Bispecific antibody, genetically modified TIL cell and construction method and application of genetically modified TIL cell

Abstract

The invention relates to the technical field of biological medicine, and discloses a bispecific antibody, a genetically modified TIL cell and a construction method and application thereof. The bispecific antibody comprises an FAP specific nano antibody VHH sequence of which the amino acid sequence is shown as SEQ ID NO: 1, a linker sequence of which the amino acid sequence is shown as SEQ ID NO: 2, and a CD3 specific antibody scFV sequence of which the amino acid sequence is shown as SEQ ID NO: 3. The FAP * CD3 bispecific antibody provided by the invention can effectively remove FAP positive tumor related fibroblasts and improve the tumor microenvironment, and can improve the effectiveness of solid tumor treatment by combining with the characteristic that the tumor antigen specificity of TIL cells is relatively high.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a bispecific antibody, a genetically modified TIL cell, and a construction method and application thereof. Background Art

[0002] TIL cells originate from within tumors and naturally possess the ability to recognize and target multiple tumor antigens. This characteristic enables TIL cell therapy to more comprehensively attack tumor cells, effectively overcoming the treatment challenges presented by tumor heterogeneity. However, while TIL cells have made some progress in some clinical studies, their efficacy against some types of solid tumors is unsatisfactory. Further improvements are needed to further enhance the effectiveness of TIL cells. Summary of the Invention

[0003] The purpose of the present invention is to overcome the problem that TIL cells in the prior art have unsatisfactory efficacy against some types of solid tumors, and to provide a bispecific antibody, a genetically modified TIL cell, and a construction method and application thereof.

[0004] In order to achieve the above objectives, the present invention provides a bispecific antibody on the one hand, which comprises a FAP-specific nanoantibody VHH sequence as shown in SEQ ID NO: 1, a linker sequence as shown in SEQ ID NO: 2, and a CD3-specific antibody scFV sequence as shown in SEQ ID NO: 3.

[0005] Preferably, the amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 4.

[0006] A second aspect of the present invention provides a genetically modified TIL cell capable of secreting a bispecific antibody, wherein the bispecific antibody comprises a FAP-specific nanoantibody VHH sequence as shown in SEQ ID NO: 1, a linker sequence as shown in SEQ ID NO: 2, and a CD3-specific antibody scFV sequence as shown in SEQ ID NO: 3.

[0007] Preferably, the TIL cells knock out the regenase-1 gene through a sgRNA sequence; wherein the sgRNA sequence is selected from at least one of the sequences shown in SEQ ID NO: 5 to 9, preferably the sequence shown in SEQ ID NO: 6.

[0008] The third aspect of the present invention provides a method for constructing TIL cells as described above, the method comprising the following steps:

[0009] (1) constructing an AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector, the nucleotide sequence of which is shown in SEQ ID NO: 17, and transfecting the vector, pAAV-RC2, and pAAV-helper plasmids into 293T cells to obtain an AAV virus containing a Regnase-1 homology arm and capable of encoding a bispecific antibody;

[0010] (2) Dissociate and digest the tumor tissue, then separate the cells, add 200-1000 UI / ml IL-2 for initial in vitro expansion, and collect TIL cells;

[0011] (3) Adding the AAV virus encoding the bispecific antibody obtained in step (1) to the TIL cells for infection, centrifuging and removing the supernatant 4 to 12 hours after infection, adding the Cas9 RNP complex, and performing gene editing on the TIL cells by electroporation.

[0012] Preferably, step (1) specifically includes:

[0013] A1. Construction of AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector;

[0014] A2. The vector, pAAV-RC2, and pAAV-helper plasmids are transfected into 293T cells. The resulting cells are subjected to virus collection and impurity removal. Finally, the virus is purified and concentrated to obtain an AAV virus containing the Reganse-1 homology arm that can encode a bispecific antibody.

[0015] Preferably, the method further comprises: adding Human T-Activator CD3 / CD28 / CD137 magnetic beads to the gene-edited TIL cells obtained in step (3), and continuing to expand in vitro for 10 to 15 days.

[0016] Preferably, the Cas9 RNP complex comprises the sgRNA sequence shown in SEQ ID NO:6.

[0017] A fourth aspect of the present invention provides the use of the bispecific antibody as described above, the genetically modified TIL cells as described above, or the genetically modified TIL cells constructed by the construction method as described above in the preparation of a drug for treating tumors.

[0018] Preferably, the tumor is a solid tumor.

