WO2017150762A1 - Anticorps anti-cd3γε et son utilisation - Google Patents

Anticorps anti-cd3γε et son utilisation Download PDF

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WO2017150762A1
WO2017150762A1 PCT/KR2016/003357 KR2016003357W WO2017150762A1 WO 2017150762 A1 WO2017150762 A1 WO 2017150762A1 KR 2016003357 W KR2016003357 W KR 2016003357W WO 2017150762 A1 WO2017150762 A1 WO 2017150762A1
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antibody
seq
antigen
sequence
binding fragment
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Korean (ko)
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김선택
류기혁
김혜진
박상래
정현호
양기혁
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(주)메디톡스
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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
    • 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
    • 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

Definitions

  • the present invention provides an antibody against CD3 ⁇ or an antigen-binding fragment thereof, a nucleic acid encoding the same, a vector comprising the nucleic acid, a cell transformed with the vector, a method for producing the antibody or an antigen-binding fragment thereof, and a T cell mediator comprising the same.
  • a composition for preventing or treating a disease and a method for preventing or treating a disease are provided.
  • the human adaptive immune system is a very sophisticated system that can specifically remove not only various infectious diseases but also cancer cells.
  • T cells it plays a role in determining cellular adaptive immunity and recognizes and removes cells when exposed to non-self or abnormal antigens.
  • a monoclonal T cell receptor complex (TCR complex) is expressed on the cell membrane to generate a major histocompatibility complex (peptide, pMHC) of an antigen presenting cell (APC).
  • pMHC major histocompatibility complex
  • APC antigen presenting cell
  • Such cellular adaptive immunity must be operated with great sophistication to effectively remove infectious diseases and cancer cells of the human body. If the antigen specific adaptive immune system does not work, it poses serious problems with infectious disease response and cancer cell clearance. On the other hand, when cellular adaptive immunity is activated by recognizing human self antigens, autoimmune diseases that destroy self cells and tissues are induced.
  • the ⁇ TCR complex consists of an ⁇ heterodimer that specifically recognizes an antigen and CD3 molecules (CD3 ⁇ , CD3 ⁇ , CD3 ⁇ ) for signaling.
  • ⁇ heterodimer has a structure similar to that of an antibody Fab, and CD3 ⁇ , ⁇ , and ⁇ each have an Ig domain in an extracellular configuration.
  • Specific antigen recognition of the [alpha] [beta] heterodimer is mediated by the CD3 molecule to signal the cell.
  • This signaling is known to be T-cell activation by intracellular tyrosine kinase (LCK, ZAP70, LAT, PLC ⁇ ) phosphorylation, cytoplasmic Ca ++ concentration changes and intracellular transcriptional system changes.
  • each domain structure was determined by NMR and X-ray structure determination, and structural information on quaternary structure binding on T cell membranes was revealed one by one.
  • the specific binding of the extracellular domain is very weak, while the electrical binding of charged amino acids in the cell membrane is important, suggesting that it can be amplified by the environment within the cell membrane.
  • TCR signal transduction may vary not simply by antibody binding capacity or number of binding antibodies, but also by the mode of binding of antibodies that bind CD3 (a recognized epitope).
  • TCR complex is a highly sensitive sensor that recognizes the small changes caused by the binding of pMHC and may show different signaling results by antibodies showing different binding.
  • the TCR complex is the academic basis for the mechanical force mediating of the binding of T cells and APCs and microspecific changes.
  • T1DM type 1 diabetes mellitus
  • the inventors of the present application tried to develop an antibody that specifically binds CD3 ⁇ .
  • the present inventors have developed an anti-CD3 ⁇ antibody that selects antibody-expressing B cells from mice immunized with CD3 ⁇ and binds CD3 ⁇ with high affinity using phage display technology. It was confirmed that it could be treated, and the present invention was completed.
  • Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • Another object of the present invention is to provide a vector comprising the nucleic acid, a cell transformed with the vector, and a method of manufacturing the same.
  • Another object of the present invention to provide a composition for the prevention or treatment of T cell mediated diseases comprising the antibody or antigen-binding fragment thereof.
  • the present invention provides an antibody or antigen-binding fragment thereof that binds to CD3 ⁇ having the amino acid sequence of SEQ ID NO: 1.
  • the present invention also provides a nucleic acid encoding a heavy chain variable region of the antibody or antigen-binding fragment thereof.
  • the present invention also provides a vector comprising the nucleic acid.
  • the present invention also provides a cell transformed with the vector.
  • the present invention also provides a method for producing the antibody or antigen-binding fragment thereof comprising the following steps: (a) culturing the cells; And (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.
