Mutants of il-21 and methods of making and using the same

Abstract

This application discloses a mutant of IL-21, its preparation method, and its application. This mutant has an amino acid mutation at least at position 37 compared to wild-type IL-21. In some embodiments, the druggability of IL-21 is optimized by designing artificial disulfide bonds and removing exposed furin cleavage sites, ultimately resulting in a fusion protein of PD-1 antibody and IL-21 mutant molecule with improved stability, good druggability, high efficacy, and low toxicity.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to mutants of IL-21, their preparation methods, and applications. Background Technology

[0002] Immune checkpoint inhibitors (PD-1 / PD-L1 antibodies) are currently the most widely used cancer immunotherapy. By blocking the PD-1 / PD-L1 signaling pathway, they allow the immune system to directly kill cancer cells {Trends in Molecular Medicine, 2015, 21(1): 24-33.}. Sales of PD-1 / PD-L1 drugs reached $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.}, indicating that simply relieving immunosuppression is not effective.

[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 secreted by activated CD4+ T cells, NK cells, TFH cells, and Th17 cells, enhancing the antigen-specific response of immune cells. IL-21 can promote the anti-tumor activity of CD8+ T cells and NK cells, and plays a key role in B cell differentiation and germinal cell development, making it a potential target for developing tumor immunotherapy. {Front Immunol. 2020; 11: 832. Current Opinion in Immunology, 23(5), 598-604. Biochem Biophys ResCommun. 2003; 300(2): 291-296. Nature Reviews Drug Discovery, 2014, 13(5): 379-395.} However, IL-21 immune activation can also cause inflammatory diseases similar to autoimmune diseases in normal people and can aggravate the systemic side effects of autoimmune diseases {Nature Reviews Drug Discovery, 2014, 13(5): 379-395.}.

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

[0005] Furthermore, the antitumor 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 its albumin-binding domain have been used to prolong the half-life of cytokines. In mice, the serum half-life of the IL-21-αHSA fusion protein (t1 / 2 = 15.48 h) is significantly longer than that of rhIL-21 (t1 / 2 = 0.61 h). In addition, the Cmax and AUC of IL-21-αHSA are nearly 60-fold and 300-fold higher than those of rhIL-21, respectively (single dose 0.15 mg / kg). In cynomolgus monkeys, compared with rhIL-21, IL-21-αHSA had a significantly prolonged half-life and exposure time, with its t1 / 2 and AUC being 10 and 50 times that of rhIL-21, respectively (single dose 0.5 mg / kg) {Adv.Therap.2021,4,2100035}.

[0006] IL-21 exhibits synergistic antitumor activity when combined with immune checkpoint inhibitors. In the B16-F10 mouse model of lung metastatic melanoma, the combination of mIL-21 with mCTLA-4 or mPD-1 significantly reduced the number of melanoma metastases in the lungs. In the MC38 colon cancer model, compared with single-agent therapy, the combination of IL-21 with mCTLA-4 or mPD-1 showed significant synergistic antitumor activity and significantly increased the percentage of CD8+ lymphocytes and Ki67+CD8+ T cells. In conclusion, the combination of IL-21 can significantly enhance the antitumor activity of immune checkpoint inhibitors (PD-1 / CTLA-4) {Oncoimmunology.(2017)7:e1377873.}

[0007] IL-21 binds to its unique receptor IL-21R and shares a γC receptor, and its affinity for the IL-21R receptor is very strong. IL-21R is widely expressed on T cells, B cells, NK cells, and bone marrow cells, making it susceptible to widespread activation of these cells by IL-21, leading to severe toxicity {Namre Reviews Drug Discovery, 2014, 13(5): 379-395.}. Therefore, to date, no drugs utilizing recombinant IL-21 as a monotherapy or in combination with checkpoint inhibitors have achieved clinical success. Therefore, it is necessary to consider targeting IL-21 specifically to activated T cells in the tumor microenvironment rather than other normal cells in the blood, thereby avoiding widespread systemic toxicity.

