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WO2025007703A1 - Anticorps anti-tfr1 et leurs utilisations - Google Patents

Anticorps anti-tfr1 et leurs utilisations Download PDF

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Publication number
WO2025007703A1
WO2025007703A1 PCT/CN2024/098331 CN2024098331W WO2025007703A1 WO 2025007703 A1 WO2025007703 A1 WO 2025007703A1 CN 2024098331 W CN2024098331 W CN 2024098331W WO 2025007703 A1 WO2025007703 A1 WO 2025007703A1
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Prior art keywords
antibody
amino acid
acid sequence
seq
heavy chain
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Inventor
Ran WU
Jianxiu GUO
Fei Zhang
Bingshi GUO
Xin Zheng
Mingchen CHEN
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Beijing Neox Biotech Ltd
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Beijing Neox Biotech Ltd
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Publication of WO2025007703A1 publication Critical patent/WO2025007703A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • 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
    • C07K2317/77—Internalization into the cell
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • This application relates to antibodies binding to TfR protein, especially human TfR1 protein, and the use thereof in the treatment and diagnosis of diseases
  • a promising approach to facilitate delivery of protein therapeutics across the blood-brain barrier is to take advantage of receptor-mediated transcytosis, an endogenous endocytic process in which a ligand is transported across an endothelial cell barrier.
  • Antibodies targeting the transferrin receptor (TfR) which is highly expressed by endothelial cells that make up the BBB, have been reported to cross the BBB.
  • High affinity ensures binding to TfR and uptake into brain endothelium despite low concentrations in blood, but it also likely reduces the probability of antibody being released from the central nervous system (CNS) vasculature, and thus potentially prevents accumulation of antibody in the brain parenchyma. It is reported that, using low-affinity antibodies to TfR may increase release of antibody from brain vascular endothelium and enhance uptake and distribution in the brain.
  • Described herein are isolated antibodies that specifically bind to transferrin receptor (TfR) and the uses thereof.
  • the antibodies described herein possess the useful properties such as desired affinity, high BBB penetration efficiency and less or no competition with transferrin (Tf) , indicating an enhanced effect of brain-targeting drug delivery and safety profile.
  • Described herein in one aspect is an isolated antibody, or antigen binding fragment thereof that binds to transferrin receptor (TfR) , comprising an immunoglobulin heavy chain variable region which comprises:
  • a CDR1 comprising an amino acid sequence shown by SEQ ID NO: 1
  • a CDR2 comprising an amino acid sequence shown by SEQ ID NO: 4
  • a CDR3 comprising an amino acid sequence shown by SEQ ID NO: 6;
  • CDR complementarity determining region
  • GRTLRFSAYGMG SEQ ID NO: 1
  • CDR2 comprising an amino acid sequence shown by AISQWGVGNTYYADSVKG (SEQ ID NO: 4)
  • CDR3 comprising an amino acid sequence shown by DTSPVTWYPADSYHYDA (SEQ ID NO: 25)
  • the CDR1 ranges from amino acid residue H26 to H35B
  • the CDR2 ranges from amino acid residue H50 to H65
  • the CDR3 ranges from amino acid residue H95 to H102
  • residue numbering follows Kabat numbering scheme.
  • the immunoglobulin heavy chain variable region comprises a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the TfR is TfR1.
  • the TfR is a human TfR.
  • the TfR is a human TfR1 (hTfR1) .
  • the antibody is a VHH antibody, a chimeric antibody, a humanized antibody or a human antibody.
  • Described herein in another aspect is an isolated antibody, or antigen binding fragment thereof that binds to transferrin receptor (TfR) , comprising:
  • a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence of a CDR1, a CDR2, and a CDR3, respectively, of a heavy chain variable region having an amino acid sequence of:
  • a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence of a CDR1, a CDR2, and a CDR3, respectively, of a heavy chain variable region having an amino acid sequence of:
  • a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence of a CDR1, a CDR2, and a CDR3, respectively, of a heavy chain variable region having an amino acid sequence of:
  • a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence of a CDR1, a CDR2, and a CDR3, respectively, of a heavy chain variable region having an amino acid sequence of:
  • a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence of a CDR1, a CDR2, and a CDR3, respectively, of a heavy chain variable region having an amino acid sequence of:
  • a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence of a CDR1, a CDR2, and a CDR3, respectively, of a heavy chain variable region having an amino acid sequence of:
  • a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence of a CDR1, a CDR2, and a CDR3, respectively, of a heavy chain variable region having an amino acid sequence of:
  • a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence of a CDR1, a CDR2, and a CDR3, respectively, of a heavy chain variable region having an amino acid sequence of:
  • a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence of a CDR1, a CDR2, and a CDR3, respectively, of a heavy chain variable region having an amino acid sequence of:
  • (x) a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence of a CDR1, a CDR2, and a CDR3, respectively, of a heavy chain variable region having an amino acid sequence of:
  • the CDRs are defined by an CDR definition scheme selected from the group consisting of: Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition, North definition, or a hybrid scheme thereof.
  • the heavy chain variable region derives from a camelid. In some embodiments, the heavy chain variable region derives from a mammal other than a camelid. In some embodiments, the heavy chain variable region derives from a rodent or a primate. In some embodiments, the FR1 to FR4 derive from human immunoglobulin heavy chain. In some embodiments, the FR1 to FR4 derive from a subgroup III framework sequence of human immunoglobulin. In some embodiments, the FR1 to FR4 derive from IGHV3-23*01+IGHJ4*04, vh3_H, IGHV3-30*02, IGHV3-23*04, or IGHV3-64*04. In some embodiments, the FR1, FR2, FR3 and FR4 from a same species or different species.
  • the isolated antibody or antigen binding fragment thereof of the present disclosure comprises any one amino acid sequence selected from SEQ ID NO: 9 to SEQ ID NO: 17, or a conservative substitution variant thereof.
  • the isolated antibody or antigen binding fragment thereof of the present disclosure comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with any one amino acid sequence selected from SEQ ID NO: 9 to SEQ ID NO: 17.
  • the amino acid sequence of the isolated antibody is set forth in any one any one amino acid sequence selected from SEQ ID NO: 9 to SEQ ID NO: 17.
  • the isolated antibody is a single domain antibody, a full-length antibody, a IgG, a Fab, a F (ab') 2, a Fab', an scFv, or a Fv.
  • the isolated antibody further comprises a human IgG Fc domain, preferably the human IgG Fc domain is IgG1, IgG2, IgG3, or IgG4 Fc domain.
  • the human IgG Fc domain comprises an amino acid sequence shown by SEQ ID NO:18, a conservative substitution variant thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with the amino acid sequence shown by SEQ ID NO: 18.
  • the IgG Fc domain is linked to the heavy chain variable region directly or by a linker.
  • the linker is a peptide.
  • the linker is a GS linker.
  • the linker is a G4S linker.
  • the IgG Fc domain is linked to the carboxyl terminal of the heavy chain variable region.
  • the isolated antibody comprises a monovalent, bivalent, trivalent, or tetravalent TfR binding domain (s) .
  • each TfR binding domain in the bivalent, trivalent, or tetravalent TfR binding domains binds to a same epitope of the TfR.
  • the amino acid sequences of each TfR binding domains in the bivalent, trivalent, or tetravalent TfR binding domains is the same.
  • each TfR binding domain in the bivalent, trivalent, or tetravalent TfR binding domains binds to a different epitope of the TfR.
  • the amino acid sequences of each TfR binding domains in the bivalent, trivalent, or tetravalent TfR binding domains is different.
  • the isolated antibody is a bispecific antibody or a multispecific antibody further comprising a binding domain that binds to a therapeutic or diagnostic target.
  • the therapeutic or diagnostic target comprises a brain antigen.
  • the brain antigen is one or more selected from the group consisting of:
  • beta-secretase 1 BACE1 , amyloid beta (Abeta) , epidermal growth factor receptor (EGFR) , human epidermal growth factor receptor 2 (HER2) , Tau, apolipoprotein E4 (ApoE4) , alpha-synuclein, CD20, huntingtin, prion protein (PrP) , leucine rich repeat kinase 2 (LRRK2) , parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6) , amyloid precursor protein (APP) , p75 neurotrophin receptor (p75NTR) , caspase 6, G protein coupled receptor (GPCR) and glucocerebrosidase.
  • the therapeutic or diagnostic target comprises one or more selected from the group consisting of: a tumor-associated antigen, an immune-checkpoint antigen and an immune-checkpoint-associated antigen.
  • the tumor-associated antigen is selected from the group consisting of: A33; ADAM-9; ALCAM; BAGE; beta-catenin; CA125; Carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD36; CD40/CD154; CD45; CD46; CD5; CD56; CD79a/CD79b; CDK4; CEA; CTLA4; Cytokeratin 8; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2/GD3/GM2; HER-2/neu; human papillomavirus-E6; human papillomavirus-E7; JAM-3;KID3; KID31; KSA (17-1A) ; LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosaminyltransferase; Oncostatin
  • the immune-checkpoint protein is selected from the group consisting of 2B4; 4-1BB; 4-1BB ligand, B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; CD19; CD200; CD27; CD27 ligand; CD28; CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; Galectin-9; GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; TIM-3; TIGIT; VSIG8.
  • the isolated antibody is a bispecific antibody or a multispecific antibody, wherein the TfR binding domain binds or links to an antibody that comprising the binding domain that binds to a therapeutic or diagnostic target.
  • the antibody that comprising the binding domain that binds to a therapeutic or diagnostic target comprise a Fc region, and the TfR binding domain is linked to the C terminal of the Fc region.
  • the antibody that comprising the binding domain that binds to a therapeutic or diagnostic target is an anti-amyloid ⁇ antibody.
  • the anti-amyloid ⁇ antibody is Donanemab.
  • the antibody consists of a heavy chain shown by SEQ ID NO: 29, a heavy chain shown by SEQ ID NO:30, and two light chains shown by SEQ ID NO: 28. In some embodiments, the antibody consists of a heavy chain shown by SEQ ID NO: 31, a heavy chain shown by SEQ ID NO: 30, and two light chains shown by SEQ ID NO: 28.
  • fusion protein comprising an isolated antibody as described in any one of the preceding aspects or embodiments.
  • the isolated antibody is fused to a functional protein or a functional domain thereof.
  • the functional protein is selected from the group consisting of: a brain antigen binding polypeptide, a hormone, a neurotrophic factor, a neuropeptide, a cytokine, an enzyme or a mimics or functional domain thereof.
