Anti-FAP nano antibody and application thereof

Abstract

The invention provides an anti-FAP nano antibody. The anti-FAP nano antibody comprises a complementary determining region (CDR) and a framework region (FR), wherein the complementarity determining region CDR comprises complementarity determining regions CDR1-CDR3, and the complementarity determining regions CDR1-CDR3 are And the framework area FR comprises framework areas FR1-FR28. According to the invention, an alpaca phage antibody library immunized by human FAP extracellular domain protein is successfully constructed, seven new anti-FAP nano antibody VHH sequences are screened from the alpaca phage antibody library, and the nano antibody sequences not only can recognize human FAP antigens, but also can recognize mouse FAP antigens. Meanwhile, the nano antibody sequence can be prepared into chimeric antigen receptor T cells (CAR-T) and can effectively kill cells highly expressing human or mouse FAP, so that the nano antibody sequence has an immunotherapy prospect of being applied to targeted FAP positive cells including tumor and fibrosis diseases.

Description

Technical Field

[0001] The present invention relates to the field of biological genes, and in particular to an anti-FAP nano antibody and application thereof. Background Art

[0002] Fibroblast activation protein (FAP) is a type II transmembrane serine protease that plays a vital role in the metabolism of a variety of endogenous peptides and peptide drugs. It is expressed on the surface of fibrotic cells and fibroblasts in a variety of tumor environments, and can promote the occurrence and development of fibrosis and tumors caused by inflammation. FAP can affect tumor growth through a variety of mechanisms, including promoting proliferation, invasion, angiogenesis, epithelial-mesenchymal transition, stem cell differentiation, immunosuppression and drug resistance. Studies have shown that FAP can be used as a potential target for tumors and as a biological marker for the early diagnosis of diseases such as tumors and rheumatoid arthritis.

[0003] At present, preclinical studies on FAP targets include: using FAP antibody-coupled toxins to specifically deliver toxins to target cells can significantly inhibit breast tumor growth and increase lymphocyte infiltration in tumors; vaccinating FAP with dendritic cells transfected with FAP mRNA can inhibit the growth of transplanted tumors and intravenously injected tumors; oral anti-FAP DNA vaccines have been shown to significantly inhibit angiogenesis, tumor growth and metastasis of breast cancer cells injected in situ, and can greatly increase the uptake of drugs in tumors after the addition of doxorubicin; FAP-specific CAR-T cells can eliminate most FAP antigen-positive cells, including cancer associated fibroblasts (CAF), and inhibit the generation of tumor stroma, thereby improving the uptake of chemotherapeutic drugs and anti-tumor effects.

[0004] At present, the clinical research on FAP targets includes: inhibiting the protease activity of FAP with small molecules or antibodies, using the protease activity of FAP to cut tumor drugs attached to FAP targeting peptides, vaccination against FAP, and chimeric antigen receptor T cell (CAR-T) therapy. Since FAP antibodies can target tumor stroma, thereby improving the tumor microenvironment and allowing drugs or CAR-T cells to effectively enter the tissue, the development of new and efficient FAP antibodies plays an important role in targeting FAP-positive fibroblasts. However, most of the FAP antibodies reported so far are single chain antibodies (scFv) composed of heavy chain variable regions and light chain variable regions, and have not shown good therapeutic effects.

[0005] Heavy chain antibodies are a new type of antibody found in camelids in 1989 that lacks the constant domain of heavy chain 1 (CH1) of the light chain. Nano antibodies are cloned from the variable domain of heavy chain of heavy chain antibody (VHH), with a crystal diameter of only 2.5nm and a molecular weight of 12-15KDa, making them the smallest known antibodies. Compared with traditional antibodies, nano antibodies have the following advantages: 1. Nano antibodies have a longer third antigen complementary determining region, maintain antigen binding ability similar to that of normal double-chain antibodies, and can better bind to antigen epitopes. 2. Inter-ring disulfide bonds are common inside nano antibodies, which are more stable at high temperatures and higher concentrations of organic solvents. 3. The second framework region of nano antibodies contains four conservative hydrophilic amino acid mutations, which makes them hydrophilic and not easy to aggregate, affecting antigen recognition. 4. Nano antibodies are small in size and can better penetrate dense tumor tissues and are used in the treatment of solid tumors. 5. Nanobodies only have heavy chain variable regions. When multiple nanobodies are used in combination, there will be no cross-reaction between the heavy chain and the light chain, which will affect the therapeutic effect of the antibody. 6. Nanobodies come from the camel family and are highly similar to human antibodies. They have low immunogenicity and will not produce neutralizing antibodies during treatment.

[0006] Phage display technology is the most commonly used method for producing nanoantibodies. cDNA is obtained from natural, immune or synthetic antibody libraries through genetic engineering; the DNA is inserted into the appropriate position of the phage coat protein to form a fusion protein with the coat protein and expressed on the phage surface; antibodies are screened using an immune antibody library, which has high specificity and makes it easier to obtain high-affinity antibodies.

