High specificity of engineered T cells with third generation CAR (CD28-4-1BB-CD3-ζ) based on biotin-bound monomeric streptavidin for potential tumor immunotherapy

Abstract

Introduction: Immunotherapy has revolutionized cancer treatment, and Chimeric Antigen Receptor T cell therapy (CAR-T) is a groundbreaking approach. Traditional second-generation CAR-T therapies have achieved remarkable success in hematological malignancies, but there is still room for improvement, particularly in developing new targeting strategies. To address this limitation, engineering T cells with multi-target universal CARs (UniCARs) based on monomeric streptavidin has emerged as a versatile approach in the field of anti-tumor immunotherapy. However, no studies have been conducted on the importance of the intracellular signaling domains of such CARs and their impact on efficiency and specificity.

Method: Here, we developed second-generation and third-generation UniCARs based on an extracellular domain comprising an affinity-enhanced monomeric streptavidin, in addition to CD28 and 4-1BB co-stimulatory intracellular domains. These UniCAR structures rely on a biotinylated intermediary, such as an antibody, for recognizing target antigens. In co-culture assays, we performed a functional comparison between the third-generation UniCAR construct and two second-generation UniCAR variants, each incorporating either the CD28 or 4-1BB as co-stimulatory domain.

Results: We observed that components in culture media could inhibit the binding of biotinylated antibodies to monomeric streptavidin-CARs, potentially compromising their efficacy. Furthermore, third-generation UniCAR-T cells showed robust cytolytic activity against cancer cell lines upon exposure to specific biotinylated antibodies like anti-CD19 and anti-CD20, underscoring their capability for multi-targeting. Importantly, when assessing engineered UniCAR-T cell activation upon encountering their target cells, third-generation UniCAR-T cells exhibited significantly enhanced specificity compared to second-generation CAR-T cells.

Discussion: First, optimizing culture conditions would be essential before deploying UniCAR-T cells clinically. Moreover, we propose that third-generation UniCAR-T cells are excellent candidates for preclinical research due to their high specificity and multi-target anti-tumor cytotoxicity.

Keywords: Chimeric Antigen Receptor; Treg; engineered cells; immunosuppression; streptavidin-based CAR.

Figure 1

Schematic design of the vectors, UniCARs and their recognition system. (A) Design of the lentiviral expression construct encoding the 3rd generation UniCAR-28-BB, both 2nd generation UniCARCD28 and UniCAR41BB, and the eGFP. (B) Representation of the extracellular, transmembrane and intracellular elements that compose the 3rd generation UniCAR-28-BB. (C) Representation of a transduced cell expressing eGFP (green dots) and 3rd generation UniCAR-28-BB, which is able to recognize and bind to a biotinylated intermediate, such as a biotinylated monoclonal antibody. The biotinylated intermediate recognizes the antigens at the surface of the target cell. Figure created with BioRender.

Figure 2

Components of culture media can neutralize the binding capacity of UniCAR to biotin. (A) Flow cytometry dot plots depicting non-transduced (NT) and transduced Jurkat cells with lentivectors encoding 3rd generation UniCAR-28-BB. Cells cultured in RPMI 1640 + 10% FBS were labeled with Atto-655 and followed by eGFP expression. These dot plots serve as a representative example from n=11. (B) Graph illustrating eGFP expression in UniCAR-28-BB Jurkat cells cultured in RPMI 1640 + 10% FBS and in Xvivo15 + 5% ABS, along with their mean + SEM. Ns= non-significant difference as determined by the Mann-Whitney comparison test. (C) Graph depicting the frequencies of Atto-655-Biotin-labeled UniCAR-28-BB-Jurkat cells when cultured in RPMI 1640 + 10% FBS or + 5% ABS and in Xvivo15 + 10% FBS or + 5% ABS, with their mean ± SEM. *Significant difference between conditions with p<0.05, as determined by the one-factor ANOVA multiple comparisons test. (D) Graph depicting the change in frequencies of Atto-655-Biotin-labeled UniCAR-28-BB Jurkat cells which had been cultured in RPMI 1640 + 10% FBS and moved to Xvivo15 + 10% FBS or had been cultured in Xvivo15 + 10% FBS and moved to RPMI 1640 + 10% FBS, with their mean ± SEM. *Significant difference between conditions with p<0.05, as determined by the 2way ANOVA multiple comparisons test.

Figure 3

Viability and frequency of effector T cell transduction. (A) Flow cytometry dot plots representing the viability (visualized by the 7AAD negative signal) of Non-transduced (NT), UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 transduced T cells. These dot plots are a representative example of n=6. (B) Box and whiskers representing the frequency of viability of NT, UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells, n=6. (C) Flow cytometry dot plots representing the frequencies of the eGFP and Atto-655 detection of NT, UniCAR-28-BB, UniCARCD28, and UniCAR41BB T cells before sorting. (D) Flow cytometry dot plots representing the frequencies of the eGFP and Atto-655 detection on NT, UniCAR-28-BB, UniCARCD28, and UniCAR41BB T cells after sorting. These dot plots are a representative example of n=6. (E) Box and whiskers representing the frequency of eGFP expression on NT, UniCAR-28-BB, UniCARCD28, and UniCAR41BB T cells, n=6. (F) Flow cytometry dot plots representing the frequency of CD19-FITC expression on NT and CAR19 T cells. These dot plots are a representative example of n=6. (G) Box and whiskers (10-90 percentile, the + symbol is the mean) representing the frequency expression of CD19-FITC on NT and CAR19 T cells, n=6.

