Single-chain antibody fragment targeting KRAS g12v, chimeric antigen receptor car and uses

Abstract

A single-chain antibody fragment targeting KRAS G12V, a chimeric antigen receptor (CAR) and uses. A TCR is modified on the basis of a KRAS G12V target, an extracellular signal region of the TCR for recognizing a tumor specific antigen is reserved, and is connected in series to an extracellular spacer region, a transmembrane region and a CD3ζ intracellular signal region in a conventional CAR structure, so that the modified CAR can specifically recognize KRAS G12V mutant polypeptides presented by HLA-A*02:01. Moreover, by means of the advantages of CAR-T / NK cell therapy, a potential new tumor treatment scheme is explored, and a foundation is laid for clinical tests.

Description

A KRAS G12V-targeted single-chain antibody fragment, chimeric antigen receptor CAR, and its application Technical Field

[0001] The present invention relates to a KRAS G12V-targeted single-chain antibody fragment, a chimeric antigen receptor CAR and applications thereof, and belongs to the field of biomedicine. Background Art

[0002] Cell therapy technology has become one of the most eye-catching fields in recent years. It has shown good therapeutic prospects in the fields of cancer, blood diseases, cardiovascular diseases, diabetes and neurological diseases. It has played an important role in various diseases, especially in tumor immunotherapy. Currently, more than ten immune cell therapies are on the market. Although immune cell therapy has shown good therapeutic effects in blood and lymphatic system tumors, it still has certain limitations in the treatment of solid tumors. Most of the antigens currently used in clinical trials are tumor-associated antigens (TAA), such as mesothelin (MSLN), epidermal growth factor receptor (EGFR), tight junction protein Claudin18.2 (CLDN18.2), etc., but TAA is also expressed in some normal tissues, which brings off-target risks and safety issues to cell therapy. Therefore, the selection of highly specific targets for solid tumors is a primary issue that needs to be addressed urgently.

[0003] Tumor antigens are divided into tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). Tumor-associated antigens (TAAs) are antigens expressed by both tumor cells and normal tissues, but their levels increase significantly when cells become cancerous. These antigens only exhibit quantitative changes and lack strict tumor specificity. Tumor-specific antigens (TSAs) are antigens that are unique to tumor cells or present only in certain tumor cells but not in normal cells. Neoantigens are tumor-specific antigens derived from non-synonymous mutations and are very attractive targets for tumor immunotherapy. Unlike TAAs, TSAs / neoantigens are mutated peptides derived from genetic alterations in the tumor genome. They are specifically expressed in tumor cells and absent in normal tissues, demonstrating high tumor specificity and reducing off-target toxicity. Personalized immunotherapy based on neoantigens can stimulate a powerful anti-tumor immune response against tumor cells.

[0004] RAS was the first human tumor gene discovered and is a commonly mutated gene in tumors. There are three main subtypes: HRAS, KRAS, and NRAS. KRAS is the most prevalent mutated form of the RAS family and is associated with 22% of human tumors. It is the most frequently mutated gene in the three most lethal human cancers: lung cancer (17%), colorectal cancer (33%), and pancreatic cancer (61%). In healthy cells, KRAS acts as a switch that regulates cell growth and plays a vital role in various cellular signaling events, including cell proliferation. However, when the KRAS gene mutates, KRAS becomes stuck in the "on" position, causing uncontrolled cell growth and activating downstream pathways, leading to malignant cell proliferation and cancer development and metastasis. 97% of KRAS mutations occur at codons 12 and 13, with the most common KRAS mutations being G12C, G12D, G12V, and G13D. Clinical studies have shown that the G12V mutation is highly promising in immunotherapy for multiple cancer types, making it an ideal target for tumor immunotherapy.

[0005] Chimeric antigen receptors (CARs) are synthetic receptors that can guide immune cells to specifically track, recognize, and eliminate tumor cells expressing their target ligands. They typically consist of an extracellular binding domain (typically derived from the antigen-binding region of a monoclonal antibody, a scFv fragment) that recognizes tumor-associated antigens, a hinge domain, a transmembrane domain, and the intracellular signaling domain of a TCR molecule, resulting in a highly targeted response. CARs bind to tumor antigens in an antigen-dependent, non-MHC (Major Histocompatibility Complex)-restricted manner, enabling CAR-modified T cells to recognize a wider range of tumor cells compared to natural T cell surface receptors (TCRs).

