Immune T cell preparation and application thereof in tumor treatment

Abstract

The invention provides an immune T cell preparation and application thereof in tumor treatment, and belongs to the technical field of cell biology. According to the method, human peripheral blood CD8 positive T cells are used as starting cells, and sequence-improved fusion peptide DerB-MA-1 is used for in-vitro induction. Experimental results show that T cells pretreated by DerB-MA-1 show extremely high killing activity under a normal oxygen condition, and the killing activity is remarkably superior to that of other fusion peptides. More importantly, under the hypoxia condition of simulating the tumor microenvironment, the preparation c can still maintain the potent killing rate. Therefore, the invention successfully overcomes the technical bottleneck of function decline of T cells in an anoxic environment, realizes obvious breakthrough of T cell functions, and has important clinical application value in immune cell therapy of solid tumors such as melanoma and the like.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, and in particular relates to an immune T-cell preparation and its application in tumor treatment. Background Technology

[0002] Malignant tumors are among the leading diseases threatening human life and health. With an aging population and changing lifestyles, their incidence and mortality rates continue to rise. Traditional cancer treatments, including surgery, radiotherapy, and chemotherapy, while extending patient survival to some extent, still have unsatisfactory overall efficacy and are accompanied by severe toxic side effects. In recent years, T-cell therapy has made groundbreaking progress, demonstrating extremely high complete remission rates in various hematological malignancies and is considered one of the most promising methods for curing malignant tumors. However, when this therapy is applied to solid tumors, its efficacy significantly decreases, becoming the biggest bottleneck currently facing tumor immunotherapy.

[0003] The high heterogeneity and immunosuppressive properties of the tumor microenvironment (TME) are major reasons for the failure of T-cell therapy. Among these factors, the widespread hypoxia within tumor tissue is considered a key limiting factor. Due to abnormal tumor proliferation leading to vascular structural and functional abnormalities, large areas of hypoxia often form within solid tumors. Studies have shown that hypoxia inhibits T-cell activation and effector function. Therefore, finding a solution that addresses the root cause of T-cell dysfunction while maintaining efficient proliferation and killing function in a hypoxic microenvironment is a pressing issue. Summary of the Invention

[0004] The purpose of this invention is to provide an immune T-cell preparation and its application in tumor treatment.

[0005] In a first aspect, the present invention provides a method for preparing an immune T cell preparation with tolerance to tumor hypoxic microenvironment and high killing activity, wherein the immune T cell preparation is an immune T cell preparation pretreated with the fusion peptide DerB-MA-1, the amino acid sequence of which is shown in SEQ ID NO.3.

[0006] Preferably, the preparation method of the immune T cell preparation is as follows: (1) Isolate CD8-positive T cells derived from human peripheral blood; (2) Prepare an inactivated A375 cell suspension using mitomycin C; (3) Mix CD8 positive T cells with inactivated A375 cells at a ratio of 5:1, add the fusion peptide DerB-MA-1, and add IL-7 and IL-15 to culture for 3 days; (4) Add IL-2 and incubate until day 7; (5) Replace half of the old culture medium, add fresh culture medium and IL-2, and culture until day 10; (6) Add A375 cells at a ratio of 10:1, along with the fusion peptides DerB-MA-1 and IL-2. Culture until day 14 to obtain the corresponding immune T cell preparation.

[0007] Preferably, in step (3), the seeding density of the CD8 positive T cells is 1×10⁻⁶. 6 cells / mL; The concentration of the fusion peptide DerB-MA-1 is 5 μg / mL, the concentration of IL-7 is 10 ng / mL, and the concentration of IL-15 is 50 ng / mL.

[0008] Preferably, in step (4), the concentration of IL-2 is 50 IU / mL; In step (5), the concentration of IL-2 is 150 IU / mL; In step (6), the concentration of the fusion peptide DerB-MA-1 is 5 μg / mL, and the concentration of IL-2 is 150 IU / mL.

