CN116554322A - Antibody targeting IL-18 Rbeta and application thereof - Google Patents

Antibody targeting IL-18 Rbeta and application thereof Download PDF

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Publication number
CN116554322A
CN116554322A CN202210108473.8A CN202210108473A CN116554322A CN 116554322 A CN116554322 A CN 116554322A CN 202210108473 A CN202210108473 A CN 202210108473A CN 116554322 A CN116554322 A CN 116554322A
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antibody
seq
antigen
binding fragment
ser
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杜勇
陈永锋
刘淑素
张玉华
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Hejing Pharmaceutical Technology Shanghai Co ltd
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Hejing Pharmaceutical Technology Shanghai Co ltd
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Priority to CN202210108473.8A priority Critical patent/CN116554322A/en
Priority to CN202380011140.6A priority patent/CN117177999A/en
Priority to PCT/CN2023/072444 priority patent/WO2023134767A1/en
Publication of CN116554322A publication Critical patent/CN116554322A/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The invention provides an antibody or an antigen binding fragment thereof aiming at IL-18 Rbeta, a preparation method and application thereof. In particular, the invention also provides nucleic acid molecules encoding the antibodies or antigen binding fragments thereof, corresponding expression vectors and host cells capable of expressing the antibodies or antigen binding fragments thereof, and methods for producing and uses of the antibodies or antigen binding fragments thereof. The antibody or antigen binding fragment thereof aiming at IL-18R beta can specifically bind to human IL-18R beta, has much higher affinity than a wild type antibody, and has good IL-18/IL-18R blocking activity. The antibody or the antigen binding fragment thereof provides a new technical means for preventing/diagnosing/treating diseases related to the abnormal expression of IL-18 receptor or related to the high activity of IL-18 signaling pathway.

