WO2024163530A2 - Nouveaux anticorps anti-ccr4 et produits dérivés - Google Patents

Nouveaux anticorps anti-ccr4 et produits dérivés Download PDF

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WO2024163530A2
WO2024163530A2 PCT/US2024/013629 US2024013629W WO2024163530A2 WO 2024163530 A2 WO2024163530 A2 WO 2024163530A2 US 2024013629 W US2024013629 W US 2024013629W WO 2024163530 A2 WO2024163530 A2 WO 2024163530A2
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antibody
antigen
cancer
binding fragment
binding
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WO2024163530A3 (fr
Inventor
Ziyong Sun
Yunping LIU
Huanhuan Zhang
Chunqing Zhang
Yongyu QIAN
Chengcheng WANG
Jayant Thatte
John Gu
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Crown Bioscience Inc
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Crown Bioscience Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates generally to the fields of medicine, oncology, and immunology. More particular, the disclosure relates to antibodies and antigen-binding fragments thereof that bind to human CC chemokine receptor 4 (CCR4), and the methods of use thereof.
  • CCR4 human CC chemokine receptor 4
  • CCR4 also called CD 194, is a seven membrane-spanning G protein-coupled chemokine receptor which binds specifically to its ligands CCL17 and CCL22.
  • CCR4 is expressed on functionally distinct subsets of T cells, including T helper type 2 cells (Th2), and the majority of regulatory T cells (Tregs) (Bonecchi R, et al., 1998 J Exp Med 187: 129; Imai T, et al., 1999 Int Immunol. 11 :81; Yoshie O, et al., 2015 Int Immunol. 27: 11).
  • CCR4 is essential for the migration of Tregs to non-lymphoid tissues, and Tregs that scarcely express CCR4 lack the ability to migrate (Sather BD, et al., 2007 J Exp Med 204: 1335; Ge X, et al.,
  • CCR4 is also abundantly expressed on tumor-associated Tregs in various types of cancer, including breast, bladder, colorectal, ovarian, oral squamous cancer and T-cell leukemia/lymphoma (Ishida T, et al., 2006 Cancer Science 97: 1139; Marshall LA, et al.,
  • CCR4 and its ligands play a key role in the development and progression of solid tumors through orchestrating the recruitment and trafficking of immune cells, such as the immunosuppressive FoxP3+CD25+CD4+ Tregs into the lymphoid infiltrates surrounding the tumor, which inhibit the activation of T and NK cells (Imai T, et al., 1997 J Biol Chem 272: 15036; Imai T, et al., 1998 J Biol Chem 273: 1764; Menetrier-Caux C, et al., 2009 Cancer Res 69:7895; Liu W, et al., 2017 Biochem Biophys Res Commun 488: 196).
  • immune cells such as the immunosuppressive FoxP3+CD25+CD4+ Tregs into the lymphoid infiltrates surrounding the tumor.
  • the present disclosure provides anti-CCR4 antibodies and antigen-binding fragment thereof, amino acid and nucleotide sequences thereof, and uses thereof.
  • the present disclosure provides an isolated anti-CCR4 antibody or an antigen-binding fragment thereof.
  • the anti-CCR4 antibody or an antigen-binding fragment comprises a heavy chain (HC) variable region (VH) and a light chain (LC) variable region (VL), wherein the VH and VL comprise clone -paired complementarity determining region (CDR) sequences as set forth in Table 1, and variants thereof wherein one or more of the HC-CDRs and/or LC-CDRs has one, two, or three amino acid substitutions, additions, deletions or a combination thereof.
  • CDR complementarity determining region
  • the VH and VL have amino acid sequences at least 90% or 95% identical to clone-paired sequences of Table 2. In some embodiments, the VH and VL have amino acid sequences identical to clone-paired sequences of Table 2.
  • the isolated antibody is a murine, a rodent, a rabbit, a chimeric, a humanized, or a human antibody.
  • the antigen-binding fragment is a recombinant ScFv
  • single chain fragment variable antibody single chain fragment variable antibody
  • Fab fragment single chain fragment variable antibody
  • F(ab’)2 fragment single chain fragment variable antibody
  • Fv fragment single chain fragment variable antibody
  • the isolated antibody or an antigen-binding fragment thereof disclosed herein further comprises an immunoglobulin constant region, optionally a constant region of Ig, or optionally a constant region of human IgG.
  • the isolated antibody or an antigen-binding fragment thereof disclosed herein is multi-specific. In some embodiments, the isolated antibody or an antigen-binding fragment thereof disclosed herein binds specifically to a second antigen selected from PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, Fc receptors, FCRL(l-6), A2AR, CD160, 2B4, TGF-P, TGF-PR, VISTA, BTLA, TIGIT, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA(l-6), 0X40, CD2, CD27, CD28, CD30, CD40, CD47, SIRPA, CLEC-1, clever- 1/stabilin-l, ADGRE, TREM1, TREM2, CD122, ICAM-1, IDO, NKG2D/C, SLAMF7, MS4A4A SIGLEC(7-15), NKp80, NKG2A, CD160, CD161,
  • the isolated antibody or an antigen-binding fragment thereof disclosed herein is linked to one or more conjugate moieties.
  • the conjugate moiety is an immune-modulatory agent, an anti-tumor drug, a clearancemodifying agent, a toxin, a detectable label, an RNA, a DNA, a cytokine, or purification moiety.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antibody or an antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier.
  • the present disclosure provides an isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof disclosed herein.
  • the present disclosure provides a vector comprising the isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof disclosed herein.
  • the present disclosure provides a host cell comprising the vector disclosed herein.
  • the present disclosure provides a method of producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell disclosed herein under the condition at which the antibody or antigen -binding fragment thereof is expressed, and recovering the antibody or antigen-binding fragment thereof.
  • the present disclosure provides a method of treating or ameliorating the effect of a cancer in a subject.
  • the method comprises administering to the subject a therapeutically effective amount of the antibody or antigenbinding fragment thereof disclosed herein, or the pharmaceutical composition disclosed herein.
  • the cancer is selected from the group consisting of adrenal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer.
  • the subject is human.
  • the antibody or an antigen-binding fragment thereof is administered intravenously, intra-arterially, intra-tumorally, or subcutaneously.
  • the present disclosure provides use of the antibody or antigen-binding fragment thereof disclosed herein in the manufacture of a medication for treating cancer in a subject.
  • the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof disclosed herein, useful in detecting CCR4.
  • FIG. 1 shows the binding EC50 of the murine hybridoma anti-CCR4 antibodies to human CCR4 N-terminal peptide as measured by ELISA.
  • the binding of mlgGl is shown as a negative control.
  • FIG. 2 shows the binding EC50 of the murine hybridoma anti-CCR4 antibodies to human CCR4 on CCR4-293T cells as measured by FACS.
  • FIG. 3 shows the binding EC50 of the chimeric anti- CCR4 antibodies to human CCR4 N-terminal peptide as measured by ELISA.
  • FIG. 4 shows the binding EC50 of the chimeric anti- CCR4 antibodies to human CCR4 on 293 T- CCR4 cells as measured by FACS.
  • FIG. 5 shows the effect of chimeric anti-CCR4 antibodies on the ADCC of CCR4+ cells mediated by NK cells.
  • FIG. 6 shows the binding EC50 of the humanized anti-CCR4 antibodies to human CCR4 N-terminal peptide as measured by ELISA.
  • FIG. 7 shows the binding EC50 of the humanized anti-CCR4 antibodies to human CCR4 on 293T-CCR4 cells as measured by FACS.
  • FIG. 8 shows the effect of humanized anti-CCR4 antibodies on the ADCC of CCR4 positive cells mediated byNK cells.
  • FIG. 9 shows the binding of anti-CCR4 antibodies to 293T-mouse CCR4 as measured by FACS.
  • FIG. 10 shows the effect of anti-CCR4 antibodies on mouse platelet aggregation.
  • antibody includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multi- specific antibody, or bispecific antibody that binds to a specific antigen.
  • a native intact antibody comprises two heavy (H) chains and two light (L) chains.
  • Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable domain (VH) and a constant region including a first, second, and third constant domain (CHI, CH2, CHS, respectively); mammalian light chains are classified as X or K, while each light chain consists of a variable domain (VL) and a constant domain (CL).
  • a typical IgG antibody has a “Y” shape, with the stem of the Y typically consisting of the second and third constant domains of two heavy chains bound together via disulfide bonding.
  • Each arm of the Y includes the variable domain and first constant domain of a single heavy chain bound to the variable and constant domains of a single light chain.
  • the variable domains of the light and heavy chains are responsible for antigen binding.
  • the variable domains in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3).
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigenbinding fragments disclosed herein may be defined or identified by the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. (1985) 186(3):651 -63 ; Chothia, C. and Lesk, A.M., J.Mol.Biol. (1987) 196:901; Chothia, C.
  • the three CDRs are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant domains of the heavy and light chains are not involved in antigen-binding but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively.
  • IgGl gammal heavy chain
  • IgG2 gamma2 heavy chain
  • IgG3 gamma3 heavy chain
  • IgG4 gamma4 heavy chain
  • IgAl alpha 1 heavy chain
  • an antigen refers to a substance capable of inducing adaptive immune responses.
