ANTI-TfR1 ANTIBODIES AND USES THEREOF

Abstract

The present application provides an anti-TfR antibody that promotes endocytosis mediated by a transferrin receptor and does not interfere with normal transferrin transport. Uses of the anti-TfR antibodies are also provided.

Description

Cross-references to related applications

[0001] This application claims priority to international patent application PCT / CN2023 / 105681, filed on July 4, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to antibodies that bind to transferrin receptor (TfR) proteins, specifically human TfR1 protein, and their use in disease treatment and diagnosis. Background Technology

[0003] Receptor-mediated endocytosis (an endogenous endocytosis process in which ligands are transported across the endothelial cell barrier) is a promising approach to facilitate the delivery of protein therapies across the blood-brain barrier (BBB). Antibodies targeting the transferrin receptor (TfR), which is highly expressed on endothelial cells constituting the BBB, have been reported to cross the BBB. High affinity ensures that antibodies bind to TfR and are taken up by brain endothelial cells even at low blood concentrations, but may also reduce the probability of antibody release from central nervous system (CNS) blood vessels, potentially preventing antibody accumulation in the brain parenchyma. The use of antibodies with low affinity for TfR has been reported to increase antibody release from cerebral vascular endothelial cells and enhance intracerebral uptake and distribution. Invention Overview This article describes isolated antibodies that specifically bind to the transferrin receptor (TfR) and their applications. The antibodies described in this article possess advantages such as ideal affinity, high blood-brain barrier penetration efficiency, and minimal or no competition with transferrin (Tf), indicating their potential to enhance the efficacy and safety of brain-targeted drug delivery.

[0005] This article describes, in one aspect, a separated antibody or antigen-binding fragment thereof that binds to the transferrin receptor (TfR), comprising an immunoglobulin heavy chain variable region, said variable region comprising: 1) CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR2 containing the amino acid sequence shown in SEQ ID NO: 4, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; 2) CDR1 containing the amino acid sequence shown in SEQ ID NO: 2, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 7; 3) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 8; or 4) Complementarity-determining region (CDR) 1 containing the amino acid sequence GRTLRVSAYGMG (SEQ ID NO: 1), CDR 2 containing the amino acid sequence AISQWGVGNTYYADSVKG (SEQ ID NO: 4), and CDR 3 containing the amino acid sequence DTSPVTWYPASGYHYDA (SEQ ID NO: 25); CDR1 ranges from amino acid residues H26 to H35B, CDR2 ranges from amino acid residues H50 to H65, and CDR3 ranges from amino acid residues H95 to H102, with residue numbering following the Kabat numbering scheme.

[0006] In some embodiments, the immunoglobulin heavy chain variable region comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In some embodiments, the TfR is TfR1. In some embodiments, the TfR is human TfR. In some embodiments, the TfR is human TfR1 (hTfR1).

[0007] In some implementations, the antibody is a VHH antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0008] This article also describes a separated antibody or its antigen-binding fragment that binds to the transferrin receptor (TfR), comprising: (i) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWVRQAPGKERELVAAISQWGVGNTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 9); (ii) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKELELVSAISQWGVGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 10); (iii) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKGRELVSAISQWGVGNTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 11); (iv) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKGLELVSAISQWGVGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYVCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 12); (v) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKELELVSAISQWGVGNTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYVCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 13); (vi) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKGRELVSAISQWGVGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYVCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 14); (vii) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASSRTFSRIQMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCGGSFGPEWDVGGYDYWGQGTQVTVSS (SEQ ID NO: 15); (viii) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASSRTFSSLQMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCGGSWGPEWDQGGYDYWGQGTQVTVSS (SEQ ID NO: 16); (ix) The heavy chain variable region containing CDR1, CDR2, and CDR3, with the following amino acid sequence: QVQLQESGGGLVQAGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKERELVAAISQWGVGNTYYADSVKGRFTISRDNAKNTVYLQLNSLKPEDTAVYVCAADTSPVTWYPADGYHYDAWGQGTQVTVSS (SEQ ID NO: 17); or (x) The heavy chain variable region containing CDR1, CDR2, and CDR3 has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKGLELVSAISQWGVGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYVCAADTSPVTWYPASGYHYDAWGQGTLVTVSS (SEQ ID NO: 26); and in which The CDR is defined by a CDR definition scheme selected from the following group: Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition, North definition, or a combination thereof.

[0009] In some embodiments, the heavy chain variable region is derived from camels. In some embodiments, the heavy chain variable region is derived from mammals other than camels. In some embodiments, the heavy chain variable region is derived from rodents or primates. In some embodiments, FR1 to FR4 are derived from the human immunoglobulin heavy chain. In some embodiments, FR1 to FR4 are derived from the III subgroup framework sequence of human immunoglobulin. In some embodiments, FR1 to FR4 are derived from IGHV3-23*01+IGHJ4*04, vh3_H, IGHV3-30*02, IGHV3-23*04, or IGHV3-64*04. In some embodiments, FR1, FR2, FR3, and FR4 may be from the same species or different species.

[0010] In some embodiments, the isolated antibody or antigen-binding fragment thereof of this disclosure comprises any amino acid sequence selected from SEQ ID NO:9 to SEQ ID NO:17 or a conserved substitution variant thereof. In some embodiments, the isolated antibody or antigen-binding fragment thereof of this 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 amino acid sequence selected from SEQ ID NO:9 to SEQ ID NO:17. In some embodiments, the amino acid sequence of the isolated antibody is as shown by any amino acid sequence selected from SEQ ID NO:9 to SEQ ID NO:17.

[0011] In some implementations, the isolated antibody is a single-domain antibody, a full-length antibody, IgG, Fab, F(ab')2, Fab', scFv, or Fv.

[0012] In some embodiments, the isolated antibody further comprises a human IgG Fc domain, preferably an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the human IgG Fc domain comprises an amino acid sequence as shown in SEQ ID NO: 18, its conserved substitution variants, 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 in SEQ ID NO: 18. In some embodiments, the IgG Fc domain is linked to the heavy chain variable region directly or via a linker. In some embodiments, the linker is a peptide. In some embodiments, the linker is a GS linker. In some embodiments, the linker is a G4S linker. In some embodiments, the IgG Fc domain is linked to the carboxyl terminus of the heavy chain variable region.

[0013] In some embodiments, the isolated antibody comprises monovalent, bivalent, trivalent, or tetravalent TfR binding domains. In some embodiments, each TfR binding domain in the bivalent, trivalent, or tetravalent TfR binding domain binds to the same epitope of the TfR. In some embodiments, the amino acid sequences of each TfR binding domain in the bivalent, trivalent, or tetravalent TfR binding domain are identical. In some embodiments, each TfR binding domain in the bivalent, trivalent, or tetravalent TfR binding domain binds to a different epitope of the TfR. In some embodiments, the amino acid sequences of each TfR binding domain in the bivalent, trivalent, or tetravalent TfR binding domain are different.

[0014] In some embodiments, the isolated antibody is a bispecific or multispecific antibody that also includes a binding domain for binding to a therapeutic or diagnostic target. In some embodiments, the therapeutic or diagnostic target includes a brain antigen.

[0015] In some implementations, the brain antigen is selected from one or more of the following groups: β-secretase 1 (BACE1), β-amyloid protein (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau protein, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin protein, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin protein, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, G protein-coupled receptor (GPCR), and glucocerebrosidase.

[0016] In some implementations, the therapeutic or diagnostic target includes one or more selected from the group consisting of tumor-associated antigens, immune checkpoint antigens, and immune checkpoint-associated antigens.

[0017] In some implementations, tumor-associated antigens are selected from the following group: A33; ADAM-9; ALCAM; BAGE; β-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; GAG E-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-acetylglucosamine transferase; tumor suppressor M; p15; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-α receptor; TNF-γ receptor; transferrin receptor; and VEGF receptor.

[0018] In some implementations, the immune checkpoint proteins are selected from the following group: 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; galactoglobulin-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.

[0019] In some embodiments, the isolated antibody is a bispecific or multispecific antibody, wherein a TfR binding domain binds to or is linked to an antibody containing a binding domain that binds to a therapeutic or diagnostic target. In some embodiments, the antibody containing a binding domain that binds to a therapeutic or diagnostic target includes an Fc region, and the TfR binding domain is linked to the C-terminus of the Fc region. In some embodiments, the antibody containing a binding domain that binds to a therapeutic or diagnostic target is an anti-amyloid β antibody. In some embodiments, the anti-amyloid β antibody is donepemab. In some embodiments, the antibody consists of the heavy chain shown in SEQ ID NO: 29, the heavy chain shown in SEQ ID NO: 30, and the two light chains shown in SEQ ID NO: 28. In some embodiments, the antibody consists of the heavy chain shown in SEQ ID NO: 31, the heavy chain shown in SEQ ID NO: 30, and the two light chains shown in SEQ ID NO: 28.

[0020] Another aspect of this document describes a fusion protein comprising isolated antibodies as described in any of the foregoing aspects or embodiments.

[0021] In some embodiments of the fusion protein, the isolated antibody is fused with a functional protein or its functional domain. In some embodiments of the fusion protein, the functional protein is selected from the group consisting of: brain antigen-binding peptides, hormones, neurotrophic factors, neuropeptides, cytokines, enzymes, or their mimics or functional domains. In some embodiments, the brain antigen-binding peptide is an antibody that binds to one or more molecules selected from the group consisting of: β-secretase 1 (BACE1), β-amyloid (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau protein, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin protein, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin protein, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), G protein-coupled receptor (GPCR), and caspase 6. In some embodiments, 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). In some embodiments, the neuropeptide is selected from the group consisting of: substance P, neuropeptide Y, vasoactive intestinal peptide (VIP), gamma-aminobutyric acid (GABA), dopamine, cholecystokinin (CCK), endorphins, enkephalins, and thyrotropin-releasing hormone (TRH).

[0022] In some embodiments of the fusion protein, the functional protein or its functional domain is linked to an isolated antibody directly or via a linker. In some embodiments, the linker is a peptide. In some embodiments, the linker is a GS linker. In some embodiments, the linker is a G4S linker. In some embodiments, the functional protein is linked to the carboxyl terminus of an isolated antibody. In some embodiments, the fusion protein comprises a structure consisting of, from the amino terminus to the carboxyl terminus, a heavy chain variable region-linker-Fc-linker-functional protein or its functional domain. In some embodiments, the structure is a heavy chain variable region-GS linker-Fc-GS linker-functional protein or its functional domain. In some embodiments, the structure is a heavy chain variable region-(G4S)3-Fc-(G4S)3-functional protein or its functional domain. In some embodiments, the functional protein is neurotensin. In some embodiments, the fusion protein comprises or is composed of the amino acids shown in SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.

[0023] Another aspect of this document describes a conjugate comprising an isolated antibody as described in any of the foregoing aspects or embodiments, wherein the isolated antibody is conjugated to a compound selected from the group consisting of nucleic acids, peptides, radionuclides, and small molecule compounds. In some embodiments, the isolated antibody is conjugated to a small molecule compound.

[0024] In some embodiments of the conjugate, the nucleic acid is selected from the group consisting of: mRNA, siRNA, short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA oligonucleotide, guide RNA (gRNA), and phosphoramidolinyl oligomer (PMO).

[0025] In some embodiments of the conjugate, the small molecule compound is a cytotoxic drug or an immunomodulator.

[0026] In some embodiments of the conjugate, the polypeptide is selected from the group consisting of soluble receptors, secretory proteins, growth factors, cytokines, hormones, neurotransmitters, or enzymes.

[0027] Another aspect of this article describes a nucleic acid comprising a sequence encoding an isolated antibody or fusion protein as described in any of the foregoing aspects or embodiments, or its antisense strand.

[0028] Another aspect of this document describes a vector comprising a nucleic acid or its antisense strand encoding an isolated antibody or fusion protein as described in any of the foregoing aspects or embodiments.

[0029] Another aspect of this document describes a host cell comprising a nucleic acid encoding vector as described in any of the foregoing aspects or embodiments. In some embodiments, the host cell is a prokaryotic or eukaryotic cell.

[0030] Another aspect of this article describes a pharmaceutical composition comprising isolated antibodies, fusion proteins, conjugates, or nucleic acids as described in any of the foregoing aspects or embodiments.

[0031] Another aspect of this document describes a method for delivering a therapeutic, diagnostic, or signaling agent across the blood-brain barrier (BBB) ​​of a subject in need, comprising administering to the subject a compound comprising the therapeutic, diagnostic, or signaling agent, said therapeutic, diagnostic, or signaling agent being conjugated to an isolated antibody as described in any of the foregoing aspects or embodiments.

[0032] Another aspect of this document describes the use of the isolated antibodies, fusion proteins, compounds, or nucleic acids described in any of the foregoing aspects or embodiments in the preparation of medicaments for the treatment of neurological disorders. In some embodiments, the neurological disorders are selected from the group consisting of: neuropathy, neurodegenerative diseases, cancer, eye diseases, epilepsy, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, central nervous system inflammation, Alzheimer's disease, Parkinson's disease, multiple sclerosis, or solid or liquid tumors with brain metastases. Attached Figure Description

[0033] Figure 1A-1B The analyzed antigen-binding fragment and human TFR ( Figure 1A ) and mouse TFR ( Figure 1B () binding activity.

[0034] Figure 2 This demonstrates the compatibility of NX56 with hCMEC.

[0035] Figure 3 The results of a competitive ELISA between anti-TfR antibody and Holo-Tf are shown.

[0036] Figure 4 The endocytosis of NX56 into hCMEC cells and CHO cells (hTFR negative) was demonstrated.

[0037] Figure 5 The internalization and transmembrane effects of NX56 on hCMEC cells were demonstrated.

[0038] Figure 6A and 6BBody temperature was observed in WT and hTFRKI mice injected with the NX56-Fc-NT fusion protein. Body temperature was measured at 0, 0.5, 1, and 2 hours (mean ± SD, N=3). NX56-Fc-NT induced hypothermia in a non-dose-dependent manner over 2 hours. Figure 6A Neurotensin (NT) was delivered to the brains of WT and hTFRKI mice via NX56-Fc-NT to lower body temperature. Figure 6A and 6B Body temperature was measured in each group, and two-way ANOVA and Dunnett's test were used to determine differences between groups. * indicates P < 0.05, ** indicates P < 0.01.