  • the present invention also provides a composition for the prevention or treatment of T cell mediated diseases comprising the antibody or antigen-binding fragment thereof as an active ingredient.
  • the present invention also provides a composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof as an active ingredient.
  • the present invention also provides a composition for preventing or treating autoimmune diseases comprising the antibody or antigen-binding fragment thereof as an active ingredient.
  • the present invention also provides a method for preventing or treating a T cell mediated disease comprising administering to the patient a therapeutically effective amount of the antibody or antigen-binding fragment thereof.
  • the present invention also provides a method for preventing or treating cancer comprising administering to the patient a therapeutically effective amount of the antibody or antigen-binding fragment thereof.
  • the present invention further provides a method for preventing or treating autoimmune disease, comprising administering to the patient a therapeutically effective amount of the antibody or antigen-binding fragment thereof.
  • FIG. 1 shows a single chain CD3 ⁇ and a single chain CD3 ⁇ fusion protein connecting two extracellular CD3 domains with a 26aa length linker.
  • Figures 2a to 2c shows the result of purification by inserting the scCD3 ⁇ gene and scCD3 ⁇ gene in the pET21a vector, which is an expression plasmid for Escherichia coli.
  • FIG. 3 is a schematic diagram showing a process for selecting a desired antibody that binds to CD3 ⁇ using antigen baiting.
  • Figure 4 shows the results of measuring the binding force of the antibody (MTI3) using a human T cell line (Jurkat).
  • the calculated KD value is about 2.75 nM based on the cell binding force measurement result of the concentration of MTI3.
  • Figure 5 shows the result of measuring the specific binding capacity of the antibody (MTI3) after binding the recombinant CD3 ⁇ protein to the beads.
  • CD3 ⁇ specific antibody UCHT1 binds both CD3 ⁇ and CD3 ⁇ while MTI3 shows only CD3 ⁇ specific binding.
  • FIG. 6 shows the results of measuring the T cell activation capacity of CD3 ⁇ specific antibody (MTI3).
  • Figure 7 shows the results confirmed that MTI3 effectively inhibits the activation ability of antigen-specific T cells by analyzing T cell activation at the ex vivo level by ELISPOT method.
  • FIG 8 shows the results showing that the nonspecific activation of T cells by MTI3 and administration of peripheral blood mononuclear cells (PBMC) in an animal model.
  • PBMC peripheral blood mononuclear cells
  • the present invention relates to an antibody or antigen-binding fragment thereof that binds to CD3 ⁇ having the amino acid sequence of SEQ ID NO: 1.
  • T cell immunity is regulated by targeting T cell antigen receptor complex signaling, in particular human CD3 monoclonal antibodies (mAbs) are widely used clinically in immunosuppressive therapy.
  • CD3 ⁇ specific mAb OKT3 was the first mAb licensed for use in humans and was used as an immunosuppressive agent in clinical implantation. Since OKT3 has a time-dependent tendency and causes cytokine release not only by immunosuppressive activity but also by T cell activity, there is a very high limit on the dose to be administered for clinical use.
  • CD3 antibodies including OKT3, such as UCHT1, and Leu-4 are monoclonal antibodies that recognize CD3 ⁇ and bind to CD3 ⁇ of CD3 ⁇ and CD3 ⁇ of a single T cell antigen receptor complex and have two binding sites. T cell antigen receptor complexes are exposed in the form of complexes of CD3 ⁇ and CD3 ⁇ on the surface of T cells, respectively. Most of them bind to and recognize only CD3 ⁇ epitopes of T-cell antigen-receptive complexes using conventional limited antigen discovery techniques.
  • CD3 expresses ⁇ , ⁇ , and ⁇ distinct proteins to later form T cell receptor complexes.
  • CD3 ⁇ gene was recombinantly expressed as a single fusion protein (single chain form) from the beginning. Based on known facts, two extracellular domains of CD3 (except cysteine rich stem) were linked with a 26aa length linker (SEQ ID NOS: 1 and 2, see FIG. 1).
  • CD3 ⁇ is one of the CD3 molecules that transmits a signal from an ⁇ TCR heterodimer that specifically recognizes an antigen in a TCR complex structure, and includes an Ig domain in an extracellular configuration, and after recognition of a specific antigen of ⁇ heterodimer. Signaling takes place into cells via CD3.
  • antibody that binds to CD3 ⁇ unlike antibodies that recognize CD3 ⁇ , binds CD3 ⁇ as an epitope (epitope) and binds to CD3 ⁇ and hardly or not binds to CD3 ⁇ or ⁇ or CD3 ⁇ rather than CD3 ⁇ . May mean an antibody.
