# US 11,111,308 B2 · Anti-human transferrin receptor antibody capable of penetrating blood-brain barrier

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- Number: US 11,111,308 B2
- Document ID: US11111308B2
- Status: Granted
- Kind: B2
- Application number: 16/909,398
- Filed: 2020-06-23
- Published: 2020-10-08
- Granted: 2021-09-07
- Earliest claimed: 2016-12-26
- Assignees: JCR Pharmaceuticals Co., Ltd.
- Applicants: JCR Pharmaceuticals Co., Ltd.
- Inventors: Hiroyuki Sonoda; Kenichi Takahashi
- Web page: https://patentgrep.com/patent/US11111308B2

## CPC

- A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- C07K 16/2881: {against CD71}
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 47/6809: {Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin}
- A61K 47/6813: {the drug being a peptidic cytokine, e.g. an interleukin or interferon}
- A61K 47/6815: {Enzymes}
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- C07K 16/2806: {against CD2}
- C07K 16/46: Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00)
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 5/10: Cells modified by introduction of foreign genetic material
- C12N 9/16: acting on ester bonds (3.1)
- C12N 15/09: Recombinant DNA-technology
- C12N 15/62: DNA sequences coding for fusion proteins
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
- C07K 2317/55: Fab or Fab'
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 9/24: acting on glycosyl compounds (3.2)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided is an anti-human transferrin receptor antibody or an analog thereof, wherein in the heavy chain variable region of the antibody, (a) CDR1 comprises the amino acid sequence set forth as SEQ ID NO: 62 or SEQ ID NO: 63, (b) CDR2 comprises the amino acid sequence set forth as SEQ ID NO: 13 or SEQ ID NO: 14, and (c) CDR3 comprises the amino acid sequence set forth as SEQ ID NO: 15 or SEQ ID NO: 16, and an analogue thereof.

## Claims

### Claim 1

An anti-human transferrin receptor antibody, wherein the heavy chain variable region of the antibody comprises,
(a) CDR1 comprising the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63,
(b) CDR2 comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and
(c) CDR3 comprising the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16, and the light chain variable region of the antibody comprises,
(a) CDR1 comprising the amino acid sequence of SEQ ID NO: 6,
(b) CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and
(c) CDR3 comprising the amino acid sequence of SEQ ID NO: 10.

### Claim 2 (depends on 1)

The antibody according to claim 1, wherein the framework region 3 of the heavy chain comprises the amino acid sequence of SEQ ID NO: 64.

### Claim 3 (depends on 2)

The antibody according to claim 2, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 65.

### Claim 4 (depends on 3)

The antibody according to claim 3, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68.

### Claim 5 (depends on 1)

The antibody according to claim 1, wherein the antibody comprises an amino acid sequence having sequence identity not lower than 80% to the amino acid sequence of the light chain variable region of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, and in the light chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 6,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10.

### Claim 6 (depends on 1)

The antibody according to claim 1, wherein the antibody comprises an amino acid sequence having a sequence identity not lower than 90% to the amino acid sequence of the light chain variable region of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, and in the light chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 6,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10.

### Claim 7 (depends on 1)

The antibody according to claim 1, wherein the antibody comprises an amino acid sequence modified from the amino acid sequence of the light chain variable region of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 by the substitution, deletion or addition of 1 to 5 amino acids, in place thereof and in the light chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 6,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10.

### Claim 8 (depends on 1)

The antibody according to claim 1, wherein the antibody comprises an amino acid sequence modified from the amino acid sequence of the light chain variable region of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 by the substitution, deletion or addition of 1 to 3 amino acids, and in the light chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 6,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10.

### Claim 9 (depends on 1)

The antibody according to claim 1, wherein the antibody comprises an amino acid sequence having sequence identity not lower than 80% to the amino acid sequence of the light chain of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 29, and in the light chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 6,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10.

### Claim 10 (depends on 1)

The antibody according to claim 1, wherein the antibody comprises an amino acid sequence having sequence identity not lower than 90% to the amino acid sequence of the light chain of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 29, and in the light chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 6,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10.

