Nanobody targeting fibroblast activation protein-α, and use thereof

Abstract

Disclosed are a nanobody targeting fibroblast activation protein-α, and the use thereof. An amino acid sequence of the nanobody comprises antigenic determinant complementary regions and framework regions with special structures. Firstly, alpacas are immunized with a recombinant FAPα antigen protein constructed to obtain a cell construct of alpaca PBMCs; secondly, an antibody targeting FAPα is screened by means of yeast display technology; and finally, an antibody having high sensitivity and specificity is obtained by means of functional detection and sequencing result analysis. The provided nanobody has the ability to specifically recognize and bind to FAPα, and has the characteristics of small molecular weight, low immunogenicity, better stability, a significant inhibitory effect on FAPα-expressing cells or tissues, etc., thus providing a potential therapeutic strategy for a disease involving FAPα expression. When the nanobody is used for developing or screening a diagnostic or therapeutic drug for FAPα-expressing cells or tissues, the obtained drug has excellent specificity and affinity, and has a significant inhibitory effect on FAPα-expressing cells or tissues.

Description

Nanobody targeting fibroblast activation protein alpha and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a nanobody targeting fibroblast activation protein alpha and application thereof. BACKGROUND

[0002] Fibroblast activation protein alpha (FAP alpha) is a 97 kDa type II transmembrane serine protease containing dipeptidyl peptidase and endopeptidase activity. FAP alpha is 760 amino acids in length, with only 4 amino acids in the intracellular domain, 21 amino acids in the transmembrane domain, and a long 735 amino acid extracellular domain. FAP alpha exists as a homodimer. In addition, FAP alpha can also exist in a soluble form in the plasma.

[0003] Under physiological conditions, the expression level of FAP alpha in most adult tissues is below the detection level. However, the expression level of FAP alpha is increased in different tumor types, with the highest median in pancreatic cancer and breast cancer. In most epithelial cancers, FAP alpha is mainly expressed in tumor-associated fibroblasts (CAFs) in the interstitium. In addition, FAP alpha is also expressed in some tumor cells such as sarcoma, glioma, mesothelioma and esophageal epithelial tumors. FAP alpha affects tumor growth through various mechanisms, including promoting proliferation, invasion, angiogenesis, epithelial-mesenchymal transition, stem cell promotion, immunosuppression and drug resistance.

[0004] Current clinical treatments using FAP alpha include: inhibiting the protease activity of FAP alpha with small molecules or antibodies, using the protease activity of FAP alpha to cleave tumor drugs attached to FAP alpha-targeting peptides, vaccination against FAP alpha, and CAR-T cell therapy.

[0005] FAPI imaging is a series of quinoline-based FAPα inhibitors (Fibroblast Activation Protein Inhibitors, FAPIs) developed for FAPα. However, as small molecule drugs, FAPα inhibitors can have non-specific inhibitory effects on other serine proteases in addition to FAPα, reducing specificity and selectivity and thus affecting normal cells and tissues. In contrast, antibody drugs directly target FAPα-expressing cells through highly specific antigen-antibody interactions, reducing the impact on normal tissues and reducing the risk of side effects. Therefore, in the field of cancer treatment, the development of antibodies against FAPα and their conjugates has become a research hotspot. These antibodies and their conjugates have shown promising results in preclinical studies, especially radioimmunoconjugates for the diagnosis and treatment of patients with FAPα-expressing tumors. Previous major studies have focused on the F19 monoclonal antibody and its humanized form sibrotuzumab, both of which can successfully accumulate at tumor sites.

[0006] However, despite this, traditional antibodies such as F19 and sibrotuzumab can have limitations in tissue penetration in vivo due to their large molecular size; and the production of traditional antibodies involves complex mammalian cell expression systems, with high production costs and time-consuming; in addition, although sibrotuzumab has been humanized, as a full-length antibody, it still has a considerable proportion of immune reactions in long-term clinical treatment.

[0007] In view of the limitations of FAPα inhibitors and traditional antibodies in specificity, tissue penetration and production costs, there is an urgent need in the scientific community to develop more efficient and economical treatment options. In this context, nanobodies, as a cutting-edge biomedical technology, have become a strong candidate for breaking through the limitations of traditional treatment due to their small molecule advantages, high degree of customization and excellent biological activity. SUMMARY

[0008] In view of the defects and deficiencies of the prior art, the present application aims to provide a nanobody targeting fibroblast activation protein alpha and its application, which has specific recognition and binding ability to FAPα, has the characteristics of small molecular weight (12.43-14.09 kDa), low immunogenicity, better stability, obvious tumor inhibition effect, etc., and provides a potential treatment strategy for diseases expressing FAPα.

[0009] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0010] A first object of the present application is to provide a fibroblast activation protein alpha targeting nanobody, comprising a heavy chain variable region CDR sequence selected from at least one of the following or an amino acid sequence having at least 80% identity thereto: SEQ ID NO. 1-27.

[0011] Preferably, the nanobody comprises:

[0012] a heavy chain variable region CDR1 sequence as shown in the amino acid sequence of SEQ ID NO. 1-9 or an amino acid sequence having at least 80% identity to SEQ ID NO. 1-9;

[0013] a heavy chain variable region CDR2 sequence as shown in the amino acid sequence of SEQ ID NO. 10-18 or an amino acid sequence having at least 80% identity to SEQ ID NO. 10-18;

[0014] a heavy chain variable region CDR3 sequence as shown in the amino acid sequence of SEQ ID NO. 19-27 or an amino acid sequence having at least 80% identity to SEQ ID NO. 19-27.

[0015] Preferably, the CDR of the fibroblast activation protein alpha targeting nanobody comprises CDR1-CDR3 as shown in the amino acid sequence as follows:

[0016] (1) CDR1 with the amino acid sequence as shown in SEQ ID NO. 1, CDR2 with the amino acid sequence as shown in SEQ ID NO. 10, and CDR3 with the amino acid as shown in SEQ ID NO. 19; or

[0017] (2) CDR1 with the amino acid sequence as shown in SEQ ID NO. 2, CDR2 with the amino acid sequence as shown in SEQ ID NO. 11, and CDR3 with the amino acid as shown in SEQ ID NO. 20; or

[0018] (3) CDR1 with the amino acid sequence as shown in SEQ ID NO. 2, CDR2 with the amino acid sequence as shown in SEQ ID NO. 12, and CDR3 with the amino acid as shown in SEQ ID NO. 20; or

[0019] (4) CDR1 with the amino acid sequence as shown in SEQ ID NO. 3, CDR2 with the amino acid sequence as shown in SEQ ID NO. 11, and CDR3 with the amino acid as shown in SEQ ID NO. 20; or

