TW202244058A - Human il-15 mutant and application thereof - Google Patents

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

Human IL-15 mutants and uses thereof

The present invention relates to a human IL-15 mutant, a nucleic acid encoding it, a fusion protein containing the IL-15 mutant and a combined mutation, a pharmaceutical composition, and related uses of the pharmaceutical composition for treating tumors.

Interleukin-15 (interleukin-15, IL-15) is an important soluble cytokine discovered and named by Grabstein in 1994 when detecting the culture supernatant of monkey kidney intradermal cell line CV-1/EBNA. Various cells and tissues can express IL-15, for example, monocytes/macrophages, lymphocytes, epithelial cells, etc. IL-15 biological activity

IL-15-mediated signaling pathway

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

Binding of IL15 to receptors: IL15Rα is mainly expressed in DCs and monocytes. In most cases, IL15/IL15Rα binds to the receptor in a trans-presented form. That is, after IL-15 and IL-15Ra are expressed in the same cell, intracellular IL-15 binds to the sushi domain of IL-15Rα with high affinity and transports to the membrane surface, and then interacts with reactive cells (such as T cells or NK βγ heterodimeric complexes or αβγ heterotrimeric complexes on the membrane surface of cells bind, β and γ receptors can activate downstream Jak1 and Jak3, respectively, leading to the activation of STAT-3 and STAT-5, and the activation cascade response, inducing specific gene expression. When IL15 acts on effector cells in an autocrine form, it can interact with IL15 receptors in the form of cis to activate downstream signals to generate effector functions.

Immunomodulatory effect of IL-15

IL-15 has a wide range of immunoregulatory effects, and is involved in regulating the activity, proliferation and function of various immune cells. (1) Regulatory effect on T cells: promote the activation and proliferation of T cells, promote the generation of memory CD8+ T cells, and also play an important role in maintaining the number of memory CD8+ T cells in the body, even in Treg In the presence of cells, IL-15 can also maintain the function and quantity of CD8+ T cells well. (2) Regulatory effect on 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 in 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

IL-15 achieves anti-tumor effects based on its ability to amplify and activate a variety of 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, small molecule, and high renal clearance rate, multiple injections per day or subcutaneous administration are extremely inconvenient in use. Therefore, the simple application of wild-type recombinant IL-15 is also limited in tumor therapy.

Studies have shown that reducing the activity of IL-15 on the proliferation of T cells and NK cells can increase the half-life of IL-15 while reducing toxicity. In addition, the combination of IL-15 and antibodies targeting tumor-associated antigens into a fusion protein can increase the specificity of IL-15, increase the concentration of IL-15 in the tumor microenvironment, and reduce the toxicity of IL-15. Therefore, the development of IL-15 mutants with reduced activity has the potential to improve the dose-effect relationship of IL-15 in treating tumors, and to expand the clinical anti-tumor application of IL-15, which has great social and economic significance.

The present invention provides IL-15 mutants, nucleic acids encoding the mutants, fusion proteins and pharmaceutical compositions containing the mutants, as well as their functions for killing tumor cells and their use for treating tumors.

In a first aspect, the present disclosure provides an IL-15 mutant polypeptide comprising one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15 mutation.

In a second aspect, the present disclosure provides a polypeptide comprising an IL-15 mutant comprising one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15 mutation at the base.

In one embodiment, the IL-15 mutant polypeptide comprises mutations at least two, three, or four amino acid residues in Val3, Ile6, Asp8, or His105.

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

In a specific embodiment, said mutation is selected from amino acid substitutions in 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).

In a specific embodiment, the IL-15 mutant polypeptide or the polypeptide comprising the IL-15 mutant comprises the following mutation or combination of mutations: (1) Asp8Glu; (2) Asp8Gln; (3) Asp8Arg; (4) (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.

In a specific embodiment, said IL-15 mutant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In a specific embodiment, the amino acid sequence of the IL-15 mutant is such as 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 Shown in 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.

In a specific embodiment, the IL-15 mutant polypeptide or the polypeptide comprising the IL-15 mutant has the following characteristics: (1) mediating the proliferation of human CD8+ T cells; (2) mediating the proliferation of human NK cells proliferation; and/or, (3) inhibiting tumor growth.

In a specific embodiment, the activity of the IL-15 mutant polypeptide or a polypeptide comprising an IL-15 mutant in mediating proliferation/expansion of CD8+ T and/or NK cells is lower than that of a polypeptide comprising wild-type IL-15 .

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

In a third aspect, the present disclosure provides a protein comprising the aforementioned IL-15 mutant polypeptide or a polypeptide comprising an IL-15 mutant; and an immunoglobulin fused to the IL-15 mutant Protein molecules or parts thereof, and/or IL-15Rα.

In one embodiment, said immunoglobulin molecule is an antibody or an antigen-binding fragment; said portion of an immunoglobulin molecule is an Fc region of an immunoglobulin.

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

In a 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.

In a 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 comprising a human or murine antibody IgG1, IgG2, IgG3 Or the sequence of the constant region of IgG4; preferably, the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO.73.

In another embodiment, the IL-15 mutant is fused to an immunoglobulin molecule or part 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 use the connecting peptide shown in SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO.69 or SEQ ID NO.71.

In another embodiment, the IL-15 mutant is fused with IL-15Rα with or without a connecting peptide, and then fused with an immunoglobulin molecule or part thereof; preferably using a connecting peptide; preferably using SEQ ID NO.65, SEQ ID The connecting peptide shown in NO.69 or SEQ ID NO.71.

In a specific embodiment, the connection order of each structural domain is from N-terminus to C-terminus:

(1) Immunoglobulin molecules or parts thereof, IL-15Rα, IL-15 mutants;

(2) Immunoglobulin molecules or parts thereof, IL-15 mutants, IL-15Rα;

(3) IL-15 mutants, IL-15Rα, immunoglobulin molecules or parts thereof;

(4) IL-15Rα, IL-15 mutants, immunoglobulin molecules or parts thereof;

(5) IL-15 mutants, immunoglobulin molecules or parts thereof;

(6) Immunoglobulin molecules or parts thereof, IL-15 Rα;

(7) IL-15Rα, immunoglobulin molecules or parts thereof;

(8) IL-15 mutant, IL-15Rα; or,

(9) IL-15Rα, IL-15 mutants.

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

In a fourth aspect, the present disclosure provides a protein or antibody fusion construct/complex comprising the following four parts:

(1) Immunoglobulin heavy chain;

(2) Immunoglobulin light chain;

(3) IL-15Rα; and,

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

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

In one embodiment, 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.

In a specific embodiment, the protein is a homodimer comprising monomers composed of moieties (1)-(4).

In a fifth aspect, the disclosure of the present invention provides a protein or Fc fusion construct comprising the following three parts:

(1) Immunoglobulin Fc region;

(2) IL-15Rα; and,

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

In one embodiment, IL-15Rα is fused to the N-terminal or C-terminal of the IL-15 mutant polypeptide through or without a connecting peptide, and then fused to the N-terminal or C-terminal of the immunoglobulin Fc region through or not through a connecting peptide .

In a specific embodiment, the protein is a homodimer comprising monomers composed of moieties (1)-(3).

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

In a specific embodiment, an anti-PD-L1 antibody comprises a heavy chain variable region and a light chain variable region having SEQ ID NO: 99 and SEQ ID NO: The sequence shown in 100, or have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more compared with the sequence shown in SEQ ID NO: 99 and SEQ ID NO: 100 Highly concordant sequences; or,

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

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

In a sixth aspect, the present disclosure provides 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 are identical to the sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100 Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to each other.

In a 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 1.8×10-9M, which is similar to that of programmed death ligand-1 (PD-L1) in cynomolgus monkeys. The dissociation constant (KD) of death ligand-1 (PD-L1) binding is not greater than 9.4×10-10M;

Or, optionally, the antibody or antigen-binding fragment binds or does not bind to monkey PD-L1;

Optionally, the antibody or antigen-binding fragment may or may not bind to murine PD-L1.

In a specific embodiment, the anti-PD-L1 antibody competitively binds to PD-L1 or its epitope, and has the following characteristics:

(1) Specifically binding to PD-L1 recombinant protein and cells expressing PD-L1;

(2) Block the combination of PD-L1 and PD-1 protein;

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

(4) Enhance T cell activity; or/and

(5) Inhibit tumor growth.

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 human or murine antibody IgG1, IgG2, The sequence of the constant region of IgG3 or IgG4.

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.

In a seventh aspect, the present disclosure provides an isolated nucleic acid molecule encoding the polypeptide, protein, antigen or antigen-binding fragment of any one of the aforementioned first to sixth aspects.

In an eighth aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule isolated in the aforementioned seventh aspect.

In a ninth aspect, the present disclosure provides a host cell, the host cell comprising the isolated nucleic acid molecule in the aforementioned seventh aspect, or the expression vector described in the aforementioned eighth aspect; preferably, the host cell It 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 ).

In the tenth aspect, the present invention provides a method for preparing a polypeptide or protein, comprising culturing the host cell described in the ninth aspect under appropriate conditions, and isolating the polypeptide or protein.

In the eleventh aspect, the present disclosure provides a pharmaceutical composition comprising the polypeptide, protein, antigen or antigen-binding fragment described in any one of the aforementioned first to sixth aspects, the aforementioned seventh aspect The isolated nucleic acid molecule in the aforementioned eighth aspect, the expression vector described in the ninth aspect, or the product prepared by the method described in the tenth aspect; and a pharmaceutically acceptable carrier; preferably, the The pharmaceutical compositions also contain additional antineoplastic agents.

In the twelfth aspect, the present disclosure provides the polypeptide, protein, antigen or antigen-binding fragment described in any one of the aforementioned first to sixth aspects, the isolated nucleic acid molecule in the aforementioned seventh aspect, the aforementioned eighth aspect The expression vector described in the 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 is used to prevent and/or treat diseases in individuals Use in medicine; said disease is preferably a tumor.

In the thirteenth aspect, the present invention provides a method for preventing and/or treating a disease in an individual, comprising administering the polypeptide, protein, protein, The antigen or antigen-binding fragment, the isolated nucleic acid molecule in the seventh aspect, the expression vector in the eighth aspect, the cell in the ninth aspect, the product prepared by the method in the tenth aspect, or the tenth aspect In one aspect, the pharmaceutical composition; the disease is preferably a tumor.

Definitions and Explanations of Terms

Unless otherwise specified, the terms used herein have the meanings commonly understood by those of ordinary skill in the art. For a term explicitly defined herein, the meaning of that term shall prevail.

The term "IL-15" or "IL15" in the present invention, interleukin 15 (interleukin-15, IL-15) is a pleiotropic cytokine that activates T cells, B cells and NK cells, and can mediate proliferation and survival of these cells. In addition, IL-15 can activate, maintain and expand CD8+ memory T cells. The "IL-15" or "IL-15 peptide" or "IL-15 polypeptide" described in the present invention can be any IL-15 (interleukin 15) or its mutants, such as human IL-15 or non-human mammal IL-15 or non-mammalian IL-15. Exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, etc. Preferably human interleukin 15 mature molecule, see database UniProtKB, accession number P40933, 49-162aa.

In the present invention, the term "IL-15 wild type" or "wild type IL-15" refers to natural source human IL-15 or non-human mammal IL-15 or non-mammal IL-15; IL-15 polypeptides that have been commonly used in the field.

The term "IL-15 mutant" in the present invention refers to an increase or decrease in the affinity between IL-15 and its receptor obtained by one or more amino acid substitutions, additions or deletion mutations, or it stimulates a specific cell line , a mutant molecule that increases or decreases the proliferative activity of T cells or NK cells and the activity of cytokine release.

