Anti-EGFR antibody or antigen-binding fragment thereof, and use thereof

Abstract

The present invention belongs to the technical field of antibody drugs. Specifically, the present disclosure relates to an anti-EGFR antibody or an antigen-binding fragment thereof, a coding sequence thereof, a corresponding expression vector and host cell, and a preparation method therefor. The present disclosure also relates to the use of the anti-EGFR antibody or the antigen-binding fragment thereof.

Description

Anti-EGFR antibodies or their antigen-binding fragments and their uses

[0001] This application claims priority to Chinese Patent Application No. 202510384925.9, filed on March 28, 2025, and Chinese Patent Application No. 202510697029.8, filed on May 28, 2025, both of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention belongs to the field of antibody drug technology. Specifically, this disclosure relates to an EGFR antibody or its antigen-binding fragment, its coding sequence, a corresponding expression vector and host cell, and a method for preparation. This disclosure also relates to the use of the said EGFR antibody or its antigen-binding fragment. Background Technology

[0003] Epidermal growth factor receptor (EGFR) is a transmembrane receptor encoded by the c-erbB proto-oncogene. With a molecular weight of approximately 170 kDa, it is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. Upon binding to its specific ligands, including epidermal growth factor (EGF) and transforming growth factor α (TGFα), EGFR dimers, further stimulating intracellular protein tyrosine kinase activity and triggering downstream signaling cascades, leading to DNA synthesis and cell proliferation. EGFR also participates in phenotypic regulation of cell migration, adhesion, and proliferation. EGFR overexpression is associated with many malignant tumors, and its overexpression is significantly associated with poor patient prognosis.

[0004] Currently, EGFR-targeted drug development is mainly based on functional blocking agents, such as small molecule drugs based on intracellular signal transduction synthesis of tyrosine kinase inhibitors, and ligand blockers based on monoclonal antibody drugs that block extracellular ligand signal transduction. Antibody conjugation can deliver cytotoxins to EGFR-expressing tumor cells and kill them, ensuring the effectiveness of targeted therapy.

[0005] Single-domain antibodies possess numerous advantages, including small size, high stability, low immunogenicity, strong tissue penetration, and low production cost. Therefore, developing EGFR single-domain antibodies and subsequently related drugs has significant pharmaceutical and clinical implications. Summary of the Invention

[0006] This invention provides antibodies or antigen-binding fragments thereof capable of specifically binding to EGFR and their uses.

[0007] The present invention provides an anti-EGFR antibody or its antigen-binding fragment thereof, which comprises a single antigen domain, wherein the single antigen domain comprises CDR1, CDR2 and CDR3 of any single antigen domain in SEQ ID NO:1-20 and 187-189.

[0008] In some implementations, the CDR is a CDR defined according to Kabat, AbM, Chothia, North, IMGT, or Contact, or a combination thereof.

[0009] In some implementations, the single antigen domain is VHH.

[0010] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment comprises at least one (e.g., one or more) of the single antigen domain.

[0011] In some embodiments, the single antigen domain comprises CDR1, CDR2, and CDR3; wherein CDR1 comprises an amino acid sequence as shown in any one of SEQ ID NO: 22, 25, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59; CDR2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 23, 26, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 62, 186; and CDR3 comprises an amino acid sequence as shown in any one of SEQ ID NO: 24, 27, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 63.

[0012] In some embodiments, the single antigen domain comprises CDR1, CDR2, and CDR3, wherein

[0013] (1) CDR1 contains the amino acid sequence shown in SEQ ID NO:22, CDR2 contains the amino acid sequence shown in SEQ ID NO:23, and CDR3 contains the amino acid sequence shown in SEQ ID NO:24; or

[0014] (2) CDR1 contains the amino acid sequence shown in SEQ ID NO:25, CDR2 contains the amino acid sequence shown in SEQ ID NO:26, and CDR3 contains the amino acid sequence shown in SEQ ID NO:27; or

[0015] (3) CDR1 contains the amino acid sequence shown in SEQ ID NO:25, CDR2 contains the amino acid sequence shown in SEQ ID NO:26, and CDR3 contains the amino acid sequence shown in SEQ ID NO:28; or

[0016] (4) CDR1 contains the amino acid sequence shown in SEQ ID NO:29, CDR2 contains the amino acid sequence shown in SEQ ID NO:30, and CDR3 contains the amino acid sequence shown in SEQ ID NO:31; or

[0017] (5) CDR1 comprises the amino acid sequence shown in SEQ ID NO:32, CDR2 comprises the amino acid sequence shown in SEQ ID NO:33, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:34; or

[0018] (6) CDR1 comprises the amino acid sequence shown in SEQ ID NO:35, CDR2 comprises the amino acid sequence shown in SEQ ID NO:36, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:37; or

[0019] (7) CDR1 contains the amino acid sequence shown in SEQ ID NO:38, CDR2 contains the amino acid sequence shown in SEQ ID NO:39, and CDR3 contains the amino acid sequence shown in SEQ ID NO:40; or

[0020] (8) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:41, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:42, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:43; or

[0021] (9) CDR1 comprises the amino acid sequence shown in SEQ ID NO:44, CDR2 comprises the amino acid sequence shown in SEQ ID NO:45, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:46; or

[0022] (10) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:7, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:48, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:49; or

[0023] (11) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:50, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:51, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:52; or

[0024] (12) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:53, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:54, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:55; or

[0025] (13) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:56, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:57, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:58; or

[0026] (14) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:59, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:60, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:61; or

[0027] (15) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:56, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:62, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:63; or

[0028] (16) The CDR1 contains the amino acid sequence shown in SEQ ID NO:29, the CDR2 contains the amino acid sequence shown in SEQ ID NO:186, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:31.

[0029] In some embodiments, any amino acid sequence in the above complementarity-determining region (CDR) sequence further includes a derived sequence that has optionally been added, deleted, modified, and / or substituted at least one amino acid and is capable of retaining the activity of specifically binding to EGFR.

[0030] In some embodiments, the number of added, deleted, modified and / or substituted amino acids is 1-5 (e.g., 1-3, preferably 1-2, more preferably 1).

[0031] In some embodiments, the derived sequence, which has been added to, deleted from, modified, and / or substituted with at least one amino acid and is capable of retaining specific EGFR binding activity, is an amino acid sequence with at least 90% homology or sequence similarity, preferably at least 95%, more preferably at least 99%.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof described in this invention is humanized, for example, partially or fully humanized. In some embodiments, the single antigen domain, such as VHH, is humanized, for example, partially or fully humanized. In some embodiments, the PTM (post-translational modification) site in the single antigen domain, such as VHH, is removed.

[0033] In some embodiments, the CDR1, CDR2, and CDR3 of the antibody or its antigen-binding fragment described in this invention are separated by frame regions FR1, FR2, FR3, and FR4, for example, by frame regions FR1, FR2, FR3, and FR4 from VHH.

[0034] In some implementations, the single antigen domain

[0035] (1) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:1, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:1, or consisting of SEQ ID NO:1; or

[0036] (2) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:2, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:2, or consisting of SEQ ID NO:2; or

[0037] (3) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:3, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:3, or composed of SEQ ID NO:3; or

[0038] (4) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:4, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:4, or consisting of SEQ ID NO:4; or

[0039] (5) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:5, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:5, or consisting of SEQ ID NO:5; or

[0040] (6) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:6, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:6, or consisting of SEQ ID NO:6; or

[0041] (7) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:7, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:7, or consisting of SEQ ID NO:7; or

[0042] (8) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:8, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:8, or consisting of SEQ ID NO:8; or

[0043] (9) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:9, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:9, or consisting of SEQ ID NO:9; or

[0044] (10) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:10, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:10, or consisting of SEQ ID NO:10; or

[0045] (11) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:11, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:11, or consisting of SEQ ID NO:11; or

[0046] (12) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:12, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:12, or composed of SEQ ID NO:12; or

[0047] (13) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:13, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:13, or composed of SEQ ID NO:13; or

[0048] (14) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:14, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:14, or composed of SEQ ID NO:14; or

[0049] (15) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:15, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:15, or consisting of SEQ ID NO:15; or

[0050] (16) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:16, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:16, or consisting of SEQ ID NO:16; or

[0051] (17) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:17, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:17, or consisting of SEQ ID NO:17; or

[0052] (18) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:18, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:18, or consisting of SEQ ID NO:18; or

[0053] (19) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:19, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:19, or consisting of SEQ ID NO:19; or

[0054] (20) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:20, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:20, or consisting of SEQ ID NO:20; or

[0055] (21) An amino acid sequence comprising any of the amino acid sequences shown in SEQ ID NO:187-189, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the amino acid sequences in SEQ ID NO:187-189, or composed of any of the amino acid sequences in SEQ ID NO:187-189.

[0056] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment is a single-domain antibody or its antigen-binding fragment targeting EGFR.

[0057] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment is a monomer (monovalent antibody), a bivalent (bivalent antibody), or a multivalent (multivalent antibody).

[0058] In some embodiments, the single antigen domain comprises an amino acid sequence as shown in any of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 187-189, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 187-189.

[0059] In some embodiments, the single antigen domain comprises an amino acid sequence as shown in SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 or 132.

[0060] In some embodiments, the single antigen domain comprises an amino acid sequence as shown in SEQ ID NO: 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 187, 188 or 189.

[0061] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment comprises one or more VHHs having an amino acid sequence as shown in any of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 187-189.

[0062] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment comprises one or more VHHs having an amino acid sequence as shown in SEQ ID NO:119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 or 132.

[0063] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment comprises one or more VHHs having an amino acid sequence as shown in SEQ ID NO:152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 187, 188 or 189.

[0064] In some embodiments, the VHH in the anti-EGFR antibody is as shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0065] In some embodiments, the VHH in the anti-EGFR antibody is as shown in SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 or 132.

[0066] In some embodiments, the VHH in the anti-EGFR antibody is as shown in SEQ ID NO: 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 or 162.

[0067] In some embodiments, the VHH in the anti-EGFR antibody is as shown in SEQ ID NO:187, 188 or 189.

[0068] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment further comprises a frame region FR; wherein the frame region FR includes FR1, FR2, FR3, and FR4. In some embodiments, a single antigen domain of the anti-EGFR antibody or its antigen-binding fragment further comprises a frame region FR; wherein the frame region FR includes FR1, FR2, FR3, and FR4.

[0069] In some embodiments, CDR1, CDR2, and CDR3 in the single antigen domain of the present invention are separated by frame regions FR1, FR2, FR3, and FR4.

[0070] In some embodiments, the frame region FR1 includes an amino acid sequence as shown in any one of SEQ ID NO: 64, 68, 72, 73, 76, 79, 82, 86, 92, 98, 102, 106, 110, 113, 114, 116, 133, 134, 163, 164, 165, 166, 167, 181.