[0020] (5) CDR1 as shown in SEQ ID NO. 4, CDR2 as shown in SEQ ID NO. 13, CDR3 as shown in SEQ ID NO. 21; or

[0021] (6) CDR1 as shown in SEQ ID NO. 2, CDR2 as shown in SEQ ID NO. 11, CDR3 as shown in SEQ ID NO. 22; or

[0022] (7) CDR1 as shown in SEQ ID NO. 5, CDR2 as shown in SEQ ID NO. 14, CDR3 as shown in SEQ ID NO. 23; or

[0023] (8) CDR1 as shown in SEQ ID NO. 6, CDR2 as shown in SEQ ID NO. 15, CDR3 as shown in SEQ ID NO. 24; or

[0024] (9) CDR1 as shown in SEQ ID NO. 7, CDR2 as shown in SEQ ID NO. 16, CDR3 as shown in SEQ ID NO. 25; or

[0025] (10) CDR1 as shown in SEQ ID NO. 8, CDR2 as shown in SEQ ID NO. 17, CDR3 as shown in SEQ ID NO. 26; or

[0026] (11) CDR1 as shown in SEQ ID NO. 9, CDR2 as shown in SEQ ID NO. 18, CDR3 as shown in SEQ ID NO. 27.

[0027] Preferably, the Nanobody comprises a framework region FR sequence selected from at least one of the following: SEQ ID NO. 28-59, or an amino acid sequence at least 80% identical thereto.

[0028] Preferably, the Nanobody comprises:

[0029] a framework region FR1 sequence as shown in SEQ ID NO. 28-36, or an amino acid sequence at least 80% identical to SEQ ID NO. 28-36;

[0030] a framework region FR2 sequence as set forth in any one of SEQ ID NOs. 37-46 or an amino acid sequence having at least 80% identity to SEQ ID NOs. 37-46;

[0031] a framework region FR3 sequence as set forth in any one of SEQ ID NOs. 47-57 or an amino acid sequence having at least 80% identity to SEQ ID NOs. 47-57;

[0032] a framework region FR4 sequence as set forth in any one of SEQ ID NOs. 58-59 or an amino acid sequence having at least 80% identity to SEQ ID NOs. 58-59.

[0033] Preferably, the fibroblast activation protein alpha targeting Nanobody comprises a framework region FR comprising FR1-FR4 as set forth in the following:

[0034] (1) FR1 as set forth in SEQ ID NO. 28, FR2 as set forth in SEQ ID NO. 37, FR3 as set forth in SEQ ID NO. 47, and FR4 as set forth in SEQ ID NO. 58; or

[0035] (2) FR1 as set forth in SEQ ID NO. 29, FR2 as set forth in SEQ ID NO. 38, FR3 as set forth in SEQ ID NO. 48, and FR4 as set forth in SEQ ID NO. 58; or

[0036] (3) FR1 as set forth in SEQ ID NO. 29, FR2 as set forth in SEQ ID NO. 38, FR3 as set forth in SEQ ID NO. 49, and FR4 as set forth in SEQ ID NO. 58; or

[0037] (4) FR1 as set forth in SEQ ID NO. 30, FR2 as set forth in SEQ ID NO. 39, FR3 as set forth in SEQ ID NO. 50, and FR4 as set forth in SEQ ID NO. 58; or

[0038] (5) FR1 as set forth in SEQ ID NO. 31, FR2 as set forth in SEQ ID NO. 40, FR3 as set forth in SEQ ID NO. 51, and FR4 as set forth in SEQ ID NO. 59; or

[0039] (6) FR1 as shown in SEQ ID NO. 32, FR2 as shown in SEQ ID NO. 41, FR3 as shown in SEQ ID NO. 52, and FR4 as shown in SEQ ID NO. 58; or

[0040] (7) FR1 as shown in SEQ ID NO. 33, FR2 as shown in SEQ ID NO. 42, FR3 as shown in SEQ ID NO. 53, and FR4 as shown in SEQ ID NO. 58; or

[0041] (8) FR1 as shown in SEQ ID NO. 34, FR2 as shown in SEQ ID NO. 43, FR3 as shown in SEQ ID NO. 54, and FR4 as shown in SEQ ID NO. 58; or

[0042] (9) FR1 as shown in SEQ ID NO. 35, FR2 as shown in SEQ ID NO. 44, FR3 as shown in SEQ ID NO. 55, and FR4 as shown in SEQ ID NO. 58; or

[0043] (10) FR1 as shown in SEQ ID NO. 36, FR2 as shown in SEQ ID NO. 45, FR3 as shown in SEQ ID NO. 56, and FR4 as shown in SEQ ID NO. 58; or

[0044] (11) FR1 as shown in SEQ ID NO. 35, FR2 as shown in SEQ ID NO. 46, FR3 as shown in SEQ ID NO. 57, and FR4 as shown in SEQ ID NO. 58.

[0045] A second object of the present application is to provide a nanobody targeting fibroblast activation protein alpha, the amino acid sequence of which is selected from at least one of the following sequences or an amino acid sequence having at least 80% identity thereto: SEQ ID NO. 60 to 71.

[0046] Another object of the present application is to provide a polypeptide comprising the above-mentioned nanobody.

[0047] Another object of the present application is to provide a nucleic acid molecule encoding the above-mentioned nanobody.

[0048] Another object of the present application is to provide an expression vector comprising the above-mentioned nucleic acid molecule.

[0049] Another object of the present application is to provide a host cell transformed or transfected with the above-mentioned expression vector.

[0050] Another object of the present application is to provide a pharmaceutical composition comprising the above-mentioned Nanobody and a pharmaceutically acceptable carrier and / or excipient.

[0051] Another object of the present application is to provide the use of the above-mentioned Nanobody, polypeptide, nucleic acid molecule, expression vector, host cell, pharmaceutical composition in the preparation or screening of a diagnostic or therapeutic drug or kit for a disease.

[0052] Preferably, the disease expresses fibroblast activation protein alpha.

[0053] Compared with the prior art, the present application has the following beneficial effects:

[0054] (1) The present application firstly immunizes a llama with a fibroblast activation protein alpha antigen to obtain a llama PBMC cell construct; secondly, a fibroblast activation protein alpha-targeted antibody is screened through a yeast display technology; and finally, an antibody with high sensitivity and specificity is obtained through functional detection and sequencing result analysis. The Nanobody provided by the present application has specific recognition and binding capacity for fibroblast activation protein alpha, has a molecular weight of 12.4-14.7 kDa, small immunogenicity, better stability, obvious tumor inhibition effect, etc., and provides a potential treatment strategy for a tumor disease expressing fibroblast activation protein alpha.

