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WO2021233260A1 - 人il-15突变体及其用途 - Google Patents

人il-15突变体及其用途 Download PDF

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WO2021233260A1
WO2021233260A1 PCT/CN2021/094167 CN2021094167W WO2021233260A1 WO 2021233260 A1 WO2021233260 A1 WO 2021233260A1 CN 2021094167 W CN2021094167 W CN 2021094167W WO 2021233260 A1 WO2021233260 A1 WO 2021233260A1
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seq
antibody
cell
mutant
immunoglobulin
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PCT/CN2021/094167
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English (en)
French (fr)
Inventor
邓婧
卢士强
刘杨
曹卓晓
唐任宏
任晋生
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Simcere Shanghai Pharmaceutical Co Ltd
Jiangsu Simcere Pharmaceutical Co Ltd
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Simcere Shanghai Pharmaceutical Co Ltd
Jiangsu Simcere Pharmaceutical Co Ltd
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Application filed by Simcere Shanghai Pharmaceutical Co Ltd, Jiangsu Simcere Pharmaceutical Co Ltd filed Critical Simcere Shanghai Pharmaceutical Co Ltd
Priority to EP21808872.2A priority Critical patent/EP4155316A4/en
Priority to CN202510591692.XA priority patent/CN120554478A/zh
Priority to JP2022570565A priority patent/JP7547507B2/ja
Priority to CA3179066A priority patent/CA3179066A1/en
Priority to CN202180036927.9A priority patent/CN115916809A/zh
Priority to AU2021273789A priority patent/AU2021273789B2/en
Priority to KR1020227044162A priority patent/KR20230012568A/ko
Priority to US17/925,547 priority patent/US20230192797A1/en
Publication of WO2021233260A1 publication Critical patent/WO2021233260A1/zh
Anticipated expiration legal-status Critical
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
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Definitions

  • the present invention relates to human IL-15 mutants, nucleic acids encoding them, fusion proteins containing IL-15 mutants and combined mutations, pharmaceutical compositions, and related uses of the pharmaceutical compositions for the treatment of tumors.
  • Interleukin-15 is an important soluble cytokine discovered and named by Grabstein in 1994 in the culture supernatant of the monkey kidney intradermal cell line CV-1/EBNA.
  • a variety of cells and tissues can express IL-15, for example, monocytes/macrophages, lymphocytes, epithelial cells, etc.
  • Interleukin 15 receptor belongs to the hematopoietic factor superfamily, which consists of three subunits: ⁇ , ⁇ (also known as CD122), and ⁇ (also known as CD132, common gamma chain, ⁇ c).
  • IL2 and IL15 share beta chain receptors.
  • IL2, IL4, IL7, IL9, IL15 and IL21 share the gamma chain receptor.
  • the receptors of IL2 and IL15 share ⁇ and ⁇ c chains, but both IL2 and IL15 have their own specific ⁇ receptor chains.
  • IL15R ⁇ people belonging to type I transmembrane proteins, IL2R ⁇ IL15R ⁇ and has a conserved binding motif (sushi domain).
  • IL15 has a relatively high affinity with IL15R ⁇ (Kd ⁇ 10 -11 M), but it does not transmit signals.
  • the affinity of IL15 and IL15 ⁇ heterodimer is relatively medium (Kd ⁇ 10 -9 M) and can conduct signals; IL15 and IL15 ⁇ heterotrimer have similar affinity to the former (Kd ⁇ 10 -9 M) and can conduct signals. Because the receptors of IL2 and IL15 share ⁇ and ⁇ chains, there are many similar biological functions between IL2 and IL15, such as promoting the proliferation of T cells and NK cells.
  • IL15R ⁇ The binding of IL15 to the receptor: IL15R ⁇ is mainly expressed in DC and monocytes. In most cases, IL15/IL15R ⁇ binds to the receptor in a trans-presented form. That is, after IL-15 and IL-15Ra are expressed in the same cell, the intracellular IL-15 binds to the sushi domain of IL-15R ⁇ with high affinity and is transported to the membrane surface, and then reacts with cells (such as T cells or NK). Binding of ⁇ heterodimer complex or ⁇ heterotrimer complex on the cell) membrane surface, ⁇ and ⁇ receptors can activate downstream Jak1 and Jak3, respectively, leading to activation of STAT-3 and STAT-5, and cascade activation Response, induce 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 produce effector functions.
  • IL-15 has a wide range of immunomodulatory effects and participates in regulating the activity, proliferation and function of a variety of immune cells.
  • Regulation of T cells Promote the activation and proliferation of T cells, promote the production 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 number of CD8 + T cells.
  • Regulating 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.
  • 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 DC cells to express costimulatory factors and IFN- ⁇ , 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.
  • IL-15 realizes its anti-tumor effect based on its ability to expand 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, its small molecule, and high renal clearance, it is extremely inconvenient to use it by multiple injections per day or subcutaneously. Therefore, the pure application of wild-type recombinant IL-15 is also limited in tumor treatment.
  • IL-15 mutants with reduced activity has the potential to improve the dose-effect relationship of IL-15 in the treatment of tumors, and 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, and their functions for killing tumor cells, and their use in the treatment of tumors.
  • the present disclosure provides an IL-15 mutant polypeptide, the polypeptide comprising one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15 mutation.
  • the present disclosure provides a polypeptide comprising an IL-15 mutant, the polypeptide comprising one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15 Mutation.
  • the IL-15 mutant polypeptide contains at least two, three, or four amino acid residue mutations in Val3, Ile6, Asp8, or His105.
  • the mutation is a substitution, insertion or deletion.
  • the mutation is selected from the group of amino acid substitutions: Val3Leu(V3L), Ile6Asp(I6D), Ile6Pro(I6P), Asp8Glu(D8E), Asp8Gln(D8Q), Asp8Arg(D8R), Asp8Ser( D8S), Asp8Val (D8V), Asp8Gly (D8G), Asp8Ile (D8I), Asp8Leu (D8L), Asp8Thr (D8T), His105Asn (H105N), and/or His105Lys (H105K).
  • the IL-15 mutant polypeptide or the IL-15 mutant-containing polypeptide comprises the following mutations or combinations of mutations: (1) Asp8Glu; (2) Asp8Gln; (3) Asp8Arg; (4) Asp8Ser; (5) Asp8Val; (6) Val3Leu; (7) Ile6Asp; (8) His105Lys; (9) His105Asn; (10) Asp8Gly; (11) Asp8Ile; (12) Asp8Leu; (13) Ile6Pro; (14) Asp8Thr; (15) Asp8Glu and Val3Leu; (16) Asp8Glu and Ile6Asp; (17) Val3Leu and Ile6Asp; (18) Ile6Asp and His105Lys; (19) Asp8Ser and His105Lys; (20) Asp8Val and Leu (21)Asn3, , Ile6Asp and His105Lys.
  • the IL-15 mutant and human wild-type IL-15 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the amino acid sequence of the IL-15 mutant is 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 SEQ ID NO. 47 are shown.
  • the IL-15 mutant polypeptide or the IL-15 mutant-containing polypeptide has the following characteristics: (1) mediate the proliferation of human CD8 + T cells; (2) mediate the proliferation of human NK cells Proliferation; and/or, (3) inhibit tumor growth.
  • the IL-15 mutant polypeptide or the IL-15 mutant-containing polypeptide has lower activity in mediating CD8+T and/or NK cell proliferation/expansion than the wild-type IL-15-containing polypeptide .
  • amino acid sequence of the wild-type IL-15 is shown in SEQ ID NO.1.
  • the present disclosure provides a protein comprising the aforementioned IL-15 mutant polypeptide or a polypeptide comprising an IL-15 mutant; and also comprising an immune system fused to the IL-15 mutant.
  • the immunoglobulin molecule is an antibody or an antigen-binding fragment; the immunoglobulin molecule part is an immunoglobulin Fc region.
  • the antibody or antigen-binding fragment is selected from: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or, (3) a fully human antibody or fragment thereof.
  • 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.
  • 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 human or murine antibodies 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.
  • the IL-15 mutant is fused to the immunoglobulin molecule or part thereof with or without a linker peptide, or the IL-15 mutant is fused to IL-15R ⁇ with or without a linker peptide; preferably a linker peptide is used ;
  • the connecting peptide shown in SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69 or SEQ ID NO. 71 is used.
  • the IL-15 mutant is fused with IL-15R ⁇ through or without a linker peptide, and then fused with an immunoglobulin molecule or part thereof; preferably, the linker peptide is used; preferably, SEQ ID NO. 65, SEQ ID are used.
  • connection sequence of each domain from N-terminus to C-terminus is:
  • IL-15R ⁇ immunoglobulin molecules or parts thereof
  • IL-15R ⁇ or IL-15 mutant when fused with an immunoglobulin molecule, it is fused to the N-terminus of the variable region of the heavy chain of the immunoglobulin molecule or the C-terminus of the immunoglobulin Fc region; when When IL-15R ⁇ or IL-15 mutant is fused with the immunoglobulin Fc region, it is fused to the N-terminus or C-terminus of the immunoglobulin Fc region.
  • the present disclosure provides a protein or antibody fusion construct/complex, which comprises the following 4 parts:
  • IL-15R ⁇ is fused to the N-terminus of the immunoglobulin heavy chain variable region or the C-terminus of the immunoglobulin Fc region with or without a linking peptide.
  • the IL-15 mutant polypeptide is non-covalently linked to IL-15R ⁇ , or the IL-15 mutant is fused to the other end of IL-15R ⁇ with or without a connecting peptide.
  • the protein is a homodimer comprising monomers composed of parts (1)-(4).
  • the present disclosure provides a protein or Fc fusion construct comprising the following three parts:
  • IL-15R ⁇ is fused to the N-terminus or C-terminus of the IL-15 mutant polypeptide with or without a connecting peptide, and then fused to the N-terminus or C-terminus of the immunoglobulin Fc region with or without a connecting peptide.
  • the protein is a homodimer comprising monomers composed of parts (1)-(3).
  • the immunoglobulin is selected from an anti-PD-L1 antibody; the anti-PD-L1 antibody is preferably Tecentriq, KN-035, 794-h1-71.
  • the anti-PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and light chain variable region having SEQ ID NO: 99 and SEQ ID NO: 100, or 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 consistent sequence; or,
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 97 and SEQ ID NO: 98, or have at least the sequence shown in SEQ ID NO: 97 and SEQ ID NO: 98. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity sequence.
  • the IL-15R ⁇ is selected from IL-15R ⁇ -sushi; preferably the amino acid sequence of IL-15R ⁇ -sushi is SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, or SEQ ID NO.55 is shown.
  • 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 ;
  • the heavy chain variable region and the light chain variable region respectively have the sequence shown in SEQ ID NO: 99 and SEQ ID NO: 100, or the sequence shown in SEQ ID NO: 99 and SEQ ID NO: 100 Compared with sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity.
  • the dissociation constant (KD) of the antibody or antigen-binding fragment binding to human programmed death ligand-1 (PD-L1) is not greater than 1.8 ⁇ 10 -9 M, which is comparable to that of cynomolgus monkeys.
  • the dissociation constant (KD) of programmed death ligand-1 (PD-L1) binding is not more than 9.4 ⁇ 10 -10 M;
  • the antibody or antigen-binding fragment binds or does not bind to monkey PD-L1;
  • the antibody or antigen-binding fragment binds or does not bind to murine PD-L1.
  • the anti-PD-L1 antibody competitively binds to PD-L1 or its epitope, and has the following characteristics:
  • the anti-PD-L1 antibody comprises a constant region selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably comprises human or murine antibodies IgG1, IgG2, The sequence of the constant region of IgG3 or IgG4.
  • the PD-L1 antibody is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, and bispecific antibodies.
  • the present disclosure provides an isolated nucleic acid molecule that encodes the polypeptide, protein, antigen or antigen-binding fragment of any one of the first to sixth aspects.
  • the present disclosure provides an expression vector comprising the nucleic acid molecule isolated in the seventh aspect.
  • the present disclosure provides a host cell comprising the isolated nucleic acid molecule of the aforementioned seventh aspect or the expression vector of the aforementioned eighth aspect; preferably, the host The cells are eukaryotic cells or prokaryotic cells; 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).
  • the present invention provides a method for preparing a polypeptide or protein, culturing the host cell described in the ninth aspect under appropriate conditions, and isolating the polypeptide or protein.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the polypeptide, protein, antigen or antigen-binding fragment according to any one of the first to sixth aspects, and the seventh
  • the present disclosure provides the polypeptide, protein, antigen or antigen-binding fragment according to any one of the foregoing first to sixth aspects, the isolated nucleic acid molecule in the foregoing seventh aspect, and the foregoing eighth aspect.
  • the expression vector of the aspect, the cell of the ninth aspect, or the product prepared by the method of the tenth aspect, or the pharmaceutical composition of the eleventh aspect is useful for preventing and/or treating diseases in an individual Use in medicine; the disease is preferably a tumor.
  • the present invention provides a method for preventing and/or treating a disease in an individual, comprising administering the polypeptide or protein according to any one of the first to sixth aspects to a patient in need thereof , 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 The pharmaceutical composition of the eleventh aspect; the disease is preferably a tumor.
  • interleukin-15 is a pleiotropic cytokine that can activate T cells, B cells and NK cells, and can mediate Direct the proliferation and survival of these cells.
  • IL-15 can activate, maintain and expand CD8 + memory T cells.
  • the "IL-15” or “IL-15 peptide” or “IL-15 polypeptide” in the present invention can be any IL-15 (interleukin 15) or its mutants, such as human IL-15 or non-human mammals IL-15 or non-mammalian IL-15.
  • non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, and the like.
  • Preferred human interleukin 15 mature molecules see the database UniProtKB, accession number P40933, 49-162aa.
  • IL-15 wild-type or wild-type IL-15 refers to human IL-15 or non-human mammalian IL-15 or non-mammalian IL-15 from natural sources; it can also refer to this
  • the IL-15 polypeptide has been commonly used in the field.
  • 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 deletions, or to stimulate specific cell lines.
  • IL-15R ⁇ in the present invention can be any species of IL-15R ⁇ or its functional fragments, such as human IL-15R ⁇ or non-human mammalian IL-15R ⁇ or non-mammalian IL-15R ⁇ .
  • exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, and the like.
  • IL-15R ⁇ variant in the present invention refers to a functional mutant formed by one or more amino acid deletion, insertion or substitution mutations on IL-15R ⁇ that has the ability to bind to its ligand molecule such as IL15, preferably human IL15R ⁇
  • the molecule more preferably a shortened form of human IL-15R ⁇ extracellular domain fragment, that is, a molecule with human interleukin 15 receptor ⁇ activity obtained from the C-terminus of the extracellular domain fragment through one or more amino acid deletion mutations, preferably retaining 65-120
  • a deletion mutation form of 6 amino acids more preferably a shortened form of deletion mutation that retains 65-102 amino acids, such as IL-15R ⁇ -sushi; preferably IL-15R ⁇ -sushi, see Table 3 for details.
  • the term "immunoglobulin Fc region” means that the constant region of an immunoglobulin chain, especially the carboxyl terminal or part of the constant region of an immunoglobulin heavy chain, has no antigen-binding activity and is a combination of antibody molecules and effector molecules. The site of cell interaction.
  • the "immunoglobulin Fc region” of the present invention can be any Fc or variants thereof, derived from human or non-human mammals.
  • the immunoglobulin Fc region may include a combination of two or more domains of the heavy chain CH1, CH2, CH3, CH4 and the immunoglobulin hinge region.
  • Fc can be derived from different species, preferably human immunoglobulin.
  • immunoglobulins can be divided into different types. There are mainly five types of immunoglobulins: IgA, IgD, IgE, IgG and IgM. Some of them can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG-4; IgA-1 and IgA-2.
  • the "Fc region” preferably includes at least one immunoglobulin hinge region, and the CH2 and CH3 domains of IgG. More preferably, it includes a CH2 domain, a CH3 domain and an immunoglobulin hinge region of IgG1, and the starting amino acid position of the hinge region can be changed.
  • Fc variant in the present invention refers to a change in the structure or function of the Fc caused by the presence of one or more amino acid substitutions, insertions or deletion mutations at a suitable site on the Fc.
