IL-2 mutant and fusion protein of IL-2 mutant and anti-PD-1 antibody

Abstract

The invention discloses an IL-2 mutant and a fusion protein of the IL-2 mutant and an anti-PD-1 antibody. In particular to an IL-2 mutant protein with the following mutation sites: Q11R, E15D, D20E, K35E, L36E, T37L, R38E, M39E, L40K, R81Q and / or Q126R, and a fusion protein containing the IL-2 mutant protein and a PD-1 antibody. The fusion protein provided by the invention reduces the affinity with an IL-2 receptor, can significantly inhibit the growth of tumors, and has good safety. The fusion protein PD-1 / IL2m disclosed by the invention can be effective on tumors which are ineffective in PD-1 treatment, meanwhile, through mutation of IL-2, the safety is improved while the activity of the PD-1 / IL-2 fusion protein is ensured, and the fusion protein has a wide clinical application prospect in the anti-tumor field.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an IL-2 mutant and a fusion protein thereof with an anti-PD-1 antibody. Background Art

[0002] Interleukin-2 (IL-2) is a protein containing 133 amino acid residues with a molecular weight of 15.5 kDa. IL-2 is composed of a four-alpha-helical core and a beta segment. It is primarily produced by antigen-activated Th1 CD4+ T cells, and to a lesser extent by CD8+ T cells and NK cells. Interleukin-2 (IL-2), also known as T cell growth factor, is a growth factor for all T cell subsets and promotes the proliferation of activated B cells. Therefore, it is a key factor in regulating immune responses and is also involved in antibody responses, hematopoiesis, and tumor surveillance. IL-2's target cells include T cells, NK cells, B cells, and monocytes and macrophages. IL-2 has complex functions and plays a crucial role in a range of processes, including immune cell maturation, activation, proliferation, and immunoregulation. IL-2 also participates in a variety of physiological and pathological responses in the body. Clinically, it is primarily used to treat viral infections, immunodeficiency, and autoimmune disorders. IL-2 can also be used to treat tumors and has a certain therapeutic effect. However, interleukin-2 has certain side effects, including fever, vomiting, metabolic disorders, and liver and kidney damage. How to reduce the toxicity of IL-2 is also one of the research directions of biomedicine.

[0003] The IL-2 receptor is a heteromer composed of three chains: α, β, and γ. Specifically, the IL-2R consists of IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). β and γ are essential for activation of downstream signaling pathways, while α primarily facilitates their binding. IL-2 exerts its signaling regulatory effects by binding to its receptor. IL-2Rγ (CD132) is expressed on all lymphocytes, while IL-2Rβ (CD122) is constitutively expressed on natural killer (NK) and CD8 memory T cells, and is also expressed on naive T cells after antigen recognition. IL-2Rγ (CD132) and IL-2Rβ (CD122) are subunits capable of intracellular signaling, while IL-2Rα (CD25) is not involved in signaling. IL-2 binds to the IL-2 receptor (IL-2R) with varying affinities, depending on the specific subunit combination of the IL-2 receptor.

[0004] Different combinations of IL-2 receptor subunits determine the affinity of IL-2 for the receptor. Monomeric IL-2Rα (CD25) is expressed on the surface of activated lymphocytes and has a low binding affinity for IL-2. Dimeric IL-2Rβ / γ is present on the surface of CD8 memory T cells, naive CD8 T cells, and natural killer cells (NK cells), and has a moderate binding affinity for IL-2. Trimeric IL-2Rα / β / γ is co-expressed on activated T cells and regulatory T cells (Tregs), and has the highest affinity for IL-2. Low concentrations of IL-2 preferentially bind to the high-affinity ternary complex receptor (IL-2Rα / βγ / ) constitutively expressed on regulatory T cells (Tregs). Therefore, low-dose IL-2 can activate Tregs to induce immune tolerance, and is used to treat autoimmune diseases such as type 1 diabetes and lupus erythematosus. After saturating Tregs with high concentrations of IL-2, it will bind to the medium-affinity binary complex receptor (IL2Rβγ) constitutively expressed on NK cells and CD8 T cells (also called cytotoxic T cells, which have a killing effect on antigenic substances such as viruses and tumor cells), activating NK cells and CD8+ T cells and increasing anti-cancer activity. When the IL-2Rα / CD25 subunit exists alone, its affinity for IL-2 is only one percent of that of the ternary complex receptor (IL2Rαβγ). Therefore, IL-2 can be mutated according to different clinical needs to change the binding of IL-2 to IL2Rα or IL2Rβγ, thereby obtaining IL-2 mutants with different clinical application functions.

[0005] PD-1 (programmed death-1) was first discovered in apoptotic T-cell hybridomas. Due to its association with apoptosis, it was named the programmed death-1 receptor. PD-1 is an important immunosuppressive molecule. PD-1 is primarily expressed on activated T and B cells. PD-1 has two ligands: PD-L1 (B7-H1) and PD-L2 (B7-DC). The tumor microenvironment in the body induces high expression of PD-1 by infiltrating T cells, and tumor cells also overexpress PD-1 ligands PD-L1 and PD-L2. When PD-L1 on the surface of tumor cells binds to PD-1 on the surface of T cells, T cell function is suppressed, preventing the immune system from signaling to attack the tumor. PD-1 / PD-L1 inhibitors can block the binding of PD-1 to PD-L1, interrupting the negative regulatory signal and restoring T cell activity, thereby enhancing the immune response. PD-1 and PD-L1 inhibitors have shown significant efficacy in a variety of tumors. However, PD-1 / PD-L1 inhibitors are only effective in 20%-40% of solid tumors, and most cancer patients respond poorly or quickly develop drug resistance. Treating cancer patients for whom PD-1 is ineffective or ineffective has long been a hot topic in biomedical research.

[0006] In view of this, researching and developing new, safer and more effective IL-2 and PD-1 antibody fusion proteins is of great significance for optimizing tumor treatment strategies and improving patients' clinical benefits. Summary of the Invention

[0007] The present invention aims to provide a novel IL-2 and PD-1 antibody fusion protein and its application. The technical problems to be solved are not limited to the technical subject matter described herein. Those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0008] To achieve the above object, the present invention first provides a mutant protein, wherein the amino acid sequence of the mutant protein may have any of the following mutations relative to the amino acid sequence of IL-2:

[0009] A1) Q11R, E15D, D20E, K35E, L36E, T37L, R38E, M39E, L40K, R81Q, and / or Q126R;

[0010] A2)K35E / L36E / T37L / R38E / M39E / L40K;

[0011] A3)K35E / L36E / T37L / R38E / M39E / L40K / Q126R;

[0012] A4)E15D / K35E / L36E / T37L / R38E / M39E / L40K / R81Q;

[0013] A5)Q11R / D20E / K35E / L36E / T37L / R38E / M39E / L40K;

[0014] A6)E15D / K35E / L36E / T37L / R38E / M39E / L40K / R81Q / Q126R;

[0015] A7)Q11R / D20E / K35E / L36E / T37L / R38E / M39E / L40K / Q126R;

[0016] A8)E15D / R81Q / Q126R;

[0017] A9)Q11R / D20E / Q126R;

[0018] The amino acid sequence of the IL-2 may be any of the following:

[0019] B1) SEQ ID NO: 1;

[0020] B2) SEQ ID NO: 2;

[0021] B3) an amino acid sequence obtained by mutating Cys at position 125 of SEQ ID NO: 1 to Ser, while keeping the other amino acids unchanged;

[0022] B4) An amino acid sequence that is 95% or more identical to the amino acid sequence shown in B1), B2) or B3) and has the same function.

[0023] The amino acid sequence shown in SEQ ID NO: 1 may be the amino acid sequence of wild-type IL-2.

[0024] The amino acid sequence shown in SEQ ID NO: 2 may be the amino acid sequence of recombinant human interleukin-2 (Aldesleukin).

[0025] The amino acid sequence shown in B3) is different from that in SEQ ID NO: 1 in the amino acid at position 125. Whether the amino acid at position 125 is Cys or Ser, it does not affect the activity of IL-2.

[0026] Furthermore, the amino acid sequence of the mutant protein may be as shown in any one of SEQ ID NO: 3 to SEQ ID NO: 10.

[0027] Compared with the IL-2 (B1), B2), B3) or B4), the mutant protein has at least one of the following characteristics:

[0028] (1) The mutant protein has reduced ability to bind to IL-2Rβ / γ;

[0029] (2) The mutant protein has reduced ability to bind to IL-2Rα (CD25).

[0030] The mutant protein (IL-2 mutant) can reduce the binding ability of IL-2 to receptor Alpha and / or IL2 receptor beta / gamma complex through mutation, thereby reducing the side effects of IL-2 while ensuring the efficacy.