The term "IL-15Rα" in the present invention can be any species of IL-15Rα or its functional fragments, such as human IL-15Rα or non-human mammal IL-15Rα or non-mammal IL-15Rα. Exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, etc. Preferably human IL-15Rα; preferably human interleukin-15 receptor α extracellular domain fragment, referred to as IL-15Rα ECD; preferably IL-15Rα-sushi, see Table 1 for details.

The term "IL-15Rα variant" in the present invention refers to a functional mutant with the ability to bind to its ligand molecule such as IL15 formed by one or more amino acid deletion, insertion or substitution mutations on IL-15Rα, preferably human The IL15Rα molecule, more preferably the shortened form of the human IL-15Rα ectodomain fragment, that is, the molecule with human interleukin-15 receptor α activity obtained from the C-terminus of the ectodomain fragment through one or more amino acid deletion mutations, preferably A deletion mutant form retaining 65-120 amino acids, more preferably a shortened form of deletion mutation retaining 65-102 amino acids, such as IL-15Rα-sushi; preferably IL-15Rα-sushi, see Table 3 for details.

The term "immunoglobulin Fc region" in the present invention refers to the constant region of the immunoglobulin chain, especially the carboxy-terminal or part of the constant region of the heavy chain of the immunoglobulin, which has no antigen-binding activity and is an important component of antibody molecules and effector molecules and site of cell interaction. The "immunoglobulin Fc region" of the present invention may be any Fc or its variants, derived from human or non-human mammals. For example, an immunoglobulin Fc region may comprise two or more domains of heavy chains CH1, CH2, CH3, CH4 in combination with an immunoglobulin hinge region. Fc can be derived from different species, preferably human immunoglobulins. According to the amino acid sequence of the heavy chain constant region, immunoglobulins can be divided into different classes, and there are mainly 5 classes of immunoglobulins: 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. An "Fc region" preferably includes at least one immunoglobulin hinge region, and the CH2 and CH3 domains of IgG. More preferably, it includes a CH2 domain of IgG1, a CH3 domain and an immunoglobulin hinge region, and the starting amino acid position of the hinge region can be changed.

The term "Fc variant" in the present invention refers to changes in the structure or function of Fc caused by one or more amino acid substitutions, insertions or deletion mutations at appropriate positions on Fc. "Interaction between Fc variants" refers to the space-filling effect, electrostatic guidance, hydrogen bond interaction, hydrophobic interaction, etc. between Fc variants designed by mutation. Interactions between Fc variants contribute to the formation of stable heterodimeric proteins. A preferred mutation design is a "Knob-into-Hole" style mutation design.

The mutation design technology of Fc variants has been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion proteins. The representative one is the "Knob-into-Hole" form proposed by Cater et al. (Protein Engineering vol.9 no.7 pp .617-621, 1996); The heterodimer form (US 20100286374 A1); the heterodimer form (SEEDbodies formed by IgG/IgA chain exchange) proposed by Jonathan H.Davis et al. (Protein Engineering, Design&Selection pp.1-8, 2010) ); Genmab's DuoBody (Science, 2007.317 (5844)) platform technology to form a bispecific molecule; Xencor's technicians integrated structure calculations and Fc amino acid mutations, and integrated different modes of action to form heterodimeric protein forms ( mAbs 3: 6, 546-557; November/December 2011); Suzhou Corning Jerry's Fc modification method based on charge network (CN201110459100.7) to obtain heterodimeric protein form; and other changes based on Fc amino acids Or functional transformation means to achieve the genetic engineering method of forming heterodimer functional protein. The Knob/Hole structure on the Fc variant fragments of the present invention means that the two Fc fragments are mutated respectively, and can be combined in a "Knob-into-Hole" form after the mutations. It is preferred to use the "knob-into-hole" model of Cater et al. to carry out site mutation modification on the Fc region, so that the obtained first Fc variant and the second Fc variant can be in the form of "knob-into-hole" Combine together to form heterodimers. The selection of particular immunoglobulin Fc regions from particular immunoglobulin classes and subclasses is within the purview of those skilled in the art. The Fc region of human antibody IgG1, IgG2, IgG3, IgG4 is preferred, and the Fc region of human antibody IgG1 is more preferred. One of the first Fc variant or the second Fc variant is randomly selected for knob mutation and the other for hole mutation.

The term "antibody" (Antibody, Ab) in the present invention refers to an immunoglobulin molecule that specifically binds to a target antigen or has immunoreactivity, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including But not limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (such as bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies), antibody conjugates and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments). Furthermore, unless otherwise indicated, the term "monoclonal antibody" (mAb) is intended to include intact antibody molecules capable of specifically binding a target protein as well as incomplete antibody fragments (such as Fab and F(ab')2 fragments, which lack Fc fragment of intact antibody (clears more quickly from circulation in animals) and thus lacks Fc-mediated effector function (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated by reference This article).

The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody). Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.

The term "antibody conjugate" refers to a couple/conjugate formed by chemically bonding an antibody molecule to another molecule either directly or through a linker. Examples include antibody-drug conjugates (ADCs), where the drug molecule is said other molecule.

The term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation by the method by which the antibody was produced.

The term "fusion protein" in the present invention refers to the protein obtained by linking the coding regions of two or more genes by gene recombination methods, chemical methods or other appropriate methods, and expressing them under the control of the same regulatory sequence product. In the fusion protein of the present invention, the coding regions of two or more genes may be fused at one or several positions by sequences encoding peptide linkers or connecting peptides. Peptide linkers or connecting peptides can also be used to construct fusion proteins of the invention. The term "fusion protein" of the present invention further includes an antibody/Fc fusion protein construct/complex, or a composition of an antibody/Fc fusion protein construct/complex formed in a non-covalent manner, for example, the present invention The fusion protein can be displayed as the following structure:

(1) IL-15 fusion protein, which is a homodimer comprising two monomers; said monomer comprises antibody heavy chain, antibody light chain, IL-15 and IL-15Rα sushi; for example, antibody heavy chain Chain Fc fusion IL-15Rα sushi, and co-expression with 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 linkage;

(2) IL-15 fusion protein, which is a homodimer comprising two monomers; the monomer comprises antibody heavy chain, antibody light chain, IL-15 and IL-15Rα sushi; for example, linker Antibody heavy chain Fc was fused with IL-15Rα sushi and IL-15-WT or IL-15 mutant in series, and co-expressed with antibody light chain;

(3) IL-15 fusion protein, which is a homodimer comprising two monomers; said monomer comprises Fc, IL-15 and IL-15Rα sushi; for example, IL-15-WT or IL-15 The mutant is connected to IL15-Rαsushi through a linker, and IL15-Rαsushi is connected to Fc through a linker; or,

(4) IL-15 fusion protein, which is a homodimer comprising two monomers; the monomer comprises Fc, IL-15 and IL-15Rα sushi; for example, IL15-Rα sushi passes linker and IL-15 -WT or IL-15 mutants were connected, and IL-15 was connected to Fc through a linker.

The term "peptide linker/connecting peptide (Linker)" in the present invention refers to the peptide used to link IL-15 with another protein molecule or protein fragment in the present invention to ensure correct folding and stability of the protein. Said another molecule includes without limitation IL-15Rα, Fc, Fc variants, antibodies and the like. The "connecting peptide" of the present invention is preferably (GGGGS)n, wherein n can be 0, 1, 2, 3, 4, 5 or more, preferably n is 2-4; or preferably SGGSGGGGSGGGSGGGGSLQ. If the connecting peptide sequence is too short, it may affect the folding of the high-level structure of the two proteins, thereby interfering with each other; if the connecting peptide sequence is too long, it will involve the issue of immunogenicity, because the connecting peptide sequence itself is a new antigen.

The term "heterodimeric protein" in the present invention refers to a protein formed by combining two different monomeric proteins. In the present invention, 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.

The term "homodimeric protein" in the present invention refers to a protein formed by combining two identical monomeric proteins. In the present invention, 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.

The "monomeric protein" constituting the heterodimeric protein or homodimeric protein in the present invention may be a fusion protein or a non-fusion protein.

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

In the present 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 "part" and/or "anti-PD-L1 antibody fragment" etc. refer to any immunoglobulin molecule comprising at least a part (such as but not limited to at least one complementarity determining region of a heavy chain or a light chain) capable of specifically binding to PD-L1 (CDR) or its ligand-binding portion, heavy or light chain variable region, heavy or light chain constant region, framework region or any portion thereof). PD-L1 antibodies also include antibody-like protein scaffolds such as the tenth fibronectin type III domain (10Fn3), which contain BC, DE, and FG structural loops that are similar in structure and solvent accessibility to antibody CDRs. The tertiary structure of the 10Fn3 domain is similar to the tertiary structure of the IgG heavy chain variable region, and by using the residues of the BC, DE and FG loops of 10Fn3 with CDR-H1, CDR-H2 from the PD-L1 monoclonal antibody Or residue replacement in the CDR-H3 region, those skilled in the art can graft the CDR of the PD-L1 monoclonal antibody onto the fibronectin scaffold.

The term "coexpression" in the present invention means that multiple genes are expressed together in a cell, and their products appear simultaneously. These genes can be present simultaneously and controlled expression separately or jointly. In the present invention, it is preferable to co-express both genes in one eukaryotic cell. The gene expression product obtained by co-expression is conducive to efficient and simple complex formation; in the present invention, it is beneficial to form heterodimeric protein or homodimeric protein.

The term "percent (%) sequence identity" refers to the sequence identity after aligning sequences for maximum percent sequence identity and introducing gaps, if necessary (e.g., for optimal alignment, one of the candidate and reference sequences can be or both, and for comparison purposes, non-homologous sequences can be ignored), the amino acid (or nucleotide) residues of the candidate sequence are different from the amino acid (or nucleotide) residues of the reference sequence ) percentage of residues identical. For purposes of determining percent sequence identity, alignment can be achieved in a number of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits a 50% to 100% sequence identity. The length of the candidate sequence aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence . When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

The term "specific binding" in the context of the present invention refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, such as those specified by antibodies or antigen-binding fragments thereof sexual identification. An antibody or antigen-binding fragment thereof that specifically binds an antigen will bind the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds an antigen will bind the antigen with a KD as high as 100 nM (eg, between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a K for that particular antigen or epitope thereof of greater than 100 nM (eg, greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM). Antibodies that are specifically immunoreactive with a particular protein or carbohydrate can be selected using a variety of immunoassay formats. For example, solid-phase ELISA immunoassays are routinely used to select for antibodies that are specifically immunoreactive with proteins or carbohydrates. 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 Immunoreactivity immunoassay format and conditions.

The term "vector" in the present invention includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses or other suitable replicons (such as viral vectors). A variety of vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. Expression vectors of the invention contain polynucleotide sequences together with additional sequence elements, eg, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the invention include plasmids that contain regulatory sequences, such as promoter and enhancer regions, that direct transcription of the gene. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA resulting from transcription of the genes. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosomal entry sites (IRES), and polyadenylation signal sites in order to direct the efficient transcription of genes carried on expression vectors. The expression vector of the present invention may also contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourthricin.

The terms "subject", "subject" and "patient" in the present invention refer to an organism receiving treatment for a particular disease or condition, such as cancer or an infectious disease, as described herein. Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, members of the bovid family (cattle, bison, buffalo, elk, and yak, etc.), sheep, and horses.

