Engineered immune effector cell as well as composition and application thereof

Abstract

The present application relates to genetically modified immune cells to express a recombinant human CD16 protein having an enhanced anti-shear ability with the presence of addition, deletion, substitution, or any combination of addition, deletion, substitution of one or more amino acids compared to a wild-type amino acid sequence.

Description

Engineered immune effector cells and their compositions and uses

[0001] Related applications

[0002] This application claims priority to Chinese patent application 202211198709.8 filed on September 29, 2022 and Chinese patent application 202310311716.2 filed on March 24, 2023, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] The present application relates to the field of cells, and in particular, to engineered immune effector cells. Background Art

[0004] CD16, also known as FcγRIII (low affinity immunoglobulin gamma Fc region receptor III), is a cell surface antigen (cluster of differentiation) found on immune cells. It is a type III Fcγ receptor that binds to the Fc fragment of immunoglobulin G (IgG). CD16 is divided into two proteins: CD16a (Fc region receptor III-A, FCGR3A) and CD16b (Fc region receptor III-B, FCGR3B). CD16a and CD16b share 96% sequence similarity in their extracellular antibody-binding domains. The amino acid sequence of CD16a is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2. CD16a is expressed on the surface of natural killer cells (NK cells), mast cells, monocytes, and macrophages, while CD16b is expressed only on neutrophils. The amino acid sequence of CD16a is 254 amino acids in length (see SEQ ID NO: 1), and is composed of a signal peptide, an extracellular domain containing two Ig-like domains, a transmembrane region, and an intracellular domain.

[0005] As a member of the immunoglobulin superfamily (IgSF), CD16 is a low-affinity IgG receptor containing two extracellular Ig-like domains that complement the receptor-binding region on the antibody Fc receptor. Binding stimulates immune cells to initiate immune responses such as phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and degranulation, attacking target cells such as cancer cells and virus-infected cells. Antibody binding to CD16a on NK cells stimulates the production of CD25 and cytokines such as interferon-γ and tumor necrosis factor-α, thereby initiating ADCC. CD16a can also activate NK cells independently, lysing target cells, even without antibody binding. Cytokine activation of NK cells, target cell interactions, and / or tumor infiltration can lead to CD16a cleavage and impair ADCC activity.

[0006] Summary of the Invention

[0007] This application describes the preparation of immune cells expressing recombinant CD16 protein. Compared to existing technologies, this recombinant CD16 protein is resistant to shearing, possesses excellent binding activity to the Fc region of human IgG1 antibodies, and can induce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0008] In a first aspect, the present application provides a cell, wherein the cell is genetically modified to contain or express an amino acid-modified CD16 protein. In some embodiments, the CD16 protein is derived from humans, rats, mice, monkeys, pigs, dogs, etc.

[0009] In some embodiments, the wild-type amino acid sequence of the CD16 protein can be selected from SEQ ID NO: 1 or 3.

[0010] In some embodiments, the amino acid-modified CD16 protein is a CD16 protein in which one or more amino acids are mutated compared to a wild-type CD16 protein.

[0011] In some embodiments, the amino acid-modified CD16 protein includes one or more amino acid additions, deletions, substitutions, or any combination of additions, deletions, and substitutions compared to the wild-type CD16 protein amino acid sequence.

[0012] In some embodiments, the one or more amino acid replacements include replacement of the glutamine residue at position 192; replacement of the leucine residue at position 194; replacement of the valine residue at position 196; replacement of the threonine residue at position 198; replacement of the isoleucine residue at position 199; and / or replacement of the serine residue at position 200 of SEQ ID NO:3.

[0013] In some embodiments, the one or more amino acid replacements include Q192P, L194P, V196P, T198P, I199P, S200P, L194Y, L194V, L194K, L194I, A195V, V196E, V196D, V196K, V196N, V196G, V196R, V196Q, V196M, V196H, T191S, Q192N, Q192K, A195G, V196S, T198S, I199L and / or S200T.

[0014] In some embodiments, the amino acid-modified CD16 protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-22, 24-32, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 4-22, 24-32.

[0015] In some embodiments, the cell further expresses a chimeric antigen receptor (CAR); preferably, the CAR specifically targets BCMA and GPRC5D; preferably, the CAR targeting BCMA and GPRC5D comprises the amino acid sequence shown in SEQ ID NO:46.

[0016] In some embodiments, the cells further express IL-15 protein; preferably, the IL-15 protein comprises the amino acid sequence shown in SEQ ID NO:47.

[0017] In some embodiments, the cells express a fusion polypeptide comprising a CAR targeting BCMA and GPRC5D and a CD16 protein, wherein the fusion polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 51-62, 64-66, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 51-62, 64-66.

[0018] In some embodiments, the cell is a T cell, a natural killer (NK) cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic cell, a pluripotent stem cell, or an embryonic stem cell.

[0019] In some embodiments, the cell is a NK cell. In a preferred embodiment, the cell is a NK92 cell.

[0020] In some embodiments, the amino acid-modified CD16 protein is resistant to cleavage.

[0021] In some embodiments, the amino acid modified CD16 protein has ADAM17

[0022] In a second aspect, the present application provides a therapeutic composition comprising the cells described in any one of the above items or a cell population comprising the cells.

[0023] In some embodiments, the therapeutic composition further comprises an iPSC cell population, a NK cell population, a NK92 cell population, or a T cell population.

[0024] In some embodiments, the iPSC cell population, NK cell population, NK92 cell population, or T cell population is genetically modified to (i) specifically recognize a tumor antigen; or (ii) specifically recognize a viral target.

[0025] In some embodiments, the therapeutic composition further comprises an additional therapeutic agent; preferably, the additional therapeutic agent is an anti-tumor agent; more preferably, the anti-tumor agent is a monoclonal antibody; more preferably, the monoclonal antibody is Daratumumab or Cetuximab.

