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WO2025230233A1 - Anticorps spécifique contre cd3 humain et son utilisation - Google Patents

Anticorps spécifique contre cd3 humain et son utilisation

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Publication number
WO2025230233A1
WO2025230233A1 PCT/KR2025/005624 KR2025005624W WO2025230233A1 WO 2025230233 A1 WO2025230233 A1 WO 2025230233A1 KR 2025005624 W KR2025005624 W KR 2025005624W WO 2025230233 A1 WO2025230233 A1 WO 2025230233A1
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WIPO (PCT)
Prior art keywords
amino acid
seq
acid sequence
antibody
variable region
Prior art date
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Pending
Application number
PCT/KR2025/005624
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English (en)
Korean (ko)
Inventor
이석묵
양하림
김지웅
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Kookmin University
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Kookmin University
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Publication date
Priority claimed from KR1020250005727A external-priority patent/KR20250160316A/ko
Application filed by Kookmin University filed Critical Kookmin University
Publication of WO2025230233A1 publication Critical patent/WO2025230233A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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

Definitions

  • the present invention was made under the support of the Ministry of Health and Welfare of the Republic of Korea under project number 1465039676, and the research management specialized institution of the project is the Korea Health Industry Development Institute, the research project name is "Infectious Disease Prevention and Treatment Technology Development Project”, the research project name is "Development of Peptide-Fusion Antibody Source Technology for Preemptive Response and Effective Treatment of Coronavirus", the main institution is Kookmin University, and the research period is from January 1, 2023 to December 31, 2023.
  • T cells play a crucial role in recognizing and responding to antigens through highly specialized functions.
  • T cells can specifically identify and target pathogens such as viruses and bacteria, as well as abnormal cells, including tumor cells.
  • Regulation of T cell activity is essential for maintaining immune homeostasis and preventing insufficient immune responses or harmful, excessive inflammation.
  • T cells provide a highly specific and finely orchestrated response. Their activation is tightly regulated through complex molecular interactions and signaling pathways, including co-stimulatory molecules, cytokines, and transcription factors, ensuring a precise response to potential threats.
  • Dysregulation of T cell activation is a hallmark of various immune disorders, including autoimmune diseases. Therefore, understanding the molecular mechanisms that regulate T cell activation not only provides insights into immune regulation but also offers potential therapeutic strategies for diseases caused by dysregulated T cell activity.
  • CD3 ⁇ is a critical component of the T cell receptor (TCR) complex and plays a role in initiating T cell activation.
  • CD3 ⁇ forms a complex with the TCR to recognize antigen fragments presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs).
  • MHC major histocompatibility complex
  • APCs antigen-presenting cells
  • the CD3 complex consists of ⁇ and ⁇ heterodimers and ⁇ homodimers, and these subunits form a signaling platform required for T cell activation.
  • MHC major histocompatibility complex
  • APCs antigen-presenting cells
  • CD3 ⁇ has emerged as an attractive therapeutic target for modulating immune responses in clinical applications, including organ transplantation, cancer immunotherapy, and the treatment of autoimmune diseases.
  • Targeting CD3 ⁇ has the potential to modulate T cell activity and offer new therapeutic possibilities for diseases associated with dysregulated T cell activity.
  • Monoclonal antibodies (mAbs) are considered invaluable tools for both research and treatment due to their exceptional target specificity and therapeutic efficacy.
  • Muromonab-CD3 (OKT3) a well-known anti-CD3 ⁇ antibody, was the first therapeutic antibody targeting CD3 ⁇ to receive approval from the US Food and Drug Administration (US FDA) for clinical use.
  • OKT3 is particularly effective in preventing acute rejection of transplanted organs by activating T cells and inducing apoptosis.
  • the use of these antibodies can cause serious side effects, such as high immunogenicity, increased susceptibility to opportunistic infections, and cytokine release syndrome (CRS), a systemic inflammatory response caused by excessive cytokine production.
  • CRS cytokine release syndrome
  • new generations of CD3 ⁇ -specific antibodies such as teplizumab, visilizumab, and otelixizumab, have been developed, containing humanized structures and modified Fc domains to reduce immunogenicity.
  • each antibody fails to completely prevent T cell activation and cytokine release, resulting in side effects such as fever, headache, and gastrointestinal symptoms.
  • the present inventors have devoted extensive research efforts to develop a novel anti-CD3 antibody, specifically a novel anti-CD3 ⁇ antibody, that can provide enhanced therapeutic efficacy while minimizing side effects.
  • the present invention was completed by identifying a novel antibody that binds to a CD3 ⁇ target epitope distinct from the binding site of conventional anti-CD3 antibodies and induces downregulation of cell membrane CD3 ⁇ through subsequent internalization, thereby more selectively inhibiting T cell activation while simultaneously improving side effects by not inducing T cell activation.
  • Another object of the present invention is to provide a recombinant vector comprising the nucleic acid molecule.
  • Another object of the present invention is to provide a host cell comprising the recombinant vector.
  • the present invention provides the following inventions 1 to 16.
  • the antibody or antigen-binding fragment thereof binds to a CD3 ⁇ subunit.
  • the antibody or antigen-binding fragment thereof binds to an epitope comprising the amino acid sequence of SEQ ID NO: 41.
  • a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6;
  • a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 14;
  • a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 22;
  • a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 27; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
  • an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 33, HCDR2 comprising the amino acid sequence of SEQ ID NO: 34, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 35; and a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 36, LCDR2 comprising the amino acid sequence of SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 38.
  • an antibody or antigen-binding fragment thereof wherein the antibody or antigen-binding fragment is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a scFv, a Fab, F(ab), and F(ab) 2 comprising a heavy chain variable region and a light chain variable region.
  • a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof according to any one of 1 to 6.
  • a recombinant vector comprising a nucleic acid molecule of 7.
  • a pharmaceutical composition for preventing or treating a disease related to T cell activity dysregulation comprising an antibody or an antigen-binding fragment thereof according to any one of 1 to 6.
  • a pharmaceutical composition for preventing or treating a disease related to T cell activity dysregulation wherein the disease related to T cell activity dysregulation in 10 is selected from the group consisting of autoimmune diseases, transplant rejection reactions, cancer, infectious diseases, and inflammatory diseases.
  • the pharmaceutical composition for prevention or treatment is for the prevention or treatment of a disease related to increased or abnormal expression of CD3, a pharmaceutical composition for the prevention or treatment of a disease related to T cell activity regulation abnormality.
  • a method for preventing or treating a disease associated with T cell activity dysregulation comprising administering to a subject an antibody or antigen-binding fragment thereof according to any one of 1 to 6, or a pharmaceutical composition according to any one of 10 to 12.
  • a method for inhibiting CD3 internalization or expression comprising administering to a subject or sample an antibody or antigen-binding fragment thereof according to any one of 1 to 6, or a composition comprising the same.
  • the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the CD3 protein.
  • the present inventors have diligently researched and developed a novel anti-CD3 antibody, specifically a novel anti-CD3 ⁇ antibody, that can provide enhanced therapeutic efficacy while minimizing side effects.
