Single-domain antibody targeting tfr1 and use thereof

Abstract

Provided in the present invention are a single-domain antibody targeting TfR1 and the use thereof. Specifically, provided in the present invention are a corresponding anti-TfR1 antibody, a chimeric antigen receptor, a fusion protein, a recombinant protein and a coding nucleic acid thereof, an expression vector, a host cell, etc. In addition, an anti-TfR1 single-domain antibody-PMO conjugate is prepared. The single-domain antibody has properties such as binding activity and stability, and can be used for tumor therapy and immunodetection.

Description

A single-domain antibody targeting TfR1 and its application Technical Field

[0001] The present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to a single-domain antibody targeting TfR1 and applications thereof. Background Art

[0002] The transferrin receptor (TfR) is a type II transmembrane glycoprotein receptor composed of two single-pass transmembrane glycoprotein subunits that exists as a homodimer. Each dimer's extracellular domain (EDC) binds to two transferrin (TF) molecules, mediating the transport of iron ions from the extracellular space to the intracellular space to meet the needs of cell growth and activity. There are two families of TfRs: TfR1 and TfR2. Studies have shown that TfR1 is expressed at basal levels in most tissue cells. High expression of TfR1 is often associated with rapid cell proliferation, including in a variety of highly metastatic tumor tissues, such as brain tumors, nasopharyngeal carcinoma, prostate cancer, breast cancer, ovarian cancer, liver cancer, leukemia, colon cancer, and lung cancer. Furthermore, high TfR1 expression is associated with cellular activity, for example, being highly expressed on the surface of muscle cells, which have a high iron demand, and on the surface of brain endothelial cells, where it maintains intracellular iron homeostasis.

[0003] Single-domain antibodies (SDAs) are a new type of antibody molecule discovered in camelids by the research group of Belgian immunologist Hamers-Casterman. Compared to traditional antibodies, they have a smaller molecular weight, one-tenth the size of traditional IgG antibodies. Therefore, they have a simple structure, are easy to modify, have strong tissue penetration, and have low immunogenicity. Single-domain antibodies also lack an Fc domain and, when used as targeting molecules, do not mediate ADCC effects and cause cytotoxicity. Furthermore, they have the advantages of good stability, resistance to high temperatures and extreme pH environments, and low production costs. Therefore, single-domain antibodies are excellent targeting molecules with great value in targeted drug development.

[0004] With the development of immunotherapy, antibody drugs have derived a variety of conjugated forms, including antibody drug conjugates (ADCs), antibody oligonucleotide conjugates (AOCs), and immunostimulating antibody conjugates (ISACs). By leveraging the targeting characteristics of antibodies, pharmacodynamic molecules, such as toxins, antisense nucleic acids, and immune receptor agonists, are delivered to the vicinity of the target, thereby improving the utilization rate of pharmacodynamic molecules and reducing side effects. Compared with conjugates prepared from traditional antibody fragments such as IgG, Fab, and scFv, single-domain antibody conjugates are simpler in the conjugation process due to their smaller molecular weight and greater stability. Once they enter the body, they have stronger tissue penetration and better efficacy. Compared with membrane-penetrating peptide conjugates with the same smaller molecular weight, such as peptide-PMO, single-domain antibody conjugates have better targeting and obvious advantages.

[0005] Based on the above background, there is an urgent need in this field to develop a single-domain antibody targeting TfR1 and to develop corresponding anti-tumor drugs, and targeted drugs for muscle and brain diseases. Summary of the Invention

[0006] The purpose of the present invention is to provide a single-domain antibody targeting TfR1 and its application.

[0007] In a first aspect of the present invention, an anti-TfR1 single domain antibody is provided, wherein the anti-TfR1 single domain antibody has one or more complementarity determining regions (CDRs) selected from the following group:

[0008] (1) CDR1 shown in SEQ ID NO: 51, CDR2 shown in SEQ ID NO: 73, and CDR3 shown in SEQ ID NO: 98;

[0009] (2) CDR1 shown in SEQ ID NO: 54, CDR2 shown in SEQ ID NO: 61, and CDR3 shown in SEQ ID NO: 53;

[0010] (3) CDR1 shown in SEQ ID NO: 54, CDR2 shown in SEQ ID NO: 61, and CDR3 shown in SEQ ID NO: 56;

[0011] (4) CDR1 shown in SEQ ID NO: 60, CDR2 shown in SEQ ID NO: 67, and CDR3 shown in SEQ ID NO: 71;

[0012] (5) CDR1 shown in SEQ ID NO: 69, CDR2 shown in SEQ ID NO: 64, and CDR3 shown in SEQ ID NO: 74;

[0013] (6) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:50;

[0014] (7) CDR1 set forth in SEQ ID NO:66, CDR2 set forth in SEQ ID NO:116, and CDR3 set forth in SEQ ID NO:119;

[0015] (8) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:100, and CDR3 shown in SEQ ID NO:50;

[0016] (9) CDR1 shown in SEQ ID NO: 51, CDR2 shown in SEQ ID NO: 46, and CDR3 shown in SEQ ID NO: 118;

[0017] (10) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:46, and CDR3 shown in SEQ ID NO:47;

[0018] (11) CDR1 shown in SEQ ID NO: 57, CDR2 shown in SEQ ID NO: 76, and CDR3 shown in SEQ ID NO: 77;

[0019] (12) CDR1 shown in SEQ ID NO: 81, CDR2 shown in SEQ ID NO: 79, and CDR3 shown in SEQ ID NO: 80;

[0020] (13) CDR1 shown in SEQ ID NO:96, CDR2 shown in SEQ ID NO:70, and CDR3 shown in SEQ ID NO:83;

[0021] (14) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:86;

[0022] (15) CDR1 shown in SEQ ID NO:93, CDR2 shown in SEQ ID NO:82, and CDR3 shown in SEQ ID NO:89;

[0023] (16) CDR1 shown in SEQ ID NO:75, CDR2 shown in SEQ ID NO:58, and CDR3 shown in SEQ ID NO:68;

[0024] (17) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:92;

[0025] (18) CDR1 shown in SEQ ID NO: 111, CDR2 shown in SEQ ID NO: 85, and CDR3 shown in SEQ ID NO: 62;

[0026] (19) CDR1 shown in SEQ ID NO:75, CDR2 shown in SEQ ID NO:58, and CDR3 shown in SEQ ID NO:95;

[0027] (20) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:47;

[0028] (21) CDR1 shown in SEQ ID NO: 63, CDR2 shown in SEQ ID NO: 55, and CDR3 shown in SEQ ID NO: 65;

[0029] (22) CDR1 shown in SEQ ID NO: 108, CDR2 shown in SEQ ID NO: 88, and CDR3 shown in SEQ ID NO: 101;

[0030] (23) CDR1 shown in SEQ ID NO: 87, CDR2 shown in SEQ ID NO: 91, and CDR3 shown in SEQ ID NO: 62;

[0031] (24) CDR1 shown in SEQ ID NO:90, CDR2 shown in SEQ ID NO:94, and CDR3 shown in SEQ ID NO:104;

[0032] (25) CDR1 shown in SEQ ID NO: 102, CDR2 shown in SEQ ID NO: 97, and CDR3 shown in SEQ ID NO: 59;

[0033] (26) CDR1 shown in SEQ ID NO:84, CDR2 shown in SEQ ID NO:103, and CDR3 shown in SEQ ID NO:107;

[0034] (27) CDR1 shown in SEQ ID NO:99, CDR2 shown in SEQ ID NO:106, and CDR3 shown in SEQ ID NO:110;

[0035] (28) CDR1 shown in SEQ ID NO: 105, CDR2 shown in SEQ ID NO: 109, and CDR3 shown in SEQ ID NO: 113;

[0036] (29) CDR1 shown in SEQ ID NO: 78, CDR2 shown in SEQ ID NO: 58, and CDR3 shown in SEQ ID NO: 68;

[0037] (30) CDR1 shown in SEQ ID NO:96, CDR2 shown in SEQ ID NO:112, and CDR3 shown in SEQ ID NO:115;

[0038] (31) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:114, and CDR3 shown in SEQ ID NO:117;

[0039] (32) CDR1 shown in SEQ ID NO:72, CDR2 shown in SEQ ID NO:64, and CDR3 shown in SEQ ID NO:74.

[0040] In another preferred example, the complementarity determining regions (CDRs) of the anti-TfR1 single domain antibody are divided according to the IMGT rules.

[0041] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain the ability to specifically bind to TfR1.

[0042] In another preferred embodiment, the derivative sequence that has been added, deleted, modified and / or substituted with at least one amino acid and can retain the ability to specifically bind to TfR1 is an amino acid sequence with a homology or sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0043] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated in sequence by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain respectively.

[0044] In another preferred embodiment, the anti-TfR1 single domain antibody has a structure shown in formula (I):

[0045] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Formula (I)

[0046] Wherein, the "-"s are each independently a connecting peptide or a peptide bond.

[0047] In another preferred embodiment, there is an inter-loop disulfide bond between the CDR1 region and the CDR3 region of the anti-TfR1 single-domain antibody.

[0048] In another preferred embodiment, the anti-TfR1 single-domain antibody further includes a framework region FR.

[0049] In another preferred embodiment, the framework region FR includes a human FR region, a mouse FR region, a rabbit FR region, a monkey FR region, an alpaca FR region or a camel FR region.

[0050] In another preferred embodiment, the framework region FR is one or more selected from the following groups:

[0051] (1) FR1 set forth in SEQ ID NO: 133, FR2 set forth in SEQ ID NO: 134, FR3 set forth in SEQ ID NO: 135, and FR4 set forth in SEQ ID NO: 136;

[0052] (2) FR1 set forth in SEQ ID NO: 137, FR2 set forth in SEQ ID NO: 138, FR3 set forth in SEQ ID NO: 139, and FR4 set forth in SEQ ID NO: 140;

[0053] (3) FR1 set forth in SEQ ID NO: 141, FR2 set forth in SEQ ID NO: 138, FR3 set forth in SEQ ID NO: 139, and FR4 set forth in SEQ ID NO: 140;

[0054] (4) FR1 set forth in SEQ ID NO: 141, FR2 set forth in SEQ ID NO: 167, FR3 set forth in SEQ ID NO: 168, and FR4 set forth in SEQ ID NO: 136;

[0055] (5) FR1 set forth in SEQ ID NO: 158, FR2 set forth in SEQ ID NO: 151, FR3 set forth in SEQ ID NO: 152, and FR4 set forth in SEQ ID NO: 159;

[0056] (6) FR1 set forth in SEQ ID NO: 120, FR2 set forth in SEQ ID NO: 162, FR3 set forth in SEQ ID NO: 150, and FR4 set forth in SEQ ID NO: 136;

[0057] (7) FR1 set forth in SEQ ID NO: 206, FR2 set forth in SEQ ID NO: 207, FR3 set forth in SEQ ID NO: 208, and FR4 set forth in SEQ ID NO: 127;

[0058] (8) FR1 set forth in SEQ ID NO: 133, FR2 set forth in SEQ ID NO: 134, FR3 set forth in SEQ ID NO: 183, and FR4 set forth in SEQ ID NO: 136;

[0059] (9) FR1 set forth in SEQ ID NO: 120, FR2 set forth in SEQ ID NO: 134, FR3 set forth in SEQ ID NO: 135, and FR4 set forth in SEQ ID NO: 136;

[0060] (10) FR1 shown in SEQ ID NO: 141, FR2 shown in SEQ ID NO: 134, FR3 shown in SEQ ID NO: 160, and FR4 shown in SEQ ID NO: 136;

[0061] (11) FR1 shown in SEQ ID NO: 120, FR2 shown in SEQ ID NO: 121, FR3 shown in SEQ ID NO: 122, and FR4 shown in SEQ ID NO: 123;

[0062] (12) FR1 shown in SEQ ID NO: 124, FR2 shown in SEQ ID NO: 125, FR3 shown in SEQ ID NO: 126, and FR4 shown in SEQ ID NO: 127;

