IL10 mutant antibody fusion protein and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses an IL10 mutant antibody fusion protein and application thereof. Specifically, the invention provides a fusion protein which comprises a PD1 binding unit and an IL10 unit, and the fusion protein disclosed by the invention has good targeting property and safety and can be used for treating tumors or inflammatory diseases.

Description

[0001] Priority declaration

[0002] This application claims priority to Chinese invention patent application with application number 202311302688.4, filing date October 9, 2023, and invention name “IL10 mutant antibody fusion protein and its application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of biotechnology, and in particular, to an IL10 mutant antibody fusion protein and applications thereof. Background Art

[0004] Programmed cell death protein 1 (PD1, PD-1) is a member of the CD28 family and is expressed on activated B cells, T cells and myeloid cells. Human PD1 is encoded by the gene Pdcd1, located at 2q37.3, with a total length of 9.6kb, consisting of 5 exons and 4 introns, and a 663bp promoter upstream. PD1 is a 55KDa type I transmembrane protein, and its molecular structure consists of an extracellular region, a transmembrane region and an intracellular region. The extracellular region contains an immunoglobulin variable region IgV domain, and the intracellular region contains an immunoreceptor tyrosine inhibitory motif (ITIM) and an immunoreceptor tyrosine switch module (ITSM). The amino acid sequence of the PD-1 extracellular region has 24% homology with CTLA-4 and 28% homology with CD28. After T cells are activated, PD-1 mainly aggregates tyrosine phospholipase SHP2 through ITIM, resulting in dephosphorylation of downstream effector molecules. PD-1 has two ligands: PD-L1 and PD-L2. Both PD-L1 and PD-L2 are B7 homologs. The PDL gene is located at the 9P24.2 site of human chromosome, with a size of 42kb. Its molecular structure contains an immunoglobulin-like variable region domain, a constant region-like domain, a transmembrane region and a short cytoplasmic tail. PD-1 can downregulate T cell activation after binding to PD-L1 and PD-L2. PD-L1 is expressed on the surface of a variety of tumor cells, including lung cancer, gastric cancer, liver cancer, esophageal cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, skin cancer, colon cancer, bladder cancer, glioma, head and neck cancer, and oral squamous cell carcinoma. In addition, a large number of CD8+T cells expressing PD1 were found around these cancers. Clinical results show that high expression levels of PD-L1 on tumor cells are associated with poor prognosis in cancer patients.

[0005] Exhausted CD8+T cells play an important role in the process of immune checkpoint therapy resistance. Under continuous antigen stimulation and the action of various inhibitory molecules, T cells will be transformed from an activated state to an exhausted state. Exhausted CD8+T cells have significantly reduced proliferation ability, cytokine secretion ability, and tumor killing ability, and cannot provide effective anti-tumor immunity. Even if anti-PD-1 antibodies are given to CD8+T cells in an exhausted state to block the PD-1 / PD-L1 pathway, the exhausted CD8+T cells cannot return to an activated state. Summary of the invention

[0006] The present disclosure provides a fusion protein, which includes a PD1 binding unit portion and an IL10 unit portion.

[0007] In some embodiments, the present disclosure discloses a fusion protein, which includes a PD1 binding unit portion and an IL10 unit portion, wherein the PD1 binding unit portion is an anti-PD1 antibody, and the IL10 unit portion is an IL10 mutant; wherein the IL10 mutant has an amino acid mutation at at least one of the following sites relative to SEQ ID NO.21: L46, P20, R24, R27, K34, T35, Q38, D41, L48, E50, S141, D144, I145, N148, T155 and R159.

[0008] In some embodiments, the fusion protein as described in any of the above items, the IL10 mutant has an amino acid mutation at at least one of the following positions relative to SEQ ID NO. 21: L46, P20, R24, T35, D41, L48, E50, S141, I145, T155 and R159;

[0009] In some embodiments, the fusion protein as described in any of the above items, the IL10 mutant has an amino acid mutation at at least one of the following positions relative to SEQ ID NO. 21: L46, K34, Q38, D144, N148 and I145;

[0010] In some embodiments, the fusion protein as described in any of the above items, the IL10 mutant has an amino acid mutation at the following positions relative to SEQ ID NO.21: L46;

[0011] In some embodiments, in the fusion protein as described in any of the above items, the amino acid mutation is an amino acid substitution.

[0012] In some embodiments, the fusion protein as described in any of the above items, wherein the IL10 mutant, the IL10 mutant relative to SEQ ID NO.21 has at least one of the following amino acid mutations: L46K, L46G, P20G, P20F, R24P, R27D, R27G, K34S, K34D, T35R, Q38P, D41W, L48G, E50G, S141F, S141Q, S141L, D144S, N148E, T155R, R159E, R159H, I145G, I145A, I145R, I145L, I145K, I145N, I145M, I145D, I145F, I145C, I145P, I145Q, I145S, I145E, I145T, I145W, I145H, I145Y, and I145V.

[0013] In some embodiments, the fusion protein as described in any of the above items, the IL10 mutant has at least one of the following amino acid mutations relative to SEQ ID NO.21: L46K, L46G, P20G, P20F, R24G, R24P, R27D, R27G, T35R, D41W, L48G, E50G, S141L, S141F, S141Q, T155R, R159E, R159H.

[0014] In some embodiments, in the fusion protein as described in any of the above items, the IL10 mutant has the following mutations relative to SEQ ID NO.21:

[0015] (a) L46K;

[0016] (b) R24G;

[0017] (c)D41W;

[0018] (d) L46G;

[0019] (e) L48G;

[0020] (f) E50G;

[0021] (g) S141F;

[0022] (h) P20G / R24G;

[0023] (i) K34S / L46G;

[0024] (j) S141Q / N148E;

[0025] (k) P20F;

[0026] (l) T35R;

[0027] (m)R24P;

[0028] (n) R27D;

[0029] (o)R27G;

[0030] (p)S141L;

[0031] (q)I145G;

[0032] (r)R159E;

[0033] (s)R159H;

[0034] (t)R24G / R27A / S141F;

[0035] (u)R24G / R27D;

[0036] (v) R24G / Q38P;

[0037] (w)R27D / K34D;

[0038] (x) S141F / D144S;

[0039] (y) T155R; or

[0040] (z)P20G.

[0041] In some embodiments, in the fusion protein as described in any of the above items, the IL10 mutant has the following amino acid mutation relative to SEQ ID NO.21: L46K.

[0042] In some embodiments, the fusion protein as described in any of the above items, the IL10 mutant, the IL10 mutant has at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO.21;

[0043] In some embodiments, in the fusion protein as described in any of the above items, the amino acid sequence of the IL10 mutant at other sites other than the above mutation site is the same as SEQ ID NO.21.

[0044] In some embodiments, the fusion protein as described in any of the above items, the IL10 mutant has similar or reduced binding activity to the IL10 receptor compared to the wild-type IL10 shown in SEQ ID NO.21; in some embodiments, the fusion protein as described in any of the above items, the IL10 mutant has reduced binding activity to the IL10 receptor compared to the wild-type IL10 shown in SEQ ID NO.21; in some embodiments, the fusion protein as described in any of the above items, the IL10 mutant can bind to the IL10 receptor; in some embodiments, the IL10 receptor is selected from IL10Rα and IL10Rβ; in some embodiments, the IL10 receptor is selected from IL10Rα; in some embodiments, the IL10 receptor is selected from IL10Rβ. In some embodiments, the fusion protein as described in any of the above items, the IL10 mutant has similar IL10Rβ binding activity to the wild-type IL10 shown in SEQ ID NO.21.

[0045] In some embodiments, the fusion protein as described in any of the above items, wherein the IL10 mutant has a spacer peptide inserted between positions 116 and 117 relative to the wild-type IL10 shown in SEQ ID NO.21; in some embodiments, the length of the spacer peptide is 5-10 amino acids; in some embodiments, the spacer peptide contains amino acid residues G and S; in some embodiments, the amino acid sequence of the spacer peptide is SEQ ID NO.22;

[0046] In some embodiments, in the fusion protein as described in any of the above items, the IL10 mutant further lacks the 1st and 2nd amino acid residues at its N-terminus relative to the wild-type IL10 shown in SEQ ID NO.21;

[0047] In some embodiments, in the fusion protein described in any of the above items, the IL10 mutant is an amino acid sequence selected from any one of SEQ ID NO. 23-53, or an amino acid sequence having at least 80% sequence identity therewith;

[0048] In some embodiments, in the fusion protein as described in any of the above items, the amino acid sequence of the IL10 mutant is shown as SEQ ID NO.23.

[0049] In some embodiments, the fusion protein as described in any of the above items, wherein the PD1 binding unit portion is an anti-PD1 antibody, and the IL10 unit portion is an IL10 mutant; wherein the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 in the variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.1, 3, 5 or 7, and / or the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in the variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.2, 4, 6 or 8;

[0050] In some embodiments, the fusion protein as described in any of the above, wherein the anti-PD1 antibody, wherein

[0051] A. The heavy chain variable region of the anti-PD1 antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO.1, and the light chain variable region of the anti-PD1 antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO.2;

[0052] B. The heavy chain variable region of the anti-PD1 antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO.3, and the light chain variable region of the anti-PD1 antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO.4;

[0053] C. the heavy chain variable region of the anti-PD1 antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO.5, and the light chain variable region of the anti-PD1 antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO.6; or

[0054] D. The heavy chain variable region of the anti-PD1 antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO.7, and the light chain variable region of the anti-PD1 antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO.8.

[0055] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.

[0056] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system.

[0057] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the IMGT numbering system.

[0058] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody, the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.9, HCDR2 comprises the amino acid sequence of SEQ ID NO.10, 15 or 16, and HCDR3 comprises the amino acid sequence of SEQ ID NO.11; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.12, LCDR2 comprises the amino acid sequence of SEQ ID NO.13, and LCDR3 comprises the amino acid sequence of SEQ ID NO.14.

[0059] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0060] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.9, SEQ ID NO.15 and SEQ ID NO.11, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0061] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.9, SEQ ID NO.16 and SEQ ID NO.11, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0062] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody is a humanized antibody, a murine antibody or a chimeric antibody.

[0063] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody, wherein the heavy chain variable region of the anti-PD1 antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.1, 3, 5 or 7, and / or the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.2, 4, 6 or 8.

[0064] In some embodiments, a fusion protein as described in any of the above items, wherein the anti-PD1 antibody, wherein the heavy chain variable region of the anti-PD1 antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO.1, 3, 5 or 7, and the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO.2, 4, 6 or 8.

[0065] In some embodiments, the fusion protein as described in any of the above, wherein the anti-PD1 antibody, wherein

[0066] (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.2;

[0067] (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.4;

[0068] (c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.6; or

[0069] (d) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.8;

[0070] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody comprises a heavy chain variable region as shown in SEQ ID NO.1, and a light chain variable region as shown in SEQ ID NO.2.

[0071] In some embodiments, a fusion protein is disclosed, which includes a PD1 binding unit portion and an IL10 unit portion, wherein the PD1 binding unit is an anti-PD1 antibody, and the IL10 unit is an IL10 mutant; wherein,

[0072] (a) the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.1, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.2;

[0073] (b) the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.3, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.4;

[0074] (c) the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.5, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.6;

[0075] (d) the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has at least 95% sequence identity with SEQ ID NO.7, and the light chain variable region has at least 95% sequence identity with SEQ ID NO.8; or

[0076] (e) The anti-PD1 antibody is an antibody that competes with the anti-PD1 antibody described in (a), (b), (c) or (d) for binding to the PD1 antigen or binds to the same epitope.

[0077] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody comprises a heavy chain variable region as shown in SEQ ID NO.1, and a light chain variable region as shown in SEQ ID NO.2.

[0078] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody comprises a constant region.

[0079] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody, the heavy chain constant region of the antibody is selected from the heavy chain constant region of IgG1, IgG2, IgG3 and IgG4, and / or the light chain constant region is selected from the constant region of κ or λ chain;

[0080] In some embodiments, in the fusion protein as described in any of the above items, the anti-PD1 antibody, the species origin of the constant region is mouse or human;

[0081] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody, the heavy chain constant region is a mouse IgG2a constant region, a mouse IgG1 constant region, a human IgG1 constant region or a human IgG4 constant region, and / or the light chain constant region is a mouse κ constant region or a human κ constant region.

[0082] In some embodiments, a fusion protein as described in any of the above items, wherein the anti-PD1 antibody, the heavy chain constant region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO.19, and / or the heavy chain constant region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO.20.

[0083] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO.19, and the light chain constant region comprises the amino acid sequence of SEQ ID NO.20.

[0084] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody, the heavy chain of the anti-PD1 antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO.17, and the light chain of the anti-PD1 antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO.18.

[0085] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody comprises a heavy chain as shown in SEQ ID NO.17 and a light chain as shown in SEQ ID NO.18.

[0086] In some embodiments, the fusion protein as described in any of the above items, wherein the anti-PD1 antibody is a full-length antibody or any one antigen-binding fragment selected from F(ab')2, Fab'-SH, Fab', Fab, scFab, dsFv, (dsFv)2, Fv and scFv.

[0087] In some embodiments, the fusion protein as described in any of the above items, wherein the IL10 mutant is directly or via a connecting peptide connected to the heavy chain or light chain of the anti-PD1 antibody; in some embodiments, the N-terminus of the IL10 mutant is connected to the C-terminus of the light chain of the anti-PD1 antibody via a connecting peptide;

[0088] In some embodiments, the fusion protein as described in any of the above items, wherein the fusion protein comprises two identical first chains represented by formula P1 and two identical second chains represented by formula P2:

[0089] P1: [Light chain of anti-PD1 antibody]-[Second connecting peptide]-[IL10 mutant],

[0090] P2: heavy chain of anti-PD1 antibody;

[0091] In some embodiments, in the formula P1, the second connecting peptide is a flexible connecting peptide; in some embodiments, the second connecting peptide is as shown in SEQ ID NO.53;

[0092] In some embodiments, a fusion protein as described in any of the above items, the fusion protein comprises a first chain of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO.54, and a second chain of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO.17;

[0093] In some embodiments, the fusion protein as described in any of the above items comprises a first chain with two amino acid sequences as shown in SEQ ID NO.54, and a second chain with two amino acid sequences as shown in SEQ ID NO.17.

