WO2019080909A1 - Anticorps thérapeutique ciblant rankl - Google Patents

Anticorps thérapeutique ciblant rankl

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Publication number
WO2019080909A1
WO2019080909A1 PCT/CN2018/111961 CN2018111961W WO2019080909A1 WO 2019080909 A1 WO2019080909 A1 WO 2019080909A1 CN 2018111961 W CN2018111961 W CN 2018111961W WO 2019080909 A1 WO2019080909 A1 WO 2019080909A1
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Prior art keywords
antibody
rankl
binding fragment
antigen binding
seq
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PCT/CN2018/111961
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English (en)
Inventor
Jieying Liu
Jing Li
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Wuxi Biologics (Shanghai) Co., Ltd.
Wuxi Biologics (Cayman) Inc.
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Application filed by Wuxi Biologics (Shanghai) Co., Ltd., Wuxi Biologics (Cayman) Inc. filed Critical Wuxi Biologics (Shanghai) Co., Ltd.
Publication of WO2019080909A1 publication Critical patent/WO2019080909A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
    • A61P5/16—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4 for decreasing, blocking or antagonising the activity of the thyroid hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates generally to antibodies against RankL and compositions thereof, and therapy in the treatment of a disease associated with bone loss using anti-RankL antibodies.
  • Receptor activator of NF kappa-B ligand also known as osteoprotegerin ligand (OPGL) and tumor necrosis factor ligand superfamily member 11 (TNFSF11)
  • OPGL osteoprotegerin ligand
  • TNFSF11 tumor necrosis factor ligand superfamily member 11
  • RANKL can be expressed on cell surface or in soluble form (Findlay DM, et. al, Osteoporos Int 22 (10) : 2597-2602) and mediated osteoclast formation, activation and survival through binding to its receptor RANK on the osteoclasts and their precursors (Hsu H, et.
  • Denosumab an FDA-approved fully human monoclonal antibody to RANKL, have shown significant reductions in vertebral, nonvertebral, and hip fractures, and provided evidence compatible with the use of denosumab in postmenopausal women with osteoporosis (David W. Dempster, Clin Ther. 2012; 34: 521-536) .
  • Three pivotal, randomized phase III trials also showed that Denosumab was superior to zoledronic acid in preventing skeleton related events (SRE) with favourable safety and convenience in patients with bone metastases from advanced cancer (Clin Ther. 2012; 34: 521-536; Prasad Narayanan, Journal of Cancer. 2013; 2 (4) : 272-277) .
  • the present invention provides isolated antibodies, in particular monoclonal antibodies or humanized monoclonal antibodies.
  • the present invention provides an antibody or an antigen binding-fragment thereof, wherein the antibody or the antigen binding-fragment binds to human, monkey and mouse RANKL.
  • the present invention provides an antibody or an antigen binding fragment thereof, wherein the antibody or the antigen binding-fragment
  • a) binds to human RANKL with a K D of between 6.98E-10 M and 6.33E-11 M and to monkey RANKL with a K D of between 5.32E-11 M and 9.03E-12 M;
  • the present invention provides an antibody or an antigen binding fragment thereof, comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%or 99%homologous to a sequence selected from a group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14,
  • the antibody or the antigen binding fragment specifically binds to RANKL.
  • the present invention provides an antibody or an antigen binding fragment thereof, comprising an amino acid sequence selected from a group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14,
  • the antibody or the antigen binding fragment specifically binds to RANKL.
  • the present invention provides an antibody, or an antigen-binding fragment thereof, comprising:
  • variable region of a heavy chain having an amino acid sequence that is at least 70%, 80%, 90%or 95%homologous to a sequence selected from a group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7;
  • variable region of a light chain having an amino acid sequence that is at least 70%, 80%, 90%or 95%homologous to a sequence selected from a group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14,
  • the antibody or the antigen binding fragment specifically binds to RANKL.
  • the present invention provides an antibody or an antigen binding fragment thereof, comprising:
  • variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7;
  • variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14,
  • the antibody or the antigen binding fragment specifically binds to RANKL.
