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WO2023090704A1 - Anticorps spécifique pour cd22 humanisé et récepteur antigénique chimérique l'utilisant - Google Patents

Anticorps spécifique pour cd22 humanisé et récepteur antigénique chimérique l'utilisant Download PDF

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WO2023090704A1
WO2023090704A1 PCT/KR2022/017092 KR2022017092W WO2023090704A1 WO 2023090704 A1 WO2023090704 A1 WO 2023090704A1 KR 2022017092 W KR2022017092 W KR 2022017092W WO 2023090704 A1 WO2023090704 A1 WO 2023090704A1
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seq
antibody
cells
chain variable
variable region
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김승구
김기태
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Innobation Bio Co Ltd
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • 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
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
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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
    • 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
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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
    • C07K2317/90—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
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a humanized CD22-specific antibody and a chimeric antigen receptor using the same, and more particularly, to an antibody that specifically binds to CD22 (4F5 antibody) and a humanized antibody thereof, comprising the antibody or the CD19xCD22 antibody It relates to a chimeric antigen receptor, a CAR-T cell expressing the chimeric antigen receptor, and a pharmaceutical composition for preventing or treating diseases mediated by B cells including the chimeric antigen receptor.
  • CD22 is associated with NHL, acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (B-CLL) and especially acute non-lymphocytic leukemia (ANLL), It is expressed in most B-cell leukemias and lymphomas.
  • B-ALL acute lymphoblastic leukemia
  • B-CLL chronic lymphocytic leukemia
  • ANLL acute non-lymphocytic leukemia
  • Antibodies specific to CD22 are being developed for the treatment or diagnosis of diseases related to CD22 expression.
  • International Publication No. WO1998-041641 discloses a recombinant anti-CD22 antibody having cysteine residues at positions V H 44 and V L 100
  • International Publication No. WO 1998-042378 discloses an antibody for the treatment of B-cell malignancies. -CD22 antibody is disclosed.
  • mice As described above, for the production of antibodies for treatment, monoclonal antibodies are mainly produced using mice. However, since non-human antibodies such as mouse-derived monoclonal antibodies are regarded as foreign antigens in the human body, they induce an immune response and have a short half-life, so their therapeutic effect is limited.
  • humanized antibodies have been developed in which the remaining parts of the antibody except for the antigen-binding portion are substituted with human antibodies.
  • the most similar human antibody gene for the antibody to be substituted is selected, and only the CDR region of the mouse antibody is replaced with the human antibody CDR position by a method called CDR transplantation.
  • Such humanized antibodies have the advantage of reducing the immune response in the human body because most of the genes are humanized.
  • CD19 is generally a target in addition to the CD22 antigen.
  • Clinical trials on CAR-T cells targeting these antigens are also in progress.
  • there is a difference in the amount of target antigen expression depending on cells such as leukemia that do not express CD19, and single CAR or single CAR-T cell therapy that targets only one antigen loses the target antigen due to the immune evasion strategy of tumor cells problems may arise.
  • CAR-T cells targeting dual or multiple antigens are being studied, and simultaneous targeting of two antigens can reduce the possibility of antigen-loss variants.
  • an antibody that binds to CD22 was selected, and humanized anti-CD22 antibodies (4F5 (V4) and 4F5 (V11)) were prepared using the antibody.
  • a humanized anti-CD22 antibody of the invention was used to generate chimeric antigen receptors and CAR-T cells targeting CD22.
  • bispecific chimeric antigen receptor (Bivalent CAR or Bispecific CAR) targeting CD19 as well as CD22 and a bispecific CAR-T cell using the same were prepared, and the CD22-CAR-T cell prepared in the present invention and the dual It was confirmed that the chimeric antigen receptor was normally expressed in specific CD19xCD22-CAR-T cells, and the present invention was completed.
  • an object of the present invention is to provide an antibody specific for CD22 or a humanized antibody specific for CD22, a polynucleotide encoding the antibody, a vector expressing the antibody, and a recombinant cell transformed with the vector.
  • Another object of the present invention is a chimeric antigen receptor comprising the CD22-specific antibody or CD22-specific humanized antibody, a polynucleotide encoding the chimeric antigen receptor, a vector comprising the polynucleotide, and the polynucleotide or It is to provide an immune effector cell expressing a chimeric antigen receptor containing a vector.
  • Another object of the present invention is a bispecific chimeric antigen receptor comprising antibodies specific for CD19 and CD22, a polynucleotide encoding the bispecific chimeric antigen receptor, a vector comprising the polynucleotide, and the polynucleotide Or to provide an immune effector cell expressing a bispecific chimeric antigen receptor comprising a vector.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases mediated by B cells, including the immune effector cells.
  • the present invention relates to a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a CDR3 region represented by the amino acids of SEQ ID NO: 3, and a heavy chain variable region comprising the amino acids represented by SEQ ID NO: 4
  • a CDR1 region represented by the amino acids of SEQ ID NO: 1 a CDR2 region represented by the amino acids of SEQ ID NO: 2
  • a CDR3 region represented by the amino acids of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acids represented by SEQ ID NO: 4
  • an antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region, a CDR2 region represented by the amino acids of SEQ ID NO: 5, and a CDR3 region represented by the amino acids of SEQ ID NO: 6.
  • the antibody specifically binding to CD22 is composed of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12.
  • the present invention provides a polynucleotide encoding the antibody or fragment thereof that specifically binds to CD22.
  • the present invention provides a vector comprising a polynucleotide encoding the antibody or fragment thereof that specifically binds to CD22.
  • the present invention provides a recombinant cell that produces an antibody or fragment thereof that specifically binds to CD22 transformed with the vector.
  • the present invention relates to a CD22-binding domain; transmembrane domain; costimulatory domain; And a chimeric antigen receptor (CAR) comprising an intracellular signal transduction domain,
  • the CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4
  • An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6
  • the antibody specifically binding to CD22 is composed of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12.
  • the transmembrane domain is a protein selected from the group consisting of CD8 ⁇ , CD4, CD28, CD137, CD80, CD86, CD152 and PD1, and the costimulatory domain is CD28, 4-1BB, It is a protein selected from the group consisting of OX-40 and ICOS, and the signaling domain may be derived from CD3 ⁇ .
  • it may further include a hinge region located between the C-terminus of the CD22-binding domain and the N-terminus of the transmembrane domain, wherein the hinge region is CD8 ⁇ may be of origin.
  • the present invention provides a polynucleotide encoding the chimeric antigen receptor.
  • the present invention provides a vector comprising a polynucleotide encoding the chimeric antigen receptor.
  • the present invention provides an immune effector cell comprising a polynucleotide encoding the chimeric antigen receptor or a vector containing the polynucleotide and expressing the chimeric antigen receptor.
  • the present invention relates to a CD19-binding domain and a CD22-binding domain
  • CAR bispecific chimeric antigen receptor
  • the CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4
  • An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6
  • a bispecific chimeric antigen receptor targeting CD19 and CD22 Provided is a bispecific chimeric antigen receptor targeting CD19 and CD22.
  • the CD19-binding domain and the CD22-binding domain are the light chain variable region of an antibody that specifically binds to CD19 - the heavy chain variable region of an antibody that specifically binds to CD22 - CD22
  • the light chain variable region of an antibody that specifically binds to the heavy chain variable region of an antibody that specifically binds to CD19 may be linked in sequence.
  • the light chain variable region of the antibody specifically binding to CD19 is a CDR1 region (QDISKY) represented by amino acids of SEQ ID NO: 44, a CDR2 region represented by amino acids of SEQ ID NO: 45 ( HTS) and a CDR3 region (QQGNTLPYT) represented by the amino acids of SEQ ID NO: 46, preferably represented by the amino acid sequence of SEQ ID NO: 48.
  • QDISKY CDR1 region represented by amino acids of SEQ ID NO: 44
  • HTS CDR2 region represented by amino acids of SEQ ID NO: 45
  • QQGNTLPYT CDR3 region represented by the amino acids of SEQ ID NO: 46, preferably represented by the amino acid sequence of SEQ ID NO: 48.