  • Epitope refers to a protein determinant to which an antibody can specifically bind.
  • Epitopes usually consist of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics. Three-dimensional epitopes and non-stereo epitopes are distinguished in that the binding to the former is lost but not to the latter in the presence of a denatured solvent.
  • antibody refers to an anti-CD3 ⁇ antibody that specifically binds to CD3 ⁇ .
  • the scope of the present invention includes not only complete antibody forms that specifically bind CD3 ⁇ , but also antigen binding fragments of such antibody molecules.
  • a complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds.
  • the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) and epsilon ( ⁇ ) types and subclasses gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), and gamma 3 ( ⁇ 3). ), Gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
  • the constant regions of the light chains have kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • An antigen binding fragment or antibody fragment of an antibody means a fragment having an antigen binding function and includes Fab, F (ab '), F (ab') 2, Fv and the like.
  • Fab in the antibody fragment has a structure having a variable region of the light and heavy chains, a constant region of the light chain and the first constant region (CH1) of the heavy chain has one antigen binding site.
  • F (ab ') 2 antibodies are produced when the cysteine residues of the hinge region of Fab' form disulfide bonds.
  • Double-chain Fv is a non-covalent bond in which a heavy chain variable region and a light chain variable region are linked, and a single chain Fv (single-chain Fv, scFv) is generally a variable region of the heavy chain and the light chain through a peptide linker.
  • This covalent linkage or the C-terminus is directly linked to form a dimer-like structure such as a double-chain Fv.
  • Such antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain yields Fab and cleavage with pepsin yields F (ab ') 2 fragments). It can also be produced by recombinant technology.
  • the antibody according to the invention is in Fv form (eg scFv) or is in the form of a complete antibody.
  • the heavy chain constant region may be selected from any one isotype of gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) or epsilon ( ⁇ ).
  • the constant region is gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4).
  • the light chain constant region may be of kappa or lambda type.
  • the term “heavy chain” refers to a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and a full length heavy chain comprising three constant region domains CH1, CH2 and CH3 And fragments thereof.
  • the term “light chain” as used herein refers to a full-length light chain and fragment thereof comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. All means.
  • Antibodies of the invention include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide-binding Fvs (sdFV) And anti-idiotype (anti-Id) antibodies, or epitope-binding fragments of the antibodies, and the like.
  • Said monoclonal antibody refers to the same except for possible naturally occurring mutations in which antibodies obtained from substantially homogeneous antibody populations, ie, individual antibodies in the population, may be present in trace amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site.
  • Non-human (eg murine) antibodies of the “humanized” form are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins.
  • humanized antibodies are non-human species (donor antibodies) that retain the desired specificity, affinity, and capacity for residues from the hypervariable region of the recipient, for example mice, rats, rabbits, or non-humans.
  • donor antibodies non-human species
  • Human immunoglobulins (receptor antibodies) replaced with residues from the hypervariable regions of primates.
  • human antibody refers to a molecule derived from human immunoglobulin, in which all of the amino acid sequences constituting the antibody including complementarity determining regions and structural regions are composed of human immunoglobulins.
  • While the heavy and / or light chain portions are the same or homologous to the corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, the remaining chain (s) are derived from another species or another antibody class or Included are "chimeric" antibodies (immunoglobulins) that are identical or homologous to the corresponding sequences in antibodies belonging to the subclass, as well as fragments of such antibodies that exhibit the desired biological activity.
  • antibody variable domain refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of complementarity determining regions (CDRs; ie CDR1, CDR2, and CDR3), and framework regions (FR). .
  • CDRs complementarity determining regions
  • FR framework regions
  • VH refers to the variable domain of the heavy chain.
  • VL refers to the variable domain of the light chain.
  • CDRs Complementarity determining regions
  • Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.
  • FRs Framework regions
  • Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4.
  • the present invention provides an antibody or antigen-binding fragment thereof comprising the following heavy chain CDRs and light chain CDRs: complementarity determining region H1 having the sequence of SEQ ID NO: 3, sequence A heavy chain variable region comprising a CDRH2 having a sequence of SEQ ID NO: 4, and a CDRH3 having a sequence of SEQ ID NO: 5; And a light chain variable region comprising CDRL1 having a sequence of SEQ ID NO: 6, CDRL2 having a sequence of SEQ ID NO: 7, and CDRL3 having a sequence of SEQ ID NO: 8.