  • the antibody or an antigen binding fragment thereof comprises:
  • variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 1;
  • variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8,
  • the antibody or the antigen binding-fragment specifically binds to RANKL
  • variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 2;
  • variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 9,
  • the antibody or the antigen binding-fragment specifically binds to RANKL
  • variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 3;
  • variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 10,
  • the antibody or the antigen binding-fragment specifically binds to RANKL
  • variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4;
  • variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 11,
  • the antibody or the antigen binding-fragment specifically binds to RANKL
  • variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 5;
  • variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12,
  • the antibody or the antigen binding-fragment specifically binds to RANKL
  • variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6;
  • variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 13,
  • the antibody or the antigen binding-fragment specifically binds to RANKL
  • variable region of a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 7;
  • variable region of a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14,
  • the antibody or the antigen binding-fragment specifically binds to RANKL
  • the invention provides an antibody or an antigen binding fragment thereof, comprising a complementarity-determining region (CDR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-36,
  • the antibody or the antigen binding fragment specifically binds to RANKL.
  • the invention provides an antibody, or an antigen binding fragment thereof, comprising: a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences; and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences,
  • heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from a group consisting of SEQ ID NOs: 15, 16, and 17, and conservative modifications thereof,
  • the antibody or the antigen binding-fragment specifically binds to RANKL.
  • the light chain variable region CDR3 sequence of the aforesaid antibody or antigen binding fragment thereof comprises an amino acid sequence selected from a group consisting of SEQ ID NOs: 18, 19, 20, and 21, and conservative modifications thereof.
  • the heavy chain variable region CDR2 sequence of the aforesaid antibody or antigen binding fragment thereof comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 22, 23 24, and 25, and conservative modifications thereof.
  • the light chain variable region CDR2 sequence of the aforesaid antibody or antigen binding fragment thereof comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 26, 27, 28, and 29, and conservative modifications thereof.
  • the heavy chain variable region CDR1 sequence of the aforesaid antibody or antigen binding fragment thereof comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 30, 31, and 32, and conservative modifications thereof.
  • the antibody of this invention wherein the light chain variable region CDR1 sequence of the aforesaid antibody or antigen binding fragment thereof comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 33, 34, 35, and 36, and conservative modifications thereof.
  • the invention provides an antibody, or an antigen binding fragment thereof, wherein the antibody or antigen binding fragment specifically binds to RANKL and comprises: a heavy chain variable region that comprises CDR1, CDR2, and CDR3 sequences; and a light chain variable region that comprises CDR 1, CDR2, and CDR3 sequences, wherein:
  • the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 30, 31, and 32
  • CDR2 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 22, 23, 24, and 25
  • CDR3 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 15, 16, and 17;
  • CDR1 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 33, 34, 35, and 36
  • CDR2 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 26, 27, 28, and 29
  • CDR3 sequence comprises an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOs: 18, 19, 20, and 21,
  • the antibody or the antigen binding-fragment specifically binds to RANKL.
  • a preferred antibody or an antigen binding fragment thereof comprises:
  • a heavy chain variable region CDR1 comprising SEQ ID NO: 30;
  • the antibody or the antigen binding-fragment specifically binds to RANKL.
  • Another preferred antibody or an antigen binding fragment thereof comprises:
  • the antibody or the antigen binding-fragment specifically binds to RANKL.
  • Another preferred antibody or an antigen binding fragment thereof comprises:
  • the antibody or the antigen binding-fragment specifically binds to RANKL.
  • Another preferred antibody or an antigen binding fragment thereof comprises:
  • a heavy chain variable region CDR1 comprising SEQ ID NO: 30;
  • the antibody specifically binds to RANKL.
  • Another preferred antibody or an antigen binding fragment thereof comprises:
  • a heavy chain variable region CDR1 comprising SEQ ID NO: 30;
  • the antibody or the antigen binding-fragment specifically binds to RANKL.
  • Another preferred antibody or an antigen binding fragment thereof comprises:
  • the antibody or the antigen binding-fragment specifically binds to RANKL.
  • Another preferred antibody or an antigen binding fragment thereof comprises:
  • the antibody or the antigen binding-fragment specifically binds to RANKL.
  • the antibodies of the invention can be chimeric antibody.
  • the antibodies of the invention can be humanized antibody.
  • the antibodies of the invention can be fully human antibody.
  • the antibodies of the invention can be rat antibody.
  • the antibodies or the antigen binding fragment thereof of the invention can exhibit at least one of the following properties:
  • a) binds to human RANKL with a K D of between 6.98E-10 M and 6.33E-11 M and to monkey RANKL with a K D of between 5.32E-11 M and 9.03E-12 M;
  • the invention provides a nucleic acid molecule encoding the antibody, or antigen binding fragment thereof.
