Antigen binding proteins targeting transferrin receptor 1 and uses thereof

Abstract

This application relates to an antigen-binding protein targeting transferrin receptor 1 (TfR1) and its application, wherein the antigen-binding protein comprises a heavy chain variable region (VH), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises an amino acid sequence as shown in SEQ ID NO:92 (RX2X3DMFYDY), wherein X2 is selected from A or F or I or T or V, and X3 is selected from D or G or H or Q or V or Y.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to an antigen-binding protein that targets transferrin receptor 1 (TfR1) and its applications. Background Technology

[0002] Transferrin receptor 1 (TfR1) mediates transferrin binding and endocytosis, transporting iron ions into cells and participating in key life activities such as DNA synthesis and mitochondrial metabolism. TfR1 is widely expressed in various cell types, especially in tissues or cells with high iron requirements, including erythroid precursor cells, tumor cells, and brain microvascular endothelial cells.

[0003] In the field of intracerebral drug delivery, receptor-mediated transcytosis (RMT) has become one of the most promising pathways for delivering large molecule drugs into the brain, with TfR1 being a representative receptor for this pathway. Roche's Trontinemab is an engineered bispecific antibody that binds to the pathogenic target β-amyloid (Aβ) at one end and TfR1 at the other, utilizing TfR1-mediated trans-blood-brain barrier transport to enhance antibody exposure in the brain for Alzheimer's disease treatment. Denali Therapeutics' TfR1-targeted Transport Vehicle (TV) technology platform modifies the antibody's Fc region to enable TfR1 binding, thus achieving highly efficient intracerebral delivery. Besides intracerebral delivery, drugs targeting TfR1 also show promise in areas such as tumor therapy and muscle-targeted delivery.

[0004] Despite the promising prospects for drug development targeting TfR1 for intrabrain delivery, key technological challenges remain, including the need for precise optimization of targeting affinity and binding valence, capture by peripherally expressed TfR1 tissues, and insufficient brain delivery efficiency. There is an urgent need to develop novel anti-TfR1 antibodies with lower toxicity, better pharmacokinetic properties, and higher brain delivery efficiency to meet the growing clinical demand. Summary of the Invention

[0005] This application provides an antigen-binding protein capable of specifically binding to transferrin receptor 1 (TfR1), which has one or more of the following beneficial effects:

[0006] (1) It has a high binding affinity for TfR1;

[0007] (2) It has cross-binding activity and can bind to human TfR1, mouse TfR1 and / or TfR1 derived from cynomolgus monkeys;

[0008] (3) It can bind to TfR1 simultaneously with transferrin;

[0009] (4) It can efficiently mediate transcytosis and cross the blood-brain barrier (BBB).

[0010] (5) It can effectively deliver therapeutic drugs to the central nervous system.

[0011] On one hand, this application provides an antigen-binding protein that can specifically bind to transferrin receptor 1, the antigen-binding protein comprising a heavy chain variable region (VH), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 92 (RX2X3DMFYDY), wherein X2 is selected from A or F or I or T or V, and X3 is selected from D or G or H or Q or V or Y.

[0012] In some embodiments, the VH includes heavy chain complementarity determination regions HCDR1, HCDR2, and HCDR3, wherein HCDR2 includes, for example, SEQ ID NO: 91 (X1X2X3X5X6X7X8X9X...). 10 X 11 X 12 The amino acid sequence shown in SVKG, wherein X1 is selected from A, I, L, T, or V; X2 is selected from A, F, I, Q, R, T, or V; X3 is selected from R, S, or T; X5 is selected from S or K; X6 is selected from E, G, L, or Q; X7 is selected from A, D, I, L, S, or V; X8 is selected from A, R, or T; X9 is selected from A, Y, or V; X 10 Selected from Y, L, T, X 11 Choose from A, E, or S, and X 12 Selected from D, R, or F.

[0013] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 contains an amino acid sequence as shown in SEQ ID NO: 90 (DAX3YELYD), and X3 is selected from T or P.

[0014] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 2-3.

[0015] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR2 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 4-27.

[0016] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR3 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 29-40.

[0017] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2; wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 91 (X1X2X3X5X6X7X8X9X... 10 X 11 X 12 The amino acid sequence shown in SVKG, wherein X1 is selected from A, I, L, T, or V; X2 is selected from A, F, I, Q, R, T, or V; X3 is selected from R, S, or T; X5 is selected from S or K; X6 is selected from E, G, or Q; X7 is selected from A, D, I, L, S, or V; X8 is selected from A, R, or T; X9 is selected from Y or V; X 10 Selected from Y, L, T, X 11 Choose from A, E, or S, and X 12 The amino acid sequence is selected from D, R, or F; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 92 (RX2X3DMFYDY), wherein X2 is selected from A, F, I, T, or V, and X3 is selected from D, H, Q, V, or Y.

[0018] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes an amino acid sequence as shown in SEQ ID NO: 2; wherein HCDR2 includes an amino acid sequence selected from any one of SEQ ID NO: 4-12, SEQ ID NO: 14-22, and SEQ ID NO: 24-27; and wherein HCDR3 includes an amino acid sequence selected from any one of SEQ ID NO: 29-33, SEQ ID NO: 35, and SEQ ID NO: 37-40.

[0019] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any combination of the following:

[0020] 1)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 5;HCDR3: SEQ ID NO: 29;

[0021] 2)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 4;HCDR3: SEQ ID NO: 30;

[0022] 3)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 7;HCDR3: SEQ ID NO: 29;

[0023] 4)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 8;HCDR3: SEQ ID NO: 29;

[0024] 5)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 9;HCDR3: SEQ ID NO: 31;

[0025] 6)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 10;HCDR3: SEQ ID NO: 29;

[0026] 7)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 4;HCDR3: SEQ ID NO: 32;

[0027] 8)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 11;HCDR3: SEQ ID NO: 33;

[0028] 9)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 12;HCDR3: SEQ ID NO: 33;

[0029] 10)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 35;

[0030] 11)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 14;HCDR3: SEQ ID NO: 32;

[0031] 12)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 15;HCDR3: SEQ ID NO: 32;

[0032] 13)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 16;HCDR3: SEQ ID NO: 32;

[0033] 14)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 17;HCDR3: SEQ ID NO: 37;

[0034] 15)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 18;HCDR3: SEQ ID NO: 32;

[0035] 16)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 19;HCDR3: SEQ ID NO: 33;

[0036] 17)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 20;HCDR3: SEQ ID NO: 37;

[0037] 18)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 21;HCDR3: SEQ ID NO: 37;

[0038] 19)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 22;HCDR3: SEQ ID NO: 38;

[0039] 20)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 24;HCDR3: SEQ ID NO: 37;

[0040] 21)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 4;HCDR3: SEQ ID NO: 39;

[0041] 22)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 25;HCDR3: SEQ ID NO: 33;

[0042] 23) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 26; HCDR3: SEQ ID NO: 40; and

[0043] 24) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 27; HCDR3: SEQ ID NO: 37.

[0044] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 3; wherein HCDR2 contains the amino acid sequence shown in SEQ ID NO: 95 (AITRSX6X7TX9YADSVKG), wherein X6 is selected from G or L, X7 is selected from S or V, and X9 is selected from Y or A; and wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO: 96 (RAX3DMFYDY), wherein X3 is selected from D or Y or V or G.

[0045] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes an amino acid sequence as shown in SEQ ID NO: 3; wherein HCDR2 includes an amino acid sequence selected from any one of SEQ ID NO: 6, SEQ ID NO: 13, and SEQ ID NO: 23; and wherein HCDR3 includes an amino acid sequence selected from any one of SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 36.

[0046] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any combination of the following:

[0047] 1) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 29;

[0048] 2) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 13; HCDR3: SEQ ID NO: 34;

[0049] 3) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 36; and

[0050] 4) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 23; HCDR3: SEQ ID NO: 33.

[0051] In some embodiments, the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 90 (DAX3YELYD), wherein X3 is selected from T or P; wherein HCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 97 (X1X2TRX5GX7X8YX). 10 The amino acid sequence shown in ADSVKG is as follows, wherein X1 is selected from A, V, or I; X2 is selected from I, T, or V; X5 is selected from S or K; X7 is selected from S or V; X8 is selected from A or T; and X... 10 The amino acid sequence is selected from Y or T; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 98 (RX2X3DMFYDY), wherein X2 is selected from A or V and X3 is selected from Y or Q.

[0052] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 contains an amino acid sequence as shown in any one of SEQ ID NO: 2-3; wherein HCDR2 contains an amino acid sequence selected from any one of SEQ ID NO: 4-10; and wherein HCDR3 contains an amino acid sequence selected from any one of SEQ ID NO: 29-31.

[0053] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any combination of the following:

[0054] 1) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 5; HCDR3: SEQ ID NO: 29;

[0055] 2) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 30;

[0056] 3) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 29;

[0057] 4) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 7; HCDR3: SEQ ID NO: 29;

[0058] 5) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 8; HCDR3: SEQ ID NO: 29;

[0059] 6) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 9; HCDR3: SEQ ID NO: 31; and

[0060] 7) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 10; HCDR3: SEQ ID NO: 29.

[0061] In some embodiments, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any combination of the following:

[0062] 1) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 5; HCDR3: SEQ ID NO: 29;

[0063] 2) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 30;

[0064] 3) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 29;

[0065] 4) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 7; HCDR3: SEQ ID NO: 29;

[0066] 5) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 8; HCDR3: SEQ ID NO: 29;

[0067] 6)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 9;HCDR3: SEQ ID NO: 31;

[0068] 7)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 10;HCDR3: SEQ ID NO: 29;

[0069] 8)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 4;HCDR3: SEQ ID NO: 32;

[0070] 9)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 11;HCDR3: SEQ ID NO: 33;

[0071] 10)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 12;HCDR3: SEQ ID NO: 33;

[0072] 11)HCDR1: SEQ ID NO:3;HCDR2: SEQ ID NO: 13;HCDR3: SEQ ID NO: 34;

[0073] 12)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 35;

[0074] 13)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 14;HCDR3: SEQ ID NO: 32;

[0075] 14)HCDR1: SEQ ID NO:3;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 36;

[0076] 15)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 15;HCDR3: SEQ ID NO: 32;

[0077] 16)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 16;HCDR3: SEQ ID NO: 32;

[0078] 17) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 17; HCDR3: SEQ ID NO: 37;

[0079] 18) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 18; HCDR3: SEQ ID NO: 32;

[0080] 19) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 19; HCDR3: SEQ ID NO: 33;

[0081] 20) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 20; HCDR3: SEQ ID NO: 37;

[0082] 21) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 21; HCDR3: SEQ ID NO: 37;

[0083] 22) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 22; HCDR3: SEQ ID NO: 38;

[0084] 23) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 23; HCDR3: SEQ ID NO: 33;

[0085] 24) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 24; HCDR3: SEQ ID NO: 37;

[0086] 25) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 39;

[0087] 26) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 25; HCDR3: SEQ ID NO: 33;

[0088] 27) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 26; HCDR3: SEQ ID NO: 40; and

[0089] 28) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 27; HCDR3: SEQ ID NO: 37.

[0090] In some embodiments, the antigen-binding protein is an antibody or its antigen-binding fragment.

[0091] In some embodiments, the antigen-binding fragment is (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment.

[0092] In some embodiments, the antigen-binding protein is VHH.

[0093] In some implementations, the TfR1 is a human, mouse, or monkey-derived TfR1.

[0094] In some embodiments, the VHH comprises an amino acid sequence as shown in any one of SEQ ID NO: 48-75, or the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with an amino acid sequence as shown in any one of SEQ ID NO: 48-75.

[0095] On the other hand, this application provides a bispecific antibody comprising a first antigen-binding domain capable of specifically binding to TfR1, wherein the first antigen-binding domain specifically binding to TfR1 comprises the antigen-binding protein described in this application.

[0096] In some embodiments, the bispecific antibody further includes a second antigen-binding domain capable of specifically binding to galactoglobulin-3 (Gal-3).

[0097] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 76, wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 77, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 78; and wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 79, wherein LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 80, and wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 81.

[0098] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 82, and the VL comprises the amino acid sequence shown in SEQ ID NO: 83.

[0099] In some embodiments, the first antigen-binding domain that specifically binds to TfR1 is directly or indirectly connected to the second antigen-binding domain that specifically binds to Gal-3.

[0100] In some embodiments, the first antigen-binding domain that specifically binds to TfR1 is indirectly connected to the second antigen-binding domain that specifically binds to Gal-3 via a linker.

[0101] In some embodiments, the linker is a peptide linker, wherein the peptide linker is (GGGGS)n, and n is an integer selected from 1 to 10.

[0102] In some embodiments, the N-terminus of the first antigen-binding domain that specifically binds to TfR1 is directly or indirectly connected to the C-terminus of the second antigen-binding domain that specifically binds to Gal-3, or the C-terminus of the first antigen-binding domain that specifically binds to TfR1 is directly or indirectly connected to the N-terminus of the second antigen-binding domain that specifically binds to Gal-3.

[0103] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 is an antigen-binding fragment or an antibody.

[0104] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 is an antigen-binding fragment, wherein the antigen-binding fragment is scFv, Fab, or VHH.

[0105] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 is an antibody, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0106] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 is an antibody, wherein the second antigen-binding domain that specifically binds to Gal-3 further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises Fc.

[0107] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 comprises a full-length heavy chain (HC) and a full-length light chain (LC), wherein the HC comprises the amino acid sequence shown in SEQ ID NO: 84, and the LC comprises the amino acid sequence shown in SEQ ID NO: 85.

[0108] In some embodiments, the N-terminus of the first antigen-binding protein is directly or indirectly connected to the C-terminus of the Fc of the second antigen-binding domain that specifically binds to Gal-3.

