Michela Cesco-Gaspere

Targeting the Wilms Tumor Antigen 1 by TCR Gene Transfer: TCR Variants Improve Tetramer Binding but Not the Function of Gene Modified Human T Cells

We have previously described the functional activity of a human TCR specific for an HLA-A2-presented peptide derived from the Wilms tumor Ag 1 (WT1). Recent studies showed that the expression and function of human TCR was improved by the introduction of an additional disulfide bond between the α- and β-chains or by the exchange of the human constant region for murine sequences. In this study, we analyzed the functional activity of WT1-TCR variants expressed in Jurkat cells and in primary T cells. The introduction of cysteine residues or murine constant sequences into the WT1-TCR did not result in a global reduction of mispairing with wild-type TCR chains. Instead, the level of mispairing was affected by the variable region sequences of the wild-type TCR chains. The analysis of freshly transduced peripheral blood T cells showed that the transfer of modified TCR constructs generated a higher frequency of Ag-responsive T cells than the transfer of the wild-type TCR. After several rounds of peptide stimulation this difference was no longer observed, as all transduced T cell populations accumulated ∼90% of Ag-responsive T cells. Although the Ag-responsive T cells expressing the modified TCR bound the HLA-A2/WT1 tetramer more efficiently than T cells expressing the wild-type TCR, this did not improve the avidity of transduced T cells nor did it result in a measurable enhancement in IFN-γ production and cytotoxic activity. This indicated that the enhanced tetramer binding of modified WT1-TCR variants was not associated with improved WT1-specific T cell function.

Retroviral transfer of a dominant TCR prevents surface expression of a large proportion of the endogenous TCR repertoire in human T cells

The latent membrane protein-2 (LMP2) of Epstein–Barr virus is a potential target for T-cell receptor (TCR) gene therapy of Hodgkin lymphoma and nasopharyngeal carcinoma. Here, we modified a human leukocyte antigen-A2-restricted, LMP2-specific TCR to achieve efficient expression following retroviral TCR gene transfer. The unmodified TCR was poorly expressed in primary human T cells, suggesting that it competed inefficiently with endogenous TCR chains for cell surface expression. In order to improve this TCR, we replaced the human constant region with murine sequences, linked the two TCR genes using a self-cleaving 2A sequence and finally, codon optimized the TCR-α-2A-β cassette for efficient translation in human cells. Retroviral transfer of the modified TCR resulted in efficient surface expression and HLA-A2/LMP2 pentamer binding. The transduced cells showed peptide-specific interferon-γ and interleukin-2 production and killed target cells displaying the LMP2 peptide. Importantly, the introduced LMP2-TCR suppressed the cell surface expression of a large proportion of endogenous TCR combinations present in primary human T cells. The design of dominant TCR is likely to improve TCR gene therapy by reducing the risk of potential autoreactivity of endogenous and mispaired TCR combinations.

Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer

T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNγ and TNFα in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.

T-cell receptor gene therapy for cancer: the progress to date and future objectives

In the last decade research has begun into the use of T-cell receptor (TCR) gene therapy as a means to control and eradicate malignancies. There is now a large body of evidence to demonstrate that through the use of this technology one can redirect T-cell antigen specificity to produce both cytotoxic and helper T cells, which are functionally competent both in vitro and in vivo and show promising antitumour effects in humans. This review focuses on the means by which TCR gene transfer is achieved and the recent advances to modify the TCRs and vector delivery systems which aim to enhance the efficiency and safety of TCR gene transfer protocols.

Immunomodulation in the treatment of haematological malignancies

Despite the continuous advances in immunology and cancer biology, haematological malignancies are often incurable. Conventional chemotherapy and radiation are efficacious for some lymphoma and leukaemia, however relapse and progressive disease often occurs. The evidence that the immune system can play an essential role in controlling cancer progression provide a basis for the development of active therapies, such as immunization, aimed to evoke or amplify a tumour-specific immune response. However, the inability of the patient’s own immune system to mount effective responses against tumour antigens is a major limit of vaccination approaches. The adoptive transfer of effectors of the adaptive immune system is an attractive strategy to circumvent the limitations of autologous immune responses. Donor lymphocyte infusion and the transfer of monoclonal antibodies (MoAbs) have been the first forms of adoptive therapy approved for clinical use and are still fundamental components of immunotherapy of haematological malignancies. Due to the continuous characterization of tumour-specific antigen, the development of tumour-tailored therapies that exploit the specificity of antibodies and T cell receptors (TCRs) is progressing rapidly. This review highlights the current advances in the field of adoptive immunotherapy of haematological malignancies, starting by elucidating the ongoing progress in passive transfer of MoAbs. We will also discuss recent advances in the adoptive transfer with tumour-specific high avidity T cells, which can be generated ex vivo by the transfer of gene constructs encoding single chain antibodies or TCRs, thus redirecting T cell specificity to selected tumour antigens. The ability to produce gene-modified T cells of desired specificity and defined functional activity may improve in the future T cell based immunotherapy of cancer.