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WO2025152667A1 - Anticorps monoclonal anti-cd117 humain et son utilisation - Google Patents

Anticorps monoclonal anti-cd117 humain et son utilisation

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Publication number
WO2025152667A1
WO2025152667A1 PCT/CN2024/138805 CN2024138805W WO2025152667A1 WO 2025152667 A1 WO2025152667 A1 WO 2025152667A1 CN 2024138805 W CN2024138805 W CN 2024138805W WO 2025152667 A1 WO2025152667 A1 WO 2025152667A1
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WIPO (PCT)
Prior art keywords
seq
cdr
amino acid
human
acid sequence
Prior art date
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Pending
Application number
PCT/CN2024/138805
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English (en)
Chinese (zh)
Inventor
裘霁宛
孔永�
陈卫
周毅
刘玉枝
李望
孙蕊
陈涛
乔怀耀
吴亦亮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Qyuns Therapeutics Co Ltd
Original Assignee
Jiangsu Qyuns Therapeutics Co Ltd
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Publication of WO2025152667A1 publication Critical patent/WO2025152667A1/fr
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Classifications

    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • 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
    • 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
    • A61P37/08—Antiallergic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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
    • 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 application relates to the field of antibody drugs. Specifically, the present application relates to monoclonal antibodies against human CD117 and their applications.
  • Chronic urticaria is a common disease that is primarily driven by mast cells and has a significant negative impact on the patient's quality of life.
  • the signs and symptoms of chronic urticaria are caused by the activation and degranulation of skin mast cells (MCs) and the subsequent release of mediators that cause sensory nerve activation, vasodilation, extravasation, and recruitment of circulating inflammatory cells (including eosinophils and basophils).
  • Mast cells are bone marrow-derived cells that are present in many organs and tissues of the human body. They contain a large number of pre-existing and newly formed secretory granules and have unique pleiotropic effects.
  • CD117 also known as c-Kit
  • c-Kit is a cell membrane protein encoded by the c-kit proto-oncogene and belongs to the type III receptor tyrosine kinase family. It is mainly distributed on the surface of hematopoietic stem cells, mast cells, and most gastrointestinal stromal tumor cells, melanoma cells, and other cells.
  • the CD117 protein contains 976 amino acids, and its structure consists of an extracellular region, a transmembrane region, and a cytoplasmic region. The extracellular region includes five immunoglobulin-like (Immunoglobulin, Ig) domains.
  • SCF stem cell factor
  • CD117 When CD117 binds to SCF, it homodimerizes and activates its intrinsic tyrosine kinase activity, which then triggers the initiation of multiple signal transduction pathways, namely the phosphatidylinositol 3-kinase (PI3-K) pathway, the Janus kinase pathway, the signal transducer and activator of transcription (STAT) pathway, and the mitogen-activated protein kinase (MAPK) pathway.
  • PI3-K phosphatidylinositol 3-kinase
  • STAT signal transducer and activator of transcription
  • MAPK mitogen-activated protein kinase
  • the c-Kit protein When the c-kit gene is abnormally expressed in mast cells, the c-Kit protein can be spontaneously phosphorylated even without binding to SCF, causing abnormal activation of the SCF/c-Kit signal, regulating the maturation, migration and proliferation, degranulation, and release of inflammatory mediators of mast cells, mediating pathological inflammatory responses, and participating in the pathological process of chronic inflammatory and autoimmune diseases such as urticaria, nodular prurigo, and eosinophilic esophagitis.
  • chronic inflammatory and autoimmune diseases such as urticaria, nodular prurigo, and eosinophilic esophagitis.
  • CDX-0159 Celldex Therapeutics' Barzolvolimab
  • CSU chronic spontaneous urticaria
  • the purpose of the present application is to provide a novel anti-human CD117 monoclonal antibody, a pharmaceutical composition comprising the monoclonal antibody, and the pharmaceutical use of the monoclonal antibody.
