WO2021160138A1

WO2021160138A1 - Antigen binding protein of anti-epidermal growth factor receptor and application thereof

Info

Publication number: WO2021160138A1
Application number: PCT/CN2021/076393
Authority: WO
Inventors: 孙非; 张喜田; 梁重阳
Original assignee: 张喜田; 孙非; 张欣
Priority date: 2020-02-10
Filing date: 2021-02-09
Publication date: 2021-08-19
Also published as: CN111018990A

Abstract

Provided is an antigen binding protein capable of specifically binding to an epidermal growth factor receptor (EGFR) on a cell membrane, said protein can bind to an amino acid epitope of domain II of EGFR so as to internalize a complex. Further provided are a method for treating tumors by using the antigen binding protein and a use thereof in the preparation of a drug.

Description

Anti-epidermal growth factor receptor antigen binding protein and its application

Technical field

This application relates to the field of biomedicine, in particular to an anti-epidermal growth factor receptor antigen binding protein. In addition, this application also relates to the preparation method and use of the antibody.

Background technique

Under the control of abnormally active growth factor receptors, cells are prone to lose normal proliferation, thereby turning into cancer cells and causing cancer. This loss of control may be caused by many factors, such as the overexpression of growth factors or the overexpression of growth factor receptors, and the spontaneous activation of biochemical pathways regulated by growth factors. Growth factor receptors involved in carcinogenesis include epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR), platelet-derived growth factor receptor (PDGFR), insulin-like growth factor receptor (IGFG), nerve growth factor receptor ( NGFR) and fibroblast growth factor receptor (FGF). The high expression of EGFR may promote tumor cell proliferation, angiogenesis, adhesion, invasion and metastasis, inhibit tumor cell apoptosis, and lead to low survival rate of tumor patients, poor prognosis, poor curative effect, high possibility of tumor metastasis, and easy to cause tumor cells Resistance to various cytotoxic drugs.

In view of the proliferation and differentiation of cancer cells related to the high expression of EGFR, the currently marketed EGFR inhibitors are mainly small molecules and antibody drugs. They target the EGF binding site of the extracellular part of EGFR or the active site of tyrosine kinase in the intracellular part of EGFR. However, there are problems of strong toxic and side effects and easy development of drug resistance. Therefore, there is an urgent need to develop highly efficient and novel EGFR antibodies.

Summary of the invention

The present application provides an isolated antigen binding protein, which can specifically bind to the epidermal growth factor receptor (EGFR) on the cell membrane to internalize the complex and enter the cell.

In one aspect, the present application provides an isolated antigen binding protein, which can specifically bind to the epidermal growth factor receptor (EGFR) on the cell membrane to internalize the complex.

In some embodiments, the isolated antigen binding protein can specifically bind to the II domain of EGFR, and the II domain of EGFR comprises the amino acid sequence shown in SEQ ID NO: 13.

In some embodiments, the isolated antigen binding protein can specifically bind to at least one amino acid selected from the group consisting of Ser196, Ser222, Lys269 and Ser282 on the II domain of EGFR.

In some embodiments, the isolated antigen binding protein can competitively bind to the EGFR with the LZ-8 protein.

In some embodiments, the isolated antigen binding protein can compete with a reference antibody for binding to the EGFR, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, and the reference The VH of the antibody includes HCDR1, HCDR2, and HCDR3, the VL of the reference antibody includes LCDR1, LCDR2, and LCDR3, and the HCDR1 of the reference antibody includes SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43 The amino acid sequence shown in any one of the HCDR2 of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30 and SEQ ID NO: 44, the reference antibody The HCDR3 of the reference antibody includes the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 31 and SEQ ID NO: 45, and the LCDR1 of the reference antibody includes SEQ ID NO: 18, SEQ ID NO: 32 and The amino acid sequence shown in any one of SEQ ID NO: 46, the LCDR2 of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33 and SEQ ID NO: 47, The LCDR3 of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 20, SEQ ID NO: 34 and SEQ ID NO: 48.

In some embodiments, the isolated antigen binding protein includes an antibody or antigen binding fragment thereof.

In some embodiments, the antigen-binding fragment includes Fab, Fab', F(ab) ₂ , Fv fragment, F(ab') ₂ , scFv, di-scFv and/or dAb.

In some embodiments, the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. For example, the antibody may be a monoclonal antibody.

In some embodiments, the isolated antigen binding protein comprises at least one CDR in a heavy chain variable region VH, and the VH comprises any of SEQ ID NO: 68, SEQ ID NO: 72 and SEQ ID NO: 76 The amino acid sequence shown in one item.

In some embodiments, the isolated antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 31, and SEQ ID NO: 45.

In some embodiments, the isolated antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30, and SEQ ID NO: 44.

In some embodiments, the isolated antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43.

In some embodiments, the isolated antigen binding protein includes HCDR1, HCDR2, and HCDR3, and the HCDR1 includes any one of SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43. The amino acid sequence, the HCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30 and SEQ ID NO: 44, and the HCDR3 includes SEQ ID NO: 17, SEQ ID NO: 31 and The amino acid sequence shown in any one of SEQ ID NO: 45.

In some embodiments, the isolated antigen binding protein comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 are selected from any of the following amino acid sequences:

(1) HCDR1: SEQ ID NO: 15, HCDR2: SEQ ID NO: 16 and HCDR3: SEQ ID NO: 17;

(2) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 30 and HCDR3: SEQ ID NO: 31;

with,

(3) HCDR1: SEQ ID NO: 43, HCDR2: SEQ ID NO: 44 and HCDR3: SEQ ID NO: 45.

In certain embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH, wherein the VH includes a framework region H-FR1, and the C-terminus of the H-FR1 is directly or directly from the N-terminus of the HCDR1. Are connected indirectly, and the H-FR1 includes the amino acid sequence shown in any one of SEQ ID NO: 21, SEQ ID NO: 35 and SEQ ID NO: 49.

In certain embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH, wherein the VH includes a framework region H-FR2, and the H-FR2 is located between the HCDR1 and the HCDR2, and The H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 22, SEQ ID NO: 36 and SEQ ID NO: 50.

In certain embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH, wherein the VH includes a framework region H-FR3, and the H-FR3 is located between the HCDR2 and the HCDR3, and The H-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 23, SEQ ID NO: 37 and SEQ ID NO: 51.

In certain embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH, wherein the VH includes a framework region H-FR4, and the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, And the H-FR4 includes the amino acid sequence shown in any one of SEQ ID NO: 24, SEQ ID NO: 38, and SEQ ID NO: 52.

In certain embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein the H-FR1 comprises SEQ ID NO: 21, SEQ ID NO: 35 and The amino acid sequence shown in any one of SEQ ID NO: 49, and the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 22, SEQ ID NO: 36 and SEQ ID NO: 50, the H-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 23, SEQ ID NO: 37 and SEQ ID NO: 51, and the H-FR4 includes SEQ ID NO: 24, SEQ ID NO: 38 and SEQ ID NO: the amino acid sequence shown in any one of 52.

In some embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, and the H-FR1, H-FR2, H-FR3, and H-FR4 are selected from From any of the following amino acid sequences:

(1) F-FR1: SEQ ID NO: 21, H-FR2: SEQ ID NO: 22, H-FR3: SEQ ID NO: 23 and H-FR4: SEQ ID NO: 24;

(2) H-FR1: SEQ ID NO: 35, H-FR2: SEQ ID NO: 36, H-FR3: SEQ ID NO: 37 and H-FR4: SEQ ID NO: 38; and,

(3) H-FR1: SEQ ID NO: 49, H-FR2: SEQ ID NO: 50, H-FR3: SEQ ID NO: 51 and H-FR4: SEQ ID NO: 52.

In some embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises any of SEQ ID NO: 68, SEQ ID NO: 72 and SEQ ID NO: 76 The amino acid sequence shown in one item.

In certain embodiments, the isolated antigen binding protein includes a heavy chain constant region, and the heavy chain constant region is derived from IgG1.

In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 57 or SEQ ID NO: 58.

In some embodiments, the isolated antigen binding protein comprises an antibody heavy chain, and the antibody heavy chain comprises the one shown in any one of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64 Amino acid sequence.

In some embodiments, the isolated antigen binding protein comprises at least one CDR in the light chain variable region VL, and the VL comprises any of SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77 The amino acid sequence shown in one item.

In some embodiments, the isolated antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 20, SEQ ID NO: 34, and SEQ ID NO: 48.

In some embodiments, the isolated antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33, and SEQ ID NO: 47.

In some embodiments, the isolated antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 18, SEQ ID NO: 32, and SEQ ID NO: 46.

In some embodiments, the isolated antigen binding protein includes LCDR1, LCDR2, and LCDR3, and the LCDR1 includes SEQ ID NO: 18, SEQ ID NO: 32, and SEQ ID NO: 46. An amino acid sequence, the LCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33 and SEQ ID NO: 47, and the LCDR3 includes SEQ ID NO: 20, SEQ ID NO: 34 and The amino acid sequence shown in any one of SEQ ID NO: 48.

In some embodiments, the isolated antigen binding protein comprises LCDR1, LCDR2, and LCDR3, and the LCDR1, LCDR2, and LCDR3 are selected from any of the following amino acid sequences:

(1) LCDR1: SEQ ID NO: 18, LCDR2: SEQ ID NO: 19 and LCDR3: SEQ ID NO: 20;

(2) LCDR1: SEQ ID NO: 32, LCDR2: SEQ ID NO: 33 and LCDR3: SEQ ID NO: 34;

with,

(3) LCDR1: SEQ ID NO: 46, LCDR2: SEQ ID NO: 47 and LCDR3: SEQ ID NO: 48.

In certain embodiments, the isolated antigen binding protein comprises a light chain variable region VL, wherein the VL includes a framework region L-FR1, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 are directly or Are connected indirectly, and the L-FR1 includes the amino acid sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 39 and SEQ ID NO: 53.

In certain embodiments, the isolated antigen binding protein comprises a light chain variable region VL, wherein the VL comprises a framework region L-FR2, and the L-FR2 is located between the LCDR1 and the LCDR2, and The L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 26, SEQ ID NO: 40, and SEQ ID NO: 54.

In certain embodiments, the isolated antigen binding protein comprises a light chain variable region VL, wherein the VL comprises a framework region L-FR3, and the L-FR3 is located between the LCDR2 and the LCDR3, and The L-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 27, SEQ ID NO: 41 and SEQ ID NO: 55.

In certain embodiments, the isolated antigen binding protein comprises a light chain variable region VL, wherein the VL comprises a framework region L-FR4, and the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, And the L-FR4 includes the amino acid sequence shown in any one of SEQ ID NO: 28, SEQ ID NO: 42 and SEQ ID NO: 56.

In certain embodiments, the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3, and L-FR4, wherein the L-FR1 comprises SEQ ID NO: 25, SEQ ID NO: 39 and The amino acid sequence shown in any one of SEQ ID NO: 53, the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 26, SEQ ID NO: 40 and SEQ ID NO: 54, L-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 27, SEQ ID NO: 41 and SEQ ID NO: 55, and the L-FR4 includes SEQ ID NO: 28, SEQ ID NO: 42 and SEQ The amino acid sequence shown in any one of ID NO:56.

In some embodiments, the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3, and L-FR4, and the L-FR1, L-FR2, L-FR3, and L-FR4 are selected from From any of the following amino acid sequences:

(1) F-FR1: SEQ ID NO: 39, L-FR2: SEQ ID NO: 40, L-FR3: SEQ ID NO: 41 and L-FR4: SEQ ID NO: 42;

(2) L-FR1: SEQ ID NO: 53, L-FR2: SEQ ID NO: 54, L-FR3: SEQ ID NO: 55 and L-FR4: SEQ ID NO: 56; and,

(3) L-FR1: SEQ ID NO: 25, L-FR2: SEQ ID NO: 26, L-FR3: SEQ ID NO: 27 and L-FR4: SEQ ID NO: 28.