[0094] In some embodiments, the fusion protein as described in any of the above items, wherein the fusion protein has at least one of the following properties:

[0095] A. The fusion protein can specifically bind to human PD1; optionally, the fusion protein can bind to human PD1 protein with an EC50 value of ≤10nM (e.g., ≤10nM, ≤5nM, ≤4.50nM, ≤3.00nM, ≤2.00nM, ≤1.00nM, ≤0.9nM, ≤0.8nM, ≤0.7nM, ≤0.6nM, ≤0.5nM, ≤0.4nM, ≤0.3nM, ≤0.2nM, ≤0.1nM, ≤0.05nM, ≤0.01nM, ≤0.005nM, ≤0.001nM or less), wherein the EC50 value is determined by the FACS method; in some embodiments, the EC50 value is determined by the method of Example 3 of the present application;

[0096] B. The fusion protein has low or no blood toxicity; in some embodiments, the blood toxicity is determined by the method of Example 3 of the present application;

[0097] C. The fusion protein can enhance the killing activity of CD8+T cells; in some embodiments, the blood toxicity is determined by the method of Example 5 of the present application;

[0098] D. The fusion protein can inhibit tumor growth; in some embodiments, the inhibition of tumor growth is determined by the method of Example 6 of the present application.

[0099] The present disclosure also provides a nucleic acid encoding any of the above fusion proteins. In some embodiments, the nucleic acid is an isolated nucleic acid.

[0100] The present disclosure also provides a vector, which contains any of the above nucleic acids.

[0101] The present disclosure also provides a cell, which contains any of the above nucleic acids or vectors.

[0102] The present disclosure also provides a pharmaceutical composition, comprising: the fusion protein, nucleic acid, vector or cell described in any of the above items. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

[0103] The present disclosure also provides the use of any of the above fusion proteins, nucleic acids, vectors, cells or pharmaceutical compositions in the preparation of drugs for treating or preventing tumors or inflammatory diseases. In some embodiments, the tumor or inflammatory disease is a disease in which human PD1 and or PDL1 are highly expressed. In some embodiments, the tumor is a PDL1 highly expressed tumor. In some embodiments, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma and blood system cancer.

[0104] In some embodiments, the present disclosure provides a method for treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of any of the foregoing fusion proteins, nucleic acids, vectors, cells or pharmaceutical compositions. In some embodiments, the disease is a tumor or an inflammatory disease; in some embodiments, the tumor or inflammatory disease is a disease in which human PD1 and or PDL1 are highly expressed. In some embodiments, the tumor is a PDL1-highly expressed tumor. In some embodiments, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma and hematological cancer.

[0105] In another aspect, the present disclosure also provides any of the foregoing fusion proteins, nucleic acids, vectors, cells or pharmaceutical compositions used as drugs. In some embodiments, the drug is used to treat a disease. In some embodiments, the disease is a tumor or an inflammatory disease; in some embodiments, the tumor or inflammatory disease is a disease in which human PD1 and or PDL1 are highly expressed. In some embodiments, the tumor is a PDL1-highly expressed tumor. In some embodiments, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma and hematological cancer.

[0106] In some embodiments, the treatment described in any of the above further comprises administering to the subject an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent.

[0107] The present disclosure also provides a kit, which comprises the fusion protein, nucleic acid, vector, cell or pharmaceutical composition as described in any of the preceding items; in some embodiments, the kit is used to detect or measure human PD1.

[0108] The present disclosure also provides a method for preparing the fusion protein as described in any of the preceding items, comprising the steps of culturing the cells as described in any of the preceding items, and then isolating and purifying to obtain the fusion protein.

[0109] The present disclosure provides a new anti-PD1-IL10 fusion protein. In some embodiments, the anti-PD1-IL10 fusion protein disclosed herein can relieve the immunosuppression of PD-1 / PD-L1 through anti-PD1 antibodies on the one hand, and can directly enrich on the surface of exhausted CD8+T cells with high expression of PD1 on the other hand, and hinder further exhaustion of CD8+T cells by means of IL10 mutants, enhance the killing activity of CD8+T cells, and inhibit activation-induced apoptosis, thereby enhancing anti-tumor immune response. In some embodiments, compared with the significant hematological toxicity of natural IL10 in crab-eating monkeys, the anti-PD1-IL10 fusion protein disclosed herein has no hematological toxicity and liver damage during administration, solving the problem of hematological toxicity of IL10 in clinical practice, resulting in too low a dose and limited efficacy. And then solve the problem of treating patients with PD1 / PDL1 resistance, enhance the body's anti-tumor immune response, and improve the survival rate of tumor patients.

[0110] In some embodiments, the anti-PD1-IL10 fusion protein (e.g., R1153) disclosed herein also has good stability, for example, it has good stability under different conditions such as being placed at a high temperature of 40° for 7 days and 14 days, placed at a low pH (3.5) for 1h, 2h, and 4h, placed at room temperature for 7 days, and placed at 4° for 7 days, and the binding activity of the anti-PD1-IL10 fusion protein is basically unaffected. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0112] Figure 1 : Schematic diagram of the structure of anti-PD1-IL10 fusion protein;

[0113] Figure 2 : Electrophoresis of anti-PD1-IL10 fusion protein;

[0114] Figure 3 : Detection results of the binding activity of anti-PD1-IL10 fusion protein with human PD1 antigen;

[0115] Figure 4 : Result diagram of the detection of binding activity of anti-PD1-IL10 fusion protein to human IL10R;

[0116] Figure 5 : The results of the IL10 activity test using the reporter gene method to detect proteins (R1153, R1152);

[0117] Figure 6 : The results of the IL10 activity test using the reporter gene method to detect proteins (R1153, R1152+CHO-hPD1);

[0118] Figure 7 : The results of the PD1 activity test using the reporter gene method to detect proteins (R1153, R1152);

[0119] Figure 8 : Anti-PD1-IL10 fusion protein CD8+T cell apoptosis detection experiment results;

[0120] Fig. 9 :The results of IFN-γ secretion detection in the anti-PD1-IL10 fusion protein CD8+T cell apoptosis experiment;

[0121] Fig.10a To detect the binding activity of some anti-PD1-IL10 fusion proteins with hIL10Rα (human IL10 receptor α) by FCM;

[0122] Fig.10b To detect the binding activity of some anti-PD1-IL10 fusion proteins with hIL10Rα (human IL10 receptor α) by FCM;

[0123] Fig.10c To detect the binding activity of some anti-PD1-IL10 fusion proteins with hIL10Rα (human IL10 receptor α) by FCM;

[0124] Fig.10d To detect the binding activity of some anti-PD1-IL10 fusion proteins with hIL10Rα (human IL10 receptor α) by FCM;

[0125] Fig.11The results of IL10 activity detection of the anti-PD1-IL10 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);

[0126] Fig.12 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1742 in the system with and without target cells; solid-no target cells, hollow-presence of target cells;

[0127] Fig.13 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1743 in the system with and without target cells; solid - no target cells, hollow - presence of target cells;

[0128] Fig.14 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1748 in the presence and absence of target cells; solid - no target cells, hollow - presence of target cells;

[0129] Fig.15 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1749 in the system with and without target cells; solid - no target cells, hollow - presence of target cells;

[0130] Fig.16 The results of IL10 activity detection of anti-PD1-IL10 fusion protein R1750 in the presence and absence of target cells; solid - no target cells, hollow - presence of target cells;

[0131] Fig.17 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1751 in the presence and absence of target cells; solid - no target cells, hollow - presence of target cells;

[0132] Fig.18 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1752 in the presence and absence of target cells; solid - no target cells, hollow - presence of target cells;

[0133] Fig.19 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1753 in the presence and absence of target cells; solid - no target cells, hollow - presence of target cells;

[0134] Fig. 20The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1754 in the presence and absence of target cells; solid - no target cells, hollow - presence of target cells;

[0135] Fig.21 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1755 in the presence and absence of target cells; solid - no target cells, hollow - presence of target cells;

[0136] Fig. 22 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1758 in the system with and without target cells; solid-no target cells, hollow-presence of target cells;

[0137] Fig.23 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1759 in the system with and without target cells; solid-no target cells, hollow-presence of target cells;

[0138] Fig.24 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1762 in the system with and without target cells; solid - no target cells, hollow - presence of target cells;

[0139] Fig.25 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1764 in the system with and without target cells; solid - no target cells, hollow - presence of target cells;

[0140] Fig.26 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1766 in the system with and without target cells; solid - no target cells, hollow - presence of target cells;

[0141] Fig. 27 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1768 in the presence and absence of target cells; solid - no target cells, hollow - presence of target cells;

[0142] Fig.28 The results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1769 in the presence and absence of target cells; solid - no target cells, hollow - presence of target cells;

[0143] Fig.29 The results of IL10 activity detection of anti-PD1-IL10 fusion protein R1770 in the presence and absence of target cells; solid - no target cells, hollow - presence of target cells;

[0144] Fig.30 These are the results of IL10 activity detection of the anti-PD1-IL10 fusion protein R1771 in the system with and without target cells; solid: no target cells present, hollow: target cells present. DETAILED DESCRIPTION

[0145] In order to make the purpose, technical scheme and advantages of the embodiments of the present disclosure clearer, the technical scheme in the embodiments of the present disclosure is clearly and completely described below, but the description and embodiments should not be interpreted as limiting the scope of the present disclosure. If the specific conditions are not specified in the embodiments, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. Unless clearly defined in the present disclosure, the technical terms and scientific terms used in the present disclosure have the meanings commonly understood by ordinary technicians in the field to which the present disclosure belongs.

[0146] The terms "a / an", "a", "said", "above", and "aforementioned" in the present disclosure include plural references unless the context clearly indicates otherwise. For example, "an antibody" means one antibody or more than one antibody. In addition, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. The term "plurality" means at least two, such as 2, 3, etc., unless otherwise clearly and specifically indicated otherwise.

[0147] Unless the context clearly indicates otherwise, in the specification and claims of the present disclosure, the words "include" or "have" should be understood as "include" rather than the exclusive or exhaustive meaning; that is, the meaning of "include but not limited to". For example, "include A" means "include A, but not limited to A".

[0148] Unless the context clearly indicates otherwise, in the description and claims of the present disclosure, "optional", "optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, that is, it includes occasions where the event or circumstance occurs or does not occur.

[0149] "About" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless otherwise expressly stated in the examples or elsewhere in the specification, "about" means within one standard deviation, or up to a range of 5%, as is customary in the art.

[0150] The term "and / or" refers to each of the two specific features or a combination thereof, which includes or excludes another specific feature. For example, "A and / or B" means including any of the following situations: A and B, A or B, A (alone) and B (alone); "A, B and / or C" means covering any of the following situations: A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0151] The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0152] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and modified amino acids thereof, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Common natural amino acids are, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to that of a naturally occurring amino acid.

[0153] The term "antibody" in the present disclosure is used in the broadest sense and covers various antibody structures, including but not limited to: monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, tetraspecific antibodies), murine antibodies, chimeric antibodies, humanized antibodies, human antibodies, full-length antibodies, antigen-binding fragments (also referred to as antigen-binding portions, or antibody fragments), etc., as long as they exhibit the desired antigen-binding activity. .

[0154] "Native antibodies" are naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain of an IgG antibody has a heavy chain variable region (VH, also called a variable heavy domain or a heavy chain variable domain), followed by a heavy chain constant region (CH, also called a constant heavy domain), which includes three constant domains (CH1, CH2, and CH3); similarly, from N to C-terminus, each light chain has a light chain variable region (VL, also called a variable light domain, or a light chain variable domain), followed by a light chain constant region (CL, also called a constant light domain). Depending on whether the antibody contains α, δ, ε, γ and μ heavy chains, antibodies can be divided into five isotypes: IgA, IgD, IgE, IgG and IgM. Isotype antibodies can be further divided into different subclasses. For example, IgG isotypes include four subtypes: IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain) and IgG4 (γ4 heavy chain). IgA isotypes are divided into two subtypes: IgA1 (α1 heavy chain) and IgA2 (α2 heavy chain).

[0155] The terms "full length antibody", "intact antibody" and "whole antibody" in the present disclosure are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure, or an antibody having a heavy chain including an Fc region.

[0156] An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody can be purified to a purity greater than 90% or 99%, which can be purified and assayed by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0157] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in antibody binding to an antigen. The heavy chain variable region (VH) and light chain variable region (VL) of a natural antibody, each variable region comprises four conserved framework regions (FR) and three hypervariable regions (also called complementarity determining regions, HVR or CDR).

[0158] The terms "complementarity determining region", "hypervariable region" or "CDR" refer to the major regions within the variable domain that contribute to antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues in the variable region. VH contains three CDR regions: HCDR1, HCDR2 and HCDR3; VL contains three CDR regions: LCDR1, LCDR2 and LCDR3. Each VH and VL is usually composed of three CDRs and four FRs arranged in the following order (from amino terminus to carboxyl terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid sequence boundaries of CDRs can be determined by methods well known to those skilled in the art, for example: "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art.

[0159] The term "light chain" includes a light chain variable region (VL) and a light chain constant region (CL). VL is at the amino terminus of the light chain, and CL is at the carboxyl amino terminus of the light chain. The light chain may be a kappa chain or a lambda chain, etc.

[0160] The term "heavy chain" includes a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain variable region is at the amino terminus of the heavy chain, and the heavy chain constant region is at the carboxyl amino terminus of the heavy chain. The IgG antibody heavy chain constant region includes three constant region domains: CH1, CH2, and CH3. The heavy chain may belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgD, IgM, and IgE, etc.

[0161] The term "constant region" or "constant domain" refers to the carboxyl terminal portion of the light chain and the heavy chain, which is not directly involved in the binding of the antibody to the antigen, and has a relatively conserved amino acid sequence compared to VH and VL.

