Enhancing the in vivo expansion of adoptively transferred EBV-specific CTL with lymphodepleting CD45 monoclonal antibodies in NPC patients

Abstract

Treatment of Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) with EBV-specific cytotoxic T cells (EBV-specific CTL) has been promising, producing clinical responses. However, infused EBV-specific CTL did not expand in vivo, likely limiting their antitumor activity. Lymphodepleting patients with chemotherapy before T-cell transfer enhances in vivo T-cell expansion, but results in nonspecific destruction of the resident immune system and can have significant toxicity. To evaluate if monoclonal antibodies (mAbs) can produce a more selective lymphodepletion, we conducted a clinical study in which NPC patients received a pair of lymphodepleting mAbs targeted to the CD45 antigen (CD45 mAbs) before EBV-specific CTL infusion. Eight patients with recurrent NPC received CD45 mAbs followed by escalating doses of autologous EBV-specific CTL. Infusion of CD45 mAbs resulted in transient lymphopenia in all patients and an increase in interleukin-15 (IL-15) levels in 6 out 8 patients. All patients had an increase in their peripheral blood frequency of EBV-specific T cells after CTL infusion. Three patients with a persistent increase had clinical benefits including 1 complete response (> 24 months) and 2 with stable disease (for 12 and 15 months). Lymphodepleting mAbs prior CTL transfer may represent an alternative to chemotherapy to enhance expansion of infused CTL. This study is registered at (http://www.clinialtrials.gov) as NCT00608257.

Figure 1

CD45 mAb infusion induces transient neutropenia and lymphodepletion. Counts are shown of (A) neutrophils, (B) B cells, (C) NK cells, (D) CD8^pos T cells, and (E) CD4^pos T cells (*P < .05).

Figure 2

Increase of EBV-specific T cells after lymphodepletion and EBV-specific CTL infusion. (A) The EBV-specific T-cell precursor frequency in patients' PBMCs was determined with IFN-γ ELISPOT assays using autologous LCL. (B) EBV peptide-specific T-cell responses were compared with CMVpp65-specific T-cell responses with IFN-γ ELISPOT assays using peptides (*EBV peptide-specific T cells before mAb vs 8 weeks, P = .001; **CMVpp65-specific T cells before mAb vs 8 weeks, P not significant).

Figure 3

Reversal of T-cell anergy after lymphodepletion and EBV-specific CTL infusion. (A) CD45 mAb-mediated lymphodepletion results in transient decrease of CD4⁺, CD25^bright, and CD69⁻ T cells (*P < .05). (B) The frequency of pentamer-positive and IFN-γ–secreting T cells specific for the HLA-A11–restricted EBNA3B peptide AVF were determined before lymphodepletion and 6 weeks after EBV-specific CTL infusion. Whereas the frequency of AVF-specific T cells was similar by pentamer analysis at both time points, AVF-specific T cells only secreted IFN-γ upon AVF peptide stimulation 6 weeks after CTL infusion.

Figure 4

CD45 mAb infusion induces a transient increase of IL-15 and EBV-DNA. Plasma (A) IL-15 and (B) IL-7 levels were determined with ELISAs. IL-15 levels increased significantly, whereas IL-7 levels remained unchanged. (C) EBV-DNA load increased transiently in 4 patients. (D) Patients with the lowest EBV-specific T-cell precursor frequency had the highest increase in EBV-DNA load.

Figure 5

Complete response after lymphodepletion and EBV-specific CTL infusion. PET images of patient (P1740) before and 8 weeks after CD45 mAbs/EBV-CTL infusion.