Monoclonal antibody targeting human folate receptor alpha and use thereof

Abstract

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

Description

TECHNICAL FIELD

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

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

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

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

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

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

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

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

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

[0010] (1) a heavy chain variable region comprising the following three CDRs:

[0011] HCDR1 having an amino acid sequence as set forth in SEQ ID NO. 1,

[0012] HCDR2 having an amino acid sequence as set forth in SEQ ID NO. 2,

[0013] HCDR3 having an amino acid sequence as set forth in SEQ ID NO. 3; and

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

[0015] LCDR1 having an amino acid sequence as set forth in SEQ ID NO. 4,

[0016] LCDR2 having an amino acid sequence as set forth in SEQ ID NO. 5,

[0017] LCDR3 having an amino acid sequence as set forth in SEQ ID NO. 6; or

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

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

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

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

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

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

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

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

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

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

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

[0029] In another preferred embodiment, the antibody comprises a monospecific, bispecific, trispecific, or 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), antibody Fc fragment-mediated opsonization, or complement-mediated opsonization.

[0030] In a second aspect of the present application, a fusion protein is provided, the fusion protein comprising:

[0031] (i) an antibody or antigen-binding fragment thereof as described in the first aspect of the present application; and

[0032] (ii) a fusion moiety fused to the antibody or antigen-binding fragment thereof, the fusion moiety selected from the group consisting of a tag sequence, a signal peptide, and / or a functional protein.

[0033] In another preferred embodiment, the tag sequence comprises a 6His tag, a GGGS sequence, a FLAG tag.

[0034] In a third aspect of the present application, a nucleic acid is provided, the polynucleotide encoding an antibody or antigen-binding fragment thereof as described in the first aspect of the present application, or a fusion protein as described in the second aspect of the present application.

[0035] In a fourth aspect of the present application, a vector is provided, the vector comprising a nucleic acid as described in the third aspect of the present application.

[0036] In another preferred embodiment, the vector is selected from the group consisting of a plasmid, a virus (such as a lentivirus, an adenovirus, an AAV virus, a retrovirus), a cosmid, or a combination thereof.

[0037] In another preferred embodiment, the vector is a plasmid expression vector.

[0038] In a fifth aspect of the present application, a host cell is provided, the host cell comprising a vector as described in the fourth aspect of the present application, or having integrated into its genome an exogenous nucleic acid as described in the third aspect of the present application. In another preferred embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, a Fv, a single-chain Fv antibody ("scFv"), a bis-scFv, a (scFv)2, a minibody, a bifunctional antibody, a trifunctional antibody, a tetrafunctional antibody, a disulfide stabilized Fv protein ("dsFv"), or a combination thereof.

[0039] In another preferred embodiment, the host cell is E. coli.

[0040] In another preferred embodiment, the cell is an isolated cell, and / or a genetically engineered cell.

[0041] In another preferred embodiment, the cell is a somatic cell.

[0042] In another preferred embodiment, the cell is a mammalian cell.

[0043] In a sixth aspect of the application, there is provided an immunoconjugate comprising:

[0044] (a) an antibody moiety selected from the group consisting of an antibody or antigen binding fragment thereof as defined in the first aspect of the application, or a fusion protein as defined in the second aspect of the application; and

[0045] (b) a conjugated moiety conjugated to the antibody moiety, the conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0046] In another preferred embodiment, the conjugate is selected from the group consisting of a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biological toxin, a cytokine (such as IL-2 and the like), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle.

[0047] In another preferred embodiment, the conjugate is an antibody-small molecule toxin.

[0048] In another preferred embodiment, the small molecule toxin is VcMMAE.

[0049] In another preferred embodiment, the conjugate has a killing effect on cervical cancer, ovarian cancer, breast cancer, and / or lung cancer.

[0050] In a seventh aspect of the application, there is provided a chimeric antigen receptor (CAR) having an antigen binding domain comprising a single chain variable region sequence (scFv) targeting FR1, the scFv comprising a heavy chain variable region and a light chain variable region as defined in the first aspect of the application.

[0051] In an eighth aspect of the present application, there is provided a CAR-NK cell or a CAR-T cell or a CAR-macrophage, which expresses the chimeric antigen receptor of the seventh aspect of the present application.

