EA046422B1 - ANTIBODIES AGAINST FOLATE 1 RECEPTOR AND THEIR APPLICATIONS - Google Patents

Description

Cross reference to related applications

This application claims priority to U.S. Provisional Patent Application No. 62/642213, filed March 13, 2018, the entire disclosure of which is incorporated herein by reference.

Field of technology to which the invention relates

The present invention relates to monoclonal antibodies against folate receptor 1 (FOLR1), to nucleic acids and expression vectors encoding antibodies, to recombinant cells containing vectors, and to compositions containing antibodies. The present invention also relates to methods for producing antibodies and to methods of using antibodies for the treatment of diseases, including malignant tumors.

Link to sequence list accepted in electronic form

This application contains a sequence listing received electronically via EFS-Web as a sequence listing in ASCII format with file name 689204-11WO Sequence Listing and creation date March 6, 2019 and having a size of 64 kB. The sequence listing received via EFS-Web forms part of the specification and its entire contents are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Folate receptor 1 (FOLR1), also known as folate receptor α (FRa) or folate binding protein (FBP), is a glycophosphatidylinositol (GPI)-anchored cell surface membrane protein that has high affinity for the active form of folate, 5-methyltetrahydrofolate (5- MTF), and transports it and its derivatives into cells (Salazar and Ratnam, Cancer Metastasis Rev 2007;26:141-52). FOLR1 has become a target in oncology because it is overexpressed in specific solid tumors, such as ovary, lung, and breast (Toffoli et al., Int J Cancer 1997;74:193-198 and Boogerd et al., Oncotarget 2016;7: 17442-17454), but its expression is at low levels in a limited number of normal human tissues (Weitman, et al., Cancer Res 1992; 52:3396-3401). Consistent with this observation, phase 1 clinical studies conducted to date with FOLR1-targeted small and large molecules have found the drug to be well tolerated (Cheung et al., Oncotarget 2016;7:52553-52574). Thus, FOLR1 is a tumor-associated/tumor-specific antigen, and monoclonal antibodies (rnAbs) against FOLR1 can be used as potential anticancer therapeutics.

Summary of the invention

In one general aspect, the invention provides isolated monoclonal antibodies or antigen binding fragments thereof that specifically bind FOLR1.

The present invention relates to isolated monoclonal antibodies or antigen binding fragments thereof containing heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, having polypeptide sequences of:

a) SEQ ID NO: 38, 39, 40, 62, 63 and 64, respectively;

b) SEQ ID NO: 17, 18, 19, 41, 42 and 43, respectively;

c) SEQ ID NO: 20, 21, 22, 44, 45 and 46, respectively;

d) SEQ ID NO: 23, 24, 25, 47, 48 and 49, respectively;

e) SEQ ID NO: 26, 27, 28, 50, 51 and 52, respectively;

f) SEQ ID NO: 29, 30, 31, 53, 54 and 55, respectively;

g) SEQ ID NO: 32, 33, 34, 56, 57 and 58, respectively; or

h) SEQ ID NO: 35, 36, 37, 59, 60 and 61, respectively;

wherein the antibody or antigen binding fragment thereof specifically binds FOLR1, preferably human FOLR1.

The present invention relates to isolated monoclonal antibodies or antigen binding fragments thereof containing heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, having polypeptide sequences of:

a) SEQ ID NO: 86, 87, 88, 110, 111 and 112, respectively;

b) SEQ ID NO: 65, 66, 67, 89, 90 and 91, respectively;

c) SEQ ID NO: 68, 69, 70, 92, 93 and 94, respectively;

d) SEQ ID NO: 71, 72, 73, 95, 96 and 97, respectively;

e) SEQ ID NO: 74, 75, 76, 98, 99 and 100, respectively;

f) SEQ ID NO:77, 78, 79, 101, 102 and 103, respectively;

g) SEQ ID NO: 80, 81, 82, 104, 105 and 106, respectively; or

h) SEQ ID NO: 83, 84, 85, 107, 108 and 109, respectively;

wherein the antibody or antigen binding fragment thereof specifically binds FOLR1, preferably human FOLR1.

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In specific embodiments, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:15, 1, 3, 5, 7, 9, 11 or 13, or a light chain variable region having a polypeptide sequence of at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16, 2, 4, 6, 8, 10, 12 or 14.

In specific embodiments, the isolated monoclonal antibody or antigen binding fragment thereof comprises:

a) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15, and a light chain variable region having the polypeptide sequence of SEQ ID NO:16;

b) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:1, and a light chain variable region having the polypeptide sequence of SEQ ID NO:2;

c) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:3, and a light chain variable region having the polypeptide sequence of SEQ ID NO:4;

d) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:5, and a light chain variable region having the polypeptide sequence of SEQ ID NO:6;

e) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7, and a light chain variable region having the polypeptide sequence of SEQ ID NO:8;

f) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9, and a light chain variable region having the polypeptide sequence of SEQ ID NO:10;

g) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:11, and a light chain variable region having the polypeptide sequence of SEQ ID NO:12; or

h) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:13, and a light chain variable region having the polypeptide sequence of SEQ ID NO:14.

In certain embodiments, the isolated monoclonal antibody or antigen binding fragment thereof is chimeric.

In specific embodiments, the isolated monoclonal antibody or antigen binding fragment thereof is human or humanized. In specific embodiments, the humanized monoclonal antibody or antigen binding fragment thereof comprises:

(1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:113, and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:113 and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

(3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:114 and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:114 and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

(5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:115, and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:115, and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

(7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:116 and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:116 and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

- 2 046422 (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:119 and a light chain variable region having the polypeptide sequence of SEQ ID NO:121;

(10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:119 and a light chain variable region having the polypeptide sequence of SEQ ID NO:122;

(11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:119, and a light chain variable region having the polypeptide sequence of SEQ ID NO:123;

(12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:120, and a light chain variable region having the polypeptide sequence of SEQ ID NO:121;

(13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:120, and a light chain variable region having the polypeptide sequence of SEQ ID NO:122;

(14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:120 and a light chain variable region having the polypeptide sequence of SEQ ID NO:123;

(15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:124 and a light chain variable region having the polypeptide sequence of SEQ ID NO:126;

(16) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:124 and a light chain variable region having the polypeptide sequence of SEQ ID NO:127;

(17) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:131;

(18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:132;

(19) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:133;

(20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:134;

(21) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:131;

(22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:132;

(23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:133;

(24) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:134;

(25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130 and a light chain variable region having the polypeptide sequence of SEQ ID NO:131;

(26) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130 and a light chain variable region having the polypeptide sequence of SEQ ID NO:132;

(27) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130 and a light chain variable region having the polypeptide sequence of SEQ ID NO:133; or (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130, and a light chain variable region having the polypeptide sequence of SEQ ID NO:134.

In specific embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof binds FOLR1 and is capable of inducing an indirect effect.

- 3 046422 ramie lysis of tumor cells.

The present invention also relates to isolated nucleic acids encoding monoclonal antibodies or antigen binding fragments thereof according to the invention.

The present invention also relates to vectors containing isolated nucleic acids encoding monoclonal antibodies or antigen binding fragments thereof according to the invention.

The present invention also relates to host cells containing vectors containing isolated nucleic acids encoding monoclonal antibodies or antigen binding fragments thereof according to the invention.

In specific embodiments, the present invention provides a pharmaceutical composition comprising an isolated monoclonal antibody or antigen binding fragment thereof of the invention and a pharmaceutically acceptable carrier.

The present invention also relates to methods of treating a malignant tumor in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention. The malignant tumor may be any liquid or solid malignant tumor, for example, it may be selected from, but not limited to, lung cancer, gastric cancer, colon cancer, hepatic cell carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer gland cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, and non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

The present invention also relates to methods for producing a monoclonal antibody or antigen binding fragment thereof according to the invention.

The methods include culturing a cell containing a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof under conditions for producing the monoclonal antibody or an antigen-binding fragment thereof, and isolating the antibody or an antigen-binding fragment thereof from the cell or culture.

The present invention also relates to methods for preparing a pharmaceutical composition containing a monoclonal antibody or antigen binding fragment according to the invention. The methods include combining a monoclonal antibody or an antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

The present invention also provides methods for determining the level of FOLR1 in a subject.

The methods include (a) obtaining a sample from a subject; (b) contacting the sample with an isolated monoclonal antibody or antigen binding fragment thereof according to the invention; and (c) determining the level of FOLR1 in the subject.

The sample may, for example, be a tissue sample or a blood sample. The tissue sample may, for example, be a cancer tissue sample.

Brief description of drawings

The foregoing summary of the invention, as well as the following detailed description of preferred embodiments of the present invention, will be better understood when read in conjunction with the accompanying drawings. It should be understood, however, that the invention is not limited to the exact embodiments shown in the drawings.

In fig. 1A-1B show the binding of purified mouse anti-FOLR1 mAbs to CHO cells stably expressing human FOLR1 as determined by FACS analysis.

In fig. 2A-2C show the results for dose-dependent binding of chimeric anti-FOLR1 mAbs to the immobilized extracellular domain of FOLR1 in an ELISA assay.

In fig. 3A-3D show results for dose-dependent binding of chimeric anti-FOLR1 mAbs to SK-OV-3 cells in a FACS assay.

In fig. 4A-4C show the results for dose-dependent binding of humanized mAb F5 to the immobilized extracellular domain of FOLR1 in an ELISA assay.

In fig. 5A-5B show results for dose-dependent binding of humanized mAb F10 to the immobilized extracellular domain of FOLR1 in an ELISA assay.

In fig. 6A-6B show results for dose-dependent binding of humanized mAb F17 to the immobilized extracellular domain of FOLR1 in an ELISA assay.

In fig. 7A-7D show results for dose-dependent binding of humanized mAb F20 to the immobilized extracellular domain of FOLR1 in an ELISA assay.

In fig. 8A-8J show results for dose-dependent binding of humanized mAbs to SK-OV-3 cells. Fig. 8A, data for humanized mAb F5; fig. 8B-8D, data for humanized mAb F10; fig. 8E, data for humanized mAb F17 mAb; fig. 8F-8J, data for humanized mAb F20.