[0009] Secondly, the present invention provides an immune T cell preparation with tolerance to tumor hypoxic microenvironment and high killing activity, wherein the immune T cell preparation is prepared by the above-described method.

[0010] Thirdly, the present invention provides the application of a fusion peptide in the preparation of a drug that enhances the tolerance of T cells to the tumor hypoxic microenvironment, wherein the fusion peptide is the fusion peptide DerB-MA-1, and the amino acid sequence of the fusion peptide DerB-MA-1 is shown in SEQ ID NO.3.

[0011] Preferably, the tumor is a melanoma.

[0012] Fourthly, the present invention provides the application of a fusion peptide in the preparation of a drug that enhances the tumor cell killing ability of T cells, wherein the fusion peptide is the fusion peptide DerB-MA-1, and the amino acid sequence of the fusion peptide DerB-MA-1 is shown in SEQ ID NO.3.

[0013] Preferably, the tumor cells are melanoma cells A375.

[0014] The beneficial effects of this invention are: The core inventive aspect of this invention lies in the unique construction and sequence modification of the fusion peptide DerB-MA-1. This fusion peptide not only achieves a general enhancement of T cell function but also produces unexpected and significant technical effects: extremely high hypoxia tolerance, an effect significantly superior to other fusion peptides. Therefore, the immune T cell preparation of this invention has extremely high clinical application prospects and inventiveness. The high specific activation and hypoxia tolerance exhibited by the preparation addresses the pain point of poor efficacy of current cell therapies for treating the core regions of solid tumors. Attached Figure Description

[0015] Figure 1 The results show the interferon-γ secretion levels of each group of immune T cell preparations under normoxic conditions. Figure 2 The results show the interferon-γ secretion levels of each group of immune T cell preparations under hypoxic conditions. Figure 3 The results show the cell killing rate of each group of immune T cell preparations under normoxic conditions; Figure 4 The results show the cell killing rate of each group of immune T cell preparations under hypoxic conditions. Detailed Implementation

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0017] Existing research has shown that many antimicrobial peptides have immunomodulatory activity and can act as molecular adjuvants. Therefore, this invention attempts to prepare a fusion peptide by combining a modified peptide of Dermaseptin B2 with MART−1 to solve the problem of reduced immune activity of T cells under hypoxic conditions.

[0018] The fusion peptide sequence involved in this invention is as follows: Fusion peptide Der-MA-1 Peptide sequence: ALWKTLLKKVLKGLGAVLSKVLGGLGALLEAGKL- GGGGSGGGGSGGGGS-AAGIGILTV; Fusion peptide DerA-MA-1 Peptide sequence: KLWKRLLKKVLKRLGARLSKVLGGLGARLEAGKL- GGGGSGGGGSGGGGS-AAGIGILTV; Fusion peptide DerB-MA-1 Peptide sequence: ALWKTSLKKVLKGLGAVLSKSLGGLGASLEAGKL- GGGGSGGGGSGGGGS-AAGIGILTV; Fusion peptide DerC-MA-1 Peptide sequence: ALWKRLLKKVLKGLGAVLSKVLGGLGAYLEAGKK- GGGGSGGGGSGGGGS-AAGIGILTV; The aforementioned fusion peptide was synthesized using a solid-phase peptide synthesis method on a fully automated peptide synthesizer by Hefei Guotai Biotechnology Co., Ltd., with a purity >95%.