[0008] Combining IL-21 with PD-1 antibodies into a fusion protein can effectively target IL-21 to tumor sites, showing superior efficacy compared to using both alone, while also reducing the toxic side effects of IL-21 activity and prolonging its half-life. The Institute of Biophysics, Chinese Academy of Sciences, fused a non-covalent homodimer of an anti-PD-1 single-chain antibody fragment (scFv) with IL-21 to generate the PD-1Ab21 fusion protein. In CT26 and MC38 mouse tumor models, the PD-1Ab21 fusion protein exhibited superior antitumor activity compared to the combination of PD-1 blockers and IL-21 {Nat Commun. 2021; 12: 951.}. However, the use of wild-type IL-21 in the PD-1Ab21 fusion protein, which has a very high affinity for IL-21R (27 pM) {Front. Immunol. 11: 832.}, suggests that the PD-1Ab21 fusion protein may still target and activate immune cells in the blood and normal tissues, potentially causing side effects. In a mouse model of PD-1 monoclonal antibody-refractory tumors, Amgen administered a fusion protein of anti-PD-1 monoclonal antibody and mutant IL-21 (reducing the affinity of IL-21 for IL-21R). Compared to the PD-1 monoclonal antibody, this fusion protein significantly inhibited tumor growth and improved overall survival. Furthermore, the PD-1 monoclonal antibody targeted and activated PD-1-expressing T cells in the tumor microenvironment with mutant IL-21, greatly reducing toxicity and extending its half-life to 41 hours in cynomolgus monkeys {Front. Immuno1.11:832.}. However, Amgen's PD-1 monoclonal antibody-mutant IL-21 fusion protein did not optimize the drug-like properties of IL-21, such as stability.

[0009] In summary, there is an urgent need for new IL-21 drugs in this field. Summary of the Invention

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

[0011] In a first aspect of this application, a mutant of interleukin-21 (IL-21) is provided, the mutant having an amino acid mutation at least at 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); and the mutation at position 39 is asparagine (N), glycine (G), serine (S), leucine (L), arginine (R), or tryptophan (w).

[0012] In a second aspect of this application, a conjugate is provided, comprising the mutant as described in the first aspect, and at least one other portion.

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

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

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

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

[0017] In a seventh aspect of this application, a kit is provided comprising one or more of the following: a mutant as described in the first aspect, a conjugate 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.

[0018] In an eighth aspect of this application, there is provided the use of a mutant as described in the first aspect, a conjugate 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, or a host cell as described in the fifth aspect in the preparation of a medicament for use against tumors.

[0019] This application reduces the affinity of IL-21 for its receptor IL-21R and γC, resulting in multiple mutants with reduced affinity and activity in IL-21R(+) cells. In some embodiments, the druggability of IL-21 is optimized by designing artificial disulfide bonds and removing exposed furin cleavage sites, ultimately leading to the fusion of IL-21 mutant PD-1 antibodies with improved stability, good druggability, high efficacy, and low toxicity. For example, compared to AMG256, CRB2110A, CRB2110B, and CRB2110C molecules exhibit stronger IL-21 signaling pathway activation activity and can more effectively activate cells with different PD-1 expression levels. They also show significant improvements in solubility, thermal stability, and accelerated stability; pharmacokinetic parameters are significantly improved, with a half-life 1.5-1.7 times that of AMG256 and 4.4-5.1 times that of the original mutant. 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 This invention illustrates the detection of IL-21 biological activity in Baf3-stat3-IL21R-Luc cells with an IL-21 mutation according to one embodiment of the present application.

[0022] Figure 2 This application shows the structure of a PD-1 antibody and IL-21 mutant fusion protein according to one embodiment of the present application.

[0023] Figure 3A , Figure 3B and Figure 3C This application illustrates the detection of affinity between the PD-1 antibody and the IL-21 mutant fusion protein and the IL-21 receptor in an embodiment of this application.

[0024] Figure 4 This application illustrates the detection of IL-21 biological activity of the PD-1 antibody and IL-21 mutant fusion protein in the PD-1(-) Baf3-stat3-IL21R-Luc cell line according to the embodiments of this application.

[0025] Figure 5 This invention illustrates the detection of IL-21 biological activity in Baf3-stat3-IL21R-Luc cell lines with different PD-1 expression levels in the embodiments of this application.

