Cetuximab mutant of biased targeting S492R or G465R mutant EGFR and application of cetuximab mutant

Abstract

The invention discloses a cetuximab mutant of biased targeting S492R or G465R mutant EGFR (Epidermal Growth Factor Receptor) and application of the cetuximab mutant, and relates to the technical field of antibody drugs. Compared with an antibody in the prior art, the binding force of the antibody to a wild type EGFR is stronger than that of the antibody to a mutant type EGFR, and the antibody does not have point mutation bias; according to the invention, a Cetuximab mutant with an amino acid sequence shown as SEQ ID NO.1 in an antibody heavy chain variable region obtained by combined mutation can be biased to be combined with S492R and G465R mutant EGFR, and the selectivity of the antibody drug on tumor cells of an anti-EGFR therapy is enhanced by the point mutation biased property of the antibody drug, so that the treatment is more accurate and effective.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody drugs, and in particular to a cetuximab mutant biasedly targeting S492R or G465R mutant EGFR and an application thereof. Background Art

[0002] Colorectal cancer (CRC) is one of the major diseases that threaten human life and health. Globally and in China, colorectal cancer is one of the top three most common cancers, and the incidence is on the rise among young people under the age of 50. Among colorectal cancer patients, patients with metastatic colorectal cancer (mCRC) have limited effective treatment options, a higher risk of death, and a 5-year survival rate of less than 20%. Currently, the treatment options for metastatic colorectal cancer are mainly individualized based on tumor biomarkers. Cetuximab and panitumumab are monoclonal antibodies (mAbs) targeting epidermal growth factor receptor (EGFR). Since the US Food and Drug Administration (FDA) approved cetuximab for the treatment of mCRC patients in 2004, cetuximab has jumped from third-line drugs to first-line drugs. According to the 2022 NCCN guidelines, cetuximab or panitumumab combined with chemotherapy is the first-line treatment for patients with RAS / RAF wild-type mCRC whose tumors are located on the left side. This therapy can effectively alleviate the disease, including prolonging progression-free survival (PFS) and overall survival (OS), and improving the objective response rate (ORR). In addition, cetuximab combined with the RAF kinase inhibitor encorafenib was also approved by the US FDA in 2020 for the treatment of patients with BRAFV600E mutant mCRC. This therapy can also prolong patients' PFS and OS and improve ORR, providing more treatment options for patients with BRAF V600E mutant mCRC cancer with extremely poor prognosis, and also expanding the applicable population of anti-EGFR therapy in mCRC patients. The successful application of anti-EGFR therapy in the treatment of patients with metastatic colorectal cancer has established the important position of this therapy in the treatment of metastatic colorectal cancer.

[0003] As anti-EGFR therapy becomes more and more important in the treatment of metastatic colorectal cancer, drug resistance is also increasing. The main resistance mechanisms found in clinical practice include mutations in the EGFR extracellular domain and mutations in signaling molecules in the downstream signaling pathway of EGFR. Among them, the acquired resistance point mutations in the EGFR extracellular region that completely lose the binding activity of cetuximab or panitumumab are more malignant. Because in the case of mutation resistance of signaling molecules in the downstream signaling pathway of EGFR, the effects of these resistance mechanisms on the efficacy of cetuximab can still be offset by targeted combination therapy. For example, in patients with BRAF V600E mutant mCRC, cetuximab can be treated by combining RAF kinase inhibitors. However, when point mutations occur in the extracellular region of EGFR, cetuximab cannot bind to it, and the possibility of combined therapy is lost. Mutations in the EGFR extracellular domain are mainly concentrated in exon 12 of EGFR, where ECD domain III is located, and where EGFR binds to cetuximab or panitumumab. EGFR extracellular domain mutations are mostly secondary resistance point mutations that occur under the pressure of cetuximab and panitumumab. The resistance mechanism is to prevent cetuximab or panitumumab from binding to EGFR with resistance point mutations, thereby making cetuximab or panitumumab lose its efficacy. EGFR extracellular domain point mutations often occur at S492, I491, K489, K467, ​​G465, S464L, I462, R451, S442, and V441 sites of EGFR ECD domain III. Among these sites, the frequency of S492R and G465R mutations is relatively high, and the G465R mutation can cause both cetuximab and panitumumab to lose their binding ability. Therefore, S492R and G465R mutations are important tumor resistance markers and have important clinical significance for anti-EGFR therapy. In addition, EGFR, as a tumor-associated antigen, is highly expressed on the surface of tumor cells and also expressed on many normal cells, such as the skin and gastrointestinal mucosa, which leads to frequent skin and gastrointestinal side effects in patients receiving cetuximab treatment. Therefore, overcoming the high-frequency point mutation resistance of the EGFR extracellular region and improving the tumor selectivity of anti-EGFR therapy are clinical problems that urgently need to be solved.

