WO2025031274A1 - Sushi domain mutant, fusion protein, and pharmaceutical composition and use - Google Patents

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 mutant, fusion protein, pharmaceutical composition and use

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), and the entire contents of the priority patent application are incorporated by reference into the present disclosure.

Technical Field

The invention belongs to the field of biomedicine and relates to a Sushi domain mutant, a fusion protein, a pharmaceutical composition and uses.

Background Art

Interleukin-15 (IL-15 or IL15) is a pleiotropic cytokine that connects innate and adaptive immune processes in vivo. IL-15 is a type 1 four-alpha helical cytokine. It can form 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 NK cells. They also promote B cells to induce immunoglobulin synthesis and regulate lymphoid homeostasis.

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 the 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 PI3K/Akt and Ras/Raf/MAPK signaling cascades. These pathways lead to the subsequent expression of Bcl2, Myc and Fos/Jun and NFkB activation.

IL-15 has a wide range of immunomodulatory activities and can participate in regulating the survival, proliferation and function of a variety of immune cells, including NK cells, memory CD8+T cells, NKT cells, etc. Because IL-15 needs to bind to IL-15Rα first 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 used after being combined with IL-15Rα. Its immune cell stimulating activity is more than 100 times that of free IL-15 alone. Since free IL-15 has the disadvantages of short half-life and limited in vivo activity, and IL15 has better biological activity after binding to IL-15Rα, the complex of IL-15 and IL-15Rα is called a super agonist. The researchers found that the use of the sushi domain in IL-15Rα without the full length of IL-15Rα can also form a superagonist complex with IL-15, and the resulting superagonist activity is equivalent to that of the full length of IL-15Rα. Therefore, the complex composed of IL-15 and the sushi domain through covalent or non-covalent bonding (such as electrostatic force) is also called an IL-15 superagonist.

Since IL-15 and its receptor IL-15Rα are easily dissociated, the stability of IL-15 superagonists is relatively poor. Therefore, current IL-15 superagonists use a series method to connect IL-15 and IL-15Rα using a linker, or use a bispecific antibody method to construct IL-15 and IL-15Rα on the two chains of Fc to form 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 worse. At present, there is still a need to develop new IL-15 superagonists.

Summary of the invention

After in-depth research and creative work, the inventors obtained a mutant of the sushi domain, and further obtained a fusion protein based on it. The sushi domain mutant or fusion protein of the present invention has higher stability and good biological activity, and has good application prospects. The following invention is provided:

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:

Selected from positions 1, 2, 15, 28, 46, 50 of the wild-type sushi domain One or more of positions 51, 53 and 55 are mutated to water-soluble amino acids;

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

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

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

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

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

The second position is mutated to lysine or arginine; and

One, two, three, four, five or six of the following sites are mutated to lysine or arginine:

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

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

Position 2 is mutated to lysine or arginine; and

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

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

Positions 2 and 28 were mutated to lysine or arginine.

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

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

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

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

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

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

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

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

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

No. 51 and No. 53.

In some embodiments of the present invention, any of the above-mentioned 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;

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

In some embodiments of the present invention, the sushi domain mutant, except for the mutated amino acid position described in any of the above items, has the same amino acids as the wild-type sushi domain or only has 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 shown in SEQ ID NO: 30. In certain embodiments, the sushi domain mutant, except for the mutated amino acid position described in any of the above items, has the same amino acids as the wild-type sushi domain.

In some embodiments, the sushi domain mutant has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more) amino acid differences from the wild-type sushi domain. In some embodiments, the amino acid residues that differ between the sushi domain mutant and 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 that differ between the sushi domain mutant and the wild-type sushi domain (e.g., 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 that differ between the sushi domain mutant and the wild-type sushi domain are mutated to lysine or arginine. In some embodiments, the amino acid residue that is different 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.

In some embodiments of the present invention, the amino acid sequence of the sushi domain mutant of any of the preceding items is (SEQ ID NO: 44):

_{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 at least one of the amino acid residues _of X1 _{- X9} is _K , R or Q;

In some embodiments, in the amino acid sequence of any of the above sushi domain mutants, _X2 is K;

In some embodiments, in the amino acid sequence of the sushi domain mutant described in any of the above items, _X3 is K;

In some embodiments, in the amino acid sequence of any of the above sushi domain mutants, _X2 is K, _X4 is R and _X9 is K;

In some embodiments, in the amino acid sequence of any of the above 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;

In some embodiments, in the amino acid sequence of any of the above 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;

In some embodiments, the amino acid sequence of the sushi domain mutant, _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;

In some embodiments, the amino acid sequence of the sushi domain mutant, _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;

In some embodiments, the amino acid sequence of the sushi domain mutant, _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;

In some embodiments, the amino acid sequence of the sushi domain mutant, _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;

In some embodiments, in the amino acid sequence of any of the above sushi domain mutants, _X1 is 1, _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.

