IL-21 mutant, fusion protein containing IL-21 mutant, nucleic acid, recombinant expression vector, host cell as well as preparation method and application of IL-21 mutant

Abstract

The invention discloses an IL-21 mutant, a fusion protein containing the IL-21 mutant, nucleic acid, a recombinant expression vector, a host cell and a preparation method and application of the IL-21 mutant. Compared with a wild type IL-21, the IL-21 mutant comprises one or more of the following mutation pairs: a mutation pair R5C and P79C; a mutation pair of H6C and T81C; the V24C and the K105C are changed into pairs; the mutation pair is L32C and A53C; a mutation pair S57C and E64C, or S57C and I67C; the mutation pair is V28C and A58C; a mutation pair I16C and K73C; and a mutation pair of D26C and N63C. The above mutation pairs can be combined with IL-21 mutation R9E / R76A, and the combination still has biological activity, so that the druggability of the finally obtained mutant is further improved.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to IL-21 mutants, fusion proteins, nucleic acids, recombinant expression vectors, host cells containing the same, and preparation methods and uses thereof. Background Art

[0002] Cytokines are molecular messengers for intercellular communication in the immune system and play an important role in tumor immunity. Currently available cytokine drugs include recombinant human interferon α-2b injection, recombinant human interleukin-2 for injection, recombinant modified human tumor necrosis factor for injection, and recombinant human granulocyte stimulating factor injection interferon (IFN). In addition, there are many cytokines, especially IL-2 family-related cytokines such as IL-2, IL-7, IL-15 and IL-21, which are undergoing clinical studies to evaluate their anti-tumor potential.

[0003] Interleukin-21 is an immune activating factor belonging to the IL-2 family and a four-helix cytokine. IL-21 is mainly produced by activated CD4 + Secreted by T cells, NK cells, TFH cells, and Th17 cells, it enhances the antigen-specific response of immune cells. IL-21 can promote CD8 +The anti-tumor activity of T cells and NK cells plays a key role in B cell differentiation and germinal cell development, and is a potential target for the development of tumor immunotherapy. IL-21 binds to its receptor (IL-21R) and γC receptor to initiate an immune response, mainly through the protein tyrosine kinase JAK / signal transducer and transcription activator SATA pathway, activates JAK (JAK1 and JAK3), then phosphorylates STAT1, STAT3, STAT4 and STAT5, and finally enters the cell nucleus to regulate the expression of corresponding genes. IL-21 has multiple functions such as inducing the proliferation, differentiation and maturation of NK, NKT, and CD8+T cells, enhancing cytotoxicity and anti-tumor activity; inhibiting the proliferation and survival of Treg cells; enhancing macrophage phagocytosis; inducing B cell proliferation or apoptosis, plasma cell differentiation, and immunoglobulin secretion; inducing TFH and TH17 proliferation and differentiation; promoting the generation of memory stem T cells (TSCM); and activating members of the mitogen-activated protein kinase MAPK family. By analyzing the Cancer Genome Atlas database, it was found that the high expression of IL-21 in the tumor microenvironment of patients with skin melanoma and head and neck squamous cell carcinoma was positively correlated with their improved survival rate. Eleven days after tumor inoculation in MC38-cEGFR tumor-bearing mice, the same dose of cetuximab (Erb)-IL21 or Erb-IL2 fusion protein was injected, and it was found that Erb-IL21 had the same anti-tumor effect as Erb-IL2 but with lower toxicity. IL-21 also has significant clinical efficacy. A Phase I clinical trial of ZymoGenetics using intravenous IL-21 for the treatment of renal cell carcinoma (RCC) showed that the overall disease control rate (DCR) was 89% in 19 RCC patients. Another Phase II clinical trial of the company using intravenous IL-21 for the treatment of metastatic melanoma (MM) showed that the DCR was 62.5% in the 40 patients included in the trial.

[0004] Although cytokines have the advantages of strong physiological activity, low immunogenicity, and high efficacy, they have poor stability, short half-life, high clearance rate, and are easily degraded by the human body, which limits their development and application. Clinical studies have shown that the plasma half-life of IL-21 is short, only 0.61h in mice and only about 3.09 hours in humans. Therefore, modifying the molecular structure of cytokines is fundamental to changing their physicochemical properties and pharmacokinetic properties. The current methods mainly include chemical modifications such as PEG, fusion proteins with HSA / Fc / antibodies, and the construction of artificial disulfide bonds to improve their stability or extend their half-life. Junshi Biosciences fuses IL-21 with HSA nanoantibodies, and the serum half-life of a single dose of 0.15mg / kg IL-21-αHSA fusion protein in mice (t1 / 2=15.48h) is much greater than that of rhIL-21 (t1 / 2=0.61h). In addition, the Cmax and AUC of IL-21-αHSA are nearly 60 times and 300 times higher than those of rhIL-21, respectively. In cynomolgus monkeys, the half-life and exposure time of a single dose of 0.5 mg / kg IL-21-αHSA were significantly prolonged compared with rhIL-21, and its t1 / 2 and AUC were 10 times and 50 times that of rhIL-21, respectively. Amgen fused PD-1 monoclonal antibody with IL-21 and reduced the affinity of IL-21. In a mouse tumor model refractory to PD-1 monoclonal antibody monotherapy, anti-PD-1 monoclonal antibody and mutant IL-21 (reduced activity) fusion protein were administered, which had a more significant inhibitory effect on tumor growth than PD-1 monoclonal antibody and improved overall survival. And in cynomolgus monkeys, the half-life was extended to 41h. However, whether it is fused with HSA nanoantibodies or fused with PD-1 antibodies, the improvement in the half-life of IL-21 is still limited.