### Claim 11 (depends on 1)

The antibody according to claim 1, wherein the antibody comprises an amino acid sequence modified from the amino acid sequence of the light chain of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 29 by the substitution, deletion or addition of 1 to 5 amino acids, and in the light chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 6,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10.

### Claim 12 (depends on 1)

The antibody according to claim 1, wherein the antibody comprises an amino acid sequence modified from the amino acid sequence of the light chain of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 29 by the substitution, deletion or addition of 1 to 3 amino acids, and in the light chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 6,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10.

### Claim 13 (depends on 1)

The antibody according to claim 1, wherein the antibody has an affinity to both the extracellular region of human transferrin receptor and the extracellular region of monkey transferrin receptor.

### Claim 14 (depends on 13)

The antibody according to claim 13, wherein the dissociation constant of its complex with the extracellular region of human transferrin receptor is not greater than 1×10−10 M, and the dissociation constant of its complex with the extracellular region of monkey transferrin receptor is not greater than 1×10−10 M.

### Claim 15 (depends on 1)

The antibody according to claim 1, wherein the antibody is Fab antibody, F(ab′)2 antibody, or F(ab′) antibody.

### Claim 16 (depends on 1)

The anti-human transferrin receptor antibody according to claim 1, wherein the antibody is a single-chain antibody selected from the group consisting of scFab, scF(ab′), scF(ab′)2 and scFv.

### Claim 17 (depends on 16)

The antibody according to claim 16, wherein the light chain and the heavy chain thereof are linked via a linker sequence.

### Claim 18 (depends on 17)

The antibody according to claim 17, wherein the heavy chain is linked, via a linker sequence, to the light chain on the C-terminal side of the light chain.

### Claim 19 (depends on 17)

The antibody according to claim 17, wherein the linker sequence consists of g 2 to 50 amino acid residues.

### Claim 20 (depends on 19)

The antibody according to claim 19, wherein the linker sequence is selected from the group consisting of the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequences set forth as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, the amino acid sequence consisting of three consecutively linked amino acid sequences each set forth as SEQ ID NO: 3, and the amino acid sequence consisting of 2 to 10 of any of said amino acid sequences that are consecutively linked.

### Claim 21 (depends on 16)

The antibody according to claim 16, wherein the light chain is linked, via a linker sequence, to the heavy chain on the C-terminal side of the heavy chain.

### Claim 22 (depends on 1)

A fusion protein comprising an anti-human transferrin receptor antibody and a different protein (A), wherein the anti-human transferrin receptor antibody is the antibody according to claim 1, and the protein (A) is linked to the light chain of the antibody on the C-terminal side or the N-terminal side of the light chain.

### Claim 23 (depends on 22)

The fusion protein according to claim 22, wherein the protein (A) is linked, directly or via a linker, to the light chain on the C-terminal side or the N-terminal side of the light chain.

### Claim 24 (depends on 22)

The fusion protein according to claim 22, wherein the protein (A) is linked, via a linker, to the light chain on the C-terminal side or the N-terminal side of the light chain.

### Claim 25 (depends on 24)

The fusion protein according to claim 24, wherein the linker is a peptide consisting of 1 to 50 amino acid residues.

### Claim 26 (depends on 25)

The fusion protein according to claim 25, wherein the linker is a peptide comprising an amino acid sequence selected from the group consisting of a single glycine, a single serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence consisting of 2 to 10 of any of said amino acid sequences that are consecutively linked.

### Claim 27 (depends on 22)

A method for the production of a fusion protein according to claim 22, comprising a step of culturing mammalian cells transformed with an expression vector comprising a DNA fragment that is incorporated therein encoding the amino acid sequence of the fusion protein to let the mammalian cells secrete the fusion protein in the culture medium.

### Claim 28 (depends on 1)

A fusion protein of an anti-human transferrin receptor antibody and a different protein (A), wherein the anti-human transferrin receptor antibody is the antibody according to claim 1, and the protein (A) is linked to the heavy chain of the antibody on the C-terminal side or the N-terminal side of the heavy chain.

### Claim 29 (depends on 28)

The fusion protein according to claim 28, wherein the protein (A) is linked, directly or via a linker, to the heavy chain on the C-terminal side or the N-terminal side of the heavy chain.

