WO2023216218A1 - Anticorps se liant de manière spécifique à cd22, son procédé de préparation et son utilisation - Google Patents

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

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WO2023216218A1
WO2023216218A1 PCT/CN2022/092634 CN2022092634W WO2023216218A1 WO 2023216218 A1 WO2023216218 A1 WO 2023216218A1 CN 2022092634 W CN2022092634 W CN 2022092634W WO 2023216218 A1 WO2023216218 A1 WO 2023216218A1
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antibody
antigen
cancer
binding fragment
lymphoma
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PCT/CN2022/092634
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Chinese (zh)
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杜靓
万婷婷
徐溜溜
张红艳
郭晓月
金理娜
陈亚莉
汤荣
金夷
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上海吉倍生物技术有限公司
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Publication of WO2023216218A1 publication Critical patent/WO2023216218A1/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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase

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 preparation methods and uses of the antibodies and antigen-binding fragments thereof.
  • Hematoma is a type of cancer that affects blood cells and includes three main types: leukemia, lymphoma, and myeloma. Of these, leukemia is found in the blood and bone marrow, lymphoma primarily affects the lymphatic system, and myeloma is a blood disorder that specifically targets plasma cells. Each year, these types of cancer account for approximately 10% of all new cancer diagnoses. In the United States, someone is diagnosed with blood cancer every 3 minutes. Leukemia is the most common cancer in children, accounting for 28% of all childhood cancers. To date, more than 1.2 million people in the United States have blood cancer or are in remission.
  • 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 involved in regulating B cell migration, B lymphocyte survival, signal transduction, and B cell Homing and cell adhesion, and are closely related to B cell development, differentiation and function.
  • CD22 is a 140KDa single-pass transmembrane glycoprotein on the surface of B cells. Its extracellular domain (ECD) consists of 7 immunoglobulin (Ig) domains (d1-d7) and contains 12 N-linked glycosylation. site.
  • CD22 is restrictedly expressed on the surface of mature B cells and most malignant B lymphoma cells, such as refractory and relapsed B acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma ( NHL) and hairy cell leukemia, but is not expressed in normal tissues, including hematopoietic stem cells and hematopoietic precursors of B lymphocytes, and can be transferred to the plasma membrane as B cells mature until plasma cells differentiate, but plasma cells do not express this molecule .
  • B lymphoma cells such as refractory and relapsed B acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma ( NHL) and hairy cell leukemia
  • ALL refractory and relapsed B acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • NHL non-Hodgkin lymphoma
  • CD22 is a better internalized antigen.
  • Anti-CD22 antibodies can be rapidly internalized after binding to CD22.
  • antibodies are often used as targeting moieties to selectively Deliver radionuclides, toxins or ribonucleases into CD22 tumor cells.
  • CD22 is expressed in more than 90% of patients with refractory and relapsed B-acute lymphoblastic leukemia (ALL), and current complete response rates for adult refractory and relapsed acute B-cell leukemia are 60-90% . However, many patients with complete remission will relapse, and only about 30-50% will have disease-free survival of 3 years or more.
  • Pfizer launched Besponsa (Inotuzumab Ozogamicin) in December 2021, an anti-CD22 antibody conjugated with calicheamicin. The complete response rate of this treatment group for refractory and relapsed acute B-cell leukemia was significantly higher than that of standard chemotherapy.
  • the Inotuzumab Ozogamicin group had a higher percentage below the threshold of minimal residual disease (0.01% bone marrow blasts) (78.4% vs. 28.1%, P ⁇ 0.001 ); the response period was longer in the Inotuzumab Ozogamicin group (median, 4.6 months vs. 3.1 months); in a survival analysis including 326 patients, the progression-free survival period in the Inotuzumab Ozogamicin group was significantly longer (median , 5.0 months vs. 1.8 months); the median overall survival was 7.7 months and 6.7 months.
  • 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. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901- 917; Definition of Chothia et al. (1989) Nature 342:878-883.
  • 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 consists 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 control 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.
  • the anti-CD22 antibody Inotuzumab (the antibody sequence is derived from Inotuzumab Ozogamicin, an antibody-conjugated drug, purchased from Pfizer, KR20170049617) was used as the benchmark antibody.
  • Inotuzumab the antibody sequence is derived from Inotuzumab Ozogamicin, an antibody-conjugated drug, purchased from Pfizer, KR20170049617) was used as the benchmark antibody.
  • In vitro activity testing showed that the antibody of the present invention was more effective than the benchmark antibody. It shows outstanding advantages in combination, internalization and killing. Therefore, this antibody has great application potential in the treatment of CD22-expressing tumors.
  • 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; or
  • 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:13; and/or, The following three light chain CDRs: VL CDR1 as shown in SEQ ID NO:6, VL CDR2 as shown in SEQ ID NO:7, and VL CDR3 as shown in SEQ ID NO:8.
