In silico analysis and in planta production of recombinant ccl21/IL1β protein and characterization of its in vitro anti-tumor and immunogenic activity

Abstract

CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1β have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1β as a recombinant protein and characterize its in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1β gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1β protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1β were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1β was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1β in stimulating the proliferation and migration of MCF7 cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1β, and IC50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1β has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1β could be considered an effective adjuvant in future in vivo and clinical tests.

Cytokine-Based Multi-Epitope Protein for Binding to Ccr7-Positive Cells

“…To produce and manufacture the multi-epitope vaccine and to increase the ability of the vaccine to bind to the CCR7 receptor, a gene construct was designed and manufactured artificially. To make the vaccine, the amino acid sequences of CCL21, CCL19, IL-Iβ, and GMCSF were taken from NCBI database and were identified by the relevant BCpreds (http://ailab-projects1.ist.psu.edu:8080/bcpred/),BepiPred2.0(https://services.healthtech.dtu.dk/service.php?BepiPred2.0),ABCpreds(http://crdd.osdd.net/raghava/abcpred/),SVMTrip(http://sysbio.unl.edu/SVMTriP/), and MAPPP online servers,(http://mendel.stanford.edu/SidowLab/downloads/MAPP/index.html), B-cell and T-cell epitopes(15, 17). Peptide signal regions were selected using Signal P software and deleted to shorten the sequence of the gene construct.…”

“…In this reaction, the impurities are removed and the purified proteins are dissolved by histidine tag by imidazole or by lowering the pH of the solution. Purified multiepitope protein was measured by standard protein dot blot assay(17). So 10μl of purified recombinant protein from transgenic strain was dotted on the nitrocellulose membrane, and it was incubated with BSA for 1 hour.…”

“…As a result, CCL19 and CCL21, together with the CCR7 receptor, are generally essential in strengthening the immune system and self-balance in physiological and pathophysiological situations(11, 12). Also, CCL21 now recognizes as an effective drug in the treatment of AIDS and cancer in international cancer institutes (13). However, these immunotherapy agents have several limitations.…”

“…Therefore, in this project, to treat and prevent cancer and viral diseases, the production of recombinant multi-epitope drugs has been designed and produced. In addition to the CCL21 sequence, it contains fragments of IL1β, CCL19, and GMCSF amino acid sequences(17). By placing epitopes related to CTLs and B lymphocytes in the desired structure to activate B and T lymphocytes(18), neutrophils and macrophages simultaneously to use as a potent adjuvant for the treatment of breast cancer, especially in combination with Her2 / neu gene expression in inducing Th1 immune response, which affects most drugs improve the disease and increase immunity(16, 19).…”

Regulation of Extracellular Vesicle-Mediated Immune Responses against Antigen-Specific Presentation

“…In the cancer immune cycle, cancer antigens are released from cancer cells that have experienced cell death and are taken up by DCs [ 244 , 245 ]. These DCs mature, migrate to the lymph nodes, and present the captured cancer antigen to the MHCI molecules, thereby activating T cells [ 246 , 247 , 248 , 249 ], which in turn migrate and infiltrate the tumour tissue to recognise and eliminate tumour cells. The interaction between T cell-expressed integrin alpha L (LFA1) and ICAM-1 on the vascular endothelium initiates T cell infiltration into tumours that are suppressed by vascular endothelial growth factor A (VEGF-A), which is produced by cancer cells, further releasing new cancer antigens and continuing the cancer immune cycle [ 250 , 251 , 252 ].…”