Immunotherapy with Anti-CD3 x Anti-CMV Bispecific Antibody (CMVBi) Armed Donor Derived Activated T Cells (ATC) – A Novel Strategy against Cytomegalovirus (CMV) Post Allogeneic Stem Cell Transplantation (SCT).
Authors: Ramesh Mayur S , Thakur Archana , Pellett Philip , Das Subhendu , Al-Kadhimi Zaid S , Lum Lawrence G 
Published: October 2019

Abstract
Abstract 1157
Poster Board I-179

Introduction
CMV reactivation and infection can cause profound negative outcome post allogeneic SCT. Current management strategies against CMV are suboptimal and are associated with adverse effects. Induction of anti-CMV T cell responses by vaccination has not been helpful in immunocompromised hosts. Immunotherapy with CMV specific donor-derived cytotoxic T lymphocytes (CTL) is effective after allografting, but it is expensive, labor intensive, and diffcult to replicate in most centers. Non-toxic targeted therapy is needed to improve clinical outcomes. In earlier studies, we generated anti-CD3 chemically heteroconjugated with anti-Her2/neu or anti-CD20 bispecific antibody (BiAb). Ex vivo expanded anti-CD3 activated T cells (ATC) and armed with BiAb exhibit high levels of specific cytotoxicity directed at the respective tumor antigens. We propose a novel strategy of using ATC armed ex-vivo with engineered CMVBi to target CMV antigens. We tested the strategy initially in an in vitro cell culture model using CMV-infected fibroblasts and T cells from normal human donors. We hypothesize that donor anti-CD3 ATC armed with CMVBi will target and eliminate CMV-infected target cells.

Materials and methods
Normal donor peripheral blood mononuclear cells (PBMC) were used to generate ATC by activation with anti-CD3 (OKT3) and interleukin 2 (IL-2). CMVBi was created by chemical heteroconjugation of OKT3 (murine IgG2a) monoclonal antibody and polyclonal anti-CMV (Cytogam®). Specific cytotoxicity was tested by 51Cr release assay using CMV-infected or uninfected human fibroblasts as target cells. Effector populations tested included CMVBi armed ATC and ATC alone; additional controls included CMVBi alone, Cytogam® alone, CMVBi armed, and unarmed PBMC. Cytotoxicity was assessed for CMVBi and an irrelevant BiAb at arming doses ranging from 1 to 500 ng/106 ATC with effector to target ratios (E:T) ranging from 25:1 to 3.125:1. Interferon gamma (IFNγ) EliSpots were used to determine cytokine response after exposing CMV-infected and uninfected fibroblasts to unarmed and CMVBi-armed ATC.

Results
CMVBi arming with as little as 1 ng/106 ATC was significantly more cytotoxic for target cells than unarmed ATC. There was an incremental increase in cytotoxicity with CMVBi armed ATC as the mutiplicity of infection (MOI) was increased in target cells. At all E:T ratios (25:1, 12.5:1, 6.25:1, 3.125:1 ), ATC armed with a dose of 50 ng/106 CMVBi demonstrated markedly enhanced killing of CMV-infected targets (MOI 1) compared to unarmed ATC (see table). In the uninfected control cells, both unarmed and armed ATC lysis was barely detectable over spontaneous lysis. Immunofluorescent studies showed that CMVBi-armed ATC specifically aggregated around GFP fluorescence-marked CMV-infected fibroblasts, whereas unarmed ATC did not aggregate. Cytokine secretion analyzed by IFNγ EliSpot confirmed the superior cytotoxicity of CMVBi-armed ATC.

Conclusion
We used polyclonal Cytogam® to make CMVBi-armed ATC that were able to kill target cells expressing various CMV antigens with high specificity. CMVBi-armed PBMC was only minimally cytotoxic in comparision to CMVBi-armed ATC. Hence, infusion of CMVBi alone to patients is unlikely to be as effective as infusion of ATC armed ex-vivo with CMVBi. We demonstrated similar degree of cytotoxicity using different donor ATC including CMV sero-negative donors. This effect is independent of MHC mediated antigen presentation hence, overcomes CMV immune escape. This non-MHC restricted specific killing strategy could be easily adapted for the prevention and/or treatment of CMV infection/disease after allogeneic SCT using donor-derived ATC. Our current use of BiAb-armed ATC for cancer immunotherapy in humans illustrates the feasibility of adoptive immunotherapy with CMVBi-armed ATC in SCT.

Disclosures
Lum: Transtarget Corporation: Founder.