  • the heavy chain variable region of the antibody specifically binding to CD19 is the CDR1 region (GVSLPDYG) represented by the amino acids of SEQ ID NO: 41 and the CDR2 region represented by the amino acids of SEQ ID NO: 42.
  • IWGSETT the CDR3 region
  • AKHYYYGGSYAMDY the amino acids of SEQ ID NO: 43, preferably represented by the amino acid sequence of SEQ ID NO: 47.
  • the antibody specifically binding to CD22 is CD22 composed of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12.
  • the transmembrane domain is a protein selected from the group consisting of CD8 ⁇ , CD4, CD28, CD137, CD80, CD86, CD152 and PD1, and the costimulatory domain is CD28, 4-1BB , OX-40 and ICOS, and the signaling domain may be derived from CD3 ⁇ .
  • it may further include a hinge region located between the C-terminus of the CD22-binding domain and the N-terminus of the transmembrane domain, wherein the hinge region is CD8 ⁇ may be of origin.
  • the present invention also provides a polynucleotide encoding the bispecific chimeric antigen receptor.
  • the present invention provides a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor.
  • the present invention provides an immune effector cell comprising a polynucleotide encoding the bispecific chimeric antigen receptor or a vector containing the polynucleotide and expressing the bispecific chimeric antigen receptor.
  • the present invention relates to an antibody or fragment thereof that specifically binds to the CD22;
  • a pharmaceutical composition for preventing or treating a disease mediated by B cells including the immune effector cells is provided.
  • the disease mediated by B cells is a tumor, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent delayed NHL, refractory NHL, refractory delayed NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) , Burkitt's lymphoma and mantle cell lymphoma.
  • NHL non-Hogkins lymphoma
  • aggressive NHL recurrent aggressive NHL
  • recurrent delayed NHL recurrent delayed NHL
  • refractory NHL refractory delayed NHL
  • CLL chronic lymphocytic leukemia
  • small lymphocytic lymphoma small lymphocytic lymphoma
  • leukemia hairy cell leukemia
  • HCL hairy cell leukemia
  • ALL acute lymphocytic leukemia
  • an antibody that specifically binds to CD22 and a humanized antibody thereof were prepared, and using them, a single CAR-T cell targeting CD22 and a bispecific CAR-T cell targeting CD19 and CD22 were prepared .
  • CD22-CAR-T cells and the bispecific CD19xCD22-CAR-T cells prepared in the present invention effectively recognized the CD22 antigen and activated the CAR-T cells, and effectively killed the cells expressing CD22. Confirmed.
  • the CD22-CAR-T cells based on the humanized antibody specifically binding to CD22 of the present invention and the bispecific CD19xCD22-CAR-T cells CAR-T cells can be usefully utilized as a composition for preventing or treating a disease related to CD22 (or CD19) expression or a disease related to B cells.
  • Figure 1 is data confirming the binding ability to CD22 of the 4F5 antibody (mouse) and the humanized 4F5 antibodies (4F5 (V4) and 4F5 (V11)) selected in the present invention by FACS.
  • FIG. 2 is a schematic diagram showing a chimeric antigen receptor (single CAR) targeting CD22.
  • FIG. 3 is a schematic diagram showing a method for preparing CD22-CAR-T cells using a lentiviral vector expressing CD22-CAR.
  • FIG. 4 is a schematic diagram showing (a) a method for preparing CD22-CART cells using peripheral blood mononuclear cells (PBMC) and (b) a method for confirming the CD22 peptide-binding ability of the prepared CD22-CAR-T cells.
  • PBMC peripheral blood mononuclear cells
  • Figure 5a is data confirming the CD22 binding ability of CD22-CAR-T cells prepared using the 4F5 antibody.
  • 5B is data confirming the CD22 binding ability of CD22-CAR-T cells based on humanized anti-CD22 antibodies, 4F5 (V4) and 4F5 (V11).
  • Figure 7a is data confirming the killing effect of U2932 cells (CD22 expressing cells) and K562 cells (CD22 non-expressing cells) by CD22-CAR-T cells prepared using the 4F5 antibody.
  • Figure 7b is data confirming the killing effect of U2932 cells (CD22 expressing cells) and K562 cells (CD22 non-expressing cells) by CD22-CAR-T cells based on 4F5 (V4) and 4F5 (V11), which are humanized anti-CD22 antibodies.
  • FIG. 8 is a schematic diagram showing a bispecific chimeric antigen receptor (Bispecific CAR) targeting CD19 and CD22.
  • FIG. 9 is a schematic diagram showing a method for preparing CD19xCD22-CAR expressing cells using a lentivirus expressing a bispecific CD19xCD22-CAR targeting CD19 and CD22.
  • FIG. 10 shows (a) a method for transfecting a HEK293 cell line with a lentiviral vector expressing CD19xCD22-CAR and (b) a method for confirming the CD19 peptide and CD22 peptide binding ability and CD19xCD22-CAR expression level of the transformed HEK293 cells It is a schematic diagram showing
  • FIG. 11 is data confirming the expression levels of CD19x4F5-CAR, CD19x4F5(V4)-CAR and CD19x4F5(V11)-CAR in HEK293FT cells transfected with a lentiviral vector expressing CD19xCD22-CAR.
  • FIG. 12 is a schematic diagram showing a method for preparing CD19xCD22-CAR-T cells using a lentiviral vector expressing CD19xCD22-CAR.
  • FIG. 13 shows (a) a method for preparing CD19xCD22-CAR-T cells using peripheral blood mononuclear cells (PBMC) and (b) a method for confirming the CD19 peptide and CD22 peptide binding ability of the prepared CD19xCD22-CAR-T cells. It is also a model.
  • PBMC peripheral blood mononuclear cells
  • CD19xCD22-CAR-T cells is data confirming the activation of CD19xCD22-CAR-T cells, and it was confirmed that CD19xCD22-CAR-T cells simultaneously bind to CD22 peptide and CD19 peptide in CD3+ CD19xCD22-CAR-T cells.
  • K562 cells CD19 and CD22 non-expressing cells
  • K562-CD19 cells CD19 expressing cells
  • K562-CD22 cells CD22 expressing cells
  • K562-CD19/CD22 cells CD19/CD22 cells
  • the present invention is a heavy chain variable region comprising a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a CDR3 region represented by the amino acids of SEQ ID NO: 3, and SEQ ID NO: 4.
  • An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 will be.
  • the antibody or fragment thereof that specifically binds to CD22 is specifically composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 12. a humanized antibody or fragment thereof that binds; or
  • It may be a humanized antibody or fragment thereof that specifically binds to CD22 composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
  • the antibody may be a monoclonal antibody.
  • the term "monoclonal antibody” is also called a monoclonal antibody or a monoclonal antibody, and is an antibody produced by a single antibody-forming cell, characterized by a uniform primary structure (amino acid sequence). It recognizes only one antigenic determinant and is generally produced by culturing a hybridoma cell in which cancer cells and antibody-producing cells are fused.
  • the antibody of the present invention can be prepared as a humanized antibody with increased similarity with a human antibody by making the remaining parts, except for the CDR region, which is a key part for antigen binding, an amino acid sequence corresponding to an antibody produced by humans.
  • the most common method for humanizing an antibody is a CDR-grafting method in which a CDR region of an animal antibody is grafted onto a human antibody, but is not limited thereto, and a method known in the art is used.
  • Humanized antibodies can be prepared using
  • CDR complementarity determining region
  • antibody can be used not only in its complete form having two full-length light chains and two full-length heavy chains, but also fragments of antibody molecules.
  • a fragment of an antibody molecule refers to a fragment having at least a peptide tag (epitope) binding function, and includes scFv, Fab, F(ab'), F(ab') 2 , single domain, and the like.
  • Fab has a structure having variable regions of light and heavy chains, constant regions of light chains, and a first constant region (CH1) of heavy chains, and has one antigen-binding site.