[0006] The HLA-A*02 allele is expressed in 44% of the Caucasian population and as high as 37.7% of the Chinese population. HLA-A*02:01 is expressed in over 10% of the Chinese population. The NetMHCpan-4.0 bioinformatics algorithm predicted the affinity of proteins expressing HLA-A*02:01 and the RAS G12V mutation. Traditional TCR-T cells can recognize both intracellular and extracellular tumor antigens presented by MHC, with hypersensitivity for intracellular antigens with low mutation levels, but are MHC-restricted. CAR-T / NK cells, on the other hand, can only recognize antigens on the surface of tumor cells.

[0007] Summary of the Invention

[0008] To solve the above technical problems, the first object of the present invention is to provide a single-chain antibody fragment targeting KRAS G12V, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3;

[0009] wherein the amino acid sequence of VH-CDR1 is the sequence shown in SEQ ID NO: 3, the amino acid sequence of VH-CDR2 is the sequence shown in SEQ ID NO: 4, and the amino acid sequence of VH-CDR3 is the sequence shown in SEQ ID NO: 5;

[0010] SEQ ID NO: 3: GSYIH;

[0011] SEQ ID NO: 4: YIDPEYGYSRYADSVKG;

[0012] SEQ ID NO: 5: DSDSDAMDV;

[0013] The amino acid sequence of VL-CDR1 is the sequence shown in SEQ ID NO: 8, the amino acid sequence of VL-CDR2 is the sequence shown in SEQ ID NO: 9, and the amino acid sequence of VL-CDR3 is the sequence shown in SEQ ID NO: 10 (QQYYYPYPT).

[0014] SEQ ID NO: 8: RASQDVNVAVA;

[0015] SEQ ID NO: 9: SSSFLYS;

[0016] SEQ ID NO: 10: QQYYYPYPT.

[0017] Furthermore, the full-length amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 6;

[0018] SEQ ID NO: 6:

[0019] or the amino acid sequence shown in SEQ ID NO: 7;

[0020] SEQ ID NO: 7:

[0021] The full-length amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 11;

[0022] SEQ ID NO: 11:

[0023] Or the amino acid sequence shown in SEQ ID NO: 12.

[0024] SEQ ID NO: 12:

[0025] Furthermore, the heavy chain variable region and the light chain variable region are connected by a connecting region, which is composed of three tetrapeptide polymers, and the amino acid sequence is shown in SEQ ID NO: 13.

[0026] SEQ ID NO: 13: GGGSGGGGSGGG.

[0027] Furthermore, the amino acid sequence of the variable region of the single-chain antibody is as shown in SEQ ID NO: 14 or a sequence with at least 80% homology;

[0028] SEQ ID NO: 14:

[0029] Or as shown in SEQ ID NO: 15 or a sequence with at least 80% homology.

[0030] SEQ ID NO: 15:

[0031] The second object of the present invention is to provide a chimeric antigen receptor CAR, comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain; wherein the antigen domain comprises a signal peptide, a Myc tag, and a single-chain antibody fragment as shown in claims 1-3 that recognizes the antigen.

[0032] Furthermore, the signal peptide is CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or a combination thereof.

[0033] Furthermore, the signal peptide is CD8, and the amino acid sequence is shown in SEQ ID NO: 1.

[0034] SEQ ID NO: 1: MALPVTALLLPLALLLHAARP;

[0035] Furthermore, the Myc tag is an epitope tag derived from the c-myc gene, which can be fused to the N-terminus or C-terminus of the single-chain antibody fragment as a fusion protein, and its amino acid sequence is shown in SEQ ID NO: 2.

[0036] SEQ ID NO: 2: EQKLISEEDL.

[0037] Furthermore, the Myc tag is fused to the N-terminus of the single-chain antibody fragment.

