CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells

Abstract

Chimeric antigen receptor (CAR)-modified T cells brought a paradigm shift in the treatment of chemotherapy-resistant lymphomas. Conversely, clinical experience with CAR T cells targeting solid tumors has been disheartening, indicating the necessity of their molecular-level optimization. While incorporating CD28 or 41BB costimulatory domains into CARs in addition to the CD3z signaling domain improved the long-term efficacy of T cell products, their influence on early tumor engagement has yet to be elucidated. We studied the antigen-independent self-association and membrane diffusion kinetics of first- (.z), second- (CD28.z, 41BB.z), and third- (CD28.41BB.z) generation HER2-specific CARs in the resting T cell membrane using super-resolution AiryScan microscopy and fluorescence correlation spectroscopy, in correlation with RoseTTAFold-based structure prediction and assessment of oligomerization in native Western blot. While .z and CD28.z CARs formed large, high-density submicron clusters of dimers, 41BB-containing CARs formed higher oligomers that assembled into smaller but more numerous membrane clusters. The first-, second-, and third-generation CARs showed progressively increasing lateral diffusion as the distance of their CD3z domain from the membrane plane increased. Confocal microscopy analysis of immunological synapses showed that both small clusters of highly mobile CD28.41BB.z and large clusters of less mobile .z CAR induced more efficient CD3ζ and pLck phosphorylation than CD28.z or 41BB.z CARs of intermediate mobility. However, electric cell-substrate impedance sensing revealed that the CD28.41BB.z CAR performs worst in sequential short-term elimination of adherent tumor cells, while the .z CAR is superior to all others. We conclude that the molecular structure, membrane organization, and mobility of CARs are critical design parameters that can predict the development of an effective immune synapse. Therefore, they need to be taken into account alongside the long-term biological effects of costimulatory domains to achieve an optimal therapeutic effect.

Keywords: cell therapy; chimeric antigen receptor (CAR); electric cell-substrate impedance sensing; fluorescence correlation spectroscopy (FCS); immunological synapse; immunotherapy; receptor clustering; receptor mobility; structure prediction.

Figure 1

Generation of CAR T cell products from first to third generation. (a) Representative flow cytometry histogram of non-transduced (NT) T cells, and HER2.z, HER2.CD28.z, HER2.41BB.z and HER2.CD28.41BB.z CAR T cells. (b) Expression of the HER2-CARwas quantified with indirect labeling using a HER2-Fc fusion protein and an Alexa Fluor 647-conjugated anti-human IgG (N = 3). Histograms represent mean ± SD.

Figure 2

Assessing the oligomerization state and surface organization of various chimeric antigen receptors in the unstimulated CAR T cell membrane. (a) Western blot analysis of HER2.z, HER2.CD28.z, HER2.41BB.z and HER2.CD28.41BB.z CAR T cells. The membranes were probed for anti-human CD3ζ. (b) Spot intensities were quantified using ImageJ/Fiji, and the proportion of dimer/oligomer states was determined. (c) Representative raw and segmented AiryScan images of fluorescently tagged CARs. Clusters were segregated by watershedding and quantified. The scale bar represents 2 µm. (d) Total intensity of the apical membrane slices. (e) Average number of clusters per 10 µm² apical membrane surface. (f) Distribution of individual cluster areas. (g) Integrated intensity of individual clusters. The charts represent mean ± SEM; nHER2.z = 54, 4 donors; nHER2.CD28.z = 44, 3 donors; nHER2.41BB.z = 47, 4 donors; nHER2.CD28.41BB.z = 54, 4 donors; * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Membrane diffusion analysis and tertiary structure modeling of HER2-specific chimeric antigen receptors. (a) Representative fluorescence fluctuation intensity traces of 10-s FCS runs. (b) Pooled autocorrelation curve for each CAR. Position of triplet lifetime (τ_Triplet), diffusion correlation times of the detached Alexa Fluor 647 conjugated monomer HER2 ECD (τ_A647HER2) and the various CAR species (τ._z; τ._CD28._z; τ._41BB._z; τ._CD28._41BB._z) are indicated by arrows. (c) Diffusion coefficients of CAR constructs. The chart represents mean ± SEM, nHER2.z = 300, 4 donors; nHER2.CD28.z = 260, 3 donors; nHER2.41BB.z = 276, 4 donors; nHER2.CD28.41BB.z = 289, 4 donors; *** p < 0.001. (d) Schematic diagram illustrating the tertiary structure of the native TCR and the CAR constructs. TCR: T Cell Receptor complex extracellular domains; CD3z TM: CD3ζ transmembrane segment; CD3z IC: CD3ζ intracellular domain; FRP5 scFv: single chain variable fragment of FRP5 antibody targeting the HER2 ECD; IgG1 SH: short hinge linker derived from IgG1; CD28 TM: transmembrane segment derived from CD28; CD28 IC: costimulatory domain from CD28; 41BB IC: costimulatory domain from 41BB. Confidence scores of the predicted models and intramolecular disulfide bonds are indicated.

Figure 4

Early activation signaling events were determined by measuring CD3ζ and Lck phosphorylation. (a) Representative microscopic images following 15 min of effector–tumor co-culturing. (b) Quantitative image analysis of CD3ζ and Lck phosphorylation. The area of the immune synapse, the average and the integrated intensity of the phospho-specific label in the synapse are plotted showing the distribution of individual data points as well as their mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Kinetic analysis of in vitro killing of the various CAR T cells. Killing of JIMT-1 target cells was determined using the Electric Cell-substrate Impedance Sensing method. The effector/target ratio was 1:1. Impedance was monitored for 25 h. Averaged traces were normalized to impedance measured at the start of treatment, and these normalized impedances were normalized to the corresponding value of the non-transduced (NT) T cell control at every time point. The graph represents mean ± SD from N = 2 independent experiments with 2 technical replicates.