Anti-frα antibody or antigen-binding fragment, and antibody drug conjugate and use thereof

Abstract

Provided are an anti-FRα antibody or antigen-binding fragment, and an antibody drug conjugate and a use thereof. Specifically, provided is an anti-FRα antibody or an antigen-binding fragment thereof that is capable of effectively binding to human FRα. Also provided is an antibody drug conjugate or the like comprising the antibody or the antigen-binding fragment, which can be applied to the preparation of drugs for treating tumors expressing or overexpressing FRα, and has good application prospects.

Description

Anti-FRα antibodies or antigen-binding fragments, antibody-drug conjugates and their applications Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to anti-FRα antibodies or antigen-binding fragments, antibody-drug conjugates and their applications. Background Technology

[0002] FRα expression is restricted in normal tissues, with extremely low expression only in non-malignant tissues such as the choroid plexus, thyroid gland, salivary gland, breast, colon, and bladder. However, it is highly expressed on the surface of various malignant tumor cells, including serous and endometrioid epithelial ovarian cancer, endometrial adenocarcinoma, choriocarcinoma, adenocarcinoma subtype of non-small cell lung cancer, mesothelioma, and some triple-negative breast cancer cells.

[0003] The ability of FRα to internalize large molecules and its high expression on the surface of certain tumor cells, coupled with its restricted expression in normal tissues, has attracted the development of antibody-drug conjugates (ADCs) targeting this antigen. ADCs consist of a monoclonal antibody, a biologically active small-molecule toxin, and a linker. The antibody portion specifically recognizes the target antigen highly expressed on the surface of target cells. The ADC-antigen complex then enters the tumor cell via endocytosis, releasing the biologically active small-molecule toxin in the acidic environment of the lysosome. The toxin primarily destroys DNA strands, inhibits microtubules, and suppresses DNA topoisomerase or RNA polymerase, ultimately leading to tumor cell death.

[0004] Currently, the American company ImmunoGen is developing... (mirvetuximab soravtansine-gynx, MIRV, research code: IMGN853) has been approved for marketing in the treatment of adult patients with folate receptor α (FRα)-positive platinum-resistant epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer who have received 1-3 lines of prior systemic therapy. ELAHERE consists of the humanized monoclonal antibody M9346A, a cleavable sulfo-SPDB linker, and the maytansine alkaloid DM4.

[0005] Currently, the small molecule toxins conjugated to several FRα-ADCs under development are mainly tubulin inhibitors, with a DAR range of 2–4. However, their efficacy is easily reduced during use due to tumor tissue heterogeneity and the rapid development of drug resistance during treatment.

[0006] Therefore, designing differentiated antibody-drug conjugates is of great clinical significance in this field. Summary of the Invention

[0007] This invention provides a differentiated anti-FRα antibody or antigen-binding fragment, antibody-drug conjugate, and its application.

[0008] In a first aspect of the invention, an anti-FRα antibody or an antigen-binding fragment thereof is provided, said antibody or antigen-binding fragment having three complementarity-determining regions (CDRs) of the heavy chain variable region and three complementarity-determining regions (CDRs) of the light chain variable region selected from the group consisting of:

[0009] (1) HCDR1 shown in SEQ ID NO:6,

[0010] HCDR2, as shown in SEQ ID NO:8,

[0011] HCDR3, as shown in SEQ ID NO:10,

[0012] LCDR1 shown in SEQ ID NO:13,

[0013] LCDR2 shown in SEQ ID NO:15,

[0014] LCDR3 as shown in SEQ ID NO:17;

[0015] (2) HCDR1 shown in SEQ ID NO:6,

[0016] HCDR2, as shown in SEQ ID NO:20,

[0017] HCDR3, as shown in SEQ ID NO:10,

[0018] LCDR1 shown in SEQ ID NO:13,

[0019] LCDR2 shown in SEQ ID NO:15,

[0020] LCDR3 as shown in SEQ ID NO:17.

[0021] In another preferred embodiment, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0022] In another preferred embodiment, the antigen-binding fragment includes the Fab fragment, the F(ab')2 fragment, and the Fv fragment.

[0023] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-FRα antibody or its antigen-binding fragment is as shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:4; and / or

[0024] The amino acid sequence of the heavy chain variable region of the anti-FRα antibody or its antigen-binding fragment is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21.

[0025] In another preferred embodiment, the heavy chain of the antibody or its antigen-binding fragment further includes a heavy chain constant region; the light chain of the antibody or its antigen-binding fragment further includes a light chain constant region.

[0026] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment.

[0027] In another preferred embodiment, the antibody is a humanized antibody, a murine antibody, or a chimeric antibody.

[0028] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0029] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0030] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0031] In another preferred embodiment, the antibody is a monoclonal antibody.

[0032] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0033] In another preferred embodiment, the antibody further comprises a linker peptide located between the heavy chain variable region and the light chain variable region.

[0034] In a second aspect of the invention, an antibody-drug conjugate is provided, the antibody-drug conjugate comprising:

[0035] (a) an antibody portion comprising an antibody or an antigen-binding fragment thereof as described in the first aspect of the invention; and

[0036] (b) A coupling portion conjugated to the antibody or its antigen-binding fragment, the coupling portion being selected from the group consisting of: detectable markers, cytotoxic drugs, toxins, or combinations thereof.

[0037] In another preferred embodiment, the expression for the antibody-drug conjugate is: mAb-(XY)n;

[0038] in,

[0039] mAb is the anti-FRα antibody or its antigen-binding fragment;

[0040] X is a connector;

[0041] Y represents the coupling portion, which is a cytotoxic drug;

[0042] n is a positive integer ≤ 8;

[0043] The conjugation portion is conjugated to the anti-FRα antibody or its antigen-binding fragment via a linker.

[0044] In another preferred embodiment, the cytotoxic drug includes a DNA topoisomerase I inhibitor (Dxd).

[0045] In another preferred embodiment, the antibody portion or the anti-FRα antibody or its antigen-binding fragment is coupled to the detectable marker via a chemical bond or linker.

[0046] In another preferred embodiment, the detectable marker is selected from the group consisting of: fluorescent or luminescent markers, biotin, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, gold nanoparticles / nanorobars, magnetic nanoparticles, or any form of nanoparticles capable of producing a detectable product.

[0047] In another preferred embodiment, the detectable marker is biotin.

[0048] In another preferred embodiment, the drug is a cytotoxic drug or toxin.

[0049] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors (e.g., Dxd), vinca alkaloids, or combinations thereof.

[0050] In another preferred embodiment, the toxin is selected from the group consisting of: ostatins (e.g., ostatin E, ostatin F, MMAE, and MMAF), chlortetracycline, methamphetamine, pyrine, pyrine A-chain, cobustatin, docalimicin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxychloroquine. Anthraxone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE)A, PE40, abrin, abrin A chain, saccharin A chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, chachomycin, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0051] In a third aspect of the invention, a recombinant protein is provided, the recombinant protein having:

[0052] (i) the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0053] (ii) Tag sequences that optionally assist in expression and / or purification.

[0054] In another preferred embodiment, the label includes an Fc label, a FLAG label, a 6His label, or a combination thereof.

[0055] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0056] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0057] In a fourth aspect of the invention, a nucleotide molecule is provided, said nucleotide molecule encoding the anti-FRα antibody or antigen-binding fragment thereof described in the first aspect of the invention.