[0026] In a third aspect, the present application provides a method comprising administering to a patient in need of such treatment a therapy comprising administering to the patient the cells described in the first aspect, or the therapeutic composition described in the second aspect. In some embodiments, the present application provides a method for treating a patient in need thereof, comprising administering to the patient the cells described in the first aspect, or the therapeutic composition described in the second aspect.

[0027] In a fourth aspect, the present application further provides use of the cell described in the first aspect or the therapeutic composition described in the second aspect in the preparation of a medicament for use in the method described in the third aspect.

[0028] In some embodiments, the method of the third aspect or the drug of the fourth aspect is used to inhibit tumor cell proliferation; in some embodiments, the tumor cells are solid tumor cells or blood tumor cells.

[0029] In some embodiments, the method described in the third aspect further comprises administering a therapeutic drug to the patient; in some embodiments, the drug can be selected from monoclonal antibodies, polyclonal antibodies, small molecule therapeutic agents, antibody-drug conjugates, cytokines, etc.; in some embodiments, the drug inhibits tumor cell proliferation.

[0030] In some embodiments, the drug described in the fourth aspect further includes other therapeutic agents. Preferably, the agent is selected from monoclonal antibodies, polyclonal antibodies, small molecule therapeutic agents, antibody-drug conjugates or cytokines; more preferably, the agent inhibits tumor cell proliferation.

[0031] In a fifth aspect, the present application provides a drug or kit comprising the cell described in the first aspect or the composition described in the second aspect.

[0032] In a sixth aspect, the present application provides a shear-resistant recombinant CD16 protein, wherein the recombinant CD16 protein has one or more amino acid additions, deletions, substitutions, or any combination of additions, deletions, and substitutions compared to the wild-type CD16 protein amino acid sequence.

[0033] In some embodiments, the CD16 protein is derived from humans, rats, mice, monkeys, pigs, or dogs.

[0034] In some embodiments, the wild-type amino acid sequence of the CD16 protein can be selected from SEQ ID NO: 1 or 3.

[0035] In some embodiments, the one or more amino acid replacements include replacement of the glutamine residue at position 192; replacement of the leucine residue at position 194; replacement of the valine residue at position 196; replacement of the threonine residue at position 198; replacement of the isoleucine residue at position 199; and / or replacement of the serine residue at position 200 of SEQ ID NO:3.

[0036] In some embodiments, the one or more amino acid replacements include Q192P, L194P, V196P, T198P, I199P, S200P, L194Y, L194V, L194K, L194I, A195V, V196E, V196D, V196K, V196N, V196G, V196R, V196Q, V196M, V196H, T191S, Q192N, Q192K, A195G, V196S, T198S, I199L and / or S200T.

[0037] In some embodiments, the recombinant CD16 protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-22, 24-32, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 4-22, 24-32.

[0038] In a seventh aspect, the present application provides a fusion polypeptide, wherein the fusion polypeptide comprises any one of the recombinant CD16 proteins described in the sixth aspect.

[0039] In some embodiments, the fusion polypeptide further comprises a CAR sequence targeting BCMA and GPRC5D; preferably, the CAR sequence targeting BCMA and GPRC5D comprises the amino acid sequence shown in SEQ ID NO:46.

[0040] In some embodiments, the fusion polypeptide further comprises an IL-15 protein sequence; preferably, the IL-15 protein sequence comprises the amino acid sequence shown in SEQ ID NO:47.

[0041] In some embodiments, the fusion polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 51-62, 64-66, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 51-62, 64-66.

[0042] In an eighth aspect, the present application provides a nucleic acid molecule encoding the recombinant CD16 protein described in the sixth aspect or the fusion polypeptide described in the seventh aspect.

[0043] In the ninth aspect, the present application provides an expression vector comprising the nucleic acid molecule described in the eighth aspect.

[0044] In the tenth aspect, the present application provides a host cell comprising the expression vector described in the ninth aspect; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

[0045] Definitions and Explanations of Terms

[0046] Unless otherwise defined herein, scientific and technical terms related to the present application shall have the meanings understood by those of ordinary skill in the art.

[0047] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0048] The terms "comprising," "including," and "having" are used interchangeably herein to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "comprising," "including," and "having" in this document also provides for "consisting of" solutions. For example, "a composition comprising A and B" should be understood to include the following technical solutions: a composition consisting of A and B, as well as a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition."

[0049] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

[0050] The term "genetic modification" herein has its clear and common meaning, and may include, but is not limited to, for example, the process of modifying an organism or cell (such as bacteria), lymphocytes (such as T cells or NK cells), bacterial cells, eukaryotic cells, insects, plants or mammals with genetic material (such as nucleic acids) that has been changed using genetic engineering techniques. For example, the target genetic material can be first isolated and copied using molecular cloning methods to produce a DNA sequence, or nucleic acids (such as DNA) can be inserted into the host genome by synthesizing DNA and then inserting the construct into the host organism. Genes and gene expression can also be removed or "knocked out" using gene editing. Those skilled in the art will appreciate the many techniques for knocking out genes. Without limitation, genes and / or gene expression can be knocked out using techniques such as RNA interference, CRISPR or TALEN. Gene targeting is a different technique that uses homologous recombination to change endogenous genes, and can be used to delete genes, remove exons, add genes or introduce point mutations. The term "genetic modification" as used herein also includes cells or genes that are engineered or naturally mutated to express proteins different from wild-type proteins.

[0051] Genetic modification by transduction is described herein. When read according to the specification sheets, "transduction" has its clear and common meaning and may include, but is not limited to, for example, a method for transferring genetic material (such as DNA or RNA) to a cell by a vector. Common techniques use viral vectors, electroporation, and chemical reagents to increase cell permeability. DNA can be transferred by virus or by viral vectors. As described herein, methods for modifying immune cells (such as natural killer cells) are provided. Viral vectors can be derived from adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus.

