A monoclonal antibody targeting human folate receptor alpha

Abstract

The present application provides a monoclonal antibody targeting folate receptor 1 (FR1) of human. Specifically, the present application immunizes mice with FR1 recombinant protein, and screens a monoclonal antibody with high affinity to FR1. In addition, the present application also provides an amino acid sequence of the monoclonal antibody, a nucleic acid containing the sequence, a vector or conjugate containing the nucleic acid, and application of the monoclonal antibody in tumors / cancers with overexpression of FR1.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and more particularly, the present application relates to a monoclonal antibody targeting human folate receptor alpha. BACKGROUND

[0002] Folic acid (vitamin B9) is an important single-carbon donor for the synthesis of purines and thymidylate, and indirectly provides a donor for protein and DNA methylation modification through S-adenosyl methionine. In adult normal tissues, folic acid is mainly absorbed by Reduced Folate Carrier (RFC). RFC is a ubiquitous anion channel expressed in the human body, responsible for the transport of folic acid by most normal cells, but has low affinity for folic acid binding (Km = 1-10 M).

[0003] Folate receptors (FR, gene name FOLR) are a class of cysteine-rich glycoproteins that bind folic acid with high affinity to mediate the absorption of folic acid, and are divided into alpha, beta, and gamma subtypes. Folate receptor alpha (referred to as FR1, gene name FOLR1) is a glycosylphosphatidylinositol (GPI) anchored membrane protein composed of a single chain polypeptide, containing a signal peptide, a folic acid binding domain, and a GPI anchor signal sequence. Among them, the folic acid binding domain is the main site of folic acid binding. FR1 is a key protein involved in the uptake of folic acid by cells. Folic acid is essential for DNA synthesis, repair, and methylation processes, so FR1 is essential for rapidly dividing cells, such as developing embryonic cells and some cancer cells.

[0004] FR1 is essential for folic acid homeostasis and has important implications in oncology, neurology, and developmental biology. Although FR1 has very low expression levels in most normal tissues, it is highly expressed in many cancer cells of epithelial origin, which are used to seize the limited folic acid molecules in the environment to meet the needs of rapid division and growth of cancer cells. Cerebral folic acid deficiency (CFD) is a rare metabolic encephalopathy caused by FOLR1 gene mutation, characterized by significant folate deficiency in the central nervous system, and clinical symptoms include seizures, motor development delay, and intellectual impairment. Its pathogenesis is closely related to the dysfunction of FOLR1-mediated folate transport across the blood-brain barrier.

[0005] The overexpression of FR1 in cancer and its role in CFD highlight its dual significance as a therapeutic target and diagnostic marker. FOLR1 is highly expressed on the surface of various tumor cells, such as ovarian cancer, lung cancer, breast cancer, etc., while the expression level in normal tissues is low or not expressed. This differential expression in normal cells and cancer cells provides an important basis for targeting FR1 for molecular diagnosis and treatment. There are various forms of means for treating and diagnosing tumors by targeting folate receptors, including high-affinity antifolates, small-molecule conjugates of folate with drugs or toxins, antibodies against folate receptor alpha (FR1), and FR1-ADC, etc. In addition, folate-based imaging agents are also used in clinical diagnosis for the imaging tracing of FR1 -high-expressing cancers.

[0006] Among the various forms of FR1 -targeting drugs, only one antibody conjugate drug targeting folate receptors (Mirvetuximab soravtansine, MIRV) has been successfully used in clinical treatment of ovarian cancer. According to the results of the phase III clinical trial of SORAYA (NCT04296890), the US FDA accelerated the approval of MIRV for the treatment of FR1 -positive, platinum-resistant epithelial ovarian cancer in November 2022. As a receptor on the surface of cancer cells, FR1 is not only highly expressed in ovarian cancer, but also in other epithelial cell-derived cancers, including non-small cell lung cancer, renal cancer, endometrial cancer, colorectal cancer, head and neck cancer, and breast cancer, showing the clinical potential of FR1 -targeting ADC drugs for the treatment of various solid tumors.

[0007] Currently, there are still many deficiencies in the research on FOLR1 monoclonal antibodies, and there is an urgent need in the field to develop new monoclonal antibodies against FR1 and explore their potential applications in various tumor-targeted therapies, diagnostic markers, and other medical fields. SUMMARY

[0008] The purpose of the present application is to provide a new monoclonal antibody against FR1.

