[go: up one dir, main page]

WO2024243819A1 - 抗白介素18受体的纳米抗体及其应用 - Google Patents

抗白介素18受体的纳米抗体及其应用 Download PDF

Info

Publication number
WO2024243819A1
WO2024243819A1 PCT/CN2023/097235 CN2023097235W WO2024243819A1 WO 2024243819 A1 WO2024243819 A1 WO 2024243819A1 CN 2023097235 W CN2023097235 W CN 2023097235W WO 2024243819 A1 WO2024243819 A1 WO 2024243819A1
Authority
WO
WIPO (PCT)
Prior art keywords
nanobody
antibody
present
seq
fusion protein
Prior art date
Application number
PCT/CN2023/097235
Other languages
English (en)
French (fr)
Inventor
万亚坤
朱敏
盖军伟
李光辉
Original Assignee
上海洛启生物医药技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海洛启生物医药技术有限公司 filed Critical 上海洛启生物医药技术有限公司
Priority to PCT/CN2023/097235 priority Critical patent/WO2024243819A1/zh
Publication of WO2024243819A1 publication Critical patent/WO2024243819A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of antibody drugs, and in particular to a nano antibody targeting interleukin 18 receptor and an application thereof.
  • Interleukin-18 is a potent pro-inflammatory cytokine produced by multiple cell types, including hematopoietic and non-hematopoietic cells, and is involved in host defense against infection, as well as in regulating innate and acquired immune responses. IL-18 can regulate innate and adaptive immunity, and its abnormal regulation may lead to autoimmune or inflammatory diseases.
  • IL-18 in the blood of patients with some immune-related diseases is elevated, and the severity of the disease is positively correlated with the level of IL-18, such as rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, and inflammatory bowel disease.
  • RA rheumatoid arthritis
  • Clinical studies have shown that by inhibiting the IL-18 signaling pathway, rare diseases such as hemophagocytic lymphohistiocytosis (HLH) and adult Still's disease can be effectively treated, and chronic pulmonary sarcoidosis may also have a good effect.
  • the IL-18 receptor consists of an inducible component, interleukin-18 receptor 1 (IL-18R1), which binds mature IL-18 with low affinity and a constitutively expressed co-receptor, interleukin-18 receptor 2 (IL-18R2).
  • IL-18 binds to the ligand receptor IL-18R1, inducing the recruitment of IL-18R2 to form a high-affinity complex that signals through the toll/interleukin-1 receptor (TIR) domain.
  • TIR toll/interleukin-1 receptor
  • IL-18 activity can be inhibited by the bound extracellular interleukin-18 binding protein (IL-18BP), which binds soluble IL-18 with higher affinity than IL-18R1, thus preventing IL-18 from binding to the IL-18 receptor.
  • IL-18BP extracellular interleukin-18 binding protein
  • Clinical studies have shown that using IL-18BP to block the signaling pathway caused by IL-18 has shown good therapeutic effects in hemophagocytic lymphohistiocytosis and adult-onset Still's disease; at the same time, the drug showed good tolerability and safety, achieving the primary endpoint of the Phase II clinical study.
  • IL-18 inhibitors include IL-18R1 monoclonal antibodies and IL-18BP, and there are no reports of nanobodies targeting this target. Therefore, it is of certain significance to study and obtain new therapeutic IL-18/IL-18R1 blocking nanobodies, which may provide new clinical treatment ideas.
  • the object of the present invention is to provide a nanobody targeting interleukin-18 receptor 1, and the use of the nanobody in detection and disease treatment.
  • an anti-IL-18R1 nanobody is provided, wherein the nanobody is capable of specifically binding to IL-18R1, and the complementarity determining region (CDR) of the VHH chain in the nanobody is one or more selected from the following group:
  • the VHH chain of the anti-IL-18R1 nanobody further comprises a framework region (FR), and the framework region (FR) comprises human FR and camel FR.
  • the framework region (FR) is one or more selected from the following group:
  • FR1 shown in SEQ ID NO:25 FR2 shown in SEQ ID NO:20, FR3 shown in SEQ ID NO:21, and FR4 shown in SEQ ID NO:26;
  • amino acid sequence of the VHH chain of the anti-IL-18R1 nanoantibody is selected from the following group: SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO: 1, or a combination thereof.
  • the VHH chain of the anti-IL-18R1 nanoantibody has an amino acid sequence that has at least 75%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in any of SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1.
  • the anti-IL-18R1 nanobody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).
  • the anti-IL-18R1 nanobody includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.
  • the anti-IL-18R1 nanobody can recognize and bind to IL-18R1 of humans and cynomolgus monkeys.
  • the anti-IL-18R1 nanobody can effectively block the interaction between IL-18 and IL-18R1.
  • the anti-IL-18R1 nanobody has good blocking activity on the IL-18/IL-18R pathway, which is better than the blocking activity of interleukin 18 binding protein (IL-18BP) on the IL-18/IL-18R pathway.
  • IL-18BP interleukin 18 binding protein
  • the second aspect of the present invention provides an anti-IL-18R1 antibody, which comprises the VHH chain of the anti-IL-18R1 nanobody as described in the first aspect of the present invention.
  • the anti-IL-18R1 antibody comprises one or more sequences having SEQ ID NO: 4, The VHH chain of the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1.
  • the anti-IL-18R1 antibody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).
  • an anti-IL-18R1 nanobody Fc fusion protein is provided, wherein the structure of the fusion protein from N-terminus to C-terminus is as shown in Formula Ia or Ib:
  • A is one or more anti-IL-18R1 Nanobodies as described in the first aspect of the present invention.
  • B is the Fc fragment of IgG.
  • L is no or flexible joint.
  • the flexible linker is a connecting peptide.
  • the IgG Fc fragment includes the Fc fragment of human IgG.
  • the IgG Fc fragment is selected from the following group: Fc fragments of IgG1, IgG2, IgG3, IgG4, or a combination thereof.
  • the fourth aspect of the present invention provides a polynucleotide, which encodes a protein selected from the following group: the anti-IL-18R1 nanobody as described in the first aspect of the present invention, the anti-IL-18R1 antibody as described in the second aspect of the present invention, or the anti-IL-18R1 nanobody Fc fusion protein as described in the third aspect of the present invention.
  • the polynucleotide includes DNA or RNA.
  • the polynucleotide includes a sequence selected from the following group: a nucleotide sequence shown in SEQ ID NO:8, SEQ ID NO:7, SEQ ID NO:6 or SEQ ID NO:5.
  • the fifth aspect of the present invention provides an expression vector, wherein the expression vector contains the polynucleotide as described in the fourth aspect of the present invention.
  • the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof.
  • the expression vector comprises a viral vector, such as a lentivirus, an adenovirus, an AAV virus, a retrovirus, or a combination thereof.
  • a viral vector such as a lentivirus, an adenovirus, an AAV virus, a retrovirus, or a combination thereof.
  • a host cell contains the The expression vector of the first aspect of the present invention, or the polynucleotide of the fourth aspect of the present invention is integrated into its genome.
  • the host cell includes a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from the following group: Escherichia coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or a combination thereof.
  • the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof.
  • the host cell is Pichia pastoris.
  • the seventh aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:
  • a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.
  • the eighth aspect of the present invention provides a pharmaceutical composition, comprising:
  • the pharmaceutical composition further contains other drugs for treating autoimmune diseases or inflammatory diseases.
  • the pharmaceutical composition is used to prepare a drug for preventing and/or treating diseases or conditions related to the IL-18/IL-18R pathway.
  • the diseases or conditions include rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, inflammatory bowel disease, as well as hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), adult Still's disease, systemic juvenile idiopathic arthritis (SJIA), etc.
  • RA rheumatoid arthritis
  • HHLH hemophagocytic lymphohistiocytosis
  • MAS macrophage activation syndrome
  • SJIA systemic juvenile idiopathic arthritis
  • the ninth aspect of the present invention provides a recombinant protein, comprising:
  • the tag sequence includes (but is not limited to) Flag tag, HA tag and 6His tag.
  • the diseases or conditions include rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, inflammatory bowel disease, as well as hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), adult Still's disease, systemic juvenile idiopathic arthritis (SJIA), etc.
  • RA rheumatoid arthritis
  • HHLH hemophagocytic lymphohistiocytosis
  • MAS macrophage activation syndrome
