CN114788870B - Composition for targeting immune cells to supplement arginine and neutralize acid environment for long time and application - Google Patents

Abstract

The invention provides a composition and application for long-term supplementation of arginine targeting immune cells and neutralizing the acid environment. It consists of L-arginine, cationic amino acid transporter 2 inhibitor and pharmaceutically acceptable slow-release drug storage carrier. The invention prepares an antitumor drug by co-loading L-arginine and cationic amino acid transporter 2 inhibitors in a slow-release drug storage. The present invention inhibits the cationic amino acid transporter 2 of cancer cells and immunosuppressive cells by local injection at the tumor site, and at the same time releases alkaline L-arginine in a long-term and sustained manner in the tumor to realize the targeted tumor-killing immune cell L- Arginine supplementation and L-arginine starvation of tumor cells and immunosuppressive cells, and the use of the alkalinity of L-arginine to neutralize the acidic environment, complete the immune-promoting improvement of the tumor microenvironment at the acidic and nutritional levels, It can significantly enhance the anti-tumor immune response and improve the therapeutic effect of tumors.

Description

Composition and application for long-term supplementation of arginine targeting immune cells and neutralizing acid environment

technical field

The invention belongs to the field of pharmaceutical preparations, and relates to a composition for long-term supplementation of arginine targeting immune cells and neutralizing acid environment and its application, which is mainly used for preparing antitumor drugs.

Background technique

In the case of sufficient oxygen, tumor cells still prefer the sugar metabolism mode of anaerobic glycolysis to synthesize the energy they need, and then produce a large amount of lactic acid. This phenomenon is called the "Warburg" effect. To maintain intracellular homeostasis, tumor cells will efflux the produced lactic acid into the interstitium, resulting in acidification of the interstitium. The resulting weakly acidic environment is thought to be highly associated with the high invasiveness and metastases of cancer cells, as well as their resistance to various therapies including chemotherapy and immunotherapy. More importantly, a low pH value is toxic to lymphocytes and inhibits interleukin-2 (IL-2)-stimulated lymphocyte proliferation, and it has been demonstrated that cytotoxic T lymphocytes are effective against various tumor cell lines under acidic conditions. The killing effect is weakened. Reduction of environmental pH to 6.0–6.5 was sufficient to induce anergic state T lymphocytes in human and mouse tumor-specific CD8+ T cells with impaired cytolytic activity and cytokine secretion, IL-2 regulated Reduced expression of IL-2Rα and T cell receptor (TCR), as well as decreased activation of signal transducer and activator of transcription 5 (STAT5) and extracellular signal-regulated kinase (ERK) following TCR activation. The immunosuppressive effect caused by the acidic environment also acts on natural killer cells (NK cells) and macrophages. Therefore, neutralizing the acidic tumor microenvironment can provide an environmental basis for immunotherapy (CancerRes. 2016 Mar15; 76(6): 1381-90).

L-arginine (L-arg) is a basic amino acid that acts as a link in the synthesis of various metabolites, including NO, polyamines, amino acids and pyrimidines, which are essential for cell survival and proliferation few key substances. One of the characteristics of the tumor environment is L-arg deficiency. On the one hand, due to abnormal angiogenesis in tumor tissue, vascular malformation, abnormal blood perfusion and material transport obstacles result in insufficient supply of L-arg. On the other hand, rapidly proliferating cancer cells in tumor tissue, myeloid-derived suppressor cells (MDSCs) and M2 macrophages that highly express arginase 1 (ARG1) will cause high consumption of L-arg. In the absence of L-arg, T cells will have reduced expression of CD3ζ chain and impaired ability to produce cytokines, and will also cause cell cycle arrest of T cells that enter the division phase after activation through the GCN2/eIF2α pathway proliferation inhibition. In contrast, sufficient L-arg levels induced a change in the overall metabolic pattern of T cells, manifested by a shift in energy production in activated T cells from glycolysis to oxidative phosphorylation, which in turn promoted central memory cells with higher viability The generation of the gene can show stronger anti-tumor activity in the mouse model. In addition, L-arg is also the substrate of inducible nitric oxide synthase (iNOS) in M1 macrophages, and the catalysis of L-arg to nitric oxide (NO) by iNOS is an important way for M1 cells to exert immune function. Insufficient L-arg will limit the ability of M1 to generate NO.

