Sushi domain mutant, fusion protein, and pharmaceutical composition and use

Abstract

The present invention relates to the field of biomedicine, relates to a Sushi domain mutant, a fusion protein, a pharmaceutical composition and a use. Specifically, the present invention relates to a Sushi domain mutant, which is a mutant of a wild-type Sushi domain, and comprises the following mutation: mutation of one or more sites selected from the first site, the second site, the 15th site, the 28th site, the 46th site, the 50th site, the 51th site, the 53th site, and the 55th site of the wild-type Sushi domain into water-soluble amino acids. The present invention also relates to a fusion protein comprising the Sushi domain mutant. The Sushi domain mutant or the fusion protein has higher stability and good biological activity, and has good application prospects.

Description

Sushi domain mutants, fusion proteins, pharmaceutical compositions and uses

[0001] This application claims priority to a Chinese patent application filed on August 4, 2023 (application number: CN202310983709.7, invention name: Sushi domain mutants, fusion proteins, pharmaceutical compositions and uses). The entire contents of the priority patent application are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biomedicine and relates to a Sushi domain mutant, a fusion protein, a pharmaceutical composition and uses. Background Art

[0003] Interleukin-15 (IL-15 or IL15) is a pleiotropic cytokine that bridges innate and adaptive immune processes. IL-15 is a type 1 quadruple α-helical cytokine. It forms receptor complexes with IL-15Rα, IL-2Rβ, and IL-2Rγ to stimulate the proliferation of activated T cells, generate cytotoxic effector T cells, and activate and maintain natural killer (NK) cells. It also promotes immunoglobulin synthesis induction by B cells and regulates lymphoid homeostasis.

[0004] The main mechanism of the IL-15 signaling pathway is that after IL-15 binds to the IL-15Rα subunit on the cell, IL-15 stays on the cell surface and forms an immune synapse with the IL-2R / IL-15Rβ-γc on nearby effector NK cells or T cells, activating the JAK1 / JAK3 and STAT3 / STAT5 pathways, Syk kinase and phospholipase C (PLC)γ, Lck kinase and Shc, leading to the activation of the PI3K / Akt and Ras / Raf / MAPK signaling cascades. These pathways lead to the subsequent expression of Bcl2, Myc, Fos / Jun and NFkB activation.

[0005] IL-15 has a wide range of immunomodulatory activities and can participate in regulating the survival, proliferation, and function of various immune cells, including NK cells, memory CD8+ T cells, NKT cells, and so on. Because IL-15 needs to first bind to IL-15Rα before it can bind to IL-2R / IL-15Rβ-γc to activate immune cells, the activity of IL-15 alone in activating immune cells is weak. In order to achieve better immune cell activation, IL-15 is combined with IL-15Rα to form a complex, and its immune cell stimulating activity is more than 100 times that of free IL-15 alone. Since free IL-15 has disadvantages such as a short half-life and limited in vivo activity, IL-15 has better biological activity after binding to IL-15Rα, so the complex of IL-15 and IL-15Rα is called a superagonist. Researchers have found that the sushi domain of IL-15Rα can form a superagonist complex with IL-15 without using the full-length IL-15Rα, and the resulting superagonist activity is comparable to that of the full-length IL-15Rα. Therefore, the complex formed by IL-15 and the sushi domain through covalent or non-covalent bonding (such as electrostatic forces) is also called an IL-15 superagonist.

[0006] Because IL-15 and its receptor IL-15Rα easily dissociate, the stability of IL-15 superagonists is relatively poor. Therefore, current IL-15 superagonists use a tandem approach to connect IL-15 and IL-15Rα using a linker, or use a bispecific antibody approach to construct IL-15 and IL-15Rα on the two chains of the Fc, forming a parallel structure. In addition, the solubility of IL-15 superagonists is poor. One manifestation is that severe precipitation will occur when the concentration exceeds a certain range during the production process; another manifestation is that the solubility of tumor-specific agonists composed of superagonists and other TAA antibodies will become even worse. At present, there is still a need to develop new IL-15 superagonists.

[0007] Summary of the Invention

[0008] Through in-depth research and creative work, the present inventors have developed a sushi domain mutant and, based on this, a fusion protein. The sushi domain mutant or fusion protein of the present invention exhibits enhanced stability and excellent biological activity, and has promising application prospects. The following invention is provided:

[0009] One aspect of the present invention relates to a sushi domain mutant, which is a mutant of a wild-type sushi domain and comprises the following mutations:

[0010] One or more positions selected from positions 1, 2, 15, 28, 46, 50, 51, 53, and 55 of the wild-type sushi domain are mutated to a water-soluble amino acid;

[0011] Optionally, the amino acid sequence of the wild-type sushi domain is shown in SEQ ID NO: 30;

[0012] Optionally, the water-soluble amino acid after each site mutation is independently selected from lysine, arginine, glutamine and asparagine.

[0013] In some embodiments of the present invention, the sushi domain mutant comprises the following mutation: the second position is mutated to lysine or arginine.

[0014] In some embodiments of the present invention, the sushi domain mutant comprises the following mutation: the second position is mutated to lysine.

[0015] In some embodiments of the present invention, the sushi domain mutant comprises the following mutations:

[0016] The second position is mutated to lysine or arginine; and

[0017] One, two, three, four, five or six sites selected from the following are mutated to lysine or arginine:

[0018] 15th, 28th, 50th, 51st, 53rd and 55th.

[0019] In some embodiments of the present invention, the sushi domain mutant comprises the following mutations:

[0020] Position 2 is mutated to lysine or arginine; and

[0021] Position 53 and / or position 55 is mutated to lysine or arginine.

[0022] In some embodiments of the present invention, the sushi domain mutant comprises the following mutations:

[0023] Positions 2 and 28 were mutated to lysine or arginine.

[0024] In some embodiments of the present invention, the sushi domain mutant comprises the following mutations:

[0025] Positions 2, 28, and 55 were mutated to lysine or arginine.

[0026] In some embodiments of the present invention, the sushi domain mutant comprises the following mutations:

[0027] Positions 2, 15, 28, and 55 were mutated to lysine or arginine.

[0028] In some embodiments of the present invention, the sushi domain mutant further comprises the following mutation:

[0029] Position 50 and / or position 51 is mutated to lysine or arginine.

[0030] In some embodiments of the present invention, the sushi domain mutant comprises the following mutations:

[0031] The 46th position is mutated to lysine or arginine; and

[0032] One or two of the following sites are mutated to lysine or arginine:

[0033] 51st and 53rd.

[0034] In some embodiments of the present invention, any of the aforementioned sushi domain mutants comprises the following mutations: amino acids at positions 2, 28, and 55 are mutated to lysine or arginine, and at least one (e.g., 1, 2, or 3) of amino acids at positions 15, 50, and 51 are mutated to lysine or arginine. In some embodiments, the sushi domain mutant comprises the following mutations: amino acids at positions 2 and 55 are mutated to lysine, amino acid at position 28 is mutated to arginine, and amino acids at positions 15, 50, and / or 51 are mutated to lysine. In some embodiments, the sushi domain mutant comprises the following mutations: amino acids at positions 2, 15, and 55 are mutated to lysine, amino acid at position 28 is mutated to arginine, and amino acids at positions 50 and / or 51 are mutated to lysine; in some embodiments, the sushi domain mutant comprises the following mutations: amino acids at positions 2, 15, 51, and 55 are mutated to lysine, and amino acid at position 28 is mutated to arginine; in some embodiments, the sushi domain mutant comprises the following mutations: amino acids at positions 2, 15, 50, and 55 are mutated to lysine, and amino acid at position 28 is mutated to arginine; in some embodiments, the sushi domain mutant comprises the following mutations: amino acids at positions 2, 15, 50, 51, and 55 are mutated to lysine, and amino acid at position 28 is mutated to arginine;

[0035] In some embodiments of the present invention, the sushi domain mutant comprises the following mutations: amino acid position 15 is mutated to lysine, and at least one (e.g., 1, 2, 3, 4, or 5) of amino acids at positions 2, 28, 50, 51, and 55 is mutated to lysine or arginine; in some embodiments, the sushi domain mutant comprises the following mutations: amino acids at positions 15, 2, and 55 are mutated to lysine, amino acid position 28 is mutated to arginine, and amino acids at positions 50 and / or 51 are mutated to lysine; in some embodiments, The sushi domain mutant comprises the following mutations: amino acids at positions 15, 2, 51, and 55 are mutated to lysine, and amino acid position 28 is mutated to arginine. In some embodiments, the sushi domain mutant comprises the following mutations: amino acids at positions 15, 2, 50, and 55 are mutated to lysine, and amino acid position 28 is mutated to arginine. In some embodiments, the sushi domain mutant comprises the following mutations: amino acids at positions 15, 2, 50, 51, and 55 are mutated to lysine, and amino acid position 28 is mutated to arginine.

[0036] In some embodiments of the present invention, the sushi domain mutant, except for the mutated amino acid positions described in any of the preceding items, has amino acids identical to those of the wild-type sushi domain or has only conservative substitutions (i.e., no deletions, additions, or non-conservative mutations); in certain embodiments, the amino acid sequence of the wild-type sushi domain is as shown in SEQ ID NO: 30. In certain embodiments, the sushi domain mutant, except for the mutated amino acid positions described in any of the preceding items, has amino acids identical to those of the wild-type sushi domain.

