Monoclonal antibody targeting human folate receptor alpha

Abstract

The invention provides a monoclonal antibody of a targeted human folate receptor alpha (FR1). Specifically, a mouse is immunized through FR1 recombinant protein, and a monoclonal antibody with high affinity to FR1 is screened out. In addition, the invention also provides an amino acid sequence of the monoclonal antibody, nucleic acid containing the sequence, a carrier or a conjugate containing the nucleic acid, and application of the monoclonal antibody in FR1 overexpressed tumors / cancers.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a monoclonal antibody targeting human folate receptor α. Background Art

[0002] Folic acid (vitamin B9) is a key one-carbon donor for purines and thymidine, essential building blocks of nucleic acid synthesis. It also indirectly contributes to the methylation of proteins and DNA through S-adenosylmethionine. In normal adult tissues, folate is primarily absorbed by the Reduced Folate Carrier (RFC). The RFC is a ubiquitous anion channel responsible for folate transport in most normal cells, but it has a low binding affinity for folate (Km = 1-10 mM).

[0003] Folate receptors (FR, FOLR) are a class of cysteine-rich glycoproteins that bind folate with high affinity, mediating folate absorption. They are divided into three subtypes: α, β, and γ. Folate receptor α (FR1, FOLR1) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein composed of a single polypeptide chain containing a signal peptide, a folate-binding domain, and a GPI-anchored signal sequence. The folate-binding domain is the primary site of folate binding. FR1 is a key protein involved in cellular folate uptake. Folate is essential for DNA synthesis, repair, and methylation, making FR1 crucial for rapidly dividing cells, such as developing embryonic cells and certain cancer cells.

[0004] FR1 is crucial for folate homeostasis and holds important implications in oncology, neurology, and developmental biology. Although FR1 is expressed at very low levels in most normal tissues, it is highly expressed in many epithelial cancer cells, where it competes for limited folate in the environment to meet the demands of rapid cancer cell division and growth. Cerebral folate deficiency (CFD) is a rare metabolic encephalopathy caused by mutations in the FOLR1 gene. It manifests as a significant folate deficiency in the central nervous system, with clinical symptoms including epilepsy, motor delay, and intellectual disability. Its pathogenesis is closely linked to dysfunction of FOLR1-mediated folate transport across the blood-brain barrier.

[0005] FR1's overexpression in cancer and its role in CFD highlight its dual significance as a therapeutic target and diagnostic biomarker. FOLR1 is highly expressed on the cell surface of various tumors, such as ovarian, lung, and breast cancers, while its expression is low or absent in normal tissues. This differential expression between normal and cancer cells provides important evidence for targeting FR1 for molecular diagnosis and therapy. Targeting the folate receptor for tumor treatment and diagnosis involves a variety of approaches, including high-affinity antifolates, small molecule conjugates of folate with drugs or toxins, antibodies against folate receptor α (FR1), and anti-FR1 antibody-toxin drug conjugates (FR1-ADCs). Furthermore, folate-based imaging agents are also used in clinical diagnosis to visualize and track cancers with high FR1 expression.

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

[0007] At present, there are still many shortcomings in the research on FOLR1 monoclonal antibodies. The field urgently needs to develop new monoclonal antibodies targeting FR1 and explore their potential applications in various medical fields such as targeted tumor treatment and diagnostic markers. Summary of the Invention

[0008] The object of the present invention is to provide a novel monoclonal antibody against FR1.

[0009] In a first aspect of the present invention, an antibody or antigen-binding fragment thereof targeting folate receptor α (FR1) is provided, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region are selected from the following group: A heavy chain variable region comprising the following three CDRs: HCDR1 of the amino acid sequence shown in SEQ ID NO. 7, HCDR2 of the amino acid sequence shown in SEQ ID NO. 8, HCDR3 having the amino acid sequence shown in SEQ ID NO. 9; and A light chain variable region comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO. 10, LCDR2 having the amino acid sequence shown in SEQ ID NO. 11, LCDR3 having the amino acid sequence shown in SEQ ID NO. 12.

[0010] In another preferred embodiment, the heavy chain sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO. 21.

[0011] In another preferred embodiment, the heavy chain of the antibody or antigen-binding fragment thereof has a sequence that has at least 90% sequence identity with the sequence shown in SEQ ID NO. 21.

[0012] In another preferred embodiment, the light chain sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO. 22.