Therefore, adding alkaline L-arg to tumor tissue can provide the necessary nutritional support for immune cells, and at the same time use its alkaline to modify the original inhibitory acidic environment, thereby further promoting the immune response. Studies have shown that oral supplementation of L-arg can improve the effect of immunotherapy (Cancer Biol Ther.2017 Feb; 18(2):94-100), which is beneficial to the prognosis of surgery (Clin Nutr.2014 Dec; 33(6):951 -7). However, the dose of L-arg required for oral administration is extremely high, which is not easily tolerated by the patient, and its alkalinity cannot be utilized, while local solution injection cannot achieve a sustained increase in L-arg levels due to rapid diffusion (Nature.2021 Oct; 598(7882) :662-666). Therefore, preparations that can locally and continuously release basic L-arginine in tumors are more suitable for L-arg supplementation therapy.

In addition, L-arg is also an essential nutrient for the proliferation of tumor cells. Although the previously designed starvation strategy of artificially consuming whole body L-arg can inhibit the growth of some tumors, its therapeutic effect has a serious impact on tumor genotypes. Dependence (JAMA Oncol.2017 Jan 1; 3(1):58-66) and has a significant inhibitory effect on the immune system (Cancer Res.2015 Jan 15; 75(2):275-83), which is not conducive to treatment. Taken together, in order to maximize the effectiveness of this strategy, it is necessary to prevent cancer cells from benefiting from an improved environment, that is, to achieve nutrient starvation of tumor cells in the context of L-arg supplementation.

The availability of cells to the environment L-arg is related to the expression of transporters. The L-arg transport of mammalian cells is mainly composed of CAT-1 (SLC7a1) and CAT-2 ( SLC7a2) dominates. During the activation phase of T cells, the expression of CAT-1 will be up-regulated to meet their own needs for L-arg, and it will be enhanced in the absence of L-arg, but it will not up-regulate CAT-2 or other transporters (Eur J Immunol.2016 Jan; 46(1):92-103). However, melanoma cells resistant to BRAF inhibitors showed a metabolic shift from glucose to arginine dependence accompanied by increased expression of CAT-2, and silencing CAT-2 expression significantly weakened their proliferative ability (Mol Oncol.2017 Dec;11(12):1806-1825). MDSCs at local inflammation and tumor sites exhibited a synergistic upregulation of CAT-2, ARG1, and NOS2, and suppressed T cell immune responses by depleting environmental L-arg. Attenuated ability to control T cell immunity in cancer models (J Immunol. 2015 Dec 1;195(11):5237-50.). Similarly, increased activation of dendritic cells and increased numbers of memory T cells were observed in a pneumonia model of CAT-2-deficient mice (Proc Natl Acad Sci US A. 2006 Oct 3;103(40):14895-900) . This differential expression of CAT-1 and CAT-2 has not been utilized in the regulation of the tumor environment. By inhibiting CAT-2 in the tumor environment, it is expected to replenish L-arg for tumor-killing immune cells while achieving tumor Cell-specific L-arg starvation.

Contents of the invention

The purpose of the present invention is to provide a long-term supplementation of arginine targeting immune cells and neutralizing the acid environment composition, the composition is composed of L-arginine, cationic amino acid transporter 2 inhibitor and pharmaceutically acceptable Sustained-release drug reservoir carrier composition.

Wherein the concentration of L-arginine is 10-160 mg/mL, the cationic amino acid transporter 2 inhibitor can be inhibitory polypeptide and antibody, or shRNA or siRNA based on the cationic amino acid transporter 2 gene sequence. The shRNA is a short hairpin RNA based on the cationic amino acid transporter 2 gene sequence, and its sequence can be:

(1) gctttatgccctatggctttatcaagagataaagccataggggcataaagctttttt;

(2) cgtccttacttgtctgctttattcaagagataaagcagacaagtaaggacgttttttt;

(3) cggcctttgctatgctgaatttcaagagaattcagcatagcaaaggccgtttttt.

The siRNA is a small interfering RNA based on the cationic amino acid transporter 2 gene sequence, and its sequence can be:

(1) cgaaauacgggauaccgaaau;

(2) gcaggaaugaacagacgaaau;

(3) gccggaaacgauacgacuua.

