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PD1-CD28 fusion protein enables CD4+ T cell help for adoptive T cell therapy in models of pancreatic cancer and non-Hodgkinlymphoma

von Fabian B.T. Kraus, Felicitas Rataj, Stefan Endres and Sebastian Kobold

Cytotoxic T cells are specialized immune cells specifically recognizing tumor antigens presented on major histocompatibility complex-1 (MHC-I). After binding to the tumor antigen in the context of MHC, T cells are activated which results in the secretion of cytotoxic factors and target cell lysis. [1]

This concept is utilized therapeutically for adoptive T cell therapy(ACT). Patient-derived T cells are isolated, expanded and subsequently infused back to the patient in therapeutic intention. Typically, ACT is performed with a mixture of CD4 and CD8 Tcells. [2] Although CD8+ T cells are in general considered to be more central to ACT efficacy, CD4+ T cells have a distinct functional and secretarial phenotype from CD8+ T cells which is not redundant and not overlapping. While it is established that CD4+ T cells can be cytotoxic on their own, a major function lays in regulating trafficking, activation, proliferation, differentiation and persistence of tumor-infiltrating cytotoxic CD8+ T cells. [3]

Despite its therapeutic potential, the anti-tumor efficacy of both, CD8+ and CD4+ T cells is inherently limited by the expression and activation of inhibitory immune checkpoints, such as programmed death receptor 1 (PD-1). Interaction of PD-1 with its ligand, PD-L1, suppresses T cell activity and permits tumors to evade T cell mediated immune-surveillance. [4] We recently demonstrated that antigen-specific CD8+ T cells transduced with a PD1-CD28 fusion protein are protected from PD-1-mediated inhibition. [5] In a second step, we are now investigating the potential of PD1-CD28 fusion protein transduced CD4+ T cells alone or incombination with CD8+ T cells for immunotherapy of pancreatic cancer and non-hodgkinlymphoma.

Therefore, OVA-specific CD4+ and CD8+ were retrovirally transduced with PD1-CD28 fusion protein. Cytokine release, proliferation, cytotoxic activity and phenotype of transduced T cells was assessed in the context of Panc02-OVA (murine pancreatic cancer model) and E.G7-PD-L1 (murine T cell lymphoma model) cells. Mice transgenic for a T cell receptor specific for ovalbumine (OT-1 or OT-2) served as T cell donors for primary murine T cell transduction.

Our results indicate that stimulation of PD1-CD28 fusion protein transduced CD4+ T cells with anti-CD3 and recombinant PD-L1 proteins induced specific T cell activation measured by IFN-g release and proliferation. Coculture with Panc02-OVA or E.G7-PD-L1 tumor cells also mediated specific activation of CD4+ T cells. Cytokine release and T cell proliferation was most effective, when tumor cells simultaneously encountered genetically engineered CD4+ and CD8+ T cells. Synergy between both cell populations was also observed forspecific tumor cell lysis. T cell cytotoxicity was mediated via Granzyme B release.

Transduced CD4+ and CD8+ T cells in co-culture with tumor cells developed a predominant central memory phenotype over time. Different ratios of CD4+ and CD8+ transduced T cells led to a significant increase of IFN-g and IL-2 secretion positively correlating with CD4+ T cell numbers used. Mechanistically, IL-2 mediated synergistic activity of CD4+ and CD8+ T cells, as neutralization of IL-2 prevented crosstalk between these cell populations.

In conclusion, we could show that CD4+ transduced T cells significantly improved anti-tumor efficacy of CD8+ fusion receptor transduced T cells by the means of T cell proliferation and viability, cytokine release and killing activity. Interestingly, this could further be ameliorated by enhancing the CD4+ to CD8+ T cell ratio, which is in line with previous clinical observations in this field. Mechanistically, IL-2 derived from CD4+ T cells mediates the synergistic effect of PD1-CD28 fusion receptor-transduced CD4+ and CD8+ T cells. Our results indicate that PD1-CD28 fusion protein transduced CD4+ T cells have the potential to overcome the PD-1 - PD-L1 immunosuppressive axis in pancreatic cancer and non-Hodgkinlymphoma.



[1] Ioannides, C. G. and T. L. Whiteside (1993). "T cell recognition of human tumors: implications for molecularimmunotherapy of cancer." Clin Immunol Immunopathol 66(2): 91-106.
[2] Gardner, R. A., O. Finney, C. Annesley, H. Brakke, C. Summers, K. Leger, M. Bleakley, C. Brown, S.Mgebroff, K. S. Kelly-Spratt, V. Hoglund, C. Lindgren, A. P. Oron, D. Li, S. R. Riddell, J. R. Park and M. C. Jensen(2017). "Intent-to-treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children andyoung adults." Blood 129(25): 3322-3331.
[3] Haabeth, O. A., A. A. Tveita, M. Fauskanger, F. Schjesvold, K. B. Lorvik, P. O. Hofgaard, H. Omholt, L. A.Munthe, Z. Dembic, A. Corthay and B. Bogen (2014). "How Do CD4(+) T Cells Detect and Eliminate Tumor CellsThat Either Lack or Express MHC Class II Molecules?" Front Immunol 5: 174.
[4] Arasanz, H., M. Gato-Canas, M. Zuazo, M. Ibanez-Vea, K. Breckpot, G. Kochan and D. Escors (2017). "PD1signal transduction pathways in T cells." Oncotarget 8(31): 51936-51945.
[5] Kobold, S., S. Grassmann, M. Chaloupka, C. Lampert, S. Wenk, F. Kraus, M. Rapp, P. Duwell, Y. Zeng, J. C.Schmollinger, M. Schnurr, S. Endres and S. Rothenfusser (2015). "Impact of a New Fusion Receptor on PD-1-Mediated Immunosuppression in Adoptive T Cell Therapy." J Natl Cancer Inst 107(8).

Fabian Kraus, IDK I-Target

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