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

Abstract

Provided are an anti-EGFR antibody or an antigen-binding fragment thereof and a use thereof. The antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3. The light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 1, LCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 2, and LCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 3. Further provided are a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, a vector and transformant for expressing the antibody or the antigen-binding fragment thereof, and a treatment and diagnosis / detection method and use of the antibody or the antigen-binding fragment thereof.

Description

An anti-EGFR antibody or its antigen-binding fragment and its uses

[0001] This application claims priority to Chinese Patent Application No. 2024118722129, filed on December 18, 2024, and Chinese Patent Application No. 2025118817636, filed on December 12, 2025. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This invention relates to the field of monoclonal antibodies, and more specifically to an anti-EGFR antibody or its antigen-binding fragment and its uses. Background Technology

[0003] EGFR (epidermal growth factor receptor) is a transmembrane glycoprotein widely found in various epithelial tissues and solid tumors, such as epidermal carcinoma, lung cancer, breast cancer, and esophageal squamous cell carcinoma. EGFR regulates cell growth, proliferation, and differentiation by activating multiple signal transduction pathways, increasing tumor cell invasiveness, promoting angiogenesis, and inhibiting tumor cell apoptosis. Therefore, inhibiting or reducing EGFR activity has become an important anti-tumor therapeutic strategy.

[0004] Currently, monoclonal antibody drugs targeting EGFR are mainly divided into two categories: EGFR monoclonal antibodies and small molecule tyrosine kinase antagonists. EGFR monoclonal antibodies inhibit the proliferation and spread of tumor cells by binding to EGFR and blocking its signaling pathway. For example, cetuximab and panitumumab are two marketed anti-EGFR monoclonal antibodies that have been shown to have significant efficacy in the treatment of metastatic colorectal cancer and head and neck cancers.

[0005] Furthermore, with technological advancements, new anti-EGFR monoclonal antibodies are constantly being developed. Although some anti-EGFR monoclonal antibodies are already in clinical use, they remain in high demand to enhance antibody specificity and tumor selectivity, thereby improving antibody function. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an anti-EGFR antibody or its antigen-binding fragment and its uses.

[0007] Specifically, the present invention provides an anti-EGFR antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3.

[0008] This invention uses the Kabat definition method to determine the CDR sequence.

[0009] In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

[0010] In some implementations, the heavy chain variable region is selected from the following:

[0011] The HCDR1 contains the amino acid sequence shown in SEQ ID NO:79, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:80, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:82.

[0012] Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:10, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:11, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:8.

[0013] Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:15, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:16, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:17.

[0014] Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:21, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:22, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:23.

[0015] Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:6, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:24, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:23.

[0016] Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:25, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:26, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:23.

[0017] Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:33, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:34, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:35.

[0018] Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:12, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:13, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:14.

[0019] Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:30, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:31, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:32.

[0020] Alternatively, HCDR1 may contain an amino acid sequence as shown in SEQ ID NO:18, HCDR2 may contain an amino acid sequence as shown in SEQ ID NO:19, and HCDR3 may contain an amino acid sequence as shown in SEQ ID NO:20.

[0021] Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:27, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:28, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:29.

[0022] In some specific implementations, the heavy chain variable region is selected from the following:

[0023] The HCDR1 contains the amino acid sequence shown in SEQ ID NO:79, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:80, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:81.

[0024] In some specific embodiments, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or,

[0025] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:9, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0026] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:8, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0027] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0028] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0029] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0030] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0031] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:24, and SEQ ID NO:23, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0032] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:23, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0033] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0034] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0035] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0036] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:8, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0037] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:38, and SEQ ID NO:8, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or...

[0038] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:39, and SEQ ID NO:8, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or

[0039] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:40, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[0040] In some embodiments, LCDR1, LCDR2 and LCDR3 are LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:4.

[0041] In some embodiments, HCDR1, HCDR2 and HCDR3 are respectively HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:41-57.

[0042] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO:41-57.

[0043] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:4.

[0044] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any of SEQ ID NO:41-57.

[0045] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0046] In some embodiments, the amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity does not involve changes to the CDR sequence.

[0047] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0048] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:42, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0049] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0050] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:44, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0051] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:45, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0052] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:46, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0053] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:47, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0054] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:48, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0055] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0056] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0057] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:51, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0058] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:52, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0059] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:53, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0060] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0061] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:55, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0062] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:56, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0063] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:57, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0064] In some embodiments, the antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0065] In some implementations, the antibody or its antigen-binding fragment is a humanized antibody.

[0066] In some embodiments, the antibody or its antigen-binding fragment is selected from one or more of the following:

[0067] (1) Full-length antibody, Fab, Fab', F(ab')2, Fv, scFv or sdAb;

[0068] (2) Monoclonal or polyclonal antibodies;

[0069] (3) Monospecific antibodies, bispecific antibodies or multispecific antibodies.

[0070] In some embodiments, the antibody is a full-length antibody that further includes at least one constant region of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA, IgM, IgD, and IgE, and variants thereof.

[0071] In some embodiments, the antibody is a full-length antibody that also includes a constant region of human IgG1 or a variant thereof.

[0072] In some embodiments, the antibody or its antigen-binding fragment is selected from any of the following:

[0073] (I) The heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO:58-74; and / or, the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5; or

[0074] (II) The heavy chain of the antibody comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:58-74; and / or, the light chain of the antibody comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5.

[0075] In some embodiments, the amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity does not involve changes to the CDR sequence.

[0076] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:58, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0077] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:59, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0078] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:60, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0079] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:61, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0080] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:62, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0081] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:63, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0082] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:64, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0083] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:65, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0084] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:66, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0085] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:67, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0086] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:68, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0087] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:69, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0088] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:70, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0089] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:71, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0090] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:72, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0091] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:73, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0092] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:74, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0093] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence that differs from the amino acid sequence shown in any one of SEQ ID NO:58-74 by 1, 2 or 3 amino acids in the Fc region, and / or the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:5.

[0094] In some specific embodiments, the heavy chain of the antibody comprises an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:58 or 59 by 1, 2 or 3 amino acids in the Fc region, and / or the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0095] In some embodiments, the amino acid differences include L234A and / or L235A (using Kabat numbering).

[0096] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:77 or 78, and / or the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0097] The present invention also provides an isolated nucleic acid that encodes the antibody or an antigen-binding fragment thereof.

[0098] The present invention also provides a recombinant expression vector comprising the aforementioned nucleic acid.

[0099] In some implementations, the recombinant expression vector is selected from viral vectors and non-viral vectors.

[0100] In some specific implementations, the non-viral vector is selected from: plasmids, linear DNA fragments, and RNA;

[0101] In some specific implementations, the recombinant expression vector is a plasmid.

[0102] In some specific implementations, the backbone plasmid of the recombinant expression vector is pCDNA3.1.

