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WO2024000259A1 - Anticorps se liant de manière spécifique à cd22, son procédé de préparation et son utilisation sur un cart bispécifique - Google Patents

Anticorps se liant de manière spécifique à cd22, son procédé de préparation et son utilisation sur un cart bispécifique Download PDF

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Publication number
WO2024000259A1
WO2024000259A1 PCT/CN2022/102313 CN2022102313W WO2024000259A1 WO 2024000259 A1 WO2024000259 A1 WO 2024000259A1 CN 2022102313 W CN2022102313 W CN 2022102313W WO 2024000259 A1 WO2024000259 A1 WO 2024000259A1
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Prior art keywords
antigen
antibody
cancer
binding fragment
lymphoma
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English (en)
Chinese (zh)
Inventor
杜靓
牟男
万婷婷
于跃
徐溜溜
张红艳
金夷
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Shanghai Genbase Biotechnology Co Ltd
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Shanghai Genbase Biotechnology Co Ltd
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Priority to PCT/CN2022/102313 priority Critical patent/WO2024000259A1/fr
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • This application belongs to the field of biological immunity technology, and specifically relates to monoclonal antibodies that can specifically bind to human CD22 and their antigen-binding fragments.
  • the present application also relates to bispecific chimeric antigen receptors and preparation methods and uses thereof.
  • Immune cell therapy uses the tumor patient's autologous immune cells, such as T cells, which are amplified and cultured in vitro and genetically modified and then infused back into the cancer patient to eliminate the tumor.
  • successful immune cell therapies include tumor infiltrating T cell therapy (TIL, Tumor Infiltrating T cell therapy), T cell receptor therapy (TCR-T, T Cell Receptor T cell therapy) and chimeric antigen receptor T cell therapy (TCR-T, T Cell Receptor T cell therapy).
  • TIL Tumor Infiltrating T cell therapy
  • TCR-T T Cell receptor therapy
  • TCR-T T Cell Receptor T cell therapy
  • CART Chimeric Antigen Receptor T cell therapy
  • CART is a new type of anti-cancer therapy. Cancer patients' T cells are cultured and expanded in vitro and genetically engineered using viral vectors to generate CART cells.
  • CART cells can recognize and activate tumor-specific antigens (such as CD19), and specifically lyse tumor cells to kill them.
  • tumor-specific antigens such as CD19
  • HLA Human Leukocyte Antigen
  • CD19 is a cell surface antigen specifically expressed on various differentiation stages of B lymphocytes.
  • Most malignant tumors derived from the B lineage including B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and non-Hodgkin lymphoma cells, express CD19. , so it is considered an ideal target for CAR-T treatment of B-cell tumors.
  • Clinical trial results show that the cure rate of CD19 CAR-T for acute B-lymphoblastic leukemia (B-ALL) has reached 90%. Facing the threat of CAR-T cell therapy, some tumor cells will regulate the CD19 expressed by cancer cells. Level up and escape the attack.
  • CD22 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglecs) family and one of the inhibitory co-receptors on the surface of B cells. It is closely related to the development, differentiation and function of B cells. CD22 is restrictedly expressed on the surface of mature B cells and most malignant B lymphoma cells, making it one of the popular targets for the treatment of autoimmune diseases and B cell malignancies.
  • immunotherapy drugs that target CD22 include monoclonal antibody drugs, antibody conjugates (ADCs) and CAR-T therapy.
  • CD22 CAR-T cell therapy (dose 1: 3 ⁇ 10 5 /kg; dose 2: 1 ⁇ 10 6 /kg; dose 3: 3 ⁇ 10 6 /kg).
  • HHLH hemophagocytic lymphohistiocytosis
  • MAS macrophage activation syndrome
  • chimeric antigen receptor refers to a cell surface receptor that can recognize a specific antigen (e.g., a tumor antigen) and contains an extracellular domain capable of recognizing the specific antigen (e.g., recognizing and binding Antigen-binding fragments of antibodies to specific antigens) and intracellular domains capable of transmitting extracellular signals to the interior of the cell (also known as signaling domains, such as the zeta chain of CD3 or the intracellular portion of Fc ⁇ RI ⁇ ).
  • a specific antigen e.g., a tumor antigen
  • intracellular domains capable of transmitting extracellular signals to the interior of the cell
  • signaling domains such as the zeta chain of CD3 or the intracellular portion of Fc ⁇ RI ⁇
  • CAR-T cells T cells that carry and express such chimeric antigen receptors are called CAR-T cells, which are able to recognize and bind to specific antigens and cells expressing the specific antigens (such as tumor cells) through the extracellular domain, and through intracellular
  • the signal transduction effect of the domain activates the immune response, releases a large number of multiple effectors, and efficiently kills cells expressing the specific antigen (such as tumor cells), thereby exerting a therapeutic effect (such as treating tumors).
  • bispecific chimeric antigen receptor or “dual-target chimeric antigen receptor” have the same meaning and refer to a cell surface that can recognize two specific antigens (e.g., tumor antigens) Receptors, which typically comprise a first antigen-binding domain (e.g., an antigen-binding fragment) for a first antigen, a second antigen-binding domain (e.g., an antigen-binding fragment) for a second antigen, and a hinge region (spacer) ), transmembrane domain, costimulatory domain (cytoplasmic region), etc.