[0009] In a first aspect of the present application, an antibody or antigen-binding fragment thereof targeting folate receptor alpha (FR1) is provided, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), said heavy chain variable region and light chain variable region being selected from the group consisting of:

[0010] a heavy chain variable region comprising the following three CDRs:

[0011] HCDR1 of the amino acid sequence as shown in SEQ ID NO. 1,

[0012] HCDR2 of the amino acid sequence set forth in SEQ ID NO. 2,

[0013] HCDR3 of the amino acid sequence set forth in SEQ ID NO. 3; and

[0014] a light chain variable region comprising the following three CDRs:

[0015] LCDR1 of the amino acid sequence set forth in SEQ ID NO. 4,

[0016] LCDR2 of the amino acid sequence set forth in SEQ ID NO. 5,

[0017] LCDR3 of the amino acid sequence set forth in SEQ ID NO. 6.

[0018] In another preferred embodiment, the heavy chain variable region sequence of the antibody or antigen binding fragment thereof is set forth in SEQ ID NO. 7.

[0019] In another preferred embodiment, the heavy chain variable region of the antibody or antigen binding fragment thereof has a sequence with at least 90% sequence identity to the sequence set forth in SEQ ID NO. 7.

[0020] In another preferred embodiment, the light chain variable region sequence of the antibody or antigen binding fragment thereof is set forth in SEQ ID NO. 8.

[0021] In another preferred embodiment, the light chain variable region of the antibody or antigen binding fragment thereof has a sequence with at least 90% sequence identity to the sequence set forth in SEQ ID NO. 8.

[0022] In another preferred embodiment, the antibody or antigen binding fragment thereof specifically binds to a human FOLR1 protein.

[0023] In another preferred embodiment, the antibody further comprises a constant region.

[0024] In another preferred embodiment, the constant region of the antibody is a humanized constant region.

[0025] In another preferred embodiment, the heavy chain variable region comprises the three heavy chain CDRs and a human heavy chain framework region for connecting the heavy chain CDRs.

[0026] In another preferred embodiment, the light chain variable region comprises the three light chain CDRs and a human light chain framework region for connecting the light chain CDRs.

[0027] In another preferred embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, a Fv, a single-chain Fv antibody ("scFv"), a bis-scFv, a (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), or a combination thereof.

[0028] In another preferred embodiment, the antibody is a murine antibody, a murine human chimeric antibody, or a humanized IgGl antibody.

[0029] In another preferred embodiment, the antibody comprises a mono-specific, bi-specific, tri-specific antibody, or a multi-specific antibody. In another preferred embodiment, the antibody mediates a biological effect selected from the group consisting of antigen neutralization, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody Fc fragment-mediated opsonization, or complement-mediated opsonization.

[0030] In a second aspect of the present application, there is provided a nucleic acid, the polynucleotide encoding the antibody or antigen-binding fragment thereof of the first aspect of the present application.

[0031] In a third aspect of the present application, there is provided a vector, the vector comprising the nucleic acid of the second aspect of the present application.

[0032] In another preferred embodiment, the vector is selected from the group consisting of a plasmid, a virus (such as a lentivirus, an adenovirus, an AAV virus, a retrovirus), a cosmid, or a combination thereof.

[0033] In another preferred embodiment, the vector is a plasmid expression vector.

[0034] In a fourth aspect of the present application, there is provided a host cell, the host cell comprising the vector of the third aspect of the present application, or having integrated into its genome an exogenous nucleic acid of the second aspect of the present application.

[0035] In another preferred embodiment, the host cell is an Escherichia coli.

[0036] In another preferred embodiment, the cell is an isolated cell, and / or a genetically engineered cell.

[0037] In another preferred embodiment, the cell is a somatic cell.

[0038] In another preferred embodiment, the cell is a mammalian cell.

[0039] In a fifth aspect of the present application, there is provided a pharmaceutical composition, the pharmaceutical composition comprising:

[0040] 1) the antibody or antigen-binding fragment thereof of the first aspect of the present application, the nucleic acid of the second aspect of the present application, the vector of the third aspect of the present application, the host cell of the fourth aspect of the present application, or a combination thereof; and

[0041] 2) a pharmaceutically acceptable carrier.

[0042] In a sixth aspect of the present application, there is provided a use of the antibody or antigen-binding fragment thereof of the first aspect of the present application, the nucleic acid of the second aspect of the present application, the vector of the third aspect of the present application, or the host cell of the fourth aspect of the present application, for the manufacture of a medicament for treating a FR1-overexpressing tumor / cancer.

[0043] In another preferred embodiment, the tumor / cancer is selected from the group consisting of ovarian cancer, non-small cell lung cancer, or a combination thereof.

[0044] In a seventh aspect of the present application, there is provided a method for treating a FR1-overexpressing tumor / cancer, the method comprising: administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of the first aspect of the present application, the nucleic acid of the second aspect of the present application, the vector of the third aspect of the present application, and / or the host cell of the fourth aspect of the present application.

