TW202426471A - Interleukin 10 mutant, fusion protein and medicament - Google Patents

Abstract

The present application provides an Interleukin 10 (IL10) mutant, a fusion protein, and a medicament, and relates to the technical field of biomedicine. The IL10 mutant in the present application includes an amino acid mutation relative to the wild-type IL10. The IL10 mutant has weak binding activity to an IL10 receptor.

Description

IL10 mutants, fusion proteins and drugs

[Priority Claim]

This application claims priority and benefits to the Chinese patent application number 202211667237.6 filed with the National Intellectual Property Administration of China on December 24, 2022, entitled "IL10 mutants, fusion proteins and drugs", and the entire text of which is incorporated into this application by reference.

The present invention relates to the field of biopharmaceutical technology, and in particular to an IL10 mutant, a fusion protein and a drug.

Interleukin 10 (IL10), also known as human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine and a homodimeric secretion. It has a very wide range of biological activities. IL10 has the ability to inhibit the activation and effector functions of monocytes, macrophages and T cells, and also has pro-inflammatory functions. Currently, there are safety issues with clinical IL10 proteins. A small amount of naturally dimerized IL10 molecules bind to its receptor IL10Rα with high affinity, and then further bind to IL10Rβ to form a hexameric complex and activate downstream signals, causing biological functional responses.

The current clinical IL10 drug molecules lack targeting, and the drug molecules are prone to off-target and produce side effects; most of the IL10 antibody fusion proteins under development use the antibody targeting effect to guide IL10 to the local environment to exert its effect. The IL10 in these fusion protein molecules often forms a normal IL10 dimer structure, but because the IL10 dimer binds to IL10Rα with high affinity, it will affect the targeting effect of the antibody end. In the absence of the antibody target, IL10 activity will also be generated, bringing side effects, and if the antibody end requires a large dose of activity, the IL10 dose will also increase accordingly. In this case, the side effects of the IL10 end are still a difficult problem.

The present invention provides an IL10 mutant, a fusion protein and a drug. Specifically, the present invention provides an IL10 mutant, a fusion protein, a nucleic acid molecule, a recombinant vector, a recombinant cell, a drug composition, and a treatment method.

Specifically, in one aspect, the present invention provides an IL10 mutant, which has an amino acid mutation at at least one of the following positions relative to SEQ ID NO. 1: P20, R24, R27, K34, T35, Q38, D41, L46, L48, E50, S141, D144, I145, N148, T155, and R159. In some embodiments, it has an amino acid mutation at at least one of the following positions relative to SEQ ID NO. 1: P20, R24, T35, D41, L46, L48, E50, S141, I145, T155, and R159.

In another aspect, the present invention also provides a fusion protein comprising the aforementioned IL10 mutant.

In another aspect, the present invention also provides an isolated nucleic acid molecule encoding the aforementioned IL10 mutant or fusion protein.

In another aspect, the present invention also provides a recombinant vector comprising the aforementioned nucleic acid molecule.

In another aspect, the present invention also provides a recombinant cell containing the aforementioned nucleic acid molecule or recombinant vector.

In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned IL10 mutant, fusion protein, nucleic acid molecule, recombinant vector, or recombinant cell.

In another aspect, the present invention provides a method for treating a tumor, comprising administering a therapeutically effective amount of the aforementioned pharmaceutical composition to a subject in need of treatment.

In another aspect, the present invention provides the use of the aforementioned IL10 mutants, fusion proteins, nucleic acid molecules, recombinant vectors, and recombinant cells in the preparation of a medicament for preventing or treating tumors.

The beneficial effects of the IL10 mutant provided by the present invention include: its binding activity to the IL10 receptor is weak, and when it is fused with an antibody, the fusion protein only shows IL10 activity in the presence of the antibody target, the side effects are reduced, and the safety is improved.

Unless otherwise specified, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs. The following definitions are provided for a better understanding of the teachings of the present invention through further guidance. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J.Gait, 1984); Animal Cell Culture (R.I.Freshney, 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M.Ausubel et al., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001), etc.

The term "amino acid" refers to naturally occurring amino acids and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. The naturally encoded amino acids are the 20 common amino acids, as shown in the following table: Chinese name English name Three-letter abbreviation Single letter abbreviation Alanine Alanine Ala A Arginine Arginine Arg R Asparagine Asparagine Asn N Aspartic acid Aspartic acid Asp D Cysteine Cysteine Cys C Glutamine Glutamate Glu E Glutathione Glutamine Gln Q Glycine Glycine Gly G Histidine Histidine His H Isoleucine Isoleucine Ile I Leucine Leucine Leu L Lysine Lysine Lys K Methionine Methionine Met M Phenylalanine Phenylalanine Phe F Proline Proline Pro P Serine Serine Ser S Threonine Threonine Thr T Tryptophan Tryptophan Trp W Tyrosine Tyrosine Tyr Y Valine Valine Val V

Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (for example, an α-carbon bound to a hydrogen, carboxyl, amino group, and R group). These analogs may have modified R groups (for example, norleucine) or may have a modified peptide backbone while still retaining the same basic chemical structure as naturally occurring amino acids. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfur oxide, methionine methyl thionine, and the like.

In the present invention, the term "IL10" is a pleiotropic immunomodulatory cytokine. The IL-10 molecule in the natural state usually exists in a dimer structure (including two natural IL-10 monomers). A single natural IL-10 monomer (i.e., the wild-type IL10 (IL10 WT) described in the embodiments of the present invention) has an amino acid sequence as shown in SEQ ID NO: 1. Low-dose IL10 has good tolerance, but high-dose IL10 causes side effects such as inflammatory reactions. When IL0 is fused with an antibody, the required dose of the bifunctional molecule antibody end is usually higher than the IL10 tolerance value, which is inconsistent with the side effects caused by high-dose IL10.

Based on this, the present invention provides an IL10 mutant that reduces the affinity of IL10 to its receptor, thereby increasing the IL10 tolerance value, reducing side effects, and improving safety, so that it can be better used in combination with antibodies to exert the target end enrichment effect and better exert the anti-tumor effect.

In a first aspect, the present invention provides an IL10 mutant having an amino acid mutation at at least one of the following positions relative to SEQ ID NO. 1: P20, R24, R27, K34, T35, Q38, D41, L46, L48, E50, S141, D144, I145, N148, T155, and R159.

It should be noted that the mutation sites described herein are amino acid residue sites with respect to the natural sequence number of the sequence SEQ ID NO.1, that is, represented by the natural sequence number of SEQ ID NO.1.

Alternatively, in some embodiments, the IL10 mutant has an amino acid mutation at at least one of the following positions relative to SEQ ID NO. 1: P20, R24, T35, D41, L46, L48, E50, S141, I145, T155, and R159.

Alternatively, in some embodiments, the above-mentioned amino acid mutation refers to amino acid substitution, insertion, or deletion.

Alternatively, in some embodiments, the above-mentioned amino acid mutation is an amino acid substitution.

Alternatively, in some embodiments, the amino acid mutation at each site is a natural amino acid mutation, and the amino acid mutation at each site can be any one of the mutation types shown in the following table: Mutated amino acid residue Mutation site A G D E N Q R K I L M V F Y W S T C P H P20 + + + + + + + + + + + + + + + + + + + R24 + + + + + + + + + + + + + + + + + + + R27 + + + + + + + + + + + + + + + + + + + K34 + + + + + + + + + + + + + + + + + + + T35 + + + + + + + + + + + + + + + + + + + Q38 + + + + + + + + + + + + + + + + + + + D41 + + + + + + + + + + + + + + + + + + + L46 + + + + + + + + + + + + + + + + + + + L48 + + + + + + + + + + + + + + + + + + + + E50 + + + + + + + + + + + + + + + + + + + S141 + + + + + + + + + + + + + + + + + + + D144 + + + + + + + + + + + + + + + + + + + N148 + + + + + + + + + + + + + + + + + + + I145 + + + + + + + + + + + + + + + + + + + T155 + + + + + + + + + + + + + + + + + + + Note: + represents the type of amino acid residue after mutation.

Alternatively, in some embodiments, the IL10 mutant is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.1.

