WO2024046396A1

WO2024046396A1 - Anti-meegfr antibody, antigen-binding fragment thereof, and use thereof

Info

Publication number: WO2024046396A1
Application number: PCT/CN2023/115936
Authority: WO
Inventors: 洪明奇; 王绍椿; 山口浩史; 许荣茂; 沈宜君; 李浤维; 林佑哲
Original assignee: 洪明奇
Priority date: 2022-08-31
Filing date: 2023-08-30
Publication date: 2024-03-07
Also published as: CN117624361A

Abstract

The present invention relates to an anti-methylated epidermal growth factor receptor (meEGFR) antibody or an antigen-binding fragment thereof, a detection kit, a method for detecting a meEGFR positive cancer, a pharmaceutical composition for treatment of a cancer, a meEGFR-specific chimeric antigen receptor, a nucleic acid encoding the meEGFR-specific chimeric antigen receptor, a meEGFR-specific chimeric antigen receptor expressing cell, an innate cell engager, and an antibody conjugate. The anti-meEGFR antibody or the antigen-binding fragment thereof specifically binds to the meEGFR, and the meEGFR has asymmetric dimethylation modification at R198 and R200 sites; therefore, the anti-meEGFR antibody or the antigen-binding fragment thereof may be used as a detection kit for detecting a meEGFR positive cancer, and can be applied to the treatment of the meEGFR positive cancer.

Description

Anti-meEGFR antibodies, antigen-binding fragments thereof and uses thereof

Technical field

The present invention relates to an anti-epidermal growth factor receptor (EGFR) antibody or an antigen-binding fragment thereof and its use, in particular to an anti-methylated epidermal growth factor receptor (meEGFR) antibody or an antigen-binding fragment thereof and its use. use.

Background technique

Cancer, also known as malignant tumors, is an abnormal proliferation of cells, and these proliferated cells may invade other parts of the body. It is a disease caused by an abnormality in the mechanism that controls cell division and proliferation. The number of people suffering from cancer is increasing all over the world, and cancer has been among the top ten causes of death for 27 consecutive years. Cancer treatment mainly includes radiotherapy, chemotherapy, targeted therapy and surgery. Despite recent advances in drugs and surgical techniques, the five-year survival rate of patients with advanced disease is still quite low, highlighting the importance of developing new treatment strategies.

Epidermal growth factor receptor (EGFR) is a type of receptor-type tyrosine kinase. Mutations or overexpression of EGFR can be observed in various cancer types. It is related to tumor proliferation, angiogenesis, and tumor metastasis. etc. related. EGFR-targeted therapies that have been developed and used clinically include EGFR-tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib, as well as Monoclonal antibodies to cetuximab and panitumumab. The overexpression rate of EGFR in triple negative breast cancer (TNBC) tumors is as high as 70%, which is much higher than other breast cancer molecular subtypes. High expression of EGFR is also significantly associated with poor progression-free survival and overall survival in TNBC. Therefore, EGFR is a potential therapeutic target for TNBC and advanced breast cancer.

A variety of EGFR-TKIs have been approved for EGFR, but secondary drug resistance mutations are a very important issue in the treatment process. The first three generations of TKIs all target the intracellular ATP binding site and inhibit EGFR activity by blocking its phosphorylation. However, to date, many clinical trials evaluating the effects of EGFR-targeted drugs in the treatment of TNBC patients have failed to show or confirm their therapeutic benefits. Therefore, new treatment strategies need to be developed to replace traditional EGFR-targeted drugs. Post-translational modification is an important mechanism for regulating the physiological functions of proteins. However, there are no antibody drugs targeting specific protein post-translational modifications in current clinical treatments and on the market. Targeting other post-translational modification pathways of EGFR is an important way to improve the response to EGFR-TKI. Potential development directions for drug resistance.

Contents of the invention

In view of this, the purpose of the present invention is to provide an anti-meEGFR antibody or an antigen-binding fragment thereof, which has high specificity and high affinity for methylated epidermal growth factor receptor (methylated epidermal growth factor receptor, meEGFR), It can be used as an ideal clinical detection tool for meEGFR-positive cancers. Therefore, a detection kit including the anti-meEGFR antibody or antigen-binding fragment thereof of the present invention, and a method for detecting meEGFR-positive cancer using the detection kit of the present invention, can As an aid to subsequent medical decisions. In addition, because the anti-meEGFR antibody or its antigen-binding fragment of the present invention has the characteristics of high specificity for meEGFR and low potential toxicity, the anti-meEGFR antibody or its antigen-binding fragment can be used as a pharmaceutical composition for treating cancer, meEGFR Specific chimeric antigen receptors, nucleic acids encoding meEGFR-specific chimeric antigen receptors, meEGFR-specific chimeric antigen receptor-expressing cells, innate cell adapters and antibody conjugates are used in the treatment of meEGFR-positive cancers to achieve breakthroughs Current clinical dilemmas in treating meEGFR-positive cancers.

One aspect of the present invention provides an anti-meEGFR antibody or an antigen-binding fragment thereof, which specifically binds to methylated epidermal growth factor receptor (meEGFR), and meEGFR binds to R198 and R200 The site carries asymmetric dimethylation modification. The anti-meEGFR antibody or antigen-binding fragment thereof includes a heavy chain variable region (V _H ) and a light chain variable region (V _L ), wherein V _H includes a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), V _L includes light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3). The sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from the group consisting of: HCDR1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8, HCDR3 of SEQ ID NO:9, SEQ ID NO:10 LCDR1 of SEQ ID NO:11 and LCDR3 of SEQ ID NO:12; HCDR1 of SEQ ID NO:13, HCDR2 of SEQ ID NO:14, HCDR3 of SEQ ID NO:15, LCDR1 of SEQ ID NO:16 , LCDR2 of SEQ ID NO:17 and LCDR3 of SEQ ID NO:18; and HCDR1 of SEQ ID NO:19, HCDR2 of SEQ ID NO:20, HCDR3 of SEQ ID NO:21, LCDR1 of SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO:24.

According to the aforementioned anti-meEGFR antibody or antigen-binding fragment thereof, the sequences of _VH and _VL can be selected from the group consisting of: _VH comprising SEQ ID NO:1 and _VL comprising SEQ ID NO:2; VH comprising SEQ ID NO:3 and _VL comprising SEQ ID NO:4; and _VH comprising SEQ ID NO: ₅ and VL comprising SEQ ID NO: ₆ .

According to the aforementioned anti-meEGFR antibody or antigen-binding fragment thereof, the anti-meEGFR antibody or antigen-binding fragment thereof can be selected from single domain antibodies, humanized antibodies, multimeric antibodies, single-chain variable fragments (scFv), Fab fragments, A group consisting of Fab' fragments and F(ab')2 fragments.

Another aspect of the present invention is to provide a detection kit for detecting meEGFR-positive cancer, the detection kit comprising the anti-meEGFR antibody or antigen-binding fragment thereof as described in the previous paragraph.

According to the aforementioned detection kit, the anti-meEGFR antibody or antigen-binding fragment thereof can be combined with a label, and the label can be a fluorescent label, a chemiluminescent label, a radioisotope label, an enzyme label, or a biotin label. or combination thereof.

Another aspect of the present invention provides a method for detecting meEGFR-positive cancer, which includes the following steps: providing a sample to be tested, providing a detection kit as described in the previous paragraph, performing a binding step, and performing a detection step. The binding step is to contact the sample to be tested with an anti-meEGFR antibody or its antigen-binding fragment and perform a binding reaction, and the detection step is to detect whether the sample to be tested contains antibody cancer cell complexes.

According to the aforementioned method for detecting meEGFR-positive cancer, the meEGFR-positive cancer can be breast cancer, colorectal cancer, prostate cancer, lung cancer or pancreatic cancer.

Another aspect of the present invention is to provide a pharmaceutical composition for treating cancer, comprising the anti-meEGFR antibody or antigen-binding fragment thereof as described in the previous paragraph and a pharmaceutically acceptable carrier.

According to the aforementioned pharmaceutical composition for treating cancer, it may further include chemotherapeutic agents, immunomodulators, targeted therapy drugs, antibody drugs, or combinations thereof, wherein the antibody drugs are different from anti-meEGFR antibodies or antigen-binding fragments thereof.

Another aspect of the present invention provides a meEGFR-specific chimeric antigen receptor, which includes an extracellular domain and an intracellular information transmission domain. The extracellular domain is used to recognize methylated epidermal growth factor receptor (meEGFR), and meEGFR has asymmetric dimethylation modifications at R198 and R200 positions. The extracellular domain includes as in the previous paragraph The anti-meEGFR antibody or antigen-binding fragment thereof.

