A nanobody against FAP and its application

Abstract

The present invention provides an anti-FAP nanobody, which includes complementary determining regions (CDRs) and framework regions (FRs); wherein the complementary determining regions (CDRs) include complementary determining regions (CDR1-CDR3); and the framework regions (FRs) include framework regions (FR1-FR28). 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 nanobody VHH sequences from it. The nanobody sequence can recognize both human FAP antigens and mouse FAP antigens. At the same time, the nanobody sequence can be prepared into chimeric antigen receptor T cells (CAR-T) and can effectively kill cells that highly express human or mouse FAP. Therefore, it has the prospect of being applied to immunotherapy targeting FAP-positive cells, including tumors and fibrotic diseases.

Description

Technical Field

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

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

[0003] Current preclinical studies targeting FAP include: using FAP antibodies coupled to 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 in situ injected breast cancer cells, and can greatly increase drug uptake in tumors after the addition of doxorubicin; FAP-specific CAR-T cells can eliminate most FAP antigen-positive cells, including cancer associated fibroblasts (CAFs), and inhibit the generation of tumor stroma, thereby improving the uptake of chemotherapy drugs and the anti-tumor effect.

[0004] Current clinical research on FAP targets includes: inhibiting the protease activity of FAP with small molecules or antibodies, utilizing the protease activity of FAP to cleave tumor drugs attached to FAP targeting peptides, vaccination against FAP, and chimeric antigen receptor T cell (CAR-T) therapy. Since FAP antibodies can target the tumor stroma, thereby improving the tumor microenvironment and enabling drugs or CAR-T cells to effectively enter the tissue, the development of new and highly effective 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 discovered in camelids in 1989 that lack both the light chain and the constant domain of the heavy chain (CH1). Nanobodies, cloned from the variable domain of the heavy chain (VHH) of a heavy-chain antibody, have a crystal diameter of only 2.5 nm and a molecular weight of 12-15 kDa, making them the smallest known antibodies. Nanobodies have the following advantages over traditional antibodies: 1. They have a longer third antigen-complementarity-determining region, maintaining antigen-binding ability similar to that of normal two-chain antibodies and enabling better binding to antigenic epitopes. 2. Inter-ring disulfide bonds are prevalent within nanobodies, making them more stable at high temperatures and in higher concentrations of organic solvents. 3. Four conserved hydrophilic amino acid mutations in the second framework region of nanobodies make them hydrophilic, less prone to aggregation, and thus impairing antigen recognition. 4. Nanobodies are small in size, enabling better penetration into dense tumor tissues and enabling their application in the treatment of solid tumors. 5. Nanobodies only have the heavy chain variable region. When multiple nanobodies are used in combination, there will be no cross-reaction between the heavy and light chains, which would affect the therapeutic effect of the antibody. 6. Nanobodies are derived from the camelid 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, there is an urgent need to develop a new technical solution to solve the problems existing in the existing technology. Summary of the Invention

[0008] Leveraging the advantages of nanobodies, the present invention team successfully constructed an alpaca phage antibody library immunized with the extracellular domain of human FAP and screened seven new anti-FAP nanobody VHH sequences from this library. Our experiments revealed that these nanobody sequences recognize both human and mouse FAP antigens. Furthermore, these nanobody sequences can be formulated into chimeric antigen receptor T cells (CAR-T) and effectively kill cells that overexpress human or mouse FAP. Therefore, these cells hold promise for immunotherapy targeting FAP-positive cells, including tumors and fibrotic diseases.

[0009] As used herein, the term "chimeric antigen receptor" refers to an artificial receptor that has been genetically engineered to confer upon immune cells (such as T lymphocytes) the ability 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 a single receptor.

[0010] The term "heavy chain CDR" involved in the present invention, that is, 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" used in the present invention refers to the sequence excluding the CDR region in a 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 regions CDR include complementary determining regions CDR1-CDR3;

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

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

[0017] The complementary determining regions CDR3 are represented by 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] Furthermore, 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 the 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 includes the nucleic acid sequence SEQ ID No.57-SEQ ID No.63.

[0027] Another object of the present invention is to provide a carrier comprising 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 applications of the carrier in antigen detection, adsorption of antigen reagents and immunotherapy.