[0031] The present invention also provides a fusion protein, which may be named PD-1 / IL2m, and the fusion protein comprises a PD-1 antibody and the mutant protein.

[0032] Furthermore, the mutant protein can be directly connected to the C-terminus of the PD-1 antibody or connected via a linker.

[0033] Furthermore, the mutant protein can be linked to the C-terminus of one of the heavy chain constant regions of the PD-1 antibody.

[0034] The linker may be a flexible peptide linker, for example, a peptide linker comprising glycine, serine, proline, and / or lysine residues. The peptide linker may be composed of 1-40 amino acids. The linker includes, but is not limited to, SGGGGS, (GxS)n, (GSSGG)n, (GGSGG)n, (GSGGSG)n, (GSGSGS)n, and (EAAAK)n, and various combinations thereof. Wherein: n can be any integer between 1 and 10; x can be any integer between 1 and 6.

[0035] Furthermore, the linker may be (GGGGS)n. n is preferably 3 or 4. In one or more embodiments of the present invention, the linker is (GGGGS)3.

[0036] It should be understood that the presence of a linker is optional and that the length of the flexible linker can be adjusted to allow for the correct folding of the fusion protein or to achieve optimal biological activity. The characteristics of the linker and its suitability for a specific purpose are known in the art, and those skilled in the art can independently select and / or optimize each peptide linker.

[0037] The PD-1 antibody can be any antibody capable of binding to PD-1 (including anti-PD-1 antibodies known to those skilled in the art). The antibody includes but is not limited to monoclonal antibodies, bispecific antibodies, multispecific antibodies, humanized antibodies (including chimeric antibodies, CDR-grafted antibodies, and SDR-grafted antibodies), and fully human antibodies.

[0038] The constant region of the PD-1 antibody can be selected from the heavy chain constant region of IgG, IgA, IgM, IgD or IgE or variants thereof, kappa (κ) or lambda (λ) type light chain constant region or variants thereof, and can also be selected from the CH1, Fc and CH3 domains or variants thereof, but is not limited thereto. Furthermore, the constant region of the PD-1 antibody can be selected from the heavy chain constant region, CH1, Fc and CH3 domains or variants thereof of human IgG subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, but is not limited thereto.

[0039] Furthermore, the constant region of the PD-1 antibody can be selected from the Fc region of IgG1, or a variant thereof (e.g., a variant that weakens antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC)).

[0040] Furthermore, the PD-1 antibody may be an anti-human PD-1 antibody or an anti-human PD-1 antibody with a knob-in-hole structure.

[0041] The "knob-into-hole" structure is well known to those skilled in the art. It involves modifying the antibody constant region (CH3 domain) so that one heavy chain constant region is mutated to form a protruding "knob" structure, while the other heavy chain constant region is mutated to form a recessed "hole" structure. This knob-into-hole structure enables the two different heavy chains to specifically bind to form a stable heterodimer.

[0042] Knob into hole mutant pairings include but are not limited to: i) one Fc region amino acid sequence contains a T366W mutation, while the other Fc amino acid sequence contains T366S, L368A, and Y407V mutations, or ii) one Fc region amino acid sequence contains T366W and Y349C mutations, while the other Fc amino acid sequence contains T366S, L368A, Y407V, and S354C mutations, or c) one Fc region amino acid sequence contains T366W and S354C mutations, while the other Fc amino acid sequence contains T366S, L368A, Y407V, and S349C mutations.

[0043] In some embodiments of the present invention, one Fc region amino acid sequence comprises a "knob" mutation: T366W; and the other Fc region amino acid sequence comprises a "hole" mutation: T366S, L368A, and Y407V.

[0044] In some embodiments, the PD-1 antibody comprises:

[0045] 1) a heavy chain containing a "knob" mutation, whose amino acid sequence may be as shown in SEQ ID NO: 11;

[0046] 2) a heavy chain containing a "hole" mutation, whose amino acid sequence may be as shown in positions 1-447 of SEQ ID NO: 15, positions 1-447 of SEQ ID NO: 16, positions 1-447 of SEQ ID NO: 17, positions 1-447 of SEQ ID NO: 18, positions 1-447 of SEQ ID NO: 19, positions 1-447 of SEQ ID NO: 20, positions 1-447 of SEQ ID NO: 21, or positions 1-447 of SEQ ID NO: 22;

[0047] 3) two light chains, wherein the amino acid sequence of the light chains may be as shown in SEQ ID NO: 12.

[0048] Furthermore, the fusion protein may comprise two light chains, a first heavy chain and a second heavy chain,

[0049] The amino acid sequence of the light chain may comprise SEQ ID NO: 12, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 12 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 12;

[0050] The amino acid sequence of the first heavy chain may comprise SEQ ID NO: 11, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 11 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 11;

[0051] The amino acid sequence of the second heavy chain may comprise any one of the following:

[0052] C1) SEQ ID NO: 15, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 15 and having an identity of 95% or more to SEQ ID NO: 15;

[0053] C2) SEQ ID NO: 16, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 16 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 16;

[0054] C3) SEQ ID NO: 17, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 17 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 17;

[0055] C4) SEQ ID NO: 18, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 18 and having an identity of 95% or more to SEQ ID NO: 18;

[0056] C5) SEQ ID NO: 19, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 19 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 19;

[0057] C6) SEQ ID NO: 20, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 20 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 20;

[0058] C7) SEQ ID NO: 21, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 21 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 21;

[0059] C8) SEQ ID NO: 22, or an amino acid sequence obtained by substituting, deleting and / or adding amino acid residues in the amino acid sequence of SEQ ID NO: 22 and having an identity of 95% or more to SEQ ID NO: 22.

[0060] The substitutions described herein may be conservative substitutions.

[0061] In some embodiments, amino acid residue substitutions, deletions, and / or additions may occur in any of the fusion proteins of the present invention, as long as such changes do not substantially alter the function and activity of the fusion protein. For example, conservative substitutions (as is well known to those skilled in the art, conservative substitutions of amino acids do not alter the properties and function of the protein) may be made in non-functional regions of the fusion protein (such as framework regions) without substantially reducing binding affinity; such substitutions do not include substitutions of amino acid residues at the mutation sites described herein. In some embodiments, the linker in any of the fusion proteins of the present invention may be replaced with other linkers well known to those skilled in the art.

[0062] The fusion protein PD-1 / IL2m of the present invention comprises two parts (such as Figure 1 (as shown): a first monomer and a second monomer. The first monomer comprises the light chain and the first heavy chain of the PD-1 antibody, wherein the first heavy chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region and a heavy chain constant region Fc1 (containing a "knob" mutation). The second monomer comprises the light chain and the second heavy chain of the PD-1 antibody, wherein the second heavy chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region, a heavy chain constant region Fc2 (containing a "hole" mutation), a flexible linker, and an IL-2 mutant.

[0063] The first monomer and the second monomer can form a heterodimer through a knob-into-hole structure. This connection pairing method is well known to those skilled in the art. The knob-into-hole structure can prevent the first monomer or the second monomer from forming a homodimer.

[0064] The fusion protein PD-1 / IL2m of the present invention may further include a signal peptide. The signal peptide may be at the N-terminus of the fusion protein. The signal peptide is non-restrictive and may be any signal peptide that can effectively secrete the fusion protein PD-1 / IL2m of the present invention. It is known to those skilled in the art that the function of the signal peptide is to guide the newly synthesized protein to the secretory pathway and is often removed during this process. By introducing a signal peptide, the fusion protein PD-1 / IL2m of the present invention can be guided to cross the cell membrane for secretory expression. Signal peptides are known to those skilled in the art, and those skilled in the art can select suitable signal peptides as needed. Suitable signal peptides can be found at http: / / www.signalpeptide.de / . The signal peptide may be a signal peptide derived from mammals (including humans).

[0065] In order to facilitate the separation, purification, detection and / or positioning of the fusion protein PD-1 / IL2m of the present invention, a tag protein can be connected to the N-terminus and / or C-terminus of the fusion protein PD-1 / IL2m. The tag protein includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, Trx (thioredoxin) tag protein, nitrogen utilization substrate A (NusA) tag protein, His tag protein (His-tag), Strep tag protein, MBP (maltose binding protein) tag protein, Flag tag protein, SUMO (small molecule ubiquitin-like modifier protein) tag protein, HA (influenza hemagglutinin) tag protein, Myc tag protein, LacZ tag protein, CBD (cellulose binding domain) tag protein, bacteriophage T7 protein kinase (T7PK) tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein, or a combination of the above tag proteins. Those skilled in the art know how to select a suitable tag protein according to the desired purpose. The use of tags does not alter the function of the recombinant protein; its purpose is to separate, purify, detect, and / or trace the protein. Therefore, the tagged proteins suitable for this application are not limited to specific types. Tagged proteins can be separated from the recombinant protein by chemical cleavage or enzymatic methods known in the art (e.g., introducing a protease cleavage site to remove the tag using TEV protease).

