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WO2020048454A1 - 融合蛋白及其在制备用于治疗肿瘤和/或病毒感染的药物中的应用 - Google Patents

融合蛋白及其在制备用于治疗肿瘤和/或病毒感染的药物中的应用 Download PDF

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Publication number
WO2020048454A1
WO2020048454A1 PCT/CN2019/104217 CN2019104217W WO2020048454A1 WO 2020048454 A1 WO2020048454 A1 WO 2020048454A1 CN 2019104217 W CN2019104217 W CN 2019104217W WO 2020048454 A1 WO2020048454 A1 WO 2020048454A1
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antibody
fusion protein
umy02
seq
human
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PCT/CN2019/104217
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English (en)
French (fr)
Inventor
莫世甫
赵勇
徐伟
王志超
杨洁
顾莉蕴
丁栋
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南京优迈生物科技有限公司
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Priority to EP19857108.5A priority Critical patent/EP3848397A4/en
Priority to JP2021510232A priority patent/JP7404343B2/ja
Priority to CN201980054796.XA priority patent/CN112585169A/zh
Priority to KR1020217006850A priority patent/KR20210056344A/ko
Priority to AU2019336426A priority patent/AU2019336426A1/en
Publication of WO2020048454A1 publication Critical patent/WO2020048454A1/zh
Priority to US17/192,399 priority patent/US20210198375A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • the invention belongs to the field of biomedicine. Specifically, the present invention relates to a fusion protein and use thereof. More specifically, the present invention relates to a fusion protein containing an anti-OX40 antibody and human interferon and its use in the preparation of a medicament for treating tumors and / or viral infections.
  • Human OX40 is mainly expressed on activated T cells, including CD4, CD8, Th and Treg cells, etc. [1]. in The expression of OX40 on T cells is very low, but its expression level is up-regulated and reaches its peak within 12 hours to 5-6 days after antigen-induced stimulation. Similarly, the expression of OX40L is also affected by the state of cell activation [1]. APC cells can detect the expression of OX40L 1-3 days after antigen stimulation. Interestingly, in addition to immune cells, muscle cells also express OX40L under the stimulation of inflammatory factors [2,3], suggesting that the OX40L-OX40 signaling pathway may widely affect the body's inflammatory response.
  • OX40L / OX40 co-stimulatory molecules The activation of antigen-dependent OX40L / OX40 co-stimulatory molecules is coupled to multiple signaling pathways in T cells. Crystal structure studies have shown that the combination of OX40L and OX40 can induce the trimerization of OX40-OX40L complex [4], thereby forming a binding site with receptor-associated factor (TRAF) in the cell. The latter (TRAF2, 5) can further activate the NF- ⁇ B signaling pathway and inhibit T cell apoptosis [2,5,6].
  • OX40 activation can lead to high expression of Bcl-2 and Bcl-xL [7], suggesting that OX40 may induce the expression of anti-apoptotic proteins through the NF- ⁇ B signaling pathway and achieve its function of inhibiting T cell apoptosis.
  • PKB / PI3K is another important signaling pathway downstream of OX40.
  • the co-stimulatory signal of OX40 on T cells is necessary for maintaining PKB activation
  • constitutively activated PKB can antagonize the down-regulation of anti-apoptotic proteins in T cells caused by OX40 deficiency.
  • the OX40 co-stimulatory signal can maintain Survivin protein expression through the PKB / PI3K signaling pathway [8].
  • TCR and OX40 on T cells can also cause the activation of calcium flux and NFAT signaling pathway, and regulate the expression of cytokines including IL-2, IL-4, IL-5 and IFN- ⁇ [9] .
  • the above studies indicate that the activation of OX40 can regulate the proliferation, apoptosis and cytokine secretion of T cells through the NF- ⁇ B signaling pathway, PKB / PI3K signaling pathway and NFAT signaling pathway, thereby achieving the effect of enhancing the vitality of the immune system.
  • OX40 has become an important target for immunotherapy. Numerous pre-clinical and clinical studies have suggested that OX40 can be an important target for tumor immunotherapy. Interestingly, recent studies have also found a role for the OX40 signaling pathway in suppressing hepatitis B virus infection [10], suggesting that OX40 agonists may be a potential means of treating antiviral infections, such as patients with hepatitis B.
  • Interferon is a class of highly active and versatile glycoproteins. on the one hand. Interferon can exert a strong anti-tumor effect by regulating tumor cell proliferation, inhibiting tumor metastasis and angiogenesis, and activating anti-tumor immune response; on the other hand, by regulating the human immune system, interferon has important clinical application value in anti-virus For example, interferon has become one of the important methods for clinical treatment of hepatitis B virus infection.
  • OX40 agonist and interferon have important application value or potential in antitumor and antiviral aspects, but the existing evidence shows that both have insufficient patient response rates and efficacy. Therefore, there is currently a need for OX40 agonists and interferons that have better therapeutic effects.
  • the object of the present invention is to provide a fusion protein comprising an antibody or an antigen-binding fragment thereof that specifically binds human OX40 and human interferon.
  • the invention also provides the use of the fusion protein for treating tumors and / or viral infections.
  • the present invention adopts the following technical solutions:
  • the invention provides a fusion protein comprising:
  • human interferon is connected to the carboxyl or amino terminus of the light or heavy chain of the antibody directly or through a peptide linker.
  • the fusion protein according to the present invention wherein the antibody or antigen-binding fragment thereof that specifically binds human OX40 comprises:
  • Antibody heavy chain variable region comprising VHCDR1 having the amino acid sequence of SEQ ID NO: 1, VHCDR2 having the amino acid sequence of SEQ ID NO: 2, and amino acid sequence having SEQ ID NO: 3 VHCDR3;
  • Antibody light chain variable region comprising VL CDR1 having the amino acid sequence of SEQ ID NO: 4, VL2 having the amino acid sequence of SEQ ID NO: 5, CDR2, and the amino acid sequence having SEQ ID NO: 6 VL CDR3.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8.
  • the fusion protein according to the present invention wherein the antibody or antigen-binding fragment thereof that specifically binds human OX40 is a camelized single domain antibody, scFv, scFv dimer, BsFv, dsFv, dsFv2, dsFv-dsFv ', Fv Fragment, Fab, Fab ', F (ab') 2, ds bifunctional antibody, Nanobody, domain antibody or bivalent domain antibody.
  • the fusion protein according to the present invention wherein the antibody further comprises an immunoglobulin constant region, such as a constant region of human IgG1, IgG2, or IgG4.
  • an immunoglobulin constant region such as a constant region of human IgG1, IgG2, or IgG4.
  • the human interferon is selected from the group consisting of type I human interferon, type II human interferon, and type III human interferon.
  • the human interferon is IFN ⁇ 2a, IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ 2b; more preferably, the human interferon is IFN ⁇ 2b, and its amino acid sequence is shown in SEQ ID NO: 9.
  • the human interferon is a mutant of IFN ⁇ 2b, which has one or more mutations selected from the following amino acid sequences shown in SEQ ID NO: 9: T106A, R149A, A145G, A145D, R120A , L117A;
  • the mutant of IFN ⁇ 2b has a double mutation selected from the following in the amino acid sequence shown in SEQ ID NO: 9:
  • T106A / A145D T106A / R149A, T106A / A145G, T106A / L117A, T106A / R120A.
  • the peptide linker is selected from (G) n, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSAGSAAGSGEF, (GGGGS) n, (GGSGG) n; preferably, the peptide linker is (GGGGS) n, Wherein n is an integer between 0-5; preferably, n is an integer between 1-3.
  • fusion protein according to the present invention, wherein the fusion protein is selected from:
  • UMY02-L1 which contains the amino acid sequence shown in SEQ ID NO: 10 and the amino acid sequence shown in SEQ ID NO: 11, wherein the heavy chain, peptide linker and human of the antibody whose amino acid sequence shown in SEQ ID NO: 10 is OX40 Interferon, the light chain of an antibody whose amino acid sequence shown in SEQ ID NO: 11 is OX40;
  • UMY02-L2 which contains the amino acid sequence shown in SEQ ID NO: 12 and the amino acid sequence shown in SEQ ID NO: 13,
  • the heavy chain of the antibody whose amino acid sequence is shown in SEQ ID NO: 12 is OX40
  • the light chain, peptide linker and human interferon of the antibody whose amino acid sequence is shown in SEQ ID NO: 13 is OX40;
  • UMY02-L3 which contains the amino acid sequence shown in SEQ ID NO: 14 and the amino acid sequence shown in SEQ ID NO: 13, wherein the amino acid sequence shown in SEQ ID NO: 14 is the heavy chain of the antibody of OX40, SEQ ID The light chain, peptide linker and human interferon of the antibody whose amino acid sequence is NO: 13 is OX40;
  • UMY02-L4 where the heavy chain is shown in SEQ ID NO: 14, the light chain and human interferon are shown in SEQ ID ID NO: 13, and the human interferon has no peptide linker;
  • UMY02-L5 where the heavy chain is shown as SEQ ID NO: 14, the light chain and human interferon are shown as light chain and human interferon in SEQ ID NO: 13, and the peptide linker is GGGGS;
  • UMY02-L6 where the heavy chain is shown as SEQ ID NO: 14, the light chain and human interferon are shown as light chain and human interferon in SEQ ID NO: 13, and the peptide linker is (GGGGS) 2;
  • UMY02-L7 where the heavy chain is shown in SEQ ID NO: 15, the light chain and human interferon are shown in SEQ ID ID: 13, and the peptide linker is GGGGS;
  • UMY02-L8 wherein the heavy chain is shown in SEQ ID NO: 15, the light chain and human interferon are shown in SEQ ID ID: 13 and the human interferon, and the peptide linker is (GGGGS) 2;
  • UMY02-L13 where the heavy chain is shown as SEQ ID NO: 15, the light chain and human interferon are shown as light chain and human interferon in SEQ ID NO: 13, the peptide linker is (GGGGS) 2, and the interferon is in For example, it is mutated to T106A / A145D based on SEQ ID NO: 9;
  • UMY02-L14 where the heavy chain is shown as SEQ ID NO: 15, the light chain and human interferon are shown as light chain and human interferon in SEQ ID NO: 13, the peptide linker is (GGGGS) 2, and the interferon is in For example, it is mutated to T106A / R149A based on SEQ ID NO: 9;
  • UMY02-L15 where the heavy chain is shown as SEQ ID NO: 15, the light chain and human interferon are shown as the light chain and human interferon in SEQ ID NO: 13, the peptide linker is (GGGGS) 2, and the interferon is in For example, it is mutated into T106A / R120A based on SEQ ID NO: 9;
  • UMY02-L16 where the heavy chain is shown as SEQ ID NO: 15, the light chain and human interferon are shown as the light chain and human interferon in SEQ ID NO: 13, the peptide linker is (GGGGS) 2, and the interferon is
  • the basic mutation of SEQ ID NO: 9 is T106A / A145G;
  • UMY02-L17 where the heavy chain is shown as SEQ ID NO: 15, the light chain and human interferon are shown as light chain and human interferon in SEQ ID NO: 13, the peptide linker is (GGGGS) 3, and the interferon is
  • the basic mutation of SEQ ID NO: 9 is T106A / R149A;
  • UMY02-L18 where the heavy chain is shown as SEQ ID NO: 15, the light chain and human interferon are shown as light chain and human interferon in SEQ ID NO: 13, the peptide linker is (GGGGS) 2, and the interferon is
  • the basic mutation of SEQ ID NO: 9 is T106A / L117A;
  • Fusion proteins OX40IFN- ⁇ 2a, OX40-IFN ⁇ , OX40-IFN ⁇ , OX40-IFN ⁇ 3, among which OX40IFN- ⁇ 2a, OX40-IFN ⁇ , OX40-IFN ⁇ , OX40-IFN ⁇ 3 heavy chain is shown as SEQ ID ID NO: 15;
  • the peptide linker and interferon are shown in SEQ ID NO: 16;
  • the OX40IFN- ⁇ light chain, the peptide linker and interferon are shown in SEQ ID NO: 17;
  • the OX40IFN- ⁇ light chain, the peptide linker and interferon are shown in SEQ ID NO : 18;
  • OX40IFN- ⁇ 3 light chain, peptide linker and interferon are shown in SEQ ID NO: 19.
  • the present invention provides an isolated polynucleotide encoding the fusion protein.
  • the invention provides a vector comprising the isolated polynucleotide.
  • the present invention provides a host cell including the vector.
  • the present invention also provides a method for expressing the fusion protein, which comprises culturing the host cell under conditions capable of expressing an isolated polynucleotide.
  • the invention also provides a kit comprising the fusion protein.
  • the invention also provides a pharmaceutical composition comprising the fusion protein and a pharmaceutically acceptable carrier.
  • the invention also provides the use of the fusion protein in the preparation of a medicament for the treatment of conditions that can benefit from enhanced immune response and / or exposure to interferon.
  • the condition is a cancer or a viral infection, such as a hepatitis B virus infection.
  • the present invention provides a method of treating a condition that can benefit by enhancing an immune response and / or by being exposed to interferon, said method comprising administering to a subject in need thereof a therapeutically effective amount of said fusion protein; preferably Where the condition is a cancer or viral infection, such as a hepatitis B virus infection.
  • the invention provides a fusion protein that treats a condition that can benefit by enhancing an immune response and / or that can benefit from exposure to interferon; preferably, wherein the condition is a cancer or a viral infection, such as a hepatitis B virus infection.
  • the present invention provides a fusion protein of OX40 agonistic antibody and interferon, which simultaneously combines the different mechanisms of action of the two, and forms a synergistically enhanced effect: on the one hand, when the interferon portion of the fusion protein binds to a highly expressed interferon receptor,
  • the surface of somatic cells may enhance the activity of OX40-activating antibodies in a receptor-mediated mode, thereby better enhancing the activity of the immune system;
  • the fusion protein of the present invention is more disturbing The half-life of the hormone molecule is greatly prolonged, and it can be used at a lower frequency in the clinic. Compared with the current interferon which requires daily injection in the clinic, it has a great advantage.
  • the fusion protein of the present invention may show unique advantages in terms of efficacy and compliance in antitumor and antiviral treatment.
  • Figure 1 shows a schematic diagram of the structure of a fusion protein according to the present invention.
  • Figure 2 shows the results of an ELISA binding experiment of a fusion protein according to the present invention with human OX40 protein.
  • FIG. 3 FACS test results show that MT01-L1 antibody binds to both human and cynomolgus monkey OX40, but not to mouse OX40.
  • Fig. 4 shows the proliferation inhibitory effect of the fusion protein according to the present invention on Daudi cells.
  • Figure 5 shows that the fusion protein according to the present invention activates NF-kB signaling pathway activity in Jurkat cells.
  • FIG. 6 shows that the fusion protein according to the present invention can promote the OX40 signaling pathway activating activity of MT01-C1 antibody.
  • the OX40 monoclonal antibody (MT01-C1 or MT01-C1 (G2)) alone or the interferon molecule IFN ⁇ 2b has no or only weak activation of the OX40 signaling pathway in Jurkat cells in this experimental system (6a).
  • the fusion proteins (UMY02-L1, UMY02-L2, and UMY02-L3) have significant activation activity on the OX40 signaling pathway under the same conditions (6b).
  • Figure 7 shows the pharmacokinetic curves of UMY02-L1 and UMY02-L3 in mice (7a).
  • Figure 7b shows the pharmacokinetic curves of MT01-C1 in mice.
  • Fig. 8 shows the killing effect of the fusion protein according to the present invention on tumor cells.
  • antibody in the present invention includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that can bind to a specific antigen.
  • a natural intact antibody contains two heavy chains and two light chains. Each heavy chain is composed of a variable region and first, second, and third constant regions; each light chain is composed of a variable region and a constant region. Mammalian heavy chains can be divided into ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , and mammalian light chains can be divided into ⁇ or ⁇ .
  • the antibody is "Y" -shaped, and the neck of the Y-type structure consists of the second and third constant regions of the two heavy chains, which are bound by disulfide bonds.
  • Each arm of the "Y" structure includes the variable region of one of the heavy chains and a first constant region that binds to the variable and constant regions of a light chain.
  • the variable regions of the light and heavy chains determine antigen binding.
  • the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDR).
  • the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3.
  • the CDR boundaries of the antibodies and antigen-binding fragments disclosed in the present invention can be named or identified by Kabat, Chothia or Al-Lazikani nomenclature.
  • the three CDRs are separated by a side continuous portion called a framework region (FR), which is more highly conserved than the CDRs and forms a scaffold to support the hypervariable loop.
  • FR framework region
  • the constant regions of the heavy and light chains have nothing to do with antigen binding, but have multiple effector functions.
  • Antibodies can be divided into several classes based on the amino acid sequence of the constant region of the heavy chain. Depending on whether they contain ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, antibodies can be divided into five major classifications or isomers: IgA, IgD, IgE, IgG, and IgM.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • antigen-binding fragment refers to an antibody fragment formed from an antibody portion containing one or more CDRs or any other antibody fragment that binds an antigen but does not have a complete antibody structure.
  • antigen-binding fragments include, but are not limited to, such as bifunctional antibodies (diabody), Fab, Fab ', F (ab') 2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2, Bispecific dsFv (dsFv-dsFv '), disulfide-stabilized bifunctional antibody (dsdiabody), single chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), bivalent single chain antibody (BsFv), multispecific antibodies, camelized single domain antibodies, Nanobodies, domain antibodies, and bivalent domain antibodies.
  • the antigen-binding fragment can bind the same antigen as the parent antibody.
  • an antigen-binding fragment can bind the same anti
  • the "Fab" fragment of an antibody refers to the portion of the antibody molecule that is composed of a light chain (including a variable region and a constant region) and a variable region and a portion of the constant region of a heavy chain that are bound by disulfide bonds.
  • Fab ' refers to a Fab fragment that contains a partial hinge region.
  • F (ab ') 2 refers to a dimer of Fab.
  • the Fc segment of an antibody is responsible for many different effector functions such as ADCC and CDC, but does not participate in antigen binding.
  • the "Fv” segment of an antibody refers to the smallest antibody fragment that contains a complete antigen-binding site.
  • the Fv fragment consists of a variable region of a light chain and a variable region of a heavy chain.
  • a “fusion protein” refers to a recombinant protein that connects a cDNA encoding a protein of interest with a cDNA encoding an antibody or an antibody fragment at the gene level and expresses it in a eukaryotic or prokaryotic expression system.
  • Linker refers to a peptide chain or a derivative thereof consisting of 1 to 50 amino acids that have formed a peptide bond, and the N-terminus and C-terminus of the peptide chain or a derivative thereof are covalently formed with any one of an anti-OX40 antibody or an interferon Bond, thereby binding the anti-OX40 antibody to interferon.
  • the anti-OX40 antibody and interferon can be integrated by binding the N-terminus or C-terminus of the interferon through a linker sequence or directly using a peptide bond on the C- or N-terminal side of the heavy or light chain of the anti-OX40 antibody, respectively. .
  • fusion protein of interferon and anti-OX40 antibody a fusion protein obtained by binding the C-terminus of the heavy or light chain of the anti-OX40 antibody to the N-terminus of the interferon via a linker sequence; or, A fusion protein in which the N-terminus of the heavy or light chain of an anti-OX40 antibody is bound to the C-terminus of an interferon via a linker sequence.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody in which the light chain variable region and the heavy chain variable region are directly connected or connected through a peptide chain (Huston, JS, etc., Proc Natl Acad Sci USA, 85: 5879 (1988)).
  • Single-chain antibody Fv-Fc or “scFv-Fc” refers to an engineered antibody composed of scFv linked to the Fc segment of an antibody.
  • HCAb Heavy-chain-only antibodies, HCAb
  • heavy chain antibodies were originally derived from the camel family (camel, dromedary, and llama).
  • camelized antibodies have the full function of confirmed antigen binding (Hamers, Casterman, C. et al., Nature 363 (6428): 446-8 (1993); Nguyen, VK., Et al., "Heavy-chain antibodies” in Camelidae: a case of evolutionary innovation, Immunogenetics. 54 (1): 39-47 (2002); Nguyen VK. Et al., Immunology. 109 (1): 93-101 (2003)).
  • Variable regions of heavy chain antibodies ( VH domain) is the smallest known antigen-binding unit for adaptive immunity (Koch-Nolte F. et al., FASEB J. 21 (13): 3490-8. Epub (2007)).
  • Nanobody refers to an antibody fragment that consists of a VH domain from a heavy chain antibody and two constant regions, CH2 and CH3.
  • a “diabody” includes a small antibody fragment with two antigen-binding sites, where the fragment contains a VH domain and a VL domain connected on the same polypeptide chain (see Holliger P. et al., Proc Natl Acad. Sci. A. 90 (14): 6444-8 (1993); EP404097; W093 / 11161).
  • the adapter between the two domains is very short, so that the two domains on the same chain cannot pair with each other, thus forcing the two domains to pair with the complementary domains of the other two chains to form two antibody binding sites.
  • These two antibody binding sites can be targeted to bind the same or different antigens (or epitopes).
  • a “domain antibody” refers to an antibody fragment that contains only one heavy chain variable region or one light chain variable region.
  • two or more VH domains are covalently bound by a polypeptide adapter and form a bivalent domain antibody.
  • the two VH domains of a bivalent domain antibody can target the same or different antigens.
  • "(dsFv) 2" contains three peptide chains: two VH genes are connected by a polypeptide adapter and are bound to two VL groups via a disulfide bond.
  • a "bispecific ds bifunctional antibody” contains VL1-VH2 (connected by two polypeptide adapters) and VH1-VL2 (also connected by two polypeptide adapters), both of which are between VH1 and VL1 Binding via disulfide bonds.
  • a "scFv dimer” is a bivalent bifunctional antibody or a bivalent single chain antibody (BsFv) that contains two VH-VL (linked by a polypeptide adapter) group that is dimerized, wherein The VH of the two groups cooperates with the VL of the other group to form two binding sites, which can be targeted to bind the same antigen (or epitope) or different antigens (or epitopes).
  • the "scFv dimer” is a bispecific bifunctional antibody that contains V L1- V H2 (connected by a peptide adapter) and V H1- V L2 (connected by a peptide adapter) interconnected Among them, V H1 and V L1 cooperate, V H2 and V L2 cooperate, and each cooperative pair has different antigen specificity.
  • whole-human origin used in this application when used in an antibody or an antigen-binding fragment means that the antibody or antigen-binding fragment has a certain amino acid sequence or consists of the amino acid sequence, and the amino acid sequence corresponds to a human Or an amino acid sequence of an antibody produced by a human immune cell or derived from a non-human source such as a transgenic non-human animal using a human antibody library, or another sequence encoding a human-derived antibody.
  • fully human antibodies do not contain amino acid residues (especially antigen-binding residues) derived from non-human antibodies.
  • humanized when used in antibodies or antigen-binding fragments, is meant to include CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (when applicable) ) Antibodies or antigen-binding fragments. Because humanized antibodies or antigen-binding fragments have reduced immunogenicity, they can be used as human therapeutics in certain embodiments.
  • the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig or hamster.
  • the humanized antibody or antigen-binding fragment consists essentially of human-derived sequences, except that the CDR sequences are of non-human origin.
  • the human-derived FR region may include the same amino acid sequence as the human-derived antibody from which it is derived, or it may include some amino acid changes, for example, no more than 10, 9, 8, 7, 6, 5, , 4, 3, 2 or 1 amino acid change.
  • the amino acid change may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains.
  • the humanized antibodies include human FRl-3 and human JH and JK.
  • chimeric refers to having a portion of the heavy and / or light chain derived from one species, and the rest of the heavy and / or light chain is derived from antibodies or antigens from different species. Fragment.
  • a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse.
  • OX40 refers to a receptor that binds to OX40L. It is a type I membrane protein belonging to the TNF receptor family. Other names are ACT-4, OX40L receptor, CD134 antigen, ACT35 antigen, TNFRSF4. It has a molecular weight of 50 kDa and is stored in SwissProt under the accession number P43489.
  • human interferon used in this application refers to a class of highly active, multifunctional secreted glycoproteins, which have antiviral, immunomodulatory and antitumor effects. According to its gene sequence and receptor specificity, it can be divided into type I, type II and type III IFNs.
  • Human type I interferons include 13 subtypes of IFN ⁇ and IFN ⁇ , IFN ⁇ , IFN ⁇ , and IFN ⁇ .
  • Type I IFNs have a common cell surface receptor, IFNAR, consisting of two subunits, IFNAR1 and IFNAR2.
  • the cell surface receptor of IFN ⁇ is IFNGR, which consists of two subunits, IFNGR1 and IFNGR2.
  • Type III IFNs include IFN ⁇ 1, IFN ⁇ 2, IFN ⁇ 3, and IFN ⁇ 4. Interferons in the fusion proteins of the present application also include interferon variants known in the art.
  • telomere binding refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen.
  • the antibodies or antigen-binding fragments thereof of the present application specifically bind to human and / or monkey OX40, and their binding affinity (K D ) is ⁇ 10 ⁇ 6 M.
  • the K D in the present application refers to the ratio (k off / k on ) of the dissociation speed to the binding speed, which can be measured by a surface plasmon resonance method, for example, using an instrument such as Biacore.
  • UY02-L1 refers to a heavy chain, a peptide linker, and a human interferon as shown in SEQ ID NO: 10, and a light chain as shown in SEQ ID NO: 11, in which the heavy chain carboxyl terminus A fusion protein of human interferon IFN ⁇ 2b as shown in SEQ ID NO: 9 was linked via a peptide linker.
  • UY02-L2 used in this application refers to a heavy chain as shown in SEQ ID NO: 12, a light chain as shown in SEQ ID NO: 13, a peptide linker, and human interferon, in which the light chain carboxyl terminus A fusion protein of human interferon IFN ⁇ 2b as shown in SEQ ID NO: 9 was linked via a peptide linker.
  • UY02-L3 refers to a heavy chain as shown in SEQ ID NO: 14, a light chain as shown in SEQ ID NO: 13, a peptide linker, and human interferon, in which the light chain carboxyl terminus A fusion protein of human interferon IFN ⁇ 2b as shown in SEQ ID NO: 9 was linked via a peptide linker.
  • MMT01-C1 used in this application means that it has the same VH (SEQ ID NO: 7) and VL (SEQ ID NO: 8) sequences as UMY02-L1, UMY02-L2, and UMY02-L3, and the heavy and light chains are constant. Regions are monoclonal antibodies against human IgG1 and kappa chains, respectively.
  • MT01-C1 (G2) used in this application means that it has the same VH (SEQ ID NO: 7) and VL (SEQ ID ID NO: 8) sequences as UMY02-L1, UMY02-L2, and UMY02-L3, and repeats
  • VH SEQ ID NO: 7
  • VL SEQ ID NO: 8
  • the light chain constant regions are monoclonal antibodies to human IgG2 and kappa chains, respectively.
  • MMT01-L1 used in this application means that it has the same heavy and light chain CDR sequences (SEQ ID NO: 1-6) as UMY02-L1, UMY02-L2, and UMY02-L3, and the heavy and light chain constant regions are human Human-mouse chimeric antibodies to IgG1 and kappa chains.
  • the "MT01-C1", “MT01-C1 (G2)”, and “MT01-L1” are all derived from the Chinese patent 201711476160.3, and the specific sequences are shown in Table 1 and the sequence table, which are incorporated by reference in their entirety. This article.
  • conservative substitution when “conservative substitution” is used in an amino acid sequence, it means that one amino acid residue is replaced with another amino acid residue in a side chain having similar physicochemical properties. For example, between hydrophobic side chain amino acid residues (such as Met, Ala, VaL, Leu, and Ile), neutral hydrophilic side chain residues (such as Cys, Ser, Thr, Asn, and Gln), acidic side chain residues Conservative substitutions are made between bases (e.g. Asp, Glu), basic side chain amino acids (e.g. His, Lys and Arg) or aromatic side chain residues (e.g. Trp, Tyr and Phe). It is known in the art that conservative substitutions generally do not cause significant changes in the conformational structure of the protein and are therefore capable of retaining the biological activity of the protein.
  • bases e.g. Asp, Glu
  • basic side chain amino acids e.g. His, Lys and Arg
  • aromatic side chain residues e.g. Trp, Tyr and Phe
  • percent sequence identity when “percent sequence identity” is used for an amino acid sequence (or nucleic acid sequence), it means that after the sequence alignment is performed, and if necessary, a gap is introduced to maximize the number of identical amino acids (or nucleic acids), in the candidate sequence, the The percentage of amino acid (or nucleic acid) residues with the same sequence as the candidate sequence. Conservative substitutions of the amino acid residues may or may not be considered the same residues. Sequences can be aligned by tools disclosed in the art to determine the percent sequence identity of amino acid (or nucleic acid) sequences. Those skilled in the art can use the default parameters of the tool or adjust the parameters appropriately according to the needs of the comparison, for example, by selecting an appropriate algorithm.
  • T cells used in the present application include CD4 + T cells, CD8 + T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and suppressor T cells.
  • Effective function refers to the biological activity of an antibody's Fc region that binds to its effectors, such as the C1 complex and Fc receptor.
  • exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by the interaction of antibodies with C1q on the C1 complex, antibody-dependent cell-mediated cells induced by binding of the Fc region of the antibody to Fc receptors on effector cells Toxicity (ADCC) and phagocytosis.
  • cancer or “cancer condition” refers to any medical condition that is mediated by the growth, proliferation, or metastasis of tumors or malignant cells and causes solid tumors and non-solid tumors such as leukemia.
  • tumor in the present invention refers to a solid substance of a tumor and / or a malignant cell.
  • viral infection refers to a pathogenic process in which a virus invades a human body through various pathways and proliferates in human cells, causing damage to the body, including chronic viral infections, such as hepatitis B, hepatitis C, and herpes viruses , EB (Epstein-Barr) virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type 2, human papilloma virus, adenovirus virus infection, Kaposi's sarcoma-associated herpes virus epidemic , Torquetenovirus, JC virus or BK virus.
  • chronic viral infections such as hepatitis B, hepatitis C, and herpes viruses , EB (Epstein-Barr) virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type 2, human papilloma virus, adenovirus virus infection, Kaposi's sarcoma-associated herpes virus epidemic
  • Treatment or “therapy” of a condition includes preventing or reducing a condition, reducing the rate at which a condition arises or developing, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition , Reduce or terminate the symptoms associated with a condition, produce a complete or partial reversal of a condition, cure a condition, or a combination of the above.
  • treatment or “therapy” can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination of the above.
  • treatment or “therapy” includes clearing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination of the above.
  • isolated matter has been artificially altered from its natural state. If some "isolated” substance or component appears in nature, it has been altered or removed from its original state, or both.
  • a naturally occurring polynucleotide or polypeptide in a living animal is not isolated, but if these polynucleotides or polypeptides are sufficiently separated from substances that coexist in their natural state and exist in a sufficiently pure state, they can We think that it is "separated”.
  • the purity of the antibody and antigen-binding fragment is at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, which is determined by electrophoresis methods (such as SDS-PAGE, isoelectric focusing) , Capillary electrophoresis), or chromatography (such as ion exchange chromatography or reversed-phase HPLC).
  • electrophoresis methods such as SDS-PAGE, isoelectric focusing
  • Capillary electrophoresis Capillary electrophoresis
  • chromatography such as ion exchange chromatography or reversed-phase HPLC.
  • a "vector” refers to a vehicle that can be operatively inserted into a polynucleotide encoding a protein and obtain expression of the protein.
  • Vectors can be used to transform, transduce, or transfect host cells so that the genetic material elements they carry are expressed within the host cells.
  • vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses.
  • vectors The types of animal viruses used as vectors are retroviruses (including lentivirus, adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), pox virus, baculovirus, papilloma virus, papilloma vacuolar virus (such as SV40)).
  • Vectors can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may contain a replication initiation site.
  • a vector may also include components that assist it in entering cells, including, but not limited to, viral particles, liposomes, or protein shells.
  • a "host cell” in the present invention is a cell that is directed into an exogenous polynucleotide and / or vector.
  • the "therapeutically effective amount” or “effective dose” in the present invention refers to the dose or concentration of a certain drug to effectively treat a disease.
  • the therapeutically effective amount is that at the dose or concentration, the antibody or antigen-conjugate can clear all or part of the tumor, inhibit or slow tumor growth, and inhibit Tumor cell metastases, alleviate any symptoms or markers associated with a tumor or cancer condition, prevent or delay the development of a tumor or cancer condition, inhibit or clear a virus or virus-infected cells, or some combination of the above.
  • “Pharmaceutically acceptable” means the carrier, vehicle, diluent, excipient, and / or salt referred to, and is generally chemically and / or physically compatible with the other ingredients in the formulation, and Physiologically compatible with the recipient.
  • the present application provides exemplary fusion proteins UMY02-L1, UMY02-L2, and / or UMY02-L3.
  • CDR sequences can be modified to include substitutions of one or more amino acids, thereby resulting in improved biological activity such as improved binding affinity to human OX40.
  • phage display technology can be used to produce and express a library of antibody variants (eg, Fab or FcFv variants), and then screen for antibodies that have an affinity for human OX40.
  • computer software can be used to simulate the binding of the antibody to human OX40 and identify the amino acid residues on the antibody that form the binding interface. Substitutions of these residues can be avoided to prevent reduced binding affinity, or these residues can be targeted for substitution to form stronger binding.
  • at least one (or all) substitutions in a CDR sequence are conservative substitutions.
  • the fusion protein and antigen-binding fragment include one or more CDR sequences, which have the same fusion proteins as the exemplary fusion proteins UMY02-L1, UMY02-L2, and / or UMY02-L3 provided in the present application.
  • Sequence identity of at least 80% e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
  • the parent antibody has substantially the same sequence, but its corresponding CDR sequence is 100% sequence identical to the listed sequence Sex.
  • the fusion protein described herein is capable of specifically binding to human OX40 with a binding affinity (K D ) of ⁇ 10 ⁇ 7 M, which is measured by surface plasmon resonance method.
  • the binding affinity value can be expressed as a K D value, which is calculated by the ratio of the dissociation rate to the binding rate (k off / k on ) when the binding of the antigen and the antigen-binding molecule reaches equilibrium.
  • the antigen binding affinity e.g., K D
  • K D may be suitably determined by suitable methods known in the art, for example, including the use of instruments such as the Biacore plasmon resonance binding assay.
  • the fusion protein described herein binds to human OX40 at an EC50 (ie, half binding concentration) of 10 ng / mL to 10 ⁇ g / mL.
  • the binding of the antibody or fusion protein to human OX40 can be determined by methods known in the art such as sandwich methods such as ELISA, Western blot, FACS or other binding assays.
  • the test antibody ie, the primary antibody
  • the unbound antibody is washed away, and a labeled secondary antibody is introduced, which can bind to the primary antibody.
  • Able to detect bound secondary antibodies The detection can be performed on a microplate reader when using immobilized OX40, or can be performed using FACS analysis when using cells expressing human OX40.
  • the fusion protein described herein binds human OX40 at an EC50 (ie, an effective concentration of 50%) of 0.1 ⁇ g / mL to 10 ⁇ g / mL (determined using FACS analysis).
  • the fusion proteins described herein can activate the human OX40 signaling pathway in an FcR-mediated or interferon receptor-mediated manner, and thus provide methods including, for example, inducing activated T cells to produce cytokines (such as CD4 + T cells and CD8 + T cells), induces the proliferation of activated T cells (such as CD4 + T cells and CD8 + T cells), and reverses the biological activity of inhibitory functions of regulatory Tregs.
  • cytokines such as CD4 + T cells and CD8 + T cells
  • activated T cells such as CD4 + T cells and CD8 + T cells
  • the fusion protein is specific for human OX40.
  • the fusion protein does not bind to murine OX40, but binds to monkey OX40 with a binding affinity similar to human OX40.
  • the binding of a monoclonal antibody MT01-L1 having the same CDR sequence as the fusion protein of the present invention to murine OX40 cannot be detected by conventional binding assay methods such as FACS analysis, and FACS detects that both MT01-L1 and monkey OX40 and human OX40 There are combinations.
  • the fusion protein has a constant region of the IgG2 isotype, which has a reduced or eliminated effector function.
  • Effector functions such as ADCC and CDC can cause cytotoxicity to cells expressing OX40.
  • Some normal cells are capable of expressing OX40.
  • certain embodiments of the antibodies of the invention have reduced or even eliminated effector functions.
  • Many tests are known to estimate ADCC or CDC activity, such as Fc receptor binding assay, complement Clq binding assay, and cell lysis method, which can be easily selected by those skilled in the art. Not wishing to be bound by theory, but it is believed that antibodies with reduced or eliminated effector functions such as ADCC and CDC will not cause or minimize cytotoxicity to cells expressing OX40, such as those normal cells, Therefore, unwanted side effects are avoided.
  • the fusion protein described in the present application has a prolonged action time in the organism than the interferon molecule. This is due to the longer half-life and drug retention time of the fusion protein in the animal. This property is beneficial to reduce the number of medications for patients and improve the efficacy of the drugs.
  • the fusion proteins described herein have reduced side effects.
  • the anti-OX40 antibody and its antigen-binding fragment may have a fully human IgG sequence, and therefore its immunogenicity is lower than that of a humanized antibody.
  • the fusion protein and its antigen-binding fragment may have the form of IgG2 or IgG4 to eliminate ADCC and CDC.
  • the fusion protein described herein has the advantage that it can be used in combination with substances having immunogenicity, such as tumor cells, purified tumor antigens, and cells transfected with encoded immunostimulatory factors, tumor vaccines.
  • the anti-OX40 antibody and its antigen-binding fragment may be included in a combination therapy, including standard chemotherapy and radiation therapy, target-based small molecule therapy, and other emerging immune checkpoint modulator therapies.
  • the antibody and its antigen-binding fragment can be used as a base molecule for an antibody-drug conjugate, a bispecific, or a multivalent antibody.
  • the fusion protein and the antigen-binding fragment thereof described herein are a camelized single domain antibody, a bifunctional antibody (diabody), scFv, scFv dimer, BsFv, dsFv, (dsFv) 2, dsFv-dsFv ', Fv fragment, Fab, Fab', F (ab ') 2, ds diabody, Nanobody, domain antibody or bivalent domain antibody.
  • the fusion proteins described herein include an immunoglobulin constant region.
  • the immunoglobulin constant regions include heavy and / or light chain constant regions.
  • the heavy chain constant region includes a CH1, CH1-CH2, or CH1-CH3 region.
  • the immunoglobulin constant region may further include one or more modifications to obtain the desired properties.
  • the constant region can be modified to reduce or eliminate one or more effector functions to enhance FcRn receptor binding or introduce one or more cysteine residues.
  • the antibodies and antigen-binding fragments thereof further comprise a conjugate.
  • the antibodies or antigen-binding fragments thereof of the present invention may be linked to various conjugates (see, for example, "Conjugate Vaccines", Contributions, Microbiology, Immunology, JMCruse, and RELewis, Jr. (eds.), Carger Press New York (1989)).
  • conjugates can be linked to the antibody or antigen conjugate by other means such as covalent binding, affinity binding, intercalation, coordinate binding, complexation, binding, mixing, or addition.
  • the antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites other than the epitope-binding portion, and these sites can be used to bind one or more conjugates.
  • a site may contain one or more reactive amino acid residues, such as cysteine and histidine residues, to assist in covalent attachment to the conjugate.
  • the antibody may be attached to the conjugate indirectly, or through another conjugate.
  • the antibody or antigen-binding fragment thereof can bind biotin and then indirectly bind a second conjugate, which is linked to avidin.
  • the conjugate may be a detectable label, a pharmacokinetic modification moiety, a purification moiety, or a cytotoxic moiety.
  • detectable labels may include fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin or Texas red), enzyme substrate labels (e.g. horseradish peroxidase, alkaline phosphatase , Luciferase, glucoamylase, lysozyme, sugar oxidase or ⁇ -D galactosidase), stable isotope or radioisotope, chromophore moiety, digoxin, biotin / avidin, DNA molecule Or gold for testing.
  • the conjugate may be a pharmacokinetic modification moiety such as PEG, which helps extend the half-life of the antibody.
  • conjugate may be a purified portion such as a magnetic bead.
  • a "cytotoxic moiety" can be any agent that is harmful to the cell or that may damage or kill the cell.
  • cytotoxic fractions include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, turpentine, mitomycin, etopoxil, tenipogan, vincristine, Vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithromycin, actinomycin D, l-dehydrotestosterone, glucocorticoids, Procaine, tetracaine, lidocaine, prochalorol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-mercaptopurine, Acacia Cytidine, 5fluorouracil dacarbaba), alkylating agents (e.g.
  • cyclophosphamide Busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (DDP) cisplatin), anthracycline antibiotics (e.g. daunorubicin (formerly Daunorubicin ) And doxorubicin), antibiotics (such as dactinomycin (formerly known as actinomycin), bleomycin, mithromycin, and ampicillin (AMC)), and antimitotic agents (such as vincristine Alkali and Changchun ).
  • DDP cis-dichlorodiamine platinum
  • the amino acid sequence in the fusion protein of the present application can be converted into the corresponding DNA coding sequence. Due to the degeneracy of the genetic code, the transformed DNA sequences can be completely identical, while the encoded protein sequence remains unchanged.
  • a vector including a polynucleotide encoding the fusion protein can be introduced into a host cell for cloning (amplifying DNA) or gene expression.
  • the fusion protein can be prepared by a method known in the art for homologous recombination.
  • vectors are available.
  • Vector components typically include, but are not limited to, two or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancement sequence, a promoter (for example: SV40, CMV, EF-1a), and Transcription termination sequence.
  • the vector system includes mammals, bacteria, yeast systems, etc., and will include plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pELpGEMEX, pGEX, pCLpCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2 and other vectors are available from laboratories or are commercially available.
  • Suitable vectors may include plasmid or viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).
  • a vector including a polynucleotide encoding the fusion protein can be introduced into a host cell for cloning or gene expression.
  • the host cell suitable for cloning or expressing the DNA in the vector in the present invention is a prokaryotic cell, a yeast, or the above-mentioned higher eukaryotic cell.
  • Prokaryotic cells suitable for use in the present invention include Eubacteria such as Gram-negative or Gram-positive bacteria, for example, Enterobacteriaceae, such as E.
  • eukaryotic microorganisms such as filamentous fungi or yeast can also be used as host cells to clone or express vectors encoding fusion proteins.
  • Saccharomyces cerevisiae, or baker's yeast, are the most commonly used lower eukaryotic host microorganisms.
  • Kluyveromyces hosts such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12, 424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces welchii (ATCC 24,178), Kluyveromyces kluyveri (ATCC 56,500), Kluyveromyces drosophila (ATCC 36,906) Kluyveromyces thermolyticus and Kluyveromyces marxianus: Yarrowia lipolytica (EP 402,226); Pichia pastoris (EP 183,070); Candida: Trichoderma reesei (EP 244,234); Alternaria Mildew; Western Schwann yeast, such as: Western Schwann yeast; and filamentous fungi, such as: Neurospora, Penicillium, Curvularia and Asper
  • the host cells provided in the present invention and suitable for expressing glycosylated antibodies or antigen-binding fragments thereof are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • a variety of baculoviral strains and their variants, as well as corresponding permissive insect host cells, have been discovered from hosts such as: Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito) ), Aedes albopictus (mosquitoes), Drosophila melanogaster (drosophila) and silkworm.
  • a number of virus strains for transfection are publicly available, such as the Bm-5 variants of Spodoptera litura nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, all of which can be used in the present invention, especially For transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potatoes, soybeans, petunias, tomatoes and tobacco can also be used as hosts.
  • spinal cells are of most interest, and the culture (tissue culture) of spinal cells has become a routine operation.
  • Examples of usable mammalian host cells are SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or subculture 293 cell subclones, Graham et al.,]. Gen.Virol. 36:59 (1977)); young hamster kidney cells (B blood, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77 : 4216 (1980)); mouse testis support cells (TM4, Mather, Biol.Reprod.
  • monkey kidney cells (CV1ATCC CCL70); African green monkey kidney cells (VERO-76 ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); Buffalo rat liver cells (BRL3A, ATCC CRL1442); human lung cells ( W138, ATCC (CCL75); human hepatocytes (Hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL51); TRI cells (Matherther et al. Annals NYAcad. Sci. 383: 44-68 (1982) ); MRC 5 cells; FS4 cells; and human liver cancer cell line (HepG2).
  • the host cell is a 293F cell.
  • the host cell is transformed with the expression or cloning vector that can produce the fusion protein, and cultured in a conventional nutrient medium, which is modified to be suitable for inducing a promoter, selecting transformed cells, or expanding.
  • a gene encoding a sequence of interest is modified to be suitable for inducing a promoter, selecting transformed cells, or expanding.
  • the host cells used to produce the fusion protein in the present invention can be cultured in a variety of media known in the art.
  • the medium may also contain any other necessary additives at appropriate concentrations known in the art.
  • the conditions of the medium such as temperature, pH, and the like, are the conditions previously used to select host cells for expression, and are well known to those of ordinary skill.
  • the antibodies can be produced intracellularly, in the parietal space, or secreted directly into the culture medium. If the antibody is produced intracellularly, the host cell or particle debris of the lysed fragment is first removed, for example, by centrifugation or ultrasound. Carter et al., Bio / Technology 10: 163-167 (1992) describes a method for isolating antibodies secreted into the membrane space of E. coli. Briefly, the cell paste is opened in the presence of uranium acetate (pH 3.5), EDTA, and benzylsulfonium fluoride (PMSF) for about 30 minutes or more. Centrifuge to remove cell debris.
  • uranium acetate pH 3.5
  • EDTA EDTA
  • PMSF benzylsulfonium fluoride
  • a commercially available protein concentration filter such as Amicon or Millipore Pellicon ultrafiltration unit is usually first used to concentrate the supernatant of the expression system.
  • Protease inhibitors such as PMSF can be added in any of the foregoing steps to inhibit protein degradation and antibiotics to prevent the growth of accidental contaminants.
  • Antibodies produced from the cells can be purified using purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography columns, ammonium sulfate precipitation, salting out, and affinity chromatography, where Affinity chromatography is the preferred purification technique.
  • the type of antibody and the presence of any immunoglobulin Fc domain in the antibody determine whether protein A is suitable as an affinity ligand.
  • Protein A can be used to purify antibodies based on human ⁇ 1, ⁇ 2 or ⁇ 4 heavy chains (Lindmark et al., J. lmmunol. Meth. 62: Bu 13 (1983)).
  • Protein G is applicable to all murine isomers and human gamma 3 (Guss et al., EMBO J. 5: 1567 1575 (1986)).
  • Agarose is the most commonly used affinity ligand attachment matrix, but other matrices are also available.
  • Mechanically stable matrices such as controlled-porosity glass or poly (styrene) benzene can achieve faster flow rates and shorter processing times than with agarose. If the antibody contains a CH3 domain, it can be purified with Bakerbond ABX.TM resin (J.T. Baker, Phillipsburg, N.J.).
  • proteins purification can also be determined based on the antibodies obtained, such as fractionation in ion exchange columns, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, anion or cation exchange resin-based heparin agarose gel chromatography (such as polyaspart Lysine column), chromatography focus, SDS-PAGE, and ammonium sulfate precipitation.
  • low pH hydrophobic interaction chromatography can be used to treat a mixture containing antibodies and impurities of interest, using an elution buffer at a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., (From about 0 to 0.25M salt concentration).
  • the application provides a kit comprising the fusion protein.
  • the kit is used to detect the presence or level of OX40 in a biological sample.
  • the biological sample may include cells or tissues.
  • the kit includes a fusion protein conjugated to a detectable label.
  • the kit includes an unlabeled fusion protein, and further includes a secondary antibody capable of binding to the unlabeled fusion protein.
  • the kit may further include instructions for use and a package that separates each component in the kit.
  • the fusion protein is linked to a substrate or instrument for a sandwich assay such as an ELISA or immunochromatographic assay.
  • a substrate or instrument for a sandwich assay such as an ELISA or immunochromatographic assay.
  • Suitable substrates or instruments can be, for example, microplates and test strips.
  • the application further provides a pharmaceutical composition comprising the fusion protein and one or more pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, etc. Osmotic substances, buffers, antioxidants, anesthetics, suspending agents / dispersing agents, integrating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art or a combination of the above.
  • Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers such as Sugar and cyclodextrin.
  • Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylmethyl Anisole, butylated hydroxytoluene and / or propyl gallate.
  • Including one or more antioxidants such as methionine in a composition containing the fusion protein disclosed in the present invention can reduce the oxidation of the fusion protein.
  • a reduction in oxidation prevents or reduces a decrease in binding affinity, thereby increasing antibody stability and extending shelf life.
  • a pharmaceutically acceptable carrier may include, for example, an aqueous medium such as sodium chloride injection, Ringer's solution injection, isotonic glucose injection solution, sterile water injection solution, or glucose and lactic acid forest.
  • non-aqueous media such as: non-volatile oils of plant origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacterial or fungal inhibitory concentrations, isotonic agents such as sodium chloride or glucose Buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl Methylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (Tween-80), integration agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis (2-aminoaminoe
  • Antibacterial agents as carriers can be added to pharmaceutical compositions in multiple-dose containers, including phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methyl and propyl parabens, Thimerosa, ammonium chlorobenzyl and chlorophenethyl ammonium.
  • Suitable excipients may include, for example, water, salt, glucose, glycerol or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or the like, such as sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrin. substance.
  • the pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release preparation or powder.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • the pharmaceutical composition is formulated as an injectable composition.
  • injectable pharmaceutical compositions can be prepared in any conventional form, for example, liquid solvents, suspensions, emulsifiers or solid forms suitable for producing liquid solvents, suspensions or emulsifiers.
  • Injectable preparations may include ready-to-use sterile and / or pyrogen-free solutions, sterile dry solubles that are combined with solvents before use, such as lyophilized powder, including subcutaneous tablets, sterile suspensions for injection, Sterile dry insoluble products that are combined with the medium before use, and sterile and / or pyrogen-free emulsions.
  • the solvent may be an aqueous phase or a non-aqueous phase.
  • the unit-dose injectable preparation is packaged in an ampoule, a tube, or a syringe with a needle. It is known in the art that all preparations for injection should be sterile and pyrogen-free.
  • sterile lyophilized powders can be prepared by dissolving the antibodies or antigen-binding fragments thereof disclosed herein in a suitable solvent.
  • the solvent may contain a powder or a reconstituted solution prepared from the powder, or other pharmacological components to improve the stability. Suitable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, brown sugar, or other suitable substances.
  • the solvent may contain a buffer, such as a citrate buffer, a sodium or potassium phosphate buffer, or other buffers known to those skilled in the art. In one embodiment, the pH of the buffer is neutral.
  • each vial can contain a single dose or multiple doses of the anti-OX40 antibody or antigen-binding fragment thereof or a combination thereof.
  • the amount of filling in each vial can be slightly higher than that required for each dose or multiple doses (e.g., a 10% excess), thereby ensuring accurate sampling and precise administration.
  • the lyophilized powder can be stored under appropriate conditions, such as in the range of about 4 ° C to room temperature.
  • the lyophilized powder was reconstituted with water for injection to obtain a preparation for injection administration.
  • the lyophilized powder can be redissolved in sterile pyrogen-free water or other suitable liquid carriers. The exact amount is determined by the chosen therapy and can be determined based on experience.
  • Methods of treatment comprising administering a therapeutically effective amount of a fusion protein described herein to a subject in need thereof.
  • the therapeutically effective dose of the fusion protein provided in this application depends on a variety of factors known in the art, such as weight, age, past medical history, current treatment, subject's health and potential for cross-infection, allergies, hypersensitivity, and side effects, and Route of administration and extent of tumor development. Those skilled in the art, such as a doctor or veterinarian, can proportionally reduce or increase the dose according to these or other conditions or requirements.
  • the fusion proteins provided herein can be administered at a therapeutically effective dose of between about 0.01 mg / kg and about 100 mg / kg. In certain embodiments, the fusion protein is administered at a dose of about 50 mg / kg or less, and in certain embodiments, the dosage is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less or 0.1 mg / kg or less.
  • a particular dose can be administered at multiple intervals, such as once a day, twice a day or more, twice a month or more, once a week, once every two weeks, once every three weeks, once a month, or every two Once a month or more.
  • the dosage administered may vary with the course of treatment.
  • the initial administered dose may be higher than the subsequent administered dose.
  • the administered dose is adjusted during the course of treatment based on the response of the subject to be administered.
  • the dosing regimen can be adjusted to achieve an optimal response (eg, a therapeutic response). For example, it may be administered in a single dose or in divided doses over two periods.
  • the fusion protein disclosed in the present invention can be administered by means known in the art, such as injection (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular or intradermal injection) or non-injection Administration (eg, oral, nasal, sublingual, rectal, or topical).
  • injection e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular or intradermal injection
  • non-injection Administration eg, oral, nasal, sublingual, rectal, or topical.
  • the fusion protein can be used to treat disorders related to its molecular mechanism, including tumors and cancers, such as non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreas Dirty cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, bone marrow Neoplasms, mycoses fungoids, Merkel cell carcinoma and other malignant hematological diseases such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T cells / histiocytic cells Of B-cell-rich lymphomas, EBV-positive and negative PTLD and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lympho
  • CHL
  • the conditions treated by the fusion protein include chronic viral infections, such as hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, simplex Herpesvirus type 2, human papilloma virus, adenovirus viral infection, Kaposi's sarcoma-associated herpes virus epidemic, Torquetenovirus, JC virus, or BK virus, etc.
  • chronic viral infections such as hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, simplex Herpesvirus type 2, human papilloma virus, adenovirus viral infection, Kaposi's sarcoma-associated herpes virus epidemic, Torquetenovirus, JC virus, or BK virus, etc.
  • the application further provides methods for using the fusion proteins.
  • the application provides a method of treating a condition or disorder associated with the fusion protein mechanism in an individual, comprising administering a therapeutically effective amount of the fusion protein described herein.
  • the fusion protein disclosed in the present invention can be administered alone or in combination with one or more other treatment means or substances.
  • the fusion protein disclosed in the present invention can be used in combination with chemotherapy, radiation therapy, cancer treatment surgery (such as tumor resection), antiviral drugs, one or more antiemetic drugs or other chemotherapy-induced complications, or any other application.
  • chemotherapy radiation therapy
  • cancer treatment surgery such as tumor resection
  • antiviral drugs one or more antiemetic drugs or other chemotherapy-induced complications, or any other application.
  • the fusion protein disclosed in the present invention when used in combination with one or more therapeutic substances, it may be administered simultaneously with the one or more therapeutic substances.
  • the fusion protein can be administered simultaneously as part of the same pharmaceutical composition.
  • fusion proteins that are "combined" with other therapeutic substances need not be administered at the same time or in the same composition as the therapeutic substance.
  • a fusion protein administered before or after another therapeutic substance is also considered to be “combined” with the therapeutic substance, even if the fusion protein is administered differently from the second substance.
  • Drug administration can be used in accordance with the methods of the product description of the other therapeutic substance, or refer to the surgeon's desk reference book 2003 (Physicians' Desk Reference, 57th Ed Medical; Economics Company; ISBN: 1563634457; 57th Edition (November 2002)), or refer to other methods known in the art.
  • the therapeutic substance is capable of inducing or enhancing an immune response against cancer.
  • tumor vaccines can be used to induce an immune response to certain tumors or cancers.
  • Cytokine therapy can be used to improve the presentation of tumor antigens to the immune system.
  • cytokine therapy examples include, but are not limited to, interferons such as interferon ⁇ , ⁇ , and ⁇ , colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF, and granulocyte CSF, and interleukins such as 1L-L, 1L-1a , 1L-2, 1L3, 1L-4, 1L-5, 1L-6, 1L-7, 1L-8, 1L-9, 1L-10, 1L-ll and 1L-12, tumor necrosis factors such as TNF- ⁇ and TNF- ⁇ .
  • interferons such as interferon ⁇ , ⁇ , and ⁇
  • colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF, and granulocyte CSF
  • interleukins such as 1L-L, 1L-1a , 1L-2, 1L3, 1L-4, 1L-5, 1L-6, 1L-7, 1L-8, 1L-9, 1L-10, 1L-ll and 1
  • agents that inactivate immunosuppressive targets such as PD-L1 / PD-1 antibodies, TGF- ⁇ inhibitors, IL-10 inhibitors, and Fas ligand inhibitors can also be used.
  • Another group of agents includes those that activate an immune response against tumors or cancer cells, such as those that increase T cell activation (such as T cell co-stimulatory agonists such as CTLA-4, ICOS), and improve dendritic cell function And antigen-presenting ones.
  • Example 1 Preparation of the fusion protein of the present invention
  • This example exemplifies the design and expression of several anti-OX40 antibody-human interferon fusion proteins.
  • the heavy and light chain sequences of the anti-human OX40 activating antibody are derived from MT01-C1 or MT01-C1 (G2) in Chinese patent 201711476160.3, where "MT01-C1" refers to UMY02-L1, UMY02-L2 and UMY02-L3 has the same VH (SEQ ID NO: 7) and VL (SEQ ID NO: 8) sequences, and the heavy and light chain constant regions are monoclonal antibodies of human IgG1 and ⁇ chains, respectively.
  • MT01-C1 (G2) means that it has the same VH (SEQ ID ID NO: 7) and VL (SEQ ID ID NO: 8) sequences as UMY02-L1, UMY02-L2, and UMY02-L3, and the heavy and light chain constant regions, respectively Monoclonal antibodies to human IgG2 and kappa chains.
  • the interferon IFN ⁇ 2b sequence is taken from human interferon IFN ⁇ 2b (NP_000596.2), and its amino acid sequence is shown in SEQ ID NO: 9.
  • UY02-L1 refers to a heavy chain, a peptide linker, and a human interferon as shown in SEQ ID NO: 10, and a light chain as shown in SEQ ID NO: 11 in which a peptide linker is passed through the carboxyl end of the heavy chain.
  • a fusion protein of human interferon IFN ⁇ 2b as shown in SEQ ID NO: 9 was linked.
  • UY02-L2 refers to a heavy chain as shown in SEQ ID NO: 12, a light chain as shown in SEQ ID NO: 13, a peptide linker, and human interferon, in which the carboxyl end of the light chain is connected by a peptide linker
  • UY02-L3 refers to a heavy chain as shown in SEQ ID NO: 14, a light chain as shown in SEQ ID NO: 13, a peptide linker and human interferon, in which the carboxyl end of the light chain is connected by a peptide linker
  • the fusion protein of human interferon IFN ⁇ 2b as shown in SEQ ID NO: 9.