  • the invention provides a cloning or expression vector comprising the nucleic acid molecule encoding the antibody, or antigen binding fragment thereof.
  • the invention also provides a host cell comprising one or more cloning or expression vectors.
  • the invention provides a process, comprising culturing the foresaid host cell and isolating the antibody;
  • the antibody is prepared through immunization in a mouse with human RankL protein.
  • the invention provides a transgenic animal such as mouse comprising human immunoglobulin heavy and light chain transgenes, wherein the animal expresses the antibody of this invention.
  • the invention provides hybridoma prepared from the animal of this invention, wherein the hybridoma produces said antibody.
  • the invention provides pharmaceutical composition
  • pharmaceutical composition comprising the antibody, or the antigen binding fragment of said antibody in the invention, and one or more of a pharmaceutically acceptable excipient, a diluent or a carrier.
  • the invention also provides a method for preparing an anti-RankL antibody or an antigen-binding fragment thereof comprising:
  • a heavy chain variable region antibody sequence comprising a CDR1 sequence that is selected from a group consisting of SEQ ID NOs: 30-32, a CDR2 sequence that is selected from a group consisting of SEQ ID NOs: 22-25; and a CDR3 sequence that is selected from the group consisting of SEQ ID NOs: 15-17; and/or
  • a light chain variable region antibody sequence comprising a CDR1 sequence that is selected from the group consisting of SEQ ID NOs: 33-36, a CDR2 sequence that is selected from the group consisting of SEQ ID NOs: 26-29, and a CDR3 sequence that is selected from the group consisting of SEQ ID NOs: 18-21;
  • the invention also provides a method of preventing or treating a disease associated with bone loss in a subject comprising administering to a subject in need a therapeutically effective amount of foresaid antibody or antigen-binding fragment in this invention.
  • the invention also provides a combined method of preventing or treating a disease associated with bone loss in a subject comprising administering to a subject in need a therapeutically effective amount of foresaid antibody or antigen-binding fragment in the invention and administering to the subject a therapeutically effective amount of immune checkpoint antibody.
  • the immune checkpoint antibody in the invention includes CTLA-4 antibody, PD-1 antibody, or PD-L1 antibody.
  • the invention also provides the use of said antibody or the antigen binding fragment thereof in the manufacture of a medicament for the prevention or treatment of a disease associated with bone loss.
  • Said disease includes Crohn Disease, Male Infertility, Chronic Kidney Disease, Gout, Rheumatoid Arthritis, Anorexia Nervosa, Thalassemia Major, Hyperthyroidism or cancer.
  • Said cancer is selected from a group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, and rectal cancer.
  • the antibodies reported in this invention have high binding affinity, specifically binding to both human and monkey RANKL protein; and potent preventing or treating a disease associated with bone loss.
  • One of the antibodies not only bound to human and monkey RANKL, but also bound to murine RANKL, which could greatly facilitate preclinical validation of its efficacy in mouse tumor models.
  • Figure 1 shows the binding of antibody WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20, WBP114_7.80 and Prolia to human RANKL.
  • Figure 2 shows the blocking assay of antibody WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20, WBP114_7.80 and Prolia.
  • Figure 3 shows the does-dependent binding of antibody WBP114_5.20 to mouse RANKL.
  • Figure 4 shows NF-kB reporter gene assay of selected antibodies.
  • Figure 5 shows RAW264.7 cell differentiation assay of selected antibodies.
  • Figure 6 shows Epitope binning of selected antibodies against Prolia.
  • Figure 7 shows ELISA binding assay of humanized antibodies.
  • Figure 8 shows ELISA blocking assay of humanized antibodies.
  • Figure 9 shows NF-kB reporter gene assay of humanized antibodies.
  • FIG. 10 shows RAW264.7 cell differentiation assay of humanized antibodies.
  • Receptor Activator for Nuclear Factor- ⁇ B Ligand “RANKL” , “TNF-related activation-induced cytokine” , “TRANCE” , “osteoprotegerin ligand” , “OPGL” , “osteoclast differentiation factor” , “ODF” are used interchangeably, and include variants, isoforms, species homologs of human RANKL or RANKL of other species, and analogs having at least one common epitope with RANKL.