[0066] The fusion protein PD-1 / IL2m described herein has at least one of the following characteristics:

[0067] (1) Compared with wild-type IL-2, it has a reduced affinity for the IL-2 receptor; (2) Compared with wild-type IL-2, it has a reduced affinity for IL-2Rβ / γ; (3) Compared with wild-type IL-2, it has a reduced affinity for IL-2Rα (CD25); (4) It can specifically bind to the PD-1 protein; (5) It has a strong tumor cell killing ability; (6) It has a good ability to inhibit tumor growth; (7) It has high safety.

[0068] The present invention also provides a biomaterial, which may be any of the following:

[0069] D1) a nucleic acid molecule encoding the mutant protein;

[0070] D2) a nucleic acid molecule encoding any one of the fusion proteins described herein;

[0071] D3) an expression cassette containing the nucleic acid molecule described in D1) or D2);

[0072] D4) a recombinant vector containing the nucleic acid molecule described in D1) or D2), or a recombinant vector containing the expression cassette described in D3);

[0073] D5) a recombinant microorganism containing the nucleic acid molecule described in D1) or D2), or a recombinant microorganism containing the expression cassette described in D3), or a recombinant microorganism containing the recombinant vector described in D4);

[0074] D6) A recombinant host cell containing the nucleic acid molecule described in D1) or D2), or a recombinant host cell containing the expression cassette described in D3), or a recombinant host cell containing the recombinant vector described in D4).

[0075] In the above biological materials, the recombinant vector can be a cloning vector or an expression vector.

[0076] The recombinant vector can be constructed using an expression vector. The structure of the expression vector is well known to those skilled in the art, and the expression vector generally contains elements required for the expression of the target gene such as promoter, multiple cloning site, terminator, ribosome binding site, etc., and can also contain screening marker genes (such as kanamycin resistance gene kanr, neomycin resistance gene neo, hygromycin resistance gene hyg, chloramphenicol resistance gene cat, streptomycin resistance gene str, bleomycin resistance gene ble, etc.). The expression vector can be constructed using any method known in the art (such as recombinant technology, synthetic technology, etc.), or can be purchased commercially, and those skilled in the art can select suitable expression vectors as needed.

[0077] Furthermore, the recombinant vector can be a recombinant expression vector obtained by cloning the gene encoding the fusion protein PD-1 / IL2m described in the present invention into an expression vector (including a prokaryotic expression vector and a eukaryotic expression vector). Although the expression vector used in the examples provided herein is the pCDNA3.4 vector, the present invention is not limited to this specific vector. Those skilled in the art may use other suitable vectors, as long as the vector is capable of expressing the fusion protein PD-1 / IL2m.

[0078] The gene encoding the fusion protein PD-1 / IL2m described herein can be any gene capable of encoding the fusion protein PD-1 / IL2m described herein. Taking into account codon degeneracy and codon preferences across species, those skilled in the art can utilize codons suitable for expression in a particular species as needed.

[0079] In the above biological material, the nucleic acid molecule can be selected from SEQ ID NO:24, SEQ ID NO:23, SEQ ID NO:27-SEQ ID NO:34.

[0080] The present invention also provides the use of the mutant protein, the fusion protein, or the biomaterial described herein in the preparation of a product for preventing and / or treating tumors or PD-1 target-related diseases.

[0081] The products described herein include, but are not limited to, reagents, kits (eg, therapeutic kits), formulations, medicaments, or pharmaceutical compositions.

[0082] The present invention also provides a pharmaceutical composition comprising the mutant protein or the fusion protein and one or more pharmaceutically acceptable carriers.

[0083] The pharmaceutical composition may have at least one of the following uses: (1) for preventing and / or treating tumors; (2) for preventing and / or treating PD-1 target-related diseases; (3) for inhibiting tumor growth; and (4) for killing tumor cells.

[0084] The pharmaceutically acceptable carrier is selected from excipients, preservatives, protective agents, cosolvents, diluents (such as water, physiological saline, PBS (phosphate buffer), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc.), wetting agents, disintegrants (such as dry starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, etc.), lubricants (such as sorbitan trioleate, soybean lecithin, lecithin, oleic acid, magnesium stearate, sodium lauryl sulfate, etc.), fillers (such as starch, dextrin, etc.), adhesives (such as gelatin, pectin, gum arabic, hydroxypropyl cellulose (CP), PVP, CMC-Na, etc.), penetration enhancers (such as Brij-78), pH regulator, stabilizer (such as sodium sulfite, citric acid, tartaric acid, EDTA, etc.), surfactant (such as Tween, Span, eucalyptus oil, polysorbate-80, sodium lauryl sulfate, soybean lecithin, sodium cholate, sodium deoxycholate, etc.), absorption accelerator (such as chitosan), thickener (such as sodium hyaluronate, sodium carboxymethyl cellulose, polyvinyl alcohol, etc.), antioxidant (such as sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, vitamin C, etc.), plasticizer (such as glycerol, sorbitol, phthalate, etc.), propellant (such as hydrofluoroalkane, dimethyl ether, etc.), aerosolizing agent, suspending agent, dispersant, colorant (such as TiO2, pigment, etc.) and flavoring agent. Those skilled in the art know that a carrier usually has multiple functions. For example, starch can be used as a disintegrant and a binder. Those skilled in the art can make a conventional selection of the above-mentioned carrier according to the properties of the drug and the route of administration.

[0085] Excipients are usually used in medicines to shape the medicine, change the physical state of the medicine, and play a supporting role. Excipients include but are not limited to: (1) Excipients for injections: such as solvent water, alcohols, ethers, amides, sulfonates, esters, etc.; (2) Excipients for injectable powders: such as sucrose, lactose, mannitol, etc.; (3) Excipients for sprays: such as soy lecithin, propylene glycol, borneol, ethanol, phenol, etc.; (4) Excipients for tablets: such as starch, sucrose, dextrin, methylcellulose, gelatin, polyethylene glycol, tartaric acid, boric acid, etc.; (5) Excipients for eye drops: such as sodium hyaluronate, disodium ethylenediaminetetraacetic acid (EDTA-Na2), etc.; (6) Excipients for suppositories: such as cocoa butter, semi-synthetic or fully synthetic fatty acid glycerides, glycerol gelatin, polyethylene glycol, etc.; (7) Excipients for granules: such as corn starch, bentonite, zeolite powder, etc.; (8) Excipients for capsules: such as gelatin, etc.; (9) Excipients for gels: such as gelatin, pectin, gum arabic, etc.; (10) Excipients for ointments: such as vaseline, paraffin, liquid paraffin, lanolin, lanolin alcohol, beeswax, lard, vegetable oil, silicone oil, silicone, soaps, higher fatty alcohols, fatty alcohol sulfates, polyols, polyethylene glycol, FAPG, etc.; (11) Excipients for patches: such as ethylene-vinyl acetate copolymer (EVA), pressure-sensitive adhesive (PSA), etc.; (12) Excipients for films: such as gelatin, shellac, gum arabic, polyvinyl alcohol compounds, acrylic acid copolymers, etc.

[0086] Biopharmaceutical preparations are susceptible to contamination by exogenous microorganisms during production, transportation, and storage, which can affect drug quality. Therefore, preservatives are typically added to inhibit the growth and reproduction of microorganisms. These preservatives include, but are not limited to, thimerosal, formaldehyde, phenol, and m-cresol.

[0087] The dosage forms of the pharmaceutical composition include, but are not limited to, injections (including injection solutions and powders for injection), gels, eye drops (including eye drops and intraocular injection solutions), oral solutions, suppositories, effervescent tablets, capsules, ointments, creams, sprays, aerosols, topical solutions, tablets, powders, pills, granules, drops, paints, patches, and long-acting sustained-release preparations. Those skilled in the art will appreciate that the various dosage forms described above can be prepared using the active ingredients, combined with suitable pharmaceutically acceptable carriers, and according to conventional preparation processes.

[0088] Furthermore, the pharmaceutical composition may be in the form of an injection preparation.

[0089] Injection generally refers to a solution or emulsion for injection into the body made after extraction and purification of the original drug, as well as a sterile powder prepared into a solution before use, which includes injection and powder for injection. The preparation method of injection is well known to those skilled in the art, and for example, powder for injection can be prepared by vacuum freeze drying technology, spray drying technology, spray freeze drying technology. It is also possible to prepare a solution by a concentrated or diluted method by mixing the drug with a suitable diluent, cosolvent and / or wetting agent, and then preparing the injection by steps such as filtration (such as surface filtration and / or deep filtration), embedding, and sterilization.