  • a novel antibody was identified that binds to a CD3 ⁇ target epitope different from the binding site of conventional anti-CD3 antibodies, more selectively inhibits T cell activation, and does not induce T cell activation, ultimately improving side effects.
  • antibody refers to a specific antibody against CD3, including complete antibody forms as well as antigen-binding fragments of antibody molecules.
  • a complete antibody has two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond.
  • the heavy-chain constant region is of the gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ), and epsilon ( ⁇ ) types, and has subclasses of gamma1 ( ⁇ 1), gamma2 ( ⁇ 2), gamma3 ( ⁇ 3), gamma4 ( ⁇ 4), alpha1 ( ⁇ 1), and alpha2 ( ⁇ 2).
  • the constant regions of the light chain are of the kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50, W. B. Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).
  • the term "antigen-binding fragment” refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2, chemically linked F(ab')2, and Fv.
  • Fab has one antigen-binding site in a structure having variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1) of the heavy chain.
  • Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
  • F(ab')2 antibodies are generated when cysteine residues in the hinge region of Fab' form a disulfide bond.
  • Fv is the minimum antibody fragment that has only the heavy chain variable region and the light chain variable region, and the recombinant technology for producing Fv fragments is disclosed in PCT International Patent Publication Nos. WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086, and WO 88/09344.
  • a two-chain Fv has a heavy chain variable region and a light chain variable region linked by a non-covalent bond
  • a single-chain Fv generally has a heavy chain variable region and a single chain variable region covalently linked by a peptide linker or directly linked at the C-terminus, so that they can form a dimer-like structure like a two-chain Fv.
  • antibody fragments can be obtained using proteolytic enzymes (for example, restriction digestion of whole antibodies with papain yields Fab fragments, and digestion with pepsin yields F(ab')2 fragments), or they can be produced using genetic recombination techniques.
  • proteolytic enzymes for example, restriction digestion of whole antibodies with papain yields Fab fragments, and digestion with pepsin yields F(ab')2 fragments
  • the antibody is preferably in the form of scFv or a complete IgG antibody.
  • the heavy chain constant region can be selected from any one of the isotypes of gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) or epsilon ( ⁇ ).
  • the constant region is gamma 1 (IgG1), gamma 3 (IgG3) and gamma 4 (IgG4), and most preferably gamma 1 (IgG1) isotype.
  • the light chain constant region can be of the kappa or lambda type, and is preferably lambda type. Therefore, the preferred antibody of the present invention is an scFv form or an IgG1 form having a lambda (kappa) light chain and a gamma 1 (gamma1) heavy chain.
  • the term “heavy chain” refers to both a full-length heavy chain and fragments thereof, comprising a variable region domain VH and three constant region domains CH1, CH2 and CH3, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.
  • the term “light chain” as used herein refers to both a full-length light chain and fragments thereof, comprising a variable region domain VL and a constant region domain CL, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.
  • CDR complementarity determining region
  • Antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv) and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of the above antibodies.
  • multispecific antibodies e.g., bispecific antibodies
  • human antibodies e.g., humanized antibodies
  • chimeric antibodies single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv) and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of the above antibodies.
  • Antibodies of the present invention include minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibodies (scFv), scFv-Fc, bispecific antibodies, multispecific antibodies, other modified configurations of immunoglobulin molecules comprising an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies and covalently modified antibodies.
  • modified antibodies and antigen-binding fragments thereof include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engagers), TandAbs (tandem diabodies), DAFs (dual acting Fabs), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulins), CovX-bodies (peptide modified antibodies), duobodies, and triomAbs.
  • the list of such antibodies and antigen-binding fragments thereof is not limited to the above.
  • FR refers to variable domain residues other than hypervariable region (HVR) residues.
  • the FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in the following order in the VH (or VL/Vk):
  • FRL1 Framework region 1 of Light chain
  • CDRL1 complementarity determining region 1 of Light chain
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen.
  • the variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR).
  • FR conserved framework regions
  • HVR hypervariable regions
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind to a specific antigen can be isolated from antibodies that bind to the antigen and screened against a library of complementary VL or VH domains, respectively, using the VH or VL domain.
  • the term "specifically binds" or the like means that an antibody or antigen-binding fragment thereof, or other component such as scFv, specifically interacts with an antigen or a corresponding substance to produce an immunological response.
  • Specific binding is at least about 1 x 10 -6 M or less (e.g., 9 x 10 -7 M, 8 x 10 -7 M, 7 x 10 -7 M, 6 x 10 -7 M, 5 x 10 -7 M, 4 x 10 -7 M, 3 x 10 -7 M, 2 x 10 -7 M, or 1 x 10 -7 M), preferably 1 x 10 -7 M or less (e.g., 9 x 10 -8 M, 8 x 10 -8 M, 7 x 10 -8 M, 6 x 10 -8 M, 5 x 10 -8 M, 4 x 10 -8 M, 3 x 10 -8 M, 2 x 10 -8 M, or 1 x 10 -8 M), more preferably 1 x 10 -8 M or less
  • affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to the intrinsic binding affinity, which reflects direct protein-protein binding between members of a binding pair (e.g., an antibody and an antigen).
  • Kd dissociation constant
  • human antibody refers to an antibody produced by a human or human cell, or an antibody derived from a non-human source utilizing human antibody repertoires or other human antibody coding sequences, which possesses an amino acid sequence corresponding to the amino acid sequence of that antibody. This definition of a human antibody excludes humanized antibodies that contain non-human antigen-binding residues.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, and the remainder of the heavy and/or light chain is derived from a different source or species.
  • humanized antibody refers to a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin (e.g., mouse) antibody.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementarity-determining region
  • a humanized antibody may comprise residues that are not found in the recipient antibody or in the imported CDR or framework sequences. Such modifications are made to further improve and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions have the sequence of an FR region of a human immunoglobulin.
  • the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc region) sequence, up to substantially a human immunoglobulin constant region (Fc region) sequence.
  • the CD3-specific, specifically CD3 ⁇ -specific, antibody or antigen-binding fragment thereof of the present invention may include amino acid sequence variants within the range capable of specifically recognizing CD3 ⁇ , as will be appreciated by those skilled in the art.
  • the amino acid sequence of the antibody may be changed to improve the binding affinity and/or other biological properties of the antibody.
  • modifications include, for example, deletions, insertions, and/or substitutions of amino acid sequence residues of the antibody.
  • the variants are said to have "substantial similarity", meaning that the two peptide sequences share at least about 90% sequence identity, and more preferably at least about 95%, 98%, or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights.
  • the non-identical residue positions differ by conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).
  • conservative amino acid substitutions do not substantially alter the functionality of the protein.
  • the percent or degree of similarity may be adjusted upward to compensate for the conservative nature of the substitutions.
  • amino acid mutations are based on the relative similarity of the amino acid side-chain substituents, such as hydrophobicity, hydrophilicity, charge, and size.