[0026] Figure 6 This application illustrates the serum stability analysis of the PD-1 antibody and IL-21 mutant fusion protein in the embodiments described herein.

[0027] Figure 7 This application demonstrates the accelerated stability of the PD-1 antibody and IL-21 mutant fusion protein in one embodiment of the present application.

[0028] Figure 8 This shows the mouse PK time curve of the PD-1 antibody and IL-21 mutant fusion protein according to one embodiment of this application. Detailed Implementation

[0029] 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.

[0030] 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.

[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 other terms that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0032] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” used in this application include all suitable combinations of any two or more of the listed items.

[0033] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be determined by whether it can implement the technical solution of this application, solve the technical problem of this application, or achieve the expected technical effect of this application.

[0034] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate a connection in the coverage of different technical solutions presented earlier and later, 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.

[0035] 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."

[0036] 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.

[0037] 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.

[0038] In this application, the exemplary descriptions involving "in some implementations (or embodiments)" or "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.

[0039] 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.

[0040] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, 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 "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0041] 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.

[0042] This application provides a mutant of interleukin-21 (IL-21), wherein the mutant has an amino acid mutation at least at 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); and 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 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.

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

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

[0046] For example, 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 ​​substitution occurs at positions 16 and 73.

[0048] In this application, the wild-type IL-21 may comprise an amino acid fragment with 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] For example, 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 of the following combinations of mutation sites:

[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 NO: 11 to 47.

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

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

[0061] Unless otherwise specified, the term "operably linked" in this application refers to the functional relationship between two regions of the conjugate, i.e., other parts, and the IL-21 mutant; wherein the two regions are linked to produce the conjugate.

[0062] In this application, the antigen-binding molecule may be an antibody, such as 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 comprises 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 K, each consisting of a variable region (VL) and a constant region. Antibodies are Y-shaped, wherein the stem of the Y-shaped structure consists 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 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 the antibody-antigen binding fragments disclosed in this application can be defined or identified by the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, *Journal of Molecular Biology*, 273(4), 927 (1997); Chothia, C. et al., *Journal of Molecular Biology*, Dec 5; 186(3): 651-63 (1985); Chothia, C. and Lesk, AM, *Journal of Molecular Biology*, 196, 901 (1987); Chothia, C. et al., *Nature*, Dec 21-28; 342(6252): 877-83 (1989); Kabat, E.A. et al., *Sequences of Proteins*, 2009-2009). of immunological interest), 5th edition, 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 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).

[0064] The term "Fab" in antibody refers to the portion of an antibody composed of a single light chain (variable region and constant region) linked to the variable region and first constant region of a single heavy chain via disulfide bonds.

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

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

[0067] In some embodiments, the mutant is operatively 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 with an amino acid sequence as shown in SEQ ID NO: 50, a light chain with an amino acid sequence as shown in SEQ ID NO: 49 or 52, and a heavy chain 2 with an amino acid sequence as shown in SEQ ID NO: 51 or 53.

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

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

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

[0072] Furthermore, this application also provides functional variants of the conjugates (e.g., fusion proteins) 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 conjugate (e.g., fusion protein), wherein the functional variant retains the biological activity of the conjugate (e.g., 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 conjugate (e.g., fusion protein).

[0073] The functional variant may comprise, for example, the amino acid sequence of a parent conjugate (e.g., a fusion protein) having at least one conserved amino acid substitution. Alternatively or additionally, the functional variant may comprise the amino acid sequence of a parent conjugate (e.g., a 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 an increase in the biological activity of the functional variant compared to the parent conjugate (e.g., a fusion protein).

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

[0075] Conjugates (e.g., fusion proteins) of embodiments of this application (including the functional portion and functional variants of this application) may contain 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, 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.

[0076] Mutants that have a certain degree of amino acid homology with the amino acid sequence of the conjugate (e.g., fusion protein) as described above, such as 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.

[0077] 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)), and ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., *Methods of Enzymology*). The alignments used to determine the percentage of identity of amino acid (or nucleic acid) sequences can be implemented using software such as ALIGN or Megalign (DNASTAR). 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.

[0078] 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.

[0079]

[0080]

[0081] This application also provides a nucleic acid molecule that encodes the mutant or conjugate described above.

