Antigen-binding molecule specifically binding to tfr1 and medical use thereof

Abstract

An antigen-binding molecule specifically binding to TfR1 and a medical use thereof. Particularly, the present invention relates to a use of the antigen-binding molecule specifically binding to TfR1 in the preparation of a drug for preventing or treating nervous system diseases.

Description

Antigen-binding molecules that specifically bind to TfR1 and their pharmaceutical applications

[0001] This application claims priority to Chinese patent application CN202411951984.1 filed on December 27, 2024 and Chinese patent application CN202510750952.3 filed on June 6, 2025. Technical Field

[0002] This disclosure pertains to the field of biotechnology, and more specifically, to antigen-binding molecules that specifically bind to TfR1 and their pharmaceutical uses. Background Technology

[0003] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0004] Transferrin receptor protein 1 (TfR1), also known as CD71 or TFRC, is a type II transmembrane protein and the most important membrane protein regulating intracellular iron transport (Pierre VC, et al. Frontiers in immunology, 2021, 12:607692). Under normal physiological conditions, the primary function of TfR1 is to bind to transferrin (TF) and mediate cellular iron uptake through endocytosis. Therefore, the TF-TfR1 system is considered an important pathway for the body to acquire iron ions. TfR1 is a membrane protein widely expressed in almost all cell and tissue types in the human body, and is widely distributed in various tissues such as the immune system, hematopoietic system, nervous system, liver, and kidneys (Kawabata H. Free radical biology & medicine, 2019, 133:46–54). The blood-brain barrier (BBB) ​​in the circulatory and central nervous systems makes drug delivery to the brain difficult, posing a significant challenge to the treatment of central nervous system diseases (Neuwelt, E., et al. The Lancet Neurology, 2008, 7(1):84–96). Studies have shown that the brain uptake of traditional IgG antibodies is only 0.1%–0.2% of the injected dose (Yu, YJ, et al. Neurootherapeutics, 10(3):459–472). To improve the efficiency of intracranial drug delivery, endocytosis of certain receptors or transporters highly expressed on brain capillary endothelial cells (BCECs) is utilized to cross the blood-brain barrier. TfR1 is a receptor highly expressed on BCECs and can serve as a good cross-BBB transport vehicle for targeted delivery of therapeutic drugs to the brain (Johnsen, KB, et al. Progress in neurobiology, 2019, 181:101665). Bispecific antibody drugs targeting TfR1 have been shown to significantly increase antibody exposure in the brain of non-human primates, by 4-18 times compared to monoclonal antibody drugs (Grimm, HP, et al. mAbs, 2023, 15(1):2261509). WO1993010819A1 discloses a murine anti-TfR1 antibody ALK 128.1, while WO2021092482A1, WO2023287238A1, and WO2024155066A1 disclose various humanized anti-TfR1 antibodies based on ALK 128.1.This disclosure establishes a central nervous system (CNS) drug delivery platform by developing antigen-binding molecules that specifically bind to TfR1, which can be used to prepare other types of drugs to overcome the BBB barrier, thereby improving the efficacy of drugs for treating CNS diseases. Summary of the Invention

[0005] <Antigen-binding molecules that specifically bind to TfR1>

[0006] This disclosure provides an antigen-binding molecule that specifically binds to TfR1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0007] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 3, HCDR2 contains the amino acid sequence of SEQ ID NO: 11, and HCDR3 contains the amino acid sequence of SEQ ID NO: 36; the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 8, wherein:

[0008] GYX1YYX2LDY SEQ ID NO: 36;

[0009] X1 can be S or Y, with S being preferred; X2 can be T or S, with T being preferred.

[0010] This disclosure provides an antigen-binding molecule that specifically binds to TfR1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0011] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 27, 12, or 26, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 13, 14, 15, or 16.

[0012] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0013] The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 27, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 13.

[0014] In some embodiments, the antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions and the LCDR1, LCDR2, and LCDR3 of the light chain variable regions are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions and the LCDR1, LCDR2, and LCDR3 of the light chain variable regions are defined according to the Kabat numbering rule. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions and the LCDR1, LCDR2, and LCDR3 of the light chain variable regions are defined according to the IMGT numbering rule. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions and the LCDR1, LCDR2, and LCDR3 of the light chain variable regions are defined according to the Chothia numbering rule. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the light chain variable regions LCDR1, LCDR2, and LCDR3, are defined according to the AbM numbering rules. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the light chain variable regions LCDR1, LCDR2, and LCDR3, are defined according to the Contact numbering rules.

[0015] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0016] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 3, HCDR2 contains the amino acid sequence of SEQ ID NO: 11, and HCDR3 contains the amino acid sequence of SEQ ID NO: 25, 5, or 24. The light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 8.

[0017] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0018] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 3, HCDR2 contains the amino acid sequence of SEQ ID NO: 11, and HCDR3 contains the amino acid sequence of SEQ ID NO: 25; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 8; or

[0019] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 3, HCDR2 contains the amino acid sequence of SEQ ID NO: 11, and HCDR3 contains the amino acid sequence of SEQ ID NO: 5; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 8; or

[0020] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 3, HCDR2 contains the amino acid sequence of SEQ ID NO: 11, and HCDR3 contains the amino acid sequence of SEQ ID NO: 24, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 8.

[0021] In some embodiments, the antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding embodiments includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein:

[0022] The amino acid sequences of the heavy chain variable regions HCDR1 are shown in SEQ ID NO: 3, HCDR2 in SEQ ID NO: 11, and HCDR3 in SEQ ID NO: 25; the amino acid sequences of the light chain variable regions LCDR1 in SEQ ID NO: 9, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 8; or

[0023] The amino acid sequences of the heavy chain variable regions HCDR1 are shown in SEQ ID NO: 3, HCDR2 in SEQ ID NO: 11, and HCDR3 in SEQ ID NO: 5; the amino acid sequences of the light chain variable regions LCDR1 in SEQ ID NO: 9, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 8; or

[0024] The amino acid sequences of the heavy chain variable regions HCDR1 are shown in SEQ ID NO: 3, HCDR2 are shown in SEQ ID NO: 11, and HCDR3 are shown in SEQ ID NO: 24. The amino acid sequences of the light chain variable regions LCDR1 are shown in SEQ ID NO: 9, LCDR2 are shown in SEQ ID NO: 10, and LCDR3 are shown in SEQ ID NO: 8.

[0025] In some embodiments, the antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding embodiments, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules.

[0026] In some embodiments, the antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, is an antibody or antibody fusion protein. In some embodiments, the antigen-binding molecule that specifically binds to TfR1 is a monoclonal antibody. In some embodiments, the antigen-binding molecule that specifically binds to TfR1 is a monospecific antibody. In some embodiments, the antigen-binding molecule that specifically binds to TfR1 is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the antigen-binding molecule that specifically binds to TfR1 is a humanized antibody.

[0027] In some implementations, the antigen-binding molecule that specifically binds to TfR1, as described in the preceding one, includes the frame region (FR) of the human antibody.

[0028] In some embodiments, the antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding embodiments, wherein the heavy chain variable region has FR1, FR2, FR3 fragments derived from IGHV1-2*06 and FR4 fragments derived from IGHJ6*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 1E, 48I, 69L, 71V and / or 73K; and / or the light chain variable region has FR1, FR2, FR3 fragments derived from IGKV3-11*01 and FR4 fragments derived from IGKJ4*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 46R, 47W and / or 58V. In some embodiments, the antigen-binding molecule that specifically binds to TfR1, wherein the heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, HCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 25, 5, or 24, and the FR of the heavy chain variable region is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 48I, 69L, 71V, and / or 73K; and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and the FR of the light chain variable region is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 46R, 47W, and / or 58V. In some embodiments, the above-mentioned variable regions and CDRs are defined according to the Kabat numbering rules.

[0029] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0030] The heavy chain variable region comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 27, 12, or 26, and the light chain variable region comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 13, 14, 15, or 16.

[0031] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0032] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27, 12 or 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 13, 14, 15 or 16.

[0033] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0034] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; or

[0035] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; or

[0036] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 13, 14, 15 or 16.

[0037] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0038] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; or

[0039] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; or

[0040] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15; or

[0041] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16; or

[0042] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; or

[0043] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; or

[0044] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15; or

[0045] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16; or

[0046] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; or

[0047] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; or

[0048] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15; or

[0049] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 16.

[0050] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0051] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 13; or

[0052] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; or

[0053] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 15; or

[0054] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16; or

[0055] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 13; or

[0056] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; or

[0057] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 15; or

[0058] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16; or

[0059] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 13; or

[0060] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; or

[0061] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 15; or

[0062] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

[0063] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein the antigen-binding molecule that specifically binds to TfR1 is humanized, reversed mutation, affinity maturation, T-cell epitope removal, antibody deamidation reduction, and / or antibody isomerization reduction modification.

[0064] In some embodiments, the antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is an antibody fragment. In some embodiments, the antigen-binding fragment is selected from Fab, Fab′, F(ab′)2, Fv, scFv, and dsFv.

[0065] In some embodiments, the antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding embodiments comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is human IgG1, IgG2, IgG3, IgG4, or a variant thereof, and the light chain constant region is human κ chain, λ chain, or a variant thereof. In some embodiments, the heavy chain constant region is human IgG1 heavy chain constant region or a variant thereof, and the light chain constant region is human κ light chain constant region or a variant thereof. In some embodiments, the heavy chain constant region is human IgG1 heavy chain constant region, and the light chain constant region is human κ light chain constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 17, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 18.

[0066] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, comprises a heavy chain and a light chain, wherein:

[0067] The heavy chain comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 29, 19, or 28, and the light chain comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 20, 21, 22, or 23.

[0068] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0069] The heavy chain comprises the amino acid sequence of SEQ ID NO: 29, 19 or 28, and the light chain comprises the amino acid sequence of SEQ ID NO: 20, 21, 22 or 23.

[0070] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0071] The heavy chain comprises the amino acid sequence of SEQ ID NO: 29, and the light chain comprises the amino acid sequence of SEQ ID NO: 20, 21, 22, or 23; or

[0072] The heavy chain comprises the amino acid sequence of SEQ ID NO: 19, and the light chain comprises the amino acid sequence of SEQ ID NO: 20, 21, 22, or 23; or

[0073] The heavy chain contains the amino acid sequence of SEQ ID NO: 28, and the light chain contains the amino acid sequence of SEQ ID NO: 20, 21, 22 or 23.

[0074] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0075] The heavy chain comprises the amino acid sequence of SEQ ID NO: 29, and the light chain comprises the amino acid sequence of SEQ ID NO: 20; or

[0076] The heavy chain comprises the amino acid sequence of SEQ ID NO: 29, and the light chain comprises the amino acid sequence of SEQ ID NO: 21; or

[0077] The heavy chain comprises the amino acid sequence of SEQ ID NO: 29, and the light chain comprises the amino acid sequence of SEQ ID NO: 22; or

[0078] The heavy chain comprises the amino acid sequence of SEQ ID NO: 29, and the light chain comprises the amino acid sequence of SEQ ID NO: 23; or

[0079] The heavy chain comprises the amino acid sequence of SEQ ID NO: 19, and the light chain comprises the amino acid sequence of SEQ ID NO: 20; or

[0080] The heavy chain comprises the amino acid sequence of SEQ ID NO: 19, and the light chain comprises the amino acid sequence of SEQ ID NO: 21; or

[0081] The heavy chain comprises the amino acid sequence of SEQ ID NO: 19, and the light chain comprises the amino acid sequence of SEQ ID NO: 22; or

[0082] The heavy chain comprises the amino acid sequence of SEQ ID NO: 19, and the light chain comprises the amino acid sequence of SEQ ID NO: 23; or

[0083] The heavy chain comprises the amino acid sequence of SEQ ID NO: 28, and the light chain comprises the amino acid sequence of SEQ ID NO: 20; or

[0084] The heavy chain comprises the amino acid sequence of SEQ ID NO: 28, and the light chain comprises the amino acid sequence of SEQ ID NO: 21; or

[0085] The heavy chain comprises the amino acid sequence of SEQ ID NO: 28, and the light chain comprises the amino acid sequence of SEQ ID NO: 22; or

[0086] The heavy chain contains the amino acid sequence of SEQ ID NO: 28, and the light chain contains the amino acid sequence of SEQ ID NO: 23.

[0087] In some embodiments, an antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, wherein:

[0088] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20; or

[0089] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain is shown in SEQ ID NO: 21; or

[0090] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain is shown in SEQ ID NO: 22; or

[0091] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain is shown in SEQ ID NO: 23; or

[0092] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20; or

[0093] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is shown in SEQ ID NO: 21; or

[0094] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is shown in SEQ ID NO: 22; or

[0095] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is shown in SEQ ID NO: 23; or

[0096] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20; or

[0097] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain is shown in SEQ ID NO: 21; or

[0098] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain is shown in SEQ ID NO: 22; or

[0099] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain is shown in SEQ ID NO: 23.

[0100] In some embodiments, the antigen-binding molecules that specifically bind to TfR1 provided in this disclosure are in EC5 concentrations of less than 10 nM (e.g., less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05 nM, less than 0.01 nM, less than 0.001 nM). 50 The value is combined with human TfR1, the EC 50 The value was measured using ELISA.

[0101] In some embodiments, the antigen-binding molecules that specifically bind to TfR1 provided in this disclosure do not block or have an IC50 value greater than 3.5 nM (e.g., greater than 4 nM, greater than 5 nM, greater than 10 nM, greater than 100 nM, greater than 1000 nM, greater than 1000 nM). 50 The IC blocks the binding of TF and TfR1. 50 The value was measured using ELISA.

