WO2019096026A1 - 抗il-17抗体/tnfr ecd融合蛋白及其用途 - Google Patents

抗il-17抗体/tnfr ecd融合蛋白及其用途 Download PDF

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WO2019096026A1
WO2019096026A1 PCT/CN2018/114079 CN2018114079W WO2019096026A1 WO 2019096026 A1 WO2019096026 A1 WO 2019096026A1 CN 2018114079 W CN2018114079 W CN 2018114079W WO 2019096026 A1 WO2019096026 A1 WO 2019096026A1
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fusion protein
seq
antibody
subunit
sequence
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French (fr)
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胡品良
邹敬
洪伟东
何芸
白洁
宋凌云
杨文第
吕志坚
向少云
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北京比洋生物技术有限公司
杭州多域生物技术有限公司
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Priority to US16/764,788 priority Critical patent/US11667704B2/en
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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Definitions

  • the invention relates to the field of medical biotechnology. Specifically, the present invention relates to a fusion protein of an interleukin-17 (IL-17) antibody and a tumor necrosis factor receptor extracellular domain (TNFR ECD), encoding the fusion A polynucleotide of a protein, a vector comprising the polynucleotide, a host cell comprising the polynucleotide or vector, and the use of the fusion protein in treating, preventing and/or diagnosing a disease in an individual.
  • IL-17 interleukin-17
  • TNFR ECD tumor necrosis factor receptor extracellular domain
  • TNF tumor necrosis factor
  • TNF- ⁇ acts as an immunoregulatory cytokine, it is essential for the prevention of infectious diseases and cancerous diseases.
  • IMID Immune-mediated inflammatory diseases
  • RA rheumatoid arthritis
  • IBD inflammatory bowel
  • PsA psoriatic arthritis
  • vasculitis ankylosing spondylitis
  • JCA juvenile chronic arthritis
  • TNF activates signal transduction through its two receptors, TNFR1 and TNFR2.
  • TNFR1 and TNFR2 activate signal transduction pathways in different cells.
  • Type I TNF-R also known as TNFR1, CD120a, p55
  • TNFR1, CD120a, p55 has 439 amino acid residues, a molecular weight of 55kDa, and its corresponding mRNA is 4.5Kbp, which can be expressed on all types of cells, mainly in cytolytic activity. effect.
  • Type II TNFR also known as TNFR2, CD120b, p75
  • TNFR2 has 426 amino acid residues with a molecular weight of 75 kDa, and its corresponding mRNA is 3 Kbp, which is only expressed on immune and endothelial cells, and is involved in signal transduction and T cell proliferation.
  • Both types of TNFR are glycoproteins, which include three parts: the extracellular region, the transmembrane region, and the intracellular region.
  • the extracellular regions of type I and type II TNFR have 28% homology but no homology in the intracellular region.
  • the structural feature of the TNFR receptor family is that the extracellular domain is composed of four functional domains.
  • Each functional domain contains six cysteine CRDs, also known as PLAD, which is a complex of TNFR.
  • CRD2 and CRD3 are the binding regions of TNF, which bind with high affinity to TNF-like factors, and the CRD4 region and other non-functional extracellular domains are not involved in the binding of TNF-like factors (Mukai Y et al.: Solution of the structure of the TNF-TNFR2 complex. Sci Signal. 2010 Nov 16; 3(148): ra83).
  • the equilibrium dissociation constant (Kd) of TNFR1 and TNF- ⁇ is only 0.2-0.5 nM, and the equilibrium dissociation constant (Kd) of TNFR2 and TNF- ⁇ is 0.03-0.07 nM. It can be seen that the affinity of TNFR2 to TNF- ⁇ is More than ten times that of TNFR1 (Dembic Z et al: Two human TNF receptors have similar extracellular, but distinct intracellular, domain sequences. Cytokine. 1990 Jul; 2(4): 231-7.).
  • sTNFR soluble TNF receptor
  • Anti-TNF drugs have been successfully used in many autoimmune diseases such as RA, AS, PsA and Behcet's disease.
  • infliximab infliximab
  • adalimumab adalimumab
  • etanercept etanercept
  • certolizumab pegol and golimumab have been approved for clinical treatment.
  • Infliximab is an anti-TNF- ⁇ human-mouse chimeric monoclonal antibody consisting of a murine variable region and a human constant region IgG1 (kappa). It has high affinity and specificity for both soluble and transmembrane TNF- ⁇ . Infliximab binds to transmembrane TNF- ⁇ (mTNF- ⁇ ) and mediates programmed cell death with high specificity, thus reducing non-specific effects on other biological pathways. Infliximab can be used for the treatment of rheumatoid arthritis, vasculitis, ankylosing spondylitis, psoriatic arthritis, ulcerative colitis and chronic severe plaque psoriasis.
  • Etanercept (commercial generic product under the trade name "Yisep”) is a combination of the full-length extracellular domain of two human TNFR2 (p75TNF receptor) and the Fc fragment (CH2 and CH3 domain) of human IgG1. And inhibition of TNF- ⁇ can effectively reduce the amount of TNF- ⁇ bound to membrane receptors. By competitive inhibition, the arms of the two TNF-R2s bind to two of the three receptor binding sites on the TNF-[alpha] trimer. Binding of TNF- ⁇ to cell surface receptors is prevented (Spencer-Green G et al., Etanercept (Enbrel): update on therapeutic use. Ann Rheum Dis.
  • Etanercept can be used for the treatment of rheumatoid arthritis, psoriatic arthritis, vasculitis, ankylosing spondylitis and juvenile chronic arthritis.
  • Adalimumab is a monoclonal antibody raised against human TNF- ⁇ . It binds to soluble TNF- ⁇ and transmembrane TNF- ⁇ and blocks the binding of TNF- ⁇ to its receptor. In vitro studies have demonstrated that it binds to transmembrane TNF- ⁇ and has an effect on cell lysis and apoptosis induction (Shen C, Assche GV, Colpaert S, Maerten P, Geboes K, Rutgeerts P, Ceuppens JL.
  • Adalimumab induces apoptosis of human monocytes :a comparative study with infliximab and etanercept.Aliment Pharmacol Ther 2005Feb 1;21(3):251–258).
  • Adalimumab can be used for the treatment of rheumatoid arthritis, psoriatic arthritis, vasculitis, ankylosing spondylitis and Behcet's disease.
  • Golimumab Chinese is called Golimumab, a monoclonal antibody against human TNF- ⁇ , which can be used for the treatment of rheumatoid arthritis, vasculitis, ankylosing spondylitis, psoriatic arthritis and ulcerative colitis. .
  • Certolizumab Chinese name is certolizumab, a Fab fragment of anti-TNF- ⁇ IgG1 monoclonal antibody, no Fc fragment, hinge region is covalently linked with two cross-linked 20kD PEG; can be used for Certolizumab rheumatoid arthritis The treatment of psoriatic arthritis.
  • Interleukin-17 is a newly discovered inflammatory cytokine with many biological activities and is one of the important factors for the occurrence and development of certain diseases.
  • IL-17 has powerful recruitment of granulocytes and promotes the release of various inflammatory cytokines. It participates in the development and development of various inflammatory and immune diseases in the body, especially with rheumatoid arthritis, asthma, and lungs. Infections, tumors, contact dermatitis and other diseases are closely related.
  • the IL-17 receptor (IL-17R) is widely present in various tissues and cells and binds to IL-17 to produce a pro-inflammatory response. The research of IL-17 family and its receptors has gradually become a hot spot in medical and molecular biology research.
  • IL-17 was cloned for the first time in 1995 (Yao Z et al, Herpesvirus saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor. Immunity. 1995, 3(6): 811-821.). It is now known that at least six IL-17 family members (IL-17A-F) are present in humans and mice. IL-17A was originally named Cytotoxic T Lymphocytes Antigen 8, CTLA-8. Five other hIL-17 family members (Moseleyl TA, et al, InterIeukin-17 family and IL-17 receptors [J]. Cytokine & Growth Factor Reviews.
  • IL-17B IL-17C
  • IL-17D IL-17F
  • IL-17E also known as IL-25
  • the IL-17 receptor (IL-17R) family includes five members (IL-17RA, B, C, D, and E). Functional receptors for the IL-17 family of cytokines are thought to be composed of homodimers or heterodimers.
  • a heterodimer composed of IL-17RA and IL-17RC is a homodimer and a heterodimer of IL-17 and IL-17F
  • IL-17RA and IL-17RB are composed of
  • a heterodimer is a receptor for IL-17E. Both IL-17B and IL-17E bind to IL-17RB.
  • IL-17A is often referred to as IL-17, and it was first mentioned in 1993 (O'Shea JJ, et al., Mechanisms underlying lineage commitment and plasticity of helper CD4+T cells.Science.2010, 327 (5969).
  • IL-17A and IL-17F are core players in adaptive immune responses, especially against bacteria and fungi (Kolls JK, et al. Interleukin-17 family members and inflammation. Immunity. 2004, 21(4): 467-476.). Little is known about the function of IL-17B, IL-17C and IL-17D.
  • the main role of IL-17 is to induce the production of chemokines and other cytokines (such as TNF- ⁇ ), which absorb central granulocytes and monocytes at the T cell activation site.
  • IL-17 also contributes to the formation of granulocytes by increasing the production and secretion of GM-CSF (granulocyte-macrophage colony stimulating factor) and its receptors.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • IL-17 can also stimulate antibacterial proteins (AMP) of neutrophils and other cells (such as LL37) (Lin AM et al, Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis, J. Immunol. 2011 , 187(1): 490-500.). Under different physiological and disease conditions, the immunogen is prone to produce Th17 cytokines.
  • AMP antibacterial proteins
  • lymphocytes including CD8+ ⁇ T cells, ⁇ T cells (H.Takatori et al, Lymphoid tissue inducer-like cells are an innate source of IL-17 and IL-22,J). .Exp.Med.2009,206(1):35-41.), LTi-like natural lymphocytes (ILCs) (NKCrellin et al, Human Cells and LTi-like cells Lineage distinct from conventional natural killer cells, J. Exp. Med.
  • ILCs LTi-like natural lymphocytes
  • NK natural killer cells
  • M.Cella et al. A human natural killer cell subset provides an innate source Of IL-22 for mucosal immunity, Nature. 2009, 457 (7230): 722-725.).
  • IL-17 congenital myeloid immune cells produce IL-17 and localize to barrier tissues such as lung, intestine, skin, and peripheral lymph nodes, which respond quickly to pathogens and allow immediate response, but also activate and amplify adaptively.
  • barrier tissues such as lung, intestine, skin, and peripheral lymph nodes, which respond quickly to pathogens and allow immediate response, but also activate and amplify adaptively.
  • Immune response Intestinal mononuclear cells and macrophages in Crohn's disease and ulcerative colitis, neutrophils in systemic vasculitis, mast cells in psoriatic lesions, and synovial hypertrophy in rheumatoid arthritis Cells are good examples.
  • IL-17 is produced by congenital cells or by Th17 adaptive cells does not alter the central role of this cytokine in the pathology of psoriasis and psoriatic arthritis (SPRaychaudhuri et al., Role of IL- 17 in psoriasis and psoriatic arthritis, Clin. Rev. Allergy Immunol. 2013, 44 (2): 183-193.). Under these conditions, anti-IL-17 drugs are therapeutic tools.
  • the market-targeted IL-17 signaling pathways include Brodalumab, Ixekizumab, Secukinumab, ABT-122 (bispecific antibodies of IL-17 and TNF- ⁇ ), CNTO 6785, CJM112, COVA322 (IL-17). And TNF- ⁇ bifunctional antibody), ALX-0761, Bimekizumab and SCH-900117.
  • Brodalumab is an antibody to the IL-17 receptor, while ixekizumab and secukinumab are neutralizing antibodies to IL-17A, wherein secukinumab is a fully humanized monoclonal antibody and ixekizumab is a humanized monoclonal antibody.
  • Secukinumab (Cosentyx, Sukinimab), the world's first marketed IL-17A monoclonal antibody, achieved $1.128 billion in the second year of FDA approval.
  • Currently approved indications include plaque-like silver shavings Disease, psoriatic arthritis, and mandatory spondylitis are the most marketable drugs in Novartis' new drug market in recent years.
  • Cosentyx is superior to Stelara (IL-12/23 antibody) in maintaining long-term plaque clearance, and patients only need to use a dosing syringe to inject once a month.
  • Stelara IL-12/23 antibody
  • it has been approved in more than 70 countries, exceeding the global scale. More than 100,000 patients benefit and will become the new standard treatment for psoriasis.
  • Johnson's Stelara Uttrazumab, anti-IL-12/23
  • a clinical standard treatment for psoriasis hit a record high of $3.232 billion in 2016.
  • Ixekizumab was superior to Amgen's heavy anti-inflammatory drug Enbrel (Enli, generic name: etanercept, etanercept) and placebo in all skin lesion regression indicators, and the data were statistically significant.
  • Enbrel generic name: etanercept, etanercept
  • IL-17A plays an important role in driving hyperproliferation and activation of keratinocytes (skin cells).
  • Ixekizumab does not bind to the cytokines IL-17B, IL-17C, IL-17D, IL-17E or IL-17F.
  • brodalumab does not have any clinical benefit for the rheumatoid arthritis test group.
  • the efficacy of ixekizumab and secukinumab in the treatment of rheumatoid arthritis is not as good as TNF- ⁇ biologics (Kalden JR: Emerging Therapies for Rheumatoid Arthritis. Rheumatol Ther (2016) 3:31–42).
  • TNF- ⁇ biologics Korean JR: Emerging Therapies for Rheumatoid Arthritis. Rheumatol Ther (2016) 3:31–42.
  • anti-IL-17 and TNF- ⁇ bispecific antibodies are better at inhibiting the development of inflammation, bone and hoof tissue in mice than with anti-IL-17 and TNF alone.
  • - ⁇ antibody treatment Fischer JA, Hueber AJ, Wilson S et al, Combined inhibition of tumor necrosis factor a and interleukin-17 as a therapeutic opportunity in rheumatoid arthritis: development and characterization of a novel bispecific antibody. Arthritis Rheumatol.2015;67 (1): 51–62.).
  • the IL-17 inhibitor and the TNF- ⁇ inhibitor are administered simultaneously, it is necessary to separately prepare the two separate products; then, for example, the two products are separately injected, and of course, the co-formulation of the two products can be injected in a single injection.
  • the dose and timing can be flexibly selected when the injection is separately, it is inconvenient for the patient due to pain and economic reasons.
  • Co-formulations can also be used to a certain degree of flexibility, but due to the different molecular characteristics of the two different products, the discovery of the formulation conditions that allow the chemical and physical stability of the two products is quite challenging, or simply impossible. of.
  • co-administration or co-formulation involves the cumulative cost of two different drug therapies, which will increase patient and/or payer costs, while a single formulation (eg, a bispecific antibody or fusion protein) allows for optimal price and its administration. Chemical.
  • drugs for example, monoclonal antibody drugs
  • drugs for example, monoclonal antibody drugs
  • it is now often used in a "one-time production process" to achieve sterile fluid transport between pharmaceutical devices in different rooms, which can achieve different rooms and different cleanliness requirements.
  • the mutual isolation between devices effectively reduce the risk of environmental pollution caused by related equipment; at the same time save verification costs, verification time and related human and material resources.
  • a "one-time production process” system is expensive.
  • two drugs for example, an IL-17 inhibitor and a TNF- ⁇ inhibitor, are separately prepared, and the huge increase in cost is obvious. .
  • bispecific antibodies have become a trend.
  • there are many limitations in the production of bispecific antibodies For example, nearly 10-20% of the bispecific antibodies previously obtained by Genentech using Knobs-into-Holes heterodimerization technology are unwanted homodimers; the dual variable structure that Abbott relies on for a long time.
  • the dual-specific antibody produced by the domain Ig (Dual-variable domains Ig, DVD-Ig) technology has a nearly 10-fold reduction in the affinity of the internal variable domain binding antigen; and the molecular weight is large, nearly 200Kd, and interferes with each other when combined with different antigens.
  • EpimAb's FIT-Ig technology produces a bispecific antibody with a molecular weight of 250Kd, which is difficult to penetrate into hypertonic tissues; at least half of the expressed protein forms a useless Fab structure; Antibody fragments are assembled to severely affect expression levels. It can be seen that in the prior art, it is not easy to obtain the desired bispecific antibody.
  • An attempt related to the present invention is a Chinese invention patent application, the authorization number is CN 104311670 B.
  • This patent application relates to an IL-17scfv/sTNFR1 fusion protein, and since the antibody used therein is IL-17scfv, such a fusion protein has a low affinity with IL-17, and is only ten corresponding to the normal structure anti-IL-17 antibody. About one point.
  • the TNF- ⁇ inhibitor in this patent application is TNFR1, and its affinity for TNF- ⁇ is much lower than that of TNFR2 and TNF- ⁇ .
  • the IL-17scfv/sTNFR1 fusion protein in the patent application is developed into a product, it can be expected that the therapeutic effect is relatively low, which inevitably requires an increase in the dose to be used (for example, an increase of about 10 times), resulting in a prominent side effect. .
  • the present invention therefore seeks to address one or more of the above problems by obtaining a novel fusion protein of an anti-IL-17 antibody and a TNF- ⁇ receptor to improve the treatment of current autoimmune diseases.
  • the inventors encountered a series of thorny problems associated with chemical stability and physical stability, requiring many new improvements, including stabilizing the VH/VL interface of the variable region of the antibody, increasing heat. Stability, reduced aggregation, rebalancing the electrostatic distribution in the binding surface of the fusion protein, and maintaining binding affinity for both targets.
  • the inventors have fully analyzed the interaction between TNFR and TNF- ⁇ through the latest research results such as bioinformatics on the basis of anti-IL-17 antibody, and developed a group of anti-IL-17 antibodies and TNFR. Fusion protein.
  • the structure of the fusion protein sufficiently ensures a suitable physical spatial distance for binding of the fusion protein to its target.
  • the specific binding of the fusion protein having such a structure to one of its target molecules does not affect the specific binding of the fusion protein to another target, that is, the fusion protein of the present invention can have high affinity with IL-17 and TNF- ⁇ , respectively.
  • the combination while inhibiting the biological functions of IL-17 and TNF- ⁇ , has the potential to treat autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, psoriasis and ulcerative colitis.
  • the production process of the fusion protein of the present invention is convenient, simple, and low in production cost, but the affinity of the obtained fusion protein is high, and its affinity with IL-17 and TNF is respectively compared with the use of the anti-IL-17 antibody and TNFR alone.
  • the activity of IL-17 and/or TNF- ⁇ can be sufficiently inhibited. That is, the fusion protein of the present invention can be prepared to obtain a single active ingredient with low time and money cost, and is free from the formulation conditions and convenient for administration when used in the pharmaceutical field.
  • the preparation cost of the fusion protein of the invention can be reduced by at least half, and the cost of storage, transportation and the like can be correspondingly decreased; meanwhile, when applied to an individual, only need to be used. A single drug that makes administration easier.
  • the fusion protein of the present invention is more potent, and the dosage is at most about 1/10, which significantly reduces possible side effects. .
  • the present invention discloses a novel fusion protein targeting IL-17 and TNF- ⁇ , a polynucleotide encoding the fusion protein, a vector comprising the polynucleotide, a host cell comprising the polynucleotide or vector, and Use of the dual targeting fusion protein in treating, preventing, and/or diagnosing a disease associated with IL-17 and TNF-[alpha] activity in an individual.
  • the structure of the fusion protein designed by the invention fully ensures the suitable physical spatial distance of the fusion protein to bind to its target.
  • the fusion protein of this structure specifically binds to one target molecule, and does not affect the fusion protein and another target. Specific binding.
  • the invention provides a fusion protein comprising an anti-human IL-17 antibody or fragment thereof and a portion that binds to TNF- ⁇ , the fusion protein inhibits binding of IL-17 to its receptor, and inhibits TNF- ⁇ from exerting its biological function .
  • the invention provides a fusion protein comprising an anti-human IL-17 antibody or fragment thereof and a portion that binds TNF-[alpha].
  • the invention provides a fusion protein comprising an anti-human IL-17 antibody or antigen-binding fragment thereof and a portion that binds TNF-[alpha].
  • the anti-IL-17 antibody in the fusion protein of the invention may be any anti-IL-17 antibody, as long as it is capable of inhibiting or reducing the binding of IL-17 to its ligand, including in the prior art.
  • the anti-IL-17 antibody in the fusion protein of the invention further comprises a variant of an amino acid sequence of an anti-IL-17 antibody, an antibody that competes with IL-17 antibody of the invention for binding to IL-17, And an antibody that binds to the same epitope of IL-17 as any of the anti-IL-17 antibodies of the invention.
  • the portion of the fusion protein of the invention that binds to TNF-[alpha] is TNFR, which may be TNFRl or TNFR2.
  • the anti-IL-17 antibody in the fusion protein of the invention binds to any of TNFRl or TNFR2.
  • the anti-IL-17 antibody in the fusion protein of the invention binds to TNFR2 since the affinity of TNFR2 to TNF-[alpha] is 10-fold that of TNFRl.
  • the present invention relates to a fusion protein of an anti-IL-17 antibody and TNFR which inhibits the biological function of IL-17 and TNF- ⁇ , comprising (i) an antigen-binding fragment derived from an anti-IL-17 antibody; Ii) an immunoglobulin constant region domain; and (iii) a TNFR extracellular domain.
  • the anti-IL-17 antibody in the fusion protein of the invention is an anti-human IL-17 antibody.