In some embodiments, the isolated antigen binding protein comprises a light chain variable region VL, and the light chain variable region VL comprises SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77 The amino acid sequence shown in any item.

In some embodiments, the isolated antigen binding protein includes a light chain constant region, and the light chain constant region includes the amino acid sequence shown in SEQ ID NO:59.

In some embodiments, the isolated antigen-binding protein comprises an antibody light chain, and the antibody light chain comprises any one of SEQ ID NO: 61, SEQ ID NO: 63, and SEQ ID NO: 65 Amino acid sequence.

In one aspect, the application provides a polypeptide comprising the isolated antigen binding protein.

In one aspect, the application provides an immunoconjugate, which comprises the isolated antigen binding protein or the polypeptide, and a small molecule compound.

In some embodiments, the small molecule compound contains a cytotoxic agent and/or a marker.

In some embodiments, the small molecule compound is coupled to the primary amine group of the upper amino acid residue of the isolated antigen binding protein.

In certain embodiments, the immunoconjugate is capable of internalizing cells.

In one aspect, the application provides isolated one or more nucleic acid molecules, which encode the isolated antigen binding protein.

In some embodiments, the nucleic acid molecule includes SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO : 67, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, and SEQ ID NO: 75.

In one aspect, the application provides a vector, which comprises the nucleic acid molecule.

In one aspect, the application provides a cell, which contains the nucleic acid molecule or is based on the vector.

In one aspect, the present application provides a method for preparing the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the isolated antigen binding protein is expressed.

In one aspect, the application provides a pharmaceutical composition comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the carrier and/or the cell, and optionally地 pharmaceutically acceptable carrier.

In one aspect, the application provides that the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition are used in the preparation of a medicine The medicine is used to prevent, alleviate and/or treat diseases related to EGFR activity.

In some embodiments, the disease associated with EGFR activity includes tumors.

In some embodiments, the tumor includes a tumor associated with abnormal expression of EGFR. In some embodiments, the tumors related to abnormal expression of EGFR include tumors related to overexpression of EGFR.

In certain embodiments, the tumor comprises a solid tumor.

In certain embodiments, the tumor includes glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer, and/or gastric cancer.

In one aspect, the present application provides a method of preventing, alleviating or treating diseases related to EGFR activity, the method comprising administering the isolated antigen binding protein, the polypeptide, the immunoconjugate to a subject in need Substance, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.

In some embodiments, the disease associated with EGFR activity includes tumors.

In certain embodiments, the tumor comprises a solid tumor.

In one aspect, the application provides the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition for preventing , Relieve or treat diseases related to EGFR activity.

In some embodiments, the disease associated with EGFR activity includes tumors.

In certain embodiments, the tumor comprises a solid tumor.

In one aspect, the present application provides a method for detecting the presence and/or content of EGFR, which comprises administering the isolated antigen binding protein or the polypeptide.

In one aspect, the application provides a kit comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the drug combination Things.

In one aspect, the application provides a method for internalizing a cell, which comprises administering the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the vector, the cell And/or the pharmaceutical composition.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in this application. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary, and not restrictive.

Description of the drawings

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

Figure 1 shows the protein electrophoresis and western blotting of the cell supernatant 5 days after transfection (lane 1: reducing conditions, lane 2: non-reducing conditions, lane P: human IgG1 as a positive control).

Figure 2 shows the results of an isothermal calorimetric titration experiment (ITC) for the extracellular domain of EGFR and LZ8.

Figure 3 shows the separation of the EGFR extracellular domain and LZ8 complex using SDS-PAGE.

Figure 4 shows the determination of the interaction interface between EGFR and LZ8 using chemical cross-linking coupled mass spectrometry, and the amino acid residues involved in the interaction are represented by spheres.

Figure 5 shows the optimal docking configuration of the extracellular domain of EGFR and LZ8. The key amino acid residues are labeled D20, K41 and S222, K269, S282, and S196 respectively.

Figure 6 shows the structure of Alexa Fluor 568 NHS Ester.

Figure 7 shows the purification of the 1Z4-AF568 conjugate.

Figure 8 shows the internalization of the 1Z4-AF568 conjugate. The ellipse on the left is the nucleus, and the vesicle structure formed by the conjugate is shown on the right.

Figure 9 shows the flow cytometry analysis of the binding of 1Z4 to EGFR positive and negative cells. The cells corresponding to 92.8 and 99.2 are MD-MB-453, and the cells corresponding to 9660 and 17420 are MD-MB-468.

Figure 10 shows the comparative analysis of the internalization intensity of the antigen binding protein 1Z4 described in this application, cetuximab and mAb806, and the oval shape is the nucleus.

Figure 11 shows the result of the interaction between the antigen binding protein 1Z4 described in this application and the NIH3T3 EGFR wild-type cell line, and the oval shape is the nucleus.

Figure 12 shows the result of the interaction of the antigen binding protein 1Z4 described in this application with the NIH3T3 EGFR mutant cell line, and the oval shape is the nucleus.

Figure 13 shows the result of the internalization of the antigen binding protein 1Z4 antibody described in the present application into the cell, and the oval shape is the nucleus.

Figure 14 shows the result of the internalization of the antigen binding protein 6F2 antibody described in the present application into the cell, and the elliptical shape is the nucleus.

Figure 15 shows the result of the internalization of the antigen binding protein 4B1 antibody described in the present application into the cell, and the oval shape is the nucleus.

Detailed ways

The following specific examples illustrate the implementation of the invention of the present application, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "epidermal growth factor receptor (EGFR)" can also be referred to as "HER-1" or "Erb-B1", which belongs to the ErbB (ErbB 1-4) family. EGFR is composed of three parts: the ligand binding domain outside the cell, the hydrophobic transmembrane domain and the kinase domain inside the cell. The intracellular structure of EGFR contains a tyrosine kinase domain (tyrosine kinase domain) and a carboxy-terminal tail with multiple autophosphorylation sites, belonging to the receptor tyrosine kinase family (receptor tyrosine kinase, RTKs); EGFR The extracellular region is composed of ligand binding sites and two cysteine-rich regions. The extracellular domain of EGFR can be further divided into four domains: I, II (amino acids 165-310), III, and IV. EGFR can bind to a variety of ligands with agonistic functions, mainly epidermal growth factor, EGF), transforming growth factor (transforming growth factor a, TGFa), EGFR ligands also include AREG, epigen (EPGN), BTC (betacellulin), eregregulin (EREG) and/or HBEGF (heparin-binding EGF). EGFR regulates multiple cellular processes through signal transduction pathways mediated by tyrosine kinases, including but not limited to signal transduction pathways that activate and control cell proliferation, differentiation, cell survival, apoptosis, angiogenesis, mitogenesis, and metastasis .

The term "EGFR" covers any natural EGFR or modified EGFR derived from any vertebrate, including fragments thereof and related polypeptides. Related polypeptides include, but are not limited to, allelic variants, splice variants, derivative variants, substitution variants, Deletion variants and/or insertion variants (including the addition of N-terminal methionine), fusion polypeptides and interspecies homologues, any of the vertebrate origins including mammals, such as primates (e.g., humans or monkeys) and rodents Class (e.g., mouse or rat). The term encompasses "full-length", unprocessed EGFR, any form of EGFR produced by processing in cells, as well as its functional fragments and functional variants (e.g., variable RNA transcripts, truncated, polymorphic Sex, etc.). EGFR can exist as a transmembrane protein or as a soluble protein. The term also encompasses naturally occurring variants of EGFR, such as splice variants or allelic variants. There are four splice variants of EGFR. The EGFR sequence is known in the art. Exemplary amino acid sequences of human EGFR proteins can be found under UniProt accession numbers P00533-1, P00533-2, P00533-3 and/or P00533-4.

In this application, the term "EGFR II domain" generally refers to the extracellular domain II of EGFR. The II domain of EGFR is usually rich in cystine, and its amino acid sequence usually includes the 165th to 310th amino acids of EGFR. In the present application, the amino acid sequence of the II domain of EGFR may include amino acids 196 to 287 of EGFR. In the present application, the amino acid sequence of the II domain of EGFR may include the amino acid sequence shown in SEQ ID NO: 13.

In this application, the terms "LZ-8" and "LZ8" are also called Ling Zhi-8 or Ganoderma lucidum protein 8, and generally refer to an immunomodulatory protein derived from Ganoderma lucidum. LZ-8 also includes its allelic variants, splice variants, derivative variants, substitution variants, deletion variants and/or insertion variants (including the addition of N-terminal methionine), fusion polypeptides and interspecies homologs . In this application, the LZ-8 may refer to an immunomodulatory protein derived from Ganoderma lucidum. In this application, the LZ-8 may include the amino acid sequence shown in SEQ ID NO: 14.

In this application, the term "competitive binding" generally means that the first molecule binds to the epitope in a manner sufficiently similar to the binding of the second molecule, so that compared to the binding of the first molecule in the absence of the second molecule, In the presence of the second molecule, the result of the binding of the first molecule to its homologous epitope is detectably reduced. For example, the antigen-binding protein of the present application binds to EGFR in a binding manner sufficiently similar to that of LZ-8. Compared with the antigen-binding protein of the present application in which LZ-8 does not exist, in the presence of LZ-8, the antigen-binding protein of the present application The result of the binding of the binding protein to EGFR is detectably reduced; or, compared with the LZ-8 in which the antigen binding protein of the present application does not exist, in the presence of the antigen binding protein of the present application, the binding of LZ-8 to EGFR is The result is a detectable reduction. The degree to which the first molecule can interfere with the binding of the second molecule to the target can be determined using a competitive binding test, for example, a FACS test, an ELISA or a BIACORE test. Generally speaking, when the second molecule is present in excess, it will inhibit the specific binding of the first molecule to the common epitope by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. %. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

In this application, the term "antigen-binding protein" generally refers to a protein that includes an antigen-binding portion, and optionally a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen. The antigen binding protein may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both, and functional fragments thereof. The variable regions of the heavy and light chains contain binding domains that interact with antigens. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments, immunoconjugates, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they show The required antigen binding activity can be obtained.

In this application, the term "antibody" generally refers to an immunoglobulin that is reactive to a specified protein or peptide or fragment thereof. Antibodies can be antibodies from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). The antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody may also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibodies of the application can be derived from any species.

In this application, the term "antigen-binding fragment" generally refers to a certain part of an antibody molecule that contains amino acid residues that interact with an antigen and confer specificity and affinity for the antibody to the antigen. Examples of antigen-binding fragments may include, but are not limited to, Fab, Fab', F(ab) ₂ , Fv fragment, F(ab') ₂ , scFv, di-scFv and/or dAb. In this application, the term "Fab" generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. ; The term "Fab'" usually refers to a fragment that is different from Fab by adding a small number of residues (including one or more cysteine from the hinge region of an antibody) to the carboxyl end of the CH1 domain of the heavy chain; the term "F(ab ') ₂ "generally refers to Fab' dimer antibody fragments comprising two Fab fragments by a disulfide bridge at the hinge region. The term "Fv" generally refers to the smallest antibody fragment that contains a complete antigen recognition and binding site. In some cases, the fragment may consist of a dimer in which a heavy chain variable region and a light chain variable region are tightly non-covalently bound; the term "dsFv" usually refers to a disulfide bond-stabilized Fv fragment, The bond between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. The term "dAb fragment" generally refers to an antibody fragment composed of a VH domain. In this application, the term "scFv" generally refers to a monovalent molecule formed by covalently connecting and pairing a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have general Structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH.

