Chimeric region for constructing chimeric protein, construction method, chimeric protein and application of chimeric protein

Abstract

The invention discloses a chimeric protein. The chimeric protein comprises a cell factor and a cell factor receptor extracellular domain, the cytokine is embedded into the chimeric region of the cytokine receptor extracellular domain, and the chimeric region is located in the flexible connection region of the cytokine receptor extracellular domain, or the cytokine receptor extracellular domain is embedded into the chimeric region of the cytokine, and the chimeric region is located in the flexible connection region of the cytokine. Proper chimeric regions are selected on CD25 and IL-2 proteins, the two proteins are divided into different peptide fragments, the peptide fragments are connected to form a peptide chain, and the peptide chain is expressed to form the chimeric protein. The present invention relates to compounds having eliminated or reduced affinity to high affinity IL-2 receptors (IL-2R [alpha] [beta] [gamma]) and retaining / enhancing affinity to medium affinity IL-2 receptors (IL-2R [beta] [gamma]). The invention further discloses a chimeric protein of the IL15RA and the IL-15 and a chimeric protein of the IL21R and the IL21. The invention also discloses a conjugate, a pharmaceutical composition, a nucleic acid molecule, an expression vector, a host cell, a production method, a kit and application of the chimeric protein.

Description

[0001] Divisional Statement

[0002] This application is a divisional application of the Chinese application with the application number of 2023101713128 and the title of “Chimeric region for constructing chimeric protein, construction method, chimeric protein and application thereof”, which has a priority claim to the Chinese application with the application number of 202210185656.X and the title of “Chimeric region for constructing chimeric protein, construction method, chimeric protein and application thereof”, filed on February 28, 2022, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine. Specifically, the present application relates to a chimeric region for constructing a chimeric protein, a construction method, a chimeric protein, a preparation method and an application. The present application also relates to a derivative, a conjugate, a pharmaceutical combination, a nucleic acid molecule, an expression vector and a host cell of the chimeric protein. BACKGROUND

[0004] Cytokines (CK) are low molecular weight soluble proteins produced by cells, which can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemotactic factors, growth factors, etc. according to their functions. Cytokines mediate a variety of biological functions, including immune regulation, hematopoiesis, cell growth and repair of damaged tissues, by binding to the corresponding cytokine receptors on the cell surface and transmitting intracellular signal transduction. Therefore, cytokine-based drugs are an important field in drug development. However, many natural cytokines are not suitable as drugs. Because a cytokine can usually bind to multiple receptors expressed on the surface of different cells, transmitting different signals, and in addition, the same receptor can also bind to different cytokines, which leads to the multi-effectiveness, overlapping, antagonism, synergy and other physiological properties of cytokines. In clinical treatment, a specific signal activated by a specific cytokine is needed to participate in, without activating unnecessary signals, to further perform the expected function, and to achieve the purpose of good therapeutic effect and small side effects. Most cytokine receptors are transmembrane proteins, consisting of extracellular domain, transmembrane region and cytoplasmic domain.

[0005] For example, taking interleukin 2 as an example, interleukin 2 (IL-2), also known as T cell growth factor (TCGF), is a 15.5 kDa globular glycoprotein with a length of 133 amino acids. The structure of IL-2 is composed of four anti-parallel, amphipathic alpha helices and some linking sequences (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). IL-2 is mainly derived from activated CD4+ T cells, activated CD8+ T cells, natural killer (NK) cells, dendritic cells and macrophages, and can regulate its action by binding to the IL-2 receptor on the cell surface.

[0006] The IL-2 receptor is a complex of three subunits, IL-2Ra (i.e., CD25), IL-2Rβ (i.e., CD122), and IL-2Rγ (i.e., CD132). The expression of these three subunits and their affinity for IL-2 vary. The heterodimeric IL-2 receptor formed by the IL-2Rβ and IL-2Rγ subunits is mainly expressed by cytotoxic CD8+ T cells and NK cells, binds IL-2 with intermediate affinity, and is referred to as the intermediate affinity IL-2 receptor (IL-2Rβγ). The heterotrimeric IL-2 receptor formed by the IL-2Ra, IL-2Rβ, and IL-2Rγ subunits is mainly expressed on regulatory T cells (Tregs) (Byman, O., and Sprent, J. Nat. Rev. Immunol. 12, 180-190 (2012)) and binds IL-2 with high affinity (about 100-fold higher than the affinity of the dimeric receptor), and is referred to as the high affinity IL-2 receptor (IL-2Raβγ). In addition, some endothelial cells are also found to express the receptor a (CD25) subunit of IL-2 on their surface. IL-2Rβ and IL-2Rγ are necessary for the activation of the downstream signaling pathway of IL-2, and when IL-2 binds to IL-2Rβ and IL-2Rγ at the same time, the two receptor subunits form a heterodimer, phosphorylate STAT5 in the cell, enter the nucleus, and lead to the transcription and expression of the corresponding genes; IL-2Ra is not necessary for the signal, but can promote the binding of IL-2 to IL-2Rβ and IL-2Rγ. IL-2Rγ is expressed in all immune cells; IL-2Rβ is expressed in CD8+ T cells, NK cells, and regulatory T cells, and the expression level is also increased after the T cells are activated; IL-2Ra is continuously highly expressed in regulatory T cells, and is transiently expressed in activated CD8+ T cells, and then the expression level is down-regulated. Because the effector T cells and NK cells in the resting state do not have IL-2Ra on the cell surface, they are relatively insensitive to IL-2. Treg cells consistently express the highest level of IL-2Ra in the body, so under normal circumstances, IL-2 will preferentially stimulate Treg cell proliferation.

[0007] IL-2 has the ability to expand lymphocyte populations and enhance the effector functions of these cells in vivo, especially the proliferation and activation of CD8+ T cells and NK cells, which endow IL-2 with anti-tumor ability. IL-2 is the first cytokine used for tumor immunotherapy in history, and its anti-tumor effect has been clinically proven. High-dose IL-2 therapy has been approved for patients with metastatic renal cell carcinoma and malignant melanoma. After years of clinical application, people have a deeper understanding of IL-2, and the clinical application of IL-2 has also brought many problems.

[0008] One concern with IL-2 immunotherapy is the side effects from recombinant human IL-2 treatment. Patients receiving high dose IL-2 treatment develop vascular (or capillary) leak syndrome (VLS), a pathologic increase in vascular permeability that leads to fluid extravasation in multiple organs (causing, for example, pulmonary and cutaneous edema and hepatocyte injury) and intravascular fluid depletion (causing blood pressure drop and compensatory heart rate increase). VLS is not treated other than by discontinuing IL-2. It has been found that the development of VLS can be related to the binding of IL-2 to IL-2RA expressed by endothelial cells (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)). Second, the number and activity of peripheral Treg cells are maintained by IL-2 binding to high affinity IL-2 receptors expressed on suppressive Treg cells (Treg) (Maloy and Powrie, Nature Immunol 6, 1171-72 (2005)). Treg cells suppress effector T cells from destroying their targets; or inhibit T cell help and activation via cell-cell contact; or deplete IL-2 induced anti-tumor immunity via release of immunosuppressive cytokines such as IL-10 or TGF-β (Imai et al., Cancer Sci 98, 416-23 (2007)). In addition, due to its small molecular weight, IL-2 has a short half-life in vivo, requiring continuous administration to maintain high concentrations of IL-2 in vivo.

[0009] Inhibiting IL-2 binding to high affinity IL2 receptors (IL-2RaPy), retaining or enhancing binding to intermediate affinity IL2 receptors (IL-2RPy), and prolonging its half-life are keys to overcoming the problems of IL-2 in tumor immunotherapy. There are now a variety of approaches developed to overcome these problems associated with IL-2 immunotherapy. For example, by mutating IL-2 to change the affinity specificity of IL-2 to different receptors. Such as Merck's mutant (R38W, F42K, WO2008003473A2), which reduces the interaction with the a receptor subunit to achieve effector T cell activation to enhance efficacy; while Roche's IL-2 mutant (F42A, Y45A and L72G, US2016 / 0208017A1), which does not bind to the a receptor but can normally bind to the b and g receptor subunit complex and can play an effect, is currently in clinical trials. There are also methods to block IL-2 binding to IL-2Ra by developing antibodies to IL-2, while retaining binding to IL-2RPy. But most of these developments are still in the research stage, and there is still a need for IL-2 or derivatives with biased activation of IL-2RPy for tumor immunotherapy in the clinic.

[0010] Therefore, it is necessary to develop long-acting cytokines or proteins that can preferentially activate the intermediate affinity IL2 receptor (IL-2Rβγ) and thus better serve as clinical drugs, especially for tumor immunotherapy.

[0011] For example, IL-15 is another member of the IL-2 family with anti-tumor activity, and its receptor is composed of three receptor subunits: IL-15 receptor alpha (IL15RA or IL-15Rα), IL-2 receptor beta (IL-2Rβ, also known as IL-15Rβ or CD122), and yc (also known as CD132). IL-15 is structurally similar to IL-2 and belongs to the helical cytokine family. The heterotrimeric receptor of IL-15 shares the IL-2R / IL-15Rβ (CD122) and common yc chain (CD132) with the IL-2 receptor.

[0012] IL-15Rα is a unique component of the IL-15 receptor complex and is mainly expressed on monocytes and dendritic cells. Unlike other yc family cytokines, IL-15 first binds to IL-15Rα-expressing cells as a cytokine, and then the IL-15 / IL-15Rα complex is presented to IL-2 / 15Rβ and yc on activated T cells or NK cells. This limits the activity of IL-15. The dimeric protein of IL-15 mutant (IL-15N72D) and IL-15RαSu / Fc has been shown to have excellent anti-tumor activity in mouse models. However, there are still disadvantages such as high clearance rate and activation of peripheral immune cells leading to toxicity. In clinical use, a biased IL-15 that can directly bind to the IL-15Rβγ receptor and directly activate T cells / NK cells while avoiding the stimulation of regulatory T cells (Tregs) is needed.

[0013] IL-21 was first discovered in 2000 and belongs to the cytokine receptor gamma chain family, which is a four-alpha helix bundle type I cytokine. IL-21 is mainly secreted by activated CD4+ T cells, NK cells, TFH cells, and Th17 cells. It signals through a receptor complex composed of IL-21R and common gamma chain / IL-2Rγ (also known as CD132). In clinical practice, IL-21 with reduced affinity for IL-21R is needed to better avoid potential toxicity problems.

