CD8 T-cells recognize peptide fragments in complex with Major Histocompatibility Complex Class I (MHCI) molecules. Recognition of peptide-MHCI (pMHCI) involves the simultaneous binding of two receptors (T-cell receptor; TCR and CD8). CD8 T-cells can be engineered to express TCRs that recognize cancer antigens and administered in vivo to achieve tumour regression. However, barriers to the widespread use of this approach still exist. One issue is that "natural" anti-cancer TCRs are often characterized by weak affinities. A strategy that could be used to overcome this is to co-express weak anti-cancer TCRs with a high affinity variant of CD8. In addition, our data suggests that the optimal pMHCI/CD8 interaction strength varies between different anti-cancer TCRs. As such, we will: 1) design and biophysically characterize a range of novel high affinity human CD8 molecules; then define their functional profile when expressed at the human T-cell surface; 2) identify the optimal high affinity CD8 pairing for a range of different anti-cancer TCRs ; 3) use combinatorial peptide libraries to examine the consequences of high affinity CD8 on T-cell crossreactivity; 4) gain a mechanistic understanding of the beneficial effects of high affinity CD8 using FRET analysis.

Aptamers are single-stranded oligonucleotide ligands that exhibit high affinities and specificities for their targets. These molecules have been extensively used in therapeutics. Several aptamers have recently been described to modulate immune receptors in cancer immunotherapy. We were pioneers in the development of the first-in-class agonistic aptamers against costimulatory receptors. In this project we aim at identifying anti-CD3 and TCR aptamers that could function as allosteric regulators of CD3 and TCR. We will study in collaboration other groups of the consortium how the anti CD3 and TCR aptamers affect at the TCR nanocluster formation. We expect to potentiate the TCR signal provided by weak tumor-antigen derived peptides presented in MCH-I or MCH-II. In this way we hope to increase the activation threshold of tumor-antigen specific lymphocytes for cancer immunotherapy. We would also plan to combine the anti-CD3 aptamers with other costimulatory aptamers that have already been selected, such as 4-1BB, OX40 and CD28, as well as with new aptamers that would be selected against ICOS and CD27. All this immuno-stimulatory aptamers would be anchored together into a biomaterial scaffold to enhance the signalosome activation on the surface of T lymphocytes. We plan to analyze and quantify how the aptamer scaffold interaction affect at the recognition of the T lymphocyte with the antigen presenting cell in collaboration with other groups of the consortium. We plan to test these therapeutic approaches in different relevant murine tumor models of melanoma and colon cancer. We would also assess the nature of the immune response that is elicited with this type of therapeutic intervention.

CAR T cells can respond very differently to identical stimuli, and build up synapses of highly heterogeneous phenotypes. We will develop a microfluidic biochip prototype allowing for highly automated analysis of individual cells, including targeted delivery to a microscope platform, characterization by fluorescence microscopy and removal of interesting cells for further characterization by other techniques. The project includes design of the biochip and i/o system, mastering of the biochip, hot embossing into thermoplastics, integration of the biochip in the i/o system and microscopy platform, and the ultimate application for our cell biological experiments.

There is a plethora of proteins that contribute to the identification of antigens by a T cell and to its specific response. Interestingly, even the most paradigmatic interaction process – the binding of the TCR to a peptide-loaded MHC – is far from being understood; a mechanistic understanding of the role of co-receptors is completely missing. We will use single molecule tracking and superresolution microscopy to quantify the interactions between the different membrane proteins at milliseconds time resolution. The single molecule approach offers a set of key advantages over conventional ensemble techniques: i) from the brightness of the individual spots it is possible to determine the stoichiometry of the participating molecules; ii) the position of the dye-labelled proteins can be determined at very high accuracy down to a few tens nanometers; iii) two-color single molecule tracking allows for directly recording interaction lifetimes. The high-resolution techniques will be employed for a detailed investigation of the protein interactions in CAR-T cells under resting conditions. Experiments will be performed in close collaboration with Johannes Huppa (MUW). Ultimately, targeted delivery of cells to the imaging platform will enhance throughput in single molecule experiments. We thus assist STRATEC in the development and implementation of a microfluidics system.

Little is known how CAR T cells recognize antigens. Knowledge of recognition events is needed for the rational design of safe and more selective CARs. We will devise a planar glass‐supported molecular imaging platform, which serves as a surrogate target cell for CAR T cells, to quantify synaptic receptor binding and signaling events in living CAR T cells, and resolve membrane architecture with single molecule resolution. Together with PhD6 and PhD7 we will extend this system for its use in automated microfluidics devices as a means to select individual CAR T cells based on synaptic and cytoplasmic signaling and effector functions and profile them in depth for T cell differentiation and gene transcription.

Therapeutic reactivity of antibody-mediated immunotherapy and chimeric antigen receptor (CAR) engineered T-cells is based on targeting of extracellular target antigens. Recent clinical studies demonstrate very promising clinical benefit, however these therapies also demonstrate that escape variants, not expressing the targeted antigen, result in relapse of the malignancy. In addition, for many malignancies no tumor-specific membrane associated target antigens are present to be able to specifically target these tumors. Therefore, new immunotherapeutic strategies have to be explored to increase the number of cancer patients that can be treated with adoptive immune therapy. Since T-cell receptors (TCR) are able to recognize antigens derived from both extracellular and intracellular proteins in the context of HLA, the potential target antigen array useful for TCR gene therapy is much broader. However, the widespread application of TCR gene therapy is hampered by lack of tumor-specific TCRs. Negative selection during thymic development deletes T-cells carrying high affinity TCRs for self/tumor-antigens in the context of autologous (self) HLA. In contrast, self-antigens presented in the context of allogeneic (non-self) HLA can elicit strong immune responses as was demonstrated by our group by the isolation of PRAME- and B-cell lineage specific T-cells (Amir et al CCR 2011, Jahn et al Oncotarget 2016). This research proposal focusses on identifying high affinity tumor-specific TCR by exploiting the immunogenicity of foreign HLA molecules combined with an in-house generated HLA -peptide elution library and high throughput T-cell enrichments. Using this strategy a library of potentially useful high affinity TCRs will be selected that exhibit potent anti-tumor reactivity and exert a stringent safety profile.