In this application, the term "variable region" or "variable domain" generally refers to the domain of an antibody heavy chain or light chain involved in the binding of an antibody to an antigen. In this application, the term "variable" generally refers to that certain parts of the sequence of the variable domain of an antibody change strongly, forming the binding and specificity of various specific antibodies to their specific antigens. The variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in the three segments of the light chain variable region and the heavy chain variable region, called the complementarity determining region (CDR) or hypervariable region (HVR), which are LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3. The more highly conserved parts of variable domains are called framework regions (FR). The variable domains of the natural heavy chain and light chain each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) Most of them adopt β-sheet configuration and are connected by three CDR structure loop regions. The CDRs in each chain are closely brought together by the FR region, and together with the CDRs from the other chain form the antigen binding site of the antibody.

In the art, a variety of methods can be used to encode the variable region of an antibody or divide the CDR of an antibody, such as the Kabat numbering scheme and definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Bethesda, Maryland (1991)), Chothia numbering scheme and definition rules based on the location of structural loop regions (see, A1-Lazikani et al., JMol Biol 273:927-48, 1997 ), efranc et al.'s IMGT numbering plan and definition rules based on the germline V gene amino acid sequence alignment, as well as Honneger's numbering plan (AHo's), Martin numbering plan, Gelfand numbering plan, etc., see Mathieu Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition, Front.Immunol., 16 October 2018.

In this application, the term "monoclonal antibody" generally refers to antibodies obtained from a population of substantially homogeneous antibodies, that is, the antibodies constituting the population are the same, except for possible naturally occurring mutations and/or Post-translational modification (eg isomerization, amidation) outside. Monoclonal antibodies are highly specific and are directed against a single antigenic site.

In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Generally, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody"), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is compared with the parental (e.g., mouse-derived) antibody in a human individual The possibility of triggering an adverse immune response is reduced.

In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulin. In the CDR regions, the addition, deletion, insertion, substitution or modification of amino acids may also be allowed, as long as they still retain the ability of the antibody to bind to a specific antigen. The humanized antibody may optionally comprise at least a portion of the constant region of a human immunoglobulin. "Humanized antibodies" retain antigen specificity similar to the original antibodies. "Humanized" forms of non-human (e.g., murine) antibodies may contain minimally chimeric antibodies derived from non-human immunoglobulin sequences. In some cases, the CDR region residues in human immunoglobulin (acceptor antibody) can be equipped with non-human species (donor antibody) (such as mouse, rat) with the desired properties, affinity, and/or ability. , Rabbit or non-human primate) residues in the CDR region. In some cases, the FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications that are not in the recipient antibody or in the donor antibody.

In this application, the term "fully human antibody" generally refers to an antibody whose all parts (including the variable and constant regions of the antibody) are encoded by genes of human origin. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology, RNA-polypeptide technology, and the like.

In this application, the terms "binding", "specific binding" or "specific to" generally refer to a measurable and reproducible interaction, such as the binding between an antigen and an antibody, which can determine the presence of a molecule The presence of targets in the context of heterogeneous populations (including biological molecules). For example, an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, affinity, easier and/or longer duration than it binds to other targets. When an antibody binds to an epitope through its antigen binding domain more easily than it will bind to a random, unrelated epitope, the antibody is said to "specifically bind" to that antigen.

In this application, the terms "polypeptide" or "protein" are used interchangeably and generally refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term can also include modified amino acid polymers, for example, by adding sugar residues to form glycoproteins or being phosphorylated. Polypeptides and proteins can be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and can contain molecules with the amino acid sequence of a natural protein, or deletions or additions of one or more amino acids of the natural sequence. And/or substituted molecules. The terms "polypeptide" and "protein" especially include the deletion, addition and/or substitution sequence of one or more amino acids of the antigen binding protein described in the present application.

In this application, the term "isolated" generally refers to a biological material (such as a virus, nucleic acid, or protein) that is substantially free of components that normally accompany or interact with it in the environment in which it naturally occurs. The isolated biological material optionally contains additional materials that the biological material has not been found to possess in its natural environment (e.g., nucleic acid or protein). In this application, when referring to a protein, "isolation" generally means that the molecule is separated and separated from the entire organism in which the molecule is found to occur naturally, or that there are substantially no other biological macromolecules of the same type. When a nucleic acid molecule is involved, it is completely or partially separated from the sequence with which it is naturally bound, or the nucleic acid has a heterologous sequence bound to it, or the calculation is separated from the chromosome.

In this application, the term "immunoconjugate" generally refers to a substance formed by linking an antigen binding protein with other active agents. Other active agents can be small molecule active agents, such as chemotherapeutics, toxins, immunotherapeutics, imaging probes Or spectroscopic probe.

In this application, the term "nucleic acid" molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogs of any length isolated from their natural environment or artificially synthesized.

In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation. The carrier also includes a carrier having a variety of the above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.

In this application, the term "cell" generally refers to individual cells, cell lines or cell cultures that can contain or already contain the nucleic acid molecule described in this application, or can express the antigen binding protein described in this application. Things. The cell may include the progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells and the original parent cells may not necessarily be identical in morphology or genome, but they can express the antibodies or antigen-binding fragments described in this application. The cells can be obtained by transfecting cells in vitro using the vectors described in this application. The cell may be a prokaryotic cell (such as Escherichia coli), or a eukaryotic cell (such as yeast cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or Myeloma cells). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell.

In the present application, the term "pharmaceutical composition" generally refers to a preparation that exists in a form that allows the biological activity of the active ingredient to be effective, and does not contain unacceptable toxicity to the subject to which the composition will be administered. Additional ingredients.

In this application, the term "treatment" generally refers to the desire to change the natural course of the individual to be treated, and may be a clinical intervention to achieve prevention and treatment or in the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis. In some cases, antibodies (e.g., anti-PD-1 antibodies) can be used to delay or slow disease progression.

In this application, the term "administering" generally refers to the subject (eg, patient) administering a certain dose of a compound (eg, an anticancer therapeutic agent) or a pharmaceutical composition (eg, a pharmaceutical composition containing an anticancer therapeutic agent) Methods. Administration can be carried out by any suitable means, including parenteral, intrapulmonary and intranasal, and (if local treatment is required) intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

In this application, the term "tumor" generally refers to all neoplastic cell growth and proliferation (whether malignant or benign) as well as all precancerous and cancerous cells and tissues. In the present application, the tumor may be a tumor in which the EGFR of cells and tissues is abnormally expressed. In the present application, the tumor may be a tumor overexpressing EGFR in cells and tissues. Tumors may include solid tumors and/or non-solid tumors (e.g., hematoma, lymphoma).

Diseases related to EGFR activity include tumor cell proliferation, pathological new blood vessel formation (which promotes the growth of solid tumors), new blood vessel formation in the eye (diabetic retinopathy, age-induced macular degeneration, etc.) and inflammation (psoriasis, Rheumatoid arthritis, etc.). Clinical studies have shown that many types of tumors such as glioma, breast cancer, lung cancer, ovarian cancer, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer, gastric cancer, etc. have high levels of EGFR Express. "Abnormal expression of EGFR" generally refers to a measurable higher or lower level of EGFR on the surface of a certain cell compared with normal cells of the same tissue type. "EGFR overexpression" generally refers to a measurable higher level of EGFR on the surface of a certain cell compared to normal cells of the same tissue type. This abnormal expression (e.g., overexpression) can be caused by gene amplification or increased transcription or translation. In this application, abnormal expression of EGFR (for example, overexpression) may be caused by mutations in EGFR, abnormal expression (for example, overexpression) of EGFR ligands (for example, EGF and TGFα) leads to excessive activation of the EGFR signal transduction system, or EGFR itself Abnormal expression (for example, overexpression). Abnormal expression (e.g., overexpression) can refer to abnormal expression (e.g., overexpression) at the protein and nucleic acid levels (due to increased transcription, post-transcriptional processing, translation, post-translational processing, altered stability and altered protein degradation), As well as local abnormal expression (for example, increased nuclear localization) and enhanced functional activity caused by changes in protein transport mode, for example, such as increased enzymatic hydrolysis of substrates. EGFR expression (or, overexpression) can be determined in diagnostic or prognostic assays by evaluating the level of EGFR present on the cell surface or in cell lysates by techniques known in the art: for example, immunohistochemical assays, immunofluorescence assays , Immunoenzyme assay, ELISA, flow cytometry, radioimmunoassay, Western blot, ligand binding and/or kinase activity, etc. In addition, whether overexpression can be assessed by measuring the level of nucleic acid molecules encoding EGFR in the cells, for example, by fluorescence in situ hybridization, Southern blotting, or PCR technology. The level of EGFR in normal cells is compared with the level of cells affected by cell proliferation disorders (such as tumors) to determine whether EGFR is abnormally expressed. Abnormal expression may be about 50%, 60%, 70%, 80%, 90% or more difference in EGFR expression level from normal cells or comparison cells. Overexpression can be about 50%, 60%, 70%, 80%, 90% or more of EGFR expression levels higher than normal cells or comparative cells.

In this application, the term "internalization" generally refers to the process by which extracellular substances (for example, proteins, nucleic acids, or small molecules) pass through cell membranes (for example, plasma membrane, endosomal membrane, and endoplasmic reticulum membrane). The internalization includes energy-dependent (ie, active) transport mechanisms (such as phagocytosis, endocytosis, pinocytosis, ligand internalization, and antibody internalization) and energy-independent (ie, passive) transport mechanisms (such as diffusion). In this application, internalization may refer to receptor-mediated endocytosis, in which proteins or polypeptides are absorbed by cells by binding to receptors on the surface of cell membranes.

In this application, the term "between" usually means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and the N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment. Indirect connection. In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly connected to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly connected to the N-terminus of the LCDR2. For another example, the N-terminus of the L-FR3 is directly or indirectly connected to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly connected to the N-terminus of the LCDR3. In the heavy chain, for example, the N-terminus of the H-FR2 is directly or indirectly connected to the C-terminus of the HCDR1, and the C-terminus of the H-FR2 is directly or indirectly connected to the N-terminus of the HCDR2. For another example, the N-terminus of the H-FR3 is directly or indirectly connected to the C-terminus of the HCDR2, and the C-terminus of the H-FR3 is directly or indirectly connected to the N-terminus of the HCDR3. In this application, the "first amino acid fragment" and the "second amino acid fragment" can be any amino acid fragment that is the same or different.

In this application, the term "including" usually means including, inclusive, containing or encompassing. In some cases, it also means "to" and "consisting of".

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

Detailed description of the invention

Antigen binding protein

In one aspect, the present application provides an isolated antigen binding protein, which can specifically bind to the epidermal growth factor receptor (EGFR) on the cell membrane to internalize the cell.

In the present application, the isolated antigen binding protein can specifically bind to the II domain of EGFR, and the II domain of EGFR may include the amino acid sequence shown in SEQ ID NO: 13.

In the present application, the isolated antigen binding protein can specifically bind to at least one amino acid selected from the group consisting of Ser196, Ser222, Lys269 and Ser282 on the II domain of EGFR.

