Cetuximab variants targeting epidermal growth factor receptor resistance mutants

Abstract

The invention discloses a cetuximab variant CtxD103R of a targeted epidermal growth factor receptor drug-resistant mutant EGFRK489E (epidermal growth factor receptor). The cetuximab variant is obtained based on the rational design of wild type cetuximab. According to the present invention, the specific amino acid mutation D103R is performed on the heavy chain sequence of the wild type cetuximab, and the affinity experiment and the functional experiment are combined to research the influence of the mutated cetuximab variant CtxD103R on the drug resistance mutation EGFRK489E so as to finally prove that the cetuximab variant CtxD103R can be well combined with the EGFRK489E mutant, such that the EGFR phosphorylation can be hindered, and the EGFRK489E can be inhibited; the influence of the drug-resistant mutation K489E is weakened. The cetuximab variant CtxD103R disclosed by the invention lays a good foundation for research on resistance to drug-resistant mutation. The antibody has a wide application prospect in the field of tumor prevention and / or treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a cetuximab variant obtained by modifying a wild-type cetuximab by amino acid mutation, and using the cetuximab variant to combat an epidermal growth factor receptor drug-resistant mutation. Background Art

[0002] Epidermal growth factor receptor is a key therapeutic target for the treatment of metastatic colorectal cancer. Cetuximab is an IgG1 monoclonal antibody targeting epidermal growth factor receptor, which can inhibit the proliferation and migration of tumor cells and exert a strong anti-tumor effect. However, the selective pressure of anti-epidermal growth factor receptor therapy can cause mutations in genes related to metastatic colorectal cancer, resulting in acquired drug resistance. This may greatly reduce the efficacy of cetuximab and may even make it ineffective.

[0003] There are different treatment options for different types of drug-resistant mutations (G465R, S492R, etc.), such as the use of other antibodies (Sym004, MM-151, etc.), or mutation modification of wild-type cetuximab (Ctx-W52D, Ctx-VY, Ctx-Y104D, etc.), but current research does not involve treatments for the K489E drug-resistant mutation. Summary of the invention

[0004] In view of the defects of the above-mentioned background technology, the present invention designs a cetuximab variant Ctx targeting the drug-resistant mutation K489E of the epidermal growth factor receptor. D103R This cetuximab variant binds to the EGFR resistance mutant K489E It performed better than wild-type cetuximab and showed high efficiency in inhibiting EGFR K489E Effects of phosphorylation on mutants.

[0005] The present invention adopts the following technical solutions:

[0006] The invention performs amino acid mutation on the wild-type cetuximab Ctx heavy chain sequence, and combines affinity experiments and functional experiments to study the cetuximab variant Ctx obtained by mutation. D103R Drug-resistant EGFR mutations K489E The impact of Ctx D103R Able to bind EGFR with higher affinity K489E Mutants that block EGFR K489E Phosphorylation weakens the effect of the drug resistance mutation K489E. The cetuximab variant Ctx of the present invention D103R This has laid a good foundation for the study of anti-resistance mutations and is of great significance for the prevention and / or treatment of drug-resistant metastatic colorectal cancer.

[0007] In the above technical solution, the drug resistance mutation targeting epidermal growth factor receptor is a mutation of lysine K at position 489 of EGFR extracellular domain III to glutamic acid E. The cetuximab variant is obtained by a specific amino acid mutation at position 103 of the wild-type cetuximab heavy chain, where position 103 of the wild-type cetuximab heavy chain is aspartic acid D, and the target of the amino acid mutation is arginine R.

[0008] The cetuximab variant designed by the present invention has arginine R at position 103 of the heavy chain. D103R The amino acid sequence of the heavy chain is shown in SEQ ID NO.1, and the amino acid sequence of the light chain is shown in SEQ ID NO.2.

[0009] The present invention also provides a cetuximab variant Ctx D103R The coding gene of the heavy chain is shown in SEQ ID NO.3, and the nucleotide sequence of the light chain is shown in SEQ ID NO.4.

[0010] The present invention also provides a method for preparing a cetuximab variant Ctx D103R The method specifically comprises introducing the above encoding gene into host cells and culturing them to obtain the antibody.

[0011] The present invention also provides a drug for preventing and / or treating tumors, the active ingredient of which is the cetuximab variant Ctx D103R and / or the encoding gene; the tumor is colorectal cancer.

[0012] The present invention also provides a use of the above antibody and / or the above encoding gene in preparing a drug for preventing and / or treating tumors; the tumor is colorectal cancer.

[0013] The beneficial effects of the present invention are as follows:

[0014] The present invention performs amino acid mutation on wild-type cetuximab, and combines affinity experiments and functional experiments to verify the cetuximab variant Ctx D103R EGFR resistance mutation K489E The cetuximab variants can better bind to EGFR K489E Mutants that block EGFR K489E Phosphorylation weakens the effect of the drug-resistant mutation K489E, laying a good foundation for the study of anti-drug-resistant mutations. In addition, the antibody of the present invention has a wide range of application scenarios in the field of preventing and / or treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention is further described below in conjunction with the accompanying drawings;

[0016] Figure 1 The wild-type cetuximab and the cetuximab variant Ctx designed by the present invention are D103R EGFR K489E The results of affinity experiments.

[0017] Figure 2 Ctuximab variant Ctx D103R Inhibits EGFR resistance mutant K489E Functional phosphorylation assay results. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with embodiments.

