CAR T Cell Therapy Cytokine Response—ELISA Kits and Multiplex Immunoassays

Cellular immunotherapy or adoptive cell therapy (ACT) involves the use of autologous (self) or allogenic (donor) cells infused into patients for the treatment of cancers. Immune cells that have been utilized or are currently being investigated for adoptive transfer include tumor-infiltrating lymphocytes (TILs), engineered T cell receptor cells (TCR) or chimeric antigen receptor (CAR) T cells, regulatory T cells (Tregs), and other cells such as natural killer (NK) cells. The workflow below highlights five-step process involving CAR generation, functional validation of molecular properties, and testing their efficacy in a variety of mouse models (Figure 1).

Figure 1. CAR T cell workflow.
 

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Cytokines play an important role in CAR T cell therapy, as they are required for in vitro expansion and persistence in delivered T cell therapeutic doses. By incorporating cytokine genes into the vectors that encode CARs during manufacturing, further optimization is achieved. In one such case, the use of IL-2 to improve the effectiveness of autologous TIL therapy in the treatment of metastatic malignant melanoma has been documented [1]. The importance of cytokines in enhancing adoptive cell therapy is also demonstrated by the fact that lymphopenia-induced elevation of homeostatic cytokines, such as IL-7 and IL-15, are key to proliferation and survival of adoptively transferred T cells [2, 3].

Understanding cytokine profiles in context of the tissue microenvironment is also a critical factor to developing effective cellular immunotherapies for solid tumors. In one study, the development of an inverted cytokine receptor (ICR) demonstrated the enhancement of the antitumor potency of T cells in the presence of a rich IL-4 immunosuppressive environment. This type of amplified immune response has also been reported in CAR T cells that secrete IL-12 or IL-18, which help increase half-life of the therapies along with modulating the tumor microenvironments [4, 5, 6].

Cellular immunotherapy can lead to adverse events such as cytokine release syndrome (CRS) or cytokine storms, cardiac and neuro toxicities, and hypotension. These adverse events can be triggered by the adoptively transferred cells and/or other host cells in response to the therapy and is mediated through the release of cytokines. Therefore, the evaluation of serum biomarkers such as pro-inflammatory cytokines in response to cellular immunotherapy can help determine the risk for subsequent toxicity and early intervention. Monitoring cytokines and other factors associated with CAR T therapy include those involved in cytotoxic activity, immunomodulation to support antitumor activity and inflammation (Table 1)[7].

Table 1. Examples of biomarkers implicated in CAR T cell therapy research and treatment.

Potency tests such as in vitro IFN-γ production and cytotoxicity assays are used to determine whether CAR T cells have the expected therapeutic efficacy and safety profiles. While these individual potency assays provide important information, they can fall short of predicting clinical efficacy and safety. This could be due to the nature of cellular immunotherapeutic products where single potency test may be insufficient to predict complex features and thus necessitates a more comprehensive analysis with a diverse set of assays.

The use of multiplexed immunoassays to measure cytokines provides an indirect but convenient and comprehensive way to test for potency of CAR T cells. Potency assessments can be measured indirectly using ProcartaPlex panels, which used in combination with cytotoxicity assays show that CAR T cells produced higher cytokine levels than untransduced controls after overnight stimulation with CD19-expressing K562 cells (Figure 4, Tab 4, ProcartaPlex multiplex assays).

Along with the overall potency of CAR T therapies, the expression of T cell exhaustion markers is an important variable that can be characterized via immunoassays. Solid tumors have dense microenvironments which can reduce CAR T efficacy, and by monitoring the amount of cytokines released, the overall effect of the therapeutics can be assessed. Several studies report how immunosuppressive cytokines such as TGF-B can rapidly increase the exhaustion of CAR T cells [8]. This type of research is important for designing therapies that deliver the optimal amount of cytokines without adverse effects or premature exhaustion. In one such instance, while IL-2 is noted as a strong inducer for T cell proliferation, it also increases the rate of exhaustion. Substitution with IL-15 or IL-7 showed improved survival in pre-clinical models, which was measured though secretion of cytokines and chemokines using ProcartaPlex multiplex immunoassays [9].