[0005] Analysis of the structure of IL-21 shows that IL-21 is mainly composed of a four-helix bundle structure, and its third helical segment (i.e., helix C) is observed to exist in two different and mutually convertible states. In one conformation, the helix C fragment presents a regular α-helix stable conformation, while in the other conformation, the fragment is largely disordered and unstable. By replacing the longer CD loop region of IL-21 with the shorter CD loop region of human IL-4, an L-21 / 4 chimeric protein was designed. This new IL-21 / 4 chimeric protein presents a single stable conformation, and the cell activity is increased by 10 times. Therefore, only by designing a more stable IL-21 structure from a structural point of view can the half-life of IL-21 be increased and its drugability be improved.

[0006] Improving the stability of proteins or cytokines by designing artificial disulfide bonds is a relatively effective method. For example, existing technologies can significantly improve the stability, yield, and Tagg value of IL-2 by introducing disulfide bonds into wild-type IL-2. There is also technology that improves the conformational stability of IL-15 by introducing a pair of disulfide bonds into the wild-type IL-15 molecule, significantly enhancing its affinity and biological activity for β and γ receptors and high production efficiency without relying on the sushi domain. Haike Molecular (Beijing) Technology Co., Ltd. stably displayed interleukin 21 (IL21) by using a mammalian cell surface protein stable display system controlled by protein conformation in vitro, and through a design based on protein structure analysis, the 16th ILE and 70th SER of wild-type IL21 were mutated to CYS, and a disulfide bond was formed between the two mutated CYS. Compared with IL21-Herceptin, the half-life of the 16c-IL21-Herceptin fusion protein with disulfide bond mutation increased by nearly 4 times, the Tm value increased to nearly 8°C, and retained biological activity.

[0007] In addition, IL-21 has a high affinity and is highly toxic. Amgen has improved safety by reducing the affinity of IL-21, and obtained a relatively good mutation combination R9E / R76A. Amgen fused IL-21 (R9E / R76A) with PD-1 antibodies to create the innovative drug Latikafusp (AMG256), which showed good safety and anti-tumor activity on tumors where PD-1 antibodies were ineffective, and has advanced to Phase I clinical trials.

[0008] However, it is now found that the functional activity of IL-21 is completely lost after combining 16c-IL21 with R9E / R76A, suggesting that it cannot be used as a drug molecular framework for IL-21R9E / R76A affinity-reduced mutant molecules. Therefore, it is necessary to develop more mutants with better adaptability for IL-21. Summary of the invention

[0009] Based on this, it is necessary to provide at least one IL-21 mutant, a fusion protein containing the same, a nucleic acid, a recombinant expression vector, a host cell, and a preparation method and use thereof.

[0010] In the first aspect of the present application, an interleukin 21 (IL-21) mutant is provided, which comprises one or more of the following mutation pairs compared to wild-type IL-21: mutation pair R5C and P79C; mutation pair H6C and T81C; mutation pair V24C and K105C; mutation pair L32C and A53C; mutation pair S57C and E64C, or S57C and I67C; mutation pair V28C and A58C; mutation pair 116C and K73C; and mutation pair D26C and N63C; wherein the amino acid sequence of the wild-type IL-21 comprises the sequence shown in SEQ ID NO: 42.

[0011] In a second aspect of the present application, a fusion protein comprising the IL-21 mutant as described in the first aspect is provided.

[0012] In the third aspect of the present application, a nucleic acid molecule encoding the IL-21 mutant described in the first aspect or the fusion protein described in the second aspect is provided.

[0013] In the fourth aspect of the present application, a recombinant expression vector is provided, which comprises the nucleic acid molecule as described in the second aspect.

[0014] In the fifth aspect of the present application, a host cell is provided, which expresses the IL-21 mutant as described in the first aspect or the fusion protein as described in the second aspect.

[0015] In the sixth aspect of the present application, a pharmaceutical composition is provided, comprising the IL-21 mutant as described in the first aspect or the fusion protein as described in the second aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0016] In the seventh aspect of the present application, a kit is provided, comprising one or more of the IL-21 mutant as described in the first aspect, the fusion protein as described in the second aspect, the nucleic acid molecule as described in the third aspect, the recombinant expression vector as described in the fourth aspect, the host cell as described in the fifth aspect, and the pharmaceutical composition as described in the sixth aspect, and a container.

[0017] In an eighth aspect of the present application, a method for preparing the IL-21 mutant as described in the first aspect or the fusion protein as described in the second aspect is provided.

[0018] In the ninth aspect of the present application, there is provided a use of the IL-21 mutant described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the recombinant expression vector described in the fourth aspect, or the host cell described in the fifth aspect in the preparation of an anti-tumor drug.

[0019] The present application designs artificial disulfide bonds for IL-21, and screens for disulfide bond mutant molecules that do not break during expression, have improved stability, and have improved half-life (for example, can be increased to 6 times the half-life of wild-type IL-21), while still retaining biological activity, greatly improving the drugability of IL-21. In addition, the above disulfide bond mutant molecules can be combined with IL-21 mutations R9E / R76A, and the combination still has biological activity, further improving drugability, and providing multiple stable IL-21 molecular skeletons for IL-21-based drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0021] Figure 1 In one embodiment of the present application, the molecular structures of Pembrolizumab fused to IL-21 wild type and Pembrolizumab fused to IL-21 disulfide bond mutant are shown, and IL-21v represents IL-21WT or IL-21 disulfide bond mutant molecule.

[0022] Figure 2 This is the reduced SDS-PAGE result of Pembrolizumab fused to IL-21WT and IL-21 disulfide bond mutant molecules in one embodiment of the present application.

[0023] Figure 3 This is the detection of the biological activity of the IL-21 signaling pathway of Pembrolizumab fused with IL-21WT and IL-21 disulfide bond mutant molecules in Baf3-stat3-IL21R-Luc cells in one embodiment of the present application.