  • first antigen-binding domain e.g., an antigen-binding fragment
  • second antigen-binding domain e.g., an antigen-binding fragment
  • a hinge region spacer
  • transmembrane domain e.g., an antigen-binding fragment
  • costimulatory domain cytoplasmic region
  • the term "spacer” or "hinge region” refers to a polypeptide linking region that provides a flexible structure that can connect the antigen-binding region to a transmembrane domain. Generally, they are flexible enough to allow the antigen-binding region to be oriented in different directions and bind to the antigen.
  • the hinge region may be naturally occurring or non-naturally occurring, including but not limited to altered hinge regions as described in U.S. Patent No. 5,677,425. In certain embodiments, the hinge region may comprise an entire hinge region derived from an antibody of a different class or subclass than that of the CH1 domain antibody.
  • transmembrane domain is a hydrophobic alpha helix that spans a membrane. Different transmembrane domains can lead to different receptor stabilities.
  • the transmembrane domain is inserted between the spacer domain and the costimulatory domain (cytoplasmic domain). In some embodiments, the transmembrane domain is inserted between the spacer and one or more costimulatory regions. In some embodiments, the linker is between a transmembrane domain and one or more costimulatory regions.
  • cytoplasmic domain or “costimulatory domain” refers to the region through which, following antigen recognition, receptors assemble and signals are transmitted to the cell.
  • the costimulatory domain is part of the cytoplasmic domain.
  • the term "signaling domain” refers to a region that provides a distinct and detectable signal (e.g., by a cell of one or more species) in response to activation by binding of an antigen to an antigen-binding domain. Increased production of various cytokines; changes in target gene transcription; changes in protein activity; changes in cell behavior, such as cell death; cell proliferation; cell differentiation; cell survival; regulation of cell signaling responses, etc.).
  • the signaling domain acts together with a costimulatory domain to transmit a signal.
  • antibody refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC).
  • Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
  • VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site.
  • the assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342:878-883 definition.
  • CDR complementarity determining region
  • the variable regions of the heavy chain and light chain each contain three CDRs, named CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art.
  • the CDRs contained in the antibodies of the invention, or antigen-binding fragments thereof are preferably determined by the Kabat, Chothia, or IMGT numbering systems.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • antibody is not limited to any particular method of producing the antibody. This includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • antigen-binding fragment of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody Specific binding to an antigen, which is also called an "antigen-binding moiety.”
  • an antigen-binding moiety which is also called an "antigen-binding moiety.”
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabody, single domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), probody and such polypeptides, which contain sufficient antigen to confer specificity to the polypeptide At least a portion of an antibody with binding capacity.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
  • full-length antibody means an antibody consisting of two “full-length heavy chains” and two “full-length light chains.”
  • “full-length heavy chain” refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), and a heavy chain in the direction from the N end to the C end. It is composed of a constant region CH2 domain and a heavy chain constant region CH3 domain; and, when the full-length antibody is of IgE isotype, optionally also includes a heavy chain constant region CH4 domain.
  • a "full-length heavy chain” is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction.
  • a "full-length light chain” is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction.
  • the two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains.
  • the full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody.
  • the full-length antibody of the present invention contains two antigen-binding sites formed by VH and VL pairs respectively, and these two antigen-binding sites specifically recognize/bind the same antigen.
  • the term “Fd” means an antibody fragment consisting of VH and CH1 domains
  • the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 ( 1989));
  • the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains;
  • the term “F(ab') 2 fragment” means an antibody fragment consisting of two fragments connected by a disulfide bridge on the hinge region An antibody fragment of a Fab fragment;
  • the term “Fab'fragment” means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd of the heavy chain. Fragment (consisting of VH and CH1 domains).
  • Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.
  • Fc means a region formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain.
  • Antibody fragments The Fc fragment of an antibody has many different functions but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.
  • scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody.
  • scFv can form (scFv) 2 , which refers to two or more individual scFvs joining in parallel to form an antibody.
  • the term "diabody” means one whose VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains of the same chain, This forces the domain to pair with the complementary domain of the other chain and creates two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).
  • single-domain antibody has the meaning commonly understood by those skilled in the art, which refers to an antibody composed of a single monomeric variable domain (e.g., a single heavy chain variable An antibody fragment consisting of a region) that retains the ability to specifically bind to the same antigen that the full-length antibody binds.
  • Single domain antibodies are also called nanobodies.
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as for intact antibodies.
  • antibody includes not only intact antibodies but also antigen-binding fragments of the antibodies, unless the context clearly indicates otherwise.
  • chimeric antibody refers to an antibody in which a portion of the light chain or/and heavy chain is derived from an antibody (which may originate from a specific species or belong to a specific species). a specific antibody class or subclass), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but regardless of However, it still retains the binding activity to the target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • the term “chimeric antibody” may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody and the heavy and light chain constant regions of the antibody are derived from a second antibody.
  • the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the first amino acid sequence or nucleic acid sequence to best match the second amino acid or nucleic acid sequence). Good comparison).
  • the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. Molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence.
  • Determination of percent identity between two sequences can also be accomplished using mathematical algorithms.
  • One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Improved in .Acad.Sci.U.S.A.90:5873-5877.
  • Such algorithms were integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.
  • variant in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term “variant” also refers to a polypeptide or peptide that has been modified (ie, by covalently linking any type of molecule to the polypeptide or peptide).