[0109] On the other hand, this application provides a fusion protein comprising the antigen-binding protein or the bispecific antibody described in this application.

[0110] On the other hand, this application provides a nucleic acid conjugate comprising the antigen-binding protein, the bispecific antibody, and / or the fusion protein described in this application.

[0111] On the other hand, this application provides a chimeric antigen receptor comprising the antigen-binding protein, the bispecific antibody, and / or the fusion protein described in this application.

[0112] On the other hand, this application provides an immune conjugate comprising the antigen-binding protein, the bispecific antibody, and / or the fusion protein described in this application.

[0113] On the other hand, this application provides a nucleic acid molecule that encodes the antigen-binding protein, the bispecific antibody, the fusion protein, and / or the chimeric antigen receptor described in this application.

[0114] On the other hand, this application provides a carrier containing the nucleic acid molecules described in this application.

[0115] On the other hand, this application provides a cell comprising the antigen-binding protein, the bispecific antibody, the nucleic acid conjugate, the fusion protein, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, and / or the vector described in this application.

[0116] On the other hand, this application provides a pharmaceutical composition comprising the antigen-binding protein, the bispecific antibody, the nucleic acid conjugate, the fusion protein, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, the carrier, and / or the cell, as described in this application, and optionally a pharmaceutically acceptable carrier.

[0117] On the other hand, this application provides a kit comprising the antigen-binding protein described in this application, the bispecific antibody described in this application, the nucleic acid conjugate described in this application, the fusion protein described in this application, the chimeric antigen receptor described in this application, the immunoconjugate described in this application, the nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, and / or the pharmaceutical composition described in this application.

[0118] On the other hand, this application provides the use of the antigen-binding protein delivery therapeutic entity described in this application to cross the blood-brain barrier.

[0119] In some embodiments, the antigen-binding protein delivers the therapeutic entity to cells, tissues, or organs expressing TfR1.

[0120] In some embodiments, the therapeutic entity is an entity that has the function of treating or preventing disease, and the therapeutic entity is a small molecule compound, protein, nucleic acid molecule or any fragment thereof.

[0121] On the other hand, this application provides the use of the bispecific antibody, fusion protein, nucleic acid conjugate, chimeric antigen receptor, immunoconjugate, nucleic acid molecule, carrier, cell, and / or pharmaceutical composition described in this application in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0122] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0123] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0124] Figure 1 The image shows the SDS-PAGE results of the recombinant human TfR1 protein expressed in this application.

[0125] Figure 2 The results shown are the ELISA results of the human TfR1 recombinant protein described in this application and the control antibody.

[0126] Figure 3 The diagram shown is a schematic of the structure of the bispecific antibody used for evaluation.

[0127] Figure 4A The figure shows the intrabrain delivery efficiency of nanobodies h5m35, h5m102, and h5m106.

[0128] Figure 4B The figure shows the intrabrain delivery efficiency of nanobodies h5m94, h5m84, h5m47, and h5m39. Detailed Implementation

[0129] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0130] Terminology Definition

[0131] In this application, the term "transferrin receptor 1" generally refers to a cell surface receptor protein, also known as CD71, that specifically binds to transferrin and mediates its endocytosis. It is a type II transmembrane glycoprotein composed of an intracellular domain, a transmembrane domain, and an extracellular domain, existing on the cell membrane as a homodimer. TfR1 mediates the transport of iron ions into the cell. The typical process involves transferrin binding to TfR1, entering the cell via clathrin-dependent endocytosis, releasing iron ions in acidic endosomes, and then the deferrferrin-TfR1 complex cycling back to the cell surface and dissociating, thus achieving receptor recycling. In this application, the TfR1 protein can be the complete TfR1 and its functionally active fragments, homologs, analogs, variants, or derivatives. For example, the TfR1 protein can be the full-length TfR1 or a truncated TfR1 that retains its functional activity; for example, the TfR1 protein can be the extracellular domain of TfR1. In this application, the TfR1 can be of any species origin, such as primates, non-human primates, or rodents. In this application, the TfR1 can be human TfR1, cynomolgus monkey-derived TfR1, or mouse-derived TfR1. In this application, the TfR1 protein can be wild-type or artificially modified. For example, the TfR1 can be a modified TfR1. In some embodiments, the TfR1 protein can be an extracellular domain, wherein the amino acid sequence of the TfR1 extracellular domain can be the amino acid sequence shown in SEQ ID NO: 89.

[0132] In this application, the term "antigen-binding protein" generally refers to a protein with antigen-binding ability. For example, an antigen-binding protein may include an isolated antigen-binding protein. In this application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability that has been removed from its naturally occurring state. In this application, the "isolated antigen-binding protein" may include a portion that binds to an antigen and optionally, allow the antigen-binding portion to employ a framework or structural portion that promotes the antigen-binding portion to bind to the antigen. The antigen-binding protein may include, for example, an antibody-derived protein framework region (FR) or an alternative protein framework region or artificial framework region having a transplanted CDR or CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic framework regions containing, for example, biocompatible polymers. In this application, antigen-binding proteins may include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH, and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, as long as they exhibit the desired antigen-binding activity. In this application, the antibody may be a chimeric antibody, a humanized antibody, or a fully human antibody. In this application, the antibody may be a recombinant, hybrid, mutated, or transplanted antibody. In this application, the antigen-binding protein may be VHH. In this application, the antigen-binding protein may specifically bind to TfR1. In this application, the antigen-binding protein may be a bispecific antibody, wherein the bispecific antibody may comprise a first antigen-binding domain specifically binding to TfR1 and a second antigen-binding domain specifically binding to TfR1, wherein the second antigen-binding domain may specifically bind to TfR1 or to different antigen proteins other than TfR1, such as Gal-3.

[0133] As used herein, the term "antigen" generally refers to a molecule or fragment that can be specifically bound by an antigen-binding protein (e.g., an antibody). In some embodiments, the antigen may be used in an animal to generate a protein (e.g., an antibody) that can bind to the antigen. An antigen may have one or more epitopes that can interact with different binding proteins (e.g., antibodies). The antigen described in this application may include TfR1 or Gal-3.

[0134] In this application, the term "VHH" generally refers to the heavy chain variable region (VH) of a heavy chain antibody. In this application, the VHH may consist of the VH of a heavy chain antibody. In this application, the VHH may contain a complementarity-determining region (CDR) and a framework region (FR). In this application, the VHH contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In this application, the VHH may encompass the full-length VHH domain as well as modified or engineered forms, including but not limited to mutants, fragments (e.g., truncated versions), variants, isotypes, and homologs, as long as they retain their binding activity to the target antigen. The VHH may also exist as a monomer, polymer, or fusion protein, for example, linked with an Fc fragment, other antibody domains, or functional protein to form an antibody, bispecific, or multispecific molecule containing a constant region. In this application, the VHH can be of animal origin or non-animal origin; for example, the VHH is a camel-derived VHH or a humanized VHH.

[0135] In this application, the term "heavy chain antibody" generally refers to a naturally occurring immunoglobulin molecule composed solely of a heavy chain and lacking a light chain. The heavy chain antibody includes heavy chain antibodies (HCAbs) derived from camelids and immunoglobulin neoantigen receptors (IgNARs) derived from cartilaginous fish (such as sharks). The constant region of the heavy chain antibody does not contain a CH1 domain. The heavy chain antibody also includes its mutants, isotypes, and homologs, as well as engineered functional derivatives, provided they retain antigen-binding activity mediated solely by the variable region of the heavy chain. In some embodiments, the constant region of the heavy chain antibody is a human Fc domain. In some embodiments, the constant region of the heavy chain antibody is a human IgG domain.

[0136] In this application, the terms "nanobody" or "VHH antibody" generally refer to a heavy chain antibody that does not contain a heavy chain constant region domain (e.g., CH2, CH3), or an antigen-binding fragment containing a VHH. The nanobody may contain a natural VHH domain or its engineered form; for example, the nanobody may contain one or more VHHs, which may target one or more antigens.

[0137] In this application, the term "antibody" includes immunoglobulin molecules capable of specifically binding to a particular antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, multimers (e.g., dimers), monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that the immunoglobulin molecule can exhibit the desired biological activity (e.g., specific binding to a particular antigen). The antibody can be derived from any suitable species; for example, "antibody" includes both antibodies derived from conventional immunoglobulin structures (e.g., full-length antibodies or fragments thereof consisting of heavy and light chains) and heavy-chain antibodies derived from camelids, which typically contain only the heavy-chain variable region (VH) capable of maintaining antigen-binding ability independently of the light chain. Optionally, the antibody or its binding fragment can exist with other peptides, proteins, and / or chemical groups in the form of fusion proteins or conjugates. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, or IgA), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass of the above antibodies. In some embodiments, the antibody is a heavy chain antibody. In some embodiments, the antibody is a bispecific antibody.

[0138] In this application, the terms "bispecific antibody" and "BsAb" are generally used interchangeably, typically referring to an antibody or antibody construct having dual specificity in its binding arm. In this application, the bispecific antibody may be obtained from two different sources and constructed using recombinant DNA or cell fusion technology. In this application, the bispecific antibody may have two different antigen-binding sites. In this application, the bispecific antibody may interact with the same or two cell surface antigens. In this application, the bispecific antibody may simultaneously block two different mediators / pathways that play unique or overlapping roles in the pathogenesis. In this application, the bispecific antibody may contain a first antigen-binding domain and a second antigen-binding domain. In this application, the first antigen-binding domain and / or the second antigen-binding domain may be an antigen-binding protein. In this application, the first antigen-binding domain and / or the second antigen-binding domain may contain an antibody or an antigen-binding fragment thereof. For example, the first targeting portion may contain a VHH. For example, the second targeting portion may contain an antibody. In this application, the bispecific antibody may contain a first polypeptide chain and a second polypeptide chain. In this application, the bispecific antibody may also contain a constant region. For example, the bispecific antibody may also contain a human IgG constant region.

[0139] In this application, the term "antigen-binding fragment" comprises a portion of a complete antibody that retains the same specific binding ability as the complete antibody; that is, the antigen-binding fragment can bind antigens or antigen fragments that the complete antibody can bind. Antigen-binding fragments can be generated through DNA recombination technology or enzymatic or chemical cleavage of the complete antibody. In some embodiments, antigen-binding fragments include VHH, dAb, Fab, Fab', F(ab')2, F(ab)2, Fv, complementarity-determining region (CDR) fragments, single-chain antibodies (such as scFv), chimeric antibodies, diantibodies, and peptides, wherein at least a portion of the complete antibody is contained, such that the aforementioned molecules retain the same specific binding ability as the complete antibody.

[0140] In this application, the term "CDR," also known as "complementarity-determining region," typically refers to a region within the variable structural domain of an antibody whose sequence is highly variable and / or forms a structurally defining loop. Generally, an antibody includes six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In some embodiments, naturally occurring camel-derived antibodies consisting only of heavy chains can function normally and stably even in the absence of light chains; therefore, such antibodies include CDRs in three VHs (HCDR1, HCDR2, HCDR3). Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia, etc. Different coding schemes may define CDRs with slight differences. For example, the antibody's CDR may be determined by Kabat. Alternatively, the antibody's CDR may be determined by IMGT. In some embodiments, the antibody's CDR may encompass CDR sequences defined by different coding systems for a given VH or VL.

[0141] In this application, the term "FR" generally refers to a more highly conserved portion of the antibody variable domain, referred to as the frame region. Typically, the variable domains of the natural heavy and light chains each contain four FR regions: four in the VH (H-FR1, H-FR2, H-FR3, and H-FR4) and four in the VL (L-FR1, L-FR2, L-FR3, and L-FR4). In this application, the amino acid sequence of the FR can be a FR of any species origin. For example, the FR can be a FR of mouse, rabbit, goat, alpaca, or human origin. For example, the FR can be a mouse FR. For example, the FR can be a human FR.

[0142] In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of the antibody heavy chain and / or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL" (or "VH" and "VL" respectively). These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0143] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, an intact antibody can be digested using papain. After digestion with papain, the antibody produces two identical antigen-binding fragments, namely the "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region, as above). The Fab fragment may consist of a complete L chain with a variable region of a heavy chain and a first constant region (CH1) of the H chain (VH).

[0144] In this application, the terms "immunoglobulin constant region" and "constant region" are generally used interchangeably, referring to a region of the antibody's non-variable region. In this application, the constant region does not directly participate in antigen binding but exhibits various effector functions. In this application, the constant region can be the entire antibody non-variable region or a portion of it. For example, the constant region can be a heavy chain constant region. For example, the constant region can be an Fc region. In this application, the constant region can be derived from animals. For example, the constant region can be derived from humans, goats, rabbits, rats, or guinea pigs. For example, the Fc region can be a human Fc region.

[0145] In this application, the term "monoclonal antibody" generally refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules constituting the group have the same amino acid sequence, except for possible naturally occurring, albeit small, mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. In contrast, polyclonal antibody preparations typically comprise many different antibodies with different amino acid sequences in their variable domains, particularly the CDR, and are generally specific to different epitopes.

[0146] In this application, the term "chimeric antibody" generally refers to an antibody comprising a variable region from one source or species and at least a portion of a constant region from different sources or species, typically prepared using recombinant DNA technology.

[0147] In this application, the term "mouse antibody" generally refers to an antibody whose variable region framework and CDR region are derived from mouse germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. The mouse antibodies of this application may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced through in vitro random or point mutations or through in vivo somatic mutations.

[0148] In this application, the term "humanized antibody" generally refers to an antibody or its variants, derivatives, analogs, or fragments that bind specifically to a target antigen and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity-determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of CDR means that the amino acid sequence of the CDR of a humanized antibody has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of the CDR of a non-human antibody.