[0024] Figure 4 In one embodiment of the present application, the molecular structures of Pembrolizumab fused to IL-21 wild type and Pembrolizumab fused to IL-21 disulfide bond mutant are shown, and IL-21v represents IL-21WT or IL-21 disulfide bond mutant molecule.

[0025] Figure 5This is the reduced SDS-PAGE result of Pembrolizumab fused to IL-21 disulfide bond and R9E / R76A combined mutant molecules in one embodiment of the present application.

[0026] Figure 6 This is the detection of the biological activity of the IL-21 signaling pathway in Baf3-stat3-IL21R-Luc cells of Pembrolizumab fused to IL-21 disulfide bond and R9E / R76A combination mutant molecules in one embodiment of the present application.

[0027] Figure 7 This is the detection of the biological activity of the IL-21 signaling pathway in Baf3-IL21R-hPD1-H03 cells of Pembrolizumab fused with IL-21 disulfide bond and R9E / R76A combination mutant molecules in one embodiment of the present application. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0030] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, which represent "one or more" in other ways, are also understood in the same way unless otherwise specified.

[0031] The "combinations thereof", "any combinations thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0032] In the present application, the “suitable” in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present application, solve the technical problems of the present application, and achieve the expected technical effects of the present application.

[0033] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent technical solutions are related in terms of the content covered, but should not be understood as limiting the previous technical solution, nor can they be understood as limiting the scope of protection of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0034] In this application, "optionally", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel solutions of "yes" or "no". If there are multiple "options" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application take "optionally include" as an example, which means "may include or not include".

[0035] The terms "comprising", "including" and "comprising" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions, timing, states, etc. for the occurrence of actions.

[0036] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0037] In the present application, exemplary descriptions such as "in some embodiments (or examples)", "in one embodiment (or example)", etc. may include but not be limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0038] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0039] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, including the two endpoint integers of the numerical range, and each integer between the two endpoints, is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0040] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0041] The inventors creatively designed a series of disulfide bonds for the IL-21 molecule to improve the stability and half-life of IL-21 and enhance its drugability. Compared with wild-type IL-21, the disulfide bonds of the present invention can make IL-21 not break during the expression process, improve thermal stability, improve half-life, and still retain biological activity. At the same time, the disulfide bonds of the present invention can also be combined with the IL-21 mutation R9E / R76A, and still have biological activity, providing multiple stable IL-21 molecular skeletons for IL-21-based drug development.

[0042] In a first aspect of the present application, there is provided an interleukin 21 (IL-21) mutant, which comprises one or more of the following mutation pairs compared to wild-type IL-21:

[0043] Mutation pair R5C and P79C (e.g. IL21-C2 in the table below);

[0044] Mutation pair H6C and T81C (e.g. IL21-C3 in the table below);

[0045] Mutation pair V24C and K105C (e.g. IL21-C4 in the table below);

[0046] Mutation pair L32C and A53C (e.g. IL21-C6 in the table below);

[0047] The mutation pair S57C and E64C (e.g., IL21-C12 in the table below), or the mutation pair S57C and I67C (e.g., IL21-C13 in the table below);

[0048] Mutation pair V28C and A58C (e.g. IL21-C15 in the table below);

[0049] Mutation pair I16C and K73C (eg, IL21-C17 in the table below); and,

[0050] Mutation pair D26C and N63C (e.g. IL21-C23 in the table below);

[0051] Wherein, the amino acid sequence of the wild-type IL-21 comprises the sequence shown in SEQ ID NO:42.

[0052] In some embodiments, the IL-21 mutant comprises one or more of the following mutation pairs compared to wild-type IL-21:

[0053] mutation pairs R5C and P79C;

[0054] mutation pairs H6C and T81C;

[0055] Mutation pairs V24C and K105C; and,

[0056] Mutation pairs S57C and E64C, or S57C and I67C.

[0057] Unless otherwise specified, the term "mutant" in the present application refers to a protein or fragment obtained by performing at least one or more of substitution, deletion and replacement on a wild-type protein or fragment, which retains at least part or all of the functions of the wild-type protein or fragment, for example, based on part or all of the functions of the wild-type protein or fragment, it also has other functions; or, after mutation, the function of the original wild-type protein or fragment is improved, etc.

[0058] In some embodiments, the IL-21 mutant further comprises substitutions R9E and R76A.

[0059] The second aspect of the present application provides a fusion protein comprising the IL-21 mutant as described in the first aspect.

[0060] The functional region to be fused with the IL-21 mutant may be, for example, an antibody or a mutant thereof.

[0061] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen. A natural complete antibody includes two heavy (H) chains and two light (L) chains. The heavy chains of mammals are classified as α, δ, ε, γ and μ, each heavy chain consisting of a variable region (VH) and a first constant region, a second constant region, a third constant region and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); the light chains of mammals are classified as λ or κ, and each light chain consists of a variable region (VL) and a constant region. The antibody is in a "Y" shape, wherein the stem of the Y-shaped structure consists of the second constant region and the third constant region of two heavy chains bound together by disulfide bonds. Each arm of the Y includes a variable region and a first constant region of a single heavy chain that is bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the two chains usually include three highly variable loops, called complementarity determining regions (CDRs) (the light chain CDRs include LCDR1, LCDR2 and LCDR3, and the heavy chain CDRs include HCDR1, HCDR2 and HCDR3).The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the Kabat, IMGT, Chothia or Al-Lazikani conventions (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989); Kabat E.A. et al., Sequences of Proteins of Immunological Significance. of immunological interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Chapter 26, 481-514, (2015)). The three CDRs are separated by side segments called framework regions (FRs) (light chain FRs include LFR1, LFR2, LFR3 and LFR4, and heavy chain FRs include HFR1, HFR2, HFR3 and HFR4), which are more highly conserved than CDRs and form a scaffold to support highly variable loops. The constant regions of heavy and light chains are not related to antigen binding, but exhibit a variety of effector functions. Antibodies can be divided into several categories based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, which are characterized by the presence of α, δ, ε, γ and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0062] The types of antibodies described in the present application are not limited, and may be, for example, anti-PD-1 antibodies or other antibodies that can act separately, jointly or synergistically with IL-21 or its mutants.

