Monoclonal antibody targeting human folate receptor alpha and application thereof

Abstract

The invention provides a monoclonal antibody targeting a human folate receptor alpha subtype. 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] This invention belongs to the field of biomedical technology, and more specifically, this invention relates to a monoclonal antibody targeting human folic acid receptor α and its application. Background Art

[0002] Folic acid (vitamin B9) is an important single-carbon donor of purines and thymidines, essential components in nucleic acid synthesis, and indirectly provides a donor for protein and DNA methylation modification via S-adenosylmethionine. In normal adult tissues, folic acid is primarily absorbed by Reduced Folate Carriers (RFCs). RFCs are anion channels widely expressed in the human body, responsible for the transport of folic acid in most normal cells, but they have a low affinity for folic acid (Km = 1-10). M).

[0003] Folic acid receptors (FRs, genetically named FOLR) are a class of cysteine-rich glycoproteins that mediate folate absorption by binding to folate with high affinity. They are classified into three subtypes: α, β, and γ. Folate receptor α (FR1, genetically named FOLR1) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein composed of a single-chain polypeptide 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 processes; therefore, FR1 is crucial for rapidly dividing cells, such as developing embryonic cells and certain cancer cells.

[0004] FR1 is crucial for folate homeostasis and plays a significant role in oncology, neurology, and developmental biology. Although FR1 expression levels are very low in most normal tissues, it is highly expressed in many epithelial-derived cancer cells, where it competes for limited folate molecules in the environment to meet the needs of rapid cancer cell division and growth. Cerebral folate deficiency (CFD) is a rare metabolic encephalopathy caused by mutations in the FOLR1 gene, characterized by a significant deficiency of folate in the central nervous system. Clinical symptoms include epilepsy, developmental delay, and intellectual disability. Its pathogenesis is closely related to FOLR1-mediated impairment of 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 biomarker. FR1 is highly expressed on the surface of various tumor cells, such as ovarian cancer, lung cancer, and breast cancer, while its expression level is low or absent in normal tissues. This differential expression between normal and cancer cells provides important evidence for molecular diagnosis and treatment targeting FR1. Various methods exist for tumor treatment and diagnosis targeting the folate receptor, including high-affinity antifolates, small molecule conjugates of folate with drugs or toxins, antibodies against folate receptor α (FR1), and anti-FR1 antibody-drug conjugates (FR1-ADCs), etc. In addition, folate-based imaging agents are also used in clinical diagnosis for the imaging and tracing of cancers with high FR1 expression.

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

[0007] Currently, there are still many shortcomings in the research on FR1 monoclonal antibodies. There is an urgent need in this field to develop novel monoclonal antibodies against FR1 and explore their potential applications in various medical fields such as targeted cancer therapy and diagnostic biomarkers. Summary of the Invention

[0008] The purpose of this invention is to provide a novel monoclonal antibody against FR1.

[0009] In a first aspect of the invention, an antibody or antigen-binding fragment thereof targeting folate receptor α (FR1) is provided, said antibody or antigen-binding fragment 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: (1) Heavy chain variable regions containing the following three CDRs: HCDR1, having the amino acid sequence shown in SEQ ID NO. 1, HCDR2 has the amino acid sequence shown in SEQ ID NO. 2. HCDR3 having the amino acid sequence shown in SEQ ID NO. 3; and Light chain variable regions containing the following three CDRs: LCDR1, having the amino acid sequence shown in SEQ ID NO. 4, LCDR2 has the amino acid sequence shown in SEQ ID NO. 5. LCDR3 having the amino acid sequence shown in SEQ ID NO. 6; or In another preferred embodiment, the heavy chain sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO. 7.

[0010] In another preferred embodiment, the heavy chain of the antibody or its antigen-binding fragment has a sequence that is at least 90% sequence identical to the sequence shown in SEQ ID NO. 7.

[0011] In another preferred embodiment, the light chain sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO. 8.

[0012] In another preferred embodiment, the light chain of the antibody or its antigen-binding fragment has a sequence that is at least 90% sequence identical to the sequence shown in SEQ ID NO. 8.

[0013] In another preferred embodiment, the antibody or its antigen-binding fragment specifically binds to the human FOLR1 protein.

[0014] In another preferred embodiment, the antibody further includes a constant region.

[0015] In another preferred embodiment, the constant region of the antibody is a humanized constant region.

