WO2020259536A1 - 单克隆抗体-细胞因子融合蛋白二聚体及其应用 - Google Patents

单克隆抗体-细胞因子融合蛋白二聚体及其应用 Download PDF

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WO2020259536A1
WO2020259536A1 PCT/CN2020/097927 CN2020097927W WO2020259536A1 WO 2020259536 A1 WO2020259536 A1 WO 2020259536A1 CN 2020097927 W CN2020097927 W CN 2020097927W WO 2020259536 A1 WO2020259536 A1 WO 2020259536A1
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heavy chain
seq
amino acid
acid sequence
monoclonal antibody
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French (fr)
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李中道
殷刘松
柳振宇
周铁林
方卓
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南京金斯瑞生物科技有限公司
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Priority to EP20832735.3A priority Critical patent/EP3988576A4/en
Priority to CN202080045773.5A priority patent/CN114008082A/zh
Priority to US17/617,156 priority patent/US20220235133A1/en
Publication of WO2020259536A1 publication Critical patent/WO2020259536A1/zh

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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C07K16/2818—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 CD28 or CD152
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C07K16/2827—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 B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to a fusion protein, in particular a protein dimer based on the fusion of a monoclonal antibody and a cytokine and its application.
  • the immune system is a set of defense systems in mammals that can resist the invasion of various pathogens and eliminate tumors.
  • Antibody molecules play a very important role in the immune system, such as recognizing tumor antigens and activating immune responses.
  • cytokines as immune regulatory factors, can simultaneously regulate innate immunity and adaptive immunity, and are of great importance in cancer immunotherapy. This type of regulatory factors participates in almost all aspects of the immune system, and exercises powerful functions in regulating and balancing the immune system. Interferon and IL-2 were first selected as cytokines for immunotherapy.
  • IFN- ⁇ is used to treat various cancers, such as melanoma, kidney cancer, follicular lymphoma, and chronic myeloid leukemia; and IL-2 was approved by the FDA in 1998 for the treatment of advanced metastatic melanoma and metastasis Kidney cancer and so on.
  • Other cytokines such as IL-7, IL-10, IL-12, IL-15 and IL-21, are also in clinical testing. Therefore, cytokines have certain potential for immunotherapy.
  • cytokine-based immunotherapy has been severely hindered, mainly due to severe side effects and poor pharmacokinetic properties. Therefore, in order to improve the therapeutic effect of cytokines, it is often necessary to modify cytokines.
  • the most commonly used method is to use antibody fusion to target cytokines to specific sites through antibodies, thereby effectively reducing cytotoxicity, improving pharmacokinetic properties, and then enhancing immune regulation. Nevertheless, some cytokines fused to the antibody will reduce the activity of the antibody itself, thus affecting the overall effect.
  • some cytokines need to be used in combination to exert their best effects, and the existing technology cannot meet this requirement.
  • the activation and suppression of immune response in immune regulation are mainly regulated by two independent signal pathways.
  • the first signal is antigen-mediated.
  • T cell receptors specifically recognize and bind to antigen peptides presented by MHC on the surface of antigen presenting cells (APC)
  • APC antigen presenting cells
  • the second signal is provided by the interaction between antigen-presenting cells and costimulatory molecules expressed on the surface of T cells.
  • the second signal pathway is very important for activating immune cells.
  • co-stimulatory and co-inhibitory receptors participate in the second signal pathway, which performs immune response and regulation of antigen-receptor presentation, while maintaining autoantigen immune tolerance, while balancing positive and negative signals to maximize Improve the immune response to invaders.
  • CD28 is a member of the CD28 family and is a major T cell costimulatory receptor, constitutively expressed on naive CD4+ and CD8+ T cells.
  • the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) molecule is also a member of the CD28 receptor family, but it is a co-inhibitory receptor.
  • This molecule is constitutively expressed in regulatory T cells (Treg) or activated by CD28 Produced after T cells. Both CD28 or CTLA-4 can bind to B7.1 (CD80) or B7.2 (CD86) ligands to transmit activation or inhibitory signals in T cells, respectively.
  • CD28 receptor ligands include CD80, CD86, programmed death 1 ligand (PD-L1), programmed death 2 ligand (PD-L2) and so on.
  • PD-L1 is a transmembrane protein, which binds to the inhibitory checkpoint molecule of PD-1, and suppresses the adaptive immune response by transmitting the suppressed "don't find me” signal.
  • the PD-1/PD-L1 signaling pathway plays a vital role in the development of immune tolerance, preventing overreaction of the immune system, thereby avoiding the occurrence of autoimmune diseases. However, this is usually deregulated as the cancer progresses, allowing tumor cells to bypass protective mechanisms by disguising themselves as healthy tissue.
  • Tumor cells overexpressing PD-L1 can evade T cell-mediated death by activating PD-1/PD-L1 inhibitory signaling pathways and inhibit the anti-tumor adaptive immune response.
  • the overexpression of PD-1 in human tumor-associated macrophages (TAMS) has also been shown to inhibit phagocytosis and tumor immunity.
  • TAMS tumor-associated macrophages
  • anti-PD-1 or anti-PD-L1 monoclonal antibodies that block the interaction of PD-1/PD-L1 are effective in cancer treatment.
  • anti-PD-1 or anti-PD-L1 monoclonal antibodies such as keytruda, opdivo, and tecentriq for the treatment of advanced cancer
  • these anti-tumor drugs only responded to some patients, suggesting a single inhibition
  • the blockade of sexual signaling pathway is not enough to activate the immune response, and there are other mechanisms to suppress the immune system.
  • Lymphocyte activation gene 3 (LAG-3) is a transmembrane protein expressed in activated T cells, natural killer cells, B cells and plasmacytoid dendritic cells.
  • LAG-3 like PD-1, is one of the immune checkpoint receptors on APC that bind to MHC II and negatively regulate T cell receptor signaling. Since LAG-3 is also expressed on Treg cells, blocking LAG-3 can inhibit the activity of Treg cells and enhance their anti-tumor immune function. Preclinical experiments have shown that blocking the LAG-3 signaling pathway has anti-tumor effects.
  • TIGIT the full name of T cell immunoglobulin and ITIM domain protein, is an inhibitory receptor shared by T cells and NK cells containing Ig and ITIM domains. TIGIT is highly expressed in T cells and natural killer (NK) cells. TIGIT, CD96, CD226 and related ligands together form an immunomodulatory signaling pathway.
  • the CD226/TIGIT/CD96 signaling pathway also contains costimulatory receptors and co-inhibitory receptors, and these receptors share some or all of the ligands, of which CD226 is a costimulatory receptor , When combined with a ligand, it transmits a stimulus signal, while TIGIT and CD96 are co-inhibitory receptors, and when combined with a related ligand, it transmits an inhibitory signal.
  • TIGIT has two ligands, CD155 and CD122, the latter two are also CD226 ligands. These two ligands are expressed in APC cells, T cells and tumor cells.
  • the ligands of CD96 include CD155 and CD111.
  • TIGIT is significantly higher than that of TIGIT and CD122 ligand, and it is also significantly higher than that of CD226 or CD96 and ligand CD155. Similar to PD-1 and CTLA-4 receptors, TIGIT is also an important inhibitory immune receptor. Inhibition of TIGIT can promote the proliferation and function of T cells; blocking TIGIT can also enhance the anti-tumor immune response mediated by NK cells. Thus inhibiting tumor growth. Therefore, monoclonal antibodies targeting the inhibitory receptor TIGIT can significantly enhance the effect of tumor immunotherapy.
  • the present invention provides a monoclonal antibody-cytokine fusion protein dimer, the monoclonal antibody includes two heavy chains, and the fusion protein dimer includes a first heavy chain polypeptide Chain and a second heavy chain polypeptide chain, wherein one heavy chain of the monoclonal antibody is linked to a cytokine to form the first heavy chain polypeptide chain, and the other heavy chain of the monoclonal antibody is optionally linked to another cell The factors are linked to form a second heavy chain polypeptide chain.
  • the monoclonal antibody is an antibody against an immune checkpoint molecule.
  • the monoclonal antibody is selected from an anti-LAG-3 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody or an anti-PD-1 antibody.
  • the monoclonal antibody is an anti-LAG-3 antibody.
  • the monoclonal antibody is an anti-PD-L1 antibody.
  • the monoclonal antibody is an anti-TIGIT antibody.
  • the monoclonal antibody is an anti-PD-1 antibody.
  • the two cytokines are selected from IL-2, IL-12, GM-CSF, IL-2 mutants, and combinations thereof.
  • the two cytokines are selected from IL-2, IL-12 or IL-2 mutants and combinations thereof.
  • the two subunits of IL-12, P35 and P40 are connected by a linker sequence to form an IL-12 single-chain protein and are present in the fusion protein dimer.
  • the two subunits P35 and P40 of IL-12 are connected by a linker sequence to form an IL-12 single-chain protein and one heavy chain of a monoclonal antibody are connected to form the first or second heavy chain polypeptide chain.
  • the monoclonal antibody and the cytokine are connected through a linker sequence or directly fused.
  • the linker sequence is selected from (S(G4S) 1-3 ), (G4S) 1-3 , KRVAPELLGGPS, ASTKG, NSPPAA, EPKSSDKTHTSPPSP, ERKSSVESPPS, and ESKYGPPSPPSP.
  • the linker sequence is (S(G4S) 1-3 ). More preferably, the linker sequence is (S(G4S) 3 ) (SEQ ID NO: 63).
  • the monoclonal antibody is linked to the cytokine through the Fc region of the heavy chain.
  • the Fc regions of the two heavy chains of the monoclonal antibody are different, and they have an asymmetric complementary structure between them.
  • the asymmetric complementary structure can be formed, for example, by KiH (knobs-into-holes) technology.
  • the combination with mutations in the Fc regions of the two heavy chains of the monoclonal antibody is selected from the mutation combination T366W/S354C and the mutation combination T366S/L368A/Y407V/Y349C.
  • the Fc region of any heavy chain of the monoclonal antibody has the mutation combination T366W/S354C
  • the Fc region of the complementary heavy chain has the mutation combination T366S/L368A/Y407V/Y349C.
  • the Fc region of the heavy chain of the monoclonal antibody is selected from a mutant of human IgG1 and a mutant of human IgG4.
  • one heavy chain of the monoclonal antibody is linked to the cytokine IL-12 to form a first heavy chain polypeptide chain
  • the other heavy chain of the monoclonal antibody is linked to the cytokine IL-2 or IL-2.
  • the mutant is connected to form the second heavy chain polypeptide chain; or one heavy chain of the monoclonal antibody is connected to the cytokine IL-2 or IL-2 mutant to form the first heavy chain polypeptide chain, and the other of the monoclonal antibody
  • the heavy chain is connected with the cytokine IL-12 to form a second heavy chain polypeptide chain.
  • one heavy chain of the monoclonal antibody is linked to the cytokine IL-12, IL-2, or IL-2 mutant to form a first heavy chain polypeptide chain, and the other heavy chain of the monoclonal antibody The cytokine is not connected as the second heavy chain polypeptide chain.
  • one heavy chain of the monoclonal antibody is connected to the cytokine IL-12 to form a first heavy chain polypeptide chain, and the other heavy chain of the monoclonal antibody is not connected to the cytokine as the second heavy chain polypeptide chain.
  • one heavy chain of the monoclonal antibody is connected to the cytokine IL-2 or IL-2 mutant to form a first heavy chain polypeptide chain, and the other heavy chain of the monoclonal antibody is not connected to the cytokine As the second heavy chain polypeptide chain.
  • the cytokine is linked to the C-terminus of the Fc region of the monoclonal antibody heavy chain through a linker sequence. In other embodiments, the cytokine is directly linked to the C-terminus of the Fc region of the monoclonal antibody heavy chain.
  • the heavy chain of the anti-LAG-3 monoclonal antibody has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97% identical to that shown in SEQ ID NOs: 8, 12 or 14. %, 98%, or 99% identity, the light chain of the anti-LAG-3 monoclonal antibody has at least 80%, 85%, 90%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 10 , 97%, 98% or 99% identity sequence.
  • the heavy chain of the anti-LAG-3 monoclonal antibody has the amino acid sequence shown in SEQ ID NOs: 8, 12 or 14, and the light chain of the LAG-3 monoclonal antibody has the amino acid sequence shown in SEQ ID NOs: ID NO: The amino acid sequence shown in 10.
  • the heavy chain of the anti-PD-L1 monoclonal antibody has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97% identical to that shown in SEQ ID NOs: 22, 26 or 28. %, 98%, or 99% identity
  • the light chain of the anti-PD-L1 monoclonal antibody has at least 80%, 85%, 90%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 24 , 97%, 98% or 99% identity sequence.
  • the heavy chain of the anti-PD-L1 monoclonal antibody has the amino acid sequence shown in SEQ ID NOs: 22, 26 or 28 and the light chain of the anti-PD-L1 monoclonal antibody has the amino acid sequence shown in SEQ ID NOs: ID NO: 24 amino acid sequence.
  • the heavy chain of the anti-TIGIT monoclonal antibody has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 97%, and SEQ ID NOs: 36, 40, or 42 98% or 99% identical sequence
  • the light chain of the anti-TIGIT monoclonal antibody has at least 80%, 85%, 90%, 95%, 96%, 97%, and the amino acid sequence shown in SEQ ID NO: 38 98% or 99% identity sequence.
  • the heavy chain of the anti-TIGIT monoclonal antibody has an amino acid sequence as shown in SEQ ID NOs: 36, 40 or 42
  • the light chain of the anti-TIGIT monoclonal antibody has an amino acid sequence as shown in SEQ ID NO: 38. The amino acid sequence shown.
  • the heavy chain of the anti-PD-1 monoclonal antibody has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97% identical to that shown in SEQ ID NOs: 50, 54 or 56. %, 98%, or 99% identity, the light chain of the anti-PD-1 monoclonal antibody has at least 80%, 85%, 90%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 52 , 97%, 98% or 99% identity sequence.
