IL-21 mutants, fusion proteins containing the same, nucleic acids, recombinant expression vectors, host cells, and methods of making and using the same

Abstract

The present application discloses an 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. The IL-21 mutant comprises mutation pairs D26C and N63C compared with wild-type IL-21. The above mutation pairs can be combined with IL-21 mutation R9E / R76A, and the combination still has biological activity, further improving the drugability of the finally obtained mutant.

Description

Technical Field

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

[0002] Cytokines are molecular messengers for intercellular communication in the immune system and play a crucial role in tumor immunity. Currently marketed 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 colony-stimulating factor injection (IFN). In addition, various other cytokines, especially IL-2 family-related cytokines such as IL-2, IL-7, IL-15, and IL-21, are undergoing clinical trials to evaluate their anti-tumor potential.

[0003] Interleukin-21 (IL-21) is an immune activating factor, belonging to the IL-2 family, and is a four-helix cytokine. IL-21 is mainly produced by activated CD4+. + T cells, NK cells, TFH cells, and Th17 cells secrete IL-21, enhancing the antigen-specific response of immune cells. IL-21 can promote CD8+. +T cells and NK cells play a crucial role in the anti-tumor activity of B cells and germinal cell development, making them potential targets for developing tumor immunotherapy. IL-21, after binding to its receptor (IL-21R) and γC receptor, initiates an immune response, primarily through the protein tyrosine kinase JAK / signal transduction and transcriptional agonist SATA pathways. It activates JAKs (JAK1 and JAK3), subsequently phosphorylating STAT1, STAT3, STAT4, and STAT5, and finally enters the nucleus to regulate the expression of corresponding genes. IL-21 has multiple functions, including inducing the proliferation, differentiation, and maturation of NK, NKT, and CD8+ T cells, enhancing cytotoxicity and anti-tumor activity; inhibiting Treg cell proliferation and survival; 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 mitogen-activated protein kinase MAPK family members. Analysis of the Cancer Genome Atlas database revealed a positive correlation between high IL-21 expression in the tumor microenvironment of patients with cutaneous melanoma and head and neck squamous cell carcinoma and improved survival rates. In MC38-cEGFR tumor-bearing mice, injection of the same dose of cetuximab (Erb)-IL21 or Erb-IL2 fusion protein 11 days after tumor inoculation showed that Erb-IL21 had the same anti-tumor activity as Erb-IL2 but with lower toxicity. IL-21 also demonstrates significant clinical efficacy. A phase I clinical trial by ZymoGenetics using intravenous IL-21 to treat renal cell carcinoma (RCC) showed an overall disease control rate (DCR) of 89% in 19 RCC patients. Another phase II clinical trial by the same company using intravenous IL-21 to treat metastatic melanoma (MM) showed a DCR of 62.5% in 40 enrolled patients.

[0004] Although cytokines possess advantages such as strong physiological activity, low immunogenicity, and high efficacy, their poor stability, short half-life, high clearance rate, and easy degradation by the human body limit their development and application. Clinical studies have shown that IL-21 has a short plasma half-life, only 0.61 hours in mice and approximately 3.09 hours in humans. Therefore, modifying the molecular structure of cytokines is fundamental to altering their physicochemical and pharmacokinetic properties. Current methods mainly involve chemical modification such as PEG, forming fusion proteins with HSA / Fc / antibodies, and constructing artificial disulfide bonds to improve stability or prolong half-life. Junshi Biosciences, through the fusion of IL-21 with HSA nanobodies, demonstrated that a single dose of 0.15 mg / kg IL-21-αHSA fusion protein in mice resulted in a serum half-life (t1 / 2 = 15.48 h) significantly longer than that of rhIL-21 (t1 / 2 = 0.61 h). Furthermore, the Cmax and AUC of IL-21-αHSA were nearly 60-fold and 300-fold higher than those of rhIL-21, respectively. In cynomolgus monkeys, compared with rhIL-21, a single dose of 0.5 mg / kg IL-21-αHSA significantly prolonged the half-life and exposure time, with its t1 / 2 and AUC being 10-fold and 50-fold higher, respectively. Amgen fused PD-1 monoclonal antibodies with IL-21 while reducing the affinity for IL-21. In a mouse model of PD-1 monoclonal antibody-refractory tumors, administration of the anti-PD-1 monoclonal antibody fusion protein with mutant IL-21 (reduced activity) showed a more significant inhibitory effect on tumor growth and improved overall survival compared to PD-1 monoclonal antibody. Furthermore, in cynomolgus monkeys, the half-life was extended to 41 hours. However, regardless of whether fused with HSA nanobodies or PD-1 antibodies, the improvement in the half-life of IL-21 remained limited.

