An approved in vitro approach to preclinical safety and efficacy evaluation of engineered T cell receptor anti-CD3 bispecific (ImmTAC) molecules.

Robust preclinical testing is essential to predict clinical safety and efficacy and provide data to determine safe dose for first-in-man studies. There are a growing number of examples where the preclinical development of drugs failed to adequately predict clinical adverse events in part due to their assessment with inappropriate preclinical models. Preclinical investigations of T cell receptor (TCR)-based immunotherapies prove particularly challenging as these biologics are human-specific and thus the conventional testing in animal models is inadequate. As these molecules harness the full force of the immune system, and demonstrate tremendous potency, we set out to design a preclinical package that would ensure adequate evaluation of these therapeutics. Immune Mobilising Monoclonal TCR Against Cancer (ImmTAC) molecules are bi-specific biologics formed of an affinity-enhanced TCR fused to an anti-CD3 effector function. ImmTAC molecules are designed to activate human T lymphocytes and target peptides within the context of a human leukocyte antigen (HLA), thus require an intact human immune system and peptidome for suitable preclinical screening. Here we draw upon the preclinical testing of four ImmTAC molecules, including IMCgp100, the first ImmTAC molecule to reach the clinic, to present our comprehensive, informative and robust approach to in vitro preclinical efficacy and safety screening. This package comprises a broad range of cellular and molecular assays using human tissues and cultured cells to test efficacy, safety and specificity, and hence predict human responses in clinical trials. We propose that this entirely in vitro package offers a potential model to be applied to screening other TCR-based biologics.

Isolation of human MHC class II-restricted T cell receptors from the autologous T-cell repertoire with potent anti-leukaemic reactivity.

Adoptive transfer of T cells genetically modified with tumour-specific T-cell receptors (TCR) is a promising novel approach in the treatment of cancer. We have previously isolated an allorestricted MHC class I-restricted TCR with specificity for Formin-like protein 1 (FMNL1) with potent activity against chronic lymphocytic leukaemia cells. CD4(+) T cells have been described to be highly important for tumour elimination although TCR derived from CD4(+) T cells with anti-tumour reactivity have been only rarely described. In this study we aimed to isolate MHC class-II-restricted CD4(+) T cells and TCR with specificity for leukaemia antigens. We used professional antigen-presenting cells pulsed with the leukaemia-associated and tumour-associated antigen FMNL1 for stimulation of autologous T cells in vitro. We isolated two CD4(+) HLA-DR-restricted T-cell clones and T-cell-derived TCR with so far unknown specificity but high reactivity against lymphoma cells and native malignant cells derived from HLA-matched patients with diverse leukaemias. Moreover, characterization of the TCR after TCR gene transfer revealed that specific characteristics of isolated TCR as reactivity in response to Toll-like receptors were transferable on effector cells. Our results have a major impact on the development of novel immunotherapies. They demonstrate that TCR with potent HLA-DR-restricted anti-leukaemic reactivity against so far undefined self-restricted antigens can be isolated from the healthy autorestricted CD4(+) T-cell repertoire and these TCR are highly interesting candidate tools for novel immunotherapies.

A single TCR alpha-chain with dominant peptide recognition in the allorestricted HER2/neu-specific T cell repertoire.

T cells can recognize tumor cells specifically by their TCR and the transfer of TCR-engineered T cells is a promising novel tool in anticancer therapies. We isolated and characterized four allorestricted TCRs with specificity for the HER2/neu-derived peptide 369 (HER2(369)) demonstrating high peptide specificity. PBMCs transduced with especially one TCR, HER2-1, mediated specific tumor reactivity after TCR optimization suggesting that this TCR represents a potential candidate for targeting HER2 by TCR-transduced effector cells. Another TCR showed high-peptide specificity without tumor reactivity. However, the TCR alpha-chain of this TCR specifically recognized HER2(369) not only in combination with the original beta-chain but also with four other beta-chains of the same variable family deriving from TCRs with diverse specificities. Pairing with one beta-chain derived from another HER2(369)-specific TCR potentiated the chimeric TCRs in regard to functional avidity, CD8 independency, and tumor reactivity. Although the frequency of such TCR single chains with dominant peptide recognition is currently unknown, they may represent interesting tools for TCR optimization resulting in enhanced functionality when paired to novel partner chains. However, undirected mispairing with novel partner chains may also result in enhanced cross-reactivity and self-reactivity. These results may have an important impact on the further design of strategies for adoptive transfer using TCR-transduced T cells.

Formin-like 1 (FMNL1) is regulated by N-terminal myristoylation and induces polarized membrane blebbing.

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1gamma) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1gamma is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1DeltaDAD), indicating that deregulation of autoinhibition is effective in FMNL1gamma. Expression of both FMNL1gamma and FMNL1DeltaDAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.