Polypeptide and conjugate and application thereof

Abstract

The invention provides a polypeptide as well as a conjugate and application thereof. Wherein the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1, and the amino acid represented by X at the 14th site and / or the 39th site has the following characteristics: the amino acid can be coupled with a modifier, and can be uncoupled with the modifier in a tumor microenvironment to release the polypeptide; wherein the binding capacity of the polypeptide which is not coupled with the modifier and the CD122 is marked as the first binding capacity, the binding capacity of the polypeptide which is coupled with the modifier and the CD122 is marked as the second binding capacity, the ratio of the first binding capacity to the second binding capacity is marked as n, and n is larger than or equal to 5. Under the action of a tumor specific enzyme, the polypeptide is prevented from activating NK cells and CD8 + T cells in peripheral circulation due to shielding of the modifier, and under the action of the tumor specific enzyme, the polypeptide is uncoupled from the conjugate and recovers activation of an IL2-R beta gamma receptor, so that a specific killing effect is achieved at a tumor part.

Description

Peptides and their conjugates and applications

[0001] This application is based on the Chinese application with CN application number 202310593255.2 and application date May 24, 2023, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of genetic engineering, and in particular to a polypeptide and a conjugate thereof and applications thereof. Background Art

[0003] With the aging population, cancer has become a major contributing factor to global mortality. Advances in biology have led to new breakthroughs in immune-based cancer therapies. The complex immune system comprises immune organs, cells, and molecules. Immune-based therapies include CAR-T therapy, which relies on immune cells. However, due to the regulatory effects of immune molecules on immune cells and their multifaceted, cross-functional, and selective nature, they have become a primary target for scientists to adapt and utilize. Currently, the FDA has approved 11 cancer immunotherapies, the majority of which are monoclonal antibody checkpoint inhibitors, such as anti-CTLA-4 and anti-PD-1 / PD-L1. Due to the limitations of CAR-T therapy for solid tumors and the limited use of immune checkpoint inhibition in "cold" tumors, the use of chemically synthesized small molecules or cytokines to directly manipulate the tumor microenvironment and modulate adaptive immune pathways offers a broader and more comprehensive approach to cancer treatment. In November 1984, a patient with metastatic melanoma received high-dose IL-2 treatment for several months, and all tumors disappeared throughout his body and did not recur for the next 29 years. This was the first time that the importance of activating immune cells through immune molecules in eliminating tumors was demonstrated. Therefore, the FDA approved high-dose IL-2 for the treatment of advanced renal cancer and malignant melanoma in 1992 and 1998, which also triggered a wave of research on IL-2.

[0004] IL-2 is a multifunctional cytokine activated by antigens. It can regulate the differentiation and development of various lymphocytes and play an important role in immune activation and maintaining immune homeostasis. IL-2 activates downstream signals by binding to its receptors. The IL-2 receptor is composed of three subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). The β and γ subunits have a medium affinity (Kd≈10 -9 M) dimer receptors initiate target gene transcription by activating STAT5, PI3K-AKT and MAPK pathways; in addition, CD25 has a lower affinity for IL-2 (Kd≈10 -8M) can capture IL-2 and then present it to the dimeric receptor through conformational changes, thus forming a high-affinity trimeric receptor (Kd≈10 -11 M) to achieve signal transduction, in which CD25 is not necessary for signal transduction. Due to the difference in affinity between dimer and trimer receptors, low concentrations of IL-2 are more likely to bind to Treg cells expressing trimeric receptors, thereby showing immunosuppressive activity, while under high concentrations, they can bind to CD8 Treg cells expressing more dimer receptors. + T and NK cells act to activate immune system.

[0005] IL-2 is a key cytokine for regulating Treg cells. Low-dose treatment (0.5-1 million IU / m 2 ) plays an important role in a variety of autoimmune diseases, including vasculitis, inflammatory myopathy and systemic lupus erythematosus. In order to achieve the purpose of treating tumors, the use of a dosage of up to 600,000 IU / kg not only greatly increases the manufacturing cost, but also induces toxic side effects such as capillary permeability syndrome, interstitial lung infiltration and liver cell damage. In order to avoid the activation of Treg cells by low concentrations of IL-2 and the toxicity caused by high concentrations, existing modifications mainly achieve the weakening or enhancement of CD25 affinity through site mutation (super cytokine IL-2), non-site (NKTR-214) or site-specific (THOR-707) coupling of polyethylene glycol to change the preference for affinity to dimeric receptors.

[0006] However, the above IL-2-based modifications all have the problem of extremely low yield due to the instability of the natural protein, and cannot completely block CD25. In the existing technology, IL-2 mutants, such as Neo-2 / 15, while eliminating the affinity of CD25, enhance the binding to the β subunit (CD122 subunit), thus activating only the immune cells CD8 + The goal is to activate T and NK cells without affecting Tregs. However, given their increased affinity for the CD122 subunit, which leads to NK cell activation, safety and dosing window have been major obstacles for existing IL-2 mutants to fully exert their functions. Furthermore, as small proteins, existing IL-2 mutants have a short half-life, requiring daily dosing in mouse models to ensure anti-tumor effects. This high frequency of dosing significantly reduces patient well-being.

