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

Abstract

The application discloses a kind of chimeric proteins, including cytokine and cytokine receptor extracellular domain;Cytokine is embedded in the chimeric region of cytokine receptor extracellular domain, and the chimeric region is located in the flexible connection region of cytokine receptor extracellular domain, or the chimeric region is located in the flexible connection region of the cytokine of cytokine receptor extracellular domain embedded in this cytokine.The application discloses that by selecting suitable chimeric region on CD25 and IL-2 protein, two proteins are divided into different peptide segments, are connected to form a peptide chain, and are expressed to form chimeric protein.It has eliminated or reduced affinity for high-affinity IL-2 receptor (IL-2Rαβγ), and retains / enhances the affinity for medium-affinity IL-2 receptor (IL-2Rβγ).The application also discloses chimeric proteins of IL15RA and IL-15 and chimeric proteins of IL21R and IL21.The application also discloses conjugates, pharmaceutical compositions, nucleic acid molecules, expression vectors, host cells, production methods, kits of the chimeric protein, and uses thereof.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine. In particular, 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

[0002] Cytokines 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 play 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 mediating intracellular signal transduction. Therefore, cytokine-based drugs are an important field in drug development. However, many natural cytokines are not suitable for use as drugs. Because one cytokine can 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 pleiotropic, overlapping, antagonistic, synergistic 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 intended function, and to achieve good therapeutic effect and small side effects. Most cytokine receptors are transmembrane proteins, consisting of an extracellular domain, a transmembrane region and a cytoplasmic domain.

[0003] For example, taking interleukin 2 as an example, interleukin 2 (IL-2) is also known as T cell growth factor (TCGF), which 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, amphiphilic alpha helices and some loop 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.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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, which can antagonize the interaction between the cytokine and itself, and constructs a chimeric protein by embedding the cytokine and the extracellular domain of the cytokine receptor, so that the chimeric protein is biased to bind to other cytokine receptors and thus performs specific functions.

[0017] In some specific embodiments, the cytokine is embedded in the chimeric region of the extracellular domain of the cytokine receptor, which 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-terminal to C-terminal:

[0018] C1-I-C2;

[0019] 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 the cytokine or its variant, modification, truncation or derivative.

[0020] 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.

[0021] Further, in some embodiments, C1 and C2 are indirectly connected to I via linker elements; the chimeric protein has a polypeptide structure formula from N-terminus to C-terminus as follows: C1-L1-I-L3-C2; wherein L1, L3 are each independently linker elements.

[0022] In some embodiments, I in the polypeptide structure formula C1-L1-I-L3-C2 has two parts of amino acid sequences I1 and I2 before or after the chimeric site in the cytokine chimeric region, which are connected via linker element L2, i.e. the chimeric protein has a polypeptide structure formula from N-terminus to C-terminus as follows: C1-L1-I1-L2-I2-L3-C2; wherein I1 is an amino acid sequence before or after any chimeric site in the cytokine chimeric region or a variant, modification, truncation or derivative thereof, and I2 is an amino acid sequence other than I1 in the cytokine or a variant, modification, truncation or derivative thereof.

[0023] In some specific embodiments, the extracellular domain of a cytokine receptor can be embedded in the chimeric region of the cytokine, which is located in the flexible linker region of the cytokine; the chimeric protein has a polypeptide structure formula from N-terminus to C-terminus as follows:

[0024] I1-C-I2;

[0025] wherein I1 is an amino acid sequence before or after any chimeric site in the cytokine chimeric region or a variant, modification, truncation or derivative thereof, I2 is an amino acid sequence other than I1 in the cytokine or a variant, modification, truncation or derivative thereof, and C is the extracellular domain of a cytokine receptor or a variant, modification, truncation or derivative thereof.

[0026] wherein I1 and I2 each contain at least three amino acids or at least one independent protein secondary structure unit such as an α-helix or a β-sheet, etc.

[0027] Further, I1 and I2 are indirectly connected to C via linker elements; the chimeric protein has a polypeptide structure formula from N-terminus to C-terminus as follows: I1-L1-C-L3-I2; wherein L1, L3 are each independently linker elements.

[0028] Further, the C in the polypeptide structure I1-L1-C-L3-I2 is connected with the two parts of amino acid sequence C1 and C2 before or after the chimeric site by linker element L2, i.e. the chimeric protein has the following polypeptide structure from N terminal to C terminal: I1-L1-C1-L2-C2-L3-I2; wherein C1 is the amino acid sequence or its variant, modification, truncation or derivative before or after any chimeric site in the extracellular domain of the cytokine receptor, and C2 is the amino acid sequence or its variant, modification, truncation or derivative except C1 in the extracellular domain of the cytokine receptor.

[0029] In the above embodiments, the linker elements L1, L2 and L3 can be the same or different sequences, which can be selected from the sequences of 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 commonly used linker elements with similar functions in the art.

[0030] In some embodiments, the chimeric protein further comprises an immunoglobulin Fc region, which can be connected at the N terminal or C terminal of the chimeric protein, directly or indirectly through a linker element. The immunoglobulin Fc region can make the molecule form a dimer, and at the same time prolong the in vivo half-life of the molecule. The Fc region that can be used in the present application can be from different subtypes of immunoglobulin, for example, IgG (such as IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM. In some embodiments, mutations can be introduced on the wild type Fc sequence for changing the Fc mediated related activities. The mutations include but are not limited to: a) mutations that change the Fc mediated CDC activity; b) mutations that change the Fc mediated ADCC activity; or c) mutations that change the Fc Rn mediated in vivo half-life. Such mutations are described in the following references: Leonard G Presta, Current Opinion in Immunology 2008, 20: 460-470; Esohe E. Idusogie et al., J Immunol 2000, 164: 4178-4184; RAPHAEL A. CLYNES et al., Nature Medicine, 2000, Volume 6, Number 4: 443-446; Paul R. Hinton et al., J Immunol, 2006, 176: 346-356.

[0031] In some embodiments, mutations can be introduced on the Fc sequence, so that the mutated Fc is more likely to form homodimers or heterodimers. The knob-hole model, which utilizes the steric effect of the side chain groups of the amino acids at the Fc contact interface, makes it easier for different Fc mutations to form heterodimers, as mentioned in Ridgway, Presta et al. 1996 and Carter 2001; or by changing the charge carried by the amino acids at the Fc contact interface, thereby changing the ionic interaction force between the Fc contact interfaces, so that different Fc mutations are more likely to form heterodimers (CN102558355A), or Fc with the same mutation is more likely to form homodimers (CN103388013A).

[0032] In some specific embodiments, the immunoglobulin Fc region is preferably a human immunoglobulin Fc region, more preferably the Fc region of human IgG1. In some specific embodiments, the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO: 20.

[0033] In some specific embodiments, it also includes derivatives of the chimeric protein.

[0034] In some specific embodiments, the cytokine receptor is CD25, and the cytokine is IL-2, which is embedded in the chimeric region of the extracellular domain of CD25 to form a CD25 / IL-2 chimeric protein. The CD25 / IL-2 chimeric protein or its derivatives have at least one of the following characteristics:

[0035] (a) capable of naturally forming a three-dimensional conformation similar to the binding of IL-2 to CD25;

[0036] (b) greatly reduced or no binding to CD25 compared to wild-type IL-2;

[0037] (c) capable of retaining or enhancing binding to CD122 compared to wild-type IL-2;

[0038] (d) larger molecular weight and longer half-life in vivo compared to wild-type IL-2;

[0039] (e) capable of effectively activating intermediate affinity IL-2 receptor (IL-2Rβγ) mediated signaling;

[0040] (f) promoting the activation of CD8+ T cells and NK cells;

[0041] (g) promoting the killing of tumor cells by immune cells.