  • the anti-IL-17 antibody in the fusion protein of the invention is an IgG class antibody, preferably the anti-IL-17 antibody is an IgG 1 subclass, an IgG 2 subclass, an IgG 4 subclass antibody.
  • the amino acid sequence of the heavy chain constant region of the IgG 1 subclass anti-IL-17 antibody is represented by SEQ ID NO: 19; the IgG 2 subclass anti-IL-17 antibody is heavy.
  • the amino acid sequence of the constant region of the chain is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain constant region of the IgG 4 subclass of anti-IL-17 antibody is set forth in SEQ ID NO:21.
  • the anti-IL-17 antibody in the fusion protein of the invention is an IgG 4 subclass antibody.
  • an amino acid substitution is included at position S228 (where S228 is the amino acid residue number of the Kabat database) in the Fc domain, which is preferably S228P.
  • the light chain of the anti-IL-17 antibody in the fusion protein of the invention is a kappa or lambda, preferably a kappa.
  • the amino acid sequence of the kappa antibody light chain constant region is represented by SEQ ID NO: 17; and the lambda light chain constant region amino acid sequence is represented by SEQ ID NO: 18. .
  • (i) of the fusion protein of the invention is an antigen-binding fragment derived from a human anti-IL-17 antibody.
  • (i) of the fusion protein of the invention is a Fab, Fab', F(ab) 2 , F(ab') 2 , Fv, single-chain Fv derived from an anti-IL-17 antibody; It is a Fab, Fab', F(ab) 2 , F(ab') 2 , Fv, single-chain Fv derived from a human anti-IL-17 antibody.
  • an antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention has the ability to bind IL-17.
  • the (i) antigen-binding fragment derived from the anti-IL-17 antibody in the fusion protein of the invention comprises a SEQ ID NO: 1/2, 3/4, 5/6, 7/ One to three of the three heavy chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequence of 8, 9/10, 11/12, 13/14 and 15/16, and One to three of the three light chain CDRs contained.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises the paired heavy chain variable region sequence of SEQ ID NO: 1/2 or 7/8 One to three of the three heavy chain CDRs contained in the /light chain variable region sequence, and one to three of the three light chain CDRs contained.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises and is selected from the group consisting of SEQ ID NO: 1/2, 3/4, 5/6, 7
  • the paired heavy chain variable region sequence/light chain variable region sequences of /8, 9/10, 11/12, 13/14 and 15/16 have at least 90%, 91%, 92%, 93%, 94%
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention is comprised in a plurality selected from the group consisting of SEQ ID NO: 1/2, 3/4, 5/6, 7
  • One or more (preferably no more than 5) of the paired heavy chain variable region sequence/light chain variable region sequences of /8, 9/10, 11/12, 13/14 and 15/16 have amino acids
  • the three, preferably the amino acid substitutions of no more than 5 amino acids are substitutions of no more than 5 conserved amino acids.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises a pair of heavy chains selected from the group consisting of SEQ ID NO: 1/2 or 7/8
  • the variable sequence/light chain variable region sequence has a pair of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises a pair of heavy chains selected from the group consisting of SEQ ID NO: 1/2 or 7/8.
  • One or more (preferably no more than 5) of the variable sequence/light chain variable region sequences have amino acid substitutions, deletions or insertions in the paired heavy chain variable region sequence/light chain variable region sequence
  • the (i) antigen-binding fragment derived from the anti-IL-17 antibody in the fusion protein of the invention comprises a SEQ ID NO: 1/2, 3/4, 5/6, 7/ Paired heavy chain variable region sequence/light chain variable region sequences of 8, 9/10, 11/12, 13/14 and 15/16.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises a variable pair of heavy chains selected from the group consisting of SEQ ID NO: 1/2 or 7/8 Sequence/light chain variable region sequences.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises and is selected from the group consisting of SEQ ID NO: 1/2, 3/4, 5/6, 7
  • the paired heavy chain variable region sequence/light chain variable region sequences of /8, 9/10, 11/12, 13/14 and 15/16 have at least 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or more sequence identity of the paired heavy chain variable region sequence/light chain variable region sequence.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention is comprised in a plurality selected from the group consisting of SEQ ID NO: 1/2, 3/4, 5/6, 7
  • One or more (preferably no more than 5) of the paired heavy chain variable region sequence/light chain variable region sequences of /8, 9/10, 11/12, 13/14 and 15/16 have amino acids
  • the paired heavy chain variable region sequence/light chain variable region sequence substituted, deleted or inserted, preferably the amino acid substitution of no more than 5 amino acids is a substitution of no more than 5 conservative amino acids.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises a pair of heavy chains selected from the group consisting of SEQ ID NO: 1/2 or 7/8
  • the variable sequence/light chain variable region sequence has a pair of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity Heavy chain variable region sequence / light chain variable region sequence.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises a pair of heavy chains selected from the group consisting of SEQ ID NO: 1/2 or 7/8.
  • One or more (preferably no more than 5) of the variable sequence/light chain variable region sequences have a pair of heavy chain variable region sequences/light chain variable region sequences with amino acid substitutions, deletions or insertions, preferably An amino acid substitution of no more than 5 amino acids is a substitution of no more than 5 conserved amino acids.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises a plurality selected from the group consisting of SEQ ID NOs: 60/62, 64/66, 68/70 and 74/ One to three of the three heavy chain CDRs contained in the paired first subunit/second subunit sequence of 72, and one to three of the three light chain CDRs contained.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises and is selected from the group consisting of SEQ ID NOs: 60/62, 64/66, 68/70 and 74
  • the paired first subunit/second subunit sequence of /72 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention is selected from the group consisting of SEQ ID NOs: 60/62, 64/66, 68/70 and 74
  • One or more (preferably no more than 5) of the paired first subunit/second subunit sequence of /72 has an amino acid substitution, deletion or insertion in the paired first subunit/second subunit sequence
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises a plurality selected from the group consisting of SEQ ID NOs: 60/62, 64/66, 68/70 and 74/ The paired heavy chain variable region sequence/light chain variable region sequence contained in the paired first subunit/second subunit sequence of 72.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention comprises and is selected from the group consisting of SEQ ID NOs: 60/62, 64/66, 68/70 and 74
  • the paired first subunit/second subunit sequence of /72 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence A pair of heavy chain variable region sequence/light chain variable region sequences contained in a pair of identical first subunit/second subunit sequences.
  • the (i) antigen-binding fragment derived from an anti-IL-17 antibody in the fusion protein of the invention is selected from the group consisting of SEQ ID NOs: 60/62, 64/66, 68/70 and 74
  • One or more (preferably no more than 5) of the paired first subunit/second subunit sequence of /72 has an amino acid substitution, deletion or insertion in the paired first subunit/second subunit sequence
  • the paired heavy chain variable region sequence/light chain variable region sequence contained, preferably the amino acid substitution of no more than 5 amino acids is a substitution of no more than 5 conservative amino acids.
  • the (ii) immunoglobulin constant region domain of the fusion protein of the invention may be the constant region domain of any immunoglobulin, in particular, (ii) is a human immunoglobulin Constant region domain.
  • the (ii) immunoglobulin constant region domain in the fusion protein of the invention may be the Fc domain of any immunoglobulin, in particular, (ii) is the Fc of a human immunoglobulin Domain.
  • the (ii) immunoglobulin heavy chain constant region domain of the fusion protein of the invention is the Fc domain of an IgG class antibody, in particular an IgG 1 subclass, an IgG 2 subclass or an IgG
  • the Fc domain of the 4 subclass antibody more particularly, the amino acid sequence of the IgG 1 subclass anti-IL-17 antibody heavy chain constant region Fc domain is set forth in SEQ ID NO: 75; the IgG 2 subclass is resistant to IL-
  • the amino acid sequence of the 17 antibody heavy chain constant region Fc domain is set forth in SEQ ID NO: 76; the amino acid sequence of the IgG 4 subclass anti-IL-17 antibody heavy chain constant region Fc domain is set forth in SEQ ID NO:77.
  • the (ii) immunoglobulin Fc domain of the fusion protein of the invention is the Fc domain of a human IgG 4 subclass antibody, wherein the IgG 4 subclass antibody is at position S228 in the Fc region (wherein S228 is the heavy chain constant region amino acid residue number of the Kabat database) contains an amino acid substitution, particularly an amino acid substitution S228P.
  • the (ii) immunoglobulin constant region domain of the fusion protein of the invention comprises the constant region domain set forth in SEQ ID NO: 19, 20 or 21; SEQ ID NO: 75, 76 or The Fc domain (CH2-CH3) shown at 77; and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with SEQ ID NO: 19, 20 or 21. a 99% or more sequence identity constant region domain; or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 with SEQ ID NO: 75, 76 or 77 %, 98%, 99% or more Fc domains of sequence identity.
  • the (ii) immunoglobulin constant region domain of the fusion protein of the invention comprises a constant region domain (CH1-CH2-CH3) amino acid set forth in SEQ ID NO: 19, 20 or 21.
  • One or more (preferably no more than 5) of the constant region domains of the amino acid sequence having an amino acid substitution, deletion or insertion, preferably the amino acid substitution of no more than 5 amino acids is a substitution of no more than 5 conservative amino acids ;
  • the (iii) extracellular domain of TNFR in the fusion protein of the invention is the intact extracellular region of TNF receptor (TNFR) or a portion thereof.
  • the (iii) TNFR extracellular region of the fusion protein of the invention is the entire extracellular region of TNFRl or TNFR2 or a portion thereof, more preferably the intact extracellular region of TNFR2 or a portion thereof.
  • the (iii) TNFR extracellular domain of the fusion protein of the invention is the entire extracellular region of human TNFR1 or human TNFR2 or a portion thereof, more preferably the entire extracellular region of human TNFR2 or a portion thereof .
  • the (iii) intact extracellular domain of the TNFR in the extracellular domain of TNFR in the fusion protein of the invention consists of four functional domains containing six cysteines (CRD1-CRD4)
  • the composition; the portion of the extracellular region of the TNFR may be CRD1-CRD4; or a combination of one or several of CRD1, CRD2, CRD3, CRD4; a portion of the extracellular region of the TNFR is preferably a fragment of CRD1-CRD2-CRD3-CRD4.
  • the (iii) TNFR extracellular region of the fusion protein of the invention comprises the amino acid sequence set forth in SEQ ID NO: 22, 23, 24 or 25.
  • the (iii) TNFR extracellular region of the fusion protein of the invention comprises at least 90%, 91%, 92% of the amino acid sequence set forth in SEQ ID NO: 22, 23, 24 or 25. Amino acid sequence of 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • the (iii) TNFR extracellular region of the fusion protein of the invention comprises one or more of the amino acid sequences set forth in SEQ ID NO: 22, 23, 24 or 25 (preferably not The amino acid sequence of the amino acid sequence having more than 5 amino acid substitutions, deletions or insertions, preferably the amino acid substitution of not more than 5 amino acids is a substitution of no more than 5 conservative amino acids.
  • the above anti-IL-17 antibody and variant thereof have substantially the same biological function as the anti-IL-17 antibody; the above-mentioned fragments derived from the anti-IL-17 antibody, antigen-binding fragments and variants thereof have anti-IL-17
  • the antibody has substantially the same antigen binding ability; the immunoglobulin Fc domain and variants thereof have substantially the same biological function as the immunoglobulin Fc domain; the TNFR extracellular region, its fragments and variants thereof have TNFR cells.
  • the outer zone has essentially the same biological function.
  • the fusion protein of the invention further comprises a linker peptide between (i), (ii) and/or (iii); the linker peptide preferably comprises one or more amino acids, the linker peptide more preferably comprising At least 5 amino acids, the linker peptide most preferably comprises a linker peptide selected from the group consisting of SEQ ID NOs: 30-58.
  • the fusion proteins of the invention are operably linked in the order of (i), (ii), and (iii) from the N-terminus to the C-terminus; operably linked in the order of (iii), (i), and (ii) Or operatively connected in the order of (iii), (ii), and (i).
  • the fusion protein of the invention is operably linked in the order of (i), (ii) and (iii) from the N-terminus to the C-terminus, preferably, (i) the C-terminus of the heavy chain portion of The N-terminus of (ii) is operably linked; or the C-terminus of the light chain portion of (i) is operably linked to the N-terminus of (ii).
  • the fusion protein of the invention is operably linked from the N-terminus to the C-terminus in the order of (iii), (ii) and (i), preferably wherein the C-terminus of (ii) is (iii) The N-terminus of each of the two heavy chains is joined, or the C-terminus of (ii) is linked to the N-terminus of each of the two light chains in (iii).
  • the anti-IL-17 antibody of the fusion protein of the present invention may be located at the N-terminus, and the extracellular domain of TNFR is linked to the C-terminus of the heavy chain of the anti-IL-17 antibody by a linker peptide; TNFR-Fc fusion protein, C-terminally linked to the anti-IL-17 antibody Fab: the heavy chain portion of the anti-IL-17 antibody Fab is located at the C-terminus of the TNFR-Fc fusion protein, and the anti-IL-17 antibody Fab free light chain portion is passed through The sulfur bond is linked to its heavy chain portion; or the light chain portion of the anti-IL-17 antibody Fab is located at the C-terminus of the TNFR-Fc fusion protein, and the Fab free heavy chain portion of the anti-IL-17 antibody is linked to its light chain portion via a disulfide bond .
  • the fusion protein of the invention is a fusion protein comprising a first subunit of SEQ ID NO: 60 and a second subunit of SEQ ID NO: 62, hereinafter referred to as fusion protein BY 19.3, It comprises a human anti-IL-17 antibody (IgG4, ⁇ , S228P) from the N-terminus to the C-terminus, and a portion of the human TNFR2 extracellular domain (CRD1-CRD2-CRD3-CRD4) operably linked to the C-terminus of the antibody heavy chain by a linker peptide Fragment).
  • a human anti-IL-17 antibody IgG4, ⁇ , S228P
  • CCD1-CRD2-CRD3-CRD4 a portion of the human TNFR2 extracellular domain operably linked to the C-terminus of the antibody heavy chain by a linker peptide Fragment
  • the fusion protein of the invention is a fusion protein comprising a first subunit of SEQ ID NO: 64 and a second subunit of SEQ ID NO: 66, hereinafter referred to as fusion protein BY 19.5, It comprises a human anti-IL-17 antibody (IgG4, kappa, S228P) from the N-terminus to the C-terminus, and an intact human TNFR2 extracellular domain operably linked to the C-terminus of the antibody heavy chain by a linker peptide.
  • fusion protein BY 19.5 a fusion protein comprising a first subunit of SEQ ID NO: 64 and a second subunit of SEQ ID NO: 66, hereinafter referred to as fusion protein BY 19.5, It comprises a human anti-IL-17 antibody (IgG4, kappa, S228P) from the N-terminus to the C-terminus, and an intact human TNFR2 extracellular domain operably linked to the C-terminus of the antibody heavy chain by a linker
  • the fusion protein of the invention is a fusion protein comprising a first subunit of SEQ ID NO: 68 and a second subunit of SEQ ID NO: 70, hereinafter referred to as fusion protein BY19.6, It comprises a portion of the extracellular domain of human TNFR2 (a fragment of CRD1-CRD2-CRD3-CRD4) from the N-terminus to the C-terminus, an Fc (CH2-CH3 direction) in which the N-terminus is operably linked to the C-terminus of the extracellular domain of human TNFR2, and human resistance.
  • a Fab of an IL-17 antibody (IgG4, kappa, S228P) wherein the heavy chain portion of the Fab is operably linked to the C-terminus of the Fc, the Fab free light chain portion being operably linked to its heavy chain portion by a disulfide bond.
  • the fusion protein of the invention is a fusion protein comprising a first subunit of SEQ ID NO: 74 and a second subunit of SEQ ID NO: 72, hereinafter referred to as fusion protein BY19.7.
  • a portion of the human TNFR2 extracellular domain (a fragment of CRD1-CRD2-CRD3-CRD4), an Fc operably linked to the C-terminus of the extracellular domain of the human TNFR2, and a human anti-IL-17 antibody (IgG4, A Fab of ⁇ , S228P) wherein the light chain portion of the Fab is operably linked to the C-terminus of the Fc, the Fab free heavy chain portion being operably linked to its light chain portion by a disulfide bond.
  • a human anti-IL-17 antibody IgG4, A Fab of ⁇ , S228P
  • the invention relates to polynucleotides encoding fusion proteins of the invention.
  • the invention provides a polynucleotide encoding a fusion protein of the invention, a vector comprising a polynucleotide encoding a fusion protein of the invention, preferably an expression vector, most preferably a glutamine synthesis having a double expression cassette Enzyme expression vector.
  • the invention features a vector comprising the above polynucleotide.
  • the invention features a host cell comprising the polynucleotide or vector described above.
  • the host cell is a CHO, HEK293 or NSO cell.
  • the individual is a mammal, preferably a human.
  • the invention provides a diagnostic kit or pharmaceutical composition comprising a fusion protein of the invention for use in treating or preventing rheumatoid arthritis, ankylosing spondylitis, psoriasis and ulceration in an individual colitis.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds.
  • the Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antibody fragments, see: Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)).
  • the Fv fragment consisting of V L and V H domains of a single arm of an antibody composition.
  • the two domains of the Fv fragment, V L and V H encoded by a separate gene, using recombinant methods may be able by these two domains as a synthetic linker to produce a single protein chain is connected, the The VL region and the VH region in a single protein chain are paired to form a single chain Fv.
  • the antigen-binding fragment of the antibody can be obtained by chemical methods, recombinant DNA methods or protease digestion.
  • antibody light chain refers to the lesser of the two types of polypeptide chains present in an antibody molecule.
  • the kappa light chain and the lambda light chain refer to two major antibody light chain isoforms.
  • variable region refers to a domain of an antibody heavy or light chain that is involved in binding of an antibody to an antigen.
  • the heavy chain variable domain (VH) and light chain variable domain (VL) of a native antibody typically have a similar structure, wherein each domain comprises four conserved framework regions (FR) and three complementarity determining regions.
  • FR conserved framework regions
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • the light chain variable region and the heavy chain variable region typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminus to the C-terminus.
  • each immunoglobulin light chain has a variable region (VL), also known as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL)
  • VL variable region
  • CL constant light chain
  • a domain also known as a "light chain constant region (domain).”
  • binding and “specifically bind” refer to an antibody or antigen-binding portion that binds to an epitope in an in vitro assay, preferably in biophotometric interferometry (ForteBio) using purified wild-type antigen.
  • an antibody or antigen binding portion is referred to as a specific binding antigen when the antibody or antigen binding portion preferably recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • This specific binding can be by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Analysis of agalacto-IgG). In rheumatoid arthritis using surface plasmon resonance, Glyco J., 2000, 17, 323-329).
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • a therapeutically effective dose of a fusion protein of the invention will typically range from about 0.1 mg/kg body weight to about 100 mg/kg body weight.
  • N-terminus refers to the last amino acid at the N-terminus
  • C-terminus refers to the last amino acid at the C-terminus
  • treatment refers to the clinical intervention intended to alter the natural course of disease in an individual being treated. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating the symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, ameliorating or mitigating the disease state, and alleviating or improving the prognosis.
  • the fusion proteins of the invention are used to delay disease progression or to slow the progression of the disease.
  • autoimmune disease refers to a disease caused by the body's immune response to its own components resulting in damage to its own tissues. In the present invention, it is especially referred to as rheumatoid arthritis, ankylosing spondylitis, psoriasis and ulcerative colitis.
  • Interleukin is a cytokine.
  • Human IL-17 (CTLA-8, Swiss Prot Q16552) is a pro-inflammatory cytokine produced by a subset of memory T cells (called Th17) involved in the pathogenesis of MS, capable of inducing epithelial cells, endothelial cells, and fibroblasts.
  • the cells synthesize and secrete IL-6, IL-8, G-CSF, and PGE2 to promote the expression of ICAM-1.
  • IL-17 plays a role in the induction of other inflammatory cytokines, chemokines and adhesion molecules. Treatment of animals with IL-17 neutralizing antibodies reduces disease incidence and severity in autoimmune encephalomyelitis ( Komiyama, Y.
  • anti-IL-17 antibody refers to an antibody which binds IL with sufficient affinity. -17 or a fragment thereof such that the antibody can be used as a diagnostic and/or therapeutic agent in targeting IL-17.
  • the anti-IL-17 antibody binds to an unrelated, non-IL-17 protein to a lesser extent than about 10% of the antibody binds to IL-17, as measured, for example, by radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • the anti-IL-17 antibody provided herein has a dissociation constant (Kd) ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (eg, 10 - Below 8 M, for example 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M).
  • Kd dissociation constant
  • the anti-IL-17 antibody is a multispecific antibody, such as a bispecific antibody.
  • the level of expression or activity of IL-17 is reduced by at least 50% in the presence of the IL-17 antibody compared to the level of expression or activity of IL-17 in the absence of the IL-17 antibody herein (eg, a decrease of 55%, 60) %, 75%, 80%, 85% or 90%), at this time, the IL-17 antibody is considered to be capable of significantly inhibiting the expression or activity of IL-17.
  • the antibody When the activity is reduced as compared to the activity measured in the absence of the antibody, the antibody "suppresses" the activity induced by or associated with the antigen, such as IL-17-induced activity.
  • the antibody inhibits the activity of the antigen in the presence of the antibody by at least 10% compared to the activity in the absence of the antibody. In some embodiments, the antibody inhibits activity by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or 100%.
  • the antibody is "neutralized" to the antigen or its associated activity when the activity is reduced by at least 50% in the presence of the antibody compared to the activity in the absence of the antibody.
  • cysteine engineered antibody such as a "thiocarba" wherein one or more residues of the antibody are substituted with a cysteine residue.
