Anti-FOLR1 nano antibody and related application thereof

Abstract

The invention provides an anti-FOLR1 nano antibody and related application thereof, and relates to the technical field of biological medicines. The nano antibody can be combined with an FOLR1 antigen with high affinity and specificity. An in-vitro experiment verifies that the FOLR1-CD3 bispecific antibody constructed on the basis of the FOLR1-CD3 bispecific antibody shows remarkable killing activity on various FOLR1 positive tumor cells, can be used for treating cancers and other diseases related to FOLR1 expression, and provides an important basis for developing an immunotherapy strategy with efficient and lasting anti-tumor activity.

Description

[0001] This application claims priority to Chinese Patent Application No. 2025116436856, filed on November 11, 2025, entitled “An anti-FOLR1 nanobody and its related applications”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of antibodies, and more specifically, to an anti-FOLR1 nanobody and its related applications. Background Technology

[0003] Folic acid receptor alpha (FOLR1) is a glycosylated phosphatidylinositol-anchored membrane protein. Its expression is highly restricted in normal tissues, primarily distributed in the kidneys, placenta, and choroid plexus. However, it exhibits significant overexpression in various epithelial tumors, such as ovarian cancer, lung cancer, breast cancer, endometrial cancer, and renal cell carcinoma. FOLR1 efficiently mediates the endocytosis of 5-methyltetrahydrofolate, meeting the demands of rapid tumor cell proliferation for nucleotide synthesis and methylation, making it an ideal target for tumor-targeted therapy. Its tumor-specific high expression minimizes off-target toxicity to normal tissues, thus expanding the therapeutic window. Therefore, antibody drugs, antibody-drug conjugates (ADCs), and cell therapy targeting FOLR1 have become research hotspots in the field of tumor immunotherapy.

[0004] Currently, targeted therapies for FOLR1 mainly focus on monoclonal antibodies (such as faretuzumab), antibody-drug conjugates (ADCs) such as IMGN853 (Mirvetuximab Soravtansine), and CAR-T cell therapy. Among these, Mirvetuximab Soravtansine received accelerated approval from the FDA in 2022 for the treatment of platinum-resistant ovarian cancer with high folate receptor expression, marking a significant breakthrough in FOLR1 targeted therapy. However, existing therapies still face many challenges: monoclonal antibodies have large molecular weights and limited intratumoral penetration; the efficacy of ADCs depends on payload delivery and may lead to drug resistance and toxicity; and the application of CAR-T therapy in solid tumors is still limited by issues such as tumor microenvironment suppression and high costs.

[0005] Therefore, developing novel FOLR1-targeting molecules is of great significance.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an anti-FOLR1 nanobody and its related applications.

[0008] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide an anti-FOLR1 nanobody, comprising: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:4.

[0009] Secondly, embodiments of the present invention provide an antibody comprising: the anti-FOLR1 nanobody described in the foregoing embodiments.

[0010] Thirdly, embodiments of the present invention provide an antibody conjugate comprising: the anti-FOLR1 nanobody described in the foregoing embodiments or the antibody described in the foregoing embodiments.

[0011] Fourthly, embodiments of the present invention provide a reagent or kit comprising: the anti-FOLR1 nanobody described in the foregoing embodiments, the antibody described in the foregoing embodiments, or the antibody conjugate described in the foregoing embodiments.

[0012] Fifthly, embodiments of the present invention provide an immunoconjugate or pharmaceutical composition comprising the anti-FOLR1 nanobody or the antibody described in the foregoing embodiments.

[0013] Sixthly, embodiments of the present invention provide the use of anti-FOLR1 nanobodies, antibodies, antibody conjugates, or immunoconjugates or pharmaceutical compositions as described in the foregoing embodiments in the preparation of products for the prevention or treatment of FOLR1-positive tumors, the preparation of products for the detection of FOLR1, or the detection of FOLR1 for non-disease treatment or diagnostic purposes.

[0014] In a seventh aspect, embodiments of the present invention provide an isolated nucleic acid or a carrier containing said nucleic acid, which encodes the anti-FOLR1 nanobody or the antibody described in the foregoing embodiments.

[0015] Eighthly, embodiments of the present invention provide a recombinant cell containing the isolated nucleic acid described in the foregoing embodiments or a vector containing the nucleic acid.