[0045] In an eighth aspect of the present application, there is provided a pharmaceutical combination comprising:

[0046] 1) the antibody or antigen-binding fragment thereof of the first aspect of the present application, the nucleic acid of the second aspect of the present application, the vector of the third aspect of the present application, or the host cell of the fourth aspect of the present application as a first medicament; and

[0047] 2) another tumor-targeting drug or a chemotherapeutic drug as a second medicament.

[0048] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features specifically described hereinafter (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 PCR amplification results of antibody light chain and heavy chain of the obtained single B cells are shown: lane 1 is DL5000 marker; lane 2 corresponds to FR1-102 light chain and heavy chain variable region PCR band; lane 6 is FR1-106 light chain and heavy chain variable region PCR band.

[0050] Figure 2 The binding of the three antibodies found in the ELISA test to the FR1 antigen is shown.

[0051] Figure 3 The ELISA test shows the affinity of antibody 102 to FR1 antigen. The fitting degree R of the curve 2 = 0.98.

[0052] Figure 4 The flow test shows the binding of different antibodies to Hela cells.

[0053] Figure 5 The flow method shows the affinity of antibody 102 to Hela cells. DETAILED DESCRIPTION

[0054] The present inventors have developed a monoclonal antibody targeting human folate receptor alpha isoform (FR1) through extensive and in-depth research. The present application immunizes mice with FR1 recombinant protein, isolates antigen-specific B cells from the spleen and lymph nodes, and purifies monoclonal antibodies with high affinity to FR1. The monoclonal antibody can be used to treat tumors / cancers overexpressing FR1.

[0055] TERMS

[0056] For easier understanding of the present application, certain technical and scientific terms are defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present application belongs. Before describing the present application, it should be understood that the present application is not limited to the particular methodology and experimental conditions described, as such methodology and conditions can vary.

[0057] As used herein, the terms "comprise", "comprising", "contain", "containing", "include", "including" and "includes" are interchangeable and are meant to be non-limiting.

[0058] In the present application, the terms "antibody 106", "106 antibody" and "FR1-106" are used interchangeably and all refer to antibody No. 106 targeting FR1.

[0059] In the present application, the terms "antibody 102", "102 antibody" and "FR1-102" are used interchangeably and all refer to antibody No. 102 targeting FR1.

[0060] In the present application, the terms "antibody 122", "122 antibody" and "FR1-122" are used interchangeably and all refer to antibody No. 122 targeting FR1.

[0061] As used herein, the term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin which specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each L chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each of VHand VLcomprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0062] As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

[0063] As used herein, the term "light chain variable region" is used interchangeably with "VL".

[0064] In a given antibody's light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one of a number of well-known antibody CDR assignment systems, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the international Immuno GeneTics database (IMGT), the EU numbering system, and Chothia definition based on loop structure position.

[0065] It is to be understood that the precise amino acid sequence boundaries of the CDRs in the application can optionally be defined utilizing the different assignment systems mentioned above. Preferably, unless otherwise specified, in the present application, when referring to residue positions in an antibody variable region, including heavy chain variable region residues and light chain variable region residues, the numbering of the positions is according to the Kabat numbering system.

[0066] As used herein, the term "variable" refers to certain portions of the variable regions of the antibodies that differ in sequence among the various particular antibodies and are responsible for binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable regions of the antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more highly conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, joined by three CDRs, which are generally termed CDR1, CDR2 and CDR3, in sequence from amino- to carboxy-terminus. The CDRs in each chain are aligned by the FR regions, which generally form a β-sheet; the CDRs are then held in place with respect to each other by the FR regions. The CDRs are largely responsible for binding the antibody to an antigen, while the framework regions affect the variable region's overall structure (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647- 669 (1991)). The constant regions of the antibodies are not directly involved in binding to an antigen but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0067] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates of drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules with the antibodies of the present application or fragments thereof.

[0068] In a preferred embodiment of the present application, the heavy chain variable region and the light chain variable region of the antibody each comprise three complementarity determining regions CDR1, CDR2, and CDR3.

[0069] The present application includes not only intact antibodies, but also fragments of the antibodies that are immunologically active or fusions of the antibodies with other sequences. Thus, the present application also includes fragments, derivatives and analogs of the antibodies.

[0070] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present application. The polypeptide fragments, derivatives or analogs of the present application can be (i) polypeptides having one or more conservative or non-conservative amino acid substitutions, preferably conservative amino acid substitutions, and such substituted amino acids can or can not be encoded by the genetic code, or (ii) polypeptides having a substitution group at one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide to another compound, such as a compound that increases the half-life of the polypeptide, for example, polyethylene glycol, or (iv) polypeptides formed by fusing additional amino acid sequences to the polypeptide, such as leader or secretion sequences, or sequences or proteins for purification of the polypeptide, or protein tags, such as a 6His tag. These fragments, derivatives and analogs are within the scope of those skilled in the art in light of the teachings herein.