[0005] Structural analysis of IL-21 revealed that it is primarily composed of a four-helix bundle, with the third helical segment (helix C) exhibiting two distinct and interconvertible states. In one conformation, the helix C segment presents a stable, regular α-helix conformation, while in the other, it is largely a disordered and unstable conformation. An L-21 / 4 chimeric protein was designed by replacing the longer CD loop region of IL-21 with the shorter CD loop region of human IL-4. This novel IL-21 / 4 chimeric protein exhibited a single, stable conformation and showed a 10-fold increase in cellular activity. Therefore, only by structurally designing a more stable IL-21 structure can its half-life be improved, thereby enhancing its drug-like properties.

[0006] Improving the stability of proteins or cytokines by designing artificial disulfide bonds is a relatively effective method. For example, existing techniques have shown that introducing disulfide bonds into wild-type IL-2 can significantly improve the stability, yield, and Tagg value of IL-2. Other techniques have improved the conformational stability of IL-15 by introducing a pair of disulfide bonds into the wild-type IL-15 molecule, significantly enhancing its affinity for β and γ receptors, biological activity, and high production efficiency without relying on the sushi domain. Haike Molecular (Beijing) Technology Co., Ltd. has achieved stable display of interleukin-21 (IL21) using a mammalian cell surface protein stability display system with in vitro controlled protein conformation. Through protein structure analysis-based design, CYS mutations were created at positions 16 (ILE) and 70 (SER) in wild-type IL21, forming a disulfide bond between the two mutated CYS molecules. Compared to IL21-Herceptin, the 16c-IL21-Herceptin fusion protein with the disulfide bond mutation showed a nearly four-fold increase in half-life, a Tm value of nearly 8°C, and retained biological activity.

[0007] Furthermore, IL-21 has a high affinity and significant toxicity. Amgen improved safety by reducing the affinity of IL-21, obtaining a favorable mutant combination of R9E / R76A. Amgen fused IL-21 (R9E / R76A) with a PD-1 antibody to create the innovative drug Latikafusp (AMG256), which demonstrated good safety and anti-tumor activity in tumors unresponsive to PD-1 antibodies and has advanced to Phase I clinical trials.

[0008] However, it has been found that combining 16c-IL21 with R9E / R76A completely eliminates the functional activity of IL-21, suggesting that it cannot be used as a drug molecule scaffold for IL-21 R9E / R76A-reduced affinity mutants. Therefore, it is necessary to develop more mutants with better compatibility targeting IL-21. Summary of the Invention

[0009] Therefore, it is necessary to provide at least one IL-21 mutant, a fusion protein containing it, a nucleic acid, a recombinant expression vector, a host cell, and its preparation method and uses.

[0010] In a first aspect of this application, an interleukin-21 (IL-21) mutant is provided, which, compared to wild-type IL-21, comprises one or more of the following mutation pairs: 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 I16C 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 this application, a fusion protein comprising the IL-21 mutant as described in the first aspect is provided.

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

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

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

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

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

[0017] In an eighth aspect of this 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 a ninth aspect of this application, there is provided the use 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, or the host cell as described in the fifth aspect in the preparation of a drug for antitumor purposes.

[0019] This application utilizes artificial disulfide bond design in IL-21 to screen for disulfide bond mutant molecules that exhibit no breakage during expression, enhanced stability, and an increased half-life (e.g., up to 6 times the half-life of wild-type IL-21), while retaining biological activity, thus significantly improving the druggability of IL-21. Furthermore, these disulfide bond mutant molecules can be combined with the IL-21 mutant R9E / R76A, and the combined combination retains biological activity, further enhancing druggability and providing multiple stable IL-21 molecular backbones for IL-21-based drug development. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. 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 illustrating this application.

[0021] Figure 1 The present invention relates to the molecular structures of Pembrolizumab fused with wild-type IL-21 and Pembrolizumab fused with IL-21 disulfide bond mutant in one embodiment of the present application, where IL-21v represents IL-21WT or IL-21 disulfide bond mutant molecule.

[0022] Figure 2 The SDS-PAGE results of the reduction of Pembrolizumab fused with IL-21WT and IL-21 disulfide bond mutant molecules in one embodiment of this application are shown.

[0023] Figure 3 This study aims to detect the biological activity of Pembrolizumab fused with IL-21WT and IL-21 disulfide mutant molecules in the IL-21 signaling pathway in Baf3-stat3-IL21R-Luc cells, as described in one embodiment of this application.

[0024] Figure 4 The present invention relates to the molecular structures of Pembrolizumab fused with wild-type IL-21 and Pembrolizumab fused with IL-21 disulfide bond mutant in one embodiment of the present application, where IL-21v represents IL-21WT or IL-21 disulfide bond mutant molecule.

[0025] Figure 5The image shows the reduced SDS-PAGE results of Pembrolizumab fused with IL-21 disulfide bonds and the R9E / R76A combined mutant molecule in one embodiment of this application.

[0026] Figure 6 This study aims to detect the biological activity of Pembrolizumab fused with an IL-21 disulfide bond and the R9E / R76A combined mutant molecule in the IL-21 signaling pathway of Baf3-stat3-IL21R-Luc cells, as described in one embodiment of this application.

