Anti-folr1 nanobody and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an anti-FOLR1 nanobody and a preparation method and application thereof. The anti-FOLR1 nanobody comprises a framework region and a complementarity determining region, and the complementarity determining region amino acid comprises CDR1, CDR2 and CDR3. The application adopts automatic panning and a mammalian expression system, significantly improves the screening efficiency and antibody expression quality, greatly shortens the development cycle, and provides an efficient tool for FOLR1 targeted diagnosis and treatment. The obtained anti-FOLR1 nanobody exhibits excellent specific recognition and binding capacity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-FOLR1 nanobody, its preparation method, and its application. Background Technology

[0002] Folic acid is a B vitamin that plays a crucial role in cellular single-carbon metabolism. In the human body, folic acid receptors (FRs) are high-affinity, low-flux transport proteins, typically expressed on the cell surface. The transport process involves folic acid binding to FRs on the cell membrane, forming endocytic vesicles through receptor-mediated endocytosis. These vesicles then enter and circulate in early endosomes. The acidic environment (low pH) within the endosome compartment effectively promotes the dissociation of folic acid from the receptor, allowing it to be released into the cytoplasm for utilization. [1] .

[0003] Four FOLR genes have been identified: FOLR1-4 (also known as folate receptors α, β, γ, and δ; historically called folate-binding protein, FBP). Among them, FOLR1 (encoding FRα) and FOLR2 (encoding FRβ) are the receptors that primarily bind folate on the cell surface. After binding, they carry folate into the cell via endocytosis. Importantly, these two receptors, especially FOLR1, have been shown to be overexpressed in various cancers, and this overexpression is generally associated with accelerated cancer progression and poor patient prognosis. [2] Furthermore, the study also found that FOLR1 is not only associated with the development and progression of cancer, but the loss of function of its folate receptor α (FOLR1) protein can also lead to a rare neurological disorder—FOLR1-associated folate transport deficiency in the brain (FOLR1-CFTD). [3] .

[0004] Recent advances in molecular targeted therapy have brought revolutionary breakthroughs to cancer detection and treatment. Folate receptors (FRs), especially alpha folate receptor 1 (FOLR1), are promising cancer biomarkers, showing outstanding performance in the detection of ovarian and breast cancer due to their significantly higher expression levels in cancer cells compared to normal cells. The overexpression of FRs in cancer cells enables folic acid-coupled drugs to achieve precise targeted therapy. This treatment approach can specifically attack cancer cells without damaging healthy cells, thereby reducing side effects and improving treatment efficacy. [4] In vivo studies using a mouse tumor xenograft model expressing human folate receptor α (FRα) have shown that anti-FRα monoclonal antibodies can inhibit tumor growth. In these experiments, researchers transplanted human cancer cells overexpressing FRα into mice and subsequently treated the mice with anti-FRα monoclonal antibodies. [5]Although folate receptor α (FOLR1) is typically expressed only on the apical membrane surface of lung, kidney, and choroid plexus epithelial cells in healthy individuals, it is overexpressed in various solid tumors, including high-grade osteosarcoma, breast cancer, ovarian cancer, and non-small cell lung cancer. Therefore, FOLR1 has become a highly attractive target in cancer detection and treatment, especially for cancers prevalent in women. Several FOLR1-targeted cancer treatment strategies have been developed, including FOLR1-targeting imaging agents for cancer diagnosis and therapies that deliver cytotoxic drugs to FOLR1-overexpressing cancer cells via folate conjugates. [6] .

[0005] However, existing FOLR1-targeted therapies, especially those based on traditional monoclonal antibodies, still face several challenges. Traditional antibodies have large molecular weights, which may lead to insufficient penetration into solid tumors; their complex structures result in high production costs; and there are potential risks of immunogenicity. Therefore, there is an urgent need in the field to develop a novel, high-affinity FOLR1-binding molecule with better tissue penetration, lower immunogenicity, and easier production, to overcome the limitations of existing technologies and provide a better tool for cancer diagnosis and treatment.

