WO2020233571A1 - 一种双特异性分子及其制备与用途 - Google Patents

一种双特异性分子及其制备与用途 Download PDF

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WO2020233571A1
WO2020233571A1 PCT/CN2020/091060 CN2020091060W WO2020233571A1 WO 2020233571 A1 WO2020233571 A1 WO 2020233571A1 CN 2020091060 W CN2020091060 W CN 2020091060W WO 2020233571 A1 WO2020233571 A1 WO 2020233571A1
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bispecific molecule
antibody
amino acid
acid sequence
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French (fr)
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王峰
郑花鸯
张雨菡
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Shanghai Yichen Biomed CoLtd
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Shanghai Yichen Biomed CoLtd
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Priority to US17/612,585 priority Critical patent/US20220233710A1/en
Priority to CN202080009752.8A priority patent/CN113316587B/zh
Priority to EP20809148.8A priority patent/EP3974445A4/en
Publication of WO2020233571A1 publication Critical patent/WO2020233571A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/245—IL-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the invention belongs to the field of biopharmaceuticals, and specifically relates to a bispecific molecule that specifically binds interleukin 1 receptor (IL-1R) and free inflammatory factors, and its preparation and use.
  • IL-1R interleukin 1 receptor
  • the use of recombinant anti-inflammatory cytokines, recombinant soluble receptors or neutralizing antibodies targeting inflammatory cytokines to treat inflammatory diseases has achieved good clinical results.
  • TNF- ⁇ is a pro-inflammatory cytokine produced by the body in the early stage of trauma or bacterial infection.
  • TNFR signaling pathway is closely related to various inflammatory diseases such as RA (rheumatoid arthritis), Crohn's disease, atherosclerosis, psoriasis, sepsis, diabetes, obesity, etc.
  • Etanercept etanercept
  • Infliximab infliximab
  • TNF- ⁇ was also approved by the FDA in 1998 for the treatment of Crohn's disease.
  • Infliximab binds monomeric (non-activated state) or trimerized (activated state) TNF- ⁇ , while Etanercept mainly binds activated TNF- ⁇ trimer.
  • Adalimumab The fully human Adalimumab (Adalimumab) was approved by the FDA in 2002 for the treatment of rheumatoid arthritis.
  • TNF- ⁇ is the main factor against intracellular bacterial infections. Therefore, while the above-mentioned TNF- ⁇ inhibitors show good clinical effects, they also inevitably increase the reactivation of various infections such as tuberculosis and HBV. The risk of herpes zoster virus (HZV).
  • HZV herpes zoster virus
  • the imbalance between the IL-1 family's pro-inflammatory signals and inhibiting inflammatory signals is also closely related to the occurrence of various chronic diseases, such as inflammatory bowel disease, rheumatoid arthritis, and IL-1 ⁇ Express related autoinflammatory diseases, etc.
  • 3 drugs targeting the IL-1 pathway (Anakinra (Anakinra), Canakinumab (Canakinumab)) and Rilonacept (Linacept) have been approved by the FDA for marketing. Similar to Anakinra, Canakinumab and Rilonacept can effectively reduce the symptoms of inflammatory diseases.
  • Canakinumab also shows good efficacy in the treatment of SJIA (systemic juvenile idiopathic arthritis).
  • SJIA systemic juvenile idiopathic arthritis
  • a meta-analysis of 4 clinical trials for the treatment of RA found that the incidence of gangrene has increased significantly. Therefore, the above-mentioned drugs for the IL-1 pathway are not recommended for patients with pneumonia, osteomyelitis, cellulitis, bursitis, herpes zoster, bunions, and dangerous patients who are treated with Anakinra with high infection risk.
  • IL-6 is another pluripotent pro-inflammatory factor in the immune system. It is secreted by a variety of cells and can stimulate B cells, NK cells, osteoclasts, and tumor cells. Therefore, it is used in autoimmune diseases such as rheumatoid arthritis. This plays an important role.
  • Tocilizumab a humanized antibody targeting IL-6, was approved for the treatment of RA and SJIA in 2010 and 2011, respectively.
  • Siltuximab (Stuximab) antibody targeting IL-6R is approved by the FDA for immunodeficiency virus (HIV) negative and herpes virus (HHV-8) negative polycentric Castleman disease (Castellman disease) .
  • the invention provides a bispecific molecule and its preparation and use.
  • drugs targeting pro-inflammatory factors are aimed at free inflammatory factors in the body. These drugs combine with inflammatory factors and circulate throughout the body with body fluids. Therefore, they may cause systemic adverse reactions to the body and increase the risk of infection.
  • the present inventors found that by using molecules that specifically bind to cell surface inflammatory factor receptors as mediators, confining antibodies targeting free inflammatory factors to or near the cell surface can exert a synergistic and local anti-inflammatory effect of the two. While enhancing the anti-inflammatory effect, it can effectively avoid possible adverse reactions of bispecific molecules in the systemic circulation.
  • the present invention provides a bispecific molecule that includes a molecule that specifically binds IL-1R and an antibody that targets free inflammatory factors, wherein the specific binding IL-1R The molecule and the antibody targeting the inflammatory factor are linked by a linker peptide.
  • the bispecific molecule of the present invention aggregates the bispecific molecule on or near the surface of cells expressing IL-1R by specifically binding IL-1R molecules to prevent it from circulating in the body with body fluids;
  • Antibodies targeting free inflammatory factors can further neutralize free inflammatory factors that are locally increased due to inflammation on the surface of or near IL-1R-expressing cells, while synergistically enhancing the anti-inflammatory response of molecules that specifically bind IL-1R, and avoid Because free inflammatory factors are widely expressed in the body, antibodies that target free inflammatory factors produce adverse reactions to the body.
  • the molecule that specifically binds IL-1R is a non-immunoglobulin polypeptide.
  • the non-immunoglobulin polypeptide that specifically binds IL-1R is IL-1RA.
  • the IL-1RA that specifically binds to IL-1R has the nucleotide sequence shown in SEQ ID NO: 49 and or has the amino acid sequence shown in SEQ ID NO: 50.
  • the non-immunoglobulin polypeptide that specifically binds IL-1R is composed of an amino acid sequence having at least 85%-99% homology with SEQ ID NO:50.
  • the molecule that specifically binds IL-1R is an anti-IL-1R antibody.
  • the antibody that specifically binds to IL-1R is a single domain antibody, a chimeric antibody, a humanized antibody, a human antibody, or a recombinant modified part of the foregoing antibodies.
  • the antibody that specifically binds IL-1R includes the CDR group: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 have the following SEQ ID NO: 52, SEQ ID The HCDR1, HCDR2, and HCDR3 sequences contained in the amino acid sequence shown in NO: 56 or SEQ ID NO: 58; LCDR1, LCDR2, and LCDR3 have the amino acid sequence shown in SEQ ID NO: 54 or SEQ ID NO: 60 Contains LCDR1, LCDR2 and LCDR3 sequence.
  • the antibody that specifically binds to L-1R is composed of the following heavy and light chains: the heavy chain shown in SEQ ID NO: 52 and the light chain shown in SEQ ID NO: 54; SEQ ID NO: The heavy chain shown in 56 and the light chain shown in SEQ ID NO: 54; or the heavy chain shown in SEQ ID NO: 58 and the light chain shown in SEQ ID NO: 60.
  • the free inflammatory factor targeted by the bispecific molecule is selected from (not limited to) one of the following: IL-1 superfamily (IL-1 ⁇ , IL-1 ⁇ , IL-18, IL- 33, IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ ), IL-4, IL-13, IL-17A, IL-17E, IL-6, IL-12, IL-23, TNF superfamily (TNF ⁇ , TNF ⁇ , TNF ⁇ , OX40L (TNFSF4), CD40L (CD154), FasL (CD178, CD95L), CD27L (CD70), CD30L (CD153), 4-1BBL, CD253 (APO-2L), CD254, APO-3L (DR3L), CD256 (TALL-2), CD257 (BlyS), LIGHT (CD258), TL1 (TNFSF18, AITRL), ED1-A1, BAFF, IFN or GM-CSF.
  • IL-1 superfamily IL-1 ⁇ , IL-1 ⁇ ,
  • the antibodies that target the aforementioned free inflammatory factors are chimeric antibodies, humanized antibodies, human antibodies, or recombinantly modified parts of these antibodies.
  • the free inflammatory factor targeted by the bispecific molecule is IL-1 ⁇ .
  • the antibody targeting free inflammatory factor IL-1 ⁇ has the HCDR1, HCDR2, and HCDR3 sequence contained in the heavy chain amino acid sequence shown in SEQ ID NO: 2, and the sequence shown in SEQ ID NO: The LCDR1, LCDR2 and LCDR3 sequences contained in the light chain amino acid sequence shown in 4; or the HCDR1, HCDR2 and HCDR3 sequences contained in the heavy chain amino acid sequence shown in SEQ ID NO: 6, and the sequences shown in SEQ ID NO: : The LCDR1, LCDR2, and LCDR3 sequences contained in the light chain amino acid sequence shown in 8.
  • the free inflammatory factor targeted by the bispecific molecule is IL-17A.
  • the targeted free inflammatory factor IL-17 has the HCDR1, HCDR2, and HCDR3 sequences contained in the heavy chain amino acid sequence shown in SEQ ID NO: 26, and the sequence shown in SEQ ID NO: 28 The LCDR1, LCDR2 and LCDR3 sequences contained in the amino acid sequence of the light chain shown.
  • the free inflammatory factor targeted by the bispecific molecule is IL-6.
  • the antibody targeting free inflammatory factor IL-6 has the HCDR1, HCDR2, and HCDR3 sequence contained in the heavy chain amino acid sequence shown in SEQ ID NO: 38, and the sequence shown in SEQ ID NO: The LCDR1, LCDR2, and LCDR3 sequences contained in the light chain amino acid sequence shown in 40.
  • the bispecific molecule of the present invention includes a molecule that specifically binds to IL-1R and an antibody that targets free inflammatory factor IL-1 ⁇ , and the molecule that specifically binds to IL-1R is related to The antibody to the free inflammatory factor IL-1 ⁇ is linked by a connecting peptide.
  • the humanized antibody targeting free inflammatory factor IL-1 ⁇ has a heavy chain shown in SEQ ID NO: 2 and a light chain shown in SEQ ID NO: 4.
  • the humanized antibody targeting free inflammatory factor IL-1 ⁇ has a heavy chain as shown in SEQ ID NO: 6 and a light chain as shown in SEQ ID NO: 8.
  • the molecule that specifically binds IL-1R is a non-immunoglobulin polypeptide.
  • the non-immunoglobulin polypeptide of a molecule that specifically binds IL-1R is IL-1RA.
  • the non-immunoglobulin polypeptide that specifically binds IL-1R has the nucleotide sequence shown in SEQ ID NO: 49 and/or has the amino acid sequence shown in SEQ ID NO: 50.