  • the heavy chain of the anti-PD-1 monoclonal antibody has the amino acid sequence shown in SEQ ID NOs: 50, 54 or 56
  • the light chain of the anti-PD-1 monoclonal antibody has the amino acid sequence shown in SEQ ID NOs: ID NO: 52 amino acid sequence.
  • the IL-12 single-chain protein has an amino acid sequence as shown in SEQ ID NO: 4.
  • the IL-2 has an amino acid sequence as shown in SEQ ID NO: 8.
  • the fusion protein dimer provided by the present invention includes two light chains of a monoclonal antibody.
  • the first and second heavy chain polypeptide chains of the fusion protein are selected from those with SEQ ID NOs: 12, 14, 16, 18, 20, 26, 28, 30, 32, 34, 40,
  • the amino acid sequence of 42, 44, 46, 48, 54, 56, 58, 60, or 62 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.
  • the first and second heavy chain polypeptide chains of the fusion protein are selected from those having SEQ ID NOs: 12, 14, 16, 18, 20, 26, 28, 30, 32, 34, 40, 42, 44, 46, 48, 54, 56, 58, 60 or 62 amino acid sequence.
  • the monoclonal antibody is an anti-LAG-3 antibody, and the first and second heavy chain polypeptide chains are selected from those having the amino acid sequence shown in SEQ ID NOs: 12, 14, 16, 18, or 20. A sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
  • the monoclonal antibody is an anti-LAG-3 antibody, and the first and second heavy chain polypeptide chains are selected from those having SEQ ID NOs: 12, 14, 16, 18, or 20. Amino acid sequence.
  • the monoclonal antibody is an anti-LAG-3 antibody, wherein the first heavy chain polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 18, and the second heavy chain polypeptide chain Has the amino acid sequence shown in SEQ ID NO: 16; the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 16, and the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 14. Or the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 20, and the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 14.
  • the present invention provides an anti-LAG-3 monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain and two anti-LAG-3 antibodies
  • the light chain of the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 18, and the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 16, the light chain It has the amino acid sequence shown in SEQ ID NO: 8.
  • the present invention provides an anti-LAG-3 monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain, and two anti-LAG-3
  • the light chain of an antibody the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 16
  • the second heavy chain polypeptide chain has the amino acid sequence shown in SED ID NO: 14
  • the light chain has the amino acid sequence shown in SEQ ID The amino acid sequence shown as NO: 8.
  • the present invention provides an anti-LAG-3 monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain and two anti-LAG-3 antibodies
  • the light chain of the first heavy chain polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 20
  • the second heavy chain polypeptide chain has an amino acid sequence as shown in SED ID NO: 14
  • the light chain has an amino acid sequence as shown in SEQ ID NO :
  • the monoclonal antibody is an anti-PD-L1 antibody, and the first and second heavy chain polypeptide chains are selected from those having the amino acid sequence shown in SEQ ID NOs: 26, 28, 30, 32, or 34 A sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
  • the monoclonal antibody is an anti-PD-L1 antibody, and the first and second heavy chain polypeptide chains are selected from those having SEQ ID NOs: 26, 28, 30, 32, or 34. Amino acid sequence.
  • the monoclonal antibody is an anti-PD-L1 antibody
  • the first heavy chain polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 32, wherein: the second heavy chain polypeptide chain It has the amino acid sequence shown in SEQ ID NO: 30; the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 30, and the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 28.
  • the monoclonal antibody is an anti-PD-L1 antibody, the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 34, and the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 28 amino acid sequence.
  • the present invention provides an anti-PD-L1 monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain and two anti-PD-L1 antibodies
  • the light chain of the first heavy chain has the amino acid sequence shown in SEQ ID NO: 32
  • the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 30, and the light chain It has the amino acid sequence shown in SEQ ID NO: 24.
  • the present invention provides an anti-PD-L1 monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain and two anti-PD-L1
  • the first heavy chain polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 30
  • the second heavy chain polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 28.
  • the chain has an amino acid sequence as shown in SEQ ID NO: 24.
  • the present invention provides an anti-PD-L1 monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain and two anti-PD-L1
  • the light chain of an antibody the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 34
  • the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 28, and the light The chain has an amino acid sequence as shown in SEQ ID NO: 24.
  • the monoclonal antibody is an anti-TIGIT antibody
  • the first and second heavy chain polypeptide chains are selected from those having the amino acid sequence shown in SEQ ID NOs: 40, 42, 44, 46 or 48 at least 80%. %, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity sequence.
  • the monoclonal antibody is an anti-TIGIT antibody
  • the first and second heavy chain polypeptide chains are selected from those having the amino acid sequence shown in SEQ ID NOs: 40, 42, 44, 46, or 48 .
  • the monoclonal antibody is an anti-TIGIT antibody, wherein: the first heavy chain polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 46, and the second heavy chain polypeptide chain has The amino acid sequence shown in SEQ ID NO: 44; the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 44, and the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 42 Amino acid sequence; or the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 48, and the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 42.
  • the present invention provides an anti-TIGIT monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain and two light chains of an anti-TIGIT antibody,
  • the first heavy chain polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 46
  • the second heavy chain polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 44
  • the light chain has an amino acid sequence as shown in SEQ ID.
  • the amino acid sequence shown as NO:38 The amino acid sequence shown as NO:38.
  • the present invention provides an anti-TIGIT monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain and two light chains of an anti-TIGIT antibody
  • the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 44
  • the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 42
  • the light chain has the amino acid sequence shown in SEQ ID NO: 42.
  • ID NO: 38 amino acid sequence amino acid sequence shown in SEQ ID NO:
  • the present invention provides an anti-TIGIT monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain and two light chains of an anti-TIGIT antibody
  • the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 48
  • the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 42
  • the light chain has the amino acid sequence shown in SEQ ID NO: 42.
  • ID NO: 38 amino acid sequence amino acid sequence shown in SEQ ID NO: 38 amino acid sequence.
  • the monoclonal antibody is an anti-PD-1 antibody
  • the first and second heavy chain polypeptide chains are selected from those having the amino acid sequence shown in SEQ ID NOs: 54, 56, 58, 60, or 62. A sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
  • the monoclonal antibody is an anti-PD-1 antibody
  • the first and second heavy chain polypeptide chains are selected from those having SEQ ID NO: 54, 56, 58, 60, or 62. Amino acid sequence.
  • the monoclonal antibody is an anti-PD-1 antibody, wherein: the first heavy chain polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 60, and the second heavy chain polypeptide chain Has the amino acid sequence shown in SEQ ID NO: 58; the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 58, and the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 56 Or the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 62, and the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 56.
  • the present invention provides an anti-PD-1 monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain and two anti-PD-1 antibodies
  • the light chain of the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 60
  • the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 58
  • the light chain It has the amino acid sequence shown in SEQ ID NO: 52.
  • the present invention provides an anti-PD-1 monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain and two anti-PD-1
  • the light chain of an antibody the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 58
  • the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 56
  • the light chain has an amino acid sequence as shown in SEQ ID NO:52.
  • the present invention provides an anti-PD-1 monoclonal antibody-cytokine fusion protein dimer, comprising a first heavy chain polypeptide chain, a second heavy chain polypeptide chain and two anti-PD-1
  • the first heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 62
  • the second heavy chain polypeptide chain has the amino acid sequence shown in SEQ ID NO: 56.
  • the chain has an amino acid sequence as shown in SEQ ID NO:52.
  • the present invention provides an isolated polynucleotide encoding the first or second heavy chain polypeptide chain of the fusion protein dimer of the present invention.
  • the polynucleotide has SEQ ID NO: 11, 13, 15, 17, 19, 25, 27, 29, 31, 33, 39, 41, 43, 45, 47, 53, The nucleotide sequence shown as 55, 57, 59 or 61.
  • the present invention provides an expression vector containing the polynucleotide.
  • the present invention provides a host cell containing the expression vector.
  • the present invention provides the application of the fusion protein dimer, polynucleotide, expression vector or host cell in the preparation of anti-tumor drugs.
  • the tumor is melanoma, gastric cancer, kidney cancer, Hodgkin's lymphoma, head and neck cancer, bladder cancer, or non-small cell lung cancer.
  • the tumor is melanoma, gastric cancer or kidney cancer.
  • the present invention provides an anti-tumor pharmaceutical composition, the pharmaceutical composition comprising the fusion protein dimer and a pharmaceutically acceptable carrier.
  • the present invention provides a method for treating tumors, comprising administering to a subject a therapeutically effective amount of the fusion protein dimer or a pharmaceutical composition containing the fusion protein dimer.
  • the anti-tumor pharmaceutical composition provided by the present invention can be administered to a subject through at least one route selected from the group consisting of: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, abdominal Internal, intracapsular, intracartilage, intracavity, intracorporeal, intracerebellar, intracerebroventricular, intracolon, intracervix, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, Prostate, lung, rectum, kidney, retina, spinal column, synovial membrane, intrathoracic cavity, tympanum, intrauterine, intravesical, intravitreal, rapid injection, subconjunctival, transvaginal, transrectal, Cheek, sublingual, intranasal, intratumoral and transdermal.
  • the anti-tumor pharmaceutical composition is administered to the subject intratumorally or intravenously.
  • the present invention uses antibody Fc heterodimer technology to provide a fusion protein with anti-tumor activity
  • the fusion protein includes a monoclonal antibody and one or more cytokines.
  • “monoclonal antibody-cytokine fusion protein”, “antibody/cytokine fusion protein” and “monoclonal antibody-cytokine fusion protein” all refer to monoclonal antibody-cytokine based on antibody Fc heterodimer technology
  • the fusion protein includes one or more cytokines connected through the Fc region of the antibody heavy chain to form a fusion protein.
  • the monoclonal antibody and the cytokine are connected to the C-terminus of the Fc region of the antibody heavy chain through a linker sequence.
  • antibody refers to a large "Y"-shaped protein secreted by plasma cells (effector B cells) and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. In the past 10 years, more and more monoclonal antibodies have been widely used in tumor treatment. Antibodies are usually tetramers composed of two identical heavy chains and two identical light chains connected to each other by disulfide bonds.
  • the "anti-immune checkpoint molecule antibody” referred to in the present invention refers to an antibody that specifically binds to an immune checkpoint molecule.
  • the immune checkpoint molecule includes but not limited to LAG-3, PD-L1, TIGIT, PD-1 or CTLA-4, the antibody refers to a monoclonal antibody with two heavy chains and two light chains.
  • antibody Fc region refers to a "Y"-shaped handle region, that is, a fragment crystallizable (Fc), including the second and third constant domains (CH2 and CH3 domains) of the heavy chain. ).
  • the antibody Fc region can be obtained by hydrolyzing antibody molecules by proteolytic enzymes (such as papain).
  • the dimeric protein involved in the present invention refers to the process of protein formation. If two subunits/monomers are the same, it is called a homodimer (homo-), if it is not exactly the same subunit/monomer The combination is called a heterodimer (hetero-).
  • antibody Fc heterodimer refers to a protein composed of two different subunits/monomers, and each subunit/monomer contains an antibody Fc fragment. The key is that the two antibody Fc fragments have different amino acid site mutations that can form complementary protein spatial structures, so that two different subunits/monomers can be combined correctly.
  • KiH (knobs-into-holes) technology refers to a technology that facilitates assembly between two heterologous antibody heavy chains.
  • the smaller threonine (T) at position 366 of the CH3 region of an antibody can be mutated to the larger tyrosine (Y) to form a prominent "knobs" structure (T366Y);
  • the larger tyrosine (Y) residue at position 407 in the CH3 region of another antibody was mutated to a smaller threonine (T) to form a recessed "holes" type structure (Y407T); use this "knobs”
  • the steric hindrance effect of the "into-holes" structure ie, asymmetric complementary structure
  • the invention achieves a better assembly effect by combining multiple site mutations in the Fc regions of the two antibodies.
  • cytokine is a low-molecular-weight soluble protein produced by immunogens, mitogens or other stimulants induced by various cells. In the organism, it combines with its specific cell surface receptors to transmit intracellular signals, thereby changing cell functions, and has multiple functions such as regulating innate immunity and adaptive immunity, hematogenesis, cell growth, APSC pluripotent cells, and damaged tissue repair .
  • interleukins such as IL-2 or IL-12
  • Cytokines can not only function alone, but also can be fused with antibodies to form antibody-cytokine fusion proteins.
  • This new protein form organically combines the specific targeting of antibodies with the immune regulation of cytokines, thereby enhancing the immunotherapy effect of antibodies. More importantly, the fused cytokine is transported and enriched to the tumor site through the targeting of the antibody, thereby effectively avoiding the side effects caused by the high-dose cytokine alone.
  • isolated polynucleotide refers to a polynucleotide that does not occur naturally in nature, including polynucleotides isolated from nature (including living organisms) through biological technology, and also includes artificially synthesized polynucleotides.
  • the isolated polynucleotide can be genomic DNA, cDNA, mRNA or other synthetic RNA, or a combination thereof.
  • This article provides a number of nucleotide sequences for encoding the fusion protein dimer and other polypeptide fragments of the present invention. It should be pointed out that those skilled in the art can use the amino acid sequence provided herein based on the codon degeneracy. , Design a nucleotide sequence that is not exactly the same as the nucleotide sequence provided above, but all encode the same amino acid sequence. These modified nucleotide sequences are also included in the scope of the present invention.
  • vector When referring to polynucleotides, the term “vector” is used to refer to any molecule (for example, nucleic acid, plasmid, virus, etc.) used to transfer nucleotide coding information into a host cell.
  • expression vector refers to a vector suitable for expressing a target gene (nucleotide sequence to be expressed) in a host cell, and usually includes a target gene, a promoter, a terminator, a marker gene and other parts.
  • host cell refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a selected gene of interest.
