Table 1.

Methods and tools to assess efficacy of engineered T cells

Assessment	Methods and tools	Advantages	Limitations	Future directions
CAR-T or TCR-T cell functionality	Killing, Antigen stress assay, proliferation, Functional avidity cytokine release	Optimized, easy-to-use methods to address functionality Suitable for TCR and/or CAR candidate selection and validation of T-cell engineering approaches	Difficult to correlate with clinical outcome Very limited relevance to TME	Perform these assays in normoxia versus hypoxia or in low glucose or low pH conditions Optimization of these techniques to be used in 3D co-culture assays
	Planar glass-supported lipid bilayers, molecular imaging	Highly quantitative methodology to assess CAR-T sensitivity towards antigen and CAR-proximal downstream signaling	Requires special expertize and equipment for implementation Reductionist approach	Broaden access to research and development (R&D) Tight collaboration between experts in biophysics and synthetic biology
	Human organoids	Recapitulates many aspects of the TME, including intratumor heterogeneity	Lack of immune cells Tumor/stroma structure not always preserved Readouts at single time points	Addition of immunosuppressive or stimulating cells Optimization of readouts to analyze engineered T-cell functions in real time
	Tumor slices	Test T-cell efficacy in a preserved human TME Identify obstacles to early T-cell responses (min to hours)	Limited availability and viability of fresh tissue slices Readouts at single time points Allogeneic responses if using non-autologous T-cells	Incorporation of microfluidic to improve ex vivo culture & model in vivo T infiltration into solid tumors. Optimization of culture conditions and readouts to analyze engineered T-cell functions in real-time
	Microphysiological 3D tumors	Simulates invasive growth of tumor cells Engineered -T cells enter arterial medium flow actively Assessment of T cell adherence and infiltration	Requires special expertize and equipment for implementation Costly Dependence on growth behavior of primary material or cell lines	Setup with patient-derived primary tumor cells or antigen-density- modified cell lines to reflect tumor heterogeneity Implementation of TME components such as immunosuppressive cells
	RNA sequencing	Deep characterization of T-cells (can be done at single cell level)	Costly Complex sample preparation workflows Requires bioinformatics expertize for data analysis	Broaden application to post-infusion patient samples Reduction of costs Simplification of data analysis
	Nanostring	Easy to use, sensitive method to analyze up to 800 RNA targets without cDNA conversion or library prep	Costly Requires access to specific equipment	Reduction of costs Development of specific panels to analyze dysfunctional vs effective post-infusion T-cell patient samples
	Polyfunctionality (Isoplexis)	Polyfunctional assessment of engineered T cells Correlates with patient outcome in CD19-specific CAR-T cell trials.	Costly Limited access to the required equipment	Reduction of costs Validation of their predictive efficacy value in more clinical trials
	Spatial RNA	RNA expression according to T-cell spatial position within the tumor tissue	Costly Requires bioinformatics expertize for data analysis	Reduction of costs Simplification of data analysis
Efficacy and in vivo persistence of human CAR-T and TCR-T	NSG animals	Easy engraftment of tumors and T-cells of human origin Study of human engineered T cells, prepared as those used in clinical trials, before regulatory approval	Difficult to test combination therapies since NSG are sensitive to irradiation and chemotherapy No human TME, could skew tumor characteristics The lack of human cytokines released by innate cells limits T-cell persistence	Use of tumor cells expressing different levels of antigen expression to mimic tumor heterogeneity Inclusion of immunosuppressive immune cells into tumors Stable expression of genes encoding for human cytokines
	Humanized SGM3 mice	Reconstitution of human TME Xeno-tolerant T cells, with less risk of GVHD	Lack of stromal component Residual lymph nodes	Transplantation with artificial lymph nodes Further humanization Colonization with human microbiota
	HLA-A2 transgenic mice	Murine tumor cells with human HLA-A2 can present peptides to human or murine T cells expressing human transgenic TCRs	Differences in antigen processing and presentation mechanisms between mice and humans	Development of multiple HLA-type transgenic mice to broaden applicability for testing of TCR-T cells
	Intravital imaging	Real-time assessment of T cell trafficking and killing dynamics in vivo Characterization of functional heterogeneity within engineered-T cells in vivo Help to guide rationale choice of the composition of the infusion product	Restricted to a few hours of continuous observation.	Improvement in technology to accurately visualize deeper organs/tissues Use of imaging chambers
Lymphodepletion regimens	Syngeneic models	Intact host immune system, recapitulation of the TME Prediction of on-target off-tumor toxicities Effects of lymphodepletion in epitope spreading	Differences between mouse and human engineered T cells Engineering of murine T cells can be challenging Limited translation to clinical setting Novel combinations may not be able to be tested in mice due to lack of target-expression or toxicity	Optimize methods to generate mouse engineered T-cells Use of transgenic mice expressing human target antigens
Role of the tumor microenvironment Epitope spreading	Syngeneic models	Responsive host immunity, including TME and reactivity of endogenous, tumor-specific cells Synergy with other immunotherapies can be assessed	Use of mouse CAR constructs. Differences between mouse and human T cells Engineering of murine T cells can be challenging	Optimization of protocols to engineer and freeze murine T-cell Assessing the impact of human microbiota on TME and engineered T-cell efficacy
Allogeneic host vs graft rejection	Allogeneic mixed lymphocyte reactions Proliferation Cytokine release	Allow testing immunosuppressive drugs Efficacy of gene editing to resist lymphodepleting drugs or suppress MHC expression	Lack of systematic evaluation of NK cell impact	Addition of allogeneic NK cells to the assays

3D, three-dimensional; GvHD, graft versus host disease; HLA, human leukocyte antigen; MHC, major histocompatibility complex; NSG, NOD/SCID/Il2rγc-/-; TME, tumor microenvironment.