Anti-tfr1 antibodies, preparation methods and uses thereof

Abstract

Provided are anti-TFR1 antibodies or antigen-binding fragments thereof, multispecific antibodies comprising an anti-TFR1 antibody or antigen-binding fragment thereof, and methods of making and uses thereof.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480082928.0 (22) Application Date 2024.12.27 (66) Domestic Priority Data PCT / CN2023 / 143182 2023.12.29 CN (85) PCT International Application Entering National Phase Date 2026.06.29 (86) PCT International Application Application Data PCT / CN2024 / 143098 2024.12.27 (87) PCT International Application Publication Data WO2025 / 140522 EN 2025.07.03 (71) Applicant Nona Bio (Suzhou) Co., Ltd. Address 215000 Unit 202, Building A3, No. 218 Xinghu Street, Suzhou Industrial Park, Suzhou City, Jiangsu Province (72) Inventors: Wang Ming, Pan Qiuming, Li Yan, Wang Chengen, Wang Yuandong, Zheng Hailin, Yang Yuanyuan, Yin Yuexiang (74) Patent Agency: Beijing Yongxin Tongchuang Intellectual Property Agency Co., Ltd. 11376 Patent Attorneys: Luan Xingming, Cheng Dajun (51) Int.Cl. C07K 16 / 28 (2006.01) C07K 19 / 00 (2006.01) C07K 16 / 46 (2006.01) C12N 15 / 13 (2006.01) C12N 15 / 63 (2006.01) C12N 5 / 10 (2006.01) A61K 38 / 00 (2006.01) A61K 39 / 00 (2006.01) A61P 35 / 00(2006.01) (54) Invention Title Anti-TFR1 Antibody, Preparation Method and Uses (57) Abstract Provides an anti-TFR1 antibody or its antigen-binding fragment, a multispecific antibody containing an anti-TFR1 antibody or its antigen-binding fragment, and its preparation method and uses.Claims (5 pages), Description (52 pages), Sequence Listing (electronic publication), Drawings (11 pages), CN 122535623 A, 2026.08.07, CN 1 22 53 56 23 A. 1. An anti-transferrin receptor 1 (TFR1) antibody or its antigen-binding fragment, the anti-transferrin receptor 1 antibody or its antigen-binding fragment comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises an amino acid sequence different from SEQ ID NO: 8 in that no more than two amino acids are added, deleted, or substituted; VH CDR2 comprises an amino acid sequence different from SEQ ID NO: 16 in that no more than two amino acids are added, deleted, or substituted; and / or VH CDR3 comprises an amino acid sequence different from SEQ ID NO: 24 in that no more than two amino acids are added, deleted, or substituted. 2. The anti-TFR1 antibody or antigen-binding fragment of claim 1, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 8, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the VH CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 24 in that no more than two amino acids are added, deleted, or substituted; preferably, the VH CDR3 comprises a non-conserved amino acid substitution at the position corresponding to amino acid 2, 4, 5, or 7 of SEQ ID NO: 24. 3. The anti-TFR1 antibody or antigen-binding fragment of claim 1 or 2, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 8, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 24, 79, 80, 81, or 82. 4. The anti-TFR1 antibody or antigen-binding fragment of claim 1, wherein the heavy chain variable domain comprises VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequences of SEQ ID NO: 52, 98, 99, 100, or 101.5. The anti-TFR1 antibody or antigen-binding fragment according to any one of claims 1-4, wherein the heavy chain variable domain comprises: (A) an amino acid sequence of SEQ ID NO: 52, 98, 99, 100, or 101; or (B) an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 52, 98, 99, 100, or 101; or (C) an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions compared to the amino acid sequence of SEQ ID NO: 52, 98, 99, 100, or 101, wherein preferably, the amino acid additions, deletions, and / or substitutions do not occur in the CDR region. 6. The anti-TFR1 antibody or antigen-binding fragment of any one of claims 1-5, wherein the anti-TFR1 antibody or antigen-binding fragment (1) binds to human TFR1 and non-human primate TFR1; and / or (2) substantially does not affect the binding of transferrin to TFR1; and / or (3) binds to the transferrin-TFR1 complex. 7. The anti-TFR1 antibody or antigen-binding fragment of any one of claims 1-6, wherein the anti-TFR1 antibody or antigen-binding fragment is a single-domain antibody or a heavy-chain-only antibody, and optionally is a fully human antibody. 8. The anti-TFR1 antibody or antigen-binding fragment of any one of claims 1-7, wherein the anti-TFR1 antibody or antigen-binding fragment further comprises an Fc region, and optionally the Fc region comprises one or more modifications to reduce effector function and / or enhance FcRn binding. 9. An anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-8, wherein the anti-TFR1 antibody or antigen-binding fragment comprises a heavy chain having an amino acid sequence having SEQ ID NO: 59, 107, 108, 109, or 110. 10. A transferrin receptor 1 (TFR1) binder comprising an anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9 conjugated to a further molecule. 11. The TFR1 binder as described in claim 10, wherein the further molecule is a therapeutic agent for treating diseases or disorders selected from: cancer, central nervous system cancers, Alzheimer's disease, Parkinson's disease, Huntington's disease, Hunt syndrome, schizophrenia, antidepressants, multiple sclerosis, amyotrophic lateral sclerosis, lysosomal storage diseases with encephalopathy, glycogen storage diseases, muscular dystrophy, cerebral ischemia, prion diseases, traumatic central nervous system disorders, viral and bacterial central nervous system diseases.12. The TFR1 binding agent of claim 10, wherein the additional molecule is an imaging agent selected from: radionuclides, biotin, fluorescent proteins, fluorophores, horseradish peroxidase, and alkaline phosphatase. 13. A multispecific antibody comprising a first antigen-binding moiety binding to TFR1 and a second antigen-binding moiety binding to a second antigen, wherein the first antigen-binding moiety comprises a heavy chain variable domain as defined in any one of claims 1-6. 14. A multispecific antibody comprising a first antigen-binding moiety binding to TFR1 and a second antigen-binding moiety binding to BACE1, wherein the first antigen-binding moiety comprises a heavy chain variable domain as defined in any one of claims 1-6, and the second antigen-binding moiety comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as contained in the amino acid sequence of SEQ ID NO: 102, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as contained in the amino acid sequence of SEQ ID NO: 104; preferably, HCDR1 comprises the amino acid sequence of SEQ ID NO: 67, HCDR2 comprises the amino acid sequence of SEQ ID NO: 73, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 83; and LCDR1 comprises the amino acid sequence of SEQ ID NO: 88, LCDR2 comprises the amino acid sequence of SEQ ID NO: 91, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 95. 15. The multispecific antibody of claim 14, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 102; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 104.16. A multispecific antibody comprising a first antigen-binding moiety binding to TFR1 and a second antigen-binding moiety binding to amyloid β (Aβ), wherein the first antigen-binding moiety comprises a heavy chain variable domain as defined in any one of claims 1-6, and the second antigen-binding moiety comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as contained in the amino acid sequence of SEQ ID NO: 103, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as contained in the amino acid sequence of SEQ ID NO: 105; preferably, HCDR1 comprises the amino acid sequence of SEQ ID NO: 68, HCDR2 comprises the amino acid sequence of SEQ ID NO: 74, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 84; and LCDR1 comprises the amino acid sequence of SEQ ID NO: 89, LCDR2 comprises the amino acid sequence of SEQ ID NO: 92, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 96. 17. The multispecific antibody of claim 16, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 103; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 105. 18. The multispecific antibody of any one of claims 13-17, wherein the second antigen-binding moiety comprises dsFv, scFv, scdsFv, di-scFv, Fab, scFab, or F(ab')2. 19. The multispecific antibody of any one of claims 13-18, wherein the multispecific antibody comprises only one heavy chain variable domain as defined in any one of claims 1-6. 20. The multispecific antibody of any one of claims 13-19, further comprising an Fc region, and optionally the Fc region comprising one or more modifications to reduce effector function and / or enhance FcRn binding. 21. The multispecific antibody of claim 20, wherein the Fc region is a heterodimeric Fc region comprising a first Fc region subunit and a second Fc region subunit, wherein one of the first Fc region subunit and the second Fc region subunit contains a club-shaped mutation and the other contains a mortar-shaped mutation.22. The multispecific antibody of claim 21, wherein the club mutation comprises one or more mutations selected from S354C and T366W; and / or the mortise mutation comprises one or more mutations selected from Y349C, T366S, L368A, and Y407V; preferably, the club mutation comprises S354C and T366W, and the mortise mutation comprises Y349C, T366S, L368A, and Y407V. 23. The multispecific antibody of claim 21 or 22, wherein the second antigen-binding portion comprises a heavy chain variable region and a light chain variable region, and the multispecific antibody comprises: (a) (1) a first polypeptide comprising the heavy chain variable region, a CH1 domain and a first Fc subunit, (2) a second polypeptide comprising the light chain variable region and a light chain constant region, and (3) a third polypeptide comprising the heavy chain variable domain and the second Fc subunit, or (b) (1) a first polypeptide comprising the heavy chain variable region, a CH1 domain, a first Fc subunit, an optional peptide linker and the heavy chain variable domain, (2) a second polypeptide comprising the heavy chain variable region, a CH1 domain and a second Fc subunit, and (3) a third polypeptide and a fourth polypeptide each comprising the light chain variable region and the light chain constant region, wherein the first Fc subunit and the second Fc subunit form the heterodimeric Fc region. 24. The multispecific antibody of claim 14, wherein the multispecific antibody comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 122, a second polypeptide having the amino acid sequence of SEQ ID NO: 113, and a third polypeptide having the amino acid sequence of SEQ ID NO: 129, 130, 131, 132, or 133. 25. The multispecific antibody of claim 16, wherein the multispecific antibody comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 119, 125, 126, 127, or 128, a second polypeptide having the amino acid sequence of SEQ ID NO: 117, and a third polypeptide and a fourth polypeptide each having the amino acid sequence of SEQ ID NO: 115. 26. A pharmaceutical composition or kit comprising an anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9, a TFR1 binder as described in any one of claims 10-12, or a multispecific antibody as described in any one of claims 13-25. 27. A nucleic acid encoding an anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9, or a multispecific antibody as described in any one of claims 13-25. 28. A vector comprising the nucleic acid as described in claim 27. 29. A host cell comprising the nucleic acid as described in claim 27 or the vector as described in claim 28.30. Use of the anti-TFR1 antibody or antigen-binding fragment as claimed in any one of claims 1-9 in the manufacture of a pharmaceutical agent for transporting a compound across the blood-brain barrier. 31. Use of the anti-TFR1 antibody or antigen-binding fragment as claimed in any one of claims 1-9 in the manufacture of a medicament for treating cancer. 32. Use of the anti-TFR1 antibody or antigen-binding fragment as claimed in any one of claims 1-9 in the manufacture of a medicament for treating a neurological disease or disorder, wherein the anti-TFR1 antibody or antigen-binding fragment is conjugated to a therapeutic agent for treating the neurological disease or disorder. 33. Use of the multispecific antibody as claimed in any one of claims 14-25 in the manufacture of a medicament for treating a neurological disease or disorder; preferably, the neurological disease or disorder is selected from Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, and glaucoma. 34. Use of the multispecific antibody of any one of claims 14-25 in the manufacture of a pharmaceutical agent or kit for diagnosing Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, or glaucoma. 35. Use of the multispecific antibody of any one of claims 14-25 in the manufacture of a medicament for inhibiting and / or reducing the formation of amyloid plaques in the brain. 36. A method of transporting a compound across the blood-brain barrier in a subject, the method comprising conjugating an anti-TFR1 antibody or antigen-binding fragment of any one of claims 1-9 to the compound and transporting the conjugate across the blood-brain barrier. 37. A method of treating cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an anti-TFR1 antibody or antigen-binding fragment of any one of claims 1-9. 38. A method of treating a neurological disease or disorder in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an anti-TFR1 antibody or antigen-binding fragment of any one of claims 1-9 conjugated to a therapeutic agent for treating the neurological disease or disorder. 39. A method for treating a neurological disease or disorder in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the multispecific antibody as described in any one of claims 14-25; preferably, wherein the neurological disease or disorder is selected from Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, and glaucoma. 40. A method for inhibiting and / or reducing the formation of amyloid plaques in the brain of a subject in need, the method comprising administering to the subject a therapeutically effective amount of the multispecific antibody as described in any one of claims 14-25 to inhibit and / or reduce the formation of amyloid plaques in the brain.41. The anti-TFR1 antibody or antigen-binding fragment of any one of claims 1-9, for use as a medicine, wherein the anti-TFR1 antibody or antigen-binding fragment transports a compound across the blood-brain barrier. 42. The anti-TFR1 antibody or antigen-binding fragment of any one of claims 1-9, for use in the treatment of cancer. 43. The anti-TFR1 antibody or antigen-binding fragment of any one of claims 1-9, for use as a medicine for the treatment of neurological diseases or disorders, wherein the anti-TFR1 antibody or antigen-binding fragment is conjugated to a therapeutic agent for the treatment of the neurological disease or disorder. 44. The multispecific antibody of any one of claims 14-25, for use in the treatment of neurological diseases or disorders, optionally wherein the neurological disease or disorder is selected from Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, and glaucoma. Claims 4 / 5 Page 5 CN 122535623 A 45. The multispecific antibody as claimed in any one of claims 14-25, for use in the diagnosis of Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, or glaucoma. 46. The multispecific antibody as claimed in any one of claims 14-25, for use in inhibiting and / or reducing the formation of amyloid plaques in the brain. Claims 5 / 5 Page 6 CN 122535623 A Cross-Reference to Related Applications Regarding Anti-TFR1 Antibody, Method of Preparation Thereof, and Use Thereof

[0001] This application claims priority to International Application No. PCT / CN2023 / 143182, filed on December 29, 2023, the contents of which are hereby incorporated by reference in their entirety. Technical Field

[0002] This disclosure relates to the fields of molecular biology and immunology, and particularly to anti-TFR1 antibodies or antigen-binding fragments thereof, multispecific antibodies comprising anti-TFR1 antibodies or antigen-binding fragments thereof, and methods of their preparation and uses. Background Art

[0003] As people's lifespans increase, the percentage of patients suffering from neurological diseases is gradually increasing. Therefore, the development of effective neurological drugs is crucial and urgent.

[0004] Monoclonal antibodies and other biological therapeutic agents have great therapeutic potential for treating central nervous system diseases. Unfortunately, many promising candidates have failed to show the expected effects because they cannot cross the blood-brain barrier (BBB), the dynamic interface that separates the central nervous system (CNS) from the circulatory system, limiting the ability to treat neurological diseases. Under normal human conditions, several essential nutrients and carrier proteins are thought to cross the BBB via receptors expressed on brain endothelial cells through a process called receptor-mediated endocytosis.Therefore, restoring BBB integrity to maintain brain homeostasis and temporarily opening the BBB to allow for efficient drug delivery to the CNS under pathological conditions are potential treatment options for patients with these disorders.

[0005] Transferrin receptor 1 (TFR1), also known as differentiation group 71 (CD71), is a type II transmembrane glycoprotein highly expressed on brain endothelial cells that binds transferrin (Tf) and plays a key role in cellular iron uptake through its interaction with iron-bound Tf. Iron is known to dissociate from Tf in acidified endosomes, and the Tf-TFR1 complex is recycled back into the plasma membrane. In this way, it is involved in the control of cellular iron homeostasis.

[0006] Utilizing this mechanism, anti-TFR antibodies have been used as shuttles to deliver target proteins across the BBB without affecting iron homeostasis. Currently available anti-TFR1 antibody-based neuropharmaceuticals include pabinafuspa alfa (a fusion protein containing a humanized anti-human transferrin receptor (TFR) antibody and human iduronate-2-sulfatase) for treating CNS symptoms of MPS-II (Hunter's syndrome) and trontinemab (a bispecific antibody targeting amyloid-β (Aβ) and TFR1) for treating Alzheimer's disease.

[0007] To date, there is a lack of fully human antibodies that do not affect the binding of TFR to TF. Furthermore, fully human heavy chain antibodies have a lower molecular weight and are more likely to cross the blood-brain barrier compared to conventional antibodies.

[0008] In one aspect, an anti-transferrin receptor 1 (TFR1) antibody or its antigen-binding fragment is provided, wherein the anti-TFR1 antibody or antigen-binding fragment comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1 comprises an amino acid sequence that differs from SEQ ID NO: 8 in that no more than 2 amino acids are added, deleted or substituted; VH CDR2 comprises an amino acid sequence that differs from SEQ ID NO: 16 in that no more than 2 amino acids are added, deleted or substituted; and / or VH CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 24 in that no more than 2 amino acids are added, deleted or substituted.

[0009] In some embodiments, the VH CDR1 contains the amino acid sequence of SEQ ID NO: 8, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 16, and the VH CDR3 contains an amino acid sequence that differs from SEQ ID NO: 24 in that no more than two amino acids are added, deleted, or substituted.In some preferred embodiments, the VH CDR3 contains a non-conservative amino acid substitution at position 2, 4, 5, or 7 of amino acid SEQ ID NO: 24. In some embodiments, the VH CDR1 contains the amino acid sequence of SEQ ID NO: 8, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 16, and the VH CDR3 contains the amino acid sequence of SEQ ID NO: 24, 79, 80, 81, or 82.

[0010] In some embodiments, the heavy chain variable domain contains VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequences of SEQ ID NO: 52, 98, 99, 100, or 101.

[0011] In some embodiments, the heavy chain variable domain comprises: (A) an amino acid sequence of SEQ ID NO: 52, 98, 99, 100, or 101; or (B) an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 52, 98, 99, 100, or 101; or (C) an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions compared to the amino acid sequence of SEQ ID NO: 52, 98, 99, 100, or 101. In some preferred embodiments, the amino acid additions, deletions, and / or substitutions do not occur in the CDR region.

[0012] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment disclosed herein (1) binds to human TFR1 and non-human primate TFR1; and / or (2) substantially does not affect the binding of transferrin to TFR1; and / or (3) binds to the transferrin-TFR1 complex.

[0013] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment disclosed herein is a single-domain antibody or a heavy-chain-only antibody, and optionally is a fully human antibody.

[0014] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment disclosed herein further comprises an Fc region, and optionally the Fc region comprises one or more modifications to reduce effector function and / or enhance FcRn binding.

[0015] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment disclosed herein comprises a heavy chain having an amino acid sequence having SEQ ID NO: 59, 107, 108, 109, or 110.

[0016] In another aspect, a transferrin receptor 1 (TFR1) binder is provided, which comprises an anti-TFR1 antibody or antigen-binding fragment of the present disclosure conjugated to an additional molecule.

[0017] In some embodiments, the additional molecule is a therapeutic agent for treating diseases or disorders selected from: cancer, central nervous system cancers, Alzheimer's disease, Parkinson's disease, Huntington's disease, Hunt syndrome, schizophrenia, antidepressants, multiple sclerosis, amyotrophic lateral sclerosis, lysosomal storage diseases with encephalopathy, glycogen storage diseases, muscular dystrophy, cerebral ischemia, prion diseases, traumatic central nervous system disorders, viral and bacterial central nervous system diseases.

[0018] In some embodiments, the additional molecule is an imaging agent selected from: radionuclides, biotin, fluorescent proteins, fluorophores, horseradish peroxidase, and alkaline phosphatase.