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.

In some embodiments of the present invention, 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 with any one of SEQ ID NO:1 to SEQ ID NO:13, and the sushi domain mutant has sushi domain function.

In some embodiments of the present invention, the sushi domain mutant includes SEQ ID NO:13, SEQ ID NO:12, SEQ ID NO:11, SEQ ID NO:10 or SEQ ID NO:3.

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

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

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

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

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

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.

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.

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;

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

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

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

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.

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.

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 sequence from the N-terminus.

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.

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 sequence in SEQ ID NO:14 to SEQ ID NO:26.

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 not on the same peptide chain.

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.

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.

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;

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;

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

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.

In some embodiments of the present invention, the fusion protein comprises 4 polypeptide chains:

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

Chain 2: Antibody heavy chain,

Chain 3 and Chain 4: Antibody light chain;

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

In some embodiments of the present invention, the HCDR1, HCDR2 and HCDR3 of the antibody are the same as the CDR in SEQ ID NO:42, and the LCDR1, LCDR2 and LCDR3 of the antibody are the same as the CDR in SEQ ID NO:43.

In some embodiments of the present 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.

In certain embodiments, the antibody of any of the preceding items, the VH/VL, and the 3 CDRs (HCDR1, HCDR2, and HCDR3) contained in the VH and the 3 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 3 CDRs (HCDR1, HCDR2, and HCDR3) contained in the VH and the 3 CDRs (LCDR1, LCDR2, and LCDR3) contained in the VL are defined by the Kabat numbering system.

In some embodiments of the present invention, the fusion protein comprises four polypeptide chains: the amino acid sequence of chain 1 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 SEQ ID NO: 37, 38, 39, 40 or 41, and the amino acid sequence of chain 2 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 SEQ ID NO: 42. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity or is identical, and the amino acid sequences of chains 3 and 4 have 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 are identical to SEQ ID NO:43.

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:

A) has the function of binding to cells expressing IL15 receptors; optionally, it can bind to KARPASS-299 with an _EC50 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); optionally, the _EC50 value is 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% (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 17% or less);

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 (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

F) has a higher 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).

The sushi domain mutant according to any one of the present invention or the fusion protein according to any one of the present invention, for use in 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.

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

Another aspect of the present invention relates to a recombinant vector, which comprises the isolated nucleic acid molecule of the present invention; optionally, the recombinant vector is a recombinant expression vector.

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.

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.

The pharmaceutical composition according to any one of the present invention is used 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.

Another aspect of the present invention relates to the use of the sushi domain mutant described in any one of the present invention, the fusion protein described in any one of the present invention or the pharmaceutical composition described in any one of the present invention in the preparation of a method for treating or preventing tumors, immunodeficiency or infection. Use in drugs for sexually transmitted 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.

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

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.

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;

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

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

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;

In some embodiments, the tumor is an advanced solid tumor;

In some embodiments, 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;

In some embodiments, the immunodeficiency is an immunodeficiency induced by a side effect of a specific treatment of anti-tumor therapy or pre-transplantation therapy;

In some embodiments, the immunodeficiency is HIV-induced immunodeficiency;

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

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

As used herein, when referring to the amino acid sequence of IL-15 (interleukin-15), it includes but It is not limited to the full length of IL-15 protein, or contains a full length fragment of 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, those skilled in the art understand 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 natural or artificial variants thereof. In addition, when describing a sequence fragment of IL-15, it also includes the corresponding sequence fragments in its natural or artificial variants.

As used herein, when referring to the amino acid sequence of IL-15Rα (interleukin-15 receptor α) or sushi domain, it includes but is not limited to the full length of IL-15Rα protein, or the extracellular fragment ECD of IL-15Rα or a fragment containing IL-15RαECD, such as sushi domain; it also includes fusion proteins of IL-15RαECD 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 in the amino acid sequence of IL-15Rα or sushi domain, mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "IL-15Rα" or "sushi domain" shall include all such sequences, including natural or artificial variants thereof. Moreover, when describing a sequence fragment of IL-15Rα or sushi domain, it also includes the corresponding sequence fragments in its natural or artificial variants.