[0052] In a ninth aspect of the present application, there is provided a pharmaceutical composition, which comprises:

[0053] 1) the antibody or antigen-binding fragment thereof of the first aspect of the present application, the fusion protein of the second aspect of the present application, the nucleic acid of the third aspect of the present application, the vector of the fourth aspect of the present application, the immunoconjugate of the sixth aspect of the present application, the cell of the eighth aspect of the present application, or a combination thereof; and

[0054] 2) a pharmaceutically acceptable carrier.

[0055] In a tenth aspect of the present application, there is provided a use of the antibody or antigen-binding fragment thereof of the first aspect of the present application, the fusion protein of the second aspect of the present application, the nucleic acid of the third aspect of the present application, the vector of the fourth aspect of the present application, the immunoconjugate of the sixth aspect of the present application, or the cell of the eighth aspect of the present application, for the manufacture of a medicament for treating a tumor / cancer overexpressing FR1.

[0056] In another preferred embodiment, the tumor / cancer is selected from the group consisting of ovarian cancer, lung cancer, breast cancer, folate deficiency, kidney cancer, endometrial cancer, liver cancer, colorectal cancer, pancreatic cancer, gastric cancer, prostate cancer, or a combination thereof.

[0057] In an eleventh aspect of the present application, there is provided a method for treating a tumor / cancer overexpressing FR1, which comprises administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of the first aspect of the present application, the fusion protein of the second aspect of the present application, the nucleic acid of the third aspect of the present application, the vector of the fourth aspect of the present application, the immunoconjugate of the sixth aspect of the present application, and / or the cell of the eighth aspect of the present application.

[0058] In a twelfth aspect of the present application, there is provided a pharmaceutical combination, which comprises:

[0059] 1) the antibody or antigen-binding fragment thereof of the first aspect of the present application, the fusion protein of the second aspect of the present application, the immunoconjugate of the sixth aspect of the present application, or the CAR-NK cell or CAR-T cell or CAR-macrophage of the eighth aspect of the present application as a first medicament; and

[0060] 2) another tumor-targeting drug or a chemotherapeutic drug as a second medicament.

[0061] It should be understood that, in the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 PCR amplification results of antibody light chain and heavy chain of the obtained single B cells are shown: lane 1 is DL5000 marker; lane 6 is FR1-106 light chain and heavy chain variable region PCR band.

[0063] Figure 2 The binding of antibody 106 to FR1 antigen is shown by ELISA method.

[0064] Figure 3 The affinity of antibody 106 to FR1 antigen is shown by ELISA, and the fitting degree R of the curve is 0.98. 2

[0065] Figure 4 The binding of antibody 106 to Hela cells is shown by flow method.

[0066] Figure 5 The binding of different concentrations of antibody 106 to Hela cells is shown by flow method.

[0067] Figure 6 HIC hydrophobic column chart of 106-antibody and 106-ADC is shown.

[0068] Figure 7 The internalization activity of antibody 106 is shown by flow method.

[0069] Figures 8-1 to 8-6 The killing effect of antibody 106-ADC drug on different cells is shown.

[0070] Figure 9 The effect of antibody 106-ADC drug on the tumor volume of Hela tumor-bearing mice is shown.

[0071] Figure 10 The effect of antibody 106-ADC drug on the body weight of Hela tumor-bearing mice is shown.

[0072] Figure 11 The effect of antibody 106-ADC drug on the tumor weight of Hela tumor-bearing mice is shown. DETAILED DESCRIPTION

[0073] ​Through extensive and in-depth research, the present application develops a monoclonal antibody targeting human folate receptor alpha subtype (FR1). The present application immunizes mice with FR1 recombinant protein, separates antigen-specific B cells from the spleen and lymph nodes, and purifies monoclonal antibodies with high affinity to FR1. The monoclonal antibody can be used for treating tumors / cancers overexpressing FR1. In addition, an antibody-small molecule toxin immunoconjugate is prepared, which has a killing effect on various tumors and inhibits tumor growth. On this basis, the present application is completed.

[0074] Definitions

[0075] For easier understanding of the present application, certain technical and scientific terms are defined below. Unless otherwise clearly defined in this text, all other technical and scientific terms used in this text have the meanings generally understood by those skilled in the art to which the present application belongs. Before describing the present application, it should be understood that the present application is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary.

[0076] As used herein, the terms "comprise", "include", "contain" are used interchangeably and include not only the closed definition, but also the semi-closed and open definition. In other words, the terms include "consist of", "consist essentially of".

[0077] In the present application, the terms "antibody 106", "106-Antibody", "106 antibody" and "FR1-106" are used interchangeably, all of which refer to the 106th antibody targeting FR1.