[0019] In another aspect, a multispecific antibody is provided comprising a first antigen-binding moiety binding to TFR1 and a second antigen-binding moiety binding to a second antigen, wherein the first antigen-binding moiety comprises a heavy chain variable domain as described herein.

[0020] In a particular aspect, a multispecific antibody (“anti-TFR1 / BACE1 antibody”) that binds to TFR1 and BACE1 is provided. In some embodiments, the anti-TFR1 / BACE1 antibody comprises a first antigen-binding moiety binding to TFR1 and a second antigen-binding moiety binding to BACE1, wherein the first antigen-binding moiety comprises a heavy chain variable domain as described herein, and the second antigen-binding moiety comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as contained in the amino acid sequence of SEQ ID NO: 102, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as contained in the amino acid sequence of SEQ ID NO: 104, as described on page 2 / 52 of the specification, CN 122535623 A. In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 67, HCDR2 comprises the amino acid sequence of SEQ ID NO: 73, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 83; and LCDR1 comprises the amino acid sequence of SEQ ID NO: 88, LCDR2 comprises the amino acid sequence of SEQ ID NO: 91, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 102; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 104.

[0021] In another particular aspect, a multispecific antibody (“anti-TFR1 / Aβ antibody”) that binds to TFR1 and amyloid β (Aβ) is provided.In some embodiments, the anti-TFR1 / Aβ antibody comprises a first antigen-binding moiety binding to TFR1 and a second antigen-binding moiety binding to Aβ, wherein the first antigen-binding moiety comprises a heavy chain variable domain as described herein, and the second antigen-binding moiety comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as contained in the amino acid sequence of SEQ ID NO: 103, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as contained in the amino acid sequence of SEQ ID NO: 105. In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 68, HCDR2 comprises the amino acid sequence of SEQ ID NO: 74, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 84; and LCDR1 comprises the amino acid sequence of SEQ ID NO: 89, LCDR2 comprises the amino acid sequence of SEQ ID NO: 92, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 103; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 105.

[0022] In some embodiments, the second antigen-binding portion comprises dsFv, scFv, scdsFv, di-scFv, Fab, scFab, or F(ab')2.

[0023] In some embodiments, the multispecific antibody disclosed herein comprises only one heavy chain variable domain as described herein.

[0024] In some embodiments, the multispecific antibody disclosed herein further comprises an Fc region, and optionally the Fc region comprises one or more modifications to reduce effector function and / or enhance FcRn binding. In some embodiments, the Fc region is a heterodimeric Fc region comprising a first Fc region subunit and a second Fc region subunit, wherein one of the first Fc region subunit and the second Fc region subunit comprises a club-shaped mutation, and the other comprises a mortar-shaped mutation. In some embodiments, the pestle mutation comprises one or more mutations selected from S354C and T366W; and / or the mortar mutation comprises one or more mutations selected from Y349C, T366S, L368A, and Y407V. In some preferred embodiments, the pestle mutation comprises S354C and T366W, and the mortar mutation comprises Y349C, T366S, L368A, and Y407V.

[0025] In some embodiments, the second antigen-binding portion comprises a heavy chain variable region and a light chain variable region, and the multispecific antibody comprises: (a) (1) a first polypeptide comprising the heavy chain variable region, a CH1 domain and a first Fc region subunit, (2) a second polypeptide comprising the light chain variable region and a light chain constant region, and (3) a third polypeptide comprising the heavy chain variable domain and the second Fc region subunit, or (b) (1) a first polypeptide comprising the heavy chain variable region, a CH1 domain, a first Fc region subunit, an optional peptide linker and the heavy chain variable domain, (2) a second polypeptide comprising the heavy chain variable region, a CH1 domain and a second Fc region subunit, and (3) a third polypeptide and a fourth polypeptide each comprising the light chain variable region and the light chain constant region, wherein the first Fc region subunit and the second Fc region subunit form the heterodimeric Fc region.

[0026] In some embodiments, the anti-TFR1 / BACE1 antibody comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 122, a second polypeptide having the amino acid sequence of SEQ ID NO: 113, and a third polypeptide having the amino acid sequence of SEQ ID NO: 129, 130, 131, 132, or 133.

[0027] In some embodiments, the anti-TFR1 / Aβ antibody comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 119, 125, 126, 127, or 128, a second polypeptide having the amino acid sequence of SEQ ID NO: 117, and a third polypeptide and a fourth polypeptide each having the amino acid sequence of SEQ ID NO: 115.

[0028] Further, pharmaceutical compositions or kits are provided comprising the anti-TFR1 antibody or antigen-binding fragment, TFR1 binder, or multispecific antibody disclosed herein.

[0029] Further provided are nucleic acids encoding the anti-TFR1 antibody or antigen-binding fragment or multispecific antibody disclosed herein, vectors containing the nucleic acids disclosed herein, and host cells containing the nucleic acids disclosed herein or the vectors.

[0030] In another aspect, use of the anti-TFR1 antibody or antigen-binding fragment disclosed herein is provided in the manufacture of pharmaceutical agents for transporting compounds across the blood-brain barrier.

[0031] In yet another aspect, use of the anti-TFR1 antibody or antigen-binding fragment disclosed herein is provided in the manufacture of medicaments for treating cancer.

[0032] Further provided are use of the anti-TFR1 antibody or antigen-binding fragment disclosed herein in the manufacture of medicaments for treating neurological diseases or disorders, wherein the anti-TFR1 antibody or antigen-binding fragment is conjugated to a therapeutic agent for treating the neurological disease or disorder.

[0033] In another aspect, the use of the multispecific antibody disclosed herein is provided in the manufacture of a medicament for treating a neurological disease or disorder; preferably, the neurological disease or disorder is selected from Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, and glaucoma.

[0034] Further, the use of the multispecific antibody disclosed herein is provided in the manufacture of a medicament or kit for diagnosing Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, or glaucoma.

[0035] Further, the use of the multispecific antibody disclosed herein is provided in the manufacture of a medicament for inhibiting and / or reducing the formation of amyloid plaques in the brain.

[0036] In yet another aspect, a method for transporting a compound across the blood-brain barrier in a subject is provided, the method comprising conjugating the anti-TFR1 antibody or antigen-binding fragment of the present disclosure to the compound, and transporting the conjugate across the blood-brain barrier.

[0037] In another aspect, a method is provided for treating a neurological disease or disorder in a subject of need, the method comprising administering to the subject a therapeutically effective amount of an anti-TFR1 antibody or antigen-binding fragment of the present disclosure conjugated to a therapeutic agent for treating the neurological disease or disorder.

[0038] In a particular aspect, a method is provided for treating a neurological disease or disorder in a subject of need, the method comprising administering to the subject a therapeutically effective amount of a multispecific antibody of the present disclosure. In some embodiments, the neurological disease or disorder is selected from Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, and glaucoma.

[0039] Further, a method is provided for inhibiting and / or reducing the formation of amyloid plaques in the brain of a subject of need, the method comprising administering to the subject a therapeutically effective amount of a multispecific antibody of the present disclosure to inhibit and / or reduce the formation of amyloid plaques in the brain.

[0040] In another aspect, the anti-TFR1 antibody or antigen-binding fragment disclosed herein is provided for use as a medicament, wherein the anti-TFR1 antibody or antigen-binding fragment transports a compound across the blood-brain barrier.

[0041] In yet another aspect, the anti-TFR1 antibody or antigen-binding fragment disclosed herein is provided for use in the treatment of cancer. Specification 4 / 52 pages 10 CN 122535623 A

[0042] In another aspect, the anti-TFR1 antibody or antigen-binding fragment disclosed herein is provided for use as a medicament for the treatment of neurological diseases or disorders, wherein the anti-TFR1 antibody or antigen-binding fragment is conjugated to a therapeutic agent for the treatment of the neurological disease or disorder.

[0043] In a particular aspect, multispecific antibodies of this disclosure are provided for use in the treatment of neurological diseases or disorders, optionally wherein the neurological disease or disorder is selected from Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, and glaucoma.

[0044] Further, multispecific antibodies of this disclosure are provided for use in the diagnosis of Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, or glaucoma.

[0045] Multispecific antibodies of this disclosure are also provided for use in inhibiting and / or reducing the formation of amyloid plaques in the brain. Brief Description of the Drawings

[0046] Figure 1 illustrates the binding affinity of the anti-TFR1 HCAb antibody to human TFR1-His protein.

[0047] Figure 2 illustrates the binding affinity of the anti-TFR1 HCAb antibody to cyno TFR1-His protein.

[0048] Figure 3 shows the binding affinity of anti-TFR1 HCAb antibody to HEK293T-huTFR1 cells.

[0049] Figure 4 shows the binding affinity of anti-TFR1 HCAb antibody to HEK293T-cyno TFR1 cells.

[0050] Figure 5 shows the binding affinity of anti-TFR1 HCAb antibody to CHOK1-hCD40 cells.

[0051] Figure 6 shows the epitope binning of anti-TFR1 HCAb antibody obtained by competitive assay.

[0052] Figure 7 shows the blocking effect of anti-TFR1 antibody obtained by ELISA on the binding of human TF protein to human TFR1.

[0053] Figure 8 shows the activity of anti-TFR1 antibody obtained by FACS in blocking the binding of human TF to TFR1.

[0054] Figure 9 shows the binding affinity of PR012592 mutant to human TFR1-His protein.

[0055] Figure 10 shows the binding affinity of the PR012592 mutant to the cyno TFR1-His protein.

[0056] Figure 11 shows the binding affinity of the PR012592 mutant to HEK293T-huTFR1 cells.

[0057] Figure 12 shows a schematic diagram of the BACE1×TFR1 BsAb structure.

[0058] Figure 13 shows a schematic diagram of the Aβ×TFR1 BsAb structure.

[0059] Figure 14 shows the binding affinity of Aβ×TFR1 BsAb to human TFR1-His protein.

[0060] Figure 15 shows the binding affinity of BACE1×TFR1 BsAb to human TFR1-His protein.

[0061] Figure 16 shows the binding affinity of Aβ×TFR1 BsAb to HEK293T-huTFR1 cells.

[0062] Figure 17 shows the binding affinity of BACE1×TFR1 BsAb to HEK293T-huTFR1 cells.

[0063] Figure 18 shows the binding affinity of BACE1×TFR1 BsAb to CHOK1-cyno TFR1 cells.

[0064] Figure 19 shows a schematic diagram of the in vivo study design.

[0065] Figure 20 shows the relative drug concentration in the brain parenchyma.

[0066] Figure 21 shows the relative drug concentration in the whole brain.

[0067] Figure 22 shows the relative drug concentration in serum.

[0068] Figure 23 shows IHC staining of the cerebral cortex. Detailed Definitions

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Furthermore, terms and experimental procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology are those widely used in the art. In addition, for better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0070] As used herein, the expressions “at least one” or “one or more” mean one, two, three, four, five, six or more. As used herein, unless explicitly indicated to the contrary, “one (a / an)” should be understood to mean “at least one”.

[0071] As used herein, the term “antigen (Ag)” is any substance that can trigger an immune response in vivo, particularly triggering antibody production. As used herein, the term refers to any substance containing one or more epitopes (i.e., one or more antigenic determinants) that are specifically bound by an antibody. Antigens can be polypeptides, lipids, carbohydrates, polynucleotides, etc. In a particular embodiment, an antigen refers to a TFR1 protein or a fragment thereof containing an epitope bound by an antibody or antigen-binding fragment of the present disclosure. TFR1 proteins or fragments thereof can belong to any species, such as humans, mice, and rabbits.

[0072] As used herein, the term "antibody (Ab)" is an immunoglobulin (Ig) molecule or fragment thereof that specifically binds to an epitope of an antigen through at least one antigen-binding site (i.e., complementary site). Antibodies can be derived from various species, including but not limited to mice, rats, rabbits, guinea pigs, and humans.

[0073] As used herein, the definition of "antibody" encompasses conventional antibodies, single-domain antibodies, heavy-chain-only antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), monoclonal antibodies, polyclonal antibodies, fully human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, biantibodies, anti-idiotype antibodies, and antigen-binding fragments.The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0074] As used herein, the terms “heavy chain only antibody” or “heavy chain antibody” or “HCAb” refer to an antibody consisting of only two heavy chains and lacking the two light chains typically found in conventional antibodies. Compared to conventional antibodies, HCAbs require only the variable domain (also known as “VHH”) of the heavy chain for antigen binding. That is, the antigen-binding site of an HCAb is primarily determined by the three CDRs of the VHH. Typically, the heavy chain of an HCAb lacks the CH1 domain typically found in the heavy chain of conventional antibodies. HCAbs can be obtained through hybridoma technology, or phage libraries or eukaryotic cell libraries that display or secrete HCAbs. HCAbs can also be obtained from transgenic animals, such as transgenic mice capable of producing fully human HCAbs (see, for example, WO 2007 / 096779).

[0075] As used herein, the term "single-domain antibody" or "nanobody" refers to an antibody consisting of only a single variable domain. The single variable domain can be a heavy chain single variable domain or a light chain single variable domain.

[0076] "Conventional" or "full-length" antibodies typically consist of four polypeptides: two heavy chains (HC) and two light chains (LC). Each light chain (from the amino terminus to the carboxyl terminus) contains a light chain variable region (VL) and a light chain constant region (CL). Each heavy chain (from the amino terminus to the carboxyl terminus) contains a heavy chain variable region (VH) and one or more heavy chain constant regions (CH), which may contain CH1, CH2, CH3, and CH4.

[0077] As used herein, the term "monoclonal antibody" refers to a formulation of an antibody molecule having a single molecular composition. Monoclonal antibodies exhibit single binding specificity and affinity. In one embodiment, the monoclonal antibody is produced by a hybridoma containing B cells fused to immortalized cells from a non-human animal (e.g., a mouse).

[0078] As used herein, the term "recombinant antibody" includes all antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from hybridomas prepared from or that are transgenic or transchromosomally modified animals (e.g., mice) for immunoglobulin genes, (b) antibodies isolated from host cells transformed for antibody expression (e.g., from transfected tumors), (c) antibodies isolated from recombinant combined antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing immunoglobulin gene sequences to other DNA sequences.Specification 6 / 52 pages 12 CN 122535623 A

[0079] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). The definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0080] The term "chimeric antibody" refers to those antibodies in which a portion of each amino acid sequence of the heavy and light chains is homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular class, while the remaining segments of the chains are homologous to a corresponding sequence in an antibody of another species or class.

[0081] As used herein, a "humanized antibody" is an antibody derived from a non-human antibody whose sequence has been modified to increase sequence similarity to human immunoglobulin molecules.

[0082] As used herein, the term "fully human antibody" refers to an antibody that contains only the sequence of human immunoglobulins. A fully human antibody may contain mouse carbohydrate chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells.

[0083] As used herein, an "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that contains at least a portion of a variable domain (e.g., one or more CDRs) of the antibody and specifically binds to the same homologous antigen as the antigen bound by the full-length antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, scFv, dsFv, scdsFv, biantibodies, single variable domain (e.g., VHH), Fd, di-scFv, Fab, Fab', scFab, F(ab')2, and other fragments. Antigen-binding fragments typically contain at least or about 50 amino acids, and typically at least or about 200 amino acids.

[0084] A single-chain variable fragment (scFv) is a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin are linked by a peptide linker. The peptide linker can link the N-terminus of the VH to the C-terminus of the VL, or vice versa.

[0085] Fv or scFv can be further stabilized by introducing an interchain disulfide bond between the VL and VH to produce a disulfide-bonded variable fragment (dsFv) or a single-chain disulfide-bonded variable fragment (scdsFv, with a peptide linker and an interchain disulfide bond), respectively.

[0086] Two single-chain variable fragments can be linked by a peptide linker to produce a covalently linked divalent single chain (referred to as "sc(Fv)2"). A "biantibody" is a non-covalent dimer of scFv.

[0087] The term “di-scFv” refers to a fusion protein comprising two light chain variable regions (VL) and two heavy chain variable regions (VH), wherein the two VLs and two VHs are optionally sequentially linked via peptide linkers and are capable of being expressed as a single-chain polypeptide. The two VLs and two VHs are optionally fused via peptide linkers to form a divalent molecule, such as in the order of VL1-linker-VH1-linker-VL2-linker-VH2 or VL1-linker-VH1-linker-VH2-linker-VL2 or VL1-linker-VL2-linker-VH2-linker-VH1 or VL1-linker-VH2-linker-VL2-linker-VH1, to form two antigen-binding domains.

[0088] The Fab fragment essentially comprises a light chain and a variable region and a constant region (i.e., VH-CH1, also known as the difficult fragment (Fd)) of the heavy chain, wherein VL and VH form antigen-binding sites (also known as the variable region fragment, Fv), and CL and CH1 are covalently linked by disulfide bonds.

[0089] Single-chain Fab (scFab) fragments can be generated by linking the light chain and Fd fragment with peptide linkers.

[0090] The F(ab')2 fragment essentially contains two Fab fragments linked by disulfide bonds in the hinge region. The Fab' fragment is half of the F(ab')2 fragment, formed by reducing disulfide bonds in the hinge region of the F(ab')2 fragment.

[0091] As used herein, the term "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain, which contains at least a portion of the constant region of the heavy chain. Preferably, the Fc region contains at least a CH3 domain, such as a CH3 domain, a CH2 domain, and a CH3 domain, or preferably a hinge region, a CH2 domain, and a CH3 domain. This term encompasses both native and variant Fc regions. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system (EU index), as described in Kabat et al., Sequences, 7 / 52, page 13, CN 122535623, A of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0092] In some embodiments, the antibody or antigen-binding fragment as described herein contains an Fc region. In some embodiments, the Fc region contains a CH3 domain. In some embodiments, the Fc region contains both a CH2 domain and a CH3 domain.In some embodiments, the Fc region comprises at least a portion of the hinge region, a CH2 domain, and a CH3 domain. In some embodiments, for example, where the Fc region is fused to the C-terminus of a heavy chain variable domain that binds to the TFR1 disclosed herein, the Fc region lacks the CH1 domain. The Fc region may be a monomeric Fc region, a soluble monomeric Fc region (see, for example, Wang C et al., Engineered Soluble Monomeric IgG1 Fc with Significantly Decreased Non-Specific Binding. Front Immunol. 2017 Nov 13; 8:1545), or a dimer Fc region comprising two identical (homodimer) or different (heterodimer) Fc region subunits.

[0093] The Fc region that can be used may be derived from any immunoglobulin subtype or subclass, such as IgG, IgM, IgA, IgD, and IgE. Preferably, the Fc region is derived from human IgG, such as human IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc region is the human IgG1 Fc region. In some embodiments, the human IgG1 Fc region may extend from Glu216, or Cys220, or Asp221, or Cys226, or Pro230 to the carboxyl terminus of the heavy chain, while the C-terminal lysine (Lys447) of the heavy chain may or may not be present. In some embodiments, a variable domain of the heavy chain is fused to the C-terminus of the Fc region, and the C-terminal Lys447 of the heavy chain may be missing.

[0094] The Fc region of the antibody may be modified to obtain an antibody with desired properties. For example, one or more cysteine ​​residues may be introduced into or removed from the hinge region (e.g., by site-directed mutagenesis) to promote or weaken dimerization. For example, the hinge region, CH2 and / or CH3 can be modified to prolong or shorten the half-life, promote cell internalization or tissue penetration, increase or decrease binding to FcγRIIB (see, for example, WO 2008150494 A1), increase or decrease Fc effector functions, including but not limited to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis and complement-dependent cytotoxicity (CDC) (see, for example, Caron, P. C., et al., J. Exp. Med. [Journal of Experimental Medicine] 176: 1191-1195 (1992); and Shopes BJ Immunol. [Immunology] 148 2918-2922 (1992)).