[0039] Figure 7 The results of affinity assays for hCMEC cells using NX759 and NX788 are shown.

[0040] Figure 8 The results of the endocytosis assay of anti-TfR antibodies NX759 and NX788 in hCMEC cells are shown.

[0041] Figure 9 The results show the delivery of neurotensin (NT) to the mouse brain via anti-TfR antibodies NX759 and NX788.

[0042] Figure 10 The structure of a bispecific antibody containing anti-TfR VHH antibody is shown.

[0043] Figure 11 The results of in vivo blood-brain barrier penetration assays of donepemab conjugated with anti-TfR antibody are shown. Invention Details This application relates to an isolated antibody that binds to the transferrin receptor (TfR) and its uses. Specifically, this application relates to a variable domain of a single variable domain on a heavy chain (VHH) antibody that can bind to TfR on the surface of a cell membrane, such as the blood-brain barrier (BBB), and its uses, for example, to transport molecules of therapeutic or diagnostic value to cells of the central nervous system or tissues or organs expressing TfR (such as malignant tumors).

[0045] It should be understood that this application is not limited to the aspects described herein, and therefore, the specific circumstances may certainly differ. It should also be understood that the terminology used herein is for describing particular aspects only and is not intended to be limiting, as the scope of this application is limited only by the appended claims.

[0046] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Unless otherwise stated, those skilled in the art will employ conventional techniques such as tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, all of which fall within the scope of this art. See, for example, Sambrook and Russell (eds.), *Molecular Cloning: A Laboratory Manual*, 3rd ed. (2001); Harlow and Lane (eds.), *Antibodies, A Laboratory Manual. MONOCLONAL ANTIBODIES: APRACTICAL APPROACH* (Shepherd, P. et al. Eds., 2000), Oxford University Press, USA, New York, NY.

[0047] As used herein, the term "transferrin receptor" or "TfR" refers to a cell surface receptor essential for iron uptake by cells via receptor-mediated endocytosis. The transferrin receptor (TfR) is involved in iron uptake in vertebrates and is regulated by intracellular iron concentration. It internalizes the transferrin-iron complex via receptor-mediated endocytosis, thereby taking up iron. Two transferrin receptors have been identified in humans: transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2). Both receptors are transmembrane glycoproteins. TfR1, also known as CD71, is a high-affinity, universally expressed receptor. In some embodiments, TfR is human TfR1, encoded by the gene with gene ID 7037 in the NCBI database. In some embodiments, TfR1 comprises the amino acid sequence shown in SEQ ID NO: 22.

[0048] The blood-brain barrier (BBB) ​​is the physiological barrier between the peripheral circulation and the brain and spinal cord. It is formed by tight junctions within the cell membranes of brain capillary endothelial cells, creating a tight barrier that restricts molecules from entering the brain. The BBB can even restrict very small molecules, such as urea (60 Daltons), from entering the brain. Examples of the BBB include the blood-brain barrier within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina; these are all continuous capillary barriers within the central nervous system (CNS). The BBB also includes the blood-cerebrospinal fluid (CSF) barrier (choroid plexus), where the barrier is composed of ependymal cells rather than capillary endothelial cells.

[0049] The "central nervous system" or "CNS" refers to the complex of neural tissues that control bodily functions, including the brain and spinal cord.

[0050] As used herein, "nervous system disease" refers to a disease or disorder affecting the central nervous system and / or originating from the central nervous system. Exemplary central nervous system diseases or disorders include, but are not limited to: neuropathy, amyloidosis, cancer, eye diseases or disorders, viral or microbial infections, inflammation, ischemia, neurodegenerative diseases, epilepsy, behavioral disorders, and lysosomal storage diseases. For the purposes of this application, the central nervous system should be understood to include the eyes, which are normally isolated from the rest of the body by the blood-retinal barrier. Specific examples of neurological diseases include, but are not limited to: neurodegenerative diseases (including but not limited to Lewy body disease, post-poliomyelitis syndrome, Shay-Dreg syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatal substantia nigra degeneration, spinocerebellar ataxia, spinal muscular atrophy), tau protein diseases (including but not limited to Alzheimer's disease and supranuclear palsy), prions (including but not limited to bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob disease, kuru, Gerstmann syndrome, chronic wasting diseases, and fatal familial insomnia), bulbar palsy, motor neuron diseases, and other neurological disorders. Heterogeneous degenerative diseases (including but not limited to Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette syndrome, Menkes curly hair syndrome, Cockayne syndrome, Hallewarden-Spatz syndrome, Lafra disease, Rett syndrome, Wilson's disease, Leisch-Niehan syndrome, and Onfried-Lundberg syndrome), dementia (including but not limited to Pick's disease and spinocerebellar ataxia), and cancer (e.g., cancers of the central nervous system and / or brain, including brain metastases caused by cancers in other parts of the body, such as CD20-positive cancer with brain metastases, or Her2-positive cancer with brain metastases).

[0051] "Drugs for neurological diseases" refers to drugs or therapeutic agents used to treat or alleviate symptoms of one or more neurological diseases. The drugs for neurological diseases in this application include, but are not limited to: small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more central nervous system targets, modified versions of natural ligands of one or more central nervous system targets, nucleic acid aptamers, inhibitory nucleic acids (i.e., small inhibitory RNA (siRNA) and short hairpin RNA (shRNA)), ribozymes, or active fragments of any of the above substances. The exemplary neurological disease drugs described in this application include, but are not limited to: antibodies, nucleic acid aptamers, proteins, peptides, inhibitory nucleic acids and small molecules, and active fragments of any of the above substances, which themselves or are capable of specifically recognizing and / or acting on (i.e., inhibiting, activating or detecting) central nervous system antigens or target molecules, such as, but not limited to: amyloid precursor protein or portions thereof, β-amyloid protein, β-secretase, γ-secretase, Tau protein, α-synuclein, Parkin protein, Huntington's protein, DR6, presenilin, ApoE, glioma or other central nervous system cancer markers, and neurotrophic factors. Non-limiting examples of drugs for treating neurological diseases and their corresponding diseases include: brain-derived neurotrophic factor (BDNF), chromium-induced brain injury (Neurogenes 1s), fibroblast growth factor 2 (FGF-2), anti-epidermal growth factor receptor (EGFR) antibody (for treating brain cancer), glial cell line-derived neurotrophic factor (GDNF) (for treating Parkinson's disease), brain-derived neurotrophic factor (BDNF) (for treating amyotrophic lateral sclerosis and depression), lysosomal enzymes (for treating lysosomal storage diseases), ciliary neurotrophic factor (CNTF) (for treating amyotrophic lateral sclerosis), neuromodulatory protein-1 (for treating schizophrenia), and anti-HER2 antibodies such as trastuzumab (for treating brain metastases from HER2-positive cancers).

[0052] As used herein, the term "target antigen" or "brain target" refers to antigens and / or molecules expressed in the central nervous system (including the brain) that can be targeted with antibodies or small molecules. Examples of such antigens and / or molecules include, but are not limited to: β-secretase 1 (BACE1), β-amyloid (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau protein, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin protein, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin protein, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), and caspase 6. In some embodiments, the target antigen is BACE1. In some embodiments, the target antigen is Tau.

[0053] As used herein, “antibody” is used in the broadest sense, specifically including full-length monoclonal antibodies, polyclonal antibodies, and (unless otherwise stated or contradicted by context) antigen-binding fragments, antibody variants, and their multispecific molecules, provided they exhibit the desired biological activity. Typically, a full-length antibody is a glycoprotein containing at least two heavy chains and two light chains linked together by disulfide bonds, or its antigen-binding portion. The term antibody is intended to encompass immunoglobulin molecules and immunoglobulin molecules with immunoactive fragments, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass. Therefore, an “antibody” can also be a single variable region (VHH) antibody on a heavy chain, also known as a heavy-chain-only antibody (HcAb), which does not contain a light chain and can be naturally produced by camels or sharks. The antigen-binding portion of an HcAb consists of a VHH fragment. In this application, unless otherwise specified, the terms “VHH antibody” or “HcAb” cover naturally occurring and synthetic heavy-chain-only antibodies, such as humanized heavy-chain-only antibodies having a CDR derived from a natural VHH fragment and a FR derived from a human.

[0054] The terms “heavy chain” (“CH”), “light chain” (“CL”), “light chain variable region” (“VL”), “heavy chain variable region” (“VH”), and “framework region” (“FR”) refer to the structural domains in natural immunoglobulins and the corresponding structural domains in synthetic (e.g., recombinant) binding proteins (e.g., humanized antibodies). The basic structural unit of natural immunoglobulins (e.g., IgG) is a tetramer, which has two light chains and two heavy chains. The amino-terminal (“N”) portion of each chain contains a variable region consisting of about 100-110 or more amino acids, which is primarily responsible for antigen recognition. The carboxyl-terminal (“C”) portion of each chain defines a constant region; the light chain has one constant region, while the heavy chain typically has three constant regions and a hinge region. Therefore, the structure of the light chain of the naturally occurring IgG molecule is N-VL-CL-C, and the structure of the IgG heavy chain is N-VH-CH1-H-CH2-CH3-C (where H is the hinge region). The variable region of an IgG molecule consists of a complementarity-determining region (CDR) and non-CDR fragments. The CDR contains residues that contact the antigen, while the non-CDR fragments (called the framework region) maintain the structure of the IgG molecule and determine the position of the CDR loop. Therefore, the VL and VH domains have the structure N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.

[0055] As used herein, the term "antigen-binding fragment" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a TfR1 protein, such as TfR1 V1). "Antigen-binding fragment" and all its grammatical variations are defined as a portion of the complete antibody, including the antigen-binding site or variable region of the complete antibody, and in some cases, the portion does not contain the constant heavy chain domains (i.e., CH2, CH3, and / or CH4, depending on the antibody isotype) of the Fc region of the complete antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; biantibodies; any antibody fragment of a polypeptide having a primary structure consisting of a continuous sequence of amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including but not limited to: (1) a single-chain Fv (scFv) molecule; (2) a single-chain polypeptide containing only one light chain variable region or a fragment thereof containing three CDRs of the light chain variable region and excluding the associated heavy chain portion; and (3) a single-chain polypeptide containing only one heavy chain variable region or a fragment thereof containing three CDRs of the heavy chain variable region and excluding the associated light chain portion; and multispecific or multivalent structures formed from antibody fragments. In an antibody fragment containing one or more heavy chains, the heavy chain may contain any constant region sequence found in the non-Fc region of the intact antibody (e.g., CH1 in the IgG isotype), and / or may contain any hinge region sequence found in the intact antibody, and / or may contain a leucine zipper sequence fused to or located therein with the hinge region sequence or constant region sequence of the heavy chain.

[0056] Papain digests antibodies to produce two identical antigen-binding fragments, called "Fab" fragments, each with an antigen-binding site, and a residual Fc fragment, the name reflecting its ease of crystallization. The "Fab" fragment also contains a constant region of the light chain and the first constant region (CH1) of the heavy chain. The "Fab'" fragment differs from the Fab fragment in that several residues are added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. "Fab'-SH" is a notation for Fab' where the cysteine ​​residues in the constant region have free thiol groups. The "F(ab')" fragment is generated by the cleavage of the disulfide bond at the cysteine ​​residue of the "F(ab')2" hinge. This is a product of pepsin digestion. "Fv" is the smallest antibody fragment containing both complete antigen recognition and binding sites. In the double-stranded Fv material, the region consists of a dimer formed by the tight binding of a heavy chain variable region and a light chain variable region, and this binding is non-covalent. In single-chain Fv substances (scFv), a heavy chain variable region and a light chain variable region are covalently linked by a flexible peptide linker, allowing the light and heavy chains to combine into a “dimer” structure similar to that of double-chain Fv substances. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. The six CDRs collectively confer the specificity of antibody-antigen binding. However, even a single variable region (or half of an Fv containing only three antigen-specific CDRs) can recognize and bind antigens, although its affinity is lower than that of the entire binding site. See, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994).

[0057] The “Fd” segment consists of VH and CH1 domains. The “dAb” segment (Ward et al., (1989) Nature 341:544-546) consists of either VH or VL domains. A separate complementarity-determining region (CDR) and combinations of two or more separate CDRs can optionally be linked together by synthetic linkers.

[0058] The term "biantibody" refers to a small antibody fragment with two antigen-binding sites, contained within a heavy chain variable region (VH) linked to a light chain variable region (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short, the two domains on the same chain cannot pair, thus forcing these two domains to pair with complementary domains on the other chain, forming two antigen-binding sites. For a more complete description of biantibodies, see, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-48 (1993).

[0059] These antibody fragments were obtained using conventional techniques known to those skilled in the art, such as recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0060] A "human receptor framework" refers to a framework that contains the amino acid sequence of a light chain variable region (VL) framework or a heavy chain variable region (VH) framework derived from the human immunoglobulin framework or the human common framework. A human receptor framework derived from the human immunoglobulin framework or the human common framework may contain the same amino acid sequence or may contain variations in the amino acid sequence. In some embodiments, the number of amino acid variations is 1-10, 2-9, 3-8, 4-7, or 5-6.

[0061] The “human common framework” refers to a framework that is the most frequently selected amino acid residue from the human immunoglobulin VL or VH framework sequence. Typically, the selection of the human immunoglobulin VL or VH sequence comes from a subgroup of the variable domain sequence. Generally, the sequence subgroups are described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In some embodiments, for VL, the subgroup is subgroup κ I as described by Kabat et al. (above). In some embodiments, for VH, the subgroup is subgroup III as described by Kabat et al. (above).

