Abstract B099: Immortalized HMy2/solid tumor hybridomas induce two different tumor antigen-specific T cell clones in vitro
Authors: Mohamed Yehia , Browning Michael 
Published: May 2016

Abstract
Introduction: Adoptive T-cell immunotherapy represents a promising approach, as a mean of cancer immunotherapy, for management of different types of malignant tumors. This immunotherapeutic approach has been used with significant clinical benefits in EBV-associated lymphoid disorders, myeloma, and leukemia, in addition to a group of solid tumors. Different studies have demonstrated the probability of generating cytotoxic T-lymphocytes (CTLs) lines to high manufacturing standards, as well as the tolerability and therapeutic benefit of the approach.
In our previous studies we verified that the EBV-associated B lymphoblastoid cell line (LCL), HMy2, fusion to hematologic tumor cells and cell lines in vitro produced stable hybridoma cells that grew spontaneously and immortally in tissue culture, expressed relevant tumor partner antigens, and showed an enhanced ability to stimulate allogeneic and semi-autologous T cell responses. Moreover, these studies prospered in the induction of hematologic tumor antigen-specific CTLs in peripheral blood lymphocytes (PBL) from healthy donors, and semi-autologous cancer patients as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity.
In the current study, we aimed to further investigate newly generated solid/HMy2 hybrids for their abilities to stimulate naive HLA-A2+ T cells of healthy donors to induce new relevant TAA-specific, HLA-A2-restricted T cell clones of therapeutic value in management of hepatocellular and pancreatic adenocarcinomas.
Materials and methods: Two tumor cell lines (HepG2; HCC cell line, and Panc-1; pancreatic tumor cell line) were used separately in generation of two distinct hybrids by fusion to HMy2, using PEG/ DMSO as fusogen and fusion protocol was followed as mentioned before. Fusion cells' (FCs) growth was selected physically using deferential detachment capabilities and then chemically by 3 days incubation in HAT supplemented growth media (RPMI-1640). Moreover, relevant CD markers and surface TAAs expression of the FCs were investigated to evaluate the fusion efficiency. Hybrid cells' viability under different tissue culture conditions, phenotypic properties (including CD markers, HLAs, and co-stimulatory molecules expression) and relevant TAAs expression profile were assessed using flowcytometric analysis and RT-PCR respectively. Two immunogenic, HLA-A2-restricted and high expressed antigens were selected (GPC3 and CEA for HCC and Panc-1 hybrids respectively) and targeted to induce T cell response against. Subsequently, depending on their phenotype, two FC clones were selected for stimulation of naïve allogeneic T cells, isolated from healthy HLA-A2+ donors, in vitro for several rounds, and the outgrowing T cells were phenotyped and analysed regarding T cell subtype, differentiation pattern, & γ-IFN release, cellular cytotoxic avidity and efficacy as well as HLA-A2-restricted peptide-specificity.
Results: The generated hybrid cell lines grew continuously in tissue culture in loosely adherent pattern and expressed CD40, CD80, CD86, HLA class I and HLA class II and the level of expression were higher in the hybrid cell lines than in the parent tumor cells. T cell proliferation assays showed enhanced ability of both fusion clones to stimulate both CD4 and CD8 subtypes of both central and effector cells, however, with slight dominance of effector than central counterparts
Two HLA-A2-restricted peptide-specific cytotoxic T cell clones (against HLA-A*02:01-GPC3 144-152 and HLA-A*02:01-CEA 694-702) were produced for the first time using this technique and demonstrated abundant & γ-IFN production and cytotoxic activity upon recognition of their target cells.
Conclusion: Hybrid cell lines, such as those described, could be used as cost effective and feasible in vitro stimulators of semi-autologous, antigen-specific CTLs for adoptive T cell immunotherapy for not only hematologic but also for certain solid tumors.
Citation Format: Yehia Mohamed, Michael Browning. Immortalized HMy2/solid tumor hybridomas induce two different tumor antigen-specific T cell clones in vitro. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B099.