  • the "interaction between Fc variants” refers to the space-filling effect, electrostatic guidance, hydrogen bonding, hydrophobic interaction, etc. between Fc variants designed by mutation. Interactions between Fc variants contribute to the formation of stable heterodimeric proteins.
  • the preferred mutation design is a "Knob-into-Hole" form of mutation design.
  • Fc variants have been widely used in the art to prepare bispecific antibodies or heterodimeric Fc fusion protein forms.
  • the representative one is the "Knob-into-Hole" format proposed by Cater et al. (Protein Engineering vol. 9no. 7pp. 617-621, 1996); Amgen’s technicians use Electrostatic Steering to form an Fc-containing heterogeneous Source dimer form (US 20100286374A1); Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp.
  • the Knob/Hole structure on the Fc variant fragments of the present invention means that the two Fc fragments are each mutated, and the mutation can be combined through the "Knob-into-Hole" format.
  • the "knob-into-hole" model of Cater et al. is used to carry out site mutation modification on the Fc region, so that the obtained first Fc variant and second Fc variant can be in the form of "knob-into-hole" Combine together to form a heterodimer.
  • the selection of a specific immunoglobulin Fc region from specific immunoglobulin classes and subclasses is within the grasp of those skilled in the art.
  • the Fc region of human antibodies IgG1, IgG2, IgG3, and IgG4 is preferred, and the Fc region of human antibody IgG1 is more preferred. Randomly choose one of the first Fc variant or the second Fc variant to make a knob mutation and the other to make a hole mutation.
  • antibody 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 Objects) and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments).
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology with the sequence of a human antibody.
  • 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 human source.
  • 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 improve immune cell activity, and ability to enhance immune response.
  • antibody conjugate refers to a conjugate/conjugate formed by chemically bonding an antibody molecule to another molecule directly or through a linker.
  • ADC antibody-drug conjugates
  • the term "monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), and is not limited to the method of producing the antibody.
  • fusion protein refers to the gene recombination method, chemical method or other appropriate method to link the coding regions of two or more genes, and express the protein obtained by gene recombination under the control of the same regulatory sequence product.
  • the coding regions of two or more genes can be fused at one or several positions by a sequence encoding a peptide linker or a connecting peptide.
  • Peptide linkers or connecting peptides can also be used to construct the fusion protein of the present invention.
  • fusion protein of the present invention further includes antibody/Fc fusion protein constructs/complexes, or combinations of antibody/Fc fusion protein constructs/complexes formed by a non-covalent manner, for example, as described in the present invention
  • the fusion protein can be displayed as the following structure:
  • IL-15 fusion protein which is a homodimer comprising two monomers; the monomers comprise antibody heavy chain, antibody light chain, IL-15 and IL-15R ⁇ sushi; for example, the antibody heavy chain Fc is fused with IL-15R ⁇ sushi, and co-expressed with the antibody light chain, and IL-15-WT (wild type) or IL-15 mutant, so that IL-15 and IL-15R ⁇ sushi form a non-covalent connection;
  • IL-15 fusion protein which is a homodimer comprising two monomers; the monomers include antibody heavy chain, antibody light chain, IL-15 and IL-15R ⁇ sushi; for example, the anti- The heavy chain Fc is expressed in tandem with IL-15R ⁇ sushi and IL-15-WT or IL-15 mutant, and co-expressed with the antibody light chain;
  • IL-15 fusion protein which is a homodimer comprising two monomers; the monomers comprise Fc, IL-15 and IL-15R ⁇ sushi; for example, IL-15-WT or IL-15 mutation
  • the body is connected to IL15-R ⁇ sushi through linker, and IL15-R ⁇ sushi is connected to Fc through linker; or,
  • IL-15 fusion protein which is a homodimer comprising two monomers; the monomers include Fc, IL-15 and IL-15R ⁇ sushi; for example, IL15-R ⁇ sushi passes linker and IL-15- WT or IL-15 mutant is connected, and IL-15 is connected to Fc through linker.
  • peptide linker/linker in the present invention refers to a peptide used to connect IL-15 to another protein molecule or protein fragment in the present invention to ensure the correct folding and stability of the protein.
  • the other molecule includes, but is not limited to, IL-15R ⁇ , Fc, Fc variants, antibodies and the like.
  • the "connecting peptide” of the present invention is preferably (GGGGS)n, where n can be 0, 1, 2, 3, 4, 5 or more, preferably n is 2-4; or preferably SGGSGGGGSGGGSGGGGSLQ.
  • connecting peptide sequence is too short, it may affect the folding of the high-level structures of the two proteins, thereby interfering with each other; if the connecting peptide sequence is too long, immunogenicity is also involved, because the connecting peptide sequence itself is a new antigen.
  • heterodimeric protein in the present invention refers to a protein formed by combining two different monomeric proteins.
  • 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.
  • homodimeric protein in the present invention refers to a protein formed by combining two identical monomeric proteins.
  • two identical monomeric proteins each contain an Fc fragment or an Fc variant fragment, and form a homodimer protein through the interaction of the Fc fragment or the Fc variant fragment.
  • the "monomer protein" constituting the heterodimer protein or homodimer protein in the present invention can be a fusion protein or a non-fusion protein.
  • 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.
  • CD279 cluster of differentiation 279
  • the PD-L1 is preferably human PD-L1.
  • anti-programmed death ligand-1 antibody refers to any immunoglobulin molecule containing 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 PD-L1 (CDR) or its ligand binding part, heavy or light chain variable region, heavy or light chain constant region, framework region or any part thereof) protein or peptide-containing molecule.
  • CDR complementarity determining region of a heavy chain or a light chain
  • the PD-L1 antibody also includes an antibody-like protein scaffold (such as the tenth fibronectin type III domain (10Fn3)), which contains BC, DE, and FG structural loops that are similar in structure and solvent accessibility to the antibody CDR.
  • the tertiary structure of the 10Fn3 domain is similar to the tertiary structure of the variable region of the IgG heavy chain. Or substitution of residues in the CDR-H3 region, those skilled in the art can graft, for example, the CDR of the PD-L1 monoclonal antibody onto the fibronectin scaffold.
  • coexpression means that multiple genes are expressed together in a cell and their products appear at the same time. These genes can be present at the same time but controlled expression separately or together. In the present invention, it is preferable to co-express two genes in one eukaryotic cell.
  • the gene expression product obtained by co-expression is conducive to the efficient and simple formation of a complex; in the present invention, it is conducive to the formation of a heterodimer protein or a homodimer protein.
  • percent (%) sequence identity refers to the alignment of sequences and the introduction of gaps (if necessary) in order to achieve the maximum percent sequence identity (for example, for optimal alignment, one of the candidate and reference sequences can be Or after introducing a gap between the two, and for the purpose of comparison, non-homologous sequences can be ignored), the amino acid (or nucleotide) residues of the candidate sequence are the same as the amino acid (or nucleotide) residues of the reference sequence Percentage.
  • percent sequence identity the alignment can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software, such as BLAST, ALIGN or Megalign (DNASTAIi) software.
  • a reference sequence used for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% of the total length of the candidate sequence or a selected portion of consecutive amino acid (or nucleotide) residues of the candidate sequence.
  • 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. .
  • 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 the same at that position.
  • the term "specific binding" in the present invention refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, for example, the protein and other biomolecules are bound by antibodies or their antigens. Fragment specific recognition.
  • the antibody or antigen-binding fragment thereof that specifically binds to the antigen will bind to the antigen with a KD of less than 100 nM.
  • an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD as high as 100 nM (for example, between 1 pM and 100 nM).
  • An antibody or antigen-binding fragment thereof that does not show specific binding to a specific antigen or epitope thereof will show a KD of greater than 100 nM (for example, greater than 500 nM, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM) for the specific antigen or epitope.
  • a variety of immunoassay methods can be used to select antibodies that specifically immunoreact with specific proteins or carbohydrates.
  • solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins or carbohydrates.
  • 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).
  • DNA vectors such as plasmids
  • RNA vectors RNA vectors
  • viruses or other suitable replicons such as viral vectors.
  • Various vectors have been developed for delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells.
  • the expression vector of the present invention contains polynucleotide sequences and, for example, additional sequence elements 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 present invention include plasmids containing regulatory sequences (such as promoter and enhancer regions) that direct gene transcription.
  • kits for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear output of mRNA produced by gene transcription. These sequence elements include, for example, 5'and 3'untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector.
  • the expression vector of the present invention may also contain the following polynucleotide, which encodes a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.
  • subject refers to an organism that receives treatment for a specific disease or condition (such as cancer or infectious disease) as described herein.
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, receiving treatment for diseases or disorders (e.g., cell proliferative disorders, such as cancer or infectious diseases).
  • Guinea pigs, members of the bovid family such as cattle, bison, buffalo, elk and yak, etc.), sheep and horses.
  • treatment refers to surgical or therapeutic treatment, and its purpose is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the subject to be treated, such as cell proliferative disorders (such as cancer or Infectious diseases).
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduced disease severity, stable disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of disease state, and alleviation (whether Partial or complete remission), whether detectable or undetectable.
  • the subjects in need of treatment include those who already have a condition or disease, as well as those who are susceptible to or who intend to prevent the condition or disease.
  • mitigation, mitigation, weakening, mitigation, mitigation, etc. their meaning also includes elimination, disappearance, and non-occurrence.
  • immunological disorders include, for example, pathological inflammation, inflammatory disorders, and autoimmune diseases or diseases.
  • Immunune disorders also refer to infections, persistent infections, and proliferative disorders, such as cancer, tumors, and angiogenesis.
  • Constant conditions include, for example, cancer, cancer cells, tumors, angiogenesis, and precancerous conditions, such as dysplasia.
  • pharmaceutical composition in the present invention refers to a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiological/ Pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • Size Exclusion Chromatograph is a liquid chromatographic technique for separation according to the molecular size of the component cvn to be tested.
  • the surface of the chromatographic column filler is distributed with pores of different sizes. After the sample enters the chromatographic column, different components of them enter the corresponding pore size according to their molecular size. Molecules larger than all pores cannot enter the filler particles.
  • the retention time is short; the molecules smaller than all the pores can freely enter all the pores of the filler surface, and the retention time in the chromatographic column is longer, which is manifested as a longer retention time; the remaining molecules are eluted in order of molecular size.
  • “Optional” or “optionally” means that the event or environment described later can but does not have to occur, and the description includes the occasion where the event or environment occurs or does not occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions may but need not be present; when present, they may be 1, 2, or 3.
  • the step of transforming host cells with recombinant DNA described in the present invention can be performed 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 mediums.
  • the host cell is cultured under conditions suitable for the growth of the host cell.
  • Figure 1 Results of blocking the binding of PD-L1 and PD-1 by humanized PD-L1 antibody, where the positive control is Tecentriq.
  • Figure 2 FACS determination of the ability of humanized PD-L1 antibody to bind to cell-level PD-L1, where the positive control is Avelumab.
  • the Jurkat-PD-1/CHO-PD-L1-NFAT system tests the blocking ability of humanized PD-L1 antibody against PD-L1/PD-1, and the positive control is Avelumab.
  • Figure 4 Humanized anti-PD-L1 antibody promotes the secretion of IFN- ⁇ in the mixed lymphocyte reaction.
  • the negative control is anti-Hel antibody and the positive control is Avelumab.
  • IL-15 wild-type or mutant is first connected with IL-15R ⁇ sushi through linker, and IL-15R ⁇ sushi is then assembled through the tandem fusion of linker and Fc;
  • IL-15R ⁇ sushi is first connected to IL-15 wild-type or mutant through linker, and IL-15 wild-type or mutant is expressed and assembled through linker and Fc in tandem fusion.
  • FIGs 6A-6D IL15 mutant promotes the proliferation of Mo7e cells: the structure of the fusion protein is shown in Figure 5A. Tecentriq is fused with IL-15R ⁇ sushi. The IL15 mutant and IL-15R ⁇ sushi are non-covalently connected without linker connection.
  • Figure 7A The structure of the fusion protein is shown in Figure 5A.
  • Tecentriq is fused with IL-15R ⁇ sushi.
  • the IL15 mutant and IL-15R ⁇ sushi are non-covalently connected, without linker connection;
  • Figure 7B-7G, 7M-7N The structure of the fusion protein is shown in Figure 5B. Self-developed PD-L1 monoclonal antibody is fused with IL-15R ⁇ sushi and IL15 mutant, and the IL15 mutant and IL-15R ⁇ sushi are expressed in tandem through a linker connection; Drug0 It represents a negative control without any protein added to the reaction system;
  • Figure 7H-7K The structure of the fusion protein is shown in Figure 5D.
  • IL-15R ⁇ sushi is first connected to IL-15 wild-type or mutant through linker, and IL-15 wild-type or mutant is expressed and assembled through linker and Fc tandem fusion;
  • Fig. 7L the fusion protein is shown in Fig. 5C, IL-15 wild-type or mutant is first connected with IL-15R ⁇ sushi through linker, and IL-15R ⁇ sushi is then expressed and assembled through linker and Fc tandem fusion.
  • Figure 8A The structure of the fusion protein is shown in 5A.
  • Tecentriq is fused with IL-15R ⁇ sushi.
  • the IL15 mutant and IL-15R ⁇ sushi are non-covalently connected, without 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-15R ⁇ sushi and IL15 mutant, and the IL15 mutant and IL-15R ⁇ sushi are expressed in tandem through a linker connection;
  • Drug0 A negative control that represents the reaction system without adding any protein.
  • FIG. 8G-8J The fusion protein is shown in Figure 5D.
  • IL-15R ⁇ sushi is first connected to IL-15 wild-type or mutant through linker, and IL-15 wild-type or mutant is expressed and assembled through linker and Fc tandem fusion expression.
  • Figure 8K The fusion protein is shown in Figure 5C. IL-15 wild-type or mutant is first connected to IL-15R ⁇ sushi through linker, and IL-15R ⁇ sushi is then expressed and assembled through linker and Fc tandem fusion.
  • Figure 9A-9C The efficacy of hPBMC-A375 mice in vivo.
  • the structure of the fusion protein is shown in Figure 5B.
  • FIGS 10A-10C The efficacy of hPBMC-A375 mice in vivo.
  • the structure of the fusion protein is shown in Figure 5C or 5D.
  • Figure 10D Body weight changes of hPBMC-A375 mice after administration.
  • CDRs transplantation the classic "CDRs transplantation" method is adopted for antibody humanization, that is, the human antibody with the highest homology is selected through sequence to provide antibody framework regions (FRs), and the complementarity determining regions of the antigen-binding fragments of the target antibody based on the Kabat naming method are selected. (CDRs), transplanted to the former to form humanized antibodies.
  • MOE software is used to model and analyze the structure of the antibody based on the following: 1). Select the amino acid residues of the antibody framework that are located at the VH-VL interface, close to or directly interact with CDRs for back mutation, Such amino acid residues are more important to maintain the conformation of the CDRs; 2).
  • 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.
  • ExpiCHO-S cells (Thermo fisher, A29127) were seeded in fresh ExpiCHO Expression medium (Invitech, A29100-01) at a density of 2.5 ⁇ 10 6 to 4 ⁇ 10 6 cells/mL. Incubate overnight in a shaker. On the day of transfection, the ExpiCHO-S cell suspension cultured overnight was counted. The cell viability was greater than 95%, and the density was between 7 ⁇ 10 6 -10 ⁇ 10 6 viable cells/mL. Take the required cell suspension, dilute it with ExpiCHO Expression medium (Invitech, A29100-01) to a density of 6 ⁇ 10 6 cells/mL, and place it on a shaker for later use.
  • the chromatographic medium is protein A filler Mbaselect Sure (GE, 17543803) that interacts with Fc
  • the equilibration buffer is PBS (2.5g/L Na 2 HPO 4 ⁇ 12H 2 O, 0.408g/L NaH 2 PO 4 , 8.76g) /L NaCl, pH7.2)
  • PBS 2.5g/L Na 2 HPO 4 ⁇ 12H 2 O, 0.408g/L NaH 2 PO 4 , 8.76g
  • the flow rate is controlled so that the retention time of the sample on the column is ⁇ 5min.
  • Biacore T200 (GE Healthcare) was used to determine the binding affinity of PD-L1 antibody to human and cynomolgus PD-L1-His protein.
  • Immobilize anti-human IgG Fc (Genway, Cat.GWB-20A705) on a CM5 chip (GE Healthcare, Cat.BR-1005-30) at 25°C.