[0030] Furthermore, the immunotherapy is an 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 enzyme-labeled plate to display the correct spatial structure of the protein. In this form of antigen, phage display technology is used to screen the nanoantibody gene library (camel heavy chain antibody phage display gene library) immune to the human FAP extracellular segment, thereby obtaining a FAP-specific nanoantibody gene, and transferring this gene into Escherichia coli to establish a nanoantibody strain that can be efficiently expressed in Escherichia coli. Our preliminary experiments found that the FAP nanoantibody screened by the method of the present invention has the characteristics of a separate immune reaction with human and mouse or human FAP antigens, and has good specificity and high affinity. It can be used to prepare FAP detection reagents or anti-tumor drugs, etc. For example, the anti-human FAP nanoantibody 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 figure shows the results of screening positive phages by using phage supernatant flow cytometry to detect binding to FAP antigen;

[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 Shown is an SDS-PAGE image of crude purification of anti-FAP Nanobodies.

[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 seven 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 seven FAP-VHH-mIgG-Fc fusion proteins at different volumes to 3T3-hFAP cells overexpressing human FAP.

[0043] Figure 5 The figure shows the killing efficiency of FAP-VHH-CAR-T cells against different cell lines detected by LDH.

[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 solutions of the present invention, the following examples are given. Unless otherwise stated, the raw materials, reactions and post-processing methods mentioned 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 regions CDR include complementary determining regions CDR1-CDR3;

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

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

[0052] The complementary determining regions CDR3 are represented by 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 comprises 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, immunize alpacas by injecting FAP antigen multiple times on both sides near the alpaca's cervical lymph nodes, 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 determined to be qualified if the dilution multiple exceeds 1:32000 and the color is displayed; then extract the alpaca peripheral blood lymphocytes (PBMCs) 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 the RNA extraction kit RC11 from Novozymes.

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

[0066] (3) PCR: Design VHH primers and perform PCR amplification 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 is completed, the target band of about 350 bp is recovered by agarose gel electrophoresis. Then, the first round product is used as a template for 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) Connect the product and electrotransform: Connect the VHH and phagemid vector through Goldgate to obtain the target recombinant vector; add the connected product to the TG1 competent medium, and electroporate at 2.5KV for 5ms; then add 2mL of warm bath medium and incubate at 37℃ and 220rpm for 1h. Draw out part of the bacterial solution and dilute it to 10 -5 , 10 -6 , 10 -7 , respectively, spread onto 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 were randomly selected from the plates and inoculated into a centrifuge tube containing 1 mL of 2YT liquid medium (Amp) with Amp resistance. The cells were cultured at 37°C for 3 h. PCR 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, let it stand at 37℃ for 30 minutes, incubate at 220 rpm for 1 hour, and then centrifuge; the pellet after centrifugation was resuspended with an equal volume of 2YT (A&K) and incubated at 30℃ and 220 rpm overnight.

[0080] (3) Centrifuge, transfer the supernatant to a clean tube, add 1 / 5 volume of PEG-NaCl, let stand at 4°C for 2 h, collect the precipitate by centrifugation, dissolve it with PBS, and centrifuge it 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 and dissolve the precipitate with an appropriate amount of PBS. Collect the precipitate into a 1.5 mL centrifuge tube and store at 4°C.

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

[0083] S3-2. Antigen Panning 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 three times with PBST, and 5 mL of 4% bovine serum albumin (BSA) was added for blocking. 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 discarding the phage, the cells were 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, 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 pellet in 20 mL of 2YTAG, and shake at 37°C and 220 rpm until the OD600 reaches 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, transfer to 150 mL of 2YTAK medium, and culture overnight at 28°C and 220 rpm.

[0089] (6) Collect the phage 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 in 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 into a 1.5 mL centrifuge tube and store at 4°C.

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

[0092] S3-3. Elisa Verification

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

[0094] (2) Single clones were picked and transferred to a 96-well plate containing 200 μL of 2YTAG medium. The plates were shaken 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 was taken for preservation. Helper phage M13K07 was added to the remaining bacterial solution, and the solution was allowed to stand at 37°C for 0.5 h. Centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. The precipitate was resuspended in 600 μL of 2YTAK, and the phage was induced at 28°C and 220 rpm overnight. The supernatant was retained after centrifugation at 4000 rpm for 10 min.

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

[0097] (5) Wash with PBST three times, then add 4% MPBS for blocking at 37°C for 1 hour.

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

[0099] (7) After washing 4 times with PBST, 100 μL of diluted anti-M13 antibody was added and reacted at 37°C for 1 h.

[0100] (8) After washing with PBST for 5 times, 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 three times with PBS.