[0038] Furthermore, the antigen binding domain and the transmembrane domain are connected by a hinge region, and the hinge region is selected from a derivative of the extracellular domain of CD8α, CD28, an IgG-based hinge region composed of the FC part of IgG1 or IgG4 or the CH2 / CH3 domain of the Fc part, or DAP12.

[0039] Furthermore, the hinge region is CD8α, and its amino acid sequence is shown in SEQ ID NO: 16.

[0040] SEQ ID NO: 16:

[0041] Furthermore, the transmembrane domain is selected from one of CD3ζ, CD8, CD28, NKG2D, 2B4, and DNAM1.

[0042] Furthermore, the transmembrane domain is CD28 or CD8; wherein the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO: 17;

[0043] SEQ ID NO: 17:

[0044] or / and

[0045] The amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO:18.

[0046] SEQ ID NO: 18: IYIWAPLAGTCGVLLLSLVITLYC.

[0047] Furthermore, the intracellular signaling domain is selected from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28 family, DAP10, DAP12, NKp44, NKG2D, TNFR family or SLAM-related receptor family.

[0048] Furthermore, the intracellular signaling domain is a combination of 4-1BB and CD3ζ; wherein the amino acid sequence of 4-1BB is shown in SEQ ID NO: 19, and the amino acid sequence of CD3ζ is shown in SEQ ID NO: 20.

[0049] SEQ ID NO: 19:

[0050] SEQ ID NO: 20:

[0051] The third object of the present invention is to provide a CAR-T cell expressing the above-mentioned chimeric antigen receptor CAR.

[0052] The fourth object of the present invention is to provide a use of the above-mentioned KRAS G12V-targeting single-chain antibody fragment, chimeric antigen receptor CAR, and CAR-T cells in the preparation of anti-tumor drugs or products.

[0053] The beneficial technical effects achieved by the present invention are as follows: The present invention improves a KRAS G12V-targeting single-chain antibody fragment, chimeric antigen receptor CAR, and its application, and modifies TCR based on the KRAS G12V target. The extracellular signaling region of the TCR that recognizes tumor-specific antigens is retained and connected in series with the extracellular spacer, transmembrane region, and CD3ζ intracellular signaling domain in the traditional CAR structure, enabling the modified chimeric antigen receptor CAR to specifically recognize the KRAS G12V mutant polypeptide presented by HLA-A*02:01. At the same time, leveraging the advantages of CAR-T / NK cell therapy, potential new tumor treatment options are explored, laying the foundation for clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1. Schematic diagram of the KGV1 CAR and KGV2 CAR structures.

[0055] Figure 2 Flow cytometry detection of KGV1 CAR-T and KGV2 CAR-T lentiviral infection efficiency;

[0056] Figure 3. Sorting efficiency of positive KGV1 CAR-T and KGV2 CAR-T cells;

[0057] Figure 4 Schematic diagram of the structures of KRAS G12V plasmid, HLA-A*02:01 plasmid and Luciferase plasmid;

[0058] Figure 5. The proportion of KRAS G12V mutation and HLA*02:01 expressed in exogenous target cells K562#4;

[0059] Figure 6 KGV1 CAR-T and KGV2 CAR-T kill exogenous target cells;

[0060] Figure 7. Secretion of cytokines TNF-α and IFN-γ during exogenous killing;

[0061] Figure 8 Phenotypic identification of endogenous target cells;

[0062] Figure 9 Killing of endogenous target cells by KGV1 CAR-T and KGV2 CAR-T;

[0063] Figure 10. Secretion of cytokines TNF-α and IFN-γ during endogenous killing. DETAILED DESCRIPTION

[0064] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0065] The present invention is further described below with reference to the accompanying drawings and embodiments.

[0066] Example 1 Sequence Design and Vector Construction of Chimeric Antigen Receptor CAR

[0067] The signal peptide, Myc tag, CD8α hinge region, CD8 transmembrane domain, 4-1BB and CD3ζ intracellular domain sequences of CAR are all from the public database Addgene, and the single-chain antibody variable region (single-chain variable fragment, scFv) fragment is obtained by screening the phage antibody mutation library shown in Example 3. According to the order of signal peptide, Myc tag, KRAS G12V targeting single-chain antibody fragment, CD8α hinge region, CD8 transmembrane domain, 4-1BB and CD3ζ intracellular signaling domain, the master plasmid is constructed between the EcoR I and Sal I restriction sites of the vector PCDH-EF1α, named KGV1 CAR and KGV2 CAR, and the structural model diagram of the master plasmid is shown in Figure 1. The specific construction process of the master plasmid can be a common method in the prior art and will not be repeated here.