[0019] Example 1 Obtain CD8 positive T cells (1) Draw 50 mL of fasting peripheral venous blood from our healthy volunteers. Add the blood to a 50 mL centrifuge tube at a volume of 10 mL per tube, add an equal volume of PBS, and mix gently. (2) Take another new 50 mL centrifuge tube, add 15 mL of room temperature Ficoll to each tube, slowly add the diluted blood to the surface of the Ficoll liquid, centrifuge at 800g×30 min×20℃ with the highest acceleration and 0 deceleration; (3) After centrifugation, carefully aspirate the white film layer and transfer it into a 50mL centrifuge tube; (4) Add PBS to 50 mL, centrifuge at 400 g × 8 min × 4℃, discard the supernatant (leave about 5 mL of residual liquid), add 45 mL of PBS again, centrifuge at 400 g × 8 min × 4℃, and resuspend in 5 mL of MACS buffer. (5) After cell counting, take 1×10 8 Transfer PBMC to a centrifuge tube and centrifuge at 400g for 5 min. (6) After centrifugation, aspirate the supernatant, add 80 μL of pre-cooled MACS buffer to resuspend, add 20 μL of CD8 MicroBeads, mix gently, and then incubate in an ice box for 15 min. (7) Add 4 mL of pre-cooled MACS buffer, centrifuge at 400 g for 5 min, and then aspirate the supernatant. (8) Add 500 μL of pre-cooled MACS buffer and resuspend completely by pipetting 10 times. (9) The LS column was mounted on a QuadroMACS magnet. After washing once with 3 mL of MACS buffer, 500 μL of cell suspension was added to the column at once and allowed to flow out naturally. (10) After washing the column three times with 3 mL of pre-cooled MACS buffer, remove the LS column, place it on a clean 15 mL centrifuge tube, add 5 mL of pre-cooled MACS buffer, and push it to the bottom quickly using the matching stopcock. (11) Add 5 mL of pre-cooled MACS buffer again and repeat the push once more to obtain CD8 positive T cells; (12) After centrifugation at 300 g × 8 min × 4℃, discard the supernatant and resuspend in 2 mL of RPMI 1640 containing 10% fetal bovine serum for later use and freezing.

[0020] Example 2 Preparation of immune T cell preparations a (1) After digesting A375 cells with trypsin, resuspend them in serum-free culture medium, add 25 μg / mL mitomycin C for 45 minutes, wash thoroughly with PBS 3 times to obtain an inactivated A375 cell suspension. (2) Adjust the CD8 positive T cells prepared in Example 1 to 1×10 6 The cells / mL density was mixed with inactivated A375 cells at a ratio of 5:1 and seeded into T cell culture flasks; (3) Add 5 μg / mL Der-MA-1, and at the same time add 10 ng / mL IL-7 and 50 ng / mL IL-15, and incubate in a 37℃ incubator for 3 days; (4) Add 50 IU / mL IL-2 and continue culturing until day 7; (5) Replace half of the old culture medium, add fresh culture medium and 150 IU / mL IL-2, and culture until day 10; (6) Add inactivated A375 cells at a ratio of 10:1, along with 5 μg / mL Der-MA-1 and 150 IU / mL IL-2. Culture for 14 days to obtain immune T cell preparation a.

[0021] Example 3 Preparation of immune T cell preparations b (1) After digesting A375 cells with trypsin, resuspend them in serum-free culture medium, add 25 μg / mL mitomycin C for 45 minutes, wash thoroughly with PBS 3 times to obtain an inactivated A375 cell suspension. (2) Adjust the CD8 positive T cells prepared in Example 1 to 1×10 6 The cells / mL density was mixed with inactivated A375 cells at a ratio of 5:1 and seeded into T cell culture flasks; (3) Add 5 μg / mL DerA-MA-1, and at the same time add 10 ng / mL IL-7 and 50 ng / mL IL-15, and incubate in a 37℃ incubator for 3 days; (4) Add 50 IU / mL IL-2 and continue culturing until day 7; (5) Replace half of the old culture medium, add fresh culture medium and 150 IU / mL IL-2, and culture until day 10; (6) Add inactivated A375 cells at a ratio of 10:1, along with 5 μg / mL Der-MA-1 and 150 IU / mL IL-2. Culture for 14 days to obtain immune T cell preparation b.