[0149] In this application, the term "fully human antibody" generally refers to an antibody whose entire amino acid sequence is derived from the human immunoglobulin gene and which contains no amino acid sequences from mouse or other non-human species. In some embodiments, fully human antibodies have lower immunogenicity.

[0150] In this application, the terms "peptide molecule" and "peptide" are used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two parts covalently linked together. Each part can be a polypeptide with different properties. These properties can be biological properties, such as in vitro or in vivo activity. They can also be simple chemical or physical properties, such as binding to a target molecule, catalysis of a reaction, etc. The two parts can be directly linked by a single peptide bond or through a peptide linker.

[0151] In this application, the term "nucleic acid conjugate" generally refers to a conjugate formed by the coupling of a nucleic acid molecule with an antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment delivers the nucleic acid molecule to target cells / tissues / organs by binding to a target antigen. In some embodiments, the nucleic acid molecule and the antibody or its antigen-binding fragment can be directly or indirectly linked. In some embodiments, the nucleic acid molecule and the antibody or its antigen-binding fragment can be covalently or non-covalently linked. In some embodiments, the nucleic acid molecule and the antibody or its antigen-binding fragment can be covalently linked. In some embodiments, the nucleic acid molecule and the antibody or its antigen-binding fragment can be indirectly linked via a linker, wherein the linker can be a cleavable linker or a non-cleavable linker.

[0152] In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating the other reagents (e.g., chemotherapeutic agents, radioactive elements, cell growth inhibitors, and cytotoxic agents) with the antibody or its antigen-binding fragment (e.g., covalently linked by a linker molecule), which can deliver the other reagents to target cells / tissues / organs by specifically binding the antibody or its antigen-binding fragment to the target antigen.

[0153] In this application, the term "chimeric antigen receptor (CAR)" generally refers to a fusion protein recombinantly expressed in immune effector cells, comprising an extracellular binding domain capable of binding antigens and at least one intracellular domain capable of activating immune effector cells. A chimeric antigen receptor may comprise a signal peptide, an antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain.

[0154] In this application, the term "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in an isolated form, or an analogue isolated from its natural environment or synthesized artificially.

[0155] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein is inserted, thereby enabling the protein to be expressed. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cells. For example, vectors can include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors can include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. The carrier may also include components that help it enter the cell, such as viral particles, liposomes, or protein coats, but not only these substances.

[0156] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that may be or is already a recipient of a subject plasmid or vector, including the nucleic acid molecules or vectors described in this application. Cells may include the progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome). Cells may include cells transfected in vitro using the vectors described in this application. Cells may be bacterial cells (e.g., *E. coli*), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, and NSO cells. The cells may also include engineered cells.

[0157] In this application, the term "pharmaceutical composition" generally refers to a composition used for the prevention / treatment of a disease or condition. Pharmaceutical compositions described in this application include, but are not limited to, liquid, freeze-dried, and lyophilized compositions. In this application, the term "pharmaceuticalally acceptable adjuvant" generally includes pharmaceutically acceptable stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives that are non-toxic to cells or mammals exposed to them at the doses and concentrations employed.

[0158] In this application, the terms “prevention” and / or “treatment” include not only the prevention and / or treatment of a disease, but also generally include preventing the onset of the disease, slowing or reversing the course of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated with it, and / or preventing further increase in the severity of the disease and / or any symptoms associated with it, and preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial to the patient being treated.

[0159] In this application, the terms "therapeutic effective amount" or "effective amount" generally refer to the amount of the antibody of this application sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer). Therapeutic effective amount is related to the disease being treated, whereby a person skilled in the art can readily determine the actual effective amount.

[0160] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0161] In this application, the proteins, peptides, and / or amino acid sequences involved should also be understood to include at least the following range: variants or homologs having the same or similar functions as the said protein or peptide. In this application, the variant can be, for example, a protein or peptide that has undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein and / or the peptide (e.g., an antibody that specifically binds to TfR1 protein or its antigen-binding fragment). For example, the functional variant may comprise a protein or peptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant can substantially retain the biological properties of the protein or peptide before the alteration (e.g., substitution, deletion, or addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the protein or peptide before the alteration. For example, the substitution can be a conserved substitution.

[0162] In this application, the homolog can be a protein or polypeptide that has at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody that specifically binds to the TfR1 protein or its antigen-binding fragment).

[0163] In this application, homology generally refers to the similarity, resemblance, or association between two or more sequences. The "sequence homology percentage" can be calculated as follows: two sequences to be aligned are compared in a comparison window, and the number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is determined to obtain the number of matching positions. The number of matching positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to produce the sequence homology percentage. Alignment for determining the sequence homology percentage can be performed in various ways known in the art.

[0164] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0165] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. Invention Details

[0167] antigen-binding proteins

[0168] On one hand, this application provides an antigen-binding protein that specifically binds to transferrin receptor 1 (TfR1). In some embodiments, the antigen-binding protein can bind to TfR1 with high affinity; for example, the antigen-binding protein provided in this application can bind to TfR1 with a KD value of less than 10. -5 M (for example, can be less than 9 × 10) - 5 M, 8×10 -5 M, 7×10 -5 M, 6×10 -5 M, 5×10 -5 M, 4×10 -5 M, 3×10 -5 M, 2×10 -5 M, 1×10 -6 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 (M or smaller) binds to TfR1. In some embodiments, the antigen-binding protein can bind to TfR1 simultaneously with transferrin.

[0169] In some embodiments, the TfR1 is human TfR1. In some embodiments, the TfR1 is mouse TfR1. In some embodiments, the TfR1 is cynomolgus monkey TfR1. In some embodiments, the TfR1 is an extracellular domain. In some embodiments, the TfR1 is an extracellular domain of human TfR1. In some embodiments, the TfR1 comprises the amino acid sequence shown in SEQ ID NO: 89.

[0170] In some embodiments, the antigen-binding protein may include a heavy chain variable region (VH), wherein the VH may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding protein may include HCDR1, HCDR2, and HCDR3 in the VH, wherein the VH contains an amino acid sequence as shown in any one of SEQ ID NO: 47-75.

[0171] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 may contain the amino acid sequence shown in SEQ ID NO: 1, wherein HCDR2 may contain the amino acid sequence shown in SEQ ID NO: 4, and wherein HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 28.

[0172] In some embodiments, the antigen-binding protein can be obtained from a parent antigen-binding protein through affinity maturation. In this application, the term "affinity maturation" refers to the process of modifying and screening the amino acid sequence of an antibody in vitro using genetic engineering methods to enhance the binding affinity between the antibody and its target antigen. Methods for antibody affinity maturation include, but are not limited to: random mutation; phage display; yeast display, etc. In some embodiments, the antigen-binding protein described in this application can be an antigen-binding protein with enhanced affinity obtained from a parent antigen-binding protein through affinity maturation, wherein the CDR1-3 of the parent antigen-binding protein has the following combination: wherein HCDR1 may contain the amino acid sequence shown in SEQ ID NO: 1, wherein HCDR2 may contain the amino acid sequence shown in SEQ ID NO: 4, and wherein HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 28.

[0173] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR3 may contain an amino acid sequence as shown in SEQ ID NO: 92 (RX2X3DMFYDY), wherein X2 is selected from A, F, I, T, or V, and X3 is selected from D, G, H, Q, V, or Y.

[0174] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR2 may contain components such as SEQ ID NO: 91 (X1X2X3X5X6X7X8X9X). 10 X 11 X 12The amino acid sequence shown in SVKG, wherein X1 is selected from A, I, L, T, or V; X2 is selected from A, F, I, Q, R, T, or V; X3 is selected from R, S, or T; X5 is selected from S or K; X6 is selected from E, G, L, or Q; X7 is selected from A, D, I, L, S, or V; X8 is selected from A, R, or T; X9 is selected from A, Y, or V; X 10 Selected from Y, L, T, X 11 Choose from A, E, or S, and X 12 Selected from D, R, or F.

[0175] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 may contain an amino acid sequence as shown in SEQ ID NO: 90 (DAX3YELYD), where X3 is selected from T or P.

[0176] In some embodiments, the antigen-binding protein may comprise heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR3 may comprise an amino acid sequence as shown in SEQ ID NO: 92 (RX2X3DMFYDY), where X2 is selected from A, F, I, T, or V, and X3 is selected from D, G, H, Q, V, or Y; wherein HCDR2 may comprise an amino acid sequence as shown in SEQ ID NO: 91 (X1X2X3RX5X6X7X8X9X... 10 X 11 X 12 The amino acid sequence shown in SVKG, wherein X1 is selected from A, I, L, T, or V; X2 is selected from A, F, I, Q, R, T, or V; X3 is selected from R, S, or T; X5 is selected from S or K; X6 is selected from E, G, L, or Q; X7 is selected from A, D, I, L, S, or V; X8 is selected from A, R, or T; X9 is selected from A, Y, or V; X 10 Selected from Y, L, T, X 11 Choose from A, E, or S, and X 12 Selected from D, R, or F; wherein the HCDR1 may contain an amino acid sequence as shown in SEQ ID NO: 90 (DAX3YELYD), wherein X3 is selected from T or P.

[0177] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 2-3.

[0178] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR2 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 4-27.

[0179] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR3 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 29-40.

[0180] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 may contain the amino acid sequence shown in SEQ ID NO: 2; wherein HCDR2 contains the amino acid sequence shown in SEQ ID NO: 93 (X1X2X3X5X6X7X8X9X...). 10 X 11 X 12 The amino acid sequence shown in SVKG, wherein X1 is selected from A, I, L, T, or V; X2 is selected from A, F, I, Q, R, T, or V; X3 is selected from R, S, or T; X5 is selected from S or K; X6 is selected from E, G, or Q; X7 is selected from A, D, I, L, S, or V; X8 is selected from A, R, or T; X9 is selected from Y or V; X 10 Selected from Y, L, T, X 11 Choose from A, E, or S, and X 12 The amino acid sequence is selected from D, R, or F; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 94 (RX2X3DMFYDY), wherein X2 is selected from A, F, I, T, or V, and X3 is selected from D, H, Q, V, or Y.

[0181] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 2; wherein HCDR2 comprises an amino acid sequence selected from any one of SEQ ID NO: 4-12, SEQ ID NO: 14-22, and SEQ ID NO: 24-27; and wherein HCDR3 comprises an amino acid sequence selected from any one of SEQ ID NO: 29-33, SEQ ID NO: 35, and SEQ ID NO: 37-40.

[0182] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any of the combinations of HCDR1-3 listed in Table 1 below:

[0183] Table 1. Exemplary CDR Combinations Provided in This Application

[0184]

[0185] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 may contain the amino acid sequence shown in SEQ ID NO: 3; wherein HCDR2 may contain the amino acid sequence shown in SEQ ID NO: 95 (AITRSX6X7TX9YADSVKG), wherein X6 is selected from G or L, X7 is selected from S or V, and X9 is selected from Y or A; and wherein HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 96 (RAX3DMFYDY), wherein X3 is selected from D, Y, V, or G.

[0186] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 may contain an amino acid sequence as shown in SEQ ID NO: 3; wherein HCDR2 may contain an amino acid sequence selected from any one of SEQ ID NO: 6, SEQ ID NO: 13, and SEQ ID NO: 23; and wherein HCDR3 may contain an amino acid sequence selected from any one of SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 36.

[0187] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any of the combinations of HCDR1-3 listed in Table 2 below:

[0188] Table 2. Exemplary CDR Combinations Provided in This Application

[0189]

[0190] In some embodiments, the antigen-binding protein may comprise heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 90 (DAX3YELYD), where X3 is selected from T or P; wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 97 (X1X2TRX5GX7X8YX). 10 The amino acid sequence shown in ADSVKG is as follows, wherein X1 is selected from A, V, or I; X2 is selected from I, T, or V; X5 is selected from S or K; X7 is selected from S or V; X8 is selected from A or T; and X... 10 The amino acid sequence is selected from Y or T; and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 98 (RX2X3DMFYDY), wherein X2 is selected from A or V and X3 is selected from Y or Q.

[0191] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 may contain amino acid sequences as shown in SEQ ID NO: 2-3; wherein HCDR2 may contain amino acid sequences selected from any one of SEQ ID NO: 4-10; and wherein HCDR3 may contain amino acid sequences selected from any one of SEQ ID NO: 29-31.

[0192] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any of the following combinations:

[0193] (1) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 5; HCDR3: SEQ ID NO: 29;

[0194] (2) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 30;

[0195] (3) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 29;

[0196] (4) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 7; HCDR3: SEQ ID NO: 29;

[0197] (5) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 8; HCDR3: SEQ ID NO: 29;

[0198] (6) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 9; HCDR3: SEQ ID NO: 31; and

[0199] (7) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 10; HCDR3: SEQ ID NO: 29.