[0027] Figure 7 This study aims to detect the biological activity of Pembrolizumab fused with IL-21 disulfide bonds and the R9E / R76A combined mutant molecule in the IL-21 signaling pathway of Baf3-IL21R-hPD1-H03 cells in one embodiment of this application. Detailed Implementation

[0028] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated 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 other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

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

[0032] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0033] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates 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," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0035] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0036] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0037] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

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

[0039] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every 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 ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0040] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0041] The inventors have creatively designed a series of disulfide bonds in the IL-21 molecule to improve its stability, half-life, and druggability. Compared to wild-type IL-21, the disulfide bonds of this invention enable IL-21 to remain intact during expression, exhibit improved thermal stability and a longer half-life, while retaining its biological activity. Furthermore, the disulfide bonds of this invention can also combine with the IL-21 mutant R9E / R76A without retaining biological activity, providing multiple stable IL-21 molecular backbones for IL-21-based drug development.

[0042] A first aspect of this application provides an interleukin-21 (IL-21) mutant, which, compared to wild-type IL-21, comprises one or more of the following mutation pairs:

[0043] Mutations against R5C and P79C (e.g., IL21-C2 in the table below);

[0044] Mutations against H6C and T81C (e.g., IL21-C3 in the table below);

[0045] Mutations against V24C and K105C (e.g., IL21-C4 in the table below);

[0046] Mutations against L32C and A53C (e.g., IL21-C6 in the table below);

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

[0048] Mutations against V28C and A58C (e.g., IL21-C15 in the table below);

[0049] Mutations against I16C and K73C (e.g., IL21-C17 in the table below); and,

[0050] Mutations against D26C and N63C (e.g., IL21-C23 in the table below);

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

[0052] Unless otherwise specified, "mutant" in this application refers to a protein or fragment obtained by at least one or more of substitution, deletion, and replacement of a wild-type protein or fragment, which retains at least some or all of the functions of the wild-type protein or fragment, such as having other functions based on some or all of the functions of the wild-type protein or fragment; or, after mutation, the function of the original wild-type protein or fragment is improved.

[0053] In some embodiments, the IL-21 mutant also includes replacements for R9E and R76A.

[0054] A second aspect of this application provides a fusion protein comprising the IL-21 mutant as described in the first aspect.

[0055] The functional region used for fusion with the IL-21 mutant may be, for example, an antibody or a mutant thereof.

[0056] 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 consists of two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, each consisting of a variable region (VH) and a first, second, third, and optionally fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are classified as λ or κ, each consisting of a variable region (VL) and a constant region. Antibodies are Y-shaped, with the stem of the Y-structure consisting of the second and third constant regions of two heavy chains linked together by disulfide bonds. Each arm of the Y includes a variable region and a first constant region of a single heavy chain that binds to the variable and constant regions of a single light chain. The variable regions of both the light and heavy chains are responsible for antigen binding. The variable regions of two chains typically include three highly variable rings, called complementarity-determining regions (CDRs) (the CDRs for light chains include LCDR1, LCDR2, and LCDR3, and the CDRs for heavy chains include HCDR1, HCDR2, and HCDR3).The CDR boundaries of antibody-antigen binding fragments disclosed in this article can be defined or identified according to the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J.Mol.Biol., 273(4), 927 (1997); Chothia, C. et al., J.Mol.Biol., 186(3):651-63 (1985); Chothia, C. and Lesk, AM, J.Mol.Biol., 196, 901 (1987); Chothia, C. et al., Nature, 21-28; 342(6252):877-83 (1989); Kabat E.A. et al., Sequences of Proteins of Immunological Significance). of immunological interest), 5th edition, 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 edition), 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; heavy chain FRs include HFR1, HFR2, HFR3, and HFR4). These framework regions are more conserved than the CDRs and form a scaffold to support the highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies can be classified into several classes 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, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further subdivided 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).

[0057] The type of antibody described in this application is not limited. For example, it may be an anti-PD-1 antibody or other antibody that can work separately, together or synergistically with IL-21 or its mutants.

[0058] The anti-PD-1 antibody includes, but is not limited to, pembrolizumab or its mutants.

[0059] In some embodiments, the anti-PD-1 antibody included 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 also comprises an Fc. In some embodiments, the anti-PD-1 antibody may be a full-length antibody.

[0060] In some embodiments, the fusion protein comprises a mutant of the aforementioned anti-PD-1 antibody.

[0061] Taking Pembrolizumab as an example, its mutation proposals 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.

[0062] The connection method (e.g., location, use of linkers, etc.) between the IL-21 mutant and the antibody or its mutant can be a conventional connection method in the art.

[0063] In some embodiments, the antibody or a mutant thereof is operatively linked to the IL-21 mutant.