  • an antigen is a substance specifically bound by antibodies or T lymphocyte antigen receptors.
  • Antigens are usually proteins and polysaccharides, less frequently also lipids. Suitable antigens include without limitation parts of bacteria (coats, capsules, cell walls, flagella, fimbrai, and toxins), viruses, and other microorganisms.
  • Antigens also include tumor antigens, e.g., antigens generated by mutations in tumors.
  • antigens also include immunogens and haptens.
  • antigen-binding fragment refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure.
  • antigen-binding fragment examples include, without limitation, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv 1 ), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a bispecific antibody, a multi - specific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody.
  • An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds.
  • a “Fab fragment” comprises one light chain and the CHI and variable domains of one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a “Fab' fragment” comprises one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.
  • a “F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • a F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • “Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen-binding site.
  • An Fv fragment consists of the variable domain of a single light chain bound to the variable domain of a single heavy chain.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody consisting of a light chain variable domain and a heavy chain variable domain connected to one another directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA (1988) 85:5879).
  • An “Fc” region comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • the Fc region of the antibody is responsible for various effector functions such as antibody-dependent cell- mediated cytotoxicity (ADCC), and complement dependent cytotoxicity (CDC), but does not function in antigen binding.
  • ADCC antibody-dependent cell- mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • Single-chain Fv-Fc antibody or “scFv-Fc” refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.
  • a “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable domain of a single light chain and the variable domain of a single heavy chain is a disulfide bond.
  • a “(dsFv ’ or “(dsFv-dsFv 1 )” comprises three peptide chains: two VH domains linked by a peptide linker (e.g., a long flexible linker) and bound to two VL domains, respectively, via disulfide bridges.
  • dsFv- dsFv' is bispecific in which each disulfide paired heavy and light chain has a different antigen specificity.
  • Camelized single domain antibody refers to an antibody that contains two VH domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1 -2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94/04678; WO94/25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas).
  • VHH domain represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. (2007) 21 :3490-8).
  • a “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.
  • “Diabodies” or “dAbs” include small antibody fragments with two antigenbinding sites, wherein the fragments comprise a VH domain connected to a VL domain in the same polypeptide chain (VH-VL or VL-VH) (see, e.g., Holliger P. et al., Proc Natl Acad Sci U S A. Jul 15;90(14):6444-8 (1993); EP404097; WO93/11161).
  • the antigen-binding sites may target the same or different antigens (or epitopes).
  • a “bispecific ds diabody” is a diabody target two different antigens (or epitopes).
  • an “scFv dimer” is divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) that can be engineered by linking two scFvs.
  • an “scFv dimer” is a bispecific diabody comprising VHI-VL2 (linked by a peptide linker) associated with VLI-V H2 (also linked by a peptide linker) such that VRI and VLI coordinate and VH2 and VL2 coordinate and each coordinated pair has a different antigen specificity.
  • a “domain antibody” refers to an antibody fragment containing only the variable domain of a heavy chain or the variable domain of a light chain.
  • two or more VH domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody.
  • the two VH domains of a bivalent domain antibody may target the same or different antigens.
  • “Valent” as used herein refers to the presence of a specified number of antigen binding sites in a given molecule.
  • the term “monovalent” refers to an antibody or an antigen-binding fragment having only one single antigen-binding site; and the term “multivalent” refers to an antibody or antigen-binding fragment having multiple antigen-binding sites.
  • the terms “bivalent”, “tetravalent”, and “hexavalent” denote the presence of two antigen-binding sites, four antigen-binding sites, and six antigen-binding sites, respectively, in an antigen-binding molecule.
  • the antibody or antigen-binding fragment thereof is bivalent.
  • a “bispecific” antibody refers to an artificial antibody which has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes.
  • the two epitopes may present on the same antigen, or they may present on two different antigens.
  • Cancer refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis, and includes both solid tumors and non-solid cancers (hematologic malignancies) such as leukemia.
  • solid tumor refers to a solid mass of neoplastic and/or malignant cells.
  • cancer or tumors include hematological malignancies, oral carcinomas (for example of the lip, tongue or pharynx), digestive organs (for example esophagus, stomach, small intestine, colon, large intestine, or rectum), peritoneum, liver and biliary passages, pancreas, respiratory system such as larynx or lung (small cell and non-small cell), bone, connective tissue, skin (e.g., melanoma), breast, reproductive organs (fallopian tube, uterus, cervix, testicles, ovary, or prostate), urinary tract (e.g., bladder or kidney), brain and endocrine glands such as the thyroid.
  • oral carcinomas for example of the lip, tongue or pharynx
  • digestive organs for example esophagus, stomach, small intestine, colon, large intestine, or rectum
  • peritoneum liver and biliary passages
  • pancreas respiratory system
  • respiratory system such
  • the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, renal cancer, bladder cancer, hepatocellular cancer, and colorectal cancer. In certain embodiments, the cancer is selected from a lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and B-cell lymphoma.
  • chimeric means an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species.
  • a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human animal, such as from mouse or rabbit.
  • the nonhuman animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.
  • the term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen.
  • the antibodies or antigen-binding fragments provided herein specifically bind to human CCR4 with a binding affinity (K D ) of ⁇ 10' 6 M (e.g., ⁇ 5x1 O' 7 M, ⁇ 2x1 O' 7 M, ⁇ 10’ 7 M, ⁇ 5x1 O’ 8 M, ⁇ 2x1 O’ 8 M, ⁇ 10’ 8 M, ⁇ 5x1 O’ 9 M, ⁇ 4xlO’ 9 M, ⁇ 3X10' 9 M, ⁇ 2x1 O' 9 M, or ⁇ 10' 9 M).
  • K D binding affinity
  • KD used herein refers to the ratio of the dissociation rate to the association rate (k 0 g/k 0 n), which may be determined by using any conventional method known in the art, including but are not limited to surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method.
  • the K D value can be appropriately determined by using flow cytometry.
  • the ability to “block binding” or to “compete for the same epitope” as used herein refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human CCR4 and an anti-CCR4 antibody) to any detectable degree.
  • an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 85%, or greater than 90%.
  • a given antibody binds to the same epitope as the antibody of present disclosure by ascertaining whether the former prevents the latter from binding to a CCR4 antigen polypeptide. If the given antibody competes with the antibody of present disclosure, as shown by a decrease in binding by the antibody of present disclosure to the CCR4 antigen polypeptide, then the two antibodies bind to the same, or a closely related, epitope. Or if the binding of a given antibody to the CCR4 antigen polypeptide was inhibited by the antibody of present disclosure, then the two antibodies bind to the same, or a closely related, epitope.
  • CCR4 refers to CCR4 derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats).
  • the term “CCR4” as used herein is intended to encompass any form of human CCR4, for example, 1) native unprocessed CCR4 molecule, “full-length” CCR4 chain or naturally occurring variants of CCR4, including, for example, splice variants or allelic variants; 2) any form of CCR4 that results from processing in the cell; or 3) full length, a fragment (e.g., a truncated form, an extracellular/transmembrane domain) or a modified form (e.g. a mutated form, a glycosylated/PEGylated, a His-tag/immunofluorescence fused form) of CCR4 subunit generated through recombinant method.
  • a fragment e.g., a trunc
  • anti-CCR4 antibody refers to an antibody that is capable of specifically binding to CCR4 (e.g., human CCR4).
  • CCR4-related disease or condition refers to any disease or condition caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of CCR4.
  • the CCR4 related condition is immune-related disorder, such as, for example, cancer, autoimmune disease, inflammatory disease or infectious disease.
  • a “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties.
  • conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g., Met, Ala, Vai, Leu, and He), among residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn and Gin), among residues with acidic side chains (e.g., Asp, Glu), among amino acids with basic side chains (e.g., His, Lys, and Arg), or among residues with aromatic side chains (e.g., Trp, Tyr, and Phe).
  • conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.
  • Appector functions refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as Cl complex and Fc receptor.
  • exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and Clq on the Cl complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell; and phagocytosis.
  • epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody binds.
  • Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen. For example, if an antibody or antigen-binding fragment blocks binding of a reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding fragment may be considered to bind the same/closely related epitope as the reference antibody.
  • homologue and “homologous” as used herein are interchangeable and refer to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequence when optimally aligned.
  • host cell refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.
  • humanized as used herein means that the antibody or antigenbinding fragment comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human.
  • an “isolated” substance has been altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide is “isolated” if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state.
  • An “isolated nucleic acid sequence” refers to the sequence of an isolated nucleic acid molecule.
  • an “isolated antibody or antigen-binding fragment thereof’ refers to the antibody or antigenbinding fragments having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC).
  • electrophoretic methods such as SDS-PAGE, isoelectric focusing, capillary electrophoresis
  • chromatographic methods such as ion exchange chromatography or reverse phase HPLC.
  • leader peptide refers to a peptide having a length of about 5-30 amino acids that is present at the N-terminus of newly synthesized proteins that form part of the secretory pathway. Proteins of the secretory pathway include, but are not limited to proteins that reside either inside certain organelles (the endoplasmic reticulum, Golgi or endosomes), are secreted from the cell, or are inserted into a cellular membrane. In some embodiments, the leader peptide forms part of the transmembrane domain of a protein.
