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WO2025044998A1 - Anticorps à domaine unique ciblant le polypeptide prame et son utilisation - Google Patents

Anticorps à domaine unique ciblant le polypeptide prame et son utilisation Download PDF

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Publication number
WO2025044998A1
WO2025044998A1 PCT/CN2024/114621 CN2024114621W WO2025044998A1 WO 2025044998 A1 WO2025044998 A1 WO 2025044998A1 CN 2024114621 W CN2024114621 W CN 2024114621W WO 2025044998 A1 WO2025044998 A1 WO 2025044998A1
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Prior art keywords
prame
antibody targeting
prame polypeptide
single domain
seq
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Chinese (zh)
Inventor
王文博
刘洪川
汤贝贝
冯爱华
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Liling Biopharmaceuticals Co Ltd
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Liling Biopharmaceuticals Co Ltd
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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
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001189—PRAME
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K16/2809—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 against the T-cell receptor (TcR)-CD3 complex
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/50—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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the field of biotechnology. More specifically, the present invention relates to a single domain antibody targeting a PRAME polypeptide and uses thereof.
  • PRAME PReferentially expressed Antigen in MElanoma
  • PRAME is an intracellular protein encoded by the PRAME gene.
  • PRAME is expressed at high levels in many tumors, such as melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc., and is rarely expressed in normal human tissues. These characteristics make PRAME an ideal target for tumor targeted therapy (Am J Surg Pathol. 2018Nov; 42(11):1456-1465.).
  • the PRAME protein After being processed by the intracellular antigen presentation system, the PRAME protein has a special polypeptide sequence: SLLQHLIGL, which can be presented to the cell surface by the major histocompatibility antigen HLA-A02 molecule.
  • the HLA-A02 complex bound to the PRAME polypeptide can be used as a cell membrane target to develop T cell receptor (T cell receptor, TCR) or TCR-like antibody-related therapies.
  • Single-domain antibodies are a special type of antibody with a smaller molecular weight.
  • Single-domain antibodies are composed of only two identical heavy chains. Compared with the molecular weight of 150-160kDa of traditional double-chain antibodies, the molecular weight of single-domain antibodies is about 110KD.
  • Single-domain antibodies generally have high specificity, high affinity, low immunogenicity, and good permeability.
  • the antigen binding region of a single-domain antibody is composed of only one chain, and the variable region (VHH) of a single-domain antibody is only 12-15kDa.
  • single-domain antibodies as the antigen recognition region of bispecific antibodies or chimeric antigen receptor T cells (Chimeric Antigen Receptor-T cells, CAR-T) is one of the future development trends (Serge Muyldermans. Annu. Rev. Biochem. 82: 775-797 (2013)).
  • Single domain antibodies can recognize cell membrane proteins, or peptides derived from intracellular proteins presented to the cell surface by major histocompatibility antigens. Using the HLA-A02 complex bound to the PRAME peptide as an antigen, specific single domain antibody molecules can be screened. These candidate molecules can be used to develop bispecific antibodies, CAR-T or ADC (Antibody drug conjugate) and other biological drugs.
  • the object of the present invention is to provide a specific single domain antibody targeting PRAME polypeptide and corresponding specific humanized single domain antibody, bispecific antibody, chimeric antigen receptor, chimeric antigen receptor-T cell and ADC targeting PRAME polypeptide.
  • the present invention also aims to provide the use of the single domain antibody, humanized single domain antibody, bispecific antibody, chimeric antigen receptor, chimeric antigen receptor-T cell and ADC in treating tumors or preparing drugs for treating tumors.
  • the present invention provides a VHH chain of a single domain antibody targeting a PRAME polypeptide, wherein the VHH chain comprises CDR1, CDR2 and CDR3 as shown in the following table:
  • amino acid sequence of the PRAME polypeptide is: SLLQHLIGL (SEQ ID NO: 121).
  • any one of the above amino acid sequences further comprises a derivative sequence which is optionally subjected to addition, deletion, modification and/or substitution of at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and can retain high affinity binding to the PRAME polypeptide.
  • a derivative sequence which is optionally subjected to addition, deletion, modification and/or substitution of at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and can retain high affinity binding to the PRAME polypeptide.
  • the VHH chain further comprises framework regions FR1, FR2, FR3 and FR4.
  • amino acid sequence of the VHH chain of the single domain antibody targeting the PRAME polypeptide is shown in the following table:
  • the present invention provides a heavy chain variable region of an antibody targeting a PRAME polypeptide, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the following table:
  • amino acid sequence of the heavy chain variable region of the antibody targeting the PRAME polypeptide is shown in the following table:
  • the present invention provides a single domain antibody targeting a PRAME polypeptide, which has the VHH chain described in the first aspect.
  • the present invention provides a humanized VHH chain of a single domain antibody targeting a PRAME polypeptide, wherein the framework regions FR1, FR2, FR3 and FR4 are humanized based on the VHH chain described in the first aspect.
  • variable region sequence of the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide is as follows:
  • the present invention provides an antibody targeting a PRAME polypeptide, wherein the antibody comprises one or more VHH chains of the single domain antibody targeting a PRAME polypeptide according to the first aspect or the VHH chain of the humanized single domain antibody targeting a PRAME polypeptide according to claim 4.
