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Review
. 2020 Aug 25;18(1):134.
doi: 10.1186/s12964-020-00617-7.

The Role of Immunological Synapse in Predicting the Efficacy of Chimeric Antigen Receptor (CAR) Immunotherapy

Dongfang Liu  1   2 , Saiaditya Badeti  3 , Gianpietro Dotti  4 , Jie-Gen Jiang  3 , He Wang  3 , James Dermody  5 , Patricia Soteropoulos  5 , Deanna Streck  5 , Raymond B Birge  6 , Chen Liu  3   7
Affiliations
Review

The Role of Immunological Synapse in Predicting the Efficacy of Chimeric Antigen Receptor (CAR) Immunotherapy

Dongfang Liu et al. Cell Commun Signal. .

Abstract

Chimeric Antigen Receptor (CAR) immunotherapy utilizes genetically-engineered immune cells that express a unique cell surface receptor that combines tumor antigen specificity with immune cell activation. In recent clinical trials, the adoptive transfer of CAR-modified immune cells (including CAR-T and CAR-NK cells) into patients has been remarkably successful in treating multiple refractory blood cancers. To improve safety and efficacy, and expand potential applicability to other cancer types, CARs with different target specificities and sequence modifications are being developed and tested by many laboratories. Despite the overall progress in CAR immunotherapy, conventional tools to design and evaluate the efficacy and safety of CAR immunotherapies can be inaccurate, time-consuming, costly, and labor-intensive. Furthermore, existing tools cannot always determine how responsive individual patients will be to a particular CAR immunotherapy. Recent work in our laboratory suggests that the quality of the immunological synapse (IS) can accurately predict CAR-modified cell efficacy (and toxicity) that can correlate with clinical outcomes. Here we review current efforts to develop a Synapse Predicts Efficacy (SPE) system for easy, rapid and cost-effective evaluation of CAR-modified immune cell immunotherapy. Ultimately, we hypothesize the conceptual basis and clinical application of SPE will serve as an important parameter in evaluating CAR immunotherapy and significantly advance precision cancer immunotherapy. Video abstract Graphic abstract for manuscript CCAS-D-20-00136 by Liu, D., et al., 'The Role of Immunological Synapse in Predicting the Efficacy of Chimeric Antigen Receptor (CAR) Immunotherapy". The various branches of evaluating cancer immunotherapy metaphorically represented as a Rubik's cube. The development of a novel approach to predict the effectiveness of Chimeric Antigen Receptor (CAR)-modified cells by quantifying the quality of CAR IS will introduce a new parameter to the rapidly expanding field of cancer immunotherapy. Currently, no single parameter can predict the clinical outcome or efficacy of a specific type of CAR-modified cell. IS quality will serve as a quantifiable measure to evaluate CAR products and can be used in conjunction with other conventional parameters to form a composite clinical predictor. Much like a Rubik's cube has countless configurations, several methods and combinations of clinical metrics have arisen for evaluating the ability of a given immunotherapeutic strategy to treat cancer. The quality of IS depicting cancer immunotherapy is metaphorically expressed as a Rubik's cube. Each face/color represents one aspect of cancer therapy. Each grid in one face indicates one factor within that aspect of cancer therapy. For example, the green color represents the tumor microenvironment, and one out of the nine grids in the green color indicates suppressor cells (suppressors in green). Changes in one factor may completely alter the entire strategy of cancer therapy. However, the quality of IS (illuminated center red grid) makes the effectiveness of CAR immunotherapy predictable.

Keywords: and cancer; chimeric antigen receptor; immunological synapse; immunotherapy.

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Conflict of interest statement

The authors declare no competing interests. No pharmaceutical company was involved in this manuscript.