As used herein, the term "Fc", "Fc segment" or "Fc fragment" is also called a crystallizable fragment. Generally, the 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 the Fc segment of human IgG. In some embodiments of the present invention, the Fc segment of human IgG is the Fc segment of human IgG1.

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.

In the present invention, unless otherwise specified, the letters before the site represent the amino acid before mutation, and the letters after the site represent the amino acid after mutation. For example, "L234A" means that the amino acid residue at position 234 mutates from the original "L" to "A".

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 usually 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 activity. In addition, 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.

Currently, the most important flexible linker peptide is composed of Gly and Ser residues ("GS" linker). The most widely used flexible linker peptide sequence is (GGGGS)n. By adjusting the value of n (number of repeats), the length of the GS linker peptide can be changed so that two proteins can be optimally connected or allowed to interact. In some embodiments of the present invention, the linker peptide comprises the amino acid sequence (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5 and 6.

As used herein, the term _EC50 refers to the concentration for 50% of maximal effect, which refers to the concentration that can induce 50% of the maximal effect.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined 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 regions of antibodies mediate 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, termed complementarity determining regions (CDRs), interspersed with more conserved regions. The variable regions of each heavy chain/light chain pair (VH and VL) form the site where the antibody binds to the antigen. The assignment of amino acids to various regions or domains follows the Kabat numbering system definition (Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987and 1991))), or the Chothia numbering system definition (Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883), or the IMGT numbering system definition (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 definition (Martin et al. Nature 1989; 342: 878-883). 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.).

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 an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

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 natural mutations that may occur spontaneously. 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, which usually recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique 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 recombinant DNA technology (see, for example, US Patent 4,816,567).

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 binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. 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 that contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

As used herein, the term "Fd fragment" means an antibody fragment consisting of _VH and C _H 1 domains; the term "Fv fragment" means an antibody fragment consisting of _VL and _VH domains of a single arm of an antibody; the term "dAb fragment" means an antibody fragment consisting of a _VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of _VL , _VH , C _L and C _H 1 domains; the term "F(ab') ₂ fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

As used herein, the term "single chain antibody (single chain fragment variable, 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. Wherein 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 a general structure: NH2-VL-linked fragment-VH-COOH or NH2-VH-linked fragment-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. Description.

As used herein, the terms "single domain antibody (VHH)" and "nanobody" have the same meaning, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, 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 one heavy chain variable region.

As used herein, the term "isolated" or "isolated" refers to that obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated 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.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are 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 (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. 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, papillomaviruses (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. In addition, the vector may also contain a replication initiation site.

As used herein, the term "host cell" refers to cells 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.

As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., 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, 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 non-ionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

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 who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount 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.

Advantageous Effects of the Invention

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

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

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

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

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

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

A) has the function of binding to cells expressing IL15 receptors; optionally, it can bind to KARPASS-299 with an _EC50 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); optionally, the _EC50 value is 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% (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 17% or less);

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 (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

F) has a higher 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

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

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

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

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

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

Figure 6: Changes in iCIEF after sushi-Fc-WT fusion protein was treated at 40°C for 2 weeks.

FIG7 : iCIEF changes of sushi-T2K-V53K fusion protein after treatment at 40° C. for 2 weeks.

Figure 8: Changes in iCIEF after sushi-T2K-I28R-H55K fusion protein was treated at 40°C for 2 weeks.

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

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

Figure 11: iCIEF changes after sushi-T2K-W15K-I28R-A50K-T51K-H55K fusion protein was treated at 40°C for 2 weeks;

Figure 12: Result of the fusion protein binding test on KapaaS299 receptor cells;

Figure 13: Result of fusion protein NFAT luciferase reporter experiment.

DETAILED DESCRIPTION

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

Table A: Partial sequences of the present invention

In Table A: the mutation sites of the sushi domain are marked with boxes; the sequences of the sushi domain in the fusion protein are marked with single underlines.

The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

Preparation Example 1: Design of sushi domain mutants

The present inventors designed mutants shown in SEQ ID NO: 1 to SEQ ID NO: 13 for IL-15 receptor Sushi domain_31-95_human (SEQ ID NO: 30). Please refer to Table A above for the specific amino acid sequences and mutant nomenclature.

