Table 1

Methods and tools to assess efficacy of engineered T cells

AssessmentMethods and toolsAdvantagesLimitationsFuture directions
CAR-T or TCR-T cell functionalityKilling,
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-TNSG 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 regimensSyngeneic 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 rejectionAllogeneic 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.