[0078] In the present application, the term "106-ADC" refers to the conjugate of antibody 106 and VcMMAE (brand: MCE; catalog number: HY-15575) small molecule toxin reagent.

[0079] As used herein, the term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin which specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each L chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each of VHand VLcomprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0080] As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

[0081] As used herein, the term "light chain variable region" is used interchangeably with "VL".

[0082] 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 of a number of well-known antibody CDR assignment systems, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the international Immuno GeneTics database (IMGT), the EU numbering system, and Chothia definition based on loop structure position.

[0083] It is to be understood that the precise amino acid sequence boundaries of the CDRs in the application can optionally be defined utilizing the different assignment systems mentioned above. Preferably, unless otherwise specified, in the present application, when referring to residue positions in an antibody variable region, including heavy chain variable region residues and light chain variable region residues, the numbering of the positions is according to the Kabat numbering system.

[0084] As used herein, the term "variable" refers to certain portions of the variable regions of the antibodies that differ in sequence among the various particular antibodies and are responsible for binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable regions of the antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more highly conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, largely in β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to an antigen, but exhibit various effector functions, such as participation in antibody- dependent cellular cytotoxicity.

[0085] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates of drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules with the antibodies of the present application or fragments thereof.

[0086] In a preferred embodiment of the present application, the heavy chain variable region and the light chain variable region of the antibody each comprise three complementarity determining regions CDR1, CDR2, and CDR3.

[0087] The present application includes not only intact antibodies, but also fragments of the antibodies that are immunologically active or fusions of the antibodies with other sequences. Thus, the present application also includes fragments, derivatives and analogs of the antibodies.

[0088] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present application. The polypeptide fragments, derivatives or analogs of the present application can be (i) polypeptides having one or more conservative or non-conservative amino acid substitutions, preferably conservative amino acid substitutions, and such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) polypeptides having a substitution group at one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide to another compound, such as a compound that extends the half-life of the polypeptide, for example, polyethylene glycol, or (iv) polypeptides formed by fusing additional amino acid sequences to the polypeptide sequence, such as a leader or secretory sequence, or a sequence for purification of the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. These fragments, derivatives and analogs are within the scope of those skilled in the art in light of the teachings herein.

[0089] The term "antibody" of the present application refers to a polypeptide having FOLRl binding activity comprising the CDR regions described above. The term also includes variants of polypeptides comprising the CDR regions described above which have the same function as the antibodies of the present application. These variants include, but are not limited to, deletion, insertion, and / or substitution of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or more (typically 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer) amino acids at the C-terminus and / or the N-terminus. For example, in the art, substitution of similar or identical amino acids typically does not change the function of the protein. Also, for example, addition of one or more amino acids at the C-terminus and / or the N-terminus typically does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present application.

[0090] The variants of the polypeptides include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to the DNA encoding the antibodies of the present application under conditions of high or low stringency, and polypeptides or proteins obtained using antisera against the antibodies of the present application.

[0091] The present application also provides other polypeptides, such as fusion proteins comprising the antibodies or fragments thereof. In addition to the almost full-length polypeptides, the present application also includes fragments of the antibodies of the present application. Typically, the fragments have at least about 50 contiguous amino acids of the antibodies of the present application, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, most preferably at least about 100 contiguous amino acids.

[0092] In the present application, "conservative variants of the antibodies of the present application" refer to polypeptides in which up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by similar or identical amino acids as compared to the amino acid sequence of the antibodies of the present application. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table A.

[0093] Table A

[0094]

[0095] The present application also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof described above. The polynucleotides of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0096] The polynucleotide encoding the mature polypeptide of the present application includes: a coding sequence encoding only the mature polypeptide; a coding sequence encoding the mature polypeptide and various additional coding sequences; a coding sequence encoding the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0097] The term "polynucleotide encoding a polypeptide" can be a polynucleotide comprising a coding sequence encoding the polypeptide, or a polynucleotide further comprising additional coding and / or non-coding sequences.

[0098] The nucleotide full-length sequence of the antibody of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. One possible method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is short. Generally, a long fragment can be obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0099] Once the relevant sequence is obtained, it can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then transforming it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules in isolated form.

[0100] At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application can be obtained entirely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present application by chemical synthesis.

[0101] The present application also relates to vectors containing the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express the protein.