[0016] In a ninth aspect, embodiments of the present invention provide a method for preparing anti-FOLR1 nanobodies or antibodies as described in the foregoing embodiments, comprising: culturing recombinant cells as described in the foregoing embodiments.

[0017] The present invention has the following beneficial effects: The anti-FOLR1 nanobody provided by this invention exhibits excellent antigen-binding activity, high specificity, and good affinity, with an affinity KD of 10. -9The FOLR1 nanobody and the anti-CD3 antibody OKT3 were used to construct a bispecific antibody. Through in vivo and in vitro experiments, it was verified that the FOLR1-CD3 bispecific antibody constructed based on the nanobody has significant killing activity against a variety of FOLR1-positive tumor cells. It can be used to treat cancers and other diseases related to FOLR1 expression, and provides a key material for developing immunotherapy strategies with efficient and durable anti-tumor activity. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 SDS-PAGE results of the recombinant nanobody targeting FOLR1 (Nb-FR5); Figure 2 To determine the reactivity of Nb-FR5 recombinant nanobodies with antigens using ELISA; Figure 3 For immunofluorescence detection of recombinant nanobodies and HeLa (FOLR1) + The binding status of cells; Figure 4 To detect the binding of recombinant nanobodies to FOLR1-expressing tumor cell lines and FOLR1-negative cell lines by flow cytometry; Figure 5 To detect the in vitro antitumor activity of FOLR1 and CD3 bispecific antibody (BiTE) using a label-free kill assay (RTCA). Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. "Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (JEColigan et al., eds., 1991), each of which is explicitly incorporated herein by reference.

[0022] Nanobodies, a class of single-domain antibodies (VHHs) derived from camel heavy chain antibodies, possess unique advantages such as small molecular weight (approximately 15 kDa), strong tissue penetration, high stability, excellent affinity, and ease of engineering, demonstrating great potential in targeted cancer therapy. Developing FOLR1-specific nanobodies can overcome the shortcomings of traditional antibodies, providing key molecular tools for novel FOLR1-targeted therapies such as CAR-T, ADC, RDC, and bispecific antibodies, further advancing the clinical translation of FOLR1-targeted cancer therapy.

[0023] This invention, through appropriate screening techniques and large-scale screening, ultimately obtained a specific anti-FOLR1 nanobody. This anti-FOLR1 nanobody and an anti-CD3 single-chain antibody were then used to construct a bispecific antibody, which still maintains good antigen-binding activity against FOLR1. In vivo and in vitro experiments confirmed that this bispecific antibody has strong killing activity against FOLR1-overexpressing tumor cells, effectively improving therapeutic efficacy and providing a key material for the subsequent development of immunotherapies with highly efficient and durable anti-tumor activity.

[0024] Definition of noun The term "antibody" as used herein is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antigen-binding fragments, provided they exhibit the desired biological activity, such as specific binding to the FOLR1 antigen or fragments thereof. An antigen-binding fragment, also known as a functional fragment of an antibody, typically has the same binding specificity as the antibody from which it originates. Antigen-binding fragments include any one selected from F(ab')2, Fab', Fab, Fv, and scFv of antibodies. Those skilled in the art will readily understand from the description of this invention that the aforementioned functional fragments of antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. The aforementioned antigen-binding fragments can also be obtained by recombinant genetic techniques, also known to those skilled in the art, or by synthesis using, for example, automated peptide synthesizers, such as those sold by Applied BioSystems.

[0025] In this article, the term "CDR" stands for "complementarity-determining region," which refers to the highly variable region of the heavy and light chains of an immunoglobulin, specifically the region containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes.

[0026] The term "backbone region" in this article, synonymous with "framework region" or "FR region," refers to the region of the antibody's heavy chain variable region excluding the CDR region. The heavy chain backbone region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4), including the HFR1, HFR2, HFR3, and HFR4 backbone regions. The heavy chain variable region is obtained by arranging and connecting the following numbered CDRs with FRs (from the amino terminus to the carboxyl terminus): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0027] The “multivalent antibody” in this article is a polymer of a monovalent antibody that recognizes the same epitope and has a higher antigen affinity than the corresponding monovalent antibody.