  • SJIA systemic juvenile idiopathic arthritis
  • the reagent is a diagnostic reagent.
  • the reagent is used to detect IL-18R1 protein in a sample.
  • the IL-18R1 includes human IL-18R1 and cynomolgus monkey IL-18R1.
  • a multispecific antibody which comprises the anti-IL-18R1 nanobody as described in the first aspect of the present invention, or the anti-IL-18R1 antibody as described in the second aspect of the present invention.
  • the multispecific antibody further comprises a second antigen binding region targeting a target selected from the group consisting of IL-4R, IL-4R ⁇ , IL-5, IL-5R, IL-13, IL-13R, IL-11, IL-11R, or a combination thereof.
  • the second antigen binding region is a nanobody.
  • the multispecific antibody comprises one or more second antigen binding regions.
  • the multispecific antibody further comprises an antibody Fc segment.
  • the anti-IL-18R1 nanobody as described in the first aspect of the present invention or the anti-IL-18R1 antibody as described in the second aspect of the present invention, or the anti-IL-18R1 nanobody Fc fusion protein as described in the third aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention, for detecting IL-18R1 protein in a sample, or for treating and/or preventing diseases or conditions related to the IL-18/IL-18R pathway.
  • the detection includes flow cytometry detection, ELISA detection, and cell immunofluorescence detection.
  • the diseases or conditions include rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, inflammatory bowel disease, as well as hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), adult Still's disease, systemic juvenile idiopathic arthritis (SJIA), etc.
  • RA rheumatoid arthritis
  • HHLH hemophagocytic lymphohistiocytosis
  • MAS macrophage activation syndrome
  • SJIA systemic juvenile idiopathic arthritis
  • the use is diagnostic and/or non-diagnostic, and/or therapeutic and/or non-therapeutic.
  • the thirteenth aspect of the present invention provides a method for detecting IL-18R1 protein in a sample, the method comprising the steps of:
  • the method is a non-diagnostic and non-therapeutic method.
  • an IL-18R1 protein detection reagent comprising:
  • the conjugated portion of the immunoconjugate is a diagnostic isotope.
  • test-acceptable carrier is a non-toxic, inert aqueous carrier medium.
  • the detection reagent is one or more reagents selected from the following group: isotope tracers, contrast agents, flow cytometry detection reagents, cell immunofluorescence detection reagents, nanomagnetic particles and imaging agents.
  • the detection reagent is used for in vivo detection.
  • the dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet, buccal preparation, inhaler).
  • the fifteenth aspect of the present invention provides a kit for detecting IL-18R1 protein, wherein the kit contains the detection reagent as described in the fourteenth aspect of the present invention, and instructions.
  • the instructions state that the kit is used for non-invasively detecting the expression of IL-18R1 in a subject to be tested.
  • an IL-18 inhibitor which comprises the anti-IL-18R1 nanobody as described in the first aspect of the present invention, or the anti-IL-18R1 antibody as described in the second aspect of the present invention, or the anti-IL-18R1 nanobody Fc fusion protein as described in the third aspect of the present invention.
  • the IL-18 inhibitor blocks the interaction between IL-18 and IL-18R1. Blocking the IL-18/IL-18R pathway inhibits the functional activity of IL-18.
  • a method for preventing and/or treating diseases or disorders associated with the IL-18/IL-18R pathway comprising the steps of administering to a subject in need thereof an anti-IL-18R1 nanobody as described in the first aspect of the present invention, or an anti-IL-18R1 antibody as described in the second aspect of the present invention, or an anti-IL-18R1 nanobody Fc fusion protein as described in the third aspect of the present invention, or an immunoconjugate as described in the seventh aspect of the present invention, or a pharmaceutical composition as described in the eighth aspect of the present invention.
  • the diseases or conditions include rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, inflammatory bowel disease, as well as hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), adult Still's disease, systemic juvenile idiopathic arthritis (SJIA), etc.
  • RA rheumatoid arthritis
  • HHLH hemophagocytic lymphohistiocytosis
  • MAS macrophage activation syndrome
  • SJIA systemic juvenile idiopathic arthritis
  • the subject in need thereof is a human or a non-human mammal (eg, cynomolgus monkey).
  • a chimeric antigen receptor (CAR) is provided, the antigen binding domain of the chimeric antigen receptor comprising the anti-IL-18R1 nanobody as described in the first aspect of the present invention, or the anti-IL-18R1 antibody as described in the second aspect of the present invention.
  • an engineered cell expressing the chimeric antigen receptor is also provided.
  • the cells are immune cells, such as T cells and NK cells.
  • engineered cells for preparing a drug for preventing and/or treating a disease or condition associated with the IL-18/IL-18R pathway; or a method for preventing and/or treating a disease or condition associated with the IL-18/IL-18R pathway.
  • the diseases or conditions include rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, inflammatory bowel disease, as well as hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), adult Still's disease, systemic juvenile idiopathic arthritis (SJIA), etc.
  • RA rheumatoid arthritis
  • HHLH hemophagocytic lymphohistiocytosis
  • MAS macrophage activation syndrome
  • SJIA systemic juvenile idiopathic arthritis
  • Figure 1 shows the inhibitory effect of candidate IL-18R1 nanobodies on reporter gene cell signaling pathways.
  • Figure 2 shows the binding activity of humanized Nanobody HuNb7 to IL-18R1 (ELISA).
  • Figure 3 shows the binding activity of humanized Nanobody HuNb24 to IL-18R1 (ELISA).
  • Figure 4 shows the binding activity of humanized nanobody HuNb7 to human IL-18R1 stably transfected cell lines (FACS).
  • Figure 5 shows the binding activity (FACS) of humanized nanobody HuNb7 to cynomolgus monkey IL-18R1 stably transfected cell line.
  • FIG6 shows the binding activity (FACS) of humanized nanobody HuNb24 to human IL-18R1 stably transfected cell lines.
  • FIG. 7 shows the binding activity (FACS) of humanized nanobody HuNb24 to cynomolgus monkey IL-18R1 stably transfected cell lines.
  • the inventors unexpectedly discovered a class of IL-18R1 nano antibodies for the first time.
  • the experimental results show that the IL-18R1 nano antibodies of the present invention can specifically recognize the IL-18R1 of humans and cynomolgus monkeys, and have good specificity and binding activity; and the nano antibodies of the present invention have good IL-18/IL-18R pathway blocking activity, and the blocking activity is better than interleukin 18 binding protein (IL-18BP), therefore, it can be used to prepare effective antibody drugs for treating diseases related to the IL-18/IL-18R pathway.
  • IL-18BP interleukin 18 binding protein
  • Nanoantibodies of the present invention As used herein, the terms “Nanoantibodies of the present invention”, “anti-IL-18R1 Nanoantibodies of the present invention”, “IL-18R1 Nanoantibodies of the present invention”, “anti-IL-18R1 Nanoantibodies”, and “IL-18R1 Nanoantibodies” have the same meaning and are used interchangeably, all referring to Nanoantibodies that specifically recognize and bind to IL-18R1 (including human IL-18R1 and cynomolgus monkey IL-18R1).
  • antibody or "immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain.
  • VL variable region
  • Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
  • single domain antibody As used herein, the terms “single domain antibody”, “nanobody” and “single domain antibody (sdAb)” have the same meaning and are used interchangeably, referring to cloning the variable region of the heavy chain of an antibody to construct a nanobody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function, the VHH chain. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody consisting of only one heavy chain variable region.
  • CH1 light chain and heavy chain constant region 1
  • variable means that certain parts of the variable region of an antibody differ in sequence, which accounts for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in the light chain and heavy chain variable regions called the complementarity determining regions (CDRs). Or three segments in the hypervariable region. The more conservative part of the variable region is called the framework region (FR).
  • the variable region of the natural heavy chain and light chain each contains four FR regions, which are generally in a b-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial b-folded structure.
  • the CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)).
  • the constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments thereof of the present invention to form conjugates.