The term "treatment" in the present invention refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesired physiological changes or lesions in the subject of treatment, such as cell proliferative disorders (such as cancer or infectious disease). Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, lessening of disease extent, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable. Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented. When referring to the terms slow down, lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.

"Immune disorder" or "immune disorder" in the present invention includes, for example, pathological inflammation, inflammatory disorder and autoimmune disease or disease. "Immune disorder" also refers to infections, persistent infections and proliferative disorders such as cancer, tumors and angiogenesis. A "cancerous condition" includes, for example, cancer, cancer cells, tumors, angiogenesis, and precancerous conditions, such as dysplasia.

The term "pharmaceutical composition" in the present invention refers to a mixture containing one or more compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiological/pharmaceutical Pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.

The term "size exclusion chromatography" (Size Exclusion Chromatograph, referred to as SEC) in the present invention is a liquid chromatography technique for separating the cvn components to be measured according to their molecular sizes. There are pores of different sizes distributed on the surface of the chromatographic column filler. After the sample enters the chromatographic column, different components in them enter the corresponding pore diameters according to their molecular sizes. Molecules larger than all the pore diameters cannot enter the interior of the filler particles. Molecules smaller than all pore sizes can freely enter all pore sizes on the surface of the filler, and have a longer retention time in the chromatographic column, which is expressed as a longer retention time; the rest of the molecules are eluted in sequence according to their molecular size.

"Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions may but need not be present; when present, there may be 1, 2 or 3.

The step of transforming host cells with recombinant DNA described in the present invention can be carried out by conventional techniques well known to those skilled in the art. The obtained transformants can be cultured by conventional methods, and express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The host cells are cultured under conditions suitable for the growth of the host cells. detailed description

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The embodiments of the present invention are merely exemplary, and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 , antibody humanization

First, the classic "CDRs transplantation" method is used for antibody humanization, that is, the human antibody with the highest homology is selected through sequence to provide antibody framework regions (FRs), and the antigen-binding fragment complementarity-determining region of the target antibody based on the Kabat naming method (CDRs), transplanted to the former to form humanized antibodies. Secondly, in order to better maintain antibody activity and affinity, MOE software is used to analyze antibody structure modeling: 1). Select the amino acid residues in the antibody framework region that are located at the VH-VL interface, close to or have direct interaction with CDRs for recovery Mutations, such amino acid residues are more important for maintaining the conformation of the CDRs region; 2). Considering immunogenicity, try to select the amino acids embedded in the protein for back mutation; 3). Considering the stability of the antibody and expression levels, molecular energy-reducing mutations were prioritized. By testing the affinity of humanized antibodies containing different mutations to human PD-L1 and binding to cells expressing PD-L1 on the surface, screen for human antibodies with comparable or better affinity, antibody characterization, and activity to murine PD-L1 antibodies derivatized antibodies.

Among them, 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 murine PD-L1 antibody PDL1-794 is shown in Table 1 below.

surface 1. Mouse and humanized antibodies PD-L1 Specific sequence information of antibody heavy chain variable region and light chain variable region 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 sequence ( VL ) PDL1-794 SEQ ID NO.98 EIVMTQSPSSLAVSVGEKVTLSCKSSQSLLYSSNQKNSLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFLTISSVKAEDLAVYYCQQYYGYPYTFGGGTKLEIK 794-h1-71 SEQ ID NO.100 EIVMTQSPPTLSLSPGERVTLSCKSSQSLLYSSNQKNSLAWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQYYGYPYTFGQGTKLEIK

Example 2 , Humanized antibody expression and purification

2.1 Expression of humanized antibody

One day before transfection, inoculate ExpiCHO-S cells (Thermo fisher, A29127) in fresh ExpiCHO Expression medium (Invitrogen, A29100-01) at a density of 2.5×106~4×106 cells/mL, and place on a shaker Incubate overnight. On the day of transfection, the overnight cultured ExpiCHO-S cell suspension was counted. The cell viability was >95%, and the density was between 7×106 - 10×106 viable cells/mL. Take the required cell suspension, dilute it to a density of 6×106cells/mL with ExpiCHO Expression Medium (Invitrogen, A29100-01), and place it on a shaker for later use. Add the prepared humanized antibody expression plasmids to the culture medium to dilute, shake the centrifuge tube gently to mix, add to OptiPRO™ SFM-DNA Diluent (Invitrogen, 12309-019), shake gently Rotate the centrifuge tube to make it evenly mixed and let it stand at room temperature for 1-5 mins. The above-mentioned plasmid complex is slowly dropped into the cell suspension to be transfected, and the shake flask is shaken during the dropping process. After transfection, the cells were cultured overnight on a shaker. Add 0.6% of ExpiFectamine™ CHO Enhancer (Invitrogen, A29129) and 16% of ExpiCHO™ Feed (Invitrogen, A29129) equivalent to the cell volume to the cells on the first day after transfection. The shaker flask was shaken gently, and the cells were transferred to a shaker for 4 days. On the 5th day after transfection, add ExpiCHO™ Feed (Invitrogen, A29129) equivalent to 16% of the cell volume to the transfected cells, and shake the flask gently during the addition. On the 12th day after transfection, 9000 g of the culture medium was taken, centrifuged for 10 mins, and the supernatant was harvested.

2.2 Purification of humanized antibody

The cell culture supernatant collected in Example 2.1 was centrifuged at high speed, passed through a 0.45 μm + 0.22 μm filter membrane, and purified in the first step by affinity chromatography. The chromatography medium is protein A filler Mbaseselect Sure (GE, 17543803) that interacts with Fc, and the equilibration buffer is PBS (2.5g/L Na2HPO4·12H2O, 0.408g/L NaH2PO4, 8.76g/L NaCl, pH7.2) , after equilibrating 4 times the column volume, the cell supernatant was loaded and combined, and the flow rate was controlled so that the retention time of the sample on the column was ≥ 5min. After loading the sample, wash the column with PBS (pH7.2) until the A280 UV absorbance drops to the baseline. Then wash with 20mM PB+1 M NaCl (pH6.0) for 2 times the column volume. Then wash the column with PBS (pH7.2) until the A280 UV absorption and conductivity reach the baseline. Finally, wash the chromatography column with 20mM citric acid (pH3.4) elution buffer, collect the elution peak according to the A280 UV absorption peak, and neutralize the collected elution sample with 1 M Tris-HCl (pH9.0) sex.

Example 3 , antibodies and human and cynomolgus monkeys PD-L1 recombinant protein-bound KD determination

The binding affinities of PD-L1 antibodies to human and cynomolgus monkey PD-L1-His proteins were determined using a Biacore T200 (GE Healthcare). Anti-human IgG Fc (Genway, Cat. GWB-20A705) was immobilized on a CM5 chip (GE Healthcare, Cat. BR-1005-30) at 25°C. Anti-human IgG Fc was diluted to 20 μg/mL with Acetate pH5.0 (GE Healthcare, BR-1003-51). Immobilization was performed using the Amine method in the Immobilization method. Or use a commercial Protein A (GE Healthcare, Cat. 29127556) chip for detection. At 25°C, a multi-cycle kinetic method was used to determine the affinity of the antibody to the antigen. In each cycle, the antibody to be tested was first captured onto the immobilized CM5 chip, and then injected into recombinant human PD-L1-His (Novoprotein, Cat. 315 ) and cynomolgus monkey PD-L1-His protein (Sino Biological, Cat. 90251-C08H), and finally regenerated with Glycine pH1.5 (Shanghai Test, 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 for 30 seconds. Biacore T200 Evaluation Software (version 3.0) was used to analyze the experimental data with a 1:1 binding model, and the equilibrium dissociation constant KD of the antibody antigen was fitted to determine the association rate constant ka and dissociation rate constant kd.

From the results, it can be seen that the tested PD-L1 antibodies showed an affinity of nM or higher for the binding of human PD-L1 recombinant protein and cynomolgus monkey PD-L1 recombinant protein, as shown in Table 2 below.

surface 2. Humanization PD-L1 Antibody Biacore binding affinity KD The measurement results Antibody number people PD-L1 ( m ) cynomolgus monkey PD-L1 ( m ) 794-h1-71 1.793E-09 9.372E-10

Example 4 , antibody blocking PD-L1 with PD-1 interaction IC50 determination

The IC50 of blocking the binding of PD-L1 protein to PD-1 protein by anti-PD-L1 antibody was determined by 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 with a final concentration of 1 μg/ml. Block with PBS solution containing 3% BSA, add serially diluted anti-PD-L1 antibody (40nM~0.02nM) and human PD-1-His recombinant protein (Sino Biological, Cat.10377-H08H) for co-incubation, add HRP-labeled anti-His tag antibody (MBL, Cat.D291-7), TMB (Thermo, Cat.34029) for color development, read OD value (dual wavelength 450nm-630nm) after termination of 1M sulfuric acid. Comparing the antibody concentration with the OD value, the competition binding curve of the test antibody can be drawn, and the IC50 value can be calculated. Figure 1 shows the competition binding curve of anti-PD-L1 antibody to human PD-L1 recombinant protein. The results showed that the tested 794-h1-71 antibody could effectively block the interaction between human PD-L1 protein and human PD-1 protein, with IC50 of 0.8488 nM, and the positive control Tecentriq (Genetech, lot: H0172) was 0.8486 nM .

Example 5 , FACS determination PD-L1 Antibody to cell surface PD-L1 combined EC50

Mix the antibody to be detected at gradient concentrations (antibody concentration: 10000ng/ml-0.1ng/ml) with CHO-PD-L1 cells that highly express PD-L1 on the cell surface (Nanjing Yongshan Biotechnology Co., Ltd., 105 cells/well), Incubate together at 4°C for 30 min. After the incubation, add anti-human IgG PE fluorescent antibody (eBioscience, Cat. 12-4998-8) diluted 1:250, and incubate together at 4°C for 30 minutes, the fluorescent antibody will specifically bind to the Fc segment of the antibody to be detected, The ability of the antibody to be detected to bind to the highly expressed PD-L1 protein on the cell surface was analyzed by detecting the fluorescence intensity of PE by FACS. The results in Figure 2 show that the EC50 of the 794-h1-71 antibody is 38.44ng/ml, which is similar to the positive control Avelumab in this experiment (EC50 is ~72ng/ml). This assay quantitatively demonstrates the ability of the 794-h1-71 antibody to bind in a dose-dependent manner to PD-L1 targets on the cell surface. Average fluorescence intensity change fold (MFI fold) = MFI value of the experimental group/MFI value of the control group without drug addition.

Example 6 , PD-1/PD-L1-NFAT reporter gene test against PD-L1 antibody suppression PD-1:PD-L1 binding and signaling

Use the Jurkat cell line stably transfected with PD-1 (GenScript, Cat.00612) and the CHO cell line stably transfected with PD-L1 (GenScript, Cat. M00613) to compare the effect of PD-L1 antibody on PD-1/PD-L1 protein Antagonism of interactions and their signaling pathways. When the inhibitory signaling pathway is inhibited, the expression of the luminescent reporter gene controlled by NFAT is enhanced, and the value of the luminescent signal increases. The strength of the antibody's blocking effect on PD-L1 is reflected by the intensity of the luminescence reading (relative light units, RLU).

