Cell Therapy Testing Assays

Charles River has translational assays for CAR-T cellular therapy testing of on target efficacy and off-target killing, using human primary cells or iPSC-derived subsets as well as in vivo models. The range of tissues and cells assessed will be determined not only by key cell types where a lack of off-target killing needs to be demonstrated but also using information about the predicted expression pattern of the target of the cellular therapy within the body. This supports proof of concept and minimizes risk of cytotoxic effects.

In Vitro CAR-T Cell Therapy Testing Assays

Diagram showing in vitro assays for CAR T cell therapy testing. Tumor cells in monolayer, spheroid, or suspension format are labelled and co-cultured with the cellular therapy.
Figure 1. Diagram of in vitro CAR-T cell therapy testing assays.

Our in vitro assessment of on-target tumor killing assays includes live cell imaging of solid tumor types, flow cytometry-based analysis of diffuse tumors, and analysis of soluble factors produced by effector immune cell subsets. Adherent tumor cells can be standard tumor cell lines or PDX/CDX cells and can be run in monolayer or spheroid formats when evaluating the ability of a cellular therapeutic to target tumor cell killing. The killing of non-adherent tumor cells can be analyzed by flow cytometry and may be combined with simultaneous phenotyping of tumor cells and/or cellular therapeutic. A variety of options are available for analyzing cytokines, chemokines, or other soluble factors present in supernatant.

Watch the video below to learn more about in vitro cytotoxicity testing of CAR T cells and how this can aid your program:



Sanne Demandt, Group Leader for Biology at Charles River Leiden, discusses in vitro cytotoxicity testing of CAR-T cells.

In Vitro Pharmacology

CAR-T or cell therapies will need to be assessed for their efficacy, either during early development and lead optimization or to investigate various clinical indications for your lead therapy. In vitro co-culture of the CAR-T cell with positive and negative target expressing cell types, where T cell activation and cell death of your target cells is quantified, can provide insight into the pharmacology of your therapy. During your in vitro pharmacology assessments, it is important to consider your cellular therapy’s activity, specificity, and potency using assay data (see example below).

DATA: T cell-mediated killing and CAR-T cell activation

Two graphs showing T cell mediated killing and CAR T cell activation.

Figure 2. In vitro CAR-T assays showing efficacy against a non-target expressing and high-target expressing cell line.
Percentage of target cell death was quantified using Hoechst/PI staining on Flow Cytometry, with Bortezomib being a toxicity control. T cell activation was quantified by IFNy expression by MSD.

During early development cell products with variation in binders, spacers, and linkers can be compared in activity and specificity through a co-culture with non-target expressing cells and mid to high target antigen-expressing cells. Cell lines exhibiting expressing close homologues to the target antigen can also be included to determine any potential off-target binding, which if present can contribute to lead optimization or be de-risked during future off-target cytotoxicity experiments utilizing healthy primary cells.

Potency of the cell therapies can be established through inclusion of multiple effector target ratios and timepoints to identify the lower and higher activity limits of the CAR-T cell therapy. An important consideration to keep in mind during efficacy assessments is that the most potent CAR-T cell might not be your best product for long persistence or safety aspects. More information on validated release testing can be found on our bioactivity and potency testing information page.

The activity and potency of your CAR-T cells against various cancer types can be an important factor to determine the clinical indication of the therapy. A large selection of cancer cell lines (multiple disease types) can be screened for target expression; either in silico or via quantification of protein expression. A sub-selection of cancer cell lines representing various commercial indication can then be screened for differences in potency of the CAR T cells through co-culture experiments quantifying target cell death and T cell activation.

Selecting the correct CAR-T product for efficacy, persistence, safety, and clinical indication is essential to the success of your therapy.