[0082] 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 herein comprises a continuous open reading frame encoding an antibody or fragment, derivative, mutant protein, or variant thereof provided herein.

[0083] This application also provides a recombinant expression vector comprising the nucleic acid molecules described above.

[0084] 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.

[0085] This application also provides a host cell that expresses the mutant or conjugate described above.

[0086] 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.

[0087] 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.

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

[0089] 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.

[0090] 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 their analogues. 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.

[0091] This application also provides a kit comprising one or more of the mutant, conjugate, nucleic acid molecule, recombinant expression vector, host cell, and pharmaceutical composition as described above, as well as a container.

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

[0093] Culture medium is obtained by culturing host cells as described above; and,

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

[0095] This application also provides a kit comprising the mutant, conjugate, nucleic acid molecule, recombinant expression vector, or host cell as described above for use in the preparation of a drug for antitumor purposes.

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

[0097] "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, and contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "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.

[0098] The following are some examples.

[0099] 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.

[0100] Example 1: Design, preparation, and affinity detection of mutations that reduce IL-21 affinity, improve stability, and enhance drug-likeness.

[0101] Using the publicly available crystal structure of the IL-21:IL-211R:γC complex (PDB ID: 8ENT) as a structural template, mutations were designed to reduce the affinity of IL-21 and improve its stability and drug-likeness using artificial intelligence and computer-aided design methods. 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 molecule was 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 and a Sartorius Octet RH96 sensor, biotinylated IL21R&hγC dimer receptor (Acro biosystems, ILA-H52W9) was captured via an SA sensor. The affinity of each antibody for the receptor was then measured at a concentration of 25 nM, with baselines of 200 s for Baseline 1, 60 s for Baseline 2, binding at 600 s, and dissociation at 3600 s. The results are shown in Table 1. Most antibodies showed reduced binding to the IL21R&hγC dimer receptor, either very weakly or not at all.

[0104] Table 1. Results of mutations leading to decreased IL-21 affinity, increased stability, and improved drug-likeness.

[0105]

[0106]

[0107]

[0108]

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

[0110] The reporter gene assay was used to detect the activation of the STAT3 signaling pathway in Baf3-stat3-IL21R-Luc cells by IL-21 mutant molecules. The Baf3-stat3-IL21R-Luc cells (H_IL-21Reporter Cell Line, GM, cat: GM-C15762) used in this experiment were Baf3 cells that stably expressed human IL21R and STAT3-induced luciferase reporter genes. Specific procedures: Collect H_IL-21 Reporter Cell Line cells in logarithmic growth phase, centrifuge, resuspend in analysis 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 the IL-21 mutant molecule, dilute it to different concentrations with analysis buffer (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.

[0111] 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 of each sequence of the PD-1 antibody and IL-21 mutant fusion protein, they 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. The structure and sequence of the PD-1 antibody and IL-21 mutant fusion protein are shown below. Figure 2 And Table 2.

[0114] Table 2. Sequence of PD-1 antibody and IL-21 mutant fusion protein

[0115]

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

[0117] Using a Biacore 8K microarray with a CM5 chip, the amino-coupled antigen hPD1-his (Acro, PD1-H5221) was analyzed, yielding approximately 16 RU or 60 RU (RU stands for response unit or resonance unit). Literature on surface plasmon resonance (SPR) technology indicates that, when using a CM5 chip for protein-protein interaction analysis, 1 RU roughly corresponds to 1 pg / mm² of surface plasmon resonance (SPR) activity. 2 Concentration changes. The response value of the Biacore system should always be in units of RU, not in units of concentration. ); then bind the antibody at concentrations of 50, 25, 12.5, 6.25, 3.125, 1.56, and 0 nM, binding for 100 s, dissociation for 180 s, regeneration with Glycine 1.5 (Cytiva) for 30 s, at a rate of 30 μL / min. The results are shown in Table 3. The results show that at conjugation amounts of 16 RU and 60 RU, Pembrolizumab tends to bind both monovalently and bivalently, while AMG256 tends to bind bivalently (monovalent binding affinity is 1.67e-09M). The affinity of CRB2110A and CRB2110C molecules is in the nM range, while the CRB2110B molecule, through mutation of the CDR region of Pembrolizumab, increases affinity {CN202311797802.5}.

