WO2021190553A1 - ANTI-IL-1β ANTIBODY, AND PHARMACEUTICAL COMPOSITION CONTAINING SAME, AND USE THEREOF - Google Patents

ANTI-IL-1β ANTIBODY, AND PHARMACEUTICAL COMPOSITION CONTAINING SAME, AND USE THEREOF Download PDF

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WO2021190553A1
WO2021190553A1 PCT/CN2021/082692 CN2021082692W WO2021190553A1 WO 2021190553 A1 WO2021190553 A1 WO 2021190553A1 CN 2021082692 W CN2021082692 W CN 2021082692W WO 2021190553 A1 WO2021190553 A1 WO 2021190553A1
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antibody
seq
region
antigen
drug
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Chinese (zh)
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夏瑜
李百勇
王忠民
陈娜
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中山康方生物医药有限公司
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Definitions

  • the invention belongs to the field of immunology, and relates to an anti-IL-1 ⁇ antibody, its pharmaceutical composition and its use. Specifically, the present invention relates to an anti-human IL-1 ⁇ antibody; more specifically, the present invention relates to an anti-human IL-1 ⁇ monoclonal antibody; in particular, the present invention relates to an anti-human IL-1 ⁇ antibody Humanized monoclonal antibody.
  • IL-1 ⁇ is a member of the IL-1 family, which plays a key role in innate immunity and inflammation.
  • IL-1 ⁇ family members include 7 cytokines (IL-1 ⁇ , IL-1 ⁇ , IL-18, IL-33, IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ , IL-37) and three specific receptors Antagonists (IL-1Ra, IL-36Ra and IL-38).
  • IL-1 ⁇ is produced by enzymatic hydrolysis of the inactive precursor protein ProIL-1 ⁇ . This process is mainly mediated by the inflammasome NLRP3 (NOD-like receptor protein 3) (JesusAA and Goldbach-Mansky R.IL-1). blockade in autoinflammatory syndromes.Annu Rev Med 2014).
  • the receptors of IL-1 ⁇ and IL-1 ⁇ are IL-1R1 and IL-1RAcP heterodimers, and IL-1R1 is the receptor subunit that IL-1 ⁇ and IL-1 ⁇ specifically bind, IL-1RAcP (interleukin- 1-receptor co-protein) is a common component of receptors of other members of the IL-1 family, such as IL-33 and IL-36.
  • IL-1R1 is a transmembrane receptor that can bind to IL-1 ⁇ and IL-1RAcP to form a receptor complex, activate downstream intracellular signaling pathways, and mediate IL-1 ⁇ -related biological effects.
  • IL-1 receptor antagonists There are natural negative regulators of IL-1 ⁇ , including IL-1R2, soluble IL-1R1 and IL-1R2, and IL-1 receptor antagonist (IL-1Ra).
  • IL-R2 can bind to IL-1 ⁇ , but because the intracellular segment is short, it cannot activate downstream pathways. The binding of soluble IL-1R1 and IL-1R2 to IL-1 prevents it from further binding to membrane-localized receptors; IL-1Ra can interact with The binding of IL-1R1 blocks the binding of IL-1 ⁇ and IL-1 ⁇ to the corresponding receptors (Boraschi D, Italiani P, Weil S, Martin MU. The family of the interleukin-1 receptors. Immunol Rev. 2018; 281(1) ):197-232).
  • IL-1 ⁇ is mainly produced by monocytes and macrophages. After IL-1 ⁇ binds to its receptor, it activates the downstream NF- ⁇ B and MAPK-dependent pro-inflammatory intracellular signal cascade. IL-1 ⁇ is an effective inducer of COX-2 expression. It produces a large amount of prostaglandin E2 (PGE2) to inhibit the production of IFN- ⁇ , thereby inhibiting Th1-type T cell-led Th1-type immune response against viruses and intracellular parasites ( Napolitani G, Acosta-Rodriguez EV, Lanzavecchia A, Sallusto F.
  • PGE2 prostaglandin E2
  • Prostaglandin E2 enhances Th17 responses via modulation of IL-17 and IFN-gamma production by memory CD4+T cells.Europeanimmunology: 1 301; 2009; –12.). Another key effect of IL-1 ⁇ is to promote the secretion of IL-6. IL-6 is a key factor in inducing the differentiation of naive T cells into Th17 T cells (Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al.
  • IL-6 can also inhibit the immunosuppressive function of regulatory T cells and prevent Th17 T cells from transforming into immunosuppressive Treg T cells (Korn T, Mitsdoerffer M, Croxford AL, Awasthi A, Dardalhon VA, Galileos G, et al. IL-6 Controls Th17 immunity in vivo by inhibiting the conversion of conventional T cells of the National Cells of the Academy of Foxp3+regulators. of Sciences of the United States of America.2008; 105(47):18460-5.Pasare C, Medzhitov R.
  • IL-1 ⁇ -mediated Th17 immune response inhibits the killing of virus-infected cells by CD8+ cytotoxic T cells, and enhances the survival rate of virus-infected cells, which may reduce the body's defense against viruses (Hou W, Kang HS, Kim BS.Th17 cells enhancement viral persistence and inhibition T cell cytotoxicity in a model of chronic virus infection. The Journal of experimental medicine.2009;206(2):313-28.).
  • IL-1 ⁇ increases the secretion of GM-CSF by inducing the proliferation and activation of Th17 cells
  • Th17 cells Mufazalov IA, Schelmbauer C, Regen T, Kuschmann J, Wanke F, Gabriel LA, Hauptmann J, Müller W, Pinteaux E, Kurschus FC ,Waisman A.IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+Th17 cells.EMBO J.2017 4; 36(1):102-115); in addition to immune cells, endothelial cells, fibroblasts Cells, chondrocytes and smooth muscle cells can be produced under the induction of IL-1 and TNF- ⁇ (GM-CSF S. Matthew Fitzgerald, David S. Chi, H.
  • IL-1 ⁇ plays a key role in the fatal systemic inflammatory response caused by viral infection.
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • studies have confirmed that SARS-CoV 3a protein can directly activate the NLRP3 inflammasome in macrophages and mediate the secretion of IL-1 ⁇ , which may be The IL-1 ⁇ observed in SARS and its downstream IL-6 increase mechanism (Chen IY, Moriyama M, Chang MF, Ichinohe T. Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3Inflammasome. Front Microbiol.eCollection 2019).
  • IL-1 ⁇ signaling pathway inhibition may be used to treat such diseases.
  • anti-IL-6 antibody has been annotated for the treatment of cytokine release syndrome caused by CAT-T therapy.
  • inhibiting IL-1 ⁇ -related signaling pathways may be more advantageous for treating excessive and fatal inflammatory responses caused by viral infections, tumors or cellular immunotherapy.
  • IL-1 receptor antagonist Anakinra a recombinant human IL-1Ra
  • CAR-T chimeric antigen receptor T cells
  • pathogenic microorganisms viruses, bacteria or fungi or other infections, burns, pancreatitis, blood transfusion or allogeneic blood cell transplantation, trauma, drugs (antibodies, immunoagonists, or cytokines), chimeric antigen cell therapy, surgery, Radiotherapy, chemotherapy, etc.
  • systemic inflammation which is manifested by increased secretion of IL-1 ⁇ , and even caused cytokine release syndrome characterized by increased secretion of IL-1 ⁇ , manifested by multiple organ damages including acute kidney injury and acute breathing System damage such as acute respiratory distress syndrome (ARDS), intravascular diffuse coagulation, methemorinemia, nervous system inflammation and so on.
  • ARDS acute respiratory distress syndrome
  • IL-1 receptor antagonists such as Anakinra have been used clinically, due to its fast metabolism in the body and a half-life of only a few hours, it needs to be injected daily to maintain drug efficacy. Therefore, anti-IL with stable pharmacokinetic characteristics has been developed. -1 ⁇ antibody drugs have great clinical application potential.
  • the inventors used the mammalian cell expression system to express recombinant IL-1 ⁇ -His as an antigen to immunize mice, and obtain hybridoma cells by fusion of mouse spleen cells and myeloma cells.
  • the inventors screened a large number of samples and obtained the following hybridoma cell lines:
  • Hybridoma cell line LT010 which was deposited in the China Center for Type Culture Collection (CCTCC) on June 21, 2018, and the deposit number is CCTCC NO: C2018133.
  • the hybridoma cell line LT010 can secrete and produce a specific monoclonal antibody (named 3H6) that specifically binds to human IL-1 ⁇ , and the monoclonal antibody can effectively block the binding of IL-1 ⁇ to IL-1R1;
  • the present inventors creatively prepared humanized antibodies against human IL-1 ⁇ (named 3H6 H1L1, 3H6 H2L2, 3H6 H3L3, 3H6 H4L1), which can effectively bind to human IL-1 ⁇ and block IL-
  • the binding of 1 ⁇ to its receptor IL-1R1 inhibits the activation of IL-1 ⁇ downstream signaling pathways; it can be used for preparation to reduce, prevent or treat pathogenic microorganisms (viruses, bacteria or fungi or other) infections, burns, pancreatitis, blood transfusions or foreign bodies Systemic inflammation caused by blood cell transplantation, trauma, drugs (antibodies, immune agonists, or cytokines), chimeric antigen cell therapy, surgery, radiotherapy, and chemotherapy.
  • pathogenic microorganisms viruses, bacteria or fungi or other
  • One aspect of the present invention relates to an anti-IL-1 ⁇ antibody or antigen-binding fragment thereof, wherein
  • the heavy chain variable region of the antibody comprises: the amino acid sequences of HCDR1-HCDR3 shown in SEQ ID NO: 17-SEQ ID NO: 19, respectively;
  • the light chain variable region of the antibody includes LCDR1-LCDR3 with amino acid sequences as shown in SEQ ID NO: 20-SEQ ID NO: 22, respectively.
  • the IL-1 ⁇ is human IL-1 ⁇ .
  • variable region of the heavy chain and the variable region of the light chain determine the binding of the antigen; the variable region of each chain contains three hypervariable regions, called the complementarity determining region (CDR) (the CDR of the heavy chain (H) includes HCDR1, HCDR2 , HCDR3, the CDR of the light chain (L) includes LCDR1, LCDR2, LCDR3; it is named by Kabat et al., see Sequences of Proteins of Immunological Interest.Fifth Edition (1991), Vols 1-3, NIH Publication 91-3242, Bethesda Md).
  • CDR complementarity determining region
  • amino acid sequences of the three HCDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFSLSTSGMG (SEQ ID NO: 17),
  • HCDR2 IYWDDDK (SEQ ID NO: 18),
  • HCDR3 ARSAYYSFAY (SEQ ID NO: 19);
  • amino acid sequences of the 3 CDR regions of the light chain variable region are as follows:
  • LCDR1 QDVDTD (SEQ ID NO: 20),
  • LCDR2 WAS (SEQ ID NO: 21),
  • LCDR3 QQYSSYPT (SEQ ID NO: 22).
  • amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, or with SEQ ID NO: 2, SEQ ID NO: 6 , SEQ ID NO: 10 and SEQ ID NO: 14 have sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity, respectively; and
  • the amino acid sequence of the variable region of the light chain of the antibody is selected from SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16, or with SEQ ID NO: 4 and SEQ ID NO: 8.
  • the sequences of SEQ ID NO: 12 and SEQ ID NO: 16 respectively have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical sequences.
  • the antibody is selected from:
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragments, single-chain antibodies (e.g. , ScFv), humanized antibody, chimeric antibody or diabody.
  • the antibody has a concentration of less than 10 -5 M, for example, less than 10 -6 M, less than 10 -7 M, less than 10 -8 M, less than 10 -9 M or A K D of less than 10 -10 M or less binds to IL-1 ⁇ protein; preferably, the K D is measured by a Biacore molecular interaction instrument; preferably, the K D is measured by a Fortebio molecular interaction instrument.
  • the antibody is less than about 100 nM, for example, less than about 10 nM, less than about 1 nM, less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about
  • the EC 50 is measured by an indirect ELISA method.
  • the antibody includes a non-CDR region, and the non-CDR region is from a species other than murine, for example, from a human antibody.
  • the constant region of the antibody is humanized.
  • the constant region of the heavy chain uses Ig gamma-1 chain C region, such as ACCESSION: P01857 or Ig gamma-4 chain C region, such as ACCESSION : P01861.1; Ig kappa chain C region is used for the light chain constant region, such as ACCESSION: P01834.
  • the antibody or antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody produced by a hybridoma cell line LT010, and the hybridoma cell line LT010 is deposited in China. Center for the Collection of Cultures (CCTCC), the deposit number is CCTCC NO: C2018133.
  • CTCC Center for the Collection of Cultures
  • the antibody is a monoclonal antibody.
  • an antibody-drug conjugate (antibody-drug conjugate, ADC), which includes an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or an antigen-binding fragment thereof is in the present invention
  • ADC antibody-drug conjugate
  • the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a chemotherapeutic drug.
  • the chemotherapeutic drugs may be conventional tumor chemotherapeutics, such as alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones, immunosuppressants and the like.
  • the antibody or antigen-binding fragment thereof is connected to the small molecule drug through a linker;
  • the linker may be a linker known to those skilled in the art, for example, the linker It is a hydrazone bond, a disulfide bond or a peptide bond.
  • the antibody or antigen-binding fragment thereof and the small molecule drug are linked at a certain molar ratio; for example, the molar ratio is 1: (2-4).
  • bispecific antibody also called a bifunctional antibody
  • first protein functional region and a second protein functional region, wherein:
  • the first protein functional region targets IL-1 ⁇
  • the second protein functional region targets a target different from IL-1 ⁇ , such as IL-17A;
  • the first protein functional region is the antibody or antigen-binding fragment thereof according to any one of the present invention.
  • the bispecific antibody is in IgG-scFv mode
  • the first protein functional region is the antibody or antigen-binding fragment thereof according to any one of the present invention, and the second protein functional region is a single-chain antibody;
  • the first protein functional region is a single-chain antibody, and its heavy chain variable region contains the amino acid sequence of HCDR1-HCDR3 shown in SEQ ID NO: 17-SEQ ID NO: 19, and its light chain variable region contains The amino acid sequence is LCDR1 to LCDR3 shown in SEQ ID NO: 20 to SEQ ID NO: 22, and the second protein functional region is an antibody (for example, a monoclonal antibody).
  • the bispecific antibody wherein the first protein functional region and the second protein functional region are directly connected or connected by a linking fragment
  • connection fragment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;
  • connection fragment is SS(GGGGS)n, where n is a positive integer, such as 1, 2, 3, 4, 5 or 6.
  • amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, or with SEQ ID NO: 2, SEQ ID NO :6, SEQ ID NO: 10 and SEQ ID NO: 14 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical, respectively; and
  • amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16, or with SEQ ID NO: 4, SEQ ID NO :8, SEQ ID NO: 12 and SEQ ID NO: 16 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical, respectively.
  • amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO: 2 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the same as SEQ ID NO: 2 A sequence of at least 99% identity, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO: 4 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 4, 96%, 97%, 98% or at least 99% identical sequences; or
  • the amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO: 6 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the same as SEQ ID NO: 6
  • the sequence is at least 99% identical, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 8 or has at least 80%, 85%, 90%, 95% with SEQ ID NO: 8 %, 96%, 97%, 98% or at least 99% identical sequences; or
  • the amino acid sequence of the variable region of the heavy chain of the single-chain antibody is SEQ ID NO: 10 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the same as SEQ ID NO: 10
  • the sequence is at least 99% identical
  • the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO: 12 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 12, 96%, 97%, 98% or at least 99% identical sequences; or
  • the amino acid sequence of the variable region of the heavy chain of the single-chain antibody is SEQ ID NO: 14 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the same as SEQ ID NO: 14 A sequence of at least 99% identity, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO: 16 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 16, Sequences that are 96%, 97%, 98%, or at least 99% identical.
  • the first protein functional region and the second protein functional region of the bispecific antibody are independently 1, 2, or 2 or more.
  • the constant region of the monoclonal antibody is selected from the constant region of human IgG1, IgG2, IgG3, or IgG4.
  • the single-chain antibody of the bispecific antibody is attached to the C-terminus of the heavy chain of the antibody or monoclonal antibody.
  • Yet another aspect of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a variable region of an antibody heavy chain and a nucleic acid sequence encoding a variable region of an antibody light chain, wherein,
  • variable region of the heavy chain of the antibody comprises the amino acid sequence of HCDR1-HCDR3 shown in SEQ ID NO: 17-ID NO: 19, and the variable region of the light chain of the antibody comprises the amino acid sequence of the amino acid sequence shown in SEQ ID NO, respectively. :20-LCDR1-LCDR3 shown in SEQ ID NO: 22;
  • the amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, or with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical to the antibody
  • the amino acid sequence of the variable region of the light chain is selected from SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16, or with SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical;
  • the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 2 or is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% with SEQ ID NO: 2 % Or at least 99% identity sequence
  • the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 4 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 4, 96%, 97%, 98% or at least 99% identical sequence
  • the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 6 or at least 80%, 85% with SEQ ID NO: 2 , 90%, 95%, 96%, 97%, 98% or at least 99% identical sequence
  • the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 8 or the same as SEQ ID NO: 8 A sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; or the amino acid sequence of the heavy chain variable region of the heavy chain
  • the isolated nucleic acid molecule may be one nucleic acid molecule or multiple nucleic acid molecules, for example, two nucleic acid molecules.
  • the variable region of the heavy chain and the variable region of the light chain of the antibody can be expressed by the same nucleic acid molecule, for example, by the same or different expression cassettes located on the same nucleic acid molecule.
  • the variable region of the heavy chain and the variable region of the light chain of the antibody can be expressed separately by different nucleic acid molecules.
  • Yet another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.
  • the recombinant vector can be one or more.
  • the multiple (for example, two) nucleic acid molecules can be expressed by the same recombinant vector, or can be expressed by different recombinant vectors respectively.
  • Another aspect of the present invention relates to a host cell, which contains the isolated nucleic acid molecule of the present invention, or contains the recombinant vector of the present invention.
  • Another aspect of the present invention relates to a method for preparing the antibody or antigen-binding fragment thereof according to any one of the present invention, which comprises culturing the host cell of the present invention under suitable conditions, and recovering all cells from the cell culture. The steps of the antibody or its antigen-binding fragment.
  • Another aspect of the present invention relates to a hybridoma cell line LT010, which is deposited in the China Center for Type Culture Collection (CCTCC), and the deposit number is CCTCC NO: C2018133.
  • CTCC China Center for Type Culture Collection
  • Yet another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment thereof according to any one of the present invention, the antibody-drug conjugate of the present invention or the bispecific antibody of the present invention
  • it also includes a pharmaceutically acceptable carrier and/or excipient.
  • Another aspect of the present invention relates to an effective amount of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention or the bispecific antibody of the present invention in the preparation of therapeutic and/or prophylactic IL.
  • systemic inflammation includes systemic inflammation syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS), and acute respiratory distress syndrome;
  • systemic inflammation syndrome cytokine release syndrome
  • MODS multiple organ dysfunction syndrome
  • acute respiratory distress syndrome cytokine release syndrome
  • the systemic inflammation is caused by pathogenic microorganism infection
  • the pathogenic microorganisms are viruses, bacteria, fungi, rickettsiae, chlamydia, mycoplasma, parasites, prions;
  • the viruses include RNA viruses and DNA viruses;
  • the RNA virus includes a coronavirus of the coronavirus family
  • the coronavirus family virus includes 2019 new coronavirus (2019-nCoV or SARS-CoV-2, which causes new coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 , SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing Middle East respiratory syndrome).
  • 2019 new coronavirus 2019-nCoV or SARS-CoV-2, which causes new coronavirus pneumonia COVID-19
  • HCoV-229E HCoV-OC43
  • HCoV-NL63 HCoV-NL63
  • HCoV-HKU1 HCoV-HKU1
  • SARS-CoV causing severe acute respiratory syndrome
  • MERS-CoV Middle East respiratory syndrome
  • Another aspect of the present invention is the use of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention, or the bispecific antibody of the present invention in the preparation of the following drugs:
  • the human IL-1R1 and/or human IL-1R2 is human IL-1R1 and/or human IL-1R2 on the cell surface.
  • the use is for non-therapeutic and/or non-diagnostic purposes.
  • the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention or the bispecific antibody of the present invention is used for the preparation of treatment and/or prevention (IL -1 ⁇ abnormal secretion is characterized by the use of drugs for systemic inflammation;
  • the systemic inflammation includes systemic inflammatory syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS), and acute respiratory distress syndrome;
  • systemic inflammatory syndrome cytokine release syndrome
  • MODS multiple organ dysfunction syndrome
  • acute respiratory distress syndrome cytokine release syndrome
  • the systemic inflammation is caused by pathogenic microorganism infection
  • the pathogenic microorganisms are viruses, bacteria, fungi, rickettsiae, chlamydia, mycoplasma, parasites, prions;
  • the viruses include RNA viruses and DNA viruses;
  • the RNA virus includes a coronavirus of the coronavirus family
  • the coronavirus family virus includes 2019 new coronavirus (2019-nCoV or SARS-CoV-2, which causes new coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 , SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing Middle East respiratory syndrome),
  • 2019 new coronavirus 2019-nCoV or SARS-CoV-2, which causes new coronavirus pneumonia COVID-19
  • HCoV-229E HCoV-OC43
  • HCoV-NL63 HCoV-HKU1
  • SARS-CoV causing severe acute respiratory syndrome
  • MERS-CoV causing Middle East respiratory syndrome
  • the antibody or antigen-binding fragment thereof is used alone or in combination with one or more antiviral drugs;
  • the antiviral drug is an antiretroviral drug or interferon
  • the antiretroviral drug wherein: the antiretroviral drug is an RNA-dependent RNA polymerase (RdRp) inhibitor, or a nucleoside analog, or a peptoid, or a human immunodeficiency virus type 1 (HIV-1) Integrase chain transfer inhibitor (INSTI), or HIV protease inhibitor.