[0090] The administration methods of the pharmaceutical composition include, but are not limited to, injection (e.g., administration in the form of an injection), mucosal administration (e.g., administration in the form of a spray, aerosol, tablets, eye drops, suppositories, granules, capsules, etc.), and transdermal administration (e.g., administration in the form of a gel, ointment, patch, film, etc.).

[0091] The administration methods of the pharmaceutical composition include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, transdermal injection, intravenous injection, arterial injection, intraperitoneal injection, intraperitoneal injection, intrathecal injection, microneedle injection, intratumor injection, intracranial injection, mucosal administration, oral administration, skin application, oral and nasal spraying, aerosol inhalation, in vivo implantation, and in vitro device administration.

[0092] The active ingredient of the pharmaceutical composition may be the mutant protein or the fusion protein PD-1 / IL2m described herein.

[0093] The present invention also provides a method for preparing a fusion protein, which comprises expressing any fusion protein described herein in a host cell, and recovering or isolating the fusion protein.

[0094] Furthermore, the preparation method may include the following steps: cloning the coding gene of the fusion protein PD-1 / IL2m of the present invention into an expression vector (such as a prokaryotic expression vector, a eukaryotic expression vector and a viral expression vector) to obtain a recombinant expression vector; introducing the recombinant expression vector into a host cell to obtain a recombinant host cell expressing the fusion protein PD-1 / IL2m; culturing the recombinant host cell, and recovering or isolating the fusion protein PD-1 / IL2m from the cultured recombinant host cell culture.

[0095] The prokaryotic expression vector can be selected from Escherichia coli expression vectors (such as pET series vectors, etc.). The eukaryotic expression vector can be selected from yeast expression vectors (such as pYES2, pPICZaA, pUG6, etc.), insect cell expression vectors (such as pFastBac1, pMT-Bip-V5-HisA, pAc5.1, etc.) and mammalian cell expression vectors (such as pVAX1, pCAGGS, pGX0001, pCMV3, pcDNA3.1, pcDNA3.4, etc.). The viral expression vector can be selected from adeno-associated virus (AAV) vectors, adenovirus vectors, herpes simplex virus (HSV) vectors, lentivirus (LV) vectors, poxvirus vectors, retrovirus vectors, rhabdovirus (baculovirus) vectors, papillomavirus vectors, Sendai virus vectors and simian virus (Simianvirus) expression vectors.

[0096] The host cell may be a mammalian cell.

[0097] The present invention also provides a method for preventing or treating tumors or PD-1 target-related diseases, which comprises administering the mutant protein, the fusion protein, or the pharmaceutical composition described herein to a subject suffering from a tumor or a PD-1 target-related disease.

[0098] In the above method, the PD-1 target-related diseases may include tumors, immune-related diseases and infectious diseases.

[0099] The present invention also provides the mutant protein, or the fusion protein, or the pharmaceutical composition for preventing or treating tumors or PD-1 target-related diseases.

[0100] Among them, the PD-1 target-related diseases may include tumors, immune-related diseases and infectious diseases.

[0101] The tumors described herein include PD-1 positive or PD-L1 positive tumors.

[0102] The tumors described herein include, but are not limited to, lung cancer, colorectal cancer, pancreatic cancer, breast cancer, head and neck squamous cell carcinoma, gastric cancer, esophageal squamous cell carcinoma, urothelial carcinoma, melanoma, liver cancer, lymphoma, kidney cancer, esophageal cancer, cervical cancer, and ovarian cancer.

[0103] When referring to an IL-2 mutant (or mutein) herein, its amino acid sequence is determined by reference to the amino acid sequence of wild-type IL-2 (SEQ ID NO: 1) or recombinant human interleukin-2 (Aldesleukin, SEQ ID NO: 2) or the amino acid sequence set forth in B3) herein. When referring to a mutation, the mutation is described using the following format: "single-letter abbreviation of the amino acid before mutation, position of mutation, and single-letter abbreviation of the amino acid after mutation." For example, "Q11R" indicates that at position 11 of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or B3), the amino acid is mutated from glutamine (Q) to arginine (R).

[0104] Multiple mutations can also be separated by " / ", for example, "Q11R / D20E / Q126R", "Q11R / D20E / Q126R" means that at positions 11, 20 and 126 of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or B3), the amino acids are mutated from glutamine (Q), aspartic acid (D) and glutamine (Q) to arginine (R), glutamic acid (E) and arginine (R), respectively.

[0105] The present invention first uses computer-aided design and screening to identify IL-2 mutants that can reduce binding to IL-2Rα (CD25) and IL-2Rβ / γ to varying degrees. Based on this, the fusion protein PD-1 / IL2m was further designed. Experiments have shown that after mutating IL-2, the fusion protein PD-1 / IL2m of the present invention has reduced affinity for the IL-2 receptor, significantly reducing its affinity for both IL-2Rβ / γ and IL-2Rα (CD25), while maintaining good affinity for the PD-1 protein. The fusion protein PD-1 / IL2m of the present invention also has strong tumor cell killing ability and can significantly inhibit tumor growth. In vivo toxicity evaluation experiments have demonstrated that the fusion protein PD-1 / IL2m of the present invention has a good safety profile. The fusion protein PD-1 / IL2m of the present invention is effective against tumors resistant to PD-1 therapy. Furthermore, by mutating IL-2, the activity of the PD-1 / IL-2 fusion protein is maintained while also improving its safety, suggesting broad clinical application prospects in the anti-tumor field.

[0106] Definition of terms

[0107] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0108] The term "host cell," also referred to as a recipient cell, generally refers to any type of cell into which a vector can be introduced, such as plant cells and animal cells. The term "host cell" is understood to refer not only to a specific recipient cell but also to the progeny of such a cell. Due to natural, accidental, or intentional mutations and / or changes, such progeny may not necessarily be completely identical to the original parent cell, but are still included within the scope of host cells. Suitable host cells are known in the art, wherein: the plant cell can be, but is not limited to, plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum); the animal cell can be a mammalian cell (e.g., Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell substrain (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells, or NK cells), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., African clawed frog (Xenopus laevis) cells, or giant salamander (Andrias davidianus) cells). davidianus) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells, Sf-9 cells or Hi-5 cells), etc., but are not limited thereto.

[0109] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0110] The term "link" generally refers to the association of two or more molecules. The link can be covalent or non-covalent. The link described herein can be directly connected by a peptide bond or connected by a linker (joint).

[0111] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is generally expressed as a percentage. Identity as described herein may refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies having exactly the same sequence have 100% identity. Those skilled in the art will appreciate that the identity of an amino acid sequence or a nucleotide sequence can be determined using an identity search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a search to calculate the identity of the amino acid sequence, the value (%) of identity can then be obtained. In addition, sequence analysis software (such as CLC Main Workbench and MegAlign TM ), for example, using the computer program BLAST with default parameters, in particular BLASTP or TBLASTN. The 95% or greater identity described herein may be at least 95%, 96%, 97%, 98% or 99% or greater identity.

[0112] The term "conservative substitution" generally refers to replacing an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between aromatic side chain residues (e.g., Trp, Tyr, and Phe). It is known in the art that conservative substitutions generally do not cause significant changes in the conformational structure of the protein and do not substantially alter the biological activity of the protein. Conservative substitutions in a protein sequence that are expected to have little or no effect on the protein structure or function can be easily designed by one of ordinary skill in the art.

[0113] The term "introduction" generally refers to the transfer of exogenous genes into recipient cells such as eukaryotic recipient cells or prokaryotic recipient cells. The method of introduction is not particularly limited, and any known transformation method can be used as long as it can transfer the target gene into the recipient cell. The method of introduction may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria by chemical transformation methods (such as Ca ion-induced transformation methods, polyethylene glycol-mediated transformation methods, or metal cation-mediated transformation methods, etc.) or physical transformation methods (such as electroporation transformation methods). (2) transducing the target gene into the host bacteria by phage transduction methods. (3) transferring the target gene into plant recipient cells by physical or chemical methods, such as gene gun method (also known as microparticle bombardment method or biological missile method), chemical stimulation method, electric shock method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasonic method, air gun method and vortex method, etc. (4) Using vectors as a medium to transfer the target gene into plant recipient cells, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method (Agrobacterium mediated method), plant virus vector mediated transformation method, etc. (5) The target gene is introduced into isolated animal cells (transfection) by calcium phosphate coprecipitation method, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation method (i.e. electrotransfection method), microinjection, gene gun method or virus-mediated method (such as retrovirus infection method, adenovirus infection method, lentivirus infection method), etc.

[0114] The term "prevent" generally refers to methods performed to prevent or delay the onset of a disease, disorder, or symptom in a subject.

