Glycine and serine restrictions in diet for cancer treatment

Abstract

The present invention provides a food composition comprising a plurality of amino acids or salts thereof wherein the amino acids or salts thereof comprise from about 0.0016 to 0.6 wt% of glycine or salts thereof and from about 0.00109 to 1.2 wt% of serine or salts thereof. Also provided is a method of treating cancer in a subject comprising partially or completely replacing or replacing the normal diet of the subject with the food composition, and optionally administering to the subject an effective therapeutic amount of an anti-PD-1 antibody or an anti-PD-L1 antibody.

Description

[0001] Incorporation by reference

[0002] All documents cited or referenced herein (including but not limited to all literature documents, patents, published patent applications cited herein) (“documents cited herein”), all documents cited or referenced in the documents cited herein, and any manufacturer's instructions, specifications, product specifications, and product sheets of any product mentioned in this document or incorporated herein by reference are hereby incorporated by reference and may be used in the practice of the present invention. More specifically, all cited documents are incorporated by reference as if each individual document had been specifically and individually indicated to be incorporated in the form of reference. Any Genbank sequence mentioned in the present invention is incorporated by reference, and the Genbank sequence is the Genbank sequence as of the earliest effective filing date of the present invention. Technical field

[0003] The present invention provides a food composition comprising a plurality of amino acids, wherein the plurality of amino acids comprises 0.0016 - 0.6 wt% glycine and 0.00109 - 1.2 wt% serine. Such a food composition can be used as a meal replacement for cancer patients, particularly a complete meal replacement. There is also provided a method for treating cancer in a subject in need thereof, comprising controlling the intake of glycine and serine by the subject such that the glycine and serine ingested by the subject account for about 0.0016 - 0.6 wt% and about 0.00109 - 1.2 wt% of the total amino acids ingested by the subject, respectively. Background art

[0004] Cancer is a disease in which cells grow uncontrollably and spread to other parts of the body. The proliferation and survival of cancer cells depend on the rapid and selective biosynthesis of proteins, nucleotides, and lipids. Serine and glycine are two non-essential amino acids that play important roles in this biosynthesis. They are also key for generating antioxidant defenses (Tajan M et al., (2021) Serine synthesis pathway inhibition cooperates with dietary serine and glycine limitation for cancer therapy. Nat Commun. 12(1):366).

[0005] Cancer cells require sufficient amounts of glycine and serine. They can upregulate aerobic glycolysis and convert 3-phosphoglycerate (3-PG) to serine catalyzed by PHGDH, PSAT1, and PSPH. De novo synthesized serine can then be converted to glycine via SHMT1 / 2. Inhibiting PHGDH, and thus inhibiting de novo synthesis of serine and glycine, has been shown to impair, for example, breast cancer cell proliferation, and increased PHGDH expression has been found to be associated with poor prognosis in, for example, breast, lung, and gastric cancers (Geeraerts SL et al., (2021) The ins and outs of serine and glycine metabolism in cancer. Nat Metab. 3(2):131-141).

[0006] Cancer cells can also rely on exogenous serine and glycine in the environment for optimal growth (Yang, M., Vousden, K. (2016) Serine and one-carbon metabolism in cancer. Nat Rev Cancer 16:650-662). Nutrients in the tumor microenvironment can be manipulated to halt cancer development. For example, the inhibitory effect of dietary serine and glycine restriction on tumor development in mice has been demonstrated. A diet lacking glycine and serine has no negative impact on mouse health, reduces tumor growth, and increases mouse survival (Maddocks et al., (2017) Modulating the therapeutic response of tumours to dietary serine and glycine starvation. Nature 544:372-376). When provided to people, this diet would consist of protein-free or low-protein foods and amino acid shakes. Optionally, cancer patients can consume meal replacements that do not contain glycine and serine, such as complete meal replacements.

[0007] However, the lack of glycine and serine may have unexpected side effects. For example, tumor cells may adapt to these extreme conditions or initiate alternative metabolic pathways. Removing these two amino acids from food may also have an adverse effect on non-tumor cells because serine and glycine are involved in normal cell metabolic pathways.

[0008] Citing or identifying any document in this application does not mean admitting that the document can be used as prior art for the present invention. Summary of the Invention

[0009] The inventors of the present application surprisingly found that when cancer cells are cultured in a medium without any glycine or serine, although the growth or proliferation rate is low, they show relatively high migratory ability. When the levels of glycine and serine in the medium increase, cancer cell migration decreases, but cell growth or proliferation is enhanced to a certain extent. Therefore, keeping cancer cells in an environment with appropriate levels of glycine and serine may be the best way to control tumor development. Other non-essential amino acids in the tumor environment have less or even negligible effects on tumor growth or metastasis in vitro. Feeding tumor-bearing mice with a diet having a glycine to total amino acid ratio in the range of 0.0016 - 0.6 wt% and a serine to total amino acid ratio in the range of 0.00109 - 1.2 wt% can inhibit tumor growth.

[0010] The inventors of the present invention also found that a glycine- and serine-free diet can increase the PD-L1 molecules on the surface of tumor cells by promoting the lactylation modification of PD-L1 (data not shown). When a diet having a glycine to total amino acid ratio of 0.0016 - 0.6 wt% and a serine to total amino acid ratio of 0.00109 - 1.2 wt% is provided to tumor-bearing mice, dietary restriction synergizes with anti-PD-1 therapy, and anti-PD-1 therapy alone has a minimal effect on, for example, the size of pMMR / MSS colorectal cancer. In subsequent clinical trials, a reduction in tumor size was observed in tumor patients receiving the same dietary restriction of glycine and serine in combination with anti-PD-1 treatment.

[0011] In addition, in a tumor microenvironment such as this, it has been found that simply restricting glycine and serine in the diet can promote the growth, proliferation, and survival of some immune cells (including CD4 + T cells and CD8 + T cells) and reduce regulatory T cells.

[0012] Accordingly, in a first aspect, the present invention provides a composition that can comprise a plurality of amino acids or their salts, wherein the plurality of amino acids or their salts can comprise about 0.0016 - 0.6 wt% of glycine or its salt and about 0.00109 - 1.2 wt% of serine or its salt.

[0013] The composition can be a food composition.

[0014] The amino acid or its salt may contain glycine or its salt in an amount of more than about 0.0016 wt%, more than about 0.2 wt%, or more than about 0.4 wt%. The amino acid or its salt may contain glycine or its salt in an amount of about 0.6 wt% or less, or about 0.4 wt% or less. In certain embodiments, the amino acid or its salt may contain about 0.2 - 0.6 wt% of glycine or its salt. In certain embodiments, the amino acid or its salt may contain about 0.41 wt% of glycine or its salt.

[0015] The amino acid or its salt may contain serine or its salt in an amount of more than about 0.00109 wt%, more than about 0.2 wt%, more than about 0.5 wt%, or more than about 0.8 wt%. The amino acid or its salt may contain serine or its salt in an amount of about 1.2 wt% or less, or about 0.8 wt% or less. In certain embodiments, the amino acid or its salt may contain about 0.2 - 1.2 wt% or about 0.5 - 1.2 wt% of serine or its salt. In certain embodiments, the amino acid or its salt may contain about 0.81 wt% of serine or its salt.

[0016] The amino acids in the food composition may include essential amino acids and non - essential amino acids, or their salts, each at an appropriate level.

[0017] The amino acids in the food composition may include essential amino acids or their salts, and the essential amino acids are selected from histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. In certain embodiments, the amino acids in the food composition may include all essential amino acids or their salts.