When the cationic amino acid transporter 2 inhibitor is inhibitory polypeptide and antibody, its concentration is 1-20mg/mL; when the cationic amino acid transporter 2 inhibitor is shRNA, its concentration is 0.1-5mg/mL; when cationic amino acid transporter When the protein 2 inhibitor is siRNA, its concentration is 10-200nmol/mL. When the cationic amino acid transporter 2 inhibitor is shRNA or siRNA, it is necessary to use gene delivery vehicles in combination, including but not limited to cationic liposomes, cationic nanoemulsions, and solid lipid nanoparticles. The types of sustained-release drug depot vehicles include, but are not limited to, microspheres, gels, and multivesicular liposomes.

Another object of the present invention is to provide the application of the composition in the preparation of antitumor drugs, and the tumor is melanoma.

The anti-tumor in the present invention is realized by administering the composition drug alone, or combining the composition with immunotherapy, surgical treatment, chemotherapy and radiotherapy.

The anti-tumor effect of the present invention is to inhibit the function of cationic amino acid transporter 2 in tumor tissue through the inhibitor of cationic amino acid transporter 2, limit the ability of tumor cells and immunosuppressive cells to utilize L-arginine in the tumor environment, and avoid It benefits from an improved environment that even forces it to be L-arginine starved. Utilizing the basicity of L-arginine and the release characteristics of the slow-release drug reservoir, it can achieve sustained targeted supplementation of L-arginine to tumor-killing immune cells and neutralization of weak acid environment, from both pH environment and nutrition At the same time, it can improve the tumor microenvironment and realize the enhancement of anti-tumor immune response.

The present invention designs a composition for long-term supplementation of arginine targeting immune cells and neutralizing the acid environment. The composition inhibits CAT-2 in tumor tissue, restricts the uptake and utilization of L-arg in the environment by tumor cells and immunosuppressive cells, and combined with local alkaline L-arginine slow-release supplementation, can improve tumor microbiology for a long time. The nutritional conditions and pH environment in the environment can simultaneously achieve the effects of strengthening anti-tumor immunity, starving tumors and weakening immunosuppression, and can be used for adjuvant immunotherapy, surgical treatment, chemotherapy and radiotherapy, and has good potential for clinical application.

The invention prepares the antitumor drug by co-loading L-arginine and cationic amino acid transporter 2 inhibitor in the slow-release drug storage. The present invention inhibits the cationic amino acid transporter 2 of cancer cells and immunosuppressive cells by local injection in the tumor site, and at the same time releases alkaline L-arginine in a long-term and sustained manner in the tumor to realize the targeted tumor-killing immune cell L- Arginine supplementation and L-arginine starvation of tumor cells and immunosuppressive cells, and the use of the alkalinity of L-arginine to neutralize the acidic environment, complete the immune-promoting improvement of the tumor microenvironment at the acidic and nutritional levels, It can significantly enhance the anti-tumor immune response and improve the therapeutic effect of tumors.

The innovation of the present invention lies in the use of sustained-release preparation technology to complete the encapsulation of high-concentration alkaline L-arginine and cationic amino acid transporter 2 inhibitors, to achieve the neutralization of the weak acid tumor microenvironment, and to fight against tumor immunity Targeted L-arginine supplementation of cells, specific L-arginine starvation of tumor cells and immunosuppressive cells. The composition of the present invention breaks through the double immunosuppressive effect mediated by environment and nutrition in tumor tissue, can significantly improve the response of the body's anti-tumor immunity, and can also be used for adjuvant chemotherapy, radiotherapy, immunotherapy and Surgical treatment has good prospects for clinical application. The preparation technology of L-arginine drug storage is simple and mature, the cost of materials is low, stable and reliable, and can be used for commercial production.

Description of drawings

Figure 1 is the in vitro release profile of the composition (microsphere dosage form).

Figure 2 is the in vitro release profile of the composition (multivesicular liposome model).

Figure 3 is the in vitro release profile of the composition (gel dosage form).

Figure 4 shows that the CAT-2 shRNA plasmid/Lipo8000 complex inhibits the CAT-2 protein of B16 cells.

Figure 5 is the effect of CAT-2 protein inhibition on the proliferation of B16 cells.

Figure 6 shows the effect of CAT-2 protein inhibition on the proliferation of CD8+ T cells.

Figure 7 shows the effect of CAT-2 protein inhibition on the differentiation of CD8+ T cells.

Figure 8 is the effect of the composition on neutralizing the acidic microenvironment of the tumor.