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

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).

N-803 control sample: shown in SEQ ID NO: 27. Its structural components are: human IL-15 mutant (SEQ ID NO: 34), G4S Linker (SEQ ID NO: 32), sushi domain wild type (SEQ ID NO: 30), human IgG1Fc-LALA (SEQ ID NO: 31).

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), sushi domain wild type (SEQ ID NO: 30), human IgG1Fc-LALA (SEQ ID NO: 31).

The above 13 fusion protein samples and 2 control samples were expressed using the ExpiCHO cell expression system. The expression and purification steps are as follows:

The pcDNA3.4 vector was used as a special vector for expressing the fusion protein. The nucleotide sequences of various fusion proteins were obtained by gene synthesis, and the vectors were double-digested with Hind III and Xho I. After recovery, they were seamlessly cloned by DNA homologous recombination enzymes, and transformed into Escherichia coli competent cells DH5a. Positive clones were selected and plasmids were extracted and sequenced for verification. The plasmids were extracted and transfected separately. One day before transfection, the suspension-cultured ExpiCHO cells were cultured with serum-free ExpiCHO expression medium (Gibco). On the day of transfection, the cell density was adjusted to a concentration of 6×10 ⁶ cells per milliliter with fresh ExpiCHO expression medium, and 1 μg of Plasmid DNA and 3.2μl ExpiFectamine were mixed with 76.8μl OptiPROSFM medium and finally added to ExpiCHO cells; feed was added 18-22h after transfection, and after 8-10d of culture, the supernatant was collected and purified separately. The purification was carried out on an AKTA pure protein purifier, including the following two steps:

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

(2) The molecular sieve purification column HiLoad 16/600 Superdex 200 pg was used for the second step of purification. Thirteen fusion protein samples and two control samples were obtained after the second step of purification.

Experimental Example 1: Protein Purity and Endotoxin Detection

1. Experimental samples

13 fusion protein samples and 2 control samples obtained in step (1) of the above-mentioned Preparation Example 2 after the first step of purification;

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

2. Experimental Methods

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

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

3. Experimental results

The SDS-PAGE and SEC-HPLC assays showed that the molecular weight of the obtained fusion protein was correct and the purity was greater than 95%. The specific results are shown in Table 1 and Figure 1 below.

Table 1

In Table 1: ^a indicates that the yield is significantly improved relative to the control sample; ^b indicates that the purity is significantly improved relative to the control sample.

The results show:

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

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.

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.

Experimental Example 2: Melting temperature (Tm) determination experiment

1. Experimental samples:

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

2. Experimental Methods

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

The melting temperature of the protein was calculated by detecting the change of fluorescent dye signal during protein heating using a fluorescent quantitative PCR instrument. Take 58.5 μL of sample (1.0 mg/mL) and 1.5 μL of 200×SYPRO orange solution (Sigma-Aldrich/S5692, specification: 5000×, dilute it to 200× with sample buffer before sample preparation on the machine), mix well, take 25 μL of the mixed solution and test on the machine, in duplicate. Experimental parameters: real-time fluorescent quantitative PCR instrument (Thermo Fisher scientific/QuantStudio 6Pro), detection temperature: 25℃ to 99℃, heating rate of 0.05℃/s. Use Protein Thermal Shift software (Thermo Fisher scientific) for analysis.

3. Experimental results

The results are shown in Table 2 below.

Table 2

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 is close to Tm1 and is combined into the same Tm.

The results showed that the fusion proteins of the present application had comparable or even better thermal stability than the control samples. For example, the Tm2 values of 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 sushi-V53K fusion protein, sushi-T2K-V53K fusion protein, and sushi-V46K-V53K fusion protein exceeded 80°C, which was comparable to the control samples. Tm1 is clearly different.

Experimental Example 3: Detection of receptor binding ability of fusion proteins containing sushi domain mutants (activity Detection experiment 1)

1. Experimental samples:

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

2. Experimental Methods

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

Prepare FACS buffer, add 2% FBS to PBS and mix well for later use. Centrifuge the receptor cells KARPASS-299 (Nanjing Kebai, CBP60271) at 300g for 5 minutes, remove the culture medium, resuspend the cells with FACS buffer, count, adjust the cell density to 2x10 ⁶ cells/mL, and add the cell suspension to a V-bottom 96-well plate at 50μL/well. Add the fusion protein sample to a final concentration of 100nM, and dilute 8 concentration gradients 5 times, with concentrations of 100nM, 20nM, 4nM, 0.8nM, 0.16nM, 0.032nM, 0.0064nM, and 0.00128nM, respectively. React the cells and samples 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. Add 100μL of 1:500 diluted fluorescent secondary antibody (Alexa 647goat anti-human IgG Fc specific, Jackson, 20221222). Incubate in a 4°C refrigerator away from light for 30 minutes. Centrifuge at 300 g for 5 minutes to remove the supernatant, wash twice with FACS buffer, resuspend the cells with 50 μL FACS buffer, and use flow cytometer (iQue3Plus) for fluorescence shift detection.