"Percentage (%) of amino acid sequence identity" with respect to protein sequences is defined as the percentage of amino acid residues in a candidate sequence (e.g., IL10 mutants herein) that are identical to amino acid residues in a reference sequence (e.g., SEQ ID NO. 1, wild-type IL10 in this application), after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as MOE, DANMAN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR), which are common software in the art. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

In a second aspect, the present invention provides an IL10 mutant having the following amino acid mutation at the following position relative to SEQ ID NO.1: I145.

Optionally, in some embodiments, the amino acid mutation at I145 is selected from: I145G, I145A, I145R, I145L, I145K, I145N, I145M, I145D, I145F, I145C, I145P, I145Q, I145S, I145E, I145T, I145W, I145H, I145Y, or I145V.

Optionally, in some embodiments, the above-mentioned IL-10 mutant further has the following amino acid mutations at at least one of the following positions relative to SEQ ID NO.1: P20, R24, R27, K34, T35, Q38, D41, L46, L48, E50, S141, D144, N148, T155, and R159.

Alternatively, in some embodiments, the amino acid mutation at each of the above sites is a natural amino acid mutation, which can be any one of the substitution mutation types shown in the following table: Amino acid residue after mutation Mutation site A G D E N Q R K I L M V F Y W S T C P H P20 + + + + + + + + + + + + + + + + + + + R24 + + + + + + + + + + + + + + + + + + + R27 + + + + + + + + + + + + + + + + + + + K34 + + + + + + + + + + + + + + + + + + + T35 + + + + + + + + + + + + + + + + + + + Q38 + + + + + + + + + + + + + + + + + + + D41 + + + + + + + + + + + + + + + + + + + L46 + + + + + + + + + + + + + + + + + + + L48 + + + + + + + + + + + + + + + + + + + + E50 + + + + + + + + + + + + + + + + + + + S141 + + + + + + + + + + + + + + + + + + + D144 + + + + + + + + + + + + + + + + + + + N148 + + + + + + + + + + + + + + + + + + + T155 + + + + + + + + + + + + + + + + + + + Note: + represents the type of amino acid residue after mutation.

Alternatively, in some embodiments, the IL-10 mutant has at least one of the following amino acid mutations relative to SEQ ID NO.1:

P20G, P20F, R24P, R27D, R27G, K34S, K34D, T35R, Q38P, D41W, L46G, L46K, L48G, E50G, S141F, S141Q, S141L, D144S, N148E, T155R, R159E, and R159H.

Alternatively, in some embodiments, the IL10 mutant is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.1.

In a third aspect, the present invention provides an IL10 mutant having at least one of the following amino acid mutations relative to SEQ ID NO. 1: P20G, P20F, R24G, R24P, R27D, R27G, T35R, D41W, L46G, L48G, E50G, S141L, S141F, S141Q, T155R, R159E, and R159H.

Alternatively, in some embodiments, the above IL-10 mutant further has the following amino acid mutations at at least one of the following positions relative to SEQ ID NO.1: K34, Q38, L46, D144, N148, and I145.

Optionally, in some embodiments, the above-mentioned IL-10 mutant further has at least one of the following amino acid mutations relative to SEQ ID NO.1: K34S, K34D, Q38P, L46K, D144S, N148E, I145G, I145A, I145R, I145L, I145K, I145N, I145M, I145D, I145F, I145C, I145P, I145Q, I145S, I145E, I145T, I145W, I145H, I145Y, and I145V.

Optionally, in some embodiments, the IL-10 mutant has the following amino acid mutations relative to SEQ ID NO.1: (a) P20G; (b) R24G; (c) D41W; (d) L46G; (e) L48G; (f) E50G; (g) S141F; (h) P20G/R24G (i.e., P20G and R24G); (i) K34S/L46G (i.e., K34S and L46G); (j) S141Q/N148E (i.e., S141Q and N148E); (k) P20F; (l) T35R; (m) R24P; (n) R27D; (o) R27G; (p) S141L; (q) I145G; (r) R159E; (s) R159H; (t) R24G/R27A/S141F (i.e. R24G, R27A and S141F); (u) R24G/R27D (i.e. R24G and R27D); (v) R24G/Q38P (i.e. R24G and Q38P); (w) R27D/K34D (i.e. R27D and K34D); (x) S141F/D144S (i.e. S141F and D144S); or (y) T155R.

Alternatively, in some embodiments, the IL10 mutant is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.1.

It should be noted that the sequences of the IL10 mutants provided in various aspects of the present invention at other sites other than the above mutation sites may be the same as SEQ ID NO.1; or may be different from SEQ ID NO.1 (may be substituted, deleted or inserted). When the sequences other than these mutation sites are different from SEQ ID NO.1, the IL10 mutant has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO.1.

It should be understood that, based on the above mutation sites disclosed in the present invention, a person skilled in the art can easily conceive of making substitutions, such as conservative substitutions, at other sites other than the above mutation sites based on SEQ ID NO.1, or making amino acid mutations to obtain similar IL10 mutations without affecting the effects brought about by the above mutation functions. Such IL10 mutants have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO.1 and should also fall within the scope of protection of the present invention.

Alternatively, in some embodiments, the IL10 mutants provided in the first aspect, the second aspect, and the third aspect have reduced binding activity to the IL10 receptor compared to the wild-type IL10 shown in SEQ ID NO.1.

As used herein, "wild type" refers to an amino acid sequence or nucleotide sequence found in nature. A wild type protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

Alternatively, in some embodiments, the IL10 receptor is selected from IL10Rα, IL10Rβ, and IL10Rα/IL10Rβ.

Alternatively, in some embodiments, the IL10 receptor is selected from IL10Rα.

Alternatively, in some embodiments, the IL10 receptor is selected from IL10Rβ.

Alternatively, in some embodiments, the IL10 receptor is selected from IL10Rα/IL10Rβ.

Alternatively, in some embodiments, the IL10 mutants provided in the first aspect, the second aspect, and the third aspect are selected from monomers or dimers.

Alternatively, in some embodiments, the above-mentioned IL10 mutants are selected from monomers.

Alternatively, in some embodiments, the above IL10 mutants are selected from dimers.

A spacer peptide is inserted relative to the wild-type IL10 shown in SEQ ID NO. 1. The insertion of the spacer peptide is beneficial to prevent adjacent IL10 mutants from forming a dimer structure.

Optionally, the IL10 mutant has a spacer peptide inserted between positions 116 and 117 relative to the wild-type IL10 shown in SEQ ID NO.1.

Optionally, in some embodiments, the length of the above-mentioned spacer peptide is 5-10 amino acids in length.

Optionally, in some embodiments, the above-mentioned spacer peptide contains amino acid residues G and S.

Optionally, in some embodiments, the amino acid sequence of the above-mentioned spacer peptide is SEQ ID NO.2 (GGGSGG).

Optionally, in some embodiments, the IL10 mutants provided in the first aspect, the second aspect, and the third aspect further lack the first and second amino acids at the N-terminus relative to the wild-type IL10 shown in SEQ ID NO.1.

Optionally, the amino acid sequence of the IL10 mutant is selected from any one of SEQ ID NO. 3-32.

In another aspect, the present invention provides a fusion protein, which comprises the IL10 mutant provided by the first aspect, the second aspect, or the third aspect.

As used herein, "fusion protein" refers to a covalent linkage of at least two proteins or protein domains.

Alternatively, in some embodiments, the fusion protein comprises an antigen binding molecule, and the IL10 mutant is covalently linked to the antigen binding molecule.

Alternatively, in some embodiments, the antigen-binding molecules specifically bind to tumor-specific antigens, immune checkpoint molecules, or a combination of the two, or other antigen molecules.

Alternatively, in some embodiments, the tumor-specific antigen is a solid tumor-specific antigen or a hematological tumor-specific antigen.

The term "solid tumor" generally refers to a tangible tumor that can be detected by clinical examination (such as X-ray, CT scan, B-ultrasound or palpation, etc.), including but not limited to mesothelioma, gastrointestinal adenoma, hepatocellular carcinoma, glioma, bladder cancer, colorectal cancer, neuroblastoma, glioblastoma, gastric cancer, colorectal cancer, esophageal cancer, pancreatic cancer, Adenocarcinoma, cervical cancer, urothelial carcinoma, kidney cancer, endometrial cancer, prostate cancer, ovarian cancer, metastatic cancer, bile duct cancer, lymphoma, melanoma, thyroid cancer, sarcoma, lung cancer, ovarian clear cell carcinoma, astrocytoma, liver cancer, colon cancer, yolk sac cancer, retinoblastoma, squamous cell carcinoma of the lung, thymic carcinoma, and breast cancer, etc.