[0022] Example 4 Preparation of immune T cell preparations c (1) After digesting A375 cells with trypsin, resuspend them in serum-free culture medium, add 25 μg / mL mitomycin C for 45 minutes, wash thoroughly with PBS 3 times to obtain an inactivated A375 cell suspension. (2) Adjust the CD8 positive T cells prepared in Example 1 to 1×10 6 The cells / mL density was mixed with inactivated A375 cells at a ratio of 5:1 and seeded into T cell culture flasks; (3) Add 5 μg / mL DerB-MA-1, and at the same time add 10 ng / mL IL-7 and 50 ng / mL IL-15, and incubate in a 37℃ incubator for 3 days; (4) Add 50 IU / mL IL-2 and continue culturing until day 7; (5) Replace half of the old culture medium, add fresh culture medium and 150 IU / mL IL-2, and culture until day 10; (6) Add inactivated A375 cells at a ratio of 10:1, along with 5 μg / mL Der-MA-1 and 150 IU / mL IL-2. Culture for 14 days to obtain immune T cell preparation c.

[0023] Example 5 Preparation of immune T cell preparations d (1) After digesting A375 cells with trypsin, resuspend them in serum-free culture medium, add 25 μg / mL mitomycin C for 45 minutes, wash thoroughly with PBS 3 times to obtain an inactivated A375 cell suspension. (2) Adjust the CD8 positive T cells prepared in Example 1 to 1×10 6 The cells / mL density was mixed with inactivated A375 cells at a ratio of 5:1 and seeded into T cell culture flasks; (3) Add 5 μg / mL DerC-MA-1, and simultaneously add 10 ng / mL IL-7 and 50 ng / mL IL-15, and incubate in a 37℃ incubator for 3 days; (4) Add 50 IU / mL IL-2 and continue culturing until day 7; (5) Replace half of the old culture medium, add fresh culture medium and 150 IU / mL IL-2, and culture until day 10; (6) Add inactivated A375 cells at a ratio of 10:1, along with 5 μg / mL Der-MA-1 and 150 IU / mL IL-2. Culture for 14 days to obtain immune T cell preparation d.

[0024] Example 6 Interferon-γ (IFN-γ) is a core indicator for evaluating cytotoxic T lymphocytes. Therefore, this invention tested the interferon-γ secretion capacity of immune T cells treated with different fusion peptides.

[0025] (1) Arrange A375 cells at a ratio of 1×10⁶ cells per well. 5 Cells / well (100 μL) were seeded into 96-well plates and incubated overnight at 37°C; (2) The immune T cell preparations a, b, c, and d harvested in Examples 2-5, as well as ordinary untreated T cells, were adjusted to 1×10⁻⁶ using RPMI 1640 complete medium containing 100 IU / mL IL-2. 6 Cell suspension with cells / mL; (3) Discard the A375 medium and add cells according to the following groups: Control group: 100 μL of standard T cell preparation was added; Formulation a group: 100 μL of immune T cell formulation a was added; Formulation b group: Add 100μL of immune T cell formulation b; Formulation C group: Add 100μL of immune T cell formulation c; Group d: Add 100 μL of immune T cell preparation d; (4) Place the 96 plates in a normal oxygen incubator and a pre-equilibrated 1% O2 hypoxic incubator respectively, and continue to incubate for 48 hours; (5) After the culture is completed, the supernatant is aspirated, centrifuged at 300g for 5 minutes to remove cell debris, and the supernatant is diluted 5 times. The IFN-γ content is detected using an IFN-γ ELISA kit, and the actual secretion amount is calculated based on the dilution factor.

[0026] Results of IFN-γ secretion under normoxic conditions are as follows Figure 1As shown, the IFN-γ secretion level in the control group was 168±23 pg / mL, the IFN-γ secretion level in the normotropic preparation a group was 843±87 pg / mL, the IFN-γ secretion level in the normotropic preparation b group was 1257±100 pg / mL, the IFN-γ secretion level in the normotropic preparation c group was 2979±251 pg / mL, and the IFN-γ secretion level in the normotropic preparation d group was 2247±221 pg / mL.