  • this application involves the following:
  • An anti-human CD117 monoclonal antibody comprising three heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and three light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, wherein:
  • CDR-H1 represents heavy chain CDR1
  • NKDVMG amino acid sequence of CDR-H1 (in this specification, CDR-H1 represents heavy chain CDR1)
  • CDR-H2 represents heavy chain CDR2
  • SEQ ID No: 2 GIYTGSGSTYYASWAKG
  • CDR-H3 represents heavy chain CDR3
  • SEQ ID No: 3 (DLFYSNYYNL)
  • CDR-L1 represents light chain CDR1
  • SEQ ID No: 4 QASESISNYLS
  • CDR-L2 represents light chain CDR2
  • SEQ ID No: 5 KASTLAS
  • CDR-L3 represents light chain CDR3
  • SEQ ID No:6 QNTYVSSGSIT
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:7 (EVQLVESGGGLVQPGGSLRLSCAASGFSFSNKDVMGWVRQAPGKGLEWIAGIYTGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLFYSNYYNLWGQGTLVTVSS), or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No:7;
  • the amino acid sequence of the light chain variable region is as shown in SEQ ID No:8 (DIQMTQSPSSVSASVGDRVTITCQASESISNYLSWYQQKPGKAPERLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNTYVSSGSITFGGGTKVEIK), or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No:8.
  • the amino acid sequence of the light chain is as shown in SEQ ID No:11 (DIQMTQSPSSVSASVGDRVTITCQASESISNYLSWYQQKPGKAPERLIYKASTLASGVPSRFSGSGTDFTLTISSLQPEDFATYYCQNTYVSSGSITFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC), or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No:11.
  • a host cell comprising the nucleic acid described in item 4.
  • a method for producing a monoclonal antibody comprising culturing the host cell according to item 5 to thereby produce the monoclonal antibody according to any one of items 1 to 3.
  • a method for treating a disease associated with signal transduction mediated by human CD117 protein comprising the step of administering a therapeutically effective amount of the monoclonal antibody described in item 1 to a patient suffering from the disease.
  • the present application provides a new anti-human CD117 monoclonal antibody, which has better affinity and biological activity with human CD117 protein than the CD117 monoclonal antibodies in the prior art, such as CDX-0159 (CDX-0159 is a monoclonal antibody drug targeting CD117 developed by Celldex Therapeutics, which can produce rapid, deep and lasting responses in a Phase 1b clinical trial of moderate to severe chronic spontaneous urticaria, and has good safety).
  • CDX-0159 is a monoclonal antibody drug targeting CD117 developed by Celldex Therapeutics, which can produce rapid, deep and lasting responses in a Phase 1b clinical trial of moderate to severe chronic spontaneous urticaria, and has good safety).
  • the monoclonal antibody provided in the present application shows biological activity at the cellular level that is superior to the CD117 monoclonal antibodies in the prior art, and is expected to show good clinical effects in the prevention and treatment of related diseases.
  • Figure 1 is a schematic diagram of the nucleic acid electrophoresis results of the QX013N transient expression plasmid, where M: Marker; Band 1: 524VH-Hu35, HindIII/EcoRI; Band 2: pQXHC, HindIII/EcoRI; Band 3: PCR product 524VK-Hu17; Band 4: pQX2.3, HindIII/BsiWI.
  • FIG. 2 is a flowchart of transient expression.
  • FIG3 is a schematic diagram of the electrophoresis detection results of QX013N (HZD524-61).
  • FIG5 is a schematic diagram of the results of the neutralization activity analysis of QX013N (reporter gene method - NF-kB signal transduction).
  • Figure 6 is a schematic diagram of the results of QX013N neutralization activity analysis (FACS Blocking method-LUVA cells).
  • FIG. 7 is a schematic diagram of the binding activity of QX013N and CDX-0159 to porcine CD117.
  • an “isolated” antibody is an antibody that has been separated from the components of its natural environment.
  • the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC).
  • electrophoresis e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reversed phase HPLC.
  • monoclonal antibody means an antibody derived from a colony of substantially homologous antibodies, that is, each antibody constituting the colony is identical and/or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced in the production process of monoclonal antibody products), such variants are usually present in trace amounts.
  • polyclonal antibody products that generally include different antibodies for different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen.
  • the modifier "monoclonal” indicates that the antibody is derived from a substantially homologous antibody colony, and should not be construed as requiring the antibody to be produced by any ad hoc method.
  • the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using all or part of a transgenic animal comprising a human immunoglobulin locus, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.
  • affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by an equilibrium dissociation constant ( KD ). Affinity can be measured by common methods known in the art.
  • human CD-117 protein (also referred to as C-kit in some cases) refers to a membrane receptor derived from humans, and the amino acid sequence of its extracellular region is shown in SEQ ID NO: 9, wherein the underlined portion represents a signal peptide.
  • anti-human CD-117 monoclonal antibody means a monoclonal antibody that can bind to human CD-117 with sufficient affinity so that the monoclonal antibody can be used as a diagnostic agent and/or therapeutic agent targeting human CD-117.
