Sa Xue

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.

Exploiting T cell receptor genes for cancer immunotherapy

Adoptive antigen‐specific immunotherapy is an attractive concept for the treatment of cancer because it does not require immunocompetence of patients, and the specificity of transferred lymphocytes can be targeted against tumour‐associated antigens that are poorly immunogenic and thus fail to effectively trigger autologous T cell responses. As the isolation and in vitro expansion of antigen‐specific lymphocytes is difficult, ‘conventional’ adoptive T cell therapy can only be carried out in specialized centres in small numbers of patients. However, T cell receptor (TCR) genes isolated from antigen‐specific T cells can be exploited as generic therapeutic molecules for ‘unconventional’ antigen‐specific immunotherapy. Retroviral TCR gene transfer into patient T cells can readily produce populations of antigen‐specific lymphocytes after a single round of polyclonal T cell stimulation. TCR gene modified lymphocytes are functionally competent in vitro, and can have therapeutic efficacy in murine models in vivo. TCR gene expression is stable and modified lymphocytes can develop into memory T cells. Introduction of TCR genes into CD8+u2003and CD4+ lymphocytes provides an opportunity to use the same TCR specificity to produce antigen‐specific killer and helper T lymphocytes. Thus, TCR gene therapy provides an attractive strategy to develop antigen‐specific immunotherapy with autologous lymphocytes as a generic treatment option.

Effect of CD3 on the efficacy of TCR gene therapy in vivo.

e13047 Background: T cell receptor (TCR) gene transfer is an efficient method by which large numbers of antigen specific T cells can be generated for cancer immunotherapy. The function of TCR gene modified T cells is dependent on efficient surface expression of the introduced TCR alpha/beta heterodimer. In order for TCR to be expressed they must form a complex with CD3 chains. We therefore tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T cell function.nnnMETHODSnThe TCR used were specific for the Wilms tumour antigen 1 peptide (WT1) and the influenza A virus NP peptide (F5). The 58α-β- murine T cell line and splenocytes from C57BL/6 mice were transduced with either TCR alone or TCR plus additional CD3 (CD3+TCR). Tetramers were used to measure TCR expression. In vitro functional assays (antigen specific cytokine production, proliferation and killing) were performed in peptide titration experiments. In vivo T cell tracking and tumour growth were measured using luciferase+ T cells and luciferase+ tumour cells, respectively, for bioluminescence imaging.nnnRESULTSnWe show that co-transfer of CD3 and TCR genes into a T cell line and primary murine T cells enhanced TCR expression and antigen-specific T cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognised lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that CD3+TCR gene modified T cells infiltrated tumours faster and in larger numbers, which resulted in more rapid tumour elimination. Following tumour clearance, CD3+TCR T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen.nnnCONCLUSIONSnEndogenous CD3 chains are rate-limiting for TCR expression and antigen-specific function in gene modified T cells.The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumour activity and memory formation of TCR gene modified T cells in vivo.