Mutant of IL-21 as well as preparation method and application thereof

Abstract

The invention discloses a mutant of IL-21 as well as a preparation method and application thereof. Compared with a wild type IL-21, the mutant has amino acid mutation at least at the 37th site. In some embodiments, artificial disulfide bond design is performed on IL-21, exposed furin cleavage sites are removed to optimize the druggability of IL-21, and finally the IL-21 is fused with the PD-1 antibody to obtain the PD-1 antibody and IL-21 mutant molecule fusion protein with improved stability, good druggability, good drug effect and low toxic and side effects.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to mutants of IL-21 and preparation methods and applications thereof. Background Art

[0002] Immune checkpoint inhibitors (PD-1 / PD-L1 antibodies) are currently the most widely used cancer immunotherapy, which blocks the PD-1 / PD-L1 signaling pathway and allows the immune system to directly kill cancer cells {Trends in Molecular Medicine, 2015, 21(1): 24-33.}. PD-1 / PD-L1 drug sales will reach $45.8 billion in 2023. However, the average ORR of most tumor PD-1 / PD-L1 antibody monotherapy is only 19.56% {Front Oncol. 2021: 11: 562315.}, and the effect of removing immunosuppression alone is not good.

[0003] Interleukin IL-21 is an immune activating factor belonging to the IL-2 family and a four-helix bundle cytokine. IL-21 is mainly secreted by activated CD4+T cells, NK cells, TFH cells, and Th17 cells to enhance the antigen-specific response of immune cells. IL-21 can promote the anti-tumor activity of CD8+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 {Front Immunol. 2020; 11: 832. Current Opinion in Immunology, 23(5), 598-604. Biochem Biophys Res Commun. 2003; 300(2): 291-296. Nature Reviews Drug Discovery, 2014, 13(5): 379-395.}. However, IL-21 immune activation can also cause autoimmune-like inflammatory diseases in normal people and aggravate the systemic side effects of autoimmune diseases {Nature Reviews Drug Discovery, 2014, 13(5): 379-395.}.

[0004] 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. A Phase I clinical trial of ZymoGenetics using intravenous IL-21 to treat renal cell carcinoma (RCC) showed that the overall disease control rate (PR+SD) was 89% in 19 RCC patients {Clin Cancer Res. 2010, 16 (21); 5312-5319.}. In addition, a Phase II clinical trial of the company using intravenous IL-21 to treat metastatic melanoma (MM) showed that the disease control rate (DCR) was 62.5% among the 40 patients included in the trial, and the dose-limiting toxicity was 30ug / kg {J Clin Oncol. 2012, 30: 3396-3401.}. The above-mentioned Phase I and Phase II clinical data showed that IL-21 has significant efficacy, but more than 20% of patients experienced fatigue, fever, chills, itching and rash as side effects, and all dose groups had grade 3 (severe) rash as side effects. Therefore, non-targeted IL-21 has significant efficacy, but has greater side effects.

[0005] In addition, the anti-tumor efficacy of IL-21 in clinical trials is limited by its short half-life. Currently, fusion with immunoglobulin Fc, pegylation, and fusion with human serum albumin (HSA) or albumin binding domain have been used to extend the half-life of cytokines. In mice, the serum half-life of IL-21-αHSA fusion protein (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 (single dose 0.15mg / kg). In cynomolgus monkeys, the half-life and exposure time of IL-21-αHSA were significantly prolonged compared with rhIL-21, with its t1 / 2 and AUC being 10 and 50 times that of rhIL-21, respectively (single dose of 0.5 mg / kg) {Adv.Therap.2021,4,2100035}.

[0006] IL-21 has synergistic antitumor activity in combination with immune checkpoint blockers. In the B16-F 10 lung metastatic melanoma mouse model, the combination of mIL-21 with mCTLA-4 monoclonal antibody or mPD-1 monoclonal antibody significantly reduced the number of melanoma metastases in the lung. In the MC38 colon cancer model, compared with single drugs, the combination of IL-21 with mCTLA-4 monoclonal antibody or mPD-1 monoclonal antibody had significant synergistic antitumor activity and significantly increased the percentage of CD8+ lymphocytes and Ki67+CD8+T cells. In summary, the combination of IL-21 can significantly enhance the antitumor activity of immune checkpoint blockers (PD-1 monoclonal antibody / CTLA-4 monoclonal antibody) {Oncoimmunology. (2017) 7: e1377873.}.