  • the anti-human CD-117 monoclonal antibody of the present application may not bind to proteins unrelated to the target.
  • unrelated proteins refer to proteins other than human CD-117 as the target; here, “unrelated” means: when the binding ability of the anti-human CD-117 monoclonal antibody of the present application to human CD-117 as its target is taken as 100%, the binding ability of the anti-human CD-117 monoclonal antibody of the present application to the unrelated protein is less than 10%, for example, less than 5%, 1%, 0.1%, 0.01%, 0.001%, or 0.
  • the human CD-117 monoclonal antibody of the present application has an equilibrium dissociation constant (K D ) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 50 nM, or ⁇ 40 nM.
  • the experimental results show that the anti-human CD-117 monoclonal antibody of the present application can specifically bind to human CD-117.
  • the anti-human CD-117 monoclonal antibody of the present application can bind to CD-117 of, for example, marmosets, cynomolgus monkeys, rhesus monkeys, cats, dogs, and pigs, but may not bind to CD-117 of, for example, rats and mice.
  • the anti-human CD-117 monoclonal antibody of the present application is comparable to or superior to similar monoclonal antibody products reported on the market in many aspects of biological activity, including, for example, neutralizing the binding activity of recombinant human SCF and CD-117.
  • an anti-human CD117 monoclonal antibody which comprises three heavy chain complementary determining regions CDR-H1 (heavy chain CDR1), CDR-H2 (heavy chain CDR2), CDR-H3 (heavy chain CDR3) and three light chain complementary determining regions CDR-L1 (light chain CDR1), CDR-L2 (light chain CDR2), CDR-L3 (light chain CDR3), wherein: the amino acid sequence of CDR-H1 is as shown in SEQ ID No: 1 (NKDVMG); the amino acid sequence of CDR-H2 is as shown in SEQ ID No: 2 (NKDVMG); The sequence is shown in SEQ ID No: 2 (GIYTGSGSTYYASWAKG); the amino acid sequence of CDR-H3 is shown in SEQ ID No: 3 (DLFYSNYYNL); the amino acid sequence of CDR-L1 is shown in SEQ ID No: 4 (QASESISNYLS); the amino acid sequence of CDR-L2 is shown in
  • the anti-human CD-117 monoclonal antibody described in the present application comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein:
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:7 (EVQLVESGGGLVQPGGSLRLSCAASGFSFSNKDVMGWVRQAPGKGLEWIAGIYTGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLFYSNYYNLWGQGTLVTVSS), or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No:7;
  • the amino acid sequence of the light chain variable region is as shown in SEQ ID No:8 (DIQMTQSPSSVSASVGDRVTITCQASESISNYLSWYQQKPGKAPERLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNTYVSSGSITFGGGTKVEIK), or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No:8.
  • the anti-human CD-117 monoclonal antibody described in the present application comprises a heavy chain and a light chain, wherein:
  • amino acid sequence of the heavy chain is as shown in SEQ ID No: 10, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No: 10;
  • amino acid sequence of the light chain is as shown in SEQ ID No:11, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No:11.
  • isolated nucleic acid means a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain nucleic acid molecules, but the nucleic acid molecules are present outside the chromosome or at a chromosomal location different from its natural chromosomal location.
  • isolated nucleic acid encoding human CD-117 monoclonal antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.
  • vector means a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • host cell refers to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells.
  • Host cells include “transformants” and “transformed cells”, which include the primary transformed cells and the offspring derived therefrom (regardless of the number of generations). Offspring may not be identical to the parental cell in terms of nucleic acid content, but may contain mutations. Offspring of mutants screened or selected for the initially transformed cells with the same function or biological activity are included in this specification.
  • pharmaceutical composition means a preparation which is in a form that enables the biological activity of the active ingredient contained therein to exert its effect and which does not contain additional components that are unacceptably toxic to the subject to which the preparation is to be administered.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition other than an active ingredient, which is non-toxic to a subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • “monoclonal antibody” generally refers to human antibodies, which can be prepared using techniques known to those skilled in the art.
  • human antibodies are generally described in van Dijk, M.A. and van de Winkel, J.G., Curr. Opin. Pharmacol. 5:368-374 (2001) and Lonberg, N., Curr. Opin. Immunol. 20:450-459 (2008).
  • Antibodies can be prepared by administering immunogens to transgenic animals that have been modified to stimulate the production of complete human antibodies or complete antibodies with human variable regions in response to antigenic challenge. These animals typically contain a portion or all of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or are present outside the chromosomes or randomly integrated into the animal. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated.