[0006] References:

[0007] [1]Zhao R, Matherly LH, Goldman ID. Mechanisms of membrane transport of folates into cells and across epithelia. Annu Rev Nutr31, 177–201.10.1146 / annurev-nutr-072610-145133 [PubMed: 21568705]

[0008] [2]Nunez S, Matherly LH, Wilson MR, Hou Z. Molecular targeting of cancer-associated folate receptors in folate deprivation: A novel approach to enhance anticancer therapy. Trends Endocrinol Metab. 2022;33(3):159-174. doi:10.1016 / j.tem.2021.12.003. PMID: 35094917.

[0009] [3]Baker PR II, Watkins PA. *HSD17B4-Related Disorders*. In: Adam MP, Feldman J, Mirzaa GM, et al., eds. GeneReviews® [Internet]. University of Washington, Seattle; August 15, 2019. Updated January 11, 2024. Accessed.PMID: 38224559.

[0010] [4]Clinical translation of folate receptor-targeted therapeutics,Expert Opin. Drug Deliv. 9 (8) (2012) 901–908, https: / / doi.org / 10.1517 / 17425247.2012.694863.

[0011] [5]Identification of a peptide for folate receptor alpha by phagedisplay and its tumor targeting activity in ovary cancer xenograft, Sci. Rep. 8 (1) (2018) 8426, https: / / doi.org / 10.1038 / s41598-018-26683-z.

[0012] [6]Life Sci. 2023 Aug 1;326:121802. doi: 10.1016 / j.lfs.2023.121802.Epub 2023 May 25.PMID: 37244363 Summary of the Invention

[0013] The purpose of this invention is to overcome the shortcomings of the prior art and provide an anti-FOLR1 nanobody, its preparation method, and its application.

[0014] The technical solution of this invention to solve the technical problem is as follows:

[0015] In a first aspect of the invention, an anti-FOLR1 nanobody is provided, the anti-FOLR1 nanobody comprising a framework region and a complementarity-determining region, the complementarity-determining region comprising CDR1, CDR2, and CDR3; the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 are selected from group 1) or group 2).

[0016] 1) The complementary decision region CDR1 sequence is shown in SEQ ID NO: 1, the complementary decision region CDR2 sequence is shown in SEQ ID NO: 2, and the complementary decision region CDR3 sequence is shown in SEQ ID NO: 3;

[0017] 2) The amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 are at least 90% identical to the amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 described in group 1), and retain the ability to bind FOLR1.

[0018] Furthermore, the amino acid sequence of the heavy chain variable region of the anti-FOLR1 nanobody is shown in SEQ ID NO:4.

[0019] In a second aspect of the invention, a nucleic acid molecule is provided that encodes an anti-FOLR1 nanobody as described in the first aspect.

[0020] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 5.

[0021] In a third aspect of the invention, a carrier is provided, the carrier comprising the nucleic acid molecule described in the second aspect.

[0022] In a fourth aspect of the invention, a host cell is provided, the host cell comprising the vector described in the third aspect. Preferably, the host cell is a eukaryotic cell.

[0023] In a fifth aspect of the present invention, a method for preparing an anti-FOLR1 nanobody is provided, comprising the following steps:

[0024] S1. Based on the protein and gene sequence information of FOLR1, analyze and design immune antigens;

[0025] S2. Immunize alpacas with the antigen obtained in step S1, test the titer of the serum separated from the immunized alpacas, and then extract alpaca PBMC cells from the peripheral blood.

[0026] S3. Using the alpaca PBMC cells obtained in step S2 as raw materials, total RNA was extracted, and cDNA fragments were obtained by reverse transcription PCR. The VHH gene fragment was amplified using this as a template. The target gene fragment was then cloned into a phage vector and transformed into competent cells to construct a phage library.

[0027] S4. Using the phage library constructed in step S3, phages are packaged, and phages are enriched by panning with the assistance of automated equipment. Single clones are then selected for initial screening, positive clones are sent for testing, and sequencing analysis is performed to select the correct antibody sequence for eukaryotic expression.