  • link refers to the association via intramolecular interaction, e.g., covalent bonds, metallic bonds, and/or ionic bonding, or inter-molecular interaction, e.g., hydrogen bond or noncovalent bonds.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given signal peptide that is operably linked to a polypeptide directs the secretion of the polypeptide from a cell.
  • a promoter that is operably linked to a coding sequence will direct the expression of the coding sequence.
  • the promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
  • Percent (%) sequence identity with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI), see also, Altschul S.F.
  • polynucleotide or “nucleic acid” includes both single- stranded and double-stranded nucleotide polymers.
  • the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2', 3 '-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • polypeptide or “protein” means a string of at least two amino acids linked to one another by peptide bonds. Polypeptides and proteins may include moieties in addition to amino acids (e.g., may be glycosylated) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “polypeptide” or “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a functional portion thereof. Those of ordinary skill will further appreciate that a polypeptide or protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally-occurring amino acid and polymers.
  • pharmaceutically acceptable indicates that the designated carrier, vehicle, diluent, excipient(s), and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
  • the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • a human includes pre- and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term “subject” is used herein interchangeably with “individual” or “patient.”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • a therapeutically effective amount refers to the dosage or concentration of a drug effective to treat a disease or condition.
  • a therapeutically effective amount is the dosage or concentration of the monoclonal antibody or antigen-binding fragment thereof capable of reducing the tumor volume, eradicating all or part of a tumor, inhibiting or slowing tumor growth or cancer cell infiltration into other organs, inhibiting growth or proliferation of cells mediating a cancerous condition, inhibiting or slowing tumor cell metastasis, ameliorating any symptom or marker associated with a tumor or cancerous condition, preventing or delaying the development of a tumor or cancerous condition, or some combination thereof.
  • Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.
  • vector refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein.
  • a vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell.
  • vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses.
  • a vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication.
  • a vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.
  • a vector can be an expression vector or a cloning vector.
  • the present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-la) operably linked to the nucleic acid sequence, and at least one selection marker.
  • promoter e.g., SV40, CMV, EF-la
  • vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40), lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, p VITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWP
  • the present disclosure in one aspect provides an anti-CCR4 antibody and antigen-binding fragment thereof that has a high binding affinity to human CCR4.
  • the antibody or antigen-binding fragment when bound to CCR4, can specifically interfere with, block or reduce the interaction between CCR4 and its ligand, thus increasing immune response.
  • Binding affinity of the antibody and antigen-binding fragment provided herein can be represented by KD value, which represents the ratio of dissociation rate to association rate (k 0 g/k 0 n) when the binding between the antigen and antigen-binding molecule reaches equilibrium.
  • the antigen-binding affinity e.g., K D
  • K D can be appropriately determined using suitable methods known in the art, including, for example, bio -layer interferometry.
  • Binding of the antibodies to human CCR4 can also be represented by “half maximal effective concentration” (EC 50) value, which refers to the concentration of an antibody where 50% of its maximal effect (e.g., binding or inhibition etc.) is observed.
  • the EC50 value can be measured by methods known in the art, for example, sandwich assay such as ELISA, Western Blot, flow cytometry assay, and other binding assays.
  • the present disclosure in one aspect provides an anti-CCR4 antibody and antigen-binding fragment thereof comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of an anti-CCR4 antibody disclosed herein.
  • CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs in anti-CCR4 antibody disclosed herein, yet substantially retain the specific binding affinity to CCR4.
  • the anti-CCR4 antibody has a CDR sequence as listed in Table 1 below.
  • the anti-CCR4 antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, as long as the antibodies and antigen-binding fragments thereof can specifically bind to CCR4.
  • FR framework region
  • the CDR sequences provided in Table 1 can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art such as recombinant techniques.
  • the antibodies and antigen-binding fragments thereof provided herein are humanized.
  • a humanized antibody or antigen-binding fragment is desirable in its reduced immunogenicity in human.
  • a humanized antibody is chimeric in its variable regions, as non-human CDR sequences are grafted to human or substantially human FR sequences.
  • Humanization of an antibody or antigen-binding fragment can be essentially performed by substituting the non-human (such as murine) CDR genes for the corresponding human CDR genes in a human immunoglobulin gene (see, for example, Jones et al., Nature (1986) 321 :522-525; Riechmann et al., Nature (1988) 332:323-327; Verhoeyen et al., Science (1988) 239:1534-1536).
  • Suitable human heavy chain and light chain variable domains can be selected to achieve this purpose using methods known in the art.
  • “best -fit” approach can be used, where a non-human (e.g., rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as the human scaffold for grafting the non-human CDR sequences (see, for example, Sims et al., J. Immunol. (1993) 151 :2296; Chothia et al., J. Mot. Biol. (1987) 196:901).
  • a framework derived from the consensus sequence of all human antibodies may be used for the grafting of the non-human CDRs (see, for example, Carter et at. Proc. Natl. Acad. Sci. USA (1992) 89:4285; Presta et al., J. Immunol. (1993) 151 :2623).
  • the humanized antibodies or antigen-binding fragments provided herein are composed of substantially all human sequences except for the CDR sequences which are non-human.
  • the variable region FRs, and constant regions if present are entirely or substantially from human immunoglobulin sequences.
  • the human FR sequences and human constant region sequences may be derived different human immunoglobulin genes, for example, FR sequences derived from one human antibody and constant region from another human antibody.
  • the anti-CCR4 antibodies and antigen-binding fragments thereof provided herein comprise paired heavy chain and light chain variable region amino acid sequences as provided in Table 2 below.
  • the anti-CCR4 antibodies and the antigen-binding fragments provided herein comprise all or a portion of the heavy chain variable domain and/or all or a portion of the light chain variable domain.
  • the anti-CCR4 antibodies and the antigen-binding fragments provided herein is a single domain antibody which consists of all or a portion of the heavy chain variable domain provided herein. More information of such a single domain antibody is available in the art (see, e.g., U. S. Pat. No. 6,248,516).
  • the anti-CCR4 antibodies and the fragments thereof provided herein further comprise an immunoglobulin constant region.
  • an immunoglobulin constant region comprises a heavy chain and/or a light chain constant region.
  • the heavy chain constant region comprises CHI, hinge, and/or CH2-CH3 regions.
  • the heavy chain constant region comprises an Fc region.
  • the light chain constant region comprises CK or C .
  • the anti-CCR4 antibody and antigen-binding fragments thereof provided herein comprise paired heavy chain and light chain constant region amino acid sequences as provided in Table 3 below.
  • the antibodies or antigen-binding fragments thereof provided herein can be a monoclonal antibody, polyclonal antibody, humanized antibody, chimeric antibody, recombinant antibody, bispecific antibody, labeled antibody, bivalent antibody, or anti- idiotypic antibody.
  • a recombinant antibody is an antibody prepared in vitro using recombinant methods rather than in animals.
  • the antibodies and antigen-binding fragments thereof provided herein also encompass various variants thereof.
  • the antibodies and antigenbinding fragments thereof encompasses various types of variants of an exemplary antibody provided herein.
  • the antibody variants comprise one or more modifications or substitutions in one or more CDR sequences as provided in Table 1, one or more variable region sequences (but not in any of the CDR sequences) provided herein, and/or the constant region (e.g., Fc region).
  • Such variants retain specific binding affinity to CCR4 of their parent antibodies, but have one or more desirable properties conferred by the modification(s) or substitution(s).
  • the antibody variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamidation or deamination, improved or increased effector function(s), reduced or depleted effector function(s), improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility to conjugation (e.g., one or more introduced cysteine residues).
  • the parent antibody sequence may be screened to identify suitable or preferred residues to be modified or substituted, using methods known in the art, for example “alanine scanning mutagenesis” (see, for example, Cunningham and Wells (1989) Science, 244: 1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine), and the modified antibodies are produced and screened for the interested property. If substitution at a particular amino acid location demonstrates an interested functional change, then the position can be identified as a potential residue for modification or substitution. The potential residues may be further assessed by substituting with a different type of residue (e.g. cysteine residue, positively charged residue, etc.).
  • alanine scanning mutagenesis see, for example, Cunningham and Wells (1989) Science, 244: 1081-1085.
  • target residues e.g.
  • Affinity variant may contain modifications or substitutions in one or more CDR sequences, one or more FR sequences, or the heavy or light chain variable region sequences provided herein.
  • the affinity variants retain specific binding affinity to CCR4 of the parent antibody, or even have improved CCR4 specific binding affinity over the parent antibody.
  • a library of antibody variants (such as Fab or scFv variants) can be generated and expressed with phage display technology, and then screened for the binding affinity to human CCR4.
  • computer software can be used to virtually simulate the binding of the antibodies to human CCR4 and identify the amino acid residues on the antibodies which form the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity or targeted for substitution to provide for a stronger binding.
  • the humanized antibody or antigen-binding fragment provided herein comprises one or more amino acid residue substitutions in one or more CDR sequences, and/or one or more FR sequences.
  • an affinity variant comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in the CDR sequences and/or FR sequences in total.
  • the anti-CCR4 antibodies and antigen-binding fragments thereof comprise 1, 2, or 3 CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 1, and in the meantime retain the binding affinity to CCR4 at a level similar to or even higher than its parent antibody.