[0014] Given the widespread functional diversity and limited activity of cytokines in therapeutic applications, there is an urgent need to develop a method for constructing functionally biased cytokines to address the problems in clinical applications. The present application provides a chimeric protein with biased cytokine function and a method for constructing the same, and based on this platform, functionally biased cytokines can be constructed to overcome the problems in the process of cytokine drug development. SUMMARY

[0015] The present application aims to construct a chimeric protein comprising a cytokine and a cytokine receptor, so that the chimeric protein can be biased to bind to other cytokine receptors and thus perform specific functions, which can be used in clinical drugs.

[0016] In the first aspect, the present application provides a chimeric region for constructing a chimeric protein and a construction method. In some embodiments, the chimeric region is located in the flexible linker region of the extracellular domain of the cytokine receptor, and the chimeric protein is constructed by directly or indirectly (through a linker element) embedding a cytokine in the flexible linker region.

[0017] In some embodiments, the chimeric region is located in the flexible linker region of the cytokine, and the chimeric protein is constructed by directly or indirectly (through a linker element) embedding the extracellular domain of the cytokine receptor in the flexible linker region.

[0018] In the second aspect, based on the above-mentioned chimeric region for constructing a chimeric protein, the present application provides a chimeric protein comprising a cytokine and an extracellular domain of a cytokine receptor; the chimeric protein takes advantage of the natural antagonist characteristics of the extracellular domain of the cytokine receptor that can antagonize the interaction between the cytokine and itself, and by chimerizing the cytokine and the extracellular domain of the cytokine receptor into a chimeric protein, the chimeric protein is biased to bind to other cytokine receptors and thus performs specific functions.

[0019] In some specific embodiments, the cytokine is embedded in the chimeric region of the extracellular domain of the cytokine receptor, the chimeric region is located in the flexible linker region of the extracellular domain of the cytokine receptor, and the chimeric protein has the following polypeptide structure formula from N-terminus to C-terminus:

[0020] C1-I-C2;

[0021] In the formula, C1 is the amino acid sequence or its variant, modification, truncation or derivative before or after any chimeric site in the chimeric region of the extracellular domain of the cytokine receptor, C2 is the amino acid sequence or its variant, modification, truncation or derivative in the extracellular domain of the cytokine receptor except C1; I is a cytokine or a variant, modification, truncation or derivative of a cytokine.

[0022] Among them, C1 and C2 contain at least three amino acids, or at least one independent protein secondary structure unit such as alpha helix or beta fold.

[0023] Furthermore, in some embodiments, C1 and C2 are indirectly connected to I via a connector element; the chimeric protein has the following polypeptide structure from the N-terminus to the C-terminus: C1-L1-I-L3-C2; where L1 and L3 are independent connector elements.

[0024] In some embodiments, the two amino acid sequences I1 and I2 before or after the chimeric site in the polypeptide structure C1-L1-I-L3-C2 are connected by a linker element L2, that is, the chimeric protein has the following polypeptide structure from the N-terminus to the C-terminus: C1-L1-I1-L2-I2-L3-C2; where I1 is the amino acid sequence before or after any chimeric site in the cytokine chimeric region or its variants, modified forms, truncated forms or derivatives, and I2 is the amino acid sequence in the cytokine other than I1 or its variants, modified forms, truncated forms or derivatives.

[0025] In some specific implementations, the extracellular domain of the cytokine receptor may be embedded in the chimeric region of the cytokine, wherein the chimeric region is located in the flexible linker region of the cytokine; the chimeric protein has the following polypeptide structure from the N-terminus to the C-terminus:

[0026] I1-C-I2;

[0027] In the formula, I1 is the amino acid sequence before or after any chimeric site in the cytokine chimeric region, or its variants, modified forms, truncated forms, or derivatives thereof; I2 is the amino acid sequence in the cytokine other than I1, or its variants, modified forms, truncated forms, or derivatives thereof; and C is the extracellular domain of the cytokine receptor or a variant, modified form, truncated form, or derivative thereof.

[0028] Among them, I1 and I2 contain at least three amino acids, or at least one independent protein secondary structure unit, such as α-helix or β-sheet.

[0029] Furthermore, I1 and I2 are indirectly connected to C through a linker element, and the chimeric protein has the following polypeptide structure from the N-terminus to the C-terminus: I1-L1-C-L3-I2; where L1 and L3 are independent linker elements.

[0030] Furthermore, in the polypeptide structure I1-L1-C-L3-I2, the two amino acid sequences C1 and C2 before or after the chimeric site are connected by the linker element L2, that is, the chimeric protein has the following polypeptide structure from the N-terminus to the C-terminus: I1-L1-C1-L2-C2-L3-I2; where C1 is the amino acid sequence before or after any chimeric site in the extracellular domain chimeric region of the cytokine receptor, or its variants, modified versions, truncated versions, or derivatives, and C2 is the amino acid sequence in the extracellular domain of the cytokine receptor other than C1, or its variants, modified versions, truncated versions, or derivatives.

[0031] In the above embodiments, connector elements L1, L2, and L3 may be the same or different sequences, and may be selected from sequences SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or other connector elements commonly used in the art that have similar functions.

[0032] In some embodiments, the chimeric protein further includes an immunoglobulin Fc region, which can be attached to the N-terminus or C-terminus of the chimeric protein, and can be directly attached or indirectly attached via a linker element. The immunoglobulin Fc region can cause the molecule to form a dimer and prolong the molecule's in vivo half-life. The Fc region used in this invention can be derived from different immunoglobulin subtypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM. In some embodiments, mutations can be introduced into the wild-type Fc sequence to alter Fc-mediated related activities. These mutations include, but are not limited to: a) mutations altering Fc-mediated CDC activity; b) mutations altering Fc-mediated ADCC activity; or c) mutations altering FcRn-mediated in vivo half-life. Such mutations are described in the following literature: Leonard G. P. Resta, Current Opinion in Immunology 2008, 20:460–470; Esohe E. Idusogie et al., J Immunol 2000, 164:4178-4184; RAPHAELA. CLYNE et al., Nature Medicine, 2000, Volume 6, Number 4:443-446; Paul R. Hintone et al., J Immunol, 2006, 176:346-356.

[0033] In some implementations, mutations can be introduced into the Fc sequence, making the mutated Fc more likely to form homodimers or heterodimers. For example, the knob-hole model, which utilizes the spatial interaction of amino acid side chain groups at the Fc contact interface, as mentioned in Ridgway, Presta et al. (1996) and Carter (2001), makes it easier for different Fc mutations to form heterodimers. Another example is CN102558355 or CN103388013A, where the charge of the amino acids at the Fc contact interface is changed, thereby altering the ionic interaction forces between the Fc contact interfaces, making it easier for different Fc mutation pairs to form heterodimers (CN102558355A), or for Fcs with the same mutation to form homodimers (CN103388013A).

[0034] In some specific embodiments, the immunoglobulin Fc region is preferably the human immunoglobulin Fc region, and more preferably the human IgG1 Fc region. In some specific embodiments, the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO:20.

[0035] In some specific implementations, derivatives of the chimeric protein are also included.

[0036] In some specific embodiments, the cytokine receptor is CD25, the cytokine is IL-2, and IL-2 is intercalated into a chimeric region of the extracellular domain of CD25 to synthesize a CD25 / IL-2 chimeric protein. This CD25 / IL-2 chimeric protein or a derivative thereof has at least one of the following characteristics:

[0037] (a) It can naturally form a three-dimensional conformation similar to the combination of IL-2 and CD25;

[0038] (b) Compared with wild-type IL-2, it significantly reduces binding to CD25 or does not bind to CD25;

[0039] (c) Compared with wild-type IL-2, it can retain or enhance the binding to CD122;

[0040] (d) Compared with wild-type IL-2, it has a larger molecular weight and a longer half-life in vivo;

[0041] (e) It can effectively activate signaling mediated by the intermediate affinity IL-2 receptor (IL-2Rβγ);

[0042] (f) Promotes the activation of CD8+ T cells and NK cells;

[0043] (g) Promotes immune cells to kill tumor cells.

[0044] In some specific embodiments, the cytokine receptor is CD25, the cytokine is IL-2, and the extracellular domain of CD25 is embedded into the chimeric region of IL-2 to form an IL2 / CD25 chimeric protein. This IL2 / CD25 chimeric protein or a derivative thereof has at least one of the following characteristics:

[0045] (a) It can naturally form a three-dimensional conformation similar to the combination of IL-2 and CD25;

[0046] (b) Compared with wild-type IL-2, it has a larger molecular weight and a longer half-life in vivo.

[0047] In some specific embodiments, the cytokine receptor is IL15RA, the cytokine is IL-15, and they are intercalated to form an IL15RA / IL-15 chimeric protein. This IL15RA / IL-15 chimeric protein or a derivative thereof has at least one of the following characteristics:

[0048] (a) It can naturally form a three-dimensional conformation similar to the combination of IL-15 and IL15RA;

[0049] (b) Compared with wild-type IL-15, it has a larger molecular weight and a longer half-life in vivo.

[0050] (c) Significantly reduced binding to IL15RA compared to wild-type IL-15;

[0051] (d) Compared with wild-type IL-15, it can retain or enhance the binding to IL15Rβ(CD122);

[0052] (e) Promotes the activation of NK cells.

[0053] In some specific embodiments, the cytokine receptor is IL-21R, the cytokine is IL-21, and they are intercalated to form an IL-21RA / IL-21 chimeric protein. This IL-21RA / IL-21 chimeric protein or a derivative thereof has at least one of the following characteristics:

[0054] (a) It can naturally form a three-dimensional conformation similar to the combination of IL-21 and IL21RA;

[0055] (b) Compared with wild-type IL-21, it has a larger molecular weight and a longer half-life in vivo.

[0056] Thirdly, the present invention provides a conjugate comprising the above-described chimeric protein or its derivatives, wherein the chimeric protein or its derivatives are directly or indirectly connected to other modules via connector elements.

[0057] In some embodiments, the other modules include an antigen-binding module, a cytotoxin, a radioactive isotope, a bioactive protein, a detectable marker, a drug, a toxin, a cytokine, gold nanoparticles / nanorobars, magnetic nanoparticles, a viral capsid protein or VLP, any one or a combination thereof.

[0058] More preferably, the antigen-binding module is an antibody or an antigen-binding fragment; most preferably, the antibody or antigen-binding fragment targets antigens presented on tumor cells or in the tumor cell environment.

[0059] In some embodiments, the antigen-binding module targets antigens presented on tumor cells or in the tumor cell environment. In some embodiments, the antigen-binding module targets antigens on functional cells (e.g., CD8+ T cells, NK cells, CIK cells, TIL cells, macrophages, DC cells, etc.).