[0007] As people's requirements for clinical drug use continue to increase, in order to improve the frequency and dosage of administration, how to prolong the half-life of IL-2 mutants while reducing toxicity has become a goal that needs to be solved.

[0008] Summary of the Invention

[0009] The main purpose of the present invention is to provide a polypeptide and its conjugate and application, so as to solve the problem in the prior art that IL-2 mutants activate NK cells and / or CD8 in the peripheral circulation due to their high affinity with CD122 (IL-2Rβ subunit). + The problem of systemic toxic side effects caused by T cells.

[0010] To achieve the above objectives, according to a first aspect of the present invention, a polypeptide is provided, comprising an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid represented by X at position 14 and / or position 39 has the following characteristics: being capable of being coupled to a modifier and being capable of being uncoupled from the modifier in a tumor microenvironment to release the polypeptide; wherein the binding ability of the polypeptide uncoupled to the modifier to CD122 is recorded as a first binding ability, the binding ability of the polypeptide coupled to the modifier to CD122 is recorded as a second binding ability, and the ratio of the first binding ability to the second binding ability is recorded as n, wherein n ≥ 5.

[0011] Furthermore, the amino acid represented by X at position 14 and / or position 39 is any amino acid having a thiol side chain; preferably, the amino acid represented by X at position 14 and / or position 39 forms an intramolecular covalent bond directly or through a linker; preferably, the linker comprises mercaptopyridine; preferably, the amino acid represented by X at position 14 and / or position 39 comprises cysteine.

[0012] To achieve the above objectives, according to a second aspect of the present invention, a polypeptide conjugate is provided, comprising a polypeptide and a modifier conjugated to the polypeptide, wherein the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid represented by X at position 14 and / or position 39 has the following characteristics: can be conjugated to the modifier and can be uncoupled from the modifier in a tumor microenvironment to release the polypeptide; wherein the binding ability of the polypeptide unconjugated to the modifier to CD122 is recorded as a first binding ability, the binding ability of the polypeptide conjugated to the modifier to CD122 is recorded as a second binding ability, and the ratio of the first binding ability to the second binding ability is recorded as n, wherein n ≥ 5.

[0013] Further, the modifier includes a polymer or a fatty acid; preferably, the polymer includes PEG; preferably, the fatty acid includes an unsaturated fatty acid or a saturated fatty acid; preferably, the unsaturated fatty acid or the saturated fatty acid is selected from a fatty acid with a C atom number of 6-22; preferably, the saturated fatty acid is selected from any one of the following: 17-carboxyheptadecanoic acid, 19-carboxynonadecanoic acid, 4-[N-(2-carboxyethyl)-N-15-carboxypentadecanoyl)amino)methyl]benzoic acid or [2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetic acid.

[0014] Furthermore, the amino acid represented by X at position 14 and / or position 39 of the polypeptide is any amino acid having a sulfhydryl side chain; preferably, the modifier is coupled to the sulfhydryl side chain on the amino acid at position 14 and / or position 39 of the polypeptide via a disulfide bond; preferably, the modifier is coupled to the sulfhydryl side chain on the amino acid at position 14 and / or position 39 of the polypeptide via a disulfide bond via mercaptopyridine; preferably, the modifier comprises PEG; preferably, the amino acid represented by X at position 14 and / or position 39 of the polypeptide comprises cysteine.

[0015] In order to achieve the above object, according to the third aspect of the present invention, a DNA molecule is provided, which encodes the above polypeptide or the polypeptide in the polypeptide conjugate.

[0016] In order to achieve the above object, according to a fourth aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above DNA molecule.

[0017] In order to achieve the above object, according to a fifth aspect of the present invention, a host cell is provided, into which the above recombinant plasmid is transformed.

[0018] In order to achieve the above object, according to the sixth aspect of the present invention, there is provided a method for preparing a method for specifically activating CD8 in a tumor microenvironment by using the above polypeptide or the above polypeptide conjugate. + Use of T cells and / or NK cells in medicine.

[0019] Furthermore, the above-mentioned use includes use in preparing drugs for promoting immunity.

[0020] Furthermore, the above-mentioned use includes use in the preparation of a medicament for preventing and / or treating solid tumors or blood tumors; preferably, the solid tumor is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer or prostate cancer; preferably, the blood tumor is chronic lymphocytic leukemia, small lymphocytic lymphoma, follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Waldenstrom macroglobulin The patient may have leukemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, thymic large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[0021] The technical solution of the present invention has an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acids represented by X at positions 14 and 39 can be coupled with a modifier with a certain steric hindrance. The polypeptide retains its existing property of not binding to CD25 (IL-2Rα subunit), and because of its weakened binding to CD122, it avoids the problem in the prior art that IL-2 mutants aggravate the proliferation of NK cells and CD8 + The activation of T cells can lead to abnormal increase in cytokines or abnormal weight loss, etc. In addition, the peptide also provides a site for conjugation, which facilitates the preparation of subsequent conjugates.