Figure 9 is the inhibitory effect of the composition on tumor growth.

Figure 10 is the effect of the composition on the body weight of mice.

Figure 11 is the effect of the composition on the survival of mice.

Figure 12 is the effect of the composition on the number of infiltrating CD8+ T cells in the tumor.

Figure 13 is the effect of the composition on the differentiation of infiltrating CD8+ T cells in tumors.

Figure 14 is the effect of the composition on the polarization of tumor-associated macrophages.

Figure 15 is the effect of the composition on cytokines in tumor tissue.

Figure 16 shows the inhibitory effect of the composition combined with anti-PD-1 therapy on tumor growth.

Figure 17 shows the effect of the composition combined with anti-PD-1 treatment on the body weight of mice.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments. It must be pointed out that the following embodiments are only used to explain and illustrate the present invention, not limit the invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the invention still belong to the protection scope of the present invention.

Embodiment 1 A kind of preparation method of composition (microsphere dosage form)

The composition of the composition (microsphere dosage form):

L-Arginine 5mg

CAT-2 siRNA/Lipo8000 complex 7nmol (based on siRNA content)

Poly(lactic-co-glycolic) acid, 1.5w, 50:50 200mg.

Dissolve the prescribed amount of L-arginine and CAT-2 siRNA/Lipo8000 complex in 200 μL of pure water as the inner aqueous phase; dissolve the prescribed amount of Poly(lactic-co-glycolic) acid in 4 mL of dichloromethane , as the oil phase. Add the inner water phase to the oil phase, vortex for 30 seconds, and then ultrasonic emulsify with a 100W probe for 2 minutes. Gained W/O emulsion was added to 40 mL of 2% PVA-containing aqueous solution, and after high-speed stirring, it was added to 100 mL of 0.2% PVA-containing aqueous solution, and stirred overnight at low speed to remove dichloromethane and complete microsphere solidification. After the obtained suspension is centrifuged at high speed, the bottom precipitate is collected to obtain the composition (microsphere dosage form). After washing three times with ultrapure water, store at 4°C.

The in vitro release of embodiment 2 composition (microsphere dosage form)

In this example, on the basis of Example 1, the composition (microsphere dosage form) was taken, resuspended in PBS solution with a pH of 7.4, placed in a constant temperature shaking box at 37°C, sampled at a set time, and collected after high-speed centrifugation In the supernatant, the L-arginine content was determined by HPLC, and the in vitro release behavior of the composition (microsphere dosage form) was investigated. The release curve shows that, under the condition of 37°C, the composition (microsphere dosage form) exhibits a sustained release capability of up to 21 days in a PBS solution with a pH of 7.4 (Fig. 1).

The HPLC conditions used were:

Mobile phase: methanol: 0.01M sodium pentanesulfonate=(10:90), 1 mL/min; detection wavelength: 206nm; chromatographic column: Agilent ZORBAX C18 column (4.6×250mm, 5μm).

Embodiment 3 A kind of preparation method of composition (multivesicular liposome dosage form)

Composition of the composition (multivesicular liposomal dosage form):

Dissolve the prescribed amount of L-arg and CAT-2 shRNA plasmid/Lipo8000 complex in 1 mL of pure water as the first aqueous phase; dissolve the prescribed amount of egg yolk lecithin E80, cholesterol and α-tocopherol in 2 mL of dichloromethane In, as the oil phase. The first water phase was added to the oil phase, and a 200W probe was ultrasonically emulsified for 4 minutes to prepare a W/O emulsion. Use 5 mg/mL sodium chloride aqueous solution containing 2% sodium lauryl sulfate as the second water phase, add the prepared W/O emulsion into 10 mL of the second water phase, and stir at a low speed to obtain W/O/W Type double emulsion, 20 mL of 5 mg/mL sodium chloride solution was added thereto, stirred in a water bath at 25°C for 5 hours, and the organic solvent was volatilized to remove the milky white homogeneous composition (multivesicular liposome formulation) suspension. The resulting suspension was centrifuged at low speed to enrich the composition (multivesicular liposome formulation), the supernatant was discarded, and 20 mL of 5 mg/mL sodium chloride solution was added for washing, and this was repeated 3 times. After washing, the composition (multivesicular liposome formulation) was collected and stored at 4°C.