In fig. 9 shows results for antibody-dependent cellular cytotoxicity (ADCC) activity of four humanized anti-FOLR1 mAbs.

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Detailed Description of the Invention

Various publications, articles and patents are cited or described in the prior art section and throughout the description; the entire contents of each of these references are incorporated herein by reference. The discussion of documents, acts, materials, devices, articles, or the like that are included herein is provided for the purpose of providing context on the invention. Such discussion is not an admission that any or all of this subject matter constitutes part of the prior art with respect to any inventions described or claimed.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one skilled in the art to which the present invention relates. Otherwise, specific terms used in this specification have the meanings as defined in the specification.

It should be noted that, as used in the present specification and in the accompanying claims, the singular forms include the plural entities of a reference unless the context clearly requires otherwise.

Unless otherwise indicated, any numerical values, such as concentration or concentration range, described herein are to be understood as modified in all cases by the term approximately. Thus, the numerical value typically includes ±10% of the specified value. For example, a concentration of 1 mg/ml includes 0.9-1.1 mg/ml. Likewise, the concentration range of 1-10% (w/v) includes 0.9-11% (w/v). For the purposes of the invention, the use of a numeric value expressly includes all possible subranges, all individual numeric values within that range, including integers within such ranges and fractions of values, unless the context clearly indicates otherwise.

Unless otherwise specified, the term at least preceding a series of elements should be understood to refer to each element in the series. One skilled in the art will know, or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be included in the invention.

For the purposes of the invention, the terms contains, contains, includes, includes, has, has, contains or contains, or any other variations thereof, are understood to imply the inclusion of the specified whole or group of wholes, but not as the exclusion of any other whole or group of wholes, and they are intended to be non-exclusive or non-limiting. For example, a composition, mixture, process, method, article or device that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent in such composition, mixture, process, method, article or device . Also, unless expressly stated to the contrary, or refers to the inclusive or rather than the exclusive or. For example, condition A or B is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and A and B both are true (or present).

Within the scope of the invention, the connecting term and/or between a plurality of listed elements is understood to include both individual and combined variants. For example, when two elements are connected by and/or, the first option refers to the applicability of the first element in the absence of the second. The second option refers to the applicability of the second element in the absence of the first. The third option relates to the applicability of the first and second elements together. Any of these options is understood to be included in the meaning, and thus satisfy the requirement of the term and/or, within the scope of the invention. The simultaneous applicability of more than one of the options is also understood to be included in the meaning, and thus satisfy the requirement of the term and/or.

As used throughout the specification and claims, the term consists of, or variations thereof such as consist of or consisting of, as used throughout the specification and claims, indicates the inclusion of any of the specified integer or group of integers, but that no additional integer or group of integers cannot be added to the specified method, structure or composition.

As used throughout the specification and claims, the term primarily consists of, or variations such as primarily consists of, or essentially consists of, as used throughout the specification and claims, indicates the inclusion of any of the specified integer or group of integers, and the optional inclusion of any of the specified a whole or group of wholes that do not significantly change the basic or novel properties of the specified method, structure or composition. See M.R.E.R. § 2111.03.

For purposes of the invention, subject is any animal, preferably a mammal, most preferably a human. The term mammal, as used herein, includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

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The terms right, left, below and above indicate directions in the drawings referred to.

It should also be understood that the terms approximately, approximately, generally, substantially and similar terms used herein when applied to a measurement or characteristic of a component of a preferred invention indicate that the described measurement/characteristic is not an exact boundary or parameter, and do not exclude minor deviations from them, which are functionally the same or similar, as understood by one skilled in the art. At a minimum, such references involving a numerical parameter include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), do not change the smallest significant figure.

The terms identical or percentage identity, in the context of two or more nucleic acid sequences or polypeptide sequences (eg, anti-FOLR1 antibodies and polynucleotides encoding them, FOLR1 polypeptides and FOLR1 polynucleotides encoding them), refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum consistency, as measured using one of the following sequence comparison algorithms, or by visual inspection.

For sequence comparisons, typically one sequence serves as a reference sequence to which the test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into the computer, subsequence coordinates are assigned, if necessary, and the algorithm parameters of the sequence comparison program are assigned. The sequence comparison algorithm then calculates the percentage sequence identity for the test sequence(s), relative to the reference sequence, based on the assigned program parameters.

Optimal alignment of sequences for comparison can be achieved, for example, using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), through the homology region alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), through the Pearson and Lipman similarity search method, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), through computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI) or through visual inspection (see, in In general, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, respectively. BLAST analysis software is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high similarity score sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive threshold score T when aligned with a word of the same length in the database sequence. T denotes the threshold score for the similarity of neighboring words (Altschul et al, supra). These initial adjacent word hits act as seeds to initiate searches to find longer HSPs containing them. Word hits are then expanded in both directions along each sequence until the cumulative alignment score can increase.

Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward for a pair of matching residues; always >0) and N (penalty for mismatched residues; always <0). For amino acid sequences, the scoring matrix is used to calculate the cumulative score. Word hit expansion in each direction stops when: the cumulative alignment score decreases by an X value from its maximum achieved value; the cumulative score tends to zero or lower due to the accumulation of one or more negatively valued residual alignments; or the end of any of the sequences has been reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses a default word length (W) of 11, expectation (E) of 10, M=5, N=-4, and comparison of both strands. For amino acid sequences, BLASTP uses a default word length (W) of 3 and expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) .

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In addition to calculating percent sequence identity, the BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One of the similarity measures provided by the BLAST algorithm is the least probability sum (P(N)), which indicates the probability with which a match between two nucleotide or amino acid sequences could occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum of probabilities when comparing the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or two polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is typically substantially identical to the second polypeptide, for example when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are essentially identical is that the two molecules hybridize to each other under stringent conditions.

Antibodies.

The invention generally relates to isolated anti-FOLR1 antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions containing the antibodies. The invention also relates to methods for producing antibodies and methods of using antibodies for the treatment of diseases, including cancer. Antibodies of the invention have one or more desirable functional properties, including, but not limited to, high affinity binding to FOLR1, high specificity for FOLR1, and the ability to inhibit tumor growth in patients in need and in animal models, when administered alone or in combination with other types of anticancer therapy.

In a General aspect, the invention relates to isolated monoclonal antibodies or antigen binding fragments thereof that specifically bind FOLR1.

As used herein, the term antibody is used in a broad sense to include immunoglobulin molecules or antibodies, including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. Typically, antibodies are protein or peptide chains that have binding specificity for a specific antigen. The structures of antibodies are well known. Immunoglobulins can be classified into five major classes (ie, IgA, IgD, IgE, IgG, and IgM), depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subclassified as isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Accordingly, the antibodies of the invention may belong to any of the five main classes or corresponding subclasses. Preferably, the antibodies of the invention are IgG1, IgG2, IgG3 or IgG4. Antibody light chains from vertebrate species can be assigned to one of two distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Accordingly, the antibodies of the invention may contain a kappa or lambda light chain constant domain. In accordance with specific embodiments, the antibodies of the invention include heavy and/or light chain constant regions from rat or human antibodies. In addition to the heavy and light constant domains, antibodies contain an antigen binding region consisting of a light chain variable region and a heavy chain variable region, each containing three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2 and CDR3) . The light chain variable region domains are alternatively designated LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively designated HCDR1, HCDR2, and HCDR3.

As used herein, the term isolated antibody refers to an antibody that is substantially free of other antibodies having a different antigenic specificity (eg, an isolated antibody that specifically binds FOLR1 is substantially free of antibodies that do not bind FOLR1). In addition, the isolated antibody is substantially free of other cellular material and/or chemicals.

As used herein, the term monoclonal antibody refers to an antibody derived from a population of substantially homogeneous antibodies, i.e. The individual antibodies that make up the population are identical, except for possible natural mutations that may be present in minute quantities. The monoclonal antibodies of the invention can be produced by the hybridoma method, phage display technology, gene cloning technology from individual lymphocytes, or recombinant DNA methods. For example, monoclonal antibodies can be produced by a hybridoma comprising a B cell derived from a transgenic non-human animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.

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As used herein, the term antigen-binding fragment refers to an antibody fragment, such as a diabody, Fab, Fab', F(ab') ₂ , Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) ₂ , bispecific dsFv ( dsFv-dsFv'), disulfide bridge-stabilized diabody (ds diabody), single chain antibody molecule (scFv), single domain antibody (sdab), scFv dimer (divalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelized single domain antibody, nanoantibody, domain antibody, divalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen that the parent antibody or parent antibody fragment binds to. In certain embodiments, the antigen binding fragment comprises a light chain variable region, a light chain constant region, and a heavy chain Fd fragment. In accordance with other specific embodiments, the antigen binding fragment comprises Fab and F(ab').

As used herein, the term single chain antibody refers to a single chain antibody conventional in the art that comprises a heavy chain variable region and a light chain variable region linked by a short peptide of about 15 to about 20 amino acids. As used herein, the term single-domain antibody refers to a single-domain antibody conventional in the art that contains a heavy chain variable region and a heavy chain constant region, or that contains only a heavy chain variable region.

As used herein, the term human antibody refers to an antibody produced by a human, or an antibody having an amino acid sequence corresponding to an antibody produced by a human, produced using any method known in the art. This definition of human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies containing at least one human heavy and/or light chain polypeptide.

As used herein, the term humanized antibody refers to a non-human antibody modified to increase sequence homology to that of a human antibody such that the antigen-binding properties of the antibody are maintained but its antigenicity in the human body is reduced.

As used herein, the term chimeric antibody refers to an antibody where the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable region of both the light and heavy chains often corresponds to the variable region of an antibody derived from a single mammalian species (eg, mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions match antibody sequences originating from another animal species (such as a human) to avoid eliciting an immune response in that species.