  • the antibody targeting the PRAME polypeptide comprises a monomer, a bivalent antibody, and/or a multivalent antibody.
  • the present invention provides a bispecific antibody, comprising a first antibody and a second antibody, wherein the first antibody comprises the VHH chain of the single domain antibody targeting the PRAME polypeptide described in the first aspect, or the heavy chain variable region of the antibody targeting the PRAME polypeptide described in the second aspect, or the single domain antibody targeting the PRAME polypeptide described in the third aspect, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide described in the fourth aspect, or the antibody targeting the PRAME polypeptide described in the fifth aspect.
  • the second antibody may bind to the same or a different antigen as the first antibody, or bind to a different epitope on the same antigen as the first antibody.
  • the second antibody is a single domain antibody, a single chain antibody or a double chain antibody.
  • the bispecific antibody comprises 2-4 single-domain antibodies targeting PRAME polypeptide; preferably, it comprises 2 single-domain antibodies targeting PRAME polypeptide; more preferably, the 2 single-domain antibodies targeting PRAME polypeptide form a single-domain antibody dimer targeting PRAME polypeptide.
  • the sequence of the bispecific antibody is shown in the following table:
  • the present invention provides a fusion protein, the fusion protein comprising the VHH chain of the single-domain antibody targeting the PRAME polypeptide according to the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide according to the second aspect, the single-domain antibody targeting the PRAME polypeptide according to the third aspect, and the humanized PRAME targeting antibody according to the fourth aspect.
  • the VHH chain of a single domain antibody of the polypeptide, or the antibody targeting the PRAME polypeptide as described in the fifth aspect an optional linker sequence and an Fc fragment or a half-life extension domain of an immunoglobulin.
  • the immunoglobulin is IgG1, IgG2, IgG3, IgG4; preferably IgG4.
  • the present invention provides a chimeric antigen receptor, which is made of the VHH chain of the single domain antibody targeting the PRAME polypeptide described in the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide described in the second aspect, the single domain antibody targeting the PRAME polypeptide described in the third aspect, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide described in the fourth aspect, or the antibody targeting the PRAME polypeptide described in the fifth aspect.
  • amino acid sequence of the chimeric antigen receptor is as follows:
  • the present invention provides an immune effector cell, wherein the immune effector cell expresses the chimeric antigen receptor described in the eighth aspect.
  • the immune effector cells include but are not limited to: T cells, NK cells, TIL cells; preferably T cells.
  • the present invention provides a nucleic acid molecule, which encodes the VHH chain of the single domain antibody targeting the PRAME polypeptide described in the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide described in the second aspect, the single domain antibody targeting the PRAME polypeptide described in the third aspect, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide described in the fourth aspect, the antibody targeting the PRAME polypeptide described in the fifth aspect, the bispecific antibody described in the sixth aspect, the fusion protein described in the seventh aspect, or the chimeric antigen receptor described in the eighth aspect.
  • the present invention provides an expression vector comprising the nucleic acid molecule described in the tenth aspect.
  • the present invention provides a host cell, wherein the host cell comprises the expression vector described in the eleventh aspect, or the nucleic acid molecule described in the tenth aspect is integrated into its genome.
  • the present invention provides a method for preparing the VHH chain of the single domain antibody targeting the PRAME polypeptide of the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide of the second aspect, the single domain antibody targeting the PRAME polypeptide of the third aspect, the humanized VHH chain of the single domain antibody targeting the PRAME polypeptide of the fourth aspect, the antibody targeting the PRAME polypeptide of the fifth aspect, the bispecific antibody of the sixth aspect, or the fusion protein of the seventh aspect, the method comprising the following steps:
  • the present invention provides an immunoconjugate, the immunoconjugate comprising:
  • a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
  • the conjugated moiety is a drug or a toxin.
  • the conjugated moiety is a detectable label.
  • the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.
  • fluorescent or luminescent markers for example, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments
  • the immunoconjugate contains: a multivalent (e.g., bivalent) VHH chain of a single domain antibody targeting a PRAME polypeptide as described in the first aspect, a heavy chain variable region of an antibody targeting a PRAME polypeptide as described in the second aspect, a single domain antibody targeting a PRAME polypeptide as described in the third aspect, a humanized VHH chain of a single domain antibody targeting a PRAME polypeptide as described in the fourth aspect, an antibody targeting a PRAME polypeptide as described in the fifth aspect, a bispecific antibody as described in the sixth aspect, or a fusion protein as described in the seventh aspect.
  • a multivalent (e.g., bivalent) VHH chain of a single domain antibody targeting a PRAME polypeptide as described in the first aspect a heavy chain variable region of an antibody targeting a PRAME polypeptide as described in the second aspect
  • a single domain antibody targeting a PRAME polypeptide as described in the third aspect a humanized VHH chain of a single domain antibody targeting
  • the multivalency refers to the presence of multiple repeating parts in the amino acid sequence of the immunoconjugate.