  • the brain antigen binding polypeptide is an antibody binding to one or more molecules selected from the group consisting of: beta-secretase 1 (BACE1) , amyloid beta (Abeta) , epidermal growth factor receptor (EGFR) , human epidermal growth factor receptor 2 (HER2) , Tau, apolipoprotein E4 (ApoE4) , alpha-synuclein, CD20, huntingtin, prion protein (PrP) , leucine rich repeat kinase 2 (LRRK2) , parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6) , amyloid precursor protem (APP) , p75 neurotrophin receptor (p75NTR) , G protein coupled receptors (GPCR) and caspase 6.
  • BACE1 beta-secretase 1
  • Abeta amyloid beta
  • EGFR epidermal growth factor receptor
  • HER2 human epidermal growth factor receptor
  • the neurotrophic factor is selected from the group consisting of: nerve growth factor (NGF) , brain derived neurotrophic factor (BDNF) , ciliary neurotrophic factor (CNTF) , glial cell-line neurotrophic factor (GDNF) and insulin-like growth factor (IGF) .
  • the neuropeptide is selected from the group consisting of: Substance P, neuropeptide Y, vasoactive intestinal peptide (VIP) , gamma-amino-butyric acid (GABA) , dopamine, cholecystokinin (CCK) , endorphins, enkephalins and thyrotropin releasing hormone (TRH) .
  • the functional protein or a functional domain thereof is linked to the isolated antibody directly or by a linker.
  • the linker is a peptide.
  • the linker is a GS linker.
  • the linker is a G4S linker.
  • the functional protein is linked to the carboxyl terminal of the isolated antibody.
  • the fusion protein comprises a structure of, from amino terminal to carboxyl terminal, the heavy chain variable region-linker-Fc-linker-he functional protein or a functional domain thereof.
  • the structure is the heavy chain variable region-GS linker-Fc-GS linker-the functional protein or a functional domain thereof.
  • the structure is the heavy chain variable region- (G4S) 3-Fc- (G4S) 3-the functional protein or a functional domain thereof.
  • the functional protein is a neurotensin.
  • the fusion protein comprises or consists of an amino acid set forth by SEQ ID NO: 21, SEQ ID NO:22, or SEQ ID NO: 23.
  • Described herein in another aspect is a conjugate comprising an isolated antibody as described in any one of the preceding aspects or embodiments, wherein the isolated antibody is coupled to a chemical compound selected from the group consisting of: a nucleic acid, a polypeptide, a radionuclide, and a small molecule compound.
  • the isolated antibody is conjugated to a small molecule compound.
  • the nucleic acid is selected from the group consisting of: an mRNA, a siRNA, short hairpin RNAs (shRNA) , microRNA (miRNA) , an antisense oligonucleotide, guide RNA (gRNA) , and a phosphoroamidate morpholino oligomer (PMO) .
  • the small molecule compound is a cytotoxic drug or an immune modulator.
  • the polypeptide is selected from the group consisting of: a soluble receptor, a secreted protein, a growth factor, a cytokine, a hormone, a neurotransmitter, or an enzyme.
  • nucleic acid comprising a sequence encoding the isolated antibody or the fusion protein as described in any one of the preceding aspects or embodiments, or an antisense strand thereof.
  • Described herein in another aspect is a vector comprising the nucleic acid encoding the isolated antibody or the fusion protein as described in any one of the preceding aspects or embodiments or an antisense strand thereof .
  • a host cell comprising the encoding nucleic acid or the vector as described in any one of the preceding aspects or embodiments.
  • the host cell is a prokaryocyte or a eukaryocyte.
  • Described herein in another aspect is a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antibody, the fusion protein, the conjugate, or the nucleic acid as described in any one of the preceding aspects or embodiments.
  • Described herein in another aspect is a method of delivering a therapeutic, diagnostic or signaling agent across the blood-brain barrier (BBB) of a subject in need thereof, comprising administering to the subject a compound comprising the therapeutic, diagnostic or signaling agent conjugated to the isolated antibody as described in any one of the preceding aspects or embodiments.
  • BBB blood-brain barrier
  • the neurological disorder is selected from the group consisting of: a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, CNS inflammation, Alzheimer's Disease, Parkinson's Disease, multiple sclerosis, or solid or liquid tumor with brain metastases.
  • FIGs. 1A-1B show the binding activities of the analyzed antigen-binding fragments to human TFR (1A) and mouse TFR (1B) .
  • FIG. 2 shows the binding ability of NX56 to hCMEC.
  • FIG. 3 shows the result of the competitive ELISA for anti-TfR antibody and Holo-Tf.
  • FIG. 4 shows the endocytosis of NX56 into hCMEC cells and CHO cells (hTFR negative) .
  • FIG. 5 shows the internalization and transmembrane effect of NX56 on hCMEC cells.
  • FIG. 7. shows the affinity assay results of NX759 and NX788 to hCMEC cells.
  • FIG. 8. shows the results of endocytosis assay of anti-TfR antibodies NX759 and NX788 in hCMEC cells.
  • FIG. 9 shows the results of neurotensin (NT) delivery to mouse brain by anti-TfR antibodies NX759 and NX788.
  • FIG. 10 shows the structure of the bispecific antibodies comprising an anti-TfR VHH antibody.
  • FIG. 11 shows results of in vivo blood-brain barrier penetration assay of anti-TfR antibody-conjugated Donanemab.
  • the application relates to an isolated antibody that binds to transferrin receptor (TfR) and the uses thereof.
  • TfR transferrin receptor
  • the application relates to variable domain of a single variable domain on a heavy chain (VHH) antibody, which bind TfR at the surface of cell membranes such as the blood-brain barrier (BBB) , and the uses thereof e.g., to transport molecules of pharmaceutical or diagnostic interest into cells of the central nervous system or TfR-expressing tissues or organs, such as malignant tumors.
  • VHH heavy chain
  • BBB blood-brain barrier
  • TfR transferrin receptor
  • a TfR is involved in iron uptake in vertebrates and is regulated in response to intracellular iron concentration. It imports iron by internalizing the transferrin-iron complex through receptor-mediated endocytosis.
  • TfR1 and TfR2 transferrin receptor 2
  • TfRl also known as CD71, is a high affinity ubiquitously expressed receptor.
  • the TfR is a human TfR1 encoded by the gene with the Gene ID 7037 in NCBI data base.
  • the TfR1 comprises an amino acid sequence set forth in SEQ ID NO: 22.
  • blood-brain barrier refers a physiological barrier between the peripheral circulation and the brain and spinal cord which is formed by tight junctions within the brain capillary endothelial plasma membranes, creating a tight barrier that restricts the transport of molecules into the brain.
  • the BBB can restrict the transport of even very small molecules such as urea (60 Daltons) into the brain.
  • examples of the BBB include the BBB within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina, all of which are contiguous capillary barriers within the CNS.
  • the BBB also encompasses the blood-CSF barrier (choroid plexus) where the barrier is comprised of ependymal cells rather than capillary endothelial cells.
  • central nervous system or “CNS” refers to the complex of nerve tissues that control bodily function, and includes the brain and spinal cord.
  • a “neurological disorder” as used herein refers to a disease or disorder which affects the CNS and/or which has an etiology in the CNS.
  • exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, an ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, and a lysosomal storage disease.
  • the CNS will be understood to include the eye, which is normally sequestered from the rest of the body by the blood-retina barrier.
  • neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy) , tauopathies (including, but not limited to, Alzheimer disease and supranuclear palsy) , prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutz-feldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia) , bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes
  • a “neurological disorder drug” is a drug or therapeutic agent useful in treating or ameliorating the effects of one or more neurological disorder (s) .
  • Neurological disorder drugs of the application include, but are not limited to, small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more CNS target (s) , modified versions of natural ligands of one or more CNS target (s) , aptamers, inhibitory nucleic acids (i.e., small inhibitory RNAs (siRNA) and short hairpin RN As (shRNA) ) , ribozymes, or active fragments of any of the foregoing.
  • siRNA small inhibitory RNAs
  • shRNA short hairpin RN As
  • Exemplary neurological disorder drugs of the application include, but are not limited to: antibodies, aptamers, proteins, peptides, inhibitory nucleic acids and small molecules and active fragments of any of the foregoing that either are themselves or specifically recognize and/or act upon (i.e., inhibit, activate, or detect) a CNS antigen or target molecule such as, but not limited to, amyloid precursor protein or portions thereof, amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophins.
  • a CNS antigen or target molecule such as, but not limited to, amyloid precursor protein or portions thereof, amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE
  • BDNF Brain-derived neurotrophic factor
  • Neurogenes1s Chrome brain injury
  • FGF-2 Fibroblast growth factor 2
  • EGFR Anti-Epidermal Growth Factor
  • target antigen refers to an antigen and/or molecule expressed in the CNS, including the brain, which can be targeted with an antibody or small molecule.
  • antigens and/or molecules include, without limitation: beta-secretase 1 (BACE1) , amyloid beta (Abeta) , epidermal growth factor receptor (EGFR) , human epidermal growth factor receptor 2 (HER2) , Tau, apolipoprotein E4 (ApoE4) , alpha-synuclein, CD20, huntingtin, prion protein (PrP) , leucine rich repeat kinase 2 (LRRK2) , parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6) , amyloid precursor protein (APP) , p75 neurotrophin receptor (p75NTR) , and caspase 6.
  • BACE1 beta-secretase 1
  • Abeta amyloid beta
  • EGFR epidermal
  • antibody herein is used in the broadest sense and specifically includes fulllength monoclonal antibodies, polyclonal antibodies, and, unless otherwise stated or contradicted by context, antigen-binding fragments, antibody variants, and multispecific molecules thereof, so long as they exhibit the desired biological activity.
  • a full-length antibody is a glycoprotein comprising at least two heavy chains and two light chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
  • the term antibody is intended to encompass immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
  • an "antibody” can also be a single variable domain on a heavy chain (VHH) antibody, also referred to as a heavy chain only antibody (HcAb) , which are devoid of light chains and can be naturally produced by camelids or sharks.
  • the antigen binding portion of the HcAb is comprised of a VHH fragment.
  • VHH antibody or “HcAb” encompasses both naturally produced and synthesized heavy chain only antibody, for example humanized heavy chain only antibody with CDRs from natural VHH fragment and FRs from human being.
  • heterogeneous chain refers to domains in naturally occurring immunoglobulins and the corresponding domains of synthetic (e.g., recombinant) binding proteins (e.g., humanized antibodies) .
  • the basic structural unit of naturally occurring immunoglobulins e.g., IgG is a tetramer having two light chains and two heavy chains.
