Pharmaceutical composition containing NK cells and application of pharmaceutical composition in preparation of antitumor drugs

Abstract

The invention discloses an engineered NK cell and a preparation method thereof. The antitumor activity of the NK cell is enhanced through synergistic modification of a fusion polypeptide and an anti-TIGIT monoclonal antibody. The amino acid sequence of the fusion polypeptide is shown as SEQ ID NO.1. The fusion polypeptide can specifically target tumor cells and activate the proliferation and killing functions of NK cells. And the anti-TIGIT monoclonal antibody further relieves the immunosuppression by blocking a TIGIT-CD155 signal channel. In-vitro experiments show that the killing rate of the combined modified NK cell on various solid tumors is obviously higher than that of the traditional CAR-NK cell. A PDX model shows that the engineered NK cells significantly inhibit tumor growth and enhance NK cell infiltration in a tumor microenvironment. The invention provides a new strategy with high efficiency and low toxicity for immunotherapy of solid tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a pharmaceutical composition containing NK cells and its use in the preparation of anti-tumor drugs. Background Art

[0002] Natural killer (NK) cells are an important part of the innate immune system, capable of directly recognizing and killing tumor cells, and regulating anti-tumor immunity by secreting cytokines (such as IFN-γ). However, in the treatment of solid tumors, NK cells face multiple biological barriers, mainly including:

[0003] Immunosuppression in the tumor microenvironment (TME): The TME highly inhibits the activity of NK cells, and the main mechanisms are as follows: (1) NK cells express a variety of inhibitory receptors on their surface, such as TIGIT, TIM-3, NKG2A, etc. After these receptors bind to ligands (such as CD155, Galectin-9, HLA-E) on the surface of tumor cells or immunosuppressive cells, the degranulation ability and cytotoxicity of NK cells can be significantly reduced. (2) Metabolic competition leads to functional exhaustion: The TME presents a state of high metabolic competition, and the environment of low glucose, low glutamine, and high lactate weakens the energy metabolism of NK cells. (3) Chemokine mismatch leads to insufficient infiltration of NK cells: The expression level of chemokines in tumor tissues is usually low, while the high expression of chemokines recruits regulatory T cells instead, further inhibiting the function of NK cells.

[0004] Limitations of existing NK cell modification strategies: (1) Limitations of single-target modification strategies: For example, the escape rate of NK cells modified with EGFR-specific antibodies in colorectal cancer models is as high as 67%, mainly because of the heterogeneity of tumor antigen expression, resulting in the inability of some tumor cells to be recognized by NK cells. (2) Side effects of traditional cytokine support: Cytokines such as IL-2 or IL-15 can enhance the activity of NK cells, but are prone to cause severe systemic toxicity. (3) Limited efficacy of blocking antibody monotherapy: For example, the TIGIT monoclonal antibody Tiragolumab showed in the trial that the ORR of monotherapy was less than 20%, indicating that only blocking inhibitory signals is not sufficient to significantly enhance the function of NK cells.

[0005] Although immunotherapy strategies targeting NK cells have been continuously developed in recent years, the following key issues still exist in the treatment of solid tumors: (1) It is difficult for a single strategy to overcome the complex immunosuppressive mechanism of TME: simply blocking inhibitory receptors or providing cytokine support cannot simultaneously improve the metabolic adaptability and tumor infiltration ability of NK cells. (2) Tumor heterogeneity leads to inefficient NK cell targeted recognition: Tumor antigen expression is highly heterogeneous, resulting in poor efficacy of traditional targeted antibodies or CAR-NK cells in some patients. (3) The toxicity of cytokine support strategies limits clinical application: systemic IL-2 or IL-15 support therapy often leads to serious side effects, such as immune storm and autoimmune activation. Summary of the invention

[0006] In response to the above challenges, the present invention proposes a peptide-antibody synergistic system based on structural biology design. This system can enhance the survival, infiltration and anti-tumor ability of NK cells in TME. To this end, the present invention provides an engineered NK cell and a method for its construction, the core of which includes:

[0007] 1. Fusion polypeptide (T-MAP): integrating tumor-targeted penetrating peptide (TPP), MMP-2 responsive connecting peptide, IL-21 / IL-15 dual signaling factor and PD-1 trans-co-stimulatory domain to enhance NK cell function through synergistic action;

[0008] 2. Anti-TIGIT monoclonal antibody: relieves immunosuppression in the tumor microenvironment by blocking TIGIT-CD155 inhibitory signals;

[0009] 3. Combined modification strategy: Through membrane-anchored T-MAP and loaded anti-TIGIT monoclonal antibody, multi-dimensional enhancement of NK cell targeting, activation and anti-immune escape capabilities can be achieved.