[0016] In another preferred embodiment, the heavy chain variable region includes the three heavy chain CDRs and the human-derived heavy chain framework region for connecting the heavy chain CDRs.

[0017] In another preferred embodiment, the light chain variable region includes the three light chain CDRs and a human-sourced light chain framework region for connecting the light chain CDRs.

[0018] In another preferred embodiment, the antibody or its antigen-binding fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody (“scFv”), bisscFv, (scFv)2, microantibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide-stabilized Fv protein (“dsFv”), or combinations thereof.

[0019] In another preferred embodiment, the antibody is a murine antibody, a murine-human chimeric antibody, or a humanized IgG1 antibody.

[0020] In another preferred embodiment, the antibody includes monospecific, bispecific, trispecific, or multispecific antibodies. In another preferred embodiment, the antibody mediates a biological effect selected from the group consisting of: antigen neutralization, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), antibody Fc fragment-mediated opsonization, or complement-mediated opsonization.

[0021] In a second aspect of the invention, a fusion protein is provided, the fusion protein comprising: (i) an antibody or an antigen-binding fragment thereof as described in the first aspect of the invention; and (ii) A fusion portion fused to the antibody or its antigen-binding fragment, the fusion portion being selected from the group consisting of: tag sequences, signal peptides, and / or functional proteins.

[0022] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.

[0023] In a third aspect of the invention, a nucleic acid is provided, the polynucleotide encoding an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, or a fusion protein as described in the second aspect of the invention.

[0024] In a fourth aspect of the invention, a carrier is provided, said carrier containing nucleic acid as described in the third aspect of the invention.

[0025] In another preferred embodiment, the vector is selected from the group consisting of plasmids, viruses (such as lentiviruses, adenoviruses, AAV viruses, retroviruses), granules, or combinations thereof.

[0026] In another preferred embodiment, the vector is a plasmid expression vector.

[0027] In a fifth aspect of the invention, a host cell is provided, the host cell containing a vector as described in the fourth aspect of the invention, or having exogenous nucleic acids as described in the third aspect of the invention integrated into its genome.

[0028] In another preferred embodiment, the host cell is Escherichia coli.

[0029] In another preferred embodiment, the cells are isolated cells and / or genetically engineered cells.

[0030] In another preferred embodiment, the cell is a somatic cell.

[0031] In another preferred embodiment, the cell is a mammalian cell.

[0032] In a sixth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising: (a) An antibody portion, said antibody portion being selected from the group consisting of: antibodies or antigen-binding fragments thereof as described in the first aspect of the invention, or fusion proteins as described in the second aspect of the invention; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0033] In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle.

[0034] In another preferred embodiment, the conjugate is an antibody-small molecule toxin.

[0035] In another preferred embodiment, the small molecule toxin is VcMMAE.

[0036] In another preferred embodiment, the conjugate has a killing effect on cervical cancer, ovarian cancer, breast cancer and / or lung cancer.

[0037] In a seventh aspect of the invention, a chimeric antigen receptor CAR is provided, wherein the antigen-binding domain of the chimeric antigen receptor contains a single-chain variable region sequence scFv targeting FR1, wherein the heavy chain variable region and the light chain variable region included in the scFv are as defined in the first aspect of the invention.

[0038] In an eighth aspect of the invention, a CAR-NK cell, CAR-T cell, or CAR-macrophage is provided, wherein the CAR-NK cell, CAR-T cell, or CAR-macrophage expresses the chimeric antigen receptor described in the seventh aspect of the invention.

[0039] In a ninth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising: 1) The antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the nucleic acid described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, the immunoconjugate described in the sixth aspect of the present invention, the cell described in the eighth aspect of the present invention, or a combination thereof; and 2) Pharmaceutically acceptable carrier.

[0040] In a tenth aspect of the invention, there is provided the use of the antibody or antigen-binding fragment thereof described in the first aspect of the invention, the fusion protein described in the second aspect of the invention, the nucleic acid described in the third aspect of the invention, the vector described in the fourth aspect of the invention, the immunoconjugate described in the sixth aspect of the invention, or the cell described in the eighth aspect of the invention, for the preparation of a medicament for treating FR1-overexpressing tumors / cancers.

[0041] In another preferred embodiment, the tumor / cancer is selected from the group consisting of: ovarian cancer, lung cancer, breast cancer, folic acid deficiency, kidney cancer, endometrial cancer, liver cancer, colorectal cancer, pancreatic cancer, stomach cancer, prostate cancer, or combinations thereof.