  • 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: 11 and VL having the sequence shown in SEQ ID NO: 12.
  • 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 variant of the heavy chain constant region (CH) may have one or more conservative substitutions of amino acids compared to the sequence from which it is derived. In such embodiments, the variant of the heavy chain constant region (CH) may have the same or substantially the same effector function as the wild-type sequence from which it is derived.
  • 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:9.
  • 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: 10.
  • 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 as described above, wherein 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.
  • 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: 14 or 16. In certain embodiments, the nucleic acid molecule comprises a sequence set forth in SEQ ID NO: 15 or 17.
  • the heavy chain variable region has the sequence set forth in SEQ ID NO: 14 or 16.
  • the light chain variable region has the sequence set forth in SEQ ID NO: 15 or 17.
  • 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) having a second binding specificity for a second target.
  • the application provides immunoconjugates comprising an antibody or 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 application provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described above, or a multispecific molecule as described above, or an immunoconjugate as described above, and a pharmaceutically acceptable carriers and/or excipients.
  • 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 or immunoconjugate 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 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 chimeric antigen receptor comprising an antigen-binding domain of an antibody as described above, or an antigen-binding fragment thereof.
  • the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragment thereof as described above.
  • the antigen binding domain is a scFv.
  • the antigen-binding receptor comprises an antigen-binding fragment of an antibody as described above.
  • the antigen-binding receptor is expressed by immune effector cells (eg, T cells).
  • immune effector cells eg, T cells
  • the application provides an isolated nucleic acid molecule encoding a 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.
  • the application provides a host cell comprising a vector as described above.
  • the present application also provides a method for reducing the expression level of CD22 on the cell surface, which includes combining the cells with the antibody or antigen-binding fragment thereof, or the multispecific molecule , or the immunoconjugate, or the pharmaceutical composition, or the chimeric antigen receptor, or the host cell contact, such that the expression level of CD22 on the cell surface is reduced; wherein, The cells express CD22 on their surface.
  • the cells are CD22-expressing tumor cells.
  • 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 multispecific molecules as described above, or immunoconjugates as described above, or pharmaceutical compositions as described above, or chimeric antigen receptor contacts as described above.
  • 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 is a multispecific molecule as described above, an immunoconjugate as described above, a pharmaceutical composition as described above, or a chimeric antigen receptor as described above.
  • the tumor expresses CD22.
  • the tumor involves CD22-expressing tumor cells.
  • the CD22 is expressed on the surface of the tumor cells.
  • 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 method further includes administering an additional 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.
  • an additional 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 methods further include administering additional anti-tumor therapy, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.
  • additional anti-tumor therapy such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.
  • the present application provides an antibody or an antigen-binding fragment thereof as described above, or a multispecific molecule as described above, or an immunoconjugate as described above, or a drug as described above.
  • the drug also contains an additional pharmaceutically active agent.
  • 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 tumor expresses CD22.
  • the tumor involves CD22-expressing tumor cells.
  • the CD22 is expressed on the surface of the tumor cells.
  • 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 methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic non-diagnostic purposes.
  • the method is an immunological assay, such as a western blot, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.
  • immunological assay such as a western blot, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.
  • 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 the antibody or 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
  • the monoclonal antibodies of the present application can bind to CD22 protein or cells expressing CD22 protein with high specificity.
  • the antibody hu13G9 of the present application has higher antigen-binding activity, stronger internalization ability on tumor cells, and can effectively induce the killing of CD22-expressing tumor cells. Therefore, the monoclonal antibody of the present application has high clinical application value and high application potential in targeted therapy of tumors.
  • Figure 1 shows the application of mouse antibody 13G9 and CD22-expressing cells Daudi (Figure 1A), NALM6 (Figure 1B), CHOS-hCD22 (Figure 1C), HEK293-hCD22 ( Figure 1D), CHOS ( Figure 1E) and HEK293 ( Figure 1F).
  • Figure 2 shows the binding activity of antibodies (the application's antibody hu13G9 and the standard antibody Inotuzumab) to Raji cells.