  • the amino-terminal ( “N” ) portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal ( “C” portion of each chain defines a constant region, with light chains having a single constant domain and heavy chains usually having three constant domains and a hinge region.
  • the structure of the light chains of a naturally occurring IgG molecule is N-VL-CL-C and the structure of IgG heavy chains is N-VH-CH1-H-CH2-CH3-C (where H is the hinge region) .
  • variable region of an IgG molecule consists of the complementarity determining regions (CDRs) , which contain the residues in contact with antigen and non-CDR segments, referred to as framework segments, which maintain the structure and determine the positioning of the CDR loops.
  • CDRs complementarity determining regions
  • framework segments which maintain the structure and determine the positioning of the CDR loops.
  • the VL and VH domains have the structure N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.
  • an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TfR1 protein, such as TfR1 V1) .
  • an “antigen binding fragment” and all grammatical variants thereof, are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody which, in certain instances, is free of the constant heavy chain domains (i.e., CH2, CH3, and/or CH4, depending on antibody isotype) of the Fc region of the intact antibody.
  • antibody fragments include Fab, Fab’ , Fab’ -SH, F(ab’ ) 2 , and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a “single-chain antibody fragment” or “single chain polypeptide” ) , including without limitation (1) single-chain Fv (scFv) molecules, (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety, and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific or multivalent structures formed from antibody fragments.
  • single-chain antibody fragment single-chain Fv
  • scFv single chain polypeptides containing only one light chain variable domain, or a fragment
  • the heavy chain (s) can contain any constant domain sequence (e.g., CH1 in the IgG isotype) found in a non-Fc region of an intact antibody, and/or can contain any hinge region sequence found in an intact antibody, and/or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain (s) .
  • any constant domain sequence e.g., CH1 in the IgG isotype
  • Papain digestion of antibodies produces two identical antigen binding fragments, called “Fab” fragments, each with a single antigen binding site, and a residual Fc fragment, whose name reflects its ability to crystallize readily.
  • the “Fab” fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • “Fab'” fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • “Fab'-SH” is the designation for Fab' in which the cysteine residue (s) of the constant domains have a free thiol group.
  • F (ab') ” fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the “F (ab') 2” which is pepsin digestion product.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy-and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species (scFv) , one heavy-and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species.
  • variable domain interacts to define an antigen-binding site on the surface of the VH-VL dimer.
  • the six CDRs confer antigen-binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three CDRs specific for an antigen has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. See, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994) .
  • a “Fd” fragment consists of the VH and CH1 domains.
  • a “dAb” fragment (Ward et al., (1989) Nature 341: 544-546) consists of a VH or VL domain.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH -VL) .
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • Diabodies are described more fully in, for example, EP 404, 097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-48 (1993) .
  • antibody fragments are obtained using conventional techniques known to those with skill in the art, for example, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.
  • human acceptor framework means a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a human acceptor framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 1-10, 2-9, 3-8, 4-7 or 5-6.
  • a “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991) , vols. 1-3.
  • the subgroup is subgroup kappa I as in Kabat et al, supra.
  • the subgroup is subgroup III as in Kabat et al, supra.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains.
  • CDRs complementarity-determining regions
  • the 3 CDRs from N terminal to C terminal on a heavy chain is referred to as HCDR1, HCDR2, and HCDR3
  • LCDR1, LCDR2, and LCDR3 the 3 CDRs from N terminal to C terminal on a light chain.
  • CDR1 to CDR3 represents LCDR1 to LCDR3 or HCDR1 to HCDR3 respectively.
  • the more highly conserved portions of variable domains are called the framework (FR) .
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • HFR1, HFR2, HFR3, and HFR4 the 4 FRs from N terminal to C terminal on a heavy chain
  • FR1 to FR4 represents LFR1 to LFR4 or HFR1 to HFR4 respectively.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991) .
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • Variable region sequences of interest include the humanized variable region sequences for TfR1 antibodies described in detail elsewhere herein.
  • CDR complementarity determining region
  • HVR hypervariable region
  • “Numbering according to Kabat” may refer to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra.
  • the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR/HVR of the variable domain.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the Kabat numbering is used when referring to a residue in the variable domains (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain)
  • the EU numbering system or index e.g., the EU index as in Kabat, numbering according to EU IgG1
  • EU index is generally used when referring to a residue in the heavy chain constant region.
  • CDR/HVR definitions/delineations are known.
  • the CDRs were defined according to a combination of Kabat and Chothia CDR definition, in which CDR1 ranges from amino acid residue H26 to H35B, the CDR2 ranges from amino acid residue H50 to H65, and the CDR3 ranges from amino acid residue H95 to H102, and the residue numbering follows Kabat numbering scheme.
  • the Kabat Complementarity Determining Regions are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) ) . Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987) ) .
  • the AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.
  • the “contact” HVRs are based on an analysis of the available complex crystal structures.
  • Extended CDRs are also known: 24-36 or 24-34 (L1) , 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1) , 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH (Kabat numbering) .
  • a “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., substantially identical but allowing for minor levels of background mutations and/or modifications. “Monoclonal” denotes the substantially homogeneous character of antibodies, and does not require production of the antibody by any particular method.
  • a monoclonal antibody is selected by its CDR/HVR, VH, and/or VL sequences and/or binding properties, e.g., selected from a pool of clones (e.g., recombinant, hybridoma, or phage-derived) .
  • a monoclonal antibody may be engineered to include one or more mutations, e.g., to affect binding affinity or other properties of the antibody, create a humanized or chimeric antibody, improve antibody production and/or homogeneity, engineer a multispecific antibody, resultant antibodies of which are still considered to be monoclonal in nature.
  • a population of monoclonal antibodies may be distinguished from polyclonal antibodies as the individual monoclonal antibodies of the population recognize the same antigenic site.
  • phage-display technologies see, e.g., Clackson et al., Nature, 352: 624-628 (1991) ; Marks et al., J. Mol. Biol. 222: 581-597 (1992) ; Sidhu et al., J. Mol. Biol. 338 (2) : 299-310 (2004) ; Lee et al., J. Mol. Biol. 340 (5) : 1073-1093 (2004) ; Fellouse, Proc. Natl. Acad. Sci. USA 101 (34) : 12467-12472 (2004) ; and Lee et al., J. Immunol.
  • Methods 284 (1-2) 119-132 (2004) , and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993) ; Jakobovits et al., Nature 362: 255-258 (1993) ; Bruggemann et al., Year in Immunol. 7: 33 (1993) ; U.S. Pat. Nos.
  • bispecific antibody or “bispecific antigen binding antibody” or “bifunctional antibody” is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • the term “native antibodies and immunoglobulins” are usually heterotetrameric glycoproteins of about 150, 000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond (also termed a “VH/VL pair” ) , while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • VH variable domain
  • Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light-and heavy-chain variable domains. See, e.g., Chothia et al., J. Mol. Biol., 186: 651 (1985) ; Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A., 82: 4592 (1985) .
  • Chimeric antibodies may refer to an antibody with one portion of the heavy and/or light chain from a particular isotype, class, or organism and another portion from another isotype, class, or organism.
  • the variable region will be from one source or organism, and the constant region will be from another.
  • Humanized antibodies may refer to antibodies with predominantly human sequence and a minimal amount of non-human (e.g., mouse or chicken) sequence.
  • a humanized antibody has one or more CDR sequences (bearing a binding specificity of interest) from an antibody derived from a non-human (e.g., mouse or chicken) organism grafted onto a human recipient antibody framework (FR) .
  • non-human residues are further grafted onto the human framework (not present in either source or recipient antibodies) , e.g., to improve antibody properties.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. See Jones et al., Nature 321: 522-525 (1986) ; Riechmann et al., Nature 332: 323-329 (1988) ; and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992) .
  • a “human antibody” may refer to an antibody having an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991) ; Marks et al., J. Mol. Biol., 222: 581 (1991) ; preparation of human monoclonal antibodies as described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) ; Boerner et al., J.
  • immunized xenomice see, e.g., U.S. Pat. Nos. 6,075,181 and 6, 150, 584 regarding XENOMOUSE TM technology
  • chickens with human immunoglobulin sequence (s) see, e.g., WO2012162422, WO2011019844, and WO2013059159
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) , e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • epitopic determinants means any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • conjugate refers to a protein covalently linked to one or more heterologous molecule (s) , including but not limited to a therapeutic peptide or protein, an antibody, a label, or a neurological disorder drug.
  • Coupled refers to the joining or connection of two or more objects together, and the connection can be non-covalently (e.g., by ionic, hydrogen, hydrophobic or Van der Waals bonds) and/or covalently.
  • “coupled” equals "conjugated” .
  • an antibody of the application can be coupled with a peptide of interest to form an antibody coupled peptide.
  • An antibody coupled peptide can be formed through specific chemical reactions designed to conjugate the antibody to the peptide.
  • an antibody of the application can be covalently coupled with a peptide of the application through a linker.
  • the linker can, for example, be first covalently connected to the antibody or the peptide, then covalently connected to the peptide or the antibody.
  • blocking antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds.
  • Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
  • isolated refers to molecules or biological or cellular materials being substantially free from other materials.
  • a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • Binding affinity refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen) . Affinity can be measured by common methods known in the art, including, for example, Biacore, radioimmunoassay (RIA) and ELISA.
  • the affinity of a molecule X for its partner Y can generally be represented by equilibrium dissociation constant (K D ) , calculated as the ratio koff/kon (kd/ka) .
  • K D equilibrium dissociation constant
  • Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • the “dissociation rate (kd) ” is measured by using surface plasmon resonance assays.
  • an “on-rate” or “rate of association” or “association rate (ka) ” or “kon” can also be determined with the same surface plasmon resonance technique and calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software) by simultaneous fitting the association and dissociation sensor gram.
  • EC 50 refers to the concentration of an antibody or an antigen-binding fragment thereof, which binds to the antigen and/or induces a response, either in an in vitro or an in vivo assay, which is 50%of the maximal binding or response, i.e., halfway between the maximal binding or response and the baseline.
  • cancer or “neoplasm” and “tumor” can be used interchangeably in the present application, referring to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells that makes them pathological to the host organism.
  • cancer refers to a benign tumor, which has remained localized.
  • cancer refers to a malignant tumor, which has invaded and destroyed neighboring body structures and spread to distant sites.
  • the cancer is associated with a specific cancer antigen.
