Header Section Image

SAFETY

by

martin headshot
igor headshot
chris headshot

et al.

To address safety concerns about using living cells as therapy, we conceived a caspase inducable killswitch for our cells as well as four other safety layers to ensure controllability. In collaboration with Team McGill we worked on resources enabling future teams to develop similar therapeutic approaches.

The safety concerns with cell therapies

B-cells generally require a specific physiological milieu and cannot survive for much longer than a few days, usually hours. For that reason we were less concerned about an accidental release into the environment than about possible side effects within patients. The focus was on introducing several layers of safety mechanisms to control BEE-cells and prevent harmful side effects.

Our solution

In order to combat unexpected side effects we implemented a cascade of safety measures that were validated through several talks with experts such as professor Ulrike Protzer or iGEM alumni Arne Zimmermann. Most of them have already been established in clinical trials but not in the context of B-cells.

Our safety stages include:

Caspase inducable kill switch

Our (iCasp9) kill switch originates from the lab of professor Ulrike Protzer and has already been established in CAR T-cell therapy. It is induced via small molecule activation and serves as highly selective countermeasure should any of our BEE-cells cause unintended side effects. 1

Synthetic chimeric B-cell receptor

A single chain variable fragment is combined with the Igβ signalling domain of the B-Cell receptor (BCR). This additional chimeric BCR activates the same downstream singalling as the regular antigen binding to BCR.2 This can be used as an activation switch to complement the kill switch. This allows for a highly adjustable form of cell therapy depending on the patients individual needs.

Depletion factor

We are introducing an epidermal growth factor receptor into the cells. This EGFR is targeted with a tyrosine kinase inhibitor and can be used to either induce apoptosis or for in vivo tracking. Just like the kill switch it has already been used in T-cells but never before in B-cells. It attacks our BEE-cells but also normal B-cells. On top of that it enables therapeutic drug monitoring of BEE-cells since blood from several body regions can be observed. It is also possible to determine where BEE-cells proliferate and in which condition they are or whether memory cells have formed.

The endogenous EGFR usually consists of four domains: An extracellular domain, a transmembrane domain, a juxtamembrane domain and a kinase domain. Our version is truncated that does not encompass the extracellular binding domains and intracellular receptor tyrosine kinase activity as to not overboost proliferation while still keeping the receptor on the surface for selection.The drug monitoring is enabled by a cell surface marker for in vivo tracking. 3,4

Targeted B-cell depletion using Rituximab antibody

BEE-cells are engineered to overexpress CD20 which can be targeted with Rituximab, a human/murine chimeric anti-CD20 monoclonal antibody. While Rituximab will attack all cells that express CD20 it will mainly target BEE-cells and has been used to treat B-cell malignancies for 20 years. 5

Immunosuppressives

Our last line of defense will be immunosuppressives. This will shutdown or reduce the overall immunesystem but is a common way to counteract therapies gone wrong.

Experiments

To determine the effectiveness of our killswitch the modified B-cells will be measured after caspase induced destruction through the previously discussed kill switch. The survival rate under normal lab conditions as well as random allocation on our bench will show whether our setup is working in practice.

Collaboration with iGEM Team McGill

To properly work on each of our four targets we consulted other labs that specialized on them. That way we were able to plan for individual safety aspects for each set of experiments. Our team also co-authored a white paper with the iGEM team McGill concerning itself with risk factors of cell- and gene therapy. In our section we argue that B-cell therapy offers, when compared to traditional antibody therapy, a customized treatment with constant titers. Our project thereby shows great potential for safer therapies due to constant effectiveness. Common drawbacks of antibody therapy such as migraines could also be less likely.

Available from URL: https://ashpublications.org/blood/article/118/5/1255/28972/A-transgene-encoded-cell-surface-polypeptide-for

Footnotes

  1. Zhou, X., Di Stasi, A., Brenner, M.K. (2015, cited 2023 October 12th). iCaspase 9 Suicide Gene System. In: Walther, W., Stein, U. (eds) Gene Therapy of Solid Cancers. Methods in Molecular Biology, vol 1317. Humana Press, New York, NY.https://doi.org/10.1007/978-1-4939-2727-2_6 ↩

  2. Pesch T, Bonati L, Kelton W, Parola C, Ehling RA, Csepregi L, Kitamura D, Reddy ST. Molecular Design, Optimization, and Genomic Integration of Chimeric B Cell Receptors in Murine B Cells. Front Immunol. 2019 Nov 14 (cited 2023 October 12th);10:2630. doi: 10.3389/fimmu.2019.02630. PMID: 31798579; PMCID: PMC6868064. Available from URL: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868064/ ↩

  3. Amelia T, Kartasasmita RE, Ohwada T, Tjahjono DH. Structural Insight and Development of EGFR Tyrosine Kinase Inhibitors. Molecules. 2022 Jan 26 (cited 2023 October 12th);27(3):819. doi: 10.3390/molecules27030819. PMID: 35164092; PMCID: PMC8838133. Available from URL: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8838133/ ↩

  4. Xiuli Wang, Wen-Chung Chang, ChingLam W. Wong, David Colcher, Mark Sherman, Julie R. Ostberg, Stephen J. Forman, Stanley R. Riddell, Michael C. Jensen; A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood 2011 (cited 2023 October 12th); 118 (5): 1255–1263. doi: https://doi.org/10.1182/blood-2011-02-337360 ↩

  5. Salles G, Barrett M, Foà R, Maurer J, O’Brien S, Valente N, Wenger M, Maloney DG. Rituximab in B-Cell Hematologic Malignancies: A Review of 20 Years of Clinical Experience. Adv Ther. 2017 Oct (cited 2023 October 12th);34(10):2232-2273. doi: 10.1007/s12325-017-0612-x. Epub 2017 Oct 5. PMID: 28983798; PMCID: PMC5656728. Available from URL: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656728/ ↩