[0028] S5. Using the antibody expressed in step S4, perform antigen protein and overexpression cell line binding detection to screen for antibodies with high specificity and high affinity, thereby obtaining the anti-FOLR1 nanobody.

[0029] Furthermore, the eukaryotic expression vector described in step S4 is the PcDNA3.4 vector.

[0030] In a sixth aspect of the invention, the use of the anti-FOLR1 nanobody as described in the first aspect is provided in the preparation of products for detection, affinity purification or binding to FOLR1; said products include detection reagents or kits.

[0031] Compared with existing technologies, the anti-FOLR1 nanobody provided by this invention has the following advantages:

[0032] (1) The core of this invention provides a novel anti-FOLR1 nanobody (number: FOLR1-27). This antibody has high affinity, and the obtained anti-FOLR1 nanobody exhibits excellent specific recognition and binding ability, as well as high sensitivity, providing a new and effective option for targeted therapy.

[0033] (2) This invention employs automated equipment to assist in phage panning, precisely controlling the number of washes and oscillation time through programmed control of multiple washing processes. This method effectively reduces the adsorption of non-specific phages and significantly improves the screening and enrichment efficiency of target nanobodies. The high-throughput operation mode greatly shortens the screening cycle, reduces manual operation time, and allows multiple rounds of screening to be completed in a shorter time, while significantly improving the reproducibility and accuracy of the experiment.

[0034] (3) This invention utilizes a mammalian cell expression system to achieve efficient expression of FOLR1 nanobodies, significantly reducing antibody development and production costs, shortening the expression cycle, and simultaneously improving production throughput and overall efficiency. This screening and expression system effectively shortens antibody development time, saves costs, and provides a reliable pathway for the large-scale preparation of nanobodies. Attached Figure Description

[0035] Figure 1 For serum titer testing;

[0036] Figure 2 Image showing the enrichment results;

[0037] Figure 3 This is a graph showing the initial screening results for monoclonal antibodies.

[0038] Figure 4 , Figure 5 , Figure 6 This is a graph showing the results of ELISA antigen-antibody detection.

[0039] Figure 7 , Figure 8 This is a graph showing the results of flow cytometry analysis. Detailed Implementation

[0040] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention. Where specific techniques or conditions are not specified in this embodiment, they shall be operated in accordance with conventional technical methods and instrument manuals in the art; where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0041] Example 1

[0042] The preparation method of the anti-FOLR1 nanobody includes the following steps:

[0043] 1. Preparation of immunogen: Based on the gene sequence and protein sequence information of FOLR1 (Accession # P15328, 25-233AA), the immunogen FOLR1 is expressed and a His-tag is linked to its C-terminus to obtain the modified antigen for subsequent purification and detection.

[0044] 2. Alpaca Immunization:

[0045] The modified antigen obtained in step S1 was mixed with Freund's adjuvant at a 1:1 ratio, emulsified, and injected subcutaneously at multiple sites. Five mL of blood was collected on day 7, and the serum was used to monitor the immune response. On days 14, 21, and 28, human FOLR1 / His protein and Freund's adjuvant were used to boost the immunization. One week after each immunization, blood was collected to detect the serum titer, and then alpaca PBMC cells were extracted from the peripheral blood.

[0046] 3. The serum titer of FOLR1 / His was detected by ELISA. The detection procedure was as follows: The ELISA plate was coated with FOLR1 / His protein at a concentration of 2 μg / mL. 100 μL of serum (controlled with pre-immunized alpaca serum) diluted 2-fold was added to each well. The plate was incubated at 25°C for 1 h. After washing 5 times, 100 μL of horseradish peroxidase-labeled Goat anti-Alpaca IgG (H+L) secondary antibody was added to each well. The plate was incubated at 25°C for 1 h. After washing 5 times, 100 μL of TMB substrate was added. The plate was incubated at 25°C. The reaction was terminated with 50 μL of 1M HCl. The OD was measured at 450 nm.