  • Analysis of the size, shape, and type of amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine are similar in size; and phenylalanine, tryptophan, and tyrosine are similar in shape. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.
  • hydrophobicity index of an amino acid can be considered.
  • Each amino acid is assigned a hydrophobicity index based on 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).
  • hydrophobic amino acid index is crucial for imparting interactive biological functions to proteins. It is well known that amino acids with similar hydrophobic indices must be substituted to retain similar biological activity. When introducing mutations based on hydrophobic indices, substitutions are preferably made between amino acids with a difference in hydrophobicity index of ⁇ 2, more preferably ⁇ 1, or even ⁇ 0.5.
  • Amino acid exchanges in proteins that do not alter the overall activity of the molecule are well known in the art (H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979).
  • the most common exchanges are between amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.
  • the drug can be conjugated to the antibody or antigen-binding fragment thereof that specifically binds to CD3, specifically CD3 ⁇ , of the present invention by covalent bonding via a chemical linker.
  • the "linker” refers to any moiety that links, connects, or associates a binding agent (e.g., an antibody or antigen-binding fragment thereof) with a drug described herein.
  • a binding agent linker suitable for the antibody conjugate is one that is sufficiently stable to utilize the circulating half-life of the antibody while simultaneously allowing its drug to be released following antigen-mediated internalization of the conjugate.
  • the linker can be cleavable or non-cleavable.
  • a cleavable linker is a linker that is cleaved by intracellular metabolism, such as hydrolysis, reduction, or enzymatic cleavage, and then internalized.
  • a non-cleavable linker is a linker that releases the attached drug through lysosomal degradation of the antibody, and then internalized.
  • Suitable linkers include, but are not limited to, acid-cleavable linkers, enzymatically cleavable linkers, reduction-cleavable linkers, self-immolative linkers, and non-cleavable linkers.
  • Suitable linkers also include, but are not limited to, those that are or include peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units.
  • PEG polyethylene glycol
  • PAB para-aminobenzyl
  • CD3 refers to a cell surface protein complex that is an essential component of the T cell receptor (TCR) complex and plays a crucial role in T cell activation.
  • the CD3 complex is composed of ⁇ and ⁇ heterodimers and ⁇ homodimers, each of which maintains the stability of the TCR and mediates signal transduction.
  • CD3 binds to the TCR and recognizes antigens presented by the major histocompatibility complex (MHC) of antigen-presenting cells (APCs), thereby enabling T cells to respond specifically to the antigen.
  • MHC major histocompatibility complex
  • APCs antigen-presenting cells
  • the antibody or antigen-binding fragment thereof binds to a CD3 ⁇ subunit.
  • CD3 is an essential component of the T-cell receptor (TCR) complex, a cell surface protein complex composed of multiple subunits.
  • the CD3 complex consists of ⁇ heterodimers, ⁇ heterodimers, and ⁇ homodimers, each of which plays a crucial role in TCR stability and signal transduction.
  • CD3 subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their intracellular domains, which are essential for transducing T-cell activation signals.
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • CD3 interacts with the TCR and recognizes antigen-MHC complexes presented by antigen-presenting cells, initiating intracellular signaling to induce T-cell activation. This signaling process is a key step in regulating T-cell proliferation, effector function, and immune responses.
  • CD3 ⁇ is a component of the CD3 complex, which is present in the ⁇ and ⁇ heterodimers, respectively.
  • CD3 ⁇ plays a crucial role in signal transduction, mediating the initial signal transduction when the TCR recognizes the antigen-MHC complex.
  • the intracellular domain of CD3 ⁇ contains ITAMs, which recruit and activate molecules such as protein tyrosine kinases in the early stages of signal transduction. This process is a key step in T cell activation, enabling T cells to carry out immune responses such as proliferation and cytokine production.
  • CD3 ⁇ maintains the structural stability of the TCR-CD3 complex and ensures the accuracy and efficiency of signal transduction.
  • the antibody or antigen-binding fragment thereof binds to an epitope comprising the amino acid sequence of SEQ ID NO: 41.
  • epitope refers to a localized portion of an antigen to which an antibody or a fragment thereof can specifically bind.
  • a continuous amino acid sequence in a polypeptide, which is an antigen may be an epitope, and two or more non-consecutive portions of the polypeptide may be combined to form an epitope by three-dimensional structural folding.
  • An epitope may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen consecutive or non-consecutive amino acids in the unique three-dimensional structure of the antigen.
  • Epitopes are typically composed of surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural properties and specific charge characteristics. Epitopes may include amino acid residues directly involved in binding (also referred to as the immunogenic component of the epitope) and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide (i.e., the amino acid residues are within the footprint of the specific antigen-binding peptide).
  • the epitope containing the amino acid at the above position may be used in a form bound to a carrier when used as a vaccine or composition to maintain its structure.
  • the carrier according to the present invention is not particularly limited as long as it is biocompatible and can achieve the desired effect of the present invention, but may preferably be selected from the group consisting of peptides, serum albumin, immunoglobulins, hemocyanins, and polysaccharides.
  • the antibody or antigen-binding fragment thereof in one embodiment, the antibody or antigen-binding fragment thereof,
  • a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6;
  • a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 14;
  • a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 22;
  • a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 27; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
  • a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 34, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 35; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 36, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 37, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 38.
  • the antibody or antigen-binding fragment thereof in one embodiment, the antibody or antigen-binding fragment thereof,
  • (v) It comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 39 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 40.
  • the antibody or antigen-binding fragment is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a scFv, a Fab, F(ab), and F(ab) 2 comprising a heavy chain variable region and a light chain variable region.
  • the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or an antigen-binding fragment thereof.
  • nucleic acid molecule has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base portion is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).
  • the nucleic acid molecule of the present invention encoding the amino acid sequence constituting the antibody or antigen-binding fragment thereof is interpreted to also include a sequence showing substantial identity therewith.
  • the substantial identity means a sequence showing at least 60% homology, more specifically 70% homology, even more specifically 80% homology, even more specifically 90% homology, and most specifically 95% homology, when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J.
  • the present invention provides a recombinant vector comprising the nucleic acid molecule.
  • vector refers to a means for expressing a target gene in a host cell, including but not limited to plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.
  • the nucleic acid molecule encoding the light chain variable region and the nucleic acid molecule encoding the heavy chain variable region are operatively linked to a promoter.
  • operatively linked refers to a functional association between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and/or translation of the other nucleic acid sequence.
  • a nucleic acid expression regulatory sequence e.g., a promoter, a signal sequence, or an array of transcription factor binding sites
  • the recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.
  • a promoter derived from the genome of a mammalian cell e.g., metallothionine promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter
  • a promoter derived from a mammalian virus e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)
  • MMTV mouse mammary tumor virus
  • LTR promoter of HIV promoter of Moloney virus
  • RSV Rous sarcoma virus
  • the vector of the present invention may be fused with other sequences to facilitate purification of antibodies expressed therefrom.
  • fusion sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).
