Human il-15 mutant and application thereof

Abstract

The invention discloses mutants of human IL-15 molecules and fusion proteins containing IL-15 mutants and combined mutations. The fusion proteins can mediate the activation and amplification of immune cells and can be used for the treatment of tumor diseases.

Description

[Technical Field]

[0001] This invention relates to human IL-15 mutants, nucleic acids encoding them, fusion proteins containing IL-15 mutants and combined mutants, pharmaceutical compositions, and related uses of the pharmaceutical compositions for the treatment of tumors. [Previous Technology]

[0002] Interleukin-15 (IL-15) is an important soluble cytokine discovered and named by Grabstein in 1994 in the culture supernatant of monkey kidney intraepithelial cell line CV-1 / EBNA. IL-15 can be expressed in various cells and tissues, such as monocytes / macrophages, lymphocytes, and epithelial cells. IL-15 biological activity...

[0003] IL-15-mediated signaling pathways

[0004] Interleukin-15 receptor (IL15R): IL15R belongs to the hematopoietic factor superfamily and is composed of three subunits: α, β (also known as CD122), and γ (also known as CD132, common gamma chain, γc). IL2 and IL15 share the β chain receptor. IL2, IL4, IL7, IL9, IL15, and IL21 share the γ chain receptor. IL2 and IL15 share the β and γc chains, but both IL2 and IL15 have their own specific α receptor chains. Human IL15Rα is a type I transmembrane protein. Both IL2Rα and IL15Rα have a conserved protein-binding motif (sushi domain). IL15 has a high affinity for IL15Rα (Kd ~ 10⁻¹¹ M), but does not transmit signals. IL15 has a moderate affinity for the IL15βγ heterodimer (Kd ~ 10⁻⁹ M) and can transduce signals; IL15 has a similar affinity for the IL15αβγ heterotrimer (Kd ~ 10⁻⁹ M) and can transduce signals. Because IL2 and IL15 share the same β and γ chains as their receptors, they share many similar biological functions, such as both promoting the proliferation of T cells and NK cells.

[0005] IL15 Binding to Receptors: IL15Rα is mainly expressed in dendritic cells (DCs) and monocytes. In most cases, IL15 / IL15Rα binds to its receptor in a trans-presented form. That is, after IL-15 and IL-15Rα are expressed in the same cells, intracellular IL-15 binds to the sushi domain of IL-15Rα with high affinity and is then transported to the membrane surface. There, it binds to the βγ heterodimer complex or αβγ heterotrimer complex on the membrane surface of reactive cells (such as T cells or NK cells). β and γ receptors can activate downstream Jak1 and Jak3, respectively, leading to STAT-3 and STAT-5 activation, triggering a cascade reaction, and inducing specific gene expression. When IL15 acts on effector cells in an autocrine form, it can interact with the IL15 receptor in a cis form, activating downstream signaling to produce effector functions.

[0006] Immunomodulatory effects of IL-15

[0007] IL-15 has a wide range of immunomodulatory effects, participating in the regulation of the activity, proliferation and function of various immune cells. (1) Regulation of T cells: It promotes the activation and proliferation of T cells, promotes the production of memory CD8+ T cells, and plays an important role in maintaining the number of memory CD8+ T cells in the body. Even in the presence of Treg cells, IL-15 can maintain the function and number of CD8+ T cells well. (2) Regulation of NK cells: IL-15 plays an important role in the activation and proliferation of NK cells and can improve the ADCC killing ability of NK cells. (3) Regulation of other immune cells: IL-15 also plays an important role in the functional maturation of DC cells and macrophages. IL-15 can promote the expression of co-stimulatory factors and IFN-γ by DC cells and improve the ability of DC cells to activate CD8+ T cells and NK cells. In addition, IL-15 can promote the proliferation of neutrophils. Anti-tumor effect of IL-15

[0008] IL-15 exerts its anti-tumor effect based on its ability to expand and activate various immune cells. Clinical studies have confirmed that IL-15 has excellent anti-tumor efficacy. However, due to the short half-life of wild-type IL-15, its small molecular size, and high renal clearance rate, multiple daily injections or subcutaneous administration are extremely inconvenient. Therefore, the use of wild-type recombinant IL-15 alone is also limited in tumor treatment.

[0009] Studies have shown that reducing the proliferative activity of IL-15 on T cells and NK cells can increase the half-life of IL-15 while reducing its toxicity. Furthermore, combining IL-15 with antibodies targeting tumor-associated antigens to form fusion proteins can increase the specificity of IL-15, raise its concentration in the tumor microenvironment, and reduce its toxicity. Therefore, developing IL-15 mutants with reduced activity has the potential to improve the dose-response relationship of IL-15 in tumor treatment and expand the clinical application of IL-15 in anti-tumor therapy, which has significant social and economic implications. [Summary of the Invention]

[0010] The present invention provides an IL-15 mutant, a nucleic acid encoding the mutant, a fusion protein comprising the mutant and a pharmaceutical composition, and their functions for killing tumor cells and their use in treating tumors.

[0011] In a first aspect, the present invention discloses an IL-15 mutant polypeptide comprising a mutation at one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15.

[0012] In a second aspect, the present invention discloses a polypeptide comprising an IL-15 mutant, said polypeptide comprising a mutation at one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15.

[0013] In one embodiment, the IL-15 mutant polypeptide contains mutations at two, three, or four amino acid residues of Val3, Ile6, Asp8, or His105.

[0014] In one embodiment, the mutation is a substitution, insertion, or deletion.

[0015] In one specific embodiment, the mutation is selected from the amino acid substitutions of the group consisting of: Val3Leu (V3L), Ile6Asp (I6D), Ile6Pro (I6P), Asp8Glu (D8E), Asp8Gln (D8Q), Asp8Arg (D8R), Asp8Ser (D8S), Asp8Val (D8V), Asp8Gly (D8G), Asp8 Ile (D8I), Asp8Leu (D8L), Asp8Thr (D8T), His105Asn (H105N), and / or His105Lys (H105K).

[0016] In one specific embodiment, the IL-15 mutant polypeptide or the polypeptide containing the IL-15 mutant comprises the following mutations or combinations of mutations: (1) Asp8Glu; (2) Asp8Gln; (3) Asp8Arg; (4) Asp8Ser; (5) Asp8Val; (6) Val3Leu; (7) Ile6Asp; (8) His105Lys; (9) His105Asn; (10) Asp8Gly; (11) Asp8Ile; (12) Asp8Leu; (13) Ile6Pro; (14) Asp8Thr; (15) Asp8Glu and Val3Leu; (16) Asp8Glu and Ile6Asp; (17) Val3Leu and Ile6Asp; (18) Ile6Asp and His105Lys; (19) Asp8Ser And His105Lys;

[0017] (20) Asp8Ser and His105Asn; or, (21) Val3Leu, Ile6Asp and His105Lys.

[0018] In one specific embodiment, the IL-15 mutant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with human wild-type IL-15.

[0019] In one specific embodiment, the amino acid sequence of the IL-15 mutant is as shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.43, SEQ ID NO.45 or SEQ ID NO.47.

[0020] In one specific embodiment, the IL-15 mutant polypeptide or the polypeptide containing the IL-15 mutant has the following characteristics: (1) mediates the proliferation of human CD8+ T cells; (2) mediates the proliferation of human NK cells; and / or, (3) inhibits tumor growth.

[0021] In one specific embodiment, the IL-15 mutant peptide or peptide containing the IL-15 mutant has lower activity in mediating CD8+ T and / or NK cell proliferation / expansion than peptide containing wild-type IL-15.

[0022] In one specific embodiment, the amino acid sequence of the wild-type IL-15 is shown in SEQ ID NO.1.

[0023] In a third aspect, the present invention discloses a protein comprising the aforementioned IL-15 mutant polypeptide or a polypeptide comprising an IL-15 mutant; and further comprising an immunoglobulin molecule or a portion thereof fused with the IL-15 mutant, and / or IL-15Rα.

[0024] In one embodiment, the immunoglobulin molecule is an antibody or antigen-binding fragment; the immunoglobulin molecule portion is an immunoglobulin Fc region.

[0025] In one embodiment, the antibody or antigen-binding fragment is selected from: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or, (3) a fully human antibody or a fragment thereof.

[0026] In one specific embodiment, the antibody or antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody, and antibody minimum recognition unit.

[0027] In one specific embodiment, the immunoglobulin Fc region is selected from the Fc region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, it contains the sequence of the constant region of human or mouse antibody IgG1, IgG2, IgG3 or IgG4; preferably, the amino acid sequence of the immunoglobulin Fc region is as shown in SEQ ID NO.73.

[0028] In another embodiment, the IL-15 mutant is fused to an immunoglobulin molecule or a portion thereof with or without a linker peptide, or the IL-15 mutant is fused to IL-15Rα with or without a linker peptide; a linker peptide is preferred; the linker peptides shown in SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69 or SEQ ID NO. 71 are preferred.

[0029] In another embodiment, the IL-15 mutant is fused to IL-15Rα with or without a linker peptide, and then fused to an immunoglobulin molecule or a portion thereof; preferably, a linker peptide is used; preferably, the linker peptide shown in SEQ ID NO.65, SEQ ID NO.69 or SEQ ID NO.71 is used.

[0030] In one specific implementation, the connection order of each structural domain from the N end to the C end is as follows:

[0031] (1) Immunoglobulin molecules or portions thereof, IL-15Rα, IL-15 mutants;

[0032] (2)Immunoglobulin molecules or portions thereof, IL-15 mutants, IL-15Rα;

[0033] (3)IL-15 mutant, IL-15Rα, immunoglobulin molecule or part thereof;

[0034] (4)IL-15Rα, IL-15 mutant, immunoglobulin molecule or part thereof;

[0035] (5)IL-15 mutant, immunoglobulin molecule or part thereof;

[0036] (6) Immunoglobulin molecules or portions thereof, IL-15 Rα;

[0037] (7)IL-15Rα, immunoglobulin molecules or portions thereof;

[0038] (8)IL-15 mutant, IL-15Rα; or,

[0039] (9)IL-15Rα,IL-15 mutant.

[0040] In another embodiment, when IL-15Rα or IL-15 mutant is fused with an immunoglobulin molecule, it is fused to the N-terminus of the variable region of the heavy chain of the immunoglobulin molecule or the C-terminus of the Fc region of the immunoglobulin; when IL-15Rα or IL-15 mutant is fused with the Fc region of the immunoglobulin, it is fused to the N-terminus or the C-terminus of the Fc region of the immunoglobulin.

[0041] In a fourth aspect, the present invention discloses a protein or antibody fusion construct / complex comprising the following four parts:

[0042] (1) Immunoglobulin heavy chain;

[0043] (2)Immunoglobulin light chains;

[0044] (3)IL-15Rα; and,

[0045] (4)The IL-15 mutant polypeptide as described in the first and second aspects above.

[0046] In one embodiment, IL-15Rα is fused to the N-terminus of the variable region of the immunoglobulin heavy chain or the C-terminus of the Fc region of the immunoglobulin, with or without a linker peptide.

[0047] In one embodiment, the IL-15 mutant peptide is non-covalently linked to IL-15Rα, or the IL-15 mutant is fused to the other end of IL-15Rα with or without a linker peptide.

[0048] In one specific embodiment, the protein is a homodimer comprising a monomer consisting of (1)-(4) parts.

[0049] In a fifth aspect, the present invention discloses a protein or Fc fusion construct comprising the following three parts:

[0050] (1) Immunoglobulin Fc region;

[0051] (2)IL-15Rα; and,

[0052] (3)The IL-15 mutant polypeptide as described in the first and second aspects above.

[0053] In one embodiment, IL-15Rα is fused to the N-terminus or C-terminus of the IL-15 mutant polypeptide via or without a linker peptide, and then fused to the N-terminus or C-terminus of the immunoglobulin Fc region via or without a linker peptide.

[0054] In one specific embodiment, the protein is a homodimer comprising a monomer consisting of (1)-(3) parts.

[0055] In another preferred embodiment, the immunoglobulin is selected from anti-PD-L1 antibodies; the anti-PD-L1 antibodies are preferably Tecentriq, KN-035, or 794-h1-71.

[0056] In one specific embodiment, the anti-PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100, respectively, or have a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher homology to the sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100; or,

[0057] The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 97 and SEQ ID NO: 98, respectively, or have a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher consistency with the sequences shown in SEQ ID NO: 97 and SEQ ID NO: 98.

[0058] In another preferred embodiment, the IL-15Rα is selected from IL-15Rα-sushi; preferably, the amino acid sequence of IL-15Rα-sushi is shown in SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO.53, or SEQ ID NO.55.

[0059] In a sixth aspect, the present invention discloses an antibody or antigen-binding fragment that specifically binds to PD-L1, the anti-PD-L1 antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100, respectively, or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher homology with the sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100.

[0060] In one specific embodiment, the dissociation constant (KD) of the antibody or antigen binding fragment binding to human programmed death ligand-1 (PD-L1) is not greater than 1.8 × 10-9 M, and the dissociation constant (KD) of the cynomolgus monkey programmed death ligand-1 (PD-L1) binding to it is not greater than 9.4 × 10-10 M.

[0061] Alternatively, the antibody or antigen-binding fragment may or may not bind to monkey PD-L1;

[0062] Optionally, the antibody or antigen-binding fragment may or may not bind to mouse PD-L1.