[0095] The term "hinge region" generally refers to the portion between the CH1 and CH2 domains of a conventional antibody, or the portion between the VHH and CH2 domains of a heavy chain antibody or its functional equivalent (e.g., the hinge region in a T-cell receptor (TCR)). The hinge region can be an entire hinge region or a portion thereof.

[0096] Variable regions (or "variable domains") allow for specific antigen recognition and binding. Each variable domain has three highly variable "complementarity-determining regions (CDRs)" and four relatively conserved "framework regions (FWRs)," arranged in the following order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus): FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. Those skilled in the art know and can identify CDRs and FWRs using methods in the art (e.g., the Kabat or Chothia numbering scheme) (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).

[0097] Each CDR forms a CDR loop and contributes to antigen-binding specificity and affinity. Framework residues form a scaffold and maintain the overall structure of the antigen-binding domain (e.g., Fv or VHH). Combinations of CDR loops (e.g., three CDRs from each of VL and VH, or three CDRs from VHH) form a three-dimensional structure with the aid of the FWR, which defines the complementary site of the antibody or the antigen-binding site. Depending on its position in VL or VH (or VHH), the CDR is designated as a heavy chain variable region CDR (HCDR or VH CDR), such as HCDR1 (or VH CDR1), HCDR2 (or VH CDR2), HCDR3 (or VH CDR3); or a light chain variable region CDR (LCDR or VL CDR), such as LCDR1 (or VL CDR1), LCDR2 (or VL CDR2), LCDR3 (or VL CDR3).

[0098] In a given amino acid sequence of the variable region of an immunoglobulin, the precise amino acid sequence boundary of each CDR can be determined by using any of a number of well-known antibody numbering schemes or combinations thereof, including, for example, Chothia (Chothia et al., (1989) Nature 342: 877-883), Al-Lazikani et al., “Standard Conformations for the Canonical Structures of Immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of The databases used include Bath (University College London), Contact (University College London), the International ImMunoGeneTics Database (IMGT) (imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. In the anti-TFR1 antibodies or antigen-binding fragments disclosed herein, the amino acid sequence of the CDR region is defined using the Chothia numbering scheme, and this does not mean that this disclosure protects only antibody molecules represented by CDR regions defined by the Chothia numbering scheme. It should be noted that the boundaries of CDRs in the variable region of the same antibody obtained based on different numbering schemes can be different; that is, the CDR sequences of the variable region in the same antibody defined under different numbering schemes can be different. Once a variable region (e.g., VH or VL) is given, those skilled in the art will understand that CDRs within the variable region can be defined by different numbering systems or combinations thereof. The CDR sequences defined under these different numbering schemes and the antibody molecules characterized thereby are also intended to be protected by this disclosure.

[0099] Those skilled in the art will be able to determine the positions of CDR, FWR, VHH, VH, CH1, hinge region, CH2, CH3, VL, and CL in an antibody, for example by means of algorithms or software (see, for example, William R. Strohl, Lila M. Strohl, (2012), Antibody structure-function relationships, Woodhead Publishing Series in Biomedicine, Therapeutic Antibody Engineering, Woodhead Publishing, pp. 37-56).

[0100] The term “multispecific antibody” is used in the broadest sense and specifically covers antibodies capable of specifically binding to two or more different epitopes. The two or more different epitopes may be present on the same biomolecule or on two or more different biomolecules. Multispecific antibodies may be multivalent (e.g., bivalent, trivalent, tetravalent, pentavalent, or more) antibodies. Multispecific antibodies can be bispecific, trispecific, or tetraspecific antibodies that can specifically bind to 2, 3, or 4 different epitopes. Exemplary multispecific antibodies can bind to both TFR1 and brain antigens. In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the multispecific antibody binds to TFR1 and BACE1 (“anti-TFR1 / BACE1 antibody”) or TFR1 and Aβ (“anti-TFR1 / Aβ antibody”).

[0101] Multispecific antibodies include, but are not limited to, full-length antibodies, antibody fragments (such as Fab, Fv, dsFv, scFv, biantibodies, single-chain biantibodies, bispecific biantibodies, and triantibodies), and antibody fragments that are covalently or non-covalently linked. Multispecific antibodies can be non-IgG-like antibodies or IgG-like antibodies. IgG-like multispecific antibodies can be symmetrical or asymmetrical. Methods for constructing multispecific antibodies using target antibodies or antigen-binding fragments are well known to those skilled in the art (see, for example, WO 93 / 08829; Suresh et al., (1986) Methods in Enzymology, 121: 210; and Traunecker et al., (1991) EMBO, 10: 3655-3659). Multispecific antibodies can be generated and isolated using a variety of methods known in the art.For example, multispecific antibodies can be expressed from a suitable host, isolated from the specification page 9 / 52 15 CN 122535623 A, or generated by chemical or enzymatic conjugation of different portions of the multispecific antibody.

[0102] As used herein, a “bispecific antibody” is a multispecific antibody capable of specifically binding to two different epitopes. The two different epitopes may be present on the same biomolecule or on two different biomolecules.

[0103] As used herein, the term “mortar and pestle structure” or “KiH” technology refers to a technique for guiding two polypeptides to pair together in vitro or in vivo by introducing a protrusion (mortar) into one polypeptide and a cavity (pothole) into the other polypeptide at the interface of two polypeptide interactions. For example, KiH can be introduced into the Fc:Fc binding interface, CL:CH1 interface, or VH / VL interface of an antibody (see, for example, US 2011 / 0287009, US 2007 / 0178552, WO 96 / 027011, WO 98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, KiH drives two different heavy chains to pair together during the manufacture of multispecific antibodies.

[0104] As used herein, the term “affinity” is a measure of the strength of the non-covalent interaction between an antibody and its antigen.

[0105] Affinity can be measured using a variety of methods known in the art. Such methods may include, but are not limited to, biolayer interferometry (BLI, e.g., using the Octet Fortebio system), radioimmunoassay (RIA), surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), flow cytometry (also known as “FACS”), and other kinetic interaction assays known in the art (see, for example, Paul, WE, Fundamental Immunology, 2nd ed., Raven Press, New York, pp. 332-336 (1989); also see U.S. Patent No. US 7,229,619). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (e.g., BiaCore 2000, Biacore AB, Uppsala, Sweden).

[0106] Typically, antibody affinity can be measured and reported by the equilibrium dissociation constant KD. KD can be calculated from kinetic analysis using the following equation: KD = Kd / Ka, where Kd and Ka are the dissociation rate constant and association rate constant between the antigen and antibody, respectively.

[0107] KD is inversely related to affinity, that is, the smaller the KD value, the greater the affinity.Typical KD values ​​for antibody or antigen-binding fragments range from μM (10⁻⁶ M) to nM (10⁻⁷ to 10⁻⁹ M). In some embodiments, the KD values ​​described herein are measured using biolayer interferometry, as illustrated in the examples of this disclosure.

[0108] As used herein, the term “half-maximum effective concentration” or “EC50” refers to the concentration of an agent (e.g., an antibody) at which it induces a response between baseline and maximum after some specified exposure time. EC50 can be used as an alternative measure of the antibody’s affinity for its target.

[0109] As used herein, “specific binding” refers to the affinity between an antibody or antigen-binding fragment and its homologous antigen. Typically, antibodies bind specifically to their homologous antigens with high affinity, or with a KD value of 10⁻⁷ to 10⁻⁹ M or less, preferably with a KD value of 10⁻⁸ or less, 10⁻⁹ or less, 10⁻¹⁰ or less, 10⁻¹¹ or less, or 10⁻¹² or less. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment specifically binds to human TFR1.

[0110] The definition of “antibody” or “antigen-binding fragment” covers any variant derived from any antibody or antigen-binding fragment as described herein, such as amino acid sequence variants, glycosylated variants, and covalently modified variants. The definition of an antigen-binding protein falls within the definition of an antibody or antigen-binding fragment.

[0111] Compared to the exemplary antibodies described herein, an “amino acid sequence variant” may contain one or more amino acid modifications (including substitutions, insertions, and / or deletions), provided that the amino acid sequence variant retains the desired antigen-binding activity. For example, an amino acid sequence variant can be obtained by substituting one or more amino acid residues with corresponding conserved residues without affecting the biological activity of the protein. Suitable conserved substitutions of amino acids are known to those skilled in the art. Those skilled in the art will recognize that, generally speaking, a single amino acid substituent in a non-essential region of a polypeptide (e.g., in the frame region of an antibody) does not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0112] Amino acid sequence variants also include “Fc variants” of any antibody or antigen-binding fragment as described herein. Such Fc variants may contain one or more modifications in the Fc region such that one or more properties of the Fc region can be altered.For example, modifications can be introduced into the Fc region to increase or decrease the effector function of the antibody (including, but not limited to, ADCC, CDC, and ADCP), increase half-life (e.g., enhanced FcRn binding), or increase stability (e.g., introducing or removing cysteine ​​residues that may be involved in disulfide bond formation).

[0113] In some embodiments, such as those relating to the treatment of neurological diseases or disorders, a reduction in the effector function of the disclosed anti-TFR1 antibody, TFR1 binder, or multispecific antibody may be desirable. One or more modifications for reducing effector function may include, but are not limited to, one or more modifications (e.g., amino acid substitutions) at positions selected from the following locations in the Fc region: 297 (e.g., N297A, N297Q, or N297G), 234 (IgG1: L234A; IgG4: F234A; IgG2: V234A), 235 (e.g., L235E, L235A), and 237 (G237A).

[0114] One or more modifications for enhancing FcRn binding may include, but are not limited to, one or more modifications (e.g., amino acid substitutions) at positions selected from the following locations in the Fc region: 252 (e.g., M252Y), 254 (e.g., S254T), 256 (e.g., T256E), 428 (e.g., M428L), 434 (e.g., N434S and N434A), and 436 (e.g., Y436I). In some embodiments, the Fc region contains modifications of M252Y, S254T, and T256E. In some embodiments, the Fc region contains modifications of N434A and Y436I.

[0115] In some embodiments, the Fc region contains a modification of C220S. In some embodiments, the Fc region contains modifications of L234A, L235A, and G237A. In some embodiments, the Fc region is a human IgG1 Fc region containing one or more modifications selected from the group consisting of C220S, L234A, L235A, and G237A. In some embodiments, the human IgG1 Fc region contains a modification of C220S. In some embodiments, the human IgG1 Fc region contains modifications of L234A, L235A, and G237A. In some embodiments, the human IgG1 Fc region contains modifications of C220S, L234A, L235A, and G237A.

[0116] As used herein, “glycosylation” refers to a post-translational modification of a protein that results in the addition of a carbohydrate or glycan moiety to the protein backbone (e.g., serine or threonine-linked glycosylation for O-linked glycans, and asparagine-linked glycosylation for N-linked glycans). Those skilled in the art can use techniques known in the art to generate glycosylated variants of therapeutic antibodies to achieve desired therapeutic efficacy.

[0117] Covalently modified variants of any antibody or antigen-binding fragment as described herein are also contemplated.As used herein, “covalently modified variants” of antibody or antigen-binding fragments can be generated by introducing natural or non-natural amino acids, compounds, or peptide linkers via covalent linkage. For example, additional peptides can be linked to antibody or antigen-binding fragments as described herein to confer desired properties or reduce undesirable properties, such as increasing half-life, solubility, or uptake (e.g., facilitating transport across phospholipid membranes or organelles); and / or reducing immunogenicity, toxicity, or side effects. Other peptides can be, for example, peptides that can be used to guide the expression and secretion of antibody or antigen-binding fragments from host cells, or to facilitate the detection and / or isolation of antibody or antigen-binding fragments. Examples of such peptides may include, but are not limited to, signal transduction peptides (lead sequences), affinity tags (e.g., multihistidine tags (His6) or glutathione S-transferase (GST) tags), peptides containing protease cleavage sites, and reporter tags (e.g., fluorescent proteins). Other additional peptides can be bioactive peptides, such as peptides or proteins having therapeutic, binding, or enzymatic activity.

[0118] As used herein, the term “transferrin receptor 1” or “TFR1” refers to any naturally occurring TFR1 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term covers “full-length” TFR1 as well as fragments of TFR1. The term also covers naturally occurring variants of TFR1, such as splice variants or allelic variants. In some embodiments, TFR1 is human TFR1 (“hTFR1” or “huTFR1”), for example, human TFR1 containing the amino acid sequence shown in Uniprot accession number: P02786. In another embodiment, TFR1 is cynomolgus monkey TFR1 (“cynoTFR1”), for example, cynomolgus monkey TFR1 containing the amino acid sequence shown in Uniprot accession number: G8F602.

[0119] As used herein, the term “blood-brain barrier” or “BBB” refers to the physiological barrier between the peripheral circulation and the brain and spinal cord (i.e., the CNS), which is formed by tight junctions within the cortical membranes of the brain capillaries, creating a tight barrier to restrict the transport of molecules, even very small molecules such as urea (60 Daltons), into the brain. The blood-brain barrier in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina are continuous capillary barriers within the CNS and are collectively referred to herein as the BBB. The definition of BBB also includes the blood-cerebrospinal fluid (CSF) barrier, in which the barrier is formed by ependymal cells rather than capillary endothelial cells.

[0120] As used herein, “brain antigen” refers to an antigen (or target) expressed in the CNS (including the brain) that can be targeted with antibodies or small molecules.Examples of brain antigens may include, but are not limited to: β-secretase 1 (BACE1), amyloid precursor protein (APP), amyloid β (Abeta or Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prions, leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), p75 neurotrophic factor receptor (p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1), interleukin 1β (IL1β), and caspase 6.

[0121] Unless otherwise indicated, as used herein, the term “BACE1” refers to any native β-secretase 1 derived from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses “full-length” untreated BACE1 as well as any form of BACE1 produced by treatment in the cell. The term also encompasses naturally occurring variants of BACE1, such as splice variants or allelic variants.

[0122] As used herein, the term “amyloid precursor protein” or “APP” refers to a protein that can be proteolytically processed or cleaved by one or more processing or cleavage reactions to produce Aβ. APP includes all isotypes produced by alternative splicing.

[0123] The terms “amyloid β” and “Abeta (Aβ)” are used interchangeably to refer to fragments of amyloid precursor protein (“APP”) produced after BACE1 cleavage of APP, as well as its modifications, fragments, and any functional equivalents, including but not limited to Aβ1-40 and Aβ1-42.

[0124] As used herein, the term “amyloid plaque” refers to an insoluble, fibrous protein aggregate containing amyloid β. Amyloid plaques can be found in the CNS (e.g., the brain), for example, around dystrophic neurites, axonal terminals and dendrites, microglia and fibrous astrocytes.

[0125] As used herein, “nervous system disease or disorder” means a disease or disorder affecting the central nervous system (CNS) and / or having an etiology in the CNS. Exemplary nervous system diseases or disorders include, but are not limited to, neuropathy, amyloidosis, brain cancer, eye diseases or disorders, viral or microbial infections, inflammation, ischemia, neurodegenerative diseases, epilepsy, behavioral disorders and lysosomal storage diseases.

[0126] Examples of neurological diseases or disorders may include, but are not limited to: neurodegenerative diseases (e.g., Lewy body disease, post-poliomyelitis syndrome, Chailly-Dregger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy), striatal substantia nigra degeneration, tau protein diseases (e.g., Alzheimer's disease and supranuclear palsy), prion diseases (e.g., bovine spongiform encephalopathy, scrapie, Kreutzfeld-Jacob syndrome, kuru disease, Gerstmann-Straussler-Scharinkel disease, chronic wasting diseases and fatal familial insomnia), bulbar palsy, motor neuron disease and neurological disorders. (12 / 52 pages, 18 CN 122535623 A) Qualitative degenerative disorders (e.g., Canavan disease, Huntington's disease, neuronal ceroid lipofuscin deposition, Alexander disease, Tourette syndrome, Menkes curly hair syndrome, Cockayne syndrome, Hallewarden-Schpattz syndrome, Lafra disease, Rett syndrome, Wilson's disease, Lesch-Nair syndrome, and Ong-Lon syndrome), dementia (e.g., Pick's disease and spinocerebellar ataxia), and central nervous system cancers (including primary brain tumors and brain metastases from cancers in other parts of the body).

[0127] As used herein, the term "peptide" refers to two or more amino acids covalently linked. The terms "peptide" and "protein" are used interchangeably herein.

[0128] As used herein, the terms "first polypeptide (chain)" and similar terms like "second polypeptide (chain)," "third polypeptide (chain)," and "fourth polypeptide (chain)" are used only for the purpose of distinguishing polypeptides (chains) and are not intended to constrain polypeptides (chains) in any way. If desired, the antibody or antigen-binding fragment may comprise two or more first / second / third / fourth polypeptide (chains), which may be identical or different. These terms may refer to the same or different polypeptide (chains) when used in different embodiments.

[0129] As used herein, the term “cancer” refers to a proliferative disease. This term encompasses solid cancers and hematopoietic malignancies. Examples of solid cancers may include, but are not limited to, esophageal squamous cell carcinoma, breast cancer, ovarian cancer, lung cancer, cervical cancer, bladder cancer, osteosarcoma, pancreatic cancer, bile duct cancer, renal cell carcinoma, hepatocellular carcinoma, adrenocortical carcinoma, and nervous system cancers. Examples of hematopoietic malignancies may include, but are not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemias (e.g., acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic myeloid leukemia, chronic lymphocytic leukemia, and chronic lymphoid leukemia), and multiple myeloma.

[0130] As used herein, “nucleic acid” or “polynucleotide” means a polymer of at least two nucleotides or nucleotide derivatives linked together by a phosphodiester bond, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Exogenous nucleic acids can be introduced into host cells in the form of a vector.For the purpose of treating a disease, therapeutic nucleic acids may be introduced into a subject.

[0131] “Host cell” can be any prokaryotic or eukaryotic cell containing exogenous polynucleotides.

[0132] As used herein, the term “expression” refers to the production of RNA polynucleotides from transcription or the production of polypeptides from translation. The expression level of a polypeptide in a biological sample can be examined using any method known in the art. Such methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, immunofluorescence imaging, and immunohistochemistry using antibodies that specifically bind to the polypeptide to be examined.

[0133] As used herein, “vector” is a medium for transferring exogenous nucleic acids into host cells, wherein the exogenous nucleic acid is amplified or expressed. As used herein, the definition of “vector” encompasses plasmids, linearized plasmids, viral vectors, granules, phage vectors, phage particles, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc. A vector can be expressed and / or replicated within a host cell, meaning that the vector is capable of expressing RNA polynucleotides or polypeptides in the host cell and / or producing multiple copies of the vector. In order to be “expressible” or “reproducible,” a vector may contain a nucleic acid sequence or element operatively linked to a promoter.

[0134] As used herein, “operatively linked” in relation to a nucleic acid sequence or element means that these nucleic acid sequences are functionally related to each other. For example, a promoter may be operatively linked to a nucleic acid sequence encoding a polypeptide, thereby regulating or mediating the transcription of the nucleic acid. Those skilled in the art can select and use appropriate vectors for specific purposes.