[0103] The present invention also provides a transformant comprising the nucleic acid or the recombinant expression vector described above, wherein the transformant is a non-animal or non-plant variety.

[0104] In some implementations, the transformant is a eukaryotic cell.

[0105] In some specific implementations, the eukaryotic cells are mammalian cells.

[0106] The present invention also provides a method for preparing an anti-EGFR antibody or an antigen-binding fragment thereof, the method comprising culturing the transformant and obtaining the antibody or an antigen-binding fragment thereof from the culture.

[0107] The present invention also provides a chimeric antigen receptor comprising the antibody or its antigen-binding fragment.

[0108] In some implementations, the chimeric antigen receptor further includes a co-stimulatory domain and a signal transduction domain.

[0109] The present invention also provides a genetically modified cell that expresses the chimeric antigen receptor.

[0110] In some implementations, the genetically modified cells are T cells or NK cells.

[0111] The present invention also provides an antibody-drug conjugate comprising a cytotoxic agent or tag, and the antibody or its antigen-binding fragment thereof.

[0112] The present invention also provides a pharmaceutical composition comprising the antibody or its antigen-binding fragment, the nucleic acid, the recombinant expression vector, the transformant, the genetically modified cell or the antibody-drug conjugate, and pharmaceutically acceptable carriers and / or excipients.

[0113] The present invention also provides a method for detecting EGFR, which includes the step of contacting the antibody or its antigen-binding fragment with a sample to be tested.

[0114] In some implementations, the detection is for non-diagnostic and / or therapeutic purposes.

[0115] The present invention also provides the use of the antibody or its antigen-binding fragment, the genetically modified cell, the antibody-drug conjugate, or the pharmaceutical composition thereof in the preparation of a medicament for the treatment and / or prevention of EGFR-mediated diseases.

[0116] In some implementations, the disease is cancer.

[0117] In some specific implementations, the cancer is selected from one or more of epidermal carcinoma, lung cancer, breast cancer, and esophageal squamous cell carcinoma.

[0118] The present invention also provides a method for treating and / or preventing EGFR-mediated diseases or conditions, the method comprising administering to a subject in need a therapeutic or preventative amount of the antibody or its antigen-binding fragment, the genetically modified cell, the antibody-drug conjugate, or the pharmaceutical composition.

[0119] In some implementations, the disease is cancer.

[0120] In some specific implementations, the cancer is selected from one or more of epidermal carcinoma, lung cancer, breast cancer, and esophageal squamous cell carcinoma.

[0121] The present invention also provides the antibody or its antigen-binding fragment, the genetically modified cell, the antibody-drug conjugate, or the pharmaceutical composition thereof for the prevention and / or treatment of EGFR-mediated diseases.

[0122] In some implementations, the disease is cancer.

[0123] In some specific implementations, the cancer is selected from one or more of epidermal carcinoma, lung cancer, breast cancer, and esophageal squamous cell carcinoma.

[0124] The present invention also provides a kit comprising the antibody or its antigen-binding fragment, the nucleic acid, the recombinant expression vector, the transformant, the genetically modified cells, the antibody-drug conjugate, or the pharmaceutical composition thereof.

[0125] Terminology Definition

[0126] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0127] To facilitate a better understanding of this invention, certain technical terms are specifically defined as follows. Unless otherwise expressly defined elsewhere in this document, the technical terms or expressions used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0128] For definitions and terminology in this field, those skilled in the art may refer at least in part to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues follow the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids. Singular forms used herein (including the claims) include their corresponding plural forms unless otherwise expressly specified herein.

[0129] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0130] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0131] As used herein, the term “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted inclusively, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, such as “only one” or “exactly one” or when “consisting of” is used in the claims, will it refer to only one number or one element of the list.

[0132] Unless the context clearly indicates otherwise, the words “one” and “a” should be understood as “at least one” as used in this article.

[0133] The term "EGFR" as used herein refers to the epidermal growth factor receptor (or ErbB-1 or HER1) and may refer to the wild-type receptor or a receptor containing one or more mutations. The terms "activity" or "biological activity," or "biological properties" or "biological characteristics," are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize EGFR activity in vivo or in vitro, and IC50. 50 The in vivo stability and immunogenic properties of antibodies. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics are observed, measured, or evaluated using techniques known in the art, including but not limited to ELISA, FACS, or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from various sources (including human, primate, or any other source).

[0134] The term "antibody" refers to any form of antibody that has the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camel-derived single-domain antibodies.

[0135] The term "isolated antibody" refers to the purified state of a binding compound, and in this context, it means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. The term "isolated" does not mean the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic application of the binding compound described herein.

[0136] The term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations, which may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies through any particular method.

[0137] The term "full-length antibody" refers to an immunoglobulin molecule that, in its natural state, contains at least four peptide chains: two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region (abbreviated as CH in this document). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity-determining regions (CDRs) and regions separated by more conserved regions called framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0138] The term "antigen-binding fragment" of an antibody ("parental antibody") includes a fragment or derivative of an antibody, typically comprising at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments well known in the art; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. In some preferred embodiments of the invention, the antigen-binding fragments of the invention are selected from Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. When antigen-binding activity is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative maintains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher of the antigen-binding affinity of the parent antibody. It is also anticipated that the antigen-binding fragment of the antibody may include conserved or non-conserved amino acid substitutions that do not significantly alter its biological activity (referred to as “conserved variants” or “functionally conserved variants” of the antibody). The term “binding compound” refers to both the antibody and its binding fragment.

[0139] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment containing both VH and VL domains, which are located within a single polypeptide chain. Fv polypeptides typically also include a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired structure for antigen binding.

[0140] The term "domain antibody" refers to an immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0141] The term "fully human antibody" refers to an antibody that contains only the sequence of human immunoglobulin proteins. If produced in mice, in mouse cells, or in hybridomas derived from mouse cells, a fully human antibody may contain mouse glycans. Similarly, a "mouse antibody" refers to an antibody that contains only the sequence of mouse immunoglobulins. Alternatively, if produced in rats, in rat cells, or in hybridomas derived from rat cells, a fully human antibody may contain rat glycans. Likewise, a "rat antibody" refers to an antibody that contains only the sequence of rat immunoglobulins.

[0142] The term "Fc region" in this document is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is based on the EU numbering system, also known as the EU index, e.g., Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0143] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes are typically composed of various chemically active surface molecules such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational and non-conformational epitopes lies in the fact that, in the presence of denaturing solvents, the former loses binding, rather than the latter.

[0144] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. In some embodiments of the invention, affinity is measured using surface plasmon resonance (SPR) technology, such as the affinity between the antibody and antigen of the present invention. In some preferred embodiments of the invention, a specific method for measuring affinity is the BIAcore method described herein.