As used herein, the term multispecific antibody refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein the sequence of a first immunoglobulin variable domain of the plurality has binding specificity for a first epitope, and the sequence of a second immunoglobulin variable domain of the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, on the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or are substantially overlapped. In one embodiment, the first and second epitopes do not overlap or are substantially non-overlapping. In one embodiment, the first and second epitopes are on different antigens, for example, on different proteins (or on different subunits of a multimeric protein). In one embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

As used herein, the term bispecific antibody refers to a multispecific antibody that binds no more than two epitopes or two antigens. The bispecific antibody is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, on the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or are substantially overlapped. In one embodiment, the first and second epitopes are on different antigens, for example, on different proteins (or on different subunits of a multimeric protein). In one embodiment, the bispecific antibody comprises a heavy chain variable domain sequence and a leg variable domain sequence.

- 8 046422 chain having binding specificity for the first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for the second epitope. In one embodiment, the bispecific antibody comprises a half-antibody or fragment thereof having binding specificity for a first epitope and a half-antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody comprises a scFv or fragment thereof having binding specificity for a first epitope, and a scFv or fragment thereof having binding specificity for a second epitope. In one embodiment, the first epitope is located on FOLR1, and the second epitope is located on PD-1, PD-L1, LAG-3, TIM-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD47, CD73, apelin, DLL3, claudin 18.2, TIP-1, CD3 and/or other tumor-associated immunosuppressive or surface antigens.

As used herein, the term FOLR1 refers to folate receptor 1 (FOLR1), also known as folate receptor α (FRa) or folate binding protein (FBP), which is a glycophosphatidylinositol (GPI)-anchored cell surface membrane protein that has high affinity for the active form of folate, 5-methyltetrahydrofolate (5-MTF), and transports it and its derivatives into cells (Salazar and Ratnam, Cancer Metastasis Rev 2007;26:141-52). FOLR1 has become a target in oncology because it is overexpressed in specific solid tumors, such as ovary, lung, and breast (Toffoli et al., Int J Cancer 1997;74:193-198 and Boogerd et al., Oncotarget 2016;7: 17442-17454), but its expression is at low levels in a limited number of normal human tissues (Weitman, et al., Cancer Res 1992; 52:3396-3401). Consistent with this observation, phase 1 clinical studies conducted to date with FOLR1-targeted small and large molecules have shown the drug to be well tolerated (Cheung et al., Oncotarget 2016;7:52553-52574). Thus, FOLR1 is a tumor-associated/tumor-specific antigen, and monoclonal antibodies (mAbs) against FOLR1 may be potential anticancer therapeutics. In addition, FOLR1 can be used to specifically target therapeutic molecules to malignant tumor cells. An exemplary amino acid sequence of human FOLR1 is represented by No. GenBank accession NP_057937 (SEQ ID NO:135).

Within the scope of the invention, an antibody that specifically binds FOLR1 refers to an antibody that binds FOLR1, preferably human FOLR1, with a KD of 1 x 10 ^-7 M or less, preferably 1 x 10 ^-8 M or less, more preferably 5 x 10 ^-9 M or less, 1x10 ^-9 M or less, 5x10 ^-10 M or less, or 1x 10 ^-10 M or less. The term KD refers to the dissociation constant, which is derived from the ratio of Kd to Ka (ie Kd/Ka) and expressed as molar concentration (M). KD values for antibodies can be determined using methods accepted in the art, in light of the present disclosure. For example, the KD of antibodies can be determined using surface plasmon resonance, for example, using a biosensor system, such as the Biacore® system, or using biolayer interferometry technology, such as the Octet RED96 system.

The lower the KD value of an antibody, the higher the affinity with which the antibody binds to the target antigen.

In accordance with a specific aspect, the invention relates to an isolated monoclonal antibody or antigen binding fragment thereof comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, having polypeptide sequences from :

a) SEQ ID NO: 38, 39, 40, 62, 63 and 64, respectively;

b) SEQ ID NO: 17, 18, 19, 41, 42 and 43, respectively;

c) SEQ ID NO: 20, 21, 22, 44, 45 and 46, respectively;

d) SEQ ID NO: 23, 24, 25, 47, 48 and 49, respectively;

e) SEQ ID NO: 26, 27, 28, 50, 51 and 52, respectively;

f) SEQ ID NO: 29, 30, 31, 53, 54 and 55, respectively;

g) SEQ ID NO: 32, 33, 34, 56, 57 and 58, respectively; or

h) SEQ ID NO: 35, 36, 37, 59, 60 and 61, respectively;

wherein the antibody or antigen binding fragment thereof specifically binds FOLR1, preferably human FOLR1.

In accordance with a specific aspect, the invention relates to an isolated monoclonal antibody or antigen binding fragment thereof comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, having polypeptide sequences from :

a) SEQ ID NO: 86, 87, 88, 110, 111 and 112, respectively;

b) SEQ ID NO: 65, 66, 67, 89, 90 and 91, respectively;

c) SEQ ID NO: 68, 69, 70, 92, 93 and 94, respectively;

d) SEQ ID NO: 71, 72, 73, 95, 96 and 97, respectively;

- 9 046422

e) SEQ ID NO: 74, 75, 76, 98, 99 and 100, respectively;

f) SEQ ID NO:77, 78, 79, 101, 102 and 103, respectively;

g) SEQ ID NO: 80, 81, 82, 104, 105 and 106, respectively; or

h) SEQ ID NO: 83, 84, 85, 107, 108 and 109, respectively;

wherein the antibody or antigen binding fragment thereof specifically binds FOLR1, preferably human FOLR1.

According to another specific aspect, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example, 95, 96, 97, 98 or 99% identical to one of SEQ ID NO: 15, 1, 3, 5, 7, 9, 11 or 13, or a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96, 97, 98 or 99% identical to one of SEQ ID NO: 16, 2, 4, 6, 8, 10, 12 or 14. According to one preferred embodiment, the isolated monoclonal antibody or antigen binding fragment thereof of the invention comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 15, 1, 3, 5, 7, 9, 11 or 13, and a light chain variable region having a polypeptide sequence, at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96, 97, 98 or 99% identical to SEQ ID NO:16, 2, 4, 6, 8 , 10, 12 or 14, respectively.

In accordance with another specific aspect, the invention relates to an isolated monoclonal antibody or antigen binding fragment thereof according to the invention, comprising:

a) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15, and a light chain variable region having the polypeptide sequence of SEQ ID NO:16;

b) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:1, and a light chain variable region having the polypeptide sequence of SEQ ID NO:2;

c) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:3, and a light chain variable region having the polypeptide sequence of SEQ ID NO:4;

d) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:5, and a light chain variable region having the polypeptide sequence of SEQ ID NO:6;

e) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7, and a light chain variable region having the polypeptide sequence of SEQ ID NO:8;

f) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9, and a light chain variable region having the polypeptide sequence of SEQ ID NO:10;

g) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:11, and a light chain variable region having the polypeptide sequence of SEQ ID NO:12; or

h) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:13, and a light chain variable region having the polypeptide sequence of SEQ ID NO:14.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 38, 39, 40, 62, 63 and 64, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:15, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:16. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15; and a light chain variable region having the polypeptide sequence of SEQ ID NO:16.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and

- 10 046422

LCDR3 having the polypeptide sequences of SEQ ID NO: 17, 18, 19, 41, 42 and 43, respectively. In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:1, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:2. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1; and a light chain variable region having the polypeptide sequence of SEQ ID NO:2.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO:20, 21, 22, 44, 45 and 46, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:3, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:4. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:3; and a light chain variable region having the polypeptide sequence of SEQ ID NO:4.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 23, 24, 25, 47, 48 and 49, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:5, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:6. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:5; and a light chain variable region having the polypeptide sequence of SEQ ID NO:6.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 26, 27, 28, 50, 51 and 52, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:7, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:8. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7; and a light chain variable region having the polypeptide sequence of SEQ ID NO:8.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 29, 30, 31, 53, 54 and 55, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:9, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:10. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9; and a light chain variable region having the polypeptide sequence of SEQ ID

- 11 046422

NO:10.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 32, 33, 34, 56, 57 and 58, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:11, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:12. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:11; and a light chain variable region having the polypeptide sequence of SEQ ID NO:12.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 35, 36, 37, 59, 60 and 61, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:13, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:14. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:13; and a light chain variable region having the polypeptide sequence of SEQ ID NO:14.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NOs: 86, 87, 88, 110, 111 and 112, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:15, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:16. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15; and a light chain variable region having the polypeptide sequence of SEQ ID NO:16.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NOs: 65, 66, 67, 89, 90 and 91, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:1, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:2. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:1; and a light chain variable region having the polypeptide sequence of SEQ ID NO:2.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 68, 69, 70, 92, 93 and 94, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:3, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID

- 12 046422

NO:4. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:3; and a light chain variable region having the polypeptide sequence of SEQ ID NO:4.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 71, 72, 73, 95, 96 and 97, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:5, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:6. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:5; and a light chain variable region having the polypeptide sequence of SEQ ID NO:6.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO: 74, 75, 76, 98, 99 and 100, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:7, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:8. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7; and a light chain variable region having the polypeptide sequence of SEQ ID NO:8.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO:77, 78, 79, 101, 102 and 103, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:9, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:10. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9; and a light chain variable region having the polypeptide sequence of SEQ ID NO:10.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO:80, 81, 82, 104, 105 and 106, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96 , 97, 98 or 99% identical to SEQ ID NO:11, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, e.g. 95, 96, 97, 98 or 99% identical to SEQ ID NO:12. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:11; and a light chain variable region having the polypeptide sequence of SEQ ID NO:12.

In one embodiment, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO:83, 84, 85, 107, 108 and 109, respectively . In another embodiment, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or

- 13 046422 more, for example, 95, 96, 97, 98 or 99% identical to SEQ ID NO:13, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95, 96, 97, 98 or 99% identical to SEQ ID NO:14. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:13; and a light chain variable region having the polypeptide sequence of SEQ ID NO:14.

According to another specific aspect, the invention relates to an isolated monoclonal antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof is chimeric.

According to another specific aspect, the invention relates to an isolated monoclonal antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof is human or humanized.

