Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor - PubMed (original) (raw)

. 2015 Jun 24;5(2):e1062969.

doi: 10.1080/2162402X.2015.1062969. eCollection 2016 Feb.

Daniela S Thommen 2, Petra Herzig 1, Marina Bacac 3, Christian Klein 3, Andreas Roller 4, Anton Belousov 4, Victor Levitsky 3, Spasenija Savic 5, Wolfgang Moersig 6, Franziska Uhlenbrock 1, Viola A Heinzelmann-Schwarz 7, Pablo Umana 3, Pavel Pisa 3, M von Bergwelt-Baildon 8, Didier Lardinois 6, Philipp Müller 1, Vaios Karanikas 3, Alfred Zippelius 2

Affiliations

Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor

Jens Schreiner et al. Oncoimmunology. 2015.

Abstract

T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction.

Keywords: Cancer; T-cell bispecific antibodies; T-cell exhaustion; immune checkpoints; tumor-infiltrating T-cells.

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Figures

Figure 1.

Figure 1.

Activation of CD8+ T-cells in tumor samples and peripheral blood T-cells from healthy donors upon exposure to FolR1-TCB. FolR1+ tumor digests and malignant effusions were cultured for 24h in the presence or absence of FolR1-TCB. As comparison, PBMC from healthy donors were co-cultured with the Skov3 tumor cell line and stimulated with FolR1-TCB. (A) The expression of activation markers on CD8+ T-cells upon FolR1-TCB stimulation was determined by flow cytometry. The FACS plots show FolR1-TCB-induced T-cell activation in a representative patient. The graphs represent the increase in marker expression after FolR1-TCB treatment with mean and standard deviations. (B) IFNγ, IL-2, TNF and perforin in the cell culture supernatants was determined by Cytometric Bead Array or ELISA and normalized to the amount of 1×105 CD3+ T-cells (IFNγ, TNF, IL-2) or CD3+ CD8+ T-cells (perforin) in the culture. The _P_-values were calculated using the unpaired Mann–Whitney test.

Figure 2.

Figure 2.

FolR1-TCB-induced tumor cell killing varies largely in tumor digests and malignant effusions. FolR1 positive and negative tumor digests, malignant effusions or PBMCs from healthy donors were co-cultured with exogenously added fluorescently labeled FolR1+ Skov3 cells at an E:T ratio of 1:1 for 24 h in the presence or absence of FolR1-TCB. The FolR1-TCB-induced specific killing of the Skov3 cells was determined by flow cytometry by measuring activated caspase 3 and the live/dead marker Live/Dead-near-IR. FolR1-TCB-mediated killing was calculated as follows: % specific killing = 100 – [(% of Skov3 live cells in FolR1-TCB treated sample / % of Skov3 live cells in untreated sample) × 100]. FACS plots show FolR1-TCB-induced killing in a representative patient. The _p_-value was calculated using the unpaired Mann–Whitney test.

Figure 3.

Figure 3.

Pattern of inhibitory receptor expression on intratumoral CD8+ T-cells. (A) The expression level of the inhibitory receptors PD-1, Tim-3, CTLA-4, Lag-3, and BTLA was determined by flow cytometry on CD8+ T-cells from tumor digests or malignant effusions. (B) Shows the gating strategy for identification of PD-1hi, PD-1int, and PD-1neg CD8+ subsets of T-cells from two representative patients and their distribution in the tumor samples analyzed. (C) Co-expression of Tim-3, CTLA-4, Lag-3, and BTLA on PD-1hi, PD-1int, and PD-1neg CD8+ T-cells. The _P_-values were calculated using one-way ANOVA with Bonferroni post-hoc-test. (D) FolR1+ tumor samples were divided according to the percentage of PD-1hi expressing CD8+ cells in two groups with PD-1hi scarce and abundant expression, respectively.

Figure 4.

Figure 4.

FolR1-TCB-induced T-cell functions depend on the PD-1 expression level of CD8+ T-cells. FolR1+ tumor digests and malignant effusions were cultured for 24h in the presence or absence of FolR1-TCB. The increase in the expression of activation markers on CD8+ T-cells (A) and the increase in the effector cytokines IFNγ, IL-2, TNF, and perforin (B) was determined in PD-1hi scarce and abundant tumors. (C) Both FolR1 positive and negative tumor samples were adjusted by addition of the FolR1+ Skov3 cell line to an E:T ratio of 1:1 and killing was compared in PD-1hi scarce and abundant tumors. _P_-values were calculated using the unpaired Mann–Whitney test.

Figure 5.

Figure 5.

PD-1 blockade increases cytokine production but not their cytolytic function in T-cells from PD-1hi scarce tumors only. (A) FolR1+ tumor digests or malignant effusions were cultured for 24h with FolR1-TCB in the presence or absence of a PD-1 blocking antibody. IFNγ, IL-2, TNF, and perforin in the cell culture supernatants were determined by Cytometric Bead Array or ELISA and normalized to the amount of 1 × 105 CD3+ T-cells (IFNγ, IL-2, TNF) or CD3+ CD8+ T-cells (perforin). The increase in cytokine secretion upon combined FolR1-TCB and anti-PD-1 treatment compared with FolR1-TCB alone was determined in PD-1hi scarce and abundant tumors. (B) