A bispecific antibody, genetically modified TIL cells, and methods of construction and use thereof

Abstract

The application relates to the technical field of biological medicine, and discloses a kind of bispecific antibody, genetically modified TIL cell and its construction method and application, wherein the bispecific antibody includes FAP-specific nanobody VHH sequence with amino acid sequence as shown in SEQ ID NO:1, linker sequence with amino acid sequence as shown in SEQ ID NO:2 and CD3-specific antibody scFV sequence with amino acid sequence as shown in SEQ ID NO:3.The FAP*CD3 bispecific antibody provided in the application can effectively eliminate FAP-positive tumor-related fibroblasts, improve tumor microenvironment, and, in combination with the strong tumor antigen specificity of TIL cells, can improve the effectiveness of solid tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a bispecific antibody, a genetically modified TIL cell and a construction method and application thereof. BACKGROUND

[0002] TIL cells are derived from inside the tumor and naturally have the ability to recognize and target multiple tumor antigens. This feature enables TIL cell therapy to more comprehensively attack tumor cells and effectively overcome the treatment difficulties caused by tumor heterogeneity. However, although TIL cells have made some progress in some clinical studies, the therapeutic effect on some types of solid tumors is not satisfactory, and TIL cells still need to be further improved to continue to improve the effectiveness of TIL cells. SUMMARY

[0003] The purpose of the present application is to overcome the problem of unsatisfactory therapeutic effect of TIL cells on some types of solid tumors in the prior art, 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-mentioned purpose, one aspect of the present application provides a bispecific antibody, which comprises a FAP-specific nanobody VHH sequence with an amino acid sequence as shown in SEQ ID NO: 1, a linker sequence with an amino acid sequence as shown in SEQ ID NO: 2, and a CD3-specific antibody scFV sequence with an amino acid 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] The second aspect of the present application provides a genetically modified TIL cell capable of secreting a bispecific antibody, wherein the bispecific antibody comprises a FAP-specific nanobody VHH sequence with an amino acid sequence as shown in SEQ ID NO: 1, a linker sequence with an amino acid sequence as shown in SEQ ID NO: 2, and a CD3-specific antibody scFV sequence with an amino acid sequence as shown in SEQ ID NO: 3.

[0007] Preferably, the TIL cell is knocked out of the regenase-1 gene by sgRNA sequence; wherein the sgRNA sequence is selected from at least one of the sequences shown in SEQ ID NO: 5-9, and is preferably the sequence shown in SEQ ID NO: 6.

[0008] The third aspect of the present application provides a construction method of the TIL cell as described above, which comprises the following steps:

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

[0010] (2) dissociating and digesting tumor tissues, then performing cell separation, adding 200-1000 UI / ml IL-2 for initial expansion in vitro, and collecting TIL cells;

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

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

[0013] A1, constructing an AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector;

[0014] A2, transfecting the vector, pAAV-RC2 and pAAV-helper auxiliary plasmid into 293T cells, performing virus collection and impurity removal on the obtained cells, and finally performing virus purification and concentration to obtain an AAV virus containing a Reganse-1 homologous arm and capable of encoding 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 10-15 days.

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

[0017] The fourth aspect of the present application provides the use of the bispecific antibody as described above, the gene-modified TIL cells as described above or the gene-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 application can effectively eliminate FAP-positive tumor-related fibroblasts, improve the tumor microenvironment, and combine the strong specificity of the tumor antigen of the TIL cell itself, thereby more effectively improving the effectiveness of solid tumor treatment.

[0021] The genetically modified TIL cell provided by the application can secrete FAP*CD3 bispecific antibody, thereby effectively eliminating FAP-positive tumor-related fibroblasts, improving the tumor microenvironment, combining the strong specificity of the tumor antigen of the TIL cell itself, and improving the effectiveness of solid tumor treatment.

