Abstract A32: Combining early heat shock protein vaccination with directed IL-2 leads to effective and persistent antitumor immunity in recipients of experimental autologous hematopoietic cell transplantation.
Authors: Newman Robert G. , Podack Eckhard R. , Levy Robert B. 
Published: August 2014

Abstract
Tumor relapse remains the major cause of morbidity and mortality in patients with hematologic cancers after aggressive chemo-radiotherapy and autologous hematopoietic cell transplant (HCT) rescue. Therefore, vaccination strategies can be employed early post-HCT during ‘reboot’ of the immune system to mold anti-tumor immunity by taking advantage of both minimal residual disease and lymphopenia. The pre-clinical studies here developed a new strategy uniquely suitable to this environment. We reasoned that by introducing antigen as a heat shock protein (hsp) vaccine, tumor specific CD8 T cells would be rapidly elicited. Subsequently, administration of IL-2 ‘directed’ to CD8 T cells would augment vaccine induced expansion and survival of lymphoma bearing HCT recipients. Recipient B6 mice were therefore conditioned with myeloablative total body irradiation prior to syngeneic HCT. To more precisely model clinical transplants, T cells were obtained from syngeneic donors bearing progressively growing lymphoma (EG7). Transplanted mice were inoculated with a low number of EG7 cells to simulate tumor relapse post-HCT. A multiple vaccination protocol was employed utilizing irradiated EG7 cells transfected to secrete the hsp fusion protein gp96-Ig (EG7-gp96-Ig). To ‘direct’ IL-2 to CD8 T cells, we infused complexes of IL-2 pre-bound to αIL-2 mAb clone S4B6 (IL-2S4B6).
Transplanted animals not vaccinated or vaccinated with parental EG7 cells exhibited an MST of ~1 month with 100% lethality. In contrast, vaccination with EG7-gp96-Ig cells significantly extended MST and increased overall survival to 20% (&gt;100 d post-HCT). Enhanced MST was dependent on transplant of donor T cells since vaccination failed to prolong survival in recipients of T cell depleted marrow grafts. Strikingly, combination therapy with EG7-gp96-Ig and IL-2S4B6 complex resulted in a dramatic increase of overall survival (60%) and MST. Treatment with either: a) unbound ‘free’ IL-2 in combination with vaccine, or b) IL-2S4B6 complex monotherapy (no vaccine), did not induce a survival advantage vs. vaccine therapy alone. Experiments are ongoing to determine which components of the donor graft are required for prolonged survival. Surviving mice (&gt;100 d post-HCT) were re-challenged with a lethal number of EG7 cells. Most HCT recipients (n = 16) vaccinated with EG7-gp96-Ig cells alone or in combination with IL-2S4B6 complex rejected the tumor (87.5%), illustrating the presence of long term anti-tumor memory. To monitor tumor specific T cells, CD8 OT-I were transferred to some donor mice preceding tumor inoculation. This resulted in ~1000 OT-I present in the 2x106 donor T cell inoculum used for HCT. Importantly, no effect on overall survival of HCT recipients was observed regardless of the presence or absence of the transplanted OT-I. Treatment with hsp vaccine and ‘directed’ IL-2 resulted in ~36,000x expansion of OT-I within 2 wk of HCT, compared to ~7000x after vaccination alone. OT-I at the vaccine site produced IFN-γ and TNF-α after ex vivo peptide stimulation. Notably, the combination strategy elicited large increases in both donor CD8 T and NK cells, populations essential for pathogen immunity and vaccine efficacy. Experiments are ongoing to determine if a 2 cycle protocol, administered in part during de novo T cell genesis (&gt;3 wk post-HCT), can elicit additional immunity from these newly derived T cells.
Vaccination approaches employing tumor cells secreting gp96-Ig are presently in clinical trials for lung cancer utilizing an ‘off the shelf’ modified allogeneic lung cancer cell line. Interestingly, others have engineered a human IL-2 “superkine”, resulting in a binding pattern similar to the cytokine/antibody complex used in the present studies. Thus, the combinatorial regimen developed here represents a promising strategy with translational applications for patients with hematologic cancers.
Citation Format: Robert G. Newman, Eckhard R. Podack, Robert B. Levy. Combining early heat shock protein vaccination with directed IL-2 leads to effective and persistent antitumor immunity in recipients of experimental autologous hematopoietic cell transplantation. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A32.