[0055] (2) The Nanobody provided by the present application can be used for preparing or screening a drug for the diagnosis or treatment of a tumor (such as: pancreatic cancer, gastric cancer, colorectal cancer, liver cancer, bile duct cancer, esophageal cancer, head and neck tumor, breast cancer, lung cancer, bone and soft tissue sarcoma, cervical cancer, ovarian cancer, bladder cancer, prostate cancer, kidney cancer, glioma, mesothelioma, etc.) and a non-tumorous disease (such as: cirrhosis, interstitial pneumonia, pulmonary fibrosis, heart failure, etc.) expressing fibroblast activation protein alpha, has good specificity and affinity, and has an obvious inhibitory effect on cells or tissues expressing fibroblast activation protein alpha. BRIEF DESCRIPTION OF DRAWINGS

[0056] FIG. 1 is an SDS-PAGE detection result of a FAPa recombinant protein;

[0057] FIG. 2 is a result of amplification of a VHH fragment in B cells of immunized llama PBMC;

[0058] FIG. 3 is a flow cytometry detection result of the binding of enriched yeast to FAPa protein after one round of magnetic bead sorting or one round of flow sorting;

[0059] Figure 4 is the binding of randomly selected yeast monoclonal after magnetic bead sorting with FAPa protein;

[0060] Figure 5 is the binding of randomly selected yeast monoclonal after flow sorting with FAPa protein;

[0061] Figure 6 is the expression of 293T cells overexpressing hFAPa detected by flow cytometry;

[0062] Figure 7 is the FCM detection result graph of anti-FAPa nanobody selectively recognizing hFAPa-293T cells overexpressing;

[0063] Figure 8 is the plasmid map and structure mode graph of CAR vector constructed based on FAPa nanobody;

[0064] Figure 9 is the transduction efficiency of CAR-T cells constructed based on FAPa nanobody;

[0065] Figure 10 is the detection result graph of CAR-T cells constructed based on FAPa nanobody killing hFAPa + target cells. DETAILED DESCRIPTION

[0066] The above content of the present application will be further explained in detail by the following specific embodiments in the form of examples. However, it should not be understood that the above-mentioned subject matter of the present application is limited to the following examples only.

[0067] 1. Reagents:

[0068] Table 1

[0069] 2. Instruments:

[0070] Table 2

[0071] 3. Solution preparation:

[0072] Table 3 SDCAA medium

[0073] Table 4 SGCAA medium

[0074] Design principle or design idea of the present application:

[0075] The camel is immunized with an antigen, peripheral blood mononuclear cells (PBMC) are isolated, and total RNA is extracted for reverse transcription. The variable domain of the heavy-chain of heavy chain antibody (VHH) is amplified by using the reverse transcription product as a template and is electroporated into yeast competent cells together with a yeast display vector to construct a yeast display library.

[0076] The yeast display library is screened by a screening method of one round of magnetic bead sorting and one round of flow sorting of the biotinylated FAP alpha antigen, and specific anti-FAP alpha nanobodies are obtained. Twelve yeast monoclonal strains capable of binding to FAP alpha recombinant protein are obtained by original library presentation and screening and identification.

[0077] The binding efficiency of the nanobodies and the FAP alpha antigen is detected by ELISA, and it is known that the screened VHH can effectively bind to the monomeric FAP alpha protein; the binding efficiency of the nanobodies and the target cells is detected by flow cytometry, and it is known that 30 VHHs recognize the FAP alpha protein expressed on the cell surface, proving that the FAP alpha-targeted nanobodies provided by the application can effectively bind to the FAP alpha antigen protein in the form of a dimer on the cell membrane surface; the killing of the target cells by the CAR-T cells is detected by the luciferase activity report method, and it is known that the CAR-T cells constructed based on the FAP alpha nanobodies can effectively kill the target cells, proving that the FAP alpha-targeted nanobodies provided by the application have the potential to be developed into downstream products. Therefore, the nanobodies provided by the application have the effect of efficiently and specifically binding to the FAP alpha target protein.

[0078] Example 1 Expression and purification of target protein

[0079] The preparation of the FAP alpha recombinant protein is entrusted to Suzhou Aikangde Company, including the following steps:

[0080] The extracellular segment sequence information (Leu 26-Asp760) of Human FAP alpha (Q12884-1) is retrieved from the UniProt database, and the amino acid sequence is shown as SEQ ID NO. 72. A His tag is added at the C terminal, and the gene is synthesized by Suzhou Aikangde Company according to the human codon bias optimization and subcloned into the pcDNA3.4 vector, as shown in SEQ ID NO. 73 and SEQ ID NO. 74. After Sanger sequencing verification, the plasmid is extracted for standby use. The constructed protein eukaryotic expression vector is transiently transfected into 293F cells, the protein expression supernatant is collected, and the target protein is purified; the protein purity is detected by SDS-PAGE experiment, and the SDS-PAGE is performed according to the laboratory standard operation procedure.

[0081] As shown in Figure 1, the SDS-PAGE detection results of the FAPa recombinant protein show that the purity of the Human FAPa recombinant protein is greater than 95%, which can be used for immunization of alpacas and screening of antibodies.

[0082] Human FAPa extracellular segment sequence information:

[0083] Optimized FAPa-His sequence:

[0084] pcDNA3.4-FAPa-His vector sequence:

[0085] Example 2: Construction of yeast nanobody library

[0086] The alpaca was immunized twice with the FAPa recombinant protein prepared in Example 1, and then the peripheral blood lymphocytes were separated, the total RNA was extracted, the RNA was reverse transcribed into cDNA, and then the nanobody VHH sequence was amplified using primers. The VHH sequence was used to construct a yeast display nanobody library.

[0087] The specific steps are as follows:

[0088] 1. Alpaca immunization:

[0089] The alpaca (Alpaca) was immunized with the recombinant protein prepared above, the immunization adjuvant was Gerbu, the immunization interval was 21 days, and the immunization was performed 2 times by subcutaneous multi-point injection. Ten days after the last immunization, part of the peripheral blood was collected, the serum was separated, and the immune effect was detected by ELISA.

[0090] 2. Detection of immune titer:

[0091] a) Collect 5 mL of peripheral blood, and place the centrifuge tube containing the blood sample in a 37°C incubator for 1 hour; then transfer the blood sample to 4°C overnight;

[0092] b) Place the centrifuge tube containing the blood sample in a centrifuge, centrifuge at 5000 rpm for 20 min; separate the upper serum, and transfer the serum to a new sterile centrifuge tube to collect the immune serum.

[0093] c) Dilute the target recombinant protein to a final concentration of 1 pg / mL using sterile CBS (carbonate buffer). Take a new 96-well enzyme-labeled plate, and add 100 pL / well 4°C coated overnight.

[0094] d) Remove the antigen coating solution, wash the plate 5 times with PBST (0.05% Tween 20).

