Chimeric antigen receptor (CAR) T-cell therapy is making major headway in the drug development world. By harnessing and enhancing the powers of the immune system, it offers the ability to destroy tumor cells and provide a durable antitumor immune response—all by using genetically modified versions of patients’ own T-cells.
In a recent article on supporting the bioanalysis of CAR-T therapies, I discussed why this approach to cancer treatment requires novel methods to monitor and quantify the therapeutic’s progression and effects. I also touched on the fact that new and modified forms of CAR-T therapies are being developed to help address the shortcomings seen with the treatment to date, to make them safer and more effective for a wider range of oncological applications. Now I would like to explore more closely what some of the next generation of CAR-T cell therapies look like, and the impact these evolutions have on bioanalytical methods to support their development:
Improving efficacy of CAR-T cells in solid tumors
While CAR-T cell therapy has shown remarkable success in treating hematological cancers such as chronic lymphoblastic leukemia (CLL), pediatric relapsed or refractory acute lymphoblastic leukemia (ALL), and adult relapsed or refractory diffuse large B cell lymphoma (DLBCL), the same magnitude of clinical gains have yet to be realized in targeting solid tumors. There are many reasons why is it more challenging to target solid tumors with CAR-T cells, including difficulty in identifying an ideal target antigen, T cell exhaustion that results in loss of effector and memory phenotypes, and the hostile tumor microenvironment (TME) to name a few. To address some of these challenges, novel CAR-T cells are being engineered to more effectively find, enter, and survive in the tumor. These include the use of dual CAR designs that recognize multiple antigens at once, and “armored” CAR-T cells that secrete specific cytokines and chemokines shown to improve survival and persistence of CAR-T cells once administered, and that activate other T cells in the patient to amplify the anti-tumor effect. For example, CAR-T cells engineered to express interleukin 7 (IL-7), known to enhance proliferation of CAR-T cells, and CCL19, a chemoattractant for T cells, are showing superior anti-tumor activity compared to conventional CAR-T cells in a solid tumor mouse model. CCL19 recruits patient T cells and dendritic cells to the tumor site to collaborate with the CAR-T cells and exert a greater anti-tumor effect. CAR-T cells can also be genetically modified to secrete other stimulatory cytokines such as IL-15, IL-12, and IL-18.
For traditional CAR-T cells, the target antigen must be expressed on the cell surface in order to engage with a T cell, leaving a plethora of potential tumor target antigens inaccessible to a CAR-T cell. T cell receptors (TCRs) recognize both extra- and intra-cellular antigens, and therefore TCR-CARs that recognize antigen in combination with major histocompatibility complex (MHC) can target both types of antigens that TCRs can. Combining CARs with checkpoint blockade or depletion of other suppressive factors in the TME has also shown very promising results to mitigate the phenomenon of T cell exhaustion. Finally, identifying and overcoming mechanisms associated with dysfunction in CAR-T cells is of vital importance to generating CAR-T cells that can proliferate and successfully eliminate tumor cells. The structure and costimulatory domains chosen for the CAR may play an important role in the overall function of CAR-T cells in the TME. There is evidence that CAR-T cell persistence can be enhanced against solid tumors by transducing CD4 and CD8 T cells with CARs encoding different costimulatory domains.
What it means from a bioanalytical perspective
There are new questions that will need to be asked and answered when assessing these novel types of CAR-T cell therapies such as: is there an immune response involving a neutralizing antibody that could potentially neutralize the endogenous levels of the engineered secreted cytokines such as IL7 and CCL19 in the patient? These responses can be detrimental to patient safety and must be carefully evaluated. We must also consider if the cytokines/chemokines being secreted by the CAR-T cells differ in any way from the endogenous molecules, and how to distinguish them. Differences can lead to new epitopes that may be immunogenic and that cross-react with endogenous forms.
The next generations of CAR constructs and CRISPR
First-generation CARs contained only the CD3ζ signaling domain, while second generation CARs contain an additional costimulatory signaling molecule to enhance their antitumor properties and persistence, such as 4-1BB, CD28, CD27, OX40. Third- and fourth- generation CARs contain two or more signaling domains. Fourth generation CAR-T cells incorporate a third stimulatory signal, cytokine expression cassette, referred to as “TRUCK” T cells redirected for universal cytokine-mediated killing. TRUCKs can strengthen T cell activation and attract innate immune cells to the targeted lesion to eradicate antigen-negative tumor cells by releasing anti-tumor cytokines, thus producing better tumoricidal effects, especially on solid tumors. They have also been shown to improve CAR-T cell expansion and persistence while rendering them resistant to the immunosuppressive TME. Fourth-generation CARs can also harbor an inducible suicide gene (inducible caspase 9) to rapidly eliminate CAR-T cells from the patient with a pharmacological reagent should severe toxicity ensue.
In addition to modification of CAR constructs and viral vector-mediated random insertion of additional genes, newer strategies take advantage of more targeted gene editing technologies such as TALEN and CRISPR-Cas systems to modify the T cell genome. Such technologies enable the knock-out of negative T cell regulators through targeted gene disruption, as well as the knock-in of transgenes.
What it means from a bioanalytical perspective
Significant research has been done with CAR-T cells to identify target antigens, avoid toxicity, improve CAR-T cell trafficking and entry into the tumor site, and promote better signaling, less exhaustion, and memory phenotypes in solid tumors—but as always, more work is needed. With the development of more complex CAR-T cell therapies will come a need for more sophisticated bioanalytical methodologies to ensure the efficacy and safety of these promising cancer therapeutics. It will require scientific collaboration as well as ingenuity to develop advanced, personalized bioanalytical packages tailored to the specifics of each CAR-T therapeutic.
Corinna Fiorotti, Ph.D., is Scientific Officer & Key Accounts Director at BioAgilytix, a bioanalytical lab specializing in large molecule bioanalysis.