[0037] In some embodiments, the sushi domain mutant differs from the wild-type sushi domain in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more) amino acids. In some embodiments, the amino acid residues at which the sushi domain mutant differs from the wild-type sushi domain are amino acid residues 1, 2, 15, 28, 46, 50, 51, 53, and 55; in some embodiments, the amino acid residues at which the sushi domain mutant differs from the wild-type sushi domain (e.g., positions 1, 2, 15, 28, 46, 50, 51, 53, and / or 55) are mutated to water-soluble amino acids (e.g., selected from lysine, arginine, glutamine, or asparagine); in some embodiments, the amino acid residues at which the sushi domain mutant differs from the wild-type sushi domain are mutated to lysine or arginine. In some embodiments, the amino acid residue that differs between the sushi domain mutant and the wild-type sushi domain is the amino acid residue at the mutation site described in any one of the above items.

[0038] In some embodiments of the present invention, the amino acid sequence of the sushi domain mutant described in any of the above items is (SEQ ID NO: 44):

[0039] X1X2CPPPMSVEHADIX3VKSYSLYSRERYX4CNSGFKRKAGTSSLTECX5LNKX6X7NX8AX9WTTPSLKCIR, where X1 is I or R, X2 is T, K or Q, X3 is W or K, X4 is I or R, X5 is V or K, X6 is A or K, X7 is T or K, X8 is V or K, X9 is H or K, and X 1～ At least one amino acid residue of X9 is K, R or Q;

[0040] In some embodiments, in the amino acid sequence of any of the above sushi domain mutants, X2 is K;

[0041] In some embodiments, in the amino acid sequence of any of the above sushi domain mutants, X3 is K;

[0042] In some embodiments, in the amino acid sequence of any of the foregoing sushi domain mutants, X2 is K, X4 is R, and X9 is K;

[0043] In some embodiments, in the amino acid sequence of any of the preceding sushi domain mutants, X1 is I, X2 is K, X3 is W, X4 is R, X5 is V, X6 is A or K, X7 is T or K, X8 is V, and X9 is K;

[0044] In some embodiments, in the amino acid sequence of any of the preceding sushi domain mutants, X1 is I, X2 is K, X3 is K, X4 is R, X5 is V, X6 is A or K, X7 is T or K, X8 is V, and X9 is K;

[0045] In some embodiments, the amino acid sequence of the sushi domain mutant is such that X1 is I, X2 is K, X3 is W, X4 is I, X5 is V, X6 is A, X7 is T, X8 is K, and X9 is H;

[0046] In some embodiments, the amino acid sequence of the sushi domain mutant is such that X1 is I, X2 is K, X3 is W, X4 is R, X5 is V, X6 is A, X7 is T, X8 is V, and X9 is K;

[0047] In some embodiments, the amino acid sequence of the sushi domain mutant is such that X1 is I, X2 is K, X3 is K, X4 is R, X5 is V, X6 is A, X7 is T, X8 is V, and X9 is K;

[0048] In some embodiments, the amino acid sequence of the sushi domain mutant is such that X1 is I, X2 is K, X3 is K, X4 is R, X5 is V, X6 is A, X7 is K, X8 is V, and X9 is K;

[0049] In some embodiments, in the amino acid sequence of any of the foregoing sushi domain mutants, X1 is I, X2 is K, X3 is K, X4 is R, X5 is V, X6 is K, X7 is K, X8 is V, and X9 is K.

[0050] In some embodiments of the present invention, the amino acid sequence of the sushi domain mutant is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 13.

[0051] In some embodiments of the present invention, the sushi domain mutant has an amino acid sequence that is ≥80%, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥98.5% or ≥99% identical to any one of SEQ ID NO:1 to SEQ ID NO:13, and the sushi domain mutant has sushi domain function.

[0052] In some embodiments of the invention, the sushi domain mutant comprises SEQ ID NO: 13, SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 10, or SEQ ID NO: 3.

[0053] In some embodiments of the present invention, the amino acid sequence of the sushi domain mutant is shown in SEQ ID NO: 13;

[0054] In some embodiments of the present invention, the amino acid sequence of the sushi domain mutant is shown in SEQ ID NO: 12;

[0055] In some embodiments of the present invention, the amino acid sequence of the sushi domain mutant is shown in SEQ ID NO: 11;

[0056] In some embodiments of the present invention, the amino acid sequence of the sushi domain mutant is shown in SEQ ID NO: 10;

[0057] In some embodiments of the present invention, the amino acid sequence of the sushi domain mutant is shown in SEQ ID NO: 3.

[0058] Another aspect of the present invention relates to a fusion protein comprising the sushi domain mutant according to any one of the present invention and the Fc segment of IL-15 and / or human IgG.

[0059] In some embodiments of the present invention, the fusion protein, wherein the amino acid sequence of the IL-15 is as shown in SEQ ID NO: 29 or SEQ ID NO: 34.

[0060] In some embodiments of the present invention, the fusion protein, wherein the Fc segment of human IgG is the Fc segment of human IgG1, IgG2, IgG3 or IgG4;

[0061] Optionally, the amino acid sequence of the Fc segment of human IgG is shown in SEQ ID NO: 33.

[0062] In some embodiments of the present invention, the fusion protein, wherein the Fc segment of the human IgG comprises a LALA mutation;

[0063] Optionally, the amino acid sequence of the Fc segment of human IgG is shown in SEQ ID NO: 31.

[0064] In some embodiments of the present invention, the fusion protein comprises IL-15, a sushi domain mutant, and the Fc segment of human IgG in sequence from the N-terminus.

[0065] In some embodiments of the present invention, the fusion protein, wherein IL-15 and the sushi domain mutant, and / or the sushi domain mutant and the Fc segment of human IgG are directly connected or connected through a connecting peptide; optionally, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO:32.

[0066] In some embodiments of the present invention, the fusion protein comprises IL-15, a connecting peptide, a sushi domain mutant and the Fc segment of human IgG in order from the N-terminus.

[0067] In some embodiments of the present invention, the fusion protein, wherein the amino acid sequence of the fusion protein is shown in any one of SEQ ID NO: 14 to SEQ ID NO: 26.

[0068] In some embodiments of the present invention, the amino acid sequence of the fusion protein is a sequence that has ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥98.5%, ≥99%, ≥99.3%, ≥99.5% or ≥99.7% identity with any one of SEQ ID NO:14 to SEQ ID NO:26.

[0069] In some embodiments of the present invention, in the fusion protein, the sushi domain mutant and IL-15 are on the same peptide chain or on different peptide chains.

[0070] In some embodiments of the present invention, the fusion protein, when the sushi domain mutant and IL-15 are on different peptide chains, is linked via one or more disulfide bonds.

[0071] In some embodiments of the present invention, in the fusion protein, when the sushi domain mutant and IL-15 are on the same peptide chain, the sushi domain mutant and IL-15 are directly connected or connected through a connecting peptide.

[0072] In some embodiments of the present invention, the fusion protein further comprises a protein functional region targeting a tumor-associated antigen and / or targeting an immune checkpoint;

[0073] Optionally, the tumor-associated antigen is selected from one or more of PD-L1, CD19, CD20, EGFR, Claudin18.2, BCMA, HER2, CD19, CD20 and Nectin-4;

[0074] Optionally, the immune checkpoint is selected from one or more of PD-1, CTLA-4, Lag-3 and Tim-3.

[0075] In some embodiments of the present invention, the fusion protein, wherein the protein functional region is an antibody or an antigen-binding fragment thereof, such as a VHH, scFv, Fv fragment, Fab fragment, or F(ab')2 fragment. In some embodiments of the present invention, the fusion protein is in the form of a monoclonal antibody or a bispecific antibody.

[0076] In some embodiments of the present invention, the fusion protein comprises four polypeptide chains:

[0077] Chain 1: From N-terminus to C-terminus: antibody heavy chain-connector peptide-sushi domain mutant-connector peptide-IL15,

[0078] Chain 2: Antibody heavy chain,

[0079] Chains 3 and 4: antibody light chains;

[0080] In some embodiments of the present invention, the antibody is an anti-PD-1 antibody;

[0081] In some embodiments of the invention, the HCDR1, HCDR2 and HCDR3 of the antibody are identical to the CDRs in SEQ ID NO:42, and the LCDR1, LCDR2 and LCDR3 of the antibody are identical to the CDRs in SEQ ID NO:43.

[0082] In some embodiments of the invention, the heavy chain variable region (VH) of the antibody has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity or is identical to the VH in SEQ ID NO:42, and the light chain variable region (VL) of the antibody has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity or is identical to the VL in SEQ ID NO:43.

[0083] In certain embodiments, the antibodies of any of the foregoing, the VH / VL, and the three CDRs (HCDR1, HCDR2, and HCDR3) contained in the VH and the three CDRs (LCDR1, LCDR2, and LCDR3) contained in the VL are defined by the Kabat, IMGT, Chothia, Contact, or Abm numbering systems. In certain embodiments, the three CDRs (HCDR1, HCDR2, and HCDR3) contained in the VH and the three CDRs (LCDR1, LCDR2, and LCDR3) contained in the VL are defined by the Kabat numbering system.

[0084] In some embodiments of the invention, the fusion protein comprises four polypeptide chains: chain 1 has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical or identical to SEQ ID NO: 37, 38, 39, 40 or 41, chain 2 has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical or identical to SEQ ID NO: 42, and chain 3 and chain 4 have an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical or identical to SEQ ID NO: NO:43 has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity or is identical.