[0058] In a fifth aspect of the invention, a carrier is provided, the carrier containing the nucleotide molecules described in the fourth aspect of the invention.

[0059] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0060] In another preferred embodiment, the vector is a eukaryotic expression vector.

[0061] In a sixth aspect of the invention, a host cell is provided, the host cell containing the vector described in the fifth aspect of the invention, or having nucleotide molecules described in the fourth aspect of the invention integrated into its genome, or expressing the anti-FRα antibody or its antigen-binding fragment described in the first aspect of the invention.

[0062] In another preferred embodiment, the cell is a eukaryotic cell or a prokaryotic cell.

[0063] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0064] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0065] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0066] In another preferred embodiment, the cell is an immune cell, and its surface simultaneously expresses a chimeric antigen receptor.

[0067] In another preferred embodiment, the immune cells are T cells, NK cells, or a combination thereof.

[0068] In another preferred embodiment, the immune cells are chimeric antigen receptor T cells (CAR-T cells).

[0069] In another preferred embodiment, the chimeric antigen receptor is FRα.

[0070] In a seventh aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0071] (i) an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, an antibody-drug conjugate as described in the second aspect of the present invention, or a recombinant protein as described in the third aspect of the present invention; and

[0072] (ii) Pharmaceutically acceptable carriers.

[0073] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0074] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating tumors selected from the group consisting of serous and endometrioid epithelial ovarian cancer, endometrial adenocarcinoma, choriocarcinoma, adenocarcinoma subtype of non-small cell lung cancer, mesothelioma, triple-negative breast cancer, or combinations thereof.

[0075] In an eighth aspect of the invention, the use of the antibody or antigen-binding fragment thereof as described in the first aspect of the invention, the antibody-drug conjugate as described in the second aspect of the invention, or the recombinant protein as described in the third aspect of the invention is provided for the preparation of pharmaceuticals, reagents, detection plates or kits.

[0076] The reagents, detection plates, or kits are used to detect FRα protein in samples;

[0077] The drug is used to treat or prevent tumors that express or overexpress the FRα protein.

[0078] In another preferred embodiment, the tumor is selected from the group consisting of serous and endometrioid epithelial ovarian cancer, endometrial adenocarcinoma, choriocarcinoma, adenocarcinoma subtype of non-small cell lung cancer, mesothelioma, triple-negative breast cancer, or combinations thereof.

[0079] In a ninth aspect of the present invention, a method for preparing the anti-FRα antibody or its antigen-binding fragment as described in the first aspect of the present invention is provided, the method comprising the following steps:

[0080] (a) Under expression conditions, host cells as described in the sixth aspect of the present invention are cultured to express the anti-FRα antibody or its antigen-binding fragment thereof;

[0081] (b) Isolate and purify the anti-FRα antibody or its antigen-binding fragment described in (a).

[0082] In a tenth aspect of the present invention, a method for detecting FRα protein in a sample is provided, the method comprising the steps of:

[0083] (1) Contact the sample with an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0084] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of FRα protein in the sample.

[0085] In another preferred embodiment, the sample includes: human or animal tissue samples, tumor resection samples, and exfoliated cell samples.

[0086] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0087] In another preferred embodiment, the method is an in vitro method.

[0088] In another preferred embodiment, the method further includes step (3) analyzing the affinity between the antibody and the antigen.

[0089] In an eleventh aspect of the present invention, a detection plate is provided, the detection plate comprising a substrate (support plate) and a test strip, the test strip containing an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, or an antibody-drug conjugate as described in the second aspect of the present invention.

[0090] In another preferred embodiment, the test strip also contains an antigen spotting area.

[0091] In another preferred embodiment, the test strip is composed of filter paper, chromatography material, nitrocellulose membrane and absorbent paper stacked in sequence.

[0092] In a twelfth aspect of the invention, a kit is provided, the kit comprising:

[0093] (1) A first container containing an antibody or an antigen-binding fragment thereof as described in the first aspect of the invention; and / or

[0094] (2) A second container containing a secondary antibody against the antibody or antigen-binding fragment thereof as described in the first aspect of the invention; and / or

[0095] (3) A third container containing a cell lysis reagent;

[0096] or,

[0097] The kit contains a detection plate as described in the eleventh aspect of the present invention.

[0098] In another preferred embodiment, the antibody in the first container is labeled with a detectable tag.

[0099] In another preferred embodiment, the antibody in the second container is labeled with a detectable tag.

[0100] In a thirteenth aspect of the invention, a method for treating FRα-related diseases is provided, comprising the steps of administering to a subject requiring treatment a therapeutically effective amount of the anti-FRα antibody or its antigen-binding fragment as described in the first aspect of the invention, the host cell as described in the sixth aspect of the invention, or the pharmaceutical composition as described in the seventh aspect of the invention.

[0101] In another preferred embodiment, the disease is cancer or a tumor.

[0102] In another preferred embodiment, the tumor is a tumor that overexpresses FRα.

[0103] In another preferred embodiment, the tumor is selected from the group consisting of serous and endometrioid epithelial ovarian cancer, endometrial adenocarcinoma, choriocarcinoma, adenocarcinoma subtype of non-small cell lung cancer, mesothelioma, triple-negative breast cancer, or combinations thereof.

[0104] In a fourteenth aspect of the invention, a CAR construct is provided, wherein the scFv segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region specifically binding to FRα, and wherein the scFv has three complementarity-determining regions (CDRs) selected from the group consisting of three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region:

[0105] (1) HCDR1 shown in SEQ ID NO:6,

[0106] HCDR2, as shown in SEQ ID NO:8,

[0107] HCDR3, as shown in SEQ ID NO:10,

[0108] LCDR1 shown in SEQ ID NO:13,

[0109] LCDR2 shown in SEQ ID NO:15,

[0110] LCDR3 as shown in SEQ ID NO:17;

[0111] (2) HCDR1 shown in SEQ ID NO:6,

[0112] HCDR2, as shown in SEQ ID NO:20,

[0113] HCDR3, as shown in SEQ ID NO:10,

[0114] LCDR1 shown in SEQ ID NO:13,

[0115] LCDR2 shown in SEQ ID NO:15,

[0116] LCDR3 as shown in SEQ ID NO:17.

[0117] In a fifteenth aspect of the invention, a recombinant immune cell is provided, said immune cell expressing an exogenous CAR construct as described in a fourteenth aspect of the invention.

[0118] 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

[0119] Figure 1 shows the species-specific binding activity assay results for mab29, mab101, and mab107.

[0120] Figures 2A, 2B, and 2C show the comparison of OVCAR3 cell endocytosis results of mab29, mab101, and mab107 antibodies under different temperature conditions (M1: fluorescence shift of primary antibody incubation at 37℃; M2: fluorescence shift of primary antibody incubation at 4℃).

[0121] Figure 3 shows the results of the binding activity of the humanized antibody to human FRα protein.

[0122] Figure 4 shows the selection of mutation sites for the FRα protein.

[0123] Figure 5 shows the binding activity results of the humanized antibody with the mutated FRα protein.

[0124] Figure 6 shows the results of the assay for the binding activity of humanized antibodies to target cells.

[0125] Figure 7 shows the proliferation inhibitory activity of hu107: (A) proliferation inhibitory activity of hu107 on SW620 cells; (B) proliferation inhibitory activity of hu107 on SW480 cells; (C) proliferation inhibitory activity of hu107 on OVCAR3 cells.