[0095] e) Add 200 μL / well of 3% MPBS 37°C blocking for 2 hours;

[0096] f) After removing the blocking buffer, wash the plate 5 times with PBST;

[0097] g) Add 100 μL of gradient-diluted serum (100 μL / well), incubate at room temperature for 1 hour, and use PBS for the control wells;

[0098] h) Remove the liquid in the wells, and wash 5 times with PBST;

[0099] i) Add 100 μL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution), incubate at room temperature for 1 hour;

[0100] j) After removing the liquid in the wells, wash the plate 5 times with PBST;

[0101] k) Add 100 μL / well of TMB developing solution;

[0102] l) Incubate at room temperature for 10-15 minutes in the dark;

[0103] m) Add 50 μL / well of stop solution;

[0104] n) Read the OD450 value in the wells using a microplate reader.

[0105] The results of the detection of the post-immune titer of the immunized alpaca against FAPa are shown in Table 5. The immune serum can bind to the FAPa recombinant protein, and the OD value changes in a gradient manner with the gradient dilution of the immune serum. The immune titer reaches 1:64K or above. 100 mL of peripheral blood is collected to prepare a cDNA library for the construction of an antibody display library.

[0106] Table 5. Results of the detection of the post-immune titer of the immunized alpaca against FAPa

[0107] 3. PBMC separation and VHH antibody fragment cloning:

[0108] a) Collect 100 mL of peripheral blood anticoagulant sample, and separate PBMC cells using lymphocyte separation medium.

[0109] b) Extract RNA according to the steps in the instructions, and use the extracted total RNA as a template to perform reverse transcription using a PrimeScript TM II 1st Strand cDNA Synthesis Kit kit to prepare cDNA.

[0110] The following reaction mixture Mixl was prepared in 200 μL PCR:

[0111] Table 6

[0112] After incubation at 65 °C for 5 min, quickly cool on ice.

[0113] The following reaction solution was prepared in the above PCR tube:

[0114] Table 7

[0115] After mixing by blowing, 80 μL / tube was aliquoted, placed in a PCR instrument at 42 °C for 1 hour, and then heat inactivated at 70 °C for 15 min. Finally, the cDNA sample was placed on ice or stored at -20 °C for long-term preservation.

[0116] c) Amplification of VHH fragments

[0117] Table 8 Two-round PCR reaction primers

[0118] The first-round PCR experiment was performed using specific primers and cDNA as template, and the specific method was as follows:

[0119] 1) Preparation of first-round PCR reaction system (50 μL / tube)

[0120] Table 9

[0121] After preparation of the PCR reaction system, the PCR instrument was set according to the following program:

[0122] Table 10

[0123] 2) Agarose electrophoresis of PCR products

[0124] The PCR products were analyzed by electrophoresis using 1% agarose, and the fragments with a molecular weight of about 750 bp were separated. The PCR products were recovered using a gel recovery kit, and the concentration was determined using NanoDrop.

[0125] 3) Preparation of second-round PCR reaction system (50 μL / tube)

[0126] Table 11

[0127] After preparation of the PCR reaction system, the PCR instrument was set according to the following program:

[0128] Table 12

[0129] 4) Agarose electrophoresis analysis of second-round PCR products

[0130] The PCR product was analyzed by electrophoresis using 1% agarose gel, and the VHH fragment with a molecular weight of about 400 bp was separated. The VHH PCR product was recovered using a gel recovery kit and the concentration was determined by NanoDrop.

[0131] As shown in Figure 2, the first round of PCR obtained PCR bands of about 1000 bp and 750 bp, respectively, and the 750 bp fragment was recovered as a template for the second round of PCR. The second round of PCR obtained a band of about 400 bp, which was a VHH fragment, and the band was recovered for subsequent electroporation of yeast and recombination into the yeast display vector pDisplay.

[0132] 4. Construction of single-domain antibody yeast display library:

[0133] a) Linearization of yeast display vector pDisplay, enzyme digestion system as follows:

[0134] Table 13

[0135] b) Use Sfil to digest pDisplay vector, 100 μL / tube, 50°C enzyme digestion overnight;

[0136] c) Use 1% agarose gel to separate pDisplay vector fragments, cut 5000 bp of the vector fragments for gel recovery, and determine the concentration by NanoDrop;

[0137] d) Divide the recovered pDisplay enzyme digestion product into 200 μL per 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3M sodium acetate, 1 μg / μL glycogen, mix well by blowing and sucking, add 880 μL of anhydrous ethanol, mix well by inverting, and store at -80°C.

[0138] 5. Electroporation to construct yeast display library:

[0139] The -80°C frozen yeast competent strain was streaked onto YPD solid medium plates and incubated at 30°C for 3-5 days;

[0140] Inoculate single colony yeast competent into 50 mL YPD medium, 250 rpm, 30°C, shake culture for 1-2 days;

[0141] Prepare the yeast competent strain. After mixing the linearized vector fragment and the PCR product, add it to the electroporation cup, and then perform electroporation. The yeast competent after electroporation was transferred to a culture bottle and incubated at 220 rpm and 30°C for 1 h;

[0142] Take 20 μL of the resuspension, dilute 5000-fold with SDCAA, take 100 μL, coat SDCAA plates, and incubate for 2-3 days. Calculate the library capacity, and continue to incubate the remaining bacterial solution for 24 h.

[0143] Bacterial preservation: collect the remaining bacterial solution into a 50 mL centrifuge tube, centrifuge at 3000 g for 5 min, discard the supernatant, add 10 mL of SDCAA resuspension, mix with 50% glycerol:resuspension = 1:1, and store at -80°C.

[0144] Finally, a library with a capacity of 6.56 x 10 8 camel anti-FAP antibody immune library was constructed for specific anti-FAP alpha nanobody screening.

[0145] Example 3: panning of the yeast antibody library

[0146] Using yeast display technology, the single-domain antibody yeast display library obtained in Example 2 was subjected to one round of magnetic bead sorting and one round of flow sorting to obtain positive clones that bind to FAP alpha recombinant protein. The specific steps are as follows:

[0147] 1. Magnetic sorting of yeast display library

[0148] Take the yeast cultured in the above-mentioned SDCAA of Example 2 and add it to a 250 mL shake flask containing 50 mL of SGCAA medium. Incubate at 30°C, 240 rpm, for 16 h on a shaker.

[0149] After centrifugation, discard the supernatant, resuspend with 1 mL of 0.5% PBSA, add to a 1.5 mL centrifuge tube, centrifuge at 3000 g for 5 min, and discard the supernatant. Wash again with 0.5% PBSA.

[0150] Wash the streptavidin magnetic beads that have been incubated with the antigen twice with 0.5% PBSA (4°C rotation incubation for 5 min each time), place them on a magnetic stand for 5 min, and discard the supernatant.