[0071] The term "antibody" of the present application refers to a polypeptide having FOLRl binding activity comprising the CDR regions described above. The term also includes variants of polypeptides comprising the CDR regions described above which have the same function as the antibodies of the present application. These variants include, but are not limited to, deletion, insertion, and / or substitution of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, as well as addition of one or more (typically 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer) amino acids at the C-terminus and / or the N-terminus. For example, in the art, substitution of similar or identical amino acids typically does not change the function of the protein. Also, addition of one or more amino acids at the C-terminus and / or the N-terminus typically does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present application.

[0072] The variants of the polypeptides include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to the DNA encoding the antibodies of the present application under high or low stringency conditions, and polypeptides or proteins obtained using antisera against the antibodies of the present application.

[0073] The present application also provides other polypeptides, such as fusion proteins comprising the antibodies or fragments thereof. In addition to the almost full-length polypeptides, the present application also includes fragments of the antibodies of the present application. Typically, the fragments have at least about 50 contiguous amino acids of the antibodies of the present application, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, most preferably at least about 100 contiguous amino acids.

[0074] In the present application, "conservative variants of the antibodies of the present application" refer to polypeptides in which up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by similar or identical amino acids compared to the amino acid sequence of the antibodies of the present application. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table A.

[0075] Table A

[0076]

[0077] The present application also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof described above. The polynucleotides of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0078] Polynucleotides encoding the mature polypeptides of the present application include: a coding sequence encoding only the mature polypeptide; a coding sequence encoding the mature polypeptide and various additional coding sequences; a coding sequence encoding the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0079] The term "polynucleotide encoding a polypeptide" can be a polynucleotide comprising a coding sequence encoding the polypeptide, or a polynucleotide comprising an additional coding and / or non-coding sequence.

[0080] The nucleotide full-length sequence of the antibody of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. One possible method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is short. Generally, a long fragment can be obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0081] Once the relevant sequence is obtained, it can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then transforming it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules in isolated form.

[0082] At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present application by chemical synthesis.

[0083] The present application also relates to vectors comprising the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express the protein.

[0084] The host cells can be prokaryotic cells such as bacterial cells, or lower eukaryotic cells such as yeast cells, or higher eukaryotic cells such as mammalian cells. Representative examples include: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0085] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation. If necessary, the transformation can be performed by electroporation. When the host is a eukaryote, transformation techniques such as calcium phosphate co-precipitation, conventional mechanical procedures such as microinjection, electroporation, and the like, can be used.

[0086] The transformants obtained can be cultured in conventional media using standard procedures to express the polypeptides encoded by the genes of the application. The medium used to culture the host cells is selected to provide an appropriate growth medium for the host cell. Once the host cells have been cultured to a density appropriate for the selected promoter, the cells are induced by appropriate means (e.g., temperature shift or chemical induction) to express the recombinant polypeptides.

[0087] The recombinant polypeptides in the above methods can be expressed intracellularly, or intramembrane, or secreted from the cell. If desired, the recombinant proteins can be isolated and purified using various separation and purification methods well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional procedures such as renaturation, treatment with a protein precipitant (salting out), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

[0088] The antibodies of the application can be used alone or in combination or conjugation with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties, or any combination of the above.

[0089] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing detectable products.

[0090] Therapeutic agents that can be combined or conjugated with the antibodies of the application include, but are not limited to, 1. radionuclides; 2. biotoxins; 3. cytokines such as IL-2 and the like; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or benzyl-hydrolyzing enzyme-like protein (BPHL)), and the like.

[0091] The present application also provides a composition. In preferred embodiments, the composition is a pharmaceutical composition comprising the antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding immune cell described above, and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably the pH is about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the disease to be treated.

[0092] The prepared pharmaceutical composition can be administered by conventional routes, including but not limited to intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the administration route of the pharmaceutical composition of the present application is preferably injection or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, etc. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, which can be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a granule, an injection or an infusion, etc.

[0093] The antibody of the present application can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0094] The pharmaceutical composition of the present application contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the monoclonal antibody (or conjugate thereof) described above and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as needle, solution is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents.

[0095] In the present application, preferably, the pharmaceutical composition of the present application further comprises one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers are conventional pharmaceutically acceptable carriers in the art, and the pharmaceutically acceptable carriers can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical adjuvant is conventional pharmaceutical adjuvant in the art, and preferably comprises pharmaceutically acceptable excipients, fillers or diluents, etc. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and 0.01-99.99% of the pharmaceutically acceptable carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0096] In the present application, preferably, the administration amount of the pharmaceutical composition is an effective amount, and the effective amount is an amount capable of alleviating or delaying the progression of a disease, a degenerative or an injury condition. The effective amount can be determined on an individual basis, and will be based in part on the condition to be treated and the considerations of the sought result. The effective amount can be determined by a person skilled in the art by using the above-mentioned factors such as individual basis and using no more than conventional experiments.

