Single-domain antibodies against folr1 and their derived proteins and uses

Abstract

The application belongs to the field of immunology and relates to a single-domain antibody against FOLR1, a derivative protein thereof and application. The single-domain antibody is composed of a heavy chain, the heavy chain comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3; the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are sequence combinations of (1)-(7) or sequences with high homology. The application uses biological genetic engineering technology to screen specific single-domain antibodies against FOLR1, the antibodies have obvious preliminary affinity, can block the release of cytokines by specific cells, have good binding activity through prokaryotic expression and have certain drugability.

Description

Technical Field

[0001] This invention belongs to the field of immunology and relates to single-domain antibodies against FOLR1, their derived proteins, and their applications. Background Technology

[0002] OLR is a member of the human folate receptor protein family, including three subtypes: FOLR1, FOLR2, and FOLR3. Among them, FOLR1 (Folate Binding Protein 1) is a glycoprotein coupled with glycosylated phosphatidyl alcohol, with a relative molecular mass of 38-40 kDa. It is encoded by the FOLR1 gene, is completely exposed on the cell membrane, and can mediate the entry of folate into the cell through the endocytosis mechanism. It participates in DNA replication and damage repair, and plays a very important role in cell division, proliferation, and tissue growth.

[0003] In recent years, many studies have reported that FOLR1 is expressed at low levels in normal tissues, but is overexpressed in many tumor cells such as ovarian cancer, breast cancer, kidney cancer, colon cancer, lung cancer, testicular cancer, tympanic membrane tumor, and choroidal tumor. Therefore, FOLR1 can be used as a diagnostic indicator for these malignant tumors. Studies have found that FOLR1 can enter the bloodstream from free receptors shed from tumors through their own pores, suggesting that serum FOLR1 can serve as an early tumor marker.

[0004] In recent years, research on FOLR1 in ovarian malignancies has been a hot topic. Ovarian malignancies rank fourth in mortality among all female malignancies and are showing a gradually increasing trend. Epithelial ovarian cancer (EOC) accounts for 85-90% of ovarian malignancies and is the leading cause of gynecological cancer death. Due to poor early screening, prevention and early diagnosis are challenging. Current first-line treatments include surgery and chemotherapy, but the five-year survival rate for EOC remains difficult to improve due to metastasis, residual lesions after surgery, and chemotherapy resistance. Therefore, developing new treatment methods and strategies is of great significance for EOC patients. Studies have indicated that serum FOLR1 may be a highly specific diagnostic biomarker for EOC and holds promise as a reference indicator for the diagnosis, targeted therapy, and monitoring of chemotherapy efficacy in epithelial ovarian cancer.

[0005] CA125 is the most widely used serum tumor marker for ovarian cancer, approved by the FDA for monitoring chemotherapy efficacy and differential diagnosis of patients with pelvic masses. However, elevated serum CA125 levels are often associated with other pathological conditions, such as endometriosis, adenomyosis, uterine fibroids, pelvic inflammatory disease, and benign ovarian tumors. Furthermore, only half of patients with early-stage ovarian cancer show elevated serum CA125 levels. Therefore, CA125 has poor sensitivity for early-stage ovarian cancer and poor overall specificity for ovarian cancer. For these reasons, CA125 is neither a biomarker for ovarian cancer screening nor a biomarker for early diagnosis of ovarian cancer. In patients with ovarian malignancies, CA125 has higher diagnostic power than CA125 for differentiating epithelial ovarian cancer. Researchers, based on different research methods and specimens from patients with ovarian malignancies, have proposed that FOLR1 could be used as a diagnostic marker for early-stage ovarian malignancies. Current research results indicate a close clinical relationship between FOLR1 and ovarian malignancies. Currently, some FOLR1-targeted therapies are undergoing clinical trials, which may be useful for monitoring chemotherapy efficacy.

[0006] To improve the sensitivity of ovarian cancer diagnosis, researchers investigated the correlation between serum high-mobility group box 2 (HMGA2), interleukin-22 (IL-22), and folate receptor 1 (FOLR1) levels and clinical indicators in ovarian cancer patients with end-stage renal disease (EOC). The results showed that the combined use of serum HMGA2, IL-22, and FOLR1 for diagnosis had better sensitivity (95%) and specificity (96.7%). Furthermore, studies explored the interaction between FOLR1 and MSLN in EOC pathogenesis and the combined detection of both to assess the prognosis of EOC patients. The results indicated that EOC patients with high FOLR1 expression had a better prognosis, while those with high MSLN expression had a poorer prognosis. These two proteins may jointly participate in the progression of EOC. The combined detection of FOLR1 and MSLN expression may aid in the prognosis of EOC, providing a new approach for the diagnosis, treatment, and prognosis of ovarian cancer.

[0007] Paclitaxel is a drug extracted from the yew tree (Taxus brevis) that possesses in vitro antitumor activity and is a cytotoxic agent that inhibits microtubule depolymerization. Paclitaxel was approved for clinical use as a new drug for the treatment of ovarian cancer in [year missing]. Currently, numerous studies have confirmed that paclitaxel can arrest tumor cells in the G2 / M phase. Tumor cells at this stage are sensitive to radiotherapy, thus paclitaxel can have a radiosensitizing effect. Therefore, paclitaxel is increasingly widely used in concurrent chemoradiotherapy for nasopharyngeal carcinoma and has achieved ideal efficacy. However, like other chemotherapeutic drugs, long-term use of paclitaxel can lead to tumor cell resistance, resulting in chemotherapy failure. Studies have shown that the deoxyribonuclease DRzE targeting FOLR1 can significantly increase the sensitivity of paclitaxel-resistant nasopharyngeal carcinoma cells to paclitaxel. On the other hand, a recent study showed that high expression of FOLR1 increases the sensitivity of ovarian cancer cells to cisplatin treatment, which may be related to improved patient prognosis. Based on the current large-sample analysis of TCGA and the latest reported mechanism studies (FOLR1 increases cisplatin sensitivity), researchers believe that high FOLR1 expression is a favorable prognostic factor for ovarian cancer. The reason may be that patients with high FOLR1 expression have increased sensitivity to chemotherapy, thereby improving their prognosis.

[0008] In recent years, the development of tumor immunotherapy has been rapid. The commercial application of PD-1 (programmed death-1), PD-L1 (programmed death-ligan1), and CTLA-4 (cytotoxic T-lymphocyte associated protein 4) antibodies, and the significant success of CAR-T technology in hematologic malignancies (such as CD19-CAR in leukemia), have led to its widespread adoption. 2017 was dubbed the "Year Zero" of CAR-T therapy, with the FDA approving two CAR-T therapies for acute lymphoblastic leukemia and lymphoma. High remission rates in clinical applications, coupled with commercial promotion, have brought adoptive cell transfer therapy (ACT) into the spotlight. The most significant characteristic of CAR-T therapy is that it directly endows T cells with targeted recognition capabilities, making it a "living drug" in clinical applications.

[0009] Domestic researchers have studied the construction of tandem CAR-T cells targeting folate receptor α and mesothelin and their application in epithelial ovarian cancer. They constructed a humanized FOLR1-CAR lentiviral plasmid containing two co-stimulatory molecules (CD28 / 4-1BB) and the IL-12 gene, packaged the lentivirus, and infected CD3 cells. The results screened and validated its highly expressed TAA in EOC, successfully constructing a fourth-generation CAR-T cell targeting FOLR1. Compared to single-target CAR-T cells, it exhibits stronger killing efficacy in vitro and secretes high levels of cytokines. The B-NDG mouse tumor-bearing model demonstrated that FOLR1-CAR still possesses anti-tumor capabilities in vivo.

[0010] Currently, more than 500 CAR-T clinical registration trials have been approved worldwide. Although CAR-T has achieved certain efficacy in solid tumors, the complete response rate (CR) and partial response rate (PR) are still low, which limits its large-scale clinical application. The harm caused to patients by its side effect, cytokine release syndrome, should not be underestimated.