Figures

Fig. 1
Fig. 1
Representative CAR good and poor IS formed on the lipid bilayer. (a) Diagram of a lipid bilayer model to study GD2-CAR IS formation and GD2-CAR-T cell activation using idiotypic antibody (clone, 1A7) against the GD2-CAR to trigger CAR signaling on the glass-supported planar lipid bilayer system. (b) Schematic representation of three-dimensional (3D) confocal image reconstitutuin. Briefly, primary GD2-CAR-T cells were stimulated on the SLB containing biotinylated fluorescently labeled anti-GD2-CAR (yellow), fixed, permeabilized, and then stained with phalloidin (blue), anti-F-actin (green), and perforin (red). An individual cell was imaged under the 3D Olympus confocal setting, and then the reconstitution of these 3D confocal images . (c & d) Comparison of good CAR IS (c) and poor CAR IS (d) is illustrated by these reconstituted 3D confocal images. The x-y focal plane represents the lipid bilayer surface. The Z focal plane represents the CAR T cell position on the top of lipid bilayer. Scale bars, 5μm. Notably, stronger F-actin accumulation, lytic granule polarization, and CAR molecular accumulation is associated with optimal CAR IS
Fig. 2
Fig. 2
Select the best CAR from different company products for a particular patient. Blood is collected by a lab professional into a 10 ml tube with anticoagulant as a regular specimen collection procedure in any certified blood testing laboratory. The tube can be sent directly to the synapse testing lab without transferring to a secondary tube. PBMCs, plasma, and tumor cells can be enriched. Plasma can be used later for mimicking tumor microenvironment by adding the patient’s plasma to the imaging system on both the lipid bilayer system and VCP system. Meanwhile, the enriched PBMCs are placed in culture and expanded. The viral vectors containing CAR1, CAR2, CAR3, CAR4, etc. are added to generate different versions of CAR products. These different CAR products are subjected to IS quality testing. Both the lipid bilayer chip and VCP device can be used to evaluate IS quality. IS quality ranking reports can be presented to physicians who can use the data to prescribe the best CAR product for a particular patient. A manufacturing company then generates this prescribed CAR with proper quality control release testing and quality assurance review. The final product is cryopreserved and delivered to distant infusion sites, where the CAR T medicine is infused
Fig. 3
Fig. 3
Optimizing CAR design for translational research. Blood is collected by a lab professional into a 10 ml tube with anticoagulant as a regular specimen collection procedure in any certified blood testing laboratory. The tube can be sent directly to the IS testing lab without transferring to a secondary tube. PBMCs can be enriched. The enriched PBMCs are placed in culture and expanded in vitro. The viral vectors containing CAR1, CAR2, CAR3, etc. are added to generate different version of CAR constructs generated by a research laboratory. These different CAR constructs are optimized to enhance the functions of CAR T. However, it is impractical to put every single construct into a preclinical study (e.g., in vivo animal model). The CAR T cells are subjected to IS quality testing using both the lipid bilayer chip and VCP device. IS quality ranking reports can be presented to scientists who can select the best CAR construct for a preclinical animal study, and ultimately, the optimal CAR design for downstream clinical applications. The SPE system can identify the best CAR construct from numerous constructs by screening the IS quality
Fig. 4
Fig. 4
Select patients who will respond to treatment with universal CAR-modified cells or a particular CAR product. To select responders for a particular CAR treatment, tumor cells or plasma can be isolated from 8 ml of peripheral blood from each patient. CAR cell IS quality can be assessed by lipid bilayer and VCP device. Briefly, blood is collected into a 10 ml tube with anticoagulant as a regular specimen collection procedure. The tube can be sent directly to the synapse testing lab without transferring to a secondary tube. PBMCs, plasma, and tumor cells can be enriched. Plasma can be used later for mimicking the tumor microenvironment by adding the patient’s plasma to the imaging system on both the lipid bilayer system and VCP system. Meanwhile, the enriched PBMCs are placed in culture and expanded. The viral