[0063] In one specific embodiment, the anti-PD-L1 antibody competitively binds to PD-L1 or its antigenic epitope, and has the following characteristics:

[0064] (1) Specifically binds to recombinant PD-L1 protein and cells expressing PD-L1;

[0065] (2) Block the binding of PD-L1 to PD-1 protein;

[0066] (3)Inhibit the binding of PD-1 to PD-L1 expressed on the cell surface;

[0067] (4) Enhance T cell activity; or / and

[0068] (5)Inhibit tumor growth.

[0069] In a preferred embodiment, the anti-PD-L1 antibody comprises a constant region selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, it comprises a sequence containing a constant region of human or mouse antibody IgG1, IgG2, IgG3 or IgG4.

[0070] In another preferred embodiment, the PD-L1 antibody is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, and bispecific antibodies.

[0071] In a seventh aspect, the present invention discloses an isolated nucleic acid molecule that encodes a polypeptide, protein, antigen or antigen-binding fragment as described in any one of the first to sixth aspects.

[0072] In an eighth aspect, the present invention discloses an expression vector comprising the nucleic acid molecules isolated in the aforementioned seventh aspect.

[0073] In a ninth aspect, the present invention discloses a host cell comprising the isolated nucleic acid molecule described in the seventh aspect above, or the expression vector described in the eighth aspect above; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from Chinese hamster ovary cells (CHO).

[0074] In a tenth aspect, the present invention provides a method for preparing a polypeptide or protein, wherein the host cell described in the ninth aspect is cultured under appropriate conditions and the polypeptide or protein is isolated.

[0075] In an eleventh aspect, the present invention discloses a pharmaceutical composition comprising a polypeptide, protein, antigen or antigen-binding fragment as described in any one of the first to sixth aspects, an isolated nucleic acid molecule as described in the seventh aspect, an expression vector as described in the eighth aspect, a cell as described in the ninth aspect, or a product prepared by the method described in the tenth aspect; and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition further comprises an additional antitumor agent.

[0076] In a twelfth aspect, the present invention discloses the use of the polypeptide, protein, antigen or antigen-binding fragment described in any one of the first to sixth aspects, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or the product prepared by the method described in the tenth aspect, or the pharmaceutical composition described in the eleventh aspect, in the preparation of a medicament for the prevention and / or treatment of a disease in an individual; wherein the disease is preferably a tumor.

[0077] In a thirteenth aspect, the present invention provides a method for preventing and / or treating a disease in an individual, comprising administering to a patient in need a polypeptide, protein, antigen or antigen-binding fragment as described in any of the first to sixth aspects above, an isolated nucleic acid molecule as described in the seventh aspect above, an expression vector as described in the eighth aspect above, a cell as described in the ninth aspect above, a product prepared by the method described in the tenth aspect above, or a pharmaceutical composition as described in the eleventh aspect above; wherein the disease is preferably a tumor.

Implementation Method

[0079] Term Definitions and Explanations

[0080] Unless otherwise stated, the terms used herein have the meanings commonly understood by one of ordinary skill in the art. For terms explicitly defined herein, the meaning of the term shall be as defined herein.

[0081] In this invention, the term "IL-15" or "IL15" refers to interleukin-15 (IL-15), a pleiotropic cytokine that activates T cells, B cells, and NK cells and mediates their proliferation and survival. Furthermore, IL-15 can activate, maintain, and expand CD8+ memory T cells. The "IL-15" or "IL-15 peptide" or "IL-15 polypeptide" described in this invention can be any IL-15 (interleukin 15) or its mutants, such as human IL-15 or non-human mammalian IL-15 or non-mammal IL-15. Exemplary non-human mammals include pigs, rabbits, monkeys, chimpanzees, mice, etc., and non-mammals include chickens, etc. Preferably, mature human interleukin 15 molecules are used, as seen in the UniProtKB database, accession number P40933, 49-162aa.

[0082] In this invention, the term "IL-15 wild type" or "wild-type IL-15" refers to human IL-15 or non-human mammalian IL-15 or non-mammal IL-15 of natural origin; it may also refer to IL-15 peptides that are already commonly used in the art.

[0083] In this invention, the term "IL-15 mutant" refers to a mutant molecule that, through one or more amino acid substitution, addition, or deletion mutations, increases or decreases the affinity between IL-15 and its receptor, or increases or decreases the activity of T cell or NK cell proliferation or cytokine release in a specific cell line.

[0084] In this invention, the term "IL-15Rα" can refer to IL-15Rα or its functional fragment from any species, such as human IL-15Rα, non-human mammalian IL-15Rα, or non-mammal IL-15Rα. Exemplary non-human mammals include pigs, rabbits, monkeys, chimpanzees, and mice, while non-mammals include chickens. Human IL-15Rα is preferred; the extracellular domain fragment of human interleukin-15 receptor α, abbreviated as IL-15Rα ECD, is preferred; IL-15Rα-sushi is also preferred, as detailed in Table 1.

[0085] In this invention, the term "IL-15Rα variant" refers to a functional mutant of IL-15Rα formed by mutations in the deletion, insertion, or substitution of one or more amino acids, which has the ability to bind to its ligand molecule such as IL15. Preferably, it is a human IL15Rα molecule, more preferably a shortened form of the extracellular domain segment of human IL-15Rα, that is, a molecule with human interleukin-15 receptor α activity obtained by mutations in the deletion of one or more amino acids starting from the C-terminus of the extracellular domain segment. Preferably, it is a deletion mutation form retaining 65-120 amino acids, more preferably a shortened deletion mutation form retaining 65-102 amino acids, such as IL-15Rα-sushi; preferably IL-15Rα-sushi, see Table 3 for details.

[0086] In this invention, the term "immunoglobulin Fc region" refers to the constant region of the immunoglobulin chain, particularly the carboxyl terminus or a portion thereof of the constant region of the immunoglobulin heavy chain, which has no antigen-binding activity and is the site where antibody molecules interact with effector molecules and cells. The "immunoglobulin Fc region" described in this invention can be any Fc or its variants, derived from humans or non-human mammals. For example, the immunoglobulin Fc region may include a combination of two or more domains of the heavy chain CH1, CH2, CH3, and CH4 with the immunoglobulin hinge region. Fc can originate from different species, preferably human immunoglobulins. Based on the amino acid sequence of the heavy chain constant region, immunoglobulins can be classified into different types, mainly five classes: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG-4; IgA-1 and IgA-2. The "Fc region" preferably includes at least one immunoglobulin hinge region, as well as the CH2 and CH3 domains of IgG. More preferably, it includes a CH2 domain, a CH3 domain, and an immunoglobulin hinge region of IgG1, wherein the position of the initiating amino acid in the hinge region can be varied.

[0087] In this invention, the term "Fc variant" refers to a change in the structure or function of an Fc caused by one or more amino acid substitutions, insertions, or deletions at appropriate sites. "Inter-Fc variant interaction" refers to the potential for space-filling effects, electrostatic attraction, hydrogen bonding, hydrophobic interactions, etc., between mutant-designed Fc variants. Inter-Fc variant interactions contribute to the formation of stable heterodimeric proteins. A preferred mutant design is a "Knob-into-Hole" type mutant design.

[0088] The mutation design technology of Fc variants has been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion protein forms. Representative examples include the "Knob-into-Hole" form proposed by Cater et al. (Protein Engineering vol.9 no.7 pp.617-621, 1996); the Fc-containing heterodimer form formed by Amgen engineers using electrostatic steering (US 20100286374 A1); the heterodimer form (SEEDbodies) formed through IgG / IgA chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp.1-8, 2010); the bispecific molecule formed by Genmab's DuoBody (Science, 2007, 317(5844)) platform technology; and the heterodimer protein form formed by Xencor engineers through a combination of structural calculations and Fc amino acid mutations, combining different modes of action (mAbs 3:6, 546-557; November / December). (2011); Suzhou Corning Jerry Co., Ltd.'s charge network-based Fc modification method (CN201110459100.7) to obtain heterodimeric protein forms; and other genetic engineering methods based on Fc amino acid changes or functional modifications to achieve the formation of heterodimeric functional proteins. The Knob / Hole structure on the Fc variant fragments described in this invention refers to the mutation of each of the two Fc fragments, which can bind together in a "knob-into-hole" manner after mutation. Preferably, the "knob-into-hole" model of Cater et al. is used to modify the Fc region by site mutation, so that the resulting first Fc variant and second Fc variant can combine together in a "knob-into-hole" manner to form a heterodimer. Selecting specific immunoglobulin Fc regions from specific immunoglobulin classes and subclasses is within the scope of those skilled in the art. Preferably, the Fc regions of human antibodies IgG1, IgG2, IgG3, and IgG4 are used, more preferably the Fc region of human antibody IgG1. Randomly select either the first Fc variant or the second Fc variant to perform a knot mutation and the other to perform a hole mutation.

[0089] In this invention, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive to a target antigen, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including, but not limited to, chimeric antibodies, humanized antibodies, fully human antibodies, heterologous conjugates (e.g., bispecific, trispecific, and tetraspecific antibodies, biantibodies, triantibodies, and tetraantibodies), antibody conjugates) and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments). Furthermore, unless otherwise stated, the term "monoclonal antibody" (mAb) means both complete antibody molecules capable of specifically binding to target proteins and incomplete antibody fragments (e.g., Fab and F(ab')2 fragments, which lack the Fc fragment of the complete antibody (which is cleared more quickly from animal circulation) and therefore lack Fc-mediated effector function (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated herein by reference).

[0090] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody originates from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) originates from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance immune responses.

[0091] The term "antibody conjugate" refers to a conjugate / complex formed by the direct or chemical bonding of an antibody molecule to another molecule via a linker. For example, an antibody-drug conjugate (ADC) is where the drug molecule is the other molecule.

[0092] The term “monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), but is not limited to the method of producing the antibody.

[0093] In this invention, the term "fusion protein" refers to a protein product obtained by linking the coding regions of two or more genes through gene recombination, chemical methods, or other suitable methods, and expressing the gene recombination under the control of the same regulatory sequence. In the fusion protein of this invention, the coding regions of two or more genes may be fused at one or more positions by sequences encoding peptide linkers or linking peptides. Peptide linkers or linking peptides can also be used to construct the fusion protein of this invention. The term "fusion protein" in this invention further includes antibody / Fc fusion protein constructs / complexes, or compositions of antibody / Fc fusion protein constructs / complexes formed non-covalently. For example, the fusion protein of this invention may exhibit the following structure:

[0094] (1) IL-15 fusion protein, which is a homodimer containing two monomers; said monomers include an antibody heavy chain, an antibody light chain, IL-15 and IL-15Rα sushi; for example, the antibody heavy chain Fc is fused with IL-15Rα sushi and co-expressed with the antibody light chain and IL-15-WT (wild type) or IL-15 mutant, so that IL-15 and IL-15Rα sushi form a non-covalent link;

[0095] (2) IL-15 fusion protein, which is a homodimer containing two monomers; the monomers contain an antibody heavy chain, an antibody light chain, IL-15 and IL-15Rα sushi; for example, the antibody heavy chain Fc is tandemly fused with IL-15Rα sushi and IL-15-WT or IL-15 mutant via a linker and expressed in combination with the antibody light chain;

[0096] (3) An IL-15 fusion protein, which is a homodimer comprising two monomers; said monomers comprising Fc, IL-15, and IL-15Rαsushi; for example, IL-15-WT or an IL-15 mutant is linked to IL15-Rαsushi via a linker, and IL15-Rαsushi is then linked to Fc via a linker; or,

[0097] (4) IL-15 fusion protein, which is a homodimer containing two monomers; the monomers include Fc, IL-15 and IL-15Rαsushi; for example, IL15-Rαsushi is linked to IL-15-WT or IL-15 mutant via a linker, and IL-15 is linked to Fc via a linker.

[0098] In this invention, the term "peptide linker" refers to a peptide used to link IL-15 to another protein molecule or fragment to ensure proper protein folding and stability. The other molecule includes, but is not limited to, IL-15Rα, Fc, Fc variants, antibodies, etc. The "linker peptide" of this invention is preferably (GGGGS)n, where n can be 0, 1, 2, 3, 4, 5, or more, preferably 2-4; or preferably SGGSGGGGSGGGGSGGGSLQ. If the linker peptide sequence is too short, it may affect the folding of the higher-order structures of the two proteins, thus interfering with each other; if the linker peptide sequence is too long, it raises immunogenicity issues, because the linker peptide sequence itself is a new antigen.

[0099] In this invention, the term "heterodimeric protein" refers to a protein formed by the combination of two different monomeric proteins. In this invention, the two different monomeric proteins each contain an Fc fragment or an Fc variant fragment, and form a heterodimeric protein through the interaction of the Fc fragment or the Fc variant fragment.

[0100] In this invention, the term "homodimeric protein" refers to a protein formed by the combination of two identical monomeric proteins. In this invention, the two identical monomeric proteins each contain an Fc fragment or an Fc variant fragment, and form a homodimeric protein through the interaction of the Fc fragment or the Fc variant fragment.

[0101] In this invention, the "monomer protein" that makes up the heterodimeric protein or homodimeric protein can be a fusion protein or a non-fusion protein.

[0102] In this invention, the term "PD-L1" refers to programmed death-ligand-1, also known as CD279 (differentiation cluster 279), which is an important immunosuppressive molecule. The PD-L1 is preferably human PD-L1.

