The Effect of Ligand Mobility on the Cellular Interaction of Multivalent Nanoparticles

Multivalent nanoparticle binding to cells can be of picomolar avidity making such interactions almost as intense as those seen with antibodies. However, reducing nanoparticle design exclusively to avidity optimization by the choice of ligand and its surface density does not sufficiently account for controlling and understanding cell-particle interactions. Cell uptake, for example, is of paramount significance for a plethora of biomedical applications and does not exclusively depend on the intensity of multivalency. In this study, it is shown that the mobility of ligands tethered to particle surfaces has a substantial impact on particle fate upon binding. Nanoparticles carrying angiotensin-II tethered to highly mobile 5 kDa long poly(ethylene glycol) (PEG) chains separated by ligand-free 2 kDa short PEG chains show a superior accumulation in angiotensin-II receptor type 1 positive cells. In contrast, when ligand mobility is constrained by densely packing the nanoparticle surface with 5 kDa PEG chains only, cell uptake decreases by 50%. Remarkably, irrespective of ligand mobility and density both particle types have similar EC50 values in the 1-3 x 10(-9) m range. These findings demonstrate that ligand mobility on the nanoparticle corona is an indispensable attribute to be considered in particle design to achieve optimal cell uptake via multivalent interactions

Genetic Control of MTOR to Improve Adoptive T Cell Therapy of Tumours

Adoptive T cell therapy to treat cancer in combination with re-directing specificity through T cell receptor (TCR) gene transfer, represents an effective therapeutic option. However, reduced effector responses due to the immunosuppressive tumour microenvironment and insufficient long-term engraftment of transferred cells represent two potential limitations. Tumours often employ mechanisms to inhibit T cell responses including secretion of TGFβ and depleting the tumour microenvironment of amino acids. The main aim of this PhD project was to develop a strategy to enhance T cell function for tumour therapy. The mammalian target of rapamycin (mTOR) pathway regulates CD8 T cell differentiation such that high mTOR activation leads to enhanced effector whilst low mTOR activation leads to increased T cell memory formation. Two retrovirus constructs have been designed whereby one expresses the positive mTOR regulator Rheb and the other expresses the negative mTOR regulator Pras40. Rheb transduction into CD8 T cells resulted in enhanced activation of mTOR, increased effector functions and partial resistance to TGFβ and low arginine concentrations. Pras40 overexpression led to a decrease in the activation of mTOR and reduced effector functions. Rheb transduced CD8 T cells expanded efficiently upon antigen encounter in vivo, followed by pronounced T cell contraction. Pras40 transduced T cells were unable to expand in vivo, but persisted at low numbers and acquired a central memory phenotype. Tumour bearing mice treated with TCR re-directed CD8 T cells transduced with Rheb showed improved tumour protection. Pras40 overexpression resulted in the loss of the protective function of TCR re-directed T cells. Together, the data show that gene transfer can be used to regulate mTOR activity in T cells. Enhancing mTOR activity led to improved tumour control despite reducing memory formation. Permanent mTOR inhibition, on the other hand, preserved some memory characteristcs of T cells but deteriorated their tumour protective functions