  • the cDNA sequences encoding the antibody heavy and light chains of the fusion protein were cloned into mammalian cell expression vector pcDNA3.4, respectively.
  • the heavy chain expression plasmid and light chain expression plasmid were transfected into HEK293 cells with Lipofectamine 2000 transfection reagent (Invitrogen) at a molar ratio of 2: 1, and cultured at 37 ° C and 5% carbon dioxide for 7 days. The culture supernatant was collected, and the antibody in the supernatant was purified by Protein A affinity chromatography. The purified antibody was dialyzed against PBS solution, freeze-dried and concentrated, and then stored at -20 ° C.
  • a 96-well high affinity plate was coated with a human OX40 protein solution at a concentration of 1 ⁇ g / mL at 100 ⁇ L / well, and shaken overnight at 4 ° C. The next day was washed three times with 300 ⁇ L of PBST (Tween 20: 0.5 ⁇ ), and then blocked with 100 ⁇ L / well of 5% BSA / PBS for 2 hours and shaken at room temperature. 300 ⁇ L PBST was washed 3 times. A gradient dilution solution of the fusion protein sample was prepared in PBS. Add 100 ⁇ L / well to a 96-well plate and shake at room temperature for 1 hour. 300 ⁇ L PBST was washed 3 times.
  • a secondary antibody goat anti-human IgG HRP solution was prepared, added to a 96-well plate at 100 ⁇ L / well, and shaken at room temperature for 1 hour. 300 ⁇ L PBST was washed 4 times. Add 100 ⁇ L / well TMB and develop color for 20 min. 100 ⁇ L / well of 0.6 N H 2 SO 4 was added to terminate the color development, and the OD 450 nm was detected.
  • Example 3 Binding to human, cynomolgus monkey and mouse OX40 (FACS)
  • CHO cells were transfected with an expression plasmid encoding human, cynomolgus monkey or mouse OX40 protein and cultured for 48 hours.
  • the OX40 antibody MT01-L1 concentration gradient solution was prepared with PBS to prepare a final concentration of 10 ⁇ working solution.
  • CHO-hOX40 cells were collected, washed and counted in PBS, diluted to 2 * 10 6 / ml cell suspension; 10 ⁇ L of OX40 antibody MT01-L1 working solution was added to 100 ⁇ L of cell suspension, and incubated at 4 ° C in the dark for 30 min; washed with PBS After 2 times, add the secondary antibody, incubate at 4 ° C in the dark for 30 min, wash once with PBS, and then suspend it in 400 ⁇ L FACS buffer, and then test on the machine. As shown in FIG. 3, the results show that MT01-L1 is bound to human OX40 (hsOX40), and its EC50 is 0.66 ⁇ g / mL.
  • the inventors tested the binding of the antibody to CHO cells expressing mouse OX40, and found that MT01-L1 did not bind to mouse OX40 (msOX40).
  • the inventors of the present application tested the binding of the antibody to CHO cells expressing cynomolgus monkey OX40, and found that MT01-L1 significantly binds to cynomolgus monkey OX40 (cyOX40).
  • Interferon receptors are highly expressed on Daudi cells (ATCC), so interferons have biological activity on them.
  • Daudi cells were plated at 20,000 cells / 90 ⁇ L / well in a 96-well plate.
  • the sample to be tested was prepared into 10 ⁇ working solution with a concentration gradient dilution, and added to the 96-well plate at 10 ⁇ L / well, and placed in a 37 ° C incubator.
  • CCK8 detects OD450 and calculates the proliferation inhibition rate of cells in each well. This inhibition rate reflects the activity of the interferon in the sample.
  • the experimental results showed that the fusion proteins UMY02-L1, UMY02-L2, and UMY02-L3 have inhibitory activity (IC50) on Daudi cells of 9.549, 9.152, and 27.44 pM ( Figure 4).
  • the inventors of the present application have constructed a cell experimental system for detecting OX40 activator. Specifically, the inventors of the present application constructed a "Jurkat-OX40-NF ⁇ B-luciferase reporter gene" stable transfected cell line, and after mixing the OX40 activating antibody with the stable transduced cell line and HEK293 cells overexpressing FcR Can activate the expression of the NF ⁇ B-luciferase reporter gene.
  • the fusion protein concentration gradient solution was prepared with PBS to prepare a final concentration of 2 ⁇ working solution, which was operated on ice.
  • Jurkat-NFkB-luc-OX40 cells and HEK293 cells overexpressing FcR were collected, resuspended in culture medium after centrifugation, and plated into 384-well plates.
  • Add the fusion protein working solution and an appropriate amount of cell suspension After incubation for 5 hours, add the One-Glo (Promega) detection reagent. After mixing, use the Pherastar autofocus fluorescence luminometer microplate reader to detect the fluorescence signal.
  • the OX40 antibody MT01-C1 As shown in Figure 5, after detection, the OX40 antibody MT01-C1, the fusion proteins UMY02-L1, UMY02-L2, and UMY02-L3 activated the NF ⁇ B-luciferase reporter genes in the above experimental system with EC50 of 0.04523, 0.02437, and 0.02837, respectively. And 0.02907ng / mL.
  • the results showed that FcR-mediated OX40 activation activities of UMY02-L1, UMY02-L2, and UMY02-L3 were stronger than MT01-C1.
  • the "Jurkat-OX40-NF ⁇ B-luciferase reporter gene" stably transformed cell line was plated at 10,000 cells / well into a 384-well plate.
  • PBS was used to configure the fusion protein concentration gradient solution or the control solution to prepare a final concentration of 2 ⁇ working solution, which was operated on ice.
  • a 384-well plate add the fusion protein or interferon working solution and an appropriate amount of cell suspension. After 5 hours of incubation, add the One-Glo (Promega) detection reagent. After mixing, use the Pherastar autofocus fluorescence luminometer and microplate reader to detect the fluorescence. signal.
  • the final concentration of all antibodies or interferons in the experiment was 10 nM.
  • the OX40 antibody MT01-C1 or MT01-C1 (G2) has no activation effect on the OX40 signaling pathway in Jurkat cells, and the addition of interferon IFN ⁇ 2b alone or the simultaneous addition of OX40 antibodies MT01-C1 and IFN ⁇ 2b is only less than 40% compared with the control group. Activation effect.
  • the fusion proteins UMY02-L1, UMY02-L2, or UMY02-L3 were added, the OX40 signaling pathway of Jurkat cells was significantly activated, and the degree of activation was significantly greater than that of the interferon group alone ( Figure 6b).
  • mice Eighteen female C57BL / 6 mice, 6-8 weeks old, were divided into 3 groups of 6 mice each. They were given intravenous injection of UMY02-L1, UMY02-L3, and MT01-C1. The dose was 5 mg / kg.
  • UMY02-L1 and UMY02-L3 collect the peripheral venous blood of animals before and after administration, and 1, 2, 6, 24, 48, 72, 96, 174, 220, and 288 hours after administration;
  • MT01-C1 collect Peripheral venous blood of the animals before, and 1, 2, 6, 24, 48, 72, 96, 192, and 312 hours after administration; serum was collected by centrifugation. Three animal serums were collected at each time point, and six animals in each group were collected blood samples at different time points.
  • a human OX40 protein solution at a concentration of 1 ⁇ g / mL was coated with a 96-well high affinity plate at 100 ⁇ L / well, and shaken at 4 ° C. overnight. The next day was washed three times with 300 ⁇ L of PBST (Tween 20: 0.5 ⁇ ), and then blocked with 100 ⁇ L / well of 5% BSA / PBS for 1 hour and shaken at room temperature. 300 ⁇ L PBST was washed 4 times. PBS was used to prepare 100-fold dilutions of the serum samples to be tested and the serum solutions of different concentrations of reference substance. Add 100 ⁇ L / well to a 96-well plate and shake at room temperature for 1.5 hours.
  • 300 ⁇ L PBST was washed 4 times.
  • a secondary antibody donkey anti-human IgG HRP Jackson ImmunoResearch, article number 709-035-149
  • 300 ⁇ L PBST was washed 4 times.
  • 100 ⁇ L / well of 0.6 N H 2 SO 4 was added to stop the color development, and the OD 450 nm was detected.
  • a 0.5 ⁇ g / mL rabbit anti-human IFN (abcam, article number ab222552) solution was prepared, added to a 96-well plate at 100 ⁇ L / well, and shaken at room temperature for 1.5 hours. 300 ⁇ L PBST was washed 4 times.
  • a secondary antibody goat anti-rabbit IgG HRP (Kingsui Biotechnology, Cat. No. A00098) solution was prepared, added to a 96-well plate at 100 ⁇ L / well, and shaken at room temperature for 1 hour. 300 ⁇ L PBST was washed 4 times. Add 100 ⁇ L / well TMB and develop color for 20 min.
  • mice were intravenously injected with 5 mg / kg of UMY02-L3 and UMY02-L1. After 7 days of administration, the serum drug concentration of UMY02-L3 was still above 10 ⁇ g / mL. After 12 days of administration, UMY02- L1 serum drug concentration was still above 10 ⁇ g / mL. It is shown that the fusion proteins UMY02-L3 and UMY02-L1 have similar pharmacokinetic properties in mice as the antibody drug MT01-C1, which greatly prolongs the half-life of 2-3 hours compared to IFN ⁇ 2b (see the product manual of Merck's product Intron® A ).
  • Example 8 Effect of antibody subtype and peptide linker length on antibody fusion protein activity
  • the heavy chains of the fusion proteins UMY02-L4, UMY02-L5 and UMY02-L6 are shown in SEQ ID NO: 14, and the light chain and human interferon are shown in SEQ ID ID NO: 13 and the light chain and human interferon, The only difference is the number of peptide linkers, as shown in Table 1 below.
  • the heavy chains of the fusion proteins UMY02-L7 and UMY02-L8 are shown in SEQ ID NO: 15, and the light chain and human interferon are shown in SEQ ID ID NO: 13, and the difference is only in the peptide
  • the number of connectors is shown in Table 1 below.
  • Example 4 According to the method of Example 4, the effects of these antibody fusion proteins on the proliferation of Daudi cells were detected. As a result, it was found that different peptide linker lengths had a significant effect on the interferon activity of antibody fusion proteins. The shorter the peptide linker length, the lower the interferon activity. (See Table 1).
  • OX40 antibody MT01-C1 and each antibody fusion protein can effectively activate the OX40 signaling pathway of Jurkat-OX40 cells.
  • OX40 activating antibodies can also be fused to interferon mutants. Compared with wild-type interferon, these mutant interferons have lower specific activity, so they can work together with OX40 antibody at the same drug concentration, while avoiding toxic side effects caused by excessive interferon activity.
  • the heavy chains of fusion proteins UMY02-L13, UMY02-L14, UMY02-L15, UMY02-L16, UMY02-L17 and UMY02-L18 are shown in SEQ ID NO: 15, and the light chain and human interferon are shown in SEQ ID ID NO:
  • the light chain and human interferon shown in 13 are only different:
  • the peptide linkers of UMY02-L13, UMY02-L14, UMY02-L15, UMY02-L16, and UMY02-L18 are (GGGGS) 2, and the peptide linkers of UMY02-L17 are (GGGGS) 3;
  • Interferons of the fusion proteins UMY02-L13, UMY02-L14, UMY02-L15, UMY02-L16, UMY02-L17 and UMY02-L18 are mutated as shown in Table 2 on the basis of SEQ ID ID NO: 9.
  • Example 4 According to the method of Example 4, the effects of these antibody fusion proteins on the proliferation of Daudi cells were detected. As a result, it was found that the interferon activity of the mutant antibody fusion protein was significantly lower than that of wild-type IFN ⁇ 2b (Table 2). At the same time, the activation effect of these antibody fusion proteins on the OX40 signaling pathway of Jurkat cells was detected according to the method of Example 5, and the results showed that the OX40 activation activity of these mutant antibody fusion proteins was basically unchanged (Table 2).
  • the amino acid position number of the mutation is indicated by referring to the amino acid sequence SEQ ID NO: 9 of the wild-type human interferon IFN ⁇ 2b.
  • IFN ⁇ 2b The activity of IFN ⁇ 2b is defined as 1.
  • the inventors fused the heavy chain of the OX40 antibody with different types of interferons to prepare different fusion proteins, among which the heavy chains of OX40IFN- ⁇ 2a, OX40-IFN ⁇ , OX40-IFN ⁇ , and OX40-IFN ⁇ 3 are as shown in SEQ ID ID NO: 15; OX40IFN- ⁇ 2a light chain, peptide linker and interferon are shown in SEQ ID NO: 16; OX40IFN- ⁇ light chain, peptide linker and interferon are shown in SEQ ID NO: 17; OX40IFN- ⁇ light chain, The peptide linker and interferon are shown in SEQ ID NO: 18; the OX40IFN- ⁇ 3 light chain, peptide linker and interferon are shown in SEQ ID NO: 19.
  • the killing effect of these antibody fusion proteins on tumor cells was tested.
  • the inventors mixed and co-cultured human-derived PBMCs and ovarian cancer SKOV3 cells at a ratio of 20: 1, and administered PBS (control) or 0.1nM OX40 mAb MT01-C1 and different antibody fusion proteins (OX40IFN - ⁇ 2a, OX40-IFN ⁇ , OX40-IFN ⁇ , OX40-IFN ⁇ 3) stimulation.
  • the CCK8 kit was used to detect the cytotoxic effect of PBMC on SKOV3 cells.
  • FIG. 8 shows that PBMC has no inhibitory effect on SKOV3 cell proliferation without stimulation; OX40 monoclonal antibody stimulates SKOV3 inhibition rate of about 28% by PBMC; and cytotoxicity of PBMC is strong under stimulation of fusion protein Stimulated by OX40 monoclonal antibody, OX40 monoclonal antibody MT01-C1-IFN ⁇ 2a and OX40 monoclonal antibody MT01-C1-FN ⁇ were significantly enhanced, with inhibition rates of 60% and 63%, respectively.