  • the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising a therapeutically or diagnostically effective amount of the VHH chain of the single-domain antibody targeting the PRAME polypeptide according to the first aspect, the VHH chain of the single-domain antibody targeting the PRAME polypeptide according to the second aspect, The heavy chain variable region of an antibody targeting a PRAME polypeptide, the single domain antibody targeting a PRAME polypeptide as described in the third aspect, the VHH chain of the humanized single domain antibody targeting a PRAME polypeptide as described in the fourth aspect, the antibody targeting a PRAME polypeptide as described in the fifth aspect, the bispecific antibody as described in the sixth aspect, the fusion protein as described in the seventh aspect, the chimeric antigen receptor as described in the eighth aspect, the immune effector cell as described in the ninth aspect, or the immunoconjugate as described in the fourteenth aspect, and optional pharmaceutically acceptable excipients.
  • the pharmaceutical composition is used to treat tumors, and the tumors are PRAME polypeptide-related tumors; preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc.
  • the present invention provides the use of the VHH chain of the single domain antibody targeting the PRAME polypeptide of the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide of the second aspect, the single domain antibody targeting the PRAME polypeptide of the third aspect, the humanized VHH chain of the single domain antibody targeting the PRAME polypeptide of the fourth aspect, the antibody targeting the PRAME polypeptide of the fifth aspect, the bispecific antibody of the sixth aspect, the fusion protein of the seventh aspect, the chimeric antigen receptor of the eighth aspect, the immune effector cell of the ninth aspect or the immunoconjugate of the fourteenth aspect for preparing the following reagents:
  • the tumor is a PRAME polypeptide-associated tumor; preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc.
  • the present invention provides a kit, comprising:
  • the present invention provides a method for detecting PRAME polypeptide protein in a sample, the method comprising the steps of:
  • the sample to be tested is mixed with the VHH chain of the single domain antibody targeting the PRAME polypeptide described in the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide described in the second aspect, the single domain antibody targeting the PRAME polypeptide described in the third aspect, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide described in the fourth aspect, the antibody targeting the PRAME polypeptide described in the fifth aspect, the bispecific antibody described in the sixth aspect, the fusion protein described in the seventh aspect, or the Contacting with the immunoconjugate of aspect 14;
  • the present invention provides a method for treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the VHH chain of the single domain antibody targeting the PRAME polypeptide of the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide of the second aspect, the single domain antibody targeting the PRAME polypeptide of the third aspect, the humanized VHH chain of the single domain antibody targeting the PRAME polypeptide of the fourth aspect, the antibody targeting the PRAME polypeptide of the fifth aspect, the bispecific antibody of the sixth aspect, the fusion protein of the seventh aspect, the chimeric antigen receptor of the eighth aspect, the immune effector cell of the ninth aspect, the immunoconjugate of the fourteenth aspect, or the pharmaceutical composition of the fifteenth aspect.
  • the subject comprises a mammal; preferably a human.
  • the disease is a PRAME polypeptide-related disease; preferably a PRAME polypeptide-related tumor; more preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc.
  • FIG1 shows that the 14 single-domain antibodies of the present invention are all able to bind to protein antigens with high affinity
  • FIG2 shows that the 14 single domain antibodies of the present invention are all able to bind to antigens at the cellular level and have high affinity
  • FIG3 shows that the single domain antibody molecules of the present invention have good specificity
  • FIG4 shows that the bispecific antibody molecule of the present invention has good cell killing activity
  • FIG5 shows that the CAR molecules of the present invention can be efficiently expressed on the surface of T cells
  • FIG6 shows the killing effect of candidate CAR-T cells on target cells under different effector-target ratios
  • FIG7 shows that the CAR-T cells of the present invention can effectively recognize T2 loaded with target polypeptide, activate and transmit immune signals, and secrete IFN- ⁇ cytokine;
  • Figure 8 shows that the humanized single-domain antibodies obtained after humanization of the LL-PR001 molecule are all able to bind to protein antigens with high affinity
  • FIG9 shows that the humanized single-domain antibodies obtained after humanization of the LL-PR004 molecule are all able to bind to protein antigens with high affinity.
  • the inventors unexpectedly discovered a class of single-domain antibodies targeting PRAME polypeptide.
  • the single domain antibody of the present invention can bind to the PRAME polypeptide with high affinity and has good specificity.
  • the present invention also provides bispecific antibodies, chimeric antigen receptors, chimeric antigen receptor-T cells, and ADCs prepared using the single domain antibody. The present invention is completed on this basis.
  • single domain antibody Single domain antibody
  • single domain antibody single domain antibody
  • VHH single domain antibody
  • the single-domain antibody VHH chain targeting the PRAME polypeptide of the present invention further comprises framework regions FR1, FR2, FR3 and FR4.
  • the present inventors On the basis of the single domain antibody VHH chain targeting the PRAME polypeptide of the present invention, the present inventors also humanized the VHH chain, thereby obtaining a humanized single domain antibody VHH chain targeting the PRAME polypeptide.
  • the present invention also provides an antibody targeting the PRAME polypeptide, which includes one or more VHH chains of the single-domain antibody targeting the PRAME polypeptide or the VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide.
  • the present invention also provides a bispecific antibody, the bispecific antibody includes a first antibody and a second antibody, the first antibody can be the VHH chain of the single-domain antibody targeting the PRAME polypeptide of the present invention, or the humanized VHH chain.
  • the bispecific antibody includes a first antibody and a second antibody
  • the first antibody can be the VHH chain of the single-domain antibody targeting the PRAME polypeptide of the present invention, or the humanized VHH chain.