Description

Antibody targeting IL-18 Rbeta and application thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an antibody specifically targeting IL-18 Rbeta, an antigen binding fragment thereof, a preparation method and application thereof.
Background
Interleukin 18 (IL-18), also known as an interferon-gamma inducer, regulates many types of immune cell-mediated inflammatory responses. IL-18 not only activates Thl cells and NK cells to promote IFN-gamma release, but also regulates activation of Th2, th17 and macrophages, which is an important inflammation promoting factor.
IL-18 has been found by many studies to be closely associated with various autoimmune diseases such as hemophagocytic lymphoproliferative disorder (Hemophagocytic lymphohistiocytosis, HLH), macrophage activation syndrome, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (Systemic juvenile idiopathic arthritis, sJIA), systemic red Bai Langchuang (Systemic lupus erythematosus, SLE), and inflammatory enteritis.
IL-18 receptors are heterodimeric transmembrane proteins that consist of an IL-18R alpha (IL-18R alpha) subunit that binds to a ligand and an IL-18R beta (IL-18R beta) subunit that is critical for functional signaling.
IL-18 acts upon the cell surface IL-18 receptor after hydrolysis to the active form by the protease Caspas-1, activating downstream pro-inflammatory signaling pathways. IL-18 binds with lower affinity to IL-18Rα to form a dimer, but does not transmit a signal, and only this dimer binds to IL-18Rβ to form a high affinity receptor complex that transmits a pro-inflammatory signal downstream. Blocking the formation of ternary complexes of IL-18 and its receptor, and effectively inhibiting the inflammatory signaling pathway downstream of IL-18, is a promising approach to the current treatment of autoimmune and inflammatory diseases.
IL-18Rα is widely expressed and can also bind to the inflammation inhibitor IL-37, which is complex in the regulation of inflammation. IL-18Rβ is specifically expressed in immune cells and is a receptor necessary for IL-18 signaling, and thus targeting IL-18Rβ antibodies specifically and effectively inhibits IL-18 signaling. Targeting IL-18 itself is affected in many ways and the mechanism of action is complex due to the high affinity decoy receptor IL-18BP naturally occurring in vivo, as well as the presence of membrane-bound forms of IL-18. In combination, the targeted IL-18Rβ antibodies have advantages in specificity, efficacy, and safety over other components of the IL-18 signaling pathway.
Therefore, there is an urgent need in the art to develop an antibody specifically targeting IL-18rβ for the treatment of autoimmune diseases, inflammatory diseases, and the like.
Disclosure of Invention
The present invention aims to provide a novel therapeutic means for autoimmune diseases, inflammatory diseases, etc.
It is a further object of the invention to provide an antibody directed against IL-18Rβ and uses thereof.
In a first aspect of the invention, there is provided an antibody or antigen-binding fragment thereof directed against IL-18rβ.
In another preferred embodiment, the antibody or antigen binding fragment thereof is capable of specifically binding to IL-18rβ.
In another preferred embodiment, the antibody or antigen binding fragment thereof comprises:
(a) The heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, wherein the amino acid sequences of the CDRH1, CDRH2 and CDRH3 are respectively shown as SEQ ID NO. 2, 3 and 4, or are respectively shown as SEQ ID NO. 10, 3 and 11, or are respectively shown as SEQ ID NO. 2, 3 and 11; and
(b) The amino acid sequences of the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 are shown as SEQ ID NOS 6, 7 and 8, respectively, or as SEQ ID NOS 13, 7 and 8, respectively, or as SEQ ID NOS 16, 7 and 8, respectively, or as SEQ ID NOS 22, 7 and 8, respectively.
In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence that is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-4) amino acid and that retains the ability to specifically bind to IL-18Rβ.
In another preferred embodiment, the antibody or antigen binding fragment thereof has 6 CDRs (CDRH 1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL 3) of an a034, a035, a037, a038, or a039 antibody in table 1.
In another preferred embodiment, the derivative sequence having at least one amino acid added, deleted, modified and/or substituted and capable of retaining the ability to specifically bind to IL-18Rβ is an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or sequence identity.
In another preferred embodiment, the antibody or antigen binding fragment thereof may comprise a beneficial mutation selected from the group consisting of the wild-type heavy chain variable region shown in SEQ ID NO. 18 and/or the wild-type light chain variable region shown in SEQ ID NO. 20: F27L, F27I, F V, S99A, S31F, A34D, A34E, or a combination thereof.
In another preferred embodiment, the antibody or antigen binding fragment thereof may comprise a beneficial mutation based on the wild-type heavy chain variable region shown in SEQ ID NO. 18 selected from the group consisting of: F27L, F27I, F V, S99A, or a combination thereof.
In another preferred embodiment, the antibody or antigen binding fragment thereof may comprise a beneficial mutation based on the wild-type light chain variable region shown in SEQ ID NO. 20 selected from the group consisting of: S31F, A34D, A E, or a combination thereof.
In another preferred embodiment, the antibody or antigen binding fragment thereof has a beneficial mutation selected from the group consisting of the wild-type heavy chain variable region set forth in SEQ ID NO. 18 and the wild-type light chain variable region set forth in SEQ ID NO. 20:
S31F、A34E、F27L；
S31F、A34E、F27I、S99A；
A34D、F27I、S99A；
S31F、A34D、F27L；
A34E、F27I；
A34D、F27I；
A34E、F27L；
S31F、A34D、F27V、S99A；
a34E, F27L, S a; or (b)
S31F、A34E、F27L。
In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR 4.
In another preferred embodiment, the antibody or antigen binding fragment thereof further comprises a framework region FR.
In another preferred embodiment, the antibody or antigen binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO. 1, 9 or 14 and has a light chain variable region as set forth in SEQ ID NO. 5, 12, 15 or 17.
In another preferred embodiment, the antibody or antigen binding fragment thereof may comprise a monomer, a bivalent antibody, and/or a multivalent antibody.
In another preferred embodiment, the bivalent antibody may also be a bispecific antibody.
In another preferred embodiment, the multivalent antibody may also be a multispecific antibody.
In another preferred embodiment, the antigen binding fragment is selected from scFv, fab, fab ', F (ab') 2 Fv fragment, disulfide linked Fv (dsFv).
In another preferred embodiment, the heavy chain variable region and the light chain variable region of the antibody or antigen binding fragment thereof have amino acid sequences as shown in SEQ ID NO. 9 and SEQ ID NO. 5, or as shown in SEQ ID NO. 1 and SEQ ID NO. 5, or as shown in SEQ ID NO. 9 and SEQ ID NO. 12, or as shown in SEQ ID NO. 14 and SEQ ID NO. 17, or as shown in SEQ ID NO. 14 and SEQ ID NO. 15, respectively.
In another preferred embodiment, the heavy chain constant region of the antibody or antigen binding fragment thereof is selected from the group consisting of the heavy chain constant region of human IgG1, igG2, igG3 or IgG 4.
In a second aspect of the present invention, there is provided a recombinant protein having:
(i) An antibody or antigen-binding fragment thereof according to the first aspect of the invention; and
(ii) Optionally a tag sequence to assist expression and/or purification.
In another preferred embodiment, the tag sequence comprises an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.
In another preferred embodiment, the recombinant protein specifically binds IL-18Rβ.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In a third aspect of the invention there is provided a nucleotide molecule, said polynucleotide encoding a protein selected from the group consisting of: the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the recombinant protein according to the second aspect of the invention.
In another preferred embodiment, the nucleic acid of the invention may be RNA, DNA or cDNA.
In a fourth aspect of the invention there is provided an expression vector comprising a nucleotide molecule according to the third aspect of the invention.
In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
In another preferred embodiment, the expression vector is selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJM, pHCMV, pLBS.CAG, pHR, pLV, etc.
In another preferred embodiment, the expression vector further comprises a gene selected from the group consisting of: promoters, transcription enhancing elements WPRE, long terminal repeat LTR, and the like.
In a fifth aspect of the invention there is provided a host cell comprising an expression vector according to the fourth aspect of the invention, or having incorporated into its genome a nucleotide molecule according to the third aspect of the invention.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of: coli, yeast cells, mammalian cells.
In a sixth aspect of the invention there is provided a chimeric antigen receptor CAR, the antigen binding region scFv fragment of the CAR being a binding region that specifically binds to IL-18rβ, and the heavy chain variable region of the scFv comprising:
The heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, wherein the amino acid sequences of the CDRH1, CDRH2 and CDRH3 are respectively shown as SEQ ID NO. 2, 3 and 4, or are respectively shown as SEQ ID NO. 10, 3 and 11, or are respectively shown as SEQ ID NO. 2, 3 and 11; and/or
The light chain variable region of the scFv comprises:
the amino acid sequences of the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 are shown as SEQ ID NOS 6, 7 and 8, respectively, or as SEQ ID NOS 13, 7 and 8, respectively, or as SEQ ID NOS 16, 7 and 8, respectively, or as SEQ ID NOS 22, 7 and 8, respectively.
In another preferred embodiment, the CAR further comprises a signal peptide.
In another preferred embodiment, the CAR further comprises an additional exogenous protein.
In another preferred embodiment, the CAR has a structure according to formula Ia:
L-scFv-H-TM-C-CD3ζ (Ia)
in the method, in the process of the invention,
l is a none or signal peptide sequence;
scFv are domains that specifically bind IL-18rβ;
h is the no or hinge region;
TM is a transmembrane domain;
c is a costimulatory signaling domain;
cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ (including wild-type, or mutant/modification thereof);
the "-" is a connecting peptide or peptide bond.
In another preferred embodiment, the L is selected from the group consisting of signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or a mutant/modification thereof, or a combination thereof.
In another preferred embodiment, the scFv targets IL-18Rβ.
In another preferred embodiment, the scFv is an IL-18Rβ antibody or antigen binding fragment thereof.
In another preferred embodiment, the H is selected from the group consisting of the hinge regions of: CD8, CD28, CD137, igG, or a combination thereof.
In another preferred embodiment, the TM is selected from the transmembrane region of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutations/modifications thereof, or combinations thereof.
In another preferred embodiment, the C is selected from the group consisting of the costimulatory domains of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD-1, dap10, LIGHT, NKG2C, B-H3, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), NKG2D, GITR, OX L, 2B4, TLR, or mutations/modifications thereof, or combinations thereof.
In a seventh aspect of the invention, there is provided an engineered immune cell expressing an exogenous CAR according to the sixth aspect of the invention.
In another preferred embodiment, the engineered immune cell is selected from the group consisting of:
(i) Chimeric antigen receptor alpha beta T cells (CAR-T cells);
(ii) Chimeric antigen receptor γδ T cells (CAR-T cells);
(iii) Chimeric antigen receptor NKT cells (CAR-NKT cells);
(iv) Chimeric antigen receptor NK cells (CAR-NK cells).
In another preferred embodiment, the engineered immune cells comprise autologous or allogeneic αβ T cells, γδ T cells, NKT cells, NK cells, or a combination thereof.
In another preferred embodiment, the engineered immune cell is a CAR-T cell.
In an eighth aspect of the invention, there is provided a method of producing an antibody or antigen-binding fragment thereof directed against IL-18rβ comprising the steps of:
(a) Culturing a host cell according to the fifth aspect of the invention under suitable conditions, thereby obtaining a culture comprising an antibody to IL-18rβ or an antigen binding fragment thereof;
(b) Isolating and/or recovering the antibody or antigen binding fragment thereof directed against IL-18rβ from the culture; and
(c) Optionally, purifying and/or modifying the antibody or antigen binding fragment thereof against IL-18rβ obtained in step (b).
In a ninth aspect of the invention, there is provided an immunoconjugate comprising:
(a) An antibody moiety comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention; and
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, gold nanoparticle/nanorod, nanomagnetic particle, viral coat protein, or VLP, or a combination thereof.
In another preferred embodiment, said moiety (a) is coupled to said coupling moiety by a chemical bond or a linker.
In another preferred embodiment, the radionuclide comprises:
(i) A diagnostic isotope selected from the group consisting of: tc-99m, ga-68, F-18, I-123, I-125, I-131, in-111, ga-67, cu-64, zr-89, C-11, lu-177, re-188, or a combination thereof; and/or
(ii) A therapeutic isotope selected from the group consisting of: lu-177, Y-90, ac-225, as-211, bi-212, bi-213, cs-137, cr-51, co-60, dy-165, er-169, fm-255, au-198, ho-166, I-125, I-131, ir-192, fe-59, pb-212, mo-99, pd-103, P-32, K-42, re-186, re-188, sm-153, ra223, ru-106, na24, sr89, tb-149, th-227, xe-133, yb-169, yb-177, or combinations thereof.
In another preferred embodiment, the coupling moiety is a drug or a toxin.
In another preferred embodiment, the agent is a drug targeted to treat an IL-18 related disorder.
In another preferred embodiment, the IL-18 related disorder comprises a disorder in which IL-18 is highly expressed, a disorder associated with aberrant expression of IL-18 receptors or a disorder associated with high activity of IL-18 signaling pathways.
In another preferred embodiment, the IL-18 related disorder is an autoimmune disorder or an inflammatory disorder.
In another preferred embodiment, the IL-18 high expression disorder is selected from the group consisting of: tumors, leukemia, diabetic nephropathy, alzheimer's disease, parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis, psoriatic arthritis, crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, grave's disease, hashimoto's thyroiditis, addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphocytosis, macrophage activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyoderma, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory Bowel Disease (IBD), adult steven-ston's, chronic Obstructive Pulmonary Disease (COPD), or a combination thereof.
In another preferred embodiment, the tumor is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.
In another preferred embodiment, the drug is a cytotoxic drug.
In another preferred embodiment, the cytotoxic agent is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs including, for example, auristatins (Auristatins), camptothecins (Camptothecins), duocarmycin/duocarmycin (Duocarmycins), etoposides (Etoposides), maytansinoids (Maytansines) and Maytansinoids (Maytansinoids) (e.g., DM1 and DM 4), taxanes (Taxanes), benzodiazepines (Benzodiazepines), or benzodiazepine-containing drugs (Benzodiazepine containing drugs) (e.g., pyrrolo [1,4] Benzodiazepines (PBDs), indoline Benzodiazepines (indomethacins) and oxazolidobenzodiazepines (oxazodiazepines)), vinca alkaloids (vilos), or combinations thereof.
In another preferred embodiment, the toxin is selected from the group consisting of: auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), aureomycin, mestaneol, ricin a-chain, combretastatin, docamicin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide (Tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthrax, diketo, actinomycin, diphtheria toxin, pseudomonas Exotoxin (PE) A, PE, abrin a chain, a-sarcina, gelonin, mitogellin, restrictocin (retstricin), phenol, enomycin, curcin (curcin), crotonin, calicheamicin, saporin (Sapaonaria officinalis), a glucocorticoid, or a combination thereof.
In another preferred embodiment, the coupling moiety is a detectable label.
In another preferred embodiment, the coupling moiety is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)) or any form of nanoparticle.
In another preferred embodiment, the immunoconjugate comprises: multivalent (e.g. bivalent) antibodies or antigen binding fragments thereof according to the first aspect of the invention.
In another preferred embodiment, the multivalent means that the same or different antibodies or antigen binding fragments thereof according to the first aspect of the invention comprise a plurality of repeats in the amino acid sequence of the immunoconjugate.
In a tenth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) An active ingredient selected from the group consisting of: an antibody or antigen binding fragment thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an engineered immune cell according to the seventh aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier, diluent or excipient.
In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injection and freeze-dried preparation.
In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the antibody or antigen binding fragment thereof according to the first aspect of the invention, or the recombinant protein according to the second aspect of the invention, or the engineered immune cell according to the seventh aspect of the invention, or the immunoconjugate according to the ninth aspect of the invention, or a combination thereof, and 0.01 to 99.99% of a pharmaceutically acceptable carrier, said percentages being mass percentages of the pharmaceutical composition.
In another preferred embodiment, the concentration of the engineered immune cells in the active ingredient is 1X 10 3 -1×10 8 Individual cells/mL, preferably 1X 10 4 -1×10 7 Individual cells/mL.