[0151] Add the yeast bacterial solution to the magnetic beads combined with the antigen, and incubate at 4°C for 60 min. Place it on a magnetic stand for 15 min.

[0152] Discard the yeast bacterial solution and leave the magnetic beads. Wash them three times with 0.5% PBSA (4°C rotation incubation for 5 min each time).

[0153] Resuspend the magnetic beads with 1 mL of SDCAA medium, take 0.5-5 μL of the resuspension to 100 μL of SDCAA medium for plating, divide the resuspension into two equal parts, one part is added with 500 μL of 50% glycerol (-80°C storage); the other part is added to a shake tube, supplemented with 2 mL of SDCAA medium, incubated at 30°C, 240 rpm, for 16 h.

[0154] Transfer the bacteria liquid in the shake tube to 50mL SDCAA medium (250mL shake flask), culture at 30℃, 240rpm overnight.

[0155] Measure the OD600 value of the bacteria liquid, centrifuge a part of the bacteria liquid according to the OD600 value, resuspend with SGCAA, transfer to 50mL SGCAA medium, make the final OD600 value 1, culture at 30℃, 240rpm overnight, resuspend the remaining bacteria liquid with SDCAA: 50% glycerol = 1:1, and freeze at -80℃.

[0156] 2. Flow sorting. After magnetic sorting, the yeast cells are plated and cultured, and after induction of expression, incubated with Human FAPα-His-Biotin for 1h, the secondary antibody is APC Streptavidin, and a round of flow analysis is performed for detection, and positive clones are sorted out.

[0157] The results are shown in Figure 3. According to the flow detection results, after one round of magnetic sorting and one round of flow sorting, the V5 display is about 30%, the antigen binding rate increases to 86.6% of the proportion of V5 positive clones, and the positive clones are significantly enriched. The sorted product is directly plated on an SDCAA plate, and single clones are selected for flow detection.

[0158] 3. Yeast single clone flow detection

[0159] After sorting, the yeast bacteria liquid is plated on an SDCAA plate, and single clones are selected for culture. After 48h of induction and expression, incubation is performed with Biotin-antigen, the secondary antibody is PE-Streptavidin, and flow detection is performed after incubation to verify the binding of the enriched yeast display antibody to the antigen. Yeast clones that bind to the target antigen are lysed using 0.2% SDS (incubated at 95℃ for 10min), centrifuged to obtain the bacterial liquid supernatant, and 0.5μL is used as a template for PCR amplification for sequencing (the remaining bacteria liquid is stored at -20℃). The sequencing is entrusted to Beijing Qikong Biotechnology Co., Ltd., and the primer information is shown in SEQ ID NO. 77 and SEQ ID NO. 78. Finally, different clones are selected.

[0160] The test results are shown in Figures 4 and 5. Figure 4 shows the binding of single clones to FAPα antigen after magnetic bead sorting, and Figure 5 shows the binding of single clones to FAPα antigen after flow sorting. The positive clones are sequenced, and single clones with sequence differences are selected for subsequent experiments.

[0161] The names of the 12 potential positive clones screened by the application are shown in the following table:

[0162] Table 14 Names of positive clones

[0163] The sequences of the clones screened by sequencing verification are:

[0164] (1) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 1A10 nanobody are: CDR1 with the amino acid sequence as shown in SEQ ID NO. 1, CDR2 with the amino acid sequence as shown in SEQ ID NO. 10, and CDR3 with the amino acid as shown in SEQ ID NO. 19; the framework region is FR1 with the amino acid sequence as shown in SEQ ID NO. 28, FR2 with the amino acid as shown in SEQ ID NO. 37, FR3 with the amino acid as shown in SEQ ID NO. 47, and FR4 with the amino acid as shown in SEQ ID NO. 58; the amino acid sequence of the 1A10 nanobody is as shown in SEQ ID NO. 60.

[0165] (2) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 1D1 nanobody are: CDR1 with the amino acid sequence as shown in SEQ ID NO. 2, CDR2 with the amino acid sequence as shown in SEQ ID NO. 11, and CDR3 with the amino acid as shown in SEQ ID NO. 20; the framework region is FR1 with the amino acid sequence as shown in SEQ ID NO. 29, FR2 with the amino acid as shown in SEQ ID NO. 38, FR3 with the amino acid as shown in SEQ ID NO. 48, and FR4 with the amino acid as shown in SEQ ID NO. 58; the amino acid sequence of the 1D1 nanobody is as shown in SEQ ID NO. 61.

[0166] (3) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 1E10 nanobody are: CDR1 with the amino acid sequence as shown in SEQ ID NO. 2, CDR2 with the amino acid sequence as shown in SEQ ID NO. 12, and CDR3 with the amino acid as shown in SEQ ID NO. 20; the framework region is FR1 with the amino acid sequence as shown in SEQ ID NO. 29, FR2 with the amino acid as shown in SEQ ID NO. 38, FR3 with the amino acid as shown in SEQ ID NO. 49, and FR4 with the amino acid as shown in SEQ ID NO. 58; the amino acid sequence of the 1E10 nanobody is as shown in SEQ ID NO. 62.

[0167] (4) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 1G1 nanobody are: CDR1 with the amino acid sequence shown as SEQ ID NO. 3, CDR2 with the amino acid sequence shown as SEQ ID NO. 11, and CDR3 with the amino acid shown as SEQ ID NO. 20; the framework regions are FR1 with the amino acid sequence shown as SEQ ID NO. 29, FR2 with the amino acid shown as SEQ ID NO. 38, FR3 with the amino acid shown as SEQ ID NO. 49, and FR4 with the amino acid shown as SEQ ID NO. 58; the amino acid sequence of the 1G1 nanobody is shown as SEQ ID NO. 63.

[0168] (5) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 1G6 nanobody are: CDR1 with the amino acid sequence shown as SEQ ID NO. 4, CDR2 with the amino acid sequence shown as SEQ ID NO. 13, and CDR3 with the amino acid shown as SEQ ID NO. 21; the framework regions are FR1 with the amino acid sequence shown as SEQ ID NO. 30, FR2 with the amino acid shown as SEQ ID NO. 39, FR3 with the amino acid shown as SEQ ID NO. 50, and FR4 with the amino acid shown as SEQ ID NO. 58; the amino acid sequence of the 1G6 nanobody is shown as SEQ ID NO. 64.

[0169] (6) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 1G9 nanobody are: CDR1 with the amino acid sequence shown as SEQ ID NO. 2, CDR2 with the amino acid sequence shown as SEQ ID NO. 11, and CDR3 with the amino acid shown as SEQ ID NO. 22; the framework regions are FR1 with the amino acid sequence shown as SEQ ID NO. 31, FR2 with the amino acid shown as SEQ ID NO. 40, FR3 with the amino acid shown as SEQ ID NO. 51, and FR4 with the amino acid shown as SEQ ID NO. 59; the amino acid sequence of the 1G9 nanobody is shown as SEQ ID NO. 65.