[0007] In summary, it is urgent to develop a new technical solution to solve the problems existing in the prior art. Summary of the invention

[0008] Based on the advantages of nano-antibodies, the team of the present invention successfully constructed an alpaca phage antibody library immunized with the extracellular segment protein of human FAP, and screened out 7 new anti-FAP nano-antibody VHH sequences. Our experiments found that the nano-antibody sequence can recognize both human FAP antigen and mouse FAP antigen. At the same time, the nano-antibody sequence can be prepared into chimeric antigen receptor T cells (CAR-T), and can effectively kill cells that highly express human or mouse FAP positive, so it has the prospect of being applied to immunotherapy targeting FAP positive cells including tumors and fibrotic diseases.

[0009] The term "chimeric antigen receptor" as used herein refers to an artificial receptor that has been genetically engineered to give immune cells (such as T lymphocytes) new abilities to target specific antigens. These receptors are chimeric because they combine the antigen-specific recognition domain of an antibody with the T cell activation domain into one receptor.

[0010] The term "heavy chain CDR" involved in the present invention, i.e., the complementarity determining region of the heavy chain, refers to the core conserved structure of the antibody heavy chain variable region, which is the key region that determines the antibody binding to the antigen, including CDR1, CDR2 and CDR3.

[0011] The term "framework region FR" involved in the present invention refers to the sequence excluding the CDR region in the VHH antibody.

[0012] One object of the present invention is to provide an anti-FAP nanobody, wherein the anti-FAP nanobody comprises a complementarity determining region CDR and a framework region FR;

[0013] in,

[0014] The complementary determining region CDR includes complementary determining regions CDR1-CDR3;

[0015] The complementary determining regions CDR1 are shown in amino acid sequences 1, 4, 7, 10, 13, 16 and 19 respectively;

[0016] The complementary determining regions CDR2 are shown in amino acid sequences 2, 5, 8, 11, 14, 17 and 20 respectively;

[0017] The complementary determining regions CDR3 are shown in amino acid sequences 3, 6, 9, 12, 15, 18 and 21 respectively;

[0018] The amino acid sequences 1-21 are shown as SEQ ID No.1-SEQ ID No.21.

[0019] Further, the framework region FR includes framework regions FR1-FR28;

[0020] in,

[0021] The framework regions FR1-FR28 are shown in amino acid sequences 22-49, respectively;

[0022] The amino acid sequences 22-49 are shown as SEQ ID No.22-SEQ ID No.49.

[0023] Furthermore, the amino acid sequence of the anti-FAP nanobody is FAP-VHH1 to FAP-VHH7; and FAP-VHH1 to FAP-VHH7 are shown in SEQ ID No.50-SEQ ID No.56.

[0024] Furthermore, the anti-FAP nanobody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies or a combination thereof.

[0025] Another object of the present invention is to provide a nucleic acid molecule encoding the above-mentioned anti-FAP nanobody.

[0026] Furthermore, the nucleic acid molecule comprises the nucleic acid sequence SEQ ID No.57-SEQ ID No.63.

[0027] Another object of the present invention is to provide a carrier, which comprises the above-mentioned anti-FAP nanobody.

[0028] Furthermore, the vector is selected from a DNA molecule, an mRNA molecule or a cell.

[0029] Another object of the present invention is to provide the use of the carrier in antigen detection, adsorption of antigen reagents and immunotherapy.

[0030] Furthermore, the immunotherapy is immunotherapy targeting FAP-positive cells including tumors or fibrotic diseases.

[0031] The present invention has the following beneficial effects:

[0032] The present invention discloses an anti-FAP nanobody, which includes heavy chain CDR1-CDR3. The present invention first expresses and purifies the human FAP extracellular segment polypeptide and makes it immunogenic, then couples the human FAP extracellular segment polypeptide to an ELISA plate to display the correct spatial structure of the protein, and uses phage display technology to screen the human FAP extracellular segment immune nanobody gene library (camel heavy chain antibody phage display gene library) using this form of antigen, thereby obtaining a FAP-specific nanobody gene, and transferring this gene to Escherichia coli, thereby establishing a nanobody strain that can be efficiently expressed in Escherichia coli; our preliminary experiments found that the FAP nanobody screened by the method of the present invention has the characteristics of immune reaction with human and mouse or human FAP antigen alone, and has good specificity and high affinity, and can be used to prepare FAP detection reagents or anti-tumor drugs, etc. For example, the anti-human FAP nanobody provided by the present invention can be used as the antigen recognition domain of CAR to construct CAR-T cells, which has a significant killing effect on cell lines expressing FAP antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of screening positive phages by using phage supernatant flow detection to bind to FAP antigen are shown;

[0034] in,

[0035] Figure 1 (a) shows the 3T3-FAP flow cytometry results;

[0036] Figure 1 (b) shows the CHO-FAP flow cytometry results.

[0037] Figure 2 The SDS-PAGE image of crude purification of anti-FAP Nanobody is shown.

[0038] Figure 3 A Western-blot analysis of the crude purified anti-FAP nanobody is shown.

[0039] Figure 4 The flow cytometry analysis shows the specific binding ability of FAP-VHH-mIgG-Fc fusion protein to human and mouse FAP-expressing positive cells;

[0040] in,

[0041] Figure 4 (a) shows the binding ability of 7 FAP-VHH-mIgG-Fc fusion proteins to 3T3, 3T3-hFAP cells overexpressing human FAP, and 3T3-mFAP cells overexpressing mouse FAP;

[0042] Figure 4 (b) shows the binding ability of 7 FAP-VHH-mIgG-Fc fusion proteins at different volumes to 3T3-hFAP cells overexpressing human FAP.