  • the anti-CCR4 antibodies and antigen-binding fragments thereof comprise one or more variable region sequences having at least 80% (e.g. , at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) provided herein, and in the meantime retain the binding affinity to CCR4 at a level similar to or even higher than its parent antibody.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs).
  • the antibody comprises a particular glycosylation pattern.
  • an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation).
  • the glycosylation pattern of an antibody may be altered to, for example, increase the affinity or avidity of the antibody for an antigen.
  • modifications can be accomplished by, for example, altering one or more of the glycosylation sites within the antibody sequence.
  • one or more amino acid substitutions can be made that result removal of one or more of the variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity or avidity of the antibody for antigen. See, e.g., U.S. Patents 5,714,350 and 6,350,861.
  • an antibody may also be made in which the glycosylation pattern includes hypofucosylated or afucosylated glycans, such as a hypofucosylated antibodies or afucosylated antibodies have reduced amounts of fucosyl residues on the glycan.
  • the antibodies may also include glycans having an increased amount of bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such modifications can be accomplished by, for example, expressing the antibodies in a host cell in which the glycosylation pathway was been genetically engineered to produce glycoproteins with particular glycosylation patterns.
  • the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (a (l,6)-fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates.
  • FUT8 a (l,6)-fucosyltransferase
  • the Ms704, Ms705, and Ms709 FUT8-/- cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704.
  • EP 1 176 195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the a-1,6 bond-related enzyme.
  • EP 1 176 195 also describes cell lines which have a low enzyme activity for adding fucose to the N- acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662).
  • PCT Publication WO 2003/035835 describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell.
  • Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication WO 06/089231.
  • antibodies with a modified glycosylation profile can be produced in plant cells, such as Lemna (US Patent 7,632,983). Methods for production of antibodies in a plant system are disclosed in the U.S. Patents 6,998,267 and 7,388,081.
  • PCT Publication WO 1999/054342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., P(l,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies. Hypofucosylation is also called afiicosylation when fiicosylation is minimal on antibodies.
  • glycoprotein-modifying glycosyl transferases e.g., P(l,4)-N-acetylglucosaminyltransferase III (GnTIII)
  • GnTIII glycoprotein-modifying glycosyl transferases
  • Hypofucosylation is also called afiicosylation when fiicosylation is minimal on antibodies.
  • the fucose residues of the antibodies can be cleaved off using a fucosidase enzyme; e.g., the fiicosidase a-L-fucosidase removes fiicosyl residues from antibodies.
  • a fucosidase enzyme e.g., the fiicosidase a-L-fucosidase removes fiicosyl residues from antibodies.
  • Antibodies disclosed herein further include those produced in lower eukaryote host cells, in particular fungal host cells such as yeast and filamentous fungi have been genetically engineered to produce glycoproteins that have mammalian- or human-like glycosylation patterns.
  • a particular advantage of these genetically modified host cells over currently used mammalian cell lines is the ability to control the glycosylation profile of glycoproteins that are produced in the cells such that compositions of glycoproteins can be produced wherein a particular N-glycan structure predominates (see, e.g., U.S. Patents 7,029,872 and 7,449,308).
  • These genetically modified host cells have been used to produce antibodies that have predominantly particular N-glycan structures.
  • fungi such as yeast or filamentous fungi lack the ability to produce fiicosylated glycoproteins
  • antibodies produced in such cells will lack fucose unless the cells are further modified to include the enzymatic pathway for producing fiicosylated glycoproteins (See for example, PCT Publication W02008112092).
  • the antibodies disclosed herein further include those produced in lower eukaryotic host cells and which comprise fiicosylated and nonfiicosylated hybrid and complex N-glycans, including bisected and multiantennary species, including but not limited to N-glycans such as GlcNAc(l-4)Man3GlcNAc2; Gal(l-4)GlcNAc(l-4)Man3GlcNAc2; NANA(l-4)Gal(l-4)GlcNAc(l-4)Man3GlcNAc2.
  • N-glycans such as GlcNAc(l-4)Man3GlcNAc2; Gal(l-4)GlcNAc(l-4)Man3GlcNAc2; NANA(l-4)Gal(l-4)GlcNAc(l-4)Man3GlcNAc2.
  • the antibody compositions provided herein may comprise antibodies having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2; GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2.
  • the hybrid N-glycan is the predominant N-glycan species in the composition.
  • the hybrid N-glycan is a particular N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the hybrid N-glycans in the composition.
  • the antibody compositions provided herein comprise antibodies having at least one complex N-glycan selected from the group consisting of GlcNAcMan3GlcNAc2; GalGlcNAcMan3GlcNAc2; NANAGalGlcNAcMan3GlcNAc2; GlcNAc2Man3GlcNAc2; GalGlcNAc2Man3GlcNAc2; Gal2GlcNAc2Man3GlcNAc2; NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2.
  • N-glycan selected from the group consisting of GlcNAcMan3GlcNAc2; GalGlcNAcMan3GlcNAc2; NANAGalGlcNAcMan3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2.
  • the complex N-glycan is the predominant N-glycan species in the composition.
  • the complex N-glycan is a particular N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycans in the composition.
  • the N-glycan is fusosylated.
  • the fucose is in an al, 3 -linkage with the GlcNAc at the reducing end of the N-glycan, an al,6-linkage with the GlcNAc at the reducing end of the N-glycan, an al,2-linkage with the Gal at the non-reducing end of the N-glycan, an al, 3 -linkage with the GlcNac at the non-reducing end of the N-glycan, or an al,4-linkage with a GlcNAc at the non-reducing end of the N-glycan.
  • the glycoform is in an al,3-linkage or al,6-linkage fucose to produce a glycoform selected from the group consisting of Man5GlcNAc2(Fuc), GlcNAcMan5GlcNAc2(Fuc), Man3GlcNAc2(Fuc), GlcNAcMan3 GlcNAc2(Fuc), GlcNAc2Man3 GlcNAc2(Fuc),
  • the antibodies comprise high mannose N-glycans, including but not limited to, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or N-glycans that consist of the Man3GlcNAc2 N-glycan structure.
  • the complex N-glycans further include fucosylated and non- fucosylated (or afucosylated) bisected and multiantennary species.
  • N-glycan and “glycoform” are used interchangeably and refer to an N-linked oligosaccharide, for example, one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide.
  • N-linked glycoproteins contain an N- acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein.
  • the anti-CCR4 antibodies and antigen-binding fragments provided herein also encompass a glycosylation variant, which can be obtained to either increase or decrease the extent of glycosylation of the antibody or antigen binding fragment.
  • the antibody or antigen binding fragment thereof may comprise one or more amino acid residues with a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached.
  • Glycosylation of antibodies is typically either N- linked or O-linked.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline.
  • O-linked glycosylation refers to the attachment of one of the sugars N- aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence in the is substituted. A new glycosylation site can be created in a similar way by introducing such a tripeptide sequence or serine or threonine residue.
  • the anti-CCR4 antibodies and antigen-binding fragments provided herein also encompass a cysteine-engineered variant, which comprises one or more introduced free cysteine amino acid residues.
  • a free cysteine residue is one which is not part of a disulfide bridge.
  • a cysteine-engineered variant is useful for conjugation with for example, a cytotoxic and/or imaging compound, a label, or a radioisoptype among others, at the site of the engineered cysteine, through for example a maleimide or haloacetyl.
  • Methods for engineering antibodies or antigen-binding fragments to introduce free cysteine residues are known in the art, see, for example, W02006/034488.
  • the antibodies disclosed herein can also be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or effector function (e.g., antigen-dependent cellular cytotoxicity).
  • the antibodies disclosed herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • the numbering of residues in the Fc region is that of the EU index of Kabat.
  • the antibodies disclosed herein also include antibodies with modified (or blocked) Fc regions to provide altered effector functions.
  • Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent dosing and thus increased convenience and decreased use of material. This mutation has been reported to abolish the heterogeneity of inter-heavy chain disulfide bridges in the hinge region.
  • the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is increased or decreased.
  • the number of cysteine residues in the hinge region of CHI is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the antibody is modified to increase its biological half-life.
  • one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent 6,277,375.
  • the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patents 5,869,046 and 6,121,022.
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector fimction(s) of the antibodies.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Patents 5,624,821 and 5,648,260.
  • one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 1994/029351.
  • the Fc region is modified to increase or decrease the ability of the antibodies to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase or decrease the affinity of the antibodies for an Fey receptor by modifying one or more amino acids at the following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338,
  • the Fc region is modified to decrease the ability of the antibodies to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243 and 264.
  • the Fc region of the antibody is modified by changing the residues at positions 243 and 264 to alanine.
  • the Fc region is modified to decrease the ability of the antibody to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.
  • the Fc region is modified to abolish the ability of the antibodies to mediate effector function by modifying residues 234, 235 and 329 to alanine or glycine (L234A-L235A-P329G).
  • the anti-CCR4 antibodies and antigen-binding fragments provided herein also encompass an Fc variant, which comprises one or more amino acid residue modifications or substitutions at its Fc region and/or hinge region.
  • the anti-CCR4 antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution(s) that improves pH-dependent binding to neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell.