[0126] Figure 8 shows the endocytic activity of the humanized antibody in the target cells.

[0127] Figure 9 shows the killing effect of small molecule toxins on tumor cells: (A) Killing activity of small molecule toxins on SW620 cells; (B) Killing activity of small molecule toxins on SW480 cells; (C) Killing activity of small molecule toxins on OVCAR3 cells.

[0128] Figures 10A and 10B show the HIC-HPLC DAR values ​​of hu107-08Dxd and hu107-09Dxd, respectively.

[0129] Figure 11 shows the ADC binding activity targeting FRα.

[0130] Figures 12A, 12B, and 12C show the binding activity of antibody-drug conjugates (ADCs) to FRα on the surface of different target cells: (A) ADC binding to FRα on the surface of SW620 cells; (B) ADC binding to FRα on the surface of SW480 cells; and (C) ADC binding to FRα on the surface of OVCAR3 cells.

[0131] Figure 13 shows the endocytosis rate of antibody-drug conjugates in SW620, SW480, and OVCAR3 cells.

[0132] Figures 14A, 14B, and 14C show the killing activity of hu107-08Dxd against target cells SW620, SW480, and OVCAR3, respectively.

[0133] Figures 15A, 15B, and 15C show the killing activity of hu107-09Dxd against SW620, SW480, and OVCAR3 tumor cells, respectively.

[0134] Figure 16 shows the efficacy of hu107-09Dxd in the SW620 nude mouse xenograft model.

[0135] Figure 17 shows the efficacy of hu107-08Dxd in the CAOV3 nude mouse xenograft model. Detailed Implementation

[0136] The inventors, through extensive and in-depth research and numerous screenings, obtained an anti-FRα antibody. Experimental results show that the anti-FRα antibody and its antibody-drug conjugate of this invention possess high affinity and good biological activity. Furthermore, the murine monoclonal antibody of this invention also exhibits cross-reactivity with human FRα and cynomolgus monkey FRα. Compared to the anti-FRα antibody, the antibody-drug conjugate of the anti-FRα antibody shows increased endocytosis rate and better tumor cell killing activity, significantly inhibiting tumor growth. Based on these findings, this invention was completed.

[0137] the term

[0138] To facilitate understanding of the 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 the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0139] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0140] 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”.

[0141] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0142] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0143] FRα

[0144] FRα (folate receptor α) is a cell membrane surface glycoprotein encoded by FOLR1 and anchored to glycosylphosphatidylinositol with a relative molecular weight of 38,000–40,000. It has a high affinity for reduced folate (such as 5-methyltetrahydrofolate and tetrahydrofolate) and folate, but compared to other folate transporters, FRα provides lower folate uptake efficiency. Folate transport by FRα is generally believed to occur via a non-classical lipid raft-mediated endocytosis pathway, i.e., endocytosis, which does not involve clathrin-coated pits but is associated with small pitted vesicles.

[0145] The binding of folic acid to FRα promotes the aggregation of the receptor-ligand complex on the cell membrane, which then forms intracellular vesicles through invagination and budding. Once internalized, the vesicles uncoat and combine individually to form early endosomes. These endosomes undergo acidification and subsequently fuse with lysosomes to release folic acid for single-carbon metabolic reactions. FRα can not only transport folic acid but also mediate the entry of antibodies and folic acid conjugates into cells via endocytosis.

[0146] FRα expression is restricted in normal tissues, with extremely low expression only in non-malignant tissues such as the choroid plexus, thyroid gland, salivary gland, breast, colon, and bladder. However, it is highly expressed on the surface of various malignant tumor cells, including serous and endometrioid epithelial ovarian cancer, endometrial adenocarcinoma, choriocarcinoma, adenocarcinoma subtype of non-small cell lung cancer, mesothelioma, and some triple-negative breast cancer cells.

[0147] Multiple studies have shown that FRα plays a role in regulating cell growth and signal transduction during tumor cell proliferation and invasion. After folic acid binds to FRα, it can continuously activate signal transduction and transcription activator 3 (FRα) through the GP130 co-receptor-mediated JAK-STAT signaling pathway, resulting in FRα remaining in an activated state in the cell nucleus and continuously activating target genes, thus promoting tumor cell proliferation. In ovarian cancer cell lines, FRα knockout leads to reduced tumor cell division, inhibited non-anchored growth, and decreased cancer cell adhesion properties; this effect may be related to the downregulation of cadherin E expression.

[0148] Furthermore, FRα can also promote the progression of ovarian cancer by inhibiting the expression of caveolin-1. In cervical cancer, FRα can regulate the growth of cervical cancer cells by phosphorylating key factors in the ERK signaling pathway, ERK1 / 2, c-Fos, and c-Jun. In vitro, downregulating FRα expression can inhibit cell proliferation, promote apoptosis, and induce cell cycle arrest in the G0 / G1 phase, while simultaneously reducing the expression of p-ERK1 / 2, pc-Fos, and pc-Jun proteins.

[0149] The above studies indicate that FRα plays an important role in the progression of tumors and is a potential anti-tumor target.

[0150] Antibody

[0151] In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region, which consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region including a domain CL; the light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 isotypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions.

[0152] In this invention, the terms "Fab" and "Fc" refer to the ability of papain to cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of its light chain. The Fc fragment, or crystallizable fragment, consists of the antibody's CH2 and CH3 domains. The Fc fragment lacks antigen-binding activity and is the site of interaction between the antibody and effector molecules or cells.

[0153] In this invention, the term "scFv" refers to a single-chain antibody fragment (scFv), which is composed of the variable regions of the antibody heavy chain and the variable regions of the light chain, typically linked by a short peptide (linker) of 15 to 25 amino acids.

[0154] In this invention, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the heavy and light chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frame regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partial β-sheet structure. The CDRs in each chain are closely packed together through 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)).

[0155] As used herein, the term "frame region" (FR) refers to the amino acid sequence inserted between CDRs, specifically those portions of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved among different immunoglobulins within a single species. Each immunoglobulin light and heavy chain has four FRs, designated L-FR1, L-FR2, L-FR3, L-FR4 and H-FR1, H-FR2, H-FR3, H-FR4, respectively. Accordingly, the light chain variable domain can thus be represented as (L-FR1)-(L-CDR1)-(L-FR2)-(L-CDR2)-(L-FR3)-(L-CDR3)-(L-FR4), and the heavy chain variable domain can thus be represented as (H-FR1)-(H-CDR1)-(H-FR2)-(H-CDR2)-(H-FR3)-(H-CDR3)-(H-FR4). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, wherein the derivative of the human antibody FR is substantially identical to the naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0156] Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the framework regions L-FR1, L-FR2, L-FR3, L-FR4 and / or H-FR1, H-FR2, H-FR3, H-FR4.

[0157] As used herein, the term "human frame region" is a frame region that is substantially identical (approximately 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to the frame region of a naturally occurring human antibody.

[0158] As used herein, the term "linker" refers to an insertion into an immunoglobulin domain that provides sufficient mobility for the light and heavy chains to fold into one or more amino acid residues of an exchangeable dual variable region immunoglobulin. In this invention, preferred linkers are Linker1 and Linker2, wherein Linker1 links the VH and VL of a single-chain antibody (scFv), while Linker2 is used to link the scFv to the heavy chain of another antibody.