  • polypeptides may be modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, Attached to cellular ligands or other proteins, etc.
  • Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
  • a variant has a similar, identical or improved function to the polypeptide or peptide from which it is derived.
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473).
  • K D , kon and kdis values can be measured by any valid method.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
  • bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
  • a detectable label of the invention may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means.
  • labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., 3H , 125I , 35S , 14C , or 32P ), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa750)), luminescent substances (such as chemiluminescent substances, such as
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses poxviruses
  • baculoviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyomavacuolating viruses such as SV40.
  • a vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as E. coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers Agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.
  • the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions).
  • such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • dextrose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • prevention refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no worsening) of the state of the disease, delaying or slowing the progression of the disease, ameliorating or alleviating the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
  • treatment may also refer to prolonging survival compared to expected survival if not receiving treatment.
  • the term "effective amount" refers to an amount sufficient to obtain, at least in part, the desired effect.
  • a disease-preventing effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of the disease
  • a disease-treating effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already suffer from the disease. quantity. Determining such effective amounts is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.
  • This application uses a CD22 high-expressing cell line for immunization.
  • the binding of antibodies and CD22 at the cellular level is examined, and this is used as a basis for screening to screen out antibodies that specifically bind CD22.
  • a CD19 and CD22 bispecific CART was constructed based on the development of CD22 antibodies.
  • CD19 and CD22 bispecific CART can improve the killing efficiency, amplification efficiency and persistence of CD19 low-expressing tumor cells. It can expand the indications and suitable groups of existing hematoma pipeline products, improve drug efficacy, and reduce the recurrence rate after treatment. Of great significance.
  • the present application provides an antibody or an antigen-binding fragment thereof that specifically binds to CD22 protein, the antibody or an antigen-binding fragment thereof comprising:
  • VH Heavy chain variable region containing the following three complementarity determining regions (CDRs):
  • VH CDR1 which consists of the following sequence: SEQ ID NO: 3, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) sequence,
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 4, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or addition), and
  • VL light chain variable region containing the following three complementarity determining regions (CDRs):
  • VL CDR1 which consists of the following sequence: SEQ ID NO: 6, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) sequence,
  • VL CDR2 which consists of the following sequence: SEQ ID NO: 7, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or addition) sequence
  • VL CDR3 which consists of the following sequence: SEQ ID NO: 8, or has one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions).
  • substitutions of any one of (i)-(vi) are conservative substitutions.
  • the CDRs described in any of (i)-(vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.
  • the CDRs of any of (i)-(vi) are defined according to the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH CDR1 as shown in SEQ ID NO:3, VH CDR2 as shown in SEQ ID NO:4, VH CDR3 as shown in SEQ ID NO:5; and/or, the following 3 Light chain CDRs: VL CDR1 as shown in SEQ ID NO:6, VL CDR2 as shown in SEQ ID NO:7, VL CDR3 as shown in SEQ ID NO:8.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH Heavy chain variable region comprising an amino acid sequence selected from the following:
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH having the sequence shown in SEQ ID NO:1 and VL having the sequence shown in SEQ ID NO:2, or
  • VH having the sequence shown in SEQ ID NO: 10 and VL having the sequence shown in SEQ ID NO: 11.
  • the antibody or antigen-binding fragment thereof comprises a constant region derived from a human immunoglobulin or a variant thereof.
  • the antibody or antigen-binding fragment thereof comprises:
  • CH heavy chain constant region
  • a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived (e.g. , substitution, deletion or addition of up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids. addition or any combination thereof); and/or
  • the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.
  • the antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) set forth in SEQ ID NO: 12.
  • the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.
  • the antibody or antigen-binding fragment thereof comprises the light chain constant region (CL) set forth in SEQ ID NO: 13.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, BsFv, dsFv, (dsFv) 2 , dsFv-dsFv', scFv, scFv dimer, camelized single chain domain antibody, diabody, ds diabody, nanobody, single domain antibody (sdAb), bivalent domain antibody; and/ Or, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a multispecific antibody.
  • the antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibody of the invention.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab’ fragments can be obtained directly from host cells; Fab’ fragments can be chemically coupled to form F(ab’)2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab’)2 fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.
  • the antibody or antigen-binding fragment thereof is labeled.
  • the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or Biotin.
  • an enzyme e.g., horseradish peroxidase
  • a radionuclide e.g., a radionuclide
  • a fluorescent dye e.g., a fluorescent dye
  • a luminescent substance e.g., a chemiluminescent substance
  • the present application provides an isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described above, or a heavy chain variable region and/or a light chain variable region thereof.
  • the nucleic acid molecule comprises a sequence set forth in SEQ ID NO: 16 or 18. In certain embodiments, the nucleic acid molecule comprises a sequence set forth in SEQ ID NO: 17 or 19.
  • the heavy chain variable region has the sequence set forth in SEQ ID NO: 16 or 18.
  • the light chain variable region has the sequence set forth in SEQ ID NO: 17 or 19.
  • the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a first nucleotide sequence encoding said antibody or antigen-binding fragment thereof A second nucleotide sequence of a light chain or light chain variable region, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different separate nucleic acid molecules.
  • the isolated nucleic acid molecule of the present invention includes a third nucleotide sequence containing the first nucleotide sequence. a nucleic acid molecule and a second nucleic acid molecule containing said second nucleotide sequence.