[0022] In a preferred embodiment, the present invention further couples the polypeptide with a modifier, and the obtained polypeptide conjugate greatly reduces its binding ability to CD122 (IL-2Rβ subunit) due to the shielding of the modifier, thereby avoiding the binding of NK cells and CD8 + Activation of T cells; in the tumor microenvironment, tumor microenvironment-specific enzymes can specifically decouple the peptide conjugate to release the peptide. The released peptide resumes activation of the IL2-Rβγ receptor, specifically activating NK cells and T cells in the tumor microenvironment, exerting a specific killing effect on the tumor, limiting the specific killing to the tumor, improving specificity and effectiveness, and avoiding toxic side effects in the peripheral circulation. At the same time, the conjugation of modifiers with significant steric hindrance greatly prolongs the half-life of the peptide in vivo, thereby helping to reduce the frequency of clinical dosing and improve patient compliance. It can achieve better therapeutic effects while reducing the frequency and dosage of dosing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0024] FIG1 shows a structural diagram of IL-2 and its receptor in Example 1 of the present invention;

[0025] FIG2 shows the proliferation-promoting activity of the polypeptides after single mutation of different sites to cysteine ​​in Example 1 of the present invention on CTLL2;

[0026] FIG3 shows the proliferation-promoting activity of the polypeptide (14C) in Example 2 of the present invention on MO7E;

[0027] FIG4 shows the proliferation-promoting activity of the polypeptide (39C) in Example 2 of the present invention on MO7E;

[0028] FIG5 shows a schematic diagram of the reaction between OPSS-PEG and cysteine ​​in Example 2 of the present invention;

[0029] FIG6 shows the proliferative activity of polypeptide (39C)-PEG of Example 2 of the present invention on CTLL2 and MO7E;

[0030] FIG7 shows the SDS-PAGE results of CC-PEG in Example 2 of the present invention;

[0031] FIG8 shows the size exclusion chromatography results of CC-PEG in Example 2 of the present invention;

[0032] FIG9 shows the uncoupling of CC-PEG under 0.3 mM GSH over time in Example 3 of the present invention;

[0033] FIG10 shows the uncoupling of CC-PEG under 3 mM GSH over time in Example 3 of the present invention;

[0034] FIG11 shows the pro-proliferative activity of CC on CTLL2 and MO7E in Example 3 of the present invention;

[0035] FIG12 shows the stability of CC-PEG in Example 4 of the present invention after different periods of time in plasma;

[0036] FIG13 shows the proliferative activity of Neo-2 / 15, CC-PEG, and CC-PEG and GSH on CTLL2 and Mo7e in Example 5 of the present invention;

[0037] FIG14 shows the effect of Q1D administration of Neo-2 / 15 and Q4D administration of CC-PEG 0.15 mg / kg on mouse body weight and anti-tumor effect in Example 7 of the present invention;

[0038] FIG15 shows the analysis of the proportion of lymphocytes in different tissues of mice at sacrifice after Q1D administration of Neo-2 / 15 and Q4D administration of CC-PEG 0.15 mg / kg in Example 7 of the present invention;

[0039] FIG16A shows the effects of different doses of CC-PEG administered Q4D on the body weight of mice in Example 8 of the present invention;

[0040] FIG16B shows the effect of different doses of CC-PEG administered Q4D on the proportion of body weight change in mice in Example 8 of the present invention;

[0041] FIG17 shows the anti-tumor effects of different doses of CC-PEG administered Q4D on mice in Example 8 of the present invention;

[0042] FIG18A shows the effects of Q1D administration of Neo-2 / 15 and Q4D administration of CC-PEG 1 mg / kg on mouse body weight in Example 9 of the present invention;

[0043] FIG18B shows the effect of Q1D administration of Neo-2 / 15 and Q4D administration of CC-PEG 1 mg / kg on the proportion of body weight change in mice in Example 9 of the present invention;

[0044] FIG19 shows the effects of Q1D administration of Neo-2 / 15 and Q4D administration of CC-PEG 1 mg / kg on the anti-tumor effects in mice in Example 9 of the present invention;

[0045] FIG20 shows the results of IFN-γ production in mice induced by Q1D administration of Neo-2 / 15 and Q4D administration of CC-PEG 1 mg / kg in Example 9 of the present invention. DETAILED DESCRIPTION

[0046] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0047] As mentioned in the background art, IL-2, as a pleiotropic cytokine after antigen activation, can regulate the differentiation and development of various lymphocytes, and plays an important role in immune activation and maintaining immune homeostasis. The prior art has IL-2 mutants that do not bind to CD25 and at the same time enhance the affinity for dimers. Although this mutant avoids the immunosuppression that may be caused by the activation of Tregs, it has a certain potential toxicity to cells due to its high affinity for the β subunit. Therefore, the inventors of this application provide a polypeptide conjugate with an extended half-life, which can specifically act in tumor tissue and has reduced toxicity.

[0048] In a first typical embodiment of the present application, a polypeptide is provided, which has an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid represented by X at position 14 and / or position 39 has the following characteristics: it can be coupled to a modifier and can be uncoupled from the modifier in a tumor microenvironment to release the polypeptide; wherein the binding ability of the polypeptide not coupled to the modifier to CD122 is recorded as a first binding ability, the binding ability of the polypeptide coupled to the modifier to CD122 is recorded as a second binding ability, and the ratio of the first binding ability to the second binding ability is recorded as n, wherein n ≥ 5.