[0159] Suitable examples of linkers include monoglycine (Gly) or serine (Ser) residues, and the identification and sequence of amino acid residues in the linker can vary depending on the type of secondary structural element that needs to be achieved in the linker.

[0160] In this invention, the antibody also includes its conserved variants, which are polypeptides 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 bispecific antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0161] Table A

[0162] In this invention, the terms "antibody," "binding," and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The antibody binds to the antigen with an equilibrium dissociation constant (KD) of M or smaller. In this invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of antibody-antigen binding.

[0163] In this invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitopes of this invention are regions of an antigen that are bound to antibodies.

[0164] 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.

[0165] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0166] 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.

[0167] Pharmaceutical Compositions and Applications

[0168] This invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein, 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 may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavitary injection. In this invention, the term "pharmaceutical composition" refers to a pharmaceutical formulation composition in which the bispecific antibody of this invention, together with a pharmaceutically acceptable carrier, can be formed to exert its therapeutic effect more stably. These formulations ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation). 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 bispecific 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 solutions, 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 using conventional methods with 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 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the bispecific antibody of the present invention can also be used with other therapeutic agents.

[0169] When using a pharmaceutical composition, a safe and effective amount of the bispecific antibody or its immunoconjugate is administered to a mammal. 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 between about 10 micrograms per kilogram of body weight and about 10 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.

[0170] Antibody-drug conjugates (ADCs)

[0171] The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0172] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0173] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0174] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0175] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0176] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0177] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0178] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In this invention, the drug-linker can be used to form an ADC in a single, simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in two or more steps. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in subsequent steps, functional groups on the linker react with the drug to form an ADC.

[0179] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0180] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0181] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0182] In some implementations, the antibody-drug conjugate (ADC) has the following molecular formula:

[0183] in:

[0184] Ab is an antibody.

[0185] LU stands for connector;

[0186] D is a drug;

[0187] Furthermore, the subscript p is a value selected from 1 to 8.

[0188] Detection uses and kits

[0189] The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0190] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of organs.

[0191] The samples used in this invention include fixed or preserved cell or tissue samples.

[0192] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0193] application

[0194] This invention provides uses for the antibodies of this invention, such as in the preparation of diagnostic agents or in the preparation of medicaments for the prevention and / or treatment of FRα-related diseases. FRα-related diseases include tumors.

[0195] In a preferred embodiment, the tumor is a tumor that expresses or overexpresses FRα. In another preferred embodiment, the tumor includes: serous and endometrioid epithelial ovarian cancer, endometrial adenocarcinoma, choriocarcinoma, adenocarcinoma subtype of non-small cell lung cancer, mesothelioma, triple-negative breast cancer, or combinations thereof.

[0196] The main advantages of this invention include:

[0197] (a) The anti-FRα antibody of the present invention has highly specific binding activity to human FRα and strong endocytic activity after binding to FRα on the surface of tumor cells.

[0198] (b) The anti-FRα antibody-drug conjugate of the present invention has good in vitro and in vivo antitumor effects and has good prospects for clinical application.

[0199] 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.

[0200] Example 1: Immunogen Preparation

[0201] In this embodiment, the extracellular segment W28-S257 (P15328, Uniprot) encoding the human folate receptor α (FRα) and the mouse IgG1 Fc gene were synthesized by Shanghai Platinum Biotech and cloned into the pTT5 eukaryotic expression vector (available from BioVector NTCC Inc.), named pTT5-FRα-mFc; the nucleotide sequence encoding the extracellular segment of the human folate receptor α was cloned into the pSGHV0 vector (GenBank: AF285183.1) and fused with hGH for expression, named pSGHV0-hGH-His-FRα.

[0202] Take 100 mL of Expi293-F cells in logarithmic growth phase, and adjust the cell volume to 90 mL and the cell density to 1.5 × 10⁻⁶ cells 24 hours before transfection. 6 After incubating the cells overnight on a cell shaker, the cell density was adjusted to 3 × 10⁶ cells / mL. 6 pTT5-FRα-mFc plasmid and pSGHV0-hGH-His-FRα plasmid were mixed with PEI at a ratio of 3:1 and transfected into Expi293-F cells. FRα-mFc protein was purified using Protein A 5–7 days after transfection, and hGH-FRα protein was purified using a Ni column.

[0203] In this embodiment, the same method was used to express and purify hFc-FRβ protein, rat FRα (hFc-FRα / R), and cynomolgus monkey FRα (hFc-FRα / M).

[0204] Example 2 Animal Immunization

[0205] Balb / c mice used in the experiment were purchased from Shanghai Xipu-Bikai Laboratory. All immunization mice were 4-6 weeks old, female, standardized, disease-free, and healthy purebred mice.

[0206] Ten Balb / c mice were divided into two groups, with five mice immunized with FRα-mFc and five mice with hGH-FRα. On day 0, FRα-mFc and hGH-FRα were mixed with 1 mg / mL aluminum adjuvant at a 1:1 volume ratio, and each mouse was immunized with 50 μg of antigen via multiple injections on the back and subcutaneous injection in the paw. On days 14, 28, and 42, each mouse was injected with 20 μg of immunogen for booster immunization using the same method. Three days before fusion, a booster immunization was performed by intravenous injection of 20 μg of immunogen in the tail vein. On the day of fusion, blood was collected from the eyeballs of the mice and the spleen was removed. Single-cell suspensions were prepared and electrofused with SP20 cells (mouse myeloma cells) to obtain hybridoma plates.

[0207] Example 3 Hybridoma Screening

[0208] In this embodiment, after 14 days of static culture of hybridomas, 100 μL of supernatant was taken from each well and added to a 96-well ELISA plate pre-coated with hGH-FRα protein. Positive and negative control wells were included. The positive control was serum from the eyeballs of fused mice diluted 1000-fold, and the negative control was serum from the eyeballs of unimmunized mice diluted 1000-fold. Detection was performed using a standard ELISA method. OD was selected. 450 Supernatant with a reading greater than or equal to half that of the positive control was considered a positive clone. The selected final positive clones were then subjected to limiting dilution for subclonal screening, and the final selected positive cells were used for subsequent experiments.

[0209] After three rounds of subcloning, it was found that the murine monoclonal antibodies mab 29, mab101, and mab107 have strong binding activity to the antigen hGH-FRα. After preparing ascites fluid and purifying the antibodies, ELISA was performed. The results are shown in Table 1.

[0210] Table 1. Results of binding activity between mouse monoclonal antibody and antigen hGH-FRα

[0211] Example 4 Antibody Subtype Detection

[0212] In this embodiment, the SBA Clonotyping System-HRP subtype detection kit was used to detect the mouse monoclonal antibody subtype in the supernatant that specifically binds to FRα positive clones. The operation steps were performed according to the kit instructions.

[0213] The results are shown in Table 2. The heavy chain subtype of the murine monoclonal antibody was IgG1, and the light chain subtype was Kappa.

[0214] Table 2. Results of mouse monoclonal antibody light and heavy chain antibody subtype detection

[0215] Example 5: Species-Specific Binding Activity of Anti-Human FRα Mouse Monoclonal Antibody

[0216] In this embodiment, ELISA is used to detect the specific binding activity of the candidate antibody.