[0007] IL-21 combines its unique receptor IL-21R and shared γC receptor, and its affinity with IL-21R receptor is very strong. IL-21R is widely expressed on T cells, B cells, NK cells and bone marrow cells, which easily causes IL-21 to widely activate the above cells and cause serious toxicity {Namre Reviews Drug Discovery, 2014, 13 (5): 379-395.}. Therefore, up to now, the above-mentioned utilization of recombinant IL-21 as a monotherapy or the drug combined with checkpoint inhibitors has not been successful clinically. Therefore, it is necessary to consider making IL-21 specifically target the activated T cells in the tumor microenvironment rather than other normal cells in the blood, so as to avoid extensive systemic toxicity.

[0008] By combining IL-21 and PD-1 antibodies into a fusion protein, effective IL-21 can be targeted to the tumor site, which is better than the combination of the two, and can reduce the active toxic side effects of IL-21 while prolonging the half-life. The Institute of Biophysics, Chinese Academy of Sciences, produced the PD-1Ab21 fusion protein by fusing the non-covalent homodimer (diabody) of the anti-PD-1 single-chain antibody fragment (scFv) with IL-21. In the CT26 and MC38 mouse tumor models, the PD-1Ab21 fusion protein was superior to the anti-tumor activity of the combination of PD-1 blockers and IL-21 {Nat Commun. 2021; 12: 951.}. However, the PD-1Ab21 fusion protein uses wild-type IL-21, and the affinity of wild-type IL-21 to IL-21R is very high, reaching 27pM {Front. Immunol. 11: 832.}. It can be foreseen that the PD-1Ab21 fusion protein will still target and activate immune cells in the blood and normal tissues, causing side effects. In a mouse tumor model that is refractory to PD-1 monoclonal antibody monotherapy, Amgen administered a fusion protein of anti-PD-1 monoclonal antibody and mutant IL-21 (reducing the affinity of IL-21 and IL-21R). Compared with PD-1 monoclonal antibody, it has a more significant inhibitory effect on tumor growth and can improve overall survival rate. In addition, PD-1 monoclonal antibody targets mutant IL-21 to activate T cells expressing PD-1 in the tumor microenvironment, greatly reducing toxic side effects, and its half-life in crab-eating monkeys is extended to 41h {Front.Immuno1.11:832.}. However, Amgen's PD-1 monoclonal antibody and mutant IL-21 fusion protein did not optimize the drugability of IL-21, such as stability.

[0009] In summary, new IL-21 drugs are urgently needed in this field. Summary of the invention

[0010] Based on this, it is necessary to provide at least one IL-21 mutant and its preparation method and application.

[0011] In the first aspect of the present application, a mutant of interleukin-21 (IL-21) is provided, which has an amino acid mutation at least in one or more of positions 36, 37 and 39 compared to wild-type IL-21; wherein: the mutation at position 36 is valine (V), isoleucine (I), arginine (R), tyrosine (Y), threonine (T) or tryptophan (w); the mutation at position 37 is glycine (G), valine (V), proline (P), serine (S), lysine (K) or phenylalanine (F); the mutation at position 39 is asparagine (N), glycine (G), serine (S), leucine (L), arginine (R) or tryptophan (w).

[0012] In the second aspect of the present application, a conjugate is provided, which comprises the mutant as described in the first aspect, and at least one other part.

[0013] In the third aspect of the present application, a nucleic acid molecule is provided, which encodes the mutant as described in the first aspect or the conjugate as described in the second aspect.

[0014] In the fourth aspect of the present application, a recombinant expression vector is provided, which comprises the nucleic acid molecule as described in the third aspect.

[0015] In the fifth aspect of the present application, a host cell is provided, which expresses the mutant as described in the first aspect or the conjugate as described in the second aspect.

[0016] In the sixth aspect of the present application, a pharmaceutical composition is provided, comprising the mutant as described in the first aspect or the conjugate as described in the second aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0017] In the seventh aspect of the present application, a kit is provided, comprising one or more of the mutant described in the first aspect, the conjugate described in the second aspect, the nucleic acid molecule described in the third aspect, the recombinant expression vector described in the fourth aspect, the host cell described in the fifth aspect, and the pharmaceutical composition described in the sixth aspect, and a container.