[0097] When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, and preferably the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dose will also take into account the route of administration, the health condition of the patient, etc., which are within the skill of a skilled physician.

[0098] Compared with the prior art, the main advantages of the present application include:

[0099] 1. The monoclonal antibody of the present application can specifically bind to folate receptor alpha (FR1).

[0100] 2. The monoclonal antibody of the present application can be used for treating tumors / cancers overexpressing FOLR1.

[0101] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0102] Example 1. Monoclonal antibody screening

[0103] 1. Monoclonal B cell screening

[0104] 1.1 Mouse immunization

[0105] Prepare 3 Balb / c mice, mix the recombinant protein human FOLR1 (uniprot: P15328; protein expression sequence Arg 25 - Met 233) with adjuvant, and then perform intraperitoneal injection, 12 μg of protein per mouse, every 3 days, 3 days after the 5th intraperitoneal injection, collect 100 μL of peripheral blood from the mouse eyeball, and detect the immune titer. Select the mouse with the best immune titer, and perform boost immunization by intraperitoneal injection of 20 μg of protein. Three days after immunization, separate the mouse spleen and lymph nodes for single B cell sorting.

[0106] 1.2 Immune titer detection

[0107] 1) Take the mouse out of the cage, disinfect the mouse's eyes with a 75% medical alcohol cotton ball, and use a 5 mm blood collection needle to make a small incision in the mouse's eyeball;

[0108] 2) Collect blood drops (100 μL of plasma need to be prepared) using a capillary glass blood collection tube;

[0109] 3) After collecting the blood, use a dry sterile cotton ball to gently press the blood collection point to stop bleeding, and then return the mouse to the cage for observation;

[0110] 4) Place the centrifuge tube containing the blood sample in a 37°C incubator for 1 hour, then transfer the blood sample to 4°C overnight.

[0111] 5) Separate the serum from the blood clot and transfer it to a new sterile centrifuge tube, centrifuge at 4°C, 10000xg for 10 min;

[0112] 6) Transfer the serum to a new sterile centrifuge tube and detect the immune titer by ELISA.

[0113] 1.3 ELISA detection

[0114] 1) Dilute the human FOLR1 his recombinant protein to a final concentration of 0.5 μg / mL using sterile carbonate buffer solution (CBS). Take a new 96-well plate, add 100 μL / well and incubate overnight at 4°C.

[0115] 2) Remove the antigen coating solution and wash 3 times with PBST (containing 0.5% Tween).

[0116] 3) Add 200 μL / well of 3% BSA and incubate at 37°C for 1 hour;

[0117] 4) After removing the blocking buffer, wash the plate 3 times with PBST;

[0118] 5) Add 100 μL mouse serum (diluted 300 times at most, 3 times gradient dilution), incubate at room temperature for 1 hour, and the control wells are 1% BSA;

[0119] 6) Remove the liquid in the wells, and wash 3 times with PBST;

[0120] 7) Add 100 μL HRP mouse IgG (1:10000 dilution), incubate at room temperature for 1 hour;

[0121] 8) After removing the liquid in the wells, wash the plate 3 times with PBST;

[0122] 9) Add 100 μL / well TMB developing solution;

[0123] 10) Incubate at room temperature for 5 minutes in the dark;

[0124] 11) Add 50 μL / well stop solution;

[0125] 12) Read the OD450 value in the wells using a microplate reader.

[0126] 2. Single B cell sorting

[0127] 2.1 Take the best mouse for immune titer detection, dislocate the neck to kill the mouse, obtain the mouse spleen and lymph nodes under sterile conditions, prepare a B cell single cell suspension, and count.

[0128] 2.2 According to the number of cells, add human FOLR1 antigen with biotin label (1 μg / 10 7Cell staining buffer wash cells, resuspend cells in 1 mL cell staining buffer, APC-Cy7 CD19 (1:150), FITC anti-mouse IgG1 (1:150), PerCP / Cy5.5 anti-mouse / human GL7 (1:150), PE / Cy7 anti-mouse CD38 (1:300), APC-streptavidin (1:500), PE-streptavidin (1:500) staining; 4°C, dark incubation for 30 min; take immune BSA protein control mouse cells to set up isotype control. At the same time, set up single positive tube, adjust the compensation between each channel. Cell staining buffer wash cells, resuspend cells in 1 mL cell staining buffer, add DAPI, room temperature, dark incubation for 5 min. Cell sorting fluid rinse cells, 1000 rpm / min, 4°C centrifugation for 5 min, discard supernatant. Resuspend cells in 500 μL PBS buffer, use Beckman Coulter cytoflex SRT flow cytometer to sort human FOLR1 antigen-specific single B cells.