  • RdRp RNA-dependent RNA polymerase
  • INSTI human immunodeficiency virus type 1 Integrase chain transfer inhibitor
  • HIV protease inhibitor HIV-dependent RNA polymerase
  • the antiretroviral drugs are favipiravir, Remdesivir, Beclabuvir, Saquinavir, Bictegravir, Lopinavir ), Dolutegravir (Dolutegravir);
  • the drug is favipiravir.
  • the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention or the bispecific antibody of the present invention is used for:
  • the human IL-1R1 and/or human IL-1R2 is human IL-1R1 and/or human IL-1R2 on the cell surface.
  • Yet another aspect of the present invention relates to an in vivo or in vitro method, comprising administering cells in an effective amount of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention, or the antibody-drug conjugate of the present invention.
  • the step of bispecific antibody, the method is selected from the following:
  • the human IL-1R1 and/or human IL-1R2 is human IL-1R1 and/or human IL-1R2 on the cell surface.
  • the in vitro method is for non-therapeutic and/or non-diagnostic purposes.
  • Another aspect of the present invention relates to a method for treating and/or preventing treatment and/or prevention of systemic inflammation characterized by abnormal secretion of IL-1 ⁇ , comprising administering to a subject or patient in need an effective amount of the present invention
  • the systemic inflammation includes systemic inflammatory syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS), and acute respiratory distress syndrome;
  • systemic inflammatory syndrome cytokine release syndrome
  • MODS multiple organ dysfunction syndrome
  • acute respiratory distress syndrome cytokine release syndrome
  • the systemic inflammation is caused by pathogenic microorganism infection
  • the pathogenic microorganisms are viruses, bacteria, fungi, rickettsiae, chlamydia, mycoplasma, parasites, prions;
  • the viruses include RNA viruses and DNA viruses;
  • the RNA virus includes a coronavirus of the coronavirus family
  • the coronavirus family virus includes 2019 new coronavirus (2019-nCoV or SARS-CoV-2, which causes new coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 , SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing Middle East respiratory syndrome).
  • 2019 new coronavirus 2019-nCoV or SARS-CoV-2, which causes new coronavirus pneumonia COVID-19
  • HCoV-229E HCoV-OC43
  • HCoV-NL63 HCoV-NL63
  • HCoV-HKU1 HCoV-HKU1
  • SARS-CoV causing severe acute respiratory syndrome
  • MERS-CoV Middle East respiratory syndrome
  • the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody can be used alone or in combination with one or more antiviral drugs use.
  • kits comprising an effective amount (for example, 0.001 mg-1000 mg) of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody, And optionally, it also contains an effective amount of one or more antiviral drugs (for example, 100-2400 mg), wherein the antiviral drugs are as described above.
  • a single drug dosage unit which contains 0.001 mg-1000 mg of the antibody or antigen-binding fragment thereof of the present invention, preferably 0.001 mg-900 mg, 0.001 mg-800 mg, 0.001 mg-700 mg, 0.001 mg -600mg, 0.001mg-500mg, 0.001mg-400mg, 0.001mg-300mg, 0.001mg-200mg, 0.001mg-100mg, more preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg
  • the antibody or antigen-binding fragment thereof of the present invention preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg
  • the antibody or antigen-binding fragment thereof of the present invention preferably 0.001 mg-1000 mg of the antibody or antigen-binding fragment thereof
  • the effective amount of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody of the present invention is 0.001 mg-1000 mg, preferably 0.001 mg- 900mg, 0.001mg-800mg, 0.001mg-700mg, 0.001mg-600mg, 0.001mg-500mg, 0.001mg-400mg, 0.001mg-300mg, 0.001mg-200mg, 0.001mg-100mg, more preferably 100mg, 200mg, 300mg , 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg.
  • the effective amount of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody of the present invention is 0.1-100mg/kg, preferably 1-90mg/kg, 1-80mg/kg, 1-70mg/kg, 1-60mg/kg, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1 -20mg/kg or 1-10mg/kg.
  • the effective amount of one or more antiviral drugs is 100-2400mg, preferably 100mg-2300mg, 100mg-2200mg, 100mg-2100mg, 100mg-2000mg, 100mg-1900mg, 100mg-1800mg, 100mg-1700mg, 100mg-1600mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, more preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg , 700mg, 800mg, 900mg, 1000mg.
  • one or more antiviral drugs is 100-2400mg, preferably 100mg-2300mg, 100mg-2200mg, 100mg-2100mg, 100mg-2000mg, 100mg-1900mg, 100mg-1800mg, 100mg-1700mg, 100mg-1600mg, 100mg
  • the effective amount of the antiviral drug is 0.1-100 mg/kg, preferably 1-90 mg/kg, 1-80 mg/kg, 1-70mg/kg, 1-60mg/kg, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1-20mg/kg or 1-10mg/kg.
  • single drug dosage unit means the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate of the present invention to be administered to the subject or patient at the time of the dosing schedule Or the single-dose dosage form of the bispecific antibody (or the pharmaceutical composition containing the same), such as one ampoule as a unit.
  • IL-1 ⁇ when referring to the amino acid sequence of IL-1 ⁇ , it includes the full length of IL-1 ⁇ protein (GenBank ID: NP_000567.1), and also includes IL-1 ⁇ fusion proteins, such as those with mice or Human IgG Fc protein fragments (mFc or hFc) or fragments of multiple His fusions.
  • IL-1 ⁇ fusion proteins
  • mutations or variations including but not limited to substitutions, deletions and/or additions
  • the term "IL-1 ⁇ ” shall include all such sequences, and their natural or artificial variants.
  • sequence fragments of IL-1 ⁇ protein it includes not only the sequence fragments, but also the corresponding sequence fragments in its natural or artificial variants.
  • IL-1R1 when referring to the amino acid sequence of IL-1R1, it includes the full length of IL-1R1 protein (GenBank ID: NP_000868), and also includes IL-1R1 fusion proteins, such as mouse or human IgG Fc protein fragments (mFc or hFc) or fragments of multiple His fusions.
  • IL-1R1 fusion proteins, such as mouse or human IgG Fc protein fragments (mFc or hFc) or fragments of multiple His fusions.
  • mutations or variations including but not limited to substitutions, deletions and/or additions
  • the term "IL-1R1" shall include all such sequences and their natural or artificial variants.
  • sequence fragment of IL-1R1 protein when describing the sequence fragment of IL-1R1 protein, it includes the IL-1R1 sequence fragment and the corresponding sequence fragment in its natural or artificial variant.
  • IL-1R2 when referring to the amino acid sequence of IL-1R2, it includes the full length of IL-1R2 protein (GenBank ID: CAA42441.1), and also includes IL-1R2 fusion proteins, such as those with mice or Human IgG Fc protein fragments (mFc or hFc) or fragments of multiple His fusions.
  • IL-1R2 when referring to the amino acid sequence of IL-1R2 protein, it includes the full length of IL-1R2 protein (GenBank ID: CAA42441.1), and also includes IL-1R2 fusion proteins, such as those with mice or Human IgG Fc protein fragments (mFc or hFc) or fragments of multiple His fusions.
  • IL-1R2 mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally or artificially introduced without affecting its biological functions. Therefore, in the present invention, the term "IL-1R2" shall include all such sequences and their natural or artificial variants.
  • EC 50 refers to the concentration for 50% of maximal effect (concentration for 50% of maximal effect), which refers to the concentration that can cause 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair has a "light” (L) chain and a “heavy” (H) chain) .
  • Antibody light chains can be classified into kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions or CDRs), interspersed with more conservative regions called framework regions (FR).
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antibody binding site.
  • the assignment of amino acids to each region or domain follows Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917 ; Definition of Chothia et al. (1989) Nature 342:878-883.
  • antibody is not limited by any specific method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody It is also called “antigen binding part” for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Recombinant DNA technology can be used. Or through the enzymatic or chemical cleavage of intact antibodies to produce antigen-binding fragments of antibodies.
  • antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining regions (CDR) Fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and such polypeptides, which comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • CDR complementarity determining regions
  • the antigen-binding fragment of the antibody is a single-chain antibody (e.g., scFv), where the VL and VH domains pair to form a monovalent molecule by pairing a linker that enables it to be produced as a single polypeptide chain (see, e.g., Bird et al. , Science 242: 423 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879 5883 (1988)).
  • scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the antigen-binding fragments of antibodies are diabodies, ie, diabodies, in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short is used to allow two structures in the same chain Pairing between the domains, thereby forcing the domain to pair with the complementary domain of the other chain and creating two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).
  • bifunctional antibodies also known as bispecific antibodies, are specific drugs that target two different antigens at the same time, and they can be produced by immunosorting and purification. In addition, it can also be obtained through genetic engineering. Genetic engineering has corresponding flexibility in terms of optimization of binding sites, consideration of synthetic forms, and yield, so it has certain advantages. At present, there are more than 45 forms of its existence (Müller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24: 89-98). Many bispecific antibodies that have been developed so far are in the form of IgG-ScFv, the Morrison model (1997 Coloma MJ, Morrison SL.
  • a given antibody for example, the monoclonal antibody 3H6, 3H6H1L1, 3H6H2L2, 3H6 H3L3 or 3H6H4L1 provided by the present invention.
  • An antigen-binding fragment of an antibody for example, the above-mentioned antibody fragment is obtained, and the antigen-binding fragment of the antibody is specifically screened in the same manner as that used for intact antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, in addition to natural mutations that may occur spontaneously, A group of identical antibody molecules.
  • the monoclonal antibody has high specificity for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which usually include at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen.
  • Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (for example, see U.S.P 4,816,567).
  • humanized antibody means that all or part of the CDR region of a human immunoglobulin (acceptor antibody) is replaced by a CDR region of a non-human antibody (donor antibody).
  • the donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody with the expected specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by corresponding non-human antibody amino acid residues, or by other antibody amino acid residues, to further improve or optimize the performance of the antibody.
  • isolated refers to those obtained from the natural state by artificial means. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolation a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • the term "vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 fruit fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 fruit fly cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • bispecific As used in the present invention, the terms "bispecific", “dual specific” or “bifunctional” antigen binding protein or antibody are hybrid antigen binding proteins or antibodies each having two different antigen binding sites.
  • a bispecific antibody is a multispecific antigen binding protein or multispecific antibody, and can be produced by a variety of methods, including, but not limited to, the fusion of hybridomas or the linking of Fab' fragments. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79: 315-321; Kostelny et al. 1992, J. Immunol. 148: 1547-1553.
  • the two binding sites of the bispecific antigen binding protein or antibody will bind to two different epitopes that are present on the same or different protein targets.
  • an antibody that specifically binds to a certain antigen means that the antibody has a concentration of less than about 10 -5 M, for example, less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, or less than about 10 -10 M or less with an affinity (K D ) that binds the antigen.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen.
  • the antibody for example, the monoclonal antibody 3H6, 3H6H1L1, 3H6H2L2, or 3H6H3L3 of the present invention
  • the antibody is less than about 10 -5 M, such as less than about 10 -6 M, less than about 10 -7 M, or less than about 10 -8 M.
  • K D The dissociation equilibrium constant (K D ) of less than about 10 -9 M or less than about 10 -10 M or less binds to the antigen (e.g., IL-1 ⁇ protein).
  • K D can be determined using methods known to those skilled in the art, for example, using a Biacore molecular interaction analyzer.
  • amino acids are usually represented by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala.
  • hybridoma and “hybridoma cell line” are used interchangeably, and when referring to the terms “hybridoma” and “hybridoma cell line”, it also includes subclones of hybridomas And progeny cells. For example, when referring to the hybridoma cell line LT010, it also refers to the subclones and progeny cells of the hybridoma cell line LT010.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject or patient and the active ingredient Agents, which are well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH regulators, surfactants, adjuvants, ions Strength enhancer.
  • pH adjusting agents include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (such as RA);
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease in a patient who has already suffered from the disease. And the amount of its complications.
  • the anti-IL-1 ⁇ antibody of the present invention especially the humanized anti-IL-1 ⁇ antibody, has one or more of the following technical effects:
  • Figure 1 Detection results of the binding activity of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 to human IL-1 ⁇ -His-Bio.
  • Figure 2 Detection results of the binding activity of 3H6H4L1 and human IL-1 ⁇ -His-Bio.
  • Figure 3 3H6, 3H6H1L1, 3H6H2L2, and 3H6H3L3 compete with human IL-1R1(1-332)-his to bind to human IL-1 ⁇ -hFc.
  • Figure 7 Inhibition of 3H6H4L1 on IL-1 ⁇ -induced IL-6 secretion by MRC-5.
  • Figure 8 Neutralizing biological activity of IL-1 ⁇ at different doses of 3H6H4L1.
  • Figure 9 The dose-response curve of 3H6H4L1 blocking IL-1 ⁇ .
  • the BALB/c mice used were purchased from Guangdong Medical Experimental Animal Center.
  • IL-1 ⁇ (Genbank ID: NP_000567.1) and IL-1R1 (Genbank ID: NP_000868, amino acids 1-332) through the NCBI GenBank protein database.
  • the amino acid sequences of human IL-1 ⁇ and IL-1R1 are respectively fused with His tag sequence (6 His) and human IgG Fc purification tag sequence (Ig gamma-1chain C region, GenbankID: P01857 position 114-330);
  • the fusion proteins were abbreviated as human IL-1 ⁇ -His, IL-1R1(1-332)-His, IL-1 ⁇ -hFc.
  • the quality of the protein sample was qualified by SDS-PAGE.
  • the prepared fusion protein was used in the following examples.
  • Human IL-1 ⁇ -his was used as the antigen to immunize BALB/c mice (purchased from Guangdong Medical Experimental Animal Center), and the spleen cells of the immunized mice were fused with mouse myeloma cells to make hybridoma cells.
  • IL-1 ⁇ -His-Bio as an antigen, hybridoma cells were screened by ELISA method to obtain hybridoma cells capable of secreting antibodies specifically binding to IL-1 ⁇ -His-Bio.
  • hybridoma cells screened by ELISA the hybridoma cells that can secrete antibodies that compete with the receptor IL-1R1(1-332)-His to bind IL-1 ⁇ -hFc were screened by competitive ELISA, and stabilized by the limiting dilution method.
  • Hybridoma cell line The method of preparing hybridoma cells refers to the established methods (for example, Stewart, SJ, "Monoclonal Antibody Production", in Basic Methods in antibody Production and Characterization, Eds. GC Howard and DRBethell, Boca Raton: CRC Press, 2000) .
  • Hybridoma cell line LT010 (IL-1 ⁇ -3H6), which was deposited in the China Center for Type Culture Collection (CCTCC) on June 21, 2018, with the deposit number CCTCC NO: C2018133, and the deposit address is Wuhan University, Wuhan, China , Zip Code: 430072.
  • Hybridoma serum-free medium containing 1% penicillin and 4% Glutamax, cell culture at 5% CO 2 , 37°C Cell culture supernatant was collected after 7 days, and purified by high-speed centrifugation, vacuum filtration with microporous membrane and HiTrap protein AHP column to prepare antibody 3H6.
  • the purified 3H6 sample was qualified by SDS-PAGE electrophoresis.
  • RNA was extracted from the LT010 cell line cultured in Example 1.
  • mRNA was extracted from the LT010 cell line cultured in Example 1.
  • Invitrogen III First-Strand Synthesis System for RT-PCR kit instructions Synthesize cDNA and perform PCR amplification.
  • the PCR amplification product is directly subjected to TA cloning, and the specific operation refers to the pEASY-T1 Cloning Kit (Transgen CT101) kit instructions.
  • the TA cloned product was directly sequenced, and the sequencing results are as follows:
  • amino acid sequence of the heavy chain variable region of antibody 3H6 is as follows: (118aa, the underlined amino acid sequence is the CDR region)
  • amino acid sequence of the light chain variable region of antibody 3H6 is as follows: (106aa, where the underlined amino acid sequence is the CDR region)
  • sequences of the heavy chain variable region and the light chain variable region of the humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 are as follows:
  • amino acid sequence of the variable region of the heavy chain of the antibody 3H6H1L1 is as follows: (118aa, the underlined amino acid sequence is the CDR region)
  • amino acid sequence of the light chain variable region of the antibody 3H6H1L1 is as follows: (106aa, the underlined amino acid sequence is the CDR region)
  • amino acid sequence of the variable region of the heavy chain of antibody 3H6H2L2 is as follows: (118aa, the underlined amino acid sequence is the CDR region)
  • amino acid sequence of the light chain variable region of the antibody 3H6H2L2 is as follows: (106aa, the underlined amino acid sequence is the CDR region)
  • amino acid sequence of the variable region of the heavy chain of the antibody 3H6H3L3 is as follows: (118aa, the underlined amino acid sequence is the CDR region)
  • amino acid sequence of the light chain variable region of the antibody 3H6H3L3 is as follows: (106aa, the underlined amino acid sequence is the CDR region)
  • the nucleic acid sequence encoding the variable region of the heavy chain of the antibody 3H6H4L1 is shown in SEQ ID NO: 5.
  • amino acid sequence of the heavy chain variable region of the antibody 3H6H4L1 is shown in SEQ ID NO: 6.
  • the nucleic acid sequence encoding the light chain variable region of the antibody 3H6H4L1 is shown in SEQ ID NO: 7.
  • amino acid sequence of the light chain variable region of the antibody 3H6H4L1 is shown in SEQ ID NO: 8.
  • the heavy chain constant regions of 3H6H1L1, 3H6H2L2 and 3H6H3L3 all use Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant regions all use Ig kappa chain C region, ACCESSION: P01834;
  • the heavy chain constant region of 3H6H4L1 is Ig gamma-4 chain C region, ACCESSION: P01861.1; the light chain constant region is Ig kappa chain C region, ACCESSION: P01834.
  • 3H6H1L1 heavy chain cDNA and light chain cDNA, 3H6H2L2 heavy chain cDNA and light chain cDNA, 3H6 H3L3 heavy chain cDNA and light chain cDNA, and 3H6H4L1 heavy chain cDNA and light chain cDNA were cloned into pUC57simple vector (gold (Provided by Sirui), eight recombinant plasmids were obtained, namely pUC57simple-3H6H1 and pUC57simple-3H6L1; pUC57simple3H6H2 and pUC57simple-3H6L2; pUC57simple-3H6H3 and pUC57simple-3H6L3; pUC57simple-3H6H4 and pUC57simple-3H6H4 and pUC57simple-3H6L2.
  • Example 4 Antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 and human Study on the binding activity of IL-1 ⁇ -his-bio (ELISA method)
  • 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 can effectively bind human IL-1 ⁇ -His-Bio, and the binding efficiency is in a dose-dependent relationship;
  • Example 5 Antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 and Study on the activity of human IL-1R1(1-332)-his in binding to human IL-1 ⁇ -hFc (ELISA method)
  • the ELISA plate was coated with 4 ⁇ g/mL human IL-1 ⁇ -hFc, 50 ⁇ L per well, and incubated overnight at 4°C. Wash the plate once and remove the remaining liquid, add 300 ⁇ L 1% BSA solution (dissolved in PBS) to each well to block, incubate at 37°C for 2 hours, wash the plate three times and remove the remaining liquid.
  • the antibody was diluted to 2 ⁇ g/mL (final concentration 1 ⁇ g/mL) as the starting concentration, and a 1:3 gradient dilution was carried out for a total of 7 concentrations, and a blank control was set up. Two replicate wells were made, each with a volume of 50 ⁇ L, and incubated for 10 at room temperature. minute.
  • 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 can effectively block the binding of antigen human IL-1 ⁇ -hFc and its receptor human IL-1R1(1-332)-his, and the blocking efficiency is dose-dependent, competing for binding
  • the activity is better than that of Canakinumab, an already marketed drug with the same target.
  • the Biacore molecular interaction instrument was used to detect the affinity constant of the antibody and human IL-1 ⁇ -his. Using PBST as a buffer, the antibody was immobilized on the surface of the CM5 chip by amino coupling, and the immobilized signal value was about 1000 RU. The antibody binds to human IL-1 ⁇ , the IL-1 ⁇ concentration is 1.56-25nM (two-fold dilution), the flow rate is 30 ⁇ l/min, the binding time is 120s, and the dissociation time is 600s. The chip is regenerated with 3M MgCl 2 at a flow rate of 30 ⁇ l/min and a time of 30s. Biacore Control 2.0 software was used for data collection, and Biacore T200 Evaluation 2.0 software was used for data analysis. The results are shown in Table 5, Figure 5 and Figure 6.
  • Human MRC-5 cells (purchased from the Cell Center of the Chinese Academy of Sciences) are routinely digested and counted. 7,500 cells/well are seeded in a flat-bottomed 96-well plate and placed in a cell culture incubator; 24h later (when the cell growth reaches 80% confluence), Dosing treatment: antibody is set to 4 concentrations (0.003nM, 0.03nM, 0.3nM, 3nM), IL-1 ⁇ (purchased from Sino Biological, item number: 10139-HNAE) is set to three concentrations (5pM, 50pM, 500pM), Antibody group IL-1 ⁇ with a concentration of 50pM (antibody and IL-1 ⁇ were incubated at 37°C for 30min), and a blank control group and an isotype control group were set at the same time; after adding the drug, it was placed in a cell incubator for 24 hours; the cells were collected Clear, use IL-6ELISAKit (purchased from Dakwei Biotechnology Co., Ltd., article number:
  • IL-1 ⁇ can induce IL-6 secretion by MRC-5 cells in a gradient
  • 3H6 H4L1 can significantly inhibit IL-1 ⁇ 's induction of IL-6 secretion and release by MRC-5, and it has a significant dose-dependent relationship.
  • 3H6 H4L1 can specifically bind IL-1 ⁇ to block IL-1 ⁇ -mediated secretion of the immune factor IL-6, and its blocking efficacy is equivalent to that of the target control antibody Canakinumab.
  • the fluorescent reporter gene method was used to detect the neutralizing biological activity of 3H6 H4L1 blocking IL-1 ⁇ to activate the NF- ⁇ B signaling pathway.