[0118] Table 3. Affinity detection of PD-1 antibody and IL-21 mutant fusion protein with human PD-1 antigen.

[0119]

[0120] Using BLI technology and a Sartorius Octect RH96, biotinylated hIL21R receptors (Acrobiosystems, ILR-H5226), IL21R & hγC dimer receptors (Acrobiosystems, ILA-H52W9), and cIL21R receptors (Acrobiosystems, ILR-C5259) were captured via SA sensors. The affinities with each antibody were then measured: CRB2110W at concentrations of 10, 3.33, 1.11, and 0.37 nM, and other antibodies at 300 nM. Baseline 1 was 200 s, Baseline 2 was 600 s, binding was 600 s, and dissociation was 3600 s. Results are as follows: Figure 3A , Figure 3B and Figure 3CAs shown, the affinity of CRB2110W (unmutated IL-21) for the hIL21R receptor, the IL21R&hγC dimer receptor, and the cIL21R receptor were 15 pM, 25 pM, and 26 pM, respectively. Pembrolizumab served as a negative control and did not bind to the above receptors. The IL-21 affinity of the CRB2110A, CRB2110B, and CRB2110C fusion proteins was reduced to the same level as AMG256, all greater than 300 nM.

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

[0122] 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 the fusion protein of PD-1 antibody and IL-21 mutant. The PD-1+ Baf3-stat3-IL21R-Luc cells (Baf3-IL21R-hPD1-H03) used 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-21ReporterCell Line, GM-C15762) used in this assay were Baf3 cells stably expressing human IL21R and STAT3-induced luciferase reporter genes. Specific procedures: Collect logarithmically growing Baf3-IL21R-hPD1-H03 or H_IL-21ReporterCell Line cells, centrifuge, resuspend in analysis 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 the PD-1 antibody and IL-21 mutant fusion protein, dilute with analysis 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 AssaySystem (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.

[0123] 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 4 and Figure 5 As shown, on PD-1 negative cells, the activities of CRB2110A, CRB2110B, and AMG256 were all relatively low (EC50 > 4E6 pM), while CRB2110C was slightly higher (~7.5E5 pM). IL-21 signaling pathway activation was observed in PD-1 positive cells with varying expression levels: CRB2110C > CRB2110B > CRB2110A > AMG256. Furthermore, as the expression level of PD-1 on the cell surface decreased, the activities of all antibody fusion proteins also decreased to varying degrees. However, AMG256 showed no activity in cells with low PD-1 expression or low PD-1 expression, while CRB2110A, CRB2110B, and CRB2110C all retained some activity. Therefore, compared with AMG256, CRB2110A, CRB2110B, and CRB2110C molecules have stronger IL-21 signaling pathway activation activity and can more effectively activate IL-21 signaling activity in 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 cynomolgus monkey serum were aseptically filtered with PBS + 1% BSA buffer (PBSA). CRB2110W, AMG256, Pembrolizumab, CRB2110A, CRB2110B, and CRB2110C molecules were added to the serum at an initial concentration of 0.2 mg / ml. The samples were aseptically aliquoted into 0.6 ml sterile centrifuge tubes at 50 μL / tube and incubated at 37°C for 0 h, 1 d, 3 d, 5 d, 7 d, 10 d, and 14 d. The samples were then stored at -80°C. The concentration of intact molecules was determined using a sandwich assay with anti-PD1 and anti-IL-21 antibodies.

[0126] The results are as follows Figure 6 As shown, CRB2110W exhibits poor plasma stability, with its concentration significantly decreasing when placed in sera from three different species and in PBS + 1% BSA buffer. CRB2110A, CRB2110B, and CRB2110C show significantly improved stability in the sera of the three different species compared to CRB2110W, and in human serum, CRB2110B demonstrates superior stability compared to AMG256.

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

[0128] During the concentration and solution change process of the prepared CRB2110A, CRB2110B, CRB2110C, and AMG256, CRB2110A, CRB2110B, and CRB2110C molecules could be concentrated to 100 mg / ml in the PBS system, while AMG256 molecules became gel-like and clogged the membrane after being concentrated to more than 8 mg / ml in the PBS system. Changing the solution to the histidine system did not improve the situation, and the concentration could only be reduced to 8 mg / ml in the end.