[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 vectors

[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 the target FAP-VHH fragment

[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 1 μL EcoRI 1 μL <![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 ligated using the Infusion enzyme (refer to the instructions). The ligation product was added to 100 μL of competent stabl3 cells, pipetted to mix thoroughly, and allowed to stand on ice for 30 minutes. Heat in a 42°C water bath for 90 seconds, and then immediately placed on ice for 2 minutes. In a clean bench, the product was evenly spread onto a solid LB plate containing ampicillin using a spreader rod. Incubate at 37°C overnight. After 16 hours, a single colony was picked and placed in liquid LB medium containing ampicillin and incubated overnight at 37°C on a shaker at 220 rpm. The plasmid was extracted using a DNA extraction kit and sequenced to obtain the correct fusion protein expression vector.

[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 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 culture medium was evenly added dropwise to a 10 cm dish, gently shaken evenly, 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 protein before and after concentration, add 5× loading buffer, pipette and mix thoroughly, and boil at 100°C for 5 min to denature the protein.

[0128] (2) Gel preparation: 10% separation gel and 5% stacking gel were prepared using SDS-PAGE Gel Kit (Kangwei Century).

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

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

[0131] (5) Blocking: After transfer, block with 5% skim milk powder for 1 hour.

[0132] (6) Antibody incubation: dilute horseradish enzyme-labeled goat anti-mouse IgG (H+L) (ZB2305) at 1:5000, incubate at room temperature for 2 h, wash three times with TBST, each time for 10 min, and develop.

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

[0134] The gel preparation and electrophoresis were similar to those for western blot. After 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 became transparent.

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

[0136] 1×10 cells of each type were taken 6 , resuspended in PBS, centrifuged at 3500rpm for 5min, and discarded the supernatant. Each cell group was divided into 4 groups: NTC group: no antibody was added, and the cells were resuspended in 100μL PBS; primary antibody group: the cells were resuspended in 100μL PBS, 5μL FAP-VHH-mIgG-Fc fusion protein was added, incubated at 4℃ in the dark for 30min, and then 1mL PBS was added, washed twice, centrifuged at 3500rpm for 5min, the supernatant was discarded, and the cells were resuspended in 100μL PBS; secondary antibody group: diluted iFluor TM 488Conjugated Goat anti-mouse IgG (HA1125) 100 μL, incubated at 4°C in the dark for 30 min, then added 1 mL PBS, washed twice, centrifuged at 3500 rpm for 5 min, discarded the supernatant, and resuspended in 100 μL PBS; Experimental group: cells were resuspended in 100 μL PBS, added 5 μL FAP-VHH-mIgG-Fc fusion protein, incubated at 4°C in the dark for 30 min, then added 1 mL PBS, washed twice, centrifuged at 3500 rpm for 5 min, discarded the supernatant, and added diluted iFluor TM 488 Conjugated Goat anti-mouse IgG (HA1125) (100 μL) was incubated at 4°C in the dark for 30 minutes, followed by addition of 1 mL of PBS. The cells were washed twice, centrifuged at 3500 rpm for 5 minutes, the supernatant discarded, and the cells resuspended in 100 μL of PBS. Flow cytometry was used for analysis. Experimental groups with different primary antibody volumes received different volumes of primary antibody: 0.0001, 0.001, 0.01, 0.1, and 1 μL. All 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 amplify the target FAP-VHH fragment using PCR. 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 kit instructions.

[0140] (2) Linearize the CAR lentiviral vector by enzyme digestion. The conditions are shown in Table 6.

[0141] Table 6 CAR lentiviral vector linearization conditions

[0142] Element volume Vector template 1 μg 10×rCutsmart buffer 5μL XB 1 μL BamHI 1 μL <![CDATA[ddH2O]]> Up to 50μL

[0143] (3) The target fragment is connected to the CAR lentiviral vector

[0144] The recovered target fragment and vector fragment were ligated using the Infusion enzyme (refer to the instructions). The ligation product was added to 100 μL of competent Stabl3 cells, pipetted to mix thoroughly, and placed on ice for 30 minutes. Heat in a 42°C water bath for 90 seconds, and then immediately placed on ice for 2 minutes. In a clean bench, the product was evenly spread onto a solid LB plate containing ampicillin using a spreader rod and incubated overnight at 37°C. After 16 hours, a single colony was picked and placed in liquid LB medium containing ampicillin and incubated overnight at 37°C on a shaker at 220 rpm. The plasmid was extracted using 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 culture medium was evenly added dropwise to a 10 cm dish, gently shaken evenly, 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 the culture medium was resuspended in x-vivo containing 10% FBS.