According to the aforementioned meEGFR-specific chimeric antigen receptor, it may also include a transmembrane domain, which connects the extracellular domain and the intracellular information transmission domain.

According to the aforementioned meEGFR-specific chimeric antigen receptor, the intracellular information transmission domain may include a costimulatory domain and a primary information transmission domain.

Another aspect of the present invention is to provide a nucleic acid encoding the meEGFR-specific chimeric antigen receptor as described in the previous paragraph.

Another aspect of the present invention is to provide a meEGFR-specific chimeric antigen receptor-expressing cell, including immune cells and the nucleic acid as described in the previous paragraph. The meEGFR-specific chimeric antigen receptor-expressing cell is a nucleic acid-transducing cell. obtained by infecting immune cells.

According to the aforementioned meEGFR-specific chimeric antigen receptor expressing cells, the immune cells can be T cells or natural killer cells.

Another aspect of the present invention is to provide a pharmaceutical composition for treating cancer, comprising the meEGFR-specific chimeric antigen receptor expressing cells as described in the previous paragraph and a pharmaceutically acceptable carrier.

Another aspect of the present invention is to provide an innate cell adapter, including a meEGFR antigen recognition domain and at least one immune cell receptor binding domain. The meEGFR antigen recognition domain includes an anti-meEGFR antibody or an antigen-binding fragment thereof as described in the previous paragraph. , at least one immune cell receptor binding domain has binding specificity for one or more of CD3, CD8, CD16, CD19 or NKG2D.

According to the aforementioned innate cell adapter, the innate cell adapter can be a bispecific T cell adapter, a trispecific T cell adapter or a multispecific T cell adapter.

According to the aforementioned innate cell adapter, the innate cell adapter may be a bispecific natural killer cell adapter, a trispecific natural killer cell adapter, or a multispecific natural killer cell adapter.

Another aspect of the present invention is to provide an antibody conjugate, comprising the anti-meEGFR antibody or its antigen-binding fragment as described in the previous paragraph and an effector molecule. The effector molecule is coupled to the anti-meEGFR antibody or its antigen-binding fragment through a chemical bond or a linker. Antigen-binding fragments, wherein the effector molecule is a toxin, growth inhibitor, toxic protein, radionuclide, radioisotope, chemotherapeutic drug, or a combination thereof.

The above summary of the invention is intended to provide a simplified summary of the invention to provide the reader with a basic understanding of the invention. This summary is not an extensive overview of the invention and it is not intended to identify key/critical elements of the invention or to delineate the scope of the invention.

Description of drawings

In order to make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the accompanying drawings are described as follows:

Figure 1 is a schematic diagram of phage display technology for screening an aspect of anti-meEGFR antibodies or antigen-binding fragments thereof of the present invention;

Figures 2A and 2B are diagrams showing the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of an aspect of the anti-meEGFR antibody or its antigen-binding fragment of the present invention;

Figure 3A, Figure 3B and Figure 3C are diagrams showing the results of the binding affinity analysis between the anti-meEGFR antibody or its antigen-binding fragment and the target peptide according to one embodiment of the present invention;

Figure 4 is a flow chart of steps of a method for detecting meEGFR-positive cancer in another aspect of the present invention;

Figures 5A and 5B are diagrams showing the binding specificity analysis results of another aspect of the detection kit of the present invention;

Figure 6A, Figure 6B and Figure 6C are the analysis results of meEGFR expression in breast cancer patients;

Figure 6D is a diagram showing the analysis results of the overall survival of meEGFR-positive and meEGFR-negative breast cancer patients;

Figure 6E is a graph showing the analysis results of disease-free survival of meEGFR-positive and meEGFR-negative breast cancer patients;

Figures 7A, 7B, 7C, 7D, 7E and 7F are analysis results of tumor cell death induced by a pharmaceutical composition for treating cancer according to another aspect of the present invention;

Figures 8A and 8B are diagrams showing the analysis results of tumor cell death induced by a pharmaceutical composition for treating cancer according to another embodiment of the present invention;

Figure 9 is a schematic diagram of the theoretical structure of yet another aspect of the meEGFR-specific chimeric antigen receptor of the present invention;

Figures 10A and 10B are diagrams showing the analysis results of specific lysis of tumor cells by meEGFR-specific chimeric antigen receptor-expressing cells in yet another aspect of the present invention;

Figures 11A and 11B are analysis results of internalization of antibody conjugates by tumor cells according to another embodiment of the present invention; and

Figure 12 is a diagram showing the analysis results of tumor cell death induced by an antibody conjugate according to another embodiment of the present invention.

Among them, the reference signs are:

100: Methods to detect meEGFR-positive cancers

110,120,130,140: steps

200:meEGFR-specific chimeric antigen receptor

210: Extracellular domain

220: Transmembrane domain

230: Intracellular information transmission domain

231:Co-stimulation domain

232: Primary information transfer domain

Detailed ways

Unless otherwise stated, all technical terms, symbols or other scientific terms or terms used in this specification have the meanings commonly understood by those skilled in the art to which this invention belongs, unless the context in which they are used indicates otherwise. In some cases, terms with conventional meanings are defined herein for purposes of clarity and/or immediate reference, and these definitions incorporated in this specification should be construed as not necessarily congruent with the conventional meanings in the art. There are substantial differences. Many of the techniques and procedures described or referenced herein are well known and are routinely used by those skilled in the art. Where appropriate, commercially available kits and reagents are generally used in accordance with the instructions and/or parameters defined by the manufacturer, unless otherwise stated.

Two specific arginine (R198 and R200) sites in the extracellular structural region of the epidermal growth factor receptor (EGFR) can be used by protein arginine methyltransferase 1 (PRMT1) ) methylation modification. The "methylated epidermal growth factor receptor (meEGFR)" described in this specification is EGFR with asymmetric dimethylation modification at the R198 and R200 sites in the extracellular structural region, R198 Asymmetric methylation modification of R200 and meEGFR will lead to continuous activation of meEGFR, thereby accelerating cancer cell proliferation and developing resistance to various EGFR inhibitors. For example, some cancer patients use the FDA-approved anti-EGFR antibody cetuximab ( Cetuximab) failed in clinical trials and developed resistance to cetuximab in these cancer patients.

The "antibody" mentioned in this manual refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by inter-chain disulfide bonds. The amino acid composition and arrangement order of the heavy chain constant region ( _CH ) of immunoglobulins are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain and γ chain respectively. , α chain, and ε chain. The same type of Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have either a kappa chain or a lambda chain.

The anti-meEGFR antibody or antigen-binding fragment thereof of the present invention contains a "functional part". The functional part refers to a part that shares at least one characteristic with the antibody, and the number is not necessarily the same. same. Functional moieties are capable of binding to the same antigen, although perhaps to different extents. The functional moiety typically contains at least one heavy chain variable domain ( _VH ) and one light chain variable domain ( _VL ). In some cases, the functional part contains only a V _H .

The anti-meEGFR antibody or antigen-binding fragment thereof of the present invention is a human antibody, wherein V _H may further comprise _CH , and the _CH comprises human IgG1, IgG2, IgG3, IgG4 or variants thereof. _VL may further comprise a light chain constant region ( _CL ₎ comprising a human kappa, lambda chain or a variant thereof.

The anti-meEGFR antibodies of the present invention may include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

The technical term "mouse-derived antibody" in the present invention refers to a monoclonal antibody specific for human meEGFR prepared according to the knowledge and skills in the art. During preparation, meEGFR is used as an antigen to inject test subjects, and then hybridomas expressing antibodies with desired sequence or functional properties are isolated. In a preferred embodiment of the present invention, the murine anti-meEGFR antibody may further comprise the _CL of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1, IgG2, IgG3 or variants thereof. Body _CH .

The technical term "chimeric antibody" is an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody. It can reduce the immune response induced by mouse antibodies. To establish chimeric antibodies, we must first establish hybridomas that secrete mouse-specific monoclonal antibodies, then select and colonize variable region genes from mouse hybridoma cells, and then select and colonize the constant region genes of human antibodies as needed. The mice can be The variable region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. In a preferred embodiment of the present invention, the antibody light chain of the chimeric antibody further comprises the _CL of human kappa, lambda chains or variants thereof. The antibody heavy chain of the chimeric antibody further includes _CH of human IgG1, IgG2, IgG3, IgG4 or variants thereof. The constant region of the human antibody may be selected from _CH of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising _CH of human IgG2 or IgG4.