[0043] Figure 5 The killing efficiency of FAP-VHH-CAR-T cells on different cell lines detected by LDH is shown.

[0044] Figure 6 The figure shows the amount of IFN-γ released during CAR-T cell killing detected by ELISA. DETAILED DESCRIPTION

[0045] In order to more clearly illustrate the technical solution of the present invention, the following examples are listed. Unless otherwise stated, the raw materials, reactions and post-treatment methods shown in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0046] Example

[0047] An anti-FAP nanobody, comprising a complementarity determining region CDR and a framework region FR;

[0048] in,

[0049] The complementary determining region CDR includes complementary determining regions CDR1-CDR3;

[0050] The complementary determining regions CDR1 are shown in amino acid sequences 1, 4, 7, 10, 13, 16 and 19 respectively;

[0051] The complementary determining regions CDR2 are shown in amino acid sequences 2, 5, 8, 11, 14, 17 and 20 respectively;

[0052] The complementary determining regions CDR3 are shown in amino acid sequences 3, 6, 9, 12, 15, 18 and 21 respectively;

[0053] The amino acid sequences 1-21 are shown as SEQ ID No.1-SEQ ID No.21;

[0054] The framework region FR includes framework regions FR1-FR28;

[0055] The framework regions FR1-FR28 are shown in amino acid sequences 22-49, respectively;

[0056] The amino acid sequences 22-49 are shown as SEQ ID No.22-SEQ ID No.49;

[0057] The amino acid sequence of the anti-FAP Nanobody is FAP-VHH1 to FAP-VHH7;

[0058] The FAP-VHH1 to FAP-VHH7 are shown as SEQ ID No.50 to SEQ ID No.56.

[0059] A nucleic acid molecule encoding the above-mentioned anti-FAP nanobody, comprising the nucleic acid sequence SEQ ID No.57-SEQ IDNo.63.

[0060] The method for preparing the above-mentioned anti-FAP nanobody comprises the following steps:

[0061] S1. Alpaca immunization with FAP antigen and antiserum validation

[0062] Prepare FAP antigen, inject FAP antigen multiple times on the left and right sides near the alpaca's cervical lymph nodes to immunize the alpaca, collect blood samples, and separate serum for subsequent antibody titer detection; use NaHCO3 coating solution to coat the antigen, dilute the serum sample in multiple ratios and add it to the corresponding antigen well for ELISA verification, and the titer is qualified when the dilution multiple exceeds 1:32000; then extract the alpaca peripheral blood lymphocytes PBMC and construct a phage library.

[0063] S2. Phage library construction

[0064] (1) RNA extraction: Total RNA from PBMC samples was extracted according to the instructions of RNA extraction kit RC11 from Novozymes.

[0065] (2) Reverse transcription: Reverse transcription was performed according to the instructions of the Novozyme RNA Reverse Transcription Kit R232; the reverse transcribed cDNA was stored at -20°C.

[0066] (3) PCR: VHH primers were designed and PCR amplification was performed using the reverse transcribed cDNA as a template to obtain a large number of target fragments. The amplification reaction conditions are shown in Table 1. After the PCR reaction was completed, the target band of about 350 bp was recovered by agarose gel electrophoresis. Then, the first round product was used as a template to perform a second round of PCR reaction to obtain the VHH fragment.

[0067] Table 1 PCR amplification reaction conditions for phage vector construction

[0068]

[0069] (4) Ligation product and electroporation: Connect VHH and phagemid vector through Goldgate to obtain the target recombinant vector; add the ligation product to TG1 competent medium, and electroporate at 2.5KV for 5ms; then add 2mL of warm bath culture medium and incubate at 37℃ and 220rpm for 1h. Draw out part of the bacterial solution and dilute to 10 -5 , 10 -6 , 10 -7 , respectively, spread on 2YTAG (1% glucose (W / v) and 100 μg / mL ampicillin) plates for overnight culture, pick single colonies, and perform PCR amplification and sequencing to detect library sequence diversity.

[0070] (5) Library diversity analysis: Monoclonal bacteria on the plate were randomly selected and inoculated into a centrifuge tube containing 1 mL of 2YT liquid culture medium (Amp) with Amp resistance, and cultured at 37°C for 3 h. PCR reaction reagents were prepared according to Table 2 and single colony PCR was performed.

[0071] Table 2 PCR reaction reagent preparation

[0072]

[0073]

[0074] PCR reaction: 95°C for 3 min; 95°C for 15 s, 56°C for 15 s, 72°C for 20 s, 32 cycles; 72°C for 5 min; 4°C∞.

[0075] The single colony PCR product was subjected to 1% agarose gel electrophoresis, and the colony with the target band fragment size of 500 bp was identified as the positive clone; several clones were randomly selected from the positive clones detected by colony PCR and sent for sequencing to detect the sequence diversity in the bacterial library.

[0076] S3. Selection and validation of anti-FAP nanobodies

[0077] S3-1. Preparation of anti-FAP phage antibody library

[0078] (1) Add glycerol bacteria to 2YT (A&G) medium at 1 OD / 100 mL and culture at 37°C and 220 rpm until the OD value is 0.4-0.6.