[0129] CRB2110A, CRB2110B, CRB2110C, and AMG256 were replaced with 20 mM histidine buffer (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 all higher than those of AMG256, exhibiting better thermal stability.

[0130] Table 4. Tm and Tagg temperatures of PD-1 antibody and IL-21 mutant fusion protein

[0131]

[0132] CRB2110A, CRB2110B, CRB2110C, and AMG256 were replaced with 20 mM histidine buffer (pH 6.2) and incubated at 45°C for one week. Changes in appearance and turbidity were compared. The results are as follows: Figure 7 As shown, CRB2110A and CRB2110B exhibited good stability, CRB2110C showed slight turbidity, and AMG256 showed severe colloid formation and precipitation. Therefore, compared to AMG256, CRB2110A and CRB2110B demonstrated better accelerated stability.

[0133] Example 8: Mouse PK detection of PD-1 antibody and IL-21 mutant fusion protein

[0134] Forty-eight male C57BL mice with a uniform body weight of 20-24g and an age of 7-9 weeks were randomly divided into 16 subgroups (n=3 per subgroup) to administer different candidate molecules. Two subgroups were administered each test molecule, with crossover blood collection. The dosage for each candidate molecule was uniformly 10 mg / kg, with a 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 0.5h, 6h, 24h, 72h, 144h, 240h, 360h, and 480h 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 hours of collection and centrifuged at 4000g for 10 min at room temperature. After centrifugation, the collected serum samples were stored at -80℃. After blood collection, the drug concentration in the serum sample was measured, and a drug concentration-time curve was plotted. The main metabolic kinetic parameters t1 / 2, Cmax, AUC, Vd, etc. were calculated.

[0135] The drug-time curve and pharmacokinetic parameters are shown in the following figures. Figure 8 As shown in Table 5, CRB2110A, CRB2110B, and CRB2110C all showed significantly increased in vivo exposure and half-life compared to CRB2110W. The half-lives of CRB2110A, CRB2110B, and CRB2110C were 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 the PD-1 antibody-IL-21 mutant fusion protein

[0137]

[0138] 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.

[0139] 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. A mutant of interleukin-21 (IL-21), characterized in that, The mutant has a unique combination of mutation sites compared to wild-type IL-21: IN76E, D37P, I16C, and K73C; IN76E indicates the insertion of glutamic acid before the 76th amino acid; The amino acid sequence of the wild-type IL-21 is shown in SEQ ID NO:

58.

2. The IL-21 mutant as described in claim 1, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:

37.

3. A conjugate, characterized in that, Its structure is as follows: The IL-21 mutant as described in claim 1 or 2 is operatively linked to the C-terminus of the heavy chain in an antigen-binding molecule, and the conjugate consists of two different heavy chains, heavy chain 1 and heavy chain 2, and a light chain. The amino acid sequence of the heavy chain 1 is shown in SEQ ID NO: 50, the amino acid sequence of the light chain is shown in SEQ ID NO: 49, and the amino acid sequence of the heavy chain 2 is shown in SEQ ID NO:

53.

4. A nucleic acid molecule, characterized in that, Its encoding is the mutant as described in any one of claims 1 to 2 or the conjugate 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 is a viral vector.

7. A host cell, characterized in that, It expresses the mutant as described in any one of claims 1 to 2 or the conjugate as described in claim 3.

8. A pharmaceutical composition, characterized in that, It comprises the mutant as described in any one of claims 1 to 2 or the conjugate as described in claim 3, and a pharmaceutically acceptable carrier; Pharmaceutically acceptable carriers include excipients.

9. A reagent kit, characterized in that, It comprises one or more of the mutants as described in any one of claims 1 to 2, the conjugates as described in claim 3, the nucleic acid molecules as described in claim 4, the recombinant expression vectors as described in claim 5 or 6, the host cells as described in claim 7, and the pharmaceutical compositions as described in claim 8, as well as a container.

10. A method for preparing a mutant as described in any one of claims 1-2 or a conjugate 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, The mutant or the conjugate is isolated from the culture medium.

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

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