[0103] In this invention, the terms "anti-programmed death-ligand-1 antibody," "programmed death-ligand-1 antibody," "anti-PD-L1 antibody," "PD-L1 antibody," "anti-PD-L1 antibody fraction," and / or "anti-PD-L1 antibody fragment," etc., refer to any protein or peptide molecule containing at least a portion of an immunoglobulin molecule capable of specifically binding to PD-L1 (e.g., but not limited to at least one complementarity-determining region (CDR) of the heavy or light chain or its ligand-binding portion, variable region of the heavy or light chain, constant region of the heavy or light chain, framework region, or any portion thereof). PD-L1 antibodies also include antibody-like protein scaffolds (such as the tenth fibronectin type III domain (10Fn3)) containing BC, DE, and FG structural loops similar in structure and solvent accessibility to the antibody CDR. The tertiary structure of the 10Fn3 domain is similar to that of the variable region of the IgG heavy chain, and by replacing the residues of the BC, DE, and FG loops of 10Fn3 with residues from the CDR-H1, CDR-H2, or CDR-H3 regions of PD-L1 monoclonal antibodies, those skilled in the art can graft, for example, the CDR of PD-L1 monoclonal antibodies onto fibronectin scaffolds.

[0104] In this invention, the term "coexpression" refers to the simultaneous expression of multiple genes in a single cell, resulting in the simultaneous appearance of their products. These genes may coexist and be expressed individually or jointly under controlled conditions. In this invention, it is preferable to coexpress two genes in a single eukaryotic cell. The gene expression products obtained from coexpression facilitate the efficient and simple formation of complexes; in this invention, it is beneficial to form heterodimeric or homodimeric proteins.

[0105] The term "percentage (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment and the introduction of gaps (if necessary) to achieve maximum percentage sequence identity (e.g., gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment can be performed in a variety of ways well known to those skilled in the art for the purpose of determining percentage sequence identity, such as using publicly available computer software like BLAST, ALIGN, or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms that require maximum alignment across the full length of the sequences being compared. For example, a reference sequence used for alignment against a candidate sequence may show sequence identity from 50% to 100% across the full length of the candidate sequence or selected portions of consecutive amino acid (or nucleotide) residues in the candidate sequence. The length of candidate sequences compared for comparative purposes can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. Molecules are considered identical at that position when a position in the candidate sequence is occupied by an amino acid (or nucleotide) residue that is the same as the corresponding position in the reference sequence.

[0106] In this invention, the term "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, such as those specifically recognized by an antibody or its antigen-binding fragment. An antibody or its antigen-binding fragment that specifically binds to an antigen will bind with a KD of less than 100 nM. For example, an antibody or its antigen-binding fragment that specifically binds to an antigen will bind with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). Antibodies or their antigen-binding fragments that do not show specific binding to a particular antigen or its epitope will show a KD greater than 100 nM (e.g., greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or its epitope. Various immunoassays can be used to select antibodies that specifically react with a particular protein or carbohydrate. For example, solid-phase ELISA is conventionally used to select antibodies that specifically react with a protein or carbohydrate. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describe the immunoassay methods and conditions that can be used to determine specific immune reactivity.

[0107] In this invention, the term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vectors of this invention contain polynucleotide sequences and additional sequence elements, for example, for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that can be used to express antibodies and antibody fragments of this invention include plasmids containing regulatory sequences (e.g., promoter and enhancer regions) that guide gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to guide efficient transcription of the gene carried on the expression vector. The expression vectors of this invention may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or norsocrine.

[0108] In this invention, the terms “subject,” “object,” and “patient” refer to an organism receiving treatment for a specific disease or condition (such as cancer or an infectious disease) as described herein. Examples of objects and patients include mammals receiving treatment for diseases or conditions (such as proliferative conditions like cancer or infectious diseases), such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk, and yaks), sheep, and horses.

[0109] In this invention, the term "treatment" refers to surgical or therapeutic treatment aimed at preventing, slowing (reducing) undesirable physiological changes or lesions in the treated individual, such as the progression of proliferative disorders (e.g., cancer or infectious diseases). Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Individuals requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as slowing, reducing, weakening, mitigating, or alleviating are used, they also include elimination, disappearance, and non-occurrence.

[0110] In this invention, "immunological disease" or "immune disorder" includes, for example, pathological inflammation, inflammatory conditions, and autoimmune diseases or disorders. "Immune disease" also refers to infections, persistent infections, and proliferative conditions, such as cancer, tumors, and angiogenesis. "Cancer disease" includes, for example, cancer cells, tumors, angiogenesis, and precancerous conditions, such as developmental abnormalities.

[0111] In this invention, the term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, thereby promoting the absorption of the active ingredient and enabling it to exert its biological activity.

[0112] In this invention, the term "size exclusion chromatography" (SEC) refers to a liquid chromatography technique that separates analytes based on their molecular size (cvn). The surface of the chromatographic column packing material has pores of different sizes. After the sample enters the column, different components enter the corresponding pores according to their molecular size. Molecules larger than all pore sizes cannot enter the packing particles and are not retained during chromatography, resulting in a short retention time. Molecules smaller than all pore sizes can freely enter all pores on the packing surface, resulting in a longer residence time in the column. The remaining molecules are eluted sequentially according to their molecular size.

[0113] “Optional” or “optionally” means that the event or circumstances described below may but do not have to occur, including the possibility that the event or circumstances may or may not occur. For example, “optionally contains 1-3 antibody heavy chain variable regions” means that antibody heavy chain variable regions may but do not have to be present; if present, there may be 1, 2 or 3.

[0114] The step of transforming host cells with recombinant DNA as described in this invention can be performed using conventional techniques well known to those skilled in the art. The obtained transformants can be cultured using conventional methods and express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium used in the culture can be selected from various conventional culture media. The host cells are cultured under conditions suitable for host cell growth. Detailed Embodiments

[0115] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. Unless otherwise specified in the embodiments, conditions are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0116] The embodiments of the present invention are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0117] Example 1: Antibody Humanization

[0118] First, the classic "CDR transplantation" method was used for antibody humanization. This involved selecting the human antibody with the highest homology to provide the antibody backbone region (FRs), and then transplanting the complementarity-determining region (CDR) of the target antibody (based on Kabat nomenclature) into the former to form a humanized antibody. Second, to better maintain antibody activity and affinity, antibody structure modeling analysis was performed using MOE software: 1) Reverse mutations were performed on amino acid residues located at the VH-VL interface, close to, or directly interacting with CDRs in the antibody backbone region. These amino acid residues are often important for maintaining the conformation of the CDR region; 2) Considering immunogenicity, amino acids embedded within the protein were preferred for reverse mutations; 3) Considering antibody stability and expression levels, molecular energy reduction mutations were prioritized. By testing the affinity of humanized antibodies with different mutations for human PD-L1 and their binding to cells expressing PD-L1, humanized antibodies with affinity, antibody characterization, and activity comparable to or better than mouse PD-L1 antibodies were screened.

[0119] The amino acid sequence information of the heavy chain and light chain variable regions of the preferred candidate antibody molecule 794-h1-71 after humanization of the mouse PD-L1 antibody PDL1-794 is shown in Table 1 below.

[0120] Table 1. Specific sequence information of the heavy chain variable region and light chain variable region of murine and humanized anti-PD-L1 antibodies. Antibody number Serial Number Heavy chain variable region sequence (VH) PDL1-794 SEQ ID NO.97 EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGSTYYNPFLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCAKMGDWLAWFAYWGQGTTVTVSS 794-h1-71 SEQ ID NO.99 QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSS Antibody number Serial Number Light chain variable region (VL) sequence PDL1-794 SEQ ID NO.98 EIVMTQSPSSLAVSVGEKVTLSCKSSQSLLYSSNQKNSLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYGYPYTFGGGTKLEIK 794-h1-71 SEQ ID NO.100 EIVMTQSPPTLSLSPGERVTLSCKSSQSLLYSSNQKNSLAWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQYYGYPYTFGQGTKLEIK

[0121] Example 2: Expression and purification of humanized antibodies

[0122] 2.1 Expression of humanized antibodies

[0123] One day before transfection, ExpiCHO-S cells (Thermo Fisher, A29127) were seeded at a density of 2.5×10⁶~4×10⁶ cells / mL in fresh ExpiCHO Expression medium (Invitrogen, A29100-01) and cultured overnight on a shaker. On the day of transfection, the overnight cultured ExpiCHO-S cell suspension was counted; cell viability was >95%, and the density was between 7×10⁶ and 10×10⁶ viable cells / mL. The required cell suspension was taken and diluted with ExpiCHO Expression medium (Invitrogen, A29100-01) to a density of 6×10⁶ cells / mL, and then placed on a shaker for later use. Dilute the prepared humanized antibody expression plasmid in culture medium, gently swirl the centrifuge tube to mix, and add it to OptiPRO™ SFM-DNA dilution buffer (Invitrogen, 12309-019). Gently swirl the centrifuge tube to mix and incubate at room temperature for 1–5 mins. Slowly add the plasmid complex to the cell suspension to be transfected, shaking the flask during the addition. After transfection, incubate the cells overnight on a shaker. On day 1 post-transfection, add 0.6% ExpiFectamine™ CHO Enhancer (Invitrogen, A29129) and 16% ExpiCHO™ Feed (Invitrogen, A29129) by cell volume to the transfected cells, gently swirl the flask during the addition, and transfer the cells to a shaker for 4 days. On day 5 post-transfection, add 16% ExpiCHO™ Feed (Invitrogen, A29129) by cell volume to the transfected cells, gently swirl the flask during the addition. On day 12 post-transfection, 9000g of culture medium was collected, centrifuged for 10 minutes, and the supernatant was harvested.

[0124] 2.2 Purification of humanized antibodies

[0125] The cell culture supernatant collected in Example 2.1 was centrifuged at high speed and filtered through 0.45 μm + 0.22 μm filters for the first step of purification using affinity chromatography. The chromatography medium was Mbaselect Sure (GE, 17543803), a protein A packing material that interacts with Fc. The equilibration buffer was PBS (2.5 g / L Na2HPO4·12H2O, 0.408 g / L NaH2PO4, 8.76 g / L NaCl, pH 7.2). After equilibration to 4 column volumes, the cell supernatant was loaded, with the flow rate controlled so that the sample retention time on the column was ≥5 min. After loading, the column was washed with PBS (pH 7.2) until the A280 UV absorbance dropped to baseline. Then, 2 column volumes of PBS were washed with 20 mM PB + 1 M NaCl (pH 6.0). The column was then washed again with PBS (pH 7.2) until the A280 UV absorbance and conductivity reached baseline. Finally, the column was washed with elution buffer containing 20 mM citric acid (pH 3.4), and the elution peak was collected based on the A280 UV absorption peak. The collected elution sample was neutralized to neutral with 1 M Tris-HCl (pH 9.0).

[0126] Example 3: KD determination of antibody binding to human and cynomolgus monkey PD-L1 recombinant protein

[0127] The binding affinity of PD-L1 antibodies to human and cynomolgus monkey PD-L1-His proteins was determined using a Biacore T200 (GE Healthcare) chip. Anti-human IgG Fc (Genway, Cat. GWB-20A705) was immobilized on a CM5 chip (GE Healthcare, Cat. BR-1005-30) at 25°C. The anti-human IgG Fc was diluted to 20 μg / mL with Acetate pH 5.0 (GE Healthcare, BR-1003-51). Immobilization was performed using the Amine method in the Immobilization method. Alternatively, detection was performed using a commercial Protein A chip (GE Healthcare, Cat. 29127556). The affinity between antibody and antigen was determined using a multi-cycle kinetic method at 25℃. In each cycle, the antibody to be tested was first captured onto a fixed CM5 chip, then recombinant human PD-L1-His (Novoprotein, Cat. 315) and cynomolgus monkey PD-L1-His protein (Sino Biological, Cat. 90251-C08H) were injected, and finally regenerated with Glycine pH 1.5 (Shanghai Experimental, Cat. 62011516). The mobile phase was HBS-EP+Buffer (GE Healthcare, Cat. BR-1006-69), the flow rate was 30 μL / min, and the binding time was 300 seconds. The regeneration flow rate was 30 μL / min, and the time was 30 seconds. Using Biacore T200 Evaluation Software (version 3.0), with a 1:1 binding model, the experimental data were analyzed to fit the equilibrium dissociation constant KD of the antibody and antigen, and the binding rate constant ka and dissociation rate constant kd were determined.

[0128] The results show that the tested PD-L1 antibodies exhibited nM or higher affinity for both human PD-L1 recombinant protein and cynomolgus monkey PD-L1 recombinant protein, as detailed in Table 2 below.