  • the present invention also includes cell surface markers or antigens combined with the anti-IL-18R1 antibodies or fragments thereof.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody comprises the above-mentioned heavy chain variable region and heavy chain constant region.
  • antibodies of the present invention are used interchangeably, and all refer to polypeptides that specifically bind to IL-18R1 protein, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.
  • the present invention also provides other proteins or fusion expression products having the antibodies of the present invention.
  • the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.
  • variable region This segment is divided into four framework regions (FR).
  • FR framework regions
  • the amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the ⁇ -folds formed by the FRs in between are close to each other in spatial structure.
  • the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody.
  • the amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.
  • variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.
  • the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention.
  • the polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a
  • the antibody of the present invention refers to a polypeptide having IL-18R1 binding activity and including the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and optimally 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • amino acids with similar or similar properties are substituted, the function of the protein is generally not changed.
  • adding one or several amino acids to the C-terminus and/or N-terminus generally does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the present invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof.
  • the invention also includes fragments of nanobodies of the invention.
  • the fragment has at least about 50 consecutive amino acids of an antibody of the invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.
  • “conservative variants of the antibodies of the present invention” refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.
  • the present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein.
  • the polynucleotide of the present invention can be in the form of DNA or RNA.
  • the DNA form includes cDNA, genomic DNA or artificially synthesized DNA.
  • the DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may include additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short.
  • a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • biomolecules nucleic acids, proteins, etc.
  • the biomolecules involved in the present invention include biomolecules in isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method, the steps used are well known in the art. Another method is to use MgCl 2. If necessary, transformation can also be carried out by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.
  • Therapeutic agents that can be combined or coupled with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • amino acid sequence of the VHH chain of the anti-IL-18R1 nanoantibody is selected from one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.
  • Nb24 amino acid sequence (SEQ ID NO:2)
  • An exemplary nucleotide sequence encoding the VHH chain of the above-mentioned IL-18R1 Nanobody is as follows:
  • Nb7 nucleotide sequence (SEQ ID NO:5)
  • Nb24 nucleotide sequence (SEQ ID NO:6)
  • HuNb7 nucleotide sequence SEQ ID NO:7
  • HuNb24 nucleotide sequence (SEQ ID NO:8)
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind to the IL-18R1 protein molecule and block the IL-18/IL-18R pathway, and thus can be used to treat diseases related to the IL-18/IL-18R pathway, including autoimmune or inflammatory diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, inflammatory bowel disease, etc., as well as rare diseases such as hemophagocytic lymphohistiocytosis (HLH) and adult Still's disease.
  • RA rheumatoid arthritis
  • HSH hemophagocytic lymphohistiocytosis
  • other therapeutic agents can also be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned nanoantibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptide of the present invention can also
  • a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 milligrams/kg body weight, preferably the dosage is about 10 micrograms/kg body weight to about 10 milligrams/kg body weight.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.
  • the nanobody carries a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label or a fluorescent label.
  • colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the nanobody against IL-18R1 is labeled with colloidal gold to obtain a colloidal gold labeled nanobody.
  • the present invention also relates to a method for detecting IL-18R1 protein.
  • the method generally comprises the following steps: obtaining a cell and/or tissue sample; dissolving the sample in a medium; and detecting the level of IL-18R1 protein in the dissolved sample.
  • the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage solution.
  • the present invention also provides a kit containing the antibody (or fragment thereof) or the detection plate of the present invention.
  • the kit further includes a container, instructions for use, a buffer, and the like.
  • the present invention also provides a detection kit for detecting IL-18R1 levels, which includes an antibody that recognizes IL-18R1 protein, a lysis medium for dissolving a sample, and universal reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc.
  • the detection kit can be an in vitro diagnostic device.
  • the nanobodies of the present invention have a wide range of biological and clinical application values, and their applications involve the diagnosis and treatment of diseases related to the IL-18/IL-18R pathway, basic medical research, biological research, and other fields.
  • a preferred application is for clinical diagnosis and targeted therapy of IL-18R1.
  • Example 1 Screening and identification of IL-18R1 nanobodies
  • the IL-18R1 extracellular segment gene (Uniprot: Q13478AA22-329) was cloned and synthesized into the pFUSE vector according to the human codon-optimized base sequence, and then the vector was transfected into HEK293F cells and purified by protein A affinity chromatography to obtain the human IL-18R1ECD-Fc fusion protein.
  • the highly purified protein was mixed with an immune adjuvant and immunized two Xinjiang Bactrian camels once a week.
  • phage display technology was used to screen IL-18R1-specific nanoantibodies (see Example 2 of patent CN110144009B for the screening method). After four rounds of "adsorption-washing-enrichment", more than 15 times of specific nanoantibody phage enrichment appeared in both libraries. 600 clones were randomly selected for ELISA detection, all positive clones were sequenced and analyzed, and all antibodies of different families (CDR3 differences) were expressed.
  • Example 2 Construction and expression of humanized IL-18R1 nanobody
  • Example 3 Detection of the inhibitory effect of IL-18R1 nanobody on cell signaling pathways
  • HEK293IL-18R reporter gene cells were used to detect the blocking effect of candidate antibodies on IL-18 and IL-18R expressed by cells.
  • the number of HEK293IL-18R reporter gene cells required for each sample was adjusted to 1 ⁇ 10 4 , and IL-18 factor with a working concentration of 40ng/mL, or gradient dilution of candidate antibodies or IL-18BP (4-fold gradient, 10 points) were added, and incubated at 37°C for 24 hours. Then, the luciferase reporter gene detection substrate was added, and the fluorescence intensity was detected on the machine after incubation in the dark for 5 minutes.
  • Example 4 ELISA detection of the binding activity of IL-18R1 nanobody
  • ELISA was used to detect the binding activity of candidate nanobodies.
  • the human IL-18R1 extracellular segment protein was coated on a 96-well ELISA plate at 37°C for 2 hours; after washing with PBST, BSA blocking solution was added and sealed at 37°C for 2 hours; after washing with PBST, gradient diluted nanobodies were added and incubated at 37°C for 1 hour; after washing with PBST, diluted biotinylated anti-his antibody was added and incubated, followed by diluted SA-HRP; after washing with PBST, color development solution and stop solution were added, and the absorbance was detected by an ELISA reader.
  • Example 5 FACS detection of the binding activity of IL-18R1 nanobody
  • Transfected cell line 293T-CynoIL-18R1 was dispensed into 96-well plates, 3E5 cells per well. After adding gradient diluted antibodies, incubate at room temperature for 30 minutes. Wash the cells twice with PBS, add diluted anti-HA antibody-APC and incubate at room temperature for 30 minutes. Wash the cells again with PBS and then detect them on the machine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明提供了IL-18R1纳米抗体及其应用。具体地，本发明提供了一种抗IL-18R1纳米抗体，还提供了编码所述纳米抗体的编码序列、相应的表达载体和能够表达该纳米抗体的宿主细胞。本发明的IL-18R1纳米抗体能够特异性识别人和食蟹猴的IL-18R1，具有良好的特异性和结合活性；并且，本发明的纳米抗体具有良好的IL-18/IL-18R通路阻断活性，且阻断活性优于白介素18结合蛋白(IL-18BP)，因此，可用于制备治疗与IL-18/IL-18R通路相关疾病的药物。