[0102] <A bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ>

[0103] In some embodiments, the antigen-binding molecule that specifically binds to TfR1, as described in any of the preceding embodiments, is a multispecific antibody. In some embodiments, the antigen-binding molecule that specifically binds to TfR1 is a bispecific antibody. In some embodiments, the antigen-binding molecule that specifically binds to TfR1 is a bivalent bispecific antibody. In some embodiments, the antigen-binding molecule that specifically binds to TfR1 is a bispecific antibody that specifically binds to both TfR1 and N3pGlu Aβ. In some embodiments, the antigen-binding molecule that specifically binds to TfR1 is a bivalent bispecific antibody that specifically binds to both TfR1 and N3pGlu Aβ.

[0104] This disclosure provides a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, comprising a first antigen-binding domain that specifically binds to TfR1 and a second antigen-binding domain that specifically binds to N3pGlu Aβ.

[0105] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0106] The first antigen-binding domain that specifically binds to TfR1 comprises a heavy chain variable region (TfR1-VH) and a light chain variable region (TfR1-VL), wherein TfR1-VH comprises HCDR1, HCDR2, and HCDR3, and TfR1-VL comprises LCDR1, LCDR2, and LCDR3, wherein:

[0107] The TfR1-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 3, HCDR2 containing the amino acid sequence of SEQ ID NO: 11, and HCDR3 containing the amino acid sequence of SEQ ID NO: 36; the TfR1-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 10, and LCDR3 containing the amino acid sequence of SEQ ID NO: 8, wherein:

[0108] GYX1YYX2LDY SEQ ID NO: 36;

[0109] X1 is S or Y, preferably S; X2 is T or S, preferably T; and

[0110] The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises a heavy chain variable region (N3pGlu Aβ-VH) and a light chain variable region (N3pGlu Aβ-VL), wherein N3pGlu Aβ-VH comprises HCDR1, HCDR2, and HCDR3, and N3pGlu Aβ-VL comprises LCDR1, LCDR2, and LCDR3, wherein:

[0111] The N3pGlu Aβ-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 46, HCDR2 containing the amino acid sequence of SEQ ID NO: 47, and HCDR3 containing the amino acid sequence of SEQ ID NO: 48. The N3pGlu Aβ-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 49, LCDR2 containing the amino acid sequence of SEQ ID NO: 50, and LCDR3 containing the amino acid sequence of SEQ ID NO: 51.

[0112] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0113] The first antigen-binding domain that specifically binds to TfR1 comprises a heavy chain variable region (TfR1-VH) and a light chain variable region (TfR1-VL), wherein TfR1-VH comprises HCDR1, HCDR2, and HCDR3, and TfR1-VL comprises LCDR1, LCDR2, and LCDR3, wherein:

[0114] The amino acid sequences of HCDR1 in TfR1-VH are shown in SEQ ID NO: 3, HCDR2 in SEQ ID NO: 11, and HCDR3 in SEQ ID NO: 36; the amino acid sequences of LCDR1 in TfR1-VL are shown in SEQ ID NO: 9, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 8, wherein:

[0115] GYX1YYX2LDY SEQ ID NO: 36;

[0116] X1 is S or Y, preferably S; X2 is T or S, preferably T; and

[0117] The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises a heavy chain variable region (N3pGlu Aβ-VH) and a light chain variable region (N3pGlu Aβ-VL), wherein N3pGlu Aβ-VH comprises HCDR1, HCDR2, and HCDR3, and N3pGlu Aβ-VL comprises LCDR1, LCDR2, and LCDR3, wherein:

[0118] The amino acid sequences of HCDR1 of N3pGlu Aβ-VH are shown in SEQ ID NO: 46, HCDR2 in SEQ ID NO: 47, and HCDR3 in SEQ ID NO: 48. The amino acid sequences of LCDR1 of N3pGlu Aβ-VL are shown in SEQ ID NO: 49, LCDR2 in SEQ ID NO: 50, and LCDR3 in SEQ ID NO: 51.

[0119] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0120] The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein:

[0121] The TfR1-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 3, HCDR2 containing the amino acid sequence of SEQ ID NO: 11, and HCDR3 containing the amino acid sequence of SEQ ID NO: 25, 5, or 24; and the TfR1-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 10, and LCDR3 containing the amino acid sequence of SEQ ID NO: 8; and

[0122] The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein:

[0123] The N3pGlu Aβ-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 46, HCDR2 containing the amino acid sequence of SEQ ID NO: 47, and HCDR3 containing the amino acid sequence of SEQ ID NO: 48. The N3pGlu Aβ-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 49, LCDR2 containing the amino acid sequence of SEQ ID NO: 50, and LCDR3 containing the amino acid sequence of SEQ ID NO: 51.

[0124] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0125] The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein:

[0126] The amino acid sequences of HCDR1 of TfR1-VH are shown in SEQ ID NO: 3, HCDR2 in SEQ ID NO: 11, and HCDR3 in SEQ ID NO: 25, 5, or 24; the amino acid sequences of LCDR1 of TfR1-VL are shown in SEQ ID NO: 9, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 8; and

[0127] The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein:

[0128] The amino acid sequences of HCDR1 of N3pGlu Aβ-VH are shown in SEQ ID NO: 46, HCDR2 in SEQ ID NO: 47, and HCDR3 in SEQ ID NO: 48. The amino acid sequences of LCDR1 of N3pGlu Aβ-VL are shown in SEQ ID NO: 49, LCDR2 in SEQ ID NO: 50, and LCDR3 in SEQ ID NO: 51.

[0129] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0130] The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein:

[0131] The TfR1-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 3, HCDR2 containing the amino acid sequence of SEQ ID NO: 11, and HCDR3 containing the amino acid sequence of SEQ ID NO: 25; and the TfR1-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 10, and LCDR3 containing the amino acid sequence of SEQ ID NO: 8; and

[0132] The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein:

[0133] The N3pGlu Aβ-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 46, HCDR2 containing the amino acid sequence of SEQ ID NO: 47, and HCDR3 containing the amino acid sequence of SEQ ID NO: 48. The N3pGlu Aβ-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 49, LCDR2 containing the amino acid sequence of SEQ ID NO: 50, and LCDR3 containing the amino acid sequence of SEQ ID NO: 51.

[0134] In some embodiments, the bispecific antibodies that specifically bind TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, wherein the HCDR1, HCDR2 and HCDR3 of TfR1-VH and the LCDR1, LCDR2 and LCDR3 of TfR1-VL are defined according to the Kabat numbering rules.

[0135] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0136] The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein:

[0137] The TfR1-VH comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 27, 12, or 26, and the TfR1-VL comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 13, 14, 15, or 16; and

[0138] The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein:

[0139] The N3pGlu Aβ-VH comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 52, and the N3pGlu Aβ-VL comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 53.

[0140] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0141] The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein:

[0142] The TfR1-VH comprises the amino acid sequence of SEQ ID NO: 27, 12, or 26, and the TfR1-VL comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; and

[0143] The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein:

[0144] The N3pGlu Aβ-VH contains the amino acid sequence of SEQ ID NO: 52, and the N3pGlu Aβ-VL contains the amino acid sequence of SEQ ID NO: 53.

[0145] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0146] The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein:

[0147] The TfR1-VH contains the amino acid sequence of SEQ ID NO: 27, and the TfR1-VL contains the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; or

[0148] The TfR1-VH contains the amino acid sequence of SEQ ID NO: 12, and the TfR1-VL contains the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; or

[0149] The TfR1-VH contains the amino acid sequence of SEQ ID NO: 26, and the TfR1-VL contains the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; and

[0150] The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein:

[0151] The N3pGlu Aβ-VH contains the amino acid sequence of SEQ ID NO: 52, and the N3pGlu Aβ-VL contains the amino acid sequence of SEQ ID NO: 53.

[0152] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0153] The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein:

[0154] The amino acid sequence of TfR1-VH is shown in SEQ ID NO: 27, and the amino acid sequence of TfR1-VL is shown in SEQ ID NO: 13, 14, 15 or 16; or

[0155] The amino acid sequence of TfR1-VH is shown in SEQ ID NO: 12, and the amino acid sequence of TfR1-VL is shown in SEQ ID NO: 13, 14, 15 or 16; or

[0156] The amino acid sequence of TfR1-VH is shown in SEQ ID NO: 26, and the amino acid sequence of TfR1-VL is shown in SEQ ID NO: 13, 14, 15 or 16; and

[0157] The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein:

[0158] The amino acid sequence of N3pGlu Aβ-VH is shown in SEQ ID NO: 52, and the amino acid sequence of N3pGlu Aβ-VL is shown in SEQ ID NO: 53.

[0159] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0160] The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein:

[0161] The TfR1-VH contains the amino acid sequence of SEQ ID NO: 27, and the TfR1-VL contains the amino acid sequence of SEQ ID NO: 13; and

[0162] The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein:

[0163] The N3pGlu Aβ-VH contains the amino acid sequence of SEQ ID NO: 52, and the N3pGlu Aβ-VL contains the amino acid sequence of SEQ ID NO: 53.

[0164] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0165] The first antigen-binding domain that specifically binds to TfR1 and the second antigen-binding domain that specifically binds to N3pGlu Aβ may be the same or different, and each is independently selected from Fab, Fab′, F(ab′)2, Fv, scFv and dsFv.

[0166] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein:

[0167] Both the first antigen-binding domain that specifically binds to TfR1 and the second antigen-binding domain that specifically binds to N3pGlu Aβ are Fab domains.

[0168] In some embodiments, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, comprises a heavy chain constant region CH1 and a light chain constant region CL, wherein the heavy chain constant region is human IgG1, IgG2, IgG3, IgG4 or variants thereof, and the light chain constant region is human κ chain, λ chain or variants thereof. In some embodiments, the heavy chain constant region is human IgG1 heavy chain constant region or variants thereof, and the light chain constant region is human κ light chain constant region or variants thereof. In some embodiments, the heavy chain constant region is human IgG1 heavy chain constant region, and the light chain constant region is human κ light chain constant region. In some embodiments, the heavy chain constant region CH1 comprises SEQ ID NO: 56, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the light chain constant region CL comprises SEQ ID NO: 18, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith. In some embodiments, the amino acid sequence of the heavy chain constant region CH1 is as shown in SEQ ID NO: 56, and the amino acid sequence of the light chain constant region CL is as shown in SEQ ID NO: 18.

[0169] In some embodiments, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding embodiments includes an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other.

[0170] In some embodiments, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, wherein the Fc region is derived from the Fc region of human IgG1, IgG2, IgG3, or IgG4.

[0171] In some embodiments, such as the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, wherein the Fc region is an Fc region derived from human IgG1; wherein Fc1 contains amino acid mutations of L351I and D356K, and Fc2 contains amino acid mutations of L351I and K439E, numbered according to the EU index.

[0172] In some embodiments, the bispecific antibody that specifically binds TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, wherein Fc1 comprises SEQ ID NO: 61, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and Fc2 comprises SEQ ID NO: 62, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith.

[0173] In some embodiments, a bispecific antibody that specifically binds TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, wherein the amino acid sequence of Fc1 is shown as SEQ ID NO: 61, and the amino acid sequence of Fc2 is shown as SEQ ID NO: 62.

[0174] In some embodiments, the bispecific antibody that specifically binds TfR1 and N3pGlu Aβ as described in any of the preceding embodiments comprises a first half-antibody and a second half-antibody, the first half-antibody comprising a Fab and Fc1 region for binding TfR1, and the second half-antibody comprising a Fab and Fc2 region for binding N3pGlu Aβ; and the Fc1 and Fc2 are associated with each other, and optionally, the Fc1 and Fc2 are interchangeable.

[0175] In some embodiments, the bispecific antibody that specifically binds TfR1 and N3pGlu Aβ as described in any of the preceding embodiments comprises a first hapten and a second hapten, the first hapten comprising a Fab and Fc1 region for binding TfR1 from the N-terminus to the C-terminus, and the second hapten comprising a Fab and Fc2 region for binding N3pGlu Aβ from the N-terminus to the C-terminus; and the Fc1 and Fc2 are associated with each other, and optionally, the Fc1 and Fc2 are interchangeable.

[0176] In some implementations, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, comprises a first chain, a second chain, a third chain, and a fourth chain, specifically as follows from the N-terminus to the C-terminus:

[0177] The first chain sequentially comprises TfR1-VH, CH1 and Fc1, wherein the C-terminus of TfR1-VH and the N-terminus of CH1 are operably connected, and the C-terminus of CH1 and the N-terminus of Fc1 are operably connected.

[0178] The second chain comprises TfR1-VL and CL in sequence, wherein the C-terminus of TfR1-VL and the N-terminus of CL are operatively connected;

[0179] The third chain sequentially comprises N3pGlu Aβ-VH, CH1 and Fc2, wherein the C-terminus of N3pGlu Aβ-VH and the N-terminus of CH1 are operatively connected, and the C-terminus of CH1 and the N-terminus of Fc2 are operatively connected.

[0180] The fourth chain comprises N3pGlu Aβ-VL and CL in sequence, wherein the C-terminus of N3pGlu Aβ-VL and the N-terminus of CL are operatively connected;

[0181] Optionally, Fc1 and Fc2 are interchanged.