[0063] Wherein, the anti-PD-1 antibody includes but is not limited to Pembrolizumab (English name: Pembrolizumab) or its mutants.

[0064] In some embodiments, the anti-PD-1 antibody contained in the fusion protein comprises at least one Fab of pembrolizumab. In some embodiments, the anti-PD-1 antibody comprises one Fab. In some embodiments, the anti-PD-1 antibody comprises two Fabs. In some embodiments, the anti-PD-1 antibody further comprises an Fc. In some embodiments, the anti-PD-1 antibody may be a full-length antibody.

[0065] In some embodiments, the fusion protein comprises a mutant of the above-mentioned anti-PD-1 antibody.

[0066] Taking Pembrolizumab as an example, its mutation profile may include one or more of the following mutations: replacement of the heavy chain constant region with the IgG1 subtype, replacement of N297G, and deletion of the last amino acid lysine at the C-terminus.

[0067] The connection method (eg, position, use of linkers, etc.) of the IL-21 mutant and the antibody or its mutant can be a conventional connection method in the art.

[0068] In some embodiments, the antibody or mutant thereof is operably linked to the IL-21 mutant.

[0069] If not specifically stated, the term "operably linked" in the present application refers to the functional relationship between two regions of a fusion protein, ie, an antibody or a mutant thereof and an IL-21 mutant; wherein the two regions are linked to produce a fusion protein.

[0070] Exemplarily, the IL-21 mutant is linked to the C-terminus of the heavy chain of the anti-PD-1 antibody.

[0071] In some embodiments, the fusion protein comprises two identical heavy chains and two identical light chains. Further optionally, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 3, 4, 5, 7, 12-16, 18 or 24, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 26.

[0072] In some embodiments, the fusion protein comprises two different heavy chains, heavy chain 1 and heavy chain 2; and a light chain; further optionally, the amino acid sequence of heavy chain 1 is as shown in SEQ ID NO: 27, the amino acid sequence of heavy chain 2 is as shown in any one of SEQ ID NOs: 30 to 34 and 37 to 41, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 26.

[0073] In addition, the present application also provides functional variants of the present application fusion protein described in the present application within the scope of the present application. The term "functional variant" used in the present application refers to a recombinant protein, polypeptide or protein with a large amount of or significant sequence identity or similarity with the parent fusion protein, and the functional variant retains the biological activity of the fusion protein. Functional variants encompass, and the amino acid sequence of the functional variant and the amino acid sequence of the parent fusion protein may have, for example, at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher identity.

[0074] The functional variant may comprise, for example, an amino acid sequence of a parent fusion protein having at least one conservative amino acid substitution. Alternatively or additionally, the functional variant may comprise an amino acid sequence of a parent fusion protein having at least one non-conservative amino acid substitution. In this case, non-conservative amino acid substitutions that do not interfere with or inhibit the biological activity of the functional variant are preferred. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent fusion protein.

[0075] The amino acid substitutions of the fusion protein of the present application are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with certain physical and / or chemical properties is exchanged for another amino acid with the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain with another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Tip, Cys, Val, etc.), a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain with another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid with a β-branched side chain with another amino acid with a β-branched side chain (e.g., Ile, Thr and Val), an amino acid with an aromatic side chain with another amino acid with an aromatic side chain (e.g., His, Phe, Trp and Tyr), etc.

[0076] The fusion protein of the embodiment of the present application (including the functional part and functional variant of the present application) may include synthetic amino acids replacing one or more naturally occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxylphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2- carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0077] Mutants having a certain amino acid homology with the amino acid sequence of the fusion protein as described above, for example, a homology between 70% and 99%, a further homology between 80% and 99%, a further homology between 90% and 99%, and a homology of 99%, should also fall within the scope of protection of the present application.

[0078] "Homology" (percentage (%) of sequence identity) of an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percentage (%) of sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence to which it is compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. For example, publicly available tools such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., Journal of Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), Clustal W2 (available on the website of the European Bioinformatics Institute, see also Higgins et al., DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin M.A. et al., Bioinformatics (Cambridge, England), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software are used to determine the comparison of the percentage of amino acid (or nucleic acid) sequence identity. Those skilled in the art can use the default parameters provided by the tool or can appropriately customize the parameters according to the needs of the comparison, for example, by selecting a suitable algorithm.

[0079] As used herein, the term "amino acid" refers to an organic compound that includes amino (-NH2) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. Amino acid names are also represented in this disclosure as standard single-letter or three-letter codes, summarized below.

[0080]

[0081]

[0082] The third aspect of the present application provides a nucleic acid molecule encoding the IL-21 mutant or fusion protein of the present application.

[0083] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably throughout the text and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs generated using nucleotide analogs (e.g., peptide nucleic acids and non-natural nucleotide analogs), and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules herein comprise a continuous open reading frame encoding an antibody or fragment, derivative, mutein or variant thereof provided herein.

[0084] The fourth aspect of the present application provides a recombinant expression vector comprising the nucleic acid molecule as described in the present application.