[0042] In some specific embodiments, the cytokine receptor is CD25, the cytokine is IL-2, and the CD25 ectodomain is embedded in the chimeric region of IL-2 to form an IL2 / CD25 chimeric protein. The IL2 / CD25 chimeric protein or its derivative has at least one of the following characteristics:

[0043] (a) capable of naturally forming a three-dimensional conformation similar to that of IL-2 binding to CD25;

[0044] (b) has a larger molecular weight and a longer half-life in vivo compared to wild-type IL-2.

[0045] In some specific embodiments, the cytokine receptor is IL15RA, the cytokine is IL-15, and the chimeric protein is an IL15RA / IL-15 chimeric protein. The IL15RA / IL-15 chimeric protein or its derivative has at least one of the following characteristics:

[0046] (a) capable of naturally forming a three-dimensional conformation similar to that of IL-15 binding to IL15RA;

[0047] (b) has a larger molecular weight and a longer half-life in vivo compared to wild-type IL-15.

[0048] (c) has a greatly reduced binding to IL15RA compared to wild-type IL-15;

[0049] (d) can retain or enhance binding to IL15Rβ (CD122) compared to wild-type IL-15;

[0050] (e) promotes the activation of NK cells.

[0051] In some specific embodiments, the cytokine receptor is IL-21R, the cytokine is IL-21, and the chimeric protein is an IL-21RA / IL-21 chimeric protein. The IL-21RA / IL-21 chimeric protein or its derivative has at least one of the following characteristics:

[0052] (a) capable of naturally forming a three-dimensional conformation similar to that of IL-21 binding to IL21RA;

[0053] (b) has a larger molecular weight and a longer half-life in vivo compared to wild-type IL-21.

[0054] In a third aspect, the present application provides a conjugate comprising the above-mentioned chimeric protein or its derivative, which is directly or indirectly linked to other modules through a linker element.

[0055] In some embodiments, the other module comprises an antigen binding module, a cytotoxin, a radioisotope, a biologically active protein, a detectable label, a drug, a toxin, a cytokine, an enzyme gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or VLP, any one or a combination thereof.

[0056] More preferably, the antigen binding module is an antibody or an antigen binding fragment; most preferably, the antibody or antigen binding fragment targets an antigen presented on a tumor cell or in the tumor cell environment.

[0057] In some embodiments, the antigen binding module targets an antigen presented on a tumor cell or in the tumor cell environment. In some embodiments, the antigen binding module targets an antigen on a functional cell (e.g., CD8+ T cell, NK cell, CIK cell, TIL cell, macrophage, DC cell, etc.).

[0058] In some embodiments, the chimeric protein is linked to at least one other module. In some embodiments, the chimeric protein and the other module form a fusion protein, i.e., the chimeric protein shares a peptide bond with the other module. In some embodiments, the chimeric protein is linked to at least one other module, e.g., a first and a second other module. In some embodiments, the other module is an antigen-binding module. In some embodiments, the chimeric protein shares an amino- or carboxy-terminal peptide bond with a first antigen-binding module, and a second antigen-binding module shares an amino- or carboxy-terminal peptide bond with i) the chimeric protein or ii) the first antigen-binding module. In some particular embodiments, the chimeric protein shares a carboxy-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 can be an antibody or an antigen-binding fragment, including but not limited to an immunoglobulin molecule (e.g., an IgG (e.g., IgG1) class immunoglobulin molecule), an antibody or an antigen-binding fragment thereof. In some particular embodiments, the antibody or antigen-binding fragment is selected from the group consisting of a polypeptide complex comprising an antibody heavy chain variable region and an antibody light chain variable region, a Fab, a Fv, a sFv, a F(ab')2, a linear antibody, a single-chain antibody, a scFv, a sdAb, a sdFv, a nanobody, a peptibody, a domain antibody, a multispecific antibody (a bispecific antibody, a diabody, a triabody, and a tetrabody, a tandem di-scFv, a tandem tri-scFv), a receptor binding domain, and an interacting protein binding domain. In the case where the chimeric protein is linked to more than one antigen-binding module, e.g., a first and a second antigen-binding module, each antigen-binding module can be independently selected from various forms of antibodies and antigen-binding fragments, e.g., the first antigen-binding module can be a nanobody molecule and the second antigen-binding module can be a scFv molecule, or each of the first and second antigen-binding modules is a nanobody molecule, or each of the first and second antigen-binding modules is a Fab molecule. In some embodiments, in the case where the chimeric protein is linked to more than one antigen-binding module, e.g., a first and a second antigen-binding module, the antigen against which each antigen-binding module is directed can be independently selected, e.g., the first and the second antigen-binding module are directed against different antigens or are directed against the same antigen.

[0059] In some embodiments, the antigen binding moiety binds an antigen selected from the group consisting of Al domain of tenascin C (TNC Al), A2 domain of tenascin C (TNC A2), Extra Domain B (EDB) of fibronectin, carcinoembryonic antigen (CEA), and melanoma-associated chondroitin sulfate proteoglycan (MCSP).In some embodiments, the tumor antigen includes, but is not limited to, MAGE, MART-1 / Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV), Adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)-C017-1A / GA733, Carcinoembryonic antigen (CEA) and immunogenic epitopes thereof CAP-1 and CAP-2, etv6, aml1, Prostate specific antigen (PSA) and immunogenic epitopes thereof PSA-1, PSA-2 and PSA-3, Prostate specific membrane antigen (PSMA), T cell receptors / CD3-zeta chain, 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), GAGE family of tumor antigens (e.g., GAGE-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, alpha-fetoprotein, E-cadherin, alpha-, beta-, and gamma-catenin, p120ctn, gplOO Pmel 117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coliprotein (APC), fodrin, Connexin 37, Ig idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of 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 hemagglutinin, Epstein-Barr virus LMP-1, Hepatitis C virus E2 glycoprotein, HIV gpl60, and HIV gpl20.In some embodiments, non-limiting examples of ECM antigens include syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, EGF-type laminin, lectin, fibronectin, notch, tenascin, and matrixin.

[0060] In a fourth aspect, the present application discloses a pharmaceutical composition comprising the chimeric protein or derivative thereof described above or the conjugate described above and a pharmaceutically acceptable diluent, carrier or adjuvant. The pharmaceutical composition can be a lyophilized preparation or an injectable solution.

[0061] In a fifth aspect, the present application provides a nucleic acid molecule encoding the chimeric protein or derivative thereof described above. The nucleic acid of the present application can be RNA, DNA or cDNA.

[0062] In one embodiment, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 24, and the corresponding amino acid sequence is as shown in SEQ ID NO: 10;

[0063] In another embodiment, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 25, and the corresponding amino acid sequence is as shown in SEQ ID NO: 11;

[0064] In another embodiment, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 26, and the corresponding amino acid sequence is as shown in SEQ ID NO: 12;

[0065] In a sixth aspect, the present application provides an expression vector containing the nucleic acid sequence of the chimeric protein or derivative thereof described above. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.

[0066] In one embodiment, the CD25 / IL-2 chimeric protein on the expression vector can comprise an N-terminal signal peptide sequence, such as sequence SEQ ID NO: 19, and an Fc sequence with a hinge region at the C-terminal, such as sequence SEQ ID NO: 20.

[0067] In some embodiments, the expression vector is a viral vector, which can produce viruses with physiological functions, such as some common oncolytic viruses: herpes simplex virus (HSV), adenovirus, vaccinia virus and reovirus, etc.

[0068] In a seventh aspect, the present application relates to a host cell expressing or capable of expressing one or more chimeric proteins of the present application and / or containing a nucleic acid or vector of the present application. The preferred host cell of the present application is a bacterial cell, a fungal cell or a mammalian cell.