  • treating or “treatment” of a disease in a subject refers to an approach for obtaining beneficial or desired results, including one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease) , stabilized (i.e., not worsening) state of a condition (including disease) , delay or slowing of condition (including disease) , progression, amelioration or palliation of the condition (including disease) , states and remission (whether partial or total) , whether detectable or undetectable.
  • a “pharmaceutically acceptable carrier” is a carrier with which an active ingredient constitutes a pharmaceutical formulation.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • package insert is used to refer to instructions customarily included in commercial package of a therapeutic product. Generally, there is information about the use of the therapeutic product on the package insert such as indications, usage, dosage, administration, combination therapy, contraindications and/or warnings.
  • an “effective amount” is at least the minimum amount required to affect a measurable improvement or prevention of a particular disease (e.g., cancer) .
  • An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of a therapeutic agent (or combination of therapeutic agents) to elicit a desired response in the individual.
  • An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects.
  • beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival.
  • an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting (i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and desirably stop) tumor metastasis; inhibiting to some extent tumor growth; and/or relieving to some extent one or more of the symptoms associated with the disorder.
  • An effective amount can be administered in one or more administrations.
  • an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish therapeutic treatment either directly or indirectly.
  • an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
  • an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
  • the term “subject” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
  • the mammal is human.
  • percent of “homology” or “identity” is used in the context of two or more nucleic acids or polypeptide sequences, referring to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 80%identity, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein) .
  • Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • the alignment and the percent homology or sequence identity can be determined using software programs known in the art. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following Internet address: ncbi. nlm. nih. gov/cgi-bin/BLAST.
  • this application encompasses an isolated anti-TfR1 antibody.
  • the isolated anti-TfR1 antibody is a VHH antibody, a chimeric antibody, a humanized antibody, or a human antibody.
  • the anti-TfR antibody binds to TfR1 molecule expressed on cells (for example, coronary microvascular endothelial cells) with appropriate affinity, facilitating the transferrin receptor mediated endocytosis, increasing release from brain vascular endothelium and BBB transmembrane transport of the antibody and cargos coupled to the anti-TfR antibody in vitro and in vivo.
  • the antibody of the present application does not compete or substantively does not compete with Holo-Tf for binding hTfR, thus not interfering with normal transferrin transport.
  • the isolated anti-TfR1 antibody is a monospecific, bispecific or multispecific antibody. In some embodiments, the anti-TfR1 antibody is a monoclonal antibody or polyclonal antibody. In some embodiments, the anti-TfR1 antibody is a monomer, dimer, or multimer. In some embodiments, the monomers in the dimer or the multimer are homologous or heterogenous. In some embodiments, the anti-TfR1 antibody may further comprise a stabilizing group to increase the plasma half-life of the anti-TfR1 antibody. The stabilizing group may be any group known to have substantial plasma half-life (e.g. at least 1 hour) and essentially no adverse biological activity.
  • stabilizing group examples include, for instance, a Fc fragment of an immunoglobulin or variants thereof, large human serum proteins such as albumin, HSA, or IgGs or PEGs molecules.
  • the stabilizing group is a Fc fragment of a human IgG1. More preferably, the stabilizing group is a glycosylated Fc fragment of an IgG1.
  • the heavy chain variable region (VH) of the anti-TfR1 antibody may be conjugated N-ter or C-ter of the stabilizing group, or both.
  • VH heavy chain variable region
  • conjugation is typically by genetic fusion.
  • the resulting protein may remain as a monomeric agent, or multimerize, depending on the nature of the stabilizing group.
  • Fc-VH or VH-Fc usually forms homodimers.
  • VH is linked to Fc fragment directly or via a peptide linker (e.g., G4S linker) .
  • polynucleotides including DNA, RNA or a heterozygous molecule of DNA and RNA
  • vectors or host cells comprising the coding sequence of the anti-TfR1 antibodies of the present application.
  • the polynucleotides can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells.
  • this application encompasses an isolated anti-TfR1 antibody which is a VHH antibody, a polynucleotide or nucleic acid comprising sequence encoding the VHH antibody.
  • the VHH antibody binds to TfR1 protein with an affinity constant (K D ) from 7.09E-10 M to 1.37E-08 (e.g., from 5.05E-09 to 1.90E-08 M) as determined by BLI analysis.
  • the VHH antibody binds to human TfR1 protein with a K D about 7E-10 M (e.g., from 6E-10 to 8E-10 M) , about 5.05E-09 M (e.g., from 4E-9 to 6E-9 M) , or about 1.90E-08 M (e.g., from 9 E-09 to 4E-08 M) , as determined by BLI analysis.
  • the VHH antibody binds to mouse TfR1 with a K D about 1.37E-08 (e.g., from 1E-08 to 2E-08) as determined by BLI analysis.
  • the VHH antibody binds to human TfR1 protein with a K D about 7E-10 M (e.g., from 6E-10 to 8E-10 M) , about 5.05E-09 M (e.g., from 4E-9 to 6E-9 M) , or about 1.90E-08 M (e.g., from 9 E-09 to 4E-08 M) , as determined by BLI analysis and binds to mouse TfR1 with a K D about 1.37E-08 (e.g. from 1E-08 to 2E-08) as determined by BLI analysis.
  • the solated anti-TfR1 antibody binds to TfR1 at a different site from Holo-transferrin (Holo-Tf) .
  • the VHH antibody binds to human TfR1 at a different site from human Holo-Tf.
  • the VHH antibody comprises a VHH fragment corresponding to the variable region of a heavy chain only camelid antibody that are naturally devoid of light chains.
  • the VHH fragment has a very small size of around 15 kDa.
  • a VHH antibody contains a single chain molecule that can bind its cognate antigen using a single domain.
  • the antigen-binding surface of a VHH antibody is usually more convex (or protruding) than those of conventional antibodies, which are usually flat or concave.
  • a VHH fragment is composed of 4 Framework Regions (or FRs) whose sequences and structures are defined as conserved, and three Complementarity Determining Regions (or CDRs) showing high variability both in sequence content and structure conformation, which are involved in antigen binding and provide antigen specificity.
  • FRs Framework Regions
  • CDRs Complementarity Determining Regions
  • VHH fragment of the application typically comprises or consists of the formula:
  • FRn designates framework regions and CDRn designates complementarity determining regions.
  • the CDR1 comprises an amino acid sequence shown by SEQ ID NO: 1
  • the CDR2 comprises an amino acid sequence shown by SEQ ID NO: 4
  • the CDR3 comprising an amino acid sequence shown by SEQ ID NO: 6.
  • the CDR1 comprises an amino acid sequence shown by SEQ ID NO: 1
  • the CDR2 comprises an amino acid sequence shown by SEQ ID NO: 4
  • the CDR3 comprises an amino acid sequence shown by SEQ ID NO: 6.
  • the CDR1 comprises an amino acid sequence shown by SEQ ID NO: 2
  • the CDR2 comprises an amino acid sequence shown by SEQ ID NO: 5
  • the CDR3 comprises an amino acid sequence shown by SEQ ID NO: 7.
  • the CDR1 comprises an amino acid sequence shown by SEQ ID NO: 3
  • the CDR2 comprises an amino acid sequence shown by SEQ ID NO: 5
  • the CDR3 comprises an amino acid sequence shown by SEQ ID NO: 8.
  • the CDR1 comprises an amino acid sequence shown by SEQ ID NO: 1
  • the CDR2 comprises an amino acid sequence shown by SEQ ID NO: 4
  • the CDR3 comprises an amino acid sequence shown by SEQ ID NO: 25.
  • VHH antibody comprises an amino acid sequence shown by SEQ ID NO: 17, a conservative substitution variant thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with the amino acid sequence shown by SEQ ID NO: 17.
  • the VHH antibody further comprises an IgG Fc domain.
  • the IgG Fc domain is a human IgG Fc domain.
  • the IgG Fc domain is a human IgG Fc domain.
  • the IgG Fc domain is a human IgG1, IgG2, IgG3, or IgG4 Fc domain.
  • the IgG Fc domain comprises an amino acid sequence shown by SEQ ID NO: 18, a conservative substitution variant thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with the amino acid sequence shown by SEQ ID NO: 18.
  • the isolated anti-TfR1 antibody is a chimeric antibody, humanized antibody, or human antibody, comprising an immunoglobulin heavy chain variable region with the formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FRn designates framework regions derived from a mammal other than camelids and CDRn designates complementarity determining regions. In some embodiments, the FRn designates framework regions derived from homo sapiens.
  • a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al (1986) Nature 321: 522-525; Riechmann et al (1988) Nature 332: 323-327; Verhoeyen et al (1988) Science 239: 1534-1536) , by substituting hypervariable region sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in camelid antibodies.
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity.
  • the sequence of the variable domain of a camelid antibody is screened against the entire library of known human variable-domain sequences.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for TfR1, is achieved.
  • Transgenic animals e.g., mice
  • mice that are also capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • JH antibody heavy-chain joining region
  • transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Nature, 362: 255 (1993) ; Bruggermann et al, Year in Immunol, 7: 33 (1993) .
  • Gene shuffling can also be used to derive human antibodies from non-human, e.g., camelid, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody.
  • this method which is also called "epitope imprinting"
  • either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described above is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras.
  • immunoglobulin heavy chain variable region further comprises a human acceptor framework.
  • the human acceptor framework is derived from a human immunoglobulin framework or a human consensus framework.
  • the human acceptor framework comprises subgroup III framework sequences for VH. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991) , vols. 1-3.
  • the human acceptor framework derives from any immunoglobulin heavy chain variable region germline selected from the group consisting of: IGHV3-64, IGHV3-23, IGHV3-30, vh3_H (described in Knappik A, Ge L, Honegger A, et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol. 2000; 296 (1) : 57-86. doi: 10.1006/jmbi. 1999.3444) and variants thereof.
  • the human acceptor framework derives from any immunoglobulin heavy chain variable region germline selected from the group consisting of: IGHV3-23*01+IGHJ4*04, vh3_H, IGHV3-30*02, IGHV3-23*04, and IGHV3-64*04.
  • the antibody or antigen binding fragment thereof comprises human consensus framework.
  • the immunoglobulin heavy chain variable region comprises human consensus framework with amino acid sequence changes, for example, 1-15, 1-10, 2-9, 3-8, 4-7 or 5-6 amino acid changes.
  • the immunoglobulin heavy chain variable region comprises a CDR1 comprising an amino acid sequence shown by SEQ ID NO: 1, a CDR2 comprising an amino acid sequence shown by SEQ ID NO: 4, and a CDR3 comprising an amino acid sequence shown by SEQ ID NO: 6.