  • the antibodies provided herein can be further modified to contain other non-protein portions known in the art and readily available.
  • Portions suitable for antibody derivatization include, but are not limited to, water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly- 1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-vinyl) Pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerin), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glyco
  • VH paired heavy chain variable region
  • VL light chain variable region
  • amino acid sequences of the light chain constant region and the heavy chain constant region of the anti-IL-17 antibody contained in the fusion protein of the present invention are provided herein below in Table 2-3.
  • Fc region is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is according to, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • the EU numbering system described in is also referred to as the EU index.
  • immunoglobulin Fc domain in a fusion protein of the invention comprises all amino acid residues of a naturally occurring immunoglobulin Fc domain or a portion of an amino acid residue comprising a naturally occurring immunoglobulin Fc domain.
  • the immunoglobulin Fc domain provides advantageous pharmacokinetic properties to the fusion proteins of the invention, including but not limited to long serum half-life.
  • the immunoglobulin Fc domain also makes it possible to purify the fusion protein of the present invention by, for example, protein A affinity chromatography.
  • the immunoglobulin Fc domain is typically a dimeric molecule that can be produced by papain digestion or trypsin digestion of intact (full length) immunoglobulin or can be produced recombinantly, comprising a CH2 domain, a CH3 domain, and optionally CH4 domain.
  • amino acid sequence of the heavy chain Fc in the antigen-binding fragment of the anti-IL-17 antibody contained in the fusion protein of the present invention is provided in Table 4 below.
  • TNFR refers to a receptor for tumor necrosis factor (TNF), a glycoprotein.
  • extracellular region refers to a portion of TNFR that is outside the cell membrane in the natural environment.
  • the TNFR domain in the fusion protein of the anti-IL-17 antibody and the extracellular domain of TNFR may be the full length of the extracellular domain of TNFR: CRD1-CRD2-CRD3-CRD4, or a fragment of the extracellular domain: CRD1-CRD2-CRD3-CRD4 .
  • amino acid sequences of the extracellular region of TNFR and portions thereof contained in the fusion protein of the present invention are provided herein below in Table 5.
  • linker peptide refers to a short peptide that can link two polypeptide sequences and is about 1-100 amino acid residues in length.
  • linker peptide wherein the amino acid sequence of the linker peptide may be selected from any one of the following:
  • AKTTPKLEEGEFSEARV (SEQ ID NO: 31);
  • AKTTPKLGG (SEQ ID NO: 32);
  • RADAAP SEQ ID NO: 35
  • RADAAAAGGPGS (SEQ ID NO: 37);
  • ADAAP SEQ ID NO: 40
  • AKTTPPSVTPLAP (SEQ ID NO: 47);
  • AKTTAP (SEQ ID NO: 48);
  • AKTTAPSVYPLAP (SEQ ID NO: 49);
  • GHEAAAVMQVQYPAS (SEQ ID NO: 55);
  • GQGTKVEIKRGGSGGGGSG SEQ ID NO: 57
  • GQGTLVTVSSGGGGSGGGGS SEQ ID NO: 58.
  • the fusion protein of the anti-IL-17 antibody of the present invention and TNFR has a function of binding to IL-17 and TNF- ⁇ , respectively, that is, after binding to one of IL-17 and TNF- ⁇ , it is possible to bind another target.
  • the fusion protein of anti-IL-17 antibody and TNFR of the invention synergistically inhibits the biological function of IL-17 and TNF- ⁇ , inhibits the release of inflammatory mediators such as chemokines and lymphokines, and inhibits the effect of anti-IL-17 antibody and TNF.
  • the combination of the - ⁇ inhibitors is comparable to the use of anti-IL-17 antibodies or TNF- ⁇ inhibitors alone.
  • the fusion protein of the anti-IL-17 antibody of the present invention and TNFR can be used for the treatment of autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, psoriasis and ulcerative colitis.
  • the fusion proteins of the invention can be obtained, for example, by solid peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production.
  • a polynucleotide encoding a first subunit of the fusion protein and/or a polynucleotide encoding a second subunit of the fusion protein is isolated and inserted into one or more vectors for further cloning in a host cell And / or expression.
  • the polynucleotide can be easily isolated and sequenced using conventional methods.
  • a vector, preferably an expression vector, comprising one or more polynucleotides of the invention is provided.
  • Expression vectors can be constructed using methods well known to those of skill in the art.
  • Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC).
  • YAC yeast artificial chromosomes
  • a glutamine synthetase high expression vector having a dual expression cassette is used.
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a variety of techniques can be used to accomplish this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection, or other conventional techniques.
  • a method of producing a fusion protein of the invention comprises culturing a host cell as provided herein, comprising a multinuclear encoding the fusion protein, under conditions suitable for expression of the fusion protein Glucuronide, and the fusion protein is recovered from the host cell (or host cell culture medium).
  • the fusion protein prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography and the like.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art.
  • the purity of the fusion protein of the present invention can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high performance liquid chromatography, and the like.
  • the physical/chemical properties and/or biological activities of the fusion proteins provided herein can be identified, screened or characterized by a variety of assays known in the art.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like which are physiologically compatible.
  • the pharmaceutical composition of the present invention may comprise the fusion protein of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions can be included in a kit, such as a diagnostic kit.
  • Pharmaceutically acceptable carriers suitable for use in the present invention may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk , glycerin, propylene, glycol, water, ethanol, etc.
  • compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • these compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.
  • Preparation can be made by mixing a fusion protein of the invention of the desired purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. Ed. (1980)).
  • the pharmaceutical preparation of the fusion protein is preferably in the form of a lyophilized preparation or an aqueous solution.
  • Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958.
  • Aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and WO2006/044908, the latter including a histidine-acetate buffer.
  • compositions or formulations of the present invention may further comprise more than one active ingredient which is required for the particular indication being treated, preferably those active ingredients which do not adversely affect each other's complementary activities, the active ingredient It is suitably combined in an amount effective for the intended use.
  • sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles, such as films or microcapsules.
  • compositions of the invention may be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories.
  • liquid solutions e.g., injectable solutions and infusible solutions
  • dispersions or suspensions e.g., liposomes, and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use.
  • a common preferred composition is in the form of an injectable solution or an infusible solution.
  • a preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection.
  • the fusion protein is administered by intravenous infusion or injection.
  • the fusion protein is administered by intramuscular, intraperitoneal or subcutaneous injection.
  • parenteral administration and “parenteral administration” as used herein mean modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, Intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.
  • compositions should generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, dispersions, liposomes or lyophilized forms.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the fusion protein) in a suitable amount in a suitable solvent, followed by filter sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle containing base dispersion medium and other ingredients.
  • a coating agent such as lecithin or the like can be used.
  • the proper fluidity of the solution can be maintained by the use of surfactants.
  • Prolonged absorption of the injectable compositions can be brought about by the inclusion in the compositions of the compositions which delay the absorption, such as the monostearate and gelatin.
  • the fusion proteins of the invention can be administered orally, for example, orally with an inert diluent or an edible carrier.
  • the fusion proteins of the invention may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets or incorporated directly into the individual's diet.
  • the compound can be incorporated with excipients and in ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, glutinous rice sacs Used in the form of a wax or the like.
  • Therapeutic compositions can also be administered using medical devices known in the art.
  • compositions of the invention may comprise a "therapeutically effective amount” or a “prophylactically effective amount” of a fusion protein of the invention.
  • “Therapeutically effective amount” means an amount effective to achieve the desired therapeutic result at the desired dosage and for the period of time required.
  • the therapeutically effective amount can vary depending on various factors such as the disease state, the age, sex, and weight of the individual.
  • a therapeutically effective amount is any amount that is toxic or detrimental to a therapeutically beneficial effect.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (eg, a tumor growth rate) of at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably, relative to an untreated individual. At least about 80%.
  • the ability of the fusion proteins of the invention to inhibit measurable parameters e.g., tumor volume
  • prophylactically effective amount is meant an amount effective to achieve the desired prophylactic result at the desired dosage and for the period of time required. Generally, a prophylactically effective amount is less than a therapeutically effective amount because the prophylactic dose is administered to the individual prior to the earlier stage of the disease or at an earlier stage of the disease.
  • Kits comprising the fusion proteins herein are also within the scope of the invention.
  • the kit may contain one or more additional elements including, for example, instructions for use; other reagents, such as labels or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to an individual.
  • the fusion proteins disclosed herein have diagnostic and therapeutic and prophylactic uses in vitro and in vivo.
  • these molecules can be administered to cultured cells in vitro or ex vivo or to an individual, eg, a human subject, to treat, prevent, and/or diagnose a variety of diseases associated with the biological activity of IL-17 and TNF-a, For example, autoimmune diseases.
  • the invention provides a diagnostic method for detecting the presence of IL-17 and TNF-[alpha] in a biological sample, such as serum, semen or urine or a tissue biopsy sample (eg, from a hyperproliferative or cancerous lesion), in vitro or in vivo.
  • the diagnostic method comprises: (i) contacting a sample (and optionally a control sample) with a fusion protein as described herein or administering the fusion protein to an individual under conditions that allow interaction to occur and (ii) detecting the fusion Formation of a complex between the protein and the sample (and optionally, the control sample). Formation of the complex indicates the presence of IL-17 and TNF-[alpha] and may indicate the suitability or need for the treatment and/or prevention described herein.
  • IL-17 and TNF-[alpha] are detected prior to treatment, for example, prior to initiation of treatment or prior to treatment after the treatment interval.
  • Detection methods that can be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR-technology (eg, RT-PCR) or in vivo imaging techniques.
  • fusion proteins used in in vivo and in vitro assays are labeled, directly or indirectly, with a detectable substance to facilitate detection of bound or unbound conjugates.
  • Suitable detectable materials include a variety of biologically active enzymes, prosthetic groups, fluorescent materials, luminescent materials, paramagnetic (eg, nuclear magnetic resonance) materials, and radioactive materials.
  • the level and/or distribution of IL-17 and TNF-[alpha] is determined in vivo, eg, in a non-invasive manner (eg, by using a suitable imaging technique (eg, positron emission tomography (PET)) Scanning) detecting a detectably labeled fusion protein of the invention.
  • a suitable imaging technique eg, positron emission tomography (PET)
  • PET positron emission tomography
  • Scanning detecting a detectably labeled fusion protein of the invention.
  • PET reagent eg, 18 F-fluorodeoxyglucose (FDG)
  • the invention provides a diagnostic kit comprising the fusion protein described herein and instructions for use.
  • the invention relates to the use of a fusion protein in vivo for the treatment or prevention of a disease in which the biological activity of IL-17 and TNF- ⁇ is required to be enhanced in an individual, thereby inhibiting or reducing the occurrence of a related disease such as an autoimmune disease or relapse.
  • the fusion protein can be used alone to inhibit the production of or prevent the occurrence of an autoimmune disease.
  • the fusion protein can be administered in combination with other autoimmune disease therapeutic/preventive agents.
  • the combination can be administered in any order or simultaneously.
  • the invention provides a method of inhibiting the development of an autoimmune disease in an individual, the method comprising administering to the individual a therapeutically effective amount of a fusion protein described herein.
  • the invention provides a method of preventing the production or recurrence of an autoimmune disease in an individual, the method comprising administering to the individual a prophylactically effective amount of a fusion protein described herein.
  • autoimmune diseases treated and/or prevented with the fusion proteins of the invention include, but are not limited to, autoimmune rheumatoid arthritis, lupus, myasthenia gravis, ankylosing spondylitis, hyperthyroidism, Hypothyroidism, ulcerative colitis, Crohn's disease, valvular heart disease, multiple sclerosis, scleroderma and autoimmune hepatitis, more preferably rheumatoid arthritis, ankylosing spondylitis, psoriasis and Ulcerative colitis.
  • the disease associated with IL-17 biological activity in the present invention means a disease associated with excessive IL-17 levels or activity, wherein atypical symptoms may be manifested locally and/or systemically in vivo due to IL-17 levels or activity.
  • diseases related to IL-17 biological activity include: atopic dermatitis, allergic rhinitis, asthma, fibrosis, inflammatory bowel disease, Crohn's disease, pneumonia disease, pulmonary fibrosis, idiopathic pulmonary fiber (IPF), chronic obstructive pulmonary disease (COPD), liver fibrosis, respiratory disease, cancer, glioblastoma, and non-Hodgkin's lymphoma.
  • the respiratory disease can be selected from the group consisting of asthma, allergic asthma, non-allergic asthma, bronchitis, chronic bronchitis, chronic obstructive pulmonary disease (COPD), emphysema, cigarette-induced emphysema, Airway inflammation, cystic fibrosis, pulmonary fibrosis, allergic rhinitis and bronchiectasis.
  • COPD chronic obstructive pulmonary disease
  • Example 1 Construction of a high expression vector for glutamine synthetase containing a gene of interest
  • the light chain (BY19.4L) nucleotide sequence of BY19.4 (SEQ ID NO: 26):
  • the first subunit light chain (BY19.4L) amino acid sequence of BY19.4 (SEQ ID NO: 27):
  • the second subunit heavy chain (BY19.4H) nucleotide sequence of BY 19.4 (SEQ ID NO: 28):
  • the second subunit heavy chain (BY19.4H) amino acid sequence of BY19.4 (SEQ ID NO: 29):
  • METDTLLLWVLLLWVPGSTG is a signal peptide sequence.
  • the BY19.4L coding nucleotide double-digested with XbaI-SaII was ligated into the glutamine synthetase high-efficiency expression vector with double expression cassette digested with XbaI-SaII by ligase, and BY19.4L was introduced.
  • the glutamine synthetase high-efficiency expression vector with double expression cassette was digested with XhoI-EcoRI, and the BY19.4H coding nucleotide double-digested with XhoI-EcoRI was ligated into the XhoI-EcoRI double restriction enzyme by ligase.
  • Express cassette glutamine synthetase high expression vector The expression was verified by sequencing and the expression vector of anti-IL-17 antibody BY19.4 was obtained.
  • the BY19.4L coding nucleotide can also be ligated into a glutamine synthetase high expression vector having a double expression cassette into which a BY19.4H coding nucleotide has been introduced, and the expression of the antibody BY19.4 can be expressed and obtained. Carrier.
  • the heavy chain variable region and light chain variable region sequences of the anti-IL-17 antibody in Table 1 the light chain constant region sequence of the antibody in Table 2, the heavy chain constant region sequence of the antibody in Table 3, and the TNFR cell in Table 4
  • the sequence of the outer region, as well as the ligation peptide sequence of SEQ ID NO: 30-58 was optimized to be a nucleotide sequence suitable for expression in Chinese hamster ovarian cancer cells (CHO), and was commissioned by Shanghai Jierui Bioengineering Co., Ltd. to synthesize the following SEQ. ID NO: polynucleotide sequences shown at 59, 61, 63, 65, 67, 69, 71, and 73.
  • the second subunit nucleotide sequence of the fusion protein BY19.3 ( ⁇ , IgG4) (SEQ ID NO: 61):
  • the second subunit amino acid sequence of the fusion protein BY19.3 ( ⁇ , IgG4) (SEQ ID NO: 62):
  • the first subunit nucleotide sequence of the fusion protein BY19.5 ( ⁇ , IgG1) (SEQ ID NO: 63):
  • the second subunit nucleotide sequence of the fusion protein BY19.5 ( ⁇ , IgG1) (SEQ ID NO: 65):
  • the second subunit amino acid sequence of the fusion protein BY19.5 ( ⁇ , IgG1) (SEQ ID NO: 66):
  • the first subunit nucleotide sequence of the fusion protein BY19.6 ( ⁇ , IgG1) (SEQ ID NO: 67):
  • the second subunit nucleotide sequence of the fusion protein BY19.6 ( ⁇ , IgG1) (SEQ ID NO: 69):
  • the second subunit amino acid sequence of the fusion protein BY19.6 ( ⁇ , IgG1) (SEQ ID NO: 70):
  • the second subunit nucleotide sequence of the fusion protein BY19.7 ( ⁇ , IgG1) (SEQ ID NO: 71):
  • the second subunit amino acid sequence of the fusion protein BY19.7 ( ⁇ , IgG1) (SEQ ID NO: 72):
  • the first subunit nucleotide sequence of the fusion protein BY19.7 ( ⁇ , IgG1) (SEQ ID NO: 73):
  • the first subunit amino acid sequence of the fusion protein BY19.7 ( ⁇ , IgG1) (SEQ ID NO: 74):
  • the coding nucleotides (SEQ ID NO: 59, 63, 67, 73) of the above first subunit were ligated to have a double expression by XbaI-SaII double digestion.
  • the cassette glutamine synthetase high-efficiency expression vector (patent authorization number: CN104195173B, obtained from Beijing Biyang Biotechnology Co., Ltd.); the nucleotide of the above second subunit is further digested by XhoI-EcoRI (SEQ ID NO: 61, 65, 69, 71) were respectively cloned into a glutamine synthetase high-efficiency expression vector having a double expression cassette to which the corresponding fusion protein first subunit coding nucleotide has been ligated; or vice versa.
  • the recombinant vector was sequenced and verified for expression.
  • the expressed fusion proteins were named as fusion proteins BY19.3, BY19.5, BY19.6, BY19.7, respectively.
  • the recombinant expression vector of the fusion protein BY19.3-BY19.7 prepared in Example 1 was plasmid DNA 250 ug and polyethylenimine (PEI) (Sigma, catalog number: 408727).
  • transient expression produced antibodies BY19.3, BY19.4, BY19.5, BY19.6, BY19.7 as controls.
  • the fusion protein present in the culture supernatant collected in the above Example 2 (1) was purified using a HiTrap MabSelect SuRe 1 ml column (GE Healthcare Life Sciences product, catalog number: 11-0034-93) equilibrated with a pH 7.4 PBS solution. Briefly, a HiTrap MabSelect SuRe 1 ml column was equilibrated with a pH 7.4 PBS solution at a volume of 10 bed volumes at a flow rate of 0.5 ml/min; the culture supernatant collected in the above Example 2 (1) was filtered through a 0.45 ⁇ m filter.
  • the purity and molecular weight of the fusion protein were analyzed by SDS-PAGE and staining with Coomassie blue in the presence of a reducing agent (5 mM 1,4-dithiothreitol). The result is shown in Figure 2.
  • the predicted values of the molecular weight theory and the actual measured values are shown in Table 6. Because of the glycosylation of proteins in eukaryotic expression systems, the actual measured molecular weight is slightly higher than the theoretical prediction.
  • Example 3 Determination of the affinity of the fusion protein of the present invention for TNF- ⁇ and IL-17 using Biacore T100
  • an anti-IgG antibody (GE Healthcare Life Sciences, catalog number: BR-1008-39) was covalently immobilized on a CM5 chip by amide coupling.
  • the CM5 chip was activated with 60 ⁇ l of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 60 ⁇ l of N-hydroxysuccinimide (NHS), followed by 5 ⁇ l of anti- IgG antibody was added to 95 ⁇ l of the dilution buffer HBST (0.1 M HEPES, 1.5 M NaCl, pH 7.4, plus 0.005% Tween 20, filtered through a 0.2 um filter), and the anti-IgG antibody was covalently immobilized in CM5 by amide coupling.
  • HBST 0.1 M HEPES, 1.5 M NaCl, pH 7.4, plus 0.005% Tween 20, filtered through a 0.2 um filter
  • a capture system of approximately 9,000-14,000 resonance units
  • the fusion protein of the present invention prepared in Example 2 (BY19.3, BY19.5, BY19.6, BY19.7), Yisaipu (TNFR2-Fc fusion protein, Shanghai CITIC Guojian Pharmaceutical Co., Ltd. Preparation) and control antibody BY19.4 were diluted to 5 ⁇ g/ml, respectively, and the dilution was injected at a flow rate of 10 ⁇ L/min for 2 minutes to non-covalently bind the above-described fusion protein of the present invention and antibody BY19.4 to the respective Fc regions.
  • the IgG antibody was captured on a CM5 chip, yielding approximately 1600 RU.
  • Recombinant human TNF- ⁇ (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 10602-HNAE) and recombinant human IL-17A (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 12047-H07Y) will be combined with antigen.
  • the concentration gradients were prepared as follows: 7 nM, 22 nM, 66 nM, 200 nM, 600 nM. Binding was measured by injecting each concentration for 180 seconds at a flow rate of 30 ⁇ l/min with a dissociation time of 600 seconds.
  • the surface was regenerated by washing with a 3 M MgCl 2 solution at a flow rate of 10 ⁇ L/min for 30 seconds.
  • Data analysis was performed using BIA evaluation software (BIAevaluation 4.1 software, from GE Healthcare Biosciences AB, Sweden), and the affinity data shown in Table 7 below was obtained.
  • the fusion proteins BY19.3 and BY19.5 of the TNFR2 of the present invention at the C-terminus were capable of binding TNF- ⁇ and IL-17A with high affinity.
  • Antibody BY 19.4 does not bind to TNF- ⁇ .
  • the fusion protein BY19.3 and the antibody BY19.4 have the same N-terminal structure, and the Biacore results indicate that the fusion protein BY19.3 is similar to the BY19.4 antibody binding IL-17A (within 1 fold).
  • the C-terminus of the fusion protein BY19.5 and the fusion protein BY19.3 is the full length of the extracellular domain of TNFR2 (BY19.5), and the N-terminal fragment of CRD1-CRD2-CRD3-CRD4 (truncated TNFR2, BY19.3). ), the two combined with TNF- ⁇ affinity is similar (less than 1 fold). It is illustrated that the truncated form of TNFR2, which is easier to express and purify in the present invention, does not affect the affinity for TNF-[alpha].