[0200] In some embodiments, the antigen-binding protein may include heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any of the following combinations:

[0201] (1) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 5; HCDR3: SEQ ID NO: 29;

[0202] (2) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 30;

[0203] (3) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 29;

[0204] (4) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 7; HCDR3: SEQ ID NO: 29;

[0205] (5) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 8; HCDR3: SEQ ID NO: 29;

[0206] (6) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 9; HCDR3: SEQ ID NO: 31;

[0207] (7) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 10; HCDR3: SEQ ID NO: 29;

[0208] (8)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 4;HCDR3: SEQ ID NO: 32;

[0209] (9)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 11;HCDR3: SEQ ID NO: 33;

[0210] (10)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 12;HCDR3: SEQ ID NO: 33;

[0211] (11)HCDR1: SEQ ID NO:3;HCDR2: SEQ ID NO: 13;HCDR3: SEQ ID NO: 34;

[0212] (12)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 35;

[0213] (13)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 14;HCDR3: SEQ ID NO: 32;

[0214] (14)HCDR1: SEQ ID NO:3;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 36;

[0215] (15)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 15;HCDR3: SEQ ID NO: 32;

[0216] (16)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 16;HCDR3: SEQ ID NO: 32;

[0217] (17)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 17;HCDR3: SEQ ID NO: 37;

[0218] (18)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 18;HCDR3: SEQ ID NO: 32;

[0219] (19) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 19; HCDR3: SEQ ID NO: 33;

[0220] (20) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 20; HCDR3: SEQ ID NO: 37;

[0221] (21) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 21; HCDR3: SEQ ID NO: 37;

[0222] (22) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 22; HCDR3: SEQ ID NO: 38;

[0223] (23) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 23; HCDR3: SEQ ID NO: 33;

[0224] (24) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 24; HCDR3: SEQ ID NO: 37;

[0225] (25) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 39;

[0226] (26) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 25; HCDR3: SEQ ID NO: 33;

[0227] (27) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 26; HCDR3: SEQ ID NO: 40; and

[0228] (28) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 27; HCDR3: SEQ ID NO: 37.

[0229] In some embodiments, the antibody CDR can be determined using multiple coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ when using different coding systems. In some embodiments, the CDR encompasses the CDR sequence partitioned according to any CDR partitioning method; it also encompasses its variants, including variations in the amino acid sequence of the CDR that have been substituted, deleted, and / or added to one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; also covering its homologs, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology with the amino acid sequence of the CDR.

[0230] In some embodiments, the antigen-binding protein may include a heavy chain complementarity-determining region (LCM) that may include a heavy chain variable region (VH), wherein the VH may further include HFR1, HFR2, HFR3, and HFR4, wherein the C-terminus of HFR1 may be directly or indirectly linked to the N-terminus of HCDR1, HFR2 may be located between HCDR1 and HCDR2, HFR3 may be located between HCDR2 and HCDR3, and the N-terminus of HFR4 may be directly or indirectly linked to the C-terminus of HCDR3.

[0231] In some embodiments, the antigen-binding protein may include a heavy chain variable region (VH), wherein the VH may also include FRs, specifically HFR1, HFR2, HFR3 and HFR4, wherein the VH contains HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 sequentially from the N-terminus to the C-terminus.

[0232] In some embodiments, the amino acid sequence of the FR can be a FR from any species. For example, the FR can be a FR from mouse, rabbit, goat, alpaca, or human. For example, the FR can be a camel-derived FR. For example, the FR can be a human-derived FR.

[0233] In some embodiments, the amino acid sequence of the FR can be obtained by humanizing camel-derived antibodies or mouse-derived antibodies to obtain humanized FRs. In this application, the term "humanization" generally refers to the process of modifying the amino acid sequence of a non-human antibody (e.g., antibodies from mouse, rabbit, camel, or other non-human mammalian sources) through genetic engineering and recombinant DNA technology to reduce its immunogenicity in the human body while preserving its binding specificity and affinity for the target antigen as much as possible. Antibodies obtained through humanization are called "humanized antibodies." Humanization methods may include, but are not limited to: CDR grafting, which typically refers to grafting the CDR sequence of a non-human antibody into a common human antibody FR, combining the CDR sequence of the non-human antibody with the framework sequence of the human antibody, thereby minimizing immunogenicity while preserving the antigen-binding specificity of the non-human antibody. This may be accompanied by reversion mutations of some amino acid residues in the FR to restore or optimize the binding affinity between the antibody and the antigen.

[0234] In some embodiments, the HFR1 comprises the amino acid sequence shown in SEQ ID NO: 41, the HFR2 comprises the amino acid sequences shown in SEQ ID NO: 42-44, the HFR3 comprises the amino acid sequence shown in SEQ ID NO: 45, and the HFR4 comprises the amino acid sequence shown in SEQ ID NO: 46.

[0235] In some implementations, the HFR1-4 may comprise the following combinations:

[0236] (1) HFR1: SEQ ID NO: 41; HFR2: SEQ ID NO: 42; HFR3: SEQ ID NO: 45; and HFR4: SEQ ID NO: 46;

[0237] (2) HFR1: SEQ ID NO: 41; HFR2: SEQ ID NO: 43; HFR3: SEQ ID NO: 45; and HFR4: SEQ ID NO: 46; and

[0238] (3) HFR1: SEQ ID NO: 41; HFR2: SEQ ID NO: 44; HFR3: SEQ ID NO: 45; and HFR4: SEQ ID NO: 46;.

[0239] In some embodiments, the antigen-binding protein may include a heavy chain variable region (VH), wherein the VH may contain an amino acid sequence as shown in any of SEQ ID NO: 47-75, or the VH may contain an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to any of SEQ ID NO: 47-75.

[0240] In some embodiments, the antigen-binding protein may be a VHH antibody. In some embodiments, the VHH antibody may comprise an amino acid sequence as shown in any one of SEQ ID NO: 47-75, or the VH may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to an amino acid sequence as shown in any one of SEQ ID NO: 47-75.

[0241] Table 3. Exemplary VHH sequences provided in this application

[0242]

[0243]

[0244] In some embodiments, the antigen-binding protein is a separated antigen-binding protein.

[0245] In some embodiments, the antigen-binding protein may include an antibody or an antigen-binding fragment thereof.

[0246] In some embodiments, the antigen-binding protein may be an antigen-binding fragment, wherein the antigen-binding fragment may be a VHH, dAb, Fab, Fab', F(ab')2, F(ab)2, Fv fragment, complementarity-determining region (CDR) fragment, single-chain antibody (such as scFv), chimeric antibody, diantibody, or peptide.

[0247] In some embodiments, the antigen-binding fragment may be VHH.

[0248] In some embodiments, the antibody may include a monoclonal antibody, a monospecific antibody, a bispecific antibody, and / or a multispecific antibody.

[0249] In some embodiments, the antibody may include camel-derived antibodies, mouse-derived antibodies, humanized antibodies, and / or fully human antibodies.

[0250] In some embodiments, the antibody may include a single-chain antibody, a nanobody, a heavy-chain antibody, and / or a chimeric antibody.

[0251] In some embodiments, the antibody may be a VHH antibody or a heavy chain antibody.

[0252] In some embodiments, the antigen-binding protein may be a VHH antibody, wherein the VHH antibody may be a camel-derived antibody, a mouse-derived antibody, or a humanized antibody.

[0253] The antigen-binding protein described in this application may comprise heavy and / or light chain sequences with one or more conserved sequence modifications. A "conserved sequence modification" refers to an amino acid modification that does not significantly affect or alter antibody binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding protein described in this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. A conserved amino acid substitution involves replacing an amino acid residue with an amino acid residue having a similar side chain. A set of amino acid residues with similar side chains is known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the heavy chain variable region and / or light chain variable region of the isolated antigen-binding protein described in this application may be replaced with other amino acid residues from the same side chain group. Those skilled in the art will recognize that some conserved sequence modifications will not cause the antigen binding to disappear.

[0254] Bispecific antibodies

[0255] In some embodiments, the antigen-binding protein may be a multispecific antibody, such as a bispecific antibody or a trispecific antibody.

[0256] In some embodiments, the antigen-binding protein is a bispecific antibody, wherein it may include a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain can specifically bind to TfR1, and the first antigen-binding domain may include the antigen-binding protein that specifically binds to TfR1 as described in this application.

[0257] In some embodiments, the first antigen-binding domain that specifically binds to TfR1 may be a VHH. In some embodiments, the VHH may comprise the antigen-binding protein, which may comprise heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any combination of the following:

[0258] (1) HCDR1: SEQ ID NO: 1; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 28;

[0259] (2) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 5; HCDR3: SEQ ID NO: 29;

[0260] (3) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 30;

[0261] (4) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 29;

[0262] (5) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 7; HCDR3: SEQ ID NO: 29;

[0263] (6) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 8; HCDR3: SEQ ID NO: 29;

[0264] (7) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 9; HCDR3: SEQ ID NO: 31;

[0265] (8) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 10; HCDR3: SEQ ID NO: 29;

[0266] (9) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 32;

[0267] (10)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 11;HCDR3: SEQ ID NO: 33;

[0268] (11)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 12;HCDR3: SEQ ID NO: 33;

[0269] (12)HCDR1: SEQ ID NO:3;HCDR2: SEQ ID NO: 13;HCDR3: SEQ ID NO: 34;

[0270] (13)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 35;

[0271] (14)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 14;HCDR3: SEQ ID NO: 32;

[0272] (15)HCDR1: SEQ ID NO:3;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 36;

[0273] (16)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 15;HCDR3: SEQ ID NO: 32;

[0274] (17)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 16;HCDR3: SEQ ID NO: 32;

[0275] (18)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 17;HCDR3: SEQ ID NO: 37;

[0276] (19)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 18;HCDR3: SEQ ID NO: 32;

[0277] (20)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 19;HCDR3: SEQ ID NO: 33;

[0278] (21) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 20; HCDR3: SEQ ID NO: 37;

[0279] (22) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 21; HCDR3: SEQ ID NO: 37;

[0280] (23) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 22; HCDR3: SEQ ID NO: 38;

[0281] (24) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 23; HCDR3: SEQ ID NO: 33;

[0282] (25) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 24; HCDR3: SEQ ID NO: 37;

[0283] (26) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 39;

[0284] (27) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 25; HCDR3: SEQ ID NO: 33;

[0285] (28) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 26; HCDR3: SEQ ID NO: 40; and

[0286] (29) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 27; HCDR3: SEQ ID NO: 37.

[0287] In some embodiments, the first antigen-binding domain that specifically binds to TfR1 may be a VHH. In some embodiments, the VHH may comprise an amino acid sequence as shown in any of SEQ ID NO: 47-75.

[0288] In some embodiments, the second antigen-binding domain may specifically bind to targets associated with the central nervous system (CNS). In some embodiments, the second antigen-binding domain may be a second antigen-binding domain that specifically binds to galactolectin-3 (Gal-3).

[0289] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 may comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH may comprise heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the VL may comprise heavy chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 76, wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 77, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 78; and wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 79, wherein LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 80, and wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 81.

[0290] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 may comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH may comprise the amino acid sequence shown in SEQ ID NO: 82, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 83.

[0291] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 can be an antibody or an antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 can be an antigen-binding fragment, wherein the antigen-binding fragment can be a VHH, dAb, Fab, Fab', F(ab')2, F(ab)2, Fv fragment, complementarity-determining region (CDR) fragment, single-chain antibody (such as scFv), chimeric antibody, diantibody, or peptide. In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 can be an antibody, wherein the antibody can be a monoclonal antibody, murine antibody, chimeric antibody, humanized antibody, or fully human antibody.

[0292] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 may be an antibody. In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 may comprise a heavy chain constant region and a light chain constant region, wherein the constant region may comprise CH1, CH2, and CH3, and wherein the light chain constant region may comprise CL. In some embodiments, the heavy chain constant region may comprise Fc.

[0293] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 may comprise a heavy chain (HC) and a light chain (LC), wherein the HC may comprise the amino acid sequence shown in SEQ ID NO: 84, and the LC may comprise the amino acid sequence shown in SEQ ID NO: 85.

[0294] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 may comprise two heavy chains (HC) and two light chains (LC), wherein the amino acid sequences of the two HCs may be identical, and the amino acid sequences of the two LCs may be identical.

[0295] In some embodiments, the second antigen-binding domain that specifically binds to Gal-3 may comprise two heavy chains (HC) and two light chains (LC), wherein the amino acid sequences of the two LCs may be identical, and the amino acid sequences of the two HCs may be different. Mutations may be introduced into the Fc of the two HCs to promote the formation of heterodimers. For example, the mutation may be a knock-in-hole mutation (also known as KIH, see Ridgway JB, Presta LG, Carter P. Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996 Jul; 9(7):617-21.). In some embodiments, the first HC of the two HCs may contain a knob mutation, and the second HC of the two HCs may contain a hole mutation corresponding to the knob, wherein the knob mutation may be T366W, and the hole mutation corresponding to the knob may be T366S, L368A and / or Y407V, wherein the amino acid residues are numbered according to the EU index shown in Kabat.

[0296] In some embodiments, the first antigen-binding domain specifically binding to TfR1 and the second antigen-binding domain specifically binding to Gal-3 can be directly connected. In some embodiments, the N-terminus of the first antigen-binding domain specifically binding to TfR1 can be directly connected to the C-terminus of the second antigen-binding domain specifically binding to Gal-3. In some embodiments, the C-terminus of the first antigen-binding domain specifically binding to TfR1 can be directly connected to the N-terminus of the second antigen-binding domain specifically binding to Gal-3. In some embodiments, the N-terminus of the first antigen-binding domain specifically binding to TfR1 can be directly connected to the C-terminus of the Fc region of the heavy chain constant region of the second antigen-binding domain specifically binding to Gal-3.

[0297] In some embodiments, the first antigen-binding domain specifically binding to TfR1 and the second antigen-binding domain specifically binding to Gal-3 can be indirectly connected. For example, the first antigen-binding domain specifically binding to TfR1 and the second antigen-binding domain specifically binding to Gal-3 can be indirectly connected via a linker. In some embodiments, the N-terminus of the first antigen-binding domain specifically binding to TfR1 can be indirectly connected to the C-terminus of the second antigen-binding domain specifically binding to Gal-3 via a linker. In some embodiments, the C-terminus of the first antigen-binding domain specifically binding to TfR1 can be indirectly connected to the N-terminus of the second antigen-binding domain specifically binding to Gal-3 via a linker. In some embodiments, the N-terminus of the first antigen-binding domain specifically binding to TfR1 can be indirectly connected to the C-terminus of the Fc region of the heavy chain constant region of the second antigen-binding domain specifically binding to Gal-3 via a linker.