The technical term "humanized antibody", also known as CDR-grafted antibody, refers to transplanting the mouse complementary determining region (CDR) sequence into the human antibody variable region framework, that is, Antibodies produced in different types of human germline antibody framework sequences. Chimeric antibodies can overcome the strong immune response induced by carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences. In order to avoid the decrease in activity caused by the decrease in immunogenicity of humanized antibodies, the humanized antibody can be treated with Minimal reverse mutations or backmutations are performed on the framework sequences of the variable region of the antibody to maintain activity. The humanized antibodies of the present invention also include humanized antibodies in which CDRs are further affinity matured by phage display.

The technical term "polymeric antibody" refers to an anti-meEGFR antibody containing two or more basic units, which can be a dimer, trimer or tetramer.

The "antigen-binding fragment" mentioned in the present invention refers to Fab fragments, Fab' fragments or F(ab')2 fragments with antigen-binding activity, as well as variable fragments (fragment variable, Fv) and single fragments that can bind to meEGFR. Chain variable fragment (single-chain variable fragment, scFv); which contains one or more CDRs selected from SEQ ID NO: 7 to SEQ ID NO: 24 of the anti-meEGFR antibody of the present invention or its antigen-binding fragment.

The technical term "Fab fragment" refers to a light chain and a heavy chain _CH 1 and variable regions. The heavy chain of the Fab fragment cannot form disulfide bonds with another heavy chain molecule.

The technical term "Fab'fragment" refers to the portion of a heavy chain that contains a light chain and a region between the V _H and CH ₁ domains and the CH ₁ and _CH 2 domains. An interchain disulfide bond is formed between the two heavy chains of the two Fab' fragments to form the F(ab')2 fragment.

The technical term "F(ab')2 fragment" refers to the part containing two light chains and two heavy chains, where the two heavy chains include the constant region between the _CH 1 domain and the _CH 2 domain. This forms an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The technical term "variable fragment (Fv)" refers to the smallest antibody fragment that contains the _VH and _VL of the antibody, but no constant region, and has all the antigen-binding sites. Typically, an Fv also contains a polypeptide linker between the _VH and _VL domains and is capable of forming the structure required for antigen binding. Different linkers can also be used to connect two antibody variable regions into a polypeptide chain, called a single-chain variable fragment (scFv) or single-chain Fv (sFv).

The technical term "single domain antibody (sdAb)" refers to a type of antibody that lacks the antibody light chain and only has _VH . Because a complete antibody contains two immunoglobulin light chains and two heavy chains, the molecular weight of a complete antibody is approximately 150-160 kDa. In contrast, the molecular weight of single-domain antibodies is only about 12-15kDa. Because of its small molecular weight, single-domain antibodies are also called nanobodies. Although single-domain antibodies have a simple structure, they can still achieve specific antigen-binding affinity that is comparable to or even higher than that of intact antibodies.

The term "binding to meEGFR" refers to the ability to interact with human meEGFR, and the term "binding to meEGFR" refers to the ability to interact with human meEGFR. "Antigen-binding site" refers to a discontinuous three-dimensional site on an antigen recognized by the anti-meEGFR antibody or its antigen-binding fragment of the present invention.

The "pharmaceutical composition" mentioned in this specification means a mixture containing one or more anti-meEGFR antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable carrier, such as physiological/ Pharmaceutically acceptable carriers and excipients. The purpose of pharmaceutical compositions is to facilitate administration to living organisms and facilitate the absorption of active ingredients to exert biological activity.

The following specific test examples are hereby further demonstrated to illustrate the present invention, so that those with common knowledge in the technical field to which the present invention belongs can fully utilize and practice the present invention without over-interpretation, and these test examples should not be regarded as These are not intended to limit the scope of the invention but are intended to illustrate the materials and methods of practicing the invention.

1. Anti-meEGFR antibodies or antigen-binding fragments thereof

The anti-meEGFR antibody or antigen-binding fragment thereof of the present invention specifically binds to meEGFR, and the meEGFR has asymmetric dimethylation modification at the R198 and R200 positions. The anti-meEGFR antibody or antigen-binding fragment thereof includes a heavy chain variable region (V _H ) and a light chain variable region (V _L ), wherein V _H includes a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), V _L includes light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3). The sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from the group consisting of: HCDR1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8, HCDR3 of SEQ ID NO:9, SEQ ID NO:10 LCDR1 of SEQ ID NO:11 and LCDR3 of SEQ ID NO:12; HCDR1 of SEQ ID NO:13, HCDR2 of SEQ ID NO:14, HCDR3 of SEQ ID NO:15, LCDR1 of SEQ ID NO:16 , LCDR2 of SEQ ID NO:17 and LCDR3 of SEQ ID NO:18; and HCDR1 of SEQ ID NO:19, HCDR2 of SEQ ID NO:20, HCDR3 of SEQ ID NO:21, LCDR1 of SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO:24.

Further, in the anti-meEGFR antibody or antigen-binding fragment thereof of the present invention, the sequences of V _H and V _L can be selected from the group consisting of: V _H comprising SEQ ID NO: 1 and V comprising SEQ ID NO: 2 _L ; VH comprising SEQ ID NO:3 and _VL comprising SEQ ID NO:4; and _VH comprising SEQ ID NO:5 and _VL comprising SEQ ID NO: ₆ .

The anti-meEGFR antibody or antigen-binding fragment thereof of the present invention can be selected from single domain antibodies, humanized A group consisting of antibodies, multimeric antibodies, single-chain variable fragments, Fab fragments, Fab' fragments, and F(ab')2 fragments. Furthermore, anti-meEGFR antibodies of the invention may be, for example, IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD or IgE isotype antibodies.

Please refer to Figure 1, which illustrates a schematic diagram of phage display technology for screening an aspect of anti-meEGFR antibodies or antigen-binding fragments thereof of the present invention. As shown in Figure 1, the experiment used phage display technology derived from human IgG library and through step-by-step screening with R198/R200 asymmetric dimethylated peptides (hereinafter referred to as "target peptides"), a total of 3 pairs were isolated. MeEGFR-specific anti-meEGFR antibodies or antigen-binding fragments thereof (hereinafter referred to as "Example 1", "Example 2", and "Example 3" respectively), V _H and V _L of Examples 1-3 The sequences of are shown in Table 1, and the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of Examples 1-3 are shown in Table 2.

Table 1. Sequences of V _H and V _L in Examples 1-3

Table 2. Sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of Examples 1-3

Please refer to Figure 2A and Figure 2B for the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis results of Examples 1-3, in which "M" is a protein molecular weight marker, and the numbers shown on the left side of the figure are are the corresponding molecular weights. Lanes 1-3 are the results of Examples 1-3 respectively. Figure 2A is the reduction SDS-PAGE results of V _H and V _L in Examples 1-3. Figure 2B is the Example. Non-reducing SDS-PAGE results for 1-3.

In order to further verify the affinity of Examples 1-3 for meEGFR, the binding affinity of Examples 1-3 to the target peptide and multiple different control peptides was further tested through ELISA analysis. The sequences and methylation modification positions of the target peptide and control peptides 1-5 are shown in Table 3. The target peptide has asymmetric dimethylation modifications at the R198 and R200 positions, while the control peptide 1 does not. With methylation modification, control peptide 2 has a monomethylation modification at the R198 position, control peptide 3 has a monomethylation modification at the R200 position, and control peptide 4 has a symmetrical dimethylation at the R198 position Modification, control peptide 5 has a symmetrical dimethylation modification at the R200 position.

Table 3. Sequences of target peptides and control peptides

Please refer to Figures 3A to 3C and Table 4. Figure 3A is a graph showing the results of the binding affinity analysis between Example 1 and the target peptide. Figure 3B is a graph showing the results of the binding affinity analysis between Example 2 and the target peptide. Figure 3C is a graph showing the results of the binding affinity analysis between Example 1 and the target peptide. The results of the binding affinity analysis between Example 3 and the target peptide are shown in Table 4. The results of the specificity and affinity analysis between Examples 1-3 and the target peptide are shown in Table 4.

Table 4. Specificity and affinity analysis results of Examples 1-3 and target peptides

The results show that Example 1, Example 2 and Example 3 all have high specificity for the target peptide, while for control peptide 4 (with symmetric dimethylation modification at R198) and control peptide 5 (at R200 has symmetrical dimethylation modification), it has very low specificity, low specificity or medium specificity, and the EC ₅₀ of Example 1, Example 2 and Example 3 for the target peptide is 1.712 respectively. nM, 0.03589nM and 0.02582nM, showing that Example 1, Example 2 and Example 3 have high specificity and high affinity for meEGFR. Since Example 1 has the best specificity, Example 1 was selected for subsequent testing.