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Abstract

本发明提供了融合蛋白及其在制备用于治疗肿瘤和/或病毒感染的药物中的应用，所述融合蛋白包含特异性结合人OX40的抗体或其抗原结合片段和人干扰素。

Description

融合蛋白及其在制备用于治疗肿瘤和/或病毒感染的药物中的应用 技术领域
本发明属于生物医药领域。具体地，本发明涉及一种融合蛋白及其用途。更具体地，本发明涉及含有抗OX40抗体和人干扰素的融合蛋白及其在制备用于治疗肿瘤和/或病毒感染的药物中的应用。
背景技术
人OX40主要表达于活化的T细胞上，包括CD4、CD8、Th和Treg细胞等[1]。在
Figure PCTCN2019104217-appb-000001
T细胞上OX40的表达很低，但是经过抗原诱导刺激后其表达水平上调并在12小时至5-6天内达到峰值。类似的，OX40L的表达也受到细胞活化状态的影响[1]。APC细胞在抗原刺激后1-3天可以检测到OX40L的表达。有趣的是，除了免疫细胞外，肌肉细胞在炎症因子的刺激下也会表达OX40L[2,3]，提示OX40L-OX40信号通路可能广泛作用于机体的炎症反应。
抗原依赖的OX40L/OX40共刺激分子的活化与T细胞内多个信号通路相偶联。晶体结构研究表明，OX40L与OX40的结合可诱导OX40-OX40L复合物三聚体化[4]，从而在胞内形成与受体相关因子(receptor-associated factor，TRAF)的结合位点。后者(TRAF2、5)则可进而激活NF-κB信号通路，抑制T细胞凋亡[2,5,6]。有研究发现，OX40活化可以导致Bcl-2和Bcl-xL高表达[7]，提示OX40可能通过NF-κB信号通路诱导抗凋亡蛋白表达而实现其抑制T细胞凋亡的功能。
PKB/PI3K是OX40下游的另一个重要信号通路。研究发现，一方面，T细胞上OX40的共刺激信号是维持PKB活化的必要条件，另一方面，组成型活化的PKB可以拮抗OX40缺陷导致的T细胞中抗凋亡蛋白的下调。OX40共刺激信号可以通过PKB/PI3K信号通路维持Survivin蛋白的表达[8]。
最后，T细胞上TCR和OX40的活化还可以协同引起钙流和NFAT信号通路的活化，调节包括IL-2、IL-4、IL-5和IFN-γ在内的细胞因子的表达[9]。
综上，上述研究表明OX40的活化可以通过NF-κB信号通路、PKB/PI3K信号通路和NFAT信号通路来调节T细胞的增殖、凋亡和细胞因子分泌活性，从而达到增强免疫系统活力的效果。
基于上述发现，OX40成为免疫疗法的一个重要靶点，众多的临床前和 临床研究都提示OX40可以作为肿瘤免疫疗法的一个重要靶点。有趣的是，最近的研究还发现OX40信号通路在抑制乙肝病毒感染中的作用[10]，提示OX40激动剂可能作为抗病毒感染，例如乙肝病人治疗的潜在手段。
干扰素是一类高活性多功能的糖蛋白。一方面。干扰素能够通过调控肿瘤细胞增殖、抑制肿瘤转移及血管新生、激活抗肿瘤免疫反应发挥强大的抗肿瘤作用；另一方面，通过调节人体免疫系统，干扰素在抗病毒方面有重要的临床应用价值，比如干扰素已经成为临床治疗乙肝病毒感染的重要手段之一。
OX40激动剂及干扰素都在抗肿瘤和抗病毒方面有重要的应用价值或潜力，但是现有的证据表明两者在病人响应率和药效方面都有不足。因此，当前对治疗效果更好的OX40激动剂及干扰素存在需求。
发明内容
本发明的目的在于提供一种融合蛋白，所述融合蛋白包含特异性结合人OX40的抗体或其抗原结合片段和人干扰素。本发明还提供了所述融合蛋白用于治疗肿瘤和/或病毒感染的用途。
为实现上述目的，本发明采用了以下技术方案：
一方面，本发明提供了一种融合蛋白，其包含：
a)特异性结合人OX40的抗体或其抗原结合片段；和
b)人干扰素；
其中所述人干扰素直接或通过肽接头连接至所述抗体的轻链或重链的羧基端或氨基端。
根据本发明所述的融合蛋白，其中所述特异性结合人OX40的抗体或其抗原结合片段包含：
抗体重链可变区，该重链可变区包含具有SEQ ID NO:1的氨基酸序列的VH CDR1、具有SEQ ID NO:2的氨基酸序列的VH CDR2、和具有SEQ ID NO:3的氨基酸序列的VH CDR3；和
抗体轻链可变区，该轻链可变区包含具有SEQ ID NO:4的氨基酸序列的VL CDR1、具有SEQ ID NO:5的氨基酸序列的VL CDR2、和具有SEQ ID NO:6的氨基酸序列的VL CDR3。
根据本发明所述的融合蛋白，其中所述重链可变区包含SEQ ID NO:7所示的氨基酸序列，所述轻链可变区包含SEQ ID NO:8所示的氨基酸序列。
根据本发明所述的融合蛋白，其中所述特异性结合人OX40的抗体或其抗原结合片段是骆驼化单域抗体、scFv、scFv二聚体、BsFv、dsFv、dsFv2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab')2、ds双功能抗体、纳米抗体、域抗 体或双价域抗体。
根据本发明所述的融合蛋白，其中所述抗体进一步包含免疫球蛋白恒定区，例如人IgG1、IgG2或IgG4的恒定区。
根据本发明所述的融合蛋白，其中所述人干扰素选自I型人干扰素、II型人干扰素和III型人干扰素，优选地，所述人干扰素为IFNα2a、IFNβ、IFNγ、IFNλ、IFNα2b；更优选地，所述人干扰素为IFNα2b，其氨基酸序列如SEQ ID NO:9所示。
进一步优选地，所述人干扰素为IFNα2b的突变体，其在SEQ ID NO:9所示的氨基酸序列上，具有选自以下的一种或多种突变：T106A、R149A、A145G、A145D、R120A、L117A；
更优选地，所述IFNα2b的突变体在SEQ ID NO:9所示的氨基酸序列上具有选自以下的双突变：
T106A/A145D、T106A/R149A、T106A/A145G、T106A/L117A、T106A/R120A。
根据本发明所述的融合蛋白，其中所述肽接头选自(G)n、KESGSVSSEQLAQFRSLD、EGKSSGSGSESKST、GSAGSAAGSGEF、(GGGGS)n、(GGSGG)n；优选地，所述肽接头为(GGGGS)n，其中n为0-5之间的整数；优选地，n为1-3之间的整数。
根据本发明所述的融合蛋白，其中所述融合蛋白选自：
UMY02-L1，其包含SEQ ID NO:10所示氨基酸序列和SEQ ID NO:11所示的氨基酸序列，其中，SEQ ID NO:10所示氨基酸序列为OX40的抗体的重链、肽接头和人干扰素，SEQ ID NO:11所示的氨基酸序列为OX40的抗体的轻链；
UMY02-L2，其包含SEQ ID NO:12所示的氨基酸序列和SEQ ID NO:13所示的氨基酸序列，
其中，SEQ ID NO:12所示的氨基酸序列为OX40的抗体的重链，SEQ ID NO:13所示的氨基酸序列为OX40的抗体的轻链、肽接头和人干扰素；或
UMY02-L3，其包含SEQ ID NO:14所示的氨基酸序列和SEQ ID NO:13所示的氨基酸序列，其中，SEQ ID NO:14所示的氨基酸序列为OX40的抗体的重链，SEQ ID NO:13所示的氨基酸序列为OX40的抗体的轻链、肽接头和人干扰素；
UMY02-L4，其中重链如SEQ ID NO:14所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，无肽接头；
UMY02-L5，其中重链如SEQ ID NO:14所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，肽接头为GGGGS；
UMY02-L6，其中重链如SEQ ID NO:14所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，肽接头为(GGGGS)2；
UMY02-L7，其中重链如SEQ ID NO:15所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，肽接头为GGGGS；
UMY02-L8，其中重链如SEQ ID NO:15所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，肽接头为(GGGGS)2；
UMY02-L13，其中重链如SEQ ID NO:15所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，肽接头为(GGGGS)2，干扰素在如SEQ ID NO:9的基础上突变为T106A/A145D；
UMY02-L14，其中重链如SEQ ID NO:15所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，肽接头为(GGGGS)2，干扰素在如SEQ ID NO:9的基础上突变为T106A/R149A；
UMY02-L15，其中重链如SEQ ID NO:15所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，肽接头为(GGGGS)2，干扰素在如SEQ ID NO:9的基础上突变为T106A/R120A；
UMY02-L16，其中重链如SEQ ID NO:15所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，肽接头为(GGGGS)2，干扰素在如SEQ ID NO:9的基础突变为T106A/A145G；
UMY02-L17，其中重链如SEQ ID NO:15所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，肽接头为(GGGGS)3，干扰素在如SEQ ID NO:9的基础突变为T106A/R149A；
UMY02-L18，其中重链如SEQ ID NO:15所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，肽接头为(GGGGS)2，干扰素在如SEQ ID NO:9的基础突变为T106A/L117A；
融合蛋白OX40IFN-α2a，OX40-IFNβ，OX40-IFNγ，OX40-IFNλ3，其中OX40IFN-α2a，OX40-IFNβ，OX40-IFNγ，OX40-IFNλ3重链如SEQ ID NO:15所示；OX40IFN-α2a轻链、肽接头和干扰素如SEQ ID NO:16所示；OX40IFN-β轻链、肽接头和干扰素如SEQ ID NO:17所示；OX40IFN-γ轻链、肽接头和干扰素如SEQ ID NO:18所示；OX40IFN-λ3轻链、肽接头和干扰素如SEQ ID NO:19所示。
另一方面，本发明提供了一种分离的多核苷酸，其编码所述的融合蛋白。
再一方面，本发明提供了一种载体，其包括所述的分离的多核苷酸。
再一方面，本发明提供了一种宿主细胞，其包括所述的载体。
本发明还提供了一种表达所述的融合蛋白的方法，其包括在能够表达根据分离的多核苷酸的条件下培养所述的宿主细胞。
本发明还提供了一种试剂盒，其包括所述的融合蛋白。
本发明还提供了一种药物组合物，其包括所述的融合蛋白以及药学上可接受的载体。
本发明还提供了所述的融合蛋白在制备药物中的用途，所述药物用于治疗能通过增强免疫应答和/或能通过暴露于干扰素而受益的病况。
优选地，所述病况是癌症或病毒感染，例如乙肝病毒感染。
本发明提供一种治疗能通过增强免疫应答和/或能通过暴露于干扰素而受益的病况的方法，所述方法包括给予有需要的受试者治疗有效量的所述的融合蛋白；优选地，其中所述病况是癌症或病毒感染，例如乙肝病毒感染。
本发明提供一种治疗能通过增强免疫应答和/或能通过暴露于干扰素而受益的病况的融合蛋白；优选地，其中所述病况是癌症或病毒感染，例如乙肝病毒感染。
本发明提供了OX40激动性抗体与干扰素的融合蛋白，同时结合了两者的不同作用机制，并且形成了协同增强的效果：一方面当融合蛋白中的干扰素部分结合到高表达干扰素受体的细胞(例如肿瘤或病毒感染的细胞)表面，可能以受体介导的模式增强OX40激活性抗体的活性，从而更好的增强免疫系统活性；另一方面，本发明的融合蛋白较干扰素分子的半衰期大大延长，在临床上可以以更低的频次用药，相较于目前临床上需每天注射的干扰素，具有很大的优势。
综上，本发明的融合蛋白有可能在抗肿瘤和抗病毒治疗方面显示出独特的药效和依从性方面的优势。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1表示根据本发明的融合蛋白的结构示意图。
图2表示根据本发明的融合蛋白与人OX40蛋白的ELISA结合实验结果。
图3FACS检测结果显示MT01-L1抗体与人和食蟹猴OX40都有结合，而与小鼠OX40没有结合。
图4表示根据本发明的融合蛋白对Daudi细胞的增殖抑制作用。
图5表示根据本发明的融合蛋白激活Jurkat细胞NF-kB信号通路活性。
图6表示根据本发明的融合蛋白可以促进MT01-C1抗体的OX40信号通路激活活性。单独的OX40单抗(MT01-C1或MT01-C1(G2))或干扰素分子IFNα2b在本实验体系中对Jurkat细胞的OX40信号通路没有或仅有微弱的激活作用(6a)。但是融合蛋白(UMY02-L1、UMY02-L2和UMY02-L3) 在同样条件下对OX40的信号通路有显著的活化活性(6b)。
图7表示UMY02-L1和UMY02-L3在小鼠上的药代动力学曲线(7a)。作为对比，图7b显示了MT01-C1在小鼠上的药代动力学曲线。
图8表示根据本发明的融合蛋白对肿瘤细胞的杀伤作用。
实施发明的最佳方式
本申请的以下描述只为说明本申请的多种实施方式。因此，此处讨论的具体修改方式不应理解为对申请范围的限制。本领域的技术人员在不偏离本申请范围的情况下即可很容易地得出多种等同方式、变化和修改，应理解这样的等同实施方式包括在本发明范围内。在本申请中引用的所有文献，包括公开出版物、专利和专利申请都通过引用的方式全文并入。
定义
本发明中的"抗体"一词包括任意可结合某特定抗原的免疫球蛋白、单克隆抗体、多克隆抗体、多特异性抗体或双特异性(双价)抗体。一个天然的完整抗体包含两条重链和两条轻链。每条重链由一可变区和第一、第二、第三恒定区组成；每条轻链由一可变区和一恒定区组成。哺乳动物的重链可分为α、δ、ε、γ和μ，哺乳动物的轻链可分为λ或κ。抗体呈"Y"型，Y型结构的颈部由两条重链的第二和第三恒定区组成，其通过二硫键结合。"Y"型结构的每条臂包括其中一条重链的可变区和第一恒定区，其与一条轻链的可变区和恒定区结合。轻链和重链的可变区决定抗原的结合。每条链的可变区均含有三个高变区，称互补决定区(CDR)。轻链(L)的CDR包含LCDR1、LCDR2、LCDR3，重链(H)的CDR包含HCDR1，HCDR2、HCDR3。本发明中公开的抗体和抗原结合片段的CDR边界可通过Kabat，Chothia或Al-Lazikani命名法命名或识别。(AI-Lazikani,B.,Chothia,C.,Lesk,A.M.,J.Mol.Biol.,273(4)：927(1997)；Chothia,C.等，J.Mol.Biol.,186(3)：651-63(1985)；Chothia,C.and Lesk,A.M.,J.Mol.Biol.,196：901(1987)；Chothia，C.等，Nature,342(6252)：877-83(1989)；Kabat,E.A.等，National Institutes of Health,Bethesda,Md.(1991))。其中，三个CDR由被称为框架区(FR)的侧面连续部分间隔开，框架区比CDR更加高度保守并形成一个支架支撑超变环。重链和轻链的恒定区与抗原结合无关，但具有多种效应功能。抗体依据重链恒定区的氨基酸序列可以分成几类。根据是否含有α、δ、ε、γ和μ重链，抗体可分别分为五个主要的分类或异构体：IgA、IgD、IgE、IgG和IgM。几个主要的抗体分类还可分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)等。
本申请中的"抗原结合片段"一词，指由含有一个或多个CDR的抗体部分或者任何其他结合抗原但不具有完整抗体结构的抗体片段所形成的一种抗体片段。抗原结合片段的例子包括，但不限于，如双功能抗体(diabody)、Fab、Fab'、F(ab')2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双功能抗体(ds diabody)、单链抗体分子(scFv)、scFv二聚体(双价的双功能抗体)、双价单链抗体(BsFv)、多特异性抗体、骆驼化单域抗体(camelized single domain antibody)、纳米抗体、域抗体和双价域抗体。抗原结合片段可以与母体抗体结合相同的抗原。在某些实施方式中，抗原结合片段可以含有来自某特定人抗体的一个或多个CDR，移接至来自一个或多个不同人抗体的框架区。
抗体的"Fab"片段是指由一条轻链(包括可变区和恒定区)和一条重链的可变区和部分恒定区经二硫键结合起来的那部分抗体分子。
"Fab’"片段是指包含了部分绞链区的Fab片段。
"F(ab')2"指的是Fab的二聚体。
抗体的Fc段负责多种不同的效应功能如ADCC和CDC，但不参与抗原的结合。
抗体的"Fv"段指的是含有完整抗原结合位点的最小抗体片段。Fv片段由一条轻链的可变区和一条重链的可变区组成。
“融合蛋白”指在基因水平上将编码目的蛋白的cDNA与编码抗体或抗体片段的cDNA相连接，并在真核或原核表达系统中表达的重组蛋白。
"接头(linker)"是指由形成了肽键的1～50个氨基酸构成的肽链或其衍生物，其N末端和C末端分别与抗OX40抗体或干扰素中的任意一者形成共价键，从而将抗OX40抗体与干扰素结合。可以通过在抗OX40抗体的重链或轻链的C末端侧或N末端侧通过接头序列或直接地利用肽键分别结合干扰素的N末端或C末端，从而使抗OX40抗体和干扰素一体化。作为干扰素与抗OX40抗体的融合蛋白的优选的实施方式，可以列举：在抗OX40抗体的重链或轻链的C末端通过接头序列结合干扰素的N末端而得到的融合蛋白；或者，在抗OX40抗体的重链或轻链的N末端通过接头序列结合干扰素的C末端而得到的融合蛋白。
"单链Fv抗体"或"scFv"是指由轻链可变区与重链可变区直接相连或通过一个肽链连接而成的工程抗体(Huston JS等，Proc Natl Acad Sci USA,85：5879(1988))。
"单链抗体Fv-Fc"或"scFv-Fc"是指由连接到某抗体Fc段的scFv组成的工程抗体。
"骆驼化单域抗体(Camelized single domain antibody)"，"重链抗体" 或"HCAb(Heavy-chain-only antibodies，HCAb)"都是指含有两个VH域而不含有轻链的抗体(Riechmann L.和Muyldermans S.,J Immunol Methods.231(1-2)：25-38(1999)；Muyldermans S.,J Biotechnol.74(4)：277-302(2001)；W094/04678；W094/25591；U.S.Patent No.6,005,079)。重链抗体最初从驼科(骆驼、单峰驼和美洲驼)衍生得到。虽然缺失轻链，骆驼化抗体(camelized antibodies)有确证的抗原结合全部功能(Hamers Casterman C.等，Nature 363(6428)：446-8(1993)；Nguyen VK.等，"Heavy-chain antibodies in Camelidae：a case of evolutionary innovation,Immunogenetics.54(1)：39-47(2002)；Nguyen VK.等，Immunology.109(1)：93-101(2003))。重链抗体的可变区(VH域)是己知的最小的获得性免疫产生的抗原结合单位(Koch-Nolte F.等，FASEB J.21(13)：3490-8.Epub(2007))。
"纳米抗体"是指一种抗体片段，其由一个来自重链抗体的VH域和两个恒定区CH2和CH3组成。
"双功能抗体(diabody)"包括带有两个抗原结合位点的小抗体片段，其中该片段含有在同一条多肽链上相连的VH域和VL域(请参见，Holliger P.等，Proc Natl Acad Sci U S A.90(14)：6444-8(1993)；EP404097；W093/11161)。两个域之间衔接物很短，使同一条链上的两个域不能互相配对，从而迫使两个域与另二条链的互补域配对，形成两个抗体结合位点。这两个抗体结合位点可靶向结合相同或不同的抗原(或抗原表位)。