The in vitro release of embodiment 4 composition (multivesicular liposome dosage form)

In this example, on the basis of Example 3, take the composition (multivesicular liposome formulation), dilute it in a PBS solution with a pH of 7.4, place it in a constant temperature shaking box at 37°C, take samples at a set time, and centrifuge at a low speed Discard the supernatant and dissolve the collected composition with methanol, measure the L-arginine content with the HPLC conditions in Example 2, and detect the in vitro release behavior of the composition (multivesicular liposome formulation). The release curve showed that under the condition of 37°C, the composition (multivesicular liposome dosage form) exhibited a sustained release capability of up to 216 hours in a PBS solution with a pH of 7.4 (Fig. 2).

Embodiment 5 A kind of preparation method of composition (gel dosage form)

Composition of the composition (gel dosage form):

After mixing the prescribed amount of Poly(lactic-co-glycolic)acid and N-methylpyrrolidone, let it stand for 2 hours. After it was completely swollen, the prescribed amount of L-arginine and anti-CAT-2 antibody was added, and mixed to obtain a composition (gel formulation) precursor solution, which was stored at 4°C.

The in vitro release of embodiment 6 composition (gel dosage form)

In this embodiment, on the basis of Example 5, the precursor solution of the composition (gel dosage form) is taken and injected into a dialysis bag, and the dialysis bag containing the gel precursor solution is suspended in a PBS solution with a pH of 7.4, and placed In a constant temperature shaking box at 37°C, samples were taken from the external release medium at a set time, and the content of L-arginine was determined by HPLC conditions in Example 2 to detect the in vitro release behavior of the composition (gel dosage form). The release curve shows that the composition (gel dosage form) exhibits a sustained release capability of up to 30 days in a PBS solution with a pH of 7.4 at 37°C ( FIG. 3 ).

Example 7 Inhibition of Tumor Cell Cationic Amino Acid Transporter 2 (CAT-2)

In the in vitro cell experiments described in the examples, the CAT-2 shRNA plasmid was used at 1 μg/mL, and the dosage of the cationic carrier used was calculated according to the ratio of the plasmid to the carrier in the instructions.

B16 cells were inoculated in 6-well plates, and after overnight adherence, they were divided into 3 groups and treated as follows: ① Add normal saline (Blank) and incubate for 24 hours; ② Add CAT-2 shRNA plasmid/Lipo8000 complex (shRNA) for co-incubation. Incubate for 24 hours, ③ add CAT-2 shRNA plasmid/Lipo8000 complex (shRNA) and incubate for 24 hours, and then select by puromycin (shRNA-p) for 24 hours. After the treatment was completed, the cells were collected to extract proteins, and the changes of CAT-2 expression were detected by western blot. The experimental results show that shRNA (CAT-2 shRNA plasmid-Lipo8000 complex) can down-regulate the expression level of CAT-2 of B16 by about 50%. No compensatory increase in CAT-1 expression was seen (Figure 4).

B16 cells were inoculated in 96-well plates, and after overnight adherence, they were divided into 4 groups and treated as follows: ① add normal saline (Blank), ② add Lipo8000 (Lipo), ③ scramble shRNA plasmid/Lipo8000 complex (Scramble), ④ CAT -2 shRNA plasmid/Lipo8000 complexes (shRNA), after co-incubating for 24 hours, the cell viability was detected by MTT method. After down-regulation of CAT-2 expression in B16, its proliferative activity when L-arg is sufficient becomes similar to that when L-arg is insufficient (Figure 5), which proves that CAT-2 protein is more dependent on L-arg of B16 cells transporter, inhibition of this protein enables starvation of tumors in an arginine-rich environment.

Example 8 Inhibiting the Effect of Tumor Cell Cationic Amino Acid Transporter 2 on Immune Cells

In this example, on the basis of Example 7, the Transwell system was used to inoculate B16 cells in the lower chamber, and activated CD8+ T cells in the upper chamber, with 200,000 cells each. The concentration of L-arginine in the culture system was adjusted to 50 μM or 100 μM, and physiological saline or CAT-2 shRNA plasmid/Lipo8000 complex was added to the lower chamber for co-cultivation for 48 hours. The results showed that the proliferation of T cells was severely inhibited when co-cultured with insufficient L-arg (50 μM), supplementing L-arg to the environment could alleviate this inhibitory effect, and the downregulation of CAT-2 in tumor cells also It has the effect of promoting the proliferation of T cells, and the two effects can be superimposed to achieve a better immune promotion effect (Figure 6). And the combined use of the two significantly increased the number of effector T cells (CD44+CD62L-) and memory T cells (CD44+CD62L+), suggesting a synergistic immune-promoting effect (Figure 7).