[0047] A positive antiserum titer is defined as an OD450 value of the sample being tested that is more than three times that of unimmunized alpaca serum. ELISA test results are as follows: Figure 1 As shown, the antiserum titer reached 12800 after the 3rd and 4th immunizations. This indicates that the antigen can induce alpacas to produce high-titer antiserum specifically targeting the FOLR1 protein. Since the titer after the 4th immunization did not significantly increase compared to the 3rd immunization, it indicates that the immune response had reached a plateau. The diversity of the phage library was higher after the 4th immunization than after the 4th immunization, and the probability of screening for functional antibodies with different epitopes was higher. Therefore, alpaca peripheral blood after the 3rd immunization was used to isolate PBMCs and construct a phage library.

[0048] 4. Using the alpaca PBMC cells obtained in step 3 as raw materials, total RNA was extracted using the TRIZOL method, and cDNA fragments were obtained by reverse transcription PCR. The VHH gene fragment was amplified using this fragment as a template. The target gene fragment was then cloned into a phage vector and transformed into competent cells to construct a phage library.

[0049] 5. Phage library construction:

[0050] (1) RNA extraction and reverse transcription

[0051] RNA was extracted from PBMCs using the Trizol method, and then reverse transcribed into cDNA using TAKARA's PrimeScript™ II Reverse Transcriptase kit, which served as a template for VHH amplification.

[0052] (2) VHH gene amplification and electroporation

[0053] 1) The VHH fragment was amplified by two rounds of PCR using cDNA as a template. The amplified products were digested, ligated, and electroporated into competent cells to obtain a phage library with an effective volume of 1.20E+08 cfu / mL. After the library passed the sequencing analysis, phage library panning was performed.

[0054] 2) Validation of the constructed phage library: A protein panning strategy was adopted, and automated equipment was used to assist in the panning. The enrichment status was judged based on the increase of the output / input ratio in each round. In addition, single clones were selected for initial screening of single phages. The correct alpaca VHH gene was cloned into the eukaryotic expression vector pCDNA3.4 and expressed in high throughput through a mammalian expression system to obtain a large number of expressed antibodies.

[0055] 6. Phage library selection:

[0056] Protein solid-phase panning was used. In the first round, human FOLR1 / His protein was coated onto 96-well microplates as the positive sieve plate (5 μg / well, 4 wells). The negative sieve plate was coated with 1% BSA at 100 μL / well and incubated overnight at 4°C. The next day, both the positive and negative sieve plates were washed three times with 0.1% Tween-PBS (hereinafter referred to as PBST), and blocked with 200 μL / well of 3% BSA (prepared with 1×PBS). Input phage (1E+12pfu) was added and incubated at room temperature for 1 hour. After washing three times with PBST, 100 μL of the blocked phage was added to each well of the negative sieve plate and incubated for 1 hour. The positive sieve plate was washed three times with PBST and twice with 1×PBS. The supernatant from the negative sieve plate was transferred to the positive sieve wells and incubated for 1 hour. Wash 10 times with PBST, then 4 times with 1×PBS. Add 100 μL / well of 0.25% EDTA-trypsin and elute for 10 min. Infect with SS320 and incubate at 37℃ for 30 min. Centrifuge at 3000 g for 5 min, enrich the bacterial cells, and plate. The next day, scrape off the bacteria for the second round of phage packaging and selection. Second round selection: 96-well microplates coated with humanFOLR1 / His protein were used as positive screening plates. Coat 4 wells at 3 μg / well. Input phage (5E + 10 pfu) was added and incubated at room temperature for 1 h. After positive screening, wash 14 times with PBST, then 4 times with 1×PBS. Add 100 μL / well of 0.25% EDTA-trypsin and elute for 10 min to obtain the second round of output phage.

[0057] The results of the selection enrichment are as follows Figure 2 As shown, a significant increase in the Output Phage / Input Phage titer was observed between the two rounds of panning, indicating that the second round of panning enriched the product by 140 times compared to the first round. Therefore, subsequent experiments were based on the product from the second round of panning.