[0182] In some implementations, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, comprises a first chain, a second chain, a third chain, and a fourth chain, specifically as follows from the N-terminus to the C-terminus:

[0183] The first chain sequentially includes TfR1-VH, CH1 and Fc1, wherein the C-terminus of TfR1-VH is directly fused to the N-terminus of CH1, and the C-terminus of CH1 is fused to the N-terminus of Fc1 through a connector;

[0184] The second chain contains TfR1-VL and CL in sequence, wherein the C-terminus of TfR1-VL is directly fused to the N-terminus of CL;

[0185] The third chain sequentially comprises N3pGlu Aβ-VH, CH1, and Fc2, wherein the C-terminus of N3pGlu Aβ-VH is directly fused to the N-terminus of CH1, and the C-terminus of CH1 is fused to the N-terminus of Fc2 through a linker;

[0186] The fourth chain comprises N3pGlu Aβ-VL and CL in sequence, wherein the C-terminus of N3pGlu Aβ-VL is directly fused to the N-terminus of CL;

[0187] Optionally, Fc1 and Fc2 are interchanged.

[0188] In some embodiments, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, comprises:

[0189] One first chain as shown in equation (a), one second chain as shown in equation (b), one third chain as shown in equation (c), and one fourth chain as shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0190] (a)[TfR1-VH]-[CH1]-[connector 2]-[connector 1]-[connector 3]-[Fc1];

[0191] (b)[TfR1-VL]-[CL];

[0192] (c)[N3pGlu Aβ-VH]-[CH1]-[linker 4]-[Fc2];

[0193] (d)[N3pGlu Aβ-VL]-[CL];

[0194] The linker 1, linker 2, linker 3 and / or linker 4 may be the same or different peptide linkers, or the linker 1, linker 2, linker 3 and / or linker 4 may not exist;

[0195] The TfR1-VH and TfR1-VL are as defined in any of the preceding items;

[0196] The N3pGlu Aβ-VH and N3pGlu Aβ-VL are as defined in any of the preceding items;

[0197] CH1 and CL are as defined in any of the preceding items;

[0198] Fc1 and Fc2 are as defined in any of the preceding items, and optionally, Fc1 and Fc2 are interchanged;

[0199] The first and second chains bind to each other, the third and fourth chains bind to each other, and the first and third chains bind to each other to form a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ.

[0200] In some embodiments, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, wherein the linker is a peptide linker. In some embodiments, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, wherein linker 1, linker 2, linker 3, and linker 4 are peptide linkers. In this disclosure, linker 1, linker 2, linker 3, and linker 4 are used only to distinguish linker positions and are not limiting of linker sequences. In some embodiments, the peptide linker may be a flexible peptide containing 1-50 or 1-20 amino acid residues. In some embodiments, each of the peptide linkers independently has L1-(GGGGS). n The structure is -L2, wherein L1 is a bond, A, G, GS, GGG, GGS, GGGS (SEQ ID NO: 72) or GGGG (SEQ ID NO: 73), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is a bond, G, GG, GGG or GGGG, and the peptide linker is not a bond. In some embodiments, the peptide linker is 2-20 amino acid residues in length. In some embodiments, the peptide linker is as follows: (GS) a (GGS) b (GGGS) c (GGGGS) d (GGGGG) e a, b, c, d, and e are independent integers greater than or equal to 0; or the peptide linker is selected from: (EAAAK)3 (SEQ ID NO: 74), (EAAAR)3 (SEQ ID NO: 75), (EGGGK)3 (SEQ ID NO: 76), (EGGGR)3 (SEQ ID NO: 77), (DAAAR)3 (SEQ ID NO: 78), (DAAAK)3 (SEQ ID NO: 79), (DGGGR)3 (SEQ ID NO: 80) or (DGGGK)3 (SEQ ID NO: 81), SS, GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 82), EPKSS (SEQ ID NO: 83), RTVAA (SEQ ID NO: 84), ASEPKSS (SEQ ID NO: 85); or the peptide linker is (G x S) yWhere x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS, GGGGS (SEQ ID NO: 86), GGGGSGGGGS (SEQ ID NO: 87), GGGGSGGGGSGGGGS (SEQ ID NO: 88), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 63); or the peptide linker is GGGGG (SEQ ID NO: 89). In some embodiments, the peptide linker is EPKSC (SEQ ID NO: 58). In some embodiments, the peptide linker is DKTHTCPPCP (SEQ ID NO: 59). In some embodiments, the peptide linker is EPKSCDKTHTCPPCP (SEQ ID NO: 57). In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 57, 58, 59, or 63. In some embodiments, the amino acid sequence of the peptide linker is as shown in SEQ ID NO: 57, 58, 59, or 63.

[0201] In some embodiments, a bispecific antibody that specifically binds TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, wherein linker 1 comprises the amino acid sequence of SEQ ID NO: 63, linker 2 comprises the amino acid sequence of SEQ ID NO: 58, linker 3 comprises the amino acid sequence of SEQ ID NO: 59, and linker 4 comprises the amino acid sequence of SEQ ID NO: 57.

[0202] In some embodiments, a bispecific antibody that specifically binds TfR1 and N3pGlu Aβ as described in any of the preceding embodiments is wherein the amino acid sequence of linker 1 is as shown in SEQ ID NO: 63, the amino acid sequence of linker 2 is as shown in SEQ ID NO: 58, the amino acid sequence of linker 3 is as shown in SEQ ID NO: 59, and the amino acid sequence of linker 4 is as shown in SEQ ID NO: 57.

[0203] In some embodiments, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, comprises:

[0204] A first strand comprising SEQ ID NO: 66, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; a second strand comprising SEQ ID NO: 20, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; a third strand comprising SEQ ID NO: 67, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and a third strand comprising SEQ ID NO: 67. NO: 55, or the fourth strand having an amino acid sequence that is at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0205] In some embodiments, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, comprises:

[0206] One first strand containing the amino acid sequence of SEQ ID NO: 66, one second strand containing the amino acid sequence of SEQ ID NO: 20, one third strand containing the amino acid sequence of SEQ ID NO: 67, and one fourth strand containing the amino acid sequence of SEQ ID NO: 55;

[0207] The first and second chains bind to each other, the third and fourth chains bind to each other, and the first and third chains bind to each other to form a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ.

[0208] In some embodiments, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as described in any of the preceding embodiments, comprises:

[0209] One first chain with an amino acid sequence as shown in SEQ ID NO: 66, one second chain with an amino acid sequence as shown in SEQ ID NO: 20, one third chain with an amino acid sequence as shown in SEQ ID NO: 67, and one fourth chain with an amino acid sequence as shown in SEQ ID NO: 55;

[0210] The first and second chains bind to each other, the third and fourth chains bind to each other, and the first and third chains bind to each other to form a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ.

[0211] Unless otherwise stated, the terms "first," "second," "third," and "fourth" in this disclosure are general identifiers only and should not be construed as identifying specific or particular portions of the antigen-binding molecules provided in this disclosure. The terms "first," "second," "third," and "fourth" may be arbitrarily reversed in any implementation of this disclosure.

[0212] In this disclosure, the first antigen-binding domain and the second antigen-binding domain are used only to distinguish the positions of the binding domains and are not intended to limit specific sequences. In some embodiments, such as the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims, the first antigen-binding domain and the second antigen-binding domain are interchangeable.

[0213] In this disclosure, the terms "first hapten" and "second hapten" are used only to distinguish the position of the hapten and do not limit specific sequences. In some embodiments, such as the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims, the first hapten and the second hapten are interchangeable.

[0214] In this disclosure, the first subunit Fc1 and the second subunit Fc2 are used only to distinguish the position of Fc and do not limit the specific sequence. In some embodiments, such as the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims, wherein Fc1 and Fc2 are interchangeable.

[0215] In this disclosure, the terms "first chain," "second chain," "third chain," and "fourth chain" are used only to distinguish the positions of the chains and do not limit specific sequences. In some embodiments, such as the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims, the first chain, second chain, third chain, and fourth chain are interchangeable.

[0216] In some implementations, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ provided in this disclosure has a higher exposure to the hTfR1 transgenic mouse brain than positive antibodies (e.g., Trotinemab).

[0217] In some implementations, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ provided in this disclosure has a higher distribution in the brain tissue of hTfR1 transgenic mice than positive antibodies (e.g., Trotinemab).

[0218] In some implementations, the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ provided in this disclosure has a longer serum half-life in cynomolgus monkeys than positive antibodies (e.g., Trotinemab).

[0219] On the other hand, this disclosure provides a conjugate comprising a payload and an antigen-binding molecule specifically binding to TfR1 as described in any of the preceding claims, or a bispecific antibody specifically binding to TfR1 and N3pGlu Aβ as described in any of the preceding claims. In some embodiments, the payload of the conjugate as described above is selected from oligonucleotides, cytotoxic drugs, antitumor agents, peptides, immunomodulators, bioresponse modifiers, lectins, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof. In some embodiments, the payload of the conjugate as described in any of the preceding claims is an oligonucleotide. In some embodiments, the conjugate as described in any of the preceding claims is an antibody oligonucleotide conjugate (AOC).

[0220] On the other hand, this disclosure provides an antigen-binding molecule that specifically binds to TfR1, which competes with an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding claims, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims, or a conjugate as described in any of the preceding claims, for binding to human TfR1.

[0221] On the other hand, this disclosure provides an antigen-binding molecule that specifically binds to TfR1, which, together with an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding claims, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims, or a conjugate as described in any of the preceding claims, binds to the same human TfR1 antigenic epitope.

[0222] On the other hand, this disclosure provides a pharmaceutical composition comprising an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding claims, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims, or a conjugate as described in any of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0223] In some embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01-99.99% of an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding embodiments, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, or a conjugate as described in any of the preceding embodiments. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding embodiments, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, or a conjugate as described in any of the preceding embodiments. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding embodiments, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, or a conjugate as described in any of the preceding embodiments. In some embodiments, the pharmaceutical composition contains 1%-99% of an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding embodiments, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, or a conjugate as described in any of the preceding embodiments. In some embodiments, the pharmaceutical composition contains 2%-98% of an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding embodiments, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding embodiments, or a conjugate as described in any of the preceding embodiments.

[0224] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable carriers, diluents, or excipients based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable carriers, diluents, or excipients.

[0225] On the other hand, this disclosure provides a nucleic acid that encodes an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding claims, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims.

[0226] On the other hand, this disclosure provides a carrier containing the nucleic acid as described above.

[0227] On the other hand, this disclosure provides a host cell containing the nucleic acid as described above, or the vector as described above.

[0228] On the other hand, this disclosure provides a method for producing an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding claims, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims, the method comprising culturing host cells as described in the preceding claims in a culture medium to form and accumulate an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding claims, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims, and recovering the antigen-binding molecule from the culture.

[0229] On the other hand, this disclosure provides the use of an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding claims, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims, or a conjugate as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims, in the preparation of a medicament for the prevention or treatment of a disease or condition.

[0230] On the other hand, this disclosure provides a method for preventing or treating a disease or condition, the method comprising administering to a subject a preventive or therapeutically effective amount of an antigen-binding molecule that specifically binds to TfR1 as described in any of the preceding claims, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in any of the preceding claims, or a conjugate as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims.

[0231] On the other hand, this disclosure provides an antigen-binding molecule that specifically binds to TfR1 as described in the preceding claim, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as described in the preceding claim, or a conjugate as described in the preceding claim, or a pharmaceutical composition as described in the preceding claim. In some embodiments, the pharmaceutical remedy is used to prevent or treat a disease or condition.

[0232] In some implementations, the disease or condition described in the preceding one is associated with TfR1 and / or N3pGlu Aβ.

[0233] In some implementations, the disease or condition described in the preceding one is a neurological disease.

[0234] In some implementations, the disease or condition described in the preceding one is a central nervous system disease.

[0235] In some implementations, the disease or condition described in the preceding one is a neurodegenerative disease.

[0236] In some implementations, the disease or condition described in the preceding one is Alzheimer's disease. Attached Figure Description

[0237] Figure 1A: Schematic diagram of the structure of the maternal monoclonal antibody that specifically binds to TfR1.

[0238] Figure 1B: Schematic diagram of the structure of the parent monoclonal antibody that specifically binds to N3pGlu Aβ.

[0239] Figure 2: Schematic diagram of the structure of a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ. Detailed Implementation

[0240] the term

[0241] To facilitate understanding of this disclosure, certain technical and scientific terms are described below. Unless otherwise expressly defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0242] The singular forms “a,” “an,” and “the” used in the specification and claims include plural references unless the context clearly indicates otherwise.

[0243] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0244] The term "and / or" implies both "and" and "or". For example, the phrase "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0245] The terms “TfR1,” “CD71,” and “TFRC” are used interchangeably to include variants, isotypes, homologues, orthologs, and paralogs of TfR1. Human TfR1 has the amino acid sequence Uniprot: P02786. Although specific database accession numbers are given, those skilled in the art will understand that TfR1 as referred to herein also encompasses the corresponding sequences reported in other databases or literature.

[0246] The terms "Aβ", "Abeta", "amyloid β", and "β-amyloid" are used interchangeably to refer to the fragments generated from amyloid precursor protein (APP) after cleavage by β-secretase 1 (BACE1), as well as their modified fragments and any functional equivalents, including but not limited to N3pGlu Aβ, Aβ1-40, and Aβ1-42. Aβ monomers can combine to form oligomers, protofibrils, fibrils, or plaques. The term "Aβ" includes variants, isotypes, homologues, orthologs, and paralogs of Aβ.