"域抗体"是指仅含有一条重链可变区或一条轻链可变区的抗体片段。在某些情况下，两个或多个VH域由一个多肽衔接物共价结合并形成双价域抗体。双价域抗体的两个VH域可靶向作用于相同或不同的抗原。
在某些实施方式中，"(dsFv)2"含有三条肽链：两个VH基因间通过一条多肽衔接物相连，并通过二硫键与两个VL基团结合。
在某些实施方式中"双特异性ds双功能抗体"含有VL1-VH2(由二个多肽衔接物相连)和VH1-VL2(也是由二个多肽衔接物相连)，两者在VH1和VLl间通过二硫键结合。
"双特异性dsFv"或"dsFv-dsFv"含有三条多肽链：VH1-VH2基团，其中两者的重链通过多肽衔接物(如：长的弹性衔接物)相连，并通过二硫键分别与VL1和VL2基团结合，每对通过二硫键配对的重链轻链具有不同的抗原特异性。
在某些实施方式中，"scFv二聚体"是双价双功能抗体或双价单链抗体(BsFv)，含有二聚化的两个VH-VL(由多肽衔接物连接)基团，其中二个基团的VH与另一个基团的VL协作形成两个结合位点，这两个结合位点可靶向结合相同抗原(或抗原表位)或不同抗原(或抗原表位)。在另一些 实施方式中，"scFv二聚体"是双特异性双功能抗体，含有相互连接的V L1-V H2(由多肽衔接物连接)和V H1-V L2(由多肽衔接物连接)，其中V H1和V L1协作，V H2和V L2协作，且每个协作的配对具有不同的抗原特异性。
本申请中使用的术语"全人源"当用于抗体或抗原结合片段时，是指所述抗体或抗原结合片段具有某氨基酸序列或由所述氨基酸序列组成，所述氨基酸序列对应于由人或人免疫细胞生产的、或从例如利用人源抗体库的转基因非人动物等非人来源衍生的抗体的氨基酸序列，或者其他编码人源抗体的序列。在某些实施方式中，全人源抗体不包含来源于非人抗体的氨基酸残基(特别是抗原结合残基)。
本申请中使用的术语"人源化"当用于抗体或抗原结合片段时，是指包括来源于非人动物的CDR、来源于人的FR区，以及来源于人的恒定区(当适用时)的抗体或抗原结合片段。由于人源化的抗体或抗原结合片段具有降低的免疫原性，其在某些实施方式中可用作人的治疗剂。在一些实施方式中，所述非人动物是哺乳动物例如小鼠、大鼠、兔、山羊、绵羊、豚鼠或仓鼠。在一些实施方式中，所述人源化抗体或抗原结合片段除了CDR序列是非人源的以外，基本上全部由人源序列组成。在一些实施方式中，所述来源于人的FR区可以包括与其来自的人源抗体相同的氨基酸序列，或其可以包括一些氨基酸改变，例如，不超过10、9、8、7、6、5、4、3、2或1个氨基酸改变。在一些实施方式中，该氨基酸改变可以仅存在于重链FR区、仅存在于轻链FR区或同时存在于两个链中。在一些优选实施方式中，所述人源化抗体包括人源FRl-3和人源JH和JK。
本申请中使用的术语"嵌合"是指具有来源于一种物种的重链和/或轻链的一部分，和所述重链和/或轻链其余部分来源于不同物种的抗体或抗原结合片段。在一个示例性的例子中，嵌合抗体可以包括来源于人的恒定区和来源于非人动物例如小鼠的可变区。
术语“OX40”是指与OX40L结合的受体。它是属于TNF受体家族的I型膜蛋白。其它的命名为ACT-4，OX40L受体，CD134抗原，ACT35抗原，TNFRSF4。它具有50kDa的分子量，并且存储在SwissProt中，登记号为P43489。
本申请中使用的"人干扰素"是指一类高活性、多功能的分泌型糖蛋白，具有抗病毒、免疫调节和抗肿瘤等作用。根据其基因序列、受体特异性等可分为I型、II型和III型IFNs。人类I型干扰素包括有13种亚型的IFNα及IFNβ、IFNε、IFNκ、IFNω。Ⅰ型IFNs具有一个共同的细胞表面受体IFNAR，由IFNAR1和IFNAR2两个亚基组成。II型干扰素只有1种，即IFNγ。IFNγ的细胞表面受体为IFNGR，由IFNGR1和IFNGR2两个亚基组成。III型IFNs 包括IFNλ1、IFNλ2、IFNλ3和IFNλ4。本申请的融合蛋白中的干扰素还包括本领域已知的干扰素变体。
本申请中的"特异性结合"或"特异性的结合"是指两分子间的非随机结合反应，如抗体和抗原间的反应。在某些实施方式中，本申请的抗体或其抗原结合片段与人和/或猴OX40特异性结合，并且其结合亲和力(K D)≤10 -6M。本申请中的K D是指解离速度与结合速度的比值(k off/k on)，可通过表面等离子共振的方法测定，例如使用如Biacore的仪器。
本申请中使用的"UMY02-L1"是指具有如SEQ ID NO:10所示的重链、肽接头和人干扰素，如SEQ ID NO:11所示的轻链，其中在重链羧基端通过肽接头连接了如SEQ ID NO:9所示的人干扰素IFNα2b的融合蛋白。
本申请中使用的"UMY02-L2"是指具有如SEQ ID NO:12所示的重链、如SEQ ID NO:13所示的轻链、肽接头和人干扰素，其中在轻链羧基端通过肽接头连接了如SEQ ID NO:9所示的人干扰素IFNα2b的融合蛋白。
本申请中使用的"UMY02-L3"是指具有如SEQ ID NO:14所示的重链、如SEQ ID NO:13所示的轻链、肽接头和人干扰素，其中在轻链羧基端通过肽接头连接了如SEQ ID NO:9所示的人干扰素IFNα2b的融合蛋白。
本申请中使用的"MT01-C1"是指与UMY02-L1、UMY02-L2及UMY02-L3具有相同VH(SEQ ID NO:7)和VL(SEQ ID NO:8)序列，且重轻链恒定区分别为人IgG1和κ链的单克隆抗体。
本申请中使用的"MT01-C1(G2)"是指与UMY02-L1、UMY02-L2及UMY02-L3具有相同VH(SEQ ID NO:7)和VL(SEQ ID NO:8)序列，且重轻链恒定区分别为人IgG2和κ链的单克隆抗体。
本申请中使用的"MT01-L1"是指与UMY02-L1、UMY02-L2及UMY02-L3具有相同重链和轻链CDR序列(SEQ ID NO:1-6)且重轻链恒定区分别为人IgG1和κ链的人-鼠嵌合抗体。
其中，所述"MT01-C1"、"MT01-C1(G2)"、"MT01-L1"均来自于中国专利201711476160.3，其具体序列如表1及序列表所示，通过引用将其整体并入本文。
在本申请中当"保守替代"用于氨基酸序列时，是指将一个氨基酸残基用另一个具有相似理化性质的侧链的氨基酸残基替代。例如，可以在疏水侧链氨基酸残基间(例如Met、Ala、VaL、Leu和Ile)、中性亲水侧链残基间(例如Cys、Ser，Thr、Asn和Gln)、酸性侧链残基间(例如Asp、Glu)、碱性侧链氨基酸间(例如His、Lys和Arg)或芳香侧链残基间(例如Trp、Tyr和Phe)进行保守替代。本领域己知保守替代通常不会引起蛋白构象结构的显著变化，因此能够保留蛋白质的生物活性。
当"百分比序列同一性"用于氨基酸序列(或核酸序列)时，是指在进行序列比对，并且必要时引入间隔使相同氨基酸(或核酸)数目达到最多后，在候选序列中，与参比序列相同的氨基酸(或核酸)残基占所述候选序列的氨基酸(或核酸)残基的百分比。所述氨基酸残基的保守替代可以认为或可以不认为是相同残基。可以通过本领域公开的工具对序列进行比对以确定氨基酸(或核酸)序列的百分比序列同一性。本领域技术人员可以使用所述工具的默认参数或根据比对的需要适当调整参数，例如通过挑选合适的算法。
本申请中使用的“T细胞”包括CD4+T细胞、CD8+T细胞、T辅助1型T细胞、T辅助2型T细胞、T辅助17型T细胞和抑制性T细胞。
本申请中使用的"效应功能"是指抗体的Fc区与其效应器例如C1复合物和Fc受体结合的生物活性。示例性的效应功能包括抗体与C1复合物上的C1q相互作用诱导的补体依赖性细胞毒性(CDC)、抗体的Fc区与效应细胞上的Fc受体结合诱导的抗体依赖性细胞介导的细胞毒性(ADCC)以及吞噬。
本申请中的“癌症”或“癌状况”是指任何由肿瘤或恶性细胞生长、增殖或转移所介导，并引发实体瘤和非实体瘤如白血病的医学状况。本发明中的"肿瘤"是指肿瘤和/或恶性细胞的实体物质。
本申请中的“病毒感染”是指病毒通过多种途径侵入人类机体，并在人细胞中增殖，导致机体损伤的致病过程，括慢性病毒感染，例如乙型肝炎、丙型肝炎、疱疹病毒、EB(Epstein-Barr)病毒、艾滋病毒、巨细胞病毒、单纯疱疹病毒I型、单纯疱疹病毒2型、人乳头状瘤病毒、腺病毒的病毒感染、卡波西肉瘤相关的疱疹病毒流行病、薄环病毒(Torquetenovirus)、JC病毒或BK病毒等。
对某种状况的"治疗"或"疗法"包括预防或减轻某种状况，降低某种状况兴起或发展的速度，减少发展出某种状况的风险，预防或延迟与某种状况相关的症状发展，减少或终止与某种状况相关的症状，产生某种状况的完全或部分的逆转，治愈某种状况，或以上的组合。对于癌症来说"治疗"或"疗法"可以指抑制或减缓肿瘤或恶性细胞生长，繁殖，或转移，或以上的某些组合。对于肿瘤来说"治疗"或"疗法"包括清除全部或部分的肿瘤，抑制或减缓肿瘤生长和转移，预防或延缓肿瘤的发展，或以上的某些组合。
"被分离"的物质已经经人工由自然状态改变。如果自然界中出现某种"被分离"的物质或成分，那么其已经被改变或脱离其原始状态，或二者均有发生。例如，某一活体动物体内天然存在的多核苷酸或多肽是未被分离的，但如果这些多核苷酸或多肽与之在天然状态下共存的物质足够分离并以足够纯的状态存在，则可以认为是"被分离"。在某些实施方式中，抗体和抗原 结合片段的纯度为至少90％、93％、95％、96％、97％、98％、99％，其由电泳方法(如SDS-PAGE、等电聚焦、毛细管电泳)，或色谱法(如离子交换色谱法或反相HPLC)确定。
本发明中"载体"是指，可将编码某蛋白的多核苷酸操作性地插入其中并使该蛋白获得表达的一种运载工具。载体可用于转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。举例来说，载体包括：质粒、噬菌粒、柯斯质粒、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1衍生的人工染色体(PAC)、噬菌体如λ噬茵体或M13噬菌体，以及动物病毒等。用作载体的动物病毒种类有逆转录病毒(包括慢病毒、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40))。载体可含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还可包括协助其进入细胞的成分，包括但不限于，病毒颗粒、脂质体或蛋白外壳。
本发明中"宿主细胞"是指导入外源多核苷酸和/或载体的细胞。
本发明中的"治疗有效量"或"有效剂量"是指，某种药物有效治疗疾病的剂量或浓度。例如，对于本发明中公开的抗体或其抗原结合片段的用途来说，治疗有效量是在该剂量或浓度下，该抗体或抗原结合物可以清除全部或部分肿瘤、抑制或减缓肿瘤生长、抑制肿瘤细胞转移、减轻任何与肿瘤或癌状况相关的症状或标记，预防或延缓肿瘤或癌状况的发展，抑制或清除病毒或病毒感染的细胞，或以上的某些组合。
"药用可接受的"是指所指的载剂、溶媒、稀释剂、辅料和/或盐，总的来说在化学上和/或在物理上与制剂中的其他配料相兼容，并在生理上与接受者相兼容。
关于本发明中的融合蛋白
在某些实施方式中，本申请提供了示例性的融合蛋白UMY02-L1、UMY02-L2和/或UMY02-L3。
本领域技术人员应理解可以将前述CDR序列进行修饰以包含一个或更多氨基酸的取代，由此得到提高的生物学活性例如提高的与人OX40的结合亲和性。例如，可以利用噬菌体展示技术生产并表达抗体变体库(例如Fab或FcFv变体)，随后筛选与人OX40有亲和性的抗体。另一个例子中，可以用计算机软件模拟所述抗体与人OX40的结合并鉴别抗体上形成结合界面的氨基酸残基。可以避免这些残基的替代以防止结合亲和性降低，或可以靶向这些残基进行替代以形成更强的结合。在某些实施方式中，CDR序列中的至少一个(或全部)取代是保守替代。
在某些实施方式中，所述融合蛋白和抗原结合片段包括一个或多个CDR序列，这些序列具有与本申请提供了示例性的融合蛋白UMY02-L1、UMY02-L2和/或UMY02-L3中所列的序列至少80％(例如至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)的序列同一性，并且同时保留了与其亲本抗体相似或甚至高于其的与人OX40的结合亲和性，所述亲本抗体具有基本相同的序列，但其相应的CDR序列与所列的序列具有100％序列同一性。
在一些实施方式中，本申请所述的融合蛋白能够以≤10 -7M的结合亲和性(K D)与人OX40特异性结合，其通过表面等离子共振法测量。结合亲和性值可以用K D值表示，其通过当抗原和抗原结合分子的结合达到平衡时的解离速率与结合速率的比值(k off/k on)计算得到。所述抗原结合亲和性(例如K D)可以通过本领域己知的适宜方法适宜地确定，例如包括使用仪器如Biacore的等离子共振结合法。
在某些实施方式中，本申请所述融合蛋白与人OX40以10ng/mL-10μg/mL的EC50(即半数结合浓度)结合。抗体或融合蛋白与人OX40的结合可以通过本领域己知的方法如夹心法如ELISA，Western印迹，FACS或其他结合试验测定。在示例性的例子中，将待测抗体(即一抗)与固定化的人OX40或表达人OX40的细胞结合，随后洗掉未结合抗体，引入标记的二抗，其能够与一抗结合因此能够检测出结合的二抗。当使用固定化的OX40时可在酶标仪板上进行所述检测，或当使用表达人OX40的细胞时可使用FACS分析进行所述检测。
在某些实施方式中，本申请所述融合蛋白以0.1μg/mL至10μg/mL(使用FACS分析测定)的EC50(即50％的有效浓度)与人OX40结合。
在某些实施方式中，本申请所述融合蛋白可以通过FcR介导或干扰素受体介导的方式激活人OX40信号通路，并由此提供了包括例如诱导活化的T细胞产生细胞因子(如CD4+T细胞和CD8+T细胞)、诱导活化的T细胞的增殖(如CD4+T细胞和CD8+T细胞)和逆转调节性Treg的抑制性功能的生物学活性。
所述融合蛋白是人OX40特异性的。在某些实施方式中，所述融合蛋白不与鼠OX40结合，但与猴OX40以与人OX40相似的结合亲和性结合。例如，与本发明融合蛋白具有相同CDR序列的单克隆抗体MT01-L1与鼠OX40的结合用常规结合测定方法如FACS分析无法检出，而FACS检测出MT01-L1与猴OX40以及与人OX40都有结合。
在一些实施方式中，所述的融合蛋白具有IgG2同种型的恒定区，其具有降低的或消除的效应功能。例如ADCC和CDC等效应功能能够导致对表 达OX40的细胞的细胞毒性。一些正常的细胞能够表达OX40。为了避免对这些正常的细胞产生潜在的不希望的毒性，本发明所述的抗体的某些实施方式具有降低的或甚至消除的效应功能。己知有许多测试用来估测ADCC或CDC活性，例如Fc受体结合试验、补体Clq结合实验和细胞裂解法，本领域技术人员能够容易选择。不希望受到理论的束缚，但据信具有降低的或消除的效应功能如ADCC和CDC的抗体不会引起对表达OX40的细胞(例如那些正常的细胞)的细胞毒性或将之降低到最小程度，因此避免了不希望的副作用。
在一些实施方式中，本申请所述的融合蛋白在生物体内较干扰素分子具有延长的作用时间。这是由于融合蛋白在动物体内具有较长的半衰期和药物滞留时间。这一性质有利于减少病人的用药次数及提高药物的药效。
在一些实施方式中，本申请所述的融合蛋白具有降低的副作用。例如所述的抗OX40抗体和其抗原结合片段可以具有全人源IgG序列，因此其免疫原性低于人源化的抗体。再例如，所述的融合蛋白和其抗原结合片段可以具有IgG2或IgG4形式以消除ADCC和CDC。
在一些实施方式中，本申请所述的融合蛋白的优势在于其能与具有免疫原性的物质联用，如肿瘤细胞、纯化的肿瘤抗原和用编码免疫刺激因子转染的细胞、肿瘤疫苗。此外，所述抗OX40抗体和其抗原结合片段可以包括在联用治疗中，包括标准化学疗法和放射疗法、基于靶点的小分子疗法、其他新兴免疫检查点调节剂疗法。在一些实施方式中，所述抗体和其抗原结合片段可以用作抗体一药物缀合物、双特异性或多价抗体的基础分子。
在一些实施方式中，本申请所述的融合蛋白和其抗原结合片段是骆驼化单域抗体(camelized single chain domain antibody)、双功能抗体(diabody)、scFv、scFv二聚体、BsFv、dsFv、(dsFv)2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab')2、ds双功能抗体(ds diabody)、纳米抗体、域抗体或双价域抗体。
在一些实施方式中，本申请所述的融合蛋白包括免疫球蛋白恒定区。在一些实施方式中，免疫球蛋白恒定区包括重链和/或轻链恒定区。所述重链恒定区包括CH1、CH1-CH2或CH1-CH3区。在一些实施方式中，免疫球蛋白恒定区可以进一步包括一个或多个修饰以获得所需的性质。例如，可以将所述恒定区修饰以降低的或消除一种或多种效应功能以增强FcRn受体结合或引入一个或多个半胱氨酸残基。
在某些实施方式中，所述抗体及其抗原结合片段进一步包含缀合物。可以设想，本发明中的抗体或其抗原结合片段可与多种缀合物连接(见例如"Conjugate Vaccines"、Contributions to Microbiology and Immunology、 J.M.Cruse and R.E.Lewis、Jr.(eds.)、Carger Press、New York(1989))。这些缀合物可以通过共价结合、亲和结合、嵌入、同等结合(coordinate binding)、络合、结合、混合或加入等其他方式与所述抗体或抗原结合物连接。在某些实施方式中，本发明公开的抗体和抗原结合片段可以通过工程的方法使其含有表位结合部分以外的特定位点，这些位点可用来结合一种或多种缀合物。例如，这样的位点可包含一种或多种反应性氨基酸残基，例如半胱氨酸残基和组氨酸残基，用于协助与结合物的共价连接。在某些实施方式中，抗体可间接连于缀合物，或通过另一个缀合物相连。例如，所述抗体或其抗原结合片段可结合生物素，然后间接结合第二个缀合物，其与亲和素相连。所述缀合物可以是可检测的标记、药代动力学修饰部分、纯化部分或细胞毒性部分。可检测的标记的例子可以包括荧光标记(例如荧光素、罗丹明、丹酰、藻红蛋白或德克萨斯红)、酶底物标记物(例如辣根过氧化物酶、碱性磷酸酶、荧光素酶、葡糖淀粉酶、溶菌酶、糖氧化酶或β-D半乳糖昔酶)、稳定同位素或者放射性同位素、发色团部分、地高辛、生物素/亲和素、DNA分子或金以进行检测。在某些实施方式中，所述缀合物可以是药代动力学修饰部分如PEG，其帮助延长抗体的半衰期。其他适宜的聚合物包括例如竣甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、乙二醇/丙二醇共聚物等。在某些实施方式中，所述缀合物可以是纯化部分例如磁珠。"细胞毒性部分"可以是对细胞有害的或可能损坏或杀死细胞的任何试剂。细胞毒性部分的示例包括，但不限于，紫杉醇、细胞松弛素B、短杆菌肽D、溴化乙绽、吐根碱、丝裂霉素、依托泊昔、替尼泊甘、长春新碱、长春碱、秋水仙碱、阿霉素、柔红霉素、二羟基炭疽菌素二酮、米托蒽醌、光神霉素、放线菌素D、l-去氢睾酮、糖皮质激素、普鲁卡因、丁卡因、利多卡因、普茶洛尔、嘌呤霉素及其类似物、抗代谢物(例如，甲氨喋呤、6-巯基嘌呤、6-巯鸟瞟岭、阿糖胞苷、5氟尿嘧啶达卡巴)、烷化剂(例如氮芥、塞替派苯丁酸氮芥、美法仑、卡莫司汀(BSNU)和洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露醇、链脲霉素、丝裂霉素C和顺-二氯二胺铂(DDP)顺铂)、蒽环类抗生素(例如柔红霉素(以前的道诺霉素)和阿霉素)、抗生素(例如更生霉素(以前称为放线菌素)、博来霉素、光神霉素和氨茵霉素(AMC))以及抗有丝分裂剂(例如长春新碱和长春碱)。
多核苷酸和重组方法
使用本领域公知的遗传工程学技术，可以将本申请的融合蛋白中的氨基酸序列转换成相应的DNA编码序列。由于遗传密码的简并性，转换所得的DNA序列可以完全一致，而编码的蛋白序列保持不变。
使用本领域公知的重组技术，可以将包括编码所述融合蛋白的多核苷酸的载体引入宿主细胞用于克隆(扩增DNA)或基因表达。在另一实施方式中，所述融合蛋白可通过本领域公知的同源重组的方法制得。多种载体可供选择。载体组分通常包括，但不限于，以下的二种或多种：信号序列、复制起始点、一种或多种标记基因、增强序列、启动子(例如：SV40，CMV，EF-1a)和转录终止序列。