[0010] Therefore, the present invention discloses a fusion polypeptide in one aspect. The fusion polypeptide is T-MAP, and its amino acid sequence is shown in SEQ ID NO.1.

[0011] Preferably, the nucleotide sequence of the fusion polypeptide of the present invention after codon optimization is as shown in SEQ ID NO.2.

[0012] In one aspect, the present invention also discloses an engineered NK cell, wherein the engineered NK cell membrane anchors the T-MAP and is loaded with an anti-TIGIT monoclonal antibody.

[0013] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TIGIT monoclonal antibody of the present invention are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0014] Preferably, the EC50 of the anti-TIGIT monoclonal antibody of the present invention is 3.5 ± 0.4 ng / mL, and the IC50 is 0.87 ± 0.05 nM.

[0015] In one aspect, the present invention also discloses the use of the engineered NK cells as described above in the preparation of anti-solid tumor drugs.

[0016] The beneficial effects of the present invention are summarized as follows:

[0017] 1. Precise targeting and microenvironment response: T-MAP targets tumor cells through the TPP module (the binding efficiency is increased by 13.2 - 16.5 times), and MMP-2-responsive cleavage ensures the local release of the drug in the tumor, reducing systemic toxicity (the cleavage efficiency ≥ 92%).

[0018] 2. Dual-signal synergistic activation of NK cells: The IL-21 / IL-15 hybrid cytokine promotes the proliferation and cytotoxicity of NK cells. The PD-1 domain blocks the PD-L1 signal and triggers a co-stimulatory signal, significantly enhancing the killing ability of NK cells (the killing rate is increased by 30% compared with traditional CAR-NK).

[0019] 3. Synergistic effect of anti-TIGIT monoclonal antibody: The anti-TIGIT monoclonal antibody (IC50 = 0.87 nM) blocks the TIGIT-CD155 pathway, relieves immunosuppression, and synergizes with T-MAP to increase the killing rate of NK cells against solid tumors to 92.1%.

[0020] 4. High efficiency in anti-tumor and inhibition of metastasis: In the PDX model, the engineered NK cells reduce the tumor weight by 83.1% and the number of metastatic foci by 86.2%, and significantly enhance the infiltration of NK cells into the tumor.

[0021] 5. High safety: The modification process has no significant effect on the survival rate of NK cells (≥ 96%), and has no targeted toxicity to normal cells (the MFI of HUVEC binding is only increased by 1.9 times). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 SDS-PAGE detection results of the fusion polypeptide (T-MAP), where 1 is the fusion polypeptide (T-MAP).

[0023] Figure 2 SDS-PAGE detection results of the anti-TIGIT monoclonal antibody, where 1 is the anti-TIGIT monoclonal antibody. DETAILED DESCRIPTION OF THE INVENTION

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0025] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0026] Example 1: Design, Preparation, and Verification of the Fusion Polypeptide (T-MAP)

[0027] I. Design of the Fusion Polypeptide (T-MAP):

[0028] 1. N-terminal targeting module: A tumor-penetrating peptide (TPP, amino acid sequence: KLAKLAKKLAKLAK) modified from the scorpion toxin BmK CT targets the negatively charged membrane of tumor cells through a cationic structure.

[0029] 2. Microenvironment-responsive module: An MMP-2-cleavable linker peptide (amino acid sequence: PLGLAG).

[0030] 3. Dual-signal core:

[0031] (1) An IL-21 / IL-15 hybrid cytokine, in which the amino acid sequence is: the α-helical region of IL-21 (AA45 - 70) + the IL-15 mutant (T72D indicates that the T at position 72 is mutated to D).

[0032] (2) The PD-1 transcostimulatory domain: Truncated PD-1 (AA62 - 127) is used to bind to PD-L1 on tumor cells and trigger the CD28 signal of NK cells.