  • the application provides a vector comprising a nucleic acid molecule as described above.
  • the vector is a cloning vector or an expression vector.
  • the vector comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a light chain encoding said antibody or antigen-binding fragment thereof. or a second nucleotide sequence of the light chain variable region, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different vectors.
  • the vector of the present invention includes a first vector containing the first nucleotide sequence and a vector containing the A second vector for a second nucleotide sequence.
  • the application also provides a host cell comprising a nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., small mouse cells, human cells, etc.).
  • the application provides a method for preparing an antibody or an antigen-binding fragment thereof as described above, which includes culturing a host cell as described above under conditions that allow expression of the antibody or an antigen-binding fragment thereof, and The antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.
  • the application provides multispecific molecules comprising an antibody or antigen-binding fragment thereof as described above.
  • the multispecific molecule specifically binds CD22 and additionally specifically binds one or more other targets.
  • the multispecific molecule further comprises at least one molecule (eg, a second antibody or antigen-binding fragment thereof) with a second binding specificity for a second target (eg, CD19).
  • at least one molecule eg, a second antibody or antigen-binding fragment thereof
  • a second binding specificity for a second target eg, CD19
  • the present application provides a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD22 protein, the antigen-binding domain comprising an antibody or an antigen-binding fragment thereof as described above.
  • the chimeric antigen receptor further comprises one or more domains selected from:
  • the chimeric antigen receptor includes, from N-terminus to C-terminus, in order: VH of the antigen-binding domain, VL of the antigen-binding domain, hinge region, transmembrane domain, and co-stimulatory domain, and signaling domains.
  • the present application provides a bispecific chimeric antigen receptor comprising a first antigen-binding domain of an antibody or an antigen-binding fragment thereof as described above.
  • the bispecific chimeric antigen receptor further comprises a second antigen binding domain that specifically binds a second target (e.g., CD19); wherein the first antigen binding domain optionally is connected to the N-terminus and/or C-terminus (eg N-terminus) of the second antigen binding domain through a linker.
  • a second target e.g., CD19
  • the first antigen binding domain optionally is connected to the N-terminus and/or C-terminus (eg N-terminus) of the second antigen binding domain through a linker.
  • the second antigen-binding domain is an antibody or antigen-binding fragment thereof that specifically binds CD19.
  • the bispecific chimeric antigen receptor further comprises one or more domains selected from:
  • the bispecific chimeric antigen receptor comprises, from the N-terminus to the C-terminus, in order: VL of the second antigen-binding domain, VH of the first antigen-binding domain, and the first antigen-binding domain.
  • the VL of the second antigen binding domain and the VH of the first antigen binding domain are connected by a linker. In certain embodiments, the VH of the first antigen binding domain and the VL of the first antigen binding domain are connected by a linker. In certain embodiments, the VL of the first antigen binding domain and the VH of the second antigen binding domain are connected by a linker.
  • the VH of the second antigen binding domain is set forth in SEQ ID NO: 14. In certain embodiments, the VL of the second antigen binding domain is set forth in SEQ ID NO: 15.
  • the sequence of the linker can refer to the sequence disclosed in the prior art, for example, Mare W, Bell B A, Sheau-Line F, et al.
  • the linker has the sequence set forth in SEQ ID NO: 24 or 25.
  • the dual specific chimeric antigen receptor contains the sequence set forth in SEQ ID NO: 9 or 26.
  • the hinge region is a hinge region selected from the following proteins: CD8, CD28, 4-1BB, or any combination thereof. In certain embodiments, the hinge region contains or consists of the amino acid sequence set forth in SEQ ID NO:20.
  • the transmembrane domain is a transmembrane domain selected from the following proteins: CD8, CD28, CD3 ⁇ , CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB, CD154, or any combination thereof.
  • the transmembrane domain contains or consists of the amino acid sequence set forth in SEQ ID NO: 21.
  • the costimulatory domain contains or consists of the amino acid sequence set forth in SEQ ID NO: 22.
  • the signaling domain is an intracellular domain selected from the group consisting of: CD3 ⁇ , CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB, PD1, DAPLO, CDS, ICAM-1, LFA-1(CDLLA/CD18), ICOS(CD278), NKG2D, GITR, TLR2, or any combination thereof.
  • the signaling domain is the intracellular domain of CD3 ⁇ .
  • the signaling domain contains or consists of the amino acid sequence set forth in SEQ ID NO: 23.
  • the application provides an isolated nucleic acid molecule encoding a bispecific chimeric antigen receptor as described above.
  • the application provides a vector comprising a nucleic acid molecule as described above.
  • the vector is a cloning vector or an expression vector. In certain embodiments, the vector is a lentiviral, adenoviral, or retroviral vector.
  • the present application provides a host cell comprising a nucleic acid molecule as described above or a vector as described above.
  • the host cells are engineered immune cells.
  • the engineered immune cells secretively express a bispecific chimeric antigen receptor as described above.
  • the engineered immune cells are selected from T cells, NK cells, ⁇ T cells, NKT cells, or any combination thereof.
  • the host cells are obtained from (eg, isolated from) peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the present application provides a method for preparing a host cell as described above, which includes: (1) providing immune cells; (2) converting the isolated nucleic acid molecule as described above or the vector as described above Introduce the immune cells.
