E; wherein the antibody is covalently conjugated to at least one therapeutic agent.

5. The antibody-drug conjugate of claim 4, wherein the antibody and the therapeutic agent are conjugated through a linker, and optionally the linker is a cleavable or non-cleavable linker, for example (a) the linker is a cleavable linker selected from a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker; (b) the linker is a protease-sensitive linker comprising a peptide sequence of 2-5 amino acids, and optionally (i) the 2-5 amino acids are selected from naturally occurring amino acid residues, non-naturally occurring amino acid residues, or a combination thereof. or a combination thereof; or (ii) the peptide sequence comprises valine-citrulline; (c) the linker is a non-cleavable linker comprising an optionally substituted alkane or a thioether; (d) the linker comprises a functional group that forms a covalent bond with the antibody and the linker, e.g., the functional group comprises a maleimide group, an iodoacetamide group, a vinylsulfone group, an acrylate group, an acrylamide group, an acrylonitrile group, or a methacrylate group; and / or (e) the linker is represented by Formula I: 【Chemistry 1】 During the ceremony, R1 is an optionally substituted C1-6 alkyl, an optionally substituted phenyl, an optionally substituted C2-6 alkylene, an optionally substituted C2-6 alkenylene, an optionally substituted C2-6 alkynylene, or an optionally substituted triazole; and X is O, S, or N; The antibody-drug conjugate further comprises a molecular spacer represented by:

6. below: (i) an extracellular domain comprising the antigen-binding fragment of claim 2, which binds to FOLR1; (ii) a transmembrane domain, and (iii) one or more intracellular stimulatory domains. A chimeric antigen receptor comprising:

7. 7. The chimeric antigen receptor of claim 6, wherein (a) the transmembrane domain comprises a transmembrane domain derived from a CD28 or CD8 receptor; and / or (b) the one or more intracellular stimulatory domains comprise a signaling domain from CD3zeta; and / or (c) the one or more intracellular stimulatory domains comprise a costimulatory signaling domain, e.g., the costimulatory signaling domain is from a 4-1BB, CD7, CD27, CD28, CD40, OX40, ICOS, GITR, HVEM, TIM1, or LFA-1 receptor. The chimeric antigen receptor.

8. A nucleic acid comprising a nucleotide sequence encoding the chimeric antigen receptor of claim 6 or 7; or a vector comprising said nucleic acid.

9. A host cell, optionally an immune cell, e.g. a T cell, expressing a chimeric receptor according to claim 6 or 7.

10. A pharmaceutical composition comprising: (i) an antibody according to claim 1 or 2, a nucleic acid or a set of nucleic acids according to claim 3, an antibody-drug conjugate according to claim 4 or 5, or a host cell according to claim 9; and (ii) a pharma- ceutically acceptable carrier.

11. 11. The pharmaceutical composition of claim 10, for use in a method for reducing the number of FOLR1+ cells in a subject in need thereof, the method comprising administering an effective amount of the pharmaceutical composition to the subject, optionally wherein the subject has or is suspected of having cancer.

12. 1. A method for detecting the presence of FOLR1+ cells, comprising: i. contacting a sample suspected of having FOLR1+ cells with an antibody according to claim 1 or 2, wherein the antibody is conjugated with a labeling agent; and ii. Detecting the presence of FOLR1+ cells in the sample based on binding of the antibody to the cells in the sample. The method comprising:

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