  • Fab' is different from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
  • the F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'.
  • Fv is a minimal antibody fragment that has only the heavy chain variable region and the light chain variable region.
  • Double-chain Fv has a heavy chain variable region and light chain variable region connected by a disulfide bond
  • single-chain Fv (scFv) is generally a peptide linker
  • the variable region of the heavy chain and the variable region of the light chain are covalently linked via.
  • the monoclonal antibody specifically binding to CD22 of the present invention can be prepared using all or a partial peptide of CD22 protein as an immunogen (or antigen). More specifically, first, as an immunogen, CD22 protein, a fusion protein containing CD22 protein, or a carrier containing CD22 protein is administered to humans together with an adjuvant (eg, Freund adjuvant) as an immune enhancer, if necessary. Immunization is performed by injecting once or more subcutaneously, intramuscularly, intravenously, balboloxal, or intraperitoneally in mammals other than those excluded.
  • an adjuvant eg, Freund adjuvant
  • Mammals other than humans are preferably mice, rats, hamsters, marmots, chickens, rabbits, cats, dogs, pigs, goats, sheep, donkeys, horses or cows (transgenic mice that produce human antibodies). (including transgenic animals engineered to produce antibodies derived from other animals such as), more preferably mice, rats, hamsters, marmots, chickens or rabbits.
  • 1 to 4 immunizations are performed about every 1 to 21 days from the first immunization, and about 1 to 10 days after the final immunization, antibody-producing cells can be obtained from immunosensitized mammals. The number of immunizations and time intervals can be appropriately changed depending on the characteristics of the immunogen to be used.
  • Preparation of a hybridoma secreting a monoclonal antibody can be performed according to the method of Keira and Mirstein et al. (Nature, 1975, Vol. 256, p. 495-497) and a method similar thereto. Any one selected from the group consisting of spleen, lymph node, bone marrow, or tonsil collected from animals other than humans immunosensitized as described above, preferably derived from a mammal that does not have the ability to produce an antibody and an antibody-producing cell contained in the spleen.
  • a hybridoma can be prepared by cell fusion of myeloma cells.
  • the mammal may be a mouse, rat, marmot, hamster, chicken, rabbit or human, preferably a mouse, rat, chicken or human.
  • a fusion promoter such as polyethylene glycol or Sendai virus or a method using an electric pulse is used.
  • a fusion medium containing a fusion promoter antibody-producing cells and mammalian-derived cells capable of immortal growth are used. is suspended at a ratio of about 1:1 to 1:10, and incubated in this state at about 30 to 40°C for about 1 to 5 minutes.
  • the fusion medium for example, MEM medium, RPMI1640 medium, and Iscove's Modified Dulbecco's Medium may be used, and serum types such as bovine serum are preferably excluded.
  • the method for screening hybridoma clones producing the monoclonal antibody is, first, transfer the fused cells obtained as described above to a selection medium such as HAT medium, and incubate at about 30 to 40 ° C. for about 3 days to 3 weeks Cells other than hybridomas are killed. Subsequently, after culturing the hybridomas in a microtiter plate, etc., the part with increased reactivity between the immunogen used for the immune response of animals other than humans described above and the culture supernatant was prepared as RIA (radioactive substance-marked immunoassay). antibody) or an immunoassay method such as ELISA (Enzyme-Linked Immunosorbent Assay). In addition, the clone producing the monoclonal antibody found above shows specific binding ability to the immunogen.
  • the monoclonal antibody of the present invention can be obtained by culturing such a hybridoma in vitro or in vivo.
  • a conventional method for culturing mammalian-derived cells is used, and for collecting a monoclonal antibody from a culture or the like, a conventional method in this field for purifying an antibody in general is used.
  • a conventional method in this field for purifying an antibody in general is used.
  • each method for example, salting out, dialysis, filtration, concentration, centrifugation, fractional precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, high-speed liquid chromatography, gel electrophoresis and isoelectric point electrophoresis. These are applied in combination as needed.
  • the purified monoclonal antibody is then concentrated and dried to be in a liquid or solid state depending on the application.
  • the monoclonal antibody of the present invention includes DNA encoding heavy chain and light chain variable regions, respectively, and known DNA encoding heavy and light chain constant regions (eg, Japan 2007-252372 (Refer to Publication No.) and each ligated gene are synthesized by the PCR method or chemical synthesis, and transplanted into a known expression vector (pcDNA 3.1 (sold by Invitrogen)) or the like that enables the expression of the gene to obtain a transformant. It can be obtained by preparing and expressing in a host such as CHO cells or Escherichia coli to produce an antibody, and purifying the antibody from this culture solution using a Protein A or G column or the like.
  • pcDNA 3.1 sold by Invitrogen
  • a hybridoma that produces an anti-CD22 antibody is prepared and screened to obtain an antibody (scFv) that specifically binds to CD22. It was selected and named as 4F5.
  • the 4F5 antibody is a heavy chain variable comprising a CDR1 region (GFSLTIYG) represented by the amino acids of SEQ ID NO: 1, a CDR2 region (MWSGGST) represented by the amino acids of SEQ ID NO: 2, and a CDR3 region (ARNDGYYWFAY) represented by the amino acids of SEQ ID NO: 3. region and a CDR1 region (QSLVHNNGNTY) represented by the amino acids of SEQ ID NO: 4, a CDR2 region (KVS) represented by the amino acids of SEQ ID NO: 5, and a CDR3 region (SQSTHVPYS) represented by the amino acids of SEQ ID NO: 6. configuration was confirmed.
  • the 4F5 antibody is composed of a heavy chain variable region represented by the amino acids of SEQ ID NO: 7 and a light chain variable region represented by the amino acids of SEQ ID NO: 8, wherein the heavy chain variable region has the nucleotide sequence of SEQ ID NO: 9, the light chain variable region was confirmed to be encoded by the nucleotide sequence of SEQ ID NO: 10.
  • humanized antibodies were prepared by changing the anti-CD22 antibody 4F5 to a structure corresponding to human, which was named 4F5 (V4) antibody and 4F5 (V11) antibody did
  • the CDRs of the heavy chain variable region and the CDRs of the light chain variable region of the 4F5 (V4) antibody and the 4F5 (V11) antibody were the same as those of 4F5, and the remaining portions except for the CDR were humanized.
  • the 4F5 (V4) antibody is composed of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12, and the heavy chain variable region of the 4F5 (V4) antibody is SEQ ID NO: 13
  • the light chain variable region may be encoded by the nucleotide sequence of SEQ ID NO: 14.
  • the 4F5 (V11) antibody is composed of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16, and the heavy chain variable region of the 4F5 (V11) antibody is SEQ ID NO: 17
  • the light chain variable region may be encoded by the nucleotide sequence of SEQ ID NO: 18.
  • the CD22-specific antibody of the present invention is preferably a scFv (single chain variable fragment), which can be prepared through genetic recombination technology so that the heavy chain variable region and the light chain variable region can be connected by a linker.
  • the linker is preferably represented by the amino acid sequence of SEQ ID NO: 19, or may be encoded by the nucleotide sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, but is not limited thereto.
  • the 4F5 antibody When linked by light chain variable region-linker-heavy chain variable region, the 4F5 antibody (mouse antibody) may be represented by the amino acid sequence of SEQ ID NO: 23 or encoded by the nucleotide sequence of SEQ ID NO: 24, and the 4F5 (V4) antibody of SEQ ID NO: 25 It may be represented by the amino acid sequence or encoded by the nucleotide sequence of SEQ ID NO: 26, and the 4F5 (V11) antibody may be represented by the amino acid sequence of SEQ ID NO: 27 or encoded by the nucleotide sequence of SEQ ID NO: 28.
  • the present invention relates to a polynucleotide encoding an antibody or fragment thereof that specifically binds to CD22.
  • polynucleotide generally refers to nucleic acid molecules, deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated of any length.