  • the term includes the offspring of the parent cell, regardless of whether the offspring and the original parent cell are identical in morphology or genetic composition, as long as the offspring has the selected target gene.
  • Commonly used host cells include bacteria, yeast, mammalian cells, etc., such as CHO cells.
  • the term "pharmaceutically acceptable carrier” is used to refer to solid or liquid diluents, fillers, antioxidants, stabilizers and other substances that can be safely administered. These substances are suitable for humans and/or It is administered to animals without excessive side effects and is suitable for maintaining the vitality of the drugs or active agents located therein. According to the route of administration, various carriers well-known in the art can be administered, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, and polyols.
  • the pharmaceutical composition provided by the present invention can be made into clinically acceptable dosage forms such as powder and injection.
  • the pharmaceutical composition of the present invention can be administered to the subject by any appropriate route, for example, oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or inhalation, transdermal, etc. Route administration.
  • terapéuticaally effective amount refers to the amount of the active compound sufficient to cause the biological or medical response desired by the clinician in the subject.
  • the “therapeutically effective amount” of the fusion protein of the present invention can be determined by those skilled in the art according to factors such as the route of administration, the subject's weight, age, and disease conditions. For example, a typical daily dosage may range from 0.01 mg to 100 mg of active ingredient per kg body weight.
  • amino acid sequence identity is defined as comparing sequences and introducing gaps when necessary to obtain the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity, and the candidate sequence is similar to a specific peptide or polypeptide sequence. The percentage of amino acid residues with the same amino acid residues. Sequence comparisons can be performed in a variety of ways within the skill of the art to determine percent amino acid sequence identity, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required to obtain the maximum comparison over the entire length of the sequence being compared.
  • the present invention uses the advantages of immune cytokines and monoclonal antibodies to improve the effect of immunotherapy by fusing cytokines to anti-immune checkpoint molecular antibodies, such as anti-LAG-3 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies or anti-PD -1 at the C-terminus of the Fc region of the heavy chain of the antibody, developed anti-immune checkpoint protein antibody-cytokine fusion protein dimers.
  • anti-immune checkpoint molecular antibodies such as anti-LAG-3 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies or anti-PD -1 at the C-terminus of the Fc region of the heavy chain of the antibody, developed anti-immune checkpoint protein antibody-cytokine fusion protein dimers.
  • the activity of these fusion proteins was evaluated by in vitro activity analysis. The study found that these fusion protein dimers Not only fully retains the biological activity of the antibody, but also significantly improves the biological activity of the cytokine.
  • the cytokines mentioned in the present invention include interleukins, such as interleukin-2 (IL-2), interleukin-12 (IL-12) and granulocyte-macrophage colony stimulating factor (GM-CSF) Among them, interleukin-2 and interleukin-12 participate in all aspects of immune regulation, thereby enhancing the immune response. More importantly, the present invention utilizes the targeting specificity of antibody molecules to enhance the immune response of immune cells and weaken the toxicity of cytokines, thereby ensuring the safety of medication on the premise of improving the efficacy of the drug.
  • interleukins such as interleukin-2 (IL-2), interleukin-12 (IL-12) and granulocyte-macrophage colony stimulating factor (GM-CSF)
  • IL-2 and interleukin-12 participate in all aspects of immune regulation, thereby enhancing the immune response.
  • the present invention utilizes the targeting specificity of antibody molecules to enhance the immune response of immune cells and weaken the toxicity of cytokines,
  • Example 1 Design and construction of monoclonal antibody-cytokine fusion protein and its expression and purification in CHO cells
  • the cytokine IL-2 or IL-12 can be linked to the C-terminus of the heavy chain of the monoclonal antibody through a linker sequence (Ser(Gly4Ser)3) (ie S(G4S)3, SEQ ID NO: 63), the resulting fusion protein Maintain the dual effects of antibodies and cytokines.
  • IL-12 Since functional IL-12 is composed of two subunits, p35 and p40, IL-12 will be fused to the antibody Fc separately in the form of two subunits, or the two subunits of IL-12 will be first passed through the linker sequence ( S(G4S)3) forms a single-chain protein, which is then connected to the C-terminus of the heavy chain of the monoclonal antibody through a linker sequence (S(G4S)3).
  • S(G4S)3 linker sequence
  • the cytokine In the traditional antibody-cytokine fusion protein structure, the cytokine is connected to the two heavy chains or two light chains of the antibody at the same time, and thus exists in the form of homodimer. However, the pharmacokinetic properties of this immune cytokine based on IgG platform fusion are poor.
  • the core of antibody heterodimer technology is to separately transform the two heavy chains of monoclonal antibodies to produce an asymmetric complementary structure, so that the two modified chains can be combined and the production of homodimers can be avoided.
  • the principle of transformation is based on the following aspects: hydrophobic/spatial complementarity (such as KiH and ZW1), electrostatic complementation (such as DD-KK), spatial complementation + electrostatic interaction (such as EW-RVT), and spatial complementation + hydrogen bond complementation (such as A107) Wait.
  • hydrophobic/spatial complementarity such as KiH and ZW1
  • electrostatic complementation such as DD-KK
  • spatial complementation + electrostatic interaction such as EW-RVT
  • spatial complementation + hydrogen bond complementation such as A107
  • the other heavy chain is not connected to the cytokine; or one cytokine is connected to the heavy chain Fm1, and the other cytokine is connected to the heavy chain Fm2, so that the final heterodimer is formed
  • the number of each cytokine contained is one instead of two in homodimers.
  • the monoclonal antibody-cytokine fusion protein constructed by the above method will be expressed in CHO-3E7 cells, and then purified by Protein A affinity chromatography and molecular sieve to obtain the protein for in vitro analysis.
  • Sample dilution Dilute the purified fusion protein solution sample and IL-2 control (100 ⁇ g/ml) in a gradient, with 9 concentration gradients for each sample and 3 replicate wells for each concentration.
  • Sample dilution Dissolve and dilute the fusion protein solution sample and IL-12 control (100 ⁇ g/ml) obtained by the purification of the test, and set 9 concentration gradients for each sample and 3 replicate holes for each concentration.
  • Sample collection Take out the 96-well plate from the incubator, centrifuge at 3700 rpm 4 degrees for 20 minutes, aspirate the supernatant, mark it, and store at -20°C.
  • Detection Take out the frozen samples, and detect the content of human IFN gamma in the samples according to the operating steps of the human IFN gamma ELISA MAX TM Standard kit. Place the 96-well plate on the microplate reader and detect the OD value at 450nm wavelength.
  • the experiment to detect the in vitro function of anti-PD-1/PD-L1 monoclonal antibody-cytokine fusion protein samples used Promega's PD-1/PD-L1 blocking function reporter gene kit (PD-1 /PD-L1 Blockade Bioassay, Promega kit product number is J1250).
  • the kit detection system consists of two genetically engineered cell lines.
  • the stimulating cell line is PD-L1 aAPC/CHO-K1 cells, which stably express human PD-L1 and a cell surface protein that can activate homologous TCR in an antigen-independent manner.
  • the effector cell line is a Jurkat T cell line, which stably expresses human PD-1 and NFAT-induced luciferase reporter gene.
  • the PD-1/PD-L1 interaction inhibits TCR signal transduction and NFAT-mediated luciferase activity.
  • the addition of anti-PD-1/PD-L1 antibody can block the binding of PD-1 and PD-L1, thereby enabling the activation of the TCR signaling pathway and the enhancement of NFAT-mediated luciferase activity and generating chemiluminescence (Luminescence).
  • the effector cell line Jurkat T cells were spread in a 96-well plate, and then the anti-PD-1/PD-L1 antibody-cytokine fusion protein sample and the stimulation cell line PD-L1aAPC/CHO-K1 cells were added. The system was incubated at 37°C for 6 hours. Then add Bio-Glo TM fluorescence detection reagent, and incubate at room temperature for 5-10 minutes. Finally, use a chemical fluorescence signal plate reader to read the fluorescence signal in the 96-well plate.
  • This experiment uses the form of 8-concentration triple wells, the relative fluorescence value is taken as the y-axis, and the concentration of the antibody sample is taken as the x-axis, and a four-parameter curve is drawn.
  • GraphPad Prism software was used to analyze the curve and obtain the EC 50 value of the anti-PD-1/PD-L1 antibody-cytokine fusion protein sample.
  • the experiment to detect the in vitro function of anti-TIGIT monoclonal antibody-cytokine fusion protein samples used Promega's TIGIT/CD155 Blockade Reporter Gene Kit (TIGIT/CD155 Blockade Bioassay, Promega kit product) The number is J2201).
  • the kit detection system consists of two genetically engineered cell lines.
  • the stimulating cell line is CD155 aAPC/CHO-K1 cells, which express human CD155 and a cell surface protein that can activate the TCR complex in an antigen-independent manner.
  • the effector cell line is a Jurkat T cell line that expresses human TIGIT and a luciferase reporter gene driven by a natural promoter that responds to TCR activation and CD226 costimulation.
  • TIGIT inhibits CD226 activation and promoter-mediated luminescence.
  • anti-TIGIT antibody can block the interaction between TIGIT and CD155 or inhibit the ability of TIGIT to prevent CD226 from forming homodimers, thereby restoring the promoter-mediated chemiluminescence (Luminescence).
  • the effector cell line Jurkat T cells were plated in a 96-well plate, and then the anti-TIGIT antibody-cytokine fusion protein sample and the stimulation cell line CD155 aAPC/CHO-K1 cells were added. The system was incubated at 37°C for 6 hours. Then add Bio-Glo TM fluorescence detection reagent, and incubate at room temperature for 5-10 minutes. Finally, use a chemical fluorescence signal plate reader to read the fluorescence signal in the 96-well plate. This experiment uses the form of 8-concentration triple wells, the relative fluorescence value is taken as the y-axis, and the concentration of the antibody sample is taken as the x-axis, and a four-parameter curve is drawn. GraphPad Prism software was used to analyze the curve and obtain the EC 50 value of the anti-TIGIT antibody-cytokine fusion protein sample.
  • the experiment to detect the in vitro function of anti-LAG3 monoclonal antibody-cytokine fusion protein samples used Promega's LAG3 Blockade Reporter Gene Kit (LAG3 Blockade Bioassay, Promega Kit product number is CS194804) .
  • the kit detection system consists of two genetically engineered cell lines.
  • the stimulating cell line is aAPC/Raji cell, which expresses MHC II and can activate TCR complex.
  • the effector cell line is a Jurkat T cell line that expresses human LAG3 and a luciferase reporter gene driven by a natural promoter that responds to TCR activation.
  • LAG3 inhibits MHCII activation and promoter-mediated luminescence.
  • the addition of anti-LAG3 antibody can block the interaction between LAG3 and MHCII, thereby restoring the promoter-mediated chemiluminescence (Luminescence).
  • the effector cell line Jurkat T cells were plated in a 96-well plate, and then the anti-LAG-3 antibody-cytokine fusion protein sample and the stimulation cell line aAPC/Raji cells were added. The system was incubated at 37°C for 6 hours. Then add Bio-Glo TM fluorescence detection reagent, and incubate at room temperature for 5-10 minutes. Finally, use a chemical fluorescence signal plate reader to read the fluorescence signal in the 96-well plate. This experiment uses the form of 8-concentration triple wells, the relative fluorescence value is taken as the y-axis, and the concentration of the antibody sample is taken as the x-axis, and a four-parameter curve is drawn. GraphPad Prism software was used to analyze the curve and obtain the EC 50 value of the anti-LAG3 antibody-cytokine fusion protein sample.
  • the components used to construct the fusion protein are monoclonal antibodies, cytokines IL-12 and IL-2.
  • the four monoclonal antibodies are used in the present invention to test whether the technology platform is suitable for most monoclonal antibodies.
  • the four monoclonal antibodies are anti-LAG-3 monoclonal antibodies, anti-PD-L1 monoclonal antibodies, anti-TIGIT monoclonal antibodies and anti-P-1 monoclonal antibodies.
  • the anti-LAG-3 monoclonal antibody is Relatlimab from BMS, which belongs to human IgG4 antibody.
  • the anti-PD-L1 monoclonal antibody is Atezolizumab from Roche, which is a human IgG1 antibody.
  • the anti-TIGIT monoclonal antibody is Tiragolumab from Roche, which belongs to human IgG1 antibody.
  • the anti-PD-1 monoclonal antibody comes from the patent application WO2018119474 and belongs to human IgG4 antibody.
  • the heavy or light chain coding sequence of each monoclonal antibody was inserted into the multiple cloning site EcoRI and HindIII of the pTT5 expression vector, and at the same time, the Kozak sequence GCCGCCACC and the signal peptide sequence (SEQ ID NO: 2) Helps to secrete protein out of the cell.
  • knob-into-holes technology to transform the Fc region of the monoclonal antibody heavy chain.
  • One of the mutation sites of the heavy chain Fc chain is T366W/S354C, and the other heavy chain
  • the mutation site combination of Fc chain is T366S/L368A/Y407V/Y349C.
  • K the last amino acid at the C-terminus of the two modified heavy chains
  • the DNA sequences encoding IL-12 and IL-2 are respectively connected to the C-terminus of the heavy chain through a linker sequence (S(G4S)3) using the Gibson assembly method, thereby generating plasmids encoding different heavy chain fusion proteins.
  • the anti-LAG-3 monoclonal antibody is composed of heavy chain H1 and light chain L1.
  • the heavy chain H1 was transformed by KIH technology to produce two heavy chain mutants H2 and H3.
  • the IL-12 sequence is connected to the C-terminus of the heavy chain H2 through a linker sequence (S(G4S)3) to produce a new polypeptide called H2a.
  • the IL-2 sequence is linked to the C-terminus of the heavy chain H2 and H3 through a linker sequence (S(G4S)3), respectively, to produce new polypeptides called H2b and H3a.
  • the anti-PD-L1 monoclonal antibody is composed of heavy chain H4 and light chain L2.
  • the heavy chain H4 was transformed by KIH technology to produce two heavy chain mutants H5 and H6.