[0049] SEQ ID NO: 1:

[0050] The above polypeptide does not bind to CD25, thus avoiding the immunosuppression caused by possible activation of Treg. At the same time, the binding ability to CD122 (IL-2Rβ subunit) is greatly weakened, thus avoiding the high binding affinity of IL-2 mutants to CD122 in the prior art, thereby activating the immune cells CD8 in the peripheral blood. + T cells and NK cells may cause systemic toxicity. In addition, the polypeptide provides a coupling site for subsequent conjugates, that is, any one of the 14th and 39th amino acids of the above-mentioned polypeptide or at the same time can be coupled with the modifier, which can further expand the steric hindrance and provide a possibility for the preparation of conjugate drugs, so that the polypeptide can be further utilized to play a role in clinical practice. In order to make the polypeptide specific after the amino acid residues at positions 14 and / or 39 are coupled to the modifier to decouple from the modifier in the tumor microenvironment and play its function, and not play the polypeptide function in the peripheral blood environment, it is possible to use a coupling method and a modifier in which the binding force of the polypeptide to CD122 decreases by more than 5 times after coupling.

[0051] The amino acid represented by X in the above-mentioned polypeptide can be coupled with a modifier to further expand its steric hindrance. When the type of modifier is different, the type of chemical bond for coupling with the polypeptide is also different, and thus the amino acid represented by X is also different, so that the coupling connection between the two can be specifically uncoupled in the tumor microenvironment. In a preferred embodiment, the amino acid represented by X at position 14 and / or position 39 is any amino acid with a sulfhydryl side chain. There is no special limitation on the specific type of any amino acid with a sulfhydryl side chain. The amino acid represented by X can be other natural or non-natural amino acids, or modified amino acids suitable for chemical coupling; in a preferred embodiment, the amino acid represented by X at position 14 and / or position 39 includes cysteine ​​(i.e., polypeptide (14C), polypeptide (39C) or polypeptide (14C+39C)CC).

[0052] The formation of intramolecular chemical bonds within the polypeptide can also contribute to the subsequent preparation of polypeptide conjugates with greater steric hindrance to avoid activating Tregs. In a preferred embodiment, the amino acid represented by X at position 14 and / or position 39 forms an intramolecular covalent bond directly or through a linker. Any linker capable of forming an intramolecular covalent bond is suitable for the present application. In a preferred embodiment, the linker includes mercaptopyridine. The linker has a thiol group that can form a disulfide bond with the thiol side chain of the amino acid represented by X at position 14 and / or position 39 of the polypeptide, and its covalent bond can be specifically disconnected in the tumor microenvironment without affecting the function of the polypeptide itself.

[0053] In a second typical embodiment of the present application, a polypeptide conjugate is provided, comprising a polypeptide and a modifier conjugated to the polypeptide, wherein the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid represented by X at position 14 and / or position 39 has the following characteristics: can be conjugated to the modifier, and can be uncoupled from the modifier in the tumor microenvironment to release the polypeptide; wherein the binding ability of the polypeptide not conjugated to the modifier to CD122 is recorded as the first binding ability, the binding ability of the polypeptide conjugated to the modifier to CD122 is recorded as the second binding ability, and the ratio of the first binding ability to the second binding ability is recorded as n, wherein n ≥ 5.

[0054] The above-mentioned peptide conjugates are coupled on the basis of peptides, and while retaining the ability of the peptide not to bind to CD25, they partially or completely block its binding to CD122 (IL-2Rβ subunit), and due to the shielding of the modifier, it cannot activate immune cells CD8 in the peripheral blood circulation. + T cells and NK cells, and in tumor tissue, they are decoupled by specific enzymes in the tumor microenvironment and play a role, achieving a specific function that only works in tumor tissue, greatly reducing the possibility of systemic toxicity caused by the high binding affinity of IL-2 mutants to CD122 in the existing technology. Furthermore, unlike the existing technology in which IL-2 mutants significantly increase the proportion of NK cells in peripheral blood and may induce inflammation, the peptide conjugate enhances the infiltration of lymphocytes in tumor tissue, greatly increasing the number of NK cells and CD8 + The proportion of T cells limits its tumor-killing effect to the interior of the tumor, making it extremely safe and effective. In addition, the half-life of the conjugate is extended, reducing the high frequency of daily dosing required for IL-2 mutants in the existing technology. On the basis of reducing toxicity, the frequency of dosing can be greatly reduced during clinical use, thereby improving patient compliance with medication.

[0055] The modifier in the polypeptide conjugate of the present application plays a role in shielding the polypeptide in the peripheral blood circulation to prevent the polypeptide from activating immune cells CD8 in the peripheral blood circulation. + The effects of T cells and NK cells. Therefore, any modifier that can achieve the above technical effects is applicable to this application. In a preferred embodiment, the modifier includes a polymer or a fatty acid. In a preferred embodiment, the polymer includes PEG. Any PEG that can achieve the technical effects of the above modifier is applicable to this application. In a preferred embodiment, the PEG is thiopyridine-PEG, and its molecular weight is 9kDa to 11kDa. Specifically, it can be 9kDa, 10kDa, or 11kDa.