[0011] Nanobodies, based on the unique structure of VHH single-domain antibodies derived from camel heavy chain antibodies, combine the advantages of traditional antibodies and small molecule drugs, almost perfectly overcoming the shortcomings of traditional antibodies such as long development cycles, low stability, and demanding storage conditions. Single-domain antibodies (sdAbs) are fragments containing a single variable domain of the antibody, representing the smallest antigen-binding unit of the antibody molecule, approximately 12–15 kDa. Although small, they are identical to complete antibodies, selectively binding to specific antigens. Furthermore, they offer advantages not found in traditional antibodies, such as improved screening, improved separation techniques, and the elimination of animal sacrifice. Due to their small molecular weight, they can penetrate the blood-brain barrier, giving them a natural advantage in biopharmaceutical development, potentially becoming the best drugs for treating malignant tumors such as ovarian cancer. The field still needs anti-FOLR1 antibodies, especially anti-FOLR1 heavy chain single-domain antibodies, that can bind with high affinity to FOLR1, mediate ADCC or undergo internalization, and possess good druggability. Summary of the Invention

[0012] To overcome the above-mentioned defects, the purpose of this invention is to provide single-domain antibodies against FOLR1 and their derived proteins and applications. Bioengineering technology is used to screen for single-domain antibodies specifically targeting FOLR1. These antibodies have obvious initial affinity and can block the release of cytokines from specific cells. They also have good binding activity when expressed in prokaryotes and have certain drug-like properties.

[0013] In a first aspect, the present invention provides a single-domain antibody against FOLR1, said single-domain antibody being composed of a heavy chain, the heavy chain including heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;

[0014] The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(7):

[0015] (1) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:22, and CDR3 shown in SEQ ID NO:32;

[0016] (2) CDR1 shown in SEQ ID NO:15, CDR2 shown in SEQ ID NO:24, and CDR3 shown in SEQ ID NO:29;

[0017] (3) CDR1 shown in SEQ ID NO:19, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:34;

[0018] (4) CDR1 shown in SEQ ID NO:20, CDR2 shown in SEQ ID NO:25, and CDR3 shown in SEQ ID NO:33;

[0019] (5) CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:28, and CDR3 shown in SEQ ID NO:34;

[0020] (6) CDR1 shown in SEQ ID NO:17, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:31;

[0021] (7) CDR1 shown in SEQ ID NO:21, CDR2 shown in SEQ ID NO:26, and CDR3 shown in SEQ ID NO:30.

[0022] That is, the heavy chain includes a complementarity-determining region (CDR); the CDR includes the amino acid sequences of heavy chain CDR1, CDR2, and CDR3. The CDR sequences (1)-(7) correspond to SEQ ID NO. 1-7 in sequence. All of the above sequences can be replaced with sequences that have "at least 80% homology" or sequences that replace only one or a few amino acids; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0023] In a preferred embodiment, the sequence of the single-domain antibody further includes a frame region FR; the frame region FR includes the amino acid sequences of FR1, FR2, FR3 and FR4;

[0024] The frame region FR sequence of the single-domain antibody is one of the following (a)-(g);

[0025] (a) FR1 shown in SEQ ID NO:36, FR2 shown in SEQ ID NO:39, FR3 shown in SEQ ID NO:49, FR4 shown in SEQ ID NO:52, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR;

[0026] (b) FR1 shown in SEQ ID NO:36, FR2 shown in SEQ ID NO:44, FR3 shown in SEQ ID NO:51, FR4 shown in SEQ ID NO:52, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR;

[0027] (c) FR1 shown in SEQ ID NO:36, FR2 shown in SEQ ID NO:42, FR3 shown in SEQ ID NO:48, FR4 shown in SEQ ID NO:52, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR;

[0028] (d) FR1 shown in SEQ ID NO:36, FR2 shown in SEQ ID NO:40, FR3 shown in SEQ ID NO:50, FR4 shown in SEQ ID NO:52, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR;

[0029] (e) FR1 shown in SEQ ID NO:38, FR2 shown in SEQ ID NO:42, FR3 shown in SEQ ID NO:47, FR4 shown in SEQ ID NO:52, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR;

[0030] (f) FR1 shown in SEQ ID NO:35, FR2 shown in SEQ ID NO:43, FR3 shown in SEQ ID NO:45, FR4 shown in SEQ ID NO:52, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR;

[0031] (g) FR1 shown in SEQ ID NO:37, FR2 shown in SEQ ID NO:41, FR3 shown in SEQ ID NO:46, FR4 shown in SEQ ID NO:52, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR.

[0032] In one embodiment, the single-domain antibody against FOLR1 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with an amino acid sequence selected from SEQ ID NO: 1-7, and is capable of specifically binding to the FOLR1 antigen.

[0033] In another preferred embodiment, the single-domain antibody against FOLR1 has at least 95% sequence homology with the amino acid sequences selected from SEQ ID NO: 1-7 and is capable of specifically binding to the FOLR1 antigen.

[0034] A second aspect of the invention is to provide single-domain antibodies against FOLR1, said single-domain antibodies being as shown in SEQ ID NO. 1-7, or said single-domain antibodies having at least 95% sequence homology with the amino acid sequences of SEQ ID NO. 1-7.

[0035] In one embodiment, the nucleic acid molecule encoding the single-domain antibody against FOLR1 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with nucleotide sequences selected from SEQ ID NO: 8-14, and the single-domain antibody against FOLR1 encoded therein is capable of specifically binding to the FOLR1 antigen.

[0036] Preferably, the coding sequences of the single-domain antibodies are as shown in SEQ ID NO. 8-14, or have at least 95% sequence homology with SEQ ID NO. 8-14.

[0037] A third aspect of the present invention is to provide an Fc fusion antibody or humanized antibody of the aforementioned single-domain antibody against FOLR1.

[0038] A fourth aspect of the invention is to provide nucleotide molecules encoding the aforementioned single-domain antibody against FOLR1, the nucleotide sequences of which are shown in SEQ ID NO: 8-14, or have at least 95% sequence homology with SEQ ID NO: 8-14.

[0039] A fifth aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding the aforementioned single-domain antibody or the aforementioned Fc fusion antibody.

[0040] A sixth aspect of the present invention is to provide a host cell that can express the aforementioned single-domain antibody against FOLR1, or an expression vector comprising the aforementioned expression vector.

[0041] The present invention also provides a method for generating a single-domain antibody against FOLR1 or an Fc fusion antibody thereto, comprising the steps of: (a) culturing the aforementioned host cells under conditions suitable for generating the single-domain antibody or an Fc fusion antibody thereto, thereby obtaining a culture containing the single-domain antibody against FOLR1 or an Fc fusion antibody thereto; (b) isolating or recovering the single-domain antibody against FOLR1 or an Fc fusion antibody thereto from the culture; and (c) optionally, purifying and / or modifying the single-domain antibody against FOLR1 or an Fc fusion antibody thereto obtained in step (b).

[0042] A seventh aspect of the present invention is to provide a pharmaceutical composition comprising: (i) a single-domain antibody against FOLR1 as described above, or an Fc fusion antibody of a single-domain antibody against FOLR1 as described above; and (ii) one or more pharmaceutically acceptable excipients.

[0043] This invention also provides the use of the aforementioned single-domain antibody against FOLR1 in the preparation of drugs or antitumor drugs that inhibit FOLR1 gene expression. Drugs that inhibit FOLR1 gene expression are applicable to any condition with high FOLR1 gene expression. Preferably, the tumors include, but are not limited to, ovarian cancer, endometrial cancer, breast cancer, and cervical cancer.

[0044] This invention also provides the use of single-domain antibodies against FOLR1 in the preparation of formulations that mediate cell internalization, ADCC, or CDC.

[0045] The present invention also provides the use of the aforementioned single-domain antibody against FOLR1, or the aforementioned Fc fusion antibody against FOLR1, for the preparation of reagents, detection plates or kits; wherein the reagents, detection plates or kits are used to detect the presence and / or content of FOLR1 protein in a sample.