Plant the CHO cell strain stably transfected with PD-L1 on a 96-well white bottom plate, with 40,000 cells per well, 100 μl/well, and put it back into the incubator overnight; -1 cell line and the PD-L1 antibody to be tested were co-incubated. The sample volume of PD-1 cells was 16,000 cells/well, and the antibody was serially diluted. Each dose was repeated in 3 wells, and the incubation volume was 100 μl/well. The duration is 6 hours. When the incubation is completed, take out the well plate, add an equal volume (100 μl) of the luminescence detection reagent, and read the value. According to the detected values, the 4-parameter analysis was performed with Graphpad to make a regression curve, and the EC50 value of each antibody was obtained. Figure 3 shows that the EC50 (166.2ng/ml) of the 794-h1-71 antibody is similar to the EC50 (184.3ng/ml) of the positive control Avelumab. This assay quantitatively confirms that the 794-h1-71 antibody has a dose-dependent inhibitory ability to inhibit T cell activity caused by the interaction of PD-1:PD-L1 on the cell surface, thereby dose-dependently enhancing the expression of the reporter gene in Jurkat cells active.

Example 7 , ELISA detection of mixed lymphocyte reaction T secreted by cells IFN-γ

The activity of PD-L1 monoclonal antibody to enhance T cells was measured by mixed lymphocyte reaction (MLR). CD4+ monocytes were isolated from peripheral blood mononuclear cells (PBMC) of healthy human donor 1, and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech, Cat.300- 03) and recombinant human interleukin 4 (rhIL-4, Peprotech, Cat.200-04) were induced to differentiate into dendritic cells (dendritic cells, DC) in vitro, and LPS (Sigma, Cat: L4516) was added on the 6th day of culture Stimulate mature DC. On the 7th day, DC cells from donor 1 and CD4+ T cells enriched from PBMC from healthy donor 2 were mixed and co-cultured. The ratio of DC: CD4+ T cells was 1:10, and the antibody to be tested was added. , negative control antibody anti-Hel (Baiying Biosynthesis) and positive control antibody Avelumab (antibody concentration: 7nM-0.28nM), co-cultured for 4 days. After 4 days, the cell culture supernatant was collected, and the content of IFN-γ in the supernatant was detected by ELISA method. As shown in Figure 4, 794-h1-71 and the positive control antibody Avelumab can significantly enhance the ability of CD4+ T cells to secrete IFN-γ in the MLR experiment compared with the anti-Hel monoclonal antibody negative control group, and along with PD-L1 Antibody drug concentration decreased, increasing the activity of secreting IFN-γ also decreased. The results indicated that the 794-h1-71 antibody could enhance the function of T cells in a dose-dependent manner (T-test, *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001.).

Example 8 , IL-15 Construction of fusion protein

Use the MOE software to simulate the key amino acid sites of the interaction between human IL-15 and the corresponding receptor βγchain. They are D8 and V3, I6, H105 respectively. According to MOE software simulation, the following IL-15 mutant sequences were designed and synthesized, the amino acid sequence is shown in Table 3, and the coding nucleic acid sequence is shown in Table 4.

There are four modes for the construction of IL-15 fusion protein (antibody/Fc fusion construct/complex):

(1) The structure shown in Figure 5A, IL-15 fusion protein, which is a homodimer comprising two monomers; the monomers comprise antibody heavy chain, antibody light chain, IL-15 and IL-15Rα sushi; Fc end of antibody heavy chain is fused to IL-15Rα sushi, and co-expressed with monoclonal antibody light chain, IL-15-WT (wild type) or IL-15 mutant, so that IL-15 and IL-15Rα sushi form non-covalent linkage;

(2) The structure shown in Figure 5B, IL-15 fusion protein, which is a homodimer comprising two monomers; the monomers comprise antibody heavy chain, antibody light chain, IL-15 and IL-15Rα Sushi: The Fc end of the heavy chain of the antibody is fused in series with IL-15Rα sushi and IL-15-WT or IL-15 mutant through a linker, and co-expressed with the light chain of the antibody;

(3) As shown in Figure 5C (defined as V5), the IL-15 fusion protein is a homodimer containing two monomers; the monomer contains Fc, IL-15 and IL-15Rα sushi ; IL-15-WT or IL-15 mutants are connected to IL15-Rαsushi through a linker, and IL15-Rαsushi is connected to Fc through a linker;

(4) As shown in Figure 5D (defined as V9), the IL-15 fusion protein is a homodimer containing two monomers; the monomer contains Fc, IL-15 and IL-15Rα sushi ; IL15-Rαsushi is connected to IL-15-WT or IL-15 mutant through a linker, and IL-15 is connected to Fc through a linker.

The numbering rules for PD-L1 antibody and IL-15 fusion protein are as follows: "Antibody name-IL-15 (wild type/mutant)-fusion protein construction mode";

For example: "T-IL15-xx-1": "T" means Tecentriq; "IL15-xx" means IL15-WT or IL15 mutant; "1" means the structural pattern shown in Figure 5A;

For example: "794-IL15-xx-2": "794" means 794-h1-71 monoclonal antibody; "IL15-xx" means IL15-WT or IL15 mutant; "2" means as shown in Figure 5B Structural patterns.

Please refer to Table 5 and Table 6 for various IL-15 fusion protein designs. According to the above four construction modes, the nucleic acid sequence encoding the fusion protein was constructed into the pTT5 plasmid respectively.

Table 3. Amino acid sequence information related to IL-15 fusion protein serial number sequence serial number IL15-WT NW V NV I S D LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV H IVQMFINTS SEQ ID NO.1 IL15-7 (D8E) NWVNVIS E LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.3 IL15-8 (D8Q) NWVNVIS Q LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.5 IL15-9 (D8R) NWVNVIS R LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.7 IL15-10 (D8S) NWVNVIS S LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.9 IL15-11 (D8V) NWVNVIS V LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.11 IL15-26 (V3L) NW L NVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.13 IL15-29 (I6D) NWVNV D SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.15 IL15-42 (H105K) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV K IVQMFINTS SEQ ID NO.17 IL15-43 (H105N) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV N IVQMFINTS SEQ ID NO.19 IL15-61 (D8G) NW V NV I S G LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV H IVQMFINTS SEQ ID NO.21 IL15-62 (D8I) NWVNVISILKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.23 IL15-63 (D8L) NW V NV I S L LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV H IVQMFINTS SEQ ID NO.25 IL15-64 (I6P) NW V NV P S D LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV H IVQMFINTS SEQ ID NO.27 IL15-65 (D8T) NW V NV I S T LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV H IVQMFINTS SEQ ID NO.29 P22339 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.31 ALT803 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIREFEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.33 IL15-com1 (D8E/V3L) NW L NVIS E LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.35 IL15-com2 (D8E/I6D) NWVNV D S E LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.37 IL15-com3 (V3L/I6D) NW L NV D SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.39 IL15-com4 (V3L/I6D/H105K) NW L NV D SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV K IVQMFINTS SEQ ID NO.41 IL15-com5 (I6D/H105K) NWVNV D SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVK IVQMFINTS SEQ ID NO.43 IL15-com6 (D8S/H105K) NWVNVIS S LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVK IVQMFINTS SEQ ID NO.45 IL15-com7 (D8S/H105N) NWVNVIS S LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVN IVQMFINTS SEQ ID NO.47 IL15Rαsushi-1 CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLETCVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SEQ ID NO.49 IL15Rαsushi-2 CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR SEQ ID NO.51 IL15Rαsushi-3 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLETCVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SEQ ID NO.53 IL15Rαsushi-4 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR SEQ ID NO.55 Tecentriq heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.57 Tecentriq Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLGSKADYEVECKQGLSS SEQ ID NO.59 794-h1-71 heavy chain QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.61 794-h1-71 light chain EIVMTQSPPTLSLSPGERVTLSCKSSQSLLYSSNQKNSLAWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSGTDFLTISSLQPEDFAVYYCQQYYGYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSKTLTLKGLNSKEVGECSS 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-IL15Rαsushi-Linker1-IL15-WT) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.77 794-IL15-7-2 (794-h1-71 heavy chain-IL15Rαsushi-Linker1-IL15-7) (D8E) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVIS E LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.79 794-IL15-65-2 (794-h1-71 heavy chain-IL15Rαsushi-Linker1-IL15-65) (D8T) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVIS T LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.81 794-IL15-64-2 (794-h1-71 heavy chain-IL15Rαsushi-Linker1-IL15-64) (I6P) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNV P SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.83 794-IL15-com1-2 (794-h1-71 heavy chain-IL15Rα sushi- Linker1- IL15-com1) (D8E/V3L) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNW L NVIS E LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.85 794-IL15-com3-2 (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-com3) (I6D/V3L) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNW L NV D SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO.87 794-IL15-com4-2 (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-com4) (I6D/V3L/H105K) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNW L NV D SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV K IVQMFINTS SEQ ID NO.89 794-IL15-com5-2 (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-com5) (I6D/H105K) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNV D SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV K IVQMFINTS SEQ ID NO.91 794-IL15-com6-2 (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-com6) (D8S/H105K) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVIS S LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV K IVQMFINTS SEQ ID NO.93 794-IL15-com7-2 (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-com7) (D8S/H105N) QVQLQQSGPGLVKPSQTLSLTCAVSGDSITSGYWNWIRKFPSRGLEYMGYISYSGSTYYNPFLKSRISINRDTSKNQYYLQLNSVTPEDTAVYYCAKMGDWLAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVIS S LKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV N IVQMFINTS SEQ ID NO.95 Note: The numbering rules for IL-15 mutant fusion proteins are as follows:

For example: "T-IL15-xx-1": "T" means Tecentriq; "IL15-xx" means IL15-WT or IL15 mutant; "1" means the structural pattern shown in Figure 5A;

For example: "794-IL15-xx-2": "794" means 794-h1-71 monoclonal antibody; "IL15-xx" means IL15-WT or IL15 mutant; "2" means as shown in Figure 5B Structural patterns.