  • the polynucleotides of the invention can be used for (1) in-vitro amplification, such as polymerase chain reaction (PCR) amplification; (2) cloning and recombination; (3) purification such as digestion and gel electrophoretic separation; (4) It can be produced through synthesis such as chemical synthesis, and preferably the isolated polynucleotide is produced by recombinant DNA technology.
  • PCR polymerase chain reaction
  • nucleic acids for encoding antibodies or antigen-binding fragments thereof are prepared by various methods known in the art, including but not limited to, restriction fragment operation of synthetic oligonucleotides or application of SOE PCR. can be manufactured.
  • the present invention relates to a vector comprising a polynucleotide encoding an antibody or fragment thereof that specifically binds to CD22, and a recombinant cell transformed with the vector.
  • the term "expression vector” is a gene product containing essential regulatory elements such as a promoter so that a target gene can be expressed in an appropriate host cell.
  • Vectors may be selected from one or more of plasmids, retroviral vectors and lentiviral vectors. Once transformed into a suitable host, the vector can replicate and function independently of the host genome or, in some cases, can integrate into the genome itself.
  • vectors may contain expression control elements that allow for correct expression of the coding region in a suitable host.
  • regulatory elements are well known to those skilled in the art and include, for example, promoters, ribosome-binding sites, enhancers and other regulatory elements for regulating gene transcription or mRNA translation. can do.
  • the specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes 5' ratios that participate in transcription initiation and translation initiation, such as TATA boxes, capped sequences, CAAT sequences, etc., respectively. -contains a transcribed sequence, and a 5' or 3' non-translated sequence.
  • a 5' non-transcribed expression control sequence can include a promoter region that can include promoter sequences for transcribing and regulating functionally linked nucleic acids.
  • promoter refers to a minimal sequence sufficient to direct transcription.
  • promoter constructs sufficient to allow expression of a regulatable promoter dependent gene induced by cell type specific or external signals or agents may be included, and such constructs may be located on the 5' or 3' portion of the gene. . Both conserved promoters and inducible promoters are included. Promoter sequences may be of prokaryotic, eukaryotic or viral origin.
  • the term "transformant” refers to a cell transformed by introducing a vector having a polynucleotide encoding one or more target proteins into a host cell, and preparing a transformant by introducing an expression vector into a host cell.
  • the calcium phosphate method or the calcium chloride/rubidium chloride method described in the literature (Sambrook, J., et al. , Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1. 74, 1989) , electroporation, electroinjection, chemical treatment methods such as PEG, methods using a gene gun, and the like.
  • antibody protein When the transformant expressing the vector is cultured in a nutrient medium, antibody protein can be produced and isolated in large quantities.
  • Media and culture conditions can be appropriately selected and used according to the host cell. Conditions such as temperature, medium pH, and incubation time should be appropriately adjusted so as to be suitable for cell growth and mass production of proteins during culture.
  • the vector according to the present invention can be transformed into a host cell, preferably a mammalian cell, for antibody production.
  • Suitable host cells capable of expressing fully glycosylated proteins include COS-1 (eg ATCC CRL 1650), COS-7 (eg ATCC CRL-1651), HEK293, BHK21 (eg ATCC CRL-10), CHO (eg ATCC CRL 1610) and BSC-1 (eg ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0 -Agl4, 293 cells, HeLa cells, etc., and these cells are readily available, for example, from the American Type Culture Collection (ATCC, USA).
  • COS-1 eg ATCC CRL 1650
  • COS-7 eg ATCC CRL-1651
  • HEK293, BHK21 eg ATCC CRL-10
  • CHO eg ATCC CRL 1610
  • BSC-1 eg
  • a chimeric antigen receptor (CAR) containing an intracellular signal transduction domain containing an intracellular signal transduction domain
  • the CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4
  • An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 It relates to a chimeric antigen receptor targeting CD22.
  • the antibody specifically binding to CD22 is a humanized antibody specifically binding to CD22 composed of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12. antibodies or fragments thereof; or
  • It may be a humanized antibody or fragment thereof that specifically binds to CD22 composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
  • chimeric antigen receptor generally refers to a fusion protein containing an antigen and an extracellular domain that has the ability to bind one or more intracellular domains.
  • a CAR is a key part of a chimeric antigen receptor T cell (CAR-T) and can include an antigen binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain.
  • a CAR may be combined with a T cell receptor-activating intracellular domain based on the antibody's antigenic (eg CD22) specificity. Genetically modified CAR-expressing T cells can specifically identify and eliminate target antigen-expressing malignant cells.
  • CD22-binding domain generally refers to a domain capable of specifically binding to the CD22 protein.
  • the CD22-binding domain may contain an anti-CD22 antibody or fragment thereof capable of specifically binding to a human CD22 polypeptide or fragment thereof expressed on B cells.
  • binding domain includes “extracellular domain”, “extracellular binding domain”, “antigen-specific binding domain” and “Extracellular antigen-specific binding domain” may be used interchangeably and refers to a CAR domain or fragment that has the ability to specifically bind to a target antigen (eg CD22) do.
  • target antigen eg CD22
  • the anti-CD22 antibody or fragment thereof is the aforementioned anti-CD22 antibody, which is a monoclonal antibody, preferably a single chain variable fragment (scFv). Specifically, it can be prepared using the 4F5 (V4) or 4F5 (V11) antibody, which is a humanized antibody specific to CD22 of the present invention.
  • the chimeric antigen receptor may be a bispecific chimeric antigen receptor further comprising a B cell surface marker-binding domain in addition to the CD22-binding domain, and the B cell surface markers are CD10, CD19, and CD20. , CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85 or CD86, preferably CD19.
  • a signal peptide may be further included at the N-terminus of the CD22-binding domain, and the "signal peptide” generally refers to a peptide chain for guiding protein transduction .
  • the signal peptide may be a short peptide having a length of 5 to 30 amino acids, and the amino acid sequence of SEQ ID NO: 36 is preferably used in the present invention.
  • it may further include a hinge region located between the C-terminus of the CD22-binding domain and the N-terminus of the transmembrane domain, and the hinge region is derived from CD8 ⁇ , preferably SEQ ID NO: 37 amino acid sequence.
  • the "hinge region” generally refers to the junction region between an antigen-binding region and an immune cell Fc receptor (FcR)-binding region.
  • transmembrane domain generally refers to a domain of a CAR that passes through a cell membrane and is connected to an intracellular signaling domain to play a role in signal transduction.
  • the transmembrane domain may be derived from a protein selected from the group consisting of CD8 ⁇ , CD4, CD28, CD137, CD80, CD86, CD152 and PD1, and may preferably be represented by the amino acid sequence of SEQ ID NO: 38.
  • costimulatory domain generally refers to an intracellular domain capable of providing immune stimulatory molecules, which are cell surface molecules required for effective response of lymphocytes to antigens.
  • the costimulatory domain described above may include a costimulatory domain of CD28, and may include a costimulatory domain of the TNF receptor family, such as the costimulatory domains of OX40 and 4-1BB, preferably SEQ ID NO: It may be 4-1BB represented by the amino acid sequence of 39.
  • intracellular signal transduction domain generally refers to a domain located inside a cell and capable of transmitting a signal.
  • the intracellular signaling domain is an intracellular signaling domain of a chimeric antigen receptor.
  • the intracellular signaling domain may be selected from the CD3 ⁇ intracellular domain, the CD28 intracellular domain, the CD28 intracellular domain, the 4-1BB intracellular domain and the OX40 intracellular domain, preferably comprising the amino acids of SEQ ID NO: 40 It may be CD3 ⁇ represented by the sequence.
  • the present invention relates to a polynucleotide encoding the chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the polynucleotide encoding the chimeric antigen receptor (CAR) is a polynucleotide encoding a CD22-binding domain; polynucleotides encoding transmembrane domains; polynucleotides coating the co-stimulatory domain; And it may include a polynucleotide encoding an intracellular signaling domain.
  • the polynucleotide encoding the CD22-binding domain may be a 4F5 antibody specific for CD22, a humanized 4F5(V4) antibody specific for CD22, or a 4F5(V11) antibody specific for CD22 of the present invention, and the light chain variable region and the heavy chain variable region are linkers.