  • the protein expressed by the vector of the present invention is an antibody
  • the expressed antibody can be easily purified through a protein A column or the like without an additional sequence for purification.
  • the present invention provides a host cell comprising the recombinant vector.
  • Any host cell capable of stably and continuously cloning and expressing the vector of the present invention is known in the art and can be used.
  • suitable eukaryotic host cells for the vector include, but are not limited to, monkey kidney cells 7 (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK-293 cells.
  • the method of transporting the vector of the present invention into a host cell can be carried out by the CaCl 2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114(1973)), the Hanahan method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114(1973); and Hanahan, D., J. Mol. Biol., 166:557-580(1983)), and the electroporation method (Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145(1988)), when the host cell is a prokaryotic cell.
  • an appropriate inducer can be added to the medium, if necessary.
  • the expression vector contains a lac promoter
  • IPTG can be added to the medium, and if it contains a trp promoter, indoleacrylic acid can be added to the medium.
  • prevention refers to the prevention or protective treatment of a disease or disease state.
  • treatment refers to the reduction, suppression, alleviation, or eradication of a disease state. For example, it refers to minimizing the spread or worsening of a disease caused by administering a therapeutic agent to a subject.
  • subject or “patient” in this specification refers to an animal, preferably a mammal, including a human, pig, chimpanzee, dog, cat, cow, mouse, rabbit or rat.
  • the pharmaceutical composition of the present invention may be formulated as a powder, granules, tablets, coated tablets, pills, dragees, capsules, liquids, suspensions, gels, syrups, slurries, suppositories, enemas, emulsions, pastes, ointments, creams, lotions, powders, sprays, or suspensions, but is not limited thereto.
  • the pharmaceutical composition of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.
  • suitable carrier excipient or diluent commonly used in the manufacture of pharmaceutical compositions.
  • suitable carrier include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, mannitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.
  • a pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined based on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field.
  • the amount of the composition used may vary depending on the patient's age, sex, and weight, but the peptide can be administered once or several times a day in an amount sufficient to achieve a blood concentration useful for the treatment of cancer.
  • the dosage of the above composition may be increased or decreased depending on the route of administration, severity of the disease, gender, weight, age, etc. Therefore, the above dosage does not limit the scope of the present invention in any way.
  • the dosage of the pharmaceutical composition of the present invention is preferably 0.001 to 100 mg/kg (body weight) per day, and the pharmaceutical composition at the dosage described above can be applied locally to the desired area, depending on the purpose of application.
  • the pharmaceutical composition of the present invention can be administered to a subject via various routes. Any route of administration is conceivable, including intracerebral administration, oral ingestion, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.
  • the method is intravenous injection.
  • the pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.
  • the disease associated with T cell activity dysregulation is selected from the group consisting of autoimmune diseases, transplant rejection, cancer, infectious diseases, and inflammatory diseases.
  • the present invention is not limited thereto, and includes any disease caused by T cell activity dysregulation or exhibiting the characteristics of T cell activity dysregulation.
  • the autoimmune disease may be, but is not limited to, type 1 diabetes, rheumatoid arthritis, multiple sclerosis, lupus, Crohn's disease, ulcerative colitis, psoriasis, Sjogren's syndrome, Behcet's disease, ankylosing spondylitis, pemphigus, systemic sclerosis, Hashimoto's thyroiditis, Graves' disease, autoimmune hepatitis, myasthenia gravis, Guillain-Barré syndrome, thrombocytopenic purpura, alopecia areata, autoimmune gastritis, severe combined immunodeficiency, sarcoidosis, scleroderma, polymyositis, dermatomyositis, or diffuse large B-cell lymphoma, for example.
  • the cancer is selected from the group consisting of, for example, breast cancer, ovarian cancer, stomach cancer, lung cancer, non-small cell lung cancer, liver cancer, hepatocellular cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, rectal cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, parathyroid cancer, squamous cell carcinoma, peritoneal cancer, kidney cancer, testicular cancer, esophageal cancer, and ureteral cancer.
  • the inflammatory disease may be, but is not limited to, for example, Cytokine Release Syndrome (CRS), Sepsis, Systemic Inflammatory Response Syndrome (SIRS), Acute Respiratory Distress Syndrome, allergic and hypersensitivity reactions, autoimmune inflammatory diseases, infection-related inflammatory diseases, and other chronic inflammatory diseases.
  • CRS Cytokine Release Syndrome
  • SIRS Systemic Inflammatory Response Syndrome
  • Acute Respiratory Distress Syndrome allergic and hypersensitivity reactions
  • autoimmune inflammatory diseases infection-related inflammatory diseases
  • other chronic inflammatory diseases chronic inflammatory diseases.
  • the above T prevention or treatment method has something in common with the contents described in the CD3-specific antibody or composition in that it administers a CD3-specific antibody or a composition containing the same, so the description of the common parts is omitted to prevent excessive redundant description in the specification.
  • the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CD3 protein.
  • the present invention provides a recombinant vector comprising the nucleic acid molecule.
  • the present invention provides a host cell comprising the recombinant vector.
  • the present invention provides a method for preventing or treating a disease related to T cell activity dysregulation, comprising a step of administering to a subject the antibody or an antigen-binding fragment thereof, or a composition comprising the same.
  • T cell activation can be more selectively suppressed and side effects can be improved compared to conventional CD3 ⁇ antibodies by not inducing T cell activation.
  • Figure 1 shows the results of screening hCD3 ⁇ -specific scFv clones from a human scFv phage display library.
  • Figure 1a Schematic diagram of the biopanning process for screening hCD3 ⁇ -specific scFv clones from a human scFv library using phage display technology. Biopanning was performed using magnetic beads coated with rhCD3 ⁇ , and the selected scFvs were converted to scFv-Fc antibodies (K108.1 to K108.5).
  • Figure 1b Phage ELISA results showing the hCD3 ⁇ -binding ability of 96 randomly selected phage clones. BSA was used as a negative control.
  • Figure 2 shows the results of biophysicochemical characterization of the selected scFv-Fc antibodies.
  • Figure 2a A bar graph showing the final production yield purified by affinity column chromatography for each scFv-Fc antibody. The efficiency of the purification process is highlighted for each antibody.
  • Figure 2b SDS-PAGE analysis and Coomassie Brilliant Blue staining results confirmed that the purity of the selected scFv-Fc antibodies was 90% or higher.
  • Figure 2c SPR sensorgrams showed binding data for K108.1, K108.2, K108.3, K108.4, and K108.5 antibodies interacting with rhCD3 ⁇ immobilized on the sensor chip at different concentrations (16, 32, 64, 128, and 256 nM).
  • Figure 3 shows the results of evaluating the thermal stability of selected antibodies using a protein thermal shift assay.
  • the thermal stability of hCD3 ⁇ -specific scFv antibodies was assessed using a protein thermal shift assay. Fluorescence intensity was plotted against temperature, and distinct peaks representing the T m (melting points) of K108.1, K108.2, K108.3, K108.4, and K108.5 antibodies were identified.