  • the fusion protein of TNFR2 at the N-terminus Yisaipu, BY19.6 and BY19.7 have higher affinity with TNF- ⁇ than BY19.3 and BY19.5, reaching 10 -12 M; but BY19.6 and BY19.7 and IL
  • the affinity of -17A is lower than BY19.4, BY19.3 and BY19.5, which is 10 -9 M. This may be related to the Fab structure of BY19.6 and BY19.7.
  • the affinity of Fab structure to IL-17 is lower than that of full-length antibody and IL-17.
  • Example 4 Determination of the interaction of anti-IL-17/TNFR fusion protein with TNF- ⁇ and IL-17 using Biacore T100
  • the CM5 chip was pretreated in the same manner as in Example 3, and an anti-IgG antibody (GE Healthcare Life Sciences, catalog number: BR-1008-39) was covalently immobilized on a CM5 chip. Then, the BY19.3 protein was diluted to 10 ⁇ g/ml, and injected at a flow rate of 10 ⁇ L/min for 1 minute. The anti-IgG antibody captured the BY19.3 protein to produce 1600 RU; the recombinant human TNF- ⁇ (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.
  • Biacore T100 assay showed that the BY19.3 protein captured by the anti-IgG antibody could still bind to IL-17A after reaching TNF- ⁇ and reaching saturation, and reached saturation. It can be seen that the BY19.3 protein does not interfere with the binding of TNF- ⁇ and IL-17A.
  • the HT-29 cells in culture (purchased from the Chinese Academy of Sciences cell bank) were digested with trypsin-EDTA (purchased from Hyclone) one day before the experiment, and the digestion products were collected; centrifuged at 1500 rpm for 5 min, and the supernatant was discarded; the cells were resuspended in 10 ml DMEM. /F12 medium (purchased from Hyclone); cell density was counted; 96 wells were plated at 100 ⁇ l/well (cell 2 ⁇ 10 4 /well); cultured overnight at 37 ° C in a 5% CO 2 incubator.
  • the final concentration of recombinant human TNF- ⁇ was 300 pM, and the final concentration of recombinant human IL-17A was 10 nM. Incubate in an incubator (37 ° C, 5% CO 2 ) for 48 h.
  • CXCL1 ELISA kit Cat. No. EK0722
  • IL-8 kit Cat. No. EK0413
  • the concentration of CXCL1 in the negative control group was 10.6 ⁇ 0.6pg/ml, 93.2 ⁇ 22.8pg/ml in the TNF- ⁇ group, 127.2 ⁇ 30.8pg/ml in the IL-17 group, and 1111.4 ⁇ 223.8pg in the TNF- ⁇ +IL-17 group. /ml.
  • the concentrations of CXCL1 in the TNF- ⁇ group, IL-17 group and TNF- ⁇ +IL-17 group were higher than those in the negative control group, and the difference was significant (p ⁇ 0.05).
  • the concentration of CXCL1 in TNF- ⁇ +IL-17 group was higher than that in TNF- ⁇ group and IL-17 group (p ⁇ 0.05).
  • the IL-8 concentration in the negative control group was 29.0 ⁇ 3.6pg/ml
  • the TNF- ⁇ group was 223.0 ⁇ 41.3pg/ml
  • the IL-17 group was 43.7 ⁇ 5.2pg/ml
  • the TNF- ⁇ +IL-17 group was 442.9 ⁇ 56.9 pg/ml.
  • the IL-8 concentrations in the TNF- ⁇ group, IL-17 group and TNF- ⁇ +IL-17 group were higher than those in the negative control group, and there was significant difference (p ⁇ 0.05).
  • the IL-8 concentration in the TNF- ⁇ +IL-17 group was significantly higher than that in the TNF- ⁇ group and the IL-17 group (p ⁇ 0.05).
  • TNF- ⁇ and IL-17 promote the secretion of CXCL1 and IL-8 by HT-29 cells when they are present alone, and both of them have a significant effect on the secretion of CXCL1 and IL-8 by HT-29 cells, and Have a synergistic effect.
  • TNF- ⁇ , IL-17, CXCL1 and IL-8 are all immunostimulating factors, which promote the activation of lymphocytes, the chemotaxis of neutrophils and the release of inflammatory factors, all of which are related to autoimmune diseases. Development is closely related. This provides a basis for clinical development of the present invention.
  • the main reagents and materials of the experiment were basically the same as those in Example 5.
  • CXCL1 ELISA kit Cat. No. EK0722
  • IL-8 kit Cat. No. EK0413
  • the concentration of CXCL1 in BY19.4 and Yisaipu group was higher than that in the negative control group, and there was significant difference (p ⁇ 0.05). There was no significant difference in the concentration of CXCL1 between the negative control group, BY19.3 group, BY19.6 group, BY19.7 group and BY19.4+ Yisaipu group (p ⁇ 0.05).
  • the IL-8 concentrations in each group were: 35.9 ⁇ 1.9 pg/ml in BY19.3, 37.9 ⁇ 3.7 pg/ml in BY19.6, and BY19.7 in BY19.7. 30.6 ⁇ 2.9pg/ml, BY4.64+ Yisaipu group was 34.6 ⁇ 2.2pg/ml, BY19.4 (ixekizumab) group was 185.1 ⁇ 21.4pg/ml, and Yisaipu group was 40.3 ⁇ 3.4pg/ml.
  • the negative control group had an IL-8 concentration of 29.0 ⁇ 3.6 pg/ml.
  • BY19.3, BY19.6 and BY19.7 have significant inhibitory effects on the inhibition of CXCL1 and IL-8 expression in HT-29 cells, and their effects are consistent with the combination of BY19.4 and Yisaipu, and the inhibition efficiency is better than BY19. .4 and Yisaipu use the group separately.
  • the anti-IL-17 antibody and TNFR fusion protein of the present invention have great potential for treating autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, psoriasis and ulcerative colitis.

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Abstract

提供了抗白细胞介素-17抗体与肿瘤坏死因子受体胞外区的融合蛋白、编码所述融合蛋白的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、以及所述融合蛋白在个体中治疗、预防和/或诊断相关疾病中的用途。

Description

抗IL-17抗体/TNFR ECD融合蛋白及其用途 技术领域
本发明涉及医药生物技术领域。具体而言，本发明涉及抗白细胞介素-17(interleukin-17，IL-17)抗体与肿瘤坏死因子受体胞外区(tumour necrosis factor receptor extracellular domain，TNFR ECD)的融合蛋白、编码该融合蛋白的多核苷酸、包含该多核苷酸的载体、包含该多核苷酸或载体的宿主细胞、以及该融合蛋白在个体中治疗、预防和/或诊断相关疾病中的用途。
背景技术
1975年，Carswell发现被细菌感染后的小鼠血清中有一种蛋白类物质可致肿瘤出血，并能抑制、杀伤体外培养的肿瘤细胞，被命名为肿瘤坏死因子(tumour necrosis factor，TNF)，又称为恶病质因子(cachectin)(Carswell EA等，An endotoxin-induced serum factor that causes necrosis of tumor.Proc Natl Sci U S A.1975Sep；72(9):3666-3670)。TNF按其结构分两型:TNF-α和TNF-β，其中由活化的巨噬细胞、单核细胞和T细胞产生的能使肿瘤坏死的细胞因子称为TNF-α(旧称TNF)，又称恶病质因子(cachectin)，有可溶和跨膜两种形式；而由活化的T细胞和NK细胞产生的淋巴毒素(lymphotoxin，LT)称为TNF-β，目前对其功能所知有限。目前研究较多的是TNF-α，它是一种由157个氨基酸组成、相对分子质量为17,000的可溶性多肽，以二聚体、三聚体或五聚体的形式存在于溶液中，成熟型TNF-α的活性形式为三聚体。虽然TNF-α作为一种免疫调节细胞因子，是防御感染性疾病和癌病变所必需的。然而，当TNF超过一定数量，就会引起各种各样的自身免疫性疾病(Immune-mediated inflammatory diseases，IMID)，如类风湿性关节炎(rheumatoid arthritis，RA)，炎症性肠病(inflammatory bowel disease，IBD)，银屑病关节炎(psoriatic arthritis，PsA)、脉管炎强直性脊柱炎(vasculitis，ankylosing spondylitis，AS)和幼年型慢性关节炎(juvenile chronic arthritis，JCA)(Scott DL等，Tumor necrosis factor inhibitors for rheumatoid arthritis.N Engl J Med.2006 Aug 17；355(7):704–712)。
TNF通过它的两个受体-TNFR1和TNFR2来激活信号转导。TNFR1和TNFR2能激活不同细胞的信号转导通路。I型TNF-R(又称TNFR1、CD120a、p55)，具有439个氨基酸残基，分子量为55kDa，其对应的mRNA有4.5Kbp，可表达于所有类型的细胞上，在溶细胞活性上起主要作用。II型TNFR(又称TNFR2、CD120b、p75)具有426个氨基酸残基，分子量为75kDa，其对应的 mRNA有3Kbp，仅表达于免疫和内皮细胞上，与信号传递和T细胞增殖有关。两型TNFR都为糖蛋白，均包括胞外区、跨膜区和胞内区三个部分。I型和II型TNFR的胞外区有28％的同源，但在胞内区无同源性。TNFR受体家族的结构特点是胞外区由4个功能结构域组成，每个功能结构域(CRD1-CRD4)含有6个半胱氨酸的CRD，CRD1也称PLAD，是TNFR形成复合体的基础，CRD2和CRD3是TNF的结合区，与TNF类因子高亲和性结合，CRD4区及胞外其他非功能结构域部分不参与TNF类因子的结合(Mukai Y等:Solution of the structure of the TNF-TNFR2 complex.Sci Signal.2010 Nov 16；3(148):ra83)。TNFR1与TNF-α的平衡解离常数(Kd)仅为0.2-0.5nM，TNFR2与TNF-α的平衡解离常数(Kd)达到为0.03-0.07nM，可见，TNFR2与TNF-α的亲和力是TNFR1的十倍以上(Dembic Z等:Two human TNF receptors have similar extracellular，but distinct intracellular，domain sequences.Cytokine.1990Jul；2(4):231-7.)。
另外，机体内部分TNFR的胞外区从细胞膜解离下来，游离于血液中，成为可溶性TNF受体(sTNFR)。sTNFR不介导信号传导，但仍可以与TNF-α结合，中和TNF-α的活性，抑制TNF-α诱导的细胞毒性和自身免疫反应，是TNF-α的天然拮抗剂。
抗TNF的药物已经成功的用于很多自身免疫疾病，例如RA、AS、PsA和Behcet氏疾病等。目前，infliximab(英夫利昔单抗)、adalimumab(阿达木单抗)、etanercept(依那西普)、certolizumab pegol和golimumab已被批准用于临床治疗。
Infliximab是抗TNF-α的人鼠嵌合型单克隆抗体，由鼠可变区和人的恒定区IgG1(к型)组成。它与可溶性和跨膜TNF-α均具有高亲和力和特异性。Infliximab与跨膜TNF-α(mTNF-α)结合，可介导细胞程序性死亡，其特异性高，因此能够减少对其他生物学途径的非特异性影响。Infliximab可用于类风湿关节炎、脉管炎强直性脊柱炎、银屑病关节炎、溃疡性结肠炎和慢性重度斑块型银屑病的治疗。
Etanercept(国内仿制药商品名为“益赛普”)是将人的两个TNFR2(p75TNF受体)的全长胞外区与人IgG1的Fc片段(CH2和CH3结构域)连接得到的，结合并抑制TNF-α，可以有效地减少与膜受体结合的TNF-α的数量。通过竞争抑制，两个TNF-R2的臂能结合TNF-α三聚体上三个受体结合位点中的两个。TNF-α与细胞表面受体的结合被阻止(Spencer-Green G等，Etanercept(Enbrel):update on therapeutic use.Ann Rheum Dis.2000 Nov；59Suppl 1:i46–i49)，信号转导受阻，因此抑制了TNF-α介导的促炎性活性。Etanercept可用于类风湿性关节 炎、银屑病关节炎、脉管炎强直性脊柱炎和幼年型慢性关节炎的治疗。
Adalimumab是全人源抗TNF-α的单克隆抗体。它能与可溶性TNF-α和跨膜TNF-α亲和力结合，阻止TNF-α与其受体的结合。体外研究证明，它与跨膜TNF-α结合后对细胞裂解和凋亡诱导具有影响(Shen C，Assche GV，Colpaert S，Maerten P，Geboes K，Rutgeerts P，Ceuppens JL.Adalimumab induces apoptosis of human monocytes:a comparative study with infliximab and etanercept.Aliment Pharmacol Ther 2005Feb 1；21(3):251–258)。Adalimumab可用于类风湿性关节炎、银屑病关节炎、脉管炎强直性脊柱炎和Behcet氏疾病的治疗。
Golimumab中文名为戈利木单抗，是全人源抗TNF-α的单克隆抗体，可用于类风湿关节炎、脉管炎强直性脊柱炎、银屑病关节炎和溃疡性结肠炎的治疗。
Certolizumab中文名为赛妥珠单抗，是抗TNF-αIgG1单克隆抗体的Fab片段，没有Fc片段，铰链区是与两个交联的20kD的PEG共价链接的；可用于Certolizumab类风湿关节炎、银屑病关节炎的治疗。
白细胞介素-17(interleukin-17，IL-17)是近年来新发现的炎性细胞因子，具有多种生物学活性，是某些疾病发生和发展的重要因素之一。IL-17具有强大的募集中性粒细胞及促进多种炎性细胞因子释放的作用，参与机体多种炎症和免疫性疾病的发生和发展，特别是与类风湿性关节炎、哮喘、肺部感染、肿瘤、接触性皮炎等疾病关系密切。IL-17受体(IL-17R)广泛存在于各种组织和细胞中，与IL-17结合产生促炎症反应。IL-17家族及其受体的研究已渐成为医学及分子生物学研究的热点。
IL-17于1995年第1次克隆得到(Yao Z等，Herpesvirus saimiri encodes a new cytokine，IL-17，which binds to a novel cytokine receptor.Immunity.1995，3(6):811-821.)。现已知在人体及鼠中至少存在6个IL-17家族成员(IL-17A-F)。IL-17A最初命名为细胞毒T淋巴细胞相关抗原8(Cytotoxic T Lymphocytes Antigen 8，CTLA-8)。5个其他的hIL-17家族成员(Moseleyl T A，等，InterIeukin-17 family and IL-17 receptors[J].Cytokine&Growth Factor Reviews.2003，14:155-174.)分别是IL-17B、IL-17C、IL-17D、IL-17F、IL-17E(也被称为IL-25)，它们与IL-17A有20％-50％的同源性，以IL-17F同源性最高，IL-17B、IL-17C、IL-17D、IL-17F、IL-17E均以同源二聚体的形式显示其保守的羧基端区域。IL-17受体(IL-17R)家族包括5个成员(IL-17RA、B、C、D和E)。IL-17家族细胞因子的功能受体被认为是由同源二聚体或异源二聚体构成。例如，IL-17RA与IL-17RC组成的异源二聚体是IL-17及IL-17F的同源二聚体及异源二聚体的受体，而 IL-17RA及IL-17RB组成的异源二聚体是IL-17E的受体。IL-17B与IL-17E均与IL-17RB结合。IL-17A常被称作为IL-17，它第一次被提及是在1993年(O'Shea JJ，等，Mechanisms underlying lineage commitment and plasticity of helper CD4+T cells.Science.2010，327(5969):1098-1102.)，在人外周血中作为重要的促炎性因子，对抗胞外微生物和不同自身免疫疾病的发病机制中起关键作用。在IL-17家族中，在自适应免疫反应中，尤其是在对抗细菌和真菌中，IL-17A和IL-17F都是核心参与者(Kolls JK，et al.Interleukin-17 family members and inflammation.Immunity.2004，21(4):467-476.)。而对IL-17B、IL-17C和IL-17D的功能了解的很少。IL-17的主要作用是诱导产生趋化因子和其他细胞因子(如TNF-α)，在T细胞活化部位吸纳中心粒细胞和单核细胞。IL-17也能通过增加GM-CSF(granulocyte-macrophage colony stimulating factor，粒细胞-巨噬细胞集落刺激因子)及其受体的生产和分泌而有助于粒细胞的形成。此外，IL-17还能刺激中性粒细胞和其他细胞的抗菌蛋白(AMP)(如LL37)(Lin AM等，Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis，J.Immunol.2011，187(1):490-500.)。在不同的生理和疾病条件下，免疫原容易产生Th17细胞因子。近来已经证实，多种因素能够诱导其他淋巴细胞产生IL-17，包括CD8+αβT细胞、γδT细胞(H.Takatori等，Lymphoid tissue inducer-like cells are an innate source of IL-17and IL-22，J.Exp.Med.2009，206(1):35-41.)、LTi-类似的天生的淋巴细胞(ILCs)(N.K.Crellin等，Human
Figure PCTCN2018114079-appb-000001
cells and LTi-like cells constitute a stable
Figure PCTCN2018114079-appb-000002
lineage distinct from conventional natural killer cells，J.Exp.Med.2010，207:281-290.)、自然杀伤细胞(NK)(]M.L.Michel等，Identification of an IL-17-producing NK1.1(neg)iNKT cell population involved in airway neutrophilia，J.Exp.Med.2007，204(5):995-1001.)和人或鼠的CD3+自然杀伤细胞(M.Cella等，A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity，Nature.2009，457(7230):722-725.)。此外，不同的先天性髓样免疫细胞能够产生IL-17，且定位于屏障组织，如肺、肠、皮肤和外周淋巴结，可以迅速对病原体作出反应并允许立即响应，但还激活和放大自适应免疫应答。像Crohn氏疾病和溃疡性结肠炎中的肠道单核细胞和巨噬细胞、系统血管炎中的中性粒细胞、银屑病皮损中的肥大细胞和类风湿关节炎中的滑膜肥大细胞都是很好的例子。