在一些实施方式中，所述载体系统包括哺乳动物、细菌、酵母系统等，并将包括质粒例如但不限于pALTER、pBAD、pcDNA、pCal、pL、pELpGEMEX、pGEX、pCLpCMV、pEGFP、pEGFT，pSV2、pFUSE、pVITRO，pVIVO、pMAL、pMONO、pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS420、pLexA、pACT2等其他可从实验室获得或市售的载体。适宜的载体可以包括质粒或病毒载体(例如，复制缺陷型逆转录病毒、腺病毒和腺相关病毒)。
可以将包括编码所述融合蛋白的多核苷酸的载体引入宿主细胞用于克隆或基因表达。本发明中适用于克隆或表达所述载体中的DNA的宿主细胞为原核细胞、酵母或上述高级真核细胞。适用于本发明用途的原核细胞包括真细菌如，革兰氏阴性菌或革兰氏阳性菌，例如，肠杆菌科，如，大肠杆菌，肠杆菌属，欧文氏菌属，克雷白氏杆菌属，变形杆菌属，沙门氏菌属，如，鼠伤寒沙门(氏)杆菌，沙雷氏菌属，如，粘质沙雷氏菌，以及志贺氏菌属，及杆菌属如，枯草芽孢杆菌和地衣芽孢杆菌，假单胞菌如，绿肽杆菌和链霉菌。
除了原核细胞以外，真核微生物如丝状真菌或酵母也可作宿主细胞克隆或表达编码融合蛋白的载体。酿酒酵母，或面包酵母是最常用的低等真核宿主微生物。但是，许多其他属、种和株都比较常用且在本发明中适用，如粟酒裂殖酵母；克鲁维酵母属宿主如，乳酸克鲁维酵母、脆壁克鲁维酵母(ATCC 12，424)、保加利亚克鲁维酵母(ATCC 16，045)、魏氏克鲁维酵母(ATCC 24，178)、克鲁雄酵母(ATCC 56，500)、果蝇克鲁维酵母(ATCC36，906)、耐热克鲁维酵母和马克斯克鲁维酵母：解脂耶氏酵母(EP 402,226)；巳斯德毕赤酵母(EP 183,070)；假丝酵母：里氏木霉(EP 244,234)；链孢霉；西方许旺酵母，如：西方许旺酵母；和丝状真菌，如：脉孢菌、青霉菌、弯颈霉和曲霉菌，如：钩巢曲霉和黑曲霉。
本发明中提供的适用于表达糖基化抗体或其抗原结合片段的宿主细胞由多细胞生物衍生得到。无脊椎细胞的实例包括植物和昆虫细胞。己发现多种杆状病毒株(baculoviral strains)及其变体以及对应的许可性昆虫宿主细胞(permissive insect host cells)，来自于诸如以下的宿主：草地夜蛾(毛虫)、 埃及斑蚊(蚊子)、白纹伊蚊(蚊子)、黑腹果蝇(果蝇)及家蚕。多种用于转染的病毒株为公众可得，例如苜蓿银纹夜蛾核型多角体病毒和家蚕核型多角体病毒的Bm-5变种，这些病毒都可在本发明中使用，特别是用于转染草地夜蛾细胞。棉花、玉米、土豆、大豆、矮牵牛花、西红柿和烟草的植物细胞培养也可用作宿主。
但是，最感兴趣的是脊椎细胞，且脊椎细胞的培养(组织培养)己经成为常规操作。可用的哺乳动物宿主细胞实例有，SV40转化的猴肾细胞CV1系(COS-7，ATCC CRL 1651)；人胚胎肾细胞系(293或悬浮培养的293细胞亚克隆，Graham et al.,].Gen Virol.36：59(1977))；幼地鼠肾细胞(B血，ATCC CCL 10)；中国仓鼠卵巢细胞/-DHFR(CHO，Urlaub et al.,Proc.Natl.Acad.Sci.USA 77：4216(1980))；小鼠睾丸支持细胞(TM4，Ma ther,Biol.Reprod.23：243-251(1980))；猴肾细胞(CV1ATCC CCL 70)；非洲绿猴肾细胞(VERO-76,ATCC CRL-1587)；人宫颈癌细胞(HELA，ATCC CCL 2)；犬肾细胞(MDCK,ATCC CCL 34)；布法罗大鼠肝细胞(BRL 3A,ATCC CRL 1442)；人肺细胞(W138，ATCC CCL75)；人肝细胞(Hep G2,HB 8065)；小鼠乳腺瘤(MMT 060562,ATCC CCL51)；TRI细胞(Ma ther等，Annals N.Y.Acad.Sci.383：44-68(1982))；MRC 5细胞；FS4细胞；及人肝癌细胞系(HepG2)。在某些优选的实施方式中，所述宿主细胞是293F细胞。
用上述的可产生所述融合蛋白的表达或克隆载体转化宿主细胞，并将其在常规的营养培养基中培养，所述营养培养基经修饰后适宜于诱导启动子、选择转化细胞或扩增编码目的序列的基因。
本发明中用于产生所述融合蛋白的宿主细胞可在多种本领域公知的培养基中培养。所述培养基还可含有本领域公知的适当浓度的任何其他必要的添加剂。所述培养基的条件，如温度、pH值等类似条件，为选择用于表达的宿主细胞此前所使用的条件，为普通技术人员所熟知。
在使用重组技术时，所述抗体可在胞内、壁膜空间生成，或直接分泌到培养基中。如果所述抗体在胞内生成，首先除去宿主细胞或裂解片断的颗粒残骸，例如，可通过离心或超声的方法。Carter et al.,Bio/Technology 10：163-167(1992)描述了将分泌到大肠杆菌壁膜空间的抗体分离的方法。简要地说，在醋酸铀(pH 3.5)、EDTA和苯甲磺酣氟(PMSF)存在的条件下化开细胞糊(cell paste)约30分钟以上。离心除去细胞碎片。如所述抗体分泌到培养基中，则通常首先使用市售的蛋白浓度过滤器，如lAmicon或Millipore Pellicon ultrafiltration unit，浓缩该表达系统的上清液。在任何前述的步骤中都可加入蛋白酶抑制剂如PMSF以抑制蛋白降解，以及抗生素以防止偶然污 染物的生长。
从所述细胞中制得的抗体可采用纯化方法进行纯化，例如羟磷灰石色谱、凝胶电泳、透析、DEAE一纤维素离子交换色谱柱、硫酸铵沉淀、盐析以及亲和色谱，其中亲和色谱为优选的纯化技术。所述抗体的种类以及所述抗体中存在任何免疫球蛋白的Fc结构域决定了蛋白A作为亲和配体是否适合。蛋白A可用于纯化基于人γ1，γ2或γ4重链的抗体(Lindmark et al.，J.lmmunol.Meth.62：卜13(1983))。蛋白G适用于所有鼠源异构体和人γ3(Guss et al.,EMBO J.5：1567 1575(1986))。琼脂糖是最常用的亲和配体附着基质，但也可选用其他基质。机械力稳定的基质如可控孔度玻璃或聚(苯乙烯)苯与用琼脂糖相比可实现更快的流速和更短的处理时间。如该抗体含有CH3结构域，则可用Bakerbond ABX.TM树脂进行纯化(J.T.Baker，Phillipsburg，N.J.)。也可根据需要获得的抗体确定其他蛋白纯化的技术，如离子交换柱中的分馏、乙醇沉淀、反相HPLC、硅胶色谱、基于阴离子或阳离子交换树脂的肝素琼脂糖凝胶色谱(如聚天冬氨酸柱)、层析聚焦、SDS-PAGE、以及硫酸铵沉淀。
在任意初步纯化步骤之后，可用低pH疏水相互作用色谱的方法处理含有感兴趣的抗体和杂质的混合物，用pH约2.5-4.5的洗脱缓冲液，优选地在低盐浓度下进行(例如，从约0到0.25M盐浓度)。
试剂盒
本申请提供了包括所述融合蛋白的试剂盒。在一些实施方式中，所述试剂盒用于检测在生物样品中的OX40的存在情况或水平。所述生物样品可以包括细胞或组织。
在一些实施方式中，所述试剂盒包括与可检测标记缀合的融合蛋白。在一些实施方式中，所述试剂盒包括未标记的融合蛋白，并进一步包括能够与未标记的融合蛋白结合标记的二抗。所述试剂盒可以进一步包括使用说明和在试剂盒中将每个组件分隔开的包装。
在一些实施方式中，所述融合蛋白与底物或仪器连接用于夹心测定如ELISA或免疫色谱测定。适用的底物或仪器可以是例如微孔板和试纸。
药物组合物和治疗方法
本申请进一步提供了包括所述融合蛋白的药物组合物和一个或多个药学上可接受的载体。
用在本申请公开的药物组合物中的药用可接受载剂可包括，例如，药用可接受的液体、凝胶或固体载剂、水相介质、非水相介质、抗微生物物质、等渗物质、缓冲液、抗氧剂、麻醉剂、悬浮剂/分散剂、整合剂、稀释剂、佐剂、辅料或无毒辅助物质，其他本领域公知的组分或以上的多种组合。
适用的组分可包括，例如，抗氧剂、填充剂、粘合剂、崩解剂、缓冲液、防腐剂、润滑剂、矫味剂、增稠剂、着色剂、乳化剂或稳定剂例如糖和环糊精。适用的抗氧剂可包括，例如，甲硫氨酸、抗坏血酸、EDTA、硫代硫酸纳、铂、过氧化氢酶、柠檬酸、半胱氨酸、巯基甘油、巯基乙酸、巯基山梨醇、丁基甲基茴香醚、丁基化羟基甲苯和/或没食子酸丙酯。在一种含有本发明公开的融合蛋白的组合物中包括一种或多种抗氧剂如甲硫氨酸，可将降低所述融合蛋白的氧化。对氧化作用的减少可防止或减少结合亲和力的降低，从而提高抗体稳定性并延长保质期。
进一步的说，药用可接受的载剂可包括，例如，水相介质如氯化钠注射液、林格氏液注射液、等渗葡萄糖注射液、无菌水注射液、或葡萄糖和乳酸林格注射液、非水介质例如：植物来源的不挥发性油、棉花子油、玉米油、芝麻油、或者花生油、细菌抑制或真菌抑制浓度下的抗菌物质、等渗剂如：氯化钠或葡萄糖、缓冲液如：磷酸盐或枸橼酸盐缓冲液，抗氧化剂如：硫酸氢钠，局部麻醉剂如：盐酸普鲁卡因，助悬剂和分散剂如：羧甲基纤维素钠、羟丙基甲基纤维素或聚乙烯吡咯烷酮，乳化剂如：聚山梨醇酯80(吐温-80)、整合试剂如EDTA(乙二胺四乙酸)或EGTA(乙二醇双(2一氨基乙基醚)四乙酸)、乙醇、聚乙二醇、丙二醇、氢氧化钠、盐酸、柠檬酸或乳酸。作为载剂的抗菌剂可加入多次剂量容器中的药物组合物中，其包括酚类或甲酚、汞制剂、苯甲醇、氯代丁醇、甲基和丙基对羟基苯甲酸酯、噻汞撒、氯苯甲烷铵和氯苯乙铵。适用的辅料可包括，例如，水、盐、葡萄糖、甘油或乙醇。适用的无毒辅助物质可包括，例如，乳化剂、pH值缓冲剂、稳定剂、增溶剂，或者醋酸钠、去水山梨糖醇月桂酸酯、三乙醇胺油酸酯或者环糊精之类的物质。
所述药物组合物可以是液体溶液、悬浮液、乳剂、丸剂、胶囊、片剂、持续释放制剂或粉末。口服制剂可以包括标准载体如药物级的甘露醇、乳糖、淀粉、硬脂酸镁、聚乙烯吡咯皖酮、糖精钠、纤维素、碳酸镁等。
在某些实施方式中，所述药物组合物被制剂成可注射的组合物。可注射的药物组合物可以任何常规的形式制备，例如，液体溶剂、悬浮剂、乳化剂或适用于产生液体溶剂、悬浮剂或乳化剂的固体形式。注射制剂可包括现用的无菌和/或无热原溶液、使用前现与溶剂结合的无菌干燥的可溶物，如冻干粉，包括皮下片、注射即用的无菌悬浮剂、使用前现与介质结合的无菌干燥不溶产品，和无菌和/或无热原的乳剂。溶剂可以为水相或非水相。
在某些实施方式中，单位剂量的注射制剂包装在一个安瓿、一支管或一支带有针的针筒中。本领域悉知，所有注射给药的制剂应为无菌无热原。
在某些实施方式中，通过将本申请公开的抗体或其抗原结合片段溶解于 某适当的溶剂中可制备无菌冻干的粉末。所述溶剂可含有一种可提高粉或由粉末制得的重组溶液的稳定性，或改善粉末或重组溶液的其他药理组分。适用的辅料包括，但不限于，水、葡萄糖、三梨糖醇、果糖、玉米糖浆、木糖醇、甘油、葡萄糖、黑糖或其他适用的物质。溶剂可含有缓冲液，如枸橼酸缓冲液、磷酸钠或磷酸钾缓冲液或其他本技术熟练人员公知的缓冲液，在一种实施方式中，缓冲液的pH为中性。在本领域公知的标准条件下进行对所述溶液进行随后的过滤除菌，然后冻干制得理想的制剂。在一种实施方式中，将所得的溶剂分装至小管中冻干。每支小管可容纳单次剂量或多次剂量的所述抗OX40抗体或其抗原结合片段或其组合物。每支小管中的装入量可略微高于每次剂量所需或多次剂量所需(例如10％过量)，从而保证取样精确和给药精确。冻干粉可在适当的条件下储存，如在约4℃到室温范围。
用注射用水将冻干粉重溶得到用于注射给药的制剂。在一种实施方式中，可将冻干粉加至无菌无热原水或其他适用的液体载剂中重溶。精确的量由选择的疗法决定，可根据经验值决定。
还提供了治疗方法，包括将治疗有效量的本申请所述的融合蛋白施用给需要其的受试者。
本申请中提供的融合蛋白的治疗有效剂量依赖于本领域公知的多种因素，例如体重、年龄、过往病史、现用治疗、对象的健康状况和交叉感染的潜力、过敏、超敏和副作用，以及给药途径和肿瘤发展的程度。本领域熟练人员(例如医生或兽医)可根据这些或其它条件或要求按比例降低或升高剂量。
在某些实施方式中，本发明提供的融合蛋白可在治疗有效剂量约0.0lmg/kg到约100mg/kg之间给药。在某些实施方式中，所述融合蛋白以约50mg/kg或更少的剂量给药，在某些实施方式中，给药剂量为10mg/kg或更少、5mg/kg或更少、1mg/kg或更少、0.5mg/kg或更少或0.1mg/kg或更少。某特定剂量可在多个间隔给药，例如每天一次、每天两次或更多、每月两次或更多、每周一次、每两周一次、每三周一次、每月一次或每两月或更多月一次。在某些实施方式中，给药剂量可随治疗进程变化。例如，在某些实施方式中，初始给药剂量可比后续给药剂量高。在某些实施方式中，给药剂量在治疗进程中根据给药对象的反应进行调整。
给药方案可通过调整达到最优反应(如治疗反应)。例如，可进行单剂量给药或在二段时间分多个分隔的剂量给药。
本发明中公开的融合蛋白可通过本领域公知的给药方式给药，例如注射给药(如，皮下注射、腹腔注射、静脉注射，包括静脉滴注，肌肉注射或皮内注射)或非注射给药(如，口服给药、鼻腔给药、舌下给药、直肠给药或 外用给药)。
在某些实施方式中，所述融合蛋白可用于治疗与其分子机制相关的病症，包括肿瘤和癌症，例如非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、草样肉芽肿(mycoses fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。在某些实施方式中，所述融合蛋白治疗的病症包括慢性病毒感染、例如乙型肝炎、丙型肝炎、疱疹病毒、Epstein-Barr病毒、艾滋病毒、巨细胞病毒、单纯疱疹病毒I型、单纯疱疹病毒2型、人乳头状瘤病毒、腺病毒的病毒感染、卡波西肉瘤相关的疱疹病毒流行病、薄环病毒(Torquetenovirus)、JC病毒或BK病毒等。
使用方法
本申请进一步提供了使用所述融合蛋白的方法。
在一些实施方式中，本申请提供了在个体中治疗与所述融合蛋白机制相关的状况或病症的方法，包括施用治疗有效量的本申请所述的融合蛋白。
本发明公开的融合蛋白可单独给药或与一种或多种其他治疗手段或物质联合给药。例如，本发明公开的融合蛋白可与化疗、放疗、癌症治疗手术(如肿瘤切除术)、抗病毒药物、一种或多种抗呕吐药或其他化疗导致的并发症的疗法、或任何其他用于癌症或病毒的治疗物质进行联用。在某些这样的实施方式中，本发明公开的融合蛋白与一种或多种治疗物质联用时，可与所述的一种或多种治疗物质同时给药，在某些这样的实施方式中，所述的融合蛋白可作为同一个药物组合物的一部分同时给药。但是，与其他治疗物质"联用"的融合蛋白不需要同时给药或与该治疗物质在同一组合物中给药。本发明中"联用"的含义还包括在另一个治疗物质之前或之后给药的融合蛋白也被认为是与该治疗物质"联用"，即使所述融合蛋白与第二种物质通过不同给药方式给药。在可能的情况下，与本发明公开的融合蛋白联用的其他治疗物质可参照该其他治疗物质的产品说明书的方法用药，或参照、外科医生的案头参考书2003(Physicians'Desk Reference,57th Ed；Medical Economics Company；ISBN：1563634457；第57版(2002年11月))，或参照 其他本领域公知的方法。
在某些实施方式中，所述治疗物质能够诱导或增强针对癌症的免疫反应。例如，肿瘤疫苗可以用于诱导对某些肿瘤或癌症的免疫应答。细胞因子治疗可以用于提高将肿瘤抗原向免疫系统的递呈。细胞因子治疗的示例包括但不限于干扰素如干扰素α、β和γ，集落剌激因子如巨噬细胞CSF、粒细胞巨噬细胞CSF和粒细胞CSF，白介素如1L-L、1L-1a、1L-2、1L 3、1L-4、1L-5、1L-6、1L-7，1L-8、1L-9、1L-10、1L-ll和1L-12，肿瘤坏死因子如TNF-α和TNF-β。还可以使用灭活免疫抑制目标的试剂，如PD-L1/PD-1抗体、TGF-β抑制剂、IL-10抑制剂和Fas配体抑制剂。另一组试剂包括激活针对肿瘤或癌细胞的免疫响应的那些试剂，例如，提高T细胞激活(如T细胞共剌激分子激动剂如CTLA-4、ICOS)的那些，以及提高树突细胞功能和抗原递呈的那些。
以下实施例旨在更好地说明本发明，且不应理解为限制本发明的范围。所有下述的特定组合物、材料和方法，其整体或部分，都在本发明的范围内。这些特定的组合物、材料和方法不是为了限制本发明，而只是为说明特定的实施方式在本发明的范围内。本领域熟练技术人员可不添加创造性及不偏离本发明范围而开发出等同的组合物、材料和方法。应理解，在对本发明的方法作出的多种改动可以仍然包括在本发明范围内。发明人意在将这样的变动包括在本发明的范围内。
实施例1：本发明的融合蛋白的制备
本实施例示例性的说明了几种抗OX40抗体-人干扰素融合蛋白的设计及表达。其中抗人OX40激活性抗体的重链和轻链序列来自于中国专利201711476160.3中的MT01-C1或MT01-C1(G2)，其中，"MT01-C1"是指与UMY02-L1、UMY02-L2及UMY02-L3具有相同VH(SEQ ID NO:7)和VL(SEQ ID NO:8)序列，且重轻链恒定区分别为人IgG1和κ链的单克隆抗体。"MT01-C1(G2)"是指与UMY02-L1、UMY02-L2及UMY02-L3具有相同VH(SEQ ID NO:7)和VL(SEQ ID NO:8)序列，且重轻链恒定区分别为人IgG2和κ链的单克隆抗体。
干扰素IFNα2b序列取自人干扰素IFNα2b(NP_000596.2)，其氨基酸序列如SEQ ID NO:9所示。
示例性的融合蛋白设计结构如图1所示。其中，"UMY02-L1"是指具有如SEQ ID NO:10所示的重链、肽接头和人干扰素，如SEQ ID NO:11所示的轻链，其中在重链羧基端通过肽接头连接了如SEQ ID NO:9所示的人干扰素IFNα2b的融合蛋白。"UMY02-L2"是指具有如SEQ ID NO:12所示的重 链、如SEQ ID NO:13所示的轻链、肽接头和人干扰素，其中在轻链羧基端通过肽接头连接了如SEQ ID NO:9所示的人干扰素IFNα2b的融合蛋白。"UMY02-L3"是指具有如SEQ ID NO:14所示的重链、如SEQ ID NO:13所示的轻链、肽接头和人干扰素，其中在轻链羧基端通过肽接头连接了如SEQ ID NO:9所示的人干扰素IFNα2b的融合蛋白。
将编码融合蛋白的抗体重链和轻链的cDNA序列分别克隆到哺乳动物细胞表达载体pcDNA3.4上。将重链表达质粒和轻链表达质粒按2:1的摩尔比用Lipofectamine 2000转染试剂(Invitrogen)转染入HEK293细胞，并在37℃、5％二氧化碳条件下培养7天。收集培养液上清，并用Protein A亲和层析法提纯上清中的抗体。纯化后的抗体经PBS溶液透析和冷冻干燥浓缩后，保存于-20℃。
实施例2：ELISA结合实验
将浓度为1μg/mL的人OX40蛋白溶液以100μL/孔包被96孔高亲和力板，4℃，振荡过夜。第二天先以300μL PBST(Tween20：0.5‰)洗涤3次，之后用100μL/孔的5％BSA/PBS封闭2小时，室温振荡。300μL PBST洗涤3次。用PBS配制融合蛋白样品的梯度稀释溶液。以100μL/孔加入96孔板，室温振荡1小时。300μL PBST洗涤3次。配制二抗羊抗人IgG HRP溶液，以100μL/孔加入96孔板，室温振荡1小时。300μLPBST洗涤4次。加入100μL/孔TMB，显色20min。加入100μL/孔0.6N H 2SO 4，终止显色，检测OD450nm。
经检测，结果如图2所示，融合蛋白UMY02-L1、UMY02-L2和UMY02-L3的ELISA结合EC50分别为0.7626、0.3948和0.3177μg/mL，与OX40抗体MT01-C1的EC50(0.2977μg/mL)相当(图2)。
实施例3：与人、食蟹猴和小鼠OX40的结合(FACS)
用编码了人、食蟹猴或是小鼠OX40蛋白的表达质粒转染CHO细胞后培养48小时。用PBS配制OX40抗体MT01-L1浓度梯度溶液，配制成终浓度的10×工作液。收集CHO-hOX40细胞，PBS洗涤一遍后计数，稀释成2*10 6/ml细胞悬液；分别取10μL OX40抗体MT01-L1工作液加入100μL细胞悬液中，4℃避光孵育30min；PBS洗涤2次后，加入二抗，4℃避光孵育30min，PBS洗涤一次后，以400μL FACS缓冲液悬起，上机检测。如图3所示，结果显示MT01-L1与人OX40(hsOX40)有结合，其EC50为0.66μg/mL。
类似的，本申请发明人检测了抗体与表达了小鼠OX40的CHO细胞的结合，发现MT01-L1与小鼠OX40(msOX40)没有结合。
类似的，本申请发明人检测了抗体与表达了食蟹猴OX40的CHO细胞 的结合，发现MT01-L1与食蟹猴OX40(cyOX40)有明显结合。
