HERV-e based therapeutic vaccine for renal cancer and method of making same

Abstract

The present application relates to a kind of renal cancer therapeutic DC vaccine based on the immune peptide activation of HERV-E protein source and its preparation method, and the preparation method includes the following steps: obtaining immature DC cell;Synthesis HERV-E protein source immune peptide;Immune peptide and immature DC are co-cultured in DC activation culture medium for 1 day to DC maturation.The renal cancer therapeutic DC vaccine obtained by the way of immature DC loaded with HERV-E immune peptide is more stable in character, and has strong anti-tumor capacity, can effectively inhibit the progress of renal cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation and its preparation method. Background Technology

[0002] Renal cell carcinoma (RCC), also known as kidney cancer, is the most common solid malignant tumor of the kidney (accounting for approximately 90%) and about 3% of all malignant tumors, ranking third among genitourinary tumors (after prostate and bladder cancer). Because RCC has an insidious onset, it often lacks early clinical manifestations. Approximately 30% of RCC patients have already metastasized at the time of diagnosis, and 20%-30% of RCC patients who undergo radical resection also develop metastases. RCC is not sensitive to radiotherapy, chemotherapy, or cytokine therapy, and advanced stages are prone to distant metastasis. The 5-year survival rate for patients with metastatic RCC is only 12%. Therefore, there is an urgent need for more effective new treatment options for advanced RCC.

[0003] Because renal cell carcinoma is a highly immunogenic tumor, immunotherapy has unique advantages in the treatment of advanced renal cell carcinoma. Currently, DC-tumor cell fusion vaccines are commonly used. However, DC therapeutic vaccines prepared using this method have many drawbacks. For example, the antigenic information carried by tumor cells is not as complete and comprehensive as that released into the extracellular matrix by various renal cell carcinoma cells carrying comprehensive antigenic information. This leads to a weaker immune response and less potent tumor-killing ability.

[0004] HERV (Human endogenous retroviruses) are endogenous viral components in the genome, highly similar to retroviruses, and can be produced by retroviruses. They are the offspring of exogenous retroviruses that accidentally invade germ cells. HERV sequences occupy approximately 8% of the 3 billion base pairs in the human genome. During the period from the 1990s to the early 21st century, Richard W. Childs' team at the National Institutes of Health discovered an LTR2C-like HERV-E sequence specifically expressed in RCC during their research on advanced renal cell carcinoma GVT, naming it CT-RCC. The full-length CT-RCC HERV-E sequence is located at locus 6q15 on chromosome 6. It currently has three known transcripts: CT-RCC-8 (EU137846), CT-RCC-9 (EU137847), and CT-RCC-Env (JQ733905), which are specifically expressed in clear cell renal cell carcinoma. Studies have shown that the deletion or mutation of the VHL gene allele is the reason for the specific expression of CT-RCC HERV-E in clear cell renal cell carcinoma. It is estimated that approximately 80% of clinical cases of clear cell renal cell carcinoma show CT-RCC HERV-E expression. Research has found that the transmembrane component of the capsule protein encoded by the CT-RCC-Env transcript can induce a strong human immune response. Another study identified a highly immunogenic decapeptide, CT-RCC-1, with high affinity for HLA-A*1101 from the shared sequences of three transcripts. Preclinical experiments showed that CT-RCC-1-induced CTL cells exhibited significant killing and inhibitory activity against HLA*1101-positive ccRCC tumor cells and tumor animal models. Through TCR sequence analysis of CT-RCC1-specific CTLs, researchers at NHLBI and Loyola University Medical Center developed a TCR-T cell therapy for CT-RCC HERV-E-positive clear cell renal cell carcinoma, currently in Phase I clinical trials. This demonstrates that HERV-E protein is a highly promising antigen source for renal cancer immunotherapy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a therapeutic DC vaccine for renal cancer based on HERV-E protein-derived immune peptides that has strong immunogenicity and specificity, and a method for preparing the same, in response to the defects of existing renal cancer vaccines such as weak immune response and poor tumor killing ability.

[0006] The technical solution adopted by this invention to solve its technical problem is: to provide a method for preparing a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation, the preparation method comprising the following steps:

[0007] 1. Obtain immature dendritic cells (DCs);

[0008] 2. Artificially synthesized immune peptides derived from HERV-E protein;

[0009] 3. Co-culture the immunopeptides and immature DCs in DC activation medium for 1 day until the DCs mature.