[0135] As used herein, “treating” a disease or disorder means curing the disease or disorder, or stopping, alleviating, improving, or eliminating the symptoms of the disease or disorder. Thus, treatment encompasses prevention, therapy, and / or cure. “Prevention” means preventing an underlying disease and / or preventing the worsening or progression of the symptoms of the disease.

[0136] As used herein, “therapeutic effective amount” means an amount of a pharmaceutical agent, compound, or composition containing one or more active agents, as specified in the specification 13 / 52 page 19 CN 122535623 A, which, when administered to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, stop, or partially stop the symptoms of a disease or disorder.

[0137] As used herein, the term “subject” refers to an animal (e.g., a mammal), including but not limited to humans, non-human primates, dogs, cats, rabbits, rodents, etc. In some embodiments, the subject is a human.

[0138] Antibody identification numbers or identifiers (e.g., PR012592, PR012838, PR012839, PR013071, PR013072, PR013356, PR013357, PR013358, PR013359, PR013360, PR012932, PR013352, PR013353, PR013354, PR013355, etc.) are used only for the purpose of distinguishing or designating different antibodies or products, and are not intended to limit the characteristics of the antibodies or products disclosed herein. Similarly, the use of identification numbers or identifiers in the examples is merely for convenience and brevity. The characteristics of the antibodies or products disclosed herein are defined by those described in the appended claims. Anti-TFR1 Antibody or Antigen-Binding Fragment Thereof

[0139] In one aspect, an anti-transferrin receptor 1 (TFR1) antibody or an antigen-binding fragment thereof is provided.

[0140] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment binds to human TFR1 and non-human primate (e.g., cynomolgus monkey) TFR1. In some embodiments, the anti-TFR1 antibody or antigen-binding fragment substantially does not affect the binding of transferrin to TFR1. The term "substantially does not affect" includes not inhibiting / reducing and not increasing the binding of transferrin to TFR1. In some embodiments, the anti-TFR1 antibody or antigen-binding fragment binds to the transferrin-TFR1 complex.

[0141] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment comprises a heavy chain variable domain. In some embodiments, the heavy chain variable domain is a heavy chain monovariable domain.

[0142] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment is a single-domain antibody. In some specific embodiments, the anti-TFR1 antibody or antigen-binding fragment is a heavy chain-only antibody.

[0143] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment is a fully human antibody. In some embodiments, the anti-TFR1 antibody or antigen-binding fragment is a fully human single-domain antibody. In some embodiments, the anti-TFR1 antibody or antigen-binding fragment is a fully human heavy-chain-only antibody.

[0144] In some embodiments, the heavy-chain variable domain comprises VH CDR1, VH CDR2, and VH CDR3.

[0145] In some embodiments, VH CDR1 comprises an amino acid sequence that differs from SEQ ID NO: 8 in that no more than two amino acids are added, deleted, or substituted.

[0146] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 8.

[0147] In some embodiments, VH CDR2 comprises an amino acid sequence that differs from SEQ ID NO: 16 in that no more than two amino acids are added, deleted, or substituted.

[0148] In some embodiments, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16.

[0149] In some embodiments, VH CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 24 in that no more than two amino acids are added, deleted, or substituted.

[0150] In some embodiments, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 24.

[0151] In some embodiments, VH CDR3 comprises a non-conservative amino acid substitution at positions 2, 4, 5, or 7 of amino acids corresponding to SEQ ID NO: 24. In some embodiments, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 82.

[0152] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 8, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the VH CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 24 in that no more than two amino acids are added, deleted, or substituted. Specification 14 / 52 pages 20 CN 122535623 A

[0153] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 8, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the VH CDR3 comprises a non-conservative amino acid substitution at the position corresponding to amino acid 2, 4, 5, or 7 of SEQ ID NO: 24.

[0154] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 8, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 24, 79, 80, 81, or 82.

[0155] In some embodiments, the heavy chain variable domain includes VH CDR1, VH CDR2, and VH CDR3 as included in SEQ ID NO: 52. In some embodiments, VH CDR1 includes the amino acid sequence of SEQ ID NO: 8, VH CDR2 includes the amino acid sequence of SEQ ID NO: 16, and VH CDR3 includes the amino acid sequence of SEQ ID NO: 24.

[0156] In some embodiments, the heavy chain variable domain comprises VH CDR1, VH CDR2, and VH CDR3, as contained in the amino acid sequence of SEQ ID NO: 98. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 8, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 79.

[0157] In some embodiments, the heavy chain variable domain comprises VH CDR1, VH CDR2, and VH CDR3, as contained in the amino acid sequence of SEQ ID NO: 99. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 8, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 80.

[0158] In some embodiments, the heavy chain variable domain comprises VH CDR1, VH CDR2, and VH CDR3, as contained in the amino acid sequence of SEQ ID NO: 100. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 8, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 81.

[0159] In some embodiments, the heavy chain variable domain comprises VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 101. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 8, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 82.

[0160] In some embodiments, the heavy chain variable domain further comprises frame regions 1 (FWR1), FWR2, FWR3, and FWR4. The frame regions may be derived from immunoglobulins of any species. In some preferred embodiments, the frame regions are derived from human immunoglobulins.

[0161] In some embodiments, FWR1 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, FWR2 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, FWR3 comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, FWR4 comprises the amino acid sequence of SEQ ID NO: 27.

[0162] In some embodiments, FWR1 comprises the amino acid sequence of SEQ ID NO: 4, and / or FWR2 comprises the amino acid sequence of SEQ ID NO: 12, and / or FWR3 comprises the amino acid sequence of SEQ ID NO: 20, and / or FWR4 comprises the amino acid sequence of SEQ ID NO: 27.

[0163] In some embodiments, FWR1 comprises the amino acid sequence of SEQ ID NO: 4, FWR2 comprises the amino acid sequence of SEQ ID NO: 12, FWR3 comprises the amino acid sequence of SEQ ID NO: 20, and FWR4 comprises the amino acid sequence of SEQ ID NO: 27.

[0164] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequences of SEQ ID NO: 52, 98, 99, 100, or 101. In some such embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 8, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 24, 79, 80, 81, or 82.

[0165] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having one or more (e.g., 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids added, deleted, and / or substituted compared to the amino acid sequence of SEQ ID NO: 52, 98, 99, 100, or 101. In some preferred embodiments, the amino acid addition, deletion, and / or substitution does not occur in the CDR region.

[0166] In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 100. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 101.

[0167] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment disclosed herein further comprises an Fc region.

[0168] In some embodiments, the Fc region comprises one or more modifications to reduce effector function. In some other embodiments, the Fc region comprises one or more modifications to enhance effector function.In some embodiments, the effector function includes at least one selected from ADCC, CDC, and ADCP.

[0169] In some embodiments, the Fc region includes one or more modifications to enhance FcRn binding.

[0170] In some embodiments, the Fc region includes one or more modifications to reduce effector function and enhance FcRn binding. In some embodiments, the Fc region includes one or more modifications to enhance effector function and enhance FcRn binding.

[0171] In some embodiments, the Fc region is a monomeric Fc region. In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the Fc region is a heterodimeric Fc region.

[0172] In some embodiments, the Fc region is directly fused to the C-terminus of the heavy chain variable domain. In some embodiments, the Fc region is optionally fused to the N-terminus of the heavy chain variable domain via a peptide linker.

[0173] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment includes a heavy chain that includes a heavy chain variable domain as described herein fused to the monomeric Fc region.

[0174] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment comprises two heavy chains, each heavy chain comprising a heavy chain variable domain fused to an Fc region subunit as described herein. In some embodiments, the two heavy chains are identical. In some embodiments, the two heavy chains are different.

[0175] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 59. In some embodiments, the anti-TFR1 antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 107. In some embodiments, the anti-TFR1 antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 108. In some embodiments, the anti-TFR1 antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 109. In some embodiments, the anti-TFR1 antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 110.

[0176] In a particular aspect, the anti-TFR1 antibody or antigen-binding fragment is antibody PR012592 as described herein. In some embodiments, antibody PR012592 includes a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 52, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 24.In some embodiments, antibody PR012592 comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 52. In some embodiments, antibody PR012592 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 59. Specification 16 / 52 pages 22 CN 122535623 A

[0177] In a particular aspect, an anti-TFR1 antibody or antigen-binding fragment is antibody PR012838 as described herein. In some embodiments, antibody PR012838 comprises a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as included in the amino acid sequence of SEQ ID NO: 98, for example, VH CDR1 including the amino acid sequence of SEQ ID NO: 8, VH CDR2 including the amino acid sequence of SEQ ID NO: 16, and VH CDR3 including the amino acid sequence of SEQ ID NO: 79. In some embodiments, antibody PR012838 comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 98. In some embodiments, antibody PR012838 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 107.

[0178] In a particular aspect, the anti-TFR1 antibody or antigen-binding fragment is antibody PR012839 as described herein. In some embodiments, antibody PR012839 comprises a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as included in the amino acid sequence of SEQ ID NO: 99, for example, VH CDR1 including the amino acid sequence of SEQ ID NO: 8, VH CDR2 including the amino acid sequence of SEQ ID NO: 16, and VH CDR3 including the amino acid sequence of SEQ ID NO: 80. In some embodiments, antibody PR012839 comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 99. In some embodiments, antibody PR012839 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 108.

[0179] In a particular aspect, the anti-TFR1 antibody or antigen-binding fragment is antibody PR013071 as described herein.In some embodiments, antibody PR013071 comprises a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 100, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 81. In some embodiments, antibody PR013071 comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 100. In some embodiments, antibody PR013071 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 109.

[0180] In a particular aspect, the anti-TFR1 antibody or antigen-binding fragment is antibody PR013072 as described herein. In some embodiments, antibody PR013072 includes a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 101, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 82. In some embodiments, antibody PR013072 includes a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 101. In some embodiments, antibody PR013072 includes a heavy chain having the amino acid sequence of SEQ ID NO: 110.

[0181] In some aspects, affinity-modified variants of any antibody or antigen-binding fragment as described herein are provided. Compared to any antibody or antigen-binding fragment as described herein, “affinity-modified variants” may have increased or decreased antigen-binding affinity but still specifically bind to TFR1 (e.g., hTFR1 or cynoTFR1). In some specific embodiments, affinity-modified variants have decreased antigen-binding affinity compared to antibody PR012592.

[0182] To obtain affinity-modified variants, one or more amino acid modifications may be introduced into one or more CDR regions and / or frame regions of an antibody. Affinity-modified variants can be obtained using methods known in the art, such as mutagenesis by scanning alanine in the CDR region and / or frame region, and by measuring the affinity of the alanine-substituted variant for TFR1. Examples of such anti-TFR1 antibodies or antigen-binding fragments may include antibodies PR012838, PR012839, PR013071, and PR013072 or antigen-binding fragments thereof.

[0183] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment binds to human TFR1 and non-human primate (e.g., cynomolgus monkey) TFR1 at a KD of about 5 × 10⁻⁸ M or less. In some preferred embodiments, the anti-TFR1 antibody or antigen-binding fragment binds to human TFR1 at a KD of about 1 × 10⁻⁸ M or less, and to non-human primate (e.g., cynomolgus monkey) TFR1 at a KD of about 5 × 10⁻⁸ M or less.

[0184] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment binds to human TFR1 with a KD ranging from about 5×10⁻⁹ M to about 1×10⁻¹² M, from about 5×10⁻⁹ M to about 1×10⁻¹¹ M, from about 5×10⁻⁹ M to about 1×10⁻¹⁰ M, from about 1×10⁻⁹ M to about 1×10⁻¹² M, from about 1×10⁻⁹ M to about 1×10⁻¹¹ M, or from about 1×10⁻⁹ M to about 1×10⁻¹⁰ M. In some embodiments, the anti-TFR1 antibody or antigen-binding fragment binds to human TFR1 at a KD of about 9 × 10⁻¹⁰ M, about 8 × 10⁻¹⁰ M, about 7 × 10⁻¹⁰ M, about 6 × 10⁻¹⁰ M, 5 × 10⁻¹⁰ M, about 4 × 10⁻¹⁰ M, about 3 × 10⁻¹⁰ M, about 2 × 10⁻¹⁰ M, or about 1 × 10⁻¹⁰ M. In some embodiments, the KD value is measured by biolayer interferometry. TFR1 binder

[0185] In another aspect, a TFR1 binder is provided comprising the anti-TFR1 antibody or antigen-binding fragment of this disclosure conjugated to an additional molecule. In some embodiments, the TFR1 binder is capable of crossing the blood-brain barrier.

[0186] As used herein, “conjugating” or “conjugation” means that two or more parts are associated by covalent or non-covalent interactions, preferably by covalent interactions.

[0187] Other molecules may be proteins (such as antibodies), small compounds, or nucleic acids.

[0188] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment disclosed herein may be conjugated to a therapeutic agent for treating cancer or neurological diseases or disorders.

[0189] In some embodiments, the additional molecule is selected from the following anticancer drugs: maytansine-like substances (e.g., maytansine), auristatin (e.g., MMAF, MMAE, MMAD), dosstatin, cantharidin, vinblastine alkaloids (e.g., vincristine), colchicine, sea haretoxin, taxane, paclitaxel, docetaxel, carbazide, enediyne antibiotics, pinocembrin, camptothecin, anthracycline antibiotics (e.g., donomycin, dihydroxyanthridine, doxorubicin), cytotoxic antibiotics (e.g., mitomycin, actinomycin, pyromycin (e.g., CC-1065), chlortetracycline, polymycin, kazimidoxam, endorphin, etc.). Phenytocin, doxorubicin, erythromycin, chachomycin, cisplatin, ethidium bromide, bleomycin, mitomycin, photomycin, praldidine, podophyllotoxin, etoposide, mitoxantrone, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nerabine, carmustine, lomustine, methotrexate, melphalan, teniposide, and glucocorticoids.

[0190] In some embodiments, the additional molecule is a therapeutic agent for treating diseases or disorders selected from: cancer (e.g., central nervous system cancers, including brain tumors), Alzheimer's disease, Parkinson's disease, Huntington's disease, Hunt syndrome (also known as "type II mucopolysaccharidosis," which includes symptoms in the CNS), schizophrenia, antidepressants, multiple sclerosis, amyotrophic lateral sclerosis, lysosomal storage diseases with encephalopathy, glycogen storage diseases, muscular dystrophy, cerebral ischemia, prion diseases, traumatic central nervous system disorders (including traumatic brain injury and traumatic spinal cord injury), viral and bacterial central nervous system diseases. In some embodiments, the therapeutic agent is iduronate-2-sulfatase. In some embodiments, the therapeutic agent is an antibody for treating Alzheimer's disease.

[0191] In some embodiments, the anti-TFR1 antibody or antigen-binding fragment disclosed herein may be conjugated with an imaging agent, for example, for use in the diagnosis of cancer or neurological diseases or disorders.

[0192] An “imaging agent” is a compound having one or more properties that allow for the direct or indirect detection of its presence and / or location. An imaging agent can be any agent used for detection, including but not limited to radionuclides (e.g., 212Bi, 213Bi, 11C, 18F, 1131, 1125, 111In, 177Lu, 13N, 15O, 186Re, 188Re, 153Sm, and 90Y), biotin, fluorescent proteins, fluorophores (e.g., FITC, Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Cy3, Texas Red, Cy5, and Rhodamine), and enzymes.Specification 18 / 52 pages 24 CN 122535623 A

[0193] In some embodiments, the additional molecule is an imaging agent selected from radionuclides, biotin, fluorescent proteins, fluorophores, horseradish peroxidase, and alkaline phosphatase.

[0194] The additional molecule (e.g., a therapeutic agent or imaging agent) may be directly or indirectly (e.g., via a linker) linked to an anti-TFR1 antibody or antigen-binding fragment or multispecific antibody.

[0195] The linker may contain an active group for covalent coupling, such as an amine, hydroxylamine, maleimide, carboxyl, phenyl, thiol, or hydroxyl group.

[0196] In some embodiments, the linker is a peptide linker. The peptide linker may contain an amino acid sequence of any length. In particular, the peptide linker may contain an amino acid sequence of 1-50, preferably 1-30, for example, 1-10 amino acids. Exemplary peptide linkers may include, but are not limited to, polyglycine, polyalanine, polyserine, and combinations thereof. Other suitable peptide linkers may include hinge regions or their functional equivalents.

[0197] Other suitable linkers may be organic compounds or polymers suitable for therapeutic proteins, such as polyethylene glycol. Multispecific Antibodies

[0198] In another aspect, a multispecific antibody is provided comprising a first antigen-binding moiety that binds to TFR1, wherein the first antigen-binding moiety comprises a heavy chain variable domain that binds to TFR1 as described herein. In some embodiments, the first antigen-binding moiety comprises an anti-TFR1 antibody or antigen-binding fragment disclosed herein.

[0199] In some embodiments, the multispecific antibody further comprises a second antigen-binding moiety that binds to a second antigen. The second antigen may be an antigen different from TFR1. The second antigen may also be TFR1, and the second antigen-binding moiety binds to an epitope different from the epitope bound by the anti-TFR1 antibody or antigen-binding fragment disclosed herein. A variety of methods known in the art can be used to determine whether two antibodies bind to the same epitope, for example, by measuring whether two antibodies compete to bind to the same epitope by ELISA, FACS, or SPR.

[0200] In some preferred embodiments, the second antigen is a brain antigen. In some embodiments, the brain antigen is selected from: BACE1, amyloid precursor protein (APP), amyloid β (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein, leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), p75 neurotrophic factor receptor (p75NTR), interleukin-6 receptor (IL6R), TNF receptor 1 (TNFR1), and caspase 6.

[0201] As used herein, the term “antigen-binding moiety” refers to an amino acid sequence that contains an antigen-binding site and is capable of binding to an epitope. The definition of an antigen-binding moiety falls within the definition of an antibody or antigen-binding fragment. An antigen-binding moiety may contain one or more (e.g., 1, 2, 3, or 4) antigen-binding domains / sites. In some embodiments, an antigen-binding moiety contains one antigen-binding domain / site. In some other embodiments, an antigen-binding moiety contains two antigen-binding domains / sites.

[0202] A first antigen-binding moiety may be an antibody or antigen-binding fragment in any form, including but not limited to single variable domain and heavy chain antibodies. In some embodiments, a first antigen-binding moiety contains a heavy chain variable domain that binds to TFR1 as described herein. In some embodiments, a multispecific antibody contains only one heavy chain variable domain that binds to TFR1 as described herein.

[0203] A second antigen-binding moiety may be any antibody or antigen-binding fragment in any form. In some embodiments, a second antigen-binding moiety contains a heavy chain variable region and a light chain variable region that bind to a second antigen. In some embodiments, the second antigen-binding portion comprises dsFv, scFv, scdsFv, di-scFv, Fab, Fab', scFab, or F(ab')2. In some embodiments, the antigen-binding portion comprises Fab. In some embodiments, the antigen-binding portion comprises F(ab')2. In some other embodiments, the second antigen-binding portion comprises a single variable domain that binds to a second antigen (such as VHH).

[0204] In some embodiments, the first antigen-binding portion and the second antigen-binding portion may optionally be linked by a linker (e.g., a peptide linker).

[0205] In some embodiments, the multispecific antibody comprises (scFv)2-VHH, wherein the second antigen-binding portion comprises (scFv)2, and the first antigen-binding portion comprises VHH.

[0206] In some embodiments, the multispecific antibody further comprises an Fc region.

