Application of nutritional agent for removing BCAA in medicine preparation

Abstract

The invention discloses application of a nutritional agent for removing BCAA in preparation of a medicine, belongs to the technical field of biological medicine, and provides application of the nutritional agent for removing BCAA in preparation of the medicine, and the medicine is a medicine for enhancing tumor immunity and resisting PD-L1. The invention further provides application of the nutritional agent for removing BCAA in preparation of tumor immunotherapy drugs. The invention finds that the nutritional preparation without BCAA can improve the anti-PD-1 / PD-L1 tumor immunotherapy effect by reducing the expression of PD-L1 protein. Therefore, the deletion of BCAA is an effective target for down-regulating PD-L1 expression and improving the curative effect of anti-PD-1 / PD-L1 therapy. The biological function of the BCAA branched chain amino acid is widened, and a new target is provided for tumor immunotherapy and drug development.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of BCAA-removed nutritional agents in the preparation of medicines. Background Art

[0002] Both PD-1 and PD-L1 are immune checkpoint proteins. PD-1 is expressed on tumor-infiltrating immune cells, while PD-L1 is expressed on antigen-presenting cells and tumor cells. Both can negatively regulate adaptive anti-tumor immune responses. PD-L1 expression enables tumor cells to escape immune surveillance, and inhibition of PD-L1 can promote anti-tumor immunity. Anti-PD-1 / PD-L1 monoclonal antibodies have changed the treatment landscape of many advanced cancers, including melanoma, lung cancer, breast cancer, kidney cancer, and many other cancers. More and more studies have shown that anti-PD-1 / PD-L1 tumor immunotherapy has achieved good long-term effects; however, only 20% to 40% of patients will respond, and even fewer patients will achieve long-term remission of the disease. The key reason is the sustainable and stable expression of PD-L1. Therefore, it is particularly urgent to reduce PD-L1 levels and improve the anti-PD-1 / PD-L1 tumor immunity effect by taking other approaches, and to achieve anti-tumor effects through combined therapy.

[0003] Branched-chain amino acids (BCAAs) are three common amino acids in proteins, namely leucine, valine and isoleucine, collectively known as branched-chain amino acids. BCAA metabolic reprogramming can not only meet the nutritional needs of tumor cells, but also regulate many tumorigenesis processes and ultimately lead to their malignant transformation. BCAAs are not only directly involved in protein synthesis and provide nitrogen sources for the generation of macromolecules such as nucleic acids, but are also indispensable substances for the growth of human tumor cells. They can also be linked to other metabolic pathways involved in tumorigenesis through degradation of substances such as glutamate. BCAA metabolic reprogramming is very important in many human tumors, including malignant gliomas, pancreatic ductal adenocarcinoma (PDAC), leukemia, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, renal clear cell carcinoma (ccRCC), osteosarcoma, endometrial cancer and hepatocellular carcinoma (HCC). BCAA metabolic changes are critical in tumor development, stem cell maintenance and drug resistance. BCAA metabolism as a target is a very promising tumor treatment method. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes the use of a nutrient preparation with BCAA (branched-chain amino acids) removed in the preparation of a drug, wherein the drug is a drug for enhancing tumor immunity against PD-L1, and the removal of BCAA to reduce PD-L1 protein expression is used as an entry point. The nutrient preparation with BCAA removed is used as a complementary application to improve anti-PD-1 / PD-L1 tumor immunotherapy, which not only broadens the biological function of BCAA, but also provides a new method for tumor immunotherapy.

[0005] To achieve the above objectives, the present invention provides the use of a nutrient agent from which BCAA is removed in the preparation of a drug, wherein the drug is a drug for enhancing tumor immunity against PD-L1.

[0006] Preferably, the BCAA-removing nutrient specifically removes BCAA composed of leucine, isoleucine and valine.

[0007] Preferably, the BCAA-removed nutritional agent comprises pharmaceutically acceptable excipients.

[0008] Preferably, the nutrient agent from which BCAA is removed is one of powder, granule or liquid preparation, and the nutrient agent from which BCAA is removed is an oral preparation or an intravenous preparation.

[0009] Preferably, the BCAA-removed nutrient improves the anti-PD-1 / PD-L1 tumor immunotherapy efficacy by downregulating PD-L1 protein expression.