[0063] (13) FR1 shown in SEQ ID NO: 128, FR2 shown in SEQ ID NO: 129, FR3 shown in SEQ ID NO: 130, and FR4 shown in SEQ ID NO: 127;

[0064] (14) FR1 shown in SEQ ID NO: 131, FR2 shown in SEQ ID NO: 125, FR3 shown in SEQ ID NO: 132, and FR4 shown in SEQ ID NO: 127;

[0065] (15) FR1 shown in SEQ ID NO: 142, FR2 shown in SEQ ID NO: 143, FR3 shown in SEQ ID NO: 144, and FR4 shown in SEQ ID NO: 127;

[0066] (16) FR1 shown in SEQ ID NO: 145, FR2 shown in SEQ ID NO: 146, FR3 shown in SEQ ID NO: 147, and FR4 shown in SEQ ID NO: 148;

[0067] (17) FR1 shown in SEQ ID NO: 141, FR2 shown in SEQ ID NO: 149, FR3 shown in SEQ ID NO: 150, and FR4 shown in SEQ ID NO: 127;

[0068] (18) FR1 shown in SEQ ID NO: 128, FR2 shown in SEQ ID NO: 151, FR3 shown in SEQ ID NO: 152, and FR4 shown in SEQ ID NO: 153;

[0069] (19) FR1 shown in SEQ ID NO: 154, FR2 shown in SEQ ID NO: 155, FR3 shown in SEQ ID NO: 156, and FR4 shown in SEQ ID NO: 127;

[0070] (20) FR1 set forth in SEQ ID NO: 157, FR2 set forth in SEQ ID NO: 146, FR3 set forth in SEQ ID NO: 147, and FR4 set forth in SEQ ID NO: 148;

[0071] (21) FR1 shown in SEQ ID NO: 161, FR2 shown in SEQ ID NO: 134, FR3 shown in SEQ ID NO: 135, and FR4 shown in SEQ ID NO: 136;

[0072] (22) FR1 shown in SEQ ID NO: 163, FR2 shown in SEQ ID NO: 134, FR3 shown in SEQ ID NO: 135, and FR4 shown in SEQ ID NO: 136;

[0073] (23) FR1 shown in SEQ ID NO: 141, FR2 shown in SEQ ID NO: 138, FR3 shown in SEQ ID NO: 139, and FR4 shown in SEQ ID NO: 164;

[0074] (24) FR1 shown in SEQ ID NO: 142, FR2 shown in SEQ ID NO: 165, FR3 shown in SEQ ID NO: 166, and FR4 shown in SEQ ID NO: 127;

[0075] (25) FR1 shown in SEQ ID NO: 169, FR2 shown in SEQ ID NO: 170, FR3 shown in SEQ ID NO: 171, and FR4 shown in SEQ ID NO: 172;

[0076] (26) FR1 shown in SEQ ID NO: 173, FR2 shown in SEQ ID NO: 174, FR3 shown in SEQ ID NO: 175, and FR4 shown in SEQ ID NO: 127;

[0077] (27) FR1 shown in SEQ ID NO: 176, FR2 shown in SEQ ID NO: 177, FR3 shown in SEQ ID NO: 178, and FR4 shown in SEQ ID NO: 179;

[0078] (28) FR1 shown in SEQ ID NO: 180, FR2 shown in SEQ ID NO: 181, FR3 shown in SEQ ID NO: 182, and FR4 shown in SEQ ID NO: 127;

[0079] (29) FR1 shown in SEQ ID NO: 163, FR2 shown in SEQ ID NO: 162, FR3 shown in SEQ ID NO: 150, and FR4 shown in SEQ ID NO: 136;

[0080] (30) FR1 shown in SEQ ID NO: 184, FR2 shown in SEQ ID NO: 185, FR3 shown in SEQ ID NO: 186, and FR4 shown in SEQ ID NO: 127;

[0081] (31) FR1 shown in SEQ ID NO: 187, FR2 shown in SEQ ID NO: 146, FR3 shown in SEQ ID NO: 147, and FR4 shown in SEQ ID NO: 148;

[0082] (32) FR1 shown in SEQ ID NO: 188, FR2 shown in SEQ ID NO: 151, FR3 shown in SEQ ID NO: 152, and FR4 shown in SEQ ID NO: 159;

[0083] (33) FR1 shown in SEQ ID NO: 189, FR2 shown in SEQ ID NO: 190, FR3 shown in SEQ ID NO: 191, and FR4 shown in SEQ ID NO: 127;

[0084] (34) FR1 shown in SEQ ID NO: 120, FR2 shown in SEQ ID NO: 192, FR3 shown in SEQ ID NO: 193, and FR4 shown in SEQ ID NO: 140;

[0085] (35) FR1 shown in SEQ ID NO: 194, FR2 shown in SEQ ID NO: 195, FR3 shown in SEQ ID NO: 196, and FR4 shown in SEQ ID NO: 127;

[0086] (36) FR1 shown in SEQ ID NO: 197, FR2 shown in SEQ ID NO: 198, FR3 shown in SEQ ID NO: 147, and FR4 shown in SEQ ID NO: 148;

[0087] (37) FR1 shown in SEQ ID NO: 163, FR2 shown in SEQ ID NO: 138, FR3 shown in SEQ ID NO: 199, and FR4 shown in SEQ ID NO: 140;

[0088] (38) FR1 shown in SEQ ID NO: 200, FR2 shown in SEQ ID NO: 201, FR3 shown in SEQ ID NO: 202, and FR4 shown in SEQ ID NO: 127;

[0089] (39) FR1 shown in SEQ ID NO: 120, FR2 shown in SEQ ID NO: 203, FR3 shown in SEQ ID NO: 204, and FR4 shown in SEQ ID NO: 127;

[0090] (40) FR1 set forth in SEQ ID NO: 205, FR2 set forth in SEQ ID NO: 151, FR3 set forth in SEQ ID NO: 152, and FR4 set forth in SEQ ID NO: 159;

[0091] (41) FR1 shown in SEQ ID NO: 209, FR2 shown in SEQ ID NO: 167, FR3 shown in SEQ ID NO: 168, and FR4 shown in SEQ ID NO: 136;

[0092] (42) FR1 shown in SEQ ID NO: 128, FR2 shown in SEQ ID NO: 165, FR3 shown in SEQ ID NO: 166, and FR4 shown in SEQ ID NO: 127;

[0093] (43) FR1 shown in SEQ ID NO: 133, FR2 shown in SEQ ID NO: 134, FR3 shown in SEQ ID NO: 135, and FR4 shown in SEQ ID NO: 148;

[0094] (44) FR1 shown in SEQ ID NO: 133, FR2 shown in SEQ ID NO: 134, FR3 shown in SEQ ID NO: 216, and FR4 shown in SEQ ID NO: 127;

[0095] (45) FR1 shown in SEQ ID NO: 133, FR2 shown in SEQ ID NO: 134, FR3 shown in SEQ ID NO: 216, and FR4 shown in SEQ ID NO: 217;

[0096] (46) FR1 shown in SEQ ID NO: 133, FR2 shown in SEQ ID NO: 134, FR3 shown in SEQ ID NO: 216, and FR4 shown in SEQ ID NO: 148;

[0097] (47) FR1 shown in SEQ ID NO: 141, FR2 shown in SEQ ID NO: 134, FR3 shown in SEQ ID NO: 216, and FR4 shown in SEQ ID NO: 217;

[0098] (48) FR1 shown in SEQ ID NO: 141, FR2 shown in SEQ ID NO: 134, FR3 shown in SEQ ID NO: 216, and FR4 shown in SEQ ID NO: 148.

[0099] In another preferred example, the amino acid sequence of the VHH chain of the anti-TfR1 single-domain antibody is selected from one or more of SEQ ID NOs: 1-44, 210-215.

[0100] In another preferred embodiment, the anti-TfR1 single-domain antibody includes a humanized antibody, an animal-derived antibody, and a chimeric antibody.

[0101] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, monkey, alpaca, or camel.

[0102] In another preferred embodiment, the anti-TfR1 single-domain antibody is a partially or fully humanized antibody.

[0103] In another preferred embodiment, the anti-TfR1 single domain antibody has one or more characteristics selected from the following group:

[0104] (a) specifically binds to human and / or alpaca TfR1;

[0105] (b) specifically binds to monkey TfR1;

[0106] (c) does not bind or weakly binds to mouse TfR1;

[0107] (d) does not bind or weakly binds to monkey TfR1;

[0108] (e) blocking the binding of TfR1 to transferrin;

[0109] (f) does not block the binding of TfR1 to transferrin;

[0110] (g) does not bind TfR2; and / or

[0111] (h) Does not block the binding of TfR1 to HFE protein.

[0112] In another preferred embodiment, the monkey is a rhesus monkey.

[0113] In the second aspect of the present invention, an anti-TfR1 antibody is provided, wherein the antibody comprises one or more VHH chains of the anti-TfR1 single domain antibody according to the first aspect of the present invention.

[0114] In another preferred example, the amino acid sequence of the VHH chain of the anti-TfR1 single-domain antibody is selected from one or more of SEQ ID NOs: 1-4944, 210-215.

[0115] In another preferred embodiment, the anti-TfR1 antibody may be a monomer, a bivalent antibody, and / or a multivalent antibody.

[0116] In another preferred embodiment, the anti-TfR1 antibody is a bivalent antibody.

[0117] In another preferred embodiment, the anti-TfR1 antibody includes a humanized antibody, an animal-derived antibody, or a chimeric antibody.

[0118] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, monkey, alpaca, or camel.

[0119] In another preferred embodiment, the anti-TfR1 antibody is a partially or fully humanized antibody.

[0120] In another preferred embodiment, the anti-TfR1 antibody has one or more characteristics selected from the following group:

[0121] (a) specifically binds to human and / or alpaca TfR1;

[0122] (b) specifically binds to monkey TfR1;

[0123] (c) does not bind or weakly binds to mouse TfR1;

[0124] (d) does not bind or weakly binds to monkey TfR1;

[0125] (e) blocking the binding of TfR1 to transferrin;

[0126] (f) does not block the binding of TfR1 to transferrin;

[0127] (g) does not bind TfR2; and / or

[0128] (h) Does not block the binding of TfR1 to HFE protein.

[0129] In the third aspect of the present invention, a chimeric antigen receptor (CAR) is provided, wherein the CAR comprises an extracellular domain, wherein the extracellular domain comprises the anti-TfR1 single domain antibody as described in the first aspect of the present invention, or the anti-TfR1 antibody as described in the second aspect of the present invention.

[0130] In another preferred embodiment, the extracellular domain further includes a signal peptide.

[0131] In another preferred embodiment, the extracellular domain also includes other exogenous proteins.

[0132] In another preferred embodiment, the CAR has the structure shown in Formula Ia:

[0133] L-Nb-H-TM-C-CD3ζ (Ia)

[0134] Where,

[0135] L is none or a signal peptide sequence;

[0136] Nb is the specific binding domain;

[0137] H is none or hinge region;

[0138] TM is the transmembrane domain;

[0139] C is the costimulatory signaling domain;

[0140] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type, or mutants / modified forms thereof);

[0141] The "-" is a connecting peptide or peptide bond.

[0142] In another preferred embodiment, the L is selected from the signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or a combination thereof.

[0143] In another preferred embodiment, the Nb targets TfR1.

[0144] In another preferred embodiment, the Nb is an anti-TfR1 single domain antibody.

[0145] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0146] In another preferred embodiment, the H is a human IgG1 Fc hinge region.

[0147] In another preferred embodiment, the TM is selected from the transmembrane region of the following histones: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutants / modified forms thereof, or combinations thereof.

[0148] In another preferred embodiment, the C is selected from the costimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants / modified forms thereof, or combinations thereof.

[0149] In the fourth aspect of the present invention, a multispecific antibody is provided, wherein the first antigen binding region of the multispecific antibody comprises: the anti-TfR1 single domain antibody as described in the first aspect of the present invention, or the anti-TfR1 antibody as described in the second aspect of the present invention.