In the present application, the isolated antigen binding protein can specifically bind to at least one amino acid on the II domain of EGFR, and the amino acid is selected from the group consisting of Ser196, Ser222, Lys269 and Ser282. In the present application, the isolated antigen binding protein can specifically bind to at least two amino acids on the II domain of EGFR, and the amino acids are selected from the group consisting of Ser196, Ser222, Lys269 and Ser282. In this application, the isolated antigen binding protein can specifically bind to three amino acids on the II domain of EGFR, and the amino acids are selected from the group consisting of Ser196, Ser222, Lys269 and Ser282. In this application, the isolated antigen binding protein can specifically bind to at least four amino acids on the II domain of EGFR, and the amino acids are selected from the group consisting of Ser196, Ser222, Lys269 and Ser282.

In the present application, the isolated antigen binding protein can competitively bind to the EGFR with the LZ-8 protein. The LZ-8 protein may include the amino acid sequence shown in SEQ ID NO: 14.

In this application, the isolated antigen binding protein can compete with a reference antibody for binding to the EGFR, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, and the reference antibody VH includes HCDR1, HCDR2, and HCDR3, the VL of the reference antibody includes LCDR1, LCDR2, and LCDR3, and the HCDR1 of the reference antibody includes any of SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43 The amino acid sequence shown in one item, the HCDR2 of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30 and SEQ ID NO: 44, and the HCDR3 of the reference antibody Contains the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 31 and SEQ ID NO: 45, and the LCDR1 of the reference antibody includes SEQ ID NO: 18, SEQ ID NO: 32 and SEQ ID The amino acid sequence shown in any one of NO: 46, the LCDR2 of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33 and SEQ ID NO: 47, the The LCDR3 of the reference antibody includes the amino acid sequence shown in any one of SEQ ID NO: 20, SEQ ID NO: 34, and SEQ ID NO: 48.

In this application, the reference antibody may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the amino acid sequence of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 is selected from any of the following groups: (1 ) HCDR1: SEQ ID NO: 15, HCDR2: SEQ ID NO: 16, HCDR3: SEQ ID NO: 17, LCDR1: SEQ ID NO: 18, LCDR2: SEQ ID NO: 19 and LCDR3: SEQ ID NO: 20; ( 2) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 30, HCDR3: SEQ ID NO: 31, LCDR1: SEQ ID NO: 32, LCDR2: SEQ ID NO: 33 and LCDR3: SEQ ID NO: 34; And, (3) HCDR1: SEQ ID NO: 43, HCDR2: SEQ ID NO: 44, HCDR3: SEQ ID NO: 45, SEQ ID NO: 46, LCDR2: SEQ ID NO: 47 and LCDR3: SEQ ID NO: 48 .

In the present application, the isolated antigen binding protein may include CDR3 in the heavy chain variable region VH, and the VH may include any one of SEQ ID NO: 68, SEQ ID NO: 72, and SEQ ID NO: 76 Amino acid sequence shown

In the present application, the isolated antigen binding protein may include HCDR3, and the HCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 31, and SEQ ID NO: 45.

In this application, the isolated antigen binding protein may include CDR2 in the heavy chain variable region VH, and the VH may include any one of SEQ ID NO: 68, SEQ ID NO: 72, and SEQ ID NO: 76 Amino acid sequence shown

In the present application, the isolated antigen binding protein may include HCDR2, and the HCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30, and SEQ ID NO: 44.

In the present application, the isolated antigen binding protein may include CDR1 in the heavy chain variable region VH, and the VH may include any one of SEQ ID NO: 68, SEQ ID NO: 72, and SEQ ID NO: 76 Amino acid sequence shown

In the present application, the isolated antigen binding protein may include HCDR1, and the HCDR1 may include the amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43.

In the present application, the isolated antigen binding protein may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include any one of SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43. An amino acid sequence, the HCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30 and SEQ ID NO: 44, and the HCDR3 may include SEQ ID NO: 17, SEQ ID NO: The amino acid sequence shown in any one of 31 and SEQ ID NO: 45.

In this application, the isolated antigen binding protein may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 15, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 16. Amino acid sequence, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 17.

In this application, the isolated antigen binding protein may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 29, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 30. Amino acid sequence, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 31.

In this application, the isolated antigen binding protein may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 43, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 44 Amino acid sequence, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:45.

In the present application, the isolated binding protein may include the framework region H-FR1, and the H-FR1 may include any one of SEQ ID NO: 21, SEQ ID NO: 35, and SEQ ID NO: 49 The amino acid sequence.

In the present application, the isolated binding protein may include the framework region H-FR2, and the H-FR2 may include any one of SEQ ID NO: 22, SEQ ID NO: 36, and SEQ ID NO: 50. The amino acid sequence.

In this application, the isolated binding protein may include the framework region H-FR3, and the H-FR3 may include any one of SEQ ID NO: 23, SEQ ID NO: 37, and SEQ ID NO: 51. The amino acid sequence.

In this application, the isolated binding protein may include the framework region H-FR4, and the H-FR4 may include any one of SEQ ID NO: 24, SEQ ID NO: 38, and SEQ ID NO: 52. The amino acid sequence.

In this application, the isolated antigen binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4, and the H-FR1 may include SEQ ID NO: 21, SEQ ID NO: 35 and The amino acid sequence shown in any one of SEQ ID NO: 49, the H-FR2 may include the amino acid sequence shown in any one of SEQ ID NO: 22, SEQ ID NO: 36, and SEQ ID NO: 50, so The H-FR3 may include the amino acid sequence shown in any one of SEQ ID NO: 23, SEQ ID NO: 37 and SEQ ID NO: 51, and the H-FR4 may include SEQ ID NO: 24, SEQ ID NO: The amino acid sequence shown in any one of 38 and SEQ ID NO: 52.

In this application, the isolated antigen binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 21, so The H-FR2 may include the amino acid sequence shown in SEQ ID NO: 22, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 23, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 24 Amino acid sequence.

In this application, the isolated antigen binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 35, so The H-FR2 may include the amino acid sequence shown in SEQ ID NO: 36, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 37, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 38 Amino acid sequence.

In the present application, the isolated antigen binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 49, so The H-FR2 may include the amino acid sequence shown in SEQ ID NO: 50, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 51, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 52 Amino acid sequence.

In the present application, the isolated antigen binding protein may include a heavy chain variable region VH, and the VH may include any one of SEQ ID NO: 68, SEQ ID NO: 72, and SEQ ID NO: 76 The amino acid sequence.

For example, the isolated antigen binding protein may include the heavy chain variable region VH, and the VH may include the amino acid sequence shown in SEQ ID NO: 68.

For example, the isolated antigen binding protein may include a heavy chain variable region VH, and the VH may include the amino acid sequence shown in SEQ ID NO: 72.

For example, the isolated antigen binding protein may include the heavy chain variable region VH, and the VH may include the amino acid sequence shown in SEQ ID NO: 76.

In this application, the isolated antigen binding protein may include a heavy chain constant region, and the heavy chain constant may be derived from IgG1.

In the present application, the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 57 or SEQ ID NO: 58.

In the present application, the isolated antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include any one of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64. Amino acid sequence.

In the present application, the isolated antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include any one of SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 11. Amino acid sequence.

In the present application, the isolated antigen binding protein may include CDR3 in the light chain variable region VL, and the VL may include any one of SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77 Amino acid sequence shown

In the present application, the isolated antigen binding protein may include LCDR3, and the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 20, SEQ ID NO: 34, and SEQ ID NO: 48.

In the present application, the isolated antigen binding protein may include CDR2 in the light chain variable region VL, and the VL may include any one of SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77 Amino acid sequence shown

In the present application, the isolated antigen binding protein may include LCDR2, and the LCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33, and SEQ ID NO: 47.

In the present application, the isolated antigen binding protein may include CDR1 in the light chain variable region VL, and the VL may include any one of SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77 Amino acid sequence shown

In the present application, the isolated antigen binding protein may include LCDR1, and the LCDR1 may include the amino acid sequence shown in any one of SEQ ID NO: 18, SEQ ID NO: 32, and SEQ ID NO: 46.

In the present application, the isolated antigen binding protein may include LCDR1, LCDR2, and LCDR3, and the LCDR1 includes the amino acid shown in any one of SEQ ID NO: 18, SEQ ID NO: 32, and SEQ ID NO: 46 The LCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33 and SEQ ID NO: 47, and the LCDR3 includes SEQ ID NO: 20, SEQ ID NO: 34 and SEQ ID NO: the amino acid sequence shown in any one of 48.

In the present application, the isolated antigen binding protein may include LCDR1, LCDR2, and LCDR3, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 18, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 19. Amino acid sequence, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO:20.

In this application, the isolated antigen binding protein may include LCDR1, LCDR2, and LCDR3, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 32, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 33 Amino acid sequence, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 34.

In the present application, the isolated antigen binding protein may include LCDR1, LCDR2, and LCDR3, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 46, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 47 Amino acid sequence, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 48.

In the present application, the isolated binding protein may include the framework region L-FR1, and the L-FR1 may include any one of SEQ ID NO: 25, SEQ ID NO: 39, and SEQ ID NO: 53 The amino acid sequence.

In the present application, the isolated binding protein may include the framework region L-FR2, and the L-FR2 may include any one of SEQ ID NO: 26, SEQ ID NO: 40, and SEQ ID NO: 54 The amino acid sequence.

In the present application, the isolated binding protein may include the framework region L-FR3, and the L-FR3 may include any one of SEQ ID NO: 27, SEQ ID NO: 41, and SEQ ID NO: 55 The amino acid sequence.

In this application, the isolated binding protein may include the framework region L-FR4, and the L-FR4 may include any one of SEQ ID NO: 28, SEQ ID NO: 42 and SEQ ID NO: 56 The amino acid sequence.

In this application, the isolated antigen binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4, and the L-FR1 includes SEQ ID NO: 25, SEQ ID NO: 39 And the amino acid sequence shown in any one of SEQ ID NO: 53, the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 26, SEQ ID NO: 40 and SEQ ID NO: 54, so The L-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 27, SEQ ID NO: 41, and SEQ ID NO: 55, and the L-FR4 includes SEQ ID NO: 28, SEQ ID NO: 42 and The amino acid sequence shown in any one of SEQ ID NO: 56.

In the present application, the isolated antigen binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4, and the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 39, so The L-FR2 may include the amino acid sequence shown in SEQ ID NO: 40, the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 41, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 42 Amino acid sequence.

In this application, the isolated antigen binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4, and the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 53, so The L-FR2 may include the amino acid sequence shown in SEQ ID NO: 54, the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 55, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 56 Amino acid sequence.

In the present application, the isolated antigen binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4, and the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 25, so The L-FR2 may include the amino acid sequence shown in SEQ ID NO: 26, the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 27, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 28. Amino acid sequence.

In the present application, the isolated antigen binding protein may include the light chain variable region VL, and the VL may include any one of SEQ ID NO: 69, SEQ ID NO: 73, and SEQ ID NO: 77 The amino acid sequence.

For example, the isolated antigen binding protein may include the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 69.

For example, the isolated antigen binding protein may include the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 73.

For example, the isolated antigen binding protein may include the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 77.

In the present application, the isolated antigen binding protein may include a light chain constant region, and the light chain constant region may include the amino acid sequence shown in SEQ ID NO:59.

In the present application, the isolated antigen binding protein may include an antibody light chain, and the antibody light chain may include any one of SEQ ID NO: 61, SEQ ID NO: 63, and SEQ ID NO: 65. Amino acid sequence.

In the present application, the isolated antigen binding protein may include an antibody light chain, and the antibody light chain may include any one of SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 12. Amino acid sequence.