[0085] In some embodiments of the present invention, the sushi domain mutant or fusion protein described in any of the above items has at least one of the following functions:

[0086] A) has the function of binding to cells expressing IL15 receptors; optionally, it can bind to cells expressing IL15 receptors with an EC value of less than 10 nM (e.g., less than 10 nM, less than 5 nM, less than 1 nM, less than 0.8 nM, less than 0.5 nM, less than 0.4 nM or less). 50 Value in combination with KARPASS-299; Optionally, the EC 50 The values ​​were determined by flow cytometry fluorescence sorting technology;

[0087] B) having the function of activating NK cells; optionally, the activated NK cell function is detected by flow cytometry;

[0088] C) having good thermal stability; optionally, the sushi domain mutant or fusion protein is placed in a 40° C. water bath for 2 weeks, and the percentage of fusion protein aggregates is less than 50% (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 17% or less);

[0089] D) having good charge stability; optionally, the sushi domain mutant or fusion protein exhibits less charge heterogeneity than a protein comprising a wild-type sushi domain when placed in a 40°C water bath for 2 weeks; optionally, the charge heterogeneity change is determined by whole-column imaging capillary isoelectric focusing electrophoresis analysis;

[0090] E) having a high yield; optionally, the sushi domain mutant or fusion protein is expressed using an ExpiCHO cell expression system, and the protein expression level is greater than 30 mg / L (e.g., greater than 30 mg / L, greater than 35 mg / L, greater than 40 mg / L, greater than 45 mg / L, greater than 50 mg / L or greater); and / or

[0091] F) having high purity; optionally, the sushi domain mutant or fusion protein is expressed using the ExpiCHO cell expression system, and after two purifications using MabSelect PrismA and HiLoad 16 / 600 Superdex 200pg purification columns, the protein purity is greater than 40% (e.g., greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75% or greater).

[0092] The sushi domain mutant according to any one of the present invention or the fusion protein according to any one of the present invention is used for treating or preventing tumors, immunodeficiency or infectious diseases;

[0093] Optionally, the tumor is one or more selected from melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancies;

[0094] Optionally, the tumor is an advanced solid tumor;

[0095] Optionally, the tumor is an advanced tumor selected from one or more tumors selected from melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancies;

[0096] Optionally, the immunodeficiency is an immunodeficiency induced by a side effect of a specific treatment such as anti-tumor therapy or pre-transplantation therapy;

[0097] Optionally, the immunodeficiency is HIV-induced immunodeficiency;

[0098] Optionally, the infectious disease is a disease caused by viral, bacterial, yeast or fungal infection.

[0099] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the sushi domain mutant according to any one of the present invention, or encoding the fusion protein according to any one of the present invention.

[0100] Another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention; optionally, the recombinant vector is a recombinant expression vector.

[0101] Another aspect of the present invention relates to a recombinant host cell comprising the isolated nucleic acid molecule of the present invention, or comprising the recombinant vector of the present invention.

[0102] Another aspect of the present invention relates to a pharmaceutical composition comprising the sushi domain mutant according to any one of the present invention, or the fusion protein according to any one of the present invention, and one or more pharmaceutically acceptable excipients.

[0103] The pharmaceutical composition according to any one of the present invention is used for treating or preventing tumors, immunodeficiency or infectious diseases;

[0104] Optionally, the tumor is one or more selected from melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancies;

[0105] Optionally, the tumor is an advanced solid tumor;

[0106] Optionally, the tumor is an advanced tumor selected from one or more tumors selected from melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancies;

[0107] Optionally, the immunodeficiency is an immunodeficiency induced by a side effect of a specific treatment such as anti-tumor therapy or pre-transplantation therapy;

[0108] Optionally, the immunodeficiency is HIV-induced immunodeficiency;

[0109] Optionally, the infectious disease is a disease caused by viral, bacterial, yeast or fungal infection.

[0110] Another aspect of the present invention relates to use of any one of the sushi domain mutants, any one of the fusion proteins, or the pharmaceutical composition of the present invention in the preparation of a medicament for treating or preventing tumors, immunodeficiency, or infectious diseases.

[0111] Optionally, the tumor is one or more selected from melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancies;

[0112] Optionally, the tumor is an advanced solid tumor;

[0113] Optionally, the tumor is an advanced tumor selected from one or more tumors selected from melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancies;

[0114] Optionally, the immunodeficiency is an immunodeficiency induced by a side effect of a specific treatment such as anti-tumor therapy or pre-transplantation therapy;

[0115] Optionally, the immunodeficiency is HIV-induced immunodeficiency;

[0116] Optionally, the infectious disease is a disease caused by viral, bacterial, yeast or fungal infection.

[0117] Another aspect of the present invention relates to a method for treating or preventing tumors, immunodeficiency or infectious diseases, comprising the step of administering to a subject in need thereof an effective amount of any one of the sushi domain mutants of the present invention, any one of the fusion proteins of the present invention, or the pharmaceutical composition of the present invention;

[0118] Optionally, the tumor is one or more selected from melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancies;

[0119] Optionally, the tumor is an advanced solid tumor;

[0120] Optionally, the tumor is an advanced tumor selected from one or more tumors selected from melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancies;

[0121] Optionally, the immunodeficiency is an immunodeficiency induced by a side effect of a specific treatment such as anti-tumor therapy or pre-transplantation therapy;

[0122] Optionally, the immunodeficiency is HIV-induced immunodeficiency;

[0123] Optionally, the infectious disease is a disease caused by viral, bacterial, yeast or fungal infection.

[0124] Another aspect of the present invention relates to the use of any of the above-mentioned sushi domain mutants, fusion proteins, nucleic acid molecules, recombinant vectors, recombinant host cells, or pharmaceutical compositions in the preparation of drugs for treating or preventing tumors, immunodeficiency or infectious diseases;

[0125] Another aspect of the present invention relates to any of the above-mentioned sushi domain mutants, fusion proteins, nucleic acid molecules, recombinant vectors, recombinant host cells, or pharmaceutical compositions for use as a medicament for treating or preventing tumors, immunodeficiency or infectious diseases;

[0126] Another aspect of the present invention relates to a method for treating or preventing tumors, immunodeficiency or infectious diseases, comprising administering to a subject in need thereof a therapeutically effective amount of any of the foregoing sushi domain mutants, fusion proteins, nucleic acid molecules, recombinant vectors, recombinant host cells, or pharmaceutical compositions;

[0127] In some embodiments, the tumor is one or more selected from melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor, and hematological malignancies;

[0128] In some embodiments, the tumor is an advanced solid tumor;

[0129] In some embodiments, the tumor is an advanced stage tumor selected from one or more tumors of melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor, and hematological malignancies;

[0130] In some embodiments, the immunodeficiency is an immunodeficiency induced by a side effect of a specific treatment such as anti-tumor therapy or pre-transplant therapy;

[0131] In some embodiments, the immunodeficiency is HIV-induced immunodeficiency;

[0132] In some embodiments, the infectious disease is a disease caused by a viral, bacterial, yeast, or fungal infection.

[0133] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0134] As used herein, when referring to the amino acid sequence of IL-15 (interleukin-15), it includes but is not limited to the full-length IL-15 protein, or a full-length fragment containing the IL-15 protein; it also includes fusion proteins containing IL-15, such as fragments fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally produced or artificially introduced into the amino acid sequence of IL-15 without affecting its biological function. Therefore, in the present invention, the term "IL-15" should include all such sequences, including their natural or artificial variants. In addition, when describing a sequence fragment of IL-15, it also includes the corresponding sequence fragments in its natural or artificial variants.

[0135] As used herein, when referring to the amino acid sequence of IL-15Rα (interleukin-15 receptor α) or the sushi domain, it includes but is not limited to the full length of the IL-15Rα protein, or the extracellular fragment ECD of IL-15Rα or a fragment comprising the IL-15Rα ECD, such as the sushi domain; it also includes fusion proteins of the IL-15Rα ECD or sushi domain, such as fragments fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally or artificially introduced into the amino acid sequence of IL-15Rα or the sushi domain without affecting its biological function. Therefore, in the present invention, the term "IL-15Rα" or "sushi domain" should include all such sequences, including natural or artificial variants thereof. Furthermore, when describing a sequence fragment of IL-15Rα or the sushi domain, it also includes the corresponding sequence fragments in its natural or artificial variants.

[0136] As used herein, the terms "Fc," "Fc segment," or "Fc fragment" are also referred to as a crystallizable fragment. Generally, an Fc segment comprises domain 2 (CH2) and domain 3 (CH3) of the heavy chain constant region. In some embodiments of the present invention, the Fc segment is that of human IgG. In some embodiments of the present invention, the Fc segment of human IgG is that of human IgG1.

[0137] As used herein, the term "LALA" or "LALA mutation refers to" the L234A mutation and the L235A mutation on the heavy chain constant region or Fc segment according to the EU numbering system.

[0138] In the present invention, unless otherwise specified, the letters before the position represent the amino acid before the mutation, and the letters after the position represent the amino acid after the mutation. For example, "L234A" means that the amino acid residue at position 234 is mutated from "L" to "A".

[0139] As used herein, the term "linker" refers to a peptide segment used to connect two proteins or polypeptides when constructing a fusion protein. Linkers include flexible linkers and rigid linkers. Flexible linkers typically contain small amino acids, non-polar amino acids such as Gly, and polar amino acids such as Ser and Thr. These smaller amino acids provide flexibility to the linker, allowing the two connected proteins to have a certain degree of mobility. Furthermore, the addition of Ser and Thr allows the linker to form hydrogen bonds with water molecules, giving the linker stability in aqueous solution, thereby reducing the interaction between the linker and the two proteins before and after.

[0140] Currently, the most common flexible linker peptides consist of Gly and Ser residues ("GS" linkers). The most widely used flexible linker peptide sequence is (GGGGS)n. By adjusting the value (number of repeats) of n, the length of the GS linker peptide can be varied to optimally connect two proteins or enable their interaction. In some embodiments of the present invention, the linker peptide comprises the amino acid sequence (GGGGS)n, where n is selected from 1, 2, 3, 4, 5, and 6.

[0141] As used herein, the term EC 50 It refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximum effect.