[0028] The "multispecific antibody" in this article is a polymer of monovalent antibodies that bind to different targets or different binding regions on the same target, and has a stronger antigen recognition ability than the corresponding monovalent antibody.

[0029] The “chimeric antibody” mentioned in this article is usually an antibody formed by fusing the variable region of a non-human antibody with the constant region or backbone region of a human antibody, which can reduce the immune response induced by non-human antibodies.

[0030] The term "fusion antibody" in this article includes fusion nanobodies, including but not limited to those formed by combining with other structures (such as BSA, IgG-Fc, etc.) through genetic engineering techniques to form new fusion molecules, such as enzymes, antimicrobial peptides, or imaging substances that can extend their half-life.

[0031] The term "treatment" in this invention includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0032] For cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0033] On one hand, embodiments of the present invention provide an anti-FOLR1 nanobody, which includes HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:4.

[0034] In some embodiments, HCDR1, HCDR2, and HCDR3 are defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

[0035] In some embodiments, the antibody comprises: HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1~3.

[0036] In some embodiments, the nanobody is at least one of monovalent nanobody, multivalent nanobody, multispecific antibody, and fusion nanobody. Nanobodies, due to their small molecular weight, are encoded by a single gene, are easily genetically engineered, and multiple nanobodies can be aggregated through short linker sequences to form multivalent or multispecific antibody structures.

[0037] In some embodiments, the heavy chain variable region further includes a skeleton region.

[0038] In some embodiments, the amino acid sequence of the heavy chain variable region of the nanobody is shown in SEQ ID NO:4.

[0039] On the other hand, embodiments of the present invention provide an antibody comprising: the anti-FOLR1 nanobody described in any of the foregoing embodiments.

[0040] In some embodiments, the antibody includes any one of the following: full-length antibody, heavy chain antibody, chimeric antibody, multivalent antibody (bivalent, trivalent, tetravalent, pentavalent or hexavalent), multispecific antibody and fusion antibody.

[0041] In some embodiments, the multispecific antibody includes any one of bispecific antibodies, trispecific antibodies, and tetraspecific antibodies.

[0042] Bispecific antibodies (BsAbs) are artificial antibodies that can simultaneously target two antigens or different epitopes of the same antigen. Bispecific antibodies can be IgG-like (full-length bispecific antibodies) or non-IgG-like bispecific antibodies that are non-full-length antibody constructs. Full-length bispecific antibodies typically retain the structure of a traditional monoclonal antibody (mAb) with two Fab arms and an Fc region, but the two Fab sites bind to different antigens. Non-full-length bispecific antibodies may lack the entire Fc region. These include chemically linked Fabs, Fab regions alone, and various types of bivalent and trivalent single-chain variable fragments (scFvs). Fusion proteins that mimic the variable domains of two antibodies also exist.

[0043] In some embodiments, bispecific antibodies include bispecific T-cell conjugating antibodies (BiTEs). The core function of bispecific T-cell conjugating antibodies (BiTEs) is to simultaneously target CD3 molecules on the surface of T cells and specific antigens on the surface of tumor cells through two different antigen-binding domains, thereby directly "recruiting" T cells to the vicinity of tumor cells, activating the killing function of T cells, and achieving specific clearance of tumor cells.

[0044] In some embodiments, when the antibody is a bispecific antibody (bispecific T-cell conjugating antibody), the bispecific antibody may further include an anti-CD3 antibody.

[0045] Verification has shown that the bispecific antibody obtained by constructing anti-FOLR1 nanobodies and anti-CD3 antibodies has good anti-tumor activity and can be used to prepare drugs for the prevention, diagnosis and treatment of at least one of colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, breast cancer, endometrial cancer and renal cell carcinoma.

[0046] In some embodiments, the anti-FOLR1 nanobody and the anti-CD3 antibody are linked by a linker peptide.

[0047] In some embodiments, the anti-CD3 antibody comprises a single-chain antibody against CD3.

[0048] In some embodiments, the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO:5.

[0049] In some embodiments, the linker peptide is (G4S)n, where n is a non-zero natural number.

[0050] In some embodiments, n is 1 to 20.

[0051] In some embodiments, n is 3 or 4.