  • antibody as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion” ) or single chains thereof.
  • An “antibody” refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) .
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • antibody refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen-binding site, regardless whether it is produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies.
  • antibody also includes antibody fragments such as Fab, F (ab′) 2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to bind RANKL specifically. Typically, such fragments would comprise an antigen-binding fragment.
  • antigen-binding fragment refers to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. In instances, where an antigen is large, the antigen-binding fragment may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding fragment is referred to as “epitope” or “antigenic determinant. "
  • An antigen-binding fragment typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) , however, it does not necessarily have to comprise both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • Fd antibody fragment consists only of a VH domain, but still retains some antigen-binding function of the intact antibody.
  • epitope defines an antigenic determinant, which is specifically bound/identified by a binding fragment as defined above.
  • the binding fragment may specifically bind to/interact with conformational or continuous epitopes, which are unique for the target structure, e.g. the human RANKL and murine RANKL.
  • a conformational or discontinuous epitope is characterized for polypeptide antigens by the presence of two or more discrete amino acid residues which are separated in the primary sequence, but come together on the surface of the molecule when the polypeptide folds into the native protein/antigen. The two or more discrete amino acid residues contributing to the epitope are present on separate sections of one or more polypeptide chain (s) .
  • a continuous or linear epitope consists of two or more discrete amino acid residues, which are present in a single linear segment of a polypeptide chain.
  • cross-reactivity refers to binding of an antigen fragment described herein to the same target molecule in human, monkey, and/or murine (mouse or rat) .
  • cross-reactivity is to be understood as an interspecies reactivity to the same molecule X expressed in different species, but not to a molecule other than X.
  • Cross-species specificity of a monoclonal antibody recognizing e.g. human RANKL, to monkey, and/or to a murine (mouse or rat) RANKL can be determined, for instance, by FACS analysis.
  • the term “subject” includes any human or nonhuman animal.
  • nonhuman animal includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms “patient” or “subject” are used interchangeably.
  • treatment and “therapeutic method” refer to both therapeutic treatment and prophylactic/preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder.
  • conservative modifications i.e., nucleotide and amino acid sequence modifications which do not significantly affect or alter the binding characteristics of the antibody encoded by the nucleotide sequence or containing the amino acid sequence.
  • conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can be introduced into the sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • mice were injected with human RankL protein via foot pads approximately every 3 days. First titer test was performed after 6 times injection.
  • ELISA assay was used to measure titers of antibody in mouse serum.
  • plates Nunc
  • human RankL at 1 ⁇ g/mL overnight at 4 °C
  • blocking buffer (1 ⁇ PBS/2%BSA)
  • Rat serum was 1 ⁇ 3 titrated starting at 1 ⁇ 100 dilution in blocking buffer and incubated for 1 h at room temperature.
  • the plates were then washed and subsequently incubated with secondary antibody goat anti-mouse IgG Fc HRP for 1 h. After washing, TMB substrate was added and the interaction was stopped by 2M HCl.
  • the absorbance at 450 nm was read using a microplate reader (Molecular Device) .
  • mice with serum titer of 312500 or higher were selected for hybridoma fusion.
  • Lymph nodes and spleen were collected from immunized mice under sterile condition, and lymphocytes were prepared using Ficoll-Paque PLUS gradient centrifugation. The isolated cells were then fused with myeloma cell P3 at a ratio of 1 ⁇ 1 using electro fusion device (BTX ECM2001) . Cells were transferred to 1/2 HA media after fusion. 5x10 5 cells were seeded per 96-well plate.
  • Hybridoma supernatant was used for primary screen.
  • the antigen specific hybridomas were than screened by ELISA blocking assay.
  • Binding assay by ELISA Plates (Nunc) were coated with human RankL at 1 ⁇ g/mL overnight at 4 °C. After blocking and washing, hybridoma supernatants were transferred to the plates and incubate at room temperature for 1 h. The plates were then washed and subsequently incubated with secondary antibody goat anti-mouse IgG Fc HRP for 1 h. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device) .
  • Blocking assay by ELISA Plates (Nunc) were coated with 5 ⁇ g/mL Rank-Fc fusion protein overnight at 4 °C. Hybridoma supernatants were mixed with 250 ng/mL RankL-His and incubated at 4 °C overnight. Prolia was used as a positive control. After blocking and washing, the mixture was added to the plates and incubated for 1 h. The plates were then washed and subsequently incubated with anti-His Ab-HRP. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device) .