[0170] (7) the CDR sequences of the heavy chain variable region of the amino acid sequence of the 2A2 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 5, CDR2 with an amino acid sequence as shown in SEQ ID NO. 14, CDR3 with an amino acid as shown in SEQ ID NO. 23; the framework region is FR1 with an amino acid sequence as shown in SEQ ID NO. 32, FR2 with an amino acid as shown in SEQ ID NO. 41, FR3 with an amino acid as shown in SEQ ID NO. 52, FR4 with an amino acid as shown in SEQ ID NO. 58; the amino acid sequence of the 2A2 nanobody is shown in SEQ ID NO. 66.

[0171] (8) the CDR sequences of the heavy chain variable region of the amino acid sequence of the 2B11 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 6, CDR2 with an amino acid sequence as shown in SEQ ID NO. 15, CDR3 with an amino acid as shown in SEQ ID NO. 24; the framework region is FR1 with an amino acid sequence as shown in SEQ ID NO. 33, FR2 with an amino acid as shown in SEQ ID NO. 42, FR3 with an amino acid as shown in SEQ ID NO. 53, FR4 with an amino acid as shown in SEQ ID NO. 58; the amino acid sequence of the 2B11 nanobody is shown in SEQ ID NO. 67.

[0172] (9) the CDR sequences of the heavy chain variable region of the amino acid sequence of the 2C1 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 7, CDR2 with an amino acid sequence as shown in SEQ ID NO. 16, CDR3 with an amino acid as shown in SEQ ID NO. 25; the framework region is FR1 with an amino acid sequence as shown in SEQ ID NO. 34, FR2 with an amino acid as shown in SEQ ID NO. 43, FR3 with an amino acid as shown in SEQ ID NO. 54, FR4 with an amino acid as shown in SEQ ID NO. 58; the amino acid sequence of the 2C1 nanobody is shown in SEQ ID NO. 68.

[0173] (10) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 2C2 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 1, CDR2 with an amino acid sequence as shown in SEQ ID NO. 10, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 19; the framework regions are FR1 with an amino acid sequence as shown in SEQ ID NO. 35, FR2 with an amino acid sequence as shown in SEQ ID NO. 44, FR3 with an amino acid sequence as shown in SEQ ID NO. 55, and FR4 with an amino acid sequence as shown in SEQ ID NO. 58; the amino acid sequence of the 2C2 nanobody is shown in SEQ ID NO. 69.

[0174] (11) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 2C8 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 8, CDR2 with an amino acid sequence as shown in SEQ ID NO. 17, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 26; the framework regions are FR1 with an amino acid sequence as shown in SEQ ID NO. 36, FR2 with an amino acid sequence as shown in SEQ ID NO. 45, FR3 with an amino acid sequence as shown in SEQ ID NO. 56, and FR4 with an amino acid sequence as shown in SEQ ID NO. 58; the amino acid sequence of the 2C8 nanobody is shown in SEQ ID NO. 70.

[0175] (12) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 2F4 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 9, CDR2 with an amino acid sequence as shown in SEQ ID NO. 18, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 27; the framework regions are FR1 with an amino acid sequence as shown in SEQ ID NO. 35, FR2 with an amino acid sequence as shown in SEQ ID NO. 46, FR3 with an amino acid sequence as shown in SEQ ID NO. 57, and FR4 with an amino acid sequence as shown in SEQ ID NO. 58; the amino acid sequence of the 2F4 nanobody is shown in SEQ ID NO. 71.

[0176] 4. Amplification of overlap PCR products, transient transfection and detection

[0177] First round of PCR: amplification of CMV, VHH and Fc

[0178] PCR reaction system was configured 【50 μL system / reaction】(in which, CMV fragment amplification primer CMV-F and PCom-R1 were as shown in SEQ ID NO. 79-80; VHH fragment amplification primer PCom-F1 and PCom-R2 were as shown in SEQ ID NO. 81-82; FC fragment amplification primer PCom-F2 and PGK-R were as shown in SEQ ID NO. 83-84)

[0179] Table 15

[0180] CMV-F primer sequence information:

[0181] PCom-R1 primer sequence information:

[0182] PCom-F1 primer sequence information:

[0183] PCom-R2 primer sequence information:

[0184] PCom-F2 primer sequence information:

[0185] PGK-R primer sequence information:

[0186] PCR reaction program was as follows:

[0187] 50 μL of PCR product was taken, 1 / 10 volume of 10x loading buffer was added, 1% agarose was used for electrophoresis analysis, the band size of CMV and FC was about 750 bp, and the band size of VHH was about 560 bp.

[0188] The target band was cut off from the gel, the PCR product was purified, and the concentration was determined by NanoDrop (if the concentration is too high, it can be diluted for subsequent reaction).

[0189] Second round of PCR: Overlap Extension PCR connecting CMV, VHH and FC

[0190] PCR reaction system was configured:

[0191] Table 16

[0192] PCR reaction program was as follows:

[0193] Add 2ul of primer CMV-F and PGK-R respectively.

[0194] PCR reaction program is as follows:

[0195] The overlap PCR product is purified using a DNA fragment recovery kit and the concentration is measured by NanoDrop. At least 10ug of PCR product is required for subsequent cell transfection verification.

[0196] The PCR product is transiently transfected into 293F cells, and the supernatant is collected for ELISA detection.

[0197] 5. ELISA detection of the binding of recombinant antibody to target protein

[0198] Specifically includes the following steps: dilute the recombinant protein to a final concentration of 2ug / mL using sterile CBS, take a new 96-well enzyme-labeled plate, add 100ul to each well, and coat overnight at 4℃. Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20). Add 200ul / well of 3% MPBS and incubate at 37℃ for 2 hours; after removing the blocking buffer, wash the plate 5 times with PBST; add the expressed recombinant antibody, transfection supernatant 100ul / well, incubate at room temperature for 1 hour, and the control well is PBS; remove the liquid in the well and wash 5 times with PBST; add 100ul / well of HRP-Protein A antibody (1:50000 dilution), incubate at room temperature for 1 hour; after removing the liquid in the well, wash the plate 5 times with PBST; add 100ul / well of TMB developing solution; incubate at room temperature for 10-15 minutes in the dark; add 50ul / well of stop solution; use an enzyme-labeled instrument to read the OD 450 value in the well.

[0199] The ELISA detection results of the binding of the screened VHH to FAPa antigen are shown in Table 17. Except for 1-E4, 1-H4, and 1-B4, the remaining clones all bind to hFAPa antigen, and 2-E09 binds to hFAPa antigen with weak binding.