surface 4. IL-15 Fusion protein coding nucleic acid sequence information serial number Nucleotide sequence serial number IL15-WT AACTGG GTG AATGTG ATC TCT GAC CTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTG CAT ATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.2 IL15-7 (D8E) AACTGGGTGAATGTGATCTCT GAG CTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.4 IL15-8 (D8Q) AACTGGGTGAATGTGATCTCT CAG CTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.6 IL15-9 (D8R) AACTGGGTGAATGTGATCTCT AGG CTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.8 IL15-10 (D8S) AACTGGGTGAATGTGATCTCT AGC CTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.10 IL15-11 (D8V) AACTGGGTGAATGTGATCTCT GTG CTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.12 IL15-26 (V3L) AACTGG CTG AATGTGATCTCT GAC CTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.14 IL15-29 (I6D) AACTGG GTG AATGTG GAC TCT GAC CTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTGCATATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.16 IL15-42 (H105K) AACTGG GTG AATGTG ATC TCT GAC CTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTG AAG ATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.18 IL15-43 (H105N) AACTGG GTG AATGTG ATC TCT GAC CTGAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGACGCCACCCTGTACACAGAGAGCGATGTGCATCCCTCTTGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCTAACAATTCCCTGTCCAGCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTTGTG AAC ATCGTGCAGATGTTTATCAATACATCT 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 TGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTATCATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTACTGGACCACACCTTCCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCT SEQ ID NO.50 IL15Rαsushi-2 TGTCCTCCTCCTATGTCTGTGGAGCACGCCGATATCTGGGTGAAGTCTTACTCTCTGTACTCTAGAGAGAGATACATCTGTAACTCTGGCTTCAAGAGAAAGGCCGGCACCTCTTCTCTGACCGAGTGTGTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCTTCTCTGAAGTGTATCAGA SEQ ID NO.52 IL15Rαsushi-3 ATCACATGTCCTCCTCCTATGTCTGTGGAGCACGCTGATATTGGGTGAAGTCTTACTCTCTGTACTCTAGAGAAAGATATTTTTGTAATTCTGGCTTTAAGAGAAAGGCTGGAACATCTTCCTGACAGAGTGTGTGCTGAATAAGGCTACAAACGTGGCTCATTGGACAACACCTTCCTGAAGTGTATTAGAGATCCTGCCCTGGTGCACCAGACCTGCTCC SEQ ID NO.54 IL15Rαsushi-4 ATCACATGTCCTCCTCCTATGTCTGTGGAGCACGCTGATATCTGGGTGAAGTCTTACTCTCTGTACTCTAGAGAGAGATACATCTGTAATTCTGGCTTTAAGAGAAAGGCTGGAACATCTTCCTCTGACAGAGTGTGTGCTGAATAAGGCTACAAATGTGGCTCACTGGACAACACCTTCTCTGAAGTGTATCAGA SEQ ID NO.56 Tecentriq Heavy Chain GAGGTGCAGCTGGTGGAGTCCGGAGGAGGACTGGTGCAGCCAGGAGGATCCCTGAGGCTGTCTTGCGCAGCAAGCGGCTTCACCTTTTCTGACAGCTGGATCCACTGGGTGCGCCAGGCACCAGGCAAGGGACTGGAGTGGGTGGCATGGATCAGCCCTTACGGCGGCTCCACCTACTATGCCGACTCTGTGAAGGGCCGGTTCACAATCTCCGCCGATACCTCTAAGAACACAGCCTATCTGCAGATGAATAGCCTGAGGGCCGAGGACACAGCCGTGTACTATTGTGCACGGAGACACTGGCCAGGAGGATTTGATTACTGGGGCCAGGGCACCCTGGTGACAGTGAGCTCCGCTTCCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGAGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACGCCTCCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCG AAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG SEQ ID NO.58 Tecentriq Light Chain GACATCCAGATGACCCAGTCCCCTAGCTCCCTGTCCGCCTCTGTGGGCGACAGGGTGACCATCACATGCAGAGCCTCTCAGGATGTGAGCACAGCAGTGGCATGGTACCAGCAGAAGCCAGGCAAGGCCCCTAAGCTGCTGATCTACAGCGCCTCCTTCCTGTATTCCGGCGTGCCCTCTCGGTTTTCTGGAAGCGGATCCGGAACCGACTTCACCCTGACAATCTCTAGCCTGCAGCCAGAGGATTTTGCCACATACTATTGTCAGCAGTACCTGTATCACCCCGCCACCTTCGGCCAGGGCACAAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGC SEQ ID NO.60 794-h1-71 heavy chain CAGGTGCAGCTGCAGCAGTCTGGACCAGGACTGGTGAAGCCTAGCCAGACCCTGTCTCTGACATGCGCCGTGTCTGGCGACTCCATCACCAGCGGCTATTGGAACTGGATCAGGAAGTTCCCATCCCGGGGCCTGGAGTACATGGGCTATATCTCTTACTCCGGCAGCACCTACTATAACCCCTTTCTGAAGTCTAGAATCTCCATCAACCGCGATACATCCAAGAATCAGTACTATCTGCAGCTGAATAGCGTGACCCCCGAGGACACAGCCGTGTACTATTGTGCTAAGATGGGCGATTGGCTGGCCTGGTTCGCTTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCAGCGCTTCCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGAGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCG AAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 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 the (Tecentriq heavy chain - IL15Rα sushi) GAGGTGCAGCTGGTGGAGTCCGGAGGAGGACTGGTGCAGCCAGGAGGATCCCTGAGGCTGTCTTGCGCAGCAAGCGGCTTCACCTTTTCTGACAGCTGGATCCACTGGGTGCGCCAGGCACCAGGCAAGGGACTGGAGTGGGTGGCATGGATCAGCCCTTACGGCGGCTCCACCTACTATGCCGACTCTGTGAAGGGCCGGTTCACAATCTCCGCCGATACCTCTAAGAACACAGCCTATCTGCAGATGAATAGCCTGAGGGCCGAGGACACAGCCGTGTACTATTGTGCACGGAGACACTGGCCAGGAGGATTTGATTACTGGGGCCAGGGCACCCTGGTGACAGTGAGCTCCGCTTCCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGAGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCG AAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAGCTGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCT SEQ ID NO.76 794-IL15-WT-2 the (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-WT) the CAAGTGCAGCTGCAGCAGTCTGGCCCTGGCCTGGTGAAGCCTTCTCAGACCCTGTCTCTGACCTGTGCCGTGAGCGGCGATTCTATCACCTCTGGCTACTGGAACTGGATCAGAAAGTTTCCTTCTAGAGGCCTGGAGTACATGGGCTACATCTCTTACTCTGGCTCTACCTACTACAACCCTTTCCTGAAGTCTAGAATCTCTATCAACAGAGATACCTCTAAGAACCAGTACTACCTGCAGCTGAACTCTGTGACCCCTGAGGATACCGCCGTGTACTACTGTGCCAAGATGGGCGATTGGCTGGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAGCTGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCTAGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCTGCAGAACTGGGTGAACGTGATCTCTGATCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCAGCATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGTCTTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.78 794-IL15-7-2 the (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-7) the (D8E) CAAGTGCAGCTGCAGCAGTCTGGCCCTGGCCTGGTGAAGCCTTCTCAGACCCTGTCTCTGACCTGTGCCGTGAGCGGCGATTCTATCACCTCTGGCTACTGGAACTGGATCAGAAAGTTTCCTTCTAGAGGCCTGGAGTACATGGGCTACATCTCTTACTCTGGCTCTACCTACTACAACCCTTTCCTGAAGTCTAGAATCTCTATCAACAGAGATACCTCTAAGAACCAGTACTACCTGCAGCTGAACTCTGTGACCCCTGAGGATACCGCCGTGTACTACTGTGCCAAGATGGGCGATTGGCTGGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAGCTGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCTAGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCTGCAGAACTGGGTGAACGTGATCTCTGAGCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTAGCTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGTCCTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCTTTCGTGCACATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.80 794-IL15-65-2 (794-h1-71 heavy chain-IL15Ra sushi-linker1-IL15-65) D8T CAAGTGCAGCTGCAGCAGTCTGGCCCTGGCCTGGTGAAGCCTTCTCAGACCCTGTCTCTGACCTGTGCCGTGAGCGGCGATTCTATCACCTCTGGCTACTGGAACTGGATCAGAAAGTTTCCTTCTAGAGGCCTGGAGTACATGGGCTACATCTCTTACTCTGGCTCTACCTACTACAACCCTTTCCTGAAGTCTAGAATCTCTATCAACAGAGATACCTCTAAGAACCAGTACTACCTGCAGCTGAACTCTGTGACCCCTGAGGATACCGCCGTGTACTACTGTGCCAAGATGGGCGATTGGCTGGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAGCTGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCTAGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCTGCAGAACTGGGTGAACGTGATCTCTACCCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCAGCATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGTCTTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.82 794-IL15-64-2 (794-h1-71 heavy chain-IL15Ra sushi-linker1-IL15-64) I6P CAAGTGCAGCTGCAGCAGTCTGGCCCTGGCCTGGTGAAGCCTTCTCAGACCCTGTCTCTGACCTGTGCCGTGAGCGGCGATTCTATCACCTCTGGCTACTGGAACTGGATCAGAAAGTTTCCTTCTAGAGGCCTGGAGTACATGGGCTACATCTCTTACTCTGGCTCTACCTACTACAACCCTTTCCTGAAGTCTAGAATCTCTATCAACAGAGATACCTCTAAGAACCAGTACTACCTGCAGCTGAACTCTGTGACCCCTGAGGATACCGCCGTGTACTACTGTGCCAAGATGGGCGATTGGCTGGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAGCTGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCTAGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCTGCAGAACTGGGTGAACGTGCCTTCTGATCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCAGCATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGTCTTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.84 794-IL15-com1-2 the (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-com1) the (D8E/V3L) CAAGTGCAGCTGCAGCAGTCTGGCCCTGGCCTGGTGAAGCCTTCTCAGACCCTGTCTCTGACCTGTGCCGTGAGCGGCGATTCTATCACCTCTGGCTACTGGAACTGGATCAGAAAGTTTCCTTCTAGAGGCCTGGAGTACATGGGCTACATCTCTTACTCTGGCTCTACCTACTACAACCCTTTCCTGAAGTCTAGAATCTCTATCAACAGAGATACCTCTAAGAACCAGTACTACCTGCAGCTGAACTCTGTGACCCCTGAGGATACCGCCGTGTACTACTGTGCCAAGATGGGCGATTGGCTGGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAGCTGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCTAGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCTGCAGAACTGGCTGAACGTCATCAGTGAGCTGAAGAAGATCGAGGACCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTCACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGAGCTCTAACGGCAACGTAACCGAGTCTGGGTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAATATCAAGGAGTTTCTGCAGTCTTTTGTGCACATCGTGCAGATGTTTATCAATACATCT SEQ ID NO.86 794-IL15-com3-2 the (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-com3) the (I6D/V3L) CAAGTGCAGCTGCAGCAGTCTGGCCCTGGCCTGGTGAAGCCTTCTCAGACCCTGTCTCTGACCTGTGCCGTGAGCGGCGATTCTATCACCTCTGGCTACTGGAACTGGATCAGAAAGTTTCCTTCTAGAGGCCTGGAGTACATGGGCTACATCTCTTACTCTGGCTCTACCTACTACAACCCTTTCCTGAAGTCTAGAATCTCTATCAACAGAGATACCTCTAAGAACCAGTACTACCTGCAGCTGAACTCTGTGACCCCTGAGGATACCGCCGTGTACTACTGTGCCAAGATGGGCGATTGGCTGGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAGCTGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCTAGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCTGCAGAACTGGCTGAACGTGGATTCTGATCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACACTGTACACAGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTTCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGAGCTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAGTCTTTTGTGCACATCGTGCAGATGTTTATCAACACATCT SEQ ID NO.88 794-IL15-com4-2 the (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-com4) the (I6D/V3L/H105K) CAAGTGCAGCTGCAGCAGTCTGGCCCTGGCCTGGTGAAGCCTTCTCAGACCCTGTCTCTGACCTGTGCCGTGAGCGGCGATTCTATCACCTCTGGCTACTGGAACTGGATCAGAAAGTTTCCTTCTAGAGGCCTGGAGTACATGGGCTACATCTCTTACTCTGGCTCTACCTACTACAACCCTTTCCTGAAGTCTAGAATCTCTATCAACAGAGATACCTCTAAGAACCAGTACTACCTGCAGCTGAACTCTGTGACCCCTGAGGATACCGCCGTGTACTACTGTGCCAAGATGGGCGATTGGCTGGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAGCTGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCTAGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCTGCAGAACTGGCTGAACGTGGATTCTGATCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGTCTAGCAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCTTTCGTGAAGATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.90 794-IL15-com5-2 the (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-com5) the (I6D/H105K) CAAGTGCAGCTGCAGCAGTCTGGCCCTGGCCTGGTGAAGCCTTCTCAGACCCTGTCTCTGACCTGTGCCGTGAGCGGCGATTCTATCACCTCTGGCTACTGGAACTGGATCAGAAAGTTTCCTTCTAGAGGCCTGGAGTACATGGGCTACATCTCTTACTCTGGCTCTACCTACTACAACCCTTTCCTGAAGTCTAGAATCTCTATCAACAGAGATACCTCTAAGAACCAGTACTACCTGCAGCTGAACTCTGTGACCCCTGAGGATACCGCCGTGTACTACTGTGCCAAGATGGGCGATTGGCTGGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAGCTGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCTAGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCTGCAGAACTGGGTGAACGTGGATTCTGATCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTTCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACAGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGAGCTCTAACGGCAACGTGACAGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAGTCTTTTGTGAAGATCGTGCAGATGTTTATCAACACCTCT SEQ ID NO.92 794-IL15-com6-2 the (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-com6) the (D8S/H105K) CAAGTGCAGCTGCAGCAGTCTGGCCCTGGCCTGGTGAAGCCTTCTCAGACCCTGTCTCTGACCTGTGCCGTGAGCGGCGATTCTATCACCTCTGGCTACTGGAACTGGATCAGAAAGTTTCCTTCTAGAGGCCTGGAGTACATGGGCTACATCTCTTACTCTGGCTCTACCTACTACAACCCTTTCCTGAAGTCTAGAATCTCTATCAACAGAGATACCTCTAAGAACCAGTACTACCTGCAGCTGAACTCTGTGACCCCTGAGGATACCGCCGTGTACTACTGTGCCAAGATGGGCGATTGGCTGGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAGCTGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCTAGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCTGCAGAACTGGGTGAACGTGATCTCTTCTCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGAGCTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCTTTCGTGAAGATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.94 794-IL15-com7-2 the (794-h1-71 heavy chain-IL15Rα sushi-Linker1-IL15-com7) the (D8S/H105N) CAAGTGCAGCTGCAGCAGTCTGGCCCTGGCCTGGTGAAGCCTTCTCAGACCCTGTCTCTGACCTGTGCCGTGAGCGGCGATTCTATCACCTCTGGCTACTGGAACTGGATCAGAAAGTTTCCTTCTAGAGGCCTGGAGTACATGGGCTACATCTCTTACTCTGGCTCTACCTACTACAACCCTTTCCTGAAGTCTAGAATCTCTATCAACAGAGATACCTCTAAGAACCAGTACTACCTGCAGCTGAACTCTGTGACCCCTGAGGATACCGCCGTGTACTACTGTGCCAAGATGGGCGATTGGCTGGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAGCTGCCCCCCTCCAATGTCTGTGGAGCACGCCGACATCTGGGTGAAGTCTTACTCCCTGTATTCCAGGGAGCGGTACATCTGCAACAGCGGCTTCAAGAGGAAGGCTGGCACCTCCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTTCTCTGAAGTGTATCAGAGATCCAGCCCTGGTGCATCAGCGCCCCGCTCCCCCTAGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCTGCAGAACTGGGTGAACGTGATCTCTTCTCTGAAGAAGATCGAGGATCTGATCCAGTCTATGCACATCGATGCCACCCTGTACACCGAGTCTGATGTGCACCCTTCTTGTAAGGTGACCGCCATGAAGTGTTTCCTGCTGGAGCTGCAGGTCATCTCTCTGGAGTCTGGCGATGCCTCTATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAACTCTCTGAGCTCTAACGGCAACGTGACCGAGTCTGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCTTTCGTGAACATCGTGCAGATGTTCATCAACACCTCT SEQ ID NO.96