[0115] The term "treatment" generally refers to a method implemented to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in disease severity, reduction in disease extent, stabilization of the disease (i.e., no longer worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Additionally, treatment may also refer to prolonging the subject's survival compared to the expected survival if they were not receiving treatment.

[0116] The term "comprising" is not intended to be limiting, but rather inclusive and means that there may be additional elements other than the listed elements, and can be interpreted as "including, but not limited to." The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of." The terms "comprising" and "including" are used interchangeably herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 Schematic diagram of the PD-1 / IL2m molecular structure.

[0118] Figure 2 This is a diagram showing the proliferation of CTLL2 cells stimulated by different PD-1 / IL2m molecules.

[0119] Figure 3 This is a diagram showing the proliferation of Mo7e cells stimulated by different PD-1 / IL2m molecules.

[0120] Figure 4A and Figure 4B The effects of different PD-1 / IL2m molecules on p-Stat5 of T cells in PBMCs.

[0121] Figure 5 The figure shows the tumor inhibition effects of different PD-1 / IL2m molecules in mouse transplanted tumor models.

[0122] Figure 6 The effects of different PD-1 / IL2m molecules on the body weight of normal mice. DETAILED DESCRIPTION

[0123] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0124] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0125] Example 1. Design of IL-2 mutants and fusion proteins

[0126] The present invention designs a fusion protein of IL-2 mutant and anti-PD-1 antibody (hereinafter referred to as PD-1 / IL2m). The anti-PD-1 antibody in the fusion protein can specifically block the binding of PD-1 on the surface of T cells and PD-L1 on the surface of tumor cells, thereby relieving the immunosuppression of T cells against tumor cells. IL-2 can stimulate the proliferation of immune cells in the tumor microenvironment, thereby further enhancing the killing effect of immune cells on tumors. The fusion protein PD-1 / IL2m of the present invention is a fusion protein formed by connecting PD-1 antibody and IL-2 mutant through a flexible linker. The structure is shown in FIG. Figure 1 .

[0127] The fusion protein PD-1 / IL2m consists of two parts. The first monomer comprises the light chain and the first heavy chain of the PD-1 antibody. The first heavy chain comprises, from the N-terminus to the C-terminus, the heavy chain variable region and the heavy chain constant region Fc1. The second monomer comprises the light chain and the second heavy chain of the PD-1 antibody. The second heavy chain comprises, from the N-terminus to the C-terminus, the heavy chain variable region, the heavy chain constant region Fc2, a flexible linker, and an IL-2 mutant.

[0128] The first monomer and the second monomer form a heterodimer through a knob-into-hole structure.

[0129] The knob-and-hole structure is a modification of the antibody constant region (CH3 domain) such that one heavy chain constant region is mutated to form a protruding "knob" structure, while the other heavy chain constant region is mutated to form a recessed "hole" structure. This knob-and-hole structure enables the two different heavy chains to specifically bind to form a stable heterodimer. Knob-and-hole structures are well known to those skilled in the art.

[0130] Flexible linkers (also called linkers) are non-restrictive and are intended to prevent steric hindrance. Those skilled in the art are familiar with other replaceable linkers, as long as the linker has a certain flexibility to allow the polypeptides on both sides to perform their respective independent functions. Flexible linkers can be peptide linkers comprising glycine, serine, proline and / or lysine residues. Including but not limited to: (G)n, (S)n, (GxS)n, (SxG)n, (GSSGG)n, (GGSGG)n, (GSGGSG)n, (GSGSGS)n, (GGQGG)n and (EAAAK)n, and various combinations thereof. Wherein: n can be any integer between 1-10; x can be any integer between 1-6. The present invention selects the flexible linker (GGGGS)n, preferably n=3 or 4.

[0131] Specifically, the flexible linker used in this embodiment is (GGGGS)3, and its amino acid sequence is: GGGGSGGGGSGGGGS.

[0132] The IL-2 mutants in the second monomer are IL-2 mutants obtained through computer-aided design and experimental screening based on the crystal structure of the IL-2 and IL-2 receptor α / β / γ complex. These IL2 mutants have weakened binding to IL-2Rα (CD25) and IL-2Rβ / γ to varying degrees.

[0133] The amino acid position of the mutation site in the IL-2 mutant can be determined based on the amino acid sequence of wild-type IL-2 (SEQ ID NO: 1) or the amino acid sequence of Aldesleukin (a recombinant human interleukin-2) (SEQ ID NO: 2). The two IL-2s represented by SEQ ID NO: 1 and SEQ ID NO: 2 differ in amino acid position 125. Whether Cys or Ser at position 125 is present, it does not affect the activity of IL-2.

[0134] The relevant IL-2 sequences and mutation sites are shown in Table 1. The mutation sites in Table 1 can be mutated based on either SEQ ID NO: 1 or SEQ ID NO: 2 or other IL-2 sequences.

[0135] Table 1. IL-2 mutants and control molecules

[0136]

[0137] The exemplary IL-2 mutants of the present invention (SEQ ID NO: 3 to SEQ ID NO: 10) are mutated based on SEQ ID NO: 2. Furthermore, as known to those skilled in the art, the first amino acid, Met, of SEQ ID NO: 2 (Aldesleukin) is removed upon expression. Therefore, when constructing the fusion protein, the first amino acid, Met, was replaced with the first amino acid, Ala, of the wild-type.

[0138] Example 2. Construction and purification of expression plasmids for anti-PD-1 antibody and IL-2 mutant fusion protein

[0139] Based on the amino acid sequences in Table 2, GenScript Biotech Co., Ltd. optimized and synthesized deoxynucleotide sequences encoding the corresponding amino acid sequences. GenScript also constructed different anti-PD-1 antibody and IL-2 mutant fusion proteins (701-710) and 2149 molecules of mammalian cell secretory expression plasmids.

[0140] The 2149 molecule (an immunoconjugate of PD-1 and IL-2, used as a comparative example) is described in the patent application "Interleukin-2 mutant and its fusion protein" with publication number CN118119635A. The 2149 molecule contains three polypeptide chains: (1) the light chain of the PD-1 antibody, with an amino acid sequence of SEQ ID NO: 35 and a nucleotide sequence of SEQ ID NO: 38; (2) a polypeptide chain fused to the Fc region of the IL-2 mutant and the PD-1 antibody, with an amino acid sequence of SEQ ID NO: 36 and a nucleotide sequence of SEQ ID NO: 39; and (3) the heavy chain of the PD-1 antibody, with an amino acid sequence of SEQ ID NO: 37 and a nucleotide sequence of SEQ ID NO: 40.

[0141] The first monomer (light chain and first heavy chain) and the second monomer (light chain and second heavy chain) of the fusion protein (701-710) were separately constructed into the pCDNA3.4 vector. According to the amino acid information of the PD-1 and IL2 immunoconjugate 2149 molecules in the patent (publication number CN118119635A), three polypeptide chains were synthesized and the corresponding mammalian cell secretion expression plasmid was constructed. The 2149 molecules of the first monomer (SEQ ID NO: 38 and SEQ ID NO: 40) and the second monomer (SEQ ID NO: 39) were separately constructed into the vector pCDNA3.4 vector.

[0142] Table 2. Anti-PD-1 antibody and IL-2 fusion protein (PD-1 / IL2m)

[0143]

[0144] According to Thermofisher's ExpiCHO TM According to the Expression System Kit instruction manual, transfection of different recombinant expression plasmids corresponding to the PD-1 / IL2m or 2149 fusion proteins (two expression plasmids for each molecule) was performed, using 200 mL of each plasmid at a dosage of 0.7 μg / mL. The culture was continued for 10 days. Cell density increased significantly during the initial stages of culture, then slowly declined. Cell viability remained high, reaching 80% when samples were collected on day 10.

[0145] The supernatant was collected by centrifugation, filtered with a 0.22 μm filter, and purified by MabSelect SuRe LX (manufacturer Cytiva, product number 17-5474-02) (CIP buffer: 0.1 M NaOH; equilibration buffer: 20 mM PB, 0.15 M NaCl, pH 7.0; wash buffer 1: 20 mM PB, 1 M NaCl, pH 7.0; wash buffer 2: 50 mM citric acid-sodium citrate, pH 5.5, elution buffer: 50 mM citric acid-sodium citrate, pH 3.5.); the eluate was collected and concentrated, and then purified by Superdex 200 (manufacturer Cytiva and product number V521276) at a volume of 3% (equilibration and eluent: 20 mmol / L PB, 150 mmol / L NaCl, pH 7.0), collect the required peaks, detect the purity by SEC-HPLC, and determine the protein concentration by UV spectrophotometer based on the theoretical extinction coefficient of each protein.