[0207] In some embodiments, the Fc region comprises one or more modifications to reduce effector function. In some other embodiments, the Fc region comprises one or more modifications to enhance effector function. In some embodiments, the effector function includes at least one selected from ADCC, CDC, and ADCP.

[0208] In some embodiments, the Fc region includes one or more modifications to enhance FcRn binding.

[0209] In some embodiments, the Fc region includes one or more modifications to reduce effector function and enhance FcRn binding. In some embodiments, the Fc region includes one or more modifications to enhance effector function and enhance FcRn binding.

[0210] In some embodiments, the Fc region is a monolithic Fc region.

[0211] In some embodiments, the Fc region is a heterodimeric Fc region comprising a first Fc region subunit and a second Fc region subunit, wherein one of the first Fc region subunit and the second Fc region subunit contains a club-shaped mutation and the other contains a mortar-shaped mutation. In some embodiments, the first Fc region subunit contains a club-shaped mutation and the second Fc region subunit contains a mortar-shaped mutation. In some other embodiments, the first Fc region subunit contains a mortar-shaped mutation and the second Fc region subunit contains a club-shaped mutation.

[0212] In some embodiments, the club-shaped mutation comprises one or more mutations selected from S354C and T366W. In some embodiments, the mortar-shaped mutation comprises one or more mutations selected from Y349C, T366S, L368A, and Y407V. In some embodiments, the club-shaped mutation comprises S354C and the mortar-shaped mutation comprises Y349C. In some embodiments, the club-shaped mutation comprises T366W and the mortar-shaped mutation comprises T366S, L368A, and Y407V. In some specific embodiments, the acetabular mutation includes S354C and T366W, and the acetabular mutation includes Y349C, T366S, L368A, and Y407V.

[0213] In some embodiments, the multispecific antibody comprises: a first polypeptide comprising a heavy chain variable region, a CH1 domain, and a first Fc region subunit; a second polypeptide comprising a light chain variable region and a light chain constant region; and a third polypeptide comprising a heavy chain variable domain and a second Fc region subunit that bind to TFR1 as described herein, wherein the heavy chain variable region and the light chain variable region bind to a second antigen, and the first Fc region subunit and the second Fc region subunit form a heterodimeric Fc region. In some preferred embodiments, the multispecific antibody may have the conformation shown in FIG12.

[0214] In some other embodiments, the multispecific antibody comprises: a first polypeptide comprising a heavy chain variable region, a CH1 domain, a first Fc subunit, an optional peptide linker, and a heavy chain variable domain that binds to TFR1 as described herein; a second polypeptide comprising a heavy chain variable region, a CH1 domain, and a second Fc subunit; and a third and a fourth polypeptide each comprising a light chain variable region and a light chain constant region, wherein the heavy chain variable region and the light chain variable region bind to a second antigen, and the first Fc subunit and the second Fc subunit form a heterodimeric Fc region. In some preferred embodiments, the multispecific antibody may have the configuration shown in FIG13.

[0215] In some embodiments, the brain antigen is BACE1. In a particular aspect, a multispecific antibody that binds to TFR1 and BACE1 is provided, wherein the multispecific antibody comprises the heavy chain variable domain of the anti-TFR1 antibody disclosed herein and an antigen-binding moiety that binds to BACE1.

[0216] In some embodiments, the antigen-binding portion that binds to BACE1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as contained in the amino acid sequence of SEQ ID NO: 102, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as contained in the amino acid sequence of SEQ ID NO: 104. In some preferred embodiments, as described on page 20 / 52 of the specification, CN 122535623 A, HCDR1 includes the amino acid sequence of SEQ ID NO: 67, HCDR2 includes the amino acid sequence of SEQ ID NO: 73, and HCDR3 includes the amino acid sequence of SEQ ID NO: 83; and LCDR1 includes the amino acid sequence of SEQ ID NO: 88, LCDR2 includes the amino acid sequence of SEQ ID NO: 91, and LCDR3 includes the amino acid sequence of SEQ ID NO: 95.

[0217] In some specific embodiments, the antigen-binding portion binding to BACE1 includes a heavy chain variable region having the amino acid sequence of SEQ ID NO: 102. In some specific embodiments, the antigen-binding portion binding to BACE1 includes a light chain variable region having the amino acid sequence of SEQ ID NO: 104. In some specific embodiments, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 102; and the light chain variable region includes the amino acid sequence of SEQ ID NO: 104.

[0218] In some embodiments, the multispecific antibody binding to TFR1 and BACE1 includes a first polypeptide having the amino acid sequence of SEQ ID NO: 122.

[0219] In some embodiments, the multispecific antibody binding to TFR1 and BACE1 includes a second polypeptide having the amino acid sequence of SEQ ID NO: 113.

[0220] In some embodiments, the multispecific antibody binding to TFR1 and BACE1 includes a third polypeptide having the amino acid sequence of SEQ ID NO: 129, 130, 131, 132, or 133.

[0221] In some embodiments, the multispecific antibody binding to TFR1 and BACE1 comprises a first polypeptide having the amino acid sequence SEQ ID NO: 122, a second polypeptide having the amino acid sequence SEQ ID NO: 113, and a third polypeptide having the amino acid sequence SEQ ID NO: 129, 130, 131, 132, or 133.

[0222] In a particular aspect, the multispecific antibody binding to TFR1 and BACE1 is antibody PR013356 as described herein.In some embodiments, antibody PR013356 comprises a first antigen-binding portion containing a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 52, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, antibody PR013356 comprises a first binding portion containing a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 52. In some embodiments, antibody PR013356 comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 122, a second polypeptide having the amino acid sequence of SEQ ID NO: 113, and a third polypeptide having the amino acid sequence of SEQ ID NO: 129.

[0223] In a particular aspect, the multispecific antibody binding to TFR1 and BACE1 is antibody PR013357 as described herein. In some embodiments, antibody PR013357 comprises a first antigen-binding moiety containing a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 98, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 79. In some embodiments, antibody PR013357 comprises a first antigen-binding moiety containing a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 98. In some embodiments, antibody PR013357 comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 122, a second polypeptide having the amino acid sequence of SEQ ID NO: 113, and a third polypeptide having the amino acid sequence of SEQ ID NO: 130.

[0224] In a particular aspect, the multispecific antibody that binds to TFR1 and BACE1 is antibody PR013358 as described herein.In some embodiments, antibody PR013358 comprises a first antigen-binding moiety containing a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 99, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 80. In some embodiments, antibody PR013358 comprises a first antigen-binding moiety containing a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 99. In some embodiments, antibody PR013358 comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 122, a second polypeptide having the amino acid sequence of SEQ ID NO: 113, and a third polypeptide having the amino acid sequence of SEQ ID NO: 131.

[0225] In a particular aspect, the multispecific antibody that binds to TFR1 and BACE1 is antibody PR013359 as described herein. In some embodiments, antibody PR013359 comprises a first antigen-binding moiety containing a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 100, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 81. In some embodiments, antibody PR013359 comprises a first antigen-binding moiety containing a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 100. In some embodiments, antibody PR013359 comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 122, a second polypeptide having the amino acid sequence of SEQ ID NO: 113, and a third polypeptide having the amino acid sequence of SEQ ID NO: 132.

[0226] In a particular aspect, the multispecific antibody that binds to TFR1 and BACE1 is antibody PR013360 as described herein.In some embodiments, antibody PR013360 comprises a first antigen-binding moiety containing a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 101, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 82. In some embodiments, antibody PR013360 comprises a first antigen-binding moiety containing a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 101. In some embodiments, antibody PR013360 comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 122, a second polypeptide having the amino acid sequence of SEQ ID NO: 113, and a third polypeptide having the amino acid sequence of SEQ ID NO: 133.

[0227] In some embodiments, the brain antigen is Aβ. In a particular aspect, a multispecific antibody that binds to TFR1 and Aβ is provided, wherein the multispecific antibody comprises a heavy chain variable domain of the anti-TFR1 antibody disclosed herein and an antigen-binding moiety that binds to Aβ.

[0228] In some embodiments, the antigen-binding moiety that binds to Aβ comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as contained in the amino acid sequence of SEQ ID NO: 103, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as contained in the amino acid sequence of SEQ ID NO: 105. In some preferred embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 68, HCDR2 comprises the amino acid sequence of SEQ ID NO: 74, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 84; and LCDR1 comprises the amino acid sequence of SEQ ID NO: 89, LCDR2 comprises the amino acid sequence of SEQ ID NO: 92, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 96.

[0229] In some specific embodiments, the antigen-binding portion that binds to Aβ includes a heavy chain variable region having the amino acid sequence of SEQ ID NO: 103. In some specific embodiments, the antigen-binding portion that binds to Aβ includes a light chain variable region having the amino acid sequence of SEQ ID NO: 105. In some specific embodiments, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 103; and the light chain variable region includes the amino acid sequence of SEQ ID NO: 105.

[0230] In some embodiments, the multispecific antibody binding to TFR1 and Aβ comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 119, 125, 126, 127, or 128. Specification 22 / 52 pages 28 CN 122535623 A

[0231] In some embodiments, the multispecific antibody binding to TFR1 and Aβ comprises a second polypeptide having an amino acid sequence of SEQ ID NO: 117.

[0232] In some embodiments, the multispecific antibody binding to TFR1 and Aβ comprises a third polypeptide having an amino acid sequence of SEQ ID NO: 115. In some embodiments, the multispecific antibody binding to TFR1 and Aβ comprises a third polypeptide and a fourth polypeptide, wherein each of the third polypeptide and the fourth polypeptide has an amino acid sequence of SEQ ID NO: 115.

[0233] In some embodiments, the multispecific antibody binding to TFR1 and Aβ comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 119, 125, 126, 127 or 128, a second polypeptide having an amino acid sequence of SEQ ID NO: 117, and a third and a fourth polypeptide each having an amino acid sequence of SEQ ID NO: 115.

[0234] In a particular aspect, the multispecific antibody binding to TFR1 and Aβ is antibody PR012932 as described herein. In some embodiments, antibody PR012932 comprises a first antigen-binding moiety containing a heavy chain variable domain having VH CDR1, VH CDR2 and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 52, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16 and VH CDR3 containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, antibody PR012932 comprises a first binding portion comprising a heavy chain variable domain having an amino acid sequence having SEQ ID NO: 52. In some embodiments, antibody PR012932 comprises a first polypeptide having an amino acid sequence having SEQ ID NO: 119, a second polypeptide having an amino acid sequence having SEQ ID NO: 117, and a third polypeptide and a fourth polypeptide each having an amino acid sequence having SEQ ID NO: 115.

[0235] In a particular aspect, the multispecific antibody binding to TFR1 and Aβ is antibody PR013352 as described herein.In some embodiments, antibody PR013352 comprises a first antigen-binding moiety containing a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 98, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 79. In some embodiments, antibody PR013352 comprises a first antigen-binding moiety containing a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 98. In some embodiments, antibody PR013352 comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 125, a second polypeptide having the amino acid sequence of SEQ ID NO: 117, and a third and fourth polypeptide each having the amino acid sequence of SEQ ID NO: 115.

[0236] In a particular aspect, the multispecific antibody that binds to TFR1 and Aβ is antibody PR013353 as described herein. In some embodiments, antibody PR013353 comprises a first antigen-binding moiety containing a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 99, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 80. In some embodiments, antibody PR013353 comprises a first antigen-binding moiety containing a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 99. In some embodiments, antibody PR013353 comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 126, a second polypeptide having the amino acid sequence of SEQ ID NO: 117, and a third and fourth polypeptide each having the amino acid sequence of SEQ ID NO: 115.

[0237] In a particular aspect, the multispecific antibody that binds to TFR1 and Aβ is antibody PR013354 as described herein.In some embodiments, antibody PR013354 comprises a first antigen-binding moiety containing a heavy chain variable domain having, for example, VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 100, such as VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 81. In some embodiments, antibody PR013354 comprises a first antigen-binding moiety containing a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 100. In some embodiments, antibody PR013354 comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 127, a second polypeptide having the amino acid sequence of SEQ ID NO: 117, and a third and fourth polypeptide each having the amino acid sequence of SEQ ID NO: 115.

[0238] In a particular aspect, the multispecific antibody that binds to TFR1 and Aβ is antibody PR013355 as described herein. In some embodiments, antibody PR013355 comprises a first antigen-binding moiety containing a heavy chain variable domain having VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequence of SEQ ID NO: 101, for example, VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 82. In some embodiments, antibody PR013355 comprises a first antigen-binding moiety containing a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 101. In some embodiments, antibody PR013355 comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 128, a second polypeptide having the amino acid sequence of SEQ ID NO: 117, and a third and fourth polypeptide each having the amino acid sequence of SEQ ID NO: 115. Pharmaceutical Composition

[0239] A pharmaceutical composition comprising the anti-TFR1 antibody or antigen-binding fragment disclosed herein, a TFR1 binder, or a multispecific antibody (e.g., an anti-TFR1 / BACE1 antibody or an anti-TFR1 / Aβ antibody).

[0240] The pharmaceutical compositions provided herein may be in various dosage forms, such as solid, semi-solid, liquid, powder, aqueous, or lyophilized forms.Depending on the dosage form, the pharmaceutical composition may further contain pharmaceutically acceptable excipients (see, in general, Alfonso R. Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, Baltimore, MD: Lippincott Williams & Wilkins).

[0241] Excipients that can be used in the preparation of pharmaceutical compositions in solid dosage forms include, but are not limited to: diluents (e.g., lactose, microcrystalline cellulose, and dextrose), binders and adhesives (e.g., gum arabic, gelatin, starch paste, and carboxymethyl cellulose), lubricants (e.g., polyethylene glycol, calcium stearate, and stearic acid), disintegrants (e.g., starch, cellulose, and crosslinking polymers), preservatives, mediators, flow aids (e.g., corn starch), sweeteners (e.g., mannitol and saccharin), coating materials (e.g., povidone, ethyl cellulose, and synthetic polymers), and plasticizers (e.g., castor oil, diacetylated monoglycerides, and polyethylene glycol). Excipients that can be used in the preparation of pharmaceutical compositions in a semi-solid dosage form include, but are not limited to: structure-forming excipients (e.g., cetearyl alcohol and mineral oil), preservatives (e.g., benzyl alcohol, propylparaben, and sodium benzoate), antioxidants (e.g., butylated hydroxytoluene and butylated hydroxyanisole), solubilizers (e.g., lanolin and cholesterol), gelling agents (e.g., carboxymethyl cellulose, hydroxypropyl cellulose, and xanthan gum), and emollients (e.g., glycerin, mineral oil, petrolatum, and isopropyl palmitate). Excipients that may be used in the preparation of pharmaceutical compositions in liquid dosage forms include, but are not limited to: solvents (e.g., water, alcohol, acetic acid, and syrup), buffers (e.g., phosphate buffer and acetate buffer), antimicrobial preservatives (e.g., benzyl alcohol, butylparaben, phenol, and thimerosal), antioxidants (e.g., ascorbic acid, sodium bisulfate, thiourea, and butylated hydroxytoluene), chelating agents (e.g., disodium EDTA, dihydroxyethylglycine, and citric acid), and emulsifiers (e.g., sodium lauryl sulfate, trimethylammonium bromide, and polyethylene glycol ester).

[0242] The pharmaceutical compositions provided herein may be administered to a subject by any method known in the art (e.g., by systemic or local administration). Routes of administration include, but are not limited to, parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intracavitary), local, epidural, or mucosal (e.g., intranasal or oral). The exact dose to be administered will depend on a variety of factors, such as the treatment objective, route of administration, and the condition of the subject, such as the patient’s health, weight, sex, diet, etc. (Instructions for Use, page 24 / 52, 30 CN 122535623 A)Therefore, therapists need to gradually adjust the dosage of the pharmaceutical composition and modify the route of administration as needed to achieve the best therapeutic effect. Generally, the dosage range used to administer the pharmaceutical compositions provided herein is those ranges that are large enough to produce the desired effect.

[0243] In a preferred embodiment, the pharmaceutical compositions disclosed herein may be prepared from the anti-TFR1 antibody or antigen-binding fragment, TFR1 binder or multispecific antibody disclosed herein and administered to a human subject in a therapeutically effective amount. The therapeutic dose of the antibody may, for example, preferably be between 0.1-25 mg / kg body weight / single treatment dose, and most preferably between 0.1-10 mg / kg body weight / single treatment dose. In a particular embodiment, the anti-TFR1 antibody or antigen-binding fragment, TFR1 binder or multispecific antibody disclosed herein may be formulated according to conventional practice for administration via any suitable route and may generally be in liquid form (e.g., a solution of the antibody in a sterile, physiologically acceptable buffer) for administration via, for example, intravenous, intraperitoneal, subcutaneous or intramuscular routes. Uses and Methods

[0244] In one aspect, use of the anti-TFR1 antibody or antigen-binding fragment of this disclosure in the manufacture of a pharmaceutical agent for transporting a compound across the blood-brain barrier (BBB) ​​is provided. In some embodiments, the pharmaceutical agent (e.g., the TFR1 binder or multispecific antibody of this disclosure) may be used for therapeutic or diagnostic purposes.

[0245] A method for transporting a compound across the blood-brain barrier in a subject is also provided, the method comprising conjugating the anti-TFR1 antibody or antigen-binding fragment of this disclosure to a compound and transporting the conjugate across the blood-brain barrier.

[0246] Further, the anti-TFR1 antibody or antigen-binding fragment of this disclosure is provided as a pharmaceutical agent, wherein the anti-TFR1 antibody or antigen-binding fragment transports a compound across the blood-brain barrier.

[0247] The compound to be transported across the BBB may be a therapeutic agent or imaging agent as described herein. The compound to be transported across the BBB may be conjugated to the anti-TFR1 antibody or antigen-binding fragment of this disclosure using methods well known in the art (e.g., chemical conjugation, enzyme conjugation, and recombinant DNA techniques).

[0248] The anti-TFR1 antibody or antigen-binding fragment, TFR1 binder, multispecific antibody (e.g., anti-TFR1 / BACE1 antibody or anti-TFR1 / Aβ antibody), nucleic acid or carrier, or pharmaceutical composition disclosed herein can be used for therapy or diagnosis, such as for treating cancer in a subject of need or for treating or diagnosing a neurological disease or disorder in a subject of need.

[0249] In a particular aspect, use of the anti-TFR1 antibody or antigen-binding fragment in the manufacture of a medicament for treating cancer is provided.

[0250] A method of treating cancer in a subject of need is also provided, the method comprising administering to the subject a therapeutically effective amount of the anti-TFR1 antibody or antigen-binding fragment disclosed herein.

[0251] Further, the anti-TFR1 antibody or antigen-binding fragment disclosed herein is provided for use in the treatment of cancer.

[0252] TFR1 expression has been reported to be elevated in many different types of cancer cells, including but not limited to solid cancers (such as esophageal squamous cell carcinoma, breast cancer, ovarian cancer, lung cancer, cervical cancer, bladder cancer, osteosarcoma, pancreatic cancer, bile duct cancer, renal cell carcinoma, hepatocellular carcinoma, adrenocortical carcinoma, and nervous system cancers) and hematopoietic malignancies (such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL)). Furthermore, TFR1's extracellular accessibility, internalization capacity, and central role in cancer cell pathology make it an attractive target for antibody-mediated therapies.