[0056] Fatty acid refers to a chemical molecule consisting of a hydrocarbon chain ending in a carboxylic acid group, typically having 6-22 carbon atoms. For the present invention, various fatty acid derivatives are also considered to be fatty acids because they can be coupled to the amino acid represented by X. Fatty acids and their derivatives are the main components of lipids and are hydrophobic. The length and saturation of the hydrocarbon chain vary from fatty acid to fatty acid, determining the relevant physical properties. The types of fatty acids include unsaturated fatty acids (polyunsaturated and monounsaturated) and saturated fatty acids; saturated fatty acids are hydrogen-saturated and are mostly straight hydrocarbon chains with an even number of carbon atoms. In a preferred embodiment, the fatty acids include unsaturated fatty acids or saturated fatty acids, and the unsaturated fatty acids or saturated fatty acids are selected from fatty acids having a C atomic number of 6-22. In a preferred embodiment, the saturated fatty acid is selected from any one of the following: 17-carboxyheptadecanoic acid, 19-carboxynonadecanoic acid, 4-[N-(2-carboxyethyl)-N-15-carboxypentadecanoyl)amino)methyl]benzoic acid or [2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetic acid.

[0057] The conjugation method of the modifier of the present invention is not limited. Any chemical group that can be decoupled in the tumor microenvironment is applicable to this application. In a preferred embodiment, the amino acid represented by X at position 14 and / or position 39 of the above-mentioned polypeptide is any amino acid with a sulfhydryl side chain. The chemical bond between the amino acid with a sulfhydryl side chain and the modifier can be specifically decomposed in the tumor microenvironment. In a preferred embodiment, the modifier is conjugated to the sulfhydryl side chain on the amino acid at position 14 and / or position 39 of the polypeptide via a disulfide bond. The disulfide bond of the conjugate is decomposed by relevant enzymes in the tumor microenvironment, allowing the polypeptide in the conjugate to be released and exert its function. In a preferred embodiment, the above-mentioned modifier is conjugated to the sulfhydryl side chain on the cysteine ​​at position 14 and / or position 39 of the polypeptide via a disulfide bond via thiopyridine. Any modifier capable of the above-mentioned conjugation connection is applicable to this application. In a preferred embodiment, the modifier includes PEG. There is no particular limitation on the specific type of any amino acid having a thiol side chain. In a preferred embodiment, the amino acid represented by X at position 14 and / or position 39 of the polypeptide includes cysteine.

[0058] In a third typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned polypeptide or the polypeptide in the above-mentioned polypeptide conjugate.

[0059] In a fourth typical embodiment of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

[0060] In a fifth exemplary embodiment of the present application, a host cell is provided, into which the aforementioned recombinant plasmid is transformed. Utilizing the aforementioned host cell, the recombinant plasmid can be replicated within the host cell, and the DNA molecules carried on the recombinant plasmid can be transcribed and translated to obtain a large number of polypeptides.

[0061] In a sixth typical embodiment of the present application, there is provided a method for preparing a method for specifically activating CD8 in a tumor microenvironment by using the above-mentioned polypeptide or the above-mentioned polypeptide conjugate. + Use of T cells and / or NK cells in medicine.

[0062] Also, the steric hindrance effect of the peptide conjugate combined with PEG blocks its binding to the β subunit, thus preventing it from activating immune cells NK cells and CD8 +The systemic toxicity caused by T cells is eliminated. In the tumor microenvironment, the disulfide bonds in the polypeptide conjugate can be specifically uncoupled and exert their effects under the action of tumor-specific GSH, achieving aggregation in the tumor environment, thereby reducing the minimum dosage of the dosing window. The use of the polypeptide conjugate of the present application can greatly reduce the original high frequency of daily dosing, and the dosage of each dose is also reduced, providing a positive impact on the tumor treatment process.

[0063] In a preferred embodiment, the above-mentioned use includes use in preparing a drug for promoting immunity.

[0064] In a preferred embodiment, the above-mentioned use includes use in the preparation of drugs for preventing and / or treating solid tumors and blood tumors.

[0065] In a preferred embodiment, the solid tumor is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer or prostate cancer. In a preferred embodiment, the above-mentioned blood tumor is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma or lymphomatoid granulomatosis.

[0066] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0067] Example 1 Design and screening of polypeptides

[0068] To achieve the goal of reducing the toxicity of IL-2 mutants in the existing technology in the peripheral blood environment and enabling them to function in the tumor microenvironment, we designed to couple two polyethylene glycols through disulfide bonds at the interface between IL-2 and its β receptor. Polyethylene glycol is a hydrophilic, low-immunogenic biocompatible polymer. On the one hand, the large steric hindrance of PEG is used to block the binding of CD122. On the other hand, the disulfide bond can be opened in a high glutathione (GSH) environment to achieve specific release in the tumor microenvironment and thus exert activity.

[0069] Based on the spatial structure of IL-2 and β receptors, 3-5 sites were selected in each of the two α-helices facing CD122 for cysteine ​​mutation. The cell proliferation activity of the mutated sites on CTLL2 was compared with that of the IL-2 mutant in the existing technology, Neo-2 / 15. Site 14, whose spatial site faces the receptor binding surface, and site 39, which has less effect on cell activity, were selected. These two sites were finally selected as mutation sites for subsequent experiments (Figures 1-2).