Example 9 Composition neutralizing effect of tumor acidic microenvironment

In this example, on the basis of Example 3, 1×10 ⁶ B16 cells were subcutaneously inoculated into adult female C57b/c mice to establish a tumor model. Seven days after inoculation, the mice were randomly divided into 2 groups (n=5) , respectively received the following treatment: ① saline group (Saline), intratumoral injection of saline 50 μL; ② composition group (Com), intratumoral injection of composition (polyvesicular liposome formulation) 50 μL (L-arg dose: 20mg /kg; CAT-2 shRNA plasmid dose: 0.7mg/kg). After one treatment, the pH changes in the tumor tissue were measured with a needle pH meter. The monitoring results of the pH value in the tumor tissue of the mouse show that the composition can continuously neutralize the acidic environment inside the tumor tissue and raise the pH of the environment to a neutral level during the release period ( FIG. 8 ).

Examination of the inhibitory effect of the composition of Example 10 on tumor growth

In this example, on the basis of Example 3, 1×10 ⁶ B16 cells were subcutaneously inoculated into adult female C57b/c mice to establish a tumor model. Seven days after inoculation, the mice were randomly divided into 4 groups (n=5) , received the following treatments: ① saline group (Saline), intratumoral injection of saline 50 μL, once a week; ② pure L-arginine composition group (L-arg), intratumoral injection without cationic amino acid transporter 2 The pure L-arginine composition of the inhibitor (multivesicular liposome dosage form) 50 μ L (L-arg dose: 20mg/kg), once a week; ③ pure cationic amino acid transporter 2 inhibitor composition group (iCAT-2 ), intratumoral injection of 50 μL of pure cationic amino acid transporter 2 inhibitor composition (multivesicular liposome dosage form) without L-arginine (CAT-2 shRNA plasmid dose: 0.7 mg/kg), once a week; ④ Combination Composition group (Com), intratumoral injection composition (multivesicular liposome formulation) 50μL (L-arg dose: 20mg/kg; CAT-2 shRNA plasmid dose: 0.7mg/kg), once a week, a total of two treatments cycle. Body weight and tumor volume changes were recorded during treatment, and when the body weight loss rate exceeded 10% or the tumor volume was greater than 1500 mm ³ , the mice were judged to be dead. The tumor volume curve shows that neither L-arg nor iCAT-2 treatment alone can cause effective tumor rejection, because the former has the effect of supporting tumor growth, while the latter cannot improve the immunosuppressive environment, but the combination effectively inhibits melanin Tumor growth (Figure 9). During the treatment, the body weight of the mice in the composition group did not decrease significantly ( FIG. 10 ), and the survival period was greatly prolonged ( FIG. 11 ). The above results show that the composition of the present invention can inhibit tumor growth from the aspects of environmental improvement, nutrient supplementation and limitation of cancer cell competitiveness.

The research of embodiment 11 composition anti-tumor mechanism

T细胞(CD44-CD62L+，绿)(图12，13)。流式结果说明，组合物改变了肿瘤组织中巨噬细胞的极化模式，M1型巨噬细胞比例显著提高，具有免疫抑制和促瘤发展的M2型巨噬细胞比例降低(图14)。细胞因子水平结果与上述实验结果相符，干扰素γ(IFN-γ)及NO水平在组合物组中明显提高，提示组合物产生了显著的抗肿瘤免疫反应，且白介素4(IL-4)水平没有明显变化，说明没有显著的免疫抑制反应发生(图15)。In this example, on the basis of Example 9, the tumor tissues of each group of mice were taken and sectioned for immunofluorescent staining to observe the infiltration and differentiation of T cells in the tissues. Tissue samples were taken, processed into a single cell suspension, and the ratio changes of M1 macrophages and M2 macrophages were analyzed by flow cytometry. After the tissue sample was homogenized, the supernatant was collected by centrifugation, and the changes of cytokines in the tissue were detected by an ELISA kit. Sectional results showed that the composition significantly increased the number of CD8+ T cells in tumor tissue, and they were mostly effector cells (CD44+CD62L-, red) and memory cells (CD44+CD62L+, yellow), not observed in the other three groups Arrived