[0018] The amino acids in the food composition may further include non - essential amino acids or their salts, and the non - essential amino acids are selected from alanine, arginine, aspartic acid, cysteine, glutamine, proline, tyrosine, asparagine, and glutamic acid.

[0019] The amino acid may contain about 1 - 5 wt% of histidine. The amino acid may contain about 3 - 7 wt% of isoleucine. The amino acid may contain about 6 - 13 wt% of leucine. The amino acid may contain about 4 - 10 wt% of lysine. The amino acid may contain about 1 - 6 wt% of methionine. The amino acid may contain about 3 - 6 wt% of phenylalanine. The amino acid may contain about 2 - 7 wt% of threonine. The amino acid may contain about 0.5 - 4 wt% of tryptophan. The amino acid may contain about 3 - 7 wt% of valine.

[0020] The amino acids may comprise about 3-7 wt% alanine. The amino acids may comprise about 3-8 wt% arginine. The amino acids may comprise about 5-14 wt% aspartic acid. The amino acids may comprise about 1-4 wt% cysteine. The amino acids may comprise about 5-20 wt% glutamine. The amino acids may comprise about 3-6 wt% proline. The amino acids may comprise about 2-5 wt% tyrosine. The amino acids may comprise about 2-6 wt% asparagine. The amino acids may comprise about 10-15 wt% glutamic acid.

[0021] In certain embodiments, the amino acids may comprise about 18.34 wt% glutamine, about 10.57 wt% leucine, about 9.30 wt% aspartic acid, about 8.59 wt% lysine, about 7.18 wt% arginine, about 5.24 wt% valine, about 4.36 wt% phenylalanine, about 6.21 wt% alanine, about 4.75 wt% proline, about 5.61 wt% isoleucine, about 4.41 wt% threonine, about 2.23 wt% histidine, about 3.98 wt% tyrosine, about 2.62 wt% methionine, about 2.58 wt% tryptophan, and about 2.81 wt% cysteine, about 0.81 wt% serine and about 0.41 wt% glycine.

[0022] In certain embodiments, the amino acids may comprise about 4.86 wt% glutamine, about 9.82 wt% leucine, about 10.02 wt% aspartic acid, about 5.1 wt% lysine, and about 6.2 wt% arginine, about 6.1 wt% valine, about 4.2 wt% phenylalanine, about 4.8 wt% alanine, about 4.4 wt% proline, about 4.63 wt% isoleucine, about 4.5 wt% threonine, about 2.9 wt% histidine, about 3.0 wt% tyrosine, about 5.9 wt% methionine, about 3.5 wt% tryptophan, about 1.4 wt% cysteine, about 0.81 wt% serine, about 0.41 wt% glycine, about 12.85 wt% glutamic acid and about 4.6 wt% asparagine.

[0023] In certain embodiments, the amino acids may comprise about 6.1 wt% glutamine, about 11.08 wt% leucine, about 12.9 wt% aspartic acid, about 4.35 wt% lysine, about 6.4 wt% valine, about 4.1 wt% phenylalanine, about 6.35 wt% alanine, about 4.8 wt% proline, about 5.6 wt% isoleucine, about 5.5 wt% threonine, about 2.87 wt% histidine, about 3.4 wt% tyrosine, about 4.8 wt% methionine, about 2.07 wt% tryptophan, about 1.47 wt% cysteine, about 0.5 wt% serine, about 0.5 wt% glycine, about 13.2 wt% glutamic acid and about 4.01 wt% asparagine.

[0024] The amino acids in the food composition of the present invention may be L-amino acids or D-amino acids. In certain embodiments, the amino acids in the food composition of the present invention may be L-amino acids. The glycine contained in the food composition may be L-glycine. The serine contained in the food composition may be L-serine.

[0025] The food composition of the present invention can be formulated to provide at least the recommended daily essential amino acid intake (except methionine) based on the average daily total protein or amino acid consumption.

[0026] The food composition may further comprise one or more macronutrients and / or micronutrients. In certain embodiments, the food composition may comprise carbohydrates, fiber and / or fat as macronutrients. In certain embodiments, the food composition may comprise vitamins and / or minerals as micronutrients.

[0027] In certain embodiments, the food composition may comprise 15 - 35 wt% amino acids, about 30 - 70 wt% carbohydrates and about 10 - 50 wt% fat. In certain embodiments, the food composition may comprise 12.4 wt% amino acids, 14 wt% fat, 34.6 wt% carbohydrates and 5 wt% fiber.

[0028] The food composition may be in the form of a solid or a beverage.

[0029] The food composition may be a meal replacement, such as a complete meal replacement. The food composition can be used to partially or completely replace the normal diet of a subject.

[0030] The food composition may not be a natural food.

[0031] The food composition can be formulated to be administered one to eight times a day. In certain embodiments, the food composition can be formulated to be administered three to six times a day.

[0032] The present invention also provides a meal replacement, which may comprise the food composition of the present invention. The meal replacement may be a complete meal replacement.

[0033] In a second aspect, the present invention provides a pharmaceutical composition, which may comprise the food composition of the present invention and a pharmaceutically acceptable carrier.

[0034] The pharmaceutical composition may further comprise an anti-tumor agent.

[0035] In certain embodiments, the pharmaceutical composition may further comprise an anti-PD-1 antibody (such as an antagonistic anti-PD-1 antibody) or an anti-PD-L1 antibody (such as an antagonistic anti-PD-L1 antibody).

[0036] In a third aspect, the present invention provides a method for treating cancer in a subject, which may comprise controlling the intake of glycine and serine by the subject, wherein the glycine ingested by the subject may account for about 0.0016 - 0.6 wt% of the total amino acids ingested by the subject, and the serine ingested by the subject may account for about 0.00109 - 1.2 wt% of the total amino acids ingested by the subject.

[0037] The method may comprise administering the food composition of the present invention to the subject.

[0038] The method may comprise partially or completely substituting or replacing the normal diet of the subject with the food composition of the present invention. In certain embodiments, the method may comprise completely substituting or replacing the normal diet of the subject with the food composition of the present invention.

[0039] The method may comprise partially or completely substituting or replacing the normal diet of the subject within a time period of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or until a treatment endpoint is observed (such as tumor shrinkage).

[0040] The method may further comprise administering to the subject an effective therapeutic amount of an anti-PD-1 antibody (such as an antagonistic anti-PD-1 antibody) or an anti-PD-L1 antibody (such as an antagonistic anti-PD-L1 antibody).

[0041] The anti-PD-1 antibody may be nivolumab or pembrolizumab.

[0042] The anti-PD-L1 antibody may be atezolizumab, avelumab or durvalumab.

[0043] The subject may be human.

[0044] Cancer can be solid tumors, including but not limited to colorectal cancer (e.g., pMMR / MSS colorectal cancer), esophageal cancer, and nasopharyngeal cancer.

[0045] The present invention also provides the use of the food composition of the present invention in treating cancer or in preparing a medicament for treating cancer.

[0046] In a fourth aspect, the present invention provides a method for sensitizing tumor cells to anti-PD-1 or anti-PD-L1 therapy in a subject in need thereof, which may include controlling the glycine and serine intake of the subject, wherein the glycine ingested by the subject may account for about 0.0016 - 0.6 wt% of the total amino acids ingested by the subject, and the serine ingested by the subject may account for about 0.00109 - 1.2 wt% of the total amino acids ingested by the subject.

[0047] The method may include administering the food composition of the present invention to the subject.

[0048] The method may include partially or completely substituting or replacing the normal diet of the subject with the food composition of the present invention. In certain embodiments, the method may include completely substituting or replacing the normal diet of the subject with the food composition of the present invention.