  • Those skilled in the art can select the second antibody in the bispecific antibody according to actual needs.
  • the second antibody can bind to the same or different antigen as the first antibody; if the second antibody binds to the same antigen as the first antibody, it is preferably bound to different epitopes.
  • the second antibody can be a single-domain antibody, a single-chain antibody or a double-chain antibody.
  • the single-domain antibody VHH chain or humanized VHH chain targeting the PRAME polypeptide of the present invention into a fusion protein, for example, a fusion protein further comprising an Fc fragment of an immunoglobulin or a half-life extension domain.
  • the fusion protein thus obtained not only has the biological activity of the single-domain antibody VHH chain itself, but also has other characteristics conferred by the Fc fragment of the immunoglobulin, such as extended plasma half-life, reduced immunogenicity, improved stability, and the like.
  • the fusion protein comprises the VHH chain or humanized VHH chain of the single-domain antibody targeting the PRAME polypeptide of the present invention, an optional linker sequence, and an Fc fragment of an immunoglobulin.
  • the immunoglobulin is IgG1, IgG2, IgG3, IgG4; preferably IgG4.
  • the half-life extension domain is as shown in the following amino acid sequence:
  • the present invention includes not only complete antibodies, but also fragments, derivatives and analogs of the antibodies.
  • fragments refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention.
  • polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.
  • the antibody of the present invention refers to a polypeptide having PRAME polypeptide protein binding activity and including the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • amino acids with similar or similar properties are substituted, the function of the protein is usually not changed.
  • adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the present invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.
  • the present invention also includes fragments of the single domain antibodies of the present invention.
  • the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.
  • “conservative variants of the antibodies of the present invention” refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to the following table.
  • the present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein.
  • the polynucleotide of the present invention can be in the form of DNA or RNA.
  • the DNA form includes cDNA, genomic DNA or artificially synthesized DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that only encodes a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and a non-coding sequence.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may include additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short.
  • a fragment with a very long sequence can be obtained by first synthesizing multiple small fragments and then connecting them.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination.
  • the biological molecules (nucleic acids, proteins, etc.) involved in the present invention include biological molecules in an isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence.
  • These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl2 .
  • the steps used are well known in the art.
  • Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • the antibodies of the present invention can be used alone or in combination or conjugated with a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or any combination of these substances.
  • Detectable markers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product.
  • Therapeutic agents that can be combined or coupled to the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines, such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. drug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • Bispecific antibodies are recombinant antibodies that have been engineered through protein engineering. Bispecific antibodies can simultaneously target two different antibody binding epitopes, which can be from different antigens or from the same antigen. Many current studies have shown that bispecific antibodies have great therapeutic potential in the treatment of diseases such as tumors, autoimmune diseases, and viral infections. Compared with monoclonal antibodies, the main advantage of bispecific antibodies is that they can mediate the spatial effect of two recognition epitopes and the synergistic effect of dual targeting, producing biological effects that cannot be achieved by the combined use of two antibodies.
  • a relatively special bispecific antibody is called a T cell engager, which can simultaneously bind to targets and T cells on the surface of tumor cells, activate endogenous T cells, and cause tumor cell lysis, thereby achieving the purpose of treating tumors.
  • T cell engagers have been shown to be used to treat tumors.
  • Bispecific T cell engagers targeting CD20 and CD19 have been approved by the FDA for marketing (Nat Rev Clin Oncol. 2020 Jul; 17(7): 418-434.). Because of their complexity, which is different from monoclonal antibodies, bispecific antibodies have higher technical barriers and R&D costs.
  • immune cells and immune effector cells have the same meaning and are generally understood by those skilled in the art. The same as explained above. It refers to cells involved in or related to the immune response, including lymphocytes and phagocytes.
  • the immune cell refers to a lymphocyte that can recognize an antigen and thus produce a specific immune response.
  • the lymphocytes are mainly T lymphocytes, B lymphocytes, K lymphocytes and NK lymphocytes.
  • cells involved in the immune response also include plasma cells, granulocytes, mast cells, antigen presenting cells and cells of the mononuclear phagocyte system (e.g., macrophages).
  • Chimeric antigen receptor T cell therapy is a very promising cellular immunotherapy.
  • CAR-T cells express CAR (Chimeric Antigen Receptor) molecules.
  • the structure of CAR is divided into: antigen binding region, hinge region, transmembrane domain and intracellular signal transduction domain.
  • Current CAR-T cells usually use scFv (Single Chain Fragment Variables) segments derived from single-chain modification of the antigen binding region of monoclonal antibodies as antigen binding regions.
  • scFv Single Chain Fragment Variables
  • problems such as reduced affinity and changed specificity are prone to occur.
  • scFv has a relatively large molecular weight and is easy to form multimers, which affects the function of CAR (Nat Rev Cancer.
  • CAR containing an antigen binding region with a novel structure.
  • the variable region of the single-domain antibody can be directly connected to the CAR structure, which is simple and convenient to design.
  • ADC is an antibody carrying cytotoxic drugs, which can deliver cytotoxic drugs to tumor cells to achieve specific killing of tumor cells.
  • ADC drugs targeting targets such as HER2, CD30 and Trop2 have shown good clinical efficacy and safety in clinical studies (Nat Rev Clin Oncol. 2021 Jun; 18(6): 327-344.).