[0068] Example 2 Construction of phage antibody mutation library

[0069] PBMCs were isolated from 1000 healthy individuals, and mRNA was extracted and reverse-transcribed into cDNA. Based on known antibody sequences, the target fragment was amplified using error-prone PCR. The PCR product was subjected to 2% agarose gel electrophoresis, and the target band of approximately 500 bp was excised and recovered from the gel. The PCR product and the phagemid vector pCANTAB5E were double-digested with Sfi I and Not I, respectively, recovered from the gel, and ligated with T4 ligase at a vector:DNA base ratio of 1:3. The ligation was performed in a PCR instrument overnight at 16°C. Five microliters of the ligation product was added to 100 μl of TG1 competent medium, pre-chilled on ice, and transferred to a pre-chilled electroporation cuvette. The electroporation voltage was adjusted to 2.5 kV for 5 ms. After electroporation, 0.9 ml of culture medium was quickly added and incubated at 37°C with shaking for 2 hours. 10 μl of serial dilutions were plated onto SOBAG plates, and the library volume was calculated. Randomly selected single clones were sequenced to verify the diversity of the mutant antibody library.

[0070] Example 3 Phage Antibody Mutation Library Screening

[0071] 1.5 mL of the above mutant antibody library was inoculated into 300 mL of culture medium, cultured with shaking at 37°C for about 1.5 h, and helper phage M13KO7 was added at 5 times the volume for superfection, and cultured with shaking at 37°C for about 1 h; centrifuged at 4000 rpm and 15°C for 15 min, and the culture medium was removed; 200 mL of culture medium (100 μg / mL Amp, 50 μg / mL Kan) was added to resuspend the bacteria, and cultured at 37°C for 2 h; centrifuged at 10000 rpm for 20 min to remove the precipitate; 40 mL of PEG / NaCl was added to the supernatant to precipitate the phage, and the mixture was ice-bathed overnight; centrifuged at 10000 rpm for 20 min, and the supernatant was removed; the phage was resuspended in 0.6 mL of culture medium and stored at 4°C for later use. ELISA microplates are coated with His Bind Resin to bind to the antigen protein, with the target protein as the stationary phase and the phage display library as the mobile phase. After a 2-hour incubation, unbound free phage are washed away, and then phage bound to the target molecule are eluted with acid. The eluted phage are infected with TG1 host bacteria, propagated and amplified, and then the next round of elution is performed. After five rounds of "adsorption-elution-amplification", specific antibodies are enriched. Single clones are plated and selected for ELISA verification. Ultimately, two single-chain antibodies targeting KRAS G12V were successfully screened. The amino acid sequences of the variable regions of these two single-chain antibodies are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0072] SEQ ID NO: 14:

[0073] SEQ ID NO: 15:

[0074] Example 4 Lentivirus Preparation

[0075] (1) 5×10 5 HEK293F cells were seeded into a 30 ml shake flask and the cell density reached 4.0–5.0 × 10 6 When the concentration of cells / ml is approximately 1:1, proceed with lentiviral packaging. Add 1.5 ml of antibiotic- and serum-free culture medium, followed by 37.5 μg of the master plasmid and 37.5 μg of the packaging plasmid mixture in a 1:1 ratio, and gently pipette to mix thoroughly. Add 180 μl of transfection reagent to 1.5 ml of antibiotic- and serum-free culture medium and gently mix thoroughly. After mixing the plasmid and transfection reagent, incubate at room temperature for 10 minutes. Slowly transfer the transfection reagent-DNA mixture to a shake flask, gently shake to mix thoroughly, and place in a 37°C incubator with 5% CO2.

[0076] (2) 48 hours after transfection, the culture supernatant was collected and centrifuged at 3000 rpm / min and 4°C for 10 min. The precipitate was discarded and the supernatant was filtered through a 0.45 μm filter membrane.