[0042] In an eleventh aspect of the present invention, a method for treating FR1-overexpressing tumors / cancers is provided, the method comprising: administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in the first aspect of the present invention, a fusion protein described in the second aspect of the present invention, a nucleic acid described in the third aspect of the present invention, a vector described in the fourth aspect of the present invention, an immunoconjugate described in the sixth aspect of the present invention, and / or cells described in the eighth aspect of the present invention to a subject.

[0043] In a twelfth aspect of the invention, a pharmaceutical combination is provided, the pharmaceutical combination comprising: 1) The antibody or its antigen-binding fragment as described in the first aspect of the present invention, the fusion protein as described in the second aspect of the present invention, the immunoconjugate as described in the sixth aspect of the present invention, or the CAR-NK cells, CAR-T cells, or CAR-macrophages as described in the eighth aspect of the present invention, as the first drug; and 2) Other targeted cancer drugs or chemotherapy drugs as second drugs.

[0044] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described 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 described in detail here. Attached Figure Description

[0045] Figure 1 The results of PCR amplification of the antibody light and heavy chains on the obtained single B cells are shown: lane 1 is the DL5000 marker; lane 6 is the PCR band of the FR1-106 light and heavy chain variable regions.

[0046] Figure 2 The results show the binding of antibody 106 to the FR1 antigen as tested by the ELISA method.

[0047] Figure 3 The curve shows the affinity of antibody 106 for the FR1 antigen detected by ELISA, and the goodness of fit R. 2 = 0.98.

[0048] Figure 4 The flow cytometry results show the binding of antibody 106 to HeLa cells.

[0049] Figure 5 The flow cytometry method was used to test the binding of different concentrations of antibody 106 to HeLa cells.

[0050] Figure 6 The HIC hydrophobic column plots of 106-antibody and 106-ADC are shown.

[0051] Figure 7 The flow cytometry method was used to detect the internalization activity of antibody 106.

[0052] Figures 8-1 to 8-6 The study demonstrated the cytotoxic effects of antibody 106-ADC on different cells.

[0053] Figure 9 The effect of antibody 106-ADC on tumor volume in HeLa tumor-bearing mice was shown.

[0054] Figure 10 The effect of antibody 106-ADC on body weight in HeLa tumor-bearing mice was shown.

[0055] Figure 11 The effect of antibody 106-ADC on tumor weight in Hela tumor-bearing mice was shown. Detailed Implementation

[0056] Through extensive and in-depth research, this invention has developed a monoclonal antibody targeting the human folate receptor α subtype (FR1). The invention involves immunizing mice with recombinant FR1 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 overexpressing FR1. Furthermore, an antibody-small molecule toxin immunoconjugate was prepared, which exhibits killing activity against various tumors and inhibits tumor growth. This invention is based on these findings.

[0057] the term To facilitate understanding of this 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 one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0058] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0059] In this invention, the terms "antibody 106", "106-Antibody", "106 antibody" and "FR1-106" are used interchangeably and all refer to antibody 106 targeting FR1.

[0060] In this invention, the term "106-ADC" refers to the conjugate of antibody 106 and VcMMAE (brand: MCE; catalog number: HY-15575) small molecule toxin reagent.

[0061] As used herein, the term "antibody" (Ab) includes, but is not limited to, immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or their antigen-binding portions. Each H chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region contains one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the 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.

[0062] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0063] As used in this article, the terms “light chain variable zone” and “VL” are used interchangeably.

[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 of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia based on the antibody's three-dimensional structure and the topology of the CDR loop; Kabat (Kabat, E., et al., Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983)) 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 definitions based on loop structural positions.

[0065] It should be understood that the precise amino acid sequence boundaries of the CDR in this invention can optionally be defined using the different assignment systems mentioned above. Preferably, unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position according to the Kabat numbering system.

[0066] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheeted and linked by three CDRs forming a linking loop, and in some cases, partially β-sheeted structures. The CDRs in each chain are tightly packed together by the FR regions 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. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0067] As those skilled in the art will know, immunoconjugates and fusion expression products include conjugates formed by combining drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules with the antibodies or fragments thereof of the present invention.

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

[0069] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0070] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0071] The term "antibody of the present invention" refers to a polypeptide having FOLR1 binding activity and including the aforementioned CDR region. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of the present invention.