[0247] The term "N3pGlu Aβ" refers to an Aβ peptide in which the glutamate at the N-terminus 3rd position is pyroglutamicized, and its written forms include, but are not limited to, N3pE Aβ, 3pE Aβ, 3pGlu Aβ, pGlu3 Aβ, and pE3 Aβ. N3pGlu Aβ includes truncated forms of the Aβ peptide, including, but not limited to, N3pGlu-42 Aβ and N3pGlu-40 Aβ. Exemplarily, the N3pGlu Aβ used in this disclosure lacks the first two amino acid residues (aspartic acid and alanine) at the N-terminus of the Aβ peptide and undergoes pyroglutamicization at the N-terminus 3rd position of the Aβ peptide. This N3pGlu Aβ is more prone to aggregation and is present in Alzheimer's amyloid deposits.

[0248] The term "central nervous system (CNS)" refers to a complex of neural tissues that control bodily functions and includes the brain and spinal cord.

[0249] The term "blood-brain barrier" (BBB) ​​refers to the physiological barrier between the peripheral circulation and the brain and spinal cord. It consists of tight junctions of the brain capillary endothelial plasma membranes, forming an extremely tight barrier that restricts the transport of molecules into the brain, even very small molecules such as urea (60 Da). The blood-brain barrier within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina form a continuous capillary barrier within the central nervous system, collectively known as the blood-brain barrier. The blood-brain barrier also includes the blood-cerebrospinal fluid barrier (choroid plexus), which is composed of ependymal cells rather than capillary endothelial cells.

[0250] The term "blood-brain barrier receptor (BBBR)" refers to a class of extracellular membrane-linked receptor proteins expressed on brain endothelial cells that can transport molecules across the blood-brain barrier or be used to transport exogenously administered molecules. Exemplary BBBRs include, but are not limited to, TfR1.

[0251] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0252] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0253] The term "amino acid mutation" includes amino acid substitution (also known as amino acid replacement), deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced or absent binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide chain. A specific amino acid mutation can be an amino acid substitution. In some embodiments, an amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid that has a different structure and / or chemical properties. Amino acid substitution includes substitution by non-naturally occurring amino acids or by derivatives of 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering that alter amino acid side chain groups, such as chemical modification, are also expected to be available. Various names may be used herein to refer to the same amino acid mutation. In this document, the amino acid residue at a specific site can be represented by the format "position + amino acid residue". For example, 356K indicates that the amino acid residue at position 356 is K. D356K indicates that the amino acid residue at position 356 has mutated from D to K. It should be understood that when the amino acid sequence is defined by position + residue in the claims, the amino acid before the mutation at that site does not constitute a limitation on the technical solution. In this document, "the Fc region contains the amino acid mutations of 356K and 349S" means that the amino acid mutation in the Fc region includes a mutation at position 356 to lysine (K) and a mutation at position 349 to serine (S).

[0254] The terms “polypeptide” and “protein” are used interchangeably in this document and refer to polymers of amino acid residues.

[0255] The term "oligonucleotide" refers to oligonucleotide compounds that are typically less than about 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNA interference (RNAi) oligonucleotides (e.g., antisense oligonucleotides (ASO), siRNAs, shRNAs, dsRNAs, etc.), microRNAs (miRNAs), spacers, mixers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, and guide RNAs (e.g., Cas9 guide RNAs). Oligonucleotides can be single-stranded or double-stranded. Oligonucleotides can be sense or antisense.

[0256] The term "antigen-binding molecule" is used in the broadest sense to encompass molecules that specifically bind antigens, including but not limited to antibodies, oligonucleotides, other peptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of the two, as well as any molecule containing the aforementioned antibodies, oligonucleotides, peptides, or antibody fusion proteins, provided they exhibit the desired antigen-binding activity. Exemplarily, the antigen-binding molecules described herein are monoclonal antibodies, monospecific antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies).

[0257] The term “antibody” is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, antibody fragments, and antigen-binding fragments (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

[0258] The term "antigen-binding fragment" encompasses full-length antibodies, Fab, modified Fab, Fab', Fab'-SH, modified Fab', F(ab')2, Fv, dsFv, Fab-Fv, Fab-dsFv, Fd, single-domain antibodies (sdAb, e.g., VH, VL, or VHH), single-chain Fab (scFab), single-chain antibodies (e.g., scFv, sc(Fv)2), biantibodies, linear antibodies, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabetic, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above. Methods for generating and preparing these antigen-binding fragments are well known in the art.

[0259] The term "antibody fragment" refers to a molecule that is distinct from the intact antibody but contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, dsFv, Fab, Fab′, Fab′-SH, F(ab′)2, Fd, single-domain antibodies (sdAb, such as VH, VL, or VHH), single-chain Fab (scFab), biantibodies, linear antibodies, single-chain antibodies (such as scFv, sc(Fv)2); and multispecific antibodies formed from antibody fragments.

[0260] The term "natural antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by a heavy chain constant region. The natural IgG heavy chain constant region typically contains three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0261] The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably in this document, referring to antibodies with a structure substantially similar to that of natural antibodies or with a heavy chain containing the Fc region as defined herein. The light chain of a natural intact antibody includes a variable region (VL) and a constant region (CL), with VL located at the amino terminus of the light chain. The constant region includes the κ and λ chains. The heavy chain includes a variable region (VH) and constant regions (CH1, CH2, and CH3), with VH located at the amino terminus of the heavy chain and the constant region located at the carboxyl terminus. CH3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0262] The term "multispecific antibody" refers to an antibody (including an antibody or its antigen-binding fragment) capable of specifically binding to multiple different antigens or at least two different antigenic epitopes of the same antigen. The term "multispecific antibody" is used in the broadest sense in this disclosure and specifically covers antibodies exhibiting multi-epitope specificity. Multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), where VH and VL together constitute an antigen-binding domain (where VH / VL exhibits multi-epitope specificity), antibodies having two or more VH and VL domains (each VH / VL unit binding to a different epitope), antibodies having two or more single variable domains (each single variable domain binding to a different epitope), full-length antibodies, and antibodies comprising one or more antibody fragments, as well as antibodies comprising antibody fragments covalently or non-covalently linked. Multispecific antibodies can be bispecific antibodies, trispecific antibodies, biantibodies, or similar molecules.

[0263] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. Suitable Fc regions for the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may also vary, for example, by omitting the C-terminal lysine (residue 447 according to the EU numbering system) or omitting both the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system). Unless otherwise stated, the numbering rule for the Fc region is the EU numbering system, also known as the EU index. Although a numbering system (such as EU) is used to define amino acid residues in a particular embodiment, corresponding technical solutions using other numbering systems are considered equivalent technical solutions.

[0264] The Fc region can be appropriately obtained by partially digesting IgG monoclonal antibodies with proteolytic enzymes such as pepsin, followed by eluting the components adsorbed on the protein A or protein G column. As the proteolytic enzyme, any enzyme capable of restrictively digesting full-length antibodies to produce Fab and F(ab')2 by appropriately setting the enzyme reaction conditions such as pH is acceptable; there is no particular limitation, and examples include pepsin and papain.

[0265] In this disclosure, the term "Fc region" or "Fc domain" refers to an antibody region that contains at least a CH2 domain and a CH3 domain. In this disclosure, the term "CH2 region" or "CH2 domain" is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering system (according to the IMGT website). However, the CH2 region can also be any other antibody isotype as described in this disclosure.

[0266] In this disclosure, the terms “CH3 region,” “CH3 domain,” or “CH3 structural domain” are intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system (according to the IMGT website). However, the CH3 region can also be any other antibody isotype as described in this disclosure.

[0267] The term "maternal antibody" refers to an antibody that serves as the source of one or more antibody fragments. A maternal antibody may contain a natural or wild-type sequence. A maternal antibody may contain an amino acid sequence in which one or more amino acid residues are replaced by one or more cysteine ​​residues. Compared to other natural, wild-type, or modified forms of antibodies, a maternal antibody may have pre-existing amino acid sequence modifications (such as additions, deletions, and / or substitutions). A maternal antibody may target a specific antigen, such as a biologically important peptide. In some specific embodiments, the maternal antibody is a maternal monoclonal antibody, which may target non-peptide antigens.

[0268] The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In this disclosure, the term "operably linked" means linking two or more amino acid segments to produce a functional polypeptide. For example, in the context of antigen-binding molecules of this disclosure, individual antigen-binding domains may be directly linked or linked via peptide linkers. In the context of nucleic acids encoding fusion proteins, such as polypeptide chains of antigen-binding molecules of this disclosure, "operably linked" means linking two nucleic acids such that the amino acid sequences encoded by the two nucleic acids remain within the frame.

[0269] The term "association" in this disclosure refers to a functional relationship between two or more chains. Specifically, the term "association" means that two or more polypeptides associate with each other, for example, through non-covalent association via molecular interactions or through covalent association via one or more disulfide bridges or chemical crosslinks, thereby generating a functional antigen-binding molecule (e.g., a bispecific antibody), wherein a first antigen-binding domain and a second antigen-binding domain can bind their respective targets. Examples of association that may exist in the antigen-binding molecules of this disclosure include, but are not limited to, association between Fc regions in the Fc domain, association between the VH and VL regions in the Fab or Fv domain, and association between CH1 and CL in the Fab domain.

[0270] In the context of antigen-binding molecules (e.g., bispecific antibodies), the term "bivalent" refers to an antigen-binding molecule that has two antigen-binding domains.

[0271] In the context of antigen-binding molecules (e.g., bispecific antibodies), the term "trivalent" refers to an antigen-binding molecule that has three antigen-binding domains.

[0272] The term "half-antibody" refers to a molecule that contains at least one antigen-binding domain and can associate with another molecule containing an antigen-binding domain via, for example, a disulfide bridge or molecular interactions (e.g., club-and-mortar interactions between Fc heterodimers, hydrogen bonds, electrostatic interactions, or salt bridges). A half-antibody may consist of one or more polypeptide chains (e.g., two polypeptide chains of Fab). In some embodiments, the half-antibody further comprises an Fc region.

[0273] Therefore, an antigen-binding molecule may contain at least one, more typically one, two, or even more than two half antibodies, and a half antibody may contain one or more antigen-binding domains.

[0274] In some embodiments, the first hapten will associate with the second hapten, for example, through heterodimerization. In other embodiments, the first hapten will be covalently linked to the second hapten, for example, through disulfide bridging, molecular interactions (e.g., club-and-mortar interactions between Fc heterodimers, hydrogen bonds, electrostatic interactions, or salt bridges), or chemical crosslinking. In one specific embodiment, the first hapten will associate with the second hapten through covalent attachment and non-covalent interactions, such as disulfide bridging and / or electrostatic interactions.

[0275] The term "half-antibody" is intended for descriptive purposes only and does not indicate a specific configuration or method of manufacture. The descriptions of half-antibodies as "first" half-antibody, "second" half-antibody, "left" half-antibody, "right" half-antibody, etc., are merely for convenience and descriptive purposes.

[0276] The term "C-terminus," also known as, but not limited to, carboxyl-terminus, C-terminus, C-tail, C-terminus, or COOH-terminus, is the terminal end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH). When a protein is translated from messenger RNA, it is produced from the N-terminus to the C-terminus. The convention for writing peptide sequences is to place the C-terminus on the right and write the sequence from the N-terminus to the C-terminus. In some embodiments, the C-terminus of a polypeptide includes the last amino acid residue of the polypeptide, which contributes its amino group to form a peptide bond with the carboxyl group of its adjacent amino acid residue.

[0277] The term "N-terminus," also known as, but not limited to, amino-terminus, amine-terminus, N-terminus, N-tail, N-terminus, or NH2-terminus, is the start of an amino acid chain (protein or polypeptide) beginning with a free amino group (-NH2). Normally, the amino group bonds to another carboxyl group in the protein to form a chain, but since only one of the two ends of a protein is chained, the free amino group refers to the N-terminus. As mentioned above, by convention, peptide sequences in LTR language are written from N-terminus to C-terminus, from left to right. This associates the translation direction with the text direction (because when a protein is translated from messenger RNA, it is produced from N-terminus to C-terminus—an amino acid is added to the carbonyl terminus). In some embodiments, the N-terminus of a polypeptide contains the first amino acid of the polypeptide, which contributes its carboxyl group to form a peptide bond with the amino group of its adjacent amino acid residue.

[0278] The term "linker" refers to a linking unit that connects two polypeptide fragments. In this disclosure, linkers appearing in the same structural formula may be the same or different. A linker may be a "peptide linker" containing one or more amino acids, typically about 1-50, about 1-30, 2-24, or 3-15 amino acids. Linkers used in this disclosure may be the same or different.

[0279] When "-" appears in a structural formula, it indicates that the units on both sides are directly connected by covalent bonds.

[0280] The term "variable region" or "variable domain" in an antibody refers to the domain in the antibody heavy or light chain involved in antibody binding to the antigen. In this paper, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0281] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. The correspondence between various numbering systems is well known to those skilled in the art and is exemplified as shown in Table A below.

[0282] Table A. Relationships between CDR numbering systems

[0283] Unless otherwise stated, the variable areas and CDRs in this disclosure embodiment are subject to the "Kabat" numbering rule.

[0284] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0285] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0286] The terms "human antibody," "humanized antibody," "fully human antibody," and "completely human antibody" are used interchangeably, referring to antibodies whose variable and constant regions are human sequences. This term encompasses antibodies derived from human genes but with sequence alterations, such as reduced potential immunogenicity, increased affinity, or the elimination of cysteine ​​or glycosylation sites that might cause undesirable folding. This term also covers antibodies recombined in non-human cells (which may confer glycosylations not characteristic of human cells). The term also includes antibodies produced in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The term "human antibody" explicitly excludes humanized antibodies.

[0287] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art, including those described herein.