  • the antibody or antigen-binding fragment thereof of the invention may comprise a framework region (FR) having one or more sequences selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 16, specifically SEQ ID NO: It may include a heavy chain variable region comprising a heavy chain framework region (FR) having one sequence selected from the group consisting of the sequence of 9 to SEQ ID NO: 12.
  • the heavy chain FR1 having the sequence of SEQ ID NO: 9 the heavy chain FR2 having the sequence of SEQ ID NO: 10
  • the heavy chain FR3 having the sequence of SEQ ID NO: 11 heavy chain FR4 having the sequence of SEQ ID NO: 12 may be included.
  • the antibody or antigen-binding fragment thereof of the present invention may comprise a light chain variable region comprising a light chain FR having one sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 16.
  • the light chain FR1 having the sequence of SEQ ID NO: 13, the light chain FR2 having the sequence of SEQ ID NO: 14, the light chain FR3 having the sequence of SEQ ID NO: 15, and the light chain FR4 having the sequence of SEQ ID NO: 16 may be included.
  • the antibody or antigen-binding fragment thereof of the invention may comprise a heavy chain variable region having the sequence of SEQ ID NO: 31 and / or comprise a light chain variable region having the sequence of SEQ ID NO: 32.
  • Fv fragments are antibody fragments containing complete antibody recognition and binding sites. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly and covalently associated, for example, with scFv.
  • Fab fragments contain the variable and constant domains of the light chain and the variable and first constant domains (CH1) of the heavy chain.
  • F (ab ') 2 antibody fragments generally comprise a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.
  • Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of an antibody, which domains are present in a single polypeptide chain.
  • the Fv polypeptide may further comprise a polypeptide linker between the VH domain and the VL domain that allows the scFv to form the desired structure for antigen binding.
  • Phase display is a technique for displaying variant polypeptides as phage proteins, such as fusion proteins with at least a portion of the envelope protein on the surface of fibrous phage particles.
  • the utility of phage display lies in the fact that a large library of randomized protein variants can be targeted to quickly and efficiently classify sequences that bind with high affinity with a target antigen. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides to identify polypeptides with specific binding properties.
  • Phage display technology provided a powerful tool for generating and selecting new proteins that bind specific ligands (eg antigens). Phage display technology can be used to generate large libraries of protein variants and to quickly sort sequences that bind with high affinity to target antigens.
  • Nucleic acids encoding variant polypeptides are fused with nucleic acid sequences encoding viral envelope proteins, eg, gene III protein or gene VIII protein.
  • Monovalent phage display systems have been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused with a nucleic acid sequence encoding a portion of a gene III protein. In monovalent phage display systems, gene fusions are expressed at low levels and wild type Gene III proteins are also expressed to maintain particle infectivity.
  • Phage display technology has several advantages over conventional hybridoma and recombinant methods for preparing antibodies with the desired characteristics. This technique allows the production of large antibody libraries with various sequences in a short time without the use of animals. The preparation of hybridomas or the production of humanized antibodies may require months of preparation. In addition, since no immunity is required at all, phage antibody libraries can produce antibodies against antigens that are toxic or low antigenic. Phage antibody libraries can also be used to generate and identify novel therapeutic antibodies.
  • Techniques for generating human antibodies from immunized, non-immunized human, germline sequences, or na ⁇ ve B cell Ig repertory using immunized phage display libraries can be used.
  • Various lymphoid tissues can be used to prepare na ⁇ ve or non-immune antigen binding libraries.
  • the ability to identify and isolate high affinity antibodies from phage display libraries is important for the isolation of novel therapeutic antibodies. Separation of high affinity antibodies from the library may depend on the size of the library, the efficiency of production in bacterial cells, and the diversity of the library.
  • the size of the library is reduced by inefficient folding of the antibody or antigen binding protein and inefficient production due to the presence of stop codons. Expression in bacterial cells can be inhibited if the antibody or antigen binding domain is not properly folded. Expression can be improved by alternately mutating residues at the surface of the variable / constant interface or selected CDR residues.
  • the sequence of the backbone region is one element to provide proper folding when generating antibody phage libraries in bacterial cells.
  • CDR3 regions have been found to often participate in antigen binding.
  • the CDR3 regions on the heavy chains vary considerably in size, sequence, and structural conformation, and thus can be used to prepare a variety of libraries.
  • diversity can be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position.
  • the use of all twenty amino acids can result in highly variable variant antibody sequences and increase the chance of identifying new antibodies.
  • Antibodies or antibody fragments of the present invention may include not only the sequences of the anti-CD3 ⁇ antibodies of the present invention described herein, but also their biological equivalents within the scope of specific recognition of CD3 ⁇ .
  • further changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody.
  • Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues of the antibody.
  • Such amino acid variations are made based on the relative similarity of amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like.
  • arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape.
  • arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.
  • each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).
  • the hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is well known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobicity index, substitutions are made between amino acids which exhibit a hydrophobicity index difference of preferably within ⁇ 2, more preferably within ⁇ 1, even more preferably within ⁇ 0.5.
  • the antibody or nucleic acid molecule encoding the same of the present invention is interpreted to include a sequence that exhibits substantial identity with the sequence described in SEQ ID NO.
  • the above substantial identity is at least 61% when the sequence of the present invention is aligned as closely as possible with any other sequence, and the aligned sequence is analyzed using algorithms commonly used in the art.
  • a sequence that shows homology more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art.
  • BLAST The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI and the like and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet.
  • BLSAT is accessible at www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparisons using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
  • the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • the nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention can be isolated to recombinantly produce the antibody or antigen-binding fragment thereof.
  • the nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or for further expression. Based on this, the present invention relates to a vector comprising the nucleic acid in another aspect.
  • Nucleic acid is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides that are the basic building blocks of nucleic acids include natural nucleotides as well as analogs with modified sugar or base sites. .
  • the sequences of nucleic acids encoding heavy and light chain variable regions of the invention can be modified. Such modifications include addition, deletion, or non-conservative or conservative substitutions of nucleotides.
  • the nucleic acid encoding the variable region of the antibody or antigen-binding site thereof according to the present invention may comprise one or more of the sequence selected from the group consisting of SEQ ID NO: 17 to SEQ ID NO: 22.
  • CDRL1 having a sequence of SEQ ID NO: 20 comprising a complementarity determining region (CDR) H1 having a sequence of SEQ ID NO: 17, a CDRH2 having a sequence of SEQ ID NO: 18, and a CDRH3 coding nucleic acid having a sequence of SEQ ID NO: 19, CDRL2 having a sequence of SEQ ID NO: 21, and CDRL3 having a sequence of SEQ ID NO: 22 may comprise a nucleic acid.
  • the nucleic acid may comprise a framework region (FR) coding nucleic acid having one or more sequences selected from the group consisting of SEQ ID NOs: 23 to 30, specifically SEQ ID NOs: 23 to 26 It may comprise a framework region (FR) coding nucleic acid of the heavy chain variable region having one sequence selected from the group consisting of the sequence of.
  • FR framework region
  • the heavy chain FR1 coding nucleic acid having the sequence of SEQ ID NO: 23, the heavy chain FR2 coding nucleic acid having the sequence of SEQ ID NO: 24, the heavy chain FR3 coding nucleic acid having the sequence of SEQ ID NO: 25, the heavy chain FR4 coding nucleic acid having the sequence of SEQ ID NO: 26 It may include.
  • the antibody or antigen-binding fragment thereof of the present invention may include a framework region coding nucleic acid of the light chain variable region having one sequence selected from the group consisting of SEQ ID NO: 27 to SEQ ID NO: 30.
  • the light chain FR1 coding nucleic acid having the sequence of SEQ ID NO: 27, the light chain FR2 coding nucleic acid having the sequence of SEQ ID NO: 28, the light chain FR3 coding nucleic acid having the sequence of SEQ ID NO: 29, the light chain FR4 coding nucleic acid having the sequence of SEQ ID NO: 30 It may include.
  • the nucleic acid may have a sequence of SEQ ID NO: 33 or SEQ ID NO: 34, wherein the nucleic acid encoding the heavy chain variable region may be SEQ ID NO: 33, and the nucleic acid encoding the light chain variable region may be SEQ ID NO: 34 .
  • Nucleic acids of the invention are also construed to include nucleotide sequences that exhibit substantial identity to the nucleotide sequence. Substantial identity is at least 80% homology when aligning the nucleotide sequence of the present invention with any other sequence as closely as possible and analyzing the aligned sequence using algorithms commonly used in the art. Preferably a nucleotide sequence that exhibits at least 90% homology, most preferably at least 95% homology.
  • the DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the DNA encoding the heavy and light chains of the antibody).
  • Many vectors are available.
  • Vector components generally include, but are not limited to, one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.
  • the term "vector” refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Cosmid vector; Viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, and the like.
  • the nucleic acid encoding the antibody in the vector is operably linked with a promoter.
  • “Operatively linked” means a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulatory sequence is the other nucleic acid. To control transcription and / or translation of the sequence.
  • a nucleic acid expression control sequence eg, an array of promoters, signal sequences, or transcriptional regulator binding sites
  • promoters capable of promoting transcription e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.