  • Methods of engineering an antibody and antigen-binding fragment thereof to improve binding affinity with FcRn are well-known in the art, see, for example, Vaughn, D. et al., Structure, 6(1): 63- 73, 1998; Kontermann, R.
  • the anti-CCR4 antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution(s) that alters the antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • Certain amino acid residues at CH2 domain of the Fc region can be substituted to provide for enhanced ADCC activity.
  • carbohydrate structures on the antibody can be changed to enhance ADCC activity.
  • the anti-CCR4 antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution(s) that alters Complement Dependent Cytotoxicity (CDC), for example, by improving or diminishing Clq binding and/or CDC (see, for example, WO99/51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821); and WO 1994/029351 concerning other examples of Fe region variants.
  • CDC Complement Dependent Cytotoxicity
  • the anti-CCR4 antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution(s) in the interface of the Fc region to facilitate and/or promote heterodimerization.
  • These modifications comprise introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance can be positioned in the cavity so as to promote interaction of the first and second Fc polypeptides to form a heterodimer or a complex.
  • Methods of generating antibodies with these modifications are known in the art, e.g., as described in U.S. Pat. No. 5,731, 168.
  • anti-CCR4 antigen-binding fragments are also provided herein.
  • Various types of antigen-binding fragments are known in the art and can be developed based on the anti- CCR4 antibodies provided herein, including for example, the exemplary antibodies whose CDR and variable sequences are provided herein, and their different variants (such as affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants and so on).
  • an anti-CCR4 antigen-binding fragment is a camelized single domain antibody, a diabody, a single chain Fv fragment (scFv), an scFv dimer, a BsFv, a dsFv, a (dsFv)2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F(ab')2, a bispecific antibody, a ds diabody, a nanobody, a domain antibody, a single domain antibody, or a bivalent domain antibody.
  • scFv single chain Fv fragment
  • scFv dimer a BsFv, a dsFv, a (dsFv)2, a dsFv-dsFv'
  • Fv fragment a Fab, a Fab', a F(ab')2, a bispecific antibody, a ds diabody, a nanobody
  • a Single Chain Variable Fragment is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker.
  • This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered.
  • These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide.
  • scFv can be created directly from subcloned heavy and light chains derived from a hybridoma.
  • Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.
  • Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alanine, serine and glycine. However, other residues can function as well.
  • Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries.
  • a random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition.
  • the scFv repertoire (approx. 5 * 10 6 different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity.
  • the recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors.
  • sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain.
  • Another multimerization domain is the Gal4 dimerization domain.
  • the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.
  • a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit.
  • the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge).
  • Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stablizing and coagulating agent.
  • a stablizing and coagulating agent e.g., a stablizing and coagulating agent.
  • dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created.
  • hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.
  • An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.).
  • primary amine group e.g., N-hydroxy succinimide
  • a thiol group e.g., pyridyl disulfide, maleimides, halogens, etc.
  • the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).
  • cross-linker having reasonable stability in blood will be employed.
  • Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents.
  • Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.
  • SMPT cross-linking reagent
  • Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is “sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.
  • thiolate anions such as glutathione which can be present in tissues and blood
  • the SMPT cross-linking reagent lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine).
  • Another possible type of crosslinker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl- 1,3 '-dithiopropionate.
  • the N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.
  • non-hindered linkers In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith.
  • Other useful cross-linkers include SATA, SPDP and 2 -iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.
  • U.S. Patent 4,680,338 describes bifrmctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like.
  • U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.
  • U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies.
  • the linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation.
  • U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques.
  • Various techniques can be used for the production of such antigen-binding fragments.
  • Illustrative methods include, enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods (1992) 24: 107-117; and Brennan et al., Science (1985) 229:81), recombinant expression by host cells such as E. Coli (e.g. for Fab, Fv and ScFv antibody fragments), screening from a phase display library as discussed above (e.g.
  • the antigen-binding fragment is a scFv.
  • Generation of scFv is described in, for example, WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458.
  • scFv may be fused to an effector protein at either the amino or the carboxyl terminus to provide for a fusion protein (see, for example, Antibody Engineering, ed. Borrebaeck).
  • the anti-CCR4 antibodies and antigen-binding fragments thereof provided herein are multi-specific.
  • the term “multi-specific” as used herein encompasses molecules having more than one specificity, e.g., bispecific, tri-specific, tetra-specific.
  • the multi-specific antibodies and antigen-binding fragments thereof provided herein are capable of specifically binding to a first and a second epitopes of CCR4, while the first epitope and the second epitopes of CCR4 are distinct from each other or non-overlapping.
  • the multi -specific antibodies and antigen-binding fragments thereof provided herein is capable of specifically binding to CCR4 and a second antigen different from CCR4.
  • the second antigen is an immune related target.
  • An immune related target as used herein encompasses a biological molecule that is involved in the stimulation, inhibition or modulation of an immune response, optionally, cellular immune responses.
  • An example of the immune related target is an immune modulator molecule that is expressed by a cancer cell, a stromal cell (fibroblast, vascular cell, etc.) or an immune cell.
  • the immune modulator molecule can mediate co-stimulatory signal to augment immune response or can mediate co-inhibitory signals to suppress immune response. Therefore, in some embodiment, the second antigen is an immune modulator molecule.
  • the immune modulator molecule is PD-1, PD-L1, PD- L2, CTLA-4, TIM-3, Fc receptors, FCRL(l-6), A2AR, CD160, 2B4, TGF-P, TGF-PR, VISTA, BTLA, TIGIT, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA(l-6), 0X40, CD2, CD27, CD28, CD30, CD40, CD47, SIRPA, CLEC-L clever- l/stabilin-1, ADGRE, TREM1, TREM2, CD122, ICAM-1, IDO, NKG2D/C, SLAMF7, MS4A4A, SIGLEC(7-15), NKp80, NKG2A, CD160, CD161, CD300, CD163, B7-H3, LFA-1, ICOS, 4- 1BB, GITR, BAFFR, HVEM, CD7, LIGHT, TNFR2,
  • the second antigen comprises a tumor antigen.
  • Tumor antigen refers to tumor specific antigens (e.g., those unique to tumor cells and normally not found on non-tumor cells), and tumor-associated antigens (e.g., found in both tumor and non-tumor cells but expressed differently in tumor cells, or found in tumor microenvironment). Tumor specific antigens can also include tumor neo-antigens (e.g., that are expressed in cancer cells because of somatic mutations that change the protein sequence or create fusion proteins between two unrelated sequences).
  • tumor antigens include, without limitation, prostate specific antigen (PSA), CA-125, gangliosides G(D2), G(M2) and G(D3), CD20, CD52, CD33, Ep- CAM, CEA, bombesin-like peptides, HER2/neu, epidermal growth factor receptor (EGFR), erbB2, erbB3/HER3, erbB4, FGFR2b, CD44v6, cancer-associated mucin, VEGF, VEGFRs (e.g., VEGFR3), estrogen receptors, Lewis-Y antigen, TGFpi, IGF-1 receptor, EGFa, c-Kit receptor, transferrin receptor, Claudin 18.2, GPC-3, Nectin-4, ROR1, methothelin, BCMA, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, BCR-ABL, E2APRL, H4-RET, IGH- IGK, MYL-R
  • Multi-specific antibodies and antigen-binding fragments thereof provided herein can be in a suitable format known in the art.
  • an exemplary bispecific format can be bispecific diabodies, scFv-based bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), BiTE, CrossMab, CrossFab, Duobody, SEEDbody, leucine zipper, dual acting Fab (DAF)-IgG, and Mab 2 bispecific formats (see, e.g., Brinkmann et al. 2017, Mabs, 9(2): 182-212).
  • the bispecific molecules can be in symmetric or asymmetric architecture.
  • the multi-specific antibodies and antigen-binding fragments provided herein can be made with any suitable methods known in the art.
  • two immunoglobulin heavy chain-light chain pairs having different antigenic specificities are coexpressed in a host cell to produce bispecific antibodies in a recombinant way (see, for example, Milstein and Cuello, Nature, 305: 537 (1983)), followed by purification by affinity chromatography.
  • the anti-CCR4 antibodies and antigen-binding fragments thereof further comprise a conjugate moiety.
  • the conjugate moiety can be linked to the antibodies and antigen-binding fragments thereof.
  • a conjugate moiety is a proteinaceous or non-proteinaceous moiety that can be attached to the antibody or antigenbinding fragment thereof. It is contemplated that a variety of conjugate moieties may be linked to the antibodies or antigen-binding fragments provided herein (see, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties may be linked to the antibodies or antigen-binding fragments by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.
  • the antibodies and antigen -binding fragments disclosed herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugate moieties.
  • a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate moiety.
  • the antibodies may be linked to a conjugate moiety indirectly, or through another conjugate moiety.
  • the antibody or antigen-binding fragments may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin.
  • conjugate moiety examples include without a limitation an immune- modulatory agent, an anti-tumor drug, a STING (Stimulator of Interferon Genes) agonist, a cytokine, a clearance-modifying agent, a toxin (e.g., a chemotherapeutic agent), an immune cell stimulator (e.g., a TLR agonist), a detectable label (e.g., a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label), a DNA, an RNA, or purification moiety.