[0064] Unless otherwise specified, the term "operably linked" in this application refers to the functional relationship between two regions of a fusion protein, namely, the antibody or its mutant and the IL-21 mutant, wherein the two regions are linked to produce a fusion protein.

[0065] For example, the IL-21 mutant is linked to the C-terminus of the heavy chain of the anti-PD-1 antibody.

[0066] 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 chains is as shown in SEQ ID NO:3, 4, 5, 7, 12-16, 18 or 24, and the amino acid sequence of the light chains is as shown in SEQ ID NO:26.

[0067] In some embodiments, the fusion protein comprises two distinct 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 NO:30-34 and 37-41, and the amino acid sequence of the light chain is as shown in SEQ ID NO:26.

[0068] Furthermore, this application also provides functional variants of the fusion protein described herein that are within the scope of this application. As used herein, the term "functional variant" refers to a recombinant protein, polypeptide, or protein having a substantial or significant sequence identity or similarity to the parental fusion protein, wherein the functional variant retains the biological activity of the fusion protein. The functional variant encompasses an amino acid sequence that 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 with the amino acid sequence of the parental fusion protein.

[0069] The functional variant may comprise, for example, the amino acid sequence of a parental fusion protein having at least one conserved amino acid substitution. Alternatively or additionally, the functional variant may comprise the amino acid sequence of a parental fusion protein having at least one non-conserved amino acid substitution. In this case, non-conserved amino acid substitutions that do not interfere with or inhibit the biological activity of the functional variant are preferred. Non-conserved amino acid substitutions can enhance the biological activity of the functional variant, resulting in increased biological activity of the functional variant compared to the parental fusion protein.

[0070] The amino acid substitutions in the fusion protein of this application are preferably conservative amino acid substitutions. Conservative amino acid substitutions are those known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can include replacing an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), replacing an amino acid with a nonpolar side chain with another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Tip, Cys, Val, etc.), replacing a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), replacing 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.), replacing 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), and replacing 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.).

[0071] The fusion proteins of embodiments of this application (including the functional portions and functional variants of this application) may comprise synthetic amino acids that replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2- Carboxylic acids, 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-norborneane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0072] Mutants that have a certain degree of amino acid homology with the amino acid sequence of the fusion protein described above, for example, homology between 70% and 99%, further homology between 80% and 99%, further homology between 90% and 99%, and homology of 99%, should also fall within the scope of protection of this application.

[0073] The "homology" (sequence identity percentage) 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 those in a reference sequence after sequence alignment, and, where necessary, the introduction of vacancies to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the sequence identity percentage (%) 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 by the total number of amino acid residues (or bases) in the candidate or reference sequence (whichever is shorter). Conservative substitutions of amino acid residues may or may not be considered identical residues. For example, publicly available tools can be used, 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 Molecular Biology 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., *Bioinformatics* (Cambridge, UK), 23(21):2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software can be used to perform alignments to determine the percentage of identity between amino acid (or nucleic acid) sequences. Those skilled in the art can use the default parameters provided by the tools or can appropriately customize the parameters as needed for the alignment, for example by selecting a suitable algorithm.

[0074] As used in this application, the term "amino acid" refers to an organic compound that includes amino (-NH2) and carboxyl (-COOH) functional groups, as well as the side chain characteristic of each amino acid. Amino acid names are also represented in this disclosure as standard single-letter or three-letter codes, summarized below.

[0075] Amino acid name Three-letter code Single-letter codes alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C glutamic acid Glu E glutamine Gln Q glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F proline Pro P Serine Ser S threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0076] A third aspect of this application provides a nucleic acid molecule that encodes the IL-21 mutant or fusion protein described in this application.

[0077] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” may be used interchangeably throughout the text and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs derived using nucleotide analogs (e.g., peptide nucleic acids and non-natural nucleotide analogs), and their hybrids. Nucleic acid molecules may be single-stranded or double-stranded. In one embodiment, the nucleic acid molecule described herein comprises a continuous open reading frame encoding an antibody or fragment, derivative, mutant protein, or variant thereof provided herein.

[0078] A fourth aspect of this application provides a recombinant expression vector comprising nucleic acid molecules as described in this application.

[0079] As used herein, the term "vector" refers to a medium in which a genetic element can be operatively inserted to induce expression of the genetic element, resulting in the production of a protein, RNA, or DNA encoded by the genetic element, or the replication of the genetic element. Vectors can be used to transform, transduce, or transfect host cells to induce expression of the genetic element they carry within the host cells. Examples of vectors include plasmids, phage particles, granules, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), etc.), bacteriophages (such as λ phage or M13 phage, etc.), and animal viruses. Vectors may include a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, vectors may include an origin of replication. Vectors may also include materials that facilitate their entry into the cell, including, but not limited to, viral particles, liposomes, or protein coatings. Vectors may be expression vectors (e.g., viral vectors) or cloning vectors. This disclosure provides vectors (e.g., expression vectors) comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof provided in this application, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF-1α), and at least one selection marker.