In the present application, the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1 may include SEQ ID NO: 15, SEQ ID NO: 29 and SEQ ID NO: 43. The amino acid sequence shown in any item, the HCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30 and SEQ ID NO: 44, and the HCDR3 may include SEQ ID NO: 17. The amino acid sequence shown in any one of SEQ ID NO: 31 and SEQ ID NO: 45, the LCDR1 may include any one of SEQ ID NO: 18, SEQ ID NO: 32 and SEQ ID NO: 46 The LCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33 and SEQ ID NO: 47, and the LCDR3 may include SEQ ID NO: 20, SEQ ID The amino acid sequence shown in any one of NO:34 and SEQ ID NO:48.

In the present application, the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 15, and the HCDR2 may include SEQ ID The amino acid sequence shown in NO: 16, the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 17, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 18, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 19, the LCDR3 may include the amino acid sequence shown in SEQ ID NO:20.

In the present application, the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 29, and the HCDR2 may include SEQ ID The amino acid sequence shown in NO: 30, the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 31, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 32, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 33, the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 34.

In the present application, the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 43, and the HCDR2 may include SEQ ID The amino acid sequence shown in NO: 44, the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 45, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 46, and the LCDR2 may include SEQ ID NO: 47. The LCDR3 may include the amino acid sequence shown in SEQ ID NO: 48.

In this application, the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, and the VH may include SEQ ID NO: 68, SEQ ID NO: 72, and SEQ ID NO: 76 The amino acid sequence shown in any one of the VL may include the amino acid sequence shown in any one of SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77.

In the present application, the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 68, and the VL may include SEQ ID NO: 69 amino acid sequence.

In the present application, the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 72, and the VL may include SEQ ID NO: The amino acid sequence shown in 73.

In the present application, the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 76, and the VL may include SEQ ID NO: The amino acid sequence shown in 77.

In the present application, the isolated antigen binding protein may include a heavy chain and a light chain, and the heavy chain may include any one of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64. An amino acid sequence, the light chain may include the amino acid sequence shown in any one of SEQ ID NO: 61, SEQ ID NO: 63, and SEQ ID NO: 65.

In this application, the isolated antigen binding protein may include a heavy chain and a light chain, the heavy chain may include the amino acid sequence shown in SEQ ID NO: 60, and the light chain may include the amino acid sequence shown in SEQ ID NO: 61 The amino acid sequence.

In the present application, the isolated antigen binding protein may include a heavy chain and a light chain, the heavy chain may include the amino acid sequence shown in SEQ ID NO: 62, and the light chain may include the amino acid sequence shown in SEQ ID NO: 63 The amino acid sequence.

In the present application, the isolated antigen binding protein may include a heavy chain and a light chain, the heavy chain may include the amino acid sequence shown in SEQ ID NO: 64, and the light chain may include the amino acid sequence shown in SEQ ID NO: 65 The amino acid sequence.

In the present application, the isolated antigen binding protein may include a heavy chain and a light chain, and the heavy chain may include any one of SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 11. An amino acid sequence, the light chain may include the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 12.

In the present application, the isolated antigen binding protein may include a heavy chain and a light chain, the heavy chain may include the amino acid sequence shown in SEQ ID NO: 3, and the light chain may include the amino acid sequence shown in SEQ ID NO: 4 The amino acid sequence.

In this application, the isolated antigen binding protein may include a heavy chain and a light chain, the heavy chain may include the amino acid sequence shown in SEQ ID NO: 7, and the light chain may include the amino acid sequence shown in SEQ ID NO: 8 The amino acid sequence.

In this application, the isolated antigen binding protein may include a heavy chain and a light chain, the heavy chain may include the amino acid sequence shown in SEQ ID NO: 11, and the light chain may include the amino acid sequence shown in SEQ ID NO: 12 The amino acid sequence.

Nucleic acid, vector, host cell and preparation method

In another aspect, the present application also provides isolated one or more nucleic acid molecules, which can encode the antigen binding protein described in the present application. For example, each nucleic acid molecule in the one or more nucleic acid molecules may encode the entire antigen binding protein, or may encode part of it (for example, HCDR1-3, LCDR1-3, VL, VH, light chain Or one or more of the heavy chain).

The nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction enzyme digestion and gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

In another aspect, this application provides one or more vectors, which comprise one or more nucleic acid molecules described in this application. Each vector may contain one or more of the nucleic acid molecules. In addition, the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions. In addition, the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host. For example, the vector is an expression vector.

In another aspect, the application provides a host cell, which may comprise one or more nucleic acid molecules described in this application and/or one or more vectors described in this application. In certain embodiments, each or each host cell may contain one or one of the nucleic acid molecules or vectors described in this application. In certain embodiments, each or each host cell may contain multiple (e.g., 2 or more) or multiple (e.g., 2 or more) nucleic acid molecules or vectors described in the present application

In another aspect, the application provides a method for preparing the antigen-binding fragment. The method may include culturing the host cell described in the present application under conditions such that the antibody or antigen-binding fragment thereof is expressed. For example, it is possible to use an appropriate medium, an appropriate temperature, a culture time, etc., and these methods are understood by those of ordinary skill in the art.

Pharmaceutical composition, method, use

On the other hand, the present application provides an immunoconjugate, which may include the isolated antigen binding protein or the polypeptide, and a small molecule compound. Immunoconjugates usually refer to the use of specific linkers to connect antibodies and small molecule cytotoxic drugs. Its main components can include antibodies, linkers and small molecule cytotoxic drugs. In this application, the small molecule compound may contain a cytotoxic agent and/or a marker. In the present application, the small molecule compound can be coupled to the primary amine group of the upper amino acid residue of the isolated antigen binding protein. In this application, the immunoconjugate is capable of internalizing cells.

On the other hand, this application provides a kit, which may contain the antigen binding protein, chimeric antigen receptor, genetically modified cell, immunoconjugate, and/or the drug described in this application combination. It can include the antigen binding protein, chimeric antigen receptor, genetically modified cell, and/or immunoconjugate described in the present application in a single common container, and can optionally be combined with one or more therapeutic agents , Optionally formulated together in a pharmaceutical composition.

On the other hand, this application provides a drug delivery device, which can be used to administer the antigen binding protein described in this application or a pharmaceutical composition thereof.

In another aspect, the application provides a pharmaceutical composition, which may comprise the antigen binding protein, the polypeptide, the nucleic acid molecule, the vector, the host cell, and any Select a pharmaceutically acceptable carrier. The pharmaceutically acceptable adjuvant is non-toxic to the recipient at the dose and concentration used. The pharmaceutical composition in this application may also contain more than one active compound, usually those that do not adversely affect each other. Those active compounds with complementary activities.

The antigen binding protein, the polypeptide, the nucleic acid molecule, the carrier, the host cell, the immunoconjugate and/or the pharmaceutical composition can be used for the treatment of EGFR activity related disease. For example, it can be used to treat tumors or inhibit tumor growth. For example, the pharmaceutical composition of the present application can inhibit or delay the development or progression of the disease, can reduce the tumor size (or even substantially eliminate the tumor), and/or can reduce and/or stabilize the disease state.

The pharmaceutical composition described in the present application may comprise a preventive and/or therapeutically effective amount of the antigen binding protein, the polypeptide, the nucleic acid molecule, the carrier, the host cell, and the immunoconjugate And/or the pharmaceutical composition. The prophylactic and/or therapeutically effective amount is a dose required to prevent and/or treat (at least partially treat) a disease or disorder and/or any complications thereof in a subject suffering from or at risk of development.

In another aspect, the present application provides the antigen binding protein, the polypeptide, the nucleic acid molecule, the carrier, the host cell, the immunoconjugate and/or the pharmaceutical composition in Use in the preparation of medicines.

The drug is used to treat diseases related to EGFR activity. In certain embodiments, the tumor includes a tumor in which EGFR is abnormally expressed (e.g., overexpressed). In some embodiments, the disease associated with EGFR activity includes tumors. In the present application, the tumor may include tumors related to abnormal expression (for example, overexpression) of EGFR. In the present application, the tumor may include solid tumors and/or non-solid tumors. For example, the tumor is a solid tumor. In this application, the tumor may include glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer and/or gastric cancer.

In one aspect, the application provides a method for internalizing a cell, which comprises administering the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the vector, the cell And/or the pharmaceutical composition. For example, the method can be an ex vivo or in vitro method. For example, the method may be a method for non-therapeutic purposes.

In another aspect, the present application provides a method for detecting the presence and/or content of EGFR protein, which comprises administering the isolated antigen binding protein and/or the polypeptide. For example, the method can be an ex vivo or in vitro method. For example, the method may be a method for non-therapeutic purposes.

The application also provides the use of an antigen binding protein in a method for diagnosing a subject suffering from a tumor or cancer, the method comprising: contacting a sample with the antigen binding protein of the application and detecting the presence of bound antibody The presence or expression level of EGFR in a sample obtained from a subject.

On the other hand, this application also provides the following implementation solutions:

1. A mouse-derived anti-epidermal growth factor monoclonal antibody, characterized in that the antibody can bind to EGFR on the cell membrane and cause the antigen-antibody complex to vigorously internalize into tumor cells.

2. The monoclonal antibody described in embodiment 1 can specifically bind to the II domain (amino acid Ser196-Cys287) of EGFR, causing the internalization of the antigen-antibody complex.

3. The monoclonal antibody according to embodiment 2, which can at least specifically bind to the following amino acids on the II domain of EGFR: Ser196, Ser222, Lys269, Ser282.

4. The monoclonal antibody according to embodiment 2, which comprises a heavy chain variable region and a light chain variable region, characterized in that the heavy chain variable region has SEQ ID NO: 3, SEQ ID NO: 7, The amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12.

5. The heavy chain variable region according to embodiment 4, characterized in that the heavy chain variable region contains the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9 .

6. The light chain variable region according to embodiment 4, characterized in that the light chain variable region contains the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 .

7. The monoclonal antibody described in embodiment 2, which can competitively bind to EGFR with LZ-8.

8. The monoclonal antibody according to embodiment 2 can be prepared from hybridoma cells obtained by fusion of mouse spleen cells immunized with human EGFR protein and myeloma cells.

9. The hybridoma cell according to embodiment 8, characterized in that it can stably secrete the monoclonal antibody according to embodiment 2.

10. The hybridoma cells according to embodiment 9, which can be obtained by screening for competitive binding with EGFR with LZ-8.

11. The method of screening hybridoma cells according to embodiment 10, including but not limited to competitive ELISA screening by LZ-8.

12. The monoclonal antibody as described in embodiment 2, which can be conjugated with a small molecule compound to form an antibody-small molecule compound conjugate.

13. The antibody-small molecule compound conjugate according to embodiment 12, the coupling method includes but is not limited to the primary amine group of the amino acid residue on the antibody.

14. The antibody-small molecule compound conjugate according to embodiment 12 can be internalized into tumor cells.

Not to be limited by any theory, the following embodiments are only used to illustrate the various technical solutions of the invention of the present application, and are not used to limit the scope of the invention of the present application.

Example

Example 1 Preparation and screening of antigen binding protein

The preparation materials are shown in Table 1.

Table 1 Experimental materials

抗原名称Antigen name	提供方provider	数量quantity	目的Purpose
人EGFR蛋白Human EGFR protein	GenScirptGenScirpt	3mg，(>85％purity,>0.5mg/ml)3mg, (>85％purity,>0.5mg/ml)	免疫/筛选Immunization/screening
LZ8LZ8	本实验室This laboratory	1mg，(>85％purity,>0.5mg/ml)1mg, (>85％purity,>0.5mg/ml)	筛选(竞争)Screening (competition)

Balb/C and C57BL/6 mice were immunized with human EGFR protein. The specific method is as follows:

1. Animal immunization: EGFR antigen is emulsified with complete Freund's adjuvant, and mice are immunized by subcutaneous or intraperitoneal injection. Five groups of animals (Balb/C mice) will be immunized as shown in Table 2. After immunization, tail blood was collected and the serum titer was determined by ELISA method. The mouse with the highest antibody titer was selected for cell fusion.