  • 3H6 H4L1 blocks IL-1 ⁇ to activate the neutralization of the NF- ⁇ B signaling pathway.
  • the 293T-NF- ⁇ B-Luc cells are routinely digested, and 20,000 cells per well are seeded in a 96-well plate. After the cells adhere to the wall, add IL-1 ⁇ to a final concentration of 1.65ng/mL, and set a blank control. Add antibodies at the same time, Canakinumab, 3H6 H4L1, 8 gradients for each antibody, the final concentrations are: 1600ng/mL, 800ng/mL, 200ng/mL, 50ng/mL, 12.5ng/mL, 3.125ng/mL, 0.3125ng/ mL, 0.03125ng/mL. After 4 hours of co-cultivation, remove the supernatant, add 50 ⁇ L PBS, and then add 50 ⁇ L Bright-Glo TM substrate (Promega, catalog number: E2620), react for 5 minutes, and test on the computer.
  • IL-1 ⁇ can activate the NF- ⁇ B signaling pathway of 293T-NF ⁇ B-Luc cells in a dose-dependent manner, and then initiate the expression of the reporter gene.
  • This system can be used to evaluate the neutralizing biological activity of 3H6H4L1 on IL-1 ⁇ .
  • the results of this study show that 3H6H4L1 specifically binds to IL-1 ⁇ and can effectively inhibit the activation of IL-1 ⁇ -mediated signaling pathways, showing the neutralizing biological activity of 3H6H4L1 on IL-1 ⁇ , and the neutralizing activity EC50 is 0.0966nM.
  • the neutralizing activity EC50 of the same target drug Canakinumab is 0.0801nM, which is equivalent in activity.

Abstract

Disclosed in the present invention are an anti-IL-1β antibody, a pharmaceutical composition containing same, and the use thereof. The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains HCDR1-HCDR3 having amino acid sequences as shown in SEQ ID NO: 17 to SEQ ID NO: 19 respectively, and the light chain variable region contains LCDR1-LCDR3 having amino acid sequences as shown in SEQ ID NO: 20 to SEQ ID NO: 22 respectively.

Description

抗IL-1β的抗体、其药物组合物及其用途Anti-IL-1β antibody, its pharmaceutical composition and its use 技术领域Technical field
本发明属于免疫学领域，涉及一种抗IL-1β的抗体、其药物组合物及其用途。具体地，本发明涉及一种抗人IL-1β的抗体；更具体地，本发明涉及一种抗人IL-1β的单克隆抗体；特别具体地，本发明涉及一种抗人IL-1β的人源化单克隆抗体。The invention belongs to the field of immunology, and relates to an anti-IL-1β antibody, its pharmaceutical composition and its use. Specifically, the present invention relates to an anti-human IL-1β antibody; more specifically, the present invention relates to an anti-human IL-1β monoclonal antibody; in particular, the present invention relates to an anti-human IL-1β antibody Humanized monoclonal antibody.
背景技术Background technique
IL-1β是IL-1家族成员，其在先天免疫和炎症发生中发挥关键作用。IL-1β家族成员包含7个细胞因子(IL-1α，IL-1β，IL-18，IL-33，IL-36α，IL-36β，IL-36γ，IL-37)和三种特异性受体拮抗物(IL-1Ra，IL-36Ra和IL-38)。IL-1β是由无活性的前体蛋白ProIL-1β酶解生成，这一过程主要由炎性小体NLRP3(NOD‐like receptor protein 3)介导(Jesus AA and Goldbach-Mansky R.IL-1 blockade in autoinflammatory syndromes.Annu Rev Med 2014)。IL-1β is a member of the IL-1 family, which plays a key role in innate immunity and inflammation. IL-1β family members include 7 cytokines (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-37) and three specific receptors Antagonists (IL-1Ra, IL-36Ra and IL-38). IL-1β is produced by enzymatic hydrolysis of the inactive precursor protein ProIL-1β. This process is mainly mediated by the inflammasome NLRP3 (NOD-like receptor protein 3) (JesusAA and Goldbach-Mansky R.IL-1). blockade in autoinflammatory syndromes.Annu Rev Med 2014).
IL-1β及IL-1α的受体是IL-1R1和IL-1RAcP异二聚体，其中IL-1R1为IL-1β及IL-1α特异性结合的受体亚基，IL-1RAcP(白介素-1-受体协同蛋白)是IL-1家族其它多个成员如IL-33、IL-36等的受体共有组成部分。IL-1R1为跨膜受体，可与IL-1β结合并与IL-1RAcP结合形成受体复合物，激活下游胞内相关信号通路，介导IL-1β相关生物学效应。IL-1β存在天然的负性调节分子，包括IL-1R2，可溶性IL-1R1和IL-1R2，以及IL-1受体拮抗物(IL-1Ra)。IL-R2可与IL-1β结合，然而由于胞内段较短因此无法激活下游通路，可溶性IL-1R1和IL-1R2结合IL-1阻止其与膜定位受体进一步结合；IL-1Ra可与IL-1R1结合进而阻断IL-1α和IL-1β与相应受体的结合(Boraschi D,Italiani P,Weil S,Martin MU.The family of the interleukin-1 receptors.Immunol Rev.2018；281(1):197-232)。The receptors of IL-1β and IL-1α are IL-1R1 and IL-1RAcP heterodimers, and IL-1R1 is the receptor subunit that IL-1β and IL-1α specifically bind, IL-1RAcP (interleukin- 1-receptor co-protein) is a common component of receptors of other members of the IL-1 family, such as IL-33 and IL-36. IL-1R1 is a transmembrane receptor that can bind to IL-1β and IL-1RAcP to form a receptor complex, activate downstream intracellular signaling pathways, and mediate IL-1β-related biological effects. There are natural negative regulators of IL-1β, including IL-1R2, soluble IL-1R1 and IL-1R2, and IL-1 receptor antagonist (IL-1Ra). IL-R2 can bind to IL-1β, but because the intracellular segment is short, it cannot activate downstream pathways. The binding of soluble IL-1R1 and IL-1R2 to IL-1 prevents it from further binding to membrane-localized receptors; IL-1Ra can interact with The binding of IL-1R1 blocks the binding of IL-1α and IL-1β to the corresponding receptors (Boraschi D, Italiani P, Weil S, Martin MU. The family of the interleukin-1 receptors. Immunol Rev. 2018; 281(1) ):197-232).
IL-1β主要由单核细胞和巨噬细胞产生，IL-1β与其受体结合后激活下游NF-κB和MAPK依赖性的促炎性细胞内信号级联反应。IL-1β是COX-2表达的有效诱导剂，产生大量的前列腺素E2(PGE2)抑制IFN-γ的产生，进而抑制Th1型T细胞主导的针对病毒和胞内寄生菌的Th1型免疫反应(Napolitani G, Acosta-Rodriguez EV,Lanzavecchia A,Sallusto F.Prostaglandin E2 enhances Th17 responses via modulation of IL-17 and IFN-gamma production by memory CD4+T cells.European journal of immunology.2009；39(5):1301–12.)。IL-1β的另一关键效应是促进IL-6的分泌。IL-6是诱导幼稚T细胞分化为Th17型T细胞的关键因素(Bettelli E,Carrier Y,Gao W,Korn T,Strom TB,Oukka M,et al.Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells.Nature.2006；441(7090):235–8.)；IL-6还可以抑制调节性T细胞的免疫抑制功能，并阻止Th17型T细胞转化为免疫抑制性的Treg型T细胞(Korn T,Mitsdoerffer M,Croxford AL,Awasthi A,Dardalhon VA,Galileos G,et al.IL-6 controls Th17 immunity in vivo by inhibiting the conversion of conventional T cells into Foxp3+regulatory T cells.Proceedings of the National Academy of Sciences of the United States of America.2008；105(47):18460–5.Pasare C,Medzhitov R.Toll pathway-dependent blockade of CD4+CD25+T cell-mediated suppression by dendritic cells.Science.2003；299(5609):1033–6)。IL-1β介导的Th17型免疫反应抑制了CD8+细胞毒性T细胞对病毒感染细胞的杀伤，并增强了病毒感染细胞的存活率，从而可能降低了机体对病毒的防御(Hou W,Kang HS,Kim BS.Th17 cells enhance viral persistence and inhibit T cell cytotoxicity in a model of chronic virus infection.The Journal of experimental medicine.2009；206(2):313–28.)。此外，IL-1β通过诱导Th17细胞的增殖和活化，增加GM-CSF的分泌(Mufazalov IA,Schelmbauer C,Regen T,Kuschmann J,Wanke F,Gabriel LA,Hauptmann J,Müller W,Pinteaux E,Kurschus FC,Waisman A.IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+Th17 cells.EMBO J.2017 4；36(1):102-115)；除免疫细胞以外，内皮细胞、成纤维细胞、软骨细胞和平滑肌细胞等在IL-1和TNF-α的诱导下均可产生(GM-CSF S.Matthew Fitzgerald,David S.Chi,H.Kenton Hall,Scott A.Reynolds,Omolola Aramide,Steven A.Lee,and Guha Krishnaswamy.GM-CSF Induction in Human Lung Fibroblasts by IL-1β,TNF-α,and Macrophage Contact.Journal of Interferon&Cytokine ResearchVol.23,No.2.A.A.M.A.Baqui,Timothy F.Meiller,Jennifer J.Chon,Been-Foo Turng,and William A.Falkler,Jr. Granulocyte-Macrophage Colony-Stimulating Factor Amplification of Interleukin-1β and Tumor Necrosis Factor Alpha Production in THP-1 Human Monocytic Cells Stimulated with Lipopolysaccharide of Oral Microorganisms.Clin Diagn Lab Immunol.1998 May；5(3):341–347)。IL-1β is mainly produced by monocytes and macrophages. After IL-1β binds to its receptor, it activates the downstream NF-κB and MAPK-dependent pro-inflammatory intracellular signal cascade. IL-1β is an effective inducer of COX-2 expression. It produces a large amount of prostaglandin E2 (PGE2) to inhibit the production of IFN-γ, thereby inhibiting Th1-type T cell-led Th1-type immune response against viruses and intracellular parasites ( Napolitani G, Acosta-Rodriguez EV, Lanzavecchia A, Sallusto F. Prostaglandin E2 enhances Th17 responses via modulation of IL-17 and IFN-gamma production by memory CD4+T cells.Europeanimmunology: 1 301; 2009; –12.). Another key effect of IL-1β is to promote the secretion of IL-6. IL-6 is a key factor in inducing the differentiation of naive T cells into Th17 T cells (Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al. Reciprocal developmental pathways for the generation of pathogenic effect TH17 and regulatory T cells.Nature.2006;441(7090):235-8.); IL-6 can also inhibit the immunosuppressive function of regulatory T cells and prevent Th17 T cells from transforming into immunosuppressive Treg T cells (Korn T, Mitsdoerffer M, Croxford AL, Awasthi A, Dardalhon VA, Galileos G, et al. IL-6 Controls Th17 immunity in vivo by inhibiting the conversion of conventional T cells of the National Cells of the Academy of Foxp3+regulators. of Sciences of the United States of America.2008; 105(47):18460-5.Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+T cell-mediated suppression by dendritic cells.Science.2003; 299 (5609):1033–6). IL-1β-mediated Th17 immune response inhibits the killing of virus-infected cells by CD8+ cytotoxic T cells, and enhances the survival rate of virus-infected cells, which may reduce the body's defense against viruses (Hou W, Kang HS, Kim BS.Th17 cells enhancement viral persistence and inhibition T cell cytotoxicity in a model of chronic virus infection. The Journal of experimental medicine.2009;206(2):313-28.). In addition, IL-1β increases the secretion of GM-CSF by inducing the proliferation and activation of Th17 cells (Mufazalov IA, Schelmbauer C, Regen T, Kuschmann J, Wanke F, Gabriel LA, Hauptmann J, Müller W, Pinteaux E, Kurschus FC ,Waisman A.IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+Th17 cells.EMBO J.2017 4; 36(1):102-115); in addition to immune cells, endothelial cells, fibroblasts Cells, chondrocytes and smooth muscle cells can be produced under the induction of IL-1 and TNF-α (GM-CSF S. Matthew Fitzgerald, David S. Chi, H. Kenton Hall, Scott A. Reynolds, Omolola Aramide, Steven A. Lee, and Guha Krishnaswamy.GM-CSF Induction in Human Lung Fibroblasts by IL-1β, TNF-α, and Macrophage Contact.Journal of Interferon&Cytokine ResearchVol.23,No.2.AAMABaqui,Timothy F.MeJiller,Jennifer .Chon, Been-Foo Turn, and William A. Falkler, Jr. Granulocyte-Macrophage Colony-Stimulating Factor Amplification of Interleukin-1β and Tumor Necrosis Factor Alpha Production in THP-1 Human Poly-Organic Monopolysaccharide with Cells Organizing Lab Immunol. 1998 May; 5(3):341–347).
现有证据证实，IL-1β在病毒感染引起的致命性系统炎症反应中发挥关键作用。在严重急性呼吸系统综合症冠状病毒(SARS-CoV)感染中，研究证实SARS-CoV 3a蛋白可直接激活巨噬细胞中的NLRP3炎性小体，介导的IL-1β的分泌，这可能是SARS中观察到的IL-1β，及其下游IL-6升高的机制(Chen IY,Moriyama M,Chang MF,Ichinohe T.Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3Inflammasome.Front Microbiol.eCollection 2019)。在同属冠状病毒科病毒感染所致的新型冠状病毒肺炎(COVID-19)死亡患者尸检中发现，患者血液中的CD4+和CD8+T细胞数量显著减少，但是Th17细胞比例显著增加(Xu Z,Shi L,Wang YJ,Zhang JY,Huang L,Zhang C et al.Pathological findings of COVID-19 associated with acute respiratory distress syndrome.The Lancet Respiratory Medicine.2020)。多项研究报道在新型冠状肺炎重症患者中IL-6、GM-CSF在重症及危重症患者中显著升高(Characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia(NCP).Wan SX,Yi QJ,Fan SB,Lv JL,Zhang XX,Guo L,Lang CH,Xiao Q,Xiao KH,et al.medRxiv 2020.02.10.20021832；陈蕾,刘辉国,刘威等.2019新型冠状病毒肺炎29例临床特征分析.中华结核和呼吸杂志,2020,43:E005)。以上均提示患者出现过度的、致命性的炎症反应，而IL-1β信号通路抑制则可能用于治疗此类疾病。目前抗IL-6抗体已被批注用于治疗CAT-T疗法发生的细胞因子释放综合征。然而，作为IL-6的上游分子，抑制IL-1β相关信号通路可能对于治疗病毒感染、肿瘤或者细胞免疫疗法引起的过度的、致命性的炎症反应更为有优势。Norelli等人报道，在嵌合抗原受体T细胞(CAR-T)治疗淋巴瘤小鼠模型中，IL-1受体拮抗剂Anakinra，一种重组人IL-1Ra，可有效缓解CAT-T细胞治疗所引起的细胞因子释放综合症临床症状，减轻CAT-T疗法的神经系统毒性，从而大大延长了实验动物的无病生存期；而抗IL-6抗体未能保护小鼠免受以神经系统炎症为特征的 延迟致死性神经毒性(Norelli M,Camisa B,Barbiera G,Falcone L,Purevdorj A,Genua M,Sanvito F,Ponzoni M,Doglioni C,Cristofori P,Traversari C,Bordignon C,Ciceri F,Ostuni R,Bonini C,Casucci M,Bondanza A.Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells.Nat Med.2018 Jun；24(6):739-748)。Existing evidence confirms that IL-1β plays a key role in the fatal systemic inflammatory response caused by viral infection. In severe acute respiratory syndrome coronavirus (SARS-CoV) infection, studies have confirmed that SARS-CoV 3a protein can directly activate the NLRP3 inflammasome in macrophages and mediate the secretion of IL-1β, which may be The IL-1β observed in SARS and its downstream IL-6 increase mechanism (Chen IY, Moriyama M, Chang MF, Ichinohe T. Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3Inflammasome. Front Microbiol.eCollection 2019). In the autopsy of patients who died of new coronavirus pneumonia (COVID-19) caused by virus infection of the same coronavirus family, it was found that the number of CD4+ and CD8+ T cells in the patient's blood was significantly reduced, but the proportion of Th17 cells was significantly increased (Xu Z, Shi L, Wang YJ, Zhang JY, Huang L, Zhang C et al. Pathological findings of COVID-19 associated with acute respiratory distress syndrome. The Lancet Respiratory Medicine. 2020). Multiple studies have reported that IL-6 and GM-CSF are significantly increased in severe and critically ill patients with new coronary pneumonia (Characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (NCP) .Wan SX, Yi QJ, Fan SB, Lv JL, Zhang XX, Guo L, Lang CH, Xiao Q, Xiao KH, et al. medRxiv 2020.02.10.20021832; Chen Lei, Liu Huiguo, Liu Wei, etc. 2019 Novel Coronavirus Analysis of the clinical characteristics of 29 cases of pneumonia. Chinese Journal of Tuberculosis and Respiratory Medicine, 2020, 43: E005). All of the above suggest that patients have an excessive and fatal inflammatory response, and IL-1β signaling pathway inhibition may be used to treat such diseases. At present, anti-IL-6 antibody has been annotated for the treatment of cytokine release syndrome caused by CAT-T therapy. However, as an upstream molecule of IL-6, inhibiting IL-1β-related signaling pathways may be more advantageous for treating excessive and fatal inflammatory responses caused by viral infections, tumors or cellular immunotherapy. Norelli et al. reported that the IL-1 receptor antagonist Anakinra, a recombinant human IL-1Ra, can effectively alleviate CAT-T cells in a mouse model of lymphoma treated with chimeric antigen receptor T cells (CAR-T) The clinical symptoms of cytokine release syndrome caused by treatment can reduce the nervous system toxicity of CAT-T therapy, thereby greatly extending the disease-free survival period of experimental animals; while anti-IL-6 antibodies failed to protect mice from the nervous system Delayed lethal neurotoxicity characterized by inflammation (Norelli M, Camisa B, Barbiera G, Falcone L, Purevdorj A, Genua M, Sanvito F, Ponzoni M, Dogliani C, Cristofori P, Traversari C, Bordignon C, Civiceri F, Ostuni R, Bonini C, Casucci M, Bondanza A. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat Med. 2018 Jun; 24(6): 739- 748).
如前述，病原微生物(病毒、细菌或真菌或其它)感染、烧伤、胰腺炎、输血或异体血细胞移植、外伤、药物(抗体、免疫激动剂、或细胞因子)、嵌合抗原细胞疗法、手术、放疗、化疗等均可导致系统性炎症反应，表现为IL-1β的分泌增加，甚至引起IL-1β分泌增加为特征的细胞因子释放综合征，表现为机体多器官损伤包括急性肾损伤，急性呼吸系统损伤如急性呼吸窘迫综合征(ARDS)，血管内弥散性凝血，高铁血蛋白症，神经系统炎症等等。As mentioned above, pathogenic microorganisms (viruses, bacteria or fungi or other) infections, burns, pancreatitis, blood transfusion or allogeneic blood cell transplantation, trauma, drugs (antibodies, immunoagonists, or cytokines), chimeric antigen cell therapy, surgery, Radiotherapy, chemotherapy, etc. can lead to systemic inflammation, which is manifested by increased secretion of IL-1β, and even caused cytokine release syndrome characterized by increased secretion of IL-1β, manifested by multiple organ damages including acute kidney injury and acute breathing System damage such as acute respiratory distress syndrome (ARDS), intravascular diffuse coagulation, methemorinemia, nervous system inflammation and so on.
虽然IL-1受体拮抗剂如Anakinra目前已在临床得到应用，然而由于其体内代谢速率快，半衰期仅为数小时，需要每天注射以维持药效，所以开发具有稳定药代动力学特征的抗IL-1β的抗体药物具有很大临床应用潜力。Although IL-1 receptor antagonists such as Anakinra have been used clinically, due to its fast metabolism in the body and a half-life of only a few hours, it needs to be injected daily to maintain drug efficacy. Therefore, anti-IL with stable pharmacokinetic characteristics has been developed. -1β antibody drugs have great clinical application potential.
发明内容Summary of the invention
本发明人经过深入的研究和创造性的劳动，利用哺乳动物细胞表达系统表达出重组的IL-1β-His作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。发明人通过对大量样本进行筛选，得到了如下的杂交瘤细胞株：After in-depth research and creative work, the inventors used the mammalian cell expression system to express recombinant IL-1β-His as an antigen to immunize mice, and obtain hybridoma cells by fusion of mouse spleen cells and myeloma cells. The inventors screened a large number of samples and obtained the following hybridoma cell lines:
杂交瘤细胞株LT010，其于2018年6月21日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018133。Hybridoma cell line LT010, which was deposited in the China Center for Type Culture Collection (CCTCC) on June 21, 2018, and the deposit number is CCTCC NO: C2018133.
本发明人惊奇地发现：The inventor surprisingly found that:
杂交瘤细胞株LT010能够分泌产生与人IL-1β特异性结合的特异性单克隆抗体(命名为3H6)，并且该单克隆抗体能够十分有效地阻断IL-1β与IL-1R1的结合；The hybridoma cell line LT010 can secrete and produce a specific monoclonal antibody (named 3H6) that specifically binds to human IL-1β, and the monoclonal antibody can effectively block the binding of IL-1β to IL-1R1;
进一步地，本发明人创造性地制得了抗人IL-1β的人源化抗体(分别命名为3H6 H1L1，3H6 H2L2，3H6 H3L3，3H6 H4L1)，能有效地结合人IL-1β，阻断IL-1β与其受体IL-1R1的结合，抑制IL-1β下游信号通路的激活；具有用 于制备降低、预防或治疗病原微生物(病毒、细菌或真菌或其它)感染、烧伤、胰腺炎、输血或异体血细胞移植、外伤、药物(抗体、免疫激动剂、或细胞因子)、嵌合抗原细胞疗法、手术、放疗、化疗所引起的系统性炎症反应。Furthermore, the present inventors creatively prepared humanized antibodies against human IL-1β (named 3H6 H1L1, 3H6 H2L2, 3H6 H3L3, 3H6 H4L1), which can effectively bind to human IL-1β and block IL- The binding of 1β to its receptor IL-1R1 inhibits the activation of IL-1β downstream signaling pathways; it can be used for preparation to reduce, prevent or treat pathogenic microorganisms (viruses, bacteria or fungi or other) infections, burns, pancreatitis, blood transfusions or foreign bodies Systemic inflammation caused by blood cell transplantation, trauma, drugs (antibodies, immune agonists, or cytokines), chimeric antigen cell therapy, surgery, radiotherapy, and chemotherapy.
