Clinical scale electroloading of mature dendritic cells with melanoma whole tumor cell lysate is superior to conventional lysate co-incubation in triggering robust in vitro expansion of functional antigen-specific CTL

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Abstract

Recent commercial approval of cancer vaccine, demonstrating statistically significant improvement in overall survival of prostate cancer patients has spurred renewed interest in active immunotherapies; specifically, strategies that lead to enhanced biological activity and robust efficacy for dendritic cell vaccines. A simple, widely used approach to generating multivalent cancer vaccines is to load tumor whole cell lysates into dendritic cells (DCs). Current DC vaccine manufacturing processes require co-incubation of tumor lysate antigens with immature DCs and their subsequent maturation. We compared electroloading of tumor cell lysates directly into mature DCs with the traditional method of lysate co-incubation with immature DCs. Electroloaded mature DCs were more potent in vitro, as judged by their ability to elicit significantly (p < 0.05) greater expansion of peptide antigen-specific CD8+ T cells, than either lysate-electroloaded immature DCs or lysate-co-incubated immature DCs, both of which must be subsequently matured. Expanded CD8+ T cells were functional as judged by their ability to produce IFN-γ upon antigen-specific re-stimulation. The electroloading technology used herein is an automated, scalable, functionally closed cGMP-compliant manufacturing technology supported by a Master File at CBER, FDA and represents an opportunity for translation of enhanced potency DC vaccines at clinical/commercial scale.

Highlights

► Generate potent DC in eliciting specific CD8+ T cells. ► Lower tumor lysate amount required for generating potent DC ► Use automated, scalable and closed FDA-approved manufacturing technology.

Introduction

The advantage of DC vaccine has been revealed, especially through the FDA's approval of Dendreon's Provenge. Enhancing further the potency of DC vaccines and perfecting antigen loading procedure are highly demanded. Generally, three ways are used to deliver antigen to DC: peptide pulsing (targeting DC membrane HLA molecules), passive delivery through co-incubation of antigen with immature DC (imDC) to make use of the endogenous endocytosis function of DC, and the active delivery of antigen through transfection/infection methods [1], [2], [3], [4]. Specific or non-specific whole antigens have been used. Loading synthetic mRNA encoding one or a few specific antigens dramatically increasing the antigen concentration inside DC and loading whole molecule antigens derived from synthetic mRNA, tumor whole cell lysates, or tumor cell apoptotic bodies become more favorable [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Active delivery of antigens through transfection of antigen-encoding mRNA demonstrated the efficiency in antigenicity, but the lack of GMP-compliant system with capacity of delivery of large number of cell overburdens the vaccine development/production process, therefore it limits the development of DC vaccine. The comparison study of active [3] and passive deliveries with whole antigens [16], [17], [18] by a GMP-compliant procedure at a clinical scale level is urgently needed to make the full use of the advantage of DC vaccine approach.

In this report, we applied our previous understanding of whole antigen approach in mouse DC study [3] to matured human DC and used a GMP-compliant electroporation-based system with scalable capacity from a fraction of millions of cells to dozens of billions of cells in transfection. We showed that the active delivery was more efficient than passive co-culture delivery, needs less material than passive co-culture delivery, is superior in antigen-specific-CD8-T-cell generation to passive co-culture delivery, and is scalable linearly from 15 million cells to 1 billion of cells, a commonly reported clinical comparable level.

Section snippets

Media and cytokines

Therapeutic grade AIM-V medium was purchased from Invitrogen (Carlsbad, CA, cat. no. 87-0112DK). Research grade recombinant human IL-4 was purchased from CellGenix (Antioch, IL, cat. no. 1403). Clinical grade recombinant human GM-CSF was purchased from Bayer (Leukine®). The following additional cytokines were also used in this study: rhIL-2 (Peprotech, Rocky Hill, NJ, cat. no. 200-02); rhIL-7 (Peprotech, cat. no. 200-07); TNF-α (Peprotech, cat. no. 300-01A); PGE-2 (Sigma-Aldrich, cat. no.

Robust level of M1 protein is detected in melanoma tumor cell lines electroloaded with plasmid DNA encoding influenza virus M1 protein

In clinical practice, autologous tumor cell lysates are frequently used to pulse patients' DCs with tumor antigens. In our studies, to establish proof of concept, we used an allogeneic melanoma tumor cell line, JCOCB, to prepare whole cell lysates and DCs from healthy volunteer donors. This tumor cell line was determined to express abundant MART-1 protein (Fig. 1A). However, previous attempts by others to expand MART-126–35-specific CD8+ T cell populations have met with varying degrees of

Discussion

The focus of this study was to translate our previously reported pre-clinical studies using whole tumor cell lysate-electroloaded murine DCs to develop a robust, efficient and regulatory-compliant process for human clinical and commercial applications. The aims of this study were: [1] to provide proof of concept that loading human DCs with cell lysates using electroporation is superior to the current practice of lysate co-incubation; and [2] to demonstrate that this process is reproducible and

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    1

    Co-first authors/these authors contributed equally.

    2

    Current address: Amplimmune, Inc., Rockville, MD, 20850, USA.

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