[0060] In some embodiments, the chimeric protein is attached to at least one other module. In some embodiments, the chimeric protein and other modules form a fusion protein, i.e., the chimeric protein shares peptide bonds with the other modules. In some embodiments, the chimeric protein is attached to at least one other module, such as first and second other modules. In some embodiments, the other module is an antigen-binding module. In some embodiments, the chimeric protein shares an amino or carboxyl-terminal peptide bond with the first antigen-binding module, and the second antigen-binding module shares an amino or carboxyl-terminal peptide bond with: i) the chimeric protein or ii) the first antigen-binding module. In some specific embodiments, the chimeric protein shares a carboxyl-terminal peptide bond with the first other module and an amino-terminal peptide bond with the second other module. In some embodiments, the other module is an antigen-binding module. The antigen-binding module may be an antibody or an antigen-binding fragment, including but not limited to immunoglobulin molecules (e.g., IgG (e.g., IgG1) class immunoglobulin molecules), antibodies, or antigen-binding fragments thereof. In some specific embodiments, the antibody or antigen-binding fragment is selected from polypeptide complexes containing variable regions of the antibody heavy chain and antibody light chain, Fab, Fv, sFv, F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, domain antibodies, multispecific antibodies (bispecific antibodies, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv), receptor-binding regions, and interacting protein-binding regions. In cases where the chimeric protein is linked to more than one antigen-binding module, such as first and second antigen-binding modules, each antigen-binding module can be independently selected from various forms of antibodies and antigen-binding fragments. For example, the first antigen-binding module may be a nanobody molecule, while the second antigen-binding module may be an scFv molecule, or each of the first and second antigen-binding modules may be a nanobody molecule, or each of the first and second antigen-binding modules may be a Fab molecule. In some implementations, when the chimeric protein is attached to more than one antigen-binding module, such as a first or second antigen-binding module, the antigen targeted by each antigen-binding module can be selected independently. For example, the first and second antigen-binding modules may target different antigens or the same antigen.

[0061] In some embodiments, the antigen bound by the antigen binding module may be selected from the group consisting of: the A1 domain (TNCA1) of tendinin C, the A2 domain (TNCA2) of tendinin C, the extradomain (EDB) of fibronectin, carcinoembryonic antigen (CEA), and melanoma-associated chondroitin sulfate proteoglycan (MCSP).In some embodiments, tumor antigens include, but are not limited to, MAGE, MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3- Zeta chains, the MAGE family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), and the GAGE ​​family of tumor antigens (e.g., G... AGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenoid nodules Adenomatous polyposis protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, Smad family tumor antigens, lmp-1, p1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2. In some embodiments, non-limiting examples of viral antigens include influenza virus hemagglutinin, Epstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gp160, and HIV gp120.In some implementations, non-limiting examples of ECM antigens include syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, EGF-type laminin, lectin, fibronectin, notch, tendinin, and matrixin.

[0062] Fourthly, the present invention discloses a pharmaceutical composition comprising the chimeric protein described above or its derivatives or the conjugates described above, as well as a pharmaceutically acceptable diluent, carrier, or adjuvant. The pharmaceutical composition may be a lyophilized formulation or an injectable solution.

[0063] Fifthly, the present invention provides a nucleic acid molecule encoding the above-mentioned chimeric protein or a derivative thereof. The nucleic acid of the present invention may be RNA, DNA, or cDNA.

[0064] In one embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:24, and the corresponding amino acid sequence is shown in SEQ ID NO:10;

[0065] In another embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:25, and the corresponding amino acid sequence is shown in SEQ ID NO:11;

[0066] In another embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:26, and the corresponding amino acid sequence is shown in SEQ ID NO:12;

[0067] In a sixth aspect, the present invention provides an expression vector containing a nucleic acid sequence of the above-mentioned chimeric protein or its derivative. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.

[0068] In one embodiment, the CD25 / IL-2 chimeric protein on the expression vector may include an N-terminal signal peptide sequence, such as sequence SEQ ID NO:19, and a C-terminal Fc sequence containing a hinge region, such as sequence SEQ ID NO:20.

[0069] In some implementations, the expression vector is a viral vector that can produce a virus with physiological functions, such as some common oncolytic viruses: herpes simplex virus (HSV), adenovirus, vaccinia virus, and reovirus.

[0070] In a seventh aspect, the present invention relates to host cells that express or are capable of expressing one or more chimeric proteins of the present invention and / or contain nucleic acids or vectors of the present invention. Preferred host cells of the present invention are bacterial cells, fungal cells, or mammalian cells.

[0071] Suitable bacterial cells include, but are not limited to, cells of Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus).

[0072] Suitable fungal cells include, but are not limited to, cells of species from the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells of species from the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichiapastoris* and *Pichiamethanolica*), and *Hansenula*.

[0073] Suitable mammalian cells include, but are not limited to, HEK293 cells, CHO cells, BHK cells, HeLa cells, and COS cells.

[0074] However, the present invention may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0075] In some embodiments, the host cell is a functional cell capable of expressing the chimeric protein or its derivatives of the present invention (e.g., CAR-T, CAR-NK, CD8+ T cells, NK cells, CIK cells, TIL cells, macrophages, DC cells, etc.), and the functional cell has any one or more of the following physiological functions: tumor killing, pathogen clearance, immune effect, etc.

[0076] Eighthly, the present invention provides the use of the chimeric protein or its derivatives, nucleic acid molecules, host cells, immune conjugates, and pharmaceutical compositions described herein in the preparation of medicaments for treating related diseases (such as proliferative diseases, immune diseases, etc.), regulating T cell-mediated immune responses, and stimulating the individual's immune system. The proliferative diseases may be tumors or cancers (e.g., metastatic tumors or cancers), or solid tumors (e.g., metastatic renal cell carcinoma and malignant melanoma).

[0077] In some embodiments, the chimeric proteins or their derivatives, immune conjugates, and drug combinations disclosed in this invention can be used to treat disease conditions in which the host's immune system is stimulated to benefit, particularly conditions where enhanced cellular immune responses are desired, including disease conditions where the host immune response is insufficient or deficient. In some embodiments, disease conditions for which the chimeric proteins or their derivatives and immune conjugates are administered include tumors or infections where cellular immune responses are a key mechanism of specific immunity, such as cancer (e.g., renal cell carcinoma or melanoma), immunodeficiency (e.g., in HIV-positive patients, immunosuppressed patients), chronic infections, etc. In some embodiments, enhancing cellular immune responses can include any one or more of the following: general elevation of immune function, elevation of T cell function, elevation of B cell function, restoration of lymphocyte function, increased IL-2 receptor expression, enhanced T cell responsiveness, increased natural killer cell activity or lymphokine-activated killer (LAK) cell activity, etc.

[0078] In some embodiments, the chimeric proteins or derivatives thereof, immune conjugates, and pharmaceutical combinations disclosed in this invention are used to treat proliferative conditions, such as cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other proliferative conditions that can be treated with the chimeric proteins or derivatives thereof disclosed herein include, but are not limited to, neoplasms located in the following areas: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included. In some embodiments, the cancer is selected from the group consisting of: renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, and head and neck cancer. Similarly, other cell proliferation disorders can be treated with the chimeric protein or its derivatives disclosed herein, including but not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary syndrome, Waldenstron's macroglobulinemia, Gaucher's disease, histiocytosis, and any other cell proliferation disorders outside of neoplasmosis in the organ systems listed above. In other embodiments, the diseases involve autoimmune diseases, transplant rejection, post-traumatic immune responses, and infectious diseases (e.g., HIV).

[0079] In some embodiments, a method is provided in which a chimeric protein or its derivative, or an immunoconjugate, is administered to a subject at least twice daily, at least once daily, at least once every 48 hours, at least once every 72 hours, at least once weekly, at least once every two weeks, at least once monthly, at least once every two months, or at least once every three months. The chimeric protein or its derivative, or the immunoconjugate, can be administered via any effective route. In some embodiments, the chimeric protein or its derivative, or the immunoconjugate, is administered via parenteral injection, including subcutaneous injection. Specific embodiments relate to pharmaceutical compositions comprising a pharmaceutically acceptable amount of a chimeric protein or its derivative, or an immunoconjugate (e.g., a therapeutically effective amount) (including those reagents described above) together with one or more pharmaceutically acceptable diluents, carriers, or excipients (e.g., isotonic injection solutions). The pharmaceutical composition is generally a pharmaceutical composition suitable for human administration. Furthermore, in some embodiments, the pharmaceutical composition comprises at least one additional prophylactic or therapeutic agent. Some embodiments contain a sterile container comprising one of the above-described pharmaceutical compositions and optionally one or more additional components.

[0080] In a ninth aspect, the present invention provides a kit comprising the chimeric protein of the present invention, its derivatives, conjugates, pharmaceutical compositions, nucleic acid molecules, expression vectors or host cells, and instructions for use. The kit generally includes a label indicating the intended use of the kit contents. The term "label" includes any written or documented material provided on or with the kit or otherwise accompanied by the kit.

[0081] In a tenth aspect, the present invention provides a method for constructing and producing a chimeric protein or a derivative thereof, comprising expressing the chimeric protein or a derivative thereof under conditions suitable for expression, or using the aforementioned nucleic acid molecules, or using the aforementioned expression vector, or using the aforementioned host cells. Attached Figure Description

[0082] Figure 1 This refers to the chimeric region and site of the CD25-IL-2 chimeric protein (the chimeric region of the extracellular domain of CD25).

[0083] Figure 2 This refers to the chimeric region and site of the CD25-IL-2 chimeric protein (the chimeric region of IL-2).

[0084] Figure 3 A schematic diagram and predicted three-dimensional conformation of the CD25 / IL-2chimera1 chimeric protein.

[0085] Figure 4 A schematic diagram and predicted three-dimensional conformation of the CD25 / IL-2chimera2 chimeric protein.

[0086] Figure 5A schematic diagram and predicted three-dimensional conformation of the CD25 / IL-2chimera3 chimeric protein.

[0087] Figure 6 Purification for expression of CD25 / IL-2 chimeric protein.

[0088] Figure 7 This shows the binding status of the CD25 / IL-2 chimeric protein to CD25.

[0089] Figure 8 The combination of Human IL-2-Fc and CD25.

[0090] Figure 9 The binding of the CD25 / IL-2 chimeric protein to CD122 is shown in the bar chart.

[0091] Figure 10 The binding of the CD25 / IL-2 chimeric protein to CD122 is shown in the graph.