[0046] The single-domain antibody is VHH, which contains only the antibody heavy chain and not the antibody light chain.

[0047] Compared to existing technologies, this invention uses bioengineering technology to screen for single-domain antibodies specifically targeting FOLR1. These antibodies exhibit significant initial affinity and can block the release of cytokines from specific cells. They also demonstrate good binding activity upon prokaryotic expression and possess certain drug-like properties. These single-domain antibodies have the following advantages:

[0048] (1) These single-domain antibodies have flexible expression systems that can be expressed in prokaryotic systems as well as in eukaryotic systems of yeast cells or mammalian cells. Moreover, the expression cost in prokaryotic systems is low, which can reduce the production cost in the later stages.

[0049] (2) Since single-domain antibodies are single-domain antibodies, it is easier to modify their multiple combination forms. Multivalent and multispecific antibodies can be obtained by simple tandem through genetic engineering. Moreover, their immune heterogeneity is very low, and they will not produce a strong immune response without humanization.

[0050] (3) As reported in many literatures, single-domain antibodies have a wider affinity range. Before affinity maturation, their affinity range can be from nM to pM, providing multiple options for antibodies for different purposes later. Attached Figure Description

[0051] Figure 1 SDS-PAGE analysis of recombinant human FOLR1 protein;

[0052] Figure 2 VHH sequence insertion rate analysis;

[0053] Figure 3 Enrichment of the FOLR1-targeted library;

[0054] Figure 4 SDS-PAGE of FOLR1 target partial prokaryotic expression antibody;

[0055] Figure 5 SDS-PAGE of FOLR1 target partial eukaryotic expression antibody;

[0056] Figure 6 FOLR1 target antibody antigen-binding activity;

[0057] Figure 7 FOLR1 target tool antibody antigen binding activity;

[0058] Figure 8 FOLR1 target antibody species cross-reactivity antigen binding activity;

[0059] Figure 9 FOLR1 target antibody ADCC activity;

[0060] Figure 10 FOLR1-targeted antibody CDC activity;

[0061] Figure 11 FOLR1 target antibody internalization activity. Detailed Implementation

[0062] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0063] Single-domain antibodies (sdAbs, also referred to as nanobodies or VHHs by the developer Ablynx) are well known to those skilled in the art. A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Therefore, a single-domain antibody contains a single complementarity-determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies include antibodies containing only the heavy chain (which naturally does not contain a light chain), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0064] Single-domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species such as camels, dromedaries, llamas, and guanacos. Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. Single-domain antibodies may contain only variable domains of the immunoglobulin chain, which have CDR1, CDR2, and CDR3, as well as a frame region.

[0065] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% homology.

[0066] In this invention, sequences with high homology to the CDR1-3 sequences disclosed in this invention can also be used to obtain nanobodies targeting FOLR1. In some embodiments, sequences with "at least 80% homology", or "at least 85% homology", "at least 90% homology", "at least 95% homology", or "at least 98% homology" with the sequences in (1)-(7) can achieve the purpose of the invention (i.e., derived proteins).

[0067] In some embodiments, the inventive objective can also be achieved by replacing only one or a few amino acids compared to the sequences in (1)-(7), for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art can consider so-called “conserved” amino acid substitutions, in which case the substitution will preferably be a conserved amino acid substitution, which can generally be described as an amino acid residue being replaced by another amino acid residue having a similar chemical structure, and the substitution having little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. The particularly preferred conserved amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. Furthermore, those skilled in the art will understand that the inventiveness of the single-domain antibody lies in the CDR1-3 regions, while the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0068] The preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0069] This patent involves preparing a target protein and a truncated form of the target protein using genetic engineering technology. The obtained antigen protein is then used to immunize Bactrian camels in Alashan, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. Through genetic engineering, the variable region coding sequence of the camel-derived antibody is recombined into a phage display vector. Specific antibodies against the antigen protein are screened using phage display technology, and their ability to bind to the antigen and their application in the treatment of autoimmune diseases are further tested.

[0070] The above technical solution will now be broken down and explained in detail, and described with specific embodiments:

[0071] Example 1: Preparation of recombinant human FOLR1 extracellular domain protein:

[0072] The human recombinant extracellular domain protein used in this patent was expressed and purified by the company itself. The specific design scheme of the expression vector for the human recombinant FOLR1 protein is as follows:

[0073] (1) The coding sequence of FOLR1 was obtained by searching in NCBI. Its accession number is NM_000802.3. The accession number of the amino acid sequence encoded by this sequence is NP_000793.1, and the Uniprot ID is P15328.

[0074] (2) The amino acid sequence corresponding to NP_000793.1 was analyzed for transmembrane region and extracellular terminus using TMHMM and SMART websites, respectively.

[0075] (3) The analysis results show that the extracellular terminus of the FOLR1 protein is amino acid 1-233, of which position 1-24 is the signal peptide of the protein.

[0076] (4) The nucleotide sequence encoding the FOLR1 protein from amino acid 1 to 233 was cloned into the vector pcDNA3.4 using gene synthesis.

[0077] (5) The constructed vector was subjected to Sanger sequencing. After comparing with the original sequence and confirming that there were no errors, the recombinant plasmid was extracted in batches, endotoxin was removed, and it was transfected into suspension 293F for expression and purification of the target protein. The SDS-PAGE analysis results of the purified FOLR1 recombinant protein are as follows: Figure 1 As shown, the purified protein has a purity of up to 90%, which meets the requirements for animal immunity.

[0078] Example 2: Construction of a single-domain antibody library targeting the FOLR1 protein:

[0079] One mg of the purified human recombinant FOLR1 protein obtained in step 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alashan, Inner Mongolia. The camel was immunized once a week for a total of 7 weeks. Except for the first immunization, the remaining six immunizations were performed by mixing one mg of FOLR1 protein with an equal volume of Freund's incomplete adjuvant. This immunization process was intended to concentrate the stimulation of the camel to produce antibodies against the FOLR1 protein.

[0080] After animal immunization, 150 mL of peripheral blood lymphocytes were collected from camels, and RNA was extracted from the cells. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified using nested PCR with the cDNA as a template.

[0081] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments were ligated to the vector. The ligated fragments were electroporated into competent TG1 cells to construct a phage display library of the FOLR1 protein, and the library size was determined to be approximately 1 × 10⁻⁶. 9 Simultaneously, the correct insertion rate of the target fragment in the library was detected by colony PCR identification, and the results are as follows: Figure 2 As shown.

[0082] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 28 clones were able to amplify a band of 600 bp (predicted size), while 2 clones amplified an incorrect band. Therefore, the correct insertion rate was 28 ÷ 30 × 100% ≈ 86.6%.

[0083] Example 3: Screening for single-domain antibodies against the FOLR1 protein:

[0084] Take 200 μL of the recombinant TG1 cells from step 2 and culture them in 2×TY medium. During this period, add 40 μL of helper phage VCSM13 to infect the TG1 cells and culture them overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and collect the amplified phage by centrifugation.

[0085] 500 μg of FOLR1 protein diluted in 100 mM pH 8.3 NaHCO3 was coupled onto an ELISA plate and incubated overnight at 4°C, with a negative control well included. The next day, 200 μL of 3% skim milk was added, and the plate was blocked at room temperature for 2 hours. After blocking, 100 μL of the amplified phage library (approximately 2 × 10⁻⁶) was added. 11 (1 phage particle), incubate at room temperature for 1 hour; after 1 hour, wash 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0086] Phages specifically binding to the FOLR1 protein were dissociated using trypsin at a final concentration of 25 mg / mL and then used to infect *E. coli* TG1 cells in logarithmic growth phase. The cells were cultured at 37°C for 1 hour, and the resulting phages were collected for the next round of screening. This screening process was repeated once to gradually enrich the cells. When the enrichment factor reached 10-fold or greater, the enrichment effect was as follows: Figure 3 As shown.