  • ribosome binding sites for initiation of translation e.g., amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.
  • a promoter derived from the genome of the mammalian cell e.g., a metallothionine promoter, a ⁇ -actin promoter, a human heroglobin promoter and a human muscle creatine promoter
  • a mammal Promoters derived from animal viruses e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse breast tumor virus (MMTV) promoter, LTR promoter of HIV
  • a promoter derived from the genome of the mammalian cell e.g., a metallothionine promoter, a ⁇ -actin promoter, a human heroglobin promoter and a human muscle creatine promoter
  • a mammal Promoters derived from animal viruses e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter
  • the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom.
  • Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).
  • Such vectors include antibiotic resistance genes commonly used in the art as selectable markers and include, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene.
  • the present invention relates to a cell transformed with the above-mentioned vector.
  • the cells used to produce the antibodies of the invention can be prokaryote, yeast or higher eukaryote cells, but are not limited thereto.
  • Bacillus strains such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (eg, Staphylocus carnosus) can be used.
  • Pseudomonas e.g. Pseudomonas putida
  • Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (eg, Staphylocus carnosus) can be used.
  • examples of useful host cell lines are COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0 , U20S, or HT1080, but is not limited thereto.
  • the present invention (a) culturing the cells; And (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.
  • the cells can be cultured in various media. It can be used as a culture medium without limitation among commercial media. All other necessary supplements known to those skilled in the art may be included at appropriate concentrations. Culture conditions, such as temperature, pH, and the like, are already in use with host cells selected for expression, which will be apparent to those skilled in the art.
  • the recovery of the antibody or antigen-binding fragment thereof can be removed by, for example, centrifugation or ultrafiltration, and the resultant can be purified using, for example, affinity chromatography or the like. Further other purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and the like can be used.
  • the present invention relates to a composition for the prevention or treatment of T cell mediated diseases comprising the antibody as an active ingredient.
  • the present invention provides, for example, (a) a pharmaceutically effective amount of an antibody against CD3 ⁇ or an antigen-binding fragment thereof according to the present invention; And (b) it may be a pharmaceutical composition for the prevention or treatment of T cell mediated diseases, such as cancer, including a pharmaceutically acceptable carrier.
  • the invention also relates to a method for the prevention or treatment of a T cell mediated disease such as cancer comprising administering to a patient an effective amount necessary for an antibody against CD3 ⁇ or an antigen-binding fragment thereof according to the invention.
  • the present invention also provides, for example, (a) a pharmaceutically effective amount of an antibody against CD3 ⁇ or an antigen binding fragment thereof according to the present invention; And (b) it may be a pharmaceutical composition for the prevention or treatment of T cell mediated diseases such as autoimmune diseases, including a pharmaceutically acceptable carrier.
  • the invention also relates to a method for the prevention or treatment of a T cell mediated disease such as an autoimmune disease comprising administering to a patient an effective amount necessary for an antibody against CD3 ⁇ or an antigen-binding fragment thereof according to the invention.
  • composition uses the above-described anti-CD3 ⁇ antibody or antigen-binding fragment thereof as an active ingredient, the descriptions common between the two are omitted.
  • Prevention means any action that inhibits or delays the progression of a T cell mediated condition, e.g., a T cell mediated cancer or an autoimmune disease, by administration of a composition according to the present invention, and "treatment” means a T cell mediated condition Inhibition of development, alleviation or elimination of T cell mediated pathology.
  • the T cell mediated condition applied to the composition may be, for example, a T cell mediated cancer or an autoimmune disease.
  • compositions of the present invention are those commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
  • the composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.
  • composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration Or the like.
  • oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach.
  • the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.
  • Suitable dosages of the compositions according to the invention vary depending on factors such as the method of formulation, mode of administration, age, weight, sex, morbidity, condition of the patient, food, time of administration, route of administration, rate of excretion and reaction sensitivity, and usually The skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.
  • the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg.
  • pharmaceutically effective amount as used herein means an amount sufficient to prevent or treat cancer.
  • compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of extracts, powders, suppositories, powders, granules, tablets, or capsules, and may further include a dispersant or stabilizer.
  • compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents.
  • CD3 In order to express human CD3 in Escherichia coli, it was cloned into the pET plasmid in a fused form of the CD3 gene.
  • Human CD3 antigen sequences (CD3 ⁇ 'NM000073', CD3 ⁇ 'NM000732', CD3 ⁇ 'NM000733', National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/) for standard protocols
  • NCBI National Center for Biotechnology Information
  • the synthetic gene was obtained by gene synthesis. Two extracellular domains of CD3 (except cysteine rich stem) were linked with a 26aa length linker (underlined below).