Description

抗白介素18受体的纳米抗体及其应用 技术领域
本发明涉及抗体药物领域，具体地，涉及一种靶向白介素18受体的纳米抗体及其应用。
背景技术
白细胞介素-18(IL-18)是一种由造血细胞和非造血细胞等多种细胞类型产生的强有力的促炎细胞因子，参与宿主防御感染，以及调节先天和获得性免疫反应。IL-18可以调节先天和适应性免疫和其调节异常可能会导致自身免疫或炎症性疾病。已有一些临床报道一些免疫相关疾病患者的血液中IL-18水平升高，其发病程度与IL-18水平正相关，如类风湿关节炎(RA)、系统性红斑狼疮、1型糖尿病、特应性皮炎、银屑病和炎症性肠病等。已有临床研究表明，通过抑制IL-18信号通路，可有效治疗嗜血细胞性淋巴组织细胞增生症(HLH)及成人still病等罕见病，也对慢性肺结节病可能有良好的疗效。
IL-18受体由可诱导成分白介素18受体1(IL-18R1)组成，该成分以低亲和力结合成熟的IL-18和组成型表达的共受体白介素18受体2(IL-18R2)。IL-18与配体受体IL-18R1结合，诱导IL-18R2募集形成高亲和力复合物，该复合物通过toll/interleukin-1受体(TIR)域发出信号。该信号结构域可激活促炎程序和NF-κB途径的MyD88衔接蛋白。IL-18的活性可以被结合的细胞外白介素18结合蛋白(IL-18BP)抑制，可溶性IL-18的亲和力高于IL-18R1，因此可阻止IL-18与IL-18受体结合。已有临床研究表明，利用IL-18BP阻断IL-18引起的信号通路，在嗜血细胞性淋巴组织细胞增生症和成人still病中表现出良好的疗效；同时该药物表现出良好的耐受性和安全性，达到临床II期研究主要终点。
现有的IL-18抑制剂包括IL-18R1单克隆抗体和IL-18BP，还未有靶向该靶点的纳米抗体报道。因此，研究获得新型治疗性IL-18/IL-18R1阻断型纳米抗体具有一定的意义，可能提供新的临床治疗思路。
发明内容
本发明的目的在于提供一种靶向白介素18受体1的纳米抗体，以及所述纳米抗体在检测和疾病治疗中的用途。
本发明的第一方面，提供了一种抗IL-18R1纳米抗体，所述纳米抗体能够特异性结合IL-18R1，所述纳米抗体中的VHH链的互补决定区(CDR)为选自下组的一种或多种：
(1)SEQ ID NO:12所示的CDR1、SEQ ID NO:13所示的CDR2和SEQ ID NO: 14所示的CDR3；和
(2)SEQ ID NO:9所示的CDR1、SEQ ID NO:10所示的CDR2和SEQ ID NO:11所示的CDR3。
在另一优选例中，所述抗IL-18R1纳米抗体的VHH链还包括框架区(FR)，所述框架区(FR)包括人源FR和骆驼源FR。
在另一优选例中，所述的框架区(FR)为选自下组的一种或多种：
(1)SEQ ID NO:25所示的FR1、SEQ ID NO:20所示的FR2、SEQ ID NO:21所示的FR3和SEQ ID NO:26所示的FR4；
(2)SEQ ID NO:19所示的FR1、SEQ ID NO:20所示的FR2、SEQ ID NO:21所示的FR3和SEQ ID NO:22所示的FR4；
(3)SEQ ID NO:23所示的FR1、SEQ ID NO:16所示的FR2、SEQ ID NO:17所示的FR3，和SEQ ID NO:24所示的FR4；和
(4)SEQ ID NO:15所示的FR1、SEQ ID NO:16所示的FR2、SEQ ID NO:17所示的FR3，和SEQ ID NO:18所示的FR4。
在另一优选例中，所述抗IL-18R1纳米抗体的VHH链的氨基酸序列选自下组：SEQ ID NO:4、SEQ ID NO:3、SEQ ID NO:2、SEQ ID NO:1、或其组合。
在另一优选例中，所述抗IL-18R1纳米抗体的VHH链具有与SEQ ID NO:4、SEQ ID NO:3、SEQ ID NO:2或SEQ ID NO:1中任一所示的氨基酸序列具有至少75％，优选至少80％、85％、90％、91％、92％、93％、94％、95％、95％、96％、97％、98％、99％或100％的序列同一性的氨基酸序列。
在另一优选例中，所述抗IL-18R1纳米抗体包括单体、二价体(二价抗体)、四价体(四价抗体)、和/或多价体(多价抗体)。
在另一优选例中，所述的抗IL-18R1纳米抗体包括人源化抗体、骆驼源抗体、嵌合抗体。
在另一优选例中，所述的抗IL-18R1纳米抗体能够识别并结合人和食蟹猴的IL-18R1。
在另一优选例中，所述的抗IL-18R1纳米抗体能够有效阻断IL-18与IL-18R1的相互作用。
在另一优选例中，所述的抗IL-18R1纳米抗体对IL-18/IL-18R通路具有良好的阻断活性，优于白介素18结合蛋白(IL-18BP)对IL-18/IL-18R通路的阻断活性。
本发明的第二方面，提供了一种抗IL-18R1抗体，所述抗体包含如本发明第一方面所述的抗IL-18R1纳米抗体的VHH链。
在另一优选例中，所述抗IL-18R1抗体包括一条或多条具有如SEQ ID NO:4、 SEQ ID NO:3、SEQ ID NO:2或SEQ ID NO:1所示的氨基酸序列的VHH链。
在另一优选例中，所述抗IL-18R1抗体包括单体、二价体(二价抗体)、四价体(四价抗体)、和/或多价体(多价抗体)。
本发明的第三方面，提供了一种抗IL-18R1纳米抗体Fc融合蛋白，所述融合蛋白从N端到C端的结构如式Ia或Ib所示：
A-L-B(Ia)；
B-L-A(Ib)；
其中，
“-”为肽键；
A为一个或多个如本发明第一方面所述的抗IL-18R1纳米抗体；
B为IgG的Fc片段；和
L为无或柔性接头。
在另一优选例中，所述的柔性接头为连接肽。
在另一优选例中，所述IgG的Fc片段包括人的IgG的Fc片段。
在另一优选例中，所述IgG的Fc片段选自下组：IgG1、IgG2、IgG3、IgG4的Fc片段、或其组合。
在另一优选例中，所述连接肽选自以下序列：(GaSb)x—(GmSn)y，其中a，b，m，n，x，y＝0或1或2或3或4或5或6或7或8或9或10(较佳地，a＝4而b＝1，m＝3而n＝1)。
本发明的第四方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的蛋白：如本发明第一方面所述的抗IL-18R1纳米抗体、如本发明第二方面所述的抗IL-18R1抗体或如本发明第三方面所述的抗IL-18R1纳米抗体Fc融合蛋白。
在另一优选例中，所述的多核苷酸包括DNA或RNA。
在另一优选例中，所述的多核苷酸包括选自下组的序列：如SEQ ID NO:8、SEQ ID NO:7、SEQ ID NO:6或SEQ ID NO:5所示的核苷酸序列。
本发明的第五方面，提供了一种表达载体，所述表达载体含有如本发明第四方面所述的多核苷酸。
在另一优选例中，所述的表达载体选自下组：DNA、RNA、病毒载体、质粒、转座子、其他基因转移系统、或其组合。
优选地，所述表达载体包括病毒载体，如慢病毒、腺病毒、AAV病毒、逆转录病毒、或其组合。
本发明的第六方面，提供了一种宿主细胞，所述宿主细胞含有如本发明第五 方面所述的表达载体，或其基因组中整合有如本发明第四方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、哺乳动物细胞、噬菌体、或其组合。
在另一优选例中，所述原核细胞选自下组：大肠杆菌、枯草杆菌、乳酸菌、链霉菌、奇异变形菌、或其组合。
在另一优选例中，所述真核细胞选自下组：毕赤酵母、酿酒酵母、裂殖酵母、木霉、或其组合。
在另一优选例中，所述的宿主细胞为毕赤酵母。
本发明的第七方面，提供了一种免疫偶联物，所述免疫偶联物包含：
(a)如本发明第一方面所述的抗IL-18R1纳米抗体、或如本发明第二方面所述的抗IL-18R1抗体、或如本发明第三方面所述的抗IL-18R1纳米抗体Fc融合蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
本发明的第八方面，提供了一种药物组合物，所述药物组合物包含：
(i)如本发明第一方面所述的抗IL-18R1纳米抗体、或如本发明第二方面所述的抗IL-18R1抗体、或如本发明第三方面所述的抗IL-18R1纳米抗体Fc融合蛋白、或如本发明第七方面所述的免疫偶联物；和
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物中还含有治疗自身免疫病或炎症性疾病的其他药物。
在另一优选例中，所述的药物组合物用于制备预防和/或治疗与IL-18/IL-18R通路相关的疾病或病症的药物。
在另一优选例中，所述疾病或病症包括类风湿关节炎(RA)、系统性红斑狼疮、1型糖尿病、特应性皮炎、银屑病、炎症性肠病，以及嗜血细胞性淋巴组织细胞增生症(HLH)、巨噬细胞活化综合征(MAS)、成人still病、系统性青少年特发性关节炎(SJIA)等。
本发明的第九方面，提供了一种重组蛋白，所述重组蛋白包含：
(Z1)如本发明第一方面所述的抗IL-18R1纳米抗体、或如本发明第二方面所述的抗IL-18R1抗体、或如本发明第三方面所述的抗IL-18R1纳米抗体Fc融合蛋白；以及
(Z2)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括(但不限于)Flag标签、HA标签和6His标签。
