Anti-FRα antibody or antigen-binding fragment, antibody-drug conjugate and its application

Abstract

The present invention provides an anti-FRα antibody or antigen-binding fragment, an antibody-drug conjugate and its application. Specifically, an anti-FRα antibody or antigen-binding fragment thereof is provided, which can effectively bind to human FRα. The present invention also provides an antibody-drug conjugate containing the antibody or antigen-binding fragment, which can be used to prepare a drug for treating tumors expressing or overexpressing FRα, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an anti-FRα antibody or antigen-binding fragment, an antibody-drug conjugate and applications thereof. Background Art

[0002] FRα expression is restricted in normal tissues, with only extremely low expression in non-malignant tissues such as the choroid plexus, thyroid, 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 subtypes of non-small cell lung cancer, mesothelioma, and some triple-negative breast cancers.

[0003] FRα's ability to internalize large molecules, coupled with its high expression on the surface of certain tumor cells and limited expression in normal tissues, has attracted the development of antibody-drug conjugates (ADCs) targeting this target. ADCs consist of a monoclonal antibody, a bioactive small molecule toxin, and a linker. The antibody portion specifically recognizes the target antigen, which is highly expressed on the surface of target cells. The ADC-antigen complex then enters the tumor cell through endocytosis, releasing the bioactive small molecule toxin within the acidic environment of the lysosome. The toxin molecules primarily disrupt DNA chains, inhibit tubulin, and inhibit DNA topoisomerase or RNA polymerase, ultimately leading to tumor cell death.

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

[0005] The small molecule toxins coupled to several FRα-targeted ADCs currently under development are mainly tubulin inhibitors, with a DAR range of 2 to 4. However, their efficacy is easily reduced during use due to the heterogeneity of tumor tissues and the drug resistance that is easily generated during treatment.

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

[0007] The present invention provides a differentiated anti-FRα antibody or antigen-binding fragment, an antibody-drug conjugate and applications thereof.

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

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

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

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

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

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

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

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

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

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

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

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

[0020] LCDR3 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 a Fab fragment, a F(ab')2 fragment, and a Fv fragment.

[0023] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-FRα antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 example, the heavy chain of the antibody or antigen-binding fragment thereof further includes a heavy chain constant region; the light chain of the antibody or antigen-binding fragment thereof 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 connecting peptide between the heavy chain variable region and the light chain variable region.

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

[0035] (a) an antibody portion, said antibody portion comprising the antibody or antigen-binding fragment thereof according to the first aspect of the invention; and

[0036] (b) a conjugated moiety conjugated to the antibody or antigen-binding fragment thereof, wherein the conjugated moiety is selected from the group consisting of a detectable label, a cytotoxic drug, a toxin, or a combination thereof.

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

[0038] in,

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

[0040] X is a linker;

[0041] Y is a coupling moiety, and the coupling moiety is a cytotoxic drug;

[0042] n is a positive integer ≤ 8;

[0043] The coupling moiety is coupled to the anti-FRα antibody or the antigen-binding fragment thereof 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 antigen-binding fragment thereof is coupled to the detectable label via a chemical bond or a linker.

[0046] In another preferred embodiment, the detectable marker is selected from the following group: fluorescent or luminescent markers, biotin, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, gold nanoparticles / nanorods, nanomagnetic particles, or any form of nanoparticles.

[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 following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors (such as Dxd), vinca alkaloids, or a combination thereof.

[0050] In another preferred embodiment, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansin, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxybenzoate, daptomycin, acetaminophen ... anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, a Sapaonaria officinalis inhibitor, a glucocorticoid, or a combination thereof.

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

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

[0053] (ii) Optionally, a tag sequence to facilitate expression and / or purification.

[0054] In another preferred embodiment, the tag includes an Fc tag, a FLAG tag, a 6His tag, 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 multimer.

[0057] In a fourth aspect of the present invention, a nucleotide molecule is provided, wherein the nucleotide molecule encodes the anti-FRα antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0058] In the fifth aspect of the present invention, a vector is provided, wherein the vector contains the nucleotide molecule described in the fourth aspect of the present invention.

[0059] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

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

[0061] In the sixth aspect of the present invention, a host cell is provided, which contains the vector described in the fifth aspect of the present invention, or has the nucleotide molecule described in the fourth aspect of the present invention integrated into its genome, or expresses the anti-FRα antibody or antigen-binding fragment thereof described in the first aspect of the present 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 a chimeric antigen receptor is expressed on its surface.

[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 present invention, a pharmaceutical composition is provided, comprising:

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

[0072] (ii) a pharmaceutically acceptable carrier.

[0073] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

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

[0075] In an eighth aspect of the present invention, there is provided a use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the antibody-drug conjugate according to the second aspect of the present invention, or the recombinant protein according to the third aspect of the present invention for preparing a medicament, reagent, test plate, or kit;

[0076] The reagent, detection plate or kit is used to: detect FRα protein in a sample;

[0077] The medicament is used for treating or preventing tumors expressing or overexpressing 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 a combination thereof.

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

[0080] (a) culturing the host cell according to the sixth aspect of the present invention under expression conditions to express the anti-FRα antibody or antigen-binding fragment thereof;

[0081] (b) isolating and purifying the anti-FRα antibody or antigen-binding fragment thereof 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) contacting a sample with the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0084] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the 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 comprises step (3) analyzing the affinity between the antibody and the antigen.

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

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

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

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

[0093] (1) a first container containing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and / or

[0094] (2) a second container containing a second antibody against the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and / or

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

[0096] or,

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

[0098] In another preferred embodiment, the antibody in the first container is detectably labeled.

[0099] In another preferred embodiment, the antibody in the second container is detectably labeled.

[0100] In the thirteenth aspect of the present invention, a method for treating a disease associated with FRα is provided, comprising the steps of administering to a subject in need of treatment a therapeutically effective amount of the anti-FRα antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the host cell described in the sixth aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention.

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

[0104] In a fourteenth aspect of the present 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 that specifically binds to FRα, and the scFv has three complementarity determining regions CDR (HCDR) of the heavy chain variable region and three complementarity determining regions CDR (LCDR) of the light chain variable region selected from the following group:

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

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

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

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

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

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

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

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

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

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

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

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

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

[0118] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 The results of species-specific binding activity assays for mab29, mab101, and mab107 are shown.