[0052] Various transduction techniques have been developed that utilize recombinant infectious viral particles for delivery. This represents a currently preferred cell transduction method. Viral vectors that can be used for transduction can include viral vectors derived from simian virus 40, adenovirus, adeno-associated virus (AAV), slow virus vectors and retroviruses. Therefore, gene transfer and expression methods are many, but basically play the role of introducing and expressing genetic material in mammalian cells. Several of the above-mentioned technologies can be used for transducing cells, including calcium phosphate transfection, protoplast fusion, electroporation and infection with recombinant adenovirus, adeno-associated virus, slow virus or retrovirus vectors. Lymphocytes have been successfully transduced by electroporation and retrovirus or slow virus infection. Therefore, retrovirus and slow virus vectors can provide an efficient method for gene transfer in eukaryotic cells. Retrovirus and slow virus vectors provide an efficient method for gene transfer in lymphocytes (such as T cells and NK cells). In addition, retrovirus or slow virus integration occurs in a controlled manner and causes the stable integration of the new genetic information of one or several copies of each cell.

[0053] As used herein, the term "amino acid modification" includes mutations of one or more amino acids, such as additions, deletions, substitutions, or any combination thereof. The term "amino acid-modified protein" herein refers to a protein whose amino acid sequence has been altered, such as one or more amino acid residues have been substituted, one or more amino acid residues have been added, or one or more amino acid residues have been deleted.

[0054] The term "PMA" in this article, which stands for Phorbol-12-myristate-13-acetate or 12-O-Tetradecanoylphorbol 13-acetate, is the most commonly used phorbol ester and can cause the cleavage or shedding of cell surface proteins, including CD16a.

[0055] The term "CD16" herein is a low affinity Fc receptor found on the surface of immune cells, such as natural killer cells, neutrophils, monocytes; or pluripotent stem cells or differentiated cells generated therefrom.

[0056] The term "natural killer cells" or "NK cells" herein has its clear and ordinary meaning and can include, but is not limited to, for example, natural killer cells from any tissue source and also includes natural killer cells produced using methods such as those described herein.

[0057] The term "hematopoietic cell" herein has its clear and ordinary meaning and may include, but is not limited to, for example, hematopoietic stem cells and hematopoietic progenitor cells.

[0058] The term "pluripotent" herein has its clear and ordinary meaning and may include, but is not limited to, for example, when referring to a cell, a cell that has the ability to differentiate into a cell of another cell type. In certain alternative embodiments, a "pluripotent stem cell" is a cell that has the ability to grow into a subset of the approximately 260 cell types of the mammalian body. Unlike totipotent cells, pluripotent stem cells do not have the ability to form all cell types.

[0059] As used herein, the term "therapeutic composition" refers to a preparation that is in a form effective to permit the biological activity of the active ingredient contained therein, and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0060] The term "inhibiting tumor cell proliferation" herein has its clear and ordinary meaning and can include, but is not limited to, for example, slowing the growth of a tumor cell population, such as by killing one or more tumor cells in the tumor cell population, such as by contacting a cell or cell population described herein or contacting an adjacent tumor cell population with a cell or cell population described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1. Schematic diagram of the plasmid structure for expressing recombinant CD16 protein.

[0062] Figure 2A-2A continued. Shear resistance results of different point mutations in recombinant CD16 protein. Flow cytometry was used to assess the proportion of CD16a positive cells on the surface of NK cells, and to analyze changes in CD16a expression on the cell surface before and after PMA treatment. Darker colors represent CD16a detection results before PMA treatment, while lighter colors represent detection results after PMA treatment.

[0063] Figure 2B. Statistics of CD16a positive rates on the cell surface before and after PMA treatment, where dark colors represent CD16a detection results before PMA treatment and light colors represent detection results after PMA treatment.

[0064] Figure 2C. Changes in the positive rate of CD16a on the cell surface before and after PMA treatment.

[0065] Figure 3. Schematic diagram of the plasmid structure of human BCMA / GPRC5D CAR targeting human BCMA / GPRC5D (A); and a schematic diagram of the plasmid structure of human BCMA / GPRC5D CAR targeting human BCMA / GPRC5D co-expressing recombinant CD16a protein containing point mutations (B).

[0066] Figure 4A. Flow cytometric detection results of the expression level of BCMA / GPRC5D CAR targeting human BCMA / GPRC5D on the surface of CA-NK cells co-expressing recombinant CD16a protein containing point mutations.

[0067] Figure 4B. Statistical results of BCMA / GPRC5D CAR expression on the surface of human BCMA / GPRC5D CA-NK cells co-expressing recombinant CD16a protein containing point mutations.

[0068] Figure 4C-4C continued. Shear resistance results of recombinant CD16 proteins with different point mutations. Flow cytometry was used to assess the proportion of CD16a-positive cells on the surface of human BCMA / GPRC5D-targeted CA-NK cells co-expressing recombinant CD16a proteins containing point mutations. Changes in cell surface CD16a expression before and after PMA treatment were analyzed. Darker colors represent CD16a detection results after PMA treatment, while lighter colors represent those before PMA treatment.

[0069] FIG5A . ADCC activity detection results of human BCMA / GPRC5D CAR-NK cells co-expressing recombinant CD16a protein containing point mutations after co-culture under E:T=1:1 conditions for 4 hours.

[0070] FIG5B . ADCC activity detection results of human BCMA / GPRC5D CAR-NK cells co-expressing recombinant CD16a protein containing point mutations after co-culture under E:T=5:1 for 4 hours.

[0071] Figure 5C. ADCC activity detection results of human BCMA / GPRC5D CAR-NK cells co-expressing recombinant CD16a protein containing point mutations after co-culture under E:T=1:1 conditions for 24 hours.

[0072] Figure 6A. Flow cytometry detection results of the expression of recombinant CD16a protein containing point mutations on the surface of NK92 cells.

[0073] FIG6B . ADCC activity detection results of NK92 cells expressing recombinant CD16a protein containing point mutations after co-culture under E:T=1:1 condition for 24 hours.

[0074] Figure 7. Schematic diagram of the fusion polypeptide structure targeting human CD19CAR and recombinant CD16a containing point mutations.