[0298] In some embodiments, the linker may be a polypeptide or a chemical linker. In some embodiments, the linker may be a polypeptide, wherein the polypeptide may comprise n repeating amino acid fragments. In some embodiments, the linker may be (GGGS)n, where n is an integer selected from 1 to 10. In some embodiments, the linker may be (GGGGS)n, where n is an integer selected from 1 to 10. In some embodiments, the linker may be GGGS, (GGGS)2, (GGGS)3, (GGGS)4, (GGGS)5, (GGGS)6, (GGGS)7, (GGGS)8, (GGGS)9, or (GGGS)10. In some implementations, the connector can be GGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, (GGGGS)6, (GGGGS)7, (GGGGS)8, (GGGGS)9 or (GGGGS)10.

[0299] In some embodiments, the bispecific antibody may comprise a first peptide chain and a second peptide chain. In this application, the first peptide chain may comprise an antibody heavy chain variable region (VH) capable of binding Gal-3 and a VHH capable of binding TfR1, and the second peptide chain may comprise an antibody light chain variable region (VL) capable of binding Gal-3. In some embodiments, in the first peptide chain, the Gal-3-binding heavy chain variable region (VH) is located at the N-terminus of the TfR1-binding VHH. In some embodiments, in the first peptide chain, the Gal-3-binding heavy chain variable region (VH) is located at the C-terminus of the TfR1-binding VHH.

[0300] In some embodiments, the first peptide chain may further include a heavy chain constant region CH. For example, the heavy chain constant region may be an IgG constant region. For example, the IgG constant region may be a human IgG constant region. For example, the IgG constant region may be a human IgG1, IgG2, or IgG4 constant region. For example, the IgG constant region may be a human IgG1 constant region. For example, the CH may be located at the C-terminus of the heavy chain variable region VH capable of binding Gal-3 and at the N-terminus of the VHH capable of binding TfR1. For example, the CH may be located at the C-terminus of the heavy chain variable region VH capable of binding Gal-3, and the VH may be located at the C-terminus of the VHH capable of binding Gal-3. For example, the first peptide chain may further include a linker. For example, the heavy chain constant region CH and the VHH capable of binding TfR1 are connected by a linker.

[0301] In some embodiments, the second peptide chain may further include a light chain constant region CL. In some embodiments, the light chain constant region CL may be a human Igκ constant region or a human Igλ constant region. In some embodiments, the light chain constant region CL may be a human Igκ constant region. For example, the light chain constant region CL may be located at the C-terminus of the light chain variable region VL capable of binding Gal-3.

[0302] In some embodiments, the bispecific antibody may comprise a first peptide chain, a second peptide chain, a third peptide chain, and a fourth peptide chain, wherein the second and fourth peptide chains are identical, and wherein the first peptide chain, from N-terminus to C-terminus, may sequentially comprise: a heavy chain HC that specifically binds to the second antigen-binding domain of Gal-3 and a VHH that specifically binds to the first antigen-binding domain of TfR1; wherein the second and fourth peptide chains, from N-terminus to C-terminus, may sequentially comprise: an LC that specifically binds to the second antigen-binding domain of Gal-3; wherein the third peptide chain, from N-terminus to C-terminus, may sequentially comprise: a heavy chain HC that specifically binds to the second antigen-binding domain of Gal-3. For example, the bispecific antibody may be as shown in the appendix. Figure 3 The structure shown.

[0303] Coupled

[0304] On the other hand, this application provides a conjugate comprising one or more of the antigen-binding proteins and therapeutic entities described in this application.

[0305] In some embodiments, the therapeutic entity may be an entity with disease-treating or disease-preventing functions. In some embodiments, the therapeutic entity may be a small molecule compound, protein, nucleic acid molecule, or any fragment thereof.

[0306] In some embodiments, the therapeutic entity may be a small molecule compound, such as a chelating agent, antibiotic, antiviral agent, immunomodulator, antitumor drug, anti-inflammatory drug, or adjuvant.

[0307] In some embodiments, the therapeutic entity may be a protein, such as an enzyme, hormone, neurotrophic factor, neuropeptide, cytokine, apolipoprotein, growth factor, antigen, antibody, or antibody fragment.

[0308] In some embodiments, the therapeutic entity may be a nucleic acid molecule, such as mRNA, ribozyme, or oligonucleotide, wherein the oligonucleotide is selected from any single-stranded or double-stranded oligonucleotide, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmer oligonucleotide, antisense oligonucleotide (ASO), short hairpin RNA (shRNA), microRNA (miRNA), aptamer RNA, bridging nucleic acid (BNA), etc.

[0309] In some embodiments, the conjugate is a nucleic acid conjugate. In some embodiments, the nucleic acid conjugate comprises the antigen-binding protein and the nucleic acid molecule described in this application.

[0310] Peptides, chimeric antigen receptors and immune conjugates

[0311] On the other hand, this application provides a polypeptide molecule that includes the antigen-binding protein described above.

[0312] On the other hand, this application provides a chimeric antigen receptor comprising the antigen-binding protein described above.

[0313] On the other hand, this application provides an immunoconjugate comprising the antigen-binding protein described above.

[0314] Nucleic acids, vectors and cells

[0315] On the other hand, this application provides a nucleic acid molecule that encodes the antigen-binding protein, the polypeptide molecule, or the chimeric antigen receptor.

[0316] This application provides one or more nucleic acid molecules that can encode the antigen-binding protein or a functional fragment thereof described in this application. For example, each of the one or more nucleic acid molecules can encode the complete antigen-binding protein or a portion thereof.

[0317] The nucleic acid molecules described in this application encompass polynucleotides containing only the coding sequence of the antigen-binding protein, as well as polynucleotides containing additional coding and / or non-coding sequences.

[0318] The nucleic acid molecules described in this application can be isolated. For example, they can be produced or synthesized by: (i) in vitro amplification, such as by polymerase chain reaction (PCR), (ii) clonal recombination, (iii) purification, such as separation by enzyme digestion and gel electrophoresis, or (iv) synthesis, such as chemical synthesis. The isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA technology. In this application, nucleic acids encoding the antibody and its antigen-binding fragment can be prepared by a variety of methods known in the art.

[0319] The nucleic acid molecules provided in this application can be in the form of RNA or DNA. The DNA can be cDNA, genomic DNA, or synthetic DNA, and can be double-stranded or single-stranded. Single-stranded DNA can be a coding strand or a non-coding (antisense) strand. The nucleic acid molecules provided in this application can be mRNA.

[0320] On the other hand, this application provides a carrier containing the aforementioned nucleic acid molecules.

[0321] Each vector may contain one or more of the aforementioned nucleic acid molecules. Furthermore, the vector may contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. The expression control sequence may be a tunable element. The specific structure of the expression control sequence may vary depending on the species or cell type and function, but typically includes 5' non-transcriptional sequences and 5' and 3' non-translational sequences, respectively, involved in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcriptional expression control sequence may contain a promoter region, which may contain a promoter sequence for transcriptionally controlling the functional linker nucleic acid. The expression control sequence may also include enhancer sequences or upstream activator sequences. In this application, suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, human U6RNA promoters, CMV promoters, and EF1a promoters. One or more nucleic acid molecules described in this application may be operatively linked to the expression control element. The vector may include mammalian vectors, such as vectors expressing the antigen-binding proteins described in this application in human cells. The vector may include viral vectors, such as retroviral vectors, lentiviral vectors, etc.

[0322] On the other hand, this application provides a cell comprising the antigen-binding protein, the polypeptide molecule, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, and / or the carrier. The nucleic acid molecule described in this application can be introduced into the cell using methods known in the art, such as electroporation, liposome transfection, viral infection, etc. In some embodiments, the cell provided in this application can be a mammalian cell. In some embodiments, the cell provided in this application can be a human cell.

[0323] Pharmaceutical Composition

[0324] On the other hand, this application provides a pharmaceutical composition comprising the antigen-binding protein, the bispecific antibody, the nucleic acid conjugate, the polypeptide molecule, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, the carrier, and / or the cell, and optionally a pharmaceutically acceptable adjuvant.

[0325] In some embodiments, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions described in this application include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0326] In some embodiments, the pharmaceutical composition may also contain more than one active compound, typically those with complementary activities that do not adversely affect each other. The type and effective amount of such a drug may depend, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.

[0327] In some embodiments, the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents that are compatible with drug administration and are generally safe and non-toxic.

[0328] In some embodiments, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally. In some embodiments, the pharmaceutical composition may be administered continuously.

[0329] Preparation method

[0330] On the other hand, this application provides a method for preparing the antigen-binding protein. The preparation method may include culturing the cells described in this application under conditions that allow the antigen-binding protein to be expressed. For example, this can be achieved by using appropriate culture media, appropriate temperatures, and culture times, methods known to those skilled in the art.

[0331] use

[0332] On the other hand, this application provides the use of the antigen-binding protein that specifically binds to TfR1 described in this application to deliver a therapeutic entity to cells, tissues or organs expressing TfR1.

[0333] On the other hand, this application provides the use of the antigen-binding protein that specifically binds to TfR1 described in this application for the delivery of therapeutic entities across the blood-brain barrier (BBB).

[0334] On the other hand, this application also provides a method for delivering a therapeutic entity to cells, tissues, or organs expressing TfR1.

[0335] On the other hand, this application also provides a method for delivering a therapeutic entity across the blood-brain barrier (BBB).

[0336] In some embodiments, the therapeutic entity may be an entity with disease-treating or disease-preventing functions. In some embodiments, the therapeutic entity may be a small molecule compound, a protein, a nucleic acid molecule, or any fragment thereof.

[0337] In some embodiments, the therapeutic entity may be a small molecule compound, such as a chelating agent, antibiotic, antiviral agent, immunomodulator, antitumor drug, anti-inflammatory drug, or adjuvant.

[0338] In some embodiments, the therapeutic entity may be a protein, such as an enzyme, hormone, neurotrophic factor, neuropeptide, cytokine, apolipoprotein, growth factor, antigen, antibody, or antibody fragment.

[0339] In some embodiments, the therapeutic entity may be a nucleic acid molecule, such as mRNA, ribozyme, or oligonucleotide, wherein the oligonucleotide may be selected from any single-stranded or double-stranded oligonucleotide, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmer oligonucleotide, antisense oligonucleotide (ASO), short hairpin RNA (shRNA), microRNA (miRNA), aptamer RNA, bridging nucleic acid (BNA), etc.

[0340] In some embodiments, the therapeutic entity may be coupled to an antigen-binding protein that specifically binds to TfR1 as described in this application.

[0341] On the other hand, this application provides the use of the bispecific antibodies, polypeptide molecules, nucleic acid conjugates, chimeric antigen receptors, immunoconjugates, nucleic acid molecules, carriers, cells, and / or pharmaceutical compositions described in this application for the preparation of preventive and / or therapeutic drugs and / or diseases.

[0342] On the other hand, this application provides a method for preventing and / or treating diseases and / or conditions, the method comprising administering to a subject in need the bispecific antibody described in this application, the nucleic acid conjugate described in this application, the polypeptide molecule described in this application, the chimeric antigen receptor described in this application, the immunoconjugate described in this application, the nucleic acid molecule described in this application, the carrier described in this application, the cell described in this application, and / or the pharmaceutical composition described in this application.

[0343] In some embodiments, the disease and / or condition may include diseases and / or conditions related to the nervous system (e.g., the central nervous system). In some embodiments, the subject may include a human or other mammal.

[0344] In some implementations, the application can be performed in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal application.

[0345] Implementation

[0346] 1. An antigen-binding protein capable of specifically binding to transferrin receptor 1 (TfR1), the antigen-binding protein comprising a heavy chain variable region (VH), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 92 (RX2X3DMFYDY), wherein X2 is selected from A or F or I or T or V, and X3 is selected from D or G or H or Q or V or Y.

[0347] 2. The antigen-binding protein according to Embodiment 1, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR2 comprises as shown in SEQ ID NO: 91 (X1X2X3X5X6X7X8X9X... 10 X 11 X 12 The amino acid sequence shown in SVKG, wherein X1 is selected from A, I, L, T, or V; X2 is selected from A, F, I, Q, R, T, or V; X3 is selected from R, S, or T; X5 is selected from S or K; X6 is selected from E, G, L, or Q; X7 is selected from A, D, I, L, S, or V; X8 is selected from A, R, or T; X9 is selected from A, Y, or V; X 10 Selected from Y, L, T, X 11 Choose from A, E, or S, and X 12 Selected from D, R, or F.

[0348] 3. The antigen-binding protein according to embodiment 1 or 2, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 90 (DAX3YELYD), wherein X3 is selected from T or P.

[0349] 4. The antigen-binding protein according to any one of embodiments 1-3, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 2-3.

[0350] 5. The antigen-binding protein according to any one of embodiments 1-4, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR2 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 4-27.

[0351] 6. The antigen-binding protein according to any one of embodiments 1-5, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 29-40.

[0352] 7. The antigen-binding protein according to any one of embodiments 1-6, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2;

[0353] The HCDR2 mentioned above includes, for example, SEQ ID NO: 93 (X1X2X3X5X6X7X8X9X). 10 X 11 X 12 The amino acid sequence shown in SVKG, wherein X1 is selected from A, I, L, T, or V; X2 is selected from A, F, I, Q, R, T, or V; X3 is selected from R, S, or T; X5 is selected from S or K; X6 is selected from E, G, or Q; X7 is selected from A, D, I, L, S, or V; X8 is selected from A, R, or T; X9 is selected from Y or V; X 10 Selected from Y, L, T, X 11 Choose from A, E, or S, and X 12 Selected from D, R, or F;

[0354] Furthermore, the HCDR3 contains an amino acid sequence as shown in SEQ ID NO: 94 (RX2X3DMFYDY), wherein X2 is selected from A, F, I, T, or V, and X3 is selected from D, H, Q, V, or Y.