[0217] Specifically, human FRα (hGH-FRα), human FRβ (hFc-FRβ) protein, rat FRα (hFc-FRα / R), and cynomolgus monkey FRα (hFc-FRα / M) proteins were diluted to 1 μg / mL with coating buffer, and 100 μL of each protein was used to coat 96-well microplates and incubated overnight at 4°C. After blocking with skim milk, purified mab107 was diluted to 10 μg / mL, and three dilutions were performed using this as the initial concentration, with 100 μL added to each well in a 4-fold gradient. PBS was used as a blank control, and the plates were incubated at 37°C for 45 min. The buffer was discarded, and the plates were washed five times with 300 μL of PBST. 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:8000 with 5% skim milk was added to each well, and the plates were incubated at 37°C for 35 min. The secondary antibody was discarded, and the plates were washed three times with 300 μL of PBST and patted dry. Add 100 μL TMB of colorimetric reagent to each well and develop at room temperature for 5 min. Add 100 μL of stop solution to terminate the color development. OD 450 reading.

[0218] The results are shown in Figure 1. The results indicate that the murine monoclonal antibodies mab29, mab101, and mab107 cross-react with human FRα and cynomolgus monkey FRα, but have no binding activity with human FRβ and rat FRα.

[0219] Therefore, the monoclonal antibody of this invention helps to validate the efficacy and safety of antibodies in animal models, broadening the scope of antibody applications. Furthermore, since the physiological characteristics of cynomolgus monkeys are similar to those of humans, drug experiments conducted on cynomolgus monkey models may more accurately predict the efficacy and safety of drugs in human subjects, reducing the cost of drug development.

[0220] Example 6: Determination of the endocytic capacity of anti-human FRα mouse monoclonal antibody in OVCAR3 cells

[0221] In this embodiment, flow cytometry was used to detect the endocytic capacity of the candidate antibody.

[0222] Specifically, OVCAR3 cells (human ovarian cancer cells) in the logarithmic growth phase were digested and the concentration of the single-cell suspension was adjusted to 1×10⁻⁶ cells using 2% PBA (PBS containing 2% fetal bovine serum). 6 Add 1 mL of single-cell suspension to each 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.

[0223] Dilute the anti-human FRα mouse monoclonal antibody to 10 μg / mL with pre-cooled PBS. Add two tubes of each antibody strain, and add 200 μL of the diluted antibody to each tube. Gently pipette to mix the cells and incubate at 4°C and 37°C for 40 minutes each.

[0224] Simultaneously, blank control and positive serum polyclonal antibody control were performed.

[0225] Centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Wash once with 2% PBA, and add 200 μL of diluted 1 μg / mL FITC-labeled goat anti-mouse IgG Fc antibody to each tube. Gently pipette to mix the cells and incubate at 4°C in the dark on ice for 30 minutes. Wash twice with pre-chilled 2% PBA. Resuspend the cells in 300 μL PBS, gently pipette to mix, and transfer to flow cytometry tubes. In the dark, analyze using a flow cytometer.

[0226] By incubating purified mab107 mouse monoclonal antibodies at different temperatures and comparing the fluorescence shift intensity at different temperatures, it was concluded that mouse monoclonal antibodies with a larger shift ratio after incubation at 37℃ have strong endocytic ability.

[0227] The results are shown in Figure 2 and Table 3. The results indicate that after incubation at 4℃ and 37℃ for 40 minutes, the fluorescence shift ratio of mab107 was M2 / M1 = 8.0, indicating strong endocytic activity.

[0228] Table 3 Results of mouse monoclonal antibody endocytosis activity

[0229] Example 7: Obtaining the variable region of a hybridoma

[0230] Total mRNA was extracted from mab107 hybridoma using the TakaRa MiniBEST Universal RNA Extraction Kit (Takara, 9767). The variable region sequence of the murine antibody was obtained by PCR using the SMARTer RACE 5' / 3' Kit (Clontech, 634858), and a chimeric antibody named ch107 was constructed.

[0231] >Mab107 Heavy Chain Variable Region Nucleotide Sequence

[0232] >Mab107 Heavy Chain Variable Region Amino Acid Sequence

[0233] >Mab107 light chain variable region nucleotide sequence

[0234] >Mab107 light chain variable region amino acid sequence

[0235] Example 8: Humanization of anti-human FRα antibody mab107

[0236] In order to reduce the immunogenicity of the antibody and reduce the effect of human anti-mouse antibody (HAMA) produced after the antibody is introduced into the body, the mab107 antibody was humanized in this embodiment.

[0237] Specifically, the variable region sequence of the mab107 antibody in Example 7 was used to design a humanized sequence. The amino acid sequences of the heavy and light chains were encoded according to the Kabat coding system. Sequence homology was compared using NCBI Igblast (https: / / www.ncbi.nlm.nih.gov / igblast / ), and a highly homologous human antibody sequence was selected as the backbone. Mouse antibody CDRs were then transplanted, and some core amino acids in the backbone region underwent reversion mutations. Simultaneously, to avoid the formation of free thiol groups from cysteine ​​in the CDR region, the cysteine ​​(C) at position 52A of the heavy chain CDR2 region was changed to alanine (A).

[0238] The FR and CDR sequences in the heavy chain variable region of the mab107 antibody, as defined by Kabat, are shown in Table 4.

[0239] Table 4

[0240] The FR and CDR sequences in the light chain variable region of the mab107 antibody, as defined by Kabat, are shown in Table 5.

[0241] Table 5

[0242] The amino acid sequence of humanized mab107 is as follows:

[0243] The amino acid sequence of the heavy chain variable region of the humanized antibody hu107

[0244] In the amino acid sequence of the heavy chain variable region, the H-CDR2 sequence after point mutation is as follows:

[0245] The amino acid sequence of the light chain variable region of the humanized antibody hu107

[0246] The underlined part is the CDR area.

[0247] Example 9: Expression and purification of chimeric and humanized antibodies

[0248] The humanized antibody was sent to Sangon Biotech to synthesize the variable region sequence. The variable regions of the heavy and light chains of the humanized antibody and the heavy and light chains of the mouse monoclonal antibody were respectively constructed into the expression vector PTT5 (available from BioVector NTCC Inc.) containing the constant region of the human IgG1 / κ heavy and light chains, to obtain the heavy and light chain expression vectors of the humanized antibody and the chimeric antibody.

[0249] Plasmids were extracted using a commercial plasmid extraction kit. The density of Expi293F cells in logarithmic growth phase was adjusted to 1.0–2.0 × 10⁶ cells one day before transfection. 6 After culturing for 24 hours, transfection was performed with a total plasmid concentration of 1 μg / mL and a heavy chain plasmid: light chain plasmid: PEI ratio of 1:1:3.

[0250] After 7 days, the cells were centrifuged to collect the culture supernatant, and the antibody was purified using a Protein A affinity column.

[0251] Example 10 Humanized Antibody Binding Activity

[0252] ELISA plates were coated with FRα antigen at a concentration of 1 μg / mL and blocked with 5% skim milk. Chimeric antibody ch107 and humanized antibody hu107 were diluted 2-fold (starting from 20 μg) in 15 gradients, with 100 μL added to each well of the antigen-coated 96-well plate. After incubation at 37°C for 1 h, the plates were washed 5 times with PBST. HRP-labeled goat anti-human IgG secondary antibody (1:3000 dilution) was added, and the plates were incubated at 37°C for 45 min. After washing 5 times with PBST, the plates were developed with 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic buffer for 5 min, and the reaction was terminated with 2M H₂SO₄. The OD values ​​were read using a microplate reader. 450 The value was calculated using GraphPad Prism 8 software to fit a synchrograph. 50 value.