[0010] In the method for preparing a DC-based therapeutic vaccine for renal cell carcinoma provided by the present invention, the DC activation culture medium is a 1640 culture medium containing 5% autologous plasma, 50 ng / mL GM-CSF, 50 ng / mL IL4, 2 μg / mL CD40 and 10 μg / mL Polyinosinic-polycytidylic acid (Poly I:C).

[0011] In the preparation method of the renal cell carcinoma therapeutic DC vaccine based on the activation of immune peptides derived from HERV-E protein provided by the present invention, the sequence of the artificially synthesized immune peptide derived from HERV-E protein is HWKTGENVNLGIDGTGLDPRVNLLIQ.

[0012] In the preparation method of the renal cell carcinoma therapeutic DC vaccine based on HERV-E protein-derived immune peptide activation provided by the present invention, the concentration of DC is (2-5) ×106 cells / mL.

[0013] In the preparation method of the renal cell carcinoma therapeutic DC vaccine based on HERV-E protein-derived immune peptide activation provided by the present invention, the process of obtaining the immature DC includes the following steps: peripheral blood is taken, diluted, and lymphocyte separation medium and anticoagulated blood are added. After centrifugation, the white turbid layer liquid is taken. After centrifugation of the white turbid layer, the supernatant is discarded, thus separating mononuclear cells. The mononuclear cells are resuspended in enrichment medium to a density of (4-6)×106 / mL. The mononuclear cells are cultured in vitro in enrichment medium for 1-1.5 hours, and the enrichment medium is removed. Then, DC induction medium is added and cultured for 48 hours to induce differentiation into immature DC.

[0014] In the preparation method of the renal cell carcinoma therapeutic DC vaccine based on HERV-E protein-derived immune peptide activation provided by the present invention, the enrichment medium is 1640 medium containing 5% autologous plasma.

[0015] In the preparation method of the renal cell carcinoma therapeutic DC vaccine based on HERV-E protein-derived immune peptide activation provided by the present invention, the DC induction medium is 1640 medium containing 5% autologous plasma, 50 ng / mL GM-CSF, and 50 ng / mL IL4.

[0016] The present invention also provides a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation, obtained by the above-described preparation method of the therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation.

[0017] The therapeutic DC vaccine for renal cell carcinoma activated by HERV-E protein-derived immune peptides and its preparation method provided by this invention can achieve the following beneficial effects: The therapeutic vaccine for renal cell carcinoma obtained by loading immature DCs with HERV-E protein-derived immune peptides is not only more stable in nature, but also has stronger immunogenicity and specificity, stronger tumor-killing ability, and can effectively regulate the body's immune mechanism and improve the phenomenon of renal immune tolerance caused by renal cell carcinoma. Attached Figure Description

[0018] Figure 1 This is a comparison chart of the anti-tumor effects of the DC vaccine in the experimental and control groups. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] This invention provides a method for preparing a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation, the method comprising the following steps:

[0021] S1, Obtain immature DC cells;

[0022] S2, an immune peptide derived from artificially synthesized HERV-E protein;

[0023] S3, the immature DCs obtained in step S1 and the immunopeptides synthesized in step S2 are co-cultured in DC activation medium for 1 day until the DCs mature.

[0024] Specifically, in a preferred embodiment of the present invention, the immature DCs in step S1 are obtained by inducing monocytes, and the specific steps are as follows:

[0025] S11 PBMC cells were isolated from peripheral blood;

[0026] S12 was incubated by resuspending PBMCs in enrichment medium;

[0027] S13 Remove the enrichment medium from step S12, and induce DC cells using DC induction medium to obtain immature DC cells.

[0028] In step S11, the time of peripheral blood ex vivo affects the quality of subsequent immature DC cells. Therefore, in this invention, it is preferable to separate PBMC cells from fresh human peripheral blood within 30 minutes of ex vivo for subsequent steps. The shorter the cell ex vivo time, the less the cell activity and state are weakened, which is more conducive to obtaining high-quality DC cells.

[0029] In step S12, the enrichment medium is preferably 1640 medium containing 5% autologous plasma.

[0030] In step S13, the DC induction medium is preferably 1640 medium containing 5% autologous plasma, 50 ng / mL GM-CSF, and 50 ng / mL IL4.

[0031] It is understood, of course, that the method of obtaining immature DCs in this invention is not limited to this, and immature DCs obtained by inducing monocytes through other induction methods are also applicable to this invention.

[0032] Specifically, in a preferred embodiment of the present invention, the immune peptide derived from HERV-E protein in step S2 is synthesized by Nanjing Genscript Biotech Co., Ltd., and the preferred purification method is HPLC purification with a purity ≥95%.