  • the specific nucleotide sequence is as described above.
  • the polynucleotide encoding the chimeric antigen receptor (CAR) of the present invention is a 4F5 antibody represented by the nucleotide sequence of SEQ ID NO: 24, a 4F5 (V4) antibody represented by the nucleotide sequence of SEQ ID NO: 26, or SEQ ID NO: 28 4F5 (V11) antibody represented by the nucleotide sequence of;
  • transmembrane domain represented by the nucleotide sequence of SEQ ID NO: 32;
  • 4-1BB (co-stimulatory domain) represented by the nucleotide sequence of SEQ ID NO: 33;
  • CD3 ⁇ intracellular signaling domain
  • SEQ ID NO: 34 The nucleotide sequence of SEQ ID NO: 34.
  • a signal peptide represented by the nucleotide sequence of SEQ ID NO: 30 may be additionally included.
  • a polynucleotide encoding a hinge region may be further included, preferably a CD8 hinge region represented by the nucleotide sequence of SEQ ID NO: 31 can
  • the present invention relates to a vector comprising a polynucleotide encoding the chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the vector is a recombinant viral vector, preferably a lentiviral vector, comprising an operably linked EF1 ⁇ promoter; polynucleotides encoding signal peptides; a polynucleotide encoding a CD22-binding domain; polynucleotides encoding transmembrane domains; It includes a polynucleotide encoding an intracellular signaling domain, and may further include a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to increase protein expression (FIG. 3).
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • the EF1 ⁇ promoter may be represented by the nucleotide sequence of SEQ ID NO: 29, and if necessary, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more of the nucleotide sequence of SEQ ID NO: 27 , or sequences that are at least 99% identical.
  • the promoter is operably linked to drive expression of a CD22-binding domain, an anti-CD22 antibody (scFv).
  • a lentiviral vector into which a polynucleotide coating CD22-CAR was inserted was prepared, and the prepared vector was transformed into 293FT cells to prepare CD22-CAR expressing cells.
  • Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors.
  • Viral vectors, and particularly retroviral vectors have become the most widely used method for inserting genes into mammalian, eg human, cells.
  • Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses and adeno-associated viruses, and the like.
  • Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (eg, artificial membrane vesicles).
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction (in vitro, ex vivo or in vivo) of nucleic acids into host cells.
  • a nucleic acid can be associated with a lipid.
  • Nucleic acids associated with lipids may be encapsulated in the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules associated with both liposomes and oligonucleotides, entrapped within liposomes, complexed with liposomes, or , dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • the lipid, lipid/DNA or lipid/expression vector associated composition is not limited to any particular structure in solution.
  • the present invention includes a polynucleotide encoding a chimeric antigen receptor (CAR) based on an antibody specific for the CD22 or a vector comprising a polynucleotide encoding the chimeric antigen receptor (CAR), wherein the CD22 It relates to immune effector cells expressing an antibody-based chimeric antigen receptor (CAR) specific for
  • the immune effector cells may be mammalian-derived isolated cells, preferably T cells, B cells, natural killer (NK) cells, dendritic cells, bone marrow cells, monocytes, or macrophages, and more Preferably it may be a T cell.
  • immune effector cells expressing the chimeric antigen receptor can be prepared by introducing the CAR vector of the present invention into immune effector cells, for example, T cells or NK cells.
  • the CAR vector may be introduced into cells by a method known in the art, such as electroporation or lipofectamine (lipofectamine 2000, Invitrogen).
  • immune effector cells can be transformed with a lentiviral vector to integrate the viral genome carrying the CAR molecule into the host genome to ensure long-term and stable expression of the target gene.
  • a transposon can be used to introduce a CAR carrier plasmid (transposon) and a transposase carrier plasmid into a target cell.
  • CAR molecules can be added to the genome by gene editing methods (eg, CRISPRCas9).
  • Immune effector cells for producing immune effector cells expressing a chimeric antigen receptor (CAR) can be obtained from a subject, wherein the “subject” is a living organism (eg, a mammal) from which an immune response can be elicited.
  • the “subject” is a living organism (eg, a mammal) from which an immune response can be elicited.
  • subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • the T cells can be obtained from a unit of blood collected from a subject using any of a number of techniques known to those skilled in the art, such as FicollTM separation.
  • Cells from blood are obtained by apheresis, and the apheresis product typically contains T cells, monocytes, granulocytes, lymphocytes including B cells, other nucleated leukocytes, red blood cells, and platelets.
  • T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, eg, by centrifugation over a PERCOLLTM gradient or by countercurrent centrifugation.
  • the CD22-CAR lentiviral vector is applied to the T cells.
  • CD22-CAR-T cells were prepared by transduction, and specifically, CD22-CAR-T cells were prepared using a 4F5 antibody, a humanized 4F5 (V4) antibody, and a 4F5 (V11) antibody, respectively.
  • CD3, CD4 or CD8-activated CD22-CAR-T cells were examined for their ability to bind to CD22 peptides. As shown in Figures 5a and 5b, it was confirmed that the CD22-CAR-T cells prepared in the present invention bind to the CD22 peptide.
  • CD22-CAR-T cells are U2932 expressing CD22. It was confirmed that the cell-specific killing effect was shown.
  • the anti-CD22 antibody of the present invention the 4F5 antibody, the 4F5(V4) antibody, or the 4F5(V11) antibody-based chimeric antigen receptor, and the CAR-T cell using the same are a composition for preventing or treating diseases related to B cell or CD22 expression can be put to good use.
  • Bispecific Chimeric Antigen Receptor Targeting CD19 and CD22
  • CD19-binding domain and CD22-binding domain CD19-binding domain and CD22-binding domain
  • CAR bispecific chimeric antigen receptor
  • the CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4
  • An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 It relates to a bispecific chimeric antigen receptor (CD19xCD22 bispecific CAR).
  • the antibody specifically binding to CD22 is an anti-CD22 antibody composed of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 7 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 8;
  • a humanized anti-CD22 antibody composed of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12; or
  • It may be a humanized anti-CD22 antibody composed of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
  • the bispecific (bispecific or bivalent) chimeric antigen receptor is a CAR capable of simultaneously binding two different types of antigens, and in the present invention, the bispecific chimeric antigen receptor preferably targets both CD19 and CD22 A receptor was prepared, and the CD19-binding domain may use a known anti-CD19 antibody sequence without limitation.
  • the chimeric antigen receptor Details of the chimeric antigen receptor are the same as described above, and the CD19-binding domain and the CD22-binding domain of the bispecific chimeric antigen receptor were connected in a loop (LoopCAR) form as shown in FIG. 7 . That is, the light chain variable region of an antibody that specifically binds to CD19 (CD19VL) - the heavy chain variable region of an antibody that specifically binds to CD22 (CD22VH) - the light chain variable region of an antibody that specifically binds to CD22 (CD22VL) - It may be linked in sequence to the heavy chain variable region (CD19VH) of an antibody that specifically binds to CD19.
  • the light chain variable region of the antibody specifically binding to CD19 is represented by the amino acid sequence of SEQ ID NO: 48, and the heavy chain variable region of the antibody specifically binding to CD19 is represented by the amino acid sequence of SEQ ID NO: 47. .
  • the CD19-binding domain and the CD22-binding domain can be prepared through genetic recombination technology so that they can be linked by a linker, and preferably CD19VL and CD22VH or CD22VL and CD19VH are linkers represented by the amino acid sequence of SEQ ID NO: 51 (linker in FIG. 8). 1, and CD22VH and CD22VL may be linked by a linker represented by the amino acid sequence of SEQ ID NO: 54 (linker 6 in FIG. 8), but is not limited thereto, and does not affect antibody activity Peptide consisting of any amino acid sequence can be used.
  • the present invention relates to a polynucleotide encoding a bispecific chimeric antigen receptor targeting CD19 and CD22.