  • Figure 3 shows the binding activity of antibodies (the application's antibody hu13G9 and the reference antibody Inotuzumab) to HT cells.
  • Figure 4 shows the binding activity of antibodies (the application's antibody hu13G9 and the standard antibody Inotuzumab) to K562-CD22 cells.
  • Figure 5 shows the internalization ability of antibodies (the application's antibody hu13G9 and the reference antibody Inotuzumab) on NALM6 cells.
  • Figure 6 shows the internalization ability of antibodies (the application's antibody hu13G9 and the reference antibody Inotuzumab) on Raji cells.
  • Figure 7 shows the internalization ability of antibodies (the application's antibody hu13G9 and the reference antibody Inotuzumab) on HT cells.
  • Figure 8 shows the internalization ability of antibodies (the application's antibody hu13G9 and the reference antibody Inotuzumab) on K562-CD19L-CD22H cells.
  • Figure 9 shows the internalization and killing ability of antibodies (the application's antibody hu13G9 and the standard antibody Inotuzumab) on NALM6 cells.
  • Figure 10 shows the internalization and killing ability of antibodies (the application's antibody hu13G9 and the standard antibody Inotuzumab) on Raji cells.
  • the molecular biology experimental methods and immunoassays used in this application basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method was carried out as described in F. M. Ausubel et al., Compiled Experimental Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases was in accordance with the conditions recommended by the product manufacturer. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially. Those skilled in the art will appreciate that the embodiments describe the present application by way of example and are not intended to limit the scope of protection claimed by the present application.
  • 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.
  • 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 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 13G9 mouse 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.
  • 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 the mouse antibody 13G9 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 13G9, which was named hu13G9.
  • the heavy chain variable region and light chain variable region of hu13G9 are as shown in SEQ ID NO: 11 and 12 respectively.
  • the sequence of the antibody heavy chain CDR3N-S mutation is SEQ ID NO:13
  • the heavy chain constant region of the antibody is SEQ ID NO:9
  • the light chain constant region is SEQ ID NO:10.
  • Anti-CD22 antibody-mediated CD22 internalization was tested by flow cytometry. Determine the relationship between internalization caused by the antibody and time; incubate the antibody to be tested with pHAB Reactive Dyes (Promega, G9845) at room temperature for 1 hour, remove the uncoupled dye by ultrafiltration, and remove the CD22-expressing cells at 37°C.
  • pHAB Reactive Dyes Promega, G9845
  • Tumor cells NALM6 (ATCC), Raji (Chinese Academy of Sciences), HT (ATCC), K562-CD19L-CD22H (self-constructed, the construction method is the same as CHOS-hCD22 and HEK293-hCD22) were incubated with labeled antibodies for different times, and then analyzed by flow cytometry Cytometric analysis of the proportion of CD22 internalization mediated by anti-CD22 antibodies.
  • Target cells used CD22 naturally expressing tumor cells NALM6 (ATCC) and Raji (Chinese Academy of Sciences), and the experiment was performed in a 96-well flat-bottomed cell plate (Corning 3903). Serially diluted antibodies were added to Fab-Zap (Advanced Targeting Systems, IT-51) and incubated at 37°C for 15 minutes. Add 3,000 tumor cells to each well and react at 37°C for 72 hours. At the end of the reaction, CellTiter-GloTM reagent (Promega, G755A) is added for fluorescence development. The cell plate is measured with a Tecan Spark10 microplate reader for luminescence readings. Data analysis uses GraphPad.
  • the abscissa uses the logarithm of the antibody molar concentration.
  • the ordinate uses the cell killing percentage.
  • the cell killing percentage 100-100 ⁇ sample luminescence reading/(maximum luminescence reading - minimum luminescence reading), where: max.
  • the luminescence reading value is the well with Fab-Zap and cells added, and the minimum luminescence reading value is the well without cells.
  • the EC50 of internalization killing induced by anti-CD22 antibody is fitted according to the curve.
  • the anti-CD22 antibody hu13G9 and the benchmark antibody Inotuzumab induce internalization and killing effects on two types of tumor cells (NALM6, Raji) that naturally express human CD22, as shown in Figure 9 and Figure 10 respectively.
  • Table 4 lists the EC50 and maximum value (Top value) of internalized killing of antibodies in different tumor cells. The results show that 13G9VH1(N-G)/VL1 can effectively induce the killing of CD22-expressing tumor cells and is stronger than the benchmark antibody Inotuzumab.
  • the CD22 antigen selected in this example is human Siglec-2/CD22 Protein (KACTUS, SIG-HM122-100ug). Anti-CD22 antibody binding to human CD22 protein was measured using probeLife's Gator label-free analyzer device. The biosensor uses a protein A probe, loaded with a target antibody whose concentration is confirmed by A280, and then loaded with CD22 protein. The kinetic association and dissociation rates were determined at 5-6 concentrations for CD22 protein at pH 7.4, and the KD was determined.
  • the binding affinity results of anti-CD22 antibodies are shown in Table 5, which lists the binding and dissociation constants of anti-CD22 antibodies and the corresponding KD values. The results showed that the binding affinity constant of humanized antibody hu13G9 to human CD22 protein was stronger than that of the standard antibody Inotuzumab.