[0033] 4. The full-length amino acid sequence of the fusion polypeptide (named T-MAP) after the above design is shown below, and its detailed amino acid sequence is shown in SEQ ID NO.1.

[0034] II. Construction and Expression of the Fusion Polypeptide (T-MAP)

[0035] 1. Gene synthesis and cloning: According to the amino acid sequence of T-MAP (SEQ ID NO.1), a 6His tag was added to its C-terminus for purification, and then codon optimization for Escherichia coli was performed. The optimized nucleotide sequence is shown in SEQ ID NO.2. The optimized sequence was submitted to GenScript for synthesis, and NdeI / XhoI restriction sites were added to both ends for cloning into the pET-28a(+) vector. The pET-28a(+) vector was digested with NdeI / XhoI (37 °C, 2 h), and the linearized vector was recovered. T-MAP was inserted into the vector using T4 DNA ligase (16 °C, 16 h). The ligation product was transformed into E. coli DH5α, and plated on LB-Kan (50 μg / mL) plates and cultured overnight at 37 °C. Single colonies were picked and the inserted fragment was verified by PCR. After confirming the correct clone, sequencing was performed, and the sequencing result matched the target sequence 100% without mutations.

[0036] 2. Recombinant protein expression: The correctly identified pET-28a(+)-T-MAP plasmid was transformed into E. coli BL21(DE3), single colonies were picked and cultured to OD600 = 0.6. IPTG (0.5 mM) was added and induced at 16 °C for 12 h. Soluble expression was detected by SDS-PAGE. The cells were collected (6000 rpm, 10 min), lysed by sonication (power 300 W, pulse 10 s on / 10 s off, for a total of 30 min), centrifuged at 12,000 rpm for 20 min, and the supernatant (soluble fraction) was taken for SDS-PAGE analysis. The results showed that the protein was expressed in the supernatant.

[0037] 3. Purification of recombinant protein (purification by Ni-NTA affinity chromatography): The binding buffer was 20 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 8.0; the elution buffer was 300 mM imidazole, pH 8.0; imidazole was removed by dialysis, filtered through a 220 nm filter to sterilize, and aliquoted and stored at -80 °C for later use.

[0038] 4. SDS-PAGE detection of the purified recombinant protein showed that ( Figure 1 ) the size of T-MAP was approximately 32 kDa, which was consistent with the theoretical value, and its purity was above 95%.

[0039] II. Verification of the fusion polypeptide (T-MAP)

[0040] 1. Detection of tumor cell targeting

[0041] (1) Cell lines: A549 (lung cancer), MCF-7 (breast cancer), PANC-1 (pancreatic cancer), HUVEC (normal vascular endothelial cells), 293T (normal renal cells).

[0042] (2) Cell culture: RPMI-1640 + 10% FBS, 37°C, 5% CO2.

[0043] (3) T-MAP-FITC labeling, removing unbound dye (dialysis, MWCO 3 kDa).

[0044] (4) 1×10 6 cells were incubated with T-MAP-FITC (100 nM) for 30 min. After washing with PBS, the MFI (Mean Fluorescence Intensity) was detected by flow cytometry.

[0045] (5) The experimental results showed (Table 1) that in the cell lines of A549 (lung cancer), MCF-7 (breast cancer), and PANC-1 (pancreatic cancer), the binding ability of T-MAP was significantly enhanced, and the MFI increased by 13.2 - 16.5 times, indicating its targeting to tumor cells. In HUVEC (normal vascular endothelial cells) and 293T (normal renal cells), the increase in MFI was not significant, indicating that T-MAP did not bind to normal cells non-specifically. The competitive inhibition experiment (free KLAKLAK peptide) could significantly reduce the binding ability of T-MAP, and the MFI decreased by about 70%, proving that the targeting effect was mainly mediated by the TPP module.

[0046] Table 1 Detection results of tumor cell targeting

[0047]

[0048] 2. Verification of MMP-2 responsiveness

[0049] (1) Experimental procedure: 200 nM MMP-2 was mixed with 10 μg T-MAP protein and incubated at 37°C (0, 1, 2, 4, 6 h). The cleavage products were analyzed by HPLC (detection wavelength 220 nm).