  • a multi-cycle kinetics method is used to determine the affinity between the antibody and the antigen at 25°C.
  • the antibody to be tested is first captured on the fixed 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 is HBS-EP+Buffer (GE Healthcare, Cat.BR-1006-69), the flow rate is 30 ⁇ L/min, and the binding time is 300 seconds.
  • the regeneration flow rate is 30 ⁇ L/min, and the time is 30 seconds.
  • Biacore T200 Evaluation Software version 3.0
  • the tested PD-L1 antibodies have an affinity of nM or higher for the binding of human PD-L1 recombinant protein and cynomolgus PD-L1 recombinant protein, as shown in Table 2 below.
  • the IC50 of anti-PD-L1 antibody blocking the binding of PD-L1 protein and PD-1 protein was determined by competitive ELISA method. Dilute the human PD-L1 recombinant protein (Sino Biological, Cat.10084-H05H) with carbonate buffer and add it to a 96-well microtiter plate with a final concentration of 1 ⁇ g/ml.
  • Block with 3% BSA in PBS solution 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 the OD value after 1M sulfuric acid termination (dual wavelength 450nm-630nm). Corresponding to the antibody concentration and the OD value, the competitive binding curve of the test antibody can be drawn, and the IC50 value can be calculated.
  • Figure 1 shows the competitive binding curve of anti-PD-L1 antibody and human PD-L1 recombinant protein.
  • the results show that the tested 794-h1-71 antibody can effectively block the interaction between human PD-L1 protein and human PD-1 protein, with an IC50 of 0.8488nM, and a positive control Tecentriq (Genetech, lot:H0172) of 0.8486nM .
  • the concentration gradient of antibody to be detected (antibody concentration: 10000ng / ml-0.1ng / ml ) to cell surface PD-L1 expression in CHO-PD-L1 cells (Nanjing Yong Hill Biotechnology Co., Ltd., 10 5 / well) , Incubate together for 30 min at 4°C. After incubation, add 1:250 diluted anti-human IgG PE fluorescent antibody (eBioscience, Cat.12-4998-8), and incubate for 30 minutes at 4°C. The fluorescent antibody will specifically bind to the Fc section of the antibody to be detected.
  • the fluorescence intensity of PE is detected by FACS and the ability of the antibody to be detected to bind to the PD-L1 protein highly expressed on the cell surface is analyzed.
  • Figure 2 shows that the EC50 of the 794-h1-71 antibody is 38.44ng/ml, which is similar to the positive control Avelumab (EC50 of ⁇ 72ng/ml) in this experiment.
  • Avelumab EC50 of ⁇ 72ng/ml
  • the PD-1 cell line and the PD-L1 antibody to be tested are incubated together.
  • the sample amount of PD-1 cells is 16000/well, and the antibody is diluted in a gradient. Each dose is 3 multiple wells, and the incubation volume is 100 ⁇ l/well.
  • the incubation time is 6 hours. When the incubation is completed, remove the well plate, add an equal volume (100 ⁇ l) to the luminescence detection reagent, and read the value.
  • FIG. 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.
  • Example 7 ELISA detection of IFN- ⁇ secreted by T cells in the mixed lymphocyte reaction
  • the PD-L1 monoclonal antibody enhances the activity of T cells through the 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 (DC) in vitro, and LPS (Sigma, Cat: L4516) was added on the 6th day of culture. ) Stimulate mature DC.
  • PBMC peripheral blood mononuclear cells
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • rhIL-4 recombinant human interleukin 4
  • DC cells from donor 1 and CD4 + T cells enriched from PBMC from healthy donor 2 are mixed and co-cultured.
  • the ratio of DC:CD4 + T cells is 1:10, and the test is added Antibody, negative control antibody anti-Hel (Bioin Biosynthesis) and positive control antibody Avelumab (antibody concentration: 7nM-0.28nM), cultured for 4 days. After 4 days, the cell culture supernatant was collected, and the content of IFN- ⁇ in the supernatant was detected by ELISA.
  • 794-h1-71 and the positive control antibody Avelumab can significantly enhance the ability of CD4 + T cells to secrete IFN- ⁇ in the MLR experiment compared with the anti-Hel monoclonal antibody negative control group.
  • concentration of antibody drug decreases, the activity to increase the secretion of IFN- ⁇ also decreases.
  • the results indicate that the 794-h1-71 antibody can enhance the function of T cells and is dose-dependent (T-test, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.).
  • MOE software to simulate the key amino acid sites of human IL-15 interaction with the corresponding receptor ⁇ chain. They are D8 and V3, I6, and H105 respectively. According to MOE software simulation, the following IL-15 mutant sequence was designed and synthesized. The amino acid sequence is shown in Table 3, and the coding nucleic acid sequence is shown in Table 4.
  • IL-15 fusion protein antibody/Fc fusion construct/complex
  • IL-15 fusion protein which is a homodimer comprising two monomers; the monomers include antibody heavy chain, antibody light chain, IL-15 and IL-15R ⁇ sushi ; Fusion of the Fc end of the antibody heavy chain with IL-15R ⁇ sushi, and co-expression with the monoclonal antibody light chain, and IL-15-WT (wild type) or IL-15 mutant, so that IL-15 and IL-15R ⁇ sushi form non-covalent connect;
  • IL-15 fusion protein which is a homodimer comprising two monomers; the monomers include antibody heavy chain, antibody light chain, IL-15 and IL-15R ⁇ sushi ; Through linker, the Fc end of the antibody heavy chain is fused with IL-15R ⁇ sushi and IL-15-WT or IL-15 mutant three parts in sequence and expressed in tandem, and co-expressed with the antibody light chain;
  • IL-15 fusion protein which is a homodimer comprising two monomers; the monomers comprise Fc, IL-15 and IL-15R ⁇ sushi; IL-15-WT or IL-15 mutant is connected to IL15-R ⁇ sushi through linker, and IL15-R ⁇ sushi is connected to Fc through linker;
  • IL-15 fusion protein which is a homodimer comprising two monomers; the monomers comprise Fc, IL-15 and IL-15R ⁇ sushi; IL15-R ⁇ sushi is connected to IL-15-WT or IL-15 mutant through linker, and IL-15 is connected to Fc through linker.
  • the numbering rules of PD-L1 antibody and IL-15 fusion protein are as follows: "Antibody code name-IL-15 (wild type/mutant)-fusion protein construction mode";
  • T-IL15-xx-1 means Tecentriq
  • IL15-xx means IL15-WT or IL15 mutant
  • 1 means the structure pattern shown in Figure 5A;
  • T-IL15-xx-1 means Tecentriq
  • IL15-xx means IL15-WT or IL15 mutant
  • 1 means the structure pattern shown in Figure 5A;
  • PD-L1-IL-15 mutant fusion protein numbering rules are as follows:
  • T-IL15-xx-1 "T", Tecentriq;
  • IL15-xx means IL15-WT or IL15 mutant;
  • 1 means the structure pattern shown in Figure 5A;
  • 794-IL15-xx-2 "794", 794-h1-71 monoclonal antibody
  • IL15-xx means IL15-WT or IL15 mutant
  • 2 means the structure shown in Figure 5B model.
  • ExpiCHO expression system for transient protein expression.
  • the host cells ExpiCHO-S (Cat no. A29127) passaged on ExpiCHOTM Expression Medium (Cat no. A2910001) were diluted to a suitable density, and placed on a shaker (100 rpm, 37° C., 8% CO 2 ) for transfection.
  • the vector carrying the nucleic acid sequence encoding the fusion protein is added to the OptiPRO TM SFM (Cat no. 12309019) medium, so that the final concentration of the vector is 0.5-1.0 ⁇ g/mL.
  • ExpiFectamine TM CHO Reagent Cat no.A29129
  • OptiPRO TM SFM medium containing DNA to form a DNA-ExpiFectamine TM CHO Reagent complex. After standing at room temperature for 1 to 5 minutes, slowly drip the complex into the transfected medium. Cell suspension. On the first day after transfection, a certain amount of ExpiFectamine TM CHO Enhancer (Cat no. A29129) and ExpiCHO TM Feed (Cat no. A29129) were added and the temperature was lowered to 32°C for culture. On the 4th to 6th day after transfection, a certain amount of ExpiCHO TM Feed (Cat no.A29129) was added. From 10 to 12 days after transfection, the supernatant was harvested by centrifugation at 5000 g for 30 min.
  • 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 discarding the supernatant, wash the magnetic beads 3 times with PBS, and finally add pH3.0 citrate to elute the IL-15 fusion protein. After neutralizing the sample with 1M Tris-Hcl, measure the protein concentration with NanoDrop One.
  • Example 11 Determination of the purity of IL-15 fusion protein by size exclusion chromatography
  • a TSKgel G3000SWXL chromatographic column (TOSOH, 0008541) and a pre-column Tskgel guard columnSWXL (TOSOH, 0008543) are used to perform size exclusion chromatography (SEC) to determine the purity of the IL-15 mutant fusion protein of the present invention.
  • the mobile phase 50 mM PB, 300 mM NaCl, pH 6.8, equilibrated the column with a flow rate of 1 mL/min. UV detection wavelength is 280nm. The results are shown in Table 7.
  • Example 12 IL-15 mutant fusion protein induces Mo7e cell proliferation experiment
  • Mo7e is a human megakaryocyte 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 10 5 cells/ml, add 50 ⁇ l mutant culture medium and 50 ⁇ l Mo7e cell suspension to a 96-well U bottom plate, mix well and incubate at 37°C with 5% CO 2. After 72h, the culture plate was taken out, and 100 ⁇ l Cell Titer Glo luminescent cell viability detection reagent (Promega, G7571) was added to detect the luminescence fluorescence intensity, which is proportional to the cell proliferation ability.
  • T-IL15-10-1 and T-IL15-10-1 The EC50 of IL15-26-1 was 28076pM and 290.9pM, respectively, and the corresponding EC50 of T-IL15-WT-1 was 143.3pM (Figure 6B).
  • the activity of T-IL15-29-1, T-IL15-42-1 mutant was lower than that of T-IL15-WT-1, EC50 was 285.2pM and 194.5pM, T-IL15-43-1 mutant Compared with T-IL15-WT-1, the activity was enhanced, with an EC50 of 103.2pM, and an EC50 of T-IL15-WT-1 of 150.7pM (Figure 6C).
  • T-IL15-com1-1 and T-IL15-com2-1 are lower than that of T-IL15-WT-1;
  • Tecentriq control group has no effect on the proliferation of Mo7e cells, that is, anti-PD-L1 antibody has no effect on Mo7e cells.
  • induced proliferative activity (FIG. 6D), on the proliferative activity of IL-15 is produced by the.
  • Example 13 IL15 mutant fusion protein induces CD8+ T cell proliferation
  • this example uses Ki67 as a proliferation marker, and different concentrations of IL-15 mutant fusion protein were tested The proliferation ratio of CD8+ T cells after stimulating human PBMC cells for three days.
  • RPMI1640 medium Gibco, Cat: 72400047
  • FBS Gibco, Cat: 10099141
  • penicillin-streptomycin Gibco, Cat: 15140122
  • the IL-15 mutant fusion protein was started from 500 nM, diluted by 4 times, a total of 11 gradients, and then 100 ⁇ l/well of each concentration of the sample was added to the 96-well U bottom plate that had been added to human PBMC, and the mixture was evenly mixed. Put it in a 37°C, 5% CO 2 incubator for three days.
  • the cultured cells use 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 then flow cytometry was used to detect the proportion of Ki67+ cells in CD3+CD8+ T cells stimulated with different concentrations of IL-15 mutant fusion protein.
  • Figure 7A-7N shows the gradient dilution of T-IL15-7-1, T-IL15-8-1, T-IL15-9-1, T-IL15-10-1, T-IL15-11-1, T-IL15-11-1, -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.
  • T-IL15-8-1, T-IL15-9-1, and T-IL15-11-1 significantly reduce the proliferation ability of CD8+ T cells.
  • T-IL15-WT-1, 794-IL15-WT-2 are wild-type IL-15 controls, P22339 is mutant IL-15 control; 794-h1-71 monoclonal antibody is anti-PD-L1 antibody control; Drug0 is Negative control without adding any protein.
  • the IL15 mutant had weaker proliferation of CD8+ T cells.
  • 794-h1-71 control mAb on CD8 + T cells had no effect on proliferation, i.e. an anti-PD-L1 antibodies without induction of CD8 + T cell proliferation (FIG. 7B), on cell proliferation activity of IL-15 is produced by Of .
  • Example 14 IL15 mutant fusion protein induces NK cell proliferation
  • Ki67 was used as a proliferation marker, and different concentrations of IL-15 mutant fusion protein stimulated human PBMC cell proliferation ratio of NK cells after three days.
  • RPMI1640 medium Gibco, Cat: 72400047
  • FBS Gibco, Cat: 10099141
  • penicillin-streptomycin Gibco, Cat: 15140122
  • the IL-15 mutant fusion protein was started from a concentration of 500 nM, and was diluted 4-fold with a total of 11 gradients. Subsequently, 100 ⁇ l/well of each gradient sample was added to the 96-well U bottom plate that had been added with human PBMC, and after the mixture was uniformly mixed, it was placed in a 37° C., 5% CO 2 incubator for three days.
  • the cultured cells use LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen, Cat: L34964), CD3-AF700 (BD, Cat: 557943), CD56-PE (BD, Cat: 555516), APC-Ki67 (Biolegend, Cat: 350514) antibody was stained, and then the ratio of Ki67+ cells in NK cells after stimulation with different concentrations of IL-15 mutant fusion protein was detected by Invitrogen Attune NxT flow cytometer.
  • Figure 8A-8M shows the gradient dilution of T-IL15-7-1, T-IL15-8-1, T-IL15-9-1, T-IL15-10-1, T-IL15-11-1, T-IL15-11-1, -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 CD3-CD56+ NK cell proliferation ratio, and the EC50 value of each mutant stimulated proliferation .
  • T-IL15-8-1, T-IL15-9-1, T-IL15-11-1, V9-IL15-com6, V9-IL15-62, and V9-IL15-63 have significant effects on the proliferation of NK cells Decrease, before reaching the plateau, the EC50 value fitting is not accurate; but the above-mentioned mutant combination has a significantly lower proliferation activity on NK cells than the control group T-IL15-WT-1 or P22339.
  • T-IL15-WT-1, 794-IL15-WT-2 are wild-type IL-15 controls, P22339 is mutant IL-15 control; 794-h1-71 monoclonal antibody is anti-PD-L1 antibody control; Drug0 is Negative control without adding any protein.
  • the IL-15 mutant Compared with the wild-type IL-15 control, the IL-15 mutant had weaker proliferation of NK cells. 794-h1-71 mAb controls had no effect on the proliferation of NK cells, i.e., anti-PD-L1 antibodies without induction of NK cell proliferation (FIG. 8B), on the cell proliferative activity of IL-15 is produced by the.
  • Example 15 In vivo efficacy of humanized anti-PD-L1 antibody-IL15 bifunctional molecule/fusion protein in mice
  • mice were randomly divided into PBS group and Tecentriq group (10 mg /kg), 794-IL15-WT-2 group (1mg/kg), 794-IL15-com6-2 group (4mg/kg), a total of 4 groups, each with 8 animals, a total of 32 animals.
  • the mode of administration is intraperitoneal administration, according to the groups 794-IL15-WT-2 group (1mg/kg) and 794-IL15-com6-2 group (4mg/kg), once a week, a total of 1 administration ; PBS group and Tecentriq group (10mg/kg) were administered twice a week for a total of 5 administrations.
  • the tumor volume was measured 3 times a week and the data was recorded.
  • TGI TV (%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100%; Ti: mean tumor volume of the treatment group on day i, T0: mean tumor volume of the treatment group on day 0; Vi: mean tumor volume of the solvent control group on day i, V0: the mean tumor volume of the solvent control group on day 0 of administration).
  • the administration group had a significant inhibitory effect on tumor volume, with statistical difference (P ⁇ 0.05), and the 794-IL15-WT-2 group and the 794-IL15-com6-2 group Compared with the Tecentriq group, it has a significant inhibitory effect on tumor volume (P ⁇ 0.05). See Figures 9A-9C, Table 8.