  • HER2, CD30 and Trop2 have shown good clinical efficacy and safety in clinical studies (Nat Rev Clin Oncol. 2021 Jun; 18(6): 327-344.).
  • a number of ADC drugs have been approved by the FDA for marketing.
  • the targets of ADC drugs are currently cell membrane proteins, and ADC drugs targeting intracellular proteins are one of the hot directions for future development.
  • ADC drugs can specifically target target cells and deliver cytotoxic molecules into target cells, thereby producing a specific killing effect.
  • Cell killing induced by cytotoxic molecules can break and improve the tumor suppressive microenvironment, and has the potential to improve the efficacy of other therapies such as immunotherapy.
  • the single-domain antibody of the present invention can be internalized into cells, so there is hope for the development of ADC drugs based on the single-domain antibody of the present invention in the future.
  • the present invention also provides an immunoconjugate, which contains the VHH chain of the single domain antibody targeting the PRAME polypeptide of the present invention, the humanized VHH chain, etc. and a coupling part.
  • the coupling part can be a detectable marker, a drug, a toxin, a cytokine, a radionuclide or an enzyme, etc., so as to achieve the purpose of diagnosis, detection or treatment, etc.
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these substances can be formulated into
  • the pharmaceutical composition is prepared in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the substance being formulated and the condition to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.
  • the pharmaceutical composition of the present invention can directly target the PRAME polypeptide expressed by tumor cells. Therefore, the pharmaceutical composition of the present invention can be used to treat tumors.
  • the tumor is a PRAME polypeptide-related tumor.
  • the tumor is melanoma, non-small cell lung cancer, ovarian cancer or breast cancer, etc.
  • the pharmaceutical composition of the present invention can also be used in combination with other therapeutic agents.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned single domain antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 10 ⁇ g/kg body weight to about 50 mg/kg body weight per day.
  • a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 ⁇ g/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dosage is about 10 ⁇ g/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.
  • the present invention also provides a kit containing the VHH chain of the single domain antibody targeting the PRAME polypeptide of the present invention, or the humanized VHH chain, antibody, fusion protein or immunoconjugate, etc.
  • the kit further comprises a container, instructions for use, a buffer, etc.
  • the single domain antibody targeting the PRAME polypeptide of the present invention binds to the PRAME polypeptide with high affinity
  • the single domain antibody targeting PRAME polypeptide of the present invention has good specificity
  • the single-domain antibody targeting the PRAME polypeptide of the present invention can be further used to prepare bispecific antibodies, chimeric antigen receptors, chimeric antigen receptor-T cells, and ADCs, thereby laying a new material foundation for the development of therapeutic or diagnostic drugs targeting the PRAME polypeptide.
  • pMHC peptide-Major histocompatibility complex
  • pMHC peptide-Major histocompatibility complex
  • the yeast library was screened using recombinantly expressed protein antigens or T2 cells loaded with target polypeptides.
  • the present invention uses a pMHC recombinant protein carrying the target polypeptide as a positive antigen and a pMHC recombinant protein carrying a negative polypeptide as a negative antigen. After three rounds of positive screening and three rounds of negative screening, a single-domain antibody clone that specifically binds to the positive antigen but not to the negative antigen was obtained.
  • the single domain antibody clones were subjected to Sanger sequencing to obtain the full-length sequence of the single domain antibody that specifically binds to the target protein of the present invention.
  • a total of 14 different single domain antibody clones were obtained in the present invention, and they were named: LL-PR001-LL-PR014.
  • the full-length amino acid sequence of the single domain antibody and the amino acid sequence of the CDR region are shown in Table 1.
  • the present invention uses conventional monoclonal antibody expression and purification methods to express and purify the single-domain antibodies in the present invention.
  • a single-domain antibody expression vector is constructed using conventional molecular cloning technology. After the vector is successfully constructed, the single-domain antibody is expressed by transient transfection of HEK293 suspension cell lines.
  • the protein concentration and total protein amount of different antibodies are detected by ultraviolet spectrophotometry, and then the expression amount of each double antibody is calculated according to the expression volume.
  • the purity of different antibodies is evaluated using the conventional gel electrophoresis SDS-PAGE method.
  • the basic steps are as follows: The samples are electrophoretically separated using the Invitrogen electrophoresis tank and SDS-PAGE gradient gel.
  • the sample is diluted to about 1mg/mL, and an appropriate amount of reducing agent, loading buffer and pure water are added. After mixing, it is heated at 70°C for about 10 minutes, the sample amount is 2-10 ⁇ g, the electrophoresis voltage is about 200V, and the electrophoresis time is about 35 minutes. After that, the gel is stained and destained respectively. After decolorization, a conventional gel imaging system is used to take pictures and analyze and calculate the purity of the main band. In addition, the purity of different antibodies was evaluated by size exclusion high performance liquid chromatography (SEC-HPLC).
  • the basic steps are as follows: dilute the sample to about 1.0 mg/mL, use a TSKgel G3000SWXL column, set the column temperature to 25°C, use 100 mM phosphate buffer, 100 mM sodium sulfate, pH 7.0 ⁇ 0.2 as the mobile phase, the injection volume is 20-50 ⁇ L, at a flow rate of 1.0 mL/min, isocratic elution for 20 min, detection at a wavelength of 280 nm, and the peak area normalization method is used to obtain the monomer content.