[0019] Preferably, the tumor includes pancreatic cancer, breast cancer, esophageal cancer, sarcoma, mesothelioma, liver cancer, lung cancer, gastric cancer and colorectal cancer.

[0020] The FAP*CD3 bispecific antibody provided by the present invention can effectively eliminate FAP-positive tumor-associated fibroblasts, improve the tumor microenvironment, and combine with the strong tumor antigen specificity of TIL cells themselves to more effectively improve the effectiveness of solid tumor treatment.

[0021] The genetically modified TIL cells provided by the present invention can secrete FAP*CD3 bispecific antibodies, thereby effectively eliminating FAP-positive tumor-associated fibroblasts and improving the tumor microenvironment. Combined with the strong tumor antigen specificity of TIL cells themselves, the effectiveness of solid tumor treatment can be improved.

[0022] Furthermore, the gene-modified TIL cells, in addition to secreting the FAP*CD3 bispecific antibody, also have the regenase-1 gene knocked out. The depletion of REGNASE-1 protein can prolong the survival time of anti-tumor CD8 T cells and enhance the function of T. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector constructed in Example 1 of the present invention;

[0024] Figure 2 is the test result of screening sgRNA in Example 1 of the present invention;

[0025] Figure 3 The results of the tumor efficacy test on mice injected with different types of TIL cells in Test Example 1 of the present invention;

[0026] Figure 4 These are the experimental results of the binding of FAP nanobodies to human and mouse FAP proteins in Test Example 2 of the present invention;

[0027] Figure 5 This is the result of the FAP*CD3 bispecific antibody binding experiment with Jurkat cells in Test Example 2 of the present invention;

[0028] Figure 6 This is a graph showing the results of the FAP*CD3 bispecific antibody inhibiting the growth of SNU387 tumor cells in Test Example 3 of the present invention. DETAILED DESCRIPTION

[0029] The following describes the specific embodiments of the present invention in detail with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0030] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0031] TIL cells originate from within the tumor and naturally have the ability to recognize and target a variety of tumor antigens. This characteristic enables TIL cell therapy to attack tumor cells more comprehensively, effectively overcoming the treatment difficulties brought about by tumor heterogeneity. Although TIL cell therapy has made significant progress in the treatment of some solid tumors, how to further improve the tumor-killing ability of TIL cells and the effectiveness of cell therapy is an important direction for technological development. The tumor microenvironment of solid tumors is an important factor affecting the efficacy of TIL cells. Among them, cancer-associated fibroblasts, a cell type that plays a vital role in the tumor microenvironment, are often regarded as the driving force behind the shaping of tumor growth and metastasis. They not only affect the proliferation and differentiation of tumor cells by secreting a variety of growth factors and cytokines, but also actively participate in building a microenvironment that is conducive to tumor growth.

[0032] Research has discovered that FAP, or fibroblast activation protein-α, is a 97 kDa type II transmembrane serine protease involved in multiple biological functions. It can influence tumor growth through various mechanisms, including promoting proliferation, invasion, angiogenesis, epithelial-to-mesenchymal transition, stem cell promotion, immunosuppression, and drug resistance. Under physiological conditions, FAP expression is low in most adult tissues, but it is often highly expressed on cancer-associated fibroblasts (CAFs). In addition, some tumor cells also express FAP on their surface. Therefore, utilizing the protease activity of FAP to selectively activate prodrugs at the tumor site to enhance drug efficacy and reduce toxicity is a promising approach.

[0033] In view of this, the present invention provides a bispecific antibody, which comprises a FAP-specific nanobody VHH sequence as shown in SEQ ID NO: 1, a linker sequence as shown in SEQ ID NO: 2, and a CD3-specific antibody scFV sequence as shown in SEQ ID NO: 3.

[0034] In the present invention, the antibodies in the bispecific antibody are CD3 antibodies and FAP antibodies, and therefore, it is also called FAP*CD3 bispecific antibody. This FAP*CD3 bispecific antibody can effectively eliminate FAP-positive tumor-associated fibroblasts, improve the tumor microenvironment, and combine with the strong tumor antigen specificity of TIL cells themselves to more effectively improve the effectiveness of solid tumor treatment.

[0035] In the present invention, the sequence of the FAP-specific Nanobody VHH (SEQ ID NO: 1) is shown below:

[0036] DVQLQESGGGLVHPGGSLRLSCAASEDTLEYYAIGWFRQAPGKEREGVS CIAIGGVASNYTDSVKGRFTISRDNTKHTVSLQMNSLKPEDTAIYYCAASAEIT VGATGQFTCSLSDYDFWGQGTQVTVSS.