[0077] (3) Virus concentrate and the above-mentioned virus supernatant filtrate were added sequentially at a ratio of 1:4. The virus supernatant filtrate was slowly added, separated by stratification, and centrifuged at 10,000 g / min at 4°C for 4 h to concentrate the virus. The supernatant was discarded, and the virus pellet was retained. Culture medium without antibiotics and serum was added at a ratio of 250:1 of virus supernatant filtrate to culture medium to dissolve the pellet. The pellet was then aliquoted and stored at -80°C until further use.

[0078] Example 5 T cell preparation

[0079] T cells were obtained from healthy human peripheral blood. To eight 50ml centrifuge tubes, add 15ml of lymphocyte separation buffer, previously cooled to room temperature. Then, dilute 100ml of heparinized blood with saline in a 1:1 ratio and mix thoroughly. Using a pipette, slowly add 25ml of the diluted blood along the tube wall onto the lymphocyte separation buffer. Centrifuge at 650g / min, 20°C, for 20 minutes. To ensure separation, set the centrifuge speed to the lowest setting. After centrifugation, four layers should be visible within the tube: plasma, buffy coat (PBMCs), lymphocyte separation buffer, granulocytes, and erythrocytes. Discard the top plasma layer, leaving approximately 1ml. Use a pipette to aspirate the buffy coat (PBMCs) along the tube wall and transfer the remaining 1ml to another 50ml centrifuge tube. Add saline and centrifuge at 470g for 10 minutes. Discard the supernatant. If there is a lot of red material at the bottom, add erythrocyte lysis buffer to lyse the erythrocytes. After lysis is complete, add normal saline and centrifuge at 300g / min for 10 minutes. Discard the supernatant, wash once with normal saline, and centrifuge at 300g / min for 5 minutes. Discard the supernatant, add 1640 complete medium preheated at 37°C, resuspend the cells, count, and calculate cell viability. PBMCs are isolated using this density gradient centrifugation method, and T cells are activated by adding the cell-stimulating factors CD3 and CD28 at a concentration of 200ng / ml. After 24 hours, 10ng / ml of IL-7, 20ng / ml of IL-15, and 20ng / ml of IL-21 are added to continue expansion and culture.

[0080] Example 6 Preparation of CAR-T cells

[0081] CAR-T cells were prepared using a lentiviral system. Activated T cells were transduced with lentiviral concentrate at an MOI of 10, and 5 μg / ml of Polybrene dye was added. After 24 hours, the medium was changed and cultured again. Flow cytometry analysis revealed lentiviral infection efficiencies of 77.45% and 76.01%, respectively, as shown in Figure 2.

[0082] Example 7 Sorting of positive CAR-T cells

[0083] Positive CAR-T cells were sorted using Myc-Tag (9B11) Mouse mAb (PE Conjugate) flow cytometry antibody (purchased from Cell Signaling Technology) and Anti-PE MicroBeads (purchased from Miltenyi Biotec) magnetic beads. The specific method is as follows: 5μl Myc-Tag (9B11) Mouse mAb (PE Conjugate) antibody was added to every 10 million cells and incubated at 4°C for 30 minutes. After washing three times with PBS, 80μl sorting buffer and 20μl Anti-PE MicroBeads were added and incubated at 4°C for 15 minutes. After washing three times with PBS, the cells were passed through an LS column. The cells eluted from the LS column are positive CAR-T cells. The positive rate of KGV1 CAR-T and KGV2 CAR-T cells after sorting reached over 98%, as shown in Figure 3.

[0084] Example 8 Construction and Verification of Exogenously Expressing KRAS G12V Mutation and HLA*02:01 Target Cells

[0085] K562 cells were selected as exogenous target cells because they do not express KRAS or endogenous HLA. KRAS G12V and HLA*02:01 plasmids were stably transfected into K562 cells via lentiviral infection. Expression was detected after 72 hours, and after 14 days of culture, a stable transgenic line was established. Double-positive cells were flow-sorted, expanded in culture, and phenotypes were identified by flow cytometry. The results are shown in Figure 5. A schematic diagram of the KRAS G12V plasmid, HLA*02:01 plasmid, and luciferase plasmid structures is shown in Figure 4. Simultaneously, a transposon plasmid expressing luciferase was electroporated into double-positive cells for subsequent cytotoxicity assays. This cell, designated K562#4, was used for subsequent cytotoxicity assays. The results are shown in Table 1.

