Cell therapies, often termed ‘living drugs’, are seen as the next generation of clinical treatments. They enable the treatment of diseases for which there is no available treatment or cases where all treatment options have been exhausted. In general, cell therapies consist of genetically engineered patient derived cells. Cells are mass-produced and reintroduced into patients.
One of the most promising cell therapies is the Chimeric Antigen Receptor T-cell (CAR-T) therapy. CAR-T therapy takes advantages of T-cells, a subtype of white blood cells, and reprograms them into cancer fighting machines. In fact, early usage of CAR-T in the clinic has resulted in complete or partial cancer remissions of between 70 and 94% of patients.
Source: Dana-Farber Cancer Institute
CAR-T therapy involves taking T-cells from the patient's blood and genetically altering them in the laboratory such that they can recognize and attack cancer cells. Genetic alterations of T-cells are typically conducted using custom retroviruses, which will randomly insert a chimeric antigen receptor (CAR) gene into the T-cell genome. Although retroviruses are simple to use, they randomly insert into the genome, which results in heterogeneous CAR expression in the T-cell population. To overcome this bottleneck, CRISPR-Cas9 mediated CAR insertion is currently being studied for its ability to precisely insert CAR at a specific genomic location, thus generating homogenous CAR-T cells with higher therapeutic efficacy.
CAR-T cells have went through multiple iterations in the clinic. The first generation of CAR molecules comprised of a fusion protein between an extracellular cancer cell sensing domain and an intracellular signaling domain. The extracellular domain is typically an antibody fragment that effectively recognizes proteins expressed on the surface of cancer cells. Second and third generation CAR molecules added single and multiple co-stimulatory domains, respectively, to the intracellular portion of CAR molecules to amplify the signal. These domains boost the therapeutic efficacy of CAR-T cells by enhancing the immune response and decreasing T-cell exhaustion.
Protein Engineering – The Driver Behind Cell Therapies
Protein engineering is a multifaceted discipline that aims to develop proteins and enzymes useful for medicine, agriculture, food and a number of global industries. The two primary methods for protein engineering are rational protein design and directed evolution. In rational protein design, researchers rely on structural knowledge to generate candidates to test for desired protein functions or outputs. The general theory of rational design is to identify amino acid substitutions at precise computed positions that will improve or modulate the properties of the target protein. In practice, however, it is difficult to gauge when these precise amino acid substitutions will alter protein properties, even with a deep understanding of the protein’s biology. Further, performance of these rationalized proteins rarely lives up to expectations. Often the natural protein will outperform the designed candidates as decades of evolution have optimized the natural protein for its intended purposes.
With directed evolution, the protein is subjected to random or comprehensive mutagenesis. The process mimics and compresses natural evolution so that it can be practically carried out in a laboratory environment. A library of tens of millions of variants can be generated through iterative rounds of mutagenesis. When coupled with an appropriate high-throughput screening assay, where the large volume of variants competes on a particular function, researchers can easily isolate variants exhibiting the desired function in a practical time frame. The larger and better quality the library of variants, the more likely a valuable protein will be found.
CAR-T therapies rely on high-throughput protein engineering for their success in saving lives by enabling the desired CAR variants to be isolated within a few weeks. This in turn improves CAR-T functionality and eventually increases a patient’s chances of survival. CAR proteins have been engineered to bind to a number of cancer specific markers, with the most popular being the CD19 antigen found on tumor cells associated with lymphocytic leukemia.
Source: Han et al., 2017
Protein engineering has played a key role in expanding the utility of CAR-T therapy in clinics. One drawback of conventional CAR-T therapy is the usage of single chain antibody fragments (scFv). Unless the scFv has undergone humanization processes, there is a risk of scFv-embedded CAR molecules to induce an immunogenic attack in patients, which in turn decreases the efficacy of CAR-T therapy and risks patient safety. To overcome this bottleneck, researchers are engineering human proteins to serve as the extracellular antigen targeting domain. In a study from the University of Southern California, researchers replaced the CD19 targeting scFv with Adnectin, a domain from the human fibronectin protein. Since Adnectin is a natural human protein, there is a less likely chance of Adnectin inducing an immunogenic attack. Adnectin was created in using a directed evolution approach, where 10e13 variants of Adnectin were screened for enhanced binding to EGFR using high-throughput mRNA display. Cell-based studies demonstrated that Adnectin CARs can be expressed on human T-cells and that they can recognize EGFR cancer cells. These findings open the door to other types of extracellular targeting domains that may broaden the usage of CAR-T therapy across different cancer and diseases.
The Toxic Risks & Safety Profile of CAR-T Therapy
As with all therapies, there is a risk of adverse reactions and CAR-T therapy is no exception. In fact, adverse reactions from CAR-T therapy can be long-lasting and lethal since infused T-cells can multiply, overloading the body’s immune system and causing cytokine release syndrome (CRS). Symptoms of CRS range from fever to breathing difficulties, low blood pressure and organ swelling. These so-called “cytokine storms” can be fatal. Another grave consequence observed in CAR-T patients is fatal cerebral edema, of which the cause is still unknown. Other adverse symptoms include memory loss, hallucinations and systemic inflammation.
Improving long term safety profiles is a high priority for the next generation of CAR-T therapy development. Not surprisingly, protein engineering will be critical for creating the next wave of safe and effective CAR proteins. Using protein engineering, researchers are working to create CAR proteins that are only active at cancer sites, undergo inducible self-destruction, bind multiple targets on cancer cell for increased specificity and much more. These will be much less toxic, more persistent, and ready to use much sooner. Therapies like CAR-T will likely one day become as widespread in medicine as surgery and pathology is today. Cell therapies are exciting and potent new weapons in the medical armory of tomorrow.
References:
Han X, Cinay GE, Zhao Y, Guo Y, Zhang X, Wang P. Adnectin-Based Design of Chimeric Antigen Receptor for T Cell Engineering. Mol Ther. 2017;25(11):2466-2476.
Emanuel SL, Engle LJ, Chao G, et al. A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor. MAbs. 2011;3(1):38-48.
