Herstellung und funktionelle Analyse von Immunzytokinen, die Interleukin-2 (IL-2) oder Granulozyten-Makrophagen Kolonie-stimulierenden Faktor (GM-CSF) enthalten, zur Verbesserung einer NDV-modifizierten Tumorvakzine

by

The presented thesis deals with the production and functional analysis of new IL-2 or GM- CSF containing bi- and trispecific single-chain antibodies (immunocytokines) for the improvement of antitumoral immune responses in cancer patients. Thematically, this work is involved in a project concerning the development of an activespecific immunotherapy (ASI) against tumors. As part of this therapy, inactivated, autologous tumor cells (deriving from the surgically resected patient’s primary tumor) are infected with the Newcastle Disease Virus (NDV, strain Ulster), before the autologous tumor vaccine cells (ATV-NDV) are transferred back into the patient following a specific protocol. The two bispecific constructs bs HN-CD3 and bs HN-CD28, produced by recombinant technology, have already been described and are made in such a way that they bind with one arm to the viral antigen hemagglutinin-neuraminidase (HN) on the surface of the vaccine cells. The second arm, however, is directed either against the CD3 molecule, which is associated with the T-cell receptor, or against the costimulatory CD28 receptor. Both signals recruit and activate T-cells at the vaccination site. The bi- and trispecific antibodies presented in this work additionally contain the cytokines IL- 2 and/or GM-CSF and therefore, by introducing supplementary immunomodulating signals, they should improve the efficacy of the vaccine. At first, it was demonstrated that the stable production of the new antibodies in CHO cells was possible. The purified antibodies, which can be directed against both HN and the viral fusion protein (F0), showed all binding characteristics as well as biological activities to ensure their stimulation capacities. In the presence of a suboptimal dose of the CD3 signal, antibodies with an IL-2 cytokine portion in addition to the CD28 signal induced a stronger and more prolonged activation of T- cells, which were isolated from peripheral blood mononuclear cells (PBMC) of healthy donors, than the CD28 signal or IL-2 alone. This improved antitumoral effect was demonstrated using the newly established in vitro tumor neutralisation assay (TNA). Especially naïve T-cells, which in contrast to memory T-cells have not yet met their cognate antigen, were strongly stimulated by these new antibodies and were cytotoxic. In this context, a new aspect was the finding that IL-2 had a similar impact on naïve T-cell costimulation as the CD28 signal. Furthermore, it was shown that CD14+ monocytes could be activated by GM-CSF containing fusion proteins and that they contributed to the antitumor effect in the TNA. In conclusion, the data presented in this thesis suggests that the clinical application of a combined therapy – as an extension of the basic vaccine ATV-NDV by attachment of CD3 and CD28 signals together with IL-2 and GM-CSF containing immunocytokines – is promising. xviii