  • an immune- modulatory agent an anti-tumor drug
  • STING Stimulator of Interferon Genes
  • cytokine e.g., a chemotherapeutic agent
  • an immune cell stimulator e.g., a TLR agonist
  • detectable label e.g., a radioactive isotope, a lanthanide,
  • immune modulatory agent examples include without limitation an immune modulator molecule disclosed herein (e.g., PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, Fc receptors, FCRL(l-6), A2AR, CD160, 2B4, TGF-P, TGF-PR, VISTA, BTLA, TIGIT, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA(l-6), 0X40, CD2, CD27, CD28, CD30, CD40, CD47, SIRPA, CLEC-1, clever- 1/stabilin-l, ADGRE, TREM1, TREM2, CD 122, ICAM-1, IDO, NKG2D/C, SLAMF7, MS4A4A, SIGLEC(7-15), NKp80, NKG2A, CD160, CD161, CD300, CD163, B7-H3, LFA-1, ICOS, 4-1BB, GITR, BAFFR,
  • the immune modulatory agent linked to the antibodies and antigen-binding fragments disclosed herein is a ligand-binding protein (e.g., ligand trapper) specific to an immune modulatory receptor.
  • a ligand-binding protein e.g., ligand trapper
  • anti-tumor drugs include without limitation a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an agent used in radiation therapy, an anti-angiogenesis agent, a cancer immunotherapeutic agent, a apoptotic agent, an anti-tubulin agent, an anti-HER-2 antibody, an anti-CD20 antibody, an epidermal growth factor receptor (EGFR) antagonist, HER1/EGFR inhibitor, a platelet derived growth factor inhibitor, a COX- 2 inhibitor, an interferon, a CTLA4 inhibitor (e.g., anti-CTLA antibody ipilimumab (YERVOY®), or tremelimumab), a PD-1 or PD-L1 inhibitor (e.g., OPDIVO® or nivolumab, KEYTRUDA® or pembrolizumab, TECENTRIQ® or atezolizumab, BAVENCIO® or avelumab, IMFINZI
  • a “toxin” can be any agent that is detrimental to cells or that can damage or kill cells.
  • toxin include, without limitation, taxol, cytochalasin B, deruxtecan, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), mertansine, emtansine, DM1, maytansinoid DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 - dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate), e
  • detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme -substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or P-D-galactosidase), radioisotopes (e.g.
  • the conjugate moiety can be a clearance-modifying agent which helps increase half-life of the antibody.
  • Illustrative examples include water- soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymers are attached, they can be the same or different molecules.
  • the conjugate moiety can be a purification moiety such as a magnetic bead.
  • the antibodies and antigen-binding fragments thereof provided herein is used for a base for a conjugate.
  • the present disclosure provides isolated polynucleotides that encode the anti- CCR4 antibodies and antigen-binding fragments thereof disclosed herein.
  • the isolated polynucleotides comprise one or more nucleotide sequences that encodes the variable region of the exemplary antibodies listed in Table 2.
  • the isolated polynucleotides comprise one or more nucleotide sequences that encodes the constant region of the exemplary antibodies listed in Table 3.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). The encoding DNA may also be obtained by synthetic methods.
  • the isolated polynucleotide that encodes the anti-CCR4 antibodies and antigen-binding fragments can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art.
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-la), and a transcription termination sequence.
  • the present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the antibodies or antigen-binding fragments, at least one promoter (e.g., SV40, CMV, EF-la) operably linked to the nucleic acid sequence, and at least one selection marker.
  • promoter e.g., SV40, CMV, EF-la
  • vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40), lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWP
  • Vectors comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment can be introduced to a host cell for cloning or gene expression.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E.
  • coli Enterobacter , Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces .
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-CCR4 antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • Schizosaccharomyces pombe ' Kluyveromyces hosts such as, e.g., K lactis, K fragilis (ATCC 12,424), K bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.
  • waltii ATCC 56,500
  • K. drosophilarum ATCC 36,906
  • K. thermotolerans K marxianus
  • Pichia pastoris EP 183,070
  • Candida Trichoderma reesia
  • Neurospora crassa ' Schwanniomyces such as Schwanniomyces occidentalism
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated antibodies or antigenfragment provided here are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • mice sertoli cells TM4, Mather, Biol. Reprod. (1980) 23:243-251; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. (1982) 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • the host cell is 293F cell.
  • Host cells are transformed with the above -described expression or cloning vectors for anti-CCR4 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the antibody may be produced by homologous recombination known in the art.
  • the host cells used to produce the antibodies or antigen-binding fragments provided herein may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing the host cells.
  • MEM Minimal Essential Medium
  • RPMI-1640 Sigma
  • DMEM Dulbecco's Modified Eagle's Medium
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology (1992) 10: 163-167 describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the anti-CCR4 antibodies and antigen-binding fragments thereof prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody and antigen-binding fragment thereof.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human gamma 1, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. (1983) 62: 1-13). Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. (1986)5: 1567-75).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the Bakerbond ABXTM resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
  • Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).
  • the antibodies of the present disclosure may be purified.
  • purified is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally - obtainable state.
  • a purified protein therefore also refers to a protein, free from the environment in which it may naturally occur.
  • substantially purified is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing.
  • protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.
  • polypeptide in purifying an antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions.
  • the polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide.
  • affinity column which binds to a tagged portion of the polypeptide.
  • antibodies are fractionated utilizing agents (z.e., protein A) that bind the Fc portion of the antibody.
  • agents z.e., protein A
  • antigens may be used to simultaneously purify and select appropriate antibodies.
  • Such methods often utilize the selection agent bound to a support, such as a column, filter or bead.
  • the antibodies are bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc. .
  • compositions comprising the anti-CCR4 antibodies or antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.
  • Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
  • Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins.
  • Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate.
  • compositions that comprise one or more antibodies or antigenbinding fragments as disclosed herein and one or more antioxidants such as methionine. Further provided are methods for preventing oxidation of, extending the shelf-life of, and/or improving the efficacy of an antibody or antigen-binding fragment as provided herein by mixing the antibody or antigen-binding fragment with one or more antioxidants such as methionine.
  • pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, com oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or
  • Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p -hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
  • compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the pharmaceutical compositions are formulated into an injectable composition.
  • the injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion.
  • Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
  • a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents.
  • the solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH.
  • the resulting solution will be apportioned into vials for lyophilization.
  • Each vial can contain a single dosage or multiple dosages of the anti- CCR4 antibody or antigen-binding fragment thereof or composition thereof.
  • Overfilling vials with a small amount above that needed for a dose or set of doses e.g., about 10% is acceptable so as to facilitate accurate sample withdrawal and accurate dosing.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
  • Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
  • the pharmaceutical compositions comprising the anti- CCR4 antibodies or antigen-binding fragments thereof described herein further comprise one or more additional therapeutic agents that are co-administered with the anti-CCR4 antibodies or antigen-binding fragments thereof.
  • the candidates of the additional therapeutic agents are disclosed infra in Section IV. It can be understood that the additional therapeutic agents can be co-formulated with the anti-CCR4 antibodies or antigen-binding fragments thereof, or be mixed with the anti-CCR4 antibodies or antigen-binding fragments thereof right before the administration, such as in the IV infusion bag.
  • the present disclosure also provides therapeutic methods comprising: administering a therapeutically effective amount of the antibody or antigen-binding fragment as provided herein to a subject in need thereof, thereby treating or preventing a CCR4-related condition or a disorder.
  • the CCR4-related condition or a disorder is cancer, autoimmune disease, inflammatory disease or infectious disease.
  • Solid tumors include but are not limited to, non-small cell lung cancer (squamous/non-squamous), small cell lung cancer, renal cell cancer, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, multiple myeloma, mycoses fungoides, Merkel cell cancer, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma/
  • Solid tumors are characterized by multiple biologic hallmarks including sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, tumor promoting inflammation, avoiding immune destruction, genomic instability and mutation, and deregulating cellular energetics.
  • Treatment efforts have evolved from cytotoxic chemotherapies targeting rapidly dividing cells to small molecules inhibiting select signaling pathways to monoclonal antibodies targeting surface proteins. More recently the concept of cancer immunotherapy to reinvigorate endogenous immunity or cellular therapies utilizing synthetic immunity have shown promise.
  • immune checkpoint inhibitors such as anti-CTLA-4 or anti-PD-l/PD-Ll have led to long-term progression-free and overall survival in a minority of patients.
  • Hematologic malignancies include but are not limited to acute lymphocytic/lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic lymphoblastic leukemia (CLL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), classical Hodgkin lymphoma (CHL), diffuse large B-cell lymphoma (DLBCL), extranodal NK/T-cell lymphoma, hairy cell leukemia, heavy chain disease, HHV8 -associated primary effusion lymphoma, lymphoid malignancy, multiple myeloma (MM), myelodysplasia, myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma, plasmablastic lymphoma, pre
  • Autoimmune or inflammatory diseases include, but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos' disease, dermatomyositisjuvenile, discoid lupus, essential mixed cryoglobulinemia, fibro
  • Inflammatory disorders include, for example, chronic and acute inflammatory disorders.