[0013] In another preferred embodiment, the light chain of the antibody or antigen-binding fragment thereof has a sequence that has at least 90% sequence identity with the sequence shown in SEQ ID NO. 22.

[0014] In another preferred embodiment, the antibody or antigen-binding fragment thereof specifically binds to human FOLR1 protein.

[0015] In another preferred embodiment, the antibody further comprises a constant region.

[0016] In another preferred embodiment, the constant region of the antibody is a humanized constant region.

[0017] In another preferred example, the heavy chain variable region includes the three heavy chain CDRs and a human heavy chain framework region for connecting the heavy chain CDRs.

[0018] In another preferred embodiment, the light chain variable region includes the three light chain CDRs and a human light chain framework region for connecting the light chain CDRs.

[0019] In another preferred embodiment, the antibody or antigen-binding fragment thereof is selected from the following group: Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), double scFv, (scFv)2, miniantibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide bond stabilized Fv protein ("dsFv"), or a combination thereof.

[0020] In another preferred embodiment, the antibody is a murine antibody, a murine-human chimeric antibody, or a humanized IgG1 antibody.

[0021] In another preferred embodiment, the antibody comprises a monospecific, bispecific, trispecific antibody or a multispecific 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), opsonization mediated by the antibody Fc region, or complement-mediated opsonization.

[0022] In a second aspect of the present invention, a nucleic acid is provided, wherein the polynucleotide encodes the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0023] In the third aspect of the present invention, a vector is provided, wherein the vector contains the nucleic acid according to the second aspect of the present invention.

[0024] In another preferred embodiment, the vector is selected from the following group: plasmid, virus (such as lentivirus, adenovirus, AAV virus, retrovirus), cosmid, or a combination thereof.

[0025] In another preferred embodiment, the vector is a plasmid expression vector.

[0026] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector according to the third aspect of the present invention, or an exogenous nucleic acid according to the second aspect of the present invention is integrated into its genome.

[0027] In another preferred embodiment, the host cell is Escherichia coli.

[0028] In another preferred embodiment, the cells are isolated cells and / or genetically engineered cells.

[0029] In another preferred embodiment, the cells are somatic cells.

[0030] In another preferred embodiment, the cells are mammalian cells.

[0031] In a fifth aspect of the present invention, a pharmaceutical composition is provided, comprising: 1) the antibody or antigen-binding fragment thereof according to the first aspect of the invention, the nucleic acid according to the second aspect of the invention, the vector according to the third aspect of the invention, the host cell according to the fourth aspect of the invention, or a combination thereof; and 2) Pharmaceutically acceptable carrier.

[0032] In the sixth aspect of the present invention, there is provided a use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention for preparing a drug for treating tumors / cancers with overexpression of FR1.

[0033] 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.

[0034] In the seventh aspect of the present invention, a method for treating tumors / cancers with overexpression of FR1 is provided, the method comprising: administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, the vector described in the third aspect of the present invention, and / or the host cell described in the fourth aspect of the present invention to a subject.

[0035] In an eighth aspect of the present invention, a pharmaceutical combination is provided, comprising: 1) the antibody or antigen-binding fragment thereof according to the first aspect of the invention, the nucleic acid according to the second aspect of the invention, the vector according to the third aspect of the invention, or the host cell according to the fourth aspect of the invention as a first medicament; and 2) Other tumor-targeted drugs or chemotherapy drugs as second drugs.

[0036] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The results of PCR amplification of the antibody light and heavy chains of the obtained single B cells are shown: lane 1 is the DL5000 marker; lane 2 corresponds to the FR1-102 light chain and heavy chain variable region PCR bands; lane 6 is the FR1-106 light chain and heavy chain variable region PCR bands.

[0038] Figure 2 The binding of three antibodies to the FR1 antigen as detected by ELISA is shown.

[0039] Figure 3 The affinity of the ELISA test antibody 102 for the FR1 antigen is shown, and the curve fit R 2 = 0.98.

[0040] Figure 4 The flow cytometry analysis of the binding of different antibodies to Hela cells is shown.

[0041] Figure 5 The affinity of antibody 102 was detected by flow cytometry on HeLa cells. DETAILED DESCRIPTION

[0042] After extensive and in-depth research, the inventors have developed a monoclonal antibody targeting the human folate receptor α subtype (FR1). This method involves immunizing mice with FR1 recombinant protein, isolating antigen-specific B cells from the spleen and lymph nodes, and purifying a monoclonal antibody with high affinity for FR1. This monoclonal antibody can be used to treat tumors / cancers in which FR1 is overexpressed.

