A paper based on personal research done on immunotherapy drugs and their mechanisms within the cell, written for Cell Biology in Fall 2018.

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Personal Research

            Cancer immunotherapy is an area of research that I am highly interested in. One particularly interesting aspect of immunotherapy is the idea of manipulating receptors in cells to turn on or off certain pathways in order to reach a desired outcome, and being able to use that ability therapeutically to improve someone’s quality of life as well as increase their lifespan. I am interested in this area of research due to having experienced the personal loss of my maternal grandmother, who was a very influential person to me during my entire life. She was diagnosed with an unusual case of stage four lung adenocarcinoma, which consisted of a tumor growing out of the muscles of her chest wall but not having spread anywhere else. She was treated with immunotherapy after the traditional few rounds of chemotherapy and radiation – a PD-L1 inhibitor drug called atezolizumab, which blocks the interaction of the protein PD-L1 with programmed cell death protein 1 (PD-1) and CD80 receptors. The binding of PD-L1 to PD-1 normally inhibits the growth of antigen-specific T cells and reduces apoptosis in regulatory T cells (En.wikipedia.org, 2018). My family unfortunately lost my grandmother due to side effects of the atezolizumab, mainly due to severe pneumonitis (inflammation of the alveoli in the lungs) caused by her immune system, which was attacking her own tissues and, despite efforts from her team of doctors, could not be suppressed due to the presence of the drug in her body.

The study that I chose examined used the gene editing tool CRISPR-Cas9 to inhibit cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in order to increase the anti-tumor activity of cytotoxic T lymphocytes (Shi et al., 2017). CRISPR-Cas9 is a tool that allows researchers to modify parts of the genome by adding, removing, or altering parts of a DNA sequence, using the enzyme Cas9 and a piece of RNA referred to as the guide RNA (gRNA) consisting of a small piece of pre-designed RNA sequence within a larger RNA scaffold (Your Genome, 2016). The method used in this study is called gene knockout, in which specific genes are silenced by mutations in order to prevent the gene products from being produced (Bird and Spradlin, 2016). The researchers in this study edited cytotoxic T-lymphocytes (CTLs) by knocking out CTLA-4 with the CRISPR-Cas9 system in order to see the effects of T-cell activation and cytotoxicity in the presence of tumor cells. CTLA-4 is a protein receptor that is expressed in activated T cells and regulatory T cells (Shi et al., 2017). It is an inhibitory molecule that competes with CD28 and downregulates the immune response through inhibitory IL-2 production and cell cycle progression. Blocking of CTLA-4 with monoclonal antibodies has been seen to enhance the anti-tumor immune response in previous studies. This would indicate that activating CTLs through targeting of CTLA-4 would be a good strategy for immunotherapy.

The researchers in this study performed the experiment using peripheral blood samples from human patients, from which they isolated the human PBMCs (peripheral blood mononuclear cells). Human colonic adenocarcinoma cells were also cultured. The CRISPR-Cas9 tool was utilized in order to produce CTLA-4 knockout cytotoxic T-lymphocytes (CTLA-4 KO CTLs). The researchers included in their data for Figure 2 a western blot that demonstrated the gene knockout of CTLA-4. The experiments were performed individually three times each. It was found through their data that, in comparison to normal control CTLs, the CTLA-4 KO CTLs demonstrated increased anti-tumor activity. Compared to the control, CTLA-4 expression was significantly reduced in CTLs that were transduced with lentivirus expressing CTLA-4 gRNA, indicating the successful knockout of CTLA-4 from most of the CTLs with the CRISPR-Cas9 system (Shi et al., 2017). Results also indicated that the CTLA-4 knockout enhanced the cytotoxicity of CTLs, and that CTLA-4 KO CTLs enhanced apoptosis and caspase activities within the cultured tumor cells than did the control CTLs. CTLA-4 KO CTLs also had elevated cleaved caspase activation levels in tumor cells; the caspase cascade leads to apoptosis. Their data indicated also that the CTLA-4 KO CTLs had produced twice as many cytokines than the control CTLs. These cytokines include tumor necrosis factor-α, or TNF-α, and interferon-γ, or IFN-γ, both of which are associated with activation of the host’s immune response to kill virally infected cells and tumor cells (Shi et al., 2017). In Figure 4 of the data, it is shown that the levels of TNF-α were more than double that of the control, and levels of IFN-γ were higher in the knockout CTLS than the control by almost double.

Another portion of the experiment included mice that were irradiated and had marked tumor growth. A group of affected mice that were injected with CTLA-4 KO CTLs were observed to have drastically repressed tumor growth compared to mice that were not treated. The treated mice were seen to have overall improved survival rates, increased lifespan, and inhibited tumor growth compared to the untreated mice.

This study effectively disrupted CTLA-4 mediated coinhibitory signaling by use of the CRISPR-Cas9 tool to knockout the CTLA-4 gene in cytotoxic T-lymphocytes, and tested its effect on the CTLs’ anti-tumor activity. Compared to the control, CTLA-4 KO CTLs could further reduce tumor cell viability by 40% (Shi et al., 2017). It was observed that the enhanced anti-tumor effect of the CTLA-4 KO CTLs was associated with increased apoptosis and augmented caspase activities in tumor cells, and that production of cytokines (used by CTLs to kill tumor cells) was increased twofold (Shi et al., 2017). In mice afflicted with cancerous tumors, CTLA-4 KO CTL treatment inhibited tumor growth and prolonged the survival of the mice. The researchers were successful in their experiment, as CRISPR knockout CTLA-4 enhanced T-cell mediated anti-tumor immunity, making this a viable possibility in future immune checkpoint targeting in cancer treatments.

From DNA to gene expression, cell biology has many multifaceted pieces and aspects that fit together to form a big picture. This study focused mainly on the editing of a DNA sequence to silence production of CTLA-4 in order to prevent inhibition of cytotoxic T-lymphocytes, with the ultimate goal of enhancing the CTLs’ anti-tumor activity and reducing the proliferation of cancerous cells. This has everything to do with cell biology; this particular method of immunotherapy focuses entirely on modifying gene expression to inhibit growth of tumor cells. The advanced technology involved in CRISPR gene editing is a marvel, considering that treatments for disease can now begin at preventing it at the DNA and molecular level instead of simply treating the symptoms. Advances like this in research and medicine give me hope for a brighter future for many people, and I hope to someday be involved in helping to create a solution for people who may have once not held such hope.

 

Works Cited

En.wikipedia.org. (2018). PD-L1. [online] Available at: https://en.wikipedia.org/wiki/PD-L1 [Accessed 9 Dec. 2018].

Shi, L., Meng, T., Zhao, Z., Han, J., Zhang, W., Gao, F. and Cai, J. (2017). CRISPR knock out CTLA-4 enhances the anti-tumor activity of cytotoxic T lymphocytes. Gene, [online] (636), pp.36-41. Available at: http://dx.doi.org/10.1016/j.gene.2017.09.010 [Accessed 9 Dec. 2018].

Yourgenome.org. (2016). What is CRISPR-Cas9?. [online] Available at: https://www.yourgenome.org/facts/what-is-crispr-cas9 [Accessed 9 Dec. 2018].

Bird, M. and Spradlin, J. (2016). Gene Knockout and Silencing. [online] Sites.tufts.edu. Available at: http://sites.tufts.edu/innateimmunity/knock-out-and-silencing/ [Accessed 9 Dec. 2018].