  • a Mean ⁇ standard error
  • b The tumor volume of the drug treatment group and the vehicle control group (PBS) tumor volume were statistically compared on the 14th day of group drug administration. Two-way ANOVA analysis, *P ⁇ 0.05,* *P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001
  • c The tumor volume of the administration group and the positive drug Tecentriq group were statistically compared on the 14th day of group administration, Two-way ANOVA Analysis, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • the humanized anti-PD-L1 antibody-IL15 bifunctional molecule/fusion protein 794-IL15-WT-2 and 794-IL15-com6-2 both have a significant inhibitory effect on the growth of A375 tumor subcutaneously transplanted tumors (P ⁇ 0.0001 ); Compared with the Tecentriq group, the 794-IL15-WT-2 group and the 794-IL15-com6-2 group have a stronger inhibitory effect on tumor volume, and there is a significant difference (P ⁇ 0.05).
  • mice were randomly divided into PBS groups according to the tumor volume, ALT803( 0.2mg/kg), V9-IL15-61(1mg/kg), V9-IL15-61(5mg/kg), V9-IL15-com6(1mg/kg), V9-IL15-com6(5mg/kg), There were 7 groups of V5-IL15-WT (2mg/kg), 8 rats in each group, 56 rats in total.
  • the mode of administration is intraperitoneal administration, which is administered once a week according to the group, for a total of 3 administrations; the tumor volume is measured 3 times a week and the data is recorded.
  • Tu mean tumor volume of the treatment group on day i
  • T0 mean tumor volume of the treatment group on day 0
  • Vi mean tumor volume of the solvent control group on day i
  • V0 the mean tumor volume of the solvent control group on day 0 of administration
  • a Mean ⁇ standard error
  • b The tumor volume of the treatment group and the vehicle control group were statistically compared on the 14th day of group dosing. Two-way ANOVA analysis, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001
  • c Identify outliers analysis of tumor volume on Day 21.
  • One of the data in the V9-IL15-com6 5mpk group is abnormally large, so this data is eliminated.
  • a Mean ⁇ standard error
  • b The weight of the administration group and the vehicle control group were statistically compared on the 21st day of group administration, and Two-way ANOVA analysis.
  • a Mean ⁇ standard error
  • b Mice died after group administration, 1 means 1 mouse died on the same day, 0 means 1 mouse died on the same day, but anemia, Symptoms such as jaundice.

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Abstract

提供人IL-15分子的突变体和含有IL-15突变体及组合突变的融合蛋白，所述融合蛋白能介导免疫细胞的激活扩增，可用于肿瘤疾病治疗的用途。

Description

人IL-15突变体及其用途 技术领域
本发明涉及人IL-15突变体，编码其的核酸，含有IL-15突变体及组合突变的融合蛋白，药物组合物，以及药物组合物用于治疗肿瘤的相关用途。
背景技术
白细胞介素15(interleukin-15，IL-15)是1994年由Grabstein在检测猴肾皮内细胞株CV-1/EBNA的培养上清中发现并命名的重要可溶性细胞因子。多种细胞和组织可表达IL-15，例如，单核/巨噬细胞，淋巴细胞，上皮细胞等。
IL-15生物学活性
IL-15介导的信号通路
白介素15受体(IL15R)：IL15R属于造血因子超家族,由α、β(又称CD122)、γ(又称CD132，common gamma chain，γc)3个亚基构成。IL2和IL15共用β链受体。IL2，IL4，IL7，IL9，IL15以及IL21共用γ链受体。IL2和IL15的受体共用β和γc链，但IL2和IL15均有其各自特异性α受体链。人IL15Rα属于I型跨膜蛋白， IL2Rα和IL15Rα都有一个保守 的蛋白结合基序(sushi domain)。IL15与IL15Rα亲和力比较高(Kd～10 -11M)，但不传递信号。IL15与IL15βγ异源二聚体的亲和力比较中等(Kd～10 -9M)，可传导信号；IL15与IL15αβγ异源三聚体亲和力与前者相似(Kd～10 -9M)，可传导信号。由于IL2和IL15的受体共用β和γ链，因此，IL2和IL15之间有许多相似的生物学功能，如都能促进T细胞，NK细胞增殖。
IL15与受体的结合：IL15Rα主要表达于DC和单核细胞。在大多数情况下,IL15/IL15Rα以trans-presented的形式与受体结合。即IL-15和IL-15Ra在相同细胞中表达后，在胞内IL-15与IL-15Rα的sushi结构域以高亲和力结合后转运至膜表面，然后与其反应性细胞(如T细胞或NK细胞)膜表面的βγ异源二聚体复合物或αβγ异源三聚体复合物结合，β和γ受体可分别活化下游Jak1和Jak3，导致STAT-3和STAT-5活化，激活级联反应，诱导特异性基因表达。IL15以自分泌的形式作用于效应细胞时，可以cis形式与IL15受体作用，活化下游信号产生效应功能。
IL-15的免疫调节作用
IL-15具有广泛的免疫调节作用，参与调节多种免疫细胞的活性、增殖与功能。(1)对T细胞的调节作用：促进T细胞的活化增殖，促进记忆性CD8 +T细胞的产生，而且在维持体内记忆性CD8 +T细胞的数目上也起着重要的作用，即使在Treg细胞存在的情况下，IL-15也可以很好的维持CD8 +T细胞的功能和数量。(2)对NK细胞的调节作用：IL-15对NK细胞的活化与增殖起着重要作用，可以提高NK细胞的ADCC杀伤能力。(3)对其他免疫细胞的调节作用：IL-15在DC细胞及巨噬细胞的功能成熟中也起着重要作用。IL-15可以促进DC细胞表达共刺激因子及IFN-γ，提高DC细胞活化CD8 +T细胞及NK细胞的能力。此外，IL-15可以促进中性粒细胞的增殖。
IL-15的抗肿瘤作用
IL-15基于其对多种免疫细胞具备扩增激活的功能而实现抗肿瘤作用，临床上已有研究证 实，IL-15具有出色的抗肿瘤疗效。但由于野生型IL-15半衰期短，且其分子小，肾脏清除率高，每天多次注射给药，或皮下给药在使用方法上极其不便。因此，单纯应用野生型重组IL-15同样在肿瘤治疗上受限。
有研究表明，降低IL-15对T细胞，NK细胞增殖的活性，可以增加IL-15的半衰期，同时降低毒性。此外，IL-15和靶向肿瘤相关抗原的抗体组合成融合蛋白，可以增加IL-15的特异性，提高IL-15在肿瘤微环境的浓度，并降低IL-15的毒性。因此，开发具有降低活性的IL-15突变体，有潜力提高IL-15治疗肿瘤的量效关系，拓展IL-15临床抗肿瘤应用，具有重大的社会和经济意义。
发明内容
本发明提供IL-15突变体，编码所述突变体的核酸，包含所述突变体的融合蛋白和药物组合物，以及它们用于杀伤肿瘤细胞的功能，用于治疗肿瘤的用途。
在第一个方面中，本发明公开提供了一种IL-15突变体多肽，所述多肽包含对应于野生型IL-15的Val3、Ile6、Asp8或His105的一个或多个氨基酸残基处的突变。
在第二个方面中，本发明公开提供了一种包含IL-15突变体的多肽，所述多肽包含对应于野生型IL-15的Val3、Ile6、Asp8或His105的一个或多个氨基酸残基处的突变。
在一个实施方案中，IL-15突变体多肽至少包含Val3、Ile6、Asp8或His105中的两个、三个、或四个氨基酸残基处的突变。
在一个实施方案中，所述突变是替换、插入或缺失。
在一个具体实施方案中，所述突变选自以下组的氨基酸替换：Val3Leu(V3L)、Ile6Asp(I6D)、Ile6Pro(I6P)、Asp8Glu(D8E)、Asp8Gln(D8Q)、Asp8Arg(D8R)、Asp8Ser(D8S)、Asp8Val(D8V)、Asp8Gly(D8G)、Asp8Ile(D8I)、Asp8Leu(D8L)、Asp8Thr(D8T)、His105Asn(H105N)、和/或His105Lys(H105K)。
在一个具体实施方案中，所述IL-15突变体多肽或包含IL-15突变体的多肽包含下述突变或突变组合：(1)Asp8Glu；(2)Asp8Gln；(3)Asp8Arg；(4)Asp8Ser；(5)Asp8Val；(6)Val3Leu；(7)Ile6Asp；(8)His105Lys；(9)His105Asn；(10)Asp8Gly；(11)Asp8Ile；(12)Asp8Leu；(13)Ile6Pro；(14)Asp8Thr；(15)Asp8Glu和Val3Leu；(16)Asp8Glu和Ile6Asp；(17)Val3Leu和Ile6Asp；(18)Ile6Asp和His105Lys；(19)Asp8Ser和His105Lys；(20)Asp8Ser和His105Asn；或，(21)Val3Leu、Ile6Asp和His105Lys。
在一个具体实施方案中，所述IL-15突变体与人野生型IL-15具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列一致性。
在一个具体实施方案中，所述IL-15突变体的氨基酸序列如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或SEQ ID NO.47所示。
在一个具体实施方案中，所述IL-15突变体多肽或包含IL-15突变体的多肽具有如下特性：(1)介导人CD8 +T细胞的增殖；(2)介导人NK细胞的增殖；和/或，(3)抑制肿瘤生长。
在一个具体实施方案中，所述IL-15突变体多肽或包含IL-15突变体的多肽介导CD8+T 和/或NK细胞增殖/扩增的活性低于包含野生型IL-15的多肽。
在一个具体实施方案中，所述野生型IL-15的氨基酸序列如SEQ ID NO.1所示。
在第三个方面中，本发明公开提供了一种蛋白，所述蛋白包含前述IL-15突变体多肽或包含IL-15突变体的多肽；还包含与所述IL-15突变体融合的免疫球蛋白分子或其部分，和/或IL-15Rα。
在一个实施方案中，所述免疫球蛋白分子为抗体或抗原结合片段；所述免疫球蛋白分子部分为免疫球蛋白Fc区。
在一个实施方案中，所述抗体或抗原结合片段选自：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或，(3)全人源抗体或其片段。
在一个具体实施方案中，所述抗体或抗原结合片段选自F(ab)2、Fab’、Fab、Fv、scFv、双特异抗体、纳米抗体和抗体最小识别单位中的一种或多种。
在一个具体实施方案中，所述免疫球蛋白Fc区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一的Fc区；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列；优选的，所述免疫球蛋白Fc区的氨基酸序列如SEQ ID NO.73所示。
在另一个实施方案中，IL-15突变体通过或不通过连接肽与免疫球蛋白分子或其部分融合，或IL-15突变体通过或不通过连接肽与IL-15Rα融合；优选使用连接肽；优选使用SEQ ID NO.65、SEQ ID NO.67、SEQ ID NO.69或SEQ ID NO.71所示连接肽。
在另一个实施方案中，IL-15突变体通过或不通过连接肽与IL-15Rα融合，再与免疫球蛋白分子或其部分融合；优选使用连接肽；优选使用SEQ ID NO.65、SEQ ID NO.69或SEQ ID NO.71所示连接肽。
在一个具体实施方案中，各结构域的连接顺序自N端至C端为：
(1)免疫球蛋白分子或其部分、IL-15Rα、IL-15突变体；
(2)免疫球蛋白分子或其部分、IL-15突变体、IL-15Rα；
(3)IL-15突变体、IL-15Rα、免疫球蛋白分子或其部分；
(4)IL-15Rα、IL-15突变体、免疫球蛋白分子或其部分；
(5)IL-15突变体、免疫球蛋白分子或其部分；
(6)免疫球蛋白分子或其部分、IL-15Rα；
(7)IL-15Rα、免疫球蛋白分子或其部分；
(8)IL-15突变体、IL-15Rα；或，
(9)IL-15Rα、IL-15突变体。
在另一个实施方案中，当IL-15Rα或IL-15突变体与免疫球蛋白分子融合时，融合于免疫球蛋白分子重链可变区的N端或免疫球蛋白Fc区的C端；当IL-15Rα或IL-15突变体与免疫球蛋白Fc区融合时，融合于免疫球蛋白Fc区的N端或C端。
在第四个方面中，本发明公开提供了一种蛋白或抗体融合构建体/复合物，其包含如下4个部分：
(1)免疫球蛋白重链；
(2)免疫球蛋白轻链；
(3)IL-15Rα；和，
(4)如前第一、第二方面中所述IL-15突变体多肽。
在一个实施方案中，IL-15Rα通过或不通过连接肽融合于免疫球蛋白重链可变区的N端或免疫球蛋白Fc区的C端。
在一个实施方案中，IL-15突变体多肽与IL-15Rα非共价连接，或IL-15突变体通过或不通过连接肽与IL-15Rα另一端融合。
在一个具体实施方案中，所述蛋白是包含由(1)-(4)部分所构成的单体的同源二聚体。
在第五个方面中，本发明公开提供了一种蛋白或Fc融合构建体，其包含如下3个部分：
(1)免疫球蛋白Fc区；
(2)IL-15Rα；和，
(3)如前第一、第二方面中所述IL-15突变体多肽。
在一个实施方案中，IL-15Rα通过或不通过连接肽融合于IL-15突变体多肽的N端或C端，再通过或不通过连接肽融合于免疫球蛋白Fc区的N端或C端。
在一个具体实施方案中，所述蛋白是包含由(1)-(3)部分所构成的单体的同源二聚体。
在另一个优选实施方案中，所述免疫球蛋白选自抗PD-L1抗体；所述抗PD-L1抗体优选Tecentriq、KN-035、794-h1-71。
在一个具体实施方案中，抗PD-L1抗体包含重链可变区和轻链可变区，所述重链可变区和轻链可变区分别具有SEQ ID NO:99和SEQ ID NO:100所示序列，或者与SEQ ID NO:99和SEQ ID NO:100所示序列相比具有至少80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；或，