[0069] Suitable bacterial cells include, but are not limited to, cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0070] Suitable fungal cells include, but are not limited to, cells of species of Trichoderma, Neurospora, and Aspergillus; or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0071] Suitable mammalian cells include, but are not limited to, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0072] However, the present application can also use amphibian cells, insect cells, plant cells, and any other cells used in the art for expressing heterologous proteins.

[0073] In some embodiments, the host cell is a functional cell (e.g., CAR-T, CAR-NK, CD8+ T cell, NK cell, CIK cell, TIL cell, macrophage, DC cell, etc.) capable of expressing the chimeric protein or derivative thereof of the present application, which has one or more of the following physiological functions: tumor killing, pathogen clearance, immune effector, etc.

[0074] In an eighth aspect, the present application provides the use of the chimeric protein or derivative thereof, nucleic acid molecule, host cell, immunoconjugate, and pharmaceutical composition of the present application for the manufacture of a medicament for treating a related disease (e.g., a proliferative disease, an immunological disease, etc.), modulating a T cell-mediated immune response, stimulating the immune system of an individual. The proliferative disease can be a tumor or cancer (e.g., a metastatic tumor or cancer), which can be a solid tumor (e.g., metastatic renal cell carcinoma and malignant melanoma).

[0075] In some embodiments, the chimeric proteins or derivatives thereof, immunoconjugates, or pharmaceutical compositions disclosed herein can be used to treat disease situations in which the host's immune system is stimulated to benefit, particularly conditions in which an enhanced cellular immune response is desired, which can include disease situations in which the host's immune response is inadequate or defective. In some embodiments, the disease situations in which the chimeric proteins or derivatives thereof, immunoconjugates are administered include tumors or infections in which cellular immune response is 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, and the like. In some embodiments, enhancing the cellular immune response can include any one or more of: a general increase in immune function, an increase in T cell function, an increase in B cell function, restoration of lymphocyte function, an increase in IL-2 receptor expression, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0076] In some embodiments, the disease treated by the chimeric proteins or derivatives thereof, immunoconjugates, or pharmaceutical compositions of the present disclosure is a proliferative disorder, such as cancer. Non-limiting examples of cancers 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 cell proliferation disorders that can be treated using the chimeric proteins or derivatives thereof of the present disclosure include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital system. Also included are precancerous conditions or lesions and cancer metastasis. In certain 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 also be treated using the chimeric proteins or derivatives thereof of the present disclosure, 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 disease outside of neoplasia in the organ systems listed above. In other embodiments, the disease involves autoimmunity, transplant rejection, post-traumatic immune response, and infectious diseases (e.g., HIV).

[0077] 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.

[0078] 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.

[0079] 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

[0080] 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).

[0081] Figure 2 This refers to the chimeric region and site of the CD25-IL-2 chimeric protein (the chimeric region of IL-2).

[0082] Figure 3 A schematic diagram and predicted three-dimensional conformation of the CD25 / IL-2chimera1 chimeric protein.

[0083] Figure 4 A schematic diagram and predicted three-dimensional conformation of the CD25 / IL-2chimera2 chimeric protein.

[0084] Figure 5Schematic diagram and predicted three-dimensional conformation of CD25 / IL-2 chimera 3 chimeric protein.

[0085] Figure 6 Expression and purification of CD25 / IL-2 chimera chimeric protein.

[0086] Figure 7 Binding of CD25 / IL-2 chimera chimeric protein to CD25.

[0087] Figure 8 Binding of Human IL-2-Fc to CD25.

[0088] Figure 9 Binding of CD25 / IL-2 chimera chimeric protein to CD122 (bar graph).

[0089] Figure 10 Binding of CD25 / IL-2 chimera chimeric protein to CD122 (line graph).

[0090] Figure 11 Stimulation of NK92 cell activation of p-STAT5 by CD25 / IL-2 chimera chimeric protein.

[0091] Figure 12 Stimulation of CD8+ T cell activation of p-STAT5 by CD25 / IL-2 chimera chimeric protein.

[0092] Figure 13 Promotion of immune cell killing of tumor cells by CD25 / IL-2 chimera chimeric protein.

[0093] Figure 14 Schematic diagram and predicted three-dimensional conformation of IL-2 / CD25 chimera chimeric protein.

[0094] Figure 15 Schematic diagram and predicted three-dimensional conformation of IL15RA / IL-15 chimera chimeric protein.

[0095] Figure 16 Binding of IL15RA / IL-15 chimera chimeric protein to IL15RA.

[0096] Figure 17 Binding of IL15RA / IL-15 chimera chimeric protein to IL15R beta (CD122).

[0097] Figure 18 Stimulation of NK92 cell activation of p-STAT5 by IL15RA / IL-15 chimera chimeric protein

[0098] Figure 19 Schematic representation of IL21R / IL-21 chimera chimeric protein and predicted three-dimensional conformation. DETAILED DESCRIPTION

[0099] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below in combination with specific embodiments.

[0100] For the purpose of facilitating an understanding of the present application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs, reference being made to, for example, standard handbooks such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nd Ed.), Gower Medical Publishing, London, New York (1989), as well as the general state of the art referred to herein; and further, unless otherwise specified, all methods, steps, techniques and procedures that are not specifically detailed are to be performed and have been performed in a manner known per se to those skilled in the art, reference being made to, for example, standard handbooks, the general state of the art referred to above and other references cited therein.

[0101] Terminology

[0102] A "chimeric protein" refers to a protein comprising amino acid sequences originally derived from two different sources (e.g., a cytokine or a cytokine receptor extracellular domain). For example, a chimeric protein can comprise domains from at least two different naturally occurring human proteins. In some examples, a chimeric protein can comprise domains that are synthetic sequences and domains derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric protein can comprise at least two different domains that are synthetic sequences. The terms "chimera", "chimeric protein" can be used interchangeably.

[0103] "Cytokine" (CK) is intended to be construed broadly as a low molecular weight soluble protein produced by cells, which can be classified into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, etc. according to its function. Cytokine is a protein polypeptide, which plays an important role in cell signaling pathways. In the present invention, cytokine encompasses the broadest category, for example, proteinaceous hormone molecules are also included in the scope of cytokine as referred to in the present invention. The term encompasses unprocessed cytokine as well as any form of cytokine resulting from processing in a cell. The term also encompasses naturally occurring variants of cytokine, such as splice variants or allelic variants. The term also encompasses artificially engineered, modified, truncated variants of cytokine having similar functions.

[0104] "Cytokine receptor ectodomain" is intended to be construed broadly as a receptor ectodomain of cytokine as described in the above term. The term encompasses unprocessed cytokine receptor ectodomain as well as any form of cytokine receptor ectodomain resulting from processing in a cell. The term also encompasses naturally occurring variants of cytokine receptor ectodomain, such as splice variants or allelic variants. The term also encompasses artificially engineered, modified, truncated variants of cytokine receptor ectodomain having similar functions.

[0105] "Flexible linker" is intended to be construed broadly as including a linking sequence connecting between protein secondary structures (alpha helix and / or beta sheet, etc.), a flexible linking sequence connecting between domains folded into secondary structures, etc., such as various types of loops, turns, etc.

[0106] "Interleukin-2" or "IL-2" refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats). The term encompasses unprocessed IL-2 as well as any form of IL-2 resulting from processing in a cell. The term also encompasses naturally occurring variants of IL-2, such as splice variants or allelic variants. The term also encompasses IL-2 variants, modified, truncated variants having similar functions. The amino acid sequence of an exemplary wild-type human IL-2 is shown in SEQ ID NO: 2. Unprocessed human IL-2 additionally comprises a N-terminal 20 amino acid signal peptide (see UniProt entry number: P60568), which is absent in the mature IL-2 molecule. In the present invention, "interleukin-2", "interleukin 2", "leukocyte interleukin-2", "leukocyte interleukin 2", "IL2" and "IL-2" can be used interchangeably.