[0033] In step S3, preferably, the DC activation medium is 1640 medium containing 5% autologous plasma, 50 ng / mL GM-CSF, 50 ng / mL IL4, 2 μg / mL CD40 and 10 μg / mL Polyinosinic-Cyclokinin (Poly I:C).

[0034] Because DC cells grow in clusters, high concentrations can cause them to clump together, making it difficult for centrally located DC cells to mature. This can affect antigen loading and thus the immunization effect of the vaccine. Therefore, the DC cell density in step S3 should not be too high. In this step, the preferred concentration of immature DCs is (2-5) × 10⁶ cells / mL, and antigen peptides are added for loading.

[0035] In addition, Poly I:C has a strong stimulatory effect on immature DCs, which can increase the loading rate of antigen peptides and promote the maturation of DCs. Therefore, the DC activation culture medium in step S3 contains 10 μg / mL Poly I:C.

[0036] The specific implementation method is as follows:

[0037] Example 1: Preparation of DC vaccine in experimental group

[0038] S1, Obtain immature DC cells;

[0039] S11, PBMC cells were isolated from peripheral blood and counted;

[0040] S12, resuspend PBMCs in enrichment medium at a density of (4-6) × 10⁶ / mL. Spread 12 mL of this medium onto a T75 flask and incubate at 37℃ in a 5% CO₂ incubator for 1-1.5 hours. Remove the flasks from the incubator. Under a 40x microscope, a suitable cell density is indicated by an average of 100-200 cells adhering to the surface in 4-8 random fields of view. Terminate the incubation at this point.

[0041] S13, Remove the enrichment medium from step S12, add 10 mL of DC induction medium to the T75 culture flask containing enriched monocytes, and incubate at 37°C in a 5% CO2 incubator. After 48 h of induction culture, observe cell morphology. At this time, the monocytes differentiate into DC morphology, and there should be many dendritic cells, which are immature DC cells;

[0042] S2, Preparation of immune peptides;

[0043] S21, Nanjing Genscript Biotech Co., Ltd. was commissioned to artificially synthesize an immune peptide derived from HERV-E protein. The amino acid sequence of the immune peptide is HWKTGENVNLGIDGTGLDPRVNLLIQ;

[0044] S22, dissolve the peptide in DMSO to 1 mg / mL;

[0045] S3, induced by mature DCs;

[0046] S31, an antigen-loading process was carried out in immature DC cells by adding an immunopeptide derived from synthetic HERV-E protein. The final peptide concentration was 10 μg / mL, and the cells were incubated at 37°C in a 5% CO2 incubator for 1 hour.

[0047] S32, add a DC maturation inducing factor and incubate overnight for induction.

[0048] S33, after 24 hours of induction, DC phenotype and purity were identified by flow cytometry, and mature DC cell vaccines were collected.

[0049] Example 2: Preparation of DC vaccine for control group

[0050] The difference from Example 1 provided by the present invention is that, in this example, the amino acid sequence of the immune peptide synthesized in step S2 and used in step S3 is obtained by randomly scrambling the amino acid sequence of the immune peptide in Example 1, and the specific sequence is: KTHWNVEGGILNGDGTPRLDLNVQIL.

[0051] Example 3: Preparation of DC-activated CTL cells

[0052] S1, PBMC cells were isolated from peripheral blood and CD8+ T cells were obtained using magnetic bead sorting.

[0053] S2, mature DC cells from the experimental group and mature DC cells from the control group were co-cultured with CD8+ T cells at a ratio of 1:10 in KBM551 medium containing 10% autologous plasma, with IL-2 added to the medium;

[0054] S3, Day 3: Add selective medium. The selective medium formula is KBM551 + 200 U / mL IL-2 + 20 ng / mL IL-7 to maintain the cell density at (0.5~1)×106 / mL. Gently pipette the dispersed lymphocytes.

[0055] S4, from day 5 to day 7, observe the cell density and add selective medium to maintain the density at (0.5-2) ×106 / mL. When the lymphocytes are large, use a pipette to gently dissipate the lymphocytes.

[0056] S5, from day 8 to day 14, observe the cell density and add culture medium to maintain the density at (1-2)×106 / mL. When the lymphocytes are large, use a pipette to gently blow away the dispersed lymphocytes. Add IL-15 to the culture medium to a final concentration of 20ng / mL.

[0057] S6, Day 15: CTL cells were collected by centrifugation and counted for later use.