[0037] In the present invention, the linker sequence (SEQ ID NO: 2) is as follows:

[0038] GGGGSGGGGSGGGGS.

[0039] In the present invention, the CD3-specific antibody scFV sequence (SEQ ID NO: 3) is shown below:

[0040] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGG SGGSGGSGGSDGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK.

[0041] In the present invention, the amino acid sequence of the FAP*CD3 bispecific antibody (SEQ ID NO: 4) is shown below:

[0042] DVQLQESGGGLVHPGGSLRLSCAASEDTLEYYAIGWFRQAPGKEREGVSCIAIGGVASNYTDSVKGRFTISRDNTKHTVSLQMNSLKPEDTAIYYCAASAEITVGATGQFTCSLSDYDFWGQGTQVTVSSGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWI GYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGSGGSGGSGGSDGVDDIQLTQSPAI MSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK.

[0043] In a preferred embodiment, the TIL cells are targeted to knockout the regenase-1 gene via an sgRNA sequence; wherein the sgRNA sequence is selected from at least one of the sequences shown in SEQ ID NOs: 5 to 9. Specifically, the sgRNA sequence is as follows:

[0044] sgRNA-1 (SEQ ID NO: 5): GGAGTGGAAGCGCTTCATCG;

[0045] sgRNA-2 (SEQ ID NO:6): CAGGCTGGGTTCCATACCAT;

[0046] sgRNA-3 (SEQ ID NO:7): CAGCTCACCCAGCACCGTGT;

[0047] sgRNA-4 (SEQ ID NO:8): TTCACACCATCACGACGCGT;

[0048] sgRNA-5 (SEQ ID NO:9): TGAGACCAGTGGTCATCGAT.

[0049] Further preferably, the sgRNA sequence is the sequence shown in SEQ ID NO: 6 (sgRNA-2), which has the highest cutting efficiency. In this way, the survival ability of TIL cells in the body can be better prolonged, and tumor cells can be better inhibited and eliminated. At the same time, the RNP formed by sgRNA and Cas9 protein forms a DBS incision at the Regnase-1 genomic position, and the single-stranded DNA provided by AAV can be used as a template to accurately integrate the DNA sequence of the secreted bispecific antibody into this position of the TIL cell.

[0050] The genetically modified TIL cells described in the present invention can not only effectively resist the tumor microenvironment formed by tumor-associated fibroblasts, but also prolong the survival ability of TIL cells in the body and better inhibit and eliminate tumor cells.

[0051] The present invention also provides a genetically modified TIL cell capable of secreting a bispecific antibody, wherein the bispecific antibody comprises a FAP-specific nanoantibody VHH sequence as shown in SEQ ID NO: 1, a linker sequence as shown in SEQ ID NO: 2, and a CD3-specific antibody scFV sequence as shown in SEQ ID NO: 3.

[0052] Preferably, the amino acid sequence of the bispecific antibody secreted by the TIL cells is shown in SEQ ID NO: 4.

[0053] In the present invention, the genetically modified TIL cells are capable of producing FAP*CD3 bispecific antibodies, thereby effectively eliminating FAP-positive tumor-associated fibroblasts and improving the tumor microenvironment. Combined with the strong tumor antigen specificity of the TIL cells themselves, the effectiveness of solid tumor treatment is more effectively improved.

[0054] The present invention also provides a method for constructing the gene-modified TIL cells as described above, the method comprising the following steps:

[0055] (1) constructing an AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector, the nucleotide sequence of which is shown in SEQ ID NO: 17, and transfecting the vector, pAAV-RC2, and pAAV-helper plasmids into 293T cells to obtain an AAV virus containing a Reganse-1 homology arm capable of encoding a bispecific antibody;

[0056] (2) The tumor tissue is dissociated and digested, and then the cells are separated. 200-1000 UI / ml IL-2 is added for the initial in vitro expansion phase, and TIL cells are collected;

[0057] (3) Adding the AAV virus encoding the bispecific antibody obtained in step (1) to the TIL cells for infection, centrifuging and removing the supernatant 4 to 12 hours after infection, adding the Cas9 RNP complex, and performing gene editing on the TIL cells by electroporation.

[0058] In the method described in the present invention, the present invention is not limited to the specific method for constructing the AAV-EF1a-FAP VHH-CD3 ScFvbispecific Antibody vector, and it can be a conventional vector construction method in the art, as long as the nucleotide sequence of the constructed vector is as shown in SEQ ID NO: 17.