[0142] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the site where the antibody binds to the antigen.The assignment of amino acids to regions or domains follows the Kabat numbering system (Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991))), or the Chothia numbering system (Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883), or the IMGT numbering system (see Ehrenmann F, Kaas Q, Lefranc MP. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF [J]. Nucleic acids research, 2009; 38 (suppl_1): D301-D307), or the Abm numbering system (Martin et al. ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272), or Contact numbering system definition (MacCallum, RM, Martin, ACR, & Thornton, JM (1996). Antibody-antigen Interactions: Contact Analysis and Binding Site Topography. Journal of Molecular Biology, 262(5),732-745.).

[0143] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0144] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or an antibody fragment from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for possible spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies and usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256 (5517): 495), but it can also be obtained by using recombinant DNA technology (see, for example, US Patent 4,816,567).

[0145] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of an antibody can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to a polypeptide.

[0146] As used herein, the term "Fd fragment" means a fragment consisting of H and C H The term "Fv fragment" refers to an antibody fragment consisting of a V domain of a single arm of an antibody. L and V H The term "dAb fragment" refers to an antibody fragment consisting of a V H domains (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of V L 、V H 、CL and C H The term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

[0147] As used herein, the term "single chain antibody (ScFv)" refers to a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) connected by a linker. The VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain (see, e.g., Bird et al, Science 1988; 242: 423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85: 5879-5883). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al, Protein Eng. 1995; 8: 725-731, Choi et al, Eur. J. Immunol. 2001; 31: 94-106, Hu et al, Cancer Res. 1996; 56: 3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293: 41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1): 51-59.

[0148] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning and refer to cloning the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only a single heavy chain variable region. This is the smallest antigen-binding fragment with complete function. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only a single heavy chain variable region.

[0149] As used herein, the term "isolated" or "isolated" refers to something that is obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and a highly pure identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0150] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0151] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0152] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0153] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0154] Advantageous Effects of the Invention

[0155] In some embodiments, the present invention achieves one or more of the following technical effects:

[0156] (1) The sushi domain mutant of the present invention or the fusion protein of the present invention has improved stability.

[0157] (2) The sushi domain mutant or the fusion protein of the present invention maintains good activity.

[0158] (3) The sushi domain mutant or the fusion protein of the present invention has a higher yield.

[0159] (4) The sushi domain mutant or the fusion protein of the present invention has higher purity.

[0160] In some embodiments, the sushi domain mutant or fusion protein of the present invention has at least one of the following functions:

[0161] A) has the function of binding to cells expressing IL15 receptors; optionally, it can bind to cells expressing IL15 receptors with an EC value of less than 10 nM (e.g., less than 10 nM, less than 5 nM, less than 1 nM, less than 0.8 nM, less than 0.5 nM, less than 0.4 nM or less). 50 Value in combination with KARPASS-299; Optionally, the EC 50 The values ​​were determined by flow cytometry fluorescence sorting technology;

[0162] B) having the function of activating NK cells; optionally, the activated NK cell function is detected by flow cytometry;

[0163] C) having good thermal stability; optionally, the sushi domain mutant or fusion protein is placed in a 40° C. water bath for 2 weeks, and the percentage of fusion protein aggregates is less than 50% (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 17% or less);

[0164] D) having good charge stability; optionally, the sushi domain mutant or fusion protein exhibits less charge heterogeneity than a protein comprising a wild-type sushi domain when placed in a 40°C water bath for 2 weeks; optionally, the charge heterogeneity change is determined by whole-column imaging capillary isoelectric focusing electrophoresis analysis;

[0165] E) having a high yield; optionally, the sushi domain mutant or fusion protein is expressed using an ExpiCHO cell expression system, and the protein expression level is greater than 30 mg / L (e.g., greater than 30 mg / L, greater than 35 mg / L, greater than 40 mg / L, greater than 45 mg / L, greater than 50 mg / L or greater); and / or

[0166] F) having high purity; optionally, the sushi domain mutant or fusion protein is expressed using the ExpiCHO cell expression system, and after two purifications using MabSelect PrismA and HiLoad 16 / 600 Superdex 200pg purification columns, the protein purity is greater than 40% (e.g., greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75% or greater). BRIEF DESCRIPTION OF THE DRAWINGS

[0167] Figure 1: Comparison of fusion protein expression yield and purity after primary purification.

[0168] Figure 2: Binding of fusion protein to KARPAS-299 cells.

[0169] Figure 3: Test results of the fusion protein's ability to activate NK cells.

[0170] Figure 4: Aggregate growth of the fusion protein after treatment at 40°C for 2 weeks.

[0171] Figure 5: Changes in iCIEF after treatment with N-803-linker fusion protein at 40°C for 2 weeks.

[0172] Figure 6: Changes in iCIEF after treatment with sushi-Fc-WT fusion protein at 40°C for 2 weeks.

[0173] Figure 7: Changes in iCIEF after treatment of sushi-T2K-V53K fusion protein at 40°C for 2 weeks.

[0174] Figure 8: Changes in iCIEF after treatment with sushi-T2K-I28R-H55K fusion protein at 40°C for 2 weeks.

[0175] Figure 9: Changes in iCIEF after treatment with sushi-T2K-W15K-I28R-H55K fusion protein at 40°C for 2 weeks.

[0176] Figure 10: Changes in iCIEF after treatment with sushi-T2K-W15K-I28R-T51K-H55K fusion protein at 40°C for 2 weeks.

[0177] Figure 11: Changes in iCIEF after treatment with sushi-T2K-W15K-I28R-A50K-T51K-H55K fusion protein at 40°C for 2 weeks;

[0178] Figure 12: Result of the fusion protein binding assay to KapaaS299 receptor cells;

[0179] Figure 13: Result of fusion protein NFAT luciferase reporter experiment. DETAILED DESCRIPTION

[0180] The partial sequences involved in the present invention are shown in Table A below.

[0181] Table A: Partial sequences of the present invention

[0182] In Table A: the mutation sites of the sushi domain are marked with boxes; the sequence of the sushi domain in the fusion protein is marked with a single underline.

[0183] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0184] Preparation Example 1: Design of sushi domain mutants

[0185] The present inventors designed mutants of IL-15 receptor Sushi domain_31-95_human (SEQ ID NO:30) as shown in SEQ ID NO:1 to SEQ ID NO:13. Specific amino acid sequences and mutant nomenclatures are detailed in Table A above.

[0186] Preparation Example 2: Design, preparation and purification of fusion protein

[0187] Fusion protein sample design: 13 fusion proteins shown in SEQ ID NO: 14 to SEQ ID NO: 26 were designed, and their structural compositions are: human IL-15 (SEQ ID NO: 29), G4S Linker (SEQ ID NO: 32), sushi domain mutant (selected from SEQ ID NO: 1 to SEQ ID NO: 13), and human IgG1Fc-LALA (SEQ ID NO: 31).

[0188] N-803 control sample: shown in SEQ ID NO: 27. Its structural composition is: human IL-15 mutant (SEQ ID NO: 34), G4S Linker (SEQ ID NO: 32), wild-type sushi domain (SEQ ID NO: 30), and human IgG1 Fc-LALA (SEQ ID NO: 31).

[0189] Wild-type control sample: shown in SEQ ID NO: 28. Its structural composition is: human IL-15 (SEQ ID NO: 29), G4S Linker (SEQ ID NO: 32), wild-type sushi domain (SEQ ID NO: 30), and human IgG1 Fc-LALA (SEQ ID NO: 31).

[0190] The 13 fusion protein samples and two control samples were expressed using the ExpiCHO cell expression system. The expression and purification steps are as follows:

[0191] The pcDNA3.4 vector was used as a dedicated vector for expressing the fusion proteins. Nucleotide sequences for various fusion proteins were obtained by gene synthesis. The vectors were double-digested with Hind III and Xho I, recovered, and seamlessly cloned using DNA homologous recombination enzymes. The clones were then transformed into competent Escherichia coli DH5a cells. Positive clones were selected and subjected to plasmid extraction and sequencing verification. Plasmids were then extracted and transfected separately. The day before transfection, suspension-cultured ExpiCHO cells were cultured in serum-free ExpiCHO expression medium (Gibco). On the day of transfection, the cell density was adjusted to 6 × 10 cells per ml using fresh ExpiCHO expression medium. 6 At a cell concentration of 1 μg of plasmid DNA, 3.2 μl of ExpiFectamine, and 76.8 μl of OptiPROSFM medium were added per ml of culture medium and then added to ExpiCHO cells. Feed was added 18-22 hours after transfection. After 8-10 days of culture, the supernatant was collected and purified separately. Purification was performed on an AKTA pure protein purifier and included the following two steps:

[0192] (1) First, the affinity purification column MabSelect PrismA was used for the first step purification, and the purity of the protein after the first step purification was evaluated by SEC-HPLC. 13 fusion protein samples and 2 control samples were obtained after the first step purification.

[0193] (2) The second step of purification was performed using a molecular sieve purification column HiLoad 16 / 600 Superdex 200 pg. 13 fusion protein samples and 2 control samples were obtained after the second step of purification.

[0194] Experimental Example 1: Protein Purity and Endotoxin Detection

[0195] 1. Experimental samples

[0196] 13 fusion protein samples and 2 control samples obtained after the first purification step in step (1) of Preparation Example 2;

[0197] 13 fusion protein samples and 2 control samples after the second purification step obtained in step (2) of the above-mentioned Preparation Example 2.

[0198] 2. Experimental Methods

[0199] use The protein concentration (sample prepared in step (1) of Preparation Example 2) was determined by absorbance at 280 nm using N60 (IMPLEN), and the protein purity was evaluated by SDS-PAGE and SEC-HPLC.

[0200] Endotoxin detection was performed using a recombinant Factor C endotoxin detection kit (sample prepared in step (2) of Preparation Example 2).

