A functional substance for regulating Teff / Treg homeostasis balance and its application

Abstract

The present application relates to the technical field related to immunotherapy, and in particular to a functional substance for regulating Teff / Treg steady-state balance and its application; the functional substance comprises an inhibitor for inhibiting RELT-related signaling pathways and / or an activator for activating RELT-related signaling pathways; the inhibitor comprises a recombinant protein of the extracellular segment of RELT, and the recombinant protein of the extracellular segment of RELT has an amino acid sequence as shown in SEQ ID NO.1; the activator comprises an Fc fusion protein of RELT, and the Fc fusion protein has an amino acid sequence as shown in SEQ ID NO.2; the sRELT recombinant protein in the functional substance can inhibit the RELT-related signaling pathway and thus treat and / or prevent type 1 diabetes, and the Fc-RELT fusion protein in the functional substance can enhance the RELT-related signaling pathway to promote immune activation and improve the effect of anti-tumor response.

Description

Technical Field

[0001] The present application relates to the technical field related to immunotherapy, and in particular to a functional substance for regulating Teff / Treg steady-state balance and its application. Background Art

[0002] From the perspective of cell function, CD4 + T cells can be divided into two major categories: effector T cells (Teff) and regulatory T cells (Treg). Teff refers to a type of cell that can proliferate, differentiate and release cytokines after receiving antigen stimulation. The released cytokines include interleukins and / or interferons. These cytokines can enhance the interaction between various cells and exert immune effects, thereby resisting or eliminating exogenous and endogenous pathogenic factors; Treg refers to a type of T cell subset with significant immunosuppressive effects. Treg mainly limits the effector function of other immune cells by contact or non-contact means, thereby avoiding overactivation of the immune system. Therefore, the steady-state balance of Teff and Treg in the body is the key to ensuring a moderate immune response and maintaining immune homeostasis.

[0003] Current studies have shown that imbalance in Teff / Treg immune homeostasis is an important mechanism for the occurrence and development of immune-related diseases such as type 1 diabetes, malignant tumors, rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, multiple sclerosis, Hashimoto's thyroiditis, primary myxedema, hyperthyroidism, autoimmune hemolytic anemia, ulcerative colitis, atrophic gastritis, and autoimmune glomerulonephritis. Therefore, the regulation of Teff / Treg immune homeostasis is currently a research hotspot for immune-related diseases.

[0004] At present, studies have found that serum free soluble RELT (sRELT) in tumor patients increases significantly, suggesting that RELT may be a potential early diagnostic marker for tumors. Although the immune regulatory mechanism of RELT membrane-bound and free forms has relevant applications in regulating Teff / Treg immune homeostasis, most of the immune regulatory mechanisms are currently mediated by chemical agents or biological agents, and these chemical agents or biological agents have poor regulatory effects on Teff / Treg immune homeostasis. Summary of the invention

[0005] The present application provides a functional substance for regulating Teff / Treg homeostasis and its application, so as to solve the technical problem that the regulation effect of Teff / Treg immune homeostasis by chemical agents or biological agents in the prior art is poor.

[0006] In a first aspect, the present application provides a functional substance for regulating Teff / Treg homeostasis, wherein the functional substance comprises an inhibitor for inhibiting RELT-related signaling pathways and / or an activator for activating RELT-related signaling pathways;

[0007] Wherein, the inhibitor comprises a recombinant protein of the extracellular segment of RELT, and the recombinant protein of the extracellular segment of RELT has an amino acid sequence as shown in SEQ ID NO.1;

[0008] The activator includes Fc fusion protein of RELT, and the Fc fusion protein has the amino acid sequence shown in SEQ ID NO.2.

[0009] Optionally, the inhibitor further comprises at least one of the following:

[0010] RNA, neutralizing antibodies, and chemical inhibitors targeting RELT.

[0011] Optionally, the RNA targeting RELT comprises at least one of the following:

[0012] siRNA targeting RELT, shRNA targeting RELT, and miRNA targeting RELT.

[0013] Optionally, the activator further comprises at least one of the following:

[0014] RELT agonistic antibodies, chemical agonists, and overexpressing organisms.

[0015] Optionally, the overexpressed organism includes at least one of the following:

[0016] Overexpressing recombinant plasmid, RELT overexpressing viral vector and RELT overexpressing immune cells.

[0017] Optionally, the immune cells overexpressing RELT include CD4+T cells overexpressing RELT.