[0253] The results are shown in Figure 3 and Table 6. The results indicate that the humanized antibody hu107 exhibits strong binding activity to human FRα protein; the relative binding activity of hu107 compared to the chimeric antibody is 122.83%.

[0254] Table 6. Specific binding of hu107 to EC 50

[0255] Example 11: Analysis of Key Binding Sites between Humanized Antibodies and Antigens

[0256] The results of Example 5 showed that mab107 binds to human FRα but not to rat FRα. Therefore, through analysis of the FRα protein sequence and tertiary structure, the inventors selected different amino acid residues in the human and rat FRα sequences and nearby amino acid residues on the protein surface for alanine scanning mutation. A total of 17 amino acid sites were selected for point mutation on the FRα-mFc plasmid (see Figure 4), namely K52A, E55A, R58A, K62A, N69A, V78A, E91A, P94A, N131A, E140A, C146A, N161A, C169A, C175A, H179A, V187A, and N201A.

[0257] The mutated plasmid was transfected according to the antibody transfection and purification method in Example 9. The expression level in the expression supernatant was detected using goat anti-mouse IgG, and then the binding activity of the supernatant with the humanized antibody was detected.

[0258] The results showed that all 17 mutant proteins were expressed, but C175A expression was low, and no further experiments were conducted. Results of antibody binding activity analysis of the mutant protein supernatant revealed that E91A and C146A had a significant impact on the binding activity of hu107.

[0259] Therefore, this embodiment further purified the above two point mutations and successfully obtained the E91A mutant protein. The C146A mutation did not purify the protein. After coating the mutant protein with an ELISA plate, an ELISA binding assay was performed. The results are shown in Figure 5. The E91A mutation significantly reduced the binding of hu107 to FRα protein.

[0260] Example 12: Detection of the binding activity of humanized antibodies to target cells

[0261] OVCAR3 cells in good logarithmic growth phase were collected, digested with trypsin, and counted. The cell density was adjusted to 1×10⁻⁶. 6 1 mL of HBsAg / mL was added to each EP tube. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and wash twice with PBA. Add 10 μg / mL of hu107, ch107, and isotype control IgG to each tube, and incubate at 4℃ for 1 h. After incubation, wash twice with PBA (PBS + 2% FBS).

[0262] After washing, 100 μL of PE-labeled goat anti-human IgG secondary antibody diluted 1:400 with PBA was added to each group, and the mixture was pipetted and incubated at 4°C in the dark for 50 min. After incubation, the mixture was washed three times with PBA, resuspended and mixed with 300 μL of PBA, and transferred to flow cytometry tubes for later use.

[0263] The fluorescence signal intensity of PE was detected by flow cytometry, the mean fluorescence intensity (MFI) was calculated, and the binding strength of each antibody to the target cells was compared using GraphPad Prism 8 software.

[0264] The results are shown in Figure 6. The results indicate that the binding activity of the humanized antibody hu107 to human ovarian cancer OVCAR3 cells is comparable to that of the chimeric antibody ch107.

[0265] Example 13: Detection of the inhibitory activity of humanized antibodies on the proliferation of target cells

[0266] Using PrestoBlue TMCell viability was assessed using dyes. SW620, SW480, and OVCAR3 cells in logarithmic growth phase were digested and diluted to 1.0 × 10⁻⁶ cells. 5 Cell culture medium was prepared at a concentration of 100 μL / well in 96-well opaque blackbody medium and incubated overnight. After aspirating the medium, 100 μL of antibody diluted with analytical medium (ch107 and hu107) was added to each well, resulting in a final volume of 100 μL per well and a final antibody concentration of 800 nM. A two-fold serial dilution was performed for a total of 9 dilutions, with double replicates. The cell culture plates were incubated at 37°C with 5% CO2 for 5 days. After incubation, the medium in the 96-well plates was aspirated, and 100 μL of 10% Presto Blue was added to each well. TM Mix the staining solution thoroughly. Place the cell culture plate in a constant temperature incubator and continue culturing for 1.5-2.5 hours. After culturing, place the cell culture plate in a multi-functional microplate reader and measure the fluorescence intensity (FLu) value. Microplate reader parameters: excitation light: 560nm, absorption light: 590nm. Analyze the data using the microplate reader after reading.

[0267] The inhibitory activity of candidate antibodies on the proliferation of target cells was detected by the zirconia assay.

[0268] The results are shown in Figures 7A-C. The results showed that hu107 did not exhibit any proliferative inhibitory activity or tumor cell killing activity against SW620, SW480, or OVCAR3 cells at the highest concentration of 800 nM.

[0269] Example 14: Endocytotic activity of humanized antibodies in target cells

[0270] By measuring the expression of FRα on the surface of various tumor cells, SW620 cells with high FRα expression and SW480 and OVCAR3 cells with moderate FRα expression were selected for hu107 endocytosis activity detection.

[0271] Collect SW620, OVCAR3, and SW480 cells in the logarithmic growth phase, digest them, centrifuge at 1000 rpm for 5 minutes at 4°C, and discard the supernatant. Resuspend the cells in 2% PBA (PBS with 2% fetal bovine serum), count them, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Adjust the single-cell suspension concentration to 2 × 10⁻⁶ cells / mL with 2% PBA. 6 Add 1 mL of single-cell suspension to each 1.5 mL centrifuge tube. Centrifuge at 2500 rpm for 2.5 min and wash twice. Serve at 1 μg / 10⁻⁶. 6To calculate antibody levels in each cell, add 100 μL of 20 μg / mL hu107 to each cell tube and incubate at 4°C for 1 h. After incubation, wash twice with PBA. The negative control group is added to PBS. Divide the washed cells into two equal tubes, one incubated at 4°C and the other at 37°C for 3 h. After incubation, wash twice with PBA solution containing 1% sodium azide. Add 100 μL of goat anti-human IgG-PE diluted 1:400 with 1% sodium azide in each tube, mix well by pipetting, and incubate at 4°C in the dark for 50 min. Wash three times with PBA. Resuspend the cells in 300 μL of PBS, mix gently by pipetting, transfer to flow cytometry tubes, and analyze by flow cytometry. Detect fluorescence signals using a flow cytometer. The endocytosis rate is calculated using the following formula:

[0272] The results are shown in Figure 8. The results indicate that the endocytosis rate of the humanized antibody hu107 was 36.7% in SW620 cells, 20.5% in SW480 cells, and 13.1% in OVCAR3 cells.

[0273] Example 15: Sensitivity Detection of Small Molecule Chemicals

[0274] Different tumor cells exhibit varying degrees of sensitivity to small molecule toxins. This invention selected Dxd and MMAE as candidate small molecules for ADCs. Dxd is a topoisomerase I inhibitor that can induce double-strand DNA breaks, ultimately leading to apoptosis in cancer cells. MMAE can effectively inhibit mitosis by suppressing microtubule polymerization.

[0275] FRα-positive SW620, SW480, and OVCAR3 cells were selected as target cells to evaluate their sensitivity to the small molecule drugs MMAE and Dxd.