[0145] The terms “nucleic acid,” “polynucleotide,” “nucleic acid molecule,” and “polynucleotide molecule” are used interchangeably herein (unless the context otherwise indicates) and refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0146] "Construction" refers to any recombinant polynucleotide molecule (such as plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecules) derived from any source, capable of integration into the genome or autonomous replication, containing one or more polynucleotide molecules that are functionally linked (i.e., operably linked). In some preferred embodiments of the invention, the recombinant construct comprises the polynucleotides of the invention operably linked to transcription initiation regulatory sequences that drive and / or direct transcription of the polynucleotides of the invention in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to drive and / or direct the expression of the polynucleotides of the invention.

[0147] "Vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into host cells). One type of vector is the "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be ligated. Another type of vector is the viral vector, into which an additional DNA segment can be ligated into the viral genome. Some vectors can replicate autonomously in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). After introduction into the host cell, other vectors (e.g., non-free-living mammalian vectors) integrate into the host cell's genome and thus replicate along with the host genome. Furthermore, some vectors can guide the expression of operatively linked genes. This paper refers to such vectors as "expression vectors."

[0148] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a target gene upon transformation, transfection, or transduction into host cells. An expression vector contains one or more phenotypic selection markers and origins of replication to ensure vector maintenance and to provide amplification within the host when needed. In some preferred embodiments of the invention, the expression vector comprises the constructs of the invention and / or the polynucleotides of the invention.

[0149] As used herein, the term "EGFR-mediated disease" refers to a disease in which EGFR is involved in the onset, progression, or prolongation of the disease, such as involving EGFR activation (e.g., aberrant or hyperactivated activation), and where EGFR involvement directly or indirectly produces at least one of the following effects: disease initiation / occurrence; increased / deeper pathological changes in the disease; expanded site / range of disease impact; aggravated bodily damage caused by the disease; increased pain caused by the disease; insensitivity / resistance to treatment; decreased tendency for spontaneous remission; and worsened prognosis. In some embodiments of the invention, the EGFR-mediated disease is an inflammatory disease; in some specific embodiments of the invention, the EGFR-mediated disease is epidermal cancer, lung cancer, breast cancer, and esophageal squamous cell carcinoma.

[0150] As used herein, the term “treatment” or “cure” for any disease or condition, in one embodiment, means improving the disease or condition (i.e., slowing or halting or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, “treatment” or “cure” means alleviating or improving at least one bodily parameter, including those physical parameters that may not be identifiable by the patient. In yet another embodiment, “treatment” or “cure” means regulating the disease or condition physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of bodily parameters), or in both ways. Unless explicitly described herein, methods for assessing the treatment and / or prevention of disease are generally known in the art.

[0151] "Subjects" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cyno-eating macaque" refers to or is derived from the cyno-eating macaque.

[0152] "Combined" administration of one or more other therapeutic agents includes simultaneous (co-) administration and consecutive administration in any order.

[0153] "Therapeutic effective amount," "therapeutic effective dose," and "effective amount" refer to the amount of the EGFR antibody or its antigen-binding fragment of the present invention, when administered alone or in combination with other therapeutic agents to cells, tissues, or subjects, that effectively prevents or improves the symptoms of one or more diseases or conditions, or the development of such diseases or conditions. Therapeutic effective dose also refers to the amount of antibody or its antigen-binding fragment sufficient to cause symptom improvement, such as the amount that treats, cures, prevents, or improves the associated medical condition, or increases the rate of treatment, cure, prevention, or improvement of such conditions. When administered to an individual as a single active ingredient, the therapeutic effective dose refers only to that ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that cause the therapeutic effect, whether administered in combination, sequentially, or simultaneously. The effective amount of the therapeutic agent will result in an increase of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% in diagnostic criteria or parameters.

[0154] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation or composition that are non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0155] Anti-EGFR antibody

[0156] In one aspect, the present invention provides antibodies that specifically bind to EGFR or antigen-binding fragments thereof. The terms "anti-EGFR antibody," "anti-EGFR," "EGFR antibody," or "EGFR-binding antibody" refer to antibodies capable of binding to the EGFR protein or fragments thereof with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting EGFR.

[0157] In some embodiments, the antibody of the present invention binds to human or cynomolgus monkey EGFR protein. In some embodiments, the antibody of the present invention binds to A431 cells. In some embodiments, the antibody of the present invention blocks the binding of EGF to EGFR on cells. In some embodiments, the antibody of the present invention has strong endocytic capacity.

[0158] The antibodies of this invention can be generated using any suitable method for antibody production. Any suitable form of EGFR can be used as an immunogen (antigen) for antibody production. By way of example and not limitation, any EGFR variant or fragment thereof can be used as an immunogen.

[0159] In some implementations, the generated anti-EGFR antibodies can be produced by screening a phage display library.

[0160] In some embodiments, the amino acid sequences of the light and heavy chain variable regions and CDRs of the anti-EGFR antibody of the present invention are shown in Table 1. The light and heavy chain variable regions of the above antibody are formed by combining the human clone heavy chain constant region Fc and light chain constant region κ with each heavy chain variable region and light chain variable region, respectively, to form complete antibody heavy and light chains.

[0161] The precise amino acid sequence boundary of the variable region CDR of the antibody of the present invention can be determined using any of many well-known schemes, including those based on the three-dimensional structure of the antibody and the topology of the CDR loop, such as 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)), and those based on antibody sequence variability, such as Kabat et al. (Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), and the International ImMunoGeneTics database (IMGT) (1999 Nucleic Acids). Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0162] Unless otherwise stated, the CDR of the antibodies of the present invention can be determined by those skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations).

[0163] It should be noted that the boundaries of the CDRs of the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may differ. Therefore, when referring to antibodies defined with the specific CDR sequence of this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0164] Antibodies with different specificities (i.e., different binding sites against different antigens) have different CDRs. However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, a minimal overlapping region can be determined, 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 portion of the 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 or Chothia may be substituted with conserved amino acid residues.

[0165] In some implementations, anti-EGFR antibodies are obtained by engineering using standard molecular biology techniques.

[0166] The amino acid sequences of the light and heavy chain variable regions and CDRs of the anti-EGFR antibody of this invention are shown in Table 4. The light and heavy chain variable regions of the above antibody are formed by combining the human clone heavy chain constant region Fc and light chain constant region κ with each heavy chain variable region and light chain variable region, respectively, to form complete antibody heavy and light chains.

[0167] In some embodiments, amino acid changes include amino acid deletions, insertions, or substitutions. In some embodiments, the anti-EGFR antibody of the present invention or its antigen-binding fragment comprises antibodies having amino acid sequences that have been mutated by amino acid deletions, insertions, or substitutions, but still possess at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the antibodies described above (particularly in the CDR region depicted in the above sequence). In some embodiments, when the antibody of the present invention is compared with the CDR region depicted in the specific sequence, the number of amino acid mutations in the CDR region that have been mutated by amino acid deletions, insertions, or substitutions does not exceed 1, 2, 3, 4, or 5. In some embodiments, when the antibody of the present invention is compared with the frame region in the specific sequence, the number of amino acid mutations in the frame region that have been mutated by amino acid deletions, insertions, or substitutions does not exceed 1, 2, 3, 4, or 5.