  • the immunoglobulin heavy chain variable region comprises a CDR1 comprising an amino acid sequence shown by SEQ ID NO: 2, a CDR2 comprising an amino acid sequence shown by SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence shown by SEQ ID NO: 7.
  • the immunoglobulin heavy chain variable region comprises a CDR1 comprising an amino acid sequence shown by SEQ ID NO: 3, a CDR2 comprising an amino acid sequence shown by SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence shown by SEQ ID NO: 8.
  • the immunoglobulin heavy chain variable region comprises a CDR1 comprising an amino acid sequence shown by SEQ ID NO: 1, a CDR2 comprising an amino acid sequence shown by SEQ ID NO: 4, and a CDR3 comprising an amino acid sequence shown by SEQ ID NO: 25.
  • the immunoglobulin heavy chain variable region comprises a sequence shown by any one selected from SEQ ID NO: 9 to 17 and SEQ ID NO: 26, a conservative substitution variant thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with any one amino acid sequence shown by SEQ ID NO: 9 to 17 and SEQ ID NO: 26.
  • the isolated anti-TfR1 antibody further comprises an IgG Fc domain.
  • the IgG Fc domain is a human IgG Fc domain.
  • the IgG Fc domain is a human IgG Fc domain.
  • the IgG Fc domain is a human IgG1, IgG2, IgG3, or IgG4 Fc domain.
  • the IgG Fc domain comprises a amino acid sequence shown by SEQ ID NO: 18, a conservative substitution variant thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with the amino acid sequence shown by SEQ ID NO: 18
  • the isolated anti-TfR1 antibody of the present application could be a monospecific, or multispecific antibody (including bispecific antibody in this application) .
  • the isolated anti-TfR1 antibody is a full-length antibody, VH, HC, Fab, F (ab') 2, Fab', scFv, or Fv, or a monomer, dimer or multimer thereof.
  • the isolated anti-TfR1 antibody comprises a monovalent, bivalent, trivalent, or tetravalent TfR binding domain (s) .
  • Multispecific antibodies are antibodies that have binding specificities for at least two different antigens.
  • at least one of the binding specificities is for TfR1.
  • multispecific antibodies may bind to two or more different epitopes of the TfR1 protein.
  • the multispecific antibody of the present application has one or more specificities for antigens other than TfR1.
  • the multispecific antibody of the present application has a specificity for a therapeutic or diagnostic target.
  • the therapeutic or diagnostic target comprises one or more selected from the group consisting of: a brain antigen, a tumor-associated antigen, an immune-checkpoint antigen and an immune-checkpoint-associated antigen.
  • the brain antigen is selected from the group consisting of: BACE1, Abeta, EGFR, HER2, Tau, ApoE4, alpha-synuclein, CD20, huntingtin, PrP, LRRK2, parkin, presenilin 1, presenilin 2, gamma secretase, DR6, APP, p75NTR, and caspase 6.
  • the tumor-associated antigen is selected from the group consisting of: A33; ADAM-9; ALCAM; BAGE; beta-catenin; CA125; Carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD36; CD40/CD154; CD45; CD46; CD5; CD56; CD79a/CD79b; CDK4; CEA; CTLA4; Cytokeratin 8; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2/GD3/GM2; HER-2/neu; human papillomavirus-E6; human papillomavirus-E7; JAM-3; KID3; KID31; KSA (17-1A) ; LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosaminyltransferase; Oncostatin M
  • the immune checkpoint protein is selected from the group consisting of 2B4; 4-1BB; 4-1BB ligand, B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; CD19; CD200; CD27; CD27 ligand; CD28; CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; Galectin-9; GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; TIM-3; TIGIT; VSIG8.
  • Multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537, 1983) , WO 93/08829, and Traunecker et al, EMBO J. 10: 3655, 1991) , and "knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168) .
  • Multispecific antibodies can also be made by engineering electrostatic steering effects (WO 2009/089004A1) ; cross-linking two or more antibodies or fragments (see, e.g., US Patent No.
  • a multispecific antibody of the application also encompasses antibodies having three or more functional antigen binding sites, including "Octopus antibodies” or “dual-variable domain immunoglobulins” (DVDs) (see, e.g. US 2006/0025576Al, and Wu et al. Nature Biotechnology, 25(11) : 1290-7, 2007) .
  • a multispecific antibody of the application also encompasses a "Dual Acting Fab” or “DAF” comprising an antigen binding region that binds to TfR as well as the brain antigen (e.g. BACE1 or Tau) (see, US 2008/0069820, for example) .
  • DAF Double Acting Fab
  • BACE1 or Tau brain antigen
  • the anti-TfR antibody can be fused to the carboxy-and/or amino-terminus of a light and/or heavy chain of the second antibody or antigen binding fragment thereof directly or via a linker.
  • heterodimeric mutations were introduced into the Fc of the two heavy chains.
  • Fc mutation include, but are not limited to, the Zymework mutations (see, e.g., US 10,457,742) and the "knob in hole” mutations (see, e.g., Ridgway et al, Protein Eng., 9 (7) : 617-621, 1996) .
  • Other heterodimer mutations can also be used in the application.
  • a modified CH3 as described herein is used to facilitate the formation of a heterodimer between the two heavy chains.
  • the Fc region of the fusion construct or bispecific antibody further comprises one or more mutations that alter (increase or decrease) , preferably eliminate ADCC/CDC (such as the AAS mutations described herein) , and/or one or more mutations that alter (increase or decrease) , preferably increase, the binding of the fusion construct or bispecific antibody to FcRn.
  • Techniques for making multispecific antibodies can also be used to prepare homodimer or homomultimer which is also encompassed in this application, wherein the monomer constituting the homodimer or homomultimer can be any of the anti-TfR antibody of the present application.
  • bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F (ab') 2 bispecific antibodies) .
  • Methods for making bispecific antibodies are known in the art.
  • bispecific antibody traditionally, the recombinant production is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low.
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) , containing the site necessary for light chain binding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm.
  • the anti-TfR1 antibodies of the application is preferably monoclonal.
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of different antibodies.
  • the monoclonal anti-TfR1 antibodies of the application can be made using the hybridoma method or recombinant DNA methods (U.S. Patent No. 4,816,567) .
  • camelid e.g., lama
  • shark is immunized by a whole TfR1 molecule or part of the molecule, for example, a polypeptide comprising the extracellular domain of TfR1, together with an adjuvant.
  • a TfR1 molecule or a polypeptide comprising the extracellular domain of TfR1 molecule may be prepared using methods well-known in the art.
  • animals are immunized with a polypeptide that contains the extracellular domain (ECD) of TfR1 fused to the Fc portion of an immunoglobulin heavy chain.
  • animals are immunized with an TfR1-IgG1 fusion protein.
  • lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986) ) .
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • Preferred myeloma cells are those that fuse efficiently, support stable high- level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against TfR1.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) .
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the binding affinity of the monoclonal antibody can then be determined by conventional methods in the art.
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986) ) .
  • Suitable culture media for this purpose include, for example, HT containing medium, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures.
  • the anti-TfR1 antibodies of the application can be made by using combinatorial libraries to screen for synthetic antibody clones with the desired activity or activities.
  • synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (e.g., VHH) fused to phage coat protein.
  • Such phage libraries are panned by affinity chromatography against the desired antigen.
  • Clones expressing antibodies or antibody fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution.
  • any of the anti-TfR1 antibodies of the application can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-TfR1 antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991) , vols. 1-3.
  • the antibodies produced by naive libraries can be of moderate affinity, but affinity maturation can also be mimicked in vitro by constructing and reselecting from secondary libraries.
  • mutation can be introduced at random in vitro by using error-prone polymerase (reported in Leung et al., Technique, 1: 11-15 (1989) ) in the method of Hawkins et al., J. MoL Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992) .
  • affinity maturation can be performed by randomly mutating one or more CDRs, e.g., using PCR with primers carrying random sequence spanning the CDR of interest, in selected individual VHH clones and screening for higher affinity clones.
  • Another effective approach is to recombine the VH domains selected by phage display with repertoires of naturally occurring V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnol, 10: 779-783 (1992) .
  • phage antibodies of different affinities can be selected from different affinities, even with affinities that differ slightly, for TfR1.
  • random mutation of a selected antibody e.g., as performed in some of the affinity maturation techniques described above
  • phages can be incubated with excess biotinylated TfR1, but with the biotinylated TfR1 at a concentration of lower molarity than the target molar affinity constant for TfR1.
  • the high affinity-binding phages can then be captured by streptavidin-coated paramagnetic beads.
  • Such "equilibrium capture” allows the antibodies to be selected according to their affinities of binding, with sensitivity that permits isolation of mutant clones with as little as two-fold higher affinity from a great excess of phages with lower affinity.
  • Anti-TfR1 clones may be selected based on performance of activity.
  • the application provides anti-TfR1 antibodies that block the binding between TfR1 and its ligand.
  • Anti-TfR1 antibodies of the application possessing the properties described herein can be obtained by screening anti-TfR1 hybridoma clones for the desired properties by any convenient method.
  • the candidate antibody can be tested in a binding competition assay, such as a competitive binding ELISA, wherein plate wells are coated with TfR1, and a solution of antibody in an excess of TfR1 is layered onto the coated plates, and bound antibody is detected enzymatically, e.g. contacting the bound antibody with HRP-conjugated anti-Ig antibody or biotinylated anti-Ig antibody and developing the HRP color reaction., e.g. by developing plates with streptavidin-HRP and/or hydrogen peroxide and detecting the HRP color reaction by spectrophotometry at a certain wavelength with an ELISA plate reader.
  • a binding competition assay such as a competitive binding ELISA
  • a BBB penetration construct comprising the aforementioned anti-TfR antibody.
  • the anti-TfR antibody is coupled with a cargo (e.g., a molecule) in the BBB penetration construct and deliver the cargo into a cell with TfR1 on the surface or a BBB system.
  • the cargo can be a therapeutic agent, a diagnostic agent and/or a tracer, for example neurological disorder drug or agent that can be used to detect or analyze a neurological disorder.
  • such drug or agent can be, without limitation, any chemical entity such as small chemical molecules (such as an antibiotic, antiviral, immunomodulator, antineoplastic, anti-inflammatory, adjuvant, etc.