[0150] In another preferred embodiment, the multispecific antibody further comprises a second antigen binding region targeting a target selected from the group consisting of BCMA, CD28, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP-1, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-SMA, AGR2, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD11b, CD11c, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, and CD44v. 6. CD47, CD51, CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD1 23. CD125, CD132(IL-2Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5(CEA), CEAC AM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL -12R, IL-12Rβl, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / KATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA , PSGR, RAAG12, RAGE, SLC44A4, Siglecl5, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, T LR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, TRK-A, TRK-B, uPAR, VAP1, V CAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, or combinations thereof.

[0151] In another preferred embodiment, the multispecific antibody comprises one or more second antigen binding regions.

[0152] In another preferred embodiment, the second antigen binding region is a single domain antibody.

[0153] In another preferred embodiment, the multispecific antibody further comprises an antibody Fc segment.

[0154] In another preferred embodiment, the antigen binding region is an antibody or antibody fragment, and the antibody fragment includes: (i) Fab fragment; (ii) F(ab')2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv (scFv) molecule; and (vi) dAb fragment.

[0155] In a fifth aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0156] (i) the anti-TfR1 single domain antibody according to the first aspect of the present invention, the anti-TfR1 antibody according to the second aspect of the present invention, or an active fragment thereof;

[0157] (ii) optionally a polypeptide molecule or fragment having therapeutic function; and / or

[0158] (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

[0159] In another preferred embodiment, the improving the physicochemical properties or drugability of the protein includes prolonging the half-life of the anti-TfR1 single domain antibody.

[0160] In another preferred embodiment, the functional domain for improving the physicochemical properties or drugability of a protein includes an Fc segment and human serum albumin (HSA).

[0161] In another preferred embodiment, the recombinant protein further comprises (iv) an optional tag sequence for facilitating expression and / or purification.

[0162] In another preferred embodiment, the tag includes an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

[0163] In another preferred embodiment, the recombinant protein specifically binds to TfR1.

[0164] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0165] In another preferred embodiment, the tag is an Fc tag.

[0166] In another preferred embodiment, the polypeptide molecules or fragments with therapeutic functions include but are not limited to: targeting BCMA, CD28, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP-1, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-SMA, AGR2, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD3, CD6, CD9, CDlla, CDllb, CDllc, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v 6. CD47, CD51, CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD1 23. CD125, CD132(IL-2Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5(CEA), CEAC AM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL -12R, IL-12Rβl, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, P SGR, RAAG12, RAGE, SLC44A4, Siglecl5, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, or a polypeptide molecule or fragment thereof.

[0167] In another preferred embodiment, the polypeptide molecules or fragments with therapeutic functions include but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, and cytokines.

[0168] In another preferred embodiment, the recombinant protein includes a fusion protein.

[0169] In another preferred embodiment, the fusion protein has the following elements from the NC end:

[0170] AB;

[0171] Among them, the A component is a single-domain antibody against TfR1; the B component is the Fc segment or human serum albumin (HAS);

[0172] “-” represents a peptide bond.

[0173] In another preferred example, the amino acid sequence of the VHH chain of the anti-TfR1 single-domain antibody is selected from one or more of SEQ ID NOs: 1-50.

[0174] In another preferred embodiment, the Fc segment is a human IgG Fc segment (eg, a human IgG1 Fc segment).

[0175] In the sixth aspect of the present invention, a polynucleotide is provided, which encodes a protein selected from the following group: the anti-TfR1 single domain antibody as described in the first aspect of the present invention, the anti-TfR1 antibody as described in the second aspect of the present invention, the chimeric antigen receptor as described in the third aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, the recombinant protein as described in the fifth aspect of the present invention, or a combination thereof.

[0176] In another preferred embodiment, the polynucleotide is RNA, DNA or cDNA.

[0177] In the seventh aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide according to the sixth aspect of the present invention.

[0178] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or combinations thereof.

[0179] In another preferred embodiment, the expression vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

[0180] In another preferred embodiment, the expression vector further comprises a member selected from the group consisting of: a promoter, a transcription enhancer element WPRE, a long terminal repeat sequence LTR, and the like.

[0181] In the eighth aspect of the present invention, a host cell is provided, wherein the host cell contains the expression vector according to the seventh aspect of the present invention, or the polynucleotide according to the sixth aspect of the present invention is integrated into its genome.

[0182] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0183] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0184] In the ninth aspect of the present invention, an engineered immune cell is provided, wherein the engineered immune cell contains the expression vector as described in the seventh aspect of the present invention, or an exogenous polynucleotide as described in the sixth aspect of the present invention is integrated into its genome, or expresses the chimeric antigen receptor as described in the third aspect of the present invention.

[0185] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0186] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0187] (ii) chimeric antigen receptor γδ T cells (CAR-T cells);

[0188] (iii) chimeric antigen receptor NKT cells (CAR-NKT cells);

[0189] (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0190] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or a combination thereof.

[0191] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0192] In a tenth aspect of the present invention, a method for producing an anti-TfR1 single domain antibody is provided, the method comprising the steps of:

[0193] (a) culturing the host cell according to the eighth aspect of the present invention under conditions suitable for producing the single domain antibody, thereby obtaining a culture containing the anti-TfR1 single domain antibody;

[0194] (b) isolating and / or recovering the anti-TfR1 single domain antibody from the culture; and

[0195] (c) Optionally, purifying and / or modifying the anti-TfR1 single domain antibody obtained in step (b).

[0196] In the eleventh aspect of the present invention, an immunoconjugate is provided, comprising:

[0197] (a) the anti-TfR1 single domain antibody according to the first aspect of the present invention, the anti-TfR1 antibody according to the second aspect of the present invention, the multispecific antibody according to the fourth aspect of the present invention, or the recombinant protein according to the fifth aspect of the present invention; and

[0198] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein, a VLP, an oligonucleotide, a nucleic acid analog, or a combination thereof.

[0199] In another preferred embodiment, the part (a) is the anti-TfR1 single domain antibody as described in the first aspect of the present invention, or the anti-TfR1 antibody as described in the second aspect of the present invention.

[0200] In another preferred embodiment, the (a) part and the coupling part are coupled via a chemical bond or a linker.

[0201] In another preferred embodiment, the radioactive nuclides include:

[0202] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0203] (ii) therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0204] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0205] In another preferred embodiment, the drug is a drug for the targeted treatment of diseases with high TfR1 expression.

[0206] In another preferred embodiment, the coupling moiety is an oligonucleotide.

[0207] In another preferred embodiment, the oligonucleotide is capable of regulating gene expression.

[0208] In another preferred embodiment, the oligonucleotide is selected from the following group: antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), nucleic acid aptamer (Aptamer), ribozyme (ribozyme), deoxyribozyme (DNAzyme), transcription factor decoy (Decoy), antigene (antigene), CpG oligonucleotide, or a combination thereof.

[0209] In another preferred embodiment, the nucleic acid analog is selected from the following group: left-handed nucleic acid, peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), thio-modified nucleic acid, 2'-fluorine-modified nucleic acid, 5-hydroxymethylcytosine nucleic acid, bridged nucleic acid (BNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), phosphorodiamidate morpholino oligonucleotide (PMO), or a combination thereof.

[0210] In another preferred embodiment, the nucleic acid analog is a phosphorodiamidate morpholino oligonucleotide (PMO).

[0211] In another preferred embodiment, the TfR1 high expression disease is selected from: myotonic diseases (such as dystrophic myotonia), genetic diseases (such as hereditary degenerative diseases, hereditary muscle wasting diseases), musculoskeletal system diseases, muscle wasting diseases (such as muscular dystrophy, Duchenne muscular dystrophy), neuromuscular diseases, myopathies, neurological diseases (such as neurodegenerative diseases, neurodegenerative diseases (such as Alzheimer's disease)), muscular dystrophy, iron metabolism disorders, β-thalassemia, iron deficiency anemia, mucopolysaccharidosis type I, mucopolysaccharidosis type II, polycythemia vera, solid tumors (such as head and neck cancer, esophageal cancer, brain tumors, nasopharyngeal cancer, prostate cancer, breast cancer, ovarian cancer, liver cancer, leukemia, colon cancer, lung cancer), non-solid tumors (such as leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia), diffuse large B-cell lymphoma), or a combination thereof.

[0212] In another preferred embodiment, the disease with high TfR1 expression is Duchenne muscular dystrophy (DMD).

[0213] In another preferred embodiment, the drug is a cytotoxic drug.

[0214] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0215] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposide, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0216] In another preferred embodiment, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansin, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxybenzoate, daptomycin, acetaminophen, chlortetracycline, chloramphenicol, chloramphenicol, daptomycin, chlortetracycline, chloramphenicol, daptomycin, chlortetracycline, chloramphenicol, chlortetracycline ... anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, Mitogellin, Retstrictocin, phenomycin, enomycin, curcin, crotin, calicheamicin, a Sapaonaria officinalis inhibitor, a glucocorticoid, or a combination thereof.

[0217] In another preferred embodiment, the coupling moiety is a detectable label.

[0218] In another preferred embodiment, the coupling portion is selected from the following group: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticles.

[0219] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) VHH chain of the anti-TfR1 single domain antibody according to the first aspect of the present invention.

[0220] In another preferred embodiment, the multivalency refers to the inclusion of multiple repeated identical or different VHH chains of the anti-TfR1 single domain antibody as described in the first aspect of the present invention in the amino acid sequence of the immunoconjugate.

[0221] In the twelfth aspect of the present invention, there is provided a use of an active ingredient, wherein the active ingredient is selected from the group consisting of the anti-TfR1 single domain antibody according to the first aspect of the present invention, the anti-TfR1 antibody according to the second aspect of the present invention, the chimeric antigen receptor according to the third aspect of the present invention, the multispecific antibody according to the fourth aspect of the present invention, the recombinant protein according to the fifth aspect of the present invention, the host cell according to the eighth aspect of the present invention, the engineered immune cell according to the ninth aspect of the present invention, the immunoconjugate according to the eleventh aspect of the present invention, or a combination thereof, and the active ingredient is used to prepare:

[0222] (a) Drugs for preventing and / or treating diseases with high TfR1 expression;

[0223] (b) Reagents for detecting diseases with high TfR1 expression.

[0224] In another preferred embodiment, the reagent is a diagnostic reagent, preferably, the diagnostic reagent is a detection sheet or a detection plate.

[0225] In another preferred embodiment, the diagnostic reagent is used to detect TfR1 protein or a fragment thereof in a sample.

[0226] In another preferred embodiment, the TfR1 high expression disease is selected from: myotonic diseases (such as dystrophic myotonia), genetic diseases (such as hereditary degenerative diseases, hereditary muscle wasting diseases), musculoskeletal system diseases, muscle wasting diseases (such as muscular dystrophy, Duchenne muscular dystrophy), neuromuscular diseases, myopathies, neurological diseases (such as neurodegenerative diseases, neurodegenerative diseases (such as Alzheimer's disease)), muscular dystrophy, iron metabolism disorders, β-thalassemia, iron deficiency anemia, mucopolysaccharidosis type I, mucopolysaccharidosis type II, polycythemia vera, solid tumors (such as head and neck cancer, esophageal cancer, brain tumors, nasopharyngeal cancer, prostate cancer, breast cancer, ovarian cancer, liver cancer, leukemia, colon cancer, lung cancer), non-solid tumors (such as leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia), diffuse large B-cell lymphoma), or a combination thereof.

[0227] In another preferred embodiment, the disease with high TfR1 expression is Duchenne muscular dystrophy (DMD).

[0228] In a thirteenth aspect of the present invention, a method for detecting TfR1 protein or a fragment thereof in a sample in vitro is provided, the method comprising the steps of:

[0229] (1) in vitro, contacting the sample with the anti-TfR1 single domain antibody as described in the first aspect of the present invention, the anti-TfR1 antibody as described in the second aspect of the present invention, the chimeric antigen receptor as described in the third aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, the recombinant protein as described in the fifth aspect of the present invention, the host cell as described in the eighth aspect of the present invention, the engineered immune cell as described in the ninth aspect of the present invention, the immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof;

[0230] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of TfR1 protein or a fragment thereof in the sample.