[0049] The method may include partially or completely substituting or replacing the normal diet of the subject within a period of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or until a treatment endpoint is observed (e.g., tumor cells are sensitized).

[0050] The method may be carried out before anti-PD-1 or anti-PD-L1 therapy. Optionally, the method and anti-PD-1 or anti-PD-L1 therapy may be carried out simultaneously.

[0051] Tumor cells may be insensitive to anti-PD-1 or anti-PD-L1 therapy.

[0052] Tumor cells can be solid tumor cells, including but not limited to colorectal cancer (e.g., pMMR / MSS colorectal cancer), esophageal cancer, and nasopharyngeal cancer.

[0053] The present invention also provides the use of the food composition of the present invention in sensitizing tumor cells to anti-PD-1 or anti-PD-L1 therapy, or in preparing a medicament for sensitizing tumor cells to anti-PD-1 or anti-PD-L1 therapy.

[0054] In a fifth aspect, the present invention provides a pharmaceutical composition, which may comprise i) a food composition, which may comprise a plurality of amino acids (including essential and non-essential amino acids) or salts thereof, wherein the plurality of amino acids or salts thereof may comprise about 0 - 0.0015 wt% glycine or a salt thereof and about 0 - 0.00108 wt% serine or a salt thereof, and ii) an anti-PD-1 antibody (such as an antagonistic anti-PD-1 antibody) or an anti-PD-L1 antibody (such as an antagonistic anti-PD-L1 antibody).

[0055] Except for the glycine and serine levels, the food composition and the components in the composition are as described in the first aspect.

[0056] The present invention also provides a method for treating cancer in a subject, which may comprise i) controlling the intake of glycine and serine by the subject, wherein the glycine ingested by the subject may account for about 0 - 0.0015 wt% of the total amino acids ingested by the subject, and the serine ingested by the subject may account for about 0 - 0.00108 wt% of the total amino acids ingested by the subject, and ii) administering to the subject an effective therapeutically amount of an anti-PD-1 antibody (e.g., an antagonistic anti-PD-1 antibody) or an anti-PD-L1 antibody (such as an antagonistic anti-PD-L1 antibody). The method may comprise partially or completely replacing or substituting the normal diet of the subject with a food composition, wherein the amino acids or salts thereof in the food composition may comprise about 0 - 0.0015 wt% glycine or a salt thereof and about 0 - 0.00108 wt% serine or a salt thereof.

[0057] The present invention also provides a method for making tumor cells sensitive to anti-PD-1 or anti-PD-L1 therapy in a subject in need thereof, comprising controlling the intake of glycine and serine by the subject, wherein the glycine ingested by the subject may account for about 0 - 0.00015 wt% of the total amino acids ingested by the subject, and the serine ingested by the subject may account for about 0 - 0.00108 wt% of the total amino acids ingested by the subject. In certain embodiments, the method may comprise partially or completely replacing or substituting the normal diet of the subject with a food composition, wherein the amino acids or salts thereof in the food composition may comprise about 0 - 0.0015 wt% glycine or a salt thereof and about 0 - 0.00108 wt% serine or a salt thereof.

[0058] Except for the glycine and serine levels, the method is as defined in the third and fourth aspects.

[0059] By the following specific embodiments and examples, other features and advantages of the present invention will become apparent, and the embodiments and examples should not be construed as limiting. The contents of all references, Genbank entries, patents, and published patent applications cited in this application are hereby expressly incorporated herein by reference.

[0060] Accordingly, it is an object of the present invention not to encompass any previously known product, process of making a product, or method of using a product within the present invention, and thus the applicant reserves the right and hereby disclaims any previously known product, process, or method. It should also be noted that the present invention is not intended to include within the scope of the present invention any product, process, or method of making or using a product that does not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of the European Patent Convention), and thus the applicant reserves the right and hereby disclaims any previously described product, process of making a product, or method of using a product. In the practice of the present invention, it may be advantageous to comply with Article 53(c) of the European Patent Convention and Articles 28(b) and (c) of the Implementing Regulations of the European Patent Convention. All rights in any embodiments of any patented subject matter in the present application lineage or any other lineage or any previously filed application by any third party are expressly disclaimed. Nothing herein should be construed as a commitment.

[0061] It should be noted that in the present invention, particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising", etc. may have the meaning given to them by United States patent law; for example, they may mean "includes", "included", "including", etc.; and terms such as "consisting essentially of" and "consists essentially of" have the meaning given to them by United States patent law, for example, they allow elements not expressly recited, but exclude elements found in the prior art or elements that affect the basic or novel characteristics of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The following detailed description is given by way of example and is not intended to limit the present invention to the specific embodiments described, which may be best understood by reference to the accompanying drawings.

[0063] Figure 1 The % cell growth (upper panel) and % metastasis (lower panel) of A431 cells cultured in the presence or absence of different concentrations of glycine and serine are shown.

[0064] Figure 2 The % cell growth (upper panel) and % metastasis (lower panel) of MCF-7 cells cultured in the presence or absence of different concentrations of glycine and serine are shown.

[0065] Figure 3Shows the cell growth % (upper panel) and metastasis % (lower panel) of HeLa cells cultured in the presence or absence of different concentrations of glycine and serine.

[0066] Figure 4 Shows the cell growth % (upper panel) and metastasis % (lower panel) of HepG2 cells cultured in the presence or absence of different concentrations of glycine and serine.

[0067] Figure 5 Shows the cell growth % (upper panel) and metastasis % (lower panel) of PC3 cells cultured in the presence or absence of different concentrations of glycine and serine.

[0068] Figure 6 Shows the cell growth % (upper panel) and metastasis % (lower panel) of SKOV3 cells cultured in the presence or absence of different concentrations of glycine and serine.

[0069] Figure 7 Shows the cell growth % (upper panel) and metastasis % (lower panel) of A549 cells cultured in the presence or absence of different concentrations of glycine and serine.

[0070] Figure 8 Shows the cell growth % (upper panel) and metastasis % (lower panel) of U251 cells cultured in the presence or absence of different concentrations of glycine and serine.

[0071] Figure 9 Shows the cell growth % (upper panel) and metastasis % (lower panel) of K562 cells cultured in the presence or absence of different concentrations of glycine and serine.

[0072] Figure 10 Shows the cell growth % (upper panel) and metastasis % (lower panel) of Caco-2 cells cultured in the presence or absence of different concentrations of glycine and serine.

[0073] Figure 11 Shows the effect of non-essential amino acids other than glycine and serine on the growth of CT26 cells.

[0074] Figure 12 Shows the effect of non-essential amino acids other than glycine and serine on the metastasis of CT26 cells.

[0075] Figure 13 Shows the in vivo anti-tumor efficacy of glycine and serine restriction in the diet alone or in combination with anti-PD-1 therapy.

[0076] Figure 14Shows GZMB in patients before glycine and serine restriction in the diet and after 3 weeks (left panel) or 6 weeks (right panel) of this dietary intervention + CD4 + The proportion of T cells in CD4 + cells and GZMB + CD8 + The percentage of T cells in CD8 + cells. Detailed implementation manner

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, it should be understood that modifications and variations are included within the spirit and scope of the present invention.

[0078] Unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" used herein also include the plural forms. Additionally, if the terms "including", "includes", "having", "has", "with" or their variants are used in the detailed implementation manner and / or claims, these terms are intended to be inclusive in a manner similar to "comprising".

[0079] The term "about" or "approximately" means within an acceptable error range of a specific value determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, in accordance with the practice in the art, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of 20%, 15%, 10%, 5% or 1% of a given value.