  • the IL-12 sequence is connected to the C-terminus of the heavy chain H5 through a linker sequence (S(G4S)3) to produce a new polypeptide called H5a.
  • the IL-2 sequence is connected to the C-terminus of the heavy chain H5 and H6 through a linker sequence (S(G4S)3), respectively, to produce new polypeptides called H5b and H6a.
  • the anti-TIGIT monoclonal antibody is composed of heavy chain H7 and light chain L3.
  • the heavy chain H7 was transformed by KIH technology to produce two heavy chain mutants H8 and H9.
  • the IL-12 sequence is connected to the C-terminus of the heavy chain H8 through a linker sequence (S(G4S)3) to produce a new polypeptide called H8a.
  • the IL-2 sequence is connected to the C-terminus of the heavy chain H8 and H9 through a linker sequence (S(G4S)3), respectively, to produce new polypeptides called H8b and H9a.
  • the anti-PD-1 monoclonal antibody is composed of heavy chain H10 and light chain L4.
  • the heavy chain H10 was transformed by KIH technology, resulting in two heavy chain mutants H11 and H12.
  • the IL-12 sequence is connected to the C-terminus of the heavy chain H11 through a linker sequence (S(G4S)3) to produce a new polypeptide called H11a.
  • the IL-2 sequence is connected to the C-terminus of the heavy chains H11 and H12 through a linker sequence (S(G4S)3), respectively, to produce new polypeptides called H11b and H12a.
  • the light chain L1 and the two heavy chains H2a and H3a are combined to produce the antibody-cytokine fusion protein mLAG301, and the light chain L1 and the two heavy chains H2a and H3 are combined to produce the antibody-cytokine fusion protein mLAG302.
  • the light chain L1 and the two heavy chains are combined to produce the antibody-cytokine fusion protein mLAG302.
  • the combination of one heavy chain H2b and H3 produces the antibody-cytokine fusion protein mLAG303; the light chain L2 and the two heavy chains H5a and H6a are combined to produce the antibody-cytokine fusion protein mPDL101.
  • the light chain L2 and the two heavy chains H5a and H6 Combine to produce antibody-cytokine fusion protein mPDL102, combine light chain L2 and two heavy chains H5b and H6 to produce antibody-cytokine fusion protein mPDL103; combine light chain L3 and two heavy chains H8a and H9a to produce antibody-cytokine
  • the fusion protein mTIGIT01 combines the light chain L3 and the two heavy chains H8a and H9 to produce the antibody-cytokine fusion protein mTIGIT02, and combines the light chain L3 and the two heavy chains H8b and H9 to produce the antibody-cytokine fusion protein mTIGIT03;
  • the combination of chain L4 and the two heavy chains H11a and H12a produces the antibody-cytokine fusion protein mPD101, the light chain L4 and the two heavy chains H11a and H12 combine to produce the antibody-cytokine fusion protein mPD102, the light chain L4 and the two heavy chains
  • Table 1 Plasmids and proteins for constructing antibody-cytokine fusion proteins
  • the fusion protein particles constructed above on the pTT5 expression vector were transiently transfected into CHO-3E7 cells with PEI transfection reagent, and then cultured at 37°C for 6 days. The culture supernatant is collected by centrifugation, and the fusion protein is purified by a Protein A affinity column, and then further purified by a molecular sieve, and the final purity reaches more than 95%.
  • the present invention uses 4 different antibodies to construct a series of antibody-cytokine fusion proteins.
  • the antibody-cytokine fusion protein fused with IL-2 compared with the biological activity of free IL-2, the IL-2 activity of all fusion proteins was significantly enhanced ( Figures 2, 3, 4 and 5). ), which shows that the structure of monoclonal antibody-cytokine fusion protein based on antibody heterodimer technology can significantly increase the activity of IL-2.
  • the IL-12 activity of the antibody-cytokine fusion protein constructed with 4 different antibodies is enhanced or unchanged ( Figures 6, 7, 8 and 9).
  • Figures 6, 7, 8 and 9 the IL-12 activity of the fusion protein fused with IL-2 remains unchanged, indicating that the structure of the monoclonal antibody-cytokine fusion protein based on antibody heterodimer technology can well maintain the activity of IL-12 .
  • the antibody-cytokine fusion protein fused with IL-12 and IL-2 at the same time has stronger cytokine activity than the IL-12 fusion protein with only IL-12, which shows that IL-12 and IL -2 may have a synergistic effect, which further proves that the structure of monoclonal antibody-cytokine fusion protein based on antibody heterodimer technology can significantly increase the activity of IL-12.
  • a series of antibody-cytokine fusion proteins are constructed based on 4 different monoclonal antibodies, and then the antibody activity detection kit of Promega Company is used to detect the antibody activity in each fusion protein.
  • the results showed that compared with the unmodified anti-LAG-3 monoclonal antibody control mLAG300, the antibody activity of the constructed heterodimeric fusion protein (mLAG303) was almost the same ( Figure 10).
  • the antibody-cytokine fusion protein of the dimer technology will not affect the antibody activity of the fusion protein.
  • the antibody activity of the IL-12 heterodimeric fusion protein is more than 2 times stronger than that of the monoclonal antibody control ( Figure 10), which indicates that the antibody is based on the heterologous antibody.
  • the dimer technology and the antibody-cytokine fusion protein fused with IL-12 can significantly enhance the antibody activity of the fusion protein.
  • the antibody activity of the antibody-cytokine fusion protein is compared with the respective monoclonal antibody control (mPDL100, mTIGIT00 and mPD100) are similar in activity. This indicates that the antibody-cytokine fusion protein modified based on antibody heterodimer technology will not affect the performance of antibody activity, and may even enhance the antibody activity in some fusion proteins.
  • the present invention has developed a novel technology platform for antibody-cytokine fusion proteins based on antibody heterodimer technology, and constructed a series of antibody-cytokine fusion proteins using 4 different antibodies , And then through a series of antibody and cytokine activity analysis experiments confirmed that the technology platform can enhance antibody activity or cytokine activity.
  • Cytokine IL-12 DNA sequence (SEQ ID NO: 3)

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Abstract

提供了一种单克隆抗体-细胞因子融合蛋白二聚体及其应用，所述单克隆抗体包括两条重链，所述融合蛋白二聚体包括第一重链多肽链和第二重链多肽链，其中所述单克隆抗体的一条重链与一种细胞因子连接形成第一重链多肽链，所述单克隆抗体另一条重链可选择地与另一种细胞因子连接形成第二重链多肽链。提供的单克隆抗体-细胞因子融合蛋白二聚体，不仅充分保留了抗体的生物活性，而且明显改善了细胞因子的生物学活性。更重要的是，利用抗体分子的靶向特异性，增强免疫细胞的免疫应答，减弱了细胞因子的毒性，从而在提升药效的前提下保证了用药安全性。

Description

单克隆抗体-细胞因子融合蛋白二聚体及其应用 技术领域
本发明涉及一种融合蛋白，特别是基于单克隆抗体与细胞因子融合的蛋白二聚体及其应用。
背景技术
免疫系统是哺乳动物体内的一套防卫系统，能够抵御各种病原体的入侵和消除肿瘤。抗体分子在免疫系统中扮演了十分重要的角色，例如识别肿瘤抗原、激活免疫应答等。同时，细胞因子作为免疫调节因子，能够同时调控固有性免疫和适应性免疫，在癌症免疫治疗中也具有不容忽视的重要性。这一类调节因子几乎参与免疫系统的各个环节，在调节和平衡免疫系统方面行使着强大的功能。干扰素和IL-2最先被选择作为细胞因子应用到免疫治疗中。其中IFN-α被用于治疗各种癌症，比如黑色素瘤、肾癌、滤泡性淋巴瘤、慢性粒细胞白血病；而IL-2在1998年被FDA批准用于治疗晚期转移性黑色素瘤和转移性肾癌等。其他细胞因子，比如IL-7，IL-10，IL-12，IL-15和IL-21等，也正处于临床测试阶段。因此，细胞因子用于免疫治疗具有一定的潜力。