According to another specific aspect, the humanized monoclonal antibody or antigen binding fragment thereof comprises:

(1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:113, and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:113, and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

(3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:114, and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:114 and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

(5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:115, and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:115, and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

(7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:116 and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:116 and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

(9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:119, and a light chain variable region having the polypeptide sequence of SEQ ID NO:121;

(10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:119 and a light chain variable region having the polypeptide sequence of SEQ ID NO:122;

(11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:119, and a light chain variable region having the polypeptide sequence of SEQ ID NO:123;

(12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:120, and a light chain variable region having the polypeptide sequence of SEQ ID NO:121;

(13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:120, and a light chain variable region having the polypeptide sequence of SEQ ID NO:122;

(14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:120 and a light chain variable region having the polypeptide sequence of SEQ ID NO:123;

(15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:124 and a light chain variable region having the polypeptide sequence of SEQ ID NO:126;

(16) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:124, and a light chain variable region having the polypeptide sequence of SEQ ID

- 14 046422

NO:127;

(17) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:131;

(18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:132;

(19) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:133;

(20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:134;

(21) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:131;

(22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:132;

(23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:133;

(24) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:134;

(25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130 and a light chain variable region having the polypeptide sequence of SEQ ID NO:131;

(26) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130 and a light chain variable region having the polypeptide sequence of SEQ ID NO:132;

(27) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130 and a light chain variable region having the polypeptide sequence of SEQ ID NO:133; or (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130, and a light chain variable region having the polypeptide sequence of SEQ ID NO:134.

In another general aspect, the invention relates to an isolated nucleic acid encoding a monoclonal antibody or antigen binding fragment thereof according to the invention. One skilled in the art will appreciate that the coding sequence of a protein can be changed (eg, replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, one skilled in the art will appreciate that the nucleic acid sequences encoding the monoclonal antibodies or antigen binding fragments thereof of the invention can be altered without changing the amino acid sequences of the proteins.

In another general aspect, the invention relates to a vector containing an isolated nucleic acid encoding a monoclonal antibody or antigen binding fragment thereof according to the invention. Any vector known to one skilled in the art in light of the present disclosure may be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any element to perform the conventional function of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selectable marker, and an origin of replication. The promoter may be a constitutive, inducible or repressible promoter. A number of expression vectors capable of delivering nucleic acids into a cell are known in the art and can be used herein to produce an antibody or antigen binding fragment thereof in a cell. Conventional cloning techniques or artificial gene synthesis can be used to produce a recombinant expression vector in accordance with embodiments of the invention. Such methods are well known to one skilled in the art in light of the present disclosure.

In another general aspect, the invention relates to a host cell containing an isolated nucleic acid encoding a monoclonal antibody or antigen binding fragment thereof of the invention. Any host cell known to one skilled in the art in light of the present disclosure can be used for recombinant expression of antibodies or antigen-binding fragments thereof.

- 15 046422 invention notes. In some embodiments, the host cells are E. coli TGI or BL21 cells (for expression, for example, scFv or Fab antibodies), CHO-DG44 or CHO-K1 cells, or HEK293 cells (for expression, for example, full-length IgG antibodies) . In certain embodiments, the recombinant expression vector is transformed into host cells by conventional methods, such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the genome of the host cell such that the recombinant nucleic acid is efficiently expressed.

In another general aspect, the invention relates to a method for producing a monoclonal antibody or an antigen-binding fragment thereof according to the invention, comprising culturing a cell containing a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof under conditions for producing a monoclonal antibody or an antigen-binding fragment thereof according to the invention, and isolating an antibody or an antigen-binding fragment thereof from a cell or cell culture (for example, from a supernatant). The expressed antibodies or antigen binding fragments thereof can be collected from cells and purified according to conventional methods known in the art and as described herein.

Pharmaceutical compositions.

In another general aspect, the invention relates to a pharmaceutical composition containing an isolated monoclonal antibody or antigen binding fragment thereof according to the invention and a pharmaceutically acceptable carrier. The term pharmaceutical composition, as used herein, means a product containing an antibody of the invention together with a pharmaceutically acceptable carrier. The antibodies of the invention and compositions containing them can also be used in the manufacture of a medicament for the therapeutic uses mentioned herein.

As used herein, the term carrier refers to any extender, diluent, excipient, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent may depend on the route of administration for a particular application. As used herein, the term pharmaceutically acceptable carrier refers to a non-toxic material that does not interfere with the effectiveness of the composition of the invention or the biological activity of the composition of the invention. In accordance with specific embodiments, in light of the present description, any pharmaceutically acceptable carrier suitable for use in a pharmaceutical antibody composition can be used in the invention.

Formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, for example, Remington: The Science and Practice of Pharmacy (eg, 21st edition (2005), and any later editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity agents, preservatives, stabilizers and chelating agents. One or more pharmaceutically acceptable carriers can be used in the formulation of the pharmaceutical compositions of the invention.

In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid composition is an aqueous composition, i.e. composition containing water. The liquid composition may comprise a solution, suspension, emulsion, microemulsion, gel, or the like. The aqueous composition typically contains at least 50% wt./wt. water or at least 60, 70, 75, 80, 85, 90, or at least 95% wt./wt. water.

In one embodiment, the pharmaceutical composition can be formulated in an injectable form that can be injected, for example, through an injection device (eg, a syringe or infusion pump). The injection can be administered, for example, subcutaneously, intramuscularly, intraperitoneally, intravitreally or intravenously.

In another embodiment, the pharmaceutical composition is a solid composition, such as a lyophilized or spray-dried composition, that can be used as is or to which the physician or patient adds solvents and/or diluents prior to use. Solid dosage forms may include tablets, such as compressed tablets and/or coated tablets, and capsules (eg, hard or soft gelatin capsules). The pharmaceutical composition may also be in the form of, for example, sachets, dragees, powders, granules, lozenges or powders for reconstitution.

Dosage forms may be immediate release, in which case they may contain a water-soluble or dispersible carrier, or they may be delayed release, sustained release, or modified release, in which case they may contain water-insoluble polymers that control the rate of dissolution of the dosage form. in the gastrointestinal tract or under the skin.

In other embodiments, the pharmaceutical composition can be delivered intranasally, intrabuccally, or sublingually.

The pH in the aqueous composition may be between pH 3 and pH 10. In one embodiment, the iso

- 16 046422 shaving, the pH of the composition is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the composition is from about 3.0 to about 7.0.

In another embodiment of the invention, the pharmaceutical composition contains a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid , tricine and tris(hydroxymethyl)aminomethane, and mixtures thereof. The buffer may be provided individually or in combination, at a concentration of from about 0.01 mg/ml to about 50 mg/ml, for example, from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions containing each of these specific buffers constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains a preservative. Non-limiting examples of preservatives include: benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidomerea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal and mixtures thereof. The preservative may be present alone or in combination, at a concentration of from about 0.01 mg/ml to about 50 mg/ml, for example, from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions containing each of these specific preservatives constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains an isotonic agent. Non-limiting examples of the embodiment include salt (such as sodium chloride), amino acid (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan and threonine), alditol (such as glycerol, 1,2-propanediol propylene glycol), 1 ,3propanediol and 1,3-butanediol), polyethylene glycol (eg PEG400) and mixtures thereof. Another example of an isotonic agent includes sugar. Non-limiting examples of sugars may be mono-, di- or polysaccharides, or water-soluble glucans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch and sodium carboxymethylcellulose. Another example of an isotonic agent is a sugar alcohol, where the term sugar alcohol is defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol and arabitol. The isotonic agent may be presented alone or in combination, at a concentration of from about 0.01 mg/ml to about 50 mg/ml, for example, from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions containing each of these specific isotonic agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA) salts, and mixtures thereof. The chelating agent may be present alone or in combination, at a concentration of from about 0.01 mg/ml to about 50 mg/ml, for example, from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions containing each of these specific chelating agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and/or one or more protease inhibitors.

In another embodiment of the invention, the pharmaceutical composition contains a stabilizer, wherein said stabilizer is carboxy-/hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing substances such as monothioglycerol) or thioglycolic acid. The stabilizer may be provided individually or in combination, at a concentration of from about 0.01 mg/ml to about 50 mg/ml, for example, from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions containing each of these specific stabilizers constitute alternative embodiments of the invention.

In further embodiments of the invention, the pharmaceutical composition contains one or more surfactants, preferably a surfactant, at least one surfactant or two different surfactants. The term surfactant refers to any molecules or ions consisting of a water-soluble (hydrophilic) part and a fat-soluble (lipophilic) part. The surfactant may, for example, be selected from the group consisting of anionic surfactants

- 17 046422 active substances, cationic surfactants, nonionic surfactants and/or zwitterionic surfactants. The surfactant may be present alone or in combination, at a concentration of from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions containing each of these specific surfactants constitute alternative embodiments of the invention.

In a further embodiment of the invention, the pharmaceutical composition contains one or more protease inhibitors, such as EDTA and/or benzamidine hydrochloric acid (HCl). The protease inhibitor may be present alone or in combination, at a concentration of from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions containing each of these specific protease inhibitors constitute alternative embodiments of the invention.

In another general aspect, the invention relates to a method for preparing a pharmaceutical composition containing a monoclonal antibody or an antigen-binding fragment thereof according to the invention, comprising combining the monoclonal antibody or an antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

Methods of application.

In another general aspect, the invention relates to a method of targeting FOLR1 on the surface of a cancer cell in a subject, comprising administering to the subject an isolated monoclonal antibody or antigen binding fragment thereof that specifically binds FOLR1, or a pharmaceutical composition of the invention. Binding of a monoclonal antibody or an antigen-binding fragment thereof to FOLR1 may mediate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and/or antibody-dependent cellular cytotoxicity (ADCC), or other effects leading to the death of the targeted cancer cell. A monoclonal antibody or an antigen-binding fragment thereof may, for example, serve to attract conjugated drugs and/or may form a bispecific antibody with another monoclonal antibody to mediate the death of the targeted cancer cell.