[0072] The variant forms of the polypeptide include: homologous sequences, conserved 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 severity conditions, and polypeptides or proteins obtained using antiserum of the antibody of the present invention.

[0073] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0074] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0075] Table A

[0076] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0077] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes 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 a non-coding sequence.

[0078] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0079] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0080] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0081] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0082] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0083] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0084] 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*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are 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 eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0085] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0086] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0087] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0088] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0089] Therapeutic agents that can bind to or conjugate with the antibodies of this invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0090] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein or its 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 typically 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.

[0091] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration 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 solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0092] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy, such as for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0093] The pharmaceutical compositions of the present invention contain 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-described monoclonal antibody (or conjugate thereof) of the present invention, 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 formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. 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 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0094] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned protein and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0095] In this invention, preferably, the dosage of the pharmaceutical composition is 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 partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0096] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. 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 dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0097] Compared with the prior art, the main advantages of the present invention include: 1. The monoclonal antibody described in this invention can specifically bind to folate receptor α (FR1).

[0098] 2. The monoclonal antibody described in this invention can be used to treat FOLR1 overexpressing tumors / cancers.

[0099] 3. The 106-ADC drug of the present invention has a killing effect on a variety of tumors.

[0100] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0101] Example 1. Monoclonal antibody screening 1. Monoclonal B cell screening 1.1 Mouse Immunization Three Balb / c mice were prepared, and recombinant protein human FOLR1 (uniprot: P15328; protein expression sequence Arg 25 - Met 233) was mixed with adjuvant and injected intraperitoneally. Each mouse received 12 μg of protein intraperitoneally, with injections every 3 days. Three days after the 5th intraperitoneal injection, 100 μL of peripheral blood was collected from the mice via the eyeball for immunotiter testing. The mice with the best immunotiter were selected and given a pulse immunization with 20 μg of protein intraperitoneally. Three days after immunization, the spleen and lymph nodes of the mice were isolated for single B cell sorting.

[0102] 1.2 Immunotiter Detection 1) Remove the mouse from the cage, disinfect the mouse's eyes with a cotton ball soaked in 75% medical alcohol, and make a small incision in the mouse's eyeball with a 5 mm lancet. 2) Collect blood droplets using capillary glass blood collection tubes (100 μL of plasma needs to be prepared); 3) After collecting blood, gently press the blood collection point with a dry sterile cotton ball to stop the bleeding, and then return the mouse to the cage for a short 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.

[0103] 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 use ELISA to detect the immunogenicity.

[0104] 1.3 ELISA detection 1) Dilute the human FOLR1 his recombinant protein to a final concentration of 0.5 μg / mL using sterile carbonate buffer (CBS). Take a new 96-well plate, add 100 μL / well, and coat overnight at 4°C.

[0105] 2) Remove the antigen coating solution and wash three times with PBST (containing 0.5% Tween).

[0106] 3) Add 200 μL / well of 3% BSA and seal at 37℃ 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 diluted 300 times, serially diluted 3 times), incubate at room temperature for 1 hour, control wells contain 1% BSA; 6) Remove the liquid from the well and wash three times with PBST; 7) Add 100 μL of HRP mouse IgG (1:10000 dilution) and incubate at room temperature for 1 hour; 8) After removing the liquid from 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 stop solution; 12) Use an ELISA reader to read the OD450 value in the well.

[0107] 2. Single B-cell sorting 2.1 Mice with the best immunogenicity were euthanized by neck dislocation. The spleen and lymph nodes of the mice were obtained under aseptic conditions, and B cell suspensions were prepared and counted.

[0108] 2.2 Add biotin-tagged human FOLR1 antigen (1 μg / 10 cells) according to the cell count. 7Cells were incubated at 4°C in the dark for 30 min, washed with cell staining buffer, and resuspended in 1 mL of cell staining buffer. Staining was performed 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). The cells were then incubated at 4°C in the dark for 30 min. Isotype controls were set up using cells from mice immunized with BSA protein. Single-positive tubes were also prepared to adjust compensation between channels. Cells were washed with cell staining buffer, resuspended in 1 mL of cell staining buffer, and DAPI was added. The cells were incubated at room temperature in the dark for 5 min. Cells were rinsed with cell sorting buffer, centrifuged at 1000 rpm / min at 4°C for 5 min, and the supernatant was discarded. Cells were resuspended in 500 μL PBS buffer and human FOLR1 antigen-specific single B cells were sorted using a Beckman Coulter cytoflex SRT flow cytometer.