实施例4：Daudi细胞增殖抑制实验
Daudi细胞(ATCC)上高表达干扰素受体，因此干扰素对其有生物学活性。将Daudi细胞以20000细胞/90μL/孔铺96孔板，待检测样品配制成浓度梯度稀释的10×工作液，分别以10μL/孔加入96孔板，置于37℃孵箱，72小时后加CCK8检测OD450并计算各孔细胞的增殖抑制率。该抑制率反映了样品中干扰素的活性。实验结果显示融合蛋白UMY02-L1、UMY02-L2和UMY02-L3对Daudi细胞的增殖抑制活性(IC50)分别为9.549、9.152和27.44pM(图4)。
实施例5：FcR介导的OX40信号通路激活实验
本申请发明人构建了检测OX40激活剂的细胞实验体系。具体来说，本申请发明人构建了“Jurkat-OX40-NFκB-萤光素酶报告基因”稳转细胞株，当把OX40激活性抗体与该稳转细胞株及过表达FcR的HEK293细胞混合后，可以激活NFκB-luciferase报告基因的表达。
用PBS配置融合蛋白浓度梯度溶液，配制成终浓度的2×工作液，冰上操作。收集Jurkat-NFkB-luc-OX40细胞和过表达FcR的HEK293细胞，离心后重悬于培养基中，并铺入384孔板。于384孔板中，加入融合蛋白工作液和适量细胞悬液，孵育5小时后，加入One-Glo(Promega)检测试剂，混匀后用Pherastar自动聚焦荧光发光仪酶标仪检测荧光信号。
如图5所示，经检测，OX40抗体MT01-C1、融合蛋白UMY02-L1、UMY02-L2和UMY02-L3在上述实验体系中激活NFκB-荧光素酶报告基因的EC50分别为0.04523、0.02437、0.02837和0.02907ng/mL。结果表明，FcR介导的OX40激活活性UMY02-L1、UMY02-L2和UMY02-L3强于MT01-C1。
实施例6：干扰素受体介导的OX40信号通路激活实验
把“Jurkat-OX40-NFκB-萤光素酶报告基因”稳转细胞株按10000个/孔铺入384孔板。用PBS配置融合蛋白浓度梯度溶液或对照组溶液，配制成终浓度的2×工作液，冰上操作。于384孔板中，加入融合蛋白或干扰素工作液和适量细胞悬液，孵育5小时后，加入One-Glo(Promega)检测试剂，混匀后用Pherastar自动聚焦荧光发光仪酶标仪检测荧光信号。
如图6a所示，试验中所有抗体或干扰素的终浓度都为10nM。OX40抗体MT01-C1或MT01-C1(G2)对Jurkat细胞的OX40信号通路没有激活作用，单独加入干扰素IFNα2b或同时加入OX40抗体MT01-C1和IFNα2b与 对照组相比也仅有不足40％的激活作用。但是，当加入融合蛋白UMY02-L1、UMY02-L2或UMY02-L3后，Jurkat细胞的OX40信号通路被显著激活，且激活程度明显大于单独的干扰素组(图6b)。
实施例7：C57BL/6小鼠药代动力学实验
18只6-8周龄雌性C57BL/6小鼠，分为3组，每组6只，分别静脉注射给予UMY02-L1、UMY02-L3、MT01-C1。给药剂量为5mg/kg。对于UMY02-L1、UMY02-L3，采集给药前，和给药后的1、2、6、24、48、72、96、174、220、288小时动物外周静脉血；对于MT01-C1，采集给药前，和给药后的1、2、6、24、48、72、96、192、312小时动物外周静脉血；离心收集血清。每个时间点采集3只动物血清，每组6只动物不同时间点轮替采集血样。
浓度为1μg/mL的人OX40蛋白溶液以100μL/孔包被96孔高亲和力板，4℃，振荡过夜。第二天先以300μL PBST(Tween20：0.5‰)洗涤3次，之后用100μL/孔的5％BSA/PBS封闭1小时，室温振荡。300μL PBST洗涤4次。用PBS配制待测血清样品、以及不同浓度对照品血清溶液的100倍稀释溶液。以100μL/孔加入96孔板，室温振荡1.5小时。300μL PBST洗涤4次。对于MT01-C1，配制二抗驴抗人IgG HRP(Jackson ImmunoResearch，货号709-035-149)溶液，以100μL/孔加入96孔板，室温振荡1小时。300μLPBST洗涤4次。加入100μL/孔TMB，显色20min。加入100μL/孔0.6N H 2SO 4，终止显色，检测OD450nm。对于UMY02-L1、UMY02-L3，配制0.5μg/mL的兔抗人IFN(abcam，货号ab222552)溶液，以100μL/孔加入96孔板，室温振荡1.5小时。300μLPBST洗涤4次。配制二抗羊抗兔IgG HRP(金斯瑞生物科技，货号A00098)溶液，以100μL/孔加入96孔板，室温振荡1小时。300μLPBST洗涤4次。加入100μL/孔TMB，显色20min。加入100μL/孔0.6N H 2SO 4，终止显色，检测OD450nm。不同浓度的对照品溶液检测值与对照品浓度进行Logistic四参数拟合，得到标准曲线回归方程。将待测样品的检测值代入方程计算，得到不同时间点的血清药物浓度。
如图7a，7b所示，试验中小鼠静脉注射5mg/kg的UMY02-L3、UMY02-L1，给药7天后，UMY02-L3血清药物浓度仍然在10μg/mL以上，给药12天后，UMY02-L1血清药物浓度仍然在10μg/mL以上。表明融合蛋白UMY02-L3、UMY02-L1在小鼠体内有着与抗体药物MT01-C1类似的药代动力学特性，较IFNα2b的2-3小时的半衰期大大延长(参见Merck公司产品Intron A的产品说明书)。
实施例8：抗体亚型和肽接头的长度对抗体融合蛋白活性的影响
为了研究链接OX40激活性抗体和干扰素IFNα2b的肽接头长度和抗体亚型对融合蛋白的OX40信号通路激活活性的影响，我们在UMY02-L3的基础上，构建了含不同长度肽接头及不同抗体亚型的抗体融合蛋白(见表1)。
其中，融合蛋白UMY02-L4、UMY02-L5和UMY02-L6的重链如SEQ ID NO:14所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，区别仅在于肽接头的数目，如下表1所示。
其中，融合蛋白UMY02-L7和UMY02-L8的重链如SEQ ID NO:15所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，区别仅在于肽接头的数目，如下表1所示。
按照实施例4的方法，检测这些抗体融合蛋白对Daudi细胞增殖的影响，结果发现，不同肽接头长度对抗体融合蛋白的干扰素活性有较明显影响，肽接头长度越短，干扰素活性越低(见表1)。
用实施例5中的方法检测这些融合蛋白对OX40信号通路的激活活性。如表1所示，经检测，OX40抗体MT01-C1、和各个抗体融合蛋白均可有效激活Jurkat-OX40细胞的OX40信号通路。
表1 抗体亚型和linker长度对干扰素活性的影响
Figure PCTCN2019104217-appb-000002
实施例9：OX40抗体与不同IFNα2b突变蛋白融合
OX40激活性抗体还可以与干扰素突变体融合。这些突变的干扰素与野生型干扰素相比，比活更低，因此可以与OX40抗体在同等药物浓度时，共同协同发挥作用，同时避免因干扰素活性过高而造成的毒副作用。
在本实施例中，我们构建并用HEK293表达体系表达了一系列OX40抗体-突变干扰素的融合蛋白(表2)。在这些抗体融合蛋白中，OX40的抗体亚型为IgG4，同时IFNα2b部分都带有T106A突变以去除其糖基化位点。
其中，融合蛋白UMY02-L13、UMY02-L14、UMY02-L15、UMY02-L16、UMY02-L17和UMY02-L18的重链如SEQ ID NO:15所示，轻链和人干扰素如SEQ ID NO:13中的轻链和人干扰素所示，区别仅在于：
1.UMY02-L13、UMY02-L14、UMY02-L15、UMY02-L16、和UMY02-L18的肽接头为(GGGGS)2，UMY02-L17的肽接头为(GGGGS)3；
2.融合蛋白UMY02-L13、UMY02-L14、UMY02-L15、UMY02-L16、UMY02-L17和UMY02-L18的干扰素在如SEQ ID NO:9的基础上如表2突变。
按照实施例4的方法，检测这些抗体融合蛋白对Daudi细胞增殖的影响，结果发现，突变的抗体融合蛋白的干扰素活性较野生型IFNα2b有不同程度的显著下降(表2)。同时按照实施例5的方法检测这些抗体融合蛋白对Jurkat细胞OX40信号通路的激活作用，结果显示这些突变的抗体融合蛋白的OX40 激活活性基本不变(表2)。这一结果说明，通过在干扰素序列中引入特定的突变，可以获得合适的OX40抗体-IFN融合蛋白，并保证OX40激活活性和干扰素活性在同等摩尔浓度下相匹配，在人体内协同发挥两者的功能，避免由于其中之一的活性过高或过低而造成治疗效果不佳或严重毒副作用。
表2 突变的抗体融合蛋白的活性
Figure PCTCN2019104217-appb-000003
1.突变的氨基酸位置序号参照野生型人干扰素IFNα2b的氨基酸序列SEQ ID NO:9标示。
2.将IFNα2b的活性定义为1。
3.将OX40单抗MT01-C1的活性定义为1。
实施例10融合蛋白对肿瘤细胞的杀伤作用
进一步的，发明人将OX40抗体的重链与不同类型干扰素相融合，制备了不同的融合蛋白，其中OX40IFN-α2a，OX40-IFNβ，OX40-IFNγ，OX40-IFNλ3的重链如SEQ ID NO:15所示；OX40IFN-α2a轻链、肽接头和干扰素如SEQ ID NO:16所示；OX40IFN-β轻链、肽接头和干扰素如SEQ ID NO:17所示；OX40IFN-γ轻链、肽接头和干扰素如SEQ ID NO:18所示；OX40IFN-λ3轻链、肽接头和干扰素如SEQ ID NO:19所示。
并检测了这些抗体融合蛋白对肿瘤细胞的杀伤作用。具体来说，发明人将人来源PBMCs与卵巢癌SKOV3细胞按照20:1的比例混合共培养，分别给予PBS(对照)，或0.1nM浓度的OX40单抗MT01-C1以及不同抗体融合蛋白(OX40IFN-α2a，OX40-IFNβ，OX40-IFNγ，OX40-IFNλ3)刺激，在37℃，5％CO 2条件下培养48h后，使用CCK8试剂盒检测PBMC对SKOV3细胞的细胞毒作用，实验结果如图8所示，表明在无刺激情况下PBMC对SKOV3细胞增殖无抑制作用；OX40单抗刺激时，PBMC对SKOV3的抑制率约为28％；而在融合蛋白的刺激下，PBMC的细胞毒作用均强于OX40单抗刺激，其中OX40单抗MT01-C1-IFNα2a和OX40单抗MT01-C1-FNβ有显著增强，抑制率分别为60％和63％。
上述仅为本发明的优选实施例而已，并不对本发明起到任何限制作用。任何所属技术领域的技术人员，在不脱离本发明的技术方案的范围内，对本发明揭露的技术方案和技术内容做任何形式的等同替换或修改等变动，均属未脱离本发明的技术方案的内容，仍属于本发明的保护范围之内。
主要参考文献：
1.Willoughby J,Griffiths J,Tews I,Cragg MS.OX40:Structure and function-What questions remain？Mol Immunol.2017；83:13-22.doi:10.1016/j.molimm.2017.01.006.
2.Imura A,Hori T,Imada K,Ishikawa T,Tanaka Y,Maeda M,Imamura S,Uchiyama T.The human OX40/gp34 system directly mediates adhesion of activated T cells to vascular endothelial cells.J Exp Med.1996；183:2185-95.doi:
3.Burgess JK,Carlin S,Pack RA,Arndt GM,Au WW,Johnson PR,Black JL,Hunt NH.Detection and characterization of OX40 ligand expression in human airway smooth muscle cells:a possible role in asthma？J Allergy Clin Immunol.2004；113:683-9.doi:10.1016/j.jaci.2003.12.311.
4.Compaan DM,Hymowitz SG.The crystal structure of the costimulatory OX40-OX40L complex.Structure.2006；14:1321-30.doi:10.1016/j.str.2006.06.015.
5.Song J,So T,Croft M.Activation of NF-kappaB1 by OX40 contributes to antigen-driven T cell expansion and survival.J Immunol.2008；180:7240-8.doi:
6.Croft M.Control of immunity by the TNFR-related molecule OX40(CD134).Annu Rev Immunol.2010；28:57-78.doi:10.1146/annurev-immunol-030409-101243.
7.Rogers PR,Song J,Gramaglia I,Killeen N,Croft M.OX40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells.Immunity.2001；15:445-55.doi:
8.Song J,So T,Cheng M,Tang X,Croft M.Sustained survivin expression from OX40 costimulatory signals drives T cell clonal expansion.Immunity.2005；22:621-31.doi:10.1016/j.immuni.2005.03.012.
9.So T,Song J,Sugie K,Altman A,Croft M.Signals from OX40 regulate nuclear factor of activated T cells c1 and T cell helper 2 lineage commitment. Proc Natl Acad Sci U S A.2006；103:3740-5.doi:10.1073/pnas.0600205103.
10.Publicover J,Gaggar A,Jespersen JM,Halac U,Johnson AJ,Goodsell A,Avanesyan L,Nishimura SL,Holdorf M,Mansfield KG,Judge JB,Koshti A,Croft M,et al.An OX40/OX40L interaction directs successful immunity to hepatitis B virus.Sci Transl Med.2018；10.doi:10.1126/scitranslmed.aah5766.

Claims (16)

  1. 一种融合蛋白，其包含：
    a)特异性结合人OX40的抗体或其抗原结合片段；和
    b)人干扰素；
    其中所述人干扰素直接或通过肽接头连接至所述抗体的轻链或重链的羧基端或氨基端。
  2. 根据权利要求1所述的融合蛋白，其中所述特异性结合人OX40的抗体或其抗原结合片段包含：
    抗体重链可变区，该重链可变区包含具有SEQ ID NO:1的氨基酸序列的VH CDR1、具有SEQ ID NO:2的氨基酸序列的VH CDR2、和具有SEQ ID NO:3的氨基酸序列的VH CDR3；和
    抗体轻链可变区，该轻链可变区包含具有SEQ ID NO:4的氨基酸序列的VL CDR1、具有SEQ ID NO:5的氨基酸序列的VL CDR2、和具有SEQ ID NO:6的氨基酸序列的VL CDR3。
  3. 根据权利要求2所述的融合蛋白，其中所述重链可变区包含SEQ ID NO:7所示的氨基酸序列，所述轻链可变区包含SEQ ID NO:8所示的氨基酸序列。
  4. 根据权利要求1-3任一项所述的融合蛋白，其中所述特异性结合人OX40的抗体或其抗原结合片段是骆驼化单域抗体、scFv、scFv二聚体、BsFv、dsFv、dsFv2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab')2、ds双功能抗体、纳米抗体、域抗体或双价域抗体。
  5. 根据权利要求1-4任一项所述的融合蛋白，其中所述抗体进一步包含免疫球蛋白的恒定区；优选地，所述恒定区为人IgG1、IgG2或IgG4的恒定区。
  6. 根据权利要求1-5任一项所述的融合蛋白，其中所述人干扰素选自I型人干扰素、II型人干扰素和III型人干扰素；
    优选地，所述人干扰素为IFNα2a、IFNβ、IFNγ、IFNλ3、IFNα2b；
    更优选地，所述人干扰素为IFNα2b，其氨基酸序列如SEQ ID NO:9所示；
    进一步优选地，所述人干扰素为IFNα2b的突变体，其在SEQ ID NO:9所示的氨基酸序列上，具有选自以下的一种或多种突变：
    T106A、R149A、A145G、A145D、R120A、L117A；
    更进一步优选地，所述IFNα2b的突变体在SEQ ID NO:9所示的氨基酸序列上具有选自以下的双突变：
    T106A/A145D、T106A/R149A、T106A/A145G、T106A/R120A、T106A/L117A。
  7. 根据权利要求1-7任一项所述的融合蛋白，其中所述肽接头选自(G)n、KESGSVSSEQLAQFRSLD、EGKSSGSGSESKST、GSAGSAAGSGEF、(GGGGS)n、(GGSGG)n；优选地，所述肽接头为(GGGGS)n，其中n为0-5之间的整数；优选地，n为1-3之间的整数。
  8. 根据权利要求1-7任一项所述的融合蛋白，其中所述所述融合蛋白选自：
    UMY02-L1、UMY02-L2、UMY02-L3、UMY02-L4、UMY02-L5、UMY02-L6、UMY02-L7、UMY02-L8、UMY02-L13、UMY02-L14、UMY02-L15、UMY02-L16、UMY02-L17、UMY02-L18、OX40IFN-α2a、OX40-IFNβ、OX40-IFNγ、OX40-IFNλ3中的一种或多种。
  9. 一种分离的多核苷酸，其编码根据权利要求1-8中任一项所述的融合蛋白。
  10. 一种载体，其包括根据权利要求9所述的分离的多核苷酸。
  11. 一种宿主细胞，其包括根据权利要求10所述的载体。
  12. 一种表达根据权利要求1-8任一项所述的融合蛋白的方法，其包括在能够表达根据权利要求9所述的分离的多核苷酸的条件下培养根据权利要求11所述的宿主细胞。
  13. 一种试剂盒，其包括根据权利要求1-8中任一项所述的融合蛋白。
  14. 一种药物组合物，其包括根据权利要求1-8中任一项所述的融合蛋白以及药学上可接受的载体。
  15. 根据权利要求1-8中任一项所述的融合蛋白在制备药物中的用途，所述药物用于治疗能通过增强免疫应答和/或能通过暴露于干扰素而受益的病况；优选地，其中所述病况是癌症或病毒感染；更优选地，所述病毒感染为乙肝病毒感染。
  16. 一种治疗能通过增强免疫应答和/或能通过暴露于干扰素而受益的病况的方法，所述方法包括给予有需要的受试者治疗有效量的如权利要求1-8中任一项所述的融合蛋白或如权利要求14所述的药物组合物；优选地，其中所述病况是癌症或病毒感染；更优选地，其中所述病毒感染为乙肝病毒感染。
PCT/CN2019/104217 2018-09-04 2019-09-03 融合蛋白及其在制备用于治疗肿瘤和/或病毒感染的药物中的应用 WO2020048454A1 (zh)

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WO2024067785A1 (zh) * 2022-09-29 2024-04-04 中国科学院生物物理研究所 基于干扰素协同免疫检查点阻断抗体治疗慢性乙型肝炎

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