vector containing the universal CAR construct such as CD19-CAR, is added to generate CAR products. These universal CAR products are subjected to IS quality testing. Both the lipid bilayer chip and VCP device can be used to evaluate the IS quality of universal CAR products in response to tumor cells isolated from each individual patient. IS quality ranking reports can be presented to physicians who will base their decision of which CAR product to prescribe. The informed physicians can select a particular patient (responder) for a particular CAR treatment or clinical trial. Meanwhile, for non-responders, IS testing can identify another suitable CAR therapy. The manufacturing company generates the prescribed CAR for this patient with proper quality control release testing and quality assurance review. The final product is cryopreserved and delivered to infusion sites, where the CAR T medicine is infused
Fig. 5
Fig. 5
Kinetics of IS formation in live Kappa-CAR T cells under total internal reflection fluorescence microscopy (TIRF) to evaluate the dynamics of CAR IS quality. All cells were imaged by TIRF microscopy on lipid bilayers carrying Kappa protein conjugated with Alexa Fluor 488 at the indicated time points. a Kappa-41BB cells were imaged at 1 min, 3 min, 5 min, 10 min, 20 min, and 30 min. Time-lapsed differential interference contrast (DIC) (top panel) and TIRF (bottom panel) images are shown. b Kappa-CD28 cells were imaged at 1min, 3min, 5min, 10min, 20min, and 30min. Time-lapsed DIC (top panel) and TIRF (bottom panel) images are shown. c Schematic representation of recombinant retroviral vectors encoding kappa-CAR. Both Kappa-CD28-CAR and Kappa-4-1BB-CAR constructs (CD28 and 4-1BB) contain CD28 transmembrane domain and intracellular domain of CD3 zeta. The difference between Kappa-CD28-CAR and Kappa-4-1BB-CAR constructs is that Kappa-4-1BB-CAR construct contains intracellular domain of 4-1BB molecule
Fig. 6
Fig. 6
Potential Multiple Factors Determine the Quality of Immunological Synapse in Cancer immunotherapy. The development of a novel SPE approach to predict the effectiveness of Chimeric Antigen Receptor (CAR)-modified cells by quantifying the quality of immunological synapse is dependent on multiple factors. In addition to patient conditions (e.g., age, sex, tumor burden, stage of diseases, tumor antigen mutations & loss, etc.), there are three main aspects to be considered to quantify the quality IS. First, intrinsic potency of CAR-modified immune cells includes different subsets of CAR-modified immune cells, different modifications of CAR constructs, inhibitory receptor expression, and CAR tonic signaling. Second, intra-tumor heterogeneity (IHT) includes mutations of tumors, inhibitory ligand (e.g., PD-L1) expression, suppressor cells, and tumor stiffness. Third, tumor microenvironment (TME) contains cytokine milieu, metabolites, hypoxia, and collagen fibers around tumor cells
Fig. 7
Fig. 7
The various branches of evaluating cancer immunotherapy metaphorically represented as a Rubik’s cube. The development of a novel approach to predict the effectiveness of Chimeric Antigen Receptor (CAR)-modified cells by quantifying the quality of CAR IS will introduce a new parameter to the rapidly expanding field of cancer immunotherapy. Currently, no single parameter can predict the clinical outcome or efficacy of a specific type of CAR-modified cell. IS quality will serve as a quantifiable measure to evaluate CAR products and can be used in conjunction with other conventional parameters to form a composite clinical predictor. Much like a Rubik’s cube has countless configurations, several methods and combinations of clinical metrics have arisen for evaluating the ability of a given immunotherapeutic strategy to treat cancer. The quality of IS depicting cancer immunotherapy is metaphorically expressed as a Rubik’s cube. Each face/color represents one aspect of cancer therapy. Each grid in one face indicates one factor within that aspect of cancer therapy. For example, the green color represents the tumor microenvironment, and one out of the nine grids in the green color indicates suppressor cells (suppressors in green). Changes in one factor may completely alter the entire strategy of cancer therapy. However, the quality of IS (illuminated center red grid) makes the effectiveness of CAR immunotherapy predictable
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