2. Test kits and methods for detecting meEGFR-positive cancers

The detection kit of the present invention is used to detect meEGFR-positive cancer, and the detection kit includes the anti-meEGFR antibody or antigen-binding fragment thereof as described in the previous paragraph. The anti-meEGFR antibody or its antigen-binding fragment can be combined with a label. The label refers to a label that can be covalently combined with the anti-meEGFR antibody or its antigen-binding fragment of the present invention, or can be physically adsorbed on the anti-meEGFR antibody or its antigen-binding fragment of the present invention. Its resistance A substance that binds to the original binding fragment and can detect the presence of anti-meEGFR antibodies or antigen-binding fragments thereof. The label may be a fluorescent label, a chemiluminescent label, a radioisotope label, an enzyme label, a biotin label, or a combination thereof. Furthermore, fluorescent markers include but are not limited to fluorescent groups such as FAM, JOE or VIC. Chemiluminescent labels include but are not limited to electrochemiluminescent compounds or chemiluminescent compounds, such as luminol, isoluminol or acridinium salts. Radioactive isotope labels include but are not limited to ^3H , ^14C , ^32P , ^35S , ^125(131) I, and ^75Se . Enzyme labels include, but are not limited to, enzymes with detectable products, such as luciferase, peroxidase, alkaline phosphatase, β-galactosidase, and the like.

Since the detection kit of the present invention includes an anti-meEGFR antibody or an antigen-binding fragment thereof, it can be used to detect meEGFR-positive cancers, and the meEGFR-positive cancers can be breast cancer, colorectal cancer, prostate cancer, lung cancer, or pancreatic cancer. Please refer to FIG. 4 , which is a flow chart of a method 100 for detecting meEGFR-positive cancer in another aspect of the present invention. The method 100 for detecting meEGFR-positive cancer includes step 110 , step 120 , step 130 and step 140 .

Step 110 is to provide a sample to be tested and obtain the sample to be tested from the subject, where the sample to be tested may be a frozen tissue section, a tissue wax block section, or a tissue microarray.

Step 120 is to provide a detection kit of the present invention. The detection kit includes an anti-meEGFR antibody or an antigen-binding fragment thereof, wherein the anti-meEGFR antibody or an antigen-binding fragment thereof can be combined with a marker, and the marker can be a fluorescent marker, Chemiluminescent labels, radioisotope labels, enzyme labels, biotin labels or combinations thereof.

Step 130 is a binding step, where the sample to be tested is contacted with the anti-meEGFR antibody or its antigen-binding fragment and a binding reaction is performed.

Step 140 is a detection step to detect whether the sample to be tested contains an antibody cancer cell complex. When there is an antibody cancer cell complex in the sample to be tested, it can be determined that the subject who provided the sample to be tested is a meEGFR-positive cancer patient. The "antibody cancer cell complex" refers to the complex formed after the anti-meEGFR antibody or its antigen-binding fragment of the present invention specifically binds to meEGFR-expressing cancer cells. Methods for detecting the presence of antibody-cancer cell complexes include but are not limited to immunofluorescence, immunohistochemical staining, Western blotting, enzyme-linked immunosorbent assay (ELISA) or automated radiography.

After being judged by the method 100 for detecting meEGFR-positive cancer of the present invention, if the subject is not a meEGFR-positive cancer patient, he or she can still be considered for subsequent treatment with a clinically approved EGFR-TKI; however, if the subject is judged to be For patients with meEGFR-positive cancer, other treatments are needed Formula, for example, use the anti-meEGFR antibody or antigen-binding fragment thereof of the present invention, a pharmaceutical composition for treating cancer, meEGFR-specific chimeric antigen receptor expressing cells, innate cell adapters and/or antibody conjugates for treatment.

In the experiment, the detection kit of the present invention is further used to detect whether the sample to be tested has antibody cancer cell complexes, as a basis for whether the subject is a meEGFR-positive cancer patient. In the following test examples, the anti-meEGFR antibody or its antigen-binding fragment in the detection kit is Example 1, and the presence of antibody cancer cell complexes in the test sample is detected through immunohistochemical staining and immunofluorescence staining.

Please refer to Figure 5A and Figure 5B, which is a diagram of the binding specificity analysis results of another aspect of the detection kit of the present invention. Figure 5A is a diagram showing the results of the binding specificity analysis in the absence or presence of meEGFR (hot peptide) or unmethylated EGFR (cold peptide). peptide), the analysis results of immunohistochemical staining using the detection kit of the present invention on tissue samples of human breast cancer patients, Figure 5B shows the transfection vector, plasmid encoding wild-type EGFR or encoding R198/200K mutation After the plasmid of EGFR (unmethylated mutant) was introduced into 293FT cells, the analysis results of immunofluorescence staining were performed using the detection kit of the present invention. The results in Figure 5A show that the detection kit of the present invention can detect the antibody cancer cell complex in the group where meEGFR is present, while the results in Figure 5B show that when EGFR is an unmethylated mutant, the antibody cannot be detected. Cancer cell complex. The above results show that the detection kit of the present invention has high specificity and high affinity for meEGFR.

3. Pharmaceutical compositions for treating cancer

The pharmaceutical composition for treating cancer of the present invention includes the anti-meEGFR antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition for treating cancer may further comprise a chemotherapeutic agent, an immunomodulatory agent, a targeted therapy drug, an antibody drug, or a combination thereof, wherein the antibody drug is different from an anti-meEGFR antibody or an antigen-binding fragment thereof.

A total of 785 breast cancer patients were recruited in the experiment, and the tumor tissues provided by the breast cancer patients were tested using the detection kit of the present invention and the method of detecting meEGFR-positive cancers of the present invention. Please refer to Figure 6A to Figure 6C, which are analysis results of meEGFR expression in breast cancer patients. The results in Figure 6A show that about 33.8% of breast cancer patients have positive meEGFR expression in tumor tissues. Further analysis of meEGFR expression in different breast cancer patients , the results of Figure 6B and Figure 6C show that the ratio of meEGFR positive and meEGFR negative in patients with tubular type A breast cancer is 17.8%:43.7%, but in patients with tubular type B breast cancer, human epithelial factor receptor protein type 2 ( Among patients with human epidermal growth factor receptor 2 (HER2)-positive (HER2 ⁺ ) breast cancer and triple negative breast cancer (TNBC) patients, the rates of meEGFR-positive and meEGFR-negative were 5.4% respectively: 6.1%, 5.5%: 6.9% and 5.1%: 9.6%. The above results support that meEGFR is an important cancer-promoting marker in breast cancer patients.

The experiment further analyzed the correlation between the above-mentioned meEGFR expression and clinical prognosis. In this experimental example, two time variable evaluation indicators were used, namely overall survival (OS) and disease-free survival (disease-free survival). DFS). Among them, the evaluation index of overall survival is to set "death" as an event and observe the time from the entry of the clinical trial to the death of the subject, while the evaluation index of disease-free survival is to set "tumor recurrence" as an event and observe the time of the subject. The time from entry into clinical trials to tumor recurrence.

Please refer to Figure 6D and Figure 6E. Figure 6D is an analysis result of the overall survival time of meEGFR-positive breast cancer patients and meEGFR-negative breast cancer patients. Figure 6E is a disease-free survival time of meEGFR-positive breast cancer patients and meEGFR-negative breast cancer patients. The analysis results chart shows that a total of 265 meEGFR-positive breast cancer patients and a total of 520 meEGFR-negative breast cancer patients were analyzed. The results in Figure 6D and Figure 6E show that meEGFR-negative breast cancer patients have higher overall survival time and disease-free survival time than meEGFR-positive breast cancer patients. Using the log-rank test method, the p-value for overall survival is 0.003, with a p-value of 0.002 in disease-free survival. The above results once again verify that meEGFR is an important cancer-promoting marker for breast cancer patients.

Experiments further verify the therapeutic effect of the pharmaceutical composition for treating cancer of the present invention on meEGFR-positive cancer. In the following test examples, the anti-meEGFR antibody or antigen-binding fragment thereof of the pharmaceutical composition for treating cancer is Example 1. Different tumor cells were treated with Example 1 or cetuximab respectively, and the cells of the tumor cells were analyzed. Survival rate. The tumor cells used were different human breast cancer cell lines, namely SUM159PT, BT549, MCF7, MDA-MB-468, HCC1806 and HCC1954, among which SUM159PT, BT549, MDA-MB-468 and HCC1806 are estrogen receptors. triple-negative breast cancer cells that are negative for expression of receptor (ER), progesterone receptor (PR), and HER2; MCF7 is a tubular breast cancer cell that is positive for ER expression, but its EGFR is lowly expressed; HCC1954 is Breast cancer cells that overexpress HER2 but are negative for ER and PR expression. The tumor cell lines used were purchased from the American Type Culture Collection (ATCC).