但是，基于细胞因子的免疫治疗在临床使用上却受到严重阻碍，主要受制于严重的副作用和表现欠佳的药代动力学性质。因此，为了提升细胞因子的治疗效果，经常需要对细胞因子进行改造。当前，比较常用的方法是采用抗体融合的方法，通过抗体将细胞因子靶向递送到特定部位，从而有效地降低细胞毒性，并改善药代动力学性质，继而增强免疫调节功能。尽管如此，有些细胞因子融合到抗体上后，会降低抗体本身的活性，从而影响整体效果。另外，有些细胞因子需要联合使用才能发挥各自最佳的效果，而现有的技术不能满足这一要求。
免疫调节中活化和抑制免疫应答主要是由两条独立的信号通路调节的。第一信号是抗原介导的。当T细胞受体特异性的识别并结合抗原呈递细胞(APC)表面MHC提呈的抗原肽时，产生第一信号。第二信号则由抗原呈递细胞和T细胞表面表达的共刺激分子间的相互作用提供。当第一、第二信号依次被活化后，T细胞才可以杀伤肿瘤。若缺乏第二信号，T细胞将进入无反应状态或免疫耐受，甚至引起细胞程序性死亡。
如上所述，第二信号通路对于活化免疫细胞非常重要。具体来讲，共刺激和共抑制受体参与了第二信号通路，对抗原-受体的呈递进行免疫应答和调节，在保持自身抗原免疫耐受的同时，平衡阳性和阴性信号，最大限度地提高对入侵者的免疫反应。CD28是CD28家族 的一员，是一种主要的T细胞共刺激受体，在初始CD4+和CD8+T细胞上组成性表达。细胞毒性T淋巴细胞相关抗原4(CTLA-4)分子，也是CD28受体家族的一员，然而却是共抑制性受体，该分子在调节性T细胞(Treg)组成性表达或通过CD28激活T细胞后产生。CD28或CTLA-4均可与B7.1(CD80)或B7.2(CD86)配体结合，从而分别在T细胞中传递活化或抑制信号。
CD28受体的配体包括CD80、CD86、程序性死亡1配体(PD-L1)、程序性死亡2配体(PD-L2)等。其中PD-L1是一种跨膜蛋白，它与PD-1的抑制性检查点分子结合，通过传递抑制的“不要找到我”信号来抑制适应性免疫反应。PD-1/PD-L1信号通路在免疫耐受的发展中起着至关重要的作用，防止免疫系统的过度反应，从而避免自身免疫疾病的发生。然而，这通常在癌症进展过程中被解除管制，允许肿瘤细胞通过伪装成健康组织来绕过保护机制。过量表达PD-L1的肿瘤细胞通过激活PD-1/PD-L1抑制信号通路，可逃避T细胞介导的死亡，抑制抗肿瘤适应性免疫反应。人类肿瘤相关巨噬细胞(TAMS)中的PD-1过度表达也被证明能抑制吞噬和肿瘤免疫。目前，阻断PD-1/PD-L1相互作用的抗PD-1或抗PD-L1单克隆抗体在癌症治疗中效果显著。尽管美国食品和药物管理局批准keytruda、opdivo和tecentriq等抗PD-1或抗PD-L1单克隆抗体用于治疗晚期癌症，但这些抗肿瘤药物只对部分患者产生反应，这暗示着单一的抑制性信号通路的阻断不足以激活免疫应答,还存在着其他机制抑制免疫系统。
淋巴细胞活化基因3(LAG-3)是一种跨膜蛋白，表达于活化的T细胞、自然杀伤细胞、B细胞和浆细胞样树突状细胞。LAG-3与PD-1一样，是APC上与MHC II结合并负性调节T细胞受体信号传导的免疫检测点受体之一。由于LAG-3在Treg细胞上也有表达，因此，阻断LAG-3可以抑制Treg细胞的活性，增强其抗肿瘤免疫功能。临床前实验显示，通过阻断LAG-3信号通路具有抗肿瘤作用。
TIGIT,全称为T细胞免疫球蛋白和ITIM结构域蛋白，是含Ig及ITIM结构域的T细胞和NK细胞共有的抑制性受体。TIGIT在T细胞和自然杀伤(NK)细胞中高表达。TIGIT，CD96，CD226和相关配体一起组成了一个免疫调节信号通路。与CD28/CTLA-4信号通路类似，CD226/TIGIT/CD96信号通路也包含共刺激性受体和共抑制性受体，并且这些受体共享部分或全部配体，其中CD226属于共刺激性受体，与配体结合后传递刺激信号，而TIGIT和CD96属于共抑制性受体，与相关配体结合后传递抑制信号。TIGIT有两种配体，即CD155和CD122，后两者同时也是CD226的配体。这两个配体在APC细胞，T细胞和肿瘤细胞中均有表达。CD96的配体包括CD155和CD111。TIGIT与CD155配体的亲和力，显著高于TIGIT与CD122配体的亲和力，同时也明显高于CD226或CD96与配体CD155的亲和力。 与PD-1和CTLA-4受体类似,TIGIT也是一个重要的抑制性免疫受体,抑制TIGIT可促进T细胞的增殖和功能；阻断TIGIT也可增强NK细胞介导的抗肿瘤免疫应答，从而抑制肿瘤生长。因此,靶向抑制性受体TIGIT的单克隆抗体，可以明显增强肿瘤免疫治疗效果。
当前，随着PD-1/PD-L1抗体的成功上市，基于免疫检查点阻断的免疫治疗得到了迅速发展和推广。但是，这些抗体只对一部分病人有效，而且会产生耐药性。因此，如何更好的联合细胞因子与抗体解决这些问题一直是研究的热点。
发明内容
为解决上述问题，一方面，本发明提供了一种单克隆抗体-细胞因子融合蛋白二聚体，所述单克隆抗体包括两条重链，所述融合蛋白二聚体包括第一重链多肽链和第二重链多肽链，其中所述单克隆抗体的一条重链与一种细胞因子连接形成第一重链多肽链，所述单克隆抗体另一条重链可选择地与另一种细胞因子连接形成第二重链多肽链。
在一些实施方案中，所述单克隆抗体抗免疫检查点分子的抗体。优选地，所述单克隆抗体选自抗LAG-3抗体、抗PD-L1抗体、抗TIGIT抗体或抗PD-1抗体。在一些实施方案中，所述单克隆抗体为抗LAG-3抗体。在另一些实施方案中，所述单克隆抗体为抗PD-L1抗体。在一些实施方案中，所述单克隆抗体为抗TIGIT抗体。在另一些实施方案中，所述单克隆抗体为抗PD-1抗体。
在一些实施方案中，所述两种细胞因子选自IL-2、IL-12、GM-CSF、IL-2突变体以及它们的组合。优选地，所述两种细胞因子选自IL-2、IL-12或IL-2突变体以及它们的组合。
在一些实施方案中，所述IL-12的两个亚基P35和P40通过接头序列连接形成IL-12单链蛋白而存在于所述融合蛋白二聚体中。在一些具体实施方案中，IL-12的两个亚基P35和P40通过接头序列连接形成IL-12单链蛋白与单克隆抗体一条重链连接形成所述第一或第二重链多肽链。
在一些实施方案中，所述单克隆抗体与细胞因子之间通过接头序列连接或直接融合。在一些实施方案中，所述接头序列选自(S(G4S) 1-3)、(G4S) 1-3、KRVAPELLGGPS、ASTKG、NSPPAA、EPKSSDKTHTSPPSP、ERKSSVESPPS以及ESKYGPPSPPSP。优选地，所述接头序列为(S(G4S) 1-3)。更优选地，所述接头序列为(S(G4S) 3)(SEQ ID NO:63)。
在一些实施方案中，所述单克隆抗体通过重链的Fc区与细胞因子连接。在一些实施方案中，所述单克隆抗体两条重链的Fc区不同，并且它们之间具有非对称互补结构。该非对称互补结构例如可通过KiH(knobs-into-holes)技术形成。优选地，所述单克隆抗体两条重链的Fc区具有突变的组合选自突变组合T366W/S354C和突变组合T366S/L368A/Y407V/Y349C。 在一些具体实施方案中，所述单克隆抗体的任一条重链的Fc区具有突变组合T366W/S354C，互补的另一条重链的Fc区具有突变组合T366S/L368A/Y407V/Y349C。在一些具体实施方案中，所述单克隆抗体重链的Fc区选自人IgG1突变体和人IgG4的突变体。
在一些实施方案中，所述单克隆抗体的一条重链与细胞因子IL-12连接形成第一重链多肽链，所述单克隆抗体的另一条重链与细胞因子IL-2或IL-2突变体连接形成第二重链多肽链；或所述单克隆抗体的一条重链与细胞因子IL-2或IL-2突变体连接形成第一重链多肽链，所述单克隆抗体的另一条重链与细胞因子IL-12连接形成第二重链多肽链。在一些实施方案中，所述单克隆抗体的一条重链与细胞因子IL-12、IL-2或IL-2突变体连接形成第一重链多肽链，所述单克隆抗体的另一条重链不连接细胞因子作为第二重链多肽链。在一些实施方案中，所述单克隆抗体的一条重链与细胞因子IL-12连接形成第一重链多肽链，所述单克隆抗体的另一条重链不连接细胞因子作为第二重链多肽链。在一些实施方案中，所述单克隆抗体的一条重链与细胞因子IL-2或IL-2突变体连接形成第一重链多肽链，所述单克隆抗体的另一条重链不连接细胞因子作为第二重链多肽链。
在一些实施方案中，所述细胞因子通过接头序列连接至所述单克隆抗体重链Fc区的C端。在另一些实施方案中，所述细胞因子直接连接至所述单克隆抗体重链Fc区的C端。
在一些实施方案中，所述抗LAG-3单克隆抗体的重链具有与SEQ ID NOs:8、12或14所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，所述抗LAG-3单克隆抗体的轻链具有与SEQ ID NO:10所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些具体实施方案中，所述抗LAG-3单克隆抗体的重链具有如SEQ ID NOs:8、12或14所示的氨基酸序列，所述LAG-3单克隆抗体的轻链具有如SEQ ID NO:10所示的氨基酸序列。
在一些实施方案中，所述抗PD-L1单克隆抗体的重链具有与SEQ ID NOs:22、26或28所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，所述抗PD-L1单克隆抗体的轻链具有与SEQ ID NO:24所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些实施方案中，所述抗PD-L1单克隆抗体的重链具有如SEQ ID NOs:22、26或28所示的氨基酸序列，所述抗PD-L1单克隆抗体的轻链具有如SEQ ID NO:24所示的氨基酸序列。
在一些实施方案中，所述抗TIGIT单克隆抗体的重链具有与SEQ ID NOs:36、40或42所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，所述抗TIGIT单克隆抗体的轻链具有与SEQ ID NO:38所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些实施方案中，所述抗TIGIT单克隆抗体的重 链具有如SEQ ID NOs:36、40或42所示的氨基酸序列，所述抗TIGIT单克隆抗体的轻链具有如SEQ ID NO:38所示的氨基酸序列。
在一些实施方案中，所述抗PD-1单克隆抗体的重链具有与SEQ ID NOs:50、54或56所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，所述抗PD-1单克隆抗体的轻链具有与SEQ ID NO:52所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些实施方案中，所述抗PD-1单克隆抗体的重链具有如SEQ ID NOs:50、54或56所示的氨基酸序列，所述抗PD-1单克隆抗体的轻链具有如SEQ ID NO:52所示的氨基酸序列。
在一些实施方案中，所述IL-12单链蛋白具有如SEQ ID NO:4所示的氨基酸序列。
在一些实施方案中，所述IL-2具有如SEQ ID NO:8所示的氨基酸序列。
本发明提供的融合蛋白二聚体包括单克隆抗体的两条轻链。
在一些实施方案中，所述融合蛋白的第一和第二重链多肽链选自具有与SEQ ID NOs:12、14、16、18、20、26、28、30、32、34、40、42、44、46、48、54、56、58、60或62所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些具体实施方案中，所述融合蛋白第一和第二重链多肽链选自具有如SEQ ID NOs:12、14、16、18、20、26、28、30、32、34、40、42、44、46、48、54、56、58、60或62所示的氨基酸序列。
在一些实施方案中，所述单克隆抗体为抗LAG-3抗体，所述第一和第二重链多肽链选自具有与SEQ ID NOs:12、14、16、18或20所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些具体实施方案中，所述单克隆抗体为抗LAG-3抗体，所述第一和第二重链多肽链选自具有如SEQ ID NOs:12、14、16、18或20所示的氨基酸序列。在一些具体实施方案中，所述单克隆抗体为抗LAG-3抗体，其中，所述第一重链多肽链具有如SEQ ID NO:18所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:16所示的氨基酸序列；所述第一重链多肽链具有如SEQ ID NO:16所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:14所示的氨基酸序列；或所述第一重链多肽链具有如SEQ ID NO:20所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:14所示的氨基酸序列。
在一些实施方案中，本发明提供了一种抗LAG-3单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第二重链多肽链和两条抗LAG-3抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:18所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:16所示的氨基酸序列，所述轻链具有如SEQ ID NO:8所示的氨基酸序列。在另一些实施方案中，本发明提供了一种抗LAG-3单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第 二重链多肽链和两条抗LAG-3抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:16，所述第二重链多肽链具有如SED ID NO:14所示的氨基酸序列，所述轻链具有如SEQ ID NO:8所示的氨基酸序列。在又一个实施方案中，本发明提供一种抗LAG-3单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第二重链多肽链和两条抗LAG-3抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:20，所述第二重链多肽链具有如SED ID NO:14所示的氨基酸序列，所述轻链具有如SEQ ID NO:8所示的氨基酸序列。
在一些实施方案中，所述单克隆抗体为抗PD-L1抗体，所述第一和第二重链多肽链选自具有与SEQ ID NOs:26、28、30、32或34所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些具体实施方案中，所述单克隆抗体为抗PD-L1抗体，所述第一和第二重链多肽链选自具有如SEQ ID NOs:26、28、30、32或34所示的氨基酸序列。在一些具体实施方案中，所述单克隆抗体为抗PD-L1抗体，所述第一重链多肽链具有如SEQ ID NO:32所示的氨基酸序列，其中：所述第二重链多肽链具有如SEQ ID NO:30所示的氨基酸序列；所述第一重链多肽链具有如SEQ ID NO:30所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:28所示的氨基酸序列；或所述单克隆抗体为抗PD-L1抗体，所述第一重链多肽链具有如SEQ ID NO:34所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:28所示的氨基酸序列。
在一些实施方案中，本发明提供了一种抗PD-L1单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第二重链多肽链和两条抗PD-L1抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:32所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:30所示的氨基酸序列，所述轻链具有如SEQ ID NO:24所示的氨基酸序列。在另一些实施方案中，本发明提供了一种抗PD-L1单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第二重链多肽链和两条抗PD-L1抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:30所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:28所示的氨基酸序列，所述轻链具有如SEQ ID NO:24所示的氨基酸序列。在又一些实施方案中，本发明提供了一种抗PD-L1单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第二重链多肽链和两条抗PD-L1抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:34所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:28所示的氨基酸序列，所述轻链具有如SEQ ID NO:24所示的氨基酸序列。
