Research paper
Ex vivo expansion protocol for human tumor specific T cells for adoptive T cell therapy

https://doi.org/10.1016/j.jim.2010.02.004Get rights and content

Abstract

Adoptive T cell therapy is a promising treatment strategy for patients with different types of cancer. The methods used for generation of high numbers of tumor specific T cells usually require long-term ex vivo culture, which frequently lead to generation of terminally differentiated effector cells, demonstrating low persistence in vivo. Therefore, optimization of protocols for generation of T cells for adoptive cell therapy is warranted. The aim of this work was to develop a protocol for expansion of antigen-specific T cells using Dynabeads CD3/CD28 to obtain T cells expressing markers important for in vivo persistence and survival. To achieve high numbers of antigen-specific T cells following expansion, we have tested the effect of depleting regulatory T cells using Dynabeads CD25 and including a pre-stimulation step with peptide prior to the non-specific expansion with Dynabeads. Our data demonstrate that virus- and tumor specific T cells can be expanded to high numbers using Dynabeads CD3/CD28 following optimization of the culture conditions. The expansion protocol presented here results in enrichment of antigen-specific CD8+ T cells with an early/intermediate memory phenotype. This is observed even when the antigen-specific CD8+ T cells demonstrated a terminal effector phenotype prior to expansion. This protocol thus results in expanded T cells with a phenotypic profile which may increase the chance of retaining long-term persistence following adoptive transfer. Based on these data we have developed a cGMP protocol for expansion of tumor specific T cells for adoptive T cell therapy.

Introduction

Adoptive T cell therapy for malignant diseases is defined as the infusion of T lymphocytes with the objective of eradicating tumor cells and prevent relapse. Reports from early clinical trials have shown that T cell therapy is a promising strategy for cancer treatment and demonstrate the feasibility and safety of this immunotherapeutic intervention (Rosenberg et al., 2004, Dudley and Rosenberg, 2003). The impressive clinical result of using ex vivo expanded tumor infiltrating lymphocytes in patients with advanced malignant melanoma has encouraged many groups to investigate the possibility of using adoptively transferred T cells for treating other types of cancer (Dudley et al., 2002a).

To obtain adequate numbers of T cells for infusion, T cells need to be cultured and expanded ex vivo. Many of the currently used methods are laborious and time-consuming (>45 days) and require large numbers of feeder cells which may limit their clinical application (Yee et al., 2000, Dudley et al., 2002b). Moreover, some of these methods generate a final T cell product dominated by terminally differentiated T cells with a predisposition towards activation induced cell death, skewing of the T cell repertoire, and poor in vivo survival (Powell, Jr. et al., 2006). In a previous study (Huang et al., 2005), long-term persistent T cells were found to express high levels of CD28 and CD27, compared to short-term persistent T cells. Moreover, the long-term persistent T cells demonstrated low expression of the terminally differentiation marker CD57. T cell expansion protocols favoring this phenotype are therefore required.

High numbers of regulatory T cells (Tregs) are found in late stage cancer patients, both in the tumor and systemically. The presence of Tregs may thus negatively impact both the expansion of specific T cells ex vivo, and the subsequent efficacy of adoptively transferred T cells (Antony et al., 2005). Removing Tregs prior to ex vivo expansion may accordingly improve both the final yield of antigen (Ag) specific T cells after expansion and avoid possible inhibitory effects of transferring Tregs into the patient.

The frequency of tumor specific T cells in peripheral blood is generally low. This may limit the efficacy of adoptively transferred T cells expanded from peripheral blood by non-specific activation protocols. Inclusion of an Ag-specific pre-stimulation step prior to non-specific stimulation/expansion has been shown to improve the absolute number of specific T cells in the final product by increasing the frequency of such cells in the starting population (Dang et al., 2007).

Dynabeads coated with antibodies against CD3 and CD28 (Dynabeads CD3/CD28) are designed to mimic the interaction between T cells and APCs in vivo and have been demonstrated to be feasible and effective for clinical grade production of large numbers of T cells (Porter et al., 2006, Rapoport et al., 2005, Thompson et al., 2003). The aim of the present paper was to develop an improved protocol for adoptive T cell therapy based on the standard platform for non-selective expansion of Ag-specific T cells using Dynabeads CD3/CD28. A model system was selected based on the availability of pentamers to detect the frequencies of CMV or EBV specific CD8+ T cells before and after expansion. Following optimization of the expansion protocol for virus specific CD8+ T cells, we performed similar experiments on PBMC from a melanoma patient vaccinated with hTERT peptides, where hTERT specific CD8+ T cells could be detected prior to expansion.

In this study, we have optimized the culture conditions with the goal of obtaining high numbers of virus- and tumor specific T cells of desired phenotype after expansion with Dynabeads CD3/CD28. Removal of CD25+ cells, presumptively containing Tregs prior to expansion greatly increased the expansion of virus specific T cells (CMV and Flu). Virus- and tumor specific CD8+ T cells demonstrated an early/intermediate memory phenotype following expansion, dominated by high expression of CD62L and CD28 and low expression of CD57. By including a pre-stimulation step with peptide prior to non-specific expansion the numbers of Ag-specific T cells were dramatically increased. Together these modifications contribute to a profoundly improved protocol for expansion of Ag-specific T cells.

Section snippets

Culture media, reagents, Dynabeads, antibodies and flow cytometry

T cells were cultured in CellGro® Serum-free Culture Media (CellGenix, Freiburg, Germany) supplemented with 5% heat inactivated human serum (PAA Laboratories GmbH, Austria), 10 mM N-acetylcystein (Mucomyst 200 mg/mL, AstraZeneca AS, Norway), gentamycin 0.05 mg/mL (Garamycin 40 mg/mL, Schering-Plough Europe, Belgium) and 100 U/mL recombinant human interleukin-2 (IL-2) (Proleukin®, Novartis). Recombinant human interleukin-7 (IL-7) was purchased from BD Pharmingen™. Anti-CD3/anti-CD28 coupled Dynabeads

Results

With the aim of developing a protocol for expansion of human tumor specific T cells, we have tested different culture conditions in order to conserve Ag-specific T cells during polyclonal expansion with Dynabeads CD3/CD28. We have explored the effect of modifying the amount of CD3 and CD28 mAbs coated onto the Dynabeads, the effect of removing CD25 positive cells prior to expansion, and the effect of including a pre-stimulation step with peptide to increase the total amount of Ag-specific T

Discussion

Protocols for ex vivo expansion of human T cells have been developed for clinical use and adoptive transfer of expanded T cells has shown promising results in a number of cancer patients (Thompson et al., 2003, Porter et al., 2006). However, several protocols for expansion of Ag-specific T cells require long-term ex vivo expansion, generating terminally differentiated effector cells which do not persist following adoptive transfer (Huang et al., 2005, Rosenberg et al., 2003). In an attempt to

Acknowledgement

We thank Velemira Todozova Koshova for technical help.

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    Permanent address: Servicio de Hematologia, Hospital Maciel, Montevideo, Uruguay.

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