[0085] As used herein, the term "vector" refers to a medium into which a genetic element can be operably inserted so that the genetic element is expressed, so that a protein, RNA or DNA encoded by the genetic element is produced or a genetic element is replicated. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1, etc.), bacteriophages (such as lambda phages or M13 phages, etc.) and animal viruses. The vector can include a variety of elements for controlling expression, including promoter sequences, transcription start sequences, enhancer sequences, selectable elements and reporter genes. In addition, the vector can include a replication origin. The vector can also include materials that assist it in entering the cell, including but not limited to, viral particles, liposomes or protein coatings. The vector can be an expression vector (such as a viral vector) or a cloning vector. The present disclosure provides a vector (e.g., an expression vector), which includes a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof provided in the present application, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0086] In a fifth aspect of the present application, a host cell is provided, which expresses the IL-21 mutant or fusion protein of the present application.

[0087] The term "cell" or "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells.

[0088] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of passages. It should also be understood that all progeny may not be exactly identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, this is clear from the context.

[0089] In a sixth aspect of the present application, a pharmaceutical composition is provided, comprising the IL-21 mutant or fusion protein of the present application, and a pharmaceutically acceptable carrier and / or excipient.

[0090] The term "pharmaceutically acceptable" means that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0091] As used in this application, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, preservatives, stabilizers, and the like. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0092] In a seventh aspect of the present application, a kit is provided, comprising one or more of the IL-21 mutant, fusion protein, nucleic acid molecule, recombinant expression vector, host cell and pharmaceutical composition in the present application, and a container.

[0093] In an eighth aspect of the present application, a method for preparing the IL-21 mutant or fusion protein as described above is provided, comprising culturing the host cells of the present application to obtain a culture medium; and isolating the IL-21 mutant or the fusion protein from the culture medium.

[0094] The ninth aspect of the present application provides use of the IL-21 mutant, fusion protein, nucleic acid molecule, recombinant expression vector or host cell of the present application in the preparation of an anti-tumor drug.

[0095] The present application also provides a method for treating tumor-related diseases, comprising administering to a subject an effective dose of one or more of the IL-21 mutants and fusion proteins described above.

[0096] "Administering," "giving," and "treating," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, immunogenic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a binding composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications. Some examples are provided below.

[0097] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0098] Example 1: Design of IL-21 disulfide bond mutations

[0099] According to the published IL-21 tertiary structure (PDB: 3TGX), artificial intelligence and computer-aided design methods were used to select sites with bond angles and bond lengths suitable for forming disulfide bonds for disulfide bond design. The designed disulfide bonds are shown in Table 1.

[0100] Table 1 Disulfide bond mutation sites of IL-21 wild type and IL-21

[0101]

[0102]

[0103] Example 2: Preparation of Pembrolizumab Fusion IL-21WT and IL-21 Disulfide Mutant Molecules

[0104] In order to better evaluate the stability of IL-21 wild-type and IL-21 disulfide mutant molecules, Pembrolizumab fused IL-21WT and IL-21 disulfide mutant molecules were constructed. Figure 1As shown, the sequence is shown in Table 2. The IL21-WT or IL-21 disulfide bond mutant molecule is fused to the C-terminus of the Pembrolizumab antibody heavy chain (the Pembrolizumab antibody heavy chain constant region is replaced with the IgG1 subtype N297G mutation, and the last amino acid K at the C-terminus is removed) and cloned into the expression vector pCDNA3.4. The Pembrolizumab antibody light chain is cloned into the expression vector pCDNA3.4. The above-mentioned IL21-WT or IL-21 disulfide bond mutant molecule and the Pembrolizumab antibody heavy chain fusion protein expression plasmid are co-transfected with the Pembrolizumab antibody light chain expression plasmid into ExpiCHO-S cells for expression, and the supernatant is collected and purified by Protein A to obtain the candidate antibody protein.

[0105] Table 2 Molecular sequences of pembrolizumab fused to IL-21 wild type and pembrolizumab fused to IL-21 disulfide bond mutant

[0106] name IL-21 disulfide bonds Heavy chain SEQ ID NO Light chain SEQ ID NO 2v2-WT N / A 1 26 2v2-C1 116C, S70C 2 26 2v2-C2 R5C,P79C 3 26 2v2-C3 H6C, T81C 4 26 2v2-C4 V24C, K105C 5 26 2v2-C5 E43C, M118C 6 26 2v2-C6 L32C, A53C 7 26 2v2-C7 V17C, L108C 8 26 2v2-C8 114C, L115C 9 26 2v2-C9 M10C, I119C 10 26 2v2-C10 F50C, F111C 11 26 2v2-C11 Q51C, K75C 12 26 2v2-C12 S57C, E64C 13 26 2v2-C13 S57C, I67C 14 26 2v2-C14 S45C, R90C 15 26 2v2-C15 V28C, A58C 16 26 2v2-C16 I14C, K112C 17 26 2v2-C17 I16C, K73C 18 26 2v2-C18 116C, L74C 19 26 2v2-C19 Q19C, S70C 20 26 2v2-C20 L20C, S70C 21 26 2v2-C21 Y23C, N63C 22 26 2v2-C22 D26C, A58C 23 26 2v2-C23 D26C, N63C 24 26 2v2-C24 L27C, P104C 25 26

[0107] Example 3: Reducing SDS-PAGE analysis of pembrolizumab fused to IL-21WT and IL-21 disulfide bond mutant molecules

[0108] The Pembrolizumab fused IL-21WT and IL-21 disulfide bond mutant molecules prepared in Example 2 were subjected to reduced SDS-PAGE detection. The results are as follows: Figure 2 As shown. The SDS-PAGE results show that the heavy chain fusion IL-21WT peptide chain of the 2v2-WT molecule breaks during the electrophoresis due to instability, forming broken fragments. However, the 2v2-C1 heavy chain fusion IL-21 peptide segment with the I16C, S70C mutation described in patent CN111205361B has basically no breakage, indicating that increasing the I16C-S70C disulfide bond can improve the stability of IL-21. 2v2-C2, 2v2-C3, 2v2-C4, 2v2-C6, 2v2-C11, 2v2-C12, 2v2-C13, 2v2-C14, 2v2-C15, 2v2-C17, and 2v2-C23 developed in this patent can well improve the breakage of the heavy chain fusion IL-21 peptide segment, indicating that increasing the corresponding disulfide bonds of the above molecules can increase the stability of the IL-21 molecule.