  • the immune cells are selected from T cells, NK cells, ⁇ T cells, NKT cells, or any combination thereof.
  • the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of immune cells.
  • the nucleic acid molecule or vector is introduced into the host cell in step (2) by viral infection or by non-viral vector transfection.
  • step (2) is followed by a step of amplifying the immune cells obtained in step (2).
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described above, or an isolated nucleic acid molecule as described above, or a carrier as described above, or a vector as described above.
  • the host cell as described above, or the multi-characteristic molecule as described above, or the bispecific chimeric antigen receptor as described above, or the isolated nucleic acid molecule as described above, or the vector as described above, or Host cells as described previously.
  • compositions further comprise additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitosis inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • a drug with anti-tumor activity such as an alkylating agent, a mitosis inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • the antibody, or antigen-binding fragment or multispecific molecule thereof, and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.
  • the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • a sterile injectable liquid such as an aqueous or non-aqueous suspension or solution.
  • such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • the present application provides an immunoconjugate comprising an antibody or an antigen-binding fragment thereof as described above and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.
  • the therapeutic agent is selected from cytotoxic agents.
  • the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and random combination.
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the present application provides a kit containing the antibody or antigen-binding fragment thereof as described above.
  • the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or Biotin.
  • a detectable label such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or Biotin.
  • the kit further includes a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof as described above.
  • the second antibody further includes a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
  • a detectable label such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
  • the present application provides a method for inhibiting the growth of CD22-expressing tumor cells and/or killing the tumor cells, which includes combining the tumor cells with an effective amount of the aforementioned antibody or its antigen. Fragments, or isolated nucleic acid molecules as described above, or vectors as described above, or host cells as described above, or multi-characteristic molecules as described above, or bispecific embedded molecules as described above The combined antigen receptor, or the isolated nucleic acid molecule as described above, or the vector as described above, or the host cell is contacted as described above.
  • the present application provides a method for inhibiting the growth of tumor cells expressing CD22 and CD19 and/or killing the tumor cells, which comprises combining the tumor cells with an effective amount of the bispecific as described above.
  • the chimeric antigen receptor, or the isolated nucleic acid molecule as described above, or the vector as described above, or the host cell as described above, or the pharmaceutical composition as described above is contacted.
  • the present application provides a method for preventing and/or treating tumors in a subject, the method comprising administering to a subject in need thereof an effective amount of an antibody as described above or The antigen-binding fragment thereof, or the isolated nucleic acid molecule as mentioned above, or the vector as mentioned above, or the host cell as mentioned above, or the multi-characteristic molecule as mentioned above, or the dual-molecule as mentioned above Specific chimeric antigen receptor, or an isolated nucleic acid molecule as described above, or a vector as described above, or a host cell as described above, or a pharmaceutical composition as described above.
  • the tumor expresses CD22. In certain embodiments, the tumor expresses CD22 and CD19.
  • the tumor involves CD22-expressing tumor cells. In certain embodiments, the tumor involves tumor cells expressing CD22 and CD19.
  • the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma
  • CHL
  • the subject is a mammal, such as a human.
  • the application provides the antibody or antigen-binding fragment thereof as described above, or the isolated nucleic acid molecule as described above, or the vector as described above, or the host cell as described above, or The multi-characteristic molecule as mentioned above, or the bispecific chimeric antigen receptor as mentioned above, or the isolated nucleic acid molecule as mentioned above, or the vector as mentioned above, or the host as mentioned above Use of cells, or a pharmaceutical composition as described above, in the preparation of a medicament for preventing and/or treating tumors in a subject.
  • the tumor expresses CD22. In certain embodiments, the tumor expresses CD22 and CD19.
  • the tumor involves CD22-expressing tumor cells. In certain embodiments, the tumor involves tumor cells expressing CD22 and CD19.
  • the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma
  • CHL
  • the subject is a mammal, such as a human.
  • the present application provides a method for detecting the presence or amount of CD22 in a sample, which includes the following steps:
  • the antibody or antigen-binding fragment thereof is detectably labeled.
  • the CD22 is human CD22.
  • the present application provides a method for detecting whether a tumor can be treated by anti-tumor therapy targeting CD22, comprising the following steps:
  • the antibody or antigen-binding fragment thereof is detectably labeled.
  • the CD22 is human CD22.
  • the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma
  • CHL
  • the present application provides the use of an antibody or an antigen-binding fragment thereof as described above in the preparation of a kit for detecting whether a tumor can be treated by anti-tumor therapy targeting CD22 .
  • the antibody or antigen-binding fragment thereof is detectably labeled.
  • the CD22 is human CD22.
  • the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma
  • CHL
  • scFv-BBz-1 sequence the underlined parts are 7E11VL, 15E11VH, 15E11VL, 7E11VH, hinge region and costimulatory domain; the italicized parts are signal peptide, transmembrane region and CD3 ⁇ region;
  • scFv-BBz-2 sequence The underlined parts are 7E11VL, 15E11-2VH, 15E11-2VL, 7E11VH, hinge region and co-stimulatory domain; the italicized parts are signal peptide, transmembrane region and CD3 ⁇ region.