In the experiment, SUM159PT and BT549 were seeded at a density of 5×10 ³ cells/per well, HCC1954, HCC1806 and MDA-MB-468 were seeded at a density of 8×10 ³ cells/per well, and MCF7 was seeded at a density of 1×10 ⁴ cells/per well. In a 96-well plate, after culturing for 16-18 hours, the culture medium in the holes is removed and added with different concentrations of DMEM-F12 culture medium of Example 1 or cetuximab (concentrations of 1 nM, 10 nM, 100 nM and 1000 nM respectively), and the MTT test was performed after culturing for 48 hours. The steps of the MTT test are to remove the culture medium in the 96-well plate, add 100 μl of DMEM-F12 containing 5 μg/mL MTT, and incubate it in the incubator for 4 hours. Completely remove the solution in the well plate, add 100 μL of DMSO, and wait until the crystals are dissolved. Use an enzyme immunoassay analyzer to read the absorbance value at a wavelength of 570 nm.

Please refer to Figures 7A to 7F, which are analysis results of tumor cell death induced by a pharmaceutical composition for treating cancer according to another embodiment of the present invention. Figure 7A is an analysis result of SUM159PT, and Figure 7B is an analysis result of BT549. Figure 7C is the analysis result figure of MCF7, Figure 7D is the analysis result figure of MDA-MB-468, Figure 7E is the analysis result figure of HCC1806, and Figure 7F is the analysis result figure of HCC1954. The results show that when the concentration of Example 1 is 1000 nM, in SUM159PT, BT549 and MCF7, the pharmaceutical composition for treating cancer of the present invention can achieve a better inhibitory effect on tumor cell growth than cetuximab, while in MDA- Among MB-468, HCC1806 and HCC1954, it can achieve the same inhibitory effect on tumor cell growth as cetuximab.

In the experiment, Example 1 was further combined with chemotherapy drugs as a pharmaceutical composition for treating cancer according to another embodiment of the present invention, and its effect of inhibiting the growth of tumor cells was analyzed. The tumor cells used were SUM159PT, and the chemotherapy drugs used were 5-fluorouracil and gemcitabine. In the experiment, a group treated with chemotherapy drugs alone or cetuximab combined with chemotherapy drugs was used as a comparative example. The treatment strategies of Examples 4-7 and Comparative Examples 1-6 are shown in Table 5. The control group was not treated with any Antibodies and chemotherapeutic drugs SUM159PT. After SUM159PT treated Comparative Examples 1-6 and Examples 4-7 respectively, a cell survival rate test was performed.

Table 5. Treatment strategies of Examples 4-7 and Comparative Examples 1-6

Please refer to Figure 8A and Figure 8B , which is an analysis result diagram of the death of SUM159PT induced by a pharmaceutical composition for treating cancer according to another aspect of the present invention. As can be seen from the results in Figure 8A, cetuximab was combined with the treatment In Comparative Examples 2 and 3 with 5-fluorouracil, no corresponding inhibitory effect was seen when the concentration of cetuximab was increased. However, in Example 4 and Example 5, which were combined with 5-fluorouracil and Example 1, it can be seen that The effect of inhibiting the growth of SUM159PT is concentration-dependent, and among all groups, Example 5 has the best effect of inhibiting the growth of SUM159PT. The results in Figure 8B show that in Comparative Examples 5 and 6 where cetuximab and gemcitabine were combined, when the concentration of cetuximab increased, it was seen that the effect of inhibiting the growth of SUM159PT was concentration-dependent, while the combined treatment From Example 1 and Example 6 and Example 7 of gemcitabine, it can also be seen that the effect of inhibiting the growth of SUM159PT is concentration-dependent, and among all groups, Example 7 has the best effect of inhibiting the growth of SUM159PT. The above results show that simultaneous administration of the anti-meEGFR antibody or antigen-binding fragment thereof and chemotherapy drugs of the present invention has a synergistic effect and can more significantly inhibit the growth of tumor cells.

4. meEGFR-specific chimeric antigen receptor, nucleic acid, meEGFR-specific chimeric antigen receptor expressing cells and pharmaceutical compositions for treating cancer

Please refer to FIG. 9 , which illustrates a schematic diagram of the theoretical structure of yet another aspect of the meEGFR-specific chimeric antigen receptor 200 of the present invention. The meEGFR-specific chimeric antigen receptor 200 of the present invention includes an extracellular domain 210 and an intracellular information transmission domain 230.

Extracellular domain 210 is used to recognize meEGFR, and meEGFR has asymmetric dimethylation modification at R198 and R200 positions. Extracellular domain 210 includes the anti-meEGFR antibody or antigen-binding fragment thereof of the present invention.

Further, the meEGFR-specific chimeric antigen receptor 200 may further include a transmembrane domain 220, which connects the extracellular domain 210 and the intracellular information transmission domain 230. Transmembrane domain 220 may comprise a transmembrane portion of a costimulatory molecule, such as a transmembrane portion of a T cell costimulatory molecule. In detail, the transmembrane domain 220 can be CD137 (4-1BB), T cell receptor alpha chain, T cell receptor beta chain, CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, CD11a (ITGAL ), CD11b(ITGAM), CD11c(ITGAX), CD11d(ITGAD), CD18(ITGB2), CD19(B4), CD27(TNFRSF7), CD28, CD29(ITGB1), CD30(TNFRSF8), CD40(TNFRSF5), CD48 (SLAMF2), CD49a(ITGA1), CD49d(ITGA4), CD49f(ITGA6), CD66a(CEACAM1), CD66b (CEACAM8), CD66c(CEACAM6), CD66d(CEACAM3), CD66e(CEACAM5), CD69(CLEC2), CD79A, CD79B, CD84(SLAMF5), CD96(Tactile), CD100(SEMA4D), CD103(ITGAE), CD134( OX40), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2 ), CD160( BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3ζ), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276( B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6), CD353( SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulatory molecule (ICOS), LFA-1 (CD11a/CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL- 2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS(GrpL), SLP-76(LCP2), PAG1/CBP, CD83 ligand, Fcγ receptor, MHC class I molecules, MHC class II Molecules, TNF receptor proteins, immunoglobulins, interleukin receptors, integrins, activated NK cell receptors, Toll ligand receptors, or combinations thereof.

Intracellular information transfer domain 230 may include costimulatory domain 231 and primary information transfer domain 232. The intracellular information transfer domain 230 can be CD137 (4-1BB), activated NK cell receptor, CD276 (B7-H3), BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55) , CD18, CD19, CD19a, CD2, CD247, CD27, CD28, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96(Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, interleukin receptor, DAP-10, DNAM1(CD226), Fcγ receptor, GADS, GITR, HVEM(LIGHTR), IA4, ICAM-1, Igα(CD79a) , IL2Rβ, IL2Rγ, IL7Rα, immunoglobulin-like protein, inducible T cell costimulatory molecule (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT , LFA-1, ligand that specifically binds CD83, LIGHT (TNFSF14), LTBR, Ly9 (CD229), lymphoid antigen-1 (LFA-1 (CD11a/CD18)), MHC class I molecules, NKG2C, NKG2D, NKp30 , NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, CD279 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAMF1 (CD150; IPO-3) ,SLAMF4(CD244;2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, Toll ligand receptor, TRANCE/RANKL, VLA1, VLA-6, or combinations thereof.

The nucleic acid of the present invention encodes the meEGFR-specific chimeric antigen receptor as described in the previous paragraph. The "nucleic acid" may include a nucleic acid containing naturally occurring and/or non-naturally occurring nucleotides and bases, for example, a nucleic acid containing a main chain. Modified ones may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleotide sequence" refers to the linear sequence of nucleotides that make up a nucleic acid molecule or polynucleotide.

The nucleic acid of the present invention sequentially includes a fragment encoding an extracellular domain and a fragment encoding an intracellular information transmission domain from the 5' end to the 3' end. The encoding extracellular domain fragment includes a nucleic acid encoding the anti-meEGFR antibody of the present invention or an antigen-binding fragment thereof, which includes an encoding _VH fragment and an encoding _VL fragment, and the encoding _VH fragment includes, for example, SEQ ID NO: 44, SEQ ID NO: 46 Or the nucleotide sequence shown in SEQ ID NO:48, the encoding _VL fragment includes the nucleotide sequence shown in SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:49. In addition, the nucleic acid encoding the anti-meEGFR antibody or antigen-binding fragment thereof of the present invention includes one or more encoding CDR fragments selected from SEQ ID NO: 50 to SEQ ID NO: 67.

Further, the nucleic acid may further comprise a segment encoding a transmembrane domain, which connects a segment encoding an extracellular domain and a segment encoding an intracellular information transmission domain. The segment encoding the intracellular information transmission domain may include the segment encoding the costimulation domain and the segment encoding the primary information transmission domain.