[0004] The patent application with publication number CN111499749A discloses a cetuximab mutant and its application. The cetuximab mutants with the amino acid sequences of the antibody heavy chain variable region as shown in SEQ ID NO.1 to 5 obtained in the application are effective against EGFR S492R, wherein the cetuximab mutants with the amino acid sequences of the antibody heavy chain variable region as shown in SEQ ID NO.1 and 2 are better than panitumumab. The cetuximab mutants with the amino acid sequences of the antibody heavy chain variable region as shown in SEQ ID NO.6 obtained in the application are effective against EGFR G465R, and the effect is significant. The cetuximab mutants with the amino acid sequences of the antibody heavy chain variable region as shown in SEQ ID NO.1, 2, and 6 obtained by the present invention still maintain affinity for wild-type EGFR. However, the point mutation antibody of the patent application is to bind to both wild-type and mutant EGFR at the same time, and the binding force to wild-type EGFR is stronger than that to mutant EGFR, and does not have point mutation bias.

[0005] Based on the above problems existing in the clinical application of anti-EGFR therapy, using the high-frequency EGFR resistance point mutations as tumor-specific markers, designing cetuximab mutants that preferentially target the resistant point mutations of EGFR can not only improve the tumor selectivity of anti-EGFR therapy, but also overcome the EGFR point mutation resistance, which is the research direction of the new generation of anti-EGFR drugs. Summary of the invention

[0006] The present invention provides a cetuximab mutant that preferentially targets S492R or G465R mutant EGFR and its application in order to solve the problem of poor tumor selectivity of cetuximab in clinic and acquired drug resistance due to point mutation in the extracellular region of EGFR after long-term use.

[0007] In view of the above problems, the present invention adopts a rational design method based on structure guidance, and slightly modifies the 58th and 104th amino acids of the antibody variable region on the basis of cetuximab, performs combined mutations, and finds that the point mutations can be preferentially combined to obtain a cetuximab mutant that can preferentially target the S492R or G465R mutant EGFR. The cetuximab mutant that can preferentially target the S492R or G465R mutant EGFR has an antibody heavy chain variable region amino acid sequence as shown in SEQ ID NO.1.

[0008] Specifically, the present invention first provides a cetuximab mutant that preferentially targets S492R or G465R mutant EGFR, comprising a cetuximab light chain and a heavy chain with a mutation, wherein the mutation in the heavy chain is located in the heavy chain variable region, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.1; or has at least 95% sequence homology with SEQ ID NO.1 and comprises a heavy chain variable region with at least Y104D and W52D mutations.

[0009] The present invention further provides a gene encoding the cetuximab mutant.

[0010] The present invention further provides a single-chain antibody, comprising a light chain variable region of cetuximab and a heavy chain variable region with mutations, wherein the amino acid sequence of the heavy chain variable region with mutations is shown in SEQ ID NO.1; or a heavy chain variable region that has at least 95% sequence homology with SEQ ID NO.1 and comprises at least Y104D and W52D mutations.

[0011] The present invention further provides a gene encoding the single-chain antibody.

[0012] The present invention further provides a Fab fragment antibody, comprising a light chain of cetuximab, a CH1 region of a heavy chain and a heavy chain variable region with mutations, wherein the amino acid sequence of the heavy chain variable region with mutations is shown in SEQ ID NO.1; or a heavy chain variable region having at least 95% sequence homology with SEQ ID NO.1 and comprising at least Y104D and W52D mutations.

[0013] The present invention further provides a gene encoding the Fab fragment antibody.

[0014] The present invention further provides a (Fab)2 fragment antibody, each molecule of the antibody contains two molecules of the Fab fragment antibody.

[0015] The present invention also provides the use of the cetuximab mutant, the single-chain antibody, the Fab fragment antibody or the (Fab)2 fragment antibody in the preparation of a drug targeting EGFR.

[0016] Preferably, when the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.1, EGFR has a S492R or G465R mutation.

[0017] Preferably, the disease targeted by the drug is colorectal cancer.