[0107] "CD25" or "a subunit of 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 as well as any form of CD25 that results from processing in cells, and also including naturally occurring variants of CD25, e.g. splice variants or allelic variants. The term also encompasses variants, engineered, truncated forms of CD25 that have similar functionality. In certain embodiments, CD25 is human CD25 (see UniProt entry no. P01589), the exemplary sequence of the extracellular domain of which is shown as SEQ ID NO: 1. "CD25" and "IL-2Ra" and "a subunit of IL-2 receptor" are used interchangeably in the present application.

[0108] CD25 and IL-2 as used in the present application do not include signal peptide sequences, the first amino acid after removal of the signal peptide is the amino acid at position 1.

[0109] The three letter code and one letter code of amino acids used in the present application are as described in J. Biol. Chem, 243, p3558 (1968), which are standard in the art and agreed upon.

[0110] Variants of CD25 and IL-2 include mutants thereof, which include amino acid substitutions, deletions, insertions, modifications, and any combination thereof of CD25 or IL-2.

[0111] Homologous peptides refer to peptides of similar sequence and function on homologous proteins from different species.

[0112] "Derivative" is intended to be construed broadly to include any product related to the target protein. This includes, but is not limited to, human and non-human homologs, fragments or truncations of the target protein, fusion proteins (e.g. fusion with a signal peptide or other active, inactive components, e.g. an antibody or antigen binding fragment thereof), modified forms (e.g. PEGylation, glycosylation, albumin conjugation / fusion, Fc conjugation / fusion, hydroxyethylation, etc.), and conservatively modified proteins, etc.

[0113] An "immunoconjugate" is a specific conjugate comprising at least one chimeric protein or derivative thereof and at least one antigen binding moiety. In certain embodiments, the immunoconjugate comprises at least one chimeric protein or derivative thereof and at least two antigen binding moieties. A specific immunoconjugate according to the present application consists essentially of one chimeric protein or derivative thereof and an antigen binding moiety linked by one or more linker sequences. The antigen binding moiety can be linked to the chimeric protein or derivative thereof by a variety of interactions and in a variety of configurations.

[0114] "Antibody" is used in the broadest sense of the term, and encompasses various antibody structures, so long as they exhibit the desired antigen-binding activity, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen binding fragments. Antibodies can include murine antibodies, human antibodies, camelid antibodies, shark antibodies, humanized antibodies, chimeric antibodies, heavy chain antibodies, nanobodies, single domain antibodies, etc. Illustratively, an antibody can be an immunoglobulin, which is a tetrameric structure consisting of two identical heavy chains and two identical light chains connected by disulfide bonds between them. The amino acid composition and arrangement of the constant region of the heavy chain of immunoglobulin are different, so its antigenicity is different. Accordingly, immunoglobulin can be divided into five types, or called isotypes of immunoglobulin, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same class of Ig can be divided into different subclasses according to the difference of amino acid composition of hinge region, number and position of heavy chain disulfide bond, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4. The light chain is divided into κ chain or λ chain by the difference of constant region. Each of the five types of Ig can have κ chain or λ chain.

[0115] "Antigen binding fragment" refers to Fab fragments, Fab' fragments, F(ab')2 fragments, single chain Fv (i.e., sFv), nanobodies (i.e., VHH), VH / VL domains, which have antigen binding activity. Fv fragments contain the variable region of the heavy chain and the light chain of the antibody, but do not have the constant region, and have the smallest antigen binding fragment of 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 connected by different linkers to form a polypeptide chain, called single chain antibody or single chain Fv (sFv).

[0116] In comparing two amino acid sequences, the term "amino acid difference" refers to an insertion, deletion, or substitution of a specified number of amino acid residues at a position in a reference sequence as compared to another sequence. In the case of a substitution, the substitution will preferably be a conservative amino acid substitution, which means that the amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or no effect on the function, activity or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example, a conservative amino acid substitution is preferably one in which an amino acid residue in one of the following groups (i)-(v) is replaced by another amino acid residue in the same group: (i) small 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 Gin; (iii) polar positively charged residues: His, Arg, and Lys; (iv) large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (v) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala replaced by Gly or Ser; Arg replaced by Lys; Asn replaced by Gin or His; Asp replaced by Glu; Cys replaced by Ser; Gin replaced by Asn; Glu replaced by Asp; Gly replaced by Ala or Pro; His replaced by Asn or Gin; Ile replaced by Leu or Val; Leu replaced by Ile or Val; Lys replaced by Arg, Gin, or Glu; Met replaced by Leu, Tyr, or Ile; Phe replaced by Met, Leu, or Tyr; Ser replaced by Thr; Thr replaced by Ser; Trp replaced by Tyr; Tyr replaced by Trp or Phe; and Val replaced by Ile or Leu.

[0117] “Sequence identity” between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. “Sequence similarity” indicates the percentage of amino acids that are either identical or represent conservative amino acid substitutions. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those of skill in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity. See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988, Biocomputing: Informatics, and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., 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., M Stockton Press, New York, 1991, for determinations of sequence identity.

[0118] A “vector” refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is one which is capable of directing the expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector can be used to introduce the genetic material elements it carries into a host cell, where the genetic material elements are expressed. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1 -derived artificial chromosomes (PACs), bacteriophages such as lambda 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, papovaviruses (such as SV40). Vectors can comprise multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors can comprise origins of replication.

[0119] "Host cell" and "host cell line" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of the original cell that has been transformed. Host cells include "transformants" and "transformed cells," which include the original transformed cell and progeny derived therefrom, without regard to the number of passages. The progeny can not necessarily be precisely identical to the parent cell both in genetic and phenotypic characteristics, but can contain mutations. Mutant progeny that have the same function or biological activity as the screened or selected function or biological activity of the originally transformed cell are included herein. "Host cell" includes, but is not limited to, prokaryotic cells such as E. 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 fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0120] The following examples using molecules such as cytokine IL-2 and its receptor CD25, IL-15 and its receptor IL-15RA, IL-21 and its receptor IL-21R, etc. are used to further illustrate the present application.

[0121] Example 1

[0122] Design and structure prediction of CD25 / IL-2 chimeric protein

[0123] 1.1 Determination of the chimeric region

[0124] As mentioned above, in the present specific embodiments, the cytokine receptor is CD25 and the cytokine is IL-2. According to the crystal structure of IL-2 and its high affinity receptor IL-2Rαβγ (PDB ID: 2B5I) in the PDB database, we analyzed that there are multiple flexible connection regions (i.e. loop regions) on the binding surface of CD25 and IL-2 and its vicinity, such as shown in Figure 1 、 Figure 2

[0125] The sequence of CD25 extracellular domain is SEQ ID NO: 1, and the amino acid positions of CD25 extracellular domain are numbered according to SEQ ID NO: 1; the sequence of IL-2 is SEQ ID NO: 2, and the amino acid positions of IL-2 are numbered according to SEQ ID NO: 2,

[0126] In some embodiments, the chimeric region is located in the CD25 extracellular domain: the chimeric region is located in the flexible connection sequence region of the interface between CD25 extracellular domain and IL-2 (referring to structure PDB ID: 2B5I): including chimeric region Q1 and chimeric region Q2:

[0127] ​(a) Chimeric region 1 (as shown in Figure 1 I37-K38-S39-G40-S41-L42 (SEQ ID NO: 3) peptide segment or a homologous peptide segment or a derivative peptide segment having similar structure and function to the peptide segment on the CD25 extracellular domain (SEQ ID NO: 1) and the peptide segment extending 10 amino acids forward and backward (SEQ ID NO: 5);

[0128] (b) Chimeric region 2 (as shown in Figure 1 T150-H151-G152-K153-T154 (SEQ ID NO: 4) peptide segment or a homologous peptide segment or a derivative peptide segment having similar structure and function to the peptide segment on the CD25 extracellular domain (SEQ ID NO: 1) and the peptide segment extending 10 amino acids forward and backward (SEQ ID NO: 6).