[0059] In some embodiments, step (1) specifically includes:

[0060] A1. Construction of AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector;

[0061] A2. The vector, pAAV-RC2, and pAAV-helper plasmids are transfected into 293T cells. The resulting cells are subjected to virus collection and impurity removal, and finally the virus is purified and concentrated to obtain an AAV virus encoding a bispecific antibody.

[0062] In the method described in the present invention, the tumor tissue in step (2) is obtained from the patient's own tumor tissue, and is not limited to any type, as long as the tumor tissue can be obtained.

[0063] In a preferred embodiment, in step (2), the duration of the initial in vitro expansion phase is 5 to 7 days.

[0064] The present invention does not limit the specific source of the Human T-Activator CD3 / CD28 / CD137 magnetic beads, which can be a common commercial product. In a specific embodiment, it is GIBCO Dynabeads TM Human T-ActivatorCD3 / CD28 / CD137 Magnetic Beads.

[0065] In the method described in the present invention, in step (3), AAV single-stranded DNA is integrated into the TIL cell genome through homologous recombination as a repair template, enabling it to secrete the bispecific antibody.

[0066] In a preferred embodiment, in step (3), the Cas9 RNP complex comprises the sgRNA sequence shown in SEQ ID NO: 6, so that the genetically modified TIL cells ultimately constructed can better inhibit and eliminate tumor cells.

[0067] The present invention also provides gene-modified TIL cells constructed by the construction method described above.

[0068] The present invention also provides the use of the gene-modified TIL cells described above in the preparation of drugs for treating tumors.

[0069] Furthermore, the tumor is various types of solid tumors.

[0070] Furthermore, the tumors shown include pancreatic cancer, breast cancer, esophageal cancer, sarcoma, mesothelioma, liver cancer, lung cancer, gastric cancer and colorectal cancer.

[0071] The present invention will be described in detail below by way of examples, but the scope of the present invention is not limited thereto. The experimental methods in the following examples, unless otherwise specified, are conventional methods in the art. The experimental materials used in the following examples, unless otherwise specified, are commercially available products.

[0072] Example 1

[0073] This example is used to illustrate the screening of FAP nanobodies and the construction of AAV viruses encoding bispecific antibodies.

[0074] 1. FAP nanobody screening (FAP nanobody phage display library construction):

[0075] (1) FAP immunization of camels

[0076] 1 mg of FAP protein was mixed with an equal volume of Freund's adjuvant to make 5 ml and injected subcutaneously at 3-5 points on the camel's neck. Blood was collected from the marginal ear vein before immunization. Immunizations were administered monthly for a total of four times; 10 ml of peripheral blood was collected at each immunization. Lymphocytes were separated using Ficoll from blood collected before and after each immunization.

[0077] (2) Total RNA extraction and cDNA synthesis

[0078] Take frozen lymphocytes, add 1ml Trizol, let it stand at room temperature for 10 minutes, then add 0.2ml chloroform, shake vigorously, let it stand at room temperature, wait for the solution to separate (about 10 minutes), centrifuge at 12,000rpm, collect the upper aqueous phase, add an equal volume of isopropanol, mix well, let it stand at room temperature for 15 minutes, wait for the nucleic acid to precipitate, centrifuge at high speed to remove the supernatant, add 1ml of 75% ethanol (prepared with DEPC water) to wash the RNA precipitate, centrifuge at high speed to remove the supernatant, control the water, dissolve the RNA in nuclease-free water, and take 1μl for concentration and purity determination. Take 1μg of RNA and use the SuperScriptT MIII First-Strand Synthesis SuperMix (Invitrogen) kit for cDNA synthesis;

[0079] (3) Phage display library construction

[0080] The V region (VHH) of camel heavy chain antibody was amplified using the synthesized cDNA as a template by Nest-PCR. Table 2 shows the names and sequences of the Nest-PCR primers.

[0081] Table 1: Primers used for camel VHH fragment amplification

[0082]

[0083] PCR reaction conditions are as follows:

[0084] First round

[0085]

[0086] Reaction conditions: 95°C, 5 min; 94°C, 1 min; 57°C, 1 min; 72°C, 1 min per cycle; 72°C, 7 min; amplification for 35 cycles.

[0087] Second round

[0088]

[0089] Reaction conditions: 95°C, 5 min; 94°C, 45'; 60°C, 45'; 72°C, 45' per cycle; 72°C, 7 min; amplification 25 cycles.