[0109] Example 4: Detection of biological activity of IL-21 signaling pathway of pembrolizumab fused to IL-21WT and IL-21 disulfide bond mutant molecules

[0110] The reporter gene method was used to detect the activation of STAT3 signaling pathway activity in Baf3-stat3-IL21R-Luc cells by Pembrolizumab fused to IL-21WT and IL-21 disulfide bond mutant molecules. The Baf3-stat3-IL21R-Luc cells (H_IL-21Reporter Cell Line, Yoshiman, cat: GM-C15762) in this experiment are Baf3 cells that stably express human IL21R and STAT3-induced luciferase reporter genes. Specific operation: Take the H_IL-21Reporter Cell Line cells in the logarithmic growth phase, centrifuge, resuspend and count with analysis buffer (1640+1% FBS+I% PS), adjust the cell density to 2E6 / ml, add to different positions of 96-well plate, 50μl / well, and incubate at 37°C, 5% CO2 for 18-20h; take Pembrolizumab fused IL-21WT and IL-21 disulfide bond mutant molecules, dilute them with analysis buffer to different concentrations (2*1000nM starting concentration, 5-fold gradient dilution, 10 concentration points, add to 96-well plate, 50μl / well; mix with a microplate shaker or a spray gun, and incubate the 96-well plate at 37°C, 5% CO2 for 9h; add Luciferase Assay System (Vazyme), 80μl / well; incubate at room temperature for 5-10min, and analyze on a multifunctional microplate reader (MD, SpectraMax The final antibody concentration was used as the abscissa and the detected LUM signal value was used as the ordinate for nonlinear fitting to calculate the EC50 value.

[0111] The experimental results are as follows Figure 3 As shown, 2v2-C2, 2v2-C3, 2v2-C4, 2v2-C6, 2v2-C11, 2v2-C12, 2v2-C13, 2v2-C14, 2v2-C15, 2v2-C17, 2v2-C23 developed in this application all have the same IL-21 signaling pathway biological activity as before the transformation. This shows that the increase in disulfide bond mutations does not have a significant effect on the biological function of IL-21. The following exemplary selection of 2v2-C3, 2v2-C4, 2v2-C6, 2v2-C11, 2v2-C12, 2v2-C13, 2v2-C14, 2v2-C15, 2v2-C17 and 2v2-C23 for subsequent experiments.

[0112] Example 5: Disulfide bond detection of pembrolizumab fused to IL-21WT and IL-21 disulfide bond mutant molecules

[0113] The collision-induced dissociation mode of tandem mass spectrometry was used to break the peptide backbone while retaining the disulfide bonds. The disulfide bonds were located by identifying characteristic fragments and analyzing the product ions.

[0114] Take 200μg of the sample solution to be tested, add 150μL 8M guanidine hydrochloride (Thermo, product number 24115), 10μL 1MTris-HCl PH7 (Invitrogen, product number AM9851), 2μL 200mM NEM (N-ethylmaleimide, Sigma, product number E3876-5G), and make up to 200μL with ultrapure water if less than 200μL. React at 37℃ for 1h. Divide the sample into two portions (100μL each), add 5μL 0.5M DTT (Sigma, product number D0632-10G) solution to one portion, and add 5μL ultrapure water to the other portion, and incubate at 60℃ for 30min. Add 10μL 0.5MIAM (Sigma, product number I1149-5G) solution to both samples at room temperature and avoid light for 30min. The sample was replaced into 20mM Tris-HCl PH7 solution using a desalting column (Thermo, item number 89882). Add 5μg enzyme (1:20) at 37°C for about 4h. Depending on the specific sequence, another enzyme can be selected for combined digestion. If combined digestion is required, the above sample can be reacted at 95°C for 5min, and after cooling, another enzyme (1:50) is added and digested at 37°C overnight. 2μL 10% FA (Fisher, item number A117) solution was added to each solution to terminate the digestion reaction. After high-speed centrifugation, the supernatant was taken for LC-MS detection. Use the instrument's own software BioPharma Finder to search for disulfide bonds, or manually extract the reduced and non-reduced sample spectra based on the theoretical molecular weight to determine the disulfide bond formation. The results are shown in Table 3.

[0115] Table 3 Mass spectrometry disulfide bond detection of pembrolizumab fused to IL-21WT and IL-21 disulfide bond mutant molecules

[0116]

[0117]

[0118] Example 6: Preparation of Pembrolizumab Fusion IL-21WT and IL-21 Disulfide Mutant Molecules

[0119] In order to better evaluate the in vivo PK of IL-21 wild-type and IL-21 disulfide mutant molecules, Pembrolizumab fusion IL-21WT and IL-21 disulfide mutant molecules were constructed. Figure 4As shown, the sequence is shown in Table 4. IL21-WT or IL-21 disulfide bond mutant molecules were fused to the C-terminus of human IgG1-Fc and cloned into the expression vector pCDNA3.4. The light chain and heavy chain of the Pembrolizumab antibody were cloned into the expression vector pCDNA3.4 respectively. Knobs-into-holes were constructed between the two heavy chains to prevent mispairing. The above expression plasmids were co-transfected into ExpiCHO-S cells for expression (Note 1v1-WT was expressed using Expi293-F, otherwise it would break easily), the supernatant was collected, and the candidate antibody protein was obtained by ProteinA and ion exchange purification. It should be noted that, in view of the fact that the disulfide bonds introduced into the mutants IL21-C3, IL21-C12 and IL21-C13 are similar in spatial structure to several other mutants, the following exemplifies the fusion proteins composed of Pembrolizumab and several other mutants and their effects, and on this basis, combined with the R9E / R76A combined mutant molecules.