The functional activity of antibodies and antigen-binding fragments thereof that bind FOLR1 can be characterized by methods known in the art and as described herein. Methods for characterizing antibodies and antigen-binding fragments thereof that bind FOLR1 include, but are not limited to, affinity and specificity assays, including Biacore, ELISA and OctetRed assays; binding assays to detect antibody binding to FOLR1 on cancer cells or cells recombinantly expressing FOLR1 by FACS. In accordance with specific embodiments, methods for characterizing antibodies and antigen binding fragments thereof that bind FOLR1 include the methods described below.

In another general aspect, the invention relates to a method of treating a cancer in a subject in need thereof, comprising administering to the subject an isolated monoclonal antibody or antigen binding fragment thereof that specifically binds FOLR1, or a pharmaceutical composition of the invention. The malignant tumor may, for example, be selected from, but not limited to, lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, cancer head and neck, pancreatic cancer, glioma, glioblastoma and other solid tumors, and non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

In accordance with embodiments of the invention, the pharmaceutical composition contains a therapeutically effective amount of an anti-FOLR1 antibody or antigen-binding fragment thereof. As used herein, the term therapeutically effective amount refers to the amount of an active ingredient or component that produces a desired biological or medical response in a subject. The therapeutically effective amount can be determined empirically and in a generally accepted manner in relation to the intended purpose.

As used herein, in relation to anti-FOLR1 antibodies or antigen-binding fragments thereof, a therapeutically effective amount refers to the amount of an anti-FOLR1 antibody or antigen-binding fragment thereof that modulates the immune response in a subject in need thereof.

In accordance with specific embodiments, a therapeutically effective amount refers to an amount of drug that is sufficient to achieve one, two, three, four or more of the following effects: (i) reducing or alleviating the severity of the disease, disorder or condition being treated, or associated symptom; (ii) reducing the duration of the disease, disorder or condition being treated, or the symptom associated therewith; (iii) preventing the progression of a disease, disorder or associated

- 18 046422 standing to be treated, or a symptom associated with it; (iv) causing regression of the disease, disorder or condition being treated, or its associated symptom; (v) preventing the development or onset of a disease, disorder or condition being treated, or a symptom associated therewith; (vi) preventing recurrence of the disease, disorder or condition being treated, or the symptom associated therewith; (vii) reducing the incidence of hospitalization of a subject having a disease, disorder or condition being treated, or a symptom associated therewith; (viii) reducing the length of hospitalization of a subject having a disease, disorder or condition being treated, or a symptom associated therewith; (ix) increasing the survival rate of a subject with the disease, disorder or condition being treated, or an associated symptom; (xi) inhibiting or reducing the disease, disorder or condition being treated, or an associated symptom in a subject; and/or (xii) enhancing or improving the prophylactic or therapeutic effect(s) of another therapy.

The therapeutically effective amount or dose may be varied according to various factors such as the disease, disorder or condition being treated, routes of administration, target site, physiological condition of the subject (including, for example, age, body weight, general health), whether the subject is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Treatment doses are optimally titrated to optimize safety and effectiveness.

In accordance with specific embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous or intramuscular administration.

As used herein, the terms treat, treatment, and treatment are all intended to refer to the alleviation or reversal of at least one measurable physical parameter related to a cancer that is not necessarily detectable in the subject, but may be detectable in the subject. The terms treat, treat, and treat may also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In a specific embodiment, treat, treatment and treatment refer to alleviating, preventing the development or onset of, or reducing the duration of one or more symptoms associated with a disease, disorder or condition, such as a tumor or more preferably, a malignant tumor. In a specific embodiment, treat, treatment and treatment refer to the prevention of a disease, disorder or condition. In a specific embodiment, treat, treatment and treatment refer to increasing the survival of a subject having a disease, disorder or condition. In a specific embodiment, treat, treatment and treatment refer to the cessation of a disease, disorder or condition in a subject.

In accordance with specific embodiments, the present invention relates to compositions used for the treatment of cancer. For the treatment of cancer, the present compositions can be used in combination with other treatments, including, but not limited to, chemotherapy, anti-CD20 mAb, anti-CD47 mAb, anti-TIM-3 mAb, anti-LAG-3 mAb, anti-CD73 mAb, anti-apelin mAb , anti-CTLA-4 mAb, anti-PD-L1 mAb, anti-PD-1 mAb, PD-1/PD-L1 therapy, other immuno-oncology drugs, antiangiogenic agent, radiotherapy, antibody-drug conjugate (ADC), targeted therapy, or other anticancer drugs. Antibodies against FOLR1 can be used to construct bispecific antibodies with partners - mAbs against PD-1, PD-L1, LAG3, TIM-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD73, CD47, CD3, apelin, DLL-3, TIP-1, CLDN18.2 and/or other tumor surface antigens, for the treatment of malignancies/tumors expressing both FOLR1 and specific tumor-associated antigen.

As used herein, the term combination, in the context of administering two or more therapeutic agents to a subject, refers to the use of more than one therapeutic agent. Use of the term in combination does not limit the order in which the therapeutic agents are administered to a subject. For example, the first therapeutic agent (e.g., a composition described herein) may be administered before (e.g., 5, 15, 30, 45 minutes, 1, 2, 4, 6, 12, 16, 24, 48, 72, 96 h, 1, 2, 3, 4, 5, 6, 8, or 12 weeks before, simultaneously, or after (e.g., 5, 15, 30, 45 min, 1, 2, 4, 6, 12, 16, 24 48, 72, 96 hours, 1, 2, 3, 4, 5, 6, 8, or 12 weeks after) administration of the second therapeutic agent to the subject.

In another general aspect, the invention relates to a method for determining the level of FOLR1 in a subject. The methods include (a) obtaining a sample from a subject; (b) contacting the sample with a monoclonal antibody or antigen binding fragment thereof according to the invention; and (c) determining the level of FOLR1 in the subject.

As used herein, a sample refers to a biological sample isolated from a subject and may include, but is not limited to, whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies (eg, cancer tissue), lymph fluid, ascites

- 19 046422 new fluid, interstitial fluid, bone marrow, cerebrospinal fluid, saliva, mucus, sputum, sweat, urine or any other product of secretion, excretion, or other physiological fluids. A blood sample refers to whole blood or any fraction of it, including blood cells, serum and plasma.

In specific embodiments, the level of FOLR1 in a subject can be determined using assays selected from, but not limited to, Western blot analysis, ELISA analysis, and/or immunohistochemistry (IHC). Relative protein levels can be determined using Western blot analysis and immunohistochemistry (IHC), and absolute protein levels can be determined using ELISA analysis. When determining relative levels of FOLR1, FOLR1 levels can be determined in comparison between at least two samples, for example, between samples from the same subject at different time points, between samples from different tissues of the same subject, and/or between samples from various subjects. Alternatively, when determining absolute levels of FOLR1, for example, through an ELISA assay, the absolute level of FOLR1 in a sample can be determined by obtaining an ELISA assay standard prior to testing the sample. One skilled in the art will understand which analytical methods to use to determine the level of FOLR1 in a sample from a subject using the antibodies or antigen binding fragments thereof of the invention.

The use of methods for determining the level of FOLR1 in a sample from a subject can lead to the diagnosis of abnormal (increased, decreased or deficient) levels of FOLR1 in a disease and to making appropriate therapeutic decisions. Such a disease may include, for example, a malignant tumor. In addition, by monitoring FOLR1 levels in a subject, the risk of developing a disease as described above can be determined based on knowledge of FOLR1 levels in a particular disease and/or during the progression of a particular disease.

Embodiments

This invention relates to the following non-limiting embodiments.

Embodiment 1 is an isolated monoclonal antibody or antigen binding fragment thereof comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, having the polypeptide sequences of:

a) SEQ ID NO: 38, 39, 40, 62, 63 and 64, respectively;

b) SEQ ID NO: 17, 18, 19, 41, 42 and 43, respectively;

c) SEQ ID NO: 20, 21, 22, 44, 45 and 46, respectively;

d) SEQ ID NO: 23, 24, 25, 47, 48 and 49, respectively;

e) SEQ ID NO: 26, 27, 28, 50, 51 and 52, respectively;

f) SEQ ID NO: 29, 30, 31, 53, 54 and 55, respectively;

g) SEQ ID NO: 32, 33, 34, 56, 57 and 58, respectively; or

h) SEQ ID NO: 35, 36, 37, 59, 60 and 61, respectively;

wherein the antibody or antigen binding fragment thereof specifically binds FOLR1, preferably human FOLR1.

Embodiment 2 is an isolated monoclonal antibody or antigen binding fragment thereof comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, having the polypeptide sequences of:

a) SEQ ID NO: 86, 87, 88, 110, 111 and 112, respectively;

b) SEQ ID NO: 65, 66, 67, 89, 90 and 91, respectively;

c) SEQ ID NO: 68, 69, 70, 92, 93 and 94, respectively;

d) SEQ ID NO: 71, 72, 73, 95, 96 and 97, respectively;

e) SEQ ID NO: 74, 75, 76, 98, 99 and 100, respectively;

f) SEQ ID NO:77, 78, 79, 101, 102 and 103, respectively;

g) SEQ ID NO: 80, 81, 82, 104, 105 and 106, respectively; or

h) SEQ ID NO: 83, 84, 85, 107, 108 and 109, respectively;

wherein the antibody or antigen binding fragment thereof specifically binds FOLR1, preferably human FOLR1.

Embodiment 3 is an isolated monoclonal antibody or antigen binding fragment thereof from Embodiment 1 or 2, comprising a heavy chain variable region having a polypeptide sequence of at least 95%, at least 96%, at least 97%, at least 98% identical or at least 99% identical to SEQ ID NO: 15, 1, 3, 5, 7, 9, 11 or 13, or a light chain variable region having a polypeptide sequence that is at least 95% identical , at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16, 2, 4, 6, 8, 10, 12 or 14.