  • the scCD3 ⁇ gene and the scCD3 ⁇ gene were amplified by a standard PCR protocol and prepared as an insert.
  • PET21a Novagen, http://www.merckmillipore.com
  • an expression plasmid for Escherichia coli was used as a vector.
  • Vectors and inserts were cut with restriction enzymes NdeI / XhoI, respectively, and then expressed and purified by ligase (Ligase) (FIGS. 2A to 2C).
  • Antigen baiting was applied to develop monoclonal antibodies.
  • Antigen tracking is a technique for expressing antibodies by cloning antibody genes in one B cell by selecting only B cells expressing a desired antibody among several B cells (Franz B et al., BLOOD 2011; 118: 348-357). ).
  • the antigen is labeled with fluorescence and mixed with the B cells, the antigen is attached to the BCR of the B cell expressing the antibody to which the antigen is attached to have fluorescence.
  • Fluorescent B cells were selected with a FACS sorter and the antibody genes were PCR amplified to prepare scFv-type antibody libraries (FIG. 3).
  • whole cell immunization (Jurkat or HuT78 cell line) is based on the immunization of human T cell lines such as Jurkat to immunize mice with human CD3, thus maintaining the morphology of CD3 on the cell surface and producing good antibodies. ), And the antigen proteins described in Example 1 were injected into mice to immunize. ELISA and FACS analysis confirmed the production of antibodies.
  • mice After immunization, spleens of the mice were removed, and the cells were spun onto the strainer to separate splenocytes (in 2ml RPMI). A total volume of 1/10 (150 ⁇ l) of cells was used, and 150 ⁇ l of ACK buffer was added to remove RBC. 1 ⁇ 10 7 splenocytes were obtained from mice immunized with Jurkat, and 1.2 ⁇ 10 7 splenocytes were obtained from mice immunized with HuT78. After making into 4.0 ⁇ 10 6 cells / 100 ⁇ l, use 100 ⁇ l each. First, add 2.5 ⁇ l CD3GE-SA-PE + 1.25 ⁇ l CD3DE-SA-A647, and then, 2.5 ° C.
  • anti-CD19-APC-Cy7 + 1.25 ⁇ l anti-IgM-FITC (BD 553437) was added and reacted at 4 ° C. for 30 minutes and washed twice with FB (no NaN 3 ).
  • FACSAriaII was used to classify CD3GE + / CD3DE ⁇ / IgM-B cells. 37,200 B cells were immunized with Jurkat and 50,600 were immunized with HuT78.
  • PCR method was used to obtain the gene of the antibody expressing each of the B cells in the mouse spleen, and primers as shown in Table 1 were prepared as follows.
  • PCR was performed to amplify the heavy and light chains by PCR in order to form a scFv form in which the heavy and light chains were connected.
  • This was used as an insert and the pComb3X (Genbank AF268281) plasmid, which is widely used for scFv expression, was used as a vector.
  • restriction enzyme SfiI treatment gel elution was performed using Qiagen gel elution kit, and the concentration was used for connection.
  • 3 unit ligase per ⁇ g was used, and 500 ⁇ l per 10 ⁇ g was used, and after 10 hours at room temperature, the degree of connection was confirmed by hanging on the gel. After confirming that the connection band was sufficiently moved, after purification by PCR purification kit to reduce the volume of the sample, it was dissolved in a total of 150 ⁇ l (10 ⁇ g) of distilled water. In order to electroporate the ligation samples, 1 liter of E. coli SS320 cells were cultured, and then electroporation competent cells were prepared. Electroporation was performed by dividing four times using 1500 ⁇ l of the cells for transformation into electroporation and 10 ⁇ g of the total ligation sample.
  • scFv clones attached to the CD3 antigen were selected by a general panning method.
  • the VH and VL moieties were linked to the CH and CL moieties of the human antibody and put into the respective expression vectors and expressed in full IgG form.
  • antibody MTI3 was selected.
  • the binding force of the discovered antibody was measured using a human T cell line (Jurkat) (FIG. 4).
  • the surface binding capacity of the human T cell line is measured at 2.75 nM on the Kd basis.
  • the binding capacity of MTI3 was measured by binding the recombinant protein to the beads and then confirming specific binding (FIG. 5). As a result of performing CD3 molecular binding evaluation of the purified MTI3 with the existing antibody UCHT1 (anti-CD3 ⁇ ), it was confirmed that MTI3 is a CD3 ⁇ specific antibody.