[0062] As used herein, the term "variable" refers to the significant sequence differences in certain portions of an antibody's variable domain that determine the binding and specificity of each particular antibody to its specific antigen. However, this variability is not uniformly distributed across the antibody's variable region. It is concentrated in three segments within the variable regions of both the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. In this paper, the three CDRs from the N-terminus to the C-terminus on the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the three CDRs from the N-terminus to the C-terminus on the light chain are referred to as LCDR1, LCDR2, and LCDR3. CDR1 to CDR3 represent LCDR1 to LCDR3, or HCDR1 to HCDR3, respectively. Correspondingly, the highly conserved portions within the variable domain are referred to as frames (FRs). The variable regions of both the natural heavy and light chains contain four FR regions, predominantly in a β-sheet configuration, linked by three CDRs to form a loop structure connecting the β-sheet structure; in some cases, the CDRs also constitute part of the β-sheet structure. In this article, the four receptors (FRs) on the heavy chain from the N-terminus to the C-terminus are referred to as HFR1, HFR2, HFR3, and HFR4, respectively, while the four FRs on the light chain from the N-terminus to the C-terminus are referred to as LFR1, LFR2, LFR3, and LFR4, respectively. FR1 to FR4 represent LFR1 to LFR4, or HFR1 to HFR4, respectively. The receptor receptors (CDRs) in each chain are tightly bound together through the FR regions and, together with the CDRs in the other chain, constitute the antigen-binding site of the antibody. For example, see Kabat et al., Sequences of Proteins of Immunological Interest , 5th edition, National Institute of Health, Bethesda, Md. (1991). The constant region does not directly participate in antibody-antigen binding but exhibits various effector functions, such as antibody involvement in antibody-dependent cytotoxicity. The target variable region sequence includes the humanized variable region sequence of the TfR1 antibody, as described in detail elsewhere in this document.

[0063] The terms “complementarity-determining region (CDR)” or “hypervariant region (HVR)” may refer to subregions of the VH and VL domains characterized by enhanced sequence variability and / or the formation of well-defined loops. These include the three CDRs (H1, H2, and H3) in the VH domain and the three CDRs (L1, L2, and L3) in the VL domain. H3 is considered to play a crucial role in conferring fine binding specificity, with L3 and H3 exhibiting the highest diversity. See Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0064] "According to Kabat numbering" likely refers to the numbering system used for the variable regions of the heavy or light chains in the antibody compilation by Kabat et al. (above). The actual linear amino acid sequence may contain fewer or more amino acids, corresponding to shortening or insertion of the variable region FR or CDR / HVR. The Kabat number of a given antibody residue can be determined by comparing the homologous regions of the antibody sequence with a "standard" Kabat numbered sequence. Typically, Kabat numbers (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) are used when referring to residues in the variable region; while the EU numbering system or index (e.g., EU indexing as in Kabat, according to EU IgG1 numbering) is generally used when referring to residues in the heavy chain constant region.

[0065] Currently, there are several known definitions / classifications of CDR / HVR. In this application, unless otherwise specified, the definition of CDR is a combination of the Kabat and Chothia CDR definitions, wherein CDR1 ranges from amino acid residues H26 to H35B, CDR2 ranges from amino acid residues H50 to H65, and CDR3 ranges from amino acid residues H95 to H102, with residue numbering following the Kabat numbering scheme.

[0066] Kabat complementarity-determining regions (CDRs) 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 to the location of the structural loop (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by the AbM antibody modeling software from Oxford Molecular. “Contact” HVRs are determined based on analysis of existing complex crystal structures. The residues of each HVR / CDR are listed below. “Frame” or “FR” residues refer to variable domain residues other than HVR / CDR residues.

[0067] 1 Residue numbering follows the nomenclature of Kabat et al. (above); 2 Residue numbering follows the nomenclature of Chothia et al. (above); 3 Residue numbering follows the nomenclature of MacCallum et al. (above); 4 Residue numbering follows the nomenclature of Lefranc et al. (above); 5 Residue numbering follows the nomenclature of Honegger and Plückthun (above).

[0068] Extended CDRs are also referred to as: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH (Kabat number).

[0069] As used herein, "monoclonal antibody" refers to an antibody derived from a group of substantially homogeneous antibodies; for example, these antibodies are substantially identical but allow for minor background mutations and / or modifications. "Monoclonal" signifies the basic homogeneity of the antibody and does not require that the antibody be produced by any particular method. In some embodiments, monoclonal antibodies are selected based on their CDR / HVR, VH, and / or VL sequences and / or binding properties, for example, from a clonal pool (e.g., recombinant, hybridoma, or phage-derived). Monoclonal antibodies can be engineered to include one or more mutations, for example, affecting the antibody's binding affinity or other properties, creating humanized or chimeric antibodies, improving antibody yield and / or homogeneity, and constructing multispecific antibodies; the resulting antibodies are still considered monoclonal antibodies. The difference between a monoclonal antibody population and a polyclonal antibody population is that the individual monoclonal antibodies in the population recognize the same antigenic site. Several techniques for producing monoclonal antibodies are known; for example, hybridoma methods (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nded. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, for example, U.S. Patent US4,816,567), phage display technology (see, for example, 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 techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, 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); US Patents US5,545,807; US5,545,806; US5,569,825; US5,625,126; US5,633,425 and US5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al. al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995)). .

[0070] As used herein, the phrases “bispecific antibody,” “bispecific antigen-binding antibody,” or “bifunctional antibody” refer to synthetic hybrid antibodies with two pairs of different heavy / light chains and two distinct binding sites.

[0071] As used herein, the term "natural antibodies and immunoglobulins" generally refers to heterotetrameric glycoproteins with a molecular weight of approximately 150,000 Daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to one heavy chain by a covalent disulfide bond (also known as a "VH / VL pair"), although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant region of the light chain aligns with the first constant region of the heavy chain, while the variable region of the light chain aligns with the variable region of the heavy chain. Certain amino acid residues are thought to form an interface between the variable regions of the light and heavy chains. See, for example, Chothia et al., J. Mol. Biol. , 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA , 82:4592 (1985).

[0072] A "chimeric antibody" may refer to an antibody whose heavy chain and / or light chain are derived from a specific isotype, class, or organism, while another portion is derived from another isotype, class, or organism. In some embodiments, the variable region will be derived from one source or organism, while the constant region will be derived from another source or organism.

[0073] "Humanized antibody" may refer to an antibody that is primarily composed of human sequences but contains a small amount of non-human (e.g., mouse or chicken) sequences. In some embodiments, the humanized antibody has one or more CDR sequences (with binding specificity of interest) from an antibody derived from a non-human (e.g., mouse or chicken) organism, which are grafted onto a human receptor antibody framework (FR). In some embodiments, non-human residues are further grafted onto the human framework (not present in either the source antibody or the receptor antibody), for example, to improve antibody performance. Generally, a humanized antibody will contain nearly all of at least one (usually two) variable regions, wherein all or nearly all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or nearly all of the FRs are FRs of human immunoglobulin sequences. Optionally, the humanized antibody may also contain a portion of an immunoglobulin constant region (Fc), typically a constant region 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).

[0074] "Human antibody" can refer to an antibody having an amino acid sequence corresponding to that of human antibodies, and / or an antibody manufactured using any of the techniques disclosed herein for manufacturing human antibodies. Human antibodies can be produced using various techniques known in the art, including phage display libraries, see Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); preparation of human monoclonal antibodies is described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991); and by administering antigens to transgenic animals that are engineered to produce such antibodies upon antigen stimulation, but whose endogenous gene sites have been disabled, for example, immunized xenogeneic mice (see, for example, U.S. Patents US6,075,181 and US6,150,584, which relate to XENOMOUSE). TM (Technology) or chickens with human immunoglobulin sequences (e.g., see WO2012162422, WO2011019844 and WO2013059159).

[0075] Immunoglobulins are mainly classified into five classes: IgA, IgD, IgE, IgG, and IgM. Several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are known.

[0076] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which an antibody determinant can bind. Epitope determinants are typically composed of surface groups of chemically active molecules (such as amino acid or sugar side chains) and usually have specific three-dimensional structural features and specific charge features.

[0077] As used in this article, “conjugate” refers to a protein covalently linked to one or more heterologous molecules, including but not limited to therapeutic peptides or proteins, antibodies, markers, or drugs for nervous system diseases.

[0078] As used herein, the term "coupling" refers to the connection of two or more objects together, which can be non-covalent (e.g., via ionic bonds, hydrogen bonds, hydrophobic bonds, or van der Waals forces) and / or covalent. When referring to a covalent connection between two or more chemical or biological compounds, "coupling" is equivalent to "conjugation." For example (but not limited to), an antibody of this application can be coupled to a target peptide to form an antibody-coupled peptide. Antibody-coupled peptides can be formed through specific chemical reactions designed to couple the antibody to the peptide. In some embodiments, the antibody of this application can be covalently coupled to the peptide of this application via a linker. For example, the linker can be covalently linked to the antibody or peptide first, and then the linker can be covalently linked to the peptide or antibody.

[0079] "Blocking" or "antagonistic" antibodies are antibodies that can inhibit or reduce the biological activity of the antigen they bind to. Preferably, blocking or antagonistic antibodies can substantially or completely inhibit the biological activity of the antigen.

[0080] As used herein, the term "isolated" refers to molecular, biological, or cellular material that is substantially free of other substances. For example, nucleic acids or peptides produced using recombinant DNA technology are substantially free of cellular material, viral material, or culture medium; nucleic acids or peptides produced through chemical synthesis are substantially free of chemical precursors or other chemical substances. Furthermore, "isolated nucleic acids" refers to nucleic acid fragments that do not exist naturally and are not found in nature in fragment form. The term "isolated" as used herein is also used to refer to polypeptides isolated from other cellular proteins, aiming to encompass purified polypeptides and recombinant polypeptides.

[0081] “Affinity” refers to the total strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, “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 using methods commonly known in the art, such as Biacore, radioimmunoassay (RIA), and ELISA.

[0082] The affinity between molecule X and its partner Y can usually be expressed by the equilibrium dissociation constant (K). D The value is expressed as the ratio of koff / kon (kd / ka). See, for example, Chen, Y., et al., (1999) J. MoI Biol 293:865-881. Low-affinity antibodies typically bind slowly to antigens and dissociate easily, while high-affinity antibodies typically bind to antigens faster and for longer periods. In one embodiment of this application, the "dissociation rate (kd)" is measured using surface plasmon resonance analysis. According to this application, the "binding rate," "binding speed," "binding rate (ka)," or "kon" can also be determined using the same surface plasmon resonance technique and calculated using a simple one-to-one Langmuir binding model (BIAcore evaluation software) by simultaneously fitting binding and dissociation sensor maps.

[0083] As used in this article, the term "EC" 50 "" refers to the concentration of the antibody or its antigen-binding fragment that can bind to the antigen and / or induce a reaction, whether in vitro or in vivo, and the concentration is 50% of the maximum binding or reaction, i.e., the midpoint between the maximum binding or reaction and the baseline.

[0084] In this application, the terms "cancer" or "vesicle" and "tumor" are used interchangeably to refer to a growth or tumor resulting from the abnormal, uncontrolled growth of cells that causes pathological effects on the host organism. In some embodiments, cancer refers to a benign tumor confined to a localized area. In other embodiments, cancer refers to a malignant tumor that has invaded and destroyed adjacent body structures and spread to distant sites. In some embodiments, cancer is associated with a specific cancer antigen.

[0085] As used herein, “treating / treatment” of a subject’s disease refers to methods used to obtain a beneficial or anticipated outcome, including but not limited to: relief or improvement of one or more symptoms, reduction of the severity of a condition (including disease), stabilization (i.e., non-deterioration) of a condition (including disease), delay or mitigation of a condition (including disease), progression, relief or mitigation of a condition (including disease), and status and remission (whether partial or complete remission) of a condition (including disease), whether or not detectable.

[0086] "Pharmaceutically acceptable carriers" refer to carriers that bind with the active ingredient to form a pharmaceutical formulation. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0087] The term "instructions for use" refers to the instructions for use typically included in the commercial packaging of a therapeutic product. Generally, drug instructions for use contain information about the use of the therapeutic product, such as indications, usage, dosage, method of administration, combination therapy, contraindications, and / or warnings.

[0088] An "effective dose" is at least the minimum amount required to achieve measurable improvement or prevention of a specific disease (e.g., cancer). The effective dose here may vary depending on factors such as the patient's disease state, age, sex, and weight, and the ability of a therapeutic agent (or combination of therapeutic drugs) to elicit the expected response in an individual may also vary. An effective dose also refers to a dose at which any toxic or adverse effects of the treatment are offset by its therapeutic benefits. Beneficial or desired outcomes for therapeutic use include clinical outcomes such as relief of one or more symptoms caused by the disease, improvement of the patient's quality of life, reduction of the dosage of other drugs required to treat the disease, enhancement of the effects of other drugs (e.g., through targeted action), delay of disease progression, and / or prolongation of survival. For cancer or tumors, an effective dose of a drug may have the following effects: reduce the number of cancer cells; shrink tumor volume; inhibit (i.e., to some extent slow down or ideally stop) the invasion of cancer cells into peripheral organs; inhibit (i.e., to some extent slow down or ideally stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with the disease to some extent. An effective dose may be administered once or multiple times. For the purposes of this disclosure, an effective dose of a drug, compound, or pharmaceutical composition means an amount sufficient to directly or indirectly achieve therapeutic effects. In a clinical setting, it is recognized that an effective dose of a drug, compound, or drug composition may or may not be achieved when used in combination with another drug, compound, or drug composition. Therefore, an "effective dose" can be considered in the context of administering one or more therapeutic agents, and the dose of a single drug can be considered effective if the desired effect can or has been achieved when used in combination with one or more other drugs.

[0089] As used herein, the term "subject" means any animal classified as a mammal, including humans, livestock and farm animals, as well as zoo, sport or pet animals such as dogs, horses, cats, cattle, etc. Preferably, such mammal is a human.

[0090] As used herein, the percentage of “homology” or “identity” is used in the context of two or more nucleic acid or polypeptide sequences and refers to two or more sequences or subsequences being identical, or having a specific percentage of identical nucleotide or amino acid residues, for example, at least 80% identity, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity in a specific region (e.g., a nucleotide sequence encoding the amino acid sequence of an antibody described herein). Homology can be determined by comparing positions in each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, the molecules at that position are homologous. The degree of homology between sequences depends on the number of matching or homologous positions between the sequences. Alignment results and percentages of homology or sequence identity can be determined using software programs known in the art. Default parameters are preferably used during alignment. The BLAST alignment program with default parameters is preferred. The preferred programs are BLASTN and BLASTP. For detailed information about these procedures, please visit the following website: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0091] As used herein, the term "about" refers to the typical range of error for a corresponding value that is readily understood by one of ordinary skill in the art. The use of "about" as a value or parameter herein includes (and describes) implementations for that value or parameter itself.

