Bi- and tri-valent T cell engagers deplete tumour-associated macrophages in cancer patient samples

Abstract

BackgroundTumour-associated macrophages (TAMs) are often implicated in cancer progression but can also exert anti-tumour activities. Selective eradication of cancer-promoting (M2-like) TAM subsets is a highly sought-after goal. Here, we have devised a novel strategy to achieve selective TAM depletion, involving the use of T cell engagers to direct endogenous T cell cytotoxicity towards specific M2-like TAMs. To avoid “on-target off-tumour” toxicities, we have explored localising expression of the T cell engagers to the tumour with enadenotucirev (EnAd), an oncolytic adenovirus in Phase I/II clinical trials.MethodA panel of bi- and tri-valent T cell engagers (BiTEs/TriTEs) was constructed, recognising CD3ε on T cells and CD206 or folate receptor β (FRβ) on M2-like macrophages. Initial characterisation of BiTE/TriTE activity and specificity was performed with M1- and M2-polarised monocyte-derived macrophages and autologous lymphocytes from healthy human peripheral blood donors. T cell engagers were inserted into the genome of EnAd, and oncolytic activity and BiTE secretion assessed with DLD-1 tumour cells. Clinically-relevant ex vivo models (whole malignant ascites from cancer patients) were employed to assess the efficacies of the free- and virally-encoded T cell engagers.ResultsT cells activated by the CD206- and FRβ-targeting BiTEs/TriTEs preferentially killed M2- over M1-polarised autologous macrophages, with EC50 values in the nanomolar range. A TriTE with bivalent CD3ε binding – the first of its kind – demonstrated enhanced potency whilst retaining target cell selectivity, whereas a CD28-containing TriTE elicited non-specific T cell activation. In immunosuppressive malignant ascites, both free and EnAd-encoded T cell engagers triggered endogenous T cell activation and IFN-γ production, leading to increased T cell numbers and depletion of CD11b+CD64+ ascites macrophages. Strikingly, surviving macrophages exhibited a general increase in M1 marker expression, suggesting microenvironmental repolarisation towards a pro-inflammatory state.ConclusionsThis study is the first to achieve selective depletion of specific M2-like macrophage subsets, opening the possibility of eradicating cancer-supporting TAMs whilst sparing those with anti-tumour potential. Targeted TAM depletion with T cell engager-armed EnAd offers a powerful therapeutic approach combining direct cancer cell cytotoxicity with reversal of immune suppression.

External Beam Radiation Therapy and Enadenotucirev: Inhibition of the DDR and Mechanisms of Radiation-Mediated Virus Increase

“…We are also currently exploring the possibility of "arming" EnAd with different biologics to provide a two-, or in the case of combination with radiotherapy, three-pronged attack on the tumour microenvironment. In this way, armed EnAd viruses can target not only tumour cells [40], but also other tumour-promoting cells, such as fibroblasts [41] and M2-polarised, wound-healing macrophages [42] that form part of a tumour's support network. Though EnAd does not replicate in or lyse these latter cell types effectively, bi-and tri-specific T cell engagers encoded within the genome of EnAd can be secreted from infected tumour cells.…”

“…EnAd shows impressive selectivity for replication in human carcinoma cells from different types of cancers, including in a co-culture of cancer and normal cells in vitro [37], and has shown a promising targeting and safety profile in an early clinical trial [38]. EnAd can serve as an efficient vector for cancer-selective expression of immune-modulating biologics [39][40][41][42] and can be delivered via the bloodstream into the tumour following systemic administration to humans [43][44][45]. It is currently being tested in clinical trials against solid tumours in combination with several other treatment modalities [38,43].…”

Immune Modulation in Lung Cancer: Current Concepts and Future Strategies

“…3) [441], while trispecific antibodies, TriTEs and TriKEs , enhance therapeutic efficacy by providing additional costimulation through CD28, IL15R, or other activating receptors (Fig. 3) [442-444]. In terms of clinical development, BiTEs are more advanced and already in phase I clinical testing for lung cancer, for example, DLL3-directed for SCLC, and mesothelin-directed for NSCLC [445, 446], while TriTEs and TriKEs show promising preclinical results.…”

“…In terms of clinical development, BiTEs are more advanced and already in phase I clinical testing for lung cancer, for example, DLL3-directed for SCLC, and mesothelin-directed for NSCLC [445, 446], while TriTEs and TriKEs show promising preclinical results. Another potential application of these products is the depletion of immunosuppressive cells in the TME, for example, M2 macrophages can be eliminated using CD206-directed BiTEs and TriTEs [444].…”

Bi- and Tri-Specific T Cell Engager-Armed Oncolytic Viruses: Next-Generation Cancer Immunotherapy

“…In 2019, Scott et al developed both BiTE-and TriTE-armed Ad viruses and showed that they can be used to deplete tumor-associated macrophages in cancer patient samples [ 78 ]. BiTEs/TriTEs were designed to recognize CD3ε on T cells, along with CD206 or folate receptor β on M2-like macrophages.…”