  • Figure 4 shows the results of comparing the CD3 ⁇ binding sites of the selected antibodies and OKT3.
  • A Competitive ELISA results.
  • K108.1 and K108.5 independently bind to hCD3 ⁇ even in the presence of OKT3.
  • B-C upper panels
  • SPR analysis results showed that K108.1 (red) or K108.5 (blue) additionally bound to hCD3 ⁇ saturated with OKT3 (black), confirming that the binding sites of the two antibodies are different from those of OKT3.
  • B-C lower panels
  • analysis of the binding process of OKT3 (black) to hCD3 ⁇ saturated with K108.1 (red) or K108.5 (blue) further demonstrated that the binding sites of the selected antibodies are different from those of OKT3.
  • Data represent the means ( ⁇ SEM) of duplicate measurements obtained in one of two independent experiments.
  • Figure 5 shows the results of epitope mapping of K108.5 using ELISA.
  • Figure 5a Schematic diagram of the design of Fc-tag fragments encompassing the extracellular domain (ECD) of hCD3 ⁇ . Each fragment encompasses the ECD region and is composed of fragments 1 to 5.
  • Figure 5b ELISA results confirmed that OKT3 primarily bound to fragments 4 and 5.
  • Figure 5c ELISA results showed that K108.5 specifically bound to fragments 2 and 3, and no binding was detected to fragments 1, 4, or 5.
  • BSA was used as a negative control.
  • Data represent the mean ( ⁇ SEM) of three replicate measurements obtained in one of two independent experiments. Abbreviations: ECD, extracellular domain; TM, transmembrane domain; CD, cytoplasmic domain.
  • Figure 6 shows the results of the binding specificity analysis of K108.5 to CD3 ⁇ on the T cell surface.
  • the specific binding of K108.5 to CD3 ⁇ on the surface of Jurkat T cells was evaluated by flow cytometry, and the results are shown as follows: untreated (light gray), K108.5 alone (gray), and K108.5 pre-incubated with rhCD3 ⁇ (blue).
  • FIG. 7 shows the results of analyzing the effect of K108.5 on T cell activation.
  • B Time-dependent T cell activation was assessed by measuring the fluorescence intensity of GFP in Jurkat T cells treated with OKT3 or K108.5. Data are presented as the mean ⁇ SEM based on triplicate measurements from one of three independent experiments.
  • FIG. 8 shows the results of the internalization and downregulation analysis of hCD3 ⁇ on the T cell surface induced by K108.5.
  • Fig. 8a Time-dependent internalization of FabFluor-pH red-labeled control antibody (black) or K108.5 (blue) for 14 hours.
  • Fig. 8b Flow cytometric analysis of CD3 ⁇ levels in Jurkat T cells treated with K108.5 (blue) or control antibody (black) using anti-CD3 ⁇ polyclonal antibody followed by treatment with Alexa Flour 488-conjugated anti-rabbit IgG.
  • Fig. 8c Bar graph of the mean fluorescence intensity obtained from the flow cytometric data in Fig. 8b. All values are presented as the mean ⁇ SEM of duplicate measurements from one of two independent experiments. Statistical analysis was performed using a two-tailed Student's t-test (* p ⁇ 0.05, *** p ⁇ 0.001).
  • Figure 9 shows the results of a binding assay of anti-CD3 ⁇ polyclonal antibodies to CD3 ⁇ in T cells.
  • Flow cytometry was used to assess whether K108.5 interferes with the binding of anti-CD3 ⁇ polyclonal antibodies to Jurkat T cells.
  • PFA-fixed Jurkat T cells were divided into groups treated with K108.5 (blue) and untreated (dark gray), and then the binding of anti-CD3 ⁇ polyclonal antibodies was assessed.
  • a mock control (light gray) treated only with secondary antibody served as a negative control.
  • Figure 10 shows the results of analyzing the effect of K108.5 on reducing CD3 ⁇ or TCR expression on the T cell surface.
  • Jurkat T cells were divided into groups treated with K108.5 (blue) and untreated (dark gray), and the levels of CD3 ⁇ or TCR on the cell surface ( Figure 10a) or ( Figure 10b) were evaluated by flow cytometry.
  • a mock control (light gray) treated only with the secondary antibody was used as a negative control.
  • FIG 11 analyzes the inhibitory effect of K108.5 on T cell activation.
  • T cell activation was assessed by measuring GFP fluorescence intensity in OKT3-activated Jurkat T-GFP cells (A) or cells co-cultured with mDCs (B).
  • A OKT3-activated Jurkat T-GFP cells
  • B cells co-cultured with mDCs
  • Experiments were performed in the presence or absence of control antibody or K108.5, and all values are expressed as the mean ⁇ SEM of triplicate measurements from one of three independent experiments.
  • Statistical analysis was performed using a two-tailed Student's t-test, with significance indicated by * p ⁇ 0.05 and *** p ⁇ 0.001.
  • Figure 12 shows the results of an analysis of the effect of K108.5 on the inhibition of activated T cell-mediated apoptosis.
  • Jurkat T cells were divided into groups treated with K108.5 (blue) and untreated (dark gray), activated with OKT3, and then co-cultured with NCI-H146 cells. After 6 hours of co-culture, the degree of NCI-H146 cell death was assessed by measuring the fluorescence intensity of the Annexin V reagent. Statistical analysis was performed using a two-tailed Student's t-test, and significance was indicated as *p ⁇ 0.05.
  • Jurkat clone E6-1 cells (Korean Cell Line Bank, Seoul, South Korea), NF- ⁇ B/Jurkat/GFP TM transcriptional reporter cell line (System Bioscience, Palo Alto, CA, USA), THP-1 cells, and NCI-H146 human small cell lung cancer (SCLC) cells (Korean Cell Line Bank) were cultured in Roswell Park Memorial Institute medium (RPMI; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific) and 100 U/mL penicillin-streptomycin (Thermo Fisher Scientific). Cells were maintained at 37 °C under 5% CO 2 .
  • RPMI Roswell Park Memorial Institute medium
  • FBS fetal bovine serum
  • penicillin-streptomycin Thermo Fisher Scientific
  • Expi293 TM cells (Thermo Fisher Scientific) were cultured in Expi293 TM expression medium (Thermo Fisher Scientific) at 37 °C under 8% CO 2 in a shaking incubator at 125 rpm. Cell counts were measured using ADAM TM CellT (NanoEntek, Seoul, Korea).
  • THP-1 cells were seeded at 2 x 10 5 cells/mL in 6-well culture plates. The cells were then cultured for 5 days in medium containing 100 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GCF; PeproTech, Rocky Hill, NJ, USA) and 100 ng/mL recombinant human interleukin-4 (IL-4; PeproTech). During the culture period, half of the medium in each well was replaced with fresh cytokine-supplemented medium every 2 days.
  • GCF granulocyte-macrophage colony-stimulating factor
  • IL-4 human interleukin-4
  • the DNA sequence encoding the hCD3 ⁇ subunit of the TCR was synthesized by GenScript (Piscataway, NJ, USA) and amplified by polymerase chain reaction (PCR).