[0201] 3. Experimental Results

[0202] SDS-PAGE and SEC-HPLC assays showed that the obtained fusion proteins had the correct molecular weight and a purity greater than 95%. Specific results are shown in Table 1 and Figure 1 below.

[0203] Table 1

[0204] In Table 1: a Indicates that the yield is significantly improved relative to the control sample; b Indicates that the purity is significantly improved compared to the control sample.

[0205] The results show:

[0206] Compared with the N-803 control sample and the wild-type control sample, the yields of some fusion proteins containing sushi domain mutants (such as sushi-T2K-W15K-I28R-H55K fusion protein, sushi-T2K-W15K-I28R-T51K-H55K fusion protein, and sushi-T2K-W15K-I28R-A50K-T51K-H55K fusion protein) were significantly improved;

[0207] Compared with the N-803 control sample and the wild-type control sample, the purity of some fusion proteins containing sushi domain mutants (such as sushi-T2K-T51K-V53K fusion protein, sushi-T2K-I28R-H55K fusion protein, sushi-T2K-W15K-I28R-H55K fusion protein, sushi-T2K-W15K-I28R-T51K-H55K fusion protein and sushi-T2K-W15K-I28R-A50K-T51K-H55K fusion protein) was significantly improved.

[0208] In addition, endotoxin testing confirmed that the endotoxin level was less than 1EU mg -1 , meeting the standards of activity experiments and physical and chemical analysis experiments.

[0209] Experimental Example 2: Melting Temperature (Tm) Determination Experiment

[0210] 1. Experimental samples:

[0211] 13 fusion protein samples and 2 control samples after the second purification step obtained in step (2) of the above-mentioned Preparation Example 2.

[0212] 2. Experimental Methods

[0213] Determine the melting temperature (Tm). The method is as follows:

[0214] The protein melting temperature was calculated by measuring the change in fluorescent dye signal during protein heating using a fluorescence quantitative PCR instrument. 58.5 μL of sample (1.0 mg / mL) was mixed with 1.5 μL of 200× SYPRO orange solution (Sigma-Aldrich / S5692, 5000×, diluted to 200× with sample buffer before sample preparation). 25 μL of the mixture was then assayed in duplicate. Experimental parameters: Real-time fluorescence quantitative PCR instrument (Thermo Fisher scientific / QuantStudio 6Pro), assay temperature: 25°C to 99°C, ramp rate: 0.05°C / s. Analysis was performed using Protein Thermal Shift software (Thermo Fisher scientific).

[0215] 3. Experimental Results

[0216] The results are shown in Table 2 below.

[0217] Table 2

[0218] Note: In the table, Tm1 corresponds to the Tm of CH2 in the sample, Tm2 corresponds to the Tm of sushi in the sample, and "NA" means that Tm2 and Tm1 are similar and are merged into the same Tm.

[0219] The results showed that the fusion proteins of the present application had thermal stability comparable to or even better than that of the control samples. For example, the Tm2 values ​​of the sushi-T2K-W15K-I28R-H55K fusion protein, sushi-T2K-W15K-I28R-T51K-H55K fusion protein, and sushi-T2K-W15K-I28R-A50K-T51K-H55K fusion protein exceeded 82°C; the Tm2 values ​​of the sushi-V53K fusion protein, sushi-T2K-V53K fusion protein, and sushi-V46K-V53K fusion protein exceeded 80°C, which was significantly different from Tm1.

[0220] Experimental Example 3: Detection of Receptor Binding Ability of Fusion Proteins Containing Sushi Domain Mutants (Activity Detection Experiment 1)

[0221] 1. Experimental samples:

[0222] 13 fusion protein samples and 2 control samples after the second purification step obtained in step (2) of the previous Preparation Example 2.

[0223] 2. Experimental Methods

[0224] The ability of the fusion protein containing the sushi domain mutant to bind to the receptor cells was detected by flow cytometry. The detection steps are as follows:

[0225] Prepare FACS buffer by adding 2% FBS to PBS and mix well. Centrifuge the recipient cells KARPASS-299 (Nanjing Kebai, CBP60271) at 300g for 5 minutes, remove the culture medium, resuspend the cells in FACS buffer, count the cells, and adjust the cell density to 2x10 6 Cells / mL were added to a V-bottom 96-well plate at 50 μL / well of the cell suspension. Fusion protein samples were added to a final concentration of 100 nM and diluted 5-fold into 8 concentration gradients, with concentrations of 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, and 0.00128 nM, respectively. The cells and samples were reacted in a 4 ° C refrigerator for 1 hour. Centrifuge at 300 g for 5 minutes, remove the supernatant, wash the cells once with FACS buffer, and centrifuge again to remove the supernatant. 100 μL of 1:500 diluted fluorescent secondary antibody (Alexa Fluor 647goat anti-human IgG Fc specific, Jackson, 20221222). Incubate at 4°C in the dark for 30 minutes. Centrifuge at 300g for 5 minutes, remove the supernatant, wash twice with FACS buffer, and resuspend the cells in 50 μL of FACS buffer. Fluorescence shift was measured using a flow cytometer (iQue3 Plus).

[0226] 3. Experimental Results

[0227] The test results are shown in Figure 2 and Table 3 below.

[0228] Table 3

[0229] Note: EC 50 The unit is nM; fold = FACS reading at the highest concentration / FACS reading at the lowest concentration.

[0230] The results showed that the fusion protein containing the sushi domain mutant of the present application did not have reduced binding to receptor cells compared with the control sample, indicating that the mutation of the sushi domain mutant of the present application did not have a negative impact on the binding activity of the fusion protein containing it to the receptor cells.

[0231] Experimental Example 4: Detection of the NK cell activation ability of fusion proteins containing sushi domain mutants (Activity Detection Experiment 2)

[0232] 1. Experimental samples:

[0233] 13 fusion protein samples and 2 control samples after the second purification step obtained in step (2) of the above-mentioned Preparation Example 2.

[0234] 2. Experimental Methods

[0235] The activation ability of fusion proteins containing sushi domain mutants on primary NK cells was detected by flow cytometry. The experimental steps are as follows.

[0236] Resuscitated PBMC (SAILYBIO, XW0102052W) and the PBMC density was adjusted to 5x10 6 PBMC cell suspension was added to a U-bottom 96-well plate at 50 μL / well. The fusion protein to be tested was then added to a final concentration of 1 nM and 0.01 nM. The cell culture plate was placed in a cell culture incubator at 37°C and 5% CO2 and incubated overnight. The next day, K562 cells were harvested and the cell density was adjusted to 1x10 5 cells / mL. Add 100 μL / well of the K562 cell suspension to the cell culture plate that has been incubated overnight. Mix gently and continue incubation in the cell culture incubator for 4 hours. Centrifuge at 500 g for 5 minutes, remove the supernatant, and wash the cells once with FACS buffer. Add Fc blocker (Human TruStain Fix, Biolegend, 422302) and incubate at room temperature for 10 minutes to block cell surface Fc receptors. LIVE / DEAD staining reagent (Violet fluorescent reactive Dye, Invitrogen, L34964A) was added to distinguish live and dead cells. Fluorescently labeled CD3 antibodies (APC / Cyanine7 anti-human CD3 Antibody, Biolegend, 300318), fluorescently labeled CD56 antibodies (APC anti-humanCD56, Biolegend, 362504), and fluorescently labeled CD107a antibodies (PE anti-human CD107a (LAMP-1) Antibody, Biolegend, 328608) were added and incubated in a dark refrigerator at 4°C for 30 minutes. The cells were centrifuged at 500 g for 5 minutes, the supernatant removed, and the cells were washed twice with 200 μL of FACS buffer per well. The cells were then resuspended in 150 μL of FACS buffer. Fluorescence shift was measured using a flow cytometer (BD FACSCelesta). The percentage of CD107a+ cells within the CD3-CD56+ population was counted.

[0237] 3. Experimental Results

[0238] The results are shown in Figure 3 and Table 4 below.

[0239] Table 4

[0240] The results showed that compared with the control sample, the fusion protein containing the sushi domain mutant of the present application did not reduce the activation of NK cells, indicating that the mutation of the sushi domain mutant of the present application did not have a negative impact on the NK cell activation activity of the fusion protein containing it.

[0241] Experimental Example 5: Long-term stability test

[0242] 1. Experimental samples:

[0243] The fusion protein sample after the second purification obtained in step (2) of the above-mentioned Preparation Example 2 and two control samples.

[0244] 2. Experimental Methods

[0245] Long-term stability tests were conducted on some fusion proteins containing sushi domain mutants. Each sample was placed at 40°C for two weeks (14 days) and the changes in the sample properties were measured. The specific experimental steps are as follows:

[0246] On day 1, divide each sample into two aliquots, each containing 150 μg, as the T0 and 2W samples. Place the T0 sample at 4°C, and the 2W sample in a 40°C water bath. On day 14, remove the 2W sample from the 40°C water bath and place it at 4°C for testing.

[0247] First, the sample was analyzed by size exclusion chromatography (SEC). SEC is a liquid chromatography method that separates molecules based on their hydrodynamic radius. It is mainly used to detect the purity of large molecular proteins. TM Isocratic elution was performed using a PREMIER (1.7 μm, 4.6 × 150 mm) and a Waters ACQUITY UPLC Protein BEH SEC column (1.7 μm, 4.6 × 150 mm). Elution was performed with a mobile phase of 50 mM PB (pH 6.8) and 300 mM NaCl. The flow rate was 0.4 mL / min, the detection wavelength was 280 nm, and the column temperature was 30°C. A 10 μg protein injection volume was used. Antibody purity was determined using the area normalization method.

[0248] 3. Experimental Results

[0249] The results are shown in Figure 4 and Table 5 below.

[0250] Table 5: Aggregate changes

[0251] HMWs represents the peak of fusion protein aggregates. The results show that after two weeks of storage, the aggregates formed by the fusion protein comprising the sushi domain mutant of the present invention are much smaller than those of the wild type.