[0231] In another preferred embodiment, the detection includes diagnostic or non-diagnostic detection.

[0232] In a fourteenth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0233] (i) the anti-TfR1 single domain antibody as described in the first aspect of the present invention, the anti-TfR1 antibody as described in the second aspect of the present invention, the chimeric antigen receptor as described in the third aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, the recombinant protein as described in the fifth aspect of the present invention, the host cell as described in the eighth aspect of the present invention, the engineered immune cell as described in the ninth aspect of the present invention, the immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof as an active ingredient; and

[0234] (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0235] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection and lyophilized preparation.

[0236] In another preferred example, the pharmaceutical composition comprises 0.01 to 99.99% of the anti-TfR1 single domain antibody as described in the first aspect of the present invention, the anti-TfR1 antibody as described in the second aspect of the present invention, the chimeric antigen receptor as described in the third aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, the recombinant protein as described in the fifth aspect of the present invention, the host cell as described in the eighth aspect of the present invention, the engineered immune cell as described in the ninth aspect of the present invention, the immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof and 0.01 to 99.99% of a pharmaceutically acceptable carrier, and the percentages are the mass percentages of the pharmaceutical composition.

[0237] In another preferred embodiment, the concentration of the engineered immune cells in the active ingredient is 1×10 3 -1×10 8 cells / mL, preferably 1×10 4 -1×10 7 cells / mL.

[0238] A fifteenth aspect of the present invention provides a kit, comprising:

[0239] (1) a first container, comprising the anti-TfR1 single domain antibody as described in the first aspect of the present invention, the anti-TfR1 antibody as described in the second aspect of the present invention, the chimeric antigen receptor as described in the third aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, the recombinant protein as described in the fifth aspect of the present invention, the host cell as described in the eighth aspect of the present invention, the engineered immune cell as described in the ninth aspect of the present invention, the immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof; and / or

[0240] (2) a second container containing a secondary antibody against the contents of the first container;

[0241] or,

[0242] The kit contains a detection plate, which includes: a substrate (support plate) and a test strip, wherein the test strip contains the anti-TfR1 single-domain antibody as described in the first aspect of the present invention, the anti-TfR1 antibody as described in the second aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, the recombinant protein as described in the fifth aspect of the present invention, the immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof.

[0243] In another preferred embodiment, the kit further comprises an instruction manual, according to which the kit is used for non-invasively detecting TfR1 expression in a subject to be tested.

[0244] In another preferred embodiment, the kit is used for detecting diseases with high TfR1 expression.

[0245] In another preferred embodiment, the TfR1 high expression disease is selected from: myotonic diseases (such as dystrophic myotonia), genetic diseases (such as hereditary degenerative diseases, hereditary muscle wasting diseases), musculoskeletal system diseases, muscle wasting diseases (such as muscular dystrophy, Duchenne muscular dystrophy), neuromuscular diseases, myopathies, neurological diseases (such as neurodegenerative diseases, neurodegenerative diseases (such as Alzheimer's disease)), muscular dystrophy, iron metabolism disorders, β-thalassemia, iron deficiency anemia, mucopolysaccharidosis type I, mucopolysaccharidosis type II, polycythemia vera, solid tumors (such as head and neck cancer, esophageal cancer, brain tumors, nasopharyngeal cancer, prostate cancer, breast cancer, ovarian cancer, liver cancer, leukemia, colon cancer, lung cancer), non-solid tumors (such as leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia), diffuse large B-cell lymphoma), or a combination thereof.

[0246] In another preferred embodiment, the disease with high TfR1 expression is Duchenne muscular dystrophy (DMD).

[0247] In the sixteenth aspect of the present invention, a method for preventing and / or treating diseases with high TfR1 expression is provided, the method comprising: administering to a subject in need thereof an anti-TfR1 single domain antibody as described in the first aspect of the present invention, an anti-TfR1 antibody as described in the second aspect of the present invention, a chimeric antigen receptor as described in the third aspect of the present invention, a multispecific antibody as described in the fourth aspect of the present invention, a recombinant protein as described in the fifth aspect of the present invention, a host cell as described in the eighth aspect of the present invention, an engineered immune cell as described in the ninth aspect of the present invention, an immunoconjugate as described in the eleventh aspect of the present invention, a pharmaceutical composition as described in the fourteenth aspect of the present invention, or a combination thereof.

[0248] In another preferred embodiment, the subject includes mammals, such as humans.

[0249] In another preferred embodiment, the TfR1 high expression disease is selected from: myotonic diseases (such as dystrophic myotonia), genetic diseases (such as hereditary degenerative diseases, hereditary muscle wasting diseases), musculoskeletal system diseases, muscle wasting diseases (such as muscular dystrophy, Duchenne muscular dystrophy), neuromuscular diseases, myopathies, neurological diseases (such as neurodegenerative diseases, neurodegenerative diseases (such as Alzheimer's disease)), muscular dystrophy, iron metabolism disorders, β-thalassemia, iron deficiency anemia, mucopolysaccharidosis type I, mucopolysaccharidosis type II, polycythemia vera, solid tumors (such as head and neck cancer, esophageal cancer, brain tumors, nasopharyngeal cancer, prostate cancer, breast cancer, ovarian cancer, liver cancer, leukemia, colon cancer, lung cancer), non-solid tumors (such as leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia), diffuse large B-cell lymphoma), or a combination thereof.

[0250] In another preferred embodiment, the disease with high TfR1 expression is Duchenne muscular dystrophy (DMD).

[0251] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from the subject (autologous cells).

[0252] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).

[0253] In another preferred embodiment, the method can be used in combination with other treatment methods.

[0254] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy and the like.

[0255] In a seventeenth aspect of the present invention, a method for diagnosing a disease with high expression of TfR1 is provided, comprising the steps of:

[0256] (i) obtaining a sample from a diagnostic subject, and contacting the sample with the anti-TfR1 single domain antibody as described in the first aspect of the present invention, the anti-TfR1 antibody as described in the second aspect of the present invention, the chimeric antigen receptor as described in the third aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, the recombinant protein as described in the fifth aspect of the present invention, the host cell as described in the eighth aspect of the present invention, the engineered immune cell as described in the ninth aspect of the present invention, the immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof; and

[0257] (ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates that the subject is a confirmed patient of a disease with high TfR1 expression.

[0258] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues and organs.

[0259] In another preferred embodiment, the TfR1 high expression disease is selected from: myotonic diseases (such as dystrophic myotonia), genetic diseases (such as hereditary degenerative diseases, hereditary muscle wasting diseases), musculoskeletal system diseases, muscle wasting diseases (such as muscular dystrophy, Duchenne muscular dystrophy), neuromuscular diseases, myopathies, neurological diseases (such as neurodegenerative diseases, neurodegenerative diseases (such as Alzheimer's disease)), muscular dystrophy, iron metabolism disorders, β-thalassemia, iron deficiency anemia, mucopolysaccharidosis type I, mucopolysaccharidosis type II, polycythemia vera, solid tumors (such as head and neck cancer, esophageal cancer, brain tumors, nasopharyngeal cancer, prostate cancer, breast cancer, ovarian cancer, liver cancer, leukemia, colon cancer, lung cancer), non-solid tumors (such as leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia), diffuse large B-cell lymphoma), or a combination thereof.

[0260] In another preferred embodiment, the disease with high TfR1 expression is Duchenne muscular dystrophy (DMD).

[0261] In an eighteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, wherein the recombinant polypeptide is the anti-TfR1 single domain antibody according to the first aspect of the present invention, the anti-TfR1 antibody according to the second aspect of the present invention, the chimeric antigen receptor according to the third aspect of the present invention, the multispecific antibody according to the fourth aspect of the present invention, the recombinant protein according to the fifth aspect of the present invention, or a combination thereof, the method comprising:

[0262] (a) culturing the host cell according to the eighth aspect of the present invention under conditions suitable for expression; and

[0263] (b) isolating the recombinant polypeptide from the culture.

[0264] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0265] Figure 1 shows the protein sequence alignment results of TfR1 antigens in three species: human, rhesus monkey, and mouse.

[0266] Figure 2 shows the construction of an anti-TfR1 single-domain antibody yeast library. a. Library transformant counting plate; b. Library antibody sequence insertion rate detection; c. Library amino acid sequence phylogenetic tree analysis.

[0267] Figure 3 shows the expression and purification process of the anti-TfR1 single domain antibody, as assessed by SDS-PAGE. Left: Supernatant after 48 hours of induction; Right: Sample purified by nickel affinity chromatography after 72 hours of induction.

[0268] Figure 4 shows the ELISA assay for the binding ability of anti-TfR1 single domain antibodies to three species of TfR1 antigens. Upper left: binding activity to human TfR1 antigen; upper right: binding activity to monkey TfR1 antigen; lower left: binding activity to mouse TfR1 antigen.

[0269] FIG5 shows the ELISA detection of the binding ability of the anti-TfR1 single domain antibody to the human TfR1 antigen.

[0270] Figure 6 shows the ELISA assay for the binding ability of anti-TfR1 single domain antibodies to monkey and mouse TfR1 antigens. Left: Binding activity to monkey TfR1 antigen; Right: Binding activity to mouse TfR1 antigen.

[0271] Figure 7 shows flow cytometry analysis of the binding ability of anti-TfR1 single domain antibodies to CHO-K1 cells that overexpress hTfR1 and hTfR2. Left: Binding activity to hTfR1 CHO-K1 cells; Right: Binding activity to hTfR2 CHO-K1 cells.

[0272] FIG8 shows the flow cytometry detection of the binding ability of anti-TfR1 single domain antibody to hTfR1 CHO-K1 cells.

[0273] FIG9 shows the ELISA test of the binding ability of the anti-TfR1 single domain antibody to hTfR1 bound to transferrin.

[0274] FIG10 shows the sequence alignment of candidate antibody TfR1-95 with human Germline.

[0275] FIG11 shows the ELISA detection of the binding ability of humanized anti-TfR1 VHH-Fc to human TfR1 antigen.

[0276] FIG12 shows the ELISA detection of the binding ability of humanized anti-TfR1 VHH-Fc to monkey TfR1 antigen.

[0277] FIG13 shows the flow cytometric detection of the binding ability of humanized anti-TfR1 VHH-Fc to hTfR1 CHO-K1 cells.

[0278] FIG14 shows the identification of TfR1 expression on the surface of SK-OV-3 tumor cells.

[0279] FIG15 shows the ELISA test of the binding ability of humanized anti-TfR1 VHH-Fc to hTfR1 bound to recombinant human HFE protein.

[0280] FIG16 shows the chromatographic profile and SDS-PAGE verification of the anti-TfR1 single domain antibody-PMO conjugate.

[0281] FIG17 shows the agarose gel electrophoresis analysis results of wild-type DMD products and Exon 23-skipping DMD products in groups G1, G2, and G3, as well as the corresponding Exon 23-skipping rates.

[0282] FIG18 shows the verification results of sequence sequencing analysis of exon skipping bands. DETAILED DESCRIPTION

[0283] After extensive and in-depth research and a large number of screenings, the inventors unexpectedly obtained for the first time a high-affinity and high-specificity anti-TfR1 single-domain antibody. Specifically, the present invention used human TfR1 antigen to immunize healthy adult alpacas and obtained high-quality 10 8 After the yeast library, a total of 44 human and monkey TfR1 antigen-binding positive single-domain antibodies were obtained using flow cytometry, and humanized antibodies were further obtained. The binding epitope of the anti-TfR1 single-domain antibody of the present invention can be the same as or different from that of transferrin, and is non-competitive with the HFE protein. It can be used for the treatment and / or diagnosis of different types of related diseases. The drugs or preparations prepared based on the antibodies are less affected by the internal environment. In addition, compared with the control hIgG1 antibody, the anti-TfR1 single-domain antibody of the present invention has higher endocytosis activity on tumor cells and can be used to prepare drugs with higher efficacy. On this basis, the present invention is completed.

[0284] the term

[0285] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below.

[0286] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0287] The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0288] The names of the single-domain antibodies targeting TfR1 of the present invention and the corresponding sequence numbers are shown in Table A below.