[0052] On the other hand, embodiments of the present invention provide an antibody conjugate comprising: the anti-FOLR1 nanobody described in any of the foregoing embodiments or the antibody described in any of the foregoing embodiments.

[0053] In some embodiments, the antibody conjugate further includes a label, purification tag, and / or solid-phase carrier conjugated to the anti-FOLR1 nanobody or the antibody or its antigen-binding fragment.

[0054] In some embodiments, the markers include fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0055] In some embodiments, fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5). Cy5.5, Cy3 and other similar dyes, Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar dyes) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar dyes).

[0056] In some embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0057] In some embodiments, radioactive isotopes include, but are not limited to, 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0058] In some embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0059] In some embodiments, nanoparticle-based markers include, but are not limited to, nanoparticles and colloids; nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0060] In some embodiments, the solid support includes, but is not limited to, microspheres, plates, and membranes.

[0061] In some embodiments, the solid support includes any one or more of magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0062] On the other hand, embodiments of the present invention provide a reagent or kit comprising: the anti-FOLR1 nanobody described in any of the foregoing embodiments, the antibody described in any of the foregoing embodiments, or the antibody conjugate described in any of the foregoing embodiments.

[0063] On the other hand, embodiments of the present invention provide an immunoconjugate or pharmaceutical composition comprising the anti-FOLR1 nanobody or the antibody described in any of the foregoing embodiments; In some embodiments, the immune conjugate further includes a therapeutic agent.

[0064] In some embodiments, the therapeutic agent comprises at least one of the following: chemotherapeutic drugs, radionuclides, photosensitizers, photothermal agents, immune checkpoint inhibitors, toxins, factors, kinase inhibitors, antibodies that inhibit second signaling molecules, PD-L1 inhibitors, and PD-1 / PD-L1 monoclonal antibody drugs.

[0065] In some embodiments, relevant biomarkers for immune checkpoint inhibitor therapy include PD-L1, MSI / bMSI, TMB / bTMB, TNB and EGFR mutations, ALK fusions, TP53 mutations, and KRAS mutations.

[0066] In some embodiments, the chemotherapeutic drugs described above are selected from any one or more of taxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, tyrosine kinase inhibitors, fulapindo, irinotecan and its metabolite SN-38, topotecan, teniposide, etoposide, imatinib, gefitinib, danuseltide, doxorubicin, daunorubicin, mitoxantrone, methotrexate, camptothecin, and saquinavir.

[0067] In some embodiments, the photosensitizer is selected from: (1) 5-aminolevulinic acid (ALA) or its derivatives; (2) photosensitizing compounds containing a tetrapyrrole ring; (3) traditional Chinese medicine photosensitizers; or (4) combinations of ALA or its derivatives with compounds in (2) or (3), respectively.

[0068] In some embodiments, the photothermal agent is selected from IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061 and IR-26.

[0069] In some embodiments, the inhibitory second signaling molecule may be PD-1; CTLA-4; or PD-1 and CTLA-4.

[0070] In some embodiments, the PD-1 / PD-L1 monoclonal antibody drug is selected from at least one of the following groups: nivolumab, pembrolizumab, pitilizumab, lambrolizumab, BMS-936559, atezolizumab, AMP-224, AMP224, AUNP12, BGB108, MCLA134, MEDI0680, PDROO1, REGN2810, SHR1210, STIAl110, STIAl1110, TSR042, BMS-936558, BGB-A317, BCD-100, and JS001.

[0071] In some embodiments, the therapeutic agent further includes a cytotoxic agent.

[0072] In some embodiments, the pharmaceutical composition includes at least one of a pharmaceutical excipient, a carrier, and a diluent. The carrier is a pharmaceutically acceptable carrier, including but not limited to fillers, lubricants, disintegrants, binders, flow aids, etc.

[0073] In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, one or a combination of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, dicalcium phosphate, calcium stearate, sodium stearoyl fumarate, silicon dioxide, titanium dioxide, talc, and indigo.

[0074] On the other hand, embodiments of the present invention provide the use of anti-FOLR1 nanobodies, antibodies, antibody conjugates, or immunoconjugates or pharmaceutical compositions as described in any of the foregoing embodiments in the preparation of products for the prevention or treatment of FOLR1-positive tumors, the preparation of products for the detection of FOLR1, or the detection of FOLR1 for non-disease treatment or diagnostic purposes.