[0027] Results of IFN-γ secretion under hypoxic conditions are as follows Figure 2 As shown, the IFN-γ secretion level in the hypoxia control group was 61±7 pg / mL, the IFN-γ secretion level in hypoxia agent a group was 207±27 pg / mL, the IFN-γ secretion level in hypoxia agent b group was 296±24 pg / mL, the IFN-γ secretion level in hypoxia agent c group was 1925±130 pg / mL, and the IFN-γ secretion level in hypoxia agent d group was 589±64 pg / mL.

[0028] The results above show that, under normoxic conditions, the IFN-γ secretion levels of immune T cells induced by the four fusion peptides were significantly higher than those of untreated CD8-positive T cells after recognizing A375 tumor cells, indicating that the fusion peptides of the present invention all possess strong immunomodulatory activity. Among them, the IFN-γ secretion level of formulation c was significantly better than that of formulations a, b, and d, demonstrating its strongest ability to promote T cell effector function. This may be because the modified fusion peptide DerB-MA-1 can effectively regulate the metabolic remodeling of T cells, enabling them to function efficiently even under hypoxic conditions.

[0029] Under 1% hypoxia conditions simulating the tumor microenvironment, the IFN-γ secretion levels of formulations a, b, and d plummeted to 23.5%–26.2% of normoxic levels, while formulation c (DerB-MA-1) maintained a high secretion level with a hypoxia retention rate as high as 64.6%. This indicates that the sequence-modified DerB-MA-1 not only has a more significant ability to promote efficient IFN-γ secretion by T cells, but also effectively enhances the tolerance of T cells under hypoxic conditions. In contrast, the fusion peptides obtained by other modification methods have a poorer ability to improve hypoxia tolerance.

[0030] Example 6 (1) Mix A375 cells at a density of 2.5 × 10⁶ cells per well. 4 Cells / well (100 μL) were seeded into 96-well plates and incubated overnight at 37°C; (2) The immune T cell preparations a, b, c, and d harvested in Examples 2-5, as well as ordinary untreated T cells, were adjusted to 1×10⁻⁶ using RPMI 1640 complete medium containing 100 IU / mL IL-2. 6 Cell suspension with cells / mL; (3) Discard the A375 medium and treat according to the following groups (set up 6 parallel replicates): Target cell spontaneous LDH release group: only 100 μL of RPMI 1640 complete medium was added for background subtraction; Target cell maximum LDH release group: Add 10 μL Lysis Solution 45 minutes before the end of the experiment; Effector cell spontaneous LDH group: 100 μL of ordinary T cell preparation or immune T cell preparation a, b, c, d were added to cell wells that did not contain A375; Control group: 100 μL of standard T cell preparation was added; Formulation a group: 100 μL of immune T cell formulation a was added; Formulation b group: Add 100μL of immune T cell formulation b; Formulation C group: Add 100μL of immune T cell formulation c; Group d: Add 100 μL of immune T cell preparation d; (4) The 96 plates were placed in a normal oxygen incubator and a pre-equilibrated 1% O2 hypoxic incubator and cultured for 24 hours respectively. (5) After the culture is completed, aspirate the supernatant, centrifuge at 300g for 5 minutes to remove cell debris, and add 50μL of supernatant to each well of a new 96-well plate; (6) Add 50 μL of freshly prepared Working Solution to each well and react at room temperature in the dark for 30 min; (7) Add 50 μL of Stop Solution to each well and immediately read the OD value at 490 nm to calculate the kill rate.

[0031] The cell killing rates of T cells obtained under normoxic conditions using different treatment methods are as follows: Figure 3 As shown, the cell killing rate of the normoxic control group was 15.80±2.30%, the cell killing rate of normoxic preparation a group was 33.74±2.71%, the cell killing rate of normoxic preparation b group was 47.83±3.52%, the cell killing rate of normoxic preparation c group was 78.34±4.04%, and the cell killing rate of normoxic preparation d group was 72.14±2.71%.