[0355] 8. The antigen-binding protein according to Embodiment 7, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 2; wherein HCDR2 comprises an amino acid sequence selected from any one of SEQ ID NO: 4-12, SEQ ID NO: 14-22, and SEQ ID NO: 24-27; and wherein HCDR3 comprises an amino acid sequence selected from any one of SEQ ID NO: 29-33, SEQ ID NO: 35, and SEQ ID NO: 37-40.

[0356] 9. The antigen-binding protein according to embodiment 7 or 8, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any group of the following combinations:

[0357] 1) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 5; HCDR3: SEQ ID NO: 29;

[0358] 2) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 30;

[0359] 3) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 7; HCDR3: SEQ ID NO: 29;

[0360] 4) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 8; HCDR3: SEQ ID NO: 29;

[0361] 5) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 9; HCDR3: SEQ ID NO: 31;

[0362] 6) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 10; HCDR3: SEQ ID NO: 29;

[0363] 7) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 32;

[0364] 8)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 11;HCDR3: SEQ ID NO: 33;

[0365] 9)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 12;HCDR3: SEQ ID NO: 33;

[0366] 10)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 35;

[0367] 11)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 14;HCDR3: SEQ ID NO: 32;

[0368] 12)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 15;HCDR3: SEQ ID NO: 32;

[0369] 13)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 16;HCDR3: SEQ ID NO: 32;

[0370] 14)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 17;HCDR3: SEQ ID NO: 37;

[0371] 15)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 18;HCDR3: SEQ ID NO: 32;

[0372] 16)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 19;HCDR3: SEQ ID NO: 33;

[0373] 17)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 20;HCDR3: SEQ ID NO: 37;

[0374] 18)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 21;HCDR3: SEQ ID NO: 37;

[0375] 19) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 22; HCDR3: SEQ ID NO: 38;

[0376] 20) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 24; HCDR3: SEQ ID NO: 37;

[0377] 21) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 39;

[0378] 22) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 25; HCDR3: SEQ ID NO: 33;

[0379] 23) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 26; HCDR3: SEQ ID NO: 40; and

[0380] 24) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 27; HCDR3: SEQ ID NO: 37.

[0381] 10. The antigen-binding protein according to any one of embodiments 1-6, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3;

[0382] The HCDR2 contains an amino acid sequence as shown in SEQ ID NO: 95 (AITRSX6X7TX9YADSVKG), wherein X6 is selected from G or L, X7 is selected from S or V, and X9 is selected from Y or A;

[0383] Furthermore, the HCDR3 contains an amino acid sequence as shown in SEQ ID NO: 96 (RAX3DMFYDY), wherein X3 is selected from D, Y, V, or G.

[0384] 11. The antigen-binding protein according to embodiment 10, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3; wherein HCDR2 comprises the amino acid sequence selected from any one of SEQ ID NO: 6, SEQ ID NO: 13, and SEQ ID NO: 23; and wherein HCDR3 comprises the amino acid sequence selected from any one of SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 36.

[0385] 12. The antigen-binding protein according to embodiment 10 or 11, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any group of the following combinations:

[0386] 1) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 29;

[0387] 2) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 13; HCDR3: SEQ ID NO: 34;

[0388] 3) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 36; and

[0389] 4) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 23; HCDR3: SEQ ID NO: 33.

[0390] 13. The antigen-binding protein according to any one of embodiments 1-6, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 90 (DAX3YELYD), wherein X3 is selected from T or P;

[0391] The HCDR2 mentioned above contains, for example, SEQ ID NO: 97 (X1X2TRX5GX7X8YX). 10 The amino acid sequence shown in ADSVKG is as follows, wherein X1 is selected from A, V, or I; X2 is selected from I, T, or V; X5 is selected from S or K; X7 is selected from S or V; X8 is selected from A or T; and X... 10Selected from Y or T;

[0392] Furthermore, the HCDR3 contains an amino acid sequence as shown in SEQ ID NO: 98 (RX2X3DMFYDY), wherein X2 is selected from A or V, and X3 is selected from Y or Q.

[0393] 14. The antigen-binding protein according to embodiment 13, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in any one of SEQ ID NO: 2-3; wherein HCDR2 comprises an amino acid sequence selected from any one of SEQ ID NO: 4-10; wherein HCDR3 comprises an amino acid sequence selected from any one of SEQ ID NO: 29-31.

[0394] 15. The antigen-binding protein according to embodiment 13 or 14, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any group of the following combinations:

[0395] 1) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 5; HCDR3: SEQ ID NO: 29;

[0396] 2) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 30;

[0397] 3) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 29;

[0398] 4) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 7; HCDR3: SEQ ID NO: 29;

[0399] 5) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 8; HCDR3: SEQ ID NO: 29;

[0400] 6) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 9; HCDR3: SEQ ID NO: 31; and

[0401] 7) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 10; HCDR3: SEQ ID NO: 29.

[0402] 16. The antigen-binding protein according to any one of embodiments 1-15, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any group of the following combinations:

[0403] 1) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 5; HCDR3: SEQ ID NO: 29;

[0404] 2) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 30;

[0405] 3) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 29;

[0406] 4) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 7; HCDR3: SEQ ID NO: 29;

[0407] 5) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 8; HCDR3: SEQ ID NO: 29;

[0408] 6) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 9; HCDR3: SEQ ID NO: 31;

[0409] 7) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 10; HCDR3: SEQ ID NO: 29;

[0410] 8) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 32;

[0411] 9) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 11; HCDR3: SEQ ID NO: 33;

[0412] 10)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 12;HCDR3: SEQ ID NO: 33;

[0413] 11)HCDR1: SEQ ID NO:3;HCDR2: SEQ ID NO: 13;HCDR3: SEQ ID NO: 34;

[0414] 12)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 35;

[0415] 13)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 14;HCDR3: SEQ ID NO: 32;

[0416] 14)HCDR1: SEQ ID NO:3;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 36;

[0417] 15)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 15;HCDR3: SEQ ID NO: 32;

[0418] 16)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 16;HCDR3: SEQ ID NO: 32;

[0419] 17)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 17;HCDR3: SEQ ID NO: 37;

[0420] 18)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 18;HCDR3: SEQ ID NO: 32;

[0421] 19)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 19;HCDR3: SEQ ID NO: 33;

[0422] 20)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 20;HCDR3: SEQ ID NO: 37;

[0423] 21) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 21; HCDR3: SEQ ID NO: 37;

[0424] 22) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 22; HCDR3: SEQ ID NO: 38;

[0425] 23) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 23; HCDR3: SEQ ID NO: 33;

[0426] 24) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 24; HCDR3: SEQ ID NO: 37;

[0427] 25) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 39;

[0428] 26) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 25; HCDR3: SEQ ID NO: 33;

[0429] 27) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 26; HCDR3: SEQ ID NO: 40; and

[0430] 28) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 27; HCDR3: SEQ ID NO: 37.

[0431] 17. The antigen-binding protein according to any one of embodiments 1-16, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

[0432] 18. The antigen-binding protein according to embodiment 17, wherein the antigen-binding fragment is (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH or dAb fragment.

[0433] 19. The antigen-binding protein according to any one of embodiments 1-18, wherein the antigen-binding protein is VHH.

[0434] 20. The antigen-binding protein according to any one of embodiments 1-19, wherein the TfR1 is human, mouse, or monkey TfR1.

[0435] 21. The antigen-binding protein according to any one of embodiments 1-20, wherein the VHH comprises an amino acid sequence as shown in any one of SEQ ID NO: 47-75, or the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to an amino acid sequence as shown in any one of SEQ ID NO: 47-75.

[0436] 22. A bispecific antibody comprising a first antigen-binding domain capable of specifically binding to TfR1, wherein the first antigen-binding domain specifically binding to TfR1 comprises the antigen-binding protein of any one of embodiments 1-21.

[0437] 23. The bispecific antibody according to embodiment 22, wherein the bispecific antibody further comprises a second antigen-binding domain capable of specifically binding to galactolectin-3 (Gal-3).

[0438] 24. The bispecific antibody according to embodiment 22 or 23, wherein the second antigen-binding domain specifically binding to Gal-3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 76, wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 77, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 78; and wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 79, wherein LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 80, and wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 81.

[0439] 25. The bispecific antibody according to any one of embodiments 22-24, wherein the second antigen-binding domain that specifically binds to Gal-3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:82, and the VL comprises the amino acid sequence shown in SEQ ID NO:83.

[0440] 26. The bispecific antibody according to any one of embodiments 22-25, wherein the first antigen-binding domain that specifically binds to TfR1 is directly or indirectly connected to the second antigen-binding domain that specifically binds to Gal-3.

[0441] 27. The bispecific antibody according to any one of embodiments 22-26, wherein the first antigen-binding domain that specifically binds to TfR1 is indirectly connected to the second antigen-binding domain that specifically binds to Gal-3 via a linker.

[0442] 28. The bispecific antibody according to any one of Embodiment 27, wherein the linker is a peptide linker, the peptide linker is (GGGGS)n, and the n is an integer selected from 1 to 10.

[0443] 29. The bispecific antibody according to any one of embodiments 22-28, wherein the N-terminus of the first antigen-binding domain that specifically binds to TfR1 is directly or indirectly connected to the C-terminus of the second antigen-binding domain that specifically binds to Gal-3, or the C-terminus of the first antigen-binding domain that specifically binds to TfR1 is directly or indirectly connected to the N-terminus of the second antigen-binding domain that specifically binds to Gal-3.

[0444] 30. The bispecific antibody according to any one of embodiments 22-29, wherein the second antigen-binding domain that specifically binds to Gal-3 is an antigen-binding fragment or an antibody.

[0445] 31. The bispecific antibody according to embodiment 30, wherein the second antigen-binding domain that specifically binds to Gal-3 is an antigen-binding fragment, wherein the antigen-binding fragment is scFv, Fab, or VHH.

[0446] 32. The bispecific antibody according to embodiment 30, wherein the second antigen-binding domain that specifically binds to Gal-3 is an antibody, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0447] 33. The bispecific antibody according to any one of embodiments 22-32, wherein the second antigen-binding domain that specifically binds to Gal-3 is an antibody, wherein the second antigen-binding domain that specifically binds to Gal-3 further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises Fc.

[0448] 34. The bispecific antibody according to any one of embodiments 22-33, wherein the second antigen-binding domain that specifically binds to Gal-3 comprises a full-length heavy chain (HC) and a full-length light chain (LC), wherein the HC comprises the amino acid sequence shown in SEQ ID NO: 84, and the LC comprises the amino acid sequence shown in SEQ ID NO: 85.

[0449] 35. The bispecific antibody according to any one of embodiments 22-34, wherein the N-terminus of the first antigen-binding protein is directly or indirectly linked to the C-terminus of the Fc of the second antigen-binding domain that specifically binds Gal-3.

[0450] 36. A fusion protein comprising the antigen-binding protein of any one of embodiments 1-21 or the bispecific antibody of any one of embodiments 22-35.

[0451] 37. A nucleic acid conjugate comprising the antigen-binding protein of any one of embodiments 1-21, the bispecific antibody of any one of embodiments 22-35, or the fusion protein of embodiment 36.

[0452] 38. A chimeric antigen receptor comprising the antigen-binding protein of any one of embodiments 1-21, the bispecific antibody of any one of embodiments 22-35, or the fusion protein of embodiment 36.

[0453] 39. An immunoconjugate comprising the antigen-binding protein of any one of embodiments 1-21, the bispecific antibody of any one of embodiments 22-35, or the fusion protein of embodiment 36.

[0454] 40. A nucleic acid molecule encoding an antigen-binding protein of any one of embodiments 1-21, a bispecific antibody of any one of embodiments 22-35, a fusion protein of embodiment 36, a nucleic acid conjugate of embodiment 37, and / or a chimeric antigen receptor of embodiment 38.

[0455] 41. A carrier comprising the nucleic acid molecule described in embodiment 40.

[0456] 42. A cell comprising the antigen-binding protein of any one of embodiments 1-21, the bispecific antibody of any one of embodiments 22-35, the fusion protein of embodiment 36, the nucleic acid conjugate of embodiment 37, the chimeric antigen receptor of embodiment 38, the immunoconjugate of embodiment 39, the nucleic acid molecule of embodiment 40, and / or the carrier of embodiment 41.

[0457] 43. A pharmaceutical composition comprising the antigen-binding protein of any one of embodiments 1-21, the bispecific antibody of any one of embodiments 22-35, the fusion protein of embodiment 36, the nucleic acid conjugate of embodiment 37, the chimeric antigen receptor of embodiment 38, the immunoconjugate of embodiment 39, the nucleic acid molecule of embodiment 40, the carrier of embodiment 41 and / or the cell of embodiment 42, and optionally a pharmaceutically acceptable carrier.

[0458] 44. A kit comprising the antigen-binding protein of any one of Embodiments 1-21, the bispecific antibody of any one of Embodiments 22-35, the fusion protein of Embodiment 36, the nucleic acid conjugate of Embodiment 37, the chimeric antigen receptor of Embodiment 38, the immunoconjugate of Embodiment 39, the nucleic acid molecule of Embodiment 40, the carrier of Embodiment 41, the cell of Embodiment 42, and / or the pharmaceutical composition of Embodiment 43.

[0459] 45. Use of the antigen-binding protein delivered to the therapeutic entity across the blood-brain barrier according to any one of embodiments 1-21.