[0018] The currently marketed monoclonal antibody drugs Cetuximab and Panitumumab for colorectal cancer are ineffective against EGFR S492R or G465R mutations, and have non-tumor target toxicity against wild-type EGFR. Compared with the antibodies in the prior art, the binding force to wild-type EGFR is stronger than to mutant EGFR, and there is no point mutation preference; the Cetuximab mutant of the antibody heavy chain variable region amino acid sequence obtained by combined mutation in the present invention, such as SEQ ID NO.1, can preferentially bind to S492R and G465R mutant EGFR, and the point mutation preference of the antibody drug enhances its selectivity for tumor cells in anti-EGFR therapy, making the treatment more precise and effective.

[0019] The beneficial effects of point mutation bias are mainly reflected in:

[0020] Improve targeting selectivity, overcome drug resistance, enhance efficacy, reduce side effects of anti-EGFR therapy and personalized treatment, etc. By preferentially binding to S492R or G465R mutant EGFR, antibody drugs can more effectively identify and target specific tumor cells, reduce nonspecific attacks on normal cells, and thus reduce side effects and toxicity.

[0021] In addition, this design provides a new treatment option for acquired drug resistance caused by EGFR mutations, extending the treatment effect of patients. Precise targeting of tumor cells not only improves the effectiveness of treatment, which may lead to higher tumor cell mortality and better clinical outcomes, but also reduces the side effects of anti-EGFR therapy and improves the durability of treatment.

[0022] Antibodies that can preferentially target specific mutations provide a basis for personalized treatment. Doctors can choose the most suitable treatment plan based on the patient's EGFR mutation status, optimize treatment results, simplify drug combinations, and reduce dependence on other drugs, thereby reducing the patient's medication burden. Through these advantages, point mutation-biased antibodies not only improve the safety and effectiveness of treatment, but also provide new ideas and directions for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a structural analysis diagram showing that cetuximab mutants preferentially bind to S492R or G465R mutant EGFR.

[0024] Figure 2 This is the result of SDS-PAGE analysis of cetuximab mutants.

[0025] Figure 3 This is the result of ELISA testing of the affinity of cetuximab mutants to S492R or G465R mutant EGFR.

[0026] Figure 4 The results of BLI determination of the binding kinetic constants of cetuximab mutants to S492R or G465R mutant EGFR

[0027] Figure 5 This is a graph showing the results of WB detection of the preferential inhibition of wild-type, S492R or G465R mutant EGFR phosphorylation by mutants. DETAILED DESCRIPTION

[0028] Example 1

[0029] Design of cetuximab mutants that preferentially bind to S492R or G465R mutant EGFR.

[0030] This example describes the design of a cetuximab mutant targeting S492R and G465R mutant EGFR, aiming to improve the targeting specificity for these drug-resistant mutations.

[0031] Considering that the S492R and G465R mutations significantly reduce the efficacy of cetuximab or panitumumab, especially the key role of the G465R mutation in treatment failure, the present invention has optimized the structure of cetuximab. Through the crystal structure analysis of the complex of cetuximab (Cetuximab) and wild-type EGFR (PDB numbering 1YY9), two mutations were introduced into the heavy chain variable region of cetuximab: W52D and Y104D. These mutations are intended to form new electrostatic interactions with the arginine residues at sites 492 and 465 in the mutant EGFR. The salt bridge interaction between the positive charge of arginine and the negative charge of aspartic acid will enhance the binding affinity and specificity of the variant to the EGFR mutant.

[0032] The cetuximab mutant was named Ctx-DD, and the heavy chain variable region sequence was shown in SEQ ID NO. 1. The structure of the cetuximab mutant binding to the S492R and G465R mutant EGFR was predicted by Alphafold3, and the structure was shown in Figure 1 As shown, the 492R site of the S492R mutant EGFR can interact with the 104D site of Ctx-DD, and the 465R site of the G465R mutant EGFR can interact with the 104D site and the 52D site of Ctx-DD.

[0033] Example 2

[0034] Construction of cetuximab mutants that preferentially bind to S492R or G465R mutant EGFR and their transient expression in eukaryotic cells.