[0080] A fifth aspect of this application provides a host cell that expresses the IL-21 mutant or fusion protein described in this application.

[0081] The term "cell," also known as "host cell," refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of *Escherichia coli* or *Salmonella*; members of the Bacillaceae family, 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.

[0082] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Therefore, “transformant” and “transformed cell” include primary test cells and cultures derived from them, regardless of passage number. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, the context will be clear.

[0083] A sixth aspect of this application provides a pharmaceutical composition comprising the IL-21 mutant or fusion protein of this application, and a pharmaceutically acceptable carrier and / or excipient.

[0084] The term “pharmaceutically acceptable” means that the specified carrier, mediator, diluent, excipient and / or salt is generally chemically and / or physically compatible with other components including the formulation and physiologically compatible with its recipient.

[0085] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, ionic strength enhancers, agents for maintaining osmotic pressure, agents for delaying absorption, diluents, preservatives, stabilizers, etc. For example, pH adjusters 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 for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents for delaying 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), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which stabilize the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium 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.

[0086] A seventh aspect of this application provides a kit comprising one or more of the IL-21 mutant, fusion protein, nucleic acid molecule, recombinant expression vector, host cell, and pharmaceutical composition described in this application, as well as a container.

[0087] An eighth aspect of this application provides a method for preparing the IL-21 mutant or fusion protein as described above, comprising culturing the host cells of this application to obtain a culture medium; and isolating the IL-21 mutant or the fusion protein from the culture medium.

[0088] The ninth aspect of this application provides the use of the IL-21 mutant, fusion protein, nucleic acid molecule, recombinant expression vector, or host cell in the preparation of a medicament for use against tumors.

[0089] This application also provides a method for treating tumor-related diseases, the method comprising administering to a subject an effective dose of one or more of the IL-21 mutant and fusion protein as described above.

[0090] "Administration," "giving," and "treatment," 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, immunomodulator, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo, such as cells. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications. Some examples are provided below.

[0091] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0092] Example 1: Design of IL-21 disulfide bond mutation

[0093] Based on the publicly available IL-21 tertiary structure (PDB:3TGX), disulfide bonds were designed using artificial intelligence and computer-aided design methods to select bond angles and bond lengths suitable for forming disulfide bonds. The designed disulfide bonds are shown in Table 1.

[0094] Table 1. IL-21 wild-type and IL-21 disulfide bond mutation sites

[0095]

[0096]

[0097] Example 2: Preparation of Pembrolizumab fused with IL-21WT and IL-21 disulfide mutant molecules

[0098] To better evaluate the stability of wild-type IL-21 and IL-21 disulfide mutant molecules, a Pembrolizumab fusion IL-21WT and IL-21 disulfide mutant molecule was constructed, with the structure shown below. Figure 1As shown in Table 2, the sequences were fused. The IL21-WT or IL-21 disulfide mutant molecule was fused to the C-terminus of the Pembrolizumab antibody heavy chain (replacing the constant region of the Pembrolizumab antibody heavy chain 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 also cloned into the expression vector pCDNA3.4. The IL21-WT or IL-21 disulfide mutant molecule and the Pembrolizumab antibody heavy chain fusion protein expression plasmid were co-transfected with the Pembrolizumab antibody light chain expression plasmid into ExpiCHO-S cells for expression. The supernatant was collected and purified with Protein A to obtain the candidate antibody protein.

[0099] Table 2. Molecular sequences of Pembrolizumab IL-21 wild-type and Pembrolizumab IL-21 disulfide bond mutant.

[0100]

[0101] Example 3: Reduction SDS-PAGE analysis of Pembrolizumab fused with IL-21WT and IL-21 disulfide mutant molecules

[0102] The Pembrolizumab fused with IL-21WT and the IL-21 disulfide mutant molecule prepared in Example 2 were subjected to reduction SDS-PAGE analysis, and the results are as follows: Figure 2 As shown in the figure. SDS-PAGE results indicate that the heavy chain fused IL-21WT peptide of the 2v2-WT molecule breaks during electrophoresis due to instability, forming fragments. However, the 2v2-C1 heavy chain fused IL-21 peptide with the I16C,S70C mutation described in patent CN111205361B shows virtually no breakage, indicating that increasing the I16C-S70C disulfide bond can improve the stability of IL-21. The 2v2-C2, 2v2-C3, 2v2-C4, 2v2-C6, 2v2-C11, 2v2-C12, 2v2-C13, 2v2-C14, 2v2-C15, 2v2-C17, and 2v2-C23 molecules developed in this patent can significantly improve the breakage of the heavy chain fused IL-21 peptide, indicating that increasing the corresponding disulfide bonds in these molecules can increase the stability of the IL-21 molecule.