[0080] "Essential amino acids" are amino acids that cannot be synthesized in the body and must be obtained from the daily diet, including histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine; while "non-essential amino acids" refer to amino acids that can be synthesized in the body, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine.

[0081] "Meal replacement" refers to a convenient and nutritionally balanced alternative to a traditional meal, usually provided in the form of a powder, ready-to-drink meal, or bar. A complete meal replacement or full-day meal replacement is designed to replace or substitute all meals in a day, including breakfast, lunch, dinner, and snacks between meals.

[0082] The term "sensitize" or "sensitizing" means to make a person or thing responsive to certain stimuli. For example, making a cell "sensitive" to anti-PD-1 therapy means making a cell that may not respond to anti-PD-1 responsive to the therapy.

[0083] The term "macronutrient" refers to nutrients that the body requires in large amounts, including fats, carbohydrates, proteins, and fiber.

[0084] The term "micronutrient" refers to elements that the body requires in small amounts, including vitamins and minerals. Examples of micronutrients are iron, cobalt, chromium, iodine, copper, zinc, molybdenum, vitamin C, and vitamin D.

[0085] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows, and horses.

[0086] The term "effective treatment amount" refers to the amount of a molecule or composition sufficient to prevent or improve symptoms associated with a disease or disorder (such as a tumor) and / or reduce the severity of the disease or disorder. The effective treatment amount is understood to be related to the disorder being treated, and the actual effective amount is readily recognizable to those skilled in the art.

[0087] As used herein, the terms "treatment / treating" refer to methods performed to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., not exacerbating) the disease state, delaying or slowing the progression of the disease, and alleviating symptoms (partially or completely), whether or not detectable. In addition, "treatment" also refers to an extended survival period compared to the expected survival period if no treatment is received. Beneficial or desired clinical outcomes described herein may include, but are not limited to, slowing tumor progression, cancer regression, enhancing the anti-tumor immune response, reducing tumor growth or size, tumor necrosis, reducing the severity of at least one disease symptom, increasing the frequency and duration of asymptomatic periods, preventing injury or disability due to disease distress, or otherwise improving the disease symptoms of a patient.

[0088] Amino acids are the building blocks of proteins and are essential for cell survival and proliferation. Amino acids have many important functions, including providing energy to enhance the immune response, helping with tissue growth and repair, and assisting with muscle function and development. Nine of these amino acids are essential and must be obtained from the daily diet. The ingested protein is digested and broken down into amino acids. The non-essential ones can be produced in the body.

[0089] Cancer cells reprogram their metabolism to support unrestricted proliferation and thus have a higher demand for amino acids such as serine and glycine. They may upregulate aerobic glycolysis, converting 3-phosphoglycerate (3-PG) to serine and then serine to glycine. They may also take up serine and glycine from the surrounding environment for optimal growth, proliferation, and survival. Studies have shown that a deficiency of glycine and serine in the diet, i.e., completely removing glycine and serine from the ingested food, may inhibit tumor growth and prolong the lifespan of tumor-bearing mice.

[0090] The inventors of the present application cultured cancer cells in media without or with different concentrations of glycine and serine and found that i) cancer cells cultured at relatively high levels of glycine and serine showed the highest growth or proliferation rate, and ii) cancer cells cultured on media without any glycine or serine showed the greatest migratory ability. Therefore, the inventors concluded that maintaining cancer cells in an environment with appropriate levels of glycine and serine may be the best way to control tumor development.

[0091] Based on the above findings, the inventors of the present invention designed a food composition in which the ratio of glycine to total amino acids is in the range of 0.0016 - 0.6 wt%, and the ratio of serine to total amino acids is in the range of 0.00109 - 1.2 wt%. Since the food composition is intended to be used as a meal replacement, especially a complete meal replacement, and the total daily intake is somewhat restricted, cancer patients who consume the meal replacement can maintain serum glycine and serine, or glycine and serine in the tumor environment, at relatively low levels.

[0092] According to the "Amino-acid content offoods and biological data on proteins" published by the Food and Agriculture Organization of the United Nations (FAO), as well as certain review or research papers (e.g., Gorissen SHM et al., Protein content and amino acid composition of commercially available plant-based protein isolates. Amino Acids. 50(12): 1685-1695; Kang JS. (2020) Dietary restriction of amino acids for Cancer therapy. Nutr Metab (Lond). 17: 20), there is no naturally occurring food or protein with a ratio of glycine / serine to total amino acids as low as the food composition of the meal replacement of the present invention.

[0093] Use of this food composition or meal replacement can avoid extreme dietary restrictions, such as a glycine-serine-free diet, which may disrupt the body's metabolism and may impair other physiological functions.

[0094] The food composition or meal replacement of the present invention, when orally administered to animals or humans, exhibits tumor suppressive effects in animal models, as shown in Example 3 below, and increases CD4 + GZMB + CD4 + The proportion of T cells and CD8 + GZMB + CD8 + Ratio of T cells. In other studies by the inventors, a diet free of glycine and serine resulted in a significant increase in the ratio of T cells, a sharp increase in naive T cells and effector T cells, and a significant decrease in suppressive regulatory T cells (Tregs).

[0095] In another study by the inventors, it was found that a diet lacking glycine and serine increases the PD-L1 molecules on the surface of tumor cells by promoting the lactylation modification of PD-L1. The lactylation modification of PD-L1 refers to the post-translational modification in which lactic acid molecules attach to the PD-L1 protein. In tumor cells, a hypoxic (low-oxygen) environment leads to an increased production of lactic acid. This lactic acid is taken up by monocarboxylate transporters (MCTs), which can modify proteins, including PD-L1, through a process called lactylation modification. A diet without glycine and serine significantly increased the PD-L1 protein level in tumor cells, while the mRNA level did not increase correspondingly, indicating that this regulation occurs post-translationally. Further analysis showed that hypoxic conditions promote the lactylation modification of PD-L1, stabilizing the protein and preventing its degradation. That is, lactylation modification inhibits the degradation of PD-L1 through the lysosomal pathway, thus maintaining a higher level of PD-L1 on the cell surface. Subsequently, the process of lactylation modification was manipulated using chemical reagents. Increasing lactylation modification could stabilize PD-L1, while inhibiting it led to faster degradation. These results provide strong evidence that lactylation modification plays a key role in regulating PD-L1 stability, which may contribute to the ability of tumors to evade the immune response.

[0096] In view of the above, the inventors of the present invention combined the restriction of glycine and serine in the diet with anti-PD-1 therapy for tumor treatment. The results showed that a glycine-serine-free diet and a low glycine-serine diet (the ratio of glycine to total amino acids is 0.0016 - 0.6 wt%, and the ratio of serine to total amino acids is 0.00109 - 1.2 wt%) synergistically inhibited colorectal cancer and esophageal cancer in combination with anti-PD-1 therapy, as shown in Examples 3 and 5 below, such as pMMR / MSS colorectal cancer (data not shown). It is known in the art that anti-PD-1 therapy alone shows minimal effect on pMMR / MSS colorectal cancer.

[0097] Accordingly, the present invention provides a food composition, which may comprise various amino acids or their salts, wherein the amino acids or their salts may comprise about 0.0016 - 0.6 wt% of glycine or its salt and about 0.00109 - 1.2 wt% of serine or its salt.

[0098] Since the food composition of the present invention is designed to partially or completely replace the normal diet of tumor patients, the amino acids should include all essential amino acids, as well as optionally some non-essential amino acids.