[0092] It is understood that various aspects and embodiments of this disclosure include those described as "comprising," "consisting of," and "mainly consisting of." The application will be described below with reference to specific embodiments and accompanying drawings, but is not limited thereto; it is limited only by the claims. The term "comprising" as used in this specification and claims does not exclude other elements or steps. When indefinite or definite articles are used to refer to singular nouns, such as "a / an" or "the," the plural form of the noun is included unless otherwise expressly specified. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0093] anti-TfR antibody In one aspect, this application covers 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 with appropriate affinity to TfR1 molecules expressed on cells (e.g., coronary microvascular endothelial cells), thereby promoting transferrin receptor-mediated endocytosis and increasing the release of the antibody and its conjugated cargo in cerebral vascular endothelial cells and transmembrane transport across the blood-brain barrier, both in vitro and in vivo. Furthermore, the antibody in this application does not compete with or substantially does not compete with holo-Tf for binding to the human transferrin receptor (hTfR), and therefore does not interfere with normal transferrin transport.

[0094] In some embodiments, the isolated anti-TfR1 antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the anti-TfR1 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the anti-TfR1 antibody is a monomer, a dimer, or a polymer. In some embodiments, the monomer in the dimer or polymer may be homologous or heterologous. In some embodiments, the anti-TfR1 antibody may also contain a stabilizing group to increase the plasma half-life of the anti-TfR1 antibody. The stabilizing group can be any group known to have a long plasma half-life (e.g., at least 1 hour) and substantially no adverse biological activity. Examples of such stabilizing groups include, for example, Fc fragments of immunoglobulins or variants thereof, large human serum proteins (such as albumin, HSA, or IgG), or PEG molecules. In one specific embodiment, the stabilizing group is an Fc fragment of human IgG1. More preferably, the stabilizing group is a glycosylated Fc fragment of IgG1. The heavy chain variable region (VH) of an anti-TfR1 antibody can be coupled to the N-terminus or C-terminus of a stabilizing group, or both. When the stabilizing group is an Fc fragment, conjugation is typically achieved through gene fusion. The resulting protein may exist as a monomer or polymerize, depending on the nature of the stabilizing group. For Fc fragments, Fc-VH or VH-Fc structures typically form homodimers. In some embodiments, the VH is linked to the Fc fragment directly or via a peptide linker (e.g., a G4S linker).

[0095] This document also provides isolated polynucleotides (including DNA, RNA, or hybrid molecules of DNA and RNA), vectors, or host cells containing the coding sequence of the anti-TfR1 antibody of this application. The polynucleotides can be placed into an expression vector, which is then transfected into host cells such as *E. coli*. E. coli The host cells, such as zygotic COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, do not produce immunoglobulins themselves, thus enabling the synthesis of the desired monoclonal antibodies in recombinant host cells.

[0096] VHH antibody In one embodiment, this application covers isolated anti-TfR1 antibodies (which are VHH antibodies) and polynucleotides or nucleic acids containing sequences encoding the VHH antibody.

[0097] In some implementations, the affinity constant (K) between the VHH antibody and the TfR1 protein is... D The K0 of the VHH antibody is from 7.09E-10M to 1.37E-08 (e.g., 5.05E-09 to 1.90E-08M), as determined by BLI analysis. In some embodiments, the VHH antibody binds to the human TfR1 protein at a K0. D Approximately 7E-10M (e.g., 6E-10 to 8E-10M), approximately 5.05E-09M (e.g., 4E-9 to 6E-9M), or approximately 1.90E-08M (e.g., 9E-09 to 4E-08M), as determined by BLI analysis. In some embodiments, the VHH antibody binds to the K+ of mouse TfR1. D Approximately 1.37E-08 (e.g., 1E-08 to 2E-08), as determined by BLI analysis. In some embodiments, the VHH antibody binds to the K of the human TfR1 protein. D Approximately 7E-10M (e.g., 6E-10 to 8E-10M), approximately 5.05E-09M (e.g., 4E-9 to 6E-9M), or approximately 1.90E-08M (e.g., 9E-09 to 4E-08M), as determined by BLI analysis; and K binding to mouse TfR1. D Approximately 1.37E-08 (e.g., 1E-08 to 2E-08), as determined by BLI analysis.

[0098] In some embodiments, the isolated anti-TfR1 antibody binds to TfR1 at a different site than that of holo-Tf. In some embodiments, the VHH antibody binds to human TfR1 at a different site than that of human Holo-Tf.

[0099] In some embodiments, the VHH antibody comprises a VHH fragment corresponding to a variable region of a camel antibody that is naturally heavy-chain-only and lacks a light chain. In some embodiments, the VHH fragment is very small, approximately 15 kDa. The VHH antibody comprises a single-chain molecule that binds to its homologous antigen via a single domain. The antigen-binding surface of the VHH antibody is typically more convex (or more prominent) than that of a conventional antibody, which is typically flat or concave. More specifically, the VHH fragment consists of four frame regions (or FRs) whose sequences and structures are defined as conserved; and three complementation-determining regions (or CDRs) whose sequence contents and structural conformations exhibit high variability and participate in antigen binding and provide antigen specificity. Compared to conventional human antibody VH, some amino acids in the FR2 region and complementation-determining regions (CDRs) of the VHH fragment are substituted. For example, highly conserved hydrophobic amino acids (such as Val47, Gly49, Leu50, and / or Trp52) in the FR2 region are typically replaced by hydrophilic amino acids (Phe42, Glu49, Arg50, Gly52), making the overall structure more hydrophilic. This contributes to improving the stability, solubility, and anti-aggregation properties of the VHH antibody and peptides containing (or consisting mainly of) a heavy-chain-only antibody (HcAb) antigen-binding domain. The VHH fragments of this application typically contain or consist of the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0100] Where FRn represents the frame region and CDRn represents the complementarity determination region.

[0101] In some embodiments of the VHH antibody, CDR1 contains the amino acid sequence shown in SEQ ID NO: 1, CDR2 contains the amino acid sequence shown in SEQ ID NO: 4, and CDR3 contains the amino acid sequence shown in SEQ ID NO: 6.

[0102] In some embodiments of the VHH antibody, CDR1 contains the amino acid sequence shown in SEQ ID NO: 1, CDR2 contains the amino acid sequence shown in SEQ ID NO: 4, and CDR3 contains the amino acid sequence shown in SEQ ID NO: 6.

[0103] In some embodiments of the VHH antibody, CDR1 contains the amino acid sequence shown in SEQ ID NO: 2, CDR2 contains the amino acid sequence shown in SEQ ID NO: 5, and CDR3 contains the amino acid sequence shown in SEQ ID NO: 7.

[0104] In some embodiments of the VHH antibody, CDR1 contains the amino acid sequence shown in SEQ ID NO: 3, CDR2 contains the amino acid sequence shown in SEQ ID NO: 5, and CDR3 contains the amino acid sequence shown in SEQ ID NO: 8.

[0105] In some embodiments of the VHH antibody, CDR1 contains the amino acid sequence shown in SEQ ID NO: 1, CDR2 contains the amino acid sequence shown in SEQ ID NO: 4, and CDR3 contains the amino acid sequence shown in SEQ ID NO: 25.

[0106] In some embodiments, the VHH antibody comprises the amino acid sequence shown in SEQ ID NO: 17, its conserved substitution variants, 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 in SEQ ID NO: 17.

[0107] In some embodiments of the VHH antibody, the VHH antibody further comprises an IgG Fc domain. In some embodiments, the IgG Fc domain is a human IgG Fc domain. In some embodiments, the IgG Fc domain is a human IgG Fc domain. In some embodiments, the IgG Fc domain is a human IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the IgG Fc domain comprises the amino acid sequence shown in SEQ ID NO: 18, its conserved substitution variants, 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 in SEQ ID NO: 18.

[0108] Chimeric antibodies, humanized antibodies, or human antibodies In one embodiment, the isolated anti-TfR1 antibody is a chimeric antibody, a humanized antibody, or a human antibody, comprising a variable region of the immunoglobulin heavy chain having the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FRn represents a frame region derived from mammals other than camelis, and CDRn represents a complementarity-determining region. In some embodiments, FRn represents a frame region derived from Homo sapiens.

[0109] Various methods for humanizing nonhuman antibodies are known in the art. For example, humanized antibodies can introduce one or more amino acid residues from a nonhuman source. These nonhuman amino acid residues are often referred to as “introduced” residues, and they are typically derived from the “introduced” variable region. Humanization can be performed essentially as Winter et al. (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), replacing the corresponding sequence of a human antibody with a hypervariable region sequence. Thus, such a “humanized” antibody is a chimeric antibody (US Patent US4,816,567), in which a significantly smaller than complete human variable region is replaced by a corresponding sequence from a nonhuman species. In practice, humanized antibodies typically replace some hypervariable region residues and possibly some FR residues in a human antibody with residues at similar sites in a camelid antibody. In the preparation of humanized antibodies, selecting appropriate human variable regions (including light and heavy chains) is crucial for reducing antigenicity. According to the so-called "best-match" method, the sequences of variable regions of camel antibody sequences are compared with a complete library of known human variable region sequences. The human sequence that is closest to the camel sequence is then used as the human frame for the humanized antibody (Simset et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. MoI. Biol. 196:901). Another approach is to utilize specific frames derived from common sequences of all human antibodies in specific subgroups of light or heavy chains.

[0110] Furthermore, it is crucial that antibody humanization retains high affinity for the antigen and other desirable biological properties. To achieve this, humanized antibodies are prepared according to a method that analyzes parental sequences and various conceptual humanization products, and utilizes three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are common and familiar to those skilled in the art. Computer programs are available that elucidate and visualize the possible three-dimensional conformations of selected candidate immunoglobulin sequences. By examining these visualizations, the possible roles of residues in the function of the candidate immunoglobulin sequence can be analyzed, i.e., analyzing residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the receptor and the introduction sequence to obtain desired antibody properties, such as increased affinity for TfR1.

[0111] Transgenic animals (e.g., mice) can produce a full set of human antibodies after immunization, even in the absence of endogenous immunoglobulin production. For example, studies have described homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice leading to complete suppression of endogenous antibody production. Transferring human germline immunoglobulin gene arrays into such germline mutant mice will produce human antibodies upon antigen stimulation. See, for example: Jakobovits et al, Nature, 362: 255 (1993); Bruggermann et al, Year in Immunol, 7: 33 (1993).

[0112] Genetic recombination can also be used to obtain human antibodies from non-human (e.g., camel) antibodies, where the human antibodies have similar affinity and specificity to the original non-human antibody. According to this method (also known as "epitope imprinting"), the heavy or light chain variable region of a non-human antibody fragment obtained via the aforementioned phage display technology is replaced by a human V domain gene library, thereby generating a chimeric scFv or Fab population of non-human / human chains. Selection with antigens isolates the chimeric scFv or Fab of non-human / human chains, where the human chain restores the antigen-binding sites disrupted by the removal of the corresponding non-human chain in the primary phage display clone; i.e., the epitope determines (imprints) the selection of the human chain chaperone. Human antibodies are obtained by repeating this process to replace the remaining non-human chain (see PCT WO93 / 06213, published April 1, 1993). Unlike conventional humanization of non-human antibodies via CDR transplantation, this technique provides fully humanized antibodies containing no non-human FR or CDR residues.

[0113] In some embodiments, the immunoglobulin heavy chain variable region further includes the human receptor framework. In some embodiments, the human receptor framework is derived from the human immunoglobulin framework or the human common framework. In some embodiments, the human receptor framework comprises the VH subgroup III framework sequence. Typically, the sequence subgroup is described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In some embodiments, the human receptor framework is derived from any immunoglobulin heavy chain variable region lineage selected from the following group: IGHV3-64, IGHV3-23, IGHV3-30, vh3_H (described in Knappik A, Ge L, Honegger A, et al. Fully synthetic human combinatorial antibodylibraries (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. In some embodiments, the human receptor framework is derived from any immunoglobulin heavy chain variable region lineage selected from the following group: IGHV3-23*01+IGHJ4*04, vh3_H, IGHV3-30*02, IGHV3-23*04, and IGHV3-64*04. In some embodiments, the antibody or its antigen-binding fragment comprises a human common framework. In some embodiments, the immunoglobulin heavy chain variable region comprises a human common framework and has amino acid sequence variations, such as 1-15, 1-10, 2-9, 3-8, 4-7, or 5-6 amino acid variations.

[0114] In some embodiments, the variable region of the immunoglobulin heavy chain comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR2 containing the amino acid sequence shown in SEQ ID NO: 4, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the variable region of the immunoglobulin heavy chain comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 2, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the variable region of the immunoglobulin heavy chain comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 3, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the variable region of the immunoglobulin heavy chain comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR2 containing the amino acid sequence shown in SEQ ID NO: 4, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 25. In some embodiments, the immunoglobulin heavy chain variable region comprises a sequence selected from any one of SEQ ID NOs: 9-17 and SEQ ID NO: 26, its conserved substitution variants, 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 in any one of SEQ ID NOs: 9-17 and SEQ ID NO: 26.

[0115] In some embodiments, the isolated anti-TfR1 antibody further comprises an IgG Fc domain. In some embodiments, the IgG Fc domain is a human IgG Fc domain. In some embodiments, the IgG Fc domain is a human IgG Fc domain. In some embodiments, the IgG Fc domain is a human IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the IgG Fc domain comprises the amino acid sequence shown in SEQ ID NO: 18, its conserved substitution variants, 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 in SEQ ID NO: 18.

[0116] Multispecific antibodies The anti-TfR1 antibody isolated in this application may be a monospecific antibody or a multispecific antibody (including the bispecific antibody in this application). In some embodiments, 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 polymer thereof. In some embodiments, the isolated anti-TfR1 antibody contains a monovalent, bivalent, trivalent, or tetravalent TfR binding domain.