无论IL-17是由先天性细胞还是由Th17自适应细胞产生，都不会改变这种细胞因子在银屑病和银屑病关节炎病理学中的核心作用(S.P.Raychaudhuri等，Role  of IL-17 in psoriasis and psoriatic arthritis，Clin.Rev.Allergy Immunol.2013，44 (2):183-193.)。在这些条件下，抗IL-17的药物是治疗工具。目前靶向IL-17信号通路的上市和在研抗体类药物有Brodalumab、Ixekizumab、Secukinumab、ABT-122(IL-17和TNF-α的双特异抗体)、CNTO 6785、CJM112、COVA322(IL-17和TNF-α双功能抗体)、ALX-0761、Bimekizumab和SCH-900117等。
Brodalumab是IL-17受体的抗体，而ixekizumab和secukinumab则是IL-17A的中和性抗体，其中secukinumab是一个全人源化单克隆抗体，而ixekizumab是人源化的单克隆抗体。Secukinumab(Cosentyx，苏金单抗)作为全球首个上市的IL-17A单抗药物，在FDA批准的第2年就取得了11.28亿美元的业绩，目前获批的适应症包括斑块状银屑病、银屑病关节炎、强制性脊柱炎，是诺华近年上市新药中最具市场潜力的一款药物。Cosentyx的疗效优于Stelara(IL-12/23抗体)可维持长期斑块清除效果，且患者只需使用给药注射器每月自行注射一次，目前已在70余个国家获批，全球范围内超过10万多例患者受益，将成为新的银屑病标准治疗药物。强生Stelara(优特克单抗，anti-IL-12/23)作为银屑病的临床标准治疗药物，2016年创下了32.32亿美元的销售额新高。2016年3月，FDA批准ixekizumab(Taltz)注射液用于中度至重度斑块型银屑病(plaque psoriasis)成人患者的治疗。Ixekizumab在各项皮损消退指标上均优于安进的重磅抗炎药Enbrel(恩利，通用名:etanercept，依那西普)和安慰剂，数据具有统计学显著性。对于银屑病患者而言，IL-17A在驱动角化细胞(皮肤细胞)过度增殖和活化方面发挥了重要作用。Ixekizumab不会与细胞因子IL-17B、IL-17C、IL-17D、IL-17E或IL-17F相结合。
但是临床研究发现brodalumab其对类风湿关节炎的试验组没有任何临床获益，ixekizumab和secukinumab在治疗类风湿关节炎的效果没有TNF-α生物制剂好(Kalden JR:Emerging Therapies for Rheumatoid Arthritis.Rheumatol Ther(2016)3:31–42)。体外实验证实，将IL-17抑制剂和TNF-α抑制剂联合使用时抑制趋化因子、淋巴因子和酶释放效应强于单独使用。在类风湿性关节炎的小鼠模型中，抗IL-17和TNF-α的双特异抗体对抑制小鼠炎症的发生、骨和结蹄组织的破坏效果好于单独使用抗IL-17和TNF-α抗体的治疗(Fischer JA，Hueber AJ，Wilson S等，Combined inhibition of tumor necrosis factor a and interleukin-17 as a therapeutic opportunity in rheumatoid arthritis:development and characterization of a novel bispecific antibody.Arthritis Rheumatol.2015；67(1):51–62.)。
如果同时施用IL-17抑制剂和TNF-α抑制剂，则需要分别制备这两种分开的 产品；然后施用，例如分开注射两种产品，当然也可以单次注射两种产品的共同制剂。虽然分开注射时可以灵活选择剂量和时机，但出于疼痛和经济方面等原因，于患者是不方便的。共同制剂也可以在一定程度上灵活选择剂量，但由于两种不同产品的不同分子特征，允许两种产品的化学和物理稳定性的配制条件的发现是相当具有挑战性的，或者根本就是不可能的。此外，共施用或共同制剂涉及两种不同药物疗法的累积成本，这将增加患者和/或支付者成本，而单一制剂(例如，双特异性抗体或融合蛋白)允许价格及其施用的最佳化。
同时，药物(例如，单克隆抗体药物)的制备过程中，现在常常采用“一次性生产工艺”来实现位于不同房间内制药设备之间的无菌流体输送，可以实现不同房间、不同洁净要求的设备之间的相互隔离；有效减少相关设备受环境污染的风险；同时节约验证费用、验证时间和相关人力物力。但是，这样的一套“一次性生产工艺”系统价格不菲。那么，相对于制备一种药物(例如，双特异性抗体或融合蛋白)而言，分别制备两种药物，例如，IL-17抑制剂和TNF-α抑制剂，成本上的巨大增加是显而易见的。
因此，生产双特异性抗体成为一种趋势。但是，就生产双特异性抗体而言，本身又存在不少局限性。例如，最初基因泰克公司采用Knobs-into-Holes异源二聚化技术得到的双特异性抗体中近10-20％是不需要的同源二聚体；雅培公司一直依赖的的双可变结构域Ig(Dual-variable domains Ig,DVD-Ig)技术产生的双特异性抗体的内部可变结构域结合抗原的亲和力降低近10倍；且分子量较大，近200Kd，结合不同抗原时有相互干扰的现象；岸迈生物(EpimAb)的FIT-Ig技术产生的双特异性抗体的分子量达到250Kd，很难渗透到高渗的组织；至少有一半的表达蛋白形成了无用的Fab结构；需要3个抗体片段来组装，严重影响表达水平。可见，现有技术中，要得到期望的双特异性抗体并非易事。
对于产生融合蛋白，本领域也已经进行了一些尝试。与本发明相关的尝试是一篇中国发明专利申请，授权公告号为CN 104311670 B。该专利申请涉及一种IL-17scfv/sTNFR1融合蛋白，由于其中使用的抗体是IL-17scfv，所以这样的融合蛋白与IL-17的亲和力较低，仅为对应正常结构抗IL-17抗体的十分之一左右。同时该专利申请中的TNF-α抑制剂是TNFR1，其与TNF-α的亲和力远远低于TNFR2与TNF-α的亲和力。因此，如果将该专利申请中的IL-17scfv/sTNFR1融合蛋白开发成为产品，可以预期其治疗效果比较低，这势必需要加大使用的剂量(例如，增加为大约10倍)，导致副作用的凸显。
因此，就本发明所涉及的领域而言，仍然存在中和人TNF-α和人IL-17两 者的高特异性单一融合蛋白的需要，并期望这样的融合蛋白是热稳定、物理稳定、低聚集，且可以高亲和力中和人TNF-α和人IL-17，从而避免寻找合适的配制条件(必须满足两种分子的不同分子特征)的难题。
本发明因此寻求解决上述问题中的一个或多个，得到一组新的抗IL-17抗体和TNF-α受体的融合蛋白，改善目前自身免疫性疾病的治疗。但是，当构建本发明的融合蛋白时，发明人遇到与化学稳定性和物理稳定性相关的一系列棘手问题，需要许多新的改进，包括稳定抗体可变区的VH/VL界面、增加热稳定性、减少聚集、重新平衡融合蛋白的结合表面中的静电分布以及维持对于两种靶标的结合亲和力。
为此，发明人在抗IL-17抗体的基础上，通过生物信息学等一些最新的研究成果，充分分析了TNFR与TNF-α相互作用的特点，开发出一组抗IL-17抗体与TNFR的融合蛋白。该融合蛋白的结构充分保证了该融合蛋白与其靶标结合的合适物理空间距离。具有这种结构的融合蛋白与其一种靶标分子特异性结合后，不影响该融合蛋白与另一种靶标的特异性结合，即本发明的融合蛋白能够分别与IL-17和TNF-α高亲和力结合，同时抑制IL-17和TNF-α的生物学功能，故具有治疗类风湿关节炎、强直性脊柱炎、银屑病和溃疡性结肠炎等自身免疫性疾病的潜力。同时，本发明融合蛋白的生产工艺方便、简单、制造成本低，但是得到的融合蛋白的亲和力却很高，其与IL-17以及TNF的亲和力分别与单独使用抗IL-17抗体以及TNFR时与一致，可以充分抑制IL-17和/或TNF-α的活性。即，本发明的融合蛋白可以实现用低时间和金钱成本，制备得到单一的活性成分，在用于药物领域时，免于配制条件的摸索、施用方便。
与分别制备抗IL-17抗体和TNFR两种蛋白质相比，本发明融合蛋白的制备成本可以降低至少一半，且储存、运输等成本均可相应下降；同时，在对于个体施用时，只需使用一种单一的药物，这使得施用变得更为简便。
与CN 104311670 B中制备的IL-17scfv/sTNFRI融合蛋白(即便其的确具有活性)相比，本发明融合蛋白的药效更强，使用剂量至多为其的约1/10，明显降低可能的副作用。
发明概述
本发明公开了一种新型的靶向IL-17和TNF-α的融合蛋白、编码该融合蛋白的多核苷酸、包含该多核苷酸的载体、包含该多核苷酸或载体的宿主细胞、以及该双靶向融合蛋白在个体中治疗、预防和/或诊断与IL-17和TNF-α活性相关的 疾病中的用途。
本发明所设计的融合蛋白的结构充分保证了该融合蛋白与其靶标结合的合适物理空间距离，这种结构的融合蛋白与其一种靶标分子特异性结合后，不影响该融合蛋白与另一种靶标的特异性结合。
一方面，本发明提供包含抗人IL-17抗体或其片段和结合TNF-α的部分的融合蛋白，该融合蛋白抑制IL-17与其受体的结合，且抑制TNF-α发挥其生物学功能。
在一个优选的实施方案中，本发明提供包含抗人IL-17抗体或其片段和结合TNF-α的部分的融合蛋白。
在进一步优选的实施方案中，本发明提供包含抗人IL-17抗体或其抗原结合片段和结合TNF-α的部分的融合蛋白。
在一些实施方案中，本发明的融合蛋白中的抗IL-17抗体可以是任何抗IL-17抗体，只要是能够抑制或减少IL-17与其配体结合的抗体即可，包括现有技术中已知的抗IL-17抗体和将来研发出的抗IL-17抗体。
在一些实施方案中，本发明的融合蛋白中的抗IL-17抗体还涵盖抗IL-17抗体的氨基酸序列的变体、与本发明的任何抗IL-17抗体竞争结合IL-17的抗体、以及与本发明的任何抗IL-17抗体结合IL-17相同表位的抗体。
在一些实施方案中，本发明的融合蛋白中结合TNF-α的部分是TNFR，该TNFR可以是TNFR1或TNFR2。
在一个优选的实施方案中，本发明的融合蛋白中的抗IL-17抗体与TNFR1或TNFR2中的任何一种结合。
在进一步优选的实施方案中，由于TNFR2与TNF-α的亲和力是TNFR1的10倍，因此本发明的融合蛋白中的抗IL-17抗体与TNFR2结合。
一方面，本发明涉及一种抑制IL-17和TNF-α的生物学功能的抗IL-17抗体与TNFR的融合蛋白，其包含(i)衍生自抗IL-17抗体的抗原结合片段；(ii)免疫球蛋白恒定区结构域；和(iii)TNFR胞外区。
在一个优选的实施方案中，本发明的融合蛋白中的抗IL-17抗体是抗人IL-17抗体。
在一个优选的实施方案中，本发明的融合蛋白中的抗IL-17抗体是IgG类抗体，优选该抗IL-17抗体是IgG 1亚类、IgG 2亚类、IgG 4亚类抗体。
在一个优选的实施方案中，本发明的融合蛋白中，IgG 1亚类抗IL-17抗体重链恒定区的氨基酸序列为SEQ ID NO:19所示；IgG 2亚类抗IL-17抗体重链恒定 区的氨基酸序列为SEQ ID NO:20所示；IgG 4亚类抗IL-17抗体重链恒定区的氨基酸序列为SEQ ID NO:21所示。
在一个优选的实施方案中，本发明的融合蛋白中的抗IL-17抗体是IgG 4亚类抗体。为防止臂交换(arm-exchange)的发生，在Fc结构域中第S228位(其中S228是Kabat数据库的氨基酸残基编号)处包含氨基酸取代，该氨基酸取代优选是S228P。
在一些实施方案中，本发明的融合蛋白中的抗IL-17抗体的轻链型别是κ型或λ型，优选是κ型。
在一个优选的实施方案中，本发明的融合蛋白中，κ型抗体轻链恒定区的氨基酸序列为SEQ ID NO:17所示；λ型轻链恒定区氨基酸序列为SEQ ID NO:18所示。
在一些实施方案中，本发明的融合蛋白中的(i)是衍生自人抗IL-17抗体的抗原结合片段。
在一些实施方案中，本发明的融合蛋白中的(i)是衍生自抗IL-17抗体的Fab、Fab'、F(ab) 2、F(ab') 2、Fv、单链Fv；优选是衍生自人抗IL-17抗体的Fab、Fab'、F(ab) 2、F(ab') 2、Fv、单链Fv。
在一个优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段具有结合IL-17的能力。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14和15/16的成对重链可变区序列/轻链可变区序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含SEQ ID NO:1/2或7/8的成对重链可变区序列/轻链可变区序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含与选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14和15/16的成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的成对重链可变区序列/轻链可变区序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含在选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14和15/16的成对重链可变区序列/轻链可变区序列中的一处或多处(优选不超过5处)具有氨基酸取代、缺失或插入的成对重链可变区序列/轻链可变区序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个，优选该不超过5处氨基酸的氨基酸取代是不超过5处保守氨基酸的取代。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含与选自SEQ ID NO:1/2或7/8的成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的成对重链可变区序列/轻链可变区序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含在选自SEQ ID NO:1/2或7/8的成对重链可变区序列/轻链可变区序列中的一处或多处(优选不超过5处)具有氨基酸取代、缺失或插入的成对重链可变区序列/轻链可变区序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个，优选该不超过5处氨基酸的氨基酸取代是不超过5处保守氨基酸的取代。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14和15/16的成对重链可变区序列/轻链可变区序列。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含选自SEQ ID NO:1/2或7/8的成对重链可变区序列/轻链可变区序列。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含与选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14和15/16的成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的成对重链可变区序列/轻链可变区序列。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含在选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、 13/14和15/16的成对重链可变区序列/轻链可变区序列中的一处或多处(优选不超过5处)具有氨基酸取代、缺失或插入的成对重链可变区序列/轻链可变区序列，优选该不超过5处氨基酸的氨基酸取代是不超过5处保守氨基酸的取代。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含与选自SEQ ID NO:1/2或7/8的成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的成对重链可变区序列/轻链可变区序列。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含在选自SEQ ID NO:1/2或7/8的成对重链可变区序列/轻链可变区序列中的一处或多处(优选不超过5处)具有氨基酸取代、缺失或插入的成对重链可变区序列/轻链可变区序列，优选该不超过5处氨基酸的氨基酸取代是不超过5处保守氨基酸的取代。
在进一步优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含选自SEQ ID NO:60/62、64/66、68/70和74/72的成对第一亚基/第二亚基序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个。
在一个优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含与选自SEQ ID NO:60/62、64/66、68/70和74/72的成对第一亚基/第二亚基序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的成对第一亚基/第二亚基序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个。
在一个优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含在选自SEQ ID NO:60/62、64/66、68/70和74/72的成对第一亚基/第二亚基序列中的一处或多处(优选不超过5处)具有氨基酸取代、缺失或插入的成对第一亚基/第二亚基序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个，优选该不超过5处氨基酸的氨基酸取代是不超过5处保守氨基酸的取代。
在一个优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含选自SEQ ID NO:60/62、64/66、68/70和74/72的成 对第一亚基/第二亚基序列中所含的成对重链可变区序列/轻链可变区序列。
在一个优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含与选自SEQ ID NO:60/62、64/66、68/70和74/72的成对第一亚基/第二亚基序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的成对第一亚基/第二亚基序列中所含的成对重链可变区序列/轻链可变区序列。
在一个优选的实施方案中，本发明的融合蛋白中的(i)衍生自抗IL-17抗体的抗原结合片段包含在选自SEQ ID NO:60/62、64/66、68/70和74/72的成对第一亚基/第二亚基序列中的一处或多处(优选不超过5处)具有氨基酸取代、缺失或插入的成对第一亚基/第二亚基序列中所含的成对重链可变区序列/轻链可变区序列，优选该不超过5处氨基酸的氨基酸取代是不超过5处保守氨基酸的取代。
在一个优选的实施方案中，本发明的融合蛋白中的(ii)免疫球蛋白恒定区结构域可以是任何免疫球蛋白的恒定区结构域，特别地，该(ii)是人免疫球蛋白的恒定区结构域。
在一个优选的实施方案中，本发明的融合蛋白中的(ii)免疫球蛋白恒定区结构域可以是任何免疫球蛋白的Fc结构域，特别地，该(ii)是人免疫球蛋白的Fc结构域。
在一个优选的实施方案中，本发明的融合蛋白中的(ii)免疫球蛋白重链恒定区结构域是IgG类抗体的Fc结构域，特别地是IgG 1亚类、IgG 2亚类或IgG 4亚类抗体的Fc结构域，更特别地，该IgG 1亚类抗IL-17抗体重链恒定区Fc结构域的氨基酸序列为SEQ ID NO:75所示；该IgG 2亚类抗IL-17抗体重链恒定区Fc结构域的氨基酸序列为SEQ ID NO:76所示；该IgG 4亚类抗IL-17抗体重链恒定区Fc结构域的氨基酸序列为SEQ ID NO:77所示。
在一个优选的实施方案中，本发明的融合蛋白中的(ii)免疫球蛋白Fc结构域是人IgG 4亚类抗体的Fc结构域，其中该IgG 4亚类抗体在Fc区中第S228位(其中S228是Kabat数据库的重链恒定区氨基酸残基编号)处包含氨基酸取代，特别是氨基酸取代S228P。
在一个优选的实施方案中，本发明的融合蛋白中的(ii)免疫球蛋白恒定区结构域包含SEQ ID NO:19、20或21所示恒定区结构域；SEQ ID NO:75、76或77所示Fc结构域(CH2-CH3)；与SEQ ID NO:19、20或21具有至少 90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的恒定区结构域；或者与SEQ ID NO:75、76或77具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的Fc结构域。
在一个优选的实施方案中，本发明的融合蛋白中的(ii)免疫球蛋白恒定区结构域包含在SEQ ID NO:19、20或21所示恒定区结构域(CH1-CH2-CH3)氨基酸序列中的一处或多处(优选不超过5处)具有氨基酸取代、缺失或插入的氨基酸序列的恒定区结构域，优选该不超过5处氨基酸的氨基酸取代是不超过5处保守氨基酸的取代；
或者包含在SEQ ID NO:75、76或77所示Fc结构域(CH2-CH3)氨基酸序列中的一处或多处(优选不超过5处)具有氨基酸取代、缺失或插入的氨基酸序列的Fc结构域，优选该不超过5处氨基酸的氨基酸取代是不超过5处保守氨基酸的取代。
在一个优选的实施方案中，本发明的融合蛋白中的(iii)TNFR胞外区是TNF的受体(TNFR)的完整胞外区或其部分。
在一个优选的实施方案中，本发明的融合蛋白中的(iii)TNFR胞外区是TNFR1或TNFR2的完整胞外区或其部分，更优选是TNFR2的完整胞外区或其部分。
在一个优选的实施方案中，本发明的融合蛋白中的(iii)TNFR胞外区是人TNFR1或人TNFR2的完整胞外区或其部分，更优选是人TNFR2的完整胞外区或其部分。
在一个优选的实施方案中，本发明的融合蛋白中的(iii)TNFR胞外区中的该TNFR的完整胞外区由4个含有6个半胱氨酸的功能结构域(CRD1-CRD4)组成；该TNFR胞外区的部分可以是CRD1-CRD4；或CRD1、CRD2、CRD3、CRD4中一个或几个的组合；该TNFR胞外区的部分优选是CRD1-CRD2-CRD3-CRD4的片段。
在一个优选的实施方案中，本发明的融合蛋白中的(iii)TNFR胞外区包含SEQ ID NO:22、23、24或25所示的氨基酸序列。
在一个优选的实施方案中，本发明的融合蛋白中的(iii)TNFR胞外区包含与SEQ ID NO:22、23、24或25所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
在一个优选的实施方案中，本发明的融合蛋白中的(iii)TNFR胞外区包 含在SEQ ID NO:22、23、24或25所示的氨基酸序列中的一处或多处(优选不超过5处)具有氨基酸取代、缺失或插入的氨基酸序列的氨基酸序列，优选该不超过5处氨基酸的氨基酸取代是不超过5处保守氨基酸的取代。
上述抗IL-17抗体及其变体均具有与抗IL-17抗体基本相同的生物学功能；上述衍生自抗IL-17抗体的片段、抗原结合片段及其变体均具有与抗IL-17抗体基本相同的抗原结合能力；免疫球蛋白Fc结构域及其变体均具有与免疫球蛋白Fc结构域基本相同的生物学功能；TNFR胞外区、其片段及其变体均具有与TNFR胞外区基本相同的生物学功能。
在一些实施方案中，本发明的融合蛋白的上述(i)、(ii)和/或(iii)之间还包含连接肽；该连接肽优选包含一个或多个氨基酸，该连接肽更优选包含至少5个氨基酸，该连接肽最优选包含选自SEQ ID NO:30-58的连接肽。
在一些实施方案中，本发明的融合蛋白从N端至C端按照(i)、(ii)和(iii)的顺序有效连接；按照(iii)、(i)和(ii)的顺序有效连接；或者按照(iii)、(ii)和(i)的顺序有效连接。