[0090] After the PCR reaction, the PCR products were detected by 1.5% agarose gel electrophoresis. The target gene fragment of the first round of PCR was located at 700 bp, and the target band was recovered by gel excision. In the second round of PCR, the target gene fragment was located at 500 bp, and the target band, i.e., the VHH fragment, was recovered by gel excision. The VHH fragment and the vector were double-digested with NEB restriction endonucleases NotI and PstI, respectively. The reaction system was as follows:

[0091] Vector enzyme digestion system

[0092]

[0093]

[0094] Add H2O to 500 μl;

[0095] Fragment digestion system:

[0096]

[0097] Add H2O to 500 μl; digest overnight at 37°C, and after agarose gel electrophoresis, cut and recover the fragment; mix the digested products of the vector and VHH fragment, and ligate with NEB's ligase at 16°C overnight;

[0098] (4) Construction of phage display library

[0099] After the ligation product was purified by PCR Purification Kit (QIAGEN), 1 μl was taken to transform TG competent cells, recovered at 37°C for 2 h, and gradiently diluted to 10 1 , 10 2 , 10 3 300 μL was taken to spread on the plate, cultured at 37°C overnight, and the number of clones was calculated, about 10 5 clones / plate.

[0100] Use the same transformation method as above to perform large-scale transformation until the number of clones in the library reaches more than 107. Elute all clones with LB, centrifuge at 5,000g for 5 minutes, resuspend the precipitate in 2 ml LB, add an equal volume of 30% glycerol, and freeze at -80.

[0101] (5) Panning of affinity FAP nanoantibody phage library

[0102] ELISA plates were coated with either human or mouse FAP proteins and incubated overnight at 4°C. The next day, phage displaying FAP nanobodies were added and incubated at room temperature for 2 hours. The wells were washed 10 times with PBST and incubated for 30 minutes at room temperature with 100 μl of triethylamine. The collected phage represented the FAP nanobody phage library obtained by affinity panning. 10 μl of the plate was infected with TG cells and plated to determine the number of clones after screening. The remaining screened phage was used for amplification.

[0103] 2. Construction of AAV virus encoding bispecific antibodies.

[0104] A1. rAAV vector construction process

[0105] The FAP homologous left arm-EF1a-FAP VHH-CD3 scFv-FAP homologous right arm DNA sequence was synthesized, and the sequence was shown in SEQ ID NO: 16. It was cloned into the AAV expression vector ssAAV.CMV.tdTomato.WPRE.SV40pA through the MluI and NOT1 restriction sites to obtain the AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector ( Figure 1 ), the sequence of which is shown in SEQ ID NO:17.

[0106] FAP homology left arm-EF1a-FAP VHH-CD3 scFV-FAP homology right arm DNA sequence (SEQ ID NO: 16):

[0107]

[0108] AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector full sequence (SEQ ID NO: 17):

[0109]

[0110] A2. rAAV virus packaging process (293T adherence)

[0111] A21. Cell Preparation

[0112] Remove the frozen cells from the liquid nitrogen tank and quickly thaw them in a 37°C water bath. After centrifugation, add fresh culture medium to the cells and culture them at 37°C and 5% CO2. Passage the cells every 2-3 days. After the cells grow normally, transfer them to a 10 cm culture dish for adherent culture.

[0113] A22, plasmid transfection

[0114] When the cell density in step A21 reaches a confluence of approximately 80-90%, transfection can be performed. The AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody plasmid obtained above, as well as pAAV-RC2 and pAAV-helper auxiliary plasmids, are prepared in a 1:1:2 mass ratio and transfected into 293T cells using PEI transfection reagent.

[0115] A23. Virus collection and removal

[0116] After culturing the cells obtained in step A22 for 72 hours, the 293T cell pellet and culture supernatant were collected simultaneously; the supernatant was centrifuged at low temperature to remove cell debris, and then PEG8000 / NaCl solution was added for overnight coagulation; the 293T cell pellet was disrupted by ultrasound to release AAV particles; the cell pellet and culture supernatant PEG precipitate were mixed, and an appropriate amount of nuclease Benonase was added to digest at 37°C for 1 hour to remove free nucleic acids.

[0117] A24. Virus purification and concentration

[0118] The virus precipitate obtained in step A23 was resuspended with PBS and placed in an ultracentrifuge for iodixanol density gradient centrifugation; after centrifugation, the solution of the virus corresponding layer was extracted and further dialyzed overnight at 4°C using a dialysis bag; the dialysate was filtered at 0.22 μm the next day and concentrated and washed through a concentrator tube to achieve the purpose of impurity removal and concentration, and finally an AAV virus containing the Regnase-1 homology arm and capable of encoding a bispecific antibody was obtained.