[0289] Table A

[0290] Note: Each numerical value in the table represents a sequence number, i.e., "1" represents "SEQ ID NO: 1", and the sequence numbers of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4 shown in the table are the numbers of their amino acid sequences.

[0291] The above-mentioned CDRs of the anti-TfR1 single domain antibody of the present invention are divided according to the IMGT rule. Those skilled in the art should understand that when other CDR division rules, such as the Kabat rule, the AbM rule, the Chothia rule, and the Contact rule are used for division, the corresponding CDRs also fall within the scope of protection of the present invention.

[0292] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0293] As used herein, the terms "single domain antibody (sdAb)", "VHH", and "Nanobody" have the same meaning and are used interchangeably. They refer to the construction of a single domain antibody (VHH) consisting solely of a single heavy chain variable region by cloning the variable region of an antibody heavy chain. This is the smallest fully functional antigen-binding fragment. Typically, antibodies naturally lacking the light chain and heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting solely of a single heavy chain variable region.

[0294] As used herein, the term "variable" refers to certain portions of the variable region in an antibody that differ in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which are generally in a β-sheet configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partial β-sheet structure. The CDRs in each chain are closely together through the FR region and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0295] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the single domain antibodies or fragments thereof directed against TfR1.

[0296] As used herein, the terms "hypervariable region," "hypervariable region," "complementarity determining region," and "complementarity determining region (CDR)" are used interchangeably.

[0297] In a preferred embodiment of the present invention, the single-domain antibody or the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.

[0298] In a preferred embodiment of the present invention, the single-domain antibody or the heavy chain of the antibody comprises the above-mentioned heavy chain variable region and heavy chain constant region.

[0299] In the present invention, the terms "nanoantibody", "single domain antibody", "single domain antibody of the present invention", "nanoantibody of the present invention", "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably to refer to polypeptides that specifically bind to TfR1 protein, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.

[0300] The present invention also provides other proteins or fusion expression products comprising the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) comprising a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0301] Generally, an antibody's antigen-binding properties are described by three specific regions within the variable region of the heavy chain, known as the variable regions (CDRs). This region is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0302] The heavy chain variable regions of the single domain antibodies or antibodies of the present invention are particularly interesting because they are at least partially involved in binding to the antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have greater than 90% (preferably greater than 95%, and most preferably greater than 98%) homology to the CDRs identified herein.

[0303] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0304] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0305] The antibody of the present invention refers to a polypeptide having TfR1 protein binding activity and comprising the above-mentioned CDR region. The term also includes variant forms of polypeptides comprising the above-mentioned CDR region and having the same function as the antibody of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not change the function of the protein. For another example, adding one or several amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0306] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0307] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention also encompasses fragments of the antibodies of the invention. Typically, the fragments comprise at least about 50 contiguous amino acids of the antibodies of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0308] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table B.

[0309] Table B

[0310] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0311] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0312] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0313] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0314] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0315] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0316] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0317] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0318] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0319] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0320] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0321] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0322] The single domain antibodies or antibodies of the present invention can be used alone or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0323] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0324] Therapeutic agents that can be combined or coupled with the antibodies of the present invention include but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0325] Anti-TfR1 single domain antibody / nanobody

[0326] In the present invention, the anti-TfR1 single-domain antibody includes monomers, bivalents (divalent antibodies), tetravalents (tetravalent antibodies), and / or multivalents (multivalent antibodies).

[0327] In a preferred example of the present invention, the amino acid sequence of the VHH chain of the anti-TfR1 single domain antibody is selected from one or more of SEQ ID NOs: 1-44, 210-215.

[0328] The anti-TfR1 single-domain antibody of the present invention can specifically bind to human and / or alpaca TfR1, but does not bind or weakly binds to mouse TfR1; it can specifically bind to monkey TfR1, or does not bind or weakly binds to monkey TfR1.

[0329] The anti-TfR1 single-domain antibody of the present invention can block the binding of TfR1 to transferrin, or does not block the binding of TfR1 to transferrin, but does not bind to TfR2, nor does it block the binding of TfR1 to HFE protein (HFE protein affects the interaction between transferrin and transferrin receptor by interacting with transferrin receptor, thereby regulating iron balance in the body).

[0330] Labeled antibodies

[0331] In a preferred embodiment of the present invention, the single domain antibody or antibody may carry a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label, or a fluorescent label.

[0332] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, the anti-TfR1 single domain antibody or antibody is labeled with colloidal gold to obtain a colloidal gold-labeled antibody. The anti-TfR1 single domain antibody or antibody of the present invention can effectively bind to the TfR1 protein.

[0333] Detection method

[0334] The present invention also relates to a method for detecting TfR1 protein or a fragment thereof. The method generally comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of TfR1 protein in the dissolved sample.

[0335] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage medium.

[0336] Reagent test kit

[0337] The present invention also provides a kit containing the anti-TfR1 single domain antibody or antibody (or fragment thereof) or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc.

[0338] The present invention also provides a detection kit for detecting TfR1 protein levels, which includes an antibody that recognizes TfR1 protein, a lysis medium for dissolving the sample, and common reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. The detection kit can be an in vitro diagnostic device.

[0339] Pharmaceutical composition

[0340] The present invention also provides a pharmaceutical composition. It contains the anti-TfR1 single domain antibody described in the first aspect of the present invention, the anti-TfR1 antibody described in the second aspect of the present invention, the chimeric antigen receptor described in the third aspect of the present invention, the fusion protein described in the fourth aspect of the present invention, the recombinant protein described in the fifth aspect of the present invention, the host cell described in the eighth aspect of the present invention, the engineered immune cell described in the ninth aspect of the present invention, the immunoconjugate described in the eleventh aspect of the present invention, or a combination thereof as an active ingredient, and a pharmaceutically acceptable carrier.

[0341] Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the substance being formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.

[0342] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0343] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight and, in most cases, does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0344] application

[0345] As described above, the single-domain antibodies and antibodies of the present invention have broad biological and clinical application value, and their applications involve multiple fields such as diagnosis and treatment of diseases related to the TfR1 protein, basic medical research, and biological research. A preferred application is for clinical diagnosis, prevention, and treatment of TfR1 protein.

[0346] The present invention also provides a method for stimulating an immune response mediated by T cells targeting a tumor cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cells of the present invention to the mammal.

[0347] In one embodiment, the present invention comprises a type of cell therapy in which a patient's own T cells (or those of an allogeneic donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then infused back into the same patient. This approach minimizes the likelihood of a graft-versus-host reaction, as antigens are recognized by T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0348] In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and can persist for months to years. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, wherein the CAR-T cells can induce a specific immune response against tumor cells that highly express the antigen recognized by the CAR antigen binding domain. For example, the CAR-T cells of the present invention induce a specific immune response against tumor cells that highly express TfR1.

[0349] Treatable cancers include tumors that are not vascularized or substantially not vascularized, as well as vascularized tumors. Cancer types treated with the CAR of the present invention include, but are not limited to, breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small intestine cancer, colorectal cancer, bile duct cancer, cervical cancer, lymphoma, esophageal cancer, etc.

[0350] Generally, cells activated and expanded as described herein can be used to treat and prevent diseases such as tumors. Therefore, the present invention provides a method for treating cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of the CAR-T cells of the present invention.

[0351] The CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may include a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0352] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or may be determined by clinical trials.

[0353] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. The pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 7The T cell composition can be administered at a dose of 10 cells / kg body weight (including all integer values ​​within the range). The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a specific patient can be easily determined by a person skilled in the art of medicine by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0354] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous injection. The T cell composition can be injected directly into the tumor, lymph node or infection site.

[0355] In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with (e.g., before, simultaneously, or after) any number of related treatment modalities, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or efavirenz treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0356] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration may be implemented according to practices accepted in the art. Typically, 1×10 5 to 1×10 10The modified T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0357] Main advantages of the present invention

[0358] 1. The present invention provides a single-domain antibody targeting TfR1, which can specifically target the surface of cells where TfR1 is highly expressed, including tumor cells, muscle cells, and brain endothelial cells. Its binding epitope can be different from that of transferrin, without affecting TfR1's iron transport function. It can be used as a drug delivery vehicle, precisely targeting tumors and muscle cells, and helping to penetrate the blood-brain barrier. Therefore, this TfR1-targeting single-domain antibody has great potential for development as an anti-tumor drug and targeted drug for muscle and brain diseases.

[0359] 2. The anti-TfR1 single domain antibody of the present invention may also have the same binding epitope as transferrin, and can be used for the treatment and / or diagnosis of specific diseases such as iron deficiency anemia caused by excessively high transferrin levels.

[0360] 3. The anti-TfR1 single-domain antibody of the present invention is non-competitive with HFE protein. HFE protein does not affect the binding of the anti-TfR1 single-domain antibody of the present invention and its humanized VHH-Fc to human TfR1. The anti-TfR1 single-domain antibody of the present invention also does not affect the interaction between HFE protein and transferrin receptor (including TfR1). Therefore, the drug or preparation prepared based on the anti-TfR1 single-domain antibody of the present invention is less affected by the internal environment and has a lower possibility of off-target effects.

[0361] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0362] Example 1: Immunization of alpacas to induce the production of anti-TfR1 single domain antibodies and serum titer detection

[0363] For the first immunization, 0.5 mg of human TfR1 antigen (SinoBiological, Cat#11020-H07H) was mixed with an equal volume of complete Freund's adjuvant (CFA) and injected subcutaneously into healthy alpacas to stimulate the alpaca's immune system to produce corresponding anti-TfR1 antibodies. For the second booster immunization, 0.25 mg of human TfR1 antigen was mixed with an equal volume of incomplete Freund's adjuvant (IFA) and injected subcutaneously into alpacas 20 days after the first immunization. Subsequently, three and four booster immunizations were performed at intervals of 20 days, and the amount of immune antigen and adjuvant used were the same as for the second immunization. Blood was collected before immunization, one week after the second, third, and fourth immunizations, respectively, to detect the titer of target antibodies in the serum.

[0364] Serum titers were assessed using ELISA plates coated with immobilized TfR1 antigen proteins from human, rhesus macaque, and mouse species. Antibody titers against the antigens from different species were measured using an HRP-labeled goat anti-Alpaca secondary antibody. The results of serum antibody titers from the second, third, and fourth immunizations are shown in Tables 1 through 3, respectively.

[0365] Table 1 - Secondary immune serum titer detection

[0366] Table 2 - Three-times immune serum titer test

[0367] Table 3 - Four immune serum titer tests

[0368] Serum titer testing revealed that alpaca immunization with human TfR1 antigen produced high-titer anti-human TfR1 antibodies, moderate-titer anti-monkey TfR1 antibodies, and low-titer anti-mouse TfR1 antibodies in their blood. Sequence alignment of TfR1 antigens from three species, Homo sapiens, Macaca mulatta, and Mus musculus, revealed a 91% similarity between the human and monkey sequences and a 76% similarity between the human and mouse sequences. The sequence alignment is shown in Figure 1. The titers of alpaca serum antibodies met expectations, and peripheral blood from the four immunizations was suitable for antibody library construction.

[0369] Example 2: Construction and screening of anti-TfR1 single domain antibody yeast library

[0370] 50 mL of peripheral blood from alpacas after four immunizations was collected to separate PBMC (Peripheral Blood Mononuclear Cell), and total RNA was extracted using RNAiso Plus reagent. TM5 μg of total RNA was reverse transcribed into cDNA using the II 1st Strand cDNA Synthesis Kit (Takara, Cat#6210A), following the procedure in the kit's instruction manual. Using the cDNA as a template, 5 μl of a 5-fold dilution of the cDNA was used for the first round of nested PCR. The approximately 750 bp PCR product was then gel-backed and used as the template for the second round of nested PCR. After two rounds of nested PCR amplification, the single-domain antibody fragment was obtained.

[0371] After purification using the Cycle-Puer Kit, the PCR product was co-transfected into yeast competent cells with the linearized pYDC2 vector fragment by electroporation, spread on 200 mm SD-CAA plates in a total volume of 5 mL, and cultured at 30°C for 4 days.