[0109] 2.3 Flow cytometry sorting of antigen-specific single B cells In all B cells, monocyte populations were selected by screening cell size and granularity. Live cells were identified using DAPI (negative selection); B cell populations were identified using CD19 (positive selection); germinal center B cells were obtained through CD38 (negative selection) and GL7 (positive selection); germinal center B cells that secrete antibodies upon antigen stimulation were selected using IgG1 (positive selection); and antigen-specific single B cells (DAPI-CD19+GL7+CD38-IgG1+Ag biotin++) were selected 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 sorting the required number of cells (92 cells), the cells were temporarily stored at 80°C for subsequent experiments.

[0110] 3. Preparation of human FOLR1 antigen-specific monoclonal B cell antibodies 3.1 The mRNA encoding the antibody in the sorted human FOLR1 antigen-specific single B cells was reverse transcribed into cDNA.

[0111] 3.2 Using cDNA as a template, the complete coding gene fragments of the antibody heavy chain and light chain were amplified using designed primers.

[0112] 3.3 VH was cloned into a pTT5 eukaryotic expression vector containing human IgG1 Fc, and the heavy chain backbone vector was named pTT5-human IgG1 CH; VL was cloned into a pTT5 eukaryotic expression vector containing human CL, and the light chain backbone vector was named pTT5-human IgG1 CL. The antibody heavy and light chain recombinant expression vectors were co-transfected into 293F suspension cells. After transfection, the supernatant was collected and purified to obtain the monoclonal antibody encoded by the single B cell, which can be used for subsequent monoclonal antibody identification and screening.

[0113] 3.4 Amplification of cDNA from a single B cell Following the instructions for Maxima H Minus reverse transcriptase (manufacturer: Thermo Scientific, catalog number: EP0753), the mRNA (containing antibody-encoding genes) from the sorted individual B cells was reverse transcribed into cDNA.

[0114] 3.5 Amplification of the complete encoding gene of a single B-cell antibody heavy chain 1) Antibody heavy chain amplification method (Table 1): Using the amplified single B cell cDNA as a template, nested PCR was first performed using primers 5' MsVHE Fw and 3' mIgG1-2b-2c. The amplification system is shown in Table 1. The PCR amplification program was: 98℃ for 10s; 55℃ for 30s, 72℃ for 55s, 35 cycles; 72℃ for 5 min. Then, using the amplified product as a template, amplification was performed using primers 2nd-5VH-FW and 2nd-3VH-RV. The PCR amplification program was: 98℃ for 10s, 62℃ for 30s, 72℃ for 45s, 35 cycles; 72℃ for 5 min. Bands matching the target fragment size (approximately 200-400 bp) were recovered and purified.

[0115] 2) Antibody light chain amplification method (Table 2): Using the amplified single B cell cDNA as a template, nested PCR was first performed using primers 5' L-Vk mix Fw and 3' mCk. The amplification system is shown in Table 2. The PCR amplification program was: 98℃ for 10s; 55℃ for 30s, 72℃ for 55s, 35 cycles; 72℃ for 5min. Then, using the amplified product as a template, amplification was performed using primers 2nd-5VK-FW and 2nd-3VK-RV. The PCR amplification program was: 98℃ for 10s, 55℃ for 30s, 72℃ for 45s, 35 cycles; 72℃ for 5min. Bands matching the target fragment size (approximately 200-400bp) were recovered and purified.

[0116] Table 1. Conditions for heavy chain VH PCR amplification

[0117] Table 2. Conditions for PCR amplification of light chain VL

[0118] PCR amplification results of antibody light and heavy chains on single B cells are as follows: Figure 1 As shown, lane 6 contains PCR bands of the variable regions of the FR1-106 antibody light and heavy chains.

[0119] 3) The recovered and purified product was sequenced (sequencing was performed by Suzhou Genewiz Biotechnology Co., Ltd.), and the sequencing results were analyzed using NCBI Igblast (https: / / www.ncbi.nlm.nih.gov / igblast). The sequence analysis results are as follows: Cloned 106 antibody heavy chain variable region (SEQ ID NO. 7): EVQLEQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGDTKYDPKFQGKATVTADTSSNTAYLQLSSLTSEDTAVYHCAKEGIYSGAMDYWGQGISVTVSS; Cloned 106 antibody light chain variable region (SEQ ID NO. 8): NIVMTQSPASLAVSLGQRATMSCRASEGVDTYVNSFMHWYQQKPGQPPRLLIYLASNLQSGIPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPWTFGGGTKLEIK; The sequences of the heavy chain CDR and light chain CDR of antibody 106 are shown in Tables 3 and 4.