  • the non-immunoglobulin polypeptide that specifically binds IL-1R is composed of an amino acid sequence having at least 85%-99% homology with SEQ ID NO:50.
  • the molecule that specifically binds IL-1R is an anti-IL-1R antibody.
  • the antibody that specifically binds IL-1R has a CDR group comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 have the following SEQ ID NO: 52, SEQ The HCDR1, HCDR2, and HCDR3 sequences contained in the amino acid sequence shown in ID NO: 56 or SEQ ID NO: 58; LCDR1, LCDR2, and LCDR3 have the amino acid sequence shown in SEQ ID NO: 54 or SEQ ID NO: 60 The LCDR1, LCDR2 and LCDR3 sequence included.
  • the bispecific molecule that specifically binds IL-1R and IL-1 ⁇ has a heavy chain and a light chain with the following amino acid sequences: SEQ ID NO: 10 and SEQ ID NO: 4; SEQ ID NO : 2 and SEQ ID NO: 12; SEQ ID NO: 14 and SEQ ID NO: 4; SEQ ID NO: 2 and SEQ ID NO: 16; SEQ ID NO: 18 and SEQ ID NO: 4; SEQ ID NO: 2 and SEQ ID NO: 20; SEQ ID NO: 6 and SEQ ID NO: 22; SEQ ID NO: 6 and SEQ ID NO: 24; SEQ ID NO: 62 and SEQ ID NO: 4; SEQ ID NO: 64 and SEQ ID NO: 4; SEQ ID NO: 66 and SEQ ID NO: 8; SEQ ID NO: 6 and SEQ ID NO: 68; SEQ ID NO: 70 and SEQ ID NO: 4; SEQ ID NO: 72 and SEQ ID NO: 4; SEQ ID NO: 74 and SEQ ID NO:
  • the bispecific molecule of the present invention includes a molecule that specifically binds IL-1R and an antibody that targets the free inflammatory factor IL-17, and the molecule that specifically binds IL-1R and the target The antibody to the free inflammatory factor IL-17 is linked by a connecting peptide.
  • the humanized antibody targeting free inflammatory factor IL-17 has a heavy chain shown in SEQ ID NO: 26 and a light chain shown in SEQ ID NO: 28.
  • the molecule that specifically binds IL-1R is a non-immunoglobulin polypeptide.
  • the non-immunoglobulin polypeptide of a molecule that specifically binds IL-1R is IL-1RA.
  • the non-immunoglobulin polypeptide IL-1RA that specifically binds to IL-1R has the nucleotide sequence shown in SEQ ID NO: 49 and/or has the amino acid shown in SEQ ID NO: 50 sequence.
  • the non-immunoglobulin polypeptide that specifically binds IL-1R is composed of an amino acid sequence having at least 85%-99% homology with SEQ ID NO:50.
  • the molecule that specifically binds IL-1R is an anti-IL-1R antibody.
  • the antibody that specifically binds IL-1R has a CDR group comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 have the following SEQ ID NO: 52, SEQ The HCDR1, HCDR2, and HCDR3 sequences contained in the amino acid sequence shown in ID NO: 56 or SEQ ID NO: 58; LCDR1, LCDR2, and LCDR3 have the amino acid sequence shown in SEQ ID NO: 54 or SEQ ID NO: 60 The LCDR1, LCDR2 and LCDR3 sequence included.
  • the bispecific molecule that specifically binds IL-1R and IL-17 has a heavy chain and a light chain with the following amino acid sequences: SEQ ID NO: 34 and SEQ ID NO: 28; SEQ ID NO : 26 and SEQ ID NO: 36 SEQ ID NO: 30 and SEQ ID NO: 28; SEQ ID NO: 26 and SEQ ID NO: 32.
  • the bispecific molecule of the present invention includes a molecule that specifically binds to IL-1R and an antibody that targets the free inflammatory factor IL-6, and the molecule that specifically binds to IL-1R and the target The antibody to the free inflammatory factor IL-6 is linked by a connecting peptide.
  • the humanized antibody targeting free inflammatory factor IL-6 has a heavy chain shown in SEQ ID NO: 38 and a light chain shown in SEQ ID NO: 40.
  • the molecule that specifically binds IL-1R is a non-immunoglobulin polypeptide.
  • the non-immunoglobulin polypeptide of a molecule that specifically binds IL-1R is IL-1RA.
  • the non-immunoglobulin polypeptide IL-1RA that specifically binds to IL-1R has the nucleotide sequence shown in SEQ ID NO: 49 and/or has the amino acid shown in SEQ ID NO: 50 sequence.
  • the non-immunoglobulin polypeptide that specifically binds IL-1R is composed of an amino acid sequence having at least 85%-99% homology with SEQ ID NO:50.
  • the molecule that specifically binds IL-1R is an anti-IL-1R antibody.
  • the antibody that specifically binds IL-1R has a CDR group including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 have the following SEQ ID NO: 52, SEQ ID The HCDR1, HCDR2, and HCDR3 sequences contained in the amino acid sequence shown in NO: 56 or SEQ ID NO: 58; LCDR1, LCDR2, and LCDR3 have the amino acid sequence shown in SEQ ID NO: 54 or SEQ ID NO: 60 Contains LCDR1, LCDR2 and LCDR3 sequence.
  • the bispecific molecule that specifically binds IL-1R and IL-6 has a heavy chain and a light chain with the following amino acid sequences: SEQ ID NO: 46 and SEQ ID NO: 40; SEQ ID NO: 38 and SEQ ID NO: 48; SEQ ID NO: 38 and SEQ ID NO: 44; SEQ ID NO: 42 and SEQ ID NO: 40.
  • the present invention provides polynucleotides encoding the above-mentioned bispecific molecules.
  • the bispecific molecule encoding IL-1R that specifically binds to IL-1R and targets the free inflammatory factor IL-1 ⁇ has a heavy chain and a light chain with the following nucleotide sequences: SEQ ID NO: 9 and SEQ ID NO : 3; SEQ ID NO: 1 and SEQ ID NO: 11; SEQ ID NO: 13 and SEQ ID NO: 3; SEQ ID NO: 1 and SEQ ID NO: 15; SEQ ID NO: 17 and SEQ ID NO: 3 ; SEQ ID NO: 1 and SEQ ID NO: 19; SEQ ID NO: 5 and SEQ ID NO: 21; SEQ ID NO: 5 and SEQ ID NO: 23; SEQ ID NO: 61 and SEQ ID NO: 3; SEQ ID NO: 63 and SEQ ID NO: 3; SEQ ID NO: 65 and SEQ ID NO: 7; SEQ ID NO: 5 and SEQ ID NO: 67; SEQ ID NO: 69 and SEQ ID NO: 3; SEQ ID NO : 71
  • the bispecific molecule encoding IL-1R that specifically binds IL-1R and targets the free inflammatory factor IL-17 has a heavy chain and a light chain with the following nucleotide sequences: SEQ ID NO: 33 and SEQ ID NO : 27; SEQ ID NO: 25 and SEQ ID NO: 35; SEQ ID NO: 29 and SEQ ID NO: 27; SEQ ID NO: 25 and SEQ ID NO: 31.
  • the bispecific molecule encoding IL-1R that specifically binds IL-1R and targets the free inflammatory factor IL-6 has a heavy chain and a light chain with the following nucleotide sequences: SEQ ID NO: 45 and SEQ ID NO : 39; SEQ ID NO: 37 and SEQ ID NO: 47; SEQ ID NO: 37 and SEQ ID NO: 43; SEQ ID NO: 41 and SEQ ID NO: 39.
  • the present invention provides an expression vector comprising the polynucleotide of the present invention.
  • the present invention provides a host cell comprising the expression vector of the present invention.
  • the present invention provides a pharmaceutical composition comprising the bispecific molecule of the present invention and a pharmaceutically acceptable carrier or formulation.
  • the present invention also provides a method of treating inflammatory diseases and/or autoimmune diseases in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of the composition
  • the composition comprises the bispecific molecule of the present invention in a pharmaceutically acceptable form.
  • the present invention provides a bispecific molecule and its preparation and use.
  • the molecules that specifically bind IL-1R (such as non-immunoglobulin polypeptide IL-1RA, IL-1R antibody) will be linked to it.
  • Antibodies targeting free inflammatory factors (such as IL-1 ⁇ , IL-17 or IL-6) accumulate on or near the cell surface, which increases the concentration of bispecific molecules on or near the cell surface, which not only avoids the need for related technologies
  • the use of antibodies that target one or more free inflammatory factors in the systemic circulation brings about adverse reactions, and also avoids the limitation of curative effect caused by the use of targeted single inflammatory factor receptors in related technologies, and greatly improves the effectiveness of treatment. At the same time, it greatly reduces the risk of patient infection.
  • Figure 1 is the SDS-PAGE chart, where M is the protein marker, "-” means that the reducing agent DTT is not added when loading the sample, and “+” indicates that the reducing agent DTT is added when loading the sample.
  • Figure 1A is an SDS-PAGE diagram of bispecific molecules that specifically bind IL-1R and IL-1 ⁇ .
  • Lanes 1-8 are: BSI-1, BSI-2, BSI-3, BSI-4, BSI-5, BSI-6, BSI-7, BSI-8;
  • Figure 1B is an SDS-PAGE image of bispecific molecules that specifically bind IL-1R and IL-17A, where lane 1 is aIL-17AH, lanes 2-5 are: BSI -9, BSI-12, BSI-11, BSI-10;
  • Figure 1C is an SDS-PAGE of bispecific molecules that specifically bind IL-1R and IL-6, where lane 1 is aIL-6, lanes 2-5, respectively For: BSI-13, BSI-15, BSI-16, BSI-14
  • Figure 2 shows the results of gel exclusion chromatography.
  • Figure 2A shows the results of bispecific molecular gel exclusion chromatography that specifically binds IL-1R and IL-17.
  • Figure 2B shows the results of IL-1R and IL-6. Results of Bispecific Molecular Gel Exclusion Chromatography
  • Figure 3 shows the ELISA test results of bispecific molecules that bind IL-1R and IL-1 ⁇ and recombinant human IL-1 ⁇ , recombinant mouse IL-1 ⁇ , recombinant human IL-1R, and recombinant mouse IL-1R
  • Figure 4 is a bispecific molecule that specifically binds IL-1R and IL-1 ⁇ to inhibit the IL-1 ⁇ -induced HEK-Blue-IL1R cell signaling pathway.
  • 4A is the recombinant human IL-1 ⁇ -induced HEK-Blue-IL1R cell signaling pathway.
  • 4B is the bispecific molecule aIL-1 ⁇ -1 and IL-1RA used alone or in combination to inhibit the IL-1 ⁇ -induced HEK-Blue-IL1R cell signaling pathway
  • 4C-4F is the bispecific molecule (IL-1RA and aIL -1 ⁇ -1 fusion) inhibition of IL-1 ⁇ -induced HEK-Blue-IL1R cell signaling pathway
  • Figure 5 is a study on the effectiveness of bispecific molecules that specifically bind IL-1R and IL-1 ⁇ in a mouse model.