[0288] As used herein, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. For example, it can be measured using a biosensing system such as a system for measuring surface plasmon resonance (e.g., Biacore), or by measuring affinity in solution using solution equilibrium titration (SET).

[0289] The term "surface plasmon resonance" refers to the optical phenomenon of analyzing real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0290] The term "effector function" refers to biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region with amino acid sequence mutations) and that vary across antibody isotypes. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0291] The term "monoclonal antibody" refers to a group of antibodies that are essentially homogeneous, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibodies typically comprise a variety of different antibodies with varying amino acid sequences in their variable domains, and they generally target different epitopes specifically. "Monoclonal" should not be interpreted as requiring the production of the antibody through any particular method.

[0292] The term "bispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment, such as a single-chain antibody) capable of specifically binding to two different antigens or at least two different antigenic epitopes of the same antigen. Various structures of bispecific antibodies have been disclosed in the prior art. Based on the integrity of the IgG molecule, they can be classified into IgG-like bispecific antibodies and antibody fragment-based bispecific antibodies. Based on the number of antigen-binding regions, they can be classified into bivalent, trivalent, tetravalent, or more bispecific antibodies. Based on structural symmetry, they can be classified into symmetrical and asymmetrical bispecific antibodies. Among these, bispecific antibodies based on antibody fragments, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by combining two or more Fab fragments into one molecule. They exhibit lower immunogenicity, smaller molecular weight, and higher tumor tissue penetration. IgG-like bispecific antibodies (e.g., those with an Fc fragment) have a relatively larger molecular weight. The Fc fragment facilitates antibody purification and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody's serum half-life.

[0293] The term "antigen" refers to a molecule or molecular part that can be selectively bound by antigen-binding proteins, including, for example, antibodies. An antigen may have one or more epitopes that can interact with different antigen-binding proteins, such as antibodies.

[0294] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain non-continuous amino acids (conformal epitopes), such as those spatially close due to antigen folding. The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen, and cross-blocking.

[0295] The terms “antibody-dependent cell cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to mechanisms that induce cell death that rely on the interaction between antibody-coated target cells and lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies described herein can be assessed in vitro using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) from lysed cells.

[0296] The term "antibody-dependent phagocytosis (ADCP)" refers to the mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells).

[0297] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q. C1q then activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0298] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs).

[0299] Nucleic acid encoding a polypeptide refers to one or more nucleic acid molecules encoding a polypeptide, including one or more such nucleic acid molecules in a single vector or separate vectors, and one or more such nucleic acid molecules present at one or more locations in the host cell. Unless otherwise stated, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences.

[0300] The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0301] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions when optimally aligned; gaps may be introduced, where necessary, to obtain the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0302] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which an additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming host cells and containing a nucleic acid sequence that directs and / or controls (alongside the host cell) the expression of one or more heterologous coding regions operatively linked to it. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, influence RNA splicing of coding regions operatively linked to them.

[0303] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. Mutant progeny are included herein, which have the same function or biological activity as cells screened or selected in the initial transformed cells. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. The following fungi are listed: *C. polymorpha*, *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium sp.*, *Fusarium gramineum*, *Fusarium venenatum*, *Physcomitrella patens*, *Neurospora crassa*, and *Yarrowia lipolytica*.

[0304] The terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Therefore, the terms “transformation” and “transformed cell” include primary subject cells and cultures derived from them, regardless of the number of passages. It should also be understood that due to intentional or unintentional mutations, not all progeny will have exactly the same DNA contents. This includes mutant progeny that have the same function or biological activity as the original transformed cells.

[0305] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0306] The term "pharmaceutical composition" means a mixture containing one or more antibodies, immunoconjugates or antibody-drug conjugates described herein, and other chemical components, such as physiological / pharmaceutical carriers, diluents or excipients.

[0307] The term "pharmaceutically acceptable carrier, diluent, or excipient" refers to a component in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers, diluents, or excipients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0308] The terms "subject" or "individual" include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably herein. In some embodiments, the individual or subject is a human being.

[0309] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0310] The term "sample" refers to a collection (such as fluid, cells, or tissue) separated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues or organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as culture media (including conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0311] "Treatment" and "treatment" (and their grammatical variations) refer to a clinical intervention on the individual being treated, and can be implemented for prevention or during a clinicopathological process. The expected effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving the prognosis.

[0312] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or mitigate damage caused by or associated with a disease state (e.g., lung disease). In some implementations, an effective dose is a therapeutically effective dose or a preventatively effective dose. A "therapeuticly effective dose" is an amount sufficient to treat a disease state or symptom, especially a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A "preventatively effective dose" is an amount that, when given to a subject, will have a predetermined preventative effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. Complete treatment or prevention may not occur after a single dose, but may occur after a series of doses. Therefore, a therapeutically or preventatively effective dose may be administered in a single or multiple-dose manner. "Therapeutic effective dose" and "preventive effective dose" can vary depending on a number of factors, such as an individual's disease state, age, sex, and weight, as well as the ability of the treatment or combination of treatments to elicit the desired response in the individual. Exemplary indicators of an effective treatment or combination of treatments include, for example, improved health status in the patient.

[0313] This disclosure discloses antigen-binding molecules that specifically bind to TfR1.

[0314] This disclosure provides antigen-binding molecules that specifically bind to TfR1 and possess numerous advantageous properties, such as good affinity, therapeutic activity, safety, pharmacokinetic properties, and drugability (e.g., solubility, viscosity, purity, and stability).

[0315] Exemplary antigen-binding molecules that specifically bind to TfR1

[0316] For example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0317] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 3, HCDR2 in SEQ ID NO: 11, and HCDR3 in SEQ ID NO: 36; the amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 9, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 8, wherein:

[0318] GYX1YYX2LDY SEQ ID NO: 36;

[0319] X1 can be S or Y, with S being preferred; X2 can be T or S, with T being preferred.

[0320] For example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0321] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 3, HCDR2 contains the amino acid sequence of SEQ ID NO: 11, and HCDR3 contains the amino acid sequence of SEQ ID NO: 25, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 8.

[0322] For example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0323] The amino acid sequences of the heavy chain variable regions HCDR1 are shown in SEQ ID NO: 3, HCDR2 are shown in SEQ ID NO: 11, and HCDR3 are shown in SEQ ID NO: 25. The amino acid sequences of the light chain variable regions LCDR1 are shown in SEQ ID NO: 9, LCDR2 are shown in SEQ ID NO: 10, and LCDR3 are shown in SEQ ID NO: 8.

[0324] For example, the antigen-binding molecules that specifically bind to TfR1 disclosed herein, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules.

[0325] For example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0326] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 37, HCDR2 contains the amino acid sequence of SEQ ID NO: 38, and HCDR3 contains the amino acid sequence of SEQ ID NO: 39, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 40, LCDR2 contains the amino acid sequence of SEQ ID NO: 41, and LCDR3 contains the amino acid sequence of SEQ ID NO: 8.

[0327] For example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0328] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 37, HCDR2 in SEQ ID NO: 38, and HCDR3 in SEQ ID NO: 39. The amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 40, LCDR2 in SEQ ID NO: 41, and LCDR3 in SEQ ID NO: 8.

[0329] For example, the antigen-binding molecules that specifically bind to TfR1 disclosed herein, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the IMGT numbering rules.

[0330] For example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0331] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 42, HCDR2 contains the amino acid sequence of SEQ ID NO: 43, and HCDR3 contains the amino acid sequence of SEQ ID NO: 25. The light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 8. GYSFTGY SEQ ID NO: 42; NPHQGG SEQ ID NO: 43.

[0332] For example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0333] The amino acid sequences of the heavy chain variable region HCDR1 are shown in SEQ ID NO: 42, HCDR2 in SEQ ID NO: 43, and HCDR3 in SEQ ID NO: 25. The amino acid sequences of the light chain variable region LCDR1 are shown in SEQ ID NO: 9, LCDR2 in SEQ ID NO: 10, and LCDR3 in SEQ ID NO: 8.

[0334] For example, the antigen-binding molecules that specifically bind to TfR1 disclosed herein, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Chothia numbering rules.

[0335] By way of example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein:

[0336] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 13.

[0337] By way of example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein:

[0338] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 13.

[0339] By way of example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain and a light chain, wherein:

[0340] The heavy chain contains the amino acid sequence of SEQ ID NO: 29, and the light chain contains the amino acid sequence of SEQ ID NO: 20.

[0341] By way of example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain and a light chain, wherein:

[0342] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20; or

[0343] The amino acid sequence of the heavy chain is shown as amino acid residues 1 to 447 of SEQ ID NO: 29, and the amino acid sequence of the light chain is shown as amino acid residues 1 to 213 of SEQ ID NO: 20.

[0344] By way of example, the antigen-binding molecule that specifically binds to TfR1 disclosed herein comprises a heavy chain and a light chain, wherein:

[0345] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0346] The antigen-binding molecule provided in this disclosure may also contain an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the above-mentioned amino acid sequence, such as a CDR sequence, a variable region sequence, a heavy chain, or a light chain sequence.

[0347] Antigen-binding molecular structure

[0348] In some implementations, the antigen-binding molecule provided herein is a full-length antibody.

[0349] In some implementations, the antigen-binding molecule provided herein is an antigen-binding fragment.

[0350] In some embodiments, the antigen-binding molecule provided herein is an antibody fragment. In some embodiments, the antigen-binding fragment is a Fab, Fab′, Fab′-SH, or F(ab′)2 fragment, particularly a Fab fragment. “Fab” is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. A “Fab fragment” can be generated by cleavage of an antigen-binding molecule using papain. “Fab′” contains VL, CL, VH, and CH1, and also contains a region between the CH1 and CH2 domains such that interchain disulfide bonds can form between the two heavy chains of two Fab′ fragments to form an F(ab′)2 molecule. “Fab′-SH” is a Fab′ fragment in which the cysteine ​​residues in the constant region have free thiol groups. “F(ab′)2” is a divalent fragment comprising two Fab fragments linked by disulfide bonds in the hinge region.

[0351] In some implementations, the antigen-binding fragment is a biantibody, triantibody, or tetraantibody. A biantibody is an antibody fragment with two antigen-binding sites, containing linked VH and VL domains within the same polypeptide chain (VH-VL). By using a short linker that prevents pairing between two domains on the same chain, these domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The two antigens can be the same or different.

[0352] In some embodiments, the antigen-binding fragment is a single-chain Fab fragment. A “single-chain Fab fragment” or “scFab” is a polypeptide consisting of VH, CH1, VL, CL, and a linker, wherein the antigen-binding domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In some embodiments, the linker is a polypeptide having at least 30 amino acids. In some embodiments, the linker is a polypeptide having between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between CL and CH1. Additionally, these single-chain Fab molecules can be further stabilized by inserting cysteine ​​residues (e.g., at position 44 in the heavy chain variable region and position 100 in the light chain variable region, according to Kabat numbering) to create interchain disulfide bonds.

[0353] In some implementations, the antigen-binding fragment is an Fv fragment consisting of the VH and VL domains of a single arm of the antibody.

[0354] In some embodiments, the antigen-binding fragment is a single-chain variable fragment (scFv). An “scFv” is a fusion protein comprising at least one antigen-binding fragment containing a light chain variable region and at least one antigen-binding fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked by a short, flexible peptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antigen-binding molecule from which it originates. Unless otherwise specified, the scFv may have VL and VH variable regions in any order herein; for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

[0355] In some embodiments, the antigen-binding fragment is dsFv, which is obtained by linking polypeptides in which one amino acid residue in each of the VH and VL is replaced by a cysteine ​​residue via disulfide bonds between the cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on predictions of the three-dimensional structure of the antigen-binding molecule using known methods.

[0356] In some implementations, the antigen-binding fragment is a single-domain antibody (dAb). A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain.

[0357] In some embodiments, the antigen-binding molecules provided herein are chimeric antibodies. In some embodiments, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, the chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from the class or subclass of the parent antibody.

[0358] In some embodiments, the antigen-binding molecule is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable regions, wherein the CDR or a portion thereof is derived from the non-human antibody, and the FR or a portion thereof is derived from the human antibody. Optionally, the humanized antibody may also contain a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody may be replaced with corresponding residues from a non-human antibody (e.g., an antibody providing the CDR sequence).

[0359] Variants of antigen-binding molecules that specifically bind to TfR1

[0360] In some embodiments, amino acid sequence variants of antigen-binding molecules that specifically bind TfR1, as provided herein, are included. For example, improvements in binding affinity and / or other biological properties of the antigen-binding molecule may be desired. Amino acid sequence variants of the antigen-binding molecule can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antigen-binding molecule, or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions, and / or substitutions of residues within the amino acid sequence of the antigen-binding molecule that specifically binds TfR1. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen-binding properties.

[0361] Replace, insert, and delete variants

[0362] In some embodiments, antigen-binding molecule variants with one or more amino acid substitutions are provided. Substitution mutagenesis sites of interest include CDR and FR. Conserved substitutions are shown in Table B under the heading “Preferred Substitutions.” More substantial variations are provided in Table B under the heading “Exemplary Substitutions” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antigen-binding molecule of interest, and the product can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0363] Table B. Substitution of Amino Acids

[0364] Based on common side-chain characteristics, amino acids can be grouped as follows:

[0365] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0366] (2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0367] (3) Acidic: Asp, Glu;

[0368] (4) Alkaline: His, Lys, Arg;

[0369] (5) Residues that affect chain orientation: Gly, Pro;

[0370] (6) Aromatic: Trp, Tyr, Phe.