[0129] In some embodiments, any one of the amino acid sites on the peptide segments described in (a) and (b) can be selected as the chimeric site, at which a partial peptide segment of IL-2, IL-2, IL-2 mutant, IL-2 engineered, IL-2 derivative, etc. can be inserted.

[0130] In some specific embodiments, the chimeric site is preferably selected from K38 of the CD25 extracellular domain described in (a), and two chimeric proteins based on this chimeric site are SEQ ID NOs: 10-11.

[0131] In one embodiment, the chimeric site is preferably selected from H151 of the CD25 extracellular domain described in (b), and one chimeric protein based on this chimeric site is SEQ ID NO: 12.

[0132] In some other embodiments, the chimeric region is located on the cytokine IL-2: the chimeric region is selected from the flexible linker sequence region (loop region) between two adjacent alpha helices of IL-2 (as shown in Figure 2

[0133] (a) Chimeric region 3 (as shown in Figure 2 T41-F42-K43-F44-Y45-M46-P47-K48-K49-A50-T51 (SEQ ID NO: 7) or a derivative peptide segment having similar structure and function to the loop region between helix A and helix B of IL-2 (SEQ ID NO: 2);

[0134] (b) Chimeric region 4 (as shown in Figure 2 ​helix B and helix C of IL-2 (SEQ ID NO: 2) L72-A73-Q74-S75-K76-N77-F78-H79-L80 (SEQ ID NO: 8) or a derivative peptide segment having similar structure and function to the loop region;

[0135] (c) Chimeric region 5 (as shown in Figure 2 helix C and helix D of IL-2 (SEQ ID NO: 2) F103-M104-C105-E106-Y107-A108-D109-E110-T111-A112 (SEQ ID NO: 9) or a derivative peptide segment having similar structure and function to the loop region.

[0136] In some embodiments, any one of the amino acid positions on the peptide segments described in (a), (b) and (c) can be selected as the chimeric position, and a partial peptide segment of the CD25 extracellular domain, CD25 extracellular domain, CD25 extracellular domain mutant, CD25 extracellular domain construct, CD25 extracellular domain derivative, etc. can be inserted before / after the position.

[0137] In some embodiments, the chimeric position is preferably selected from the F103 position of the C’D’ loop region of IL-2.

[0138] In some embodiments, the chimeric position is preferably selected from the Y45 position of the A’B’ loop region of IL-2.

[0139] In some embodiments, the chimeric position is preferably selected from the N77 position of the B’C’ loop region of IL-2.

[0140] 1.2 Construction of the chimeric protein and structure prediction

[0141] By selecting any one of the amino acid positions in the chimeric region Q1, Q2 as the chimeric position, the amino acid sequence of C1 can be the following peptide segment: 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.

[0142] The amino acid sequence of C2 can be the following peptide segment: 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.

[0143] C1 and C2 can also be the homologous peptide segment of any of the above peptide segments in other (non-human) species; or a derivative peptide segment formed by substitution, deletion or addition of one or more (e.g., 1-10) amino acid residues to any of the above peptide segments, and having similar structure and function of the peptide segment; or an amino acid sequence having at least 80% sequence identity to the sequence of the above peptide segment.

[0144] The amino acid sequence of I1 can be the following peptide segment: 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, by taking any of the amino acid positions in the chimeric region Q3-Q5 as the chimeric point.

[0145] The amino acid sequence of I2 can be the following peptide segment: 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-Q74, 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.

[0146] I1and I2may also be homologous peptide segments of any of the above peptide segments in other (non-human) species; or a derivative peptide segment of any of the above peptide segments, which is formed by substitution, deletion or addition of one or more (e.g. 1-10) amino acid residues to the amino acid sequence of the peptide segment, and has similar structure and function to the peptide segment; or an amino acid sequence having at least 80% sequence identity to the sequence of the above peptide segment.

[0147] By chimeric connection of different peptide segments of CD25 and IL-2 in the flexible connection region through linker elements of appropriate length according to the structure of C1-L1-I1-L2-I2-L3-C2 in the present application. When the chimeric site is selected from K38 of the extracellular domain of CD25, such as SEQ ID NO: 10-11 is two chimeric proteins constructed based on this chimeric site, and when the chimeric site is H151 of the extracellular domain of CD25, such as SEQ ID NO: 12 is one chimeric protein constructed based on this chimeric site.

[0148] Further, we chimeric according to the combinations in Table 1 to form a chimeric body of a polypeptide chain: CD25 / IL-2chimera1, CD25 / IL-2chimera2, CD25 / IL-2chimera3. Use Alphafold2 to predict the protein structure of these sequences (Jumper, J et al., Nature (2021); Varadi, M et al., Nucleic Acids Research (2021).), use the complete version of the database provided during running BigFantasticDatabase (BDF), and set the training parameter --db_preset to full_dbs, and other parameters are default parameters, and use PyMol to view the predicted protein structure. The results show that the three peptide segments form a complex crystal structure similar to the binding of CD25 and IL-2 (PDB ID: 2B5I), as shown in Figures 3 to 5 .

[0149] From the protein structure, it can be seen that the CD25 epitope of the IL-2 domain in these chimeric proteins has been completely blocked by the CD25 domain, and it is speculated that these CD25 / IL-2 chimeric proteins have the potential to bind / activate the intermediate affinity IL-2 receptor (IL-2Rβγ) and weaken (or not) the binding / activation of the high affinity IL-2 receptor (IL-2Rαβγ). The subsequent examples further verify the structure-based functional speculation.

[0150] Table 1. Sequences of peptide segments and linkers of 3 chimeric proteins

[0151]

[0152]

[0153] CD25 / IL-2chimera1, CD25 / IL-2chimera2, CD25 / IL-2chimera3 were added to the immunoglobulin Fc region respectively to obtain SEQ ID NO:21 (CD25 / IL-2chimera1-Fc), SEQ ID NO:22 (CD25 / IL-2chimera2-Fc), SEQ ID NO:23 (CD25 / IL-2chimera3-Fc).

[0154] Example Two

[0155] Preparation of experimental materials

[0156] The information of the relevant experimental materials used in Examples 3-12 is provided in Table 2.

[0157] Table 2 Experimental materials

[0158]

[0159]

[0160] Example 3

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

[0162] 3.1 Vector construction

[0163] 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 Xiamen Bicai Biological Company for Expi293F cell expression in a vector (LV082-AbV-Human-IGG1-Fc-1GS) with a human IGG1-Fc tag, obtaining two expression plasmids expressing CD25 / IL-2chimera-Fc, 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 cell expression of CD25 / IL-2chimera-Fc protein

[0165] The constructed CD25 / IL-2 chimera-Fc plasmid was transfected into EXPi293F cells, and the transfection process was as follows: Expi293F cells were cultured at 37°C in an 8% CO2 incubator at 135 rpm to 4 to 5 x 10^6 cells / ml, and the cells were diluted with fresh culture medium to a concentration of 3 x 10^6 cells / ml, 30 ml / bottle. 1.5 ml of Opti-Medium + 30 ug of plasmid was pre-mixed for 5 minutes (A liquid), 1.5 ml of Opti-Medium + 60 ul of PEI (2 ug / ul) (B liquid) was pre-mixed for 5 minutes, A liquid and B liquid were mixed and incubated at room temperature for 20 minutes (dose per well), the above liquid was dropped into the culture bottle, shaken gently, and the cells were placed back into the 37°C, 8% CO2 incubator at 135 rpm for continuous culture. After 24 hours of 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. After 5 days of transfection, the cell supernatant was collected by centrifugation at 2000 rcf for 5 minutes.