  • transgenic animals see Lonberg, N., Nat. Biotech. (Nature Biotechnology) 23: 1117-1125 (2005). See also, for example, the XENOMOUSETM technology described in U.S. Pat. Nos.
  • Human antibodies can also be prepared by hybridoma-based methods.
  • Human myeloma and mouse-human hybrid myeloma cells for producing human monoclonal antibodies have been described (see, e.g., Kozbor, D., J. Immunol. 133: 3001-3005 (1984); Brodeur, B. R. et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; Boerner, P. et al., J. Immunol. 147: 86-95 (1991)).
  • Human antibodies produced via human B cell hybridoma technology are also described in Li, J. et al., Proc. Natl. Acad. Sci.
  • Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries, and such variable domain sequences can then be combined with the desired human constant domains.
  • Human antibodies can also be selected based on antibody libraries, that is, human antibodies can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods for producing phage display libraries and screening such libraries for antibodies with the desired binding characteristics are known in the art. This method is reviewed in, for example, Hoogenboom, H.R. et al., Methods in Molecular Biology 178:1-37 (2001), and is further described in, for example, McCafferty, J. et al., Nature 348:552-554 (1990); Clackson, T. et al., Nature 352:624-628 (1991); Marks, J.D. et al., J. Mol. Biol.
  • phage display methods the repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which is then screened for antigen-binding phage, as described in Winter, G. et al., Ann. Rev. Immunol. 12: 433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas.
  • PCR polymerase chain reaction
  • non-immunized repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a large number of non-self and also self-antigens without any immunization, as described by Griffiths, A.D. et al., EMBO J, 12: 725-734 (1993).
  • unimmunized libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom, H.R. and Winter, G., J. Mol. Biol. 227:381-388 (1992).
  • Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.
  • the antibody may also be a multispecific antibody, such as a bispecific antibody.
  • a bispecific antibody is a monoclonal antibody with binding specificity to at least two different sites.
  • Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of immunoglobulin heavy chain-light chains with different specificities (see Milstein, C. and Cuello, A.C., Nature 305: 537-540 (1983); WO 93/08829; and Traunecker, A. et al., EMBO J. 10: 3655-3659 (1991)) and "node-into-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168).
  • the monoclonal antibodies described herein also include engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies” (see, e.g., US 2006/0025576).
  • the antibodies herein may also include the multispecific antibodies described in WO2009/080251, WO2009/080252, WO2009/080253, WO2009/080254, WO2010/112193, WO2010/115589, WO2010/136172, WO2010/145792, WO2010/145793, WO2011/117330, WO2012/025525, WO2012/025530, WO2013/026835, WO2013/026831, WO2013/164325 or WO2013/174873.
  • the monoclonal antibodies described herein may also be antibody variants, for example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • the amino acid sequence variants of the antibody may be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and/or insertion and/or substitution of residues in the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution may be performed to obtain the final construct, as long as the final construct has the desired characteristics, such as antigen binding.
  • antibody variants with one or more amino acid substitutions are provided, and the sites of interest for substitution mutations include HVR and FR, for example, amino acid substitutions may be introduced into the antibody of interest and screened for products with desired activity, for example, retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • the reagents and instruments used in the following examples are conventional reagents and instruments in the art and can be obtained commercially.
  • the experimental methods used are conventional methods unless otherwise specified, and those skilled in the art can undoubtedly implement the scheme and obtain corresponding results according to the contents of the examples.
  • the human CD117 antigen produced by Quanxin Biotechnology was used as an immunogen to immunize New Zealand rabbits.
  • the monoclonal antibodies with antigen binding specificity were obtained using B cell cloning technology, and then the monoclonal antibodies that bind to human CD117 and have the ability to inhibit human CD117 activity were screened.
  • the target clones were selected through analysis and screening by Binding ELISA and Blocking ELISA. The above immunization and screening processes were entrusted to commercial companies.
  • clone 524 was humanized. NCBI IgBlast was used to perform homology alignment of human IgG germline sequences, and IGHV3-66*01 was selected as the heavy chain CDR transplantation template, and the CDR region of the 524# clone heavy chain (i.e., CDR-H1 (SEQ ID No: 1), CDR-H2 (SEQ ID No: 2), and CDR-H3 (SEQ ID No: 3)) was transplanted into the framework region of IGHV3-66*01; IGKV1-12*01 was selected as the light chain CDR transplantation template, and the CDR region of the 524# clone light chain (i.e., CDR-L1 (SEQ ID No: 4), CDR-L2 (SEQ ID No: 5), and CDR-L3 (SEQ ID No: 6)) was transplante
  • the heavy chain gene (SEQ ID NO: 10) was synthesized by GenScript and obtained by double digestion with HindIII and EcoRI.