[0087] Figure 3 In this context, P / N = the number of monoclonal bacteria grown from phages eluted from positive wells in the biopanning process after infecting TG1 bacteria / the number of monoclonal bacteria grown from phages eluted from negative wells after infecting TG1 bacteria. This parameter gradually increases after enrichment occurs. I / E = the total number of phages added to positive wells in each round of the biopanning process / the total number of phages eluted from positive wells in each round of the biopanning process. This parameter gradually approaches 1 after enrichment occurs.

[0088] Example 4: Screening for FOLR1-specific positive clones using phage enzyme-linked immunosorbent assay (ELISA):

[0089] According to the screening method in Example 3 above, single-domain antibodies against the antiFOLR1 protein were screened for three rounds. The phage enrichment factor against the antiFOLR1 protein reached more than 10. After screening, 384 single colonies were selected from the positive clones and inoculated into 96-well plates of 2×TY medium containing 100 μg / mL ampicillin. A blank control was set up. After incubation at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and incubated overnight at 28°C.

[0090] Crude antibody was obtained using the osmotic burst method. FOLR1 recombinant protein was released into 100 mM NaHCO3 (pH 8.3), and 100 μg of protein was coated overnight at 4°C in an ELISA plate. 100 μL of the obtained crude antibody extract was transferred to an ELISA plate containing the antigen and incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and 100 μL of Mouse Anti-HA tag Antibody (HRP) (Mouse anti-HA horseradish peroxidase labeled antibody, ThermoFisher) diluted 1:2000 was added. The plate was incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and horseradish peroxidase chromogenic solution was added. The reaction was carried out at 37°C for 15 min, and then stop solution was added. The absorbance was read at 450 nm using a microplate reader.

[0091] When the OD value of the sample well is more than 5 times that of the control well, it is determined to be a positive clone well. The bacteria in the positive clone well are transferred to LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0092] Gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while clones with different sequences were considered different clones. Finally, single-domain antibodies specifically targeting the FOLR1 protein were obtained (SEQ ID NO. 1-7 for single-domain antibodies 1D11, 2D8, 2E5, 3B3, 3H11, 4B10, and 4E11, and single-domain antibodies 1C9, 1D4, 1E5, 1E9, 2D11, 2D9, 2E3, 2E7, 3H9, 4C8, and 4D10, respectively).

[0093] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, constituting the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system to ultimately obtain the single-domain antibody protein.

[0094] The CDR, FR, and amino acid sequences of the seven single-domain antibodies are shown in Tables 1, 2, and 3, respectively.

[0095] Table 1. CDR sequences of 7 single-domain antibodies

[0096] Actual clone number CDR1 SEQ ID Actual clone number CDR2 SEQ ID 2D8-hFC1 DYTYRRYC SEQ ID NO:15 1D11-hFC1 ISSGGST SEQ ID NO:22 1D11-hFC1 GDTYSSAC SEQ ID NO:16 4B10-hFC1 ISWNGGST SEQ ID NO:23 4B10-hFC1 GFTFDDYA SEQ ID NO:17 2D8-hFC1 IYLGDGTT SEQ ID NO:24 3H11-hFC1 GHTYSRLS SEQ ID NO:18 3B3-hFC1 IYTGESMT SEQ ID NO:25 2E5-hFC1 GYPYHRVS SEQ ID NO:19 4E11-hFC1 IYTGGGST SEQ ID NO:26 3B3-hFC1 GYTYRRLS SEQ ID NO:20 2E5-hFC1 IYTGGLYTGGSMT SEQ ID NO:27 4E11-hFC1 GYTYSSNS SEQ ID NO:21 3H11-hFC1 IYTGGPSGPMT SEQ ID NO:28

[0097]

[0098] Table 2. FR sequences of 7 single-domain antibodies

[0099]

[0100]

[0101] Actual clone number FR3 SEQ ID 4B10-hFC1 FYADSVKGRFTIVRDNAKNTVYLEMNSLKPEDTAMYYC SEQ ID NO:45 4E11-hFC1 YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYC SEQ ID NO:46 3H11-hFC1 YYADSVKGRFTISQDNAQRTVYLQMNSLKPEDTAMYYC SEQ ID NO:47 2E5-hFC1 YYADSVKGRFTISQDNARNTVYLQMNSLKPEDTAMYYC SEQ ID NO:48 1D11-hFC1 YYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAMYYC SEQ ID NO:49 3B3-hFC1 YYADSVRGRFTISQDNAQNTVYLQMNSLKPEDTATYYC SEQ ID NO:50 2D8-hFC1 YYTDSVKGRFTISQENAKNTIYLQMNNLKPEDTAMYYC SEQ ID NO:51

[0102]

[0103] Table 3. Amino acid sequences of seven single-domain antibodies

[0104]

[0105] Example 5: Purification and expression of a specific single-domain antibody against the FOLR1 protein in the host bacterium *Escherichia coli*.

[0106] The plasmids (pMECS-VHH) of different clones obtained from the sequencing analysis in Example 4 were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated overnight at 37°C. Single colonies were selected and inoculated into 5 mL of LB medium containing penicillin and incubated overnight on a shaker at 37°C.

[0107] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium and incubate at 37°C in a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and incubate overnight at 28°C in a shaker. Centrifuge to collect E. coli and obtain crude antibody extract using the osmotic rupture method.

[0108] The antibody was purified by nickel column affinity chromatography. The purified single-domain antibody, such as... Figure 4 As shown, it includes VHH1 to 18. Figure 4 VHH1-18 correspond to single-domain antibodies 1C9, 1D11, 1D4, 3B3, 1E5, 1E9, 2D11, 2D8, 2D9, 2E3, 2E5, 2E7, 3H9, 4B10, 3H11, 4C8, 4D10, and 4E11, respectively. The sequences of 1D11, 2D8, 2E5, 3B3, 3H11, 4B10, and 4E11 are shown in SEQ ID NO:1-7, respectively. The sequences of the other single-domain antibodies are not shown (as they are single-domain antibodies that are not technically effective or do not need to be protected in this application).

[0109] Example 6: Construction of a eukaryotic expression vector for an Fc fusion antibody of a specific single-domain antibody against the FOLR1 protein.

[0110] (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened in Example 4 was obtained by Sanger sequencing to obtain its nucleotide sequence;

[0111] (2) The codon-optimized nucleotide sequence (SEQ ID NO. 8-14) was synthesized into the vector RJK-V4-hFC designed and modified by our company by sequence synthesis. The modification method of the vector is as described in Example 10.

[0112] (3) The recombinant eukaryotic expression vector constructed by the company was transformed into DH5α Escherichia coli, cultured for plasmid extraction, and endotoxin was removed;

[0113] (4) The plasmids after large-scale extraction were then sequenced and identified.

[0114] (5) After confirming the recombinant vector, prepare it for subsequent eukaryotic cell transfection and expression. After expressing the Fc protein of VHH by the method of Example 7 or 8, purify the above antibody by the method of Example 9.

[0115] Example 7: Expression of Fc fusion antibody of FOLR1 protein-specific single-domain antibody in suspension ExpiCHO-S cells

[0116] (1) Three days before transfection, use 2.5×10 5 / mL cell passage and expansion culture ExpiCHO-S TM Cells, the calculated desired cell volume, were transferred to 120 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium, the cell concentration was increased to approximately 4 × 10⁻⁶ cells / mL. 6 -6×10 6 live cells / mL;

[0117] (2) One day before transfection, ExpiCHO-S TM Cells were diluted to a concentration of 3.5 × 10⁻⁶. 6 Live cells / mL, incubate cells overnight;

[0118] (3) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 7 × 10⁻⁶ cells / day before transfection. 6 -10×10 6 live cells / mL;

[0119] (4) Use fresh ExpiCHO preheated to 37°C TM The expression medium was used to dilute the cells to 6 × 10⁶. 6 viable cells / mL. The calculated desired cell volume was transferred to 100 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium;

[0120] (5) Gently invert to mix ExpiFectamine TM CHO reagent, using 3.7 mL OptiPRO TM Culture medium for diluting ExpiFectamine TM CHO reagent, vortex or mix well;

[0121] (6) Use 4 mL of refrigerated OptiPRO TM Dilute the plasmid DNA with the culture medium and vortex to mix; the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the specific single-domain antibody of FOLR1 protein obtained in Example 6.