[0162] The term "antigen-binding fragment" refers to a portion that contains an intact antibody, but unlike an intact antibody, this portion can specifically bind to the antigen bound by the intact antibody. Some examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dsFv, (dsFv)2, single-chain Fab (scFab), single-chain antibodies (such as scFv), diabodies, linear antibodies, and multispecific antibodies formed by antigen-binding fragments. Among them, "Fab" is a monovalent fragment composed of VL, VH, CL and CH1 domains; "Fv" is composed of VH and VL; "Fab'" is a Fab fragment containing part of the hinge region; "F(ab')2" contains two F(ab')2 domains connected by disulfide bonds in the hinge region. ab' fragment; "scFab" is a polypeptide consisting of VH, CH1, VL, CL and a linker, wherein the antibody domain and the linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; "scFv" is a fusion protein comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are linked by a peptide linker. The scFv is connected to the head and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv herein can have VL and VH variable regions in any order, for example, from the N-terminus to the C-terminus of the scFv polypeptide, it can contain: a) VL-linker-VH or b) VH-linker-VL; "dsFv" is a disulfide bond-stabilized Fv fragment; (dsFv)2 is a dimerized dsFv; "Fab'-SH" is a cysteine ​​residue in the hinge region of the Fab' fragment carrying a free thiol group.

[0163] The term "Fc region" is the C-terminal region of an antibody heavy chain, including a natural antibody Fc region and an Fc region variant. In some embodiments, the Fc region of a human IgG heavy chain is an extension from the amino acid residue at the Cys226 position to its carboxyl terminus. In some embodiments, the Fc region of a human IgG heavy chain is an extension from Pro230 to its carboxyl terminus. The C-terminal end of the Fc region may also be, for example, a C-terminal lysine (residue 447 according to the EU numbering system) or a C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system) missing from the Fc region. In some embodiments, the composition of a complete antibody may include an antibody population lacking a K447 residue and / or a G446+K447 residue. In some embodiments, the composition of a complete antibody may include an antibody population without a K447 residue and / or a G446+K447 residue missing. In some embodiments, the composition of a complete antibody has an antibody population with and without a mixture of antibodies with a K447 residue and / or a G446+K447 residue. The Fc region used for the antibodies described in the present disclosure includes the Fc region of human IgG1, IgG2 (IgG2a, IgG2b), IgG3 and IgG4. Unless otherwise specified in the present disclosure, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0164] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular species and the remainder of the heavy chain and / or light chain is derived from another species.

[0165] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in the human body. For example, it can be achieved by retaining the CDR region of a non-human antibody and replacing the rest of the antibody with its human counterpart (i.e., the framework region of the heavy chain variable region of a human antibody, the framework region of the light chain variable region, and the human antibody constant region).

[0166] The terms "human antibody", "humanized antibody", "fully human antibody" and "fully human antibody" are used interchangeably and refer to antibodies in which the variable and constant region sequences are human sequences. Human antibodies include antibodies that have human genes but have been altered, such as cysteine ​​or glycosylation sites. Human antibodies may be antibodies recombinantly produced in non-human cells. Human antibodies include antibodies obtained from transgenic mice that express human immunoglobulin heavy and light chain loci.

[0167] The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as disclosed herein, "affinity" refers to internal binding affinity, which reflects 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to its ligand Y is usually expressed as a dissociation constant (KD). Affinity can be measured by conventional methods known in the art. The term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to a dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka), usually expressed as a molar concentration (M). The KD value of an antibody can be measured using methods well known in the art. The method for measuring the KD of an antibody includes using a biosensor system such as a system to measure surface plasmon resonance, or measuring affinity in a solution by a solution equilibrium titration method (SET).

[0168] The term "affinity matured" antibody refers to an antibody that has one or more amino acid residue changes in one or more CDRs of the antibody, resulting in an improved affinity of the antibody for the antigen compared to the parent antibody. In some embodiments, affinity matured antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies can be generated by methods known in the art. Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in Barbas et al., PNAS, 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995) and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0169] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an Fc region of amino acid sequence mutation). Examples of antibody effector functions include, but are not limited to, C1q binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0170] The term "monoclonal antibody" refers to a substantially homogeneous antibody population, i.e., the amino acid sequences of the antibody molecules contained in the population are identical (except for possible natural mutations that may be present in small amounts). In contrast, polyclonal antibodies typically contain different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes and / or different antigens. "Monoclonal" refers to an antibody obtained from a substantially homogeneous antibody population. Monoclonal antibodies can be produced by methods well known in the art, for example, they can be prepared by the hybridoma method (Kohler et al., (1975) Nature 256:495), they can be prepared by recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567), they can be prepared by isolation from phage antibody libraries (Clackson et al., (1991) Nature 352:624-628 and Marks et al., (1991) J. Mol. Biol. 222:581-597), and monoclonal antibodies can also be prepared using transgenic animals containing all or part of the human immunoglobulin loci (see Presta (2005) J. Allergy Clin. Immunol. 116:731).

[0171] The term "antigen" refers to a protein that can selectively bind to an antigen binding protein (eg, an antibody). An antigen may have one or more epitopes that interact with different antigen binding proteins (eg, antibodies).

[0172] The term "epitope" refers to an area (area or region) on an antigen that can specifically bind to an antibody. An epitope is usually composed of an antigenic determinant of a special chemical group with a certain structure. An epitope can be formed by continuous amino acid residues (linear epitope) or by non-continuous amino acid residues (conformational epitope), such as non-continuous amino acid residues that are close to each other in space due to the folding of the antigen. In some embodiments, the epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acid residues in a unique spatial conformation. The epitope can be determined by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structural determination methods (e.g., nuclear magnetic resonance spectroscopy). Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be achieved using methods known in the art, including, but not limited to, for example, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496) and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0173] An "antibody that binds to the same epitope" as a reference antibody or an "antibody that competes for binding" with a reference antibody refers to an antibody that blocks the binding of the reference antibody to the antigen by 50% or more in a competition assay, or an antibody whose binding to the antigen is blocked by the reference antibody by 50% or more. In order to determine whether a test antibody binds to the same epitope as a reference antibody, it can be tested under saturation conditions whether the reference antibody is allowed to bind to the antigen, for example, after removing excess reference antibody, the ability of the test antibody to bind to the antigen is assessed; if the test antibody is able to bind to the antigen after saturation binding of the reference antibody, then the test antibody binds to a different epitope than the reference antibody; however, if the test antibody is unable to bind to the antigen after saturation binding of the reference antibody, then the test antibody can bind to the same epitope as the reference antibody. To confirm whether the test antibody binds to the same epitope or is merely hindered from binding for steric reasons, routine experiments (e.g., peptide mutations and detection using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative method known in the art) can be used to determine that if in both settings (i.e., both antibodies are used as saturated antibodies), only the saturated antibody is able to bind to the antigen, then it can be concluded that the test antibody and the reference antibody compete for binding to the antigen. In some embodiments, in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502), if an excess (e.g., 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold amount) of an antibody inhibits the binding of the other antibody to the antigen by at least 50%, at least 75%, at least 90%, or even 99% or more, then the two antibodies are considered to bind to the same or overlapping epitope.

[0174] The term "specific binding" or "specifically binds" refers to non-random binding between two molecules, for example, an antibody binds to its corresponding antigen or epitope with a higher affinity than to other antigens or epitopes. -7 M or less (e.g., about 1×10 -8 M or smaller, about 1×10 -9 M or smaller, about 1×10 -10 M or smaller, about 1×10 -11 M or smaller, or about 1×10 -12 The antibody binds to the antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of 1% or less. In some embodiments, the KD of the antibody binding to the antigen is 10%, 1% or less of the KD of the antibody binding to a non-specific antigen (e.g., BSA, casein). The KD value can be measured using methods known in the art, such as by Surface plasmon resonance assay measurement.An antibody that specifically binds to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, for example, to corresponding antigens from other species such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp), or marmoset (Callithrixjacchus) (common marmoset, marmoset).

[0175] The terms "anti-PD1 antibody" and "antibody that binds to PD1" refer to an antibody that can bind to the PD1 antigen with sufficient affinity. In one embodiment, the extent of binding of the anti-PD1 antibody to non-PD1 proteins is less than about 10%, 1%, or less of the binding to PD1, and the binding can be Surface plasmon resonance assay. In some embodiments, the antibody that binds to PD1 has the following dissociation constant (KD): < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM or < about 0.001 nM or less. In certain embodiments, the anti-PD1 antibody binds to a conserved PD1 epitope in PD1 from different species.

[0176] The binding of the anti-PD1 antibodies provided in the present disclosure to human PD1 can also be expressed as a "half-maximal effective concentration" (EC50) value. Generally, the smaller the EC50, the better the affinity, indicating that the antibody can bind to the antigen at a lower concentration. The EC50 value can be determined by binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometric fluorescence sorting technology, and other binding assays).

[0177] In some embodiments, the anti-PD1 antibody of the present disclosure is R1149, R1150, R1151 or R1152.

[0178] The term "fusion protein" refers to a naturally occurring, synthetic, semisynthetic or recombinant protein molecule that comprises all or part of two or more heterologous polypeptides linked by peptide bonds.

[0179] In some embodiments, the fusion protein disclosed herein is an anti-PD1-IL10 fusion protein (including a PD1 binding unit portion and an IL10 unit portion), wherein the PD1 binding unit portion is an anti-PD1 antibody, and the IL10 unit portion is an IL10 mutant. In some embodiments, the IL10 unit is connected to the C-terminus and / or N-terminus of the heavy chain and / or light chain of the anti-PD1 antibody molecule, thereby constructing the anti-PD1-IL10 fusion protein. The anti-PD1-IL10 fusion protein isolates the IL10 unit through the steric hindrance of the anti-PD1 antibody structure itself. When the IL10 unit fusion protein is in a free state, even if the IL10 unit binds to IL10Rα, it cannot cause a functional response. When the antibody end binds to the target cell, the adjacent IL10 unit-IL10Rα complex is gathered through the aggregation effect, and further combined with IL10Rβ to form a complex to generate downstream signals and trigger 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 trigger 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 increased.

[0180] In some embodiments, the present disclosure proposes an anti-PD1-IL10 fusion protein, including a PD1 binding unit portion and an IL10 unit portion, wherein the PD1 binding unit portion is an anti-PD1 antibody, which includes two identical light chains and two identical heavy chains, and the IL10 unit is connected to the antibody heavy chain and / or light chain. The natural IL10 molecule is in the form of a dimer. According to some specific embodiments of the present disclosure, the IL10 molecule is in the form of a monomer before the antibody binds to the target cell, and cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell. After the antibody binds to the target cell, a large number of IL10 unit molecules aggregate, so that the two IL10 units close to each other act as dimers and bind to the IL10 receptor, thereby causing a normal biological response, so that the targeting and safety of the fusion protein are significantly improved, and the efficacy is fully exerted. According to some specific embodiments of the present disclosure, each antibody light chain of the anti-PD1 antibody includes a VL region and a CL region, and each antibody heavy chain includes a VH region, a CH1 region, a CH2 region, and a CH3 region; the N-terminus of the IL10 unit is connected to the C-terminus of the antibody light chain, or the C-terminus of the IL10 unit is connected to the N-terminus of the antibody light chain, or the N-terminus of the IL10 unit is connected to the C-terminus of the hydrogen chain VL region, the C-terminus of the IL10 unit is connected to the N-terminus of the hydrogen chain CL region, or the C-terminus of the IL10 unit is connected to the N-terminus of the antibody heavy chain, or the N-terminus of the IL10 unit is connected to the C-terminus of the heavy chain VH region, the C-terminus of the IL10 unit is connected to the N-terminus of the heavy chain CH1 region, or the N-terminus of the IL10 unit is connected to the C-terminus of the heavy chain CH2 region, and the C-terminus of the IL10 unit is connected to the N-terminus of the heavy chain CH3 region.

[0181] According to some specific embodiments of the present disclosure, the IL10 unit comprises a natural IL10 unit.

[0182] IL10 (Interleukin-10, also known as IL-10) is a cytokine with multidirectional regulatory functions. Its active form is usually a homodimer formed by two subunits connected by a disulfide bond. IL10 is mainly expressed and secreted by immune cells such as monocytes, macrophages, and DCs, and plays an important role in inhibiting the secretion of inflammatory factors such as TNF-α and IL-6. The IL10 receptor IL10R consists of two binding subunits (R1 and R2), and the IL10 / IL10Rα / IL10Rβ hexameric complex formed mediates downstream signal transduction. The IL10 receptor is mainly expressed in monocytes / macrophages, DC cells, T cells, and B cells, and it plays different roles in different cells. Studies have found that initial CD8+T cells do not express IL10 receptors. In tumor tissues, the higher the degree of CD8+T cell activation, the higher the expression of IL10 receptors. Exhausted CD8+T cells have higher expression levels than activated CD8+T cells. Therefore, IL10 plays a bidirectional regulatory role in the tumor microenvironment. On the one hand, it reduces antigen presentation by inhibiting the inflammatory response of Th1 cells, macrophages, and Th17 cells and downregulating the MHC expression of macrophages and DC cells, thereby promoting tumors; on the other hand, it can play an anti-tumor role by directly stimulating the activation of CD8+T cells infiltrating the tumor. In some specific embodiments of the present disclosure, the anti-PD1-IL10 fusion protein of the present disclosure can relieve the immunosuppression of PD-1 / PD-L1 on the one hand, and can directly enrich on the surface of exhausted CD8+T cells with high expression of PD1 on the other hand, and hinder the further exhaustion of CD8+T cells by means of IL10 polypeptide, enhance the killing activity of CD8+T cells, and inhibit activation-induced apoptosis, thereby enhancing the anti-tumor immune response.