[0129] 2.3 Flow sorting of antigen-specific single B cells

[0130] Among all B cells, by screening cell size and cell granularity, mononuclear cell population is selected, and live cells are identified by DAPI (negative selection); CD19 (positive selection) recognizes B cell population, and germinal center B cells are sorted by CD38 (negative selection) and GL7 (positive selection); IgG1 (positive selection) secretes antibodies stimulated by antigen; PE (positive selection) and APC (positive selection) sort out antigen-specific single B cells (DAPI-CD19+GL7+CD38-IgG1+Ag biotin++). Sort cells into 96-well PCR plate wells containing 4 μL sterile enzyme-free PBS, and after sorting the required number of cells (92 cells), transfer the cells to 80°C for temporary storage for subsequent experiments.

[0131] 3. Preparation of human FOLR1 antigen-specific single B cell antibodies

[0132] 3.1 Reverse transcription of mRNA encoding antibodies in sorted human FOLR1 antigen-specific single B cells into cDNA.

[0133] 3.2 Amplify antibody heavy chain and light chain complete coding gene fragments respectively by designing primers with cDNA as template.

[0134] 3.3 Clone VH into pTT5 eukaryotic expression vector containing human IgG1 Fc, name the heavy chain skeleton vector as pTT5-human IgG1 CH; clone VL into pTT5 eukaryotic expression vector containing human CL, name the light chain skeleton vector as pTT5-human IgG1 CL. Co-transfect the antibody heavy chain and light chain recombinant expression vectors into 293F suspension cells, collect the supernatant after transfection and purify, and obtain the single antibody expressed by the single B cell encoding, which can be used for subsequent monoclonal antibody identification and screening.

[0135] 3.4 Amplification of single B cell cDNA

[0136] According to the requirements of the instruction of Maxima H Minus Reverse Transcriptase (manufacturer: Thermo Scientific, product number: EP0753), the mRNA (containing antibody encoding genes) in the sorted single B cells is reverse transcribed into cDNA.

[0137] 3.5 Amplification of single B cell antibody heavy chain complete encoding gene

[0138] 1) Antibody heavy chain amplification method (Table 1): using the single B cell cDNA amplified as the template, using the nest PCR technique, first using primers 5' MsVHE Fw and 3' mIgG1-2b-2c for amplification. The amplification system is shown in Table 1, and the PCR amplification program is: 98℃ 10s; 55℃ 30s, 72℃ 55s, 35 cycles; 72℃ 5min. The band of the desired fragment size (about 200-400bp) is recovered and purified.

[0139] 2) Antibody light chain amplification method (Table 2): using the single B cell cDNA amplified as the template, using the nest PCR technique, first using primers 5' L-Vk mix Fw and 3' mCk for amplification. The amplification system is shown in Table 2, and the PCR amplification program is: 98℃ 10s; 55℃ 30s, 72℃ 55s, 35 cycles; 72℃ 5min. Using primers 2nd-5VK-FW and 2nd-3VK-RV to amplify the amplification product, the PCR amplification program is: 98℃ 10s, 55℃ 30s, 72℃ 45s, 35 cycles; 72℃ 5min. The band of the desired fragment size (about 200-400bp) is recovered and purified.

[0140] Table 1. Conditions for heavy chain VH PCR amplification

[0141]

[0142] Table 2. Conditions for light chain VK PCR amplification

[0143]

[0144] PCR amplification results of antibody light and heavy chains of single B cells are shown in Figure 1 , in which lane 2 corresponds to FR1-102 antibody light and heavy chain variable region PCR bands; lane 6 is FR1-106 antibody light and heavy chain variable region PCR bands.

[0145] 3) The recovered and purified product was sequenced (sequencing was completed by Suzhou Jw Biotech Co., Ltd.), and the sequencing results were analyzed by NCBI Igblast (https: / / www.ncbi.nlm.nih.gov / igblast), and part of the amplification results are shown in Figure 3 , and the sequence analysis results are as follows:

[0146] Cloning 102 antibody heavy chain variable region (SEQ ID NO. 7):

[0147] QVQLHYSGAELAKPGASLKVSCKASGYTFTTYCMHWVKHRPGQGLEWIGYINPTTGYTEYNQKFKDKATLTADKSSSTAYMQLSTLTSEDSAVYYCSRSMYYSTDYWFDYWGQGTTLTVSS;

[0148] Cloning 102 antibody light chain variable region (SEQ ID NO. 8):

[0149] DIVITQSPAILSVSPGERVSFSCRASQSIGTSIHWYQRRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNTWPLTFGAGTKLELK.

[0150] Table 3. Sequence of heavy chain CDR of 102 antibody

[0151]

[0152] Table 4. Sequence of light chain CDR of 102 antibody

[0153]

[0154] Example 2. Antibody verification

[0155] 1. Construction of single B cell antibody complete coding gene recombinant plasmid

[0156] According to the sequence analysis verified as IgG antibody heavy, light chain complete coding gene sequence design primer, respectively in the upstream primer 5' end and downstream primer 3' end respectively add pTT5-human IgG1 CH and pTT5-human IgG1 CL expression vector homologous sequence, upstream and downstream primer respectively named Primer-f1, Primer-b1.

[0157] PCR product as template for amplification, PCR reaction as shown in Table 5, PCR program: 95℃ 2 min; 95℃ 15s, 50℃ 30s, 72℃ 30s, 30 cycles; 72℃ 5min, as shown in Table 6. The amplified product was identified by 1.5% agarose gel electrophoresis, and the band with the desired fragment size was recovered and purified. By homologous recombination, the purified VH target fragment was ligated with the linearized pTT5-human IgG1 CH vector obtained by PmeI and NheI double digestion, and the purified VK target fragment was ligated with the linearized pTT5-human IgG1 CL vector obtained by PmeI and BsiwI double digestion. The reaction conditions were 50℃ for 60min, and the reaction system was as shown in Table 7 and Table 8.

[0158] The homologous recombination product was transformed into E. coli Top10 competent cells, and 3 clones were randomly selected for sequencing. The sequencing alignment (analyzed by Snapgene software) was performed, and the single B cell antibody complete coding gene recombinant expression plasmid was extracted by shaking bacteria with correct sequencing results.

[0159] Table 5. Cloning PCR component table

[0160]

[0161] Table 6. Cloning PCR reaction temperature

[0162]

[0163] Table 7. Heavy chain ligation reaction component table

[0164]

[0165] Table 8. Light chain ligation reaction component table

[0166]

[0167] 2. Expression of single B cell antibody

[0168] The 293F suspension cells were subcultured into 125 mL triangular flask, and when the cell density reached 2 x 10 6 / mL, the cells were transfected. As an example of one flask, the antibody heavy chain and light chain recombinant expression plasmids cloned from the same B cell were co-transfected. Prepare A solution: add 10 μg of heavy chain plasmid, 20 μg of light chain plasmid and 1 mL of SMM 293-TII medium in a 1.5 mL centrifuge tube, and mix gently. Prepare B solution: add 60 μg of PEI transfection reagent and 1 mL of SMM 293-TII medium in another 1.5 mL EP tube, and mix gently. Then slowly add A solution to B solution drop by drop, mix gently, and stand for 15 min. After that, evenly add the transfection complex to the cells, and incubate in a shaker. After 1 day of transfection, add glucose (final concentration 3 g / L) and sodium valproate (3.5 mM), and after 5 days of transfection, collect the supernatant to purify the antibody.

[0169] 3. Chimeric antibody purification

[0170] Collect the supernatant of 293F suspension cells after 5 days of transfection, centrifuge at 3000 rpm for 20 min at 4°C; discard the cell pellet and transfer the supernatant to a clean 50 mL centrifuge tube, add 300 μL of protein A beads, and rotate for incubation at 4°C for 45 min. After incubation, centrifuge at 3000 rpm for 20 min at 4°C to discard the supernatant, and transfer the beads to an affinity chromatography column. After washing the column with PBS for 5-6 times, add 700 μL of 0.1 M acetic acid to the beads to elute, and then neutralize with 100 μL of 1 M tris pH 8.0. After neutralizing the antibody, use a concentration tube to replace the buffer so that the antibody buffer is changed to PBS buffer.

[0171] 4. Verify the expressed antibody

[0172] 4.1 Flow cytometry verification of expressed recombinant antibody

[0173] 1) Take 1 x 10 5Hela cells were placed in 1.5 mL EP tubes, 100 nM purified antibodies were added, including antibodies 102, 106, and 122 screened by the application and the clinical antibody IMGN853 from ImmunoGen, and incubated at 4°C in the dark for 30 min in a 100 μL system; after incubation, centrifugation was performed at 300g at 4°C for 5 min; after resuspension in 500 μL PBS (containing 0.1% BSA) and centrifugation at 300g at 4°C for 5 min, the supernatant was discarded, and secondary antibody anti-human IgG H+L (brand: invitrogen; item number: A21445; 1:2000) was added in a 100 μL system and incubated at 4°C in the dark for 30 min; after incubation, centrifugation was performed at 300g at 4°C for 5 min; after resuspension in 500 μL PBS (containing 0.1% BSA) and centrifugation at 300g at 4°C for 5 min, the supernatant was discarded, and 300 μL PBS was added; after filtration of the cells through a 300-mesh filter, the cells were subjected to flow detection.