[0043] the term In order to facilitate understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary.

[0044] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0045] In the present invention, the terms "antibody 106", "106 antibody" and "FR1-106" are used interchangeably, and all refer to antibody 106 targeting FR1.

[0046] In the present invention, the terms "antibody 102", "102 antibody" and "FR1-102" are used interchangeably and all refer to antibody 102 targeting FR1.

[0047] In the present invention, the terms "antibody 122", "122 antibody" and "FR1-122" are used interchangeably, and all refer to antibody 122 targeting FR1.

[0048] As used herein, the term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions 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 light 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 hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0049] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0050] As used herein, the terms "light chain variable region" and "VL" are used interchangeably.

[0051] In a given antibody 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 or a combination 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 (Kabat, E., et al., USDepartment of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983), AbM (University of Bath), Contact (University College London), the International Immuno GeneTics database (IMGT), the EU numbering system, and the Chothia definition based on loop structural position.

[0052] It will be appreciated that the precise amino acid sequence boundaries of the CDRs of the present invention may optionally be defined using the various designation systems mentioned above. Preferably, unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), the numbering positions are those according to the Kabat numbering system.

[0053] As used herein, the term "variable" refers to certain portions of the variable region in an antibody that differ in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0054] As known to those skilled in the art, immunoconjugates and fusion expression products include: conjugates formed by the combination of drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules with the antibodies of the present invention or fragments thereof.

[0055] In a preferred embodiment of the present invention, the heavy chain variable region and light chain variable region of the antibody each include three complementarity determining regions CDR1, CDR2, and CDR3.

[0056] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0057] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; (ii) a polypeptide having a substituent group in one or more amino acid residues; (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, a sequence for purifying the polypeptide, a proprotein sequence, or a fusion protein with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0058] The antibodies of the present invention refer to polypeptides that have FOLR1 binding activity and include the above-mentioned CDR regions. The term also includes variant forms of polypeptides that include the above-mentioned CDR regions and have the same function as the antibodies of the present invention. These variant forms include (but are not limited to): deletion, insertion, and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not alter the function of the protein. For another example, addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0059] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antisera of the antibody of the present invention.

[0060] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention also encompasses fragments of the antibodies of the invention. Typically, the fragments comprise at least about 50 contiguous amino acids of the antibodies of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0061] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0062] Table A

[0063] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0064] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0065] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0066] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0067] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0068] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0069] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0070] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0071] 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 prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0072] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0073] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0074] The antibodies of the present invention may be used alone or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0075] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0076] Therapeutic agents that can be combined or coupled with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0077] The present invention also provides a composition. In preferred embodiments, the composition is a pharmaceutical composition comprising the above-mentioned antibody, active fragment thereof, fusion protein thereof, ADC, or corresponding immune cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0078] The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration. The injection preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules, injections or infusions, etc.

[0079] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0080] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) 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 match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0081] In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, and preferably includes a pharmaceutically acceptable excipient, filler or diluent, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the above-mentioned protein and 0.01 to 99.99% of a pharmaceutical carrier, and the percentages are the mass percentages of the pharmaceutical composition.

[0082] In the present invention, the pharmaceutical composition is preferably administered in an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative, or damaging condition. The effective amount can be determined on an individual basis and will be based in part on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by utilizing the above factors on an individual basis and using no more than routine experimentation.

[0083] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0084] Compared with the prior art, the main advantages of the present invention include: 1. The monoclonal antibody of the present invention is capable of specifically binding to folate receptor α (FR1).

[0085] 2. The monoclonal antibody of the present invention can be used to treat tumors / cancers in which FOLR1 is overexpressed.

[0086] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those 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 indicated, percentages and parts are weight percentages and parts by weight.

[0087] Example 1. Monoclonal Antibody Screening 1. Monoclonal B cell screening 1.1 Immunization of mice Three Balb / c mice were prepared and injected intraperitoneally with 12 μg of recombinant human FOLR1 (uniprot: P15328; protein expression sequence Arg 25 - Met 233) mixed with adjuvant. Each mouse received 12 μg of protein every three days. Three days after the fifth intraperitoneal injection, 100 μL of peripheral blood was collected from the eyeballs for immunohistochemistry. Mice with the highest immunohistochemistry titers were selected and pulsed with 20 μg of protein intraperitoneally. Three days after immunization, spleens and lymph nodes were isolated for single B cell isolation.