[0018] In the eighth aspect of the present application, there is provided a use of the mutant as described in the first aspect, the conjugate 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 an anti-tumor drug.

[0019] The present application reduces the affinity of IL-21 to its receptor IL-21R and γC, and obtains multiple affinities and mutants with reduced activation of IL-21R (+) cell activity. In some embodiments, IL-21 is subjected to artificial disulfide bond design and the exposed furin protease cleavage site is removed to optimize its drugability, and finally fused with PD-1 antibody to obtain PD-1 antibody and IL-21 mutant molecule fusion protein with improved stability, good drugability, good efficacy and low toxic and side effects. For example, CRB2110A, CRB2110B, and CRB2110C molecules are more active than AMG256 in IL-21 signaling pathway activation, and cells with different PD-1 expression levels can be more effectively activated, with significant improvements in solubility, thermal stability, and accelerated stability; Pharmacokinetic parameters are significantly improved, and the half-life is 1.5-1.7 times that of AMG256, which is 4.4-5.1 times before the transformation. 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 The biological activity of IL-21 mutant in Baf3-stat3-IL21R-Luc cells according to one embodiment of the present application is shown.

[0022] Figure 2 The structure of the fusion protein of PD-1 antibody and IL-21 mutation in one embodiment of the present application is shown.

[0023] Figure 3A , Figure 3B and Figure 3C The affinity detection between PD-1 antibody, IL-21 mutant fusion protein and IL-21 receptor in the embodiment of the present application is shown.

[0024] Figure 4 The IL-21 biological activity detection of the PD-1 antibody and IL-21 mutant fusion protein in the embodiment of the present application is shown on the PD-1(-) Baf3-stat3-IL21R-Luc cell line.

[0025] Figure 5 The IL-21 biological activity detection of the PD-1 antibody and IL-21 mutant fusion protein in the embodiment of the present application is shown on the Baf3-stat3-IL21R-Luc cell line with different PD-1 expression levels.

[0026] Figure 6 The serum stability analysis of the fusion protein of PD-1 antibody and IL-21 mutation in the embodiment of the present application is shown.

[0027] Figure 7 The accelerated stability of the fusion protein of PD-1 antibody and IL-21 mutation is shown in one embodiment of the present application.

[0028] Figure 8 The mouse PK drug-time curve of the fusion protein of PD-1 antibody and IL-21 mutant in one embodiment of the present application is shown. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] In the present application, the "suitable" in "suitable combination", "suitable method", "any suitable method", etc., is 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.

[0034] 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.

[0035] 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".

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The present application provides a mutant of interleukin-21 (IL-21), which has an amino acid mutation at least in one or more of positions 36, 37 and 39 compared to wild-type IL-21; wherein: the mutation at position 36 is valine (V), isoleucine (I), arginine (R), tyrosine (Y), threonine (T) or tryptophan (W); the mutation at position 37 is glycine (G), valine (V), proline (P), serine (S), lysine (K) or phenylalanine (F); the mutation at position 39 is asparagine (N), glycine (G), serine (S), leucine (L), arginine (R) or tryptophan (W).

[0043] Unless otherwise specified, "mutant" in this application refers to a protein or fragment obtained by at least replacing, deleting and substituting one or more of the wild-type protein or fragment, which at least retains 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.

[0044] In some embodiments, the mutant also has an amino acid mutation at position 76; wherein glutamic acid is inserted before the amino acid at position 76 (IN76E).

[0045] In some embodiments, the mutant further comprises at least two cysteine ​​substitutions compared to wild-type IL-21.

[0046] Illustratively, the two cysteine ​​substitutions may occur at positions 5 and 79, 6 and 81, 24 and 105, 32 and 53, 57 and 64, 57 and 67, 28 and 58, 16 and 73, or 26 and 63, respectively.

[0047] In some embodiments, the cysteine ​​substitutions occur at positions 16 and 73.

[0048] In the present application, the wild-type IL-21 may include an amino acid fragment having an amino acid sequence as shown in SEQ ID NO: 58. In some embodiments, the amino acid sequence of the wild-type IL-21 is as shown in SEQ ID NO: 58.

[0049] Illustratively, two cysteine ​​substitutions may occur at R5C and P79C, H6C and T81C, V24C and K105C, L32C and A53C, S57C and E64, S57C and I67C, V28C and A58C, I16C and K73C, or D26C and N63C.