  • the expression and purity information of different single domain antibodies are shown in Table 2.
  • the expression of the single domain antibody of the present invention using a transient expression system is in the range of 650-850 mg/L, proving that the dual antibody structure of the present invention has a higher expression level.
  • the purity of the reduced SDS-PAGE and SEC-HPLC of most single domain antibodies of the present invention is above 95%. These results prove that the single domain structure of the present invention has a good expression level and a high purity.
  • this example uses ELISA and flow cytometry methods to detect at the protein and cell levels, so as to comprehensively compare the affinity level of the candidate molecules to the target antigen.
  • the specific operation steps are as follows:
  • Peptide loading Prepare a 60 ⁇ M peptide with 1640 medium, add 50 ⁇ L/well of the peptide dilution to the well plate and mix evenly with the cells, incubate at 37°C for 2 hours to load the peptide onto the T2 cells. Add 200 ⁇ L 1640 medium to resuspend the cells, centrifuge at 400 g for 5 minutes, and remove the supernatant;
  • Antibody incubation Use antibody diluent (PBS + 0.5% FBS) to adjust the initial concentration of the original antibody to 1 ⁇ g/mL, dilute 4 times, and set 10 concentration gradients in total. Take 100 ⁇ L of the diluted antibody and add it to the previously loaded cells. After pipetting and mixing, incubate at 4°C in the dark for 60 min.
  • Antibody washing Add 200 ⁇ L of antibody diluent to the incubated antibody-cell mixture, centrifuge at 400 g for 5 min, discard the supernatant, resuspend the cells with 200 ⁇ L of antibody diluent, and repeat the washing once;
  • results are shown in FIG2 : The results show that the 14 single-domain antibodies screened by the present invention are all able to bind to antigens at the cellular level and have high affinity.
  • the present invention detects the off-target situation of all candidate molecules.
  • Figure 3 shows the off-target analysis results of some molecules.
  • the single domain antibody molecules screened by the present invention have good specificity and can be further developed.
  • Example 5 Design of bispecific antibodies based on single domain antibodies
  • the bispecific antibody molecules designed in the present invention can bind to the PRAME polypeptide-HLA-A02 complex and CD3 at the same time. Some bispecific antibodies in the present invention integrate Fc structures or half life extender structures.
  • the amino acid sequence of the CD3 antibody used in the bispecific antibody molecules designed in the present invention is:
  • the half life extender amino acid sequence used in the bispecific antibody molecule designed in the present invention is:
  • the present invention designed a total of 13 bispecific antibody molecules with different structures, and the amino acid sequences are shown in Table 3.
  • the bispecific antibody molecules designed by the present invention were expressed and purified, and the expression level and purity were detected.
  • the expression level and purity information of different bispecific antibodies are shown in Table 4.
  • the expression level of the bispecific antibody of the present invention is in the range of 600-850 mg/L, which proves that the bispecific antibody structure of the present invention has a higher expression level.
  • the monomer purity of the bispecific antibody of the present invention by SEC-HPLC is above 90%.
  • the purity of the reduced SDS-PAGE of all bispecific antibodies is above 95%.
  • the bispecific antibody molecule described in the present invention also known as a T cell engager, can simultaneously and specifically bind to the target presented by pMHC in the tumor cell and the CD3 protein on the surface of the T cell, thereby mediating the directional cruising of T cells to the vicinity of the tumor cells, endogenously activating and releasing cytokines, leading to the lysis of the tumor cells, thereby achieving the purpose of treating the tumor.
  • this example co-cultures the target cells with CD3+T cells at a certain effect-target ratio, and adds different concentrations of candidate antibody molecules to detect the antibody-mediated target cell apoptosis ratio and T cell activation and factor secretion levels; in addition, this example sets tumor cell lines NCI-H1755, HS695T, OVCAR3, U2OS with different antigen expression abundances, PRAME expression negative HLA-A2 positive T2, MCF7 and HLA-A2 and PRAME negative A549 cell lines as target cells, which can further evaluate the target-specific recognition ability of the bispecific antibody molecules, thereby more comprehensively screening out suitable bispecific antibody molecules for in-depth research.
  • the basic implementation steps are as follows:
  • Cell co-culture Use lentivirus carrying luciferase to transfect different target cells to prepare fluorescently labeled Enzyme cell lines, labeled as: NCI-H1755-GFP, HS695T-GFP, OVCAR3-GFP, MCF7-GFP and A549-GFP.
  • Different target cells and effector cells were resuspended in culture medium (1640 culture medium containing 2% FBS) at a concentration of 2 ⁇ 10 5 /mL and 1 ⁇ 10 6 /mL, respectively, and plated into 96-well flat-bottom opaque white plates at 25 ⁇ L/well, and temporarily placed at 37°C for incubation;
  • Antibody incubation dilute the antibody in a gradient manner with the culture medium, with the highest concentration being 20 nM, and dilute to 10 concentration points. Add 50 ⁇ L of the corresponding bispecific antibody to each well. Mix thoroughly and centrifuge at 500 rpm for 3 minutes. Incubate the cells at 37°C for 24 hours;
  • luciferase activity relative light unit, RLU
  • the specific steps are: take out the opaque 96-well flat-bottom plate after co-culture, add 100 ⁇ L of an equal volume of D-luciferin substrate (Thermo Fisher Scientific: 88293) to the well, mix well, protect from light and develop color for 5 minutes, and detect the fluorescence intensity in the chemiluminescence mode of the microplate reader. Since luciferase is only expressed in target cells, the remaining luciferase activity in the well is directly related to the number of live target cells in the well. In the absence of effector cells and antibodies, the maximum luciferase activity is obtained by adding culture medium to the target cells as a control;
  • bispecific antibodies of the present invention can mediate T cell activation and kill cells expressing positive target sites ( FIG. 4 ).