  • GenScript Progene, Inc., Inc.
  • PCR polymerase chain reaction
  • the PCR product was subcloned into the pcDNA3.4 mammalian expression vector (Invitrogen, Waltham, MA, USA) to produce His-tagged recombinant proteins.
  • the recombinant vector was transfected into Expi293 cells according to the manufacturer's recommendations, and the proteins were overexpressed. Subsequently, the proteins were purified from the culture medium by affinity column chromatography using HisPur Ni-NTA Resin (Thermo Fisher Scientific).
  • Human antibodies specific for hCD3 ⁇ were isolated from a human scFv antibody library using phage display technology.
  • Four ⁇ g of rhCD3 ⁇ was coated onto M-270 Epoxy Dynabeads TM (Invitrogen), and four rounds of biopanning were performed.
  • specific phages bound to the target antigen were captured and washed with phosphate-buffered saline (PBST) containing 0.05% (v/v) Tween 20 to remove unbound phages. Phage recovery and amplification were performed using helper phage (VCSM13).
  • PBST phosphate-buffered saline
  • VCSM13 helper phage
  • 96 phage clones were randomly selected from the output colonies and evaluated for reactivity against the target antigen using phage enzyme-linked immunosorbent assay (ELISA).
  • ELISA phage enzyme-linked immunosorbent assay
  • Colonies selected in the fourth round of biopanning were inoculated into 1 mL of Super Broth medium supplemented with 50 ⁇ g/mL carbenicillin in a 96-deep-well plate (Axygen, Union City, CA, USA) and incubated at 37 °C for 5 h. Then, 10 11 pfu/mL of helper phage (VCSM13) and 70 ⁇ g/mL kanamycin were added, and the plate was incubated overnight at 37 °C. The plate was then centrifuged at 3,000 ⁇ g for 30 min, and the phage supernatant was used for ELISA.
  • VCSM13 helper phage
  • a 96-well high-binding microplate (Corning, Corning, NY, USA) was coated with 0.1 ⁇ g of rhCD3 ⁇ and incubated overnight at 4 °C. After blocking with 3% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS), 100 ⁇ L of phage supernatant was added to the plate and incubated at 37 °C for 2 h. After washing three times with 0.05% (v/v) PBST, horseradish peroxidase (HRP)-conjugated anti-hemagglutinin antibody (1:3000; Bethyl Laboratories, Montgomery, TX, USA) was added and incubated at 37 °C for 1 h.
  • HRP horseradish peroxidase
  • TMB 3,3′,5,5′-tetramethylbenzidine
  • H 2 SO 4 1 M sulfuric acid
  • absorbance at 450 nm was measured using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).
  • the selected scFv clones and OKT3 scFv were subcloned into the pCEP4 mammalian expression vector, which contains the Fc region of human immunoglobulin G1 (IgG1), respectively.
  • the recombinant vectors encoding the scFv-Fc antibodies were transfected into Expi293 cells using the ExpiFectamine TM 293 transfection kit (Thermo Fisher Scientific) according to the manufacturer's instructions, and cultured at 37 °C, 125 rpm shaking, and 8% CO 2 .
  • the selected antibodies were dissolved in HBS-N buffer (10 mM HEPES and 150 mM NaCl, pH 7.4) at increasing concentrations (16, 32, 64, 128, and 256 nM) and injected at a flow rate of 10 ⁇ L/min at 25 °C. Each association step lasted 2 min, followed by a 10-min dissociation step. After each cycle, the sensor chip was regenerated with 3 M magnesium chloride to remove bound antibodies from the surface. All equilibrium dissociation constant (K D ) values were calculated using Biacore T200 Control Software Version 3.2 (Cytiva).
  • an iMSPR-mini SPR instrument (icluebio, Seongnam, South Korea) was used to verify the binding of K108.1 or K108.5 to the CD3 ⁇ -OKT3 complex.
  • RhCD3 ⁇ (Sino Biological, Beijing, China) was immobilized on a research-grade carboxylic acid (COOH) sensor chip (icluebio) using an amine coupling kit (icluebio) according to the manufacturer's instructions.
  • 500 nM OKT3 and 500 nM K108.1 or K108.5 were sequentially applied to the immobilized CD3 ⁇ at room temperature (RT) at a flow rate of 50 ⁇ L/min. Data were analyzed using iMSPR analysis software (icluebio).
  • a 96-well high-binding microplate (Corning) was coated with 0.1 ⁇ g of rhCD3 ⁇ and blocked with 3% (w/v) BSA in PBS at 37 °C for 2 h. Then, 10 ⁇ g/mL of each selected scFv-Fc antibody (K108.1, K108.2, K108.3, K108.4, or K108.5) was added to the wells and incubated at 37 °C for 2 h.
  • HRP horseradish peroxidase
  • TMB substrate solution (Thermo Fisher Scientific) was added and the reaction was allowed to proceed for 5 minutes. Finally, 100 ⁇ L of 1 M H 2 SO4 was added to terminate the reaction, and the absorbance was measured at 450 nm using a microplate reader.
  • the recombinant vector was transfected into Expi293 cells using the ExpiFectamine TM 293 transfection kit (Thermo Fisher Scientific) according to the manufacturer's instructions and cultured at 37 °C, 125 rpm shaking, and 8% CO 2 . Seven days after transfection, the supernatant was collected, and the Fc-tag fragment was purified by affinity chromatography using Protein A-Sepharose (GE Healthcare).
  • a 96-well high-binding microplate (Corning) was coated with 0.1 ⁇ g of each Fc-tag fragment and blocked with 3% (w/v) BSA in phosphate-buffered saline (PBS) at 37 °C for 2 h.
  • Horseradish peroxidase (HRP)-conjugated K108.5 or HRP-conjugated OKT3 was then added to the wells at a concentration of 14 ⁇ g/mL and incubated at 37 °C for 2 h.
  • FabFluor-pH Red antibody labeling reagent (Sartorius, G ⁇ ttingen, Germany) according to the manufacturer's instructions.
  • FabFluor reagent fluorescence is minimal in the neutral or slightly alkaline environment (pH 6.3–7.4) of the extracellular solution and in early endosomes, but significantly increases in the acidic environment of lysosomes (pH 4.5) after antibody internalization.
  • the plates were then transferred to an Incucyte SX1 real-time cell analysis instrument (Sartorius) and imaged at 10x magnification for 14 h. Fluorescence intensity was measured to assess antibody internalization.
  • Jurkat T cells (1 ⁇ 10 5 cells) were pretreated with or without 20 ⁇ g/mL K108.5 at 37 °C for 2, 4, or 8 h. Cells were then fixed with 4% (w/v) PFA and incubated with 1 ⁇ g of anti-CD3 ⁇ polyclonal antibody (Bioss, Woburn, MA, USA), anti-CD3 ⁇ polyclonal antibody (Proteintech, Rosemont, IL, USA), or anti-TCR ⁇ / ⁇ polyclonal antibody (eBioscience, San Diego, CA, USA) for 2 h at RT. Next, cells were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG (1:100, Invitrogen) for 1 h at RT. All samples were analyzed using a flow cytometer (Millipore), and data were processed using FlowJo (TreeStar).