[0022] Further, the genetically modified TIL cell further knocks out the regenase-1 gene on the basis of secreting the FAP*CD3 bispecific antibody, and the depletion of the REGNASE-1 protein can prolong the survival time of the anti-tumor CD8 T cell and enhance the function of T. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is an AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector constructed in Example 1 of the application;

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

[0025] Figure 3 is a tumor efficacy test result of injecting different types of TIL cells in mice in Test Example 1 of the application;

[0026] Figure 4 is a binding experiment result of FAP nanobody with human and mouse FAP protein in Test Example 2 of the application;

[0027] Figure 5 is a binding experiment result of FAP*CD3 bispecific antibody with jurkat cell in Test Example 2 of the application;

[0028] Figure 6 is a result graph of FAP*CD3 bispecific antibody inhibiting SNU387 tumor cell growth in Test Example 3 of the application. DETAILED DESCRIPTION

[0029] The specific embodiments of the application are described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the application, and are not used to limit the application.

[0030] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the invention. Each integer value within the range is incorporated into the disclosure as if each value is individually recited. The endpoints of the ranges and any value are provided as a separate point for each integer value within the range, but the application is not to be limited to that precise value, unless the context clearly indicates otherwise.

[0031] TIL cells are derived from inside the tumor, naturally have the ability to recognize and target multiple tumor antigens, and this feature enables TIL cell therapy to more comprehensively attack tumor cells, 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 effect of cell therapy is an important direction of technological development. The tumor microenvironment of solid tumors is an important factor affecting the efficacy of TIL cells. Among them, cancer-associated fibroblasts play a crucial role in the tumor microenvironment and are often regarded as the mastermind behind the shaping of tumor growth and metastasis. They not only affect the proliferation and differentiation of tumor cells by secreting various growth factors and cytokines, but also actively participate in constructing a microenvironment conducive to tumor growth.

[0032] It has been found that FAP, i.e., fibroblast activation protein-alpha, is a 97kDa type II transmembrane serine protease involved in various biological functions, which can affect tumor growth through various mechanisms, including promoting proliferation, invasion, angiogenesis, epithelial-mesenchymal transition, stem cell promotion, immunosuppression and drug resistance. Under physiological conditions, the expression amount of FAP in most adult tissues is low, but it is often highly expressed on cancer-associated fibroblast (CAF) cells. In addition, some tumor cells also express FAP on their surface. Therefore, it is a good method to use FAP protease activity to selectively activate prodrugs at tumor sites to improve drug efficacy and reduce toxicity.

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

[0034] In the present application, the antibody in the bispecific antibody is a CD3 antibody and a FAP antibody, and is therefore also referred to as a FAP*CD3 bispecific antibody. The FAP*CD3 bispecific antibody can effectively eliminate FAP-positive tumor-associated fibroblasts, improve the tumor microenvironment, and, in combination with the strong tumor antigen specificity of TIL cells, more effectively improve the effectiveness of solid tumor treatment.

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

[0036] DVQLQESGGGLVHPGGSLRLSCAASEDTLEYYAIGWFRQAPGKEREGVSCIAIGGVASNYTDSVKGRFTISRDNTKHTVSLQMNSLKPEDTAIYYCAASAEITVGATGQFTCSLSDYDFWGQGTQVTVSS.

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

[0038] GGGGSGGGGSGGGGS.

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

[0040] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGSGGSGGSGGSGDGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK.

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

[0042] DVQLQESGGGLVHPGGSLRLSCAASEDTLEYYAIGWFRQAPGKEREGVSCIAIGGVASNYTDSVKGRFTISRDNTKHTVSLQMNSLKPEDTAIYYCAASAEITVGATGQFTCSLSDYDFWGQGTQVTVSSGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGSGGSGGSGGSDGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK.