[0168] In some embodiments, the anti-EGFR antibodies of the present invention can mutate the Fc region, including L234A and L235A mutations (using Kabat numbering). In some preferred embodiments, the Fc regions of anti-EGFR antibodies 1 and 14 of the present invention are mutated, including L234A and L235A mutations, to produce antibodies 13 and 19.

[0169] The term "percentage (%) amino acid sequence identity," or simply "identity," is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0170] In some embodiments, the full-length antibody disclosed in this invention includes a light chain variable region and a heavy chain variable region. Those skilled in the art can also select light chain constant regions and heavy chain constant regions from different antibody sources according to actual needs, such as light chain constant regions (or conventional variants thereof) and heavy chain constant regions (or conventional variants thereof) derived from human antibodies. Furthermore, combinations of light chain variable regions and heavy chain variable regions in Table B can form single-chain antibodies (scFv), Fab, or other antigen-binding fragments containing scFv or Fab.

[0171] In some implementations, the EGFR antibody is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody or a modified form thereof, as described in the following sections.

[0172] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to produce an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) with amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0173] In some implementations, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb", in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0174] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0175] antibody expression

[0176] In another aspect, the present invention provides a polynucleotide molecule encoding the anti-EGFR antibody or its antigen-binding fragment described herein. The polynucleotide molecule may comprise a polynucleotide molecule containing an amino acid sequence encoding the light chain variable region and / or the heavy chain variable region of the antibody, or a polynucleotide molecule containing an amino acid sequence encoding the light chain and / or the heavy chain of the antibody.

[0177] In some embodiments, the polynucleotide molecule encoding the antibody of the present invention includes a polynucleotide molecule that has been mutated by nucleotide deletion, insertion or substitution, but still has at least about 60, 70, 80, 90, 95 or 100% identity with the coding region corresponding to the CDR depicted in the sequence described above.

[0178] In another aspect, the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably a eukaryotic expression vector. In some embodiments, the polynucleotide molecule as described herein is contained in one or more expression vectors.

[0179] In another aspect, the present invention provides a host cell comprising a polynucleotide molecule as described herein or an expression vector as described herein, preferably a eukaryotic cell, more preferably a mammalian cell.

[0180] In another aspect, the present invention provides a method for preparing an anti-EGFR antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or the antigen-binding fragment thereof in a host cell as described herein under conditions suitable for expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.

[0181] This invention provides mammalian host cells for expressing the recombinant antibodies of this invention, including a variety of immortalized cell lines available from the American Type Culture Collection (ATCC). These particularly include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells, and many other cell lines. Mammal host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines exhibit high expression levels.

[0182] In one embodiment, the present invention provides a method for preparing anti-EGFR antibodies, wherein the method includes, when an expression vector is introduced into a mammalian host cell, producing antibodies by culturing the host cell for a sufficient period of time to allow the antibody to be expressed in the host cell, or more preferably by secreting the antibody into a culture medium in which the host cell grows.

[0183] Antibodies can be recovered from the culture medium using standard protein purification methods. The antibody molecules prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the antibody molecules of this invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC.

[0184] Antibodies expressed in different cell lines or in transgenic animals may have different glycosylations. However, all antibodies encoded by the nucleic acid molecules provided herein or containing the amino acid sequences provided herein are part of the invention, regardless of their glycosylation. Similarly, in some embodiments, non-fucosylated antibodies are advantageous because they generally have stronger efficacy in vitro and in vivo than their fucosylated counterparts and are unlikely to be immunogenic because their sugar structure is a normal component of native human serum IgG.

[0185] Pharmaceutical compositions and pharmaceutical preparations

[0186] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-EGFR antibody or an antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector or a host cell as described herein, and a pharmaceutically acceptable carrier or excipient. It should be understood that the anti-EGFR antibody or pharmaceutical composition thereof provided by the present invention can integrate suitable carriers, excipients, and other agents in a formulation for co-administration, thereby providing improved transfer, delivery, tolerability, etc.

[0187] The term "pharmaceutical composition" refers to a formulation that allows the biologically effective form of the active ingredient contained therein to be present, and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation.

[0188] Pharmaceutical formulations containing the antiEGFR antibodies described herein can be prepared by mixing the antiEGFR antibody of the present invention, having the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. editor (1980)), preferably in the form of an aqueous solution or a lyophilized formulation.

[0189] The pharmaceutical compositions or formulations of the present invention may further comprise one or more other active ingredients required for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. In some embodiments, the other active ingredients are immune checkpoint inhibitors or growth inhibitors, which are suitably combined in amounts effective for the intended use. In some embodiments, the pharmaceutical compositions of the present invention further comprise a composition encoding a polynucleotide molecule of an anti-EGFR antibody.

[0190] In another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0191] In another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein.

[0192] Medical uses and treatment methods

[0193] Any anti-EGFR antibody or corresponding immunoconjugate provided herein may be used for therapeutic purposes. It should also be understood that, when discussing "antibody," compositions containing antibodies are also included. The anti-EGFR antibody of the present invention may be used in therapeutic or preventative amounts in any embodiment of the therapeutic or preventative method described herein.

[0194] In another aspect, the present invention provides the use of the antibodies or antigen-binding fragments thereof described herein, the pharmaceutical compositions described herein, or the pharmaceutical combinations described herein in the preparation of medicaments for treating and / or preventing EGFR-mediated diseases, preferably cancer or tumors; more preferably, the diseases are selected from epidermal carcinoma, lung cancer, breast cancer, and esophageal squamous cell carcinoma.

[0195] In another aspect, the present invention provides antibodies or antigen-binding fragments thereof described herein, pharmaceutical compositions or combinations thereof described herein, for the treatment and / or prevention of EGFR-mediated diseases, preferably cancer or tumors; more preferably, the diseases are selected from colorectal cancer, non-small cell lung cancer, squamous cell carcinoma, nasopharyngeal carcinoma, glioblastoma, ovarian cancer, pancreatic cancer, breast epidermal cancer, lung cancer, breast cancer, and esophageal squamous cell carcinoma.

[0196] In another aspect, the present invention provides a method for treating and / or preventing EGFR-mediated diseases or conditions, comprising administering to a subject in need a therapeutically or preventively effective amount of an antibody or antigen-binding fragment thereof described herein, a pharmaceutical composition described herein, or a combination of pharmaceuticals described herein, preferably the disease being cancer or a tumor; more preferably, the disease being selected from epidermal cancer, lung cancer, breast cancer, and esophageal squamous cell carcinoma.