[0120] Figure 2A 、 Figure 2B and Figure 2C The comparison of the OVCAR3 cell endocytosis results of mab29, mab101, and mab107 antibodies under different temperature conditions is shown respectively (M1: fluorescence shift of the primary antibody incubated at 37°C; M2: fluorescence shift of the primary antibody incubated at 4°C).

[0121] Figure 3 The results of the binding activity of humanized antibodies to human FRα protein are shown.

[0122] Figure 4 The selection of mutation sites of FRα protein is shown.

[0123] Figure 5 The results of the binding activity of humanized antibodies to mutated FRα proteins are shown.

[0124] Figure 6 The results of the humanized antibody binding activity test to target cells are shown.

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

[0126] Figure 8 The endocytic activity of the humanized antibody in target cells was demonstrated.

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

[0128] Figure 10A and 10B The HIC-HPLC DAR value detection results of hu107-08Dxd and hu107-09Dxd are shown respectively.

[0129] Figure 11 The binding activity of ADC targeting FRα was shown.

[0130] Figure 12A 、 Figure 12B and Figure 12C The binding activity of antibody-drug conjugates to FRα on the surface of different target cells is shown respectively: (A) ADC binding to FRα on the surface of SW620 cells; (B) ADC binding to FRα on the surface of SW480 cells; (C) ADC binding to FRα on the surface of OVCAR3 cells.

[0131] Figure 13 The internalization rate of antibody-drug conjugates in SW620, SW480, and OVCAR3 cells is shown.

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

[0133] Figure 15A 、 15B and 15C respectively showed the cytotoxic activity of hu107-09Dxd against SW620, SW480, and OVCAR3 tumor cells.

[0134] Figure 16 The results showed that hu107-09Dxd has therapeutic efficacy in SW620 nude mouse xenograft tumor model.

[0135] Figure 17 The results showed that hu107-08Dxd has therapeutic efficacy in CAOV3 nude mouse xenograft tumor model. DETAILED DESCRIPTION

[0136] The inventors conducted extensive and in-depth research and, after extensive screening, obtained an anti-FRα antibody. Experimental results demonstrated that the anti-FRα antibody and its antibody-drug conjugate of the present invention possess high affinity and excellent biological activity. Furthermore, the murine monoclonal antibody of the present invention cross-reacts with human FRα and cynomolgus monkey FRα. Compared to anti-FRα antibodies, the anti-FRα antibody-drug conjugate exhibits increased internalization rates and superior tumor cell killing activity, significantly inhibiting tumor growth. This work was completed on this basis.

[0137] the term

[0138] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0139] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. 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 "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[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 reaction), ie, has a reasonable benefit / risk ratio.

[0142] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is tolerated by humans and / or animals. Those skilled in the art will appreciate that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and combination therapy with other drugs.

[0143] FRα

[0144] FRα (folate receptor α) is a glycosylphosphatidylinositol-anchored cell membrane glycoprotein with a relative molecular weight of 38,000–40,000, encoded by FOLR1. It has a high affinity for reduced folates (such as 5-methyltetrahydrofolate and tetrahydrofolate) and folic acid, but FRα provides less efficient folate uptake than other folate transporters. It is generally believed that FRα transports folate via a non-classical lipid raft-mediated endocytosis pathway, i.e., endocytosis, which does not involve clathrin-coated pits but rather is associated with caveolae.

[0145] The binding of folate 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 the individual vesicles combine to form early endosomes, which undergo acidification and subsequently fuse with lysosomes to release folate for one-carbon metabolic reactions. FRα not only transports folate but also mediates the entry of antibodies and folate-conjugated substances into cells through endocytosis.

[0146] FRα expression is restricted in normal tissues, with only extremely low expression in non-malignant tissues such as the choroid plexus, thyroid, 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 subtypes of non-small cell lung cancer, mesothelioma, and some triple-negative breast cancers.

[0147] Multiple studies have demonstrated that FRα regulates cell growth and signal transduction during tumor cell proliferation and invasion. Folic acid binding to FRα can activate the JAK-STAT signaling pathway, mediated by the GP130 co-receptor, leading to sustained activation of signal transducer and activator of transcription factor 3 (SAT3). This activated SAT3 resides in the cell nucleus, continuously activating target genes and promoting tumor cell proliferation. In ovarian cancer cell lines, FRα knockout reduces tumor cell division, inhibits anchorage-independent growth, and reduces cancer cell adhesion. This effect may be related to downregulating E-cadherin expression.

[0148] In addition, 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 ERK1 / 2, c-Fos, and c-Jun, key factors in the ERK signaling pathway. In vitro, downregulating FRα expression can inhibit cell proliferation, promote cell apoptosis, induce cell cycle arrest at the G0 / G1 phase, and reduce the expression of p-ERK1 / 2, pc-Fos, and pc-Jun proteins.

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

[0150] Antibody

[0151] In the present invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. One end of each light chain has a variable region (VL) and the other end has a constant region. The light chain constant region includes a domain CL; the constant region of the light chain pairs with the CH1 domain of the heavy chain constant region, and the variable region of the light chain pairs with the variable region of the heavy chain. Constant regions are not directly involved in antibody-antigen binding, but they exhibit various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 subtypes; 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 an inter-polypeptide disulfide bond formed between the hinge region.

[0152] As used herein, the terms "Fab" and "Fc" refer to the fact that papain can cleave antibodies into two identical Fab fragments and one Fc fragment. The Fab fragment is composed of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of the light chain. The Fc fragment (Fc) is composed of the CH2 and CH3 domains of the antibody. The Fc fragment has no antigen-binding activity and is the site of interaction between the antibody and effector molecules or cells.

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

[0154] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ in sequence, which contributes to the binding and specificity of each particular antibody for its specific antigen. However, this variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments within the heavy and light chain variable regions, known as the complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are known as the framework regions (FRs). The variable regions of native heavy and light chains each contain four FRs, which generally have a β-pleated sheet configuration and are connected by three CDRs that form a connecting loop, which in some cases may form a partial β-pleated sheet structure. The CDRs in each chain are closely aligned by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).