3. Experimental results

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

Table 3

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

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 with receptor cells.

Experimental Example 4: Detection of the NK cell activation ability of fusion proteins containing sushi domain mutants (active Sex detection experiment 2)

1. Experimental samples:

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

2. Experimental Methods

The activation ability of the fusion protein containing the sushi domain mutant on primary NK cells was detected by flow cytometry. The experimental steps are as follows.

Resuscitate PBMC (SAILYBIO, XW0102052W), adjust the PBMC density to 5x10 ⁶ cells/mL, and add PBMC cell suspension to a U-bottom 96-well plate at 50 μL/well. Then add the fusion protein to be tested to a final concentration of 1nM and 0.01nM. Incubate the cell culture plate in a cell culture incubator at 37°C and 5% CO ₂ overnight. Collect K562 cells the next day and adjust the cell density to 1x10 ⁵ cells/mL. Add K562 cell suspension to the cell culture plate incubated overnight, 100 μL/well. Mix gently and continue to incubate in the cell culture incubator for 4 hours. Centrifuge at 500g for 5 minutes to remove the supernatant and wash the cells once with FACS buffer. Add Fc blocker (Human TruStain Fix, Biolegend, 422302) and react 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 the live and dead cells. Fluorescently labeled CD3 antibody (APC/Cyanine7 anti-human CD3 Antibody, Biolegend, 300318), fluorescently labeled CD56 antibody (APC anti-humanCD56, Biolegend, 362504) and fluorescently labeled CD107a antibody (PE anti-human CD107a (LAMP-1) Antibody, Biolegend, 328608) were added and incubated in a refrigerator at 4 degrees in the dark for 30 minutes. The supernatant was removed by centrifugation at 500g for 5 minutes, and 200μL FACS buffer was added to each well to wash the cells twice and then the cells were resuspended in 150μL FACS Buffer. Fluorescence shift detection was performed using a flow cytometer (BD FACSCelesta). The percentage of CD107a+ cells in the CD3-CD56+ cell population was counted.

3. Experimental results

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

Table 4

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.

Experimental Example 5: Long-term stability test

1. Experimental samples:

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

2. Experimental Methods

The long-term stability of some fusion proteins containing sushi domain mutants was tested. Each sample was placed at 40°C for two weeks (14 days) and the changes in the properties of the samples were measured. The specific experimental steps are as follows:

On the first day, each sample to be tested was divided into two portions, each containing 150 μg, namely T0 and 2W samples. The T0 sample was placed at 4°C, and the 2W sample was placed in a 40°C water bath. On the 14th day, the 2W sample was taken out from 40°C and placed at 4°C for testing.

First, the sample was analyzed by size exclusion chromatography (SEC). SEC is a liquid chromatography method that separates molecules based on the difference in hydrodynamic radius of the molecules to be tested. It is mainly used to detect the purity of large molecular proteins. Experimental method: An ultra-high performance liquid system (Waters/ACQUITY ^TM PREMIER) and a Waters ACQUITY UPLC Protein BEH SEC column (1.7μm, 4.6×150mm) were used. 50mM PB (pH 6.8) and 300mM NaCl were used as mobile phases for isocratic elution. The elution time was 7min, the flow rate was 0.4mL/min, the detection wavelength was 280nm, the column temperature was 30℃, and the protein injection volume was 10μg. The area normalization method was used to determine the purity of the antibody.

3. Experimental results

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

Table 5: Polymer changes

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.

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

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

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

1. Experimental samples:

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

2. Experimental Methods

The samples were analyzed by whole-column imaging capillary isoelectric focusing (iCIEF). iCIEF is based on the different charges of proteins. The variants were separated by capillary electrophoresis according to their isoelectric point (pI) characteristics, and the isoelectric points of the charged variants of the protein were determined and the relative percentages were calculated.