  • Jurkat T cells Jurkat T-GFP expressing green fluorescent protein (GFP) upon activation were seeded at a density of 2 ⁇ 10 4 cells/well in 96-well culture plates (Falcon, Corning, NY, USA). Cells were cultured for 2 h in the presence or absence of 20 ⁇ g/mL K108.5 at 37 °C. Cells were then treated with 1 ⁇ g/mL OKT3.
  • Jurkat T-GFP cells (1 ⁇ 10 4 effector cells) were co-cultured with mDCs (2 ⁇ 103, stimulator cells) at a 5:1 ratio.
  • mDCs (2 ⁇ 103, stimulator cells)
  • Jurkat T-GFP cells (1 ⁇ 10 4) were seeded alone and used as a control. All cells were cultured for 4 h at 37 °C, 5% CO 2 and then seeded in 96-well culture plates (Falcon).
  • a 96-well culture plate was coated with poly-L-lysine for 2 hours at 37 °C and then washed three times with PBS. After drying the plate, 5 ⁇ 103 NCI-H146 cells were seeded and cultured overnight at 37 °C.
  • Phage ELISA results showed that all selected scFv clones showed strong reactivity to hCD3 ⁇ (Fig. 1b) and did not react with BSA used as a negative control.
  • VH Variable domain of heavy chain sequence of selected antibodies FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 K108.1 (Sequence number 7) EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYYMS WVRQAPGKGLEWVS AISPGSSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK HLVQTQHGRLYSDDGMDV WGQGTLVTVSS K108.2 (SEQ ID NO: 15) EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYDMS WVRQAPGKGLEWVS WIYHGGGDTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR APSACGARKCSSSYAMDV WGQGTLVTVSS K108.3 (SEQ ID NO: 23) EVQLLESGGGLVQPGGSLRLSCAASGFTFS NYAMS WVRQAPGKGLEWVS AI
  • VL Variable domain of light chain sequence of selected antibodies FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 K108.1 (SEQ ID NO: 8) QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNYVT WYQQLPGTAPKLLIY DSSQRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC AAWDDSLSAYV FGGGTKLTVL K108.2 (SEQ ID NO: 16) QSVLTQPPSASGTPGQRVTISC TGSSSNIGSNYVT WYQQLPGTAPKLLIY ADSHRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC ATWDSSLNGYV FGGGTKLTVL K108.3 (SEQ ID NO: 24) QSVLTQPPSASGTPGQRVTISC TGSSSNIGSNSVT WYQQLPGTAPKLLIY SNSNRPS GVPDRFSGSKSGTSASLAISGLRSEDEAD
  • the antibodies were overexpressed and purified via affinity column chromatography using Protein A-Sepharose (Fig. 2a). The purity of each antibody was assessed by SDS-PAGE, and a purity level exceeding 90% was confirmed (Fig. 2b).
  • K D equilibrium dissociation constant
  • K a association constant
  • K d dissociation constant
  • K108.5 exhibited a K value of 2.4 nM and high thermal stability, and was identified to have a unique binding site on hCD3 ⁇ , making it a prime candidate for further study in this study.
  • Example 4 Evaluation of the effect of K108.5 on CD3 ⁇ binding and T cell activation.
  • K108.5 specifically binds to CD3 ⁇ on the Jurkat T cell surface.
  • K108.5 was pre-incubated with or without rhCD3 ⁇ and then co-incubated with the cells.
  • Flow cytometry analysis revealed that pre-incubation with rhCD3 ⁇ significantly reduced K108.5 cell surface binding, indicating that K108.5 specifically binds to CD3 ⁇ (Fig. 6).
  • K108.5 and a control antibody were conjugated to Fab-Fluor reagent.
  • Jurkat T cells were then treated with each conjugate, and internalization was quantitatively monitored for 14 hours. The results showed that K108.5 was significantly internalized, while the control antibody exhibited minimal internalization (Fig. 8a).
  • the analysis results confirmed that the surface CD3 ⁇ level was significantly reduced in T cells treated with K108.5 (Figs. 8b and 8c).
  • the surface expression levels of CD3 ⁇ and TCR were compared in Jurkat T cells treated with K108.5 and untreated control cells. As a result, it was confirmed that K108.5 specifically downregulates CD3 ⁇ on the surface of Jurkat T cells without affecting CD3 ⁇ and TCR levels (Figs. 10a and 10b).
  • an activated T cell-mediated apoptosis assay was performed to evaluate the ability of K108.5 to modulate T cell effector function.
  • NCI-H146 human small cell lung cancer (SCLC) cells were used as a model cell line to assess activated T cell-induced apoptosis.
  • Jurkat T cells were activated with OKT3, either pretreated with K108.5 or left untreated.
  • Activated Jurkat T cells were co-cultured with NCI-H146 SCLC cells together with Annexin V Red reagent (a fluorescent dye for detecting apoptosis). Quantification of SCLC cell apoptosis revealed that K108.5 suppresses activated T cell-mediated apoptosis by modulating T cell function ( Figure 12).
  • T cells are a crucial component of the immune system, and the TCR/CD3 complex plays a central role in antigen recognition and subsequent T cell activation.
  • Unregulated T cell activation is associated with various diseases, highlighting the need for effective T cell activation modulation to treat conditions requiring T cell regulation.
  • CD3 ⁇ a key component of the TCR complex, is crucial for T cell activation and may serve as a promising target for therapeutic intervention.
  • K108.5 a fully human monoclonal antibody specific for CD3 ⁇ .
  • K108.5 effectively suppresses T cell activation by inducing potent internalization and downregulation of CD3 ⁇ on the T cell surface.
  • Antibody internalization is a crucial mechanism for regulating the expression of cell surface proteins and inhibiting their interactions with signaling molecules associated with target proteins.
  • trastuzumab a well-known internalizing antibody clinically used to treat HER2-positive breast cancer, effectively downregulates HER2 expression in human breast cancer cells, thereby inhibiting oncogenic pathways in HER2-expressing cancer cells.
  • K108.5 specifically recognizes CD3 ⁇ with high affinity (2.4 nM) and exhibits potent internalization activity, which is accompanied by a concomitant decrease in CD3 ⁇ expression on the T cell surface.
  • Time-dependent internalization analysis in Jurkat T cells confirmed the internalization activity of K108.5, and flow cytometry analysis revealed a selective downregulation of CD3 ⁇ expression induced by K108.5.
  • K108.5 effectively suppressed T cell activation induced by dendritic cells or OKT3 by inhibiting TCR signaling.
  • K108.5 also suppressed activated T cell-mediated apoptosis by modulating T cell function.
  • Nck is an adaptor protein that can regulate TCR signaling pathways, and disruption of Nck recruitment to the TCR impairs T-cell antigen responses both in vitro and in vivo.