[0075] In some embodiments, the FOLR1-positive tumor includes at least one of colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, breast cancer, endometrial cancer, and renal cell carcinoma.

[0076] In some embodiments, the product includes at least one of immune cells, reagents, kits, drugs, and drug compositions.

[0077] On the other hand, embodiments of the present invention provide an isolated nucleic acid or a carrier containing said nucleic acid, which encodes the anti-FOLR1 nanobody or the antibody described in any of the foregoing embodiments.

[0078] Considering the degeneracy of codons, the gene sequence encoding the above antibodies can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; alternatively, the gene can be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0079] In some embodiments, the vector containing the nucleic acid includes a recombinant vector. The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct that can directly introduce the target DNA fragment into the host cell for target gene expression via transformation, transfection, or transduction.

[0080] In some embodiments, the recombinant vector is a plasmid or a virus; the virus is an adenovirus, adeno-associated virus, retrovirus, lentivirus, or oncolytic virus.

[0081] On the other hand, embodiments of the present invention provide a recombinant cell containing the isolated nucleic acid described in any of the foregoing embodiments or a vector containing the nucleic acid.

[0082] In some embodiments, the recombinant cells include prokaryotic or eukaryotic cells. The prokaryotic cells include, but are not limited to, bacterial cells, such as *Escherichia coli*; the eukaryotic cells include, but are not limited to, yeast cells, insect cells, animal cells, or plant cells; the yeast cells may be, but are not limited to, *Pichia pastoris* or *Saccharomyces cerevisiae* cells. Optionally, the recombinant cells may be 293 cells, 293T cells, 293FT cells, CHO cells, or Per6 cells. The 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins and are well known to those skilled in the art.

[0083] Furthermore, embodiments of the present invention provide a method for preparing anti-FOLR1 nanobodies or antibodies as described in any of the foregoing embodiments, comprising: culturing recombinant cells as described in any of the foregoing embodiments.

[0084] The present invention does not specifically limit the culture conditions of the host cells; culture conditions that enable the host cells to express and produce the antibody can be obtained based on conventional technical knowledge.

[0085] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0086] All animal experiments are conducted in accordance with animal ethics guidelines and are carried out only after being approved by the relevant institution's animal experiment ethics committee.

[0087] Example 1: Preparation of recombinant FOLR1 protein The nucleic acid sequence encoding human FOLR1 (NM_016725.1) was synthesized by Anhui General Biotechnology Co., Ltd. The extracellular domain (ECD) of FOLR1 was amplified by PCR and subcloned into a pcDNA3.1 expression vector (Invitrogen) carrying a His tag at the C-terminus. Then, the protein was transiently transfected with HEK293F, cultured in FreeStyle™ serum-free medium (Life Technologies) in shake flasks for 5–7 days, and the supernatant was collected. High-purity recombinant FOLR1 protein was obtained by affinity chromatography using an NTA-Ni column.

[0088] Example 2: Preparation of anti-FOLR1 protein-specific nanobodies This invention first involves immunizing Bactrian camels four times with a mixture of FOLR1 protein (1 mg / dose) and an equal volume of Freund's adjuvant, then collecting peripheral blood to isolate lymphocytes. Total RNA was extracted from the lymphocytes using an RNA extraction kit, and the VHH gene was amplified by RT-PCR. The amplified VHH gene was then cloned into the phage display vector pMECS, successfully constructing a VHH phage antibody library. Anti-FOLR1 nanobodies were obtained through three rounds of phage display screening. Using these nanobodies as templates, the VHH gene was amplified and constructed into the eukaryotic expression vector pcDNA3.1-hFc-His via homologous recombination. This was then transfected into HEK293F cells in logarithmic growth phase using PEI transfection reagent. After 5 days of expression, the supernatant was collected, and high-purity recombinant nanobodies (Nb-FR5) were obtained by affinity chromatography using an NTA-Ni column. SDS-PAGE results showed that the Nb-FR5 recombinant nanobodies obtained after affinity chromatography purification met the expected band sizes. Figure 1 ).