Figure 4

Cytotoxicity assay on target cell lines. (A) Histogram representing the frequencies of cell death (referred to as cytotoxicity, mean ± SEM) of CTVio+ K562 and K562-CD19 cells after 72 h of co-culture with Non-transduced T (NT), UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells, both in the absence of intermediate antibody (w/o Ab) or with biotinylated αCD19 Ab. Significant differences were determined using a 2way ANOVA multiple comparisons, corrected with the Tukey’s test. (B) Histogram representing the cytotoxicity of CTVio+ Raji cells after 72 h of co-culture with NT, UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells, in absence of intermediate antibody, with biotinylated αCD19 Ab, or with biotinylated αCD20 Ab (mean ± SEM). Significant differences were determined using a 2way ANOVA multiple comparisons, corrected with the Dunn’s test. (C) Histogram representing the frequencies of cell death (mean ± SEM) of CTVio+ K562 cells (which do not express CD19) after 72 h of co-culture with NT, UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells, stimulated with biotinylated αCD19 Ab. Significant differences were determined using a 1way ANOVA multiple comparisons (Kruskal-Wallis’s test). (D) Histogram representing the frequencies of cell death (mean ± SEM) of CTVio+ K562-CD19 cells after 72 h of co-culture with NT, UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells, stimulated with the non-biotinylated αCD19 Ab. Significant differences were determined using a 1way ANOVA multiple comparisons (Kruskal-Wallis’s test). (E) Histogram representing the frequencies of cell death (mean ± SEM) of CTVio+ K562-CD19 cells after 72 h of co-culture with NT, UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells, stimulated with the non-specific biotinylated αIgG Ab. Significant differences were determined using a 1way ANOVA multiple comparisons (Kruskal-Wallis’s test). (F) Histogram representing the frequencies of cell death (mean ± SEM) of CTVio+ Raji cells after 72 h of co-culture with NT, UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells, stimulated with the non-specific biotinylated αIgG Ab. Significant differences were determined using a 1way ANOVA multiple comparisons (Kruskal-Wallis’s test). All co-culture conditions were performed with a ratio 1:1 (effector T cell: Target cell). Each data point represents one experiment. *: Significant differences between indicated conditions are depicted by black lines, and significant differences inter-conditions are indicated by colored lines which correspond to the histogram’s color code. Inter-conditions statistics for (C–F) were depicted on Supplementary Figure S4 . Significant differences when p<0.05.

Figure 5

Activation of effector T cell subsets measured by CD25 expression. (A) Histogram representing the frequencies of CD25+ cells on NT, UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells (gated on living cells, mean ± SEM) after 72 h of co-culture with CTVio+ K562 and CTVio+ K562-CD19 cells, in the absence of antibody intermediate, or with biotinylated αCD19 Ab, non-biotinylated αCD19 Ab or with biotinylated IgG. *Significant differences between indicated conditions. Inter-conditions statistics for (A) were depicted on Supplementary Figure S6A . (B) Histogram representing the frequencies of CD25+ cells on NT, UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells (gated on living cells, mean ± SEM) after 72 h of co-culture with CTVio+ Raji cells, in the absence of intermediate antibody, or with biotinylated αCD19 Ab, biotinylated αCD20 Ab or with biotinylated IgG. *Significant differences between indicated conditions. Inter-conditions statistics for (B) were depicted on Supplementary Figure S6B . (C) Histogram representing the frequencies of CD25+ cells on NT, UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells (gated on living cells, mean ± SEM) after 72 h of culture alone in the absence of intermediate antibody, or with PHA, biotinylated αCD19 Ab, biotinylated αCD20 Ab or biotinylated IgG. *Significant differences between indicated conditions. Inter-conditions statistics for (C) were depicted on Supplementary Figure S6C . Significant differences when p<0.05, as determined by the by 2way ANOVA multiple comparisons test, corrected with the Tukey’s test. All co-culture conditions were performed with a ratio 1:1 (effector T cell: Target cell). Each data point represents one experiment.

Figure 6

Activation of effector T cell subsets measured by ICOS expression. (A) Histogram representing the frequencies of ICOS+ cells on NT, UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells (gated on living cells, mean ± SEM) after 72 h of co-culture with CTVio+ K562 and CTVio+ K562-CD19 cells, in the absence of antibody intermediate, or with biotinylated αCD19 Ab, non-biotinylated αCD19 Ab or with biotinylated IgG. *: Significant differences between indicated conditions are depicted by black brackets, and significant differences inter-conditions are indicated by colored lines which correspond to the histogram’s color code. (B) Histogram representing the Mean of Fluorescent Intensity (MFI) of ICOS+ cells on NT, UniCAR-28-BB, UniCARCD28, UniCAR41BB and CAR19 T cells (gated on living cells, mean ± SEM) after 72 h of co-culture with CTVio+ K562 and CTVio+ K562-CD19 cells, in the absence of antibody intermediate, or with biotinylated αCD19 Ab, non-biotinylated αCD19 Ab or with biotinylated IgG. Significant differences when p<0.05, as determined by the by 2way ANOVA multiple comparisons test, corrected with the Tukey’s test. All co-culture conditions were performed with a ratio 1:1 (effector T cell: Target cell). Each data point represents one experiment.

Figure 7

Correlations between effector T cell activation and cytotoxicity of target cell lines. Correlations and linear regression between the frequency of effector T cells positive for the CD25 marker and the percentage of cytotoxicity of K562 and K562-CD19 cells (left) and Raji cells (right). Correlations were determined by Pearson’s rank correlation and considered statistically significant when p < 0.05. Each symbol corresponds to one culture condition.