[0071] In some embodiments, the frame region FR2 includes an amino acid sequence as shown in any one of SEQ ID NO: 65, 69, 74, 77, 80, 83, 87, 90, 93, 99, 103, 107, 111, 117, 135, 136, 137, 138, 139, 140, 141, 142, 168, 169, 170, 171, 172, 173.

[0072] In some embodiments, the frame region FR3 includes an amino acid sequence as shown in any one of SEQ ID NO: 66, 70, 75, 78, 81, 84, 88, 91, 94, 96, 97, 100, 104, 108, 112, 115, 118, 143, 144, 145, 146, 147, 148, 174, 175, 176, 177.

[0073] In some embodiments, the frame region FR4 includes an amino acid sequence as shown in any one of SEQ ID NO: 67, 71, 85, 89, 95, 101, 105, 109, 149, 150, 151, 178.

[0074] In some implementations, the frame region FR includes:

[0075] (1) FR1 as shown in SEQ ID NO:64, FR2 as shown in SEQ ID NO:65, FR3 as shown in SEQ ID NO:66, and FR4 as shown in SEQ ID NO:67; or

[0076] (2) FR1 as shown in SEQ ID NO:68, FR2 as shown in SEQ ID NO:69, FR3 as shown in SEQ ID NO:70, and FR4 as shown in SEQ ID NO:71; or

[0077] (3) FR1 as shown in SEQ ID NO:72, FR2 as shown in SEQ ID NO:69, FR3 as shown in SEQ ID NO:70, and FR4 as shown in SEQ ID NO:71; or

[0078] (4) FR1 as shown in SEQ ID NO:73, FR2 as shown in SEQ ID NO:74, FR3 as shown in SEQ ID NO:75, and FR4 as shown in SEQ ID NO:71; or

[0079] (5) FR1 as shown in SEQ ID NO:76, FR2 as shown in SEQ ID NO:77, FR3 as shown in SEQ ID NO:78, and FR4 as shown in SEQ ID NO:71; or

[0080] (6) FR1 as shown in SEQ ID NO:79, FR2 as shown in SEQ ID NO:80, FR3 as shown in SEQ ID NO:81, and FR4 as shown in SEQ ID NO:71; or

[0081] (7) FR1 as shown in SEQ ID NO:82, FR2 as shown in SEQ ID NO:83, FR3 as shown in SEQ ID NO:84, and FR4 as shown in SEQ ID NO:85; or

[0082] (8) FR1 as shown in SEQ ID NO:86, FR2 as shown in SEQ ID NO:87, FR3 as shown in SEQ ID NO:88, and FR4 as shown in SEQ ID NO:89; or

[0083] (9) FR1 as shown in SEQ ID NO:72, FR2 as shown in SEQ ID NO:90, FR3 as shown in SEQ ID NO:91, and FR4 as shown in SEQ ID NO:71; or

[0084] (10) FR1 as shown in SEQ ID NO:92, FR2 as shown in SEQ ID NO:93, FR3 as shown in SEQ ID NO:94, and FR4 as shown in SEQ ID NO:95; or

[0085] (11) FR1 as shown in SEQ ID NO:86, FR2 as shown in SEQ ID NO:87, FR3 as shown in SEQ ID NO:96, and FR4 as shown in SEQ ID NO:89; or

[0086] (12) FR1 as shown in SEQ ID NO:64, FR2 as shown in SEQ ID NO:65, FR3 as shown in SEQ ID NO:97, and FR4 as shown in SEQ ID NO:67; or

[0087] (13) FR1 as shown in SEQ ID NO:102, FR2 as shown in SEQ ID NO:103, FR3 as shown in SEQ ID NO:104, and FR4 as shown in SEQ ID NO:105; or

[0088] (14) FR1 as shown in SEQ ID NO:106, FR2 as shown in SEQ ID NO:107, FR3 as shown in SEQ ID NO:108, and FR4 as shown in SEQ ID NO:109; or

[0089] (15) FR1 as shown in SEQ ID NO:110, FR2 as shown in SEQ ID NO:111, FR3 as shown in SEQ ID NO:112, and FR4 as shown in SEQ ID NO:101; or

[0090] (16) FR1 as shown in SEQ ID NO:113, FR2 as shown in SEQ ID NO:111, FR3 as shown in SEQ ID NO:112, and FR4 as shown in SEQ ID NO:101; or

[0091] (17) FR1 as shown in SEQ ID NO:114, FR2 as shown in SEQ ID NO:111, FR3 as shown in SEQ ID NO:115, and FR4 as shown in SEQ ID NO:101; or

[0092] (18) FR1 as shown in SEQ ID NO:116, FR2 as shown in SEQ ID NO:117, FR3 as shown in SEQ ID NO:118, and FR4 as shown in SEQ ID NO:89; or

[0093] (19) FR1 as shown in SEQ ID NO:64, FR2 as shown in SEQ ID NO:65, FR3 as shown in SEQ ID NO:97, and FR4 as shown in SEQ ID NO:67; or

[0094] (20) FR1 as shown in SEQ ID NO:98, FR2 as shown in SEQ ID NO:99, FR3 as shown in SEQ ID NO:100 and FR4 as shown in SEQ ID NO:101.

[0095] In some implementations, the frame region FR includes:

[0096] (1) FR1 as shown in SEQ ID NO:133, FR2 as shown in SEQ ID NO:135, FR3 as shown in SEQ ID NO:143, and FR4 as shown in SEQ ID NO:149; or

[0097] (2) FR1 as shown in SEQ ID NO:133, FR2 as shown in SEQ ID NO:136, FR3 as shown in SEQ ID NO:143, and FR4 as shown in SEQ ID NO:149; or

[0098] (3) FR1 as shown in SEQ ID NO:133, FR2 as shown in SEQ ID NO:137, FR3 as shown in SEQ ID NO:144, and FR4 as shown in SEQ ID NO:149; or

[0099] (4) FR1 as shown in SEQ ID NO:133, FR2 as shown in SEQ ID NO:137, FR3 as shown in SEQ ID NO:143, and FR4 as shown in SEQ ID NO:150; or

[0100] (5) FR1 as shown in SEQ ID NO:133, FR2 as shown in SEQ ID NO:138, FR3 as shown in SEQ ID NO:143, and FR4 as shown in SEQ ID NO:150; or

[0101] (6) FR1 as shown in SEQ ID NO:133, FR2 as shown in SEQ ID NO:137, FR3 as shown in SEQ ID NO:145, and FR4 as shown in SEQ ID NO:149; or

[0102] (7) FR1 as shown in SEQ ID NO:133, FR2 as shown in SEQ ID NO:137, FR3 as shown in SEQ ID NO:146, and FR4 as shown in SEQ ID NO:149; or

[0103] (8) FR1 as shown in SEQ ID NO:133, FR2 as shown in SEQ ID NO:138, FR3 as shown in SEQ ID NO:145, and FR4 as shown in SEQ ID NO:149; or

[0104] (9) FR1 as shown in SEQ ID NO:133, FR2 as shown in SEQ ID NO:138, FR3 as shown in SEQ ID NO:145, and FR4 as shown in SEQ ID NO:150; or

[0105] (10) FR1 as shown in SEQ ID NO:134, FR2 as shown in SEQ ID NO:139, FR3 as shown in SEQ ID NO:147, and FR4 as shown in SEQ ID NO:151; or

[0106] (11) FR1 as shown in SEQ ID NO:181, FR2 as shown in SEQ ID NO:140, FR3 as shown in SEQ ID NO:148, and FR4 as shown in SEQ ID NO:151; or

[0107] (12) FR1 as shown in SEQ ID NO:134, FR2 as shown in SEQ ID NO:141, FR3 as shown in SEQ ID NO:147, and FR4 as shown in SEQ ID NO:151; or

[0108] (13) FR1 as shown in SEQ ID NO:134, FR2 as shown in SEQ ID NO:142, FR3 as shown in SEQ ID NO:147, and FR4 as shown in SEQ ID NO:151; or

[0109] (14) FR1 as shown in SEQ ID NO:134, FR2 as shown in SEQ ID NO:142, FR3 as shown in SEQ ID NO:148 and FR4 as shown in SEQ ID NO:151.

[0110] In some implementations, the frame region FR includes:

[0111] (1) FR1 as shown in SEQ ID NO:163, FR2 as shown in SEQ ID NO:168, FR3 as shown in SEQ ID NO:174, and FR4 as shown in SEQ ID NO:150; or

[0112] (2) FR1 as shown in SEQ ID NO:163, FR2 as shown in SEQ ID NO:169, FR3 as shown in SEQ ID NO:174, and FR4 as shown in SEQ ID NO:150; or

[0113] (3) FR1 as shown in SEQ ID NO:164, FR2 as shown in SEQ ID NO:169, FR3 as shown in SEQ ID NO:175, and FR4 as shown in SEQ ID NO:150; or

[0114] (4) FR1 as shown in SEQ ID NO:165, FR2 as shown in SEQ ID NO:169, FR3 as shown in SEQ ID NO:175, and FR4 as shown in SEQ ID NO:150; or

[0115] (5) FR1 as shown in SEQ ID NO:166, FR2 as shown in SEQ ID NO:168, FR3 as shown in SEQ ID NO:174, and FR4 as shown in SEQ ID NO:150; or

[0116] (6) FR1 as shown in SEQ ID NO:166, FR2 as shown in SEQ ID NO:170, FR3 as shown in SEQ ID NO:175, and FR4 as shown in SEQ ID NO:150; or

[0117] (7) FR1 as shown in SEQ ID NO:166, FR2 as shown in SEQ ID NO:169, FR3 as shown in SEQ ID NO:175, and FR4 as shown in SEQ ID NO:150; or

[0118] (8) FR1 as shown in SEQ ID NO:167, FR2 as shown in SEQ ID NO:169, FR3 as shown in SEQ ID NO:175, and FR4 as shown in SEQ ID NO:150; or

[0119] (9) FR1 as shown in SEQ ID NO:166, FR2 as shown in SEQ ID NO:171, FR3 as shown in SEQ ID NO:176, and FR4 as shown in SEQ ID NO:178; or

[0120] (10) FR1 as shown in SEQ ID NO:166, FR2 as shown in SEQ ID NO:172, FR3 as shown in SEQ ID NO:177, and FR4 as shown in SEQ ID NO:178; or

[0121] (11) FR1 as shown in SEQ ID NO:167, FR2 as shown in SEQ ID NO:173, FR3 as shown in SEQ ID NO:177 and FR4 as shown in SEQ ID NO:178.

[0122] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment further comprises an Fc region of an immunoglobulin. In some preferred embodiments, the Fc region is an Fc region of a human immunoglobulin, such as the Fc region of human IgG (IgG1, IgG2, IgG3, or IgG4). Preferably, the Fc region is the Fc region of human IgG1 or human IgG4; more preferably, the Fc region comprises the amino acid sequence of SEQ ID NO:179 or SEQ ID NO:180.