[0120] Table 3. Sequence list of heavy chain CDRs of antibody 106

[0121] Table 4. Sequence list of the light chain CDR of antibody 106

[0122] Example 2. Antibody Validation 1. Construction of a recombinant plasmid encoding the complete gene of a single B-cell antibody Based on sequencing analysis, primers were designed to identify the complete coding sequences of the heavy and light chains of the IgG antibody. 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.

[0123] Amplification was performed using the recovered PCR products as templates. The PCR reaction components are shown in Table 5. The PCR program was as follows: 95℃ for 2 min; 95℃ for 15 s, 50℃ for 30 s, 72℃ for 30 s, 30 cycles; 72℃ for 5 min, as shown in Table 6. After identification by 1.5% agarose gel electrophoresis, bands matching the size of the target fragment were recovered and purified. The purified VH target fragment was ligated to the pTT5-human IgG1 CH linearized vector obtained by double digestion with PmeI and NheI via homologous recombination. Similarly, the purified VL target fragment was ligated to the pTT5-human IgG1 CL linearized vector obtained by double digestion with Pmel and BsiwI. The reaction conditions were 50℃ for 60 min, and the reaction systems are shown in Tables 7 and 8.

[0124] The homologous recombination product was transformed into E. coli Top10 competent cells, and three clones were randomly selected for sequencing. Sequencing alignment was performed (using Snapgene software for analysis). For cells with correct sequencing results, a single B cell antibody complete encoding gene recombinant expression plasmid was extracted from the culture.

[0125] Table 5. Composition of Cloned PCR

[0126] Table 6. Cloning PCR reaction temperature

[0127] Table 7. Composition of Heavy Chain Linkage Reaction

[0128] Table 8. Components of Light Chain Linkage Reaction

[0129] 2. Expression of single B cell antibodies 293F suspension cells were passaged into 125 mL Erlenmeyer flasks until the cell density reached 2 × 10⁻⁶ cells / mL. 6Transfection was performed at a concentration of / mL. Using one shake flask as an example, the heavy chain and light chain recombinant expression plasmids cloned from the same B cell were co-transfected. Preparation of Solution A: In a 1.5mL centrifuge tube, add 10 μg of heavy chain plasmid, 20 μg of light chain plasmid, and 1 mL of SMM 293-TII medium, and gently mix. Preparation of Solution B: In another 1.5mL EP tube, add 60 μg of PEI transfection reagent and 1 mL of SMM293-TII medium, and gently mix. Then, slowly add Solution A dropwise to Solution B, gently mix, and let stand for 15 minutes. Afterward, evenly add the transfection complex to the cells and incubate in a shaker. One day after transfection, add glucose (final concentration 3g / L) and sodium valproate (3.5 mM). Five days after transfection, collect the supernatant and purify the antibody.

[0130] 3. Purification of chimeric antibodies Collect the supernatant of 293F suspended cells 5 days after 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 incubate at 4°C for 45 min by rotation. After incubation, centrifuge at 3000 rpm for 20 min at 4°C and discard the supernatant. Transfer the beads to an affinity chromatography column, wash with PBS 5-6 times, then elute with 700 μL of 0.1 M acetic acid and transfer to a centrifuge tube. Neutralize with 100 μL of 1 M Tris at pH 8.0. After neutralizing the antibody, replace the antibody buffer with PBS buffer using a concentration tube.

[0131] 4. Verify the expressed antibody 4.1 Validation of the expressed recombinant antibody using ELISA 1) Dilute human FOLR1 his recombinant protein to a final concentration of 0.5 μg / mL using sterile CBS. Take a new 96-well plate, add 100 μL / well, and coat overnight at 4°C.

[0132] 2) Remove the antigen coating solution and wash three times with PBST (containing 0.5% Tween).