[0050] In some embodiments, the mutant comprises any combination of mutation sites shown below:

[0051] IN76E and E36V / I / R / Y / T / W

[0052] IN76E and D37G / V / P / S / K / F;

[0053] 1N76E and E39N / G / S / L / R / W

[0054] IN76E, E36V / I / R / Y / T / W, I16C and K73C;

[0055] IN76E, D37G / V / P / S / K / F, I16C, and K73C;

[0056] IN76E, E39N / G / S / L / R / W, I16C, and K73C; or,

[0057] IN76E, I16C and K73C.

[0058] In some embodiments, the amino acid sequence of the mutant comprises the amino acid sequence shown in any one of SEQ ID NOs: 11-47.

[0059] The present invention also provides a conjugate comprising the mutant as described above, and at least one other part.

[0060] In some embodiments, the other part is an antigen binding molecule. It should be understood that the antigen binding molecule and the mutant can be operably linked. In some embodiments, the conjugate is a fusion protein.

[0061] If not specifically stated, the term "operably linked" in the present application refers to a functional relationship between two regions of a conjugate, ie, the other part and the IL-21 mutant; wherein the two regions are linked to produce a conjugate.

[0062] In the present application, the antigen binding molecule may be an antibody, for example an antibody targeting PD-1.

[0063] As used in this application, 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 K, and each light chain consists of a variable region (VL) and a constant region. The antibody is "Y" shaped, 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).

[0064] "Fab" with respect to antibodies refers to that portion of the antibody composed of a single light chain (variable and constant regions) bound to the variable region and first constant region of a single heavy chain by disulfide bonds.

[0065] In the present application, the antigen binding molecule may comprise at least one Fab and an Fc operably linked thereto, wherein the Fab comprises HCDR1 to 3 having amino acid sequences as shown in SEQ ID NOs: 5% to 61, and LCDR1 having a sequence as shown in SEQ ID NO: 62 or 63, LCDR2 having a sequence as shown in SEQ ID NO: 64, and LCDR3 having a sequence as shown in SEQ ID NO: 65.

[0066] In some embodiments, the Fab comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 66, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 67 or 68. Further, the antigen binding molecule comprises two heavy chains having amino acid sequences as shown in SEQ ID NO: 69 and 70, respectively, and a light chain having an amino acid sequence as shown in SEQ ID NO: 71 or 72.

[0067] In some embodiments, the mutant is operably linked to the C-terminus of the heavy chain in the antigen binding molecule.

[0068] In some embodiments, the conjugate comprises a heavy chain 1 having an amino acid sequence as shown in SEQ ID NO: 50, a light chain having an amino acid sequence as shown in SEQ ID NO: 49 or 52, and a heavy chain 2 having an amino acid sequence as shown in SEQ ID NO: 51 or 53.

[0069] In some embodiments, the conjugate comprises a heavy chain 1 having an amino acid sequence as shown in SEQ ID NO:50, a light chain having an amino acid sequence as shown in SEQ ID NO:52, and a heavy chain 2 having an amino acid sequence as shown in SEQ ID NO:51.

[0070] In some embodiments, the conjugate comprises a heavy chain 1 having an amino acid sequence as shown in SEQ ID NO:50, a light chain having an amino acid sequence as shown in SEQ ID NO:49, and a heavy chain 2 having an amino acid sequence as shown in SEQ ID NO:51.

[0071] In some embodiments, the conjugate comprises a heavy chain 1 having an amino acid sequence as shown in SEQ ID NO:50, a light chain having an amino acid sequence as shown in SEQ ID NO:49, and a heavy chain 2 having an amino acid sequence as shown in SEQ ID NO:53.

[0072] In addition, the present application also provides functional variants of the conjugates (e.g., fusion proteins) of the present application 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 having a large amount or significant sequence identity or similarity with the parent conjugate (e.g., fusion protein), and the functional variant retains the biological activity of the conjugate (e.g., fusion protein). Functional variants encompass that the amino acid sequence of the functional variant 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 more identity with the amino acid sequence of the parent conjugate (e.g., fusion protein).

[0073] The functional variant may comprise, for example, an amino acid sequence of a parent conjugate (e.g., a 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 conjugate (e.g., a 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 conjugate (e.g., fusion protein).

[0074] The amino acid substitutions of the conjugates (e.g., fusion proteins) 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.