[0075] Figure 8. ADCC activity test results of targeted human CD19CAR-NK cells expressing recombinant CD16a protein containing point mutations prepared by iPSC induced editing after co-culture under different E:T conditions for 4 hours. DETAILED DESCRIPTION

[0076] The present application will be further described below with reference to specific examples, and the advantages and features of the present application will become clearer as the description proceeds. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or those recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0077] The embodiments of the present application are merely exemplary and do not constitute any limitation on the scope of the present application. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present application may be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements shall fall within the scope of protection of the present application.

[0078] Unless otherwise specified, the target cells MOLP8 (Nanjing Kebai, Cat# CBP60562) and HCT-116 (Nanjing Kebai, Cat# CBP60028) referred to in the following examples were transfected with the luciferase gene to express luciferase. Fluorescence intensity was measured using a luciferase reporter gene assay to reflect cell viability and NK cell cytotoxicity.

[0079] Example 1 Expression vector construction

[0080] 1.1 Introduction of site-directed mutagenesis of CD16a protein

[0081] Amino acid sequence of CD16a protein (NP_000560.7, NCBI database): Note: The single-line part is the signal peptide, the double-line part is the transmembrane region, the italic part is the intracellular domain, and the rest is the extracellular domain containing two Ig-like domains.

[0082] Nucleotide coding sequence of CD16a protein (NM_000569.8, NCBI database):

[0083] The introduced mutations are as follows: Q192P, L194P, V196P, T198P, I199P, S200P, L194Y, L194V, L194K, L194I, A195V, V196E, V196D, V196K, V196N, V196G, V196R, V196Q, V196M, V196H, T191S, Q192N, Q192K, A195G, V196S, T198S, I199L, and S200T. The sequences after mutation are shown in Table 1.

[0084] Table 1. Amino acid sequences of recombinant CD16a containing point mutations

[0085] 1.2 Plasmid construction

[0086] Conventional molecular biological methods in the art were used. In this example, with reference to the plasmid schematic shown in FIG. 1 , retroviral shuttle plasmids 1600, 1602, 1604, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1615, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1629, 1630, 1631, 1632, 1633, 1635, 1636, 1637, 1638, 1639, and 1640 expressing CD16a protein were constructed using retroviral vector templates.

[0087] First, the insert sequences 16, 16-2, 16-4, 16-6, 16-7, 16-8, 16-9, 16-10, 16-11, 16-12, 16-15, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 16-26, 16-27, 16-30, 16-31, 16-32, 16-33, 16-35, 16-36, 16-37, 16-38, 16-39 and 16-40 were synthesized respectively, and EcoRI and SalI restriction sites and corresponding vector homologous sequences were added at both ends. Among them, the insertion sequence of control plasmid 1600 is a recombinant CD16a protein carrying a natural F176V point mutation, control plasmid 1629 is a retroviral shuttle plasmid without an insertion sequence, and the insertion sequence 16-30 of the positive control plasmid 1630 comes from patent US2020 / 0017570Al.

[0088] The retroviral vector template was digested with restriction endonucleases EcoRI (Thermo, Cat#FD0274) and SalI (Thermo, Cat#FD0644), and the linearized plasmid was recovered and purified by agarose gel electrophoresis. The polynucleotide sequences synthesized in the above steps were ligated into the linearized vector using the recombinase 5× In-Fusion HD enzyme (TaKaRa, Cat#ST0344). The reaction system consisted of 2 μl of the synthesized polynucleotide fragment (50 ng / μl), 1 μl of the linearized plasmid (50 ng / μl), 2 μl of 5× HD In-Fusion enzyme, and 5 μl of ddH2O. The mixture was gently pipetted to mix, centrifuged briefly, and incubated at 50°C for 15 min. Add 10 μl of the recombinant reaction product to 100 μl of competent bacterial cells and place on ice for 5 minutes. Evenly spread the transformed bacterial solution on LB plates containing 50 μg / ml kanamycin and incubate inverted in a constant temperature incubator for 12-16 hours. Randomly select 3-5 clones from each plate for sequencing verification. Transfer the correctly sequenced bacterial solution to 100 ml of LB liquid medium containing 50 μg / ml kanamycin and incubate at 37°C overnight. Use the MN Endotoxin-Free Plasmid Extraction Kit (MN, Cat#740420.50) for plasmid extraction. After quantification, dilute to 1000 ng / μl with endotoxin-free ultrapure water to obtain the above-mentioned retroviral shuttle plasmid expressing the CD16a protein.

[0089] 1.3 Retrovirus Preparation

[0090] 293T cells (Cell Bank of the Chinese Academy of Sciences, Cat# GNHu17) were seeded into 100 mm culture dishes and cultured in DMEM (Gibco, Cat# 10566016) supplemented with 10% FBS (Gibco, Cat# 10099141). When the 293T cells reached approximately 70% of the dish surface, plasmid transfection was performed: a retroviral shuttle plasmid expressing CD16a protein and a packaging plasmid were mixed and added to 1.2 ml of Opti-MEM medium (Thermofisher Scientific, Cat# 31985070). 35 μl of Fugene HD (Promega, Cat# 04709691001) was added and mixed thoroughly. The mixture was incubated at room temperature for 15 minutes. The mixture was then added to the culture medium of healthy 293T cells and cultured in a constant temperature incubator (37°C, 5% CO2). After 48 hours, the 293T cell supernatant was collected, filtered through a 0.45 μm filter membrane, and concentrated for later use.