Alternatively, in some embodiments, the solid tumor specific antigens include but are not limited to GUCY2C, MSLN, Claudin18.2, GPC3, EGFR, VEGFR2, HER2, CEA, GD2, EGFRvⅢ, MUC1, PRLR, CLCA1, MUC12, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS1IE, CD207, SLC30A8 , CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISSIR, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP, EphA2, FAP, IL13-Ra2, PSMA, ROR1, VEG FR-II, FR- a, EpCAM, EGFRvII, tMUC1, PSCA, FCER2, GPR18, FCRLA, CXCR5, FCRL3, FCRL2, HTR3A, CLEC17A, TRPMI, SLC45A2, SLC24A5, DPEP3, KCNK16, LIM2, KCNV2, SLC26A4, CD171, Glypican-3, IL-13, CD79a/ b and MAGEA4.

Immune checkpoints generally refer to regulatory molecules that play an inhibitory role in the immune system. Immune checkpoints can be expressed on immune cells and inhibit the function of immune cells. Generally speaking, immune checkpoints are associated with tumor immune escape. Optionally, in some embodiments, immune checkpoint molecules are selected from PD-1, CD47, CCR8, TIGIT, PVRIG, CD137, CD134, KIR, LAG-3, PD-L1, CTLA-4, B7.1, B7H3, CCRY, OX-40 and CD40, etc.

Alternatively, in some embodiments, the above-mentioned antigen-binding molecule is an antibody or an antigen-binding fragment thereof.

The term "antibody" as used herein refers to an immunoglobulin (Ig) or a single domain antibody (VHH) that can specifically bind to an antigen, and may be a monospecific antibody or a multispecific antibody.

Optionally, in some embodiments, the antibody is selected from IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, IgD.

Optionally, in some embodiments, the above-mentioned antigen-binding fragment is selected from Fab, Fab', Fv fragment, F(ab')2, F(ab)2, and scFv. The term "Fab" refers to a part of an antibody composed of a single light chain (including a variable region and a constant region) and a single heavy chain variable region and a first constant region bound together via a disulfide bond. "Fab' fragment" refers to a Fab fragment containing a portion of the hinge region. "F(ab')2" refers to a dimer of Fab'. "Fv fragment" is composed of a single light chain variable region and/or a single heavy chain variable region. "scFv" refers to an antibody fragment formed by the light chain variable region and the heavy chain variable region being directly connected to each other or connected through a peptide linker sequence.

Alternatively, in some embodiments, the IL10 mutant is directly or indirectly linked to the N-terminus of one or both heavy chains, the C-terminus of one or both heavy chains, between the VH and CH1 of one or both heavy chains, between the CH1 and CH2 of one or both heavy chains, between the CH2 and CH3 of one or both heavy chains, the N-terminus of one or both light chains, the C-terminus of one or both light chains, and/or between the VL and CL of one or both light chains in the antibody.

Alternatively, in some embodiments, the C-termini of the two light chains of the antibody are linked to the IL10 mutant.

Alternatively, in some embodiments, the IL10 mutant is linked to the C-terminus of the light chain of the antibody via a linker peptide.

Alternatively, in some embodiments, the IL10 mutant is linked to the antibody via a flexible linker peptide containing amino acid residues G and S.

The term "linker peptide", "linker polypeptide", "linker", "linker" or "linker" refers to a linker unit that connects two polypeptide fragments, which is usually flexible and does not cause the loss of the original function of the protein domain. The linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. In some embodiments, the linker is selected from (GxS)yGzSt linker, wherein x is selected from an integer of 1-5, and y, z, and t are independently selected from integers of 0-6 (when y, z, and t are 0, it means that the corresponding amino acid residues do not exist, for example, the amino acid residue of (G4S)4G1S0 is: GGGGSGGGGSGGGGSGGGGSG), in some embodiments, the linker is selected from (GxS)y linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6.

Optionally, in some embodiments, the amino acid sequence of the flexible linker peptide is as shown in SEQ ID NO:33 (GGGGSGGGGSGGGGSGGGGSG).

Alternatively, in some embodiments, the amino acid sequence of the light chain in the antibody comprises: VL-CL-Linker-M;

Among them, VL is the light chain variable region sequence, CL is the light chain constant region sequence, Linker is the connecting peptide sequence, and M is the IL10 mutant sequence.

Alternatively, in some embodiments, the IL10 mutant has at least one of the following amino acid mutations relative to SEQ ID NO.1: K34S, K34D, Q38P, L46K, and D144S.

Optionally, in some embodiments, the antibody is an anti-PD-1 antibody, an anti-CD47 antibody, an anti-GUCY2C antibody, an anti-CD137 antibody, an anti-CD134 antibody, an anti-KIR antibody, an anti-LAG-3 antibody, an anti-PD-L1 antibody, an anti-EGFR antibody, an anti-VEGFR-II antibody, an anti-CTLA-4 antibody, an anti-B7.1 antibody, an anti-B7H3 antibody, an anti-CCRY antibody, an anti-OX-40 or an anti-CD40 antibody.

Alternatively, in some embodiments, the antibody portion in the above fusion protein is a complete antibody (i.e., immunoglobulin).

Optionally, in some embodiments, in the above-mentioned fusion protein, the C-terminus of the light chain of the above-mentioned antibody is linked to the above-mentioned IL10 mutant.

Optionally, in some embodiments, the antibody portion in the above fusion protein is an anti-PD-1 antibody.

Optionally, in some embodiments, in the above fusion protein, the C-termini of the two light chains of the anti-PD-1 antibody are linked to the IL10 mutant.

Alternatively, in some embodiments, the fusion protein disclosed herein isolates the IL10 unit by the steric hindrance of the antibody structure itself, and when the IL10 unit fusion protein is in a free state, even if the IL10 unit binds to IL10Rα, it cannot induce a functional response. When the antibody end binds to the target cell, the adjacent IL10 unit-IL10Rα complex is aggregated through the aggregation effect, and further binds to IL10Rβ to form a complex to generate downstream signals and induce biological functions, thereby preventing the IL10 molecules from interfering with the targeting of the antibody before the drug molecules reach the target site, thereby avoiding clinical side effects; and because the IL10 unit is difficult to induce biological functions, the anti-PD1-IL10 fusion protein will not produce the dosage limitation of the natural dimer IL10 molecule, so that the dosage of the fusion protein is significantly improved.

In another aspect, the present invention provides an isolated nucleic acid molecule encoding the above-mentioned IL10 mutant or the above-mentioned fusion protein.

The term "nucleic acid molecule" generally refers to isolated forms of nucleotides, either deoxyribonucleotides or ribonucleotides of any length, or analogs isolated from their natural environment or artificially synthesized.

In another aspect, the present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

The term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can transform, transduce or transfect a host cell so that the genetic material elements it carries are expressed in the host cell. A vector may contain multiple elements that control expression. In addition, a vector may also contain a replication origin site. A vector may also include components that assist it in entering the cell.

In another aspect, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule or the aforementioned recombinant vector.

The term "recombinant cell" generally refers to a single cell, a cell line or a cell culture that can be or has been a recipient of a plasmid or a vector, which includes a nucleic acid molecule as described herein or a vector as described herein.

In another aspect, the present invention provides the use of the above-mentioned IL10 mutant, the above-mentioned fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector, and the above-mentioned recombinant cell in the preparation of a drug for preventing or treating tumors.

On the other hand, the present invention provides a method for preparing the above-mentioned IL10 mutant or the above-mentioned fusion protein, which comprises: culturing cells expressing the above-mentioned IL10 mutant or the above-mentioned fusion protein, and isolating and purifying the above-mentioned IL10 mutant or the above-mentioned fusion protein from the culture product.

In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned IL10 mutant, the above-mentioned fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector, or the above-mentioned recombinant cell.

Optionally, in some embodiments, the above-mentioned pharmaceutical composition further includes a pharmaceutically acceptable carrier.