In an eleventh aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: the antibody or antigen binding fragment thereof according to the first aspect of the invention, or the recombinant protein according to the second aspect of the invention, or the engineered immune cell according to the seventh aspect of the invention, or the immunoconjugate according to the ninth aspect of the invention, or a combination thereof, the active ingredients being used for the preparation of:
(a) Medicaments for preventing and/or treating IL-18 related diseases;
(b) Reagents for detecting IL-18 related disorders.
In another preferred embodiment, the reagent is a diagnostic reagent, preferably a test strip or a test plate.
In another preferred embodiment, the diagnostic reagent is for: detecting IL-18Rβ protein or a fragment thereof in the sample.
In another preferred embodiment, the IL-18 related disorder comprises a disorder in which IL-18 is highly expressed, a disorder associated with aberrant expression of IL-18 receptors or a disorder associated with high activity of IL-18 signaling pathways.
In another preferred embodiment, the IL-18 related disorder is an autoimmune disorder or an inflammatory disorder.
In another preferred embodiment, the IL-18-expressing disorder is selected from the group consisting of: tumors, leukemia, diabetic nephropathy, alzheimer's disease, parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis, psoriatic arthritis, crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, grave's disease, hashimoto's thyroiditis, addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphocytosis, macrophage activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyoderma, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory Bowel Disease (IBD), adult steven-ston's, chronic Obstructive Pulmonary Disease (COPD), or a combination thereof.
In another preferred embodiment, the tumor is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.
In a twelfth aspect of the invention, there is provided a method for detecting IL-18rβ protein or a fragment thereof in a sample in vitro, the method comprising the steps of:
(1) Contacting the sample in vitro with an antibody or antigen binding fragment thereof according to the first aspect of the invention;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of a corresponding target of the IL-18Rβ protein or fragment thereof in the sample.
In another preferred embodiment, the detection comprises diagnostic or non-diagnostic.
In a thirteenth aspect of the present invention, there is provided a kit comprising:
(1) A first container comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an engineered immune cell according to the seventh aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention, or a pharmaceutical composition according to the tenth aspect of the invention, or a combination thereof; and/or
(2) A second container containing a second antibody against the contents of the first container;
or alternatively, the process may be performed,
the kit comprises a detection plate, wherein the detection plate comprises: a substrate (support) and a test strip comprising an antibody or antigen binding fragment thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an engineered immune cell according to the seventh aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention, or a pharmaceutical composition according to the tenth aspect of the invention, or a combination thereof.
In another preferred embodiment, the kit further comprises a description, and the kit is used for non-invasively detecting the expression of IL-18Rβ in the subject according to the description.
In another preferred embodiment, the kit is used for detection of IL-18 related diseases.
In another preferred embodiment, the IL-18 related disorder comprises a disorder of high IL-18 expression, including a disorder associated with aberrant expression of IL-18 receptors or with high activity of IL-18 signaling pathways.
In another preferred embodiment, the IL-18 high expression disorder is selected from the group consisting of: tumors, leukemia, diabetic nephropathy, alzheimer's disease, parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis, psoriatic arthritis, crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, grave's disease, hashimoto's thyroiditis, addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphocytosis, macrophage activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyoderma, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory Bowel Disease (IBD), adult steven-ston's, chronic Obstructive Pulmonary Disease (COPD), or a combination thereof.
In another preferred embodiment, the tumor is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.
In a fourteenth aspect of the invention, there is provided a method of preventing and/or treating an IL-18-associated disease, the method comprising: administering to a subject in need thereof an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, or a recombinant protein as described in the second aspect of the invention, or an engineered immune cell as described in the seventh aspect of the invention, or an immunoconjugate as described in the ninth aspect of the invention, or a pharmaceutical composition as described in the tenth aspect of the invention, or a combination thereof.
In another preferred embodiment, the subject comprises a mammal, such as a human.
In another preferred embodiment, the IL-18 related disorder comprises a disorder in which IL-18 is highly expressed, a disorder associated with aberrant expression of IL-18 receptors or a disorder associated with high activity of IL-18 signaling pathways.
In another preferred embodiment, the IL-18 related disorder is an autoimmune disorder or an inflammatory disorder.
In another preferred embodiment, the IL-18 high expression disorder is selected from the group consisting of: tumors, leukemia, diabetic nephropathy, alzheimer's disease, parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis, psoriatic arthritis, crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, grave's disease, hashimoto's thyroiditis, addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemopoietic lymphocytosis, macrophage activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyoderma, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory Bowel Disease (IBD), adult steven-ston's, chronic Obstructive Pulmonary Disease (COPD), or a combination thereof.
In another preferred embodiment, the tumor is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.
In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from the subject (autologous cells).
In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from a healthy individual (allogeneic cells).
In another preferred embodiment, the methods described can be used in combination with other therapeutic methods.
In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy, and the like.
In a fifteenth aspect of the present invention, there is provided a diagnostic method for an IL-18-associated disease comprising the steps of:
(i) Obtaining a sample from a subject, contacting said sample with an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an engineered immune cell according to the seventh aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention, or a pharmaceutical composition according to the tenth aspect of the invention; and
(ii) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates that the subject is a definitive patient for an IL-18 related disorder.
In another preferred embodiment, the sample is a blood sample or a pharyngeal swab sample, or a sample in another tissue organ.
In another preferred embodiment, the IL-18 related disorder comprises a disorder in which IL-18 is highly expressed, a disorder associated with aberrant expression of IL-18 receptors or a disorder associated with high activity of IL-18 signaling pathways.
In another preferred embodiment, the IL-18 related disorder is an autoimmune disorder or an inflammatory disorder.
In another preferred embodiment, the IL-18 high expression disorder is selected from the group consisting of: tumors, leukemia, diabetic nephropathy, alzheimer's disease, parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis, psoriatic arthritis, crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, grave's disease, hashimoto's thyroiditis, addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphocytosis, macrophage activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyoderma, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory Bowel Disease (IBD), adult steven-ston's, chronic Obstructive Pulmonary Disease (COPD), or a combination thereof.
In another preferred embodiment, the tumor is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows sequence information for IL-18RβWT antibodies.
FIG. 2 shows the concentration-response curve of anti-IL-18 Rβ antibodies against IFN- γ inhibition in KG-1 cells.
FIG. 3 shows the concentration-response curve of anti-IL-18 Rβ antibodies against IFN- γ inhibition in human PBMC cells.
Detailed Description