[0122] (7) Incubate the ExpiFectamine CHO / plasmid DNA complex at room temperature for 1-5 minutes, then gently add it to the prepared cell suspension while gently shaking the flask during the addition process;

[0123] (8) The cells were cultured with shaking in humidified air at 37°C and 8% CO2.

[0124] (9) Add 600ul of ExpiFectamine on the first day after transfection (18-22 hours later). TM CHO Enhancer and 24mLExpiCHO feed.

[0125] (10) Collect the supernatant about 8 days after transfection (when cell viability is less than 70%).

[0126] Example 8: Expression of Fc fusion antibody of FOLR1 protein-specific single-domain antibody in suspension 293F cells

[0127] Recombinant single-domain antibody expression experimental procedure (taking a 500mL shake flask as an example):

[0128] (1) Three days before transfection, use 2.5×10 5 After passage and expansion of 293F cells at / mL, the calculated desired cell volume was transferred to a 500mL shake flask containing 120mL (final volume) of fresh, preheated OPM-293CD05 Medium to achieve a cell concentration of approximately 2×10⁻⁶. 6 -3×10 6 Live cells / mL.

[0129] (2) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 2 × 10⁻⁶ cells / day before transfection. 6 -3×10 6 Live cells / mL.

[0130] (3) Dilute the cells to 1×10⁻⁵ using preheated OPM-293CD05 Medium. 6 1 live cells / mL. Calculate the required cell volume and transfer it to a 500 mL shake flask containing 100 mL (final volume) of fresh, preheated culture medium.

[0131] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, swirl or pipette to mix; dilute plasmid DNA with 4 mL Opt-MEM medium, swirl to mix, and filter through a 0.22 μm filter. Incubate at room temperature for 5 min.

[0132] (5) Add the diluted PEI reagent to the diluted DNA and mix by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15-20 minutes, then gently add it to the prepared cell suspension while gently shaking the flask during the addition process.

[0133] (6) Culture the cells at 37°C, 5% CO2, and 120 rpm with shaking.

[0134] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0135] (8) Collect the supernatant about 7 days after transfection (when cell viability is less than 70%).

[0136] Example 9: Purification of human Fc recombinant single-domain antibody

[0137] (1) The protein expression supernatant obtained in Example 7 or 8 was filtered with a 0.45 μm disposable filter to remove insoluble impurities;

[0138] (2) The above filtrate was purified by affinity chromatography using a protein purifier. The agarose packing material coupled with Protein A was used to purify the filtrate by utilizing the ability of human Fc to bind to Protein A.

[0139] (3) Pass the filtrate through a pre-packed Protein A column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the packing material.

[0140] (4) Wash the impurity proteins bound to the column with low-salt and high-salt buffers;

[0141] (5) Use a low pH buffer to systemically bind the target protein on the column;

[0142] (6) Quickly add the eluent to a Tris-HCl solution with a pH of 9.0 to neutralize it;

[0143] (7) After dialyzing the neutralized protein solution, perform SDS-PAGE analysis, such as... Figure 5 As shown, Figure 5 In the text 1-7, the single-domain antibodies 1D11, 2D8, 2E5, 3B3, 3H11, 4B10, and 4E11 are respectively. After confirming that the protein purity is above 95% and the concentration is above 0.5 mg / mL, they are stored at low temperature for later use.

[0144] Example 10: Construction of the eukaryotic expression vector RJK-V4-hFc for nanobodies

[0145] The commonly used target vector for nanobodies, RJK-V4-hFC, is the same as the commercially available vector pCDNA3.4 from Invitrogen (vector information link: [link to Invitrogen]).

[0146] This vector was modified from the human IgG heavy chain coding sequence (NCBI Accession No.: AB776838.1) by fusing the Fc region from the sequence (https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf). Specifically, this vector contains the CH2 and CH3 hinge regions of the IgG heavy chain. The specific modification scheme is as follows:

[0147] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0148] (2) Multiple cloning sites (MCS) and 6×His tags were introduced at the 5' and 3' ends of the Fc fragment coding sequence, respectively, by overlapping PCR.

[0149] (3) The above fragment was amplified by PCR using a pair of primers with XbaI and AgeI restriction sites respectively;

[0150] (4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction endonucleases XbaI and AgeI, respectively;

[0151] (5) The digested vector and the insert fragment were ligated with T4 ligase, and then the ligation product was transformed into E. coli, amplified, sequenced and verified to obtain the recombinant plasmid.

[0152] Example 11: Humanization of a single-domain antibody against FOLR1

[0153] The humanization method was completed using the nanobody humanization platform based on phage display technology, which was independently developed by our company.

[0154] The humanization process is as follows:

[0155] (1) Establish amino acid sequence databases for the heavy chain variable region of human antibodies and camel antibodies respectively;

[0156] (2) Compare the differences between the amino acid sites in the FR region of human antibodies and camel antibodies, and make statistics on the types and proportions of amino acids at different amino acid sites in each FR region.

[0157] (3) Based on the differences in the types and proportions of amino acids at each site, insert the CDR region of the sequence to synthesize the library or use computer simulation to model homology.

[0158] (4) The synthetic library and homology modeling data of the sequence are screened to select the clone with the most humanized sites and better affinity and function, which is the optimal humanized VHH.

[0159] Example 12: Determination of the binding dose-response curve of a specific single-domain antibody (prokaryotic cell) to FOLR1 protein.

[0160] (1) Coat 50 μL of 1 μg / mL FOLR1 overnight at 4℃.

[0161] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 1 hour.

[0162] (3) Dilute VHH to 2 μg / mL, and then perform a 5-fold serial dilution of the antibody to a total of 8 concentration gradients. Here, VHH refers to the specific single-domain antibody against FOLR1 protein prepared by prokaryotic expression in Example 5.

[0163] (4) Wash the plate; add 50 μL of single-domain antibody diluted in step (3), double replicates, and incubate at 37°C for 1 h.

[0164] (5) Wash the plate; add 50 μL of mouse anti-HA tag-HRP secondary antibody and incubate at 37℃ for 30 min.

[0165] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought back to room temperature, and react at room temperature in the dark for 15 min.

[0166] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0167] (8) Plot the curve and calculate EC50, as follows: Figure 6 As shown, the single-domain antibodies 2D8, 1D11, 4B10, 3H11, 2E5, 3B3, and 4E11 of the present invention exhibit excellent binding efficacy and specificity to the FOLR1 protein.

[0168] Example 13: Expression and purification of a tool antibody (Tab) targeting human FOLR1

[0169] The Tab (farletuzumab) sequence is derived from IMGT.

[0170] The searched sequences were commissioned to General Biosystems (Anhui) Co., Ltd. for codon optimization in mammalian cell expression systems and cloned into the pcDNA3.1 vector.

[0171] After resistance screening, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50).

[0172] Add 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) per 100 mL of cells and transiently transpose in 293F cells (culture medium: FreeStyle 293Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032) using PEI;

[0173] 6–24 h after transfection, add 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) and incubate with 8% CO2 at 130 rpm for about 7–8 days.

[0174] When cell viability dropped to 50%, the expression supernatant was collected and purified using a Protein A (GE, Cat#17-5438-02) gravity column.

[0175] After PBS dialysis, the concentration was determined using Nanodrop, the purity was identified by SEC, and the binding capacity was verified by indirect ELISA.

[0176] The Tab obtained by this method has a concentration of not less than 2 mg / ml and a purity greater than 94%. Its EC50 for binding with FOLR1 (ACRO, Cat#FO1-H52H1) is approximately 0.04-0.9 nM. The results are as follows: Figure 7 As shown.