[0092] Figure 11 The activation of p-STAT5 in NK92 cells by CD25 / IL-2 chimeric protein stimulation.

[0093] Figure 12 The activation of p-STAT5 in CD8+ T cells by CD25 / IL-2 chimeric protein stimulation.

[0094] Figure 13 The CD25 / IL-2 chimeric protein promotes the killing of tumor cells by immune cells.

[0095] Figure 14 A schematic diagram and predicted three-dimensional conformation of the IL-2 / CD25 chimera chimeric protein.

[0096] Figure 15 A schematic diagram and predicted three-dimensional conformation of the IL15RA / IL-15chimera chimeric protein.

[0097] Figure 16 The binding status of IL15RA / IL-15chimera chimeric protein to IL15RA.

[0098] Figure 17 The binding of IL15RA / IL-15chimera chimeric protein to IL15Rβ (CD122).

[0099] Figure 18 The activation of p-STAT5 in NK92 cells by IL15RA / IL-15chimera chimeric protein stimulation.

[0100] Figure 19 A schematic diagram and predicted three-dimensional conformation of the IL21R / IL-21chimera chimeric protein. Detailed Implementation

[0101] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0102] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined elsewhere in this invention, all other technical and scientific terms used herein shall have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains, as referenced, for example, standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), Volumes 1–3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York, (1990); and Roitt et al., “Immunology” (2nd edition), Gower Medical Publishing, London, New York (1989), and general prior art cited herein; furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein may and have been performed in a manner known per se to those skilled in the art, as well as referenced, for example, standard manuals, the aforementioned general prior art, and other references cited therein.

[0103] Terminology

[0104] "Chimeric protein" refers to a protein comprising an amino acid sequence originally derived from two different sources (e.g., cytokines or the extracellular domain of cytokine receptors). For example, a chimeric protein may include domains derived from at least two naturally occurring different human proteins. In some instances, a chimeric protein may include a domain as a synthetic sequence and a domain derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric protein may include at least two different domains as synthetic sequences. The terms "chimera" and "chimeric protein" used in this invention are interchangeable.

[0105] "Cytokine" (CK) is intended to be interpreted broadly as a low-molecular-weight, soluble protein produced by cells. Based on their function, they can be classified into interleukins, interferons, the tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc. Cytokines are protein polypeptides that play important roles in cell signaling pathways. In this invention, cytokine encompasses the broadest possible scope; for example, protein hormone molecules are also included within the scope of cytokines referred to in this invention. The term covers unprocessed cytokines as well as any form of processed cytokine derived from cells. The term also covers naturally occurring cytokine variants, such as splice variants or allelic variants. The term further covers artificially modified cytokine variants, altered forms, and truncated forms with similar functions.

[0106] "Cytokine receptor extracellular domain" is intended to be interpreted broadly to include the extracellular domains of cytokine receptors as described in the terminology above. The term encompasses both unprocessed cytokine receptor extracellular domains and any processed form of cytokine receptor extracellular domain derived from cells. The term also encompasses variants of naturally occurring cytokine receptor extracellular domains, such as splice variants or allelic variants. Furthermore, the term encompasses variants, modified forms, and truncated forms of artificially engineered cytokine receptor extracellular domains with similar functions.

[0107] "Flexible linker regions" are intended to be interpreted broadly, including: linker sequences connecting protein secondary structures (α-helices and / or β-sheets, etc.), flexible linker sequences connecting domains formed by secondary structure folds, such as various types of loops and turns.

[0108] "Interleukin-2" or "IL-2" refers to any natural IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers unprocessed IL-2 as well as any form of IL-2 derived from cells and processed forms. The term also covers naturally occurring IL-2 variants, such as splice variants or allelic variants. The term also covers IL-2 variants, engineered forms, and truncated forms with similar functions. An illustrative wild-type human IL-2 amino acid sequence is shown in SEQ ID NO: 2. Unprocessed human IL-2 additionally contains a 20-amino acid signal peptide at the N-terminus (see UniProt entry number: P60568), which is absent in the mature IL-2 molecule. In this invention, "interleukin-2," "interleukin 2," "interleukin-2," "IL2," and "IL-2" are used interchangeably.

[0109] "CD25" or "α subunit of the IL-2 receptor" refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including "full-length" unprocessed CD25 and any form of processed CD25 derived from cells, as well as naturally occurring CD25 variants, such as splice variants or allelic variants. The term also covers CD25 variants, engineered forms, and truncated forms with similar functions. In some embodiments, CD25 is human CD25 (see UniProt entry number: P01589), whose extracellular domain illustrative sequence is shown in SEQ ID NO: 1. The terms "CD25" and "IL-2Rα" and "α subunit of the IL-2 receptor" used herein are interchangeable.

[0110] The CD25 and IL-2 described in this invention do not include the signal peptide sequence, and the first amino acid after removing the signal peptide is the first amino acid.

[0111] The amino acid three-letter codes and single-letter codes used in this invention, as described in J. biol. chem, 243, p3558 (1968), are well-known and agreed standards in the art.

[0112] Variants of CD25 and IL-2 include their mutants, which include amino acid substitutions, deletions, insertions, modifications, and any combination thereof in CD25 or IL-2.

[0113] Homologous peptides refer to peptide segments with similar sequences and functions from homologous proteins of different species.

[0114] "Derivatives" is intended to be interpreted broadly to include any product related to a target protein. This includes, but is not limited to, human and non-human target protein homologues, fragments or truncated forms, fusion proteins (such as those fused with a signal peptide or other active or inactive components, such as antibodies or their antigen-binding fragments), modified forms (such as PEGylation, glycosylation, albumin conjugation / fusion, Fc conjugation / fusion, hydroxyethylation, etc.), and conserved modified proteins.

[0115] An "immunoconjugate" is a specific conjugate comprising at least one chimeric protein or a derivative thereof and at least one antigen-binding module. In some embodiments, the immunoconjugate comprises at least one chimeric protein or a derivative thereof and at least two antigen-binding modules. Specific immunoconjugates according to the invention essentially consist of a chimeric protein or a derivative thereof and an antigen-binding module linked by one or more linker sequences. The antigen-binding module can be linked to the chimeric protein or its derivative through various interactions and in various configurations.

[0116] The term "antibody" is used in the broadest sense herein and encompasses a wide range of antibody structures that exhibit the desired antigen-binding activity. These antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments. Antibodies can include murine antibodies, human antibodies, camel antibodies, shark antibodies, humanized antibodies, chimeric antibodies, heavy chain antibodies, nanobodies, single-domain antibodies, etc. By way of example, an antibody can be an immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Based on differences in the amino acid composition of their hinge region and the number and position of disulfide bonds in their heavy chain, Ig can be further divided into different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0117] "Antigen-binding fragments" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain Fv (sFv), nanobodies (VHH), and VH / VL domains that possess antigen-binding activity. Fv fragments contain variable regions of both the antibody heavy and light chains, but lack constant regions, and are the smallest antigen-binding fragments possessing all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and can form the structure required for antigen binding. Two antibody variable regions can also be linked into a single polypeptide chain using different linkers, called a single-chain antibody or single-chain Fv (sFv).

[0118] When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion, or substitution of a specified number of amino acid residues at a position in a reference sequence compared to another sequence. In the case of substitution, the substitution will preferably be a conserved amino acid substitution, which is an amino acid residue that is replaced by another amino acid residue with a similar chemical structure and has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art, and for example, a conserved amino acid substitution is preferably the substitution of one amino acid within the following groups (i)-(v) by another amino acid residue within the same group: (i) smaller aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (iii) polar positively charged residues: His, Arg, and Lys; (iv) larger aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (v) aromatic residues: Phe, Tyr, and Trp. The particularly preferred conserved amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln, or Glu; Met is substituted by Leu, Tyr, or Ile; Phe is substituted by Met, Leu, or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; Val is substituted by Ile or Leu.

[0119] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of identical or conserved amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For instance, the BLAST program in the NCBI database can be used to determine identity. For determining sequence identity, see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics, and, Genome, Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991.

[0120] A "vector" is a nucleic acid medium into which polynucleotides can be inserted. When a vector allows the expression of a protein encoded by the polynucleotides inserted therein, the vector is called an expression vector. This vector can be transformed, transduced, or transfected into host cells to express the carried genetic material elements in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (such as SV40). Vectors may contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication.

[0121] The terms "host cell" and "host cell line" are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include the initially transformed cells and their derived progeny (regardless of passage number). Progeny may not be identical to the parent cells in terms of nucleic acid contents, but may contain mutations. This text includes mutant progeny with the same function or biological activity as those screened or selected from the original transformed cells. "Host cells" include, but are not limited to, prokaryotic cells such as *Escherichia coli* or *Bacillus subtilis*, eukaryotic cells such as yeast cells or *Aspergillus*, insect cells such as S2 *Drosophila* cells or Sf9, and mammalian cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0122] The following examples use molecular pairs such as cytokine IL-2 and its receptor CD25, IL-15 and its receptor IL-15RA, and IL-21 and its receptor IL-21R to further illustrate the present invention.

[0123] Example 1

[0124] Design and structural prediction of CD25 / IL-2 chimeric protein

[0125] 1.1 Determination of the chimeric region

[0126] As mentioned above, in this specific embodiment, the cytokine receptor is CD25, and the cytokine is IL-2. Based on the crystal structure of IL-2 and its high-affinity receptor IL-2Rαβγ (PDBID: 2B5I) in the PDB database, we analyzed and found that there are multiple flexible connection regions (i.e., loop regions) at and near the CD25-IL-2 binding surface, such as... Figure 1 , Figure 2 As shown.

[0127] The sequence number of the CD25 extracellular domain is SEQ ID NO:1, and the amino acid positions of the CD25 extracellular domain are numbered according to SEQ ID NO:1; the sequence number of IL-2 is SEQ ID NO:2, and the amino acid positions of IL-2 are numbered according to SEQ ID NO:2.

[0128] In some implementations, the chimeric region is located in the CD25 extracellular domain: the chimeric region is located in the flexible connective sequence region of the CD25 extracellular domain and the IL-2 interaction interface (refer to structure PD BID: 2B5I): including chimeric region Q1 and chimeric region Q2:

[0129] (a) Chimeric region 1 (e.g.) Figure 1 The I37-K38-S39-G40-S41-L42 (SEQ ID NO:3) peptide located on the extracellular domain of CD25 (SEQ ID NO:1), or a homologous peptide of the peptide, or a derived peptide having a similar structure and function to the peptide, and a peptide extending 10 amino acids forward and backward from the peptide (SEQ ID NO:5);

[0130] (b) Chimera 2 (e.g.) Figure 1 The peptide segment T150-H151-G152-K153-T154 (SEQ ID NO:4) located on the extracellular domain of CD25 (SEQ ID NO:1), or a homologous peptide segment of the peptide segment, or a derived peptide segment having a similar structure and function to the peptide segment, and a peptide segment extending 10 amino acids forward and backward from the peptide segment (SEQ ID NO:6).