[0119] Example 2

[0120] This example is used to illustrate the genetically modified TIL cells with regenase-1 gene knockout and secreting FAP*CD3 bispecific antibodies and the preparation method thereof according to the present invention.

[0121] (1) The tumor tissue removed by surgery should be preserved in Miltenyi MACS tissue preservation medium and transported to the laboratory for TIL isolation within 24 hours to maintain cell activity.

[0122] (2) Tissue dissociation and digestion: Mechanically cut the tumor tissue into 1-3 mm pieces 3 For fragmentation, adjust the volume to 5-10 ml with RPMI-1640, add 0.05% collagenase type I and IV, 0.001% hyaluronidase and DNase, and stir at room temperature for 4-6 hours; obtain a single-cell suspension by filtering through a 100 μm cell strainer.

[0123] (3) Cell separation and expansion culture: Wash the single cell suspension twice with physiological saline, centrifuge at 1800 rpm for 8 minutes at room temperature, discard the supernatant, take the precipitate and resuspend it with X-VIVO, then inoculate it into a 24-well plate at a density of 1E6 / ml, add 200-1000 IU / ml of IL-2 and culture for 5-7 days. This stage is the initial expansion stage of TIL cells.

[0124] (4) AAV infection and cell electroporation: The TIL cells obtained in step (3) were collected by centrifugation, and the AAV virus containing the Regnase-1 homology arm and capable of encoding a bispecific antibody obtained in Example 1 was added at an MOI of 5E5 vg / cell. After 6 hours of infection, the supernatant was removed by centrifugation, and the Cas9 RNP complex was added. The TIL cells were gene-edited using the Lonza4D electroporation system;

[0125] The electroporated RNP complexes are shown in Table 2 below.

[0126] Table 2

[0127]

[0128] In Table 2, *sgRNA refers to the sequence shown in sgRNA-2 (SEQ ID NO: 6).

[0129] Among them, the sgRNA screening process (preliminary experiment) is as follows:

[0130] After ordering sgRNA-1 to sgRNA-6 from GenScript, they were incubated with Cas9 protein according to Table 1 above to prepare Cas9 RNP complexes. 293T cells in logarithmic growth phase were digested and dispersed into single cells. 1E6 293T cells were mixed with the Cas9 RNP complexes and electroporated using a Lonza 4D electroporation system. Following electroporation, cells were cultured for 24 hours, and DNA was extracted using a kit. Primers were designed 500 bases around the sgRNA cleavage site to amplify the target DNA sequence, and high-throughput sequencing of the amplicons was performed. Bioinformatics analysis allowed calculation of the cleavage efficiency of different sgRNAs.

[0131] The results are as follows Figure 2 As shown, the results show that sgRNA-2 has the highest cutting efficiency. Therefore, sgRNA-2 was used in the preparation of TIL cells in the animal experiments of this embodiment and the test examples below.

[0132] (5) TIL rapid expansion stage: Add GIBCODynabeads to the gene-edited TIL cells in step 4 TM Human T-Activator CD3 / CD28 / CD137 magnetic beads were used to continue in vitro expansion for 14 days to obtain genetically modified TIL cells that could secrete FAP*CD3 bispecific antibodies and had the regnase-1 gene knocked out.

[0133] Example 3

[0134] This example is used to illustrate the preparation and purification of the FAP*CD3 bispecific antibody of the present invention.

[0135] The DNA sequence encoding the FAP*CD3 bispecific antibody is cloned into the eukaryotic expression vector pcDNA3.1; the gene is sequenced to confirm its correctness, and the plasmid is extracted from Escherichia coli; the plasmid is then encapsulated with liposomes (such as Lipofectamine 2000) and transfected into 293T cells; the supernatant is collected 48-72 hours after transfection; cell debris is removed by centrifugation (3000×g, 20 minutes); and the bispecific antibody of interest is purified by ion exchange chromatography and molecular sieve separation.

[0136] Test Example 1

[0137] Animal model preparation

[0138] Four-week-old SPF male NOD / SCID mice weighing 18-20 g were taken and acclimated for 1 week. Tumor cells from patients in the logarithmic growth phase were collected by trypsin digestion, resuspended in sterile PBS, and counted. The cell density was adjusted to 2 × 10 7The model was established by subcutaneously injecting 100 μL of cell suspension into the back of the left upper limb of the mouse. After 14 days of modeling, the tumor volume of the mouse reached 80-100 mm 3 The above results indicate that the modeling is successful.