由此提供了下述发明：This provides the following inventions:
本发明的一个方面涉及一种抗IL-1β的抗体或其抗原结合片段，其中，One aspect of the present invention relates to an anti-IL-1β antibody or antigen-binding fragment thereof, wherein
所述抗体的重链可变区包含：氨基酸序列分别如SEQ ID NO:17－SEQ ID NO:19所示的HCDR1-HCDR3；和The heavy chain variable region of the antibody comprises: the amino acid sequences of HCDR1-HCDR3 shown in SEQ ID NO: 17-SEQ ID NO: 19, respectively; and
所述抗体的轻链可变区包含：氨基酸序列分别如SEQ ID NO:20－SEQ ID NO:22所示的LCDR1-LCDR3。The light chain variable region of the antibody includes LCDR1-LCDR3 with amino acid sequences as shown in SEQ ID NO: 20-SEQ ID NO: 22, respectively.
优选地，所述IL-1β为人IL-1β。Preferably, the IL-1β is human IL-1β.
重链可变区和轻链可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Sequences of Proteins of Immunological Interest.Fifth Edition(1991),第1-3卷,NIH Publication 91-3242,Bethesda Md)。The variable region of the heavy chain and the variable region of the light chain determine the binding of the antigen; the variable region of each chain contains three hypervariable regions, called the complementarity determining region (CDR) (the CDR of the heavy chain (H) includes HCDR1, HCDR2 , HCDR3, the CDR of the light chain (L) includes LCDR1, LCDR2, LCDR3; it is named by Kabat et al., see Sequences of Proteins of Immunological Interest.Fifth Edition (1991), Vols 1-3, NIH Publication 91-3242, Bethesda Md).
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库分析本发明的抗体3H6、3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1，可以发现其具有相同的HCDR1-3和LCDR1-3：Through technical means well known to those skilled in the art, such as analyzing the antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 of the present invention through the VBASE2 database, it can be found that they have the same HCDR1-3 and LCDR1-3:
其重链可变区的3个HCDR区的氨基酸序列如下：The amino acid sequences of the three HCDR regions of the heavy chain variable region are as follows:
HCDR1：GFSLSTSGMG(SEQ ID NO：17)，HCDR1: GFSLSTSGMG (SEQ ID NO: 17),
HCDR2：IYWDDDK(SEQ ID NO：18)，HCDR2: IYWDDDK (SEQ ID NO: 18),
HCDR3：ARSAYYSFAY(SEQ ID NO：19)；HCDR3: ARSAYYSFAY (SEQ ID NO: 19);
其轻链可变区的3个CDR区的氨基酸序列如下：The amino acid sequences of the 3 CDR regions of the light chain variable region are as follows:
LCDR1：QDVDTD(SEQ ID NO:20)，LCDR1: QDVDTD (SEQ ID NO: 20),
LCDR2：WAS(SEQ ID NO:21)，LCDR2: WAS (SEQ ID NO: 21),
LCDR3：QQYSSYPT(SEQ ID NO:22)。LCDR3: QQYSSYPT (SEQ ID NO: 22).
在本发明的一个或多个实施方案中，其中，In one or more embodiments of the present invention, wherein,
所述抗体的重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14或与SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO: 10和SEQ ID NO:14的序列分别具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；和The amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, or with SEQ ID NO: 2, SEQ ID NO: 6 , SEQ ID NO: 10 and SEQ ID NO: 14 have sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity, respectively; and
所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4和SEQ ID NO:8、SEQ ID NO:12和SEQ ID NO:16或与SEQ ID NO:4和SEQ ID NO:8、SEQ ID NO:12和SEQ ID NO:16的序列分别具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列。The amino acid sequence of the variable region of the light chain of the antibody is selected from SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16, or with SEQ ID NO: 4 and SEQ ID NO: 8. The sequences of SEQ ID NO: 12 and SEQ ID NO: 16 respectively have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical sequences.
在本发明的一些实施方案中，所述抗体选自：In some embodiments of the invention, the antibody is selected from:
(1)如SEQ ID NO:2或与SEQ ID NO:2具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VH和如SEQ ID NO:4或与SEQ ID NO:4具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VL；(1) The VH shown in SEQ ID NO: 2 or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO: 2 And the VL shown in SEQ ID NO: 4 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO: 4;
(2)如SEQ ID NO:6或与SEQ ID NO:6具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VH和如SEQ ID NO:8或与SEQ ID NO:8具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VL；(2) The VH shown in SEQ ID NO: 6 or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO: 6 And the VL shown in SEQ ID NO: 8 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO: 8;
(3)如SEQ ID NO:10或与SEQ ID NO:10具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VH和如SEQ ID NO:12或与SEQ ID NO:12具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VL；和(3) The VH shown in SEQ ID NO: 10 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO: 10 And the VL shown in SEQ ID NO: 12 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO: 12; and
(4)如SEQ ID NO:14或与SEQ ID NO:14具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VH和如SEQ ID NO:16或与SEQ ID NO:16具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VL。(4) The VH shown in SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO: 14 And VL shown in SEQ ID NO: 16 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO: 16.
在本发明的一个或多个实施方案中，所述抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双抗体。In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragments, single-chain antibodies (e.g. , ScFv), humanized antibody, chimeric antibody or diabody.
在本发明的一个或多个实施方案中，其中，所述的抗体以小于10 -5M，例如小于10 -6M、小于10 -7M、小于10 -8M、小于10 -9M或小于10 -10M或更小的K D结合IL-1β蛋白；优选地，所述K D通过Biacore分子相互作用仪测得；优选地， 所述K D通过Fortebio分子相互作用仪测得。 In one or more embodiments of the present invention, wherein the antibody has a concentration of less than 10 -5 M, for example, less than 10 -6 M, less than 10 -7 M, less than 10 -8 M, less than 10 -9 M or A K D of less than 10 -10 M or less binds to IL-1β protein; preferably, the K D is measured by a Biacore molecular interaction instrument; preferably, the K D is measured by a Fortebio molecular interaction instrument.
在本发明的一些实施方式中，其中，所述的抗体以小于大约100nM，例如小于大约10nM、小于大约1nM、小于大约0.9nM、小于大约0.8nM、小于大约0.7nM、小于大约0.6nM、小于大约0.5nM、小于大约0.4nM、小于大约0.3nM、小于大约0.2nM、小于大约0.1nM或更小的EC 50结合IL-1β蛋白。具体地，所述EC 50通过间接ELISA方法测得。 In some embodiments of the present invention, wherein the antibody is less than about 100 nM, for example, less than about 10 nM, less than about 1 nM, less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about An EC 50 of about 0.5 nM, less than about 0.4 nM, less than about 0.3 nM, less than about 0.2 nM, less than about 0.1 nM or less binds IL-1 β protein. Specifically, the EC 50 is measured by an indirect ELISA method.
在本发明的一个或多个实施方案中，其中，In one or more embodiments of the present invention, wherein,
所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。The antibody includes a non-CDR region, and the non-CDR region is from a species other than murine, for example, from a human antibody.
在本发明的一些实施方案中，所述抗体的恒定区是人源化的，例如，重链恒定区均采用Ig gamma-1chain C region，例如ACCESSION:P01857或Ig gamma-4chain C region，例如ACCESSION：P01861.1；轻链恒定区均采用Ig kappa chain C region，例如ACCESSION:P01834。In some embodiments of the present invention, the constant region of the antibody is humanized. For example, the constant region of the heavy chain uses Ig gamma-1 chain C region, such as ACCESSION: P01857 or Ig gamma-4 chain C region, such as ACCESSION : P01861.1; Ig kappa chain C region is used for the light chain constant region, such as ACCESSION: P01834.
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段，其中所述抗体是由杂交瘤细胞株LT010产生的单克隆抗体，所述杂交瘤细胞株LT010保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018133。In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody produced by a hybridoma cell line LT010, and the hybridoma cell line LT010 is deposited in China. Center for the Collection of Cultures (CCTCC), the deposit number is CCTCC NO: C2018133.
在本发明的一个或多个实施方案中，所述抗体为单克隆抗体。In one or more embodiments of the invention, the antibody is a monoclonal antibody.
本发明的另一方面涉及一种抗体药物偶联物(antibody-drug conjugate，ADC)，其包括抗体或其抗原结合片段以及小分子药物，其中，所述抗体或其抗原结合片段为本发明中任一项所述的抗体或其抗原结合片段；优选地，所述小分子药物为小分子细胞毒药物；更优选地，所述小分子药物为化疗药物。Another aspect of the present invention relates to an antibody-drug conjugate (antibody-drug conjugate, ADC), which includes an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or an antigen-binding fragment thereof is in the present invention Any one of the antibody or antigen-binding fragment thereof; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a chemotherapeutic drug.
所述化疗药物可以是常规的肿瘤化疗药物，例如烷化剂、抗代谢药、抗肿瘤抗生素、植物类抗癌药、激素、免疫抑制剂等。The chemotherapeutic drugs may be conventional tumor chemotherapeutics, such as alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones, immunosuppressants and the like.
在本发明的一个或多个实施方案中，所述抗体或其抗原结合片段通过连接子与小分子药物连接；所述连接子可以是本领域技术人员知悉的连接子，例如，所述连接子为腙键、二硫键或肽键。In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof is connected to the small molecule drug through a linker; the linker may be a linker known to those skilled in the art, for example, the linker It is a hydrazone bond, a disulfide bond or a peptide bond.
在本发明的一个或多个实施方案中，所述抗体或其抗原结合片段与小分子药物以一定的摩尔比连接；例如，所述摩尔比为1：(2－4)。In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof and the small molecule drug are linked at a certain molar ratio; for example, the molar ratio is 1: (2-4).
本发明的再一方面涉及一种双特异性抗体(又称为双功能抗体)，其包括第一蛋白功能区和第二蛋白功能区，其中：Another aspect of the present invention relates to a bispecific antibody (also called a bifunctional antibody), which includes a first protein functional region and a second protein functional region, wherein:
所述第一蛋白功能区靶向IL-1β，The first protein functional region targets IL-1β,
所述第二蛋白功能区靶向不同于IL-1β的靶点，例如IL-17A；The second protein functional region targets a target different from IL-1β, such as IL-17A;
其中,所述第一蛋白功能区为本发明中任一项所述的抗体或其抗原结合片段；Wherein, the first protein functional region is the antibody or antigen-binding fragment thereof according to any one of the present invention;
优选地，所述双特异性抗体为IgG-scFv模式；Preferably, the bispecific antibody is in IgG-scFv mode;
优选地，Preferably,
(1)所述第一蛋白功能区为本发明中任一项所述的抗体或其抗原结合片段，并且所述第二蛋白功能区为单链抗体；(1) The first protein functional region is the antibody or antigen-binding fragment thereof according to any one of the present invention, and the second protein functional region is a single-chain antibody;
或者，or,
(2)所述第一蛋白功能区为单链抗体，其重链可变区包含氨基酸序列如SEQ ID NO:17－SEQ ID NO:19所示的HCDR1－HCDR3，其轻链可变区包含氨基酸序列如SEQ ID NO:20－SEQ ID NO:22所示的LCDR1－LCDR3，并且所述第二蛋白功能区为抗体(例如单克隆抗体)。(2) The first protein functional region is a single-chain antibody, and its heavy chain variable region contains the amino acid sequence of HCDR1-HCDR3 shown in SEQ ID NO: 17-SEQ ID NO: 19, and its light chain variable region contains The amino acid sequence is LCDR1 to LCDR3 shown in SEQ ID NO: 20 to SEQ ID NO: 22, and the second protein functional region is an antibody (for example, a monoclonal antibody).
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；In some embodiments of the present invention, the bispecific antibody, wherein the first protein functional region and the second protein functional region are directly connected or connected by a linking fragment;
优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；Preferably, the connection fragment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;
优选地，所述连接片段为SS(GGGGS)n，n为正整数，例如1、2、3、4、5或6。Preferably, the connection fragment is SS(GGGGS)n, where n is a positive integer, such as 1, 2, 3, 4, 5 or 6.
在本发明的一些实施方式中，所述的双特异性抗体的第(2)项中，In some embodiments of the present invention, in item (2) of the bispecific antibody,
所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14或与SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14的序列分别具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；和The amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, or with SEQ ID NO: 2, SEQ ID NO :6, SEQ ID NO: 10 and SEQ ID NO: 14 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical, respectively; and
所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4和SEQ ID NO:8、SEQ ID NO:12和SEQ ID NO:16或与SEQ ID NO:4、SEQ ID NO:8、 SEQ ID NO:12和SEQ ID NO:16的序列分别具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列。The amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16, or with SEQ ID NO: 4, SEQ ID NO :8, SEQ ID NO: 12 and SEQ ID NO: 16 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical, respectively.
在本发明的一些实施方式中，所述的双特异性抗体的第(2)项中，In some embodiments of the present invention, in item (2) of the bispecific antibody,
所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:2或与SEQ ID NO:2具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:4或与SEQ ID NO:4具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者The amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO: 2 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the same as SEQ ID NO: 2 A sequence of at least 99% identity, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO: 4 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 4, 96%, 97%, 98% or at least 99% identical sequences; or
所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:6或与SEQ ID NO:6具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:8所示或与SEQ ID NO:8具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者The amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO: 6 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the same as SEQ ID NO: 6 The sequence is at least 99% identical, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 8 or has at least 80%, 85%, 90%, 95% with SEQ ID NO: 8 %, 96%, 97%, 98% or at least 99% identical sequences; or
所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:10或与SEQ ID NO:10具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:12或与SEQ ID NO:12具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者The amino acid sequence of the variable region of the heavy chain of the single-chain antibody is SEQ ID NO: 10 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the same as SEQ ID NO: 10 The sequence is at least 99% identical, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO: 12 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 12, 96%, 97%, 98% or at least 99% identical sequences; or
所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:14或与SEQ ID NO:14具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:16或与SEQ ID NO:16具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列。The amino acid sequence of the variable region of the heavy chain of the single-chain antibody is SEQ ID NO: 14 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the same as SEQ ID NO: 14 A sequence of at least 99% identity, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO: 16 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 16, Sequences that are 96%, 97%, 98%, or at least 99% identical.
在本发明的一些实施方式中，所述双特异性抗体的所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。In some embodiments of the present invention, the first protein functional region and the second protein functional region of the bispecific antibody are independently 1, 2, or 2 or more.
在本发明的一些实施方式中，所述双特异性抗体的(2)项中，所述单克隆抗体的恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。In some embodiments of the present invention, in item (2) of the bispecific antibody, the constant region of the monoclonal antibody is selected from the constant region of human IgG1, IgG2, IgG3, or IgG4.
在本发明的一些实施方式中，所述双特异性抗体的所述单链抗体连接在抗体或单克隆抗体的重链的C末端。In some embodiments of the present invention, the single-chain antibody of the bispecific antibody is attached to the C-terminus of the heavy chain of the antibody or monoclonal antibody.
本发明的再一方面涉及一种分离的核酸分子，其包含编码抗体重链可变区的核酸序列和编码抗体轻链可变区的核酸序列，其中，Yet another aspect of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a variable region of an antibody heavy chain and a nucleic acid sequence encoding a variable region of an antibody light chain, wherein,
所述抗体的重链可变区包含氨基酸序列分别如SEQ ID NO:17－SEQ ID NO:19所示的HCDR1-HCDR3，和所述抗体的轻链可变区包含氨基酸序列分别如SEQ ID NO:20－SEQ ID NO:22所示的LCDR1-LCDR3；The variable region of the heavy chain of the antibody comprises the amino acid sequence of HCDR1-HCDR3 shown in SEQ ID NO: 17-ID NO: 19, and the variable region of the light chain of the antibody comprises the amino acid sequence of the amino acid sequence shown in SEQ ID NO, respectively. :20-LCDR1-LCDR3 shown in SEQ ID NO: 22;
优选地，所述抗体的重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14或与SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，和所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4和SEQ ID NO:8、SEQ ID NO:12和SEQ ID NO:16或与SEQ ID NO:4和SEQ ID NO:8、SEQ ID NO:12和SEQ ID NO:16具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；Preferably, the amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, or with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical to the antibody The amino acid sequence of the variable region of the light chain is selected from SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16, or with SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical;
更优选地，所述抗体的重链可变区的氨基酸序列为SEQ ID NO:2或与SEQ ID NO:2具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO:4或与SEQ ID NO:4具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者所述抗体的重链可变区的氨基酸序列为SEQ ID NO:6或与SEQ ID NO:2具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO:8或与SEQ ID NO:8具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者所述抗体的重链可变区的氨基酸序列为SEQ ID NO:10或与SEQ ID NO:10具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO:12所示或与SEQ ID NO:12具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者所述抗体的重链可变区的氨基酸序列为SEQ ID NO:14或与SEQ ID NO:14具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO:16所示或与SEQ ID NO:16具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列进一步优选地，所述分离的核酸分子包含：More preferably, the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 2 or is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% with SEQ ID NO: 2 % Or at least 99% identity sequence, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 4 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 4, 96%, 97%, 98% or at least 99% identical sequence; or the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 6 or at least 80%, 85% with SEQ ID NO: 2 , 90%, 95%, 96%, 97%, 98% or at least 99% identical sequence, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 8 or the same as SEQ ID NO: 8 A sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; or the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 10 Or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO: 10, and the amino acid of the light chain variable region of the antibody The sequence is shown in SEQ ID NO: 12 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO: 12; or The amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 14 or is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO: 14 The amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 16 or has at least 80%, 85%, 90%, 95%, 96%, 97 %, 98% or at least 99% sequence identity. Further preferably, the isolated nucleic acid molecule comprises:
SEQ ID NO:1和SEQ ID NO:3所示的核酸序列，The nucleic acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3,
SEQ ID NO:5和SEQ ID NO:7所示的核酸序列，The nucleic acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 7,
SEQ ID NO:9和SEQ ID NO:11所示的核酸序列，或者The nucleic acid sequence shown in SEQ ID NO: 9 and SEQ ID NO: 11, or
SEQ ID NO:13和SEQ ID NO:15所示的核酸序列。The nucleic acid sequences shown in SEQ ID NO: 13 and SEQ ID NO: 15.
所述分离的核酸分子，可以是一个核酸分子，也可以是多个核酸分子，例如两个核酸分子。当是一个核酸分子时，抗体的重链可变区和轻链可变区可以通过同一个核酸分子表达，例如通过位于同一核酸分子上的相同或不同的表达框来表达。当是多个核酸分子例如两个核酸分子时，抗体的重链可变区和轻链可变区可以通过不同的核酸分子分别表达。The isolated nucleic acid molecule may be one nucleic acid molecule or multiple nucleic acid molecules, for example, two nucleic acid molecules. When it is a nucleic acid molecule, the variable region of the heavy chain and the variable region of the light chain of the antibody can be expressed by the same nucleic acid molecule, for example, by the same or different expression cassettes located on the same nucleic acid molecule. In the case of multiple nucleic acid molecules, such as two nucleic acid molecules, the variable region of the heavy chain and the variable region of the light chain of the antibody can be expressed separately by different nucleic acid molecules.
本发明的再一方面涉及一种重组载体，其包含本发明的分离的核酸分子。所述的重组载体可以是一个或多个。当所述核酸分子是多个(例如两个)时，多个(例如两个)核酸分子可以通过同一重组载体表达，也可以分别通过不同的重组载体来表达。Yet another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention. The recombinant vector can be one or more. When there are multiple (for example, two) nucleic acid molecules, the multiple (for example, two) nucleic acid molecules can be expressed by the same recombinant vector, or can be expressed by different recombinant vectors respectively.
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者包含本发明的重组载体。Another aspect of the present invention relates to a host cell, which contains the isolated nucleic acid molecule of the present invention, or contains the recombinant vector of the present invention.
本发明的再一方面涉及一种制备本发明中任一项所述的抗体或其抗原结合片段的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述抗体或其抗原结合片段的步骤。Another aspect of the present invention relates to a method for preparing the antibody or antigen-binding fragment thereof according to any one of the present invention, which comprises culturing the host cell of the present invention under suitable conditions, and recovering all cells from the cell culture. The steps of the antibody or its antigen-binding fragment.
本发明的再一方面涉及一种杂交瘤细胞株LT010，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018133。Another aspect of the present invention relates to a hybridoma cell line LT010, which is deposited in the China Center for Type Culture Collection (CCTCC), and the deposit number is CCTCC NO: C2018133.
本发明的再一方面涉及一种药物组合物，其包含有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体；可选地，其还包括药学上可接受的载体和/或赋形剂。Yet another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment thereof according to any one of the present invention, the antibody-drug conjugate of the present invention or the bispecific antibody of the present invention Optionally, it also includes a pharmaceutically acceptable carrier and/or excipient.