[0043] In a preferred embodiment, the TIL cell is knocked out of the regenase-1 gene by sgRNA sequence; wherein the sgRNA sequence is selected from at least one of the sequences shown in SEQ ID NO: 5-9. 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 cleavage efficiency, so that the survival ability of TIL cells in vivo can be better prolonged, and tumor cells can be better inhibited and eliminated; at the same time, the RNP formed by the sgRNA and the Cas9 protein forms a DBS cut at the Regnase-1 genomic position, and the single-stranded DNA provided by the AAV can be used as a template to accurately integrate the DNA sequence of the secreted bispecific antibody into the TIL cell at the position.

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

[0051] The application also provides a genetically modified TIL cell, which can secrete a bispecific antibody, the bispecific antibody comprising a FAP-specific nanobody VHH sequence with an amino acid sequence as shown in SEQ ID NO: 1, a linker sequence with an amino acid sequence as shown in SEQ ID NO: 2, and a CD3-specific antibody scFV sequence with an amino acid sequence as shown in SEQ ID NO: 3.

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

[0053] In the application, the genetically modified TIL cell can secrete FAP*CD3 bispecific antibody, so as to effectively eliminate FAP-positive tumor-associated fibroblasts, improve the tumor microenvironment, and in combination with the strong tumor antigen specificity of the TIL cell itself, more effectively improve the effectiveness of solid tumor treatment.

[0054] The application also provides a method for constructing the genetically modified TIL cell as described above, which comprises 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 auxiliary plasmid into 293T cells to obtain an AAV virus containing a Reganse-1 homologous arm capable of encoding a bispecific antibody;

[0056] (2) Dissociating and digesting the tumor tissue, then performing cell separation, adding 200-1000 UI / ml IL-2 for an initial expansion stage in vitro, and collecting TIL cells;

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

[0058] In the method described in the present application, the present application does not limit the specific method for constructing the AAV-EF1a-FAP VHH-CD3 ScFv bispecific antibody vector, and can be a conventional method for constructing a vector 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 comprises:

[0060] A1, constructing an AAV-EF1a-FAP VHH-CD3 ScFv bispecific antibody vector;

[0061] A2, transfecting the vector, pAAV-RC2 and pAAV-helper helper plasmid into 293T cells, performing virus collection and impurity removal on the obtained cells, and finally performing virus purification and concentration to obtain an AAV virus encoding a bispecific antibody.

[0062] In the method described in the present application, the tumor tissue in step (2) is obtained from the tumor tissue of the patient himself, and is not limited in type, as long as the tumor tissue can be obtained.

[0063] In a preferred embodiment, in step (2), the time for the initial expansion stage in vitro is 5-7 days.

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

[0065] In the method described in the present application, in step (3), the AAV single-stranded DNA acts as a repair template and is integrated into the TIL cell genome by homologous recombination, so that the TIL cell can secrete the bispecific antibody.

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

[0067] The application also provides the genetically modified TIL cells constructed by the construction method as described above.

[0068] The application also provides the use of the genetically modified TIL cells as described above in the preparation of a drug for treating tumors.

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

[0070] Further, the tumor includes pancreatic cancer, breast cancer, esophageal cancer, sarcoma, mesothelioma liver cancer, lung cancer, gastric cancer and colorectal cancer.

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

[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. Screening of FAP nanobodies (construction of FAP nanobody phage display library):

[0075] (1) Immunization of camels with FAP

[0076] 1 mg of FAP protein was mixed with an equal volume of Freund's adjuvant to 5 ml, and injected subcutaneously in 3-5 points of the neck of the camel. Blood was collected from the auricular vein of the camel before immunization. Immunization was performed once a month, and a total of 4 times; 10 ml of peripheral blood was collected from the camel at each immunization. The blood collected before immunization and after each immunization was separated by Ficoll to obtain lymphocytes.