[0174] The results of flow cytometry detection of the binding of the antibodies to Hela cells (naturally expressing FR1) are shown in FIG. 2, which shows that antibodies 102, 106, and 122 can all bind to Hela cells expressing FR1. Sample IMGN853 is MIRV, i.e., a control antibody, and blank is the flow signal without the addition of primary antibody. Figure 4 The affinity of antibody 102 was detected on Hela cells by flow cytometry, and the results are shown in FIG. 3, which shows that the Kd of antibody 102 is 6.1 nM. Figure 5

[0175] 4.2 Elisa verification of expressed recombinant antibodies

[0176] 1) Dilute the human FOLR1 his recombinant protein to a final concentration of 0.5 μg / mL using sterile CBS. Take a new 96-well plate, and add 100 μL / well for coating overnight at 4°C.

[0177] 2) Remove the antigen coating solution, and wash 3 times with PBST (containing 0.5% Tween).

[0178] 3) Add 200 μL / well of 3% BSA and incubate at 37°C for 1 hour;

[0179] 4) After removing the blocking buffer, wash the plate 3 times with PBST;

[0180] 5) Add 1 μg of antibody, 3-fold gradient dilution, and incubate at room temperature for 1 hour; the control well is 1% BSA;

[0181] 6) Remove the liquid in the well, and wash 3 times with PBST;

[0182] ​7) Add 100 μL HRP human IgG (1:10000 dilution) and incubate for 1 hour at room temperature;

[0183] 8) After removing the liquid from the wells, wash the plate 3 times with PBST;

[0184] 9) Add 100 μL / well TMB color developing solution;

[0185] 10) Incubate for 5 minutes at room temperature in the dark;

[0186] 11) Add 50 μL / well stop solution;

[0187] 12) Read the OD450 value in the wells using a microplate reader.

[0188] Conclusion: ELISA was used to detect the binding of antibodies 102, 106, 122 to FRl antigen. As shown in Table 1, antibodies 102 and 106 have stronger binding ability to FRl antigen. Figure 2

[0189] Subsequently, ELISA was used to detect the affinity of antibody 102 to FRl antigen. As shown in Table 2, the Kd of antibody 102 binding to FRl antigen is 0.38 μg / mL. Figure 3

[0190] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that various modifications or changes can be made to the present application by those skilled in the art after reading the above teaching of the present application, and these equivalent forms also fall within the scope of the appended claims of the present application.

[0191] The sequences involved in the present application are:

[0192] ​​

Claims

1. An antibody or antigen-binding fragment thereof targeting folate receptor alpha (FR1), characterized in that, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of: a heavy chain variable region comprising the following three CDRs: HCDR1 of the amino acid sequence as set forth in SEQ ID NO. 1, HCDR2 of the amino acid sequence as set forth in SEQ ID NO. 2, HCDR3 of the amino acid sequence as set forth in SEQ ID NO. 3; and a light chain variable region comprising the following three CDRs: LCDR1 of the amino acid sequence as set forth in SEQ ID NO. 4, LCDR2 of the amino acid sequence as set forth in SEQ ID NO. 5, LCDR3 of the amino acid sequence as set forth in SEQ ID NO.

6.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO.

7.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof has a sequence with at least 90% sequence identity to the sequence as set forth in SEQ ID NO.

7.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein, The light chain variable region sequence of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO.

8.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein, The light chain variable region sequence of the antibody or antigen-binding fragment thereof has a sequence with at least 90% sequence identity to the sequence as set forth in SEQ ID NO.

8.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein The antibody or antigen-binding fragment thereof specifically binds to human FOLR1 protein.

7. A nucleic acid, characterized in that, The nucleic acid encodes the antibody or antigen-binding fragment thereof of claim 1.

8. A vector, characterized in that, The vector comprises the nucleic acid of claim 7.

9. A host cell, characterized in that, The host cell comprises the vector of claim 8, or has integrated into its genome an exogenous nucleic acid of claim 7.

10. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: 1) the antibody or antigen-binding fragment thereof of claim 1, the nucleic acid of claim 7, the vector of claim 8, the host cell of claim 9, or a combination thereof; and 2) a pharmaceutically acceptable carrier.

11. Use of the antibody or antigen-binding fragment thereof of claim 1, the nucleic acid of claim 7, the vector of claim 8, or the cell of claim 9, characterized in that, The antibody or antigen-binding fragment thereof is used for preparing a medicament for treating a tumor / cancer overexpressing FR1, which is ovarian cancer and / or non-small cell lung cancer.

12. A drug combination, characterized in that, The pharmaceutical combination comprises: 1) the antibody or antigen-binding fragment thereof of claim 1, the nucleic acid of claim 7, the vector of claim 8, or the cell of claim 9 as a first medicament; and 2) another tumor-targeting drug or a chemotherapeutic drug as a second medicament.

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