[0058] To further verify the significant effect of the renal cell carcinoma therapeutic DC vaccine based on HERV-E protein-derived immune peptide activation (hereinafter referred to as "DC vaccine") provided by this invention, the following experiments were conducted for detection and verification.

[0059] Experimental subjects:

[0060] Experimental group: namely, CTL cells prepared according to Example 3 using the DC vaccine provided in Example 1 of this invention;

[0061] Control group: namely, CTL cells prepared according to Example 3 using the DC vaccine provided in Example 2 of this invention.

[0062] Experimental methods: In vivo anti-tumor experiment in nude mice

[0063] Human clear cell renal carcinoma 786-O cells were cultured according to the prescribed conditions. The 786-O cell line was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. 786-O cells were subcutaneously injected into the backs of female NOD-SCID immunodeficient mice at a dose of 10⁷ cells / mouse. NOD-SCID mice were purchased from Shanghai Silex Laboratory Animal Co., Ltd. Two weeks after inoculation, when the tumor size on the back of the mice reached approximately 100–150 mm³, CTL cells were injected via the tail vein at a dose of 10⁷ cells / injection, once every other day, for a total of 5 injections. Two days after the 5th CTL cell injection, the mice were sacrificed, and the back tumors were dissected. The tumor size was compared between the experimental and control groups to evaluate the antitumor effect of the DC vaccine in both groups. The results are as follows: Figure 1 As shown:

[0064] Experimental results: such as Figure 1 As shown, compared to the CTL cells activated by the DC vaccine in the control group, the CTL cells activated by the DC vaccine in the experimental group exhibited significant inhibition against subcutaneous xenografts of renal cell carcinoma cells in vivo. The tumor inhibition rate in the experimental group was 83.64% compared to the control group; this indicates that the renal cell carcinoma therapeutic DC vaccine based on HERV-E protein-derived immune peptide activation provided by this invention can effectively stimulate T cells to produce a specific killing effect against renal cell carcinoma cells.

[0065] The embodiments of the present invention have been described above. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A method for preparing a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation, characterized in that: The preparation method includes the following steps: obtaining immature DC cells; synthesizing an immune peptide derived from HERV-E protein, wherein the sequence of the immune peptide derived from HERV-E protein is HWKTGENVNLGIDGTGLDPRVNLLIQ; and co-culturing the immune peptide with immature DC cells in DC activation medium for 1 day until the DCs mature. The immature cells were obtained by inducing monocytes, and the specific steps are as follows: S1 isolates PBMC cells from peripheral blood; S2 was incubated by resuspending PBMC in enriched medium; S3 Remove the enrichment medium from step S2, and use DC induction medium to induce DC cells to obtain immature DC cells.

2. The method for preparing a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation according to claim 1, characterized in that, The DC activation medium was 1640 medium containing 5% autologous plasma, 50 ng / mL GM-CSF, 50 ng / mL IL4, 2 μg / mL CD40, and 10 μg / mL Polyinosinic-polycytidylic acid (Poly I:C).

3. The method for preparing a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation according to claim 1, characterized in that, The concentration of the immature DC cells was (2–5) × 10⁻⁶. 6 per ml.

4. The method for preparing a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation according to claim 1, characterized in that, The process of obtaining the immature DC cells includes the following steps: peripheral blood is collected, diluted, and then lymphocyte separation medium and anticoagulated blood are added. After centrifugation, the white turbid layer is collected. The supernatant is discarded after centrifugation of the white turbid layer, thus separating mononuclear cells. The mononuclear cells are resuspended in enrichment medium to a concentration of (4-6) × 10⁻⁶. 6 The density of mononuclear cells was determined by culturing them in an enrichment medium for 1–1.5 hours in vitro, then removing the enrichment medium; then DC induction medium was added and cultured for 48 hours to induce differentiation into immature DC cells.

5. The method for preparing a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation according to claim 4, characterized in that, The enrichment medium is 1640 medium containing 5% autologous plasma.

6. The method for preparing a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation according to claim 4, characterized in that, The DC induction medium was a 1640 medium containing 5% autologous plasma, 50 ng / mL GM-CSF, and 50 ng / mL IL4.

7. The method for preparing a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation according to claim 1, characterized in that, The HERV-E protein-derived immunopeptides are artificially synthesized.

8. The method for preparing a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation according to claim 7, characterized in that, The concentration of the HERV-E protein-derived immune peptide used to load immature DC cells is 10 μg / mL.

9. A therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptide activation, characterized in that, The method for preparing a therapeutic DC vaccine for renal cell carcinoma based on HERV-E protein-derived immune peptides as described in any one of claims 1-8.

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