Table 2 Immune progress table

流程Process	时间time	免疫途径Immune pathway	免疫剂量Immunization dose
免疫前采血Blood collection before immunization	T＝-4天T = -4 days	To	To
初疫First epidemic	T＝0天T = 0 days	s.c.注射s.c. injection	每只动物50μgA蛋白50μg protein A per animal
第一次加免First time exemption	T＝14天sT = 14 days s	i.p.注射i.p. injection	每只动物25μgA蛋白25μg protein A per animal
测试血清1Test serum 1	T＝21天sT = 21 days s	To	To
第二次加免Second time exemption	T＝28天sT = 28 days s	s.c.注射s.c. injection	每只动物25μgA蛋白25μg protein A per animal
测试血清2Test serum 2	T＝35天sT = 35 days s	To	To
终免Exempt	T＝56±7天T=56±7 days	i.p.注射i.p. injection	每只动物25μgA蛋白25μg protein A per animal
融合Fusion	T＝终免+4天T = Final exemption + 4 days	To	To

2. Cell Fusion and Screening

The myeloma cells were sp2/0 derived from BALB/c, which were in the logarithmic growth phase when fused; the spleens of immunized mice were taken to prepare a single cell suspension of lymphocytes; the ratio of mouse spleen lymphocytes to myeloma cells was 1:5 Mix at -1:10, add 1ml of 50% PEG (PH 8.0) at 37°C dropwise, add incomplete medium and the rest of the stop solution, centrifuge to discard the supernatant, add HAT medium to suspend and mix, and dilute MC to 50ml. Dispense them into 3.5cm petri dishes, place them in a humid box, and place them in a 37°C, 5% CO ₂ constant temperature incubator for culture.

3. Screening and competitive screening

Cell clones were selected within 7-10 days of cell fusion, and ELISA tests were performed on all parent clones against human EGFR protein, and 120 monoclonal wells with higher OD450 positive values were selected. The above single clones were subjected to the first round of subcloning in one step, subjected to limiting dilution, and subjected to competitive ELISA screening against LZ8 and antibodies, and 12 subclones were obtained that could block the binding of LZ8 to EGFR. The above subclones were subjected to the second round of subcloning, and again subjected to competitive ELISA screening against LZ8 and EGFR antibodies. Finally, according to the positive value of 12 subclones and the analysis of the antibody content in the supernatant, 3 high positive values were obtained The stable monoclonal, corresponding to the fusion plate cell lines are UMAB1Z4, UMAB4B1, UMAB6F2.

4. Preparation and Purification of Antibodies on Cells

The above 3 cell lines were cultured in a 10 cm culture dish with 15% serum-containing DMEM medium, cultured to 4×10 ⁷ hours, centrifuged at 800 rpm for 5 minutes, discarded the supernatant and transferred the cells to a 2L flask, and added serum-free Medium so that the cell density is about 3×10 ⁵ cells/ml. Continue to culture for 1 to 2 weeks. When the cell death rate reaches 60%-70%, collect the cell suspension and centrifuge at 6000 rpm for 20 minutes, take the supernatant, and purify the supernatant by affinity column chromatography. Select the corresponding column according to the antibody pressure type. Materials, monoclonal antibodies UMAB1Z4, UMAB4B1, UMAB6F2 subtypes are IgG1, and protein G is used for purification. The concentration of purified monoclonal antibody was determined and divided into aliquots (100ml/tube, concentration of 1mg/ml, stored at 4-8°C).

Example 2 Sequence determination of antigen binding protein

The anti-EGFR monoclonal cell lines of the aforementioned cell lines UMAB1Z4 (hereinafter referred to as 1Z4), UMAB4B1 (hereinafter referred to as 4B1), and UMAB6F2 (hereinafter referred to as 6F2) were sequenced. Total RNA was extracted from the obtained hybridoma cells, reverse transcribed into cDNA with universal primers for the variable region of murine antibody, and then amplified by PCR. The variable regions of the light and heavy chains obtained were entrusted to Genscript for sequencing.

SEQ ID NO: 67 and SEQ ID NO: 68 are the nucleotide coding sequences of the heavy chain variable region and the light chain variable region of the highly active and highly active monoclonal antibody 1Z4 obtained in this application; SEQ ID NO: 69 and SEQ ID NO: 70 is the amino acid sequence of the heavy chain variable region and the light chain variable region of 1Z4, respectively. SEQ ID NO: 71 and SEQ ID NO: 72 are the nucleotide coding sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 4B1, respectively; SEQ ID NO: 73 and SEQ ID NO: 74 are respectively of 4B1 The amino acid sequence of the variable region of the heavy chain and the variable region of the light chain. SEQ ID NO: 75 and SEQ ID NO: 76 are the nucleotide coding sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 6F2, respectively; SEQ ID NO: 77 and SEQ ID NO: 78 are for 6F2, respectively The amino acid sequence of the variable region of the heavy chain and the variable region of the light chain.

Example 3 Purification of antigen binding protein expression vector

The 1Z4 antibody in Example 2 was selected for further analysis, and the heavy chain and light chain variable regions of the 1Z4 antibody were inserted into the expression vector pcDNA3.1(+)EcoRI and HindIII sites by PCR amplification to construct Murine anti-EGFR monoclonal antibody gene expression vector.

In this example, HiTrapTm MabSelect SuRe protein column technology was used to purify ^{antibodies expressed in Expi293F TM} cells to obtain antibodies with a concentration of 0.5 mg/ml and a purity of more than 90%. Specific steps are as follows:

Expi293F ^™ cells were cultured in serum-free Expi293 ^™ expression medium (Thermo Fisher Science). The cells were then stored in Erlenmeyer culture flasks and placed in an incubator at 37°C and 8% carbon dioxide. When the cell concentration reached 2.0×10 ⁶ cells/mL, DNA and ExpiFectamine ^TM 293 were added to the transfected cell culture flask at a ratio of 1:2.7.

The recombinant plasmids encoding the heavy and light chains of antibody 1Z4 were co-transfected into Expi293F cells in suspension culture. About 17 hours after transfection, add ExpiFectamine ^TM 293 Transfection Enhancer 1 and ExpiFectamine ^TM 293 Transfection Enhancer 2 into the culture flask. On the 5th day after transfection, the cell density and cell viability were measured, and about 1 mL of cell culture supernatant was collected for evaluation of antibody expression. Take the cell culture supernatant harvested on the 6th day for purification.

The cell culture supernatant was collected on day 6 for antibody purification. The filtered supernatant was loaded onto HiTrapTM MabSelect Sure 5ml (GE, catalog number 11-0034-95) at 3ml/min. It was eluted with 4 column volumes of PBS buffer (pH 7.2), and eluted with 50 mM citric acid (pH 3.0). During the elution process, each 1mL component was immediately neutralized with 124mol/L 1M Tris-HCl buffer (pH9.0). Part of the peaks were mixed together, and then the buffer was exchanged to PBS buffer (pH 7.2). The molecular weight, protein yield and purity were analyzed by SDS-PAGE and Western blot (Figure 1). The concentration of the sample was determined by the A280 method, and the extinction coefficient was 1.505.

Example 4 Determination of the binding site of LZ8 and EGFR

(1) Chemical cross-linking of EGFR extracellular domain and LZ8

In order to locate the epitope that LZ8 binds to EGFR, the extracellular region of EGFR and the monoclonal antibody were incubated in phosphate buffer, and cross-linked with DSS/BS3 cross-linking agent according to the ratio (1:4) at room temperature for 1 hour, then SDS-PAGE The gel separates the cross-linked product and stores it in a 10% acetic acid solution.

(2) Liquid-mass tandem mass spectrometry analysis

Cysteine residues in the protein were reduced and alkylated with Tris-(carboxyethyl)phosphine hydrochloride (TCEP) reagent and chloroacetamide (CAA) reagent, respectively, and reacted at 56°C for 60 minutes and at room temperature for 45 minutes. Then it was digested with trypsin (Thermo Fisher Scientific) overnight at 37°C, desalted on a C18 column, and loaded into the LC/MS tandem mass spectrometer;

The peptides pass through the Nano1200, the flow rate is 300nL/min, and the elution gradient is: 5 to 60% B (60 min), 60-70% B (20 min), and 70% B (10 min). Among them, A liquid is 0.1% formal acid in water, and B liquid is 95% Acetonitrile, 0.1% formal acid in water;

Mass spectrometry data collection is performed on Orbitrap Fusion Lumos tandem mass spectrometry (Thermo Fisher Scientific). The mass-to-charge ratio range of the first-order spectrum collection is 400-2000, and the resolution is 70,000 (m/z 400). The data acquisition method of the secondary spectrum is data-dependent. The first 10 peptides with the strongest signal in the primary spectrum are further collected for secondary spectrum data with a resolution of 35,000 (m/z 400), and finally through SIM- XL identifies cross-linked peptides.

(3) Isothermal calorimetric titration (ITC) experiment

Isothermal calorimetric titration experiments were performed with MicroCal VP-ITC (GE Healthcare) instrument. Drop 400μM LZ8 into the sample pool containing 20μM EGFR extracellular domain, 3ml per drop, 19 drops in total. The reaction buffer is 20mm phosphate buffer, 150mm NaCl (pH 8.0), and the temperature is 20°C. Analyze the obtained ITC data with MicroCal Origin software to find

K, n and dissociation constant values.

The results show that the molar ratio of the extracellular domain of EGFR and LZ8 when forming a complex is 1:2, which implies that the extracellular domain of EGFR interacts with the dimer of LZ8 in the form of a monomer. The result is shown in Figure 2.

Subsequently, the EGFR extracellular domain formed by cross-linking and the LZ8 complex were separated by SDS-PAGE and treated with trypsin. The result is shown in Figure 3, and then analyzed by tandem mass spectrometry.

A total of 26 cross-linked peptides were detected by tandem mass spectrometry, of which 3 cross-linked peptides with high confidence were formed by the cross-links between the extracellular domain of EGFR and LZ8, which can reflect the interface between EGFR and LZ8. information. Then the cross-linking sites between EGFR and LZ8 were marked on the three-dimensional structure of LZ8 and EGFR extracellular domain (Figure 4). It can be seen from Figure 4 that the interaction interface between the extracellular domain of EGFR and LZ8 includes serine 169, serine 222, and lysine 269. These three positions are related to the dimerization of the extracellular domain of EGFR. The amino acid involved in the interaction on the LZ8 protein is lysine 41, and this residue is on the ring BC of the LZ8 protein. In addition, the "zero-length" cross-linking agent NHS/EDC was used to chemically cross-link the EGFR/LZ8 complex to further verify the cross-linking results obtained with BS3/DSS. The results showed that the cross-linked peptide formed between aspartic acid at position 20 of LZ8 and serine at position 282 of EGFR, and these two amino acid residues are just near the EGFR/LZ8 interaction interface identified above , Which further verified the reliability of the cross-linking results.

The results showed that the binding epitope of LZ8 and EGFR is located in the II domain of EGFR (amino acids Ser196-Cys287).

Example 5 Combining computer simulation to speculate on the key sites of LZ8 binding to EGFR

According to the structure of LZ8 (PDB: 3F3H.pdb), prepare ligand is carried out in Discovery Studio 4.0, and the structure of EGFR is downloaded in PDB bank, PDB ID: 4kro.pdb, dewatered in Discovery Studio 4.0, and then hydrogenated Prepare protein, and then perform CDOCKER docking between the ligand and the EGFR target. The parameters are set as follows: Top Hits=10, RandomConformations=10, Orientations to Refine=10, Force field=CHARMm, Use Full Potential=False, and other parameters choose default settings .