[0148] Table 7 Lentiviral packaging system

[0149]

[0150]

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

[0152] Human peripheral blood mononuclear cells (PBMC) were revived, 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. The mixture was pipetted and mixed, placed on a magnetic stand, and allowed to stand for 1 minute. The waste liquid was discarded. The cells were resuspended in 1 mL of X-vivo medium containing 10% FBS and 100 IU / mL IL-2, mixed with the 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 for resuspending and cultured in a 24-well plate. After 24 hours, the cells were collected, counted, and 1 × 10 5 Cells were plated at 1 mL / well in a 24-well plate. FAP-VHH-CAR lentivirus was added at an MOI of 1 to a final volume of 1 mL. The infection promoter F108 was added to a final concentration of 1 mg / mL. The cells were mixed by pipetting and centrifuged at 1500 g for 90 minutes at room temperature, with a speed of 3°C and a speed of 1°C. 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. The cells were then reinfected 24 hours later. Once T cells had expanded to a sufficient number, the positive rate of CAR-T cells was detected using a self-produced FAP extracellular domain protein and a fluorescent secondary antibody.

[0153] S6-4. Verification of FAP-VHH-CAR-T cell function in vitro

[0154] S6-4-1. LDH assay to detect CAR-T cell killing effect

[0155] (1) Cell plating

[0156] After digestion, tumor cells were counted, and target cells were counted at 100 μL per well, 2 × 10 4 Cells were plated in a round-bottom 96-well plate. After flow cytometry testing for CAR-T cell positivity, 100 μL of cells were added to each well at effector-target ratios of 0.5:1, 1:1, 2:1, 4:1, and 8:1. The cells were mixed by pipetting and placed in a 37°C, 5% CO2 incubator. A target cell spontaneous release well (target cells only), a target cell maximum release well (lysate added during testing), and an effector cell spontaneous release well were set as controls. The plates were then centrifuged at 250 g for 5 minutes and incubated at 37°C, 5% CO2.

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

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

[0159] S6-4-2. ELISA detection of IFN-γ released during CAR-T cell killing

[0160] (1) Sample preparation: Dilute the supernatant in S6-4-1 50-fold 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, turn it upside down on absorbent paper, and discard the 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 aspirate 100 μL to well 2, pipette to mix, and aspirate 100 μL to well 3, and so on; aspirate 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 place it 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 liquid and wash with wash buffer 3-5 times, soaking for 1 minute each time.

[0172] (13) Add diluted HRP.

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

[0174] (15) Discard the liquid 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 and incubate it with 3T3-FAP cells and CHO-FAP cells for flow cytometry detection. The results are as follows: Figure 1 shown.

[0181] Figure 1 The figure shows the results of screening positive phages by using phage supernatant flow cytometry to detect binding to FAP antigen;

[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 1As can be seen, the results of screening positive phages showed 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 Shown is an SDS-PAGE image of crude purification of anti-FAP Nanobodies.

[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 nanoantibodies 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 seven 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 seven 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-incubated 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 figure shows the killing efficiency of FAP-VHH-CAR-T cells against different cell lines detected by LDH.

[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 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 all released high levels of IFN-γ to 3T3-hFAP and 3T3-mFAP cells, and the levels decreased as the effector-target ratio decreased. However, the levels of IFN-γ release to 3T3 cells that did not express FAP were 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 invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0211] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method 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 can also be appropriately combined to form other implementation methods 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 regions CDR include complementary determining regions CDR1-CDR3; The complementary determining regions CDR1 are shown in amino acid sequence 1; The complementary determining regions CDR2 are shown in amino acid sequence 2; The complementary determining regions CDR3 are shown in amino acid sequence 3; The amino acid sequences 1-3 are shown as SEQ ID No.1-SEQ ID No.

3.

2. The anti-FAP nanobody according to claim 1, characterized in that The framework region FR includes framework regions FR1-FR4; in, The framework regions FR1-FR4 are shown in amino acid sequences 22-25, respectively; The amino acid sequences 22-25 are shown as SEQ ID No.22-SEQ ID No.

25.

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

50.

4. The anti-FAP nanobody according to claim 1, characterized in that The anti-FAP nanobody is selected from: a chimeric antibody and a humanized antibody.

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 sequence of the nucleic acid molecule is SEQ ID No.

57.

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 the preparation of a FAP antigen detection reagent or a FAP antigen adsorption reagent.

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