  • the polynucleotide encoding the bispecific chimeric antigen receptor includes a polynucleotide encoding a CD19-binding domain and a polynucleotide encoding a CD22-binding domain; polynucleotides encoding transmembrane domains; polynucleotides coating the co-stimulatory domain; And it may include a polynucleotide encoding an intracellular signaling domain.
  • the polynucleotide encoding the bispecific chimeric antigen receptor of the present invention is
  • CD19VL Light chain variable region of antibody specifically binding to CD19 (CD19VL; SEQ ID NO: 50) - heavy chain variable region of antibody specifically binding to CD22 (CD22VH; SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 17) - CD22 Bispecific consisting of the light chain variable region of the antibody specifically binding (CD22VL; SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 18) - the heavy chain variable region of the antibody specifically binding to CD19 (CD19VH; SEQ ID NO: 49) antibodies;
  • transmembrane domain represented by the nucleotide sequence of SEQ ID NO: 32;
  • 4-1BB (co-stimulatory domain) represented by the nucleotide sequence of SEQ ID NO: 33;
  • CD3 ⁇ intracellular signaling domain
  • SEQ ID NO: 34 The nucleotide sequence of SEQ ID NO: 34.
  • the polynucleotide for the linker may be represented by the nucleotide sequence of SEQ ID NO: 52 or SEQ ID NO: 53, ,
  • the polynucleotide for the linker may be represented by the nucleotide sequence of SEQ ID NO: 55, but is not limited thereto and affects antibody activity. Polynucleotides encoding any amino acid sequence not specified may be used.
  • a signal peptide represented by the nucleotide sequence of SEQ ID NO: 30 may be additionally included.
  • a polynucleotide encoding a hinge region may be further included, preferably a CD8 hinge region represented by the nucleotide sequence of SEQ ID NO: 31 can
  • the present invention relates to a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor.
  • the vector is a recombinant viral vector, preferably a lentiviral vector, comprising an operably linked EF1 ⁇ promoter; polynucleotides encoding signal peptides; polynucleotides encoding a CD19-binding domain and a CD22-binding domain; polynucleotides encoding transmembrane domains; It includes a polynucleotide encoding an intracellular signaling domain, and may further include a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to increase protein expression (FIG. 9).
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • the EF1 ⁇ promoter may be represented by the nucleotide sequence of SEQ ID NO: 29, and if necessary, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more of the nucleotide sequence of SEQ ID NO: 27 , or sequences that are at least 99% identical.
  • the promoter is operably linked to induce expression of an anti-CD19/CD22 antibody (CD19VL-CD22VH-CD22VL-CD19VH), which is a CD19xCD22-binding domain, and details of the vector are the same as described above.
  • a lentiviral vector into which a polynucleotide coating CD19xCD22-CAR was inserted was prepared, and the prepared vector was transformed into 293FT cells to express CD19xCD22-CAR cells were prepared.
  • FIG. 11 it was confirmed that the bispecific chimeric antigen receptor targeting CD19 and CD22 was expressed in the prepared CD19xCD22-CAR expressing cells.
  • the present invention includes a polynucleotide encoding the bispecific chimeric antigen receptor (CAR) or a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor (CAR), It relates to immune effector cells that express specific chimeric antigen receptors (CARs).
  • CAR bispecific chimeric antigen receptor
  • CARs immune effector cells that express specific chimeric antigen receptors
  • the immune effector cells may be mammalian-derived isolated cells, preferably T cells, B cells, natural killer (NK) cells, dendritic cells, bone marrow cells, monocytes, or macrophages, and more Preferably it may be a T cell. Further, details of the immune effector cells expressing the chimeric antigen receptor are the same as described above.
  • CD19xCD22-CAR lentiviral vector is applied to the T cells.
  • CD19xCD22-CAR-T cells were prepared by transduction, and specifically, CD19xCD22-CAR-T cells were prepared using a 4F5 antibody (mouse), a humanized 4F5 (V4) antibody, and a 4F5 (V11) antibody, respectively.
  • the binding ability of the CD3-activated CD19xCD22-CAR-T cells to the CD22 peptide and the CD19 peptide was confirmed. As shown in FIG. 14, it was confirmed that the CD19xCD22-CAR-T cells prepared in the present invention simultaneously bind to the CD22 peptide and the CD19 peptide.
  • CD19xCD22-CAR-T cells are K562-CD19 cells (CD19 expressing cells). ), K562-CD22 cells (CD22 expressing cells) and K562-CD19/CD22 cells (CD19 and CD22 expressing cells).
  • composition for preventing or treating diseases mediated by B cells or diseases associated with CD19 or CD22 expression
  • the present invention provides an antibody that specifically binds to CD22; humanized antibodies that specifically bind to CD22; immune effector cells expressing chimeric antigen receptors that target CD22; or a pharmaceutical composition for preventing or treating a disease mediated by B cells, comprising immune effector cells expressing a bispecific chimeric antigen receptor that specifically binds to CD19xCD22.
  • the B cell may preferably be a cell expressing CD19 or CD22, and the disease is tumor/cancer, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent delayed NHL, refractory NHL, refractory delayed NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute It may be selected from the group consisting of acute lymphocytic leukemia (ALL), Burkitt's lymphoma and mantle cell lymphoma.
  • NHL non-Hogkins lymphoma
  • aggressive NHL recurrent aggressive NHL
  • recurrent delayed NHL recurrent delayed NHL
  • refractory NHL refractory delayed NHL
  • CLL chronic lymphocytic leukemia
  • small lymphocytic lymphoma small lymphocytic lymphoma
  • leukemia hairy cell leukemia
  • HCL hairy cell leuk
  • the composition may include a therapeutic agent for a disease mediated by B cells, and the therapeutic agent is present in a state covalently bound to an antibody that specifically binds to CD19 or CD22, or the CD22- It can be administered in combination with CAR immune effector cells or CD19xCD22-CAR immune effector cells.
  • the therapeutic agent includes a small molecule drug, a peptide drug, a toxin (eg, cytotoxin), and the like.
  • the small molecule drug may be a compound that exhibits a pharmaceutical activity of interest and generally has a molecular weight of about 800 Da or less or 2000 Da or less.
  • Inorganic small molecules refer to molecules containing no carbon atoms, whereas organic small molecules refer to compounds containing at least one carbon atom.
  • the peptidic drug refers to an amino acid containing polymeric compound, which includes naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides and proteins, as well as peptide mimetics.
  • the peptide drug may be obtained by chemical synthesis or generated from a genetically encoded source (eg, a recombinant source).
  • the molecular weight of the peptide drug may range from 200 Da to 10 kDa or more.
  • the toxin is preferably a cytotoxin, including, but not limited to, ricin, abrin, diphtheria toxin, Pseudomonas exotoxin (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, orca.
  • cytotoxin including, but not limited to, ricin, abrin, diphtheria toxin, Pseudomonas exotoxin (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, orca.
  • PAP antiviral protein
  • botulinum toxin briodin
  • momordin momordin
  • bunin antiviral protein
  • the therapeutic agent may be an anticancer agent.
  • Anti-cancer agents reduce the proliferation of cancer cells and include non-peptidic (i.e., non-proteinaceous) compounds, including cytotoxic agents and cytostatic agents.
  • Non-limiting examples of anticancer agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids and steroid hormones. Peptidic compounds may also be used.
  • the humanized antibody, CD22-CAR immune effector cells or CD19xCD22-CAR immune effector cells that specifically bind to CD22 are the only active ingredients in the therapeutic or diagnostic composition, or, for example, anti-T cells, It can be used with other active ingredients including other antibody components such as anti-IFN ⁇ or anti-LPS antibodies, or non-antibody components such as xanthines.
  • the pharmaceutical composition preferably comprises a therapeutically effective amount of an antibody of the present invention.
  • therapeutically effective amount means the amount of a therapeutic agent required to treat, ameliorate, or prevent the target disease or condition, or to produce an appreciable therapeutic or prophylactic effect.