[0102] The host cells can be prokaryotic cells such as bacterial cells, or lower eukaryotic cells such as yeast cells, or higher eukaryotic cells such as mammalian cells. Representative examples include: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0103] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation. If necessary, the transformation can be performed by electroporation. When the host is a eukaryote, the transformation can be effected by the use of techniques such as calcium phosphate precipitation, conventional mechanical procedures such as microinjection, electroporation, lipofection, etc.

[0104] The resulting transformant can be cultured in conventional nutrient media using standard procedures known in the art. The culture medium used will vary depending on the host cell selected. Appropriate media can be selected from a variety of media known to those skilled in the art. The culture conditions, such as temperature, pH and the like, can also be varied depending on the host cell selected.

[0105] The recombinant polypeptides of the above methods can be expressed intracellularly, on the cell membrane, or secreted from the cell. If desired, the recombinant proteins can be isolated and purified using various separation and purification methods based on their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation procedures, treatment with protein precipitants (salting-out procedures), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0106] The antibodies of the present application can be used alone or in combination or conjugation with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties, or any combination of these.

[0107] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing detectable products.

[0108] Therapeutic agents that can be combined or conjugated with the antibodies of the present application include, but are not limited to, 1. radionuclides; 2. biotoxins; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or benzylphenyl-hydrolase-like protein (BPHL)), etc.

[0109] The present application also provides a composition. In preferred embodiments, the composition is a pharmaceutical composition comprising the antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding immune cell described above, and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably the pH is about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the condition to be treated.

[0110] The prepared pharmaceutical composition can be administered by conventional routes, including but not limited to intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the administration route of the pharmaceutical composition of the present application is preferably injection or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, etc. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, which can be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a granule, an injection or an infusion, etc.

[0111] The antibody of the present application can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0112] The pharmaceutical composition of the present application contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the monoclonal antibody (or conjugate thereof) described above and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as needle, solution is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents.

[0113] In the present application, preferably, the pharmaceutical composition of the present application further comprises one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers are conventional pharmaceutically acceptable carriers in the art, and the pharmaceutically acceptable carriers can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical adjuvant is conventional pharmaceutical adjuvant in the art, and preferably includes pharmaceutically acceptable excipients, fillers or diluents, etc. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and 0.01-99.99% of the pharmaceutically acceptable carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0114] In the present application, preferably, the administration amount of the pharmaceutical composition is an effective amount, and the effective amount is an amount capable of alleviating or delaying the progression of a disease, a degenerative or an injury condition. The effective amount can be determined on an individual basis and will be based in part on the condition to be treated and the considerations of the sought result. The effective amount can be determined by a person skilled in the art by using the above-mentioned factors such as individual basis and using no more than conventional experiments.

[0115] When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, and preferably the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dose should also take into account the administration route, the patient's health condition, etc., which are within the skill of a skilled physician.

[0116] Compared with the prior art, the main advantages of the present application include:

[0117] 1. The monoclonal antibody of the present application can specifically bind to folate receptor alpha (FR1).

[0118] 2. The monoclonal antibody of the present application can be used for treating tumors / cancers overexpressing FOLR1.

[0119] 3. The 106-ADC drug of the present application has a killing effect on a variety of tumors.

[0120] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0121] Example 1. Monoclonal antibody screening

[0122] 1. Monoclonal B cell screening

[0123] 1.1 Mouse immunization

[0124] Prepare 3 Balb / c mice, inject intraperitoneally with 12 μg of recombinant protein human FOLR1 (uniprot: P15328; protein expression sequence Arg 25 - Met 233) mixed with adjuvant, inject intraperitoneally every 3 days, 3 days after the 5thintraperitoneal injection, collect 100 μL of peripheral blood from the mouse eye, and detect the immune titer. Select the mouse with the best immune titer, and perform a boost immunization by injecting 20 μg of protein intraperitoneally. Three days after immunization, isolate the mouse spleen and lymph nodes for single B cell sorting.

[0125] 1.2 Immune titer detection

[0126] 1) Take the mouse out of the cage, disinfect the eye of the mouse with a 75% medical alcohol cotton ball, and use a 5 mm blood collection needle to make a small incision in the eye of the mouse;

[0127] 2) Collect blood droplets (100 μL of plasma need to be prepared) using a capillary glass blood collection tube;

[0128] 3) After collecting the blood, use a dry sterile cotton ball to gently press the blood collection point to stop the bleeding, and then return the mouse to the cage for observation;

[0129] 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.

