The Decrease of Anti-CD3 Antibody Concentration Improved the Cytotoxicity of Chimeric Antigen Receptor (CAR) T Cells in the Treatment of Chronic Lymphoblastic Leukemia (CLL)

Purpose:

Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated impressive responses in refractory and relapsed acute lymphoblastic leukemia (ALL) and non-hodgkin's lymphoma (NHL), however, the outcome among chronic lymphoblastic leukemia (CLL) seems to be inferior compared to other lymphoblastic malignancies, indicating that efficacy of CAR-T cell therapy may be attributed to inherent T cell defects that are characteristic of CLL which impaired their proliferative capacity and sustained persistence in vivo. Thereby, infusion of less-differentiated T cells which have the capacity to persist and engraft long-term in vivo may enhance the anti-tumor activity.

Materials and methods:

On day 0, cryopreserved PBMCs from healthy donors (HDs) and CLL patients were thawed and seeded on anti-CD3 antibody (0.1μg/ml vs 1μg/ml) in combination with anti-CD28 antibody (1μg/ml) coated 24-well plates. On day 3, activated T cells (ATCs) supplied with retroviral supernatant of the third-generation RV-SFG.CD19.CD28.4-1BBzeta vector were transferred into 24-well plates previously coated with retronectin. Transduction efficiencies and phenotypes of CAR-T cells were evaluated on days 7, 10 and 14 after transduction using flow cytometry analysis. On a functional level, chromium 51 (Cr-51) release assay and intracellular staining (ICS) analysis were performed to explore the altered cytotoxic capability of CAR-T cells.

Results:

We observed that the decrease of anti-CD3 antibody concentration (0.1μg/ml) showed no influence on viability, expansion, transduction efficiency of CAR-T cells generated from HDs or CLL patients compared to standard anti-CD3 antibody concentration (1μg/ml). Moreover, the decrease of anti-CD3 antibody (0.1μg/ml)-mediated T cell activation resulted in an enrichment of less-differentiated naïve (CD45RA ⁺CCR7 ⁺) and central memory (CD45RA ^-CCR7 ⁺)-like T cells both among CD4 ⁺ and CD8 ⁺ CAR-T cells. Additionally, cytokines-production (TNF-α, IFN-γ) were dramatically increased evaluated with ICS analysis from HDs and CLL patients in two different concentrations (0.1μg/ml vs. 1μg/ml) . Notably, CAR-T cells derived from HDs displayed decreased cytotoxic capability while CLL patients-derived CAR-T cells demonstrated increased cytotoxicity with lower anti-CD3 antibody concentration (0.1μg/ml) in the assessment of Cr-51 release assay, indicating that the proliferative capacity and sustained persistence of CAR-T cells derived from CLL patients were obtained in vivo.

Conclusion:

Anti-CD3 antibody-mediated activation of T cells altered anti-tumor efficiency of CAR-T cells before the transduction of ACTs with virus vectors. Consequently, when exploring the strategies to improve the efficacy of CAR-T cells, especially among CLL patients with inherent T cell defects, improvement of the functionality of T cells has to be taken into account.

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