Alternatively, in some embodiments, the pharmaceutically acceptable carrier may include water, a pharmaceutically acceptable thickener, a pH adjuster, and or a pharmaceutically acceptable preservative or antibacterial agent, an antioxidant, a stabilizer, an isotonic regulator, an absorption enhancer, a solubilizer, a formulator, a diluent, a humectant, etc., or any one or more of them.

In another aspect, the present invention provides a method for treating tumors, comprising: administering a therapeutically effective amount of the IL10 mutant, the fusion protein, or the pharmaceutical composition to a subject in need of treatment.

The term "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's general health, the method route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[ Specific implementation methods ]

In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. If no specific conditions are specified in the embodiments, the experiment is carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

The features and properties of the present invention are further described in detail below in conjunction with the embodiments.

Embodiment 1

Construction of IL10 mutants

Amino acid residues are mutated at the contact residues between wild-type IL10 (IL10 WT) and IL10Rα to weaken the binding activity with IL10Rα and reduce the effectiveness of IL10 biological function. After the obtained IL10 mutant is fused with a targeting protein such as an antibody, the IL10 mutant can aggregate to the target cell through the targeting protein to exert biological function only in the presence of the target cell (which can be specifically bound by the aforementioned antibody).

Exemplary amino acid mutations provided in this embodiment are selected from the following mutations: P20L, R24PG, R27DG, K34DS, Q38P, D41W, L46GK, L48G, E50G, S141FL, D144S, I145G, R159EH, R24G/R27A/S141F, P20G/R24G, R24G/R27D, R24G/Q38P, R27D/K34D, K34S/L46G, S141F/D144S, S141F/N148E, P20F, T35R and T155R.

The IL10 mutant (IL10MT) provided in this embodiment also lacks the first and second amino acid residues at its N-terminus relative to IL10 WT, and the amino acid mutations contained in each are shown in Table 1 below. [Table 1] IL10 mutant number Amino acid mutation IL10 mutant number Amino acid mutation MT1741 P20G MT1756 D144S MT1742 R24P MT1757 I145G MT1743 R24G MT1758 R159E MT1744 R27D MT1759 R159H MT1745 R27G MT1761 R24G/R27A/S141F MT1746 K34D MT1762 P20G/R24G MT1747 K34S MT1763 R24G/R27D MT1748 Q38P MT1764 R24G/Q38P MT1749 D41W MT1765 R27D/K34D MT1750 L46G MT1766 K34S/L46G MT1751 L46K MT1767 S141F/D144S MT1752 L48G MT1768 S141Q/N148E MT1753 E50G MT1769 P20F MT1754 S141F MT1770 T35R MT1755 S141L MT1771 T155R

The amino acid sequence of IL10 WT is as follows: SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO. 1).

Embodiment 2

Construction of IL10 mutant and antibody fusion protein

(1) Inserting a spacer peptide (SEQ ID NO.2: GGGSGG) at positions 116-117 of IL10WT to obtain IL10 monomer (IL10M, SEQ ID NO.34);

The amino acid sequence of IL10M is as follows SEQ ID NO.34: SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

Based on each IL10 mutant (IL10 MT) provided in Table 1 of Example 1, a spacer peptide (GGGSGG) was inserted at positions 116-117 to obtain a monomeric IL10 mutant (IL10MMT).

The names and sequences of the monomeric forms of the IL10 mutants corresponding to the monomeric mutations of each IL10 mutant are shown in Table 2 below (the underline indicates the position of the mutation). The sequence alignment of IL10WT, IL10M, and IL10MMT (MMT1741, MMT1761) (using MOE software with default parameters) is shown in Figure 1.

[Table 2] IL10 MT IL10 MMT Amino acid sequence of IL10 MMT SEQ MT1741 MMT1741 GQGTQSENSCTHFPGNL GNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 3 MT1742 MMT1742 GQGTQSENSCTHFPGNLPNML P DLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 4 MT1743 MMT1743 GQGTQSENSCTHFPGNLPNML G DLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 5 MT1744 MMT1744 GQGTQSENSCTHFPGNLPNMLRDL D DAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 6 MT1745 MMT1745 GQGTQSENSCTHFPGNLPNMLRDL G DAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 7 MT1746 MMT1746 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRV D TFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 8 MT1747 MMT1747 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRV S TFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 9 MT1748 MMT1748 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFF P MKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 10 MT1749 MMT1749 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMK W QLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 11 MT1750 MMT1750 GQGTQSENSCTHFPGNLPNMLGDLRDAFSRVKTFFQMKDQLDN G LLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 12 MT1751 MMT1751 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDN K LLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 13 MT1752 MMT1752 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLL GKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 14 MT1753 MMT1753 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLK G SLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 15 MT1754 MMT1754 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAM F EFDIFINYIEAYMTMKIRN 16 MT1755 MMT1755 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAM L EFDIFINYIEAYMTMKIRN 17 MT1756 MMT1756 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEF S IFINYIEAYMTMKIRN 18 MT1757 MMT1757 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFD G FINYIEAYMTMKIRN 19 MT1758 MMT1758 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKI E N 20 MT1759 MMT1759 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKI H N twenty one MT1761 MMT1761 GQGTQSENSCTHFPGNLPNML G DL A DAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMFE F DIFINYIEAYMTMKIRN twenty two MT1762 MMT1762 GQGTQSENSCTHFPGNL G NML G DLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN twenty three MT1763 MMT1763 GQGTQSENSCTHFPGNLPNML G DL D DAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN twenty four MT1764 MMT1764 GQGTQSENSCTHFPGNLPNML G DLRDAFSRVKTFF P MKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 25 MT1765 MMT1765 GQGTQSENSCTHFPGNLPNMLRDL D DAFSRV D TFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 26 MT1766 MMT1766 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVSTFFQMKDQLDN G LLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 27 MT1767 MMT1767 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAM F EF S IFINYIEAYMTMKIRN 28 MT1768 MMT1768 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAM Q EFDIFI E YIEAYMTMKIRN 29 MT1769 MMT1769 GQGTQSENSCTHFPGNL F NMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 30 MT1770 MMT1770 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVK R FFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 31 MT1771 MMT1771 GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYM R MKIRN 32

(2) Each IL10MMT is fused to the PD-1 antibody to obtain a fusion protein of the IL10 monomer mutant and the PD-1 antibody.

The structure of the fusion protein is shown in Figure 2a, which includes two identical heavy chains and two identical light chains, and IL10MMT is covalently linked to the C-terminus of the PD-1 antibody light chain through a linker (SEQ ID NO: 33: GGGGSGGGGSGGGGSGGGGSG).

The names of IL10MMT and antibodies corresponding to each fusion protein are shown in Table 3 below: [Table 3] Fusion Protein IL10MMT antibody R1741 MMT1741 PD-1 Antibody R1742 MMT1742 PD-1 Antibody R1743 MMT1743 PD-1 Antibody R1744 MMT1744 PD-1 Antibody R1745 MMT1745 PD-1 Antibody R1746 MMT1746 PD-1 Antibody R1747 MMT1747 PD-1 Antibody R1748 MMT1748 PD-1 Antibody R1749 MMT1749 PD-1 Antibody R1750 MMT1750 PD-1 Antibody R1751 MMT1751 PD-1 Antibody R1752 MMT1752 PD-1 Antibody R1753 MMT1753 PD-1 Antibody R1754 MMT1754 PD-1 Antibody R1755 MMT1755 PD-1 Antibody R1756 MMT1756 PD-1 Antibody R1757 MMT1757 PD-1 Antibody R1758 MMT1758 PD-1 Antibody R1759 MMT1759 PD-1 Antibody R1761 MMT1761 PD-1 Antibody R1762 MMT1762 PD-1 Antibody R1763 MMT1763 PD-1 Antibody R1764 MMT1764 PD-1 Antibody R1765 MMT1765 PD-1 Antibody R1766 MMT1766 PD-1 Antibody R1767 MMT1767 PD-1 Antibody R1768 MMT1768 PD-1 Antibody R1769 MMT1769 PD-1 Antibody R1770 MMT1770 PD-1 Antibody R1771 MMT1771 PD-1 Antibody

Taking the fusion protein R1741 as an example, the sequence of the fusion protein is as follows:

The heavy chain sequence of the fusion protein (R1741): VH - CH1-Hinge-CH2-CH3 (SEQ ID NO.35): QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The italic part is the heavy chain variable region of the PD-1 antibody, and the underlined part is the heavy chain constant region of the PD-1 antibody; The light chain sequence of the fusion protein (R1741): VL - CL - linker - MMT1741 (SEQ ID NO.36): DTVLTQSPALAVSLGQRVTISCKASETVSSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGGGSGGGGSGGGGSGGGGSG GQGTQSENSCTHFPGNLGNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN ; the italic part is the light chain variable region of the PD-1 antibody, and the underlined part under the dotted line is the light chain constant region of the PD-1 antibody; the wavy underlined part is the linker sequence, and the solid underlined part is the MMT1741 sequence.