[0183] According to some specific embodiments of the present disclosure, the IL10 unit is a modified IL10 mutant of natural IL10, for example, the modification includes at least one of the following (i) and (ii): (i) removing the first two amino acids at the N-terminus of the natural IL10 unit; (ii) inserting a first connecting peptide between the 116th and 117th amino acids, wherein the first connecting peptide is a flexible connecting peptide, for example, the first connecting peptide is shown in SEQ ID NO. 22. After removing the first two amino acids at the N-terminus of the natural IL10 unit, the stability of the IL10 unit is improved, and after inserting the first connecting peptide between the 116th and 117th amino acids of the IL10 unit, the IL10 unit molecule in the fusion protein becomes a closed-loop monomer molecule (the two IL10 chains of the natural IL10 dimer are staggered together, and after the linker is inserted, the IL10 monomer can independently form a domain, i.e., a closed-loop monomer molecule), and the two IL10 units in the same fusion protein will not be interconnected or otherwise interfered.

[0184] According to some specific embodiments of the present disclosure, the IL10 mutant has an amino acid mutation at at least one of the following positions relative to SEQ ID NO.21: P20, R24, R27, K34, T35, Q38, D41, L46, L48, E50, S141, D144, I145, N148, T155, and R159.

[0185] It should be noted that the mutation sites described herein are amino acid residue sites numbered relative to the natural sequence of the sequence SEQ ID NO.21, that is, represented by the natural sequence number of SEQ ID NO.21.

[0186] In some embodiments, the IL10 mutant has an amino acid mutation at at least one of the following positions relative to SEQ ID NO. 21: P20, R24, T35, D41, L46, L48, E50, S141, I145, T155, and R159.

[0187] In some embodiments, the IL10 mutant, the amino acid mutation refers to an amino acid substitution, insertion, or deletion.

[0188] In some embodiments, in the IL10 mutant, the amino acid mutation is an amino acid substitution.

[0189] In some embodiments, in the IL10 mutant, 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 Table 1 below:

[0190] Table 1. IL10 amino acid mutation residues

[0191]

[0192] Note: In the table, "+" represents the type of amino acid residue after mutation at the mutation site. For example, at mutation site L46, the amino acid residue after mutation can be A, G, D, E

[0193] , N, Q, R, K, I, M, V, F, Y, W, S, T, C, P or H;

[0194] In some embodiments, the IL10 mutant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO.21.

[0195] In some embodiments, the IL10 mutant has an amino acid mutation at the following position relative to SEQ ID NO. 21: I145.

[0196] 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.

[0197] In some embodiments, the IL10 mutant further has an amino acid mutation at at least one of the following positions relative to SEQ ID NO. 21: P20, R24, R27, K34, T35, Q38, D41, L46, L48, E50, S141, D144, N148, T155, and R159.

[0198] In some embodiments, the amino acid mutation at each of the above positions is a natural amino acid mutation, which can be any one of the substitution mutation types shown in Table 2 below:

[0199] Table 2. IL10 amino acid mutation residues

[0200]

[0201]

[0202] Note: In the table, "+" represents the type of amino acid residue after mutation at the mutation site. For example, at mutation site L46, the amino acid residue after mutation can be A, G, D, E

[0203] , N, Q, R, K, I, M, V, F, Y, W, S, T, C, P or H.

[0204] In some embodiments, the IL10 mutant has at least one of the following mutations relative to SEQ ID NO.21:

[0205] P20G, P20F, R24P, R27D, R27G, K34S, K34D, T35R, Q38P, D41W, L46G, L46K, L48G, E50G, S141F, S141Q, S141L, D144S, N148E, T155R, R159E, and R159H.

[0206] In some embodiments, the IL10 mutant is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.21.

[0207] In some embodiments, the IL10 mutant has at least one of the following amino acid mutations relative to SEQ ID NO.21: P20G, P20F, R24G, R24P, R27D, R27G, T35R, D41W, L46G, L48G, E50G, S141L, S141F, S141Q, T155R, R159E, and R159H.

[0208] In some embodiments, the IL10 mutant further has an amino acid mutation at at least one of the following positions relative to SEQ ID NO. 21: K34, Q38, L46, D144, N148, and I145.

[0209] In some embodiments, the above-mentioned IL10 mutant further has at least one of the following amino acid mutations relative to SEQ ID NO.21: 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.

[0210] In some embodiments, the IL10 mutant has the following mutations relative to SEQ ID NO.21:

[0211] (a) P20G;

[0212] (b) R24G;

[0213] (c)D41W;

[0214] (d) L46G;

[0215] (e) L48G;

[0216] (f) E50G;

[0217] (g) S141F;

[0218] (h) P20G / R24G (i.e. P20G and R24G);

[0219] (i) K34S / L46G (i.e. K34S and L46G);

[0220] (j) S141Q / N148E (i.e. S141Q and N148E);

[0221] (k) P20F;

[0222] (l) T35R;

[0223] (m)R24P;

[0224] (n) R27D;

[0225] (o)R27G;

[0226] (p)S141L;

[0227] (q)I145G;

[0228] (r)R159E;

[0229] (s)R159H;

[0230] (t) R24G / R27A / S141F (i.e. R24G, R27A and S141F);

[0231] (u) R24G / R27D (i.e. R24G and R27D);

[0232] (v) R24G / Q38P (i.e. R24G and Q38P);

[0233] (w) R27D / K34D (i.e. R27D and K34D);

[0234] (x) S141F / D144S (i.e. S141F and D144S); or

[0235] (y)T155R.

[0236] In some embodiments, 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%, 99% or more sequence identity with SEQ ID NO.21.

[0237] In some embodiments, the amino acid residues of the IL10 mutant at other sites other than the above mutation sites are the same as those of SEQ ID NO.21; in some embodiments, the amino acid residues of the IL10 mutant at other sites other than the above mutation sites are different from those of SEQ ID NO.21 (may be substitutions, deletions or insertions), and when the sequence outside these mutation sites is different from SEQ ID NO.21, 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 sequence identity with SEQ ID NO.21.

[0238] Based on the above mutation sites disclosed in the present disclosure, it is easy for those skilled in the art to conceive of making substitutions, such as conservative substitutions, at other sites other than the above mutation sites based on SEQ ID NO. 21, or making amino acid mutations without affecting the effects brought about by the above mutation functions to obtain similar IL10 mutations. 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. 21, and should also fall within the scope of protection of the present disclosure.

[0239] In some embodiments, the IL10 mutant has similar or reduced binding activity to the IL10 receptor compared to the wild-type IL10 shown in SEQ ID NO.21;

[0240] In some embodiments, the IL10 mutant has reduced binding activity to the IL10 receptor compared to the wild-type IL10 shown in SEQ ID NO.21.

[0241] In some embodiments, in the fusion protein as described in any of the above items, the IL10 mutant can bind to the IL10 receptor; in some embodiments, the IL10 receptor is selected from IL10Rα and IL10Rβ; in some embodiments, the IL10 receptor is selected from IL10Rα; in some embodiments, the IL10 receptor is selected from IL10Rβ. In some embodiments, the IL10 mutant has similar IL10Rβ binding activity to the wild-type IL10 shown in SEQ ID NO.21.

[0242] Herein, "wild type" refers to an amino acid sequence or a nucleotide sequence found in nature. A wild type protein has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.

[0243] In some embodiments, the above-mentioned IL10 receptor is selected from IL10Rα and IL10Rβ.

[0244] In some embodiments, the IL10 receptor is IL10Rα.

[0245] In some embodiments, the IL10 mutant has a spacer peptide inserted between positions 116 and 117 relative to the wild-type IL10 shown in SEQ ID NO. 21. The insertion of the spacer peptide is beneficial to prevent adjacent IL10 mutants from forming a dimer structure.

[0246] In some embodiments, the length of the above-mentioned spacer peptide is 5-10 amino acids.

[0247] In some embodiments, the above-mentioned spacer peptide contains amino acid residues G and S.

[0248] In some embodiments, the amino acid sequence of the above-mentioned spacer peptide is SEQ ID NO.22 (GGGSGG).

[0249] In some embodiments, the IL10 mutant described above further lacks the first and second amino acids at its N-terminus relative to the wild-type IL10 shown in SEQ ID NO.21.

[0250] In some embodiments, the amino acid sequence of the IL10 mutant is selected from any one of SEQ ID NO. 23-52.

[0251] According to an embodiment of the present disclosure, the IL10 unit is connected to the antibody heavy chain or light chain through a second connecting peptide. The second connecting peptide connects the IL10 monomer to the antibody heavy chain or light chain, so that after the antibody part in the fusion protein binds to the corresponding target cell, the IL10 units of the two fusion proteins close to each other are more likely to form dimers, bind to the IL10 receptor, and then play a role. According to an embodiment of the present disclosure, the N-terminus of the second connecting peptide is connected to the C-terminus of the antibody light chain, and the C-terminus of the second connecting peptide is connected to the N-terminus of the IL10 unit; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL10 unit, and the C-terminus of the second connecting peptide is connected to the N-terminus of the antibody light chain; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL10 unit, and the C-terminus of the second connecting peptide is connected to the N-terminus of the antibody light chain; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL10 unit, and the C-terminus of the second connecting peptide is connected to the N-terminus of the antibody heavy chain. According to an embodiment of the present disclosure, the IL10 unit is connected to the heavy chain terminus or the light chain terminus of the antibody through the second connecting peptide; according to an embodiment of the present disclosure, the N-terminus of the second connecting peptide is connected to the C-terminus of the antibody light chain, and the C-terminus of the second connecting peptide is connected to the N-terminus of the IL10 unit; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL10 unit, and the C-terminus of the second connecting peptide is connected to the N-terminus of the antibody light chain; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL10 unit, and the C-terminus of the second connecting peptide is connected to the N-terminus of the antibody heavy chain.

[0252] According to an embodiment of the present disclosure, the N-terminus of the second connecting peptide is connected to the C-terminus of the light chain VL region, and the C-terminus of the second connecting peptide is connected to the N-terminus of the IL10 unit; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL10 unit, and the C-terminus of the second connecting peptide is connected to the N-terminus of the light chain CL region; or the N-terminus of the second connecting peptide is connected to the C-terminus of the heavy chain VH region, and the C-terminus of the second connecting peptide is connected to the N-terminus of the IL10 unit; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL10 unit, and the C-terminus of the second connecting peptide is connected to the N-terminus of the heavy chain CH1 region; or the N-terminus of the second connecting peptide is connected to the C-terminus of the heavy chain CH2 region, and the C-terminus of the second connecting peptide is connected to the N-terminus of the IL10 unit; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL10 unit, and the C-terminus of the second connecting peptide is connected to the N-terminus of the heavy chain CH3 region.

[0253] According to an embodiment of the present disclosure, the N-terminus of the IL10 unit is connected to the C-terminus of the VL region of the light chain of the antibody through the second connecting peptide, and the C-terminus of the IL10 unit is connected to the N-terminus of the CL region of the light chain of the antibody through the second connecting peptide; or the N-terminus of the IL10 unit is connected to the C-terminus of the VH region of the heavy chain of the antibody through the second connecting peptide, and the C-terminus of the IL10 unit is connected to the N-terminus of the CH1 region of the heavy chain of the antibody through the second connecting peptide; or the N-terminus of the IL10 unit is connected to the C-terminus of the CH2 region of the heavy chain of the antibody through the second connecting peptide, and the C-terminus of the IL10 unit is connected to the N-terminus of the CH3 region of the heavy chain of the antibody through the second connecting peptide.

[0254] In some embodiments, the fusion protein disclosed herein comprises two identical first chains represented by formula P1 and two identical second chains represented by formula P2, wherein P1: [light chain of anti-PD1 antibody]-[second connecting peptide]-[IL10 mutant], P2: heavy chain of anti-PD1 antibody; in some embodiments, in formula P1, the second connecting peptide is a flexible connecting peptide; for example, the second connecting peptide is as shown in SEQ ID NO.53.

[0255] The term "connecting peptide", "connecting polypeptide", "connector", "Linker" or "linker" refers to a connecting unit connecting two polypeptide fragments, which usually has a certain flexibility, and the use of the linker will 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 residue does 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. In some embodiments, the linker is a polypeptide shown in SEQ ID NO.22 or SEQ ID NO.53.

[0256] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in the present disclosure and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or connections, and nucleic acids can be synthetic, naturally occurring, and non-naturally occurring, such as non-natural nucleic acids that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to reference nucleotides. Including but not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNA) modified nucleic acid. "Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in the following cells, which usually contain the nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome or at a chromosomal position different from its natural chromosomal position. "Nucleic acid encoding an anti-PD1 antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including one or more nucleic acid molecules in a single vector or separate vectors, and one or more nucleic acid molecules present in one or more positions in a host cell. In some embodiments, the nucleic acid sequence also includes conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. For example, degenerate codon substitutions can be obtained by generating a sequence in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0257] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0258] The terms "identity" and "sequence identity" refer to the degree (percentage) to which the amino acids (amino acid sequence identity) or nucleic acids (nucleic acid sequence identity) of the two sequences are identical at equivalent positions when the two sequences are optimally aligned. To obtain the optimal alignment of the two sequences, gaps may be introduced, if necessary, to obtain the maximum percentage of sequence identity. The percentage of amino acid sequence identity can be determined in various ways known in the art, such as software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW or CLUSTAL OMEGA, which are well known in the art. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the comparison sequence.

[0259] The term "conservatively modified variants" or "conservative substitutions" of amino acids refers to the use of other amino acids with similar characteristics (e.g., charge, side chain size, hydrophilicity / hydrophobicity, backbone morphology and rigidity, etc.) to replace the amino acids in the sequence, but does not change the biological activity of the protein. It is known to those skilled in the art that, under normal circumstances, single amino acid substitutions in non-essential regions of a polypeptide generally do not change the biological activity (see, for example, Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). The term "conservatively modified variants" or "conservative substitutions" of nucleic acids refers to nucleic acids that encode the same or substantially the same amino acid sequence, and also includes nucleic acids of substantially the same sequence when the nucleic acid does not encode the amino acid sequence. Due to the degeneracy of the genetic code, any given protein can be encoded by multiple functionally identical nucleic acids. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Therefore, at each position where a codon specifies alanine, the codon can be changed to any of the corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide in the present disclosure encompasses every possible silent variation of the nucleic acid. Each codon in a nucleic acid (except AUG, which is usually the only codon for methionine; and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule.