在一个优选的实施方案中，本发明的融合蛋白从N端至C端按照(i)、(ii)和(iii)的顺序有效连接，优选地，(i)中重链部分的C端与(ii)的N端有效连接；或者(i)中轻链部分的C端与(ii)的N端有效连接。
在一个优选的实施方案中，本发明的融合蛋白从N端至C端按照(iii)、(ii)和(i)的顺序有效连接，优选地，其中(ii)的C端与(iii)中两条重链中的每一条重链的N端连接，或者(ii)的C端与(iii)中两条轻链中的每一条轻链的N端连接。
在一个优选的实施方案中，本发明的融合蛋白中抗IL-17抗体可以位于N端，而TNFR胞外功能区通过连接肽连接于抗IL-17抗体的重链C端；或N端为TNFR-Fc融合蛋白，C端与该抗IL-17抗体Fab连接:该抗IL-17抗体Fab的重链部分位于TNFR-Fc融合蛋白C端，抗IL-17抗体Fab游离轻链部分通过二硫键与其重链部分连接；或该抗IL-17抗体Fab的轻链部分位于TNFR-Fc融合蛋白C端，抗IL-17抗体的Fab游离重链链部分通过二硫键与其轻链部分连接。
在一个优选的实施方案中，本发明的融合蛋白是包含SEQ ID NO:60的第一亚基和SEQ ID NO:62的第二亚基的融合蛋白，下文中称为融合蛋白BY19.3，其从N端到C端包含人抗IL-17抗体(IgG4，κ，S228P)，以及通过连接肽与抗 体重链C端有效连接的人TNFR2胞外区部分(CRD1-CRD2-CRD3-CRD4的片段)。
在一个优选的实施方案中，本发明的融合蛋白是包含SEQ ID NO:64的第一亚基和SEQ ID NO:66的第二亚基的融合蛋白，下文中称为融合蛋白BY19.5，其从N端到C端包含人抗IL-17抗体(IgG4，κ，S228P)，以及通过连接肽与抗体重链C端有效连接的完整人TNFR2胞外区。
在一个优选的实施方案中，本发明的融合蛋白是包含SEQ ID NO:68的第一亚基和SEQ ID NO:70的第二亚基的融合蛋白，下文中称为融合蛋白BY19.6，其从N端到C端包含人TNFR2胞外区部分(CRD1-CRD2-CRD3-CRD4的片段)、N端与人TNFR2胞外区部分C端有效连接的Fc(CH2-CH3方向)、人抗IL-17抗体(IgG4，κ，S228P)的Fab，其中该Fab的重链部分与Fc的C端有效连接，该Fab游离轻链部分通过二硫键与其重链部分有效连接。
在一个优选的实施方案中，本发明的融合蛋白是包含SEQ ID NO:74的第一亚基和SEQ ID NO:72的第二亚基的融合蛋白，下文中称为融合蛋白BY19.7其从N端到C端包含人TNFR2胞外区部分(CRD1-CRD2-CRD3-CRD4的片段)、N端与人TNFR2胞外区部分C端有效连接的Fc、人抗IL-17抗体(IgG4，κ，S228P)的Fab，其中该Fab的轻链部分与Fc的C端有效连接，该Fab游离重链部分通过二硫键与其轻链部分有效连接。
一方面，本发明涉及编码本发明的融合蛋白的多核苷酸。
在一些实施方案中，本发明提供了编码本发明融合蛋白的多核苷酸、包含编码本发明融合蛋白的多核苷酸的载体，优选地表达载体，最优选地具有双表达盒的谷氨酰胺合成酶表达载体。
另一方面，本发明涉及包含上述多核苷酸的载体。
另一方面，本发明涉及包含上述多核苷酸或载体的宿主细胞。
在一些实施方案中，该宿主细胞是CHO、HEK293或NSO细胞。
另一方面，本发明涉及用于产生本发明的融合蛋白的方法，包括步骤(i)在适于表达本发明的融合蛋白的条件下培养上述宿主细胞，和(ii)回收该融合蛋白。
另一方面，本发明涉及可以通过消除、抑制或降低IL-17活性而改善、减缓、抑制或预防疾病或病症的方法。
在另一方面，本发明的方法还涉及通过联合疗法治疗自身免疫性疾病的方法，该方法包括向个体施用有效量的本文任何融合蛋白和一种或多种其他药物。
在一些实施方案中，个体是哺乳动物，优选地人。
另一方面，本发明涉及包含本发明的融合蛋白和可药用载体的药物组合物。
在一些实施方案中，本发明提供了包含本发明融合蛋白的诊断试剂盒或药物组合物。
另一方面，本发明涉及本发明的融合蛋白和上述药物组合物的用途，用于制备在个体中治疗或预防与IL-17和TNF-α的活性相关的疾病的药物。
在一些实施方案中，本发明提供了包含本发明融合蛋白的诊断试剂盒或药物组合物，可用于治疗或预防个体中的自身免疫性疾病。
在一个优选的实施方案中，本发明提供了包含本发明融合蛋白的诊断试剂盒或药物组合物，可用于治疗或预防个体中的类风湿关节炎、强直性脊柱炎、银屑病和溃疡性结肠炎。
在一个优选的实施方案中，本发明提供了包含本发明融合蛋白的诊断试剂盒或药物组合物，可用于治疗或预防个体中的银屑病；其中个体优选是哺乳动物，更优选是人。本发明还涵盖本文任何实施方案的任意组合。本文任何实施方案或其任何组合适用于本发明的任何抗IL-17抗体或其片段与TNFR融合蛋白的制备方法和用途。
附图说明
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了部分优选的实施方案。然而，应当理解本发明不限于图中所示的实施方案。
图1A和B:例示了本发明靶向IL-17且靶向TNF-α的融合蛋白的结构示意图，其中图1A例示了从N端至C端包含抗IL-17抗体的抗原结合片段、免疫球蛋白重链恒定区结构域和TNFR ECD的融合蛋白的结构示意图；图1B例示了从N端至C端包含TNFR ECD、免疫球蛋白Fc结构域和抗IL-17抗体的抗原结合片段的融合蛋白的结构示意图。
图2:显示了实施例2中制备并纯化的本发明融合蛋白在还原剂(5mM 1,4-二硫苏糖醇)存在下通过SDS-PAGE电泳并用考马斯蓝染色电泳得到的凝胶后的结果，其中泳道1:蛋白分子量标准标志物；泳道2:融合蛋白BY19.3；泳道3:融合蛋白BY19.5；泳道4:融合蛋白BY19.6；泳道5:融合蛋白BY19.7；泳道6:融合蛋白BY19.4。
图3:显示了本发明的抗IL-17/TNFR融合蛋白分别结合TNF-α和IL-17，相互不干扰。
图4:显示了TNF-α和IL-17的对CXCL1表达的协同促进作用。
图5:显示了TNF-α和IL-17的对IL-8表达的协同促进作用。
图6:显示了本发明的抗IL-17/TNFR融合蛋白对CXCL1表达的抑制作用。
图7:显示了本发明的抗IL-17/TNFR融合蛋白对IL-8表达的抑制。
发明详述
除非另外限定，本文中所用的全部术语具有如本发明所属技术领域的普通技术人员通常理解的含义。
为了解释本说明书，将使用以下部分术语的定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文定义的部分所用术语仅是为了描述具体的实施方案，并非旨在限制。
I.定义
本文所用的术语“抗体”在本文中以最广泛的意义使用并且涵盖多种抗体结构物，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)和抗体片段，只要它们显示出所需的抗原结合活性即可。
本文所用的术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换使用，均指具有基本上与天然抗体结构相似的结构。
本文所用的术语“单克隆抗体”或“单克隆抗体组合物”指具有单一氨基酸组成的抗体分子的制备物，而不局限于其产生的方法。单克隆抗体或其抗原结合片段可以例如通过杂交瘤技术、重组技术、噬菌体展示技术、合成技术例如CDR嫁接、或其他本领域已知的技术及其组合来产生。
本文所用的术语“人抗体”指具有下述氨基酸序列的抗体，该氨基酸序列对应于由人或人细胞生成，或来源于非人来源，利用人抗体库或其他人抗体编码序列产生的抗体的氨基酸序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。
抗体可以是完整抗体分子，也可以是完整抗体分子的功能性片段，包括但不限于例如抗原结合部分、Fab、Fab'、F(ab) 2、F(ab') 2、Fv。抗体的恒定区可以经改变(例如经突变)以修饰抗体的特性，例如，可以增加或减少以下一个或多个特性:抗体糖基化、半胱氨酸残基数目、效应细胞功能或补体功能。完整抗体通常将包含至少两条全长重链和两条全长轻链，但在某些情况下可包括较少的链，例如骆驼中天然存在的抗体可仅包含重链。
本文所用的术语“抗体片段”是指不同于完整抗体的分子，其包含完整抗体的部分，该部分结合完整抗体结合的抗原。
本文所用的术语抗体的“抗原结合片段”是比完整或完全抗体的一部分或一段，其能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、酶切割或化学切割完整的抗体，来制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’) 2、Fv、单链Fv。该Fab片段是一种由V L、V H、C L和CH1结构域组成的单价片段，例如，通过木瓜蛋白酶消化完全抗体能够获得Fab片段。此外，通过胃蛋白酶在铰链区的二硫键下面消化完全抗体产生F(ab') 2，其为Fab’的二聚体，是双价片段。F(ab') 2可以在中性条件下通过破坏铰链区中的二硫键而被还原，因此将F(ab') 2二聚体转化为Fab'单体。Fab'单体基本上是具有铰链区的Fab片段(其他抗体片段的更详细的描述请参见:基础免疫学(Fundamental Immunology),W.E.Paul编辑,Raven Press,N.Y.(1993))。该Fv片段由抗体单臂的V L和V H结构域组成。另外，虽然Fv片段的两个结构域V L和V H由独立的基因编码，但是使用重组方法，可以将它们通过能够使这两个结构域作为单条蛋白链产生的合成性接头连接，在该单条蛋白链中V L区和V H区配对以形成单链Fv。可以通过化学方法、重组DNA方法或蛋白酶消化法获得该抗体的抗原结合片段。
本文所用的术语“抗体重链”指在抗体分子中存在的两种类型多肽链中的较大者，其在正常情况下决定抗体所属的类别。
本文所用的术语“抗体轻链”指在抗体分子中存在的两种类型多肽链中的较小者。κ轻链和λ轻链指两个主要的抗体轻链同种型。
取决于其重链恒定区的氨基酸序列，将抗体以“类”划分:IgA、IgD、IgE、IgG和IgM，并且这些类别中的几种可以进一步划分成亚类，如，IgG1、IgG2、IgG3和IgG4、IgA1以及IgA2。对应于不同抗体类的重链恒定区分别称作α、δ、ε、γ和μ。可以在全部五个抗体类中找到的轻链恒定区(CL)称作κ和λ。在全长轻链和重链内，通常可变区和恒定区由约12个或更多个氨基酸的“J”区连接，且重链还包括约10个以上氨基酸的“D”区。参见例如Fundamental Immunology，Ch.7(Paul，W.编辑，第二版，Raven Press，N.Y.(1989))(其为所有目的以其整体在此引作参考)。每一轻链/重链对的可变区通常形成抗原结合位点。
本文所用的术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链或轻链的结构域。天然抗体的重链可变结构域(VH)和轻链可变结构域(VL)通常具有相似的结构，其中每个结构域包含四个保守的框架区(FR)和 三个互补决定区。(参见，例如，Kindt等Kuby Immunology，6th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可足以给予抗原结合特异性。轻链可变区和重链可变区从N-末端到C-末端通常包含结构域FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。
本文所用的术语“免疫球蛋白”指具有天然存在抗体的结构的蛋白质。例如，IgG类免疫球蛋白是由二硫键结合的两条轻链和两条重链组成的约150,000道尔顿的异四聚体糖蛋白。从N端至C端，每条免疫球蛋白重链具有一个可变区(VH)，也称作可变重链域或重链可变结构域，随后是三个恒定结构域(CH1、CH2和CH3)，也称作“重链恒定区(结构域)”。类似地，从N端至C端，每条免疫球蛋白轻链具有一个可变区(VL)，也称作可变轻链域或轻链可变结构域，随后一个恒定轻链(CL)结构域，也称作“轻链恒定区(结构域)”。
本文所用的术语“人免疫球蛋白”是这样一种免疫球蛋白，其拥有对应于人或人细胞产生的免疫球蛋白的氨基酸序列或从利用人免疫球蛋白库或其他编码人免疫球蛋白的序列的非人来源衍生。
本文所用的术语“结合”和“特异性结合”指抗体或抗原结合部分在体外测定法中，优选地在采用纯化的野生型抗原的生物光干涉测量(ForteBio)中与抗原表位结合。在某些实施方案中，在抗体或抗原结合部分优选识别蛋白质和/或大分子的复杂混合物中其靶抗原时，将抗体或抗原结合部分称作特异性结合抗原。该特异性结合可以通过酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的其他技术，例如表面等离子体共振(SPR)技术(在BIAcore仪上分析)(Liljeblad等人,Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance，Glyco J.,2000，17,323-329)测量。
本文所用的术语“亲和力”是指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。除非另有说明，在用于本文时，“结合亲和力”指反映结合对的成员(例如抗体与抗原)之间1∶1相互作用的内在结合亲和力。分子X对其配偶物Y的亲和力可以通常由解离常数(K D)代表，解离常数是解离速率常数和缔合速率常数(分别是k off和k on)的比例。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是表面等离子体共振法(SPR)。
当本文所用的术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定:在该测定法中，待检测的抗原结合蛋白(例如抗体或其免疫学 功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如IL-17或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如:固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methods in Enzymology 9:242-253)。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
本文所用的术语“效应子功能”指那些可归于抗体Fc区且随抗体同种型而变化的生物学活性。抗体效应子功能的实例包括:C1q结合和补体依赖性细胞毒性(CDC)；Fc受体结合；抗体依赖性细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体(例如B细胞受体)下调；和B细胞活化。
本文所用的术语“约”在与数字联合使用时意为涵盖具有比指定数字小5％的下限和比指定数字大5％的上限的范围内的数字。
本文所用的术语“包含”或“包括”意指包括要素、整数或步骤，但是不排除任意其他要素、整数或步骤，有时也特指仅有列出的要素、整数或步骤组成。
本文所用的术语“有效量”、“治疗有效量”指本发明的抗体或抗原结合片段以单一或多次剂量施用患者后，在治疗的个体中产生预期效果的量或剂量，该预期效果包括个体病症的改善(例如，一个或多个症状的改善)和/或症状进展的延迟等。
有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定:诸如哺乳动物的物种；它的大小、年龄和一般健康；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
如上所述，在某些情况下，抗体及其靶抗原之间的相互作用会干扰靶标的功能。所需的给药量进一步取决于抗体对其特异抗原的结合亲和力，还取决于在接受给药的个体中给予的抗体的清除速率。作为非限制性示例，本发明的融合蛋白的治疗有效剂量的常见范围为从约0.lmg/kg体重至约l00mg/kg体重。在一些实施方式中，以0.lmg/kg、0.5mg/kg、lmg/kg、2mg/kg、5mg/kg、l0mg/kg、15mg/kg、20mg/kg、25mg/kg、30mg/kg、50mg/kg、75mg/kg、l00mg/kg或更高的剂量将本发明的融合蛋白给予个体。常见的剂量频率范围是例如每天两次至 每周一次、每两周一次，每三周一次，每一月一次、每二月一次、每三月一次、每半年一次。
本文所用的术语“有效连接”意指指定的各组分处于一种允许它们以预期的方式起作用的关系。
本文所用的术语“信号序列”是连接至蛋白质的N-端的氨基酸的序列，其促进蛋白质分泌至细胞外。细胞外蛋白质的成熟形式没有信号序列，其在分泌过程期间被切除。
本文所用的术语“N端”指N端的最末氨基酸，术语“C端”指C端的最末氨基酸。
本文所用的术语“融合”指将两个或多个组分由肽键直接连接或借助一个或多个连接肽有效连接。
本文所用的术语“宿主细胞”指已经向其中引入外源多核苷酸序列的细胞，包括这类细胞的子代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞(亲本细胞)和从其衍生的子代。子代在核酸含量上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括与在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体子代。宿主细胞是可以用来产生本发明融合蛋白的任何类型的细胞系统。宿主细胞包括培养的细胞，也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。
本文所用的术语“个体”或“受试者”可互换使用，指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类动物(例如，人和非人灵长类如猴)、兔和啮齿类动物(例如，小鼠和大鼠)。特别地，个体是人。
本文所用的术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其有效连接的核酸的表达。这样的载体在本文中被称为“表达载体”。
本文所用的术语“分离的”抗体是这样的抗体，其已经与其天然环境的组分分离。在一些实施方案中，将抗体纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。对于用于评估抗体纯度的方法的综述，参见，例如，Flatman等，J.Chromatogr.B848:79-87(2007)。
本文所用的术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、 减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的融合蛋白用来延缓疾病发展或用来减慢疾病的进展。
本文所用的术语“自身免疫性疾病”是指机体对自身成分发生免疫反应而导致自身组织损害所引起的疾病。本发明中尤其是指类风湿关节炎、强直性脊柱炎、银屑病和溃疡性结肠炎。
文所用的术语“变体”是指来自亲本核苷酸序列或氨基酸序列，但是本身的序列以与亲本序列不同的核苷酸序列或氨基酸序列。“变体”包括自然界中本身就存在的变体序列、通过自然过程获得的变体序列，以及通过人工方法获得的变体序列。
本文所用的术语“保守取代/置换”是指一个氨基酸经相同类别内的另一氨基酸取代/置换，例如一个酸性氨基酸经另一酸性氨基酸取代/置换，一个碱性氨基酸经另一碱性氨基酸取代/置换，或一个中性氨基酸经另一中性氨基酸取代/置换。示例性的取代如下表A所示:
表A
Figure PCTCN2018114079-appb-000003
相对于对照/参照氨基酸序列的“百分比(％)氨基酸序列同一性”定义为在将一条氨基酸序列进行比对(并在必要时导入空位，任何保守取代不视为相同的序列)，以获取最大百分比序列同一性时，该条氨基酸序列中，与对照/参照多肽序 列的氨基酸残基相同的氨基酸残基数目除以其总的氨基酸残基数目，得到的商(以百分比表示)。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。
当在本申请中提到序列同一性的百分比时，若未另外特别指出，这些百分比相对于较长序列的全长计算。相对于较长序列的全长计算适用于核酸序列和多肽序列两者。
II.融合蛋白
本文所用的术语“融合蛋白”是指由两个或多个相同或不同的多肽序列连接得到的新的多肽序列，尤其是指重组得到的，包含天然并不连接的一个或多个相同或不同的多肽序列的多肽序列。
本发明的融合蛋白以10 -8M或更小、例如以10 -9M至10 -12M的解离常数(K D)与IL-17结合；且以10 -8M或更小、例如以10 -9M至10 -12M的解离常数(K D)与TNF分子特异性结合。
本发明的融合蛋白的上述(i)、(ii)和/或(iii)任选地通过连接肽有效连接。
II-1.抗IL-17抗体
白介素(IL)是一种细胞因子。人IL-17(CTLA-8，Swiss Prot Q16552)是促炎性细胞因子，由一亚群参与MS的发病机理的记忆T细胞(称为Th17)产生，能够诱导上皮细胞、内皮细胞、成纤维细胞合成分泌IL-6、IL-8、G-CSF、PGE2，促进ICAM-1的表达。IL-17在诱导其他炎性细胞因子、趋化因子和粘附分子中发挥作用。通过IL-17中和抗体治疗动物在自身免疫性脑脊髓炎(autoimmune encephalomyelitis)中降低疾病发病率和严重性(Komiyama，Y.等，免疫学杂志(J.Immunol.)177(2006)566-573)。IL-17A在MS患者脑脊液中过量表达(Hellings，P.W.等，美国呼吸细胞分子生物学杂志(Am.J.Resp.Cell Mol.Biol.)28(2003)42-50；Matusevicius，D.等，多发性硬化(Multiple Sclerosis)5(1999)101-104；WO2005/051422)。此外，IL-17A中和抗体降低小鼠胶原蛋白诱导的关节炎RA模型的严重性和发病率，并且高水平的IL-17A可在来自RA患者的发炎关节的滑膜液中检测到(Ziolkowska，M.等，免疫学杂志(J.Immunol.)164(2000)2832-2838；Kotake，S.等，临床研究杂志(J.Clin.Invest.)103(1999)1345-1352；Hellings，P.W. 等，美国呼吸细胞分子生物学杂志(Am.J.Resp.Cell Mol.Biol.)28(2003)42-50)。
当用于本文中时，IL-17是指来自任何脊椎动物来源(包括哺乳动物如灵长类动物(例如人)和啮齿类动物(例如，小鼠和大鼠))的任何天然IL-17，除非另有说明。该术语涵盖“全长”未加工的IL-17以及由细胞内加工产生的任何形式的IL-17或其任何片段。该术语还包括天然存在的IL-17的变体，例如，剪接变体或等位变体。本文中的IL-17有时优选是指IL-17A。
本文所用的术语“抗IL-17抗体”、“抗IL-17”、“IL-17抗体”或“结合IL-17的抗体”是指这样的抗体，该抗体能够以足够的亲合力结合IL-17或其片段以致该抗体可以用作靶向IL-17中的诊断剂和/或治疗剂。在一个实施方案中，抗IL-17抗体与不相关的、非IL-17蛋白结合的程度低于该抗体与IL-17结合的约10％，如例如通过放射性免疫测定(RIA)测量的。在一些实施方案中，本文提供的抗IL-17的抗体的解离常数(Kd)≤1μM，≤100nM，≤10nM，≤1nM，≤0.1nM，≤0.01nM，或≤0.001nM(例如10 -8M以下，例如10 -8M至10 -13M，例如10 -9M至10 -13M)。在一些实施方案中，抗IL-17抗体是多特异性抗体，如双特异性抗体。
本发明的IL-17抗体或其抗原结合片段包含取代、插入或缺失。在优选的实施方案中，取代、插入或缺失发生在CDR外的区域(例如在FR中)。任选地，本发明的抗IL-17抗体包括对轻链或重链可变区、轻链或重链的翻译后修饰。
本发明提供的IL-17抗体表现出抑制活性，例如抑制IL-17的表达(如抑制T细胞对于IL-17的表达)、活性和/或信号传递，或干扰IL-17及其受体之间的相互作用。本发明提供的IL-17抗体在结合IL-17(如人IL-17)或与其相互作用后完全或部分地降低或调节IL-17的表达或活性。在抗体与人IL-17多肽和/或肽之间相互作用后，IL-17生物学功能的降低或调节是完全、显著或部分的。当与不存在同本文的抗体相互作用(如结合)时IL-17的表达或活性水平相比，存在抗体时IL-17的表达或活性水平降低了至少95％(例如降低了96％、97％、98％、99％或100％)时，该抗体被认为能够完全抑制IL-17的表达或活性。与不存在同本文的IL-17抗体结合时IL-17的表达或活性水平相比，存在IL-17抗体时IL-17的表达或活性水平降低了至少50％(例如降低了55％、60％、75％、80％、85％或90％)，此时该IL-17抗体被认为能够显著抑制IL-17的表达或活性。与不存在同本文的抗体相互作用(如结合)时IL-17的表达或活性水平相比，存在抗体时IL-17的表达或活性水平降低了少于95％(例如降低了10％、20％、25％、30％、40％、50％、60％、75％、80％、85％或90％)，此时该抗体被认 为能够部分抑制IL-17的表达或活性。