[0129] Table 2. Results of Biacore binding affinity KD assay for humanized PD-L1 antibody Antibody number Human PD-L1 (M) cynomolgus monkey PD-L1 (M) 794-h1-71 1.793E-09 9.372E-10

[0130] Example 4: IC50 determination of antibody blocking PD-L1 and PD-1 interaction

[0131] The IC50 of the anti-PD-L1 antibody blocking the binding of PD-L1 protein to PD-1 protein was determined by a competitive ELISA method. Human PD-L1 recombinant protein (Sino Biological, Cat. 10084-H05H) was diluted with carbonate buffer and added to a 96-well ELISA plate to a final concentration of 1 μg / ml. The plate was blocked with PBS containing 3% BSA, and then co-incubated with serially diluted anti-PD-L1 antibody (40 nM–0.02 nM) and human PD-1-His recombinant protein (Sino Biological, Cat. 10377-H08H). HRP-labeled anti-His tag antibody (MBL, Cat. D291-7) was added, and TMB (Thermo, Cat. 34029) was used for color development. The reaction was stopped with 1M sulfuric acid, and the OD values ​​were read (dual wavelengths 450 nm–630 nm). The competitive binding curve of the antibody was plotted by mapping the antibody concentration to the OD values, and the IC50 value was calculated. Figure 1 shows the competitive binding curves between the anti-PD-L1 antibody and recombinant human PD-L1 protein. The results indicate that the tested 794-h1-71 antibody can effectively block the interaction between human PD-L1 protein and human PD-1 protein, with an IC50 of 0.8488 nM, compared to 0.8486 nM for the positive control Tecentriq (Genetech, lot: H0172).

[0132] Example 5: FACS determination of EC50 of PD-L1 antibody binding to PD-L1 on cell surface

[0133] Gradual concentrations of the test antibody (antibody concentration: 10000 ng / ml - 0.1 ng / ml) were incubated with CHO-PD-L1 cells (Nanjing Yongshan Biotechnology Co., Ltd., 10⁵ cells / well) that highly expressed PD-L1 on their cell surface, and incubated at 4℃ for 30 min. After incubation, 1:250 diluted anti-human IgG PE fluorescent antibody (eBioscience, Cat. 12-4998-8) was added, and the cells were incubated at 4℃ for 30 min. The fluorescent antibody specifically bound to the Fc fragment of the test antibody. The ability of the test antibody to bind to the highly expressed PD-L1 protein on the cell surface was analyzed by detecting the intensity of PE fluorescence using FACS. The results in Figure 2 show that the EC50 of the 794-h1-71 antibody was 38.44 ng / ml, which is similar to that of the positive control Avelumab (EC50 of ~72 ng / ml) in this experiment. This assay quantitatively confirmed the ability of the 794-h1-71 antibody to bind to the PD-L1 target on the cell surface in a dose-dependent manner. Mean fluorescence intensity fold (MFI fold) = MFI value of the experimental group / MFI value of the control group without drug.

[0134] Example 6: PD-1 / PD-L1-NFAT reporter gene assay: Anti-PD-L1 antibody inhibits PD-1: PD-L1 binding and signal transduction.

[0135] The antagonistic effects of PD-L1 antibodies on PD-1 / PD-L1 protein interaction and its signaling pathway were compared using a Jurkat cell line stably transfected with PD-1 (GenScript, Cat. 00612) and a CHO cell line stably transfected with PD-L1 (GenScript, Cat. M00613). When the signaling pathway was inhibited, the expression of the NFAT-controlled luminescent reporter gene was enhanced, and the luminescence signal value increased. The relative light units (RLU) of the luminescence readings reflected the strength of the antibody's blocking effect on PD-L1.

[0136] Stable PD-L1-transfected CHO cell lines were seeded in 96-well white plates at 40,000 cells per well, 100 μl / well, and incubated overnight. The next day, the plates were removed, the culture medium was aspirated, and the stable PD-L1-transfected cell lines and the PD-L1 antibody to be tested were added and co-incubated. The PD-L1 cell loading was 16,000 cells / well, and the antibody was serially diluted, with each dose in 3 replicates, with an incubation volume of 100 μl / well and an incubation time of 6 hours. After incubation, the plates were removed, and an equal volume (100 μl) of luminescent detection reagent was added, and the values ​​were read. Based on the detection values, Graphpad was used to perform 4-parameter analysis to generate regression curves and obtain the EC50 values ​​of each antibody. Figure 3 shows that the EC50 of the 794-h1-71 antibody (166.2 ng / ml) is similar to that of the positive control Avelumab (184.3 ng / ml). This assay quantitatively confirmed that the 794-h1-71 antibody exhibited a dose-dependent inhibitory effect on T cell activity induced by the PD-1:PD-L1 interaction on the cell surface, thereby dose-dependently enhancing the activity of reporter genes in Jurkat cells.

[0137] Example 7: ELISA detection of IFN-γ secreted by T cells in mixed lymphocyte reaction

[0138] The activity of PD-L1 monoclonal antibody-enhanced T cells was measured by mixed lymphocyte reaction (MLR). CD4+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donor 1 and induced to differentiate into dendritic cells (DCs) in vitro using recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech, Cat.300-03) and recombinant human interleukin-4 (rhIL-4, Peprotech, Cat.200-04). On day 6 of culture, LPS (Sigma, Cat: L4516) was added to stimulate mature DCs. On day 7, DCs from donor 1 were co-cultured with CD4+ T cells enriched from PBMCs of healthy donor 2 at a DC:CD4+ T cell ratio of 1:10. The test antibody, negative control antibody anti-Hel (synthesized by Baiying Biotechnology), and positive control antibody Avelumab (antibody concentration: 7nM-0.28nM) were added and co-cultured for 4 days. Four days later, cell culture supernatants were collected, and the IFN-γ content in the supernatant was detected by ELISA. As shown in Figure 4, both 794-h1-71 and the positive control antibody Avelumab significantly enhanced the ability of CD4+ T cells to secrete IFN-γ in the MLR experiment compared with the anti-Hel monoclonal antibody negative control group. Furthermore, the activity of increasing IFN-γ secretion decreased with decreasing PD-L1 antibody concentration. These results indicate that the 794-h1-71 antibody can enhance T cell function in a dose-dependent manner (T-test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0139] Example 8: Construction of IL-15 fusion protein

[0140] The key amino acid sites for the interaction between human IL-15 and its corresponding receptor βγchain were simulated using MOE software. These sites are D8 and V3, I6, and H105, respectively. Based on the MOE software simulation, the following IL-15 mutant sequences were designed and synthesized. The amino acid sequences are detailed in Table 3, and the encoding nucleic acid sequences are detailed in Table 4.

[0141] There are four modes for constructing IL-15 fusion proteins (antibody / Fc fusion construct / complex):

[0142] (1) As shown in Figure 5A, the IL-15 fusion protein is a homodimer containing two monomers; the monomers contain an antibody heavy chain, an antibody light chain, IL-15 and IL-15Rα sushi; the Fc end of the antibody heavy chain is fused with IL-15Rα sushi and co-expressed with the monoclonal antibody light chain and IL-15-WT (wild type) or IL-15 mutant, so that IL-15 and IL-15Rα sushi form a non-covalent link;

[0143] (2)As shown in Figure 5B, the IL-15 fusion protein is a homodimer containing two monomers; the monomers contain an antibody heavy chain, an antibody light chain, IL-15 and IL-15Rα sushi; the Fc end of the antibody heavy chain is sequentially tandemly fused with IL-15Rα sushi and IL-15-WT or IL-15 mutant through a linker and co-expressed with the antibody light chain;

[0144] (3) As shown in Figure 5C (defined as V5), the IL-15 fusion protein is a homodimer containing two monomers; the monomers contain Fc, IL-15 and IL-15Rαsushi; IL-15-WT or IL-15 mutant is linked to IL15-Rαsushi through a linker, and IL15-Rαsushi is then linked to Fc through a linker;

[0145] (4)As shown in Figure 5D (defined as V9), the IL-15 fusion protein is a homodimer containing two monomers; the monomers contain Fc, IL-15 and IL-15Rαsushi; IL15-Rαsushi is linked to IL-15-WT or IL-15 mutant through a linker, and IL-15 is then linked to Fc through a linker.

[0146] The numbering rule for PD-L1 antibody and IL-15 fusion protein is as follows: "Antibody name - IL-15 (wild type / mutant) - Fusion protein construction mode";

[0147] For example: “T-IL15-xx-1”: “T” represents Tecentriq; “IL15-xx” represents IL15-WT or IL15 mutant; “1” represents the structural pattern shown in Figure 5A;

[0148] For example: “794-IL15-xx-2”: “794” means 794-h1-71 monoclonal antibody; “IL15-xx” means IL15-WT or IL15 mutant; “2” means the structural pattern shown in Figure 5B.

[0149] Please refer to Tables 5 and 6 for details on the design of various IL-15 fusion proteins. Based on the above four construction modes, the nucleic acid sequences encoding the fusion proteins were constructed into the pTT5 plasmid.

[0150] Table 3. IL-15 fusion protein related amino acid sequence information serial number sequence Serial Number IL15-WT NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.1 IL15-7 (D8E) NWVNVISELKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.3 IL15-8 (D8Q) NWVNVISQLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.5 IL15-9 (D8R) NWVNVISRLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.7 IL15-10 (D8S) NWVNVISSLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.9 IL15-11 (D8V) NWVNVISVLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.11 IL15-26 (V3L) NWLNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.13 IL15-29 (I6D) NWVNVDSDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.15 IL15-42 (H105K) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVKIVQMFINTS SEQ ID NO.17 IL15-43 (H105N) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVNIVQMFINTS SEQ ID NO.19 IL15-61 (D8G) NWVNVISGLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.21 IL15-62 (D8I) NWVNVISILKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.23 IL15-63 (D8L) NWVNVISLLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.25 IL15-64 (I6P) NWVNVPSDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.27 IL15-65 (D8T) NWVNVISTLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.29 P22339 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.31 ALT803 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIREFEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.33 IL15-com1 (D8E / V3L) NWLNVISELKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.35 IL15-com2 (D8E / I6D) NWVNVDSELKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.37 IL15-com3 (V3L / I6D) NWLNVDSDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.39 IL15-com4 (V3L / I6D / H105K) NWLNVDSDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVKIVQMFINTS SEQ ID NO.41 IL15-com5 (I6D / H105K) NWVNVDSDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVKIVQMFINTS SEQ ID NO.43 IL15-com6 (D8S / H105K) NWVNVISSLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVKIVQMFINTS SEQ ID NO.45 IL15-com7 (D8S / H105N) NWVNVISSLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVNIVQMFINTS SEQ ID NO.47 IL15Rαsushi-1 CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SEQ ID NO.49 IL15Rαsushi-2 CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR SEQ ID NO.51 IL15Rα sushi-3 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SEQ ID NO.53 IL15Rα sushi-4 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR SEQ ID NO.55 Tecentriq heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.57 Tecentriq light chain DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.59 794-h1-71 Heavy chain QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.61 794-h1-71 Light chain EIVMTQSPPTLSLSPGERVTLSCKSSQSLLYSSNQKNSLAWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQYYGYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.63 Linker-1 SGGSGGGGSGGGSGGGGSLQ SEQ ID NO.65 Linker-2 GGGGSGGGGSGGGGS SEQ ID NO.67 Linker-3 EF SEQ ID NO.69 Linker-4 SGGGSGGGGSGGGGSGGGGSGGGSLQ SEQ ID NO.71 Human Fc EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.73 T-IL15-WT-1 (Tecentriq heavy chain - IL15Rα sushi) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SEQ ID NO.75 794-IL15-WT-2 (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-WT) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.77 794-IL15-7-2 (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-7) (D8E) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISELKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.79 794-IL15-65-2 (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-65) (D8T) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISTLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.81 794-IL15-64-2 (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-64) (I6P) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVPSDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.83 794-IL15-com1-2 (794-h1-71 heavy chain - IL15Rα sushi - Linker1 - IL15-com1) (D8E / V3L) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWLNVISELKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.85 794-IL15-com3-2 (794-h1-71 heavy chain - IL15Rα sushi -Linker1-IL15-com3) (I6D / V3L) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWLNVDSDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.87 794-IL15-com4-2 (794-h1-71 heavy chain - IL15Rα sushi -Linker1-IL15-com4) (I6D / V3L / H105K) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWLNVDSDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVKIVQMFINTS SEQ ID NO.89 794-IL15-com5-2 (794-h1-71 heavy chain - IL15Rα sushi -Linker1-IL15-com5) (I6D / H105K) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVDSDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVKIVQMFINTS SEQ ID NO.91 794-IL15-com6-2 (794-h1-71 heavy chain - IL15Rα sushi -Linker1-IL15-com6) (D8S / H105K) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISSLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVKIVQMFINTS SEQ ID NO.93 794-IL15-com7-2 (794-h1-71 heavy chain - IL15Rα sushi -Linker1-IL15-com7) (D8S / H105N) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECV LNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGGSGGGSGGGGSLQNWVNVISSLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVNIVQMFINTS SEQ ID NO.95 Note: The numbering rules for IL-15 mutant fusion proteins are as follows:

[0151] For example: “T-IL15-xx-1”: “T” represents Tecentriq; “IL15-xx” represents IL15-WT or IL15 mutant; “1” represents the structural mode shown in Figure 5A;

[0152] For example: “794-IL15-xx-2”: “794” means 794-h1-71 monoclonal antibody; “IL15-xx” means IL15-WT or IL15 mutant; “2” means the structural pattern shown in Figure 5B.