[0197] In some embodiments, the administration methods of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intra-articular, intra-articular (e.g., in arthritic joints), inhalation, aerosol delivery, or local administration to the lesion.

[0198] The term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual undergoing treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any adverse feelings or direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. For the antibodies of this invention, the ability to reduce the severity of the disease in one or more ways, i.e., to exert a therapeutic effect, is manifested in one or more of the following: increased patient lifespan (survival); delayed disease progression; and reduced need for medical care.

[0199] The present invention also provides for the combined administration of a therapeutically effective amount of one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to a subject. The antibodies of the present invention can be used alone or in combination with other therapeutic agents in a therapy. In some embodiments, the antibodies of the present invention are co-administered with at least one additional therapeutic agent.

[0200] Methods for diagnosis and detection

[0201] In another aspect, the present invention provides a method for detecting the presence of EGFR in a sample using an antibody or antigen-binding fragment thereof described herein. The term "detection" as used herein includes quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In some embodiments, the biological sample comprises cells or tissue. In some embodiments, the EGFR is human EGFR or cynomolgus monkey EGFR. The method includes the steps of contacting the sample with an antibody or antigen-binding fragment thereof described herein or a detection composition containing the antibody or antigen-binding fragment thereof, and detecting the presence of a conjugate or binding signal generated by the antibody or antigen-binding fragment thereof binding to EGFR. When used for detection purposes, the antibody or antigen-binding fragment thereof described herein may be labeled to indicate whether the conjugate has been formed. In some embodiments, the method may be an in vitro or in vivo method.

[0202] In some implementations, EGFR is detected before treatment, for example, before initiating treatment or before a treatment interval. In one implementation, an anti-EGFR antibody or its antigen-binding fragment is provided for diagnostic or detection methods.

[0203] The EGFR-targeting antibody of the present invention has the following advantages:

[0204] 1. The EGFR-targeting antibodies described in this invention all exhibit significant binding activity to the antigen;

[0205] 2. The EGFR-targeting antibodies described in this invention all exhibit good binding ability to A431 cells;

[0206] 3. The EGFR-targeting antibody described in this invention has better selective binding ability to tumor cells;

[0207] 4. All EGFR-targeting antibodies described in this invention can be internalized and enter A431 cells.

[0208] This invention includes all combinations of the specific embodiments described. Further embodiments of the invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the invention, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. For all purposes, all disclosures, patents, and patent applications cited herein, including in citations, are incorporated herein by reference in their entirety. The compounds of this invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other methods, and equivalent substitutions well known to those skilled in the art, with preferred embodiments including but not limited to the embodiments of this invention. Attached Figure Description

[0209] Figure 1 shows the antibody screening antigen and control antibody activity detection.

[0210] Figures 2-4 show the ELISA method for detecting the binding activity of EGFR monoclonal antibodies against antigen proteins of different species; Figure 2 shows the binding activity of human EGFR monoclonal antibodies against human EGFR antigen proteins; Figure 3 shows the binding activity of human EGFR monoclonal antibodies against monkey EGFR antigen proteins; Figure 4 shows the binding activity of human EGFR monoclonal antibodies against mouse EGFR antigen proteins.

[0211] Figure 5 shows the FACS method for detecting the binding activity of EGFR monoclonal antibody to tumor cells.

[0212] Figures 6 and 7 show the internalization ability of EGFR monoclonal antibodies detected by the Incucyte method; Figure 6 shows the internalization time curve of human EGFR monoclonal antibody in A431 cells; Figure 7 shows the calculation of the area under the internalization curve of human EGFR monoclonal antibody in A431 cells.

[0213] Figure 8 shows the HTRF method for detecting the ability of EGFR monoclonal antibodies to block hEGF activation of EGFR signaling.

[0214] Figure 9 shows the tumor-selective binding ability of the preferred antibody.

[0215] Figure 10 shows the ELISA method for detecting the binding activity of affinity-matured antibodies to human EGFR antigen protein.

[0216] Figure 11 shows the FACS method for detecting the binding activity of affinity maturation antibodies to tumor cells.

[0217] Figures 12-13 show the internalization ability of affinity maturation antibodies as detected by the Incucyte method; Figure 12 shows the internalization ability of affinity maturation antibodies in NCI-H1975 cells; Figure 13 shows the internalization ability of affinity maturation antibodies in KYSE150 cells. Detailed Implementation

[0218] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. The detailed description below shows and describes only exemplary embodiments of this application. As those skilled in the art will recognize, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in this application are merely exemplary and not restrictive.

[0219] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0220] Example 1: Preparation of recombinant protein for detecting anti-EGFR antibody activity

[0221] The EGFR antibody screening antigens GSV0->EGFR-P00533-25-645-chis and GSV0->EGFR-P00533-25-645-Fc were synthesized internally and their activity was determined. The antigen activity assay control antigens Human EGFR his and Human EGFR Fc were purchased from ACRO, catalog numbers EGR-H5222 and EGR-H5252, respectively. The amino acid sequence of the human EGFR extracellular domain is amino acids 25-645 of Uniprot ID: P00533, used for the activity assay of the antibodies in this invention.

[0222] The antigens Mouse-EGFR-his and Rhesus macaque EGFR-His were purchased from ACRO, with catalog numbers EGR-M5224 and EGR-C52H1, respectively.

[0223] Cetuximab was used as the EGFR control antibody in this invention. This invention is a human-mouse chimeric monoclonal antibody obtained by conventional antibody expression and purification methods according to the amino acid sequence of Cetuximab (light chain as shown in SEQ ID NO:83, heavy chain as shown in SEQ ID NO:84) and with reference to US6217866B1.

[0224] The results of EGFR antibody screening of antigen GSV0->EGFR-P00533-25-645-chis and antigen activity detection of control antigen Human EGFR his and EGFR control antibody Cetuximab are shown in Figure 1.

[0225] Example 2: Screening of phage display libraries

[0226] In this embodiment, conventional phage display library technology was used, and the antigen protein prepared in Example 1 was used as the screening antigen to screen the library. Using the following screening method, multiple antibody molecules that specifically bind to human EGFR were obtained.

[0227] 2.1 Screening of antibody gene phage display libraries using magnetic bead method

[0228] Magnetic bead screening involves biotin-labeling antigen proteins and then binding them to magnetic beads conjugated with streptavidin. The process involves incubating, washing, and eluting the antigen-bound magnetic beads and antibody gene phage display libraries. Typically, 3-4 rounds of screening are performed, resulting in a large enrichment of antigen-specific monoclonal antibodies. In this example, biotin-labeled antigen proteins GSV0->EGFR-P00533-25-645-chis and GSV0->EGFR-P00533-25-645-Fc were used for phage display library screening. Three rounds of screening were performed, with each round using a 3-fold gradient of decreasing antigen concentration to obtain anti-EGFR antibody clones with high affinity.