[0117] A multispecific antibody is an antibody that has binding specificity to at least two different antigens. In the multispecific antibodies of this application, at least one binding specificity is against TfR1. In some embodiments, the multispecific antibody can bind to two or more different epitopes of the TfR1 protein. In some embodiments, the multispecific antibody of this application has one or more specificities for antigens other than TfR1. In some embodiments, the multispecific antibody of this application is specific for therapeutic or diagnostic targets. In some embodiments, the therapeutic or diagnostic targets include one or more selected from the group consisting of brain antigens, tumor-associated antigens, immune checkpoint antigens, and immune checkpoint-related antigens. In some embodiments, the brain antigen is selected from the group consisting of BACE1, Aβ, EGFR, HER2, Tau, ApoE4, α-synuclein, CD20, huntingtin, PrP, LRRK2, Parkin protein, presenilin 1, presenilin 2, γ-secretase, DR6, APP, p75NTR, and caspase 6. In some implementations, tumor-associated antigens are selected from the following group: A33; ADAM-9; ALCAM; BAGE; β-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; GAG E-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-acetylglucosamine transferase; tumor suppressor M; p15; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF receptor; TNF-γ receptor; transferrin receptor; and VEGF receptor.In some implementations, the immune checkpoint proteins are selected from the following group: 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; galactoglobulin-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.

[0118] Techniques for manufacturing multispecific antibodies include, but are not limited to: recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537, 1983, WO93 / 08829, and Traunecker et al, EMBO J. 10: 3655, 1991), and "knob-in-hole" engineering (e.g., see US Patent 5,731,168). Multispecific antibodies can also be prepared by: utilizing electrostatic redirection effects (WO2009 / 089004A1); crosslinking two or more antibodies or fragments (e.g., see US Patent 4,676,980 and Brennan et al, Science, 229: 81, 1985); using leucine zippers (e.g., see Kostelny et al, J. lmmunol., 148(5): 1547-1553, 1992); using “dual antibody” techniques (e.g., see Hollinger et al, Proc. Natl. Aerni. Sci. USA, 90:6444-6448, 1993); using single-chain Fv (sFv) dimers (e.g., Gruber et al, J. lmmunol, 152: 5368 (1994)); and preparing trispecific antibodies, as described in, for example, Tutt et al. al. J. Immunol. 147: 60, 1991. The multispecific antibodies in this application also encompass antibodies having three or more functional antigen-binding sites, including “octopus antibodies” or “dual variable domain immunoglobulins” (DVD) (e.g., see US2006 / 0025576A1, and Wu et al. Nature Biotechnology, 25(11):1290-7, 2007). The multispecific antibodies in this application also encompass “dual-acting Fab” or “DAF”, which contains an antigen-binding region capable of binding to TfR and brain antigens (e.g., BACE1 or Tau) (e.g., see US2008 / 0069820).

[0119] In some embodiments of the multispecific antibody of this application, the anti-TfR antibody may be fused directly or via a linker to the carboxyl terminus and / or amino terminus of the light chain and / or heavy chain of a second antibody or its antigen-binding fragment.

[0120] To promote the formation of heterodimers between two heavy chains, for example, one heavy chain is fused with an anti-TfR antibody while the other is not, or one heavy chain contains an Fc region of an anti-TfR arm while the other contains an Fc region of an anti-brain target arm, heterodimer mutations are introduced into the Fc regions of the two heavy chains. Examples of Fc mutations include, but are not limited to, the Zymework mutation (e.g., see US Patent 10,457,742) and the "mortar and pestle structure" mutation (e.g., see Ridgway et al, Protein Eng., 9(7): 617-621, 1996). Other heterodimer mutations may also be used in this application. In some embodiments, the modified CH3 described herein is used to promote the formation of heterodimers between the two heavy chains.

[0121] In addition to heterodimer mutations, other mutations may be introduced. In some embodiments, the Fc region of the fusion construct or bispecific antibody may also contain one or more mutations that alter (increase or decrease), preferably eliminate, ADCC / CDC mutations (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.

[0122] The techniques for preparing multispecific antibodies can also be used to prepare homodimers or homopolymers, which are also covered in this application, wherein the monomers constituting the homodimers or homopolymers can be any anti-TfR antibody in this application.

[0123] Specifically, bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibody). Methods for preparing bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities. Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (tetramomas) can produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purifying the correct molecule (usually by affinity chromatography) is quite cumbersome and yields low results. According to another, more preferred method, an antibody variable region having the desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant region sequence. This fusion is preferably fused to an immunoglobulin heavy chain constant region, which includes at least a portion of the hinge region, CH2 region, and CH3 region. Preferably, the first heavy chain constant region (CH1) is present in at least one fusion, which contains the site required for light chain binding. DNA encoding an immunoglobulin heavy chain fusion protein (and, if desired, an immunoglobulin light chain) is inserted into different expression vectors and co-transfected into a suitable host organism. This provides considerable flexibility in adjusting the ratio of the three polypeptide chains used for construction when the proportions are unequal, thereby achieving optimal yield. However, when high yields are obtained by expressing at least two polypeptide chains in equal proportions, or when the proportions are not particularly significant, the coding sequences of two or all three polypeptide chains can be inserted into a single expression vector. In a preferred embodiment of the method, the bispecific antibody consists of a mixed immunoglobulin heavy chain in one arm with a first binding specificity and a mixed immunoglobulin heavy chain-light chain pair in the other arm with a second binding specificity. Studies have found that this asymmetric structure facilitates the separation of the desired bispecific compound from the unwanted immunoglobulin chain combination, as the immunoglobulin light chain is present in half of the bispecific antibody, thus providing a simple separation method. This method is disclosed in WO94 / 04690. For more detailed information on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0124] Monoclonal antibodies The anti-TfR1 antibody of this application is preferably a monoclonal antibody. A monoclonal antibody is obtained from a group of substantially homogeneous antibodies, meaning that the individual antibodies comprising the group are identical, except for the possibility of a small number of naturally occurring mutations. Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of different antibodies. The monoclonal anti-TfR1 antibody of this application can be prepared using the hybridoma method or the recombinant DNA method (US Patent 4,816,567). In the hybridoma method, camelids (e.g., alpacas) or sharks are immunized with the complete TfR1 molecule or a portion thereof (e.g., a polypeptide containing the TfR1 extracellular domain) and an adjuvant. TfR1 molecules or polypeptides containing the TfR1 extracellular domain can be prepared using methods known in the art. In one embodiment, animals are immunized with a polypeptide containing the TfR1 extracellular domain (ECD) fused to the Fc portion of the immunoglobulin heavy chain. In one embodiment, animals are immunized with a TfR1-IgG1 fusion protein. Immunizations are performed once or multiple times, with each immunization spaced two weeks apart. Seven to fourteen days later, blood samples were collected from the animals, and the serum anti-TfR1 antibody titer was measured. The animals were then immunized until the antibody titer reached a plateau. Alternatively, lymphocytes could be immunized in vitro. The lymphocytes were then fused with myeloma cells using a suitable fusion agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0125] The prepared hybridoma cells are seeded into a suitable culture medium and cultured in it, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. Preferred myeloma cells are those capable of efficient fusion, supporting stable high-level antibody production by selected antibody-producing cells, and sensitive to culture media such as HAT medium. The culture medium in which the hybridoma cells are grown is analyzed to determine the presence of monoclonal antibodies against TfR1. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0126] The binding affinity of the monoclonal antibody can then be determined using conventional methods in the art. After identifying hybridoma cells capable of producing antibodies with the desired specificity, affinity, and / or activity, the clone can be subcloned using a limiting dilution procedure and cultured using 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 media, D-MEM, or RPMI-1640 media. Furthermore, hybridoma cells can grow into ascites tumors in animals. The monoclonal antibodies secreted by the subclones are appropriately isolated from the culture medium, ascites, or serum using standard immunoglobulin purification procedures.

[0127] The anti-TfR1 antibody of this application can be prepared by screening synthetic antibody clones with the desired activity or multiple activities using a combinatorial library. In principle, the synthetic antibody clones are selected by screening a phage library containing phages exhibiting various antibody variable region (e.g., VHH) fragments fused to the phage coat protein. Affinity chromatography is used to screen the phage library against the target antigen. Clones expressing antibodies or antibody fragments capable of binding to the target antigen are adsorbed onto the antigen, thus separating them from non-binding clones in the library. The binding clones are then eluted from the antigen and further enriched through additional antigen adsorption / elution cycles. Any anti-TfR1 antibody in this application can be obtained by designing a suitable antigen screening procedure to screen for target phage clones; and then constructing a full-length anti-TfR1 antibody clone using the Fv sequence of the target phage clone and a suitable constant region (Fc) sequence, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0128] Antibodies derived from natural or synthetic initial libraries may have moderate affinity, but affinity maturation can be mimicked in vitro by constructing and reselecting from secondary libraries. For example, mutations can be randomly introduced in vitro using a fault-prone polymerase (reported in Leung et al., Technique, 1: 11-15 (1989)), with the method described in Hawkins et al., J. MoL Biol., 226: 889-896 (1992), or Gram et al., Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992). Furthermore, affinity maturation can be performed by randomly mutating one or more CDRs, for example, by using PCR and primers carrying random sequences across the target CDR to screen for clones with higher affinity from a selected single VHH clone. Another effective approach is to display the selected VH domain on a phage, recombine it with a library of natural V domain variants obtained from an unimmunized donor, and screen for variants with higher affinity in several rounds of chain recombination, as described in Marks et al., Biotechnol, 10: 779-783 (1992).

[0129] Phage antibodies with varying affinities to TfR1 can be screened, even if these antibodies differ slightly in affinity. However, random mutation of the selected antibody (e.g., mutations performed in some affinity maturation techniques described above) may yield numerous mutants, most of which bind to the antigen, with a few exhibiting higher affinity. To preserve all higher-affinity mutants, phages can be incubated with an excess of biotinylated TfR1, but at a molar concentration below the target molar affinity constant of TfR1. The high-affinity-binding phages can then be captured using streptavidin-coated paramagnetic beads. This “balanced capture” method selects antibodies based on their binding affinity, with sufficient sensitivity to isolate mutant clones with up to two times the affinity from a large pool of lower-affinity phages.

[0130] Anti-TfR1 clones can be selected based on their activity. In one embodiment, this application provides an anti-TfR1 antibody that blocks the binding between TfR1 and its ligand. Anti-TfR1 antibodies with the properties described herein can be obtained by screening for anti-TfR1 hybridoma clones with the desired properties using any convenient method. For example, if a monoclonal antibody against TfR1 that can block or not block the binding of TfR1 to transferrin (e.g., holoterloterrin) is required, the candidate antibody can be tested in a binding competition assay (such as a competitive binding ELISA), in which TfR1 is coated onto an ELISA plate, and an antibody solution containing an excess of TfR1 is added to the coated plate, and the bound antibody is detected enzymatically. For example, the bound antibody is contacted with an HRP-labeled anti-Ig antibody or a biotin-labeled anti-Ig antibody and subjected to an HRP colorimetric reaction; or, streptavidin-HRP and / or hydrogen peroxide can be used for colorimetric development, and then the HRP colorimetric reaction can be detected spectrophotometrically at a specific wavelength using an ELISA reader.

[0131] BBB Penetration Building In a second aspect of this application, a BBB permeation construct comprising the aforementioned anti-TfR antibody is provided. The anti-TfR antibody binds to cargo (e.g., molecules) in the BBB permeation construct and delivers the cargo to cells or a BBB system having TfR1 on their surface. The cargo may be a therapeutic agent, diagnostic agent, and / or tracer, such as a drug for neurological diseases or a reagent that can be used to detect or analyze neurological diseases. For example, such drugs or reagents may include, but are not limited to, any chemical entity, such as small molecule compounds (e.g., antibiotics, antiviral drugs, immunomodulators, antitumor drugs, anti-inflammatory drugs, adjuvants, etc.); peptides, polypeptides, and proteins (e.g., enzymes); hormones; neurotrophic factors, 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); neuropeptides, including but not limited to: substance P, neuropeptide Y, vasoactive intestinal peptide (VIP), γ-aminobutyric acid (GABA), dopamine, cholecystokinin (CCK), endorphins, enkephalins, and thyrotropin-releasing hormone (TRH); cytokines, apolipoproteins, and bioactive compounds. Long factors, antigens, antibodies or portions of antibodies, adjuvants, etc.; nucleic acids (such as human, viral, animal, eukaryotic or prokaryotic, plant or synthetic RNA or DNA, including, for example, gene-coding, inhibitory nucleic acids such as ribozymes, antisense interfering nucleic acids, complete genomes or portions thereof, plasmids, etc.); lipids, viruses, markers or tracers, etc. Generally, the cargo can be any pharmaceutically active ingredient, whether a chemical compound, biochemical compound, natural compound or synthetic compound. Typically, the term "small molecule compound" refers to a molecule with a molecular weight of at most 1000 Daltons, usually 300-700 Daltons, that has pharmaceutical value. The anti-h anti-TfR antibody of this application can effectively promote the delivery of the target cargo from the blood to the brain and exert its effect in the brain.

[0132] Specifically, the goods of interest can be delivered in combination or linked to the anti-h anti-TfR antibody of this application via a parenteral route (e.g., intravenous injection). The goods can be non-covalently (e.g., via ionic bonds, hydrogen bonds, hydrophobic bonds, or van der Waals forces) to the anti-h anti-TfR antibody, and / or covalently linked to the anti-h anti-TfR antibody to form a conjugate. The conjugation of the goods to the anti-h anti-TfR antibody can be cleavable or non-cleavable in physiological media or within cells. Furthermore, the conjugation reaction can be carried out on a variety of reactive groups, particularly on one or more terminal and / or one or more internal or lateral reactive groups. Genetic engineering can also be used for conjugation. Preferably, the interaction is strong enough that the goods will not dissociate from the anti-h anti-TfR antibody before reaching its site of action.