Table 5. Molecular composition of PD-L1-IL-15 fusion protein Each domain and corresponding amino acid sequence of the PD-L1-IL-15 fusion protein molecule in Examples 9-15 Fusion protein number mAb heavy chain sequence IL15Rα sushi Linker IL-15 protein mAb 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:

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;

For example: "794-IL15-xx-2": "794", 794-h1-71 monoclonal antibody; "IL15-xx", indicates IL15-WT or IL15 mutant; "2", indicates the structure shown in Figure 5B model.

surface 6. IL-15-Fc Fusion protein molecular composition Example 9-16 middle IL-15-Fc Each domain of the fusion protein molecule and its corresponding amino acid sequence 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

Example 9 , IL-15 Expression of fusion protein

Transient protein expression was performed using the ExpiCHO expression system. Dilute the host cell ExpiCHO-S (Cat no. A29127) passaged in ExpiCHOTM Expression Medium (Cat no. A2910001) to a suitable density and place it on a shaker (100 rpm, 37°C, 8% CO2) for transfection. Add the vector carrying the nucleic acid sequence encoding the fusion protein into OptiPRO™ SFM (Cat no.12309019) medium so that the final concentration of the vector is 0.5-1.0 μg/mL. Add an appropriate amount of ExpiFectamine™ CHO Reagent (Cat no.A29129) to the DNA-containing OptiPRO™ SFM medium to form a DNA-ExpiFectamine™ CHO Reagent complex. After standing at room temperature for 1-5 minutes, slowly drop the complex into the transfection medium. in the cell suspension. On the first day after transfection, add a certain amount of ExpiFectamine™ CHO Enhancer (Cat no.A29129) and ExpiCHO™ Feed (Cat no.A29129) and cool down to 32°C for culture. On the 4th to 6th day after transfection, add a certain amount of ExpiCHO™ Feed (Cat no.A29129). On day 10-12 after transfection, the supernatant was harvested by centrifugation at 5000 g for 30 min.

Example 10 , IL-15 Purification of fusion proteins

The mutant fusion protein was purified by the magnetic bead method, and 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 hours to ensure that the IL-15 fusion protein was bound to the magnetic beads After the supernatant was discarded, the magnetic beads were washed 3 times with PBS, and finally citrate at pH 3.0 was added to elute to obtain IL-15 fusion protein. After neutralizing the sample with 1M Tris-Hcl, the protein concentration was determined with NanoDrop One.

Example 11 , Molecular Exclusion Chromatography Determination IL-15 Purity of fusion protein

In the detection method of the present invention, TSKgel G3000SWXL chromatographic column (TOSOH, 0008541) and pre-column Tskgel guard columnSWXL (TOSOH, 0008543) are used for size exclusion chromatography (SEC) to determine the purity of the IL-15 mutant fusion protein of the present invention. The mobile phase (50mM PB, 300mM NaCl, pH6.8) was used to equilibrate the column at a flow rate of 1mL/min. The ultraviolet detection wavelength is 280nm. The results are shown in Table 7.

surface 7. Determination by Size Exclusion Chromatography IL-15 Fusion protein purity 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 the the

Example 12 , IL-15 Mutant fusion protein induction Mo7e Cell Proliferation Assay

Mo7e is a human megakaryocytic leukemia cell line (Cobioer, CBP60791), which can be used to study the activity of IL-15 on cell proliferation. The IL-15 mutant fusion protein was diluted with 1640-10% FBS (RPMI1640, Gibco, 72400047; FBS, Gibco, 10099141) to a gradient concentration (final concentration 100000pM-1.28pM, 5-fold dilution), and Mo7e cells were diluted with 1640- Dilute 10% FBS to 105 cells/ml, add 50 μl mutant culture solution and 50 μl Mo7e cell suspension to 96-well U-bottom plate, mix well and incubate at 37°C with 5% CO2. After 72 hours, the culture plate was taken out, and 100 μl Cell Titer Glo luminescent cell viability detection reagent (Promega, G7571) was added to detect the fluorescence intensity of luminescence, which is proportional to the cell proliferation ability.

The results shown in Figure 6A-6D: T-IL15-7-1, T-IL15-8-1, T-IL15-9-1 mutant activity significantly decreased compared to T-IL15-WT-1, T- The EC50 of IL15-7-1 was 5735pM, and the corresponding EC50 of T-IL15-WT-1 was 133.3pM (Fig. 6A). The activities of T-IL15-10-1, T-IL15-11-1, T-IL15-26-1 mutants were significantly decreased compared with T-IL15-WT-1, T-IL15-10-1 and T- The EC50 of IL15-26-1 were 28076pM and 290.9pM, respectively, and the corresponding EC50 of T-IL15-WT-1 was 143.3pM (Fig. 6B). Compared with T-IL15-WT-1, the activities of T-IL15-29-1 and T-IL15-42-1 mutants were reduced, with EC50 of 285.2pM and 194.5pM, respectively, and the activity of T-IL15-43-1 mutants Compared with T-IL15-WT-1, the activity was enhanced with EC50 of 103.2pM, and the EC50 of T-IL15-WT-1 was 150.7pM (Fig. 6C). The combined mutation activity of T-IL15-com1-1 and T-IL15-com2-1 was lower than that of T-IL15-WT-1; the Tecentriq control group had no effect on the proliferation of Mo7e cells, that is, the anti-PD-L1 antibody had no effect on Mo7e cells Proliferative activity was induced (Fig. 6D), and the effect on cell proliferation activity was produced by IL-15.

Example 13 , IL15 Mutant fusion protein induction CD8+T Cell Proliferation

In order to detect the effect of the IL-15 mutant fusion protein on stimulating the proliferation of CD8+ T cells in human PBMC (Allcells, Lot: 1911150123), this example uses Ki67 as a proliferation marker to detect different concentrations of the IL-15 mutant fusion protein Proliferation ratio of CD8+ T cells after stimulating human PBMC cells for three days. First, human PBMCs were suspended in RPMI1640 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-bottom plate (Corning, Cat: 3799). Then, starting from 500nM, the IL-15 mutant fusion protein was diluted 4 times, with a total of 11 gradients, and then 100 μl/well of each concentration of the sample was added to the 96-well U-bottom plate that had been added to human PBMCs, and after mixing evenly , placed in a 37°C, 5% CO2 incubator for three days. The cultured cells were treated with LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen, Cat: L34964) and CD3-AF700 (BD, Cat: 557943), CD8-FITC (BD, Cat: 555366), APC-Ki67 (Biolegend, Cat: 350514) antibody was stained, and flow cytometry was used to detect the proportion of Ki67+ cells in CD3+CD8+ T cells stimulated by different concentrations of IL-15 mutant fusion protein. Figure 7A-7N shows the serially diluted 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, V9 - IL15-63, V9-IL15-com6, 794-IL15-65-2, 794-IL15-64-2, V5-IL15-WT and P22339. After stimulating human PBMC for three days, the proliferation ratio of CD8+ T cells, and the EC50 value of each mutant stimulated proliferation. Among them, the proliferation ability of T-IL15-8-1, T-IL15-9-1, and T-IL15-11-1 on CD8+ T cells was significantly reduced, and the EC50 could not be calculated by curve fitting before reaching the plateau; 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 value fitting was inaccurate; but the results showed that the above mutant combinations had lower proliferative activity on CD8+ T cells than 794-IL15-WT-2 or P22339. T-IL15-WT-1, 794-IL15-WT-2 is wild-type IL-15 control, P22339 is mutant IL-15 control; 794-h1-71 monoclonal antibody is anti-PD-L1 antibody control group; Drug0 is Negative control without adding any protein. The proliferation of CD8+ T cells was weaker in IL15 mutants relative to wild-type IL-15 controls. The 794-h1-71 monoclonal antibody control group had no effect on the proliferation of CD8+ T cells, that is, the anti-PD-L1 antibody had no activity inducing the proliferation of CD8+ T cells (Figure 7B), and the effect on cell proliferation was produced by IL-15 of.