[0155] As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between the CDRs, that is, it refers to those portions of the light and heavy chain variable regions of immunoglobulins that are relatively conserved between different immunoglobulins in a single species. The light and heavy chains of immunoglobulins each have four FRs, referred to as 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 be referred to as (L-FR1)-(L-CDR1)-(L-FR2)-(L-CDR2)-(L-FR3)-(L-CDR3)-(L-FR4), and the heavy chain variable domain can 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, i.e., the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0156] Knowing the amino acid sequence of the CDRs, one 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 framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

[0158] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the light and heavy chain domains to fold into an exchange dual variable region immunoglobulin. In the present invention, preferred linkers are Linker1 and Linker2, wherein Linker1 connects the VH and VL of a single-chain antibody (scFv), and Linker2 is used to connect the scFv to the heavy chain of another antibody.

[0159] Examples of suitable linkers include a single glycine (Gly), or serine (Ser) residue. The identity and sequence of the amino acid residues in the linker may vary depending on the type of secondary structural element desired to be achieved in the linker.

[0160] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced with amino acids having similar or similar properties compared to the amino acid sequence of the bispecific antibody of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0161] Table A

[0162]

[0163] In the present invention, the terms "anti", "binding", and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. -7 M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The equilibrium dissociation constant (KD) of M or less is combined with the antigen. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, surface plasmon resonance (SPR) is used to measure the binding affinity of the antibody to the antigen in a BIACORE instrument or the relative affinity of the antibody to the antigen is measured using ELISA.

[0164] In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitope of the present invention is a region of an antigen that is bound by an antibody.

[0165] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0166] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0167] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0168] Pharmaceutical compositions and applications

[0169] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Generally, 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 about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal injection), intracranial injection, or intracavitary injection. In the present invention, the term "pharmaceutical composition" refers to the bispecific antibody of the present invention that can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to more stably exert therapeutic effects. These preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, while also protecting the protein's multiple functional groups from degradation (including but not limited to aggregation, deamination or oxidation). The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned bispecific antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the bispecific antibody of the present invention can also be used in conjunction with other therapeutic agents.

[0170] When using the pharmaceutical composition, a safe and effective amount of the bispecific antibody or immunoconjugate thereof is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight and, in most cases, does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0171] Antibody-drug conjugates (ADCs)

[0172] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0173] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0174] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0175] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0176] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker positioned between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers typically readily degrade in the intracellular environment, for example, at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. 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 a pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug when the antibody is hydrolyzed by proteases.

[0177] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (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, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0178] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0179] 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, and the like. In the present invention, drug-linkers can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue reacts with a reactive portion of a linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0180] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached 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), phosphines (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 Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0181] The present invention also provides a method for preparing an ADC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0182] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0183] In some embodiments, the antibody drug conjugate ADC has the following molecular formula:

[0184]

[0185] Ab is an antibody.

[0186] LU is the connector;

[0187] D is for medicine;

[0188] And the subscript p is a value selected from 1 to 8.

[0189] Detection Uses and Kits

[0190] The antibodies of the present invention can be used in detection applications, for example, for detecting a sample to provide diagnostic information.

[0191] In the present invention, the samples used include cells, tissue samples and biopsy specimens. The term "biopsy" as used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention can include, for example, tissue samples prepared by endoscopic methods or puncture or needle biopsy of an organ.

[0192] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0193] 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 comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0194] application

[0195] The present invention provides uses of the antibodies of the present invention, such as for preparing diagnostic preparations or preparing drugs for preventing and / or treating FRα-related diseases, including tumors.

[0196] In a preferred embodiment, the tumor is a tumor expressing or overexpressing FRα. In a 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 a combination thereof.

[0197] The main advantages of the present invention include:

[0198] (a) The anti-FRα antibody of the present invention has highly specific binding activity to human FRα and has strong endocytosis activity after binding to FRα on the surface of tumor cells;

[0199] (b) The anti-FRα antibody-drug conjugate of the present invention has good anti-tumor effects in vitro and in vivo and has good clinical application prospects.

[0200] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0201] Example 1 Immunogen Preparation

[0202] In this example, the genes encoding the extracellular segment of human folate receptor α (FRα) W28-S257 (P15328, Uniprot) and mouse IgG1 Fc were synthesized by Shanghai Boshang Biotechnology and cloned into the pTT5 eukaryotic expression vector (available from BioVectorNTCC Inc.), named pTT5-FRα-mFc; the nucleotide sequence encoding the extracellular segment of human folate receptor α was cloned into the pSGHV0 vector (GenBank: AF285183.1) for fusion expression with hGH, named pSGHV0-hGH-His-FRα.

[0203] Take 100 mL of Expi293-F cells in the logarithmic growth phase and adjust the cell volume to 90 mL 24 hours before transfection, and adjust the cell density to 1.5×10 6 After overnight culture on a cell shaker, the cell density was adjusted to 3×10 6 pTT5-FRα-mFc plasmid and pSGHV0-hGH-His-FRα plasmid were mixed with PEI at a ratio of 3:1 and then transfected into Expi293-F cells. 5-7 days after transfection, FRα-mFc protein was purified using Protein A, and hGH-FRα protein was purified using a Ni column.

[0204] In this example, the same method was used to express and purify hFc-FRβ protein, rat FRα (hFc-FRα / R), and cynomolgus monkey FRα (hFc-FRα / M).

[0205] Example 2 Animal Immunization

[0206] Balb / c mice used in the experiment were purchased from Shanghai Xipu-Bikai Laboratory. All mice used for immunization were 4-6 weeks old, female, standardized, disease-free, and healthy purebred mice.

[0207] Ten Balb / c mice were divided into two groups, each immunized with 5 mice each of FRα-mFc and hGH-FRα. On day 0, 50 μg of antigen, FRα-mFc or hGH-FRα, was mixed with 1 mg / mL aluminum adjuvant at a 1:1 ratio and injected subcutaneously into the back and soles of the feet. Boosters of 20 μg of the immunogen were administered to each mouse using the same method on days 14, 28, and 42. Three days before fusion, a booster of 20 μg of the immunogen was administered via the tail vein. On the day of fusion, eye bleeding was performed and spleens were removed. Single-cell suspensions were prepared and electrofused with SP20 cells (mouse myeloma cells) to generate hybridomas for plating.