[0103] Example 4: Detection of biological activity of IL-21 signaling pathway in pembrolizumab fused with IL-21WT and IL-21 disulfide mutant molecules

[0104] The reporter gene assay was used to detect the activation of the STAT3 signaling pathway in Baf3-stat3-IL21R-Luc cells by Pembrolizumab fusion with IL-21WT and IL-21 disulfide mutant molecules. The Baf3-stat3-IL21R-Luc cells used in this assay (H_IL-21Reporter Cell Line, GM-C15762) were Baf3 cells stably expressing human IL21R and STAT3-induced luciferase reporter genes. Specific procedures: Collect H_IL-21Reporter Cell Line cells in logarithmic growth phase, centrifuge, resuspend in analytical buffer (1640 + 1% FBS + 1% PS) for counting, adjust cell density to 2E6 cells / ml, and add 50 μl / well to different positions in a 96-well plate. Incubate at 37℃ and 5% CO2 for 18-20 h. Take Pembrolizumab fusion IL-21WT and IL-21 disulfide mutant molecules, dilute with analytical buffer to different concentrations (2*1000 nM starting concentration, 5-fold serial dilutions, 10 concentration points), and add 50 μl / well to a 96-well plate. Mix thoroughly using a microplate shaker or pipette, and incubate the 96-well plate at 37℃ and 5% CO2 for 9 h. Add 80 μl / well of Luciferase Assay System (Vazyme). Incubate at room temperature for 5-10 min, and then transfer to a microplate reader (MD, SpectraMax). The LUM signal value was detected using i3X. A nonlinear fitting was performed with the final antibody concentration as the x-axis and the detected LUM signal value as the y-axis to calculate the EC50 value.

[0105] Experimental results are as follows Figure 3 As shown, the 2v2-C2, 2v2-C3, 2v2-C4, 2v2-C6, 2v2-C11, 2v2-C12, 2v2-C13, 2v2-C14, 2v2-C15, 2v2-C17, and 2v2-C23 mutants developed in this application all exhibit essentially the same IL-21 signaling pathway biological activity as before the modification. This indicates that adding disulfide bond mutations does not significantly affect the biological function of IL-21. The following examples demonstrate the 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.

[0106] Example 5: Disulfide bond detection of Pembrolizumab fusion with IL-21WT and IL-21 disulfide bond mutant molecules

[0107] The collision-induced dissociation mode of tandem mass spectrometry is used to break the peptide backbone while preserving disulfide bonds. By identifying characteristic fragments, the product ions are analyzed to locate the disulfide bonds.

[0108] Take 200 μg of the sample solution to be tested, add 150 μL of 8M guanidine hydrochloride (Thermo, catalog number 24115), 10 μL of 1M Tris-HCl PH7 (Invitrogen, catalog number AM9851), and 2 μL of 200 mM NEM (N-ethylmaleimide, Sigma, catalog number E3876-5G). If the volume is less than 200 μL, add ultrapure water to bring the total volume to 200 μL. Incubate at 37℃ for 1 h. Divide the sample into two portions (100 μL each). Add 5 μL of 0.5M DTT (Sigma, catalog number D0632-10G) solution to one portion, and add 5 μL of ultrapure water to the other portion. Incubate at 60℃ for 30 min. Add 10 μL of 0.5M IAM (Sigma, catalog number I1149-5G) solution to both portions and incubate at room temperature in the dark for 30 min. The samples were replaced with a desalting column (Thermo, catalog number 89882) in 20 mM Tris-HCl pH 7 solution. 5 μg of enzyme (1:20) was added and incubated at 37°C for approximately 4 hours. Depending on the specific sequence, another enzyme could be selected for combined digestion. If combined digestion is required, the samples could be reacted at 95°C for 5 minutes, cooled, and then another enzyme (1:50) added, followed by overnight digestion at 37°C. 2 μL of 10% FA (Fisher, catalog number A117) solution was added to each solution to terminate the digestion reaction. After high-speed centrifugation, the supernatant was collected for LC-MS / MS analysis. Disulfide bond searching was performed using the instrument's built-in software, BioPharma Finder, or manual extraction was performed by comparing the spectra of reduced and non-reduced samples based on theoretical molecular weights to determine disulfide bond formation. The results are shown in Table 3.

[0109] Table 3. Mass spectrometry detection of disulfide bonds in Pembrolizumab fusion with IL-21WT and IL-21 disulfide bond mutant molecules.

[0110]

[0111]

[0112] Example 6: Preparation of Pembrolizumab fused with IL-21WT and IL-21 disulfide mutant molecules

[0113] To better evaluate the in vivo pharmacokinetic (PK) of wild-type IL-21 and IL-21 disulfide mutant molecules, a pembrolizumab fusion IL-21WT and IL-21 disulfide mutant molecule were constructed, as shown in the structure below. Figure 4As shown in Table 4, the sequences were fused to the C-terminus of human IgG1-Fc and cloned into the expression vector pCDNA3.4. The light and heavy chains 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 mismatches. The above expression plasmids were co-transfected into ExpiCHO-S cells for expression (note: 1v1-WT was expressed using Expi293-F, otherwise it is prone to breakage). The supernatant was collected and purified by Protein A and ion exchange to obtain the candidate antibody protein. It should be noted that, given that the disulfide bonds introduced in mutants IL21-C3, IL21-C12, and IL21-C13 are similar in spatial structure to several other mutants, the following exemplarily illustrates the fusion proteins formed by Pembrolizumab with several other mutants and their effects, and further demonstrates the combination of the R9E / R76A mutant molecule.