Example 14 , IL15 Mutant fusion protein induction NK Cell Proliferation

In order to detect the effect of IL-15 mutant fusion protein on stimulating the proliferation of NK cells in human PBMC (Allcells, Lot: 1911150123), this example uses Ki67 as a proliferation marker to detect different concentrations of IL-15 mutant fusion protein stimulating human The proliferation ratio of NK cells after three days of PBMC cells. First, human PBMCs were suspended in RPMI1640 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-bottom plate (Corning, Cat: 3799). Then, starting from the concentration of 500nM, the IL-15 mutant fusion protein was serially diluted 4 times, with a total of 11 gradients. Then, 100 μl/well of each gradient sample was added to a 96-well U-bottom plate in which human PBMC had been added, mixed evenly, and cultured in a 37°C, 5% CO2 incubator for three days. The cultured cells were treated with LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen, Cat: L34964) and CD3-AF700 (BD, Cat: 557943), CD56-PE (BD, Cat: 555516), APC-Ki67 (Biolegend, Cat: 350514) antibody was used for staining, and then Invitrogen Attune NxT flow cytometer was used to detect the proportion of Ki67+ cells in NK cells stimulated by different concentrations of IL-15 mutant fusion protein. Figure 8A-8M shows the serially diluted 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, V9-IL15 -com6, 794-IL15-65-2, 794-IL15-64-2, V5-IL15-WT and P22339 stimulated human PBMC for three days, the proliferation ratio of CD3-CD56+ NK cells, and the EC50 of each mutant stimulated proliferation value. Among them, T-IL15-8-1, T-IL15-9-1, T-IL15-11-1, V9-IL15-com6, V9-IL15-62, V9-IL15-63 have significant proliferation ability on NK cells Decreased, did not reach the plateau, and the EC50 value fitting was not accurate; but the above-mentioned mutant combination significantly reduced the proliferation activity of NK cells compared with the control group T-IL15-WT-1 or P22339. T-IL15-WT-1, 794-IL15-WT-2 is wild-type IL-15 control, P22339 is mutant IL-15 control; 794-h1-71 monoclonal antibody is anti-PD-L1 antibody control group; Drug0 is Negative control without any added protein. IL-15 mutants had weaker proliferation of NK cells relative to wild-type IL-15 controls. The 794-h1-71 monoclonal antibody control group had no effect on the proliferation of NK cells, that is, the anti-PD-L1 antibody had no activity of inducing proliferation of NK cells (Figure 8B), and the effect on cell proliferation activity was produced by IL-15.

Example 15 , humanized anti PD-L1 Antibody -IL15 bifunctional molecule / In vivo efficacy of fusion protein in mice

Inoculate human melanoma cell A375 cells (BNCC100266) 5×106 cells/100 μL subcutaneously on the right back of NPG mice, and recover cryopreserved PBMCs (ALLCELLs, PB005F-C) at 5×106 cells/ 200 μl was injected into the tail vein of NPG mice (5-6 weeks, female; purchased from Beijing Weitongda Biotechnology Co., Ltd.). Six days after PBMC inoculation, 40 μl of blood was collected to detect the proportion of hCD45+ cells. After the tumor grew to about 80 mm3, the body weight, proportion of hCD45+ cells, and tumors that were too large or too small were removed. The mice were randomly divided into PBS group and Tecentriq group (10 mg /kg), 794-IL15-WT-2 group (1 mg/kg), 794-IL15-com6-2 group (4 mg/kg), a total of 4 groups, 8 rats in each group, 32 rats in total. The administration method is intraperitoneal administration, according to grouping 794-IL15-WT-2 group (1 mg/kg) and 794-IL15-com6-2 group (4 mg/kg), administration once a week, total administration 1 time; PBS group and Tecentriq group (10mg/kg) were administered twice a week, 5 times in total. The tumor volume was measured 3 times a week, and the data were recorded. Calculate tumor volume (long diameter × short diameter 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 treatment group on day 0 of administration; Vi: mean tumor volume of solvent control group on day i of administration, V0: The average tumor volume of the vehicle control group on the 0th day of administration). On the 14th day of group administration, the administration group had a significant inhibitory effect on the tumor volume, with a statistical difference (P<0.05), and the 794-IL15-WT-2 group and the 794-IL15-com6-2 group Compared with the Tecentriq group, there was a significant inhibitory effect on the tumor volume (P<0.05). See Figures 9A-9C, Table 8.

Table 8. Effects of test substances on A375 tumor volume in immune reconstituted NPG mice group Test substance Tumor volume ( mm ³ ) ^a P ^b P ^c Before administration Day 21 of group administration 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: Statistical comparison between the tumor volume of the administration group and the Vehicle control group (PBS) on the 14th day of group administration, Two-way ANOVA analysis, *P<0.05, * *P<0.01, ***P<0.001, ****P<0.0001; c: Statistical comparison between the tumor volume of the administration group and the tumor volume of the positive drug Tecentriq group on the 14th day of group administration, Two-way ANOVA Analysis, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

The above results indicated that the humanized anti-PD-L1 antibody-IL15 bifunctional molecule/fusion protein 794-IL15-WT-2 and 794-IL15-com6-2 had a significant inhibitory effect on the growth of A375 tumor subcutaneous xenografts (P<0.0001 ); 794-IL15-WT-2 group and 794-IL15-com6-2 group had a stronger inhibitory effect on tumor volume than Tecentriq group, and there was a significant difference (P<0.05).

Example 16 , IL15-Fc In vivo efficacy test of fusion protein in mice

Inoculate 5×106 cells/100 μL of human melanoma cell A375 (BNCC100266) subcutaneously on the right back of NPG mice, and recover frozen PBMC (ALLCELLs; PB005F-C) at 5×106 cells the next day after inoculating A375 /200 μl volume was injected into NPG mice (5-6 weeks, female; purchased from Beijing Weitongda Biotechnology Co., Ltd.) through the tail vein. Six days after PBMC inoculation, 40 μl of blood was collected to detect the proportion of hCD45+ cells. After the tumor grew to about 69 mm3, the body weight, low proportion of hCD45+ cells, too large and too small tumors were removed, and the mice were randomly divided into PBS groups according to tumor volume. 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 kg), V5-IL15-WT (2 mg/kg) in 7 groups, 8 rats in each group, 56 rats in total. The administration method was intraperitoneal administration, and the administration was performed once a week according to the grouping, and the administration was administered 3 times in total; the tumor volume was measured 3 times a week, and the data were recorded. Calculate tumor volume (long diameter × short diameter 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 treatment group on day 0 of administration; Vi: mean tumor volume of solvent control group on day i of administration, V0: The average tumor volume of the vehicle control group on the 0th day of administration). On the 21st day of group administration, compared with the PBS control group, the administration group had a significant inhibitory effect on the tumor volume, with a statistical difference (P<0.05), and the positive drug ALT803 group and V9-IL15-com6 The two groups had similar inhibitory effects on tumor volume (P>0.05). See Figures 10A-10C, Table 9.

Table 9. Effects of test substances on A375 tumor volume in immune reconstituted NPG mice group Test substance Tumor volume ( mm ³ ) ^a P ^b Before administration Day 21 of group administration TGI _TV (%) G1 PBS 70 ±5 1755±360 - - G2 ALT803 (0.2mg/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 (5mg/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: Statistical comparison between the tumor volume of the drug administration group and the tumor volume of the Vehicle control group on the 14th day of group administration, Two-way ANOVA analysis, *P<0.05, **P< 0.01, ***P<0.001, ****P<0.0001; c: The tumor volume on Day 21 was analyzed by identify outliers, one data in the V9-IL15-com6 5mpk group was abnormally large, and this data was excluded.

The experimental animals were in a good state of activity and eating during the administration period. After the administration, the body weight of the ALT803 and V5-IL15-WT administration groups showed a significant downward trend, and the animals died, indicating that the mice were sensitive to ALT803 and V5-IL15-WT in this administration. Drug frequency and dose intolerance. Animals in the V9-IL15-61 (5mpk) group and V9-IL15-com6 (1mpk) group died, but GVHD such as anemia was observed before death. It is speculated that the death of the mice was caused by GVHD and had nothing to do with the drug. See Figure 10D, Table 10, Table 11.

Table 10 Effects of test substances on the body weight of immune reconstituted A375 tumor-bearing NPG mice group Test substance body weight ( g ) ^a Body weight change after 21 days of administration ( g ) Before administration Day 21 of group administration P ^b G1 PBS 21.7±0.7 21.3±0.8 - -0.4 G2 ALT803 (0.2mg/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 (5mg/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 body weight of the administration group and the Vehicle control group were statistically compared on the 21st day of group administration, Two-way ANOVA analysis.

Table 11 Effects of test substances on the survival of immune reconstituted A375 tumor-bearing NPG mice group Test substance The number of surviving mice on the 21st day of group administration ^a mouse death time Day 15 of group administration Day 19 of group administration On the 20th day of group administration Day 21 of group administration G1 PBS 8 - - - - G2 ALT803 (0.2 mg/kg) 7 ^1b - - - G3 V9-IL15-61 (1 mg/kg) 8 - - - - G4 V9-IL15-61 (5mg/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: death of mice after group administration, 1 means 1 mouse died on the day, 0 means 1 mouse died on the day but anemia, symptoms such as jaundice.

The above results show that both IL15-Fc fusion proteins V9-IL15-61 and V9-IL15-com6 have significant inhibitory effect on the growth of human immune reconstitution A375 tumor subcutaneous xenografts; V9-IL15-com6 (5mg/kg) and positive control antibody ALT803 (0.2mg/kg), the TGI (tumor growth inhibition rate) levels of the two are comparable, and the safety is better than ALT803.

none

The foregoing aspects and other aspects of the present invention are clearly illustrated below by the detailed description of the present invention and the accompanying drawings. The drawings herein are for the purpose of illustrating some preferred embodiments of the invention, however, it is to be understood that the invention is not limited to the particular embodiments disclosed. Figure 1. Humanized PD-L1 antibody blocking PD-L1 and PD-1 binding results, where the positive control is Tecentriq. Figure 2. FACS determination of the binding ability of the humanized PD-L1 antibody to PD-L1 at the cellular level, where the positive control is Avelumab. Figure 3. Jurkat-PD-1/CHO-PD-L1-NFAT system tests the blocking ability of humanized PD-L1 antibody on PD-L1/PD-1, and the positive control is Avelumab. Figure 4. Humanized anti-PD-L1 antibody promotes the secretion of IFN-γ in mixed lymphocyte reaction, in which the negative control is anti-Hel antibody, and the positive control is Avelumab. Figure 5. Two structures of IL-15 fusion protein: A: IL-15 wild-type or mutant co-expressed with IL-15Rα sushi assembly, non-covalent linkage; B: IL-15 wild-type or mutant and IL-15Rαsushi were fused and assembled in series through a linker; C: IL-15 wild-type or mutant is connected with IL-15Rαsushi through a linker, and then IL-15Rαsushi is tandemly expressed and assembled with Fc through a linker; D: IL-15Rα sushi is connected to IL-15 wild type or mutant through linker, and IL-15 wild type or mutant is then tandemly expressed and assembled with Fc through linker. Figure 6A-6D, IL15 mutants promote the proliferation of Mo7e cells: the structure of the fusion protein is shown in Figure 5A, Tecentriq is fused with IL-15Rαsushi, IL15 mutants are non-covalently linked to IL-15Rαsushi, and there is no linker connection. Figure 7A-7N, IL-15 mutants induce CD8+T cell proliferation: Figure 7A: The structure of the fusion protein shown in Figure 5A, Tecentriq is fused with IL-15Rαsushi, IL15 mutants are non-covalently connected to IL-15Rαsushi, and there is no linker connection; Figure 7B-7G, 7M-7N: The structure of the fusion protein is shown in Figure 5B. The self-developed PD-L1 monoclonal antibody is fused with IL-15Rαsushi and IL15 mutant, and the IL15 mutant and IL-15Rαsushi are expressed in tandem through linker connection; Drug0 Represents the negative control in which no protein was added to the reaction system; Figure 7H-7K: The structure of the fusion protein is shown in Figure 5D. IL-15Rαsushi is connected to IL-15 wild type or mutant through linker, and then IL-15 wild type or mutant is tandemly expressed and assembled with Fc through linker; Figure 7L, the fusion protein shown in Figure 5C, IL-15 wild-type or mutant is connected with IL-15Rαsushi through a linker, and then IL-15Rαsushi is tandemly expressed and assembled with Fc through a linker. Figure 8A-8M, IL-15 mutants induce NK cell proliferation: Figure 8A: The structure of the fusion protein shown in 5A, Tecentriq is fused with IL-15Rαsushi, IL15 mutants are non-covalently connected to IL-15Rαsushi, and there is no linker connection; Figure 8B-8F, 8L-8M: The structure of the fusion protein is shown in Figure 5B. The self-developed PD-L1 monoclonal antibody is fused with IL-15Rαsushi and IL15 mutant, and the IL15 mutant and IL-15Rαsushi are expressed in tandem through a linker connection; Drug0 Represents the negative control in which no protein was added to the reaction system. Fig. 8G-8J: Fusion protein As shown in Fig. 5D, IL-15Rα sushi is connected with IL-15 wild type or mutant through linker first, and then IL-15 wild type or mutant is tandemly expressed and assembled with Fc through linker. Figure 8K: Fusion protein as shown in Figure 5C, IL-15 wild-type or mutant is connected with IL-15Rαsushi through a linker, and then IL-15Rαsushi is tandemly expressed and assembled with Fc through a linker. Figures 9A-9C: Drug efficacy in hPBMC-A375 mice. The structure of the fusion protein is shown in Figure 5B. Figures 10A-10C: Drug efficacy in hPBMC-A375 mice. The structure of the fusion protein is shown in Figure 5C or 5D. Fig. 10D: Changes in body weight of hPBMC-A375 mice after administration.