  • Blank is the experimental group without any drugs or human IL-1 ⁇ ; the DPBS group is injected with DPBS first. Human IL-1 ⁇ was injected 24 hours later; 1 is the aIL-1 ⁇ -1 administration group, 2 is the bispecific molecule (IL-1RA and aIL-1 ⁇ -1 fusion) administration group, and 3 is the isotype control antibody administration group , 4 is the IL-1RA and isotype antibody fusion administration group.
  • Blank is the experimental group without any drugs or human IL-1 ⁇ ; the DPBS group is injected with DPBS first. Human IL-1 ⁇ was injected 24 hours later; 1 is the aIL-1 ⁇ -1 administration group, 2 is the bispecific molecule (IL-1RA and aIL-1 ⁇ -1 fusion) administration group, and 3 is the isotype control antibody administration group , 4 is the IL-1RA and isotype antibody fusion administration group.
  • Figure 6 shows the IL-17 binding ELISA with bispecific molecules that specifically bind IL-1R and IL-17A
  • Figure 7 is the IL-6 binding ELISA with bispecific molecules that specifically bind IL-1R and IL-6
  • Figure 8 shows the amino acid sequence alignment of the heavy chain variable regions of three anti-IL-1R antibodies. Complementarity determining regions (CDRs) are underlined.
  • Figure 9 shows the amino acid sequence alignment of the light chain variable regions of three anti-IL-1R antibodies. Complementarity determining regions (CDRs) are underlined.
  • Figure 10 shows the variable regions of the heavy and light chains of aIL-1 ⁇ -1 antibody, and the complementarity determining regions (CDRs) are underlined.
  • Figure 11 shows the variable regions of the heavy and light chains of aIL-1 ⁇ -2 antibody, and the complementarity determining regions (CDRs) are underlined.
  • Figure 12 shows the variable regions of the heavy and light chains of the aIL-17A antibody, and the complementarity determining regions (CDRs) are underlined.
  • Figure 13 shows the variable regions of the heavy and light chains of aIL-16 antibody. Complementarity determining regions (CDRs) are underlined.
  • the "bispecific molecule” of the present invention is a binding molecule with two binding specificities. It can be a non-immunoglobulin polypeptide with antigen binding specificity or an antibody with antigen binding specificity.
  • the "antibody” of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of any one of them, which has the ability to specifically bind to an antigen under typical physiological conditions to induce, promote, Improve and/or regulate the physiological response related to antibody binding to antigen. Unless the context indicates otherwise or is obviously contradictory, the “antibody” of the present invention includes fragments of antibodies, which are antigen-binding fragments, that is, retain the ability to specifically bind to an antigen.
  • the "molecule that specifically binds IL-1R" in the present invention refers to a non-immunoglobulin polypeptide or antibody that can compete with IL-1 ⁇ and/or IL-1 ⁇ to bind to IL-1R and inhibit the biological activity of IL-1R .
  • Other single domain antibodies and heavy chain antibodies that can compete with IL-1 ⁇ and/or IL-1 ⁇ to bind to IL-1R and inhibit the biological activity of IL-1R are also included in the present invention.
  • inflammatory factor is a general term for cytokines involved in the occurrence and development of inflammation.
  • Free inflammatory factors refers to inflammatory factors that exist in solution in the body and are not expressed on the surface of cells. Common free inflammatory factors include but are not limited to the following examples: IL-1 superfamily (IL-1 ⁇ , IL-1 ⁇ , IL-18, IL-33, IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ ), IL-4, IL-13, IL-17A, IL-17E, IL-6, IL-12, IL-23, TNF superfamily (TNF ⁇ , TNF ⁇ , TNF ⁇ , OX40L (TNFSF4), CD40L (CD154), FasL (CD178, CD95L), CD27L (CD70), CD30L (CD153), 4-1BBL, CD253 (APO-2L) ), CD254, APO-3L (DR3L), CD256 (TALL-2), CD257 (BlyS), LIGHT (CD25), CD25 and CD25
  • CDR refers to the complementarity determining region within the immunoglobulin variable region sequence.
  • CDR1, CDR2, and CDR3 CDR1, CDR2, and CDR3.
  • CDR set refers to a set of three CDRs that appear in a single variable region capable of binding antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody or binding protein, but also provides a definition of each heavy chain or light chain The precise residue boundaries of the three CDRs in the sequence.
  • CDRs can be called Kabat CDRs. Chothia and colleagues (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 877-883) found that some sub-parts of Kabat CDR adopt almost the same peptide bone structure. Although there is great diversity at the amino acid sequence level. These sub-portions are named L1, L2 and L3 or H1, H2 and H3, where "L” and “H” refer to the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have borders that overlap with Kabat CDRs.
  • the term "homology” or “identity” refers to between two polymer molecules (for example, between two nucleic acid molecules (eg, between two DNA molecules or two RNA molecules) or between two polypeptide molecules ) Sequence identity of subunits. When the position of the secondary unit in these two molecules is occupied by the same monomeric secondary unit, for example, if a certain position in each molecule of two DNA molecules is occupied by adenine, they are homologous at that position Or the same. The identity between two sequences varies directly with the number of matching positions or homologous positions.
  • the two sequences are 50% homologous (also referred to as 50% homology or 50% identity); if 90% of the positions (for example, 9 positions out of 10) are matched or identical Source, these two sequences are 90% homologous (also said to have 90% homology or 90% identity).
  • inflammatory disease refers to a disease related to an inflammatory response.
  • inflammatory diseases include arthritis such as RA, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis and other inflammatory diseases of the joints, inflammatory bowel diseases such as ulcerative colitis, Crohn’s Disease, Barrett’s syndrome, ileitis, enteritis, gluten-sensitive enteropathy, inflammatory diseases of the respiratory system, such as asthma, adult respiratory distress syndrome, allergic rhinitis, silicosis, chronic Respiratory obstructive diseases, hypersensitivity lung diseases, bronchiectasis; inflammatory diseases of the inflammatory skin, including psoriasis, scleroderma, and inflammatory skin diseases such as eczema, atopic dermatitis, urticaria and pruritus; involved Disorders of central and external peripheral nervous system inflammation include multiple sclerosis, idiopathic demyelinating polyneuropathy, Guillain
  • inflammatory diseases include tuberculosis and chronic cholecystitis.
  • Harrison's Principles of Internal Medicine (Harrison Internal Medicine), 12th Edition, Wilson, et al., eds., McGrawill, Inc. also describe other chronic inflammatory diseases.
  • the pharmaceutical composition as described herein is prepared by mixing the bispecific antibody of the present invention with the desired purity and one or more optional pharmaceutically acceptable carriers, which is in the form of a lyophilized formulation or an aqueous solution.
  • the pharmaceutically acceptable carrier is generally non-toxic to the recipient at the dose and concentration used.
  • the present invention may be administered as an active ingredient alone, or in combination with, for example, an adjuvant or with other drugs such as immunosuppressive or immunomodulatory agents or other anti-inflammatory agents, for example for the treatment of inflammatory diseases and/or immune diseases.
  • an adjuvant or with other drugs such as immunosuppressive or immunomodulatory agents or other anti-inflammatory agents, for example for the treatment of inflammatory diseases and/or immune diseases.
  • Example 1 Construction, expression, purification and functional detection of bispecific molecules that specifically bind IL-1R and IL-1 ⁇
  • Example 1.2 Expression and purification of bispecific molecules that specifically bind IL-1R and IL-1 ⁇
  • the heavy chain and light chain of the expression vector constructed in Example 1.1 were transiently transfected into FreeStyle HEK293 cells (ThermoFisher), and the amount of heavy chain plasmid and light chain plasmid during transfection was 1:1): 28ml FreeStyle HEK293 ( A total of 3 ⁇ 10 7 cells) were inoculated into a 125ml cell culture flask, the plasmid was diluted with 1ml Opti-MEM (Invitrogen) and then added to 1ml Opti-MEM containing 60 ⁇ l 293Fectin (Invitrogen), allowed to stand at room temperature for 30 min, and the plasmid-293Fectin mixture Add to the cell culture medium 125rpm, 37°C, 5% CO 2 culture.
  • the cell culture supernatant was collected at 48h and 96h after transfection, and purified by Protein A Resin (Thermo Fisher Scientific). These proteins were analyzed by SDS-PAGE. The results are shown in Figure 1A, indicating that the expected bispecific molecule was successfully expressed.
  • Example 1.4 In vitro binding analysis of bispecific molecules that specifically bind IL-1R and IL-1 ⁇
  • the results are shown in Figure 3.
  • the bispecific molecules targeting IL-1R and IL-1 ⁇ have a binding ability to human IL-1 ⁇ or murine IL-1 ⁇ similar to aIL-1 ⁇ -2 antibody, indicating that the fusion of IL-1RA has a positive effect on aIL
  • the binding of -1 ⁇ antibody to IL-1 ⁇ has little effect.
  • the binding capacity of bispecific molecules to human IL-1 ⁇ is about 5-7 times that of mouse IL-1 ⁇ .
  • Example 1.5 Analysis of HEK-Blue-IL1R cell activation by recombinant human IL-1 ⁇
  • 5x10 4 HEK-Blue-IL1R cells (InvivoGene) were plated on a 96-well plate, and 100ul IL-1 ⁇ (R&D systems) (diluted with 5% FBS/DMEM medium) was added to each well with 100ul dilutions (0.024-50ng/ml) , Cultivate overnight.
  • 100ul IL-1 ⁇ R&D systems
  • 100ul dilutions 0.024-50ng/ml
  • IL-1 ⁇ has a strong ability to stimulate the HEK-Blue-IL1R cell pathway, and its EC50 is 1.54 ⁇ 0.35ng/ml.
  • Figure 4B-4F shows that after IL-1RA is fused to aIL-1 ⁇ antibody (aIL-1 ⁇ -1 or aIL-1 ⁇ -2), its ability to inhibit the HEK-Blue-IL1R cell signaling pathway induced by IL-1 ⁇ is stronger than that of alone
  • aIL-1 ⁇ antibody aIL-1 ⁇ -1 or aIL-1 ⁇ -2
  • Example 1.6 In vivo effectiveness study of bispecific molecules that specifically bind IL-1R and IL-1 ⁇ .
  • DPBS or bispecific molecules were injected intraperitoneally into female C57BL/6 mice (6-7 weeks). After 24 hours, each mouse was injected with 20ng of recombinant human IL- 1 ⁇ (PHC0815, ThermoFisher Scientific). After 2 hours, blood was taken from the posterior eye socket, and the mouse IL-6 ELISA MAX TM Deluxe (Cat.431306, BioLegend) was used to detect the level of mIL-6 in mouse plasma according to the instructions provided by the manufacturer. The data was drawn and analyzed with PrizmGraphpad software. Statistical analysis used unpaired, two-tailed t test.