[0018] In a second aspect, the present application provides an application of a functional substance for regulating Teff / Treg steady-state balance in regulating Teff / Treg steady-state balance, wherein the application includes using the functional substance described in the first aspect as an agent for inhibiting or activating RELT-related signals to regulate Teff / Treg steady-state balance.

[0019] In a third aspect, the present application provides an application of a functional substance for regulating Teff / Treg steady-state balance in the preparation of preventive and / or therapeutic drugs for immune diseases, wherein the application comprises using the functional substance described in the first aspect in the preparation of therapeutic drugs for immune diseases.

[0020] Optionally, the immune disease includes at least one of the following:

[0021] Type 1 diabetes, malignancy, rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, multiple sclerosis, Hashimoto's thyroiditis, primary myxedema, hyperthyroidism, autoimmune hemolytic anemia, ulcerative colitis, atrophic gastritis, and autoimmune glomerulonephritis.

[0022] In a fourth aspect, the present application provides a pharmaceutical composition for treating immune diseases, wherein the pharmaceutical composition comprises the functional substance described in the first aspect.

[0023] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0024] The embodiment of the present application provides a functional substance for regulating the steady-state balance of Teff / Treg. Since the regulatory mechanism of the RELT-related signaling pathway is mainly achieved by the transformation of RELT from a membrane-bound state to a free state, and the transformation of the membrane-bound state to the free state is mainly achieved by the shedding of the extracellular segment, the functional substance is designed for the related protein of the extracellular segment shedding, so that a functional substance with good specificity can be obtained. Based on the ligand binding domain of the RELT extracellular segment, the present application designs an inhibitor including a recombinant protein of the RELT extracellular segment and an activator including an Fc fusion protein of RELT. The recombinant protein of the RELT extracellular segment can block the binding of RELT to the RELT ligand through competitive binding, thereby inhibiting the expression of the RELT-related signaling pathway and the downstream immune activation effect of the RELT-related signaling pathway. In addition, the Fc fusion protein can specifically bind to the RELT ligand to activate the reverse signal of the RELT ligand receptor, thereby promoting the immune activation of the downstream RELT ligand. Therefore, the functional substance can specifically regulate the RELT-related signaling pathway according to actual needs, thereby regulating the steady-state balance of Teff / Treg through the regulation of the RELT-related signaling pathway by the functional substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0026] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0027] Figure 1This is a diagram showing the preparation process of the sRELT recombinant protein and Fc fusion protein provided in Example 1 of the present application, wherein: Figure 1 A is a diagram showing the preparation process of sRELT recombinant protein. Figure 1 B is a diagram showing the results of the preparation process of Fc-RELT recombinant protein;

[0028] Figure 2 This is a schematic diagram of the sRELT recombinant protein provided in Example 1 of the present application inhibiting Th1 differentiation and enhancing Treg stability;

[0029] Figure 3 The result graphs of immune activation indexes and corresponding T cell subset ratios of the RELT extracellular segment recombinant protein treatment group and the IgG control group provided in Example 1 of the present application; wherein, Figure 3 A is a schematic diagram of the disease results of mice in the sRELT treatment group and the IgG control group. Figure 3 B is a comparison of the proportion of CD44+CD62L- cells in the sRELT-treated mice and the IgG control group mice. Figure 3 C is a comparison of the proportion of CD25+Foxp3+Treg cells in the sRELT-treated mice and the IgG control group mice. Figure 3 D is a comparison of the proportion of IFN-r+ Teff cells in mice treated with sRELT and mice in the IgG control group;

[0030] Figure 4 CD4-RELT provided in the embodiments of the present application TG Methods for constructing transgenic mice;

[0031] Figure 5 CD4-RELT provided in the embodiments of the present application TG The results of gene identification of transgenic mice, among which, Figure 5 A is the result of CD4-cre identification. Figure 5 B is RELT TG Identification result diagram;

[0032] Figure 6 CD4-RELT provided in Example 3 of the present application TG The results of immune activation indicators and T cell subset ratios of mice and control NOD mice, among which, Figure 6 A is CD4-RELT TG Schematic diagram of the disease results of female mice and control group NOD female mice. Figure 6 B is CD4-RELT TG Schematic diagram of the disease results of male mice and control group NOD male mice. Figure 6 C is CD4-RELT TGComparison of the proportion of CD44+CD62L- cells in mice and control group NOD mice. Figure 6 D is CD4-RELT TG Comparison of the proportion of IFN-g+ Teff cells in mice and control group NOD mice. Figure 6 E is CD4-RELT TG Comparison of the proportion of Foxp3+ Treg cells in mice and control group NOD mice;