[0099] The amino acids present in the food composition of the present invention may be amino acids in free form, prodrug form, salt or amino acid ester. Amino acids with one or more N-terminal or C-terminal modifications, as well as homopolymers, homodimers, heteropolymers and heterodimers forms, may also be considered.

[0100] Unless otherwise specified herein, the food compositions of the present invention can be formulated to provide at least the recommended daily intake of essential amino acids based on the average daily total protein intake. Based on the average daily total protein intake, the Institute of Medicine recommends the following daily intakes of essential amino acids: histidine 18 mg / g protein; isoleucine 25 mg / g protein; leucine 55 mg / g protein; lysine 51 mg / g protein; methionine and cysteine together 25 mg / g protein; phenylalanine and tyrosine together 47 mg / g protein; threonine 27 mg / g protein; tryptophan 7 mg / g protein; and valine 32 mg / g protein.

[0101] For example, the Institute of Medicine recommends that adults consume 0.8 grams of protein per kilogram of body weight per day. The European Society for Palliative Care (ESPC) recommends that cancer patients consume at least 1.0 g / kg body weight of protein per day to maintain or regain lean body mass. During the recommended daily intake of the composition, the food composition can be formulated to provide at least 0.8 or 1.0 grams of protein per kilogram of body weight.

[0102] In addition, since the food compositions of the present invention are intended to partially or completely replace the normal diet of cancer patients, they should contain one or more macronutrients and / or micronutrients. The food composition can contain carbohydrates, fiber, and / or fat as macronutrients. The food composition can contain vitamins and / or minerals as micronutrients.

[0103] The Dietary Reference Intakes for Energy, Carbohydrates, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids, released by the Institute of Medicine in September 2002, provides guidance and recommended daily intakes for macronutrients. A non-exhaustive list of macronutrients that may be additional components of the food composition includes: carbohydrates, fiber, and fat (such as n-6 polyunsaturated fatty acids, n-3 polyunsaturated fatty acids, saturated fatty acids, and trans fatty acids, as well as cholesterol).

[0104] A non-exhaustive list of micronutrients includes vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, niacin, vitamin B6, folic acid, vitamin B12, pantothenic acid, biotin, choline, calcium, chromium, copper, fluoride, iodine, iron, magnesium, molybdenum, phosphorus, selenium, zinc, potassium, sodium, and chloride. Suitably, the food composition can be formulated to provide an acceptable or recommended daily intake, as detailed in the publication ("Dietary Reference Intakes: RDA and Al for Vitamins and Elements", NAS, IOM, Food and Nutrition Board).

[0105] The food composition can be formulated to be administered one to eight times a day, such as one to four times a day. Thus, the food composition can be formulated into suitable unit dosage forms.

[0106] The food composition of the present invention can be provided in the form of powder, gel, solution, suspension, paste, solid, liquid, liquid concentrate, reconstitutable powder, shake, concentrate, pill, bar, tablet, capsule, or ready-to-use product. It is contemplated that when the food composition is in the form of a tablet, pill, capsule, liquid, aerosol, injectable solution, or other pharmaceutically acceptable formulation, it can also be a pharmaceutical composition.

[0107] The present invention also provides a method for preparing the food composition of the present invention, wherein the amino acid is dissolved or dispersed in water and spray-dried.

[0108] The amino acid can be mixed with other ingredients, such as macronutrients and micronutrients. Binders, emulsifiers, or other ingredients suitable for human or animal consumption can be added as needed.

[0109] In another aspect, the present invention provides a method for treating cancer in a subject, which may include controlling the intake of glycine and serine by the subject, wherein the glycine ingested by the subject may account for about 0.0016 - 0.6 wt% of the total amino acids ingested by the subject, and the serine ingested by the subject may account for about 0.00109 - 1.2 wt% of the total amino acids ingested by the subject. The method may include partially or completely replacing or substituting the normal diet of the subject with the food composition of the present invention. The method can last, for example, 12 weeks.

[0110] As described above, a glycine-serine-free diet and a low glycine-serine diet (the ratio of glycine to total amino acids is 0.0016 - 0.6 wt%, and the ratio of serine to total amino acids is 0.00109 - 1.2 wt%) can increase the PD-L1 molecules on the surface of tumor cells by promoting the lactylation modification of PD-L1 and synergize with anti-PD-1 therapy.

[0111] Thus, in another aspect, the present invention provides a method for treating cancer in a subject, which may comprise i) controlling the intake of glycine and serine by the subject, wherein the glycine intake by the subject may account for about 0-0.6 wt% of the total amino acids ingested by the subject, and the serine intake by the subject may account for about 0-1.2 wt% of the total amino acids ingested by the subject, and ii) administering to the subject an effective therapeutic amount of an anti-PD-1 antibody (such as an antagonistic anti-PD-1 antibody) or an anti-PD-L1 antibody (such as an antagonistic anti-PD-L1 antibody). The method may comprise partially or completely substituting or replacing the normal diet of the subject with a food composition, wherein the amino acids or their salts in the food composition may comprise about 0-0.6 wt% of glycine or its salt and about 0-1.2 wt% of serine or its salt.

[0112] The method may comprise partially or completely substituting or replacing the normal diet of the subject within a time period of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or until a treatment endpoint is observed (such as tumor shrinkage).

[0113] The cancer may be a solid tumor, including but not limited to colorectal cancer (such as pMMR / MSS colorectal cancer), esophageal cancer, and nasopharyngeal cancer.

[0114] The present invention also provides a method for sensitizing tumor cells to anti-PD-1 or anti-PD-L1 therapy in a subject in need thereof, comprising controlling the intake of glycine and serine by the subject, wherein the glycine intake by the subject may account for about 0-0.6 wt% of the total amino acids ingested by the subject, and the serine intake by the subject may account for about 0-1.2 wt% of the total amino acids ingested by the subject. The method may comprise partially or completely substituting or replacing the normal diet of the subject with the food composition of the present invention, wherein the amino acids or their salts in the food composition may comprise about 0-0.6 wt% of glycine or its salt and about 0-1.2 wt% of serine or its salt.

[0115] The method may comprise partially or completely substituting or replacing the normal diet of the subject within a time period of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or until a treatment endpoint is observed (such as tumor cells being sensitized).

[0116] The method may be carried out before anti-PD-1 or anti-PD-L1 therapy. Alternatively, the method and anti-PD-1 or anti-PD-L1 therapy may be carried out simultaneously.

[0117] The present invention will be further illustrated in the following embodiments, which are for illustrative purposes only and are not intended to limit the present invention in any way.

[0118] Embodiment

[0119] Example 1: Effects of serine or glycine treatment on tumor cell growth and migration

[0120] The effects of serine or glycine treatment on tumor cell growth or migration were measured in 10 tumor cell lines, including A431, MCF-7, HeLa, HepG2, PC3, SKOV3, A549, U251, K562, and Caco-2.