[0088] 1.2 Immune titer detection 1) Remove the mouse from 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 wound in the mouse's eyeball; 2) Collect blood drops using capillary glass blood collection tubes (100 μL of plasma must be prepared); 3) After blood collection, gently press the blood collection site with a dry sterile cotton ball to stop bleeding, then return the mouse to its cage for observation. 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.

[0089] 5) Separate the serum from the blood clot and transfer it to a new sterile centrifuge tube. Centrifuge at 10,000 x g for 10 min at 4°C. 6) Transfer the serum to a new sterile centrifuge tube and detect the immune titer using ELISA.

[0090] 1.3 ELISA test 1) Dilute human FOLR1 his recombinant protein to a final concentration of 0.5 μg / mL in sterile carbonate buffered saline (CBS). Add 100 μL / well of this solution to a new 96-well plate and coat overnight at 4°C.

[0091] 2) Remove the antigen coating solution and wash three times with PBST (containing 0.5% Tween).

[0092] 3) Add 200 μL / well of 3% BSA and block at 37°C for 1 hour; 4) After removing the blocking buffer, wash the plate three times with PBST; 5) Add 100 μL of mouse serum (maximum concentration is 300-fold dilution, 3-fold serial dilution), incubate at room temperature for 1 hour, and the control wells contain 1% BSA; 6) Remove the liquid in the wells and wash three times with PBST; 7) Add 100 μL HRP mouse IgG (1:10,000 dilution) and incubate at room temperature for 1 hour; 8) After removing the liquid in the wells, wash the plate three times with PBST; 9) Add 100 μL / well TMB colorimetric solution; 10) Incubate at room temperature in the dark for 5 minutes; 11) Add 50 μL / well of stop solution; 12) Use a microplate reader to read the OD450 value in the well.

[0093] 2. Single B cell isolation 2.1 The mouse with the best immune titer was killed by cervical dislocation. The spleen and lymph nodes of the mouse were obtained under sterile conditions, and a single-cell suspension of B cells was prepared and counted.

[0094] 2.2 Add human FOLR1 antigen (1 μg / 10 7 Cells were incubated in the dark at 4°C for 30 minutes. After washing with cell staining buffer, cells were resuspended in 1 mL of cell staining buffer and stained with 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), and PE-streptavidin (1:500). Incubation was continued at 4°C in the dark for 30 minutes. Mouse cells were immunostained with BSA protein control to serve as isotype controls. Single-positive tubes were also set up to adjust compensation between channels. Cells were washed with cell staining buffer, resuspended in 1 mL of cell staining buffer, and DAPI was added. Incubation was continued at room temperature in the dark for 5 minutes. After rinsing with cell sorting buffer, cells were centrifuged at 1000 rpm / min at 4°C for 5 minutes, and the supernatant was discarded. The cells were resuspended in 500 μL PBS buffer and single B cells specific for the human FOLR1 antigen were sorted using a Beckman Coulter cytoflex SRT flow cytometer.

[0095] 2.3 Flow cytometry sorting of antigen-specific single B cells From all B cells, mononuclear cells were selected by screening for cell size and granularity. Live cells were identified using DAPI (negative selection). B cell populations were identified using CD19 (positive selection), and germinal center B cells were isolated using CD38 (negative selection) and GL7 (positive selection). Germinal center B cells that secrete antibodies in response to antigen stimulation were isolated using IgG1 (positive selection). Antigen-specific single B cells (DAPI-CD19+GL7+CD38-IgG1+Ag biotin++) were isolated using PE (positive selection) and APC (positive selection). Cells were sorted into 96-well PCR plates containing 4 μL of sterile, enzyme-free PBS. After the desired number of cells (92 cells) had been sorted, the cells were transferred to 80°C for storage until subsequent experiments.

[0096] 3. Preparation of Human FOLR1 Antigen-Specific Single B Cell Antibodies 3.1 Reverse transcribe the antibody-encoding mRNA in sorted human FOLR1 antigen-specific single B cells into cDNA.

[0097] 3.2 Using cDNA as a template, the designed primers were used to amplify the complete gene fragments encoding the antibody heavy chain and light chain.