如与抗体不存在下测量的活性相比，活性降低时，抗体“抑制”由抗原诱导或与之相关的活性如IL-17诱导的活性。在某些实施方案中，与抗体不存在下的活性相比，抗体抑制在抗体存在下抗原的活性至少10％。在一些实施方案中，抗体抑制活性至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％或至少90％或100％。与抗体不存在下的活性相比，在抗体存在下活性降低至少50％，则认定抗体“中和”抗原或其相关的活性。在一些实施方案中，中和抗体抑制活性至少60％、至少70％、至少80％或至少90％或100％。在某些实施方案中，IL-17诱导的活性是细胞体外或体内增殖。在某些其他实施方案中，IL-17诱导的活性是IL-17介导的炎症反应或免疫相关疾病。在其他实施方案中，IL-17诱导的活性是IL-17介导的炎性细胞浸润。
在某些实施方案中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如取代)的人Fc区序列(例如人IgGl、IgG2、IgG3或IgG4Fc区)。
在某些实施方案中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基取代。
在某些实施方案中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1,3-二烷、聚-1,3,6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇及其混合物。
本文在下表1A中提供了本发明融合蛋白中包含的抗IL-17抗体的抗原结合片段中成对重链可变区(VH)和轻链可变区(VL)的实例。
表1.本发明融合蛋白中包含的抗IL-17抗体的抗原结合片段中重链可变区和轻链可变区序列的实例
Figure PCTCN2018114079-appb-000004
Figure PCTCN2018114079-appb-000005
本文在下表2-3中提供了本发明融合蛋白中包含的抗IL-17抗体轻链恒定区和重链恒定区的氨基酸序列的实例。
表2.本发明融合蛋白中包含的抗IL-17抗体的抗原结合片段中轻链恒定区氨基酸序列的实例
Figure PCTCN2018114079-appb-000006
表3.本发明融合蛋白中包含的抗IL-17抗体的抗原结合片段中重链恒定区氨基酸序列的实例
Figure PCTCN2018114079-appb-000007
Figure PCTCN2018114079-appb-000008
II-2免疫球蛋白Fc结构域
本文所用的术语“Fc区”在本文中用来定义免疫球蛋白重链的含有至少一部分恒定区的C端区域。该术语包括天然序列Fc区和变体Fc区。在一些实施方案中，人IgG重链Fc区从Cys226或从Pro230延伸至重链的羧基端。然而，Fc区的C端赖氨酸(Lys447)可以存在或可以不存在。除非本文中另外说明，否则Fc区或恒定区中的氨基酸残基的编号根据如Kabat等人,Sequences of Proteins of Immunological Interes,第5版,Public Health Service,National Institutes of Health,Bethesda,MD,1991中所述的EU编号体系，也称作EU索引。
本发明融合蛋白中的“免疫球蛋白Fc结构域”包含天然存在的免疫球蛋白Fc结构域的全部氨基酸残基或包含天然存在的免疫球蛋白Fc结构域的一部分氨基酸残基。免疫球蛋白Fc结构域对本发明的融合蛋白提供有利的药代动力学特性，包括但不限于长血清半寿期。另外，免疫球蛋白Fc结构域还使得通过例如蛋白A亲和层析纯化本发明的融合蛋白成为可能。
免疫球蛋白Fc结构域通常是二聚体分子，可以通过木瓜蛋白酶消化或胰蛋白酶消化完整(全长)免疫球蛋白来产生或可以重组产生，其包含CH2结构域、CH3结构域和可选的CH4结构域。
本文在下表4中提供了本发明融合蛋白中包含的抗IL-17抗体的抗原结合片段中重链Fc的氨基酸序列的实例。
表4.本发明融合蛋白中包含的抗IL-17抗体的抗原结合片段中重链Fc氨基酸序列的实例
Figure PCTCN2018114079-appb-000009
Figure PCTCN2018114079-appb-000010
II-3 TNFR胞外区
本文所用的术语“TNFR”是指肿瘤坏死因子(TNF)的受体，是一种糖蛋白。本文所用的术语“胞外区”是指TNFR在天然环境中，处于细胞膜之外的部分。
抗IL-17抗体与TNFR胞外区的融合蛋白中的TNFR功能区可为TNFR胞外区全长:CRD1-CRD2-CRD3-CRD4，或胞外区部分:CRD1-CRD2-CRD3-CRD4的片段。
本文在下表5中提供了本发明融合蛋白中包含的TNFR胞外区及其部分的氨基酸序列实例。
表5.本发明融合蛋白中包含的TNFR胞外区及其部分的氨基酸序列实例。
Figure PCTCN2018114079-appb-000011
II-4连接肽
本文所用的术语“连接肽”是指可以连接两个多肽序列的短肽，长度为约1-100个氨基酸残基。
本发明的融合蛋白中抗IL-17抗体和TNFR胞外区通过连接肽连接，其中连接肽的氨基酸序列可选自如下中的任一种:
AKTTPKLEEGEFSEAR(SEQ ID NO:30)；
AKTTPKLEEGEFSEARV(SEQ ID NO:31)；
AKTTPKLGG(SEQ ID NO:32)；
SAKTTPKLGG(SEQ ID NO:33)；
SAKTTP(SEQ ID NO:34)；
RADAAP(SEQ ID NO:35)；
RADAAPTVS(SEQ ID NO:36)；
RADAAAAGGPGS(SEQ ID NO:37)；
RADAAAA(SEQ ID NO:38)；
SAKTTPKLEEGEFSEARV(SEQ ID NO:39)；
ADAAP(SEQ ID NO:40)；
DAAPTVSIFPP(SEQ ID NO:41)；
TVAAP(SEQ ID NO:42)；
TVAAPSVFIFPP(SEQ ID NO:43)；
QPKAAP(SEQ ID NO:44)；
QPKAAPSVTLFPP(SEQ ID NO:45)；
AKTTPP(SEQ ID NO:46)；
AKTTPPSVTPLAP(SEQ ID NO:47)；
AKTTAP(SEQ ID NO:48)；
AKTTAPSVYPLAP(SEQ ID NO:49)；
ASTKGP(SEQ ID NO:50)；
ASTKGPSVFPLAP(SEQ ID NO:51)；
GGGGSGGGGSGGGGS(SEQ ID NO:52)；
GENKVEYAPALMALS(SEQ ID NO:53)；
GPAKELTPLKEAKVS(SEQ ID NO:54)；
GHEAAAVMQVQYPAS(SEQ ID NO:55)；
GGGGSGGGGSGGGGSA(SEQ ID NO:56)；
GQGTKVEIKRGGSGGGGSG(SEQ ID NO:57)；
GQGTLVTVSSGGGGSGGGGS(SEQ ID NO:58)。
本发明的抗IL-17抗体与TNFR的融合蛋白具有分别结合IL-17和TNF-α的功能，即，结合IL-17和TNF-α中一个后，尚可以结合另一个靶标。
本发明的抗IL-17抗体与TNFR的融合蛋白可协同抑制IL-17和TNF-α的生物学功能，抑制趋化因子和淋巴因子等炎症介质释放，抑制效果与抗IL-17抗体与TNF-α抑制剂联合使用相当，强于单独使用抗IL-17抗体或TNF-α抑制剂。
本发明的抗IL-17抗体与TNFR的融合蛋白可用于治疗于类风湿关节炎、 强直性脊柱炎、银屑病和溃疡性结肠炎等自身免疫性疾病的治疗。
III.本发明的融合蛋白的生产和纯化
本发明的融合蛋白可以例如通过固态肽合成(例如Merrifield固相合成)或重组生产获得。为了重组生产，将编码该融合蛋白的第一亚基的多核苷酸和/或编码该融合蛋白的第二亚基的多核苷酸分离并插入一个或多个载体中以便进一步在宿主细胞中克隆和/或表达。使用常规方法，可以轻易地分离该多核苷酸并将其测序。在一个实施方案中，提供了包含本发明的一种或多种多核苷酸的载体，优选地表达载体。
可以使用本领域技术人员熟知的方法来构建表达载体。表达载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在一个优选的实施方案中，使用了具有双表达盒的谷氨酰胺合成酶高效表达载体。
一旦已经制备了用于表达的包含本发明的一种或多种多核苷酸的表达载体，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质体的转染或其他常规技术。
本领域已知在这些宿主细胞系统中表达外源基因的标准技术。在一个实施方案中，提供了产生本发明的融合蛋白的方法，其中该方法包括在适于表达该融合蛋白的条件下培养如本文中提供的宿主细胞，该宿主细胞包含编码该融合蛋白的多核苷酸，并且从宿主细胞(或宿主细胞培养基)回收该融合蛋白。
如本文所述制备的融合蛋白可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于如净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。
可以通过多种熟知分析方法中的任一种方法确定本发明的融合蛋白的纯度，该熟知分析方法包括凝胶电泳、高效液相色谱等。可以通过本领域已知的多种测定法，鉴定、筛选或表征本文提供的融合蛋白的物理/化学特性和/或生物学活性。
IV.药物组合物和试剂盒
本文所用的术语“药物组合物”指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用该制剂的个体具有不 可接受的毒性的另外的成分。本发明的药物组合物适于静脉内、肌内、皮下、肠胃外、直肠、脊髓或表皮施用(例如，通过注射或输注)。
本文所用的术语“可药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。
本发明的药物组合物可包括本发明的融合蛋白和可药用载体。这些药物组合物可包括于试剂盒中，如诊断试剂盒。
适用于本发明的药用载体可以是无菌液体，如水和油，包括那些具有石油、动物、植物或合成起源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。合适的药用赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of PharmaceuticalExcipients”,第五版,R.C.Rowe,P.J.Seskey和S.C.Owen,PharmaceuticalPress,London,Chicago。若期望的话，该组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。口服配制剂可以包含标准载体，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。
可以通过将具有所需纯度的本发明的融合蛋白与一种或多种任选的药用载体(Remington’s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合来制备包含本文所述的融合蛋白的药物制剂，优选地以冻干制剂或水溶液的形式。
示例性的冻干抗体制剂描述于美国专利号6,267,958。水性抗体制剂包括美国专利号6,171,586和WO2006/044908中所述的那些，后一种制剂包括组氨酸-乙酸盐缓冲剂。
本发明的药物组合物或制剂还可以包含超过一种活性成分，该活性成分是被治疗的特定适应证所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分，该活性成分以对于目的用途有效的量合适地组合存在。
可制备持续释放制剂。持续释放制剂的合适实例包括含有抗体的固体疏水聚合物的半渗透基质，该基质呈成形物品，例如薄膜或微囊形式。
本发明的组合物可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，可注射用溶液剂和可输注溶液剂)、分散体剂或混 悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。常见的优选组合物处于可注射用溶液剂或可输注溶液剂形式。优选的施用模式是肠胃外(例如，静脉内、皮下、腹腔(i.p.)、肌内)注射。在一个优选实施方案中，通过静脉内输注或注射施用融合蛋白。在另一个优选实施方案中，通过肌内、腹腔或皮下注射施用融合蛋白。
如本文所用的短语“肠胃外施用“和“肠胃外方式施用”意指除了肠施用和局部施用之外的施用模式，通常通过注射施用，并且包括但不限于静脉内、肌内、动脉内、皮内、腹腔、经气管、皮下注射和输注。
治疗性组合物一般应当是无菌的并且在制造和储存条件下稳定。可以将组合物配制为溶液、微乳液、分散体、脂质体或冻干形式。可以通过将活性化合物(即融合蛋白)以要求的量加入适宜的溶剂中，随后过滤消毒，制备无菌可注射溶液剂。通常，通过将该活性化合物并入无菌溶媒中来制备分散体，该无菌溶媒含有基础分散介质和其他成分。可以使用包衣剂如卵磷脂等。在分散体的情况下，可以通过使用表面活性剂来维持溶液剂的适宜流动性。可以通过在组合物中包含延迟吸收的物质例如单硬脂酸盐和明胶而引起可注射组合物的延长吸收。
在某些实施方案中，可以口服施用本发明的融合蛋白，例如随惰性稀释剂或可食用载体一起经口施用。本发明的融合蛋白也可以封闭在硬壳或软壳明胶胶囊中、压缩成片剂或直接掺入个体的膳食中。对于口服治疗施用，该化合物可以随赋形剂一起掺入并且以可摄取的片剂、颊用片剂、锭剂(troche)、胶囊剂、酏剂、混悬剂、糖浆剂、糯米纸囊剂(wafer)等形式使用。为了通过非肠胃外施用方法施用本发明的融合蛋白，可能需要将该融合蛋白与防止其失活的材料包衣或随这种材料共施用。还可以用本领域已知的医疗装置施用治疗组合物。
本发明的药物组合物可以包含“治疗有效量”或“预防有效量”的本发明的融合蛋白。“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。可以根据多种因素如疾病状态、个体的年龄、性别和重量等变动治疗有效量。治疗有效量是任何有毒或有害作用不及治疗有益作用的量。相对于未治疗的个体，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率)至少约20％、更优选地至少约40％、甚至更优选地至少约60％和仍更优选地至少约80％。可以在预示人肿瘤中的功效的动物模型系统中评价本发明的融合蛋白抑制可度量参数(例如，肿瘤体积)的能力。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果 的量。通常，由于预防性剂量在个体中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量小于治疗有效量。
包含本文的融合蛋白的试剂盒也处于本发明的范围内。试剂盒可以包含一个或多个其他要素，例如包括:使用说明书；其他试剂，例如标记物或用于偶联的试剂；可药用载体；和用于施用至个体的装置或其他材料。
V.融合蛋白的用途
本文公开的融合蛋白具有体外和体内诊断用途以及治疗性和预防性用途。例如，可以将这些分子施用至体外或离体的培养细胞或施用至个体，例如，人类个体，以治疗、预防和/或诊断多种与IL-17和TNF-α的生物活性相关的疾病，例如自身免疫性疾病。
在一个方面，本发明提供了体外或体内检测生物样品，例如血清、精液或尿或组织活检样品(例如，来自过度增生性或癌性病灶)中存在IL-17和TNF-α的诊断方法。该诊断方法包括:(i)在允许相互作用发生的条件下使样品(和任选地，对照样品)与如本文所述的融合蛋白接触或向个体施用该融合蛋白和(ii)检测该融合蛋白和样品(和任选地，对照样品)之间复合物的形成。复合物的形成表示存在IL-17和TNF-α，并且可以显示本文所述治疗和/或预防的适用性或需求。
在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测IL-17和TNF-α。可以使用的检测方法包括免疫组织化学、免疫细胞化学、FACS、ELISA测定、PCR-技术(例如，RT-PCR)或体内成像技术。一般地，体内和体外检测方法中所用的融合蛋白直接或间接地用可检测物质标记以促进检测结合的或未结合的结合物。合适的可检测物质包括多种生物学活性酶、辅基、荧光物质、发光物质、顺磁(例如，核磁共振活性)物质和放射性物质。
在一些实施方案中，体内确定IL-17和TNF-α的水平和/或分布，例如，以非侵入方式确定(例如，通过使用合适的成像技术(例如，正电子发射断层摄影术(PET)扫描)检测可检测物标记的本发明融合蛋白。在一个实施方案中，例如，通过检测用PET试剂(例如， 18F-氟脱氧葡萄糖(FDG))以可检测方式标记的本发明融合蛋白，体内测定IL-17和TNF-α的水平和/或分布。
在一个实施方案中，本发明提供了包含本文所述融合蛋白和使用说明书的诊断试剂盒。
在另一个方面，本发明涉及使用融合蛋白体内用来治疗或预防需要在个体 中增强IL-17和TNF-α的生物学活性的疾病，从而抑制或减少相关疾病如自身免疫性疾病的出现或复发。可以单独使用融合蛋白以抑制自身免疫性疾病的产生或者预防其出现。备选地，融合蛋白可以与其他自身免疫性疾病治疗剂/预防剂组合施用。当本发明的融合蛋白与一种或多种其他药物组合施用时，这种组合可以按任何顺序施用或者同时施用。
因此，在一个实施方案中，本发明提供一种抑制个体中自身免疫性疾病发生的方法，该方法包括向个体施用治疗有效量的本文所述的融合蛋白。在另一个实施方案中，本发明提供一种防止个体中自身免疫性疾病的产生或者复发的方法，该方法包括向个体施用预防有效量的本文所述的融合蛋白。
在一些实施方案中，用本发明的融合蛋白治疗和/或预防的自身免疫性疾病包括但不限于:自身免疫类风湿性关节炎、狼疮、重症肌无力、强直性脊柱炎、甲状腺机能亢进、甲状腺功能减退症、溃疡性结肠炎、克罗恩病、心脏瓣膜疾病、多发性硬化症、硬皮病和自身免疫肝炎，更优选地为类风湿关节炎、强直性脊柱炎、银屑病和溃疡性结肠炎。
本发明中与IL-17生物活性相关的疾病意指与过多IL-17水平或活性相关的疾病，其中非典型症状可以因IL-17水平或活性在体内局部和/或全身性表现。IL-17生物活性相关的疾病的例子包括:异位性皮炎、过敏性鼻炎、哮喘、纤维化、炎性肠病、节段性回肠炎、肺炎性疾病、肺纤维化、特发性肺纤维化(IPF)、慢性阻塞性肺病(COPD)、肝纤维化、呼吸道疾病、癌症、胶质母细胞瘤和非霍奇金淋巴瘤。在本文所述的任何实施方案中，呼吸道疾病可以选自哮喘、过敏性哮喘、非过敏性哮喘、支气管炎、慢性支气管炎、慢性阻塞性肺病(COPD)、气肿、香烟所致气肿、气道炎症、囊性纤维化、肺纤维化、过敏性鼻炎和支气管扩张症。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入本文作为参考。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中获得。
具体实施方式
实施例1.包含目的基因的谷氨酰胺合成酶高效表达载体的构建
(1)作为对照的抗IL-17A抗体BY19.4编码核苷酸的合成及表达载体的构建
根据International Nonproprietary Name(INN)数据库中编号为9467的ixekizumab单抗的氨基酸序列数据，优化为适合在中国仓鼠卵巢癌细胞(CHO)中表达的下述核苷酸序列，并委托上海捷瑞生物工程有限公司合成该核苷酸序列。该核苷酸序列表达后产生的抗IL-17A抗体在本文中表示为抗体BY19.4。
BY19.4的轻链(BY19.4L)核苷酸序列(SEQ ID NO:26):
Figure PCTCN2018114079-appb-000012
BY19.4的第一亚基轻链(BY19.4L)氨基酸序列(SEQ ID NO:27):
Figure PCTCN2018114079-appb-000013
BY19.4的第二亚基重链(BY19.4H)核苷酸序列(SEQ ID NO:28):
Figure PCTCN2018114079-appb-000014
BY19.4的第二亚基重链(BY19.4H)氨基酸序列(SEQ ID NO:29):
Figure PCTCN2018114079-appb-000015
其中带下划线部分“ METDTLLLWVLLLWVPGSTG”为信号肽序列。
上海捷瑞生物工程有限公司合成了上述BY19.4轻链(BY19.4L)编码核苷酸序列和BY19.4重链(BY19.4H)编码核苷酸序列。分别将BY19.4L编码核苷酸序列和具有双表达盒的谷氨酰胺合成酶高效表达载体(专利授权号:CN104195173B，获自北京比洋生物技术有限公司)用XbaI-SaII双酶切，再通过连接酶将经XbaI-SaII双酶切的BY19.4L编码核苷酸连接入经XbaI-SaII双酶切的具有双表达盒的谷氨酰胺合成酶高效表达载体，获得已导入了BY19.4L编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体；然后，分别将BY19.4H编码核苷酸用XhoI-EcoRI双酶切，将已导入了BY19.4L编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体用XhoI-EcoRI双酶切，再通过连接酶将经XhoI-EcoRI双酶切的BY19.4H编码核苷酸连接入经XhoI-EcoRI双酶切的已导入了BY19.4L编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体，由此获得了已导入BY19.4L编码核苷酸和BY19.4H编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体，经测序验证正确后表达，获得抗IL-17抗体BY19.4的表达载体。
备选地，也可以将BY19.4L编码核苷酸连接入已导入了BY19.4H编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体，表达并获得抗体BY19.4的表达载体。
(2)本发明示例性融合蛋白的编码核苷酸的合成及表达载体的构建
根据表1中抗IL-17抗体的重链可变区和轻链可变区序列、表2中抗体的轻链恒定区序列、表3中抗体的重链恒定区序列、表4中TNFR胞外区的序列，以及SEQ ID NO:30-58的连接肽序列，优化为适合在中国仓鼠卵巢癌细胞(CHO)中表达的核苷酸序列，并委托上海捷瑞生物工程有限公司合成如下SEQ ID NO:59、61、63、65、67、69、71、73所示的多核苷酸序列。
融合蛋白BY19.3(κ，IgG4)的第一亚基核苷酸序列(SEQ ID NO:59):
Figure PCTCN2018114079-appb-000016
融合蛋白BY19.3(κ，IgG4)的第一亚基氨基酸序列(SEQ ID NO:60＝SEQ ID NO:27):
Figure PCTCN2018114079-appb-000017
Figure PCTCN2018114079-appb-000018
融合蛋白BY19.3(κ，IgG4)的第二亚基核苷酸序列(SEQ ID NO:61):
Figure PCTCN2018114079-appb-000019
融合蛋白BY19.3(κ，IgG4)的第二亚基氨基酸序列(SEQ ID NO:62):
Figure PCTCN2018114079-appb-000020
融合蛋白BY19.5(κ，IgG1)的第一亚基核苷酸序列(SEQ ID NO:63):
Figure PCTCN2018114079-appb-000021
融合蛋白BY19.5(κ，IgG1)的第一亚基氨基酸序列(SEQ ID NO:64):
Figure PCTCN2018114079-appb-000022
Figure PCTCN2018114079-appb-000023
融合蛋白BY19.5(κ，IgG1)的第二亚基核苷酸序列(SEQ ID NO:65):
Figure PCTCN2018114079-appb-000024
融合蛋白BY19.5(κ，IgG1)的第二亚基氨基酸序列(SEQ ID NO:66):
Figure PCTCN2018114079-appb-000025
融合蛋白BY19.6(κ，IgG1)的第一亚基核苷酸序列(SEQ ID NO:67):
Figure PCTCN2018114079-appb-000026
Figure PCTCN2018114079-appb-000027