[0033] Figure 7 This is a schematic diagram of the effect of the Fc-RELT fusion protein provided in Example 4 of the present application on T cell activation, proliferation and differentiation;

[0034] Figure 8 This is a schematic diagram of immune activation related indicators of the Fc fusion protein treatment group and the IgG control group provided in Example 4 of the present application, wherein: Figure 8 A is a comparison of the incidence rates of mice in the Fc-RELT treatment group and the IgG control group. Figure 8 B is a comparison of the ratio of activated CD44+CD62L-T cells in vivo between mice in the Fc-RELT treatment group and mice in the IgG control group. Figure 8 C is a comparison of the proportion of CD25+Foxp3+Treg cells in the Fc-RELT treated mice and the IgG control group mice. Figure 8 D is a comparison of the proportion of Teff cells in the Fc-RELT treated mice and the IgG control group mice;

[0035] Fig. 9 A schematic diagram of a mouse tumor mass provided in Example 5 of the present application;

[0036] Fig.10 This is a flow cytometry analysis result diagram of mouse immune cells in Example 5 of the present application, wherein: Fig.10 A is the result of CD4+T cell activation level in tumor tissue of mice in the Fc-RELT treatment group. Fig.10 B is the result graph of CD8T cell activation level in tumor tissue of mice in the Fc-RELT treatment group. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0038] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0039] It should be noted that a large number of studies have shown that the imbalance of Treg and Teff homeostasis is a key feature of the development of type 1 diabetes (T1D). The occurrence of T1D is due to the disorder of the body's immune system, which attacks its own pancreatic β cells, leading to the death of a large number of pancreatic β cells, thereby causing the body to have absolute insulin deficiency. Therefore, T1D is an autoimmune metabolic disease. In the pathological process of T1D, Teff cells are the main cell population that damages pancreatic β cells and induces autoimmune reactions. Teff can directly secrete inflammatory cytokines (such as IFN-γ, TNF-α, IL-1β) and other soluble mediators (such as nitric oxide (NO)) to induce β cell death, and can also activate CD8 + T cells to promote CD8 + T cells kill pancreatic β cells by releasing granzymes, perforins or by FAS-mediated cell lysis. In normal organisms, Treg can maintain the body's immune homeostasis by inhibiting the proliferation, differentiation and cytokine secretion of Teff; however, in T1D patients, Teff / Treg is in a state of functional imbalance, which is mainly manifested in: Teff overactivation, decreased proportion of Treg cells, and a certain degree of functional abnormality of Treg cells, which in turn exacerbates the progression of T1D.

[0040] At present, studies have shown that in the tumor microenvironment (TME) of malignant tumors, the body will show a significant increase in the number of Treg cells, a significant decrease in the infiltration of Teff, and myeloid cells (such as DCs and macrophages) showing unique tolerance phenotypes. These characteristics jointly promote the immune escape of tumors. Moreover, in the body of malignant tumors, Treg cells are recruited to tumor tissues through chemokine receptor-chemokine interactions (such as CCR4-CCL17 / 22, CCR8-CCL1, CCR10-CCL28 and CXCR3-CCL9 / 10 / 11 axes, etc.), and then Treg cells inhibit the anti-tumor immune response of Teff cells by competitively binding to IL-2, secreting inhibitory cytokines, and regulating tryptophan and adenosine metabolism. Therefore, the imbalance of immune homeostasis of Teff / Treg in TME is closely related to the continuous progression of various tumors and low survival rates. As an innovative treatment method, tumor immunotherapy uses molecular targeting to specifically block inhibitory signals or amplify stimulatory signals to correct the homeostatic imbalance of Teff / Treg, and has become a cutting-edge hotspot in the field of tumors.

[0041] In addition, Teff / Treg immune homeostasis imbalance is also an important mechanism for the occurrence and development of immune-related diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, multiple sclerosis, Hashimoto's thyroiditis, primary myxedema, hyperthyroidism, autoimmune hemolytic anemia, ulcerative colitis, atrophic gastritis, autoimmune glomerulonephritis, etc. Therefore, the regulation of Teff / Treg immune homeostasis balance is currently a hot topic in the research of immune-related diseases.