[0253] The anti-TFR1 antibody or antigen-binding fragment disclosed herein can be used in cancer therapy, for example, by: (1) indirectly by conjugation to an anticancer drug (e.g., a TFR1 binder comprising an anticancer drug as described herein) and / or (2) directly by fusion to the Fc region to induce Fc effector functions (e.g., ADCC, ADCP, and / or CDC). In some embodiments, the cancer is a cancer of the central nervous system.

[0254] In some embodiments, the cancer comprises cancer cells that overexpress TFR1. In some embodiments, the cancer is selected from esophageal squamous cell carcinoma, breast cancer, ovarian cancer, lung cancer, cervical cancer, bladder cancer, osteosarcoma, pancreatic cancer, bile duct cancer, renal cell carcinoma, hepatocellular carcinoma, adrenocortical carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia (e.g., acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia, chronic lymphocytic leukemia, and chronic lymphoid leukemia), and multiple myeloma.

[0255] In a particular aspect, use of an anti-TFR1 antibody or antigen-binding fragment in the manufacture of a medicament for treating a neurological disease or disorder is provided, wherein the anti-TFR1 antibody or antigen-binding fragment is conjugated to a therapeutic agent for treating a neurological disease or disorder. In some embodiments, the therapeutic agent comprises a second antigen-binding portion that binds to BACE1 or Aβ as described herein.

[0256] A method for treating a neurological disease or disorder in a subject of need is also provided, the method comprising administering to the subject a therapeutically effective amount of an anti-TFR1 antibody or antigen-binding fragment of the present disclosure conjugated to a therapeutic agent for treating the neurological disease or disorder.

[0257] Further provided is an anti-TFR1 antibody or antigen-binding fragment of the present disclosure as a medicament for treating a neurological disease or disorder, wherein the anti-TFR1 antibody or antigen-binding fragment is conjugated to a therapeutic agent for treating a neurological disease or disorder.

[0258] The multispecific antibodies disclosed herein (e.g., anti-TFR1 / BACE1 antibodies or anti-TFR1 / Aβ antibodies) can be used to treat diseases or disorders associated with a second antigen and / or to detect a second antigen in isolated biological samples or in situ. In some embodiments, the multispecific antibody can be conjugated with an imaging agent as described herein. For example, the multispecific antibody can be conjugated with a radionuclide (e.g., positron emission tomography (PET) radiotracers such as 11C, 18F, 13N, and 15O) to form a radioimmunoconjugate for in situ detection of a second antigen, for example for use in diagnosing diseases or disorders associated with a second antigen.

[0259] In a particular aspect, the use of the multispecific antibodies disclosed herein (e.g., anti-TFR1 / BACE1 antibodies or anti-TFR1 / Aβ antibodies) in the manufacture of medicaments for treating neurological diseases or disorders is provided.

[0260] A method of treating a neurological disease or disorder in a subject of need is also provided, the method comprising administering a therapeutically effective amount of the multispecific antibody disclosed herein (e.g., an anti-TFR1 / BACE1 antibody or an anti-TFR1 / Aβ antibody).

[0261] Further, a multispecific antibody disclosed herein (e.g., an anti-TFR1 / BACE1 antibody or an anti-TFR1 / Aβ antibody) for use in the treatment of a neurological disease or disorder is provided.

[0262] In some embodiments, the neurological disease or disorder is selected from Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, and glaucoma. In some embodiments, the neurological disease or disorder is Alzheimer's disease.

[0263] In yet another specific aspect, the use of the anti-TFR1 / BACE1 antibody or anti-TFR1 / Aβ antibody disclosed herein is provided in the manufacture of a pharmaceutical agent or kit for diagnosing Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, or glaucoma. In some preferred embodiments, the agent or kit is used for in situ use.

[0264] Further, multispecific antibodies (e.g., anti-TFR1 / BACE1 antibodies or anti-TFR1 / Aβ antibodies) disclosed herein are provided for use in the diagnosis of Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, or glaucoma.

[0265] Amyloid plaques are a common feature of many neurodegenerative diseases, including Alzheimer's disease, and play a central role therein. Multispecific antibodies (e.g., anti-TFR1 / BACE1 antibodies or anti-TFR1 / Aβ antibodies) disclosed herein can be used to inhibit the formation of amyloid plaques in the brain and / or remove existing amyloid plaques in the brain.

[0266] Therefore, in yet another specific aspect, the use of the multispecific antibody disclosed herein (e.g., anti-TFR1 / BACE1 antibody or anti-TFR1 / Aβ antibody) in the manufacture of a medicament for inhibiting and / or reducing the formation of amyloid plaques in the brain is provided.

[0267] A method for inhibiting and / or reducing the formation of amyloid plaques in the brain of a subject in need is also provided, the method comprising administering to the subject a therapeutically effective amount of the multispecific antibody disclosed herein (e.g., anti-TFR1 / BACE1 antibody or anti-TFR1 / Aβ antibody) to inhibit and / or reduce the formation of amyloid plaques in the brain.

[0268] Further, a multispecific antibody (e.g., anti-TFR1 / BACE1 antibody or anti-TFR1 / Aβ antibody) is provided for use in inhibiting and / or reducing the formation of amyloid plaques in the brain. Nucleic Acids, Vectors, and Host Cells

[0269] In another aspect, a nucleic acid encoding the anti-TFR1 antibody or antigen-binding fragment or multispecific antibody disclosed herein is provided. In some embodiments, the nucleic acid disclosed herein is an isolated nucleic acid.

[0270] In another aspect, vectors comprising the nucleic acids disclosed herein are provided. In some embodiments, the vector is an expression vector, such as a plasmid for expression in bacterial, yeast, or mammalian cells, or a phage vector and phage particle vector for phage display. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes.

[0271] The nucleic acids disclosed herein may be present in one or more vectors.

[0272] The nucleic acids disclosed herein can be obtained using a variety of methods known in the art. For example, the nucleic acids disclosed herein can be isolated from or chemically synthesized from phage display libraries, yeast display libraries, immunized animals (e.g., transgenic mice or camelids), immortalized cells (e.g., mouse B-cell hybridomas, EBV immortalized B cells). The nucleic acids disclosed herein can be codon-optimized for optimal expression in host cells.

[0273] In some embodiments, the nucleic acids disclosed herein are prepared as recombinant nucleic acids comprising additional nucleic acid sequences, such as regulatory elements and nucleic acid sequences encoding desired peptides or proteins. Recombinant nucleic acids can be prepared using molecular cloning techniques well known in the art, such as chemical synthesis, site-directed mutagenesis, and polymerase chain reaction (PCR) techniques (see Sambrook, J., EF Fritsch and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory, New York). Suitable regulatory elements for use in this disclosure may include, but are not limited to, enhancers, insulators, and internal ribosome entry sites (IRES).Examples of peptides or proteins that can facilitate the detection or isolation of expressed antibodies or antigen-binding fragments include, but are not limited to, affinity tags (e.g., biotin tags, polyhistidine tags (His6), or glutathione S-transferase (GSH) tags), enzyme-cleavable peptides, and reporter proteins (e.g., fluorescent proteins). In some embodiments, the nucleic acid disclosed herein and the regulatory element and / or nucleic acid sequence encoding the desired peptide or protein may be operatively linked to a promoter. Depending on the type of host cell to be used and the purification strategy, those skilled in the art can select appropriate expression vectors, promoters, regulatory elements, and peptides or proteins.

[0274] In another aspect, host cells comprising the nucleic acids or vectors disclosed herein are provided.

[0275] The host cells described herein can be used to generate the anti-TFR1 antibody or antigen-binding fragment or multispecific antibody disclosed herein. Examples of host cells used to generate recombinant antibodies may include, but are not limited to: mammalian cells, such as HEK293 cells, Chinese hamster ovary (CHO) cells, COS cells, myeloma cells, young hamster kidney (BHK) cells, HeLa and Vero cells; insect cells, such as sf9, sf21 and Tn5; plant cells, such as plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); yeast cells, such as those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae); and bacterial cells, such as Escherichia coli or Bacillus subtilis.

[0276] Nucleic acids or vectors may be introduced into suitable host cells using methods well known in the art. Such methods may include, but are not limited to, liposome transfection, electroporation, viral transduction and calcium phosphate transfection.

[0277] In some embodiments, a single vector containing nucleic acids encoding both the heavy and light chains may be used. In a particular embodiment, two vectors are used, one encoding the light chain of the antibody and the other encoding the heavy chain of the antibody. In some embodiments, one or more expression vectors may further contain one or more selective marker genes, such as neomycin or puromycin resistance genes. In some embodiments, molecular chaperone plasmids may be introduced into the same host cell, such as in bacteria, along with the expression vectors described above, to assist in the dissolution and / or folding of the antibody. The isolation and purification of the antibody may be performed using techniques well known in the art, such as using a protein A affinity column.

[0278] A method for generating the anti-TFR1 antibody or antigen-binding fragment or multispecific antibody of the present disclosure is also provided, the method comprising: culturing the host cells of the present disclosure under suitable conditions allowing expression of the anti-TFR1 antibody or antigen-binding fragment or multispecific antibody, and optionally isolating the anti-TFR1 antibody or antigen-binding fragment or multispecific antibody from the host cells or culture medium. Kit

[0279] Kits are also provided comprising the anti-TFR1 antibody or antigen-binding fragment of the present disclosure, a TFR1 binder, a multispecific antibody, a nucleic acid or vector, or a pharmaceutical composition, and optionally instructions for use thereof. The kit may be used for therapeutic or diagnostic purposes. In some embodiments, the kit is used in an isolated biological sample or in a method for in situ detection of a second antigen as described herein.

[0280] The kit may further comprise a suitable container. In some embodiments, the kit further comprises a means for administration. Typically, the kit may further comprise a label indicating the intended use and / or method of use of the contents of the container. The term “label” includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

[0281] This disclosure provides the following exemplary embodiments:

[0282] Example 1. An antibody or antigen-binding protein comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16, and 24, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0283] Example 2. An antibody or antigen-binding protein comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16, and 79, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0284] Example 3. An antibody or antigen-binding protein comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16 and 80, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0285] Example 4. An antibody or antigen-binding protein comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16, and 81, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0286] Example 5. An antibody or antigen-binding protein comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16, and 82, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0287] Example 6. An antibody or antigen-binding protein comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 52.

[0288] Example 7. An antibody or antigen-binding protein comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 98.

[0289] Example 8. An antibody or antigen-binding protein comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 99.

[0290] Example 9. An antibody or antigen-binding protein comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 100.

[0291] Example 10. An antibody or antigen-binding protein comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 101.

[0292] Example 11. An antibody or antigen-binding protein comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 59.

[0293] Example 12. An antibody or antigen-binding protein comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 107.

[0294] Example 13. An antibody or antigen-binding protein comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 108.

[0295] Example 14. An antibody or antigen-binding protein comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 109.

[0296] Example 15. An antibody or antigen-binding protein comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 110.

[0297] Example 16. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16 and 24, respectively; wherein the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 88, 91 and 95, respectively, the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 67, 73 and 83, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0298] Example 17. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16, and 79, respectively; and the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 88, 91, and 95, respectively, as per the specification page 29 / 52, CN 122535623 A; and the VH region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 88, 91, and 95, respectively. The amino acid sequences shown are 67, 73, and 83; and the CDR region is defined in the Chothia numbering system.

[0299] Example 18. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16 and 80, respectively; wherein the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 88, 91 and 95, respectively, the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 67, 73 and 83, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0300] Example 19. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16 and 81, respectively; wherein the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 88, 91 and 95, respectively, the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 67, 73 and 83, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0301] Example 20. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16 and 82, respectively; wherein the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 88, 91 and 95, respectively, the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 67, 73 and 83, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0302] Example 21. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16 and 24, respectively; wherein the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 89, 92 and 96, respectively, the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 68, 74 and 84, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0303] Example 22. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16 and 79, respectively; wherein the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 89, 92 and 96, respectively, the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 68, 74 and 84, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0304] Example 23. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, the first antigen-binding fragment comprising a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16, and 80, respectively; the second antigen-binding fragment comprising a light chain variable region (VL) and a heavy chain variable region (VH), the VL region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 89, 92, and 96, respectively, the VH region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 89, 92, and 96, respectively, the VH region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 89, 92, and 96, respectively. The amino acid sequences shown are 68, 74, and 84; and the CDR region is defined in the Chothia numbering system.

[0305] Example 24. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16 and 81, respectively; wherein the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 89, 92 and 96, respectively, the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 68, 74 and 84, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0306] Example 25. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 16 and 82, respectively; wherein the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 89, 92 and 96, respectively, the VH region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 68, 74 and 84, respectively; and wherein the CDR region is defined in the Chothia numbering system.

[0307] Example 26. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 52; and the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL region contains the amino acid sequence shown in SEQ ID NO: 104 and the VH region contains the amino acid sequence shown in SEQ ID NO: 102.

[0308] Example 27. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 98; and the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL region contains the amino acid sequence shown in SEQ ID NO: 104, and the VH region contains the amino acid sequence shown in SEQ ID NO: 102.

[0309] Example 28. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 99; and the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL region contains the amino acid sequence shown in SEQ ID NO: 104, and the VH region contains the amino acid sequence shown in SEQ ID NO: 102.

[0310] Example 29. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 100; and the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence shown in SEQ ID NO: 104, and the VH region comprises the amino acid sequence shown in SEQ ID NO: 102. Specification 31 / 52 pages 37 CN 122535623 A

[0311] Example 30. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 101; and the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence shown in SEQ ID NO: 104, and the VH region comprises the amino acid sequence shown in SEQ ID NO: 102.

[0312] Example 31. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 52; and the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL region contains the amino acid sequence shown in SEQ ID NO: 105, and the VH region contains the amino acid sequence shown in SEQ ID NO: 103.

[0313] Example 32. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 98; and the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL region contains the amino acid sequence shown in SEQ ID NO: 105, and the VH region contains the amino acid sequence shown in SEQ ID NO: 103.

[0314] Example 33. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 99; and the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL region contains the amino acid sequence shown in SEQ ID NO: 105, and the VH region contains the amino acid sequence shown in SEQ ID NO: 103.

[0315] Example 34. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 100; and the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL region contains the amino acid sequence shown in SEQ ID NO: 105, and the VH region contains the amino acid sequence shown in SEQ ID NO: 103.

[0316] Example 35. A bispecific antibody or antigen-binding protein comprising two different antigen-binding fragments, wherein the first antigen-binding fragment comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 101; and the second antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence shown in SEQ ID NO: 105 and the VH region comprises the amino acid sequence shown in SEQ ID NO: 103.

[0317] Example 36. A bispecific antibody or antigen-binding protein comprising three polypeptides, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 122, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 113, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 129.

[0318] Example 37. A bispecific antibody or antigen-binding protein comprising three polypeptides, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 122, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 113, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 130.

[0319] Example 38. A bispecific antibody or antigen-binding protein, the bispecific antibody or antigen-binding protein specification page 32 / 52 38 CN 122535623 A comprising three polypeptides, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 122, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 113, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 131.

[0320] Example 39. A bispecific antibody or antigen-binding protein, the bispecific antibody or antigen-binding protein comprising three polypeptides, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 122, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 113, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 132.

[0321] Example 40. A bispecific antibody or antigen-binding protein comprising three polypeptides, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 122, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 113, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 133.

[0322] Example 41. A bispecific antibody or antigen-binding protein comprising three polypeptides, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 119, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 117, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 115.

[0323] Example 42. A bispecific antibody or antigen-binding protein comprising three polypeptides, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 125, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 117, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 115.

[0324] Example 43. A bispecific antibody or antigen-binding protein comprising three polypeptides, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 126, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 117, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 115.

[0325] Example 44. A bispecific antibody or antigen-binding protein comprising three polypeptides, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 127, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 117, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 115.

[0326] Example 45. A bispecific antibody or antigen-binding protein comprising three polypeptides, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 128, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 117, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 115.

[0327] Although this disclosure has been described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the application and principles of this disclosure. Modifications may be made without departing from the spirit and scope of this disclosure.Beneficial Effects

[0328] The beneficial effects of the anti-TFR1 antibody or antigen-binding fragment, TFR1 binder or multispecific antibody disclosed herein may include at least one of the following: (1) the anti-TFR1 antibody or antigen-binding fragment, TFR1 binder or multispecific antibody disclosed herein binds to human TFR1 and non-human primate TFR1; (2) the anti-TFR1 antibody or antigen-binding fragment, TFR1 binder or multispecific antibody disclosed herein substantially does not affect the binding of transferrin to TFR1; (3) the anti-TFR1 antibody or antigen-binding fragment, TFR1 binder or multispecific antibody disclosed herein binds to the transferrin-TFR1 complex; and (4) the anti-TFR1 antibody or antigen-binding fragment, TFR1 binder or multispecific antibody disclosed herein exhibits TFR1 binding characteristics (e.g., binding affinity to hTFR1 and dissociation from hTFR1), which allow efficient cross-BBB and high relative uptake in the brain.

[0329] Furthermore, the binding of the anti-TFR1 antibody or antigen-binding fragment disclosed herein to TFR1 requires only a single variable domain having a low molecular weight that is advantageous for production, synthesis, and crossing the BBB.

[0330] The anti-TFR1 antibody or antigen-binding fragment disclosed herein may be a fully human antibody. Therapeutic antibodies generated in non-human systems can be immunogenic and elicit undesirable and sometimes fatal immune responses; for example, mouse antibodies typically induce a human anti-mouse antibody response (HAMA). In addition, immunogenicity can also alter the pharmacokinetics of therapeutic antibodies by affecting clearance rates, thereby affecting in vivo efficacy. Examples

[0331] The present disclosure is further described below with reference to specific examples. It should be understood that these examples are not intended to limit the scope of the present disclosure. Specific experimental methods not mentioned in the following examples were performed according to conventional experimental methods. Unless otherwise stated, the instruments described are commercially available, and the reagents and materials are commercially available or prepared according to methods known in the art. Example 1: Preparation of Immunogen

[0332] Human TFR1 mRNA-LNP was used as an immunogen. Human TFR1 mRNA was generated in vitro from a linearized DNA template encoding hTFR1 (uniprot: P02786) using T7 RNA polymerase-mediated transcription. The template incorporated 5' and 3' untranslated regions and a multi-A tail. The hTFR1 mRNA was then encapsulated in lipid nanoparticles. The lipid nanoparticle (LNP) formulation was prepared by mixing ethanol and aqueous phases at a 1:3 volume ratio in a microfluidic device.The ethanol phase was prepared by dissolving a mixture of ionizable lipids, 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and PEG-lipids in a molar ratio of 50:10:38.5:1.5. The aqueous phase was prepared in citrate buffer containing hTFR1 mRNA. The LNP was dialyzed against PBS and concentrated to the desired concentration. Example 2: Animal Immunization Protocol

[0333] To obtain TFR1-specific antibodies (referred to as anti-TFR1 antibodies), Harbour HCAb transgenic mice were immunized with human TFR1 mRNA-LNP as an immunogen (https: / / harbourantibodies.com / ; see, for example, WO 2007 / 096779 for a detailed description). The immunization protocol is listed in Table 1 below. In short, 80 µg of immunogen was administered to each mouse via subcutaneous injection (sc) for primary immunization, and 50 µg of immunogen was administered for subsequent booster immunization. Immunization was performed every three weeks using an immunogen diluted in PBS, for a total of five times. Serum titers against human TFR1-His protein (Acro, catalog number CD1-H5243) were tested using ELISA.