### Claim 30 (depends on 28)

The fusion protein according to claim 28, wherein the protein (A) is linked, via a linker, to the heavy chain on the C-terminal side or the N-terminal side of the heavy chain.

### Claim 31 (depends on 30)

The fusion protein according to claim 30, wherein the linker sequence is a peptide consisting of 1 to 50 amino acid residues.

### Claim 32 (depends on 31)

The fusion protein according to claim 31, wherein the linker is a peptide comprising an amino acid sequence selected from the group consisting of a single glycine, a single serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence consisting of 2 to 10 of any of said amino acid sequences that are consecutively linked.

### Claim 33 (depends on 28)

The fusion protein according to claim 28, wherein the protein (A) is a protein originating from human.

### Claim 34 (depends on 28)

The fusion protein according to claim 28, wherein the protein (A) is selected from the group consisting of nerve growth factor (NGF), lysosomal enzymes, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3, neurotrophin-4/5, neurotrophin-6, neuregulin-1, erythropoietin, darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), cytokines, tumor necrosis factor α receptor (TNF-α receptor), PD-1 ligands, PD-L1, PD-L2 enzymes having β-amyloid-degrading activity, anti-β-amyloid antibody, anti-BACE antibody, anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-HER2 antibody, anti-TNF-α antibody, and anti-CTLA-4 antibody.

### Claim 35 (depends on 28)

The fusion protein according to claim 28, wherein the protein (A) is a lysosomal enzyme, and the lysosomal enzyme is selected from the group consisting of α-L-iduronidase, iduronate 2-sulfatase, human acidic α-glucosidase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acidic sphingomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl CoA:α-α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase 1, tripeptidyl-peptidase 1, hyaluronidase 1, CLN1 and CLN2.

### Claim 36 (depends on 28)

The fusion protein according to claim 28, wherein the protein (A) is human iduronate 2-sulfatase, human acidic α-glucosidase, or human α-L-iduronidase.

### Claim 37 (depends on 36)

A method for the treatment of a disease of the central nervous system accompanying Pompe's disease comprising parenterally administering a therapeutically effective amount of the fusion protein according to claim 36 to a patient with the disease.

### Claim 38 (depends on 36)

A method for the treatment of a disease of the central nervous system accompanying Hurler syndrome or Hurler-Scheie syndrome, the method comprising parenterally administering a therapeutically effective amount of the fusion protein according to claim 36 to a patient with the disease.

### Claim 39 (depends on 28)

The fusion protein according to claim 28, wherein a human IgG Fc region or part thereof is introduced between the protein (A) and the antibody.

### Claim 40 (depends on 39)

The fusion protein according to claim 39, wherein the human IgG Fc region is linked, directly or via a linker sequence, to the protein (A) on the C-terminal side of the protein (A), and the heavy chain or the light chain of the antibody is linked, directly or via a linker sequence, to the human IgG Fc region on the C-terminal side of the human IgG Fc region.

### Claim 41 (depends on 39)

The fusion protein according to claim 39, wherein the human IgG Fc region comprises the amino acid sequence of SEQ ID NO: 70.

### Claim 42 (depends on 41)

The fusion protein according to claim 41, wherein the human IgG Fc region is linked, via a linker sequence, to a Fab heavy chain consisting of the amino acid sequence of SEQ ID NO: 61, and comprises the amino acid sequence of SEQ ID NO: 71 formed thereby.

### Claim 43 (depends on 28)

A DNA fragment encoding the amino acid sequence of the fusion protein according to claim 28.

### Claim 44 (depends on 28)

A method for the production of a fusion protein according to claim 28, comprising a step of culturing mammalian cells transformed with an expression vector comprising a DNA fragment that is incorporated therein encoding the amino acid sequence of the fusion protein to let the mammalian cells secrete the fusion protein in the culture medium.

### Claim 45 (depends on 1)

A DNA fragment encoding the amino acid sequence of the anti-human transferrin receptor antibody according to claim 1.

### Claim 46 (depends on 45)

An expression vector comprising the DNA fragment according to claim 45.

### Claim 47 (depends on 46)

A mammalian cell transformed with the expression vector according to claim 46.