所述重链可变区和轻链可变区分别具有SEQ ID NO:97和SEQ ID NO:98所示序列，或者与SEQ ID NO:97和SEQ ID NO:98所示序列相比具有至少80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列。
在另一个优选实施方案中，所述IL-15Rα选自IL-15Rα-sushi；优选IL-15Rα-sushi的氨基酸序列如SEQ ID NO.49、SEQ ID NO.51、SEQ ID NO.53、或SEQ ID NO.55所示。
在第六个方面中，本发明公开提供了一种特异性结合PD-L1的抗体或抗原结合片段，所述抗PD-L1抗体或抗原结合片段包含重链可变区和轻链可变区；优选的，所述重链可变区和轻链可变区分别具有SEQ ID NO:99和SEQ ID NO:100所示序列，或者与SEQ ID NO:99和SEQ ID NO:100所示序列相比具有至少80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列。
在一个具体实施方案中，所述的抗体或抗原结合片段与人程序性死亡配体-1(PD-L1)结合的解离常数(KD)不大于1.8×10 -9M，与食蟹猴程序性死亡配体-1(PD-L1)结合的解离常数(KD)不大于9.4×10 -10M；
或，可选地，所述抗体或抗原结合片段与猴PD-L1结合或不结合；
可选地，所述抗体或抗原结合片段与鼠PD-L1结合或不结合。
在一个具体实施方案中，所述抗PD-L1抗体竞争性地结合PD-L1或其抗原表位，并且具备以下特性：
(1)特异性结合PD-L1重组蛋白及表达PD-L1的细胞；
(2)阻断PD-L1与PD-1蛋白的结合；
(3)抑制PD-1与细胞表面表达的PD-L1的结合；
(4)增强T细胞活性；或/和
(5)抑制肿瘤生长。
在一个优选实施方案中，所述抗PD-L1抗体包含选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一的恒定区；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
在另一个优选实施方案中，所述PD-L1抗体选自F(ab)2、Fab’、Fab、Fv、scFv、双特异抗体中的一种或多种。
在第七个方面中，本发明公开提供了一种分离的核酸分子，其编码前述第一至第六方面任一项所述的多肽、蛋白、抗原或抗原结合片段。
在第八个方面中，本发明公开提供了一种表达载体，所述表达载体包含前述第七方面中分离的核酸分子。
在第九个方面中，本发明公开提供了一种宿主细胞，所述宿主细胞包含前述第七方面中的分离的核酸分子、或前述第八方面中所述的表达载体；优选，所述宿主细胞是真核细胞或原核细胞；更优选，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选，所述宿主细胞选自中国仓鼠卵巢细胞(CHO)。
在第十个方面中，本发明提供了一种多肽或蛋白的制备方法，在适当的条件下培养第九方面中所述的宿主细胞，并分离所述多肽或蛋白。
在第十一个方面中，本发明公开提供了一种药物组合物，所述组合物包含前述第一至第六方面任一项所述的多肽、蛋白、抗原或抗原结合片段、前述第七方面中的分离的核酸分子、前述第八方面中所述的表达载体、第九方面所述的细胞，或第十方面中所述方法制备的产品；以及药学上可接受的载体；优选，所述药物组合物还包含额外的抗肿瘤剂。
在第十二个方面中，本发明公开提供了前述第一至第六方面任一项所述的多肽、蛋白、抗原或抗原结合片段、前述第七方面中的分离的核酸分子、前述第八方面中所述的表达载体、第九方面所述的细胞，或第十方面中所述方法制备的产品、或第十一方面所述药物组合物在制备预防和/或治疗个体中的疾病的药物中的用途；所述疾病优选为肿瘤。
在第十三个方面中，本发明提供了一种预防和/或治疗个体中的疾病的方法，包含向有此需要的患者施用前述第一至第六方面任一项所述的多肽、蛋白、抗原或抗原结合片段、前述第七方面中的分离的核酸分子、前述第八方面中所述的表达载体、第九方面所述的细胞，第十方面中所述方法制备的产品、或第十一方面所述药物组合物；所述疾病优选肿瘤。
术语定义和说明
除非另外说明，本文所用术语具有所属技术领域普通技术人员通常理解的含义。对于本文中明确定义的术语，则该术语的含义以所述定义为准。
本发明中术语“IL-15”或“IL15”，白细胞介素15(interleukin-15，IL-15)是一种多效性细胞因子，具有激活T细胞、B细胞和NK细胞，并可介导这些细胞的增殖和存活的功能。此外，IL-15能激活、维持和扩增CD8 +记忆性T细胞。本发明所述的“IL-15”或“IL-15肽段”或“IL-15多肽”可以是任何IL-15(白介素15)或其突变体，如人IL-15或非人哺乳动物IL-15或非哺乳动物IL-15。示例性非人哺乳动物如猪、兔、猴、猩猩、鼠等，非哺乳动物如鸡等。优选人的白介素15成熟分子，见数据库UniProtKB，登录号P40933，49-162aa。
本发明中术语“IL-15野生型”或“野生型IL-15”是指，天然来源的人IL-15或非人哺乳动物IL-15或非哺乳动物IL-15；也可以指代本领域已经通用的IL-15多肽。
本发明中术语“IL-15突变体”是指，通过一个或多个氨基酸替换、增加或者缺失突变获得的对IL-15与其受体间亲合力提高或者降低，或其刺激特定细胞系，T细胞或者NK细胞增殖活性、细胞因子释放的活性增加或者降低的突变体分子。
本发明中术语“IL-15Rα”可以是任何物种的IL-15Rα或者其功能性片段，如人IL-15Rα或非人哺乳动物IL-15Rα或非哺乳动物IL-15Rα。示例性非人哺乳动物如猪、兔、猴、猩猩、鼠等，非哺乳动物如鸡等。优选人的IL-15Rα；优选人的白介素15受体α胞外域片段，简称IL-15RαECD；优选IL-15Rα-sushi详见表1。
本发明中术语“IL-15Rα变体”指在IL-15Rα上通过一个或者多个氨基酸缺失、插入或替换突变形成的具有与其配体分子如IL15结合能力的功能性突变体，优选人的IL15Rα分子，更优选人的IL-15Rα胞外域片段的缩短形式，即从胞外域片段C端开始通过一个或多个氨基酸缺失突变所得的具有人白介素15受体α活性的分子，优选保留65-120个氨基酸的缺失突变形式，更优选保留65-102个氨基酸的缺失突变缩短形式，比如IL-15Rα-sushi；优选IL-15Rα-sushi，详见表3。
本发明中术语“免疫球蛋白Fc区”是指，免疫球蛋白链恒定区，特别是免疫球蛋白重链恒定区的羧基端或其中的一部分，无抗原结合活性，是抗体分子与效应分子和细胞相互作用的部位。本发明所述“免疫球蛋白Fc区”可以是任何Fc或其变体，来源于人或非人哺乳动物。例如，免疫球蛋白Fc区可包括重链CH1、CH2、CH3、CH4的两个或更多结构域与免疫球蛋白铰链区的组合。Fc可以来源于不同的种属，优选人的免疫球蛋白。根据重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类，主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM。其中一些还可进一步分成亚类(同种型)，如IgG-1、IgG-2、IgG-3、IgG-4；IgA-1和IgA-2。“Fc区”优选包括至少一个免疫球蛋白绞链区，以及IgG的CH2和CH3结构域。更优选包括IgG1的一个CH2结构域，一个CH3结构域和一个免疫球蛋白绞链区，铰链区起始氨基酸位置可以变动。
本发明中术语“Fc变体”是指，通过在Fc上合适的位点处存在一个或者多个氨基酸替换、插入或缺失突变引起Fc结构或功能的变化。“Fc变体间作用”指的是经突变设计的Fc变体之间，可以形成空间填充效应、静电导引、氢键作用、疏水作用等。Fc变体间相互作用有助于形成稳定的异源二聚体蛋白。优选的突变设计为“Knob-into-Hole”形式的突变设计。
Fc变体的突变设计技术在本领域内已经较为广泛的应用于制备双特异性抗体或者异源二聚的Fc融合蛋白形式。代表性的有Cater等人(Protein Engineering vol.9no.7pp.617-621，1996)提出的“Knob-into-Hole”形式；Amgen公司技术人员利用静电导引(Electrostatic Steering)形成含Fc的异源二聚体形式(US 20100286374A1)；Jonathan H.Davis等人(Protein Engineering，Design&Selection pp.1-8，2010)提出的通过IgG/IgA链交换形成的异源二聚体形式(SEEDbodies)；Genmab公司DuoBody(Science，2007.317(5844))平台技术形成的双特异性分子；Xencor公司的技术人员综合结构计算及Fc氨基酸突变，综合不同作用方式形成异源二聚体蛋白形式(mAbs 3：6，546-557；November/December 2011)；苏州康宁杰瑞公司的基于电荷网络的Fc改造方法(CN201110459100.7)得到异源二聚体蛋白形式；以及其它基于 Fc氨基酸变化或者功能改造手段，达到形成异源二聚体功能蛋白的基因工程方法。本发明所述的Fc变体片段上的Knob/Hole结构指两条Fc片段各自突变，突变后可以通过“Knob-into-Hole”形式进行结合。优选用Cater等人的“knob-into-hole”模型在Fc区上进行位点突变的改造，以使得到的第一Fc变体和第二Fc变体能以“knob-into-hole”的形式结合在一起形成异源二聚体。从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区在本领域技术人员所掌握的范围之内。优选人类抗体IgG1、IgG2、IgG3、IgG4的Fc区，更优选人抗体IgG1的Fc区。随机任选第一Fc变体或第二Fc变体中一个做knob的突变，另一个做hole的突变。
本发明中术语“抗体”(Antibody，Ab)是指，与目标抗原特异性结合或具有免疫反应性的免疫球蛋白分子，包括抗体的多克隆、单克隆、基因工程化和其他修饰形式(包括但不限于嵌合抗体，人源化抗体，全人源抗体，异源偶联抗体(例如双特异性、三特异性和四特异性抗体，双抗体，三抗体和四抗体)，抗体缀合物)以及抗体的抗原结合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。此外，除非另有说明，否则术语“单克隆抗体”(mAb)意指包括能够特异性结合靶蛋白的完整抗体分子以及不完整的抗体片段(例如Fab和F(ab’) 2片段，它们缺少完整抗体的Fc片段(从动物循环中更快地清除)，因此缺乏Fc介导的效应功能(effector function)(参见Wahl等人，J.Nucl.Med.24:316,1983；其内容援引加入本文)。
术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
术语“抗体缀合物”是指抗体分子直接或者通过连接接头与另一个分子化学键合而形成的偶联体/缀合物。例如抗体-药物缀合物(ADC)，其中药物分子就是所述的另一个分子。
术语“单克隆抗体”是指来源于单个克隆(包括任何真核、原核、或噬菌体克隆)的抗体，而不限于该抗体的产生方法。
本发明中术语“融合蛋白”(fusion protein)是指，通过基因重组方法、化学方法或其它适当方法将两个或多个基因的编码区连接，在同一调控序列控制下表达基因重组所得的蛋白质产物。本发明的融合蛋白中，两个或多个基因的编码区之间可由编码肽接头或连接肽的序列于一个或数个位置融合。肽接头或连接肽也可用于构建本发明的融合蛋白。本发明术语“融合蛋白”进一步还包括抗体/Fc融合蛋白构建体/复合物，或通过非共价方式形成的抗体/Fc融合蛋白构建体/复合物的组合物，例如，本发明所述的融合蛋白可展示为以下结构:
(1)IL-15融合蛋白，其为包含两条单体的同源二聚体；所述单体包含抗体重链、抗体轻链、IL-15和IL-15Rαsushi；例如，将抗体重链Fc融合IL-15Rαsushi，并与抗体轻链、和IL-15-WT(野生型)或IL-15突变体共表达，使IL-15与IL-15Rαsushi形成非共价连接；
(2)IL-15融合蛋白，其为包含两条单体的同源二聚体；所述单体包含抗体重链、抗体轻链、IL-15和IL-15Rαsushi；例如，通过linker将抗体重链Fc与IL-15Rαsushi以及IL-15-WT或IL-15突变体三部分串联融合表达，并与抗体轻链共表达；
(3)IL-15融合蛋白，其为包含两条单体的同源二聚体；所述单体包含Fc、IL-15和IL-15Rαsushi；例如，IL-15-WT或IL-15突变体通过linker和IL15-Rαsushi相连接，IL15-Rαsushi再通过linker和Fc相连；或，
(4)IL-15融合蛋白，其为包含两条单体的同源二聚体；所述单体包含Fc、IL-15和IL-15Rαsushi；例如，IL15-Rαsushi通过linker和IL-15-WT或IL-15突变体相连接，IL-15再通过linker和Fc相连。
本发明中术语“肽接头/连接肽(Linker)”是指，在本发明中用于连接IL-15与另一蛋白分子或蛋白片段，以保证蛋白的正确折叠和稳定性的肽。所述另一分子包括不限于IL-15Rα、Fc、Fc变体、抗体等。本发明的“连接肽”优选为(GGGGS)n，其中n可以为0、1、2、3、4、5或者更多，优选n为2-4；或优选SGGSGGGGSGGGSGGGGSLQ。如果连接肽序列太短，可能影响两蛋白高级结构的折叠，从而相互干扰；如果连接肽序列太长，又涉及免疫原性的问题，因为连接肽序列本身就是新的抗原。
本发明中术语“异源二聚体蛋白”是指两个不同的单体蛋白质结合而成的蛋白。在本发明中，两个不同的单体蛋白质各自含有Fc片段或Fc变体片段，并通过Fc片段或Fc变体片段相互作用形成异源二聚体蛋白。
本发明中术语“同源二聚体蛋白”是指两个相同的单体蛋白质结合而成的蛋白。在本发明中，两个相同的单体蛋白质各自含有Fc片段或Fc变体片段，并通过Fc片段或Fc变体片段相互作用形成同源二聚体蛋白。
本发明中组成异源二聚体蛋白或同源二聚体蛋白的“单体蛋白质”可以是融合蛋白或非融合蛋白。
本发明中术语“PD-L1”是指程序性死亡配体-1，也称为CD279(分化簇279)，是一种重要的免疫抑制分子。所述PD-L1优选的是人PD-L1。
本发明中术语“抗-程序性死亡配体-1抗体”、“程序性死亡配体-1抗体”、“抗PD-L1抗体”、“PD-L1抗体”、“抗-PD-L1抗体部分”和/或“抗-PD-L1抗体片段”等是指任何包含能够特异性结合PD-L1的免疫球蛋白分子的至少一部分(例如但不限于重链或轻链的至少一个互补决定区(CDR)或其配体结合部分、重链或轻链可变区、重链或轻链恒定区、框架区或其任何部分)的含蛋白质或肽的分子。PD-L1抗体还包括抗体样蛋白支架(如第十纤连蛋白III型结构域(10Fn3))，其含有与抗体CDR在结构和溶剂可及性上相似的BC、DE和FG结构环。10Fn3结构域的三级结构类似于IgG重链可变区的三级结构，并且通过将10Fn3的BC、DE和FG环的残基用来自PD-L1单克隆抗体的CDR-H1、CDR-H2或CDR-H3区的残基替换，本领域技术人员可以将例如PD-L1单克隆抗体的CDR接枝到纤连蛋白支架上。
本发明中术语“共表达”(coexpression)是指，在一个细胞中多个基因一起表达，同时出现它们的产物。这些基因可以是同时存在而分别或共同地受控表达。在本发明中，优选在一个真核细胞中共表达两种基因。共表达得到的基因表达产物有利于高效、简单地形成复合物；在本发明中，有利于形成异源二聚体蛋白或同源二聚体蛋白。
术语“百分比(％)序列一致性”是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残 基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。
本发明中术语“特异性结合”是指一种结合反应，其决定抗原在蛋白质和其他生物分子的一个异质性群体中的存在状况，所述蛋白质和其他生物分子例如被抗体或其抗原结合片段特异性识别。与抗原特异性结合的抗体或其抗原结合片段将以小于100nM的KD与抗原结合。例如，与抗原特异性结合的抗体或其抗原结合片段将以高达100nM(例如，1pM至100nM之间)的KD与抗原结合。不显示与特定抗原或其表位特异性结合的抗体或其抗原结合片段将显示对该特定抗原或其表位的大于100nM(例如，大于500nM、1μM、100μΜ、500μΜ或1mM)的KD。可以使用多种免疫测定方式来选择与特定蛋白或碳水化合物进行特异性免疫反应的抗体。例如，常规地使用固相ELISA免疫测定法来选择与蛋白质或碳水化合物进行特异性免疫反应的抗体。参见，Harlow & Lane，Antibodies,ALaboratory Manual,Cold Spring Harbor Press,NewYork(1988)以及Harlow & Lane,Using Antibodies，A Laboratory Manual,Cold Spring Harbor Press,NewYork(1999)，其描述了可以用于确定特异免疫反应性的免疫测定方式和条件。