  • the T cell activation ability of the discovered CD3 ⁇ specific antibody MTI3 was experimentally demonstrated at the in vitro level.
  • Non-specific T cell activation ability compared to CD3 ⁇ specific antibody OKT3 was confirmed by the surface expression of T cell activation markers CD3, CD69, TCR.
  • the ability to inhibit antigen-specific T cell activation of MTI3, an extracted CD3 ⁇ specific antibody, was observed. was experimentally demonstrated at the in vitro level.
  • CD8 + T cells and dendritic cells were isolated from HLA-A2 + PBMCs, and the isolated DCs presented by binding peptides to present antigens. Used as cells (APC). While repeatedly presenting peptides, it was confirmed by IFN ⁇ secreted by CD8 + cells that candidate peptides activate and induce proliferation of human CD8 + T cells ex vivo.
  • the ELISPOT method was used first to confirm the frequency of activated cells in the whole cells, and it was confirmed that MTI3 effectively inhibited the activation ability of antigen-specific T cells (FIG. 7).
  • mice Six-week-old NSG (immunodeficient mice, female) mice were used in the experiment for safety evaluation in animal models. 2 x 107 peripheral blood mononuclear cells (PBMC) per mouse were grafted for 2 weeks. After 13 days of PBMC administration and 6 hours, 24 hours after the first dose of antibody, and 6 hours, 24 hours, and 1 week after the last dose, blood analysis was performed as follows. The nonspecific activation of T cells by MTI3 was much lower than that of OKT3. This was confirmed (FIG. 8). Experimentally, the following method was carried out.
  • PBMC peripheral blood mononuclear cells
  • Anti-human CD4-V450 (RPA-T4, BD, # 560345), anti-human CD8-V500 (RPA-T8, BD, # 560774), anti-human CD3-A488 (UCHT1, BD, # 557694) in 20ul of blood )
  • Add 1.5ul of mixture block light exposure for 15 minutes at room temperature, and after dyeing, dilute the dissolved solution (10X) with 1X in distilled water (DW), add 150ul for each sample, and block light exposure for 15 minutes at room temperature. Stained.
  • FACS buffer PBS, 2% Fetal bovine serum (FBS), 0.05% sodium azide
  • MFI mean fluorescence intensity
  • the present invention provides novel antibodies or antigen binding fragments thereof that bind to CD3 ⁇ .
  • the present invention provides a medicinal use for the prevention or treatment of a T cell mediated disease of a novel antibody or antigen-binding fragment thereof that binds CD3 ⁇ .

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Abstract

La présente invention concerne : un anticorps dirigé contre CD3γε ou son fragment de liaison à l'antigène; un acide nucléique codant pour ce dernier; un vecteur comprenant l'acide nucléique; des cellules transformées au moyen du vecteur; un procédé de préparation de l'anticorps ou de son fragment de liaison à l'antigène; une composition pour prévenir ou traiter des maladies induites par les lymphocytes t, le comprenant; et un procédé de traitement des maladies induites par les lymphocytes t.
PCT/KR2016/003357 2016-02-29 2016-03-31 Anticorps anti-cd3γε et son utilisation WO2017150762A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140066600A1 (en) * 2009-01-30 2014-03-06 Ab Biosciences, Inc. Novel lowered affinity antibodies and uses therefor
US20140141020A1 (en) * 2011-06-14 2014-05-22 Mayo Foundation For Medical Education And Research Anti-cd3 therapies
WO2015181098A1 (fr) * 2014-05-28 2015-12-03 F. Hoffmann-La Roche Ag Anticorps se liant au cd3-epsilon humain et de singe cynomolgus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140066600A1 (en) * 2009-01-30 2014-03-06 Ab Biosciences, Inc. Novel lowered affinity antibodies and uses therefor
US20140141020A1 (en) * 2011-06-14 2014-05-22 Mayo Foundation For Medical Education And Research Anti-cd3 therapies
WO2015181098A1 (fr) * 2014-05-28 2015-12-03 F. Hoffmann-La Roche Ag Anticorps se liant au cd3-epsilon humain et de singe cynomolgus

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI [O] 3 August 2017 (2017-08-03), "Chain A, Crystal Structure Of Cd3gammaepsilon Heterodimer In Complex With Okt3 Fab Fragment", XP055414571, Database accession no. 1SY6_A *
KJER-NIELSEN ET AL.: "Crystal Structure of the Human T Cell Receptor CD 3 epsilon gamma Heterodimer Complexed to the Therapeutic mAb OKT3", PNAS, vol. 101, no. 20, 2004, pages 7675 - 7680, XP055329073 *

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