[0139] Thirty tumor-bearing mice were randomly divided into five groups, with six mice in each group, namely: G1: MOCK T administration group; G2: TIL cell administration group; G3: TIL cell administration group secreting FAP*CD3 bispecific antibody; G4: TIL cell administration group with regnase-1 gene knockout; G5: TIL cell administration group secreting FAP*CD3 bispecific antibody and with regnase-1 gene knockout (i.e., TIL cells obtained in Example 2). The total number of cells administered in each group was 1×10 7 The drug was administered through tail vein.

[0140] Test results Figure 3 shown.

[0141] Depend on Figure 3 It can be seen that whether the Regnase-1 gene of TIL cells is knocked out alone or the TIL cells are allowed to secrete FAP*CD3 bispecific antibodies, the inhibitory effect of TIL cells on tumor cells can be enhanced; at the same time, TIL cells that secrete FAP*CD3 bispecific antibodies and have the regnase-1 gene knocked out have the most significant inhibitory effect on tumors, indicating that knocking out the Regnase-1 gene and secreting the FAP*CD3 bispecific antibody have a synergistic effect.

[0142] Test Example 2

[0143] 1. Binding experiment of FAP nanobodies obtained by screening in Example 1 with human and mouse FAP proteins

[0144] 96-well plates were coated with human or mouse FAP protein overnight. The coated plates were washed once with PBST and then blocked with 1% BSA in a 37°C incubator for 1 hour. The plates were washed and dried. Recombinant FAP nanobody was serially diluted in 1% BSA to a gradient solution ranging from 5 μg / mL to 0.01 μg / mL. The plates were incubated at 37°C for 2 hours, washed five times, and dried. A 1:15,000 dilution of Goat anti-mouse IgG-HRP secondary antibody (100 μL / well) was added to each well. The plates were incubated at 37°C for 30 minutes, washed five times, and dried. 100 μL of TMB substrate development solution was added to each well. The plates were reacted at 37°C in the dark for 20 minutes. The reaction was terminated with 100 μL / well of stop solution. The OD values were immediately read at a wavelength of 450 nm using an Infinite F50 microplate reader.

[0145] The test results are as follows Figure 4 shown.

[0146] Depend on Figure 4 It can be seen that the FAP nanobody sequence described in the present invention can specifically bind to human or mouse FAP protein.

[0147] 3. Binding experiment of the FAP*CD3 bispecific antibody prepared in Example 3 to Jurkat cells.

[0148] (1) Take no less than 1×10 6 Transfer 100 pc / tube of Jurkat cells to a 1.5 ml microcentrifuge tube.

[0149] (2) Wash the cells once with WB (PBS + 2% FBS), centrifuge at 300×g for 5 min, and remove the supernatant.

[0150] (3) Resuspend Jurkat cells in 100 μl of Western blotting buffer, add human FAP-biotin protein (2 μl / test), and stain at room temperature for 25 min.

[0151] (4) Add 1000 μl of WB to each well, mix thoroughly by pipetting, centrifuge at 300 × g for 5 min, and aspirate the supernatant.

[0152] (5) Wash the cells once with WB (PBS + 2% FBS), centrifuge at 300×g for 5 min, and remove the supernatant.

[0153] (6) Resuspend in 100 μl of Western Blot, add APC Streptavidin (1 μl / test), and stain at 4°C for 15 min.

[0154] (7) Add 1000 μl of WB to each well, mix thoroughly by pipetting, centrifuge at 300 × g for 5 min, and aspirate the supernatant.

[0155] (8) Wash the cells once with Western blotting (PBS + 2% FBS), centrifuge at 300 × g for 5 min, and remove the supernatant.

[0156] (9) Resuspend the Jurkat cells in 150 μl of WB, pipette to mix thoroughly, and then perform flow cytometry.

[0157] The test results are as follows Figure 5 shown.

[0158] Depend on Figure 5 It can be seen that the bispecific antibody of the present invention can bind to the human CD3 target protein well.

[0159] Test Example 3

[0160] Experiment on the inhibition of SNU387 tumor cell growth by the FAP*CD3 bispecific antibody of the present invention.