Evaluating On-Target and Off-Tumor Effects of Cell Therapies

In addition to evaluating the efficacy of tumor cell killing by cellular therapeutics such as CAR-T and TCR transgenic T cells, we can assess off-target activity or on-target/off-tumor activity by screening against a panel of primary human cell types. These cover the critical organs/tissues known to be potentially susceptible to the adverse effects of cell therapies mediated by off-target cytotoxicity. Additional primary cell types can be added to the panel according to the pattern of expression of the target of the TCR or CAR-T.

On-Target and Off-Tumor Effects

Diagram of on-target and off-target effects of cellular therapies. Cell therapy testing allows these effects to be predicted in in vitro assays.
Figure 3. Diagram of on-target and off-target effects seen in in vitro CAR-T cell therapy testing assays.

These off-target effects are discriminated from potential allogeneic-driven cytotoxicity by matching MHC class I expression between cellular therapeutic and target cells as closely as possible. In many cases, on-target/off-tumor effects can also be evaluated by loading the target antigen onto cell types that would not normally express the tumor-associated antigen.

This platform is also suitable for testing the ability of immunomodulators to enhance the killing of tumor cells by endogenous immune cells. Both IncuCyte® and flow cytometry-based assays can be adapted to use PBMC from normal healthy donors as the effector cells. Specific immune subsets can either be isolated from PBMC or depleted from PBMC prior to co-culture with tumor targets, allowing for identification of those subsets most impacted by the immune modulator. Validated assays include the testing of checkpoint inhibitors, multi-specifics, and enhancers of general and specific (i.e., ADCC, phagocytosis, etc.) immune effector function.

In vitro CAR-T and cell therapy testing assays to evaluate on-target efficacy and off-target or on-target/off-tumor activity of cellular therapies:

  • IncuCyte®-based cell therapy assays (solid tumors)
    • Tumor cell number and apoptosis/necrosis readouts
    • Kinetic data allowing calculation of EC50 values for tumor killing
    • Monolayer or spheroid formats
  • Flow cytometry-based cell therapy assays (diffuse tumors)
    • Tumor cell viability can be evaluated in multiple ways
    • Tumor cells can be phenotyped in the context of immune killing/immune editing (e.g., expression of target molecules, inhibitors of immune effector function).
    • Activation state of cell therapy can be assessed for potential mechanism of action insight
  • Cytokine analysis
    • Luminex® or MSD for comprehensive cytokine profiling
    • TR-FRET for rapid, highly sensitive single cytokine analysis
    • ELISA for analysis of less common analytes
    • FC for cell-type specific analysis of cytokine production
  • Immunomodulators
    • These assays can be combined with immunomodulators (e.g., checkpoint inhibitors, multi-specifics, ADCC-enhancers) to evaluate potential synergy with SoC therapeutics currently used in the clinic
    • PBMCs isolated from normal healthy donors can be used as effectors in tumor killing assays, with specific cell types isolated or depleted prior to target co-culture

Tumor cells are labelled prior to co-culture with cellular therapeutic to allow for their identification in an effector:target co-culture system. Tumor cells may be cultured as a monolayer, as spheroids or in suspension. Cellular therapeutics may be CAR-T, TCR transgenics, endogenous PBMC, or any novel immune cell effector. Tumor killing and cellular therapeutic activation can be assessed by IncuCyte®, flow cytometry, and cytokine analysis.

DATA: Assessment of On-Target Killing

Figure showing the assessment of on-target killing in spheroid format (tumor cells were co-cultured with PBMC or PBMC + trastuzumab and spheroid size monitored over time to assess tumor cell killing) and for diffuse tumors, tumor cells were labelled and co-cultured with immune cell subsets at different E:T ratios and tumor death measured at a set time point.

Figure 4. In vitro CAR-T cell therapy testing assays for the assessment of on-target killing (A) spheroid format. Tumor cells were co-cultured with PBMC or PBMC +trastuzumab and spheroid size monitored over time to assess tumor cell killing. (B) for diffuse tumors, tumor cells were labelled and co-cultured with immune cell subsets at different E:T ratios and tumor death measured at a set time point.