  • the gene fragments encoding the heavy chain and light chain of the aIL-17 antibody were synthesized separately, and IL-1RA was fused to the N-terminal, light-chain N-terminal, and N-terminal of the heavy chain of the aIL-17 antibody through the connecting peptide L1, L2 or L3 using standard molecular biology techniques. C-terminus of heavy chain or C-terminus of light chain.
  • VH and VL of aIL-1R antibody Fuse the VH and VL of aIL-1R antibody to the N-terminus of the heavy and light chain of aIL-17 antibody by connecting peptides L4 and L5, respectively, or fuse the VH and VL of aIL-17 antibody to the heavy chain of aIL-1R antibody, respectively All sequences of the N-terminal of the light chain and the N-terminal of the light chain are verified by sequencing.
  • Example 2.2 Expression, purification and analysis of bispecific molecules that specifically bind IL-1R and IL-17
  • the heavy chain and light chain of the expression vector constructed in Example 2.1 were transiently transfected into FreeStyleHEK293 cells (ThermoFisher), and the amount of the plasmid of the heavy chain and the plasmid of the light chain during the transfection was 1:1: 28ml FreeStyle HEK 293 (total 3 ⁇ 10 7 cells) were inoculated into a 125ml cell culture flask, the plasmid was diluted with 1ml Opti-MEM (Invitrogen) and then added to 1ml Opti-MEM containing 60 ⁇ l 293Fectin (Invitrogen), allowed to stand at room temperature for 30min, and the plasmid-293Fectin mixture was added to Cell culture medium was cultured at 125 rpm, 37°C, and 5% CO 2 .
  • the cell culture supernatant was collected at 48h and 96h after transfection, and purified by Protein A Resin (Thermo Fisher Scientific). These proteins were analyzed by SDS-PAGE. The results are shown in Fig. 1B. The size of each lane is consistent with the expected, indicating that the expected bispecific molecule was successfully expressed.
  • GE's AKTA chromatography was used for gel exclusion chromatography.
  • the chromatography column used is HiLoad 16/600 Superdex 200 gel exclusion chromatography column, and the solution used for gel exclusion chromatography is DPBS buffer (0.010M phosphate buffer, 0.0027M KCl, 0.14M NaCl, pH 7.4 ), the flow rate used in gel exclusion chromatography is 0.4 ml/min.
  • the gel exclusion chromatography results of Fig. 2A show that the obtained bispecific molecules mainly exist in the form of monomers, and have high purity without obvious visible aggregation.
  • the affinity of IL-17, the affinity of bispecific molecules targeting IL-1RA and IL-17 to IL-17 is similar to that of aIL-17A antibody.
  • the gene fragments encoding the heavy and light chains of the aIL-6 antibody were synthesized separately, and IL-1RA was fused to the N-terminal, light-chain N-terminal, and N-terminal of the heavy chain of the aIL-6 antibody through the connecting peptide L1, L2 or L3 using standard molecular biology techniques.
  • the VH and VL of the aIL-1R antibody are fused to the N-terminus of the heavy and light chains of the aIL-6 antibody by connecting peptides L4 and L5, or the VH and VL was fused to the N-terminus of the heavy chain and the N-terminus of the light chain of the aIL-1R antibody, and all sequences were verified by sequencing.
  • Example 3.2 Expression, purification and analysis of bispecific molecules that specifically bind IL-1R and IL-6
  • the heavy chain and light chain of the expression vector constructed in Example 3.1 were transiently transfected into FreeStyle HEK293 cells (ThermoFisher), and the amount of the heavy chain plasmid and the light chain plasmid during transfection was 1:1: 28ml FreeStyle HEK293 (total 3 ⁇ 10 7 cells) were inoculated into a 125ml cell culture flask, the plasmid was diluted with 1ml Opti-MEM (Invitrogen) and then added to 1ml Opti-MEM containing 60 ⁇ l 293Fectin (Invitrogen), allowed to stand at room temperature for 30min, and the plasmid-293Fectin mixture was added to Cell culture medium was cultured at 125 rpm, 37°C, and 5% CO 2 .
  • the cell culture supernatant was collected at 48h and 96h after transfection, and purified by Protein A Resin (Thermo Fisher Scientific). These proteins were analyzed by SDS-PAGE. The results are shown in Figure 1C. The size of each lane is consistent with the expected, indicating that the expected bispecific molecule was successfully expressed.
  • GE's AKTA chromatography was used for gel exclusion chromatography.
  • the chromatography column used is HiLoad 16/600 Superdex 200 gel exclusion chromatography column, and the solution used for gel exclusion chromatography is DPBS buffer (0.010M phosphate buffer, 0.0027M KCl, 0.14M NaCl, pH 7.4 ), the flow rate used in gel exclusion chromatography is 0.4 ml/min.
  • the gel exclusion chromatography results of Fig. 2B show that the obtained bispecific molecules mainly exist in the form of monomers, and have high purity without obvious visible aggregation.
  • the ELISA results in Figure 7 show that the fusion of IL-1RA and aIL-6 antibody heavy or light chain does not affect the affinity of aIL-6 antibody to IL-6, and the bispecific molecular pair targeting IL-1RA and IL-6 The affinity of IL-6 is similar to that of aIL-6 antibody.

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Abstract

提供一种双特异性分子及其制备和用途，双特异性分子包括特异性结合白介素1受体(IL-1R)的分子和靶向游离炎症因子的抗体；通过特异性结合细胞表面白介素1受体(IL-1R)的分子将与之连接的靶向游离炎症因子的抗体聚集在细胞表面或其附近，使得细胞表面或其附近的双特异性分子浓度局部增高，避免了不良反应，提高治疗有效性的同时，降低了患者感染的风险。

Description

一种双特异性分子及其制备与用途 技术领域