  • Infectious disease include, but are not limited to, fungus infection, parasite/protozoan infection or chronic viral infection, for example, malaria, coccidioiodmycosis immitis, histoplasmosis, onychomycosis, aspergillosis, blastomycosis, candidiasis albicans, paracoccidiodomycosis, microsporidiosis, Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardia
  • an antibody or antigen -binding fragment as provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.
  • the antibody or antigen -binding fragment as provided herein may be administered at a therapeutically effective dosage of about 0.0001 mg/kg to about 100 mg/kg.
  • the antibody or antigen-binding fragment is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 3 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less.
  • the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.
  • the antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
  • parenteral e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection
  • non-parenteral e.g., oral, intranasal, intraocular, sublingual, rectal, or topical routes.
  • the antibodies or antigen-binding fragments disclosed herein may be administered alone or in combination with one or more additional therapeutic means or agents.
  • the antibodies or antigen-binding fragments disclosed herein may be administered in combination with another therapeutic agent, for example, a chemotherapeutic agent or an anti-cancer drug.
  • an antibody or antigen-binding fragment as disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments the antibody or antigen-binding fragment and the additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition.
  • an antibody or antigen-binding fragment administered “in combination” with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent.
  • An antibody or antigen-binding fragment administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment and second agent are administered via different routes.
  • additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Prescriber’s Digital Reference (available online only at pdr.net) or protocols well known in the art.
  • the agent for combination therapy is an anti- neoplastic composition.
  • an “anti-neoplastic composition” refers to a composition useful in treating cancer comprising at least one active therapeutic agent.
  • therapeutic agents include, but are not limited to, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, cancer immunotherapeutic agents, apoptotic agents, anti-tubulin agents, and other- agents to treat cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (Tarceva®), platelet derived growth factor inhibitors (e.g., Gleevec® (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), inter
  • EGFR epidermal growth factor receptor
  • the agent for combination therapy is a chemotherapeutic agent.
  • a “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents that can be administered in methods herein include, but are not limited to, alkylating agents such as thiotepa and Cytoxan® cyclo sphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan);
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin®doxorubicin (including morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, e
  • chemotherapeutic agents that can be administered in methods herein include anti-hormonal agents that act to regulate or inhibit hormone action on cancers such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex® tamoxifen), raloxifene, droloxifene, 4 -hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Fareston® toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, Megase® megestrol acetate, Aromasin® exemestane, formestanie, fadrozole, Rivisor® vorozole, Femara® letrozole, and Arimidex® anastrozo
  • SERMs selective estrogen receptor modul
  • the agent for combination therapy is an antiangiogenesis agent.
  • an “anti-angiogenesis agent” refers to a small molecular weight substance, a polynucleotide (including, e.g., an inhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly.
  • RNAi or siRNA inhibitory RNA
  • the anti-angiogenesis agent includes those agents that bind and block the angiogenic activity of the angiogenic factor or its receptor.
  • an anti-angiogenesis agent that can be administered in methods herein can include an antibody or other antagonist to an angiogenic agent, e.g., antibodies to VEGF-A (e.g., bevacizumab (Avastin®)) or to the VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec® (Imatinib Mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, Sutent®/SUl 1248 (sunitinib malate), AMG706, or those described in, e.g., international patent application WO 2004/113304).
  • an antibody or other antagonist to an angiogenic agent e.g., antibodies to VEGF-A (e.g., bevacizumab (Avastin®)) or to the VEGF-A receptor (e.g., KDR receptor or Flt-1
  • Anti-angiogenesis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D’Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179; Ferrara & Alitalo (1999) Nature Medicine 5(12): 1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556; and Sato (2003) Int. J. Clin. Oncol. 8:200-206.
  • native angiogenesis inhibitors e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D’Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179; Ferrara & Alitalo (1999) Nature Medicine 5(12)
  • the agent for combination therapy is a growth inhibitory agent.
  • a “growth inhibitory agent” as used herein refers to a compound or composition that inhibits growth of a cell (such as a cell expressing VEGF) either in vitro or in vivo.
  • the growth inhibitory agent that can be administered in methods herein may be one that significantly reduces the percentage of cells (such as a cell expressing VEGF) in S phase.
  • growth inhibitory agents include, but are not limited to, agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5- fluorouracil, and ara-C.
  • Taxanes are anticancer drugs both derived from the yew tree.
  • Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
  • the dose of the agent for the combination therapy can be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular agent. Typically, the attending physician will decide the dosage of the agent for the combination therapy with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, the agent be administered, route of administration, and the severity of the condition being treated.
  • the dose for the combination therapy can be about 0.0001 to about 1 g/kg body weight of the subject being treated/day, from about 0.0001 to about 0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kg bodyweight/day. Dosage units may be also expressed in mg/m 2 , which refer to the quantity in milligrams per square meter of body surface area.
  • Each therapeutic agent in the combination therapy described herein may be administered simultaneously (e.g., in the same medicament or at the same time), concurrently (i.e., in separate medicaments administered one right after the other in any order or sequentially in any order.
  • Sequential administration may be useful when the therapeutic agents in the combination therapy are in different dosage forms (one agent is a tablet or capsule and another agent is a sterile liquid) and/or are administered on different dosing schedules, e.g., a chemotherapeutic that is administered at least daily and a biotherapeutic that is administered less frequently, such as once weekly, once every two weeks, or once every three weeks.
  • the anti-CCR4 antibody of the present disclosure and the second drug are combined or co-formulated in a single dosage form.
  • the anti-CCR4 antibody of the present disclosure and the second drug are administered separately.
  • anti-cancer activity may also be achieved by subsequent administration of one compound in isolation (for example, the anti-CCR4 antibody following initial combination treatment, or alternatively, the second drug following initial combination treatment).
  • the anti-CCR4 antibody is administered before administration of the second drug, while in other embodiments, the anti-CCR4 antibody is administered after administration of the second drug.
  • At least one of the therapeutic agents in the combination therapy is administered using the same dosage regimen (dose, frequency and duration of treatment) that is typically employed when the agent is used as monotherapy for treating the same cancer.
  • the patient receives a lower total amount of at least one of the therapeutic agents in the combination therapy than when the agent is used as monotherapy, e.g., smaller doses, less frequent doses, and/or shorter treatment duration.
  • the combination therapy of the invention may be used prior to or following surgery to remove a tumor and may be used prior to, during or after radiation therapy.
  • the combination therapy of the invention may be used to treat a tumor that is large enough to be found by palpation or by imaging techniques well known in the art, such as MRI, ultrasound, or CAT scan.
  • the combination therapy of the invention is used to treat an advanced stage tumor having dimensions of at least about 200 mm 3 , 300 mm 3 , 400 mm 3 , 500 mm 3 , 750 mm 3 , or up to 1000 mm 3 .
  • the present disclosure further provides methods of using the anti-CCR4 antibodies or antigen-binding fragments thereof to detect presence or amount of CCR4 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof, and determining the presence or the amount of CCR4 in the sample.
  • the method of detecting CCR4 using an anti-CCR4 antibody includes, without limitation, ELISA, Westernblot, flow cytometry and FACS.
  • the present disclosure provides methods of diagnosing a CCR4 related disease or condition in a subject, comprising: a) contacting a sample obtained from the subject with the antibody or antigen-binding fragment thereof provided herein; b) determining presence or amount of CCR4 in the sample; and c) correlating the existence of the CCR4 to the CCR4 related disease or condition in the subject.
  • kits comprising the antibody or antigen-binding fragment thereof provided herein, optionally conjugated with a detectable moiety.
  • the kits may be useful in detection of CCR4 or diagnosis of CCR4 related disease.
  • the present disclosure also provides use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CCR4 related disease or condition in a subject, in the manufacture of a diagnostic reagent for diagnosing a CCR4 related disease or condition.
  • N-terminal peptide of CCR4 (SEQ ID NO:1) was synthesized and conjugated with keyhole limpet hemocyanin (hCCR4pep-KLH).
  • DNAs encoding the N-terminal peptide of CCR4 fused with mouse Fc (hCCR4pep-mFc, SEQ ID NO:2), and human Fc tag (hCCR4pep-hFc, SEQ ID NO:3) were obtained by PCR and subcloned into expression vector pcDNA3.1 (Invitrogen Cat. No:V- 790), respectively.
  • hCCR4pep-mFc and hCCR4pep-hFc were purified with Protein A column (GE healthcare).
  • mice were immunized subcutaneously every 2 weeks for 6 weeks with hCCR4pep-KLH (lOOpg/mouse) emulsified with an equal volume of Freund's complete/incomplete adjuvant.
  • mice were boosted by intravenous injection of the antigen without adjuvant.
  • Spleen cells (1 x 108) from immunized mouse were fused with SP2/0 myeloma cells (1.5x 107) with PEG Hybri-Max (Sigma Inc., Cat. No:7181). After fusion, the cells were distributed into 96- well plates at 0.1 ml per well and incubated at 37°C, 5% CO2 incubator.
  • This example illustrates the selection of anti-hCCR4 hybridoma antibodies based on ELISA and FACS binding assays.
  • RNA of anti-hCCR4 hybridoma clone 22C10, 69 A7, 26B1, 3B9, 40A6 or 1F7 was isolated by RNeasy Mini Kit (Qiagen, Cat. No:74104) and used as the template to synthesize first-strand cDNA with SuperScript® II Reverse Transcriptase (Life Technology, Cat. No: 18064-14) according to the manufacturer's instructions.