在一些实施方案中，所述单克隆抗体为抗TIGIT抗体，所述第一和第二重链多肽链选自具有与SEQ ID NOs:40、42、44、46或48所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些具体实施方案中，所述单克隆抗体为抗 TIGIT抗体，所述第一和第二重链多肽链选自具有如SEQ ID NOs:40、42、44、46或48所示的氨基酸序列。在一些具体实施方案中，所述单克隆抗体为抗TIGIT抗体，其中：所述第一重链多肽链具有如SEQ ID NO:46所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:44所示的氨基酸序列；所述第一重链多肽链具有如SEQ ID NO:44所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:42所示的氨基酸序列；或所述第一重链多肽链具有如SEQ ID NO:48所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:42所示的氨基酸序列。
在一些实施方案中，本发明提供了一种抗TIGIT单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第二重链多肽链和两条抗TIGIT抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:46所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:44所示的氨基酸序列，所述轻链具有如SEQ ID NO:38所示的氨基酸序列。在另一些实施方案中，本发明提供了一种抗TIGIT单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第二重链多肽链和两条抗TIGIT抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:44所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:42所示的氨基酸序列，所述轻链具有如SEQ ID NO:38所示的氨基酸序列。在又一些实施方案中，本发明提供了一种抗TIGIT单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第二重链多肽链和两条抗TIGIT抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:48所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:42所示的氨基酸序列，所述轻链具有如SEQ ID NO:38所示的氨基酸序列。
在一些实施方案中，所述单克隆抗体为抗PD-1抗体，所述第一和第二重链多肽链选自具有与SEQ ID NOs:54、56、58、60或62所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些具体实施方案中，所述单克隆抗体为抗PD-1抗体，所述第一和第二重链多肽链选自具有如SEQ ID NO:54、56、58、60或62所示的氨基酸序列。在一些具体实施方案中，所述单克隆抗体为抗PD-1抗体，其中：所述第一重链多肽链具有如SEQ ID NO:60所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:58所示的氨基酸序列；所述第一重链多肽链具有如SEQ ID NO:58所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:56所示的氨基酸序列；或所述第一重链多肽链具有如SEQ ID NO:62所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:56所示的氨基酸序列。
在一些实施方案中，本发明提供了一种抗PD-1单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第二重链多肽链和两条抗PD-1抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:60所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:58所示的氨 基酸序列，所述轻链具有如SEQ ID NO:52所示的氨基酸序列。在另一些实施方案中，本发明提供了一种抗PD-1单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第二重链多肽链和两条抗PD-1抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:58所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:56所示的氨基酸序列，所述轻链具有如SEQ ID NO:52所示的氨基酸序列。在又一些实施方案中，本发明提供了一种抗PD-1单克隆抗体-细胞因子融合蛋白二聚体，包括第一重链多肽链、第二重链多肽链和两条抗PD-1抗体的轻链，所述第一重链多肽链具有如SEQ ID NO:62所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:56所示的氨基酸序列，所述轻链具有如SEQ ID NO:52所示的氨基酸序列。
另一方面，本发明提供了分离的多核苷酸，其编码本发明所述的融合蛋白二聚体第一或第二重链多肽链。
在一些具体实施方案中，所述多核苷酸具有如SEQ ID NO:11、13、15、17、19、25、27、29、31、33、39、41、43、45、47、53、55、57、59或61所示的核苷酸序列。
另一方面，本发明提供了包含所述多核苷酸的表达载体。
另一方面，本发明提供了包含所述表达载体的宿主细胞。
另一方面，本发明提供了所述融合蛋白二聚体、多核苷酸、表达载体或宿主细胞在制备抗肿瘤药物中的应用。在一些实施方案中，所述肿瘤为黑色素瘤、胃癌、肾癌、霍奇金淋巴瘤、头颈癌、膀胱癌或非小细胞肺癌。优选地，所述肿瘤为黑色素瘤、胃癌或肾癌。
另一方面，本发明提供了抗肿瘤药物组合物，所述药物组合物包含所述融合蛋白二聚体和药学上可接受的载体。
另一方面，本发明提供了治疗肿瘤的方法，包括给予受试者治疗有效量的所述融合蛋白二聚体或含有所述融合蛋白二聚体的药物组合物。
在一些实施方案中，本发明提供的抗肿瘤药物组合物可以通过选自以下的至少一种路径施用至受试者：胃肠外、皮下、肌肉内、静脉内、关节内、支气管内、腹内、囊内、软骨内、腔内、体腔内、小脑内、脑室内、结肠内、宫颈内、胃内、肝内、心肌内、骨内、骨盆内、心包内、腹膜内、胸膜内、前列腺内、肺内、直肠内、肾内、视网膜内、脊柱内、滑膜内、胸腔内、鼓室内、子宫内、膀胱内、玻璃体内、快速注射、结膜下、经阴道、经直肠、经颊、舌下、鼻内、肿瘤内和经皮肤。优选地，所述抗肿瘤药物组合物通过肿瘤内或静脉内施用至受试者。
附图说明
图1单克隆抗体-细胞因子融合蛋白二聚体结构
图2抗LAG-3抗体-细胞因子融合蛋白的IL-2活性检测
图3抗PD-L1抗体-细胞因子融合蛋白的IL-2活性检测
图4抗TIGIT抗体-细胞因子融合蛋白的IL-2活性检测
图5抗PD-1抗体-细胞因子融合蛋白的IL-2活性检测
图6抗LAG-3抗体-细胞因子融合蛋白的IL-12活性检测
图7抗PD-L1抗体-细胞因子融合蛋白的IL-12活性检测
图8抗TIGIT抗体-细胞因子融合蛋白的IL-12活性检测
图9抗PD-1抗体-细胞因子融合蛋白的IL-12活性检测
图10抗LAG-3抗体-细胞因子融合蛋白的LAG-3阻断活性检测
图11抗PD-L1抗体-细胞因子融合蛋白的PD-L1阻断活性检测
图12抗TIGIT抗体-细胞因子融合蛋白的TIGIT阻断活性检测
图13抗PD-1抗体-细胞因子融合蛋白的PD-1阻断活性检测
具体实施方式
除非另有说明，本发明所用的技术和科学术语具有本发明所属领域的普通技术员通常所理解的含义。
本发明利用抗体Fc异源二聚体技术提供了具有抗肿瘤活性的融合蛋白，该融合蛋白包括单克隆抗体和一个或多个细胞因子。本发明中“单克隆抗体-细胞因子融合蛋白”、“抗体/细胞因子融合蛋白”及“单抗-细胞因子融合蛋白”均指基于抗体Fc异源二聚体技术的单克隆抗体-细胞因子融合蛋白，包括一个或多个细胞因子通过抗体重链Fc区连接而形成融合蛋白，可选择地，单克隆抗体和细胞因子通过接头序列连接在抗体重链Fc区的C端。
术语“抗体(antibody)”指由浆细胞(效应B细胞)分泌，被免疫系统用来鉴别与中和外来物质如细菌、病毒等的大型“Y”形蛋白质。在过去的10多年里，越来越多的单克隆抗体被广泛地应用到肿瘤治疗中。抗体通常为由2个相同重链和2个相同轻链通过二硫键相互连接组成的四聚体。本发明中涉及的“抗免疫检查点分子抗体”是指特异性结合免疫检查点分子的抗体，所述的免疫检查点分子包括但不限于LAG-3、PD-L1、TIGIT、PD-1或CTLA-4，所述抗体是指有两条重链和两条轻链的单克隆抗体。
术语“抗体Fc区”或“抗体Fc”指“Y”形的柄部区域，即可结晶片段(fragment crystallizable,Fc)，包括重链的第二和第三恒定结构域(CH2和CH3结构域)。可通过蛋白水解酶(如木瓜蛋白酶)水解抗体分子得到抗体Fc区。
本发明中涉及的二聚体蛋白，是指蛋白在形成的过程中，若两个亚基/单体相同称为同源二聚体(homo-)，若是不完全相同的亚基/单体组合而成，则称为异源二聚体 (hetero-)。术语“抗体Fc异源二聚体”是指蛋白由两个不同的亚基/单体组合而成，并且每个亚基/单体均含有一个抗体Fc片段。关键在于这两个抗体Fc片段各有不同的氨基酸位点突变能够形成互补的蛋白质空间结构，从而使得两个不同的亚基/单体能正确的组合在一起。
术语“KiH(knobs-into-holes)技术”指一种促进两种异源抗体重链之间进行装配的技术。例如，可以将一个抗体的重链CH3区366位体积较小的苏氨酸(T)突变为体积较大的酪氨酸(Y)，形成突出的“knobs”型结构(T366Y)；同时将另一个抗体重链CH3区407位较大的酪氨酸(Y)残基突变成较小的苏氨酸(T)，形成凹陷的“holes”型结构(Y407T)；利用这种“knobs-into-holes”结构(即非对称互补结构)的空间位阻效应可以实现两种不同抗体重链间的正确装配。本发明通过在两抗体Fc区中通过多个位点突变组合，实现了更佳的装配效果。
术语“细胞因子(cytokine，CK)”是免疫原、丝裂原或其它刺激剂诱导多种细胞产生的低分子量可溶性蛋白质。在生物体内通过与其特异的细胞表面受体结合，传递细胞内信号，从而改变细胞功能，具有调节固有免疫和适应性免疫、血细胞生成、细胞生长、APSC多能细胞以及损伤组织修复等多种作用。白细胞介素(比如IL-2或IL-12)作为其中的一组细胞因子，通过调控免疫系统来调节免疫应答。细胞因子不仅可以单独作用行使功能，也可以与抗体融合，形成抗体-细胞因子融合蛋白。这种新的蛋白形式将抗体特有的靶向性与细胞因子的免疫调节有机的结合了起来，从而增强了抗体的免疫治疗效果。更重要的是，融合的细胞因子通过抗体的靶向性被运输并富集到肿瘤部位，从而有效的避免了单独使用高剂量的细胞因子引起的副作用。
术语“分离的多核苷酸”指非自然界中天然存在状态的多核苷酸，包括通过生物学技术从自然界(包括生物体内)分离出的多核苷酸，也包括人工合成的多核苷酸。分离的多核苷酸可以是基因组DNA、cDNA、mRNA或合成的其它RNA，或者它们的组合。本文提供了多个用于编码本发明的融合蛋白二聚体以及其它多肽片段的核苷酸序列，需要指出的是，本领域技术人员可以根据本文所提供的氨基酸序列，基于密码子简并性，设计出与以上提供的核苷酸序列不完全相同的核苷酸序列，但都编码相同的氨基酸序列。这些经改动的核苷酸序列也包括在本发明的范围内。
当涉及多核苷酸时，所用的术语“载体”指用于将核苷酸编码信息转移到宿主细胞内的任一种分子(例如，核酸、质粒、或病毒等)。术语“表达载体”指适于在宿主细胞内表达目的基因(待表达核苷酸序列)的载体，通常包括目的基因、启动子、终止子、标记基因等部分。
术语“宿主细胞”指已经或者能够用核酸序列转化并从而表达所选的目的基因的细胞。该术语包括亲本细胞的后代，无论该后代与原来的亲本细胞在形态或基因组成上是否相同，只要后代存在所选目的基因即可。常用的宿主细胞包括细菌、酵母、哺乳动物细胞等，例如CHO细胞。
提及药物组合物，所使用的术语“药学上可接受的载体”指可以安全地进行施用的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质，这些物质适合于人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。依照给药途径，可以施用本领域众所周知的各种不同的载体，包括，但不限于糖类、淀粉、纤维素及其衍生物、麦芽糖、明胶、滑石、硫酸钙、植物油、合成油、多元醇、藻酸、磷酸缓冲液、乳化剂、等渗盐水、和/或无热原水等。本发明所提供的药物组合物可以制成粉末、注射剂等临床可接受的剂型。可以使用任何适当的途径向受试者施用本发明的药物组合物，例如可通过口服、静脉内输注、肌肉内注射、皮下注射、腹膜下、直肠、舌下，或经吸入、透皮等途径给药。
术语“治疗有效量”指足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物的量。本发明融合蛋白的“治疗有效量”可由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的日剂量范围可以为每kg体重0.01mg至100mg活性成分。
术语“氨基酸序列一致性”定义为对比序列并在必要时引入缺口以获取最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分，候选序列中与特定肽或多肽序列中的氨基酸残基相同的氨基酸残基的百分率。可以本领域技术范围内的多种方式进行序列对比以测定百分比氨基酸序列同一性，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定测量对比的适宜参数，包括对所比较的序列全长获得最大对比所需的任何算法。
用语“和/或”指该用语之前和之后的元素可以同时存在，或者仅其中一个元素存在。例如，“A和/或B”可以为A和B、仅A或者仅B。
本发明利用免疫细胞因子和单克隆抗体的优势提高免疫治疗的效果，通过将细胞因子融合到抗免疫检查点分子抗体，如抗LAG-3抗体、抗PD-L1抗体、抗TIGIT抗体或抗PD-1抗体重链Fc区的C端，开发出针对抗免疫检查点蛋白抗体-细胞因子融合蛋白二聚体，通过体外的活性分析评估这些融合蛋白的活性，研究发现，这些融合蛋白二聚体不仅充分保留了抗体的生物活性，而且明显改善了细胞因子的生物学活性。本发明中提到的细胞因子包括白细胞介素，例如白细胞介素-2(IL-2)、白细胞介素-12(IL-12)以及粒细胞-巨噬细胞集落刺激因子(GM-CSF)等，其中白细胞介素-2和白细胞介素-12参与免疫调节的各个环节，从而增强免疫应答。更重要的是，本发明利用抗体分子的靶向特异性，增强免疫细胞的免疫应答，减弱了细胞因子的毒性，从而在提升药效的前提下保证了用药安全性。
实施例1单克隆抗体-细胞因子融合蛋白的设计构建以及在CHO细胞中的表达和纯化
细胞因子IL-2或IL-12可通过接头序列(Ser(Gly4Ser)3)(即S(G4S)3,SEQ ID NO:63)连接到单克隆抗体的重链C端，所形成的融合蛋白保持了抗体和细胞因子的双重效果。由于功能性的IL-12由p35和p40两个亚基构成，IL-12将以两个亚基的形式单独融合到抗体Fc上，或者先 将IL-12的两个亚基通过接头序列(S(G4S)3)形成单链蛋白，然后再通过接头序列(S(G4S)3)连接到单克隆抗体的重链C端。
传统的抗体-细胞因子融合蛋白结构中，细胞因子同时连接到抗体的两条重链或者两条轻链上，从而以同源二聚体的形式存在。但是，这种基于IgG平台融合的免疫细胞因子的药代动力学性质较差。为了将细胞因子以单体形式融合到抗体上，同时改变抗体与细胞因子的比例，从而增强融合蛋白的靶向性，我们采用了基于抗体异源二聚体技术，将IL-2或者IL-12单独融合到一条重链上，另外一条链不连接细胞因子，形成IL-12或者IL-2单体融合蛋白，分别如图1B和图1C所示，或者将IL2和IL-12各自连接到一条重链上，产生同时具有IL-2和IL-12蛋白的融合蛋白，如图1A所示。