[0075] The conjugates (e.g., fusion proteins) of the embodiments of the present application (including the functional parts and functional variants 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-carboxyphenylalanine, 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.

[0076] Mutants having a certain amino acid homology with the amino acid sequence of the conjugate (e.g., 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.

[0077] "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 DG et al., Methods in Enzymology, 25:3389-3402 (1997)), can be used. In Enzymology, 266: 383-402 (1996); Larkin M.A. et al. Bioinformatics (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.

[0078] 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.

[0079]

[0080]

[0081] The present application also provides a nucleic acid molecule, which encodes the mutant or conjugate as described above.

[0082] 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.

[0083] The present application also provides a recombinant expression vector, which comprises the nucleic acid molecule as described above.

[0084] 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.

[0085] The present application also provides a host cell, which expresses the mutant or the conjugate as described above.

[0086] 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.

[0087] 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.

[0088] The present application also provides a pharmaceutical composition, which comprises the mutant as described above or the conjugate as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0089] 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.

[0090] 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.

[0091] The present application also provides a kit, which comprises one or more of the mutant described above, the conjugate described above, the nucleic acid molecule described above, the recombinant expression vector described above, the host cell described above, and the pharmaceutical composition described above, and a container.

[0092] The present application also provides a method for preparing the mutant or the conjugate as described above, the method comprising:

[0093] Cultivating the host cell as described above to obtain a culture fluid; and,

[0094] The mutant or the conjugate is isolated from the culture fluid.

[0095] The present application also provides a kit, which comprises the mutant as described above, the conjugate as described above, the nucleic acid molecule as described above, the recombinant expression vector as described above or the host cell as described above for use in preparing an anti-tumor drug.

[0096] 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 mutants and conjugates of IL-21 as described above.

[0097] "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.

[0098] Some examples are provided below.

[0099] 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.

[0100] Example 1: Design, preparation and affinity testing of mutations that reduce IL-21 affinity and improve stability and drugability

[0101] Using the published IL-21:IL-211R:γC complex crystal structure (PDB ID: 8ENT) as a structural template, we used artificial intelligence and computer-aided design methods to design mutations that reduce the affinity of IL-21, improve stability, and enhance drugability. The designed mutations are shown in Table 1.

[0102] The designed IL-21 mutant molecule was fused to the C-terminus of human IgG1-Fc, and a signal peptide was added to the N-terminus. After codon optimization, the proteins were cloned into the expression vector pCDNA3.4 (Thermo Fisher Scientific) and co-transfected into ExpiCHO-S cells (Thermo Fisher Scientific) for expression. The supernatant was collected and purified by Protein A (Cytiva) to obtain the candidate antibody protein.

[0103] Using BLI technology, Sartorius Octet RH96 was used to capture the biotinylated IL21R&hγC dimer receptor (Acro biosystems, ILA-H52W9) through the SA sensor, and then the affinity with each antibody was measured, the antibody concentration was 25nM, Baseline1 200s, Baseline2 60s, binding 600s, dissociation 3600s. The results are shown in Table 1, and most antibodies were reduced to very weak or no binding to the IL21R&hγC dimer receptor.

[0104] Table 1 Mutation sequences and affinity results of IL-21 with reduced affinity, improved stability and improved drugability

[0105]

[0106]

[0107]

[0108]

[0109] Example 2: Detection of biological activity of IL-21 mutants

[0110] The reporter gene method was used to detect the activation of STAT3 signaling pathway activity in Baf3-stat3-IL21R-Luc cells by IL-21 mutant molecules. The Baf3-stat3-IL21R-Luc cells (H_IL-21Reporter Cell Line, Yoshimitsu, cat: GM-C15762) in this test are Baf3 cells that stably express human IL21R and STAT3-induced luciferase reporter genes. Specific operation: Take H_IL-21Reporter Cell Line cells in logarithmic growth period, centrifuge, resuspend and count with analysis buffer (1640+1% FBS+1% PS), adjust cell density to 2E6 / ml, add to different positions of 96-well plate, 50μL / well, incubate at 37℃, 5% CO2 for 18-20h; take IL-21 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 gun, incubate 96-well plate at 37℃, 5% CO2 for 9h; add LuciferaseAssay System (Vazyme), 80μL / well; incubate at room temperature for 5-10min, 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 1 As shown, the IL-21 mutant molecules developed in this application can effectively reduce the biological activity of the IL-21 signaling pathway.