DATA: Off-Target Screening Assay

Image showing primary/IPSC derived cells labelled in an off-target screening assay. Labelling the cells allows them to be differentiated from the cellular therapy and cell numbers to be monitored over time.

Figure 5. Examples of labelled primary/iPSC-derived cells used in an off-target screening assay. Labelling the cells allows them to be differentiated from the cellular therapy and cell numbers to be monitored over time. Addition of a caspase-3 reporter gives information on number of cells undergoing apoptosis.


The Right Tools to Optimize Cell Therapies for Oncology

In Vitro CAR T testing cell therapy testing to ensure efficacy and mitigate risk factors in the treatment of cancer.

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Our in vitro CAR-T cell therapy testing assays can accelerate the progress of your project and our core panel of off-target primary cell screening can be customized to fit the needs of your program. Importantly, the assays cover both on-target efficacy, off-target safety, and on-target/off tumor killing. Combined with our other early discovery, in vivo, and clinical services we provide a complete pipeline to take your therapeutic design from concept to clinic.

How can we help you?

Frequently Asked Questions (FAQs) about In Vitro CAR-T Testing and Cell Therapy

  • Do you offer a platform for CAR-T Discovery?

    We partner with Distributed Bio to provide a platform ideally suited for CAR T Discovery. Distributed Bio’s offering is unique because of the design of the library and the subsequent functional screen, reducing timeline and reduced issues downstream with developability, immunogenicity, and thermostability. The SuperHuman library already in scFv format, and can allow panning with different antigens (mouse, NHP, human). By working with us you can save time by immediately moving into a functional screen and in vitro CAR T testing.

  • What primary cell types do I need to screen my cellular therapy against?

    The primary cells needed for in vitro CAR T Cell therapy testing assays will be determined by the nature of your cellular therapy and the antigen/target it recognizes. Most cellular therapy programs screen against a panel of primary cell types from key critical organs and these form our core panel to screen for off-target killing.

    For TCR therapies, off-target killing may be driven by recognition of unexpected or unrelated antigens. By selecting primary cells expressing the correct HLA type for your TCR this should give a window above the allogenic response that may be generated by endogenous TCR recognizing allogeneic MHC. You will have determined expression of your antigen of interest and any highly related antigens or epitopes. By surveying normal human tissue for expression, this will help determine which primary cell types should be used in the screen for on-target killing.

    For CAR-based cellular therapies, you will have performed a search for expression of the antigen and any highly related antigens your CAR is directed against and screened normal tissue for expression. Based on these findings, on-target/off-tumor killing of primary cells can be assessed alongside off-target killing which may be driven by unrelated or unexpected antigens.

  • Why should I screen my therapy using in vitro models?

    In vivo models for cellular therapy testing use immunodeficient animals bearing the relevant tumor, expressing your antigen of interest, and reconstituted with your CAR T cell therapy. This allows assessment of the efficacy of tumor killing mediated by your CAR T cell therapy. However, the cellular therapy is effectively a xenograft which will make a xenoresponse to the mouse tissue over time.

    This mismatch between human HLA-restricted T cells and mouse MHC H-2 expressing cells means that specific off-target antigen-driven responses cannot be assessed within the mouse system. Therefore, in vitro CAR T testing assays for on-target killing of human cells complement in vivo tumor efficacy models and for off-target killing in vitro assays using primary human cells are more relevant and translatable.

  • What type of cell therapies are these assays designed for?

    Any current cellular therapeutic model can be tested in our assay setup for example CAR T cells (or CAR NK, CAR-Macrophage etc.) or TCR transgenic approach, and therapeutics designed to enhance the tumor-killing capability of the patient’s own immune cells, for example checkpoint inhibitors. This platform can also be adapted for novel cell therapies.

  • What size of project do you work on for in vitro CAR-T testing?

    Projects may be as small as a single proof-of-principle experiment or as large as a multi-site endeavor spanning several years. Readouts for in vitro CAR T cell therapy testing assays are tailored to suit the needs of the individual project.