[0334] Table 1. Immunization protocol example 3: TFR1 HCAb antibody discovery

[0335] In this example, spleens from mice with high antibody titers were harvested to prepare cDNA. The variable region of HCAb cDNA was amplified by PCR using specific primers (Instructions for Use, pages 34 / 52, 40 CN 122535623 A 5'-GGTGTCCAGTGTSAGGTRCAGCTG-3', SEQ ID NO: 134; 5'-AATCCCTGGGCACTGARGAGACGGTGACCRKKGT-3', SEQ ID NO: 135) and cloned into a mammalian expression vector (pCAG) containing the Fc region of the human immunoglobulin heavy chain of the IgG1 subclass to generate an HCAb cDNA library (named "pCAG-HCAb library"). The plasmid of the pCAG-HCAb library was prepared and transfected into HEK293 cells (ATCC, catalog number CRL-1573) in 96-well plates for expression. The supernatant of HEK293-pCAG-HCAb was then harvested and transferred to different 96-well plates for screening by in vitro binding assays. The binding of the protein to the recombinant human TFR1 His tag (Bepsys, catalog number CD1-H5243) was measured by ELISA.Binding to the stable cell line HEK293T-huTFR1 expressing human TFR1 and the stable cell line HEK293T-cynoTFR1 expressing cynomolgus monkey TFR1 (UniProtKB / TrEMBL: G8F602) was tested by FACS. HEK293 cell supernatants exhibiting binding to the recombinant human TFR1 His-tag protein, HEK293T-huTFR1, and HEK293T-cynoTFR1 were sequenced. Finally, multiple HCAb clones were selected for further characterization. Example 4: Antibody Production and Purification

[0336] The recombinant plasmid encoding the target antibody was transiently transfected into HEK293-6E cells (National Research Council) using a PEI (Polyscience, catalog number 24885). After transfection, the cells were incubated at 37°C and 5% CO2 with shaking at 120 rpm. Six to seven days after transfection, the cell culture supernatant containing the target antibody was harvested by centrifugation and filtration. The monoclonal antibody was purified using protein A magnetic beads (AmMag protein A magnetic beads, Genscript, catalog number L00695).

[0337] The antibody purity was tested by SEC-HPLC (Agilent 1260 Infinity II HPLC, Welch Xtimate SEC-300 column, 1 × PBS pH 7.4 as mobile phase) and SDS-PAGE (SurePAGE, Bis-Tris, 10×8, 4%-12%, 12 wells, Genscript, catalog number M00653). The successfully expressed and purified HCAb antibody PR012592 was used for further characterization. The amino acid sequence of PR012592 is shown in Table 2 (CDR as defined by Chothia).

[0338] Table 2. Amino acid sequence of anti-TFR1 antibody (PR012592) Specification 35 / 52 pages 41 CN 122535623 A

[0339] Meanwhile, anti-TFR1 antibodies PR011073, PR011074 and PR012560 were generated according to the procedure shown above for reference, wherein the sequence information of US 10323089 B2 (clone 299; named PR011073 in this disclosure), US 20210145978 A1 (clone 21.12; named PR011074 in this disclosure) and US 20220064288 A1 (JR141; named PR012560 in this disclosure) are listed in Table 3.Specification 36 / 52 pages 42 CN 122535623 A

[0340] Table 3. SEQ ID NO of the amino acid sequence of the anti-TFR1 reference antibody used in this application Example 5: Binding affinity of anti-TFR1 antibody to TFR1 5.1 Binding affinity with human and cyno TFR1-His protein

[0341] The binding affinity of recombinant anti-TFR1 antibody to human TFR1-His protein (Bepsys, catalog number CD1-H5243) and cyno TFR1-His protein (Bepsys, catalog number TFR-C524a) was tested by ELISA. In short, 1 μg / mL antigen was added to 96-well plates at 100 μL / well. After incubating overnight at 4°C, the plate was washed three times with 1×PBST (300 μL / well) and then blocked for 1 hour at 37°C with 5% skim milk in 1×PBST (300 μL / well). Anti-TFR1 antibody was serially diluted in PBS and added to the plate. After incubating for 1 hour at 37°C, the plate was washed three times with 1×PBST (300 μL / well) and 100 μL / well of secondary antibody (goat anti-human IgG-Fc fragment HRP conjugated, Bethyl Laboratories, A80-304P) was added. After incubating for 1 hour at 37°C, the plate was washed three times with 1×PBST (300 μL / well) and 100 μL / well of TMB was added, and the plate was held for approximately 5 minutes. Finally, 100 μL / well 2 M H2SO4 was added to the plate to terminate the reaction, and the readings were taken at 450 nm using a Molecular device spectra max plus 384.

[0342] The results are shown in Figures 1, 2 and Table 4. The results indicate that anti-TFR1 HCAb PR012592 exhibits a strong binding affinity to human and cyno TFR1-His protein. These results indicate that anti-TFR1 HCAb PR012592 binds to human and cyno TFR1 with high affinity.

[0343] Table 4. Binding affinity of anti-TFR1 antibody to human and cyno TFR1-His protein as measured by ELISA 5.2 Binding affinity to cells expressing TFR1

[0344] The binding of anti-TFR1 antibody to human (HEK293T-huTFR1) or cynomolgus monkey (HEK293T-cynoTFR1) TFR1-overexpressing cells was tested by flow cytometry.

[0345] In short, the anti-TFR1 antibody was serially diluted in PBS. The antibody solution was incubated with 1×10⁵ cells at 4°C for 1 hour.Cells were washed twice with PBS, and 100 µL of a 1:1000 diluted fluorescently labeled anti-human IgG antibody (Alexa Fluor 647 AffiniPure goat anti-human IgG Fc, Jackson ImmunoResearch, catalog 109-606-008) was added to each well. After incubation at 4°C for 1 hour, cells were washed twice with staining buffer and subjected to flow cytometry. Fluorescence signal is expressed as median fluorescence intensity (MFI).

[0346] The results are shown in Figures 3, 4 and Table 5. These results show that anti-TFR1 HCAb PR012592 has a strong binding affinity for HEK293T-A huTFR1 cells and HEK293T-cynoTFR1 cells, indicating that anti-TFR1 HCAb PR012592 has cross-reactivity with cynoTFR1.

[0347] Table 5. Binding affinity of anti-TFR1 antibody to HEK293T-huTFR1 and HEK293T-cynoTFR1 cells as measured by FACS 5.3 Binding affinity to CHOK1-hCD40 cells

[0348] The binding affinity of anti-TFR1 antibody to CHOK1-hCD40 cells was tested by flow cytometry using a similar procedure as described in Example 5.2. In this example, the cell line expressing hCD40 was the CHOK1 cell line that had been transfected to express human CD40 (hCD40) on its surface.

[0349] The results are shown in Figure 5. These results show that anti-TFR1 HCAb PR012592 has no binding affinity to CHOK1-hCD40 cells, indicating that this HCAb antibody does not have cross-reactivity with human CD40. Example 6: Epitope Competitive Assay

[0350] To determine whether the anti-TFR1 antibody binds to different or similar binding epitopes on human TFR1, flow cytometry was used to perform an epitope competition assay on the anti-TFR1 antibody.

[0351] Briefly, the antibody to be tested (anti-TFR1 antibody or human IgG1 as a negative control) was serially diluted in PBS containing 2 μg / mL biotin-PR12560. The antibody solution was incubated with 1×10⁵ HEK293T-huTFR1 cells at 4°C for 1 hour. The cells were washed twice with PBS, and 100 µL of 1:1000 diluted fluorescently labeled streptavidin (Alexa Fluor 647-conjugated streptavidin, Jackson Immunological Research, catalog 016-600-084) was added to each well. After incubation at 4°C for 30 min, the cells were washed twice with staining buffer and subjected to flow cytometry.The inhibition rate was calculated using the following formula:

[0352] Inhibition rate (%) = (AB) / (AC) × 100 A: 100% signal without antibody treatment;

[0353] B: 100% signal for each tested antibody; C: Streptavidin signal only.

[0354] As shown in Figure 6, the results show that HCAb antibodies PR012592 and PR012560 share similar or overlapping epitopes. Example 7: Inhibitory activity of anti-TFR1 antibody against human TF protein binding to human TFR1 7.1 ELISA

[0355] To investigate the activity of anti-TFR1 antibody in blocking the binding of human TF to its receptor human TFR1 in vitro by ELISA, human TFR1-His protein was used to perform protein-level human TF / human TFR1 binding and blocking experiments.

[0356] In short, 0.5 μg / mL human TF-His protein (Bepsys, catalog number TRN-H82E3) was added to 96-well plates at a rate of 100 μL per well. After incubation overnight at 4°C, the plates were washed three times with 1×PBST (300 μL / well) and then blocked for 2 hours at 37°C with 5% skim milk in 1×PBST (300 μL / well). Subsequently, the plates were washed three times, and 0.5 μg / mL human TFR1-His protein was added, followed by incubation at 37°C for 1 hour. The plates were then washed three times, and serially diluted anti-TFR1 antibody in PBS was added. After incubating at 37°C for 1 hour, the plate was washed 3 times, and secondary antibody (Yama, manual page 38 / 52, 44 CN 122535623 A, goat anti-human IgG-Fc fragment HRP conjugated, Bethyl Laboratories (BETHYL), A80-304P) was added at 100 μL / well. After incubating at 37°C for 1 hour, the plate was washed 6 times, and then 100 μL / well TMB was added for about 5 min. Finally, 100 μL / well 2 M H2SO4 was added to the plate to terminate the reaction, and the readings were taken at 450 nm using a Molecular device spectra max plus 384.

[0357] The results are shown in Figure 7. The results show that PR011074 cannot bind to the TF-TFR complex, indicating that the epitope of PR011074 is located within the TF binding region. The binding signal of PR011073 to the TF-TFR complex is very low, indicating that the epitope of PR011073 overlaps with the TF binding region. PR12560 and PR012592 exhibited strong binding ability to the TF-TFR complex, indicating that the epitopes of these antibodies are far from the TF-binding region.7.2 FACS

[0358] To investigate the activity of anti-TFR1 antibody in blocking the binding of human TF to its receptor human TFR1 in vitro by flow cytometry, cell-based human TF / human TFR1 binding and blocking experiments were performed using HEK293T-huTFR1 cells.

[0359] Briefly, the antibody to be tested (anti-TFR1 antibody or human IgG1 as a negative control; final concentration 4 nM, 20 nM or 100 nM) and biotinylated human TF-His protein (Bioscience, catalog number TRN-H82E3) (final concentration 0.1 μg / mL) were incubated with 1×105 cells at 4°C for 1 hour. The cells were washed twice with PBS and 100 µL of 1:200 diluted fluorescently labeled streptavidin (eBioscience™ streptavidin PE conjugate, Invitrogen™, catalog number 12-4317-87) was added to each well. After incubation at 4°C for 30 min, the cells were washed twice with staining buffer and subjected to flow cytometry. The inhibition rate was calculated using the following formula:

[0360] Inhibition rate (%) = (AB) / (AC) × 100 A: 100% signal without antibody treatment; B: 100% signal for each test antibody; C: Streptavidin signal only.

[0361] The results are shown in Figure 8. PR011074 strongly blocked the binding of human TF to its receptor. PR011073 and PR012560 enhanced the binding of human TF to its receptor. At different concentrations, the inhibition rate for PR012592 was less than 10%. These results show that, compared with the control antibodies (PR011073, PR011074 and PR012560), PR012592 neither inhibited nor increased the binding of TF to its receptor at different concentrations, indicating that PR012592 does not affect the function of TF binding to its receptor at all. Example 8: Binding Affinity to TFR1 Protein Determined by BLI Method

[0362] In BLI (Biolayer Interferometry) analysis, anti-TFR1 antibody was diluted to 5 μg / mL using freshly prepared 1× kinetic buffer (10× kinetic buffer (ForteBio, No. 18-1105) diluted with PBS (BBI Life Sciences, No. E607016-0500) and captured on the surface of an anti-human Fc (AHC) Octet biosensor (ForteBio, No. 18-5060) to achieve a capture level between 0.8 and 1.0 nM. The captured biosensor was then immersed in wells containing serially diluted 2-fold antigen protein to detect association signals, followed by a dissociation step in wells containing 1× kinetic buffer.Human and cyno TFR1-His proteins were diluted from 80 nM to 20 nM; the association phase lasted 180 seconds, and the dissociation phase lasted 600 seconds. Sensing plots were recorded and a reference signal was subtracted, followed by curve fitting using ForteBio Data Analysis 11.0 software. The association rate (kon) and dissociation rate (kdis) were calculated using a simple one-to-one Langmuir binding model. The equilibrium dissociation constant (KD) was calculated as the ratio of kdis / kon. Table 6 summarizes the binding kinetic parameters of anti-TFR1 antibodies to human TFR1 and cyno TFR1.

[0363] Table 6. Kinetics of anti-TFR1 antibody against soluble human and cyno TFR1 protein 39 / 52 pages 45 CN 122535623 A Example 9: Antibody engineering of PR012592

[0364] PR012592 was modified in the CDR region by alanine scanning to reduce its binding affinity to human TFR1. Mutants derived from PR012592 were generated by transient expression using the procedure described in Example 4. The binding activity of the mutants to TFR1 was tested by ELISA and Fortebio Octet, and several mutants were selected for further evaluation based on their different binding affinity to TFR1. The amino acid sequences of the mutants derived from PR012592 are shown in Tables 7 and 8 (CDR as defined by Chothia).

[0365] Table 7. SEQ ID NO of amino acid sequences of PR012592 and its mutants

[0366] Table 8. Amino acid sequences of mutants derived from PR012592 Specification 40 / 52 pages 46 CN 122535623 A Specification 41 / 52 pages 47 CN 122535623 A Specification 42 / 52 pages 48 CN 122535623 A Specification 43 / 52 pages 49 CN 122535623 A Example 10: Binding affinity of mutants derived from PR012592 to TFR1 10.1 Binding affinity of PR012592 mutant to human and cynomolgus monkey TFR1 protein

[0367] Using the procedure described in Example 5.1, the recombinant anti-TFR1 antibody was tested by ELISA for binding affinity to human TFR1-His protein (Bepsys, catalog number CD1-H5243) and cyno Binding of TFR1-His protein (Bepsys, catalog number TFR-, specification page 44 / 52, 50 CN 122535623 A C524a).

[0368] The results are shown in Figures 9, 10 and Table 9. The results indicate that the mutant PR012592 exhibits different binding affinity to human and cyno TFR1-His proteins.

[0369] Table 9. Binding of PR012592 mutant to human cyno TFR1-His protein by ELISA 10.2 Binding affinity of PR012592 mutant to TFR1-expressing cell lines

[0370] The binding of PR012592 mutant to human TFR1-overexpressing cells HEK293T-cynoTFR1 was tested by flow cytometry using a similar procedure as described in Example 5.2.

[0371] The results are shown in Figure 11 and Table 10. These results show that the mutant exhibits different binding affinities to HEK293T-huTFR1 cells.

[0372] Table 10. Binding MFI of PR012592 mutant to HEK293T-huTFR1 cells by FACS Max: Calculated maximum MFI. 10.3 Binding affinity of PR012592 mutant to TFR1 protein obtained by BLI method

[0373] The binding affinity of PR012592 mutant to human and cyno TFR1 protein was analyzed by BLI using the procedure described in Example 8. The binding kinetic parameters of anti-TFR1 HCAb antibody to human TFR1 and cyno TFR1 are summarized in Table 11.

[0374] Table 11. Kinetics of anti-TFR1 antibody to soluble human and cyno TFR1 protein 45 / 52 pages 51 CN 122535623 A Example 11: BACE1×TFR1 and Aβ×TFR1 bispecific antibody

[0375] In order to evaluate the application of anti-TFR1 antibody in the context of bispecific antibody for BBB shuttle, BACE1×TFR1 and Aβ×TFR1 bispecific antibody (bsAb) were generated and evaluated.

[0376] BACE1×TFR1 bsAb was generated in the form shown in Figure 12, wherein the BACE1 binding domain (anti-BACE1 Fab) is derived from the sequence of Ab6266 (antibody ID PR013076 in this disclosure) in US 11008403 B2, and the TFR1 binding domain (anti-TFR1 VH) is derived from PR012592 and its mutants. Positive control BACE1×TFR1 bsAb PR013078 was also generated using the sequences of anti-BACE1 Ab6266 and anti-TFR1 Hu15G11 .LC92A .M108L disclosed in US 11008403 B2. Negative control BACE1×lysozyme bsAb PR013079 was also generated using the sequence of anti-BACE1 Ab6266 and the sequence of a cAb-Lys3 nanobody targeting chicken egg lysozyme. To form the heterodimer Fc of BACE1×TFR1 bsAb, a "mortar and pestle structure" technique was employed.The "palm" mutation (S354C, T366W) was introduced into the heavy chain encoding the variable region of the anti-BACE1 Fab, and the "mortar" mutation (Y349C, T366S, L368A, Y407V) was introduced into the heavy chain encoding the anti-TFR1 domain. To eliminate Fc effector function, triple mutations L234A, L235A, G237A (denoted as "AAA") were introduced into the CH2 region of both heavy chains. The same mutations were applied to BACE1×lysozyme bsAb. Table 12 lists the SEQ ID NOs of the amino acid sequences of BACE1×TFR1 and BACE1×lysozyme bsAb. Table 13 lists the SEQ ID NOs of the CDR sequence of each antigen-binding fragment in BACE1×TFR1 bsAb derived from anti-TFR1 HCAb. Table 16 lists the SEQ ID NO of the amino acid sequences of Ab6266 (PR013076) and cAb-Lys3 (PR000336).

[0377] Aβ×TFR1 bsAb was generated in the form shown in Figure 13, wherein the Aβ binding domain (anti-Aβ Fab) is derived from the sequence of erlotinib (antibody ID PR014308 in this disclosure), and the TFR1 binding domain (anti-TFR1 VH) is derived from PR012592 and its mutants. Positive control Aβ×TFR1 bsAb erlotinib (antibody ID PR012931 in this disclosure) was also generated using the disclosed sequence. Negative control Aβ×lysozyme bsAb PR013351 was also generated using the anti-Aβ Fab sequence of erlotinib and the sequence of cAb-Lys3 nanobody targeting chicken egg lysozyme. To form the heterodimer Fc of Aβ×TFR1 bsAb erlotinib, a "mortar and pestle" structure technique was employed; and to reduce chain mismatches between different VH and VL, mutations were introduced into the CH1 and CL domains of the anti-Aβ Fab. In the erlotinib structure, the anti-TFR1 Fab is attached to the C-terminus of an Fc. When Aβ×TFR1 bsAb is generated from anti-TFR1 HCAb PR012592 and its mutants, the anti-TFR1 Fab of erlotinib is directly replaced by anti-TFR1 VH. Table 14 lists the SEQ ID NOs of the amino acid sequences of Aβ×TFR1 and Aβ×lysozyme bsAb. Table 15 lists the SEQ ID NOs of the CDR sequence of each antigen-binding fragment in the Aβ×TFR1 bsAb derived from anti-TFR1 HCAb. Table 16 lists the SEQ ID NO of the amino acid sequences of erlotinib against Aβ Fab (PR014308) and cAb-Lys3 (PR000336).