[0200] Table 17

[0201] In summary, the present application finds 12 potential positive clones by constructing a yeast nanobody library and performing panning and ELISA preliminary screening. However, FAPa usually exists in the form of a dimer on the cell surface. To verify the binding of the screened VHH to FAPa on the cell membrane surface, the binding of the obtained VHH to target cells expressing FAPa is further detected by flow cytometry.

[0202] Example 4 Construction of FAPa overexpression plasmid

[0203] comprising the following steps:

[0204] Construction of hFAPα overexpression plasmid:

[0205] PCR reaction system was prepared according to Table 19 below, hFAPα CDS fragment was amplified, and primers were used as shown in the table.

[0206] Table 18

[0207] The reaction system was prepared as follows:

[0208] Table 19 reaction system

[0209] The reaction procedure was as follows:

[0210] Table 20

[0211] The above reagents were from NEB.

[0212] After the reaction was completed, the PCR product was subjected to 1% agarose gel electrophoresis, and the hFAPα CDS fragment of about 2300 bp was recovered and quantified by ultraviolet absorption.

[0213] 1 μg of the backbone plasmid and the above recovered DNA fragment were subjected to double enzyme digestion with BamHI and NotI, and after 37°C reaction for 1 h, the large fragment of the vector backbone and the small fragment of about 2300 bp were recovered by gel electrophoresis.

[0214] Table 21

[0215] The fragments recovered in the above steps were ligated with T4 ligase, the T4 ligation system was as shown in Table 21, and after the preparation was completed, the reaction was carried out at room temperature for 2 h. The conventional method was used for transformation into E. coli stbl3 competent cells, and single colonies were selected from solid culture medium for further sequencing identification. The sequencing result was consistent with the expectation.

[0216] Example 5 Preparation of hFAP overexpression lentivirus.

[0217] comprising the following steps:

[0218] (1) 8 × 10 6 The number of cells was inoculated into 15 cm culture dishes, and 293T cells were cultured at 37°C, 5% CO2 for 48 h for lentivirus packaging. The culture medium was added with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin mixture and 1% glutamine;

[0219] (2) Dissolve 22 μg of the hFAPα overexpression plasmid constructed in Example 4, 15 μg of the helper plasmid gag / pol and 15 μg of the envelope plasmid VSVg in 2500 μL of Opti-MEM medium, and mix well;

[0220] (3) Dissolve 135 μg of PEI (1 μg / μL) in 2500 μL of Opti-MEM medium, and mix well by gently blowing;

[0221] (4) Formation of the transfection complex: add the PEI mixture to the DNA mixture, and vortex mix or mix gently immediately after addition, and incubate at 25°C for 20 min;

[0222] (5) Add the transfection complex dropwise to 20 mL of medium in a 15 cm 293T cell culture dish, and replace the medium with fresh medium after 24 h;

[0223] (6) Collect the virus supernatant at 48 h and 72 h, respectively; and collect the cell supernatant containing the lentivirus.

[0224] (7) Centrifuge about 37 mL of the collected virus supernatant at 2000 g and 4°C for 10 min, filter the supernatant after centrifugation using a 0.45 μm filter to remove cell debris, and then balance using a one-thousandth balance, and further concentrate and purify in an ultracentrifuge at 25000 rpm and 4°C for 2 h. After centrifugation, discard the supernatant, and resuspend the lentivirus precipitate using 250 μL of serum-free DMEM overnight. The next day, perform infection of Jurkat T cells and titer determination, or store the virus at -80°C for long-term preservation.

[0225] Example 6 Construction of hFAP Overexpression Cell Strain

[0226] The method comprises the following steps:

[0227] (1) Digest and count the 293T cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences), and resuspend in DMEM + 10% FBS medium. Take 5 x 106cells, and add 1 mL of the hFAP overexpression lentivirus supernatant, and supplement the medium to 2 mL, and add polybrene to a final concentration of 5 μg / mL, and culture at 37°C and 5% CO2 overnight; 4 (2) Replace the medium of the 293T cells infected with the virus with fresh DMEM + 10% FBS medium 2 mL, and continue to culture at 37°C and 5% CO2;

[0228] (2) Replace the medium of the 293T cells infected with the virus with fresh DMEM + 10% FBS medium 2 mL, and continue to culture at 37°C and 5% CO2;

[0229] (3) After 3 days of culture, the expression efficiency of hFAP in 293T cells was detected by flow cytometry, and the antibody used was anti-hFAP-APC. The experimental results are shown in Figure 6. As can be seen from Figure 6, the overexpression efficiency of hFAPa-293T is about 23%.

[0230] Example 7 Flow cytometry detection of the binding of nanobodies to target cells

[0231] Since FAPa usually exists in the form of a dimer on the surface of the cell membrane, in this example, 293T cells overexpressing hFAPa were selected as target cells to detect the binding of FAPa antibodies to cell membrane surface antigens. 293T-wt cells were used as controls to detect the specificity of FAPa antibody binding.

[0232] 2.5 x 10 5 hFAPa-293T was resuspended in 100 μL of 1% FBS-PBS, and anti-FAPa VHH antibodies were added. After incubation in an ice bath for 30 min, the cells were washed with PBS. The blank control group was not added with anti-FAPa VHH antibodies, and then incubated with antibodies for 30 min. The binding efficiency of nanobodies to target cells was detected by flow cytometry.

[0233] The FCM detection results of anti-FAPa nanobodies selectively recognizing hFAPa-293T cells are shown in Figure 7, which shows that the anti-FAPa antibodies prepared by screening can recognize the FAPa antigen on the surface of the cells.

[0234] Example 8 Preparation of a lentiviral vector expressing a chimeric antigen receptor (FAPa CAR) targeting FAPa

[0235] (1) The lentiviral vector pHIV-VHH CAR-P2A-GFP carrying the FAPa CAR chimeric antigen receptor was constructed, and the vector map is shown in Figure 8A. The structural mode diagram of the chimeric antigen receptor vector is shown in Figure 8B. The amino acid sequences of the modules of the CAR are shown in SEQ ID NO. 87-90, including a CD8a signal peptide, an anti-FAPa nanobody (anti-FAPa VHH), a CD8a hinge region, a CD8a transmembrane region, a 4-1BB costimulatory domain, and an immunoreceptor tyrosine-based activation motif (CD3zeta). The amino acid sequence of the signal peptide is shown in SEQ ID NO. 87. The amino acid sequences of the CD8a hinge region and the transmembrane region are shown in SEQ ID NO. 88. The amino acid sequence of the 4-1BB intracellular region is shown in SEQ ID NO. 89. The amino acid sequence of CD3zeta is shown in SEQ ID NO. 90.

[0236] The amino acid sequence of the signal peptide is: MALPVTALLLPLALLLHAARP (SEQ ID NO. 87).