本发明的第十方面，提供了如本发明第一方面所述的抗IL-18R1纳米抗体、或如本发明第二方面所述的抗IL-18R1抗体、或如本发明第三方面所述的抗IL-18R1纳米抗体Fc融合蛋白、或如本发明第七方面所述的免疫偶联物的用途，用于：(a)制备预防和/或治疗与IL-18/IL-18R通路相关的疾病或病症的药物；(b)制备检测IL-18R1的试剂、检测板或试剂盒。
在另一优选例中，所述疾病或病症包括类风湿关节炎(RA)、系统性红斑狼疮、1型糖尿病、特应性皮炎、银屑病、炎症性肠病，以及嗜血细胞性淋巴组织细胞增生症(HLH)、巨噬细胞活化综合征(MAS)、成人still病、系统性青少年特发性关节炎(SJIA)等。
在另一优选例中，所示试剂为诊断试剂。
在另一优选例中，所述试剂用于检测样品中的IL-18R1蛋白。
在另一优选例中，所述IL-18R1包括人IL-18R1和食蟹猴IL-18R1。
本发明的第十一方面，提供了一种多特异性抗体，其包含如本发明第一方面所述的抗IL-18R1纳米抗体、或如本发明第二方面所述的抗IL-18R1抗体。
在另一优选例中，所述多特异性抗体还包括靶向选自下组的靶点的第二抗原结合区：IL-4R、IL-4Rα、IL-5、IL-5R、IL-13、IL-13R、IL-11、IL-11R、或其组合。
在另一优选例中，所述的第二抗原结合区为纳米抗体。
在另一优选例中，所述多特异性抗体包括一个或多个第二抗原结合区。
在另一优选例中，所述多特异性抗体还包含抗体的Fc段。
本发明的第十二方面，提供了如本发明第一方面所述的抗IL-18R1纳米抗体、或如本发明第二方面所述的抗IL-18R1抗体、或如本发明第三方面所述的抗IL-18R1纳米抗体Fc融合蛋白、或如本发明第七方面所述的免疫偶联物的用途，用于检测样品中的IL-18R1蛋白，或用于治疗和/或预防与IL-18/IL-18R通路相关的疾病或病症。
在另一优选例中，所述检测包括流式细胞检测、ELISA检测、细胞免疫荧光检测。
在另一优选例中，所述疾病或病症包括类风湿关节炎(RA)、系统性红斑狼疮、1型糖尿病、特应性皮炎、银屑病、炎症性肠病，以及嗜血细胞性淋巴组织细胞增生症(HLH)、巨噬细胞活化综合征(MAS)、成人still病、系统性青少年特发性关节炎(SJIA)等。
在另一优选例中，所述用途为诊断性和/或非诊断性的，和/或治疗性和/或非治疗性的。
本发明的第十三方面，提供了一种检测样品中IL-18R1蛋白的方法，所述方法包括步骤：
(1)将样品与如本发明第一方面所述的抗IL-18R1纳米抗体、或如本发明第二方面所述的抗IL-18R1抗体、或如本发明第三方面所述的抗IL-18R1纳米抗体Fc融合蛋白、或如本发明第七方面所述的免疫偶联物接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在IL-18R1蛋白。
在另一优选例中，所述方法为非诊断和非治疗性的方法。
本发明的第十四方面，提供了一种IL-18R1蛋白检测试剂，所述的检测试剂包含：
(C1)如本发明第一方面所述的抗IL-18R1纳米抗体、或如本发明第二方面所述的抗IL-18R1抗体、或如本发明第三方面所述的抗IL-18R1纳米抗体Fc融合蛋白、或如本发明第七方面所述的免疫偶联物；以及
(C2)检测学上可接受的载体。
在另一优选例中，所述的免疫偶联物的偶联部分为诊断用同位素。
在另一优选例中，所述的检测学上可接受的载体为无毒的、惰性的水性载体介质。
在另一优选例中，所述的检测试剂为选自下组的一种或多种试剂：同位素示踪剂、造影剂、流式检测试剂、细胞免疫荧光检测试剂、纳米磁粒和显像剂。
在另一优选例中，所述的检测试剂用于体内检测。
在另一优选例中，所述的检测试剂的剂型为液态或粉状(如水剂，针剂，冻干粉，片剂，含服剂，吸雾剂)。
本发明的第十五方面，提供了一种检测IL-18R1蛋白的试剂盒，所述试剂盒含有如本发明第十四方面所述的检测试剂，以及说明书。
在另一优选例中，所述的说明书记载，所述的试剂盒用于非侵入性地检测待测对象的IL-18R1表达。
本发明的第十六方面，提供了一种IL-18抑制剂，其包含如本发明第一方面所述的抗IL-18R1纳米抗体、或如本发明第二方面所述的抗IL-18R1抗体、或如本发明第三方面所述的抗IL-18R1纳米抗体Fc融合蛋白。
在另一优选例中，所述IL-18抑制剂阻断IL-18与IL-18R1的相互作用，从 而阻断IL-18/IL-18R通路，抑制IL-18的功能活性。
本发明的第十七方面，提供了一种防和/或治疗与IL-18/IL-18R通路相关的疾病或病症的方法，包括步骤：向有需要的受试者施用如本发明第一方面所述的抗IL-18R1纳米抗体、或如本发明第二方面所述的抗IL-18R1抗体、或如本发明第三方面所述的抗IL-18R1纳米抗体Fc融合蛋白、或如本发明第七方面所述的免疫偶联物、或如本发明第八方面所述的药物组合物。
在另一优选例中，所述疾病或病症包括类风湿关节炎(RA)、系统性红斑狼疮、1型糖尿病、特应性皮炎、银屑病、炎症性肠病，以及嗜血细胞性淋巴组织细胞增生症(HLH)、巨噬细胞活化综合征(MAS)、成人still病、系统性青少年特发性关节炎(SJIA)等。
在另一优选例中，所述有需要的受试者为人类或非人类哺乳动物(例如食蟹猴)。
本发明的第十八方面，提供了一种嵌合抗原受体(CAR)，所述嵌合抗原受体的抗原结合结构域包含如本发明第一方面所述的抗IL-18R1纳米抗体、或如本发明第二方面所述的抗IL-18R1抗体。
进一步地，还提供了表达所述嵌合抗原受体的工程化的细胞。
在另一优选例中，所述细胞为免疫细胞，例如T细胞、NK细胞。
进一步地，还提供了所述工程化的细胞的用途，用于制备预防和/或治疗与IL-18/IL-18R通路相关的疾病或病症的药物；或用于预防和/或治疗与IL-18/IL-18R通路相关的疾病或病症的方法。
在另一优选例中，所述疾病或病症包括类风湿关节炎(RA)、系统性红斑狼疮、1型糖尿病、特应性皮炎、银屑病、炎症性肠病，以及嗜血细胞性淋巴组织细胞增生症(HLH)、巨噬细胞活化综合征(MAS)、成人still病、系统性青少年特发性关节炎(SJIA)等。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了候选IL-18R1纳米抗体对报告基因细胞信号通路的抑制作用。
图2显示了人源化纳米抗体HuNb7与IL-18R1的结合活性(ELISA)。
图3显示了人源化纳米抗体HuNb24与IL-18R1的结合活性(ELISA)。
图4显示了人源化纳米抗体HuNb7与人IL-18R1稳转细胞株的结合活性 (FACS)。
图5显示了人源化纳米抗体HuNb7与食蟹猴IL-18R1稳转细胞株的结合活性(FACS)。
图6显示了人源化纳米抗体HuNb24与人IL-18R1稳转细胞株的结合活性(FACS)。
图7显示了人源化纳米抗体HuNb24与食蟹猴IL-18R1稳转细胞株的结合活性(FACS)。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次意外地发现一类IL-18R1纳米抗体，实验结果表明，本发明的IL-18R1纳米抗体能够特异性识别人和食蟹猴的IL-18R1，具有良好的特异性和结合活性；并且，本发明的纳米抗体具有良好的IL-18/IL-18R通路阻断活性，且阻断活性优于白介素18结合蛋白(IL-18BP)，因此，可用于制备治疗与IL-18/IL-18R通路相关疾病的有效抗体药物。在此基础上，完成了本发明。
术语
如本文所用，术语“本发明的纳米抗体”、“本发明的抗IL-18R1纳米抗体”、“本发明的IL-18R1纳米抗体”、“抗IL-18R1纳米抗体”、“IL-18R1纳米抗体”具有相同的含义，可互换使用，均指特异性识别和结合于IL-18R1(包括人IL-18R1和食蟹猴IL-18R1)的纳米抗体。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体”、“纳米抗体(nanobody)”、“重链抗体(single domain antibody,sdAb)”具有相同的含义并可互换使用，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体，它是具有完整功能的最小的抗原结合片段，VHH链。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR) 或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈b-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分b折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的抗IL-18R1抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合IL-18R1蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有IL-18R1结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含纳米抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明纳米抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A