[0050] (2) The experimental results showed that HPLC analysis indicated that the cleavage efficiency of T-MAP reached 92% within 4 hours under the action of MMP-2.

[0051] IV. Summary

[0052] 1. Strong targeting: T-MAP can specifically recognize tumor cells with little effect on normal cells.

[0053] 2. MMP-2 Responsive Release: T-MAP can be activated in the MMP-2-enriched TME, ensuring drug action at the tumor site and reducing systemic side effects.

[0054] 3. Enhancement of NK Cell Anti-tumor Activity: The IL-21 / IL-15 dual signals of T-MAP can promote NK cell proliferation and cytotoxicity, and enhance anti-tumor ability in the immune tolerance environment.

[0055] 4. Overcoming PD-L1 Immune Escape: The PD-1 trans-costimulation in T-MAP can enhance T cell immune responses, thereby overcoming tumor PD-L1-mediated immune suppression.

[0056] Example 2: Engineering Modification of NK Cells

[0057] 1. Isolation and Culture of NK Cells

[0058] (1) PBMC Extraction: Collect 50 mL of anticoagulated blood from healthy volunteers. Isolate PBMCs using Ficoll-Paque gradient centrifugation (400 g, 25 min). Wash the cells twice with PBS buffer (300 g, 10 min), and discard the supernatant.

[0059] (2) CD56 + NK Cell Sorting: Resuspend the cells in 300 μL of MACS buffer (PBS + 0.5% BSA + 2 mM EDTA). Add 100 μL of CD56 microbeads and incubate at 4°C in the dark for 15 min. After filtering the cell suspension (30 μm filter), perform sorting through an LS magnetic column. Collect CD56 + cells, wash twice with PBS, and adjust to 1×10 6 cells / mL. Use a flow cytometer to detect cell purity, and the result shows that the CD56 + purity > 95%. This indicates successful preparation of CD56 + NK cells, and expand the culture of these cells for subsequent experiments.

[0060] 2. Membrane Anchoring of T-MAP and Loading of Anti-TIGIT Monoclonal Antibody (Prepared in Example 5)

[0061] (1) Preparation of T-MAP Complex: Mix the T-MAP (1 mg / mL) prepared in Example 1 with DSPE-PEG2000-MAL (10 mg / mL) at a molar ratio of 1:5 in PBS buffer and incubate at 4°C for 2 h. Then, under 4°C conditions, dialyze in PBS buffer using a 10 kDa filter membrane for 4 h to remove free DSPE-PEG2000-MAL.

[0062] (2) NK cell membrane anchoring, experimental grouping (1×10 7 cells per group): WT-NK (wild-type NK cells) (unmodified), T-MAP-NK (modified only with T-MAP), TIGIT-NK (loaded only with anti-TIGIT monoclonal antibody), T-MAP+TIGIT-NK (combined modification).

[0063] (3) Membrane anchoring steps of T-MAP (applicable to T-MAP-NK and T-MAP+TIGIT-NK): Cells were resuspended in 500 μL PBS (without Ca 2+ , Mg 2+ ). The prepared T-MAP-DSPE complex was added at a molar ratio of 1:500, and incubated at room temperature for 30 min. Centrifuge at low speed (150 g, 5 min) to remove unbound T-MAP, and wash twice with PBS. Detect the expression level of T-MAP by flow cytometry (His-Tag-FITC).

[0064] (4) Loading steps of anti-TIGIT monoclonal antibody: Applicable to the above TIGIT-NK and T-MAP+TIGIT-NK: Cells were resuspended in 500 μL PBS (pH 6.0). Add anti-TIGIT monoclonal antibody (10 μg / mL), and incubate at 4 °C for 1 h. Centrifuge at low speed (150 g, 5 min), and wash twice with PBS. Detect the antibody binding rate by flow cytometry (anti-human IgG-FITC).

[0065] 3. Experimental tests and results

[0066] (1) Anchoring efficiency of T-MAP on NK cell membrane: The results of flow cytometry detection showed that T-MAP was successfully anchored on T-MAP-NK and T-MAP+TIGIT-NK cells. The T-MAP molecule loading amounts in the two groups were 1.7×10 4 ± 2.0×10 3 molecules / cell and 1.8×10 4 ± 2.1×10 3 molecules / cell respectively, while no T-MAP signal was detected in the WT-NK and TIGIT-NK groups, indicating that the modification process was effective and stable.