Sample preparation for the machine: First, prepare the premix solution. The composition of each component is as follows: 0.5μL each of pI marker 4.65 (ProteinSimple/102223) and pI marker 8.18 (ProteinSimple/102408), 4.0μL of Pharmalyte 3-10 (GE Healthcare/17-0456-01), 35μL of 1% methylcellulose solution (ProteinSimple/101876), 37.5μL of 8M urea and 2.5μL of ultrapure water. Replace the sample solution with ultrapure water to make the final concentration of the sample 1.0mg/mL. Take 20μL of the sample after the solution change and mix it with 80μL of the premix solution. After centrifugation at 13000rpm for 1 minute, transfer 95μL of the supernatant to a 96-well plate and centrifuge at 3000rpm for at least 5 minutes.

Capillary isoelectric focusing electrophoresis analysis parameters and data processing:

Instrument information: fully automatic protein characterization system (ProteinSimple/Maurice), sample plate temperature: 10°C, sample injection time: 60 seconds. Sample focusing voltage and time: 1500 volts for 1 minute in the first stage, 3000 volts for 5.5 minutes in the second stage. After the sample injection is completed, the spectrum is imported into Empower software for analysis.

3. Experimental results

The results are shown in Figures 5 to 11.

From the observation of charge heterogeneity, it can be seen that after the wild-type control sample was placed at 40°C for one week, the charge heterogeneity changed significantly and became 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. However, 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) were still discrete peaks after being placed at 40°C for 2 weeks, indicating that the stability in charge heterogeneity is good.

In addition, overall, after 2 weeks of storage, all samples showed a phenomenon of migration towards the acid peak. It is speculated that this is because the recombinant protein has a deamidation site, and deamidation occurs when stored at 40°C, resulting in migration towards the acid peak.

Preparation Example 3: Expression and purification of fusion protein constructed by sushi domain mutant, IL15 and antibody

The sushi domain mutant was constructed into a fusion protein with IL15 and the antibody Pembrolizumab (pembrolizumab, the heavy chain is shown in SEQ ID NO:42, and the light chain is shown in SEQ ID NO:43). The fusion protein includes 4 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). The specific sequences are shown in Table 6.

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

Table 6. Amino acid sequence of fusion protein constructed by sushi, IL-15 and Pembrolizumab

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.

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

1. Experimental samples:

The fusion protein constructed by the sushi domain mutant prepared in the previous Preparation Example 3, IL15 and Pembrolizumab.

2. Experimental Methods

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

Prepare FACS buffer, add 2% FBS to PBS and mix well for later use. Centrifuge the receptor cells KARPASS-299 (Nanjing Kebai, CBP60271) at 300g for 5 minutes, remove the culture medium, resuspend the cells with FACS buffer, count, adjust the cell density to 2x10 ⁶ cells/mL, and add the cell suspension to a V-bottom 96-well plate at 50μL/well. Add the fusion protein sample to a final concentration of 100nM, and dilute 8 concentration gradients 5 times, with concentrations of 100nM, 20nM, 4nM, 0.8nM, 0.16nM, 0.032nM, 0.0064nM, and 0.00128nM, respectively. React the cells with the sample 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. Add 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) to each well. Incubate in a 4°C refrigerator away from light for 30 minutes. Centrifuge at 300 g for 5 minutes to remove the supernatant and perform FACS. The cells were washed twice with 50 μL FACS buffer and resuspended in 50 μL FACS buffer, and fluorescence shift was detected by flow cytometer (iQue3Plus).

3. Experimental results

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.

Table 7. Results of the fusion protein binding assay with KapaaS299 receptor cells

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

Experimental Example 8: Fusion protein constructed by sushi domain mutant, IL15 and antibody and HEC293T-PD1 Cell binding ability assay

1. Experimental samples:

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

2. Experimental Methods

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

Prepare FACS buffer, add 2% FBS to PBS and mix well for later use. Centrifuge the receptor cells HEC293T-PD1 (Nanjing Kebai, CBP74042) at 300g for 5 minutes, remove the culture medium, resuspend the cells with FACS buffer, count, adjust the cell density to 2x10 ⁶ cells/mL, and add the cell suspension to a V-bottom 96-well plate at 50μL/well. Add the fusion protein sample to a final concentration of 100nM, and dilute 8 concentration gradients 5 times (the concentrations are 100nM, 20nM, 4nM, 0.8nM, 0.16nM, 0.032nM, 0.0064nM, and 0.00128nM, respectively). React the cells and samples 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. Add 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) to each well. Incubate in a 4°C refrigerator away from light for 30 minutes. Centrifuge at 300 g for 5 minutes to remove the supernatant, wash twice with FACS buffer, resuspend the cells with 50 μL FACS buffer, and use flow cytometer (iQue3Plus) for fluorescence shift detection.