[0123] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment comprises ALB from the N-terminus to the C-terminus; wherein A is the single antigen domain, B is the Fc region fragment of the immunoglobulin, and L is absent or without a linker.

[0124] In some embodiments, the single antigen domain, such as VHH, in the anti-EGFR antibody or its antigen-binding fragment of the present invention can be linked to the Fc region of an immunoglobulin via a linker.

[0125] In some implementations, the joint is a flexible joint.

[0126] In some embodiments, the linker is a peptide linker, for example, the peptide linker has 1 to 50 amino acids, preferably 1 to 20 amino acids.

[0127] In some embodiments, the connector has a structure of (GGGGS)n and / or (GS)n, where n is a positive integer from 1 to 5.

[0128] The present invention also provides a fusion protein having an ALB structure from the N-terminus to the C-terminus; wherein, A is the aforementioned single antigen domain such as VHH, B is the Fc region of an immunoglobulin such as the Fc region of IgG, and L is absent or has no linker.

[0129] In some embodiments, the connector is a flexible connector. In some embodiments, the flexible connector is a peptide connector.

[0130] In some implementations, the Fc region of the IgG includes the Fc region of human IgG.

[0131] In some embodiments, the Fc region of the IgG is selected from the group consisting of the Fc regions of IgG1, IgG2, IgG3, and IgG4, or combinations thereof, preferably the Fc region of IgG1.

[0132] In some embodiments, the fusion protein is an Fc fusion protein of a single-domain antibody targeting an EGFR epitope or its binding protein.

[0133] The present invention also provides a polynucleotide that encodes the aforementioned anti-EGFR antibody or its antigen-binding fragment, or the aforementioned fusion protein.

[0134] In some implementations, the polynucleotide may be DNA or RNA.

[0135] The present invention also provides an expression vector containing the aforementioned polynucleotides.

[0136] In some embodiments, the expression vector is selected from DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0137] In some embodiments, the expression vector is preferably a viral vector, such as lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0138] The present invention also provides a host cell containing the expression vector described above, or having the polynucleotides described above integrated into its genome.

[0139] In some implementations, the host cell includes prokaryotic cells and eukaryotic cells.

[0140] In some embodiments, the host cell is selected from Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0141] In some embodiments, the prokaryotic cells are selected from Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0142] In some embodiments, the eukaryotic cells are selected from Pichia pastoris, brewer's yeast, fissile yeast, Trichoderma, or combinations thereof.

[0143] In some embodiments, the eukaryotic cells are selected from insect cells such as armyworms, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

[0144] In some embodiments, the host cell is preferably a mammalian cell, more preferably a HEK293 cell, CHO cell, BHK cell, NSO cell, or COS cell.

[0145] The present invention also provides a method for producing an anti-EGFR antibody or an antigen-binding fragment thereof, comprising:

[0146] (a) Under suitable conditions, the host cells described above in this invention are used to obtain a culture containing the anti-EGFR antibody;

[0147] (b) Isolate or recover the anti-EGFR antibody from the culture;

[0148] (c) Optionally, purify and / or modify the anti-EGFR antibody obtained in step (b).

[0149] The present invention also provides a bispecific or multispecific antibody comprising the anti-EGFR antibody described above or its antigen-binding fragment, or the fusion protein described above.

[0150] The present invention also provides a pharmaceutical composition comprising (i) the aforementioned anti-EGFR antibody or its antigen-binding fragment thereof, or the aforementioned fusion protein or the aforementioned bispecific or multispecific antibody, and (ii) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is used for the prevention or treatment of EGFR-related diseases or conditions.

[0151] The present invention also provides the anti-EGFR antibody or its antigen-binding fragment described above and / or the fusion protein described above or the bispecific or multispecific antibody described above, for the prevention or treatment of EGFR-related diseases or conditions.

[0152] The present invention also provides the use of the anti-EGFR antibody or antigen-binding fragment thereof described above and / or the fusion protein described above or the bispecific or multispecific antibody described above in the preparation of a medicament for the prevention or treatment of EGFR-related diseases or conditions.

[0153] The present invention also provides a treatment method for EGFR-related diseases or conditions, the method comprising administering to a subject in need the aforementioned anti-EGFR antibody or its antigen-binding fragment thereof and / or the aforementioned fusion protein and / or the aforementioned bispecific or multispecific antibody of the present invention.

[0154] In some implementations, the EGFR-related disease or condition is a tumor and / or cancer that expresses the EGFR protein (i.e., EGFR positive).

[0155] In some implementations, the tumor and / or cancer is selected from: lung cancer, breast cancer, ovarian cancer, colorectal cancer, nasopharyngeal cancer, esophageal cancer, brain cancer, bladder cancer, pancreatic cancer, endometrial cancer, uterine cancer, cervical cancer, head and neck cancer, thyroid cancer, glioma, glioblastoma, chordoma, squamous cell carcinoma, liver cancer, stomach cancer, colorectal cancer, prostate cancer, lymphoma, mesothelioma, urothelial carcinoma, skin cancer, or hematologic malignancies.

[0156] It should be understood that, within the scope of this invention, the foregoing technical features and the technical features specifically described 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 described in detail here. Attached Figure Description

[0157] Figure 1 shows the SDS-PAGE detection results of the EGFR-His recombinant protein reduced according to the present invention.

[0158] Figure 2 shows the results of a single round of PCR using VHH amplification primers, and the detection of PCR products by agarose gel electrophoresis.

[0159] Figure 3 shows the results of flow cytometry sorting of the yeast display library.

[0160] Figure 4 shows the SDS-PAGE detection results of the purified antibody of this invention.

[0161] Figure 5 shows the FACS detection results of the purified antibody of this invention.

[0162] Figure 6 shows the configuration of the anti-EGFR and HER3 bispecific antibody.

[0163] Figure 7 shows the affinity of the bispecific antibody molecule for binding to BXPC-3 cells.

[0164] Figure 8 shows the affinity of the bispecific antibody molecule for binding to A375 cells.

[0165] Figure 9 shows that the bispecific antibody molecule blocks the binding of EGFR and EGF.

[0166] Figure 10 shows the results of the endocytosis experiment of bispecific antibody molecules in MDA-MB-468 cells.

[0167] Figure 11 shows the results of the endocytosis experiment of bispecific antibody molecules in HCC-1569 cells. Detailed Implementation

[0168] Through screening and research, the inventors obtained anti-EGFR single-domain antibodies or their antigen-binding fragments with excellent endocytic activity and specificity. To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0169] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0170] The following are explanations of some of the terms used in this invention:

[0171] As used herein, the term “antibody” is used in the broadest sense, encompassing monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv), conventional antibodies (tetrapeptide chain antibodies consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds), as well as Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, and domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies).

[0172] The term "Fc" region refers to two heavy chain segments containing the CH2 and CH3 domains of an antibody, held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. Various Fc constant region variants have been disclosed in the prior art, such as Fc regions of antibody heavy chain constant regions having substitutions of one or more amino acids at 238, 265, 269, 270, 297, 327, and 329 (using the EU numbering system) (US Patent No. 6,737,056), or Fc regions of antibody heavy chain constant regions having substitutions of one or more amino acids at 234, 235, 265, and 329 (using the EU numbering system). Alternatively, the Fc region of the antibody's heavy chain constant region may have one or more amino acid substitutions at positions 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (using the EU numbering system) (see U.S. Patent No. 7,371,826). These mutations have been shown to endow antibodies with new properties without altering the function of the antibody's variable region.

[0173] As used herein, the term "single antigen domain" refers to an immunoglobulin variable domain capable of specifically binding to an antigenic epitope without pairing with other immunoglobulin variable domains. An exemplary "single antigen domain" could be a domain antibody or a VHH as defined below.

[0174] As used herein, the terms "VHH," "VHH chain," "nanobody," and "single-domain antibody" have the same meaning and are used interchangeably. A VHH is a small, stable, and highly efficient antigen-recognizing unit formed by a single heavy chain variable domain, consisting of only one chain from the C-terminus to the N-terminus: FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1. VHH-specific binding to epitopes does not require recognition by other antigen-binding domains (unlike conventional tetrapeptide chain antibodies, where epitopes are recognized by the VL and VH structure pair). Nanobodies possess excellent biological properties, with a molecular weight of 12-15 kDa, one-tenth the size of a complete antibody. They exhibit excellent tissue penetration, high specificity, and good water solubility. Due to their unique structural properties, they combine the advantages of traditional antibodies and small molecule drugs, almost overcoming the shortcomings of traditional antibodies such as long development cycles, low stability, and stringent storage conditions. They are gradually becoming an emerging force in next-generation antibody therapy, showing broad application prospects in immunodiagnosis and treatment. VHH includes, but is not limited to, natural antibodies produced by camelids, or antibodies produced by camelids that have been humanized, or antibodies obtained through phage display technology.

[0175] The terms "complementarity-determining region" or "CDR region" or "CDRs" or "CDR" refer to regions within the variable domain of an antibody that are highly variable in sequence and form structurally defined loops ("hypervariant loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes and are sequentially numbered from the N-terminus as CDR1, CDR2, and CDR3. In a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; and Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (http: / / imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing methods. In one embodiment, the CDR of the single-domain antibody of the present invention is located according to the Kabat numbering scheme.

[0176] Antibodies with different specificities (i.e., different binding sites against different antigens) have different core binding receptors (CDRs). However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, AbM, IMGT, and Contact methods, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat, Chothia, AbM, IMGT, or Contact may be substituted with conserved amino acid residues.

[0177] The "percentage (%) sequence identity" of amino acid sequences has a generally accepted definition in the art, referring to the percentage of identical amino acid sequences between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a known algorithm). It can be determined using methods known to those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0178] "Originating from", "derived from", or "derived from" an amino acid sequence that is partially or entirely identical or homologous to a reference amino acid sequence.

[0179] The term "nucleic acid" refers to a nucleotide chain of any length and includes both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerases.

[0180] The term "expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of the present invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may contain any type of nucleotide, including but not limited to DNA and RNA that may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring nucleotide linkages, or both. In an exemplary aspect, modified nucleotides or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector. The expression vectors of the present invention may be any suitable expression vector, such as a recombinant expression vector, capable of being used for transformation or transfection to deliver one or more genes or sequences of interest into any suitable host cell and preferably to express said genes or sequences in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both of the above. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0181] The term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. Host cells can be eukaryotic cells, such as plants, animals, fungi, or algae; or host cells can be prokaryotic cells, such as bacteria or protozoa. As described herein, host cells can be cells originating from or obtained from an individual. Host cells can be derived from or obtained from mammals. As used herein, the term "mammal" means any mammal, including but not limited to rodents such as mice and hamsters; and lagomorphs such as rabbits. Preferably, the mammal is from the order Carnivora, including felines (cats) and canines (dogs). More preferably, the mammal is from the order Artiodactyla, including bovines (cattle) and suidae (pigs), or belongs to the order Perissodactyla, including equines (horses). Most preferably, the mammal belongs to the order Primates, Cebooids, or Simoids (monkeys) or the suborder Anthropoids (humans and apes). Humans are particularly preferred. The expression vector can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system, or TALEN system), transposons (Sleeping Beauty or PiggyBAC), gene guns, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are cultured in a medium and screened for suitable activity. The methods and conditions used to culture the resulting transfected cells and to recover the generated antibody molecules are known to those skilled in the art and can be varied or optimized based on methods known in this specification and the prior art, depending on the specific expression vector used and the mammalian host cells. Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide protrophic, biocidal (e.g., antibiotic) or heavy metal (e.g., copper) resistance to auxotrophic hosts. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0182] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. A multispecific antibody is an antibody that has binding specificity to at least two different antigenic epitopes. In one embodiment, this document provides such a multispecific antibody that has binding specificity against a first antigen and a second antigen, also referred to as a "bispecific antibody."