The structures of the other fusion proteins are basically the same as R1741, except for the different sequences of IL10MMT on the light chain.

The structures of some of the reference molecules (R0991, R1049, R0579, R1187, R0674) used in the following examples are shown in Figure 2 b-f:

R0991: The structure is basically the same as the above fusion protein, except that IL10M is used instead of IL10MMT.

R1049: PD-1 antibody, comprising two identical heavy chains and two identical light chains: The heavy chain sequence is as follows: VH - CH1-Hinge-CH2-CH3 (SEQ ID NO.35): QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK light chain sequence is as follows: VL - CL (SEQ ID NO.37): DTVLTQSPALAVSLGQRVTISCKASETVSSSMYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.

R0579: It is composed of a fusion of a PD-1 targeting antibody and IL10WT, including two identical heavy chains and two identical light chains, and the C-termini of both heavy chains are fused with IL10WT; R0579 and R1049 have the same variable regions, but different antibody subtypes; Among them, the heavy chain sequence is as follows: VH - CH1-Hinge-CH2-CH3 - linker - IL10WT (SEQ ID NO.38): QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSS AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFP APIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGA GGGGSGGGGSGGGGSGGGGSG SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN ; The light chain is as follows: VL - CL (SEQ ID NO.39): DTVLTQSP ALAVSLGQRVTISCKASETVSSSMYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKR ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC .

R1187: It is composed of a non-PD-1 targeted antibody fused with IL10M, including two identical heavy chains and two identical light chains, and the C-termini of both light chains are fused with IL10M: Among them, the heavy chain sequence is as follows: VH - CH1-Hinge-CH2-CH3 (SEQ ID NO.40): QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAAIWYDGSNKYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLRGVMYFDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The light chain is as follows: VL -CL- linker - IL10M (SEQ ID NO.41) : EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQLRNNWPLTFGGGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGGGSGGGGSGGGGSGGGGSG GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN .

R0674: consists of an Fc region and IL10 WT fused to its C-terminus, which includes two identical peptide chains, one of which is as follows: Hinge-CH2-CH3- linker - IL10WT (SEQ ID NO.42): DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GGGGSGGGGSGGGGSGGGGS SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN .

R0862 (IgG1.8 ISO), non-PD-1 targeted IgG control antibody: The heavy chain sequence is as follows: VH - CH1-Hinge-CH2-CH3 ( SEQ ID NO.40 ) : QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAAIWYDGSNKYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLRGVMYFDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ; the light chain is as follows: VL-CL (SEQ ID NO.43): EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQLRNNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC.

Embodiment 3

Preparation of fusion protein

The plasmid containing the target gene (encoding the heavy chain and light chain of the aforementioned fusion protein) - pCDDNA3.4A (see Figure 3) was constructed by conventional methods. The plasmid containing the target gene was introduced into the host cell Expi293 after forming a cationic complex with the transfection reagent PEI. While the plasmid was in the cell, the exogenous gene on the plasmid was transcribed and translated in the cell, thereby obtaining the target protein.

Expi293 was cultured at 37°C, 8% CO2, and 130 rpm. Before transfection, cells were counted and inoculated into 125 mL shake flasks at a cell density of 2.9E6 cells/mL. The culture volume was approximately 30 mL. Prepare transfection complex and prepare for transfection: first, add 75μg target plasmid to a 15mL centrifuge tube containing 1.5mL Opti-MEM reagent, mix gently, and mark it as tube A; add 0.15mg transfection reagent PEI to a 15mL centrifuge tube containing 1.5mL Opti-MEM reagent, mix gently, and incubate at room temperature for 5min, and mark it as tube B; add the PEI dilution in tube B dropwise to the DNA dilution in tube A, mix gently, and incubate at room temperature for 15min. After the incubation, add the PEI-target plasmid complex to Expi293 cells and continue to incubate in a 37℃ shaker. Collect samples after D7.

The transient cell expression solution was centrifuged at 9000rpm/20min, the supernatant was collected, and then sterilized and filtered through a 0.22μm filter membrane. ProA affinity chromatography was used for purification. The process is as follows: ProA magnetic beads plus a magnetic frame are used to purify small-volume samples in batches, and the ProA magnetic beads are balanced with at least 5CV of equilibrium buffer (10mM PBS). The treated sample and the balanced magnetic beads are mixed, and fully cultured on a uniform speed rotating instrument at room temperature for more than 1 hour to allow the target protein to adsorb on the magnetic beads. After the culture is completed, the ProA magnetic beads adsorbed with the target protein are collected with a magnetic frame and the supernatant is discarded. Wash the ProA beads again with at least 5CV of equilibration buffer (10mM PBS), collect the beads with a magnetic stand and discard the supernatant, repeat the wash twice, and discard the supernatant. Then use elution buffer (20mM NaAc, pH=3.4) to elute the target protein, adsorb the beads with a magnetic stand, collect the eluate, and add 1/50CV of neutralization buffer (1M Tris, pH8.0).

Results: The expression data of some fusion proteins are shown in Table 4. After one-step affinity purification, the samples were tested by SEC-HPLC. The target purity was above 90% for R1748, R1751, R1752, R1753 and R1754. The samples were tested by SDS-PAGE. Each fusion protein showed the correct light and heavy chain distribution, which was consistent with the SEC-HPLC test results. The electrophoresis results are shown in Figure 4. [Table 4] Fusion protein sample name Conc. after purification (mg/mL) SEC-Purity HMW% Main% LMW% R1741 0.65 15.64 82.21 2.15 R1742 0.41 43.66 50.84 5.50 R1743 0.52 15.94 82.47 1.59 R1748 1.06 3.47 96.01 0.53 R1749 0.27 18.95 77.47 3.58 R1750 0.52 14.53 84.42 1.04 R1751 0.59 1.52 97.96 0.53 R1752 0.29 3.10 96.09 0.80 R1753 0.33 2.40 97.16 0.44 R1754 0.38 4.60 94.46 0.94 R1755 0.53 20.20 77.83 1.97 R1756 0.46 36.52 58.94 4.53 R1758 0.75 12.08 87.26 0.66 R1759 0.53 16.30 82.76 0.94 R1762 0.49 16.94 80.38 2.68 R1764 0.52 14.17 83.04 2.79 R1766 0.61 23.61 75.32 1.07 R1768 0.61 41.72 55.68 2.60 R1769 0.54 21.48 77.63 0.89 R1770 0.44 29.20 70.42 0.38 R1771 0.63 17.10 82.04 0.87

Embodiment 4

In vitro activity of fusion proteins

4.1 The binding activity of each IL10 fusion protein to IL10Rα was detected by flow cytometry.

Each fusion protein molecule was diluted to an initial concentration of 200 nM with PBS buffer containing 3% BSA, a volume of 180 μL, 3-fold gradient dilution (60 μL sample + 120 μL dilution buffer), and 10 concentration gradient points. CHO-hIL10R (CHO cells expressing human IL10Rα) cells were centrifuged at 350g/5min, the supernatant was discarded, the cell density was adjusted to 2E+06 with 3% BSA PBS buffer, and the cells were evenly distributed into 96-well V-shaped plates at 100μL/well; the above-mentioned diluted molecules were added to the cells at 100μL/well and cultured at 2-8 degrees for 0.5h; the 96-well plate was taken out, centrifuged at 350g for 5min, the supernatant was carefully discarded, and 3% BSA PBS buffer 200μL/well was added, and the cells were centrifuged at 350g for 5min again, and the supernatant was carefully discarded; PBS containing 3% BSA was used to Prepare PE fluorescent secondary antibody (1:500 dilution) in buffer, add 100 μL/well to the corresponding 96-well plate, resuspend the cells, and incubate at 2-8 degrees for 30 minutes; take out the 96-well plate, centrifuge at 350g for 5 minutes, carefully remove the supernatant, add PBS buffer containing 3% BSA at 200 μL/well, centrifuge at 350g for 5 minutes again, and carefully remove the supernatant; resuspend with 1xPBS 100 μL/well, and detect by FACS.