融合蛋白BY19.6(κ，IgG1)的第一亚基氨基酸序列(SEQ ID NO:68＝SEQ ID NO:27):
Figure PCTCN2018114079-appb-000028
融合蛋白BY19.6(κ，IgG1)的第二亚基核苷酸序列(SEQ ID NO:69):
Figure PCTCN2018114079-appb-000029
融合蛋白BY19.6(κ，IgG1)的第二亚基氨基酸序列(SEQ ID NO:70):
Figure PCTCN2018114079-appb-000030
融合蛋白BY19.7(κ，IgG1)的第二亚基核苷酸序列(SEQ ID NO:71):
Figure PCTCN2018114079-appb-000031
Figure PCTCN2018114079-appb-000032
融合蛋白BY19.7(κ，IgG1)的第二亚基氨基酸序列(SEQ ID NO:72):
Figure PCTCN2018114079-appb-000033
融合蛋白BY19.7(κ，IgG1)的第一亚基核苷酸序列(SEQ ID NO:73):
Figure PCTCN2018114079-appb-000034
融合蛋白BY19.7(κ，IgG1)的第一亚基氨基酸序列(SEQ ID NO:74):
Figure PCTCN2018114079-appb-000035
使用上述实施例1(1)中相同的方法，分别通过XbaI-SaII双酶切将上述第一亚基的编码核苷酸(SEQ ID NO:59、63、67、73)连接至具有双表达盒的谷氨酰胺合成酶高效表达载体(专利授权号:CN104195173B，获自北京比洋生物技术有限公司)；再通过XhoI-EcoRI双酶切将上述第二亚基的编码核苷酸(SEQ ID  NO:61、65、69、71)分别克隆至已连接了相应的融合蛋白第一亚基编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体；或者反之亦然。将重组载体测序验证正确后用于表达。所表达的融合蛋白分别命名为融合蛋白BY19.3、BY19.5、BY19.6、BY19.7。
实施例2、融合蛋白的表达和纯化
(1)融合蛋白的瞬时表达
将293F(购自Invitrogen公司，目录号:11625-019)细胞悬浮培养于无血清CD 293培养液(购自Invitrogen公司，目录号:11913-019)中。转染前离心细胞培养物，获得细胞沉淀，用新鲜的无血清CD 293培养液悬浮细胞，将细胞浓度调整为1×10 6个细胞/ml。将细胞悬浮液置于摇瓶中。以100ml细胞悬浮液为例，分别将实施例1制备的融合蛋白BY19.3-BY19.7的重组表达载体质粒DNA 250ug和聚乙烯亚胺(polyethylenimine(PEI))(Sigma，目录号:408727)500ug加入1ml无血清CD 293培养液中混匀，室温静置8分钟后，将PEI/DNA混悬液逐滴加入放置有100ml细胞悬浮液的摇瓶中。轻轻混匀，置于5％CO 2、37℃摇床培养(120转/分钟)。5天后收集培养上清。
由此，瞬时表达产生作为对照的抗体BY19.3、BY19.4、BY19.5、BY19.6、BY19.7。
(2)表达蛋白的纯化
用pH 7.4 PBS溶液平衡的HiTrap MabSelect SuRe 1ml柱(GE Healthcare Life Sciences产品，目录号:11-0034-93)纯化上述实施例2(1)收集的培养上清中存在的融合蛋白。简言之，用pH 7.4的PBS溶液以10个柱床体积平衡HiTrap MabSelect SuRe 1ml柱，流速为0.5ml/分钟；将上述实施例2(1)收集的培养上清用0.45μm滤膜过滤后，载样至用pH 7.4 PBS溶液平衡的HiTrap MabSelect SuRe 1ml柱；装载上清液后，将该柱首先用pH 7.4的PBS溶液以流速0.5ml/分钟洗涤5-10个柱床体积，并随后用100mM柠檬酸缓冲液(pH4.0)以流速0.5ml/分钟洗脱。收集洗脱峰，目的蛋白存在于洗脱峰中。
在还原剂(5mM 1，4-二硫苏糖醇)存在下通过SDS-PAGE并用考马斯蓝染色，分析融合蛋白的纯度和分子量。结果如图2所示。分子量理论预测值与实际测定值见表6。因真核表达系统中存在对蛋白质的糖基化作用，故分子量实际测定值略高于理论预测值。
表6.经纯化的表达蛋白的分子量大小
Figure PCTCN2018114079-appb-000036
实施例3、使用Biacore T100测定本发明融合蛋白对TNF-α和IL-17的亲和力
在
Figure PCTCN2018114079-appb-000037
T100仪器(GE Healthcare Biosciences AB，瑞典)上于25℃进行表面等离子体共振测量。
首先，通过酰胺偶联将抗IgG抗体(GE Healthcare Life Sciences，目录号:BR-1008-39)共价固定在CM5芯片上。使用60μl N-乙基-N'-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和60μl N-羟基琥珀酰亚胺(NHS)活化CM5芯片，然后将5μl抗IgG抗体加95μl稀释缓冲液HBST(0.1M HEPES，1.5M NaCl，pH7.4，加0.005％吐温20，经0.2um滤膜过滤后)，通过酰胺偶联将抗IgG抗体共价固定在CM5芯片上，产生约9000-14000共振单位(RU)的捕获系统。使用120μl乙醇胺封闭CM5芯片。
然后，将实施例2制备的本发明的融合蛋白(BY19.3、BY19.5、BY19.6、BY19.7)、益赛普(TNFR2-Fc融合蛋白，上海中信国健药业股份有限公司制备)和对照抗体BY19.4分别稀释为5μg/ml，以流速10μL/分钟注射该稀释液2分钟，使上述本发明的融合蛋白和抗体BY19.4通过各自的Fc区非共价结合到抗IgG抗体上，捕获到CM5芯片上，产生约1600RU。
将结合抗原重组人TNF-α(北京义翘神州生物技术有限公司产品，目录号:10602-HNAE)和重组人IL-17A(北京义翘神州生物技术有限公司产品，目录号:12047-H07Y)分别配制为如下浓度梯度:7nM、22nM、66nM、200nM、600nM。通过以流速30μl/分钟注射每个浓度180秒，解离时间600秒，测量结合。通过用3M MgCl 2溶液以流速10μL/分钟洗涤30秒使表面再生。使用BIA评价软件(BIAevaluation 4.1software，来自GE Healthcare Biosciences AB，瑞典)进行数据分析，获得下表7所示的亲和力数据。
表7.各蛋白与PD-1的结合
Figure PCTCN2018114079-appb-000038
根据表7所示的数据可见，本发明TNFR2位于C端的融合蛋白BY19.3和BY19.5均能够以高亲和力结合TNF-α和IL-17A。抗体BY19.4不结合TNF-α。
融合蛋白BY19.3和抗体BY19.4具有相同的N端结构，Biacore结果表明了融合蛋白BY19.3与BY19.4抗体结合IL-17A亲和力相似(1倍以内)。
融合蛋白BY19.5和融合蛋白BY19.3的C端分别为TNFR2的胞外区全长(BY19.5)，以及CRD1-CRD2-CRD3-CRD4的N端片段(截短的TNFR2，BY19.3)，二者结合TNF-α亲和力相似(1倍以内)。说明本发明中更易于表达和纯化的截短形式的TNFR2不影响与TNF-α的亲和力。
TNFR2位于N端的融合蛋白:益赛普、BY19.6和BY19.7与TNF-α的亲和力较BY19.3和BY19.5高，达到10 -12M；但BY19.6和BY19.7与IL-17A的亲和力低于BY19.4、BY19.3和BY19.5，为10 -9M。这可能与BY19.6和BY19.7为Fab结构有关系，Fab结构与IL-17的亲和力要低于全长抗体与IL-17的亲和力。
实施例4、使用Biacore T100测定抗IL-17/TNFR融合蛋白对结合TNF-α和IL-17的相互影响
CM5芯片预处理同实施例3，将抗IgG抗体(GE Healthcare Life Sciences，目录号:BR-1008-39)共价固定在CM5芯片上。然后，将BY19.3蛋白稀释至10μg/ml，以流速10μL/分钟注射1分钟，抗IgG抗体捕获BY19.3蛋白，产生1600RU；将重组人TNF-α(北京义翘神州生物技术有限公司产品，目录号:10602-HNAE)稀释为80nM，以流速20μL/分钟注射2分钟，BY19.3蛋白捕获了重组人TNF-α蛋白，产生361RU，并达到饱和；再将重组人IL-17A(北京义 翘神州生物技术有限公司产品，目录号:12047-H07Y)稀释为80nM，以流速20μL/分钟注射2分钟，已结合TNF-α蛋白并达饱和状态的芯片可继续捕获重组人IL-17A蛋白，产生290RU，并达到饱和。用3M MgCl2溶液以流速10μL/分钟洗涤2分钟，使表面再生。
结果:Biacore T100检测表明，通过抗IgG抗体俘获的BY19.3蛋白在结合TNF-α并达到饱和后仍然可以结合IL-17A，并到达饱和。可见，BY19.3蛋白对TNF-α和IL-17A的结合不存在相互干扰现象。
实施例5TNF-α和IL-17对CXCL1和IL-8表达的协同作用
在实验前一天用胰酶-EDTA(购自Hyclone)将培养中的HT-29细胞(购自中国科学院细胞库)消化，收集消化产物；1500rpm离心5min，弃上清；细胞重悬于10ml DMEM/F12培养基(购自Hyclone)中；计数细胞密度；按100μl/孔(细胞2×10 4/孔个)铺96孔；置于37℃、5％CO2培养箱中过夜培养。
实验当天分别配制重组人TNF-α(北京义翘神州生物技术有限公司产品，目录号:10602-HNAE)和重组人IL-17A(北京义翘神州生物技术有限公司产品，目录号:12047-H07Y)。加入前一天所铺的HT-29细胞孔中，实验设置4个组:TNF-α、IL-17A、TNF-α+IL-17A和阴性对照组，每组6个重复孔，除阴性对照组外，重组人TNF-α终浓度为300pM，重组人IL-17A的终浓度为10nM。置培养箱(37℃、5％CO2)中培养48h。
在培养结束后，收集上清，用博士德生物工程有限公司产品CXCL1 ELISA试剂盒(货号:EK0722)和IL-8试剂盒(货号:EK0413)分别检测上清中CXCL1和IL-8的表达水平。
结果:
阴性对照组CXCL1浓度为10.6±0.6pg/ml，TNF-α组为93.2±22.8pg/ml，IL-17组为127.2±30.8pg/ml，TNF-α+IL-17组为1111.4±223.8pg/ml。TNF-α组、IL-17组和TNF-α+IL-17组CXCL1浓度均高于阴性对照组，差异显著(p<0.05)。TNF-α+IL-17组CXCL1浓度高于TNF-α组和IL-17组，差异显著(p<0.05)。
阴性对照组IL-8浓度为29.0±3.6pg/ml，TNF-α组为223.0±41.3pg/ml，IL-17组为43.7±5.2pg/ml，TNF-α+IL-17组为442.9±56.9pg/ml。TNF-α组、IL-17组和TNF-α+IL-17组IL-8浓度均高于阴性对照组，有显著差异(p<0.05)。TNF-α+IL-17组IL-8浓度高于TNF-α组和IL-17组，有显著差异(p<0.05)。
上述结果表明，TNF-α和IL-17在单独存在时促进HT-29细胞分泌CXCL1 和IL-8，二者同时存在时对HT-29细胞分泌CXCL1和IL-8有显著促进作用，并且二者具有协同作用。
TNF-α、IL-17、CXCL1和IL-8均为免疫促进因子，促进淋巴细胞的活化、中性粒细胞的趋化作用和炎症因子的释放等重要作用，这些均与自身免疫性疾病的发生发展密切相关。这为本发明的临床开发提供了依据。
实施例6抗IL-17/TNFR融合蛋白对CXCL1和IL-8表达的抑制作用
实验主要试剂和材料与实施例5基本一致。实验设置7个组:BY19.3、BY19.6、BY19.7、BY19.4、益赛普(上海中信国健药业股份有限公司制备)、BY19.4+益赛普组和阴性对照组，每组6个平行孔。除阴性对照组外，各组重组人TNF-α组的终浓度为300pM，各组重组人IL-17A组的终浓度为10nM。BY19.3、BY19.6、BY19.7、BY19.4和益赛普的终浓度均为40nM。混匀后，置培养箱(37℃、5％CO2)中培养48h。
在培养结束后，收集上清，用博士德生物工程有限公司产品CXCL1ELISA试剂盒(货号:EK0722)和IL-8试剂盒(货号:EK0413)分别检测上清中CXCL1和IL-8的表达水平。
结果:
在TNF-α和IL-17A同时存在时，各组CXCL1浓度分别是:BY19.3组为12.4±0.6pg/ml，BY19.6为14.7±1.23pg/ml，BY19.7为15.4±3.22pg/ml，BY19.4+益赛普组为10.7±0.58pg/ml，BY19.4(ixekizumab)组为42.1±5.6pg/ml，益赛普组为118.6±38.9pg/ml，阴性对照组CXCL1浓度为10.6±0.6pg/ml。BY19.4和益赛普组CXCL1浓度均高于阴性对照组，有显著差异(p<0.05)。阴性对照组、BY19.3组、BY19.6组、BY19.7组和BY19.4+益赛普组3组CXCL1浓度无显著差异(p<0.05)。
上述结果说明，BY19.4和益赛普在单独使用，尤其是益赛普单独使用，对CXCL1表达的抑制作用不如BY19.3、BY19.6和BY19.7组的抑制作用。但将BY19.4和益赛普联合使用或使用BY19.3、BY19.6和BY19.7时，对CXCL1表达有显著抑制作用。
在TNF-α和IL-17A同时存在时，各组IL-8浓度分别是:BY19.3组为35.9±1.9pg/ml，BY19.6组为37.9±3.7pg/ml，BY19.7组为30.6±2.9pg/ml，BY19.4+益赛普组为34.6±2.2pg/ml，BY19.4(ixekizumab)组为185.1±21.4pg/ml，益赛普组为40.3±3.4pg/ml，阴性对照组IL-8浓度为29.0±3.6pg/ml。BY19.3组、BY19.6 组、BY19.7组、BY19.4+益赛普组和益赛普组与阴性对照组相比IL-8浓度无显著差异(p>0.05)。使用单独BY19.4组IL-8浓度高于其他组，有显著差异(p<0.05)。
上述结果说明，BY19.4单独使用时抑制IL-8表达的作用较益赛普、BY19.4+益赛普联合或使用BY19.3、BY19.6和BY19.7时为差。
总之，BY19.3、BY19.6和BY19.7在抑制HT-29细胞表达CXCL1和IL-8方面有显著抑制作用，其作用与BY19.4和益赛普联合效果一致，抑制效率好于BY19.4和益赛普单独使用组。由此可见，本发明的抗IL-17抗体和TNFR融合蛋白在治疗类风湿关节炎、强直性脊柱炎、银屑病和溃疡性结肠炎等自身免疫性疾病有巨大的潜力。
另外，由于现有的人抗IL-17抗体，例如人IL-17scfv，不能结合鼠模型中的IL-17；人TNFR1和人TNFR2均不能结合鼠模型中的TNF-α，故发明人由于无法找到合适的动物模型，无法提供本发明的融合蛋白在动物体内的实验数据。这也是本领域的一个尚未解决的问题。

Claims (15)

  1. 一种抗IL-17抗体与TNFR的融合蛋白，包含(i)衍生自抗IL-17抗体的抗原结合片段；(ii)免疫球蛋白恒定区结构域；和(iii)TNFR胞外区。
  2. 根据权利要求1的融合蛋白，其中:
    (1)所述(i)是衍生自人抗IL-17抗体的抗原结合片段；优选包含下述序列(a)-(d)之一:
    (a)选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14和15/16的成对重链可变区序列/轻链可变区序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个；或
    与选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14和15/16的成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的成对重链可变区序列/轻链可变区序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个，或
    在选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14和15/16的成对重链可变区序列/轻链可变区序列中的一处或多处具有氨基酸取代、缺失或插入的成对重链可变区序列/轻链可变区序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个；
    (b)选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14和15/16的成对重链可变区序列/轻链可变区序列，或
    与选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14和15/16的成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的成对重链可变区序列/轻链可变区序列，或
    在选自SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14和15/16的成对重链可变区序列/轻链可变区序列中的一处或多处具有氨基酸取代、缺失或插入的成对重链可变区序列/轻链可变区序列；
    (c)选自SEQ ID NO:60/62、64/66、68/70和74/72的成对第一亚基/第二亚基序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个，或
    与选自SEQ ID NO:60/62、64/66、68/70和74/72的成对第一亚基/第二亚基序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、 98％、99％或更多序列同一性的成对第一亚基/第二亚基序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个，或
    在选自SEQ ID NO:60/62、64/66、68/70和74/72的成对第一亚基/第二亚基序列中的一处或多处具有氨基酸取代、缺失或插入的成对第一亚基/第二亚基序列中所含的三个重链CDR中的一个至三个，以及所含的三个轻链CDR中的一个至三个；
    和
    (d)选自SEQ ID NO:60/62、64/66、68/70和74/72的成对第一亚基/第二亚基序列中所含的成对重链可变区序列/轻链可变区序列，或
    与选自SEQ ID NO:60/62、64/66、68/70和74/72的成对第一亚基/第二亚基序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的成对第一亚基/第二亚基序列中所含的成对重链可变区序列/轻链可变区序列，或
    在选自SEQ ID NO:60/62、64/66、68/70和74/72的成对第一亚基/第二亚基序列中的一处或多处具有氨基酸取代、缺失或插入的成对第一亚基/第二亚基序列中所含的成对重链可变区序列/轻链可变区序列；
    (2)所述(ii)是人免疫球蛋白恒定区结构域；优选地包含下述序列之一或由下述序列之一组成:
    -SEQ ID NO:19、20或21所示氨基酸序列中的恒定区结构域，或
    -与SEQ ID NO:75、76或77所示氨基酸序列中的Fc结构域，或
    -与SEQ ID NO:19、20或21所示氨基酸序列中的恒定区结构域具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的恒定区结构域，或
    -与SEQ ID NO:75、76或77所示氨基酸序列中的Fc结构域具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的Fc结构域，或
    -在SEQ ID NO:19、20或21所示氨基酸序列中的恒定区结构域的一处或多处具有氨基酸取代、缺失或插入的氨基酸序列的恒定区结构域，或
    -在SEQ ID NO:75、76或77所示氨基酸序列中的Fc结构域的一处或多处具有氨基酸取代、缺失或插入的氨基酸序列的Fc结构域；
    (3)所述(iii)是人TNFR胞外区；优选包含下述序列之一或由下述序列之一 组成:
    -SEQ ID NO:22、23、24或25所示的氨基酸序列；或
    -与SEQ ID NO:22、23、24或25所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列，或
    -在SEQ ID NO:22、23、24或25所示的氨基酸序列中的一处或多处具有氨基酸取代、缺失或插入的氨基酸序列。
  3. 权利要求2的融合蛋白，其中所述至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性是至少95％、96％、97％、98％、99％或更多同一性。
  4. 权利要求2的融合蛋白，其中所述一处或多处氨基酸的取代、缺失或插入是至多5处氨基酸的取代、缺失或插入，其中所述至多5处氨基酸的取代优选是至多5处保守氨基酸的取代。
  5. 权利要求2的融合蛋白，其中所述(i)是衍生自人抗IL-17抗体的Fab、Fab'、F(ab) 2、F(ab') 2、Fv、单链Fv，或者
    所述(ii)是人IgG 1、IgG 2或IgG 4的重链恒定区结构域；或者
    所述(iii)包含人TNFR1胞外区或人TNFR2胞外区，更优选包含人TNFR2胞外区全长或其部分；最优选包含人TNFR2胞外区CRD1-CRD2-CRD3-CRD4的片段。
  6. 权利要求1-5中任一项所述的融合蛋白，其中所述抗IL-17抗体是抗人IL-17抗体，优选是抗人IL-17A抗体；抗体的轻链型为κ型或λ型，优选为κ型；所述抗IL-17抗体优选是IgG类抗体，更优选是IgG 1亚类、IgG 2亚类、IgG 4亚类抗体。
  7. 根据权利要求1-6中任一项所述的融合蛋白，其中所述融合蛋白包含所述(i)、(ii)和/或(iii)之间的连接肽；
    所述连接肽优选包含一个或多个氨基酸；更优选包含至少5个氨基酸；最优选包含选自SEQ ID NO:30-58的连接肽。
  8. 根据权利要求7的融合蛋白，其中所述融合蛋白从N端至C端按照以下顺序有效连接：
    (1)按照(i)、(ii)和(iii)的顺序有效连接；
    优选地，(i)中重链部分的C端与(ii)的N端有效连接；或者(i)中轻链部分的C端与(ii)的N端有效连接；
    或者
    (2)按照(iii)、(ii)和(i)的顺序有效连接，
    优选地，(ii)的C端与(iii)中两条重链部分中的每一条重链部分的N端有效连接；或者(ii)的C端与(iii)中两条轻链部分中的每一条轻链部分的N端有效连接。
  9. 根据权利要求1－8中任一项所述的融合蛋白，其中所述融合蛋白包含下述序列之一或由下述序列之一组成:
    (1)SEQ ID NO:60的第一亚基和SEQ ID NO:62的第二亚基的融合蛋白；
    (2)SEQ ID NO:64的第一亚基和SEQ ID NO:66的第二亚基的融合蛋白；
    (3)SEQ ID NO:68的第一亚基和SEQ ID NO:70的第二亚基的融合蛋白；和
    (4)SEQ ID NO:74的第一亚基和SEQ ID NO:72的第二亚基的融合蛋白。
  10. 多核苷酸，其编码权利要求1－9中任一项所述的融合蛋白。
  11. 包含权利要求10所述的多核苷酸的载体，其中所述载体优选是表达载体，更优选是具有双表达盒的谷氨酰胺合成酶表达载体。
  12. 宿主细胞，包含权利要求10所述的多核苷酸或权利要求11所述的载体，优选地，所述宿主细胞是CHO、HEK293或NSO细胞。
  13. 用于产生权利要求1－9中任一项所述的融合蛋白的方法，包括：
    步骤(i)在适于表达所述融合蛋白的条件下培养权利要求12所述的宿主细胞，和
    (ii)回收所述融合蛋白。
  14. 药物组合物，包含权利要求1－9中任一项所述的融合蛋白和可药用载体。
  15. 权利要求1－9中任一项所述的融合蛋白、权利要求14所述的药物组合物的用途，用于制备在个体中治疗或预防与IL-17和TNF-α的活性相关的疾病的药物，
    其中所述疾病优选是自身免疫性疾病，更优选是类风湿关节炎、强直性脊柱炎、银屑病和溃疡性结肠炎；最优选是银屑病；
    其中所述个体是哺乳动物，更优选是人。
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