本发明的再一方面涉及有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体在制备治疗和/或预防IL-1β异常分泌增加为特征的系统性炎症的药物中的用途；Another aspect of the present invention relates to an effective amount of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention or the bispecific antibody of the present invention in the preparation of therapeutic and/or prophylactic IL. The use of drugs for systemic inflammation characterized by abnormal secretion of -1β;
优选地，所述系统性炎症包括系统性炎症综合征，细胞因子释放综合征、多器官功能障碍综合征(multiple organ dysfunction syndrome，MODS)、急性呼 吸窘迫综合征；Preferably, the systemic inflammation includes systemic inflammation syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS), and acute respiratory distress syndrome;
优选地，所述系统性炎症是由病原微生物感染所引起的；Preferably, the systemic inflammation is caused by pathogenic microorganism infection;
优选地，所述病原微生物为病毒、细菌、真菌、立克次体、衣原体、支原体、寄生虫、朊病毒；Preferably, the pathogenic microorganisms are viruses, bacteria, fungi, rickettsiae, chlamydia, mycoplasma, parasites, prions;
优选地，所述病毒包括RNA病毒及DNA病毒；Preferably, the viruses include RNA viruses and DNA viruses;
优选地，所述RNA病毒包括冠状病毒科病毒；Preferably, the RNA virus includes a coronavirus of the coronavirus family;
优选地，所述冠状病毒科病毒包括2019新型冠状病毒(2019-nCoV或SARS-CoV-2，引发新型冠状病毒肺炎COVID-19)，HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV(引发重症急性呼吸综合征)和MERS-CoV(引发中东呼吸综合征)。Preferably, the coronavirus family virus includes 2019 new coronavirus (2019-nCoV or SARS-CoV-2, which causes new coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 , SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing Middle East respiratory syndrome).
本发明的再一方面本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体在制备如下药物中的用途：Another aspect of the present invention is the use of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention, or the bispecific antibody of the present invention in the preparation of the following drugs:
阻断人IL-1β与人IL-1R1和/或人IL-1R2结合的药物，Drugs that block the binding of human IL-1β to human IL-1R1 and/or human IL-1R2,
下调人IL-1β活性或水平的药物，或者Drugs that down-regulate the activity or level of human IL-1β, or
抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活的药物。Drugs that inhibit the activation of downstream signal transduction pathways mediated by the binding of human IL-1β and human IL-1R1 and/or human IL-1R2.
在本发明的一个实施方案中，所述人IL-1R1和/或人IL-1R2是细胞表面的人IL-1R1和/或人IL-1R2。In one embodiment of the present invention, the human IL-1R1 and/or human IL-1R2 is human IL-1R1 and/or human IL-1R2 on the cell surface.
在本发明的一个实施方案中，所述用途是非治疗目的的和/或非诊断目的的。In one embodiment of the invention, the use is for non-therapeutic and/or non-diagnostic purposes.
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体，其用于制备治疗和/或预防(IL-1β异常分泌增加为特征的)系统性炎症的药物中的用途；In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention or the bispecific antibody of the present invention is used for the preparation of treatment and/or prevention (IL -1β abnormal secretion is characterized by the use of drugs for systemic inflammation;
优选地，所述系统性炎症包括系统性炎症综合征，细胞因子释放综合征、多器官功能障碍综合征(multiple organ dysfunction syndrome，MODS)、急性呼吸窘迫综合征；Preferably, the systemic inflammation includes systemic inflammatory syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS), and acute respiratory distress syndrome;
优选地，所述系统性炎症是由病原微生物感染所引起的；Preferably, the systemic inflammation is caused by pathogenic microorganism infection;
优选地，所述病原微生物为病毒、细菌、真菌、立克次体、衣原体、支原体、寄生虫、朊病毒；Preferably, the pathogenic microorganisms are viruses, bacteria, fungi, rickettsiae, chlamydia, mycoplasma, parasites, prions;
优选地，所述病毒包括RNA病毒及DNA病毒；Preferably, the viruses include RNA viruses and DNA viruses;
优选地，所述RNA病毒包括冠状病毒科病毒；Preferably, the RNA virus includes a coronavirus of the coronavirus family;
优选地，所述冠状病毒科病毒包括2019新型冠状病毒(2019-nCoV或SARS-CoV-2，引发新型冠状病毒肺炎COVID-19)，HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV(引发重症急性呼吸综合征)和MERS-CoV(引发中东呼吸综合征)，Preferably, the coronavirus family virus includes 2019 new coronavirus (2019-nCoV or SARS-CoV-2, which causes new coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 , SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing Middle East respiratory syndrome),
优选地，所述抗体或其抗原结合片段为单独使用或者与一种或多种抗病毒药物联用；Preferably, the antibody or antigen-binding fragment thereof is used alone or in combination with one or more antiviral drugs;
优选地，所述抗病毒药物为抗逆转录病毒药物或干扰素；Preferably, the antiviral drug is an antiretroviral drug or interferon;
优选地，所述抗逆转录病毒药物其中：抗逆转录病毒药物是RNA依赖的RNA聚合酶(RdRp)抑制剂，或者是核苷类似物，或者是类肽，或者是人免疫缺陷病毒类型1(HIV-1)整合酶的链转移抑制剂(INSTI)，或者是HIV蛋白酶抑制剂。Preferably, the antiretroviral drug wherein: the antiretroviral drug is an RNA-dependent RNA polymerase (RdRp) inhibitor, or a nucleoside analog, or a peptoid, or a human immunodeficiency virus type 1 (HIV-1) Integrase chain transfer inhibitor (INSTI), or HIV protease inhibitor.
优选地，所述抗逆转录病毒药物为法匹拉韦(favipiravir)、瑞德西韦(Remdesivir)、Beclabuvir，沙奎那韦(Saquinavir)，比特拉韦(Bictegravir)，洛匹那韦(Lopinavir)，多替拉韦(Dolutegravir)；Preferably, the antiretroviral drugs are favipiravir, Remdesivir, Beclabuvir, Saquinavir, Bictegravir, Lopinavir ), Dolutegravir (Dolutegravir);
更优选地，所述药物为法匹拉韦。More preferably, the drug is favipiravir.
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体，其用于：In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention or the bispecific antibody of the present invention is used for:
阻断人IL-1β与人IL-1R1和/或人IL-1R2结合，Block the binding of human IL-1β to human IL-1R1 and/or human IL-1R2,
下调人IL-1β活性或水平，或者Down-regulate the activity or level of human IL-1β, or
抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活。Inhibit the activation of downstream signal transduction pathways mediated by the binding of human IL-1β to human IL-1R1 and/or human IL-1R2.
在本发明的一个实施方案中，所述人IL-1R1和/或人IL-1R2是细胞表面的人IL-1R1和/或人IL-1R2。In one embodiment of the present invention, the human IL-1R1 and/or human IL-1R2 is human IL-1R1 and/or human IL-1R2 on the cell surface.
本发明的再一方面涉及一种在体内或体外方法，包括施加细胞以有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体的步骤，所述方法选自如下：Yet another aspect of the present invention relates to an in vivo or in vitro method, comprising administering cells in an effective amount of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention, or the antibody-drug conjugate of the present invention. The step of bispecific antibody, the method is selected from the following:
阻断人IL-1β与人IL-1R1和/或人IL-1R2结合的方法，Methods of blocking the binding of human IL-1β to human IL-1R1 and/or human IL-1R2,
下调人IL-1β活性或水平的方法，或者Methods to down-regulate the activity or level of human IL-1β, or
抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活的方法。A method for inhibiting the activation of downstream signal transduction pathways mediated by the combination of human IL-1β and human IL-1R1 and/or human IL-1R2.
在本发明的一个实施方案中，所述人IL-1R1和/或人IL-1R2是细胞表面的人IL-1R1和/或人IL-1R2。In one embodiment of the present invention, the human IL-1R1 and/or human IL-1R2 is human IL-1R1 and/or human IL-1R2 on the cell surface.
在本发明的一个实施方案中，所述体外方法是非治疗目的的和/或非诊断目的的。In one embodiment of the invention, the in vitro method is for non-therapeutic and/or non-diagnostic purposes.
本发明的再一方面涉及一种治疗和/或预防治疗和/或预防IL-1β异常分泌增加为特征的系统性炎症的方法，包括给予有需求的受试者或患者以有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体的步骤；Another aspect of the present invention relates to a method for treating and/or preventing treatment and/or prevention of systemic inflammation characterized by abnormal secretion of IL-1β, comprising administering to a subject or patient in need an effective amount of the present invention The steps of any one of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention, or the bispecific antibody of the present invention;
优选地，所述系统性炎症包括系统性炎症综合征，细胞因子释放综合征、多器官功能障碍综合征(multiple organ dysfunction syndrome，MODS)、急性呼吸窘迫综合征；Preferably, the systemic inflammation includes systemic inflammatory syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS), and acute respiratory distress syndrome;
优选地，所述系统性炎症是由病原微生物感染所引起的；Preferably, the systemic inflammation is caused by pathogenic microorganism infection;
优选地，所述病原微生物为病毒、细菌、真菌、立克次体、衣原体、支原体、寄生虫、朊病毒；Preferably, the pathogenic microorganisms are viruses, bacteria, fungi, rickettsiae, chlamydia, mycoplasma, parasites, prions;
优选地，所述病毒包括RNA病毒及DNA病毒；Preferably, the viruses include RNA viruses and DNA viruses;
优选地，所述RNA病毒包括冠状病毒科病毒；Preferably, the RNA virus includes a coronavirus of the coronavirus family;
优选地，所述冠状病毒科病毒包括2019新型冠状病毒(2019-nCoV或SARS-CoV-2，引发新型冠状病毒肺炎COVID-19)，HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV(引发重症急性呼吸综合征)和MERS-CoV(引发中东呼吸综合征)。Preferably, the coronavirus family virus includes 2019 new coronavirus (2019-nCoV or SARS-CoV-2, which causes new coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 , SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing Middle East respiratory syndrome).
在本发明的任一个实施方案中，所述的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体可以单独使用或与一种或多种抗病毒药物联合使用。In any embodiment of the present invention, the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody can be used alone or in combination with one or more antiviral drugs use.
在本发明的一个方面，涉及试剂盒，其包含有效量(例如0.001mg-1000mg)的所述的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体，和任选地，还包含有效量的一种或多种抗病毒药物(例如100-2400mg)，其中所述抗病毒药物所上所述。In one aspect of the present invention, it relates to a kit comprising an effective amount (for example, 0.001 mg-1000 mg) of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody, And optionally, it also contains an effective amount of one or more antiviral drugs (for example, 100-2400 mg), wherein the antiviral drugs are as described above.
在本发明的另一个方面，涉及单次药物剂量单元，其包含0.001mg-1000mg的本发明的抗体或其抗原结合片段，优选0.001mg-900mg、0.001mg-800mg、0.001mg-700mg、0.001mg-600mg、0.001mg-500mg、0.001mg-400mg、0.001mg-300mg、0.001mg-200mg、0.001mg-100mg，更优选为100mg、200mg、300mg、400mg、500mg、600mg、700mg、800mg、900mg或1000mg的本发明的抗体或其抗原结合片段。In another aspect of the present invention, it relates to a single drug dosage unit, which contains 0.001 mg-1000 mg of the antibody or antigen-binding fragment thereof of the present invention, preferably 0.001 mg-900 mg, 0.001 mg-800 mg, 0.001 mg-700 mg, 0.001 mg -600mg, 0.001mg-500mg, 0.001mg-400mg, 0.001mg-300mg, 0.001mg-200mg, 0.001mg-100mg, more preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg The antibody or antigen-binding fragment thereof of the present invention.
在本发明的上述任一实施方案中，本发明的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体的有效量为0.001mg-1000mg，优选0.001mg-900mg、0.001mg-800mg、0.001mg-700mg、0.001mg-600mg、0.001mg-500mg、0.001mg-400mg、0.001mg-300mg、0.001mg-200mg、0.001mg-100mg，更优选为100mg、200mg、300mg、400mg、500mg、600mg、700mg、800mg、900mg或1000mg。或者，在本发明的上述任一实施方案中，基于受试者的体重，本发明的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体的有效量为0.1-100mg/kg，优选1-90mg/kg、1-80mg/kg、1-70mg/kg、1-60mg/kg、1-50mg/kg、1-40mg/kg、1-30mg/kg、1-20mg/kg或1-10mg/kg。In any of the above-mentioned embodiments of the present invention, the effective amount of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody of the present invention is 0.001 mg-1000 mg, preferably 0.001 mg- 900mg, 0.001mg-800mg, 0.001mg-700mg, 0.001mg-600mg, 0.001mg-500mg, 0.001mg-400mg, 0.001mg-300mg, 0.001mg-200mg, 0.001mg-100mg, more preferably 100mg, 200mg, 300mg , 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg. Alternatively, in any of the above-mentioned embodiments of the present invention, based on the weight of the subject, the effective amount of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody of the present invention is 0.1-100mg/kg, preferably 1-90mg/kg, 1-80mg/kg, 1-70mg/kg, 1-60mg/kg, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1 -20mg/kg or 1-10mg/kg.
在本发明的上述任一实施方案，一种或多种抗病毒药物(例如法匹拉韦)的有效量为100-2400mg，优选100mg-2300mg、100mg-2200mg、100mg-2100mg、100mg-2000mg、100mg-1900mg、100mg-1800mg、100mg-1700mg、100mg-1600mg、100mg-1800mg、100mg-1800mg、100mg-1800mg、100mg-1800mg、100mg-1800mg，更优选为100mg，200mg，300mg，400mg，500mg，600mg，700mg，800mg，900mg，1000mg。或者，在本发明的上述任一实施方案中，基于受试者或患者的体重，所述抗病毒药物的有效量为0.1-100mg/kg，优选1-90mg/kg、1-80mg/kg、1-70mg/kg、1-60mg/kg、1-50mg/kg、1-40mg/kg、1-30mg/kg、1-20mg/kg或1-10mg/kg。In any of the above-mentioned embodiments of the present invention, the effective amount of one or more antiviral drugs (such as favipiravir) is 100-2400mg, preferably 100mg-2300mg, 100mg-2200mg, 100mg-2100mg, 100mg-2000mg, 100mg-1900mg, 100mg-1800mg, 100mg-1700mg, 100mg-1600mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, more preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg , 700mg, 800mg, 900mg, 1000mg. Alternatively, in any of the above embodiments of the present invention, based on the weight of the subject or patient, the effective amount of the antiviral drug is 0.1-100 mg/kg, preferably 1-90 mg/kg, 1-80 mg/kg, 1-70mg/kg, 1-60mg/kg, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1-20mg/kg or 1-10mg/kg.
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本发明中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in the present invention are all routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, definitions and explanations of related terms are provided below.
在本发明中，术语“单次药物剂量单元”表示在给药方案的时刻待给药于受试者或患者的本发明所述的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体(或包含其的药物组合物)的单次药物剂型，如以一个安瓿瓶为单位。In the present invention, the term "single drug dosage unit" means the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate of the present invention to be administered to the subject or patient at the time of the dosing schedule Or the single-dose dosage form of the bispecific antibody (or the pharmaceutical composition containing the same), such as one ampoule as a unit.
如本文中所使用的，当提及IL-1β的氨基酸序列时，其包括IL-1β蛋白的全长(GenBank ID：NP_000567.1)，还包括IL-1β的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)或者多个His进行融合的片段。然而，本领域技术人员理解，在IL-1β的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“IL-1β”应包括所有此类序列，及其天然或人工的变体。并且，当描述IL-1β蛋白的序列片段时，其不仅包括序列片段，还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of IL-1β, it includes the full length of IL-1β protein (GenBank ID: NP_000567.1), and also includes IL-1β fusion proteins, such as those with mice or Human IgG Fc protein fragments (mFc or hFc) or fragments of multiple His fusions. However, those skilled in the art understand that in the amino acid sequence of IL-1β, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally or artificially introduced without affecting its biological functions. Therefore, in the present invention, the term "IL-1β" shall include all such sequences, and their natural or artificial variants. Moreover, when describing the sequence fragments of IL-1β protein, it includes not only the sequence fragments, but also the corresponding sequence fragments in its natural or artificial variants.
如本文中所使用的，当提及IL-1R1的氨基酸序列时，其包括IL-1R1蛋白的全长(GenBank ID：NP_000868)，还包括IL-1R1的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)或者多个His进行融合的片段。然而，本领域技术人员理解，在IL-1R1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“IL-1R1”应包括所有此类序列以及其天然或人工的变体。并且，当描述IL-1R1蛋白的序列片段时，包括IL-1R1序列片段，还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of IL-1R1, it includes the full length of IL-1R1 protein (GenBank ID: NP_000868), and also includes IL-1R1 fusion proteins, such as mouse or human IgG Fc protein fragments (mFc or hFc) or fragments of multiple His fusions. However, those skilled in the art understand that in the amino acid sequence of IL-1R1 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally or artificially introduced without affecting its biological functions. Therefore, in the present invention, the term "IL-1R1" shall include all such sequences and their natural or artificial variants. Moreover, when describing the sequence fragment of IL-1R1 protein, it includes the IL-1R1 sequence fragment and the corresponding sequence fragment in its natural or artificial variant.
如本文中所使用的，当提及IL-1R2的氨基酸序列时，其包括IL-1R2蛋白的全长(GenBank ID：CAA42441.1)，还包括IL-1R2的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)或者多个His进行融合的片段。然而，本领域技术人员理解，在IL-1R2蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“IL-1R2”应包括所有此类序列以及其天然或人工的变体。并且，当描述IL-1R2蛋白的序列片段时，包括IL-1R2序列片段，还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of IL-1R2, it includes the full length of IL-1R2 protein (GenBank ID: CAA42441.1), and also includes IL-1R2 fusion proteins, such as those with mice or Human IgG Fc protein fragments (mFc or hFc) or fragments of multiple His fusions. However, those skilled in the art understand that in the amino acid sequence of IL-1R2 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally or artificially introduced without affecting its biological functions. Therefore, in the present invention, the term "IL-1R2" shall include all such sequences and their natural or artificial variants. Moreover, when describing the sequence fragment of IL-1R2 protein, it includes the IL-1R2 sequence fragment and the corresponding sequence fragment in its natural or artificial variant.
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％ of maximal effect)，是指能引起50％最大效应的浓度。 As used herein, the term EC 50 refers to the concentration for 50% of maximal effect (concentration for 50% of maximal effect), which refers to the concentration that can cause 50% of the maximal effect.
如本文中所使用的，术语“抗体”是指，是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区即CDR)，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM抗体。As used herein, the term "antibody" refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair has a "light" (L) chain and a "heavy" (H) chain) . Antibody light chains can be classified into kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a domain CL. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions or CDRs), interspersed with more conservative regions called framework regions (FR). Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antibody binding site. The assignment of amino acids to each region or domain follows Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917 ; Definition of Chothia et al. (1989) Nature 342:878-883. The term "antibody" is not limited by any specific method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibodies.
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以 赋予多肽特异性抗原结合能力的抗体的至少一部分。As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody It is also called "antigen binding part" for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Recombinant DNA technology can be used. Or through the enzymatic or chemical cleavage of intact antibodies to produce antigen-binding fragments of antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining regions (CDR) Fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and such polypeptides, which comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如,Bird等人,Science 242:423 426(1988)和Huston等人,Proc.Natl.Acad.Sci.USA 85:5879 5883(1988))。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。 In some cases, the antigen-binding fragment of the antibody is a single-chain antibody (e.g., scFv), where the VL and VH domains pair to form a monovalent molecule by pairing a linker that enables it to be produced as a single polypeptide chain (see, e.g., Bird et al. , Science 242: 423 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879 5883 (1988)). Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。In some cases, the antigen-binding fragments of antibodies are diabodies, ie, diabodies, in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short is used to allow two structures in the same chain Pairing between the domains, thereby forcing the domain to pair with the complementary domain of the other chain and creating two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).
在另一些情况下，抗体的抗原结合片段是“双功能抗体”。双功能抗体亦称为双特异性抗体(Bispecific Antibody)，是同时靶向两种不同抗原的特异性药物，其可通过免疫分选纯化生产。另外，也可通过基因工程获得，基因工程在结合位点优化，合成形式的考量以及产量等方面都具有相应的灵活性，所以具有一定的优势。目前，其存在形式已被证明有超过45种(Müller D,Kontermann RE.Bispecific antibodies for cancer immunotherapy:Current perspectives.BioDrugs 2010；24:89-98)。目前已开发的多种双特异性抗体为IgG-ScFv形式即Morrison模式(1997 Coloma MJ,Morrison SL.Design and production of novel tetravalent bispecific antibodies.Nature Biotechnology,1997；15:159-163)，由于这种类似于天然存在的IgG形式，其在抗体工程、表达和纯化上所具有的优势，已被证明是双功能抗体的其中一种理想存在形式(Miller BR,Demarest SJ,et al.,Stability engineering of scFvs for the development of bispecific and multivalent  antibodies.Protein Eng Des Sel 2010；23:549-57；Fitzgerald J,Lugovskoy A.Rational engineering of antibody therapeutics targeting multiple oncogene pathways.MAbs 2011；3:299-309)。In other cases, the antigen-binding fragment of the antibody is a "bifunctional antibody." Bifunctional antibodies, also known as bispecific antibodies, are specific drugs that target two different antigens at the same time, and they can be produced by immunosorting and purification. In addition, it can also be obtained through genetic engineering. Genetic engineering has corresponding flexibility in terms of optimization of binding sites, consideration of synthetic forms, and yield, so it has certain advantages. At present, there are more than 45 forms of its existence (Müller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24: 89-98). Many bispecific antibodies that have been developed so far are in the form of IgG-ScFv, the Morrison model (1997 Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nature Biotechnology, 1997; 15:159-163). Similar to the naturally occurring IgG form, its advantages in antibody engineering, expression and purification have been proven to be one of the ideal forms of bifunctional antibodies (Miller BR, Demarest SJ, et al., Stability engineering of scFvs for the development of bispecific and multiple antibodies. Protein Eng Des Sel 2010; 23:549-57; Fitzgerald J, Lugovskoy A. Rational engineering of antibody therapeutics targeting multiple oncogene pathways. MAbs-309).
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的单克隆抗体3H6、3H6H1L1、3H6H2L2、3H6 H3L3或3H6H4L1)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。Conventional techniques known to those skilled in the art (for example, recombinant DNA technology or enzymatic or chemical fragmentation) can be used to obtain a given antibody (for example, the monoclonal antibody 3H6, 3H6H1L1, 3H6H2L2, 3H6 H3L3 or 3H6H4L1 provided by the present invention). An antigen-binding fragment of an antibody (for example, the above-mentioned antibody fragment) is obtained, and the antigen-binding fragment of the antibody is specifically screened in the same manner as that used for intact antibodies.