[0070] Example 2 Construction of IL-2 mutant coupled with double PEG

[0071] Cysteine ​​was substituted at position 14 or position 39 of SEQ ID NO: 1, respectively, to generate polypeptides (14C) and (39C), and the effects of the mutations on their activity and function were verified in CTLL2 and MO7E cells. As shown in Figures 3-4, the mutations in polypeptide (39C) had little effect on their activity and function in CTLL2 and MO7E cells, but polypeptide (14C) exhibited approximately 100-fold decreased proliferative activity against CTLL2 and MO7E cells compared to Neo-2 / 15.

[0072] The cysteine ​​thiol group then reacted with the thiol pyridine of OPSS-PEG to form a disulfide bond for coupling (Figure 5). In Figure 5, R represents a cysteine-containing peptide, and P represents a PEG with a thiol group. Through the above reaction, the single mutant was successfully coupled to a single 10KD PEG (peptide (39C)-PEG and peptide (14C)-PEG), and its activity was verified in CTLL2 and MO7E cells. The results showed that the blocking effect of peptide (39C)-PEG was approximately 10-fold weaker than that of Neo-2 / 15 (Figure 6). No relevant data for peptide (14C)-PEG are provided.

[0073] Through point mutagenesis, we successfully constructed an expression plasmid for polypeptide 14C+39C (hereinafter referred to as CC). After transformation into the BL21 expression strain, the plasmid was cultured at 37°C to an OD600 of 0.6-0.8 and induced at 25°C. The supernatant was purified by high-pressure or ultrasonication, centrifuged, and passed through a nickel column. The eluate was treated with TCEP and concentrated over molecular sieves (DPBS, pH 7.5) to obtain the CC monomer. The concentrated CC was incubated with 10 molar equivalents of thiopyridine-PEG (OPSS-PEG) (10K) at 37°C for 2-3 hours. Unreacted OPSS-PEG was directly removed by anion exchange column (Tris, pH 9.0) to obtain the pure coupling product CC-PEG (Figures 7-8), which was finally desalted and exchanged in DPBS buffer.

[0074] Example 3 Verification of CC-PEG decoupling under GSH conditions

[0075] CC-PEG was incubated with GSH at final concentrations of 0.3 mM and 3 mM at 37°C for different times (1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, and 60 h), and the uncoupling was verified by SDS. As shown in Figure 9, under the action of 0.3 mM GSH, CC coupled with two PEGs can first quickly release one of the PEGs, and then achieve simultaneous release of both PEGs over time, ultimately achieving maximum uncoupling effect within 8-12 h, and the ratio of CC-PEG with any one PEG removed to CC with both PEGs removed is approximately 2:1.

[0076] Under the action of higher concentrations of GSH (Figure 10, 3 mM GSH), CC-PEG can quickly achieve complete uncoupling and thus exert its function. The final uncoupling products are mainly peptides with both PEGs removed, with only a very small amount of peptides with only one PEG removed.

[0077] In addition, the uncoupled CC was subjected to an experiment to investigate its effect on the proliferative activity of CTLL2 and MO7E. As shown in Figure 11 , it can be seen that CC has little effect on the active functions of the two, and that CC can still maintain immune cell activation activity similar to that of Neo-2 / 15 in tumor tissues.

[0078] Example 4 CC-PEG plasma stability verification

[0079] CC-PEG was incubated in serum at 37°C for different time periods (1 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h). Western Blot analysis revealed that CC-PEG was not prematurely released from the blood within a certain period of time, thus significantly avoiding the systemic toxicity caused by highly active Neo-2 / 15 (Figure 12).

[0080] Example 5 Cell function detection

[0081] Based on this experiment, we tested the proliferation-promoting effects of Neo-2 / 15, CC-PEG, and the protein obtained by co-incubating CC-PEG with GSH in a GSH environment simulating the in vivo environment on CTLL2 cells expressing trimeric receptors and MO7E cells expressing dimeric receptors.

[0082] After the prepared proteins with different concentration gradients were co-incubated with CTLL2 for 18 hours, the readings were directly measured by Celltiter-Glo. The results are shown in Figure 13. Compared with Neo-2 / 15, the proliferation-promoting activity of CC-PEG on CTLL2 decreased by about 170 times, and the proliferation-promoting activity of CC obtained after GSH treatment on CTLL2 decreased by about 14 times compared with the activity of Neo-2 / 15, while it still maintained similar effects on immune cell proliferation as that of Neo-2 / 15. Similarly, after the proteins with different concentration gradients were co-incubated with MO7E pre-plated on a 96-well plate for 3 days, the measurement results are shown in Figure 13. Compared with Neo-2 / 15, the proliferation-promoting activity of CC-PEG on MO7E decreased by about 110 times, and the proliferation-promoting activity of CC obtained after GSH treatment on MO7E decreased by about 7 times compared with the activity of Neo-2 / 15, while it still maintained similar effects on immune cell proliferation as that of Neo-2 / 15.

[0083] In short, CC-PEG achieved effective activity blocking and activity recovery after GSH treatment on both MO7E and CTLL2 cells. Comparing the overall effects on CTLL2 and MO7E, since CC retains the property of neo-2 / 15 not binding to CD25, CC-PEG ultimately tends to act on cells expressing dimeric receptors when it finally exerts its function.

