Multiplexed Cytokine Assessment of T- and NK-Cell Responses to Tumor Cells: Parallel Assessment of Biomarkers Associated with Efficacy, Safety, and Persistence
Authors: Layman Hans , Jackson Belinda , Belkina Natalya , Ranganathan Sripriya , Wohlstadter Jacob N 
Published: December 2024

Engineered immune effector cell (IEC) therapies such as CAR T have revolutionized the management of hematological malignancies and solid tumors. Multiple CAR T therapies (eg Kymriah, Yescarta, and Breyanzi) have been approved by the FDA since 2017. Patients treated with CAR T often experience durable relapse-free survival. In a subset of responding patients, these IEC therapies demonstrate persistence, enhanced effector function, limited T cell exhaustion, and favorable safety profiles. Rational design of cellular immunotherapies must optimize for persistence and efficacy of the treatment while minimizing cellular exhaustion and adverse events resulting from excessive immune stimulation. Presently, limited human-relevant assays exist to evaluate a cellular immunotherapy's mode of action in parallel with any potential safety liabilities. Thus, we created protein-based, translationally relevant, multiplexed immunoassays to rapidly assess the potential efficacy, persistence and safety of CAR T or CAR NK cellular immunotherapies using in vitro co-culture assays.

To evaluate the translationally-relevant biomarkers of cellular immunotherapy associated with efficacy, persistence and safety, we first established a co-culture model using TALL104 cells or NK92 cells (effectors) with various target cell lines associated with solid tumors (breast: MCF7, prostate: PC3, glioma: U87MG), hematological malignancies (CML: K562) and normal healthy cells (HEK293). TALL104 cells were used as an allogeneic T cell that have lytic capabilities to numerous cell types, including healthy cells due to non-restricted human leukocyte antigen (HLA) interactions. NK92 cells were used as an allogeneic NK cell line that spares select cell types due to selectivity for regulated KIR ligands and receptors. We co-cultured the effector cells with the target cells at various effector:target (E:T) ratios for 6 and 24 hours.

Following incubation, we collected culture supernatants and cell lysates. Biomarkers of efficacy, persistence, and safety were evaluated in multiplex using the Meso SECTOR S 600MM instrument. The presence and concentration of multiple candidate biomarker proteins were assessed in multiplex using Meso Scale Discovery U-PLEX CAR-T Combos related to (1) immune activation and cytotoxicity (IFNγ, Granzyme B, IL2, IL12p70, Perforin), (2) persistence of cellular immunotherapy (IL7, IL15, IL18, CD40L), and (3) adervse events like CRS and ICANSa (IL1β, IL10, IL6, MCP1). Target cell death (via cleaved caspase 3) and total cell viability were also assessed.

We found differing magnitudes of safety, persistence, and efficacy biomarkers that were effector cell-specific. Biomarkers related to CRS and ICANS such as MCP1, IL1β and IL6 were significantly elevated in co-cultures of HEK293 with TALL104 cells compared to NK92. This may be due to the low density of KIR ligands and receptors on HEK293 versus MHC-I. Moreover, we observed a significant increase in the cytotoxicity of HEK293 when co-cultured with TALL104 from increased levels of Cleaved Caspase 3 and ATP compared to NK92. Secreted biomarkers associated with efficacy and persistence (e.g. IFNγ, Granzyme B, IL7, IL15) were also detected in co-cultures of HEK293 with TALL104 further indicating that the TALL104 cell is facilitating a cytolytic mechanism and persisting due to T cell receptor / HLA engagement with target HEK293 cells.

With tumor cell lines, efficacy and persistence biomarkers were dependent on the E:T ratio of the co-culture. Namely, levels of IFNγ, IL2, IL7, IL15, and IL18 were elevated based on the number of effector versus targets. This indicates that HLA or KIR ligand presence and avidity facilitates efficacy and persistence programs. Safety biomarkers were also elevated in co-culture conditions, but this is expected as death of tumor cells via cytolysis increases pro-inflammatory markers like IL10, IL1β, MCP1, and IL6.

Multiplexed protein biomarker assays provide a comprehensive means to evaluate a cellular immunotherapy's functional activity via persistence and killing of target cells while simultaneously assessing a safety margin. Here, we have demonstrated the utility of Meso Scale's multiplexed panels against relevant tool IECs and tumor cell co-culture models. The adaptation of these biomarker assays from in vitro screens may aid the development of new allogeneic cellular immunotherapies with improved outcomes.