[0019] Example 1: Testing of cetuximab variants Ctx D103R EGFR resistant mutant K489E Affinity

[0020] The amino acid mutation D103R at position 103 of the wild-type cetuximab heavy chain was used to obtain the cetuximab variant Ctx D103R .

[0021] Cetuximab binds to the epidermal growth factor receptor with high affinity, occupies the epidermal growth factor binding site, inhibits the proliferation and migration of tumor cells, and thus achieves anti-tumor activity. D103R The inventors conducted a study on the effect of cetuximab variant Ctx D103R EGFR resistant mutant K489E Affinity experiment.

[0022] This experiment was performed according to Open SPR TM The carboxyl chip was installed according to the instrument standard operating procedure. At the beginning, the detection buffer PBST (phosphate-buffered saline Tween buffer) was run at the maximum flow rate. After reaching the signal baseline, 200 μL of isopropanol was loaded and run for 10 seconds to remove bubbles. After reaching the baseline, the detection buffer was replaced and the flow rate was adjusted to 20 μl / min. After the chip was activated, the epidermal growth factor receptor resistant mutant EGFR diluted in activation buffer (10 mM sodium acetate, pH 5.0) was loaded. K489E , and then blocked after 4 minutes. D103RUse activation buffer to dilute to 900nM, 300nM, 100nM, and 33.3nM for analysis, keep the flow rate at 20μl / min, the binding time at 240 seconds, and the dissociation time at 360 seconds. The analysis method used in this experiment is a one-to-one analysis model. The specific parameters involved are as follows:

[0023]

[0024] As a control, the EGFR resistant mutant was tested under the same experimental conditions. K489E Binding affinity experiments with wild-type cetuximab. Figure 1 A and B show wild-type cetuximab and cetuximab variant Ctx, respectively. D103R EGFR K489E The affinity test results show that EGFR K489E Mutants and variants of cetuximab Ctx D103R The binding affinity is stronger than EGFR K489E Binding affinity of mutants to wild-type cetuximab.

[0025] Example 2: Cetuximab variant Ctx D103R Inhibits EGFR resistance mutant K489E Phosphorylation

[0026] Cetuximab achieves its anti-tumor function by occupying the epidermal growth factor binding site and inhibiting the phosphorylation of the epidermal growth factor receptor, thereby further inhibiting the activation of downstream tumor cell proliferation and migration signaling pathways. D103R The inventors conducted a study on the anti-tumor function of the cetuximab variant Ctx D103R Inhibits EGFR resistance mutant K489E Functional experiments on phosphorylation ( Figure 2 ).

[0027] The main experimental methods involved are cell culture and immunoblotting experiments. First, we constructed a cell line expressing the drug-resistant mutant of epidermal growth factor receptor EGFR K489E HEK293 cells were transiently transfected and re-plated from a 10 cm dish to a 6 cm dish 24 hours after transfection. After serum starvation (high glucose Dulbecco's modified eagle medium (DMEM) containing 2% fetal bovine serum) for 12 hours, 100 nM of the cetuximab variant Ctx was added. D103RAfter 2 hours of antibody treatment, epidermal growth factor (EGF) was added to stimulate the cells for 15 minutes and all cells were collected. D103K As a control, the cetuximab mutant Ctx D103R The same treatment was performed. All experimental groups were subsequently processed and developed using the immunoblotting method. The immunoblotting results showed that the cetuximab variant Ctx D103R Can inhibit the drug-resistant mutant EGFR K489E phosphorylation.

Claims

1. Targeting the drug-resistant mutant EGFR K489E Cetuximab variant Ctx D103R , which is characterized by: The drug resistance mutation of the epidermal growth factor receptor is that the lysine K at position 489 of the EGFR extracellular domain III is mutated to glutamic acid E; the cetuximab variant is obtained by mutating the aspartic acid D at position 103 of the wild-type cetuximab heavy chain to arginine R.

2. The method of targeting epidermal growth factor receptor drug-resistant mutant EGFR according to claim 1 K489E Cetuximab variant Ctx D103R , characterized in that, The amino acid sequence of its heavy chain is shown in SEQ ID NO.1, and the amino acid sequence of its light chain is shown in SEQ ID NO.

2.

3. The method for targeting the drug-resistant mutant EGFR of epidermal growth factor receptor as claimed in claim 1 K489E Cetuximab variant Ctx D103R The coding gene is characterized in that The nucleotide sequence of its heavy chain is shown in SEQ ID NO.3, and the nucleotide sequence of its light chain is shown in SEQ ID NO.

4.

4. A method for preparing the targeted epidermal growth factor receptor drug-resistant mutant EGFR according to claim 1 K489E Cetuximab variant Ctx D103R The method is to introduce the encoding gene described in claim 3 into a host cell, culture it, and obtain it.

5. A drug for preventing and / or treating tumors, wherein the active ingredient is the EGFR targeting resistant mutant of epidermal growth factor receptor as claimed in claim 1 or 2 K489E Cetuximab variant Ctx D103R And / or the encoding gene described in claim 3; the tumor is colorectal cancer.

6. The cetuximab variant Ctx according to claim 1 or 2 D103R And / or use of the coding gene according to claim 3 in the preparation of drugs for preventing and / or treating tumors; the tumor is colorectal cancer.

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