[0372] As shown in Figure 2a, the reservoir capacity was calculated by taking 10 μL of the electroporated yeast cell suspension and spreading it on a 90 mm plate for gradient dilution. The total number of transformants was 1 × 10 8 indivual.

[0373] As shown in Figure 2b, 48 single clones were randomly picked from the library plate for PCR identification, and the results showed that the insertion rate was 100%.

[0374] As shown in Figure 2c, 60 positive PCR products were randomly selected for sequencing. After protein translation, the amino acid sequences were aligned and a phylogenetic tree was drawn. According to the number of transformants, library insertion rate and diversity sequencing analysis results, the library capacity of the anti-TfR1 single domain antibody yeast library was 1×10 8 .

[0375] In order to obtain cross-species antibodies between humans and monkeys, the screening process used biotin-labeled rhesus macaque TfR1 antigen (SinoBiological, Cat#90253-C07H-B) for antibody screening and enrichment. The first round of enrichment used 100nM rhesus macaque TfR1 antigen and was performed by magnetic bead sorting. The yeast cell input was 1.36×10 10 The second round of enrichment was performed on the magnetic bead-enriched pool from the first round, using 100 nM rhesus macaque TfR1 antigen. The double-positive cell population was identified by flow cytometry, and the double-positive monoclonal clones were sorted.

[0376] The monoclonal clones were then sequenced and analyzed for sequence alignment. Flow cytometry was then used to further validate the binding activity of the monoclonal cells corresponding to the unique sequences for human, mouse, and monkey TfR1 antigens. Sequence alignment and flow cytometry binding validation of the monoclonal clones revealed 44 sequences that positively bound to human and monkey TfR1 antigens. No sequences that bound to mouse TfR1 were identified (Table 4). These sequences involved 23 CDR1, 25 CDR2, and 27 CDR3 sequences (Table 5).

[0377] Table 4 Sequences of human and monkey TfR1 antigen-binding positive single domain antibodies

[0378] Table 5 Human and monkey TfR1 antigen binding positive CDR sequences

[0379] Example 3: Construction of yeast expression strain for anti-TfR1 single domain antibody and protein preparation

[0380] The anti-TfR1 single-domain antibody gene sequence with a his-tag added to the N-terminus was codon-optimized and constructed into the pPICZ alpha A plasmid. The plasmid was then linearized with Pem I enzyme. 5 μL of the linearized plasmid was added to 100 μL of competent cells, and the plasmid carrying the target gene was transformed into X33 Pichia pastoris cells using an electroporator (Biorad, MicroPulser) at 1.6 kV and 4 ms.

[0381] After electroporation, Pichia pastoris cells were restored to viability using a mixed culture medium (YPD:Sorbitol = 1:1). 50 μL of Pichia pastoris cells were then plated onto YPD solid medium containing 200, 400, 600, and 800 μg / mL zeocin (Invitrogen, Cat#R25001). Zeocin concentration gradient screening was performed to obtain strains expressing high copies of the target gene. Monoclonal screening was performed using GMGY medium (Sangon, Cat#B540130) at 30°C and 250 rpm to culture the monoclonal strains. After obtaining sufficient cells, GMMY medium (Sangon, Cat#B540131) was used to induce secretory expression of the target single-domain antibody at 20°C and 250 rpm. After 24 hours, 20 μL of supernatant was collected and the expression level of each colony was analyzed by gel electrophoresis. The strain with the highest expression level was selected for bacterial maintenance and protein expression production.

[0382] The protein was expressed and purified in large quantities as follows: 400 μL of bacterial culture was inoculated into 200 mL of BMGY medium and cultured for enrichment at 30°C and 250 rpm for 3 days. Subsequently, 80 mL of BMMY medium was replaced to induce expression, supplemented with 1% methanol every 24 hours. Secretory expression of the target single-domain antibody was induced at 20°C and 250 rpm for 3 days. After induction, the supernatant was collected by centrifugation at 12,000 rpm for 15 minutes in a high-speed refrigerated centrifuge. The protein was then purified using nickel affinity chromatography (Cytiva, Cat# 17092108). The nickel affinity column was equilibrated with binding buffer before the supernatant containing the target protein was passed through the column. The His-tagged protein was retained on the column by binding to nickel ions. Nonspecific binding to the column was then removed with a buffer containing 20 mM imidazole. Finally, the target protein was eluted with an elution buffer containing 250 mM imidazole.

[0383] As shown in Figure 3, the SDS-PAGE gel image shows that the purity of the single domain antibody in the protein expression supernatant is greater than 95%. After one-step nickel ion affinity purification, the protein purity can reach more than 99%.

[0384] Example 4: Analysis of Binding Activity of Anti-TfR1 Single Domain Antibodies to Human and Monkey TfR1 Recombinant Proteins

[0385] Prepare human TfR1-His (ACRO, Cat#CD1-H5243), monkey TfR1-His (ACRO, Cat#TFR-C524A), and mouse TfR1-His (ACRO, Cat#TFR-M524B) antigen solutions in PBS to a final concentration of 1 μg / mL. Add 100 μl / well to a 96-well microtiter plate and coat overnight at 4°C. Wash three times with PBST (PBS + 0.05% Tween 20), add 200 μl / well of blocking solution (PBST + 3% BSA), and block at room temperature for 1 hour. Wash once with PBST, add 100 μl / well of serially diluted single-domain antibody solution, and incubate at room temperature for 1 hour. The cells were washed three times with PBST, and 100 μl / well of a 1:5000 dilution of horseradish peroxidase-labeled rabbit anti-camelid VHH antibody (Genscript, Cat#A02016) was added and incubated at room temperature for 1 hour. The cells were washed three times with PBST, patted dry, and 100 μl / well of the chromogenic substrate TMB solution (Beyotime, Cat#P0209) was added. The cells were allowed to develop at room temperature for 5 to 30 minutes, followed by the addition of 100 μl / well of the chromogenic stop solution (Beyotime, Cat#P0215). The absorbance at 450 nm was measured in each well using a multifunctional microplate reader (Molecular Devices, SpectraMax i3x). The EC50 value was calculated using a sigmoidal curve fitted with a 4-parameter equation using GraphPad Prism 9 software.

[0386] As shown in Figures 4-6, the experimental results show that most candidate antibodies have cross-binding activity between human and monkey species, and all candidate antibodies have no binding activity to mouse antigens.

[0387] Example 5: Holo-transferrin does not affect the binding activity of anti-TfR1 single domain antibody

[0388] Prepare human TfR1-His (ACRO, Cat#CD1-H5243) antigen solution and human Holo-transferrin (Biyuntian, Cat#ST1135) solution in PBS to final concentrations of 1 μg / mL and 100 μg / mL, respectively. Add 100 μl / well to a 96-well microtiter plate and coat overnight at 4°C. Wash three times with PBST (PBS + 0.05% Tween 20), add 200 μl / well of blocking solution (PBST + 3% BSA), and block at room temperature for 1 hour. Wash once with PBST, add 100 μl / well of human TfR1-His solution at a concentration of 1 μg / mL to the microtiter plate coated with human Holo-transferrin, and incubate at room temperature for 1 hour. After washing three times with PBST, 100 μl / well of serially diluted single-domain antibody solution was added to the microplate coated with human TfR1-His and human Holo-transferrin, and incubated at room temperature for 1 hour. After washing three times with PBST, 100 μl / well of a 1:5000 dilution of horseradish peroxidase-conjugated rabbit anti-camelid VHH antibody (Genscript, Cat#A02016) was added and incubated at room temperature for 1 hour. After washing three times with PBST, pat dry, 100 μl / well of the chromogenic substrate TMB solution (Beyotime, Cat#P0209) was added, and color was developed at room temperature for 5 to 30 minutes. Then, 100 μl / well of the color stop solution (Beyotime, Cat#P0215) was added. The absorbance of each well was measured at 450 nm using a multi-function microplate reader (Molecular Devices, SpectraMax i3x). The binding EC50 values ​​were calculated using a sigmoidal 4-parameter equation fitted with GraphPad Prism 9 software.

[0389] As shown in Figure 7, the experimental results show that the candidate antibodies TfR1-21, TfR1-23, and TfR1-134 have a non-competitive relationship with Holo-transferrin and do not affect the function of TfR1 in transporting iron ions; the candidate antibodies TfR1-74 and TfR1-132 have a competitive relationship with Holo-transferrin.

[0390] Example 6: Analysis of Binding Activity of Anti-TfR1 Single Domain Antibodies to Human TfR Highly Expressing Cells

[0391] hTfR1 CHO-K1 cells and hTfR2 CHO-K1 cells were obtained by stably transfecting CHO-K1 cells with the pIRES-Neo3 vector expressing the hμman TfR1 gene (NM_003234.4) and the hμman TfR2 gene (NM_003227.4), respectively. Cells in the logarithmic growth phase were collected, washed with flow cytometry buffer (PBS + 2% FBS), and the cell density was adjusted to 1×10 6 cells / mL, add 180 μl / well of the cell suspension to a 96-well M-bottom plate. Dilute the test sample stock solution with flow cytometry buffer to prepare a serial dilution of the antibody solution at a 10x concentration. Add 20 μl of this solution to the cell suspension in the 96-well plate, vortex to mix, and incubate the 96-well plate at 4°C for 30 minutes. Centrifuge at 1000 rpm for 5 minutes at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, then add 200 μl / well of a 1:1000 dilution of iFlμor647-conjugated rabbit anti-camelid VHH antibody (Genscript, Cat#A02019), vortex to mix, and incubate the 96-well plate at 4°C for 30 minutes. Centrifuge at 1000 rpm for 5 minutes at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, and resuspend the cells in 200 μl / well of flow cytometry buffer. Measure the mean fluorescence intensity of each sample using a flow cytometer (BD, FACSCelesta). The binding EC50 values ​​were calculated using a sigmoidal 4-parameter equation fitted with GraphPad Prism 9 software.

[0392] As shown in Figures 8-9, the experimental results showed that most candidate antibodies could bind to hTfR1 expressed on the cell surface, and all candidate antibodies did not bind to hTfR2 on the cell surface.

[0393] Example 7: Humanization of anti-TfR1 single domain antibody

[0394] The humanization of the anti-TfR1 single-domain antibody was achieved by aligning the parent sequence (the parent sequence is AS001-N037-095-P sequence, i.e., TfR1-95 sequence, whose amino acid sequence is shown in SEQ ID NO: 14) with the human Germline database. After defining the CDR and framework regions of the parent antibody, humanized sequences of varying degrees were designed based on the differential sites in the framework region.

[0395] As shown in Figure 10, sequence alignment results indicate that the parental sequence has the highest homology to the human germline IGHV3-23 sequence, containing eight camel-derived amino acid sites. Therefore, IGHV3-23, with the highest homology, was selected as the humanization design template. Six sequences with a humanization degree greater than 95% were designed through back mutation: VHH1, VHH2, VHH3, VHH4, VHH5, and VHH6, also known as AS001-N037-095-VHH1 to AS001-N037-095-VHH6 in Table 6, having the amino acid sequences shown in SEQ ID NOs: 210-215, respectively.

[0396] Table 6 Summary of humanized sequences of anti-TfR1 single domain antibodies

[0397] Example 8: Expression and purification of anti-TfR1 humanized VHH-Fc

[0398] The C-terminus of the humanized VHH sequence was fused to the human IgG1 Fc segment. After codon optimization, the fusion sequence was constructed into the pcDNA3.4 vector. The fusion expression plasmid was transiently transfected into Expi CHO cells for 7 days of expression. The expression medium was Expi CHO. TM Expression medium (Thermo fisher, Cat#A2910001), transfection kit is ExpiFectamine TM CHO Transfection Kit (Thermo Fisher, Cat# A29129). After the cells were expressed, the supernatant was centrifuged and filtered through a 0.22 μM filter membrane and then eluted with Protein A affinity filler (Cytiva, MabSelect SμRe TM ) Purification of humanized VHH-Fc protein.