The results of the fusion proteins obtained by the small-scale preparation method (Example 3) for binding activity to IL10Rα are shown in Figures 6a-6d; it can be seen that compared with the control molecule R0674, the binding activity of each fusion protein to IL10Rα is lower, indicating that the mutations in Table 1 reduce the binding activity of IL10 mutants or monomer mutants to IL10Rα.

Embodiment 5

Detection of IL10 activity of fusion protein in the presence of target cells

All the above fusion proteins were diluted to an initial concentration of 50 nM with Assay Buffer (DMEM containing 10% FBS), a volume of 360 μL, 3-fold gradient dilution (120 μL sample + 240 μL dilution buffer), and 6 concentration gradient points; Cell preparation: HEK293-hIL10 reporter gene cells (Genman Biotech, Cat. No. GM-C07927; this cell line stably expresses the IL10R and STAT3 signaling pathway reporter gene system, and can activate the expression of cell luciferase by IL10 protein stimulation) were taken out and observed under a microscope. The cells adhered normally, the particles were transparent, and the cells with moderate density could be used as effector cells for experiments.

The above cells were digested with TE, and after terminating the digestion, the supernatant was removed by centrifugation at 350g/4min, and the cells were resuspended with 1% FBS-PBS and washed again; the supernatant was discarded, and the cells were finally resuspended with culture medium. After counting the cells, the cell density was adjusted to 5E5/mL, and 50μL was plated per well, and then placed in a 37℃ incubator for 6h to adhere to the wall. Target cells R0326Fc-118 cells (CHO cells that stably express PD-1 and play an enrichment role in the system) were centrifuged at 350g/5min, the supernatant was discarded, and the cell density was adjusted to 1E+06/mL with Assay Buffer; two reaction systems were prepared in a flat-bottom plate (1 Enrichment system: According to the experimental design, the diluted fusion protein was first mixed with the target cell R0326Fc-118 at a volume of 25μL/well, and co-cultured for about 30min. Then, the target cell R0326Fc-118 and each fusion protein were carefully transferred into the adherent HEK293-hIL10 reporter gene cells; 100μl Assay Buffer was added to the Medium only wells; Buffer, add 25μL of Assay Buffer to the wells of Cell only, and the final volume of all wells is 100μL. Seal the edge wells with 200μL of sterile water, and continue to culture the 96-well plate in the incubator for 16h; 2 No enrichment system: do not add target cells (i.e. R0326Fc-118), that is, directly add the corresponding fusion protein to the HEK293 cells that have been treated for adhesion; other operations are the same as the enrichment system). Thaw the Bright-LumiTM Firefly Luciferase Assay Reagent in advance and equilibrate to room temperature. After the cells are cultured to the time point, take out the cell culture plate and equilibrate to room temperature for 10min (not more than 30min). Add 100 μL Bright-LumiTM Firefly Luciferase Detection Reagent to each well and incubate at room temperature for 5-10 minutes. Take 100 μL of the reaction system from each well and transfer it to a 96-well all-white plate; use a multifunctional enzyme labeler to detect the signal using the chemiluminescence mode.

The test results are shown in Figures 7-26. It can be seen that the reporter gene signal values of each fusion protein fused with IL10 monomer mutants in the enrichment system with target cells are all improved or reporter gene signal values appear after enrichment. Among them, R1750, R1751, R1758, R1759, R1766, R1768, and R1770 have significantly improved signal values after enrichment at the target end, which are close to or higher than the signal value of R0991. There is no significant difference in the signal value of R0674 before and after enrichment, and the signal value of R0579 decreases after enrichment, indicating that enrichment will weaken the binding with effector cells for R0579, and the signal value of R1187 before and after enrichment has basically not changed, indicating that the system is stable.

The above results indicate that the IL10MMT provided in the embodiments of the present invention has weak binding activity with IL10Rα. When it is fused with the targeted antibody, the fusion protein will exert IL10 activity only in the presence of target cells. In the absence of target cells, the IL10 activity is low or absent, which further indicates that the IL10 mutant provided in the embodiments of the present invention has low affinity for its receptor, thereby increasing the IL10 tolerance value, reducing side effects and improving safety.

Embodiment 6

ELISA was used to detect the binding activity of each IL10 fusion protein with IL10Rα

The antigen IL10Rα (IL10Rα-his) was diluted to 1μg/mL (diluent: 50 mM CB), added to a 96-well ELISA plate at 100μL/well, sealed with a sealing film, and placed in a 4°C refrigerator overnight; the next day, the plate was washed 3 times with 1×PBST and patted dry, and assay buffer (1% BSA) was added at 200μL/well, and sealed at room temperature for 1.5h; the plate was washed once with 1×PBST and patted dry, and the antibody to be tested diluted in assay buffer (60nM starting, 3-fold dilution of 11 concentration points) was added at 100μL/well, sealed with a sealing film, and incubated at room temperature with shaking (500 rpm) for 1.5h; the plate was washed 5 times with 1×PBST and patted dry, and GAH-IgG Fc diluted in assay buffer was added at 100μL/well. HRP (1:10K), sealed with sealing film, incubated at room temperature for 40 min; washed the strips 6 times with 1×PBST and patted dry, added TMB colorimetric solution at 100μL/well, incubated at room temperature for 5-10 min; added stop solution at 100μL/well, read the plate at 450 nm & 630 nm, and analyzed the data with SoftMax Pro.

The binding activity detection results are shown in Figure 27a-Figure 27g. In the figure, "Span" represents the difference between the high platform and the low platform. The larger the value, the larger the window and the higher the detection sensitivity. It can be seen that after mutations at different positions on the structure of R0991, the binding affinity of the IL10 modified molecules R1741, R1742, R1743, R1744, R1745, R1746, R1747, R1748, R1749, R1750, R1751, R1752, R1754, R1755, R1756, R1757, R1758, R1759, R1761, R1762, R1763, R1764, R1765, R1766, R1767, R1768, R1769, R1770, and R1771 to IL10Rα decreased to varying degrees compared to R0991 without mutations. It is shown that the mutations in Table 1 reduce the binding activity of the IL10 mutant or monomer mutant to IL10Rα.

Embodiment 7

ELISA was used to detect the binding activity of each IL10 fusion protein to IL10Rβ

The antibody His-Tag Mouse mAb was diluted to 1μg/mL (diluent: 50 mM CB), added to a 96-well ELISA plate at 100μL/well, sealed with a sealing film, and placed in a 4°C refrigerator overnight; the next day, the plate was washed 3 times with 1×PBST and patted dry, and assay buffer (1% BSA) was added at 200μL/well, and sealed at room temperature for 1.5h; the plate was washed once with 1×PBST and patted dry, and antigen R2050 (IL10β-his, 1μg/mL) diluted in assay buffer was added at 100μL/well, sealed with a sealing film, and incubated at room temperature with shaking (500 rpm) for 1h; the plate was washed 5 times with 1×PBST and patted dry, and assay buffer was added at 100μL/well. The antibody to be tested was diluted with assay buffer (starting at 60 nM, 3-fold dilution to 11 concentration points), sealed with a sealing film, and incubated at room temperature with shaking (500 rpm) for 1.5 h; the plates were washed 5 times with 1×PBST and tapped dry, GAH-IgG Fc HRP (1:10K) diluted with assay buffer was added at 100 μL/well, sealed with a sealing film, and incubated at room temperature for 40 min; the plates were washed 6 times with 1×PBST and tapped dry, TMB color development solution was added at 100 μL/well, and incubated at room temperature for 20-30 min; the stop solution was added at 100 μL/well, the plates were read at 450 nm & 630 nm, and the data were analyzed by SoftMax Pro.

The results of binding activity detection are shown in Figure 28a-28g; it can be seen that after mutations at different positions on the structure of R0991, the binding ability of IL10 modified molecules R1741, R1742, R1743, R1744, R1745, R1746, R1747, R1748, R1749, R1750, R1751, R1755, R1756, R1758, R1759, R1761, R1762, R1763, R1764, R1765, R1766, R1767, R1768, R1769, and R1771 to IL10Rβ all showed varying degrees of decrease compared to R0991 without mutation. It is shown that the mutations in Table 1 reduce the binding activity of the IL10 mutant or monomer mutant to IL10Rβ.