[0276] Human colon cancer cells SW620, SW480, and OVCAR3, which are in the logarithmic growth phase, were digested with trypsin, resuspended in their respective culture media, and adjusted to a density of 2 × 10⁻⁶ cells / mL. 4 / ml, seeded at 100μl / well in a 96-well microplate with a gap in the center, so that the number of cells per well is 2×10. 3 Add 200 μl of culture medium to each well of the surrounding cells. After overnight incubation at 37°C and 5% CO2, aspirate the supernatant from the cell culture plate. Use culture medium to serially dilute the linker-small molecule drug (Dxd / MMAE) in 2-fold increments starting at 400 nM, for a total of 9 concentration gradients. Add 100 μl of the diluted sample to each well. Continue incubation for 6 days for color development. Remove the cell plate and equilibrate at room temperature for 15-20 min. Add 100 μl of [unspecified medium]. Luminescent Cell Viability assay solution (purchased from Promega), shaken at 200 rpm for 2 min, protected from light for 18 min, and then read using an M5 microplate reader.

[0277] Result processing: Then, a four-parameter fitting plot was created with the logarithm of concentration on the x-axis and the lethality on the y-axis.

[0278] As shown in Figures 9A-C, at the same concentration, Dxd showed a higher killing rate against SW620, SW480, and OVCAR3 cells than MMAE; the IC50 of Dxd's killing activity against SW620, SW480, and OVCAR3 cells was [data missing]. 50 The NA values ​​are 0, 2.715 nM, and 4.534 nM. The IC50 values ​​for the killing activity of MMAE against SW620, SW480, and OVCAR3 are... 50 The values ​​are 6.237 nM, NA, and 22.88 nM (Table 7).

[0279] In summary, SW620, SW480, and OVCAR3 cells showed greater sensitivity to Dxd, and Dxd was subsequently selected as the small molecule toxin portion of the ADC.

[0280] Table 7 IC50 of small molecule drugs against tumor cells 50

[0281] Note: NA~0: Because the minimum concentration kill rate is still in the plateau phase, no result has been fitted.

[0282] Example 16 Preparation of Antibody-Drug Conjugates

[0283] The linker-drug used in this embodiment is -VASGFG-Dxd (08Dxd) or -VASGFG-Dxd (09Dxd), which was synthesized and prepared in accordance with patent WO2023098691.

[0284] Using a buffer exchange device (desalting column, ultrafiltration tube, etc.), replace HU107 with the target buffer (citrate buffer or histidine hydrochloride buffer, pH 6.0-6.5) and measure the antibody concentration. Maintain the antibody concentration at 5-20 mg / mL, mix with 0.04-0.4 mM TCEP reducing agent, and incubate at 10-37°C for 1-5 hours to allow the disulfide bonds between antibody chains to fully open. Subsequently, adjust the pH of the reduced antibody solution to 6.0 using phosphoric acid and lower the temperature to room temperature.

[0285] Dissolve the linker-small molecule drug in DMSO and calculate its concentration. Mix the linker-drug solution (08Dxd, 09Dxd) with the completely reduced antibody solution at a suitable drug / antibody molecule ratio (molar ratio > 10), maintaining a DMSO concentration not exceeding 10% (v / v), and hold for 0.5–2 hours until the reaction is complete. After filtration, purify the crude ADC product solution using a purification apparatus (desalting column, ultrafiltration tube, tangential flow system, etc.) to remove organic small molecule impurities and excess DMSO. Then, transfer the ADC product to a storage buffer.

[0286] Table 8 Connecting Substructures

[0287] Example 17 DAR Detection of Antibody-Drug Conjugate Products

[0288] The HIC-HPLC method was used for detection. Sample loading and elution were performed under the condition of pH 7.0 mobile phase, and the absorption peak area at the specified wavelength was detected.

[0289] The specific test methods are shown in Table 9, and the test results are shown in Table 10 and Figures 10A and 10B. The DAR of the antibody-drug conjugate product is between 7 and 8.

[0290] Table 9 HIC-HPLC Detection Methods

[0291] Table 10 Results of HIC-HPLC DAR Value Detection

[0292] Example 18 Binding activity of antibody-drug conjugates

[0293] In this embodiment, the binding activity of the antibody-drug conjugate to FRα protein was verified by protein ELISA. First, an FRα protein antigen plate with a concentration of 1 μg / ml was coated. Then, the initial concentrations of hu107, 107-08Dxd, and 107-09Dxd were adjusted to 125 nM and diluted 15 times at a 1:2 gradient. The OD450 values ​​were then measured.

[0294] As shown in Figure 11, the antibody-drug conjugate did not show a significant decrease in binding activity to the FRα protein compared to the naked antibody, and a certain dose-response relationship was observed.

[0295] ADC binding activity targeting FRα EC 50 As shown in Table 11, the EC of hu107 50 It is an EC of 0.424 nM and 107-08 Dxd. 50 It is an EC of 0.320nM and 107-09Dxd. 50The value is 0.428 nM, indicating that the antibody conjugation to a small molecule toxin did not significantly affect the antibody binding activity.

[0296] Table 11 ADC binding activity targeting FRα EC 50

[0297] Example 19 Flow cytometry detection of antibody-drug conjugate binding activity to target cells

[0298] The binding of hu107-08Dxd and hu107-09Dxd to FRα on the surface of SW620, SW480, and OVCAR3 cells was analyzed by flow cytometry.

[0299] The results are shown in Figures 12A, 12B, and 12C. hu107-08Dxd and hu107-09Dxd can bind to FRα on the surface of SW620, SW480, and OVCAR3 cells. Compared with the naked antibody, the binding activity of the antibody-drug conjugates did not show a significant decrease.

[0300] Example 20: Assay of the endocytic activity of antibody-drug conjugates

[0301] The binding activity and endocytosis of antibody-drug conjugates are crucial for ADCs to exert their tumor-killing effect. Hu107 has certain endocytic activity in multiple target cells. Whether the ADC structure constructed with Hu107 as the antibody moiety will affect the binding of the antibody moiety to the antigen and the endocytosis of the ADC needs further verification.

[0302] The endocytic activity of hu107-08Dxd and hu107-09Dxd in FRα-highly expressed SW620, SW480, and OVCAR3 cells was preliminarily verified by flow cytometry according to the method in Example 16.

[0303] The endocytic activity of hu107 was detected in SW620 cells with high FRα expression, SW480 cells with moderate FRα expression, and OVCAR3 cells.

[0304] As shown in Figure 13, hu107-08Dxd and hu107-09Dxd both exhibited some endocytosis in SW620, SW480, and OVCAR3 cells. Compared with hu107, the endocytosis rate of ADC was improved to some extent.

[0305] Example 21 Cell-killing activity of antibody-drug conjugates

[0306] The cytotoxic activity of hu107-08Dxd and hu107-09Dxd against target cells was detected according to the cell killing activity method in Example 17.

[0307] As shown in Figures 14A, 14B, and 14C and Table 12, 107-08Dxd exhibits specific cytotoxic activity against FRα-positive SW620, SW480, and OVCAR3 cells, showing a certain dose-response relationship; the cytotoxic activity IC50 of 107-08Dxd against SW620, SW480, and OVCAR3 cells is [not specified in the original text]. 50 The values ​​are 4.39 nM, 6.22 nM, and 24.98 nM, respectively.