  • the monoclonal antibodies of the present application can bind to CD22 protein or cells expressing CD22 protein with high specificity. Furthermore, a CD19 and CD22 bispecific CART was constructed based on the anti-CD22 antibody. Compared with the benchmark bispecific CART, the bispecific CART of the present application can improve the killing efficiency, amplification efficiency and persistence of CD19 low-expressing tumor cells, and can also inhibit tumor growth in mice. Therefore, the monoclonal antibodies and bispecific CART of the present application have high clinical application value and high application potential in targeted therapy of tumors.
  • Figure 1 shows the application of the mouse antibody 15E11 and CD22-expressing cells Daudi ( Figure 1A), NALM6 (Figure 1B), CHOS-CD22 ( Figure 1C), HEK293-CD22 ( Figure 1D), CHOS ( Figure 1E) and HEK293 ( Figure 1F).
  • Figure 2 shows a schematic structural diagram of the GV401 vector.
  • Figure 3 shows the results of IL2 release by cytokines after target cells were treated with bispecific CART 7E11-15E11 and control (MockT, bispecific CART 7E11-m971) of the present application.
  • Figure 4 shows the results of cytokine IFN ⁇ release after the dual-specific CART 7E11-15E11 and control (MockT, dual-specific CART 7E11-m971) of the present application treat target cells.
  • Figure 5 shows the binding of humanized antibody 15E11-2 to tumor cells that naturally express CD22 (Daudi).
  • Figure 6 shows the results of IL2 release by cytokines after target cells were treated with bispecific CART 7E11-15E11-2 and control (MockT, bispecific CART 7E11-m971, CART 7E11) of the present application.
  • Figure 7 shows the results of cytokine IFN ⁇ release after the dual-specific CART 7E11-15E11-2 of the present application and the control (MockT, dual-specific CART 7E11-m971, CART 7E11) treated target cells.
  • Figure 8 shows the results of tumor growth in mice after the bispecific CART 7E11-15E11-2 of the present application and the control (MockT, bispecific CART 7E11-m971, CART 7E11) treated mice.
  • the lentivirus was provided by Shanghai Gene Medical Technology Co., Ltd. After the cells were infected for 72 hours, corresponding antibiotics were added and cultured for 2-4 weeks, amplified and cryopreserved to obtain two overexpression cell lines, HEK293-hCD22 and CHOS-hCD22. , for subsequent experiments.
  • the constructed CHOS-hCD22 cells overexpressing human CD22 were used to immunize Balb/c mice (Beijing Vital River Experimental Animal Technology Co., Ltd., strain code 216); the primary immunization adjuvant was completely free His adjuvant CFA (InvivoGen Company, product number vac-cfa-60), and subsequent immune adjuvants use IFA (InvivoGen Company, product number vac-ifa-60); the immune route is subcutaneous multiple points.
  • the spleen cells of the immunized mice were fused with mouse myeloma cells SP2/0 using the polyethylene glycol method to obtain B cell fusions that can express antibodies and proliferate indefinitely in vitro, and were selectively cultured in HAT Cultured in the substrate.
  • the fused hybridoma cells were plated in a 96-well cell culture plate, and by detecting the ability of the antibodies in the supernatant to bind CD22 at the cellular level, the target positive clones were screened and 2-3 rounds of subcloning were performed.
  • High-throughput screening of mouse anti-binding cell levels Separate plating was performed in the screen by using human CD22-expressing cells (HEK293-hCD22). Dilute 6,000 cells into 100 ⁇ L of complete culture medium, use a flat-bottomed 96-well plate, and allow the cells to adhere or sink to the bottom of the well overnight. Remove the supernatant the next day. Add 100 ⁇ L of hybridoma supernatant to be screened to the cell plate and incubate at room temperature for 1 hour.
  • the imaging obtained by the fluorescence channel counts the antibody-bound cells according to the fluorescently labeled cell morphology and fluorescence intensity setting parameters.
  • the imaging obtained by the brightfield channel counts adherent cells according to the cell morphology setting parameters, and then the two sets of data are compared.
  • the percentage of cells showing fluorescence that binds to the antibody to the total number of cells is obtained. Based on this ratio, the binding effect of the antibody in the fusion tumor supernatant to CD22-expressing cells was determined.
  • Flow cytometric evaluation of mouse anti-CD22 binding 200,000 CD22-expressing cells Daudi (Chinese Academy of Sciences), NALM6 (ATCC), CHOS-hCD22, HEK293-hCD22, CHOS (Invitrogen), HEK293 (ATCC) were placed in FACS buffer (PBS+ 2% FBS)/well, add the mouse antibody to be tested and incubate at 4 degrees Celsius for 1 hour. Centrifuge to remove the supernatant, wash twice with FACS buffer, add secondary antibody (DyLight488 goat anti-mouse IgG, Abcam catalog number ab97015) and incubate at 4 degrees Celsius for 0.5 hours.
  • FACS buffer PBS+ 2% FBS
  • the experimental cells were measured and read using a flow cytometer (BD Company, model CantoII). During the measurement, first circle the cell position based on FSC and SSC, and then select the second antibody corresponding to the fluorescent channel and SSC to analyze the cells.
  • 15E11 a mouse antibody with good binding activity was obtained, which was named 15E11.
  • the binding conditions of 15E11 to cells Daudi, NALM6, CHOS-hCD22, HEK293-hCD22, CHOS and HEK293 are shown in Figure 1 respectively.