[0130] 5) Separate the serum from the blood clot and transfer it to a new sterile centrifuge tube, centrifuge at 4°C, 10000xg for 10 min;

[0131] 6) Transfer the serum to a new sterile centrifuge tube, and detect the immune titer by ELISA.

[0132] 1.3 ELISA detection

[0133] 1) Dilute the human FOLR1 his recombinant protein to a final concentration of 0.5 μg / mL using sterile carbonate buffer solution (CBS). Take a new 96-well plate, add 100 μL / well and incubate overnight at 4°C.

[0134] 2) Remove the antigen coating solution, wash 3 times with PBST (containing 0.5% Tween).

[0135] 3) Add 200 μL / well of 3% BSA 37°C blocking for 1 hour;

[0136] 4) After removing the blocking buffer, wash the well plate 3 times with PBST;

[0137] 5) Add 100 μL of mouse serum (highest concentration is diluted 300 times, 3 times gradient dilution), incubate at room temperature for 1 hour, and the control well is 1% BSA;

[0138] 6) Remove the liquid in the well, and wash 3 times with PBST;

[0139] 7) Add 100 μL of HRP mouse IgG (1:10000 dilution), incubate at room temperature for 1 hour;

[0140] 8) After removing the liquid in the well, wash the well plate 3 times with PBST;

[0141] 9) Add 100 μL / well TMB developing solution;

[0142] 10) Incubate at room temperature for 5 minutes in the dark;

[0143] 11) Add 50 μL / well stop solution;

[0144] 12) Read the OD450 value in the well using a microplate reader.

[0145] 2. Single B cell sorting

[0146] 2.1 Take the best mouse for immune titer detection from the cervical dislocation, obtain the mouse spleen and lymph nodes under sterile conditions, prepare a B cell single cell suspension, and count.

[0147] 2.2 According to the number of cells, add human FOLR1 antigen with biotin label (1 μg / 10 7Cell staining buffer wash cells, resuspend cells in 1 mL cell staining buffer, add DAPI, incubate at room temperature for 5 min in the dark. Cell sorting fluid rinse the cells, centrifuge at 1000 rpm / min for 5 min at 4°C, discard the supernatant. Resuspend the cells in 500 μL PBS buffer, use Beckman Coulter cytoflex SRT flow cytometer to sort human FOLR1 antigen-specific single B cells.

[0148] 2.3 Flow sorting of antigen-specific single B cells

[0149] Among all B cells, by screening cell size and cell granularity, mononuclear cell population was selected, and live cells were identified by DAPI (negative selection); CD19 (positive selection) recognized B cell population, and germinal center B cells were sorted by CD38 (negative selection) and GL7 (positive selection); IgG1 (positive selection) secreted antibody by antigen stimulation of germinal center B cells; PE (positive selection) and APC (positive selection) sorted out antigen-specific single B cells (DAPI-CD19+GL7+CD38-IgG1+Ag biotin++). The cells were sorted into 96-well PCR plate wells containing 4 μL sterile enzyme-free PBS, and after sorting the required number of cells (92 cells), the cells were transferred to 80°C for temporary storage for subsequent experiments.

[0150] 3. Preparation of human FOLR1 antigen-specific single B cell antibodies

[0151] 3.1 Reverse transcription of mRNA encoding antibodies in sorted human FOLR1 antigen-specific single B cells into cDNA.

[0152] 3.2 Amplify the complete coding gene fragments of antibody heavy chain and light chain respectively with cDNA as template and designed primers.

[0153] 3.3 Clone VH into pTT5 eukaryotic expression vector containing human IgG1 Fc, name the heavy chain skeleton vector as pTT5-human IgG1 CH; clone VL into pTT5 eukaryotic expression vector containing human CL, name the light chain skeleton vector as pTT5-human IgG1 CL. Co-transfect the antibody heavy chain and light chain recombinant expression vectors into 293F suspension cells, collect the supernatant after transfection and purify, and obtain the single antibody expressed by the single B cell encoding, which can be used for subsequent monoclonal antibody identification and screening.

[0154] 3.4 Amplification of single B cell cDNA

[0155] According to the requirements of the instruction of Maxima H Minus Reverse Transcriptase (manufacturer: Thermo Scientific, product number: EP0753), the mRNA (containing antibody encoding genes) in the sorted single B cells is reverse transcribed into cDNA.