[0308] Table 12. Killing activity IC50 of hu107-08Dxd against target cells SW620, SW480, and OVCAR3 50

[0309] As shown in Figures 15A, 15B, and 15C and Table 13, hu107-09Dxd exhibits specific killing activity against FRα-positive SW620, SW480, and OVCAR3 cells, with a certain dose-response relationship; the IC50 value of 107-09Dxd against SW620, SW480, and OVCAR3 cells is [not specified]. 50 The values ​​are 3.53 nM, 12.80 nM, and 36.71 nM, respectively.

[0310] Table 13. IC50 of 107-09Dxd on the killing activity of SW620, SW480, and OVCAR37 tumor cells 50

[0311] In summary, hu107-08Dxd and hu107-09Dxd can specifically kill FRα-expressing tumor cells.

[0312] Example 22: Efficacy of antibody-drug conjugate hu107-09Dxd in a xenograft model

[0313] Female SPF-grade BALB / c nude mice, 3-4 weeks old, were selected and acclimatized for 3-4 days before tumor implantation. Logarithmic growth phase SW620 cells were digested, washed twice with serum-free 1640 medium, and then implanted into the tumor. 0.2 ml of the medium was subcutaneously injected into the right axilla of each mouse, for a total of 5 × 10⁶ cells. 6 1 cell / animal. One week after inoculation, the tumor grew to 150±50 mm. 3 The subjects were randomly assigned to blocks with 5 subjects per group. The blank model group served as the PBS control.

[0314] The hu107-09Dxd mice were administered at two dose groups: 2 mg / kg and 6 mg / kg, via intraperitoneal injection. The administration frequency was once a week for four consecutive weeks, with measurements taken twice a week. Tumor volume and mouse weight were recorded before each administration.

[0315] Tumor volume calculation: V (mm) 3 ) = (W 2 x L) / 2, where W is the short diameter of the tumor near the midline, and L is the long diameter of the tumor near the midline. When the tumor volume is greater than 2000 mm... 3 Mice were euthanized by cervical dislocation when their body weight decreased by more than 20%, or when interference with important physiological functions or tumor ulceration and necrosis were observed.

[0316] As shown in Figure 16, in the SW620 mouse subcutaneous xenograft model, hu107-09Dxd significantly inhibited tumor growth at doses of 2 mg / kg and 6 mg / kg, demonstrating a dose-response effect. No significant decrease in body weight was observed in any group of mice during the administration period.

[0317] Example 23: Efficacy of antibody-drug conjugate hu107-08Dxd in a xenograft model

[0318] Female SPF-grade BALB / c nude mice, 3-4 weeks old, were selected and acclimatized for 3-4 days before tumor implantation. Logarithmic growth phase ovarian cancer cells (CAOV3 cells) were digested, washed twice with serum-free 1640 medium, and diluted to 5 × 10⁻⁶. 7 Mix 0.2 ml of the mixture with an equal volume of matrix gel and subcutaneously inject 0.2 ml into the right axilla of mice, for a total of 5 × 10⁶ ml. 6 1 cell / animal. One week after inoculation, the tumor grew to 150±50 mm. 3 The subjects were randomly assigned to blocks with 5 subjects per group. The blank model group served as the PBS control.

[0319] The mice were administered hu107-08Dxd at three doses: 1 mg / kg, 3 mg / kg, and 10 mg / kg, via intraperitoneal injection. The frequency of administration was once a week for four consecutive weeks, with tumor volume and mouse weight recorded twice weekly before each administration. Observation continued after the administration period ended.

[0320] Tumor volume calculation: V (mm) 3 ) = (W 2 x L) / 2, where W is the short diameter of the tumor near the midline, and L is the long diameter of the tumor near the midline. When the tumor volume is greater than 2000 mm... 3 Mice were euthanized by cervical dislocation when their body weight decreased by more than 20%, or when interference with important physiological functions or tumor ulceration and necrosis were observed.

[0321] As shown in Figure 17, hu107-08Dxd significantly inhibited the growth of subcutaneous xenografts in CAOV3 nude mice. At the end of administration, 1 / 5, 3 / 5, and 5 / 5 of the tumors in the 1 mg / kg, 3 mg / kg, and 10 mg / kg groups, respectively, showed complete regression. Continued observation up to day 67 showed that 1 / 5, 2 / 5, and 4 / 5 of the tumors in each dose group still showed complete regression. No significant weight loss or other symptoms were observed in any of the mice, indicating good tolerability.

[0322] 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.

Claims

1. An anti-FRα antibody or antigen-binding fragment thereof, characterized in that, the anti-FRα antibody or antigen-binding fragment thereof has three complementarity determining regions (CDRs) of a heavy chain variable region (HCDRs) and three complementarity determining regions (CDRs) of a light chain variable region (LCDRs) selected from the group consisting of: (1) a HCDR1 of SEQ ID NO: 6, a HCDR2 of SEQ ID NO: 8, a HCDR3 of SEQ ID NO: 10, a LCDR1 of SEQ ID NO: 13, a LCDR2 of SEQ ID NO: 15, a LCDR3 of SEQ ID NO: 17; (2) a HCDR1 of SEQ ID NO: 6, a HCDR2 of SEQ ID NO: 20, a HCDR3 of SEQ ID NO: 10, a LCDR1 of SEQ ID NO: 13, a LCDR2 of SEQ ID NO: 15, a LCDR3 of SEQ ID NO:

17.

2. The anti-FRα antibody or antigen-binding fragment thereof of claim 1, wherein, the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 2 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 4; and / or the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 19 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO:

21.

3. An antibody drug conjugate, characterized in that, the antibody drug conjugate comprises: (a) an antibody moiety comprising the anti-FRα antibody or antigen-binding fragment thereof of claim 1; and (b) a conjugating moiety conjugated to the anti-FRα antibody or antigen-binding fragment thereof, the conjugating moiety being selected from the group consisting of a detectable label, a cytotoxic drug, a toxin, or a combination thereof.

4. The antibody drug conjugate of claim 3, wherein, the antibody drug conjugate has a formula of: mAb-(X-Y)n; wherein, mAb is the anti-FRα antibody or antigen-binding fragment thereof; X is a linker; Y is a conjugating moiety, the conjugating moiety being a cytotoxic drug; n is a positive integer of < 8; the conjugating moiety is conjugated to the anti-FRα antibody or antigen-binding fragment thereof via the linker.

5. A recombinant protein, characterized in that, the recombinant protein has: (i) the antibody or antigen-binding fragment thereof of claim 1; and (ii) optionally a tag sequence to assist expression and / or purification.

6. A nucleotide molecule, characterized in that, the nucleotide molecule encodes the anti-FRα antibody or antigen-binding fragment thereof of claim 1 7. A vector, characterized in that, the vector contains the nucleotide molecule of claim 6.

8. A host cell, characterized in that, the host cell contains the vector of claim 7, or has the nucleotide molecule of claim 6 integrated into its genome, or expresses the anti-FRα antibody or antigen-binding fragment thereof of claim 1.

9. A pharmaceutical composition, characterized by, the pharmaceutical composition comprises: (i) the anti-FRα antibody or antigen-binding fragment thereof of claim 1, the antibody drug conjugate of claim 2, or the recombinant protein of claim 3; and (ii) a pharmaceutically acceptable carrier.

10. A method of detecting FRa protein in a sample, comprising contacting the sample with an antibody of claim 1 and detecting the formation of an antibody-antigen complex. 5 the method comprises the step of: (1) contacting a sample with an anti-FRα antibody or antigen-binding fragment thereof according to claim 1 ; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of FRα protein in the sample.

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