[0121] Briefly, 1×10 4 tumor cells were seeded on 96-well plates and cultured for 48 hours at 37°C in 200 μl of amino acid-free high-glucose DMEM medium (Gibco, A14430-01) supplemented with 200 mg / L of anhydrous calcium chloride, 0.1 mg / L of ferric nitrate nonahydrate, 400 mg / L of potassium chloride, 97.67 mg / L of anhydrous magnesium chloride, 125 mg / L of anhydrous NaH2PO4, 84 mg / L of L-arginine hydrochloride, 84 mg / L of L-cystine hydrochloride, 584 mg / L of L-glutamine, 42 mg / L of L-histidine hydrochloride, 105 mg / L of L-isoleucine, 105 mg / L of L-leucine, 146 mg / L of L-lysine hydrochloride, 95 mg / L of L-threonine, 16 mg / L of L-tryptophan, 104 mg / L of sodium tyrosine, 94 mg / L of L-valine, 4 mg / L of calcium D-pantothenate, 4 mg / L of folic acid, 7.2 mg / L of inositol, 4 mg / L of nicotinamide, 0.4 mg / L of riboflavin, 4 mg / L of ammonium chloride, 4 mg / L of pyridoxine hydrochloride, 110 mg / L of sodium pyruvate, and 15 mg / L of phenol red, (i) without serine or glycine (control), (ii) 0.00109, 0.2, 0.5, 1.2, or 1.5 wt% serine and 1.7 wt% glycine (based on the total amino acid content), (iii) 0.0016, 0.2, 0.4, 0.6, or 0.8 wt% glycine and 2.3 wt% serine (based on the total amino acid content), or (iv) 0.00109 wt% serine and 0.0016 wt% glycine, 0.2 wt% serine and 0.2 wt% glycine, 0.5 wt% serine and 0.4 wt% glycine, 1.2 wt% serine and 0.6 wt% glycine, or 1.5 wt% serine and 0.8 wt% glycine (based on the total amino acid content). All non-essential amino acids were from Sigma-Aldrich.

[0122] Then, MTT assays and Transwell migration assays were performed on the cells. Each test was conducted three times to ensure reproducibility.

[0123] According to the manufacturer's manual, the MTT assay was performed using an MTT kit (Beyotime, ST316). The absorbance at 570 nm was measured in a microplate reader to reflect the number of viable cells. The group with the highest OD570 value was set as the control group, and (OD570 of a certain group / OD570 of the control group) × 100% was calculated as the growth percentage.

[0124] According to the manufacturer's manual, insert (8.0 μm pore size, catalog number 3422) was used for the Transwell migration assay at an initial cell density of 1×10 5 cells per well. The group with the highest cell count was set as the control group, and (cell count of a certain group / cell number of the control group) × 100% was calculated as the migration or metastasis percentage.

[0125] The results are as Figures 1 to 10 shown. It can be seen that compared with the cells treated with high levels of homoserine and glycine, the tumor cells cultured with low serine levels or low glycine levels grew slowly but showed relatively high migration ability. The control group cells cultured in cell medium without serine or glycine also grew slowly but showed the highest migration activity.

[0126] The data indicate that the presence of serine or glycine at relatively low levels, rather than the absence of both amino acids, can effectively inhibit the growth, proliferation, and migration of tumor cells. In other words, the presence of serine and glycine at appropriate levels may control the development and metastasis of tumors.

[0127] Example 2: Effects of other non-essential amino acids on tumor cell growth and migration

[0128] The effects of other non-essential amino acids on tumor cell growth or migration were determined using CT26 tumor cells (ATCC, CRL-2638) in the absence of serine or glycine, or in the presence of different levels of serine and glycine.

[0129] Briefly, 1×10 4CT26 cells were seeded into 96-well plates and cultured in 200 μl of amino acid-free DMEM high-glucose medium (Gibco, A14430-01) supplemented with 10% amino acid-free fetal bovine serum (Sorfa, SX1500), 1% penicillin / streptomycin / amphotericin B, 4% MEM amino acid solution (Gibco, 11130036), and non-essential amino acids at 37 °C for 48 hours as described below. All non-essential amino acids were purchased from Sigma-Aldrich.

[0130] For the cells in Group 1 (Ctrl(+) group), the cell medium contained 9 non-essential amino acids at basal concentrations, namely L-arginine hydrochloride at 84 mg / L, L-alanine at 2.5 mg / L, L-glutamic acid at 75 mg / L, L-glutamine at 584 mg / L, L-proline at 4 mg / L, sodium tyrosine at 104 mg / L, L-cysteine hydrochloride at 84 mg / L, L-aspartic acid at 2 mg / L, and L-asparagine at 5 mg / L, plus 1 of the 9 non-essential amino acids with a 50% increase. For example, in the Ctrl(-) arginine group, the cell medium contained L-arginine hydrochloride at 126 (84 * 150%) mg / L, L-alanine at 2.5 mg / L, L-glutamic acid at 75 mg / L, L-glutamine at 584 mg / L, L-proline at 4 mg / L, sodium tyrosine at 104 mg / L, L-cysteine hydrochloride at 84 mg / L, L-aspartic acid at 2 mg / L, and L-asparagine at 5 mg / L. Serine or glycine was not added to the cells in this group.

[0131] For the cells in Group 2 (Mid-Add(+) group), the cell medium contained i) 1.2 wt% serine and 0.6 wt% glycine (both based on total amino acids), and ii) 9 non-essential amino acids at basal concentrations, plus 1 of the 9 non-essential amino acids with a 50% increase.

[0132] For the cells in Group 3 (HighAdd(+) group), the cell medium was supplemented with i) 5 wt% serine and 2.5 wt% glycine (both based on total amino acids), and ii) 9 non-essential amino acids at basal concentrations, plus 1 of the 9 non-essential amino acids with a 50% increase.

[0133] For the cells of Group 4 (Ctrl(-) group), the cell culture medium contained 8 out of the 9 non-essential amino acids (i.e., arginine, L-alanine, L-glutamic acid, L-glutamine, L-proline, tyrosine, L-cystine, L-aspartic acid, and L-asparagine) at the basal concentration as described above. For example, for the cells from the Ctrl(-) arginine group, the cell culture medium contained the 9 non-essential amino acids except arginine, i.e., 84 mg / L of L-arginine hydrochloride, 2.5 mg / L of L-alanine, 75 mg / L of L-glutamic acid, 584 mg / L of L-glutamine, 4 mg / L of L-proline, 104 mg / L of sodium tyrosine, 84 mg / L of L-cystine hydrochloride, 2 mg / L of L-aspartic acid, and 5 mg / L of L-asparagine. Serine or glycine was not added to the cells of this group.

[0134] For the cells of Group 5 (Mid-Add(-) group), the cell culture medium contained i) 1.2 wt% serine and 0.6 wt% glycine (both based on total amino acids), and ii) 8 out of the 9 non-essential amino acids at the basal concentration as described above, i.e., arginine, L-alanine, L-glutamic acid, L-glutamine, L-proline, tyrosine, L-cystine, L-aspartic acid, and L-asparagine.

[0135] For the cells of Group 6 (HighAdd(-) group), the cell culture medium contained i) 5 wt% serine and 2.5 wt% glycine (both based on total amino acids) added at the above-mentioned base concentration, and ii) 8 out of the 9 non-essential amino acids at the basal concentration as described above, i.e., arginine, L-alanine, L-glutamic acid, L-glutamine, L-proline, tyrosine, L-cystine, L-aspartic acid, and L-asparagine.

[0136] The cells were subjected to MTT assay and Transwell migration assay according to the protocol of Example 1. The experiments were performed in triplicate.

[0137] The results were summarized in Figure 11 and Figure 12 as follows.

[0138] As Figure 11 shown, there were some differences in tumor growth between the Ctrl(+) group and the Ctrl(-) group, between the Mid-Add(+) group and the Mid-Add(-) group, and between the High-Add(+) group and the High-Add(-) group, but tumor growth mainly depended on the serine and glycine levels. The increase in serine and glycine levels promoted tumor growth.

[0139] As Figure 12As shown, although there were differences between the Ctrl(+) and Ctrl(-) groups, the Mid-Add(+) and Mid-Add(-) groups, and the High-Add(+) and High-Add(-) groups, tumor cell migration decreased with increasing serine and glycine levels. Cells in the Ctrl(+) and Ctrl(-) groups showed the highest migration ability, indicating easy migration. Cells from the Mid-Add(+) and Mid-Add(-) groups showed lower migration ability than the Ctrl(+) and Ctrl(-) groups but higher than the High-Add(+) and High-Add(-) groups.