[0010] The present invention also provides a drug for enhancing tumor immunity against PD-L1, comprising the nutrient from which BCAA is removed.

[0011] Preferably, the drug for enhancing tumor immunity against PD-L1 also includes a pharmaceutically acceptable excipient.

[0012] The present invention also provides the use of the BCAA-removed nutrient in the preparation of tumor immunotherapy drugs.

[0013] The present invention also provides the use of the BCAA-removed nutrient in the preparation of breast cancer immunotherapy drugs.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention uses a nutritional preparation that removes branched-chain amino acids (BCAA) as a preparation for improving anti-PD-1 / PD-L1 tumor immunotherapy. Studies have found that nutritional preparations that remove BCAA can improve the anti-PD-1 / PD-L1 tumor immunotherapy effect by reducing the expression of PD-L1 protein. Therefore, BCAA deficiency is an effective target for downregulating PD-L1 expression and improving the efficacy of anti-PD-1 / PD-L1 therapy. The present invention broadens the biological functions of branched-chain amino acids and also provides new targets for tumor immunotherapy and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 The results of the measurement of the effect of BCAA on mouse tumor growth, where A is a graph showing the changes in tumor volume in nude mice without an immune system, B is a statistical graph showing the changes in tumor volume in nude mice without an immune system, C is a graph showing the changes in tumor volume in mice with an immune system, and D is a statistical graph showing the changes in tumor volume in mice with an immune system; Figure 2 This is a diagram showing the effect of BCAA stimulation on PD-L1 expression in breast cancer cells, where A is the Western Blot detection of BCAA-related pathway proteins BCAT1 / BCKDHA / BCKDK and PD-L1 protein, B is the correlation between BCAT1 and immune infiltration detected by a public database, C is the effect of BCAT1 and CD8+T expression levels on breast cancer survival, and D is the detection of the fluorescence expression level of PD-L1 under BCAA stimulation; Figure 3 Figure 2 is a graph showing that BCKDHA, a key enzyme in BCAA metabolism, regulates PD-L1 expression, where A is the expression of BCKDHA in breast cancer, B is the correlation between BCKDHA and breast cancer survival rate, C is the expression of PD-L1 after overexpression of BCKDHA, D is the expression of PD-L1 after knocking down BCAT1, E is the expression of PD-L1 after knocking down BCKDHA by siRNA in breast cancer cells HCC1937, F is the expression of PD-L1 after knocking down BCKDHA by siRNA in breast cancer cells MDA-MB-231, and G is the change in PD-L1 expression after knocking down BCKDHA by siRNA after BCAA stimulation; Figure 4The effects of BCAA stimulation on PD-L1 protein degradation, A is the expression of PD-L1 after BCAA stimulation or MG132 treatment, B is the effect of BCAA stimulation on PD-L1 ubiquitination and degradation in breast cancer cells HCC1937, and C is the effect of BCAA stimulation on PD-L1 ubiquitination and degradation in breast cancer cells MCF7; Figure 5 This is a graph showing the expression of BCKDHABCAT1 and PD-L1 detected by immunohistochemistry in breast cancer patients; Figure 6 This is a graph showing the expression of BCKDHA, BCAT1 and PD-L1 detected by multiple immunofluorescence in breast cancer patients; Figure 7 To remove the effect of BCAA(-) combined with atezolizumab in the treatment of triple-negative breast cancer; Figure 8 Diagram of the treatment model for tumors using BCAA inhibition combined with anti-PD-1 / PD-L1 therapy. DETAILED DESCRIPTION

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0020] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0021] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0022] Example 1. BCAA branched-chain amino acids promote tumor growth in mice via immune system dependency Babl / c nude mice (without immune system) were inoculated with MCF7 cells and divided into 4 groups with at least 5 mice in each group. The initial tumor volume was approximately 100-150mm 3 (Mouse tumor volume V = 1 / 2ab 2 , a is the longer side, b is the shorter side). In order to study the effect of BCAA on tumor growth, BCAA components are present in mouse feed, and a mouse feed with BCAA removed is customized (eliminating the effect of BCAA in the feed itself). The mice were fed with BCAA-removed feed and divided into four groups: BCAA (-); BCAA (-) + 200mM BCAA; BCAA (-) + 400mM BCAA; BCAA (-) + 800mM BCAA. The effect of BCAA on mouse tumor growth was explored by exogenous addition of different concentrations of BCAA under the condition of BCAA-removed feed. Mice were gavaged with different concentrations of BCAA three times a week for 14 days.