[0133] In some embodiments, conjugation is achieved by constructing a protein fusion (i.e., by fusing two genes encoding an anti-h anti-TfR antibody and a neurological disease drug and expressing them as a single protein). The cargo can be linked to the anti-h anti-TfR antibody of this disclosure using known methods. See, for example, Wu et al., NatBiotechnol., 23(9): 1137-46, 2005; Trail et al., Cancer lmmunol lmmunother., 52(5):328-37, 2003; Saito et al., Adv Drug Deliv Rev., 55(2): 199-215, 2003; Jones et al., Pharmaceutical Research, 24(9): 1759-1771, 2007.

[0134] In some embodiments, the cargo and the anti-TtR antibody are covalently linked (or conjugated) via a non-peptide linker or a peptide linker. For example, a BBB permeation construct is a conjugate comprising the cargo and the anti-TtR antibody. In some embodiments, the BBB permeation construct is an antibody-drug conjugate (ADC). Examples of non-peptide linkers include, but are not limited to: polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylene polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, polymeric lipids, chitin and hyaluronic acid, or derivatives thereof, or combinations thereof. A peptide linker can be a peptide chain or derivative thereof consisting of 1-50 amino acids linked by peptide bonds, the N-terminus and C-terminus of which can be covalently linked to the anti-TfR antibody.

[0135] Pharmaceutical Composition Therapeutic formulations containing the above-described anti-TfR1 antibody, polynucleotide (including DNA, RNA, or a hybrid of DNA and RNA), vector, or host cell or BBB permeation construct containing the encoding sequence of the anti-TfR1 antibody can be prepared by mixing the anti-TfR1 antibody, polynucleotide, vector, host cell, or BBB permeation construct of this disclosure with an optional physiologically acceptable vector, excipient, or stabilizer of desired purity, and stored in the form of an aqueous solution, lyophilized or other dried formulation (see Remington: The Science and Practice of Pharmacy, 20th edition (2000)). Acceptable carriers, excipients, or stabilizers that are non-toxic to the recipient at the doses and concentrations used include buffers such as phosphates, citrates, histidines, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 amino acid residues) peptides; 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes; and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0136] Depending on the specific indications being treated, this formulation may also contain more than one active compound, preferably compounds with complementary activities that do not adversely affect each other. These molecules are present in appropriate combinations and in sufficient quantities to achieve the intended purpose.

[0137] Active ingredients can also be encapsulated in microcapsules, such as microcapsules prepared by coagulation technology or interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, for use in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions.

[0138] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers containing the immunoglobulins of this application, which are in the form of molded articles, such as films or microcapsules.

[0139] Pharmacological and Detection Applications of Anti-TfR1 Antibodies On one hand, this application provides a method for transporting a therapeutic or diagnostic agent across the blood-brain barrier (BBB), comprising exposing an anti-TfR antibody conjugated to the therapeutic or diagnostic agent to the BBB and forming the aforementioned BBB-permeable construct on the BBB, such that the antibody or its antigen-binding fragment transports the drug conjugated thereto across the BBB. In one embodiment, the drug is a drug for treating a neurological disease. In another embodiment, the drug is an imaging agent or a drug for detecting a neurological disease.

[0140] In some embodiments, the BBB is located in a mammal, preferably a primate such as a human, and more preferably in a human with a neurological disease. In one embodiment, the neurological disease 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 syndrome, Liddell syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, and traumatic brain injury.

[0141] In one implementation, an anti-TfR antibody or a BBB permeation construct is used to detect neurological disorders before symptoms appear and / or assess the severity or duration of the disorder or impairment. The antibody, antigen-binding fragment, or conjugate thereof allows for the detection and / or imaging of neurological disorders, including imaging via radiographic imaging, computed tomography, or magnetic resonance imaging (MRI).

[0142] In another embodiment, the anti-TfR antibody or BBB permeation construct is used to treat a neurological disorder (e.g., Alzheimer's disease), comprising administering an effective amount of the anti-TfR antibody or BBB permeation construct to a subject requiring treatment. In some embodiments, the method further includes administering an effective amount of at least one other therapeutic agent to the subject.

[0143] In another embodiment, this application relates to the use of anti-TfR antibodies or BBB permeation constructs in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat a neurological disorder or disability. In another embodiment, the medicament is used in a method of treating a neurological disorder or disability, the method comprising administering an effective amount of the medicament to an individual suffering from a neurological disorder or disability.

[0144] Another general aspect of this application relates to a method for inducing antibody-dependent phagocytosis (ADP) without stimulating a subject to secrete pro-inflammatory cytokines: comprising administering a complex to a subject comprising a therapeutic antibody or antigen-binding fragment thereof conjugated (preferably covalently conjugated) to an anti-TfR antibody, wherein the therapeutic antibody or antigen-binding fragment thereof does not have effector function. For example, the therapeutic antibody or antigen-binding fragment thereof may contain one or more amino acid modifications to reduce or eliminate effector function, such as ADCC or CDC, for example, reducing or eliminating mutations that bind to the Fcγ receptor. In one embodiment, the therapeutic antibody or antigen-binding fragment thereof specifically binds to tau aggregates.

[0145] In some embodiments, the method further includes administering an effective amount of at least one other therapeutic agent to the subject. In some embodiments, the other therapeutic agent is a therapeutic agent capable of effectively treating the same or different neurological disease as the anti-TfR antibody. Exemplary other therapeutic agents include, but are not limited to: the various neurological drugs described above, cholinesterase inhibitors (such as donepezil, galantamine, rovastin, and tacrine), NMDA receptor antagonists (such as memantine), β-amyloid peptide aggregation inhibitors, antioxidants, γ-secretase modulators, nerve growth factor (NGF) mimics or NGF gene therapy, PPARγ agonists, HMS-CoA reductase inhibitors (statins), ampaquinone, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulins, muscarinic receptor agonists, nicotinic receptor modulators, active or passive β-amyloid peptide immunization, phosphodiesterase inhibitors, serotonin receptor antagonists, and anti-β-amyloid peptide antibodies. In some embodiments, at least one other therapeutic agent is selected to mitigate one or more side effects of the neurological drugs. Other therapeutic agents may be administered in the same or different formulations and may be administered together with or alone with anti-TfR antibodies or BBB permeation constructs. Anti-TfR antibodies or BBB permeation constructs may be administered before, concurrently with, and / or after the administration of other therapeutic agents and / or adjuvants.

[0146] Anti-TfR antibodies or BBB permeation constructs can also be used in combination with other interventional therapies, such as, but not limited to, radiotherapy, behavioral therapy, or other therapies known in the art and suitable for the treatment or prevention of neurological disorders.

[0147] For treatment using anti-TfR1 antibodies or BBB permeation constructs, the appropriate dose of the antibody (used alone or in combination with other agents) depends on the type of disease to be treated, the type of antibody, the severity and duration of the disease, whether the antibody is for prophylactic or therapeutic purposes, prior treatment history, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody may be administered to the patient once or multiple times as appropriate. Depending on the type and severity of the disease, in some embodiments, an antibody dose of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1-10 mg / kg) is a suitable dose for administration to the patient, whether by single or multiple administrations alone or by continuous infusion.

[0148] Anti-TfR antibodies or BBB permeation constructs can be administered via any suitable route, including parenteral, intrapulmonary, and intranasal administration, and may also be administered intralesionally if local treatment is required. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous injection, depending in part on whether the administration is transient or chronic.

[0149] For the prevention or treatment of disease, the appropriate dosage of anti-TfR antibodies or BBB permeation constructs (used alone or in combination with one or more other 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 its conjugate is for prophylactic or therapeutic purposes, previous treatment history, the patient's clinical history and response to the antibody, the subject's physiological state (including age, weight, health status, etc.), and the judgment of the attending physician. The therapeutic dosage is optimized to achieve the best safety and efficacy.

[0150] Example Example 1: Immunization of camel species with antigen Alpaca Immunology and Phage Library Construction 500 μg of His-tagged human transferrin R protein (hTfR) (ACRO, CD1-H5243) and His-tagged mouse transferrin R protein (ACRO, TFR-M524b) were mixed with Freund's adjuvant and subcutaneously injected into alpacas at multiple sites at 2-week intervals. After 2-4 repeated immunizations, peripheral blood mononuclear cells (PBMCs) were collected. Total RNA was extracted from PBMCs and reverse transcribed into cDNA. The antibody gene sequence was amplified using the cDNA as a template. The gene sequence was digested with restriction endonucleases and ligated into a phage vector. The phage vector was electrotransfected into TG1 competent cells to prepare a phage library.

[0151] Phage library screening The obtained phage library was subjected to two rounds of in vitro screening and three rounds of in vivo screening in mice.

[0152] In the first round of in vitro screening, biopanning was performed using a 100 nM solution of free biotinylated human transferrin R protein (ACRO, TFR-H82E5) and a 5 μg / mL solution of His-tagged human transferrin R protein bound to a solid-phase carrier (ACRO, CD1-H5243). In the second round of in vitro screening, biopanning was performed using a 50 nM solution of free biotinylated mouse transferrin R protein (ACRO, TFR-M8249) and a 2.5 μg / mL solution of His-tagged mouse transferrin R protein bound to a solid-phase carrier (ACRO, TFR-M524b). Competitive washing was performed using 100 μg / mL of total transferrin (Holo-Tf, Sigma, #T0665-50MG).

[0153] Phage libraries obtained from in vitro screening were amplified and then screened in vivo. 6-8 week old BALB / c mice (Vitollea) were administered phage at a dose of 5E+11 cfu / 100 μL via tail vein injection, and sacrificed with carbon dioxide 2 hours later. Brain tissue was collected from the mice. Surface capillaries were removed, and three volumes of homogenate (1% Triton X-100 + 1% PMSF in PBS) were added. The brain tissue was homogenized for 10 seconds using a medium-speed homogenizer, dextran was added, and the mixture was centrifuged at 14,800 g for 25 minutes at 4°C to obtain the supernatant. The supernatant was used to infect *Escherichia coli* (E. coli). E. coli TG1 was used to amplify the phage. Third and fourth rounds of in vivo screening were repeated. The in vivo screening process was repeated twice.

[0154] Monoclonal identification Single clones were selected from the screened phage library and cultured, with supernatants collected from each culture. The binding of monoclonal antibodies to human-mouse antigens and blocking agents was detected by ELISA. Monoclonal antibodies that bound to both human-mouse antigens and not to the blocking agents were selected for sequencing. Analysis of the sequencing results yielded sequences for NX17, NX54, NX56, NX96, and other unique clones.

[0155] Example 2: Detection of the binding ability of anti-TfR antigen-binding fragments to human and mouse TfR using ELISA and BLI. Each VHH sequence is linked to an Fc via a (G4S)n linker to form a full-length heavy chain with an Fc fragment (VHH-Fc). The Fc region can be a native Fc region or a variant Fc region. The variant Fc region may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) with amino acid modifications (e.g., substitutions) at one or more amino acid positions. The exemplary molecule used herein is a homodimer of VHH-Fc (IgG1).

[0156] Specifically, an example of the NX56 amino acid sequence in VHH-Fc form is given in SEQ ID NO: 19. In the experimental examples and figures below, references to monoclonal antibodies (such as “NX56”) or humanized monoclonal antibodies (such as “HuNX5601”) used in functional tests or assays refer to the VHH-Fc antibody (a homodimer of VHH-Fc) described herein, and not the VHH fragment alone.

[0157] The expression method of VHH-Fc antibody is as follows: : The gene encoding the variable region was amplified by PCR. A signal peptide was added via digestion and overlap extension PCR, and then ligated into the vector pCMV3 containing the antibody Fc (IgG1) sequence. After PCR amplification, the vector was sent to a sequencing company for sequencing. The amino acid sequence corresponding to the anti-TfR antibody DNA sequence was obtained. The homodimer was formed by the pairing of two VHH-(G4S)3-Fc monomers. Expi293 cells were transfected with the vector and cultured on a shaker at 37°C / 5% CO2 / 125 rpm. After transient expression for 3-4 days, the supernatant containing the expressed protein was collected. After 5 days of transient expression, the supernatant was quantified using the Octet® 384 method, and the antibody was purified by Protein A affinity chromatography. The antibody concentration was determined based on the extinction coefficient at A280.

[0158] (1) Human transferrin receptor binding ELISA Using 96-well plates coated with human TfR antigen (Acro#CD1-H5243, 0.5 μg / mL, 100 μL per well), different concentrations of anti-hTfR VHH-Fc antibody (preliminary experiments have shown that this antibody does not bind to human total transferrin) and control antibody V201 (see WO2020 / 144233A1, sequence shown below) were tested by ELISA. The binding of VHH-Fc antibody was analyzed and compared using GraphPad Prism 5.

[0159] The amino acid sequence of V201 is shown in SEQ ID NO: 20.

[0160] (2) Mouse transferrin receptor binding ELISA Different concentrations of anti-hTfR VHH-Fc antibody (preliminary experiments have shown that this antibody does not bind to human holotransferrin) were tested by ELISA using 96-well plates coated with mouse TfR antigen (Acro#TFR-M524b, 0.5 μg / mL, 100 μL per well). The binding of VHH-Fc antibody was analyzed and compared using a GraphPad Prism 5.

[0161] Figure 1A-1B Table 1 describes the binding activity of the analyzed antibodies. The results showed that the binding activity of the screened TfR antigen-binding fragments was similar to that of the control antibody (V201). Furthermore, NX56 exhibited better biological activity compared to the control group.

[0162] Table 1: Summary of Human EGFR Binding / Blocking ELISA Results for Anti-EGFR Antigen Binding Fragments

[0163] (3) Determine the binding of TfR in humans and mice using biological layer interferometry (BLI). The affinity of anti-TfR antibody NX56 for human and mouse antigens was determined using the Biolayer Interferometry (BLI) assay performed using the Octet® R8 Protein Analysis System (Sartorius). The assay procedure is as follows: Prepare a 1% BSA PBST (0.04% Tween 20) buffer solution and a 100 nM glycine regeneration solution (pH 1.5). Then, determine the binding kinetics of NX56 using the Octet® R8 BLI protein analysis system (Sartorius). The Protein A sensor was pre-wetted with PBST buffer for 10 min. Then, the Sartorius Octet® ProA biosensor was immersed in baseline PBST buffer for 60 s. Next, the sensor was loaded with 5 μg / mL NX56 antibody for 180 s. After loading, the sensor was immersed in a second baseline containing PBST buffer. Then, the sensor was immersed in hTfR solutions of different concentrations (250, 125, 62.5, 31.25, 15.625, 7.83, 3.91, 0 nM) for 180 s for binding, and then transferred back to PBST buffer for 180 s for dissociation. The sensor was regenerated in 10 mM glycine regeneration solution (pH 1.5) for 10 seconds, then washed by immersion in PBST buffer for 10 seconds. This step was repeated three times to completely dissociate the previously isolated protein. BLI curves obtained with hTFR concentrations in the range of 0–250 nM were analyzed, and the Ka, kdis, and KD values ​​were determined. The binding kinetics curve data were globally fitted using a 1:1 binding model and Sartorius analysis software (v12.2).

