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. 2019 Nov 21;7(1):320.
doi: 10.1186/s40425-019-0807-6.

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

Eleanor M Scott  1 , Egon J Jacobus  1 , Brian Lyons  1 , Sally Frost  1 , Joshua D Freedman  1 , Arthur Dyer  1 , Hena Khalique  1 , William K Taverner  1 , Alison Carr  2 , Brian R Champion  3 , Kerry D Fisher  1 , Len W Seymour  4 , Margaret R Duffy  1
Affiliations

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

Eleanor M Scott et al. J Immunother Cancer. .

Abstract

Background: Tumour-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.

Method: A 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.

Results: T 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.

Conclusions: This 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.

Keywords: Bispecific T cell engagers; Oncolytic virus; Tumour microenvironment; Tumour-associated macrophages.

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Conflict of interest statement

LWS, BRC and KDF own equity or share options in PsiOxus Therapeutics (Oxford, UK), which is leading clinical development of EnAd and its derivatives.

Figures

Fig. 1
Fig. 1
CD206 and folate receptor (FR) β are markers of human tumour-associated macrophages. a Representative flow cytometry plots showing CD206 and FRβ expression on primary ascites cells after gating for CD11b+/CD64+ double positivity. b, c, Percentage positivity for (b) and expression levels of (c) CD206 (PE/Cy7) and FRβ (PE) on primary CD11b+/CD64+ ascites cells from five different cancer patients, and on monocyte-derived macrophages (from healthy donors) polarised with 10 ng/mL LPS and 25 ng/mL IFN-γ (“M(IFN-γ/LPS)”), as determined by flow cytometric analysis. d Primary human monocytes from healthy donors were differentiated into macrophages through 6 days’ culture in medium containing 1% human serum. Where indicated, culture medium contained 10 ng/mL LPS and 25 ng/mL IFN-γ (“M(IFN-γ/LPS)”, added on Day 4), or 50% acellular ascitic supernatant from 11 different patients (“Patient 1″-“Patient 11″, added on Day 0). Expression levels of CD206 (PE) and FRβ (PE) were determined by flow cytometry. c, d Data show mean ± SD of biological triplicates. Statistical analysis was performed by one-way ANOVA with Dunnett’s post-hoc analysis compared with “M(IFN-γ/LPS)” (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Fig. 2
Fig. 2
CD206- and FRβ-targeting BiTEs activate primary human T cells to kill autologous M2-polarised macrophages. a Schematic representations of CD206- and FRβ-targeting BiTEs. b, Western blot analysis of supernatants from HEK293A cells 48 h after transfection with BiTE expression plasmids. Blots were probed with a mouse anti-His primary antibody, followed by an HRP-conjugated anti-mouse secondary antibody. c Human MDMs were polarised as indicated, stained with CFSE, and treated with T cells (10:1 E:T ratio) and increasing concentrations of BiTEs. Macrophage killing was assessed 96 h later by propidium iodide staining and Celigo image cytometry. d MDMs were stained with CFSE and treated with the indicated concentrations of BiTE in the presence or absence of T cells (10:1 E:T ratio). 96 h later, cytotoxicity was assessed by propidium iodide staining and analysis with a Celigo image cytometer. e T cell activation in the presence or absence of target cells was assessed by flow cytometric measurement of CD25 expression 96 h after BiTE addition. Data show mean ± SD of biological triplicates (c, d and e). Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc tests comparing with the relevant “Mock” condition (d and e) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Fig. 3
Fig. 3
Human malignant ascites supernatant suppresses CD206 BiTE activity but not FRβ BiTE activity. a CFSE-stained MDMs were co-cultured with T cells (10:1 E:T ratio) and the indicated BiTEs, in the presence or absence of 50% ascitic supernatant from three patients (Patients 1, 2 and 5). 96 h later, percentage live MDMs was determined with propidium iodide staining and a Celigo image cytometer. b T cell activation was assessed by flow cytometric analysis of CD25 expression after 96 h of co-culture with MDMs and the indicated BiTEs, in the presence or absence of 50% ascitic supernatant from three patients (Patients 1, 2 and 5). c Quantities of IL-6, IL-10 and total (active and latent) TGF-β in malignant ascites fluid from six different patients, as determined by enzyme-linked immunosorbent assay. Normal serum (NS) pooled from three healthy donors was included as a control. d Quantities of soluble CD206 in malignant ascites fluid from nine different patients was determined by enzyme-linked immunosorbent assay. Pooled NS was used as a control. Each condition was measured in biological triplicate and represented as mean ± SD (a-d). Statistical significance was assessed by one-way ANOVA with Dunnett’s post-hoc analysis compared with “Pooled NS” (c, d), or two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the “Mock” condition within the relevant group (a and b). (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, non-significant)
Fig. 4
Fig. 4