The present inventors have conducted extensive and intensive studies and, as a result, have developed, for the first time, a high affinity antibody targeting IL-18rβ and antigen binding fragments thereof. Specifically, the present invention constructs a precise mutation library using a WT antibody of IL-18Rβ as a starting material for improving affinity, and introduces saturation mutations into all 73 residues of the six CDR regions of the WT antibody. After further screening, selected mutants were ranked by dissociation rate constants determined by surface plasmon resonance, and combinatorial libraries were constructed with random combinations of beneficial mutations, resulting in antibodies of the invention and antigen binding fragments thereof with increased binding affinity. The antibody and antigen binding fragment thereof for targeting IL-18 Rbeta, which are developed by the invention, can be used as a novel therapeutic means for targeting treatment of diseases related to abnormal expression of IL-18 receptors or related to high activity of IL-18 signaling pathways.
Terminology
As used herein, the terms "antibody of the invention", "antibody of the invention against IL-18rβ", "antibody of IL-18rβ", "IL-18rβ antibody" have the same meaning, and are used interchangeably, to refer to antibodies that specifically recognize and bind to IL-18rβ proteins (including human IL-18rβ proteins).
Preferably, the antibody numbering and corresponding sequence numbering of the invention is shown in table 1 below.
TABLE 1
Note that: the values in the table represent the sequence numbers, i.e. "1" for "SEQ ID NO:1", and the sequence numbers for VH, CDRH1, CDRH2, CDRH3, VL, CDRL1, CDRL2, CDRL3 shown in the table are the amino acid sequence numbers thereof.
The term "antibody" herein is intended to include full length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. Full length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The light chain constant region is composed of one domain CL. VH and VL regions can also be divided into hypervariable regions called Complementarity Determining Regions (CDRs) which are separated by more conserved Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the traditional complement system (C1 q).
The term "antigen-binding fragment" (or antigen-binding portion) of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., IL-18rβ protein). It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of full length antibodies. Examples of binding fragments contained in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH 1; (ii) F (ab') 2 A fragment comprising a bivalent fragment of two Fab fragments disulfide-bridged at the hinge region; (iii) Is composed of VH and CH1Fd fragment of (C); (iv) Fv fragments consisting of single arm VL and VH of the antibody; (v) a dAb fragment consisting of VH; (vi) an isolated Complementarity Determining Region (CDR); and (vii) nanobodies, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, by recombinant methods, via a synthetic linker that makes both single protein chains, in which the VL and VH regions pair to form monovalent molecules) (known as single chain Fc (scFv)). These single chain antibodies are also intended to be included in the term meaning. These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as the whole antibody.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming a connecting loop, which in some cases may form part of a b-sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
Those skilled in the art can define the amino acid sequence boundaries of CDRs using any of a variety of known numbering schemes, including those set forth in the following documents: kabat et al, supra ("Kabat" numbering scheme); al-Lazikani et Al, 1997, J.mol.biol.,. 273:927-948 ("Chothia" numbering scheme); lefranc et al, dev. Comp. Immunol.,2003,27:55-77 ("IMGT" numbering scheme).
Immunoconjugates and fusion expression products include, as known to those of skill in the art: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules with antibodies or fragments thereof of the present invention. The invention also includes cell surface markers or antigens that bind to the antibodies or fragments thereof directed against the novel coronaviruses.
As used herein, the term "heavy chain variable region" is used interchangeably with "VH".
As used herein, the term "light chain variable region" is used interchangeably with "VL".
As used herein, the term "variable region" is used interchangeably with "complementarity determining region (Complementarity determining region, CDR)".
In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDRH1, CDRH2, and CDRH3.
In a preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.
In a preferred embodiment of the invention, the light chain variable region of the antibody comprises three complementarity determining regions CDRL1, CDRL2, and CDRL3.
In a preferred embodiment of the invention, the light chain of the antibody comprises the light chain variable region and the light chain constant region described above.
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably to refer to a polypeptide that specifically binds to an IL-18rβ protein, such as a protein or polypeptide having a heavy chain variable region. They may or may not contain an initiating methionine.
The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.
In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the variable region of the heavy chain, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.
The variable regions of the heavy chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to antigens. Thus, the invention includes those molecules having antibody heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.
The antibody of the present invention refers to a polypeptide having IL-18Rβ protein binding activity and comprising the above CDR regions. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
The invention also provides other polypeptides, such as fusion proteins comprising an antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table 2.
TABLE 2
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.
The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, 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 a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl 2 The process is carried out using procedures well known in the art. Another approach is to use MgCl2. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, DNA transfection may be selected from the followingThe method comprises the following steps: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, 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 coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.
Therapeutic agents that may be conjugated or coupled to an antibody of the invention include, but are not limited to: 1. a radionuclide; 2. a biotoxin; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and the like.
IL-18, IL-18R and IL-18 Rbeta
Interleukin-18 (IL 18, also known as an interferon-gamma inducer) is a protein that is encoded by the IL-18 gene in humans. The protein encoded by the gene is a pro-inflammatory cytokine. Many cell types, both hematopoietic and non-hematopoietic, have the potential to produce IL-18.
The IL-18 receptor consists of an inducible component IL-18Rα (which binds mature IL-18 with low affinity) and a constitutively expressed co-receptor IL-18Rβ. IL-18 binds to the ligand receptor IL-18Rα, inducing IL-18Rβ recruitment to form a high affinity complex that signals through the toll/interlukin-1 receptor (TIR) domain. The signal domain activates pro-inflammatory processes and MyD88 adaptor proteins of the NF-. Kappa.B pathway. IL-18Rβ is specifically expressed in immune cells and is a receptor necessary for IL-18 signaling, and thus targeting IL-18Rβ antibodies specifically and effectively inhibits IL-18 signaling. Effective inhibition of the inflammatory signaling pathway downstream of IL-18 is a promising approach to the current treatment of autoimmune and inflammatory diseases.
Pharmaceutical composition
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intraperitoneal, intravenous, or topical administration.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Antibodies to IL-18 Rbeta
In the present invention, the antibody against IL-18Rβ includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).
In a preferred embodiment of the invention, the antibody against IL-18Rβ comprises one or more heavy chains having a heavy chain variable region as shown in SEQ ID NO. 1, 9 or 14, and a light chain having a light chain variable region as shown in SEQ ID NO. 5, 12, 15 or 17.
Labeled antibodies
In a preferred embodiment of the invention, the antibody is provided with a detectable label. More preferably, the marker is selected from the group consisting of: isotopes, colloidal gold labels, colored labels, or fluorescent labels.
Colloidal gold labelling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the invention, the antibodies against IL-18Rβ protein are labeled with colloidal gold to provide colloidal gold-labeled antibodies.
The antibodies of the invention directed against IL-18Rβ are capable of binding to IL-18Rβ protein efficiently.
Detection method
The invention also relates to methods of detecting IL-18Rβ proteins or fragments thereof. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving a sample in a medium; detecting the level of IL-18rβ protein in the solubilized sample.
In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.
Kit for detecting a substance in a sample
The invention also provides a kit comprising an antibody (or fragment thereof) or assay plate of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, and the like.