[0146] Example 3. Affinity of PD-1 / IL2m with IL-2 receptor or PD-1 protein

[0147] The affinity of IL-2 mutants to the receptors IL-2Rα and IL-2Rβ / γ was measured by surface plasmon resonance (Biacore T-100).

[0148] 2.1. Determination of affinity between PD-1 / IL2m and IL-2 receptor β / γ

[0149] Human IL2-Rβ & Rγ-Fc Protein (Cat.#ILG-H5254, AcroBiosystems) was coupled to a CM5 chip (Cat.#BR100530, Cytiva) using the amino coupling method. The affinity between PD-1 / IL2m and IL-2Rβ & Rγ was analyzed using pH 7.4 buffer (solvent: water, solute: 10mM HBS-EP, 150mM NaCl, 0.05% Tween20, and 3mM EDTA). The samples (different PD-1 / IL2m) were diluted with buffer to protein solutions of 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM, 0.78125nM, and 0.390625nM, respectively. Human IL2-Rβ&Rγ-Fc (Cat.# ILG-H5254) was diluted to 10 μg / mL, with a binding level of approximately 100 RU. Kinetics analysis of the affinity constant (KD) was performed using a 1:1 binding model. Results are shown in Table 3. Molecule 703 binds to the IL-2 receptor β / γ complex at a similar rate to wild-type IL-2, while the other mutants exhibited varying degrees of reduced binding to the IL-2 receptor β / γ complex.

[0150] Table 3. Affinity of anti-PD-1 antibody and IL2 fusion protein (PD-1 / IL2m) for IL-2 receptor β / γ

[0151] Molecular name Ka(1 / Ms) Kd(1 / s) KD(M) 701 3.47E+05 3.85E-04 1.11E-09 702 2.92E+05 3.07E-04 1.05E-09 703 2.41E+05 3.97E-04 1.64E-09 704 8.12E+04 1.86E-04 2.29E-08 705 4.66E+04 1.49E-04 4.07E-08 706 3.47E+04 3.34E-04 9.63E-08 707 2.73E+04 8.25E-03 3.03E-7 708 3.39E+04 1.83E-02 5.06E-7 709 3.62E+04 9.47E-03 2.62E-7 710 4.21E+04 1.74E-02 4.13E-07

[0152] 2.2. Determination of affinity between PD-1 / IL2m and IL-2 receptor α

[0153] Human IL2-Rα Protein, His Tag (Cat. #ILA-H52H9, AcroBiosystems) was coupled to a CM5 chip using the amino coupling method. The working buffer was 10mM HBS-EP, 150mM NaCl, 0.05% Tween20, and 3mM EDTA, pH 7.4. The protein was diluted to 10ug / mL, resulting in a coupling level of approximately 100RU. Samples (different PD-1 / IL2m concentrations) were diluted in the buffer to 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM, 0.78125nM, and 0.390625nM protein solutions. The affinity of the samples (various PD-1 / IL-2 mutant fusion proteins) for Human IL2-Rα Protein was analyzed using a 1:1 binding kinetic model. The results are shown in Table 4. Different mutants exhibited varying binding to IL-2 receptor α, with molecule 703 not binding to IL-2 receptor α, and molecule 708 binding very weakly.

[0154] Table 4. Affinity of anti-PD-1 antibody and IL2 fusion protein (PD-1 / IL2m) for IL-2 receptor α

[0155] Molecular name KD(M) 701 4.23E-08 702 4.59E-8 703 No Binding 704 8.36E-07 705 3.18E-07 706 1.74E-06 707 2.16E-06 708 Weaking Binding 709 7.36E-08 710 3.18E-07

[0156] 2.3. Affinity determination of PD-1 / IL2m and human PD-1 His tag protein

[0157] The affinity of the whole antibody was determined using a capture assay. Anti-human IgG (Cat: 10702-T16, Sino Biological) was coupled to a CM5 chip surface. Each PD-1 / IL2m was diluted to ensure approximately 200 RU of antibody was captured by the anti-human IgG. A human PD-1 His tag (AcroBiosystems, Cat. No. PD1-H5221) was diluted to a concentration gradient of 50 nM, 20 nM, 8.0 nM, 3.2 nM, 1.28 nM, 0.512 nM, 0.2048 nM, 0.08192 nM, 0.032768 nM, and 0.01311 nM to determine the affinity of the antibody. The affinity between the samples (each PD-1 / IL2m) and the PD-1 His tag was analyzed using a 1:2 binding kinetic model. The results are shown in Table 5. The binding differences between different PD-1 / IL2m molecules and PD-1 are very small, and the ability of different PD-1 / IL2m to bind to PD-1 protein is similar.

[0158] Table 5. Affinity of anti-PD-1 antibody and IL2 fusion protein (PD-1 / IL2m) to PD-1

[0159] Molecular name Ka(1 / Ms) Kd(1 / s) KD(M) 701 2.056E+05 6.574E-05 3.197E-10 702 1.978E+05 8.437E-05 4.265E-10 703 2.733E+05 9.437E-05 3.453E-10 704 3.316E+05 1.042E-04 4.283E-10 705 2.327E+05 7.849E-05 3.373E-10 706 3.249E+05 1.157E-04 3.561E-10 707 2.175E+05 5.969E-05 2.744E-10 708 3.093E+05 8.976E-05 2.902E-10 709 3.734E+05 1.239E-05 3.318E-10 710 3.042E+05 9.027E-05 2.967E-10

[0160] Example 4: Effects of different PD-1 / IL2m on the proliferation activity of CTLL-2 and Mo7e cells

[0161] According to the literature Mol Cancer Ther (2012) 11 (6): 1279–1288, CTLL-2 cells have IL2-Rα / β / γ complexes, similar to Treg cells; while Mo7e cells have only IL2-β / γ receptors but no IL2-Rα receptors on their surface, similar to CD8+ T cells and NK cells. If the IL-2 mutant has a weaker affinity for the IL2-Rα receptor, its ability to stimulate the proliferation of CTLL2 (Treg cells) may theoretically be weaker. If the IL-2 mutant has a low affinity for the IL2-β / γ receptor, its ability to stimulate the proliferation of Mo7e (CD8+ T and NK cells) may theoretically be weaker.

[0162] 4.1 Effect of IPD-1 / IL2m on the proliferation activity of CTLL-2 cells

[0163] Cytokine growth-dependent CTLL-2 cells (mouse T cells) (Suzhou Vertex Biopharmaceutical Co., Ltd., Cat. No. TCM-C724) were cultured in RPMI 1640 medium supplemented with 200 IU / ml IL-2 (Beijing Sihuan Pharmaceutical Co., Ltd., National Medicine Standard S20040020) and 10% fetal bovine serum at 37°C, 5.0% CO2 until the logarithmic phase. The cells were harvested by centrifugation at 1000 rpm for 5 minutes and washed three times with phosphate buffered saline (Gibco, Cat#10010023). The cells were then washed with RPMI containing 10% fetal bovine serum. The cells were resuspended in 1640 medium and added to 96-well cell culture plates at a density of 5000 cells per well. After culturing for 4 hours (cytokine starvation), PD-1 / IL2m samples (703, 704, 705, 706, 707, 708, 709 and 710) serially diluted with phosphate buffer (100 nM, 40 nM, 16 nM1, 6.4 nM, 2.56 nM, 1.024 nM, 0.410 nM, 0.164 nM, 0.0655 nM, 0.0262 nM and 0 nM) or 701 samples (10 nM, 4 nM, 1.6 nM, 0.64 nM, 0.256 nM, 0.1024 nM, 0.0410 nM, 0.0164 nM, 0.00655 nM, 0.00262 nM and 0 nM) were added to the wells of a 96-well tissue culture plate and cultured for another 2 days. Then, 20 μl / well of CCK8 (Dojindo, Cat#CK04-500tests) was added and incubated for 2 hours at 37°C, 5.0% CO2. The cell growth was then examined by reading the absorbance at 450 nm and 630 nm.

[0164] See the results Figure 2 As shown, Figure 2 The vertical axis represents the difference in absorbance at 450 nm and 630 nm, while the horizontal axis represents protein concentration. The results show that compared to molecule 701 (IL-2 wild-type), the selected PD-1 / IL-2 mutant fusion proteins all reduced their ability to stimulate the proliferation of CTLL-2 cells. Among them, 706, 707, and 708 showed weaker CTLL-2 proliferation, while 709 exhibited higher CTLL-2 proliferation activity than the other PD-1 / IL-2 mutant fusion proteins.