[0371] Non-conservative replacement would require replacing a member of one of these categories with a member of another category.

[0372] One class of substitution variants involves replacing one or more CDR residues in a parent antigen-binding molecule (e.g., humanized or human antibody). Generally, the resulting variants selected for further research will have alterations (e.g., improvements) to certain biological properties (e.g., increased affinity, decreased affinity, decreased immunogenicity) relative to the parent antigen-binding molecule, and / or will substantially retain some of the biological properties of the parent antigen-binding molecule. An exemplary substitution variant is an affinity-matured antigen-binding molecule, which can be conveniently generated, for example, using phage display-based affinity maturation techniques (such as those described herein). In short, one or more CDR residues are mutated, the variant antigen-binding molecule is displayed on a phage, and it is screened for specific biological activities (e.g., binding affinity). Modifications (e.g., substitutions) can be made to the CDRs, for example, to improve the affinity of the antigen-binding molecule. Such alterations can be made to CDR "hotspots"—residues encoded by codons that undergo high-frequency mutations during somatic maturation—and / or residues that come into contact with antigens, while simultaneously testing the binding affinity of the resulting variants VH or VL. In some embodiments of affinity maturation, diversity is introduced into the selected variant gene for maturation using any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-guided mutagenesis. A secondary library is then created. The library is then screened to identify any antigen-binding molecule variants with the desired affinity. Another method for introducing diversity involves CDR-directed approaches, where several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. In particular, HCDR3 and LCDR3 are frequently targeted.

[0373] In some embodiments, substitution, insertion, or deletion can occur within one or more CDRs, provided that such changes do not materially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided herein) can be made to the CDRs that do not materially reduce binding affinity. Such changes can, for example, be external to the antigen-contacting residues in the CDR. In some embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0374] One method for identifying residues or regions in an antigen-binding molecule that can serve as mutagenic targets is called "alanine scanning mutagenesis." In this method, a residue or target group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine whether the interaction between the antigen-binding molecule and the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Furthermore, the contact points between the antigen-binding molecule and the antigen can be identified by studying the crystal structure of the antigen-antigen-binding molecule complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0375] Amino acid sequence insertions include fusion of the amino and / or carboxyl ends of peptides ranging in length from 1 residue to 100 or more residues, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antigen-binding molecules having an N-terminal methionyl residue. Other insertion variants of antigen-binding molecules include fusions of the N- or C-terminus of an antigen-binding molecule with an enzyme or a peptide that extends the serum half-life of the antigen-binding molecule.

[0376] Recombination method

[0377] Antigen-binding molecules that specifically bind to TfR1 and bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ can be generated using recombinant methods. For these methods, one or more isolated nucleic acids encoding antigen-binding molecules that specifically bind to TfR1 and bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ are provided.

[0378] In some embodiments, this disclosure provides isolated nucleic acids encoding an antigen-binding molecule that specifically binds to TfR1 as described above, and a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ. Such nucleic acids can be derived from independently encoding any of the aforementioned polypeptide chains. In some embodiments, this disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In some embodiments, this disclosure provides host cells comprising such nucleic acids. In some embodiments, a method for preparing a polypeptide or fusion protein is provided, wherein the method includes culturing host cells comprising nucleic acids encoding said polypeptide or fusion protein, as provided above, under conditions suitable for expression, and optionally recovering said antigen-binding molecule that specifically binds to TfR1, and the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, from the host cells (or host cell culture medium).

[0379] To recombinantly generate antigen-binding molecules that specifically bind to TfR1, and bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ, nucleic acids encoding the proteins are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be readily isolated and sequenced using standard procedures, or generated via recombinant methods or obtained through chemical synthesis.

[0380] Suitable host cells for cloning or expressing vectors encoding antigen-binding molecules that specifically bind to TfR1, or bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ, include the prokaryotic or eukaryotic cells described herein. For example, they can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. Following expression, they can be separated from bacterial cell paste in a soluble fraction and can be further purified.

[0381] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding fusion proteins, including fungal and yeast strains. Suitable host cells for expressing fusion proteins can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells; plant cell cultures can also be used as hosts, such as US5959177, US6040498, US6420548, US7125978, and US6417429; and vertebrate cells, such as mammalian cell lines adapted for growth in suspension, can also be used as hosts. Other examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293T cells); young hamster kidney cells (BHK); mouse seltoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NSO, and Sp2 / 0.

[0382] Measurement

[0383] The antigen-binding molecules that specifically bind to TfR1 and the bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ disclosed herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activities using a variety of assays known in the art. In some embodiments, the activities of the antigen-binding molecules that specifically bind to TfR1 and the bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ disclosed herein are tested, for example, by known methods such as ELISA, Western blotting, etc.

[0384] Treatment methods and routes of administration

[0385] Any antigen-binding molecule that specifically binds to TfR1, or any bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, provided in this disclosure, may be used for therapeutic purposes. In some embodiments, the use of the antigen-binding molecule that specifically binds to TfR1, or the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, provided in this disclosure, in the manufacture or preparation of a medicament.

[0386] In some embodiments, a pharmaceutical composition is provided comprising the antigen-binding molecule that specifically binds to TfR1, and a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, for example, for any of the pharmaceutical uses or therapeutic methods described above. In some embodiments, the pharmaceutical composition comprises any of the antigen-binding molecules that specifically bind to TfR1 provided herein, a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0387] The antigen-binding molecules that specifically bind to TfR1 and the bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ disclosed herein can be used alone or in combination with other agents for treatment. For example, the antigen-binding molecules disclosed herein can be co-administered with at least one other therapeutic agent.

[0388] The antigen-binding molecules that specifically bind to TfR1, the bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ (and any other therapeutic agents) disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0389] The antigen-binding molecules that specifically bind to TfR1 and the bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the reagent, the method of administration, the timing of administration, and other factors known to a medical practitioner. The antigen-binding molecules that specifically bind to TfR1 and the bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ may be formulated with or without one or more reagents currently used for the prevention or treatment of the stated condition. The effective amount of such other reagents depends on the amount present in the pharmaceutical composition, the type of condition or treatment, and other factors. These are generally used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at other dosages, and at any route determined empirically / clinically as appropriate.

[0390] For the prevention or treatment of disease, the appropriate dosage of the antigen-binding molecule that specifically binds to TfR1, or the bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ (when used alone or in combination with one or more other additional therapeutic agents), will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and duration of the disease, whether it is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is appropriately administered to the patient either as a single treatment or as part of a series of treatments.

[0391] Products

[0392] In another aspect of this disclosure, an article of manufacture is provided comprising materials that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article of manufacture comprises a container and a label or package insert on or in conjunction with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container contains a composition, alone or in combination with another composition, that is effective in treating, preventing, and / or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper puncturable by a hypodermic needle). At least one active agent in the composition is an antigen-binding molecule that specifically binds to TfR1, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, as disclosed herein. The label or package insert indicates that the use of the composition is for the treatment of the selected condition. Furthermore, the article may comprise: (a) a first container containing a composition comprising the antigen-binding molecule that specifically binds to TfR1, and a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ as disclosed herein; and (b) a second container containing a composition comprising additional cytotoxic agents or other therapeutic agents. The article in this embodiment of the present disclosure may further comprise a packaging insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer solution. From a commercial and user perspective, it may further include other materials as desired, including additional buffers, diluents, filters, needles, and syringes.

[0393] Example

[0394] The following examples and test cases further describe the invention, and these examples and test cases should not be construed as limiting the scope of the invention. The embodiments or test cases of the present invention do not include detailed descriptions of conventional methods. Experimental methods without specific conditions are generally performed under conventional conditions, as such methods are well known to those skilled in the art and are described in many publications, such as Cold Spring Harbor's Handbook of Antibody Techniques, Molecular Cloning, or according to the conditions recommended by the manufacturer of the experimental materials. Some experimental materials without a specified source are obtained commercially.

[0395] Example 1. Humanization of anti-TfR1 antibody

[0396] The variable region sequence of the murine anti-TfR1 antibody ALK 128.1 disclosed herein is derived from WO1993010819A1. The amino acid sequences of the variable region and CDR of the antibody are as follows:

[0397] ALK 128.1 Mouse-derived heavy chain variable region

[0398] ALK 128.1 Mouse-derived light chain variable region

[0399] Note: The primary structure of the above sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the background texture is determined by CDR according to the Kabat numbering system; the rest is FR.

[0400] Table 1. CDR sequences of anti-TfR1 antibodies

[0401] Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0402] In the IgBlast database, murine ALK 128.1-VH and VL sequences were compared with human IgG germline sequences for homology. Based on homology analysis, IGHV1-2*06 and IGKV3-11*01 were selected as templates for the humanization of ALK 128.1-VH and VL, respectively. The CDRs of murine ALK 128.1-VH and VL were then transplanted into their corresponding human templates. WGQGTTVTVSS (SEQ ID NO: 44) and FGGGTKVEIK (SEQ ID NO: 45) were selected as FR4 sequences for VH and VL, respectively, forming CDR-grafted VH and VL with the regional structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0403] Optionally, amino acid residues at positions 24, 46, 47, 56 and / or 58 of the light chain variable region of the humanized antibody may be substituted; and / or amino acid residues at positions 1, 48, 54, 69, 71 and / or 73 of the heavy chain variable region of the humanized antibody may be substituted.

[0404] Table 2. Humanized ALK 128.1 Antibodies

[0405] Note: L46R means that the 46th bit L is mutated to R, and so on.

[0406] The CDRs for the humanized antibody against ALK 128.1 are as follows:

[0407] Table 3. CDR of humanized ALK 128.1 antibody

[0408] The sequences of the light chain variable region and heavy chain variable region of the ALK 128.1 humanized antibody are as follows:

[0409] TVH1

[0410] TVL1

[0411] TVL2

[0412] TVL3

[0413] TVL4

[0414] Note: The primary structure of the above sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the added background is the CDR determined according to the Kabat numbering system; the italicized and bolded parts are mutation sites; the rest are FR.

[0415] The humanized heavy chain variable region and light chain variable region of ALK 128.1 were recombined with the human IgG1 heavy chain constant region (hIgG1) and the human κ light chain constant region (CL), respectively, to obtain the humanized antibodies shown in Table 4 below.

[0416] Table 4. Humanized antibodies against ALK 128.1

[0417] Note: In the table, “ALK128H1L1” indicates a humanized antibody with a heavy chain variable region of TVH1 (SEQ ID NO: 12), a light chain variable region of TVL1 (SEQ ID NO: 13), a heavy chain constant region as shown in SEQ ID NO: 17, and a light chain constant region as shown in SEQ ID NO: 18, and so on.

[0418] hIgG1

[0419] >CL

[0420] For example, the full-length sequence of a humanized antibody is as follows:

[0421] Heavy chain sequences of ALK128H1L1, ALK128H1L2, ALK128H1L3, and ALK128H1L4

[0422] The light chain sequence of ALK128H1L1

[0423] The light chain sequence of ALK128H1L2

[0424] The light chain sequence of ALK128H1L3

[0425] The light chain sequence of ALK128H1L4

[0426] Note: In the above sequence, the added background is the antibody variable region, and the rest is the antibody constant region.

[0427] Example 2. Affinity optimization of humanized TfR1 antibody

[0428] Mutations were introduced into TVH1 using site-directed mutagenesis, and Kabat markers were used to identify the positions of amino acid residues in the variable region. Point mutations were performed on the CDR region of TVH1, and optionally, amino acid residues at positions 97 and / or 100 on the heavy chain variable region of the humanized antibody were substituted to obtain different variable regions and antibodies.

[0429] Table 5. CDR after affinity optimization

[0430] Note: In the above sequences, mutation sites and CDR sequences are determined according to the Kabat numbering system; the italicized and bolded parts are mutation sites.

[0431] For example, the antibody variable region sequence is as follows:

[0432] TVH2

[0433] TVH3

[0434] Note: The primary structure of the above sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The background is the CDR determined according to the Kabat numbering system; the italicized and bolded parts are mutation sites; the rest are FR.

[0435] The aforementioned heavy chain variable region and light chain variable region were recombined with the human IgG1 heavy chain constant region (hIgG1) and the human κ light chain constant region (CL), respectively, to obtain the humanized antibodies shown in Table 6 below.

[0436] Table 6. Humanized antibodies with optimized affinity

[0437] For example, the full-length sequence of the modified humanized antibody ALK128H1L1 is as follows:

[0438] Light chain sequences of ALK128H2L1 and ALK128H3L1: Same as SEQ ID NO: 20;

[0439] The light chain sequences of ALK128H2L2 and ALK128H3L2 are identical to SEQ ID NO: 21;

[0440] Light chain sequences of ALK128H2L3 and ALK128H3L3: Same as SEQ ID NO: 22;

[0441] Light chain sequences of ALK128H2L4 and ALK128H3L4: same as SEQ ID NO: 23.

[0442] Heavy chain sequences of ALK128H2L1, ALK128H2L2, ALK128H2L3, and ALK128H2L4

[0443] Heavy chain sequences of ALK128H3L1, ALK128H3L2, ALK128H3L3, and ALK128H3L4

[0444] Note: In the above sequence, the added background is the antibody variable region, and the rest is the antibody constant region.

[0445] Example 3. Preparation of maternal monoclonal antibodies that specifically bind to TfR1 or N3pGlu Aβ

[0446] The anti-N3pGlu Aβ antibody disclosed herein can be derived from any suitable antibody. For example, the sequence of the anti-N3pGlu Aβ antibody Hu89008 is derived from patents CN202410608148.7 and PCT / CN2025 / 095336 (included herein in their entirety by reference), and the relevant amino acid sequence is as follows:

[0447] Table 7. CDR sequences of anti-N3pGlu Aβ antibodies

[0448] Hu89008 Heavy Chain Variable Region (NAVH1)

[0449] >Hu89008 Light Chain Variable Region (NAVL1)

[0450] Note: The primary structure of the above sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The background texture is CDR as determined by the Kabat numbering system, and the rest is FR.