[0460] 46. ​​According to the use described in embodiment 45, the antigen-binding protein delivers a therapeutic entity to cells, tissues, or organs expressing TfR1.

[0461] 47. The use according to embodiment 45 or 46, wherein the therapeutic entity is an entity having a function of treating or preventing disease, and wherein the therapeutic entity is a small molecule compound, protein, nucleic acid molecule and any fragment thereof.

[0462] 48. Use of any one of the bispecific antibodies of Embodiments 22-35, the fusion protein of Embodiment 36, the nucleic acid conjugate of Embodiment 37, the chimeric antigen receptor of Embodiment 38, the immunoconjugate of Embodiment 39, the nucleic acid molecule of Embodiment 40, the carrier of Embodiment 41, the cell of Embodiment 42, and / or the pharmaceutical composition of Embodiment 43 in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0463] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the antigen-binding protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0464] Example

[0465] The information on the commercial experimental materials (e.g., reagents, animals, cells, kits, strains) used in the example experiments is shown in the table below.

[0466] Table 4 Experimental Material Information

[0467]

[0468] Example 1: Recombinant Expression of Immunogen

[0469] The extracellular amino acid sequence (SEQ ID NO: 89) of wild-type human TfR1 (Uniprot: P02786) was used to add 6×His and Avi tags to the N-terminus of the protein for protein purification and biotin labeling, respectively. After codon optimization and gene synthesis, the sequence was carried into the pCDNA3.4 expression vector. Expi293 cells (ThermoFisher) were grown in Expi293 medium (ThermoFisher). Transient transfection was performed using polyethyleneimine (PEI) as the transfection reagent. After transfection, the cell supernatant was purified using nickel filler (Tiandi Renhe Biotechnology Co., Ltd.) and washed with different concentrations of imidazole. After elution, the buffer was replaced with PBS.

[0470] To confirm the size and integrity of the TfR1 recombinant protein, the fusion protein was analyzed using SDS-PAGE. The TfR1 recombinant protein was mixed with sample buffer. Non-reduced samples were directly loaded into a 4-12% Bis-Tris gel (Thermo Fisher). Reduced samples were heated at 95°C for 10 minutes after adding reducing agent before loading, and then run at 200V for 22 minutes. Staining was performed using staining solution (Yaxin Biotechnology). Pre-stained protein markers (Thermo Fisher) were used as molecular weight standards.

[0471] To evaluate the antigenicity of the TfR1 recombinant protein, its binding activity with the marketed antibody JR141 (see WO2016208695A1, where the full-length amino acid sequence of the heavy chain is shown in SEQ ID NO: 87 and the full-length amino acid sequence of the light chain is shown in SEQ ID NO: 88) was detected using an indirect ELISA method. The antigen to be evaluated was diluted to 1 μg / mL with PBS solution, and 100 μL was added to each well of a 96-well microplate. The plates were sealed with sealing film and incubated overnight at 4°C. The liquid in the microplate was discarded, and 300 μL of BSA solution was added to each well. The plates were then sealed with sealing film and incubated at 37°C for 2 h. After sealing, the liquid in the microplate was discarded, and the test samples were incubated at 37°C for 1 h. Discard the liquid in the wells. Wash the microplate with PBST solution, 300 μL per well each time, three times. Add 100 μL of diluted horseradish peroxidase (HRP)-conjugated secondary antibody to each well and incubate at 37°C for 1 h. Discard the liquid in the wells. Wash the microplate with PBST solution, 300 μL per well each time, three times. Add 100 μL of TMB chromogenic buffer (Surmodics) to each well and develop for 270 s. Then stop the reaction with 50 μL of 1 M sulfuric acid solution to each well. Place the microplate in a multi-plate reader to read the optical density signal.

[0472] Experimental results:

[0473] SDS-PAGE results showed that the produced TfR1 protein band was single with no obvious impurities, purity >95%, theoretical molecular weight of 78.21 kDa, and the band size shown in the SDS-PAGE results was as expected. Figure 1 ELISA results showed that the produced TfR1 protein could bind significantly to the JR141 antibody. Figure 2 ).

[0474] Example 2: Preparation of alpaca nanobody targeting TfR1

[0475] (A) Animal immunization and serum titer determination

[0476] Two alpacas (A and B) were used for immunization. A 10 mL blood sample was collected before immunization as a negative control. For the primary immunization, 0.5 mg of recombinant TfR1 protein was mixed with 1 mL of Freund's complete adjuvant (CFA) and injected subcutaneously. On day 21, a second immunization was performed, with 0.25 mg of recombinant TfR1 protein mixed with 1 mL of Freund's incomplete adjuvant (IFA) and injected subcutaneously. On day 42, a third immunization was performed, with 0.25 mg of recombinant TfR1 protein mixed with 1 mL of Freund's incomplete adjuvant (IFA) and injected subcutaneously. On day 63, a fourth immunization was performed, with 0.25 mg of recombinant TfR1 protein mixed with 1 mL of Freund's incomplete adjuvant (IFA) and injected subcutaneously.

[0477] Blood samples were collected one week after each booster immunization, and serum antibody titers and specificities were detected by ELISA. The results are shown in Table 5. After four immunizations, the TfR1 titers of both alpacas (A and B) were 1:1024K (OD450>0.2), which is at a high level. The blank control was 1% (w / v) BSA. The third and fourth immunizations refer to alpaca serum on the seventh day after the third and fourth immunizations, respectively. The data in the table are OD450 nm values, and K represents 1000.

[0478] Table 5. Alpaca serum titer detection

[0479]

[0480] (B) Yeast library construction

[0481] Peripheral blood cells (PBMCs) were isolated from 50 mL of blood collected after the fourth immunization, following the instructions for use of the lymphocyte separation medium. Total RNA was extracted from the PBMCs using RNAiso Plus reagent (Takara). cDNA was prepared using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara), with a total RNA transcription of 5 μg. The cDNA stock solutions from the two alpaca immunizations were mixed and diluted 5-fold for VHH fragment amplification. The VHH fragment was recovered by gel extraction and purified using a DNA product purification kit, recovering a VHH fragment of approximately 750 bp. The purified VHH fragment was used for yeast electroporation. A total of 5 electroporations were performed. Immediately after each electroporation, 1 mL of reconstitution solution (a 1:1 mixture of electroporation buffer and YPD medium) (preheated to 30°C) was added to the electroporation vessel for recovery. The electroporation product was aspirated, and the electroporation vessel was washed with reconstitution solution, yielding a total of 100 mL of recovery product. The cells were incubated statically at 30°C for 1 h, centrifuged, and resuspended in 3.5 mL of SD-CAA medium. 10 μL of each solution was serially diluted to 10^5 and 10^6 to determine the number of transformants. The transformed cells were then plated on 90 mm plates, and the remainder were plated on three 200 mm plates. On the fourth day, there were 54 clones on the plates used for transformant determination. Therefore, the number of transformants in the yeast library was 54 × 3.5 × 10^6 = 1.89 × 10^8 CFU.

[0482] (C) Screening of alpaca nanobodies targeting TfR1

[0483] 1) Take 1 mL of library cells, revive them in 1 L of SD-CAA medium for 16 h, centrifuge to collect the cells, and adjust the concentration to 5 × 10⁻⁶. ^6 Cells / mL, 1 L SG culture based on expression at 20 ℃ for 24 h, expression concentration: 3.31×10^7 cells / mL.

[0484] 2) Magnetic bead preparation: Take 300 μL of magnetic beads, wash them 3 times with PBSAT buffer on a magnetic rack, and set aside.

[0485] 3) Take 1.0×10^10 yeast library cells, wash them with PBSAT buffer, and incubate them with 100 μL of magnetic beads at 4 ℃ for 2 h (incubation volume: 5 mL) for negative sieving. Remove the magnetic beads with a magnetic rack and collect the suspended yeast cells.

[0486] 4) Incubate the target protein with 200 μL of magnetic beads at 4 °C for 2 h (incubation volume: 200 μL) for positive screening, and wash twice with PBSAT buffer.

[0487] 5) Incubate yeast cells with protein-positive sieve magnetic beads at 4 ℃ for 2 h, wash the magnetic bead yeast complex 3 times with PBSAT buffer, resuspend the magnetic bead yeast complex in 1 mL SD medium, add it to 100 mL SD medium, and amplify.

[0488] 6) Amplify cells at 30 ℃ in 100 mL SD medium for 15-16 h, remove magnetic beads from the culture medium, and store 8 vials containing 25% glycerol.

[0489] 7) Centrifuge the cells expanded in step 6), adjust the initial expression concentration to 4 × 10^6 cells / mL, and express for 16 h at 25℃ in 200 mL. The final expression concentration is 2.51 × 10^7 cells / mL. Divide into 7 aliquots (1 × 10^7 cells per aliquot), 100 μL each. Wash 3 times with PBSAT and set aside.

[0490] 8) Primary staining. Biotinylated target proteins were incubated with SA-650 (1:100 dilution) for 1 h.

[0491] 9) Secondary staining. Cells were incubated with the primary staining product and Anti-HA-488 for 1 h.

[0492] 10) After washing three times with ice-cold PBSAT and resuspending, the sample was identified by flow cytometry and then sorted by a sorting instrument. The flow cytometry sorting was performed in two rounds.

[0493] (D) Sequencing

[0494] After screening, a total of 8 human TfR1 binding positive sequences were obtained at the cell staining level.

[0495] Example 3: Construction and expression of recombinant candidate antibodies

[0496] Candidate VHH sequences were recombined into the carboxyl terminus of the heavy chain of monoclonal antibodies, and the heavy chain was modified with Knob-into-Hole technology to produce bispecific antibodies in the form of monovalent anti-TfR1. Figure 3The monoclonal form is more conducive to research on delivery across the blood-brain barrier (refer to Niewoehner, Jens et al. Neuron vol. 81, 1 2014: 49-60.). The monoclonal antibody used to construct the bispecific antibody is antibody 2665 against human galactolectin-3 (the amino acid sequence of 2665 is shown in Table 6 below). The heavy and light chain sequences are shown in SEQ ID NO:84 and SEQ ID NO:85, and the heavy chain is modified with Knob-into-Hole to reduce heavy chain mismatch. All nanobodies in this patent are evaluated in this form. ExpiCHO cells (ThermoFisher) were grown in ExpiCHO medium (ThermoFisher) and transiently transfected using a dedicated transfection reagent (ThermoFisher). After transfection, the supernatant was purified using Protein A packing material (Cytiva), eluted with 50 mM sodium citrate at pH 3.4, neutralized with 1 M tris solution, and then the buffer was replaced with PBS.

[0497] Table 6. Amino acid sequence of antibody 2665 that specifically binds to Gal-3.

[0498]

[0499] Example 4 Identification of recombinant candidate antibodies

[0500] (A) Evaluation of anti-TfR1 nanobody competitively binding to TfR1 with transferrin

[0501] Biomembrane interferometry (BLI) was used to evaluate the competitive binding of nanobodies to transferrin for TfR1. The experiments were performed using a Sartorius / ForteBio RED96e system. An anti-HIS (HIS1K) biosensor was used, capturing histidine-tagged human TfR1 (20 μg / ml, loading 0.3 nm) via an anti-histidine antibody on its surface. Then, nanobodies were sequentially bound to transferrin (with or without transferrin; if absent, PBS buffer containing 0.1% BSA + 0.05% Tween 20 was used instead) and nanobodies (binding time 60 s, transferrin concentration 5 μg / ml, binding height 500 nM). The binding signal of the nanobodies was detected with or without transferrin to determine whether the nanobodies were competing with transferrin for TfR1 binding. The chip surface was regenerated using 10 mM glycine hydrochloride solution (pH 1.5). All experimental results have been subtracted for blank (run buffer).

[0502] (B) Affinity identification of anti-TfR1 nanobodies

[0503] Biomembrane interferometry (BLI) was used to assess the binding affinity of nanobodies to TfR1, and experiments were performed using a Sartorius / ForteBio RED96e system. An anti-human Fc capture (AHC) biosensor was employed, using nanobodies containing the human Fc region (5 μg / ml, loading 0.6 nm) immobilized with anti-human Fc antibodies on its surface as ligands. Subsequently, analytes (TfR1) at different concentration gradients were bound to the immobilized nanobodies, followed by dissociation in running buffer (PBS 1× + 0.1% BSA + 0.05% Tween 20, pH 7.4) at binding and dissociation times of 600 s and 900 s, respectively. After each binding-dissociation cycle, the chip surface was regenerated using 10 mM glycine hydrochloride solution (pH 1.5). All obtained sensor maps were subtracted from the blank (running buffer) to correct for baseline drift, and a 1:1 binding model was used to fit and analyze the kinetic data.

[0504] Experimental results:

[0505] According to BLI assay, the YD01 antibody finally screened can specifically bind to human TfR1 recombinant protein, as shown in Table 7. The affinity level is moderate, at the nanomolar (nM) level, and the antibody does not compete with transferrin for binding to TfR1. Both can bind to TfR1 simultaneously.

[0506] Table 7 Affinity constants measured using the ForteBio Octet instrument

[0507]

[0508] Example 5: Humanization of Nanobodies

[0509] In clinical applications, the immunogenicity of camel-derived antibodies can affect their efficacy. Humanization design can minimize the immunogenicity of antibodies. Antibody humanization follows two principles: first, to reduce or essentially eliminate the immunogenicity of the antibody; and second, to maintain or improve the antibody's specific affinity. By comparing with the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database, germline genes with high homology to VHH nanobodies were selected as templates. The CDRs of VHH nanobodies were then transplanted into the corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As needed, key amino acids in the backbone sequence were reverted to the amino acids corresponding to VHH nanobodies to maintain the original affinity, thus obtaining humanized monoclonal antibodies.