[0086] Table 1 Firefly luciferase detection in K562-luc cells and K562#4-luc cells

[0087] Example 9 Identification of target cells endogenously expressing KRAS G12V mutation and HLA*02:01

[0088] The pancreatic cancer cell line CFPAC-1 was selected as an endogenous target cell, and sequencing and genomic PCR confirmed its endogenous KRAS G12V mutation and HLA*02:01 expression. The pancreatic cancer cell line BxPC-3 was also selected as a control target cell, and sequencing and genomic PCR confirmed its lack of endogenous KRAS G12V mutation and HLA*02:01 expression. The pancreatic cancer cell line PANC-1 was also selected as a control target cell, and sequencing and genomic PCR confirmed its endogenous KRAS G12D mutation but lack of endogenous KRAS G12V mutation, as well as low HLA*02:01 expression. The results are shown in Figure 9, Tables 2, and 3. Phenotypic characterization of the endogenous target cells is shown in Figure 8. Primer sequences for KRAS sequencing are shown in Table 4.

[0089] Table 2 KRAS sequencing results of three pancreatic cancer cell lines

[0090] Table 3 HLA-A allele sequencing results of three pancreatic cancer cell lines

[0091] Table 4 Primer sequences for KRAS sequencing

[0092] Example 10 Analysis of in vitro functional studies of CAR-T cells expressing exogenous KRAS G12V and HLA-A*02:01

[0093] CAR-T cells were co-cultured with K562#4 tumor target cells at effector-target ratios of 1:2, 1:1, 2:1, 5:1, and 10:1. After 24 hours of culture, live target cells were labeled with luciferase to calculate the killing efficiency and to measure the expression of cytokines IFN-γ and TNF-α. Blank K562 cells served as a control.

[0094] The results showed that the killing rate of the two target cells gradually increased with the increase of the effector-target ratio. K562 cells are double-negative cells that do not express KRAS G12V and HLA-A*02:01. Compared with the control group, there was no overall statistical difference (P>0.05), and the killing ability of the two CAR-T cells was similar. For the double-positive cells K562#4 that express both KRAS G12V and HLA-A*02:01, the killing efficiency of the two CAR-T cells was significantly higher than that of the control group at each effector-target ratio, and the difference was statistically significant (P<0.05). The killing ability of the two CAR-T cells was comparable, as shown in Figure 6.

[0095] In addition, we also measured the release (concentration) of cytokines in the supernatant. In the double-negative K562 cells, although cytokine release increased with increasing effector-to-target ratio, the increase was small, and no significant differences were found among the three experimental groups. However, in the double-positive K562#4 cells, the levels of TNF-α and IFN-γ increased significantly with increasing effector-to-target ratio, and there were significant differences in cytokine concentrations between the two CAR-T cell groups and the control T cell group (P < 0.05), indicating that K562#4 cells were able to activate both CAR-T cells. Comparison of the two target cell groups also revealed that the TNF-α and IFN-γ levels in the double-positive K562#4 cells far exceeded those in the K562 group, as shown in Figure 7. This in vitro functional assay demonstrates that both CARs designed and used enable T cells to specifically recognize exogenously expressed target antigens and exert a killing effect. Furthermore, the targeted antigens specifically activate T cells that recognize them, stimulating T cells to release more cytokines, which not only promotes rapid T cell expansion but also enhances the killing effect of T cells.

[0096] Example 11 Analysis of in vitro functional studies of CAR-T cells against endogenously expressing KRAS G12V and HLA-A*02:01

[0097] CAR-T cells were co-cultured with three pancreatic cancer cells at effector-target ratios of 1:5, 1::2, 1:1, 2:1, 5:1, and 10:1. After 24 hours of culture, live target cells were labeled with CCK8 to calculate the killing efficiency, and the expression of cytokines IFN-γ and TNF-α in the supernatant was detected using a kit.