Embodiment 4 is an isolated monoclonal antibody or antigen binding fragment thereof from Embodiments 1-3, comprising:

- 20 046422

a) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15, and a light chain variable region having the polypeptide sequence of SEQ ID NO:16;

b) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:1, and a light chain variable region having the polypeptide sequence of SEQ ID NO:2;

c) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:3, and a light chain variable region having the polypeptide sequence of SEQ ID NO:4;

d) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:5, and a light chain variable region having the polypeptide sequence of SEQ ID NO:6;

e) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7, and a light chain variable region having the polypeptide sequence of SEQ ID NO:8;

f) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9, and a light chain variable region having the polypeptide sequence of SEQ ID NO:10;

g) a heavy chain variable region having a polypeptide sequence from SEQID

NO:11, and a light chain variable region having the polypeptide sequence from SEQID

NO:12; or

h) a heavy chain variable region having a polypeptide sequence from SEQID

NO:13, and a light chain variable region having the polypeptide sequence from SEQID

NO:14.

Embodiment 5 is an isolated monoclonal antibody or antigen binding fragment thereof from any of embodiments 1-4, wherein the antibody or antigen binding fragment thereof is chimeric.

Embodiment 6 is an isolated monoclonal antibody or antigen-binding fragment thereof from any of Embodiments 1-5, wherein the antibody or antigen-binding fragment thereof is human or humanized.

Embodiment 7 is the isolated monoclonal antibody or antigen binding fragment thereof from any of embodiment 6, wherein the humanized monoclonal antibody or antigen binding fragment thereof comprises:

(1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:113, and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:113, and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

(3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:114, and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:114 and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

(5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:115, and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:115, and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

(7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:116 and a light chain variable region having the polypeptide sequence of SEQ ID NO:117;

(8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:116 and a light chain variable region having the polypeptide sequence of SEQ ID NO:118;

(9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:119, and a light chain variable region having the polypeptide sequence of SEQ ID NO:121;

(10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:119, and a light chain variable region having the polypeptide sequence of SEQ ID

- 21 046422

NO:122;

(11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:119, and a light chain variable region having the polypeptide sequence of SEQ ID NO:123;

(12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:120, and a light chain variable region having the polypeptide sequence of SEQ ID NO:121;

(13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:120, and a light chain variable region having the polypeptide sequence of SEQ ID NO:122;

(14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:120 and a light chain variable region having the polypeptide sequence of SEQ ID NO:123;

(15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:124 and a light chain variable region having the polypeptide sequence of SEQ ID NO:126;

(16) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:124 and a light chain variable region having the polypeptide sequence of SEQ ID NO:127;

(17) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:131;

(18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:132;

(19) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:133;

(20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:128 and a light chain variable region having the polypeptide sequence of SEQ ID NO:134;

(21) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:131;

(22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:132;

(23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:133;

(24) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:129 and a light chain variable region having the polypeptide sequence of SEQ ID NO:134;

(25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130 and a light chain variable region having the polypeptide sequence of SEQ ID NO:131;

(26) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130 and a light chain variable region having the polypeptide sequence of SEQ ID NO:132;

(27) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130 and a light chain variable region having the polypeptide sequence of SEQ ID NO:133; or (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:130, and a light chain variable region having the polypeptide sequence of SEQ ID NO:134.

Embodiment 8 is an isolated monoclonal antibody or antigen-binding fragment thereof from any of embodiments 1-7, wherein the monoclonal antibody or antigen-binding fragment thereof is capable of binding FOLR1 and inducing effector-mediated lysis of tumor cells.

Embodiment 9 is an isolated nucleic acid encoding a monoclonal antibody or an antigen binding fragment thereof from any of embodiments 1-8.

- 22 046422

Embodiment 10 is a vector containing the isolated nucleic acid from Embodiment 9.

Embodiment 11 is a host cell containing the vector from Embodiment 10.

Embodiment 12 is a pharmaceutical composition comprising the isolated monoclonal antibody or antigen binding fragment thereof from any of embodiments 1-8 and a pharmaceutically acceptable carrier.

Embodiment 13 is a method of treating a cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of Embodiment 12.

Embodiment 14 is a method of targeting FOLR1 on the surface of a cancer cell in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of Embodiment 12.

Embodiment 15 is a method of producing a monoclonal antibody or an antigen-binding fragment thereof from any of embodiments 1 to 8, comprising culturing a cell containing a nucleic acid encoding the monoclonal antibody or an antigen-binding fragment thereof under conditions for producing the monoclonal antibody or an antigen-binding fragment thereof, and isolation of an antibody or its antigen-binding fragment from a cell or culture.

Embodiment 16 is a method of preparing a pharmaceutical composition containing a monoclonal antibody or antigen binding fragment from any of embodiments 1-8, comprising combining the monoclonal antibody or antigen binding fragment thereof with a pharmaceutically acceptable carrier to form a pharmaceutical composition.

Embodiment 17 is a method of determining the level of FOLR1 in a subject, comprising:

a) obtaining a sample from the subject;

b) contacting the sample with the isolated monoclonal antibody or antigen binding fragment thereof from any of embodiments 1-8; And

c) determining the subject's FOLR1 level.

Embodiment 18 is the method of Embodiment 17, where the sample is a tissue sample.

Embodiment 19 is the method of Embodiment 18, where the tissue sample is a cancer tissue sample.

Embodiment 20 is the method of Embodiment 17, where the sample is a blood sample.

Examples

Example 1: Identification of monoclonal antibodies against FOLR1.

Mice were immunized with a fusion protein containing human FOLR1 (Arg25 to Met233) fused C-terminally to human Fc (huFOLR1-huFc). Plasma titer was determined by ELISA. After euthanasia, lymph nodes and spleens were collected to obtain hybridomas. Hybridomas were grown in 96-well culture plates, and supernatants from individual wells were screened by ELISA and FACS using CHO cells stably expressing full-length human FOLR1. The best positive clones were isolated and sequenced.

The heavy and light chain variable region sequences for anti-FOLR1 monoclonal antibodies are presented in Tables 1 and 2, and the CDR regions for anti-FOLR1 monoclonal antibodies are presented in Tables 3-6.

Table 1

Heavy chain variable region sequences for anti-FOLR1 mAbs

mAb clones VH ID F4 EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLEW VAFISSGSNTIYYADIVKGRFAISRDNAKNTLFLQMASLRSEDTALYYC ARLAEWDVAYWGQGTLVTVSA 1 F5 EVQLVESGGELVKPGGSLKLSCAVSGFTFSNYGMSWVRQTPDKRLEW VATISSGGSYTYYPDSVKGRFTISRDNDKNTLYLQMSSLKSEDTAMYY CSTQGSSGYVGYWGQGTTLTVSS 3 F7 EFQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSNGKSLEWI GVIDPNYGTTNYNQKFVGKATLTVDQSSITAYMQLNSLTAEDSAVYFC AIKGYGNPAAYWGQGTLVTVSA 5 F8 EVMLVESGGGLVKPGGSLKLSCVASGFTLSTYAMSWVRQTPEKRLEW VATISGGGGDTYHLDTVKGRFTISRDNAKNTLYLQMSSLRSEDTALYY 7

- 23 046422

CARQSHYGSSYYFDNWGQGTTLTVSS F10 QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVRQRPGKGLEW IGRIYPGDGYTHYNGMFKGKATLTADKSSSTGYMQLSSLTSEDSAVYF CTRHGDFPYWYFDVWGTGTTVTVSS 9 F17 DVQLVESGGGLVQPGGSRKLSCAASGTFSDFGMHWIRQAPERGLEW VAYMSYTPGTFHYADTVKDRFFISRDNAKNTLFLQMTSLRSDDTAMY YCARVHVGTVDYWGQGTSVTVSS eleven F19 EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPDKGLE WVAQIGNKFHNYETYYSDSVKGRFTISRDDSKSSVYLQMNSLRVEDTG IYYCTKLGRGYYVMDYWGQGTSVTVSS 13 F20 QVQLQQSGAELVKPGASVQLSCKASGYTFASYYLYWVKQRPGQGLE WIGEINPRSGGTNFNEKFKSKATVTVDKSSSTAYMQLSSLTSEDSAVYY CSRSGRLRGFYTMDYWGQGTSVTVSS 15

VH: variable heavy chain region; ID: SEQ ID NO.

table 2

Light chain variable region sequences for anti-FOLR1 mAbs

mAb clones VL ID F4 DIVLTQSPATLSVTPGDRISLSCRASQNINNNLHWYQQKSHESPRLLIKF ASQSISGIPSRFSGSGSGTDFTLNINGVETEDFGMYFCQQIYSWPQLTFG AGTRLELK 2 F5 DIQMTQSPSSLSAFLGGKVTITCKASQDITNFIGWYQHKPGKGPRLLISY TSILESGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYYNLWTFGGGT KLEIK 4 F7 DIQMTQSPSSLSASLGGKVTITCKASQDINKYLAWYQHEPGKGPRLLIR YTSILESGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYYNLWTFGG GTKLEIK 6 F8 DIQMTQSPASLSSASVGEIVTIICRVSENIDSYLAWYQQKQGKSPQLLVY AATNLADGVPSRFSGSGSGSQYSLKINSLQSEDVARYYCQHYYTTPPTF GGGTKLDIK 8 F10 DIQMTQSPASLSSASVGESVTITCRASENIDSYLAWYQQKQGKSPQLLVY AATNLAVGVPSRFSGSGSGTQYTLKINSLQSEDVARYYCQHHYSTPPTF GGGTKLEIK 10 F17 DVVLTQSPATLSVTPGDSVSLSCRASQNINNNLHWFQQKSHESPRLLIK YASQSISGIPSRFSGSGSGTDFTLSINNVETEDFGIYFCQQSNSWPALTFG TGTKVELK 12 F19 DIQMTQTTSSLSASLGDRVTLNCRASQDITNHLNWFQQKPDGTFQLLIY YTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQDSQHPWTF GGGTKLEIK 14 F20 NIVMTQSPKSMSVSVGERVTLSCKAGENVGSYVSWYQQKPEQSPELLI YGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYYCGQTYRFLT FGAGTKLELK 16

VL: variable light chain region; ID: SEQ ID NO.