本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))等。
本发明的IL-18R1纳米抗体
本发明中，所述抗IL-18R1纳米抗体的VHH链的氨基酸序列选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4中的一种或多种。
Nb7氨基酸序列(SEQ ID NO:1)
QVQLQESGGGSVHAGETLRLSCTYSGFTFDDSDMDWYRQAPGNECELVAVIDSDGSTYYADSVKGRFAISIDNAKNTVYLQMNSLKPEDTAVYYCAAEWGPA HGDYGCVGQGTQVTVSS(下划线部分依次为CDR1、CDR2和CDR3)
Nb24氨基酸序列(SEQ ID NO:2)
QVQLQESGGGSVQAGETLRLSCTASRFSFDDFDMGWFRQAPGNECELVSTISSDGSTYYADSVKGRFTISQDNAKKTVFLQMNSLKPEDTAVYYCAAEWTGN WDELQCWGQGTQVTVSS(下划线部分依次为CDR1、CDR2和CDR3)
HuNb7氨基酸序列(SEQ ID NO:3)
EVQLLESGGGLVHPGETLRLSCTYSGFTFDDSDMDWYRQAPGNECELVAVIDSDGSTYYADSVKGRFAISIDNAKNTVYLQMNSLKPEDTAVYYCAAEWGPA HGDYGCVGQGTLVTVSS(下划线部分依次为CDR1、CDR2和CDR3)
HuNb24氨基酸序列(SEQ ID NO:4)
EVQLLESGGGLVQPGETLRLSCTASRFSFDDFDMGWFRQAPGNECELVSTISSDGSTYYADSVKGRFTISQDNAKKTVFLQMNSLKPEDTAVYYCAAEWTGNW DELQCWGQGTLVTVSS(下划线部分依次为CDR1、CDR2和CDR3)
编码上述IL-18R1纳米抗体的VHH链的示例性核苷酸序列如下：
Nb7核苷酸序列(SEQ ID NO:5)
Nb24核苷酸序列(SEQ ID NO:6)
HuNb7核苷酸序列(SEQ ID NO:7)
HuNb24核苷酸序列(SEQ ID NO:8)
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合IL-18R1蛋白分子，阻断IL-18/IL-18R通路，因而可用于治疗与IL-18/IL-18R通路相关的疾病，包括自身免疫或炎症性疾病，例如类风湿关节炎(RA)、系统性红斑狼疮、1型糖尿病、特应性皮炎、银屑病、炎症性肠病等，以及嗜血细胞性淋巴组织细胞增生症(HLH)及成人still病等罕见病。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的纳米抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
标记的纳米抗体
在本发明的一个实施方式中，所述纳米抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的实施方式中，IL-18R1的纳米抗体用胶体金标记，得到胶体金标记的纳米抗体。
检测方法
本发明还涉及检测IL-18R1蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中IL-18R1蛋白的水平。
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测IL-18R1水平的检测试剂盒，该试剂盒包括识别IL-18R1蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
如上所述，本发明的纳米抗体有广泛生物应用价值和临床应用价值，其应用涉及到与IL-18/IL-18R通路相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对IL-18R1的临床诊断和靶向治疗。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。
实施例1：IL-18R1纳米抗体的筛选鉴定
将IL-18R1胞外段基因(Uniprot：Q13478AA22-329)按照人密码子优化的碱基序列克隆合成至pFUSE载体，然后将该载体转染HEK293F细胞后经protein A亲和层析纯化出人IL-18R1ECD-Fc融合蛋白。将高纯度的蛋白与免疫佐剂混合后分别免疫2只新疆双峰驼，每周一次。七次免疫后取骆驼外周血，分离RNA并转录扩增出VHH基因片段，随后将其克隆至pMECS载体，电转化至TG1感受态细胞中建立高质量噬菌体展示纳米抗体文库。经检测，两个文库的库容均达到1×109CFU以上，片段插入率均在90％以上。
然后利用噬菌体展示技术筛选IL-18R1特异性纳米抗体(筛选方法参见专利CN110144009B实施例2)。经四轮“吸附-洗涤-富集”的过程，2个文库均出现15倍以上的特异性纳米抗体噬菌体富集。从中随机挑选600个克隆进行ELISA检测，将所有阳性克隆进行测序分析，将所有不同家族的(CDR3差异)抗体进行表达。
实施例2：人源化IL-18R1纳米抗体的构建和表达
不同家族的抗体序列经人源化(人源化方法参见专利CN2018101517526中实施例4的方法)后，将其按照毕赤酵母密码子优化的碱基序列合成至pPICZaA载体上。将插入抗体基因的重组质粒线性化后电转至毕赤酵母X33感受态细胞，随后从抗性平板上挑取单克隆，经甲醇诱导表达，检测培养液中抗体的表达量。人源化IL-18R1纳米抗体表达量检测结果如表1所示。
表1人源化IL-18R1纳米抗体在毕赤酵母中的表达量统计结果
实施例3：IL-18R1纳米抗体对细胞信号通路的抑制作用检测
利用HEK293IL-18R报告基因细胞检测候选抗体对IL-18与细胞表达的IL-18R的阻断作用。每个样本所需的HEK293IL-18R报告基因细细胞数调整为1×104个，分别加入工作浓度为40ng/mL的IL-18因子，或梯度稀释的候选抗体或IL-18BP(4倍梯度，10个点)，37℃孵育24小时。然后加入荧光素酶报告基因检测底物，避光孵育5min后上机检测荧光强度。
结果表明：候选抗体对IL-18激活的HEK293IL-18R报告基因细胞信号通路具有良好的阻断活性，如图1所示，其中HuNb7和HuNb24是较好的IL-18/IL-18R通路抑制剂。