The meEGFR-specific chimeric antigen receptor-expressing cells of the present invention comprise immune cells and the nucleic acid of the present invention. The meEGFR-specific chimeric antigen receptor-expressing cells are obtained by transfecting the nucleic acid of the present invention into immune cells. The immune cells can be T cells or natural killer cells. The meEGFR-specific chimeric antigen receptor-expressing cells of the present invention can specifically recognize meEGFR on the tumor cell membrane, thereby poisoning the tumor cells. Therefore, the meEGFR-specific chimeric antigen receptor expressing cells of the present invention can be combined with a pharmaceutically acceptable carrier to serve as another aspect of the pharmaceutical composition for treating cancer of the present invention.

The experimental preparation of the meEGFR-specific chimeric antigen receptor of Example 8 (hereinafter referred to as "Example 8") is an example, which includes an IgGκ leader sequence (sequence shown in SEQ ID NO: 31), an anti-meEGFR antibody of the present invention The scFv (sequence shown in SEQ ID NO:32), CD28 hinge region (sequence shown in SEQ ID NO:33), CD28 transmembrane domain (sequence shown in SEQ ID NO:34), CD28 costimulatory domain (sequence shown in SEQ ID NO:34) The sequence is shown in SEQ ID NO: 35) and the CD3ζ primary information transmission domain (the sequence is shown in SEQ ID NO: 36), and the nucleic acid encoding Example 8 is constructed using a lentiviral vector, and then packaged into corresponding lentiviral particles. Lentiviral particles were used to transduce the nucleic acid encoding Example 8 into the NK-92 cell line (hereinafter referred to as "NK-92") to obtain the meEGFR-specific chimeric antigen receptor expressing cells of Example 9 (hereinafter referred to as "NK-92"). "Example 9"), and conduct the cytotoxicity test of Example 9.

The nucleic acid encoding Example 8 includes an IgGκ leader sequence fragment (sequence shown in SEQ ID NO:37), an scFv fragment encoding an anti-meEGFR antibody (sequence shown in SEQ ID NO:38), and a CD28 hinge region fragment (sequence shown in SEQ ID NO:38). The sequence is shown in SEQ ID NO:39), the fragment encoding the CD28 transmembrane domain (the sequence is shown in SEQ ID NO:40), the fragment encoding the CD28 co-stimulatory domain (the sequence is shown in SEQ ID NO:41) and the encoding CD3ζ primary Information transfer domain fragment (sequence shown in SEQ ID NO:42).

The experiment was divided into two groups. One group used tumor cells that were transfected with a plasmid encoding wild-type PRMT1, the human colorectal cancer cell line SW620 (hereinafter referred to as "SW620"), and the other group used tumor cells that were transfected with a plasmid encoding wild-type PRMT1. SW620, a plasmid encoding a mutant PRMT1 that has lost enzyme activity (hereinafter referred to as "mutant PRMT1"), was mixed with tumor cells and Example 9 at a ratio of 2:1. co-cultured in a humidified incubator of the Zoom System (Essen Bioscience) for 24 hours, and then followed the manufacturer's instructions for use. The Zoom System performs cytotoxicity assays through matrix detection with IncuCyte Caspase-3/7 Green Apoptosis Detection Reagent to measure the area with fluorescent signal and evaluate cell apoptosis, where the green fluorescent signal represents the apoptotic cell population.

Please refer to Figure 10A, which is an analysis result of the specific cleavage of SW620 in Example 9. The results show that introducing wild-type PRMT1 into SW620 will increase the methylation modification of EGFR, which in turn can increase the toxicity of Example 9 to SW620. killing effect, and the group into which mutant PRMT1 was introduced was the negative control group.

In the experiment, it was also tested whether Example 9 can induce the death of MDA-MB-231. In the experiment, MDA-MB-231 was first treated with shRNA targeting PRMT1 (shPRMT1) and control shRNA (shVOID). MDA-MB-231 (hereinafter referred to as "231-shPRMT1") that treated shPRMT1 was knocked out due to PRMT1. The degree of EGFR methylation modification will be reduced, while the degree of EGFR methylation modification of MDA-MB-231 (hereinafter referred to as "231-shVOID") that treats shVOID will not be affected. The night before the experiment, 231-shPRMT1 and 231-shVOID were seeded into a 96-well plate at a density of 5×10 ³ cells/well. After 16-18 hours, the culture medium was removed and 2.5×10 ³ cells were added to each well. Example 9 or NK-92 and tumor cells were co-cultured, and DMEM-F12 containing IncuCyte Caspase-3/7 green cell apoptosis detection reagent (250× dilution) was added to each well, and placed The zoom system is set to take pictures every 2 hours under white light and fluorescence conditions, and leave it for a total of 24 hours before use. The analysis software of the zoom system was used to perform analysis to confirm whether the presence of EGFR methylation modification affects the poisonous effect of Example 9 on MDA-MB-231.

Please refer to Figure 10B, which is a diagram showing the analysis results of specific cleavage of MDA-MB-231 in Example 9. The results show that Example 9 has the best poisoning effect on 231-shVOID, which has a normal degree of EGFR methylation modification, while the poisoning effect of Example 9 on 231-shPRMT1, which has a reduced degree of EGFR methylation modification, is the same as that of native NK- 92 is no different.

The above results show that the meEGFR-specific chimeric antigen receptor expressed by the meEGFR-specific chimeric antigen receptor expressing cells of the present invention can specifically bind to meEGFR on tumor cells, making the meEGFR-specific chimeric antigen receptor Antigen receptor-expressing cells have immunotoxic activity and can effectively kill tumor cells. Especially when the degree of EGFR methylation modification of tumor cells is normal, the tumor cells can be effectively made sensitive to killing by the meEGFR-specific chimeric antigen receptor expressing cells of the present invention.

5. Innate Cell Adapter

The innate cell adapter of the present invention includes a meEGFR antigen recognition domain and at least one immune cell receptor binding domain. The meEGFR antigen recognition domain includes an anti-meEGFR antibody or an antigen-binding fragment thereof of the present invention. At least one immune cell receptor binding domain is sensitive to CD3. One or more of , CD8, CD16, CD19 or NKG2D has binding specificity.

The at least one immune cell receptor binding domain can specifically bind to receptors on various immune cells. The immune cells can be selected from T cells, natural killer cells, B cells, dendritic cells, and monocytes. , macrophages, neutrophils, mesenchymal stem cells and neural stem cells.

Furthermore, the innate cell adapter of the present invention can be a bispecific T cell adapter, a trispecific T cell adapter or a multispecific T cell adapter; the innate cell adapter can also be a bispecific natural killer cell adapter. body, trispecific natural killer cell adapter or multispecific natural killer cell adapter.

The innate cell adapter of the present invention can be based on the structure of scFv, connecting VL _and /or _VH to each other through a short connecting peptide, and the connecting peptide is at the carboxyl terminus of one variable domain and the other variable domain. A bridge of approximately 3.5 nm is formed between the amine termini. For example, the meEGFR antigen recognition domain of the innate cell adapter of the present invention can be a scFv structure containing _VL -linked peptide- _VH or _VH -linked peptide- _VL . The _VH sequence can be selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, and _{the VL} sequence can be selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO: composed of 6 For example, the connecting peptide can be a repeated fragment of the sequence shown in SEQ ID NO:43, which is (GGGGS)n or a variant thereof, where n is a non-zero natural number, preferably in the range of 1 to 20.

The innate cell adapter of the present invention can be obtained by the following method: construct it using the nucleic acid encoding the anti-EGFR antibody or its antigen-binding fragment of the present invention and the nucleic acid encoding the anti-CD3 antibody and an expression vector, and introduce the constructed expression vector into CHO cells or HEK293 cells to obtain transfected cells. The obtained transfected cells are then cultured, and the culture fluid is purified by chromatography to obtain the innate cell adapter of the present invention.

6. Antibody conjugates

The antibody conjugate of the present invention includes an anti-meEGFR antibody or an antigen-binding fragment thereof of the present invention and an effector molecule. The effector molecule is coupled to the anti-meEGFR antibody or an antigen-binding fragment thereof through a chemical bond or a linker, wherein the effector molecule is a toxin, a growth factor, or a growth factor. Inhibitors, toxic proteins, radionuclides, radioisotopes, chemotherapeutic drugs, or combinations thereof.

Further, the coupling between the anti-meEGFR antibody or its antigen-binding fragment and the effector molecule of the present invention can be achieved through a coupling agent, wherein the coupling agent can be a non-selective coupling agent, a carboxyl coupling agent, a peptide chain and One or more of the disulfide coupling agents, the non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the anti-meEGFR antibody or antigen-binding fragment thereof of the present invention, such as glutaraldehyde. The carboxyl coupling agent may be one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acyl hydrazone coupling agent (the coupling site is an acyl hydrazone).