[0177] Example 14: Determination of species cross-binding dose-response curve of specific single-domain antibody against FOLR1 protein

[0178] (1) Coat 50 μL of 1 μg / mL human, cynomolgus monkey, mouse, rat and canine FOLR1, respectively, and incubate at 4℃ overnight.

[0179] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 1 hour.

[0180] (3) VHH-hFc was diluted to 2 μg / mL, and then the antibody was serially diluted 5-fold to a total of 8 concentration gradients. VHH-hFc was obtained by purifying the Fc fusion antibody of the specific single-domain antibody of FOLR1 protein prepared in Example 8 in Example 9, including single-domain antibodies 2D8, 1D11, 4B10, 3H11, 2E5, 3B3, and 4E11.

[0181] (4) Wash the plate; add 50 μL of antibody diluted in step (3), double replicates, and incubate at 37°C for 1 h.

[0182] (5) Wash the plate; add 50 μL of goat anti-human IgG-HRP secondary antibody and incubate at 37°C for 30 min.

[0183] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought back to room temperature, and react at room temperature in the dark for 15 min.

[0184] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0185] (8) Plot the curve and calculate EC50; where hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase; Tab was prepared in Example 13; the results are as follows. Figure 8 As shown, the single-domain antibodies 2D8, 1D11, 4B10, 3H11, 2E5, 3B3, and 4E11 of the present invention exhibit excellent cross-species binding efficacy against the FOLR1 protein.

[0186] Example 15: Human-specific single-domain antibody against FOLR1 and tool antibody-induced ADCC (antibody-dependent cell-mediated cytotoxicity):

[0187] After being revived and passaged for 3-4 generations, OVCAR-3 and SK-OV-3 cells were collected and seeded into 96-well plates at a rate of 20,000 cells per well.

[0188] The Tab, hIgG, and VHH-hFc samples were prepared into solutions with a maximum concentration of 10 μg / mL and serially diluted 10-fold to obtain seven concentrations. VHH-hFc was obtained by purifying the Fc fusion antibody of the specific single-domain antibody of FOLR1 protein prepared in Example 8 in Example 9, including single-domain antibodies 2D8, 1D11, 4B10, 3H11, 2E5, 3B3, and 4E11. Tab was prepared in Example 13.

[0189] Add the serially diluted antibody solution to the cell culture wells in equal volumes to the cell suspension;

[0190] For the sample wells and E / T wells (antibody concentration of 0), Jurkat-NFAT-luc-FcγRIIIa cells were collected and added to the cell culture wells at a rate of 20,000 cells per well.

[0191] After 6 hours of incubation, cell killing was detected using the One-Glo kit, and luminescence was read.

[0192] Calculate the fold of induction: (sample - BG) / (E / T - BG)

[0193] Based on the target cell killing rate and concentration, a four-parameter fitting was performed to calculate the EC50 concentration of each antibody-mediated ADCC effect. The results are as follows: Figure 9 As shown, the seven single-domain antibodies of this invention can mediate ADCC activity.

[0194] Example 16: Human-specific single-domain antibody against FOLR1 and tool antibody-induced CDC (complement-dependent cytotoxicity):

[0195] CHO-K1-FOLR1 is a stable cell line with high FOLR1 expression constructed using a lentiviral method.

[0196] After being revived and passaged for 3-4 generations, CHO-K1-FOLR1 cells were collected and seeded into 96-well plates at a rate of 5000 cells per well.

[0197] The Tab, hIgG, and VHH-hFc samples were prepared into solutions with a maximum concentration of 10 μg / mL and then serially diluted 3-fold to obtain 7 concentrations. VHH-hFc was obtained by purifying the Fc fusion antibody of the specific single-domain antibody of FOLR1 protein prepared in Example 8 in Example 9, including single-domain antibodies 2D8, 1D11, 4B10, 3H11, 2E5, 3B3, and 4E11.

[0198] The serially diluted antibodies were added to CHO-K1-FOLR1 cells and incubated for half an hour.

[0199] Add the serum to the above mixture to achieve a final concentration of 10%;

[0200] After 4 hours of incubation, cell viability was detected using a cell viability assay kit and a multi-functional microplate reader.

[0201] A dose-response curve was established by fitting four parameters to antibody concentration and luminescence intensity, and the results are as follows: Figure 10 As shown, the seven single-domain antibodies of this invention can mediate CDC activity.

[0202] Example 17: Internalization of a single-domain antibody specifically targeting FOLR1 in CHO-K1-FOLR1 cells

[0203] After being revived and passaged for 3-4 generations, CHO-K1-FOLR1 cells were washed with PBS and seeded into 96-well plates at a rate of 500,000 cells per well.

[0204] Phrodo-labeled Tab, hIgG, and single-domain antibodies were prepared into a 10 μg / mL solution; the single-domain antibodies were obtained by purifying the Fc fusion antibody of the specific single-domain antibody of FOLR1 protein prepared in Example 8 in Example 9, including single-domain antibodies 2D8, 1D11, 4B10, 3H11, 2E5, 3B3, and 4E11.

[0205] After centrifuging the CHO-K1-FOLR1 cell suspension, it was resuspended in the prepared antibody solution and incubated on ice and at 37°C for 4 hours at their respective temperatures.

[0206] The fluorescence value of the mixture from the previous step was read using a flow cytometer.

[0207] Based on the fluorescence intensity analysis results, the ratio of the 37℃ sample to the ice sample was calculated. A higher ratio indicates a higher degree of internalization, as shown in the following figures. Figure 11 As shown, single-domain antibodies specifically targeting FOLR1 can achieve internalization in CHO-K1-FOLR1 cells.