[0120] Table 4 Pembrolizumab fused with IL-21WT and IL-21 disulfide bond mutant molecular sequences

[0121]

[0122]

[0123] Example 7: PK testing of pembrolizumab fused to IL-21WT and IL-21 disulfide bond mutant molecules

[0124] 42 male C57BL mice with uniform body weight of 20-24g and age of 7-9 weeks were selected and randomly divided into 14 subgroups according to body weight, with 3 mice in each subgroup, for administration of different candidate molecules, and 2 subgroups for each test substance for cross-blood collection. The dosage of each candidate molecule was unified at 10mg / kg, the administration volume was 10mL / kg, the administration concentration was 1mg / mL, and the administration was administered by tail vein injection, and the administration was single. Blood was collected from each group of animals at 0.5h, 6h, 24h, 72h, 144h, 240h, 360h, and 480h after administration. Blood was collected alternately from 4 animals in each group. After blood collection, the centrifuge tube was placed in a centrifuge tube, which was stored at room temperature before blood collection and blood separation. Serum was separated within 2h after blood collection and centrifuged at 4000g for 10min at room temperature. After centrifugation, the collected serum samples were frozen at -80℃. After blood collection, the drug concentration in the serum sample was detected, and the main metabolic kinetic parameters such as t1 / 2, Cmax, and AUC were calculated.

[0125] The metabolic kinetic parameters are shown in Table 5. Compared with 1v1-WT, the in vivo exposure and half-life of the samples with increased disulfide bonds were significantly increased. Among them, the half-life of 1v1-C1 (with patent CN111205361B disulfide bond) was increased to 4 times that of 1v1-WT, the half-life of 1v1-C4, 1v1-C6, 1v1-C15, and 1v1-C23 was increased to 1.7-2.26 times that of 1v1-WT, and the half-life of 1v1-C17 was increased to 6.0 times that of 1v1-WT, and the AUC (0-t) was significantly better than other molecules.

[0126] Table 5 Metabolic kinetic parameters of pembrolizumab fused to IL-21WT and IL-21 disulfide bond mutant molecules

[0127] Pharmacokinetic parameters unit 1v1C-WT 1v1C-C1 1v1C-C4 1v1C-C6 1v1C-C15 1v1C-C17 1v1C-C23 AUC(0-t) mg / L*h 459.48 625.81 664.0 628.1 694.7 2390.2 781.3 t1 / 2z h 18.9 75.6 35.2 32.6 32.1 113.4 42.7 Tmax h 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Cmax mg / L 21.81 18.84 28.58 27.43 27.31 76.83 33.82

[0128] Example 7: Preparation of Pembrolizumab Fusion IL-21 Disulfide Bond and R9E / R76A Combination Mutant Molecules

[0129] The IL21-WT or IL-21 disulfide bond mutant molecules were added with R9E and R76A mutations on the IL-21 protein, respectively, fused to the C-terminus of the Pembrolizumab antibody heavy chain (replacing the Pembrolizumab antibody heavy chain constant region with the IgG1 subtype N297G mutation, and removing the last amino acid K at the C-terminus), and cloned into the expression vector pCDNA3.4. The Pembrolizumab antibody light chain was cloned into the expression vector pCDNA3.4. The above mutant molecules and the Pembrolizumab antibody heavy chain fusion protein expression plasmid were co-transfected with the Pembrolizumab antibody light chain expression plasmid to express ExpiCHO-S cells, and the supernatant was collected and purified by Protein A and ion exchange to obtain the candidate antibody protein.

[0130] Table 6 Pembrolizumab fused to IL-21 disulfide bond and R9E / R76A combined mutation molecular sequences

[0131]

[0132]

[0133] Example 8: Stability analysis of pembrolizumab fused to IL-21 disulfide bond and R9E / R76A combined mutant molecules

[0134] The Pembrolizumab fused to IL-21 disulfide bond and R9E / R76A combined mutant molecules prepared in Example 7 were subjected to reduced SDS-PAGE detection. The results are as follows: Figure 5 As shown. The SDS-PAGE results show that the heavy chain fusion IL-21 (R9E, R76A) peptide chain of the 1v1-WT-256 molecule breaks during electrophoresis due to instability, forming broken fragments. However, the 1v1-C1-256 heavy chain fusion IL-21 peptide segment with the I16C, S70C mutations described in patent CN111205361B has basically no breakage, indicating that increasing the 116C-S70C disulfide bond can improve the stability of IL-21. Other molecules developed in this patent can also improve the breakage of the heavy chain fusion IL-21 peptide segment, indicating that increasing the corresponding disulfide bonds in the above molecules can increase the stability of the IL-21 molecule.