  • Example 8 Molecular design of CAR sequence targeting PRAME and construction of lentiviral vector
  • Primers were designed to use PCR to amplify five different CAR molecules from the pUC57 vector.
  • the homology arms of the lentiviral vector should be added to the 5' ends of the forward and reverse primers.
  • the amplified PCR products were detected by agarose gel electrophoresis and then gel-recovered and purified (Nanjing Novozyme Biotechnology Co., Ltd., catalog number DC301) to obtain DNA fragments.
  • the DNA fragments recovered by enzyme digestion were cloned into the lentiviral vector by homologous recombination.
  • the sequencing primers were: Lenti-seqF: TTGAGTTGGATCTTGGTTC (SEQ ID NO: 103), Lenti-seqR: CAGCAACCAGGATTTATACA (SEQ ID NO: 104). Sanger sequencing verified that the five lentiviral vector plasmids were constructed correctly.
  • the correct lentiviral plasmids verified by sequencing were transformed into Escherichia coli stbl3 (purchased from Yisheng Biotechnology Co., Ltd.). The next day, single clones were picked from the transformed plates and placed in a 2ml liquid LB culture tube containing kanamycin (50ug/ml) and cultured on a shaker at 37°C220rpm for 8h. 1ml of the activated bacterial solution was inoculated into 250ml liquid LB culture medium containing kanamycin and cultured on a shaker at 37°C220rpm for 12-16h.
  • the frozen 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were taken out of liquid nitrogen, thawed in a 37°C water bath, and then the tube mouth was wiped with 75% alcohol and transferred to a tube containing 10 ml of preheated DMEM complete medium (90% DMEM +
  • plasmid transfection Before transfection, the culture medium was replaced with DMEM medium containing 10% FBS but without dual antibodies. Prepare the plasmid complex first: add the following plasmids into 1.5 ml Opti-MEM (Thermo Fisher Scientific; 31985-070) and mix well: 18 ⁇ g of psPAX2 plasmid (Addgene; Catalog No.: 12260), 9 ⁇ g of pMD2.G plasmid (Addgene; Catalog No.: 12259), 18 ⁇ g of lentiviral vector plasmid.
  • Opti-MEM Thermo Fisher Scientific; 31985-070
  • the lentiviral plasmids are: p-lenti-PRAME-CAR-01, p-lenti-PRAME-CAR-02, p-lenti-PRAME-CAR-03, lenti-PRAME-CAR-04, lenti-PRAME-CAR-05.
  • the transfection reagent complex according to the mass ratio of plasmid to PEI of 1:3, add 67.5 ⁇ L (2mg/mL) of PEI (polysciences: 24765) to 1.5mL Opti-MEM, mix well, and let stand at room temperature for 5min; then add the transfection reagent complex dropwise to the plasmid complex, mix well and let stand for 20min. Finally, slowly drip the transfection complex into the 293T cell culture flask, gently mix, and continue to culture in a cell culture incubator at 37°C with 5 % CO2.
  • virus collection 48 hours after transfection, collect the culture supernatant and centrifuge at 2000rpm for 10 minutes to remove cell debris.
  • a 0.45 ⁇ M filter membrane Millex-HV, catalog number SLHVR33RB
  • After discarding the supernatant use 1ml of X-VIVO-15
  • the lentivirus was resuspended in the culture medium, and the lentivirus was aliquoted and stored in an ultra-low temperature refrigerator at -80°C. According to the process, lentivirus containing PRAME-CAR-01, PRAME-CAR-02, PRAME-CAR-03, PRAME-CAR-04, and PRAME-CAR-05 were prepared respectively.
  • the prepared lentivirus containing PRAME-CAR-01, PRAME-CAR-02, PRAME-CAR-03, PRAME-CAR-04, and PRAME-CAR-05 were used to infect primary human T cells to prepare CAR-T cells carrying different CAR genes.
  • the CAR-T cells carrying the five CAR genes were named PRAME-CAR-T-01, PRAME-CAR-T-02, PRAME-CAR-T-03, PRAME-CAR-T-04, and PRAME-CAR-T-05, and the untransfected T cells were used as negative controls and named NT.
  • the specific steps are as follows:
  • CD3+T cells from healthy human peripheral blood (Miaoshun (Shanghai) Biotechnology Co., Ltd.) were resuspended in T cell culture medium containing 300 IU/mL IL-2 to a density of 1 ⁇ 10 6 /mL, and T cell activator CD3/CD28 magnetic beads (ACRO Biosystems, catalog number: MBS-C001) were added at a ratio of 1:1 between cells and magnetic beads. After thorough mixing, the cells were inoculated in 6-well plates for culture;
  • T cells were counted and inoculated into a new 24-well plate, with 500ul per well and 5 ⁇ 10 5 cells/well. After the inoculation, 100 ⁇ L of lentivirus solution carrying different CAR genes was added to infect the T cells. T cells without virus solution were used as negative control NT, and the cells were placed in the incubator for further culture.