[0077] (2) Extraction of total RNA and synthesis of cDNA

[0078] Take the frozen lymphocytes, add 1 ml Trizol, room temperature for 10 min, then add 0.2 ml chloroform, shake vigorously, room temperature, wait for the solution to stratify (about 10 min), centrifugation at 12,000 rpm, collect the upper aqueous phase, add an equal volume of isopropanol, mix well, room temperature for 15 min, wait for the nucleic acid to precipitate, high-speed centrifugation to remove the supernatant, add 1 ml of 75% ethanol (DEPC water preparation) to the RNA precipitate for washing, high-speed centrifugation to remove the supernatant, and then dry the water, dissolve the RNA with nuclease-free water, take 1 μl for concentration and purity determination. Take 1 μg of RNA, use the SuperScriptT MIII First-Strand SynthesisSuperMix (Invitrogen) kit to synthesize cDNA;

[0079] (3) Construction of phage display library

[0080] The above synthesized cDNA was used as a template to amplify the V region (VHH) of camel heavy chain antibody by Nest-PCR, and Table 2 shows the name and sequence of the Nest-PCR primer.

[0081] Table 1: Primer information used for amplification of camel VHH fragments

[0082]

[0083] The PCR reaction conditions are as follows:

[0084] First round

[0085]

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

[0087] Second round

[0088]

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

[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 at 700 bp, and the target band was recovered by cutting the gel and subjected to the second round of PCR. The target gene fragment was at 500 bp, and the target band was recovered by cutting the gel, i.e. the VHH fragment. The VHH fragment and the vector were double-digested with NEB restriction endonucleases NotI and PstI, respectively, and the reaction system was as follows:

[0091] Vector digestion system

[0092]

[0093]

[0094] Add H2O to 500 μl;

[0095] Fragment digestion system:

[0096]

[0097] Add H2O to 500 μl; 37°C, overnight digestion, after agarose gel electrophoresis, cut and recover; mix the digestion products of the vector and the VHH fragment, and use the ligation enzyme of NEB to ligate 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 of the transformed TG competent cells was taken, incubated at 37°C for 2 h, and gradient-diluted to 10 1 , 10 2 , 10 3 , respectively, 300 μL was taken for plating, incubated at 37°C overnight, and the number of clones was calculated, about 10 5 clones per plate.

[0100] The same transformation method was used for large-scale transformation until the number of clones in the library reached more than 107. All clones were eluted with LB, centrifuged at 5,000g for 5 min, the precipitate was suspended with 2 ml of LB, and an equal volume of 30% glycerol was added. -80 was stored.

[0101] (5) Affinity FAP nanobody phage library panning

[0102] FAP protein coated ELISA plates were used alternately with human or mouse, 4°C, overnight incubation. Next day, FAP Nanobody-displaying phage were added, room temperature, 2h incubation; PBST wash wells 10 times, 100μl triethylamine was added, room temperature, 30min incubation, collected phage was FAP Nanobody phage library obtained by affinity panning; 10μl of infected TG cells were plated for determination of the number of clones after screening, the remaining phage after screening was used for amplification.

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

[0104] A1. rAAV vector construction process

[0105] The full gene synthesis FAP homologous left arm-EF1a-FAP VHH-CD3 scFV-FAP homologous right arm DNA sequence, the sequence is shown as SEQ ID NO: 16, was cloned into AAV expression vector ssAAV.CMV.tdTomato.WPRE.SV40pA through MluI and NOT1 enzyme cutting sites to obtain AAV-EF1a-FAP VHH-CD3 ScFv bispecific antibody vector ( Figure 1 ), the sequence of which is shown as SEQ ID NO: 17.

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

[0107]

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

[0109]

[0110] A2, rAAV virus packaging process (293T adherent)

[0111] A21, cell preparation

[0112] The cryopreserved cells were removed from the liquid nitrogen and quickly placed in a 37°C water bath for recovery. After centrifugation, fresh culture medium was added to the cells, which were cultured at 37°C and 5% CO2. Cell passage was performed every 2-3 days. After the cells grew normally, they were transferred to a 10 cm culture dish for adherent culture.

[0113] A22, plasmid transfection

[0114] When the cell density in step A21 reached about 80-90% confluence, transfection was performed. The AAV-EF1a-FAP VHH-CD3 ScFv bispecific antibody plasmid obtained above and the pAAV-RC2, pAAV-helper helper plasmid were configured in a mass ratio of 1:1:2, and then transfected into 293T cells by PEI transfection reagent.