[0079] (2) Add M13K07 and shake well, incubate at 37°C for 30 min, incubate at 220 rpm for 1 h, and then centrifuge; resuspend the precipitate after centrifugation with an equal volume of 2YT (A&K) and incubate at 30°C and 220 rpm overnight.

[0080] (3) Centrifuge, transfer the supernatant to a clean tube, add 1 / 5 volume of PEG-NaCl, incubate at 4°C for 2 h, collect the precipitate by centrifugation, dissolve it with PBS, and centrifuge at 4°C for 5 min.

[0081] (4) Transfer the supernatant to a clean tube and add 1 / 5 volume of PEG-NaCl. Incubate at 4°C for 60 min. Discard the supernatant, dissolve the precipitate with an appropriate amount of PBS, collect it in a 1.5 mL centrifuge tube, and store it at 4°C.

[0082] (5) Titer measurement: The collected phages were diluted to 10 -9 , 10 -10 , 10 -11 After infecting the logarithmic phase TGI, spread on 2YTAG plates, culture at 37°C overnight, and count the number of colonies.

[0083] S3-2. Antigen selection and amplification

[0084] (1) The immunotube was coated with antigen and incubated at 37°C for 2 h. After the antigen was discarded, the tube was washed 3 times with PBST, and 5 mL of 4% bovine serum albumin (BSA) was added for blocking, and the tube was incubated at 37°C for 1 h.

[0085] (2) Wash 3 times with PBST and add 1×10 12 The antibody library phage was placed at 37℃ for 1 hour. After the phage was discarded, it was washed 10 times with PBST.

[0086] (3) Add 1 mL of Gly-HCL (pH 2.0) and shake at room temperature for 8 min to elute the phages, and then add 200 μL of Tris-HCL (pH 9.5) to neutralize.

[0087] (4) Take the eluate to measure the titer, and infect the rest with 5 mL of TG1. Let it stand at 37°C for 0.5 h. Centrifuge at 6000 rpm for 10 min, resuspend the precipitate in 20 mL of 2YTAG, and shake at 37°C and 220 rpm until the OD600 is about 0.5.

[0088] (5) Add helper phage M13K07 at an MOI of 20 and incubate at 37°C for 0.5 h. Centrifuge at 6000 rpm for 10 min, collect the precipitate and transfer to 150 mL of 2YTAK medium, and culture overnight at 28°C and 220 rpm.

[0089] (6) Collect phages and centrifuge. Transfer the supernatant to a clean tube and add 1 / 5 volume of PEG-NaCl. Incubate at 4°C for 2 h. Collect the precipitate by centrifugation, dissolve it with PBS, and centrifuge it at 4°C for 5 min.

[0090] (7) Transfer the supernatant to a clean tube and add 1 / 5 volume of PEG-NaCl. Incubate at 4°C for 60 min. Discard the supernatant and dissolve the precipitate with an appropriate amount of PBS. Collect the precipitate in a 1.5 mL centrifuge tube and store at 4°C.

[0091] (8) Titer measurement: The collected phages were diluted to 10 -9 , 10 -10 , 10 -11 After infecting the logarithmic phase TGI, spread on 2YTAG plates, culture at 37°C overnight, and count the number of colonies.

[0092] S3-3. Elisa Verification

[0093] (1) After the phages from the third round of panning were infected with TG1, they were spread on 2YTAG plates and cultured at 37°C overnight.

[0094] (2) Pick a single clone into a 96-deep-well plate with 200 μL of 2YTAG medium and culture at 37°C and 220 rpm.

[0095] (3) When the OD600 of the bacterial solution reaches about 0.5, 100 μL of the culture medium is taken out for preservation. Helper phage M13K07 is added to the remaining bacterial solution, and the solution is allowed to stand at 37°C for 0.5 h. Centrifuge at 4000 rpm for 10 min, discard the supernatant, and resuspend the precipitate in 600 μL 2YTAK. Induce phage overnight at 28°C and 220 rpm, and centrifuge at 4000 rpm for 10 min to retain the supernatant.

[0096] (4) Use NaHCO3 to coat the antigen on the Elisa plate at 50 ng / well and incubate at 4°C overnight.

[0097] (5) After washing three times with PBST, add 4% MPBS and block at 37°C for 1 h.

[0098] (6) After washing three times with PBST, 50 μL of phage supernatant and 4% MPBS were added and incubated at 37°C for 1 h.

[0099] (7) After washing four times with PBST, 100 μL of diluted anti-M13 antibody was added and reacted at 37°C for 1 h.

[0100] (8) After washing five times with PBST, TMB colorimetric solution was added for color development. After quenching with sulfuric acid, the plate was read at 450 nm on a microplate reader.

[0101] (9) The phage supernatant that showed positive color was retained for flow cytometry detection.

[0102] S3-4. Streaming Verification

[0103] (1) Prepare the cells and phage supernatant required for flow cytometry. Each phage sample requires 1×10 5 cells.

[0104] (2) Block cells and phages with 3% BSA-PBS at 4°C for 30 min; add positive cells to phages, incubate at 4°C for 30 min, and wash with PBS three times.

[0105] (3) After adding the labeled primary antibody, incubate at 4°C for 30 min and wash three times with PBS. After adding the corresponding fluorescent secondary antibody, incubate at 4°C for 30 min and wash three times with PBS.