  • treatment with a peptide that disrupts the Nck-CD3 ⁇ interaction inhibits T-cell activation, suggesting that targeting this interaction could be a promising approach for the development of immunosuppressive or immunomodulatory agents.
  • K108.5 on CD3 ⁇ could complement other strategies targeting Nck or related signaling pathways.
  • OKT3 the first therapeutic antibody targeting hCD3 ⁇ to receive FDA approval
  • CRS Creplizumab
  • teplizumab the first therapeutic antibody targeting hCD3 ⁇ to receive FDA approval
  • otelixizumab the first therapeutic antibody targeting hCD3 ⁇ to receive FDA approval
  • CD3 ⁇ -specific antibodies with modified Fc domains such as teplizumab, visilizumab, and otelixizumab, cannot evade T cell activation.
  • K108.5 is a fully human monoclonal antibody isolated from a human antibody library, minimizing the risk of immunogenicity. K108.5 binds to a unique site on CD3 ⁇ that distinguishes it from OKT3, suggesting that K108.5 may exhibit different biological activities compared to OKT3. Notably, unlike other CD3 ⁇ -targeting antibodies, K108.5 does not induce T cell activation and instead suppresses TCR signaling by downregulating CD3 ⁇ on the T cell surface. Collectively, these results highlight the potential of K108.5 to effectively modulate T cell activation.
  • K108.5 binds to distinct regions of CD3 ⁇ compared to OKT3.
  • epitope mapping results of the present invention demonstrated that K108.5 specifically binds to segments 2 and 3, while OKT3 primarily binds to segments 4 and 5. Identifying the epitope of K108.5 could provide valuable insights for the design of novel antibodies with enhanced efficacy for CD3 ⁇ -targeted therapies. Ultimately, this could improve therapeutic strategies for modulating T cell responses.
  • K108.5 is a novel CD3 ⁇ -specific internalizing antibody that effectively modulates T cell activation.
  • K108.5 specifically binds to CD3 ⁇ on the T cell surface, is rapidly internalized, and downregulates CD3 ⁇ , thereby modulating T cell activation.
  • K108.5 can serve as a valuable research tool for studying the complexity of TCR signaling pathways.
  • it can be utilized for antibody-mediated gene delivery for T cell-specific gene therapy without the risk of T cell activation.
  • Targeting CD3 ⁇ with K108.5 presents a promising strategy for modulating T cell activation in diseases associated with T cell dysfunction.

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Abstract

La présente invention concerne un anticorps se liant de manière spécifique à CD3 ou un fragment de liaison à l'antigène de celui-ci. L'utilisation de l'anticorps ou du fragment de liaison à l'antigène de celui-ci selon la présente invention permet d'inhiber de manière plus sélective l'activation des lymphocytes T, et de ne pas induire l'activation des lymphocytes T, les effets secondaires peuvent ainsi être réduits pour être inférieurs à ceux des anticorps CD3ε classiques.
PCT/KR2025/005624 2024-05-03 2025-04-25 Anticorps spécifique contre cd3 humain et son utilisation Pending WO2025230233A1 (fr)

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KR20240059430 2024-05-03
KR10-2024-0059430 2024-05-03
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KR20240179926 2024-12-05
KR1020250005727A KR20250160316A (ko) 2024-05-03 2025-01-14 인간 cd3에 특이적인 항체 및 이의 용도
KR10-2025-0005727 2025-01-14

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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20070038495A (ko) * 2004-06-03 2007-04-10 노비뮨 에스 에이 항-ｃｄ3 항체 및 그의 사용 방법
WO2016014974A2 (fr) * 2014-07-25 2016-01-28 Cytomx Therapeutics, Inc. Anticorps anti-cd3, anticorps anti-cd3 activables, anticorps anti-cd3 multispécifiques, anticorps anti-cd3 activables multispécifiques et procédés d'utilisation de ces anticorps
US20200190189A1 (en) * 2017-05-08 2020-06-18 Adimab, Llc Anti-cd3-binding domains and antibodies comprising them, and methods for their generation and use
KR20210012007A (ko) * 2018-05-24 2021-02-02 얀센 바이오테크 인코포레이티드 항-cd3 항체 및 이의 용도
KR20210013167A (ko) * 2018-05-23 2021-02-03 화이자 인코포레이티드 Cd3에 특이적인 항체 및 이의 용도
KR20210143192A (ko) * 2019-02-22 2021-11-26 우한 이지 바이오파마 씨오., 엘티디. 변형된 Fc 단편, 이를 포함하는 항체 및 이의 응용
US20230277662A1 (en) * 2018-12-21 2023-09-07 Hoffmann-La Roche Inc. Antibodies binding to cd3
WO2023169419A1 (fr) * 2022-03-09 2023-09-14 Antengene (Hangzhou) Biologics Co., Ltd. Nouveaux anticorps anti-cd3 et leurs utilisations
WO2023179716A1 (fr) * 2022-03-23 2023-09-28 上海济煜医药科技有限公司 Anticorps anti-cd3, son procédé de préparation et son utilisation
WO2024086617A2 (fr) * 2022-10-18 2024-04-25 Adimab, Llc Anticorps anti-cd3 dépendant du ph et procédés associés

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20070038495A (ko) * 2004-06-03 2007-04-10 노비뮨 에스 에이 항-ｃｄ3 항체 및 그의 사용 방법
WO2016014974A2 (fr) * 2014-07-25 2016-01-28 Cytomx Therapeutics, Inc. Anticorps anti-cd3, anticorps anti-cd3 activables, anticorps anti-cd3 multispécifiques, anticorps anti-cd3 activables multispécifiques et procédés d'utilisation de ces anticorps
US20200190189A1 (en) * 2017-05-08 2020-06-18 Adimab, Llc Anti-cd3-binding domains and antibodies comprising them, and methods for their generation and use
KR20210013167A (ko) * 2018-05-23 2021-02-03 화이자 인코포레이티드 Cd3에 특이적인 항체 및 이의 용도
KR20210012007A (ko) * 2018-05-24 2021-02-02 얀센 바이오테크 인코포레이티드 항-cd3 항체 및 이의 용도
US20230277662A1 (en) * 2018-12-21 2023-09-07 Hoffmann-La Roche Inc. Antibodies binding to cd3
KR20210143192A (ko) * 2019-02-22 2021-11-26 우한 이지 바이오파마 씨오., 엘티디. 변형된 Fc 단편, 이를 포함하는 항체 및 이의 응용
WO2023169419A1 (fr) * 2022-03-09 2023-09-14 Antengene (Hangzhou) Biologics Co., Ltd. Nouveaux anticorps anti-cd3 et leurs utilisations
WO2023179716A1 (fr) * 2022-03-23 2023-09-28 上海济煜医药科技有限公司 Anticorps anti-cd3, son procédé de préparation et son utilisation
WO2024086617A2 (fr) * 2022-10-18 2024-04-25 Adimab, Llc Anticorps anti-cd3 dépendant du ph et procédés associés

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