3. Experimental results

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, and the EC ₅₀ value is smaller than that of pembrolizumab.

Table 8. Results of the fusion protein binding activity test on HEC293T-PD1 receptor cells

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

Experimental Example 9: NFAT luciferase reporter experiment of IL-15/sushi and antibody fusion protein

1. Experimental samples:

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

2. Experimental Methods

The NFAT-Luciferase Reporter Assay of the fusion protein was detected by flow cytometry. The detection steps are as follows:

After the target cell CHO-PDL1 (Nanjing Kebai, CBP74066) was treated with trypsin, centrifuged at 300g for 5 minutes, the culture medium was removed, and the cells were resuspended in F12 culture medium. After counting, the cell density was adjusted to 3.5E5 cells/mL, and the cell suspension was added to a flat-bottomed 96-well plate at 100 μL/well. After 16 hours, the culture medium was removed, 50 μL of fusion protein sample was added to a final concentration of 100 nM, and 8 concentration gradients were diluted 5 times (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, respectively). After the effector cells Jurkat-NFAT-LUC-PD1 (Nanjing Kebai, CBP74018) were treated, 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 50μL of effector cells were added to each well. The samples were placed in a 37℃ incubator for 4 hours. 100μL 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. The luciferase detection was performed using an ELISA reader (Tecan, spark).

3. Experimental results

The test results are shown in FIG. 13 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.

Table 9. Results of fusion protein NFAT luciferase reporter assay

Note: EC ₅₀ is in nM; E _MAX is the reading when the maximum concentration is reached.

Experimental Example 10: Effect of fusion protein constructed by sushi domain mutant, IL15 and antibody on NK cell activation Force detection experiment

1. Experimental samples:

A fusion protein constructed by the sushi domain mutant prepared in the above-mentioned Preparation Example 3, IL15 and Pembrolizumab.

2. Experimental Methods

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

Resuscitate PBMC (SAILYBIO, NF0065), adjust the PBMC density to 5x10 ⁶ cells/mL, and pre-activate with 1μg/mL OKT3 for 24h. The next day, add 2E5 PBMC cells to a U-bottom 96-well plate at 50μL/well, and then add the fusion protein to be tested to a final concentration of 100nM and 10nM. Incubate the cell culture plate in a cell culture incubator at 37°C and 5% CO ₂ overnight. Add 1E4 OVCR3 cells to the cell culture plate incubated overnight, 100μL/well. Mix gently and continue incubating in the cell culture incubator for 4 hours. Centrifuge at 500g for 5 minutes to remove the supernatant, and wash the cells once with FACS buffer. Add LIVE/DEAD staining reagent (Violet fluorescent Reactive Dye, invitrogen, L34964A) was used to distinguish the live and dead cells. Fluorescently labeled CD3 antibody (APC/Cyanine7 anti-human CD3 Antibody, Biolegend, 300318), fluorescently labeled CD56 antibody (APC anti-humanCD56, Biolegend, 362504) and fluorescently labeled CD107a antibody (PE anti-human CD107a (LAMP-1) Antibody, Biolegend, 328608) were added and incubated in a refrigerator at 4 degrees in the dark for 30 minutes. The supernatant was removed by centrifugation at 500g for 5 minutes, and 200μL FACS buffer was added to each well to wash the cells twice and then the cells were resuspended in 150μL FACS Buffer. Fluorescence shift detection was performed using a flow cytometer (BD FACS Celesta). The percentage of CD107a+ cells in the CD3-CD56+ cell population was counted.

3. Experimental results

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 good activation ability on primary NK cells, especially when the concentration was 10 nM, CD107A (%) was greater than that of Pembrolizumab-IL15 and pembrolizumab groups.

Table 10. Results of fusion protein NK cell activation experiment

Experimental Example 11: Fusion protein constructed by sushi domain mutant, IL15 and antibody stimulates CD8+ T cells Viability test

1. Experimental samples:

The fusion protein constructed by the sushi domain mutant obtained in the above-mentioned Preparation Example 3, IL15 and Pembrolizumab.