[0161] SNU387 tumor cells and T cells were seeded in a 96-well plate at a 1:1 effector-target ratio. The FAP*CD3 bispecific antibody obtained in Example 3 was added at varying concentrations and cultured in a 37°C, 5% CO2 incubator. Tumor killing activity was assessed using the LDH assay at 24, 36, and 48 hours after incubation. The assay method included: centrifugation to remove the supernatant, adding 50 μL / well of the assay mixture, mixing thoroughly, and incubating at room temperature in the dark for 30 minutes; adding 50 μL / well of the stop solution, thoroughly vortexing, and measuring the absorbance at 492 nm using a spectrophotometer. The cell killing rate was calculated as (OD value of the experimental calibration well - OD value of the effector cell spontaneous calibration well - OD value of the target cell spontaneous calibration well) / (OD value of the target cell maximum release calibration well - OD value of the target cell spontaneous calibration well) × 100%.

[0162] The test results are as follows Figure 6 shown.

[0163] Depend on Figure 6 It can be seen that the FAP*CD3 bispecific antibody provided by the present invention can effectively mediate the killing effect of T cells on FAP-positive tumor cells in a dose-dependent manner.

[0164] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A bispecific antibody, characterized in that: The bispecific antibody comprises a FAP-specific nanobody VHH sequence as shown in SEQ ID NO: 1, a linker sequence as shown in SEQ ID NO: 2, and a CD3-specific antibody scFV sequence as shown in SEQ ID NO:

3.

2. The bispecific antibody according to claim 1, characterized in that The amino acid sequence of the bispecific antibody is shown in SEQ ID NO:

4.

3. A genetically modified TIL cell, characterized in that: The TIL cells are capable of secreting bispecific antibodies, wherein the bispecific antibodies include a FAP-specific nanoantibody VHH sequence as shown in SEQ ID NO: 1, a linker sequence as shown in SEQ ID NO: 2, and a CD3-specific antibody scFV sequence as shown in SEQ ID NO:

3.

4. The genetically modified TIL cell according to claim 3, characterized in that The TIL cells have a site-directed knockout of the regenase-1 gene through an sgRNA sequence; wherein the sgRNA sequence is selected from at least one of the sequences shown in SEQ ID NOs: 5 to 9, preferably the sequence shown in SEQ ID NO:

6.

5. A method for constructing the genetically modified TIL cells according to claim 3 or 4, characterized in that: The method comprises the following steps: (1) constructing an AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector, the nucleotide sequence of which is shown in SEQ ID NO: 17, and transfecting the vector, pAAV-RC2, and pAAV-helper plasmids into 293T cells to obtain an AAV virus containing a Reganse-1 homology arm capable of encoding a bispecific antibody; (2) The tumor tissue is dissociated and digested, and then the cells are separated. 200-1000 UI / ml IL-2 is added for the initial in vitro expansion phase, and TIL cells are collected; (3) Adding the AAV virus encoding the bispecific antibody obtained in step (1) to the TIL cells for infection, centrifuging and removing the supernatant after 4 to 12 hours of infection, adding the Cas9 RNP complex, and performing gene editing on the TIL cells by electroporation. The single-stranded DNA carried by the AAV serves as a template for homologous recombination repair at the incision formed by the Cas9 RNP, and the sequence of the secreted bispecific antibody is integrated into the TIL cell genome.

6. The construction method according to claim 5, characterized in that: Step (1) specifically includes: A1. Construction of AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector; A2. The vector, pAAV-RC2, and pAAV-helper plasmids are transfected into 293T cells. The resulting cells are subjected to virus collection and impurity removal. Finally, the virus is purified and concentrated to obtain an AAV virus containing the Reganse-1 homology arm that can encode a bispecific antibody.

7. The construction method according to claim 5 or 6, characterized in that: The method further comprises: adding Human T-Activator CD3 / CD28 / CD137 magnetic beads to the gene-edited TIL cells obtained in step (3), and continuing to amplify in vitro for 10 to 15 days; Preferably, in step (3), the Cas9 RNP complex comprises the sgRNA sequence shown in SEQ ID NO:

6.

8. Use of the bispecific antibody according to claim 1 or 2, the genetically modified TIL cell according to claim 3, or the TIL cell according to any one of claims 5 to 7 in the preparation of a medicament for treating tumors.

9. The use according to claim 8, characterized in that The tumor is a solid tumor.

10. The use according to claim 8 or 9, characterized in that: The tumors include pancreatic cancer, breast cancer, esophageal cancer, sarcoma, mesothelioma, liver cancer, lung cancer, gastric cancer and colorectal cancer.

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