CD8 T Cell Expansion Early Post-Autologous HSCT Following Vaccination with Lymphoma Cells Secreting gp96-Ig and Augmentation by IL-2 Antibody Cytokine Complex
Authors: Newman Robert G. , Podack Eckhard R. , Levy Robert B. 
Published: October 2019

Abstract
Abstract 4286
In patients with hematologic cancers, such as lymphoma, aggressive radiation and chemotherapy followed by autologous hematopoietic stem cell transplantation (auto-HSCT) can achieve a minimal residual disease state. Nonetheless, relapse remains the major cause of morbidity and mortality in such patients, and there remains a critical need for more effective anti-tumor therapy. Vaccination with tumor cells secreting the heat shock fusion protein gp96-Ig can induce robust CTL expansion in non-transplanted mice, however its efficacy in HSCT recipients where the opportunity to manipulate the immune response during hematolymphoid reconstitution has not been studied. We examined strategies to expand antigen specific CD8 T cells using lymphoma cells secreting gp96-Ig heat shock fusion protein in the early post auto-HSCT setting. B6 mice (CD45.2+) received ablative conditioning (9.5 Gy TBI) followed by the infusion of 5×106 congenic (CD45.1+) T cell depleted BMC. To investigate vaccine timing, E.G7-OVA lymphoma cells secreting gp96-Ig (E.G7-gp96-Ig: 107/120 Gy) were introduced either 2 or 7 days following auto-HSCT. Two days prior to vaccination, recipients received 106 CD8 OT-I T cells. Following single intraperitoneal E.G7-gp96-Ig vaccination, OT-I expansion occurred at the site of vaccination, reaching maximal levels 5 days post-vaccination. Expansion was clearly superior following vaccination on day 7. Multiple vaccinations (day 7 and 13) improved this response including enhancement of OT-I accumulation in the spleen and LN. Greater than 80% of the OT-I CD8 T cells displayed an antigen-experienced effector memory phenotype (CD44hiCD62Llo) after vaccination with E.G7-gp96-Ig. Notably, little OT-I expansion occurred following vaccination with parental E.G7-OVA (Fig 1), and the OT-I present displayed a central memory phenotype (CD44hiCD62Lhi). Since gp96 chaperoned peptides are cross-presented by APC, we determined the source of B7.1/2 costimulation with regard to donor and recipient APC. Interestingly, following day 7 vaccination CD8 OT-I expansion was markedly diminished when donor BMC were transplanted from B7.1/2 KO mice into WT or B7.1/2 KO recipients (Fig 1). In contrast, following day 2 vaccination, recipient B7.1/2+ APC clearly contributed to OT-I expansion. These observations are consistent with the transition to predominant donor APC presence early following auto-HSCT. To attempt to enhance vaccine induced CD8 expansion, immune complexes comprised of IL-2 and anti-IL-2 mAb clone S4B6 (IL-2/S4B6) were administered following day 7 vaccination. This specific IL-2 immune complex has been shown to interact with CD8 T cells expressing the intermediate affinity IL-2 receptor (IL-2Rβγ), which is expressed on memory CD8 T (concomitantly expressing CD44) and NK cells. Combination treatment with E.G7-gp96-Ig and IL-2/S4B6 immune complex resulted in 4x greater expansion of OT-I T cell numbers at the site of vaccination and in the spleen versus E.G7-gp96-Ig vaccination alone. Thus, 1 week following vaccination, >6×106 OT-I were identified in the compartments analyzed. Interestingly, residual host CD8 T cells were also expanded following complex administration. In summary, we hypothesize that gp96-Ig vaccination post-HSCT results in cross presentation of tumor peptides to CD8 T cells by mature or newly derived B7.1/2 expressing APC of donor origin. Addition of IL-2 via specific mAb/cytokine immune complex provides a strategy to augment gp96-Ig induced CD8 expansion. Studies are underway to address the efficacy of this approach in pre-clinical lymphoma models involving T cell replete auto-HSCT.

Disclosures:
Podack: Heat Biologics, Inc.: Consultancy, Equity Ownership.