[0229] 2.2 Screening of antibody gene phage display libraries using the immunotube method

[0230] Immunotube screening involves coating the antigen proteins GSV0->EGFR-P00533-25-645-chis and GSV0->EGFR-P00533-25-645-Fc onto the surface of an immunotube with high adsorption capacity. A phage-displaying antibody library is added to the immunotube, and the protein adsorbed on the immunotube surface is incubated, washed, and eluted. After 2-4 rounds of screening, specific monoclonal antibodies against the antigen are finally enriched. In this example, three rounds of screening are performed, with each round using a 3-fold gradient of decreasing antigen concentration to obtain anti-EGFR antibody clones with high affinity.

[0231] Example 3: Monoclonal Antibody Screening

[0232] The enrichment effect was evaluated by ELISA detection of the phage pools eluted in each round. Clones were randomly selected from the phage pools selected in each round for sequence analysis. The enrichment effect and the reproducibility ratio of the measured sequences were combined to select an appropriate round for single clone selection.

[0233] Initial ELISA monoclonal screening used the antigen protein GSV0->EGFR-P00533-25-645-chis. The obtained clones were further verified using ELISA to bind to Mouse-EGFR-his and Rhesus macaque EGFR-His, resulting in 12 specific antibodies. The amino acid sequences of the CDR regions of the obtained antibodies VH and VL are shown in Table 1. The CDR sequences were determined using the Kabat definition method.

[0234] Table 1: CDR and variable region amino acid sequences of anti-EGFR antibodies (KABAT protocol)

[0235] Example 4: Construction and expression of fully human anti-EGFR antibody

[0236] The coding sequences for the Fc region of the heavy chain and the κ region of the light chain of the human clone were introduced into the pCDNA3.1 plasmid. The coding sequences for the variable regions of the heavy and light chains of the aforementioned anti-human EGFR antibodies were synthesized. The coding sequences for the variable regions of the heavy and light chains of various anti-human EGFR antibodies were introduced into the pCDNA3.1 plasmid containing the pre-introduced constant region coding sequences, and the correct clones were confirmed by sequencing. Various chimeric heavy and light chain expression plasmids were mixed and paired for transfection into Expi CHO expression cells to obtain fully human anti-human EGFR antibodies. The Fc region of antibody 1 was mutated with L234A and L235A to obtain antibody 1-FC mutant antibody—antibody 13.

[0237] The full-length light chain sequence of the anti-human EGFR fully human antibody is shown in SEQ ID NO:5, and the full-length heavy chain sequence is shown in Table 2.

[0238] Table 2: Full-length amino acid sequence of the heavy chain of anti-EGFR antibody

[0239] Example 5: Detection of Antigen-Binding Activity of Fully Human Antibody Against Human EGFR

[0240] Dilute the antigens (GSV0->EGFR-P00533-25-645-chis, Rhesus macaque EGFR-His, Mouse-EGFR-his) with 1×PBS to a concentration of 2 μg / mL, and add 30 μL / well to a 96-well ELISA plate. Incubate overnight at 4°C. Wash the plate three times with PBST, add blocking buffer (5% PBSM), and block for 2 h at room temperature. After washing three times with PBST, add 30 μL / well of sample diluted with 1% PBSM and incubate for 60 min at room temperature. Wash the plate three times with PBST, add secondary antibody (Anti-human-IgG-Fc-HRP (purchased from abcam; catalog number: ab97225)), and incubate for 60 min at room temperature. Wash the plate three times with PBST, and add 30 μL of TMB to each well. Stop the reaction by adding 2M stop solution and simultaneously measure OD450.

[0241] The results are shown in Figures 2-4 and Table 3. The anti-human EGFR antibodies of the present invention can bind to EGFR in humans, monkeys and mice.

[0242] Table 3: Antigen-binding activity of anti-EGFR antibodies (ECG) 50 )

[0243] Example 6: Detection of the binding ability of fully human anti-EGFR antibody to A431 cells

[0244] A431 cells (human epidermal cancer cells) were prepared into a cell suspension and the density was adjusted to 1×10⁻⁶. 6 Cells / mL. Take a 96-well round-bottom plate and add 100 μL of cell suspension to each well using a 100 μL pipette. Centrifuge at 300 g / min for 5 min. Discard the supernatant. Dilute the antibody with FACS Buffer to prepare eight concentration gradients: 20.00000, 5.00000, 1.25000, 0.31250, 0.07813, 0.01953, 0.00488, and 0.00122 μg / mL. Add 100 μL of antibody dilution to each well using a 100 μL pipette and incubate at 4°C for 60 min. Wash the plate twice with FACS Buffer. Secondary antibody PE labelled anti-human IgG Fc (purchased from Abcam; catalog number: 98596) was diluted 1:300 with FACS Buffer and added to each well at 100 μL. The plates were incubated at 4°C for 30 min. The plates were washed twice with FACS buffer. Flow cytometry was used for analysis.

[0245] The results are shown in Figure 5 and Table 4. All 12 human EGFR monoclonal antibodies had the ability to bind to A431 cells.

[0246] Table 4: Binding ability of human EGFR monoclonal antibodies to A431 cells

[0247] Example 7: Detection of the internalization ability of fully human anti-EGFR antibody on A431 cells

[0248] Take a clean, sterile 3599 cell culture plate, add 80 μL of cell suspension, and allow the cells to adhere for 4 hours. Mix an equal volume of the test antibody with Fabfluor-pH Dyes (molar ratio: 1:3 of test antibody to labeling Fab), and incubate at room temperature in the dark for 15 minutes. Slowly add 20 μL of antibody-Fabfluor mix below the liquid surface, and gently shake the plate horizontally back and forth and left and right to mix the antibody-Fabfluor mix with the cells. Place the plate in an IncuCyte container. Set the imaging program and take pictures.

[0249] The results are shown in Figures 6 and 7. All 12 human EGFR monoclonal antibodies were internalized into A431 cells.

[0250] Example 8: Detection of the ability of fully human anti-EGFR antibody to block the binding of EGF and EGFR on cells.

[0251] NCI-H292 (human lung adenocarcinoma cells) were cultured routinely at 37°C and 5% CO2. Cells in the logarithmic growth phase were counted after digestion. The cell suspension was adjusted to a suitable density, and 90 μL of cell suspension was added at a density of 50,000 cells / well. The cells were incubated overnight. The next day, 1 μM EGF and 1 μM sample stock solution were prepared. The test antibody was added to the cell culture wells at a final concentration of 10 nM, and the cells were incubated for 30 minutes. Then, 10 nM EGF was added, and the cells were incubated for another 30 minutes. Samples were processed according to the HTRF HUMAN PAN PHOSPHO-EGFR DETECTION KITS: 500 tests (64HR1PEG) manufacturer's instructions. The signals at 665 nm and 620 nm were detected using an EnVision microplate reader, and the signal ratio at the two wavelengths was calculated.