抗体异源二聚体技术的核心是将单克隆抗体的两条重链分别改造，产生非对称互补结构，从而能将改造的两条链结合起来，避免了同源二聚体的产生。改造的原理基于以下方面：疏水/空间互补(比如KiH和ZW1)、静电互补(比如DD-KK)、空间互补+静电相互作用(比如EW-RVT)以及空间互补+氢键互补(比如A107)等。本研究中，我们采用疏水/空间互补改造单克隆抗体的两条重链，形成不一样的两条重链，分别命名为Fm1和Fm2(图1)，然后将一种细胞因子连接到一条重链的C端上，另一条重链不连接细胞因子；或者将一种细胞因子连接到重链Fm1上，另一种细胞因子连接到重链Fm2上，使得最终形成的异源二聚体中含有的每种细胞因子的数目为一个，而不是同源二聚体中的两个。
通过以上方法构建的单克隆抗体-细胞因子融合蛋白将在CHO-3E7细胞里面表达，然后通过Protein A亲和层析和分子筛纯化得到用于体外分析的蛋白。
实施例2单克隆抗体-细胞因子融合蛋白的体外活性分析实验
2.1:IL-2活性检测：
(1)细胞的培养与准备：将一支CTLL-2细胞于液氮罐取出复苏，于37℃5％CO2培养箱中培养，根据测活实验设计所需的总细胞数，将复苏后细胞进行传代扩大培养。
(2)铺板：将培养的CTLL-2细胞取样计数，取适量的细胞接种于96孔板。
(3)样品稀释：将纯化得到的融合蛋白溶液样品以及IL-2对照(100μg/ml)梯度稀释，每个样品设置9个浓度梯度，每个浓度设置3个复孔。
(4)加样：将融合蛋白样品及对照加入接种CTLL-2细胞的96孔板中，于37℃5％CO2培养箱中孵育70小时。
(5)显色：从培养箱中取出96孔板，关闭生物安全柜的照明灯，并注意避光，于每个实验孔中加Promega Substrate Cell Titer 96 Aqueous One Solution Reagent试剂，在避光条件下37℃反应约3h。待反应完成后，将96孔板置于酶标仪上，490nm波长下检测OD值。
2.2:IL-12活性检测
(1)细胞的培养与准备：将一支NK-92细胞于液氮罐取出复苏，于37℃5％CO2培养箱中培养，根据测活实验设计所需的总细胞数，将复苏后细胞进行传代扩大培养。
(2)铺板：将培养的NK-92细胞取样计数，取适量的细胞接种于96孔板。
(3)样品稀释：将待测纯化得到的融合蛋白溶液样品以及IL-12对照(100μg/ml)溶解并梯度稀释，每个样品设置9个浓度梯度，每个浓度设置3个复孔。
(4)加样：将融合蛋白样品及对照加入接种NK-92细胞的96孔板中，于37℃5％CO2培养箱中孵育24小时。
(5)样品收集:从培养箱中取出96孔板，3700rpm 4度离心20min，吸上清，做好标记，-20℃保存。
(6)检测：取出冻存的样品，按照human IFN gamma ELISA MAX TM Standard试剂盒的操作步骤检测样品human IFN gamma的含量。将96孔板置于酶标仪上，450nm波长下检测OD值。
2.3:PD-1/PD-L1通路阻断实验测试抗PD-1/PD-L1抗体-细胞因子融合蛋白活性
检测抗PD-1/PD-L1单抗-细胞因子融合蛋白样品体外功能的实验使用的是普洛麦格(Promega)的PD-1/PD-L1阻断功能报告基因试剂盒(PD-1/PD-L1 Blockade Bioassay，普洛麦格试剂盒产品编号为J1250)。该试剂盒检测系统由两种经基因工程改造的细胞系组成。刺激细胞系为PD-L1 aAPC/CHO-K1细胞，该细胞稳定表达人源PD-L1和一种能以不依赖于抗原方式激活同源TCR的细胞表面蛋白。效应细胞系是一个Jurkat T细胞系，该细胞系稳定表达人源PD-1和NFAT诱导的荧光素酶报告基因。当把两种类型的细胞共同培养时，PD-1/PD-L1相互作用抑制TCR信号转导以及NFAT介导的荧光素酶活性。加入抗PD-1/PD-L1抗体可以阻断PD-1和PD-L1的结合，从而使TCR信号通路激活以及NFAT介导的荧光素酶活性增强并产生化学发光(Luminescence)。
首先将效应细胞系Jurkat T细胞铺在96孔板内，随后加入抗PD-1/PD-L1抗体-细胞因子融合蛋白样品和刺激细胞系PD-L1aAPC/CHO-K1细胞。将该体系在37℃下孵育6个小时。然后加入Bio-Glo TM荧光检测试剂，并在室温下孵育5-10分钟。最后使用化学荧光信号读板机读取96孔板中的荧光信号。该实验使用8浓度三复孔的形式，相对荧光值作为y-轴，抗 体样品的浓度作为x-轴，画出四参数曲线。使用GraphPad Prism软件分析该曲线并得出抗PD-1/PD-L1抗体-细胞因子融合蛋白样品的EC 50值。
2.4:抗TIGIT抗体-细胞因子融合蛋白的体外活性分析
检测抗TIGIT单抗-细胞因子融合蛋白样品体外功能的实验使用的是普洛麦格(Promega)的TIGIT/CD155阻断功能报告基因试剂盒(TIGIT/CD155 Blockade Bioassay，普洛麦格试剂盒产品编号为J2201)。该试剂盒检测系统由两种经基因工程改造的细胞系组成。刺激细胞系为CD155 aAPC/CHO-K1细胞，该细胞表达人源的CD155以及一种能不依赖于抗原方式激活TCR复合物的细胞表面蛋白。效应细胞系是一个Jurkat T细胞系，该细胞系表达人源TIGIT以及一个对TCR激活和CD226共刺激均会产生应答的天然启动子所驱动的荧光素酶报告基因。当把两种类型的细胞共同培养时，TIGIT抑制CD226激活和启动子介导的发光。加入抗TIGIT抗体则可以阻断TIGIT与CD155的相互作用或抑制TIGIT阻止CD226形成同源二聚体的能力，从而恢复启动子介导的化学发光(Luminescence)。
首先将效应细胞系Jurkat T细胞铺在96孔板内，随后加入抗TIGIT抗体-细胞因子融合蛋白样品和刺激细胞系CD155 aAPC/CHO-K1细胞。将该体系在37℃下孵育6个小时。然后加入Bio-Glo TM荧光检测试剂，并在室温下孵育5-10分钟。最后使用化学荧光信号读板机读取96孔板中的荧光信号。该实验使用8浓度三复孔的形式，相对荧光值作为y-轴，抗体样品的浓度作为x-轴，画出四参数曲线。使用GraphPad Prism软件分析该曲线并得出抗TIGIT抗体-细胞因子融合蛋白样品的EC 50值。
2.5:抗LAG-3抗体-细胞因子融合蛋白的体外活性分析
检测抗LAG3单抗-细胞因子融合蛋白样品体外功能的实验使用的是普洛麦格(Promega)的LAG3阻断功能报告基因试剂盒(LAG3 Blockade Bioassay，普洛麦格试剂盒产品编号为CS194804)。该试剂盒检测系统由两种经基因工程改造的细胞系组成。刺激细胞系为aAPC/Raji细胞，该细胞表达MHC II并可以激活TCR复合物。效应细胞系是一个Jurkat T细胞系，该细胞系表达人源LAG3以及一个对TCR激活会产生应答的天然启动子所驱动的荧光素酶报告基因。当把两种类型的细胞共同培养时，LAG3抑制MHCII激活和启动子介导的发光。加入抗LAG3抗体则可以阻断LAG3与MHCII的相互作用，从而恢复启动子介导的化学发光(Luminescence)。
首先将效应细胞系Jurkat T细胞铺在96孔板内，随后加入抗LAG-3抗体-细胞因子融合蛋白样品和刺激细胞系aAPC/Raji细胞。将该体系在37℃下孵育6个小时。然后加入Bio-Glo TM荧光检测试剂，并在室温下孵育5-10分钟。最后使用化学荧光信号读板机读取96孔板中的荧光信号。该实验使用8浓度三复孔的形式，相对荧光值作为y-轴，抗体样品的浓度 作为x-轴，画出四参数曲线。使用GraphPad Prism软件分析该曲线并得出抗LAG3抗体-细胞因子融合蛋白样品的EC 50值。
实验结果及分析
1.基于单克隆抗体异源二聚体的免疫细胞因子融合蛋白的构建
本研究构建了一系列的基于单克隆抗体异源二聚体的抗体-细胞因子融合蛋白。其中用于构建融合蛋白的组件为单克隆抗体，细胞因子IL-12和IL-2。本发明中采用了四种不同的单克隆抗体，用于测试该技术平台是否适用于大多数单克隆抗体。这四种单克隆抗体分别是抗LAG-3的单克隆抗体，抗PD-L1的单克隆抗体，抗TIGIT的单克隆抗体和抗P-1的单克隆抗体。抗LAG-3的单克隆抗体是来自BMS公司的Relatlimab，属于人IgG4抗体。抗PD-L1的单克隆抗体是来自Roche公司的Atezolizumab，属于人IgG1抗体。抗TIGIT的单克隆抗体是来自Roche公司的Tiragolumab，属于人IgG1抗体。抗PD-1的单克隆抗体是来自专利申请WO2018119474，属于人IgG4抗体。
首先将各个单克隆抗体的重链或轻链编码序列分别插入到pTT5表达载体的多克隆位点EcoRI与HindIII之间，同时在起始密码子ATG前面还加入KOZAK序列GCCGCCACC和信号肽序列(SEQ ID NO:2)帮助把蛋白分泌到细胞外。
为了产生异源二聚体，我们利用knob-into-holes(KIH)技术进行单克隆抗体重链Fc区域的改造，其中一条重链Fc链的突变位点组合是T366W/S354C，另外一条重链Fc链的突变位点组合是T366S/L368A/Y407V/Y349C。另外，如果两条经过改造的重链C端最后一个氨基酸是K，也会被去除。编码IL-12和IL-2的DNA序列，通过接头序列(S(G4S)3)，采用Gibson组装的方法，分别连接到重链的C端，从而产生编码不同重链融合蛋白的质粒。
如上所述，抗LAG-3的单克隆抗体由重链H1和轻链L1组成。重链H1经过KIH技术改造，产生2条重链突变体H2和H3。IL-12序列通过接头序列(S(G4S)3)连接到重链H2的C端产生新的多肽，称为H2a。IL-2序列通过接头序列(S(G4S)3)分别连接到重链H2和H3的C端产生新的多肽，称为H2b和H3a。
抗PD-L1的单克隆抗体由重链H4和轻链L2组成。重链H4经过KIH技术改造，产生2条重链突变体H5和H6。IL-12序列通过接头序列(S(G4S)3)连接到重链H5的C端产生新的多肽，称为H5a。IL-2序列通过接头序列(S(G4S)3)分别连接到重链H5和H6的C端产生新的多肽，称为H5b和H6a。
抗TIGIT的单克隆抗体由重链H7和轻链L3组成。重链H7经过KIH技术改造，产生2条重链突变体H8和H9。IL-12序列通过接头序列(S(G4S)3)连接到重链H8的C端产生新的多肽，称 为H8a。IL-2序列通过接头序列(S(G4S)3)分别连接到重链H8和H9的C端产生新的多肽，称为H8b和H9a。
抗PD-1的单克隆抗体由重链H10和轻链L4组成。重链H10经过KIH技术改造，产生2条重链突变体H11和H12。IL-12序列通过接头序列(S(G4S)3)连接到重链H11的C端产生新的多肽，称为H11a。IL-2序列通过接头序列(S(G4S)3)分别连接到重链H11和H12的C端产生新的多肽，称为H11b和H12a。
将这些构建的融合蛋白，分别与未经改造的亲本轻链组合，从而产生一系列的抗体-细胞因子融合蛋白。将轻链L1和两条重链H2a和H3a组合产生抗体-细胞因子融合蛋白mLAG301,将轻链L1和两条重链H2a和H3组合产生抗体-细胞因子融合蛋白mLAG302,将轻链L1和两条重链H2b和H3组合产生抗体-细胞因子融合蛋白mLAG303；将轻链L2和两条重链H5a和H6a组合产生抗体-细胞因子融合蛋白mPDL101,将轻链L2和两条重链H5a和H6组合产生抗体-细胞因子融合蛋白mPDL102,将轻链L2和两条重链H5b和H6组合产生抗体-细胞因子融合蛋白mPDL103；将轻链L3和两条重链H8a和H9a组合产生抗体-细胞因子融合蛋白mTIGIT01,将轻链L3和两条重链H8a和H9组合产生抗体-细胞因子融合蛋白mTIGIT02,将轻链L3和两条重链H8b和H9组合产生抗体-细胞因子融合蛋白mTIGIT03；将轻链L4和两条重链H11a和H12a组合产生抗体-细胞因子融合蛋白mPD101,将轻链L4和两条重链H11a和H12组合产生抗体-细胞因子融合蛋白mPD102,将轻链L4和两条重链H11b和H12组合产生抗体-细胞因子融合蛋白mPD103。本实验中构建的质粒和蛋白信息见表1。
表1：构建抗体-细胞因子融合蛋白的质粒和蛋白
Figure PCTCN2020097927-appb-000001
Figure PCTCN2020097927-appb-000002
以上构建到pTT5表达载体上的融合蛋白质粒通过PEI转染试剂瞬转CHO-3E7细胞，然后37℃培养6天。通过离心收取培养液上清，首先通过Protein A亲和柱纯化融合蛋白，然后通过分子筛进一步纯化融合蛋白，最终纯度达到95％以上。
2.细胞因子活性检测结果分析
本发明为了证明基于抗体异源二聚体技术的单克隆抗体-细胞因子融合蛋白的技术平台的广谱性，利用4种不同的抗体构建了一系列的抗体-细胞因子融合蛋白。对于融合了IL-2的抗体-细胞因子融合蛋白来说，与游离的IL-2的生物活性相比较，所有的融合蛋白的IL-2的活性均明显增强(图2,3,4和5)，这说明基于抗体异源二聚体技术的单克隆抗体-细胞因子融合蛋白的结构能显著性的提高IL-2的活性。
对于融合了IL-12的融合蛋白来说，与游离的IL-12的生物活性相比较，利用4种不同的抗体构建的抗体-细胞因子融合蛋白的IL-12的活性均为增强或不变(图6,7,8和9)。其中只融合了IL-2的融合蛋白的IL-12的活性保持不变，说明基于抗体异源二聚体技术的单克隆抗体-细胞因子融合蛋白的结构能很好的维持IL-12的活性。更重要的是，同时融合了IL-12和IL-2的 抗体-细胞因子融合蛋白，比只融合了IL-12的融合蛋白的IL-12细胞因子活性还要强，这说明IL-12与IL-2可能存在着协同效应，也进一步证明基于抗体异源二聚体技术的单克隆抗体-细胞因子融合蛋白的结构能显著性的提高IL-12的活性。
3.抗体活性检测结果分析
本发明中基于4种不同单克隆抗体构建了一系列的抗体-细胞因子融合蛋白，然后利用Promega公司的抗体活性检测试剂盒分别检测各个融合蛋白中的抗体活性。结果显示，与未经改造的抗LAG-3单克隆抗体对照mLAG300相比较，构建的异源二聚体融合蛋白(mLAG303)的抗体活性与之几乎一样(图10)，这说明基于抗体异源二聚体技术的抗体-细胞因子融合蛋白不会影响该融合蛋白的抗体活性的发挥。更重要的是，融合了IL-12的异源二聚体融合蛋白(mLAG301和mLAG302)的抗体活性比单克隆抗体对照的抗体活性增强了2倍以上(图10)，这说明基于抗体异源二聚体技术并融合了IL-12的抗体-细胞因子融合蛋白能显著增强融合蛋白的抗体活性。类似地，基于其他三种单克隆抗体(抗PD-L1抗体，抗TIGIT抗体和抗PD-1抗体)改造而来的抗体-细胞因子融合蛋白的抗体活性与各自的单克隆抗体对照(mPDL100,mTIGIT00和mPD100)相比活性类似。这说明基于抗体异源二聚体技术改造的抗体-细胞因子融合蛋白不会影响抗体活性的发挥，甚至会增强某些融合蛋白中的抗体活性。
综上所述，本发明开发了一种新颖的基于抗体异源二聚体技术的抗体-细胞因子融合蛋白的技术平台，并利用4种不同的抗体构建了一系列的抗体-细胞因子融合蛋白，然后通过一系列的抗体和细胞因子活性分析实验证实了该技术平台能增强抗体活性或细胞因子活性。
本发明所属领域技术员应理解，以上描述的方法和材料，仅是示例性的，而不应视为限定本发明的范围。
序列信息
信号肽DNA序列(SEQ ID NO:1)：
Figure PCTCN2020097927-appb-000003
信号肽的氨基酸序列(SEQ ID NO:2)
Figure PCTCN2020097927-appb-000004
细胞因子IL-12DNA序列(SEQ ID NO:3)
Figure PCTCN2020097927-appb-000005
细胞因子IL-12全长氨基酸序列(SEQ ID NO：4)
Figure PCTCN2020097927-appb-000006
细胞因子IL-2的DNA序列(SEQ ID NO:5)
Figure PCTCN2020097927-appb-000007
细胞因子IL-2的氨基酸序列(SEQ ID NO:6)
Figure PCTCN2020097927-appb-000008
多肽链H1的DNA序列(SEQ ID NO:7)
Figure PCTCN2020097927-appb-000009
多肽链H1的氨基酸序列(SEQ ID NO:8)
Figure PCTCN2020097927-appb-000010
多肽链L1的DNA序列(SEQ ID NO:9)
Figure PCTCN2020097927-appb-000011
多肽链L1的氨基酸序列(SEQ ID NO:10)
Figure PCTCN2020097927-appb-000012
多肽链H2的DNA序列(SEQ ID NO:11)
Figure PCTCN2020097927-appb-000013
多肽链H2的氨基酸序列(SEQ ID NO:12)
Figure PCTCN2020097927-appb-000014
多肽链H3的DNA序列(SEQ ID NO:13)