本发明中术语“载体”包括核酸载体，例如DNA载体(如质粒)，RNA载体，病毒或其他适合的复制子(例如病毒载体)。已经开发了多种载体用于将编码外源蛋白质的多核苷酸递送到原核或真核细胞中。本发明的表达载体含有多核苷酸序列以及例如用于表达蛋白质和/或将这些多核苷酸序列整合到哺乳动物细胞基因组中的附加序列元件。可以用于表达本发明的抗体和抗体片段的某些载体包括含有指导基因转录的调控序列(如启动子和增强子区域)的质粒。用于表达抗体和抗体片段的其他有用的载体含有多核苷酸序列，其增强这些基因的翻译速率或改善由基因转录产生的mRNA的稳定性或核输出。这些序列元件包括例如5’和3’非翻译区、内部核糖体进入位点(IRES)和聚腺苷酸化信号位点，以便指导表达载体上携带的基因的有效转录。本发明的表达载体还可以含有以下多核苷酸，该多核苷酸编码用于选择含有这种载体的细胞的标记。适合的标记的实例包括编码抗生素(如氨苄青霉素、氯霉素、卡那霉素或诺尔丝菌素)抗性的基因。
本发明中术语“受试者”、“对象”和“患者”是指接受对如本文所述的特定疾病或病症(如癌症或传染性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症(例如细胞增殖性病症，如癌症或传染性疾病)的治疗的哺乳动物，如人、灵长类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛科家族成员(如家牛、野牛、水牛、麋鹿和牦牛等)、绵羊和马等。
本发明中术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如细胞增殖性病症(如癌 症或传染性疾病)的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本发明中“免疫病症”或“免疫障碍”包括例如病理性炎症、炎性病症和自身免疫性疾病症或疾病。“免疫病症”还指感染、持续感染和增生性病症，例如癌症、肿瘤和血管发生。“癌性病症”包括例如癌症、癌细胞、肿瘤、血管发生和癌变前病症，例如发育异常。
本发明中术语“药物组合物”是指含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其它化学组分的混合物，以及其它组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本发明中术语“分子排阻色谱法”(Size Exclusion Chromatograph，简称SEC)是根据待测组分cvn分子大小进行分离的一种液相色谱技术。色谱柱填充剂表面分布着不同尺寸的孔径，样品进入色谱柱后，它们中不同组分按其分子大小进入相应的孔径内，大于所有孔径的分子不能进入填充剂颗粒内部，在色谱过程中不被保留，保留时间较短；小于所有孔径的分子能自由进入填充剂表面的所有孔径，在色谱柱中滞留时间较长，表现为保留时间较长；其余分子则按分子大小依次被洗脱。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着抗体重链可变区可以但不必须存在；存在时，可以是1个，2个或3个。
本发明中所述的用重组DNA转化宿主细胞的步骤可用本领域技术人员熟知的常规技术进行。获得的转化子可以用常规方法培养，以及表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。宿主细胞在适于宿主细胞生长的条件下进行培养。
附图说明
下面通过对本发明的详细描述以及附图来清楚地说明本发明前面叙述的方面以及其他方面。本文中附图是为了举例说明本发明的一些优选的实施方案，然而，可以理解，本发明并不限于所公开的特定实施方案。
图1、人源化PD-L1抗体阻断PD-L1和PD-1结合结果，其中阳性对照为Tecentriq。
图2、FACS测定人源化PD-L1抗体与细胞水平PD-L1结合能力，其中阳性对照为Avelumab。
图3、Jurkat-PD-1/CHO-PD-L1-NFAT体系测试人源化PD-L1抗体对PD-L1/PD-1阻断能力，其中阳性对照为Avelumab。
图4、人源化抗PD-L1抗体促进混合淋巴细胞反应中IFN-γ的分泌结果，其中阴性对照为anti-Hel抗体，阳性对照为Avelumab。
图5、IL-15融合蛋白的两种结构：
A：IL-15野生型或突变体与IL-15Rαsushi共表达组装，非共价连接；
B：IL-15野生型或突变体与IL-15Rαsushi通过linker串联融合表达组装；
C：IL-15野生型或突变体先与IL-15Rαsushi通过linker连接，IL-15Rαsushi再通过linker与Fc串联融合表达组装；
D：IL-15Rαsushi通过linker先与IL-15野生型或突变体连接，IL-15野生型或突变体再通过linker与Fc串联融合表达组装。
图6A-6D、IL15突变体促进Mo7e细胞增殖：融合蛋白如图5A所示结构，Tecentriq融合IL-15Rαsushi，IL15突变体与IL-15Rαsushi之间非共价连接，无linker连接。
图7A-7N、IL-15突变体诱导CD8+T细胞增殖：
图7A：融合蛋白如图5A所示结构，Tecentriq融合IL-15Rαsushi，IL15突变体与IL-15Rαsushi之间非共价连接，无linker连接；
图7B-7G，7M-7N：融合蛋白如图5B所示结构，自研PD-L1单抗融合IL-15Rαsushi和IL15突变体，IL15突变体与IL-15Rαsushi之间通过linker连接串联表达；Drug0代表反应体系中未加入任何蛋白的阴性对照；
图7H-7K：融合蛋白如图5D所示结构，IL-15Rαsushi通过linker先与IL-15野生型或突变体连接，IL-15野生型或突变体再通过linker与Fc串联融合表达组装；
图7L，融合蛋白如图5C所示，IL-15野生型或突变体先与IL-15Rαsushi通过linker连接，IL-15Rαsushi再通过linker与Fc串联融合表达组装。
图8A-8M、IL-15突变体诱导NK细胞增殖：
图8A：融合蛋白如5A所示结构，Tecentriq融合IL-15Rαsushi，IL15突变体与IL-15Rαsushi之间非共价连接，无linker连接；
图8B-8F，8L-8M：融合蛋白如图5B所示结构，自研PD-L1单抗融合IL-15Rαsushi和IL15突变体，IL15突变体与IL-15Rαsushi之间通过linker连接串联表达；Drug0代表反应体系未加入任何蛋白的阴性对照。
图8G-8J：融合蛋白如图5D所示，IL-15Rαsushi通过linker先与IL-15野生型或突变体连接，IL-15野生型或突变体再通过linker与Fc串联融合表达组装。
图8K：融合蛋白如图5C所示，IL-15野生型或突变体先与IL-15Rαsushi通过linker连接，IL-15Rαsushi再通过linker与Fc串联融合表达组装。
图9A-9C：hPBMC-A375小鼠体内药效。融合蛋白如图5B所示结构。
图10A-10C：hPBMC-A375小鼠体内药效。融合蛋白如图5C或5D所示结构。
图10D：hPBMC-A375小鼠给药后体重变化。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本发明实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1.抗体人源化
首先采用经典“CDRs移植”方法进行抗体人源化，即通过序列挑选同源性最高的人源性抗体提供抗体骨架区(FRs)，把目标抗体的基于Kabat命名方法的抗原结合片段互补决定区(CDRs)，移植到前者形成人源化抗体。其次，为更好保持抗体活性和亲和力，通过MOE软件基于抗体结构建模分析：1).选择抗体骨架区位于VH-VL界面、靠近或与CDRs有直接相互作用等氨基酸残基进行回复突变，这类氨基酸残基对保持CDRs区构象多较重要；2).考虑到免疫原性，尽量选择包埋在蛋白内部的氨基酸进行回复突变；3).考虑到抗体稳定性和表达水平，优先考虑分子能量降低突变。通过测试含有不同突变的人源化抗体与人PD-L1的亲和力以及和表面表达PD-L1的细胞的结合，筛选与鼠源PD-L1抗体亲和力、抗体表征和活性功能相当或更好的人源化抗体。
其中，鼠源PD-L1抗体PDL1-794经过人源化后的优选候选抗体分子794-h1-71的重链和轻链可变区的氨基酸序列信息如下表1所示。
表1.鼠源及人源化抗PD-L1抗体重链可变区和轻链可变区的具体序列信息
Figure PCTCN2021094167-appb-000001
实施例2 人源化抗体表达和纯化
2.1人源化抗体的表达
转染前一天，将ExpiCHO-S细胞(Thermo fisher，A29127)以2.5×10 6～4×10 6cells/mL密度接种于新鲜ExpiCHO Expression培养基(英潍捷基，A29100-01)中，置于摇床中过夜培养。转染当天，取过夜培养的ExpiCHO-S细胞悬液计数，细胞活率＞95％，密度处于7×10 6-10×10 6viable cells/mL之间。取所需细胞悬液，用ExpiCHO Expression培养基(英潍捷基，A29100-01)稀释至6×10 6cells/mL的密度，置于摇床备用。将准备好的人源化抗体表达质粒加入培养基中稀释，轻轻摇转离心管使其混匀，加入到OptiPRO TM SFM-DNA稀释液(英潍捷基，12309-019)中，轻轻摇转离心管使其混匀后室温静置1～5mins。上述质粒复合物缓慢滴入待转染细胞悬液中，在滴加过程中摇转摇瓶。转染结束后，将细胞放入摇床过夜培养。向转染后第一天的细胞中补加相当于细胞体积0.6％的ExpiFectamine TM CHO Enhancer(英潍捷基，A29129)和16％的ExpiCHO TM Feed(英潍捷基，A29129)，加入过程中轻轻摇转摇 瓶，并且将细胞转移至摇床中培养4天。转染后第5天向转染的细胞中补加相当于细胞体积16％的ExpiCHO TM Feed(英潍捷基，A29129)，加入过程中轻轻摇转摇瓶。转染后第12天取9000g培养液，离心10mins后收获上清。
2.2人源化抗体的纯化
高速离心实施例2.1中收集的细胞培养液上清，过0.45μm+0.22μm滤膜，利用亲和层析进行第一步纯化。层析介质为与Fc相互作用的protein A填料Mbaselect Sure(GE，17543803)，平衡缓冲液为PBS(2.5g/L Na 2HPO 4·12H 2O，0.408g/L NaH 2PO 4，8.76g/L NaCl，pH7.2)，平衡4倍柱体积后，将细胞上清上样结合，流速控制为样品在柱上保留时间≥5min。上样结束后，用PBS(pH7.2)冲洗柱子，直至A280紫外吸收降至基线。然后用20mM PB+1M NaCl(pH6.0)洗杂2倍柱体积。再用PBS(pH7.2)冲洗柱子，直至A280紫外吸收和电导达到基线。最后用20mM柠檬酸(pH3.4)的洗脱缓冲液冲洗层析柱，根据A280紫外吸收峰收集洗脱峰，收集的洗脱样品用1M Tris-HCl(pH9.0)中和至中性。
实施例3 抗体与人以及食蟹猴PD-L1重组蛋白结合的KD测定
使用Biacore T200(GE Healthcare)测定PD-L1抗体对于人和食蟹猴PD-L1-His蛋白的结合亲和力。25℃下在CM5芯片(GE Healthcare，Cat.BR-1005-30)上固定anti-human IgG Fc(Genway，Cat.GWB-20A705)。将anti-human IgG Fc用Acetate pH5.0(GE Healthcare，BR-1003-51)稀释至20μg/mL。使用Immobilization method中Amine方法进行固定。或者使用商品化Protein A(GE Healthcare，Cat.29127556)芯片进行检测。25℃下采用多循环动力学法测定抗体与抗原的亲和力，在每一个循环中，首先将待测抗体捕获到固定好的CM5芯片，然后注入重组人PD-L1-His(Novoprotein,Cat.315)和食蟹猴PD-L1-His蛋白(Sino Biological，Cat.90251-C08H)，最后用Glycine pH1.5(沪试，Cat.62011516)再生。流动相为HBS-EP+Buffer(GE Healthcare，Cat.BR-1006-69)，流速30μL/min，结合时间为300秒。再生流速30μL/min，时间为30秒。应用Biacore T200 Evaluation Software(version 3.0)，以1：1结合模型，分析试验数据，拟合抗体抗原的平衡解离常数KD，确定结合速率常数ka和解离速率常数kd。
从结果可知所测试的PD-L1抗体对人PD-L1重组蛋白与食蟹猴PD-L1重组蛋白的结合，都表现出nM或更高的亲和力，详见下表2。
表2.人源化PD-L1抗体Biacore结合亲和力KD测定结果
抗体编号 人PD-L1(M) 食蟹猴PD-L1(M)
794-h1-71 1.793E-09 9.372E-10
实施例4 抗体阻断PD-L1和PD-1相互作用的IC50测定
通过竞争性ELISA方法确定抗PD-L1抗体阻断PD-L1蛋白与PD-1蛋白结合的IC50。使用碳酸盐缓冲液稀释人PD-L1重组蛋白(Sino Biological,Cat.10084-H05H)，加入96孔酶标板，终浓度为1μg/ml。用含3％BSA的PBS溶液封闭，加入梯度稀释的抗PD-L1抗体(40nM～0.02nM)以及人PD-1-His重组蛋白(Sino Biological,Cat.10377-H08H)进行共孵育后，加入HRP标记的抗His标签抗体(MBL,Cat.D291-7)，TMB(Thermo,Cat.34029)显色，1M硫酸终止后读取OD值(双波长450nm-630nm)。将抗体浓度与OD值对应即可绘制出测试抗体的竞争结合曲线，计算出IC50值。图1显示了抗PD-L1抗体与人PD-L1重组蛋白的竞争结合曲线。结果表明，被测试的794-h1-71抗体可以有效的阻断人PD-L1蛋白与人PD-1蛋白 的相互作用，IC50为0.8488nM，阳性对照Tecentriq(Genetech，lot:H0172)为0.8486nM。
实施例5 FACS测定PD-L1抗体对细胞表面PD-L1结合的EC50
将梯度浓度的待检测抗体(抗体浓度：10000ng/ml-0.1ng/ml)与细胞表面高表达PD-L1的CHO-PD-L1细胞(南京勇山生物科技有限公司，10 5个/孔)，4℃共同孵育30min。孵育结束后，加入1:250稀释的anti-human IgG PE荧光抗体(eBioscience，Cat.12-4998-8)，4℃下共同孵育30min，荧光抗体与待检测抗体的Fc段产生特异性结合，通过FACS检测PE荧光强度的高低而对待检测抗体的结合细胞表面高表达的PD-L1蛋白的能力进行分析。图2结果显示，794-h1-71抗体EC50为38.44ng/ml，与本次实验的阳性对照Avelumab(EC50为～72ng/ml)相近。该检测定量地证实了794-h1-71抗体对细胞表面上PD-L1靶点剂量依赖性结合的能力。平均荧光强度变化倍数(MFI fold)＝实验组MFI值/未加药物的对照组MFI值。
实施例6 PD-1/PD-L1-NFAT报告基因测试抗PD-L1抗体阻抑PD-1:PD-L1结合和信号传导
利用稳定转染PD-1的Jurkat细胞株(GenScript，Cat.00612)和稳定转染PD-L1的CHO细胞株(GenScript，Cat.M00613)比较PD-L1抗体对PD-1/PD-L1蛋白相互作用及其信号通路的拮抗作用。当抑制信号通路阻抑，NFAT控制的发光报告基因表达增强，发光信号值增加。通过发光读值的强弱(relative light units,RLU)反应抗体对PD-L1的阻断作用强弱。
将稳转PD-L1的CHO细胞株种在96孔白底板上，每孔40000个细胞，100μl/孔，放回培养箱过夜；第二天，取出孔板，吸去培养基，加入稳转PD-1的细胞株及待测的PD-L1抗体共孵育，PD-1细胞加样量为16000个/孔，抗体则做梯度稀释，每个剂量3复孔，孵育体积为100μl/孔，孵育时长为6小时，待孵育完成时，取出孔板，等体积(100μl)加入发光检测试剂，读值。根据检测值用Graphpad进行4参数分析做回归曲线，得到各抗体的EC50值。图3显示，794-h1-71抗体的EC50(166.2ng/ml)和阳性对照Avelumab的EC50(184.3ng/ml)相近。该检测定量地证实了794-h1-71抗体对细胞表面PD-1：PD-L1相互作用导致的T细胞活性抑制呈现剂量依赖性的阻抑能力，从而剂量依赖地增强Jurkat细胞内报告基因的活性。
实施例7 ELISA检测混合淋巴细胞反应中T细胞分泌的IFN-γ
通过混合淋巴细胞反应(mixed lymphocyte reaction，MLR)来测定PD-L1单抗增强T细胞的活性。从健康人供体1的外周血单核细胞(peripheral blood mononuclear cells,PBMC)中分离CD4 +单核细胞，应用重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF，Peprotech，Cat.300-03)和重组人白介素4(rhIL-4,Peprotech，Cat.200-04)进行体外诱导分化为树突状细胞(dendritic cell,DC)，于培养第6天加入LPS(Sigma，Cat：L4516)刺激成熟DC，第7天将供体1的DC细胞与从健康供体2的PBMC富集的CD4 +T细胞混合共培养，DC：CD4 +T细胞数比例为1:10，加入待测抗体、阴性对照抗体anti-Hel(百英生物合成)和阳性对照抗体Avelumab(抗体浓度：7nM-0.28nM)，共培养4天。4天后收集细胞培养上清，用ELISA方法检测上清中IFN-γ的含量。如图4显示，794-h1-71及阳性对照抗体Avelumab相比anti-Hel单抗阴性对照组，均可明显增强MLR实验中CD4 +T细胞分泌IFN-γ的能力，并且随着PD-L1抗体药物浓度降低，增加分泌IFN-γ的活性也降低。该结果表明794-h1-71抗体可增强T细胞的功能，且具有剂量依赖性(T-test，*P<0.05，**P<0.01，***P<0.001，****P<0.0001.)。
