Antigen Selection Strategy

Antigen selection was initially performed, utilizing existing available literature, focused on the analysis of the histopathology of pancreatic cancer and evaluating expression levels of certain antigens in the cancerous tissue, compared to normal tissue. In the meantime, the role of each overexpressed membrane bound antigens had to be clarified to find its association with cancer prognosis and thus detect a highly specific antigen. In this manner, an initial pool of specific antigens was chosen which subsequently underwent further investigation through utilization of the Geo2R analysis tool.

The Gene Expression Omnibus (GEO) serves as a repository database that houses a significant portion of publicly accessible high-throughput gene expression data. The analysis of gene expression represents a potent instrument for acquiring understanding regarding the mechanisms and procedures that underlie the biological and phenotypic distinctions observed among various sample groups. By leveraging the data within GEO2R, we can identify differentially expressed genes in pancreatic cancer tissues compared to normal tissue, providing insights into potential target candidates. This strategy allows for the systematic evaluation of gene expression profiles, enabling the identification of specific antigens that play pivotal roles in the progression of pancreatic cancer.

LogFC stands for "log Fold Change". It is a statistical metric employed in the examination of gene expression data, primarily when comparing gene expression levels across different sets of groups or conditions. Specifically, Fold Change (FC) is quantifying the degree of alteration in a gene's expression level between two experimental conditions. This metric helps researchers identify genes that are significantly upregulated (positive logFC) or downregulated (negative logFC) between the conditions being compared. It is a crucial indicator for understanding which genes are differentially expressed and to what extent in a given study. Positive logFC: indicates a significant upregulation of a gene in the treatment group compared to the control. Negative logFC: indicates a significant downregulation. A LogFC value in close proximity to zero signifies minimal to negligible fluctuations in gene expression between the two sample groups. Genes characterized by logFC values near zero are regarded as exhibiting similar expression levels in both groups.

Through extensive analysis of gene expression data within the database, we have consistently observed overexpression of mesothelin in pancreatic cancer tissues when compared to normal tissues. This stark contrast makes mesothelin an attractive candidate for targeted therapies, as it is an antigen that is not only abundant in cancer cells but also relatively absent in healthy tissues. The utilization of GEO2R has played a pivotal role in the discovery and validation of mesothelin as a target antigen, providing valuable insights into its potential as a therapeutic target for pancreatic cancer. From the volcano plot below we can clearly see that mesothelin is overexpressed in pancreatic cancer and this overexpression is statistically significant, making mesothelin the ideal candidate antigen.

One more element that led us to the choice of mesothelin is that the protein has been crystallized and several PDB files have been deposited in the Protein Data Bank database. This provides our Dry-Lab with the ability to perform accurate molecular simulations and docking experiments.

Plasmid Design Strategy

Proper design of our Chimeric Antigen Receptor (scFv-NKG2D-2B4-CD3z) is an essential step to prove our idea, target and kill cancer cells in pancreatic cancer. Because there was no experience in previous plasmid design we ended up with this proposed plasmid design strategy: the sequence of the desired genes that make up the receptor were found from the National Center for Biotechnology Information (NCBI) database. However, in the transfer plasmid, only a section of the complete sequence should be integrated, necessitating the identification of this specific segment. For this purpose, we utilized existing plasmids that are deposited in the Addgene repository and consist of our structural units. Then, the exact sequence of these genes that have been used in the deposited plasmids, were searched using the BLAST global alignment tool. By this method we were led in a more secure way to the total sequence of the plasmid. Below you can see the use of this strategy for the identification of the CD3z domain.

Culturing cells

Care and maintenance of cell health ensures their functionality, expansion, proper preparation for transduction and their cytotoxic efficacy. Corresponding protocols include initial thawing process, culturing and feeding. Specifically, it is detailing the thawing procedure, a critical step that significantly influences the subsequent progression of the culture, as it can lead to a potential reduction of up to 50% in the cell count. Additionally, the protocols provide instructions regarding how often to replenish nutrients, the quantity to administer, and the method for cell counting subsequent to staining with Trypan Blue. Cancer cell line AsPC-1 is also included in protocols concerning mammalian cell lines.

Testing Conditions

After receiving cells, we had the time to test different activation conditions for Natural Killer Cells obtained from a Peripheral Blood sample, provided by the laboratory we worked in. These Condition Testing Protocols were created based on bibliography research and include PBMCs isolation, Natural Killer cells selection, three different culturing conditions, further tested with Flow Cytometry. In these three conditions, we maintained a standard cytokine cocktail of IL-2, IL-25 and IL-21, adding the anti-CD2 and anti-NKp46 antibodies in cloudz condition, while the third one contained K562 feeder cells previously treated with mitomycin C and irradiation. In this way, we could detect differences in NK activation efficacy and finally use the best condition as an alternative culture medium for iPSCs-NK activation.

Preparing for Transfection

Plasmid is delivered in a lyophilized form and has to be multiplied in order to get sufficient final quantity. For this purpose we utilized the E. coli DH5α for initial plasmid transformation, followed by plasmid mini prep and mega prep, aiming to harvest high plasmid concentrations before transfecting HEK 293 cells. Preparation for transfection protocols include in detail experiments concerning culturing E. coli (DH5α) in agar, plasmid transformation, colony selection, mini prep and mega prep.

Transfection

HEK 293 cells were co-transfected with three plasmids for lentiviral particle formation. Transfection Protocol includes the detailed methodology, from preparing HEK-293 cell line, to transfection and supervision of the progress, finalized with Flow Cytometry aiming to detect eGFP, contained in transfer plasmid, transferring gene encoding our Chimeric Antigen Receptor.

Envelope Plasmid: pMD.2G

Packaging Plasmid: psPAX2

Transfer Plasmid: pLenti-IRES-eGFP-BleoR

Transduction

Induced Pluripotent Stem Cells (iPSCs)-derived NK cells and NK92 cell line, were transduced with lentiviral vectors, produced previously, aiming to the expression of our anti-mesothelin-CAR. Transduction Protocol includes previously counting of cells after staining with Trypan Blue for ensuring their health.