[0208] Example 3 Hybridoma Screening

[0209] In this example, after the hybridomas were statically cultured for 14 days, 100 μL of supernatant was collected from each well and added to a 96-well ELISA plate pre-coated with hGH-FRα protein. Positive and negative control wells were set up. The positive control was serum from the eyeballs of fused mice diluted 1000 times, and the negative control was serum from the eyeballs of non-immunized mice diluted 1000 times. The results were detected by conventional ELISA. 450 The supernatant with a reading greater than or equal to half of the positive control reading was considered a positive clone. The selected final positive clone was subcloned using the limiting dilution method, and the final positive cells screened were used for subsequent experiments.

[0210] After three rounds of subcloning, it was found that mouse monoclonal antibodies mab 29, mab101, and mab107 had strong binding activity with the antigen hGH-FRα. Antibodies were prepared from ascites and purified for ELISA testing. The results are shown in Table 1.

[0211] Table 1 Results of the binding activity of mouse monoclonal antibodies to antigen hGH-FRα

[0212] Antibody No. OD450 mab29 1.6112 mab101 2.030 mab107 1.880

[0213] Example 4 Antibody subtype detection

[0214] In this example, the SBA Clonotyping System-HRP subtype detection kit was used to detect the subtypes of mouse monoclonal antibodies in the supernatant of clones that specifically bind to FRα, and the operation steps were carried out according to the kit instructions.

[0215] The results are shown in Table 2. The heavy chain subtype of the mouse monoclonal antibodies was IgG1, and the light chain subtype was Kappa.

[0216] Table 2 Results of mouse monoclonal antibody light and heavy chain antibody subtype detection

[0217] Antibody No. Heavy chain subtype Light chain subtype mab29 IgG1 IgGκ mab101 IgG1 IgGκ mab107 IgG1 IgGκ

[0218] Example 5 Species-Specific Binding Activity of Anti-Human FRα Mouse Monoclonal Antibodies

[0219] In this example, Elisa was used to detect the specific binding activity of the candidate antibodies.

[0220] Specifically, human FRα (hGH-FRα), human FRβ (hFc-FRβ), rat FRα (hFc-FRα / R), and cynomolgus monkey FRα (hFc-FRα / M) proteins were diluted to 1 μg / mL in coating buffer, and 100 μL was applied to each well of a 96-well microtiter plate overnight at 4°C. After blocking with skim milk, purified mab107 was diluted to 10 μg / mL. This initial concentration was used as the starting concentration, and three 4-fold dilutions were added to each well. PBS was used as a blank control, and the plate was incubated at 37°C for 45 minutes. The plate was then discarded, washed five times with 300 μL of PBST, and 100 μL of HRP-conjugated goat anti-mouse IgG secondary antibody diluted 1:8000 in 5% skim milk was added to each well. The plate was incubated at 37°C for 35 minutes. The secondary antibody was discarded, and each well was washed three times with 300 μL of PBST and patted dry. 100 μL of TMB color development solution was added to each well and the color was developed at room temperature for 5 min. 100 μL of stop solution was added to stop the color development. 450 reading.

[0221] The results are as follows Figure 1 As shown, the results showed that the mouse monoclonal antibodies mab29, mab101, and mab107 had cross-reactivity with human FRα and cynomolgus monkey FRα, but had no binding activity with human FRβ and rat FRα.

[0222] Therefore, the monoclonal antibodies of the present invention help verify the efficacy and safety of antibodies in animal models, broadening the scope of antibody applications. Furthermore, because the physiological characteristics of cynomolgus macaques are similar to those of humans, drug trials conducted in cynomolgus macaque models can more accurately predict the efficacy and safety of drugs in human subjects, thereby reducing the cost of drug development.

[0223] Example 6 Determination of the endocytic ability of anti-human FRα mouse monoclonal antibody in OVCAR3 cells

[0224] In this example, flow cytometry was used to detect the endocytosis ability of candidate antibodies.

[0225] Specifically, OVCAR3 (human ovarian cancer cells) in the logarithmic growth phase were taken and digested, and the concentration of the single cell suspension was adjusted to 1×10 6 1 mL of single-cell suspension was added to each 1.5 mL centrifuge tube, and the tubes were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded.

[0226] Dilute anti-human FRα mouse monoclonal antibody to 10 μg / mL with pre-cooled PBS. Add two tubes of each antibody, add 200 μL of diluted antibody to each tube, gently pipette to mix the cells, and incubate at 4°C and 37°C for 40 minutes each.

[0227] At the same time, blank control and positive serum multi-antibody control were performed.

[0228] 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 on ice at 4°C in the dark for 30 minutes. Wash twice with pre-chilled 2% PBA. Resuspend the cells in 300 μL of PBS and gently pipette to mix. Place in a flow cytometer, protect from light, and analyze using a flow cytometer.

[0229] The purified mab107 mouse monoclonal antibody was screened by incubation at different temperatures, and the fluorescence shift intensity at different temperatures was compared. It was believed that the mouse monoclonal antibody with a larger shift ratio after incubation at 37°C had a strong endocytic ability.

[0230] The results are shown in Figure 2 and Table 3. The results showed that after incubation at 4°C and 37°C for 40 minutes, the ratio of fluorescence shift of mab107 was M2 / M1 = 8.0, indicating strong endocytic activity.

[0231] Table 3 Results of mouse monoclonal antibody endocytosis activity

[0232] Antibody No. M2 / M1 mab29 2.9 mab101 1.4 mab107 8.0

[0233] Example 7 Acquisition of Hybridoma Variable Regions

[0234] The total mRNA of mab107 hybridoma was extracted using the RNA extraction kit TakaRa MiniBEST Universal RNA Extraction Kit (Takara, 9767), and the mouse antibody variable region sequence was obtained by PCR using the SMARTer RACE 5' / 3' Kit (Clontech, 634858). A chimeric antibody was constructed and named ch107.