[0089] Example 3: ELISA identification of the reactivity of recombinant nanobodies with antigens To assess the reactivity of the recombinant nanobody-hFc fusion protein with the antigen, 200 ng / well of FOLR1 recombinant protein was pre-coated onto an ELISA plate, incubated overnight at 4°C, and then the plate was blocked. Different amounts of recombinant nanobody (dilution: 10) were then added. -5 ~10 2µg / mL, added secondary antibody for washing, color development, and reaction termination, and the optical density (OD450) at 450 nm was measured using a microplate reader. Binding ability was determined using a four-parameter nonlinear regression curve fitting. The results showed that one of the recombinant nanobodies of FOLR1, Nb-FR5, could bind specifically and strongly to the recombinant FOLR1 protein. Figure 2 ).

[0090] Example 4: Affinity determination of FOLR1 nanobody The affinity of the FOLR1 nanobody (Nb-FR5) was verified by surface plasmon resonance (SPR), and its binding kinetic constant (Kd) was determined. Using a GE Biacore™ 8K instrument, anti-mouse IgG antibody was immobilized on the surface of a CM5 chip using conjugation buffer from an amino-conjugation kit. FOLR1 (ECD)-mFc was then captured onto the CM5 chip in 2-fold serial dilutions. The purified FOLR1 nanobody was then allowed to flow through the chip surface, and the instrument read Ka (1 / Ms), kd (1 / s), and KD (M), thus measuring the affinity of the recombinant nanobody. The affinity assay results showed that the candidate FOLR1 nanobody could specifically bind to the FOLR1 antigen, with an affinity of 10-1. -9 These antibodies have a KD level or higher and are classified as high-affinity antibodies. Specific data from the detection are shown in Table 1.

[0091] Table 1 Summary of affinity data for nanobodies

[0092] Example 5: Immunofluorescence detection of recombinant nanobodies and HeLa (FOLR1) + ) cell binding The recombinant nanobody (5 μg / mL) prepared in Example 2 was incubated with HeLa cells at 37°C for 40 min. After washing three times with PBS, it was incubated with 594@goat anti-human secondary antibody. After washing three times with PBS, the cells were imaged using a fluorescence microscope. The results are as follows: Figure 3 As shown, the recombinant nanobody Nb-FR5 can bind well to the above-mentioned cells.

[0093] Example 6: Flow cytometry detection of the binding of recombinant nanobody to FOLR1-expressing tumor cell lines The recombinant nanobody Nb-FR5 and the positive control antibody prepared in Example 2 were incubated with Huh-7 cells (which do not express FOLR1) and OV-90 cells (which express FOLR1), respectively, at 37°C for 40 min. After washing three times with PBS, they were incubated with APC@goat anti-human IgG (H+L) secondary antibody. After washing three times with PBS, the results were analyzed by flow cytometry. The results are as follows: Figure 4 As shown, the recombinant nanobody exhibits good reactivity with positive cells OV-90 and does not bind to negative cells Huh-7.

[0094] Example 7: Preparation and activity detection of anti-FOLR1 and CD3 bispecific antibody (BiTE) The anti-FOLR1 nanobody (Nb-FR5) provided in this embodiment of the invention and the anti-CD3 antibody OKT3 (amino acid sequence as shown in SEQ ID NO:5) were tandemly constructed into a eukaryotic expression vector using a (G4S)3 linker, transfected into HEK293F cells for expression, and purified using a Ni column.

[0095] FOLR1-positive OV-90 cells and FOLR1-negative Huh-7 cells were added to label-free killer detection (RTCA) instrument-specific 96-well plates and ordinary 96-well cell culture plates at a ratio of 5000 cells per well and 100 μL / well (replicas), respectively. The RTCA-specific 96-well plates were placed in the instrument and cultured until the Cell Index value was between 1.0 and 2.0. Then, different concentrations (10 nM, 1 nM and 0.1 nM) of bispecific antibody prepared in Example 7 were co-incubated with activated T cells from peripheral blood of healthy donors at an effector-to-target ratio of 2:1. The RTCA monitoring time was 48 h.

[0096] The results are as follows Figure 5 As shown, the above-mentioned bispecific antibody has no killing effect on antigen-negative Huh-7 cells, but has strong killing activity on OV-90 cells, and still exhibits relatively strong killing activity at a low concentration of 0.1 nM.