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature,256:495,1975)，但也可采用重组DNA技术获得(如参见U.S.P 4,816,567)。As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, in addition to natural mutations that may occur spontaneously, A group of identical antibody molecules. The monoclonal antibody has high specificity for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which usually include at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (for example, see U.S.P 4,816,567).
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature,321:522 525(1986)；Reichmann et al.,Nature,332:323 329(1988)；Presta,Curr.Op.Struct.Biol.,2:593 596(1992)；和Clark,Immunol.Today 21:397 402(2000)。As used herein, the term "humanized antibody" means that all or part of the CDR region of a human immunoglobulin (acceptor antibody) is replaced by a CDR region of a non-human antibody (donor antibody). The donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody with the expected specificity, affinity or reactivity. In addition, some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by corresponding non-human antibody amino acid residues, or by other antibody amino acid residues, to further improve or optimize the performance of the antibody. For more details about humanized antibodies, see, for example, Jones et al., Nature, 321:522 525 (1986); Reichmann et al., Nature, 332:323 329 (1988); Presta, Curr. Op .Struct.Biol., 2:593 596 (1992); and Clark, Immunol. Today 21:397 402 (2000).
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为 分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。As used herein, the term "isolated" or "isolated" refers to those obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of. The term "isolated" or "isolated" does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses. Polyoma vacuole virus (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site.
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 fruit fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
如本发明中所使用的，术语“双特异性”、“双重特异性”或“双功能性”抗原结合蛋白或抗体是分别具有两个不同的抗原结合位点的杂交抗原结合蛋白或抗体。双特异性抗体是一种多特异性抗原结合蛋白或多特异性抗体，并且可通过多种方法产生，包括，但不限于杂交瘤的融合或Fab′片段的连接。参见，例如，Songsivilai和Lachmann，1990，Clin.Exp.Immunol.79：315-321；Kostelny等人.1992，J.Immunol.148：1547-1553。双特异性抗原结合蛋白或抗体的两个结合位点将结合两个不同的表位，所述表位存在于相同或不同的蛋白质靶标上。As used in the present invention, the terms "bispecific", "dual specific" or "bifunctional" antigen binding protein or antibody are hybrid antigen binding proteins or antibodies each having two different antigen binding sites. A bispecific antibody is a multispecific antigen binding protein or multispecific antibody, and can be produced by a variety of methods, including, but not limited to, the fusion of hybridomas or the linking of Fab' fragments. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79: 315-321; Kostelny et al. 1992, J. Immunol. 148: 1547-1553. The two binding sites of the bispecific antigen binding protein or antibody will bind to two different epitopes that are present on the same or different protein targets.
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原 的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、小于大约10 -7M、小于大约10 -8M、小于大约10 -9M或小于大约10 -10M或更小的亲和力(K D)结合该抗原。 As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) means that the antibody has a concentration of less than about 10 -5 M, for example, less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, or less than about 10 -10 M or less with an affinity (K D ) that binds the antigen.
如本文中所使用的，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体(例如，本发明的单克隆抗体3H6、3H6H1L1、3H6H2L2、或3H6H3L3)以小于大约10 -5M，例如小于大约10 -6M、小于大约10 -7M、小于大约10 -8M、小于大约10 -9M或小于大约10 -10M或更小的解离平衡常数(K D)结合抗原(例如，IL-1β蛋白)。可以使用本领域技术人员知悉的方法测定K D，例如使用Biacore分子相互作用仪测定。 As used herein, the term "K D "refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Generally, the antibody (for example, the monoclonal antibody 3H6, 3H6H1L1, 3H6H2L2, or 3H6H3L3 of the present invention) is less than about 10 -5 M, such as less than about 10 -6 M, less than about 10 -7 M, or less than about 10 -8 M. The dissociation equilibrium constant (K D ) of less than about 10 -9 M or less than about 10 -10 M or less binds to the antigen (e.g., IL-1β protein). K D can be determined using methods known to those skilled in the art, for example, using a Biacore molecular interaction analyzer.
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and can be used interchangeably. And in the present invention, amino acids are usually represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
如本文中所使用的，术语“杂交瘤”和“杂交瘤细胞株”可互换使用，并且当提及术语“杂交瘤”和“杂交瘤细胞株”时，其还包括杂交瘤的亚克隆和后代细胞。例如，当提及杂交瘤细胞株LT010时，其还指杂交瘤细胞株LT010的亚克隆和后代细胞。As used herein, the terms "hybridoma" and "hybridoma cell line" are used interchangeably, and when referring to the terms "hybridoma" and "hybridoma cell line", it also includes subclones of hybridomas And progeny cells. For example, when referring to the hybridoma cell line LT010, it also refers to the subclones and progeny cells of the hybridoma cell line LT010.
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者或患者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject or patient and the active ingredient Agents, which are well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH regulators, surfactants, adjuvants, ions Strength enhancer. For example, pH adjusting agents include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如RA)有效量是指，足以预防、阻止或延迟疾病(例如RA)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止 已患有疾病的患者的疾病和其并发症的量。As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (such as RA) refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (such as RA); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease in a patient who has already suffered from the disease. And the amount of its complications.
发明的有益效果：The beneficial effects of the invention:
本发明的抗IL-1β抗体特别是人源化的抗IL-1β抗体具有如下技术效果中的一项或多项：The anti-IL-1β antibody of the present invention, especially the humanized anti-IL-1β antibody, has one or more of the following technical effects:
(1)有效地结合人IL-1β，阻断IL-1β与其受体IL-1R1的结合；(1) Effectively bind to human IL-1β and block the binding of IL-1β to its receptor IL-1R1;
(2)抑制IL-1β下游信号通路的激活；(2) Inhibit the activation of IL-1β downstream signaling pathway;
(3)能够特异性抑制IL-1β诱导MRC-5细胞分泌IL-6的活性；(3) It can specifically inhibit the IL-1β-induced IL-6 secretion activity of MRC-5 cells;
(4)能够有效地阻断IL-1β对NF-κB的激活；(4) It can effectively block the activation of NF-κB by IL-1β;
(5)具有用于制备抑制IL-1β的药物的潜力；(5) It has the potential to be used to prepare drugs that inhibit IL-1β;
(6)具有用于制备预防和/或治疗由病原微生物感染所引起的系统性炎症，包括系统性炎症综合征，细胞因子释放综合征、多器官功能障碍综合征(multiple organ dysfunction syndrome，MODS)、急性呼吸窘迫综合征的药物的潜力。(6) It is used to prepare and prevent and/or treat systemic inflammation caused by pathogenic microorganism infection, including systemic inflammatory syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS) The potential of drugs for acute respiratory distress syndrome.
附图说明Description of the drawings
图1：3H6、3H6H1L1、3H6H2L2和3H6H3L3与人IL-1β-His-Bio的结合活性检测结果。Figure 1: Detection results of the binding activity of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 to human IL-1β-His-Bio.
图2：3H6H4L1与人IL-1β-His-Bio的结合活性检测结果。Figure 2: Detection results of the binding activity of 3H6H4L1 and human IL-1β-His-Bio.
图3：3H6、3H6H1L1、3H6H2L2和3H6H3L3与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果。Figure 3: 3H6, 3H6H1L1, 3H6H2L2, and 3H6H3L3 compete with human IL-1R1(1-332)-his to bind to human IL-1β-hFc.
图4：3H6H4L1与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果。Figure 4: 3H6H4L1 competes with human IL-1R1(1-332)-his to bind to human IL-1β-hFc activity test results.
图5：3H6H4L1与人IL-1β亲和力常数检测结果。注：曲线1～5分别表示分析物的浓度分别为25nM、12.5nM、6.25nM、3.13nM、1.56nM。Figure 5: 3H6H4L1 and human IL-1β affinity constant test results. Note: Curves 1 to 5 indicate that the analyte concentrations are 25nM, 12.5nM, 6.25nM, 3.13nM, 1.56nM, respectively.
图6：Canakinumab与人IL-1β亲和力常数检测结果。注：曲线1～5分别表示分析物的浓度分别为25nM、12.5nM、6.25nM、3.13nM、1.56nM。Figure 6: Test results of the affinity constant between Canakinumab and human IL-1β. Note: Curves 1 to 5 indicate that the analyte concentrations are 25nM, 12.5nM, 6.25nM, 3.13nM, 1.56nM, respectively.
图7：3H6H4L1对IL-1β诱导MRC-5分泌IL-6的抑制作用。Figure 7: Inhibition of 3H6H4L1 on IL-1β-induced IL-6 secretion by MRC-5.
图8：不同剂量3H6H4L1对IL-1β的中和生物学活性。Figure 8: Neutralizing biological activity of IL-1β at different doses of 3H6H4L1.
图9：3H6H4L1阻断IL-1β的剂量效应曲线。Figure 9: The dose-response curve of 3H6H4L1 blocking IL-1β.
具体实施方式Detailed ways
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。The embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, follow the techniques or conditions described in the literature in the field (for example, refer to the "Molecular Cloning Experiment Guide" translated by J. Sambrook et al., Huang Peitang et al., third edition, Science Press) or follow the product instructions. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased on the market.
在本发明的下述实施例中，使用的BALB/c小鼠购自广东省医学实验动物中心。In the following examples of the present invention, the BALB/c mice used were purchased from Guangdong Medical Experimental Animal Center.
在本发明的下述实施例中，所用的同靶点已上市药物抗体Canakinumab(商品名
Figure PCTCN2021082692-appb-000001
)作为对照抗体，购自诺华(Novartis)。 In the following examples of the present invention, the same target marketed drug antibody Canakinumab (trade name
Figure PCTCN2021082692-appb-000001
) As a control antibody, purchased from Novartis.
制备例1：融合蛋白人IL-1β-His、IL-1R1(1-332)-His、IL-1β-hFc以及人Preparation Example 1: Fusion proteins human IL-1β-His, IL-1R1(1-332)-His, IL-1β-hFc and human IL-1β-His-Bio的制备Preparation of IL-1β-His-Bio
通过NCBI GenBank蛋白数据库查找人IL-1β(Genbank ID:NP_000567.1)及IL-1R1(Genbank ID:NP_000868，1-332位氨基酸)的蛋白序列。将人IL-1β及IL-1R1的氨基酸序列分别与His标签序列(6个His)，人IgG Fc纯化标签序列(Ig gamma-1chain C region，GenbankID:P01857位置114-330)进行融合设计；上述的融合蛋白分别简写命名为人IL-1β-His、IL-1R1(1-332)-His、IL-1β-hFc。Search the protein sequences of human IL-1β (Genbank ID: NP_000567.1) and IL-1R1 (Genbank ID: NP_000868, amino acids 1-332) through the NCBI GenBank protein database. The amino acid sequences of human IL-1β and IL-1R1 are respectively fused with His tag sequence (6 His) and human IgG Fc purification tag sequence (Ig gamma-1chain C region, GenbankID: P01857 position 114-330); The fusion proteins were abbreviated as human IL-1β-His, IL-1R1(1-332)-His, IL-1β-hFc.
蛋白样品质量通过SDS-PAGE鉴定合格。The quality of the protein sample was qualified by SDS-PAGE.
使用EZ-
Figure PCTCN2021082692-appb-000002
Sulfo-NHS-LC-Biotinylation Kit(Thermo scientific)制备生物素化偶联的人IL-1β-His蛋白样品(简称为人IL-1β-His-Bio)；具体制备方法参考该试剂盒的说明书进行。 Use EZ-
Figure PCTCN2021082692-appb-000002
Sulfo-NHS-LC-Biotinylation Kit (Thermo scientific) prepares biotinylated and coupled human IL-1β-His protein samples (referred to as human IL-1β-His-Bio); the specific preparation method refers to the instructions of the kit.
制得的上述融合蛋白用于下面的实施例。The prepared fusion protein was used in the following examples.
实施例1：抗IL-1β鼠源抗体3H6的制备Example 1: Preparation of anti-IL-1β murine antibody 3H6
1.杂交瘤细胞株LT010的制备1. Preparation of hybridoma cell line LT010
以人IL-1β-his作为抗原免疫BALB/c小鼠(购自广东医学实验动物中心)，取免疫后小鼠的脾细胞与小鼠骨髓瘤细胞融合，制成杂交瘤细胞。以IL-1β-His-Bio作为抗原，对杂交瘤细胞进行ELISA法筛选，获得能够分泌和IL-1β-His-Bio特异性结合的抗体的杂交瘤细胞。对ELISA筛选得到的杂交瘤细胞，通过竞争ELISA筛选出能够分泌与受体IL-1R1(1-332)-His竞争结合IL-1β-hFc的抗体的杂交瘤细胞，并经过有限稀释法得到稳定的杂交瘤细胞株。杂交瘤细胞制备的方法参考目前已确立的方法(例如，Stewart,S.J.,“Monoclonal Antibody Production”,in Basic Methods in antibody Production and Characterization,Eds.G.C.Howard and D.R.Bethell,Boca Raton:CRC Press,2000)。Human IL-1β-his was used as the antigen to immunize BALB/c mice (purchased from Guangdong Medical Experimental Animal Center), and the spleen cells of the immunized mice were fused with mouse myeloma cells to make hybridoma cells. Using IL-1β-His-Bio as an antigen, hybridoma cells were screened by ELISA method to obtain hybridoma cells capable of secreting antibodies specifically binding to IL-1β-His-Bio. For the hybridoma cells screened by ELISA, the hybridoma cells that can secrete antibodies that compete with the receptor IL-1R1(1-332)-His to bind IL-1β-hFc were screened by competitive ELISA, and stabilized by the limiting dilution method. Hybridoma cell line. The method of preparing hybridoma cells refers to the established methods (for example, Stewart, SJ, "Monoclonal Antibody Production", in Basic Methods in antibody Production and Characterization, Eds. GC Howard and DRBethell, Boca Raton: CRC Press, 2000) .
发明人将以上杂交瘤细胞株命名为杂交瘤细胞株LT010(IL-1β-3H6)，其分泌的单克隆抗体命名为3H6。The inventor named the above hybridoma cell line hybridoma cell line LT010 (IL-1β-3H6), and the monoclonal antibody secreted by it was named 3H6.
杂交瘤细胞株LT010(IL-1β-3H6)，其于2018年6月21日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018133，保藏地址为中国.武汉.武汉大学，邮编：430072。Hybridoma cell line LT010 (IL-1β-3H6), which was deposited in the China Center for Type Culture Collection (CCTCC) on June 21, 2018, with the deposit number CCTCC NO: C2018133, and the deposit address is Wuhan University, Wuhan, China , Zip Code: 430072.
2.抗IL-1β的抗体3H6的制备2. Preparation of anti-IL-1β antibody 3H6
用含杂交瘤无血清培养基对上面制得的LT010细胞株进行培养(杂交瘤无血清培养基，内含1％青链霉素和4％的Glutamax，于5％CO 2，37℃细胞培养箱中进行培养)，7天后收集细胞培养上清，通过高速离心、微孔滤膜抽真空过滤以及HiTrap protein AHP柱进行纯化，制得抗体3H6。纯化后的3H6样品进行SDS-PAGE电泳检测合格。 Culture the LT010 cell line prepared above with hybridoma serum-free medium (hybridoma serum-free medium containing 1% penicillin and 4% Glutamax, cell culture at 5% CO 2 , 37℃ Cell culture supernatant was collected after 7 days, and purified by high-speed centrifugation, vacuum filtration with microporous membrane and HiTrap protein AHP column to prepare antibody 3H6. The purified 3H6 sample was qualified by SDS-PAGE electrophoresis.
实施例2：抗IL-1β的抗体3H6的序列分析Example 2: Sequence analysis of anti-IL-1β antibody 3H6
按照总RNA提取试剂盒(Tiangen，货号DP430)的方法，从实施例1中培养的LT010细胞株中提取mRNA。按照Invitrogen
Figure PCTCN2021082692-appb-000003
III First-Strand Synthesis System for RT-PCR试剂盒说明书合成cDNA，并进行PCR扩增。PCR扩增产物直接进行TA克隆，具体操作参考pEASY-T1 Cloning Kit(Transgen CT101)试剂盒说明书进行。 According to the method of the total RNA extraction kit (Tiangen, article number DP430), mRNA was extracted from the LT010 cell line cultured in Example 1. Follow Invitrogen
Figure PCTCN2021082692-appb-000003
III First-Strand Synthesis System for RT-PCR kit instructions Synthesize cDNA and perform PCR amplification. The PCR amplification product is directly subjected to TA cloning, and the specific operation refers to the pEASY-T1 Cloning Kit (Transgen CT101) kit instructions.
将TA克隆的产物直接进行测序，测序结果如下：The TA cloned product was directly sequenced, and the sequencing results are as follows:
编码抗体3H6的重链可变区的核酸序列：(354bp)Nucleic acid sequence encoding the variable region of the heavy chain of antibody 3H6: (354bp)
Figure PCTCN2021082692-appb-000004
Figure PCTCN2021082692-appb-000004
抗体3H6的重链可变区的氨基酸序列如下：(118aa，其中下划线标记的氨基酸序列为CDR区域)The amino acid sequence of the heavy chain variable region of antibody 3H6 is as follows: (118aa, the underlined amino acid sequence is the CDR region)
Figure PCTCN2021082692-appb-000005
Figure PCTCN2021082692-appb-000005
编码抗体3H6的轻链可变区的核酸序列：(318bp)Nucleic acid sequence encoding the light chain variable region of antibody 3H6: (318bp)
Figure PCTCN2021082692-appb-000006
Figure PCTCN2021082692-appb-000006
抗体3H6的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)The amino acid sequence of the light chain variable region of antibody 3H6 is as follows: (106aa, where the underlined amino acid sequence is the CDR region)
Figure PCTCN2021082692-appb-000007
Figure PCTCN2021082692-appb-000007
实施例3：抗IL-1β的人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和Example 3: Anti-IL-1β humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1的设计和制备Design and preparation of 3H6H4L1
1.抗IL-1β人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1的轻链和重链序列的设计1. Design of the light chain and heavy chain sequences of anti-IL-1β humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1
根据IL-1β蛋白的三维晶体结构(van Oostrum J,Priestle JP,Grütter MG,Schmitz A.The structure of murine interleukin-1 beta at 2.8 A resolution.J Struct Biol.1991，107(2):189-95.)以及实施例2获得的序列信息，设计得到人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1重链可变区的序列和轻链可变区的序列(3H6H1L1、3H6H2L2和3H6H3L3抗体恒定区序列，来自NCBI的数据库，重链恒定区为Ig gamma-1 chain C region，ACCESSION:P01857，轻链恒定区为Ig kappa chain C region，ACCESSION:P01834；3H6H4L1抗体恒定区序列，来自NCBI的数据库，重链恒定区为Ig gamma-4 chain C region，ACCESSION：P01861.1；轻链恒定区为Ig kappa chain C region，ACCESSION:P01834)。According to the three-dimensional crystal structure of IL-1β protein (van Oostrum J, Priestle JP, Grütter MG, Schmitz A. The structure of Murine interleukin-1 beta at 2.8 A resolution. J Struct Biol. 1991, 107(2): 189-95 .) and the sequence information obtained in Example 2, the humanized antibody 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 heavy chain variable region sequence and light chain variable region sequence (3H6H1L1, 3H6H2L2 and 3H6H3L3 antibody constant region sequence, From NCBI database, heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857, light chain constant region is Ig kappa chain C region, ACCESSION: P01834; 3H6H4L1 antibody constant region sequence, from NCBI database, heavy chain The constant region is Ig gamma-4 chain C region, ACCESSION: P01861.1; the light chain constant region is Ig kappa chain C region, ACCESSION: P01834).
人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1的重链可变区序列和轻链可变区序列如下：The sequences of the heavy chain variable region and the light chain variable region of the humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 are as follows:
(1)人源化单克隆抗体3H6H1L1(1) Humanized monoclonal antibody 3H6H1L1
编码抗体3H6H1L1的重链可变区的核酸序列：(354bp)Nucleic acid sequence encoding the variable region of the heavy chain of the antibody 3H6H1L1: (354bp)
Figure PCTCN2021082692-appb-000008
Figure PCTCN2021082692-appb-000008
抗体3H6H1L1的重链可变区氨基酸序列如下：(118aa，其中下划线标记的氨基酸序列为CDR区域)The amino acid sequence of the variable region of the heavy chain of the antibody 3H6H1L1 is as follows: (118aa, the underlined amino acid sequence is the CDR region)
Figure PCTCN2021082692-appb-000009
Figure PCTCN2021082692-appb-000009
编码抗体3H6H1L1的轻链可变区的核酸序列：(318bp)Nucleic acid sequence encoding the variable region of the light chain of the antibody 3H6H1L1: (318bp)
Figure PCTCN2021082692-appb-000010
Figure PCTCN2021082692-appb-000010
抗体3H6H1L1的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)The amino acid sequence of the light chain variable region of the antibody 3H6H1L1 is as follows: (106aa, the underlined amino acid sequence is the CDR region)
Figure PCTCN2021082692-appb-000011
Figure PCTCN2021082692-appb-000011
(2)人源化单克隆抗体3H6H2L2(2) Humanized monoclonal antibody 3H6H2L2
编码抗体3H6H2L2的重链可变区的核酸序列：(354bp)Nucleic acid sequence encoding the variable region of the heavy chain of the antibody 3H6H2L2: (354bp)
Figure PCTCN2021082692-appb-000012
Figure PCTCN2021082692-appb-000012
抗体3H6H2L2的重链可变区的氨基酸序列如下：(118aa，其中下划线标 记的氨基酸序列为CDR区域)The amino acid sequence of the variable region of the heavy chain of antibody 3H6H2L2 is as follows: (118aa, the underlined amino acid sequence is the CDR region)
Figure PCTCN2021082692-appb-000013
Figure PCTCN2021082692-appb-000013
编码抗体3H6H2L2的轻链可变区的核酸序列：(318bp)Nucleic acid sequence encoding the light chain variable region of antibody 3H6H2L2: (318bp)
Figure PCTCN2021082692-appb-000014
Figure PCTCN2021082692-appb-000014
抗体3H6H2L2的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)The amino acid sequence of the light chain variable region of the antibody 3H6H2L2 is as follows: (106aa, the underlined amino acid sequence is the CDR region)
Figure PCTCN2021082692-appb-000015
Figure PCTCN2021082692-appb-000015
(3)人源化单克隆抗体3H6H3L3(3) Humanized monoclonal antibody 3H6H3L3
编码抗体3H6H3L3的重链可变区的核酸序列：(354bp)Nucleic acid sequence encoding the variable region of the heavy chain of the antibody 3H6H3L3: (354bp)
Figure PCTCN2021082692-appb-000016
Figure PCTCN2021082692-appb-000016
抗体3H6H3L3的重链可变区的氨基酸序列如下：(118aa，其中下划线标记的氨基酸序列为CDR区域)The amino acid sequence of the variable region of the heavy chain of the antibody 3H6H3L3 is as follows: (118aa, the underlined amino acid sequence is the CDR region)
Figure PCTCN2021082692-appb-000017
Figure PCTCN2021082692-appb-000017
编码抗体3H6H3L3的轻链可变区的核酸序列：(318bp)Nucleic acid sequence encoding the variable region of the light chain of the antibody 3H6H3L3: (318bp)
Figure PCTCN2021082692-appb-000018
Figure PCTCN2021082692-appb-000018
抗体3H6H3L3的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)The amino acid sequence of the light chain variable region of the antibody 3H6H3L3 is as follows: (106aa, the underlined amino acid sequence is the CDR region)
Figure PCTCN2021082692-appb-000019
Figure PCTCN2021082692-appb-000019
(4)人源化单克隆抗体3H6H4L1(4) Humanized monoclonal antibody 3H6H4L1
编码抗体3H6H4L1的重链可变区的核酸序列如SEQ ID NO:5所示。The nucleic acid sequence encoding the variable region of the heavy chain of the antibody 3H6H4L1 is shown in SEQ ID NO: 5.