[0084] Example 6 PK of CC-PEG in mice

[0085] Female C57BL mice were randomly divided into two groups (6 mice / group) and injected with equal doses of Neo-2 / 15 or CC-PEG via the tail vein. Eye blood was collected at different time points. The concentrations of Neo-2 / 15 and CC-PEG in each sample were determined by ELISA, and the data were processed using GraphPad Prism. The results, shown in Table 1, demonstrate that CC-PEG has a significantly prolonged in vivo half-life compared to Neo-2 / 15.

[0086] Table 1 PK parameters of CC-PEG in mice

[0087] Example 7 Evaluation of CC-PEG efficacy in mice

[0088] Female C57BL / 6J mice were randomly divided into PBS, Neo-2 / 15, and CC-PEG groups, with 3 mice in each group. B16F10SI (B16F10SI is a mouse melanoma cell line commonly used in basic and clinical research on cancer and tumors) was injected subcutaneously. When the tumor size reached approximately 50 mm, the cells were injected with B16F10SI. 3After the volume of the tumor was increased to 100 μL, 0.15 mg / kg Neo-2 / 15 or CC-PEG was administered via intraperitoneal injection, and the day was recorded as D0. Thereafter, the Neo-2 / 15 group was continuously administered from D0 to D5, while the PBS group and CC-PEG group were administered twice, on D0 and D4, respectively. The changes in the tumor volume of the mice were recorded every two days. When the tumor volume of the mice in the PBS group was greater than 1000 mm 3 At the same time, the mice were euthanized, and the whole blood, spleen, and tumor tissue were collected to analyze the lymphocyte components.

[0089] As shown in Figure 14, compared with the PBS group, continuous administration of Neo-2 / 15 at a high frequency can play a certain role in delaying tumor growth in a short period of time, while CC-PEG administered only twice can significantly inhibit tumor growth on the basis of greatly reducing the frequency of administration.

[0090] The flow cytometry results showed (Figure 15) that CC-PEG significantly increased the expression of CD8 + / CD4 + The proportion of T cells in peripheral blood and spleen was significantly increased, while multiple administrations of Neo-2 / 15 significantly increased the proportion of NK cells in peripheral blood. As a member of the innate immune system, the significant increase in NK cells in peripheral blood may be closely related to the induction of inflammatory responses. Furthermore, in flow cytometry analysis of tumor tissue, we detected that CC-PEG can enhance lymphocyte infiltration in tumors and increase the proportion of NK cells, thereby reducing the proportion of NK cells in peripheral blood and reducing the possibility of inducing inflammation.

[0091] Example 8 Analysis of the relationship between CC-PEG dosage and anti-tumor effect in mice

[0092] Female C57BL / 6J mice were randomly divided into PBS group and three CC-PEG groups with different dosages (0.15 mg / kg, 1 mg / kg, 3 mg / kg), with 4 mice in each group, and subcutaneously injected with B16F10SI. 3 After the volume of the mice was greater than 1000 mm, the drug was administered by intraperitoneal injection. Four days later, the second administration was carried out in the same way, and the volume of the mice tumor was recorded every two days. When the tumor volume of the mice in the PBS group was greater than 1000 mm 3 When , the mice were euthanized.

[0093] As shown in Figures 16A, 16B, and 17, compared to the DPBS control group, all dose-administered groups can achieve a good tumor inhibition effect, and show a significantly enhanced anti-tumor effect with the increase in dose, which is dose-dependent. Body weight detection shows that the administration group has basically no effect on the weight of mice below 1 mg / kg, and all show a slowly increasing trend; administration of 3 mg / kg will cause a temporary decline in the weight of mice, which will recover within two days. Therefore, CC-PEG can achieve significant anti-tumor effects alone within a wide dosing window.

[0094] Example 9 Toxicity detection of CC-PEG in mice

[0095] Female C57BL / 6J mice were randomly divided into PBS group, Neo-2 / 15 group, and CC-PEG group, with 3 mice in each group, and subcutaneously injected with B16F10SI. 3 After the volume of the tumor was increased to 100 μL, 1 mg / kg Neo-2 / 15 or CC-PEG was administered by intraperitoneal injection of 100 μL PBS, and the dose was recorded as D0. Thereafter, the Neo-2 / 15 group was continuously administered from D0 to D5, while the PBS group and CC-PEG group were administered twice, on D0 and D4, respectively. The changes in the volume of the tumor in the mice were recorded every two days, and eye blood was collected 6 hours after the second administration of CC-PEG on D4 and 6 hours after the continuous administration of Neo-2 / 15 on D5 and D6. When the tumor volume of the mice in the PBS group was greater than 1000 mm 3 When , the mice were euthanized.

[0096] As shown in Figures 18A, 18B, and 19, under the condition of medium-dose administration (1 mg / kg), continuous administration of Neo-2 / 15 and Q4D administration of CC-PEG can significantly inhibit tumor growth, but the tumor growth curve shows that CC-PEG is more effective than Neo-2 / 15. Under this premise, the body weight of mice decreased after continuous administration of Neo-2 / 15 on D4, and continued to decrease with subsequent administration.