[0252] As shown in Figure 8, all 12 human EGFR monoclonal antibodies were able to block the signal activation caused by EGF to varying degrees, and their ability to block the binding of EGF to EGFR was superior to that of Cetuximab.

[0253] Example 9: Detection of tumor selective binding ability

[0254] HCC1143 cells (human breast cancer cells, triple negative) were digested, centrifuged, and resuspended in PBS. The cell density was adjusted to 1E6 cells / mL. 0.5 μM CFSE was added, and the cells were incubated at room temperature in the dark for 20 min for staining. Alternatively, HCC1143 cells were digested, centrifuged, and resuspended in PBS. The cell density was adjusted to 15E6 cells / mL. 40 μL of MCF10A was added to the CFSE-stained HCC1143 cells, achieving a cell ratio of 1:30 for both HCC1143 and MCF10A. A 100 nM antibody solution was prepared using FACS buffer (PBS + 2% FBS). 10 μL of the prepared solution was added to each well to a final concentration of 10 nM, and the cells were incubated at 4°C for 40 min. After antibody incubation, wash once with 100 μL of PBS; dilute anti-human Fc PE with FACS buffer (PBS + 2% FBS) according to the fluorescent secondary antibody instructions, add 100 μL to each well of a U-plate, and incubate at 4°C in the dark for 30 minutes. Wash once with 100 μL of PBS solution containing 2% FBS, resuspend in 120 μL of PBS solution containing 2% FBS, and then detect the mean fluorescence intensity on HCC1143 and MCF10A cells by flow cytometry. The ratio of the mean fluorescence intensity on HCC1143 and MCF10A cells is used as the ordinate to compare the selective binding ability of different samples on tumor cells and normal cells.

[0255] As shown in Figure 9, antibody 1 exhibited superior tumor-selective binding ability compared to the control antibody molecule Cetuximab.

[0256] Example 10: Construction and Screening of Antibody Affinity Maturation Library

[0257] 10.1 Library Construction

[0258] Mutation primers were designed according to the library construction strategy. Using the correctly sequenced antibody 1 plasmid as a template, single- or double-point mutations were introduced into the target gene using molecular cloning technology, and the target fragment was amplified. The antibody gene was then recombined using in vitro ligation. After obtaining the antibody gene, the vector and gene sequence were digested with enzymes, and the antibody nucleotide sequence was constructed into a phage display vector. The constructed vector was then electroporated into E. coli to obtain the phage display library. The library capacity of the five mutant antibody libraries was calculated using the dilution plating method, and the statistical results are shown in Table 5.

[0259] Table 5: Statistics on antibody 1 affinity maturation library capacity

[0260] 10.2 Document Library Selection

[0261] Phage display mutant libraries or obtained output sets are processed through a series of steps including inoculation, phage infection assistance, phage amplification, phage precipitation and resuspension to prepare enriched phages for the next round of screening. Liquid-phase screening: Biotin-labeled antigens are combined with streptavidin-conjugated magnetic beads, then incubated, washed, and eluted with the prepared phages. After 3-4 rounds of screening, specific monoclonal antibodies against the antigen are enriched. Solid-phase screening: Antigens are coated on the surface of highly absorbent immunotubes, then the prepared phages are added to the immunotubes for incubation, washing, and elution. After 3-4 rounds of screening, specific monoclonal antibodies against the antigen are enriched. Cell-based screening: The negatively screened phages and antigen-overexpressing cells are incubated, washed, and eluted. After 3-4 rounds of screening, specific monoclonal antibodies against the antigen are enriched. The enrichment effect is detected by ELISA on the selected output sets.

[0262] 10.3 Monoclonal Screening

[0263] Based on the detection results of the initial screening output set, suitable library outputs were selected for monoclonal ELISA screening. The process involved a series of steps, including target antigen coating, blocking, primary antibody incubation, secondary antibody addition, color development, termination, and OD value detection. Clones with OD values ​​higher than the parent clone were selected as preferred clones and submitted for testing. Through ELISA screening and sequencing analysis, a total of 5 specific antibodies were screened. The amino acid sequences of the CDR regions of the obtained antibodies VH and VL are shown in Table 6. The CDR sequences were determined using the Kabat definition method.

[0264] Table 6: CDR and variable region amino acid sequences of affinity maturation candidate molecules (KABAT scheme)

[0265] Example 11: Construction and expression of affinity maturation antibodies

[0266] Five affinity maturation candidates were expressed, purified, and subjected to quality testing as described in Example 4. The Fc region of antibody 14 was mutated with L234A and L235A to obtain antibody 14-FC mutant antibody—antibody 19.

[0267] The full-length light chain sequence of the anti-human EGFR fully human antibody is shown in SEQ ID NO:5, and the full-length heavy chain sequence is shown in Table 7.

[0268] Table 7: Full-length amino acid sequence of the heavy chain of affinity-matured anti-EGFR antibodies

[0269] Example 12: Detection of Affinity Mature Antibody-Antigen Binding Activity

[0270] Dilute the antigens (GSV0->EGFR-P00533-25-645-chis, Rhesus macaque EGFR-His, Mouse-EGFR-his) with 1×PBS, serially diluting from 1 μg / mL. Add 30 μL / well to each well of a 96-well ELISA plate and incubate overnight at 4°C. Wash the plate three times with PBST, add blocking buffer (5% PBSM), and block for 2 h at room temperature. After washing three times with PBST, add 30 μL / well of 1% PBSM to dilute to 1 μg / mL sample and incubate for 60 min at room temperature. Wash the plate three times with PBST, add secondary antibody (Anti-human-IgG-Fc-HRP (purchased from: abcam; catalog number: ab97225)), and incubate for 60 min at room temperature. Wash the plate three times with PBST, and add 30 μL of TMB to each well. Stop the reaction by adding 2M stop solution and simultaneously measure OD450.

[0271] The experimental results are shown in Figure 10 and Table 8. All the mature molecules with affinity showed significant binding activity to the antigen, among which antibody 14, antibody 15 and antibody 17 showed better binding activity to the antigen than antibody 1.

[0272] Table 8: Binding activity of affinity-matured antibodies to human EGFR antigen protein

[0273] Example 13: Detection of the binding ability of affinity maturation antibody to A431 cells

[0274] The binding ability of the affinity-matured antibody molecule to A431 was detected according to the method in Example 6.

[0275] The results are shown in Figure 11 and Table 9. The affinity-matured antibody molecules have a significant binding ability to A431 cells.

[0276] Table 9: Binding activity of affinity maturation antibodies to A431 cells

[0277] Example 14: Detection of Affinity Maturation Antibody Internalization Ability

[0278] The internalization ability of affinity-matured antibody molecules in NCI-H1975 (human lung adenocarcinoma cells) and KYSE150 (human esophageal squamous cell carcinoma cells) was detected using the method described in Example 7.