[0183] The term "pharmaceutically acceptable carrier" refers to any of those carriers that are conventionally used and are limited only by physicochemical considerations and by route of administration. The pharmaceutically acceptable carriers described herein, such as mediators, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. In one aspect, a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredient of a pharmaceutical composition and that does not have adverse side effects or toxicity under the conditions of use.

[0184] Therapeutic formulations suitable for carrying out the methods disclosed herein, such as peptides, polynucleotides, or antibodies, can be prepared for storage by mixing the selected composition, having the desired purity, with an optional physiologically and pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized cake or aqueous solution (Remington's Pharmaceutical Sciences, 18th ed., ARGennaro, Mack Publishing Company (1990)). Pharmaceutical compositions can be manufactured by incorporating one or more suitable carriers or adjuvants, such as water, mineral oil, polyethylene glycol, starch, talc, lactose, thickeners, stabilizers, suspending agents, etc. Such compositions can be in the form of solutions, suspensions, tablets, capsules, creams, ointments, ointments, or other conventional forms. Compositions used for in vivo administration should be sterile. Compositions intended for parenteral administration are typically stored in lyophilized or solution form.

[0185] The carrier can be a solvent or dispersion medium containing, for example, water or a suitable mixture thereof, and vegetable oil. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. The choice of carrier can be determined by the specific type of pharmaceutical composition targeting the antigen-binding molecule of EGFR and the route of administration of the pharmaceutical composition. Accordingly, various formulations of suitable pharmaceutical compositions exist.

[0186] The pharmaceutical compositions of the present invention may contain any pharmaceutically acceptable ingredients, including, for example, acidifiers, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, matrices, binders, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disinfectants, disintegrants, dispersants, solubilizers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film-forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, heat-insulating agents, lubricants, mucosal adhesives, ointment matrices, ointments, oily mediators, organic bases, lozenge matrices, pigments, plasticizers, polishing agents, preservatives, multivalent chelating agents, skin penetrants, solubilizers, solvents, stabilizers, suppository matrices, and surfactants. Agents, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tension agents, toxic agents, viscous agents, water absorbents, water-miscible cosolvents, water softeners, or wetting agents.

[0187] The pharmaceutical compositions of anti-EGFR antibodies or their antigen-binding fragments described herein are formulated for parenteral, subcutaneous, intravenous, intramuscular, intra-arterial, intrathecal, or intraperitoneal administration. The pharmaceutical compositions can be administered via nasal, spray, oral, aerosol, rectal, or vaginal administration. The compositions can also be administered by infusion, rapid injection, or via implanted device.

[0188] Those skilled in the art will understand that, in addition to the pharmaceutical compositions described above, the compositions of the present invention can be formulated into inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

[0189] The term "monoclonal antibody" refers to an antibody derived from a single cloned cell line, which is not limited to eukaryotic, prokaryotic, or bacteriophage cloned cell lines.

[0190] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain non-continuous amino acids (conformal epitopes), which become spatially proximate due to the folding of the antigen (i.e., through the tertiary folding of the antigen, which is of a protein nature).

[0191] The term "specific binding" refers to an antibody binding to an antigen or an epitope within that antigen with a higher affinity than it binds to other antigens or epitopes. Typically, antibodies bind with an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 M or smaller, approximately 1×10 -9M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or about 1×10 -12 The equilibrium dissociation constant (KD) of an antibody (M or smaller) binds to an antigen or an epitope within the antigen. In some embodiments, the KD of antibody binding to an antigen is 10% or 1% of the KD of antibody binding to a nonspecific antigen (e.g., BSA, casein). The KD can be measured using standard procedures, such as those measured by BIACORE surface plasmon resonance assay.

[0192] The terms "antibody of the invention," "protein of the invention," "single-domain antibody of the invention," or "peptide of the invention" all refer to peptides that specifically bind to EGFR proteins, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0193] The present invention also provides other proteins or fusion expression products having 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) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0194] This invention develops a nanobody molecule targeting EGFR based on alpaca nanobodies and phage display technology. This molecule has high binding and high endocytic activity to tumor cell lines, and when developed into an antibody-drug conjugate, it has good tumor cell killing effect and has the potential to be developed into an antibody drug and / or conjugate drug.

[0195] The main consumables used in this invention are: 50mL Falcon centrifuge tubes (Corning, CAT#352070); T125 shake flasks (Corning, CAT#431143); 15mL Falcon centrifuge tubes (Corning, CAT#430052); 50mL Falcon centrifuge tubes (Corning, CAT#352070); 1.5mL RNase-free EP tubes (QSP, CAT#:509-GRD-Q); 200μL RNase-free PCR tubes (Axygen, PCR-02D-C); electroporation cuvettes (Bio-Rad 0.2cm); 6-well plates (Corning, CAT#3516); and 96-well plates (Corning, CAT#3365).

[0196] The main equipment used in this invention includes: a constant temperature incubator (Shanghai Jinghong, DNP-9052); a constant temperature shaking incubator (Jingqi, CO-O6U); a clean bench (Sujing Antai, SW-CJ-1FD); a biosafety cabinet (Haier, HR40-IIA2); a flow cytometer (Thermo, Attune Nxt); a CO2 incubator (Thermo, 3111); a benchtop centrifuge (Xiangyi, L535R); an ELISA reader (ALLSHENG, AMR-100); a PCR instrument (Applied Biosystems, ABI2720); an electroporator (Gene Pulser Xcell); and a cell sorter (BD, FACSAria). TM III).

[0197] Example 1: Raw Material Preparation

[0198] 1.1 Antigen Preparation

[0199] Based on the EGFR protein amino acid sequence information (P00533-1), codon optimization was performed according to mammalian codon preferences. After synthesizing the antigen-encoding nucleic acid sequence, the EGFR nucleic acid sequence was subcloned into the pCDNA3.4 vector (Invitrogen, A14697), and a His tag was added to the C-terminus to construct a eukaryotic expression vector. This vector was transfected into 293F cells (Gibco, A14527) and cultured in OPM-293CD05 Medium (Optimum Biotech, CAT#81075-001). The supernatant was collected, and the target protein was purified using a nickel column. SDS-PAGE was performed to detect the protein purity. The results of the EGFR-His recombinant protein reduced SDS-PAGE assay are shown in Figure 1. The sequence of the EGFR-His recombinant protein (EGFR(Leu25-Ser645)) is shown in SEQ ID NO.21.

[0200] The control antibody used in this invention is cetuximab, which was constructed and expressed according to the method of patent WO9640210A1.

[0201] 1.2 Biotin-conjugated antigen protein

[0202] Prepare 1 mg of purified antigen (buffered by PBS, concentration 1 mg / mL); weigh NHS-Biotin (APExBIO, CAT#: A8002), dissolve in DMSO to prepare 10 mM NHS-Biotin; add the freshly prepared 10 mM NHS-Biotin solution to the protein sample at a molar ratio of 10:1, place the sample tube in a light-protected self-sealing bag, and couple at 180 rpm for 30 min at room temperature. Replace with PBS to remove unlabeled Biotin, and store the labeled antigen protein at -80℃. Add 100 μL of HRP-Streptavidin (Boster, CAT#: BA1088) (1:10000 dilution) to each well of the coated Biotin-conjugated antigen, and detect the Biotin conjugation effect using an ELISA assay.

[0203] The ELISA results of EGFR-His recombinant protein are shown in Table 1 below.

[0204] Table 1

[0205] The test results showed that the purity of EGFR-His was >90% and it had good binding activity with the positive control antibody, so it can be used for immunization and antibody screening.

[0206] Example 2: Detection of EGFR antigen protein in alpaca immunization and serum titer

[0207] 2.1 Animal Immunization

[0208] Immunization was administered subcutaneously to two alpacas, hereinafter designated as 38# and 47#. The immunogen used was the protein prepared in Example 1. The first immunization used 500 μg of Adjuvant immunoadjuvant (Gerbu CAT#3030).

[0209] The remaining immunizations use 250ug; immunize once every 2 weeks, for a total of 4 immunizations.

[0210] Using the recombinant protein (EGFR-His) prepared in Example 1, the immunization interval was 14 days. Starting from the second immunization, peripheral blood was collected 10 days after each immunization (i.e., days 24, 38, and 52) to monitor the immune serum titer. After the immunization was completed (i.e., day 69), 100 ml of peripheral blood was collected, and PBMCs were isolated for the construction of a single-domain antibody display library.

[0211] 2.2 Immunotiter Detection

[0212] Blood was collected using the method described in Example 2.1, with 5 mL of peripheral blood collected each time. The centrifuge tube containing the blood sample was placed in a 37°C incubator for 1 hour; then the blood sample was transferred to 4°C and incubated overnight; the centrifuge tube containing the blood sample was then placed in a centrifuge and centrifuged at 5000 rpm for 20 min; the supernatant serum was separated and transferred to a new sterile centrifuge tube to collect the immune serum. The target recombinant protein was diluted with sterile CBS (carbonate buffer) to a final concentration of 1 μg / mL. Take a new 96-well microplate, add 100 μL to each well, and coat overnight at 4°C; remove the antigen coating solution, and wash 5 times with PBST (containing 0.05% Tween 20); add 200 μL / well of 3% MPBS and block at 37°C for 2 hours, remove the blocking buffer, and wash the plate 5 times with PBST; add 100 μL of serially diluted serum (100 μL / well), incubate at room temperature for 1 hour (control wells are treated with PBS), remove the liquid in the wells, and wash 5 times with PBST; add 100 μL of HRP anti-Llama IgG (H+L) (Novus, CAT#NBP1-75095) antibody (1:50000 dilution), incubate at room temperature for 1 hour, remove the liquid in the wells, and wash the plate 5 times with PBST. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 10-15 minutes. Then add 50 μL of stop solution to each well and read the OD450 value of the well using a microplate reader.