实施例8 IL-15融合蛋白的构建
使用MOE软件模拟人的IL-15与相应的受体βγchain相互作用的关键氨基酸位点。分别为D8和V3，I6，H105。根据MOE软件模拟，设计合成以下IL-15突变体序列，氨基酸序列详见表3，编码核酸序列详见表4。
IL-15融合蛋白(抗体/Fc融合构建体/复合物)的构建有四种模式：
(1)如图5A所示结构，IL-15融合蛋白，其为包含两条单体的同源二聚体；所述单体包含抗体重链、抗体轻链、IL-15和IL-15Rαsushi；将抗体重链Fc端融合IL-15Rαsushi，并与单抗轻链、和IL-15-WT(野生型)或IL-15突变体共表达，使IL-15与IL-15Rαsushi形成非共价连接；
(2)如图5B所示结构，IL-15融合蛋白，其为包含两条单体的同源二聚体；所述单体包含抗体重链、抗体轻链、IL-15和IL-15Rαsushi；通过linker将抗体重链Fc端与IL-15Rαsushi以及IL-15-WT或IL-15突变体三部分顺序串联融合表达，并与抗体轻链共表达；
(3)如图5C(定义为V5)所示结构，IL-15融合蛋白，其为包含两条单体的同源二聚体；所述单体包含Fc、IL-15和IL-15Rαsushi；IL-15-WT或IL-15突变体通过linker和IL15-Rαsushi相连接，IL15-Rαsushi再通过linker和Fc相连；
(4)如图5D(定义为V9)所示结构，IL-15融合蛋白，其为包含两条单体的同源二聚体；所述单体包含Fc、IL-15和IL-15Rαsushi；IL15-Rαsushi通过linker和IL-15-WT或IL-15突变体相连接，IL-15再通过linker和Fc相连。
PD-L1抗体与IL-15融合蛋白编号规则如下：“抗体代称-IL-15(野生型/突变体)-融合蛋白构建模式”；
例如：“T-IL15-xx-1”：“T”，表示Tecentriq；“IL15-xx”，表示IL15-WT或IL15突变体；“1”，表示如图5A所示结构模式；
例如：“794-IL15-xx-2”：“794”，表示794-h1-71单抗；“IL15-xx”，表示IL15-WT或IL15突变体；“2”，表示如图5B所示结构模式。
各种IL-15融合蛋白设计请详见表5和表6。根据上述4种构建模式，将编码融合蛋白的核酸序列分别构建至pTT5质粒。
表3.IL-15融合蛋白相关氨基酸序列信息
Figure PCTCN2021094167-appb-000002
Figure PCTCN2021094167-appb-000003
Figure PCTCN2021094167-appb-000004
Figure PCTCN2021094167-appb-000005
Figure PCTCN2021094167-appb-000006
注：IL-15突变体融合蛋白编号规则如下：
例如：“T-IL15-xx-1”：“T”，表示Tecentriq；“IL15-xx”，表示IL15-WT或IL15突变体；“1”，表示如图5A所示结构模式；
例如：“794-IL15-xx-2”：“794”，表示794-h1-71单抗；“IL15-xx”，表示IL15-WT或IL15突变体；“2”，表示如图5B所示结构模式。
表4.IL-15融合蛋白编码核酸序列信息
Figure PCTCN2021094167-appb-000007
Figure PCTCN2021094167-appb-000008
Figure PCTCN2021094167-appb-000009
Figure PCTCN2021094167-appb-000010
Figure PCTCN2021094167-appb-000011
Figure PCTCN2021094167-appb-000012
Figure PCTCN2021094167-appb-000013
Figure PCTCN2021094167-appb-000014
Figure PCTCN2021094167-appb-000015
Figure PCTCN2021094167-appb-000016
Figure PCTCN2021094167-appb-000017
表5.PD-L1-IL-15融合蛋白分子组成
Figure PCTCN2021094167-appb-000018
注：PD-L1-IL-15突变体融合蛋白编号规则如下：
例如：“T-IL15-xx-1”：“T”，Tecentriq；“IL15-xx”，表示IL15-WT或IL15突变体；“1”，表示如图5A所示结构模式；
例如：“794-IL15-xx-2”：“794”，794-h1-71单抗；“IL15-xx”，表示IL15-WT或IL15突变体；“2”，表示如图5B所示结构模式。
表6.IL-15-Fc融合蛋白分子组成
Figure PCTCN2021094167-appb-000019
实施例9 IL-15融合蛋白的表达
利用ExpiCHO表达系统进行蛋白瞬时表达。将在ExpiCHOTM Expression Medium(Cat no.A2910001)传代的宿主细胞ExpiCHO-S(Cat no.A29127)稀释至合适密度，置于摇床(100rpm，37℃，8％CO 2)待转染。将携带编码融合蛋白核酸序列的载体加入OptiPRO TM SFM(Cat no.12309019)培养基中，使得载体终浓度为0.5～1.0μg/mL。向含有DNA的OptiPRO TM SFM培养基中加入适量的ExpiFectamine TM CHO Reagent(Cat no.A29129)形成DNA-ExpiFectamine TM CHO Reagent复合物，室温静置1～5min后将复合物缓慢滴入待转染的细胞悬液中。转染后第1天，补加一定量的ExpiFectamine TM CHO Enhancer(Cat no.A29129)和ExpiCHO TM Feed(Cat no.A29129)后降温至32℃培养。转染后第4～6天，补加一定量的ExpiCHO TM Feed(Cat no.A29129)。转染后第10～12天，5000g离心30min收获上清。
实施例10 IL-15融合蛋白的纯化
采用磁珠法纯化突变体融合蛋白，发酵上清中加入适量磁珠悬浮液(GenScript，Cat.NO.L00695)，于旋转混匀器中孵育2h以保证IL-15融合蛋白结合到磁珠上，弃上清后用PBS洗涤磁珠3次，最后加入pH3.0柠檬酸盐洗脱得到IL-15融合蛋白，1M Tris-Hcl中和样品后，用NanoDrop One测定蛋白浓度。
实施例11 分子排阻色谱法测定IL-15融合蛋白的纯度
本发明检测方法中，采用TSKgel G3000SWXL色谱柱(TOSOH，0008541)，预柱Tskgel guard columnSWXL(TOSOH，0008543)进行分子排阻色谱法(SEC)测定本发明的IL-15突变体融合蛋白的纯度。流动相(50mM PB，300mM NaCl，pH6.8)平衡色谱柱，流速为1mL/min。紫外检测波长280nm。结果见表7。
表7.分子排阻色谱法测定IL-15融合蛋白纯度
编号 SEC主峰纯度％ 编号 SEC主峰纯度％
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    
实施例12 IL-15突变体融合蛋白诱导Mo7e细胞增殖实验
Mo7e为人巨核细胞白血病细胞株(Cobioer，CBP60791)，可用于IL-15对细胞增殖活性的研究。将IL-15突变体融合蛋白用1640-10％FBS(RPMI1640，Gibco，72400047；FBS， Gibco，10099141)稀释成梯度浓度(终浓度100000pM-1.28pM，5倍稀释)，将Mo7e细胞用1640-10％FBS稀释成10 5cells/ml，在96孔U底板中加入50μl突变体培养液和50μl Mo7e细胞悬液，混匀后37℃5％CO 2孵育。72h后将培养板取出，加入100μl Cell Titer Glo luminescent cell viability检测试剂(Promega,G7571)，检测luminescence荧光强度，荧光强度与细胞增殖能力成正比。
结果显示如图6A-6D：T-IL15-7-1，T-IL15-8-1，T-IL15-9-1突变体的活性相较于T-IL15-WT-1显著降低，T-IL15-7-1的EC50为5735pM,对应的T-IL15-WT-1的EC50为133.3pM(图6A)。T-IL15-10-1，T-IL15-11-1，T-IL15-26-1突变体的活性相较于T-IL15-WT-1显著降低，T-IL15-10-1和T-IL15-26-1的EC50分别为28076pM和290.9pM,对应的T-IL15-WT-1EC50为143.3pM(图6B)。T-IL15-29-1，T-IL15-42-1突变体的活性相较于T-IL15-WT-1降低，EC50分别为285.2pM和194.5pM，T-IL15-43-1突变体的活性相较于T-IL15-WT-1增强，EC50为103.2pM,T-IL15-WT-1的EC50为150.7pM(图6C)。T-IL15-com1-1和T-IL15-com2-1组合突变活性相较于T-IL15-WT-1降低；Tecentriq对照组对Mo7e细胞增殖无影响， 即抗PD-L1抗体对Mo7e细胞无诱导增殖活性(图6D)，对细胞增殖活性的影响 是由IL-15产生的。
实施例13 IL15突变体融合蛋白诱导CD8+T细胞增殖
为了检测IL-15突变体融合蛋白对于人PBMC(Allcells，Lot:1911150123)中CD8+T细胞的刺激增殖作用，本实施例以Ki67作为增殖标志，检测了不同浓度的IL-15突变体融合蛋白刺激人PBMC细胞三天后，CD8+T细胞的增殖比例。首先，将人PBMC悬浮在含10％FBS(Gibco，Cat：10099141)和1％青-链霉素(Gibco,Cat：15140122)的RPMI1640培养基(Gibco，Cat：72400047)中，调节细胞密度为2×10 6个/ml，100μl/孔加入到96孔U底板(Corning，Cat：3799)中。然后，将IL-15突变体融合蛋白从500nM起始，4倍梯度稀释，共11个梯度，随后每个浓度的样品100μl/孔加入到已加入人PBMC的96孔U底板中，混合均匀后，放入37℃，5％CO 2培养箱中培养三天。培养后的细胞用LIVE/DEAD Fixable Violet Dead Cell Stain Kit(Invitrogen，Cat：L34964)及CD3-AF700(BD，Cat：557943)、CD8-FITC(BD，Cat：555366)、APC-Ki67(Biolegend，Cat：350514)抗体进行染色，随后用流式细胞仪检测不同浓度IL-15突变体融合蛋白刺激后CD3+CD8+T细胞中Ki67+细胞的比例。图7A-7N显示了梯度稀释的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和P22339。刺激人PBMC三天后，CD8+T细胞的增殖比例，以及各突变体刺激增殖的EC50值。其中，T-IL15-8-1，T-IL15-9-1，T-IL15-11-1对CD8+T细胞的增殖能力显著降低，未到平台，曲线无法进行拟合计算出EC50；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未到平台，EC50值拟合不准确；但结果显示上述突变体组合比794-IL15-WT-2或P22339对CD8+T细胞的增殖活性降低。T-IL15-WT-1，794-IL15-WT-2为野生型IL-15对照，P22339为突变型IL-15对照；794-h1-71单抗为抗PD-L1抗体对照组；Drug0为未加入任何蛋白的阴对 照。相对于野生型IL-15对照，IL15突变体对CD8+T细胞的增殖较弱。794-h1-71单抗对照组对CD8+T细胞增殖无影响，即 抗PD-L1抗体对CD8+T细胞无诱导增殖活性(图7B)， 对细胞增殖活性的影响是由IL-15产生的。
实施例14 IL15突变体融合蛋白诱导NK细胞增殖
为了检测IL-15突变体融合蛋白对于人PBMC(Allcells，Lot:1911150123)中NK细胞的刺激增殖作用，本实施例以Ki67作为增殖标志，检测了不同浓度的IL-15突变体融合蛋白刺激人PBMC细胞三天后，NK细胞的增殖比例。首先，将人PBMC悬浮在含10％FBS(Gibco，Cat：10099141)和1％青-链霉素(Gibco,Cat：15140122)的RPMI1640培养基(Gibco，Cat：72400047)中，调节细胞密度为2×10 6个/ml，100μl/孔加入到96孔U底板(Corning，Cat：3799)中。然后，将IL-15突变体融合蛋白从500nM浓度起始，4倍梯度稀释，共11个梯度。随后每个梯度的样品100μl/孔加入到已加入人PBMC的96孔U底板中，混合均匀后，放入37℃、5％CO 2培养箱中培养三天。培养后的细胞用LIVE/DEAD Fixable Violet Dead Cell Stain Kit(Invitrogen，Cat：L34964)及CD3-AF700(BD，Cat：557943)、CD56-PE(BD，Cat：555516)、APC-Ki67(Biolegend，Cat：350514)抗体进行染色，随后用Invitrogen Attune NxT流式细胞仪检测不同浓度IL-15突变体融合蛋白刺激后NK细胞中Ki67+细胞的比例。图8A-8M显示了梯度稀释的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和P22339刺激人PBMC三天后，CD3-CD56+的NK细胞增殖比例，以及各突变体刺激增殖的EC50值。其中，T-IL15-8-1，T-IL15-9-1，T-IL15-11-1，V9-IL15-com6，V9-IL15-62,V9-IL15-63对NK细胞的增殖能力显著降低，未到平台，EC50值拟合不准确；但上述突变体组合比对照组T-IL15-WT-1或P22339对NK细胞的增殖活性显著降低。T-IL15-WT-1，794-IL15-WT-2为野生型IL-15对照，P22339为突变型IL-15对照；794-h1-71单抗为抗PD-L1抗体对照组；Drug0为未加入任何蛋白的阴性对照。相对于野生型IL-15对照，IL-15突变体对NK细胞的增殖较弱。794-h1-71单抗对照组对NK细胞增殖无影响， 即抗PD-L1抗体对NK细胞无诱导增殖活性(图8B)，对细 胞增殖活性的影响是由IL-15产生的。
实施例15 人源化抗PD-L1抗体-IL15双功能分子/融合蛋白的小鼠体内药效
接种人黑色素瘤细胞A375细胞(北纳生物；BNCC100266)5×10 6个/100μL于NPG小鼠右后背部皮下，接种A375后次日复苏冻存PBMC(ALLCELLs，PB005F-C)以5×10 6cells/200μl量尾静脉注射到NPG小鼠(5-6周，雌性；购自北京维通达生物技术有限公司)体内。PBMC接种6天后采血40μl检测hCD45+细胞比例，待肿瘤长至约80mm 3后，去除体重、hCD45+细胞比例和肿瘤过大和过小的，按肿瘤体积将小鼠随机分为PBS组，Tecentriq组(10mg/kg)，794-IL15-WT-2组(1mg/kg)，794-IL15-com6-2组(4mg/kg)共4组，每组8只，共32只。给药方式为腹腔给药，按照分组794-IL15-WT-2组(1mg/kg)和794-IL15-com6-2组(4mg/kg)，每周一次进行给药，共给药1次；PBS组和Tecentriq组(10mg/kg)每周两次进行给药，共给药5次。每周测量3次瘤体积，记录数据。计算肿瘤体积(长径×短径 2/2)和生长抑制率(TGI TV(％)，tumor growth inhibition％,TGI TV(％)＝ [1-(Ti-T0)/(Vi-V0)]×100％；Ti：治疗组在给药第i天的肿瘤体积均值，T0：治疗组在给药第0天的肿瘤体积均值；Vi：溶剂对照组在给药第i天的肿瘤体积均值，V0：溶剂对照组在给药第0天的肿瘤体积均值)。在分组给药第14天，给药组在肿瘤体积上均有显著的抑制效果，具有统计学差异(P<0.05)，且794-IL15-WT-2组和794-IL15-com6-2组与Tecentriq组相比在肿瘤体积上有显著的抑制效果(P<0.05)。见图9A-9C，表8。
表8.受试物对免疫重建NPG小鼠A375肿瘤体积的影响
Figure PCTCN2021094167-appb-000020
注：a：平均数±标准误；b：给药组肿瘤体积与Vehicle对照组(PBS)肿瘤体积在分组给药第14天进行统计学比较，Two-way ANOVA分析，*P<0.05,**P<0.01，***P<0.001,****P<0.0001；c：给药组肿瘤体积与阳性药Tecentriq组肿瘤体积在分组给药第14天进行统计学比较，Two-way ANOVA分析，*P<0.05,**P<0.01，***P<0.001,****P<0.0001。