  • neuropeptides including, but not limited to, nerve growth factor (NGF) , brain derived neurotrophic factor (BDNF) , ciliary neurotrophic factor (CNTF) , glial cell-line neurotrophic factor (GDNF) and insulin-like growth factor (IGF) ; neuropeptide, including, but not limited to, Substance P, neuropeptide Y, vasoactive intestinal peptide (VIP) , gamma-amino-butyric acid (GABA) , dopamine, cholecystokinin (CCK) , endorphins, enkephalins and thyrotropin releasing hormone (TRH) ; cytokine, apolipoprotein, growth factor, antigen, antibody or part of an antibody, adjuvant, etc.
  • NGF nerve growth factor
  • BDNF brain derived neurotrophic factor
  • CNTF ciliary neurotrophic factor
  • GDNF glial cell-line neurotrophic factor
  • IGF insulin
  • nucleic acids such as RNA or DNA of human, viral, animal, eukaryotic or prokaryotic, plant or synthetic origin, etc., including e.g., coding genes, inhibitory nucleic acids such as ribozymes, antisense, interfering nucleic acids, full genomes or portions thereof, plasmids, etc) ; lipids, viruses, markers, or tracers, for instance.
  • the cargo can be any drug active ingredient, whether a chemical, biochemical, natural or synthetic compound.
  • small chemical molecule designates a molecule of pharmaceutical interest with a maximum molecular weight of 1000 Daltons, typically between 300 Daltons and 700 Daltons.
  • the anti-hAnti-TfR antibody of the application can be an effective means to enhance the delivery of a cargo of interest from the blood into the brain and function there.
  • a cargo of interest can be delivered in a combined form or linked to the anti-hAnti-TfR antibody of the application, parenterally, e.g., intravenously.
  • the cargo can be non-covalently coupled (e.g., by ionic, hydrogen, hydrophobic or Van der Waals bonds) to the anti-hAnti-TfR antibody and/or covalently attached to the anti-hAnti-TfR antibody to form a conjugate.
  • the coupling of the cargo and the anti-hAnti-TfR antibody can be cleavable or noncleavable in physiological medium or within cells.
  • the coupling can be made at various reactive groups, and notably at one or more terminal ends and/or at one or more internal or lateral reactive groups. Coupling can also be carried out using genetic engineering. It is preferable that the interaction is sufficiently strong so that the cargo is not dissociated from the anti-hAnti-TfR antibody before having reached its site of action.
  • the conjugation is by construction of a protein fusion (i.e., by genetic fusion of the two genes encoding the anti-hAnti-TfR antibody and a neurological disorder drug and expression as a single protein) .
  • a protein fusion i.e., by genetic fusion of the two genes encoding the anti-hAnti-TfR antibody and a neurological disorder drug and expression as a single protein.
  • Known methods can be used to link the cargo to an anti-hAnti-TfR antibody of the present disclosure.
  • the cargo and the anti-TtR antibody are covalently linked (or conjugated) via a non-peptide linker or a peptide linker, e.g., the BBB penetration construct is a conjugate comprising the cargo and the anti-TtR antibody.
  • the BBB penetration construct is an antibody-drug conjugate (ADC) .
  • non-peptide linkers include, but are not limited to, polyethylene glycol, polypropylene glycol, copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymer, polymerized lipid, chitins, and hyaluronic acid, or derivatives thereof, or combinations thereof.
  • a peptide linker can be a peptide chain consisting of 1 to 50 amino acids linked by peptide bonds or a derivative thereof, whose N terminus and C terminus can be covalently linked to the anti-TfR antibody.
  • Therapeutic formulations comprising the aforementioned anti-TfR1 antibody, polynucleotides (including DNA, RNA or a heterozygous molecule of DNA and RNA) , vectors, or host cells comprising the coding sequence of the anti-TfR1 antibody, or the BBB penetration construct are prepared for storage by mixing the anti-TfR1 antibody, polynucleotides, vectors, host cells, or the BBB penetration construct of the present disclosure with the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy 20th edition (2000) ) , in the form of aqueous solutions, lyophilized or other dried formulations.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes; and/or non-ionic surfactants such as TWEEN TM , PLURONICS TM or poly
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly- (methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulin of the application, which matrices are in the form of shaped articles, e.g., films, or microcapsule.
  • the application provides methods for transporting a therapeutic or diagnostic agent across the blood-brain barrier (BBB) comprising exposing the anti-TfR antibody coupled to a therapeutic or diagnostic agent and forming the aforementioned BBB penetration construct to the blood-brain barrier such that the antibody or antigen binding fragment thereof transports the agent coupled thereto across the blood-brain barrier.
  • the agent is a neurological disorder drug.
  • the agent is an imaging agent or an agent for detecting a neurological disorder.
  • the BBB is in a mammal, preferably a primate, such as a human, more preferably a human having a neurological disorder.
  • the neurological disorder is selected from the group consisting of Alzheimer's disease (AD) , stroke, dementia, muscular dystrophy (MD) , multiple sclerosis (MS) , amyotrophic lateral sclerosis (ALS) , cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, and traumatic brain injury.
  • the anti-TfR antibody or the BBB penetration construct is used to detect a neurological disorder before the onset of symptoms and/or to assess the severity or duration of the disease or disorder.
  • the antibody, antigen binding fragment or conjugate thereof permits detection and/or imaging of the neurological disorder, including imaging by radiography, tomography, or magnetic resonance imaging (MRI) .
  • MRI magnetic resonance imaging
  • the anti-TfR antibody or the BBB penetration construct is used in treating a neurological disorder (e.g., Alzheimer's disease) , comprising administering to a subject in need of the treatment an effective amount of the anti-TfR or the BBB penetration construct.
  • the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent.
  • the application relates to the use of the anti-TfR antibody or the BBB penetration construct in the manufacture or preparation of a medicament.
  • the medicament is for treatment of neurological disease or disorder.
  • the medicament is for use in a method of treating neurological disease or disorder comprising administering to an individual having neurological disease or disorder an effective amount of the medicament.
  • Another general aspect of the application relates to a method of inducing antibody dependent phagocytosis (ADP) without stimulating secretion of a pro-inflammatory cytokine in a subject in need thereof: comprising administering to the subject a complex comprising a therapeutic antibody or antigen binding fragment thereof coupled to, preferably covalently conjugated to the anti-TfR antibody, wherein the therapeutic antibody or antigen binding fragment thereof does not have effector function.
  • the therapeutic antibody or antigen binding fragment thereof can comprise one or more amino acid modifications that reduces or eliminates the effector function, such as the ADCC or CDC, such as mutations that reduce or abolish the binding to Fc gamma receptor.
  • the therapeutic antibody or antigen binding fragment thereof binds specifically to tau aggregates.
  • an additional therapeutic agent is a therapeutic agent effective to treat the same or a different neurological disorder as the anti-TfR antibody.
  • additional therapeutic agents include, but are not limited to: the various neurological drugs described above, cholinesterase inhibitors (such as donepezil, galantamine, rovastigmine, and tacrine) , NMDA receptor antagonists (such as memantine) , amyloid beta peptide aggregation inhibitors, antioxidants, gama-secretase modulators, nerve growth factor (NGF) mimics or NGF gene therapy, PPARy agonists, HMS-CoA reductase inhibitors (statins) , ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulin, muscarinic receptor agonists, nicrotinic receptor modulators, active or passive amy
  • the at least one additional therapeutic agent is selected for its ability to mitigate one or more side effects of the neurological drug.
  • the additional therapeutic agent can be administered in the same or separate formulations and administered together or separately with the anti-TfR antibody or the BBB penetration construct.
  • the anti-TfR antibody or the BBB penetration construct can be administered prior to, simultaneously with, and/or following, the administration of the additional therapeutic agent and/or adjuvant.
  • the anti-TfR antibody or the BBB penetration construct can also be used in combination with other interventional therapies such as, but not limited to, radiation therapy, behavioral therapy, or other therapies known in the art and appropriate for the neurological disorder to be treated or prevented.
  • other interventional therapies such as, but not limited to, radiation therapy, behavioral therapy, or other therapies known in the art and appropriate for the neurological disorder to be treated or prevented.
  • the appropriate dosage of an antibody of the application (when used alone or in combination with other agents will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one or multiple times.
  • about 1 ⁇ g/kg to 15 mg/kg (e.g., 0.1mg/kg-10mg/kg) of antibody is a intestinalte dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • the anti-TfR antibody or the BBB penetration construct can be admimstered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration, depending in part on whether the administration is brief or chronic.
  • the appropriate dosage of the anti-TfR antibody or the BBB penetration construct (when used alone or in combination with one or more other additional therapeutic agents) will depend on various factors, such as the type of disease to be treated, the type of antibody or conjugate, the severity and course of the disease, whether the antibody, antigen binding fragment or conjugate thereof is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, the physiological state of the subject (including, e.g., age, body weight, health) , and the discretion of the attending physician. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • hTfR Human Transferrin R Protein
  • ACRO Human Transferrin R Protein
  • ACRO Human Transferrin R Protein
  • ACRO Mouse Transferrin R Protein with His Tag
  • 500 ⁇ g each were mixed with Freund's adjuvant and given subcutaneously to alpacas via repetitive immunization at multiple sites at 2-week intervals.
  • PBMCs were taken.
  • Total RNA of the PBMCs was extracted and reverse transcribed to cDNA.
  • the cDNA was used as template to amplify gene sequences of antibodies.
  • the gene sequences were digest by restriction enzyme and linked to phage vector.
  • the phage vector was electrotransferred to TG1 competent cells to prepare phage library.
  • the obtained phage library was subjected to two rounds of in vitro screening and three rounds of in vivo screening in mice.
  • the phage library obtained from in vitro screening was amplified and screened in vivo.
  • 6–8-week-old BALB/c mice (Vital River) were administered with the phage at a dose of 5E+11 cfu/100 ⁇ L via tail vein injection and executed with CO2 after 2h.
  • the brain tissues of the mice were collected. Surface capillaries were removed, 3 times the volume of homogenate (1%TritonX-100+1%PMSF in PBS) was added, and brain tissue was homogenized for 10 s with a medium-speed homogenizer, added with dextran, and centrifuged at 14, 800g for 25 min at 4°C to obtain the supernatant.
  • the supernatant was used to infect E. coli TG1 for phage amplification.
  • the third and fourth rounds of in vivo screening were repeated. The in vivo screening process was repeated for another two times.
  • Single clones were selected from the screened phage library for culture, and the supernatant of each culture was taken.
  • the binding of the monoclonal to human mouse antigen and blocker was detected by ELISA, and the monoclonal that bound to both human and mouse antigens and not bound to blocker was selected for sequencing.
  • the sequencing results were analyzed and the sequences of NX17, NX54, NX56, NX96 and other unique clones were obtained.