[0213] The ELISA results for alpaca immune titer are shown in Table 2 below:

[0214] Table 2

[0215] Limiting dilutions were performed according to the dilution gradient listed in the table, and ELISA assays were conducted with antigen-pre-coated 96-well plates. ELISA results showed that the immune serum could bind to the target recombinant protein, and the OD values ​​changed gradients with the gradient dilution of the immune serum. The binding of alpaca 38# quadruple immune serum and alpaca 47# triple immune serum to EGFR-His protein was significantly increased, meeting the requirements for blood collection and library construction. 100 mL of peripheral blood was collected from each serum for antibody display library construction.

[0216] Example 3: PBMC isolation and screening of EGFR-targeting single-domain antibodies

[0217] 1. Collect 100 mL of peripheral blood and separate PBMCs using lymphocyte separation solution.

[0218] 2. Extract RNA using PrimeScript. TMII. Reverse transcription was performed using the 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#6210B) to prepare cDNA. The following reaction mixture (Mix1) was prepared in 200 μL of PCR: Oligo dT Primer (50 μM), 8 μL; dNTP Mixture (10 mM each), 8 μL; total RNA sample, 20 μg; RNase-free water was added to a total volume of 80 μL. After mixing thoroughly, 80 μL was dispensed into each tube and placed in a PCR instrument at 42°C for 1 hour, followed by heat inactivation at 70°C for 15 minutes. Finally, the cDNA samples were stored on ice or at -20°C for long-term preservation.

[0219] 3. Perform PCR amplification of the VHH fragment. Prepare the first-round PCR reaction mixture (50 μL / tube): upstream primer (5 μM), 2 μL; downstream primer (10 μM), 1 μL; NuHi Power mix (2x), 25 μL; cDNA template, 2 μL; sterile water, 20 μL. The upstream primer binds to the signal peptide, and the downstream primer binds to the CH2 region. Perform PCR. Analyze the PCR products using 1% agarose gel electrophoresis, separating fragments with a molecular weight of approximately 750 bp. Recover the PCR products using a gel extraction kit. Prepare the two-round PCR reaction system (50 μL / tube): 2 μL of 2nd F primer, 2 μL of 2nd R primer, 25 μL of NuHi Power mix (2x) (Xinhai Biotechnology, CAT#NH9303), 200 ng of the product recovered from the first round of PCR, and sterile water to a final volume of 50 μL. The upstream primer binds to the antibody FR1 region, and the downstream primer binds to the anti-Hinge and FR4 regions. The restriction enzyme site is SfiI. After preparing the PCR reaction system, perform agarose gel electrophoresis analysis on the two-round PCR products: use 1% agarose gel to analyze the PCR products and separate the VHH fragment with a molecular weight of approximately 400 bp; recover the VHH PCR product using a gel extraction kit. The recovered second-round PCR products were aliquoted into 200 μL portions in each 1.5 mL centrifuge tube. 1 / 10 volume (20 μL) of 3M sodium acetate and 1 μg / μL glycogen were added, and the mixture was thoroughly mixed by pipetting and aspiration. Then, 880 μL of anhydrous ethanol was added, and the mixture was inverted and mixed again before freezing at -80°C. Figure 2 shows the results of the first-round PCR using VHH amplification primers and the PCR products detected by agarose gel electrophoresis. Figure 2(a) is the RNA agarose gel image; Figure 2(b) shows the PCR bands of approximately 1000 bp and 750 bp obtained in the first round of PCR, respectively. The 750 bp fragment was recovered from the gel and used as the template for the second-round PCR. Figure 2(c) shows the band of approximately 500 bp obtained in the second round of PCR, which is the VHH fragment, with homologous arm fragments added. Figure 2(d) shows the VHH fragment and the yeast display vector pYDisplay co-electrotransformed into EYB100 competent cells to construct a single-domain antibody yeast display library through recombination. Figure 2(e) shows the diluted yeast cell culture plate, with a calculated cell volume of 1×10⁻⁶. 9 Twenty single clones were randomly selected for sequencing to analyze the diversity of the constructed yeast display library. Based on the sequencing results, the empty vector rate and antibody repetition rate of the yeast display library were no higher than 5%.

[0220] Example 4: Construction of a single-domain antibody yeast display library

[0221] The pYDisplay vector (iCareab) was digested with SfiI (100 μL / tube) and incubated overnight at 50°C. The pYDisplay vector fragment was separated using a 1% agarose gel, and a 5000 bp fragment was extracted for gel recovery (Gel Recovery Kit, Qiagen, CAT#28706). The recovered pYDisplay digestion product was aliquoted into 1.5 mL centrifuge tubes, each containing 200 μL. 1 / 10 volume (20 μL) of 3M sodium acetate (Sigma, CAT#126-96-5) and 1 μg / μL Glycogen were added, and the mixture was stirred by pipetting and aspiration. Then, 880 μL of anhydrous ethanol was added, and the mixture was inverted and mixed at -80°C. The linearization digestion system for the yeast display vector pYDisplay was: pYDisplay 140 μg, 10x... Add 200 μL of buffer, 1200 U (60 μL) of SfiI (NEB,CAT#R0123L), and sterile water to a final volume of 2000 μL.

[0222] Electroporation construction of a yeast display library: Strawberry competent cells frozen at -80℃ were streaked onto YPD solid medium plates and activated at 30℃ for 3-5 days. Single colonies of competent yeast were inoculated into 50mL YPD medium and incubated at 250rpm and 30℃ for 1-2 days. To prepare competent yeast cells, the linearized vector fragment and PCR product were mixed and added to an electroporation cuvette for electroporation. The electroporated competent yeast cells were then transfected into culture flasks and incubated at 220rpm and 30℃ for 1 hour. 20μL of the resuspension was diluted 5000 times with SDCAA, and 100μL was plated onto an SDCAA plate and incubated for 2-3 days. The library volume was calculated, and the remaining bacterial culture was incubated for another 24 hours. Preservation: The remaining bacterial culture was collected in 50mL centrifuge tubes, centrifuged at 3000xg for 5 minutes, the supernatant was discarded, and 10mL of SDCAA was added for resuspension. The mixture was then mixed with 50% glycerol at a 1:1 ratio and stored at -80℃.

[0223] Example 5: Screening of Yeast Display Library

[0224] Yeast display library magnetic sorting: Yeast cultured in SDCAA was added to a 250 mL shake flask containing 50 mL of SGCAA medium and cultured at 30 °C and 240 rpm for 16 h. After centrifugation, the supernatant was discarded, and the resuspended product was resuspended in 1 mL of 0.5% PBSA and added to a 1.5 mL centrifuge tube. The resuspended product was centrifuged at 3000 g for 5 min, the supernatant was discarded, and the product was washed again with 0.5% PBSA. Streptavidin beads incubated with Biotin antigen were washed twice with 0.5% PBSA, placed on a magnetic rack for 5 min, and the supernatant was discarded. Yeast culture was added to the antigen-bound magnetic beads, and the mixture was incubated at 4 °C with rotation for 60 min, then placed on a magnetic rack for 15 min. The yeast culture was discarded, and the magnetic beads were washed three times with 0.5% PBSA. Resuspend the magnetic beads in 1 mL of SDCAA medium. Transfer 0.5–5 μL of the resuspended solution to 100 μL of SDCAA medium and plate it. Divide the resuspended solution into two portions. Add 500 μL of 50% glycerol to one portion (store at -80℃); add the other portion to a shaker tube, add 2 mL of SDCAA medium, and incubate at 30℃ and 240 rpm for 16 h. Transfer the bacterial culture from the shaker tube to 50 mL of SDCAA medium and incubate overnight at 30℃ and 240 rpm. Measure the OD600 value of the bacterial culture. Based on the OD600, take a portion of the bacterial culture, centrifuge, resuspend in SGCAA, transfer to 50 mL of SGCAA medium to achieve a final OD600 value of 1, and incubate overnight at 30℃ and 240 rpm. Resuspend the remaining bacterial culture in a 1:1 ratio of SDCAA and 50% glycerol and store at -80℃. After one round of magnetic sorting using Biotin-EGFR-His, V5 (V5 Tag Antibody [FITC], mAb (iCareab)) showed approximately 54% positivity, and EGFR positivity was approximately 2.27%. Compared with the original library, there was enrichment but the positivity rate was low, so a second round of sorting was performed.

[0225] Yeast display library two-stage flow cytometry sorting: Take 1 ml of SGCAA-cultured yeast culture into a 1.5 ml centrifuge tube, centrifuge to remove the supernatant, wash twice with 1 ml PBS, resuspend in 1 ml PBS, take 100 μL of the culture into a new 1.5 ml centrifuge tube, centrifuge, and remove the supernatant. Dilute Biotin-antigen protein to 10 μg / mL with 100 μL PBS, resuspend the experimental group cells, incubate at 4°C for 60 min, centrifuge at 3000g for 3 min, discard the supernatant, and wash twice with 1 ml PBS. Resuspend the experimental group cells with APC-Streptavidin diluted 1:1000 and FITC-anti-V5 at 10 μg / mL, incubate at 4°C for 60 min, centrifuge at 3000g for 3 min, discard the supernatant, wash twice with 1 mL PBS, and resuspend in 1 mL PBS. Add the resuspended cells to flow cytometry tubes for flow cytometry sorting. Prepare 1 ml of SDCAA culture medium into a 15 ml centrifuge tube as the collection tube. Perform flow cytometry sorting to separate yeast cells that are positive for both antigen and V5 dual fluorescence. Figure 3 shows the results of the second round of flow cytometry sorting of the yeast display library. After the second round of flow cytometry sorting using Biotin-EGFR-His protein, the results show significant enrichment compared to the first round of magnetic sorting, with a positive rate of 38.8%, indicating that single clones can be selected for detection.

[0226] Example 6: Yeast Monoclonal Flow Cytometry Detection

[0227] After sorting, yeast culture was plated on SDCAA plates, and single clones were selected for culture. After 48 hours of induction for expression, the culture was incubated with Biotin-antigen. PE-Streptavidin (eBioscience, CAT#12-4317-87) or APC-Streptavidin (Biolegend, CAT#405207) was used as the secondary antibody. Flow cytometry was performed after incubation. Yeast clones bound to the target antigen were lysed by heating, centrifuged, and 1 μL of the supernatant was used as a template for PCR amplification and testing (the remaining culture was stored at -20℃). Results: Single clones were selected from 96-well plates for FACS analysis. Positive yeast clones were sequenced, yielding 20 different antibody sequences, which were then used to construct antibody expression vectors for validation.

[0228] Example 7 Construction of antibody eukaryotic expression vector

[0229] Positive yeast clones were subjected to PCR to obtain antibody sequences, which were then digested with SfiI and ligated into the eukaryotic expression vector pcDNA3.4-human IgG1Fc to construct an antibody expression vector. The eukaryotic expression vector was transiently transfected into 293F cells, and the antibody expression supernatant was collected. The binding of candidate antibodies and antigen proteins was detected by FACS. A total of 20 different sequences were obtained through three sorting processes. Eukaryotic expression vectors (with an Fc tag added to the C-terminus) were constructed and transfected into 293F cells to express antibodies for validation, as shown in SEQ ID NO:1–20 of the sequence listing.