以上结果表明人源化抗PD-L1抗体-IL15双功能分子/融合蛋白794-IL15-WT-2和794-IL15-com6-2均对A375肿瘤皮下移植瘤生长具有显著抑制作用(P<0.0001)；794-IL15-WT-2组和794-IL15-com6-2组与Tecentriq组相比在肿瘤体积上抑制效果更强，且有显著性差异(P<0.05)。
实施例16 IL15-Fc融合蛋白的小鼠体内药效检测
接种人黑色素瘤细胞A375(北纳生物；BNCC100266)细胞5×10 6个/100μL于NPG小鼠右后背部皮下，接种A375后次日复苏冻存PBMC(ALLCELLs；PB005F-C)以5×10 6个/200μl量尾静脉注射到NPG小鼠(5-6周，雌性；购自北京维通达生物技术有限公司)体内。PBMC接种6天后采血40μl检测hCD45+细胞比例，待肿瘤长至约69mm 3后，去除体重、hCD45+细胞比例过低、肿瘤过大和过小的，按肿瘤体积将小鼠随机分为PBS组，ALT803(0.2mg/kg)，V9-IL15-61(1mg/kg)，V9-IL15-61(5mg/kg)，V9-IL15-com6(1mg/kg)，V9-IL15-com6(5mg/kg)，V5-IL15-WT(2mg/kg)共7组，每组8只，共56只。给药方式为腹腔给药，按照分组每周一次进行给药，共给药3次；每周测量3次瘤体积，记录数据。计算肿瘤体积(长径×短径 2/2)和生长抑制率(TGI TV(％)，tumor growth inhibition％,TGI TV(％)＝[1-(Ti-T0)/(Vi-V0)]×100％；Ti：治疗组在给药第i天的肿瘤体积均值，T0：治疗组在给药第0天的肿瘤体积均值；Vi：溶剂对照组在给药第i天的肿瘤体积均值，V0：溶剂对照组在给药第0天的肿瘤体积均值)。在分组给药第21天，与PBS对照组相比，给药组在肿瘤体积上均有显著的抑制效果，具有统计学差异(P<0.05)，且阳性药ALT803组和V9-IL15-com6组在肿瘤体积上有相近的抑制效果(P＞0.05)。见图10A-10C、表9。
表9.受试物对免疫重建NPG小鼠A375肿瘤体积的影响
Figure PCTCN2021094167-appb-000021
注：a：平均数±标准误；b：给药组肿瘤体积与Vehicle对照组肿瘤体积在分组给药第14天进行统计学比较，Two-way ANOVA分析，*P<0.05,**P<0.01，***P<0.001,****P<0.0001；c:对Day21天瘤体积进行identify outliers分析，V9-IL15-com6 5mpk组有一个数据异常大，将该数据剔除。
实验动物在给药期间活动和进食状态良好，给药后ALT803和V5-IL15-WT给药组体重有明显下降趋势，且出现动物死亡，表明小鼠对ALT803和V5-IL15-WT在该给药频次和剂量下不耐受。V9-IL15-61(5mpk)组和V9-IL15-com6(1mpk)组出现动物死亡，但死亡前表现出了贫血等GVHD现象，推测小鼠死亡为GVHD导致，与药物无关。见图10D、表10、表11。
表10 受试物对免疫重建的A375荷瘤NPG小鼠体重的影响
Figure PCTCN2021094167-appb-000022
注：a：平均数±标准误；b：给药组体重与Vehicle对照组体重在分组给药第21天进行统计学比较，Two-way ANOVA分析。
表11 受试物对免疫重建的A375荷瘤NPG小鼠生存的影响
Figure PCTCN2021094167-appb-000023
Figure PCTCN2021094167-appb-000024
注：a：平均数±标准误；b：分组给药后出现小鼠死亡，1表示当天有1只小鼠死亡，0表示当天有1只小鼠死亡但之前有观察到小鼠出现贫血、黄疸等症状。
以上结果表明IL15-Fc融合蛋白V9-IL15-61和V9-IL15-com6均对人免疫重建A375肿瘤皮下移植瘤生长具有显著抑制作用；V9-IL15-com6(5mg/kg)与阳性对照抗体ALT803(0.2mg/kg)相比，相比两者TGI(肿瘤生长抑制率)水平相当，且安全性优于ALT803。

Claims (18)

  1. 一种IL-15突变体多肽，其特征在于，所述多肽包含对应于野生型IL-15的Val3、Ile6、Asp8或His105的一个或多个氨基酸残基处的突变。
  2. 一种包含IL-15突变体的多肽，其特征在于，所述多肽包含对应于野生型IL-15的Val3、Ile6、Asp8或His105的一个或多个氨基酸残基处的突变。
  3. 根据权利要求1或2所述多肽，其特征在于，所述突变是替换、插入或缺失；
    优选的，所述多肽至少包含Val3、Ile6、Asp8或His105中的两个、三个或四个氨基酸残基处的突变；
    优选的，所述突变选自以下组的氨基酸替换：Val3Leu(V3L)、Ile6Asp(I6D)、Ile6Pro(I6P)、Asp8Glu(D8E)、Asp8Gln(D8Q)、Asp8Arg(D8R)、Asp8Ser(D8S)、Asp8Val(D8V)、Asp8Gly(D8G)、Asp8Ile(D8I)、Asp8Leu(D8L)、Asp8Thr(D8T)、His105Asn(H105N)、和/或His105Lys(H105K)；
    优选的，所述多肽包含下述突变或突变组合：
    (1)Asp8Glu；(2)Asp8Gln；(3)Asp8Arg；(4)Asp8Ser；(5)Asp8Val；(6)Val3Leu；(7)Ile6Asp；(8)His105Lys；(9)His105Asn；(10)Asp8Gly；(11)Asp8Ile；(12)Asp8Leu；(13)Ile6Pro；(14)Asp8Thr；(15)Asp8Glu和Val3Leu；(16)Asp8Glu和Ile6Asp；(17)Val3Leu和Ile6Asp；(18)Ile6Asp和His105Lys；(19)Asp8Ser和His105Lys；(20)Asp8Ser和His105Asn；或，(21)Val3Leu、Ile6Asp和His105Lys；
    优选的，所述IL-15突变体与人野生型IL-15具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列一致性。
  4. 根据权利要求1-3任一项所述多肽，其特征在于，所述IL-15突变体的氨基酸序列如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或SEQ ID NO.47所示；
    优选的，所述多肽具有如下特性：
    (1)介导人CD8 +T细胞的增殖；
    (2)介导人NK细胞的增殖；和/或，
    (3)抑制肿瘤生长；
    优选的，所述多肽介导CD8 +T和/或NK细胞增殖/扩增的活性低于包含野生型IL-15的多肽；
    优选的，所述野生型IL-15的氨基酸序列如SEQ ID NO.1所示。
  5. 一种蛋白，其特征在于，所述蛋白包含权利要求1-4任一项所述多肽；还包含与所述IL-15突变体融合的：(1)免疫球蛋白分子或其部分，和/或(2)IL-15Rα；
    优选的，所述免疫球蛋白分子为抗体或抗原结合片段；所述免疫球蛋白分子部分为免疫球蛋白Fc区；
    优选的，所述抗体或抗原结合片段选自：
    (1)嵌合抗体或其片段；
    (2)人源化抗体或其片段；或，
    (3)全人源抗体或其片段；
    优选的，所述抗体或抗原结合片段选自F(ab)2、Fab’、Fab、Fv、scFv、双特异抗体、纳米抗体和抗体最小识别单位中的一种或多种；
    优选的，所述免疫球蛋白Fc区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一的Fc区；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列；优选的，所述免疫球蛋白Fc区的氨基酸序列如SEQ ID NO.73所示；
    优选的，IL-15突变体通过或不通过连接肽与免疫球蛋白分子或其部分融合，或IL-15突变体通过或不通过连接肽与IL-15Rα融合；优选使用连接肽；优选使用SEQ ID NO.65、SEQ ID NO.67、SEQ ID NO.69或SEQ ID NO.71所示连接肽；
    优选的，IL-15突变体通过或不通过连接肽与IL-15Rα融合，再与免疫球蛋白分子或其部分融合；优选使用连接肽；优选使用SEQ ID NO.65、SEQ ID NO.69或SEQ ID NO.71所示连接肽。
  6. 根据权利要求5所述蛋白，其特征在于，各结构域的连接顺序自N端至C端为：
    (1)免疫球蛋白分子或其部分、IL-15Rα、IL-15突变体；
    (2)免疫球蛋白分子或其部分、IL-15突变体、IL-15Rα；
    (3)IL-15突变体、IL-15Rα、免疫球蛋白分子或其部分；
    (4)IL-15Rα、IL-15突变体、免疫球蛋白分子或其部分；
    (5)IL-15突变体、免疫球蛋白分子或其部分；
    (6)免疫球蛋白分子或其部分、IL-15Rα；
    (7)IL-15Rα、免疫球蛋白分子或其部分；
    (8)IL-15突变体、IL-15Rα；或，
    (9)IL-15Rα、IL-15突变体；
    优选的，当IL-15Rα或IL-15突变体与免疫球蛋白分子融合时，融合于免疫球蛋白分子 重链可变区的N端或免疫球蛋白Fc区的C端；当IL-15Rα或IL-15突变体与免疫球蛋白Fc区融合时，融合于免疫球蛋白Fc区的N端或C端。
  7. 一种蛋白，其包含如下4个部分：
    (1)免疫球蛋白重链；
    (2)免疫球蛋白轻链；
    (3)IL-15Rα；和，
    (4)权利要求1-4任一项所述IL-15突变体多肽；
    优选的，所述蛋白是包含由(1)-(4)部分所构成的单体的同源二聚体；
    优选的，IL-15Rα通过或不通过连接肽融合于免疫球蛋白重链可变区的N端或免疫球蛋白Fc区的C端；
    优选的，IL-15突变体多肽与IL-15Rα非共价连接，或IL-15突变体通过或不通过连接肽与IL-15Rα另一端融合。
  8. 一种蛋白，其包含如下3个部分：
    (1)免疫球蛋白Fc区；
    (2)IL-15Rα；和，
    (3)权利要求1-4任一项所述IL-15突变体多肽；
    优选的，所述蛋白是包含由(1)-(3)部分构成的单体的同源二聚体；
    优选的，IL-15Rα通过或不通过连接肽融合于IL-15突变体多肽的N端或C端，再通过或不通过连接肽融合于免疫球蛋白Fc区的N端或C端。
  9. 根据权利要求5-8任一项所述蛋白，其特征在于，所述免疫球蛋白选自抗PD-L1抗体；所述抗PD-L1抗体优选Tecentriq、KN-035、794-h1-71；
    优选的，抗PD-L1抗体包含重链可变区和轻链可变区，所述重链可变区和轻链可变区分别具有SEQ ID NO:99和SEQ ID NO:100所示序列，或者与SEQ ID NO:99和SEQ ID NO:100所示序列相比具有至少80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；或，
    所述重链可变区和轻链可变区分别具有SEQ ID NO:97和SEQ ID NO:98所示序列，或者与SEQ ID NO:97和SEQ ID NO:98所示序列相比具有至少80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    优选的，所述IL-15Rα选自IL-15Rα-sushi；优选IL-15Rα-sushi的氨基酸序列如SEQ ID NO.49、SEQ ID NO.51、SEQ ID NO.53、或SEQ ID NO.55所示。
  10. 一种特异性结合PD-L1的抗体或抗原结合片段，其特征在于，所述抗PD-L1抗体或 抗原结合片段包含重链可变区和轻链可变区；优选的，所述重链可变区和轻链可变区分别具有SEQ ID NO:99和SEQ ID NO:100所示序列，或者与SEQ ID NO:99和SEQ ID NO:100所示序列相比具有至少80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    优选的，其与人程序性死亡配体-1(PD-L1)结合的解离常数(KD)不大于1.8×10 -9M，与食蟹猴程序性死亡配体-1(PD-L1)结合的解离常数(KD)不大于9.4×10 -10M；
    或，可选地，所述抗体或抗原结合片段与猴PD-L1结合或不结合；
    可选地，所述抗体或抗原结合片段与鼠PD-L1结合或不结合；
    优选的，所述抗PD-L1抗体竞争性地结合PD-L1或其抗原表位，并且具备以下特性：
    (1)特异性结合PD-L1重组蛋白及表达PD-L1的细胞；
    (2)阻断PD-L1与PD-1蛋白的结合；
    (3)抑制PD-1与细胞表面表达的PD-L1的结合；
    (4)增强T细胞活性；或/和
    (5)抑制肿瘤生长；
    优选的，所述抗PD-L1抗体包含选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一的恒定区；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列；
    优选的，所述PD-L1抗体选自F(ab)2、Fab’、Fab、Fv、scFv、双特异抗体中的一种或多种。
  11. 一种分离的核酸分子，其特征在于，其编码权利要求1-4任一项所述的多肽、权利要求5-9任一项所述的蛋白、或权利要求10所述的抗体或抗原结合片段。
  12. 一种表达载体，其特征在于，所述表达载体包含权利要求11所述分离的核酸分子。
  13. 一种宿主细胞，其特征在于，所述宿主细胞包含权利要求11所述的分离的核酸分子、或权利要求12所述的表达载体；优选，所述宿主细胞是真核细胞或原核细胞；更优选，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选，所述宿主细胞选自中国仓鼠卵巢细胞(CHO)。
  14. 一种多肽或蛋白的制备方法，其特征在于，在适当的条件下培养权利要求13所述的宿主细胞，并分离所述多肽或蛋白。
  15. 一种药物组合物，其特征在于，所述组合物包含权利要求1-4任一项所述的多肽、权利要求5-9任一项所述的蛋白、权利要求10所述的抗体或抗原结合片段、权利要求11的分离的核酸分子、权利要求12的表达载体、权利要求13的细胞，或权利要求14方法制备的产品；以及药学上可接受的载体；优选的，所述药物组合物还包含额外的抗肿瘤剂。
  16. 权利要求1-4任一项所述的多肽、权利要求5-9任一项所述的蛋白、权利要求10所述的抗体或抗原结合片段、权利要求11的分离的核酸分子、权利要求12的表达载体、权利要求13的细胞，或权利要求14方法制备的产品、或权利要求15所述的药物组合物在制备预防和/或治疗个体中的疾病的药物中的用途；所述疾病优选为肿瘤或炎性疾病；
    优选的，所述肿瘤选自胶质母细胞瘤，前列腺癌，血液癌，B细胞肿瘤，多发性骨髓瘤，B细胞淋巴瘤，B细胞非霍奇金淋巴瘤，霍奇金淋巴瘤，慢性淋巴细胞白血病，急性髓性白血病，皮肤T细胞淋巴瘤，T细胞淋巴瘤，实体瘤，尿路上皮/膀胱癌，黑色素瘤，肺癌，肾细胞癌，乳腺癌，胃癌和食道癌，前列腺癌，胰腺癌，结肠直肠癌，卵巢癌，非小细胞肺癌和鳞状细胞头颈癌。
  17. 一种预防和/或治疗个体中的疾病的方法，包含向有此需要的患者施用权利要求1-4任一项所述的多肽、权利要求5-9任一项所述的蛋白、权利要求10所述的抗体或抗原结合片段、权利要求11的分离的核酸分子、权利要求12的表达载体、权利要求13的细胞，或权利要求14方法制备的产品、或权利要求15所述的药物组合物；所述疾病优选为肿瘤或炎性疾病；
    优选的，所述肿瘤选自胶质母细胞瘤，前列腺癌，血液癌，B细胞肿瘤，多发性骨髓瘤，B细胞淋巴瘤，B细胞非霍奇金淋巴瘤，霍奇金淋巴瘤，慢性淋巴细胞白血病，急性髓性白血病，皮肤T细胞淋巴瘤，T细胞淋巴瘤，实体瘤，尿路上皮/膀胱癌，黑色素瘤，肺癌，肾细胞癌，乳腺癌，胃癌和食道癌，前列腺癌，胰腺癌，结肠直肠癌，卵巢癌，非小细胞肺癌和鳞状细胞头颈癌。
  18. 权利要求1-4任一项所述的多肽、权利要求5-9任一项所述的蛋白、权利要求10所述的抗体或抗原结合片段、权利要求11的分离的核酸分子、权利要求12的表达载体、权利要求13的细胞，或权利要求14方法制备的产品、或权利要求15所述的药物组合物，其特征在于，用于预防和/或治疗个体中的疾病；所述疾病优选为肿瘤疾病或炎性疾病；
    优选的，所述肿瘤选自胶质母细胞瘤，前列腺癌，血液癌，B细胞肿瘤，多发性骨髓瘤，B细胞淋巴瘤，B细胞非霍奇金淋巴瘤，霍奇金淋巴瘤，慢性淋巴细胞白血病，急性髓性白血病，皮肤T细胞淋巴瘤，T细胞淋巴瘤，实体瘤，尿路上皮/膀胱癌，黑色素瘤，肺癌，肾细胞癌，乳腺癌，胃癌和食道癌，前列腺癌，胰腺癌，结肠直肠癌，卵巢癌，非小细胞肺癌和鳞状细胞头颈癌。
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