2. Experimental Methods

The activation ability of the fusion protein containing the sushi domain mutant on CD8+T cells was detected by flow cytometry. The experimental steps are as follows:

Resuscitate PBMC (SAILYBIO, NF0065), adjust the PBMC density to 5x10 ⁶ cells/mL, and pre-activate with 1μg/mL OKT3 for 24h. The next day, add 2E5 PBMC cells to a U-bottom 96-well plate at 50μL/well, and then add the fusion protein to be tested to a final concentration of 100nm, 10nM and 1nM. Incubate the cell culture plate in a cell culture incubator at 37°C and 5% CO ₂ overnight. Add 1E4 OVCR3 cells to the cell culture plate incubated overnight, 100μL/well. Mix gently and continue incubation in the cell culture incubator for 4 hours. Centrifuge at 500g for 5 minutes to remove the supernatant and wash the cells once with FACS buffer. Add LIVE/DEAD staining reagent (Violet fluorescent reactive Dye, invitrogen, L34964A) to distinguish live and dead cells, add fluorescently labeled CD8 antibody (PerCP/Cyanine5.5 anti-human CD8, Biolegend, 344710), fluorescently labeled CD25 antibody (Brilliant Violet 785 ^TM anti-human CD25 Antibody, Biolegend, 302638), and incubate in a refrigerator at 4 degrees in the dark for 30 minutes. Centrifuge at 500g for 5 minutes to remove the supernatant, add 200μL FACS buffer to each well, wash the cells twice, and resuspend the cells in 150μL FACS Buffer. Use flow cytometer (BD FACS Celesta) for fluorescence shift detection. Count the percentage of CD25+ cells in the CD8+ cell population.

3. Experimental results

The results are shown in Table 11. The results show that the fusion protein constructed by the sushi mutant of the present invention and pembrolizumab has good CD8+T cell activation ability, especially at 1 nM, CD8+CD25+(%) is greater than that of pembrolizumab and Pembrolizumab-IL-15 group.

Table 11. Results of the fusion protein CD8+T cell activation experiment

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims (16)

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. The sushi domain mutant according to claim 1, comprising the following mutation: 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. The sushi domain mutant according to claim 1 or 2, comprising the following mutation: 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. 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 at least one amino acid residue _among X1 _{- X9} is K, _R or Q; Optionally, X ₂ is K; Optionally, X ₃ 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; Alternatively, 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. 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 as 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 shown in SEQ ID NO:31. The fusion protein according to claim 5, wherein the sushi domain mutant and IL-15 are on the same peptide chain or are 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. 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 as SEQ ID NO:32. The fusion protein according to any one of claims 5 to 7, wherein the amino acid sequence of the fusion protein is as 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. 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 CDR in SEQ ID NO:42, and the LCDR1, LCDR2 and LCDR3 of the antibody are identical to the CDR 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. 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) having the function of binding to cells expressing IL15 receptors; optionally, it can bind to KARPASS-299 with an _EC50 value of less than 10 nM, and the _EC50 value is 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) has a higher 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%. An isolated nucleic acid molecule encoding the sushi domain mutant according to any one of claims 1 to 4, or encoding the fusion protein according to any one of claims 5 to 9. A recombinant vector comprising the isolated nucleic acid molecule according to claim 15; optionally, the recombinant vector is a recombinant expression vector. A recombinant host cell comprising the isolated nucleic acid molecule of claim 15, or comprising the recombinant vector of claim 16. A pharmaceutical composition comprising the sushi domain mutant according to any one of claims 1 to 4, Or it comprises the fusion protein according to any one of claims 5 to 9, and one or more pharmaceutically acceptable excipients. Use of the sushi domain mutant according to any one of claims 1 to 4, the fusion protein according to any one of claims 5 to 9, the isolated nucleic acid molecule according to claim 10, the recombinant vector according to claim 11, the recombinant host cell according to claim 12, or the pharmaceutical composition according to 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. A method for treating or preventing tumors, immunodeficiency or infectious diseases, the method comprising administering to a subject in need thereof a therapeutically effective amount of the sushi domain mutant according to any one of claims 1 to 4, the fusion protein according to any one of claims 5 to 9, the isolated nucleic acid molecule according to claim 10, the recombinant vector according to claim 11, the recombinant host cell according to claim 12, or the pharmaceutical composition according to 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.