[0112] Example 3: Preparation of PD-1 antibody and IL-21 mutant fusion protein

[0113] After codon optimization, the sequences of PD-1 antibody and IL-21 mutant fusion protein were cloned into expression vector pCDNA3.4 (Thermo Fisher), co-transfected into ExpiCHO-S cells (Thermo Fisher) for expression, and the supernatant was collected and purified by Protein A (Cytiva) to obtain candidate antibody proteins. The structures and sequences of PD-1 antibody and IL-21 mutant fusion protein are shown in Figure 2 and Table 2.

[0114] Table 2 PD-1 antibody and IL-21 mutant fusion protein sequences

[0115]

[0116] Example 4: Affinity detection of PD-1 and IL-21 of fusion protein of PD-1 antibody and IL-21 mutation

[0117] Using Biacore 8K, using CM5 chip amino-coupled antigen hPD1-his (Acro, PD1-H5221), about 16RU or 60RU (RU is response unit or resonance unit, that is, response value. In the literature related to surface plasmon resonance technology, it is indicated that if the CM5 chip is used for protein-protein interaction analysis, then 1RU is approximately equivalent to 1pg / mm 2 Changes in concentration. The response value of the Biacore system should always be in RU, not in concentration. ); then bind the antibody, concentration 50, 25, 12.5, 6.25, 3.125, 1.56, 0nM, bind 100s, dissociate 180s, regenerate Glycine1.5 (Cytiva) 30s, 30μL / min. The results are shown in Table 3. The results show that Pembrolizumab tends to bind monovalently and bivalently at coupling amounts of 16RU and 60RU, while AMG256 tends to bind bivalently (monovalent binding affinity is 1.67e-09M). The affinity of CRB2110A and CRB2110C molecules is at the nM level, while the CRB2110B molecule mutates in the CDR region of Pembrolizumab to increase affinity {CN202311797802.5}.

[0118] Table 3 Affinity test of PD-1 antibody, IL-21 mutant fusion protein and human PD-1 antigen

[0119]

[0120] Using BLI technology, Sartorius Octect RH96 was used to capture biotinylated hIL21R receptor (Acrobiosystems, ILR-H5226), IL21R&hγC dimer receptor (Acro biosystems, ILA-H52W9), and cIL21R receptor (Acrobiosystems, ILR-C5259) through SA sensors; then their affinity with each antibody was measured, CRB2110W concentration was 10, 3.33, 1.11, and 0.37 nM, and the concentration of other antibodies was 300 nM, Baseline1 200s, Baseline260s, binding 600s, and dissociation 3600s. The results are shown in Figure 3A , Figure 3B and Figure 3CAs shown, the affinity of CRB2110W (unmutated IL-21) to hIL21R receptor, IL21R&hγC dimer receptor, and cIL21R receptor were 15pM, 25pM, and 26pM, respectively. Pembrolizumab was used as a negative control and did not bind to the above receptors. The affinity of the IL-21 end of CRB2110A, CRB2110B, and CRB2110C fusion proteins was reduced to the same level as AMG256, all greater than 300nM.

[0121] Example 5: Detection of IL-21 biological activity of fusion protein of PD-1 antibody and IL-21 mutation

[0122] The reporter gene method was used to detect the activity of the STAT3 signaling pathway in Baf3-stat3-IL21R-Luc cells activated by PD-1 antibody and IL-21 mutant fusion protein. The PD-1+ Baf3-stat3-IL21R-Luc cells (Baf3-IL21R-hPD1-H03) in this test are Baf3 cells that stably express the human PD-1 receptor, IL21R, and STAT3-induced luciferase reporter genes, and the PD-1- Baf3-stat3-IL21R-Luc cells (H_IL-21ReporterCell Line, Yoshimitsu, cat: GM-C15762) in this test are Baf3 cells that stably express the human IL21R and STAT3-induced luciferase reporter genes. Specific operation: Take Baf3-IL21R-hPD1-H03 or H_IL-21ReporterCell 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°C, 5% CO2 for 18-20h; take PD-1 antibody and IL-21 mutant fusion protein, 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 gun, incubate 96-well plate at 37°C, 5% CO2 for 9h; add Luciferase AssaySystem (Vazyme), 80μL / well; incubate at room temperature for 5min-10min, and incubate 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.