  • the antibodies which can block the binding of human Rank Ligand to human Rank were selected for further characterization.
  • the selected antibodies with both binding and blocking activities were purified from hybridoma supernatant.
  • the selected hybridoma lines were subcloned. Hybridoma subclones were verified by binding and blocking ELISA assay, and their isotypes were also detected.
  • Hybridoma cells of each selected lines were plated in 96-well plates at densities of 0.5, 1 and 5 cell/well. The single clones were picked and tested in binding ELISA. Three subclones of each hybridoma line were selected and froze down.
  • Antibody Isotype was identified by ELISA. Plates (Nunc) were coated with goat anti-mouse IgG1/anti-mouse IgG2a/anti-mouse IgG2b/anti-mouse IgG3/anti-mouse IgM antibodies at 1 ⁇ g/mL overnight at 4 °C. After blocking and washing, the hybridoma supernatants were transferred to the coated plates and incubate at room temperature for 1 h. The plates were then incubated with secondary antibody goat anti-mouse kappa HRP or goat anti mouse lambda HRP (Southern Biotech) for 45 min. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device) .
  • RNA from hyridoma cell using Trizol reagent (Invitrogen-15596018) .
  • cDNA was amplifiedy using 5’-RACE kit (Takara-28001488) , followed by PCR amplification using 3’-degenerated primers and 3’-adaptor primers (ExTaq: Takara-RR001B) .
  • PCR fragments was inserted into pMD18-T vector (Takara-D 101C) and sent for sequencing (Shanghai Biosune) .
  • variable region amino acid sequence of murine anti-RankL antibodies were shown in Table 4, and the variable region DNA sequence was shown in Table 5.
  • the underline sequence is CDR1-3 separately.
  • the antibodies WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20 and WBP114_7.80 exhibited sub-nanomolar binding activity and their EC 50 values are comparable with Prolia.
  • the binding EC 50 and maximum binding values were summarized in Table 6 and Figure 1.
  • the antibodies WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20 and WBP114_7.80 can blocking RANKL binding to RANK.
  • WBP114.5.2.1, WBP114_5.14.1, and WBP114_7.80 showed comparable blocking activity with Prolia.
  • the inhibition IC 50 values were summarized in Table 7 and Figure 2.
  • ELISA plates (Nunc) were coated with mouse RankL at 5 ⁇ g/mL overnight at 4 °C. After blocking and washing, 1 ⁇ g/mL antibody samples or Prolia were added and incubated for 1 h. The plates were then washed and subsequently incubated with secondary antibody anti-mouse IgG Fc HRP/anti-human IgG Fc HRP (Bethyl) for 1 h. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device) . Binding to murine RANKL was detected by ELISA.
  • Antibody WBP114_5.20 can bind to murine RANKL ( Figure 3) with an EC 50 of 0.067 nM.
  • WBP114.5.2.1, WBP114_5.14.1, WBP114_7.80 and Prolia do not bind to to mouse RANKL (data not shown) .
  • the ability of antibody to neutralize RANKL-mediated cellular function was measured using an engineered HK293 cell line that contains RANK and NF-kB-luc.
  • 293F cells were resuspended at a density of 5 ⁇ 10 5 cells/mL in FreeStyle 293 expression medium (Invitrogen-12338) . 1 mL/well of 293F cells suspension was transfered into 24-well plate.
  • pGl-NF-kB vector was diluted in Opti-MEM I Reduced Serum Medium (Invitrogen-31985) .
  • PlasFect was diluted in Opti-MEM I Reduced Serum Medium. After 5 minutes incubation, combine the diluted pGl-NF-kB and PlasFect. Mix gently and incubate for 20 minutes at room temperature. The mixture was added to the cells and Incubate at 37 °C overnight.
  • Transfected 293F-pGl-NF-kB cells were seeded in 96-well plate (Coming 3916) at a density of 5000 cells/well in FreeStyle 293 expression medium.
  • Mixture of RANKL and various concentrations of antibodies were pre-incubating at 4 °C for 30 minutes and added to the cells. The final concentration of RANKL was 300 ng/mL.
  • 50 ⁇ L/well Nano-Glo luciferase (Promega-N1120) was added to each well and Incubated at RT for 5 min (avoid light) . The fluorescence was read by MD SpectraMax M5e.