[0208] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details. sequence list <110> Nanjing Rongjiekang Biotechnology Co., Ltd. <120> Single-domain antibodies against FOLR1 and their derived proteins and applications <130> GY-03-2021-29 <141> 2021-12-13 <160> 52 <170> SIPOSequenceListing 1.0 <210> 1 <211> 126 <212> PRT <213> Artificial Sequence <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Tyr Ser Ser Ala 20 25 30 Cys Met Asp Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Gly Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Arg Arg Val Gly Arg Ile Pro Cys Arg Thr Met Leu Pro Gly Leu 100 105 110 Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 2 <211> 125 <212> PRT <213> Artificial Sequence <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Tyr Thr Tyr Arg Arg Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val ​​Ala Ala Ile Tyr Leu Gly Asp Gly Thr Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Glu Asn Ala Lys Asn Thr Ile Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Asp Arg Ser Gly Cys Ser Val Thr Trp Ser Tyr Ser Ala Phe 100 105 110 Glu Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 3 <211> 132 <212> PRT <213> Artificial Sequence <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Pro Tyr His Arg Val 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Gly Ile Tyr Thr Gly Gly Leu Tyr Thr Gly Gly Ser Met Thr Tyr 50 55 60 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala 65 70 75 80 Arg Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 85 90 95 Ala Met Tyr Tyr Cys Ala Ala Thr Pro Gln Phe Leu Ile Gly Ser Gly 100 105 110 Leu Leu Arg Pro Asp Lys Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val 115 120 125 Thr Val Ser Ser 130 <210> 4 <211> 127 <212> PRT <213> Artificial Sequence <400> 4 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Arg Arg Leu 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val 35 40 45 Gly Gly Ile Tyr Thr Gly Glu Ser Met Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Gln Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Ala Thr Pro Gln Phe Leu Ile Gly Ser Gly Leu Leu Arg Pro Asp 100 105 110 Lys Tyr Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 5 <211> 130 <212> PRT <213> Artificial Sequence <400> 5 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Pro Arg Leu Ser Cys Ala Val Ser Gly His Thr Tyr Ser Arg Leu 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Gly Ile Tyr Thr Gly Gly Pro Ser Gly Pro Met Thr Tyr Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Gln Arg 65 70 75 80 Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met 85 90 95 Tyr Tyr Cys Ala Ala Thr Pro Gln Phe Leu Ile Gly Ser Gly Leu Leu 100 105 110 Arg Pro Asp Lys Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser 130 <210> 6 <211> 127 <212> PRT <213> Artificial Sequence <400> 6 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Trp Asn Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Val Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Leu Ala Tyr Cys Ser Gly Gly His Trp Ser Pro Gly Asp Leu 100 105 110 Ser Gly Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 7 <211> 127 <212> PRT <213> Artificial Sequence <400> 7 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Thr Tyr Ser Ser Asn 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Tyr Thr Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Pro Thr Trp Tyr Asp Gly Ser Trp Ser Arg Gly Asp 100 105 110 Glu Tyr Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 8 <211> 378 <212> DNA <213> Artificial Sequence <400> 8 caagtgcaac tcgtggaatc cggcggcggg agcgtccaag ccggcggctc cctgagactg 60 agctgcgctg ctagcgggga cacctacagc agcgcctgca tggactggtt cagacaagcc 120 cctggcaagg agagagaggg cgtggccggc atcagcagcg gcggcagcac ctactacgcc 180 gacagcgtga agggcagatt caccatcagc agagacaacg ccaagaacac cctgtacctg 240 cagatgaaca gcctgaagcc cgaggacacc gccatgtact actgcgctgc ccggagagtg 300 ggcagaatcc cctgcagaac catgctgccc ggcctgcaag actactgggg ccaaggcacc 360 caagtgaccg tgagcagc 378 <210> 9 <211> 375 <212> DNA <213> Artificial Sequence <400> 9 caagtgcaac tggtcgagag cgggggcggg agcgtgcaag ccgggggcag cctgagactg 60 caagtgcaac tggtcgagag cgggggcggg agcgtgcaag ccgggggcag cctgagactg 60 agctgcgccg ctagcgacta cacctacaga agatactgca tgggctggtt cagacaagcc 120 agctgcgccg ctagcgacta cacctacaga agatactgca tgggctggtt cagacaagcc 120 cccggcaagg agagagagaa ggtggccgcc atctacctgg gcgacggcac cacctactac 180 cccggcaagg agagagagaa ggtggccgcc atctacctgg gcgacggcac cacctactac 180 accgacagcg tgaagggcag attcaccatc agccaagaga acgccaagaa caccatctac 240 accgacagcg tgaagggcag attcaccatc agccaagaga acgccaagaa caccatctac 240 ctgcagatga acaacctgaa gcccgaggac accgccatgt actactgcgc cgccgacaga 300 ctgcagatga acaacctgaa gcccgaggac accgccatgt actactgcgc cgccgacaga 300 agcggctgca gcgtgacctg gagctacagc gccttcgagg actggggcca aggcacccaa 360 agcggctgca gcgtgacctg gagctacagc gccttcgagg actggggcca aggcacccaa 360 gtgaccgtga gcagc 375 gtgaccgtga gcagc 375 <210> 10<210> 10 <211> 396<211> 396 <212> DNA<212> DNA <213> 人工序列(Artificial Sequence) <213> Artificial Sequence <400> 10 <400> 10 caagtgcaac tggtcgaaag cgggggcggc agcgtgcaag ctggggggtc cctcagactg 60 caagtgcaac tggtcgaaag cgggggcggc agcgtgcaag ctggggggtc cctcagactg 60 agctgcgccg ctagcggcta cccctaccac agagtgagca tgggctggtt cagacaagcc 120 agctgcgccg ctagcggcta cccctaccac agagtgagca tgggctggtt cagacaagcc 120 cccggcaagg agagagaggg cgtggccggc atctacaccg gcggcctcta taccgggggc 180 cccggcaagg agagagaggg cgtggccggc atctacaccg gcggcctcta taccgggggc 180 agcatgacct actacgccga cagcgtgaag ggcagattca ccatcagcca agacaacgct 240 agcatgacct actacgccga cagcgtgaag ggcagattca ccatcagcca agacaacgct 240 agaaacaccg tgtacctgca gatgaacagc ctgaagcccg aggacaccgc catgtactac 300 tgcgccgcca cccctcagtt cctgatcggc agcggcctgc tgagacccga caagtacaac 360 agctggggcc aaggcaccca agtgaccgtg agcagc 396 <210> 11 <211> 381 <212> DNA <213> Artificial Sequence <400> 11 caagtgcagc tcgtggaaag cggcggcggg tccgtgcaag ccggggggag cctgagactg 60 agctgcgccg ctagcggcta cacctacaga agactgagca tgggctggtt cagacaagcc 120 cccggcaagg agagagaggc cgtgggcggc atctacaccg gcgagagcat gacctactac 180 gccgacagcg tgagaggcag attcaccatc agccaagaca acgctcagaa caccgtgtac 240 ctgcagatga acagcctgaa gcccgaggac accgccacct actactgcgc cgccacccct 300 cagttcctga tcggcagcgg cctgctgaga cccgacaagt acgacagctg gggccaaggc 360 acccaagtga ccgtgagcag c 381 <210> 12 <211> 390 <212> DNA <213> Artificial Sequence <400> 12 caagtgcagc tggtggaatc cggcgggggc agcgtgcaaa ccggggggag ccctagactg 60 caagtgcagc tggtggaatc cggcgggggc agcgtgcaaa ccggggggag ccctagactg 60 agctgcgccg tgagcggcca cacctacagc agactcagca tggggtggtt tcggcaagcc 120 agctgcgccg tgagcggcca cacctacagc agactcagca tggggtggtt tcggcaagcc 120 cccggcaagg aaagagaggg cgtggccggc atctacaccg gcggccctag cggccccatg 180 cccggcaagg aaagagaggg cgtggccggc atctacaccg gcggccctag cggccccatg 180 acctactacg ccgacagcgt gaagggcaga ttcaccatca gccaagacaa cgctcagaga 240 acctactacg ccgacagcgt gaagggcaga ttcaccatca gccaagacaa cgctcagaga 240 accgtgtacc tgcagatgaa cagcctgaag cccgaggaca ccgccatgta ctactgcgcc 300 accgtgtacc tgcagatgaa cagcctgaag cccgaggaca ccgccatgta ctactgcgcc 300 gccacccctc agttcctgat cggcagcggc ctgctgagac ccgacaagta caacagctgg 360 gccacccctc agttcctgat cggcagcggc ctgctgagac ccgacaagta caacagctgg 360 ggccaaggca cccaagtgac cgtgagcagc 390 ggccaaggca cccaagtgac cgtgagcagc 390 <210> 13<210> 13 <211> 381<211> 381 <212> DNA<212> DNA <213> 人工序列(Artificial Sequence)<213> Artificial Sequence <400> 13<400> 13 caagtgcaac tcgtcgaatc cggcgggggc ctcgtccaag ctggggggtc cctgagactg 60 caagtgcaac tcgtcgaatc cggcgggggc ctcgtccaag ctggggggtc cctgagactg 60 agctgcaccg ctagcggctt caccttcgac gactacgcca tgggctggtt cagacaagcc 120 agctgcaccg ctagcggctt caccttcgac gactacgcca tgggctggtt cagacaagcc 120 cccggcaagg agagagaggg cgtgagctgc atcagctgga acggcggcag caccttctac 180 cccggcaagg agagagaggg cgtgagctgc atcagctgga acggcggcag caccttctac 180 gccgacagcg tgaagggcag attcaccatc gtgagagaca acgccaagaa caccgtgtac 240 gccgacagcg tgaagggcag attcaccatc gtgagagaca acgccaagaa caccgtgtac 240 ctggagatga acagcctgaa gcccgaggac accgccatgt actattgcgc cgctctcgct 300 tactgtagcg gcgggcactg gagccccggg gatctcagcg gcggctactg gggccaaggc 360 acccaagtga ccgtgagcag c 381 <210> 14 <211> 381 <212> DNA <213> Artificial Sequence <400> 14 caagtgcaac tggtggagag cggcggcggg agcgtccaag ctgggggcag cctgagactg 60 agctgcgccg tgagcggcta cacctacagc agcaacagca tggggtggtt cagacaagcc 120 cccggcaagg agagagaggg ggtcgccgct atctacaccg gcggcgggag cacctactac 180 gccgacagcg tgaagggcag attcaccatc agccaagaca acgccaagaa caccgtgtac 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccggcggc 300 cccacatggt acgacggcag ctggagcaga ggcgacgagt acacctactg gggccaaggc 360 acccaagtga ccgtgagcag c 381 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <400> 15 Asp Tyr Thr Tyr Arg Arg Tyr Cys 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <400> 16 Gly Asp Thr Tyr Ser Ser Ala Cys 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <400> 17 Gly Phe Thr Phe Asp Asp Tyr Ala 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <400> 18 Gly His Thr Tyr Ser Arg Leu Ser 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <400> 19 Gly Tyr Pro Tyr His Arg Val Ser 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <400> 20 Gly Tyr Thr Tyr Arg Arg Leu Ser 1 5 <210> twenty one <211> 8 <212> PRT <213> Artificial Sequence <400> twenty one Gly Tyr Thr Tyr Ser Ser Asn Ser 1 5 <210> twenty two <211> 7 <212> PRT <213> Artificial Sequence <400> twenty two Ile Ser Ser Gly Gly Ser Thr 1 5 <210> twenty three <211> 8 <212> PRT <213> Artificial Sequence <400> twenty three Ile Ser Trp Asn Gly Gly Ser Thr 1 5 <210> twenty four <211> 8 <212> PRT <213> Artificial Sequence <400> twenty four Ile Tyr Leu Gly Asp Gly Thr Thr 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <400> 25 Ile Tyr Thr Gly Glu Ser Met Thr 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <400> 26 Ile Tyr Thr Gly Gly Gly Ser Thr 1 5 <210> 27 <211> 13 <212> PRT <213> Artificial Sequence <400> 27 Ile Tyr Thr Gly Gly Leu Tyr Thr Gly Gly Ser Met Thr 1 5 10 <210> 28 <211> 11 <212> PRT <213> Artificial Sequence <400> 28 Ile Tyr Thr Gly Gly Pro Ser Gly Pro Met Thr 1 5 10 <210> 29 <211> 18 <212> PRT <213> Artificial Sequence <400> 29 Ala Ala Asp Arg Ser Gly Cys Ser Val Thr Trp Ser Tyr Ser Ala Phe 1 5 10 15 Glu Asp <210> 30 <211> 20 <212> PRT <213> Artificial Sequence <400> 30 Ala Ala Gly Gly Pro Thr Trp Tyr Asp Gly Ser Trp Ser Arg Gly Asp 1 5 10 15 Glu Tyr Thr Tyr 20 <210> 31 <211> 20 <212> PRT <213> Artificial Sequence <400> 31 Ala Ala Leu Ala Tyr Cys Ser Gly Gly His Trp Ser Pro Gly Asp Leu 1 5 10 15 Ser Gly Gly Tyr 20 <210> 32 <211> 20 <212> PRT <213> Artificial Sequence <400> 32 Ala Ala Arg Arg Val Gly Arg Ile Pro Cys Arg Thr Met Leu Pro Gly 1 5 10 15 Leu Gln Asp Tyr 20 <210> 33 <211> 20 <212> PRT <213> Artificial Sequence <400> 33 Ala Ala Thr Pro Gln Phe Leu Ile Gly Ser Gly Leu Leu Arg Pro Asp 1 5 10 15 Lys Tyr Asp Ser 20 <210> 34 <211> 20 <212> PRT <213> Artificial Sequence <400> 34 Ala Ala Thr Pro Gln Phe Leu Ile Gly Ser Gly Leu Leu Arg Pro Asp 1 5 10 15 Lys Tyr Asn Ser 20 <210> 35 <211> 25 <212> PRT <213> Artificial Sequence <400> 35 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 36 <211> 25 <212> PRT <213> Artificial Sequence <400> 36 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 37 <211> 25 <212> PRT <213> Artificial Sequence <400> 37 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser 20 25 <210> 38 <211> 25 <212> PRT <213> Artificial Sequence <400> 38 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Pro Arg Leu Ser Cys Ala Val Ser 20 25 <210> 39 <211> 17 <212> PRT <213> Artificial Sequence <400> 39 Met Asp Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala 1 5 10 15 Gly <210> 40 <211> 17 <212> PRT <213> Artificial Sequence <400> 40 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val Gly 1 5 10 15 Gly <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <400> 41 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala 1 5 10 15 Ala <210> 42 <211> 17 <212> PRT <213> Artificial Sequence <400> 42 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala 1 5 10 15 Gly <210> 43 <211> 17 <212> PRT <213> Artificial Sequence <400> 43 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser 1 5 10 15 Cys <210> 44 <211> 17 <212> PRT <213> Artificial Sequence <400> 44 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala 1 5 10 15 Ala <210> 45 <211> 38 <212> PRT <213> Artificial Sequence <400> 45 Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Val Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Val Tyr Leu Glu Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 46 <211> 38 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 46 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn 1 5 10 15 Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 47 <211> 38 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 47 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn 1 5 10 15 Ala Gln Arg Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 48 <211> 38 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 48 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn 1 5 10 15 Ala Arg Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 49 <211> 38 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 49 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 50 <211> 38 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 50 Tyr Tyr Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Gln Asp Asn 1 5 10 15 Ala Gln Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Thr Tyr Tyr Cys 35 <210> 51 <211> 38 <212> PRT <213> Artificial Sequence <400> 51 Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Glu Asn 1 5 10 15 Ala Lys Asn Thr Ile Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <400> 52 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10