[0091] Example 2 Detection of CD16a shearing effect induced by PMA

[0092] 2.1 NK cell culture and viral infection

[0093] Fresh PBMCs were centrifuged at 500 g for 7 minutes at room temperature, the supernatant was discarded, and NK cells were isolated according to the protocol provided by the Human NK Cell Isolation Kit (Stemcell, Cat#17955). The isolated NK cells were activated with K562 cells. The activation method was as follows: on day 0, the cells were counted using the AO / PI staining method, and the cells were mixed at a ratio of NK:K562 = 1:2. The mixed cells were added to a non-treated 6-well plate at 2 ml / well (the culture medium was NK cell culture medium containing 200 IU / ml human IL-2 (Miltenyi Biotec, Cat#130-114-429) and cultured in an incubator (37°C, 5% CO2); on day 4, 3 ml of culture medium was added to each well; on day 5, 7 μg / ml of IL-2 was used to stimulate the cells. RetroNectin reagent (Takara, Cat#T202) was coated on a 24-well plate, 500 μl per well, at 4°C overnight. On day 6, the corresponding retrovirus was added to the 24-well plate. The 24-well plate with the virus added was centrifuged at 2000g, 4-8°C for 60 minutes. The upper viral liquid was discarded. NK cells were counted and added to the 24-well plate at 3E5 cells / well. The plate was centrifuged at 400g for 5 minutes at room temperature. The 24-well plate was placed in an incubator for incubation (37°C, 5% CO2). On day 7, the transfected NK cells were transferred to a non-treated 6-well plate and continued to be cultured.

[0094] 2.2 Detection of CD16a shedding results

[0095] On day 5 after virus infection, the expression of CD16a on the cell surface was detected. Before the start of the flow cytometry experiment, 100 ng / ml PMA (STEMCELL, Cat#74044) was added to the culture medium. After 40 minutes, the cells were washed three times by centrifugation with flow cytometry staining buffer (Gibco, Cat#00-4222-26), incubated with 2 μg / ml CD16a nanobody (derived from patent PCT / CN2022 / 101713, VHH-Fc12-05, homemade) at 4°C for 1 hour, and then washed three times by centrifugation with flow cytometry staining buffer. A 1:800 diluted secondary antibody Alexa Fluor 647 AffiniPure Goat Anti-Human IgG Fcγ fragment specific antibody (Jackson ImmunoReearch, Cat#109-605-098) was added and incubated at 4°C for 1 hour. The cells were washed three times by centrifugation with flow cytometry staining buffer, and the cells were suspended with 100 μl FACS buffer. The cells were detected and analyzed using a flow cytometer (BD, CANTO II).

[0096] The results are shown in Figures 2A and 2B. After the addition of PMA, almost all CD16a on the surface of primary NK cells (Parental NK) and cells infected with empty virus 1629 was shed, while the positive rate of CD16a with introduced point mutation on the cell surface was significantly increased. As shown in Table 2, the CD16a positivity rates on the surfaces of cells 1602, 1604, 1607, 1610, 1611, 1612, 1615, 1623, 1625, 1626, 1627, 1632, 1635, and 1637 were 64.7%, 68.4%, 56.9%, 55.4%, 54.9%, 55.3%, 73.8%, 57.3%, 65.1%, 71.6%, 62.2%, 60.1%, 61.9%, and 57.1%, respectively, which were similar to or higher than the positive rate (64.9%) of the positive control 1630. Unstained cells represent the flow cytometry results of primary NK cells without the addition of primary antibody.

[0097] Table 2. CD16a expression on cell surface before and after PMA treatment

[0098] As shown in Figure 2C, after the addition of PMA, the change in the positive rate of CD16a on the cell surface with the introduced point mutation was significantly reduced. As shown in Table 3, the changes in the positive rate of CD16a on the surface of cells 1602, 1604, 1607, 1610, 1611, 1612, 1615, 1623, 1625, 1626, 1627, 1632, 1635, and 1637 were 26.7%, 22.3%, 31.2%, 35.1%, 36.2%, 35.8%, 18.9%, 33.1%, 27.8%, 21.8%, 30.0%, 31.6%, 29.5%, and 29.3%, respectively. These changes were similar to or lower than the positive rate change value of the positive control 1630 (28.6%). Unstained (Unstaining) represents the flow cytometry results of primary NK cells without the addition of primary antibody.

[0099] Table 3. Changes in CD16a positive rate on cell surface before and after PMA treatment

[0100] Therefore, we demonstrated that the introduction of point mutations Q192P, L194P, T198P, L194Y, L194V, L194K, A195V, V196G, V196Q, V196M, V196H, Q192N, L194I, or V196S into CD16a could effectively block shear-induced CD16a shedding.

[0101] Example 3 Expression and purification of a recombinant fragment of the extracellular domain of the CD16a protein containing a point mutation

[0102] The recombinant pCDNA3.4 plasmid was constructed according to the amino acid sequence shown in Table 4 and Expi293 was used. TM The protein was expressed and purified using the expression system. The purification results are shown in Table 5.

[0103] Table 4. Amino acid sequences of recombinant fragments of the extracellular domain of CD16a protein containing point mutations

[0104] As shown in Table 5, the purity of the purified recombinant proteins was greater than 90% by SEC analysis. Compared to the wild-type CD16a protein 1600WE and the S197P point mutation 1630E, the purity of most recombinant proteins containing other point mutations was significantly improved, with the 1604E protein having a purity of 99.76% and the 1615E, 1623E, 1625E, and 1626E proteins having a purity of 100%. This indicates that the introduction of L194P, A195V, V196G, V196Q, and V196M, respectively, can significantly improve the purity of the CD16a protein extracellular domain fragment.

[0105] Table 5. Purity test results of recombinant fragments of the extracellular domain of CD16a protein containing point mutations

[0106] Example 4: Affinity determination of recombinant CD16a protein containing point mutations and IgG1

[0107] Antibody anti-FITC IgG1 (homemade) was adjusted to a final concentration of 2 μg / ml using HBS-EP+ buffer (Cytiva, Cat#BR-1006-69) and added to a 96-well plate (Greiner Bio-one, Cat#210100581) at 600 μl per well. Anti-FITC IgG1 was captured using a Protein A chip (Cytiva, Cat#29-1275-56) using a Biacore 8K instrument (Cytiva, Cat#Biacore 8K). The CD16a protein purified in Example 3 was serially diluted from 6000 nM to 187.5 nM using HBS-EP+ buffer and added to a 96-well plate (Greiner Bio-one, Cat#210100581) at 600 μl per well. Affinity values ​​were determined according to the Biacore 8K Control Software instructions. The results are shown in Table 6.

