New class of dual agonistic T-cell engagers for the in-patient activation and expansion of v delta 1 T cells with broad antigen agnostic antitumour activity Free

Introduction:

Gamma Delta (γδ) T cells have gained broad scientific recognition as a third subset of lymphocytes with a distinct and evolutionary conserved role in the recognition of malignant cells. Especially γδ T cells bearing a V delta 1 T cell receptor (Vδ1 T cells) have been shown to protect from cancer in many animal models. Clinically, the presence and number of Vδ1 T cells correlates with overall and disease-free survival in various solid and hematological cancers. Especially in the latter, Vδ1 T cells are often seen expanding after hematopoietic stem cell transplantation with strong correlations with graft versus leukemia effects but reduced graft versus host disease occurrence. The anti-cancer properties of Vδ1 T cells are often innate in nature, independent of the T cell receptor (TCR) and do not require the presence of a tumor associated or specific antigen.

The clinical use of Vδ1 T cells is strongly limited due to our poor understanding of their activation requirements and low numbers in the blood with currently no means available to stimulate these cells in patients through drugs or antibody-based therapies. Thus, Vδ1 immunotherapy is currently limited to complex and costly cell therapy approaches in early clinical development whilst cell targeting approaches focus often on the more readily available anti-bacterial Vδ2 T cell subset. Cell therapies using Vδ1 T cells show great potency and translational potential in pre-clinical models especially through the addition of chimeric antigen receptors (CAR) but they will most likely require additional gene engineering to avoid rejection due to the allogeneic nature of most of these approaches.

Methods:

We have developed a new class of antibody-based dual agonistic T-cell engagers called ‘Boosters’ that target Vδ1 T cells through their TCR whilst co delivering tumor necrosis factor receptor (TNFR) ligands such as 4-1BB ligand as well as a cytokine such as IL-21. Whilst such a multi-functional antibody has no effect on Vδ1 T cells per se unless immobilized on beads or manufacturing vessels, the use of the IgG1 architecture of our Vδ1 ‘Booster’ allows for binding to CD16 and CD64 positive cells leading to cross presentation of the TCR-binder, TNFR ligand and cytokine to Vδ1 T cells enabling cross linking of the TCR whilst providing dual agonistic co-stimulation of the cognate TNFR and cytokine receptor. Avidity engineering can furthermore focus the presentation of the TNFR ligand and IL-21 to Vδ1 T cells by reducing the binding of these functional domains to non-Vδ1 T cells.

Results:

Application of a single shot (0.2nM) of our Vδ1 ‘Booster’ leads to potent activation and expansion of Vδ1 T cells in human PBMCs with no induction of growth or activation of αβ T cells, Vδ2 T cells or NK cells. Vδ1 T cells enrich more than 50-fold in less than 11 days to represent more than 10% of PBMCs (from 0.1%-3% at day 0). Despite the FC competent nature of the Vδ1 ‘Booster’, no ADCC is observed towards Vδ1 T cells nor NK cells. Activated Vδ1 T cells upregulate the innate receptors NKG2D and DNAM-1 and become CD56 bright. Vδ1 ‘Booster’ activated Vδ1 T cells produce TNF-α, IFN-γ, CCL2, GM-CSF but not IL-10 or IL-17. These hyperactivated Vδ1 T cells are furthermore capable of killing AML and Burkitt's lymphoma cell lines at low effector target ratios (1:1 and 2:1). Surprisingly, ‘booster’ activated Vδ1 T cells also target SKOV3 ovarian cancer cells which normally do not get killed by blood derived Vδ1 T cells grown under cell therapy manufacturing conditions (referred to as Delta ONE T cells or DOT) without the introduction of a chimeric antigen receptor. In all conditions tested, cytotoxicity does not rely on the presence of booster and is antigen agnostic.

Conclusion:

We present the first proof of concept that Vδ1 T cells can be activated and enriched in human PBMCs using antibody-based dual agonistic T-cell engagers. Stimulation is specific to Vδ1 T cells and through using signal 1, 2 and 3 allows for the expansion of hyper functional Vδ1 T cells that kill multiple cancer targets through antigen independent, innate mechanisms. Importantly, Vδ1 ‘Boosters’ do not activate NK cells and do not cause fratricide of NK cells or γδ T cells. The antibody-based dual agonistic T-cell engagers presented could potentially create cell therapy like numbers of highly potent anti-cancer Vδ1 T cells within patients.