[0153] Table 4. Nucleic acid sequence information encoding IL-15 fusion protein Number Nucleotide sequence Sequence number IL15-WT AACTGGGTGAATGTGATCTCTGACCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.2 IL15-7 (D8E) AACTGGGTGAATGTGATCTCTGAGCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.4 IL15-8 (D8Q) AACTGGGTGAATGTGATCTCTCAGCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.6 IL15-9 (D8R) AACTGGGTGAATGTGATCTCTAGGCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.8 IL15-10 (D8S) AACTGGGTGAATGTGATCTCTAGCCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.10 IL15-11 (D8V) AACTGGGTGAATGTGATCTCTGTGCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.12 IL15-26 (V3L) AACTGGCTGAATGTGATCTCTGACCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.14 IL15-29 (I6D) AACTGGGTGAATGTGGACTCTGACCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.16 IL15-42 (H105K) AACTGGGTGAATGTGATCTCTGACCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGAAGATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.18 IL15-43 (H105N) AACTGGGTGAATGTGATCTCTGACCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGAACATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.20 IL15-61 (D8G) AACTGGGTGAACGTGATCTCTGGCCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCAGCATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGTCTTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.22 IL15-62 (D8I) AACTGGGTGAACGTGATCTCTATCCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCAGCATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGTCTTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.24 IL15-63 (D8L) AACTGGGTGAACGTGATCTCTCTGCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCAGCATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGTCTTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.26 IL15-64 (I6P) AACTGGGTGAACGTGCCTTCTGATCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCAGCATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGTCTTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.28 IL15-65 (D8T) AACTGGGTGAACGTGATCTCTACCCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCAGCATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGTCTTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.30 P22339 AACTGGGTGAATGTGATCTCTGACCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTTGCGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT AtcacctgcccccctccaatgtctgtggagcacgccgacatctgggtgaagtcctacagcctgtatagcagggagcggtacatctgtaactctggcttcaagaggaaggctggcacctgctccctgacagagtgcgtgctgaacaaggccacaaatgtggctcactggaccacacccagcctgAagtgcatcagagatcccgccctggtgcatcagagaggcggcggcggctctggcggcggcggctccgaacccaagtcctccgacaagacccacacctgtcccccttgtcctgcccctgaactgctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccgGacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccctgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgacagtgctgcatcaggactggctgaacGgcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgaccaagaaccaggtgtccctgacctgtctcgtgaaaggcttctacccctccgataTcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtacagcaagctgaccgtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaag SEQ ID NO.32 ALT803 AACTGGGTGAATGTGATCTCTGACCTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAATGACTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT ATCACCTGCCCACCTCCAATGTCCGTGGAGCACGCTGACATCTGGGTGAAGTCTTACTCCCTGTATAGCAGGGAGCGGTACATCTGCAACTCTGGCTTCAAGAGAAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACAAATGTGGCTCATTGGACCACACCCAGCCTGAAGTGTATCCGCGAGTTTgaacccaagtcctgcgacaagacccacacctgtcccccttgtcctgcccctgaactgctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccctgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgacagtgctgcatcaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgaccaagaaccaggtgtccctgacctgtctcgtgaaaggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtacagcaagctgaccgtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaag SEQ ID NO.34 IL15-com1 (D8E / V3L) AACTGGCTGAACGTCATCAGTGAGCTGAAGAAGATCGAGGACCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTCACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGAGCTCTAACGGCAACGTAACCGAGTCTGGGTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAATATCAAGGAGTTTCTGCAGTCTTTTGTGCACATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.36 IL15-com2 (D8E / I6D) AACTGGGTGAATGTTGACTCTGAGTTGAAGAAAATTGAGGACCTAATCCAGTCCATGCATATCGACGCAACTCTGTACACTGAGTCTGACGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACAGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGAGCTCTAACGGAAACGTGACCGAGTCTGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCTTTCGTGCATATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.38 IL15-com3 (V3L / I6D) AACTGGCTGAACGTGGATTCTGATCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACACTGTACACAGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTTCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGAGCTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAGTCTTTTGTGCACATCGTGCAGATGTTTATCAACACATCT SEQ ID NO.40 IL15-com4 (V3L / I6D / H105K) AACTGGCTGAACGTGGATTCTGATCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGTCTAGCAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCTTTCGTGAAGATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.42 IL15-com5 (I6D / H105K) AACTGGGTGAACGTGGATTCTGATCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTTCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACAGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGAGCTCTAACGGCAACGTGACAGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAGTCTTTTGTGAAGATCGTGCAGATGTTTATCAACACCTCT SEQ ID NO.44 IL15-com6 (D8S / H105K) AACTGGGTGAACGTGATCTCTTCTCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGAGCTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCTTTCGTGAAGATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.46 IL15-com7 (D8S / H105N) AACTGGGTGAACGTGATCTCTTCTCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGAGCTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCTTTCGTGAACATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.48 IL15Rαsushi-1 TGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCT SEQ ID NO.50 IL15Rαsushi-2 TGTCCTCCTCCTATGTCTGTGGAGCACGCCGATATCTGGGTGAAGTCTTACTCTCTGTACTCTAGAGAGAGATACATCTGTAACTCTGGCTTCAAGAGAAAGGCCGGCACCTCTTCTCTGACCGAGTGTGTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCCTTCTCTGAAGTGTATCAGA SEQ ID NO.52 IL15Rαsushi-3 ATCACATGTCCTCCTCCTATGTCTGTGGAGCACGCTGATATTTGGGTGAAGTCTTACTCTCTGTACTCTAGAGAAAGATATATTTGTAATTCTGGCTTTAAGAGAAAGGCTGGAACATCTTCTCTGACAGAGTGTGTGCTGAATAAGGCTACAAACGTGGCTCATTGGACAACACCTTCTCTGAAGTGTATTAGAGATCCTGCCCTGGTGCACCAGAGACCTGCTCCTCCT SEQ ID NO.54 IL15Rα sushi-4 ATCACATGTCCTCCTCCTATGTCTGTGGAGCACGCTGATATCTGGGTGAAGTCTTACTCTCTGTACTCTAGAGAGAGATACATCTGTAATTCTGGCTTTAAGAGAAAGGCTGGAACATCTTCTCTGACAGAGTGTGTGCTGAATAAGGCTACAAATGTGGCTCACTGGACAACACCTTCTCTGAAGTGTATCAGA SEQ ID NO.56 Tecentriq heavy chain SEQ ID NO.58 Tecentriq light chain GACATCCAGATGACCCAGTCCCCTAGCTCCCTGTCCGCCTCTGTGGGCGACAGGGTGACCATCACATGCAGAGCCTCTCAGGATGTGAGCACAGCAGTGGCATGGTACCAGCAGAAGCCAGGCAAGGCCCCTAAGCTGCTGATCTACAGCGCCTCCTTCCTGTATTCCGGCGTGCCCTCTCGGTTTTCTGGAAGCGGATCCGGAACCGACTTCACCCTGACAATCTCTAGCCTGCAGCCAGAGGATTTTGCCACATACTATTGTCAGCAGTACCTGTATCACCCCGCCACCTTCGGCCAGGGCACAAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGC SEQ ID NO.60 794-h1-71 Heavy chain SEQ ID NO.62 794-h1-71 Light chain GAGATCGTGATGACCCAGTCCCCACCTACACTGTCCCTGAGCCCAGGAGAGAGAGTGACCCTGAGCTGCAAGTCCAGCCAGTCTCTGCTGTACTCTTCCAACCAGAAGAATTCCCTGGCCTGGTATCAGCAGAAGCCAGGACAGGCTCCAAGGCTGCTGATCTACTGGGCTTCTACCAGGGAGTCCGGAATCCCTGCTCGGTTCTCTGGATCCGGAAGCGGCACAGACTTTACCCTGACAATCAGCTCTCTGCAGCCTGAGGATTTCGCCGTGTACTATTGTCAGCAGTACTATGGCTACCCATATACCTTTGGCCAGGGCACAAAGCTGGAGATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGC SEQ ID NO.64 Linker-1 AGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCTGCAG SEQ ID NO.66 Linker-2 GGCGGCGGCGGAAGCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCT SEQ ID NO.68 Linker-3 GAGTTC SEQ ID NO.70 Linker-4 TCTGGAGGCGGCAGCGGCGGCGGCGGCTCTGGAGGCGGCGGCAGCGGCGGCGGCGGCTCTGGCGGCGGATCTCTGCAG SEQ ID NO.72 Human Fc GAGCCTAAGTCTAGCgacaagacccacacctgtcccccttgtcctgcccctgaagccgccggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccctgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgacagtgctgcatcaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgaccaagaaccaggtgtccctgacctgtctcgtgaaaggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtacagcaagctgaccgtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaag SEQ ID NO.74 T-IL15-WT-1 (Tecentriq heavy chain - IL15Rα sushi) SEQ ID NO.76 794-IL15-WT-2 (794-h1-71 heavy chain-IL15Rα sushi- Linker1- IL15-WT) SEQ ID NO.78 794-IL15-7-2 (794-h1-71 heavy chain-IL15Rα sushi -Linker1-IL15-7) (D8E) SEQ ID NO.80 794-IL15-65-2 (794-h1-71 heavy chain-IL15Ra sushi-linker1-IL15-65) D8T SEQ ID NO.82 794-IL15-64-2 (794-h1-71 heavy chain-IL15Ra sushi-linker1-IL15-64) I6P SEQ ID NO.84 794-IL15-com1-2 (794-h1-71 heavy chain-IL15Rα sushi- Linker1- IL15-com1) (D8E / V3L) SEQ ID NO.86 794-IL15-com3-2 (794-h1-71 heavy chain - IL15Rα sushi -Linker1-IL15-com3) (I6D / V3L) SEQ ID NO.88 794-IL15-com4-2 (794-h1-71 heavy chain - IL15Rα sushi -Linker1-IL15-com4) (I6D / V3L / H105K) SEQ ID NO.90 794-IL15-com5-2 (794-h1-71 heavy chain - IL15Rα sushi -Linker1-IL15-com5) (I6D / H105K) SEQ ID NO.92 794-IL15-com6-2 (794-h1-71 heavy chain - IL15Rα sushi -Linker1-IL15-com6) (D8S / H105K) SEQ ID NO.94 794-IL15-com7-2 (794-h1-71 heavy chain - IL15Rα sushi -Linker1-IL15-com7) (D8S / H105N) SEQ ID NO.96

[0154] Table 5. Molecular composition of PD-L1-IL-15 fusion protein The domains and corresponding amino acid sequences of the PD-L1-IL-15 fusion protein molecules in Examples 9-15 Fusion protein number monoclonal antibody heavy chain sequence IL15Rα sushi Linker IL-15 protein Monoclonal antibody light chain sequence T-IL15-WT-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.1 SEQ ID NO.59 T-IL15-7-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.3 SEQ ID NO.59 T-IL15-8-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.5 SEQ ID NO.59 T-IL15-9-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.7 SEQ ID NO.59 T-IL15-10-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.9 SEQ ID NO.59 T-IL15-11-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.11 SEQ ID NO.59 T-IL15-26-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.13 SEQ ID NO.59 T-IL15-29-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.15 SEQ ID NO.59 T-IL15-42-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.17 SEQ ID NO.59 T-IL15-43-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.19 SEQ ID NO.59 T-IL15-com1-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.35 SEQ ID NO.59 T-IL15-com2-1 SEQ ID NO.57 SEQ ID NO.49 NA SEQ ID NO.37 SEQ ID NO.59 794-IL15-WT-2 SEQ ID NO.61 SEQ ID NO.49 SEQ ID NO.65 SEQ ID NO.1 SEQ ID NO.63 794-IL15-7-2 SEQ ID NO.61 SEQ ID NO.49 SEQ ID NO.65 SEQ ID NO.3 SEQ ID NO.63 794-IL15-64-2 SEQ ID NO.61 SEQ ID NO.49 SEQ ID NO.65 SEQ ID NO.27 SEQ ID NO.63 794-IL15-65-2 SEQ ID NO.61 SEQ ID NO.49 SEQ ID NO.65 SEQ ID NO.29 SEQ ID NO.63 794-IL15-com1-2 SEQ ID NO.61 SEQ ID NO.49 SEQ ID NO.65 SEQ ID NO.35 SEQ ID NO.63 794-IL15-com3-2 SEQ ID NO.61 SEQ ID NO.49 SEQ ID NO.65 SEQ ID NO.39 SEQ ID NO.63 794-IL15-com4-2 SEQ ID NO.61 SEQ ID NO.49 SEQ ID NO.65 SEQ ID NO.41 SEQ ID NO.63 794-IL15-com5-2 SEQ ID NO.61 SEQ ID NO.49 SEQ ID NO.65 SEQ ID NO.43 SEQ ID NO.63 794-IL15-com6-2 SEQ ID NO.61 SEQ ID NO.49 SEQ ID NO.65 SEQ ID NO.45 SEQ ID NO.63 794-IL15-com7-2 SEQ ID NO.61 SEQ ID NO.49 SEQ ID NO.65 SEQ ID NO.47 SEQ ID NO.63 Note: The numbering rules for PD-L1-IL-15 mutant fusion proteins are as follows:

[0155] For example: “T-IL15-xx-1”: “T”, Tecentriq; “IL15-xx”, indicating IL15-WT or IL15 mutant; “1”, indicating the structural pattern shown in Figure 5A;

[0156] For example: “794-IL15-xx-2”: “794”, 794-h1-71 monoclonal antibody; “IL15-xx”, indicating IL15-WT or IL15 mutant; “2”, indicating the structural pattern shown in Figure 5B.