[0067] (2) Binding efficiency of anti-TIGIT monoclonal antibody: In the TIGIT-NK and T-MAP+TIGIT-NK groups, the binding rates of anti-TIGIT monoclonal antibody were 83.2% ± 4.3% and 83.7% ± 4.5% respectively, while no TIGIT-NK signal was detected in the WT-NK and T-MAP-NK groups, indicating that the antibody loading process was not affected by T-MAP modification, and NK cells had a high binding ability to the antibody.

[0068] (3) Cell viability: The Annexin V / PI double staining assay was used to evaluate the effect of the modification process on NK cells. The results showed (Table 2) that the viability of NK cells in each group was above 96%, and there was no significant decrease compared with the WT-NK group (p > 0.05), indicating that the T-MAP modification and anti-TIGIT monoclonal antibody loading process did not cause NK cell apoptosis and the cell state remained good.

[0069] Table 2 Detection results of cell viability

[0070]

[0071] (4) Evaluation of cytotoxic function: The 51Cr release assay was used to evaluate the killing ability of NK cells in different modified groups against K562 tumor cells. The results showed (Table 3) that at different E:T ratios (1:1, 5:1, 10:1), the killing ability of the T-MAP+TIGIT-NK group was significantly higher than that of the single modification groups (T-MAP-NK or TIGIT-NK), indicating that the synergistic effect of the two could significantly enhance the anti-tumor effect of NK cells.

[0072] Table 3 Detection results of cytotoxic function

[0073]

[0074] Example 3: In vitro killing assay

[0075] 1. Target cell preparation

[0076] Cell line: MDA-MB-231 cells (PD-L1 + HER2 - ).

[0077] Fluorescent labeling: The pLV-mCherry vector was used to construct a fluorescently labeled stable cell line. After lentiviral infection, cells were screened with 2 μg / mL puromycin for 7 days, and cells with a fluorescent positive rate > 95% were selected by flow cytometry for the experiment.

[0078] Cell culture: Cells were cultured in RPMI-1640 + 10% FBS in a 37 °C, 5% CO2 cell culture incubator.

[0079] 2. Effector cell groups

[0080] Experimental group: T-MAP+TIGIT-NK cells

[0081] Control groups: Conventional CAR-NK (targeting PD-L1), WT-NK cells (unmodified), T-MAP-NK (only modified with T-MAP), TIGIT-NK (only loaded with anti-TIGIT monoclonal antibody).

[0082] 3. In vitro killing experiment

[0083] Cell seeding: 5×10 4 MDA-MB-231-mCherry cells per well were seeded into a 96-well plate and cultured adherently for 12 hours.

[0084] Effector-to-target ratio: Three effector cell:target cell ratios of 1:1, 5:1, and 10:1 were set.

[0085] Imaging monitoring: The IncuCyte S3 live cell imaging system was used to record the fluorescence intensity of tumor cells every 2 hours.

[0086] Data analysis: Based on the attenuation of the mCherry fluorescence signal, the killing rate was calculated.

[0087] 4. The killing rate results at different effector-to-target ratios showed (Table 4) that the killing rate of T-MAP+TIGIT-NK cells was much higher than that of traditional CAR-NK cells (p<0.001). Especially at 48 h, the killing rate reached 92.1%, showing stronger anti-tumor ability. Both T-MAP modification and anti-TIGIT monoclonal antibody loading could individually enhance the killing ability of NK cells, but the killing rate was further increased after the combination of the two (T-MAP+TIGIT-NK>

[0088] T-MAP-NK / TIGIT-NK), demonstrating a synergistic enhancement effect between the two. Compared with WT-NK cells, the killing ability of T-MAP+TIGIT-NK cells was increased by about 3 times, indicating that this strategy could significantly enhance the tumor-targeted killing effect of NK cells. T-MAP+TIGIT-NK cells had extremely strong anti-tumor effects at a high effector-to-target ratio (10:1) and still showed good killing activity at lower effector-to-target ratios (1:1, 5:1), with good clinical application potential.