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Abstract

La présente demande relève du 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 en outre un procédé de préparation de l'anticorps et du fragment de liaison à l'antigène de celui-ci et l'utilisation de l'anticorps et du fragment de liaison à l'antigène de celui-ci.
PCT/CN2022/092634 2022-05-13 2022-05-13 Anticorps se liant de manière spécifique à cd22, son procédé de préparation et son utilisation WO2023216218A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101626782A (zh) * 2006-12-01 2010-01-13 梅达雷克斯公司 结合cd22的人抗体及其用途
US20130028888A1 (en) * 2006-03-06 2013-01-31 Tarran Jones Humanized anti-CD22 antibodies and their use in treatment of oncology, transplantation and autoimmune disease
CN114106176A (zh) * 2020-09-01 2022-03-01 深圳市菲鹏生物治疗股份有限公司 Cd22抗体及其应用
CN114316063A (zh) * 2020-09-29 2022-04-12 昆明赛诺制药股份有限公司 抗-cd22抗体分子或其抗原结合片段及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130028888A1 (en) * 2006-03-06 2013-01-31 Tarran Jones Humanized anti-CD22 antibodies and their use in treatment of oncology, transplantation and autoimmune disease
CN101626782A (zh) * 2006-12-01 2010-01-13 梅达雷克斯公司 结合cd22的人抗体及其用途
CN114106176A (zh) * 2020-09-01 2022-03-01 深圳市菲鹏生物治疗股份有限公司 Cd22抗体及其应用
CN114316063A (zh) * 2020-09-29 2022-04-12 昆明赛诺制药股份有限公司 抗-cd22抗体分子或其抗原结合片段及其应用

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Title
MIKIEWICZ DIANA; ŁUKASIEWICZ NATALIA; ZIELIńSKI MARCIN; CECUDA-ADAMCZEWSKA VIOLETTA; BIERCZYńSKA-KRZYSIK ANNA; ROMA: "Bacterial expression and characterization of an anti-CD22 single-chain antibody fragment", PROTEIN EXPRESSION AND PURIFICATION, ACADEMIC PRESS, SAN DIEGO, CA., vol. 170, 5 February 2020 (2020-02-05), SAN DIEGO, CA. , XP086082561, ISSN: 1046-5928, DOI: 10.1016/j.pep.2020.105594 *

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