[0131] In some embodiments, any amino acid site selected from the peptides described in (a) and (b) can be used as a chimeric site, and a portion of the IL-2 peptide, IL-2, IL-2 mutants, IL-2 modified organisms, IL-2 derivatives, etc. can be inserted before or after this site.

[0132] In some specific embodiments, the chimeric site is preferably selected from the CD25 extracellular domain K38 position described in (a), such as SEQ ID NO: 10-11, which is one of two chimeric proteins constructed based on this chimeric site.

[0133] In one embodiment, the chimeric site is preferably selected from the CD25 extracellular domain H151 position described in (b), such as SEQ ID NO: 12, which is a chimeric protein constructed based on this chimeric site.

[0134] In some other embodiments, the chimeric region is located on the cytokine IL-2: the chimeric region is selected from the flexible linker region (loop region) between two adjacent α-helices of IL-2 (e.g., Figure 2 (as shown)

[0135] (a) Chimeric region 3 (e.g.) Figure 2 (As shown): the A'B' loop region T41-F42-K43-F44-Y45-M46-P47-K48-K49-A50-T51 (SEQ ID NO:7) between helixA and helixB of IL-2 (SEQ ID NO:2) or a derived peptide having a similar structure and function to this loop region;

[0136] (b) Chimera 4 (e.g.) Figure 2(as shown): L72-A73-Q74-S75-K76-N77-F78-H79-L80 (SEQ ID NO:8) of the B'C' loop region between helixB and helixC of IL-2 (SEQ ID NO:2) or a derived peptide having a similar structure and function to this loop region;

[0137] (c) Chimera 5 (e.g.) Figure 2 (As shown): The C'D' loop region F103-M104-C105-E106-Y107-A108-D109-E110-T111-A112 sequence of IL-2 (SEQ ID NO:2) between helixC and helixD (SEQ ID NO:9) or a derived peptide with a similar structure and function to this loop region.

[0138] In some implementations, any amino acid site selected from the peptides described in (a), (b), and (c) can be used as a chimera site, and a portion of the CD25 extracellular domain peptide, CD25 extracellular domain mutant, CD25 extracellular domain modified, CD25 extracellular domain derivative, etc. can be inserted before or after this site.

[0139] In some implementations, the chimeric site is preferably selected from the F103 site of the C'D'loop region on IL-2.

[0140] In some implementations, the chimera site is preferably selected from the Y45th site of the A'B' loop region on IL-2.

[0141] In some implementations, the chimeric site is preferably selected from the N77th site of the B'C' loop region on IL-2.

[0142] 1.2 Construction of the chimeric protein and structure prediction

[0143] By using any amino acid position before or after the chimeric region Q1 or Q2 as the chimeric site, the amino acid sequence of C1 can be obtained as the following peptide segments: E1-R36, or E1-I37, or E1-K38, or E1-S39, or E1-G40, or E1-S41, or E1-L42, or E1-M149, or E1-T150, or E1-H151, or E1-G152, or E1-K153, or E1-T154.

[0144] The amino acid sequence of C2 can be one of the following peptides: I37-E217, or K38-E217, or S39-E217, or G40-E217, or S41-E217, or L42-E217, or Y43-E217, or H151-E217, or G152-E217, or K153-E217, or T154-E217, or R155-E217.

[0145] C1 and C2 may also be homologous peptides of any of the above peptides in other (non-human) species; or derivative peptides formed by the substitution, deletion or addition of one or more (e.g., 1-10) amino acid residues of any of the above peptides, and having a similar structure and function to the peptide; or amino acid sequences that have at least 80% sequence identity with the above peptide sequences.

[0146] By using any amino acid position before or after Q3-Q5 in the chimeric region as the chimeric site, the amino acid sequence of I1 can be obtained as the following peptides: T41-Y133, F42-Y133, or K43-Y133, or F44-Y133, or Y45-Y133, or M46-Y133, or P47-Y133, or K48-Y133, or K49-Y133, or A50-Y133, or T51-Y133, or L72-Y133, or A73-Y133, or Q74-Y133, or S75-Y133, or K76-Y133, or N77-Y133, or F78-Y133, or H79-Y133, or L80-Y133, or F103-Y133, or M104-Y133, or C105-Y133, or E106-Y133, or Y107-Y133, or A108-Y133, or D109-Y133, or E110-Y133, or T111-Y133, or A112-Y133.

[0147] The amino acid sequence of I2 can be one of the following peptides: P2-L40, or P2-T41, or P2-F42, or P2-K43, or P2-F44, or P2-Y45, or P2-M46, or P2-P47, or P2-K48, or P2-K49, or P2-A50, or P2-N71, or P2-L72, or P2-A73, or P2-Q 74, or P2-S75, or P2-K76, or P2-N77, or P2-F78, or P2-H79, or P2-T102, or P2-F103, or P2-M104, or P2-C105, or P2-E106, or P2-Y107, or P2-A108, or P2-D109, or P2-E110, or P2-T111.

[0148] I1 and I2 may also be homologous peptides of any of the above peptides in other (non-human) species; or derivative peptides formed by substitution, deletion or addition of one or more (e.g., 1-10) amino acid residues of any of the above peptides, and having a similar structure and function to the peptide; or amino acid sequences that have at least 80% sequence identity with the above peptide sequences.

[0149] Different peptides of CD25 and IL-2 are chimericly linked within a flexible linker region using a linker element of appropriate length, according to the C1-L1-I1-L2-I2-L3-C2 structure of this invention. When the chimeric site is selected from the K38 position of the CD25 extracellular domain, SEQ ID NO: 10-11 are two chimeric proteins constructed based on this chimeric site; and when the chimeric site is the H151 position of the CD25 extracellular domain, SEQ ID NO: 12 is a chimeric protein constructed based on this chimeric site.

[0150] Furthermore, following the combinations in Table 1, we formed chimeras of single polypeptide chains: CD25 / IL-2chimera1, CD25 / IL-2chimera2, and CD25 / IL-2chimera3. We used Alphafold2 to predict the protein structures of these sequences (Jumper, Jetal., Nature (2021); Varadi, Metal., Nucleic Acids Research (2021)). During the process, we used the provided full version of the BigFantastic Database (BDF), setting the training parameter `--db_preset` to `full_dbs`, while leaving other parameters at their default values. We used PyMol to view the predicted protein structures. The results showed that all three peptides formed a complex crystal structure similar to that formed by the binding of CD25 and IL-2 (PDBID: 2B5I), as shown below. Figures 3-5 As shown.

[0151] The protein structures reveal that the CD25 epitope of the IL-2 domain in these chimeric proteins is completely blocked by the CD25 domain. This suggests that these CD25 / IL-2 chimeric proteins may have a potential bias towards binding / activating the intermediate-affinity IL-2 receptor (IL-2Rβγ) while weakening (or not) binding / activating the high-affinity IL-2 receptor (IL-2Rαβγ). Subsequent examples further validate this structure-based functional hypothesis.

[0152] Table 1. Sequences of peptides and linkers of the three chimeric proteins.

[0153]

[0154] CD25 / IL-2chimera1, CD25 / IL-2chimera2, and CD25 / IL-2chimera3 were added to the Fc region of immunoglobulin, respectively, to obtain SEQ ID NO:21(CD25 / IL-2chimera1-Fc), SEQ ID NO:22(CD25 / IL-2chimera2-Fc), and SEQ ID NO:23(CD25 / IL-2chimera3-Fc).

[0155] Example Two

[0156] Preparation of experimental materials

[0157] Table 2 provides information on the relevant experimental materials used in Examples 3-12.

[0158] Table 2 Experimental Materials

[0159] Materials Supplier Catalogue number Recombinant human IL2 protein Coastal Biologic GMP-CD66 Recombinant human CD25-Fc protein Coastal Biologic CJ178 Recombinant human CD122-Fc Xiamen Bicai Biotechnology Co., Ltd. / Recombinant human IL15RA-Fc Xiamen Bicai Biotechnology Co., Ltd. / Alpaca VHH-Fc Anti-IL2 antibody Xiamen Bicai Biotechnology Co., Ltd. / Anti-Phospho-Stat5 (Tyr694) antibody CST 4322 Anti-actin antibody Sigma A5441 Rabbit anti-VHH (HRP) Genscript A01861-200 Goat anti-rabbit IgG (HPR) Thermo 31430 Goat anti-mouse IgG (HPR) Thermo 31460 Goat anti-human IgG (HPR) Proteintech SA00001-17 HRP-Streptavidin Bi Yun Tian A0303 TMB color developing solution Solebio PR1200-500ML NHS-biotin Helyon Biotech HS-11002038 QIAquick Gel Extraction Kit Qiagen 28704 Plasmid Miniprep Kit Tiangen DP103 Agencourt AMPure XP BECKMAN A63881 Expi293F cells Thermo A14635 Expi293 medium Thermo A14351-01 NK92 serum-free cell culture medium Haoyang Biotech TBD NK92 KIT PEI (Polyethylenimine) Polysciences 24765-1 Ficoll GE healthcare 17144002

[0160] Example 3

[0161] Expression and purification of CD25 / IL-2chimera-Fc chimera

[0162] 3.1 Carrier Construction

[0163] The CD25 / IL-2chimera1 (SEQ ID NO:10) and CD25 / IL-2chimera3 (SEQ ID NO:12) sequences were synthesized by General Biosystems (Anhui) Co., Ltd. and then inserted into the vector (LV082-AbV-Human-IGG1-Fc-1GS) with the humanIGG1-Fc tag, which is used for expression in Expi293F cells by Xiamen Baici Biotechnology Co., Ltd. Two expression plasmids for CD25 / IL-2chimera-Fc were obtained, which can be used for subsequent expression of CD25 / IL-2chimera1-Fc (SEQ ID NO:21) and CD25 / IL-2chimera3-Fc (SEQ ID NO:23) proteins, respectively.