[0378] Table 12. SEQ ID NO of the amino acid sequence of BACE1×TFR1 and BACE1×lysozyme bsAb. Specification 46 / 52 pages 52 CN 122535623 A

[0379] Table 13. SEQ ID NO of the CDR sequence of each antigen-binding fragment in BACE1×TFR1.

[0380] Table 14. SEQ ID NO of the amino acid sequence of Aβ×TFR1 and Aβ×lysozyme bsAb. Specification 47 / 52 pages 53 CN 122535623 A

[0381] Table 15. SEQ ID NO of the CDR sequence of each antigen-binding fragment in Aβ×TFR1.

[0382] Table 16. SEQ ID NO of amino acid sequences of anti-BACE1 Ab6266, terlotinib anti-Aβ Fab, and anti-lysozyme cAb-Lys3 Specification 48 / 52 pages 54 CN 122535623 A Example 12: Binding affinity of BsAb to TFR1 12.1 Binding affinity of BsAb to human TFR1 protein

[0383] The binding affinity of BsAb to human TFR1-His protein (Bepsys, catalog number CD1-H5243) was tested by ELISA using the procedure described in Example 5.1.

[0384] The results are shown in Figures 14, 15 and Table 17. The results indicate that BsAb exhibits different binding affinities to human TFR1-His protein.

[0385] Table 17. Binding of Aβ×TFR1 and BACE1×TFR1 bsAb to human TFR1-His protein by ELISA (Instruction manual 49 / 52 pages 55 CN 122535623 A 12.2 Binding affinity of BsAb to TFR1-expressing cell lines)

[0386] Using a similar procedure as described in Example 5.2, the binding of BsAb to human (HEK293T-huTFR1) or cyno (CHOK1-cynoTFR1) TFR1-overexpressing cells was tested by flow cytometry.

[0387] The results are shown in Figures 16, 17, 18 and Table 18. These results show that BsAb exhibits different binding affinities for HEK293T-huTFR1 cells and CHOK1-cynoTFR1 cells.

[0388] Table 18. Binding of Aβ×TFR1 and BACE1×TFR1 bsAb with human HEK293T-huTFR1 and CHOK1-cynoTFR1 cells obtained by FACS. Instructions for use, pages 50 / 52, 56 CN 122535623 A MFI Max: Calculated maximum MFI.Example 13: In vivo evaluation of BACE1×TFR1 BsAb

[0389] The efficacy of BACE1×TFR1 BsAb in vivo was evaluated using human TFR1 knock-in (hTFR1-KI) transgenic mice (Biocytogen). The experimental design is shown in Figure 19 and Table 19. For 6 mice in each group, brain and serum were collected from 3 mice 24 or 48 hours after administration, and half of the brain was homogenized and subjected to capillary depletion, and the drug concentration in the brain was determined by ELISA. The other half of the brain was paraffin-embedded and sectioned, and the distribution of the drug in the brain was detected by immunohistochemistry (IHC).

[0390] IHC detection of human IgG: (1) Dewaxing and hydration: Tissue sections were dewaxed in xylene I, xylene II and xylene III in a fixed order for 10 minutes each, washed in anhydrous ethanol I and anhydrous ethanol II for 5 minutes in sequence, and then hydrated in 95% ethanol, 70% ethanol and pure water for 5 minutes in sequence. (2) Antigen retrieval: Tissue sections were placed in boiling antigen retrieval solution and subjected to antigen retrieval at low heat in a microwave, and then cooled to room temperature; then the sections were washed 3 times with PBS (3 minutes each time), sealed with 3% hydrogen peroxide at room temperature for 10 minutes, washed 3 times with PBS (3 minutes each time), and blocked with antigen blocking solution at 37°C for 30 minutes. (3) Secondary antibody incubation: Incubate the slides with the secondary antibody (Jackson Immuno Research, 109-036-098) at 37°C for 30 minutes; after washing with PBS, wash the slides three times with PBS (3 minutes each time), and incubate with ABC reagent at 37°C for 30 minutes; after washing with PBS, wash the slides three times with PBS (3 minutes each time). (4) Substrate colorimetric method: Incubate the slides with the substrate colorimetric solution at room temperature for 3-10 minutes, then wash with PBS to stop the color development, and stain with hematoxylin for 15-60 seconds, then wash with PBS and rinse under tap water for 3 minutes. (5) Sealing: The slides were washed sequentially with 95% ethanol, anhydrous ethanol III and anhydrous ethanol IV for 2 minutes, and then washed with anhydrous ethanol: xylene = 1:1, anhydrous ethanol IV and anhydrous ethanol V for 1 minute and sealed with neutral resin.

[0391] Table 19. Study design.

[0392] The results of ELISA are shown in Figures 20, 21 and 22. As shown in Figures 20 and 21, BACE1xTFR1 bsAb showed differential brain penetration ability.Surprisingly, the BACE1xTFR1 bsAb PR013357, PR013359, and PR013360 derived from anti-TFR1 HCAb showed comparable or even stronger brain penetration than the positive control PR013078.

[0393] The IHC results are shown in Figure 23, with arrows pointing to neurons taking up BsAb. Consistent with the ELISA results, BACE1xTFR1 bsAb showed differential brain uptake and distribution. Surprisingly, the BACE1xTFR1 bsAb PR013357, PR013359, and PR013360 derived from anti-TFR1 HCAb showed comparable or even greater neuronal uptake and brain parenchyma distribution than the positive control PR013078.

[0394] It should be understood that the foregoing description of the embodiments is intended to be purely illustrative of the principles of this disclosure and not to exhaustively describe them, and variations and modifications will be apparent to those skilled in the art, and this disclosure is not intended to be limited except as expressly shown in the following claims. Instruction manual, pages 52 / 52, 58, CN 122535623 A, Figure 1, Figure 2; Instruction manual, Figure 1 / 11, page 59, CN 122535623 A, Figure 3, Figure 4; Instruction manual, Figure 2 / 11, page 60, CN 122535623 A, Figure 5, Figure 6; Instruction manual, Figure 3 / 11, page 61, CN 122535623 A, Figure 7, Figure 8; Instruction manual, Figure 4 / 11, page 62, CN 122535623 A, Figure 9, Figure 10; Instruction manual, Figure 5 / 11, page 63, CN 122535623 A, Figure 11, Figure 12; Instruction manual, Figure 6 / 11, page 64, CN 122535623 A, Figure 13, Figure 14; Instruction manual, Figure 7 / 11, page 65, CN 122535623 A, Figure 15, Figure 16; Instruction manual, Figure 8 / 11, page 66, CN 122535623 A, Figure 17, Figure 18. Figure 19 Figure 20 Figure 21 Figure 19 Figure 21 Figure 22 Figure 23 Figure 23 Figure 23 Figure 24 Figure 25 Figure 23 Figure 25 Figure 23 Figure 24 Figure 25 Figure 23 Figure 25 Figure 23 Figure 24 Figure 25 Figure 23 Figure 25 Figure 23 Figure 25 Figure 23 Figure 24 Figure 25 Figure 23 Figure 25 Figure 23 Figure 25 Figure 21 Figure 23 Figure 24 Figure 25 Figure 21 Figure 23 Figure 25 Figure 21 Figure 22 Figure 23 Figure 23 Figure 24 Figure 25 Figure 21 Figure 22 Figure 23 ...4 Figure 25 Figure 21 Figure 2

Claims

1. An anti-transferrin receptor 1 (TFR1) antibody or its antigen-binding fragment thereof, the anti-transferrin receptor 1 antibody or its antigen-binding fragment comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises VH CDR1, VH CDR2 and VHCDR3, wherein The VH CDR1 contains an amino acid sequence that differs from SEQ ID NO: 8 in that it has an addition, deletion or substitution of no more than two amino acids; The VH CDR2 contains an amino acid sequence that differs from SEQ ID NO: 16 in that it has an addition, deletion, or substitution of no more than two amino acids; and / or The VH CDR3 contains an amino acid sequence that differs from SEQ ID NO: 24 in that it has an addition, deletion or substitution of no more than two amino acids.

2. The anti-TFR1 antibody or antigen-binding fragment as described in claim 1, wherein... The VH CDR1 contains the amino acid sequence of SEQ ID NO:

8. The VH CDR2 contains the amino acid sequence of SEQ ID NO: 16, and The VH CDR3 contains an amino acid sequence that differs from SEQ ID NO: 24 in that no more than two amino acids are added, deleted, or substituted. Preferably, the VH CDR3 contains a non-conservative amino acid substitution at the position of amino acid 2, 4, 5 or 7 corresponding to SEQ ID NO:

24.

3. The anti-TFR1 antibody or antigen-binding fragment as described in claim 1 or 2, wherein... The VH CDR1 contains the amino acid sequence of SEQ ID NO:

8. The VH CDR2 contains the amino acid sequence of SEQ ID NO: 16, and The VH CDR3 contains the amino acid sequence of SEQ ID NO: 24, 79, 80, 81 or 82.

4. The anti-TFR1 antibody or antigen-binding fragment of claim 1, wherein the heavy chain variable domain comprises VH CDR1, VH CDR2, and VH CDR3 as contained in the amino acid sequences of SEQ ID NO: 52, 98, 99, 100, or 101.

5. The anti-TFR1 antibody or antigen-binding fragment according to any one of claims 1-4, wherein the heavy chain variable domain comprises: (A) The amino acid sequence of SEQ ID NO: 52, 98, 99, 100 or 101; or (B) An amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 52, 98, 99, 100, or 101; or (C) An amino acid sequence having one or more amino acid additions, deletions and / or substitutions compared to the amino acid sequence of SEQ ID NO: 52, 98, 99, 100 or 101, wherein preferably, the amino acid additions, deletions and / or substitutions do not occur in the CDR region.

6. The anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-5, wherein the anti-TFR1 antibody or antigen-binding fragment (1) Binding to human TFR1 and non-human primate TFR1; and / or (2) It has virtually no effect on the binding of transferrin to TFR1; and / or (3) It binds to the transferrin-TFR1 complex.

7. The anti-TFR1 antibody or antigen-binding fragment according to any one of claims 1-6, wherein the anti-TFR1 antibody or antigen-binding fragment is a single-domain antibody or a heavy-chain-only antibody, and optionally a fully human antibody.

8. The anti-TFR1 antibody or antigen-binding fragment according to any one of claims 1-7, wherein the anti-TFR1 antibody or antigen-binding fragment further comprises an Fc region, and optionally the Fc region comprises one or more modifications to reduce effector function and / or enhance FcRn binding.

9. The anti-TFR1 antibody or antigen-binding fragment according to any one of claims 1-8, wherein the anti-TFR1 antibody or antigen-binding fragment comprises a heavy chain having an amino acid sequence having SEQ ID NO: 59, 107, 108, 109 or 110.

10. A transferrin receptor 1 (TFR1) binder comprising an anti-TFR1 antibody or antigen-binding fragment conjugated to any one of claims 1-9 with an additional molecule.

11. The TFR1 binder of claim 10, wherein the additional molecule is a therapeutic agent for treating diseases or disorders selected from: cancer, central nervous system cancers, Alzheimer's disease, Parkinson's disease, Huntington's disease, Hunter syndrome, schizophrenia, antidepressants, multiple sclerosis, amyotrophic lateral sclerosis, lysosomal storage diseases with encephalopathy, glycogen storage diseases, muscular dystrophy, cerebral ischemia, prions, traumatic central nervous system disorders, viral and bacterial central nervous system diseases.

12. The TFR1 binder of claim 10, wherein the additional molecule is a developing agent selected from the group consisting of radionuclides, biotin, fluorescent proteins, fluorophores, horseradish peroxidase, and alkaline phosphatase.

13. A multispecific antibody comprising a first antigen-binding moiety binding to TFR1 and a second antigen-binding moiety binding to a second antigen, wherein the first antigen-binding moiety comprises a heavy chain variable domain as defined in any one of claims 1-6.

14. A multispecific antibody comprising a first antigen-binding moiety binding to TFR1 and a second antigen-binding moiety binding to BACE1, wherein The first antigen-binding portion includes a heavy chain variable domain as defined in any one of claims 1-6. The second antigen-binding region comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as contained in the amino acid sequence of SEQ ID NO: 102, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as contained in the amino acid sequence of SEQ ID NO:

104. Preferably, HCDR1 contains the amino acid sequence of SEQ ID NO: 67, HCDR2 contains the amino acid sequence of SEQ ID NO: 73, and HCDR3 contains the amino acid sequence of SEQ ID NO: 83; and LCDR1 contains the amino acid sequence of SEQ ID NO: 88, LCDR2 contains the amino acid sequence of SEQ ID NO: 91, and LCDR3 contains the amino acid sequence of SEQ ID NO:

95.

15. The multispecific antibody of claim 14, wherein... The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 102; and / or the light chain variable region contains the amino acid sequence of SEQ ID NO:

104.

16. A multispecific antibody comprising a first antigen-binding moiety binding to TFR1 and a second antigen-binding moiety binding to amyloid β (Aβ), wherein The first antigen-binding portion includes a heavy chain variable domain as defined in any one of claims 1-6. The second antigen-binding region comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as contained in the amino acid sequence of SEQ ID NO: 103, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as contained in the amino acid sequence of SEQ ID NO:

105. Preferably, HCDR1 contains the amino acid sequence of SEQ ID NO: 68, HCDR2 contains the amino acid sequence of SEQ ID NO: 74, and HCDR3 contains the amino acid sequence of SEQ ID NO: 84; and LCDR1 contains the amino acid sequence of SEQ ID NO: 89, LCDR2 contains the amino acid sequence of SEQ ID NO: 92, and LCDR3 contains the amino acid sequence of SEQ ID NO:

96.

17. The multispecific antibody of claim 16, wherein... The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 103; and / or The variable region of the light chain contains the amino acid sequence of SEQ ID NO:

105.

18. The multispecific antibody according to any one of claims 13-17, wherein the second antigen-binding moiety comprises dsFv, scFv, scdsFv, di-scFv, Fab, scFab, or F(ab')2.

19. The multispecific antibody of any one of claims 13-18, wherein the multispecific antibody comprises only one heavy chain variable domain as defined in any one of claims 1-6.

20. The multispecific antibody of any one of claims 13-19, wherein the multispecific antibody further comprises an Fc region, and optionally the Fc region comprises one or more modifications to reduce effector function and / or enhance FcRn binding.

21. The multispecific antibody of claim 20, wherein the Fc region is a heterodimeric Fc region comprising a first Fc region subunit and a second Fc region subunit, wherein one of the first Fc region subunit and the second Fc region subunit contains a club-shaped mutation and the other contains a mortar-shaped mutation.

22. The multispecific antibody of claim 21, wherein... This mutation includes one or more mutations selected from S354C and T366W; and / or This mutation includes one or more mutations selected from Y349C, T366S, L368A, and Y407V; Preferably, the pestle mutation includes S354C and T366W, and the mortar mutation includes Y349C, T366S, L368A, and Y407V.

23. The multispecific antibody of claim 21 or 22, wherein the second antigen-binding moiety comprises a heavy chain variable region and a light chain variable region, and the multispecific antibody comprises: (a) (1) a first polypeptide comprising the heavy chain variable region, the CH1 domain, and the first Fc subunit; (2) a second polypeptide comprising the light chain variable region and the light chain constant region; and (3) a third polypeptide comprising the heavy chain variable domain and the second Fc subunit, or (b) (1) a first polypeptide comprising the heavy chain variable region, the CH1 domain, a first Fc subunit, an optional peptide linker, and the heavy chain variable domain; (2) a second polypeptide comprising the heavy chain variable region, the CH1 domain, and a second Fc subunit; and (3) a third polypeptide and a fourth polypeptide, each comprising the light chain variable region and the light chain constant region. The first Fc region subunit and the second Fc region subunit form the heterodimer Fc region.

24. The multispecific antibody of claim 14, wherein the multispecific antibody comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 122, a second polypeptide having an amino acid sequence of SEQ ID NO: 113, and a third polypeptide having an amino acid sequence of SEQ ID NO: 129, 130, 131, 132 or 133.

25. The multispecific antibody of claim 16, wherein the multispecific antibody comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 119, 125, 126, 127 or 128, a second polypeptide having an amino acid sequence of SEQ ID NO: 117, and a third polypeptide and a fourth polypeptide each having an amino acid sequence of SEQ ID NO:

115.

26. A pharmaceutical composition or kit comprising an anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9, a TFR1 binder as described in any one of claims 10-12, or a multispecific antibody as described in any one of claims 13-25.

27. A nucleic acid encoding an anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9, or a multispecific antibody as described in any one of claims 13-25.

28. A vector comprising the nucleic acid as described in claim 27.

29. A host cell comprising the nucleic acid as described in claim 27 or the vector as described in claim 28.

30. Use of the anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9 in the manufacture of a pharmaceutical agent for transporting a compound across the blood-brain barrier.

31. Use of the anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9 in the manufacture of a medicament for treating cancer.

32. Use of the anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9 in the manufacture of a medicament for treating a neurological disease or disorder, wherein the anti-TFR1 antibody or antigen-binding fragment is conjugated to a therapeutic agent for treating the neurological disease or disorder.

33. Use of the multispecific antibody as described in any one of claims 14-25 in the manufacture of a medicament for treating a neurological disease or disorder; preferably, the neurological disease or disorder is selected from Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, and glaucoma.

34. Use of the multispecific antibody as described in any one of claims 14-25 in the manufacture of a pharmaceutical preparation or kit for the diagnosis of Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, or glaucoma.

35. Use of the multispecific antibody as described in any one of claims 14-25 in the manufacture of a medicament for inhibiting and / or reducing the formation of amyloid plaques in the brain.

36. A method for transporting a compound across the blood-brain barrier in a subject, the method comprising conjugating the compound with an anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9, and transporting the conjugate across the blood-brain barrier.

37. A method of treating cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9.

38. A method of treating a neurological disease or disorder in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an anti-TFR1 antibody or antigen-binding fragment conjugated to a therapeutic agent as described in any one of claims 1-9, the therapeutic agent being used to treat the neurological disease or disorder.

39. A method for treating a neurological disease or disorder in a subject in need, the method comprising administering to the subject a therapeutically effective amount of a multispecific antibody as described in any one of claims 14-25; preferably, wherein the neurological disease or disorder is selected from Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, and glaucoma.

40. A method for inhibiting and / or reducing the formation of amyloid plaques in the brain of a subject in need, the method comprising administering to the subject a therapeutically effective amount of a multispecific antibody as described in any one of claims 14-25 to inhibit and / or reduce the formation of amyloid plaques in the brain.

41. The anti-TFR1 antibody or antigen-binding fragment according to any one of claims 1-9, for use as a medicine, wherein the anti-TFR1 antibody or antigen-binding fragment transports the compound across the blood-brain barrier.

42. The anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9, for use in the treatment of cancer.

43. The anti-TFR1 antibody or antigen-binding fragment as described in any one of claims 1-9, for use as a medicament for treating a neurological disease or disorder, wherein the anti-TFR1 antibody or antigen-binding fragment is conjugated to a therapeutic agent for treating the neurological disease or disorder.

44. The multispecific antibody as described in any one of claims 14-25, for use in the treatment of neurological diseases or disorders, optionally wherein the neurological disease or disorder is selected from Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, and glaucoma.

45. The multispecific antibody as described in any one of claims 14-25, for use in the diagnosis of Alzheimer's disease, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, or glaucoma.

46. ​​The multispecific antibody as described in any one of claims 14-25, for use in inhibiting and / or reducing the formation of amyloid plaques in the brain.

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