[0260] The term "vector" is a vehicle capable of transporting genetic elements (e.g., nucleic acids) connected thereto. A vector can be used to transform, transduce, or transfect a host cell so that the genetic elements it carries are expressed in the host cell. Exemplarily, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or artificial chromosomes (PAC) derived from P1, bacteriophages such as lambda phages or M13 phages, and animal viruses, etc. In some embodiments, a vector is a "plasmid," which is a circular double-stranded DNA loop to which additional DNA segments can be connected. In some embodiments, a vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), and a DNA segment can be connected to a viral genome. A vector may contain a variety of elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene, a vector may also contain a replication initiation site, and a vector may also include components that assist it in entering a cell, including, but not limited to, viral particles, liposomes, or protein shells. A vector may be an expression vector. In some embodiments, a vector (eg, an expression vector) contains a nucleic acid sequence encoding an antibody of the present disclosure, a promoter (eg, SV40, CMV, EF 1α), and may further include at least one selection marker.

[0261] The term "expression vector" or "expression construct" is a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and / or controls (together with the host cell) the expression of one or more coding regions operably linked thereto. An expression vector may include, but is not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of a coding region operably linked thereto.

[0262] The term "operably linked" refers to a connection relationship in which the components referred to by the term allow them to perform their inherent functions under suitable conditions. For example, a control sequence "operably linked" to a protein coding sequence refers to a sequence that is linked to it and achieves the expression of the protein coding sequence under conditions compatible with the transcriptional activity of the control sequence.

[0263] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to parent cells in nucleic acid content, but may contain mutations. Mutant offspring with the same function or biological activity as screened or selected in the initial transformed cells are included herein. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include but are not limited to mammalian cells, insect cell lines, plant cells and fungal cells. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia puntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces cerevisiae, Hansenula polymorpha. polymorpha), Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. Pichia, any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Fusarium, Yarrowia lipolytica, and Neurospora crassa.

[0264] The term "pharmaceutical composition" refers to a mixture containing one or more drugs (eg, antibodies described herein) and other components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0265] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0266] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus), sheep, dogs, cows, chickens, amphibians and reptiles. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably in the present disclosure. Unless explicitly defined in the present disclosure, the term "cynomolgus" or "cynomolgus" refers to cynomolgus (Macaca fascicularis). In certain embodiments, the subject is a human.

[0267] "Administering" or "administering," as it applies to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.

[0268] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically removed tissues, organ cultures, or cell cultures.

[0269] The term "treatment" or "treat" refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the formation of the disease or slow the progression of the disease.

[0270] The terms "administering" or "administering" as applied to an animal, a human, a test subject, a cell, a tissue, an organ or a biological fluid, refers to the contact of an exogenous agent with the animal, the human, the subject, the cell, the tissue, the organ or the biological fluid.

[0271] The term "effective amount" generally refers to an amount sufficient to reduce the severity or frequency of symptoms, eliminate symptoms and / or potential causes, prevent symptoms and / or their potential causes from occurring, and improve the amount of damage (e.g., lung disease) associated with the disease state. In some embodiments, an effective amount may be a therapeutically effective amount or a preventive effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way. A "preventive effective amount" is an amount that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete treatment or preventive effect may not occur after administering a dose, but may occur after administering a series of doses. Thus, a therapeutically or preventive effective amount may be administered in one or more administrations. A "therapeutically effective amount" and a "prophylactically effective amount" may vary, depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic to elicit a desired response in the individual.

[0272] The terms "cancer" and "tumor" refer to diseases characterized by unregulated cell growth in mammals. Tumors include benign tumors and malignant tumors, and early tumors refer to cancers that are non-invasive or metastatic, or classified as stage 0, stage I, or stage II cancer. In some embodiments, the tumor is a tumor that expresses PD1, and examples of cancer include but are not limited to lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and blood system cancer.

[0273] In some embodiments, the present disclosure uses conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are well known in the art, to prepare or detect the antibodies of the present disclosure. Related techniques have been disclosed in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987); Chain Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), which are incorporated herein by reference.

[0274] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0275] Example 1. Construction of anti-PD1-IL10 fusion protein monomer fusion protein

[0276] The N-terminus of the IL10 monomer was connected to the C-terminus of the light chain of the anti-PD1 antibody to construct an anti-PD1-IL10 fusion protein (the structural schematic diagram of the anti-PD1-IL10 fusion protein is shown in the attached Figure 1 ).

[0277] 1. Anti-PD1 antibody. The anti-PD1 antibody can be selected from R1149, R1150, R1151, and R1152, wherein the variable region sequence of the anti-PD1 antibody is shown in Table 3 below:

[0278] Table 3. Anti-PD1 antibody variable region sequence list

[0279]

[0280] Amino acid sequences of the CDRs (defined by the Kabat numbering system) of anti-PD1 antibodies R1149, R1150, R1151, R1152:

[0281] Amino acid sequence of HCDR1: TYYMY (SEQ ID NO. 9);

[0282] Amino acid sequence of HCDR2: GINPSNGGTNYAQKFQG (SEQ ID NO.10); GINPSNGGTNFNEKFKS (SEQ ID NO.15); or GINPSNGGTNYAEKFKG (SEQ ID NO.16)

[0283] Amino acid sequence of HCDR3: RDSNYDGGFDY (SEQ ID NO. 11);

[0284] Amino acid sequence of LCDR1: RASKSVSTSGYSYMH (SEQ ID NO. 12);

[0285] Amino acid sequence of LCDR2: LAYHLES (SEQ ID NO. 13);

[0286] Amino acid sequence of LCDR3: QHSWELPIT (SEQ ID NO. 14).

[0287] The antibody VH in Table 3 above is connected to the human heavy chain constant region (SEQ ID NO.19), and the antibody VL is connected to the human light chain constant region (SEQ ID NO.20) to obtain an anti-PD1 antibody. Exemplarily, the anti-PD1 antibody is R1152, and its amino acid sequence is as follows:

[0288] The amino acid sequence of the R1152 antibody heavy chain (SEQ ID NO.17):

[0289] QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMYWVRQAPGQGLEWMGGINPSNGGTNYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCTRRDSNYDGGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0290] Amino acid sequence of the light chain of R1152 antibody (SEQ ID NO.18):

[0291] DIQMTQSPSSLSASVGDRVTITCRASKSVSTSGYSYMHWYQQKPGKAPKLLIYLAYHLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQHSWELPITFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0292] Amino acid sequence of the heavy chain constant region of the antibody (SEQ ID NO.19):

[0293] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0294] The amino acid sequence of the antibody light chain constant region (SEQ ID NO.20):

[0295] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

[0296] 2. IL10 monomer. IL10 monomer can be selected from IL10 mutants. The exemplary IL10 mutants provided in this example have amino acid mutations selected from the following on IL10 WT: P20GF, R24PG, R27DG, K34DS, Q38P, D41W, L46GK, L48G, E50G, S141FL, D144S, I145G, R159EH, R24G / R27A / S141F, P20G / R24G, R24G / R27D, R24G / Q38P, R 27D / K34D, K34S / L46G, S141F / D144S, S141F / N148E, P20F, T35R and T155R (Note: for example, P20GF means that the amino acid residue at position 20 mutates from P to G or F, and P20G / R24G means that the amino acid residue at position 20 mutates from P to G, and the amino acid residue at position 24 mutates from R to G).

[0297] Amino acid sequence of IL10 WT (SEQ ID NO.21):

[0298] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQA ENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0299] The IL10 mutant (IL10MT) provided in this example lacks the first amino acid residue "S" and the second amino acid residue "P" at the N-terminus relative to IL10 WT and has the amino acid mutations shown in Table 4 below. The mutation sites of the IL10 mutant are shown in Table 4 below:

[0300] Table 4. Mutation sites of IL10 mutants

[0301]

[0302]

[0303] Note: The amino acid mutation sites of the IL10 mutants described in the table are numbered in natural sequence relative to IL10 WT (SEQ ID NO.21); for example, the IL10 mutant numbered MT1751 (L46K mutation) is obtained by mutating the 46th amino acid residue L of IL10 WT (SEQ ID NO.21) to K, while deleting the 1st amino acid residue "S" and the 2nd amino acid residue "P" at the N-terminus.

[0304] In addition, based on the IL10 mutants provided in Table 4, a spacer peptide GGGSGG (SEQ ID NO. 22) was inserted between positions 116 and 117 (relative to the natural sequence numbering of IL10 WT) to obtain a monomeric IL10 mutant (IL10MMT). The names and sequences of the monomeric IL1 mutants corresponding to the monomeric IL10 mutants after the monomeric mutation are shown in Table 5 below:

[0305] Table 5. Sequence list of IL10 monomers with a spacer peptide inserted between positions 116 and 117

[0306]

[0307]

[0308]

[0309]

[0310] Note: In the table, for example, MMT1751 is an IL10 mutant obtained by further inserting a spacer peptide GGGSGG (SEQ ID NO.22) between positions 116 and 117 (relative to the numbering of IL10 WT (SEQ ID NO.21)) based on the IL10 mutant MT1751; that is, compared with the MT1751 sequence, MMT1751 only has an additional spacer peptide GGGSGG at positions 116 and 117.

[0311] The above IL10 monomer was covalently linked to the C-terminus of the anti-PD1 antibody light chain through the second connecting peptide: GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO.53) to construct the anti-PD1-IL10 fusion protein R2870. R2870 has two identical first chains and two identical second chains. The amino acid sequence of R2870 is as follows:

[0312] The amino acid sequence of the first chain of the anti-PD1-IL10 fusion protein R2870 (SEQ ID NO.54):

[0313] DIQMTQSPSSSLSASVGDRVTITCRASKSVSTSGYSYMHWYQQKPGKAPKLLIYLAYHLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSWELPITFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GG GGSGGGGSGGGGSGGGGSGGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNKLLKESLLEDFKGY LGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFN KLQEKGIYKAMSEFDIFINYIEAYMTMKIRN;

[0314] The amino acid sequence of the heavy chain of the anti-PD1-IL10 fusion protein R2870 is the same as the amino acid sequence of the heavy chain of the R1152 antibody, both of which are SEQ ID NO.17.

[0315] Other control molecule sequences used in the examples:

[0316] Heavy chain sequence of R0862 (SEQ ID NO.55):

[0317] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAAIWYDGSNKYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLRGVMYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0318] The light chain sequence of R0862 (SEQ ID NO.56):

[0319] EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQLRNNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0320] R0676 contains two identical peptide chains. The amino acid sequence of one of the chains (SEQ ID NO.57):

[0321] DKTHTCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGSGGGGSSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLK ESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVK NAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN ;

[0322] Heavy chain sequence of R0579 (SEQ ID NO.58):

[0323] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDT

[0324] GTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSD

[0325] LYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFV

[0326] DDVEVHTAQTKPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNF

[0327] FPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGAGGGGSGGGGSGGGG

[0328] SGGGGSGS PGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLG CQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIY KAMSEFDIFINYIEAYMTMKIRN ;

[0329] The light chain sequence of R0579 (SEQ ID NO.59):

[0330] DTVLTQSPALAVSLGQRVTISKASETVSSSMYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFC QQSWNPWTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTL TKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC;

[0331] R0674: It 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:

[0332] Hinge-CH2-CH3-linker-IL10WT(SEQ ID NO.60):

[0333] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD

[0334] WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

[0335] LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSSPGQGTQSENSCTHFPGNLPNMLRDLRD

[0336] AFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0337] Example 2, Preparation of anti-PD1-IL10 mutant fusion protein;

[0338] The plasmid containing the target gene is constructed and prepared by conventional methods. The plasmid containing the target gene is introduced into the host cell Expi293 after forming a cationic complex with the transfection reagent PEI. During the time when the plasmid is in the cell, the exogenous gene on the plasmid is transcribed and translated in the cell, thereby obtaining the target protein.

[0339] Expi293 was cultured at 37°C, 8% carbon dioxide, and 130rpm. Before transfection, 2E6 cells were inoculated into a 1L shake flask by cell counting. The culture system was about 300mL. Prepare the transfection complex for transfection: First, add 750μg of the target plasmid to a 50mL centrifuge tube containing 15mL Opti-MEM reagent, mix gently, and mark it as tube A; add 1.5mg of transfection reagent PEI to a 50mL centrifuge tube containing 15mL Opti-MEM reagent, mix gently, and incubate at room temperature for 5min, marked 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 the Expi293 cells and continue to culture in a 37°C shaker. Collect samples after D5-D10.

[0340] 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. Protein A affinity chromatography was used for purification. The process is as follows: using an AKTA avant 150 chromatography device, the chromatography column (such as MabSelectSuRe LX, GE) was equilibrated with at least 5CV equilibration buffer (10mM PBS), and the sample was loaded onto the chromatography column so that the target protein was adsorbed on the chromatography column while other impurities penetrated and separated. After the loading is completed, the chromatography column is rinsed again with at least 5CV equilibration buffer (10mM PBS), and then the target protein is eluted with elution buffer (20mM NaAc, pH=3.4). Neutralization buffer (1M Tris, pH8.0) was pre-added to the collection tube. The volume of neutralization buffer added was determined according to the estimated content of the eluted sample, and generally 10% of the elution volume was added.

[0341] The results of the anti-PD1-IL10 fusion protein expression experiment are shown in Table 6. The experimental results show that the transient expression level of R2870 reached 193.50 mg / L.

[0342] The experimental results of the sample after one-step affinity purification by SEC-HPLC are shown in Table 7. The experimental results show that the target purity reaches 94.66%. The sample is tested by SDS-PAGE, and its electrophoresis is shown in the attached Figure 2 , the experimental results showed that the PD1-IL10 fusion protein had the correct light and heavy chain pairing.

[0343] Table 6. Anti-PD1-IL10 fusion protein expression experimental results

[0344] sample Protein content mg Concentration (mg / mL) Titer(mg / L) R2870 559.30 4.36 193.50

[0345] Note: In the table, Titer refers to the transient expression concentration of the protein.