Table 3

Heavy chain CDR regions 1-3 for anti-FOLR1 mAb

mAb clones HC CDR1 ID HC CDR2 ID HC CDR3 ID F4 GFTFSDYG 17 ISSGSNTI 18 ARLAEWDVAY 19 F5 GFTFSNYG 20 ISSGGSYT 21 STQGSSGYVGY 22 F7 GYSFTDYN 23 IDPNYGTT 24 AIKGYGNPAAY 25 F8 GFTLSTYA 26 ISGGGGDT 27 ARQSHYGSSYYFDN 28 F10 GYAFSSSW 29 IYPGDGYT thirty TRHGDFPYWYFDV 31

F17 GFTFSDFG 32 MSYTPGTF 33 ARVHVGTVDY 34 F19 GFTFSDYW 35 IGNKFHNYET 36 TKLGRGYYVMDY 37 F20 GYTFASYY 38 INPRSGGT 39 SRSGRLRGFYTMDY 40

HC: heavy chain; CDR: complementarity determining region; ID: SEQ ID NO.

The HC CDR for the anti-FOLR1 mAb was determined using the IMGT method (Lefranc, M.-P. et al., Nucleic Acids Res. 1999; 27:209-212).

- 24 046422

Table 4

Light chain CDR regions 1-3 for anti-FOLR1 mAb

mAb clones LC CDR1 ID LC CDR2 ID LC CDR3 ID F4 QNINNN 41 F.A.S. 42 QQIYSWPQLT 43 F5 QDITNF 44 YTS 45 LQYYNLWT 46 F7 QDINKY 47 YTS 48 LQYYNLWT 49 F8 ENIDSY 50 AAT 51 QHYYTTPPT 52 F10 ENIDSY 53 AAT 54 QHHYSTPPT 55 F17 QNINNN 56 YAS 57 QQSNSWPALT 58 F19 QDITNH 59 YTS 60 QQDSQHPWT 61 F20 ENVGSY 62 G.A.S. 63 GQTYRFLT 64

LC: light chain; CDR: complementarity determining region; ID: SEQ ID NO LC CDR for mAb against FOLR1 was determined using the IMGT method (Lefranc, M.-P. et al., Nucleic Acids Res. 1999; 27:209-212).

Table 5

Heavy chain CDR regions 1-3 for anti-FOLR1 mAb

mAb clones HC CDR1 ID HC CDR2 ID HC CDR3 ID F4 GFTFSDYGMH 65 FISSGSNTIYYADIVKG 66 ARLAEWDVAY 67 F5 GFTFSNYGMS 68 TISSGGSYTYYPDSVKG 69 STQGSSGYVGY 70 F7 GYSFTDYNMN 71 VIDPNYGTTNYNQKFVG 72 AIKGYGNPAAY 73 F8 GFTLSTYAMS 74 TISGGGGDTYHLDTVKG 75 ARQSHYGSSYYFD N 76 F10 GYAFSSSWMN 77 RIYPGDGYTHYNGMFKG 78 FRHGDFPYWYFDV 79 F17 GFTFSDFGMH 80 YMSYTPGTFHYADTVKD 81 ARVHVGTVDY 82 F19 GFTFSDYWMN 83 QIGNKFHNYETYYSDSV KG 84 FKLGRGYYVMDY 85 F20 GYTFASYYLY 86 EINPRS GGTNFNEKFKS 87 SRSGRLRGFYTMD 88

HC: heavy chain; CDR: complementarity determining region; ID: SEQ ID NO.

The HC CDR for the anti-FOLR1 mAb was determined using the method of Kabat (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991)).

Table 6

Light chain CDR regions 1-3 for anti-FOLR1 mAbs

mAb clones LC CDR1 ID LC CDR2 ID LC CDR3 ID F4 RASQNINNNLH 89 FASQSIS 90 QQIYSWPQLT 91 F5 KASQDITNFIG 92 YTSILES 93 LQYYNLWT 94 F7 KASQDINKYLA 95 YTSILES 96 LQYYNLWT 97 F8 RVSENIDSYLA 98 AATNLAD 99 QHYYTTPPT 100 F10 RASENIDSYLA 101 AATNLAV 102 QHHYSTPPT 103 F17 RASQNINNNLH 104 YASQSIS 105 QQSNSWPALT 106 F19 RASQDITNHLN 107 YTSRLHS 108 QQDSQHPWT 109 F20 KAGENVGSYVS 110 GASNRYT 111 GQTYRFLT 112

LC: light chain; CDR: complementarity determining region; ID: SEQ ID NO.

The LC CDR for the anti-FOLR1 mAb was determined using the method of Kabat (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991)).

Example 2: mAb production and purification from hybridoma supernatants.

Mouse anti-FOLR1 mAbs were purified from hybridoma media/supernatants from positive clones using protein A affinity chromatography.

Example 3: FACS binding analysis of purified mouse anti-FOLR1 antibodies.

CHO cells stably transfected with full-length human FOLR1 were transferred to a 96-well plate at 200,000 cells/well, washed once with FACS buffer (1xPBS, pH6.8+2% FBS) and incubated with purified mouse anti-FOLR1 mAb from hybridoma supernatants. various concentrations for 15 min on ice. Cells were then centrifuged at 1000 rpm for 5 min and washed with FACS buffer three times. Cells were incubated with PE-conjugated polyclonal goat anti-mouse IgG antibodies and incubated on ice for a further 15 min. Cells were then washed using FACS buffer three times, and then resuspended in FACS buffer containing 0.1 μg/ml PI (propidium iodide) for live/dead cell selection. Cells were then processed using a FACS Caliber device and data were analyzed using Flowjo software. The binding results of purified mouse anti-FOLR1 mAbs to CHO cells stably expressing human FOLR1 are shown in FIG. 1A-1B. MFI, mean fluorescence intensity.

Example 4: Production and purification of chimeric anti-FOLR1 mAbs from culture media from transfected 293E cells.

To produce recombinant chimeric mAbs against FOLR1 using expression vectors,

- 25 046422 containing murine variable regions (VH and VL) fused to human IgG1 heavy chain and human kappa light chain constant regions, respectively, were transiently transfected into 293E cells. Recombinant antibodies produced in the transfected cell suspension were purified using protein A affinity chromatography.

Example 5: Binding ELISA assay of purified anti-FOLR1 chimeric mAbs.

Purified chimeric mAbs were tested in an ELISA assay for their ability to bind to immobilized human FOLR1. Recombinant human FOLR1 extracellular domain (Novoprotein, cat#: C784) in carbonate coating buffer was coated onto a 96-well plate at room temperature for 1 hour. The plate was blocked with 5% BSA in TBST for 1 hour at room temperature. In each well of an individual plate, mAbs at various concentrations were incubated for 1 hour at room temperature. The plate was washed and binding to FOLR1 was detected using horseradish peroxidase-conjugated anti-human IgG (hIgG-HRP) (ThermoFisher Scientific, cat#: H10007) with incubation for 1 hour at room temperature. Then, after washing, the ELISA was developed using a one-step detection solution (ThermoFisher Scientific, Cat#: 34028) and measured as absorbance at 450 nm. The results are shown in Fig. 2A2S.

Example 6: FACS binding analysis of purified anti-FOLR1 chimeric mAbs.

To assess the binding of chimeric anti-FOLR1 mAbs to cells known to express FOLR1, SK-OV-3 cells (ATCC® HTV-77™) were plated at 80,000 cells per well in a 96-well non-binding U-bottom plate (Greiner Bio-One, cat.#: 650901). In some experiments, 90,000 cells per well were used. Cells were incubated in 50 μl of FACS buffer (HBSS with 0.1% BSA and 0.05% sodium azide) containing various concentrations of mAb on ice for 15 min. After washing, cells were incubated in 50 μl FACS buffer containing 3.5 μg/ml goat anti-human Fc IgG conjugated to Alexa Fluor® 488 (Invitrogen, cat#: H10120) on ice in for 15 minutes in the dark and washed again. Cells were analyzed using an Attune NxT flow cytometer. The binding results of the chimeric anti-FOLR1 mAb to SK-OV-3 cells are shown in FIG. 3A-3D. MFI, mean fluorescence intensity.

Example 7: Biacore assay using anti-FOLR1 chimeric mAbs.

The interaction of chimeric mAbs against FOLR1 and FOLR1 was measured by surface plasmon resonance (SPR) (Biacore 8k, GE Healthcare). Briefly, anti-FOLR1 mAb was immobilized on a Protein A sensor chip (GE Healthcare, Cat#: 29-1275-56) in HBS-P buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0. 05% v/v surfactant P20) with an injection flow rate of 10 µl/ml. Recombinant FOLR1 (Novoprotein, cat.#: C784) at different concentrations was loaded at a flow rate of 30 μl/min in HBS-P buffer. After antigen loading, the surface was regenerated using 10 mM glycine-HCl (pH 1.5). Sensograms were fit to a 1:1 binding model using Biacore 8k evaluation software (GE Healthcare). The results for the binding affinity of the anti-FOLR1 mAb are shown in Table 7.

Table 7 _________KD values for anti-FOLR1 mAbs from the Biacore assay_________

Chimeric mAb KD(hM) F4 0.547 F5 0.352 F7 2.33 F8 0.721 F10 0.704 F17 0.265 F19 11.8 F20 1.00

Example 8: Humanization of anti-FOLR1 mAb.

Murine anti-FOLR1 mAbs have been humanized to reduce the potential for immunogenicity when used in human patients. The sequences of the heavy and light chain variable regions (VH and VL) were compared with human antibody sequences in the Protein Data Bank (PDB) and homology models were constructed. CDRs in both the heavy and light chains of murine mAbs were grafted into human scaffolds having the highest ability to maintain the proper structure likely required for antigen binding. Back mutations from human residues to murine residues or other mutations were developed as necessary. The sequences of the humanized VH and VL regions are shown in table. 8. The humanized VH and VL regions were fused to the human IgG1 heavy chain and human kappa light chain constant regions, respectively. Constructs corresponding to mAb sequences were used for transient transfection in CHO cells, and purified mAbs were analyzed for their ability to bind to the immobilized extracellular domain of FOLR1 in an ELISA. Isotype controls were tested in assays to confirm the absence of nonspecific

- 26 046422 sky binding above the background of the analysis. The results for the humanized F5 clones are shown in FIG. 4A-4C; results for the humanized F10 clones are shown in FIG. 5A-5B; results for the humanized F17 clones are shown in FIG. 6A-6B; results for the humanized F20 clones are shown in FIG. 7A-7D.