[0510] (A) Determination of CDR area

[0511] Antibody variable regions (VH or VL, including portions of the VDJ gene) are typically divided into framework regions and complementarity determining regions (CDRs). Commonly used CDR definition and numbering systems include the Kabat system, IMGT system, AbM system, and Chothia system. Different CDR definition systems may identify different CDR regions. Based on the results of these commonly used systems, the single-chain CDR regions of YD01 were determined, as shown in Table 8 below (underlined regions are CDRs).

[0512] Table 8. Amino acid sequence of antibody YD01

[0513]

[0514] (B) Determination of Germline

[0515] Using the sequences of camel-derived antibodies, we searched for homologous proteins in the V region of camel-derived antibodies in several human germline framework libraries (such as Kabat, Igblast, IMGT, etc.). By comparing antibody sequences from several databases, we found sequences with high homology. Then, from the sequences with high homology, we selected human germline framework sequences that were frequently used as candidate templates.

[0516] (C) CDR grafting and reversion mutation

[0517] Humanization design employs the CDR grafting method. The basic approach is to replace the camel-derived frame region (FR) with the human germline frame region (FR) selected in the preceding steps, retaining only the camel-derived CDR. However, the FR region cannot be arbitrarily replaced entirely, as its sequence and conformation often influence the spatial configuration and function of the CDR region, and sometimes even participate in antibody binding. For example, FR regions spatially close to the CDR region, typically within 5 Å or 3 Å, may exhibit intramolecular interactions with the CDR region. Therefore, simple CDR grafting often reduces the affinity for antigen-antibody binding. To mitigate the adverse effects of humanization on the antibody's spatial configuration, activity, and function, it is necessary to perform reversion mutations on some critical amino acids in the human frame region, i.e., reverting them to the corresponding amino acids in the camel-derived FR region.

[0518] By obtaining the structural files of camel-derived antibodies from antibody databases and examining the 3D structure of the antibodies, we can understand the interactions between amino acids in the CDR region and amino acids in other framework regions, such as distances, van der Waals forces, and electrostatic forces. This allows us to locate amino acids in the framework regions that have close interactions with the CDR region and list these amino acids as candidate sites for reversion mutations.

[0519] Experimental results:

[0520] Sequence analysis and alignment identified one human germline framework sequence, IGHV3-23*04, as the template for humanization of YD01.

[0521] After CDR grafting is completed, several reversion mutation sites are selected from the humanized sequences generated from each template. Based on different reversion mutation sites and combinations, several humanized sequence schemes are designed using the same template.

[0522] The nanobody molecules corresponding to the above schemes were all constructed into the bispecific antibody form in Example 3, and their affinity with recombinant human TfR1 protein was identified. The results are shown in Table 9. The humanized antibody YD01-h5 (SEQ ID NO: 47) that can effectively bind to human TfR1 was obtained through screening.

[0523] Table 9. Affinity of bispecific antibodies carrying YD01-h5

[0524]

[0525] Example 6 Affinity Modification of Nanobodies

[0526] In this embodiment, the humanized antibody YD01-h5 underwent affinity maturation modification to enhance its affinity and biological activity. Affinity maturation modification was performed using M13 phage display technology. NNK primers (in primer synthesis, a single codon is composed of NNK) were used to introduce mutations in the CDR region, resulting in the construction of four phage display libraries: Library 1 contained a single-point combination mutation of CDR1+CDR2+CDR3; Library 2 contained a double-point combination mutation of CDR1+CDR2; Library 3 contained a double-point combination mutation of CDR1+CDR3; and Library 4 contained a double-point combination mutation of CDR2+CDR3.

[0527] Using the humanized antibody YD01-h5 as a template, a single CDR region mutation fragment was obtained by PCR, and then the full-length VHH fragment was obtained by overlapping PCR. The point mutation antibody was ligated into the phage display vector by double enzyme digestion (HindIII and NotI) and double-end ligation. Finally, the VHH sequence with the mutation site was transformed into Escherichia coli SS320 by electroporation.

[0528] After the four constructed libraries were packaged into phages, solid-phase, liquid-phase, and solid-liquid cross-panning were performed. Phages displaying the full-length VHH fragment by antigen binding were used, with the pressure of reducing the amount of coated antigen and increasing the number of washes to pan for potentially high-affinity antibodies. After panning, elution, and infection with E. coli SS320, the next panning cycle was performed. After 2-3 panning cycles, monoclonal nanobodies were selected to construct the bispecific antibody form in Example 3. A total of 28 nanobodies were selected. The modified nanobodies showed varying degrees of increased affinity for human TfR1. The identification results are shown in Table 10.

[0529] Table 10 Affinity data of nanobodies after affinity modification

[0530]

[0531] Example 7 Evaluation of in vivo cross-blood-brain barrier delivery of nanobodies

[0532] The modified YD01-h5m35, YD01-h5m39, YD01-h5m47, YD01-h5m84, YD01-h5m94, YD01-h5m102, and YD01-h5m106 molecules were selected to construct the bispecific antibody form in Example 3 for in vivo evaluation of cross-blood-brain barrier delivery.

[0533] In this embodiment, human TFR1 transgenic mice (Nanmo Biotechnology) were injected with either the Yinshen monoclonal antibody (2665) or a modified bispecific antibody. Administration was intravenous (IV), with a volume of 10 mL / kg and a dose of 1 mg / kg, administered as a single dose. All animals were euthanized 24 h post-administration, and brain tissue samples were collected. Drug concentration in the brain tissue was analyzed using ELISA, and the drug penetration efficiency for each animal was calculated (brain penetration efficiency = total moles of drug in the whole brain / total moles of drug injected into the mouse).

[0534] After brain tissue sampling, two volumes of cell lysis buffer (100 mL Western and IP cell lysis buffer + 1 mL PMSF) and three grinding beads were added. The brain tissue was homogenized using a cryo-homogenizer to obtain a brain tissue homogenate. After centrifugation at 12000 g for 10 minutes at 4 °C, the supernatant was diluted 2.5 times with cell lysis buffer and centrifuged again at 12500 g for 10 minutes at 4 °C. The resulting supernatant was the 5× brain tissue homogenate. Samples were detected using the Human IgG ELISA Kit (Solepro Biosciences) at dual wavelengths of 450 / 630 nm. The theoretical standard curve concentration was plotted on the x-axis, and the mean value of the standard curve replicates (OD difference at 450 nm and 630 nm) was plotted on the y-axis. The relevant parameters of the standard curve were fitted using a four-parameter regression, and the sample concentration was calculated using SoftMax Pro 7.1.2 software.

[0535] The final drug-to-brain efficiency is shown in Figure 4A and Figure 4B The fold increase in intrabrain delivery compared to monoclonal antibodies is also marked in the figure. The results show that, compared to monoclonal antibody 2665 without anti-TfR1 antibody (which has difficulty crossing the blood-brain barrier), all bispecific antibodies carrying anti-TfR1 antibody can significantly improve brain entry efficiency and the ability to cross the blood-brain barrier; among them, YD01-h5m35, YD01-h5m102, YD01-h5m106, YD01-h5m94, and YD01-h5m84 can all achieve a fold increase in brain entry of more than 42 times, especially YD01-h5m94 and YD01-h5m84, which can reach 47.3 times and 48.3 times, respectively, effectively delivering anti-Gal-3 monoclonal antibody into the brain.

Claims

1. An antigen-binding protein capable of specifically binding to transferrin receptor 1 (TfR1), said antigen-binding protein comprising a heavy chain variable region (VH), wherein said VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein said HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 92 (RX2X3DMFYDY), wherein X2 is selected from A or F or I or T or V, and X3 is selected from D or G or H or Q or V or Y.

2. The antigen-binding protein according to claim 1, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR2 comprises as shown in SEQ ID NO: 91 (X1X2X3X5X6X7X8X9X... 10 X 11 X 12 The amino acid sequence shown in SVKG, wherein X1 is selected from A, I, L, T, or V; X2 is selected from A, F, I, Q, R, T, or V; X3 is selected from R, S, or T; X5 is selected from S or K; X6 is selected from E, G, L, or Q; X7 is selected from A, D, I, L, S, or V; X8 is selected from A, R, or T; X9 is selected from A, Y, or V; X 10 Selected from Y, L, T, X 11 Choose from A, E, or S, and X 12 Selected from D, R, or F.

3. The antigen-binding protein according to claim 1 or 2, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 90 (DAX3YELYD), wherein X3 is selected from T or P.

4. The antigen-binding protein according to any one of claims 1-3, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 2-3.

5. The antigen-binding protein according to any one of claims 1-4, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR2 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 4-27.

6. The antigen-binding protein according to any one of claims 1-5, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises one of the amino acid sequences selected from any of the following: SEQ ID NO: 29-40.

7. The antigen-binding protein according to any one of claims 1-6, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2; The HCDR2 mentioned above includes, for example, SEQ ID NO: 93 (X1X2X3X5X6X7X8X9X). 10 X 11 X 12 The amino acid sequence shown in SVKG, wherein X1 is selected from A, I, L, T, or V; X2 is selected from A, F, I, Q, R, T, or V; X3 is selected from R, S, or T; X5 is selected from S or K; X6 is selected from E, G, or Q; X7 is selected from A, D, I, L, S, or V; X8 is selected from A, R, or T; X9 is selected from Y or V; X 10 Selected from Y, L, T, X 11 Choose from A, E, or S, and X 12 Selected from D, R, or F; Furthermore, the HCDR3 contains an amino acid sequence as shown in SEQ ID NO: 94 (RX2X3DMFYDY), wherein X2 is selected from A, F, I, T, or V, and X3 is selected from D, H, Q, V, or Y.

8. The antigen-binding protein according to claim 7, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2; wherein HCDR2 comprises the amino acid sequence selected from any one of SEQ ID NO: 4-12, SEQ ID NO: 14-22, and SEQ ID NO: 24-27; and wherein HCDR3 comprises the amino acid sequence selected from any one of SEQ ID NO: 29-33, SEQ ID NO: 35, and SEQ ID NO: 37-40.

9. The antigen-binding protein according to claim 7 or 8, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any group of the following combinations: 1) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 5; HCDR3: SEQ ID NO: 29; 2) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 30; 3) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 7; HCDR3: SEQ ID NO: 29; 4) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 8; HCDR3: SEQ ID NO: 29; 5) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 9; HCDR3: SEQ ID NO: 31; 6) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 10; HCDR3: SEQ ID NO: 29; 7) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 32; 8) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 11; HCDR3: SEQ ID NO: 33; 9) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 12; HCDR3: SEQ ID NO: 33; 10) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 6; HCDR3: SEQ ID NO: 35; 11) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 14; HCDR3: SEQ ID NO: 32; 12) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 15; HCDR3: SEQ ID NO: 32; 13) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 16; HCDR3: SEQ ID NO: 32; 14) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 17; HCDR3: SEQ ID NO: 37; 15) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 18; HCDR3: SEQ ID NO: 32; 16) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 19; HCDR3: SEQ ID NO: 33; 17) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 20; HCDR3: SEQ ID NO: 37; 18) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 21; HCDR3: SEQ ID NO: 37; 19) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 22; HCDR3: SEQ ID NO: 38; 20) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 24; HCDR3: SEQ ID NO: 37; 21) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 39; 22) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 25; HCDR3: SEQ ID NO: 33; 23) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 26; HCDR3: SEQ ID NO: 40; and 24) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 27; HCDR3: SEQ ID NO:

37.

10. The antigen-binding protein according to any one of claims 1-9, wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1-3 are selected from any group of the following combinations: 1)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 5;HCDR3: SEQ ID NO: 29; 2)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 4;HCDR3: SEQ ID NO: 30; 3)HCDR1: SEQ ID NO:3;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 29; 4)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 7;HCDR3: SEQ ID NO: 29; 5)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 8;HCDR3: SEQ ID NO: 29; 6)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 9;HCDR3: SEQ ID NO: 31; 7)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 10;HCDR3: SEQ ID NO: 29; 8)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 4;HCDR3: SEQ ID NO: 32; 9)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 11;HCDR3: SEQ ID NO: 33; 10)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 12;HCDR3: SEQ ID NO: 33; 11)HCDR1: SEQ ID NO:3;HCDR2: SEQ ID NO: 13;HCDR3: SEQ ID NO: 34; 12)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 35; 13)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 14;HCDR3: SEQ ID NO: 32; 14)HCDR1: SEQ ID NO:3;HCDR2: SEQ ID NO: 6;HCDR3: SEQ ID NO: 36; 15)HCDR1: SEQ ID NO:2;HCDR2: SEQ ID NO: 15;HCDR3: SEQ ID NO: 32; 16) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 16; HCDR3: SEQ ID NO: 32; 17) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 17; HCDR3: SEQ ID NO: 37; 18) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 18; HCDR3: SEQ ID NO: 32; 19) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 19; HCDR3: SEQ ID NO: 33; 20) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 20; HCDR3: SEQ ID NO: 37; 21) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 21; HCDR3: SEQ ID NO: 37; 22) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 22; HCDR3: SEQ ID NO: 38; 23) HCDR1: SEQ ID NO: 3; HCDR2: SEQ ID NO: 23; HCDR3: SEQ ID NO: 33; 24) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 24; HCDR3: SEQ ID NO: 37; 25) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 4; HCDR3: SEQ ID NO: 39; 26) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 25; HCDR3: SEQ ID NO: 33; 27) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 26; HCDR3: SEQ ID NO: 40; and 28) HCDR1: SEQ ID NO: 2; HCDR2: SEQ ID NO: 27; HCDR3: SEQ ID NO: 37.

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