[0035] First, the light and heavy chain gene fragments of the above-mentioned cetuximab mutant (as shown in SEQ ID No. 2 and SEQ ID No. 3, respectively) were cloned into the eukaryotic expression vector pCDNA3.1 (+). Next, the DH5α competent Escherichia coli was transformed, and the monoclonal colonies were picked and sequenced correctly to prepare the transfection-grade eukaryotic expression plasmid. The plasmid was transfected into HEK-293F cells using a transfection reagent. The transfected cells were cultured in a shaker at 37°C and 5% CO2 for 4 days to achieve transient expression and secretion of the antibody. Subsequently, the culture supernatant was collected and the HiTrap Protein A column was used to separate the supernatant. The pure system purifies antibodies from culture supernatants. After purification, purity analysis was performed by SDS-PAGE and antibody concentration was determined using A280. Figure 2 The cetuximab mutants shown have complete light and heavy chain bands and are of high purity.

[0036] Example 3

[0037] Affinity detection of cetuximab mutants that preferentially bind to S492R or G465R mutant EGFR.

[0038] The wild-type and point mutant EGFR-ECD-Fc antigen proteins (WT-EGFR-ECD-Fc, S492R-EGFR-ECD-Fc, G465R-EGFR-ECD-Fc, the sequence and preparation method are consistent with the applicant's previous patent with publication number CN111499749A) were diluted to 1 μg / mL with ELISA coating solution, and added to 96-well plates at 100 μL / well, and incubated overnight at 4°C. The coating solution in the wells was discarded the next day, and the wells were washed 3 times with PBST at 200 μL / well, followed by adding 5% skim milk powder at 200 μL / well as blocking solution and blocking at 37°C for 1h. After the blocking, the blocking solution in the wells was discarded, and a series of cetuximab and cetuximab mutants diluted with blocking solution were added at 100 μL / well, and three biological replicates were set for each concentration gradient, and incubated at 37°C for 1h. After the incubation, the liquid in the wells was discarded, and the wells were washed three times with PBST at a volume of 200 μL / well. Then, a goat anti-human K light chain secondary antibody labeled with horseradish peroxidase diluted 1:1000 with blocking solution was added, and the amount added to each well was 100 μL, and incubated at 37°C for 1 hour. After the incubation, the liquid in the wells was discarded, and the wells were washed three times with PBST at a volume of 200 μL / well. Then, ELISA color development solution was added at a volume of 100 μL / well, and incubated at 37°C for 15min to 30min. After the color development was completed, ELISA stop color development solution was added at a volume of 100 μL / well, and the OD450 value was detected with an enzyme reader. The Kd value was calculated using Graphpad software, and the results are shown in Tables 1 and Figure 3 As shown, the affinity of the cetuximab mutant Ctx-DD for mutant EGFR is higher than its affinity for wild-type EGFR.

[0039] Table 1 Affinity of cetuximab and cetuximab mutants to wild-type and mutant EGFR

[0040]

[0041] Example 4

[0042] Determination of dissociation equilibrium constants for cetuximab mutants that preferentially bind to S492R or G465R mutant EGFR.

[0043] This example describes the use of the biolayer interferometry (BLI) method to determine the dissociation equilibrium constant (KD) of cetuximab mutants against wild-type and S492R and G465R mutant EGFR. First, a streptavidin sensor was immersed in a biotinylated cetuximab mutant solution to solidify the antibody on the sensor; then, it was immersed in a wild-type or mutant EGFR-ECD-Fc antigen protein solution containing different concentration gradients to allow the antigen and antibody to bind; finally, it was immersed in a blank solution containing a running buffer to dissociate the antigen and antibody; The R8 system monitors the binding and dissociation process between the antibody and the antigen in real time and collects the corresponding sensor signal data. Based on the kinetic data of binding and dissociation, the OctetAnalysis Studio CFR software is used to analyze the data and calculate K D , to evaluate the affinity of the antibody to EGFR mutants. The results are shown in Table 2 and Figure 4 As shown, the affinity of the cetuximab mutant Ctx-DD for mutant EGFR is higher than its affinity for wild-type EGFR.

[0044] Table 2 K values ​​of cetuximab mutants binding to wild-type and mutant EGFR D value

[0045]

[0046] Example 5

[0047] The inhibitory effects of cetuximab mutants that preferentially bind to S492R or G465R mutant EGFR on phosphorylation of wild-type and mutant EGFR were detected.