T cells (CD44-CD62L+, green) (Fig. 12, 13). The results of flow cytometry showed that the composition changed the polarization pattern of macrophages in tumor tissue, the proportion of M1 macrophages was significantly increased, and the proportion of M2 macrophages with immunosuppression and tumor promotion was decreased ( FIG. 14 ). The results of cytokine levels were consistent with the above experimental results. The levels of interferon gamma (IFN-γ) and NO were significantly increased in the composition group, suggesting that the composition produced a significant anti-tumor immune response, and the level of interleukin 4 (IL-4) There was no significant change, indicating that no significant immunosuppressive reaction occurred ( FIG. 15 ).

Study on the therapeutic effect of the composition of Example 12 combined with anti-PD-1 antibody

In this example, on the basis of Example 9, 1×10 ⁶ B16 cells were subcutaneously inoculated into adult female C57b/c mice to establish a tumor model. After 7 days of inoculation, the mice were randomly divided into 3 groups (n=5) , received the following treatments: ① Anti-PD-1 antibody group (anti-PD-1), intraperitoneal injection of anti-PD-1 antibody (12.5mg/kg), once a week; ② Composition group (Com), intratumoral injection of combination (multivesicular liposome dosage form) 50 μL (L-arg dose: 20 mg/kg; CAT-2 shRNA plasmid dose: 0.7 mg/kg), once a week; ③ combined group (anti-PD-1&Com), intratumoral injection Injection composition (multivesicular liposome dosage form) 50μL (L-arg dose: 20mg/kg; CAT-2 shRNA plasmid dose: 0.7mg/kg), and intraperitoneal injection of anti-PD-1 antibody (12.5mg/kg), once a week. A total of two cycles of treatment were performed. Body weight and tumor volume changes were recorded during the treatment period. When the weight loss rate exceeded 10% or the tumor volume was greater than 1500 mm ³ , the mice were determined to be dead. The tumor volume curve shows (Fig. 16) that administration of anti-PD-1 antibody alone cannot produce an effective inhibitory effect on B16 tumors, while the composition of the present invention can reverse tumor non-responsiveness to immunotherapy and produce significant inhibition of tumor growth effect, improve the effect of immunotherapy, and did not cause significant weight loss, with good safety (Figure 17).

sequence listing

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Claims (3)

1. A composition targeting immune cells to replenish arginine and neutralize the acid environment for a long time, characterized in that the composition consists of L-arginine, cationic amino acid transporter 2 inhibitor and pharmaceutically acceptable The sustained-release drug storage carrier composition, the cationic amino acid transporter 2 inhibitor is shRNA or siRNA based on the cationic amino acid transporter 2 gene sequence; Wherein the shRNA is a short hairpin RNA based on the sequence of the cationic amino acid transporter 2 gene, and its sequence is: (1) gctttatgccctatggctttatcaagagataaagccataggggcataaagctttttt; (2) cgtccttacttgtctgctttattcaagagataaagcagacaagtaaggacgttttttt; (3) cggcctttgctatgctgaatttcaagagaattcagcatagcaaaggccgtttttt; siRNA is a small interfering RNA based on the sequence of the cationic amino acid transporter 2 gene, and its sequence is: (1) cgaaauacgggauaccgaaau; (2) gcaggaaugaacagacgaaau; (3) gccggaaacgauacgacuua; The concentration of L-arginine is 10-160 mg/mL, when the cationic amino acid transporter 2 inhibitor is shRNA, its concentration is 0.1-5 mg/mL, when the cationic amino acid transporter 2 inhibitor is siRNA, its The concentration is 10-200 nmol/mL; When the cationic amino acid transporter 2 inhibitor is shRNA or siRNA, a gene delivery carrier needs to be used in combination, and the gene delivery carrier includes cationic liposomes, cationic nanoemulsions or solid lipid nanoparticles; The types of sustained-release drug depot vehicles include microspheres, gels or multivesicular liposomes. 2. The application of the composition according to claim 1 in the preparation of antitumor drugs, characterized in that the tumor is melanoma. 3. The application according to claim 2, characterized in that the anti-tumor is administered through the composition alone, or combined with immunotherapy, surgical treatment, chemotherapy and radiotherapy at the same time.

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