[0157] Table 6. Molecular composition of IL-15-Fc fusion protein The domains and corresponding amino acid sequences of the IL-15-Fc fusion protein molecules in Examples 9-16 Fusion protein number IL-15 protein Linker IL15Rα sushi Linker Human Fc V9-IL15-61 (D8G) SEQ ID NO.21 SEQ ID NO.65 SEQ ID NO.53 SEQ ID NO.67 SEQ ID NO.73 V9-IL15-com6 (D8S / H105K) SEQ ID NO.45 SEQ ID NO.65 SEQ ID NO.53 SEQ ID NO.67 SEQ ID NO.73 V9-IL15-62 (D8I) SEQ ID NO.23 SEQ ID NO.65 SEQ ID NO.53 SEQ ID NO.67 SEQ ID NO.73 V9-IL15-63 (D8L) SEQ ID NO.25 SEQ ID NO.65 SEQ ID NO.53 SEQ ID NO.67 SEQ ID NO.73 V9-IL15-64 (I6P) SEQ ID NO.27 SEQ ID NO.65 SEQ ID NO.53 SEQ ID NO.67 SEQ ID NO.73 V9-IL15-65 (D8T) SEQ ID NO.29 SEQ ID NO.65 SEQ ID NO.53 SEQ ID NO.67 SEQ ID NO.73 V5-IL15-WT SEQ ID NO.1 SEQ ID NO.71 SEQ ID NO.53 SEQ ID NO.69 SEQ ID NO.73 V5-IL15-64 (I6P) SEQ ID NO.27 SEQ ID NO.71 SEQ ID NO.53 SEQ ID NO.69 SEQ ID NO.73 V5-IL15-65 (D8T) SEQ ID NO.29 SEQ ID NO.71 SEQ ID NO.53 SEQ ID NO.69 SEQ ID NO.73

[0158] Example 9: Expression of IL-15 fusion protein

[0159] Transient protein expression was performed using the ExpiCHO expression system. ExpiCHO-S (Cat no. A29127) host cells passaged in ExpiCHO™ Expression Medium (Cat no. A2910001) were diluted to an appropriate density and placed on a shaker (100 rpm, 37°C, 8% CO2) for transfection. The vector carrying the nucleic acid sequence encoding the fusion protein was added to OptiPRO™ SFM (Cat no. 12309019) medium to a final concentration of 0.5–1.0 μg / mL. An appropriate amount of ExpiFectamine™ CHO Reagent (Cat no. A29129) was added to the DNA-containing OptiPRO™ SFM medium to form a DNA-ExpiFectamine™ CHO Reagent complex. After standing at room temperature for 1–5 min, the complex was slowly added dropwise to the cell suspension to be transfected. On day 1 post-transfection, a certain amount of ExpiFectamine™ CHO Enhancer (Cat no. A29129) and ExpiCHO™ Feed (Cat no. A29129) were added, followed by incubation at 32°C. From day 4 to day 6 post-transfection, a certain amount of ExpiCHO™ Feed (Cat no. A29129) was added again. From day 10 to day 12 post-transfection, the supernatant was harvested by centrifugation at 5000 g for 30 min.

[0160] Example 10: Purification of IL-15 fusion protein

[0161] The mutant fusion protein was purified using the magnetic bead method. An appropriate amount of magnetic bead suspension (GenScript, Cat. NO. L00695) was added to the fermentation supernatant and incubated in a rotary mixer for 2 h to ensure that the IL-15 fusion protein was bound to the magnetic beads. After discarding the supernatant, the magnetic beads were washed three times with PBS. Finally, pH 3.0 citrate was added to elute the IL-15 fusion protein. After neutralizing the sample with 1M Tris-HCl, the protein concentration was determined using NanoDrop One.

[0162] Example 11: Determination of the purity of IL-15 fusion protein by size exclusion chromatography

[0163] In the detection method of this invention, the purity of the IL-15 mutant fusion protein of this invention was determined by size exclusion chromatography (SEC) using a TSKgel G3000SWXL column (TOSOH, 0008541) and a pre-column TSKgel guard column SWXL (TOSOH, 0008543). The mobile phase (50 mM PB, 300 mM NaCl, pH 6.8) was used to equilibrate the column, and the flow rate was 1 mL / min. The UV detection wavelength was 280 nm. The results are shown in Table 7.

[0164] Table 7. Determination of IL-15 fusion protein purity by size exclusion chromatography serial number SEC main peak purity % serial number SEC main peak purity % T-IL15-7-1 96.99 794-IL15-com1-2 99.83 T-IL15-8-1 93.7 794-IL15-com3-2 99.6 T-IL15-9-1 93.4 794-IL15-com4-2 98.6 T-IL15-10-1 96.5 794-IL15-com5-2 99.3 T-IL15-11-1 94 794-IL15-com6-2 99.2 T-IL15-26-1 99 794-IL15-com7-2 99.2 T-IL15-29-1 95.6 V9-IL15-61 97.7 T-IL15-42-1 98.5 V9-IL15-com6 97.5 T-IL15-43-1 96.7 V9-IL15-62 97.7 T-IL15-WT-1 96.08 V9-IL15-63 99.11 794-IL15-WT-2 98.3 794-IL15-65-2 98.56 794-IL15-7-2 99.78 794-IL15-64-2 98.32 T-IL15-com1-1 98 V5-IL15-WT 98.23 T-IL15-com2-1 97.26

[0165] Example 12: Experiment on Mo7e cell proliferation induced by IL-15 mutant fusion protein

[0166] Mo7e is a human megakaryocyte leukemia cell line (Cobioer, CBP60791) that can be used to study the cell proliferation activity of IL-15. The IL-15 mutant fusion protein was diluted to a gradient concentration (final concentration 100000pM-1.28pM, 5-fold dilution) with 1640-10% FBS (RPMI 1640, Gibco, 72400047; FBS, Gibco, 10099141). Mo7e cells were diluted to 10⁵ cells / ml with 1640-10% FBS. 50 μl of mutant culture medium and 50 μl of Mo7e cell suspension were added to a 96-well U-plate, mixed well, and incubated at 37°C with 5% CO₂. After 72 hours, the culture plate was removed and 100 μl of Cell Titer Glo luminescent cell viability assay reagent (Promega, G7571) was added. The luminescence intensity was measured, and the fluorescence intensity was directly proportional to the cell proliferation capacity.

[0167] The results, as shown in Figures 6A-6D, indicate that the activities of the T-IL15-7-1, T-IL15-8-1, and T-IL15-9-1 mutants were significantly lower than those of T-IL15-WT-1. The EC50 of T-IL15-7-1 was 5735 pM, while the corresponding EC50 of T-IL15-WT-1 was 133.3 pM (Figure 6A). The activities of the T-IL15-10-1, T-IL15-11-1, and T-IL15-26-1 mutants were also significantly lower than those of T-IL15-WT-1. The EC50s of T-IL15-10-1 and T-IL15-26-1 were 28076 pM and 290.9 pM, respectively, while the corresponding EC50 of T-IL15-WT-1 was 143.3 pM (Figure 6B). The activities of the T-IL15-29-1 and T-IL15-42-1 mutants were lower than those of T-IL15-WT-1, with EC50 values ​​of 285.2 pM and 194.5 pM, respectively. The activity of the T-IL15-43-1 mutant was enhanced compared to T-IL15-WT-1, with an EC50 of 103.2 pM, while the EC50 of T-IL15-WT-1 was 150.7 pM (Figure 6C). The combined mutant activities of T-IL15-com1-1 and T-IL15-com2-1 were lower than those of T-IL15-WT-1. The Tecentriq control group had no effect on Mo7e cell proliferation, meaning that the anti-PD-L1 antibody had no induced proliferation activity in Mo7e cells (Figure 6D), and the effect on cell proliferation activity was generated by IL-15.

[0168] Example 13: IL15 mutant fusion protein induces CD8+ T cell proliferation

[0169] To detect the stimulatory effect of the IL-15 mutant fusion protein on the proliferation of CD8+ T cells in human PBMCs (Allcells, Lot: 1911150123), this embodiment used Ki67 as a proliferation marker to detect the proliferation rate of CD8+ T cells three days after stimulating human PBMCs with different concentrations of the IL-15 mutant fusion protein. First, human PBMCs were suspended in RPMI 1640 medium (Gibco, Cat: 72400047) containing 10% FBS (Gibco, Cat: 10099141) and 1% penicillin-streptomycin (Gibco, Cat: 15140122), and the cell density was adjusted to 2 × 106 cells / ml. 100 μl / well was added to a 96-well U-plate (Corning, Cat: 3799). Then, the IL-15 mutant fusion protein was serially diluted 4-fold starting at 500 nM for a total of 11 gradients. 100 μl of each concentration was then added to each well of a 96-well U-plate containing human PBMCs. After thorough mixing, the plates were incubated at 37°C in a 5% CO2 incubator for three days. The cultured cells were stained using the LIVE / DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen, Cat: L34964) and antibodies against CD3-AF700 (BD, Cat: 557943), CD8-FITC (BD, Cat: 555366), and APC-Ki67 (Biolegend, Cat: 350514). Flow cytometry was then used to detect the proportion of Ki67+ cells in CD3+CD8+ T cells stimulated with different concentrations of the IL-15 mutant fusion protein. Figures 7A-7N show the gradient dilutions of T-IL15-7-1, T-IL15-8-1, T-IL15-9-1, T-IL15-10-1, T-IL15-11-1, T-IL15-26-1, T-IL15-29-1, T-IL15-42-1, T-IL15-43-1, 794-IL15-7-2, 794-IL15-com1-2, 794-IL15-com3-2, 794-IL15-com4-2, 794-IL15-com5-2, 794-IL15-com6-2, 794-IL15-com7-2, V9-IL15-61, V9-IL15-62, and V9 -IL15-63, V9-IL15-com6, 794-IL15-65-2, 794-IL15-64-2, V5-IL15-WT, and P22339. The proliferation rate of CD8+ T cells and the EC50 values ​​of each mutant after stimulating human PBMCs for three days.Among them, T-IL15-8-1, T-IL15-9-1, and T-IL15-11-1 significantly reduced the proliferation ability of CD8+ T cells, and did not reach the plateau, so the EC50 could not be calculated by fitting the curve. 794-IL15-com4-2, 794-IL15-com5-2, 794-IL15-com6-2, 794-IL15-com7-2, V9-IL15-61, V9-IL15-62, V9-IL15-63, V9-IL15-com6, and 794-IL15-65-2 did not reach the plateau, and the EC50 values ​​were not accurately fitted. However, the results showed that the above mutant combinations had lower proliferation activity of CD8+ T cells than 794-IL15-WT-2 or P22339. T-IL15-WT-1 and 794-IL15-WT-2 were wild-type IL-15 controls, and P22339 was a mutant IL-15 control; 794-h1-71 monoclonal antibody was an anti-PD-L1 antibody control; Drug0 was a negative control without any added protein. Compared to the wild-type IL-15 control, the IL-15 mutant showed weaker proliferative activity against CD8+ T cells. The 794-h1-71 monoclonal antibody control group had no effect on CD8+ T cell proliferation, meaning that the anti-PD-L1 antibody had no proliferative activity against CD8+ T cells (Figure 7B), and the effect on cell proliferation was caused by IL-15.

[0170] Example 14: IL15 mutant fusion protein induces NK cell proliferation

[0171] To investigate the stimulatory effect of the IL-15 mutant fusion protein on the proliferation of NK cells in human PBMCs (Allcells, Lot: 1911150123), this embodiment used Ki67 as a proliferation marker to detect the proliferation rate of NK cells three days after stimulating human PBMCs with different concentrations of the IL-15 mutant fusion protein. First, human PBMCs were suspended in RPMI 1640 medium (Gibco, Cat: 72400047) containing 10% FBS (Gibco, Cat: 10099141) and 1% penicillin-streptomycin (Gibco, Cat: 15140122), and the cell density was adjusted to 2 × 10⁶ cells / ml. 100 μl / well was added to a 96-well U-plate (Corning, Cat: 3799). Then, the IL-15 mutant fusion protein was serially diluted 4-fold starting from 500 nM, for a total of 11 gradients. Subsequently, 100 μl / well of each gradient sample was added to a 96-well U-plate containing human PBMCs, mixed thoroughly, and incubated at 37°C in a 5% CO2 incubator for three days. The cultured cells were then stained using the LIVE / DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen, Cat: L34964) and antibodies against CD3-AF700 (BD, Cat: 557943), CD56-PE (BD, Cat: 555516), and APC-Ki67 (Biolegend, Cat: 350514). The proportion of Ki67+ cells in NK cells stimulated with different concentrations of the IL-15 mutant fusion protein was then detected using an Invitrogen Attune NxT flow cytometer. Figures 8A-8M show the gradient dilutions of T-IL15-7-1, T-IL15-8-1, T-IL15-9-1, T-IL15-10-1, T-IL15-11-1, T-IL15-26-1, T-IL15-29-1, T-IL15-42-1, T-IL15-43-1, 794-IL15-7-2, 794-IL15-com1-2, 794-IL15-com4-2, 794-IL15-com5-2, 794-IL15-com6-2, 794-IL15-com7-2, V9-IL15-61, V9-IL15-62, V9-IL15-63, and V9-IL15. Three days after stimulation of human PBMCs with -com6, 794-IL15-65-2, 794-IL15-64-2, V5-IL15-WT, and P22339, the proportion of CD3-CD56+ NK cells proliferated, and the EC50 value of each mutant stimulation.Among them, T-IL15-8-1, T-IL15-9-1, T-IL15-11-1, V9-IL15-com6, V9-IL15-62, and V9-IL15-63 significantly reduced the proliferation capacity of NK cells, failing to reach a plateau, and the EC50 values ​​did not accurately fit the data; however, the above mutant combinations significantly reduced the NK cell proliferation activity compared to the control groups T-IL15-WT-1 or P22339. T-IL15-WT-1 and 794-IL15-WT-2 were wild-type IL-15 controls, P22339 was a mutant IL-15 control; 794-h1-71 monoclonal antibody was an anti-PD-L1 antibody control; Drug0 was a negative control without any added protein. Compared to the wild-type IL-15 control, the IL-15 mutants showed weaker NK cell proliferation. The 794-h1-71 monoclonal antibody control group had no effect on NK cell proliferation, meaning that the anti-PD-L1 antibody had no induced proliferation activity in NK cells (Figure 8B). The effect on cell proliferation activity was caused by IL-15.