[0048] The constructed NIH / 3T3 cells expressing wild-type or point-mutated EGFR (WT-EGFR-NIH / 3T3, S492R-EGFR-NIH / 3T3, G465R-EGFR-NIH / 3T3) were revived and cultured to the logarithmic growth phase. The cells were digested with trypsin in a biosafety cabinet and counted. 1×10 6WT-EGFR-NIH / 3T3 cells, S492R-EGFR-NIH / 3T3 cells and G465R-EGFR-NIH / 3T3 cells were inoculated in 6-well plates and cultured overnight. The next day, gradient dilutions of cetuximab, cetuximab mutants or solvent PBS were added and incubated in a cell culture incubator at 37°C and 5% CO2 for 2 hours, followed by EGF or solvent PBS incubation for 15 minutes. After incubation, the cell culture medium was discarded, the cells were washed twice with pre-cooled PBS, 100 μL of pre-cooled cell lysis solution with PMSF and phosphatase inhibitors added was added to each well, and the cells were scraped with a cell scraper. The scraped cell suspension was collected in a 1.5 mL EP tube, vortexed for 1 minute, and ice-bathed for 30 to 40 minutes to fully lyse the cells. Finally, centrifuged at 14,000 × g in a pre-cooled centrifuge at 4°C for 10 minutes, and the supernatant was taken to determine the protein concentration using a BCA protein concentration assay kit. Take 10μg protein from each sample, dilute it to 8μL with cell lysis buffer, add 2μL of 5× loading buffer (denaturation) and mix well, boil it in boiling water for 5min to denature the protein, and then load the processed protein sample and pre-stained protein maker in turn. After loading, start electrophoresis, and the electrophoresis conditions are 90V, 30min first and then 120V, 2h. After the electrophoresis, remove the gel and cut off the gel at the location of the target protein. At the same time, cut a PVDF membrane slightly larger than the cut strip and activate it with methanol. Place the sponge, filter paper, strip, PVDF membrane, filter paper, sponge in the transfer device in order from bottom to top, and then transfer the membrane under an ice bath. The transfer conditions are set to 90V, 90min. After the transfer, take out the PVDF membrane, wash it with TBST, add 5% BSA as a blocking solution, and place it on a shaker at 70rpm to shake and oscillate at room temperature for 2h. After the blocking is completed, the blocking solution is discarded, and rabbit anti-EGFR antibody, rabbit anti-phosphorylated EGFR (Tyr1068) antibody, and mouse anti-β-actin antibody diluted 1:1000 with the primary antibody diluent are added, and the membrane is placed on a shaker at 70×rpm and incubated overnight at 4°C with shaking. The primary antibody is recovered the next day and stored in a refrigerator at 4°C, and the membrane is washed 6 times with TBST. After washing, horseradish peroxidase-labeled goat anti-mouse IgG (H+L) secondary antibody and horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) secondary antibody diluted 1:1000 with the blocking solution are added respectively, and the membrane is placed on a shaker at 70rpm and incubated with shaking at room temperature for 1 hour. After incubation, the secondary antibody is discarded, and the membrane is washed 6 times with TBST. After washing, it is developed with an ultra-sensitive ECL chemiluminescence kit and imaged on a Bio-Rad chemiluminescence imaging system. The results are as shown in Figure 5 As shown, the cetuximab mutant Ct x-DD ​​has a stronger inhibitory effect on mutant EGFR phosphorylation than wild-type EGFR phosphorylation.

Claims

1. A cetuximab mutant that preferentially targets S492R or G465R mutant EGFR, characterized in that: It comprises a light chain of cetuximab and a heavy chain with a mutation, wherein the mutation of the heavy chain is located in the heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

1.

2. A gene encoding the cetuximab mutant according to claim 1.

3. A single-chain antibody, characterized in that It comprises a light chain variable region of cetuximab and a heavy chain variable region with a mutation, and the amino acid sequence of the heavy chain variable region with a mutation is shown in SEQ ID NO.

1.

4. A gene encoding the single-chain antibody according to claim 3.

5. A Fab fragment antibody, characterized in that: It includes the light chain of cetuximab, the CH1 region of the heavy chain and the heavy chain variable region with mutations. The amino acid sequence of the heavy chain variable region with mutations is shown in SEQ ID NO.

1.

6. A gene encoding the Fab fragment antibody according to claim 5.

7. A (Fab)2 fragment antibody, characterized in that: Each antibody molecule contains two molecules of the Fab fragment antibody as claimed in claim 5.

8. Use of the cetuximab mutant according to claim 1, the single-chain antibody according to claim 3, the Fab fragment antibody according to claim 5 or the (Fab)2 fragment antibody according to claim 7 in the preparation of a drug targeting EGFR.

9. The use according to claim 8, characterized in that EGFR has S492R mutation or G465R mutation.

10. The use according to claim 8, characterized in that The disease targeted by the drug is colorectal cancer.

Images