[0166] The supernatant was incubated with ProteinA beads for 2 hours, then the liquid and beads were transferred to an empty column, the impurities were washed away with Washing Buffer, the protein was eluted with Elution Buffer, the eluted protein was dialyzed in PBS, the dialyzed protein was filtered with a 0.22 uM filter, the protein concentration was determined by the BCA method, and the protein was run on SDS-PAGE gel, and the expression of the protein was observed after staining with Coomassie brilliant blue, as shown in Figure 6 As shown in Figure 6 It can be seen that CD25 / IL-2 chimera1-Fc (SEQ ID NO: 21) and CD25 / IL-2 chimera3-Fc (SEQ ID NO: 23) can be normally expressed.

[0167] Example 4

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

[0169] CD25 recombinant protein was coated on ELISA plates at an amount of 400 ng / well, room temperature for 2 h, 5% skimmed milk powder blocking at room temperature. After blocking, wash 3 times with washing buffer, then add 100 ul of gradient dilution series of Human IL-2, CD25 / IL-2 chimera 1-Fc or CD25 / IL-2 chimera 3-Fc (0.625 nM to 1000 nM) per well, react at room temperature for 2 h, wash 3 times with washing buffer. Add Alpaca VHH-Fc Anti-IL2 antibody, react at room temperature for 1 h, wash 3 times with washing buffer. Add Rabbit anti-VHH (HRP) (Genscript, A01861-200) and incubate at room temperature for 1 h. After incubation, add TMB developing solution (Solebo, PR1200-500ML) and terminate the reaction by 1M H2SO4. Use a microplate reader (Tecan, SPARK10M) to read the absorbance value at 460 nm. Use GraphPad Prism 9 to process and analyze the data, and obtain the binding curve of wild-type Human IL-2, CD25 / IL-2 chimera 1-Fc and CD25 / IL-2 chimera 3-Fc to CD25, as shown in Figure 7 Meanwhile, the binding of Human IL-2-Fc and CD25 was checked, as shown in Figure 8

[0170] From the experimental results, we found that:

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

[0172] (2) CD25 / IL-2 chimera 1-Fc (SEQ ID NO: 21) and CD25 / IL-2 chimera 3-Fc (SEQ ID NO: 23) did not show significant binding to CD25 even at a concentration of 1000 nM, suggesting that CD25 / IL-2 chimera 1-Fc (SEQ ID NO: 21) and CD25 / IL-2 chimera 3-Fc (SEQ ID NO: 23) may not be able to bind / activate the high-affinity IL-2 receptor.

[0173] ​(3) The binding ability of Fc-fused IL-2 protein (Human IL-2-Fc) to CD25 was almost the same as that of wild-type Human IL-2, indicating that Fc did not significantly affect the binding of IL-2 to CD25, further indicating that the extremely significantly weakened binding reaction of CD25 / IL-2 chimera 1-Fc and CD25 / IL-2 chimera 3-Fc to CD25 was a specific effect of CD25 / IL-2 chimera 1 and CD25 / IL-2 chimera 3.

[0174] Example 5

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

[0176] 5.1 Biotin coupling of Human IL-2 and CD25 / IL-2 chimera

[0177] 500 μg of the relevant protein was dissolved in 500 μl of PBS to a final concentration of 1 mg / ml. 5 μl of 10 mg / ml NHS-biotin solution was added and mixed well (NHS was dissolved in DMSO). Incubation was performed at room temperature for 30 min. 50 μl of 100 mM glycine solution was added, and incubation was performed at room temperature for 10 min, and the reaction was terminated by mixing well. The coupled protein was dialyzed using PBS. After dialysis, it was filtered using a 0.22 uM filter, the protein concentration was measured using BCA, and it was stored at -80°C for use.

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

[0179] CD122 recombinant protein was coated in an ELISA plate at a dose of 400 ng / well, and then an equal amount of Biotin-coupled Human IL-2, CD25 / IL-2 chimera 1-Fc, CD25 / IL-2 chimera 3-Fc or Fc-Isotype (negative control) was added, and the binding of each protein to CD122 was detected using HRP-Streptavidin, as shown in Figure 9

[0180] ​From the experimental results, it can be seen that the wild type Human IL-2 and CD122 have weak interaction. Compared with the wild type Human IL-2, the binding of CD25 / IL-2 chimera 1-Fc and CD25 / IL-2 chimera 3-Fc to CD122 is significantly enhanced, which is likely to be the chimeric IL-2 and CD25 that enhances its affinity to CD122. The control Fc protein does not bind to CD122, which further indicates that the binding of CD25 / IL-2 chimera 1-Fc and CD25 / IL-2 chimera 3-Fc to CD122 is a specific reaction of CD25 / IL-2 chimera 1 and CD25 / IL-2 chimera 3.

[0181] In order to further confirm the binding ability of CD25 / IL-2 chimera 1-Fc and CD25 / IL-2 chimera 3-Fc to CD122, the binding curves of the two chimeras and CD122 were further detected by gradient dilution series (0.625 nM to 1000 nM) of CD25 / IL-2 chimeras binding experiment, as shown in Figure 10 Figure 10 From the above, it can be seen that CD25 / IL-2 chimera 1-Fc (SEQ ID NO: 21) and CD25 / IL-2 chimera 3-Fc (SEQ ID NO: 23) can bind to CD122 well, which indicates that the chimeras retain the ability to bind / activate the medium affinity IL-2 receptor.

[0182] Example 6

[0183] Stimulation of NK92 cells by CD25 / IL-2 chimeras

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

[0185] ​Before stimulation, NK92 cells were cultured in IL-2-free medium for 2-3 days. IL-2-starved NK92 cells were seeded in 24-well plates according to the sample size. The IL-2-starved NK92 cells were treated with PBS (negative control), Human IL-2, CD25 / IL-2 chimera 1-Fc (SEQ ID NO: 21), CD25 / IL-2 chimera 3-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; after stimulation, collect cell precipitate at 1000rcf for 5 min. Lyse cells with RIPA lysis buffer, add 5xLoading buffer, western blot to detect P-STAT5 and Actin, as shown in Figure 11

[0186] From the experimental results, CD25 / IL-2 chimera 1-Fc (SEQ ID NO: 21) and CD25 / IL-2 chimera 3-Fc (SEQ ID NO: 23) can well activate the phosphorylation of STAT5 in NK92 cells, suggesting that the chimera has the function of activating NK cells. CD25-Fc and Fc-Isotype cannot activate the phosphorylation of STAT5, further indicating that the activation of NK92 by CD25 / IL-2 chimera 1-Fc and CD25 / IL-2 chimera 3-Fc is a specific response to CD25 / IL-2 chimera 1 and CD25 / IL-2 chimera 3.

[0187] Example 7

[0188] Stimulation of CD8+T cells by CD25 / IL-2 chimera

[0189] 7.1 Isolation of mouse CD8+T cells

[0190] The CD8+T cells were obtained by negative screening method, and the non-CD8 cells such as CD4+T cells, B cells and red blood cells were removed from the obtained mouse spleen and lymph node cells. The specific process is as follows:

[0191] Take the mouse spleen and lymph nodes in 2 ml of culture medium.