HuNb7和HuNb24及其人源化前的骆驼源纳米抗体Nb7和Nb24的序列如下表2所示：
表2
实施例4：ELISA检测IL-18R1纳米抗体的结合活性
利用ELISA检测候选纳米抗体的结合活性。将人IL-18R1胞外段蛋白包被96孔酶标板于37℃包被2小时；PBST洗涤后加入BSA封闭液37℃封2小时；PBST洗涤后再加入梯度稀释的纳米抗体在37℃孵育1小时；PBST洗涤后加入稀释的生物素化的anti-his抗体孵育，随后再加入稀释的SA-HRP；PBST洗涤后加入显色液及终止液，酶标仪检测吸光值。
结果如图2和图3所示，两株人源化抗体HuNb7和HuNb24均能有效结合人和食蟹猴IL-18R1。
实施例5：FACS检测IL-18R1纳米抗体的结合活性
利用流式细胞术检测候选抗体与细胞表面人和食蟹猴IL-18R1的结合活性。将高表达人IL-18R1的稳转细胞株293T-hIL-18R1和高表达食蟹猴IL-18R1的稳 转细胞株293T-CynoIL-18R1分别分装至96孔板，每孔3E5个。加入梯度稀释的抗体后室温孵育30分钟。PBS洗涤细胞两次，加入稀释的anti-HA antibody-APC室温孵育30分钟。再次用PBS洗涤细胞后上机检测。
结果表明：如图4-7所示，候选抗体HuNb7和HuNb24均与细胞表面人IL-18R1和食蟹猴IL-18R1具有良好的结合活性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种抗IL-18R1纳米抗体，其特征在于，所述纳米抗体能够特异性结合IL-18R1，所述纳米抗体中的VHH链的互补决定区(CDR)为选自下组的一种或多种：
    (1)SEQ ID NO:12所示的CDR1、SEQ ID NO:13所示的CDR2和SEQ ID NO:14所示的CDR3和
    (2)SEQ ID NO:9所示的CDR1、SEQ ID NO:10所示的CDR2和SEQ ID NO:11所示的CDR3。
  2. 一种抗IL-18R1抗体，其特征在于，所述抗体包含如权利要求1所述的抗IL-18R1纳米抗体的VHH链。
  3. 一种抗IL-18R1纳米抗体Fc融合蛋白，所述融合蛋白从N端到C端的结构如式Ia或Ib所示：
    A-L-B(Ia)；
    B-L-A(Ib)；
    其中，
    “-”为肽键；
    A为一个或多个如权利要求1所述的抗IL-18R1纳米抗体；
    B为IgG的Fc片段；和
    L为无或柔性接头。
  4. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的蛋白：如权利要求1所述的抗IL-18R1纳米抗体、如权利要求2所述的抗IL-18R1抗体或如权利要求3所述的抗IL-18R1纳米抗体Fc融合蛋白。
  5. 一种表达载体，其特征在于，所述表达载体含有如权利要求4所述的多核苷酸。
  6. 一种宿主细胞，所述宿主细胞含有如权利要求5所述的表达载体，或其基因组中整合有如权利要求4所述的多核苷酸。
  7. 一种免疫偶联物，其特征在于，所述免疫偶联物包含：
    (a)如权利要求1所述的抗IL-18R1纳米抗体、或如权利要求2所述的抗IL-18R1抗体、或如权利要求3所述的抗IL-18R1纳米抗体Fc融合蛋白；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
  8. 一种药物组合物，其特征在于，所述药物组合物包含：
    (i)如权利要求1所述的抗IL-18R1纳米抗体、或如权利要求2所述的抗IL-18R1抗体、或如权利要求3所述的抗IL-18R1纳米抗体Fc融合蛋白、或如权利要求7所述的免疫偶联物；和
    (ii)药学上可接受的载体。
  9. 如权利要求1所述的抗IL-18R1纳米抗体、或如权利要求2所述的抗IL-18R1抗体、或如权利要求3所述的抗IL-18R1纳米抗体Fc融合蛋白、或如权利要求7所述的免疫偶联物的用途，用于：(a)制备预防和/或治疗与IL-18/IL-18R通路相关的疾病或病症的药物；(b)制备检测IL-18R1的试剂、检测板或试剂盒。
  10. 一种多特异性抗体，其包含如权利要求1所述的抗IL-18R1纳米抗体、或如权利要求2所述的抗IL-18R1抗体。
  11. 如权利要求1所述的抗IL-18R1纳米抗体、或如权利要求2所述的抗IL-18R1抗体、或如权利要求3所述的抗IL-18R1纳米抗体Fc融合蛋白、或如权利要求7所述的免疫偶联物的用途，用于检测样品中的IL-18R1蛋白，或用于治疗和/或预防与IL-18/IL-18R通路相关的疾病或病症。
  12. 一种重组蛋白，所述重组蛋白包含：
    (Z1)如权利要求1所述的抗IL-18R1纳米抗体、或如权利要求2所述的抗IL-18R1抗体、或如权利要求3所述的抗IL-18R1纳米抗体Fc融合蛋白；以及
    (Z2)任选的协助表达和/或纯化的标签序列。
  13. 一种IL-18R1蛋白检测试剂，所述的检测试剂包含：
    (C1)如权利要求1所述的抗IL-18R1纳米抗体、或如权利要求2所述的抗IL-18R1抗体、或如权利要求3所述的抗IL-18R1纳米抗体Fc融合蛋白、或如权利要求7所述的免疫偶联物；以及
    (C2)检测学上可接受的载体。
  14. 一种IL-18抑制剂，其包含如权利要求1所述的抗IL-18R1纳米抗体、或如权利要求2所述的抗IL-18R1抗体、或如权利要求3所述的抗IL-18R1纳米抗体Fc融合蛋白。
  15. 一种嵌合抗原受体，其特征在于，所述嵌合抗原受体的抗原结合结构域包含如权利要求1所述的抗IL-18R1纳米抗体、或如权利要求2所述的抗IL-18R1抗体。
PCT/CN2023/097235 2023-05-30 2023-05-30 抗白介素18受体的纳米抗体及其应用 WO2024243819A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/097235 WO2024243819A1 (zh) 2023-05-30 2023-05-30 抗白介素18受体的纳米抗体及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/097235 WO2024243819A1 (zh) 2023-05-30 2023-05-30 抗白介素18受体的纳米抗体及其应用