[0042] RELT (Receptor Expressed on Lymphoid Tissues) belongs to the TNF receptor superfamily (TNFRSF), also known as TNFRSF19L, and is mainly expressed in immune cells. Like other members of the TNF receptor superfamily, RELT exists mainly in two forms: membrane-bound and free, and the transition from membrane-bound to free is achieved by ectodomain shedding. During the ectodomain shedding process, the 137th amino acid of the extracellular membrane-proximal part of RELT can be cleaved and released to become soluble sRELT (SolubleRELT). This process is mediated by matrix metalloprotease and disintegrin family members ADAM17, ADAM9, ADAM10, ADAM19, MMP7, Elastase, Proteinase 3, etc. At present, studies have found that the serum free soluble RELT (Soluble RELT, sRELT) of tumor patients will increase significantly, suggesting that RELT may be a potential early diagnostic marker for tumors. Although studies have found the application of immune regulatory mechanisms of RELT membrane-bound and free forms in regulating Teff / Treg immune homeostasis, most of them use chemical or biological agents to inhibit or activate the immune regulatory mechanism, and these chemical or biological agents are difficult to inhibit or activate the immune regulatory mechanism specifically, resulting in poor regulatory effects of these chemical or biological agents on Teff / Treg immune homeostasis.

[0043] The embodiment of the present application provides a functional substance for regulating Teff / Treg steady-state balance, wherein the functional substance includes an inhibitor for inhibiting RELT-related signaling pathways and / or an activator for activating RELT-related signaling pathways;

[0044] Wherein, the inhibitor comprises a recombinant protein of the extracellular segment of RELT, and the recombinant protein of the extracellular segment of RELT has an amino acid sequence as shown in SEQ ID NO.1;

[0045] The activator includes Fc fusion protein of RELT, and the Fc fusion protein has the amino acid sequence shown in SEQ ID NO.2.

[0046] It should be noted that the RELT extracellular segment recombinant protein can be obtained by expressing the RELT extracellular segment ligand binding domain in vitro and then purifying the in vitro expressed product.

[0047] In some optional embodiments, the inhibitor further comprises at least one of the following:

[0048] RNA, neutralizing antibodies and chemical inhibitors targeting RELT; in these embodiments, on the basis that the inhibitor may include a recombinant protein of the extracellular segment of RELT, the inhibitor may also be at least one of RNA, neutralizing antibodies or chemical inhibitors targeting RELT, which can improve the inhibitory effect of the inhibitor on the expression of RELT-related signaling pathways, and improve the inhibitory effect of the inhibitor on the downstream immune activation of RELT-related signaling pathways.

[0049] In some optional embodiments, the RNA targeting RELT comprises at least one of the following:

[0050] siRNA targeting RELT, shRNA targeting RELT and miRNA targeting RELT; in these embodiments, RNA targeting RELT may include at least one of siRNA targeting RELT, shRNA targeting RELT and miRNA targeting RELT, which can cover most of the existing RNA targeting RELT, so that RNA targeting RELT can be combined with RELT extracellular segment recombinant protein to enhance the inhibitory effect of the inhibitor on the expression of RELT-related signaling pathways and the downstream immune activation of RELT-related signaling pathways.

[0051] In some optional embodiments, the activator further comprises at least one of the following:

[0052] RELT agonist antibodies, chemical agonists and overexpressed organisms; in these embodiments, on the basis that the activator can be an Fc fusion protein, the activator can also be at least one of RELT agonist antibodies, chemical agonists and overexpressed organisms, and these RELT agonist antibodies, chemical agonists and overexpressed organisms can be used in conjunction with Fc fusion proteins to enhance the activation effect of the reverse signal of RELT ligand receptor action, thereby further promoting the immune activation process of the downstream RELT ligand.

[0053] In some optional embodiments, the overexpressed organism comprises at least one of the following:

[0054] Overexpressing recombinant plasmids, RELT-overexpressing viral vectors and RELT-overexpressing immune cells; in these embodiments, the overexpressing organism can be at least one of the overexpressing recombinant plasmids, RELT-overexpressing viral vectors and RELT-overexpressing immune cells, which can cover most of the existing overexpressing organisms, so that the overexpressing organism can be combined with Fc fusion protein to promote the immune activation process of the downstream RELT ligand.

[0055] In some optional embodiments, the immune cells overexpressing RELT include CD4+T cells overexpressing RELT; in these embodiments, the immune cells overexpressing RELT may include CD4+T cells overexpressing RELT, and the CD4+T cells expressing RELT may be combined with Fc fusion proteins to promote the immune activation process of downstream RELT ligands.

[0056] It should be noted that the CD4+T cells overexpressing RELT can be constructed by constructing CD4+T cell-specific RELT overexpressing mouse CD4 cre -RELT TG obtained by the model.