[0230] Candidate single-domain antibodies were named using clone numbers, and the CDRs for VHH were determined using the Kabat method for defining CDRs. The CDRs and VHH sequences of exemplary anti-EGFR single-domain antibodies of this invention are shown in Table 3 below.

[0231] Table 3

[0232] Example 8: Expression, Purification, and Identification of Candidate Single-Domain Antibodies

[0233] 1. Select positive clones and prepare expression plasmids for antibodies.

[0234] 2. Thaw the LVTransm transfection reagent (iCarEab, Cat#LVTran100) and pcDNA3.4-human IgG1Fc antibody expression vector at room temperature, then mix thoroughly by pipetting. Remove the PBS buffer and warm it to room temperature. Transfer 2 mL of PBS to one well of a 6-well plate, add 20 μg of antibody expression vector, mix thoroughly by pipetting, then add 60 μL of LVTransm, immediately mix by pipetting, and let stand at room temperature for 10 minutes.

[0235] 3. Add the DNA / LVTransm complex to 20 mL of 293F cells and gently shake to mix thoroughly. Incubate the cells at 37°C, 5% CO2, 130 rpm.

[0236] 4. After continuous culture for 5-7 days, collect the supernatant by centrifugation, filter it through a 0.45μm filter membrane, transfer the filtrate to a sterile centrifuge tube, and purify the antibody using Protein A magnetic beads (Nanjing Genscript Biotech Co., Ltd., CAT#L00695).

[0237] The purified antibodies were detected using the standard SDS-PAGE method. For the preparation of the non-reducing solution: 3 μg of each obtained antibody was added to 2×SDS loading buffer, heated in a dry bath at 99°C for 5 min, cooled to room temperature, and centrifuged at 12000 rpm for 5 min to collect the supernatant. The supernatant was added to a Bis-tris 4-15% gradient gel (GenScript) for gel electrophoresis and stained with Coomassie Brilliant Blue to visualize the protein bands. The protein gel showing the visualized protein bands was photographed using an imaging system (after destaining with destaining solution until the gel background was transparent). The purity of the reduced and non-reduced bands was calculated using the built-in software. The results showed that the purified antibody purity was >90%. Figure 4 shows the SDS-PAGE detection results.

[0238] Example 9: Detection of the binding of recombinant antibody to target protein

[0239] For ELISA detection, the recombinant protein was diluted to a final concentration of 1 μg / mL using sterile CBS. 100 μL was added to each well of a 96-well microplate and incubated overnight at 4°C. The antigen coating solution was removed, and the plate was washed five times with PBST (containing 0.05% Tween 20). 200 μL / well of 3% MPBS was added, and the plate was blocked at 37°C for 2 hours. After removing the blocking buffer, the plate was washed five times with PBST. The expressed recombinant antibody was added, with 100 μL / well of purified antibody (starting at 10 μg / mL, serially diluted 3-fold for 3 spots, 100 μL / well), and incubated at room temperature for 1 hour (control wells were treated with PBS). The liquid in the wells was removed, and the plate was washed five times with PBST. Add 100 μL / well of HRP-Protein A (Boster, BA1080) antibody (1:50000 dilution) and incubate at room temperature for 1 hour; 8) After removing the liquid from the wells, wash the plate 5 times with PBST; add 100 μL / well of TMB chromogenic solution; incubate at room temperature in the dark for 10-15 minutes; add 50 μL / well of stop solution; read the OD450 value of the wells using a microplate reader. The ELISA results are shown in Table 4 below.

[0240] Table 4

[0241] Example 10: Detection of the binding of candidate antibodies to overexpressing cell lines

[0242] CHO-S and CHO-S-EGFR cell lines were resuscitated in liquid nitrogen and adjusted to the logarithmic growth phase. The cells were then divided into several fractions, each containing 3 × 10⁶ cells. 55) Add PE anti-human IgG (eBioscience, Cat#:12-4998-82) (1:5000 dilution), mix thoroughly, and incubate at room temperature for 30 minutes in the dark. Centrifuge at 800xg for 3 minutes, remove the supernatant containing the antibody, and wash the cells three times with PBS. 6) Add PE anti-human IgG (eBioscience, Cat#:12-4998-82) (1:5000 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 minutes. Centrifuge at 800xg for 3 minutes, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS. Resuspend the cells in 200μL PBS and perform flow cytometry analysis.

[0243] The FACS test results are shown in Figure 5.

[0244] Example 11: ELISA assay for blocking EGFR and EGF binding

[0245] Dilute the EGF-Fc recombinant protein to a final concentration of 0.5 μg / mL using sterile CBS. Add 100 μL to each well of a 96-well microplate and incubate overnight at 4°C. Remove the antigen coating solution and wash five times with PBST (containing 0.05% Tween 20). Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours. After removing the blocking buffer, wash the plate five times with PBST. Prepare 4 μg / mL Biotin-EGFR solution, 50 μL / well. All test antibodies, starting at 60 μg / mL, are serially diluted 5-fold to 7 spots, 50 μL / well. After co-incubation at 37℃ for 0.5 h, transfer all solutions to an EGF-Fc-coated microplate and incubate at 37℃ for 1 h. Remove the liquid from the wells and wash 5 times with PBST. Add 100 μL / well of HRP-Streptavidin antibody (1:50000 dilution) and incubate at room temperature for 1 h. Remove the liquid from the wells and wash the plate 5 times with PBST. Add 100 μL / well of TMB chromogenic buffer and incubate at room temperature in the dark for 10-15 minutes. Add 50 μL / well of stop solution and read the OD450 values ​​of the wells using a microplate reader. In this experiment, Anmai Bio's EMB-01 was selected as the positive label and prepared according to WO2017136820A2. The EGF blocking results of the purified antibody are shown in Table 5 below. The results showed that the single-domain antibody of the present invention has a significant blocking effect.

[0246] Table 5

[0247] Example 12 Single-domain antibody affinity detection

[0248] Candidate antibodies were immobilized using an AHC2 sensor at a concentration of 5 μg / ml for 30 s. The buffer was PBST (PBS + 0.02% Tween 20), diluted with EGFR(25-645)-6His to 50, 25, 12.5, 6.25, 3.13, and 0 nM. Affinity was measured: equilibration for 60 s, binding for 180 s, dissociation for 180 s, and the detection temperature was 25 °C. Kinetic characterization was performed using a ForteBio OCTET R2 system. The results show the affinity of the single-domain antibody described in this invention, and Table 6 below records the affinity detection results.

[0249] Table 6

[0250] Example 13 Tumor Cell Binding Assay

[0251] BXPC-3 cell lines (derived from the Chinese Academy of Sciences Cell Bank) were cultured in complete medium at 37℃ and 5% CO2. Cells in the logarithmic growth phase were harvested, and cell viability was assessed using the trypan blue exclusion method to ensure a viability of over 90%. After centrifugation at 200g for 5 min, the supernatant was discarded. Cells were washed once with PBS, resuspended in FACS Buffer to prepare a single-cell suspension, and the cell density was adjusted to 1x10⁻⁶. 6 cells / mL; Add 100 μL of cell suspension to each well of a 96-well plate to make the initial concentration of the working solution 1000 nM, dilute 5 times, for a total of 8 concentrations; after mixing, incubate at 4℃ for 60 min; wash the cells twice with FACS Buffer, 200 μL each time, centrifuge at 200g for 5 min, and finally resuspend the cells with 100 μL of APC-labeled secondary antibody (Allophycocyanin (APC) Affini Pure F(ab')2 Fragment Goat Anti-Human IgG, Fcγfragment specific; Jackson, 109-136-098) diluted with FACS Buffer, and incubate at 4℃ in the dark for 30 min;

[0252] Wash cells twice with 200 μL of FACS Buffer each time, centrifuge at 200g for 5 min, and finally resuspend cells with 150 μL of FACS Buffer; detect fluorescence values ​​by flow cytometry.

[0253] Table 7 Affinity of EGFR-VHH molecules to BXPC-3 cells

[0254] Example 14 Humanization and Validation of Antibodies

[0255] 14.1 CDR grafting

[0256] Humanized design utilizes a proprietary professional antibody structure simulation and performance optimization analysis platform. Homology modeling of antibody structures is used to predict antibody structures and analyze the entire CDR structure to determine the CDR. Camel-derived antibody sequences are used to search for V-region homologous proteins in several human framework libraries (such as Kabat, Igblast, and IMGT). Alignment with antibody sequences from several databases is performed to identify sequences with high homology. From these high-homology sequences, frequently used human germline framework sequences are selected as candidate templates.

[0257] Humanization design employed the CDR grafting method. The basic approach involves replacing the camel-derived frame region (FR) with the human germline frame region (FR) selected in the preceding steps, retaining only the camel-derived CDR. Simple CDR grafting often reduces the affinity of antigen-antibody binding. To mitigate the adverse effects of humanization on the antibody's spatial conformation, activity, and function, it is necessary to perform reversion mutations on some key amino acids in the human frame region, i.e., reverting them to the corresponding amino acids in the camel-derived FR region. By obtaining the structural files of camel-derived antibodies from antibody databases and examining their 3D structures, the interactions between the amino acids in the camel-derived antibody's CDR region and other frame regions—such as distances, van der Waals forces, and electrostatic forces—can be determined. This allows for the localization of amino acids within the frame region that have strong interactions with the CDR region, and these amino acids are then identified as candidate sites for reversion mutations.

[0258] Expression of humanized antibodies: Humanized heavy chain antibody genes were cloned into plasmids and expressed as VHH-Fc. After humanization, 25 modified antibodies were obtained, which, together with the parent antibodies SEQ ID NO:1 and SEQ ID NO:4, yielded a total of 27 heavy chain plasmids. Approximately 0.5 ml of electroporation buffer was added to competent cells, mixed well, and then an appropriate amount of heavy chain plasmid (concentration 500 ng / µl) was added. After thorough mixing of the cell-plasmid suspension, 1 ml was added to a 1 ml electroporation tube, and the tube was placed in an electroporator for electroporation. After electroporation, the cells in the electroporation tube were aliquoted into shake flasks containing 20 ml of culture medium and incubated statically for 40 min. After incubation, the shake flasks were cultured at 37°C, 270 rpm, and 8% CO2 for 4 days. The supernatant was collected by centrifugation (8000 rpm, 5 min) and purified using a Protein A (Cytiva, 29127556) affinity chromatography column.

[0259] The purity of each humanized hu-2-2-B1-3 antibody was determined using size exclusion chromatography (SEC) and SDS-PAGE, and the results are as follows:

[0260] The purity of each humanized hu-1G6 antibody was determined using size exclusion chromatography (SEC) and SDS-PAGE, and the results are as follows:

[0261] 14.2 PTM site removal

[0262] Analysis of high-risk PTM sites in heavy chain sequence CDRs revealed a high-risk PTM site (DG) in the heavy chain variable region CDR2.