[0346] Table 7. SEC-HPLC test results of anti-PD1-IL10 fusion protein samples after one-step purification

[0347] sample HMW% Main% LMW% R2870 4.29 94.66 1.05

[0348] Note: In the table, HMW% refers to the percentage of high molecular weight aggregates (High Molecular Weight%); LMW% refers to the percentage of low molecular weight fragments (LOW Molecular Weight%); Main% refers to the percentage of main peak.

[0349] Example 3. Anti-PD1-IL10 fusion protein binding activity detection

[0350] Flow cytometry was used to detect the binding activity of the antibody end and IL10 monomer end of the anti-PD1-IL10 fusion protein.

[0351] Weigh 6g BSA into 200mL 1×PBS, mix well and place on ice for later use; dilute the molecule to be tested with 3% BSA to an initial concentration of 125nM (configuration concentration is 250nM), volume 300μL, multiple dilution (100+200) for a total of 11 points; after counting the cells, divide them into 96-well V-shaped plates at 50μL, 2E+05 / well, add 50μL of the molecule to be tested at different concentrations to the cells, incubate at 2-8 degrees for 30min; centrifuge at 350xg for 5min, remove the supernatant, and prepare the fluorescent antibody PE Mouse anti-human IgG at 200μL / well 3% BSA; centrifuge at 350xg for 5min, remove the supernatant, and prepare the fluorescent antibody PE Mouse anti-human IgG at 3% BSA Fc (1:500x dilution), added to the corresponding 96-well plate at 100 μL / well, incubated at 2-8 degrees for 30 minutes; centrifuged at 350g for 5 minutes, removed the supernatant, and washed the cells once with 200 μL / well 3% BSA; after centrifugation at 350xg for 5 minutes, removed the supernatant, added 1×PBS at 100 μL / well to resuspend the cells; performed FACS detection on the machine according to the "CytoFLEX Flow Cytometer Standard Operating Procedure".

[0352] The experimental results are attached. Figure 3 and 4The experimental results show that compared with PD1 monoclonal antibody R1152, the binding activity of the antibody end of the anti-PD1-IL10 fusion protein has not changed significantly. This shows that the antibody and IL10 monomers constitute a fusion protein and do not affect the binding activity of the antibody end with its antigen. Compared with IL10-Fc fusion protein R0676 (a dimer fusion protein constructed by directly connecting IL10 described in SEQ ID NO.22 to the N-terminus of the human Fc constant region), the binding of the anti-PD1-IL10 fusion protein disclosed in the present invention to IL10R is significantly weaker.

[0353] Example 4. Detection of anti-PD1-IL10 fusion protein activity by reporter gene method

[0354] 4.1. Detection of the activity of the IL10 end of the anti-PD1-IL10 fusion protein by reporter gene assay

[0355] Cell preparation: Take out the HEK293-hIL10-Report gene cell (Jiman Biotechnology, catalog number GM-C07927) in culture and observe it under a microscope. The cells adhere normally, the particles are transparent, and the cells with moderate density can be used as effector cells for the experiment; digest the above cells with trypsin, centrifuge at 350g / 4min after terminating the digestion to remove the supernatant, resuspend with 1% FBS-PBS and wash again; discard the supernatant, and finally resuspend the cells with DMEM medium, adjust the cell density to 5E5 / mL after cell counting, and plate 96 flat-bottom plates at 50μL per well, which is equivalent to 2.5E4 cells per well, and then place in a 37℃ incubator for 6h of adhesion treatment. Take R0056FC2-6 cells (CHO-hPD1), centrifuge to remove the supernatant, count, adjust the cell density to 1E6 / mL, and plate 25μL per well, which is equivalent to 2.5E4 cells per well;

[0356] Dilution: Use Assay Buffer (DMEM basal medium) to dilute the molecules to be tested (R1152, R2870) to an initial and final concentration of 50 nM (dilution concentration 200 nM), a volume of 150 μL, 3-fold gradient dilution (50+100), 7 concentrations; experiments were performed under non-enrichment conditions and enrichment conditions respectively;

[0357] Under the non-enrichment condition, the diluted test molecule was directly added to the adherent cells at 25 μL / well. Under the enrichment condition, the diluted test molecule was mixed with the target cells R0056FC2-6 at a volume of 25 μL / well and incubated for about 30 minutes. Then, they were carefully transferred to the HEK293 cells that had been adherent for 6 hours. 25 μL of culture medium was added to the wells of Cell only, and the final volume of all wells was 100 μL. The edge wells were sealed with 200 μL of sterile water. The 96-well plate was cultured in the incubator for 16 hours.

[0358] The next day, thaw the Bright-LumiTM Firefly Luciferase Detection Reagent in advance and equilibrate to room temperature; after the cell culture reaches the time point, take out the cell culture plate and equilibrate to room temperature for 10 minutes (not more than 30 minutes), add 100μL Bright-LumiTM Firefly Luciferase Detection Reagent to each well, and incubate at room temperature for 5-10 minutes with shaking; then take 100μL of the system after the reaction is completed from each well to a 96-well all-white plate, and use the chemiluminescence mode to detect the signal with a multi-function microplate reader.

[0359] The test results are attached. Figure 5 and attached Figure 6 The experimental results show that the anti-PD1-IL10 fusion protein R2870 constructed in the present invention has high fluorescence activity under CHO-hPD1 enrichment conditions.

[0360] 4.2. Detection of PD1 binding activity of anti-PD1-IL10 fusion protein by reporter gene assay

[0361] Cell preparation: Count the cells, take an appropriate amount of CHO-OKT3-hPDL1 (CHO cells overexpressing OKT3 and human PDL1) and Jurkat-luc-hPD1 cells (Jurkat cells overexpressing NFAT-luc and human PD1), centrifuge at 300g for 5 min, discard the supernatant, wash once with 1640+10% FBS medium, resuspend with 1640+10% FBS medium, adjust the CHO-OKT3-hPDL1 cell density to 1E+06 cells / mL, and the Jurkat-luc-hPD1 cell density to 5E+06 cells / mL;

[0362] Dilution: dilute the molecule to be tested to an initial and final concentration of 250 nM (dilution concentration 500 nM) using 1640 + 10% FBS medium, volume 100 μL, 2-fold gradient dilution (50 + 50), 10 concentrations;

[0363] According to the experimental design, 20 μL of the test molecule dilution was added to each well of the 96-well all-leukocyte culture flat-bottom plate. CHO-OKT3-hPDL1 cells were added at 4E+04 / well, i.e., 40 μL / well. Jurkat-luc-hPD1 cells were added at 2E+05 / well, i.e., 40 μL / well. A certain volume of culture medium was added to the corresponding wells to make the total volume of all wells 100 μL. 100 μL of PBS was added to the outermost circle. The cell culture plate was placed in a CO 2Incubate in the incubator for 6 hours; thaw the Bright-LumiTM firefly luciferase detection reagent in advance and equilibrate to room temperature; after the cell culture reaches the time point, take out the cell culture plate and equilibrate to room temperature for 10 minutes (not more than 30 minutes), add 100μL Bright-LumiTM firefly luciferase detection reagent to each well, and incubate at room temperature with shaking for 5-10 minutes; perform chemiluminescence detection on the machine.

[0364] The experimental results are attached. Figure 7 The fluorescence activity of the PD1 binding end of the anti-PD1-IL10 fusion protein disclosed in the present invention is basically consistent with the fluorescence activity of the anti-PD1 monoclonal antibody R1152, indicating that the construction of the fusion protein between the anti-PD1 antibody and the IL10 monomer does not affect the fluorescence activation activity of the antibody end.

[0365] Example 5. Anti-PD1-IL10 fusion protein-mediated depletion of CD8+ T cells functional assay

[0366] Fresh healthy human blood was separated to obtain PBMC according to the operation procedure; CD8+T cells in PBMC were separated according to the method provided by Miltenyi Biotec CD8 microbeads kit, and the purity was identified by FITC anti-human CD8 flow cytometry antibody; D0-CD8+T cells were adjusted to a density of 1E6 cells / mL with 1640+10% FBS+100U / mL IL-2 medium, and CD3 / CD28 magnetic beads were added at a ratio of 1:1. After mixing, 200μL / well, equivalent to 2E5 cells / well, were inoculated in 96 flat-bottom plates and placed at 37°C, 5% CO 2 Incubator induced culture; D0-isolated CD8+T cells were identified by flow cytometry using PE antihuman IL10R (Biolegend, 308804), FITC anti human PD1 (Biolegend, 329904) and APC antihuman TIM3 (Biolegend, 364804) antibodies for initial expression levels.

[0367] D1 (D1 means the first day of the experiment, D2 means the second day... and so on) dilute PD-1-his (R2294, human PD1 protein with his tag) protein to 5 μg / mL with DPBS, add to 96 flat-bottom plates at a volume of 200 μL / well, and incubate at 4°C overnight; D2- dilute the test molecules (R2870, control molecules R0579, R1152 disclosed in the present invention, blank control group cells) with 1640+10% FBS culture medium to a final concentration of 50 nM, 5-fold dilution (3 gradients), and set aside. Discard the liquid of the plate overnight at 4°C, wash twice with sterile DPBS, then add the diluted gradient test molecules, and incubate in a 37°C incubator for 0.5h-1h. During D2-incubation, all CD8+T cells in the 96-well plate were blown down and collected and mixed. The beads were removed with a magnet and the cells were counted. Some cells were taken for surface marker detection (including IL10R, PD1, TIM3). The remaining cells were centrifuged and the density was adjusted to 1.5E6 cells / mL with 1640+10% FBS culture medium. According to the experimental requirements, the corresponding amount of cells was taken into a new centrifuge tube, and CD3 / CD28 magnetic beads were added at a ratio of 1:1. After mixing, 100 μL (1.5E5 cells) / well of cells were added to the antibody-incubated plate and placed in a 37°C incubator for incubation for 4 days. D2-After centrifugation, the remaining cells were adjusted to a density of 5E5 cells / mL with 1640+10% FBS+100U / mL IL-2 culture medium, and CD3 / CD28 magnetic beads were added at a ratio of 1:1. After mixing, the cells were re-seeded in a 96-well plate at a volume of 200μL (1E5 cells) / well and continued to be cultured and stimulated at 37°C.

[0368] Continue pre-coating and antibody incubation according to the above operations; detect markers and add induced CD8+T cells to the antibody incubated plate, and incubate in a 37℃ incubator for 4 days; D6-detect cell apoptosis after incubation for 4 days on D2: blow up the corresponding cells and transfer them to a V-bottom plate, centrifuge at 350g and discard the supernatant, wash once with Annexin-V binding buffer (200μL / well), prepare staining solution with Annexin-V binding buffer (prepare a mixed solution by adding 1μL APCannexin-V and 1μL Propidium Iodide Solution to each well), add 100μL / well, incubate at room temperature in the dark for 15 minutes, add 100μL Annexin-V binding buffer to each well and mix well, and finally detect cell apoptosis by upflow. D10-detect cell apoptosis after incubation for 4 days on D6.

[0369] Cytokine detection: The cell supernatants were collected after 4 days of incubation, and IFN-γ was detected using Human IFN-γ uncoated ELISA kit (Invitrogen, 88-7316-77).

[0370] The test results are attached. Figure 8 and Fig. 9 Compared with the Cell group, under the enrichment condition of the presence of PD-1-his protein, the apoptosis of exhausted CD8+T cells acted upon by the anti-PD1-IL10 fusion protein disclosed herein was significantly reduced; the secretion of IFN-γ factor increased, which was less than that of the positive control R0579.

[0371] Example 6. In vivo efficacy study of anti-PD1-IL10 fusion protein

[0372] Objective: To observe the anti-tumor activity of anti-PD1-IL10 fusion protein against CT-26 mouse colon cancer, and to set up isotype control group and single drug control group.

[0373] Experimental materials: hPDL1 Knock in mice, female, 6-8 weeks (Balb / c background, source: Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., animal quality certificate No. 320327230100208956); CT-26 cells (Shanghai Cell Bank, catalog number: TCM37); RPMI 1640 medium (Gibco, 11875085), FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), penicillin-streptomycin (Gibco, 15140122), DPBS (Hyclone, SH30028.02)

[0374] Instruments and equipment: electronic balance (Shanghai Sunyu Hengping Scientific Instrument Co., Ltd., JA12002), vernier caliper (Shanghai Menite Industrial Co., Ltd., MNT-150T), microscope (Chongqing Aote Optical Instrument Co., Ltd., BDS200), medical centrifuge (Hunan Xiangyi Laboratory Development Co., Ltd., L530R), digital display constant temperature water bath (Prius Machinery Co., Ltd., HH-S), carbon dioxide incubator (Panasonic Healthcare Medical Devices Co., Ltd., MCO-18AC), biological safety cabinet (Guangzhou Qianjiang Experimental Technology Co., Ltd., ACZ-451), cell counter (Shanghai Ruiyu Biological, IC1000)

[0375] Experimental methods

[0376] Cell culture: Mouse colon cancer cells (CT-26) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1:1) at 37°C and 5% CO 2 Culture in an incubator.

[0377] Inoculation: Collect CT-26 cells in the logarithmic growth phase, wash twice with DPBS, and adjust the cell concentration to 1×10 6 / mL. Female hPDL1 mice were subcutaneously inoculated with CT-26 cells at a volume of 0.1 mL / mouse, i.e., 1×10 5 / mice.

[0378] Administration: The day of inoculation was recorded as day 0 (D0). When the average tumor volume reached 80-100 mm 3 , the mice can be randomly divided into groups according to the tumor volume to ensure that the average tumor volume of each group of mice is the same or similar. On the 9th day (D9) of this experiment, the mice were randomly divided into 5 groups according to the tumor volume, with 7 mice in each group, and drug administration was started (the dosing schedule is shown in Table 8 below). The R2870 group was administered with a dose of 10 mg / kg, and the other groups were administered with an equimolar dose of R2870 (10 mg / kg).

[0379] Table 8. Dosage, method and frequency of administration in CT-26 tumor model

[0380] Group Dosage (mg / kg) Dosing volume (μL / g) Route of administration Dosing frequency Isotype control_hIgG1.8 8 10 ip Q3D*4 R0676 5 10 ip Q3D*4 R1152 7.9 10 ip Q3D*4 R2870 10 10 ip Q3D*4

[0381] Note: ip, intraperitoneal injection; Q3D, administration once every 3 days; if body weight decreases by more than 15% during administration, the dosing regimen will be adjusted.

