| المستخلص: |
Background: Molecular Investigations have led to the discovery of cancers markers and the development of more potent therapies. The human epidermal growth factor receptor (EGFR) is one of the first cancer-marker to be used as therapeutic target. Objectives of the study: We have carried the current work to investigate genetic variations in the EGFR-CR2 domain and to generate variant specific monoclonal antibodies. Materials and Methods: we first investigated mutations and polymorphisms in the CR2, extracellular ligand binding domain, with focus on the gene exons 13-16 which encode for this Cysteine-rich domain, in 6 different types of cancer in patients from the Arabian Gulf populations. Based on the mutational analysis results, we have put emphasis on the R521K variants to clone, express and purify recombinant CR2-R and CR2-K variants of the EGFR extracellular domain in E. coli BL21. These recombinant proteins along with free synthetic peptides were used to generate anti-CR2-R and anti- CR2-K specific monoclonal antibodies. Results: The mutational analysis revealed the following: in exon 13, a novel SNP (1782 C>T) was found in healthy control, colon and bladder cancer patients; with the “C” being the major allele. The 3 known SNPs: rs2227983 [R521K], rsl42429250 and rsl7336800 were also observed in our control and patients groups. Analysis of SNP rs2227983 showed a frequency of the GG genotype [homozygous RR] 52.6 in control and 49 in patients groups, while the AA genotype [Homozygous KK] frequency was 10.5, 12 respectively. In exon 14, we observed a novel [V550M] missense mutation in 3 colon cancer patients and in one patient with ovary cancer. Reported rsl7290103 SNP was observed in breast cancer sample and in healthy group. In exon 16, a new a synonymous mutation [G632G] was found in 2 colon cancer samples, in 1 ovary cancer sample and in 4 thyroid cancer samples. An additional rare novel SNP (2159 C>T) was found in 2 of healthy control samples out of 114 and reported rs2227984 SNP was observed in patients samples and control samples. In the second phase of our work, we cloned, expressed and purified recombinant CR2-R and CR2-K proteins and used them along with variants peptides to develop 3 monoclonal antibodies to EGFR; one that does not distinguish CR2 variants and 2 that selectively recognize the R and K. Conclusions: We have developed the first monoclonal antibodies that discriminate between R and K EGFR variants. These antibodies can be used to develop novel improved therapeutic tools for cancer based on the EGFR genetic make-up of the patient.
|