[0135] Example 9: Analysis of biological activity of pembrolizumab fused to IL-21 disulfide bond and R9E / R76A combined mutant molecules

[0136] The reporter gene method was used to detect the STAT3 signaling pathway activity of Baf3-stat3-IL21R-Luc cells activated by Pembrolizumab fused to IL-21 disulfide bonds and R9E / R76A combined mutant molecules in PD-1+ and PD-1-. The PD-1+ Baf3-stat3-IL21R-Luc cells (Baf3-IL21R-hPD1-H03) in this experiment are Baf3 cells that stably express human PD-1 receptor, IL21R and STAT3-induced luciferase reporter genes, and the PD-1- Baf3-stat3-IL21R-Luc cells (H_IL-21Reporter Cell Line, Yoshiman, cat: GM-C15762) in this experiment are Baf3 cells that stably express human IL21R and STAT3-induced luciferase reporter genes. Specific operation: Take Baf3-IL21R-hPD1-H03 or H_IL-21Reporter Cell Line cells in logarithmic growth phase, centrifuge, resuspend and count with analysis buffer (1640+1% FBS+1% PS), adjust the cell density to 2E6 / ml, add to different positions of 96-well plate, 50μl / well, incubate at 37℃, 5% CO2 for 18-20h; take Pembrolizumab fused to IL-21 disulfide bond and R9E / R76A combination mutant molecules, dilute to different concentrations with analysis buffer (2*1000nM starting concentration, 5-fold gradient dilution, 10 concentration points, add to 96-well plate, 50μl / well; mix with microplate shaker or spray gun, and incubate the 96-well plate at 37℃, 5% CO2 for 9h; add LuciferaseAssay System (Vazyme), 80 μl / well; incubate at room temperature for 5-10 min, and detect the LUM signal value on a multifunctional microplate reader (MD, SpectraMaxi3X). Use the final antibody concentration as the abscissa and the detected LUM signal value as the ordinate for nonlinear fitting to calculate the EC50 value.

[0137] The results of IL-21 biological activity test of PD-1 antibody and IL-21 mutant fusion protein are as follows Figure 6 and Figure 7As shown. On PD-1 negative cells, all molecules showed low activity. On PD-1 positive cells, the 1v1-WT-256 molecule broke due to IL-21 instability and had no IL-21 signaling pathway activation activity. The molecule 1v1-C1-256 using the disulfide bond of patent CN111205361B also had no IL-21 signaling pathway activation activity on PD-1 positive cells, which indicates that although the disulfide bond combined with the IL-21 mutation R9E / R76A of Amgen AMG256 can improve stability, it leads to the loss of original biological activity and cannot be used as a stable skeleton for IL-21-based drug development. After the disulfide bond developed by this patent is combined with the IL-21 mutation R9E / R76A of Amgen AMG256, it can not only improve stability on PD-1 positive cells, but most of them still retain the IL-21 signaling pathway activation activity, which can provide multiple stable IL-21 molecular skeletons for IL-21-based drug development.

[0138] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0139] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. An interleukin 21 (IL-21) mutant, characterized in that Compared to wild-type IL-21, it comprises one or more of the following mutation pairs: mutation pairs R5C and P79C; mutation pairs H6C and T81C; Mutation pairs V24C and K105C; and, mutation pairs S57C and E64C, or S57C and I67C; Wherein, the amino acid sequence of the wild-type IL-21 comprises the sequence shown in SEQ ID NO:

42.

2. The IL-21 mutant according to claim 1, characterized in that It also includes replacements for R9E and R76A.

3. A fusion protein, characterized in that It comprises the IL-21 mutant according to claim 1 or 2.

4. The fusion protein according to claim 3, characterized in that The fusion protein comprises an antibody or a mutant thereof, and the IL-21 mutant according to claim 1 or 2; Optionally, the antibody comprises an anti-PD-1 antibody; Further optionally, the anti-PD-1 antibody comprises at least one Fab of pembrolizumab or a mutant thereof; wherein, the mutant of pembrolizumab optionally comprises one or more of the following mutations compared to pembrolizumab: replacement of the heavy chain constant region with an IgG1 subtype, replacement of N297G, and deletion of the last amino acid lysine at the C-terminus.

5. The fusion protein according to claim 3 or 4, characterized in that The IL-21 mutant is linked to the C-terminus of the heavy chain of the anti-PD-1 antibody; Optionally, the fusion protein comprises two identical heavy chains and two identical light chains, and further optionally, the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 3, 4, 5 and 12 to 15, and the amino acid sequence of the light chain is shown in SEQ ID NO: 26; Optionally, the fusion protein comprises two different heavy chains, heavy chain 1 and heavy chain 2; and a light chain; further optionally, the amino acid sequence of heavy chain 1 is as shown in SEQ ID NO: 27, the amino acid sequence of heavy chain 2 is as shown in SEQ ID NO: 30 or 37, and the amino acid sequence of the light chain is as shown in SEQ ID NO:

26.

6. A nucleic acid molecule, characterized in that It encodes the IL-21 mutant according to claim 1 or 2 or the fusion protein according to any one of claims 3 to 5.

7. A recombinant expression vector, characterized in that: It comprises the nucleic acid molecule according to claim 6; Optionally, the recombinant expression vector comprises a viral vector.

8. A host cell, characterized in that It expresses the IL-21 mutant according to claim 1 or 2 or the fusion protein according to any one of claims 3 to 5.

9. A pharmaceutical composition, characterized in that It comprises the IL-21 mutant according to claim 1 or 2 or the fusion protein according to any one of claims 3 to 5, and a pharmaceutically acceptable carrier and / or excipient.

10. A kit comprising one or more of the IL-21 mutant according to claim 1 or 2, the fusion protein according to any one of claims 3 to 5, the nucleic acid molecule according to claim 6, the recombinant expression vector according to claim 7, the host cell according to claim 8 and the pharmaceutical composition according to claim 9, and a container.

11. A method for preparing the IL-21 mutant according to claim 1 or 2 or the fusion protein according to any one of claims 3 to 5, characterized in that: The method comprises: Cultivating the host cell according to claim 8 to obtain a culture fluid; and, The IL-21 mutant or the fusion protein is separated from the culture medium.

12. Use of the IL-21 mutant according to claim 1 or 2, the fusion protein according to any one of claims 3 to 5, the nucleic acid molecule according to claim 6, the recombinant expression vector according to claim 7 or the host cell according to claim 8 in the preparation of an anti-tumor drug.

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