[0399] Example 9: Stability analysis of anti-TfR1 humanized VHH-Fc

[0400] The aggregation tendency of humanized VHH-Fc was analyzed by HPLC-SEC. The column used in the experiment was XBridge BEH SEC 3.5 μm, 7.8×300 mm (Waters, 186007640), flow rate was set to 0.8 mL / min; detection wavelength was 280 nm.

[0401] The test results are shown in Table 7, which show that the monomer ratios of all humanized antibodies are higher than 98%, and the samples are relatively stable and not prone to forming aggregates.

[0402] Table 7 Summary of HPLC-SEC results of anti-TfR1 humanized VHH-Fc

[0403] Note: “ / ” represents none.

[0404] The thermal stability of humanized VHH-Fc was further analyzed using differential scanning fluorimetry (DSF). The experiment was performed using an ABI 7500 Fast Real-Time PCR instrument. The melting curve assay was selected in continuous mode, with a scanning temperature range of 25°C to 99°C. The temperature corresponding to the first peak and valley of the melting curve derivative was determined as the protein's denaturation temperature (Tm1), and the temperature corresponding to the second peak and valley was determined as the protein's denaturation temperature (Tm2). The test results are shown in Table 8.

[0405] Table 8 Summary of DSF results of anti-TfR1 humanized VHH-Fc

[0406] The results showed that the Tm1 of VHH1, 2, 4, and 6 were all reduced to varying degrees, and their stability was slightly lower than that of the parent. The Tm1 of VHH3 was consistent with the parent, and that of VHH5 was slightly higher than that of the parent.

[0407] Example 10: Analysis of Binding Activity of Humanized VHH-Fc to Human and Monkey TfR1 Proteins

[0408] The binding activity of humanized VHH-Fc to human and monkey TfR1 proteins was analyzed by enzyme-linked immunosorbent assay (ELISA). Human TfR1-His (ACRO, Cat#CD1-H5243) and monkey TfR1-His (ACRO, Cat#TFR-C524a) antigen solutions were prepared in PBS to a final concentration of 1 μg / mL. 25 μl / well was added to a 384-well ELISA plate and coated overnight at 4°C. The plates were washed three times with PBST (PBS + 0.05% Tween 20), and 50 μl / well of blocking solution (PBST + 3% BSA) was added and blocked at room temperature for 1 hour. The plates were washed once with PBST, and 25 μl / well of serially diluted VHH-Fc solutions were added and incubated at room temperature for 1 hour. The cells were washed three times with PBST, and 25 μl / well of a 1:5000 dilution of horseradish peroxidase-labeled goat anti-human IgG antibody (Yeasen, Cat#33501ES60) was added and incubated at room temperature for 1 hour. The cells were washed three times with PBST, patted dry, and 25 μl / well of the chromogenic substrate TMB solution (Beyotime, Cat#P0209) was added. The cells were allowed to develop for 5 to 30 minutes at room temperature, followed by the addition of 25 μl / well of the chromogenic stop solution (Beyotime, Cat#P0215). The absorbance at 450 nm was measured in each well using a multi-function microplate reader (Molecular Devices, SpectraMax i3x). The EC50 value was calculated using a sigmoidal curve fitted with a four-parameter equation using GraphPad Prism 9 software.

[0409] As shown in Figures 11-12, the experimental results show that all humanized VHH-Fc bind to human TfR1-His, and the binding activity is comparable to that of the parent, with VHH3 and 5 slightly stronger than the parent; VHH1, 2, 4, and 6 have lost their binding activity to monkey TfR1-His, while VHH3 and 5 retain monkey TfR1-His binding activity comparable to that of the parent.

[0410] The binding activity of humanized VHH-Fc to human TfR1 protein (ACRO, Cat#CD1-H5243) was analyzed by surface plasmon resonance (SPR). The instrument used was a biacore T200 (Cytiva). The test results are shown in Table 9.

[0411] Table 9 SPR detection results of anti-TfR1 humanized VHH-Fc

[0412] The affinity test results were consistent with the ELISA method. All humanized VHH-Fc bound to human TfR1-His, and the binding activity was comparable to that of the parent, with VHH3 and 5 slightly stronger than the parent.

[0413] Example 11: Analysis of Binding Activity of Humanized VHH-Fc to Human TfR Highly Expressing Cells

[0414] hTfR1 CHO-K1 cells in the logarithmic growth phase were collected, washed with flow cytometry buffer (PBS + 2% FBS) and the cell density was adjusted to 1×10 6 cells / mL, add 180 μl / well of the cell suspension to a 96-well M-bottom plate. Dilute the test sample stock solution with flow cytometry buffer to prepare a serial dilution of the antibody solution at a 10x concentration. Add 20 μl of this solution to the cell suspension in the 96-well plate, vortex to mix, and incubate the 96-well plate at 4°C for 30 minutes. Centrifuge at 1000 rpm for 5 minutes at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, then add 200 μl / well of a 1:1000 dilution of FITC-conjugated mouse anti-human IgG antibody (Biolegend, Cat# 410720), vortex to mix, and incubate the 96-well plate at 4°C for 30 minutes. Centrifuge at 1000 rpm for 5 minutes at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, and resuspend the cells in 200 μl / well of flow cytometry buffer. Measure the mean fluorescence intensity of each sample using a flow cytometer (BD, FACSCelesta). The binding EC50 values ​​were calculated using a sigmoidal 4-parameter equation fitted with GraphPad Prism 9 software.

[0415] As shown in Figure 13, the experimental results showed that all humanized VHH-Fc bound to hTfR1 CHO-K1 cells, the binding activity of VHH3 and 5 was comparable to that of the parent, and that of VHH1, 2, 4, and 6 was slightly inferior to that of the parent.

[0416] Example 12: Endocytic activity of humanized VHH-Fc in SK-OV-3 tumor cells

[0417] SK-OV-3 is a human ovarian cancer cell line. Flow cytometry analysis confirmed that TfR1 protein is highly expressed on its surface. The flow cytometry results are shown in Figure 14. Flow cytometry was used to detect the endocytic activity of humanized VHH-Fc in SK-OV-3 tumor cells. The experimental procedure is as follows:

[0418] SK-OV-3 cells in the logarithmic growth phase were collected, washed with flow cytometry buffer (PBS + 2% FBS) and the cell density was adjusted to 2×10 6 cells / mL, and the cell density of the blank group and the secondary antibody control group was 5×10 5Cells were centrifuged at 1 mL / tube in 1.5 mL centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 minutes at 4°C, the supernatant discarded, and the cells were resuspended in 1 mL of FITC-labeled mouse anti-human IgG antibody (Biolegend, Cat#410720) at a concentration of 15 μg / mL. The cells were incubated at 4°C for 1 hour. The cells were centrifuged at 1000 rpm for 5 minutes at 4°C, the supernatant discarded, and the cells were washed twice with flow cytometry buffer. The cells were then resuspended in 1 mL of complete culture medium (McCoy's 5A + 10% FBS) and 250 μl / tube dispensed into 1.5 mL centrifuge tubes. The cells were labeled as the non-elution group, the non-endocytosis group, the endocytosis group (-30 minutes), and the endocytosis group (-120 minutes).

[0419] The non-elution and non-endocytosis groups were incubated on ice, while the endocytosis-30-minute and endocytosis-120-minute groups were incubated in a 37°C cell culture incubator for 30 and 120 minutes, respectively. At the corresponding time points, the cells were removed from the incubator and pre-chilled on ice for 5 minutes. All samples were centrifuged at 4°C, 1000 rpm for 5 minutes, the supernatant discarded, and the cells were washed once with flow cytometry buffer. The non-endocytosis, endocytosis-30-minute, and endocytosis-120-minute groups were centrifuged at 4°C, 1000 rpm for 5 minutes, the supernatant discarded, and the cells were resuspended in 250 μl / tube of elution buffer (0.05 M glycine, 0.1 M sodium chloride, pH adjusted to 2.5 with HCl). The cells were incubated at room temperature for 8 minutes, then centrifuged at 4°C, 1000 rpm for 5 minutes, the supernatant discarded, and the cells were washed twice with flow cytometry buffer. All samples were centrifuged at 4°C, 1000 rpm for 5 minutes, the supernatant was discarded, 200 μl / tube of fixative (4% paraformaldehyde, pH 7.4) was added to resuspend the cells, and incubated at 4°C for 30 minutes.

[0420] The mean fluorescence intensity of each sample was measured using a flow cytometer (BD, FACSCelesta), and the antibody internalization percentage was calculated. Detection window = (MFI of the non-elution group - MFI of the blank control group) / (MFI of the non-endocytosis group - MFI of the blank cells). Antibody internalization percentage = (MFI of the endocytosis group - MFI of the non-endocytosis group) / (MFI of the non-elution group - MFI of the non-endocytosis group) × 100%. The results are shown in Tables 10-11.

[0421] Table 10 Summary of anti-TfR1 humanized VHH-Fc endocytosis results

[0422] Table 11 Summary of control IgG antibody endocytosis results

[0423] The control hIgG1 antibody 13E4_variant 2-ii (Avidity Bioscience, MS11028179) had a 2-hour endocytosis efficiency of 17.9% on SK-OV-3 cells, with a detection window of >6.41. The 2-hour endocytosis efficiency of the seven VHH-Fc antibodies on SK-OV-3 cells was >55%, with a detection window of >5.7, indicating significantly higher endocytic activity than the control antibody.

[0424] Example 13: HFE does not affect the binding activity of humanized VHH-Fc

[0425] Human TfR1-His (ACRO, Cat#CD1-H5243) antigen solution and recombinant human HFE (Abcam, Cat#ab139241) solution were prepared in PBS to final concentrations of 1 μg / mL and 2 μg / mL, respectively. 100 μl / well was added to a 96-well microtiter plate and coated overnight at 4°C. The plates were washed three times with PBST (PBS + 0.05% Tween 20), and 200 μl / well of blocking buffer (PBST + 3% BSA) was added and blocked at room temperature for 1 hour. The plates were washed once with PBST, and 100 μl / well of 1 μg / mL human TfR1-His solution was added to the human HFE-coated plate and incubated at room temperature for 1 hour. The plates were washed three times with PBST, and 100 μl / well of serially diluted VHH-Fc solution was added to the human TfR1-His and human HFE-coated plates and incubated at room temperature for 1 hour. The cells were washed three times with PBST, and 100 μl / well of a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-human IgG antibody (Yeasen, Cat#33501ES60) was added and incubated at room temperature for 1 hour. The cells were washed three times with PBST, patted dry, and 100 μl / well of the chromogenic substrate TMB solution (Beyotime, Cat#P0209) was added. The cells were allowed to develop at room temperature for 5 to 30 minutes, followed by the addition of 100 μl / well of the color stop solution (Beyotime, Cat#P0215). The absorbance at 450 nm was measured in each well using a multi-function microplate reader (Molecular Devices, SpectraMax i3x). The EC50 value for binding was calculated using a sigmoidal curve fitted with a four-parameter equation using GraphPad Prism 9 software.

[0426] As shown in FIG15 , the experimental results indicate that HFE is in a non-competitive relationship with humanized VHH-Fc and does not affect the binding of humanized VHH-Fc to human TfR1-His.

[0427] Example 14: Preparation of anti-TfR1 single domain antibody-PMO conjugate

[0428] Duchenne muscular dystrophy (DMD) is a hereditary muscle wasting disorder caused by a mutation in the dystrophin gene, resulting in a deficiency of the dystrophin protein. The disease primarily manifests as progressive muscle atrophy and weakness. Patients begin to experience muscle weakness between the ages of 2 and 5, becoming unable to walk between the ages of 9 and 12. Ultimately, the disease affects all smooth muscles, as well as the heart and respiratory muscles, leading to respiratory failure or cardiac dysfunction and death. The average life expectancy for patients with the disease is approximately 30 years.

[0429] PMO (Phosphorodiamidate morpholino oligomer) is a nucleic acid analog that can bind to premRNA through base complementary pairing, causing the exons of the erroneous gene to be skipped during RNA splicing, ultimately producing a shortened but functional Dystrophin protein, thereby treating the disease.