[0108] Table 6. Affinity values ​​of CD16a proteins containing different point mutations and IgG1

[0109] As shown in Table 6, compared with wild-type CD16a (KD value of 2.60E-06), the naturally occurring F176V point mutation (4.84E-07) can increase the affinity of CD16a to IgG1Fc; compared with the F176V point mutation, the additionally introduced point mutations have no effect on affinity.

[0110] Therefore, we demonstrated that the introduction of point mutations Q192P, L194P, A195V, V196G, V196Q, V196M, V196H, Q192N, L194I, or V196S into CD16a not only conferred the ability to resist shearing, but also effectively guaranteed the affinity with the Fc of IgG1 antibodies.

[0111] Example 5 Determination of ADCC activity of human BCMA / GPRC5D targeted CAR-NK cells co-expressing recombinant CD16a protein containing point mutations

[0112] 5.1 Expression vector construction

[0113] According to the method in Example 1, with reference to the plasmid diagram shown in Figure 3A, a retroviral shuttle plasmid BCAR was constructed as a negative control. The BCAR contained a CAR targeting human BCMA and GPRC5D (BCMA / GPRC5D CAR, SEQ ID NO: 46) and human IL-15 (SEQ ID NO: 47). Meanwhile, referring to the plasmid schematic shown in FIG3B, human CD16a sequences were introduced into BCAR: 16, 16-2, 16-4, 16-7, 16-10, 16-11, 16-12, 16-15, 16-23, 16-25, 16-26, 16-27, 16-30, 16-32, 16-33, 16-35, and 16-37 to construct retroviral shuttle plasmids B00, B02, B04, B07, B10, B11, B12, B15, B23, B25, B26, B27, B30, B32, B33, B35, and B37, of which B30 was a positive control. The relevant amino acid sequences are shown in Tables 7 and 8, respectively.

[0114] Table 7. Amino acid sequences of BCAR and its components

[0115] Table 8. Amino acid sequences of human BCMA / GPRC5D targeting CAR and human CD16 fusion polypeptides containing point mutations

[0116] 5.2 Detection of CD16a shedding results

[0117] Referring to the method in Example 2, cell culture, viral infection, PMA stimulation and protein expression detection were performed. The expression of BCMA / GPRC5D CAR was detected on the 5th day after infection. The expression of BCMA CAR on the surface of NK cells was detected by flow cytometry using FITC-labeled BCMA protein (Acrobiosystems, Cat#BCA-HF254), and the test results are shown in Figures 4A and 4B. The expression of BCMA / GPRC5D CAR on the surface of each CAR-NK cell was similar.

[0118] The expression of CD16a on the surface of NK cells was detected on the 6th day after viral infection. Before the CD16a shedding assay, 100 ng / ml PMA (STEMCELL, Cat#74044) was added to the culture medium. After 60 min, the cells were washed three times with flow cytometry staining buffer (Gibco, Cat#00-4222-26) by centrifugation, incubated with 2 μg / ml CD16a nanobody VHH-Fc12-05 (derived from patent PCT / CN2022 / 101713, VHH-Fc12-05, homemade) at 4°C for 1 h, and washed three times with flow cytometry staining buffer by centrifugation. A 1:800 dilution of the secondary antibody Alexa Fluor 647 AffiniPure Goat Anti-Human IgG Fcγ fragment specific (Jackson ImmunoResearch, Cat#109-605-098) was added and incubated for 1 h at 4°C, and washed three times with flow cytometry staining buffer by centrifugation. The cells were then lysed with 100 μl of PBS. The cells were suspended in FACS buffer and detected and analyzed using flow cytometry (BD, CANTO II).

[0119] The results are shown in Figure 4C. After the addition of PMA, almost all CD16a on the surface of primary NK cells (Parental NK) and cells expressing only the BCMA / GPRC5D CAR was shed. However, the positive rate of CD16a on the cell surface with the introduced point mutation was significantly increased. The positive rate of CD16a on cells B04, B15, B23, and B27 did not change significantly, and was similar to that of the positive control B30. Unstaining is the flow cytometry result of primary NK cells without the addition of primary antibody.

[0120] 5.3 ADCC activity detection

[0121] The ADCC activity of CAR-NK cells and Daratumumab (Biointron, Cat# B625101) targeting human BCMA / GPRC5D CAR-NK cells co-expressing CD16a protein with point mutations was verified. The target cells MOLP8-Luc were incubated with 10 nM Daratumumab at 4 ° C for 30 min, centrifuged and the supernatant was discarded. The MOLP8-Luc cells were resuspended in RPMI1640 medium (Gibco, Cat# 11875093) at a density of 2 × 10 4 The experiment was set at an effector-target ratio of 1:1 or 5:1. 100 μl of CAR-NK cells and 100 μl of MOLP8-Luc cells were added to an opaque 96-well plate, with duplicate wells set up. The plates were then co-cultured for 4 or 24 hours in a cell culture incubator. After co-culture, 50 μl of the luciferin bioluminescent substrate D-luciferin (Yisheng Biotechnology, Cat# 115144-35-9) was added. The cells were incubated in the dark for 10 minutes at room temperature, and then the plate was read and analyzed using a microplate reader (PE, Ensight-HH3400). The results are shown in Figure 5. Lower values ​​indicate fewer remaining target cells and stronger ADCC activity. As shown in Figure 5A, after 4 hours of co-culture under the E:T = 1:1 condition, the ADCC activity of each CAR-NK cell was similar, and there was no significant difference in the number of remaining target cells. As shown in Figure 5B, after 4 hours of co-culture under E:T = 5:1 conditions, the ADCC activity of each CAR-NK cell showed differences, with B02, B11, B23, and B27 showing stronger ADCC activity, and the number of remaining cells was similar to that of the positive control B30. As shown in Figure 5C, after 24 hours of co-culture under E:T = 1:1 conditions, the ADCC activity of each CAR-NK cell showed differences, with B02, B23, B27, and B37 showing stronger ADCC activity, and the number of remaining cells was significantly less than that of the positive control B30.