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

[0193] ​Wash filter with 8 ml of magnetic beads isolation buffer (Streptavidin Particles Plus - DM, BD Biosciences, 557812), 500 g, 5 min, 4°C.

[0194] Lysed with 3 ml of red blood cell lysis solution for 5 min.

[0195] Stop reaction by adding 5-10 ml of magnetic beads isolation buffer and centrifuge.

[0196] Count cells and dilute to 2 x 10^7 per ml.

[0197] Add 400 μl of antibody cocktail per 1 x 10^8 cells.

[0198] Prepare antibody (purchased from BioLegend) cocktail in 100 ul of magnetic beads isolation buffer as follows:

[0199]

[0200] Incubate at 4°C for 20 min, 500 g, 5 min, 4°C, centrifuge.

[0201] Wash once with 10 ml of magnetic beads isolation buffer.

[0202] Prepare two magnetic beads: 50 μl for the first one and 40 ul beads for the second one, dilute with magnetic beads isolation buffer at 3 times the volume of beads.

[0203] Resuspend the cells with the first beads and incubate for 5 min, then transfer to a flow tube placed on a magnetic stand and let it adsorb for 1 min.

[0204] Transfer the supernatant to the second beads and incubate for 5 min, then transfer to a flow tube placed on a magnetic stand and let it adsorb for 1 min.

[0205] Transfer the supernatant to another flow tube and let it adsorb for 1 min.

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

[0207] Plate in 24 well plates with culture medium without IL-2 according to the amount of cells needed and keep them ready to use.

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

[0209] PBS (negative control), Human IL-2, CD25 / IL-2 chimera 1-Fc (SEQ ID NO: 21) and CD25 / IL-2 chimera 3-Fc (SEQ ID NO: 23) were used to treat the CD8+T cells isolated in 7.1, treatment time: 20 min, 2 h; treatment concentration: 15.625 nM; after stimulation, 1000 rcf, 5 min, collect cell precipitate. Lysis cells with RIPA lysis buffer, add 5xLoading buffer, western blot detection P-STAT5 and Actin, as shown in Figure 12 The experimental results show that CD25 / IL-2 chimera 1-Fc (SEQ ID NO: 21) and CD25 / IL-2 chimera 3-Fc (SEQ ID NO: 23) can well activate the phosphorylation of STAT5 in CD8+T cells, suggesting that the chimera has the function of activating CD8+T cells.

[0210] Example 8

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

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

[0213] Fresh blood is diluted with PBS at 1:1 and gently blown up and down. Ficoll reagent is shaken well by inverting 5-10 times, and is aliquoted into centrifuge tubes for use. The diluted blood is attached to the Ficoll-containing centrifuge tube, and the centrifuge tube is transferred to the centrifuge, which is balanced at room temperature at 400g for 30 min, with the speed set to 1 and the speed set to 0. The upper serum is gently aspirated with a 1 mL pipette. After centrifugation, there are four layers from bottom to top, red blood cells, Ficoll solution, white membrane layer, and PBS. The gun head is inserted into the white membrane layer, and the white membrane layer (i.e. lymphocyte layer) is carefully aspirated and transferred to a new 15 ml centrifuge tube. Try to avoid aspirating cells from the lower or upper layers to avoid contamination. Add 10 ml PBS to the transferred lymphocytes, mix gently, and centrifuge at 100xg for 10 min. Discard the supernatant, add 1 ml of red cell lysis solution, and lyse for 3 min. Add the washing solution to stop the lysis, and centrifuge at 300xg for 5 min. Add the washing solution to make the volume to 10 ml, and wash at 100xg for 8 min. Discard the supernatant, and collect the cell precipitate for use.

[0214] 8.2 Expansion and activation of immune cells

[0215] The isolated PBMCs were added to 24-well cell culture plates, which were previously coated with 10 pg / mL anti-CD3 (novoprotein, GMP-A018) and anti-CD28 (novoprotein, GMP-A063) and kept at 4°C overnight. The anti-CD3 and anti-CD28 antibodies can promote the activation and proliferation of T cells. The PBMCs were cultured in RPMI 1640 containing 10% fetal bovine serum, containing 1:100 penicillin-streptomycin. The cells were divided into different parts, and the following were added at a final concentration of 15.625 nM: Human IL-2, CD25 / IL-2 chimera 1-Fc (SEQ ID NO: 21), CD25 / IL-2 chimera 3-Fc (SEQ ID NO: 23), CD25-Fc (control), or Fc-Isotype (control). The cells were passaged every other day, and an equal amount of fresh medium was added, and the corresponding stimulator: Human IL-2, CD25 / IL-2 chimera 1-Fc, CD25 / IL-2 chimera 3-Fc, CD25-Fc, or Fc-Isotype was added. The proliferated and activated immune cells were used for subsequent tumor killing experiments.

[0216] 8.3 RTCA esight detection of the killing ability of immune cells on tumor cells

[0217] The steps are as follows: 50 pL of 1640 complete culture medium was added to an E-Plate 96-well culture plate, which was balanced at 37°C for 1 hour to detect the impedance baseline. Tumor cells (human lung squamous carcinoma cells H226) were inoculated at a number of 5000 cells / well, and the well plate was placed horizontally at room temperature for 30 minutes to allow the cells to settle evenly at the bottom of the well plate. The target cell adhesion proliferation was detected by impedance. The impedance was collected every 15 minutes, and the image was collected every 1 hour. After the tumor cells adhered for 12 hours, the expanded effector cells (mainly T cells) in 8.2 were collected, and the prepared effector cell suspension was added to the wells containing target cells (tumor cells) at a ratio of 5:1 per well. The added well plate was placed back in the corresponding position of the xCELLigence RTCA eSight (Agilent), and balanced for 30 min. Then data collection was started, and the process of effector cells killing target cells was monitored in real time, with impedance collected every 15 min and image collected every 1 hour. The results are as follows Figure 13As shown: compared with CD25-Fc and Fc-Isotype, CD25 / IL-2chimera1-Fc and CD25 / IL-2chimera3-Fc can significantly promote immune killing of H226 tumor cells, which shows that CD25 / IL-2chimera1-Fc and CD25 / IL-2chimera3-Fc can effectively promote immune cells to kill tumors, and this activity is a specific response of CD25 / IL-2chimera1 and CD25 / IL-2chimera3.

[0218] Example 9

[0219] The present embodiment discloses a kit comprising a container, wherein the container is provided with the CD25 / IL-2chimeric protein of embodiment three. The kit of the present embodiment can promote the activation of NK cells and CD8+T cells or promote the killing of tumor cells by immune cells to promote the treatment of related diseases.

[0220] Example 10

[0221] Design and structure prediction of IL-2 / CD25chimeric protein

[0222] According to the chimeric region determined in 1.1, the different peptide segments of CD25 and IL-2 are connected according to the structure I1-L1-C-L3-I2 in the present application by a linker element of appropriate length in the flexible connection region. When the chimeric site is selected from position S75 of IL-2 (numbered according to SEQ ID NO: 2), SEQ ID NO: 27 is a chimeric protein constructed based on this chimeric site, which is called IL-2 / CD25 Chimera.

[0223] Further, we chimerized according to the combinations in Table 3 to form a chimeric body of a polypeptide chain: IL-2 / CD25Chimera. Alphafold2 was used to predict the protein structure of these sequences (Jumper, J et al., Nature (2021); Varadi, M et al., Nucleic Acids Research (2021).), and the complete version of the database BigFantasticDatabase (BDF) provided during running was used, with the training parameter --db_preset set to full_dbs, and other parameters as default parameters. PyMol was used to view the predicted protein structure. The results show that the peptide segments form a complex crystal structure similar to the combination of CD25 and IL-2 (PDB ID: 2B5I), as shown in Figure 14indicated (CD25 is numbered according to SEQ ID NO: 1, IL-2 is numbered according to SEQ ID NO: 2).