[0451] Hu89008 heavy chain

[0452] Hu89008 Light Chain

[0453] Note: In the above sequence, the added background is the antibody variable region, and the rest is the antibody constant region.

[0454] For example, using the variable regions of the anti-TfR1 antibody, the variable regions of the anti-N3pGlu Aβ antibody (e.g., the heavy chain variable region NAVH1 and the light chain variable region NAVL1 of Hu89008), CH1 (SEQ ID NO: 56), hinge region (SEQ ID NO: 57), hinge region fragment EPKSC (SEQ ID NO: 58), hinge region fragment DKTHTCPPCP (SEQ ID NO: 59), Fc-WT (SEQ ID NO: 60), human κ light chain constant region (CL, SEQ ID NO: 18), and linkers, parental monoclonal antibodies that specifically bind to TfR1 or N3pGlu Aβ are constructed using the Fc point mutations D356K+L351I or K439E+L351I in CN202311544684.7, CN202410093537.0, and PCT / CN2024 / 133232, respectively. The constructed maternal monoclonal antibody that specifically binds to TfR1 has the structure of Format 1 (Figure 1A), and the constructed maternal monoclonal antibody that specifically binds to N3pGlu Aβ has the structure of Format 2 (Figure 1B).

[0455] Format 1 contains two chains 1 and two chains 2 with the following structure:

[0456] Chain 1 is: [TfR1-VH]-[CH1]-[EPKSC]-[Connector 1]-[DKTHTCPPCP]-[Fc1];

[0457] Chain 2 is: [TfR1-VL]-[CL].

[0458] Format 2 contains two chains 3 and two chains 4 with the following structure:

[0459] Chain 3 is: [N3pGlu Aβ-VH]-[CH1]-[hinge region]-[Fc2];

[0460] Chain 4 is: [N3pGlu Aβ-VL]-[CL].

[0461] Fc1 refers to Fc-WT (SEQ ID NO: 60) with amino acid substitution of D356K+L351I, and Fc2 refers to Fc-WT (SEQ ID NO: 60) with amino acid substitution of K439E+L351I. The numbering is based on the EU index. The relevant sequences are as follows:

[0462] CH1

[0463] Hinge area

[0464] >Fc-WT

[0465] >Fc1

[0466] >Fc2

[0467] Connector 1

[0468] For example, the maternal monoclonal antibodies that specifically bind to TfR1, as shown in the table below, were constructed.

[0469] Table 8. Maternal monoclonal antibodies that specifically bind to TfR1

[0470] In Table 8, TfR1-F1-1 indicates a maternal monoclonal antibody that specifically binds to TfR1, using the Format 1 structure, with chain 1 as shown in SEQ ID NO: 64 and chain 2 as shown in SEQ ID NO: 20, and so on. The amino acid sequences of each chain are as follows:

[0471] F1-TVH1

[0472] F1-TVH2

[0473] F1-TVH3

[0474] F1-TVL1

[0475] F1-TVL2

[0476] F1-TVL3

[0477] F1-TVL4

[0478] Note: In the above sequence, the background with added shading represents the antibody variable region, the italicized part represents the hinge region or its fragments, the bolded part represents the linker, the remaining part represents the antibody constant region, and the italicized part with added shading represents the amino acid mutation of the constant region.

[0479] For example, the maternal monoclonal antibody that specifically binds to N3pGlu Aβ, as shown in the table below, was constructed.

[0480] Table 9. Maternal monoclonal antibodies that specifically bind to N3pGlu Aβ

[0481] In Table 9, N3pGlu Aβ-F2-1 represents a maternal monoclonal antibody that specifically binds to N3pGlu Aβ, employing a Format 2 structure, with chain 3 as shown in SEQ ID NO: 67 and chain 4 as shown in SEQ ID NO: 55. The amino acid sequences of each chain are as follows:

[0482] >F2-NAVH1

[0483] >F2-NAVL1

[0484] Note: In the above sequence, the added background is the antibody variable region, and the rest is the antibody constant region.

[0485] Example 4. Preparation of bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ

[0486] The parent monoclonal antibody from Example 3 was assembled in vitro into a bispecific antibody specifically binding to TfR1 and N3pGlu Aβ using the methods described in CN202311544684.7, CN202410093537.0, and PCT / CN2024 / 133232. Specifically, equal amounts of the parent monoclonal antibody specifically binding to TfR1 and the parent monoclonal antibody specifically binding to N3pGlu Aβ were mixed. A final volume of 10% 2-MEA stock solution (750 mM) was added to the mixture of anti-TfR1 antibody and anti-N3pGlu Aβ antibody, and the mixture was thoroughly mixed. The above mixture of anti-TfR1 antibody, anti-N3pGlu Aβ antibody, and 2-MEA was incubated in a 37°C water bath for 2.5 hours. After incubation, the antibody solution was dialyzed against PBS for medium replacement, and the collected sample was the in vitro recombinant bispecific antibody specifically binding to TfR1 and N3pGlu Aβ. The bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ has a Format 3 structure (Figure 2), in which...

[0487] Format 3 contains four chains with the following structure:

[0488] Chain 1 is: [TfR1-VH]-[CH1]-[EPKSC]-[Connector 1]-[DKTHTCPPCP]-[Fc1];

[0489] Chain 2 is: [TfR1-VL]-[CL];

[0490] Chain 3 is: [N3pGlu Aβ-VH]-[CH1]-[hinge region]-[Fc2];

[0491] Chain 4 is: [N3pGlu Aβ-VL]-[CL].

[0492] For example, bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ, as shown in the table below, were assembled.

[0493] Table 10. Bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ as disclosed herein

[0494] In Table 10, TNA-F3-1 indicates the use of Format 3 structure, with chain 1 as shown in SEQ ID NO: 66, chain 2 as shown in SEQ ID NO: 20, chain 3 as shown in SEQ ID NO: 67, and chain 4 as shown in SEQ ID NO: 55, which specifically binds to the bispecific antibody TfR1 and N3pGlu Aβ.

[0495] The positive control antibody used in this disclosure is ALK 128.1-hIgG1, whose variable region sequence is derived from patent WO1993010819A1. The heavy chain variable region and light chain variable region were recombined with the human IgG1 heavy chain constant region (hIgG1) and the human κ light chain constant region (CL), respectively, to obtain ALK 128.1-hIgG1. The full-length sequence is as follows:

[0496] Heavy chain sequence of ALK 128.1-hIgG1

[0497] The light chain sequence of ALK 128.1-hIgG1

[0498] The positive control molecule, trontinemab, used in this disclosure has a sequence derived from WHO Drug Information, Vol. 36, No. 3, 2022, INN: List 88. The amino acid sequences of each chain are as follows:

[0499] >Trontinemab heavy chain 1:

[0500] >Trontinemab heavy chain 2:

[0501] >Trontinemab Light Chain 1:

[0502] >Trontinemab Light Chain 2:

[0503] The positive control antibody used in this disclosure is Tro-Tmab, whose variable region sequence is derived from a fragment of the anti-TfR antibody in Trotinemab (WHO Drug Information, Vol. 36, No. 3, 2022, INN: List 88). The heavy chain variable region and light chain variable region were recombined with the human IgG1 heavy chain constant region (hIgG1) and the human κ light chain constant region (CL), respectively, to obtain Tro-Tmab. The full-length sequence is as follows:

[0504] Tro-Tmab heavy chain sequence

[0505] >Tro-Tmab light chain sequence

[0506] The negative control antibody used in this disclosure is C25-hIgG1, whose variable region sequence is derived from patent US6114143A. The heavy chain variable region and light chain variable region are recombined with the human IgG1 heavy chain constant region (hIgG1) and the human κ light chain constant region (CL), respectively, to obtain C25-hIgG1. The full-length sequence is as follows:

[0507] >C25-hIgG1 heavy chain sequence

[0508] >C25-hIgG1 light chain sequence

[0509] Note: In the above sequence, the added background shading represents the antibody variable region.

[0510] Test case

[0511] Test Example 1. ELISA detection of the binding activity of anti-TfR1 antibody to human TfR1.

[0512] Dilute recombinant human TfR1 protein (purchased from ACROBiosystems, Cat:CD1-H5243) to 0.1 μg / mL or 0.2 μg / mL with phosphate buffer (pH 7.4), and transfer this solution to 96-well microplates (Corning, CLS3590-100EA) (100 μL per well), incubate overnight at 4°C. After washing, transfer phosphate buffer containing 1% bovine serum albumin and 0.05% Tween-20 to the microplates (200 μL per well), and incubate at room temperature for 1 h to block the microplates. Serially dilute the test antibody and transfer 50 μL to each well of the above microplates, incubate at room temperature for 0.5 h. After washing, add a moderately diluted HRP-goat anti-human IgG (H+L) secondary antibody (Jackson, 109-035-003) to each well, and incubate at room temperature for 0.5 h. After washing the plate, add 50 μL of colorimetric reagent (KPL, 5120-0077) with TMB (3,3′,5,5′-Tetramethylbenzidine) as the substrate to each well and incubate at room temperature for 1–5 min. When the color development reaches an appropriate level, add 50 μL of 2M H2SO4 solution to each well to terminate the reaction. OD450 was measured using a microplate reader (Molecular Devices). Data analysis and plotting were performed using a GraphPad Prism 10. The results are shown in Table 11.

[0513] Table 11. Binding activity of anti-TfR1 antibody to human TfR1

[0514] The results showed that the binding activity of ALK128H3L1 to human TfR1 was weakened by two orders of magnitude compared to ALK 128.1-hIgG1, but its binding activity to human TfR1 was slightly stronger than that of the control antibody Tro-Tmab.

[0515] Test Example 2. ELISA detection of the blocking activity of anti-TfR1 antibody against the binding of TF to TfR1.

[0516] Human TfR1 recombinant protein (purchased from ACROBiosystems, Cat:CD1-H5243) was diluted to 1 μg / mL with phosphate buffer (pH 7.4), and this solution was transferred to 96-well microplates (Corning, CLS3590-100EA) (100 μL per well) and incubated overnight at 4°C. After washing, phosphate buffer containing 1% bovine serum albumin and 0.05% Tween-20 was transferred to the microplates (200 μL per well) and incubated at room temperature for 1 h to block the microplates. Different concentrations of antibodies were diluted with 1 μg / mL of biotinylated (Thermo, 20217) labeled Human Holo-Transferrin (TF) protein (R&D, 2914-HT-100MG) solution, and transferred to the above microplates (50 μL per well) and incubated at room temperature for 0.5 h. After washing the plate, add 50 μL of moderately diluted Streptavidin-Peroxidase Polymer secondary antibody (Sigma, S2438) to each well and incubate at room temperature for 0.5 hours. After washing the plate again, add 50 μL of colorimetric reagent (KPL, 5120-0077) with TMB (3,3′,5,5′-Tetramethylbenzidine) as the substrate to each well and incubate at room temperature for 1–5 minutes. When the color development reaches an appropriate level, stop the reaction by adding 50 μL of 2M H2SO4 solution to each well. OD450 was measured using a microplate reader (Molecular Devices). Data analysis and plotting were performed using a GraphPad Prism 10. The results are shown in Table 12.

[0517] Table 12. Blocking activity of anti-TfR1 antibodies against TF-TfR1 binding.

[0518] The results showed that the anti-TfR1 antibody disclosed in this paper could not block the binding of TF to TfR1, and theoretically did not affect the normal iron transport function of TfR1. The control antibody Tro-Tmab could block the binding of TF to TfR1.

[0519] Test Example 3. Blood-brain barrier penetrating ability of bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ in human TfR1 transgenic mice.

[0520] Female hTfR1 transgenic mice (Biocytogen, B-hTFR1 mice) were intravenously injected with the antibody drug. Whole blood and brain tissue were collected from the treatment group at 1 h, 8 h, 24 h, 48 h, and 96 h after drug administration.

[0521] (1) Collect about 300 μL of whole blood, let it stand for 15 min, centrifuge at 3500 rpm for 10 min and take about 100 μL of serum, store at -20℃; detect the antibody content in the serum;

[0522] (2) Brain tissue collection: After general anesthesia with 7 mL / kg of 20% urethane, the brain tissue was collected after perfusion of the heart with 30 mL of physiological saline and blotted dry with filter paper. The separated brain tissue was then divided into two equal parts using a scalpel. The left hemisphere of the brain tissue was used to detect the antibody content in the brain tissue. It was placed in a 1.5 mL EP tube after peeling and weighed for recording, and stored at -20℃. The right hemisphere of the brain tissue was placed face down in a cryoembryo box, fixed in dry ice, and stored at -20℃ for IF staining.