[0058] 7. Initial screening of monoclonal antibodies and recombinant expression:

[0059] Culture medium (containing tetracycline, ampicillin, kanamycin, and helper phage M13K07) was added to 96-well deep-well plates. Single clones from the second round of solid-phase panning were picked for detection and cultured overnight at 37°C in a shaker at 220 rpm to obtain single-clone phages. In 96-well ELISA plates, 100 μL of 1 μg / mL human FOLR1 / his protein was added to each well and incubated overnight at 4°C. The next day, the supernatant was discarded, and the plates were washed three times with 200 μL of PBST per well. The plates were then blocked with 200 μL / well of 1% BSA at room temperature for 1 h. The overnight bacteria were centrifuged at 3000 rpm for 5 min, and 50 μL / well of the supernatant sample was added to the corresponding well using a multichannel pipette. The plates were then incubated together with 50 μL / well of 1% BSA at room temperature for 1 h. Dilute sufficient secondary antibody (Anti-M13 Antibody (HRP), Mouse Monoclonal) at 100 µL / well to the corresponding 96-well microplate, incubate at room temperature for 45 min, wash 6 times with PBST, then add TMB at room temperature and incubate for approximately 5 min at room temperature for color development. Add TMB stop solution (450 nm, sulfuric acid-free) to stop the reaction, and read the data at OD450 using a microplate reader.

[0060] Monoclonal antibody initial screening test results ( Figure 3 The initial screening of monoclonal antibodies yielded 81 positive clones. Forty-eight clones were selected for sequencing analysis, yielding 13 unique sequences. These 13 VHH genes were cloned into expression vectors, transiently transfected into mammalian cells, and the expression supernatant was purified to obtain purified antibodies. The 13 antibodies are: FOLR1-01, FOLR1-02, FOLR1-08, FOLR1-12, FOLR1-13, FOLR1-15, FOLR1-16, FOLR1-23, FOLR1-24, FOLR1-27, FOLR1-33, FOLR1-38, and FOLR1-47. SDS-PAGE analysis showed that the purity of all purified antibodies was >95%.

[0061] 8. Detection of recombinant expressed antibodies

[0062] 1) The binding of antibodies to antigens was detected by ELISA. The specific experimental steps are as follows: In a 96-well ELISA plate, 100 μL of 2 μg / mL Human FOLR1 His Protein was added to each well, and the plate was coated overnight at 4°C. The supernatant was removed the next day, and the plate was washed three times with 200 μL of PBST per well. The plate was then blocked with 200 μL of 1% BSA per well at room temperature for 2 hours. The antibody to be tested was serially diluted 11 times in a 4-fold gradient, 100 μL per well, and incubated at room temperature for 1 hour. Sufficient secondary antibody Anti-Human IgG (Fcspecific) (1:5000) was diluted and added to the corresponding 96-well plate at 100 µL per well. The plate was incubated at room temperature for 30 minutes, washed six times with PBST, and then 100 µL of TMB at room temperature was added. The plate was incubated at room temperature for 15 minutes for color development. Add TMB for color development, then add 100µL of stop solution (450nm, sulfuric acid-free) to terminate the reaction. Read the data using an ELISA reader at OD450.

[0063] ELISA antigen and antibody test results ( Figures 4-6 The results showed that 10 out of 13 antibodies tested exhibited strong binding activity to the FOLR1 antigen. However, one antibody (FOLR1-47) showed a weak binding signal, while two antibodies (FOLR1-33 and FOLR1-15) showed no specific binding.

[0064] 2) Antibody binding activity was detected using overexpression cell lines. The specific experimental steps were as follows: The cell number was adjusted to 4.00E+06 / mL in pre-chilled MACS buffer (PBS, 2% FBS, 2mM EDTA). 50 μL of cell suspension was added to each well of a 96-well plate. The antibody was serially diluted 4-fold with MACS buffer, starting at 400 nM in the first well. After dilution, 50 μL of antibody dilution buffer was added to the cell suspension, and the mixture was thoroughly mixed. The plate was incubated at 4°C for 60 min. 100 μL of MACS buffer was added, and the plate was centrifuged at 400 g for 5 min. The supernatant was discarded. 200 μL of MACS buffer was added to wash the cells, and the plate was centrifuged at 400 g for 5 min. The supernatant was discarded, and the cells were washed twice. The cells were resuspended in 100 μL of fluorescent secondary antibody (Goat anti-Human Fc, Alexa Fluor 647, 1:1000 dilution) and incubated at 4°C for 30 min. Add 100 μL of MACS buffer, centrifuge at 400 g for 5 min, discard the supernatant, add 200 μL of MACS buffer to the wells to wash the cells, centrifuge at 400 g for 5 min, discard the supernatant, and wash twice. Resuspend the cells in 200 μL of MACS buffer and perform flow cytometry analysis.

