Combining Early Heat Shock Protein Vaccination with Directed IL-2 Leads to Effective Anti-Tumor Immunity in Autologous Hematopoietic Cell Transplantation Recipients
Authors: Newman Robert G. , Podack Eckhard R. , Levy Robert B. 
Published: October 2019

Abstract
Abstract 998
Tumor relapse is still the major cause of morbidity and mortality in patients with hematologic cancers that undergo aggressive chemo-radiotherapy followed by autologous hematopoietic cell transplantation (auto-HCT). Hence, there is a critical need for new anti-tumor therapies. Heat shock protein (HSP) based vaccines elicit innate and adaptive immune responses in murine studies and have shown promise in clinical trials. The pre-clinical studies here investigated the efficacy of vaccination with tumor cells secreting the HSP fusion gp96-Ig together with directed IL-2 in tumor bearing auto-HCT recipients. To mimic clinical T cell replete auto-HCT, transplanted donor T cells were obtained from congenic tumor bearing mice (C57BL/6 CD45.2+ CD90.1+) that had been previously inoculated intraperitoneally (ip) with 4×106 OVA expressing lymphoma cells (E.G7). Some of these donor mice received 0.5×106 CD8 T cells specific for OVA257–264 (OT-I) to allow for tumor antigen specific T cell monitoring. Three weeks later, T cells were harvested from these animals bearing progressively growing tumor for use in T cell replete auto-HCT. Recipient mice (C57BL/6 CD45.2+ CD90.2+) received 9.5 Gy TBI with subsequent infusion of 5×106 congenic T cell depleted bone marrow cells (C57BL/6 CD45.1+ CD90.2+) supplemented with 2×106 enriched T cells from the tumor bearing donors. The following day, recipients were inoculated ip with 1×105 viable E.G7 lymphoma cells. Based on our prior findings, a multiple vaccination protocol was employed utilizing 1×107 irradiated E.G7 cells transfected to secrete the HSP fusion gp96-Ig (E.G7-gp96-Ig). Some recipients were administered IL-2 via specific antibody-cytokine complexes comprised of IL-2 and αIL-2 mAb clone S4B6 (IL-2/αIL-2CD122). This specific IL-2 complex has been shown to interact with cells expressing the β chain (CD122) of the IL-2 receptor, such as memory CD8 T cells and NK cells, but not with cells expressing the α chain (CD25).
Compared to recipients of T cell replete auto-HCT vaccinated with parental E.G7 tumor cells who exhibited virtually no increase in antigen-specific CD8 T cells, marked expansion was detected in the blood after 2 vaccinations with E.G7-gp96-Ig, i.e. within 1 week of auto-HCT. This response reached a plateau after 3 vaccinations, and persisted throughout the 5 vaccine protocol. To quantitate this vaccine induced CD8 T cell expansion, analysis of the vaccine site, splenic and lymph node compartments was performed following 3 vaccinations, i.e. 2 weeks post-HCT. In contrast to the modest 25× increase observed after vaccination with parental E.G7 cells, a 175× expansion was detected following E.G7-gp96-Ig vaccination (6.8×106 vs. 3.8×104 input). Moreover, 75% of these gp96-Ig expanded CD8 T cells at the vaccine site were bifunctional, expressing IFN-γ and TNF-α following antigen specific stimulation ex vivo. Strikingly, combined treatment with vaccine cells secreting gp96-Ig together with IL-2/αIL-2CD122 complex resulted in a 1000× enhancement of antigen specific CD8 T cell numbers in all compartments analyzed. Tumor bearing auto-HCT recipients exhibited a median survival time (MST) of 1 month if not vaccinated or if vaccinated with parental E.G7 cells (Figure). However, vaccination with E.G7-gp96-Ig extended the MST by more than 2 weeks and ∼20% of recipients survived long term (>100 days). This effect was dependent on T cells since gp96-Ig vaccination alone without donor T cells resulted in no MST extension. Combination therapy with tumor cells secreting gp96-Ig and IL-2/αIL-2CD122 complex markedly elevated total CD8 T cells as well as NK cells at the vaccine site and in secondary lymphoid tissues, two populations that have been shown to facilitate HSP based vaccines. Notably, this strategy resulted in a MST >100 days with ∼60% of mice surviving indefinitely. We propose that 3 components are required together with auto-HCT to avoid relapse related mortality: (1) transplanted autologous T cells, (2) a pan-antigen vaccination approach that induces potent antigen presentation and activation of multiple antigen specific T cells, i.e. tumor cells secreting gp96-Ig, and (3) an adjuvant that potentiates this vaccine induced response, i.e. IL-2 delivered in the form of an antibody-cytokine complex. In total, this combinatorial protocol represents a promising regimen that could be translated into the clinic for patients with hematologic cancers.

Disclosures:
Podack: Heat Biologics, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.