[0172] Example 15: In vivo efficacy of humanized anti-PD-L1 antibody-IL15 bifunctional molecule / fusion protein in mice

[0173] Human melanoma cells A375 (Beina Biotechnology; BNCC100266) were injected subcutaneously into the right posterior back of NPG mice at a dose of 5×106 cells / 100μL. The day after A375 inoculation, PBMCs (ALLCELLs, PB005F-C) were thawed and cryopreserved and injected into NPG mice (5-6 weeks old, female; purchased from Beijing Vitonda Biotechnology Co., Ltd.) via the tail vein at a dose of 5×106 cells / 200μl. Six days after PBMC inoculation, 40 μl of blood was collected to detect the proportion of hCD45+ cells. Once the tumor reached approximately 80 mm³, mice were removed based on body weight, hCD45+ cell proportion, and tumor size (too large or too small). Mice were then randomly divided into four groups (n=8 per group, totaling 32 mice) according to tumor volume: PBS group, Tecentriq group (10 mg / kg), 794-IL15-WT-2 group (1 mg / kg), and 794-IL15-com6-2 group (4 mg / kg). Administration was intraperitoneal. The 794-IL15-WT-2 group (1 mg / kg) and the 794-IL15-com6-2 group (4 mg / kg) received the drug once a week for a total of one dose. The PBS group and the Tecentriq group (10 mg / kg) received the drug twice a week for a total of five doses. Tumor volume was measured three times a week, and data were recorded. Tumor volume (major diameter × minor diameter² / 2) and growth inhibition rate (TGITV (%), TGITV (%) = [1-(Ti-T0) / (Vi-V0)]×100%; Ti: mean tumor volume in the treatment group on day i of administration, T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the solvent control group on day i of administration, V0: mean tumor volume in the solvent control group on day 0 of administration) were calculated. On day 14 of administration, all treatment groups showed significant inhibition of tumor volume, with statistically significant differences (P<0.05), and the 794-IL15-WT-2 and 794-IL15-com6-2 groups showed significantly greater inhibition of tumor volume compared with the Tecentriq group (P<0.05). See Figures 9A-9C and Table 8.

[0174] Table 8. Effects of test substances on tumor volume of A375 mice in immune-reconstituted NPG mice Group test substance Tumor volume (mm) 3 ) a P b P c Before administration Group dosing Day 21 TGI TV (%) G1 PBS 82±6 427±73 - - - G2 Tecentriq 78±5 253±38 49.35 <0.0001 - G3 794-IL15-WT-2 78±4 198±35 65.08 <0.0001 0.0399 G4 794-IL15-com6-2 78±5 129±34 85.15 <0.0001 0.0125 Note: a: Mean ± standard error; b: Tumor volume in the drug-treated group and the Vehicle control group (PBS) were statistically compared on day 14 of drug administration, using two-way ANOVA analysis. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; c: Tumor volume in the drug-treated group and the Tecentriq positive drug group were statistically compared on day 14 of drug administration, using two-way ANOVA analysis. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0175] The above results indicate that the humanized anti-PD-L1 antibody-IL15 bifunctional molecule / fusion protein 794-IL15-WT-2 and 794-IL15-com6-2 both significantly inhibited the growth of subcutaneous xenografts of A375 tumors (P<0.0001); the 794-IL15-WT-2 group and the 794-IL15-com6-2 group showed stronger inhibitory effects on tumor volume compared with the Tecentriq group, and the difference was significant (P<0.05).

[0176] Example 16: In vivo efficacy detection of IL15-Fc fusion protein in mice

[0177] Human melanoma cells A375 (Beina Biotechnology; BNCC100266) were injected subcutaneously into the right posterior back of NPG mice at a dose of 5×106 cells / 100μL. The day after A375 inoculation, PBMCs (ALLCELLs; PB005F-C) were thawed and cryopreserved and injected into NPG mice (5-6 weeks old, female; purchased from Beijing Vitonda Biotechnology Co., Ltd.) via the tail vein at a dose of 5×106 cells / 200μl. Six days after PBMC inoculation, 40 μl of blood was collected to detect the proportion of hCD45+ cells. Once the tumor reached approximately 69 mm³, mice were removed based on body weight, hCD45+ cell ratio, tumor size (too large or too small), and tumor volume. Mice were then randomly divided into seven groups based on tumor volume: PBS group, ALT803 (0.2 mg / kg), V9-IL15-61 (1 mg / kg), V9-IL15-61 (5 mg / kg), V9-IL15-com6 (1 mg / kg), V9-IL15-com6 (5 mg / kg), and V5-IL15-WT (2 mg / kg), with eight mice in each group, for a total of 56 mice. Administration was intraperitoneal, administered once weekly for three weeks according to the group assignments. Tumor volume was measured three times weekly, and data were recorded. Tumor volume (long axis × short axis 2 / 2) and growth inhibition rate (TGITV (%), tumor growth inhibition %, TGITV (%) = [1-(Ti-T0) / (Vi-V0)]×100%; Ti: mean tumor volume of the treatment group on day i of administration, T0: mean tumor volume of the treatment group on day 0 of administration; Vi: mean tumor volume of the solvent control group on day i of administration, V0: mean tumor volume of the solvent control group on day 0 of administration) were calculated. On day 21 of administration, compared with the PBS control group, the treatment groups showed significant inhibition of tumor volume, with statistically significant differences (P<0.05), and the positive control group ALT803 and V9-IL15-com6 showed similar inhibition of tumor volume (P>0.05). See Figures 10A-10C and Table 9.

[0178] Table 9. Effects of test substances on tumor volume of A375 mice in immune-reconstituted NPG mice Group test substance Tumor volume (mm) 3 ) a P b Before administration Group dosing Day 21 TGI TV (%) G1 PBS 70 ±5 1755±360 - - G2 ALT803 (0.2 mg / kg) 68 ±5 788 ±144 57.3 <0.0001 G3 V9-IL15-61 (1 mg / kg) 69 ±6 1082 ±137 39.9 <0.0001 G4 V9-IL15-61 (5 mg / kg) 70 ±6 1001 ±206 44.7 <0.0001 G5 V9-IL15-com6 (1 mg / kg) 69 ±6 1144 ±298 36.2 <0.0001 G6 V9-IL15-com6 (5 mg / kg) c 67 ±6 809 ±215 56.0 <0.0001 G7 V5-IL15-WT (2 mg / kg) 69 ±6 1065 ±148 40.9 <0.0001 Note: a: Mean ± Standard Error; b: Tumor volume in the drug treatment group and the vehicle control group were statistically compared on day 14 of drug administration using a two-way ANOVA analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; c: Identify outliers analysis was performed on tumor volume on Day 21. One data point in the V9-IL15-com6 5mpk group was abnormally large and was removed.

[0179] The experimental animals were active and fed well during the administration period. After administration, the ALT803 and V5-IL15-WT groups showed a significant trend of weight loss and some animals died, indicating that the mice were intolerant to ALT803 and V5-IL15-WT at this frequency and dosage. Animals in the V9-IL15-61 (5mpk) and V9-IL15-com6 (1mpk) groups died, but they exhibited GVHD symptoms such as anemia before death. It is speculated that the deaths were caused by GVHD and were unrelated to the drugs. See Figure 10D, Table 10, and Table 11.

[0180] Table 10 Effects of test substances on body weight of immune-reconstituted A375 tumor-bearing NPG mice Group test substance Body weight (g) a 21 days after administration Weight change (g) Before administration Group dosing Day 21 P b G1 PBS 21.7±0.7 21.3±0.8 - -0.4 G2 ALT803 (0.2 mg / kg) 21.7±0.5 20.8±1.0 0.6482 -0.9 G3 V9-IL15-61 (1 mg / kg) 20.8±0.5 20.8±0.7 0.3405 0 G4 V9-IL15-61 (5 mg / kg) 21.4±0.7 19.6±0.9 0.2574 -1.8 G5 V9-IL15-com6 (1 mg / kg) 21.7±0.7 20.8±1.0 0.4836 -0.9 G6 V9-IL15-com6 (5 mg / kg) 21.3±0.8 18.4±1.1 0.0097 -2.9 G7 V5-IL15-WT (2 mg / kg) 21.5±0.3 20.0±0.7 0.0037 -1.5 Note: a: mean ± standard error; b: the weight of the drug administration group and the vehicle control group were statistically compared on day 21 of drug administration, using a two-way ANOVA analysis.

[0181] Table 11 Effects of the test substance on the survival of immune-reconstituted A375 tumor-bearing NPG mice Group test substance Number of mice surviving on day 21 after group drug administration a Time of death of mice Day 15 of group-based medication administration Day 19 of group-based medication administration Day 20 of group-based medication administration Day 21 of group-based medication administration G1 PBS 8 - - - - G2 ALT803 (0.2 mg / kg) 7 1 b - - - G3 V9-IL15-61 (1 mg / kg) 8 - - - - G4 V9-IL15-61 (5 mg / kg) 7 0 - - - G5 V9-IL15-com6 (1 mg / kg) 7 - - 0 - G6 V9-IL15-com6 (5 mg / kg) 8 - - - - G7 V5-IL15-WT (2 mg / kg) 6 - 1 - 0 Note: a: mean ± standard error; b: mouse death after group administration, 1 indicates that 1 mouse died on that day, 0 indicates that 1 mouse died on that day but previously observed to have symptoms such as anemia and jaundice.

[0182] The above results show that both IL15-Fc fusion proteins V9-IL15-61 and V9-IL15-com6 have significant inhibitory effects on the growth of human immune reconstitution A375 tumor subcutaneous xenografts; V9-IL15-com6 (5 mg / kg) and the positive control antibody ALT803 (0.2 mg / kg) have comparable TGI (tumor growth inhibition rate) levels and V9-IL15-com6 has better safety than ALT803. [Simplified Explanation of the Diagram]

[0078] The foregoing and other aspects of the present invention will be clearly illustrated below through a detailed description of the invention and the accompanying drawings. The accompanying drawings are provided to illustrate some preferred embodiments of the invention; however, it is to be understood that the invention is not limited to the specific embodiments disclosed. Figure 1: Results of humanized PD-L1 antibody blocking PD-L1 and PD-1 binding, with Tecentriq as the positive control. Figure 2: FACS assay of the binding ability of humanized PD-L1 antibody to PD-L1 at the cellular level, with Avelumab as the positive control. Figure 3: Jurkat-PD-1 / CHO-PD-L1-NFAT system test of the blocking ability of humanized PD-L1 antibody against PD-L1 / PD-1, with Avelumab as the positive control. Figure 4: Results of humanized anti-PD-L1 antibody promoting IFN-γ secretion in mixed lymphocyte reactions, with anti-Hel antibody as the negative control and Avelumab as the positive control. Figure 5. Two structures of the IL-15 fusion protein: A: IL-15 wild-type or mutant co-expressed and assembled with IL-15Rαsushi, non-covalently linked; B: IL-15 wild-type or mutant fused with IL-15Rαsushi via a linker for tandem expression and assembly; C: IL-15 wild-type or mutant first linked with IL-15Rαsushi via a linker, then IL-15Rαsushi is fused with Fc via a linker for tandem expression and assembly; D: IL-15Rαsushi first linked with IL-15 wild-type or mutant via a linker, then IL-15 wild-type or mutant is fused with Fc via a linker for tandem expression and assembly. Figures 6A-6D. IL-15 mutant promotes Mo7e cell proliferation: The fusion protein structure is shown in Figure 5A. Tecentriq is fused with IL-15Rαsushi, and the IL-15 mutant and IL-15Rαsushi are non-covalently linked, without a linker link.Figure 7A-7N, IL-15 mutant-induced CD8+ T cell proliferation: Figure 7A: The fusion protein structure is shown in Figure 5A. Tecentriq is fused with IL-15Rαsushi. The IL-15 mutant and IL-15Rαsushi are non-covalently linked without a linker. Figure 7B-7G, 7M-7N: The fusion protein structure is shown in Figure 5B. The self-developed PD-L1 monoclonal antibody is fused with IL-15Rαsushi and the IL-15 mutant. The IL-15 mutant and IL-15Rαsushi are expressed in tandem via a linker. Drug0 represents a negative control in the reaction system without any added protein. Figure 7H-7K: The fusion protein structure is shown in Figure 5D. IL-15Rαsushi is first linked to the wild-type or mutant IL-15 via a linker, and the wild-type or mutant IL-15 is then fused to Fc via a linker for tandem expression and assembly. Figure 7L shows the fusion protein as shown in Figure 5C. IL-15 wild-type or mutant is first linked to IL-15Rαsushi via a linker, and then IL-15Rαsushi is tandemly fused to Fc via a linker for expression and assembly. Figures 8A-8M show the IL-15 mutant-induced NK cell proliferation: Figure 8A: The fusion protein structure is shown in Figure 5A. Tecentriq is fused with IL-15Rαsushi. The IL-15 mutant and IL-15Rαsushi are non-covalently linked, without a linker connection. Figures 8B-8F, 8L-8M: The fusion protein structure is shown in Figure 5B. A self-developed PD-L1 monoclonal antibody is fused with IL-15Rαsushi and an IL-15 mutant. The IL-15 mutant and IL-15Rαsushi are tandemly expressed via a linker. Drug0 represents a negative control in which no protein was added to the reaction system. Figures 8G-8J: The fusion protein is shown in Figure 5D. IL-15Rαsushi is first linked to IL-15 wild-type or mutant via a linker, and then IL-15 wild-type or mutant is tandemly fused to Fc via a linker for expression and assembly. Figure 8K: The fusion protein is shown in Figure 5C. IL-15 wild-type or mutant is first linked to IL-15Rαsushi via a linker, and then IL-15Rαsushi is tandemly fused to Fc via a linker for expression and assembly. Figures 9A-9C: In vivo efficacy of hPBMC-A375 mice. The fusion protein structure is shown in Figure 5B. Figures 10A-10C: In vivo efficacy of hPBMC-A375 mice. The fusion protein structure is shown in Figure 5C or 5D. Figure 10D: Changes in body weight of hPBMC-A375 mice after drug administration. [Sequence List]

Claims

1. An IL-15 mutant polypeptide, characterized in that the polypeptide contains a mutation at one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15.