  • Inhibition rate was calculated as [V (RANKL) -V (sample) ] / [V (RANKL) -V (blank) ] *100%.
  • V (RANKL) RFU of (cell+RANKL)
  • V (sample) RFU of (cell+RANKL+Ab)
  • V (blank) RFU of cell) .
  • RAW cell differentiation assay Inhibition of osteoclast formation by antibodies was examined using RAW cell differentiation assay.
  • RAW cells can be stimulated by RANKL to differentiate into osteoclast-like cells and the differentiation can be measured by tartrate resistant acid phosphatase (TRAP) activity.
  • RANKL RANKL
  • TRIP tartrate resistant acid phosphatase
  • RAW264.7 cells were seeded in a 96-well plate at a density of 2 ⁇ 10 4 cells/well in DMEM medium containing 10%FBS. The plate was incubated at 37 °C overnight. Mixture of RANKL and various concentrations of antibodies was added to the cells. The final concentration of RANKL was 100 ng/mL. After 4 or 5 days, medium was removed and 60 ⁇ L of lysis buffer (0.1 M citric acid, 0.1 M citrate trisodium salt, 0.1%Triton X-100) was added to the cells. 40 ⁇ L of supernatant was removed and tartrate resistant acid phosphatase was detected using TRAP Assay Kit (Beyotime Biotechnology, Cat: P0332) .
  • lysis buffer 0.1 M citric acid, 0.1 M citrate trisodium salt, 0.1%Triton X-100
  • the kinetic binding affinity of selected antibodies to human and cynomolgus monkey RANKL were tested by Biacore.
  • Antibody binding affinity to human and cynomolgus monkey RankL was detected by SPR assay using Biacore T200 (GE) .
  • GE Biacore T200
  • Each antibody was captured on a Protein A immobilized CM5 sensor chip (GE) or directly immobilized to the chip.
  • Human or cynomolgus RankL at different concentrations were injected over the sensor chip at a flow rate of 30 ⁇ L/min. The chip was regenerated by Glycine (pH1.5) after each binding cycle.
  • the sensorgrams for blank surface and buffer channel are subtracted from the test sensorgrams.
  • the experimental data was fitted by 1 ⁇ 1 model using Langmiur analysis. Molecular weight of 34 KDa was used to calculate the molar concentration of analyte.
  • the SPR result showed that the affinity to human RANKL of antibody WBP114.5.2.1, WBP114_5.14.1, WBP114_7.80 is higher than Prolia.
  • the affinity of WBP114_5.20 is slightly lower than Prolia.
  • the antibodies were binned against Prolia by ELISA.
  • ELISA plates (Nunc) were coated with Prolia overnight at 4 °C.
  • Antibodies were serially diluted starting from 10 ⁇ g/mL and mixed with 20 ng/mL RankL-His protein. After blocking and washing, the mixture was added to the plate and incubated for 1 h. The plates were then washed and subsequently incubated with secondary antibody anti-His HRP. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device) .
  • the antibodies WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20 and WBP114_7.80 were tested in competitive ELISA assay against Prolia ( Figure 6) .
  • WBP114.5.2.1, WBP114_5.14.1 and WBP114_7.80 bind to RANKL at the same or very close epitope compared with Prolia.
  • the antibody WBP114_5.20 may bind to RANKL at a different or partially overlapping epitope with Prolia.
  • V-region DNA of each murine antibody was cloned into a pcDNA3.3 vector containing human constant region gene.
  • HEK293 cell was transfected with plasmids that encode antibody heavy and light chains. Supernatant from transfected cells was harvested by removing cells and filtration.
  • Antibodies were purified by Protein A column (MabSelect SuRe, GE) and buffer exchanged into PBS. Antibody concentration was detected by Nanodrop. Purity was evaluated by SDS-PAGE (Invitrogen, NuPAGE4%-12%Bis-Tris Gel) and HPLC-SEC (Agilent) .
  • “Best Fit” approach was used to humanize antibody light and heavy chains.
  • amino acid sequences of corresponding V-genes were blasted against in-house human germline V-gene database.
  • the sequence of humanized VL-gene was derived by replacing human CDR sequences in the top hit with mouse CDR sequences using Kabat CDR definition.
  • For heavy chains 4 humanized sequences were derived.
  • First sequence was derived as for light chain.