[0164] 3.2 Expi293F mammalian cells express CD25 / IL-2 chimera-Fc protein

[0165] The constructed CD25 / IL-2chimera-Fc plasmid was transfected into EXPi293F cells. The transfection procedure was as follows: EXPi293F cells were cultured at 37°C and 8% CO2 in an incubator with shaking at 135 rpm until the cell density reached 4 to 5 × 10^6 cells / ml. The cells were then diluted with fresh culture medium to a concentration of 3 × 10^6 cells / ml, 30 ml / flask. 1.5 ml of Opti-Medium + 30 μg of plasmid was premixed for 5 minutes (solution A), and 1.5 ml of Opti-Medium + 60 μl of PEI (2 μg / μl) was premixed for 5 minutes (solution B). Solutions A and B were mixed and allowed to stand at room temperature for 20 minutes (dose per well). The above liquid was then added dropwise to the culture flask, gently shaken to mix, and the cells were returned to a 37°C and 8% CO2 incubator for further culture at 135 rpm. 24 hours after transfection, D-Glucose was added to a final concentration of 4.5 g / L, and VPA was added to a final concentration of 3 mM. Five days after transfection, the cell supernatant was collected by centrifugation at 2000 rcf for 5 minutes.

[0166] The supernatant was incubated with Protein Abeads by rotation for 2 hours. Then, the liquid and beads were transferred to an empty column. Impurities were washed away with Washing Buffer, and proteins were eluted with Elution Buffer. The eluted protein was dialyzed in PBS, filtered through a 0.22 μM filter, and the protein concentration was determined using the BCA method. The protein was then run on an SDS-PAGE gel, stained with Coomassie Brilliant Blue, and the protein expression was observed. Figure 6 As shown. Figure 6 It can be seen that both CD25 / IL-2chimera1-Fc (SEQ ID NO:21) and CD25 / IL-2chimera3-Fc (SEQ ID NO:23) can be expressed normally.

[0167] Example 4

[0168] Detecting the binding of CD25 / IL-2 chimera to CD25

[0169] CD25 recombinant protein was coated onto ELISA plates at a rate of 400 ng / well and incubated at room temperature for 2 h. Blocking was performed with 5% skim milk powder at room temperature. After blocking, the plates were washed three times with washing buffer. Then, 100 μL of a serially diluted series of Human IL-2, CD25 / IL-2chimera1-Fc, or CD25 / IL-2chimera3-Fc (0.625 nM to 1000 nM) was added to each well and incubated at room temperature for 2 h, followed by three washes with washing buffer. AlpacaVHH-FcAnti-IL2 antibody was added and incubated at room temperature for 1 h, followed by three washes with washing buffer. Rabbitanti-VHH (HRP) (Genscript, A01861-200) was added and incubated at room temperature for 1 h. After incubation, TMB chromogenic buffer (Solepro, PR1200-500 mL) was added, and the reaction was terminated with 1 M H₂SO₄. The absorbance at 460 nm was read using a microplate reader (Tecan, SPARK10M). Data processing and plotting were performed using a GraphPad Prism9 to obtain binding curves of wild-type Human IL-2, CD25 / IL-2chimera1-Fc, and CD25 / IL-2chimera3-Fc with CD25, as shown below. Figure 7 As shown. The binding of Human IL-2-Fc and CD25 was also examined, as shown... Figure 8 As shown.

[0170] The experimental results show that:

[0171] (1) Wild-type Human IL-2 can bind well to CD25.

[0172] (2) CD25 / IL-2chimera1-Fc (SEQ ID NO:21) and CD25 / IL-2chimera3-Fc (SEQ ID NO:23) did not bind significantly to CD25 at a concentration of 1000 nM, suggesting that CD25 / IL-2chimera1-Fc (SEQ ID NO:21) and CD25 / IL-2chimera3-Fc (SEQ ID NO:23) may not be able to bind to / activate the high-affinity IL-2 receptor.

[0173] (3) The binding ability of the IL-2 protein fused with Fc (Human IL-2-Fc) to CD25 is similar to that of wild-type Human IL-2, indicating that Fc does not significantly affect the binding of IL-2 and CD25. This further suggests that the extremely weakened binding response of CD25 / IL-2chimera1-Fc and CD25 / IL-2chimera3-Fc to CD25 is a specific effect of CD25 / IL-2chimera1 and CD25 / IL-2chimera3.

[0174] Example 5

[0175] Detecting the binding of CD25 / IL-2 chimera to CD122

[0176] 5.1 Biotin Coupling of Human IL-2 and CD25 / IL-2 Chimera

[0177] Dissolve 500 μg of the relevant protein in 500 μl of PBS to a final concentration of 1 mg / ml. Add 5 μl of 10 mg / ml NHS-biotin solution and mix well (NHS was dissolved in DMSO). Incubate at room temperature for 30 min. Add 50 μl of 100 mM glycine solution and incubate at room temperature for 10 min, then mix well to terminate the reaction. Dialyze the conjugated protein using PBS. After dialysis, filter through a 0.22 μM filter membrane, measure the protein concentration using BCA, and store at -80°C for later use.

[0178] 5.2 ELISA detection of the binding of CD25 / IL-2 chimera to CD122

[0179] CD122 recombinant protein was coated into ELISA plates at a rate of 400 ng / well. Then, equal amounts of biotin-conjugated Human IL-2, CD25 / IL-2chimera1-Fc, CD25 / IL-2chimera3-Fc, or Fc-Isotype (negative control) were added. The binding of each protein to CD122 was detected using HRP-Streptavidin. Figure 9 As shown.

[0180] The experimental results show that wild-type Human IL-2 and CD122 have a weak interaction. Compared with wild-type Human IL-2, the binding of CD25 / IL-2chimera1-Fc and CD25 / IL-2chimera3-Fc to CD122 is significantly enhanced, which is likely due to the chimerism of IL-2 and CD25, enhancing their affinity for CD122. The control Fc protein does not bind to CD122, further indicating that the binding of CD25 / IL-2chimera1-Fc and CD25 / IL-2chimera3-Fc to CD122 is a specific response of CD25 / IL-2chimera1 and CD25 / IL-2chimera3, respectively.

[0181] To further confirm the binding affinity of CD25 / IL-2chimera1-Fc and CD25 / IL-2chimera3-Fc to CD122, binding curves of the two chimeras and CD122 were detected using a gradient dilution series (0.625 nM to 1000 nM) of the CD25 / IL-2 chimeras. Figure 10 As shown. From Figure 10 It can be seen that CD25 / IL-2chimera1-Fc (SEQ ID NO:21) and CD25 / IL-2chimera3-Fc (SEQ ID NO:23) can bind well to CD122, suggesting that this chimera retains the ability to bind / activate the intermediate affinity IL-2 receptor.

[0182] Example 6

[0183] CD25 / IL-2 chimerism stimulates NK92 cells

[0184] NK92 cells (ATCC, CRL-2407) were passaged using TBDNK92KIT medium from Tianjin Haoyang Biotechnology.

[0185] Before stimulation, NK92 cells were cultured in IL-2-free medium for 2-3 days. The required sample volume of IL-2-free NK92 cells was seeded into 24-well plates. The IL-2-starved NK92 cells were treated with PBS (negative control), Human IL-2, CD25 / IL-2chimera1-Fc (SEQ ID NO:21), CD25 / IL-2chimera3-Fc (SEQ ID NO:23), CD25-Fc (negative control), and Fc-Isotype (negative control), respectively. Treatment time: 20 min, 60 min; treatment concentration: 15.625 nM; stimulation time: 1000 rcf, 5 min, cell pellet collected. Cells were lysed with RIPAlysis buffer, and 5× Loading buffer was added. Western blot was used to detect p-STAT5 and Actin. Figure 11 As shown.

[0186] The experimental results show that CD25 / IL-2chimera1-Fc (SEQ ID NO:21) and CD25 / IL-2chimera3-Fc (SEQ ID NO:23) can effectively activate STAT5 phosphorylation in NK92 cells, suggesting that this chimera has the function of activating NK cells. Neither CD25-Fc nor Fc-Isotype can activate STAT5 phosphorylation, further indicating that the activation of NK92 cells by CD25 / IL-2chimera1-Fc and CD25 / IL-2chimera3-Fc is a response specifically stimulated by CD25 / IL-2chimera1 and CD25 / IL-2chimera3.

[0187] Example 7

[0188] CD25 / IL-2 chimerism stimulates CD8+ T cells

[0189] 7.1 MouseCD8+ T cell isolation

[0190] CD8+ T cells were isolated using a negative screening method. Non-CD8 cells, such as CD4+ T cells, B cells, and erythrocytes, were removed from mouse spleen and lymph node cells. The specific procedure is as follows:

[0191] Spleen and lymph nodes of mice were placed in 2 ml of culture medium.

[0192] Place a 70-micron filter on a 50-ml tube and grind the tissue on the filter.

[0193] Rinse the filter with 8 mL of magnetic bead separation buffer (Streptavidin Particles Plus-DM, BDBiosciences, 557812), centrifuge at 500 g for 5 min at 4 °C.

[0194] Lyse the cells with 3 ml of erythrocyte lysis buffer for 5 min.

[0195] Add 5-10 ml of magnetic bead separation buffer to terminate the reaction, then centrifuge.

[0196] Cell count, diluted to 2 x 10^7 per milliliter.

[0197] Add 400 μL of antibody cocktail per 1 × 10^8 cells.

[0198] Prepare the antibody mixture (purchased from BioLegend) in 100 μL of magnetic bead separation buffer as follows:

[0199]

[0200] Incubate at 4 degrees Celsius for 20 minutes, then centrifuge at 500g for 5 minutes at 4 degrees Celsius.

[0201] Wash once with 10 ml of magnetic bead separation buffer.

[0202] Prepare two sets of magnetic beads: the first set is 50 μL; the second set is 40 μL beads; dilute with magnetic bead separation buffer at 3 times the volume of the beads.

[0203] After resuspending the cells in the first batch of beads for 5 minutes, they were transferred to flow cytometry tubes placed on a magnetic rack and allowed to adsorb for 1 minute.

[0204] Transfer the supernatant to a second batch of beadds and incubate for 5 minutes, then transfer it to a flow cytometer placed on a magnetic rack and adsorb for 1 minute.

[0205] Then transfer the supernatant to another flow cytometer tube for one minute of adsorption.

[0206] Transfer to a 1.5 mL tube, centrifuge, wash once, resuspend in the appropriate solution, count, and analyze purity.

[0207] Based on the required cell volume, seed the cells in 24-well plates with IL-2-free culture medium.