  • the cells were aspirated from the culture wells, the magnetic beads were removed by magnetic adsorption using a magnetic stand, and the cells were collected by centrifugation and resuspended in fresh T cell culture medium.
  • This experiment uses two HLA-A2 and PRAME target expression double positive cell lines as target cells, namely non-small cell lung cancer NCI-H1755 and melanoma HS695T.
  • Two PRAME and HLA-A2 negative A549 and 293T cells are used as negative cells, and the five CAR-T cells prepared in Example 2 above are co-cultured with target cells to detect the killing effect and evaluate the biological functions of different CAR-T.
  • the specific steps are as follows:
  • GFP luciferase (GenBank: AAR29591.1) to transfect different target cells to obtain cell lines labeled with luciferase, labeled as: NCI-H1755-GFP-luc, HS695T-GFP-luc, 293T-GFP-luc and A549-GFP-luc;
  • NCI-H1755-GFP-luc, HS695T-GFP-luc, 293T-GFP-luc and A549-GFP cells were inoculated into 96-well flat-bottom opaque cell culture plates at a cell concentration of 1 ⁇ 10 5 /mL and 50 ⁇ L/well, and temporarily placed at 37°C for incubation;
  • the five CAR-T cells prepared in Example 2 and the target cells in Example 4 were co-cultured at a 1:1 effector-target ratio for 24 hours, and the levels of IFN- ⁇ and IL-2 cytokines secreted by T cells in the culture supernatant were detected.
  • the specific steps are as follows:
  • the results are shown in Figure 7.
  • the results show that the candidate CAR-T cells can effectively recognize T2 loaded with target peptides, activate and transmit immune signals, and secrete IFN- ⁇ cytokines, among which the CAR-T-01 molecule has relatively better biological activity.
  • the CAR sequence described in the present invention has good affinity activity and biological function, indicating that the CAR molecule of the present invention has the value of further development and application.
  • the present invention humanizes two single-domain antibody candidate molecules, LL-PR001 and LL-PR004, respectively.
  • LL-PR001 was used as the humanized parent to design ten candidate humanized single domain antibodies, and the antibody sequences are shown in Table 7.
  • LL-PR004 was used as the humanized parent to design six candidate humanized single domain antibodies, and the antibody sequences are shown in Table 8.
  • the humanized antibody molecule is first codon optimized to obtain a nucleotide sequence, and the full-length gene of the humanized antibody molecule is constructed into a single-domain antibody expression vector by gene synthesis. After the vector is successfully constructed, the single-domain antibody is expressed by transient transfection of HEK293 suspension cell lines. The specific steps are referred to Example 2.
  • the expression levels and purities of different humanized single domain antibodies are shown in Table 9.
  • the expression levels of the humanized single domain antibodies of the present invention using the transient expression system are in the range of 700-900 mg/L, proving that the humanized single domain antibodies of the present invention have a high expression level.
  • the purity of the reduced SDS-PAGE and SEC-HPLC of most humanized single domain antibodies is above 95%.
  • the binding ability of the humanized molecule to the target antigen is detected by ELISA to determine the affinity of the humanized molecule.
  • the results show that the 10 candidate humanized single domain antibodies (huLL-PR001-1 to huLL-PR001-10) obtained after the humanization of the LL-PR001 molecule can bind to the protein antigen and have a high affinity (the results are shown in Figure 8).
  • the affinity of the molecule after partial humanization has decreased slightly, but it still has a high affinity. It can be applied to the research and development of subsequent bispecific antibodies, CAR-T, ADC and RDC and other biopharmaceutical projects.
  • the six candidate humanized single domain antibodies (huLL-PR004-1 to huLL-PR004-6) obtained after the humanization of the LL-PR004 molecule can also bind to the protein antigen and have a high affinity.
  • the six affinities after humanization are slightly lower than those of the parent molecule, but still have a high affinity (the results are shown in Figure 9). It can be applied to the subsequent research and development of biologics projects such as bispecific antibodies, CAR-T, ADC and RDC.

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Abstract

L'invention concerne un anticorps à domaine unique ciblant un polypeptide PRAME. L'anticorps à domaine unique ciblant le polypeptide PRAME peut se lier au polypeptide PRAME avec une affinité élevée et présente une spécificité relativement bonne, ce qui permet de poser les bases d'un nouveau matériau pour le développement d'un médicament antitumoral ciblant le polypeptide PRAME. L'invention concerne en outre un anticorps bispécifique, un récepteur antigénique chimérique, un récepteur antigénique chimérique-T, un ADC, etc, préparés au moyen de l'anticorps à domaine unique.
PCT/CN2024/114621 2023-08-25 2024-08-26 Anticorps à domaine unique ciblant le polypeptide prame et son utilisation Pending WO2025044998A1 (fr)

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CN118221811B (zh) * 2023-08-25 2025-05-30 立凌生物制药(苏州)有限公司 靶向prame多肽的单域抗体及其用途

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