[0235] >mab107 heavy chain variable region nucleotide sequence

[0236] GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTAGTGAAGACTGGGGCTTCAGTGAGGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGTTACT ACATGCACTGGGTCAGGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTAGTTGTTATAATGGTGCTACTTACTACAACCAAAACTTCAAGGGCAAGGCCACATTTACTGTAGACACATCCTCCAGCACAGCCTACATGCAGTTCAACAGCCTGACATCTGAAGACTCTGCGGTCTATTACTGTGCAAGAGTGGGGGACGGTAGTAGGTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO:1)

[0237] >mab107 heavy chain variable region amino acid sequence

[0238] EVQLQQSGPELVKTGASVRISCKASGYSFT GYYMH WVRQSHGKSLEWI G YISCYNGATYYNQNFKG KATFTVDTSSSTAYMQFNSLTSEDSAVYYCAR V GDGSRSWFAY WGQGTLVTVSA(SEQ ID NO:2)

[0239] >mab107 light chain variable region nucleotide sequence

[0240] GAAATTGTGCTCACTCAGTCTCCAGCCATCACAGCTGCATCTCTGGGGCAAAAGGTCACCATCACCTGCAGTGCCAGTTCAAGTGTAACTTACATGCACTGGTACCAGCAGCAGTCTGGCACCTCCCCCAAACCATGGATTTATGAAATATCCAAACTGGCTTCTGGAGTCCCAACTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCATTTATTACTGCCAGCAGTGGAATTTTCCTCTTATCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:3)

[0241] >mab107 light chain variable region amino acid sequence

[0242] EIVLTQSPAITAASLGQKVTITC SASSSVTYMH WYQQQSGTSPKPWIY EI SKLAS GVPTRFSGSGSGTSYSLTISSMEAEDAAIYYC QQWNFPLIT FGAGTKL ELK (SEQ ID NO: 4)

[0243] Example 8 Humanization of anti-human FRα antibody mab107

[0244] In order to reduce the immunogenicity of the antibody and reduce the human anti-mouse antibody (HAMA) effect produced after the antibody is administered into the body, in this example, the mab107 antibody was humanized.

[0245] Specifically, the variable region sequence of the mab107 antibody described in Example 7 was humanized. The heavy and light chain amino acid sequences were encoded according to the Kabat coding system and compared for sequence homology using NCBI Igblast (https: / / www.ncbi.nlm.nih.gov / igblast / ). A human antibody sequence with high homology was selected as the framework for mouse antibody CDR grafting. Some core amino acids in the framework region were backmutated. To avoid the presence of cysteine ​​in the CDR region and the formation of free sulfhydryl groups, the cysteine ​​(C) at position 52A in the heavy chain CDR2 region was converted to alanine (A).

[0246] The FR and CDR sequences of the heavy chain variable region of the mab107 antibody according to the Kabat definition are shown in Table 4.

[0247] Table 4

[0248]

[0249] The FR and CDR sequences of the light chain variable region of the mAb107 antibody according to the Kabat definition are shown in Table 5.

[0250] Table 5

[0251]

[0252] The amino acid sequence of humanized mab107 is as follows:

[0253] Amino acid sequence of the heavy chain variable region of humanized antibody hu107

[0254] QVQLVQSGAEVKKPGASVKVSCKASGYSFT GYYMHWVRQAPGQGLE WMG YISAYNGATYYNQNFKG RVTMTVDTSTSTVYMELSSLRSEDTAVYYC AR VGDGSRSWFAY WGQGTLVTVSS (SEQ ID NO: 19)

[0255] In the heavy chain variable region amino acid sequence, the sequence of H-CDR2 after point mutation is:

[0256] YISAYNGATYYNQNFKG(SEQ ID NO:20)

[0257] Amino acid sequence of the light chain variable region of humanized antibody hu107

[0258] DIQMTQSPSTLSASVGDRVTITC SASSSVTYMH WYQQKPGKAPKLLIY EI SKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQWNFPLIT FGQGTKV EIK (SEQ ID NO: 21)

[0259] The underlined part is the CDR region.

[0260] Example 9 Expression and purification of chimeric and humanized antibodies

[0261] The humanized antibody was sent to Bio-Tech Biotechnology to synthesize the variable region sequence, and the heavy and light chain variable regions of the humanized antibody and the heavy and light chain variable regions of the mouse monoclonal antibody were respectively constructed into the expression vector PTT5 (available from BioVectorNTCC Inc.) containing the human IgG1 / κ heavy and light chain constant regions to obtain the humanized antibody and chimeric antibody heavy and light chain expression vectors.

[0262] Plasmids were extracted using a commercial plasmid extraction kit. The day before transfection, the density of Expi293F cells in the logarithmic growth phase was adjusted to 1.0-2.0×10 6 / mL, and after culturing for 24 h, the final concentration of the total plasmid was 1 μg / mL and the ratio of heavy chain plasmid: light chain plasmid: PEI was 1:1:3 for transfection.

[0263] After 7 days, the cells were centrifuged to collect the culture supernatant, and the antibodies were purified using a Protein A affinity column.

[0264] Example 10 Humanized Antibody Binding Activity

[0265] Elisa enzyme-labeled 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 from 20 μg in 15 gradients, and 100 μL per well was added to the antigen-coated 96-well plate. After incubation at 37°C for 1 hour, the plate was washed 5 times with PBST, and a 1:3000 diluted HRP-labeled goat anti-human IgG secondary antibody was added. After incubation at 37°C for 45 minutes, the plate was washed 5 times with PBST, and the color was developed with 3,3'5,5'-tetramethylbenzidine (TMB) color solution for 5 minutes and terminated with 2M H2SO4. The OD value was read on a microplate reader. 450 EC values ​​were calculated using GraphPad Prism 8 software. 50 value.

[0266] The results are as follows Figure 3 As shown in Table 6. The results showed that the humanized antibody hu107 had strong binding activity with human FRα protein; the relative binding activity of hu107 to the chimeric antibody was 122.83%.

[0267] Table 6 hu107 specific binding EC 50

[0268] Antibody No. <![CDATA[EC 50 (μg / mL)]]> Relative binding activity (%) hu107 0.02164 122.83 ch107 0.02658 100

[0269] Example 11 Analysis of key binding sites between humanized antibodies and antigens

[0270] The results of Example 5 showed that mab107 binds to human FRα but not to rat FRα. Therefore, by analyzing the FRα protein sequence and tertiary structure, the present inventors selected different amino acid residues in the human and rat FRα sequence and nearby amino acid residues on the protein surface for alanine scanning mutagenesis. A total of 17 amino acid sites were selected for point mutation on the FRα-mFc plasmid (see Figure 4 ), which are K52A, E55A, R58A, K62A, N69A, V78A, E91A, P94A, N131A, E140A, C146A, N161A, C169A, C175A, H179A, V187A, and N201A respectively.

[0271] The mutated plasmid was transfected according to the antibody transfection and purification method in Example 9, and 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.