[0382] Record:

[0383] Tumor volume was measured and recorded starting from D9, and then the long and short diameters of the tumor were measured with a vernier caliper twice a week. The formula was: (1 / 2) × long diameter × (short diameter) 2 The tumor volume was calculated (see Table 9). When each mouse reached the experimental endpoint (weight loss exceeded 20% or tumor volume exceeded 2000 mm 3 reached a merciful end point), CO 2 The mice were killed by asphyxiation.

[0384] Table 9. Antitumor activity of PD1-IL10 fusion protein in CT-26 mouse colon cancer model

[0385]

[0386]

[0387] Note: CR (D27) means the percentage of mice in each group whose tumors were completely eliminated on the 27th day after tumor implantation.

[0388] Data calculation, statistics and analysis: Independent sample T test can be used for comparison between two groups. One-Way ANOVA is used for comparison of more than 3 groups. If the F value shows a significant difference, post hoc analysis between multiple groups can be performed. Data were processed using PrismGraphPad, and p<0.05 indicated a statistically significant difference. Tumor volume (TV) = 0.5a×b 2 , a and b are the long and short diameters of the tumor, respectively. Tumor growth inhibition TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100, Ti is the average tumor volume of the treatment group on the i-th day, T0 is the average tumor volume of the treatment group at the beginning of treatment, Vi is the average tumor volume of the solvent control group on the i-th day, and V0 is the average tumor volume of the solvent control group at the beginning of treatment.

[0389] Experimental results: The anti-PD1-IL10 fusion protein R2870 has significant anti-tumor efficacy in the hPD1 mouse CT-26 colon cancer model, and has a clear advantage in the proportion of complete tumor elimination (CR) compared with the anti-tumor effects of PD1 monoclonal antibody R1152 and IL10 fusion protein R0676 alone.

[0390] Example 7. Early safety evaluation of anti-PD1-IL10 fusion protein and wild-type Fc-IL10 fusion protein

[0391] Experiment 1: This experiment aims to give R0674 to cynomolgus monkeys by intravenous injection. One week after the first administration, multiple administrations were carried out, twice a week, for a total of 6 times (including the first administration), to observe the toxic reactions caused by 674 and study its toxicokinetics (pharmacokinetics) characteristics, so as to provide reference for the design of subsequent experiments. In this experiment, R0674 was divided into two groups, with doses of 1 mg / kg and 3 mg / kg respectively; a solvent control substance was also set up. During the experiment, the following observations were made on the animals: death and dying, clinical observation, body weight, food intake, body temperature, hematology and coagulation index detection, serum biochemical indexes and immune function detection. The animals were euthanized on D27 and gross dissection was performed. The main organs (adrenal glands, brain, epididymis, heart, kidneys, liver, spleen, testis, thymus, thyroid and parathyroid glands) were weighed, and the liver, spleen and kidneys were subjected to histopathological examination.

[0392] During the experiment, no obvious abnormalities were found in animal death and moribundity, clinical observation, body weight, food intake, body temperature, coagulation, immune function tests, and organ weights.

[0393] Hematology: Compared with the vehicle control group, the R0674 low-dose group (1 mg / kg) showed RBC↓(-37.4%), HGB↓(-37.0%), and HCT↓(-25.6%) on D15; the high-dose group (3 mg / kg) also showed RBC↓(-13.6%), HGB↓(-15.7%), and HCT↓(-12.8%) during the same period. The above abnormal indicators are considered to be related to the test product. On D28, the above abnormal indicators basically returned to normal. (Note: "↑" indicates an increase in the corresponding value compared with the vehicle control group, and "↓" indicates a decrease in the corresponding value compared with the vehicle control group. For example, "RBC↓(-37.4%)" means that the RBC value is 37.4% lower than that of the vehicle group, and the same applies to others).

[0394] Serum biochemistry: Compared with the vehicle control group, the serum biochemical indicators of the R0674 high-dose group (3 mg / kg) on ​​D23 were ALT↑, AST↑, CK↓, BUN↑, and CREA↑.

[0395] Toxicokinetics (pharmacokinetics): [Exposure accumulation relationship] The exposure (AUC(0-t) and Cmax) of R0674 increased with the increase of dose. The proportion of the increase in animal exposure after administration on D1 was equivalent to the dose ratio. The proportion of animal exposure after administration on D22 (the third time) was higher than the dose ratio. [Accumulation] After 22 days, there was no obvious accumulation of R0674 in each dose group.

[0396] Cytokines: During the experiment, compared with the vehicle control group, there was no abnormality in the cytokines IFN-γ, IL-17A, IL-1β, IL-4, IL-5, and TNF-α in the low- and high-dose groups of R0674 before the first administration, 6 hours after the first administration, and 6 hours after D22 administration; 6 hours after the first administration and 6 hours after D22 administration, the cytokine IL-6 was detected in all dose groups of R0674. However, it was also detected in the vehicle control group, and the detection value was slightly higher than the lower limit of quantification, which was considered to be a fluctuation in the detection baseline. The detection values ​​of the cytokine IL10 in the low- and high-dose groups of R0674 were higher than the upper limit of quantification 6 hours after the first administration.

[0397] In summary, cynomolgus monkeys were given R0674 intravenously once in the first week (D1, the first day of the experiment), and then twice a week (D8, D11, D15, D18 and D22), a total of 6 times, with a dose of 1mg / kg and 3mg / kg. During the experiment, the animal's clinical observation, body weight, food intake, body temperature, coagulation and immune function tests showed no obvious abnormalities related to the test product. R0674 (≥1mg / kg) on ​​D15, red blood cell indicators decreased (RBC↓, HGB↓, HCT↓), which was considered to be related to the test product, but basically recovered on D28. In the R0674 high-dose group (3 mg / kg), ALT↑, AST↑, CK↓, BUN↑, and CREA↑ appeared; at the same time, gross anatomical observation revealed bilateral gray discoloration of the kidneys, and histopathological examination revealed moderate renal tubular casts, moderate renal tubular dilatation, mild renal tubular single cell necrosis, mild tubular nephritis, mild renal tubular mineralization, and mild interstitial mononuclear cell infiltration. At the end of the dosing period, histopathological changes that may be related to the test article were found in the eosinophilic deposition of the germinal center of the spleen white pulp in the R0674 dose group.

[0398] Experiment 2: This experiment aims to give the anti-PD1-IL10 fusion protein R2870 to crab-eating monkeys by intravenous injection, once a week for 3 consecutive weeks, for a total of 4 times (D1, D8, D15, D22). The first day of administration is defined as D1, and the characteristics of the toxic reactions caused are observed to provide a reference for the design of subsequent experiments. This experiment has 2 groups, Group 1 solvent control group, Group 2 test group D1, D8, D15, D22 dosages are 1mg / kg, 3mg / kg, 8.5mg / kg, 10mg / kg respectively; During the experiment, the following observations were made on the animals: death and dying, clinical observation, body weight, body temperature, hematology and coagulation indexes, serum biochemical indexes and immune function tests.

[0399] During the experiment, no obvious abnormalities were found in the tests of animal death and moribundity, body weight, body temperature, hematology, blood biochemistry, coagulation, and immune function (immune cell typing and cytokines).

[0400] Clinical observation: The planned dose of the third administration of D15 was 10 mg / kg. During the infusion, it was observed that the crab-eating monkey had mild foaming at the mouth and mild pale cheeks. The drug was stopped immediately. The actual infusion dose was 8.5 mg / kg. The monkey fully recovered 5 hours after stopping the drug. No abnormalities were observed on D1-D14 and D16-D50. The abnormal reaction on D15 was not related to the test product and was caused by the infusion reaction.

[0401] In summary, the anti-PD1-IL10 fusion protein R2870 was administered intravenously to cynomolgus monkeys once a week, and the dosages of D1, D8, D15 and D22 were 1 mg / kg, 3 mg / kg, 8.5 mg / kg, and 10 mg / kg, respectively. During the experiment, no abnormalities related to the test sample were found in the clinical observation of the animals, body weight, body temperature, coagulation and immune function tests (immune lymphocyte subtypes, serum cytokines). In Experiment 1, the Fc-IL10 fusion protein R0674 showed obvious abnormalities such as decreased red blood cell indicators, increased liver function indicators, and increased cytokines. In the R2870 group, the corresponding indicators were normal, which shows that the safety of the anti-PD1-IL10 fusion protein R2870 disclosed in the present invention is significantly improved compared with the control group.

[0402] Appendix I: Check parameter indicators are shown in Tables 10 to 13:

[0403] Table 10. Blood cell count ( Series blood analyzers)

[0404]

[0405] Note: This study only analyzes the above indicators, and other indicators measured by the instrument are not analyzed.

[0406] Table 11. Blood cell count ( Series fully automatic modular blood and body fluid analyzers)

[0407]

[0408] Note: This study only analyzes the above indicators, and other indicators measured by the instrument are not analyzed.

[0409] Table 12. Blood biochemical indicators (TBA-120FR fully automatic biochemical analyzer)

[0410]

[0411]

[0412] Table 13. Blood biochemical indexes (Beckman Coulter AU5800 automatic biochemical analyzer)

[0413]

[0414] Immune cell phenotyping: using BD FACSCalibur flow cytometer and / or BD LSRFortessa TM Flow cytometer and / or BD FACSCanto TM II flow cytometer was used to detect the total T lymphocytes (CD45+ CD3 + ), Th cells (CD45 + CD3 + CD4 + ), Tc cells (CD45 + CD3 + CD8 + ) and Treg cells (CD45 + CD3 + CD4 + CD25 + Foxp3 + ) and monocytes (CD14 + ) and calculate the absolute value based on the blood cell count results, and calculate CD3 + CD4 + / CD3 + CD8 + ratio.

[0415] Cytokines: BD FACSCalibur flow cytometer and / or BD LSRFortessa TM Flow cytometer and / or BD FACSCanto TM II flow cytometer, commercial kit Serum interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL10), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were tested. The detailed test methods are stored in the research materials.

[0416] Example 8. Activity detection of other anti-PD1-IL10 fusion proteins

[0417] 8.1. Preparation of other anti-PD1-IL10 fusion proteins

[0418] Each IL10MMT in Table 5 was fused to the light chain of the anti-PD1 antibody R1049 to obtain other anti-PD1-IL10 fusion proteins R1741-R1771 (the structural schematic diagram of the anti-PD1-IL10 fusion protein is shown in the attached Figure 1 ), which includes two identical heavy chains and two identical light chains, and IL10MMT is covalently linked to the C-terminus of the light chain of R1049 antibody through a linker (SEQ ID NO: 53: GGGGSGGGGSGGGGSGGGGSG). The names of IL10MMT and antibodies corresponding to each fusion protein are shown in Table 14 below:

[0419] Table 14. Other anti-PD1-IL10 fusion proteins

[0420] Fusion Protein IL10MMT Antibody R1741 MMT1741 PD1 Antibody R1742 MMT1742 PD1 Antibody R1743 MMT1743 PD1 Antibody R1744 MMT1744 PD1 Antibody R1745 MMT1745 PD1 Antibody R1746 MMT1746 PD1 Antibody R1747 MMT1747 PD1 Antibody R1748 MMT1748 PD1 Antibody R1749 MMT1749 PD1 Antibody R1750 MMT1750 PD1 Antibody R1751 MMT1751 PD1 Antibody R1752 MMT1752 PD1 Antibody R1753 MMT1753 PD1 Antibody R1754 MMT1754 PD1 Antibody R1755 MMT1755 PD1 Antibody R1756 MMT1756 PD1 Antibody R1757 MMT1757 PD1 Antibody R1758 MMT1758 PD1 Antibody R1759 MMT1759 PD1 Antibody R1761 MMT1761 PD1 Antibody R1762 MMT1762 PD1 Antibody R1763 MMT1763 PD1 Antibody R1764 MMT1764 PD1 Antibody R1765 MMT1765 PD1 Antibody R1766 MMT1766 PD1 Antibody R1767 MMT1767 PD1 Antibody R1768 MMT1768 PD1 Antibody R1769 MMT1769 PD1 Antibody R1770 MMT1770 PD1 Antibody R1771 MMT1771 PD1 Antibody

[0421] Note: The PD1 antibody in the table is R1049 antibody

[0422] Taking the fusion protein R1741 as an example, the sequence of the fusion protein is as follows:

[0423] Heavy chain sequence of fusion protein (R1741):

[0424] VH-CH1-Hinge-CH2-CH3 (SEQ ID NO.61):

[0425] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY I CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM I SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT I SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0426] Note: The italic part is the heavy chain variable region of the PD1 antibody, and the dotted underlined part is the heavy chain constant region of the PD1 antibody;

[0427] Fusion protein ( R1741 )'s light chain sequence:

[0428] VL-CL-linker- MMT1741 (SEQ ID NO.62):

[0429] DTVLTQSPALAVSLGQRVTISCKASETVSSSMYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSGGQGTQSENSCTHFPGNLGN MLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLG CQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKL QEKGIYKAMSEFDIFINYIEAYMTMKIRN;

[0430] Note: The italic part is the light chain variable region of the PD1 antibody, the dotted underlined part is the light chain constant region of the PD1 antibody; the wavy underlined part is the linker sequence, and the solid underlined part is the MMT1741 sequence.

[0431] The structures of other fusion proteins are basically the same as R1741, except that the sequence of IL10MMT on the light chain is different.

[0432] The structures of some control molecules (R0991, R1049, R1187) in the examples are as follows:

[0433] R0991: The structure is basically the same as the aforementioned fusion protein, except that IL10M is used instead of IL10MMT.