[0165] 4.2 Effect of PD-1 / IL2m on the proliferation activity of Mo7e cells

[0166] The experimental method for PD-1 / IL2m stimulation of Mo7e cells (cytomegalovirus leukemia cell line) (Zhejiang Meisen Cell Technology Co., Ltd., Cat. No. CTCC-001-0368) is similar to that for CTLL-2, with the following differences: Mo7e cells were cultured in RPMI 1640 medium supplemented with 8 ng / ml GM-CSF (PeproTech, Cat#300-03-50UG) and 10% fetal bovine serum to the logarithmic phase, the cells were collected and washed, and then RPMI without GM-CSF was used. The cells were resuspended in 1640 medium and added to a 96-well plate at a density of 20,000 cells per well. After culturing for 4 hours, PD-1 / IL2m samples (703, 704, 705, 706, 707, 708, 709, and 710) diluted in phosphate buffer (concentrations of 450 nM, 180 nM, 72 nM, 28.8 nM, 11.52 nM, 4.608 nM, and 1 .843nM, 0.737nM, 0.295nM, 0.118nM and 0nM) or sample 701 (concentrations of 100nM, 40nM, 16nM, 6.4nM, 2.56nM, 1.024nM, 0.410nM, 0.164nM, 0.0655nM and 0nM, respectively) were added to 20,000 cells in a well of a 96-well tissue culture plate and cultured at 37°C, 5.0% CO2 for 4 days. After 4 days, CCK8 (Dojindo, Cat#CK04-500tests) was added at 20μl / well and cultured at 37°C, 5.0% CO2 for 2 hours. Cell growth was then detected by reading at 450nm and 630nm.

[0167] See the results Figure 3 China A and Figure 3 Middle B, Figure 3 The vertical axis represents the difference in absorbance at 450 nm and 630 nm, and the horizontal axis represents protein concentration. The results indicate that molecules 708 and 710 have very weak activity in stimulating Mo7e proliferation, while other mutants retain the ability to stimulate Mo7e proliferation to varying degrees.

[0168] Combine Figure 2 and Figure 3, the same PD-1 / IL2m may have a significant difference in its tendency to stimulate CTLL2 and Mo7e. For example, 709 and 710 can stimulate the proliferation of CTLL2 cells very well, but the stimulation of Mo7e proliferation is weak. The 703 molecule can stimulate the proliferation of Mo7e very well. The results show that the fusion protein PD-1 / IL2m constructed by the present invention has good diversity and can selectively stimulate the proliferation of CTLL2 cells (with IL-2 receptor α / β / γ complex on the cell surface) or Mo7e cells (with IL-2 receptor β / γ complex on the cell surface). Different PD-1 / IL2m can be used to match different drug development needs.

[0169] Example 5: Flow cytometry detection of phosphorylated Stat5

[0170] After IL-2 binds to the cell surface receptor, it can activate the downstream JAK-stat5 signaling pathway. The phosphorylation level of stat5 can be used as an indicator of the activity of the JAK-stat5 pathway. The detection steps of phosphorylated Stat5 flow cytometry are as follows:

[0171] 1) The sample (PD-1 / IL2m) was diluted in RPMI1640 medium in a gradient manner, with an initial concentration of 150 nM, and then diluted 5-fold in sequence (concentrations were 150 nMol / L, 30 nM, 6 nM, 1.2 nM, 0.24 nM, 0.048 nM, 0.0096 nM, and 0.00192 nM, respectively).

[0172] 2) Resuscitate primary human PBMC cells (Beijing Red Biotech, Cat#hPB010C). After the cell suspension is completely thawed, transfer the cell suspension to a centrifuge tube containing 5 mL of culture medium and centrifuge (1500 rpm, 5 min). Discard the supernatant and resuspend the cells in 5 mL of culture medium. Centrifuge (1500 rpm, 5 min). Discard the supernatant and resuspend the cells in culture medium. Count the cells and adjust the cell concentration to 5 × 10 6 cells / mL.

[0173] 3) Inoculate cells in a 1.5 mL centrifuge tube, 5×10 5 cells / tube, 100 μL / tube.

[0174] 4) Add the diluted sample to the centrifuge tube inoculated with cells, 100 μL / tube, and then incubate in a 37°C carbon dioxide incubator for 30 minutes.

[0175] 5) Centrifuge (1500 rpm, 5 min), discard the supernatant, wash the cells twice with Cell staining Buffer (BD, Cat#554656) (0.5 mL / tube), and resuspend the cells in 100 μL Cell staining Buffer.

[0176] 6) Add FITC anti-human CD3 (BioLegend, Cat#344803), PerCP / Cyanine5.5 anti-human CD8 (BioLegend, Cat#344709), and PE anti-human CD4 Antibody (BioLegend, Cat#300507) to each tube, 5 μL / tube, and incubate at 4°C for 30 min.

[0177] 7) Centrifuge (1500 rpm, 5 min), discard the supernatant, wash the cells twice with Cell Staining Buffer (0.5 mL / tube), and resuspend the cells in 250 μl Cell Staining Buffer.

[0178] 8) Add 250 μL of Fixation Buffer (BD, Cat#554655), pipette evenly, and incubate at room temperature for 15 min.

[0179] 9) Centrifuge (350 × g, 5 min), discard the supernatant, wash the cells twice with Cell Staining Buffer (1 mL / tube), resuspend the cells in 300 μL Perm Buffer III (BD, Cat#558050), and incubate the cells on ice for 30 min.

[0180] 10) Centrifuge (1000 × g, 5 min), discard the supernatant, wash the cells twice with Cell Staining Buffer (1 mL / tube), and resuspend the cells in 100 μl Cell Staining Buffer.

[0181] 11) Add Alexa Fluor 647 Mouse Anti-Stat5 (pY694) (BD, Cat#612599) and PD-1 Antibody (Invitrogen, Cat#25-9969-42), 5 μL each per tube, and incubate at room temperature in the dark for 30 min.

[0182] 12) Centrifuge (1000 × g, 5 min), discard the supernatant, wash the cells twice with Cell Staining Buffer (500 μl / tube), and resuspend the cells in 100 μl Cell Staining Buffer.

[0183] 13) Flow cytometry and analysis of relevant results. The results (curve and Ec50 value) are as follows: Figure 4A and Figure 4B As shown, in order to show the differences conveniently, the vertical axis scales in the figures of different molecules may be different.

[0184] CD3 cells are markers of T lymphocytes. CD3 and CD4 positive T lymphocytes are helper T lymphocytes, and CD8 positive T cells are usually cytotoxic T cells that can receive signals and kill virus-infected cells or cancer cells. Figure 4A and Figure 4B Results showed that PD-1 / IL2m expressed higher levels of p-Stat5 in PD-1-positive cells. p-Stat5 is an important indicator of IL-2 signaling pathway activation, suggesting that PD-1 / IL2m can more specifically activate T cells that overexpress PD-1. Different mutants stimulated p-Stat5 activity differently in CD3+CD4+ and CD3+CD8+ T cells. For example, molecules 703 and 707 were more potent in activating cytotoxic T cells expressing PD-1 (CD3+CD8+PD1+) than helper T cells expressing PD-1 (CD3+CD4+PD1+). These findings offer insights into the selection of anti-tumor drugs.

[0185] Cytotoxic T cells have a killing effect on antigens such as viruses and tumor cells, and together with natural killer cells, they form a crucial line of defense in the body's antiviral and antitumor immunity. Therefore, the selectivity of PD-1-positive and PD-1-negative cytotoxic T cells was calculated. The calculation formula is: Fold (fold) = the ratio of the Ec50 of a molecule on CD3+CD8+PD1- T cells to the Ec50 of the molecule on CD3+CD8+PD1+ T cells. The higher the Fold (fold), the greater the selectivity of cytotoxic T cells for PD-1-positive cytotoxic cells. The results are shown in Table 6, showing that each mutant PD-1 / IL2m has a stronger selectivity for PD-1-positive cytotoxic T cells than the 701 molecule.

[0186] Table 6. Cytotoxic T cell selection by different molecules in cells expressing or not expressing PD-1

[0187] molecular 701 703 704 705 706 707 708 709 710 Fold 3.16 7.64 36.00 12.27 36.42 6.25 About 808 24.46 38.18

[0188] Example 6: In vivo pharmacodynamics experiment on LLC1 cell transplanted tumors

[0189] The experiment was completed at Sino-US Crown Biotechnology Co., Ltd. (Beijing). hPD-1huGEMM (C57B / L6b background) mice, 7-9 weeks old, female, weighing 17-23g, were purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd. LLC1 cells (murine lung cancer cell line, cultured in DMEM containing 10% fetal bovine serum, preserved by Sino-US Crown Biotechnology) were subcutaneously inoculated to establish a subcutaneous transplant tumor model of mouse colon cancer. When the tumor volume was 60-100 cubic millimeters, the mice were randomly divided into 7 groups for drug administration (the experiment was divided into 7 groups, with 5 tumor-bearing mice in each group). The first day of drug administration was day 1, and the drug administration cycle was day 1 and day 9. The drug administration volume was 10μl / g body weight. The administration method and dosage are shown in Table 7. The tumor volume was measured twice a week. Among them, the 711 molecules were constructed and expressed and purified according to the amino acid sequence of the 2149 molecules in the patent (publication number CN118119635A). Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter). The result is as follows Figure 5 The commercially available Sindizumab (Innovent Biologics, trade name: Tyvyt) was not effective in this model, and molecules 704, 706, 707, and 710 were more effective in suppressing tumors than molecule 711.