[0164] The experimental results are shown in Table 2.

[0165] Table 2. Results of Affinity Measurement of Anti-TfR Antibody NX56 with TfR

[0166] Table 2 shows that NX56 binds to both human and mouse TfR, but its affinity for both is weaker than that for the control antibody V201. Therefore, NX56 may have better potential for penetrating the blood-brain barrier.

[0167] Example 3: Binding of anti-TfR VHH to human TfR-positive hCMEC cells Cultured human coronary microvascular endothelial cells (hCMECs) were collected and resuspended in PBS at a cell density of 2 x 10⁻⁶ cells / mL. 6Cells / mL, then seed 50 μL / well of cell suspension into 96-well round-bottom plates. Dilute all expression supernatants to 5 nM, add 50 μL of each supernatant to a 96-well plate containing hCMEC cells, incubate at 4°C for 30 min, wash twice with PBS, then add 50 μL of 1:50 diluted fluorescent secondary antibody (R-phycoerythrin AffiniPure goat anti-human IgG, Fcγ fragment specific, Jackson, 109-115-098). Incubate at 4°C for 30 min, wash twice with PBS, resuspend in 100 μL PBS, and analyze on a CytoFLEX flow cytometer. Export MFI (mean fluorescence intensity) data, such as... Figure 2 As shown, the results indicate that NX56 has a better binding affinity to hCMEC than the control antibody V201.

[0168] Example 4: Competitive detection of anti-TfR antibody and holo-Tf (Holo-Tf) To avoid interfering with normal transferrin transport, the required anti-TfR antibody should not competitively bind to holo-transferrin (Holo-Tf). The competitive binding of anti-TfR antibody and holo-transferrin to human transferrin was determined using an ELISA method. First, each well was coated with 100 μL of ELISA coating buffer containing 0.5 μg / mL antigen (Acro#CD1-H5243) and incubated overnight at 4°C. After washing three times with 250 μL of 0.05% PBST, 200 μL of blocking buffer was added, and the mixture was incubated at room temperature for 1 hour. After three washes, human Holo-Tf (Sigma, #T0665-50MG) was diluted to 2.5 μM with blocking buffer (MACKLIN, #B824162).

[0169] Anti-hTfR VHH-Fc antibody was diluted in a gradient with blocking buffer (MACKLIN, #B824162) (antibody concentrations of 10, 2.5, 0.625, 0.156, 0.039, 0.00977, 0.00244, 0.00244, and 0.0024), and 2.5 μM Holo-Tf (Sigma, #T0665-50MG) was diluted separately with blocking buffer. The diluted antibodies were added to plates coated with 0.5 μg / mL antigen (Acro #CD1-H5243). The plates were incubated at room temperature for 1 hour, followed by washing three times. 100 μl of a 10,000-fold diluted goat anti-human IgG Fc HRP (Abcam #ab97225) was added to each well, and the plates were incubated at room temperature for 1 hour. After washing three times, 50 μL of TMB chromogenic buffer (solarbio#PR1200) was added to each well, and the mixture was incubated in the dark for 8 minutes. 50 μL of stop buffer was then added to each well, and the absorbance was read at 450 nm using a microplate reader. The competitive binding of anti-TfR antibody and Holo-Tf to human transferrin was analyzed using a GraphPad Prism 5. Results are as follows: Figure 3 As shown, the anti-TfR antibody NX56 does not compete with Holo-Tf for binding to hTfR.

[0170] Example 5: Internalization of hCEMC relative to anti-TfR antibody Prepare a 2 mg / mL protein solution in 10 mM PBS buffer (pH 7, containing 1 mM EDTA), add 20 molar amounts of TCEP antibody, and react at room temperature for 1 hour. Incubate the reaction mixture at room temperature for 2 hours, then add a pre-prepared 0.5 mL desalting column (Therm#89883) and centrifuge at 1000×g for 5 minutes. Determine the antibody concentration. Add 20 molar amounts of pHAb dye (Promega#G9835) to the antibody, react at room temperature for 1 hour, add a 0.5 mL desalting column (Therm#89883), and centrifuge at 1000×g for 5 minutes. After centrifugation, each collection tube contains purified dye-labeled protein. Determine the antibody concentration at 280 nm (A280) and 532 nm (A532), and calculate the DtAR (dye-to-antibody ratio) value according to Formulas I and II. The extinction coefficient of the pHAb dye is 75,000; the correction factor of the pHAb dye is 0.256.

[0171] Formula 1: Formula 2: Fc-blocked hCMEC cells were seeded into 96-well plates (Corning #3599). 20,000 cells per well were added, followed by 50 μL of complete DMEM medium, and incubated with 50 μL of 0.2 μM NX56, V201, or IgG1 (prepared in DMEM complete medium) at 37°C for 0 h, 5 h, and 24 h. Cells were then digested with trypsin, collected by centrifugation, and analyzed using a CytoFLEX S flow cytometer (BECKMAN). Figure 4 The results showed that NX56 had slightly enhanced endocytosis in hCMEC cells compared to the positive control V201, while the situation was different in CHO cells (hTFR negative), indicating hTFR targeting specificity.

[0172] Example 6: Measurement of transmembrane transport across the blood-brain barrier The culture plate (7.5 μg / cm) 2 Using rat tail collagen (Gibco) TM The cells were coated with A1048301 for 1 hour, then coated with human plasma fibronectin (Sigma#F0895) to a final concentration of 5 μg / mL for 1 hour. 150,000 hCMEC cells were seeded into 12-well plates (Corning#3460). The culture medium was changed every 2 days for 8 consecutive days, and resistance values ​​were continuously measured. On day 8 of cell culture, the base side of the 1.5 mL centrifuge tubes and Transwell chambers used for detection were coated overnight with 1% BSA PBS solution. On day 9, the base side of the 1.5 mL centrifuge tubes and Transwell chambers were coated with culture medium for 1 hour. Electrical resistance and fluorescence saturation were measured. The antibody was diluted to a final concentration of 100 nM with 500 μL of culture medium and added to the top side containing hCMECs. 1000 μL of culture medium was added to the base side. After incubation for 1 hour, the culture medium for both the top and base sides was replaced. After incubation for 1 hour, 4 hours, and 24 hours, the base side culture medium was collected for ELISA detection. The results are as follows Figure 5 As shown, the anti-TfR antibody NX56 exhibits better internalization and transmembrane effects when used on hCMEC cells, and may have a stronger ability to enter the brain.

[0173] Example 7: Blood-brain barrier penetration assay of wild-type mouse anti-TfR antibody Six 6-12 week old Balb / c mice (Viotronics) were prepared, divided into three groups of two mice each, and marked. Antibodies were injected into the mice via the tail vein at a dose of 10 mg / kg. Two hours later, the mice were sacrificed using CO2, and blood and brain tissue were collected. After removing surface capillaries, three volumes of homogenate (1% Triton X-100 + 1% PMSF in PBS) were added, homogenized for 10 seconds using a medium-speed homogenizer, and then centrifuged at 14,800 g for 25 minutes at 4°C. The supernatant was carefully aspirated, and the antibody content in the brain tissue was determined using a double-antigen sandwich ELISA method.

[0174] Antigen (anti-hFc) was coated with coating buffer at a concentration of 2 μg / ml, 100 μL per well, and incubated overnight at 4°C. The next day, the culture plate was washed three times with 0.05% PBST, and 150 μL of blocking buffer was added to each well, incubating at room temperature for 1 hour; the plate was then washed three more times with 0.05% PBST. Brain tissue homogenates were diluted 2-fold and 8-fold, and 100 μL of the dilution was added to each well of the culture plate, incubating at room temperature for 1 hour. The culture plate was washed three times with 0.05% PBST. 100 μL of the corresponding secondary antibody (1:10k) was added to each well, and incubated for 1 hour. The culture plate was washed three times with 0.05% PBST, and then 50 μL of TMB was added to each well for color development under light-protected conditions. After 10 minutes, stop solution was added. The values ​​at 450 nm were read and analyzed using a microplate reader.

[0175] The results are shown in Table 3.

[0176] Table 3: Results of in vivo blood-brain barrier penetration assay

[0177] The IgG1-isotype is the anti-HEL human IgG1-Kappa isotype control (B117901, Biointron).

[0178] Following intravenous injection of control IgG1 isotype V201 and anti-TfR antibody NX56 (10 mg / kg), the concentrations of anti-TfR antibodies in the brain and plasma of mice were measured. Within 2 hours, the antibody concentration in brain tissue was significantly higher with NX56 compared to the negative control IgG; NX56 was also significantly higher than the reference antibody V201.

[0179] Example 8: Determination of anti-TfR antibody-mediated neurotensin (NT) delivery into the mouse brain The NT was linked to the C-terminus of the Fc (IgG1) of the test antibody NX56 using a G4S linker, yielding the complete sequence (NX56-Fc-NT). The structure of this sequence is VHH-(G4S)3-Fc-(G4S)3-NT (from the N-terminus to the C-terminus). The fusion protein is a homodimer of this sequence. The amino acid sequence of NX56-Fc-NT is shown in SEQ ID NO: 21.

[0180] Nine 6-12 week old Balb / c mice were divided into three groups of three and labeled. Six 6-12 week old hTFR KI mice were divided into two groups of three and labeled. Antibodies at concentrations of 3 mg / ml and 10 mg / ml and IgG1 isotype antibodies were prepared and administered to the mice via tail vein injection. Rectal temperature was measured at 0, 0.5, 1, and 2 hours post-injection. The results are shown in Figure 6. The results indicate that delivery of NT (neurotensin) to the brain of WT and hTFRKI mice via NX56 significantly reduced body temperature. The hypothermia induced by NX56-Fc-NT within 2 hours was dose-independent.

[0181] Example 9: Design and Detection of Humanized Anti-TfR Antibodies 9.1 Humanization of anti-TfR antibody NX56 The NX56 sequence was compared with human heavy chain sequences in the database. The two human sequences with the highest sequence homology, IGHV3-23*01 and vh3_H, were selected. The CDR region of NX56 was then transplanted onto IGHV3-23*01 and vh3_H to form a variable region sequence with the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid residues were identified and annotated using the Kabat numbering system.

[0182] Following the BMSCORE scoring process described in the literature DOI: 10.1007 / 978-1-0716-2075-5_14, the transplanted sequences were scored, and amino acid sites with scores greater than 1 were selected. A reversion mutant phage library was constructed, and after two rounds of phage library screening, single clones were selected. ELISA-positive clones were sequenced to obtain unique clone sequences, and the clone sequences were analyzed and scored according to the HSC scoring method described in the literature DOI:10.1016 / j.molimm.2006.09.029.

[0183] The obtained humanized variable region sequence is shown in SEQ ID NOs: 9-14.

[0184] Table 4: Template Selection and Reversion Mutation Design for hNX56

[0185] Note: For example, G44E means that according to the Kabat numbering system, the 44th G is mutated to E.

[0186] 9.2 Preparation of humanized antibodies Antibodies were prepared by binding the constant region of human Fc-IgG1 to the variable region of VHH or its humanized version.

[0187] 9.3 Determination of the affinity between anti-TfR antibody NX56 and TfR Using a BLI instrument, the affinity of the humanized anti-TfR antibody for human and mouse antigens was determined as described in Example 2.

[0188] Table 5: Summary of the binding of anti-TfR antigen-binding fragments to human and mouse TfR as determined by bioluminescence imaging (BLI)

[0189] 9.4: Anti-TfR antibody's effect on blood-brain barrier penetration in hTFRKI mice Fourteen Balb / c mice aged 6-12 weeks were prepared, divided into 7 groups of 2 mice each, and marked. The mice were injected via the tail vein. The mice were sacrificed according to the time shown in Table 6, and blood and brain tissue were collected for ELISA testing as described in Example 7.

[0190] Table 6: Results of Blood-Brain Barrier Penetration Measurement

[0191] Example 10: Screening of AI-designed TfR combination.

[0192] Our initial hypothesis was that binders targeting similar epitopes on hTfR could serve as a good starting point for hTfR binding agent design. Based on this hypothesis, we first fragmented hTfR into peptides of length 5–20. Then, we screened for protein epitopes with sequence similarity to the peptides and examined the presence of proteins binding to these epitopes. Our search results revealed approximately 1000 complexes consisting of hTfR-like sequences and their binding proteins. We further screened these complexes using criteria including structural similarity to hTfR, the quality of binding between the two chains in the complex, and potential steric hindrance.

[0193] After rigorous screening, we identified two hTfR binding loops and transplanted them into the CDR region of the general VHH framework. We found that several VHHs had weak binding affinity to hTfR, and then designed a phage display library based on the best VHHs we selected.

[0194] These VHHs were homologously recombinated with the hIgG1 constant region gene to construct the full-length antibody sequence (VHH-Fc).

[0195] Then, NX759 (VHH amino acid sequence as shown in SEQ ID NO: 15) and NX788 (VHH amino acid sequence as shown in SEQ ID NO: 16) stood out in the phage display screening, with an affinity for hTfR reaching the nanomolar level.

[0196] Example 11: BLI assay of anti-TfR antibody affinity The affinity of anti-TfR antibodies NX759 and NX788 for human and monkey antigens was measured using the BLI instrument described in Example 2.

[0197] Table 7: Affinity assay results of anti-TfR antibodies NX759 and NX788 with TfR

[0198] Experimental results show that NX759 and NX788 have weak affinity for human transferrin receptor (TfR). Some studies have shown that TfR antibodies with relatively low affinity for TfR have strong brain penetration effects.