本发明属于生物制药领域，具体涉及一种特异性结合白介素1受体(IL-1R)和游离炎症因子的双特异性分子及其制备和用途。
背景技术
促炎症细胞因子调节着机体的各种生理过程，其不正常的上调是各种炎症性疾病发生的主要原因。利用重组抗炎症细胞因子、重组可溶性受体或靶向炎症细胞因子的中和性抗体治疗炎症性疾病已经在临床上取得了较好的效果。
TNF-α是机体在遭受创伤或细菌感染早期产生的促炎症细胞因子。TNFR信号通路传导与多种炎症性疾病如RA(类风湿性关节炎)、克罗恩病(Crohn’s disease)、动脉粥样硬化、银屑病、败血症、糖尿病、肥胖等密切相关。Etanercept(依那西普)是第一个被批准用于治疗类风湿性关节炎的TNF-α抑制剂。靶向TNF-α的Infliximab(英夫利西单抗)也于1998年被FDA批准用于治疗克罗恩病。Infliximab结合单体(非活化状态)或三聚(活化状态)的TNF-α，而Etanercept主要结合活化的TNF-α三聚体。全人源的Adalimumab(阿达木单抗)于2002年被FDA批准用于治疗类风湿性关节炎。TNF-α是对抗细胞内细菌感染的主要因子，因此上述这些TNF-α抑制剂在表现出较好的临床效果的同时，也不可避免地增加了各种感染如肺结核、HBV的重新激活、带状疱疹病毒(herpes zoster virus，HZV)等的风险。作为免疫抑制药物，TNF-α抑制剂在表现出较好临床效果的同时，也大大增加了患者恶性血液肿瘤发生的概率。
与TNF-α家族类似，IL-1家族促炎症信号和抑制炎症信号之间的失衡也与各种慢性疾病的发生密切相关，如炎症性肠病、类风湿性关节炎、与IL-1β过表达有关的自身炎症性疾病等。目前已经有3个靶向IL-1通路的药物(Anakinra(阿那白滞素)，Canakinumab(康纳单抗))和Rilonacept(利纳西普)被FDA批准上市。与Anakinra类似，Canakinumab和Rilonacept可有效减少炎症性疾病的症状，此外，Canakinumab在治疗SJIA(全身型幼年特发性关节炎)方面还表现出较好的疗效。对4个治疗RA的临床试验的荟萃分析发现， 坏疽的发生率大大增加。因此，上述这些针对IL-1通路的药物均不推荐用于具有高感染风接受Anakinra治疗的患者肺炎、骨髓炎、蜂窝织炎、滑囊炎、带状疱疹、拇趾囊肿、险的患者。
IL-6是免疫系统中另一个多能干促炎症因子，其由多种细胞分泌，并可刺激B细胞、NK细胞、破骨细胞、肿瘤细胞，因此在自身免疫性疾病如类风湿性关节炎种发挥重要作用。靶向IL-6的人源化抗体Tocilizumab(托珠单抗)分别于2010年和2011年被批准用于RA和SJIA的治疗。靶向IL-6R的Siltuximab(司妥昔单抗)抗体被FDA批准用于免疫缺陷病毒(HIV)阴性且疱疹病毒(HHV-8)阴性的多中心性Castleman病(卡斯特莱曼病)。但是，这两个靶向药物的临床应用也带了诸如TNF-α、IL-1靶向药物类似的不良反应，大大增加了患者感染的几率。靶向效应Th17细胞因子的抗体Ustekinumab(优特克单抗)、Secukinumab(苏金单抗)和ixekizumab(艾塞吉珠单抗)在治疗前被要求接受预防治疗，以避免肺结核重新激活的风险(Rider P，Carmi Y and Cohen I.Iht J Cell Biol.2016：9259646)。
开发同时靶向上述通路的双特异性抗体，各大制药公司已经进行了多种尝试，例如靶向TNF-α和IL-17A的多个双特异性抗体如ABT-12、COVA-322、LY3114062、BCD-121等在临床试验过程中发现疗效未达预期(Baker，KF.et al.Ann Rheum Dis.2018，77(2)：175-187)。Abbott公司靶向IL-1α和IL-1β的双抗ABT-981最近的II期临床结果显示，ABT-981对侵蚀性手骨关节炎(HOA)的疼痛或影像学终点无改善作用，对Western Ontario和McMaster Universities Osteoarthritis(WOMAC)疼痛改善有限，未见对滑膜炎的改善作用(Kloppengurg，M.et al.Ann Rheum Dis.2019，78：413-420和Fleischmann，RM.et al.Arthritis Rheumatol.2019，doi：10.1002/art.40840)。
因此，本领域急需一种双特异性抗体，在提高其临床治疗炎症性疾病效果的同时，大大减少其对机体带来的不良反应。
本发明简述
本发明提供了一种双特异性分子及其制备与用途。
目前针对促炎症因子的药物多数针对的是体内游离的炎症因子，这些药物与炎症因子结合后随体液在全身循环，因此可能会带来对机体的全身不良反应，增加机体感染的风险。本发明人发现，通过特异性结合细胞表面炎症因子受体的分子为媒介，将靶向游离炎症因子的抗体局限在细胞表面或其附近，可以发挥两者协同的、局部的抗炎症效应，在增强 抗炎症效应的同时，可有效避免双特异性分子在全身循环可能带来的不良反应。
基于上述发现，本发明提供了一种双特异性分子，所述双特异性分子包括特异性结合IL-1R的分子和靶向游离炎症因子的抗体，其中，所述特异性结合IL-1R的分子与所述靶向炎症因子的抗体通过连接肽连接。本发明所述的双特异性分子，通过特异性结合IL-1R的分子，将双特异性分子聚集到表达IL-1R的细胞表面或其附近，避免其在体内随体液进行全身循环；接着，靶向游离炎症因子的抗体可进一步中和表达IL-1R的细胞表面或其附近由于炎症局部增高的游离炎症因子，在协同增强特异性结合IL-1R的分子的抗炎症反应的同时，避免了因游离炎症因子在体内广泛表达而导致靶向游离炎症因子的抗体产生对机体的不良反应。
在某些实施方式中，特异性结合IL-1R的分子为非免疫球蛋白多肽。在某些实施方式中，所述特异性结合IL-1R的非免疫球蛋白多肽为IL-1RA。在某些实施方式中，所述特异性结合IL-1R的IL-1RA具有如SEQ ID NO：49所示核苷酸序列和或具有如SEQ ID NO：50所示的氨基酸序列。在某些实施方式中，所述特异性结合IL-1R的非免疫球蛋白多肽为具有与SEQ ID NO：50具有至少85％-99％同源性的氨基酸序列组成。
在某些实施方式中，特异性结合IL-1R的分子为抗IL-1R抗体。在某些实施方式中，特异性结合IL-1R的抗体为单域抗体、嵌合抗体、人源化抗体、人源抗体或上述这些抗体的重组改变部分。在某些实施方式中，所述特异性结合IL-1R的抗体包括CDR组：HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中，HCDR1、HCDR2和HCDR3具有如SEQ ID NO：52、SEQ ID NO：56或SEQ ID NO：58所示的氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列；LCDR1、LCDR2和LCDR3具有如SEQ ID NO：54或SEQ ID NO：60所示的氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列。在某些实施方式中，特异性结合L-1R的抗体由如下重链和轻链组成：SEQ ID NO：52所示的重链和SEQ ID NO：54所示的轻链；SEQ ID NO：56所示的重链和SEQ ID NO：54所示的轻链；或者，SEQ ID NO：58所示的重链和SEQ ID NO：60所示的轻链。
在某些实施方式中，所述双特异性分子靶向的游离炎症因子选自以下(不限于)其中之一：IL-1超家族(IL-1α、IL-1β、IL-18、IL-33、IL-36α、IL-36β、IL-36γ)、IL-4、IL-13、IL-17A、IL-17E、IL-6、IL-12、IL-23、TNF超家族(TNFα、TNFβ、TNFγ、OX40L(TNFSF4)、CD40L(CD154)、FasL(CD178、CD95L)、CD27L(CD70)、CD30L(CD153)、4-1BBL、CD253(APO-2L)、CD254、APO-3L(DR3L)、CD256(TALL-2)、CD257(BlyS)、LIGHT(CD258)、TL1(TNFSF18、AITRL)、ED1-A1、BAFF、IFN或 GM-CSF。
在某些实施方式中，靶向前述游离炎症因子的抗体为嵌合抗体、人源化抗体、人源抗体或上述这些抗体的重组改变部分。
在某些具体的实施方式中，所述双特异性分子靶向的游离炎症因子为IL-1β。在某些实施方式中，所述靶向游离炎症因子IL-1β的抗体具有如SEQ ID NO：2所示的重链氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO：4所示的轻链氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列；或者具有如SEQ ID NO：6所示的重链氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO：8所示的轻链氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列。
在某些具体些实施方式中，所述双特异性分子靶向的游离炎症因子为IL-17A。在某些实施方式中，所述靶向游离炎症因子IL-17具有如SEQ ID NO：26所示的重链氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO：28所示的轻链氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列。
在某些具体实施方式中，所述双特异性分子靶向的游离炎症因子为IL-6。在某些实施方式中，所述靶向游离炎症因子IL-6的抗体具有如SEQ ID NO：38所示的重链氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO：40所示的轻链氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列。
在某些实施方式中，本发明的双特异性分子包括特异性结合IL-1R的分子和靶向游离炎症因子IL-1β的抗体，所述特异性结合IL-1R的分子与所述靶向游离炎症因子IL-1β的抗体通过连接肽连接。在某些实施方式中，所述靶向游离炎症因子IL-1β的人源化抗体具有SEQ ID NO：2所示的重链和SEQ ID NO：4所示的轻链。在某些实施方式中，所述靶向游离炎症因子IL-1β的人源化抗体具有如SEQ ID NO：6所示的重链和SEQ ID NO：8所示的轻链。在某些实施方式中，所述特异性结合IL-1R的分子为非免疫球蛋白多肽。在某些实施方式中，特异性结合IL-1R的分子的非免疫球蛋白多肽为IL-1RA。在某些实施方式中，特异性结合IL-1R的非免疫球蛋白多肽具有如SEQ ID NO：49所示的核苷酸序列和/或具有如SEQ ID NO：50所示的氨基酸序列。在某些实施方式中，所述特异性结合IL-1R的非免疫球蛋白多肽为具有与SEQ ID NO：50具有至少85％-99％同源性的氨基酸序列组成。在某些实施方式中，所述特异性结合IL-1R的分子为抗IL-1R的抗体。在某些实施方式中，所述特异性结合IL-1R的抗体具有包括CDR组：HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中，HCDR1、HCDR2和HCDR3具有如SEQ ID NO：52、SEQ ID NO：56或SEQ ID NO：58所示的氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列；LCDR1、LCDR2和LCDR3具有如SEQ ID NO：54或SEQ ID NO：60所示的氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列。在某些实施方式中， 所述特异性结合IL-1R和IL-1β的双特异性分子具有如下氨基酸序列的重链和轻链：SEQ ID NO：10和SEQ ID NO：4；SEQ ID NO：2和SEQ ID NO：12；SEQ ID NO：14和SEQ ID NO：4；SEQ ID NO：2和SEQ ID NO：16；SEQ ID NO：18和SEQ ID NO：4；SEQ IDNO：2和SEQ ID NO：20；SEQ ID NO：6和SEQ ID NO：22；SEQ ID NO：6和SEQ ID NO：24；SEQ ID NO：62和SEQ ID NO：4；SEQ ID NO：64和SEQ ID NO：4；SEQ ID NO：66和SEQ ID NO：8；SEQ ID NO：6和SEQ ID NO：68；SEQ ID NO：70和SEQ ID NO：4；SEQ ID NO：72和SEQ ID NO：4；SEQ ID NO：74和SEQ ID NO：4；SEQ ID NO：76和SEQ ID NO：4。