  • Three additional sequences were created by blasting mouse frameworks against human germline V-gene database. Frameworks were defined using extended CDR definition where Kabat CDR1 was extended by 5 amino acids at N-terminus. Top three hits were used to derive sequences of humanized VH-genes.
  • Humanized genes were back-translated, codon-optimized for mammalian expression, and synthesized by GeneArt Costum Gene Synthesis (Life Technologies) . Synthetic genes were re-cloned into IgG expression vector, expressed, and purified. The variable region sequence of humanized anti-RankL antibodies were shown in Table 12 and 13.
  • the humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 exhibited comparable binding activity with Prolia.
  • the binding EC 50 values were summarized in Table 14.
  • the humanized antibodies can block RANKL binding to RANK.
  • WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 showed comparable blocking activity with Prolia.
  • the inhibition IC 50 values were summarized in Table 15.
  • the humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 maintained their original affinity to human RankL.
  • the affinity of WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 to human RankL is more than 10-fold higher than Prolia (Table 16) .
  • the humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 showed pico-molar avidity to cynomolgus monkey RankL.

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Abstract

La présente invention concerne des anticorps monoclonaux RANKL, en particulier des anticorps monoclonaux humanisés se liant de manière spécifique à RANKL avec une affinité élevée. La présente invention concerne également des anticorps monoclonaux fonctionnels ayant une réactivité croisée vis-à-vis de RANKL chez l'homme, le singe cynomolgus et la souris. La présente invention concerne en outre des séquences d'acides aminés codant pour les anticorps selon l'invention, des vecteurs de clonage ou d'expression, des cellules hôtes et des procédés pour exprimer ou isoler les anticorps. Selon l'invention, les épitopes des anticorps sont identifiés. L'invention concerne en outre des compositions thérapeutiques comprenant les anticorps selon l'invention. L'invention concerne également des méthodes de traitement de cancers et d'autres maladies avec des anticorps anti-RANKL.
PCT/CN2018/111961 2017-10-27 2018-10-25 Anticorps thérapeutique ciblant rankl WO2019080909A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
CN111793135A (zh) * 2020-05-11 2020-10-20 廊坊天光生物技术有限公司 一种用于检测血清中rankl含量的抗体对及其用途
WO2021115465A1 (fr) * 2019-12-13 2021-06-17 Biosion Inc. Anticorps se liant à rankl et leurs utilisations
CN117264071A (zh) * 2023-11-22 2023-12-22 江苏迈威康新药研发有限公司 一种抗rankl单克隆抗体或其衍生物的结合剂及其应用
CN117285637A (zh) * 2023-11-22 2023-12-26 江苏迈威康新药研发有限公司 一种抗独特型抗体及其应用

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WO2014159430A1 (fr) * 2013-03-14 2014-10-02 Apexigen, Inc. Anticorps anti-rankl et leurs procédés d'utilisation
EP3085709A1 (fr) * 2014-12-28 2016-10-26 Genor Biopharma Co., Ltd Anticorps anti-rankl humain humanisé, composition pharmaceutique et son utilisation

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WO2014159430A1 (fr) * 2013-03-14 2014-10-02 Apexigen, Inc. Anticorps anti-rankl et leurs procédés d'utilisation
EP3085709A1 (fr) * 2014-12-28 2016-10-26 Genor Biopharma Co., Ltd Anticorps anti-rankl humain humanisé, composition pharmaceutique et son utilisation

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021115465A1 (fr) * 2019-12-13 2021-06-17 Biosion Inc. Anticorps se liant à rankl et leurs utilisations
CN111793135A (zh) * 2020-05-11 2020-10-20 廊坊天光生物技术有限公司 一种用于检测血清中rankl含量的抗体对及其用途
CN117264071A (zh) * 2023-11-22 2023-12-22 江苏迈威康新药研发有限公司 一种抗rankl单克隆抗体或其衍生物的结合剂及其应用
CN117285637A (zh) * 2023-11-22 2023-12-26 江苏迈威康新药研发有限公司 一种抗独特型抗体及其应用
CN117285637B (zh) * 2023-11-22 2024-03-22 江苏迈威康新药研发有限公司 一种抗独特型抗体及其应用
CN117264071B (zh) * 2023-11-22 2024-03-22 江苏迈威康新药研发有限公司 一种抗rankl单克隆抗体或其衍生物的结合剂及其应用

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