[0224] From the protein structure, it can be seen that the CD25 epitope of the IL-2 domain in the chimeric protein has been completely blocked by the CD25 domain, and it is thus inferred that these IL-2 / CD25 chimeric proteins have the potential to preferentially bind / activate the intermediate affinity IL-2 receptor (IL-2Rβγ) and weaken (or not) the binding / activation of the high affinity IL-2 receptor (IL-2Rαβγ).

[0225] Table 3. Sequences of peptide segments and linkers of IL-2 / CD25 Chimera (SEQ ID NO: 27)

[0226]

[0227] Example 11

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

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

[0230] By chimeric connection of different peptide segments of IL15RA and IL15 in the flexible linker region through a linker element of appropriate length according to the C1-L1-I1-L2-I2-L3-C2 structure formula in the present application, when the chimeric site is selected from position A67 of the chimeric region (amino acid positions of IL15RA are numbered according to UniProt entry number: Q13261), SEQ ID NO: 28 is a chimeric protein constructed based on the chimeric site, which is called IL15RA / IL-15 Chimera.

[0231] Further, we chimerized the peptides in Table 4 to form a chimeric polypeptide chain: IL15RA / IL-15 Chimera. We used Alphafold2 to predict the protein structure of these sequences (Jumper, J et al., Nature (2021); Varadi, M et al., Nucleic Acids Research (2021).) with the full version of the database BigFantasticDatabase (BDF) provided during the running process, while setting the training parameter --db_preset to full_dbs, and other parameters to default. We used PyMol to view the predicted protein structure. The results showed that the peptide segment formed a complex crystal structure similar to the binding of IL15RA and IL-15 (PDB ID: 2Z3Q), as shown in Figure 15 IL15RA is numbered according to UniProt ID: Q13261, and IL-15 is numbered according to UniProt ID: P40933.

[0232] According to the protein structure, the IL15RA epitope of the IL-15 domain in the chimeric protein has been completely blocked by the IL15RA domain, and it is speculated that the IL15RA / IL-15 chimeric protein has the potential to bind / activate IL-15 βγ (CD122 / CD132) receptors and weaken (or not) the binding / activation of IL15Rα (IL15RA) receptors.

[0233] Table 4. Sequences of peptides and linkers of IL15RA / IL-15 Chimera (SEQ ID NO: 28)

[0234]

[0235] Further, similar to Examples 3-6, we constructed the expression vector of the IL15RA / IL-15 chimeric protein, prepared the IL15RA / IL-15 chimeric protein, and detected the binding of IL15RA / IL-15 Chimera and IL15RA by ELISA experiment Figure 16 ); detected the binding of IL15RA / IL-15 Chimera and IL15Rβ (CD122) by ELISA experiment Figure 17 ); and detected the activation of NK cells by IL15RA / IL-15 Chimera by using IL15RA / IL-15 Chimera to stimulate NK92, followed by western blot to detect the phosphorylation level of STAT5 Figure 18 ).

[0236] From the results, it can be seen that IL15RA / IL-15 Chimera greatly reduces the binding with IL15RA, enhances the binding with IL15Rβ (CD122), and can directly promote the phosphorylation of STAT5 in NK92 cells, indicating that IL15RA / IL-15 Chimera has the function of directly binding to IL-15βγ (CD122 / CD132) receptor and weakening (or not) the binding / activation of IL15Rα (IL15RA) receptor, and further can directly promote the activation of NK cells.

[0237] Example 12

[0238] Design and structure prediction of IL21R / IL-21 Chimera

[0239] As previously described, in the present specific embodiment, the cytokine receptor is IL21R and the cytokine is IL-21. According to the crystal structure of IL-21 and IL21R in the PDB database (PDB ID: 3TGX), we analyzed that there are multiple flexible linker regions on the binding surface of IL21R and IL-21 and in the vicinity thereof.

[0240] By connecting the different peptide segments of IL21R and IL-21 in the flexible linker region through a linker element of appropriate length according to the C1-L1-I-L3-C2 structure in the present application, when the chimeric site is selected from position D91 of IL21R (numbered according to UniProt entry number: Q9HBE5), SEQ ID NO: 29 is a chimeric protein constructed based on this chimeric site, called IL21R / IL-21 Chimera.

[0241] Further, we chimerized according to the combinations in Table 5 to form a chimeric body of a polypeptide chain: IL21R / IL-21 Chimera. Alphafold2 was used to predict the protein structure of these sequences (Jumper, J et al., Nature (2021); Varadi, M et al., Nucleic Acids Research (2021).), and the complete version of the database BigFantasticDatabase (BDF) provided during running was used, with the training parameter --db_preset set to full_dbs, and other parameters as default parameters. PyMol was used to view the predicted protein structure. The results show that the peptide segments form a complex crystal structure similar to that of IL21R and IL21 (PDB ID: 3TGX), as shown in Figure 19 IL21R is numbered according to UniProt ID: Q9HBE5, and IL-21 is numbered according to UniProt ID: Q9HBE4).

[0242] From the protein structure, it can be seen that the IL21R epitope of the IL-21 domain in the chimeric protein has been completely blocked by the IL21R domain, and it is thus inferred that the IL21R / IL-21 chimeric protein has the potential to weaken the binding / activation function of the L21R receptor.

[0243] Table 5. Sequence of peptide segments and linkers of IL21R / IL-21 Chimera (SEQ ID NO: 29)

[0244]

[0245] Example 13

[0246] Application of chimeric region

[0247] In addition to the chimeric extracellular domain of a cytokine receptor or a cytokine, the chimeric region of the cytokine / cytokine receptor described in the present application can also be a single-chain antibody / single-domain antibody or other functional proteins, etc.; thus, after embedding the target antibody / functional protein at a relevant position (such as an antibody targeting T cell antigens or NK cell antigens or tumor-related antigens, etc.), the binding of the cytokine to its receptor can be destroyed by steric hindrance, and at the same time, the target cytokine can be targeted to a specific location (such as T cells, NK cells or tumor cells) by the embedded antibody / functional protein, so as to achieve the effect of specifically regulating the purposeful distribution of the target cytokine in the body.

[0248] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which shall be encompassed within the protection scope of the present application.

Claims

1. A chimeric protein, characterized in that, the chimeric protein is any one of the polypeptide sequences in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:

28.

2. Conjugate, characterized in that: comprising the chimeric protein of claim 1, which is directly or indirectly linked to other modules through linker elements; the other modules are selected from any one of the following or a combination thereof: a detectable label, a nanomagnetic particle.

3. A pharmaceutical composition, characterized by: comprising the chimeric protein of claim 1 or the conjugate of claim 2 and a pharmaceutically acceptable carrier.

4. A nucleic acid molecule, characterized in that: a nucleic acid molecule encoding the chimeric protein of claim 1.

5. An expression vector, characterized by: comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the expression vector of claim 5, or the nucleic acid molecule of claim 4, or expressing the chimeric protein of claim 1 or the conjugate of claim 2; the host cell is a bacterial cell, a fungal cell, or a mammalian cell, an amphibian cell, or an insect cell.

7. A kit characterized in that: comprising the chimeric protein of claim 1, the conjugate of claim 2, the pharmaceutical composition of claim 3.

8. A method of making a chimeric protein, characterized by: expressing using the nucleic acid molecule of claim 4, or using the expression vector of claim 5, or using the host cell of claim 6 under conditions suitable for expressing the chimeric protein.

Images