[0263] After humanization of the antibodies, the PTM site was removed from 1G6-huVHH1, 1G6-huVHH6, and 1G6-huVHH10. The PTM-removed antibodies were expressed, and their affinity was tested, yielding PTM-removed humanized antibodies with affinity comparable to the humanized antibodies. The obtained 1G6-huHCDR2 sequence is shown in SEQ ID NO:186; the VHH sequences of 1G6huVHH1-DA, 1G6-huVHH6-DA, and 1G6-huVHH10-DA are shown in SEQ ID NO:187, SEQ ID NO:188, and SEQ ID NO:189.

[0264] Example 15: Affinity detection of humanized antibody hu-2-2-B1-3

[0265] The kinetics and affinity properties of the antibodies were tested using the Biacore 8K (Cytiva, 8K SPR system) platform. Single-concentration assays were performed on 14 humanized antibodies (SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132) and one camel-derived maternal antibody (SEQ ID NO: 1).

[0266] The dissociation equilibrium constant KD reflects the affinity of an antibody for its target; a smaller value indicates stronger affinity. KD is determined by the ratio of the dissociation rate constant Kd to the association rate constant Ka, i.e., KD = Kd / Ka. The ratio of the dissociation equilibrium constant KD between humanized and camel-derived antibodies is a change factor (Fold↓), which reflects the change in antibody affinity after humanization. A change factor of 1 indicates that the affinity of the humanized antibody is equal to that of the camel-derived antibody; a change factor greater than 1 indicates that the affinity of the humanized antibody is lower than that of the camel-derived antibody; and a change factor less than 1 indicates that the affinity of the humanized antibody is higher than that of the camel-derived antibody.

[0267] The results showed that the affinity of multiple humanized antibodies remained basically consistent with that of the maternal antibody, and the humanization of hu-2-2-B1-3 was successful based on the optimized safety of humanization.

[0268] Example 16 Affinity Detection of Humanized Antibody hu-1G6

[0269] Using a humanized affinity assay method similar to that in Example 15, the kinetics and affinity properties of the antibodies were tested using the Biacore 8K (Cytiva, 8K SPR system) platform. Single-concentration assays were performed on 11 humanized antibodies (SEQ ID NO: 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162) and one camel-derived maternal antibody (SEQ ID NO: 4).

[0270] The results showed that the affinity of several humanized antibodies was basically consistent with that of the parent antibody, and the affinity of SEQ ID NO:161 (i.e. hu-1G6VHH10) was even slightly improved; based on the optimization of safety through humanization, hu1G6VHH was successfully obtained.

[0271] The humanized sequences 1G6huVHH1-DA, 1G6-huVHH6-DA, and 1G6-huVHH10-DA obtained in Example 14.2 were tested using the same affinity assay. Three humanized antibodies (SEQ ID NO: 187, 188, 189) and one camel-derived maternal antibody (SEQ ID NO: 4) were also tested. The results are as follows:

[0272] The results showed that, in order to remove the high-risk PTM site DG in the heavy chain variable region CDR2, three PTM removal sequences were designed, and three antibodies were expressed. SPR affinity assays showed that the affinity of the three humanized PTM site-removed antibodies decreased by less than three-fold compared to the unmodified antibodies. The result is a humanized VHH with PTM site removal.

[0273] Example 17 Preparation of EGFR VHH / HER3 bispecific antibody

[0274] In this study, the anti-EGFR / HER3 bispecific antibody SI-1X6.4, the HER3 monoclonal antibody Patritumab, and the EGFR monoclonal antibody Cetuximab were used as positive control antibodies.

[0275] Patritumab is prepared according to the sequence disclosed in WO2007077028 (VH and VL are SEQ ID NO. 70 and SEQ ID NO. 72 in that patent, respectively). Cetuximab monoclonal antibody is prepared according to conventional methods or is commercially available. SI-1X6.4 bispecific antibody is prepared according to WO2023083381 (especially SEQ ID NO. 2 and SEQ ID NO. 4 in that patent application) and according to the method in that application.

[0276] Example 18: Construction of a bispecific antibody against EGFR VHH and HER3

[0277] 18.1 Control Antibodies and Their Preparation

[0278] This paper uses anti-HER3 VHH 7D12, HER3 monoclonal antibody Patritumab, EGFR-HER3 bispecific antibody SI-1X6.4 (also known as BL-B01D1), and EGFR monoclonal antibody Cetuximab as positive control antibodies. Patritumab was prepared according to the sequence disclosed in WO2007077028 (VH and VL are SEQ ID NO. 70 and SEQ ID NO. 72 in that patent, respectively), and SI-1X6.4 was prepared according to WO2023083381 (especially SEQ ID NO. 2 and SEQ ID NO. 4 in that patent application), and according to the method of the patent application. Cetuximab monoclonal antibody (Cetuximab antibody sequence is shown as SEQ ID NO: 182 and SEQ ID NO: 183 in this application) was commercially available or prepared according to conventional methods.

[0279] 18.2 Antibody Configuration and Construction

[0280] This embodiment describes the structure and expression vector construction of anti-EGFR and HER3 bispecific antibodies. Using VHH 1-G6, 2-2-B1-3, 4-F5, and humanized 2B13-huVHH12 of this invention, four anti-EGFR and HER3 bispecific antibodies were designed and constructed: 1G6-Pach, 2B13-Pach, 4F5-Pach, and hu2B13-12-HER3-2. Each antibody contains two identical first polypeptide chains (heavy chains) and two identical second polypeptide chains (light chains). The antibody conformation design is shown in Figure 6, and the corresponding amino acid sequences are shown in the table below. The conformation involves linking the EGFR VHH of this invention (via a linker) to the N-terminus of the HER3 antibody heavy chain, and then combining it with the HER3 antibody light chain to form an anti-EGFR and HER3 bispecific antibody. The specific antibody conformation is described below:

[0281] Using conventional expression and purification methods in the art, and identifying the bispecific antibody molecules using SEC-HPLC, the purity of the bispecific antibody monomers was greater than 95%.

[0282] Example 19: Binding activity of bispecific antibodies to overexpressing cell lines

[0283] 19.1 Binding assay with BXPC-3 cells

[0284] BXPC-3 cell line (from the Chinese Academy of Sciences Cell Bank) was cultured in complete medium at 37℃ and 5% CO2. Cells in the logarithmic growth phase were harvested, and cell viability was assessed using the trypan blue exclusion method to ensure a viability of over 90%. After centrifugation at 200g for 5 min, the supernatant was discarded. Cells were washed once with PBS, resuspended in FACS Buffer to prepare a single-cell suspension, and the cell density was adjusted to 1 x 10⁻⁶ cells / mL. 6 cells / mL; Add 100 μL of cell suspension to each well of a 96-well plate to make the working solution of the test sample (bispecific antibody constructed in Example 18) start at 1000 nM, dilute 5 times, for a total of 8 concentrations; after mixing, incubate at 4°C for 60 min; wash the cells twice with FACS Buffer, 200 μL each time, centrifuge at 200g for 5 min, and finally resuspend the cells with 100 μL of APC-labeled secondary antibody (Allophycocyanin (APC) Affini Pure F(ab')2 Fragment Goat Anti-Human IgG, Fcγfragment specific; Jackson, 109-136-098) diluted with FACS Buffer, and incubate at 4°C in the dark for 30 min;

[0285] Cells were washed twice with 200 μL of FACS Buffer each time, centrifuged at 200g for 5 min, and finally resuspended in 150 μL of FACS Buffer; fluorescence values ​​were detected by flow cytometry. Figure 7 records the affinity of the bispecific antibody molecule for BXPC-3 cells. The results show that the bispecific antibody of the present invention has good affinity.

[0286] 19.2 Binding assay with A375 cells

[0287] Using the A375 cell line (source: Shanghai Cell Bank, Chinese Academy of Sciences), the affinity of the bispecific antibody molecules for A375 cells was determined using FACS, a method similar to that in Example 19.1. Figure 8 records the MFI of the affinity of each group of bispecific antibody molecules. The results show that the bispecific antibody of the present invention has excellent affinity.

[0288] Example 20: Blocking experiment of bispecific antibody in EGFR and EGF binding

[0289] Flow cytometry (FACS) buffer was prepared by adding 2% fetal bovine serum (FBS) to phosphate-buffered saline (PBS); cells were collected by digestion with TrypLE (Gibco), washed with FACS buffer, and then resuspended in FACS buffer to a density of 2 × 10⁶ cells / mL. 6 / ml. Cells were then seeded into 96-well plates (100 μL / well), centrifuged at 300g for 5 minutes, and the supernatant was discarded. 50 μL of a mixture of diluted antibody (the bispecific antibody constructed in Example 18, final concentration 100 nM, diluted 5-fold with FACS buffer) and 50 μL of epidermal growth factor (EGF, final concentration 50 ng / ml) prepared with FACS buffer was added to each well, and the cells were incubated at 4°C in the dark for 1 hour. Cells were washed twice with flow cytometry buffer, centrifuged at 300g for 5 minutes, and the supernatant was discarded. 100 μL of fluorescently conjugated secondary antibody (APC-conjugated goat anti-human IgG Fc, prepared 1:500 with FACS buffer) diluted in FACS buffer was added to each well to stain the cells, and the cells were incubated at 4°C in the dark for 0.5 hours. Cells were washed twice with flow cytometry buffer, centrifuged at 300g for 5 minutes, and the supernatant was discarded. Cells were resuspended in 100 μL of FACS buffer, and the cell samples were analyzed by flow cytometry.

[0290] The EGF blocking results of the purified antibody are shown in Figure 9. The results show that the EGFR / HER3 bispecific antibody constructed using the VHH of this invention has a significant blocking effect.

[0291] Example 21: Detection of endocytosis of humanized bispecific antibodies

[0292] This embodiment examines the endocytic effect of the bispecific antibody drug targeting EGFR and HER3 of the present invention on MDA-MB-468 (Shanghai Cell Bank, Chinese Academy of Sciences) and HCC1569 (Nanjing Kebai Biotechnology) cells expressing EGFR and / or HER3, and compares its endocytic activity with that of the parent monoclonal antibody. Cells were co-incubated with an excess of a fixed concentration of the antibody drug, and the amount of residual antigen on the cell surface was assessed using FACS to evaluate the endocytic capacity of the antibody drug.

[0293] Experimental methods (using MDA-MB-468 as an example; the method for HCC1569 cells is the same)

[0294] 1. Cell culture: MDA-MB-468 cells were cultured in Leibovitz's L-15 (Gibco) medium with 10% FBS, while HCC-827 and HCC1569 cells were cultured in RPMI-1640 medium (Gibco) with 10% FBS.

[0295] 2. Cell preparation: Take MDA-MB-468, HCC-827, and HCC1569 cells in logarithmic growth phase, wash once with PBS, and digest for 2-3 minutes. After complete digestion, add 10-15 mL of cell culture medium to elute the digested cells. Centrifuge at 200g for 5 minutes, discard the supernatant, add cell culture medium to resuspend the cells into a single-cell suspension, and adjust the viable cell density to 1×10⁶ cells / mL. 6 cells / mL.