[0115] A23, virus collection and impurity removal

[0116] The cells obtained in step A22 were cultured for 72 hours, and then the 293T cell precipitate and culture medium supernatant were collected. The supernatant was centrifuged at low temperature to remove cell debris, and then PEG8000 / NaCl solution was added for overnight precipitation. The 293T cell precipitate was broken by ultrasonic to release the AAV particles. The cell precipitate and PEG precipitate of the culture medium supernatant were mixed, and an appropriate amount of nuclease Benonase was added for 37°C digestion 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 subjected to iodixanol density gradient centrifugation in an ultracentrifuge. After centrifugation, the solution corresponding to the virus layer was extracted, and dialysis bags were used for further dialysis at 4°C overnight. The next day, the dialysate was filtered through a 0.22um filter, and concentrated and washed through a concentration tube to remove impurities and concentrate. Finally, AAV virus containing Regnase-1 homologous arms and capable of encoding a bispecific antibody was obtained.

[0119] Example 2

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

[0121] (1) The tumor tissue removed by surgery should be preserved in Miltenyi MACS tissue preservation solution, and the time for transporting to the laboratory for TIL separation should not exceed 24 hours to maintain cell activity,

[0122] (2) Tissue dissociation and digestion: The tumor tissue is mechanically sheared into 1-3 mm 3 pieces, and the volume is adjusted to 5-10 ml with RPMI-1640, and 0.05% collagenase type I and type IV, 0.001% hyaluronidase and DNase are added, and stirred at room temperature for 4-6 h; filter the single cell suspension through a 100 μm cell strainer.

[0123] (3) Cell separation and expansion culture: Wash the single cell suspension twice with normal saline, centrifuge at 1800 rpm for 8 minutes at room temperature, discard the supernatant, and resuspend the precipitate in X-VIVO 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: Centrifuge to collect the TIL cells in step (3), add the AAV virus containing the Regnase-1 homologous arm and capable of encoding a bispecific antibody obtained in Example 1 at a MOI of 5E5 vg / cell, infect for 6 hours, centrifuge to remove the supernatant, add the Cas9 RNP complex, and perform gene editing on the TIL cells by lonza 4D electroporation system;

[0125] The RNP complex for electroporation is shown in Table 2.

[0126] Table 2

[0127]

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

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

[0130] After sgRNA-1 to sgRNA-6 were ordered from Geneart, they were incubated with Cas9 protein according to the above Table 1 to configure cas9 RNP complex; logarithmic growth phase 293T cells were digested and dispersed into single cells. 1E6 293T cells were mixed with cas9 RNP complex, and then electroporated by Lonza 4D electroporation system. After electroporation, the cells were cultured for 24 hours, and then the cell DNA was extracted by a kit. The primers were designed to amplify the target DNA sequence at the left and right of the sgRNA cleavage site, and then the amplicon was subjected to high-throughput sequencing. Through bioinformatics analysis, the cleavage efficiency of different sgRNAs can be calculated.

[0131] The results are shown in Figure 2 The results show that the sgRNA-2 has the highest cleavage efficiency. Therefore, sgRNA-2 is used in the preparation of TIL cells in the animal experiments of the present embodiment and the following test examples.

[0132] (5) TIL rapid expansion phase: GIBCO Dynabeads CD3 / CD28 / CD137 magnetic beads were added to the genetically edited TIL cells in step 4 TM Human T-Activator CD3 / CD28 / CD137 magnetic beads were added to the genetically edited TIL cells in step 4

[0133] Example 3

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

[0135] The DNA sequence encoding the FAP*CD3 bispecific antibody was cloned into the eukaryotic expression vector pcDNA3.1; the sequence was confirmed to be correct, and the plasmid was extracted by E. coli; then the plasmid was transfected into 293T cells by liposome (such as Lipofectamine 2000); the supernatant was collected 48-72 hours after transfection; the cells were removed by centrifugation (3000xg, 20 minutes); and the target bispecific antibody was purified by ion exchange chromatography and molecular sieve.