[0114] Table 4. Molecular sequences of Pembrolizumab fusion with IL-21WT and IL-21 disulfide bond mutants

[0115]

[0116]

[0117] Example 7: PK detection of Pembrolizumab fusion with IL-21WT and IL-21 disulfide mutant molecules

[0118] Forty-two male C57BL mice with a uniform body weight of 20–24 g and an age of 7–9 weeks were selected and randomly divided into 14 subgroups (n=3 per subgroup) according to body weight. Each subgroup was administered different candidate molecules, with two subgroups administered each test molecule, and crossover blood samples were collected. The dosage for each candidate molecule was uniformly 10 mg / kg, with an administration volume of 10 mL / kg and a concentration of 1 mg / mL, administered via tail vein injection as a single dose. Blood samples were collected from each group at 0.5 h, 6 h, 24 h, 72 h, 144 h, 240 h, 360 h, and 480 h post-administration. Four animals in each group underwent alternating blood collection. Blood samples were transferred to centrifuge tubes and stored at room temperature before blood collection and separation. Serum was separated within 2 h after blood collection and centrifuged at 4000 g for 10 min at room temperature. After centrifugation, the collected serum samples were frozen at -80°C. After blood collection, the drug concentration in the serum sample was measured, and the main metabolic kinetic parameters t1 / 2, Cmax, AUC, etc. were calculated.

[0119] Metabolic kinetic parameters are shown in Table 5. Compared to 1v1-WT, the in vivo exposure and half-life of the samples with added disulfide bonds were significantly increased. Specifically, the half-life of 1v1-C1 (with patent CN111205361B disulfide bond) increased to 4 times that of 1v1-WT, the half-lives of 1v1-C4, 1v1-C6, 1v1-C15, and 1v1-C23 increased to 1.7-2.26 times that of 1v1-WT, and the half-life of 1v1-C17 increased to 6.0 times that of 1v1-WT, while its AUC(0-t) was significantly better than that of other molecules.

[0120] Table 5. Metabolic kinetic parameters of Pembrolizumab fused with IL-21WT and IL-21 disulfide mutant molecules

[0121] 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

[0122] Example 7: Preparation of Pembrolizumab fused with IL-21 disulfide bonds and R9E / R76A combined mutant molecules

[0123] The IL21-WT or IL-21 disulfide bond mutant molecules were fused to the IL-21 protein with R9E and R76A mutations, respectively, and then 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 also 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 into ExpiCHO-S cells for expression. The supernatant was collected and purified by Protein A and ion exchange to obtain the candidate antibody protein.

[0124] Table 6. Sequences of Pembrolizumab fusion with IL-21 disulfide bonds and R9E / R76A combined mutant molecules

[0125]

[0126]

[0127] Example 8: Stability analysis of Pembrolizumab fused with IL-21 disulfide bonds and R9E / R76A combined mutant molecules

[0128] The Pembrolizumab fused with the IL-21 disulfide bond and the R9E / R76A combined mutant molecule prepared in Example 7 were subjected to reducing SDS-PAGE analysis, and the results are as follows: Figure 5 As shown in the figure. SDS-PAGE results indicate that the heavy chain fusion IL-21 (R9E, R76A) peptide chain of the 1v1-WT-256 molecule breaks during electrophoresis due to instability, forming fragments. However, the 1v1-C1-256 heavy chain fusion IL-21 peptide segment with the I16C,S70C mutation described in patent CN111205361B shows virtually no breakage, indicating that increasing the I16C-S70C disulfide bond can improve the stability of IL-21. Other molecules developed in this patent can also effectively improve the breakage of the heavy chain fusion IL-21 peptide segment, indicating that increasing the corresponding disulfide bonds in these molecules can increase the stability of the IL-21 molecule.