[0123] The results of IL-21 biological activity test of PD-1 antibody and IL-21 mutant fusion protein are as follows Figure 4 and Figure 5 As shown. On PD-1 negative cells, the activities of CRB2110A, CRB2110B and AMG256 are relatively low (EC50>4E6 pM), and the CRB2110C molecule is slightly higher (~7.5E5pM). The IL-21 signaling pathway activation activity can be seen on positive cells with different PD-1 expressions: CRB2110C>CRB2110B>CRB2110A>AMG256. In addition, as the expression level of PD-1 on the cell surface decreases, the activity of all antibody fusion proteins also decreases to varying degrees, but AMG256 has no activity on cells with medium and low PD-1 expression and PD-1 low expression, while CRB2110A, CRB2110B, and CRB2110C molecules still have certain activity. Therefore, compared with AMG256, CRB2110A, CRB2110B, and CRB2110C molecules have stronger IL-21 signaling pathway activation activity and can more effectively activate the IL-21 signaling activity of cells with different PD-1 expression levels.

[0124] Example 6: Serum stability analysis of PD-1 antibody and IL-21 mutant fusion protein

[0125] Commercial human, mouse, and crab-eating macaque sera and PBS+1% BSA buffer (PBSA) were sterile filtered; CRB2110W, AMG256, Pembrolizumab, CRB2110A, CRB2110B, and CRB2110C molecules were added to the serum at an initial concentration of 0.2 mg / ml, and aseptically dispensed into 0.6 ml sterile centrifuge tubes at a volume of 50 μL / tube, incubated at 37°C for 0h, 1d, 3d, 5d, 7d, 10d, and 14d, and the removed samples were stored at -80°C; the concentration of the intact molecule was detected using the double antibody sandwich method of anti-PD1 antibody and anti-IL-21 antibody.

[0126] The results are as follows Figure 6 As shown, the plasma stability of CRB2110W molecule is poor, and the concentration is significantly reduced when placed in three different species serum and PBS + 1% BSA buffer. The stability of CRB2110A, CRB2110B and CRB2110C in three different species serum is significantly improved compared with CRB2110W, and in human serum, the stability of CRB2110B is better than AMG256.

[0127] Example 7: Solubility, thermal stability and accelerated stability evaluation of PD-1 antibody and IL-21 mutant fusion protein

[0128] During the concentration and fluid exchange process of the prepared CRB2110A, CRB2110B, CRB2110C, and AMG256, the CRB2110A, CRB2110B, and CRB2110C molecules can be concentrated to 100 mg / ml in the PBS system, while the AMG256 molecule becomes gelatinous and blocks the membrane after being concentrated to more than 8 mg / ml in the PBS system. There is no improvement after the fluid is exchanged to the histidine system, and the final concentration can only be concentrated to 8 mg / ml.

[0129] CRB2110A, CRB2110B, CRB2110C, and AMG256 were replaced with 20 mM histidine buffer solution (pH 6.2), and the melting temperature (Tm) and aggregation temperature (Tagg) were detected using Uncle (Unchained labs) full spectrum fluorescence (DSF). The results are shown in Table 4. The Tm1, Tm2, ​​and Tagg temperatures of CRB2110A, CRB2110B, and CRB2110C were higher than those of AMG256, showing better thermal stability.

[0130] Table 4 Tm and Tagg temperatures of fusion proteins of PD-1 antibody and IL-21 mutation

[0131]

[0132] The solution of CRB2110A, CRB2110B, CRB2110C, and AMG256 was replaced with 20 mM histidine buffer solution (pH 6.2) and placed at 45°C for one week. The changes in appearance and turbidity were compared. The results are as follows: Figure 7 As shown in the figure, CRB2110A and CRB2110B showed good stability, CRB2110C showed slight turbidity, and AMG256 showed serious colloid and precipitation. Therefore, compared with AMG256, CRB2110A and CRB2110B showed good accelerated stability.

[0133] Example 8: PK test of PD-1 antibody and IL-21 mutant fusion protein in mice

[0134] 48 male C57BL mice with uniform body weight of 20g-24g and age of 7-9 weeks were selected and randomly divided into 16 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 as 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 drug concentration-time curve was drawn, and the main metabolic kinetic parameters such as t1 / 2, Cmax, AUC, Vd, etc. were calculated.