[0272] The results showed that all 17 mutant proteins were expressed, but the expression level of C175A was low, so further experiments were not performed. The results of the mutant protein supernatant binding activity with antibodies showed that E91A and C146A had a greater impact on the binding activity of hu107.

[0273] Therefore, this example further purified the above two point mutations and successfully obtained the E91A mutant protein. However, no protein was purified after the C146A mutation. The mutant protein was coated on an ELISA plate and then subjected to an ELISA binding experiment. The results were as follows: Figure 5 As shown, the E91A mutation significantly reduced the binding of hu107 to FRα protein.

[0274] Example 12 Detection of Binding Activity of Humanized Antibodies to Target Cells

[0275] OVCAR3 cells in good logarithmic growth phase were obtained, digested with trypsin and counted, and the cell density was adjusted to 1×10 6 1 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, incubate at 4°C for 1 hour, and wash twice with PBA (PBS + 2% FBS).

[0276] After washing, 100 μL of PE-labeled goat anti-human IgG secondary antibody diluted 1:400 in PBA was added to each tube. Mix thoroughly by pipetting and incubate at 4°C in the dark for 50 min. After incubation, wash three times with PBA, resuspend and mix thoroughly in 300 μL of PBA, and transfer to flow cytometry tubes for later use.

[0277] The PE fluorescence signal intensity 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.

[0278] The results showed that Figure 6 The results showed that the binding activity of humanized antibody hu107 to human ovarian cancer OVCAR3 cells was comparable to that of chimeric antibody ch107.

[0279] Example 13 Detection of the proliferation inhibitory activity of humanized antibodies on target cells

[0280] Using PrestoBlue TM Cell viability was detected by dye. SW620, SW480, and OVCAR3 cells in the logarithmic growth phase were digested and diluted to 1.0×10 5 / mL, plated in 96-well opaque black medium at a volume of 100μL per well, and cultured overnight. After aspirating the medium, add 100μL of antibodies diluted in analysis medium to each well, ch107 and hu107, respectively, so that the final volume of each well is 100μL, so that the final concentration of the antibody is 800nM, 2-fold gradient dilution, a total of 9 gradients, double wells. Place the cell plate in a constant temperature incubator at 37°C, 5% CO2 for 5 days, then take it out, aspirate the medium in the 96-well plate, and add 100μL 10% PrestoBlue to each well. TM Add the dye solution and mix thoroughly. Place the cell plate in a constant temperature incubator and continue incubating for 1.5-2.5 hours. After incubation, place the cell plate in a multifunctional microplate reader and measure the fluorescence intensity (FLu) using the reader. Set the reader parameters to excitation: 560 nm, absorption: 590 nm. After reading, analyze the data using the microplate reader.

[0281] The inhibitory activity of candidate antibodies on target cell proliferation was detected by resazurin assay.

[0282] The results are as follows Figure 7 AC. The results showed that at the highest concentration of 800 nM, hu107 had no proliferation inhibition or tumor cell killing activity on SW620, SW480, or OVCAR3 cells.

[0283] Example 14 Endocytic activity of humanized antibodies in target cells

[0284] 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 to detect the endocytic activity of hu107.

[0285] Take SW620, OVCAR3, and SW480 cells in the logarithmic growth phase, digest them, centrifuge them at 1000 rpm for 5 minutes at 4°C, and discard the supernatant. Resuspend them in 2% PBA (PBS plus 2% fetal bovine serum), count them, centrifuge them at 1000 rpm for 5 minutes, and discard the supernatant. Use 2% PBA to adjust the single cell suspension concentration to 2×10 6 1 mL of single cell suspension was added to each 1.5 mL centrifuge tube. Centrifuge at 2500 rpm for 2.5 min and wash twice. 6Calculate the amount of antibody per cell, add 100 μL of hu107 at a concentration of 20 μg / mL to each tube of cells, incubate at 4°C for 1 hour, and wash twice with PBA after the incubation. Add PBS to the negative control group. Divide the washed cells into two equal tubes, one tube is placed at 4°C. The other tube is placed at 37°C for 3 hours. After the incubation, wash twice with PBA solution containing 1% sodium azide. Add goat anti-human IgG-PE diluted 1:400 with 1% sodium azide PBA solution to each group, 100 μL per tube, pipette to mix the cells, and incubate at 4°C in the dark for 50 minutes. Wash three times with PBA. Resuspend the cells in 300 μL PBS, gently pipette to mix, transfer to the flow tube, detect on the machine in the dark, detect the fluorescence signal by flow cytometer, and the internalization rate is calculated as follows:

[0286]

[0287] The results are as follows Figure 8 The results showed that the internalization rate of humanized antibody hu107 was 36.7% on SW620 cells, 20.5% on SW480 cells, and 13.1% on OVCAR3 cells.

[0288] Example 15 Small molecule drug sensitivity detection

[0289] Different tumor cells have varying degrees of sensitivity to small molecule toxins. This study selected Dxd and MMAE as candidate small molecules for ADCs. Dxd is a topoisomerase I inhibitor that can induce double-stranded DNA breaks, ultimately leading to apoptosis in cancer cells. MMAE can effectively inhibit mitosis by inhibiting tubulin polymerization.

[0290] FRα-positive SW620, SW480, and OVCAR3 cells were selected as target cells to evaluate their sensitivity to the small molecule drugs MMAE and Dxd.

[0291] Human colon cancer cells SW620, SW480, and OVCAR3 in the logarithmic growth phase were digested with trypsin and resuspended in the corresponding culture medium to adjust the density to 2×10 4 / ml, and the middle of the 96-well microplate was plated with 100 μl / well to make the number of cells in each well 2×10 3200 μl of culture medium was added to each well around the remaining cells. After overnight incubation at 37°C and 5% CO2, the supernatant from the cell culture plate was aspirated and the linker-small molecule drug (Dxd / MMAE) was diluted 2-fold using culture medium starting at a concentration of 400 nM for a total of 9 concentration gradients. 100 μl of the diluted sample was added to each well. Culture was continued for 6 days for color development. The cell plate was removed and equilibrated at room temperature for 15-20 minutes. 100 μl of CellTiter- Luminescent CellViability assay solution (purchased from Promega) was used, shaken on a shaker at 200 rpm for 2 min, protected from light for 18 min, and then read using an M5 microplate reader.

