T-Cell Receptor Stimulation Enhances the Expansion and Function of CD19 Chimeric Antigen Receptor-Expressing T Cells

Abstract

Purpose: Current protocols for CD19 chimeric antigen receptor-expressing T cells (CD19.CAR-T cells) require recipients to tolerate preinfusion cytoreductive chemotherapy, and the presence of sufficient target antigen on normal or malignant B cells.

Patients and methods: We investigated whether additional stimulation of CD19.CAR-T cells through their native receptors can substitute for cytoreductive chemotherapy, inducing expansion and functional persistence of CD19.CAR-T even in patients in remission of B-cell acute lymphocytic leukemia. We infused a low dose of CD19.CAR-modified virus-specific T cells (CD19.CAR-VST) without prior cytoreductive chemotherapy into 8 patients after allogeneic stem cell transplant.

Results: Absent virus reactivation, we saw no CD19.CAR-VST expansion. In contrast, in patients with viral reactivation, up to 30,000-fold expansion of CD19.CAR-VSTs was observed, with depletion of CD19⁺ B cells. Five patients remain in remission at 42-60+ months.

Conclusions: Dual T-cell receptor and CAR stimulation can thus potentiate effector cell expansion and CAR-target cell killing, even when infusing low numbers of effector cells without cytoreduction.

Figure 1.

Outline of study design and CD19.CAR-VST cell expansion. (A) Stem cell donor blood, obtained between one and 31 days before HSCT, was used to manufacture CD19.CAR-VSTs. Cells were infused into B-ALL patients in remission of hematopoietic stem cell transplant (HSCT) between day 74 and 158 after HSCT. Immune cell reconstitution was monitored by flow cytometry and virus specificity was analyzed by INF-γ ELISPOT at the indicated times after infusion. The presence of the CD19.CAR transgene was analyzed using Q-PCR assay. (B) Expansion of CD19.CAR-VSTs in patients with EBV virus load. No expansion of CD19.CAR-VSTs was detected by Q-PCR in 5 patients without detectable EBV, CMV or adenovirus (black lines dashed lines represent individual patients and black solid line for mean values). CD19.CAR-VSTs expanded in 3 patients with reactivated EBV (red dashed lines for each patient and red solid line for mean values). (C) No significant expansion of CD19.CAR VSTs in the blood of representative patient with no detectable viral reactivation/ infections.

Figure 2.. CD19.CAR-VSTs expansion associated with EBV reactivation in patient 1.

(A) Expansion of CD19.CAR-VSTs and control of reactivated EBV. (B) CD19.CAR-VSTs were detectable by flow cytometry in patient’s peripheral blood (upper panel). CD19+ normal B cells were depleted from blood on week 2 post-infusion (lower panel). (C) Normal B-cells were completely depleted from the blood of patient from week 2 until week 38. CD19+ B cell numbers remaining low (<100 CD19+ cells/μL) at 18 months post-infusion. (D). Reactivity in PBMCs to three viruses (EBV, CMV and adenoviruses) were measured in PBMCs by IFN-γ ELISpot. EBV responses were analyzed with LCLs and overlapping peptide libraries to EBNA1, LMP1/2, BZLF1, BARF-1, EBNA3a,3b, 3c (EBVpx). Responses to CMV were analyzed using overlapping pepmixes to pp65 (CMV px); adenovirus responses were analyzed using overlapping peptide libraries for hexon and penton (Ad px). LCLs alone were plated (time-points month 9, 12, 24 and 30) as one of the controls and had no spontaneous IFN-γ release (0 SFC/well).

Figure 3.. Expansion of CD19.CAR-VSTs upon EBV reactivation in patients 3 and 4.

(A, B) Q-PCR data for the CD19.CAR and EBV in patients 3 and 4 respectively. (C) CD19.CAR-VSTs were detected by flow cytometry PBMCs from patient 4 on day 10 after infusion and CD19+ B cells were depleted coincident with CD19.CAR-VST expansion. (D) Transient depletion of CD19+B cells and expansion of CD19.CAR-VSTs in patient 4 during the study period.

Figure 4.. CD19.CAR-VSTs were cytotoxic to autologous normal B cells and expanded in vitro

(A) B cells were purified by positive selection with CD19 microbeads and co-cultured at 1:1 ratio with CD19.CAR VSTs or NT VSTs for three days. CD19.CAR-VSTs eliminated B cells (CD20+) on day 1 of culture. (B) CD19.CAR+ EBVST cell expansion in response to multiple stimulations with B cells pulsed with EBV pepmixes or B cells alone. (C) Expansion of CD19.CAR+CD3+ EBVSTs on day 7 of co-culture with B cells alone (13±10-fold cumulative expansion) or B cells pulsed with EBV pepmixes (246±115-fold). Representative results from one donor after the third stimulation are shown.

Figure 5.. VSTs are not eliminated by monocytes in cocultures.

(A). CD19.CAR VSTs or non-transduced VSTs were co-cultured with autologous CD14+monocytes for three days with or without EBV pepmixes (px) spanning EBNA1, LMP1,LMP2 antigens. Viability of CD3+T cells was assessed on day 3 by flow cytometry with 7ADD (n=3). (B) Fold of expansion of CD19.CAR-VSTs and non-transduced (NT) VSTs three days after co-culture with monocytes with or without EBV pepmixes stimulation. Cells were harvested and analyzed by flow cytometry with Dynabeads to obtain absolute cell numbers (n=3). No statistically significant difference for CAR.VSTs pulsed with EBV px was observed at early days (day 3) post-stimulation. (C) Monocytes are eliminated on day 1 of co-culture with CD19.CAR-VSTs and NT cells.