[0106] (4) On-machine testing.

[0107] (5) Flow cytometry sequencing of positive samples.

[0108] Preparation of S4.FAP-VHH-mIgG-Fc fusion protein

[0109] Construction of S4-1 and FAP-VHH-mIgG-Fc fusion protein vector

[0110] (1) PCR amplification was performed to obtain the target FAP-VHH fragment. The PCR reaction system and amplification conditions are shown in Table 3.

[0111] Table 3 PCR reaction system and amplification conditions for obtaining FAP-VHH target fragments

[0112]

[0113]

[0114] After PCR amplification, the correct DNA band was obtained by agarose gel electrophoresis and gel recovery was performed according to the kit instructions.

[0115] (2) The fusion protein expression vector was linearized by enzyme digestion. The conditions are shown in Table 4.

[0116] Table 4 Linearization conditions of fusion protein expression vector

[0117] Element volume Vector Template 1 μg 10*rCutsmart buffer 5μL XOt 1μL EcoRI 1μL <![CDATA[ddH2O]]> Up to 50μL

[0118] (3) The target fragment is connected to the fusion protein expression vector

[0119] The recovered target fragment and vector fragment were connected by Infusion enzyme (refer to the instructions), and the connection product was added to 100 μL competent cells stabl3, mixed by blowing, and placed on ice for 30 minutes, heated in a 42°C water bath for 90 seconds, and immediately placed on ice for 2 minutes; evenly spread on a solid LB plate containing ampicillin with a coating rod in an ultra-clean workbench; cultured overnight at 37°C. After 16 hours, a single colony was picked and placed in a liquid LB medium containing ampicillin, and cultured overnight at 37°C and 220rpm on a constant temperature shaker; the plasmid was extracted by a DNA extraction kit, sequenced, and the correct fusion protein expression vector was obtained.

[0120] Expression and purification of S4-2 and FAP-VHH-mIgG-Fc fusion proteins

[0121] (1) Cell plating: One day before transfection, 5×10 6 The cells were plated in a 10 cm dish.

[0122] (2) Transfection: After 24 hours, the successfully constructed target plasmid and PEI were diluted with serum-free DMEM (the fusion protein expression system is shown in Table 5), and then allowed to stand at room temperature for 5 minutes. Then, tube A was slowly added to tube B, mixed evenly, and allowed to stand at room temperature for 20 minutes. Then, the mixture was evenly added dropwise to a 10 cm dish, gently shaken, and placed in a 37°C, 5% CO2 incubator. After 12 hours, 20 mL of fresh DMEM medium containing 10% FBS was replaced. After 48 hours and 72 hours, the supernatant was collected, filtered through a 0.45 μm filter membrane, and then the protein was concentrated using a 30 KDa protein purification column.

[0123] Table 5 Fusion protein expression system

[0124]

[0125]

[0126] Western-blot identification of S4-3 and FAP-VHH-mIgG-Fc fusion protein

[0127] (1) Protein denaturation: Take 20 μL of the protein before and after concentration, add 5× loading buffer, mix well by pipetting, and boil at 100°C for 5 min to denature the protein.

[0128] (2) Gel preparation: SDS-PAGE Gel Kit (Kangwei Century) was used to prepare 10% separation gel and 5% concentration gel.

[0129] (3) Gel electrophoresis: Add equal volume of protein to the sample wells, run at 90 V until the band is 1 cm from the bottom of the glass plate.

[0130] (4) Transfer: 90 V, 90 min.

[0131] (5) Blocking: Block with 5% skimmed milk powder for 1 h after transfer.

[0132] (6) Antibody incubation: Horseradish enzyme-labeled goat anti-mouse IgG (H+L) (ZB2305) was diluted 1:5000, incubated at room temperature for 2 h, washed three times with TBST, each time for 10 min, and developed.

[0133] S4-4. SDS-PAGE identification of FAP-VHH-mIgG-Fc fusion protein

[0134] The preparation and electrophoresis of gel were the same as western-blot. After the electrophoresis, the gel with the target protein band was placed in Coomassie Brilliant Blue R250 staining solution at room temperature for 1-2 hours. The gel was destained on a shaker until the gel was transparent.

[0135] S5. Detection of the binding ability of the generated FAP nanobodies to cell lines overexpressing human or mouse FAP

[0136] Take 1×10 cells of each type 6 , resuspended with PBS, centrifuged at 3500rpm for 5min, and discarded the supernatant. Each cell group was divided into 4 groups, namely NTC group: no antibody was added, and the cells were resuspended with 100μL PBS; primary antibody group: resuspended cells with 100μL PBS, added with 5μL FAP-VHH-mIgG-Fc fusion protein, incubated at 4℃ in the dark for 30min, then added with 1mL PBS, washed twice, centrifuged at 3500rpm for 5min, discarded the supernatant, and resuspended with 100μL PBS; secondary antibody group: added diluted iFluor TM 488Conjugated Goat anti-mouse IgG (HA1125) 100 μL, incubate at 4℃ in dark for 30 min, add 1 mL PBS, wash twice, centrifuge at 3500 rpm for 5 min, discard the supernatant, and resuspend in 100 μL PBS; Experimental group: resuspend cells in 100 μL PBS, add 5 μL FAP-VHH-mIgG-Fc fusion protein, incubate at 4℃ in dark for 30 min, add 1 mL PBS, wash twice, centrifuge at 3500 rpm for 5 min, discard the supernatant, add diluted iFluor TM 488Conjugated Goat anti-mouse IgG (HA1125) 100μL, incubate at 4℃ in dark for 30min, add 1mL PBS, wash twice, centrifuge at 3500rpm for 5min, discard the supernatant, and resuspend in 100μL PBS. Detect by flow cytometry. Experimental groups with different volumes of primary antibody added different volumes of primary antibody: 0.0001, 0.001, 0.01, 0.1 and 1μL, and other experimental conditions remained unchanged.