The invention also provides a detection kit for detecting the IL-18 Rbeta protein level, which comprises an antibody for recognizing the IL-18 Rbeta protein, a lysis medium for dissolving a sample, and general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates and the like. The detection kit may be an in vitro diagnostic device.
Application of
As described above, the antibody of the present invention has a wide range of biological and clinical applications, and its application relates to the treatment of diseases associated with high expression of IL-18, and has a plurality of fields such as diagnosis, basic medical research, biological research, etc. of high expression of IL-18. One preferred application is for clinical prophylaxis and treatment against IL-18Rβ proteins.
The invention also provides a method of stimulating an immune response mediated by T cells targeted to a mammalian tumor cell population or tissue, comprising the steps of: administering the CAR-T cells of the invention to a mammal.
In one embodiment, the invention includes a class of cell therapies in which autologous T cells (or heterologous donors) from a patient are isolated, activated and genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. This approach results in a very low probability of graft versus host response, and antigen is recognized by T cells in a non-MHC restricted manner. Furthermore, a CAR-T can treat all cancers that express this antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to sustained control of tumors.
In one embodiment, the CAR-T cells of the invention can undergo stable in vivo expansion and can last from months to years. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-T cells can induce a specific immune response to highly expressing tumor cells of the antigen recognized by the CAR antigen binding domain. For example, the CAR-T cells of the invention elicit a specific immune response against tumor cells that are highly expressed by IL-18rβ.
Treatable cancers include tumors that are not vascularized or have not been substantially vascularized, as well as vascularized tumors. Types of cancers treated with the CARs of the invention include, but are not limited to: gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, etc.
In general, cells activated and expanded as described herein are useful in the treatment and prevention of diseases such as tumors. Accordingly, the invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell of the invention.
The CAR-T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the invention may comprise a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease, or may be determined by clinical trials.
When referring to an "immunologically effective amount", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). Pharmaceutical compositions comprising T cells described herein may be administered at 10 4 To 10 9 A dose of individual cells/kg body weight, preferably 10 5 To 10 7 A dose of individual cells/kg body weight (including all whole values within the range) is administered. T cell compositions may also be administered multiple times at these doses. Cells can be administered by using injection techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng. J. Of Med.319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.
Administration of the subject compositions may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by intravenous injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.
In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously with, or after) any number of relevant therapeutic modalities, including, but not limited to, treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or ertapelizumab therapy for psoriasis patients or other therapy for PML patients. In a further embodiment, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressives such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously or after) with bone marrow transplantation, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives injection of expanded immune cells of the invention after transplantation. In an additional embodiment, the expanded cells are administered pre-operatively or post-operatively.
The dose of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio administered to humans may be carried out according to accepted practices in the art. Typically, 1X 10 will be administered per treatment or per course of treatment 5 Up to 1X 10 10 The modified T cells of the invention are administered to a patient, for example, by intravenous infusion.
The main advantages of the invention include:
1. the antibody or antigen binding fragment thereof of the IL-18 Rbeta can specifically bind to the IL-18 Rbeta, has high affinity, and improves the affinity by more than ten times and up to 22 times compared with the affinity of WT.
2. The antibody or antigen binding fragment thereof of the IL-18 Rbeta has good IL-18/IL-18R blocking activity and can effectively inhibit the downstream inflammatory signal path of IL-18.
3. The antibodies to IL-18Rβ or antigen binding fragments thereof of the invention have advantages in specificity, efficacy and safety over other components of the IL-18 signaling pathway.
4. The antibody or antigen binding fragment thereof of the IL-18 Rbeta can effectively block the release of IFN-gamma in KG-1 cells stimulated by IL-18, and the activity efficacy reaches 20 times of that of WT.
5. The antibody or antigen binding fragment thereof of the IL-18 Rbeta can effectively block the IFN-gamma release of human peripheral blood mononuclear cells stimulated by IL-18, and the activity is obviously higher than that of WT.
6. The antibody or antigen binding fragment thereof of the IL-18 Rbeta of the invention contributes to the prevention/diagnosis/treatment of diseases related to the abnormal expression of IL-18 receptors or to the high activity of IL-18 signaling pathways, and provides novel methods and technical means.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Amino acid sequence
SEQ ID NO. 1A 035 heavy chain variable region
EVQLVESGGGLVQPGGSLRLSCAASGINLYYSSMHWVRQAPGKGLEWVASIYSSNGRTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARASFSHGYGWYGLDYWGQGTLVTVSS
SEQ ID NO. 2A 035, A039, A038 heavy chain complementarity determining region CDRH1
GINLYYSSMH
SEQ ID NO. 3A 035, A037, A034, A039, A038, C056-WT heavy chain complementarity determining region CDRH2
SIYSSNGRTYYADSVKG
SEQ ID NO. 4A 035 heavy chain complementarity determining region CDRH3
ASFSHGYGWYGLDY
SEQ ID NO. 5A 035, A034 light chain variable region
DIQMTQSPSSLSASVGDRVTITCRASQSVSFAVEWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIK
SEQ ID NO. 6A 035, A034 light chain complementarity determining region CDRL1
RASQSVSFAVE
SEQ ID NO. 7A 035, A037, A034, A039, A038, C056-WT light chain complementarity determining region CDRL2
SASSLYS
SEQ ID NO. 8A 035, A037, A034, A039, A038, C056-WT light chain complementarity determining region CDRL3
QQYGYHDAGLIT
SEQ ID NO. 9A 037, A034 heavy chain variable region
EVQLVESGGGLVQPGGSLRLSCAASGLNLYYSSMHWVRQAPGKGLEWVASIYSSNGRTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYWGQGTLVTVSS
SEQ ID NO. 10A 037, A034 heavy chain complementarity determining region CDRH1
GLNLYYSSMH
11A037, A034, A039, A038, C056-WT heavy chain complementarity determining region CDRH3
SSFSHGYGWYGLDY
SEQ ID NO. 12A 037 light chain variable region
DIQMTQSPSSLSASVGDRVTITCRASQSVSFAVDWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIK
CDRL1 of the light chain complementarity determining region of SEQ ID NO. 13A 037
RASQSVSFAVD
SEQ ID NO. 14A 039, A038 heavy chain variable region
EVQLVESGGGLVQPGGSLRLSCAASGINLYYSSMHWVRQAPGKGLEWVASIYSSNGRTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYWGQGTLVTVSS
15A 039 light chain variable region of SEQ ID NO
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVDWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIK
SEQ ID NO. 16A 039 light chain complementarity determining region CDRL1
RASQSVSSAVD
17A 038 light chain variable region of SEQ ID NO
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVEWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIK
SEQ ID NO. 18C 056-WT heavy chain variable region
EVQLVESGGGLVQPGGSLRLSCAASGFNLYYSSMHWVRQAPGKGLEWVASIYSSNGRTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYWGQGTLVTVSS
SEQ ID NO. 19C 056-WT heavy chain complementarity determining region CDRH1
GFNLYYSSMH
SEQ ID NO. 20C 056-WT light chain variable region
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIK
SEQ ID NO. 21C 056-WT light chain complementarity determining region CDRL1
RASQSVSSAVA
SEQ ID NO. 22A 038 light chain complementarity determining region CDRL1
RASQSVSSAVE
SEQ ID NO. 23 IL-18 Rbeta antigen sequence
1 MLCLGWIFLW LVAGERIKGF NISGCSTKKL LWTYSTRSEE EFVLFCDLPE PQKSHFCHRN
61 RLSPKQVPEH LPFMGSNDLS DVQWYQQPSN GDPLEDIRKS YPHIIQDKCT LHFLTPGVNN
121 SGSYICRPKM IKSPYDVACC VKMILEVKPQ TNASCEYSAS HKQDLLLGST GSISCPSLSC
181 QSDAQSPAVT WYKNGKLLSV ERSNRIVVDE VYDYHQGTYV CDYTQSDTVS SWTVRAVVQV
241 RTIVGDTKLK PDILDPVEDT LEVELGKPLT ISCKARFGFE RVFNPVIKWY IKDSDLEWEV
301 SVPEAKSIKS TLKDEIIERN IILEKVTQRD LRRKFVCFVQ NSIGNTTQSV QLKEKRGVVL
361 LYILLGTIGT LVAVLAASAL LYRHWIEIVL LYRTYQSKDQ TLGDKKDFDA FVSYAKWSSF
421 PSEATSSLSE EHLALSLFPD VLENKYGYSL CLLERDVAPG GVYAEDIVSI IKRSRRGIFI
481 LSPNYVNGPS IFELQAAVNL ALDDQTLKLI LIKFCYFQEP ESLPHLVKKA LRVLPTVTWR
541 GLKSVPPNSR FWAKMRYHMP VKNSQGFTWN QLRITSRIFQ WKGLSRTETT GRSSQPKEW
Example 1 preparation of anti-IL-18 Rbeta high affinity antibodies