[0122] Example 6 Determination of ADCC activity of NK92 cells expressing recombinant CD16a protein containing point mutations

[0123] 6.1 NK cell culture and viral infection

[0124] According to the method of Example 1, NK92 cells were infected with retrovirus containing plasmids 1600, 1602, 1607, 1610, 1611, 1612, 1615, 1623, 1626, 1627, 1629, 1630, 1632, 1635, and 1637 in Example 1, respectively, to prepare NK92 cells 9200, 9202, 9207, 9210, 9211, 9212, 9215, 9223, 9226, 9227, 9229, 9230, 9232, 9235, and 9237 expressing recombinant CD16a proteins containing point mutations. The expression level of CD16a was detected 5 days after infection. The results are shown in Figure 6A, showing that all proteins were expressed on the surface of NK92 cells.

[0125] Table 9 NK92 cell numbers and corresponding mutation points expressing recombinant CD16a proteins containing point mutations

[0126] 6.2 ADCC activity assay

[0127] The ADCC activity of NK92 cells expressing CD16a protein with point mutations was verified by combining NK92 cells and Cetuximab (Biointron, Cat#B139201) to kill HCT-116-Luc cells. HCT116-Luc cells were resuspended in RPMI1640 medium (Gibco, Cat#11875093) at a density of 2×10 4 The experiment was set at an effector-target ratio of 1:1. 100 μl of NK92 cells and 100 μl of HCT-116-Luc cells were added to an opaque 96-well plate, with duplicate wells set up. The cells were co-cultured in a cell culture incubator for 24 hours. After the co-culture, 50 μl of the luciferin bioluminescent substrate D-luciferin (Yishen Biotechnology, Cat#115144-35-9) was added. The cells were incubated in the dark for 10 minutes at room temperature, and then the cells were read and analyzed using a microplate reader (PE, Ensight-HH3400). The results are shown in Figure 6B. After 24 hours of co-culture at an E:T ratio of 1:1, the ADCC activity of cells in each group was significantly different. 9207, 9210, 9211, 9212, 9215, and 9223 showed stronger ADCC activity than the positive control 9230, and the cell killing rate was higher. Mutated CD16a exhibited excellent ADCC stimulation on NK92 cells.

[0128] Example 7 Determination of ADCC activity of human CD19 CAR-NK cells co-expressing recombinant CD16a protein prepared by iPSC induction editing

[0129] 7.1 CAR-NK Cell Preparation

[0130] Referring to the currently disclosed induction method of induced pluripotent stem cells (iPSC), iPSC-derived NK cells are prepared. For the induction method, see, for example, Zhu, H., Kaufman, DS (2019). An Improved Method to Produce Clinical-Scale Natural Killer Cells from Human Pluripotent Stem Cells. In: Kaneko, S. (eds) In Vitro Differentiation of T-Cells. Methods in Molecular Biology, vol 2048. The induced NK cells were further subjected to a gene editing CAR NK cell method to prepare NK cells (referred to as 1916 cells) that co-expressed recombinant CD16a protein (containing a V196G point mutation) and human CD19 CAR. The gene editing method is described in, for example, Burnight ER, et.al. CRISPR-Cas9-Mediated Correction of the 1.02kb Common Deletion in CLN3 in Induced Pluripotent Stem Cells from Patients with Batten Disease. CRISPR J. 2018 Feb; 1(1): 75-87. The inserted gene comprises nucleotides encoding a fusion polypeptide targeting human CD19CAR, human IL-15, and recombinant human CD16a, the structural diagram of which is shown in FIG7 , and the relevant amino acid sequences are shown in Table 10, wherein the amino acid sequence of recombinant human CD16a carrying a V196G point mutation is shown in SEQ ID NO. 18.

[0131] Table 10. Amino acid sequences of proteins targeting human CD19CAR and human CD16 fusion polypeptides containing point mutations

[0132] 7.2 ADCC Activity Assay

[0133] The ADCC activity of 1916 cells expressing a point-mutated CD16a protein was verified using 1916 cells alone and in combination with cetuximab against HCT-116-Luc cells. Unedited NK cells served as a negative control, and groups of NK cells alone and NK cells combined with cetuximab were established. The experiments were performed at effector-target ratios (E:T) of 10:1, 5:1, 2.5:1, and 1.25:1. The cell lysis rate in each group was measured according to the method in Example 6.2. The percentage of target cell lysis increased by 1916 cells combined with cetuximab compared to 1916 cells alone was then calculated, as well as the percentage of target cell lysis increased by unedited NK cells combined with cetuximab compared to unedited NK cells alone in the negative control. The results are shown in Figure 8.

[0134] Because CD16a complements the receptor-binding region on the Fc region of cetuximab, binding stimulates cetuximab's ADCC activity. Figure 8 shows that, under varying effector-target ratios, co-culturing 1916 cells with cetuximab for 4 hours significantly enhanced cetuximab's ADCC activity, resulting in a higher percentage of cell killing, significantly outperforming unedited iPSC-derived NK cells. This demonstrates that mutant CD16a exhibits superior ADCC stimulation on iPSC-derived NK cells.

Claims

1. A cell, wherein the cell is genetically modified to express an amino acid-modified CD16 protein.

2. The cell according to claim 1, wherein the amino acid-modified CD16 protein comprises one or more amino acid additions, deletions, substitutions, or any combination of additions, deletions, and substitutions compared to the wild-type CD16 protein amino acid sequence.

3. The cell of claim 2, wherein the one or more amino acid substitutions comprise substitution of a glutamine residue at position 192; a leucine residue at position 194; a valine residue at position 196; a threonine residue at position 198; an isoleucine residue at position 199; and / or a serine residue at position 200 of SEQ ID NO:

3.