2. A polypeptide comprising an IL-15 mutant, characterized in that the polypeptide comprises a mutation at one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15.

3. The polypeptide as claimed in claim 1 or 2, characterized in that the mutation is a substitution, insertion, or deletion; preferably, the polypeptide contains at least two, three, or four amino acid residues selected from Val3, Ile6, Asp8, or His105; preferably, the mutation is an amino acid substitution selected from the group consisting of: Val3Leu (V3L), Ile6Asp (I6D), Ile6Pro (I6P), Asp8Glu (D8E), Asp8Gln (D8Q), Asp8Arg (D8R), Asp8Ser (D8S), Asp8Val (D8V), Asp8Gly (D8G), Asp8 Ile (D8I), Asp8Leu (D8L), Asp8Thr (D8T), His105Asn (H105N), and / or His105Lys (H105K); preferably, the polypeptide contains the following mutations or combinations of mutations: (1) Asp8Glu; (2) Asp8Gln; (3) Asp8Arg; (4) Asp8Ser; (5) Asp8Val; (6) Val3Leu; (7) Ile6Asp; (8) His105 Lys; (9) His105 Asn; (10) Asp8Gly; (11) Asp8Ile; (12) Asp8Leu; (13) Ile6Pro; (14) Asp8Thr; (15) Asp8Glu and Val3Leu; (16) Asp8Glu and Ile6Asp; (17) Val3Leu and Ile6Asp; (18) Ile6Asp and His105Lys; (19) Asp8Ser and His105Lys; (20) Asp8Ser and His105 Asn; or, (21) Val3Leu, Ile6Asp and His105Lys; preferably, the IL-15 mutant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with human wild-type IL-15.

4. The polypeptide as claimed in any one of claims 1-3, characterized in that the amino acid sequence of the IL-15 mutant is as shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.43, SEQ ID NO.45 or SEQ ID NO.47; preferably, the polypeptide has the following properties: (1) mediates the proliferation of human CD8+ T cells; (2) mediates the proliferation of human NK cells; and / or, (3) inhibits tumor growth; Preferably, the peptide mediates the proliferation / expansion of CD8+ T and / or NK cells less than that of peptides containing wild-type IL-15; preferably, the amino acid sequence of the wild-type IL-15 is as shown in SEQ ID NO.

1.

5. A protein, characterized in that the protein comprises a polypeptide as described in any one of claims 1-4; and further comprises, fused with the IL-15 mutant,: (1) an immunoglobulin molecule or a portion thereof, and / or (2) IL-15Rα; preferably, the immunoglobulin molecule is an antibody or antigen-binding fragment; the immunoglobulin molecule portion is an immunoglobulin Fc region; preferably, the antibody or antigen-binding fragment is selected from: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or, (3) a fully human antibody or a fragment thereof; preferably, the antibody or antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, nanobodies, and antibody minimum recognition units; Preferably, the immunoglobulin Fc region is selected from the Fc region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; preferably, it contains the sequence of the constant region of human or mouse antibody IgG1, IgG2, IgG3, or IgG4; preferably, the amino acid sequence of the immunoglobulin Fc region is as shown in SEQ ID NO. 73; preferably, the IL-15 mutant is fused to the immunoglobulin molecule or a portion thereof with or without a linker peptide, or the IL-15 mutant is fused to IL-15Rα with or without a linker peptide; preferably, a linker peptide is used; preferably, the linker peptides shown in SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, or SEQ ID NO. 71 are used; preferably, the IL-15 mutant is fused to IL-15Rα with or without a linker peptide, and then fused to the immunoglobulin molecule or a portion thereof; preferably, a linker peptide is used; preferably, the linker peptides shown in SEQ ID NO. 65, SEQ ID NO. 69, or SEQ ID NO. 71 are used.

6. The protein as claimed in claim 5, characterized in that the connection order of each domain from the N-terminus to the C-terminus is: (1) an immunoglobulin molecule or a portion thereof, IL-15Rα, IL-15 mutant; (2) an immunoglobulin molecule or a portion thereof, IL-15 mutant, IL-15Rα; (3) IL-15 mutant, IL-15Rα, immunoglobulin molecule or a portion thereof; (4) IL-15Rα, IL-15 mutants, immunoglobulin molecules or parts thereof; (5) IL-15 mutant, immunoglobulin molecule or part thereof; (6) immunoglobulin molecule or part thereof, IL-15 Rα; (7) IL-15 Rα, immunoglobulin molecule or part thereof; (8) IL-15 mutant, IL-15Rα; or, (9) IL-15Rα, IL-15 mutant; Preferably, when IL-15Rα or IL-15 mutant is fused with an immunoglobulin molecule, it is fused at the N-terminus of the variable region of the heavy chain of the immunoglobulin molecule or the C-terminus of the Fc region of the immunoglobulin; when IL-15Rα or IL-15 mutant is fused with the Fc region of the immunoglobulin, it is fused at the N-terminus or C-terminus of the Fc region of the immunoglobulin.

7. A protein comprising the following four parts: (1) an immunoglobulin heavy chain; (2) an immunoglobulin light chain; (3) IL-15Rα; and (4) an IL-15 mutant polypeptide as claimed in any one of claims 1-4; preferably, the protein is a homodimer comprising a monomer consisting of parts (1)-(4); preferably, IL-15Rα is fused to the N-terminus of the variable region of the immunoglobulin heavy chain or the C-terminus of the Fc region of the immunoglobulin chain, with or without a linker peptide; preferably, the IL-15 mutant polypeptide is non-covalently linked to IL-15Rα, or the IL-15 mutant is fused to the other end of IL-15Rα, with or without a linker peptide.

8. A protein comprising the following three parts: (1) an immunoglobulin Fc region; (2) IL-15Rα; and (3) an IL-15 mutant polypeptide as claimed in any one of claims 1-4; preferably, the protein is a homodimer comprising monomers consisting of parts (1)-(3); preferably, IL-15Rα is fused to the N-terminus or C-terminus of the IL-15 mutant polypeptide by or without a linker peptide, and then fused to the N-terminus or C-terminus of the immunoglobulin Fc region by or without a linker peptide.

9. The protein according to any one of claims 5-8, characterized in that the immunoglobulin is selected from anti-PD-L1 antibodies; the anti-PD-L1 antibody is preferably Tecentriq, KN-035, or 794-h1-71; preferably, the anti-PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region having sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100, respectively, or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity compared to the sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100; or, the heavy chain variable region and the light chain variable region having sequences shown in SEQ ID NO: 97 and SEQ ID NO: 98, respectively, or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity compared to the sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100; or, the heavy chain variable region and the light chain variable region having sequences shown in SEQ ID NO: 97 and SEQ ID NO: 98, respectively, or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity compared to the sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100; The sequence shown in SEQ ID NO. 98 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher homology with the sequence shown in SEQ ID NO. 98; preferably, the IL-15Rα is selected from IL-15Rα-sushi; preferably, the amino acid sequence of IL-15Rα-sushi is shown in SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53 or SEQ ID NO.

55.

10. An antibody or antigen-binding fragment that specifically binds to PD-L1, characterized in that the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100, respectively, or have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity compared with the sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100; preferably, its dissociation constant (KD) for binding to human programmed death ligand-1 (PD-L1) is not greater than 1.8 × 10⁻⁹ M, and its dissociation constant (KD) for binding to cynomolgus monkey programmed death ligand-1 (PD-L1) is not greater than 9.4 × 10⁻¹⁰ M; or, optionally, the antibody or antigen-binding fragment binds to or does not bind to monkey PD-L1; Optionally, the antibody or antigen-binding fragment may or may not bind to mouse PD-L1; preferably, the anti-PD-L1 antibody competitively binds to PD-L1 or its antigenic epitope and has the following properties: (1) specifically binds to recombinant PD-L1 protein and cells expressing PD-L1; (2) blocks the binding of PD-L1 to PD-1 protein; (3) inhibits the binding of PD-1 to PD-L1 expressed on the cell surface; (4) enhances T cell activity; and / or (5) inhibits tumor growth; preferably, the anti-PD-L1 antibody contains a constant region selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, it contains a sequence containing a constant region of human or mouse antibody IgG1, IgG2, IgG3 or IgG4; preferably, the PD-L1 antibody is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies.

11. An isolated nucleic acid molecule, characterized in that it encodes a polypeptide as described in any one of claims 1-4, a protein as described in any one of claims 5-9, or an antibody or antigen-binding fragment as described in claim 10.

12. An expression vector, characterized in that the expression vector comprises a nucleic acid molecule isolated as described in claim 11.

13. A host cell, characterized in that the host cell comprises an isolated nucleic acid molecule as described in claim 11 or an expression vector as described in claim 12; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from Chinese hamster ovary cells (CHO).

14. A method for preparing a polypeptide or protein, characterized in that a host cell as described in claim 13 is cultured under appropriate conditions, and the polypeptide or protein is isolated.

15. A pharmaceutical composition, characterized in that the composition comprises a polypeptide as described in any one of claims 1-4, a protein as described in any one of claims 5-9, an antibody or antigen-binding fragment as described in claim 10, an isolated nucleic acid molecule as described in claim 11, an expression vector as described in claim 12, a cell as described in claim 13, or a product prepared by the method described in claim 14; and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition further comprises an additional antitumor agent.

16. The use of a polypeptide as claimed in any one of claims 1-4, a protein as claimed in any one of claims 5-9, an antibody or antigen-binding fragment as claimed in claim 10, an isolated nucleic acid molecule as claimed in claim 11, an expression vector as claimed in claim 12, a cell as claimed in claim 13, a product prepared by the method of claim 14, or a pharmaceutical composition as claimed in claim 15 in the preparation of a medicament for the prevention and / or treatment of a disease in an individual; wherein the disease is preferably a tumor or an inflammatory disease; preferably, the tumor is selected from glioblastoma, prostate cancer, hematologic malignancies, B-cell tumors, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, cutaneous T-cell lymphoma, T-cell lymphoma, solid tumors, urothelial / bladder cancer, melanoma, lung cancer, renal cell carcinoma, breast cancer, gastric and esophageal cancer, prostate cancer, pancreatic cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, and squamous cell head and neck cancer.

17. A method for preventing and / or treating a disease in an individual, comprising administering to a patient in need a polypeptide as described in any one of claims 1-4, a protein as described in any one of claims 5-9, an antibody or antigen-binding fragment as described in claim 10, an isolated nucleic acid molecule as described in claim 11, an expression vector as described in claim 12, a cell as described in claim 13, a product prepared by the method of claim 14, or a pharmaceutical composition as described in claim 15; wherein the disease is preferably a tumor or an inflammatory disease; Preferably, the tumor is selected from glioblastoma, prostate cancer, hematologic malignancies, B-cell tumors, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, cutaneous T-cell lymphoma, T-cell lymphoma, solid tumors, urothelial / bladder cancer, melanoma, lung cancer, renal cell carcinoma, breast cancer, gastric and esophageal cancer, prostate cancer, pancreatic cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, and squamous cell head and neck cancer.

18. A polypeptide as described in any one of claims 1-4, a protein as described in any one of claims 5-9, an antibody or antigen-binding fragment as described in claim 10, an isolated nucleic acid molecule as described in claim 11, an expression vector as described in claim 12, a cell as described in claim 13, a product prepared by the method described in claim 14, or a pharmaceutical composition as described in claim 15, for the prevention and / or treatment of a disease in an individual; said disease is preferably a neoplastic disease or an inflammatory disease; preferably, said tumor is selected from glioblastoma, prostate cancer, hematologic malignancies, B-cell tumors, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, cutaneous T-cell lymphoma, T-cell lymphoma, solid tumors, urothelial / bladder cancer, melanoma, lung cancer, renal cell carcinoma, breast cancer, gastric and esophageal cancer, prostate cancer, pancreatic cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, and squamous cell head and neck cancer.

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