[0065] Flow cytometry (FACS) results as follows Figures 7-8As shown, among the 11 preferred candidate antibodies, 10 antibodies could specifically bind to cell lines that highly express FOLR1, while the FOLR1-47 antibody did not show significant cell binding activity.

[0066] Based on the combined results of ELISA and overexpression cell line binding assays, the FOLR1-27 antibody was ultimately selected for protection. The sequence of this antibody is as follows: the CDR1 sequence of the complementarity-determining region of the FOLR1-27 antibody is shown in SEQ ID NO: 1, the CDR2 sequence is shown in SEQ ID NO: 2, the CDR3 sequence is shown in SEQ ID NO: 3, the amino acid sequence of the variable region of the heavy chain of the FOLR1-27 antibody is shown in SEQ ID NO: 4, and the corresponding nucleotide sequence is shown in SEQ ID NO: 5.

[0067] Finally, it should be noted that the above are merely embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. An anti-FOLR1 nanobody, comprising a framework region and a complementarity-determining region, characterized in that, The complementarity-determining regions include CDR1, CDR2, and CDR3; the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 are selected from group 1) or group 2). 1) The complementary decision region CDR1 sequence is shown in SEQ ID NO: 1, the complementary decision region CDR2 sequence is shown in SEQ ID NO: 2, and the complementary decision region CDR3 sequence is shown in SEQ ID NO: 3; 2) The amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 are at least 90% identical to the amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 described in group 1); and retain the ability to bind FOLR1.

2. The anti-FOLR1 nanobody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-FOLR1 nanobody is shown in SEQ ID NO:

4.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-FOLR1 nanobody as described in any one of claims 1 to 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

5.

5. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, The host cell comprises the vector as described in claim 5.

7. A method for preparing an anti-FOLR1 nanobody as described in any one of claims 1 to 2, characterized in that, Includes the following steps: S1. Based on the protein and gene sequence information of FOLR1, analyze and design immune antigens; S2. Immunize alpacas with the antigen obtained in step S1, test the titer of the serum separated from the immunized alpacas, and then extract alpaca PBMC cells from the peripheral blood. S3. Using the alpaca PBMC cells obtained in step S2 as raw materials, total RNA was extracted, and cDNA fragments were obtained by reverse transcription PCR. The VHH gene fragment was amplified using this as a template. The target gene fragment was then cloned into a phage vector and transformed into competent cells to construct a phage library. S4. Using the phage library constructed in step S3, phages are packaged, and phages are enriched by panning with the assistance of automated equipment. Single clones are then selected for initial screening, positive clones are sent for testing, and sequencing analysis is performed to select the correct antibody sequence for eukaryotic expression. S5. Using the antibody expressed in step S4, perform antigen protein and cell detection to screen for antibodies with high specificity and high affinity, thereby obtaining the anti-FOLR1 nanobody.

8. The preparation method according to claim 7, characterized in that, In step S2, alpacas were immunized with recombinant human FOLR1 protein with a histidine tag. Serum was collected after four immunizations for titer testing, and the alpaca PBMC library was constructed using the third immunized alpaca protein.

9. The preparation method according to claim 7, characterized in that, The eukaryotic expression vector described in step S4 is the PcDNA3.4 vector.

10. The use of the anti-FOLR1 nanobody as described in any one of claims 1 to 2 in the preparation of products for detection, affinity purification or binding to FOLR1; said products include detection reagents or kits.

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