Research Report 2010 - MDC

Graduate andUndergraduate StudentsMario BunseFlorian Helm*Matthias LeisegangPeter Meyerhuber*Simone ReußDaniel SommermeyerTechnical AssistantsUta FischerJanina HauchwitzKordelia HummelMartina KalupaIrmgard KüttnerMatthias Richter*SecretariatAnette Madel*part of the period reportedFigure Modifications increase the avidity of TCR genemodifiedT cells. A NY-ESO-1-reactive wild-type (wt) TCRwas modified by murinization (mu; substitution of humanTCR constant regions by mouse counterparts), introductionof a cysteine bond (cys), codon optimization (co) orcombinations of modifications. Transduction efficiency(%), TCR expression level (MFI) and fuctionality of TCRgene-modified human PBL after co-culture with antigenpresenting tumor cells (IFN-γ release) were determined.instead of completely murinized constant regions dramaticallyreduces the number of foreign residues andthereby the risk for immunogenicity of therapeutic TCRs.Designer T cells by vector optimizationMatthias Leisegang, Peter Meyerhuber, Elisa Kieback,Daniel Sommermeyer, Simone Reuß in collaboration withHans Stauss,For clinical application of TCR-redirected T cells, efficientfunctional expression of the transgenic TCR is a key prerequisite.We compared the influence of the transgenecassette on the expression and function of the murineTCR P14 (recognizing a LCMV gp33 epitope) and thehuman TCR WT-1 (recognizing an epitope of the tumorassociatedantigen WT-1). We constructed different vectors,in which TCR-α and β-chain genes were (i) linkedby an IRES element, (ii) combined by a 2A peptide or (iii)introduced into two individual retroviral constructs. Wefound that IRES-, but not 2A-employing TCR expressionis hampered in primary T cells, where the transgenicTCR has to compete with endogenous TCR chains.Differences in expression were independent of copynumber integration as shown by quantitative PCR.Thus, linking TCR-α and β-chain genes by a 2A peptideseems superior to an IRES element and to single genevectors for TCR expression and T cell function.Construction of TCR-retrovirus packaging cell linesSimone Reuß, Matthias LeisegangWe constructed suspension cell line-based packagingcells derived from human lymphoblastoid -Jurkat cellsto produce TCR-retroviruses. These cells lack endogenousTCRβ-chains and are unable to present CD3 moleculeson the cell surface. Packaging functions weretransferred into ∆β-Jurkat cells by electroporation ofplasmids encoding the gag-pol gene of murine leukemiavirus (MLV) and the GALV or MLV-10A1 env gene.Cell clones expressing high amounts of gag-pol and envgene products were determined by RT-PCR and Westernblot analysis. Upon introduction of a TCR-encodingretroviral vector, ∆β-Jurkat packaging cells shiftedthrough the expression of the transgenic TCR-chainfrom CD3-negative to CD3-positive cells. CD3 highexpressingpackaging cells were enriched by FACS sortingand produced high-titer TCR-retrovirus containingsupernatant.Selected PublicationsSommermeyer, D, Neudorfer, J, Weinhold, M, Leisegang, M, Charo, J, Engels,B, Nößner, E. Heemskerk, M, Schendel, DJ, Blankenstein, T, Bernhard, H,Uckert, W. (2006). Designer T cells by T cell receptor replacement. Eur. J.Immunol. 36, 3052-3059.Reuss, S, Biese, P, Cosset, FL, Takeuchi, Y, Uckert, W. (2007). Suspensionpackaging cell lines for the simplified generation of T cell receptor encodingretrovirus vector particles. Gene Therapy 14, 595-603.Leisegang, M, Engels, B, Meyerhuber, P, Kieback, E, Sommermeyer, D, Xue,S-A, Reuß, S, Stauss, H, Uckert, W.(2008). Enhanced functionality of T cellreceptor-redirected T cells is defined by the transgene cassette. J. Mol.Med. 86: 573-583.Kieback, E, Charo, J, Sommermeyer, D, Blankenstein, T, Uckert, W. (2008). Asafeguard eliminates T cell receptor gene-modified autoreactive T cellsafter adoptive transfer. Proc. Natl. Acad. Sci. USA, 105: 623-628.Uckert, W, Schumacher, T. (2009). TCR transgenes and transgene cassettesfor TCR gene therapy: status in 2008. Cancer Immunol. Immunother. 58:809-822.Cancer Research 125