Embodiment 8

ELISA was used to detect the binding activity of each IL10 fusion protein with IL10Rα/IL10Rβ

Antigen R2051-E7 (IL10Rα/IL10Rβ-hFc) was diluted to 1μg/mL (diluent: 50 mM CB), added to a 96-well ELISA plate at 100μL/well, sealed with a sealing film, and placed in a 4°C refrigerator overnight; the next day, the plate was washed 3 times with 1×PBST and patted dry, and assay buffer (1% BSA) was added at 200μL/well, and sealed at room temperature for 1.5h; the plate was washed once with 1×PBST and patted dry, and the antibody to be tested diluted in assay buffer (60nM starting point, 3-fold dilution of 11 concentration points) was added at 100μL/well, sealed with a sealing film, and incubated at room temperature with shaking (500 rpm) for 1.5h; the plate was washed 5 times with 1×PBST and patted dry, and assay buffer was added at 100μL/well. mPD1-His (1μg/mL) diluted in buffer was sealed with a sealing film and incubated at room temperature for 1h; the plates were washed 5 times with 1×PBST and tapped dry, anti-his HRP (1:5K) diluted in assay buffer was added at 100μL/well, the plates were sealed with a sealing film and incubated at room temperature for 40 min; the plates were washed 6 times with 1×PBST and tapped dry, TMB color development solution was added at 100μL/well, and incubated at room temperature for 10-15min; stop solution was added at 100μL/well, the plates were read at 450nm&630nm, and the data were analyzed by SoftMax Pro.

The results of binding activity detection are shown in Figure 29a-29g; it can be seen that after mutations at different positions on the structure of R0991, the IL10 modified molecules R1741, R1742, R1743, R1744, R1745, R1746, R1747, R1748, R1749, R1750, R1751, R1752, R1754, R1755, R1760, R1761, R1762, R1763, R1764, R1765, R1766, R1767, R1768, R1769, R1770, R1771, R1772, R1773, R1774, R1775 The binding ability of 55, R1756, R1757, R1758, R1759, R1761, R1762, R1763, R1764, R1765, R1766, R1767, R1768, R1769, R1770, and R1771 to IL10Rα/IL10β decreased to varying degrees compared with R0991 without mutation. This indicates that the mutations in Table 1 reduce the binding activity of IL10 mutants or monomer mutants to IL10Rα/IL10Rβ.

The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

without

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the figures required for use in the embodiments. It should be understood that the following figures only show certain embodiments of the present invention and should not be regarded as limiting the scope. For ordinary technicians in this field, other related figures can be obtained based on these figures without creative work. Figure 1 is a sequence alignment result diagram of IL10WT, IL10M, and IL10 monomer mutants (MMT1741, MMT1761), and the arrows are mutation sites. Figure 2 is a schematic diagram of the structure of the fusion protein and the control molecule in the embodiment. Figure 3 is a plasmid map of pCDDNA3.4A. Figure 4 is the SDS-PAGE electrophoresis result of the fusion protein prepared by the method of Example 3 (upper row: unreduced, lower row: reduced, the sizes of the MK bands from top to bottom are: 180Kd, 130 Kd, 100 Kd, 70 Kd, 55 Kd, 40 Kd, 35 Kd, 25 Kd, 15 Kd, 10 Kd). Figure 5 is the SDS-PAGE electrophoresis result of the fusion protein prepared by the method of Example 4 (NR: unreduced, R: reduced). Figure 6a is the result of the binding activity test of part of the fusion protein with hIL10Rα (human IL10 receptor α) by FCM (Flow Cytometry). Figure 6b is the result of the binding activity test of part of the fusion protein with hIL10Rα (human IL10 receptor α) by FCM. Figure 6c shows the results of the binding activity test of some fusion proteins with hIL10Rα (human IL10 receptor α) by FCM. Figure 6d shows the results of the binding activity test of some fusion proteins with hIL10Rα (human IL10 receptor α) by FCM. Figure 7 shows the results of the IL10 activity test of fusion protein R1741 in the system with and without target cells; solid-no target cells (i.e., no enrichment system), hollow-presence of target cells (enrichment system). Figure 8 shows the results of the IL10 activity test of fusion protein R1742 in the system with and without target cells; solid-no target cells, hollow-presence of target cells. Figure 9 shows the results of IL10 activity detection of fusion protein R1743 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 10 shows the results of IL10 activity detection of fusion protein R1748 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 11 shows the results of IL10 activity detection of fusion protein R1749 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 12 shows the results of IL10 activity detection of fusion protein R1750 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 13 shows the results of IL10 activity detection of fusion protein R1751 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 14 shows the results of IL10 activity detection of fusion protein R1752 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 15 shows the results of IL10 activity detection of fusion protein R1753 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 16 shows the results of IL10 activity detection of fusion protein R1754 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 17 shows the results of IL10 activity detection of fusion protein R1755 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 18 shows the results of IL10 activity detection of fusion protein R1758 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 19 shows the results of IL10 activity detection of fusion protein R1759 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 20 shows the results of IL10 activity detection of fusion protein R1762 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 21 shows the results of IL10 activity detection of fusion protein R1764 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 22 shows the results of IL10 activity detection of fusion protein R1766 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 23 shows the results of IL10 activity detection of fusion protein R1768 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 24 shows the results of IL10 activity detection of fusion protein R1769 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 25 shows the results of IL10 activity detection of fusion protein R1770 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 26 shows the results of IL10 activity detection of fusion protein R1771 in the presence and absence of target cells; solid-no target cells, hollow-presence of target cells. Figure 27a shows the results of activity detection of each IL10 fusion protein binding to IL10Rα by ELISA. Figure 27b shows the results of activity detection of each IL10 fusion protein binding to IL10Rα by ELISA. Figure 27c shows the results of activity detection of each IL10 fusion protein binding to IL10Rα by ELISA. Figure 27d shows the activity detection results of each IL10 fusion protein binding to IL10Rα by ELISA. Figure 27e shows the activity detection results of each IL10 fusion protein binding to IL10Rα by ELISA. Figure 27f shows the activity detection results of each IL10 fusion protein binding to IL10Rα by ELISA. Figure 27g shows the activity detection results of each IL10 fusion protein binding to IL10Rα by ELISA. Figure 28a shows the activity detection results of each IL10 fusion protein binding to IL10Rβ by ELISA. Figure 28b shows the activity detection results of each IL10 fusion protein binding to IL10Rβ by ELISA. Figure 28c shows the activity detection results of each IL10 fusion protein binding to IL10Rβ by ELISA. Figure 28d shows the activity detection results of each IL10 fusion protein binding to IL10Rβ by ELISA. Figure 28e shows the activity detection results of each IL10 fusion protein binding to IL10Rβ by ELISA. Figure 28f shows the activity detection results of each IL10 fusion protein binding to IL10Rβ by ELISA. Figure 28g shows the activity detection results of each IL10 fusion protein binding to IL10Rβ by ELISA. Figure 29a shows the activity detection results of each IL10 fusion protein binding to IL10Rα/IL10Rβ by ELISA. Figure 29b shows the activity detection results of each IL10 fusion protein binding to IL10Rα/IL10Rβ by ELISA. Figure 29c shows the activity detection results of each IL10 fusion protein binding to IL10Rα/IL10Rβ by ELISA. Figure 29d shows the activity detection results of each IL10 fusion protein binding to IL10Rα/IL10Rβ by ELISA. Figure 29e shows the activity detection results of each IL10 fusion protein binding to IL10Rα/IL10Rβ by ELISA. Figure 29f shows the activity detection results of each IL10 fusion protein binding to IL10Rα/IL10Rβ by ELISA. Figure 29g shows the activity detection results of each IL10 fusion protein binding to IL10Rα/IL10Rβ by ELISA.