[0057] Based on a general inventive concept, an embodiment of the present application provides an application of a functional substance for regulating Teff / Treg steady-state balance, wherein the application includes using the functional substance as an agent for inhibiting or activating RELT-related signals to regulate Teff / Treg steady-state balance.

[0058] This application is realized based on the above-mentioned functional substance. The specific composition and composition information of the functional substance can refer to the above-mentioned embodiment. Since this application adopts part or all of the technical solutions of the above-mentioned embodiment, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiment, which will not be described one by one here.

[0059] Based on a general inventive concept, an embodiment of the present application provides an application of a functional substance for regulating Teff / Treg steady-state balance in the preparation of preventive and / or therapeutic drugs for immune diseases, wherein the application includes using the functional substance in the preparation of therapeutic drugs for immune diseases.

[0060] The application of the functional substance for regulating Teff / Treg steady-state balance in the preparation of preventive and / or therapeutic drugs for immune diseases is realized based on the above-mentioned functional substance. The specific composition and composition information of the functional substance can refer to the above-mentioned embodiment. Since the application of the functional substance of the RELT-related signaling pathway in the preparation of preventive and / or therapeutic drugs for immune diseases adopts part or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

[0061] In some optional embodiments, the immune disease includes at least one of the following:

[0062] Type 1 diabetes, malignant tumors, rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, multiple sclerosis, Hashimoto's thyroiditis, primary myxedema, hyperthyroidism, autoimmune hemolytic anemia, ulcerative colitis, atrophic gastritis and autoimmune glomerulonephritis; in these embodiments, the immune disease may include at least one of type 1 diabetes, malignant tumors, rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, multiple sclerosis, Hashimoto's thyroiditis, primary myxedema, hyperthyroidism, autoimmune hemolytic anemia, ulcerative colitis, atrophic gastritis and autoimmune glomerulonephritis, which can cover most of the existing immune-related diseases, thereby improving the applicability of functional substances in different immune-related diseases.

[0063] Based on a general inventive concept, an embodiment of the present application provides a pharmaceutical composition for treating immune diseases, wherein the pharmaceutical composition includes the functional substance.

[0064] The pharmaceutical composition is realized based on the above-mentioned functional substances. The specific composition and composition information of the functional substances can be referred to the above-mentioned embodiments. Since the pharmaceutical composition adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

[0065] It should be noted that when using functional substances as pharmaceutical compositions for the prevention and treatment of type 1 diabetes, the functional substances can be directly injected into the body of a mammal to restore the Teff / Treg immune homeostasis, thereby achieving the purpose of preventing and treating type 1 diabetes.

[0066] It should be noted that the process of delivering the pharmaceutical composition to the corresponding subject may include at least one of the following:

[0067] Direct ingestion by mouth, intravenous, intraperitoneal administration, subcutaneous, enteral, rectal, nasal infusion (e.g., bolus infusion), oral, sublingual, intramuscular inhalation, transdermal administration, topical administration into the eye, vagina, rectum, and intracranial injection.

[0068] It should be noted that the pharmaceutical composition can be delivered subcutaneously or intravenously in the process of preventing or treating type 1 diabetes and tumor-related diseases. In addition, the pharmaceutical composition can be added with other drug adjuvants or non-drugs on the basis of functional substances.

[0069] It should be noted that in the process of preventing or treating type 1 diabetes, the pharmaceutical composition can combine the functional substance with insulin, insulin analogs, metformin, glucosidase inhibitors (such as acarbose, etc.), GLP-1, low-dose interleukin 2 (IL-2) and other immunosuppressants (such as tacrolimus, etc.).

[0070] It should be noted that the pharmaceutical composition can be combined with radiotherapy, chemotherapy and other immune checkpoint blockade therapies (such as anti-PD1 and / or anti-CTLA4, etc.) when preventing or treating tumor diseases.

[0071] It should be noted that the pharmaceutical composition may also include a pharmaceutically acceptable carrier or excipient. The carrier may be any existing carrier that can be used for drugs, and the carrier may retain its own biological activity when combined with the functional substance. The carrier may be a phosphate buffered saline solution, water, an emulsion (such as an oil / water emulsion) and various types of wetting agents.

[0072] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to industry standards. If there is no corresponding industry standard, then the conditions recommended by the manufacturer are followed.

[0073] Example 1

[0074] Mice were handled according to the Implementation Rules for the Management of Medical Laboratory Animals. NOD mice, C57BL / 6 mice, and NOD-SCID mice were maintained in an SPF-grade animal facility.