[0140] The above data indicate that appropriate levels of serine and glycine inhibit tumor development and metastasis, and the other nine non-essential amino acids have little effect on tumor growth or metastasis.

[0141] Example 3: Antitumor efficacy of dietary serine and glycine restriction alone or in combination with anti-PD-1 therapy

[0142] The inhibitory effect of serine and glycine restriction alone or in combination with anti-PD-1 therapy on tumor growth was tested in BALB / c mice.

[0143] Briefly, male BALB / c mice aged 6 - 8 weeks were divided into 8 groups and injected subcutaneously with 5×10 5 CT26 cells on the right side and started receiving the diet according to the following experimental design on day 0.

[0144] Mice in group 1 (Low_PD-1) and group 2 (Low_No_PD-1) were fed a diet (BioPike, LLC) containing serine and glycine at 0.00109% and 0.0016% of total amino acids, respectively.

[0145] Mice in group 3 (Moderate_PD-1) and group 4 (Moderate_No_PD-1) were fed a diet containing serine and glycine at 0.2% and 0.2% of total amino acids, respectively.

[0146] Mice in group 5 (Medium_PD-1) and group 6 (Medium_No_PD-1) were fed a diet containing serine and glycine at 1.2% and 0.6% of total amino acids, respectively.

[0147] Mice in group 7 (High_PD-1) and group 8 (High_No_PD-1) were fed a diet containing serine and glycine at 1.5% and 0.8% of total amino acids, respectively.

[0148] On days 7, 10, and 13, mice in groups 1, 3, 5, and 7 were further intraperitoneally injected with 100 μl of PBS containing 100 μg of anti-PD-1 antibody (Junshi Biosciences) or 100 μl of PBS (as a vehicle control).

[0149] The tumor size of the mice was measured on days 3, 7, 10, 12, and 13.

[0150] As Figure 13 shown, significant tumor suppression was observed in the mice of the Moderate_PD-1 group and the Medium_PD-1 group, followed by the mice of the Low_PD-1 group. In addition, the tumors of the mice from the Moderate_No_PD-1 group, the Medium_No_PD-1 group, and the Low_No_PD-1 group were larger than those of the mice in the above three groups, and the tumors of these three groups were much smaller than those of the Low_No_PD-1 and High_No_PD-1 groups.

[0151] The results indicate that serine and glycine restriction in the diet, rather than deficiency, can significantly inhibit tumor growth, and the anti-tumor activity of this dietary restriction can be enhanced by anti-PD-1 therapy.

[0152] Example 4: Medical food formulation and amino acid composition for dietary serine and glycine restriction

[0153] An amino acid composition with appropriate serine and glycine levels can be prepared and used to prepare a medical food composition for tumor patients. The medical food composition may contain other nutrients that the patient may need, including carbohydrates, proteins, and vitamins.

[0154] Table 1 below provides an exemplary medical food formula.

[0155] Table 1: Exemplary medical food formula

[0156]

[0157]

[0158]

[0159] The protein in the above formula may have the amino acid composition listed in Table 2.

[0160] Table 2: Exemplary amino acid composition

[0161]

[0162]

[0163] An exemplary medical food composition is obtained by: i) mixing a compound vitamin (VR24469231, DSM Vitamin (Shanghai) Co., Ltd.), a food flavor (Hasegawa Flavors (Suzhou) Co., Ltd.), sucralose (Jinhe Industrial Co., Ltd., Anhui), and maltodextrin (Cargill Bio-chemical Co., Ltd.) to obtain Premix 1, where the ratio of vitamin + food flavor + sucralose to maltodextrin is 1:10 w / w; ii) mixing a compound mineral (VR24470231, VR24471231, DSM Vitamin (Shanghai) Co., Ltd.) and maltodextrin (the same amount as in step i) or Premix 1) to obtain Premix 2; and iii) mixing Premix 1, Premix 2, a plant-derived compound amino acid powder (61B07-16B, Yunuo Biotech Co., Ltd., Dalian), pectin (Yantai Andre Pectin Co., Ltd., Yantai), and a vegetable oil microcapsule powder (40506-110, Yunuo Biotech Co., Ltd., Dalian) to obtain the final mixture. Then, the mixture is inspected for foreign objects and packaged.

[0164] The prepared medical food composition is intended for use as a meal replacement for certain cancer patients, especially for all-day meal replacement. Patients are recommended to ingest 45 - 80 g of the medical food composition each time, 5 - 8 times a day, and mix the composition with warm water at a powder-to-water ratio of 1:4 - 8 before oral administration or tube feeding (suitable for patients with nasogastric / enteral tubes or gastric / intestinal fistulas).

[0165] Example 5: Safety and efficacy of dietary serine and glycine restriction in cancer patients

[0166] Patients with solid tumors were enrolled and underwent self-controlled dietary interventions to test the safety and efficacy of serine and glycine restriction in the diet.

[0167] During the dietary intervention, patients were only allowed to orally consume the medical food composition containing Amino Acid Composition 1 prepared in Example 4, as well as the low-protein-content foods listed in Table 3, and compound vitamin and mineral tablets (Carlyle Trace Minerals, as additional supplements). The maximum daily serine intake from the foods in Table 3 is 36 mg, and the maximum daily glycine intake from the foods in Table 3 is 16 mg.

[0168] Table 3: Foods for patients undergoing serine and glycine restriction in the diet

[0169]

[0170]

[0171] This study was completed at West China Hospital of Sichuan University.

[0172] Inclusion criteria included:

[0173] · Patients aged 18 - 70 years, regardless of gender.

[0174] · BMI ≥ 18.5.

[0175] · NRS-2002 scale score < 3.

[0176] · Diagnosed with stage IIIa or higher solid tumors.

[0177] · Able to take orally or through a gastric tube and tolerate enteral nutrition.

[0178] · Willing to participate in the study and provide written informed consent.

[0179] Exclusion criteria included:

[0180] · Participated in other interventional clinical trials (including drugs, nutritional supplements, medical devices, etc.) within the previous 4 weeks before screening.

[0181] · Experienced severe diarrhea, intractable vomiting, severe malabsorption syndrome, paralysis, mechanical bowel obstruction, or active gastrointestinal bleeding.

[0182] · Allergic to the sample components.

[0183] · Currently using other nutritional supplements that may affect the validity of the experiment.

[0184] · Pregnant women, lactating female patients, or fertile women who tested positive in the baseline pregnancy test.

[0185] · Had cognitive impairment or mental illness that made it impossible to understand the study content.

[0186] · The researchers considered the subject unsuitable for participating in this study.

[0187] Withdrawal criteria included:

[0188] · The subject requested to withdraw.

[0189] · The study was terminated.

[0190] · The researchers believed that if the subject continued to participate in this study, he would face unacceptable risks.

[0191] · Other circumstances requiring withdrawal.

[0192] Suspension criteria included:

[0193] · A serious adverse event occurred during the experiment, and the researchers determined that it was necessary to terminate.

[0194] ·BMI < 18.5.

[0195] ·NRS-2002 scale score ≥ 3.

[0196] ·Other situations that require suspension.

[0197] The researchers believe that when participants withdraw their consent, their condition deteriorates, serious adverse events occur, compliance is poor, or medications are discontinued for the best interests of the patient, their dietary intervention should be terminated.