  • Collect hybridoma cells by centrifugation Add 1ml TRIzol and 0.2ml chloroform for every 5-10 ⁇ 10 6 cells. Shake vigorously for 15 seconds and leave at room temperature for 3 minutes. Centrifuge to remove the water phase and add 0.5ml isopropanol. Leave at room temperature for 10 minutes and collect the precipitate. , washed with ethanol and dried to obtain RNA. Add the template RNA and primers to the pre-cooled centrifuge tube, make sure the primers and template are correctly paired, then perform the reverse transcription process, and then perform PCR amplification. Add 2.5 ⁇ l of dNTP/ddNTP mixture to each microcentrifuge tube, and incubate the mixture at 37°C for 5 minutes and set aside.
  • VH and VL sequences of the 15E11 murine antibody are shown in Table 1. Further, the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647-669 page), the CDR sequence of the mouse monoclonal antibody was determined. The sequence information of the antibody is shown in Tables 1 and 2.
  • Example 3 In vitro activity detection of chimeric anti-CD22 and anti-CD19 bispecific CART
  • the constructed scFv-BBz-1 (its amino acid sequence is shown in SEQ ID NO:9, where the underlined parts of SEQ ID NO:9 are 7E11VL, 15E11VH, 15E11VL, 7E11VH, hinge region and costimulatory domain; The italicized parts are the signal peptide, transmembrane region and CD3 ⁇ ), which were inserted into the GV401 vector through the BamHI and EcoRI restriction sites (as shown in Figure 2).
  • the constructed dual-specific CART was named 7E11-15E11.
  • control anti-CD22 antibody was m971 (US2020283522), which is currently in the first phase of clinical trials.
  • the construction method of the control chimeric anti-CD22 and anti-CD19 bispecific CART 7E11-m971 is the same as above, except that it contains different antibody sequences.
  • the construction method of control CART 7E11 is the same as above, except that it contains different antibody sequences.
  • Lentivirus packaging The GV401 vector containing the scFv mutant BBz and the two plasmids pCMV-VSVGenv and pCMV-Gag/pol are transiently transfected into 293T cells using the calcium phosphate precipitation method at a mass ratio of 3:2:2, 48-72 Collect the supernatant after 1 hour, which contains VSVG-packaged lentiviral particles.
  • PBMC Peripheral blood mononuclear cells isolated from healthy people were cultured in X-vivo15 medium containing 200IU/ml hIL2 and 10% FBS; anti-CD3 (OKT3 clone) and CD28 antibodies ( 15E8 clone) was activated for 24 hours, and lentivirus containing 7E11-15E11 and 7E11-m971 was added. CD19 and CD22 were expressed on T cells 72 hours after infection, and the infection efficiency was detected. The specific results are shown in Table 3.
  • Cytokine release experiment Resuspend the target cells (K562, K562-CD19, K562-CD22, K562-CD19-CD22) in 1640 culture medium containing 2% FBS to 1 ⁇ 10 5 /ml, and take a U-shaped bottom 96 well plate, add 100ul; after diluting the T cell clone to 1 ⁇ 10 5 /ml, add 100ul to each well, incubate overnight, remove 50ul of the supernatant, use the FACS method to measure IL2 and IFN ⁇ cytokines, and calculate the cells by the ratio to the background release The fold change of factor release and the specific results of cytokine IL2 and IFN ⁇ release are shown in Figure 3 and Figure 4.
  • the murine antibodies provided in the above examples were humanized designed and prepared, and the murine antibodies were humanized using methods known in the art.
  • CDR regions inserted into human framework sequences see Winter, U.S. Patent Nos. 5,225,539; Queen et al., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).
  • the heavy chain and light chain CDR regions of mouse antibody 15E11 were moved to the FR framework of the corresponding humanized template, and a series of back mutations were performed on the amino acid residues in the FR region of the humanized template. , so that the humanized antibody retains the antigen-binding ability of the mouse antibody as much as possible.
  • the inventors prepared a humanized antibody of murine antibody 15E11, which was named 15E11-2.
  • the heavy chain variable region and light chain variable region of 15E11-2 are as shown in SEQ ID NO: 10 and As shown in 11, the heavy chain constant region of the antibody is SEQ ID NO:12, and the light chain constant region is SEQ ID NO:13.
  • Example 6 In vitro activity detection of humanized anti-CD22 and anti-CD19 bispecific CART
  • Anti-CD19 antibody 7E11 light chain variable region SEQ ID NO:15
  • anti-CD22 antibody 15E11-2 heavy chain variable region SEQ ID NO:10
  • Anti-CD22 antibody 15E11-2 light chain variable region SEQ ID NO:11
  • anti-CD19 antibody 7E11 heavy chain variable region SEQ ID NO:14
  • the constructed scFv-BBz-2 (amino acid sequence is shown in SEQ ID NO:26, where the underlined parts of SEQ ID NO:26 are 7E11VL, 15E11-2VH, 15E11-2VL, 7E11VH, hinge region and co-stimulation Structural domain; the italicized parts are signal peptide, transmembrane region and CD3 ⁇ ) were inserted into the GV401 vector (same as Figure 2) through the BamHI and EcoRI restriction sites.
  • the constructed dual-specific CART was named 7E11-15E11-2, and its structure is similar to The 7E11-15E11 constructed in Implementation 3 is exactly the same.
