Investigating targeted mutations in tumor Angiogenic factor (VEGF) in colorectal cancer by using edrecolomab antibody engineering
Investigating targeted mutations in tumor Angiogenic factor (VEGF) in colorectal cancer by using edrecolomab antibody engineering
Seyed Mohsen Miresmaeili,1saharshahgholi,2,*
1. Biology Department, Science and Arts university, Yazd , Iran 2. Biology Department, Science and Arts university, Yazd , Iran
Introduction: VEGF family members, not only in the development but also in the therapy of CRC, in order to fully elucidate their role in carcinogenesis, are extremely important (Dakowicz, Zajkowska et al. 2022).
VEGF family (VEGF-A, VEGF-B, VEGF-C, VEGF-D) plays a key role in the processes of blood vessel formation in embryonic development as well as in pathological angiogenesis and lymph angiogenesis, which allow the tumor to grow exponentially (Dabravolski, Khotina et al. 2022).
Upon binding their corresponding vascular endothelial growth factor receptors (VEGFRs), VEGFs promote the proliferation and migration of endothelial cells, tube formation, increase vascular permeability and vascular endothelial cell survival, altogether angiogenesis(Bokhari and Hamar 2023)
Colorectal cancer is the second leading cause of cancer deaths in men and women worldwide, with an estimated incidence of 1.9 million new cases diagnosed in 2020. The treatment of metastatic colorectal cancer (mCRC) requires multidisciplinary management, and molecular biology knowledge has enabled the incorporation of targeted therapies, such as use of anti-VEGF drugs, into combined chemotherapy regimens(Ortiz-Morales, Toledano-Fonseca et al. 2022)
The use of monoclonal antibodies in colorectal and gastric cancers showed the best outcomes when combined with chemotherapy (Bronte, Cicero et al. 2013)even though In this research, high affinity the monoclonal antibody edrecolomab to the VEGF antigen was obtained with the targeted mutations created in the amino acids of the long and small chains of the monoclonal antibody of edraclomab. which is important for angiogenesis signaling, commonly upregulated in mCRC (Hurwitz 2004).
Sorting Intolerant from Tolerant (SIFT) is an algorithm that predicts the potential impact of amino acid substitutions on protein function(Sim, Kumar et al. 2012).
In this research, we used the Sift software to create targeted mutations, so that by engineering the monoclonal antibody of edercolomab, it can increase its affinity with the VEGF antigen and be useful as a drug candidate in preventing metastatic colorectal cancer.
Methods: Antibody engineering requires the identification of antigen binding domains or variable regions (VR) unique to each antibody(Babrak, McGarvey et al. 2017)
National Center for Biotechnology Information (NCBI) URL (www.ncbi.nlm.nih.gov/gene/), light and heavy chain sequences of the monoclonal antibody edrecolomab were shown.
proABC is a web server for predicting CDR regions(Berezin, Glaser et al. 2004) in the antibody binding site that are involved in antigen recognition (http://www.biocomputing.it/proABC). The light and heavy chain sequence was included in this software.
Antibody design and amino acid replacement. The amino acids located in the antibody binding site were identified based on the results obtained from proABC web applications. At this stage, the appropriate amino acid was selected for mutation design
SIFT analysis was used for prediction (http://sift.jcvi.org/) to determine amino acid substitutions to improve protein function based on the degree of conservation of amino acid residues in sequence alignments derived from closely related sequences(Kumar, Henikoff et al. 2009).
Detailed analysis of protein-protein binding was done using HADDOCK software.
At http://haddock.science.uu.nl/services/HADDOCK 2.2/. This careful examination was used to determine the interaction and orientation between the two molecules to determine the correct binding between the antigen and the antibodies(De Vries, Van Dijk et al. 2010).
Results: In this study, by creating targeted mutations in the light and heavy chains of the monoclonal antibody of edrecolomab in the 56th amino acid of the heavy chain and the 79th and 105th amino acids of the light chain and using Haddock's software agent, the affinity of the anti-VEGF antigen was compared to the control group of - 85 to -140, and with engineering, we present this variant as the best candidate for treatment of colorectalcancer.
Conclusion: Making specific changes in the genome has been used to analyze gene function and to develop high affinity monoclonal antibody models. Targeted mutations allow the functional examination of specific domains or amino acids in a protein ) (Menke 2013).
We purposefully created mutations in the amino acid sequence of the light and heavy chains of the edrecolomab antibody.
Research has shown that mutations in the amino acids valine and Isoleucine was also found to be largely affected in the mutant, resulting in large decreases in the levels of amino acid-related fatty acids, and these changes seemed to affect the responses in the fluidity of the membrane and cold shock-related phenomena (Choi, Oh et al. 2023) .
Amino acids with low molecular weight, such as glycine, alanine, threonine, serine, Proline and cysteine, and the presence of long-chain or branched amino acids, such as tryptophan, and amino acids such as arginine and phenylalanine, despite having a higher molecular weight and replacement with the amino acid lysine. which are necessary to support growth, development and protein synthesis as well as to reduce stress, can cause high affinity in antibody engineering(Aryal, Dhakal et al. 2022).
We created targeted mutations by changing the amino acid valine in the large chain and converting it to isoleucine, changing the amino acid Proline to lysine, and changing the amino acid tryptophan to lysine in the small chain. For antibody development and engineering, antigen-targeting ability and functional characteristics, including antigen-binding affinity, target specificity, biological efficacy through epitope analysis, and develop ability characteristics by creating targeted mutations are considered(Kim, McFee et al. 2023)
In this study, targeted mutations in the tumor angiogenic factor( VGEF) in colorectal cancer using the engineering of the Edrecolomab antibody caused high affinity.