[0098] 3.3 Clone the VH into the pTT5 eukaryotic expression vector containing human IgG1 Fc, naming the heavy chain backbone vector pTT5-human IgG1 CH; clone the VL into the pTT5 eukaryotic expression vector containing human CL, naming the light chain backbone vector pTT5-human IgG1 CL. Co-transfect the heavy and light chain recombinant expression vectors into 293F suspension cells. Collect and purify the supernatant after transfection to obtain the monoclonal antibody encoded and expressed by the single B cell, which can be used for subsequent monoclonal antibody identification and screening.

[0099] 3.4 Amplification of Single B Cell cDNA According to the instructions of Maxima H Minus Reverse Transcriptase (Manufacturer: Thermo Scientific, Catalog No.: EP0753), the mRNA (including antibody-encoding genes) in the sorted single B cells was reverse transcribed into cDNA.

[0100] 3.5 Amplification of the complete antibody heavy chain encoding gene in a single B cell 1) Antibody heavy chain amplification method (Table 1): Using amplified single B cell cDNA as a template, nested PCR was performed using primers 5' MsVHE Fw and 3' mIgG1-2b-2c. The amplification system is shown in Table 1. The PCR amplification program was as follows: 98°C for 10 seconds, 55°C for 30 seconds, 72°C for 55 seconds, 35 cycles, and 72°C for 5 minutes. The amplified product was then amplified using primers 2nd-5VH-FW and 2nd-3VH-RV. The PCR amplification program was as follows: 98°C for 10 seconds, 62°C for 30 seconds, 72°C for 45 seconds, 35 cycles, and 72°C for 5 minutes. Bands of the target fragment size (approximately 200-400 bp) were recovered and purified.

[0101] 2) Antibody Light Chain Amplification Method (Table 2): Using amplified single B cell cDNA as a template, nested PCR was performed using primers 5' L-Vk mix Fw and 3' mCk. The amplification system is shown in Table 2. The PCR amplification program was as follows: 98°C for 10 s, 55°C for 30 s, 72°C for 55 s, 35 cycles, and 72°C for 5 min. The amplified product was then amplified using primers 2nd-5VK-FW and 2nd-3VK-RV. The PCR amplification program was as follows: 98°C for 10 s, 55°C for 30 s, 72°C for 45 s, 35 cycles, and 72°C for 5 min. Bands of the target fragment size (approximately 200-400 bp) were recovered and purified.

[0102] Table 1. Conditions for heavy chain VH PCR amplification

[0103] Table 2. Conditions for light chain VK PCR amplification

[0104] The results of PCR amplification of antibody light and heavy chains of single B cells are as follows Figure 1 As shown, lane 2 corresponds to the PCR bands of the light chain and heavy chain variable regions of the FR1-102 antibody; lane 6 corresponds to the PCR bands of the light chain and heavy chain variable regions of the FR1-106 antibody.

[0105] 3) The recovered and purified products were sequenced (sequencing was completed by Suzhou Jinweizhi Biotechnology Co., Ltd.), and the sequencing results were analyzed by NCBI Igblast (https: / / www.ncbi.nlm.nih.gov / igblast). Some amplification results are as follows Figure 3 The sequence analysis results are as follows: Clone 102 antibody heavy chain variable region (SEQ ID NO. 21): QVQLHYSGAELAKPGASLKVSCKASGYTFTTYCMHWVKHRPGQGLEWIGYINPTTGYTEYNQKFKDKATLTADKSSSTAYMQLSTLTSEDSAVYYCSRSMYYSTDYWFDYWGQGTTLTVSS; Clone 102 antibody light chain variable region (SEQ ID NO. 22): DIVITQSPAILSVSPGERVSFSCRASQSIGTSIHWYQRRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNTWPLTFGAGTKLELK.

[0106] Table 3. Sequences of the heavy chain CDRs of the 102 antibody Antibody H-CDR1 H-CDR2 H-CDR3 102 GYTFTTYCMH (SEQ ID NO. 7) YINPTTGYTEYNQKFKD (SEQ ID NO. 8) SRSMYYSTDYWFDY (SEQ ID NO. 9) Table 4. Sequences of the light chain CDRs of 102 antibodies Antibody L-CDR1 L-CDR2 L-CDR3 102 QSIGTSIH (SEQ ID NO. 10) YASESIS (SEQ ID NO. 11) QQSNTWPLT (SEQ ID NO. 12)

[0107] Example 2. Antibody Validation 1. Construction of a recombinant plasmid encoding a complete gene for a single B cell antibody Primers were designed based on the sequences of the complete genes encoding the heavy and light chains of IgG antibodies verified by sequencing analysis. Homologous sequences of the pTT5-human IgG1 CH and pTT5-human IgG1 CL expression vectors were added to the 5' end of the upstream primer and the 3' end of the downstream primer, respectively. The upstream and downstream primers were named Primer-f1 and Primer-b1, respectively.