[0097] ELISA detection of cytokine levels in plasma (see Figure 20) showed that no IFN-γ was produced in the DPBS group. In the two drug-treated groups, CC-PEG had a better anti-tumor effect than Neo-2 / 15. Continuous administration of Neo-2 / 15 promoted the production of more IFN-γ than CC-PEG, which may have caused the weight loss of mice, suggesting that CC-PEG is safer than Neo-2 / 15.

[0098] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the polypeptides (polypeptide (14C), polypeptide (39C), CC) and polypeptide conjugates (CC-PEG) in the present application have the characteristics of not binding to CD25 and weakening binding to CD122. Moreover, the polypeptide conjugate (CC-PEG) can specifically act only in tumor tissue, significantly inhibiting tumor growth, reducing the toxic and side effects of IL-2 variants in systemic immunity in the prior art, increasing the maximum dosage of the dosing window, and providing a wider range of choices for dosing in clinical experiments. In addition, the half-life of the polypeptide conjugate (CC-PEG) in mice is long, which greatly reduces the frequency of administration. Also, because the polypeptide conjugate (CC-PEG) can accumulate in large quantities in tumor tissue, its dosage is also greatly reduced. It can be seen that the polypeptides and polypeptide conjugates of the present application provide a positive impact on the treatment of tumors.

[0099] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A polypeptide, It is characterized in that The polypeptide has an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid represented by X at position 14 and / or position 39 has the following characteristics: it can be coupled with a modifier, and can be decoupled from the modifier in a tumor microenvironment to release the polypeptide; Among them, the binding ability of the polypeptide not coupled to the modifier to CD122 is recorded as the first binding ability, the binding ability of the polypeptide coupled to the modifier to CD122 is recorded as the second binding ability, and the ratio of the first binding ability to the second binding ability is recorded as n, wherein n≥5.

2. The polypeptide according to claim 1, It is characterized in that The amino acid represented by X at position 14 and / or position 39 is any amino acid having a thiol side chain; Preferably, the amino acid represented by X at position 14 and / or position 39 forms an intramolecular covalent bond directly or through a linker; Preferably, the linker comprises mercaptopyridine; Preferably, the amino acid represented by X at position 14 and / or position 39 includes cysteine.

3. A polypeptide conjugate, It is characterized in that The polypeptide conjugate comprises a polypeptide and a modifier coupled to the polypeptide, wherein the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid represented by X at position 14 and / or position 39 has the following characteristics: it can be coupled to the modifier and can be decoupled from the modifier in a tumor microenvironment to release the polypeptide; Among them, the binding ability of the polypeptide not coupled to the modifier to CD122 is recorded as the first binding ability, the binding ability of the polypeptide coupled to the modifier to CD122 is recorded as the second binding ability, and the ratio of the first binding ability to the second binding ability is recorded as n, wherein n≥5.

4. The polypeptide conjugate according to claim 3, It is characterized in that The modification includes polymers or fatty acids; Preferably, the polymer comprises PEG; Preferably, the fatty acid comprises an unsaturated fatty acid or a saturated fatty acid; Preferably, the unsaturated fatty acid or saturated fatty acid is selected from fatty acids with 6-22 carbon atoms; Preferably, the saturated fatty acid is selected from any one of the following: 17-carboxyheptadecanoic acid, 19-carboxynonadecanoic acid, 4-[N-(2-carboxyethyl)-N-15-carboxypentadecanoyl)amino)methyl]benzoic acid or [2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetic acid.

5. The polypeptide conjugate according to claim 4, It is characterized in that The amino acid represented by X at position 14 and / or position 39 of the polypeptide is any amino acid having a thiol side chain; Preferably, the modifier is coupled to the thiol side chain on the 14th and / or 39th amino acid of the polypeptide via a disulfide bond; Preferably, the modifier is a thiopyridine-containing compound on the 14th and / or 39th amino acid of the polypeptide. The sulfhydryl side chains are coupled with disulfide bonds; Preferably, the modifier comprises PEG; Preferably, the amino acid represented by X at position 14 and / or position 39 of the polypeptide includes cysteine.

6. A DNA molecule, It is characterized in that The DNA molecule encodes the polypeptide according to claim 1 or 2 or the polypeptide in the polypeptide conjugate according to any one of claims 3 to 5.

7. A recombinant plasmid, It is characterized in that The recombinant plasmid is connected to the DNA molecule according to claim 6.

8. A host cell, It is characterized in that The host cell is transformed with the recombinant plasmid according to claim 7.

9. The polypeptide according to claim 1 or 2 or the polypeptide conjugate according to any one of claims 3 to 5 in the preparation of a polypeptide specifically activating CD8 in a tumor microenvironment + Use of T cells and / or NK cells in medicine.

10. The use according to claim 9, It is characterized in that The use includes use in preparing drugs for promoting immunity.

11. The use according to claim 10, It is characterized in that The use includes use in preparing a drug for preventing and / or treating solid tumors or blood tumors; Preferably, the solid tumor is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer or prostate cancer; Preferably, the blood tumor is chronic lymphocytic leukemia, small lymphocytic lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, thymic large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma or lymphomatoid granulomatosis.

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