[0137] Application of S6 and FAP nanoantibodies in CAR-T cell immunotherapy

[0138] Construction of S6-1 and FAP-VHH-CAR lentiviral expression vectors

[0139] (1) Design primers containing homology arms and obtain the target FAP-VHH fragment by PCR amplification. The PCR reaction system and amplification conditions are the same as those in Table 2. After PCR amplification, obtain the correct DNA band by agarose gel electrophoresis and recover the DNA according to the instructions of the kit.

[0140] (2) Linearizing the CAR lentiviral vector by enzyme digestion, the conditions of which are shown in Table 6.

[0141] Table 6 CAR lentiviral vector linearization conditions

[0142] Element volume Vector Template 1 μg 10×rCutsmart buffer 5μL Xgf 1μL B H 1μL <![CDATA[ddH2O]]> Up to 50μL

[0143] (3) Target fragment is connected to CAR lentiviral vector

[0144] The recovered target fragment and vector fragment were connected by Infusion enzyme (refer to the instructions), and the connection product was added to 100 μL competent cells Stabl3, mixed by pipetting, placed on ice for 30 minutes, heated in a 42°C water bath for 90 seconds, and immediately placed on ice for 2 minutes; evenly spread on a solid LB plate containing ampicillin with a coating rod in an ultra-clean workbench, and cultured at 37°C overnight; after 16 hours, a single colony was picked and placed in a liquid LB medium containing ampicillin, and cultured overnight at 37°C and 220rpm on a constant temperature shaker. The plasmid was extracted by a DNA extraction kit and sequenced to obtain the correct CAR lentiviral vector.

[0145] S6-2. Preparation of FAP-VHH-CAR lentivirus

[0146] (1) Cell plating: One day before transfection, 5×10 6 The cells were plated in a 10 cm dish.

[0147] (2) Transfection: After 24 hours, the successfully constructed target plasmid and PEI were diluted with serum-free DMEM (the lentiviral packaging system is shown in Table 7), and allowed to stand at room temperature for 5 minutes. Then, tube A was slowly added to tube B, mixed evenly, and allowed to stand at room temperature for 20 minutes. Then, the mixture was evenly added dropwise to a 10 cm dish, gently shaken, and placed in a 37°C, 5% CO2 incubator. After 12 hours, 20 mL of fresh DMEM medium containing 10% FBS was replaced. After 48 hours and 72 hours, the supernatant was collected and filtered through a 0.45 μm filter membrane; centrifuged at 18500 rpm and 4°C for 90 minutes; the supernatant was discarded and resuspended in x-vivo containing 10% FBS.

[0148] Table 7 Lentivirus packaging system

[0149]

[0150]

[0151] S6-3. Preparation of FAP-VHH-CAR-T cells

[0152] Human peripheral blood mononuclear cells (PBMC) were resuscitated, and the cells were counted after 4 hours, and centrifuged at 1000 rpm for 5 minutes. At the same time, 1 mL of X-vivo medium was prepared, and CD3 / CD28 magnetic beads were added at a ratio of cells: magnetic beads = 1:3, and the mixture was mixed by pipetting, placed on a magnetic stand, and allowed to stand for 1 minute, and the waste liquid was discarded; the cells were resuspended with 1 mL of X-vivo medium containing 10% FBS and 100 IU / mL IL-2, mixed with magnetic beads, and incubated at room temperature for 1 hour at 10 rpm on a 180° flip mixer; after the incubation, the centrifuge tube was placed on a magnetic stand, allowed to stand for 1 minute, the waste liquid was discarded, and 1 mL of X-vivo medium containing 10% FBS and 100 IU / mL IL-2 was added to resuspend the cells, and cultured in a 24-well plate; after 24 hours, the cells were collected, counted, and 1×10 5 cells / well were plated in a 24-well plate, and FAP-VHH-CAR lentivirus was added at an MOI of 1, with a final volume of 1 mL. The infection-aiding reagent F108 was added to a final concentration of 1 mg / mL, and the mixture was mixed by pipetting, and centrifuged at 1500g, speed 3, speed 1, and room temperature for 90 min; after centrifugation, the cells were placed in a 37°C, 5% CO2 incubator; after 4 hours, each well was supplemented with 1 mL of X-vivo medium containing 10% FBS and 100 IU / mL IL-2; after 24 hours, the infection was repeated. When T cells were expanded to a sufficient number, the positive rate of CAR-T cells was detected using self-produced FAP extracellular segment protein and fluorescent secondary antibody.