Humanized anti-FOLR1 mAbs were tested for their ability to bind to SKOV-3 cells using FACS analysis. The data is shown in Fig. 8A-8J.

Antibody-dependent cellular cytotoxicity (ADCC) of the mAb was measured using the ADCC Reporter Bioassay Core Kit (Promega, cat. #G7010), according to the protocol provided by the manufacturer. Briefly, approximately 12,500 SK-OV-3 cells per well were mixed with various concentrations of test antibodies in ADCC assay buffer in a 96-well, half-well microplate (Corning-Costar, cat. #3696). Then, approximately 37,500 ADCC Bioassay Effector cells per well were added to a final volume of 37.5 μl and mixed. The plate was incubated at 37°C for 6 hours without shaking. To measure luciferase activity, 12.5 μL of assay mixture was removed from each well and 25 μL of BioGlo Luciferase Assay Reagent was added. The plates were incubated at room temperature for 10 min with shaking. 30 μl of the mixture per well was transferred to a white plate (BRAND, cat. #781621) for luminescence measurement in an EnVision 2102 multimode plate reader. Results for ADCC activity of the 4 humanized mAbs are shown in FIG. 9. RLU, relative light units.

Table 8 Heavy chain and light chain variable region sequences of humanized anti-FOLR1 mAbs

VH/VL SUBSEQUENCE ID: F5-H1 EVQLLESGGGLVQPGGSLRLSCAVSGFTFSNYGMSWVRQAPGKGLEWV ATISSGGSYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCST QGS SGYVGYWGQGTLVTVS S 113 F5-H2 EVQLVESGGGLVQPGGSLRLSCAVSGFTFSNYGMSWVRQAPGKGLEWV ATISSGGSYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCST QGS SGYVGYWGQGTLVTVS S 114 F5-H3 EVQLVESGGGLVQPGGSLRLSCAVSGFTFSNYGMSWVRQAPGKGLEWV ATISSGGSYTYYPDSVKGRFTISRDNSKNTLYLQMsSLRAEDTAVYYCSTQ GS SGYVGYWGQGTLVTVS S 115 F5-H4 EVQLVESGGGLVQPGGSLRLSCAVSGFTFSNYGMSWVRQAPGKGLEWV ATISSGGSYTYYPDSVKGRFTISRDNDKNTLYLQMsSLRAEDTAVYYCST QGS SGYVGYWGQGTLVTVS S 116 F5-L1 DIQMTQSPSSVSASVGDRVTITCKASQDITNFIGWYQHKPGKAPKLLISYT SILESGVPSRFSGSGSGRDYTLTISSLQPEDFATYYCLQYYNLWTFGGGTK VEIL 117 F5-L2 DIQMTQSPSSVSASVGDRVTITCKASQDITNFIGWYQHKPGKAPKLLISYT SILESGVPSRFSGSGSGtDYTLTISSLQPEDFATYYCLQYYNLWTFGGGTK VEIL 118 F10-H1 E VQLVQ SGAEVKKPGS S VK VSCK ASGYAF S S S WMNWVRQ APGQGLEWI GRIYPGDGYTHYNGMFKGRASLTADKSSTTGYMELSSLRSEDTAVFFCTR HGDFPYWYFDVWGRGTLVTVSP 119 F10-H2 E VQLVQ SGAEVKKPGS SVK VSCK ASGYAF S S S WMNWVRQ APGQGLEWI GRIYPGDGYTHYNGMFKGRASLTADKSSTTGYMELSSLRSEDTAVFFCTR HGDFP YW YFD VWGRGTLVT VS S 120 F10-L1 DIQMTQSPSTLSASVGDRVTITCRASENIDSYLAWYQQKPGRAPKLLVYA ATNLAVGVPSRFSGSGSGTEYTLTISSLQSDDFATYYCQHHYSTPPTFGQG TKLEIK 121 F10-L2 DIQMTQSPSTLSASVGDRVTITCRASENIDSYLAWYQQKPGRAPKLLVYA ATNLAVGVPSRFSGSGSGTEYTLTISSLQpDDFATYYCQHHYSTPPTFGQG TKLEIK 122 F10-L3 DIQMTQSPSTLSASVGDRVTITCRASENIDSYLAWYQQKPGRAPKLLVYA ATNLAVGVPSRFSGSGSGTEYTLTISSLQSeDFATYYCQHHYSTPPTFGQG TKLEIK 123 F17-H1 EVQLVETGGGLIQPGGSLRLSCAASGFTFSDFGMHWIRQAPGKGLEWVA YMSYTPGTFHYADTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR VHVGT VD YWGQGTLVT VS S 124 F17-H2 QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMHWIRQAPGKGLEWV AYMSYTPGTFHYADTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RVHVGTVD YWGQGTLVT VS S 125 F17-L1 EVVLTQSPATLSLSPGERATLSCRASQNINNNLHWFQQKPGQAPRLLIKYA SQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNSWPALTFGQGTK VEIL 126 F17-L2 EVVLTQSPATLSLSPGERATLSCRASQNINNNLHWFQQKPGQAPRLLIKYA SQSISGIPARFSGSGSGTDFTLTISSLEtEDFAVYFCQQSNSWPALTFGQGTK 127

-

Claims (6)

V.E.I.K. F20-H1 QVQLVQSGAEVKKPGSSVKVSCKASGYTFASYYLYWVRQAPGQGLEWI GEINPRSGGTNFNEKFKSRATVTVDKSTSTAYMELSSLRSEDTAVYYCSRS GRLRGF YTMDYWGQGTLVT VS S 128 F20-H2 QVQLVQSGAEVKKPGSSVKVSCKASGYTFASYYLYWVRQAPGQGLEWI GEINPRSGGTNFNEKFKSRATVTVDaSTSTAYMELSSLRSEDTAVYYCSRS GRLRGF YTMDYWGQGTLVT VS S 129 F20-H3 QVQLVQSGAEVKKPGSSVKVSCKASGYTFASYYLYWVRQAPGQGLEWI GEINPRSGGTNFNEKFKSKATVTVDKSTNTAYMELS SLRSEDTAVYYC SR SGRLRGF YTMDYWGQGTLVTVS S 130 F20-L1 DIVMTQSPDSLAVSLGERATINCKAGENVGSYVSWYQQKPGQPPKLLIY GASNRYTGVPDRF SGSGS ATDFTLTIS SLQ AED VAVYYCGQTYRFLTFGQ GTKVEIK 131 F20-L2 DIVMTQSPDSLAVSLGERATINCKAGENVGSYVSWYQQKPGQsPKLLIYG ASNRYTGVPDRF SGSGS ATDFTLTIS SLQ AED VAVYYCGQTYRFLTFGQGT KVEIK 132 F20-L3 DIQMTQSPSTLSASVGDRVTITCKAGENVGSYVSWYQQKPGKAPKLLIY GASNRYTGVPARFSGSGSATEFTLTISSLQPDDFATYYCGQTYRFLTFGQG TKVEVK 133 F20-L4 DIQMTQSPSTLSASVGDRVTITCKAGENVGSYVSWYQQKPGKAPKLLIY GASNRYTGVPARFSGSGSATEFTLTISSLQPeDFATYYCGQTYRFLTFGQGT KVEVK 134 One skilled in the art will appreciate that changes can be made to the embodiments described above without departing from their broad inventive spirit. It is understood, therefore, that this invention is not limited to the specific embodiments described, but is intended to include modifications within the scope and scope of the present invention as defined by the present description. CLAIM 1. An isolated monoclonal antibody or antigen-binding fragment thereof containing heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, with polypeptide sequences SEQ ID NO:29, 30, 31, 53, 54 and 55, respectively, or SEQ ID NO: 77, 78, 79, 101, 102 and 103, respectively, wherein the antibody or antigen binding fragment thereof specifically binds to human FOLR1, preferably human cFOLR1. 2. An isolated monoclonal antibody or an antigen-binding fragment thereof according to claim 1, containing a heavy chain variable region with a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98 % or at least 99% identical to SEQ ID NO:120, 9 or 119; or a light chain variable region with a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:122 , 10, 121 or 123. 3. An isolated monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, containing: a) a heavy chain variable region with the polypeptide sequence SEQ ID NO:120; and a light chain variable region with the polypeptide sequence SEQ ID NO: 122; b) a heavy chain variable region with the polypeptide sequence of SEQ ID NO:9; and a light chain variable region with the polypeptide sequence of SEQ ID NO:10; c) a heavy chain variable region with the polypeptide sequence SEQ ID NO: 119; and a light chain variable region with the polypeptide sequence SEQ ID NO: 121; d) a heavy chain variable region with the polypeptide sequence SEQ ID NO: 119; and a light chain variable region with the polypeptide sequence SEQ ID NO: 122; e) a heavy chain variable region with the polypeptide sequence SEQ ID NO: 119; and a light chain variable region with the polypeptide sequence SEQ ID NO: 123; f) a heavy chain variable region with the polypeptide sequence of SEQ ID NO:120; and a light chain variable region with the polypeptide sequence SEQ ID NO: 121; g) a heavy chain variable region with the polypeptide sequence of SEQ ID NO:120; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 123. 4. An isolated monoclonal antibody or antigen binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen binding fragment thereof is chimeric, human or humanized. 5. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the monoclonal antibody or antigen-binding fragment thereof is capable of binding to FOLR1 and inducing effector-mediated lysis of tumor cells. 6. Bispecific antibody, characterized in that it contains a monoclonal antibody or its -