After docking, it was determined that LZ8 and EGFR formed a binding interface at amino acids Ser196-Cys287, and 4 amino acid residue positions (Ser196, Ser222, Lys269, Ser282) had strong binding force with EGFR, indicating that it is an antigen-binding protein The key amino acid residues that interact with EGFR. The experimental results are shown in Figure 5.

Example 6 Antigen binding protein 1Z4 can carry small molecule compounds to internalize into tumor cells

In this example, Alexa Fluor 568NHS Ester (AF568) was coupled to 1Z4 antibody to detect the internalization ability of 1Z4 antibody after modification of small molecule compounds. AF568 is a small molecule compound with a molecular weight of 791.8, and its structure is shown in Figure 6. AF568 can react with the primary amine group (R-NH ₂ ) in the amino acid residue of the protein to couple to form a complex.

In this example, firstly, 10 mg of 1Z4 antibody was dissolved in 1 mL of 0.1 M sodium bicarbonate buffer, and 5 mg of AF568 was dissolved in 0.5 mL of DMSO. Then, while vortexing the 1Z4 antibody solution, slowly added 100 μL of AF568 DMSO solution, and Stir at room temperature for 1 hour to fully react. After the reaction, use Sephadex ^TM G-25 gel filtration chromatography column for purification. As shown in Figure 7, the product of the first peak is 1Z4-AF568 conjugate, and the second peak is excess uncoupled AF568.

The 40nM 1Z4-AF568 conjugate was incubated with HeLa cells at 4°C for 30 minutes, and then allowed to bind to cell surface receptors, and then transferred to a 374°C cell incubator for further incubation for 1 hour. The internalization of the 1Z4-AF568 conjugate was observed with an OMX ultra-high resolution microscope. The results showed that the 1Z4-AF568 conjugate can be internalized into tumor cells in the form of vesicles with a diameter of about 1 micron (Figure 8).

Example 7 The antigen binding protein of the present application can bind to EGFR on the cell membrane and trigger high-intensity internalization

7.1 Antigen binding proteins 1Z4, 6F2 and 4B1 can bind to EGFR on the cell membrane and trigger high-intensity internalization

Respectively, the 40 nM 1Z4, 6F2 and 4B1 antibodies modified by the small molecule compound Alexa Fluor 568 NHS Ester (modification method is the same as in Example 6) were incubated with HeLa cells at 4°C for 30 minutes, and then incubated at 37°C for 1 hour to make them internalized. The OMX imaging system was used to observe the internalization of 1Z4, 6F2 and 4B1 antibodies. The results of Figure 13, Figure 14 and Figure 15 indicate that the 1Z4, 6F2 and 4B1 antibodies were successfully internalized into the cells.

7.2 Antigen binding protein 1Z4 can bind to EGFR on the cell membrane and trigger high-intensity internalization

With the EGFR monoclonal antibody Cetuximab (Cetuximab) as a control, flow cytometry was used to detect the 1Z4 monoclonal antibody and the EGFR-positive cell line MD-MB-468 and the EGFR-negative cell line MD-MB-453 cell surface EGFR The combination of circumstances. The Alexa Fluor 647 NHS Ester modified 1Z4 (modification method is the same as in Example 6) was incubated with the two kinds of cells at 4° C. for 30 minutes, and then washed with a pre-cooled PBS solution to remove unbound antibodies before performing flow cytometry. The results showed that the 1Z4 monoclonal antibody can specifically bind to the EGFR-positive cell line MD-MB-468, but not to the EGFR-negative cell line MD-MB-453 (Figure 9).

With cetuximab and mAb806 as controls, the internalization intensity of 1Z4 antibody was analyzed using a high-content imaging system. Incubate the 40nM cetuximab, mAb806, and 1Z4 antibody (modification method is the same as in Example 6) with HeLa cells at 4°C for 30 minutes, and then continue to incubate at 37°C for 1 hour. Its internalization. The results showed that the internalization intensity of 1Z4 was much higher than that of cetuximab and mAb806 (Figure 10).

Example 8 Verification of the binding site of antigen binding protein 1Z4 and EGFR

The Alexa Fluor 488 was coupled to the 1Z4 monoclonal antibody (the modification method is the same as in Example 6) to compare the binding ability of the 1Z4 monoclonal antibody with mutant EGFR (S196A/S222A/K269A/S282A) and wild-type EGFR. The mutated EGFR mutation site is the key site for the binding of LZ8 to EGFR.

In this example, NIH3T3 EGFR (S196A/S222A/K269A/S282A) mutant cell lines and NIH3T3 EGFR wildtype cell lines were selected for related research. Incubate mAb1Z4-Alexa Fluor 488 and the above adherent cultured cells on ice for 30 minutes to bind to cell surface receptors, then fix with paraformaldehyde, and infect the nucleus with Hoechst 33342. A high-content system was used to analyze the average fluorescence intensity to reflect the difference in binding ability between mAb1Z4 and EGFR and its mutants at the cellular level.

The results of Figure 11 and Figure 12 show that the binding ability of 1Z4 to wild-type EGFR is significantly better than that of mutant EGFR, which indicates that S196A, S222A, K269A and S282A are the key sites that affect the binding of mAb1Z4 to EGFR. In addition, studies have shown that S196A, S222A, K269A, and S282A are located at the binding interface of LZ8 and EGFR, which is the key site for LZ8 to bind to EGFR, and mAb1Z4 is obtained through screening of competitively binding to EGFR with LZ8. In summary, the binding sites of 1Z4 and EGFR are the same as LZ8, and S196A, S222A, K269A and S282A are the key binding sites of both.

The foregoing detailed description is provided by way of explanation and examples, and is not intended to limit the scope of the appended claims. Various changes of the embodiments listed in the present application are obvious to those of ordinary skill in the art, and are reserved within the scope of the appended claims and their equivalents.

Claims

An isolated antigen binding protein that can specifically bind to the epidermal growth factor receptor (EGFR) on the cell membrane to internalize the complex.
The isolated antigen binding protein according to claim 1, which can specifically bind to the II domain of EGFR, and the II domain of EGFR comprises the amino acid sequence shown in SEQ ID NO: 13.
The isolated antigen binding protein according to claim 1 or 2, which can specifically bind to at least one amino acid selected from the group consisting of Ser196, Ser222, Lys269 and Ser282 on the II domain of EGFR.
The isolated antigen binding protein of any one of claims 1 to 3, which is capable of binding to the EGFR competitively with the LZ-8 protein.
The isolated antigen binding protein of any one of claims 1 to 4, which is capable of competing with a reference antibody for binding to the EGFR, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, the VH of the reference antibody includes HCDR1, HCDR2, and HCDR3, the VL of the reference antibody includes LCDR1, LCDR2, and LCDR3, and the HCDR1 of the reference antibody includes SEQ ID NO: 15, SEQ ID NO: 29 And the amino acid sequence shown in any one of SEQ ID NO: 43, the HCDR2 of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30 and SEQ ID NO: 44 The HCDR3 of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 31 and SEQ ID NO: 45, and the LCDR1 of the reference antibody comprises SEQ ID NO: 18, The amino acid sequence shown in any one of SEQ ID NO: 32 and SEQ ID NO: 46, the LCDR2 of the reference antibody includes any one of SEQ ID NO: 19, SEQ ID NO: 33 and SEQ ID NO: 47 As shown in the amino acid sequence, the LCDR3 of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 20, SEQ ID NO: 34 and SEQ ID NO: 48.
The isolated antigen binding protein of any one of claims 1-5, which comprises an antibody or an antigen binding fragment thereof.
The isolated antigen binding protein according to claim 6, wherein the antigen binding fragment comprises Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/ Or dAb.
The isolated antigen binding protein of any one of claims 1-7, wherein the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
The isolated antigen binding protein of any one of claims 1-8, which comprises at least one CDR in a heavy chain variable region VH, the VH comprising SEQ ID NO: 68, SEQ ID NO: 72 and SEQ ID NO: the amino acid sequence shown in any one of 76.
The isolated antigen binding protein according to any one of claims 1-9, which comprises HCDR3, and the HCDR3 comprises SEQ ID NO: 17, SEQ ID NO: 31 and SEQ ID NO: 45. The amino acid sequence shown.
The isolated antigen binding protein of any one of claims 1-10, which comprises HCDR2, and the HCDR2 comprises any one of SEQ ID NO: 16, SEQ ID NO: 30, and SEQ ID NO: 44. The amino acid sequence shown.
The isolated antigen binding protein of any one of claims 1-11, which comprises HCDR1, and the HCDR1 comprises any one of SEQ ID NO: 15, SEQ ID NO: 29 and SEQ ID NO: 43. The amino acid sequence shown.
The isolated antigen binding protein of any one of claims 1-12, which comprises HCDR1, HCDR2 and HCDR3, and said HCDR1 comprises SEQ ID NO: 15, SEQ ID NO: 29 and SEQ ID NO: 43 The amino acid sequence shown in any one of the HCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30 and SEQ ID NO: 44, and the HCDR3 includes SEQ ID NO: 17, The amino acid sequence shown in any one of SEQ ID NO: 31 and SEQ ID NO: 45.
The isolated antigen binding protein of any one of claims 1-13, which comprises HCDR1, HCDR2 and HCDR3, and said HCDR1, HCDR2 and HCDR3 are selected from any of the following amino acid sequences:

(1) HCDR1: SEQ ID NO: 15, HCDR2: SEQ ID NO: 16 and HCDR3: SEQ ID NO: 17; (2) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 30 and HCDR3: SEQ ID NO: 31; and,

(3) HCDR1: SEQ ID NO: 43, HCDR2: SEQ ID NO: 44 and HCDR3: SEQ ID NO: 45.
The isolated antigen binding protein of any one of claims 1-14, which comprises a heavy chain variable region VH, wherein the VH includes a framework region H-FR1, and the C-terminus of the H-FR1 is connected to the The N-terminus of HCDR1 is directly or indirectly connected, and the H-FR1 includes the amino acid sequence shown in any one of SEQ ID NO: 21, SEQ ID NO: 35, and SEQ ID NO: 49.
The isolated antigen binding protein of any one of claims 1-15, which comprises a heavy chain variable region VH, wherein the VH includes a framework region H-FR2, and the H-FR2 is located between the HCDR1 and the HCDR1. Between the HCDR2, and the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 22, SEQ ID NO: 36, and SEQ ID NO: 50.
The isolated antigen binding protein of any one of claims 1-16, comprising a heavy chain variable region VH, wherein the VH includes a framework region H-FR3, and the H-FR3 is located between the HCDR2 and the Between the HCDR3, and the H-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 23, SEQ ID NO: 37, and SEQ ID NO: 51.
The isolated antigen binding protein of any one of claims 1-17, comprising a heavy chain variable region VH, wherein the VH includes a framework region H-FR4, and the N-terminus of the H-FR4 is connected to the The C-terminus of the HCDR3 is connected, and the H-FR4 includes the amino acid sequence shown in any one of SEQ ID NO: 24, SEQ ID NO: 38, and SEQ ID NO: 52.
The isolated antigen binding protein of any one of claims 1-18, which comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1 comprises SEQ ID NO: 21, The amino acid sequence shown in any one of SEQ ID NO: 35 and SEQ ID NO: 49, and the H-FR2 includes any one of SEQ ID NO: 22, SEQ ID NO: 36 and SEQ ID NO: 50 The H-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 23, SEQ ID NO: 37, and SEQ ID NO: 51, and the H-FR4 includes SEQ ID NO: 24, SEQ The amino acid sequence shown in any one of ID NO: 38 and SEQ ID NO: 52.
The isolated antigen binding protein of any one of claims 1-19, comprising H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1, H-FR2, H-FR4 FR3 and H-FR4 are selected from any of the following amino acid sequences:

(1) F-FR1: SEQ ID NO: 21, H-FR2: SEQ ID NO: 22, H-FR3: SEQ ID NO: 23 and H-FR4: SEQ ID NO: 24;

(2) H-FR1: SEQ ID NO: 35, H-FR2: SEQ ID NO: 36, H-FR3: SEQ ID NO: 37 and H-FR4: SEQ ID NO: 38; and,

(3) H-FR1: SEQ ID NO: 49, H-FR2: SEQ ID NO: 50, H-FR3: SEQ ID NO: 51 and H-FR4: SEQ ID NO: 52.
The isolated antigen binding protein of any one of claims 1-20, comprising a heavy chain variable region VH, the heavy chain variable region VH comprising SEQ ID NO: 68, SEQ ID NO: 72 and SEQ ID NO: the amino acid sequence shown in any one of 76.
The isolated antigen binding protein of any one of claims 1-21, which comprises a heavy chain constant region, and the heavy chain constant region is derived from IgG1.
The isolated antigen binding protein of any one of claims 1-22, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 57 or SEQ ID NO: 58.
The isolated antigen binding protein of any one of claims 1-23, which comprises an antibody heavy chain, and the antibody heavy chain comprises SEQ ID NO: 60, SEQ ID NO: 62 and SEQ ID NO: 64 The amino acid sequence shown in any item.
The isolated antigen binding protein of any one of claims 1-24, which comprises at least one CDR in the light chain variable region VL, the VL comprising SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: the amino acid sequence shown in any one of 77.
The isolated antigen binding protein of any one of claims 1-25, which comprises LCDR3, and the LCDR3 comprises SEQ ID NO: 20, SEQ ID NO: 34, and SEQ ID NO: 48. The amino acid sequence shown.
The isolated antigen binding protein of any one of claims 1-26, which comprises LCDR2, and the LCDR2 comprises SEQ ID NO: 19, SEQ ID NO: 33 and SEQ ID NO: 47. The amino acid sequence shown.
The isolated antigen binding protein according to any one of claims 1-27, which comprises LCDR1, and the LCDR1 comprises SEQ ID NO: 18, SEQ ID NO: 32 and SEQ ID NO: 46. The amino acid sequence shown.
The isolated antigen binding protein of any one of claims 1-28, which comprises LCDR1, LCDR2 and LCDR3, and said LCDR1 comprises SEQ ID NO: 18, SEQ ID NO: 32 and SEQ ID NO: 46 The amino acid sequence shown in any one of the LCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33 and SEQ ID NO: 47, and the LCDR3 includes SEQ ID NO: 20, The amino acid sequence shown in any one of SEQ ID NO: 34 and SEQ ID NO: 48.
The isolated antigen binding protein of any one of claims 1-29, which comprises LCDR1, LCDR2, and LCDR3, and the LCDR1, LCDR2, and LCDR3 are selected from any of the following amino acid sequences:

(1) LCDR1: SEQ ID NO: 18, LCDR2: SEQ ID NO: 19 and LCDR3: SEQ ID NO: 20;

(2) LCDR1: SEQ ID NO: 32, LCDR2: SEQ ID NO: 33 and LCDR3: SEQ ID NO: 34; and,

(3) LCDR1: SEQ ID NO: 46, LCDR2: SEQ ID NO: 47 and LCDR3: SEQ ID NO: 48.
The isolated antigen binding protein of any one of claims 1-30, comprising a light chain variable region VL, wherein the VL includes a framework region L-FR1, and the C-terminus of the L-FR1 is connected to the The N-terminus of LCDR1 is directly or indirectly connected, and the L-FR1 includes the amino acid sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 39 and SEQ ID NO: 53.
The isolated antigen binding protein of any one of claims 1-31, which comprises a light chain variable region VL, wherein the VL comprises a framework region L-FR2, and the L-FR2 is located between the LCDR1 and the LCDR1. Between the LCDR2, and the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 26, SEQ ID NO: 40, and SEQ ID NO: 54.
The isolated antigen binding protein of any one of claims 1-32, which comprises a light chain variable region VL, wherein the VL comprises a framework region L-FR3, and the L-FR3 is located between the LCDR2 and the LCDR2. Between the LCDR3, and the L-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 27, SEQ ID NO: 41, and SEQ ID NO: 55.
The isolated antigen binding protein of any one of claims 1-33, which comprises a light chain variable region VL, wherein the VL comprises a framework region L-FR4, and the N-terminus of the L-FR4 is connected to the The C-terminus of LCDR3 is connected, and the L-FR4 includes the amino acid sequence shown in any one of SEQ ID NO: 28, SEQ ID NO: 42 and SEQ ID NO: 56.
The isolated antigen binding protein of any one of claims 1-34, which comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein the L-FR1 comprises SEQ ID NO: 25, The amino acid sequence shown in any one of SEQ ID NO: 39 and SEQ ID NO: 53, the L-FR2 includes any one of SEQ ID NO: 26, SEQ ID NO: 40 and SEQ ID NO: 54 The L-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 27, SEQ ID NO: 41 and SEQ ID NO: 55, and the L-FR4 includes SEQ ID NO: 28, SEQ The amino acid sequence shown in any one of ID NO: 42 and SEQ ID NO: 56.
The isolated antigen binding protein according to any one of claims 1-35, which comprises L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR1, L-FR2, L-FR4 FR3 and L-FR4 are selected from any of the following amino acid sequences:

(1) F-FR1: SEQ ID NO: 39, L-FR2: SEQ ID NO: 40, L-FR3: SEQ ID NO: 41 and L-FR4: SEQ ID NO: 42;

(2) L-FR1: SEQ ID NO: 53, L-FR2: SEQ ID NO: 54, L-FR3: SEQ ID NO: 55 and L-FR4: SEQ ID NO: 56; and,

(3) L-FR1: SEQ ID NO: 25, L-FR2: SEQ ID NO: 26, L-FR3: SEQ ID NO: 27 and L-FR4: SEQ ID NO: 28.
The isolated antigen binding protein of any one of claims 1-36, which comprises a light chain variable region VL, and the light chain variable region VL comprises SEQ ID NO: 69, SEQ ID NO: 73 and The amino acid sequence shown in any one of SEQ ID NO: 77.
The isolated antigen binding protein of any one of claims 1-37, which comprises a light chain constant region, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:59.
The isolated antigen binding protein of any one of claims 1-38, which comprises an antibody light chain, and the antibody light chain comprises SEQ ID NO: 61, SEQ ID NO: 63 and SEQ ID NO: 65 The amino acid sequence shown in any item.
A polypeptide comprising the isolated antigen binding protein of any one of claims 1-39.
An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-39 or the polypeptide of claim 40, and a small molecule compound.
The immunoconjugate according to claim 41, wherein the small molecule compound comprises a cytotoxic agent and/or a marker.
The immunoconjugate according to any one of claims 41-42, wherein the small molecule compound is coupled to the primary amine group of the upper amino acid residue of the isolated antigen binding protein.
The immunoconjugate of any one of claims 41-43, which is capable of internalizing the complex.
An isolated one or more nucleic acid molecules that encode the isolated antigen binding protein of any one of claims 1-39.
The nucleic acid molecule of claim 45, which comprises SEQ ID NO: 1, SEQID NO: 5, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 66, the nucleotide sequence shown in any one of SEQID NO: 67, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, and SEQ ID NO: 75.
A vector comprising the nucleic acid molecule according to any one of claims 45-46.
A cell comprising the nucleic acid molecule according to any one of claims 45-46 or the vector according to claim 47.
A method for preparing the isolated antigen binding protein of any one of claims 1-39, the method comprising culturing under conditions such that the isolated antigen binding protein of any one of claims 1-39 is expressed The cell of claim 48.
A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-39, the polypeptide of claim 40, the immunoconjugate of any one of claims 41-44, and The nucleic acid molecule of any one of claims 45-46, the vector of claim 47, and/or the cell of claim 48, and optionally a pharmaceutically acceptable carrier.
The isolated antigen binding protein of any one of claims 1-39, the polypeptide of claim 40, the immunoconjugate of any one of claims 41-44, any of claims 45-46 Use of one of the nucleic acid molecules, the vector of claim 47, the cells of claim 48 and/or the pharmaceutical composition of claim 50 in the preparation of medicines for prevention and relief And/or treat diseases related to EGFR activity.
The use according to claim 51, the disease related to EGFR activity includes tumor.
The use according to claim 52, wherein the tumor includes a tumor associated with abnormal expression of EGFR.
The use according to any one of claims 52-53, wherein the tumor comprises a solid tumor.
The use according to any one of claims 52-54, wherein the tumors include glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer and / Or stomach cancer.
A method for preventing, alleviating or treating a disease related to EGFR activity, the method comprising administering the isolated antigen binding protein of any one of claims 1-39, the method of claim 40 to a subject in need Polypeptide, the immunoconjugate of any one of claims 41-44, the nucleic acid molecule of any one of claims 45-46, the vector of claim 47, the cell of claim 48, and /Or the pharmaceutical composition of claim 50.
The method of claim 56, wherein the disease associated with EGFR activity includes tumor.
The method of claim 57, wherein the tumor comprises a tumor associated with abnormal expression of EGFR.
The method of any one of claims 57-58, wherein the tumor comprises a solid tumor.
The method according to any one of claims 57-59, wherein the tumor comprises glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer and / Or stomach cancer.
The isolated antigen binding protein of any one of claims 1-39, the polypeptide of claim 40, the immunoconjugate of any one of claims 41-44, any of claims 45-46 The nucleic acid molecule according to one item, the vector according to claim 47, the cell according to claim 48 and/or the pharmaceutical composition according to claim 50 are used to prevent, alleviate or treat diseases related to EGFR activity.
The isolated antigen binding protein, polypeptide, immunoconjugate, nucleic acid molecule, carrier, cell, and/or pharmaceutical composition according to claim 61, wherein the disease related to EGFR activity includes tumor.
The isolated antigen binding protein, polypeptide, immunoconjugate, nucleic acid molecule, carrier, cell, and/or pharmaceutical composition according to claim 62, wherein the tumor includes a tumor related to abnormal expression of EGFR.
The isolated antigen binding protein, polypeptide, immunoconjugate, nucleic acid molecule, carrier, cell and/or pharmaceutical composition according to any one of claims 62-63, wherein the tumor comprises a solid tumor.
The isolated antigen binding protein, polypeptide, immunoconjugate, nucleic acid molecule, carrier, cell and/or pharmaceutical composition according to any one of claims 62-64, wherein the tumor includes glioma, breast Cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer and/or stomach cancer.
A method for detecting the presence and/or content of EGFR, which comprises administering the isolated antigen binding protein of any one of claims 1-39 or the polypeptide of claim 40.
A kit comprising the isolated antigen binding protein according to any one of claims 1-39, the polypeptide according to claim 40, the immunoconjugate according to any one of claims 41-44, and claims The nucleic acid molecule of any one of 45-46, the vector of claim 47, the cell of claim 48, and/or the pharmaceutical composition of claim 50.
A method of internalizing a complex, which comprises administering the isolated antigen binding protein of any one of claims 1-39, the polypeptide of claim 40, or any one of claims 41-44 The immunoconjugate of claim 1, the nucleic acid molecule of any one of claims 45-46, the vector of claim 47, the cell of claim 48, and/or the pharmaceutical composition of claim 50.