Cetuximab variants targeting epidermal growth factor receptor resistance mutants

Abstract

This invention discloses a targeted epidermal growth factor receptor resistance mutant EGFR K489E Cetuximab variant Ctx D103R The cetuximab variant described herein is rationally designed based on wild-type cetuximab. This invention utilizes a specific amino acid mutation (D103R) in the heavy chain sequence of wild-type cetuximab, combined with affinity and functional assays, to study the resulting cetuximab variant Ctx. D103R EGFR resistance mutation K489E The effects ultimately proved the efficacy of the cetuximab variant Ctx. D103R Able to better combine EGFR K489E The mutant inhibits EGFR phosphorylation, weakening the effect of the drug-resistant mutation K489E. The cetuximab variant Ctx of this invention... D103R This invention lays a solid foundation for research on drug resistance mutations. The antibody of this invention has broad application prospects in the prevention and / or treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to cetuximab variants obtained by modifying wild-type cetuximab through amino acid mutation, and their use against epidermal growth factor receptor resistance mutations. Background Technology

[0002] Epidermal growth factor receptor (EGFR) is a key therapeutic target for metastatic colorectal cancer. Cetuximab, an IgG1 monoclonal antibody targeting EGFR, can inhibit tumor cell proliferation and migration, exerting a potent anti-tumor effect. However, the selective pressure of anti-EGFR therapy can lead to mutations in genes associated with metastatic colorectal cancer, resulting in acquired resistance. This can significantly reduce the efficacy of cetuximab, or even render it ineffective.

[0003] Different treatment options exist for different drug resistance mutations (G465R, S492R, etc.), such as using other antibodies (Sym004, MM-151, etc.) or modifying wild-type cetuximab (Ctx-W52D, Ctx-VY, Ctx-Y104D, etc.). However, current research has not covered treatment methods for the K489E drug resistance mutation. Summary of the Invention

[0004] To address the shortcomings of the aforementioned background technology, this invention designs a cetuximab variant, Ctx, that targets the epidermal growth factor receptor K489E resistance mutation. D103R This cetuximab variant binds to the epidermal growth factor receptor resistance mutant EGFR. K489E It outperformed wild-type cetuximab and demonstrated highly effective inhibition of EGFR. K489E The role of phosphorylation in mutants.

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

[0006] This invention involves amino acid mutations in the heavy chain sequence of wild-type cetuximab Ctx, and then studies the resulting cetuximab variant Ctx using affinity and functional assays. D103R EGFR resistance mutation K489E The impact, ultimately proved Ctx D103R Capable of binding EGFR with higher affinity K489E Mutant that inhibits EGFR K489E Phosphorylation weakens the effect of the drug resistance mutation K489E. The cetuximab variant Ctx of this invention... D103R This research lays a solid foundation for the study of drug-resistant 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 the epidermal growth factor receptor is a mutation of lysine K to glutamate E at position 489 of the EGFR extracellular domain III. The cetuximab variant is obtained by a specific amino acid mutation at position 103 of the wild-type cetuximab heavy chain, where the wild-type cetuximab heavy chain position 103 is aspartic acid D, and the target amino acid mutation is arginine R.

[0008] The cetuximab variant designed in this invention has arginine R at position 103 of its heavy chain. Cetuximab variant Ctx 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] This invention also provides the cetuximab variant Ctx D103R The encoding gene has a heavy chain nucleotide sequence as shown in SEQ ID NO.3 and a light chain nucleotide sequence as shown in SEQ ID NO.4.

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

[0011] The present invention also provides a medicament for the prevention and / or treatment of 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 the use of the above-mentioned antibody and / or the above-mentioned encoding gene in the preparation of a drug for the prevention and / or treatment of tumors; wherein the tumor is colorectal cancer.

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

[0014] This invention involves amino acid mutations in wild-type cetuximab, and, combined with affinity and functional assays, validates the effectiveness of the cetuximab variant Ctx. D103R EGFR resistance mutation to epidermal growth factor receptor K489E Due to the influence of cetuximab variants, they can bind to EGFR more effectively. K489E Mutant that inhibits EGFR K489E Phosphorylation weakens the effect of the drug resistance mutation K489E, laying a solid foundation for research on resistance mutations. Furthermore, the antibody of this invention has broad application prospects in the prevention and / or treatment of tumors. Attached Figure Description

[0015] The present invention will be further described below with reference to the accompanying drawings;

[0016] Figure 1 It is wild-type cetuximab and the cetuximab variant Ctx designed in this invention. D103R EGFR, a drug-resistant mutant of epidermal growth factor receptor K489E The results of the affinity experiment.

[0017] Figure 2 It is a cetuximab variant Ctx D103R EGFR, a mutant that inhibits the epidermal growth factor receptor (EGFR) K489E Functional experimental results of phosphorylation. Detailed Implementation

[0018] The present invention will be further described below with reference to the embodiments.