4. The cell of claim 3, wherein the one or more amino acid replacements comprise Q192P, L194P, V196P, T198P, I199P, S200P, L194Y, L194V, L194K, L194I, A195V, V196E, V196D, V196K, V196N, V196G, V196R, V196Q, V196M, V196H, T191S, Q192N, Q192K, A195G, V196S, T198S, I199L, and / or S200T.

5. The cell according to any one of claims 1 to 4, wherein the amino acid-modified CD16 protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 4-22 and 24-32, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 4-22 and 24-32.

6. The cell of any one of claims 1-5, wherein the cell further expresses a chimeric antigen receptor (CAR); preferably, the CAR specifically targets BCMA and GPRC5D.

7. The cell of claim 6, wherein the CAR targeting BCMA and GPRC5D comprises the amino acid sequence shown in SEQ ID NO:

46.

8. The cell according to claim 6 or 7, wherein the cell further expresses an IL-15 protein comprising the amino acid sequence shown in SEQ ID NO:

47.

9. The cell of claim 8, wherein the cell expresses a fusion polypeptide comprising a CAR targeting BCMA and GPRC5D and a CD16 protein, wherein the fusion polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 51-62, 64-66, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 51-62, 64-66.

10. The cell according to any one of claims 1 to 9, wherein the cell is a T cell, a natural killer (NK) cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic cell, a pluripotent stem cell or an embryonic stem cell; preferably, the cell is a NK cell; more preferably, the cell is a NK92 cell. The cell according to any one of claims 1 to 10, wherein the amino acid-modified CD16 protein has cleavage resistance.

12. A therapeutic composition comprising the cell of any one of claims 1 to 11 or a cell population comprising the cell.

13. The therapeutic composition of claim 12, wherein the therapeutic composition further comprises an iPSC cell population, a NK cell population or a T cell population; preferably, the iPSC cell population, the NK cell population or the T cell population is genetically modified to (i) specifically recognize a tumor antigen; or (ii) specifically recognize a viral target.

14. The therapeutic composition of claim 12 or 13, further comprising an additional therapeutic agent; preferably, the additional therapeutic agent is an anti-tumor agent; more preferably, the anti-tumor agent is a monoclonal antibody; more preferably, the monoclonal antibody is Daratumumab or Cetuximab.

15. A method for treating a patient in need thereof, comprising administering to the patient the cell of any one of claims 1-11 or the therapeutic composition of any one of claims 12-14.

16. Use of the cell of any one of claims 1 to 11 or the therapeutic composition of any one of claims 12 to 14 in the preparation of a medicament for treating a patient in need thereof.

17. The method of claim 15, wherein the method is used to inhibit tumor cell proliferation, preferably, the tumor cells are solid tumor cells or blood tumor cells; optionally, the method further comprises administering other therapeutic agents to the patient, preferably, the agents are selected from monoclonal antibodies, polyclonal antibodies, small molecule therapeutic agents, antibody drug conjugates or cytokines; more preferably, the agents inhibit tumor cell proliferation.

18. The use according to claim 16, wherein the drug is used to inhibit tumor cell proliferation, preferably, the tumor cells are solid tumor cells or blood tumor cells; optionally, the drug further comprises other therapeutic agents, preferably, the agents are selected from monoclonal antibodies, polyclonal antibodies, small molecule therapeutic agents, antibody drug conjugates or cytokines; more preferably, the agents inhibit tumor cell proliferation.

19. A medicament or kit comprising the cell of any one of claims 1-11 or the therapeutic composition of any one of claims 12-14.

20. A shear-resistant recombinant CD16 protein, wherein the recombinant CD16 protein has one or more amino acid additions, deletions, substitutions, or any combination of additions, deletions, and substitutions compared to the wild-type amino acid sequence of CD16 protein.

21. The protein according to claim 20, wherein the CD16 protein is derived from human, rat, mouse, monkey, pig or dog, and preferably, the amino acid sequence of the wild-type CD16 protein is selected from SEQ ID NO: 1 or 3.

22. The protein of claim 20 or 21, wherein the one or more amino acid substitutions comprise substitutions of the glutamine residue at position 192; substitutions of the leucine residue at position 194; substitutions of the valine residue at position 196; substitutions of the threonine residue at position 198; substitutions of the isoleucine residue at position 199; and / or substitutions of the serine residue at position 200 of SEQ ID NO:

3.

23. The protein of any one of claims 20-22, wherein the one or more amino acid replacements comprise Q192P, L194P, V196P, T198P, I199P, S200P, L194Y, L194V, L194K, L194I, A195V, V196E, V196D, V196K, V196N, V196G, V196R, V196Q, V196M, V196H, T191S, Q192N, Q192K, A195G, V196S, T198S, I199L, and / or S200T.

24. The protein of any one of claims 20-23, wherein the protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 4-22, 24-32, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 4-22, 24-32.

25. A fusion polypeptide, wherein the fusion polypeptide comprises the recombinant CD16 protein according to any one of claims 20 to 24.

26. The fusion polypeptide of claim 25, further comprising a CAR sequence targeting BCMA and GPRC5D; preferably, the CAR sequence targeting BCMA and GPRC5D comprises the amino acid sequence shown in SEQ ID NO:

46.

27. The fusion polypeptide according to claim 26, further comprising an IL-15 protein sequence; preferably, the IL-15 protein sequence comprises the amino acid sequence shown in SEQ ID NO:

47.

28. The fusion polypeptide of any one of claims 25 to 27, comprising the amino acid sequence of any one of SEQ ID NOs: 51 to 62, 64 to 66, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 51 to 62, 64 to 66.

29. A nucleic acid molecule encoding the protein according to any one of claims 20 to 24 or the fusion polypeptide according to any one of claims 25 to 28.

30. An expression vector comprising the nucleic acid molecule of claim 29.

31. A host cell comprising the vector of claim 30; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).