TW202426471A_112148262_SEQL.xml

Claims (23)

An IL10 mutant is characterized in that it has the following amino acid mutations at at least one of the following positions relative to SEQ ID NO. 1: P20, R24, R27, K34, T35, Q38, D41, L46, L48, E50, S141, D144, I145, N148, T155, and R159. The IL10 mutant as described in claim 1 is characterized in that it has the following amino acid mutations at at least one of the following positions relative to SEQ ID NO.1: P20, R24, T35, D41, L46, L48, E50, S141, I145, T155, and R159; Optionally, the amino acid mutation is an amino acid substitution. The IL10 mutant as described in claim 2 is characterized in that the IL10 mutant has the following amino acid mutation at the following position relative to SEQ ID NO.1: I145; Optionally, the amino acid mutation is selected from I145G, I145A, I145R, I145L, I145K, I145N, I145M, I145D, I145F, I145C, I145P, I145Q, I145S, I145E, I145T, I145W, I145H, I145Y, or I145V. The IL10 mutant as described in claim 3 is characterized in that the IL-10 mutant also has the following amino acid mutations at at least one of the following positions relative to SEQ ID NO.1: P20, R24, R27, K34, T35, Q38, D41, L46, L48, E50, S141, D144, N148, T155, and R159. The IL10 mutant as described in any one of claim 3-4 is characterized in that the IL-10 mutant further has at least one of the following mutations relative to SEQ ID NO.1: P20G, P20F, R24P, R27D, R27G, K34S, K34D, T35R, Q38P, D41W, L46G, L46K, L48G, E50G, S141F, S141Q, S141L, D144S, N148E, T155R, R159E, and R159H. The IL10 mutant as described in claim 1 or 2 is characterized in that the IL-10 mutant has at least one of the following amino acid mutations relative to SEQ ID NO.1: P20G, P20F, R24G, R24P, R27D, R27G, T35R, D41W, L46G, L48G, E50G, S141L, S141F, S141Q, T155R, R159E, and R159H. The IL10 mutant as described in claim 6 is characterized in that the IL-10 mutant further has the following amino acid mutations at at least one of the following positions relative to SEQ ID NO.1: K34, Q38, L46, D144, N148, and I145. The IL10 mutant as described in claim 7 is characterized in that the IL-10 mutant further has at least one of the following amino acid mutations relative to SEQ ID NO.1: K34S, K34D, Q38P, L46K, D144S, N148E, I145G, I145A, I145R, I145L, I145K, I145N, I145M, I145D, I145F, I145C, I145P, I145Q, I145S, I145E, I145T, I145W, I145H, I145Y, and I145V. The IL10 mutant as described in any one of claims 1-2, 6-8 is characterized in that the IL-10 mutant has the following amino acid mutations relative to SEQ ID NO.1: (a) P20G; (b) R24G; (c) D41W; (d) L46G; (e) L48G; (f) E50G; (g) S141F; (h) P20G/R24G; (i) K34S/L46G; (j) S141Q/N148E; (k) P20F; (l) T35R; (m) R24P; (n) R27D; (o) R27G; (p) S141L; (q) I145G; (r) R159E; (s) R159H; (t) R24G/R27A/S141F; (u) R24G/R27D; (v) R24G/Q38P; (w) R27D/K34D; (x) S141F/D144S; or (y) T155R. The IL10 mutant as described in any one of claim items 1-9 is characterized in that the IL-10 mutant has at least 80% identity with SEQ ID NO.1; Optionally, the amino acid sequence of the IL-10 mutant at other sites other than the above-mentioned mutation site is the same as SEQ ID NO.1; Optionally, the IL10 mutant has reduced binding activity to the IL10 receptor compared to the wild-type IL10 shown in SEQ ID NO.1; Optionally, the IL10 receptor is selected from IL10Rα, IL10Rβ, and IL10Rα/IL10Rβ; Optionally, the IL10 receptor is selected from IL10Rα; Optionally, the IL10 receptor is selected from IL10Rβ; Optionally, the IL10 receptor is selected from IL10Rα/IL10Rβ. The IL10 mutant as described in any one of claim items 1-10 is characterized in that the IL10 mutant is selected from a monomer or a dimer; Optionally, the IL10 mutant is selected from a monomer; Optionally, the IL10 mutant is selected from a dimer; Optionally, the IL10 mutant has a spacer peptide inserted relative to the wild-type IL10 shown in SEQ ID NO.1; Optionally, the IL10 mutant has a spacer peptide inserted between positions 116 and 117 relative to the wild-type IL10 shown in SEQ ID NO.1; Optionally, the length of the spacer peptide is 5-10 amino acids; Optionally, the spacer peptide contains amino acid residues G and S; Optionally, the amino acid sequence of the spacer peptide is SEQ ID NO.2 (GGGSGG); Optionally, the IL10 mutant also lacks the 1st and 2nd amino acids at its N-terminus relative to the wild-type IL10 shown in SEQ ID NO.1; Optionally, the IL10 mutant sequence is selected from any one of SEQ ID NO.3-32, or has at least 80% identity with any one of them. A fusion protein, characterized in that it comprises the IL10 mutant according to any one of claims 1 to 11. The fusion protein as described in claim 12 is characterized in that the fusion protein includes an antigen binding molecule, and the IL10 mutant is linked to the antigen binding molecule; Optionally, the antigen binding molecule specifically binds to a tumor-specific antigen, an immune checkpoint molecule, or a combination of the two. The fusion protein as described in claim 13 is characterized in that the immune checkpoint molecule is selected from PD-1, CD137, CD134, KIR, LAG-3, PD-L1, CTLA-4, B7.1, B7H3, CCRY, OX-40 and CD40. The fusion protein as described in claim 13 or 14 is characterized in that the antigen-binding molecule is an antibody or an antigen-binding fragment thereof; Optionally, the antibody is selected from IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, and IgD; Optionally, the IL10 mutant is directly or indirectly connected to one or both heavy chain N-termini, one or both heavy chain C-termini, between VH and CH1 of one or both heavy chains, between CH1 and CH2 of one or both heavy chains, between CH2 and CH3 of one or both heavy chains, one or both light chain N-termini, one or both light chain C-termini, and/or between VL and CL of one or both light chains in the antibody; Optionally, the C-termini of the two light chains of the antibody are connected to the IL10 mutant; Optionally, the IL10 mutant is connected to the C-termini of the light chain of the antibody via a linker peptide; Optionally, the sequence of the linker peptide contains amino acid residues G and S; Optionally, the amino acid sequence of the linker peptide is such as SEQ ID NO.33 (GGGGSGGGGSGGGGSGGGGSG); Optionally, the amino acid sequence of the light chain in the antibody includes: VL-CL-Linker-M; Wherein, VL is the light chain variable region sequence, CL is the light chain constant region sequence, Linker is the linker peptide sequence, and M is the IL10 mutant sequence. The fusion protein as described in any one of claims 12 to 15 is characterized in that the IL10 mutant has at least one of the following amino acid mutations relative to SEQ ID NO.1: K34S, K34D, Q38P, L46K, and D144S. An isolated nucleic acid molecule, characterized in that it encodes the IL10 mutant as described in any one of claims 1-11, or the fusion protein as described in any one of claims 12-16. A recombinant vector, characterized in that it contains the nucleic acid molecule of claim 17. A recombinant cell, characterized in that it contains the nucleic acid molecule of claim 17, or contains the recombinant vector of claim 18. Use of the IL10 mutant as described in any one of claims 1 to 11, the fusion protein as described in any one of claims 12 to 16, the nucleic acid molecule as described in claim 17, the recombinant vector as described in claim 18, or the recombinant cell as described in claim 19 in the preparation of a drug for preventing or treating tumors. A method for preparing the IL10 mutant as described in any one of claims 1 to 11 or the fusion protein as described in any one of claims 12 to 16, characterized in that it comprises: culturing cells expressing the IL10 mutant or the fusion protein, and isolating and purifying the IL10 mutant or the fusion protein from the culture product. A pharmaceutical composition, characterized in that it comprises the IL10 mutant as described in any one of claims 1-11, the fusion protein as described in any one of claims 12-16, the nucleic acid molecule as described in claim 17, the recombinant vector as described in claim 18, or the recombinant cell as described in claim 19. A method for treating tumors, characterized in that it comprises: administering a therapeutically effective amount of the IL10 mutant as described in any one of claims 1-11, the fusion protein as described in any one of claims 12-16, or the pharmaceutical composition as described in claim 22 to a subject in need of treatment.