[0032] The cell killing rates of T cells obtained under hypoxic conditions using different treatment methods are as follows: Figure 4 As shown, the cell killing rate of the hypoxia control group was 6.69±1.96%, the cell killing rate of hypoxia agent a group was 14.75±0.95%, the cell killing rate of hypoxia agent b group was 26.55±1.66%, the cell killing rate of hypoxia agent c group was 64.34±3.65%, and the cell killing rate of hypoxia agent d group was 32.72±4.06%.

[0033] The results above show that, under normoxic conditions, the 24-hour killing rate of A375 tumor cells induced by the four fusion peptides was significantly higher than that of untreated CD8-positive T cells. Among them, the killing rate of preparation c was the highest, reaching 78.34 ± 4.04%, which was significantly better than that of preparations a, b, and d. This further confirms that DerB-MA-1 has a significantly better effect on enhancing the killing ability of T cells than other fusion peptides in a normoxic environment.

[0034] Under 1% hypoxia conditions simulating the tumor microenvironment, the killing rates of formulations a, b, and d plummeted to 43.7%, 55.5%, and 45.4% of normoxic levels, respectively, with low retention rates. In contrast, formulation c (DerB-MA-1) maintained a potent killing rate of 64.34 ± 3.65%, with a hypoxia retention rate as high as 82.1%, 9.6 times that of the control group and 2.0–4.4 times that of the other three fusion peptides, demonstrating exceptionally strong hypoxia tolerance-promoting capabilities.

Claims

1. A method for preparing an immune T-cell preparation possessing tolerance to tumor hypoxic microenvironment and high cytotoxic activity, characterized in that, The immune T-cell preparation is an immune T-cell preparation pretreated with the fusion peptide DerB-MA-1, the amino acid sequence of which is shown in SEQ ID NO.

3.

2. The method according to claim 1, characterized in that, The preparation method of the immune T cell preparation is as follows: (1) Isolate CD8-positive T cells derived from human peripheral blood; (2) Prepare an inactivated A375 cell suspension using mitomycin C; (3) Mix CD8 positive T cells with inactivated A375 cells at a ratio of 5:1, add the fusion peptide DerB-MA-1, and add IL-7 and IL-15 to culture for 3 days; (4) Add IL-2 and incubate until day 7; (5) Replace half of the old culture medium, add fresh culture medium and IL-2, and culture until day 10; (6) Add A375 cells at a ratio of 10:1, along with the fusion peptides DerB-MA-1 and IL-2. Culture until day 14 to obtain the corresponding immune T cell preparation.

3. The method according to claim 2, characterized in that, In step (3), the seeding density of the CD8 positive T cells is 1×10⁻⁶. 6 cells / mL; The concentration of the fusion peptide DerB-MA-1 is 5 μg / mL, the concentration of IL-7 is 10 ng / mL, and the concentration of IL-15 is 50 ng / mL.

4. The method according to claim 4, characterized in that, In step (4), the concentration of IL-2 is 50 IU / mL; In step (5), the concentration of IL-2 is 150 IU / mL; In step (6), the concentration of the fusion peptide DerB-MA-1 is 5 μg / mL, and the concentration of IL-2 is 150 IU / mL.

5. An immune T-cell preparation possessing tolerance to tumor hypoxic microenvironment and high cytotoxic activity, characterized in that, The immune T-cell preparation is prepared by the method according to any one of claims 1-4.

6. The application of a fusion peptide in the preparation of a drug that enhances the tolerance of T cells to the tumor hypoxic microenvironment, characterized in that, The fusion peptide is the fusion peptide DerB-MA-1, and the amino acid sequence of the fusion peptide DerB-MA-1 is shown in SEQ ID NO.

3.

7. The application according to claim 6, characterized in that, The tumor is melanoma.

8. The application of a fusion peptide in the preparation of a drug that enhances the tumor cell killing ability of T cells, characterized in that, The fusion peptide is the fusion peptide DerB-MA-1, and the amino acid sequence of the fusion peptide DerB-MA-1 is shown in SEQ ID NO.

3.

9. The application according to claim 8, characterized in that, The tumor cells were melanoma cells A375.

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