[0208] 7.2 CD25 / IL-2 chimera stimulation of CD8+ T cells

[0209] CD8+ T cells isolated in section 7.1 were treated with PBS (negative control), Human IL-2, CD25 / IL-2chimera1-Fc (SEQ ID NO:21), and CD25 / IL-2chimera3-Fc (SEQ ID NO:23) for 20 min and 2 h, respectively. The treatment concentration was 15.625 nM. After stimulation, the cells were stimulated at 1000 rcf for 5 min, and the cell pellet was collected. Cells were lysed with RIPAlysis buffer, and 5× Loading buffer was added. Western blot was used to detect p-STAT5 and Actin. Figure 12 As shown in the figure. The experimental results show that CD25 / IL-2chimera1-Fc (SEQ ID NO:21) and CD25 / IL-2chimera3-Fc (SEQ ID NO:23) can effectively activate STAT5 phosphorylation in CD8+ T cells, suggesting that this chimera has the function of activating CD8+ T cells.

[0210] Example 8

[0211] CD25 / IL-2 chimeric protein promotes immune cells to kill tumor cells.

[0212] 8.1 Extraction of human peripheral blood mononuclear cells (PBMCs)

[0213] Dilute fresh blood with PBS at a 1:1 ratio and gently pipette. Invert the Ficoll reagent 5-10 times to mix thoroughly, then aliquot the Ficoll reagent into centrifuge tubes. Add the diluted blood to the centrifuge tube containing Ficoll, transfer the tube to a centrifuge, balance the volume, and centrifuge at 400g for 30 minutes at room temperature with an ascending speed of 1 and a descending speed of 0. Gently aspirate the upper serum layer using a 1mL pipette. After centrifugation, separate the cells into four layers from bottom to top: red blood cells, Ficoll solution, white blood cell layer, and PBS. Carefully aspirate the white blood cell layer (i.e., the lymphocyte layer) into a new 15ml centrifuge tube, avoiding aspirating cells from the lower or upper layers to prevent contamination. Add 10ml of PBS to the transferred lymphocytes, gently pipette to mix, and centrifuge at 100×g for 10 minutes. Discard the supernatant, add 1ml of erythrocyte lysis buffer, and centrifuge for 3 minutes. Add washing buffer to stop the lysis, and centrifuge at 300×g for 5 minutes. Add washing buffer to a volume of 10 ml for washing, centrifuge twice at 100×g for 8 min. Discard the supernatant and collect the cell pellet for later use.

[0214] 8.2 Expansion and activation of immune cells

[0215] The isolated PBMCs were added to 24-well cell culture dishes pre-coated with 10 μg / mL anti-CD3 (novoprotein, GMP-A018) and anti-CD28 (novoprotein, GMP-A063) and incubated overnight at 4°C. Anti-CD3 and anti-CD28 antibodies can promote T cell activation and proliferation. PBMCs were cultured in RPMI 1640 containing 10% fetal bovine serum and 1:100 penicillin-streptomycin. Cells were divided into different aliquots and added to a final concentration of 15.625 nM with: HumanIL-2, CD25 / IL-2chimera1-Fc (SEQ ID NO:21), CD25 / IL-2chimera3-Fc (SEQ ID NO:23), CD25-Fc (control), or Fc-Isotype (control). Cells were passaged in half every other day, and an equal amount of fresh culture medium was added, along with the corresponding stimulants: Human IL-2, CD25 / IL-2chimera1-Fc, CD25 / IL-2chimera3-Fc, CD25-Fc, or Fc-Isotype. The proliferated and activated immune cells were then used for subsequent tumor killing experiments.

[0216] 8.3 RTCA esight assay to detect the killing ability of immune cells against tumor cells

[0217] The steps are as follows: Add 50 μL of 1640 complete culture medium to an E-Plate 96-well plate and equilibrate at 37°C for 1 hour, then measure the baseline impedance. Seed 5000 tumor cells (human lung squamous cell carcinoma H226) per well. Place the plate horizontally at room temperature for 30 minutes to allow cells to settle evenly to the bottom of the plate. Detect target cell adhesion and proliferation using impedance measurement. Acquire impedance data every 15 minutes and image data every hour. After 12 hours of tumor cell adhesion, collect the expanded effector cells (mainly T cells) from step 8.2. Add 50 μL of the prepared effector cell suspension to each well containing target cells (tumor cells) at an effector-to-target ratio of 5:1. Place the plate back into the corresponding position on the xCELLigence RTCA eSight (Agilent) and equilibrate for 30 minutes. Then begin data acquisition to monitor the process of effector cell killing of target cells in real time. Measure impedance every 15 minutes and acquire images every hour. Results are as follows: Figure 13As shown, compared with CD25-Fc and Fc-Isotype, CD25 / IL-2chimera1-Fc and CD25 / IL-2chimera3-Fc, similar to HumanIL-2, can significantly promote immune killing of H226 tumor cells. This indicates that CD25 / IL-2chimera1-Fc and CD25 / IL-2chimera3-Fc can effectively promote immune cell killing of tumors, and this activity is a specific response of CD25 / IL-2chimera1 and CD25 / IL-2chimera3.

[0218] Example 9

[0219] This embodiment discloses a kit comprising a container containing the CD25 / IL-2 chimeric protein of Embodiment 3. Using this kit, the activation of NK cells and CD8+ T cells or the killing of tumor cells by immune cells can be promoted, thereby facilitating the treatment of related diseases.

[0220] Example 10

[0221] Design and structural prediction of IL-2 / CD25 chimeric proteins

[0222] Based on the chimeric region defined in 1.1, CD25 and different peptides of IL-2 are chimericly linked within the flexible linker region using a linker element of appropriate length, according to the I1-L1-C-L3-I2 structure of this invention. When the chimeric site is selected from the S75 position of IL-2 (numbered according to SEQ ID NO:2), such as SEQ ID NO:27, a chimeric protein constructed based on this chimeric site is called IL-2 / CD25 Chimera.

[0223] Furthermore, following the combinations in Table 3, we formed a chimera of a single polypeptide chain: IL-2 / CD25Chimera. We used Alphafold2 to predict the protein structure of these sequences (Jumper, Jetal., Nature (2021); Varadi, Metal., Nucleic Acids Research (2021)). During the process, we used the provided full version of the BigFantastic Database (BDF), setting the training parameter `--db_preset` to `full_dbs`, while leaving other parameters at their default values. We used PyMol to view the predicted protein structure. The results showed that the peptide formed a crystal structure resembling a complex formed by the binding of CD25 and IL-2 (PDB ID: 2B5I), as shown below. Figure 14As shown (CD25 is numbered according to SEQ ID NO: 1, and IL-2 is numbered according to SEQ ID NO: 2).

[0224] The protein structure shows that the CD25 epitope of the IL-2 domain in this chimeric protein has been completely blocked by the CD25 domain. It is inferred that these IL-2 / CD25 chimeric proteins have the potential to preferentially bind / activate the intermediate affinity IL-2 receptor (IL-2Rβγ) while weakening (or not) binding / activating the high affinity IL-2 receptor (IL-2Rαβγ).

[0225] Table 3. Peptide and linker sequences of IL-2 / CD25 Chimera (SEQ ID NO: 27)

[0226]

[0227] Example 11

[0228] Design and structural prediction of IL15RA / IL-15 chimeric protein

[0229] As mentioned above, in this specific embodiment, the cytokine receptor is IL15RA, and the cytokine is IL-15. Based on the crystal structures of IL-15 and IL15RA in the PDB database (PDB ID: 2Z3Q), we analyzed and found that there are multiple flexible linker regions at and near the IL15RA-IL-15 binding surface. Preferably, we selected the K66-A67-G68-T69-S70-S71 flexible linker region on the extracellular domain of IL15RA (the amino acid positions of IL15RA are numbered according to UniProt entry number: Q13261) as the chimeric region for constructing the IL15RA / IL-15 chimeric protein in this embodiment.

[0230] Within this flexible linker region, different peptides of IL15RA and IL15 are chimericly linked according to the C1-L1-I1-L2-I2-L3-C2 structure of this invention using a linker element of appropriate length. When the chimeric site is selected from position A67 of this chimeric region (the amino acid positions of IL15RA are numbered according to UniProt entry number: Q13261), a chimeric protein constructed based on this chimeric site, such as SEQ ID NO: 28, is called IL15RA / IL-15Chimera.

[0231] Furthermore, following the combinations in Table 4, we formed a chimera of a single polypeptide chain: IL15RA / IL-15Chimera. We used Alphafold2 to predict the protein structure of these sequences (Jumper, Jetal., Nature (2021); Varadi, Metal., Nucleic Acids Research (2021)). During the process, we used the provided full version of the BigFantastic Database (BDF), setting the training parameter `--db_preset` to `full_dbs`, while leaving other parameters at their default values. We used PyMol to view the predicted protein structure. The results showed that the peptide formed a crystal structure similar to a complex formed by the binding of IL15RA and IL-15 (PDB ID: 2Z3Q), as shown below. Figure 15 As shown (IL15RA is numbered according to UniProt ID: Q13261, and IL-15 is numbered according to UniProt ID: P40933).

[0232] The protein structure shows that the IL15RA epitope of the IL-15 domain in this chimeric protein has been completely blocked by the IL15RA domain. Therefore, it is speculated that this IL15RA / IL-15 chimeric protein has the potential to bias the binding / activation of IL-15βγ (CD122 / CD132) receptor while weakening (or not) binding / activating the IL15Rα (IL15RA) receptor.

[0233] Table 4. Peptide and linker sequences of IL15RA / IL-15Chimera (SEQ ID NO: 28)

[0234]

[0235] Furthermore, similar to Examples 3-6, we constructed an expression vector for the IL15RA / IL-15 chimeric protein, prepared the IL15RA / IL-15 chimeric protein, and detected the binding of IL15RA / IL-15Chimera and IL15RA using an ELISA assay. Figure 16 The binding of IL15RA / IL-15Chimera and IL15Rβ (CD122) was detected by ELISA. Figure 17 ); and by stimulating NK92 cells with IL15RA / IL-15Chimera, followed by Western blot analysis of STAT5 phosphorylation levels, the activation of NK cells by IL15RA / IL-15Chimera was assessed. Figure 18 ).

[0236] The results show that IL15RA / IL-15Chimera significantly reduced the binding to IL15RA, enhanced the binding to IL15Rβ (CD122), and could directly promote the phosphorylation of STAT5 in NK92 cells. This indicates that the IL15RA / IL-15 chimeric protein has the function of directly binding to the IL-15βγ (CD122 / CD132) receptor while weakening (or not) binding to / activating the IL15Rα (IL15RA) receptor, and can further directly promote the activation of NK cells.