在某些实施方式中，本发明的双特异性分子包括特异性结合IL-1R的分子和靶向游离炎症因子IL-17的抗体，所述特异性结合IL-1R的分子与所述靶向游离炎症因子IL-17的抗体通过连接肽连接。在某些实施方式中，所述靶向游离炎症因子IL-17的人源化抗体具有SEQ ID NO：26所示的重链和SEQ ID NO：28所示的轻链。在某些实施方式中，所述特异性结合IL-1R的分子为非免疫球蛋白多肽。在某些实施方式中，特异性结合IL-1R的分子的非免疫球蛋白多肽为IL-1RA。在某些实施方式中，特异性结合IL-1R的非免疫球蛋白多肽IL-1RA具有如SEQ ID NO：49所示的核苷酸序列和/或具有如SEQ ID NO：50所示的氨基酸序列。在某些实施方式中，所述特异性结合IL-1R的非免疫球蛋白多肽为具有与SEQ ID NO：50具有至少85％-99％同源性的氨基酸序列组成。在某些实施方式中，所述特异性结合IL-1R的分子为抗IL-1R的抗体。在某些实施方式中，所述特异性结合IL-1R的抗体具有包括CDR组：HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中，HCDR1、HCDR2和HCDR3具有如SEQ ID NO：52、SEQ ID NO：56或SEQ ID NO：58所示的氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列；LCDR1、LCDR2和LCDR3具有如SEQ ID NO：54或SEQ ID NO：60所示的氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列。在某些实施方式中，所述特异性结合IL-1R和IL-17的双特异性分子具有如下氨基酸序列的重链和轻链：SEQ ID NO：34和SEQ ID NO：28；SEQ ID NO：26和SEQ ID NO：36SEQ ID NO：30和SEQ ID NO：28；SEQ ID NO：26和SEQ ID NO：32。
在某些实施方式中，本发明的双特异性分子包括特异性结合IL-1R的分子和靶向游离炎症因子IL-6的抗体，所述特异性结合IL-1R的分子与所述靶向游离炎症因子IL-6的抗体通过连接肽连接。在某些实施方式中，所述靶向游离炎症因子IL-6的人源化抗体具有SEQ ID NO：38所示的重链和SEQ ID NO：40所示的轻链。在某些实施方式中，所述特异性结合IL-1R的分子为非免疫球蛋白多肽。在某些实施方式中，特异性结合IL-1R的分子 的非免疫球蛋白多肽为IL-1RA。在某些实施方式中，特异性结合IL-1R的非免疫球蛋白多肽IL-1RA具有如SEQ ID NO：49所示的核苷酸序列和/或具有如SEQ ID NO：50所示的氨基酸序列。在某些实施方式中，所述特异性结合IL-1R的非免疫球蛋白多肽为具有与SEQ ID NO：50具有至少85％-99％同源性的氨基酸序列组成。在某些实施方式中，所述特异性结合IL-1R的分子为抗IL-1R的抗体。在某些实施方式中，所述特异性结合IL-1R的抗体具有包括CDR组HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中，HCDR1、HCDR2和HCDR3具有如SEQ ID NO：52、SEQ ID NO：56或SEQ ID NO：58所示的氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列；LCDR1、LCDR2和LCDR3具有如SEQ ID NO：54或SEQ ID NO：60所示的氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列。在某些实施方式中，所述特异性结合IL-1R和IL-6的双特异性分子具有如下氨基酸序列的重链和轻链：SEQ ID NO：46和SEQ ID NO：40；SEQ IDNO：38和SEQ ID NO：48；SEQ ID NO：38和SEQ ID NO：44；SEQ ID NO：42和SEQ ID NO：40。
一方面，本发明提供了编码上述双特异性分子的多核苷酸。
在某些实施方式中，编码特异性结合IL-1R和靶向游离炎症因子IL-1β的双特异性分子具有如下核苷酸序列的重链和轻链：SEQ ID NO：9和SEQ ID NO：3；SEQ ID NO：1和SEQ ID NO：11；SEQ ID NO：13和SEQ ID NO：3；SEQ ID NO：1和SEQ ID NO：15；SEQ ID NO：17和SEQ ID NO：3；SEQ ID NO：1和SEQ ID NO：19；SEQ ID NO：5和SEQ ID NO：21；SEQ ID NO：5和SEQ ID NO：23；SEQ ID NO：61和SEQ ID NO：3；SEQ ID NO：63和SEQ ID NO：3；SEQ ID NO：65和SEQ ID NO：7；SEQ ID NO：5和SEQ ID NO：67；SEQ ID NO：69和SEQ ID NO：3；SEQ ID NO：71和SEQ ID NO：3；SEQ ID NO：73和SEQ ID NO：3；SEQ ID NO：75和SEQ ID NO：3。。
在某些实施方式中，编码特异性结合IL-1R和靶向游离炎症因子IL-17的双特异性分子具有如下核苷酸序列的重链和轻链：SEQ ID NO：33和SEQ ID NO：27；SEQ ID NO：25和SEQ ID NO：35；SEQ ID NO：29和SEQ ID NO：27；SEQ ID NO：25和SEQ ID NO：31。
在某些实施方式中，编码特异性结合IL-1R和靶向游离炎症因子IL-6的双特异性分子具有如下核苷酸序列的重链和轻链：SEQ ID NO：45和SEQ ID NO：39；SEQ ID NO：37和SEQ ID NO：47；SEQ ID NO：37和SEQ ID NO：43；SEQ ID NO：41和SEQ ID NO：39。
一方面，本发明提供了一种表达载体，所述表达载体包含本发明的多核苷酸。一
方面，本发明提供了一种宿主细胞，所述宿主细胞包含本发明的表达载体。
一方面，本发明提供了一种药物组合物，其包含本发明的双特异性分子和药学上 可接受的载体或制剂。
另一方面，本发明还提供了一种治疗治疗有需要的受试者的炎症性疾病和/或自身免疫性疾病的方法，所述方法包括对所述受试者施用治疗有效量的组合物，所述组合物包含药学可接受形式的本发明所述的双特异性分子。
如上所述，本发明提供了一种双特异性分子及其制备和用途，通过特异性结合IL-1R的分子(如非免疫球蛋白多肽IL-1RA、IL-1R抗体)将与之连接的靶向游离炎症因子(如IL-1β、IL-17或IL-6)的抗体聚集在细胞表面或其附近，使得细胞表面或其附近的双特异性分子浓度局部增高，不仅避免了相关技术中使用靶向一个或多个游离炎症因子的抗体在全身循环带来的不良反应，而且还避免了相关技术中使用靶向单一炎症因子受体所带来的疗效受限问题，在大大提高治疗有效性的同时，极大降低了患者感染的风险。
附图说明
构成本申请的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定，在附图中：
图1为SDS-PAGE图，其中M为蛋白marker，“-”表示上样时不加还原剂DTT，“+”表示上样时加还原剂DTT。图1A为特异性结合IL-1R和IL-1β的双特异分子SDS-PAGE图，其中泳道1-8分别为：BSI-1、BSI-2、BSI-3、BSI-4、BSI-5、BSI-6、BSI-7、BSI-8；图1B为特异性结合IL-1R和IL-17A的双特异分子SDS-PAGE图，其中泳道1为aIL-17AH，泳道2-5分别为：BSI-9、BSI-12、BSI-11、BSI-10；图1C为特异性结合IL-1R和IL-6的双特异分子SDS-PAGE图，其中泳道1为aIL-6，泳道2-5分别为：BSI-13、BSI-15、BSI-16、BSI-14
图2为凝胶排阻层析分析结果，其中图2A为特异性结合IL-1R和IL-17的双特异分子凝胶排阻层析结果，图2B为靶向IL-1R和IL-6的双特异分子凝胶排阻层析结果
图3为结合IL-1R和IL-1β的双特异性分子与重组人IL-1β、、重组鼠IL-1β、重组人IL-1R、重组鼠IL-1R ELISA检测结果
图4为特异性结合IL-1R和IL-1β的双特异分子抑制IL-1β诱导的HEK-Blue-IL1R细胞信号通路，其中4A为重组人IL-1β诱导HEK-Blue-IL1R细胞信号通路，4B为双特异性分子aIL-1β-1与IL-1RA单用或联合使用对IL-1β诱导的HEK-Blue-IL1R细胞信号通路的抑制，4C-4F为双特异分子(IL-1RA与aIL-1β-1融合)对IL-1β诱导的HEK-Blue-IL1R细胞信号通路的抑制
图5为特异性结合IL-1R和IL-1β的双特异分子在小鼠模型上的有效性研究，其中Blank为未注射任何药物或human IL-1β的实验组；DPBS组为先注射DPBS，24小时后注射human IL-1β；1为aIL-1β-1给药组，2为双特异性分子(IL-1RA与aIL-1β-1融合)给药组，3为isotype对照抗体给药组，4为IL-1RA与isotype抗体融合给药组。其中，“*”：与DPBS组相比p＜0.05；“**”：与DPBS组相比p＜0.005；“***”：与DPBS组相比p＜0.0005。
图6为特异性结合IL-1R和IL-17A的双特异分子与IL-17结合ELISA
图7为特异性结合IL-1R和IL-6的双特异分子与IL-6结合ELISA
图8显示了三个抗-IL-1R抗体重链可变区的氨基酸序列比对。互补决定区(CDRs)为下划线标出的部分。
图9显示了三个抗-IL-1R抗体轻链可变区的氨基酸序列比对。互补决定区(CDRs)为下划线标出的部分。
图10显示了aIL-1β-1抗体重链和轻链可变区，互补决定区(CDRs)为下划线标出的部分。
图11显示了aIL-1β-2抗体重链和轻链可变区，互补决定区(CDRs)为下划线标出的部分。
图12显示了aIL-17A抗体重链和轻链可变区，互补决定区(CDRs)为下划线标出的部分。
图13显示了aIL-16抗体重链和轻链可变区，互补决定区(CDRs)为下划线标出的部分。
本发明的详述
本发明在此通过对使用下述定义和实施例的引用进行详细描述。所有在本文中提及的专利和公开文献的内容，包括在这些专利和公开中披露的所有序列，明确地通过提述并入本文。
双特异性分子
本发明的“双特异性分子”是具有两种结合特异性的结合分子，其可以是具有抗原结合特异性的非免疫球蛋白多肽，也可以是具有抗原结合特异性的抗体。
抗体
本发明的“抗体”指免疫球蛋白分子、免疫球蛋白分子的片段，或其任一者的衍生物，其具有以下的能力：在典型的生理条件下特异性结合抗原，以诱导、促进、提高和/或调节与抗体结合抗原有关的生理反应。除非上下文另外指明或明显矛盾，本发明的“抗体”包括抗体的片段，其为抗原-结合片段，即，保留特异性结合抗原的能力。
结合IL-1R的分子
本发明中所述的“特异性结合IL-1R的分子”是指能与IL-1α和/或IL-1β竞争结合IL-1R，并抑制IL-1R生物活性的非免疫球蛋白多肽或抗体。其它能与IL-1α和/或IL-1β竞争结合IL-1R，并抑制IL-1R生物活性的单域抗体、重链抗体也包括在本发明中。
炎症因子
如本申请所用“炎症因子”是参与炎症发生、发展的细胞因子的总称。“游离的炎症因子”是指在体内以溶液中的形式存在的、不表达于细胞表面的炎症因子，常见的游离炎症因子包括但不限于如下所列例子：IL-1超家族(IL-1α、IL-1β、IL-18、IL-33、IL-36α、IL-36β、IL-36γ)、IL-4、IL-13、IL-17A、IL-17E、IL-6、IL-12、IL-23、TNF超家族(TNFα、TNFβ、TNFγ、OX40L(TNFSF4)、CD40L(CD154)、FasL(CD178、CD95L)、CD27L(CD70)、CD30L(CD153)、4-1BBL、CD253(APO-2L)、CD254、APO-3L(DR3L)、CD256(TALL-2)、CD257(BlyS)、LIGHT(CD258)、TL1(TNFSF18、AITRL)、ED1-A1)、BAFF、IFN或GM-CSF等。炎症因子受体是指可与炎症因子结合、且表达于细胞表面的分子。
CDR