[0252] LMWs represent the peaks produced by degradation of the fusion protein. The results show that after two weeks of storage, the degradation of the fusion protein comprising the sushi domain mutant of the present invention is much less than that of the wild type.

[0253] The Main Peak represents the peak produced by the active ingredient (the sample itself). The results show that after two weeks of storage, the proportion of active ingredients in the fusion protein comprising the sushi domain mutant of the present invention is much higher than that of the wild type.

[0254] Experimental Example 6: Whole-column imaging capillary isoelectric focusing (iCIEF) analysis

[0255] 1. Experimental samples:

[0256] The fusion protein sample after the second purification obtained in step (2) of the above-mentioned Preparation Example 2 and two control samples.

[0257] 2. Experimental Methods

[0258] The samples were analyzed by whole-column imaging capillary isoelectric focusing (iCIEF). iCIEF separates protein variants with different charge according to their isoelectric point (pI) characteristics by capillary electrophoresis, determines the pI of each protein variant, and calculates the relative percentage.

[0259] Sample preparation: First, prepare a premix containing the following components: 0.5 μL each of pI marker 4.65 (ProteinSimple / 102223) and pI marker 8.18 (ProteinSimple / 102408), 4.0 μL of Pharmalyte 3-10 (GE Healthcare / 17-0456-01), 35 μL of 1% methylcellulose solution (ProteinSimple / 101876), 37.5 μL of 8 M urea, and 2.5 μL of ultrapure water. Exchange the sample solution with ultrapure water to a final concentration of 1.0 mg / mL. Mix 20 μL of the exchanged sample with 80 μL of the premix solution and centrifuge at 13,000 rpm for 1 minute. Transfer 95 μL of the supernatant to a 96-well plate and centrifuge at 3,000 rpm for at least 5 minutes.

[0260] Capillary isoelectric focusing electrophoresis analysis parameters and data processing:

[0261] Instrument information: Fully automated protein characterization system (ProteinSimple / Maurice), sample tray temperature: 10°C, sample injection time: 60 seconds. Sample focusing voltage and time: first stage: 1500 V for 1 minute, second stage: 3000 V for 5.5 minutes. After sample injection, the spectrum was imported into Empower software for analysis.

[0262] 3. Experimental Results

[0263] The results are shown in Figures 5 to 11.

[0264] From the observation of charge heterogeneity, it can be seen that the charge heterogeneity of the wild-type control sample changed significantly after being placed at 40°C for one week, becoming a continuous peak. Although the N-803 control sample still had obvious discrete peaks after 1 week, it also became a continuous peak after 2 weeks of treatment. In contrast, multiple samples of the present application (sushi-T2K-V53K, sushi-T2K-I28R-H55K, sushi-T2K-W15K-I28R-H55K, sushi-T2K-W15K-I28R-T51K-H55K, sushi-T2K-W15K-I28R-A50K-T51K-H55K) still had discrete peaks after being placed at 40°C for 2 weeks, indicating good stability in charge heterogeneity.

[0265] In addition, overall, all samples showed migration toward the acid peak after 2 weeks of storage. This is presumably because the recombinant protein contains deamidation sites, which deamidate upon storage at 40°C, leading to migration toward the acid peak.

[0266] Preparation Example 3: Expression and purification of fusion proteins constructed from sushi domain mutants, IL15, and antibodies

[0267] The sushi domain mutant was constructed into a fusion protein with IL15 and the antibody pembrolizumab (pembrolizumab, heavy chain as shown in SEQ ID NO: 42, light chain as shown in SEQ ID NO: 43). The fusion protein includes four chains: one heavy chain 1 (VH-CH1-CH2-CH3-linker1-sushi-linker2-IL15 of pembrolizumab), one heavy chain 2 (heavy chain VH-CH1-CH2-CH3 of pembrolizumab) and two identical light chains (light chain of pembrolizumab). For specific sequences, see Table 6.

[0268] The pcDNA3.4 vector was used to express a fusion protein composed of IL-15, sushi, and pembrolizumab. DNA encoding the heavy chain 1, heavy chain 2, and light chain of the fusion protein were directionally cloned into an expression vector containing a signal peptide (purchased from Invitrogen). The nucleotide sequences of the heavy and light chains of the fusion protein were amplified by PCR. The vector was double-digested with Hind III and Xho I, recovered, seamlessly cloned using DNA homologous recombination enzymes, and transformed into competent Escherichia coli DH5a cells. Positive clones were selected, plasmids were extracted, and sequenced for verification. Plasmids were extracted and transfected. The corresponding expression plasmids were mixed in a ratio of heavy chain 1: heavy chain 2: light chain = 2:2:3. The day before transfection, suspension-cultured ExpiCHO cells were cultured in serum-free ExpiCHO expression medium (Gibco). On the day of transfection, the cell density was adjusted to 6 × 10 cells / ml using fresh ExpiCHO expression medium. 6 At a concentration of 10 cells, 1 μg of plasmid DNA and 3.2 μL ExpiFectamine were added to 76.8 μL OptiPROSFM medium per milliliter of culture medium, and then added to ExpiCHO cells; feed was added 18-22 hours (h is the abbreviation for hours) after transfection, and after culturing for 8-10 days (d is the abbreviation for days), the supernatant was collected and the next step of purification was carried out. The purification was carried out on an AKTA pure protein purifier. The first step of purification was performed using the affinity purification column MabSelect PrismA, and the purity of the protein after one step of purification was evaluated by SEC-HPLC; the second step of purification was performed using a cation exchange chromatography column RESOURCE S. The final product obtained by purification was used Protein concentration was determined by absorbance at 280 nm using N60 (IMPLEN), and protein purity was assessed by SDS-PAGE and SEC-HPLC. Endotoxin detection was performed using a recombinant Factor C endotoxin detection kit. SDS-PAGE and SEC-HPLC assays confirmed that the resulting fusion protein had the correct molecular weight and a purity greater than 90%. Endotoxin testing confirmed that the endotoxin level was less than 1 EU mg -1 , meeting the standards of activity experiments and physical and chemical analysis experiments.

[0269] Table 6. Amino acid sequences of fusion proteins constructed from sushi, IL-15, and Pembrolizumab

[0270] Note: In Table 6, the single underlined part is sushi, the dotted underlined part is IL15, the italic part is the linker (connecting peptide), the double underlined part is the CDR determined according to the Kabat numbering system, and the bold part is the heavy / light chain variable region.

[0271] Experimental Example 7: Detection of the receptor binding ability of the fusion protein constructed by sushi domain mutant, IL15 and antibody

[0272] 1. Experimental samples:

[0273] A fusion protein constructed by combining the sushi domain mutant prepared in the previous Preparation Example 3 with IL15 and Pembrolizumab.

[0274] 2. Experimental Methods

[0275] The binding ability of the fusion protein constructed by the sushi domain mutant, IL15, and Pembrolizumab to the receptor cell KARPASS-299 was detected by flow cytometry. The detection steps are as follows:

[0276] Prepare FACS buffer by adding 2% FBS to PBS and mix well. Centrifuge the recipient cells KARPASS-299 (Nanjing Kebai, CBP60271) at 300g for 5 minutes, remove the culture medium, resuspend the cells in FACS buffer, count the cells, and adjust the cell density to 2x10 6 Cells / mL were added to a V-bottom 96-well plate at 50 μL / well of the cell suspension. Fusion protein samples were added to a final concentration of 100 nM and diluted 5-fold into 8 concentration gradients, with concentrations of 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, and 0.00128 nM, respectively. The cells and samples were reacted in a 4°C refrigerator for 1 hour. Centrifuge at 300 g for 5 minutes, remove the supernatant, wash the cells once with FACS buffer, and centrifuge again to remove the supernatant. 100 μL of 1:500 diluted fluorescent secondary antibody (Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 488, Invitrogen, A-11013). Incubate in a 4°C refrigerator in the dark for 30 minutes. Centrifuge at 300 g for 5 minutes, remove the supernatant, wash twice with FACS buffer, and resuspend the cells in 50 μL FACS buffer. Fluorescence shift analysis was performed using a flow cytometer (iQue3 Plus).

[0277] 3. Experimental Results

[0278] The test results are shown in Figure 12 and Table 7. The results show that the fusion protein constructed by the sushi mutant of the present invention and pembrolizumab has good binding activity to KapaaS299 receptor cells.

[0279] Table 7. Results of the fusion protein binding assay to KapaaS299 receptor cells

[0280] Note: EC 50 The unit is nM; fold = FACS reading at the highest concentration / FACS reading at the lowest concentration.

[0281] Experimental Example 8: Detection of the binding ability of sushi domain mutants, IL15 and antibody fusion proteins to HEC293T-PD1 cells

[0282] 1. Experimental samples:

[0283] A fusion protein constructed by preparing the sushi domain mutant prepared in Example 3, IL15 and Pembrolizumab.

[0284] 2. Experimental Methods

[0285] The binding ability of the fusion protein to the receptor cell HEC293T-PD1 was detected by flow cytometry. The detection steps are as follows:

[0286] Prepare FACS buffer by adding 2% FBS to PBS and mix well. Centrifuge the recipient HEC293T-PD1 cells (Nanjing Kebai, CBP74042) at 300g for 5 minutes, remove the culture medium, resuspend the cells in FACS buffer, count the cells, and adjust the cell density to 2x10 6 Cells / mL, 50 μL / well of the cell suspension was added to a V-bottom 96-well plate. Fusion protein sample was added to a final concentration of 100 nM, and 8 concentration gradients were diluted 5-fold (the concentrations were 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, and 0.00128 nM). The cells and samples were reacted in a 4 ° C refrigerator for 1 hour. Centrifuge at 300g for 5 minutes, remove the supernatant, wash the cells once with FACS buffer and centrifuge again to remove the supernatant. 100 μL of 1:500 diluted fluorescent secondary antibody (Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM488, Invitrogen, A-11013). Incubate in a 4°C refrigerator in the dark for 30 minutes. Centrifuge at 300 g for 5 minutes, remove the supernatant, wash twice with FACS buffer, and resuspend the cells in 50 μL FACS buffer. Fluorescence shift analysis was performed using a flow cytometer (iQue3 Plus).

