OFB-SCI · Orthogonally Functionalized Binary Nanopatterned Polymeric Substrates for T cell activation and proliferation in Cancer Immunotherapy

Orthogonally Functionalized Binary Nanopatterned Polymeric Substrates for T cell activation and proliferation in Cancer Immunotherapy

The OFB-SCI project proposed the development of efficient artificial “T cell proliferation machines” based on orthogonally functionalized binary nanopatterned polymeric substrates to improve the current ex-vivo T cell expansion technology, which should facilitate the introduction of novel cancer immunotherapies into clinics, through the accomplishment of different work packages, whose main objectives were achieved as summarized below. In the first part of the project, we described a methodology to reproducibly and efficiently fabricate nanostructures consisting of rigid surfaces decorated with quasi-hexagonal arrays of Au and TiO2 nanoparticles (NPs) using block copolymer micellar lithography (BCML). Thus, we were able to prepare binary nanostructured patterns with different interparticle distances by changing the block copolymer used and the dip-coating velocity following a modification of a published protocol (Polleux et al., Nanolett., 2011, 5, 8, 6355) that enabled us to reduce the preparation time from a few weeks to only one. Moreover, the arrays of Au and TiO2 NPs were transferred to the surface of polyethylene glycol (PEG) hydrogels by a polymerization reaction. In the next step, we described a protocol to orthogonally functionalize binary Au/TiO2 patterned substrates by means of x-ray photoelectron spectroscopy (XPS) and fluorescence microscopy. In this study, we used pairs of different synthetic ligands to functionalize binary micropatterns, which triggered cell adhesion through two different kinds of integrins, the alpha v beta 3 and alpha 5 beta 1. Using this system, which was experimentally less complex than the functionalization of binary nanopatterned hydrogels for T cell activation, we demonstrated the successful orthogonal functionalization of the surface-ligand interface of binary Au/TiO2 micropatterns by XPS. In addition, fluorescence microscopy of soluble proteins and cell experiments enabled us to demonstrate the orthogonality also in the ligand-cell interface (Figure 1). The orthogonally functionalized binary micropatterns allowed us to analyze specific roles and the crosstalk between the alpha v beta 3 and alpha 5 beta 1 integrins in cell adhesion and migration giving us a better understanding of such processes which are very important for T cell activation. This work has been recently published in the peer-reviewed journal “Advanced Materials” (DOI: 10.1002/adma.201500900). Figure 1. Brightfield and fluorescence images as well as schematic representation of the substrates (not to scale) of the segregation of the immunostained alpha v beta 3 and alpha 5 beta 1 integrins on alternating functionalized Au (thin) and MxOy (thick) stripes. Alpha v beta 3 was tagged with Alexa Fluor 488 conjugated antibodies whereas alpha 5 beta 1 with Alexa Fluor 647 conjugated antibodies. Scale bar = 10 µm. In the second part of the project, we evaluated the capacity of PEG hydrogels decorated with AuNPs functionalized with anti-CD3 to activate T cells. To our initial surprise, the Au-nanopatterned hydrogels with immobilized anti-CD3 and soluble anti-CD28 showed strong problems for activating human CD4+ T cells, which were attributed to the low affinity of cells towards the PEG. Indeed, this hypothesis was proven by introducing peptides that trigger cell adhesion into the hydrogels. Thus, T cell activation and proliferation were achieved, pointing out the importance of an adhesive background in addition to presenting the essential chemical signals (anti-CD3/anti-C28). Nevertheless, the incorporation of cell adhesive molecules and the optimization of the surface stiffness which was also done by varying the polymer length and water content did not boost the T cell activation responses to the values that are needed in the clinics, nor did the orthogonal immobilization of both anti-CD3 and anti-CD28 on the two different AuNPs and TiO2NPs of the binary nanopatterned polymeric substrates. Therefore, we considered the addition of a secondary activation system, which in concert with the nanostructures, significantly enhanced T cell proliferation to values that can challenge current T cell activation systems. In summary, we focused on determining key factors for enhancing the CD4+ T cell proliferation ex-vivo, thus contributing to a better understanding of the immunological synapse in artificial substrates and introducing ideas to improve the current T cell expansion technologies.

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