[0075] 1. Preventive and therapeutic effects of RELT extracellular segment recombinant protein (sRELT) on spontaneous diabetes in NOD mice:

[0076] 8-week-old NOD mice were selected and intraperitoneally injected with sRELT (n=10) or IgG control protein (n=10) (administered at 20 mg / kg body weight, approximately 400 μg / mouse), three times a week for a total of two weeks, and then stopped after two weeks. Blood glucose was tested weekly during the administration period, and if the blood glucose level exceeded 12.8 mM for two consecutive times, it was considered that type 1 diabetes had occurred; when the incidence of one group of mice reached 80%, both groups of mice were euthanized;

[0077] The spleen of mice was taken, ground to prepare single cell suspension and detect immune activation indicators such as CD44+CD62L and the proportion of corresponding T cell subsets such as Foxp3, IFN-g, and IL-17A. The results are as follows Figure 3 shown.

[0078] Figure 3 The result diagram exemplarily shows the immune activation indexes and the corresponding T cell subpopulation ratios of the RELT extracellular segment recombinant protein treatment group and the BSA control group provided in Example 1 of the present application;

[0079] like Figure 3 As shown in A, mice in the IgG control group began to develop the disease at 13 weeks of age, and the incidence rate reached 80% at 26 weeks of age; mice in the sRELT treatment group began to develop the disease at 19 weeks of age, the onset time was postponed and the incidence rate was only 30% at 26 weeks of age. The NOD spontaneous type 1 diabetes model showed that sRELT can inhibit the progression of type 1 diabetes and has a preventive and therapeutic effect on spontaneous diabetes in NOD mice.

[0080] like Figure 3 As shown in B, there was no substantial difference in the proportion of activated CD44+CD62L- T cells in the IgG control group and the sRELT-treated group. Figure 3 C shows that the proportion of Foxp3+Treg cells in mice treated with sRELT increased. Figure 3 D shows that the proportion of IFN-r+ Teff cells in mice treated with sRELT was reduced, which fully demonstrates that sRELT recombinant protein can regulate the immune homeostasis of Teff / Treg.

[0081] Figure 1 The diagram exemplarily shows the preparation principle result of the sRELT recombinant protein and Fc fusion protein provided in Example 1 of the present application;

[0082] Combination Figure 1 As can be seen from A, the sRELT recombinant protein is obtained by in vitro expression and purification of the RELT extracellular segment ligand binding domain. The amino acid sequence of the sRELT recombinant protein is:

[0083] MGSSHHHHHHSSGLVPRGSHMTPITPWLCPPGKEPDPDPGQGTLCRTCPPGTFSASWNSYPCCPHYRCSLQKRLEAQAGTATHDTMCGDCQHGWFGPQGVPHVPCQPCSKAPPSTGGCDESGRGGGRGVEVAAGTSSNGEPRQPGNGTRAGGPEETA (SEQ ID NO. 1).

[0084] Figure 2 A schematic diagram showing exemplarily that the sRELT recombinant protein provided in Example 1 of the present application inhibits Th1 differentiation and enhances Treg stability;

[0085] Combination Figure 2 It can be seen that sRELT can inhibit the differentiation of Th1, promote the differentiation of Treg and enhance the stability of Treg. Therefore, sRELT recombinant protein can regulate Teff / Treg immune homeostasis.

[0086] Example 2

[0087] 2. CD4-RELT TG Construction of transgenic mice (NOD background):

[0088] Figure 4 The CD4-RELT provided in the embodiment of the present application is exemplified. TG Methods for constructing transgenic mice;

[0089] like Figure 4 As shown, RELT TG Mice were mated with CD4-cre transgenic mice to obtain CD4-RELT TG Mice. RELT TG The mouse construction method was based on the prior art. CD4-cre (NOD background) transgenic mice were purchased from Jackson Laboratory: NOD.Cg-Tg (Cd4-cre) 1Cwi / 2AchJ, No. 013234.

[0090] Figure 5 The CD4-RELT provided in the embodiment of the present application is exemplified. TG Figure 1 shows the results of gene identification of transgenic mice;

[0091] The constructed CD4-RELT TG The transgenic mice were used for gene identification. In this gene identification, the primer sequences used included:

[0092] CD4_Cre_F: GAGGCTCAGATTCCCAACCA (SEQ ID NO.3);

[0093] CD4_Cre_R: GGTTCTTGCGAACCTCATCAC (SEQ ID NO.4);

[0094] RELT-Tg_F: CCTCCCCCTGAACCTGAAACAT (SEQ ID NO.5);

[0095] RELT-Tg_R: ACAGCAAGGTCCGCTTCATCAT (SEQ ID NO. 6).