  • the therapeutically effective dose can be determined initially by cell culture assays or animal models, usually rodents, rabbits, dogs, pigs or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can be used to determine useful dosages and routes for human administration.
  • an effective dosage is 0.01 to 50 mg/kg, preferably 0.1 to 20 mg/kg, more preferably about 15 mg/kg.
  • compositions may be administered to a patient individually or in combination with other agents, drugs or hormones.
  • the dosage at which an antibody of the invention is administered depends on the nature of the condition being treated, the grade of the malignant lymphoma or leukemia, and whether the antibody is being used to prevent disease or to treat an existing condition.
  • the frequency of administration depends on the half-life of the antibody molecule and the persistence of the drug effect. If the antibody molecule has a short half-life (eg, 2-10 hours), it may be necessary to give one or more doses per day. Alternatively, if the antibody molecule has a long half-life (eg, 2-15 days), it may be necessary to give a dose once daily, once a week, or once every 1 or 2 months.
  • the pharmaceutical composition may contain a pharmaceutically acceptable carrier for administration of the antibody.
  • the carrier must not itself induce the production of antibodies harmful to the subject to which the composition is administered, and must be non-toxic.
  • Suitable carriers can be slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers and inactive viral particles.
  • salts are, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate and sulfate, or acetic acid, propionic acid. Salts of organic acids such as malonic acid and benzoic acid may be used.
  • Pharmaceutically acceptable carriers in therapeutic compositions may additionally include liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances such as wetting agents, emulsifying agents or pH buffering substances may be present in these compositions.
  • the carrier may be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion of the pharmaceutical composition by a patient.
  • Preferred forms for administration include forms suitable for parenteral administration, eg by injection or infusion (eg bolus injection or continuous infusion).
  • parenteral administration eg by injection or infusion (eg bolus injection or continuous infusion).
  • the product may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, which may contain such prescriptive agents as suspending agents, preservatives, stabilizing and/or dispersing agents.
  • the antibody molecule may be in anhydrous form and reconstituted with an appropriate sterile solution prior to use.
  • compositions of the present invention can be administered directly to a patient.
  • the patients to be treated may be animals. However, it is preferred that the compositions are tailored for administration to human patients.
  • the pharmaceutical composition of the present invention may be used, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (see eg WO 98/20734), subcutaneous, It may be administered by any route including intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes.
  • a hypospray can be used to administer the pharmaceutical composition of the present invention.
  • therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions.
  • solid forms suitable for solution or suspension in liquid excipients may be prepared prior to injection.
  • Direct delivery of the composition may generally be by injection, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, or may be delivered into the interstitial space of a tissue.
  • the composition may be administered to a wound site. Dosage treatment can be a single dose schedule or a multiple dose schedule.
  • the active ingredient in the composition may be an antibody molecule. As such, it can be susceptible to degradation within the gastrointestinal tract. Thus, if the composition is administered by a route using the gastrointestinal tract, the composition will need to contain an agent that protects the antibody from degradation but releases the antibody once absorbed from the gastrointestinal tract.
  • Example 1 Production and screening of antibodies that specifically bind to CD22
  • a hybridoma producing an antibody that binds to CD22 was prepared and the antibody was selected.
  • splenocytes were isolated by immunization with CD22 protein (ACRObiosystems Inc., cat#CD2-H52H8, USA), and hybridoma cells were prepared through cell fusion with mouse myeloma cells.
  • HGPRT HypoxanthineGuanidine-Phosphoribosyl-Transferase
  • a limiting dilution method was used to select hybridomas producing antibodies that bind to CD22 among the proliferated hybridomas.
  • the number of cells per 96 well was reduced to 1 or less, and then, whether the antibodies obtained from the clones proliferated from 1 cell were binding to CD22 was confirmed by ELISA, and clones binding to CD22 were selected. By repeating the above process three times, hybridomas producing antibodies binding to CD22 were selected. In this way, an antibody binding to CD22 was obtained.
  • the antibody was named 4F5, and its base sequence and amino acid sequence were analyzed. Sequence information for the heavy chain variable region and the light chain variable region of each antibody according to the sequencing results is shown in Table 1 below, and the underlined portion in Table 1 means the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Sequence information of 4F5 antibody 4F5 sequence information sequence number heavy chain variable region CDR1 GFSLTIYG SEQ ID NO: 1 heavy chain variable region CDR2 MWSGGST SEQ ID NO: 2 heavy chain variable region CDR3 ARNDGYYWFAY SEQ ID NO: 3 light chain variable region CDR1 QSLVHNNGNTY SEQ ID NO: 4 light chain variable region CDR2 KVS SEQ ID NO: 5 light chain variable region CDR3 SQSTHVPYS SEQ ID NO: 6 heavy chain variable region amino acid sequence QVQLKESGPGLVQPSQSLSITCTVS GFSLTIYG VHWIRQSPGKGLEWLGV MWSGGST DYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYC ARNDGYYWFAY WGQGTLVTVSA SEQ ID NO: 7 light chain variable region amino acid sequence DVLMTQTPLSLPVSLGDQASISCRSS QSLVHNNGNTY LHWYLQKPGQ
  • a humanized antibody was prepared by changing the 4F5 antibody selected in Example 1 to a structure corresponding to human.
  • mouse 4F5 antibody is obtained by a CDR-grafting method in which the CDR of a mouse antibody that binds to CD22 is replaced with the CDR of a human antibody using the germline sequence of a human antibody as a frame.
  • a humanized antibody was prepared.
  • the humanized antibodies were named 4F5(V4) antibody and 4F5(V11) antibody, and the amino acid sequences were analyzed.
  • Sequence information for the heavy chain variable region and the light chain variable region of the antibody according to the sequencing results are shown in Tables 2 and 3 below, and the underlined portions in Tables 2 and 3 are complementary determining regions (complementarity determining regions; CDRs).
  • CD22-expressing non-cell lymphoma U2932 (B-cell lymphoma U2932 cell) was reacted with 1x10 6 cells and 1 ⁇ g of 4F5 antibody for 30 minutes, and then, after staining the surface with a secondary antibody, flow cytometry measured.
  • PE-conjugated anti-CD22 antibody Biolegend Inc., cat# 302506, USA
  • PE-conjugated anti-mouse IgG antibody PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat# 405307, USA
  • the 4F5 antibody, the humanized 4F5(V4) antibody, and the 4F5(V11) antibody specifically bind to cells expressing CD22.
  • lentivirus expressing a chimeric antigen receptor (CAR) targeting CD22 A vector CD22-CAR lentiviral vector
  • a polynucleotide encoding the CD22-binding domain (4F5 antibody represented by the nucleotide sequence of SEQ ID NO: 24, 4F5 (V4) antibody represented by the nucleotide sequence of SEQ ID NO: 26, or 4F5 (V11) represented by the nucleotide sequence of SEQ ID NO: 28 antibody);
  • polynucleotide encoding 4-1BB (co-stimulatory domain) (SEQ ID NO: 33);
  • CAR DNA consisting of a polynucleotide (SEQ ID NO: 35) encoding WPRE was synthesized in vitro and inserted into a third-generation lentiviral vector.
  • Lentiviral vector DNA (0.5 ⁇ g) was transferred to HEK293FT cells (5 ⁇ 10 5 cells/500 ⁇ l), and 293HEK cells expressing the CD22-CAR gene were constructed.
  • Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) was used to transfer the gene to 293HEK cells, and cultured in Opti-MEM (gibco, cat# 51985-034) medium for 4 hours.
  • CD22-CAR-T cells based on 4F5 antibody, 4F5(V4) antibody and 4F5(V11) antibody were prepared by transfecting T cells with the CD22-CAR lentiviral vector prepared in Example 4 above.
  • T cell activation beads (Miltenyl Biotec, cat# 130-091-441 ) was used to activate T cells.
  • 4F5 antibody-based CD22-CAR-T cells, 4F5 (V4) antibody-based CD22-CAR-T cells, and 4F5 ( V11) antibody-based CD22-CAR-T cells were respectively prepared.