  • Example 2 ELISA and BLI assay for the binding abilities of anti-TfR antigen-binding fragments to human and mouse TfR
  • Each VHH sequence was linked to an Fc using (G4S) n linker to form full-length heavy chain with Fc fragment (VHH-Fc) .
  • the Fc region could be either native Fc regions or variant Fc regions.
  • a variant Fc region may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) with an amino acid modification (e.g., a substitution) at one or more amino acid positions.
  • the exemplary molecules used herein is a homodimer of VHH-Fc (IgG1) .
  • VHH-Fc format NX56 amino acid sequence was set forth in SEQ ID NO: 19.
  • a monoclonal antibody such as 'NX56'
  • a humanized monoclonal antibody such as 'HuNX5601'
  • VHH-Fc antibodies The method for the expression of the VHH-Fc antibodies were as follows:
  • the gene encoding the variable region was amplified using PCR, the signal peptide was added by digestion and overlap extension PCR, and ligated to the vector pCMV3 with antibody Fc (IgG1) sequence. After amplified by PCR, it was sent to a sequencing company for sequencing. The amino acid sequence corresponding to the DNA sequence of the anti-TfR antibody was obtained. The homodimer was formed by pairing two VHH- (G4S) 3 -Fc monomers.
  • Expi293 cells were transfected with the vector and cultured at 37°C/5%, CO 2 /125rpm in a shaker, and the supernatant containing the expressed proteins was collected after 3-4 days of transient expression. 5 days after the transient expression, the supernatant was quantified by 384, and purified by Protein A affinity chromatography to obtain the antibody. The antibody concentration was determined according to the extinction coefficient at A280.
  • Anti-hTfR VHH-Fc antibodies (pre-experiments have demonstrated that the antibodies do not bind to human holo-Tf) and the control antibody V201 (described in detail in WO2020/144233AI, the sequence is shown below) of different concentrations were tested by ELISA using 96-well plates coated with human TfR antigen (Acro #CD1-H5243, 0.5 ⁇ g/mL, 100 ⁇ L per well) . Binding of VHH-Fc antibodies were analyzed and compared using GraphPad Prism 5.
  • amino acid sequence of V201 is set forth in SEQ ID NO: 20.
  • Anti-hTfR VHH-Fc antibodies (pre-experiments have demonstrated that the antibodies do not bind to human holo-transferrin) of different concentrations were tested by ELISA using 96-well plates coated with mouse TfR antigen (Acro #TFR-M524b, 0.5 ⁇ g/mL, 100 ⁇ L per well) . Binding of VHH-Fc antibodies were analyzed and compared using GraphPad Prism 5.
  • FIGs. 1A-1B and Tables 1 depict the binding activities of the analyzed antibodies.
  • the results show that the screened TfR antigen-binding fragments have binding activities similar to those of the control antibodies (V201) .
  • NX56 demonstrated better biological activities relative to the control.
  • Table 1 Summary of human EGFR binding/block ELISA of the anti-EGFR antigen-binding fragments
  • 1%BSA in PBST (0.04%Tween 20) buffer solution and 100 nM glycine regeneration solution (pH 1.5) were prepared.
  • the binding kinetics of NX56 was then determined using an R8 BLI Protein Analysis System (Sartorius) .
  • the Protein A sensor was pre-wetted with the PBST buffer solution 10 min in advance. Afterwards, the Sartorius ProA Biosensors were immersed in baseline PBST buffer for 60 s. The sensors were then loaded with 5 ⁇ g/mL NX56 antibody for 180 s. After loading, the sensors were immersed in a second baseline containing PBST buffer.
  • the sensor was then immersed in different concentrations (250, 125, 62.5, 31.25, 15.625, 7.83, 3.91, 0 nM) of hTfR for 180s for the binding step and then transferred back into PBST buffer for 180s for the dissociation step.
  • the sensor was regenerated in 10 mM glycine regeneration solution (pH 1.5) for 10 s and immersed in PBST buffer for 10 s for washing, and this step was repeated three times to completely dissociate the previous proteins.
  • NX56 binds to both human-and mouse-derived TfR and has weaker affinity to both than the control antibody V201. Thus, NX56 may have a better BBB-crossing potential.
  • Example 3 Binding of anti-TfR VHH to human TfR positive hCMEC cells
  • hCMEC Human coronary microvascular endothelial cell
  • a desired anti-TfR antibody do not compete with Holo-Tf.
  • the competitive binding of anti-TfR antibody and Holo-Tf of to human transferrin was determined by ELISA. Each well was first coated with 100 ul ELISA coating buffer prepared with 0.5 ⁇ g/mL antigen (Acro #CD1-H5243) overnight at 4°C. 250 ⁇ L of 0.05%PBST was added for 3 washes, followed by adding 200 ⁇ L of blocking buffer and incubating at room temperature for 1 h. After 3 washes, human Holo-Tf (Sigma, #T0665-50MG) was diluted to 2.5 ⁇ M with blocking buffer (MACKLIN, #B824162) .
  • the anti-hTfR VHH-Fc antibody was diluted in gradient (antibody concentrations 10, 2.5, 0.625, 0.156, 0.039, 0.00977, 0.00244, 0.00244, and 0.0024) with blocking buffer (MACKLIN, #B824162) and 2.5 ⁇ M Holo-Tf (Sigma, #T0665-50MG) diluted in the blocking buffer, respectively.
  • the diluted antibody was added to plates coated with 0.5 ⁇ g/mL of antigen (Acro #CD1-H5243) . The plate was incubated at room temperature for 1 h and then washed 3 times.
  • Fc-blocked hCMEC cells were plated in 96-well plates (Corning #3599) . 20, 000 cells per well were re-placed in 50 ul of DMEM complete medium and incubated with 50 ul of 0.2 ⁇ M NX56, V201, or IgG1 (DMEM complete medium preparation) at 37°C for 0 h, 5 h, 24 h. Trypsin digestion was performed, cells were collected by centrifugation and analyzed using a CytoFLEX S flow cytometer (BECKMAN) . According to the results shown in FIG. 4, the endocytosis of NX56 in hCMEC cells is slightly enhanced when compared with that of the positive control V201, while the case is not the same in CHO cells (hTFR negative) , indicating hTFR target specificity.
  • Plates (7.5 ⁇ g/cm2) were coated with Rat tail collagen (Gibco TM A1048301) for 1 h, followed by Human plasma fibronectin (Sigma #F0895) at a final concentration of 5 ⁇ g/mL for 1 h.
  • Rat tail collagen Rat tail collagen
  • Human plasma fibronectin Human plasma fibronectin
  • 150, 000 hCMEC cells per well were inoculated in 12-well plates (Corning #3460) .
  • the culture medium was changed every 2 days for 8 consecutive days and the resistance values were measured continuously.
  • 1.5 mL centrifuge tubes and basal side of Transwell chambers for use in the assay were coated with 1%BSA in PBS overnight.
  • the 1.5 mL centrifuge tubes and the basal side of the Transwell chambers were coated with medium for 1 h.
  • Antibodies were diluted with 500 ⁇ L medium to a final concentration of 100 nM, and added to the apical side where the hCMEC present. 1000 ⁇ L of medium was added to the basal side. After 1 h of incubation, the medium at the apical and basal sides were replaced. After 1 h, 4 h and 24 h of incubation, the medium at the basal side was harvested for ELISA. The results are shown in FIG. 5, indicating that the anti-TfR antibody NX56 has better internalization and transmembrane effects when using hCMEC cells and may have a superior brain entry potential.
  • mice Six 6-12 weeks old Balb/c mice (Vital River) were prepared, divided into 3 groups of 2 and labeled; mice were injected via the tail vein with antibodies at a dose of 10 mg/kg; 2 hours later, CO2 execution was performed, and blood and brain tissue was collected. Surface capillaries were removed, 3 times the volume of homogenate (1%TritonX-100+1%PMSF in PBS) was added, and brain tissue was homogenized for 10 s with a medium-speed homogenizer, and centrifuged at 14,800g for 25 min at 4°C. The supernatant was carefully pipetted and determined for brain tissue antibody content by Double-antigen sandwich ELISA.
  • homogenate 1%TritonX-100+1%PMSF in PBS
  • the antigen (anti-hFc) was coated with coating buffer at a concentration of 2 ug/ml and 100 ul per well overnight at 4°C.
  • the plate was washed 3 times with 0.05%PBST, 150 ul of blocking buffer was added to each well, and the plate was blocked at room temperature for an hour; the plate was washed 3 times with 0.05%PBST.
  • the brain tissue homogenate was diluted 2 times and 8 times, respectively, and 100 ul of which were added to the plate per well and incubated for one hour at room temperature.
  • the plate was washed 3 times with 0.05%PBST. 100 ul/well of the corresponding secondary antibody (1: 10k) was added and incubated for one hour.
  • the plate was washed 3 times with 0.05%PBST and 50 ul of TMB was added to each well for color development under lightproof condition. After 10 minutes, the termination solution was added. Values as 450 nm wavelength was read and analyzed using a microplate reader.
  • IgG1-isotype is Anti-HEL Human IgG1-Kappa Isotype control (B117901, Biointron)
  • the anti-TfR antibody concentrations in brain and plasma of mice were measured after intravenous injection of control IgG1-isotype, V201 and anti-TfR antibody NX56 (10 mg/kg) .
  • NX56 showed a highly significant increase compared to negative control IgG, and a significant increase compared to reference antibody V201, in brain tissue antibody concentration within 2 h.
  • Example 8 Determination of neurotensin (NT) delivered to the mouse brain by anti-TfR antibodies
  • NX56-Fc-NT was linked to the C-terminus of the Fc (IgG1) of the test antibody NX56 via the G4S linker to obtain the full sequence (NX56-Fc-NT) as follows.
  • the sequence has a structure of VHH- (G4S) 3-Fc-(G4S) 3-NT (from N to C terminus) .
  • a fusion protein was a homodimer of the sequence.
  • the amino acid sequence of NX56-Fc-NT is set forth in SEQ ID NO: 21.
  • mice Nine 6-12-week-old Balb/c mice were prepared and divided into 3 groups of 3, and labeled.
  • Six 6-12-week-old hTFR KI mice were prepared, divided into 2 groups of 3, and labeled.
  • 3 mg/ml and 10 mg/ml of antibody and IgG1-isotype were prepared and inject the mice into the tail vein.
  • the anal temperature of the mice was measured at 0h, 0.5h, 1h and 2h after intravenous injection.
  • the results of the experiment are shown in FIG. 6.