[0199] Example 12: Competitive determination of anti-TfR antibody against total transferrin using bioluminescence imaging (BLI) technology Sartorius Octet® Discovery software (v12.2) was used. The Octet® SA biosensor was immersed in 1% BSA in PBST (0.04% Tween 20) buffer for 10 minutes. After probe loading, it was immersed in Sartorius PBST (0.04% Tween 20) buffer for 30 seconds. Then, the probe was immersed in 5 μg / ml hTfR solution for 120 seconds until the signal value was 1. After that, the probe was immersed in PBST buffer for 30 seconds, then immersed in 100 nM antibody solution for testing, and bound to the antibody until saturation, followed by immersion in 100 nM Holo-Tf antibody solution for binding until saturation. After the competition, the probe was regenerated in 10 mM glycine regeneration solution (pH 1.5) for 10 seconds, and then rinsed in PBST buffer for 10 seconds. This step was repeated three times to completely dissociate the previous protein. Data analysis was performed using Sartorius Octet® Analysis Studio. The percentage of inhibition in each step involving the antibody was calculated by taking the competitive antibody signal and dividing it by the maximum signal of the same antibody used in the saturation step. Multiplying this value by 100 gives the inhibition percentage; a lower percentage indicates less epitope transfer and greater competition between the two antibodies. The calculation results are shown in Table 2.

[0200] Table 8: Competitive assay results of anti-TfR antibodies NX759 and NX788 against all transferrin (Holo-Tf)

[0201] Note: Divide the competitive antibody signal by the maximum signal of the same antibody in the saturation step to calculate the inhibition percentage. 0-30 indicates complete competitive inhibition, 30-70 indicates partial competitive inhibition, and >70 indicates non-competitive inhibition.

[0202] The results showed that the anti-TfR antibodies NX759 and NX88 did not compete with Holo-Tf at all.

[0203] Example 13: Detection of the binding of anti-TfR antibody to hCEMC cells The binding activity of the anti-TfR antibody to hCEMC cells was detected by flow cytometry using the same method as in Example 3. The results are as follows: Figure 7 As shown.

[0204] Figure 7 The results showed that NX759 and NX88 could effectively bind to hCMEC cells, and their binding ability was stronger than that of the positive control V201.

[0205] Example 14: Internalization analysis of anti-TfR antibody on hCEMC cells The internalization of anti-TfR antibody in hCEMC cells was measured according to Example 4.

[0206] The results of the internalization test are as follows Figure 8 As shown.

[0207] Figure 8 The results showed that hCMEC cells exhibited stronger endocytosis of NX759 and NX788 than the positive control 201, indicating that NX759 and NX788 have better brain entry potential.

[0208] Example 15: Blood-brain barrier penetration assay of anti-TfR antibodies in hTFR KI mice Six 6-12 week old Balb / c mice were prepared, divided into three groups of two mice each, and marked; then injected via tail vein. The mice were sacrificed according to the time shown in Table 9, and blood and brain tissue were collected for ELISA detection as described in Example 7.

[0209] The results are shown in Table 9.

[0210] Table 9

[0211] Example 16: Delivery of neurotensin (NT) to the mouse brain via anti-TfR antibody The construction methods of anti-TfR antibodies NX759-Fc-NT (amino acid sequence as shown in SEQ ID NO: 23) and NX788-Fc-NT (amino acid sequence as shown in SEQ ID NO: 24) are the same as in Example 8.

[0212] Twelve hTFRKI mice aged 6-12 weeks were prepared and divided into four groups of three. All mice were tagged. Antibodies and IgG1 isotype antibodies at a concentration of 10 mg / ml were prepared and injected into the mice via the tail vein. The rectal temperature of the mice was measured at 0, 0.5, 1, and 2 hours post-injection. Results are as follows: Figure 9 As shown.

[0213] The results showed that delivering neurotensin NT to the brains of WT and hTFRKI mice via NX788 reduced the mice's body temperature.

[0214] Example 17: Design and detection of reduced affinity of anti-TfR antibodies after humanization 17.1 Design of humanized anti-TfR antibodies with reduced affinity The affinity profiles between TfR and TfR nanobodies could potentially lead to the discovery of drug candidates with higher drug delivery efficiency. To design NX56 mutants with different affinities, we modeled the structure of the huNX5604 / hTfR complex and used a computer scanning interface to search for mutations that could alter binding affinity.

[0215] The sequence of the mutant huNX5604A (VHH) is shown in SEQ ID NO: 26. The VHH-Fc antibody used for affinity assay was constructed in the same manner as in Example 2.

[0216] 17.2 Affinity assay of anti-TfR antibody huNX5604A As described in Example 2, the affinity of the humanized anti-TfR antibody for TfR was determined by BLI.

[0217] Table 10: Affinity assay results of anti-TfR antibody huNX5604A and hTfR

[0218] 17.3 Constructing bispecific antibodies by conjugating anti-TfR antibodies with donepemab Donemumab is an antibody produced in mice that targets amyloid-beta (Aβ) to clear excess protein that causes a burden on the brain and is used to slow the progression of early Alzheimer's disease. For more information, see PCT Publication WO2012021469(A1).

[0219] The sequences of donepemumab heavy chain and donepemumab light chain are shown in SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0220] Anti-TfR antibody huNX5604 and the reduced-affinity anti-TfR antibody huNX5604A were linked to the C-terminus of donepembal Fc via flexible linkers. To improve antibody purification efficiency, a "mortar and pestle structure" mutation was further introduced into the donepembal Fc fragment. A bispecific antibody was then constructed to test the transfer capability of currently available anti-TfR antibodies against the full-length antibody. The structure of the bispecific antibody is shown below. Figure 10 As shown.

[0221] The light chain sequence of the bispecific antibody is the same as that of donepemab.

[0222] The heavy chain sequences of the bispecific antibodies are shown in SEQ ID NO: 29 (Don5604A heavy chain 1 (mortar)), SEQ ID NO: 30 (Don5604A heavy chain 2 (mortar) and Don5604 heavy chain 2 (mortar)) and SEQ ID NO: 31 (Don5604 heavy chain 1 (mortar)).

[0223] 17.4 Affinity determination of bispecific antibodies with reduced affinity for TfR The affinity of bispecific antibodies containing anti-TfR antibodies was determined by BLI.

[0224] Table 11: Results of affinity assays between bispecific antibodies Don5604A and Don6604 and TfR

[0225] 17.5 In hTFR KI mice, in vivo blood-brain barrier penetration assay was performed using a bispecific antibody containing anti-TfR antibody. Ten hTFR KI mice aged 6-12 weeks were randomly divided into 5 groups of 2 mice each and marked. They were then injected via the tail vein. Six hours after intravenous injection, the heart was perfused with 25 ml PBS containing 1 Eu / ml heparin, followed by subsequent surgery. Brain tissue homogenate was collected for ELISA detection according to the method in Example 7. Results (e.g.) Figure 11 As shown in the figure, the anti-TfR antibody can effectively deliver the full-length antibody to the brain of TfR transgenic mice (hTFR knock-in mice).

[0226] sequence list

Claims

1. An isolated antibody that binds to transferrin receptor (TfR), comprising a variable region of an immunoglobulin heavy chain, said variable region comprising: 1) A complementarity-determining region (CDR) 1 containing the amino acid sequence GRTLRVSAYGMG (SEQ ID NO: 1); a CDR 2 containing the amino acid sequence AISQWGVGNTYYADSVKG (SEQ ID NO: 4); and a CDR 3 containing the amino acid sequence DTSPVTWYPADGYHYDA (SEQ ID NO: 6); 2) CDR1 containing the amino acid sequence SRTFSRIQMG (SEQ ID NO: 2), CDR2 containing the amino acid sequence AISRTGGSTYYPDSVEG (SEQ ID NO: 5), and CDR3 containing the amino acid sequence SFGPEWDVGGYDY (SEQ ID NO: 7); 3) CDR1 containing the amino acid sequence SRTFSSLQMG (SEQ ID NO: 3), CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence SWGPEWDQGGYDY (SEQ ID NO: 8); or 4) Complementarity-determining region (CDR) 1 containing the amino acid sequence GRTLRVSAYGMG (SEQ ID NO: 1), CDR 2 containing the amino acid sequence AISQWGVGNTYYADSVKG (SEQ ID NO: 4), and CDR 3 containing the amino acid sequence DTSPVTWYPASGYHYDA (SEQ ID NO: 25).

2. The isolated antibody as claimed in claim 1, wherein the antibody is a humanized antibody.

3. An isolated antibody that binds to a transferrin receptor, comprising: (i) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWVRQAPGKERELVAAISQWGVGNTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 9); (ii) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKELELVSAISQWGVGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 10); (iii) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKGRELVSAISQWGVGNTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 11); (iv) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKGLELVSAISQWGVGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYVCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 12); (v) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKELELVSAISQWGVGNTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYVCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 13); (vi) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKGRELVSAISQWGVGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYVCAADTSPVTWYPADGYHYDAWGQGTLVTVSS (SEQ ID NO: 14); (vii) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASSRTFSRIQMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCGGSFGPEWDVGGYDYWGQGTQVTVSS (SEQ ID NO: 15); (viii) The heavy chain variable region containing CDR1, CDR2 and CDR3, with the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASSRTFSSLQMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCGGSWGPEWDQGGYDYWGQGTQVTVSS (SEQ ID NO: 16); (ix) The heavy chain variable region containing CDR1, CDR2, and CDR3, with the following amino acid sequence: QVQLQESGGGLVQAGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKERELVAAISQWGVGNTYYADSVKGRFTISRDNAKNTVYLQLNSLKPEDTAVYVCAADTSPVTWYPADGYHYDAWGQGTQVTVSS (SEQ ID NO: 17); or (x) The heavy chain variable region containing CDR1, CDR2 and CDR3 has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGRTLRFSAYGMGWFRQAPGKGLELVSAISQWGVGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYVCAADTSPVTWYPASGYHYDAWGQGTLVTVSS (SEQ ID NO: 26); The CDR is defined by Kabat, Chothia, Aho, Abm, IMGT, Contact, North, or a hybrid scheme thereof.

4. The isolated antibody according to any one of claims 1-3, comprising any amino acid sequence selected from SEQ ID NO: 9-17, a conserved substitution variant of the amino acid sequence, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any amino acid sequence selected from SEQ ID NO: 9-17.

5. The antibody isolated according to any one of claims 1-4, wherein the isolated antibody is a single-domain antibody.

6. The isolated antibody according to any one of claims 1-4, wherein the isolated antibody further comprises a human IgG Fc domain, preferably the human IgG Fc domain being an IgG1, IgG2, IgG3 or IgG4 Fc domain.

7. The isolated antibody according to any one of claims 1-5, wherein the isolated antibody comprises a monovalent, bivalent, trivalent or tetravalent TfR binding domain.

8. The isolated antibody according to any one of claims 1-7, wherein the isolated antibody is a bispecific antibody or a multispecific antibody, and further comprises a binding domain for binding to a therapeutic or diagnostic target.

9. The isolated antibody of claim 7, wherein the therapeutic or diagnostic target comprises a brain antigen.

10. The isolated antibody of claim 9, wherein the brain antigen is selected from one or more of the group consisting of: β-secretase 1 (BACE1), β-amyloid protein (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau protein, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin protein, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin protein, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, G protein-coupled receptor (GPCR), and glucocerebrosidase.

11. The isolated antibody according to any one of claims 8-10, wherein the therapeutic or diagnostic target comprises one or more selected from the group consisting of tumor-associated antigens, immune checkpoint antigens, and immune checkpoint-associated antigens.

12. A fusion protein comprising the isolated antibody according to any one of claims 1-11.

13. The fusion protein of claim 12, wherein the isolated antibody is fused with a brain antigen-binding polypeptide, hormone, neurotrophic factor, cytokine, enzyme, or its mimicry or functional domain, preferably the brain antigen-binding polypeptide is an antibody that binds to one or more molecules selected from the group consisting of: β-secretase 1 (BACE1), β-amyloid protein (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau protein, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin protein, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin protein, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), G protein-coupled receptor (GPCR), and caspase 6.

14. A conjugate comprising the isolated antibody of any one of claims 1-8, wherein the isolated antibody is conjugated to a nucleic acid, polypeptide, radionuclide, or small molecule compound.

15. The conjugate of claim 14, wherein the nucleic acid is selected from the group consisting of mRNA, siRNA, antisense oligonucleotides, microRNA (miRNA), guide RNA (gRNA), and phosphoramidomorpholine oligomers (PMO).

16. The conjugate of claim 14, wherein the small molecule compound is a cytotoxic drug or an immunomodulator.

17. The conjugate of claim 14, wherein the polypeptide is selected from the group consisting of soluble receptors, secretory proteins, growth factors, cytokines, hormones, neurotransmitters, or enzymes.

18. A nucleic acid comprising a sequence encoding an antibody isolated according to any one of claims 1-11 or a fusion protein according to claim 12 or 13, or an antisense strand thereof.

19. A vector comprising the nucleic acid of claim 18.

20. A host cell comprising the nucleic acid of claim 18 or the vector of claim 19.

21. A pharmaceutical composition comprising the isolated antibody of any one of claims 1-11, the fusion protein of any one of claims 12 or 13, the conjugate of any one of claims 14-17, or the nucleic acid of claim 18.

22. A method of delivering a therapeutic agent, diagnostic agent, or signaling agent across the blood-brain barrier (BBB) ​​of a subject in need, comprising administering to the subject a compound comprising the therapeutic agent, diagnostic agent, or signaling agent, said therapeutic agent, diagnostic agent, or signaling agent being conjugated to an isolated antibody as described in any one of claims 1-11 or a fusion protein as described in claim 12 or 13.

23. Use of the isolated antibody of any one of claims 1-11, the fusion protein of claim 12 or 13, the compound of any one of claims 14-17, or the nucleic acid of claim 18 in the preparation of a medicament for treating a neurological disease, preferably the neurological disease being selected from the group consisting of: neuropathy, neurodegenerative diseases, cancer, eye diseases, epilepsy, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, central nervous system inflammation, Alzheimer's disease, Parkinson's disease, multiple sclerosis, or a solid tumor or cyst with brain metastases.

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