[0198] After enrollment, the researchers conducted nutritional risk screening and collected basic information, blood data, imaging data, current and past medical histories, and serum serine and glycine levels of the participants. The participants received a 12-week dietary intervention, and the intakes of L-serine and L-glycine were controlled at 0.81% or less than 0.81% and 0.41% or less than 0.41% respectively according to the total food amino acids consumed. The participants were followed up every 3 weeks for a total of 4 times. Nutritional, blood, immune, metabolomics, and imaging data were collected during the follow-up. Serum serine and glycine levels were compared before and after the intervention.

[0199] The dietary intervention lasted for 12 weeks. The daily protein intake of cancer patients was 1.0 - 1.5 g / kg / day, and the energy intake was 25 - 30 kcal / kg / day. Since indirect calorimetry was not used to evaluate resting energy expenditure in this study and considering the catabolic nature of tumors that require adequate nutrition, the study used 30 kcal / kg / day of energy and 1.5 g / kg / day of protein as the standard to calculate the food composition required for the participants. For example, an esophageal cancer patient with nutritional risk weighing 50 kg needs 1500 kcal and 75 g of protein per day. Before oral administration, the medical food composition was mixed with warm water (about 50 °C) at a powder-to-water ratio of 1:4 - 8.

[0200] To ensure accurate calculation of the daily appropriate amino acid intake for each patient, a food scale with a precision of 0.1 g was provided to each participant for weighing foods other than the medical food composition. The participants were required to take photos when weighing the foods to clearly show the foods and the weights displayed on the food scale.

[0201] The baseline data collected immediately after enrollment included:

[0202] ① General information: gender, age, diagnosis, TNM stage, height, weight, education level, marital status, family annual income, current medical history, past medical history, etc., were collected by the researchers through interviewing the participants and reviewing the patient medical records.

[0203] ②Nutritional risk: NRS2002 nutritional risk screening and PG-SGA were collected face-to-face by trained researchers using the NRS2002 questionnaire and the PG-SGA survey.

[0204] ③InBody body composition: including body weight, body fat content, skeletal muscle content, etc.

[0205] ④Blood indicators: serum amino acid concentration, liver and kidney function, myocardial enzyme spectrum, total protein amount, prealbumin level, CD3+, CD4+ or CD8+ cell populations.

[0206] ⑤Tumor imaging: CT, MRI or ultrasound.

[0207] ⑥Tumor markers: CEA, CA19-9, AFP, CA125, CA153, CA724.

[0208] Items ② to ⑥ above were also collected during the follow-up period. Specifically, tumor markers, blood indicators, nutritional risk data, and safety evaluation indicators were collected at weeks 3, 6, 9, and 12, and tumor imaging was performed at weeks 6 and 12. Safety evaluation indicators included the occurrence of gastrointestinal adverse reactions, including nausea, vomiting, diarrhea, abdominal pain, etc.

[0209] Statistical analysis was performed using SAS 9.4. Data with P < 0.05 generated through paired t-tests, chi-square tests, correlation analyses, linear regression, or logistic regression were considered statistically significant.

[0210] From March 2022 to September 2023, a total of 28 patients with advanced solid tumors underwent eligibility assessment, and 20 patients (esophageal cancer and colorectal cancer) participated in the single-arm trial. Among them, 5 patients withdrew midway due to inability to adapt to the designed daily diet. All patients received PD-1 treatment (Junshi Biosciences) during the trial. Finally, 20 patients completed at least one cycle of dietary restriction and were included in the statistical analysis.

[0211] The numbers of two T cells, GZMB + CD4 + T cells and GZMB + CD8 + T cells that play a central role in tumor immunity in blood samples were measured by flow cytometry, and the proportion of GZMB + CCD4 + T cells in CD4 + cells and the proportion of GZMB + CD8 + T cells in CD8 + cells were analyzed using Flowjo V_10.

[0212] Figure 14 shows the GZMB in 8 patients before food intervention (week 0 or week W0) and 3 or 6 weeks later (week W3, week W6) + CD4 + T cells and GZMB + CD8 + The proportions of T cells. After dietary restriction of glycine and serine, GZMB in most participants + CD4 + and GZMB + CD8 + T cell levels were all significantly increased, among which GZMB + CCD4 + cells had the most significant increase.

[0213] CT results showed that the tumor size decreased in several patients.

[0214] No grade 3 or 4 side effects were observed.

[0215] Example 6: Preparation of an exemplary medical food composition for dietary serine and glycine restriction

[0216] An exemplary biscuit for dietary restriction of serine and glycine was prepared, containing L-glutamine (18.34%), L-leucine (10.57%), L-aspartic acid (9.30%), L-lysine hydrochloride (8.59%), L-arginine (7.18%), L-valine (5.24%), L-phenylalanine (4.36%), L-alanine (6.21%), L-proline (4.75%), L-isoleucine (5.61%), L-threonine (4.41%), L-histidine (2.23%), L-tyrosine (3.98%), L-methionine (2.62%), L-tryptophan (2.58%), L-cysteine (2.81%), L-serine (0.81%) and L-glycine (0.41%), and the percentage of each amino acid is based on the total amino acids in the biscuit.

[0217] Briefly, 50 g of softened unsalted butter (Land O Lakes Unsalted Butter, 3 pk. / 1 lb.) and 30 g of sugar are mixed, and further 10 g of amino acid powder (amino acid composition as described above), 1 / 8 teaspoon of salt (for flavor balance), and 1 / 2 teaspoon of vanilla extract (Nielsen-Massey Madagascar Bourbon Pure Vanilla Extract) are added and mixed. Then, 100 g of corn starch (Roots Circle 100% Pure Corn Starch 17.63 oz) is gradually added to the resulting mixture with continuous stirring, and if the mixture is too dry or difficult to form a dough, about 20 - 30 mL of water is added as needed. The dough is kept at 4 °C for about 30 minutes, rolled out to a thickness of about 0.5 cm, cut into the desired shape, and baked in an oven preheated to 180 °C (350 °F) for about 10 - 15 minutes.

[0218] After the preferred embodiments of the present invention have been described in detail, it should be understood that the invention defined by the above paragraphs is not limited to the specific details set forth in the above description, as many obvious variations are possible without departing from the spirit or scope of the present invention.

Claims

1. A food composition comprising a plurality of amino acids, wherein the plurality of amino acids comprises all essential amino acids, 0.0016 - 0.6 wt% glycine, and 0.00109 - 1.2 wt% serine.

2. The food composition according to claim 1, wherein the plurality of amino acids comprises 0.2 - 0.6 wt% glycine.

3. The food composition according to claim 1, wherein the plurality of amino acids comprises 0.2 - 1.2 wt% serine.

4. The food composition according to claim 1, wherein the plurality of amino acids further comprises one or more non-essential amino acids selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, proline, and tyrosine.

5. The food composition according to claim 1, further comprising one or more macronutrients and / or one or more micronutrients.

6. The food composition according to claim 1, which is prepared as a solid.

7. A meal replacement comprising the food composition according to any one of claims 1 - 6.

8. A meal replacement consisting of the food composition according to claim 5.

9. A pharmaceutical composition comprising the food composition according to any one of claims 1 - 6 and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, further comprising an anti-PD-1 antibody or an anti-PD-L1 antibody.

11. Use of the pharmaceutical composition according to claim 9 or 10 in the preparation of a medicament for treating cancer.

12. The use according to claim 11, wherein the cancer is esophageal cancer or colorectal cancer.

13. Use of the pharmaceutical composition according to claim 9 in the preparation of a medicament for sensitizing tumor cells to PD-1 or PD-L1 therapy.

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