[0287] 3. Experimental Results

[0288] The test results are shown in Table 8. The fusion protein constructed by the sushi mutant of the present invention and pembrolizumab has good binding activity to HEC293T-PD1 receptor cells. 50 The value was smaller than that of pembrolizumab.

[0289] Table 8. Results of the fusion protein binding activity assay on HEC293T-PD1 receptor cells

[0290] Note: EC 50 The unit is nM; fold = FACS reading at the highest concentration / FACS reading at the lowest concentration.

[0291] Experimental Example 9: NFAT luciferase reporter assay using IL-15 / sushi and antibody-derived fusion protein

[0292] 1. Experimental samples:

[0293] A fusion protein constructed by preparing the sushi domain mutant prepared in Example 3, IL15 and Pembrolizumab.

[0294] 2. Experimental Methods

[0295] Detect the fusion protein using the NFAT-Luciferase Reporter Assay by flow cytometry. The detection steps are as follows:

[0296] After trypsin treatment of target CHO-PDL1 cells (Nanjing Kebai, CBP74066), centrifugation at 300g for 5 minutes, the culture medium was removed, and the cells were resuspended in F12 medium. After counting, the cell density was adjusted to 3.5E5 cells / mL and the cell suspension was added to a flat-bottom 96-well plate at 100 μL / well. After 16 hours, the culture medium was removed and 50 μL of the fusion protein sample was added to a final concentration of 100 nM, and 8 concentration gradients were diluted 5-fold (concentrations were 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, and 0.00128 nM, respectively). After treatment, Jurkat-NFAT-LUC-PD1 effector cells (Nanjing Kebai, CBP74018) were centrifuged at 300g for 5 minutes. The culture medium was removed and the cells were resuspended in RPMI-1640 medium. After counting, the cell density was adjusted to 2.8E6 cells / mL, and 50 μL of effector cells were added to each well. The samples were incubated in a 37°C incubator for 4 hours. 100 μL of one-step luciferase assay reagent (BPS, 78263-5) was added to each well and the cells were allowed to stand at room temperature for 5 minutes. Luciferase detection was performed using a microplate reader (Tecan, spark).

[0297] 3. Experimental Results

[0298] The test results are shown in FIG13 and Table 9 below, and the results show that the fusion protein constructed by the sushi mutant of the present invention and pembrolizumab can effectively bind to effector cells.

[0299] Table 9. Results of fusion protein NFAT luciferase reporter assay

[0300] Note: EC 50 The unit is nM; E MAX This is the reading when the maximum concentration is reached.

[0301] Experimental Example 10: Detection of the NK cell activation ability of fusion proteins constructed with sushi domain mutants, IL15, and antibodies

[0302] 1. Experimental samples:

[0303] A fusion protein constructed by combining the sushi domain mutant prepared in the aforementioned Preparation Example 3 with IL15 and Pembrolizumab.

[0304] 2. Experimental Methods

[0305] The activation ability of the fusion protein on primary NK cells was detected by flow cytometry. The experimental steps are as follows.

[0306] Resuscitate PBMC (SAILYBIO, NF0065) and adjust the PBMC density to 5x10 6 Cells / mL were pre-activated with 1μg / mL OKT3 for 24 hours. The next day, 2E5 PBMC cells were added to a U-bottom 96-well plate at 50μL / well, and the fusion protein to be tested was added to a final concentration of 100nM and 10nM. The cell culture plate was placed in a cell culture incubator at 37°C and 5% CO2 and incubated overnight. 1E4 OVCR3 cells were added to the cell culture plate that had been incubated overnight at 100μL / well. After gentle mixing, the cells were incubated in the cell culture incubator for another 4 hours. The supernatant was removed by centrifugation at 500g for 5 minutes, and the cells were washed once with FACS buffer. LIVE / DEAD staining reagent (Violet fluorescent reactive Dye, Invitrogen, L34964A) was added to distinguish live and dead cells. Fluorescently labeled CD3 antibodies (APC / Cyanine7 anti-human CD3 Antibody, Biolegend, 300318), fluorescently labeled CD56 antibodies (APC anti-humanCD56, Biolegend, 362504), and fluorescently labeled CD107a antibodies (PE anti-human CD107a (LAMP-1) Antibody, Biolegend, 328608) were added and incubated in a dark refrigerator at 4°C for 30 minutes. The cells were centrifuged at 500 g for 5 minutes, the supernatant removed, and the cells were washed twice with 200 μL of FACS buffer per well. The cells were then resuspended in 150 μL of FACS buffer. Fluorescence shift was measured using a flow cytometer (BD FACS Celesta). The percentage of CD107a+ cells within the CD3-CD56+ population was counted.

[0307] 3. Experimental Results

[0308] The results are shown in Table 10. The results showed that the fusion protein constructed by the sushi mutant of the present invention and pembrolizumab had a good activation ability on primary NK cells, especially at a concentration of 10 nM, the CD107A (%) was greater than that of the pembrolizumab-IL15 and pembrolizumab groups.

[0309] Table 10. Results of NK cell activation experiments with fusion proteins

[0310] Experimental Example 11: Detection of the CD8+ T cell activation ability of fusion proteins constructed with sushi domain mutants, IL15, and antibodies

[0311] 1. Experimental samples:

[0312] A fusion protein constructed by the sushi domain mutant obtained in the above Preparation Example 3, IL15 and Pembrolizumab.

[0313] 2. Experimental Methods

[0314] The ability of fusion proteins containing sushi domain mutants to activate CD8+ T cells was detected by flow cytometry. The experimental steps are as follows:

[0315] Resuscitate PBMC (SAILYBIO, NF0065) and adjust the PBMC density to 5x10 6 Cells were pre-activated with 1 μg / mL OKT3 for 24 hours. The next day, 2E5 PBMCs were added to a U-bottom 96-well plate at 50 μL / well, followed by the fusion protein to be tested, to a final concentration of 100 nM, 10 nM, and 1 nM. The cell culture plates were incubated overnight in a 37°C, 5% CO2 incubator. 1E4 OVCR3 cells were added to the incubated cell culture plate at 100 μL / well. After gentle mixing, the cells were incubated in the cell culture incubator for another 4 hours. The supernatant was removed by centrifugation at 500 g for 5 minutes, and the cells were washed once with FACS buffer. LIVE / DEAD staining reagent (Violet fluorescent reactive Dye, Invitrogen, L34964A) was added to distinguish between live and dead cells, and fluorescently labeled CD8 antibody (PerCP / Cyanine5.5 anti-human CD8, Biolegend, 344710) and fluorescently labeled CD25 antibody (Brilliant Violet 785 TM Anti-human CD25 Antibody (Biolegend, 302638) was added and incubated in a dark refrigerator at 4°C for 30 minutes. The cells were centrifuged at 500g for 5 minutes, the supernatant removed, and the cells were washed twice with 200μL of FACS buffer per well. The cells were then resuspended in 150μL of FACS buffer. Fluorescence shift was measured using a flow cytometer (BD FACS Celesta). The percentage of CD25+ cells within the CD8+ cell population was calculated.

[0316] 3. Experimental Results

[0317] The results are shown in Table 11. The results showed that the fusion protein constructed by the sushi mutant of the present invention and pembrolizumab had good CD8+ T cell activation ability, especially at 1 nM, the CD8+CD25+ (%) was greater than that of the pembrolizumab and Pembrolizumab-IL-15 groups.

[0318] Table 11. Results of the CD8+ T cell activation experiment with fusion proteins

[0319] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A sushi domain mutant, which is a mutant of a wild-type sushi domain and comprises the following mutations: The amino acid at one or more positions selected from positions 1, 2, 15, 28, 46, 50, 51, 53 and 55 of the wild-type sushi domain is mutated to a hydrophilic amino acid; Optionally, the amino acid sequence of the wild-type sushi domain is shown in SEQ ID NO: 30; Optionally, the hydrophilic amino acids are independently selected from lysine, arginine, glutamine and asparagine.

2. The sushi domain mutant according to claim 1, comprising the following mutations: The second amino acid is mutated to lysine or arginine; Optionally, the second amino acid is mutated to lysine or arginine, and Amino acid mutations at one, two, three, four, five or six positions selected from the group consisting of position 15, position 28, position 50, position 51, position 53 and position 55 to lysine or arginine; Optionally, the sushi domain mutant comprises a mutation selected from the following (1)-(5): (1) the amino acid at position 2 is mutated to lysine or arginine; and the amino acid at position 53 and / or 55 is mutated to lysine or arginine; (2) The amino acids at positions 2 and 28 are mutated to lysine or arginine; (3) amino acids at positions 2, 28, and 55 are mutated to lysine or arginine; (4) the amino acids at positions 2, 15, 28 and 55 are mutated to lysine or arginine; or (5) The amino acids at positions 2, 15, 28 and 55 are mutated to lysine or arginine; and the amino acids at positions 50 and / or 51 are mutated to lysine or arginine.

3. The sushi domain mutant according to claim 1 or 2, comprising the following mutations: The amino acid at position 46 is mutated to lysine or arginine; and The amino acid at one or two positions selected from the following is mutated to lysine or arginine: position 51 and position 53.