[0133] 3) Add 200 μL / well of 3% BSA and seal at 37℃ for 1 hour; 4) After removing the blocking buffer, wash the plate three times with PBST; 5) Add 1 μg of antibody, dilute 3-fold serially, incubate at room temperature for 1 hour, and use 1% BSA for the control well; 6) Remove the liquid from the well and wash three times with PBST; 7) Add 100 μL of HRP human IgG (1:10000 dilution) and incubate at room temperature for 1 hour; 8) After removing the liquid from 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 stop solution; 12) Use an ELISA reader to read the OD450 value in the well.

[0134] Conclusion: ELISA was used to detect the binding of antibody 106 to the FR1 antigen, such as... Figure 2 As shown, antibody 106 has a strong binding ability to the FR1 antigen.

[0135] Subsequently, the affinity of antibody 106 for the FR1 antigen was detected using ELISA, and the results were as follows: Figure 3 As shown, the Kd of antibody 106 binding to FR1 antigen is 0.2 μg / mL.

[0136] 4.2 Flow cytometry validation of the expressed recombinant antibody 1) Take 1x10 5 One HeLa cell was placed in a 1.5 mL EP tube, and 100 nM purified antibody containing antibody 106 selected in this invention and IMGN853 clinical antibody derived from ImmunoGen was added. The mixture was incubated in a 100 μL system at 4°C in the dark for 30 min. After incubation, the cells were centrifuged at 300g at 4°C for 5 min. The cells were then resuspended in 500 μL PBS (containing 0.1% BSA) and centrifuged at 300g at 4°C for 5 min. The supernatant was discarded, and the secondary antibody anti-huamn IgG H+L (brand: Invitrogen; catalog number: A21445; 1:2000) was added and incubated in a 100 μL system at 4°C in the dark for 30 min. After incubation, the cells were centrifuged at 300g at 4°C for 5 min. The cells were then resuspended in 500 μL PBS (containing 0.1% BSA) and centrifuged at 300g at 4°C for 5 min. The supernatant was discarded, and the secondary antibody anti-huamn IgG H+L (brand: Invitrogen; catalog number: A21445; 1:2000) was added and incubated in a 100 μL system at 4°C in the dark for 30 min. The cells were then centrifuged at 300g at 4°C for 5 min. The supernatant was discarded, and the secondary antibody anti-huamn IgG H+L (brand: Invitrogen; catalog number: A21445; 1:2000) was added and incubated in a 100 μL system. Cells were filtered through a 300-mesh PBS filter before being analyzed by flow cytometry.

[0137] The results of flow cytometry analysis of antibody binding to HeLa cells (naturally expressing FR1) are as follows: Figure 4 , 5 As shown, antibody 106 can bind to HeLa cells expressing FR1, and the binding to FR1 approaches saturation as the concentration of antibody 106 increases.

[0138] Example 3. Preparation of Antibody-Conjugate (ADC) 1. Preparation method 1) Reduce the FR1-106 antibody with approximately 8 times the molar concentration of TCEP (tris(2-carboxyethyl)phosphine hydrochloride) and incubate at room temperature for 3 hours; 2) Add approximately 4-8 times the molar concentration of FR1-106 antibody in VcMMAE (brand: MCE; catalog number: HY-15575) small molecule toxin reagent solution, and react at room temperature for 1-2 hours; 3) Add 8 times the molar concentration of FR1-106 antibody NAC reagent (Acetylcysteine; MCE, HY-B0215) and incubate on ice for 30 min; 4) The prepared 106-ADC was buffer-replaced with 1xPBS solution using a desalting column (Zeba™ Desalt Spin Column, 7K MWCO, 5 mL; ThermoScientific, 89891); 5) Concentrate 10⁶-ADC using a 30 kDa concentrator and determine its concentration; 6) The prepared 106-ADC was identified using an analytical HIC hydrophobic column (Proteomix HIC Butyl); Identification chart as follows Figure 6 As shown, this indicates that the 106-ADC was successfully prepared.

[0139] 2. Detection of HeLa cell-mediated internalization of anti-FR1 antibodies using FACS (flow cytometry). HeLa cells, 5×10⁻⁶, were extracted from human cervical cancer cells. 5 One tube of cells was added to each tube, with antibody diluted to 10 μg / ml. The antibody was divided into three groups (experimental group incubated for 4h, 8h, and 12h, and control group), one tube per group. The experimental group was placed in a 37℃ thermostatic incubator for 4h, 8h, and 12h, and then placed on ice. The control group was kept on ice as a negative control. After all samples had incubated, they were centrifuged at 300g, 4℃ for 5min, the supernatant was discarded, and the cells were washed once with ice-cold PBS. Secondary antibody anti-human IgG H+L (brand: Invitrogen; catalog number: A21445; 1:2000) was added, and the cells were incubated on ice for 30min. Then, they were centrifuged at 300g, 4℃ for 5min, the supernatant was discarded, and the cells were washed with ice-cold PBS. 200ul of ice-cold PBS was used to resuspend the cells, and the mean fluorescence intensity (MFI) was detected by FACS. The internalization efficiency was calculated using the following formula: .