  • CD22 peptide binding capacity of CD22-CAR-T cells was confirmed by flow cytometry.
  • the 4F5 antibody, 4F5(V4) antibody-based CD22-CAR-T cells, and 4F5(V11) antibody-based CD22-CAR-T cells prepared above were subjected to CD3, anti-CD4, and anti-CD8 antibodies.
  • CD4 or CD8 activated CD22-CAR-T cells were respectively sorted, reacted with FITC-CD22 peptide, and fluorescence intensity was measured using a FACS machine.
  • CD3, CD4, or CD8-activated CD22-CAR-T cells all bind to the CD22 peptide.
  • Example 6 Confirmation of the killing effect of CD22-CAR-T cells on CD22 expressing cells
  • K562 cells human erythroleukemic cell line
  • the degree of cell death was calculated using Equation 1 below with the measured value.
  • Target Spontaneous Luminescence value derived from the target cell only medium
  • Target Maximum Luminescence value derived from 100% lysis of target cells (using Lysis Reagent)
  • the anti-CD22 antibody of the present invention the 4F5 antibody, the 4F5(V4) antibody or the 4F5(V11) antibody, the antibody-based chimeric antigen receptor, and the CAR-T cell using the same are used to prevent or prevent diseases associated with B cell or CD22 expression. It can be usefully utilized as a therapeutic composition.
  • Example 7 Construction of a bispecific chimeric antigen receptor expression vector targeting CD19/CD22
  • a lentiviral vector (CD19xCD22-CAR lentiviral vector) expressing a bispecific chimeric antigen receptor targeting CD19 and CD22 was prepared in the same manner as in Example 4 above.
  • polynucleotide encoding 4-1BB (co-stimulatory domain) (SEQ ID NO: 33);
  • CAR DNA consisting of a polynucleotide (SEQ ID NO: 35) encoding WPRE was synthesized in vitro and inserted into a third-generation lentiviral vector.
  • the known anti-CD19 antibody (FMC63) was used for the CD19-binding domain
  • the 4F5 (mouse) antibody, 4F5 (V4) antibody and 4F5 (V11) antibody of the present invention were used for the CD22-binding domain.
  • the CD19xCD22-binding domain is the light chain variable region of an antibody that specifically binds to CD19 (CD19VL) - the heavy chain variable region of an antibody that specifically binds to CD22 (CD22VH) - the light chain variable region of an antibody that specifically binds to CD22 (CD22VL) - linked in sequence to the heavy chain variable region (CD19VH) of an antibody specifically binding to CD19 (LoopCAR), and sequence information for polynucleotides encoding them is as follows:
  • CD19x4F5 (mouse): CD19VL represented by the nucleotide sequence of SEQ ID NO: 50 - Linker represented by the nucleotide sequence of SEQ ID NO: 52 (linker 1 in FIG. 8) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 9 - nucleotide sequence of SEQ ID NO: 55 Linker represented by sequence (linker 6 in FIG. 8) - CD22VL represented by nucleotide sequence of SEQ ID NO: 10 - Linker represented by nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 8) - represented by nucleotide sequence of SEQ ID NO: 49 becoming CD19VH;
  • CD19x4F5 (V4): CD19VL represented by the nucleotide sequence of SEQ ID NO: 50 - Linker represented by the nucleotide sequence of SEQ ID NO: 52 (linker 1 in FIG. 8) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 13 - nucleotide sequence of SEQ ID NO: 55 Linker represented by sequence (linker 6 in FIG. 8) - CD22VL represented by nucleotide sequence of SEQ ID NO: 14 - Linker represented by nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 8) - represented by nucleotide sequence of SEQ ID NO: 49 becoming CD19VH;
  • CD19x4F5 (V11): CD19VL represented by the nucleotide sequence of SEQ ID NO: 50 - Linker represented by the nucleotide sequence of SEQ ID NO: 52 (linker 1 in FIG. 8) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 17 - nucleotide sequence of SEQ ID NO: 55 Linker represented by sequence (linker 6 in FIG. 8) - CD22VL represented by nucleotide sequence of SEQ ID NO: 18 - Linker represented by nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 8) - represented by nucleotide sequence of SEQ ID NO: 49 being CD19VH.
  • Lentiviral vector DNA (0.5 ⁇ g) was transferred to HEK293FT cells (5 ⁇ 10 5 cells/500 ⁇ l), and 293HEK cells expressing the CD19/CD22-CAR gene were constructed.
  • Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) was used to transfer the gene to 293HEK cells, and cultured in Opti-MEM (gibco, cat# 51985-034) medium for 4 hours (FIGS. 9 and 10). ).
  • CD19xCD22-CAR-T cells CD19x4F5, C19x4F5 (V4) and CD19x4F5 (V11), were transfected into T cells in the same manner as in Example 5 with the CD19xCD22-CAR lentiviral vector prepared in Example 7, respectively. was prepared (Figs. 12 and 13).
  • CD19xCD22-CAR-T cells (CD19x4F5, CD19x 4F5 (V4), and CD19x4F5 (V11)) were sorted into CD3-activated CD19xCD22-CAR-T cells using an anti-CD3 antibody, and then PE-CD19 peptide and FITC After reacting with the -CD22 peptide, fluorescence intensity was measured using a flow cytometer.
  • CD3-activated CD19xCD22-CAR-T cells simultaneously bind to CD19 peptide and CD22 peptide.
  • Example 9 Confirmation of the killing effect of CD19xCD22-CAR-T cells on CD22 or CD19 expressing cells
  • K562 cells (ATCC, cat# CCL-243) that do not express CD19 and CD22 were used to construct three cells K562-CD19, K562-CD22, and K562-CD19/CD22 that express CD19, CD22 or CD19xCD22. , The target cell killing effect by CD19xCD22-CAR-T cells was confirmed.
  • the CD19x4F5-CAR-T cells, CD19x4F5(V4)-CAR-T cells and CD19x4F5(V11)-CAR-T cells prepared in the present invention express CD22 or CD19 or CD19/CD22. It was confirmed that the expressing cells were specifically killed.
  • a humanized antibody that specifically binds to CD22 was prepared, and using this, single CAR-T cells targeting CD22 and bispecific CAR-T cells targeting CD19 and CD22 were prepared.
  • CD22-CAR-T cells and the bispecific CD19xCD22-CAR-T cells prepared in the present invention effectively recognized the CD22 antigen, activated the CAR-T cells, and effectively killed cells expressing CD22.
  • CD22 (or CD19) can be usefully used as a composition for preventing or treating a disease related to expression or a disease related to B cells.

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Abstract

La présente invention concerne un anticorps spécifique de CD22 et un récepteur antigénique chimérique l'utilisant et, plus particulièrement, un anticorps se liant de manière spécifique à CD22 (anticorps 4F5) et un anticorps humanisé de celui-ci, un récepteur antigénique chimérique comprenant l'anticorps ou un anticorps CD19xCD22, une cellule CAR-T exprimant le récepteur antigénique chimérique et une composition pharmaceutique le comprenant pour prévenir ou traiter des maladies médiées par les lymphocytes B. Dans la présente invention, un anticorps se liant de manière spécifique au CD22 et un anticorps humanisé de celui-ci ont été préparés et une seule cellule CAR-T ciblant CD22 et une cellule CAR-T bispécifique ciblant CD19 et CD22 ont été préparées à l'aide de celui-ci. Les cellules CD22-CAR-T et les cellules CD19xCD22-CAR-T préparées dans la présente invention ont été découvertes pour reconnaître de manière efficace l'antigène CD22 et détruire efficacement des cellules exprimant CD22, et peuvent ainsi être avantageusement utilisées en tant que composition pour prévenir ou traiter des maladies associées à l'expression de CD22 (ou CD19).
PCT/KR2022/017092 2021-11-17 2022-11-03 Anticorps spécifique pour cd22 humanisé et récepteur antigénique chimérique l'utilisant Ceased WO2023090704A1 (fr)

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