4. The sushi domain mutant according to any one of claims 1 to 3, which is selected from any one of the following groups A) to D); A) the amino acid sequence of the sushi domain mutant is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 13; B) the amino acid sequence of the sushi domain mutant is a sequence that has ≥80%, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥98.5% or ≥99% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 13, and the sushi domain mutant has a sushi domain function; C) the sushi domain mutant comprises SEQ ID NO: 13, SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 10 or SEQ ID NO: 3; D) The amino acid sequence of the sushi domain mutant is: X1X2CPPPMSVEHADIX3VKSYSLYSRERYX4CNSGFKRKAGTSSLTECX5LNKX6X7NX8AX9WTTPSLKCIR, wherein: X1 is I or R, X2 is T, K or Q, X3 is W or K, X4 is I or R, X5 is V or K, X6 is A or K, X7 is T or K, X8 is V or K, X9 is H or K, and X 1- At least one amino acid residue of X9 is K, R or Q; Optionally, X2 is K; Optionally, X3 is K; Optionally, X2 is K, X4 is R and X9 is K; Optionally, X1 is I, X2 is K, X3 is W, X4 is R, X5 is V, X6 is A or K, and X7 is T or K, X8 is V, and X9 is K; Optionally, X1 is I, X2 is K, X3 is K, X4 is R, X5 is V, X6 is A or K, X7 is T or K, X8 is V, and X9 is K; Optionally, 1) X1 is I, X2 is K, X3 is K, X4 is R, X5 is V, X6 is A, X7 is K, X8 is V, and X9 is K; II) X1 is I, X2 is K, X3 is K, X4 is R, X5 is V, X6 is K, X7 is K, X8 is V, and X9 is K; III) X1 is I, X2 is K, X3 is W, X4 is I, X5 is V, X6 is A, X7 is T, X8 is K, and X9 is H; IV) X1 is I, X2 is K, X3 is W, X4 is R, X5 is V, X6 is A, X7 is T, X8 is V, and X9 is K; or V) X1 is I, X2 is K, X3 is K, X4 is R, X5 is V, X6 is A, X7 is T, X8 is V, and X9 is K.

5. A fusion protein comprising the sushi domain mutant according to any one of claims 1 to 4, and the Fc segment of IL-15 and / or human IgG; Optionally, the amino acid sequence of the IL-15 is shown in SEQ ID NO:29 or SEQ ID NO:

34. Optionally, the Fc segment of human IgG is the Fc segment of human IgG1, IgG2, IgG3 or IgG4; Optionally, the amino acid sequence of the Fc segment of human IgG is shown in SEQ ID NO: 33; Optionally, the Fc segment of the human IgG comprises a LALA mutation; Optionally, the amino acid sequence of the Fc segment of human IgG is as shown in SEQ ID NO:

31.

6. The fusion protein according to claim 5, wherein The sushi domain mutant and IL-15 are on the same peptide chain or not on the same peptide chain; Alternatively, when the sushi domain mutant and IL-15 are on the same peptide chain, they are linked via a linker peptide or directly linked; Optionally, when the sushi domain mutant and IL-15 are not on the same peptide chain, they are linked via one or more disulfide bonds.

7. The fusion protein according to claim 5 or 6, wherein The fusion protein comprises IL-15, a sushi domain mutant and an Fc segment of human IgG in sequence from the N-terminus; Optionally, the IL-15 and the sushi domain mutant, and / or the sushi domain mutant and the Fc segment of human IgG are directly connected or connected via a connecting peptide; Optionally, the fusion protein comprises IL-15, a connecting peptide, a sushi domain mutant and an Fc segment of human IgG in order from the N-terminus; Optionally, the amino acid sequence of the connecting peptide is shown in SEQ ID NO:

32.

8. The fusion protein according to any one of claims 5 to 7, wherein The amino acid sequence of the fusion protein is shown in any one of SEQ ID NO: 14 to SEQ ID NO: 26; or The amino acid sequence of the fusion protein is a sequence that is ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥98.5%, ≥99%, ≥99.3%, ≥99.5% or ≥99.7% identical to any one of SEQ ID NO:14 to SEQ ID NO:

26.

9. The fusion protein according to any one of claims 5 to 8, comprising a protein functional region targeting a tumor-associated antigen and / or targeting an immune checkpoint; Optionally, the tumor-associated antigen is selected from PD-L1, CD19, CD20, EGFR, Claudin18.2, BCMA, One or more of HER2, CD19, CD20, and Nectin-4; Optionally, the immune checkpoint is selected from one or more of PD-1, CTLA-4, Lag-3 and Tim-3; Optionally, the protein functional region is an antibody or an antigen-binding fragment thereof, such as a VHH, scFv, Fv fragment, Fab fragment or F(ab')2 fragment; Optionally, the fusion protein is in the form of a monoclonal antibody or a bispecific antibody; Optionally, the fusion protein comprises 4 polypeptide chains: Chain 1: From N-terminus to C-terminus: antibody heavy chain, connecting peptide, sushi domain mutant, connecting peptide, IL15, Chain 2: Antibody heavy chain, Chain 3 and Chain 4: Antibody light chain; Optionally, the antibody is an anti-PD-1 antibody; Optionally, the HCDR1, HCDR2 and HCDR3 of the antibody are identical to the CDRs in SEQ ID NO:42, and the LCDR1, LCDR2 and LCDR3 of the antibody are identical to the CDRs in SEQ ID NO:43; Optionally, the heavy chain variable region (VH) of the antibody has at least 85% sequence identity or is identical to the VH in SEQ ID NO:42, and the light chain variable region (VL) of the antibody has at least 85% sequence identity or is identical to the VL in SEQ ID NO:43; Optionally, the fusion protein comprises 4 polypeptide chains: the amino acid sequence of chain 1 has at least 85% sequence identity or is identical to SEQ ID NO:37, 38, 39, 40 or 41, the amino acid sequence of chain 2 has at least 85% sequence identity or is identical to SEQ ID NO:42, and the amino acid sequences of chain 3 and chain 4 have at least 85% sequence identity or are identical to SEQ ID NO:

43.

10. The sushi domain mutant according to any one of claims 1 to 4 or the fusion protein according to any one of claims 5 to 9, which has at least one of the following functions: A) has the function of binding to cells expressing IL15 receptor; optionally, it can bind to cells expressing IL15 receptor with an EC less than 10 nM 50 value combined with KARPASS-299, the EC 50 The values ​​were determined by flow cytometry fluorescence sorting technology; B) having the function of activating NK cells; optionally, the NK cell activation function is detected by flow cytometry; C) having good thermal stability; optionally, the sushi domain mutant or fusion protein is placed in a 40° C. water bath for 2 weeks, and the percentage of fusion protein aggregates is less than 50%; D) having good charge stability; optionally, the sushi domain mutant or fusion protein has a smaller change in charge heterogeneity when placed in a water bath at 40° C. for 2 weeks compared to a protein comprising a wild-type sushi domain; optionally, the change in charge heterogeneity is determined by whole-column imaging capillary isoelectric focusing electrophoresis analysis; E) has a higher yield; optionally, the sushi domain mutant or fusion protein is expressed using an ExpiCHO cell expression system, and the protein expression amount is greater than 30 mg / L; and / or F) having a higher purity; optionally, the sushi domain mutant or fusion protein is expressed using an ExpiCHO cell expression system, and after being purified twice using MabSelect PrismA and HiLoad 16 / 600Superdex 200pg purification columns, the protein purity is greater than 40%.

11. An isolated nucleic acid molecule encoding the sushi domain mutant of any one of claims 1 to 4, or encoding the fusion protein of any one of claims 5 to 9.

12. A recombinant vector comprising the isolated nucleic acid molecule of claim 15; optionally, the recombinant vector is a recombinant expression vector.

13. A recombinant host cell comprising the isolated nucleic acid molecule of claim 15, or comprising the recombinant vector of claim 16. 14 . A pharmaceutical composition comprising the sushi domain mutant according to claim 1 , or the fusion protein according to claim 5 , and one or more pharmaceutically acceptable excipients.

15. Use of the sushi domain mutant of any one of claims 1 to 4, the fusion protein of any one of claims 5 to 9, the isolated nucleic acid molecule of claim 10, the recombinant vector of claim 11, the recombinant host cell of claim 12, or the pharmaceutical composition of claim 14 in the preparation of a medicament for treating or preventing tumors, immunodeficiency or infectious diseases; Optionally, the tumor is one or more selected from melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancies; Optionally, the tumor is an advanced solid tumor; Optionally, the tumor is an advanced tumor selected from one or more tumors of melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancy; Optionally, the immunodeficiency is an immunodeficiency induced by a side effect of a specific treatment of anti-tumor therapy or pre-transplantation therapy; Optionally, the immunodeficiency is HIV-induced immunodeficiency; Optionally, the infectious disease is a disease caused by viral, bacterial, yeast or fungal infection.

16. A method for treating or preventing a tumor, an immunodeficiency or an infectious disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the sushi domain mutant of any one of claims 1 to 4, the fusion protein of any one of claims 5 to 9, the isolated nucleic acid molecule of claim 10, the recombinant vector of claim 11, the recombinant host cell of claim 12, or the pharmaceutical composition of claim 14; Optionally, the tumor is one or more selected from melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancies; Optionally, the tumor is an advanced solid tumor; Optionally, the tumor is an advanced tumor selected from one or more tumors of melanoma, squamous cell carcinoma, breast cancer, glioma, ovarian cancer, pancreatic tumor and hematological malignancy; Optionally, the immunodeficiency is an immunodeficiency induced by a side effect of a specific treatment of anti-tumor therapy or pre-transplantation therapy; Optionally, the immunodeficiency is HIV-induced immunodeficiency; Optionally, the infectious disease is a disease caused by viral, bacterial, yeast or fungal infection.

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