[0434] Heavy chain sequence of R0991 (SEQ ID NO.63):

[0435] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0436] The light chain sequence of R0991 (SEQ ID NO.64):

[0437] DTVLTQSPALAVSLGQRVTISCKASETVSSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSGGGGSG GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLG CQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKL QEKGIYKAMSEFDIFINYIEAYMTMKIRN;

[0438] R1049: An anti-PD1 antibody, comprising two identical heavy chains and two identical light chains:

[0439] The heavy chain sequence of R1049 (SEQ ID NO.65):

[0440] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY I CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM I SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT I SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0441] Light chain sequence of R1049 (SEQ ID NO.66):

[0442] DTVLTQSPALAVSLGQRVTISCKASETVSSSMYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATY FCQQSWNPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0443] R1187: It is a fusion of a non-PD1 targeting antibody and IL10M, including two identical heavy chains and two identical light chains, and the C-termini of both light chains are fused with IL10M:

[0444] Heavy chain sequence of R1187 (SEQ ID NO.67):

[0445] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAAIWYDGSNKYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLRGVMYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD I AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0446] Light chain sequence of R1187 (SEQ ID NO.68) :

[0447] EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQLRNNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSG GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQ ALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQE KGIYKAMSEFDIFINYIEAYMTMKIRN 。

[0448] 8.2. In vitro activity detection of anti-PD1-IL10 fusion protein

[0449] Flow cytometry was used to detect the IL10Rα binding activity of the fusion protein.

[0450] Each fusion protein molecule was diluted to an initial concentration of 200 nM using PBS buffer containing 3% BSA, with a volume of 180 μL, 3-fold gradient dilution (60 samples + 120 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 100μL / well was evenly distributed into 96-well V-shaped plates; the above-mentioned diluted molecules were added to the cells, 100μL / well, and incubated at 2-8 degrees for 0.5h; the 96-well plate was removed, centrifuged at 350g for 5min, the supernatant was carefully removed, 200μL / well of 3% BSA PBS buffer was added, and centrifuged at 350g for 5min again, and the supernatant was carefully removed; PE fluorescent secondary antibody (1:500 dilution) was prepared with PBS buffer containing 3% BSA, and 100μL / well was added to the corresponding 96-well plate, the cells were resuspended, and incubated at 2-8 degrees for 30min; the 96-well plate was removed, centrifuged at 350g for 5min, the supernatant was carefully removed, and PBS containing 3% BSA was added Buffer, 200 μL / well, centrifuge again at 350g for 5 min, carefully remove the supernatant; resuspend with 1xPBS 100 μL / well, and detect by FACS.

[0451] The results of the fusion protein binding activity test on IL10Rα are shown in Figure 10a-Figure 10d It can be seen that, compared with the control molecule R0674, each fusion protein has lower binding activity to IL10Rα, indicating that the mutations in Table 4 reduce the binding activity of IL10 mutants or monomeric mutants to IL10Rα.

[0452] 8.3. Detection of IL10 activity of anti-PD1-IL10 fusion protein in the presence of target cells

[0453] 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 (Jiman Biotechnology Company, catalog number GM-C07927; this cell line stably expresses the IL10R and STAT3 signaling pathway reporter gene system, and the use of IL10 protein stimulation can activate the increase of cell luciferase expression) were taken out and observed under a microscope. Cells with normal cell adhesion, transparent particles, and moderate density can be used as effector cells for the experiment.

[0454] The above cells were digested with TE, centrifuged at 350g / 4min after digestion, and the supernatant was removed. 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 cell counting, the cell density was adjusted to 5E5 / mL, and 50μL was plated per well, and then placed in a 37°C incubator for 6h. The target cells R0326Fc-118 cells (CHO cells that stably express PD1 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 (1 Enrichment system: According to the experimental design, the diluted fusion protein was first mixed with the target cell R0326Fc-118 in an equal volume at a volume of 25 μL / well, and then incubated for about 30 minutes. Then, the target cell R0326Fc-118 and each fusion protein were carefully transferred into the adherent HEK293-hIL10 reporter gene cells; 100 μl Assay Buffer was added to the Medium only wells, and 25 μL Assay Buffer was added to the Cell only wells. The final volume of all wells was 100 μL. The edge wells were sealed with 200 μL sterile water, and the 96-well plate was continued to be cultured in the incubator for 16 hours; 2 No enrichment system: Without adding target cells (i.e. R0326Fc-118), the corresponding fusion protein is directly added 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 Detection 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 10 minutes (not more than 30 minutes). Add 100μL Bright-LumiTM Firefly Luciferase Detection Reagent to each well and incubate at room temperature for 5-10 minutes. Take 100μL of the system after the reaction is completed from each well to a 96-well all-white plate; use the multi-function microplate reader to detect the signal using the chemiluminescence mode.

[0455] Test results see Figure 11-Figure 30 It can be seen that the reporter gene signal values ​​of each fusion protein fused with IL10 monomer mutant 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, R1770, R1772, R1774, and R1776 have significantly improved signal values ​​after enrichment at the target end, which is 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 has basically not changed before and after enrichment, indicating that the system is stable.

[0456] The above results indicate that the IL10MMT provided in the embodiments of the present invention has weak binding activity to IL10Rα. When it is fused with the targeting 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.

[0457] In summary, there are safety issues with the current clinical IL10 protein, the clinical IL10 drug molecules lack targeting, the drug molecules are prone to off-target, and produce side effects. Most of the IL10 antibody fusion proteins under development use antibody targeting to introduce IL10 into the local environment to exert its effects, but these fusion protein molecules often have a normal IL10 dimer structure, and the side effects of the IL10 end are still a thorny problem. The safety of the anti-PD1-IL10 fusion protein provided by the embodiments of the present disclosure is significantly improved. Compared with the IL10 dimer (Fc-IL10), the anti-PD1-IL10 fusion protein disclosed in the present disclosure has no hematological toxicity and liver damage during administration, which helps to solve the problem of limited efficacy due to the high hematotoxicity of IL10 treatment in clinical practice, resulting in too low a dosage.

[0458] The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A fusion protein comprising a PD1 binding unit portion and an IL10 unit portion, wherein: The PD1 binding unit portion is an anti-PD1 antibody, and the IL10 unit portion is an IL10 mutant; wherein the IL10 mutant has an amino acid mutation at at least one of the following sites relative to SEQ ID NO.21: L46, P20, R24, R27, K34, T35, Q38, D41, L48, E50, S141, D144, I145, N148, T155 and R159.

2. The fusion protein according to claim 1, wherein the IL10 mutant, wherein Optionally, the IL10 mutant has an amino acid mutation at at least one of the following positions relative to SEQ ID NO. 21: L46, P20, R24, T35, D41, L48, E50, S141, I145, T155 and R159; Optionally, the IL10 mutant has an amino acid mutation at at least one of the following positions relative to SEQ ID NO. 21: L46, K34, Q38, D144, N148 and I145; Optionally, the IL10 mutant has an amino acid mutation at the following positions relative to SEQ ID NO. 21: L46; Optionally, the amino acid mutation is an amino acid substitution; Optionally, the IL10 mutant is relative to SEQ ID NO.21 has at least one of the following amino acid mutations: L46K, L46G, P20G, P20F, R24P, R27D, R27G, K34S, K34D, T35R, Q38P, D41W, L48G, E50G, S141F, S141Q, S141L, D144S, N148E, T155R, R159E, R159H, I145G, I145A, I145R, I145L, I145K, I145N, I145M, I145D, I145F, I145C, I145P, I145Q, I145S, I145E, I145T, I145W, I145H, I145Y, I145V; Optionally, the IL10 mutant has at least one of the following amino acid mutations relative to SEQ ID NO. 21: L46K, L46G, P20G, P20F, R24G, R24P, R27D, R27G, T35R, D41W, L48G, E50G, S141L, S141F, S141Q, T155R, R159E, R159H; Optionally, the IL10 mutant has the following mutation relative to SEQ ID NO.21: (a) L46K; (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; (y) T155R; or (z) P20G; Optionally, the IL10 mutant has the following amino acid mutation relative to SEQ ID NO.21: L46K.

3. The fusion protein according to claim 1 or 2, wherein the IL10 mutant has at least 80% sequence identity with SEQ ID NO.21; Optionally, the amino acid sequence of the IL10 mutant at other sites other than the above mutation site is the same as SEQ ID NO.21; Optionally, the IL10 mutant has reduced binding activity to the IL10 receptor compared to the wild-type IL10 shown in SEQ ID NO.21; Optionally, the IL10 receptor is selected from IL10Rα and IL10Rβ; Optionally, the IL10 receptor is selected from IL10Rα.

4. The fusion protein according to any one of claims 1 to 3, wherein the IL10 mutant has a spacer peptide inserted between positions 116 and 117 relative to the wild-type IL10 shown in SEQ ID NO.21; Optionally, the spacer peptide is 5-10 amino acids long; Optionally, the spacer peptide contains amino acid residues G and S; Optionally, the amino acid sequence of the spacer peptide is SEQ ID NO.22; Optionally, the IL10 mutant is further missing the first amino acid residue S and the second amino acid residue P at the N-terminus relative to the wild-type IL10 shown in SEQ ID NO.21; Optionally, the IL10 mutant is an amino acid sequence selected from any one of SEQ ID NOs. 23-52, or an amino acid sequence having at least 80% sequence identity therewith; Optionally, the amino acid sequence of the IL10 mutant is shown in SEQ ID NO.

23.

5. The fusion protein according to any one of claims 1 to 4, wherein The anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in a variable region having at least 90% sequence identity with SEQ ID NO.1, 3, 5 or 7, and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in a variable region having at least 90% sequence identity with SEQ ID NO.2, 4, 6 or 8; Optionally, A. The heavy chain variable region of the anti-PD1 antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO.1, and the light chain variable region of the anti-PD1 antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO.2; B. The heavy chain variable region of the anti-PD1 antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO.3, and the light chain variable region of the anti-PD1 antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO.4; C. the heavy chain variable region of the anti-PD1 antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO.5, and the light chain variable region of the anti-PD1 antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO.6; or D. the heavy chain variable region of the anti-PD1 antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO.7, and the light chain variable region of the anti-PD1 antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO.8; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat numbering system, or by the IMGT numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat numbering system; Optionally, the anti-PD1 antibody, wherein the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.9, HCDR2 comprises the amino acid sequence of SEQ ID NO.10, 15 or 16, and HCDR3 comprises the amino acid sequence of SEQ ID NO.11; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.12, LCDR2 comprises the amino acid sequence of SEQ ID NO.13, and LCDR3 comprises the amino acid sequence of SEQ ID NO.14; Optionally, the anti-PD1 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, respectively.

6. The fusion protein according to any one of claims 1 to 5, wherein The heavy chain variable region of the anti-PD1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 1, 3, 5 or 7, and / or the light chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 2, 4, 6 or 8; Optionally, wherein (a) the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.2; (b) the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.4; (c) the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.6; (d) the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.8; or (e) the anti-PD1 antibody is an antibody that competes with the anti-PD1 antibody described in (a), (b), (c) or (d) for binding to the PD1 antigen or binds to the same epitope; Optionally, the anti-PD1 antibody comprises a heavy chain variable region as shown in SEQ ID NO.1, and a light chain variable region as shown in SEQ ID NO.

2.

7. The fusion protein according to any one of claims 1 to 6, wherein the anti-PD1 antibody is a humanized antibody, a murine antibody or a chimeric antibody; Optionally, the anti-PD1 antibody comprises a constant region; Optionally, the heavy chain constant region of the antibody is selected from the heavy chain constant region of IgG1, IgG2, IgG3 and IgG4, and / or the light chain constant region of the antibody is selected from the κ or λ chain constant region; Optionally, the species origin of the constant region is mouse or human; Optionally, the heavy chain constant region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO.19, and / or the light chain constant region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO.20; Optionally, the heavy chain of the anti-PD1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO.17, and the light chain of the anti-PD1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO.18; Optionally, the anti-PD1 antibody comprises a heavy chain as shown in SEQ ID NO.17 and a light chain as shown in SEQ ID NO.

18.

8. The fusion protein according to any one of claims 1 to 7, wherein The IL10 mutant is directly or via a connecting peptide connected to the heavy chain or light chain of the anti-PD1 antibody; Optionally, the N-terminus of the IL10 mutant is connected to the C-terminus of the light chain of the anti-PD1 antibody via a connecting peptide; Optionally, the fusion protein comprises two identical first chains represented by formula P1 and two identical second chains represented by formula P2; P1: [Light chain of anti-PD1 antibody]-[Second connecting peptide]-[IL10 mutant], P2: heavy chain of anti-PD1 antibody; Optionally, in the formula P1, the second connecting peptide is a flexible connecting peptide; optionally, the amino acid sequence of the second connecting peptide is as shown in SEQ ID NO.53; Optionally, the fusion protein comprises a first chain of an amino acid sequence having at least 90% sequence identity with SEQ ID NO.54, and a second chain of an amino acid sequence having at least 90% sequence identity with SEQ ID NO.17; Optionally, the fusion protein includes two first chains of amino acid sequences as shown in SEQ ID NO.54, and two second chains of amino acid sequences as shown in SEQ ID NO.

17.

9. The fusion protein according to any one of claims 1 to 8, wherein the fusion protein has at least one of the following properties: A. The fusion protein can specifically bind to human PD1; optionally, the fusion protein can bind to human PD1 protein with an EC50 value of ≤10 nM, wherein: The EC50 value was determined by FACS method; B. the fusion protein has low or no blood toxicity; C. The fusion protein can enhance the killing activity of CD8+T cells; D. The fusion protein can inhibit tumor growth.

10. A nucleic acid encoding the fusion protein according to any one of claims 1 to 9. A vector comprising the nucleic acid according to claim 10.

12. A cell comprising the nucleic acid of claim 10 or the vector of claim 11.

13. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 9, the nucleic acid of claim 10, the vector of claim 11 or the cell of claim 12; optionally, further comprising one or more pharmaceutically acceptable carriers, diluents or excipients.

14. Use of the fusion protein according to any one of claims 1 to 9, the nucleic acid according to claim 10, the vector according to claim 11, the cell according to claim 12, or the pharmaceutical composition according to claim 13 in the preparation of a drug for treating or preventing tumors or inflammatory diseases; Optionally, the tumor is a tumor with high expression of PDL1; Optionally, the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma and hematological cancer.

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