[0019] Example 1: Testing the cetuximab variant Ctx D103R EGFR, a mutant resistant to epidermal growth factor receptor K489E affinity

[0020] The cetuximab variant Ctx was obtained by mutating the wild-type cetuximab heavy chain at position 103 (D103R). D103R .

[0021] Cetuximab exerts its antitumor activity by binding to the epidermal growth factor receptor (EGFR) with high affinity, thereby preempting EGFR binding sites and inhibiting tumor cell proliferation and migration. To validate the designed cetuximab variant Ctx... D103R The inventors developed the cetuximab variant Ctx to achieve the desired effect. D103R EGFR, a mutant resistant to epidermal growth factor receptor K489E Affinity experiments.

[0022] This experiment follows the Open SPR TM The standard operating procedure for the instrument is to install the carboxyl chip. Initially, run the detection buffer PBST (phosphate-Tween buffer) at maximum flow rate. After reaching the signal baseline, load 200 μL of isopropanol and run for 10 seconds to remove air bubbles. Once the baseline is reached, replace with the detection buffer and adjust the flow rate to 20 μL / min. After chip activation, load the epidermal growth factor receptor resistance mutant EGFR diluted in activation buffer (10 mM sodium acetate, pH 5.0). K489E After running for 4 minutes, the blockade was performed. The cetuximab variant Ctx was then administered. D103RThe sample was diluted with activation buffer to 900 nM, 300 nM, 100 nM, and 33.3 nM for analysis, maintaining a flow rate of 20 μl / min, a binding time of 240 seconds, and a dissociation time of 360 seconds. The analytical method used in this experiment was a one-to-one analytical model. Specific parameters involved are shown in the table below:

[0023]

[0024] As a control, the epidermal growth factor receptor resistance mutant EGFR was tested under the same experimental conditions as described above. K489E Binding affinity assay with wild-type cetuximab. Figure 1 Images A and B respectively show wild-type cetuximab and the cetuximab variant Ctx. D103R EGFR, a drug-resistant mutant of epidermal growth factor receptor K489E The affinity test results, obtained from the calculated affinity constant, show that EGFR K489E mutants and cetuximab variants Ctx D103R Its binding affinity is stronger than that of EGFR K489E Binding affinity of the mutant to wild-type cetuximab.

[0025] Example 2: Cetuximab variant Ctx D103R EGFR, a mutant that inhibits the epidermal growth factor receptor (EGFR) K489E Phosphorylation

[0026] Cetuximab exerts its anti-tumor function by preemptively occupying the epidermal growth factor (EGF) binding site, inhibiting EGF receptor phosphorylation, and thereby further inhibiting the activation of downstream signaling pathways related to tumor cell proliferation and migration. To further validate the cetuximab variant Ctx... D103R To assess its anti-tumor function, the inventors developed a cetuximab variant, Ctx. D103R EGFR, a mutant that inhibits the epidermal growth factor receptor (EGFR) K489E Functional experiments on phosphorylation ( Figure 2 ).

[0027] The main experimental methods involved are cell culture and Western blotting. First, an epidermal growth factor receptor (EGFR) resistant mutant was constructed. K489E HEK293 cells were transiently transfected for 24 hours, then re-seeded from 10 cm dishes to 6 cm dishes. After serum starvation (high-glucose Dulbecco's modified eagle medium containing 2% fetal bovine serum, DMEM medium) for 12 hours, 100 nM cetuximab variant Ctx was added. D103RAfter 2 hours of antibody treatment, epidermal growth factor (EGF) was added for 15 minutes of stimulation before all cells were harvested. Control, IgG antibody, and cetuximab variant Ctx groups were also included. D103K As a control, compared with the cetuximab mutant Ctx D103R All experimental groups underwent the same treatment. Subsequently, all experimental groups were treated and visualized using Western blotting. The Western blotting results showed that the cetuximab variant Ctx... D103R It can inhibit the epidermal growth factor receptor resistance mutant EGFR K489E Phosphorylation.

Claims

1. The use of the cetuximab variant CtxD103R and / or its encoding gene in the preparation of a tumor-treating drug, characterized in that: The CtxD103R is obtained by mutating aspartic acid D at position 103 of the heavy chain of wild-type cetuximab to arginine R. 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. The nucleotide sequence of the heavy chain of the encoding gene is shown in SEQ ID NO.3, and the nucleotide sequence of its light chain is shown in SEQ ID NO.

4. The tumor is colorectal cancer carrying a drug-resistant mutation in lysine K at position 489 of the extracellular domain III of epidermal growth factor receptor EGFR, which is mutated to glutamate E. The colorectal cancer is metastatic colorectal cancer that has acquired resistance to wild-type cetuximab.

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