ALBERT

Description: T cells are probably one of the most pivotal cell types in the human adaptive immune system. They have the capability to eradicate primary, metastatic, relapsed tumours and can ameliorate otherwise fatal viral infections. Not surprisingly therefore, activation and expansion of T cells has become one of the main focuses for immunotherapy and immune gene therapy. Sufficient T cells numbers however, are required to deliver a significant clinical impact to patients, and rapid reproducible expansion of viable T cells still remains one of the main challenges for significant improvement. One of the main concerns with adoptive immunotherapy is that it relies on one critical factor: ex-vivo cell manipulation; the problem with this is that the longer the in-vitro T cell culture, the shorter the in-vivo T cell survival after infusion. In-vivo artificial expansion systems for active immunotherapy would clearly circumvent this problem. Therefore ideally a flexible system should be constructed in order to performed both adoptive and/or active immunotherapy depending on the patients requirements. Currently there is no comprehensive artificial Antigen Presenting Cell system (aAPC) for both effective ex-vivo and in-vivo antigen specific T cell expansion. In order to address this, using nanotechnology, we have constructed a nano sized super-para-magnetic artificial targeted and traceable in-vivo APC system by coating liposomes (approved for human use) with an optimised number of MHC Class I / peptide complexes and a specific selected range of ligands for adhesion (anti LFA1), early activation (anti CD28, anti CD27), late activation (anti 4-1BB) and survival (anti CD40L) T cell receptors in the form of Fab antibody regions. We have constructed targeted liposomes (immuno-liposomes), which are also traceable in-vivo via fluorescent and Magnetic Resonance Imaging (MRI). Ex-vivo (human) and in-vivo (animal) models have been investigated showing firstly that these super-para-magnetic immuno-liposomes circulate the body safely and facilitate their own focusing to specific organs, tumour sites or body areas by applying external magnetic attraction. Secondly, in a viral (CMV) antigen specific model and measured by relevant and irrelevant tetramers, the system is capable of activating and expanding antigen specific T cells at greater levels (200 fold) than standard methods from CMV positive (memory) individuals. The system has also been able to accomplish a small successful level of T cell priming from naive CMV negative individuals. The T cells are phenotypicaly relevant and fully functional in terms of degranulation and cytokine production when specifically challenged. As mechanisms of action, we have demonstrated that the system functions directly on T cells as micro APCs and also semi-directly on the surface of natural APCs following a similar exosomes kinetics. The system is generated in less that 48 hr. Once the aAPC is created and it remains viable and stable for 7 days minimum. We have established optimal conditions for an efficient artificial APC, which embodies a superior and controllable approach and platform with enormous potential for cancer nanotechnology and T cell mediated immunotherapy.

Description: Abstract 1016 Posttranslationally modified (PTM) antigens, particularly phosphorylated antigens, comprise a significant component of tumour antigens and are thought to be represented within anti-tumour immune responses. Moreover, PTM antigens may circumvent the barrier of central tolerance thus offer a new paradigm for immunologically targeting malignant disease. Our previous work has shown that altered signal transduction in neoplastic myeloid and lymphoid cells can generate novel phosphopeptides that are uniquely and differentially presented on malignant cells by class I MHC molecules. Here, we have examined the peripheral blood CD8+ and CD4+ T cell immune responses against phosphopeptide antigens, displayed on primary haematolymphoid malignancies, in healthy donors and patients with leukaemia. Using HLA class-I and class-II tetramers in addition to cytotoxicity assays, we have found that these T cell responses are highly cytotoxic in both in vitro and in vivo preclinical models of adoptive immunotherapy. Our data suggest that recall responses are present to a significant proportion of previously identified class-I restricted phosphorylated antigens in healthy donors. Furthermore, in ex-vivo stimulation renders T cell responses to more than 90% of phosphopeptide antigens tested. These T cells recognise and kill primary tumour cells in a phosphopeptide-specific manner. Ten out of 10 HLA A2 donors and 9 out of 10 HLA B7 donors each respond to up to 7 different phosphopeptides from our pools (50 to 100 different phosphopeptides from CLL and AML). Some of the most relevant cytotoxic CD4+ T cell responses generated are directed towards CLL-specific phosphoproteins such as phosphorylated CD19, CXCR4 and CD20 whereas some of the most relevant cytotoxic CD8+ T cell responses generated target CLL specific phosphopeptides derived from LSP1, MRCL3 and NRC1I3. All these antigens are either overexpressed or uniquely expressed on CLL primary tumour cells. High affinity, tetramer-binding T cells against phosphopeptides derived from BCL2, cMyc, GFI1, MLL transcription factor, LIM, RUNX1, SKI, GRK and MAP3K, have been successfully generated from healthy donors which elicit strong cytotoxic responses against primary tumours. No responses have been elicited to the nonphosphorylated counterparts. Furthermore, some of the phosphopeptides are uniquely displayed on primary AML tissue and derived from established leukaemogenic oncoproteins making them extremely attractive targets. These data support the hypothesis that T cells specific for PTM antigens are represented within the peripheral T cell repertoire and thus are not subject to central tolerance and can be exploited to recognise and destroy neoplastic cells. As haematolymphoid malignancies do not typically downregulate MHC molecules, T cell adoptive transfer can now be envisaged while phosphopeptide vaccination strategies are also a clear objective. Disclosures: No relevant conflicts of interest to declare.