Claims

1. A single-domain antibody against FOLR1, characterized in that: The single-domain antibody is composed of heavy chains, including heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are as follows (1) or (2): (1) CDR1 shown in SEQ ID NO:21, CDR2 shown in SEQ ID NO:26, and CDR3 shown in SEQ ID NO:30; (2) CDR1 shown in SEQ ID NO:17, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:

31.

2. The single-domain antibody against FOLR1 according to claim 1, characterized in that: The single-domain antibody against FOLR1 has at least 95% sequence homology with the amino acid sequence of SEQ ID NO: 7 or 6 and is capable of specifically binding to the FOLR1 antigen.

3. The single-domain antibody against FOLR1 according to claim 1, characterized in that: The sequence of the frame region FR of the single-domain antibody is as follows (a) or (b); (a) FR1 shown in SEQ ID NO:37, FR2 shown in SEQ ID NO:41, FR3 shown in SEQ ID NO:46, FR4 shown in SEQ ID NO:52, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR; (b) FR1 shown in SEQ ID NO:35, FR2 shown in SEQ ID NO:43, FR3 shown in SEQ ID NO:45, FR4 shown in SEQ ID NO:52, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR.

4. A single-domain antibody against FOLR1, characterized in that: The amino acid sequences of the single-domain antibodies are shown in SEQ ID NO: 7 or 6, respectively.

5. The single-domain antibody against FOLR1 according to claim 4, characterized in that: The coding sequences of the single-domain antibodies are shown in SEQ ID NO: 14 or 13, respectively.

6. The Fc fusion antibody or humanized antibody of the single-domain antibody against FOLR1 as described in any one of claims 1-5.

7. A polynucleotide molecule encoding the FOLR1 single-domain antibody according to any one of claims 1-5, characterized in that: Their nucleotide sequences are shown in SEQ ID NO: 14 or 13, respectively.

8. An expression carrier, characterized in that, It comprises a polynucleotide molecule encoding a single-domain antibody as described in any one of claims 1-5, an Fc fusion antibody as described in claim 6, or a polynucleotide molecule as described in claim 7.

9. A host cell, characterized in that, It can express the single-domain antibody against FOLR1 as described in any one of claims 1-5, or the expression vector as described in claim 8.

10. The use of the single-domain antibody against FOLR1 according to any one of claims 1-5 in the preparation of an antitumor drug, characterized in that, The tumors mentioned are ovarian cancer, endometrial cancer, breast cancer, or cervical cancer.

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