抗体3H6H4L1的重链可变区的氨基酸序列如SEQ ID NO:6所示。The amino acid sequence of the heavy chain variable region of the antibody 3H6H4L1 is shown in SEQ ID NO: 6.
编码抗体3H6H4L1的轻链可变区的核酸序列如SEQ ID NO:7所示。The nucleic acid sequence encoding the light chain variable region of the antibody 3H6H4L1 is shown in SEQ ID NO: 7.
抗体3H6H4L1的轻链可变区的氨基酸序列如SEQ ID NO:8所示。The amino acid sequence of the light chain variable region of the antibody 3H6H4L1 is shown in SEQ ID NO: 8.
2.人源化抗体3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1的制备2. Preparation of humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1
3H6H1L1、3H6H2L2和3H6H3L3的重链恒定区均采用Ig gamma-1chain C region，ACCESSION:P01857；轻链恒定区均采用Ig kappa chain C region， ACCESSION:P01834；The heavy chain constant regions of 3H6H1L1, 3H6H2L2 and 3H6H3L3 all use Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant regions all use Ig kappa chain C region, ACCESSION: P01834;
3H6H4L1的重链恒定区为Ig gamma-4chain C region，ACCESSION：P01861.1；轻链恒定区为Ig kappa chain C region，ACCESSION:P01834。The heavy chain constant region of 3H6H4L1 is Ig gamma-4 chain C region, ACCESSION: P01861.1; the light chain constant region is Ig kappa chain C region, ACCESSION: P01834.
将3H6H1L1重链cDNA和轻链的cDNA、3H6H2L2的重链cDNA和轻链的cDNA、3H6 H3L3重链cDNA和轻链的cDNA，以及3H6H4L1重链cDNA和轻链的cDNA分别克隆到pUC57simple载体(金斯瑞公司提供)中，分别获得8个重组质粒即pUC57simple-3H6H1和pUC57simple-3H6L1；pUC57simple3H6H2和pUC57simple-3H6L2；pUC57simple-3H6H3和pUC57simple-3H6L3；pUC57simple-3H6H4和pUC57simple-3H6L1。并分别亚克隆到pcDNA3.1载体中。将包含重链的质粒和包含轻链的重组质粒共转染293F细胞后收集培养液进行纯化获得人源化抗体3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1；并经SDS-PAGE检测，结果正确。3H6H1L1 heavy chain cDNA and light chain cDNA, 3H6H2L2 heavy chain cDNA and light chain cDNA, 3H6 H3L3 heavy chain cDNA and light chain cDNA, and 3H6H4L1 heavy chain cDNA and light chain cDNA were cloned into pUC57simple vector (gold (Provided by Sirui), eight recombinant plasmids were obtained, namely pUC57simple-3H6H1 and pUC57simple-3H6L1; pUC57simple3H6H2 and pUC57simple-3H6L2; pUC57simple-3H6H3 and pUC57simple-3H6L3; pUC57simple-3H6H4 and pUC57simple-3H6H4 and pUC57simple-3H6L2. And subcloned into pcDNA3.1 vector respectively. After co-transfecting the plasmid containing the heavy chain and the recombinant plasmid containing the light chain into 293F cells, the culture medium was collected and purified to obtain humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1; and the results were correct by SDS-PAGE.
实施例4：抗体3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1与人Example 4: Antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 and human IL-1β-his-bio结合的活性研究(ELISA法)Study on the binding activity of IL-1β-his-bio (ELISA method)
以2μg/mL的SA(链霉亲和素)，每孔50μL包被酶标板，4℃孵育过夜。洗板一次并除去残留液体，每孔用300μL 1％BSA溶液(用PBS溶解)封闭，37℃孵育2小时。洗板三次并除去残留液体。用PBST将人IL-1β-His-Bio稀释成0.2μg/mL，50μL/孔，37℃孵育30分钟，洗板三次并除去残留液体。分别将抗体稀释至如表1的1μg/mL或如表2的0.333μg/mL作为起始浓度，进行1:3的梯度稀释共7个浓度，另设空白对照，均做2个复孔，每孔体积100μL，37℃孵育30分钟。洗板三次后通过拍打将包被好的酶标板除去残余液体，每孔加入50μL辣根过氧化物酶标记的羊抗人IgG(H+L)二抗(货号：109-035-088，厂家：Jackson)工作液或辣根过氧化物酶标记的羊抗鼠IgG(H+L)二抗(货号：115-035-062，厂家：Jackson)工作液，37℃孵育30分钟。其中，50μL辣根过氧化物酶标记的羊抗人IgG(H+L)二抗工作液(对应3H6H1L1、3H6H2L2、3H6H3L3、3H6H4L1、Canakinumab所在的孔)和50μL辣根过氧化物酶标记的羊抗鼠IgG(H+L)二抗工作液(对应3H6所在的孔)。洗板四次并除去残留液体，每孔加入50μL TMB显色液，室温避光显色5分钟后， 每孔加入50μL终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。With 2μg/mL SA (streptavidin), 50μL per well was coated with the ELISA plate, and incubated overnight at 4°C. Wash the plate once and remove the remaining liquid, block each well with 300 μL 1% BSA solution (dissolved in PBS), and incubate at 37°C for 2 hours. Wash the plate three times and remove residual liquid. Dilute human IL-1β-His-Bio with PBST to 0.2μg/mL, 50μL/well, incubate at 37°C for 30 minutes, wash the plate three times and remove residual liquid. Dilute the antibody to 1μg/mL as shown in Table 1 or 0.333μg/mL as shown in Table 2 as the starting concentration, and perform a 1:3 gradient dilution for a total of 7 concentrations. A blank control is also set, and 2 replicates are made. The volume of each well is 100μL, and incubate at 37°C for 30 minutes. After washing the plate three times, remove the remaining liquid from the coated ELISA plate by tapping, and add 50μL horseradish peroxidase-labeled goat anti-human IgG (H+L) secondary antibody (Cat. No.: 109-035-088, Manufacturer: Jackson) working solution or horseradish peroxidase-labeled goat anti-mouse IgG (H+L) secondary antibody (Cat. No.: 115-035-062, manufacturer: Jackson) working solution, incubate at 37°C for 30 minutes. Among them, 50μL horseradish peroxidase-labeled goat anti-human IgG (H+L) secondary antibody working solution (corresponding to 3H6H1L1, 3H6H2L2, 3H6H3L3, 3H6H4L1, Canakinumab) and 50μL horseradish peroxidase-labeled goat Anti-mouse IgG (H+L) secondary antibody working solution (corresponding to the well where 3H6 is located). Wash the plate four times and remove the remaining liquid. Add 50μL of TMB color developing solution to each well. After 5 minutes of color development at room temperature, avoid light, add 50μL of stop solution to each well to stop the color reaction. Immediately put the microplate in the microplate reader, select the 450nm light wavelength to read the OD value of each well of the microplate.
用SoftMax Pro 6.2.1软件对数据进行分析处理。以抗体浓度为横坐标，吸光度值为纵坐标进行4-parameter拟合曲线作图，结果分别如图1及图2所示。3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1与人IL-1β-His-Bio的结合活性检测结果分别见表1和表2。Use SoftMax Pro 6.2.1 software to analyze and process the data. Using the antibody concentration as the abscissa and the absorbance value as the ordinate, the 4-parameter fitting curve was plotted, and the results are shown in Figure 1 and Figure 2 respectively. The binding activity test results of 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 and human IL-1β-His-Bio are shown in Table 1 and Table 2, respectively.
表1：3H6、3H6H1L1、3H6H2L2以及3H6H3L3与人IL-1β-His-Bio的结合活性检测结果Table 1: Detection results of the binding activity of 3H6, 3H6H1L1, 3H6H2L2, and 3H6H3L3 with human IL-1β-His-Bio
Figure PCTCN2021082692-appb-000020
Figure PCTCN2021082692-appb-000020
表2：3H6H4L1与人IL-1β-His-Bio的结合活性检测结果Table 2: Detection results of the binding activity of 3H6H4L1 and human IL-1β-His-Bio
Figure PCTCN2021082692-appb-000021
Figure PCTCN2021082692-appb-000021
Figure PCTCN2021082692-appb-000022
Figure PCTCN2021082692-appb-000022
结果显示：The results show that:
3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1均能够有效地结合人IL-1β-His-Bio，并且结合效率呈现剂量依赖关系；3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 can effectively bind human IL-1β-His-Bio, and the binding efficiency is in a dose-dependent relationship;
在相同检测条件下，3H6 H1L1、3H6 H2L2及3H6H4L1与抗原人IL-1β-His-Bio的结合效率呈现剂量依赖关系，并且结合活性优于同靶点已上市药物Canakinumab；3H6H3L3结合活性与Canakinumab相当。Under the same detection conditions, the binding efficiency of 3H6 H1L1, 3H6 H2L2 and 3H6H4L1 to the antigen human IL-1β-His-Bio showed a dose-dependent relationship, and the binding activity was better than that of the marketed drug Canakinumab with the same target; the binding activity of 3H6H3L3 was equivalent to Canakinumab .
实施例5：抗体3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1与Example 5: Antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 and 人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性研究(ELISA法)Study on the activity of human IL-1R1(1-332)-his in binding to human IL-1β-hFc (ELISA method)
以4μg/mL的人IL-1β-hFc，每孔50μL包被酶标板，4℃孵育过夜。洗板一次并除去残留液体，每孔加入300μL 1％BSA溶液(用PBS溶解)封闭，37℃孵育2小时，洗板三次并除去残留液体。抗体稀释至2μg/mL(终浓度1μg/mL)作为起始浓度，进行1:3的梯度稀释共7个浓度，另设空白对照，均做2个复孔，每孔体积50μL，室温孵育10分钟。将0.08μg/mL(终浓度0.04μg/ml)或0.1μg/ml(终浓度0.05μg/ml)的人IL-1R1(1-332)-his加入到酶标板中，每孔体积50μL与抗体体积1:1轻柔混匀，每孔终体积100μL，37℃孵育30分钟。洗板三次并除去残留液体，每孔加入50μL抗His标签鼠单克隆抗体(HRP标记)(货号：CW0285M，厂家：康为世纪)工作液，37℃孵育30分钟。洗板四次并除去残留液体，每孔加入50μl的TMB显色液，室温避光显色 10分钟或5分钟后，每孔加入50μL终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。The ELISA plate was coated with 4μg/mL human IL-1β-hFc, 50μL per well, and incubated overnight at 4°C. Wash the plate once and remove the remaining liquid, add 300 μL 1% BSA solution (dissolved in PBS) to each well to block, incubate at 37°C for 2 hours, wash the plate three times and remove the remaining liquid. The antibody was diluted to 2μg/mL (final concentration 1μg/mL) as the starting concentration, and a 1:3 gradient dilution was carried out for a total of 7 concentrations, and a blank control was set up. Two replicate wells were made, each with a volume of 50μL, and incubated for 10 at room temperature. minute. Add 0.08μg/mL (final concentration 0.04μg/ml) or 0.1μg/ml (final concentration 0.05μg/ml) human IL-1R1(1-332)-his into the microtiter plate, the volume of each well is 50μL and The antibody volume is 1:1 and mixed gently, with a final volume of 100μL per well, and incubate at 37°C for 30 minutes. Wash the plate three times and remove the remaining liquid, add 50μL of anti-His-tagged mouse monoclonal antibody (HRP labeled) (Cat. No.: CW0285M, manufacturer: Kangwei Century) working solution to each well, and incubate at 37°C for 30 minutes. Wash the plate four times and remove the remaining liquid. Add 50μl of TMB color developing solution to each well. After 10 minutes or 5 minutes of color development in the dark at room temperature, add 50μL of stop solution to each well to stop the color reaction. Immediately put the microplate in the microplate reader, select the 450nm light wavelength to read the OD value of each well of the microplate.
用SoftMax Pro 6.2.1软件对数据进行分析处理，以抗体浓度为横坐标，吸光度值为纵坐标进行4-parameter拟合曲线作图，结果如图3及图4所示。3H6、3H6H1L1、3H6H2L2及3H6H3L3和3H6H4L1与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果见表3及表4。Use SoftMax Pro 6.2.1 software to analyze and process the data. Take the antibody concentration as the abscissa and the absorbance as the ordinate to plot the 4-parameter fitting curve. The results are shown in Figure 3 and Figure 4. 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3, and 3H6H4L1 compete with human IL-1R1(1-332)-his to bind to human IL-1β-hFc. See Table 3 and Table 4 for the activity detection results.
表3：3H6、3H6H1L1、3H6H2L2以及3H6H3L3与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果Table 3: 3H6, 3H6H1L1, 3H6H2L2, and 3H6H3L3 compete with human IL-1R1(1-332)-his to bind to human IL-1β-hFc activity test results
Figure PCTCN2021082692-appb-000023
Figure PCTCN2021082692-appb-000023
表4：3H6H4L1与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果Table 4: 3H6H4L1 and human IL-1R1(1-332)-his competition binding human IL-1β-hFc activity test results
Figure PCTCN2021082692-appb-000024
Figure PCTCN2021082692-appb-000024
Figure PCTCN2021082692-appb-000025
Figure PCTCN2021082692-appb-000025
结果显示：The results show that:
3H6、3H6H1L1、3H6H2L2及3H6H3L3和3H6H4L1均能够有效地阻断抗原人IL-1β-hFc与其受体人IL-1R1(1-332)-his的结合，且阻断效率呈现剂量依赖关系，竞争结合活性优于同靶点已上市药物Canakinumab。3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 can effectively block the binding of antigen human IL-1β-hFc and its receptor human IL-1R1(1-332)-his, and the blocking efficiency is dose-dependent, competing for binding The activity is better than that of Canakinumab, an already marketed drug with the same target.
实施例6：抗体3H6H4L1与人IL-1β的亲和力常数测定Example 6: Determination of the affinity constant of antibody 3H6H4L1 and human IL-1β
采用Biacore分子相互作用仪检测抗体与人IL-1β-his的亲和力常数。以PBST作为缓冲液，采用氨基偶联方式将抗体固定于CM5芯片表面，固定的信号值约为1000RU。抗体与人IL-1β结合，IL-1β浓度为1.56-25nM(两倍梯度稀释)，流速为30μl/min，结合的时间为120s，解离时间为600s。芯片使用3M MgCl 2再生，流速为30μl/min，时间为30s。使用Biacore Control 2.0软件进行数据采集，Biacore T200 Evaluation 2.0软件进行数据分析。结果如表5、图5和图6所示。 The Biacore molecular interaction instrument was used to detect the affinity constant of the antibody and human IL-1β-his. Using PBST as a buffer, the antibody was immobilized on the surface of the CM5 chip by amino coupling, and the immobilized signal value was about 1000 RU. The antibody binds to human IL-1β, the IL-1β concentration is 1.56-25nM (two-fold dilution), the flow rate is 30μl/min, the binding time is 120s, and the dissociation time is 600s. The chip is regenerated with 3M MgCl 2 at a flow rate of 30μl/min and a time of 30s. Biacore Control 2.0 software was used for data collection, and Biacore T200 Evaluation 2.0 software was used for data analysis. The results are shown in Table 5, Figure 5 and Figure 6.
表5：3H6H4L1与人IL-1β亲和力常数检测结果Table 5: Test results of affinity constant between 3H6H4L1 and human IL-1β
抗体名称Antibody name K D(M) K D (M) ka(1/Ms)ka(1/Ms) SE(ka)SE(ka) kd(1/s)kd(1/s) SE(kd)SE(kd) Rmax(RU)Rmax(RU)
3H6H4L13H6H4L1 8.79E-118.79E-11 1.44E+061.44E+06 3.15E+033.15E+03 1.27E-041.27E-04 1.75E-071.75E-07 112.49-122.37112.49-122.37
CanakinumabCanakinumab 9.79E-119.79E-11 5.24E+055.24E+05 6.84E+026.84E+02 5.13E-055.13E-05 1.43E-071.43E-07 76.61-86.3776.61-86.37
结果显示：3H6H4L1与人IL-1β的亲和力常数为8.79E-11M，Canakinumab与人IL-1β的亲和力常数为9.79E-11M，表明3H6H4L1与人IL-1β有较强的结合能力。The results showed that the affinity constant of 3H6H4L1 and human IL-1β was 8.79E-11M, and the affinity constant of Canakinumab and human IL-1β was 9.79E-11M, indicating that 3H6H4L1 has a strong binding ability to human IL-1β.
实施例7：抗体3H6 H4L1的细胞生物学活性研究Example 7: Study on the Cell Biological Activity of Antibody 3H6 H4L1
1.3H6 H4L1阻断IL-1β诱导MRC-5细胞分泌IL-6的细胞学活性检测1.3 Detection of the cytological activity of H6 H4L1 blocking IL-1β-induced IL-6 secretion by MRC-5 cells
人MRC-5细胞(购自中国科学院细胞中心)常规消化、计数，7,500个/孔接种于平底96孔板中，置于细胞培养箱培养；24h后(细胞生长达到80％汇合度时)，进行给药处理：抗体设4个浓度(0.003nM、0.03nM、0.3nM、3nM)，IL-1β(购自Sino Biological，货号：10139-HNAE)设三个浓度(5pM、50pM、500pM)，抗体组IL-1β用50pM的浓度(抗体和IL-1β先在37℃孵育30min)，另外同时设置空白对照组及同型对照组；加药处理后置于细胞培养箱中培养24h；收集细胞上清，用IL-6ELISAKit(购自达科为生物技术有限公司，货号：1110602)进行检测。检测结果如图7和表6所示。Human MRC-5 cells (purchased from the Cell Center of the Chinese Academy of Sciences) are routinely digested and counted. 7,500 cells/well are seeded in a flat-bottomed 96-well plate and placed in a cell culture incubator; 24h later (when the cell growth reaches 80% confluence), Dosing treatment: antibody is set to 4 concentrations (0.003nM, 0.03nM, 0.3nM, 3nM), IL-1β (purchased from Sino Biological, item number: 10139-HNAE) is set to three concentrations (5pM, 50pM, 500pM), Antibody group IL-1β with a concentration of 50pM (antibody and IL-1β were incubated at 37°C for 30min), and a blank control group and an isotype control group were set at the same time; after adding the drug, it was placed in a cell incubator for 24 hours; the cells were collected Clear, use IL-6ELISAKit (purchased from Dakwei Biotechnology Co., Ltd., article number: 1110602) for detection. The test results are shown in Figure 7 and Table 6.
表6：3H6 H4L1对IL-1β诱导MRC-5细胞分泌释放IL-6的抑制作用Table 6: Inhibition of 3H6 H4L1 on IL-1β-induced MRC-5 cell secretion and release of IL-6
Figure PCTCN2021082692-appb-000026
Figure PCTCN2021082692-appb-000026
结果表明IL-1β可梯度诱导MRC-5细胞分泌IL-6，3H6 H4L1可显著抑制IL-1β对MRC-5分泌释放IL-6的诱导作用，且具有显著剂量依赖关系。综合表明，3H6 H4L1可通过特异性结合IL-1β，阻断IL-1β介导的免疫因子IL-6 的分泌，阻断效能与同靶点对照抗体Canakinumab相当。The results show that IL-1β can induce IL-6 secretion by MRC-5 cells in a gradient, and 3H6 H4L1 can significantly inhibit IL-1β's induction of IL-6 secretion and release by MRC-5, and it has a significant dose-dependent relationship. Overall, it shows that 3H6 H4L1 can specifically bind IL-1β to block IL-1β-mediated secretion of the immune factor IL-6, and its blocking efficacy is equivalent to that of the target control antibody Canakinumab.
2.3H6 H4L1阻断IL-1β激活NF-κB信号通路2.3H6 H4L1 blocks IL-1β and activates the NF-κB signaling pathway
本实验通过荧光报告基因方法检测3H6 H4L1阻断IL-1β激活NF-κB信号通路的中和生物活性。In this experiment, the fluorescent reporter gene method was used to detect the neutralizing biological activity of 3H6 H4L1 blocking IL-1β to activate the NF-κB signaling pathway.