[0108] Amplification was performed using the recovered PCR product as a template. The PCR reaction was as shown in Table 5. The PCR program was as follows: 95°C for 2 min; 30 cycles of 95°C for 15 s, 50°C for 30 s, and 72°C for 30 s; and 72°C for 5 min, as shown in Table 6. The amplified products were identified by 1.5% agarose gel electrophoresis, and the bands corresponding to the target fragment size were recovered and purified. The recovered and purified VH target fragment was ligated via homologous recombination with the pTT5-human IgG1 CH linearized vector obtained by double digestion with PmeI and NheI. Similarly, the purified VK target fragment was ligated with the pTT5-human IgG1 CL linearized vector obtained by double digestion with PmeI and BsiwI. The reaction conditions were 50°C for 60 min. The reaction systems are shown in Tables 7 and 8.

[0109] The homologous recombination products were transformed into Escherichia coli Top10 competent cells, and 3 clones were randomly selected for sequencing and alignment (analyzed by Snapgene software). The clones with correct sequencing results were shaken to extract the recombinant expression plasmid of the complete encoding gene of the single B cell antibody.

[0110] Table 5. Cloning PCR components

[0111] Table 6. Cloning PCR reaction temperature

[0112] Table 7. Heavy chain ligation reaction components

[0113] Table 8. Light chain ligation reaction components

[0114] 2. Antibody Expression by Single B Cells The 293F suspension cells were subcultured into 125 mL triangular shake flasks and the cell density reached 2 × 10 6 Transfection was performed at 4% PE / mL. Using a single shake flask as an example, recombinant expression plasmids for the heavy and light chains of an antibody cloned from the same B cell were co-transfected. To prepare Solution A, add 10 μg of heavy chain plasmid, 20 μg of light chain plasmid, and 1 mL of SMM 293-TII medium to a 1.5 mL centrifuge tube and gently flick to mix. To prepare Solution B, add 60 μg of PEI transfection reagent and 1 mL of SMM293-TII medium to another 1.5 mL EP tube and gently flick to mix. Solution A was then slowly added dropwise to Solution B, gently flicking to mix. After allowing to stand for 15 minutes, the transfection complex was evenly added dropwise to the cells and incubated on a shaker. One day after transfection, glucose (final concentration 3 g / L) and sodium valproate (3.5 mM) were added. Five days after transfection, the supernatant was collected for antibody purification.

[0115] 3. Chimeric Antibody Purification Collect the supernatant of 293F suspended cells 5 days after transfection, centrifuge at 4°C, 3000 rpm for 20 min; discard the cell pellet and transfer the supernatant to a clean 50 mL centrifuge tube, add 300 μL protein A beads, incubate with rotation at 4°C for 45 min, centrifuge at 4°C, 3000 rpm for 20 min after incubation, discard the supernatant, transfer the beads to the affinity chromatography column, wash the column 5-6 times with PBS, add 700 μL 0.1M acetic acid to the beads and elute them into a centrifuge tube, neutralize with 100 μL 1 M tris pH8.0, and after neutralizing the antibody, use a concentration tube to replace the buffer to convert the antibody buffer to PBS buffer.

[0116] 4. Validation of expressed antibodies 4.1 Flow cytometry verification of expressed recombinant antibodies 1) Take 1x10 5 Hela cells were placed in a 1.5 mL EP tube, and 100 nM purified antibodies were added, including the antibodies screened by the present invention: No. 102, 106, 122 and the clinical antibody IMGN853 from ImmunoGen, and incubated in a 100 μL system at 4°C in the dark for 30 min; after the incubation, the cells were centrifuged at 300g for 5 min at 4°C; 500 μL PBS (containing 0.1% BSA) was added, the cells were resuspended, and the cells were centrifuged at 300g for 5 min at 4°C; the supernatant was discarded, and the secondary antibody anti-huamn IgG H+L (brand: invitrogen; product number: A21445; 1:2000) was added and incubated in a 100 μL system at 4°C in the dark for 30 min; after the incubation, the cells were centrifuged at 300g for 5 min at 4°C; 500 μL PBS (containing 0.1% BSA) was added, the cells were resuspended, and the cells were centrifuged at 300g for 5 min at 4°C; the supernatant was discarded, and 300 μL PBS; filter the cells through a 300-mesh filter and perform flow cytometry detection.