Addition of a second T cell-binding domain to the parental CD206 BiTE. a Schematic representations of CD206-targeting TriTEs. b Western blotting analysis of supernatants from HEK293A cells transfected with TriTE expression plasmids 48 h previously. Blots were probed with a mouse anti-His primary antibody and an HRP-conjugated anti-mouse secondary antibody. c BiTE and TriTE binding to recombinant CD206 protein, as determined by ELISA using a mouse anti-His primary antibody and an HRP-conjugated anti-mouse secondary antibody. d-f T cells were cultured for 96 h with the indicated BiTEs/TriTEs at a dose of 50 nM in the presence or absence of target cells. T cell activation was assessed by measuring CD25 expression by flow cytometry, with representative histograms displayed in (d) and geometric MFI values displayed in (e). IFN-γ levels in the supernatants were quantified by ELISA (f). Data show mean ± SD of biological triplicates (c, e and f). Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the “Mock” condition within the relevant group (e and f) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Fig. 5
Fig. 5
A CD206 TriTE with bivalent CD3 binding retains specificity for M2 macrophages and overcomes ascites suppression. a Monocyte-derived macrophages were polarised as indicated, CFSE-stained, and co-cultured for 96 h with T cells at increasing E:T ratios and BiTE/TriTE concentrations. % Live cells were calculated with propidium iodide staining and Celigo image cytometry, with values displayed as a heat map. b T cells were co-cultured with monocyte-derived macrophages and BiTEs/TriTEs in the presence or absence of 50% ascites fluid from seven different patients (Patients 1, 2, 3, 4, 6, 7 and 8). 96 h later, CD25 expression was determined by flow cytometry. C, CFSE-stained monocyte-derived macrophages were treated with T cells (10:1 E:T ratio) and BiTEs/TriTEs in medium alone or 50% ascites supernatant from seven different patients (Patients 1, 2, 3, 4, 6, 7 and 8). 96 h later, cells were stained with proprodium iodide and analysed with a Celigo image cytometer to calculate % live cells. Data show mean ± SD of biological triplicates (b, c). Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the relevant “Mock” condition (b, c) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Fig. 6
Fig. 6
CD206- and FRβ-targeting T cell engagers activate endogenous ascites T cells to kill ascites macrophages. a-e Total unpurified ascites cells from five different patients were cultured for five days with 50 nM BiTEs/TriTEs in medium only or 50% ascites supernatant from the same patient sample. a, b Cells were stained with anti-CD11b, anti-CD64, anti-CD80 and anti-CD86 antibodies, as well as a LIVE/DEAD fixable stain, then analysed by flow cytometry. a % Live residual CD11b+CD64+ cells were calculated relative to “Mock”-treated samples. b Fold-changes in geometric MFI values of CD64, CD80 and CD86 on live CD11b+CD64+ ascites cells were calculated relative to “Mock”-treated samples for each patient sample. c Activation of endogenous CD4+ and CD8+ ascites T cells was assessed by flow cytometric measurement of CD25 expression. d IFN-γ levels in the culture supernatants were determined by ELISA. e Numbers of CD4+ and CD8+ cells were determined through addition of counting beads to samples immediately prior to antibody staining. Fold-changes in CD4+ and CD8+ cell count were calculated relative to “Mock”-treated samples. Data show the grand mean ± SD of five individual patient means (calculated from biological triplicate). Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the relevant “Mock” condition (a, c-e) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Fig. 7
Fig. 7
EnAd expressing FRβ-targeting BiTEs activates endogenous ascites T cells to kill ascites macrophages. a DLD-1 cells were infected with parental or armed EnAd, and viability assessed four days later by MTT assay. % Live cells was calculated relative to “Mock”-treated cells. b DLD-1 cells were infected with armed EnAd at 100 vp/cell. Supernatants were harvested 72 h later and analysed for BiTE expression by western blotting using anti-His primary antibody and an HRP-conjugated anti-mouse secondary antibody. c-g Total unpurified ascites cells from five patients were infected with 100 vp/cell parental or BiTE-expressing EnAd for five days with or without autologous fluid. c Activation of endogenous CD4+ and CD8+ ascites T cells was assessed by flow cytometric measurement of CD25 expression. d CD4+ and CD8+ cell numbers were determined by adding counting beads to samples immediately prior to antibody staining. Fold-changes in CD4+ and CD8+ cell count were calculated relative to “Mock”-treated samples. e, g Cells were stained with anti-CD11b, anti-CD64, anti-CD80 and anti-CD86 antibodies, and a LIVE/DEAD fixable stain, then analysed by flow cytometry. e % Live residual CD11b+CD64+ cells were calculated relative to “Mock”-treated samples. f IFN-γ levels in the supernatants were determined by ELISA. g Fold-changes in geometric MFI values of CD64, CD80 and CD86 on live CD11b+CD64+ ascites cells were calculated relative to “Mock”-treated samples for each patient sample. c-f Data show the grand mean ± SD of five individual patient means (calculated from biological triplicate). c-f Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the relevant “Mock” condition (c-f) (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (c-g) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
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