  • sequences of 15E11VH and 15E11VL are replaced by the sequences of 15E11-2VH and 15E11-2VL respectively.
  • the dual-specific CART7E11-m971 and CART 7E11 are used as controls.
  • Lentivirus packaging The GV401 vector containing the scFv mutant BBz and the two plasmids pCMV-VSVGenv and pCMV-Gag/pol are transiently transfected into 293T cells using the calcium phosphate precipitation method at a mass ratio of 3:2:2, 48-72 Collect the supernatant after 1 hour, which contains VSVG-packaged lentiviral particles.
  • PBMC Peripheral blood mononuclear cells isolated from healthy people were cultured in X-vivo15 medium containing 200IU/ml hIL2 and 10% FBS; anti-CD3 (OKT3 clone) and CD28 antibodies ( 15E8 clone) was activated for 24 hours, and the lentivirus containing 7E11-15E11-2 and 7E11-m971 was added. The infection efficiency was detected by T cell expression of CD19 and CD22 72 hours after infection. The specific results are shown in Table 5.
  • Cytokine release experiment Resuspend the target cells (K562, K562-CD19, K562-CD22, K562-CD19-CD22, NALM6-LUC) to 1 ⁇ 10 5 /ml in 1640 medium containing 2% FBS, and take U Type-bottom 96-well plate, add 100ul; dilute the T cell clone to 1 ⁇ 10 5 /ml, add 100ul to each well, incubate overnight, remove 50ul of supernatant, use FACS method to measure IL2 and IFN ⁇ cytokines, and compare with background release Ratio, fold change of cytokine release was calculated, and the specific results of cytokine IL2 and IFN ⁇ release are shown in Figures 6 and 7.
  • Example 7 In vivo efficacy verification of humanized anti-CD22 and anti-CD19 bispecific CART
  • the tumor-bearing cells used were NALM6-LUC
  • the positive control group was bispecific CART 7E11-m971 (m971US2020283522) and CART 7E11
  • the negative control was MockT.
  • Each mouse was given 1 ⁇ 10 6 cells, and 1E7CART cells were given on day 6.
  • NALM6-LUC cells Preparation of NALM6-LUC cells:
  • the ffluc (firefly Luciferase) gene is inserted into the Ubc promoter-ffluc-IRES-PuroR reading frame through standard molecular biology methods, and the reading frame is inserted into the GV260 vector (Jikai Gene) and packaged into lentivirus , by infecting NALM6 cells and testing Luciferase expression after screening with 1ug/ml puromycin.
  • NDG mouse late-stage tumor-bearing model On the first day, 1 ⁇ 10 6 Raji-ffluc cells were resuspended in 200ul PBS solution; 4-6 weeks old NDG mice were administered via the tail vein. On day 6, 150 mg/ml luciferin was administered intraperitoneally to the mice, and the fluorescence intensity was measured (PerkinElmer IVIS Spectrum) 10 minutes later.
  • bispecific CART 7E11-15E11-2 was more effective than bispecific CART 7E11-m971 and CART 7E11 in the NALM6-LUC model.
  • the tumor growth curve is shown in Figure 8. As can be seen from Figure 8, the bispecific CART of the present application has the best inhibitory effect on tumor growth.

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Abstract

La présente demande se rapporte au domaine technique de l'immunité biologique, et concerne en particulier un anticorps monoclonal capable de se lier de manière spécifique à CD22 et un fragment de liaison à l'antigène de celui-ci. La présente demande concerne également un récepteur antigénique chimérique bispécifique, son procédé de préparation et son utilisation.
PCT/CN2022/102313 2022-06-29 2022-06-29 Anticorps se liant de manière spécifique à cd22, son procédé de préparation et son utilisation sur un cart bispécifique Ceased WO2024000259A1 (fr)

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WO2025219870A1 (fr) * 2024-04-15 2025-10-23 Biocell Innovations Pte Ltd Lymphocyte t à récepteur antigénique chimérique (car) ciblant cd19 modifié et ses utilisations

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WO2020014482A1 (fr) * 2018-07-12 2020-01-16 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps monoclonal spécifique pour cd22 à maturation par affinité et utilisations associées
CN111484562A (zh) * 2020-04-25 2020-08-04 首都医科大学附属北京朝阳医院 一种靶向cd22蛋白的抗体、嵌合抗原受体和药物
WO2021235697A1 (fr) * 2020-05-19 2021-11-25 주식회사 이노베이션바이오 Anticorps spécifique de cd22 et son utilisation
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WO2020014482A1 (fr) * 2018-07-12 2020-01-16 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps monoclonal spécifique pour cd22 à maturation par affinité et utilisations associées
CN111484562A (zh) * 2020-04-25 2020-08-04 首都医科大学附属北京朝阳医院 一种靶向cd22蛋白的抗体、嵌合抗原受体和药物
WO2021235697A1 (fr) * 2020-05-19 2021-11-25 주식회사 이노베이션바이오 Anticorps spécifique de cd22 et son utilisation
WO2022042494A1 (fr) * 2020-08-27 2022-03-03 深圳市菲鹏生物治疗股份有限公司 Anticorps anti-cd22 et son application

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025219870A1 (fr) * 2024-04-15 2025-10-23 Biocell Innovations Pte Ltd Lymphocyte t à récepteur antigénique chimérique (car) ciblant cd19 modifié et ses utilisations

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