The linker can be a degradable linker or a non-degradable linker, where degradable linkers are typically susceptible to degradation in the intracellular environment, for example at the target site, thereby releasing the drug. Suitable degradable linkers include enzymatically degradable linkers, which include peptidyl linkers or sugar linkers that can be degraded by intracellular proteases, such as lysosomal or endosomal proteases. The peptidyl linker can be a dipeptide such as valine-citrulline, phenylalanine-lysine or valine-alanine. Sugar linkers are, for example, glucoside-containing linkers that are degradable by glucosidases. Other suitable degradable linkers include pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, e.g., hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Nondegradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by proteases.

The toxin can be a small molecule toxin or an enzymatically active toxin, such as, but not limited to, diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin ricin A chain, abrin A chain A chain), modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein, Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin , phenomycin, neomycin or trichothecene.

The radionuclide may be selected from the group consisting of: ¹¹¹ In, ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ¹²⁵ I and ³ H. The radioactive isotope may be selected from the group consisting of: At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu radioactive isotopes.

The chemotherapeutic drug may be selected from the group consisting of: 5-fluorouracil, gemcitabine, maytansinoid, auristatin, dolastatin, kach calicheamicin or its derivatives, anthracycline, methotrexate, vindesine, taxanes, trichothecene, CC1065, Catharanthus roseus alkaloids, methotrexate, adriamicin, vincristine, vinblastine, etoposide, doxorubicin, Melphalan, mitomycin C, chlorambucil and daunorubicin.

First, in order to test that the antibody conjugate of the present invention can be internalized into tumor cells by tumor cells through endocytosis, Example 1 was experimentally coupled to the dye DyLight 488 to form the antibody conjugate of Example 10 ( (hereinafter referred to as "Example 10") is used as an example, and then Example 10 is added to the culture medium of MDA-MB-231 and MDA-MB-468 and cultured together for 30 minutes to observe whether Example 10 can be internalized into MDA- intracellularly of MB-231 and MDA-MB-468. Please refer to Figure 11A, which is an analysis result diagram of Example 10 internalized by MDA-MB-231 and MDA-MB-468. The results show that Example 10 can be seen in both MDA-MB-231 and MDA-MB-468. Green fluorescence signal shows that both TNBC cell lines MDA-MB-231 and MDA-MB-468 have good ability to internalize the antibody conjugate of the present invention.

In the experiment, vectors, plasmids encoding wild-type EGFR or plasmids encoding R198/200K mutant EGFR (unmethylated mutant) were transfected into SW620, and Example 10 was added to the culture medium of the above cells. After culturing for 30 minutes, observe whether Example 10 can be internalized into the cells of SW620.

Please refer to Figure 11B, which is a graph showing the analysis results of Example 10 internalized by SW620. The results show that in SW620 of the plasmid encoding wild-type EGFR, Example 10 can be seen to be internalized into SW620 cells. However, in SW620 transfected with vectors or plasmids encoding R198/200K mutant EGFR (unmethylated mutant), it can be seen that the green fluorescence signal of Example 10 is located at the periphery of the cell membrane and is not internalized into the cells of SW620. .

The above results show that the meEGFR antibody or antigen-binding fragment thereof on the antibody conjugate of the present invention has high specificity for meEGFR, and therefore can identify meEGFR-expressing tumor cells and be internalized by meEGFR-expressing tumor cells through endocytosis. in it. In this way, the effector molecules on the antibody conjugate can be internalized into the tumor cells together, so that the effector molecules can play the role of poisoning the tumor cells.

In order to test whether the antibody conjugate of the present invention can induce tumor cell death, another example is to use the anti-meEGFR antibody or antigen-binding fragment thereof in the present invention to couple with effector molecules to generate an antibody conjugate. In detail, Example 1 was coupled with the chemotherapy drug monomethyl auristatin E (MMAE) to form the antibody conjugate of Example 11 (hereinafter referred to as "Example 11"), in which the antibody conjugate based on half Example 11 of cystine (Cystenine) can be constructed using two methods: continuous coupling and in-situ coupling. The linker-MMAE payload was synthesized as a peptide-based maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol-p-nitrophenyl carbonate (Mc-Val- Cit-PABC-PNP) connectors or rebridge connectors react with MMAE.

The traditional linker-MMAE payload was constructed as follows: 18.20 μmol of MMAE, 16.38 μmol of Mc-Val-Cit-PABC-PNP, and 3.64 μmol of hydroxybenzotriazole were dissolved and mixed in 500 μL of dimethylformamide. Add 18.20 μmol of pyridine to the mixture and mix frequently after 2 min. An additional 20 μmol of trifluoroacetic acid (TFA) was added, and the reaction was completed after 24 hours.

The method of constructing the rebriging linker-MMAE payload is as follows: In the experiment, the rebriging linker was first synthesized, and 3.91 mmol of 6-aminocaproic acid and 3.91 mmol of 3,4-dibromofuran were mixed in 20 mL of acetic acid. -2,5-dione. After stirring at room temperature for 10 minutes, the solution was heated at 100°C for 18 hours, the solvent was removed under vacuum, and the linker was purified using silica gel, using 0-40% methylene chloride/ethyl acetate as the eluent. . Mix 13.55 μmol of N,N′-diisopropylcarbodiimide, 13.55 μmol of N,N-diisopropylethylamine, and 33.85 μmol of religation linker in 0.25 mL of dichloromethane. Mix frequently for 1 hour at room temperature. Add 13.55 μmol of MMAE and mix frequently for another 16 hours. The synthesized rejoiner-MMAE was solvent removed via a vacuum pump and then analyzed using a Waters HPLC system equipped with a 600 controller/pump and a 996PDA detector. Perform purification. The purification process uses C18(2) with 5μm and a 250 × 10 mm reversed-phase C18 column (Phenomenex Luna) with a gradient elution buffer of phase A (water + 0.1% TFA) and phase B (acetonitrile). Purified products were confirmed by Agilent 6500 series accurate mass Q-TOF LC/MS (Agilent Technologies).

The continuous coupling method is to reduce 5 mg/mL of anti-meEGFR antibody or its antigen-binding fragment with 1 mM dithiothreitol (DTT) in 50 mM borate buffer at pH 8.0 at 37°C for 1 hour. Repeat buffer exchanges were performed using a Pierce dialysis column containing 1mM PBS buffer. The traditional linker-MMAE payload and the religated linker-MMAE payload were mixed with the reduced anti-meEGFR antibody or its antigen-binding fragment respectively. The molar ratio of traditional linker-MMAE payload:anti-meEGFR antibody or its antigen-binding fragment was 6.6. : 1, the molar ratio of religated linker-MMAE payload: anti-meEGFR antibody or its antigen-binding fragment is 4.4:1. After 1 h of incubation at 4°C, the reaction was terminated by adding a 20-fold molar excess of cysteine over the payload. The final product was purified by G-25 gel filtration to provide Example 11.

The in situ coupling method uses 7 equivalents of tris(2-carboxyethyl)phosphine (TCEP) to reduce 5 mg/mL of anti-meEGFR antibody or its antigen-binding fragment in 50 mM borate buffer at pH 8.0. Also add legacy connector-MMAE payload or reconnect linker-MMAE payload with 7 equivalents of TCEP. After incubation at 37°C for 2 hours, purification by G-25 gel filtration gave Example 11.

The experiment further tested whether Example 11 could induce tumor cell death, and the tumor cells used were MDA-MB-468. The day before the test, MDA-MB-468 was seeded in a 96-well plate at a density of 8×10 ³ cells/well. After culturing for 16-18 hours, the culture medium in the holes was removed, and Example 1 containing different concentrations was added. , DMEM-F12 culture medium of Example 11 or MMAE (concentrations are 0.1 nM, 1 nM, 10 nM, 100 nM and 1000 nM respectively), the group adding MMAE is the positive control group, and the MTT test is performed after 48 hours of culture. The method of the MTT test is to remove the culture medium in the 96-well plate, add 100 μl of DMEM-F12 containing 5 μg/mL MTT, and incubate it in the incubator for 4 hours. Completely remove the solution in the well plate and add 100 μL of DMSO until the crystals are dissolved. Use an enzyme immunoassay analyzer to read the absorbance value at a wavelength of 570 nm.