[0430] The present invention selects a PMO sequence that can jump exon 23 on mouse DMD: its sequence is SEQ ID NO: 218.

[0431] EXON23: SEQ ID NO: 218

[0432] 5'-GGCCAAACCTCGGCTTACCTGAAAT-3'

[0433] The 3' end of PMO is modified with NH2 group for coupling to single domain antibodies.

[0434] Dissolve the 3'-terminally NH2-modified PMO single chain in phosphate buffer (50 mM NaH2PO4, 150 mM NaCl, pH 7.4) to a final concentration of 1 mM. Dissolve SM(PEG)2 (linker molecule) powder in dimethyl sulfoxide (DMSO) to prepare a fresh 250 mM SM(PEG)2 stock solution. Add a 10- to 50-fold molar amount of SM(PEG)2 to the PMO single chain stock solution, mix rapidly, and react at room temperature for 30 minutes to 2 hours. After the reaction is complete, add 10% of the volume of 1M Tris-HCl (pH 7.0), mix, and incubate at room temperature for 20 minutes to terminate the reaction with excess SM(PEG)2. After incubation, remove unreacted SM(PEG)2 linker by acetone precipitation, and purify the SM(PEG)2-PMO conjugate for later use.

[0435] A cysteine ​​mutation was introduced into the carboxyl terminus of the anti-TfR1 single domain antibody TfR1-21 (SEQ ID NO: 6) for nucleic acid coupling. The single domain antibody expression and purification methods were the same as in Example 3.

[0436] The single domain antibody was dialyzed with a dialysis buffer containing a reducing agent (20mM Tris, 15mM NaCl, pH 7.4). During the dialysis process, the sulfhydryl group at the C-terminus was reduced, and impurity small molecules such as free -SH groups were removed. The reduced single domain antibody and SM (PEG) 2-PMO single chain were mixed in a molar ratio of 1:1 to 2 and then reacted at room temperature for 2 hours. The unreacted SM (PEG) 2-PMO single chain was removed using a His tag affinity column, and the single domain antibody and the single domain antibody-PMO mixture were collected. Using Supdex TM 75increase 10 / 300GL was used to separate the single domain antibody and single domain antibody-PMO conjugate, and the purity of the final product was verified by SDS-PAGE.

[0437] As shown in Figure 16, the experimental results showed that after conjugation and two-step purification, an anti-TfR1 single domain antibody-PMO conjugate with high purity was obtained.

[0438] Example 15: DMD exon skipping efficacy analysis of anti-TfR1 single domain antibody-PMO conjugate in hTfR1 humanized mice

[0439] hTfR1 humanized C57BL / 6 mice weighing 16-24g and aged 6-8 weeks were selected. Unmodified PMO and TfR1-21-PMO prepared in Example 14 were administered intramuscularly at a single dose of 15 mg / kg. PBS was used as a control. Gastrocnemius muscle tissue samples were collected 4, 7, and 14 days after administration and analyzed for drug-induced exon skipping. Twenty-seven mice were randomly divided into three groups of nine mice each, grouped by weight.

[0440] Detailed dosing information is shown in Table 12:

[0441] Table 12 Grouping and Dosing

[0442] Note: a: Single dose means administration once on the day of grouping;

[0443] All mice underwent clinical observation daily, including but not limited to their condition and diet. Body weight was measured twice weekly, and monitoring data showed that all mice's weights remained within normal ranges during the experiment, with no adverse reactions observed. At the conclusion of the experiment or at a humane endpoint, animals were euthanized using an overdose of CO2.

[0444] On days 4, 7, and 14 after administration, bilateral gastrocnemius muscle tissue samples were collected from three mice in each group for exon skipping analysis.

[0445] After grinding each tissue, total RNA was extracted and analyzed for exon skipping. The ground tissue samples were lysed using TRIzol reagent (Thermo, Cat#15596018), and total RNA was isolated using the phenol / chloroform method. Approximately 1 μg of RNA from each of the 72 samples was reverse transcribed (with gDNA wiper) using the following PCR protocol: 37°C, 45 min; 85°C, 5 sec. A 2 μL aliquot of the reverse-transcribed cDNA product was used for the first round of PCR amplification using Ex20F Primer / Ex26R Primer (reaction system and protocol are shown in Table 13). A 1 μL aliquot of the first-round PCR product was used for the second round of nested PCR amplification using Ex20F2 Primer / Ex25R Primer (reaction system and protocol are shown in Table 14). After nested PCR, 10 μL of each sample was loaded and analyzed by 3% TAE agarose gel electrophoresis. The expected size of the wild-type DMD product is 788 bp, and the expected size of the DMD product with exon 23 skipping is 575 bp.

[0446] The primer sequences used are as follows:

[0447] Ex20F: 5'-CAGAATTCTGCCAATTGCTGAG-3' (SEQ ID NO: 219)

[0448] Ex26R: 5'-TTCTTCAGCTTGTGTCATCC-3' (SEQ ID NO: 220)

[0449] Ex20F2:5'-ACCCAGTCTACCACCCTATC-3'(SEQ ID NO:221)

[0450] Ex25R: 5'-CTCTTTATCTTCTGCCCACCTT-3' (SEQ ID NO: 222)

[0451] Table 13 Primary PCR protocol

[0452] Table 14 Nested PCR protocol

[0453] Agarose gel electrophoresis results were analyzed and quantified using Image Lab software, and a histogram was plotted using GraphPad Prism 9. As shown in Figure 17, the experimental results demonstrate that the anti-TfR1 single-domain antibody-PMO conjugate can effectively induce DMD exon 23 skipping in mouse gastrocnemius muscle tissue, and the exon skipping efficacy is significantly superior to that of unmodified PMO.

[0454] The exon-skipping bands were recovered from the gel and sequenced to verify the success of the exon skipping. As shown in Figure 18, the experimental results showed that the DMD exon 23 skipping was correct.

[0455] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anti-TfR1 single domain antibody, characterized in that The anti-TfR1 single domain antibody has one or more complementarity determining regions (CDRs) selected from the following group: (1) CDR1 shown in SEQ ID NO: 51, CDR2 shown in SEQ ID NO: 73, and CDR3 shown in SEQ ID NO: 98; (2) CDR1 shown in SEQ ID NO: 54, CDR2 shown in SEQ ID NO: 61, and CDR3 shown in SEQ ID NO: 53; (3) CDR1 shown in SEQ ID NO: 54, CDR2 shown in SEQ ID NO: 61, and CDR3 shown in SEQ ID NO: 56; (4) CDR1 shown in SEQ ID NO: 60, CDR2 shown in SEQ ID NO: 67, and CDR3 shown in SEQ ID NO: 71; (5) CDR1 shown in SEQ ID NO: 69, CDR2 shown in SEQ ID NO: 64, and CDR3 shown in SEQ ID NO: 74; (6) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:50; (7) CDR1 set forth in SEQ ID NO:66, CDR2 set forth in SEQ ID NO:116, and CDR3 set forth in SEQ ID NO:119; (8) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:100, and CDR3 shown in SEQ ID NO:50; (9) CDR1 shown in SEQ ID NO: 51, CDR2 shown in SEQ ID NO: 46, and CDR3 shown in SEQ ID NO: 118; (10) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:46, and CDR3 shown in SEQ ID NO:47; (11) CDR1 shown in SEQ ID NO: 57, CDR2 shown in SEQ ID NO: 76, and CDR3 shown in SEQ ID NO: 77; (12) CDR1 shown in SEQ ID NO: 81, CDR2 shown in SEQ ID NO: 79, and CDR3 shown in SEQ ID NO: 80; (13) CDR1 shown in SEQ ID NO:96, CDR2 shown in SEQ ID NO:70, and CDR3 shown in SEQ ID NO:83; (14) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:86; (15) CDR1 shown in SEQ ID NO:93, CDR2 shown in SEQ ID NO:82, and CDR3 shown in SEQ ID NO:89; (16) CDR1 shown in SEQ ID NO:75, CDR2 shown in SEQ ID NO:58, and CDR3 shown in SEQ ID NO:68; (17) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:92; (18) CDR1 shown in SEQ ID NO: 111, CDR2 shown in SEQ ID NO: 85, and CDR3 shown in SEQ ID NO: 62; (19) CDR1 shown in SEQ ID NO:75, CDR2 shown in SEQ ID NO:58, and CDR3 shown in SEQ ID NO:95; (20) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:47; (21) CDR1 shown in SEQ ID NO: 63, CDR2 shown in SEQ ID NO: 55, and CDR3 shown in SEQ ID NO: 65; (22) CDR1 shown in SEQ ID NO: 108, CDR2 shown in SEQ ID NO: 88, and CDR3 shown in SEQ ID NO: 101; (23) CDR1 shown in SEQ ID NO: 87, CDR2 shown in SEQ ID NO: 91, and CDR3 shown in SEQ ID NO: 62; (24) CDR1 shown in SEQ ID NO:90, CDR2 shown in SEQ ID NO:94, and CDR3 shown in SEQ ID NO:104; (25) CDR1 shown in SEQ ID NO: 102, CDR2 shown in SEQ ID NO: 97, and CDR3 shown in SEQ ID NO: 59; (26) CDR1 shown in SEQ ID NO:84, CDR2 shown in SEQ ID NO:103, and CDR3 shown in SEQ ID NO:107; (27) CDR1 shown in SEQ ID NO:99, CDR2 shown in SEQ ID NO:106, and CDR3 shown in SEQ ID NO:110; (28) CDR1 shown in SEQ ID NO: 105, CDR2 shown in SEQ ID NO: 109, and CDR3 shown in SEQ ID NO: 113; (29) CDR1 shown in SEQ ID NO: 78, CDR2 shown in SEQ ID NO: 58, and CDR3 shown in SEQ ID NO: 68; (30) CDR1 shown in SEQ ID NO:96, CDR2 shown in SEQ ID NO:112, and CDR3 shown in SEQ ID NO:115; (31) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:114, and CDR3 shown in SEQ ID NO:117; (32) CDR1 shown in SEQ ID NO:72, CDR2 shown in SEQ ID NO:64, and CDR3 shown in SEQ ID NO:

74.

2. The anti-TfR1 single domain antibody according to claim 1, wherein The amino acid sequence of the VHH chain of the anti-TfR1 single-domain antibody is selected from one or more of SEQ ID NOs: 1-44, 210-215.

3. An anti-TfR1 antibody, characterized in that The antibody comprises one or more VHH chains of the anti-TfR1 single domain antibody according to claim 1 or 2.

4. A chimeric antigen receptor (CAR), characterized in that The CAR contains an extracellular domain, and the extracellular domain comprises the anti-TfR1 single-domain antibody according to claim 1 or 2, or the anti-TfR1 antibody according to claim 3.

5. A multispecific antibody, characterized in that The first antigen-binding region of the multispecific antibody comprises: the anti-TfR1 single domain antibody according to claim 1 or 2, or the anti-TfR1 antibody according to claim 3.

6. A recombinant protein, characterized in that The recombinant protein has: (i) the anti-TfR1 single domain antibody according to claim 1 or 2, the anti-TfR1 antibody according to claim 3, or an active fragment thereof; (ii) optionally a polypeptide molecule or fragment having therapeutic function; and / or (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

7. A polynucleotide, characterized in that The polynucleotide encodes a protein selected from the following group: the anti-TfR1 single domain antibody according to claim 1 or 2, the anti-TfR1 antibody according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody according to claim 5, the recombinant protein according to claim 6, or a combination thereof.

8. An expression vector, characterized in that The expression vector contains the polynucleotide according to claim 7.

9. A host cell, characterized in that The host cell contains the expression vector according to claim 8, or the polynucleotide according to claim 7 is integrated into its genome.

10. An immunoconjugate, characterized in that The immunoconjugate contains: (a) the anti-TfR1 single domain antibody of claim 1 or 2, the anti-TfR1 antibody of claim 3, the multispecific antibody of claim 5, or the recombinant protein of claim 6; and (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein, a VLP, an oligonucleotide, a nucleic acid analog, or a combination thereof.

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