The Synthetic Construct

Cellular research is booming, particularly in cancer. Growing understanding of the tumor microenvironment and how immune cells infiltrate and kill tumor cells has spurred new research, including identifying new targets for biologics and engineering of cellular therapies to treat malignant disease.

Design iteration for CARs and screening T-cell receptors (TCRs) for tumor-associated antigen-specificity can be particularly challenging. The synthetic construction and assembly of dozens of candidate CARs and TCRs, and the introduction of these constructs into cells, can often be the most time-consuming step in the discovery process. High-throughput testing requires quick assembly and integration of constructs into viral vectors for transfection of cells used for screening assays. Traditional methods are not only time-consuming, taking several weeks to generate viral vectors for transduction, but also have limitations in quality control and are costly, limiting the number of CARs or TCRs that researchers can test in a single iteration. To expedite the design-build-test cycle for CARs and TCRs, it is essential to improve and optimize this process.

In a traditional engineered T cell workflow, a lead TCR sequence is cloned, and a viral construct is generated to transduce T cell lines or primary T cells for candidate screening assays. This process generally takes up to six weeks and often involves external service providers for process steps. The entire process can now be sped up thanks to new technologies; for example, it is possible to create multiple candidate TCR mRNAs overnight for transfection and expression, reducing the time from establishing the sequence to functional testing of a candidate TCR. Though mRNA is an attractive alternative to viral transduction to generate high levels of protein expression in primary T-cells/cell lines, generating mRNA quickly and efficiently can be tedious using traditional synthesis methods. You might perform a single-cell RNA sequencing analysis of specific sorted and/or expanded T cell populations to identify TCR sequences of interest. Based on this data, you can create heterodimeric TCR expression constructs and use these to electroporate RNA into T cell lines or primary T cells for functional screens.

Using high-throughput automation platforms for synthetic biology can help speed up synthetic assembly processes. Genes can be built based on a digital sequence, and clones, libraries, and mRNA can be created more rapidly. 

New technology can also help on the CAR front. Beyond engineered T Cells, CAR T cell therapy has shown tremendous potential for improving outcomes for several hematological cancers. Therapeutic applications are also expanding to solid tumors, autoimmune, and infectious diseases. To overcome issues with solid tumors’ immunosuppressive microenvironment and to find the best antigenic targets for therapy, research is currently focused on improving CAR designs by exploring targeting domains, improving the intracellular signaling domain, and performing other modifications to the construct, such as adding co-stimulatory domains for cytokine production.

It is well accepted that off-the-shelf therapies using allogeneic cells can reduce treatment costs compared with personalized therapies, while improving the quality of the cellular product – ultimately making treatment accessible to a larger group of patients. However, allogeneic cells risk graft-versus-host disease because they contain an original TCR that may be reactive to the patient’s tissues. To overcome this, CAR-T cells using allogeneic cells need to be constructed so that the endogenous TCRα gene is knocked out while the CAR transgenes are inserted via an adeno- or retroviral vector.

By synthesizing multiple gene fragments overnight, more CAR designs can be explored. Construct designs can be rapidly iterated to identify the optimal characteristics for the CAR T cell, and multiple CAR or costimulatory constructs can be built simultaneously to accelerate candidate screening experiments. 

In my view, automated synthetic biology solutions are the way forward for both TCR-T and CAR-T research. As a developer of novel TCR-T or CAR-T therapies, your end goal might be to develop constructs targeting various antigens to assess tumor reactivity. Your process begins by selecting sequences for these constructs and then transducing constructs into T cells for candidate screening experiments in vitro and in vivo. Traditional methods for the “build” part of this “design–build–test” cycle is often too slow for development timelines. High throughput synthetic biology platforms offer fast, accurate, hands-free, end-to-end solutions to accelerate discovery workflows.