[0156] 3.5 Amplification of single B cell antibody heavy chain complete encoding gene

[0157] 1) Antibody heavy chain amplification method (Table 1): using the single B cell cDNA amplified as the template, using the nest PCR technique, first using primers 5' MsVHE Fw and 3' mIgG1-2b-2c for amplification. The amplification system is shown in Table 1, and the PCR amplification program is: 98℃ 10s; 55℃ 30s, 72℃ 55s, 35 cycles; 72℃ 5min. The band of the desired fragment size (about 200-400bp) is recovered and purified.

[0158] 2) Antibody light chain amplification method (Table 2): using the single B cell cDNA amplified as the template, using the nest PCR technique, first using primers 5' L-Vk mix Fw and 3' mCk for amplification. The amplification system is shown in Table 2, and the PCR amplification program is: 98℃ 10s; 55℃ 30s, 72℃ 55s, 35 cycles; 72℃ 5min. Using primers 2nd-5VK-FW and 2nd-3VK-RV to amplify the amplification product, the PCR amplification program is: 98℃ 10s, 55℃ 30s, 72℃ 45s, 35 cycles; 72℃ 5min. The band of the desired fragment size (about 200-400bp) is recovered and purified.

[0159] Table 1. Conditions for heavy chain VH PCR amplification

[0160]

[0161] Table 2. Conditions for light chain VL PCR amplification

[0162]

[0163] PCR amplification results of antibody light chain and heavy chain of single B cells are shown in Figure 1 Figure 2, in which lane 6 is the FR1-106 antibody light chain and heavy chain variable region PCR band.

[0164] 3) The recovered and purified product was sequenced (sequencing was completed by Suzhou Jw Biotech Co., Ltd.), and the sequencing results were analyzed by NCBI Igblast (https: / / www.ncbi.nlm.nih.gov / igblast). The sequence analysis results are as follows:

[0165] Cloning 106 antibody heavy chain variable region (SEQ ID NO. 7):

[0166] EVQLEQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGDTKYDPKFQGKATVTADTSSNTAYLQLSSLTSEDTAVYHCAKEGIYSGAMDYWGQGISVTVSS;

[0167] Cloning 106 antibody light chain variable region (SEQ ID NO. 8):

[0168] NIVMTQSPASLAVSLGQRATMSCRASEGVDTYVNSFMHWYQQKPGQPPRLLIYLASNLQSGIPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPWTFGGGTKLEIK;

[0169] The sequences of the heavy chain CDR and light chain CDR of antibody 106 are shown in Table 3 and Table 4.

[0170] Table 3. Sequence list of heavy chain CDR of antibody 106

[0171]

[0172] Table 4. Sequence list of light chain CDR of antibody 106

[0173]

[0174] Example 2. Antibody verification

[0175] 1. Construction of single B cell antibody complete coding gene recombinant plasmid

[0176] According to the sequence analysis verification of IgG antibody heavy and light chain complete coding gene, the primer was designed at the 5' end of the upstream primer and the 3' end of the downstream primer, respectively, and the pTT5-human IgG1 CH and pTT5-human IgG1 CL expression vector homologous sequences were added. The upstream and downstream primers were named Primer-f1 and Primer-b1, respectively.

[0177] The PCR product was used as a template for amplification, and the PCR reaction components were as shown in Table 5. The PCR program was as follows: 95°C for 2 min; 95°C for 15 s, 50°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 5 min, as shown in Table 6. After the amplification product was identified by 1.5% agarose gel electrophoresis, the band with the desired fragment size was recovered and purified. The purified VH target fragment was ligated with the pTT5-human IgG1 CH linearized vector obtained by PmeI and NheI double digestion by homologous recombination, and the purified VL target fragment was ligated with the pTT5-human IgG1 CL linearized vector obtained by PmeI and BsiwI double digestion, and the reaction conditions were 50°C for 60 min. The reaction system is shown in Tables 7 and 8.

[0178] The homologous recombination product was transformed into E. coli Top10 competent cells, and 3 clones were randomly selected for sequencing. The sequencing alignment (analyzed by Snapgene software) was performed, and the single B cell antibody complete coding gene recombinant expression plasmid was extracted by shaking bacteria for the correct sequencing results.