[0237] Example 12

[0238] Design and structural prediction of IL21R / IL-21 chimeric protein

[0239] As mentioned above, in this specific embodiment, the cytokine receptor is IL21R, and the cytokine is IL-21. Based on the crystal structures of IL-21 and IL21R in the PDB database (PDB ID: 3TGX), we analyzed and found that there are multiple flexible connection regions at and near the IL21R-IL-21 binding surface.

[0240] Different peptides of IL21R and IL-21 are chimericly linked within a flexible linker region using a linker element of appropriate length, according to the C1-L1-I-L3-C2 structure described in this invention. When the chimeric site is selected from the D91 position of IL21R (numbered according to UniProt entry number: Q9HBE5), such as SEQ ID NO: 29, a chimeric protein constructed based on this chimeric site is called IL21R / IL-21Chimera.

[0241] Furthermore, following the combinations in Table 5, we formed a chimera of a single polypeptide chain: IL21R / IL-21Chimera. We used Alphafold2 to predict the protein structure of these sequences (Jumper, Jetal., Nature (2021); Varadi, Metal., Nucleic Acids Research (2021)). During the process, we used the provided full version of the BigFantastic Database (BDF), setting the training parameter `--db_preset` to `full_dbs`, while leaving other parameters at their default values. We used PyMol to view the predicted protein structure. The results showed that the peptide formed a crystal structure similar to a complex formed by the binding of IL21R and IL21 (PDB ID: 3TGX), as shown below. Figure 19 As shown (IL21R is numbered according to UniProt ID: Q9HBE5, and IL-21 is numbered according to UniProt ID: Q9HBE4).

[0242] The protein structure shows that the IL21R epitope of the IL-21 domain in this chimeric protein has been completely blocked by the IL21R domain. Therefore, it is speculated that this IL21R / IL-21 chimeric protein has the potential to weaken the binding / activation of the L21R receptor.

[0243] Table 5. Peptide and linker sequences of IL21R / IL-21Chimera (SEQ ID NO: 29)

[0244]

[0245] Example 13

[0246] Application expansion of the interlocking region

[0247] The chimeric region of the cytokine / cytokine receptor described in this invention can not only embed the extracellular domain of the cytokine receptor or cytokines, but also embed single-chain antibodies / single-domain antibodies or other functional proteins. In this way, after embedding the target antibody / functional protein (such as an antibody targeting T cell antigens, NK cell antigens, or tumor-associated antigens) at the relevant position, the binding of cytokines to their receptors is disrupted by steric hindrance. At the same time, the embedded antibody / functional protein can target the target cytokine to specific locations (such as T cells, NK cells, or tumor cells) to achieve the effect of specifically regulating the targeted distribution of the target cytokine in vivo.

[0248] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A chimeric protein, characterized in that, This includes cytokines and the extracellular domain of cytokine receptors; The cytokine is embedded in a chimeric region of the extracellular domain of the cytokine receptor, the chimeric region being located in the flexible junction region of the extracellular domain of the cytokine receptor. The chimeric protein has the following polypeptide structure from the N-terminus to the C-terminus: C1-I-C2, where C1 is the amino acid sequence before or after any chimeric site in the chimeric region of the extracellular domain of the cytokine receptor, C2 is the amino acid sequence in the extracellular domain of the cytokine receptor excluding C1, and I represents the cytokine. Alternatively, the extracellular domain of the cytokine receptor may be embedded in the chimeric region of the cytokine, wherein the chimeric region is located in the flexible linker region of the cytokine; the chimeric protein has the following polypeptide structure from the N-terminus to the C-terminus: I1-C-I2; where I1 is the amino acid sequence before or after any chimeric site in the cytokine chimeric region, I2 is the amino acid sequence of the cytokine excluding I1; and C is the extracellular domain of the cytokine receptor.

2. The chimeric protein of claim 1, characterized in that: The C1, C2, I1, and I2 contain at least three amino acids or at least one independent protein secondary structure unit; the C1 and C2 are indirectly connected to I through a linker element; the chimeric protein has the following polypeptide structure from the N-terminus to the C-terminus: C1-L1-I-L3-C2. Alternatively, I1 and I2 may be indirectly connected to C via a connector element, and the chimeric protein has the following polypeptide structure from the N-terminus to the C-terminus: I1-L1-C-L3-I2. In the above formula, L1 and L3 are independent connector elements, and L1 and L3 may be the same or different sequences.

3. The chimeric protein as described in claim 2, characterized in that: In the polypeptide structure C1-L1-I-L3-C2, the two amino acid sequences I1 and I2 before or after the chimeric site are connected by the linker element L2. That is, the chimeric protein has the following polypeptide structure from the N-terminus to the C-terminus: C1-L1-I1-L2-I2-L3-C2; where I1 is the amino acid sequence before or after any chimeric site in the cytokine chimeric region, and I2 is the amino acid sequence in the cytokine excluding I1. Alternatively, in the polypeptide structure I1-L1-C-L3-I2, the two amino acid sequences C1 and C2 before or after the chimeric site are connected by the linker element L2, that is, the chimeric protein has the following polypeptide structure from the N-terminus to the C-terminus: I1-L1-C1-L2-C2-L3-I2; where C1 is the amino acid sequence before or after any chimeric site in the extracellular domain chimeric region of the cytokine receptor, and C2 is the amino acid sequence in the extracellular domain of the cytokine receptor excluding C1; it also includes the immunoglobulin Fc region.

4. The chimeric protein according to any one of claims 1 to 3, characterized in that: The cytokine receptor is CD25, and the cytokine is IL-2; or the cytokine receptor is IL15RA, and the cytokine is IL-15; or the cytokine receptor is IL21R, and the cytokine is IL-21.

5. The chimeric protein as described in claim 4, characterized in that: The sequence number of the extracellular domain of CD25 is SEQ ID NO:1, and the amino acid positions of CD25 are numbered according to SEQ ID NO:1; C1 contains any of the following amino acid sequences: (a) The following peptides on the extracellular domain of CD25: E1-R36, or E1-I37, or E1-K38, or E1-S39, or E1-G40, or E1-S41, or E1-L42, or E1-M149, or E1-T150, or E1-H151, or E1-G152, or E1-K153, or E1-T154; (b) and (a) are homologous peptides in other non-human species; C2 contains any of the following amino acid sequences: (a) The following peptides on the extracellular domain of CD25: I37-E217, or K38-E217, or S39-E217, or G40-E217, or S41-E217, or L42-E217, or Y43-E217, or H151-E217, or G152-E217, or K153-E217, or T154-E217, or R155-E217; The peptides described in (b) and (a) are homologous peptides in other non-human species.

6. The chimeric protein as described in claim 4, characterized in that: The sequence number of IL-2 is SEQ ID NO:2, and the amino acid positions of IL-2 are numbered according to SEQ ID NO:2; I1 contains any of the following amino acid sequences: (a) The following peptides on IL-2: T41-Y133, F42-Y133, or K43-Y133, or F44-Y133, or Y45-Y133, or M46-Y133, or P47-Y133, or K48-Y133, or K49-Y133, or A50-Y133, or T51-Y133, or L72-Y133, or A73-Y133, or Q74-Y133, or S75-Y13 3, or K76-Y133, or N77-Y133, or F78-Y133, or H79-Y133, or L80-Y133, or F103-Y133, or M104-Y133, or C105-Y133, or E106-Y133, or Y107-Y133, or A108-Y133, or D109-Y133, or E110-Y133, or T111-Y133, or A112-Y133; (b) and (a) are homologous peptides in other non-human species; I2 contains any of the following amino acid sequences: (a) The following peptides on IL-2: P2-L40, or P2-T41, or P2-F42, or P2-K43, or P2-F44, or P2-Y45, or P2-M46, or P2-P47, or P2-K48, or P2-K49, or P2-A50, or P2-N71, or P2-L72, or P2-A73, or P2-Q7 4, or P2-S75, or P2-K76, or P2-N77, or P2-F78, or P2-H79, or P2-T102, or P2-F103, or P2-M104, or P2-C105, or P2-E106, or P2-Y107, or P2-A108, or P2-D109, or P2-E110, or P2-T111; The peptides described in (b) and (a) are homologous peptides in other non-human species.

7. The chimeric protein as described in claim 4, characterized in that: The chimeric protein comprises any one of the polypeptide sequences in SEQ ID NO:10-12, SEQ ID NO:21-23, SEQ ID NO:27, SEQ ID NO:28-29 or has a polypeptide sequence in the same order as any one of the polypeptide sequences in SEQ ID NO:10-12, SEQ ID NO:21-23, SEQ ID NO:27, SEQ ID NO:28-29.

8. A conjugate, characterized in that: The invention includes the chimeric protein or its derivative as described in any one of claims 1 to 7, wherein the chimeric protein or its derivative is directly or indirectly connected to other modules through a connector element; the other modules include any one or a combination of bioactive proteins, detectable markers, drugs, toxins, magnetic nanoparticles, viral capsid proteins or VLPs; the other modules are any one or a combination of antigen-binding modules, cytotoxins, radioactive isotopes, cytokines, gold enzyme nanoparticles / nanorobars.

9. A pharmaceutical composition, characterized in that: This includes the chimeric protein or its derivative as described in any one of claims 1 to 7, or the conjugate as described in any one of claims 15 to 16, and a pharmaceutically acceptable carrier.

10. A nucleic acid molecule, characterized by: It encodes the chimeric protein as described in any one of claims 1 to 7.

11. An expression vector, characterized in that: It includes the nucleic acid molecule as described in claim 10.

12. A host cell comprising the expression vector of claim 11, or the nucleic acid molecule of claim 10, or expressing the chimeric protein or its derivative as described in any one of claims 1 to 7, or the conjugate of claim 8; wherein the host cell is a bacterial cell, fungal cell, mammalian cell, amphibian cell, or insect cell.

13. Use of the chimeric protein or derivative thereof according to any one of claims 1 to 7, the conjugate of claim 8, the pharmaceutical composition of claim 9, the nucleic acid molecule of claim 10, the expression vector of claim 11, or the host cell of claim 12 in the preparation of a medicament for treating proliferative or immune diseases; wherein the proliferative disease is a tumor or cancer.

14. A reagent kit, characterized in that: It comprises the chimeric protein or its derivative as described in any one of claims 1 to 7, the conjugate as described in claim 8, the pharmaceutical composition as described in claim 9, the nucleic acid molecule as described in claim 10, the expression vector as described in claim 11, or the host cell as described in claim 12.

15. A method for preparing chimeric proteins, characterized in that: Expression may be performed using the nucleic acid molecule of claim 10, the expression vector of claim 11, or the host cell of claim 12, under conditions suitable for expressing the chimeric protein.

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