[0237] The amino acid sequences of the anti-FAP alpha VHH are shown in SEQ ID NO. 60-71.

[0238] The amino acid sequence of the CD8 alpha hinge region and transmembrane region is:

[0239] The amino acid sequence of the 4-1BB intracellular region is:

[0240] The amino acid sequence of CD3 zeta is:

[0241] The specific preparation method is as follows:

[0242] (1) The enzyme digestion reaction system (reagents are purchased from NEB) is prepared according to Table 22, and the anti-FAP alpha-VHH fragment is digested.

[0243] Table 22

[0244] The above reagents are from NEB.

[0245] After preparation, incubate at 50°C for 1 h in a PCR instrument for reaction, and subject the PCR product to 1% agarose gel electrophoresis, recover the 700 bp or so fragment, and quantitate by ultraviolet absorption method.

[0246] The 700 bp or so fragment recovered in the above step and the digested vector are connected with T4 ligase, the T4 ligation system is shown in Table 22, after preparation, react at room temperature for 2 h, and transform into E. coli stbl3 competent cells according to the conventional method, select single colonies from solid culture medium, and further identify by sequencing, and the sequencing result is consistent with the expectation.

[0247] Table 23

[0248] (2) Packaging of lentivirus: same as Example 5.

[0249] Example 9: Transduction of T lymphocytes using lentivirus

[0250] Recruit healthy volunteers to extract peripheral blood, separate PBMC, obtain human primary T lymphocytes by magnetic bead sorting, and use TexMACS T M GMP medium culture (Miletnyi Biotec), use T cell activator Transact at a ratio of 1:100 to activate for 48 h, collect activated T cells, adjust the cell density to 1x10 6 / mL, the above prepared lentivirus of Example 8 was added at a multiplicity of infection (MOI) = 30, and vector fusin was added to a final concentration of 10 μg / mL to promote transduction; after incubation at 37°C in a 5% CO2 environment overnight, fresh culture medium was replaced, and the transduction efficiency was detected after 72 h, and the experimental results are shown in Figure 9.

[0251] Example 10 Anti-hFAPα-CAR-T cells were subjected to in vitro toxicity experiments

[0252] Each group of anti-FAPα VHH-CAR-T cells and untransduced primary T cells were planted in a V-bottom 96-well plate at a ratio of 1:1, 5:1, and 10:1 with U87 cells naturally expressing FAP or HEB cells not expressing FAPα, and U87 or FAPα-293T cells without primary T cells were set as blank groups, 3 replicates were set for each group, the culture medium volume was 200 ul, and the ratio of T cell medium to target cell medium was 1:1; after 12 h of culture, the 96-well V-bottom plate was centrifuged at 350 g for 5 min to remove the culture medium, 200 ul of PBS was added to each well, and then centrifuged at 350 g for 5 min again, and the PBS was completely discarded; the lysis solution and luciferase substrate were prepared according to the instructions, 20 ul of lysis solution was added to each well, and the cells were gently blown to lyse them, and the lysed liquid was transferred to a black opaque 96-well luminescence plate, 40 ul of luciferase substrate was added to each well, the parameters were adjusted according to the instrument use instructions of the multifunctional enzyme label instrument, and the luminescence value was detected; the killing of each group of primary T cells or CAR-T cells to target cells was calculated according to the detection value. Among them: the killing ratio = (1-CAR-T co-culture well luminescence value / untransduced virus primary T cell well luminescence value) * 100%. The experimental results are shown in Figure 10, and the CAR-T cells constructed based on the nanobodies can exhibit specific killing effect.

[0253] In summary, 12 specific anti-FAPα nanobodies were screened by immunizing alpacas and combining with a yeast surface display system, and the CAR-T cells constructed based on these nanobodies can specifically kill target cells expressing FAPα.

[0254] The above examples only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. A nanobody targeting fibroblast activation protein alpha, characterized in that, comprises a heavy chain variable region CDR sequence selected from at least one of the following or an amino acid sequence at least 80% identical thereto: SEQ ID NO. 1-27.

2. The Nanobody according to claim 1, characterized in that, The nanobody comprises: a heavy chain variable region CDR1 sequence as set forth in an amino acid sequence of SEQ ID NO. 1-9, respectively, or an amino acid sequence at least 80% identical to SEQ ID NO. 1-9; a heavy chain variable region CDR2 sequence as set forth in an amino acid sequence of SEQ ID NO. 10-18, respectively, or an amino acid sequence at least 80% identical to SEQ ID NO. 10-18; a heavy chain variable region CDR3 sequence as set forth in an amino acid sequence of SEQ ID NO. 19-27, respectively, or an amino acid sequence at least 80% identical to SEQ ID NO. 19-27.

3. The fibroblast activation protein alpha targeting nanobody according to claim 1, characterized in that, The nanobody comprises a framework region FR sequence selected from at least one of the following or an amino acid sequence at least 80% identical thereto: SEQ ID NO. 28-59.

4. The Nanobody of claim 1, wherein The nanobody comprises: a framework region FR1 sequence as set forth in an amino acid sequence of SEQ ID NO. 28-36, respectively, or an amino acid sequence at least 80% identical to SEQ ID NO. 28-36; a framework region FR2 sequence as set forth in an amino acid sequence of SEQ ID NO. 37-46, respectively, or an amino acid sequence at least 80% identical to SEQ ID NO. 37-46; a framework region FR3 sequence as set forth in an amino acid sequence of SEQ ID NO. 47-57, respectively, or an amino acid sequence at least 80% identical to SEQ ID NO. 47-57; a framework region FR4 sequence as set forth in an amino acid sequence of SEQ ID NO. 58-59, respectively, or an amino acid sequence at least 80% identical to SEQ ID NO. 58-59.

5. A Nanobody targeting fibroblast activation protein alpha, characterized in that, The amino acid sequence of the nanobody is selected from at least one of the following sequences or an amino acid sequence at least 80% identical thereto: SEQ ID NO. 60-71.

6. A polypeptide, characterized in that, The nanobody of any one of claims 1-5.

7. A nucleic acid molecule encoding the nanobody of any one of claims 1-5.

8. An expression vector, characterized by, The nucleic acid molecule of claim 7.

9. A host cell transformed or transfected with the expression vector of claim 8.

10. A pharmaceutical composition, characterized by, It contains the nanobody as set forth in any one of claims 1-5, and a pharmaceutically acceptable carrier and / or excipient.

11. Use of the Nanobody according to any one of claims 1 to 5, the polypeptide according to claim 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, the pharmaceutical composition according to claim 10 for the manufacture or selection of a diagnostic or therapeutic drug or kit for the diagnosis or treatment of a disease, characterized in that, The disease expresses fibroblast activation protein alpha. The disease expresses fibroblast activation protein alpha.

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