[0089] Table 4 Detection results of killing rate at different effector-to-target ratios (10:1)

[0090]

[0091] 5. Experimental conclusion

[0092] The results of this experiment proved that the combined modification of T-MAP and anti-TIGIT monoclonal antibody could significantly enhance the tumor killing ability of NK cells. Its killing effect was better than that of traditional CAR-NK cells, and stable and potent anti-tumor activity was demonstrated at different effector-to-target ratios. This strategy could provide new ideas for the treatment of refractory solid tumors such as triple-negative breast cancer with NK cells and had good clinical translation potential.

[0093] Example 4: PDX model verification

[0094] 1. Establishment of PDX model: Select tissue samples from a mouse model of hepatocellular carcinoma (HCC) (HCC-03, PD-L1 + ), cut the tissue into tumor blocks approximately 2×2×2 mm 3 in size, wrap them with Matrigel, and transplant them into the liver of NSG mice (NOD-scid IL2Rγ - / - ). Monitor the health status of the mice after the operation, measure the body weight every 3 days, and confirm the tumor growth using a small animal imaging system 3 weeks after transplantation.

[0095] 2. Experimental grouping: A total of four groups were divided, with 6 NSG mice in each group:

[0096] (1) Engineered NK cell group (T-MAP+TIGIT-NK prepared above): Inject 5×10 6 engineered NK cells via the tail vein once a week for a total of 3 times.

[0097] (2) Conventional NK cell group (WT-NK): Inject 5×10 6 non-engineered NK cells via the tail vein once a week for a total of 3 times.

[0098] (3) PBS control group: Inject an equal volume of PBS via the tail vein once a week for a total of 3 times.

[0099] (4) Blank control group: No tumor transplantation, only used as a healthy control.

[0100] 3. Endpoint detection

[0101] (1) Tumor weight measurement: On the 21st day of cell therapy, sacrifice the mice, remove the tumor tissue and weigh it.

[0102] (2) Metastasis focus statistics: Observe the metastasis foci in the liver and lungs, and confirm them by HE staining.

[0103] (3) NK cell infiltration detection: Detect the density of CD56 + NK cells in the tumor tissue by immunofluorescence staining.

[0104] 4. Result analysis

[0105] (1) Engineered NK cells significantly inhibit tumor growth: Engineered NK cells further reduce the tumor weight compared with conventional NK cells (P<0.01), indicating that the engineering strategy significantly enhances the anti-tumor effect. The specific data are shown in Table 5.

[0106] Table 5 Tumor statistical results

[0107]

[0108] (2) Significantly reduced metastatic foci: The number of metastatic foci in the engineered NK cell group was significantly lower than that in the conventional NK cell group (P<0.01), indicating that engineered NK cells were more effective in inhibiting distal metastasis. The specific results are shown in Table 6.

[0109] Table 6 Statistical results of metastatic foci

[0110]

[0111] (3) Enhanced NK cell infiltration: The infiltration of NK cells in the tumor tissue of the engineered NK cell group was significantly higher than that in the conventional NK cell group (P<0.01), indicating that engineered NK cells had stronger tumor tropism. The specific results are shown in Table 7.

[0112] Table 7 Detection results of NK cell infiltration

[0113]

[0114] 5. Conclusion

[0115] Compared with conventional NK cells, engineered NK cells could more effectively inhibit the tumor growth of the PDX liver cancer model, and the inhibition rate increased by 33.9%. The inhibitory effect of engineered NK cells on distal metastatic foci was more significant, with a reduction of 86.2%, while conventional NK cells only reduced by 43.7%. Engineered NK cells significantly increased the infiltration density of NK cells in the tumor tissue, which was 2.5 times that of conventional NK cells, indicating that the engineering strategy improved the tumor tropism of NK cells.

[0116] The results of this study showed that the combination of T-MAP modification and anti-TIGIT antibody loading synergistically enhanced the anti-tumor activity of NK cells, providing a new strategy for liver cancer immunotherapy.