[0153] S6-4. In vitro functional verification of FAP-VHH-CAR-T cells

[0154] S6-4-1. LDH method to detect the killing effect of CAR-T cells

[0155] (1) Cell plating

[0156] After digestion, tumor cells were counted. 100 μL of target cells were added to each well, and 2×10 4 Cells were plated in a round-bottom 96-well plate. After the CAR-T cells were tested for positive rate by flow cytometry, they were added according to the effector-target ratio of 0.5:1, 1:1, 2:1, 4:1, and 8:1, 100 μL per well, mixed by pipetting, and placed in a 37°C, 5% CO2 incubator; at the same time, the target cell spontaneous release well (only target cells), the target cell maximum release well (lysate was added during detection), and the effector cell spontaneous release well were set as controls; then centrifuged at 250g for 5 minutes and placed in 37°C, 5% CO2 culture conditions for culture.

[0157] (2) After the cells have been cultured for a certain period of time, lysis solution was added to the target cell maximum release well 30 min in advance; the culture plate was centrifuged at 250 g for 5 min, 50 μL of supernatant was aspirated from each well into a new flat-bottom 96-well plate, 50 μL of working solution was added, and the cells were incubated at room temperature in the dark for 30 min; then 50 μL of stop solution was added, and the absorbance at 492 nm was detected by an ELISA reader.

[0158] (3) Analytical data: cytotoxicity or mortality % = (experimental well - spontaneous activity of target cells - spontaneous activity of effector cells) / (maximum enzyme activity well - spontaneous activity of target cells) × 100. The absorbance of the experimental wells measured should be equal to the absorbance of the background blank control wells.

[0159] S6-4-2. ELISA to detect the amount of IFN-γ released during CAR-T cell killing

[0160] (1) Sample preparation: The supernatant in S6-4-1 was diluted 50 times with PBS.

[0161] (2) Coating plate: Add 100 μL of the prepared 1× capture antibody to the ELISA plate, seal the plate, and incubate at 4°C overnight.

[0162] (3) Discard the liquid, wash three times with 250 μL Wash buffer, leaving it for 1 min each time, invert it on absorbent paper, and discard the clean liquid.

[0163] (4) Blocking: Add 200 μL ELISPOT diluent and incubate at room temperature for 1 h.

[0164] (5) Prepare the standard: add 100 μL of ELISPOT diluent to wells 2-7, add 200 μL of standard to well 1, then pipette 100 μL into well 2, pipette to mix, then pipette 100 μL into well 3, and so on; pipette at least 5 times each time.

[0165] (6) Discard the blocking solution, wash once with Wash buffer, and discard the solution.

[0166] (7) Add 100 μL of sample or standard and 100 μL of ELISPOT to the blank well.

[0167] (8) Seal the plate and incubate at room temperature for 2 hours or at 4°C overnight.

[0168] (9) Discard the liquid and wash 3-5 times.

[0169] (10) Add 100 μL of detecting antibody to each well and incubate at room temperature for 1 h.

[0170] (11) Prepare HRP.

[0171] (12) Discard the solution and wash 3-5 times with wash buffer, soaking for 1 min each time.

[0172] (13) Add diluted HRP.

[0173] (14) Seal the plate and incubate at room temperature for 30 min.

[0174] (15) Discard the solution and wash 5-7 times.

[0175] (16) Add 100 μL of TMB solution and incubate at room temperature for 15 min.

[0176] (17) Add 100 μL Stop solution.

[0177] (18) Plate reading: Read the value at 450 nm.

[0178] Test Example 1

[0179] Generation of anti-FAP nanosequences.

[0180] Test method: Take the anti-FAP positive phage supernatant, incubate it with 3T3-FAP cells and CHO-FAP cells, and perform flow cytometry detection. The results are as follows: Figure 1 shown.

[0181] Figure 1 The results of screening positive phages by using phage supernatant flow detection to bind to FAP antigen are shown;

[0182] in,

[0183] Figure 1 (a) shows the flow cytometry results of 3T3-FAP; wherein the symbols in the figure represent: C2818-3C6: VHH1; C2818-3F4: VHH2; C2818-3G9: VHH3; C2818-3G12: VHH4; C2818-3H10: VHH5; C2818-4D12: VHH6; C2818-4G11: VHH7;

[0184] Figure 1 (b) shows the CHO-FAP flow cytometry results; Figure 1 (a) The same.

[0185] Depend on Figure 1It can be seen that the results of screening positive phages have 14 positive signals, and 7 sequences were sequenced. These 7 anti-FAP nanobody sequences were subsequently named FAP-VHH1, FAP-VHH2, FAP-VHH3, FAP-VHH4, FAP-VHH5, FAP-VHH6, and FAP-VHH7.

[0186] Test Example 2

[0187] Characterization of the physicochemical properties of anti-FAP nanoantibodies.

[0188] Test method: FAP-VHH-mouse-IgG-Fc fusion protein was produced and concentrated using a 30KDa protein concentration column. After concentration, SDS-PAGE and Western-blot were performed for identification. The results were as follows: Figure 2 and Figure 3 shown.

[0189] Figure 2 The SDS-PAGE image of crude purification of anti-FAP Nanobody is shown.

[0190] Figure 3 A Western-blot analysis of the crude purified anti-FAP nanobody is shown.