[0023] The results are as follows Figure 1 Middle A and Figure 1 As shown in B, after 14 days of treatment, there was little difference in the size of tumors in each group of mice without an immune system. It was preliminarily determined that BCAA had no effect on tumor growth in mice without an immune system.

[0024] Furthermore, when Babl / c mice with preserved immune systems were inoculated with EMT6 mouse-derived breast cancer cells, the initial tumor volume was approximately 100-150 mm 3 (Mouse tumor volume V = 1 / 2ab 2 , a is the longer side, b is the shorter side). BCAA (-); BCAA (-) + 200mM BCAA; BCAA (-) + 400mM BCAA; BCAA (-) + 800mM BCAA were given respectively to explore the effect of adding different concentrations of BCAA on tumor growth in mice with retained immune system when feeding with BCAA-free feed. Mice were given different concentrations of BCAA by gavage three times a week for 14 days.

[0025] The results are as follows Figure 1 Middle C and Figure 1As shown in D, after 14 days of treatment, the tumor growth volume of mice in each group was quite different. Under the high concentration 800mM BCAA treatment condition, the tumor growth of mice was significantly accelerated, and was significantly higher than that of the other three groups. It is preliminarily judged that BCAA has the effect of promoting the proliferation of tumors in mice with immune systems.

[0026] 2. BCAA and metabolic pathway proteins BCAT1 / BCKDHA upregulate PD-L1 expression PD-L1 is mainly expressed on tumor cells. By binding to PD-1 protein on T cells, it exhausts and weakens the immune response of T cells, thereby inhibiting the immune response and promoting tumor proliferation. PD-L1 expression enables tumor cells to escape immune surveillance. Breast cancer cells MCF7 and SK-BR-3 were treated with BCAA for further immunofluorescence experiments.

[0027] The results are as follows Figure 2 As shown in A, after BCAA treatment of breast cancer cells MCF7 and HCC1937, it was found that the levels of BCKDHA, BCAT1 and BCKDK in the BCAA metabolic pathway were upregulated, and the PD-L1 level was also upregulated. Figure 2 Middle B is a public database to detect the correlation between BCAT1 and immune infiltration; Figure 2 Middle C is the effect of BCAT1 and CD8+T expression levels on breast cancer survival rate; Figure 2 Figure D in the middle is an immunofluorescence experiment that confirmed that BCAA treatment can upregulate PD-L1 levels, explaining why tumors in mice that retain their immune system can grow faster under BCAA feeding conditions. Mice that retain their immune system have T cells in their bodies, and PD-L1 expression on tumor cells is upregulated, which combines with PD-1 on immune T cells to inhibit T cell immune responses, ultimately causing tumor cells to escape immune surveillance and proliferate faster. However, nude mice do not have T cells in their bodies, and therefore cannot produce an immune response.

[0028] Figure 3 To confirm that BCKDHA / BCAT1, a key enzyme in BCAA metabolism, regulates PD-L1 expression. Figure 3 Middle A, shows the high expression of BCKDHA in breast cancer in the public database GEPIA; Figure 3 Middle B, is the correlation between BCKDHA and breast cancer survival rate in the public database GEPIA; Figure 3 Middle C, detection of PD-L1 expression after overexpression of BCKDHA in MCF7 cells; Figure 3 Middle D, downregulation of PD-L1 expression detected after knockdown of BCAT1 in HCC1937 cells; Figure 3Middle E, shows the expression of PD-L1 after knocking down BCKDHA using siRNA in breast cancer cells HCC1937; Figure 3 Middle F, shows the expression of PD-L1 after knocking down BCKDHA by siRNA in breast cancer cells MDA-MB-231. These data show that NCAA metabolism-related enzymes such as BCAT1 / BCKDHA can regulate PD-L1 expression. We further want to confirm whether BCAA stimulation depends on the BCAA metabolic pathway to upregulate PD-L1 expression, such as Figure 3 Middle G, PD-L1 expression was downregulated after BCAA stimulation and BCKDHA knockdown using siRNA, confirming that BCAA stimulation upregulates PD-L1 depending on BCKDHA metabolic enzymes.