[0129] Example 9: Biological activity analysis of Pembrolizumab fused with IL-21 disulfide bonds and R9E / R76A combined mutant molecules

[0130] The reporter gene assay was used to detect the activation of the STAT3 signaling pathway in PD-1+ and PD-1- Baf3-stat3-IL21R-Luc cells by Pembrolizumab fused to an IL-21 disulfide bond and the R9E / R76A combined mutant molecule. The PD-1+ Baf3-stat3-IL21R-Luc cells (Baf3-IL21R-hPD1-H03) in this assay were Baf3 cells stably expressing human PD-1 receptor, IL21R, and STAT3-induced luciferase reporter genes. The PD-1- Baf3-stat3-IL21R-Luc cells (H_IL-21Reporter Cell Line, GM-C15762) in this assay were Baf3 cells stably expressing human IL21R and STAT3-induced luciferase reporter genes. Specific procedures: Collect Baf3-IL21R-hPD1-H03 or H_IL-21Reporter Cell Line cells in logarithmic growth phase, centrifuge, resuspend in analytical buffer (1640 + 1% FBS + 1% PS) for counting, adjust cell density to 2E6 cells / ml, and add 50 μl / well to different positions in a 96-well plate. Incubate at 37℃ and 5% CO2 for 18-20 h. Take Pembrolizumab fused with IL-21 disulfide bonds and the R9E / R76A combined mutant molecule, dilute with analytical buffer to different concentrations (2*1000 nM starting concentration, 5-fold serial dilutions, 10 concentration points), and add 50 μl / well to a 96-well plate. Mix thoroughly using a microplate shaker or pipette, and incubate the 96-well plate at 37℃ and 5% CO2 for 9 h. Add LuciferaseAssay. System (Vazyme), 80 μl / well; incubate at room temperature for 5-10 min, then detect LUM signal values ​​using a multi-functional microplate reader (MD, SpectraMaxi3X). The EC50 value was calculated by performing a non-linear fitting with the final antibody concentration as the x-axis and the detected LUM signal value as the y-axis.

[0131] The results of the IL-21 biological activity assay of the PD-1 antibody and the IL-21 mutant fusion protein are as follows: Figure 6 and Figure 7As shown, all molecules exhibited low activity on PD-1 negative cells. On PD-1 positive cells, the 1v1-WT-256 molecule broke due to IL-21 instability and showed no IL-21 signaling pathway activation activity. The molecule 1v1-C1-256, using the disulfide bond from patent CN111205361B, also lacked IL-21 signaling pathway activation activity on PD-1 positive cells. This indicates that while combining this disulfide bond with the Amgen AMG256 IL-21 mutant R9E / R76A improves stability, it results in the loss of its original biological activity and cannot serve as a stable framework for IL-21-based drug development. The disulfide bond developed in this patent, when combined with the Amgen AMG256 IL-21 mutant R9E / R76A, not only improves stability on PD-1 positive cells but also largely retains IL-21 signaling pathway activation activity, providing multiple stable IL-21 molecular frameworks for IL-21-based drug development.

[0132] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0133] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. An interleukin-21 (IL-21) mutant, characterized in that, Compared to wild-type IL-21, it has two mutation pairs: D26C and N63C. The amino acid sequence of the wild-type IL-21 is shown in SEQ ID NO:

42.

2. An IL-21 mutant, characterized in that, It also includes substitutions for R9E and R76A in addition to the IL-21 mutant described in claim 1.

3. A fusion protein, characterized in that, It comprises the IL-21 mutant as described in claim 1 or 2 and an anti-PD-1 antibody; the IL-21 mutant is linked to the C-terminus of the heavy chain of the anti-PD-1 antibody; The fusion protein comprises two identical heavy chains and two identical light chains, the amino acid sequence of the heavy chains being shown in SEQ ID NO: 24, and the amino acid sequence of the light chains being shown in SEQ ID NO:

26. Alternatively, the fusion protein comprises two distinct heavy chains, heavy chain 1 and heavy chain 2, and a light chain; the amino acid sequence of heavy chain 1 is shown in SEQ ID NO: 27, the amino acid sequence of heavy chain 2 is shown in SEQ ID NO: 34 or 41, and the amino acid sequence of the light chain is shown in SEQ ID NO:

26.

4. A nucleic acid molecule, characterized in that, It encodes the IL-21 mutant as described in claim 1 or 2, or the fusion protein as described in claim 3.

5. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 4.

6. The recombinant expression vector as described in claim 5, characterized in that, The recombinant expression vector includes a viral vector.

7. A host cell, characterized in that, It expresses the IL-21 mutant as described in claim 1 or 2 or the fusion protein as described in claim 3.

8. A pharmaceutical composition, characterized in that, It comprises the IL-21 mutant as described in claim 1 or 2 or the fusion protein as described in claim 3, and a pharmaceutically acceptable vector.

9. A kit comprising one or more of the following: the IL-21 mutant as claimed in claim 1 or 2, the fusion protein as claimed in claim 3, the nucleic acid molecule as claimed in claim 4, the recombinant expression vector as claimed in claim 5 or 6, the host cell as claimed in claim 7, and the pharmaceutical composition as claimed in claim 8, and a container.

10. A method for preparing the IL-21 mutant as described in claim 1 or 2, or the fusion protein as described in claim 3, characterized in that, The method includes: Culture medium obtained by culturing the host cells as described in claim 7; and, Isolate the IL-21 mutant or the fusion protein from the culture medium.

11. Use of the fusion protein of claim 3 in the preparation of a medicament for antitumor purposes.

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