[0191] Depend on Figure 2 and Figure 3 It can be seen that the relative molecular weight of the anti-FAP nanobody fusion protein is about 40KD. Through SDS-PAGE and Western-blot analysis, the molecular weight of the recombinant protein in this test example is consistent with the expected size, and the protein concentration after concentration is significantly higher than that of the unconcentrated protein.

[0192] Test Example 3

[0193] FAP-VHH-mIgG-Fc recombinant protein was constructed, and the specificity and binding ability of FAP nanoantibody were verified using cell lines overexpressing human or mouse FAP.

[0194] Test method: The purified FAP-VHH-mIgG-Fc recombinant protein was used as the primary antibody, and different volumes of fluorescently labeled goat anti-mouse IgG were added as the secondary antibody. The binding ability of the FAP-VHH-mIgG to 3T3, 3T3-hFAP and 3T3-mFAP cells was verified by flow cytometry. The results are as follows: Figure 4 shown.

[0195] Figure 4 The flow cytometry analysis shows the specific binding ability of FAP-VHH-mIgG-Fc fusion protein to human and mouse FAP-expressing positive cells;

[0196] in,

[0197] Figure 4 (a) shows the binding ability of 7 FAP-VHH-mIgG-Fc fusion proteins to 3T3, 3T3-hFAP cells overexpressing human FAP, and 3T3-mFAP cells overexpressing mouse FAP;

[0198] Figure 4 (b) shows the binding ability of 7 FAP-VHH-mIgG-Fc fusion proteins at different volumes to 3T3-hFAP cells overexpressing human FAP.

[0199] Depend on Figure 4 It can be seen that VHH1 and VHH4 among the 7 FAP-VHH-mIgG-Fc fusion proteins can bind to the FAP antigen on the surface of 3T3-hFAP and 3T3-mFAP cells, the other VHHs can bind to FAP on the surface of 3T3-hFAP cells, and all VHHs have no binding to 3T3 cells.

[0200] Test Example 4

[0201] In vitro functional validation of CAR-T cells based on anti-FAP nanoantibodies.

[0202] Test method: FAP-VHH-CAR-T cells were co-cultured with 3T3, 3T3-hFAP and 3T3-mFAP cells at different effector-target ratios, and the killing efficiency of CAR-T cells was detected by LDH. The results are as follows Figure 5 shown.

[0203] Figure 5 The killing efficiency of FAP-VHH-CAR-T cells on different cell lines detected by LDH is shown.

[0204] Depend on Figure 5 It can be seen that the seven types of FAP-VHH-CAR-T cells have good killing effects on 3T3-hFAP and 3T3-mFAP, but have no killing effects on 3T3 cells.

[0205] Test Example 5

[0206] ELISA was used to detect the amount of IFN-γ released during CAR-T cell killing.

[0207] Test method: Take the supernatant of the cells for killing function verification, and use ELISA to detect the amount of IFN-γ released. The results are as follows: Figure 6 shown.

[0208] Figure 6 The figure shows the amount of IFN-γ released during CAR-T cell killing detected by ELISA.

[0209] Depend on Figure 6It can be seen that the seven types of FAP-VHH-CAR-T cells have a high level of IFN-γ release to 3T3-hFAP and 3T3-mFAP cells, and the level decreases with the decrease of the effector-target ratio, while the level of IFN-γ release to 3T3 cells that do not express FAP is very low.

[0210] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0211] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An anti-FAP nanobody, characterized in that The anti-FAP nanobody includes a complementarity determining region CDR and a framework region FR; in, The complementary determining region CDR includes complementary determining regions CDR1-CDR3; The complementary determining regions CDR1 are shown in amino acid sequences 1, 4, 7, 10, 13, 16 and 19 respectively; The complementary determining regions CDR2 are shown in amino acid sequences 2, 5, 8, 11, 14, 17 and 20 respectively; The complementary determining regions CDR3 are shown in amino acid sequences 3, 6, 9, 12, 15, 18 and 21 respectively; The amino acid sequences 1-21 are shown as SEQ ID No.1-SEQ ID No.

21.

2. The anti-FAP nanobody according to claim 1, characterized in that The framework region FR includes framework regions FR1-FR28; in, The framework regions FR1-FR28 are shown in amino acid sequences 22-49, respectively; The amino acid sequences 22-49 are shown as SEQ ID No.22-SEQ ID No.

49.

3. The anti-FAP nanobody according to claim 1, characterized in that The amino acid sequence of the anti-FAP Nanobody is FAP-VHH1 to FAP-VHH7; The FAP-VHH1 to FAP-VHH7 are shown as SEQ ID No.50 to SEQ ID No.

56.

4. The anti-FAP nanobody according to claim 1, characterized in that The anti-FAP nanobody is selected from: an animal-derived antibody, a chimeric antibody, a humanized antibody or a combination thereof.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-FAP Nanobody according to any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, characterized in that The nucleic acid molecule includes the nucleic acid sequences SEQ ID No.57-SEQ ID No.

63.

7. A carrier, characterized in that The carrier comprises the anti-FAP Nanobody according to any one of claims 1 to 4.

8. The carrier according to claim 7, characterized in that: The vector is selected from a DNA molecule, an mRNA molecule or a cell.

9. Use of the carrier according to any one of claims 7 to 8 in antigen detection, adsorption of antigen reagents and immunotherapy.

Images