[0179] Table 5. Cloning PCR component table

[0180]

[0181] Table 6. Cloning PCR reaction temperature

[0182]

[0183] Table 7. Heavy chain ligation reaction component table

[0184]

[0185] Table 8. Light chain ligation reaction component table

[0186]

[0187] 2. Expression of single B cell antibody

[0188] 293F suspension cells were passaged into 125 mL Erlenmeyer flasks until the cell density reached 2 × 10⁻⁶ cells / mL. 6 Transfection 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.

[0189] 3. Purification of chimeric antibodies

[0190] 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.

[0191] 4. Verify the expressed antibody

[0192] 4.1 Validation of the expressed recombinant antibody using ELISA

[0193] 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.

[0194] 2) Remove the antigen coating solution and wash three times with PBST (containing 0.5% Tween).

[0195] 3) Add 200 μL / well of 3% BSA and seal at 37℃ for 1 hour;

[0196] 4) After removing the blocking buffer, wash the plate three times with PBST;

[0197] 5) Add 1 μg antibody, 3-fold dilution gradient, incubate at room temperature for 1 hour, control wells are 1% BSA;

[0198] 6) Remove the liquid in the wells, and wash 3 times with PBST;

[0199] 7) Add 100 μL HRP human IgG (1:10000 dilution), incubate at room temperature for 1 hour;

[0200] 8) After removing the liquid in the wells, wash the plate 3 times with PBST;

[0201] 9) Add 100 μL / well TMB developing solution;

[0202] 10) Incubate at room temperature for 5 minutes in the dark;

[0203] 11) Add 50 μL / well stop solution;

[0204] 12) Use a microplate reader to read the OD450 value in the wells.

[0205] Conclusion: ELISA detected the binding of antibody 106 to FR1 antigen, as shown in Figure 2 Figure 2, antibody 106 has strong binding ability to FR1 antigen.

[0206] Subsequently, the affinity of antibody 106 for FR1 antigen was detected using ELISA, and the results are shown in Figure 3 Figure 3, the Kd of antibody 106 binding to FR1 antigen is 0.2 μg / mL.

[0207] 4.2 Flow verification of expressed recombinant antibodies

[0208] 1) Take 1x10 5 Hela cells into a 1.5 mL EP tube, add 100 nM purified antibody, including antibody No. 106 screened by the application and clinical antibody IMGN853 from ImmunoGen, incubate at 4°C in the dark for 30 min in a 100 μL system; after incubation, centrifuge at 300g at 4°C for 5 min; resuspend in 500 μL PBS (containing 0.1% BSA) and centrifuge at 300g at 4°C for 5 min; discard the supernatant and add secondary antibody anti huamn IgG H+L (brand: invitrogen; item number: A21445; 1:2000) in a 100 μL system, incubate at 4°C in the dark for 30 min; after incubation, centrifuge at 300g at 4°C for 5 min; resuspend in 500 μL PBS (containing 0.1% BSA) and centrifuge at 300g at 4°C for 5 min; discard the supernatant and add 300 μL PBS; filter the cells through a 300 mesh filter and perform flow detection on the machine.

[0209] The results of flow cytometry detection of the binding of the antibody to Hela cells (naturally expressing FR1) are shown in Figure 4 、 5 , indicating that the antibody 106 can bind to Hela cells expressing FR1, and the binding to FR1 tends to be saturated as the concentration of the antibody 106 increases.

[0210] Example 3. Preparation of antibody-conjugate (ADC)

[0211] 1. Preparation method

[0212] 1) Reduce the FR1-106 antibody with TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) at about 8 times the molar concentration of the FR1-106 antibody, incubate at room temperature for 3 h;

[0213] 2) Add VcMMAE (brand: MCE; catalog number: HY-15575) small molecule toxin reagent solution at about 4-8 times the molar concentration of the FR1-106 antibody, react at room temperature for 1-2 h;

[0214] 3) Add NAC reagent (Acetylcysteine; MCE, HY-B0215) at 8 times the molar concentration of the FR1-106 antibody, incubate on ice for 30 min;

[0215] 4) Perform buffer exchange on the prepared 106-ADC with 1xPBS solution using a desalting column (Zeba™ Desalt Spin Column, 7K MWCO, 5 mL; ThermoScientific, 89891);

[0216] 5) Concentrate the 106-ADC with a 30KDa concentration tube and determine the concentration;

[0217] 6) Identify the prepared 106-ADC with an analytical HIC hydrophobic column (Proteomix HIC Butyl);

[0218] The identification map is shown in Figure 6 , indicating that the 106-ADC is successfully prepared.