Please refer to Figure 12, which is an analysis result diagram of the death of MDA-MB-468 induced by Example 11. The results show that administration of Example 11 can significantly induce the death of MDA-MB-468, and its therapeutic effect is better than that of the group treated with antibody alone ( Example 1), and administration of Example 11 can achieve a therapeutic effect comparable to administration of MMAE. Although MMAE has a good therapeutic effect, it cannot be used as a separate medicine because of its toxicity. MMAE is coupled with the anti-meEGFR antibody or its antigen-binding fragment of the present invention to form an antibody conjugate, which can achieve a therapeutic effect equivalent to that of MMAE and can also reduce the toxicity of MMAE itself to organisms. In addition, the anti-meEGFR antibody or antigen-binding fragment thereof of the present invention can be coupled with other effector molecules. For example, MMAE can be replaced with chemotherapy drugs such as 5-fluorouracil or gemcitabine, which can not only reduce the toxicity of the chemotherapy drugs themselves to the organism, but also can The synergistic effect of the anti-meEGFR antibody or its antigen-binding fragment of the present invention and chemotherapy drugs can be further utilized to achieve better therapeutic effects.

In summary, the present invention provides an anti-meEGFR antibody or antigen-binding fragment thereof, a detection kit, a method for detecting meEGFR-positive cancer, a pharmaceutical composition for treating cancer, a meEGFR-specific chimeric antigen receptor, and an encoding meEGFR-specific Chimeric antigen receptor nucleic acids, meEGFR-specific chimeric antigen receptor expressing cells, innate cell adapters and antibody conjugates. The description proves that the anti-meEGFR antibody or its antigen-binding fragment of the present invention has high specificity and high affinity for meEGFR, and proves that the anti-meEGFR antibody or its antigen-binding fragment of the present invention is very suitable for detecting meEGFR in tumor tissue and can be used for Detection of meEGFR-positive cancers. The specification also proves that the pharmaceutical composition for treating cancer containing the anti-meEGFR antibody or antigen-binding fragment thereof of the present invention has excellent inhibitory effect on tumor cell growth. In addition, the cell test of tumor cells in the instructions proves that the meEGFR-specific chimeric antigen receptor and its expressing cells developed based on the anti-meEGFR antibody or its antigen-binding fragment of the present invention have excellent specific lysis of tumor cells. ability. Moreover, the anti-meEGFR antibody or its antigen-binding fragment of the present invention has good internalization ability in tumor cells. Therefore, antibody conjugates and innate cell adapters can be developed based on the anti-meEGFR antibody or its antigen-binding fragment of the present invention as clinical applications. application, overcoming and breaking through the current difficulties in clinical treatment of meEGFR-positive cancers in many aspects, providing a solution for low response rates in cancer treatment that is resistant to traditional EGFR-targeted drugs, and can also be applied to many types of meEGFR-positive cancers In the treatment of solid tumors, it is expected to greatly help cancer patients with drug selection and treatment effects. It has profound clinical significance, broad market value and global competitive advantages.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present invention. Therefore, the present invention The scope of protection shall be determined by the scope defined by the appended claims.

Claims

An anti-meEGFR antibody or an antigen-binding fragment thereof, characterized in that it specifically binds to a methylated epidermal growth factor receptor (meEGFR), and the meEGFR carries R198 and R200 sites With asymmetric dimethylation modification, the anti-meEGFR antibody or its antigen-binding fragment contains:

A heavy chain variable region (V H ) comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2) and a heavy chain complementarity determining region 3 (HCDR3); and

A light chain variable region (V L ), which includes a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2) and a light chain complementarity determining region 3 (LCDR3);

The sequences of the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are selected from the group consisting of:

The HCDR1 of SEQ ID NO:7, the HCDR2 of SEQ ID NO:8, the HCDR3 of SEQ ID NO:9, the LCDR1 of SEQ ID NO:10, the LCDR2 of SEQ ID NO:11 and SEQ ID NO:12 The LCDR3;

The HCDR1 of SEQ ID NO:13, the HCDR2 of SEQ ID NO:14, the HCDR3 of SEQ ID NO:15, the LCDR1 of SEQ ID NO:16, the LCDR2 of SEQ ID NO:17 and SEQ ID NO:18 of the LCDR3; and

The HCDR1 of SEQ ID NO:19, the HCDR2 of SEQ ID NO:20, the HCDR3 of SEQ ID NO:21, the LCDR1 of SEQ ID NO:22, the LCDR2 shown in SEQ ID NO:23 and SEQ ID NO :The LCDR3 shown in 24.
The anti-meEGFR antibody or antigen-binding fragment thereof according to claim 1, wherein the sequences of the V H and the V L are selected from the group consisting of:

The V H comprising SEQ ID NO: 1 and the V L comprising SEQ ID NO: 2;

The V H comprising SEQ ID NO: 3 and the V L comprising SEQ ID NO: 4; and

The VH comprising SEQ ID NO:5 and the VL comprising SEQ ID NO:6.
The anti-meEGFR antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-meEGFR antibody or antigen-binding fragment thereof is selected from the group consisting of a single domain antibody, a humanized antibody, a multimeric antibody, a single chain A group of variable fragments (scFv), a Fab fragment, a Fab' fragment and an F(ab')2 fragment.
A detection kit for detecting meEGFR-positive cancer, characterized in that the detection kit includes:

The anti-meEGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.
The detection kit of claim 4, wherein the anti-meEGFR antibody or antigen-binding fragment thereof is combined with a label, and the label is a fluorescent label, a chemiluminescence label, or a radioisotope label. , an enzyme label, a biotin label or a combination thereof.
A method for detecting meEGFR-positive cancer, characterized by comprising:

Provide a sample to be tested;

Provide the detection kit as claimed in claim 4;

Perform a binding step to contact the sample to be tested with the anti-meEGFR antibody or antigen-binding fragment thereof and perform a binding reaction; and

A detection step is performed to detect whether the sample to be tested has an antibody cancer cell complex.
The method for detecting meEGFR-positive cancer according to claim 6, wherein the meEGFR-positive cancer is breast cancer, colorectal cancer, prostate cancer, lung cancer or pancreatic cancer.
A pharmaceutical composition for treating cancer, characterized by containing:

The anti-meEGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; and

A pharmaceutically acceptable carrier.
The pharmaceutical composition for treating cancer according to claim 8, further comprising a chemotherapeutic agent, an immunomodulatory agent, a targeted therapy drug, an antibody drug or a combination thereof, wherein the antibody drug and the anti-cancer drug meEGFR antibodies or antigen-binding fragments thereof are different.
A meEGFR-specific chimeric antigen receptor, characterized by comprising:

An extracellular domain used to identify a methylated epidermal growth factor receptor (meEGFR), and the meEGFR has asymmetric dimethylation modifications at the R198 and R200 positions, and the extracellular domain includes as follows The anti-meEGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; and

An intracellular information transfer domain.
The meEGFR-specific chimeric antigen receptor according to claim 10, further comprising a transmembrane domain connecting the extracellular domain and the intracellular information transmission domain.
The meEGFR-specific chimeric antigen receptor according to claim 10, wherein the intracellular information transmission domain includes a co-stimulatory domain and a primary information transmission domain.
A nucleic acid, characterized in that it encodes the meEGFR-specific chimeric antigen receptor as claimed in claim 10.
A meEGFR-specific chimeric antigen receptor expressing cell, characterized by containing:

an immune cell; and

The nucleic acid as claimed in claim 13;

The meEGFR-specific chimeric antigen receptor expressing cells are obtained by transfecting the nucleic acid into the immune cells.
The meEGFR-specific chimeric antigen receptor expressing cell according to claim 14, wherein the immune cell is a T cell or a natural killer cell.
A pharmaceutical composition for treating cancer, characterized by containing:

The meEGFR-specific chimeric antigen receptor expressing cell as claimed in claim 14; and

A pharmaceutically acceptable carrier.
An innate cell adapter, characterized by containing:

A meEGFR antigen recognition domain, comprising the anti-meEGFR antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3; and

At least one immune cell receptor binding domain having binding specificity for one or more of CD3, CD8, CD16, CD19 or NKG2D.
The innate cell adapter of claim 17, wherein the innate cell adapter is a bispecific T cell adapter, a trispecific T cell adapter or a multispecific T cell adapter.
The innate cell adapter of claim 17, wherein the innate cell adapter is a bispecific natural killer cell adapter, a trispecific natural killer cell adapter or a multispecific natural killer cell adapter. .
An antibody conjugate, characterized by comprising:

The anti-meEGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; and

An effector molecule, the effector molecule is coupled to the anti-meEGFR antibody or its antigen-binding fragment through a chemical bond or a linker, wherein the effector molecule is a toxin, a growth inhibitor, a toxic protein, a radionuclide, a radioactive isotope, a chemotherapeutic drug, or a combination thereof.