Immunologically Targeting the Leukaemia Phosphoproteome

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.