Claims (18)

An IL-15 mutant polypeptide, characterized in that the polypeptide comprises mutations at one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15. A polypeptide comprising an IL-15 mutant, characterized in that the polypeptide comprises mutations at one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15. The polypeptide according to claim 1 or 2, wherein the mutation is substitution, insertion or deletion; Preferably, the polypeptide at least comprises mutations at two, three or four amino acid residues in Val3, Ile6, Asp8 or His105; Preferably, the mutation is selected from amino acid substitutions in the following group: 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 comprises 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; 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% and human wild-type IL-15 % or 99% sequence identity. The polypeptide according to any one of claims 1-3, wherein the amino acid sequence of the IL-15 mutant is such as 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 Shown in SEQ ID NO.47; Preferably, the polypeptide has the following properties: (1) Mediate the proliferation of human CD8+ T cells; (2) mediate the proliferation of human NK cells; and/or, (3) inhibit tumor growth; Preferably, the activity of the polypeptide in mediating proliferation/expansion of CD8+ T and/or NK cells is lower than that of a polypeptide comprising wild-type IL-15; Preferably, the amino acid sequence of the wild-type IL-15 is shown in SEQ ID NO.1. A protein, characterized in that the protein comprises the polypeptide described in any one of Claims 1-4; and further comprises: (1) an immunoglobulin molecule or part thereof fused to the IL-15 mutant, and/or or (2) IL-15Rα; Preferably, the immunoglobulin molecule is an antibody or an antigen-binding fragment; the immunoglobulin molecule part is an immunoglobulin Fc region; Preferably, the antibody or antigen-binding fragment is selected from: (1) Chimeric antibodies or fragments thereof; (2) Humanized antibodies or fragments thereof; or, (3) Fully human antibodies or fragments thereof; Preferably, 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; 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 comprising the constant region of human or murine antibody IgG1, IgG2, IgG3 or IgG4 The sequence of the region; preferably, the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO.73; Preferably, the IL-15 mutant is fused with an immunoglobulin molecule or part thereof with or without a linking peptide, or the IL-15 mutant is fused with IL-15Rα with or without a linking peptide; preferably using a linking peptide; preferably using SEQ The connecting peptide shown in ID NO.65, SEQ ID NO.67, SEQ ID NO.69 or SEQ ID NO.71; Preferably, the IL-15 mutant is fused with IL-15Rα with or without a connecting peptide, and then fused with an immunoglobulin molecule or part thereof; preferably using a connecting peptide; preferably using SEQ ID NO.65, SEQ ID NO.69 or Linking peptide shown in SEQ ID NO.71. The protein according to claim 5, wherein the connection sequence of each domain is from the N-terminal to the C-terminal: (1) Immunoglobulin molecules or parts thereof, IL-15Rα, IL-15 mutants; (2) Immunoglobulin molecules or parts thereof, IL-15 mutants, IL-15Rα; (3) IL-15 mutants, IL-15Rα, immunoglobulin molecules or parts thereof; (4) IL-15Rα, IL-15 mutants, immunoglobulin molecules or parts thereof; (5) IL-15 mutants, immunoglobulin molecules or parts thereof; (6) Immunoglobulin molecules or parts thereof, IL-15 Rα; (7) IL-15Rα, immunoglobulin molecules or parts thereof; (8) IL-15 mutant, IL-15Rα; or, (9) IL-15Rα, IL-15 mutants; Preferably, when IL-15Rα or IL-15 mutant is fused with an immunoglobulin molecule, it is fused to the N-terminal of the heavy chain variable region of the immunoglobulin molecule or the C-terminal of the Fc region of the immunoglobulin; when IL-15Rα or When the IL-15 mutant is fused to the Fc region of the immunoglobulin, it is fused to the N-terminal or C-terminal of the Fc region of the immunoglobulin. A protein consisting of the following 4 parts: (1) Immunoglobulin heavy chain; (2) Immunoglobulin light chain; (3) IL-15Rα; and, (4) IL-15 mutant polypeptide as described in any one of claims 1-4; Preferably, the protein is a homodimer comprising monomers composed of parts (1)-(4); Preferably, IL-15Rα is fused to the N-terminal of the variable region of the heavy chain of the immunoglobulin or the C-terminal of the Fc region of the immunoglobulin through or without connecting 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 connecting peptide. A protein that consists of the following three parts: (1) Immunoglobulin Fc region; (2) IL-15Rα; and, (3) IL-15 mutant polypeptide as described in any one of claims 1-4; Preferably, the protein is a homodimer comprising monomers composed of parts (1)-(3); Preferably, IL-15Rα is fused to the N-terminal or C-terminal of the IL-15 mutant polypeptide with or without a connecting peptide, and then fused to the N-terminal or C-terminal of the immunoglobulin Fc region with or without a connecting peptide. The protein according to any one of claims 5-8, wherein the immunoglobulin is selected from anti-PD-L1 antibodies; the anti-PD-L1 antibodies are preferably Tecentriq, KN-035, 794-h1-71; Preferably, the anti-PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, and 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 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity compared to the sequence shown in SEQ ID NO: 99 and SEQ ID NO: 100 sequence; or, The heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO: 97 and SEQ ID NO: 98, respectively, or have at least Sequences with 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity; Preferably, the IL-15Rα is selected from IL-15Rα-sushi; preferably the amino acid sequence of IL-15Rα-sushi such as SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO.53, or SEQ ID NO .55 shown. An antibody or antigen-binding fragment specifically binding 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 can be The 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 at least 80%, 85% of the sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100 , 90%, 95%, 96%, 97%, 98%, 99% or more identical sequences; Preferably, its dissociation constant (KD) for binding to human programmed death ligand-1 (PD-L1) is not greater than 1.8×10-9M, and it binds to cynomolgus monkey programmed death ligand-1 (PD-L1) The dissociation constant (KD) is not greater than 9.4×10-10M; Or, optionally, the antibody or antigen-binding fragment binds or does not bind to monkey PD-L1; Optionally, the antibody or antigen-binding fragment binds or does not bind to murine PD-L1; Preferably, the anti-PD-L1 antibody competitively binds to PD-L1 or its epitope, and has the following characteristics: (1) Specifically binding to PD-L1 recombinant protein and cells expressing PD-L1; (2) Block the combination of PD-L1 and PD-1 protein; (3) Inhibit the binding of PD-1 to PD-L1 expressed on the cell surface; (4) Enhance T cell activity; or/and (5) inhibit tumor growth; Preferably, the anti-PD-L1 antibody comprises a constant region selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably a human or murine antibody IgG1, IgG2, IgG3 or IgG4 the sequence of the constant region; Preferably, the PD-L1 antibody is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, and bispecific antibodies. An isolated nucleic acid molecule, characterized in that it encodes the polypeptide as described in any one of claims 1-4, the protein as described in any one of claims 5-9, or the antibody as described in claim 10 or Antigen-binding fragments. An expression vector, characterized in that the expression vector comprises the isolated nucleic acid molecule as described in claim 11. A host cell, characterized in that, the host cell comprises the isolated nucleic acid molecule as claimed in item 11, or the expression vector as described in claim 12; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; More preferably, the host cells are derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis; more preferably, the host cells are selected from Chinese hamster ovary cells (CHO). A method for preparing a polypeptide or protein, characterized in that the host cell according to claim 13 is cultivated under appropriate conditions, and the polypeptide or protein is isolated. A pharmaceutical composition, characterized in that the composition comprises the polypeptide according to any one of claim items 1-4, the protein according to any one of claim items 5-9, and the antibody according to claim item 10 Or an antigen-binding fragment, 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, or a product prepared by a method as claimed in claim 14; and a pharmaceutically acceptable carrier; preferred , the pharmaceutical composition further comprises an additional antineoplastic agent. The polypeptide as described in any one of claims 1-4, the protein as described in any one of claims 5-9, the antibody or antigen-binding fragment as described in claim 10, the isolated nucleic acid molecule as claimed in claim 11 , the expression vector as claimed in claim 12, the cell as claimed in claim 13, or the product prepared by the method as claimed in claim 14, or the pharmaceutical composition as described in claim 15 in the preparation of medicines for preventing and/or treating diseases in individuals The purposes in; Said disease is preferably tumor or inflammatory disease; Preferably, the tumor is selected from glioblastoma, prostate cancer, blood cancer, B-cell tumor, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphoma Cellular 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 carcinoma, colorectal cancer, ovarian cancer, non-small cell lung cancer and squamous cell head and neck cancer. A method for preventing and/or treating a disease in an individual, comprising administering the polypeptide according to any one of claims 1-4, the protein according to any one of claims 5-9, such as The antibody or antigen-binding fragment as claimed in claim 10, the isolated nucleic acid molecule as claimed in claim 11, the expression vector as claimed in claim 12, the cell as claimed in claim 13, or the product prepared by the method as claimed in claim 14, or the product as claimed in claim 14 The pharmaceutical composition described in item 15; the disease is preferably a tumor or an inflammatory disease; Preferably, the tumor is selected from glioblastoma, prostate cancer, blood cancer, B-cell tumor, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphoma Cellular 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 carcinoma, colorectal cancer, ovarian cancer, non-small cell lung cancer and squamous cell head and neck cancer. The polypeptide as described in any one of claims 1-4, the protein as described in any one of claims 5-9, the antibody or antigen-binding fragment as described in claim 10, the protein as described in claim 11 The isolated nucleic acid molecule, the expression vector as described in claim 12, the cell as described in claim 13, or the product prepared by the method as described in claim 14, or the pharmaceutical composition as described in claim 15, can be used For preventing and/or treating a disease in an individual; said disease is preferably a neoplastic disease or an inflammatory disease; Preferably, the tumor is selected from glioblastoma, prostate cancer, blood cancer, B-cell tumor, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphoma Cellular 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 carcinoma, colorectal cancer, ovarian cancer, non-small cell lung cancer and squamous cell head and neck cancer.

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