术语“CDR”是指在免疫球蛋白可变区序列内的互补决定区。对于各重和轻链可变区，在重链和轻链的各可变区中存在三个CDR，其被命名为CDR1、CDR2和CDR3。术语“CDR组”是指在能够结合抗原的单一可变区中出现的三个CDR的组。这些CDR的确切边界已根据不同系统不同地定义。由Kabat(Kabat等(1987)和(1991))描述的系统，不仅提供了可适用于抗体或结合蛋白的任何可变区的明确残基编号系统，而且还提供了定义各重链或轻链序列中的三个CDR的精确残基边界。这些CDR可以被称为Kabat CDR。Chothia和同事(Chothia和Lesk(1987)J.Mol.Biol.196：901-917；Chothia等(1989)Nature342：877-883)发现Kabat CDR内的某些亚部分采取几乎相同的肽骨架构象，尽管在氨基酸序列水平上具有大的多样性。这些亚部分被命名为L1、L2和L3或H1、H2和H3，其中“L”和“H”分别指轻链和重链区域。这些区域可以被称为ChothiaCDR，所述Chothia CDR具有与Kabat CDR重叠的边界。定义与Kabat CDR重叠的CDR的其它边界已由Padlan(1995)FASEB J.9：133-139和MacCallum(1996)J.Mol.Biol.262(5)：732-45)描述。还有其它CDR边界定义可以不严格遵循本文系统之一，但仍将与Kabat CDR重叠，尽管鉴于特定残基或残基组或甚至整个CDR不显著影响抗原结合的预测或实验发现，它们可以缩短或加长。本文使用的方法可以利用根据这些系统中的任一种定义的CDR，尽管某些实施方案使用Kabat或Chothia定义的CDR(CN105324396 A)。
术语“同源性”或“同一性”指两个聚合物分子之间(例如，在两个核酸分子(如，两个DNA分子或两个RNA分子之间)或在两个多肽分子之间)的次级单位序列同一性。当这两个分子中的次级单位位置被相同单体性次级单位占据时，例如，若两个DNA分子中每个分子中的某位置被腺嘌呤占据，则它们在该位置是同源或相同的。两个序列之间的同一性直接随匹配位置或同源位置的数目而变化，例如，如果两个序列中一半位置(例如长度为十个次级单位的聚合物中的五个位置)是同源的，则这两个序列是50％同源的(亦可称具有50％同源性或50％同一性)；如果90％的位置(例如10个位置中9个位置)是匹配或同源的，这这两个序列是90％同源的(亦可称具有90％同源性或90％同一性)。
炎症性疾病
如本申请所用的“炎症性疾病”指的是与炎症反应有关的疾病。炎症性疾病的实例包括关节炎如RA、牛皮癣银屑病关节炎、强直性脊柱炎、青少年特发性关节炎以及关节的其他炎症疾病，炎性肠疾病如溃疡性结肠炎、克罗恩氏病、巴雷特综合征(Barrett′s syndrome)，回肠炎、肠炎、谷蛋白敏感性肠病，呼吸系统的炎性病症，诸如哮喘、成人呼吸窘迫综合征、变态反应性鼻炎、矽肺、慢性呼吸道阻塞性疾病、超敏反应肺疾病、支气管扩张症；炎症性皮肤的炎症疾病，包括银屑病、硬皮病和诸如湿疹、特应性皮炎、荨麻疹和瘙痒症的炎症皮肤病；涉及中枢和外周围神经系统炎症的病症包括多发性硬化、特发性脱髓鞘性多神经病、格-巴二氏综合征(Guillain-Barre syndrome)、慢性炎症性脱髓鞘性多发性神经病和诸如阿尔茨海默氏病的神经变性疾病；系统性红斑性狼疮，免疫介导性肾脏病，例如肾小球肾炎和脊椎关节病；带有不良慢性炎症成分的疾病，诸如系统性硬化病，特发性炎症性肌病，肖格伦氏综合征(Sjogren′s syndrome)，脉管炎，肉瘤样病，甲状腺炎，痛风，耳炎，结膜炎，窦炎，肉瘤样病，贝赫切特综合征(Behcet′s syndrome)，肝胆管疾病诸如肝炎、原发性胆汁性肝硬化、肉芽肿性肝炎和硬化性胆管炎；炎症和心血管系统的缺血损伤诸如缺血性心脏病，中风和动脉粥样硬化；以及移植物排斥(graft rejection)，包括同种异体移植排斥和移植物抗宿主病。各种其它炎症疾病包括肺结核和慢性胆囊炎。例如在Harrison′s Principles of Internal Medicine(哈里森内科学)，12th Edition，Wilson，et al.，eds.，McGrawill，Inc.)中也描述了其它慢性炎症疾病。
药物组合物
通过混合具有想要纯度的本发明双特异性抗体与一种或多种任选的药学上可接受的载体来制备如本文所述的药物组合物，其为冻干制剂或水溶液的形式。药学可接受的载体在采用的剂量和浓度下对接受者一般是无毒的。
本发明可以作为单独的活性成分，或与例如佐剂或与其他药物例如免疫抑制或免疫 调节剂或其他抗炎剂组合施用，例如用于治疗炎症性疾病和/或免疫性疾病。
实施例
需要说明的是，在不冲突的情况下，本申请中的实施例仅为举例说明，不旨在对本发明造成任何方式上的限制。
实施例1特异性结合IL-1R和IL-1β的双特异分子的构建、表达、纯化和功能检测
实施例1.1特异性结合IL-1R和IL-1β的双特异分子的克隆
分别合成编码aIL-1R-1、aIL-1R-2、aIL-1R-3、aIL-1β-1、aIL-1β-2抗体的重链(HC)和轻链(LC)、IL-1RA的基因片段，采用标准分子生物学技术通过连接肽将IL-1RA融合在aIL-1β抗体(aIL-1β-1和aIL-1β-2)重链N端、轻链N端、重链C端或轻链C端，将aIL-1R抗体的VH和VL分别通过连接肽融合到aIL-1β抗体重链和轻链的N端，或者将aIL-1β抗体的VH和VL分别通过连接肽融合到aIL-1R抗体重链的N端和轻链的N端，所有的序列都通过测序验证。
表1双特异分子核酸和序列
Figure PCTCN2020091060-appb-000001
Figure PCTCN2020091060-appb-000002
Figure PCTCN2020091060-appb-000003
实施例1.2特异性结合IL-1R和IL-1β的双特异分子的表达和纯化
将实施例1.1构建好的表达载体重链和轻链瞬时转染FreeStyle HEK293细胞(ThermoFisher)，转染时重链的质粒和轻链的质粒用量为摩尔比1∶1)：将28ml FreeStyle HEK293(共3×10 7细胞)接种至125ml细胞培养瓶，质粒用1ml Opti-MEM(Invitrogen)稀释后加至1ml含60μl 293Fectin(Invitrogen)的Opti-MEM中，室温静置30min，将质粒-293Fectin mixture加至细胞培养液中125rpm，37℃，5％CO 2培养。分别于转染后48h和96h收集细胞培养上清，Protein A Resin(Thermo Fisher Scientific)纯化。通过SDS-PAGE分析这些蛋白质。结果如图1A所示，说明成功表达出了预期的双特异性分子。
实施例1.3特异性结合IL-1R和IL-1β的双特异分子质谱分析
将实施例2中获得的Protein A resin纯化后的样品与PNGase F(NEB)37℃孵育8小时后，加10mM二硫苏糖醇处理后，将样品注射进HPLC-Q-TOF-MS(Agilent，USA)的300SB-C8，2.1 x 50mm柱，进行质谱分析。如表2所示，不同融合形式的抗体融合蛋白利用质谱检测得到的分子量与理论预测值基本一致。
表2双特异分子理论分子量及MS检测分子量
Figure PCTCN2020091060-appb-000004
实施例1.4特异性结合IL-1R和IL-1β的双特异分子体外结合分析
包被重组人IL-1β(ThermoFisher Scientific)、重组小鼠IL-1β(SinoBio)、重组小鼠IL-1R(SinoBio)、重组人IL-1R(100ng/孔)(DPBS buffer，pH7.4)于96孔板，4℃孵育过夜；含2％脱脂奶粉的PBST(0.5％Tween-20in PBS)室温封闭1小时，经PBST洗3次后，分别加入梯度稀释的双特异分子(0.006nM-100nM)37℃孵育1h，PBST洗3次后，加入2％milk/PBST稀释的anti-human Fc-HRP(KPL；1∶2500)二抗室温孵育1h，PBST洗5次后，QuantaBlu荧光过氧化物酶底物(Life technologies，Cat.15169)显色后于325nm和420nm处读数，或者TMB显色试剂(BioLegend)显色后于650um处读数。Prizm Graphpad软件用non-linear regression中specific binding模型对数据进行非线性回归。
结果如图3所示，靶向IL-1R和IL-1β的双特异分子对人IL-1β或鼠IL-1β的结合能力与aIL-1β-2抗体类似，说明IL-1RA的融合对aIL-1β抗体与IL-1β的结合影响很小。双特异分子对人IL-1β的结合能力大约是对小鼠IL-1β结合能力的5-7倍。
实施例1.5重组人IL-1β激活HEK-Blue-IL1R细胞分析
5x10 4HEK-Blue-IL1R细胞(InvivoGene)铺于96孔板，每孔加100ul梯度稀释(0.024-50ng/ml)的IL-1β(R&D systems)(用5％FBS/DMEM的培养基稀释)，培养过夜。取细胞上清与QUANTI-Blue(InvivoGene)按1∶9混匀，37℃孵育4小时后，Spectramax fluorescence plate reader读取655nm波长的吸光度。数据用PrizmGraphpad软件“non-linear regression”中的log[agonist]vs response(3 parameters)模型绘制和分析。如图4A所示，IL-1β具有较强的刺激HEK-Blue-IL1R细胞通路的能力，其EC50为1.54±0.35ng/ml。图4B-4F显示，aIL-1β抗体(aIL-1β-1或aIL-1β-2)上融合IL-1RA后，其抑制IL-1β诱导的HEK-Blue-IL1R细胞信号通路的能力强于单独的IL-1RA、aIL-1β抗体或IL-1RA+aIL-1β联用，提示双特异分子的IL-1RA与aIL-1β对IL-1R具有协同抑制作用。
实施例1.6特异性结合IL-1R和IL-1β的双特异分子体内有效性研究。
将DPBS或双特异分子(两个剂量：5mg/kg，15mg/kg)腹腔注射雌性C57BL/6小鼠(6-7周)，24小时后，往上述小鼠每只注射20ng重组人IL-1β(PHC0815，ThermoFisher Scientific)。2小时后，后眼窝取血，根据厂家提供的说明书，利用Mouse IL-6 ELISA MAX TM Deluxe(Cat.431306，BioLegend)检测小鼠血浆中mIL-6的水平。数据用PrizmGraphpad软件绘 制和分析。统计分析采用非配对、双尾t检验。
结果如图5所示：与Blank组相比，DPBS组小鼠体内IL-6的水平明显上调。相比于DPBS组，给药组中，15mg/ml和5mg/ml剂量的IL-1RA与aIL-1β-1融合的双特异性分子可以显著抑制IL-1β引起的小鼠IL-6的上调；注射15mg/ml剂量的IL-1RA与isotype的融合抗体只能一定程度上抑制IL-1β引起的小鼠IL-6的上调。
实施例2特异性结合IL-1R和IL-17的双特异分子的构建、表达、纯化和功能检测实施例
2.1特异性结合IL-1R和IL-17的双特异分子的克隆
分别合成编码aIL-17抗体重链和轻链的基因片段，采用标准分子生物学技术通过连接肽L1、L2或L3将IL-1RA融合在aIL-17抗体重链N端、轻链N端、重链C端或轻链C端。通过连接肽L4和L5将aIL-1R抗体的VH和VL分别融合到aIL-17抗体重链和轻链的N端，或者将aIL-17抗体的VH和VL分别融合到aIL-1R抗体重链的N端和轻链的N端，所有的序列都通过测序验证。
实施例2.2特异性结合IL-1R和IL-17的双特异分子的表达、纯化和分析
将实施例2.1构建好的表达载体重链和轻链瞬时转染FreeStyleHEK293细胞(ThermoFisher)，转染时重链的质粒和轻链的质粒用量为摩尔比1∶1：将28ml FreeStyle HEK 293(共3×10 7细胞)接种至125ml细胞培养瓶，质粒用1ml Opti-MEM(Invitrogen)稀释后加至1ml含60μl293Fectin(Invitrogen)的Opti-MEM中，室温静置30min，将质粒-293Fectin mixture加至细胞培养液中125rpm，37℃，5％CO 2培养。分别于转染后48h和96h收集细胞培养上清，Protein A Resin(Thermo Fisher Scientific)纯化。通过SDS-PAGE分析这些蛋白质。结果如图1B所示，各泳道条带的大小与预期的一致，说明成功表达出了预期的双特异性分子。
为进一步研究双特异分子在溶液中的物理特性，用GE的AKTA chromatography进行凝胶排阻层析。所用的层析柱为HiLoad 16/600 Superdex 200凝胶排阻层析柱，凝胶排阻层析所用的溶液为DPBS缓冲液(0.010M phosphate buffer，0.0027M KCl，0.14M NaCl，pH7.4)，凝胶排阻层析所用的流速为0.4ml/min。