As shown in FIG. 1, the inventors used the WT antibody of IL-18Rβ (i.e., wild-type antibody, C056-WT in Table 1) as a starting material for improving affinity. A library of exact mutations was constructed to introduce saturation mutations into all 73 residues of the six CDR regions of the WT antibody. After further screening, selected mutants were ranked by dissociation rate constant (Dissociation rate constant) determined by surface plasmon resonance (Surface plasmon resonance, SPR). A combinatorial library is then constructed using random combinations of beneficial mutations. The lead Fab clones were also determined by SPR sequencing. All SPR screens were performed on Biacore 8K. The running buffer was HBS-EP (10mM HEPES,500mM NaCl,3mM EDTA,0.05% Tween 20, pH 6.0). A secreted Fab was adsorbed onto the SASA biosensor. After equilibration, antigen was injected for 120 seconds (binding period) and then running buffer was injected for 360 seconds (dissociation period). The sensor surface was regenerated before injection of other selected clones. Experimental data were fitted locally to the 1:1 interaction model using Biacore 8K evaluation software to obtain the off-rate of Fab clones. Finally, full-length IgG from the pilot clone was expressed in HEK293 cells and evaluated in binding and functional assays.
As shown in table 3, 44 hits were analyzed by SPR and it was determined that 7 beneficial mutants exhibited better binding affinity (i.e., > 2-fold of WT antibody affinity, as shown in table 4).
The first 10 combinatorial mutations (> 10-fold of WT antibody affinity) with improved binding affinity are listed in table 5. 5 pilot clones (a 034, a035, a037, a038 and a 039) were selected for full length antibody production. The sequence is provided below.
TABLE 3 affinity and kinetics of IL-18Rβ for 44 hit mutants
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TABLE 4 beneficial mutants identified by saturation mutagenesis screening
Table 5 the first 10 affinity-enhanced beneficial mutant combinations
Sequence number Clone numbering ka(1/Ms) kd(1/s) KD(M) Ratio (kd) Sequence analysis
1 A034 1.31E+05 4.24E-05 3.22E-10 28.07 VL-S31F/VL-A34E/VH-F27L
2 A035 1.43E+05 3.19E-05 2.24E-10 37.30 VL-S31F/VL-A34E/VH-F27I/VH-S99A
3 A036 1.07E+05 6.91E-05 6.48E-10 17.22 VL-A34D/VH-F27I/VH-S99A
4 A037 1.18E+05 1.00E-06 8.47E-12 1190.00 VL-S31F/VL-A34D/VH-F27L
5 A038 7.82E+04 5.67E-05 7.25E-10 20.99 VL-A34E/VH-F27I
6 A039 1.15E+05 3.00E-05 2.60E-10 39.67 VL-A34D/VH-F27I
7 A040 7.70E+04 8.00E-05 1.04E-09 14.88 VL-A34E/VH-F27L
8 A041 1.53E+05 6.35E-05 4.15E-10 18.74 VL-S31F/VL-A34D/VH-F27V/VH-S99A
9 A042 7.90E+04 1.06E-04 1.34E-09 11.23 VL-A34E/VH-F27L/VH-S99A
10 A043 1.28E+05 6.29E-05 4.91E-10 18.92 VL-S31F/VL-A34E/VH-F27L
C056-WT 6.66E+04 1.19E-03 1.78E-08 1.00
Example 2 evaluation of affinity of anti-IL-18 Rβ antibodies
Affinity of anti-IL-18 rβ antibodies to human IL-18rβ protein was determined using a surface plasmon resonance biosensor Biacore 8K (GE Healthcare). Measurements were performed at 25℃with running buffer HBS-EP+. Antibodies were injected as a capture onto Series S sensor chip protein a. The diluted IL-18Rβ (400, 200,100,50,25,12.5,6.25 nM) was then injected as a binding phase into the surface of flow cells 1 and 2. The bonding time was 120 seconds. The buffer stream was maintained for 420 seconds to allow separation. The surface was regenerated by injection of 10mM glycine-HCl pH 1.5 for 30 seconds. Data for dissociation (kd) rate constants and association (ka) rate constants were obtained using Biacore evaluation software. The equilibrium dissociation constant (KD) is calculated from the ratio of KD to ka.
Table 6 summarizes the binding kinetics data for anti-IL-18 Rβ antibodies. The binding affinities of A035, A037 and A039 were in the range of 0.72-0.98 nm, which was 16-22 fold improved compared to the parent antibody WT (15.8 nm).
TABLE 6 binding kinetics of anti-IL-18 Rβ antibodies
EXAMPLE 3 Activity of anti-IL-18 Rβ antibodies against blocking IL-18 stimulated IFN- γ Release in KG-1 cells
Will be 3X 10 5 KG-1 cells (ATCC, # CCL-246) were seeded into each well of the 96-well plate. Serial dilutions of the antibodies prepared in example 1 were added to wells before stimulation with 10ng/mL IL-18 for 1 h. After 24 hours incubation, cells were pelleted by centrifugation and, according to manufacturer's instructions, using an ELISA kit (R&Dsystems, #val 104C) measures IFN- γ from the supernatant. At least for the anti-IL-18 Rbeta antibody3 replicates were performed and the IC50 of each antibody is summarized in table 7.
TABLE 7 IC50 of anti-IL-18 Rβ antibodies in KG-1 assays
Antibodies to IC50(nM)
WT 8.14
A035 0.26
A037 0.27
A039 0.59
Representative experimental results are shown in figure 2, with a035, a037 and a039 antibodies being about 20-fold more potent than WT antibodies.
EXAMPLE 4 anti-IL-18 Rβ antibodies block the IL-18 stimulated activation of IFN- γ release by human peripheral blood mononuclear cells Sex characteristics
Human peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC) (TPCS, #PB025C-W) at 1X 10 5 The individual cell/well concentrations were seeded in 96-well plates. The cells were then incubated with varying concentrations of anti-IL-18 Rβ antibodies at 37℃and 5% CO under conditions of 50ng/mL IL-18 and 5ng/mL IL-12 2 And incubated for 24 hours. According to the manufacturer's instructions, a human ELISA kit (R&D Systems, #VALX104C) measures IFN-. Gamma.production.
The results are shown in figure 3, with the a035, a037 and a039 antibodies showing higher potency than the WT antibodies.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
<110> and Shanghai pharmaceutical technologies (Shanghai Co., ltd.)
<120> an antibody targeting IL-18 Rbeta and use thereof
<130> P2021-3129
<160> 23
<170> PatentIn version 3.5
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<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 20
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Tyr His Asp Ala
85 90 95
Gly Leu Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 21
<211> 11
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 21
Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala
1 5 10
<210> 22
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
CDRL1 of <223> A038
<400> 22
Arg Ala Ser Gln Ser Val Ser Ser Ala Val Glu
1 5 10
<210> 23
<211> 599
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 23
Met Leu Cys Leu Gly Trp Ile Phe Leu Trp Leu Val Ala Gly Glu Arg
1 5 10 15
Ile Lys Gly Phe Asn Ile Ser Gly Cys Ser Thr Lys Lys Leu Leu Trp
20 25 30
Thr Tyr Ser Thr Arg Ser Glu Glu Glu Phe Val Leu Phe Cys Asp Leu
35 40 45
Pro Glu Pro Gln Lys Ser His Phe Cys His Arg Asn Arg Leu Ser Pro
50 55 60
Lys Gln Val Pro Glu His Leu Pro Phe Met Gly Ser Asn Asp Leu Ser
65 70 75 80
Asp Val Gln Trp Tyr Gln Gln Pro Ser Asn Gly Asp Pro Leu Glu Asp
85 90 95
Ile Arg Lys Ser Tyr Pro His Ile Ile Gln Asp Lys Cys Thr Leu His
100 105 110
Phe Leu Thr Pro Gly Val Asn Asn Ser Gly Ser Tyr Ile Cys Arg Pro
115 120 125
Lys Met Ile Lys Ser Pro Tyr Asp Val Ala Cys Cys Val Lys Met Ile
130 135 140
Leu Glu Val Lys Pro Gln Thr Asn Ala Ser Cys Glu Tyr Ser Ala Ser
145 150 155 160
His Lys Gln Asp Leu Leu Leu Gly Ser Thr Gly Ser Ile Ser Cys Pro
165 170 175
Ser Leu Ser Cys Gln Ser Asp Ala Gln Ser Pro Ala Val Thr Trp Tyr
180 185 190
Lys Asn Gly Lys Leu Leu Ser Val Glu Arg Ser Asn Arg Ile Val Val
195 200 205
Asp Glu Val Tyr Asp Tyr His Gln Gly Thr Tyr Val Cys Asp Tyr Thr
210 215 220
Gln Ser Asp Thr Val Ser Ser Trp Thr Val Arg Ala Val Val Gln Val
225 230 235 240
Arg Thr Ile Val Gly Asp Thr Lys Leu Lys Pro Asp Ile Leu Asp Pro
245 250 255
Val Glu Asp Thr Leu Glu Val Glu Leu Gly Lys Pro Leu Thr Ile Ser
260 265 270
Cys Lys Ala Arg Phe Gly Phe Glu Arg Val Phe Asn Pro Val Ile Lys
275 280 285
Trp Tyr Ile Lys Asp Ser Asp Leu Glu Trp Glu Val Ser Val Pro Glu
290 295 300
Ala Lys Ser Ile Lys Ser Thr Leu Lys Asp Glu Ile Ile Glu Arg Asn
305 310 315 320
Ile Ile Leu Glu Lys Val Thr Gln Arg Asp Leu Arg Arg Lys Phe Val
325 330 335
Cys Phe Val Gln Asn Ser Ile Gly Asn Thr Thr Gln Ser Val Gln Leu
340 345 350
Lys Glu Lys Arg Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr Ile
355 360 365
Gly Thr Leu Val Ala Val Leu Ala Ala Ser Ala Leu Leu Tyr Arg His
370 375 380
Trp Ile Glu Ile Val Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp Gln
385 390 395 400
Thr Leu Gly Asp Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala Lys
405 410 415
Trp Ser Ser Phe Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu His
420 425 430
Leu Ala Leu Ser Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr Gly Tyr
435 440 445
Ser Leu Cys Leu Leu Glu Arg Asp Val Ala Pro Gly Gly Val Tyr Ala
450 455 460
Glu Asp Ile Val Ser Ile Ile Lys Arg Ser Arg Arg Gly Ile Phe Ile
465 470 475 480
Leu Ser Pro Asn Tyr Val Asn Gly Pro Ser Ile Phe Glu Leu Gln Ala
485 490 495
Ala Val Asn Leu Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu Ile
500 505 510
Lys Phe Cys Tyr Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val Lys
515 520 525
Lys Ala Leu Arg Val Leu Pro Thr Val Thr Trp Arg Gly Leu Lys Ser
530 535 540
Val Pro Pro Asn Ser Arg Phe Trp Ala Lys Met Arg Tyr His Met Pro
545 550 555 560
Val Lys Asn Ser Gln Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr Ser
565 570 575
Arg Ile Phe Gln Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly Arg
580 585 590
Ser Ser Gln Pro Lys Glu Trp
595