[0136] Test Example 1

[0137] Preparation of animal models

[0138] SPF male NOD / SCID mice aged 4 weeks and weighing 18-20 g were taken and adapted to the environment for 1 week. Logarithmic growth phase patient-derived tumor cells were taken, and the cells were collected by trypsin digestion, resuspended in sterile PBS, and counted to adjust the cell density to 2x10 7The cell suspension was administered subcutaneously to the left upper limb dorsal side of mice to induce a tumor model. After 14 days of modeling, the tumor volume in mice reached 80–100 mm². 3 The above indicates that the model was successfully created.

[0139] Thirty tumor-bearing mice were randomly divided into 5 groups of 6 mice each: G1: MOCK T cell administration group; G2: TIL cell administration group; G3: TIL cell administration group secreting FAP*CD3 bispecific antibody; G4: renase-1 gene knockout TIL cell administration group; G5: TIL cell administration group secreting both FAP*CD3 bispecific antibody and renase-1 gene knockout (i.e., TIL cells obtained in Example 2). The total number of cells administered to each group was 1×102. 7 One dose is administered via the tail vein.

[0140] Test Results Figure 3 As shown.

[0141] Depend on Figure 3 It can be seen that both knocking out the Regnase-1 gene in TIL cells alone and inducing TIL cells to secrete FAP*CD3 bispecific antibody can enhance the inhibitory effect of TIL cells on tumor cells. At the same time, the inhibitory effect of TIL cells secreting FAP*CD3 bispecific antibody and those with knocked-out regnase-1 gene is the most significant, indicating that knocking out the Regnase-1 gene and secreting FAP*CD3 bispecific antibody have a synergistic effect.

[0142] Test Example 2

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

[0144] 96-well plates were coated with human or mouse FAP protein and incubated overnight. After coating, the plates were washed once with PBST, then blocked with 1% BSA at 37°C for 1 hour, washed, and dried. A gradient solution of recombinant FAP nanobody was prepared by serially diluting it with 1% BSA from 5 μg / mL to 0.01 μg / mL. The plates were co-incubated at 37°C for 2 hours, washed 5 times, and dried. 100 μL of 1:15000 diluted Goat anti-mouse IgG-HRP secondary antibody was added to each well, and the plates were incubated at 37°C for 30 minutes. After washing 5 times and drying, 100 μL of TMB substrate chromogenic solution was added to each well, and the plates were incubated at 37°C in the dark for 20 minutes. The reaction was stopped by adding 100 μL of stop solution to each well, and the OD value was immediately read at 450 nm using an Infinite F50 microplate reader.

[0145] Test results are as follows Figure 4 As shown.

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

[0147] 3. FAP*CD3 bispecific antibody prepared in Example 3 and jurkat cell binding experiment.

[0148] (1) Take no less than 1x10 6 Jurkat cells to a 1.5ml microcentrifuge tube.

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

[0150] (3) Resuspend the Jurkat cells with 100ul WB, add human FAP-biotin protein, 2ul / test, and stain at room temperature for 25min.

[0151] (4) Add 1000ul WB to each well, mix by blowing, centrifuge at 300xg for 5min, and aspirate the supernatant.

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

[0153] (6) Resuspend with 100ul WB, add APC Streptavidin, 1ul / test, and stain at 4℃ for 15min.

[0154] (7) Add 1000ul WB to each well, mix by blowing, centrifuge at 300xg for 5min, and aspirate the supernatant.

[0155] (8) Wash the cells once with WB (PBS+2% FBS), centrifuge at 300xg for 5min, and aspirate the supernatant.

[0156] (9) Resuspend the Jurkat cells with 150ul WB, mix by blowing, and then perform flow detection on the machine.