  • the results showed that the delivery of NT (neurotensin) to the brain of both WT and hTFR KI mice by NX56 significantly reduced the body temperature of mice. And the hypothermia induced by NX56-Fc-NT within 2 hours was not dose-dependent.
  • the NX56 sequence was compared with the human heavy chain sequences in the database, and the two human sequences with the highest sequence homology, IGHV3-23*01 and vh3_H, were selected, and the CDR region of NX56 was Grafted onto IGHV3-23*01 and vh3_H to form a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where amino acid residues are identified and annotated by the Kabat numbering system.
  • sequences after grafting were scored according to the BM SCORE scoring process described in literature DOI: 10.1007/978-1-0716-2075-5_14, and the amino acid sites with a score greater than 1 were selected.
  • the back mutation phage library was constructed and single clones were picked after two rounds of phage library screening, and the ELISA-positive clone was selected for sequencing to obtain the unique clone sequence, and the clone sequence was analyzed and scored according to HSC scoring as described in literature Doi: 10.1016/j. molimm. 2006.09.029.
  • the obtained humanized variable region sequences were set forth in SEQ ID NO: 9 to 14.
  • G44E indicates a mutation of G at position 44 back to E according to the Kabat numbering system.
  • the antibodies were prepared by combing the constant region of human Fc-IgG1 to the variable region of VHH or humanized versions thereof.
  • Table 5 Summary of human and mouse TfR binding of the anti-TfR antigen-binding fragments by BLI
  • mice Fourteen 6-12 weeks old Balb/c mice were prepared, divided into 7 groups of 2 and labeled properly. Mice were injected via tail vein. Mice were sacrificed according to the time shown in Table 6, and blood and brain tissue were taken for ELISA assay as described in Example 7.
  • Example 10 The screening of AI designed TfR-binding.
  • hTfR binders to similar epitopes on hTfR can be a good starting point for hTfR binder design.
  • hTfR was first split into peptides with length ranging from 5 to 20. Then we blasted for protein epitopes with similar sequences to the peptides and checked if there is any protein binding to the epitopes. ⁇ 1000 of complexes consisting of hTfR-like sequence and its binder were found in our search.
  • VHHs were homologously recombined with the hIgG1 constant region gene to construct the full-length sequence of the antibody (VHH-Fc) .
  • NX759 VHH amino acid sequence shown by SEQ ID NO: 15
  • NX788 VHH amino acid sequence shown by SEQ ID NO: 16
  • Example 12 Competitive assay for Anti-TfR Antibodies with Holo-Tf determined by BLI
  • Sartorius Discovery software (v 12.2) was used.
  • the SA Biosensor was immersed in 1%BSA in PBST (0.04%Tween 20) buffer solution for 10 min. After loading the probe, it was immersed in Sartorius PBST (0.04%Tween 20) buffer solution for 30 s. The probe was then immersed in 5 ⁇ g/ml hTfR solution for 120 s until the signal value was 1. Afterwards, the probe was immersed in PBST buffer for 30 s, then in 100 nM antibody solution for test and bound the antibody until saturation, followed by 100 nM Holo-Tf antibody solution to bind until saturation.
  • the probe was regenerated in 10 mM glycine regeneration solution (pH 1.5) for 10s and the probe was rinsed by dipping into PBST buffer for 10 s. This step is repeated three times to completely dissociate the previous protein.
  • Data analysis was performed using Sartorius Analysis Studio to calculate the percentage inhibition during each step involving antibodies by taking the competitive antibody signal and dividing it by the maximum signal of the same antibody during the step used as saturation. This number was multiplied by 100 to obtain the percentage inhibition, with a lower percentage indicating a smaller epitope shift and greater competition between the two antibodies. The results of the calculation are shown in Table 2.
  • the competitive antibody signal was taken and divided by the maximum signal of the same antibody during the saturation step to calculate the percentage inhibition. 0-30 is fully competitive, 30-70 is partially competitive and >70 is not competitive.
  • Example 13 Test for the binding of Anti-TfR antibody to hCEMC cells
  • the anti-TfR antibody binding activity to hCEMC cells was detected by flow cytometry in the same way as in Example 3. The results are shown in FIG. 7.
  • FIG. 7 shows that NX759 and NX88 bind effectively to hCMEC cells, and both have stronger binding ability than the positive control V201.
  • Example 14 Internalization assay on hCEMC cells for anti-TfR antibodies
  • the internalization assay on hCEMC cells for anti-TfR antibody was performed by referring to Example 4. The results of the internalization assay are shown in FIG. 8.
  • FIG. 8 shows that hCMEC cells have stronger endocytosis of NX759 and NX788, which is significantly stronger than that of the positive control 201, suggesting that NX759 and NX788 have better brain entry potential.
  • Example 15 Anti-TfR antibodies in hTFR KI mice in vivo blood-brain barrier penetration assay
  • mice Six 6-12 weeks old Balb/c mice were prepared, divided into 3 groups of 2 and labeled; and injected via tail vein. Mice were sacrificed according to the time shown in Table 9, and blood and brain tissue were taken for ELISA assay as described in Example 7.
  • Example 16 Delivery of neurotensin (NT) to mouse brain by anti-TfR antibody
  • the anti-TfR antibodies NX759-Fc-NT (amino acid sequence shown by SEQ ID NO: 23) and NX788-Fc-NT (amino acid sequence shown by SEQ ID NO: 24) were constructed in the same way as Example 8.
  • mice Twelve 6-12 weeks old hTFR KI mice were prepared and divided into 4 groups of 3. All mice were labled. 10 mg/ml of antibody and IgG1-isotype were prepared and inject into mice via tail vein. The anal temperature of the mice was measured at 0h, 0.5h, 1h and 2h after injection. The results are shown in FIG. 9.
  • Example 17 Design and detection of reduced affinity of anti-TFR antibodies after humanization
  • a spectrum of affinity between TfR and TfR nanobody may provide the possibility to find candidates with better performance in drug delivery efficiency.
  • VHH-Fc antibody for affinity determination was constructed in the same way as in Example 2
  • the affinity of humanized anti-TfR antibody to TfR was determined by BLI as described in Example 2.
  • Donanemab is an antibody produced in mice that targets amyloid ⁇ (A ⁇ ) to remove the excess protein that causes brain burden and is used to slow the progression of early Alzheimer's disease.
  • a ⁇ amyloid ⁇
  • the anti-TfR antibody huNX5604 and the affinity-reduced anti-TfR antibody huNX5604A were linked to the C-terminus of Donanemab Fc with flexible linker, respectively.
  • “Knob in Hole” mutations were further introduced to the Donanemab Fc segment.
  • a bispecific antibody was constructed to test the transferring ability of the presently disclosed Anti-TfR antibody for full-length antibodies.
  • the structure of the bispecific antibody is shown in Figure 10.
  • the light chain sequences of the bispecific antibodies were the same as that of the Donanemab.
  • the heavy chain sequences of the bispecific antibodies were shown by SEQ ID NO: 29 (Don5604A Heavy chain 1 (knob) ) , SEQ ID NO: 30 (Don5604A Heavy chain 2 (hole) and Don5604 Heavy chain 2 (hole) ) , and SEQ ID NO: 31 (Don5604 Heavy chain 1 (knob) ) .
  • mice Ten 6-12 weeks old hTFR KI mice were prepared, divided into 5 groups of 2 and labeled; and injected through tail vein. 6 hours after intravenous injection, 25 ml of PBS containing 1 Eu/ml heparin was used for cardiac perfusion and subsequent operations. Brain tissue homogenates were taken for ELISA assay as in Example 7. The results (shown in FIG. 11) indicate that the anti-TfR antibody could effectively carry the full-length antibody into the brain of TfR transgenic mice (hTFR knocked in mice) .

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Abstract

L'invention concerne un anticorps anti-TfR, qui facilite l'endocytose médiée par le récepteur de la transferrine, et n'interfère pas avec le transport normal de la transferrine. L'invention concerne également l'utilisation de l'anticorps anti-TfR.
PCT/CN2024/098331 2023-07-04 2024-06-11 Anticorps anti-tfr1 et leurs utilisations Pending WO2025007703A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109476728A (zh) * 2016-07-14 2019-03-15 生命北极公司 脑部递送蛋白
WO2020144233A1 (fr) * 2019-01-09 2020-07-16 Vect-Horus Molécules de liaison au récepteur de transferrine, leurs conjugués et leurs utilisations
WO2022258841A1 (fr) * 2021-06-11 2022-12-15 Bioarctic Ab Molécule de liaison bispécifique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109476728A (zh) * 2016-07-14 2019-03-15 生命北极公司 脑部递送蛋白
WO2020144233A1 (fr) * 2019-01-09 2020-07-16 Vect-Horus Molécules de liaison au récepteur de transferrine, leurs conjugués et leurs utilisations
WO2022258841A1 (fr) * 2021-06-11 2022-12-15 Bioarctic Ab Molécule de liaison bispécifique

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KAWABATA HIROSHI: "Transferrin and transferrin receptors update", FREE RADICAL BIOLOGY & MEDICINE, vol. 133, 1 March 2019 (2019-03-01), US , pages 46 - 54, XP093259592, ISSN: 0891-5849, DOI: 10.1016/j.freeradbiomed.2018.06.037 *
PARDRIDGE WILLIAM M.: "Treatment of Alzheimer’s Disease and Blood–Brain Barrier Drug Delivery", PHARMACEUTICALS, vol. 13, no. 11, CH , pages 394 - 394-25, XP093259593, ISSN: 1424-8247, DOI: 10.3390/ph13110394 *
RUÉ LAURA, JASPERS TOM, DEGORS ISABELLE M. S., NOPPEN SAM, SCHOLS DOMINIQUE, DE STROOPER BART, DEWILDE MAARTEN: "Novel Human/Non-Human Primate Cross-Reactive Anti-Transferrin Receptor Nanobodies for Brain Delivery of Biologics", PHARMACEUTICS, vol. 15, no. 6, CH, pages 1748, XP093119084, ISSN: 1999-4923, DOI: 10.3390/pharmaceutics15061748 *
TONG ZHEN-HUA, QIAN NI-LIANG, ZHANG JING, CUI XIN-LING, LIU YUN-HUI, GAO XIN, SONG HAI-FENG: "Screening, Construction and Expression of Human Anti-TfR scFv Antibody", LETTERS IN BIOTECHNOLOGY, vol. 27, no. 3, 30 May 2016 (2016-05-30), CN , pages 358 - 361, XP093259598, ISSN: 1009-0002, DOI: 10.3969/j.issn.1009-0002.2016.03.013 *

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