[0279] The results are shown in Figures 12 and 13. All the maturation antibodies with different affinity were able to internalize into NCI-H1975 and KYSE150, and the internalization activity of each maturation antibody in NCI-H1975 and KYSE150 was better than that of the control antibody Cetuximab.

[0280] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments listed herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents.

[0281] Sequence list (all are amino acid sequences; see the preceding sections for sequence definitions / descriptions):

[0282] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An anti-EGFR antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region includes light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3. The LCDR1 contains the amino acid sequence shown in SEQ ID NO:1, the LCDR2 contains the amino acid sequence shown in SEQ ID NO:2, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:

3.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region is selected from the following: The HCDR1 contains the amino acid sequence shown in SEQ ID NO:79, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:80, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:

82. Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:10, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:11, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:

8. Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:15, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:16, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:

17. Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:21, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:22, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:

23. Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:6, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:24, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:

23. Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:25, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:26, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:

23. Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:33, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:34, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:

35. Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:12, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:13, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:

14. Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:30, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:31, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:

32. Alternatively, HCDR1 may contain an amino acid sequence as shown in SEQ ID NO:18, HCDR2 may contain an amino acid sequence as shown in SEQ ID NO:19, and HCDR3 may contain an amino acid sequence as shown in SEQ ID NO:

20. Alternatively, HCDR1 may contain the amino acid sequence shown in SEQ ID NO:27, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:28, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:

29. Preferably, the heavy chain variable region is selected from the following: The HCDR1 contains the amino acid sequence shown in SEQ ID NO:79, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:80, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:

81.

3. The antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:9, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:8, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:24, and SEQ ID NO:23, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:23, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:8, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:38, and SEQ ID NO:8, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:39, and SEQ ID NO:8, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:40, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

4. The antibody or antigen-binding fragment thereof as described in any one of claims 1-3, characterized in that, The antibody or its antigen-binding fragment is selected from the following: (1) The heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO:41-57; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:4; or, (2) The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:41-57; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4; preferably, the amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity does not involve changes to the CDR sequence.

5. The antibody or antigen-binding fragment thereof as described in any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

6. The antibody or antigen-binding fragment thereof as described in any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is selected from one or more of the following: (1) Full-length antibody, Fab, Fab', F(ab')2, Fv, scFv or sdAb; (2) Monoclonal or polyclonal antibodies; (3) Monospecific antibodies, bispecific antibodies, or multispecific antibodies; Preferably, the antibody or its antigen-binding fragment is a full-length antibody, and preferably includes at least one constant region selected from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA, IgM, IgD and IgE and their variants.

7. The antibody or antigen-binding fragment thereof as described in any one of claims 1-6, characterized in that, The antibody or its antigen-binding fragment is selected from any of the following: (I) The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NO:58-74; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:5; or, (II) The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:58-74; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5; Preferably, the amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity does not involve changes to the CDR sequence.

8. The antibody or its antigen-binding fragment as described in claim 7, characterized in that, The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence that differs from the amino acid sequence shown in any one of SEQ ID NO:58-74 by 1, 2 or 3 amino acids in the Fc region, and / or the light chain of the antibody comprises an amino acid sequence shown in SEQ ID NO:5; for example, the amino acid difference includes L234A and / or L235A. Preferably, the heavy chain of the antibody comprises an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:58 or 59 by 1, 2 or 3 amino acids in the Fc region, and / or the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5; for example, the amino acid difference includes L234A and / or L235A. More preferably, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:77 or 78, and / or the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:

5.

9. An isolated nucleic acid, characterized in that, The nucleic acid encodes an antibody or an antigen-binding fragment thereof as described in any one of claims 1-8.

10. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 9; Preferably, the recombinant expression vector is selected from: viral vectors and non-viral vectors; More preferably, the non-viral vector is selected from: plasmids, linear DNA fragments, and RNA; More preferably, the recombinant expression vector is a plasmid, and the backbone plasmid of the recombinant expression vector is preferably pCDNA3.

1.

11. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 9 or the recombinant expression vector as described in claim 10, and the transformant is a non-animal or non-plant variety; preferably, the transformant is a eukaryotic cell, more preferably, the eukaryotic cell is a mammalian cell.

12. A method for preparing an anti-EGFR antibody or an antigen-binding fragment thereof, the method comprising culturing a transformant as described in claim 11 and obtaining the antibody or an antigen-binding fragment thereof from the culture.

13. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an antibody or an antigen-binding fragment thereof as described in any one of claims 1-8; Preferably, the chimeric antigen receptor further includes a co-stimulatory domain and a signal transduction domain.

14. A genetically modified cell, characterized in that, The cell expresses the chimeric antigen receptor as described in claim 13; preferably, the gene-modified cell is a T cell or an NK cell.

15. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate comprises a cytotoxic agent or tag, and an antibody or antigen-binding fragment thereof as described in any one of claims 1-8.

16. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an antibody or antigen-binding fragment thereof as described in any one of claims 1-8, a nucleic acid as described in claim 9, a recombinant expression vector as described in claim 10, a transformant as described in claim 11, a genetically modified cell as described in claim 14, or an antibody-drug conjugate as described in claim 15, and a pharmaceutically acceptable carrier and / or excipients.

17. A method for detecting EGFR, characterized in that, It includes the step of contacting the sample to be tested with an antibody or an antigen-binding fragment thereof as described in any one of claims 1-8; preferably, the detection is for non-diagnostic and / or therapeutic purposes.

18. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-8, the genetically modified cell as described in claim 14, the antibody-drug conjugate as described in claim 15, or the pharmaceutical composition as described in claim 16 in the preparation of a medicament for the treatment and / or prevention of EGFR-mediated diseases; Preferably, the disease is cancer; More preferably, the cancer is selected from one or more of epidermal cancer, lung cancer, breast cancer, and esophageal squamous cell carcinoma.

19. A method for treating and / or preventing EGFR-mediated diseases or conditions, the method comprising administering to a subject in need a therapeutically or preventively effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-8, a genetically modified cell as described in claim 14, an antibody-drug conjugate as described in claim 15, or a pharmaceutical composition as described in claim 16; Preferably, the disease is cancer; More preferably, the cancer is selected from one or more of epidermal cancer, lung cancer, breast cancer, and esophageal squamous cell carcinoma.

20. A reagent kit, characterized in that, The kit comprises an antibody or antigen-binding fragment thereof as described in any one of claims 1-8, a nucleic acid as described in claim 9, a recombinant expression vector as described in claim 10, a transformant as described in claim 11, a gene-modified cell as described in claim 14, an antibody-drug conjugate as described in claim 15, or a pharmaceutical composition as described in claim 16.

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