[0117] Example 5: Preparation of anti-TIGIT monoclonal antibody

[0118] I. Preparation of monoclonal antibody

[0119] 1. Immunization protocol: Select 6-8-week-old SPF-grade Balb / c mice and immunize them according to the following protocol:

[0120] (1) Primary immunization: Recombinant human TIGIT protein (ab227414) (50 μg / mouse) + Freund's complete adjuvant (1:1), subcutaneous injection of 0.1 mL / mouse;

[0121] (2) Booster immunization (on days 14, 28, and 42): Recombinant human TIGIT protein (50 μg / mouse) + Freund's incomplete adjuvant (1:1), subcutaneous injection of 0.1 mL / mouse;

[0122] (3) Final immunization (day 49): Intravenous injection of recombinant human TIGIT protein (100 μg / animal) in PBS solution (0.1 mL / animal).

[0123] 2. Cell fusion and screening: Three days after the final immunization, spleen cells were taken and fused with SP2 / 0 myeloma cells at a ratio of 5:1 (mediated by PEG1500). Then, hybridoma clones were screened using HAT selection medium, and clones that could bind TIGIT efficiently were screened by ELISA. After this screening, a good hybridoma cell was obtained, which was expanded in culture and then stored in liquid nitrogen for future use.

[0124] 3. Monoclonal antibody expression and purification

[0125] (1) Recombinant monoclonal antibody sequence screening and optimization: The variable region genes of the screened hybridoma clones were amplified by RT-PCR, optimized for humanized sequences, and then cloned into the human IgG1-Fc framework to construct an expression vector. The expression vector was transiently transfected into HEK293F to express the antibody, and the antibody was purified using Protein A affinity chromatography. The purified antibody was detected by SDS-PAGE, and the results showed ( Figure 2 ) that the purity of the monoclonal antibody was above 95%. The amino acid sequences of the heavy chain variable region and light chain variable region of the humanized optimized anti-TIGIT monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0126] II. Functional detection of monoclonal antibody

[0127] 1. ELISA binding experiment: Recombinant human TIGIT protein was coated on an ELISA plate (100 ng / well), and then gradient-diluted anti-TIGIT monoclonal antibody (0.01 - 500 ng / mL) was added. Incubate at 37°C for 1 hour and wash 3 times; add a 5000-fold diluted HRP-labeled anti-human IgG secondary antibody and incubate at 37°C for 30 minutes; after washing, add 0.1 mL of TMB chromogenic solution and incubate at 37°C for 10 minutes; add 0.1 mL of 2M H2SO4 termination solution and immediately measure the OD450 nm value. After calculation and analysis (Table 8), the EC50 value of the anti-TIGIT monoclonal antibody of the present invention is lower, indicating that the anti-TIGIT monoclonal antibody of the present invention has a stronger binding ability.

[0128] Table 8 Results of ELISA binding experiment

[0129]

[0130] 2. Competitive binding assay: Jurkat-TIGIT+ cells (stably expressing TIGIT) were used to detect the competitive inhibition ability of anti-TIGIT antibodies against the binding of TIGIT to hCD155 by flow cytometry. After incubating CD155-Fc with anti-TIGIT monoclonal antibodies at different concentrations, the binding amount of CD155-Fc on the cell surface was detected. Through calculation and analysis (Table 9), the anti-TIGIT monoclonal antibody of the present invention has a stronger ability to inhibit CD155 binding and a better effect of blocking TIGIT signals.

[0131] Summary of experimental results in Table 9

[0132]

[0133] The above results indicate that the anti-TIGIT monoclonal antibody prepared by the present invention has good sensitivity and specificity, is better than the commercially available monoclonal antibody, and has better application prospects.

[0134] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A fusion polypeptide, characterized in that, The fusion polypeptide is T-MAP, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The fusion polypeptide according to claim 1, wherein The nucleotide sequence of the fusion polypeptide after codon optimization is shown in SEQ ID NO.

2.

3. An engineered NK cell, characterized in that, The engineered NK cell membrane anchors the T-MAP described in claim 1 and loads an anti-TIGIT monoclonal antibody.

4. The NK cell according to claim 3, wherein The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TIGIT monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

5. The NK cell according to claim 3, wherein The EC50 of the anti-TIGIT monoclonal antibody is 3.5 ± 0.4 ng / mL, and the IC50 is 0.87 ± 0.05 nM.

6. Use of the engineered NK cell according to claim 3 in the preparation of an anti-solid tumor drug.

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