[0096] The results are as follows Figure 5 As shown, Figure 5 A and Figure 5As shown in B, this indicates that the present application successfully constructed CD4-RELT TG Transgenic mice.

[0097] Example 3

[0098] The CD4-RELT constructed in Example 2 TG Based on transgenic mice (NOD background), further manipulations were performed:

[0099] CD4-RELT TG Transgenic mice (NOD background) show obvious immune activation phenotype:

[0100] CD4-RELT TG Twenty male and female mice and 20 male and female control NOD mice were kept in an SPF animal room. Blood glucose was tested weekly from the 8th week onwards. Type 1 diabetes was considered to occur when blood glucose levels exceeded 12.8 mM for two consecutive times. When the incidence rate of either group reached 80%, the mice in both groups were euthanized, spleens were taken, single cell suspensions were prepared by grinding, and immune activation indicators such as CD44 and CD62L, as well as the proportions of corresponding T cell subsets such as Foxp3, IFN-g, and IL-17A were tested. The results are shown in the figure. Figure 6 shown.

[0101] Figure 6 The CD4-RELT provided in Example 3 of the present application is exemplified. TG Results of immune activation indexes and T cell subset ratios in mice and control NOD mice;

[0102] like Figure 6 As shown in A, CD4-RELT TG Female mice began to develop the disease at 9 weeks of age, and the incidence rate reached 80% at 23 weeks of age; female mice in the control group, NOD, began to develop the disease at 13 weeks of age, the onset time was delayed, and the incidence rate reached 80% at 28 weeks of age; Figure 6 B shows CD4-RELT TG Male mice began to develop the disease at 10 weeks of age, and the incidence reached 50% at 35 weeks of age; the NOD male mice in the control group began to develop the disease at 21 weeks of age, the onset time was delayed, and the incidence was only 20% at 35 weeks of age. The results of T1D incidence showed that CD4-RELT TG It aggravated the progression of spontaneous diabetes in NOD mice and presented a clear immune activation phenotype.

[0103] like Figure 6 C shows CD4-RELT TG The proportion of CD44+CD62L- cells in mice increased significantly, showing an obvious immune activation phenotype; Figure 6 D as shown and Figure 6 As shown in E, the proportion of IFN-g+ Teff cells increased significantly, and the proportion of Foxp3+ Treg cells decreased significantly, suggesting that CD4+ T cell-specific RELT overexpression presents an immune activation phenotype.

[0104] Example 4

[0105] 4. Study on the promotion of disease progression of T1D induced by adoptive transfer of pathogenic T cells by Fc fusion protein of RELT (Fc-RELT):

[0106] 8-week-old NOD mice were intraperitoneally injected with Fc-RELT (n=10) or control IgG (n=10) (administered at 20 mg / kg body weight, approximately 400 μg / mouse), three times a week for a total of two weeks, and the administration was stopped after two weeks. Blood glucose was tested weekly during the administration period, and if the blood glucose level exceeded 12.8 mM for two consecutive times, it was considered that type 1 diabetes had occurred; when the incidence rate of one group reached 80%, the two groups of mice were euthanized, the spleens were removed, and single cell suspensions were prepared by grinding and the immune activation-related indicators were tested. The results are shown in Figure 8 shown.

[0107] Figure 8 The schematic diagram of immune activation related indicators of the Fc fusion protein treatment group and the IgG control group provided in Example 4 of the present application is exemplarily shown;

[0108] like Figure 8 As shown in A, mice in the IgG control group began to develop the disease at 14 weeks of age, and the incidence rate was 40% at 23 weeks of age. Mice in the Fc-RELT treatment group began to develop the disease at 11 weeks of age, the onset time was earlier and the incidence rate reached 90% at 23 weeks of age, which proved that Fc-RELT can aggravate the progression of spontaneous diabetes in NOD mice.

[0109] like Figure 8 As shown in B, the proportion of activated CD44+CD62L- T cells in mice treated with Fc-RELT increased. Figure 8 As shown in C, the proportion of CD25+Foxp3+Treg cells in the Fc-RELT treatment group decreased. Figure 8 As shown in D, the proportion of IFN-r+ Teff cells in the Fc-RELT treatment group increased, suggesting that Fc-RELT fusion protein can disrupt the immune homeostasis of Teff / Treg.