Publications (1)

Publication Number Publication Date
WO2024243819A1 true WO2024243819A1 (zh) 2024-12-05

Family

ID=93656433

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/097235 WO2024243819A1 (zh) 2023-05-30 2023-05-30 抗白介素18受体的纳米抗体及其应用

Country Status (1)

Country Link
WO (1) WO2024243819A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2004235595A1 (en) * 2003-04-30 2004-11-11 Japan Science And Technology Agency Human antihuman interleukin-18 antibody, fragment thereof and method of using the same
CN102206277A (zh) * 2000-02-10 2011-10-05 雅培制药有限公司 结合人白介素-18的抗体及制备和使用方法
WO2012025536A1 (en) * 2010-08-25 2012-03-01 F. Hoffmann-La Roche Ag Antibodies against il-18r1 and uses thereof
CN102659944A (zh) * 2003-11-12 2012-09-12 雅培制药有限公司 Il-18结合蛋白

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102206277A (zh) * 2000-02-10 2011-10-05 雅培制药有限公司 结合人白介素-18的抗体及制备和使用方法
AU2004235595A1 (en) * 2003-04-30 2004-11-11 Japan Science And Technology Agency Human antihuman interleukin-18 antibody, fragment thereof and method of using the same
CN1780911A (zh) * 2003-04-30 2006-05-31 独立行政法人科学技术振兴机构 人抗人白介素－18抗体及其片断和它们的利用方法
CN102659944A (zh) * 2003-11-12 2012-09-12 雅培制药有限公司 Il-18结合蛋白
WO2012025536A1 (en) * 2010-08-25 2012-03-01 F. Hoffmann-La Roche Ag Antibodies against il-18r1 and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIPINSKI BRITTA, UNMUTH LAURA, ARRAS PAUL, BECKER STEFAN, BAUER CHRISTINA, TOLEIKIS LARS, KRAH SIMON, DOERNER ACHIM, YANAKIEVA DES: "Generation and engineering of potent single domain antibody-based bispecific IL-18 mimetics resistant to IL-18BP decoy receptor inhibition", MABS, LANDES BIOSCIENCE, US, vol. 15, no. 1, 31 December 2023 (2023-12-31), US , XP093122582, ISSN: 1942-0862, DOI: 10.1080/19420862.2023.2236265 *
TAN YOUWEN, WU JIANCHENG: "Effects of the serum with anti-immunohepatofibrotic drugs of IL-18 or IL-18 antibody on apoptosis and proliferration of mice hepatic stellate cells", JIANGSU YIYAO = JIANGSU MEDICAL JOURNAL, vol. 37, no. 17, 30 September 2011 (2011-09-30), pages 1991 - 1994,1984, XP009559747, ISSN: 0253-3685, DOI: 10.19460/j.cnki.0253-3685.2011.17.004 *

Similar Documents

Publication Publication Date Title
US11466085B2 (en) Anti-PD-L1 nanobody, coding sequence and use thereof
WO2018233574A1 (zh) 一种抗pd-l1人源化纳米抗体及其应用
WO2018233575A1 (zh) 阻断型cd47纳米抗体及其用途
CN114853888B (zh) 抗tslp纳米抗体及其应用
JP7656962B2 (ja) 抗ｉｌ５ナノボディおよびその適用
CN116003598A (zh) 靶向人gprc5d的重组人源化单克隆抗体及其应用
CN118221811B (zh) 靶向prame多肽的单域抗体及其用途
US12012456B2 (en) Anti-interleukin-4 receptor (IL-4R) single-domain antibody, encoding polynucleotide and methods of making and using the antibody for IL-4R detection and disease treatment
EP4389769A1 (en) Anti-ptk7 single-domain antibody and application thereof
WO2023125842A1 (zh) 一种新型upar单域抗体的开发
WO2023134767A1 (zh) 一种靶向IL-18Rβ的抗体及其应用
EP4365202A1 (en) Anti-trop2 single-domain antibody and use thereof
WO2023125975A1 (zh) 一种新型靶向人flt3的嵌合抗原受体修饰的t细胞的构建及应用
WO2024243819A1 (zh) 抗白介素18受体的纳米抗体及其应用
CN116554322A (zh) 一种靶向IL-18Rβ的抗体及其应用
WO2024099310A1 (zh) 抗il-13长效纳米抗体序列及其应用
WO2024216505A1 (zh) 抗Notch2纳米抗体及其应用
CN118745224A (zh) Tslp鲨鱼单域抗体及其应用
WO2025031098A1 (zh) 一种分拣蛋白1特异性的纳米抗体、含有其的重组aav及应用
CN117186226A (zh) 抗cd38纳米抗体及其应用
CN115873113A (zh) 靶向CaSR的纳米抗体及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23938813

Country of ref document: EP

Kind code of ref document: A1