图2A的凝胶排阻层析结果显示，所获得的双特异分子主要以单体形式存在，并且具有较高的纯度，无明显可见聚集。
实施例2.3特异性结合IL-1R和IL-17的双特异分子体外结合分析
包被重组人IL-17(SinoBiological)(100ng/孔)(PBS buffer，pH7.4)于96孔板，4℃孵育过夜；含2％脱脂奶粉的PBST(0.5％Tween-20in PBS)室温封闭1小时，经PBST洗3次后，分别加入梯度稀释的双特异分子(0.04nM-30nM)37℃孵育1h，PBST洗3次后，加入2％milk/PBST稀释的anti-human Fc-HRP(KPL，1∶2500)二抗室温孵育1h，PBST洗5次后TMB(BioLegend)显色，Thermal VARIOSKAN FLASH plate reader在650m波长下读数，数据用PrizmGraphpad软件“non-linear regression”中的log[agonist]vs response(3 parameters)模型绘制和分析。
图6的ELISA结果显示，IL-1RA与aIL-17A抗体重链或轻链的融合并不影响aIL-17A抗体对
IL-17的亲和力，靶向IL-1RA和IL-17的双特异分子对IL-17的亲和力与aIL-17A抗体相似。
实施例3特异性结合IL-1R和IL-6的双特异分子的构建、表达、纯化和功能检测
实施例3.1特异性结合IL-1R和IL-6的双特异分子的克隆
分别合成编码aIL-6抗体重链和轻链的基因片段，采用标准分子生物学技术通过连接肽L1、L2或L3将IL-1RA融合在aIL-6抗体重链N端、轻链N端、重链C端或轻链C端，通过连接肽L4和L5将aIL-1R抗体的VH和VL分别融合到aIL-6抗体重链和轻链的N端，或者将aIL-6抗体的VH和VL分别融合到aIL-1R抗体重链的N端和轻链的N端，所有的序列都通过测序验证。
实施例3.2特异性结合IL-1R和IL-6的双特异分子的表达、纯化和分析
将实施例3.1构建好的表达载体重链和轻链瞬时转染FreeStyle HEK293细胞(ThermoFisher)，转染时重链的质粒和轻链的质粒用量为摩尔比1∶1：将28ml FreeStyle HEK293(共3×10 7细胞)接种至125ml细胞培养瓶，质粒用1ml Opti-MEM(Invitrogen)稀释后加至1ml含60μl293Fectin(Invitrogen)的Opti-MEM中，室温静置30min，将质粒-293Fectin mixture加至细胞培养液中125rpm，37℃，5％CO 2培养。分别于转染后48h和96h收集细胞培养上清，Protein A Resin(Thermo Fisher Scientific)纯化。通过SDS-PAGE分析这些蛋白质。结果如图1C所示，各泳道条带的大小与预期的一致，说明成功表达出了预期的双特异性分子。
为进一步研究双特异分子在溶液中的物理特性，用GE的AKTA chromatography进行凝胶排阻层析。所用的层析柱为HiLoad 16/600 Superdex 200凝胶排阻层析柱，凝胶排阻层析所用的溶液为DPBS缓冲液(0.010M phosphate buffer，0.0027M KCl，0.14M NaCl，pH7.4)，凝胶排阻层析所用的流速为0.4ml/min。
图2B的凝胶排阻层析结果显示，所获得的双特异分子主要以单体形式存在，并且具有较高的纯度，无明显可见聚集。
实施例3.3特异性结合IL-1R和IL-6的双特异分子体外结合分析
包被重组人IL-6(SinoBiological)(100ng/孔)(PBS buffer，pH7.4)于96孔板，4℃孵育过夜；含2％脱脂奶粉的PBST(0.5％Tween-20in PBS)室温封闭1小时，经PBST洗3次后，分别加入梯度稀释的双特异分子(0.04nM-30nM)37℃孵育1h，PBST洗3次后，加入2％milk/PBST稀释的anti-human Fc-HRP(KPL，1∶2500)二抗室温孵育1h，PBST洗5次后TMB(BioLegend)显色，Thermal VARIOSKAN FLASH plate reader在650nm波长下读数，数据用PrizmGraphpad软件的特定结合模型中的“non-linear regression”绘制和分析。
图7的ELISA结果显示，IL-1RA与aIL-6抗体重链或轻链的融合并不影响aIL-6抗体对IL-6的亲和力，靶向IL-1RA和IL-6的双特异分子对IL-6的亲和力与aIL-6抗体相似。

Claims (24)

  1. 一种双特异性分子，其包括：
    a)特异性结合IL-1R的分子；
    b)靶向游离炎症因子的抗体；
    其中，所述特异性结合IL-1R的分子与所述靶向炎症因子的抗体通过连接肽连接。
  2. 权利要求1的双特异性分子，其中，所述特异性结合IL-1R的分子为非免疫球蛋白多肽。
  3. 权利要求2的双特异性分子，其中，所述特异性结合IL-1R的非免疫球蛋白多肽为IL-1RA。
  4. 权利要求2的双特异性分子，其中，所述特异性结合IL-1R的非免疫球蛋白多肽包含与SEQ ID NO：50具有85％-99％同源性的氨基酸序列，或由这样的氨基酸序列组成。
  5. 权利要求1的双特异性分子，其中，所述特异性结合IL-1R的分子为抗IL-1R的抗体。
  6. 权利要求5的双特异性分子，其中，所述抗IL-1R的抗体为单域抗体、嵌合抗体、人源化抗体、人源抗体或上述这些抗体的重组改变部分。
  7. 权利要求5的双特异性分子，其中，所述抗IL-1R的抗体包括CDR组：HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中，
    a) HCDR1、HCDR2和HCDR3分别具有如SEQ ID NO：52序列中所含的HCDR1、HCDR2和HCDR3序列；LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：54序列中所含的LCDR1、LCDR2和LCDR3序列；
    b) HCDR1、HCDR2和HCDR3分别具有如SEQ ID NO：56序列中所含的HCDR1、HCDR2和HCDR3序列；LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：54序列中所含的LCDR1、LCDR2和LCDR3序列；或者
    c) HCDR1、HCDR2和HCDR3分别具有如SEQ ID NO：58所示的氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列；LCDR1、LCDR2和LCDR3具有如SEQ ID NO：60序列中所含的LCDR1、LCDR2和LCDR3序列。
  8. 权利要求5的双特异性分子，其中，所述抗IL-1R的抗体具有如下重链和轻链：
    a)SEQ ID NO：52所示的重链和SEQ ID NO：54所示的轻链；
    b)SEQ ID NO：56所示的重链和SEQ ID NO：54所示的轻链；或者c)SEQ ID NO：58所示的重链和SEQ ID NO：60所示的轻链。
  9. 权利要求1-6中任一项所述的双特异性分子，其中所述游离炎症因子选自以下其中之一：IL-1超家族(IL-1α、IL-1β、IL-18、IL-33、IL-36α、IL-36β、IL-36γ)、IL-4、IL-13、IL-17A、IL-17E、IL-6、IL-12、IL-23、TNF超家族(TNFα、TNFβ、TNFγ、OX40L(TNFSF4)、 CD40L(CD154)、FasL(CD178、CD95L)、CD27L(CD70)、CD30L(CD153)、4-1BBL、CD253(APO-2L)、CD254、APO-3L(DR3L)、CD256(TALL-2)、CD257(BlyS)、LIGHT(CD258)、TL1(TNFSF18、AITRL)、ED1-A1)、BAFF、IFN或GM-CSF。
  10. 权利要求9的双特异性分子，其中，所述游离炎症因子为IL-1β。
  11. 权利要求10的双特异性分子，其中，靶向所述游离炎症因子IL-1β的抗体具有如SEQ ID NO：2所示的重链氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO：4所示的轻链氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列；或者具有如SEQ ID NO：6所示的重链氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO：8所示的轻链氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列。
  12. 权利要求9的双特异性分子，其中，所述游离炎症因子为IL-17。
  13. 权利要求12的双特异性分子，其中，靶向所述游离炎症因子IL-17的抗体具有如SEQ ID NO：26所示的重链氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO：28所示的轻链氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列。
  14. 权利要求9的双特异性分子，其中，所述游离炎症因子为IL-6。
  15. 权利要求14的双特异性分子，其中，靶向所述游离炎症因子IL-6的抗体具有如SEQ ID NO：38所示的重链氨基酸序列中所含的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO：40所示的轻链氨基酸序列中所含的LCDR1、LCDR2和LCDR3序列。
  16. 权利要求1-15中任一项的双特异性分子，其中，靶向所述游离炎症因子的抗体为嵌合抗体、人源化抗体、人源抗体或上述这些抗体的重组改变部分。
  17. 权利要求2的双特异性分子，其中，所述双特异性分子包括靶向游离炎症因子IL-1β的抗体，且所述双特异性分子包含分别具有如下氨基酸序列的重链和轻链：SEQ ID NO：10和SEQ ID NO：4；SEQ ID NO：2和SEQ ID NO：12；SEQ ID NO：14和SEQ ID NO：4；SEQ ID NO：2和SEQ ID NO：16；SEQ ID NO：18和SEQ ID NO：4；SEQ ID NO：2和SEQ ID NO：20；SEQ ID NO：6和SEQ ID NO：22；SEQ ID NO：6和SEQ ID NO：24；SEQ ID NO：62和SEQ ID NO：4；SEQ ID NO：64和SEQ ID NO：4；SEQ ID NO：66和SEQ ID NO：8；SEQ ID NO：6和SEQ ID NO：68；SEQ ID NO：70和SEQ ID NO：4；SEQ ID NO：72和SEQ ID NO：4；SEQ ID NO：74和SEQ ID NO：4；SEQ ID NO：76和SEQ ID NO：4。
  18. 权利要求2的双特异性分子，其中，所述双特异性分子包括靶向游离炎症因子IL-17的抗体，且所述双特异性分子包含分别具有如下氨基酸序列的重链和轻链：SEQ ID NO：34和SEQ ID NO：28；SEQ ID NO：26和SEQ ID NO：36；SEQ ID NO：30和SEQ ID NO：28；SEQ ID NO：26和SEQ ID NO：32。
  19. 权利要求2的双特异性分子，其中，所述双特异性分子包括靶向游离炎症因子IL-6的抗体，且所述双特异性分子包含分别具有如下氨基酸序列的重链和轻链：SEQ ID NO：46和SEQ ID NO：40；SEQ ID NO：38和SEQ ID NO：48；SEQ ID NO：38和SEQ ID NO：44；SEQ ID NO：42和SEQ ID NO：40。
  20. 一种核酸，编码如权利要求1-22中任一项所述的双特异性分子，或其重链或轻链。
  21. 一种表达载体，其包含如权利要求23所述的核酸。
  22. 一种宿主细胞，其包含如权利要求24所述的表达载体。
  23. 一种药物组合物，其包含如权利要求1-22中任一项所述的双特异性分子和药学上可接受的载体或制剂。
  24. 一种治疗有需要的受试者的炎症性疾病和/或自身免疫性疾病的方法，所述方法包括对所述受试者施用治疗有效量的组合物，所述组合物包含药学可接受形式的权利要求1-19中任一项所述的双特异性分子。
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