[0117] The results of flow cytometry detection of antibody binding to Hela cells (naturally expressing FR1) are as follows Figure 4 As shown, antibodies 102, 106, and 122 can all bind to Hela cells expressing FR1. Sample IMGN853 is MIRV, i.e., the control antibody, and blank is the flow cytometry signal without the addition of primary antibody. In addition, the affinity of antibody 102 was tested on Hela cells by flow cytometry, and the results are shown in Figure 2. Figure 5 As shown, the Kd of antibody 102 is 6.1 nM.

[0118] 4.2 Elisa validation of expressed recombinant antibodies 1) Dilute human FOLR1 his recombinant protein in sterile CBS to a final concentration of 0.5 μg / mL. Add 100 μL / well to a new 96-well plate and coat overnight at 4°C.

[0119] 2) Remove the antigen coating solution and wash three times with PBST (containing 0.5% Tween).

[0120] 3) Add 200 μL / well of 3% BSA and block at 37°C for 1 hour; 4) After removing the blocking buffer, wash the plate three times with PBST; 5) Add 1 μg of antibody, dilute serially 3-fold, and incubate at room temperature for 1 hour. The control wells contain 1% BSA. 6) Remove the liquid in the wells and wash three times with PBST; 7) Add 100 μL HRP human IgG (1:10,000 dilution) and incubate at room temperature for 1 hour; 8) After removing the liquid in the wells, wash the plate three times with PBST; 9) Add 100 μL / well TMB colorimetric solution; 10) Incubate at room temperature in the dark for 5 minutes; 11) Add 50 μL / well of stop solution; 12) Use a microplate reader to read the OD450 value in the well.

[0121] Conclusion: ELISA detected the binding of antibodies 102, 106, and 122 to FR1 antigen. Figure 2 As shown, antibodies 102 and 106 have strong ability to bind to FR1 antigen.

[0122] Subsequently, the affinity of antibody 102 to FR1 antigen was tested using ELISA, and the results were as follows: Figure 3 As shown, the Kd of antibody 102 binding to FR1 antigen is 0.38 μg / mL.

[0123] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

[0124] The present invention relates to the sequence:

Claims

1. An antibody or antigen-binding fragment thereof targeting folate receptor α (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), wherein the heavy chain variable region and the light chain variable region are selected from the following group: A heavy chain variable region comprising the following three CDRs: HCDR1 of the amino acid sequence shown in SEQ ID NO. 7, HCDR2 of the amino acid sequence shown in SEQ ID NO. 8, HCDR3 having the amino acid sequence shown in SEQ ID NO. 9; and A light chain variable region comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO. 10, LCDR2 having the amino acid sequence shown in SEQ ID NO. 11, LCDR3 having the amino acid sequence shown in SEQ ID NO.

12.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO. 21, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO.

21.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The light chain sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO. 22, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO.

22.

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

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

6. A carrier, characterized in that The vector contains the nucleic acid according to claim 5.

7. A host cell, characterized in that The host cell contains the vector according to claim 6, or the exogenous nucleic acid according to claim 5 is integrated into its genome.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: 1) the antibody or antigen-binding fragment thereof of claim 1, the nucleic acid of claim 5, the vector of claim 6, the host cell of claim 7, or a combination thereof; and 2) Pharmaceutically acceptable carrier.

9. Use of the antibody or antigen-binding fragment thereof according to claim 1, the nucleic acid according to claim 5, the vector according to claim 6, or the cell according to claim 7, characterized in that: Used for preparing drugs for treating tumors / cancers with overexpression of FR1, including ovarian cancer and / or non-small cell lung cancer.

10. A pharmaceutical combination, characterized in that: The drug combination includes: 1) the antibody or antigen-binding fragment thereof of claim 1, the nucleic acid of claim 5, the vector of claim 6, or the cell of claim 7 as a first medicament; and 2) Other tumor-targeted drugs or chemotherapy drugs as second drugs.

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