  • the cDNA product was then subjected to PCR in a 50 pl volume reaction mixture using degenerate mouse IgG primers (Kettleborough CA, et al., (1993) European Journal of Immunology 23:206; Strebe N, et al., (2010) Antibody Engineering 1 :3).
  • the reaction was carried out in a S1000TM Thermal Cycler (Bio-Rad, Cat. No:184-2000) with 30 cycles of: 94°C, 1.5 minutes for denaturation; 50°C, 1 minutes for annealing; and 72°C, 1 minute for synthesis. At the end of the 30th cycle, the reaction mixture was incubated for additional 7 minutes at 72° C for extension.
  • the PCR mixture was subjected to electrophoresis in a 1% agarose/Tris-Borate gel containing 0.5 pg/ml ethidium bromide. DNA fragments having the expected sizes (approximately 450 bp for the heavy chain and the light chain) were excised from the gel and purified.
  • Antibody 22C10, 69 A7, 26B1, 3B9, 40A6 and 1F7 chimeric light chains were constructed by linking the PCR-amplified cDNAs of mouse VL regions with human kappa chain constant region (SEQ ID NO: 72), respectively, and their chimeric heavy chains were constructed by linking the cDNAs of mouse VH regions with human IgGl constant region which contained S239D/I332E mutations (Larza GA, et al., 2006 PNAS 103:4005).
  • mice cDNA sequences were modified using PCR primers designed to add a leader sequence to both light chain and heavy chain, and the DNA encoding the light chain and heavy chain of each chimeric antibody was subcloned into expression vector pcDNA3.1 (Invitrogen Cat. No: V-790), respectively.
  • Freestyle 293 cells (200 mL at l *10 A 6/mL) were transfected with 100 pg of each of the chimeric heavy and light chain expression plasmids and cultured for 6 days at 37° C.
  • the chimeric antibody in the supernatant was then purified with Protein-A column (GE healthcare). Binding of the chimeric antibody with CCR4 was measured by ELISA (FIG. 3, Table 7) and FACS (FIG. 4, Table 8), using the similar methods described above in Example 2.
  • HuT78 (2*10 A 4 cells/ well) and human NK cells (2*10 A 5 cells/well) suspended in CTL Test Medium (Cellular Technology Limited) were seeded in a 96-well plate with serially diluted CCR4 antibody (lOOOng/mL, 200ng/mL, 40ng/mL, 8ng/mL, 1.6ng/mL, 0.32ng/mL). After incubation for 4 hours at 37° C in 5% CO2, the LDH activities of cell culture supernatants were measured by using Cytotoxicity Detection KitPLUS (LDH) (Roche Applied Science). The percentage cytotoxicity was calculated as described in the manufacturer’s protocol. As shown in FIG. 5, chimeric antibody 22C10, 69A7, 26B1, 3B9, 40A6, 1F7 elicited ADCC of HuT78 in a dose dependent manner.
  • CTL Test Medium Cellular Technology Limited
  • This example illustrates the binding kinetic study of humanized CCR4 antibodies.
  • 69 A7 antibody were humanized using the CDR grafting approach as described in U.S. Pat. No.5,225,539.
  • the light chain and heavy chain variable chain sequences of the murine antibody 69A7 were compared to those available in the Research Collaboratory for Structural Bioinformatics (RCSB) protein databank by searching the NCBI database, http://www.ncbi.nlm.nih.gov/igblast/igblast.cgi.
  • the model of antibody 69A7 was generated respectively based on the VH and VL structure with the highest sequence homology.
  • Human antibody germlines which have high sequence homology with 69 A7 mouse antibody were obtained by searching the IMGT/Domain Gap Align 3D structure database, http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi.
  • the frameworks of human germline antibodies were used as template for grafting the complementary determining regions (CDRs) from the VH and VL of mouse antibodies.
  • CDRs complementary determining regions
  • CDR amino acid sequences of the aforementioned template human antibody were substituted by the amino acid sequence of CDRs of mouse 69 A7 antibody.
  • the frameworks of the above-mentioned template human antibody VH and VL were grafted with the necessary amino acid sequences from VH and VL of mouse 69A7 antibody to give a functional humanized antibody.
  • VH and VL of 69 A7 several sites of framework amino acid of the aforementioned template human antibody were back mutated to the corresponding amino acid sequences in mouse 69A7 antibody.
  • the amino acid at position 49 was mutated from Tyr (Y) to Phe (F) and the amino acid at position 100 was mutated from Gin (Q) to Ser (S); and for the heavy chain variable region of humanized 69A7 antibody, the amino acid at position 68 was mutated from Thr (T) to He (I), the amino acid at position 73 was mutated from Asn (N) to Asp (D), the amino acid at position 76 was mutated from Asn (N) to Ser (S), the amino acid at position 78 was mutated from Leu (L) to Vai (V).
  • the amino acid sequences of the variable light and variable heavy chains of humanized 69A7 antibody were designated SEQ ID NOs: 29 and 30, respectively.
  • DNA encoding humanized 69A7 antibody light chain and heavy chain was synthesized and cloned to the expression vector pcDNA3.1 (Invitrogen, Cat. No:V-790). Freestyle 293 cells (200 mL at 106/mL) were transfected with lOOpg of each of the humanized heavy and light chain expression plasmids and cultured for 6 days at 37° C. The humanized antibody in the supernatant was then purified with Protein-A column (GE healthcare). Binding of the humanized antibody and positive control antibody Poteligo with CCR4 was measured by ELISA (FIG. 6, Table 9) and FACS (FIG. 7, Table 10), and the methods used were similar to those described above in Example 2.
  • NK cells (2* 10 A 5 cells/well) were incubated with HuT78 cells (2*10 A 4 cells/ well) in the presence of series concentrations of humanized 69A7 antibody (1000 ng/mL,100 ng/mL, 10 ng/mL,l ng/mL, 0.1 ng/mL) at 37°C, 5% CO2 for 4 hours.
  • humanized 69A7 antibody 1000 ng/mL,100 ng/mL, 10 ng/mL,l ng/mL, 0.1 ng/mL
  • both chimeric and humanized 69 A7 antibody had more potent ADCC activity (FIG. 8).
  • the percentage cytotoxicity was calculated as described in the manufacturer’s protocol.
  • hCCR4pep-mFc analyte series were prepared by diluting the stocks with running buffer to lOOnM followed by 2-fold serial dilution in the same buffer down to 3.125nM. Analytes were injected in series over the reference and experiment flow cells for 3 minutes at a flow rate of 30 pL/minute. Running buffer (HBS-EP) was allowed to flow over for 10 minutes at a flow rate of 30 pL/minute. At the end of each cycle, the biosensor surface was regenerated with 30 second injection of 3M MgC12 buffer at a flow rate of 10 pL/minute.
  • binding responses obtained from the experimental biosensor surface were double referenced by subtracting simultaneously recorded responses from the reference surface followed by additional subtraction of responses from a single referenced running buffer sample.
  • the association and dissociation rate constants (ka and kd) were determined simultaneously by fitting double-referenced sensorgrams of the entire titration series to Langmuir model (1 : 1) using Biaevaluation software.
  • Mouse platelet rich plasma was obtained by centrifugation fresh blood at 185 g in plastic tubes at room temperature for 15 min. The platelets were incubated either with 69 A7 or an isotype control at a concentration of 10 pg/mL in 96 -well plate. Adenosine diphosphate (ADP) at a final concentration 3 pM was used as a positive control. Platelet - dependent thrombus formation was detected at 37°C with a microplate reader. The signal obtained with ADP was set to 100% and the baseline was defined by the platelet depleted serum, which was measured in the same 96-well plate. Both chimeric and humanized 69A7 antibody cannot induce platelet aggregation as shown in FIG. 10.
  • ADP Adenosine diphosphate

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  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des anticorps anti-CCR4 ou des fragments de liaison à l'antigène de ceux-ci, des polynucléotides isolés codant pour ceux-ci, des compositions pharmaceutiques les comprenant, et leurs utilisations.
PCT/US2024/013629 2023-02-01 2024-01-31 Nouveaux anticorps anti-ccr4 et produits dérivés Ceased WO2024163530A2 (fr)

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CNPCT/CN2023/074084 2023-02-01
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025145207A1 (fr) 2023-12-29 2025-07-03 Bristol-Myers Squibb Company Polythérapie d'inhibiteur de kras et d'agent de déplétion des treg

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3511343A1 (fr) * 2012-05-04 2019-07-17 Dana Farber Cancer Institute, Inc. Anticorps anti-ccr4 monoclonaux humanisés maturés par affinité et leurs procédés d'utilisation
JOP20170154B1 (ar) * 2016-08-01 2023-03-28 Omeros Corp تركيبات وطرق لتثبيط masp-3 لعلاج أمراض واضطرابات مختلفة
CN117396515A (zh) * 2021-05-19 2024-01-12 南京再明医药有限公司 抗msln抗体及其应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025145207A1 (fr) 2023-12-29 2025-07-03 Bristol-Myers Squibb Company Polythérapie d'inhibiteur de kras et d'agent de déplétion des treg

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