[0140] The results are as follows Figure 7 As shown, the amount of 106 antibody endocytosed by HeLa cells increases over time.

[0141] 3. Assay of the cytotoxic activity of antibody-drug conjugate 106-ADC against cells Different tumor cells (HeLa, OVCAR-3, MCF-7, NCI-H441, NCI-H3122, and NCI-H2170) were collected at a ratio of 5-8 x 10⁻⁶ cells / mL. 3 Cells were seeded per well in 96-well plates at 100 μl per well and pre-cultured at 37°C in a 5% CO2 incubator for 16-24 hours. Then, different concentrations of 106-ADC were added according to Table 9, and the plates were incubated at 37°C in a 5% CO2 incubator for 72 hours. After incubation, the plates were placed at room temperature for 30 minutes to allow them to equilibrate to room temperature. The cell-killing activity of the 106-ADC antibody-drug conjugate was detected using the Promega CellTiter-Glo™ Luminescent CellViability Assay Kit.

[0142] The results are as follows Figures 8-1 to 8-6 As shown, this indicates that 106-ADC has a killing effect on different types of solid tumor cells, including cervical cancer, ovarian cancer, breast cancer, and lung cancer.

[0143] Table 9. Concentration of 106-ADC in different cell types

[0144] 4. Pharmacodynamic evaluation of antibody-drug conjugate 106-ADC in HeLa subcutaneous tumor model Six-week-old female BALB / c-nu mice were subcutaneously inoculated with 2 x 10 6 Human cervical cancer cells (HeLa), when the tumor grows to 150 mm 3 After being placed in the left and right sides, mice were randomly divided into two groups of 6 mice each: a blank control group and a treatment group. The treatment group was given 5 mg / kg of 106-ADC via tail vein injection once a week. Tumor volume and mouse weight were measured at the same time as administration. The experiment was stopped before the tumor volume exceeded 1000 mm3 and the mice were euthanized.

[0145] The results are as follows Figure 9 , 10 As shown in Figure 11, 106-ADC significantly inhibited the growth rate of tumors and reduced the size of tumors compared with the control group, and had no significant effect on the weight of mice after administration, indicating that the antibody-drug conjugate had no significant toxicity to mice.

[0146] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0147] The sequence involved in this invention:

Claims

1. An antibody or its antigen-binding fragment targeting folate receptor α, characterized in that, The antibody or its antigen-binding fragment 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 group consisting of: (1) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 1, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 2, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 3; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 4, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 5, LCDR3, as shown in the amino acid sequence of SEQ ID NO.

6.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO. 7, or has at least 90% sequence identity with the sequence shown in SEQ ID NO.

7.

3. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The light chain sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO. 8, or has at least 90% sequence identity with the sequence shown in SEQ ID NO.

8.

4. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antibody or its antigen-binding fragment specifically binds to the human FORL1 protein.

5. A nucleic acid, characterized in that, The nucleic acid encodes the antibody or its antigen-binding fragment as described in claim 1.

6. A carrier, characterized in that, The carrier contains the nucleic acid as described in claim 5.

7. A host cell, characterized in that, The host cell contains the vector as described in claim 6, or its genome has an integrated exogenous nucleic acid as described in claim 5.

8. An immunoconjugate, said immunoconjugate comprising: (a) The antibody portion, wherein the antibody portion is selected from the group consisting of: The antibody or its antigen-binding fragment as described in claim 1; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, enzymes, or combinations thereof.

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

10. Use of the antibody or antigen-binding fragment thereof as claimed in claim 1, the nucleic acid as claimed in claim 5, the vector as claimed in claim 6, the cell as claimed in claim 7, or the immunoconjugate as claimed in claim 8, characterized in that, This is used to prepare drugs for treating tumors / cancers that overexpress FR1, including ovarian cancer and / or non-small cell lung cancer.

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