[0135] The drug-time curve and metabolic kinetic parameters are shown in Figure 8 As shown in Table 5. The in vivo exposure and half-life of CRB2110A, CRB2110B, and CRB2110C were significantly increased compared to CRB2110W. The half-life of CRB2110A, CRB2110B, and CRB2110C was 1.5 to 1.7 times that of AMG256 and 4.4 to 5.1 times that of CRB2110W.

[0136] Table 5 Metabolic kinetic parameters of fusion protein of PD-1 antibody and IL-21 mutation

[0137]

[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. A mutant of interleukin-21 (IL-21), characterized in that The mutant has an amino acid mutation at least at position 37 compared to wild-type IL-21; the mutation at position 37 is proline (P).

2. The mutant according to claim 1, characterized in that The mutant also has an amino acid mutation at position 76; wherein glutamic acid is inserted before the amino acid at position 76 (IN76E).

3. The mutant according to claim 1 or 2, characterized in that The mutant further comprises at least two cysteine ​​substitutions compared to wild-type IL-21; Optionally, the cysteine ​​substitutions occur at positions 16 and 73.

4. The mutant according to claim 1 or 2, characterized in that The wild-type IL-21 comprises an amino acid fragment having an amino acid sequence as shown in SEQ ID NO: 58; Optionally, the mutant comprises a combination of mutation sites: IN76E and D37P; or, IN76E, D37P, I16C and K73C; Further optionally, the amino acid sequence of the mutant comprises the amino acid sequence shown in SEQ ID NO: 19 or 37.

5. A conjugate, characterized in that It comprises the mutant according to any one of claims 1 to 4, and at least one other part.

6. The conjugate according to claim 5, characterized in that The other part is an antigen binding molecule; Optionally, the antigen binding molecule targets PD-1; Further optionally, the antigen binding molecule comprises at least one Fab and an Fc operably linked thereto, wherein the Fab comprises HCDR1 to HCDR3 having amino acid sequences as shown in SEQ ID NOs: 59 to 61, respectively, and LCDR1 having an amino acid sequence as shown in SEQ ID NO: 62 or SEQ ID NO: 63, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 64, and LCDR3 having an amino acid sequence as shown in SEQ ID NO: 65; Further optionally, the Fab comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 66, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 67 or SEQ ID NO: 68; Alternatively, the antigen binding molecule comprises two heavy chains whose amino acid sequences are shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively, and a light chain whose amino acid sequence is shown in SEQ ID NO: 71 or 72.

7. The conjugate according to claim 6, characterized in that The mutant is operably linked to the C-terminus of the heavy chain in the antigen-binding molecule; Optionally, the conjugate comprises a heavy chain 1 having an amino acid sequence as shown in SEQ ID NO:50, a light chain having an amino acid sequence as shown in SEQ ID NO:49, and a heavy chain 2 having an amino acid sequence as shown in SEQ ID NO:

53.

8. A nucleic acid molecule, characterized in that It encodes the mutant according to any one of claims 1 to 4 or the conjugate according to any one of claims 5 to 7.

9. A recombinant expression vector, characterized in that: It comprises the nucleic acid molecule according to claim 8; Optionally, the recombinant expression vector is a viral vector.

10. A host cell, characterized in that It expresses the mutant according to any one of claims 1 to 4 or the conjugate according to any one of claims 5 to 7.

11. A pharmaceutical composition, characterized in that It comprises the mutant according to any one of claims 1 to 4 or the conjugate according to any one of claims 5 to 7, and a pharmaceutically acceptable carrier and / or excipient.

12. A kit, characterized in that: It comprises one or more of the mutant according to any one of claims 1 to 4, the conjugate according to any one of claims 5 to 7, the nucleic acid molecule according to claim 8, the recombinant expression vector according to claim 9, the host cell according to claim 10 and the pharmaceutical composition according to claim 11, and a container.

13. A method for preparing the mutant according to any one of claims 1 to 4 or the conjugate according to any one of claims 5 to 7, characterized in that: The method comprises: Cultivating the host cell according to claim 10 to obtain a culture fluid; and, The mutant or the conjugate is isolated from the culture fluid.

14. Use of the mutant according to any one of claims 1 to 4, the conjugate according to any one of claims 5 to 7, the nucleic acid molecule according to claim 8, the recombinant expression vector according to claim 9, or the host cell according to claim 10 in the preparation of an anti-tumor drug.

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