| A01 |

Project Area A
Title

Orthotopic T cell receptor replacement by advanced non-viral cell engineering

Research Area

Antigen-specific T cell receptor identification and recombinant expression, safety evaluation

Project Summary

The T cell receptor (TCR) repertoire of healthy individuals and patients represents an almost unlimited resource for the identification of antigen-specific receptors and their subsequent use in adoptive immunotherapy. However, engineering large numbers of TCRs with different HLA restrictions for clinical applications remains a major challenge. Currently used (unphysiological) technologies for recombinant TCR expression generate highly variable, therefore unpredictable, T cell products in terms of safety and function.

Hence, new editing techniques for the production of safe and predictable TCR redirected T cell products are urgently needed for clinical translation. In addition, recent data demonstrate that a diverse repertoire containing T cells with variable TCR-ligand avidities – might be advantageous for long-term therapeutic effects. As shown recently by us (Schober & Müller et al. 2019, Nature Biomedical Engineering) and others, advanced genomic engineering tools, such as CRISPR/Cas9, allow for an effective and fast engineering of many TCRs without the application of viral vectors. Moreover, CRISPR Cas9-mediated homology-directed repair provides the means for an exact TCR replacement, i.e. to simultaneously eliminate endogenous TCR chains and insert a transgenic receptor directly into the TCR locus. Such ‘orthotopic TCR replacement’ should result in engineered T cells with physiological TCR expression, more predictable functionality and largely reduced potential for toxicity. In this project, we will precisely characterize such physiologically TCR-engineered T cells in vitro and in vivo. Furthermore, we will elaborate on the advantage of therapy with oligoclonal T cell grafts for immediate as well as enduring protection in syngeneic as well as humanized mouse models. Long-term goal of this project is to bring physiologically engineered virus specific TCR redirected T cell products into first clinical applications. Here, we focus on Cytomegalovirus (CMV), Epstein-Barr virus (EBV) and Adenovirus (AdV) post-transplant infections in patients receiving hematopoietic stem cells from virus-seronegative donors.
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Project-Related Publications

Knabel, M., Franz, T.J., Schiemann, M., Wulf, A., Villmow, B., Schmidt, B., Bernhard, H., Wagner, H., and Busch, D.H. (2002). Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer. Nat. Med. 8, 631–637.

Stemberger, C., Huster, K.M., Koffler, M., Anderl, F., Schiemann, M., Wagner, H., and Busch, D.H. (2007). A Single Naive CD8+ T Cell Precursor Can Develop into Diverse Effector and Memory Subsets. Immunity 27, 985–997.

Buchholz, V.R., Flossdorf, M., Hensel, I., Kretschmer, L., Weissbrich, B., Gräf, P., Verschoor, A., Schiemann, M., Höfer,
T., and Busch, D.H. (2013). Disparate individual fates compose robust CD8+ T cell immunity. Science 340, 630–635.

Dössinger, G., Bunse, M., Bet, J., Albrecht, J., Paszkiewicz, P.J., Weißbrich, B., Schiedewitz, I., Henkel, L., Schiemann,

M., Neuenhahn, M., Uckert, W., and Busch, D.H. (2013). MHC Multimer-Guided and Cell Culture-Independent Isolation of Functional T Cell Receptors from Single Cells Facilitates TCR Identification for Immunotherapy. PLoS One 8.

Nauerth, M., Weißbrich, B., Knall, R., Franz, T., Dössinger, G., Bet, J., Paszkiewicz, P.J., Pfeifer, L., Bunse, M., Uckert, W., Holtappels, R., Gillert-Marien, D., Neuenhahn, M., Krackhardt, A., Reddehase, M.J., Riddell, S.R., and Busch, D.H.(2013). TCR-ligand koff rate correlates with the protective capacity of antigen-specific CD8+ T cells for adoptive transfer. Sci. Transl. Med. 5, 192ra87.

Graef, P., Buchholz, V.R., Stemberger, C., Flossdorf, M., Henkel, L., Schiemann, M., Drexler, I., Höfer, T., Riddell, S.R., and Busch, D.H. (2014). Serial transfer of single-cell-derived immunocompetence reveals stemness of CD8(+) central memory T cells. Immunity 41, 116–126.

Stemberger, C., Graef, P., Odendahl, M., Albrecht, J., Dössinger, G., Anderl, F., Buchholz, V.R., Gasteiger, G., Schiemann, M., Grigoleit, G.U., Schuster, F.R., Borkhardt, A., Versluys, B., Tonn, T., Seifried, E., Einsele, H., Germeroth, L., Busch, D.H., and Neuenhahn, M. (2014). Lowest numbers of primary CD8(+) T cells can reconstitute protective immunity upon
adoptive immunotherapy. Blood 124, 628–637.

Paszkiewicz, P.J., Fräßle, S.P., Srivastava, S., Sommermeyer, D., Hudecek, M., Drexler, I., Sadelain, M., Liu, L., Jensen, M.C., Riddell, S.R., and Busch, D.H. (2016). Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia. J. Clin. Invest. 126, 4262–4272.

Neuenhahn, M., Albrecht, J., Odendahl, M., Schlott, F., Dössinger, G., Schiemann, M., Lakshmipathi, S., Martin, K., Bunjes, D., Harsdorf, S., Weissinger, E.M., Menzel, H., Verbeek, M., Uharek, L., Kröger, N., Wagner, E., Kobbe, G., Schroeder, T., Schmitt, M., Held, G., Herr, W., Germeroth, L., Bonig, H., Tonn, T., Einsele, H., Busch, D.H., and Grigoleit, G.U. (2017). Transfer of minimally manipulated CMV-specific T cells from stem cell or third-party donors to treat CMV infection after
allo-HSCT. Leukemia 1–11.

Schober, K., Müller, T.R., Gökmen, F., Grassmann, S., Effenberger, M., Poltorak, M., Stemberger, C., Schumann, K., Roth,
T.L., Marson, A., and Busch, D.H. (2019). Orthotopic replacement of T-cell receptor α- and β-chains with preservation of
near-physiological T-cell function. Nat. Biomed. Eng..

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