(1)293T-NF-κB-LUC细胞构建(1) 293T-NF-κB-LUC cell construction
胰酶消化293T细胞，传代培养；转染前2h，更换opti-DMEM培养基；在无菌EP管中，加入500μL opti-DMEM培养基，再加入3μg的质粒pNF-kB-Luc2P-hygro(Akeso)；随后用转染试剂PEI(货号:23966，Polyscience)和表达载体感染构建293T-NFκB-Luc细胞；转染8h后，更换新鲜培养基；转染24h后，加入Hygromycin B(Invitrogen，货号：10687010)，终浓度100μg/mL筛选，设对照孔293T未转染质粒。7-10天后，对照孔细胞完全死亡为准，收获筛选完的细胞扩增培养。继续加药100μg/mL维持。得到293T-NF-κB-LUC稳转细胞株。Trypsin digestion of 293T cells, subculture; 2h before transfection, replace opti-DMEM medium; in a sterile EP tube, add 500μL of opti-DMEM medium, and then add 3μg of plasmid pNF-kB-Luc2P-hygro(Akeso ); Then use the transfection reagent PEI (Cat. No.: 23966, Polyscience) and expression vector to infect and construct 293T-NFκB-Luc cells; 8h after transfection, change the fresh medium; after 24h of transfection, add Hygromycin B (Invitrogen, Cat. No.: 10687010), the final concentration was 100μg/mL for screening, and the control well 293T was not transfected with plasmid. After 7-10 days, if the cells in the control wells are completely dead, harvest the selected cells for expansion and culture. Continue to add 100μg/mL to maintain. Obtained 293T-NF-κB-LUC stably transfected cell line.
(2)3H6 H4L1阻断IL-1β激活NF-κB信号通路的中和生物活性检测常规消化293T-NF-κB-Luc细胞，按每孔20,000个细胞接种于96孔板中。待细胞贴壁后，添加IL-1β，使其终浓度1.65ng/mL，设置空白对照。同时添加抗体，Canakinumab，3H6 H4L1，每个抗体8个梯度，终浓度分别为:1600ng/mL，800ng/mL，200ng/mL，50ng/mL，12.5ng/mL，3.125ng/mL，0.3125ng/mL，0.03125ng/mL。共培养4小时后，去上清，加入50μL PBS，再加入50μL Bright-Glo TM底物(Promega，货号：E2620)，反应5min，上机检测。 (2) 3H6 H4L1 blocks IL-1β to activate the neutralization of the NF-κB signaling pathway. The 293T-NF-κB-Luc cells are routinely digested, and 20,000 cells per well are seeded in a 96-well plate. After the cells adhere to the wall, add IL-1β to a final concentration of 1.65ng/mL, and set a blank control. Add antibodies at the same time, Canakinumab, 3H6 H4L1, 8 gradients for each antibody, the final concentrations are: 1600ng/mL, 800ng/mL, 200ng/mL, 50ng/mL, 12.5ng/mL, 3.125ng/mL, 0.3125ng/ mL, 0.03125ng/mL. After 4 hours of co-cultivation, remove the supernatant, add 50 μL PBS, and then add 50 μL Bright-Glo TM substrate (Promega, catalog number: E2620), react for 5 minutes, and test on the computer.
结果如图8、图9所示。The results are shown in Figure 8 and Figure 9.
结果显示：The results show that:
IL-1β依赖的以293T-NFκB-Luc作为工作细胞的报告基因检测体系，IL-1β可呈剂量依赖性激活293T-NFκB-Luc细胞的NF-κB信号通路，进而启动报告基因的表达。该体系可用于评价3H6H4L1对IL-1β的中和生物活性。本研究结果表明，3H6H4L1特异性与IL-1β结合后可高效抑制IL-1β介导的信号通路的活化，表现3H6 H4L1对IL-1β的中和生物学活性，中和活性EC50为0.0966nM，而同靶点上市药Canakinumab中和活性EC50为0.0801nM，二者活性相当。IL-1β-dependently uses 293T-NFκB-Luc as the reporter gene detection system of working cells. IL-1β can activate the NF-κB signaling pathway of 293T-NFκB-Luc cells in a dose-dependent manner, and then initiate the expression of the reporter gene. This system can be used to evaluate the neutralizing biological activity of 3H6H4L1 on IL-1β. The results of this study show that 3H6H4L1 specifically binds to IL-1β and can effectively inhibit the activation of IL-1β-mediated signaling pathways, showing the neutralizing biological activity of 3H6H4L1 on IL-1β, and the neutralizing activity EC50 is 0.0966nM. The neutralizing activity EC50 of the same target drug Canakinumab is 0.0801nM, which is equivalent in activity.
以上对本发明的较佳实施方式进行了具体说明，但本发明创造并不限于所 述实施例，熟悉本领域的技术人员在不违背本发明精神的前提下还可做出种种的等同变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。The preferred embodiments of the present invention are described in detail above, but the invention is not limited to the embodiments. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or replacements are all included in the scope defined by the claims of this application.

Claims (18)

  1. 抗IL-1β的抗体或其抗原结合片段、抗体药物偶联物或双特异性抗体在治疗或预防(IL-1β异常分泌增加为特征的)系统性炎症或制备治疗和/或预防性治疗或预防(IL-1β异常分泌增加为特征的)系统性炎症的药物中的应用，其中，Anti-IL-1β antibodies or their antigen-binding fragments, antibody-drug conjugates or bispecific antibodies are used in the treatment or prevention of systemic inflammation (characterized by abnormally increased IL-1β secretion) or preparation of treatment and/or preventive treatment or Application of drugs to prevent systemic inflammation (characterized by abnormally increased secretion of IL-1β), in which,
    所述抗体的重链可变区包含：氨基酸序列分别如SEQ ID NO:17－SEQ ID NO:19所示的HCDR1-HCDR3；和The heavy chain variable region of the antibody comprises: the amino acid sequences of HCDR1-HCDR3 shown in SEQ ID NO: 17-SEQ ID NO: 19, respectively; and
    所述抗体的轻链可变区包含：氨基酸序列分别如SEQ ID NO:20－SEQ ID NO:22所示的LCDR1-LCDR3，The light chain variable region of the antibody includes LCDR1-LCDR3 with amino acid sequences as shown in SEQ ID NO: 20-SEQ ID NO: 22, respectively,
    优选地，所述系统性炎症包括系统性炎症综合征，细胞因子释放综合征、多器官功能障碍综合征(multiple organ dysfunction syndrome，MODS)、急性呼吸窘迫综合征；Preferably, the systemic inflammation includes systemic inflammatory syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS), and acute respiratory distress syndrome;
    优选地，所述系统性炎症是由病原微生物感染所引起的；Preferably, the systemic inflammation is caused by pathogenic microorganism infection;
    优选地，所述病原微生物为病毒、细菌、真菌、立克次体、衣原体、支原体、寄生虫、朊病毒；Preferably, the pathogenic microorganisms are viruses, bacteria, fungi, rickettsiae, chlamydia, mycoplasma, parasites, prions;
    优选地，所述病毒包括RNA病毒及DNA病毒；Preferably, the viruses include RNA viruses and DNA viruses;
    优选地，所述RNA病毒包括冠状病毒科病毒；Preferably, the RNA virus includes a coronavirus of the coronavirus family;
    优选地，所述冠状病毒科病毒包括2019新型冠状病毒(2019-nCoV或SARS-CoV-2，引发新型冠状病毒肺炎COVID-19)，HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV(引发重症急性呼吸综合征)和MERS-CoV(引发中东呼吸综合征)。Preferably, the coronavirus family virus includes 2019 new coronavirus (2019-nCoV or SARS-CoV-2, which causes new coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 , SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing Middle East respiratory syndrome).
  2. 根据权利要求1所述的应用，其中，The application according to claim 1, wherein:
    所述抗体的重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14或与SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；和The amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, or with SEQ ID NO: 2, SEQ ID NO: 6 , SEQ ID NO: 10 and SEQ ID NO: 14 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical; and
    所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4和SEQ ID NO:8、SEQ ID NO:12和SEQ ID NO:16或与SEQ ID NO:4和SEQ ID NO:8、 SEQ ID NO:12和SEQ ID NO:16具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列。The amino acid sequence of the variable region of the light chain of the antibody is selected from SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16, or with SEQ ID NO: 4 and SEQ ID NO: 8. , SEQ ID NO: 12 and SEQ ID NO: 16 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical.
  3. 根据权利要求1所述的抗体或其抗原结合片段，其中，The antibody or antigen-binding fragment thereof according to claim 1, wherein:
    所述抗体的重链可变区的氨基酸序列为SEQ ID NO:2或与SEQ ID NO:2具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO:4或与SEQ ID NO:4具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者The amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 2 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% with SEQ ID NO: 2 % Identity sequence, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 4 or at least 80%, 85%, 90%, 95%, 96%, 97 %, 98% or at least 99% sequence identity; or
    所述抗体的重链可变区的氨基酸序列为SEQ ID NO:6或与SEQ ID NO:6具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO:8或与SEQ ID NO:8具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者The amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 6 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% with SEQ ID NO: 6 % Identity sequence, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 8 or at least 80%, 85%, 90%, 95%, 96%, 97 %, 98% or at least 99% sequence identity; or
    所述抗体的重链可变区的氨基酸序列为SEQ ID NO:10或与SEQ ID NO:10具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO:12或与SEQ ID NO:12具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者The amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 10 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% with SEQ ID NO: 10 % Identity sequence, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 12 or at least 80%, 85%, 90%, 95%, 96%, 97 %, 98% or at least 99% sequence identity; or
    所述抗体的重链可变区的氨基酸序列为SEQ ID NO:14或与SEQ ID NO:14具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO:16或与SEQ ID NO:16具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列。The amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 14 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% with SEQ ID NO: 14 % Identity sequence, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 16 or at least 80%, 85%, 90%, 95%, 96%, 97 %, 98% or at least 99% sequence identity.
  4. 根据权利要求1至3中任一项所述的应用，其中，所述抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。The application according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragments, Single chain antibody, humanized antibody, chimeric antibody or diabody.
  5. 根据权利要求1至3中任一项所述的应用，其中，所述的抗体以小于10 -5M，例如小于10 -6M、小于10 -7M、小于10 -8M、小于10 -9M或小于10 -10M或更小的K D结合IL-1β蛋白；优选地，所述K D通过Biacore分子相互作用 仪测得。 Application of 1-1 according to any according to claim 3, wherein said antibody is less than 10 -5 M, such as less than 10 -6 M, less than 10 -7 M, less than 10 -8 M, less than 10 - The K D of 9 M or less than 10 -10 M or less binds to IL-1β protein; preferably, the K D is measured by a Biacore molecular interaction instrument.
  6. 根据权利要求1至3中任一项所述的应用，其中，所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。The use according to any one of claims 1 to 3, wherein the antibody includes a non-CDR region, and the non-CDR region is from a species other than murine, for example, from a human antibody.
  7. 根据权利要求1至3中任一项所述的应用，其中，所述的抗体，其恒定区来自人抗体；The use according to any one of claims 1 to 3, wherein the constant region of the antibody is derived from a human antibody;
    优选地，所述抗体的恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。Preferably, the constant region of the antibody is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4.
  8. 根据权利要求1至3中任一项所述的应用，其中，所述的抗体，其重链恒定区为Ig gamma-1 chain C region或Ig gamma-4 chain C region，并且其轻链恒定区为Ig kappa chain C region。The application according to any one of claims 1 to 3, wherein the heavy chain constant region of the antibody is Ig gamma-1 chain C region or Ig gamma-4 chain C region, and the light chain constant region It is Ig kappa chain C region.
  9. 根据权利要求1至3中任一项所述的应用，其中所述抗体是由杂交瘤细胞株LT010产生的单克隆抗体，所述杂交瘤细胞株LT010保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018133。The use according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody produced by the hybridoma cell line LT010, which is deposited at the Chinese Type Culture Collection (CCTCC) , The deposit number is CCTCC NO: C2018133.
  10. 根据权利要求1至3中任一项所述的应用，其中所述抗体药物偶联物包括所述抗体或其抗原结合片段以及小分子药物；优选地，所述小分子药物为小分子细胞毒药物；更优选地，所述小分子药物为肿瘤化疗药物。The application according to any one of claims 1 to 3, wherein the antibody drug conjugate comprises the antibody or antigen-binding fragment thereof and a small molecule drug; preferably, the small molecule drug is a small molecule cytotoxicity Drug; More preferably, the small molecule drug is a tumor chemotherapeutic drug.
  11. 根据权利要求10所述的应用，其中，所述抗体或其抗原结合片段通过连接子与小分子药物连接；例如，所述连接子为腙键、二硫键或肽键。The application according to claim 10, wherein the antibody or antigen-binding fragment thereof is connected to the small molecule drug through a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond.
  12. 根据权利要求10或11所述的应用，其中，所述抗体或其抗原结合片段与小分子药物以一定的摩尔比连接；例如，所述摩尔比为1：(2－4)。The application according to claim 10 or 11, wherein the antibody or antigen-binding fragment thereof and the small molecule drug are connected at a certain molar ratio; for example, the molar ratio is 1: (2-4).
  13. 根据权利要求1至3中任一项所述的应用，其中所述双特异性抗体包括第一蛋白功能区和第二蛋白功能区，其中：The use according to any one of claims 1 to 3, wherein the bispecific antibody comprises a first protein functional region and a second protein functional region, wherein:
    所述第一蛋白功能区靶向IL-1β，The first protein functional region targets IL-1β,
    所述第二蛋白功能区靶向不同于IL-1β的靶点(例如，IL-17A)；The second protein functional region targets a target different from IL-1β (for example, IL-17A);
    其中，所述第一蛋白功能区为权利要求1－9中任一项所述的抗体或抗原结合片段；Wherein, the first protein functional region is the antibody or antigen-binding fragment according to any one of claims 1-9;
    优选地，所述双特异性抗体为IgG-scFv模式；Preferably, the bispecific antibody is in IgG-scFv mode;
    优选地，所述第一蛋白功能区为权利要求1－9中任一项所述的抗体，并且所述第二蛋白功能区为单链抗体；或者Preferably, the first protein functional region is the antibody of any one of claims 1-9, and the second protein functional region is a single-chain antibody; or
    优选地，所述第一蛋白功能区为权利要求4所述的单链抗体，并且所 述第二蛋白功能区为抗体。Preferably, the first protein functional region is the single-chain antibody of claim 4, and the second protein functional region is an antibody.
  14. 根据权利要求13所述的应用，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；The application according to claim 13, wherein the first protein functional region and the second protein functional region are directly connected or connected by a connecting fragment;
    优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；Preferably, the connection fragment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;
    优选地，所述连接片段为SS(GGGGS)n，n为正整数，例如1、2、3、4、5或6。Preferably, the connection fragment is SS(GGGGS)n, where n is a positive integer, such as 1, 2, 3, 4, 5 or 6.
  15. 根据权利要求13所述的应用，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。The use according to claim 13, wherein the first protein functional region and the second protein functional region are independently one, two or more than two.
  16. 根据权利要求13至15中任一项所述的应用，其中，所述单链抗体连接在抗体的重链的C末端。The use according to any one of claims 13 to 15, wherein the single-chain antibody is attached to the C-terminus of the heavy chain of the antibody.
  17. 根据权利要求1-16任一项所述的应用，其中所述抗体或其抗原结合片段、所述抗原药物偶联物或所述双特异性抗体的剂量为有效量，优选所述有效量为0.001mg-1000mg，更优选0.001mg-900mg、0.001mg-800mg、0.001mg-700mg、0.001mg-600mg、0.001mg-500mg、0.001mg-400mg、0.001mg-300mg、0.001mg-200mg、0.001mg-100mg，最优选为100mg、200mg、300mg、400mg、500mg、600mg、700mg、800mg、900mg或1000mg，或者，基于受试者或患者的体重，所述有效量为0.1-100mg/kg，优选1-90mg/kg、1-80mg/kg、1-70mg/kg、1-60mg/kg、1-50mg/kg、1-40mg/kg、1-30mg/kg、1-20mg/kg或1-10mg/kg。The use according to any one of claims 1-16, wherein the dose of the antibody or antigen-binding fragment thereof, the antigen-drug conjugate or the bispecific antibody is an effective amount, preferably the effective amount is 0.001mg-1000mg, more preferably 0.001mg-900mg, 0.001mg-800mg, 0.001mg-700mg, 0.001mg-600mg, 0.001mg-500mg, 0.001mg-400mg, 0.001mg-300mg, 0.001mg-200mg, 0.001mg- 100mg, most preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg, or, based on the weight of the subject or patient, the effective amount is 0.1-100mg/kg, preferably 1- 90mg/kg, 1-80mg/kg, 1-70mg/kg, 1-60mg/kg, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1-20mg/kg or 1-10mg/ kg.
  18. 一种治疗和/或预防性治疗和/或预防系统性炎症的方法，包括给予有需求的受试者或患者以有效量的权利要求1至16中任一项所涉及的抗体或其抗原结合片段、抗体药物偶联物或者双特异性抗体，A method for treatment and/or preventive treatment and/or prevention of systemic inflammation, comprising administering to a subject or patient in need an effective amount of the antibody or antigen binding thereof according to any one of claims 1 to 16 Fragments, antibody-drug conjugates or bispecific antibodies,
    优选地，所述抗体或其抗原结合片段、抗体药物偶联物或者双特异性抗体为单独使用或者与有效量的一种或多种抗病毒药物联用；Preferably, the antibody or its antigen-binding fragment, antibody-drug conjugate or bispecific antibody is used alone or in combination with an effective amount of one or more antiviral drugs;
    优选地，所述抗病毒药物为抗逆转录病毒药物或干扰素，优选地，所述抗逆转录病毒药物其中：抗逆转录病毒药物是RNA依赖的RNA聚合酶(RdRp)抑制剂，或者是核苷类似物，或者是类肽，或者是人免疫缺陷病毒类型1(HIV-1)整合酶的链转移抑制剂(INSTI)，或者是HIV蛋白酶抑制剂。Preferably, the anti-retroviral drug is an anti-retroviral drug or an interferon. Preferably, the anti-retroviral drug is an RNA-dependent RNA polymerase (RdRp) inhibitor, or Nucleoside analogues are either peptoids, chain transfer inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase (INSTI), or HIV protease inhibitors.
    优选地，所述抗病毒药物为抗逆转录病毒药物，优选地，所述抗逆转录病毒药物为法匹拉韦(favipiravir)、瑞德西韦(Remdesivir)、Beclabuvir，沙奎那韦(Saquinavir)，比特拉韦(Bictegravir)，洛匹那韦(Lopinavir)，多替拉韦(Dolutegravir)；更优选地，所述化疗药物为法匹拉韦；Preferably, the anti-retroviral drug is an anti-retroviral drug. Preferably, the anti-retroviral drug is favipiravir, Remdesivir, Beclabuvir, Saquinavir ), Bictegravir, Lopinavir, Dolutegravir; more preferably, the chemotherapeutic drug is favipiravir;
    更优选地，所述抗体或其抗原结合片段、抗体药物偶联物或者双特异性抗体的有效量为0.001mg-1000mg，更优选0.001mg-900mg、0.001mg-800mg、0.001mg-700mg、0.001mg-600mg、0.001mg-500mg、0.001mg-400mg、0.001mg-300mg、0.001mg-200mg、0.001mg-100mg，最优选为100mg、200mg、300mg、400mg、500mg、600mg、700mg、800mg、900mg或1000mg，或者，基于受试者或患者的体重，所述抗体或其抗原结合片段、抗体药物偶联物或者双特异性抗体的有效量为0.1-100mg/kg，优选1-90mg/kg、1-80mg/kg、1-70mg/kg、1-60mg/kg、1-50mg/kg、1-40mg/kg、1-30mg/kg、1-20mg/kg或1-10mg/kg；More preferably, the effective amount of the antibody or its antigen-binding fragment, antibody-drug conjugate or bispecific antibody is 0.001 mg-1000 mg, more preferably 0.001 mg-900 mg, 0.001 mg-800 mg, 0.001 mg-700 mg, 0.001 mg-600mg, 0.001mg-500mg, 0.001mg-400mg, 0.001mg-300mg, 0.001mg-200mg, 0.001mg-100mg, most preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000 mg, or, based on the weight of the subject or patient, the effective amount of the antibody or antigen-binding fragment, antibody-drug conjugate or bispecific antibody is 0.1-100 mg/kg, preferably 1-90 mg/kg, 1 -80mg/kg, 1-70mg/kg, 1-60mg/kg, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1-20mg/kg or 1-10mg/kg;
    更优选地，所述抗病毒药物的有效量为100-2400mg，优选100mg-2300mg、100mg-2200mg、100mg-2100mg、100mg-2000mg、100mg-1900mg、100mg-1800mg、100mg-1700mg、100mg-1600mg、100mg-1800mg、100mg-1800mg、100mg-1800mg、100mg-1800mg、100mg-1800mg，更优选为100mg，200mg，300mg，400mg，500mg，600mg，700mg，800mg，900mg，1000mg，或者，基于受试者或患者的体重，所述抗病毒药物的有效量为0.1-100mg/kg，优选1-90mg/kg、1-80mg/kg、1-70mg/kg、1-60mg/kg、1-50mg/kg、1-40mg/kg、1-30mg/kg、1-20mg/kg或1-10mg/kg。More preferably, the effective amount of the antiviral drug is 100-2400mg, preferably 100mg-2300mg, 100mg-2200mg, 100mg-2100mg, 100mg-2000mg, 100mg-1900mg, 100mg-1800mg, 100mg-1700mg, 100mg-1600mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, more preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, or based on the subject or The weight of the patient, the effective amount of the antiviral drug is 0.1-100mg/kg, preferably 1-90mg/kg, 1-80mg/kg, 1-70mg/kg, 1-60mg/kg, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1-20mg/kg or 1-10mg/kg.
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