[0219] 2. FACS (flow cytometry) detection of Hela cell-mediated internalization of anti-FR1 antibody

[0220] Take human cervical cancer cells Hela, 5x10 5Cells / tube, respectively, add No. 106 antibody diluted to 10 μg / ml, the antibody is divided into three groups (incubation 4h, 8h, 12h experimental group and control group), each group 1 tube. Experimental group into 37℃ electric heating incubator, incubate 4h, 8h, 12h respectively, and then placed on ice, the control group is always ice incubation as negative control; all samples after incubation, 300g, 4℃ centrifugation 5min, discard the supernatant, ice cold PBS wash 1 times, add secondary antibody anti human IgG H+L (brand: invitrogen; item number: A21445; 1:2000), ice incubation 30min, 300g, 4℃ centrifugation 5min, discard the supernatant, ice cold PBS wash, take 200ul ice cold PBS resuspended cells, FACS detection of mean fluorescence intensity MFI, and calculate the internalization efficiency by the following formula:

[0221] .

[0222] Results as shown in Figure 7 , 106 antibody with the passage of time, the amount of endocytosis by Hela cells increases.

[0223] 3. Antibody-drug conjugate 106-ADC cell killing activity assay

[0224] Take different tumor cells (Hela, OVCAR-3, MCF-7, NCI-H441, NCI-H3122 and NCI-H2170), according to 5-8x10 3 Cells / well in 96-well plates, 100 μl per well, in 37℃, 5% CO2 incubator pre-culture 16-24 hours, according to table 9 to add different concentrations of 106-ADC, 37℃, 5% CO2 incubator incubation, incubation for 72 hours, the culture plate at room temperature for 30min to balance to room temperature, using Promega CellTiter-Glo™ Luminescent Cell Viability Assay Kit to detect 106-ADC antibody-drug conjugate on cell killing activity.

[0225] Results as shown in Figures 8-1 to 8-6 , indicating that 106-ADC on different types of solid tumor cells have killing effect, including cervical cancer, ovarian cancer, breast cancer and lung cancer.

[0226] Table 9 106-ADC in different cells to add concentration

[0227]

[0228] 4. Antibody-drug conjugate 106-ADC in Hela subcutaneous tumor model pharmacodynamic evaluation

[0229] Take 6-week-old, female BALB / c-nu mice, subcutaneously inoculate 2x10 6 Human cervical cancer cells (Hela), when the tumor grows to 150 mm 3 Randomly grouped after 6 mice / group, divided into blank control group and drug administration group, drug administration group according to 5 mg / kg intravenous injection of drug (106-ADC) once a week, drug administration at the same time measure tumor volume and mouse weight, before the tumor volume exceeds 1000mm3 stop experiment, euthanize the mice.

[0230] Results as shown in Figure 9 、 10 , 11, 106-ADC compared to the control group can significantly inhibit the growth rate of tumor, reduce the size of the tumor, and after administration on the mouse weight no significant effect, indicating that the antibody-drug conjugate on mice no significant toxicity.

[0231] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that, after reading the above teaching of the present application, those skilled in the art can make various modifications or changes to the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

[0232] The sequences involved in the present application are:

[0233]

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: (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 variable region sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO.

7.

3. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is a sequence that is at least 90% sequence-identical to the sequence shown in SEQ ID NO.

7.

4. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The light chain variable region sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO.

8.

5. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The light chain variable region sequence of the antibody or its antigen-binding fragment is a sequence that is at least 90% sequence-identical to the sequence shown in SEQ ID NO.

8.

6. 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.

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

8. A carrier, characterized in that, The carrier contains the nucleic acid as described in claim 7.

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

10. An 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, cytokines, and enzymes.

11. The immunoconjugate as described in claim 10, characterized in that, The coupling component is a toxin.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: 1) The antibody or its antigen-binding fragment as described in claim 1, the nucleic acid as described in claim 7, the vector as described in claim 8, the host cell as described in claim 9, and / or the immunoconjugate as described in claim 10; and 2) Pharmaceutically acceptable carrier.

13. Use of the antibody or antigen-binding fragment thereof as claimed in claim 1, the nucleic acid as claimed in claim 7, the vector as claimed in claim 8, the cell as claimed in claim 9, or the immunoconjugate as claimed in claim 10, characterized in that, This is used to prepare a drug for treating FOLR1-overexpressing tumors / cancers, including cervical cancer, breast cancer, ovarian cancer, and / or non-small cell lung cancer.

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