[0523] Methods for detecting antibody levels in brain tissue and serum: A complete left brain sample was weighed and placed in a 4 mL EP tube. Tissue lysis buffer (thermo, 78510) was added to the weighed sample at a solid:liquid ratio of 1:10. The sample was homogenized (-10℃, 60Hz, 90s). The homogenized tissue was centrifuged at 13000g at 4℃ for 10 min. The supernatant was transferred to a 96-well plate and stored at 4℃ for later analysis. The plate (PerkinElmer, AAAND-0001) was coated with 1 μg / mL anti-human IgG Fc (Jackson immunoresearch, 109-005-098) and incubated overnight at 4℃. After washing, blocking buffer (thermo, 37528) was added and blocked at 37℃ for 1-2 h. After washing, serum or brain tissue homogenate was added and incubated at 37℃ for 1-1.5 h. After washing the plates, goat anti-human IgG (Fab')2 (Biotin) (abcam, ab64666) was added and incubated at 37°C for 1-1.5 hours. After washing the plates again, SA-EU (PerkinElmer, 1244-360) was added and incubated at 37°C for 1-1.5 hours. After washing the plates again, DELFIA Enhancement Solution (PerkinElmer, 4001-0010) was added and incubated in the dark for 30 minutes. The absorbance values ​​were then read using an envision microplate reader. The results are shown in Table 13.

[0524] IF staining method: Brain tissue sections were placed in 4% PFA (Boster Biologics, AR1068) pre-cooled to 4℃ and fixed at 4℃ for 15 min. Then, they were washed three times with TBST for 3 minutes each time. Serum blocking solution was added and blocked for 30 min. After removing the blocking solution, 200 μL of Goat anti-human IgG (H+L) fluorescent secondary antibody (Invitrogen, A21445) was added to each section and incubated in the dark for 60 min. Then, they were washed three times with TBST for 3 minutes each time. The tissue sections were washed three times with pure water for 3 minutes each time. They were then air-dried in the dark and mounted using a DAPI-containing anti-fluorescence quenching mounting medium (Vector, H-2000). After standing for 10 minutes, images were acquired and stored at 4℃ in the dark as usual. The results are shown in Table 14.

[0525] Table 13. Bispecific antibodies that specifically bind to TfR1 and N3pGlu Aβ in brain-penetrating hTfR1 transgenic mice

[0526] The results showed that, when hTfR1 transgenic mice were administered the same molar dose, the serum antibody half-life of TNA-F3-1 was superior to that of the control antibody Trotinemab within 0-96 hours, and the antibody exposure AUC in the brain was 4 times higher than that of the control antibody Trotinemab.

[0527] Table 14. Distribution of antibody in brain tissue of hTfR1 transgenic mice 24 h after drug administration, as detected by IF staining.

[0528] In hTfR1 transgenic mice administered the same molar dose, IF staining of mouse brain tissue sections 24 hours after administration showed that the monoclonal antibody Hu89008 did not detect significant antibody signals in the mouse cerebral cortex or hippocampus. The brain-penetrating bispecific antibody TNA-F3-1 showed significant antibody distribution signals in both the mouse cerebral cortex and hippocampus. 24 hours after administration, a relatively significant diffuse signal distribution was observed in the brain parenchyma (including the cerebral cortex and hippocampus), with a positive antibody signal ratio reaching 44.1%, significantly higher than the approximately 4.17% antibody distribution of the control antibody Trotinemab in the brain parenchyma.

[0529] Although the invention has been described in detail with the aid of accompanying drawings and examples for clarity of understanding, these descriptions and examples should not be construed as limiting the scope of this disclosure. All patent and scientific literature disclosures cited herein are clearly and fully incorporated by reference.

Claims

1. An antigen-binding molecule that specifically binds to TfR1, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein: The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 3, HCDR2 contains the amino acid sequence of SEQ ID NO: 11, and HCDR3 contains the amino acid sequence of SEQ ID NO: 36; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO: 8; or The heavy chain variable regions HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 27, 12, or 26, and the light chain variable regions LCDR1, LCDR2, and LCDR3 respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 13, 14, 15, or 16.

2. The antigen-binding molecule that specifically binds to TfR1 according to claim 1, wherein: The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 3, HCDR2 contains the amino acid sequence of SEQ ID NO: 11, and HCDR3 contains the amino acid sequence of SEQ ID NO: 25, 5, or 24; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO:

8. Preferably, The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 3, HCDR2 contains the amino acid sequence of SEQ ID NO: 11, and HCDR3 contains the amino acid sequence of SEQ ID NO: 25, and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 9, LCDR2 contains the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains the amino acid sequence of SEQ ID NO:

8.

3. The antigen-binding molecule that specifically binds to TfR1 according to claim 1 or 2, wherein the antigen-binding molecule that specifically binds to TfR1 is an antibody or an antibody fusion protein; Preferably, the antigen-binding molecule that specifically binds to TfR1 is a humanized antibody.

4. The antigen-binding molecule that specifically binds to TfR1 according to any one of claims 1 to 3, wherein: The heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 27, 12 or 26, and the light chain variable region comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 13, 14, 15 or 16; Preferably, The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27, 12 or 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13, 14, 15 or 16. More preferably, The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; or The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 13, 14, 15 or 16; Most preferably, The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27, and the light chain variable region contains the amino acid sequence of SEQ ID NO:

13.

5. The antigen-binding molecule that specifically binds to TfR1 according to any one of claims 1 to 4, wherein the antigen-binding molecule that specifically binds to TfR1 comprises a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is the human IgG1 heavy chain constant region or a variant thereof, and the light chain constant region is the human κ light chain constant region or a variant thereof; More preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 17, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:

18.

6. The antigen-binding molecule that specifically binds to TfR1 according to any one of claims 1 to 4, wherein the antigen-binding molecule that specifically binds to TfR1 is an antigen-binding fragment. Preferably, the antigen-binding fragment is selected from Fab, Fab′, F(ab′)2, Fv, scFv and dsFv.

7. The antigen-binding molecule that specifically binds to TfR1 according to any one of claims 1 to 5, wherein the antigen-binding molecule that specifically binds to TfR1 comprises a heavy chain and a light chain, wherein: The heavy chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 29, 19 or 28, and the light chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 20, 21, 22 or 23; Preferably, The heavy chain comprises the amino acid sequence of SEQ ID NO: 29, 19 or 28, and the light chain comprises the amino acid sequence of SEQ ID NO: 20, 21, 22 or 23; More preferably, The heavy chain comprises the amino acid sequence of SEQ ID NO: 29, and the light chain comprises the amino acid sequence of SEQ ID NO: 20, 21, 22, or 23; or The heavy chain comprises the amino acid sequence of SEQ ID NO: 19, and the light chain comprises the amino acid sequence of SEQ ID NO: 20, 21, 22, or 23; or The heavy chain comprises the amino acid sequence of SEQ ID NO: 28, and the light chain comprises the amino acid sequence of SEQ ID NO: 20, 21, 22 or 23; Most preferably, The heavy chain contains the amino acid sequence of SEQ ID NO: 29, and the light chain contains the amino acid sequence of SEQ ID NO:

20.

8. The antigen-binding molecule that specifically binds to TfR1 according to any one of claims 1 to 6, wherein it is a multispecific antibody; Preferably, the antigen-binding molecule that specifically binds to TfR1 is a bispecific antibody; More preferably, the antigen-binding molecule that specifically binds to TfR1 is a bispecific antibody that specifically binds to both TfR1 and N3pGlu Aβ.

9. A bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ, comprising a first antigen-binding domain that specifically binds to TfR1 and a second antigen-binding domain that specifically binds to N3pGlu Aβ. Preferably, The first antigen-binding domain that specifically binds to TfR1 comprises a heavy chain variable region (TfR1-VH) and a light chain variable region (TfR1-VL), wherein TfR1-VH comprises HCDR1, HCDR2, and HCDR3, and TfR1-VL comprises LCDR1, LCDR2, and LCDR3, wherein: The TfR1-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 3, HCDR2 containing the amino acid sequence of SEQ ID NO: 11, and HCDR3 containing the amino acid sequence of SEQ ID NO: 36; and the TfR1-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 10, and LCDR3 containing the amino acid sequence of SEQ ID NO: 8; and The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises a heavy chain variable region (N3pGlu Aβ-VH) and a light chain variable region (N3pGlu Aβ-VL), wherein N3pGlu Aβ-VH comprises HCDR1, HCDR2, and HCDR3, and N3pGlu Aβ-VL comprises LCDR1, LCDR2, and LCDR3, wherein: The N3pGlu Aβ-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 46, HCDR2 containing the amino acid sequence of SEQ ID NO: 47, and HCDR3 containing the amino acid sequence of SEQ ID NO: 48; and the N3pGlu Aβ-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 49, LCDR2 containing the amino acid sequence of SEQ ID NO: 50, and LCDR3 containing the amino acid sequence of SEQ ID NO:

51. More preferably, The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein: The TfR1-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 3, HCDR2 containing the amino acid sequence of SEQ ID NO: 11, and HCDR3 containing the amino acid sequence of SEQ ID NO: 25, 5, or 24; and the TfR1-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 10, and LCDR3 containing the amino acid sequence of SEQ ID NO: 8; and The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein: The N3pGlu Aβ-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 46, HCDR2 containing the amino acid sequence of SEQ ID NO: 47, and HCDR3 containing the amino acid sequence of SEQ ID NO: 48; and the N3pGlu Aβ-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 49, LCDR2 containing the amino acid sequence of SEQ ID NO: 50, and LCDR3 containing the amino acid sequence of SEQ ID NO:

51. Most preferably, The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein: The TfR1-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 3, HCDR2 containing the amino acid sequence of SEQ ID NO: 11, and HCDR3 containing the amino acid sequence of SEQ ID NO: 25; and the TfR1-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 10, and LCDR3 containing the amino acid sequence of SEQ ID NO: 8; and The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein: The N3pGlu Aβ-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 46, HCDR2 containing the amino acid sequence of SEQ ID NO: 47, and HCDR3 containing the amino acid sequence of SEQ ID NO:

48. The N3pGlu Aβ-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 49, LCDR2 containing the amino acid sequence of SEQ ID NO: 50, and LCDR3 containing the amino acid sequence of SEQ ID NO:

51.

10. The bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ according to claim 9, wherein: The TfR1-VH comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 27, 12, or 26, and the TfR1-VL comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 13, 14, 15, or 16; and The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein: The N3pGlu Aβ-VH contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO: 52, and the N3pGlu Aβ-VL contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO: 53; Preferably, The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein: The TfR1-VH comprises the amino acid sequence of SEQ ID NO: 27, 12, or 26, and the TfR1-VL comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; and The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein: The N3pGlu Aβ-VH contains the amino acid sequence of SEQ ID NO: 52, and the N3pGlu Aβ-VL contains the amino acid sequence of SEQ ID NO: 53; More preferably, The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein: The TfR1-VH contains the amino acid sequence of SEQ ID NO: 27, and the TfR1-VL contains the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; or The TfR1-VH contains the amino acid sequence of SEQ ID NO: 12, and the TfR1-VL contains the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; or The TfR1-VH contains the amino acid sequence of SEQ ID NO: 26, and the TfR1-VL contains the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; and The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein: The N3pGlu Aβ-VH contains the amino acid sequence of SEQ ID NO: 52, and the N3pGlu Aβ-VL contains the amino acid sequence of SEQ ID NO: 53; Most preferably, The first antigen-binding domain that specifically binds to TfR1 comprises TfR1-VH and TfR1-VL, wherein: The TfR1-VH contains the amino acid sequence of SEQ ID NO: 27, and the TfR1-VL contains the amino acid sequence of SEQ ID NO: 13; and The second antigen-binding domain that specifically binds to N3pGlu Aβ comprises N3pGlu Aβ-VH and N3pGlu Aβ-VL, wherein: The N3pGlu Aβ-VH contains the amino acid sequence of SEQ ID NO: 52, and the N3pGlu Aβ-VL contains the amino acid sequence of SEQ ID NO:

53.

11. The bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ according to claim 9 or 10, comprising: One first chain containing SEQ ID NO: 66, or an amino acid sequence having at least 80% sequence identity with it; one second chain containing SEQ ID NO: 20, or an amino acid sequence having at least 80% sequence identity with it; one third chain containing SEQ ID NO: 67, or an amino acid sequence having at least 80% sequence identity with it; and one fourth chain containing SEQ ID NO: 55, or an amino acid sequence having at least 80% sequence identity with it. Preferably, The bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ comprises: One first strand containing the amino acid sequence of SEQ ID NO: 66, one second strand containing the amino acid sequence of SEQ ID NO: 20, one third strand containing the amino acid sequence of SEQ ID NO: 67, and one fourth strand containing the amino acid sequence of SEQ ID NO: 55; The first and second chains bind to each other, the third and fourth chains bind to each other, and the first and third chains bind to each other to form a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ.

12. A conjugate comprising a payload and an antigen-binding molecule specifically binding to TfR1 as described in any one of claims 1 to 8, or a bispecific antibody specifically binding to TfR1 and N3pGlu Aβ as described in any one of claims 9 to 11, conjugated to the payload. Preferably, the payload is an oligonucleotide.

13. A pharmaceutical composition comprising an antigen-binding molecule that specifically binds to TfR1 according to any one of claims 1 to 8, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ according to any one of claims 9 to 11, or a conjugate according to claim 12, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

14. A nucleic acid encoding an antigen-binding molecule that specifically binds to TfR1 according to any one of claims 1 to 8, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ according to any one of claims 9 to 11.

15. A host cell comprising the nucleic acid as described in claim 14.

16. A method for preventing or treating a disease or condition, the method comprising administering to a subject a preventive or therapeutically effective amount of an antigen-binding molecule that specifically binds to TfR1 according to any one of claims 1 to 8, or a bispecific antibody that specifically binds to TfR1 and N3pGlu Aβ according to any one of claims 9 to 11, or a conjugate according to claim 12, or a pharmaceutical composition according to claim 13. Preferably, the disease or symptom is a neurological disease; More preferably, the disease or symptom is a neurodegenerative disease; Most preferably, the disease or symptom is Alzheimer's disease.

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