Claims (25)

1. An antibody or antigen-binding fragment thereof directed against IL-18rβ, comprising:
(a) The heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, wherein the amino acid sequences of the CDRH1, CDRH2 and CDRH3 are respectively shown as SEQ ID NO. 2, 3 and 4, or are respectively shown as SEQ ID NO. 10, 3 and 11, or are respectively shown as SEQ ID NO. 2, 3 and 11; and/or
(b) The amino acid sequences of the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 are shown as SEQ ID NOS 6, 7 and 8, respectively, or as SEQ ID NOS 13, 7 and 8, respectively, or as SEQ ID NOS 16, 7 and 8, respectively, or as SEQ ID NOS 22, 7 and 8, respectively.
Preferably, any of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-4) amino acid and that retains the ability to specifically bind to IL-18Rβ.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NOs 1, 9 or 14 and has a light chain variable region as set forth in SEQ ID NOs 5, 12, 15 or 17.
3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a beneficial mutation selected from the group consisting of the wild-type heavy chain variable region set forth in SEQ ID No. 18 and/or the wild-type light chain variable region set forth in SEQ ID No. 20: F27L, F27I, F V, S99A, S31F, A34D, A34E, or a combination thereof.
4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof has a beneficial mutation selected from the group consisting of the wild-type heavy chain variable region set forth in SEQ ID No. 18 and the wild-type light chain variable region set forth in SEQ ID No. 20:
S31F、A34E、F27L；
S31F、A34E、F27I、S99A；
A34D、F27I、S99A；
S31F、A34D、F27L；
A34E、F27I；
A34D、F27I；
A34E、F27L；
S31F、A34D、F27V、S99A；
a34E, F27L, S a; or (b)
S31F、A34E、F27L。
5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a monomer, a bivalent antibody, and/or a multivalent antibody.
Preferably, the bivalent antibody may also be a bispecific antibody; preferably, the multivalent antibody may also be a multispecific antibody.
6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is selected from scFv, fab, fab ', F (ab') 2 Fv fragment, disulfide linked Fv (dsFv).
7. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof have amino acid sequences as shown in SEQ ID No. 9 and SEQ ID No. 5, or as shown in SEQ ID No. 1 and SEQ ID No. 5, or as shown in SEQ ID No. 9 and SEQ ID No. 12, or as shown in SEQ ID No. 14 and SEQ ID No. 17, or as shown in SEQ ID No. 14 and SEQ ID No. 15, respectively.
8. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain constant region of the antibody or antigen-binding fragment thereof is selected from the group consisting of the heavy chain constant region of human IgG1, igG2, igG3, or IgG 4.
9. A recombinant protein, wherein said recombinant protein comprises:
(i) The antibody or antigen-binding fragment thereof of any one of claims 1-8; and
(ii) Optionally a tag sequence to assist expression and/or purification.
10. A nucleotide molecule encoding the antibody or antigen binding fragment thereof according to any one of claims 1-8, and/or the recombinant protein according to claim 9.
11. An expression vector comprising the nucleotide molecule of claim 10.
12. A host cell comprising the expression vector of claim 11, or having incorporated into its genome the nucleotide molecule of claim 10.
13. A chimeric antigen receptor CAR, wherein an antigen binding region scFv fragment of the CAR is a binding region that specifically binds to IL-18rβ, and wherein a heavy chain variable region of the scFv comprises:
the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, wherein the amino acid sequences of the CDRH1, CDRH2 and CDRH3 are respectively shown as SEQ ID NO. 2, 3 and 4, or are respectively shown as SEQ ID NO. 10, 3 and 11, or are respectively shown as SEQ ID NO. 2, 3 and 11; and/or
The light chain variable region of the scFv comprises:
the amino acid sequences of the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 are shown as SEQ ID NOS 6, 7 and 8, respectively, or as SEQ ID NOS 13, 7 and 8, respectively, or as SEQ ID NOS 16, 7 and 8, respectively, or as SEQ ID NOS 22, 7 and 8, respectively.
14. An engineered immune cell expressing an exogenous CAR of claim 13.
15. A method of producing an antibody or antigen-binding fragment thereof directed against IL-18rβ comprising the steps of:
(a) Culturing the host cell of claim 12 under suitable conditions, thereby obtaining a culture comprising an antibody or antigen-binding fragment thereof to IL-18rβ;
(b) Isolating and/or recovering the antibody or antigen binding fragment thereof directed against IL-18rβ from the culture; and
(c) Optionally, purifying and/or modifying the antibody or antigen binding fragment thereof against IL-18rβ obtained in step (b).
16. An immunoconjugate, characterized in that the immunoconjugate comprises:
(a) An antibody moiety comprising the antibody or antigen-binding fragment thereof of any one of claims 1-8; and
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, gold nanoparticle/nanorod, nanomagnetic particle, viral coat protein, or VLP, or a combination thereof.
17. A pharmaceutical composition comprising:
(i) An active ingredient selected from the group consisting of: the antibody or antigen-binding fragment thereof of any one of claims 1-8, or the recombinant protein of claim 9, or the engineered immune cell of claim 14, or the immunoconjugate of claim 16, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier, diluent or excipient.
18. Use of an active ingredient selected from the group consisting of: the antibody or antigen-binding fragment thereof of any one of claims 1-8, or the recombinant protein of claim 9, or the engineered immune cell of claim 14, or the immunoconjugate of claim 16, or a combination thereof, wherein the active ingredient is used to prepare:
(a) A medicament for preventing and/or treating IL-18-related diseases;
(b) Reagents for detecting IL-18 related disorders.
19. A method for in vitro detection of IL-18rβ protein or a fragment thereof in a sample, the method comprising the steps of:
(1) Contacting the sample with the antibody or antigen binding fragment thereof of any one of claims 1-8 in vitro;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of a corresponding target of the IL-18Rβ protein or fragment thereof in the sample.
20. A kit, comprising:
(1) A first container comprising the antibody or antigen-binding fragment thereof of any one of claims 1-8, or the recombinant protein of claim 9, or the engineered immune cell of claim 14, or the immunoconjugate of claim 16, or the pharmaceutical composition of claim 17; and/or
(2) A second container containing a second antibody against the contents of the first container;
optionally instructions for use.
21. A method of preventing and/or treating an IL-18-associated disease, the method comprising: administering to a subject in need thereof an antibody or antigen-binding fragment thereof of claims 1-8, or a recombinant protein of claim 9, or an engineered immune cell of claim 14, or an immunoconjugate of claim 16, or a pharmaceutical composition of claim 17, or a combination thereof.
22. The use according to claim 18, wherein the IL-18 related disorder comprises a disorder of high IL-18 expression, a disorder associated with aberrant expression of IL-18 receptors or a disorder associated with high activity of IL-18 signaling pathways.
23. The use according to claim 18, wherein the IL-18 related disorder is an autoimmune disorder or an inflammatory disorder.
24. The use according to claim 18, wherein the IL-18 related disorder is selected from the group consisting of: tumors, leukemia, diabetic nephropathy, alzheimer's disease, parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis, psoriatic arthritis, crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, grave's disease, hashimoto's thyroiditis, addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphocytosis, macrophage activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyoderma, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory Bowel Disease (IBD), adult steven-ston's, chronic Obstructive Pulmonary Disease (COPD), or a combination thereof.
25. The use of claim 24, wherein the tumor is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.
CN202210108473.8A 2022-01-14 2022-01-28 Antibody targeting IL-18 Rbeta and application thereof Pending CN116554322A (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN202210108473.8A CN116554322A (en) 2022-01-28 2022-01-28 Antibody targeting IL-18 Rbeta and application thereof
CN202380011140.6A CN117177999A (en) 2022-01-14 2023-01-16 Antibody targeting IL-18 Rbeta and application thereof
PCT/CN2023/072444 WO2023134767A1 (en) 2022-01-14 2023-01-16 ANTIBODY TARGETING IL-18Rβ AND USES THEREOF

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210108473.8A CN116554322A (en) 2022-01-28 2022-01-28 Antibody targeting IL-18 Rbeta and application thereof

Publications (1)

Publication Number Publication Date
CN116554322A true CN116554322A (en) 2023-08-08

Family

ID=87486661

Family Applications (1)

Application Number Title Priority Date Filing Date
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Country Status (1)

Country Link
CN (1) CN116554322A (en)

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