Figure PCTCN2020097927-appb-000015
Figure PCTCN2020097927-appb-000016
多肽链H3的氨基酸序列(SEQ ID NO:14)
Figure PCTCN2020097927-appb-000017
多肽链H2a的DNA序列(SEQ ID NO:15)
Figure PCTCN2020097927-appb-000018
Figure PCTCN2020097927-appb-000019
多肽链H2a的氨基酸序列(SEQ ID NO:16)
Figure PCTCN2020097927-appb-000020
多肽链H3a的DNA序列(SEQ ID NO:17)
Figure PCTCN2020097927-appb-000021
Figure PCTCN2020097927-appb-000022
多肽链H3a的氨基酸序列(SEQ ID NO:18)
Figure PCTCN2020097927-appb-000023
多肽链H2b的DNA序列(SEQ ID NO:19)
Figure PCTCN2020097927-appb-000024
多肽链H2b的氨基酸序列(SEQ ID NO:20)
Figure PCTCN2020097927-appb-000025
多肽链H4的DNA序列(SEQ ID NO:21)
Figure PCTCN2020097927-appb-000026
多肽链H4的氨基酸序列(SEQ ID NO:22)
Figure PCTCN2020097927-appb-000027
多肽链L2的DNA序列(SEQ ID NO:23)
Figure PCTCN2020097927-appb-000028
多肽链L2的氨基酸序列(SEQ ID NO:24)
Figure PCTCN2020097927-appb-000029
多肽链H5的DNA序列(SEQ ID NO:25)
Figure PCTCN2020097927-appb-000030
Figure PCTCN2020097927-appb-000031
多肽链H5的氨基酸序列(SEQ ID NO:26)
Figure PCTCN2020097927-appb-000032
多肽链H6的DNA序列(SEQ ID NO:27)
Figure PCTCN2020097927-appb-000033
多肽链H6的氨基酸序列(SEQ ID NO:28)
Figure PCTCN2020097927-appb-000034
多肽链H5a的DNA序列(SEQ ID NO:29)
Figure PCTCN2020097927-appb-000035
Figure PCTCN2020097927-appb-000036
多肽链H5a的氨基酸序列(SEQ ID NO:30)
Figure PCTCN2020097927-appb-000037
多肽链H6a的DNA序列(SEQ ID NO:31)
Figure PCTCN2020097927-appb-000038
多肽链H6a的氨基酸序列(SEQ ID NO:32)
Figure PCTCN2020097927-appb-000039
Figure PCTCN2020097927-appb-000040
多肽链H5b的DNA序列(SEQ ID NO:33)
Figure PCTCN2020097927-appb-000041
多肽链H5b的氨基酸序列(SEQ ID NO:34)
Figure PCTCN2020097927-appb-000042
多肽链H7的DNA序列(SEQ ID NO:35)
Figure PCTCN2020097927-appb-000043
Figure PCTCN2020097927-appb-000044
多肽链H7的氨基酸序列(SEQ ID NO:36)
Figure PCTCN2020097927-appb-000045
多肽链L3的DNA序列(SEQ ID NO:37)
Figure PCTCN2020097927-appb-000046
多肽链L3的氨基酸序列(SEQ ID NO:38)
Figure PCTCN2020097927-appb-000047
多肽链H8的DNA序列(SEQ ID NO:39)
Figure PCTCN2020097927-appb-000048
Figure PCTCN2020097927-appb-000049
多肽链H8的氨基酸序列(SEQ ID NO:40)
Figure PCTCN2020097927-appb-000050
多肽链H9的DNA序列(SEQ ID NO:41)
Figure PCTCN2020097927-appb-000051
多肽链H9的氨基酸序列(SEQ ID NO:42)
Figure PCTCN2020097927-appb-000052
Figure PCTCN2020097927-appb-000053
多肽链H8a的DNA序列(SEQ ID NO:43)
Figure PCTCN2020097927-appb-000054
多肽链H8a的氨基酸序列(SEQ ID NO:44)
Figure PCTCN2020097927-appb-000055
多肽链H9a的DNA序列(SEQ ID NO:45)
Figure PCTCN2020097927-appb-000056
多肽链H9a的氨基酸序列(SEQ ID NO:46)
Figure PCTCN2020097927-appb-000057
Figure PCTCN2020097927-appb-000058
多肽链H8b的DNA序列(SEQ ID NO:47)
Figure PCTCN2020097927-appb-000059
多肽链H8b的氨基酸序列(SEQ ID NO:48)
Figure PCTCN2020097927-appb-000060
多肽链H10的DNA序列(SEQ ID NO:49)
Figure PCTCN2020097927-appb-000061
Figure PCTCN2020097927-appb-000062
多肽链H10的氨基酸序列(SEQ ID NO:50)
Figure PCTCN2020097927-appb-000063
多肽链L4的DNA序列(SEQ ID NO:51)
Figure PCTCN2020097927-appb-000064
多肽链L4的氨基酸序列(SEQ ID NO:52)
Figure PCTCN2020097927-appb-000065
多肽链H11的DNA序列(SEQ ID NO:53)
Figure PCTCN2020097927-appb-000066
Figure PCTCN2020097927-appb-000067
多肽链H11的氨基酸序列(SEQ ID NO:54)
Figure PCTCN2020097927-appb-000068
多肽链H12的DNA序列(SEQ ID NO:55)
Figure PCTCN2020097927-appb-000069
多肽链H12的氨基酸序列(SEQ ID NO:56)
Figure PCTCN2020097927-appb-000070
多肽链H11a的DNA序列(SEQ ID NO:57)
Figure PCTCN2020097927-appb-000071
Figure PCTCN2020097927-appb-000072
多肽链H11a的氨基酸序列(SEQ ID NO:58)
Figure PCTCN2020097927-appb-000073
Figure PCTCN2020097927-appb-000074
多肽链H12a的DNA序列(SEQ ID NO:59)
Figure PCTCN2020097927-appb-000075
多肽链H12a的氨基酸序列(SEQ ID NO:60)
Figure PCTCN2020097927-appb-000076
多肽链H11b的DNA序列(SEQ ID NO:61)
Figure PCTCN2020097927-appb-000077
Figure PCTCN2020097927-appb-000078
多肽链H11b的氨基酸序列(SEQ ID NO:62)
Figure PCTCN2020097927-appb-000079
接头序列(SEQ ID NO:63):
Figure PCTCN2020097927-appb-000080

Claims (26)

  1. 一种单克隆抗体-细胞因子融合蛋白二聚体，所述单克隆抗体包括两条重链，所述融合蛋白二聚体包括第一重链多肽链和第二重链多肽链，其中所述单克隆抗体的一条重链与一种细胞因子连接形成第一重链多肽链，所述单克隆抗体另一条重链可选择地与另一种细胞因子连接形成第二重链多肽链。
  2. 如权利要求1所述的融合蛋白二聚体，所述单克隆抗体为抗免疫检查点分子的抗体
  3. 如权利要求1或2所述的融合蛋白二聚体，所述单克隆抗体选自抗LAG-3抗体、抗PD-L1抗体、抗TIGIT抗体或抗PD-1抗体。
  4. 如权利要求1-3中任一项所述的融合蛋白二聚体，所述两种细胞因子均选自IL-2、IL-12、GM-CSF、IL-2突变体以及它们的组合。
  5. 如权利要求4所述的融合蛋白二聚体，所述IL-12的两个亚基P35和P40通过接头序列连接形成IL-12单链蛋白与单克隆抗体一条重链连接形成所述第一或第二重链多肽链。
  6. 如权利要求1-5中任一项所述的融合蛋白二聚体，所述单克隆抗体通过重链的Fc区与细胞因子连接。
  7. 如权利要求6所述的融合蛋白二聚体，所述单克隆抗体两条重链的Fc区不同，并且它们之间具有非对称互补结构。
  8. 如权利要求7所述的融合蛋白二聚体，所述单克隆抗体的任一条重链的Fc区具有突变组合T366W/S354C，互补的另一条重链的Fc区具有突变组合T366S/L368A/Y407V/Y349C。
  9. 如权利要求1-8中任一项所述的融合蛋白二聚体，所述单克隆抗体的一条重链与细胞因子IL-12连接形成第一重链多肽链，所述单克隆抗体的另一条重链与细胞因子IL-2或IL-2突变体连接形成第二重链多肽链；或所述单克隆抗体的一条重链与细胞因子IL-2或IL-2突变体连接形成第一重链多肽链，所述单克隆抗体的另一条重链与细胞因子IL-12连接形成第二重链多肽链。
  10. 如权利要求1-8中任一项所述的融合蛋白二聚体，所述单克隆抗体的一条重链与细胞因子IL-12或IL-2连接形成第一重链多肽链，所述单克隆抗体的另一条重链不连接细胞因子作为第二重链多肽链。
  11. 如权利要求3-10中任一项所述的融合蛋白二聚体，所述抗LAG-3单克隆抗体的重链具有与SEQ ID NOs:8、12或14所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，所述抗LAG-3单克隆抗体的轻链具有与SEQ ID NO:10所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。
  12. 如权利要求3-10中任一项所述的融合蛋白二聚体，所述抗PD-L1单克隆抗体的重链具有与SEQ ID NOs:22、26或28所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，所述抗PD-L1单克隆抗体的轻链具有与SEQ ID NO:24所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。
  13. 如权利要求3-10中任一项所述的融合蛋白二聚体，所述抗TIGIT单克隆抗体的重链具有与SEQ ID NOs:36、40或42所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，所述抗TIGIT单克隆抗体的轻链具有与SEQ ID NO:38所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。
  14. 如权利要求3-10中任一项所述的融合蛋白二聚体，所述抗PD-1单克隆抗体的重链具有与SEQ ID NOs:50、54或56所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，所述抗PD-1单克隆抗体的轻链具有与SEQ ID NO:52所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。
  15. 如权利要求1-14中任一项所述的融合蛋白二聚体，所述IL-12单链蛋白具有如SEQ ID NO:4所示的氨基酸序列。
  16. 如权利要求1-11中任一项所述的融合蛋白二聚体，所述单克隆抗体为抗LAG-3抗体，其中：
    所述第一重链多肽链具有如SEQ ID NO:18所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:16所示的氨基酸序列；
    所述第一重链多肽链具有如SEQ ID NO:16所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:14所示的氨基酸序列；或
    所述第一重链多肽链具有如SEQ ID NO:20所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:14所示的氨基酸序列。
  17. 如权利要求1-10及12中任一项所述的融合蛋白二聚体，所述单克隆抗体为抗PD-L1抗体，其中：
    所述第一重链多肽链具有如SEQ ID NO:32所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:30所示的氨基酸序列；
    所述第一重链多肽链具有如SEQ ID NO:30所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:28所示的氨基酸序列；或
    所述第一重链多肽链具有如SEQ ID NO:34所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:28所示的氨基酸序列。
  18. 如权利要求1-10及13中任一项所述的融合蛋白二聚体，所述单克隆抗体为抗TIGIT抗体，其中：
    所述第一重链多肽链具有如SEQ ID NO:46所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:44所示的氨基酸序列；
    所述第一重链多肽链具有如SEQ ID NO:44所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:42所示的氨基酸序列；或
    所述第一重链多肽链具有如SEQ ID NO:48所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:42所示的氨基酸序列。
  19. 如权利要求1-10及14中任一项所述的融合蛋白二聚体，所述单克隆抗体为抗PD-1抗体，其中：
    所述第一重链多肽链具有如SEQ ID NO:60所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:58所示的氨基酸序列；
    所述第一重链多肽链具有如SEQ ID NO:58所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:56所示的氨基酸序列；或
    所述第一重链多肽链具有如SEQ ID NO:62所示的氨基酸序列，所述第二重链多肽链具有如SEQ ID NO:56所示的氨基酸序列。
  20. 如前述权利要求中任一项所述的融合蛋白二聚体，所述单克隆抗体与细胞因子之间通过接头序列连接或直接融合。
  21. 如权利要求20所述的融合蛋白二聚体，所述接头序列选自(S(G4S) 1-3)、(G4S) 1-3、KRVAPELLGGPS、ASTKG、NSPPAA、EPKSSDKTHTSPPSP、ERKSSVESPPSP以及ESKYGPPSPPSP。
  22. 一种分离的多核苷酸，所述多核苷酸编码权利要求1-21中任一项所述的融合蛋白二聚体。
  23. 包含权利要求22所述多核苷酸的表达载体。
  24. 包含权利要求23所述表达载体的宿主细胞。
  25. 如权利要求1-21中任一项所述融合蛋白二聚体、权利要求22所述多核苷酸、权利要求23所述表达载体或权利要求24所述宿主细胞在制备抗肿瘤药物中的应用。
  26. 一种抗肿瘤药物组合物，所述药物组合物包含权利要求1-21中任一项所述的融合蛋白二聚体和药学上可接受的载体。
PCT/CN2020/097927 2019-06-24 2020-06-24 单克隆抗体-细胞因子融合蛋白二聚体及其应用 WO2020259536A1 (zh)

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