  • the light chain variable region gene (SEQ ID NO: 8) was obtained by PCR amplification.
  • the heavy chain expression plasmid pQXHC was double digested with HindIII and EcoRI; the light chain expression plasmid pQX2.3 was double digested with HindIII and BsiWI; the heavy chain gene was inserted into the heavy chain expression plasmid using T4 ligase to construct the heavy chain expression plasmid pQXHC-524VH-Hu35; the light chain PCR amplified gene was inserted into the light chain expression plasmid using Infusion recombinase to construct the light chain expression plasmid pQX2.3-524VK-Hu17.
  • the heavy chain expression plasmid pQXHC-524VH-Hu35 (the amino acid sequence of the expressed heavy chain is shown in SEQ ID NO: 10) and the light chain expression plasmid pQX2.3-524VK-Hu17 (the amino acid sequence of the expressed light chain is shown in SEQ ID NO: 11) with the correct sequence were co-transfected into ExpiCHO-S cells.
  • ExpiCHO-S cells were diluted to 3 ⁇ 10 6 cells/mL for pre-transfection passage.
  • the cell density was diluted to 6 ⁇ 10 6 cells/mL, and 25 mL of cells were placed in a 125 mL shake flask and waited for transfection.
  • the transfection and expression process is shown in Figure 2.
  • the affinity of QX013N (HZD524-61) to human CD117 was detected by Biacore T200, and all processes were carried out at 25°C.
  • the commercial Sensor chip CM5 chip was used to fix an appropriate amount of antibody by capture method, so that Rmax was around 50RU, and the capture flow rate was 10 ⁇ L/min.
  • the antigen was gradient diluted, and the instrument flow rate was switched to 30 ⁇ L/min.
  • the reference channel and the channel of the fixed antibody were flowed in order from low to high concentration, and the buffer was flowed as a negative control. After each binding and dissociation, the chip was regenerated with pH1.5 glycine.
  • the 1:1 binding model in the Kinetics option was selected in the instrument's own analysis software for fitting, and the antibody binding rate constant k a , dissociation rate constant k d and dissociation equilibrium constant K D values were calculated.
  • CDX-0159 a monoclonal antibody against human CD117 that is currently in Phase 2 clinical trials.
  • the detection method for known antibodies was the same as that for QX013N, and the results are shown in Table 1.
  • CDX-0159 was constructed based on the sequence provided by patent NZ630363B, and was transiently transfected into ExpiCHO-S cells to obtain the product.
  • the data in the table are: each sample was tested three times and the average value was calculated.
  • M07e cells were used to determine the cell proliferation activity induced by QX013N and CDX-0159 in neutralizing recombinant human SCF.
  • M07E was plated into 96-well plates at 4 ⁇ 10 4 cells per well, and the antibody was diluted to a concentration range of 0 to 10 ⁇ g/mL. After being added to the cells and incubated for 1 hour, 50 ng/mL of recombinant human SCF was added and mixed, and the cells were cultured at 37°C and 5% CO 2 for 24 hours.
  • Example 4 QX013N and CDX-0159 neutralize NF-kB signaling activity induced by recombinant human SCF in HEK293 cells overexpressing human CD117 reporter gene
  • Example 5 QX013N and CDX-0159 neutralize the binding activity of recombinant human SCF to CD117 on the surface of LUVA cell membrane

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Abstract

La présente invention concerne un anticorps monoclonal anti-CD117 humain et son utilisation. L'anticorps monoclonal anti-CD117 humain de la présente invention comprend trois régions déterminant la complémentarité de chaîne lourde CDR-H1, CDR-H2 et CDR-H3 ayant des séquences d'acides aminés telles que représentées dans SEQ ID NO 1-3, respectivement, et trois régions déterminant la complémentarité de chaîne légère CDR-L1, CDR-L2 et CDR-L3 ayant des séquences d'acides aminés telles que représentées dans SEQ ID NO 4-6, respectivement. L'anticorps présente une bonne activité au niveau cytologique.
PCT/CN2024/138805 2024-01-15 2024-12-12 Anticorps monoclonal anti-cd117 humain et son utilisation Pending WO2025152667A1 (fr)

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