Since most CAR-targeted “tumor associated antigens” are not only expressed by cancer cells but also by healthy cells, combinatorial recognition of two antigens on cancer cells may overcome the risk of severe auto‐immunity. Thus, the identification of molecular mechanisms which result in productive (co‐)synapse formation of two CARs of different specificities and signal complementation of these CARs is necessary. The project builds on our recently developed strategy of combinatorial antigen recognition by two CARs which deliver the primary TCR signal and the CD28 costimulation, respectively.

Our project is based on our expertise to study and understand the T cell receptor (TCR) as a macromolecular machine. Using our newest knowledge, we aim to generate a new generation of CARs and to simultaneously obtain further insight into the functioning of the TCR. This will be done by a combination of state-of-the-art engineering technologies, signaling studies and functional assays.
We were the first to show that the TCR is expressed on the surface of T cells not only as individual receptors, but also in a preclustered form (nanoclusters) (Schamel et al, 2005; Wang et al, 2016), Importantly, the TCR nanoclusters are stimulated by lower amounts of antigen than the individual TCRs and thus, the proportion of TCRs that form nanoclusters influences the sensitivity of T cells. Indeed, in collaboration with the partner of Madrid, Hisse Martien van Santen, we found that TCR nanoclustering is dynamically regulated (Kumar et al, 2011), since naïve T cells (less sensitive to antigen) contain small, and effector/memory T cells contain large nanoclusters (more sensitive to antigen).
We also found that the TCR undergoes a conformational change upon antigen binding, and that this structural rearrangement is the trigger for signaling (Gil et al, 2002; Minguet et al, 2007). More recently, we have demonstrated that the TCR is in equilibrium between two conformations, the resting and the active one; and the latter one is stabilized by antigen binding (Swamy et al, 2016; Schamel et al, 2017). Using this knowledge, we have already significantly enhance tumor killing by T cells (Dopfer et al, 2014).
Now, we plan to use our knowledge to engineer novel CARs that would incorporate new domains, in order to enhance their activity. Hence, based on currently used CARs and the expertise of this EN-ACT2ING consortium, we plan to test the effect of nanoclustering and conformational changes in signal initiation of these CARs. The sensitivity of the corresponding cells and their capacity to kill tumor cells will also be studied. Importantly, the CARs will be engineered to improve their signaling capacity and to generate fundamental knowledge on the functioning of the TCR itself.
In addition to the program offered by EN-ACT2ING, the PhD student will be part of a local team to work on novel avenues in cancer immunotherapy. He or she will be carefully supervised by ourselves.

References

Dopfer EP, Hartl FA, Oberg HH, Siegers GM, Yousefi OS, Kock S, Fiala GJ, Garcillan B, Sandstrom A, Alarcon B, Regueiro JR, Kabelitz D, Adams EJ, Minguet S, Wesch D, Fisch P, Schamel WW (2014) The CD3 Conformational Change in the gammadelta T Cell Receptor Is Not Triggered by Antigens but Can Be Enforced to Enhance Tumor Killing. Cell Reports 7: 1704-1715

Gil D, Schamel WW, Montoya M, Sanchez-Madrid F, Alarcon B (2002) Recruitment of Nck by CD3 epsilon reveals a ligand-induced conformational change essential for T cell receptor signaling and synapse formation. Cell 109: 901-912

Kumar R, Ferez M, Swamy M, Arechaga I, Rejas MT, Valpuesta JM, Schamel WW, Alarcon B, van Santen HM (2011) Increased Sensitivity of Antigen-Experienced T Cells through the Enrichment of Oligomeric T Cell Receptor Complexes. Immunity 35: 375-387

Minguet S, Swamy M, Alarcon B, Luescher IF, Schamel WW (2007) Full activation of the T cell receptor requires both clustering and conformational changes at CD3. Immunity 26: 43-54

Schamel WW, Arechaga I, Risueno RM, van Santen HM, Cabezas P, Risco C, Valpuesta JM, Alarcon B (2005) Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response. J Exp Med 202: 493-503

Schamel WW, Alarcon B, Hofer T, Minguet S (2017) The Allostery Model of TCR Regulation. J Immunol, in press

Swamy M, Beck-Garcia K, Beck-Garcia E, Hartl FA, Morath A, Yousefi OS, Dopfer EP, Molnar E, Schulze AK, Blanco R, Borroto A, Martin-Blanco N, Alarcon B, Hofer T, Minguet S, Schamel WW (2016) A Cholesterol-Based Allostery Model of T Cell Receptor Phosphorylation. Immunity 44: 1091-1101

Wang F, Beck-Garcia K, Zorzin C, Schamel WW, Davis MM (2016) Inhibition of T cell receptor signaling by cholesterol sulfate, a naturally occurring derivative of membrane cholesterol. Nat Immunol 17: 844-850