[0292] Result processing: Then a four-parameter fitting graph was drawn with the logarithm of the concentration as the horizontal axis and the killing rate as the vertical axis.

[0293] like Figure 9 As shown in AC, at the same concentration, the killing rate of Dxd on SW620, SW480, and OVCAR3 cells was higher than that of MMAE; the killing activity IC of Dxd on SW620, SW480, and OVCAR3 was 50 The IC values ​​of MMAE against SW620, SW480 and OVCAR3 are NA~0, 2.715nM and 4.534nM respectively. 50 They were 6.237 nM, NA, and 22.88 nM (Table 7).

[0294] In summary, SW620, SW480, and OVCAR3 cells are more sensitive to Dxd, and Dxd will be subsequently selected as the small molecule toxin portion of ADC.

[0295] Table 7 IC50 of small molecule drugs against tumor cells 50

[0296]

[0297] Note: NA~0: Since the killing rate at the lowest concentration is still in the plateau phase, no fitting is done.

[0298] Example 16 Preparation of Antibody-Drug Conjugates

[0299] The linker-drug used in this embodiment is -VASGFG-Dxd (08Dxd) or -VASSGFG-Dxd (09Dxd), which was synthesized with reference to patent WO2023098691.

[0300] Using a fluid 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 and incubate with a reducing agent, 0.04-0.4 mM TCEP, at 10-37°C for 1-5 hours to fully open the interchain disulfide bonds. Subsequently, adjust the pH of the reduced antibody solution to 6.0 using phosphoric acid and bring the temperature to room temperature.

[0301] Dissolve the linker-small molecule drug in DMSO and calculate its concentration. Mix the linker-drug solution (08Dxd, 09Dxd) with the fully reduced antibody solution at an appropriate drug / antibody ratio (molar ratio > 10), maintaining a DMSO ratio of no more than 10% (v / v). Maintain the reaction for 0.5-2 hours to complete. After filtering the crude ADC product solution, purify it using a purification device (desalting column, ultrafiltration tubing, tangential flow system, etc.) to remove organic small molecule impurities and excess DMSO. The ADC product is then exchanged into storage buffer.

[0302] Table 8 Connector structure

[0303]

[0304] Example 17 DAR Detection of Antibody-Drug Conjugate Products

[0305] HIC-HPLC was used for detection. Samples were loaded and eluted under the mobile phase condition of pH 7.0, and the absorption peak area at the specified wavelength was detected.

[0306] The specific test methods are shown in Table 9, and the test results are shown in Table 10 and Figure 10A and 10B As shown in Figure 2, the DAR of the antibody-drug conjugate product is between 7 and 8.

[0307] Table 9 HIC-HPLC detection method

[0308]

[0309] Table 10 HIC-HPLC DAR value detection results

[0310]

[0311] Example 18 Binding Activity of Antibody-Drug Conjugates

[0312] In this example, the binding activity of the antibody-drug conjugate to the FRα protein was verified using a protein ELISA assay. First, a plate was coated with FRα protein antigen at a concentration of 1 μg / ml. Hu107, 107-08Dxd, and 107-09Dxd were then diluted to a starting concentration of 125 nM, followed by 15 1:2 dilutions and OD450 readings.

[0313] The results are as follows Figure 11 As shown in the figure, compared with naked antibody, the antibody-drug conjugate has no significant decrease in binding activity to FRα protein, and has a certain dose-effect relationship.

[0314] ADC targeting FRα binds to active EC 50 As shown in Table 11, the EC of hu107 50 The EC of Dxd is 0.424nM and 107-08 50 The EC of 107-09Dxd is 0.320nM. 50 It is 0.428nM, indicating that the antibody binding activity is not significantly affected after the antibody is coupled with the small molecule toxin.

[0315] Table 11 Binding activity EC of ADC targeting FRα 50

[0316] Antibody hu107 hu107-08Dxd hu107-09Dxd EC50 (nM) 0.424 0.320 0.428 <![CDATA[R 2 ]]> 0.988 0.982 0.987

[0317] Example 19 Flow cytometry detection of the binding activity of antibody-drug conjugates to target cells

[0318] The binding of hu107-08Dxd, hu107-09Dxd to FRα on the surface of SW620, SW480, and OVCAR3 cells was analyzed by flow cytometry.

[0319] The results are as follows Figure 12A 、 12B As shown in Figure 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 conjugate was not significantly decreased.

[0320] Example 20 Detection of Endocytic Activity of Antibody-Drug Conjugates

[0321] The binding activity and endocytosis of antibody-drug conjugates are crucial for ADCs to exert their tumor-killing effects. hu107 has certain endocytosis activity in multiple target cells. Whether the ADC structure constructed with hu107 as the antibody part will affect the binding of the antibody part to the antigen and the internalization of the ADC requires further verification.

[0322] The endocytic activity of hu107-08Dxd and hu107-09Dxd in SW620, SW480, and OVCAR3 cells with high FRα expression was preliminarily verified by flow cytometry according to the method in Example 16.

[0323] SW620 cells with high FRα expression, SW480 cells with medium FRα expression, and OVCAR3 cells were selected to detect the endocytic activity of hu107.

[0324] The results are as follows Figure 13 As shown, hu107-08Dxd and hu107-09Dxd were internalized to a certain extent in SW620, SW480, and OVCAR3 cells, and the internalization rate of ADC was significantly increased compared with hu107.

[0325] Example 21 Cell Killing Activity of Antibody-Drug Conjugates

[0326] The cell killing activity of hu107-08Dxd and hu107-09Dxd on target cells was detected according to the cell killing activity method in Example 17.

[0327] like Figure 14A 、 14B As shown in Figure 14C and Table 12, 107-08Dxd has specific killing activity against FRα-positive SW620, SW480, and OVCAR3 cells, with a certain dose-effect relationship; the killing activity IC of hu107-08Dxd against SW620, SW480, and OVCAR3 cells is 50 They are 4.39nM, 6.22nM and 24.98nM respectively.

[0328] Table 12 The killing activity IC of hu107-08Dxd against target cells SW620, SW480 and OVCAR3 50

[0329]

[0330] like Figure 15A 、 15B As shown in Figure 15C and Table 13, hu107-09Dxd has specific killing activity against FRα-positive SW620, SW480, and OVCAR3 cells, and has a certain dose-effect relationship; the killing activity IC of 107-09Dxd against SW620, SW480, and OVCAR3 cells is 50 They are 3.53nM, 12.80nM and 36.71nM respectively.