[0097] The antibody sequences involved in the foregoing embodiments and the CDR sequences summarized by antibody sequence analysis and comparison based on the IMGT database are shown in Table 1 below.

[0098] Table 1. Antibody sequence information

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanobody against FOLR1, characterized in that, It includes: The amino acid sequences are HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:

4.

2. The nanobody according to claim 1, characterized in that, The HCDR1, HCDR2, and HCDR3 are defined by any one of the following systems: Kabat, Chothia, IMGT, AbM, or Contact. Optionally, the antibody comprises: HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1~3 in sequence; Optionally, the nanobody is at least one of monovalent nanobody, multivalent nanobody, multispecific antibody and fusion nanobody; Optionally, the amino acid sequence of the heavy chain variable region of the nanobody is shown in SEQ ID NO:

4.

3. An antibody, characterized in that, It includes: The anti-FOLR1 nanobody according to claim 1 or 2; Optionally, the antibody includes any one of the following: full-length antibody, heavy chain antibody, chimeric antibody, multivalent antibody, multispecific antibody, and fusion antibody; Optionally, the multispecific antibody includes any one of bispecific antibodies, trispecific antibodies, and tetraspecific antibodies; Optionally, the bispecific antibody includes a bispecific T-cell conjugating antibody; Optionally, when the antibody is a bispecific antibody, the bispecific antibody further includes: an anti-CD3 antibody; Optionally, the anti-FOLR1 nanobody and the anti-CD3 antibody are linked by a linker peptide; Optionally, the anti-CD3 antibody includes a single-chain antibody against CD3; Optionally, the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO:5; Optionally, the linker peptide is (G4S)n, where n is a non-zero natural number; Optionally, n is 1 to 20; Optionally, n is 3 or 4.

4. An antibody conjugate, characterized in that, It includes: The anti-FOLR1 nanobody according to claim 1 or 2, or the antibody according to claim 3; Optionally, the antibody conjugate may further include a label, purification tag, and / or solid-phase carrier conjugated to the anti-FOLR1 nanobody or the antibody or its antigen-binding fragment.

5. A reagent or kit, characterized in that, It includes: The anti-FOLR1 nanobody according to claim 1 or 2, the antibody according to claim 3, or the antibody conjugate according to claim 4.

6. An immunoconjugate or pharmaceutical composition, characterized in that, It includes the anti-FOLR1 nanobody as described in claim 1 or 2, or the antibody as described in claim 3; Optionally, the immune conjugate further includes a therapeutic agent; Optionally, the therapeutic agent includes at least one of the following: chemotherapeutic drugs, radionuclides, photosensitizers, photothermal agents, immune checkpoint inhibitors, toxins, factors, kinase inhibitors, antibodies against inhibitory second signaling molecules, PD-L1 inhibitors, and PD-1 / PD-L1 monoclonal antibody drugs; Optionally, the pharmaceutical composition may further include at least one of a pharmaceutical excipient, a carrier, and a diluent.

7. The use of the anti-FOLR1 nanobody as described in claim 1 or 2, the antibody as described in claim 3, the antibody-drug conjugate as described in claim 4, or the immunoconjugate or pharmaceutical composition as described in claim 6 in the preparation of products for the prevention or treatment of FOLR1-positive tumors, the preparation of products for the detection of FOLR1, or the detection of FOLR1 for non-disease treatment or diagnostic purposes; Optionally, the FOLR1 positive tumor includes at least one of colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, breast cancer, endometrial cancer, and renal cell carcinoma; Optionally, the product includes: At least one of immune cells, reagents, kits, drugs, and drug compositions.

8. An isolated nucleic acid or a vector containing said nucleic acid, characterized in that, It encodes the anti-FOLR1 nanobody as described in claim 1 or 2, or the antibody as described in claim 3.

9. A recombinant cell, characterized in that, It contains the isolated nucleic acid as described in claim 8 or a vector containing said nucleic acid.

10. A method for preparing the anti-FOLR1 nanobody as described in claim 1 or 2, or the antibody as described in claim 3, characterized in that, It includes: Cultivate the recombinant cells as described in claim 9.

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