[0296] 3. Cell seeding: Add 100 μL / well to a 96-well cell culture plate. Centrifuge at 200g for 5 min and discard the supernatant.

[0297] 4. Sample addition procedure: Adjust the concentration of the antibody to be tested (the bispecific antibody constructed in Example 18) to 100 nM, add 100 μL / well to a 96-well cell culture plate and resuspend the cells.

[0298] 5. Antibody incubation: After incubating the culture plate at 4°C for 1 hour, centrifuge at 200g for 5 minutes and discard the supernatant.

[0299] 6. Washing cells: Add 200 μL of PBS to a 96-well cell culture plate to resuspend the cells, centrifuge at 200g for 5 min, and discard the supernatant. Repeat the washing process once more.

[0300] 7. Antibody internalization: Add 200 μL of cell culture medium to each well of a 96-well cell culture plate and resuspend the cells. Incubate the plate at 37°C for 2 hours, then centrifuge at 200g for 5 minutes and discard the supernatant. (Note: For samples from the 0h group, skip this step and proceed directly to step 9.)

[0301] 8. Washing cells: Add 200 μL of PBS to a 96-well cell culture plate to resuspend the cells, centrifuge at 200g for 5 min, and discard the supernatant. Repeat the washing process once more.

[0302] 9. Secondary antibody incubation: Add 100 μL / well of the secondary antibody working solution (Allophycocyanin (APC), Affini Pure F(ab')2 Fragment Goat Anti-Human IgG, Fcγfragment specific; Jackson 109-136-098) to a 96-well cell culture plate and resuspend the cells. Incubate the culture plate at 4°C for 30 min, then centrifuge at 200g for 5 min and discard the supernatant.

[0303] 10. Washing cells: Add 200 μL of PBS to a 96-well cell culture plate to resuspend the cells, centrifuge at 200g for 5 min, and discard the supernatant. Repeat the washing once more.

[0304] 11. Flow cytometry: Resuspend cells in 100 μL of PBS in a 96-well cell culture plate and detect them using a flow cytometer.

[0305] 12. Data Analysis: The MFI value obtained by flow cytometry was used to calculate the endocytosis rate of the antibody drug. The calculation formula is as follows:

[0306] Internalization rate = (1 - (MFI of the sample to be tested)) t2 -Background MFI t2 ) / (MFI of the sample to be tested) t0 -Background MFI t0 ))*100, where t0 represents antibody internalization time of 0h, t2 represents antibody internalization time of 2h, and background MFI represents MFI when only secondary antibody is added.

[0307] Figure 10 shows the results of the endocytosis experiment in MDA-MB-468 cells, and Figure 11 shows the results of the endocytosis experiment in HCC-1569 cells. The results show that the EGFR / HER3 bispecific antibody constructed using the VHH of this invention exhibits excellent endocytosis activity.

[0308] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and controls without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

[0309] The sequence information discussed in this article

Claims

1. An anti-EGFR antibody or an antigen-binding fragment thereof, comprising a single antigen domain, said single antigen domain comprising CDR1, CDR2 and CDR3 of any single antigen domain in SEQ ID NO:1-20 and 187-189, preferably, said single antigen domain is VHH.

2. The anti-EGFR antibody or its antigen-binding fragment according to claim 1, characterized in that, The single antigen domain comprises complementarity-determining regions CDR1, CDR2, and CDR3; wherein CDR1 comprises an amino acid sequence according to any one of SEQ ID NO: 22, 25, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59; CDR2 comprises an amino acid sequence according to any one of SEQ ID NO: 23, 26, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 62, 186; and CDR3 comprises an amino acid sequence according to any one of SEQ ID NO: 24, 27, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 63.

3. The anti-EGFR antibody or its antigen-binding fragment according to claim 1 or 2, wherein: (1) CDR1 contains the amino acid sequence shown in SEQ ID NO:22, CDR2 contains the amino acid sequence shown in SEQ ID NO:23, and CDR3 contains the amino acid sequence shown in SEQ ID NO:24; or (2) CDR1 contains the amino acid sequence shown in SEQ ID NO:25, CDR2 contains the amino acid sequence shown in SEQ ID NO:26, and CDR3 contains the amino acid sequence shown in SEQ ID NO:27; or (3) CDR1 contains the amino acid sequence shown in SEQ ID NO:25, CDR2 contains the amino acid sequence shown in SEQ ID NO:26, and CDR3 contains the amino acid sequence shown in SEQ ID NO:28; or (4) CDR1 contains the amino acid sequence shown in SEQ ID NO:29, CDR2 contains the amino acid sequence shown in SEQ ID NO:30, and CDR3 contains the amino acid sequence shown in SEQ ID NO:31; or (5) CDR1 comprises the amino acid sequence shown in SEQ ID NO:32, CDR2 comprises the amino acid sequence shown in SEQ ID NO:33, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:34; or (6) CDR1 comprises the amino acid sequence shown in SEQ ID NO:35, CDR2 comprises the amino acid sequence shown in SEQ ID NO:36, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:37; or (7) CDR1 contains the amino acid sequence shown in SEQ ID NO:38, CDR2 contains the amino acid sequence shown in SEQ ID NO:39, and CDR3 contains the amino acid sequence shown in SEQ ID NO:40; or (8) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:41, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:42, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:43; or (9) CDR1 comprises the amino acid sequence shown in SEQ ID NO:44, CDR2 comprises the amino acid sequence shown in SEQ ID NO:45, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:46; or (10) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:47, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:48, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:49; or (11) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:50, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:51, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:52; or (12) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:53, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:54, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:55; or (13) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:56, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:57, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:58; or (14) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:59, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:60, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:61; or (15) The CDR1 comprises the amino acid sequence shown in SEQ ID NO:56, the CDR2 comprises the amino acid sequence shown in SEQ ID NO:62, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:63; or (16) The CDR1 contains the amino acid sequence shown in SEQ ID NO:29, the CDR2 contains the amino acid sequence shown in SEQ ID NO:186, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

31.

4. The anti-EGFR antibody or its antigen-binding fragment according to any one of claims 1-3, characterized in that, The single antigen domain includes: (1) An amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:1, or consisting of SEQ ID NO:1; (2) The amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:2, or consisting of SEQ ID NO:2; (3) An amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:3, or composed of SEQ ID NO:3; (4) An amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:4, or consisting of SEQ ID NO:4; (5) An amino acid sequence as shown in SEQ ID NO:5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:5, or composed of SEQ ID NO:5; (6) An amino acid sequence as shown in SEQ ID NO:6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:6, or consisting of SEQ ID NO:6; (7) An amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:7, or consisting of SEQ ID NO:7; (8) An amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:8, or consisting of SEQ ID NO:8; (9) An amino acid sequence as shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:9, or consisting of SEQ ID NO:9; (10) An amino acid sequence as shown in SEQ ID NO:10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:10, or consisting of SEQ ID NO:10; (11) An amino acid sequence as shown in SEQ ID NO:11, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:11, or composed of SEQ ID NO:11; (12) An amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:12, or composed of SEQ ID NO:12; (13) An amino acid sequence as shown in SEQ ID NO:13, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:13, or composed of SEQ ID NO:13; (14) An amino acid sequence as shown in SEQ ID NO:14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:14, or composed of SEQ ID NO:14; (15) An amino acid sequence as shown in SEQ ID NO:15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:15, or composed of SEQ ID NO:15; (16) An amino acid sequence as shown in SEQ ID NO:16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:16, or consisting of SEQ ID NO:16; (17) An amino acid sequence as shown in SEQ ID NO:17, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:17, or composed of SEQ ID NO:17; (18) An amino acid sequence as shown in SEQ ID NO:18, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:18, or consisting of SEQ ID NO:18; (19) An amino acid sequence as shown in SEQ ID NO:19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:19, or composed of SEQ ID NO:19; or (20) An amino acid sequence as shown in SEQ ID NO:20, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:20, or consisting of SEQ ID NO:

20.

5. The anti-EGFR antibody or its antigen-binding fragment according to any one of claims 1-4, characterized in that, The single antigen domain comprises: an amino acid sequence as shown in SEQ ID NO:130, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131 or 132, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:130, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131 or 132, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, or an amino acid sequence as shown in SEQ ID NO:130, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131 or 132, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, or an amino acid sequence having at least 80%, at least 85 NO: 130, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131 or 132; and / or, the single antigen domain comprises: an amino acid sequence as shown in SEQ ID NO: 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 187, 188 or 189, or in combination with SEQ ID The amino acid sequence of SEQ ID NO:152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 187, 188 or 189 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, or consists of an amino acid sequence of SEQ ID NO:152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 187, 189 or 189.

6. The anti-EGFR antibody or its antigen-binding fragment according to any one of claims 1-5, further comprising an Fc region of an immunoglobulin, preferably an Fc region of a human immunoglobulin such as IgG; more preferably, the Fc region is an Fc region of human IgG1 or human IgG4.

7. The anti-EGFR antibody or its antigen-binding fragment according to claim 6, wherein its structure from the N-terminus to the C-terminus is ALB; wherein, A represents the single antigen domain, B represents the Fc region of the immunoglobulin, and L represents the absence of a linker.

8. A polynucleotide, said polynucleotide encoding a single-domain anti-EGFR antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7.

9. An expression vector comprising the polynucleotide of claim 8.

10. A host cell comprising the expression vector of claim 9, or having the polynucleotide of claim 8 integrated into its genome.

11. A method for producing an anti-EGFR antibody or an antigen-binding fragment thereof, comprising the following steps: (a) Culturing the host cells of claim 10 under suitable conditions to obtain a culture containing the anti-EGFR antibody or its antigen-binding fragment; (b) Isolate or recover the anti-EGFR antibody or its antigen-binding fragment from the culture; (c) Optionally, purify and / or modify the anti-EGFR antibody or its antigen-binding fragment obtained in step (b).

12. A bispecific or multispecific antibody comprising an anti-EGFR antibody or an antigen-binding fragment thereof as described in any one of claims 1-7.

13. A pharmaceutical composition comprising (i) an anti-EGFR antibody as claimed in any one of claims 1-7 or an antigen-binding fragment thereof or a bispecific or multispecific antibody as claimed in claim 12; and (ii) a pharmaceutically acceptable carrier.

14. Use of the anti-EGFR antibody or antigen-binding fragment thereof as described in any one of claims 1-7 and the bispecific or multispecific antibody as described in claim 12 in the preparation of a medicament for the prevention or treatment of EGFR-related diseases or conditions.

15. A method for preventing or treating EGFR-related diseases or conditions, comprising administering to a subject in need an anti-EGFR antibody or an antigen-binding fragment thereof as described in any one of claims 1-7 and a bispecific or multispecific antibody as described in claim 12.

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