[0331] Table 13 IC values ​​of 107-09Dxd against SW620, SW480 and OVCAR37 tumor cells50

[0332]

[0333] In summary, hu107-08Dxd and hu107-09Dxd can specifically kill FRα-expressing tumor cells.

[0334] Example 22 Efficacy of Antibody-Drug Conjugate hu107-09Dxd in a Transplanted Tumor Model

[0335] Female SPF-grade BALB / c nude mice, 3-4 weeks old, were selected and adapted for 3-4 days before tumor inoculation. SW620 cells in the logarithmic growth phase were digested and washed twice with serum-free 1640 medium. 5×10 cells were subcutaneously inoculated in the right axilla of the mice with 0.2 ml of the medium. 6 cells / mouse. One week after inoculation, the tumor grew to 150±50mm 3 The mice were randomly divided into groups according to the block design, with 5 mice in each group. The blank model group served as the PBS control.

[0336] hu107-09Dxd was administered in two dose groups, 2 mg / kg and 6 mg / kg, by intraperitoneal injection. The administration frequency was once a week for four consecutive doses. Measurements were taken twice a week, and tumor volume and mouse weight data were recorded before each administration.

[0337] Tumor volume calculation: V (mm 3 )=(W 2 x L) / 2, W is the short diameter of the tumor near the midline, L is the long diameter of the tumor near the midline. When the tumor volume is greater than 2000mm 3 Mice were sacrificed by cervical dislocation when body weight loss was greater than 20% or interference with vital physiological functions or tumor ulceration and necrosis was observed.

[0338] The results are as follows Figure 16 As shown, hu107-09Dxd significantly inhibited tumor growth in the SW620 mouse subcutaneous xenograft tumor model at doses of 2 mg / kg and 6 mg / kg, demonstrating a dose-response effect. No significant weight loss was observed in any of the groups during the dosing period.

[0339] Example 23 Efficacy of Antibody-Drug Conjugate hu107-08Dxd in a Transplanted Tumor Model

[0340] Female SPF-grade BALB / c nude mice, 3 to 4 weeks old, were selected and adapted for 3-4 days before tumor inoculation. CAOV3 ovarian cancer cells in the logarithmic growth phase were digested, washed twice with serum-free 1640 medium, and diluted to 5×10 7 / ml was mixed with an equal volume of matrix gel and 0.2ml was subcutaneously inoculated into the right axilla of mice for a total of 5×106 cells / mouse. One week after inoculation, the tumor grew to 150±50mm 3 The mice were randomly divided into groups according to the block design, with 5 mice in each group. The blank model group served as the PBS control.

[0341] hu107-08Dxd was administered at three doses: 1 mg / kg, 3 mg / kg, and 10 mg / kg, via intraperitoneal injection once a week for four consecutive doses. Tumor volume and mouse body weight were measured twice weekly before each dose. Observation continued after the end of dosing.

[0342] Tumor volume calculation: V (mm 3 )=(W 2 x L) / 2, W is the short diameter of the tumor near the midline, L is the long diameter of the tumor near the midline. When the tumor volume is greater than 2000mm 3 Mice were sacrificed by cervical dislocation when body weight loss was greater than 20% or interference with vital physiological functions or tumor ulceration and necrosis was observed.

[0343] The results are as follows Figure 17 As shown, hu107-08Dxd significantly inhibited the growth of subcutaneous xenograft tumors in CAOV3 nude mice. At the end of dosing, 1 / 5, 3 / 5, and 5 / 5 of the tumors were completely regressed at 1 mg / kg, 3 mg / kg, and 10 mg / kg, respectively. At 67 days of observation, 1 / 5, 2 / 5, and 4 / 5 of the tumors in each dose group remained completely regressed. No significant weight loss or other symptoms were observed in any of the groups, demonstrating good tolerance.

[0344] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anti-FRα antibody or an antigen-binding fragment thereof, characterized in that: The anti-FRα antibody or antigen-binding fragment thereof has three complementarity determining regions HCDR of the heavy chain variable region and three complementarity determining regions LCDR of the light chain variable region of (1) or (2): (1) HCDR1 shown in SEQ ID NO:6, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:10, LCDR1 shown in SEQ ID NO:13, LCDR2 shown in SEQ ID NO:15, LCDR3 shown in SEQ ID NO: 17; (2) HCDR1 shown in SEQ ID NO:6, HCDR2 shown in SEQ ID NO:20, HCDR3 shown in SEQ ID NO:10, LCDR1 shown in SEQ ID NO:13, LCDR2 shown in SEQ ID NO:15, LCDR3 shown in SEQ ID NO:

17.

2. The anti-FRα antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the anti-FRα antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; and / or The amino acid sequence of the heavy chain variable region of the anti-FRα antibody or antigen-binding fragment thereof 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.

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

4. The antibody-drug conjugate according to claim 3, characterized in that: The coupling moiety is a cytotoxic drug.

5. The antibody-drug conjugate according to claim 4, characterized in that: The expression of the antibody drug conjugate is: mAb-(XY)n; in, mAb is the anti-FRα antibody or an antigen-binding fragment thereof; X is a linker; Y is a coupling moiety, and the coupling moiety is a cytotoxic drug; n is a positive integer ≤8; The coupling part is coupled to the anti-FRα antibody or the antigen-binding fragment thereof via a linker.

6. A recombinant protein, characterized in that The recombinant protein has: (i) the antibody or antigen-binding fragment thereof according to claim 1; and (ii) Tag sequences that facilitate expression and / or purification.

7. A nucleotide molecule, characterized in that The nucleotide molecule encodes the anti-FRα antibody or antigen-binding fragment thereof according to claim 1.

8. A carrier, characterized in that The vector contains the nucleotide molecule according to claim 7.

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

10. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (i) the anti-FRα antibody or antigen-binding fragment thereof according to claim 1, the antibody-drug conjugate according to claim 3, or the recombinant protein according to claim 6; and (ii) a pharmaceutically acceptable carrier.

11. An in vitro non-diagnostic method for detecting FRα protein in a sample, characterized in that: The method comprises the steps of: (1) contacting a sample with the 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 the complex indicates the presence of FRα protein in the sample.

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