[0157] The test results are shown in Figure 5 .

[0158] By Figure 5 It can be seen that the bispecific antibody described in the application can better bind to human CD3 target protein.

[0159] Test Example 3

[0160] The FAP*CD3 bispecific antibody described in the application inhibits SNU387 tumor cell growth experiment.

[0161] Tumor cells SNU387 and T cells were seeded in 96-well plates at an effector-to-target ratio of 1:1. Different concentrations of the FAP*CD3 bispecific antibody obtained in Example 3 were added, and the plates were incubated at 37°C in a 5% CO2 incubator. The tumor killing ability was detected by the LDH method at 24h, 36h, and 48h after culture. The specific detection method included: centrifugation to remove the supernatant, adding 50μL / well of the detection mixture, mixing thoroughly, and incubating at room temperature in the dark for 30min; adding 50μL / well of stop solution, shaking thoroughly, and detecting the absorbance at 492nm using a spectrophotometer, and calculating the cell killing rate as follows: (OD value of experimental calibration wells - OD value of effector cell spontaneous calibration wells - OD value of target cell spontaneous calibration wells) / (OD value of target cell maximum release calibration wells - OD value of target cell spontaneous calibration wells) × 100%.

[0162] Test results are as follows Figure 6 As 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; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A bispecific antibody, characterized in that, The bispecific antibody comprises the FAP-specific nanobody VHH sequence as shown in SEQ ID NO:1, the linker sequence as shown in SEQ ID NO:2, and the CD3-specific antibody scFV sequence as shown in SEQ ID NO:3; wherein the amino acid sequence of the bispecific antibody is shown in SEQ ID NO:

4.

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

4.

3. The gene-modified TIL cells according to claim 2, characterized in that, The TIL cells knocked out the regenase-1 gene at specific sites using sgRNA sequences; wherein the sgRNA sequences are selected from at least one of the sequences shown in SEQ ID NO:5 to 9.

4. The gene-modified TIL cells according to claim 3, characterized in that, The sgRNA sequence was selected from the sequence shown in SEQ ID NO:

6.

5. A method for constructing gene-modified TIL cells according to any one of claims 2-4, characterized in that, The method includes the following steps: (1) Construct the AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector, the nucleotide sequence of which is shown in SEQ ID NO:

17. Transfect the vector, pAAV-RC2 and pAAV-helper plasmid into 293T cells to obtain AAV virus containing the Reganse-1 homologous arm that can encode bispecific antibodies. (2) The tumor tissue was dissociated and digested, and then the cells were separated. 200-1000 UI / ml IL-2 was added for the initial in vitro expansion stage, and TIL cells were collected. (3) Infect the TIL cells with the AAV virus encoding bispecific antibodies obtained in step (1). After 4 to 12 hours of infection, centrifuge to remove the supernatant, add the Cas9 RNP complex, and perform gene editing on the TIL cells by electroporation. The single-stranded DNA carried by AAV serves as a homologous recombination repair template at the nick formed by Cas9 RNP, and the sequence of secreting bispecific antibodies is integrated into the TIL cell genome.

6. The construction method according to claim 5, characterized in that, Step (1) specifically includes: A1. Construct the AAV-EF1a-FAP VHH-CD3 ScFv bispecific Antibody vector; A2. The vector, pAAV-RC2, and pAAV-helper plasmid were transfected into 293T cells. The cells were then subjected to virus collection and purification. Finally, the virus was purified and concentrated to obtain an AAV virus containing the Reganse-1 homologous arm that encodes a bispecific antibody.

7. The construction method according to claim 5 or 6, characterized in that, The method also includes adding Human T-Activator CD3 / CD28 / CD137 magnetic beads to the gene-edited TIL cells obtained in step (3) and continuing to expand them in vitro for 10 to 15 days.

8. The construction method according to claim 5 or 6, characterized in that, In step (3), the Cas9 RNP complex contains the sgRNA sequence shown in SEQ ID NO:6.

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