[0098] The control group reflects the inherent cytotoxicity of T cells against target cells, with the killing rate against the three target cells slowly increasing with increasing effector-to-target ratio. Although the killing ability of KGV1 CAR-T cells and KGV2 CAR-T cells against PANC-1 and BxPC-3 cells also slowly increased with increasing effector-to-target ratio, the killing rate was close to that of the control group, and there was no statistical difference among the three groups (P>0.05). In other words, KGV1 CAR-T cells and KGV2 CAR-T cells only exerted the inherent cytotoxicity of T cells against PANC-1 and BxPC-3. In the CFPAC-1 cell group, the killing ability of both CAR-T cells was significantly higher than that of the control group (P<0.05). At an effector-to-target ratio of 10:1, they were able to almost completely eliminate all target cells, with a killing rate approaching 100%. The results are shown in Figure 9. In other words, KGV1 CAR-T and KGV2 CAR-T activate T cells through the specific recognition of KRAS G12V peptide presented by HLA-A*02:01, giving T cells killing efficacy beyond their inherent killing properties.

[0099] Overall, the levels of each cytokine increased with increasing effector-target ratios. Among them, the change in IFN-γ levels showed a very clear trend. The two CAR-T cells in the CFPAC-1 cell group released much more IFN-γ than the control group. The IFN-γ released by the CAR-T cells in the other groups was similar to that in the control group. Furthermore, the IFN-γ release by the CFPAC-1 cell-derived CAR-T cell group was much higher than that in the other two pancreatic cancer cell-derived CAR-T cell groups (P < 0.05). The results are shown in Figure 10.

[0100] Therefore, both CARs we designed and used enable T cells to specifically recognize endogenous target antigens, activate T cells, and exert a killing effect. Because T cell activation produces more cytokines, it not only promotes rapid T cell proliferation in a feedback loop, but also enhances the killing effect of T cells.

[0101] The present invention has been disclosed above with preferred embodiments, which are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. A single-chain antibody fragment targeting KRAS G12V, characterized in that: It includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes VH-CDR1, VH-CDR2, VH-CDR3 and the light chain variable region includes VL-CDR1, VL-CDR2, VL-CDR3; wherein the amino acid sequence of VH-CDR1 is the sequence shown in SEQ ID NO: 3, the amino acid sequence of VH-CDR2 is the sequence shown in SEQ ID NO: 4, and the amino acid sequence of VH-CDR3 is the sequence shown in SEQ ID NO: 5; The amino acid sequence of VL-CDR1 is the sequence shown in SEQ ID NO:8, the amino acid sequence of VL-CDR2 is the sequence shown in SEQ ID NO:9, and the amino acid sequence of VL-CDR3 is the sequence shown in SEQ ID NO:

10.

2. The KRAS G12V-targeting single-chain antibody fragment according to claim 1, characterized in that: The full-length amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 6; or the amino acid sequence shown in SEQ ID NO: 7; The full-length amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 11; Or, the amino acid sequence shown in SEQ ID NO: 12; The heavy chain variable region and the light chain variable region are connected by a connecting region, the connecting region is composed of three tetrapeptide polymers, and the amino acid sequence is shown in SEQ ID NO: 13; Or as shown in SEQ ID NO: 15 or a sequence with at least 80% homology.

3. The KRAS G12V-targeting single-chain antibody fragment according to claim 2, characterized in that: The amino acid sequence of the variable region of the single-chain antibody is as shown in SEQ ID NO: 14 or a sequence with at least 80% homology; 4. A chimeric antigen receptor CAR, characterized in that: It comprises an antigen binding domain, a transmembrane domain and an intracellular signal domain; wherein the antigen domain comprises a signal peptide, a Myc tag and the single-chain antibody fragment shown in claim 1 that recognizes the antigen.

5. A CAR-T cell expressing the chimeric antigen receptor CAR according to claim 4.

6. Use of the KRAS G12V-targeted single-chain antibody fragment according to claims 1-3, the chimeric antigen receptor CAR according to claim 4, and the CAR-T cell according to claim 5 in the preparation of endogenous KRAS G12V mutant pancreatic cancer drugs.

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