Abstract B70: Generation of MAGE-A4 230-239 peptide-specific cytotoxic T lymphocytes using an alloreactive approach.
Authors: Lövgren Tanja , Mao Yumeng , Böschen Maxi-Lu , Allerbring Eva B. , Achour Adnane , Poschke Isabel , Kiessling Rolf 
Published: August 2014

Abstract
The aim of this project was to generate cytotoxic T lymphocytes (CTL) specific for the weakly immunogenic MAGE-A4 antigen, by an alloreactive approach.
CTL may recognize and eliminate tumor cells. However, to avoid generation of autoreactive CTL, most CTL specific for tumor-associated antigens (TAA) are eliminated by negative selection during their development in the thymus. Thus, compared to virus-specific CTL, TAA-specific CTL precursors are often rare and more challenging to expand from peripheral blood.
One TAA that is difficult to raise specific CTL against is the MAGE-A4 antigen, a cancer-testis antigen expressed by many tumors but not normal tissues. A MAGE-A4 230-239 peptide is processed (Duffour et al. Eur J Immunol 1999;29;3329–37) and presented by the HLA-A2 complex (Hilling et al. J Mol Biol 2001;310:1167-76). Still, rarely have MAGE-A4 230-239 specific CTL been found after in vitro stimulation or in vivo vaccination. We speculated that the reason was elimination of basically all precursors by negative selection. To get around this, we transfected HLA-A2 into mature dendritic cells (moDC) generated from HLA-A2 negative donor monocytes. In contrast to previous studies using this approach for other TAA (Stronen et al. Scand J Immunol 2009;69:319-28), we used the HLA-A2 gene in a plasmid instead of the HLA-A2 mRNA for transfection. This approach excludes in vitro transcription and avoids RNA stability issues. The transfection efficiency with our method was between 25 to 50% after 24 hours.
We pulsed the HLA-A2 transfected moDC with peptide and co-cultured them with autologous CD8 T cells. As these CD8 T cells have not encountered HLA-A2 during development, they should contain higher frequencies of precursors specific for peptides presented by this HLA allele. Thus, the setting is allogeneic only for peptides presented by HLA-A2, and will from hereon be referred to as the HLA-A2 allogenic setting.
As a control, we used the optimized MART-1 26-35 (A27L) peptide, known to generate potent in vitro responses even in CD8 T cells from healthy HLA-A2 positive donors using an autologous setting. We managed to expand MART-1 specific CTL in vitro from 50% of healthy HLA-A2 positive donors (2/4), when using PBMC and in every donor when using moDC (3/3) as antigen-presenting cells (APC). Thus it was not surprising that we could also generate MART-1 specific cells from all donors with the HLA-A2 allogeneic setting (3/3).
In the case of MAGE-A4, we have never succeeded to generate specific CTL from HLA-A2 positive donors, neither with peptide-pulsed PBMC (0/8) nor with moDC (0/3) as APC. Therefore, it is very promising that we were able to generate MAGE-A4 specific cells from almost half of the donors (2/5) with the HLA-A2 allogenic setting. More donors will be included to determine the efficiency of the method.
The MAGE-A4 and MART-1 specific cells generated by the HLA-A2 allogeneic approach were cloned and functionally tested. The clones for both specificities were similarly capable of killing peptide-pulsed target cells, for some at a high efficiency (80% lysis). MART-1 clones generally required less peptide for lysis (picomolars) than MAGE-A4 clones (nanomolars). This may be due to superior binding of the optimized MART-1 26-35 (A27L) peptide to the HLA-A2. The clones will be further phenotyped and tested for other effector functions such as cytokine production and lysis of MAGE-A4 expressing tumor-cells.
We conclude that functional MAGE-A4 230-239 specific CTL can be generated by the HLA-A2 allogeneic method.
Citation Format: Tanja Lövgren, Yumeng Mao, Maxi-Lu Böschen, Eva B. Allerbring, Adnane Achour, Isabel Poschke, Rolf Kiessling. Generation of MAGE-A4 230-239 peptide-specific cytotoxic T lymphocytes using an alloreactive approach. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B70.