[0029] 3. BCAA stimulation can inhibit PD-L1 ubiquitination and degradation PD-L1 is upregulated after BCAA stimulation in breast cancer cells. Figure 4 As shown in Figure A, it was found that the proteasome inhibitor MG132 can upregulate the expression of PD-L1. Further, when BCAA stimulation was used to upregulate PD-L1, the protein synthesis inhibitor CHX was given and the degradation rate of PD-L1 protein was slowed down, confirming that BCAA can inhibit the degradation rate of PD-L1. Figure 4 Medium B and Figure 4 As shown in C, it was confirmed in HCC1937 and MCF7 cells, respectively.

[0030] 4. Positive correlation between BCKDHA / BCAT1 and PD-L1 expression in breast cancer patients The expression of BCKDHA, BCAT1 and PD-L1 was detected in patients with triple negative breast cancer (TNBC) and hormone receptor positive (HR+ / HER2-). Multiple immunofluorescence assay, the results are as follows Figure 5 As shown in the figure, it was confirmed that BCAT1 / BCKDHA and PD-L1 expression were significantly positively correlated, with high expression in TNBC patients and low expression in HR+ / HER2- patients, and the three were co-localized. Figure 6 As shown, all three are highly expressed in TNBC patients and lowly expressed in HR+ / HER2- patients. This confirms that BCAA metabolism (key enzymes BCKDHA and BCAT1) and PD-L1 expression are positively correlated in clinical breast cancer patients.

[0031] 5. BCAA depletion combined with anti-PD-L1 monoclonal antibody therapy inhibits tumor progression It has been previously confirmed that BCAA can affect PD-L1 expression. In addition, BCAA-depleted feed was used in combination with anti-PD-L1 monoclonal antibody Atezolizumab. BCAA-depleted mouse feed was used (eliminating the effect of BCAA in the feed itself). Furthermore, Babl / c mice with preserved immune systems were inoculated with EMT6 mouse-derived breast cancer cells. The initial tumor volume was approximately 100-150mm 3 (Mouse tumor volume V = 1 / 2ab 2 , a is the longer side, b is the shorter side). The mice were divided into four groups: control group; atezolizumab group; BCAA (-) group; BCAA (-) + atezolizumab group. The mice were treated with anti-PD-L1 monoclonal antibody in the absence of BCAA feed, and the effect of BCAA removal on anti-PD-L1 monoclonal antibody therapy in mice was investigated. Atezolizumab was intraperitoneally injected three times a week for 14 days.

[0032] The results are as follows Figure 7 As shown in the results, atezolizumab alone and BCAA removal (-) could not significantly inhibit tumor growth, but the combination of the two significantly reduced tumor volume. It was confirmed that BCAA removal can enhance the effect of immunotherapy when PD-1 / PD-L1 monoclonal antibody is used (e.g. Figure 8 ).

[0033] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a nutrient agent from which BCAA is removed in the preparation of a medicine, characterized in that: The drug is a drug that enhances tumor immunity against PD-L1.

2. The application according to claim 1, characterized in that: The BCAA-removed nutrient specifically removes BCAA composed of leucine, isoleucine and valine.

3. The application according to claim 1, characterized in that: The BCAA-removed nutritional agent includes pharmaceutically acceptable excipients.

4. The use according to claim 1, characterized in that: The BCAA-removed nutrient is a powder, a granule or a liquid preparation, and the BCAA-removed nutrient is an oral preparation or an intravenous injection preparation.

5. The use according to claim 1, characterized in that: BCAA-depleted nutrients improve the efficacy of anti-PD-1 / PD-L1 tumor immunotherapy by downregulating PD-L1 protein expression.

6. A drug for enhancing tumor immunity against PD-L1, comprising the nutrient from which BCAA is removed as claimed in claim 1.

7. The drug for enhancing tumor immunity against PD-L1 according to claim 6, characterized in that: The drug for enhancing tumor immunity against PD-L1 also includes pharmaceutically acceptable excipients.

8. Use of the nutrient agent without BCAA as claimed in claim 1 in the preparation of tumor immunotherapy drugs.

9. Use of the nutrient agent without BCAA as claimed in claim 1 in the preparation of a drug for immunotherapy of breast cancer.

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