[0110] The Fc-RELT fusion protein was prepared as follows Figure 1 As shown in B, it is mainly obtained by fusing the RELT extracellular segment ligand binding domain and the Fc segment of IgG2. The amino acid sequence of the Fc-RELT fusion protein is:

[0111] MSLQGLMMKRTLLCWPLSCLFVLLPWPLATPTPITPWLCPPGKEPDPDPGQGTLCRTCPPGTF

[0112] SASWNSYPCQPHYRCSLQKRLEAQAGTATHDTMCGDCQHGWFGPQGVPHVPCQPCSKAPP

[0113] STGGCDESGRGGGRGVEVAAGTSSNGEPRQPGNGTRAGGPEETAMVRSPRGPTIKPCPPCKC

[0114] PAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHR

[0115] EDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEE

[0116] EMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKHHHHHH (SEQ ID NO. 2).

[0117] Figure 7 The schematic diagram exemplarily shows the effect of the Fc-RELT fusion protein provided in Example 4 of the present application on T cell activation, proliferation and differentiation;

[0118] like Figure 7 As shown, the culture dish was pre-coated with Fc-RELT fusion protein and then Cell culture; The results showed that Fc-RELT fusion protein can promote T cell activation and Th1 cell polarization. In addition, Fc-RELT fusion protein can also inhibit the differentiation of Treg cells, which fully demonstrates that Fc-RELT fusion protein can regulate Teff / Treg immune homeostasis.

[0119] Example 5

[0120] 5. Fc-RELT inhibits tumor progression:

[0121] Mice were subcutaneously inoculated with prostate cancer cells (PCa) for 10 6The mice were given Fc-RELT (n=7) or control IgG (n=7) (at 20 mg / kg body weight, approximately 400 μg / mouse) by intraperitoneal injection three times a week until the size of the tumor mass in one group reached the lower limit of ethical requirements (approximately 1 cm 3 ~2cm 3 ).

[0122] Fig. 9 The schematic diagram of mouse tumor mass provided in Example 5 of the present application is exemplarily shown;

[0123] The mice were then euthanized, the tumor masses were removed, and the tumor diameters were measured. Fig. 9 As shown, it was found that the tumor tissue of mice treated with Fc-RELT was significantly reduced, suggesting that Fc-RELT can inhibit tumor progression.

[0124] Fig.10 The flow cytometry analysis results of mouse immune cells in Example 5 of the present application are shown as an example;

[0125] The mouse tumor tissue was then minced, digested with collagenase, and immune cells were extracted with lymphocyte separation fluid for flow cytometry analysis. Fig.10 As shown;

[0126] like Fig.10 As shown in A, after Fc-RELT treatment, the activation level of intratumoral CD4+T cells was enhanced, while the proportion of Foxp3+Treg cells decreased, indicating a stronger immune activation state; Fig.10 As shown in B, the activation level of CD8 T cells increased and the proportion of IFN-g+ effector CD8+ T cells increased. The above results indicate that Fc-RELT can inhibit tumor progression by regulating the immune microenvironment.

[0127] In summary, the embodiments of the present application provide a functional substance for regulating the steady-state balance of Teff / Treg. The sRELT recombinant protein in the functional substance can inhibit the RELT-related signaling pathway to treat and / or prevent type 1 diabetes, while the Fc-RELT fusion protein in the functional substance can enhance the RELT-related signaling pathway, thereby further promoting immune activation and improving the effect of anti-tumor response.

[0128] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0129] In the description of the specification of the present application, the terms "include", "comprise", etc. mean "including but not limited to". In this article, relational terms such as "first" and "second", etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A and B can be singular or plural. In this article, "at least one" means one or more, and "plurality" means two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b and c", can both mean: a, b, c, ab (i.e. a and b), ac, bc or abc, where a, b, c can be single or plural, respectively.

[0130] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A functional substance for regulating Teff / Treg homeostasis, characterized in that: The functional substances include inhibitors that inhibit RELT-related signaling pathways and activators that activate RELT-related signaling pathways; Wherein, the inhibitor is a recombinant protein of the extracellular segment of RELT, which is obtained by in vitro expression and purification of the ligand binding domain of the extracellular segment of RELT, and the amino acid sequence is shown in SEQ ID NO.1; The activator is a RELT Fc fusion protein, which is obtained by fusing the RELT extracellular segment ligand binding domain and the IgG2 Fc segment, and the amino acid sequence is shown in SEQ ID NO.2.

Images