[0190] Table 7 Grouping, medication records and methods in the LLC1 transplant tumor model

[0191] Group (sample name) Dosage Dosing days Dosage control group 5mg / Kg body weight Day 1, day 9 medication Intraperitoneal administration Sintilimab (commercially available) 5mg / Kg body weight Day 1, day 9 medication Intraperitoneal administration 704 5mg / Kg body weight Day 1, day 9 medication Intraperitoneal administration 706 5mg / Kg body weight Day 1, day 9 medication Intraperitoneal administration 707 5mg / Kg body weight Day 1, day 9 medication Intraperitoneal administration 708 5mg / Kg body weight Day 1, day 9 medication Intraperitoneal administration 710 5mg / Kg body weight Day 1, day 9 medication Intraperitoneal administration 711 5mg / Kg body weight Day 1, day 9 medication Intraperitoneal administration

[0192] The control group was injected with saline only.

[0193] Example 7: Toxicity evaluation in C57B / L6 mice

[0194] The purpose of this study was to observe the severity of toxic reactions in C57B / L6 mice after intraperitoneal injection of the test drug, including changes in the skin at the injection site and toxic reactions in organs such as the heart, liver, spleen, lungs, and kidneys. Thirty 7-8 week old male C57BL / 6 mice were acclimated for 5-7 days and weighed.

[0195] Animals were randomly divided into seven groups based on body weight, with three animals per group. The grouping date was considered PG-Day 0, and the drug was administered on the same day at a dose of 10 mg / kg. The administration site was marked after administration. Specific grouping, administration method, and dose are shown in Table 8.

[0196] Table 8. Grouping, dosing methods and dosage of C57B / L6 mice

[0197] Group (sample name) Dosage Dosing days Dosage Control group (normal saline) 10mg / kg body weight Day 0 Dosing Intraperitoneal administration 701 10mg / kg body weight Day 0 Dosing Intraperitoneal administration 704 10mg / kg body weight Day 0 Dosing Intraperitoneal administration 706 10mg / kg body weight Day 0 Dosing Intraperitoneal administration 707 10mg / kg body weight Day 0 Dosing Intraperitoneal administration 708 10mg / kg body weight Day 0 Dosing Intraperitoneal administration 710 10mg / kg body weight Day 0 Dosing Intraperitoneal administration

[0198] After the start of dosing, the animals' clinical status, food intake, activity, fur, eyes, and other appearances were observed daily, and any abnormalities were recorded. The animals were weighed three times a week. The animals were inspected daily and the time of death was recorded. If the animals' health deteriorated or became moribund (the animals had significant weight loss, with a weight loss greater than 20%), could not eat or drink normally, or had difficulty moving or were paralyzed, they were considered to be moribund and were euthanized with CO2.

[0199] Weight results as Figure 6 To show the difference, Figure 6 The lowest value of the vertical axis (weight) is 18g. Figure 6 It can be seen that the weight of the control group (saline group), groups 707, 708, and 710 was relatively stable. The weight of groups 701 and 704 decreased, accompanied by obvious abnormal conditions, manifested as arched backs, erect hair, and erythema on the abdomen. All mice in group 701 died or were dying (needing to be euthanized) on day 11. The weight of mice in group 704 did not recover throughout the experiment. The weight of group 706 decreased slightly after administration, but recovered during the experiment. Therefore, it can be seen that the toxic and side effects of the PD-1 / IL2 wild-type fusion protein are relatively large, while the side effects of the PD-1 / IL2 mutant are lower. Some PD-1 / IL2m fusion proteins did not show significant differences from the control group (saline group) in this experiment.

[0200] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A mutant protein, characterized in that The amino acid sequence of the mutant protein has any of the following mutations relative to the amino acid sequence of IL-2: A1) Q11R, E15D, D20E, K35E, L36E, T37L, R38E, M39E, L40K, R81Q, and / or Q126R; A2)K35E / L36E / T37L / R38E / M39E / L40K; A3)K35E / L36E / T37L / R38E / M39E / L40K / Q126R; A4)E15D / K35E / L36E / T37L / R38E / M39E / L40K / R81Q; A5)Q11R / D20E / K35E / L36E / T37L / R38E / M39E / L40K; A6)E15D / K35E / L36E / T37L / R38E / M39E / L40K / R81Q / Q126R; A7)Q11R / D20E / K35E / L36E / T37L / R38E / M39E / L40K / Q126R; A8)E15D / R81Q / Q126R; A9)Q11R / D20E / Q126R; The amino acid sequence of the IL-2 is any one of the following: B1) SEQ ID NO: 1; B2) SEQ ID NO: 2; B3) an amino acid sequence obtained by mutating Cys at position 125 of SEQ ID NO: 1 to Ser, while keeping the other amino acids unchanged; B4) An amino acid sequence that is 95% or more identical to the amino acid sequence shown in B1), B2) or B3) and has the same function.

2. The mutant protein according to claim 1, characterized in that The amino acid sequence of the mutant protein is shown in any one of SEQ ID NO: 3 to SEQ ID NO:

10.

3. A fusion protein, characterized in that The fusion protein comprises a PD-1 antibody and the mutant protein according to claim 1 or 2.

4. The fusion protein according to claim 3, characterized in that The mutant protein is directly connected to the C-terminus of the PD-1 antibody or connected via a linker.

5. The fusion protein according to claim 3 or 4, characterized in that The PD-1 antibody is an anti-human PD-1 antibody or an anti-human PD-1 antibody with a knob-and-hole structure.

6. The fusion protein according to any one of claims 3 to 5, characterized in that The fusion protein comprises two light chains, a first heavy chain and a second heavy chain, The amino acid sequence of the light chain comprises SEQ ID NO: 12, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 12 and having an identity of 95% or more to SEQ ID NO: 12; The amino acid sequence of the first heavy chain comprises SEQ ID NO: 11, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 11 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 11; The amino acid sequence of the second heavy chain comprises any one of the following: C1) SEQ ID NO: 15, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 15 and having an identity of 95% or more to SEQ ID NO: 15; C2) SEQ ID NO: 16, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 16 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 16; C3) SEQ ID NO: 17, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 17 and having an identity of 95% or more to SEQ ID NO: 17; C4) SEQ ID NO: 18, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 18 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 18; C5) SEQ ID NO: 19, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 19 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 19; C6) SEQ ID NO: 20, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 20 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 20; C7) SEQ ID NO: 21, or an amino acid sequence obtained by substituting, deleting, and / or adding amino acid residues of the amino acid sequence of SEQ ID NO: 21 and having an amino acid sequence with greater than 95% identity to SEQ ID NO: 21; C8) SEQ ID NO: 22, or an amino acid sequence obtained by substituting, deleting and / or adding amino acid residues in the amino acid sequence of SEQ ID NO: 22 and having an identity of 95% or more to SEQ ID NO:

22.

7. Biomaterial, characterized in that The biological material is any one of the following: D1) a nucleic acid molecule encoding the mutant protein according to claim 1 or 2; D2) a nucleic acid molecule encoding the fusion protein according to any one of claims 3 to 6; D3) an expression cassette containing the nucleic acid molecule described in D1) or D2); D4) a recombinant vector containing the nucleic acid molecule described in D1) or D2), or a recombinant vector containing the expression cassette described in D3); D5) a recombinant microorganism containing the nucleic acid molecule described in D1) or D2), or a recombinant microorganism containing the expression cassette described in D3), or a recombinant microorganism containing the recombinant vector described in D4); D6) A recombinant host cell containing the nucleic acid molecule described in D1) or D2), or a recombinant host cell containing the expression cassette described in D3), or a recombinant host cell containing the recombinant vector described in D4).

8. The biomaterial according to claim 7, characterized in that The nucleic acid molecule is selected from SEQ ID NO:24, SEQ ID NO:23, SEQ ID NO:27-SEQ ID NO:

34.

9. Use of the mutant protein according to claim 1 or 2, the fusion protein according to any one of claims 3 to 6, or the biomaterial according to claim 7 or 8 in the preparation of a product for preventing and / or treating tumors or PD-1 target-related diseases.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the mutant protein according to claim 1 or 2, or the fusion protein according to any one of claims 3 to 6, and one or more pharmaceutically acceptable carriers.

Images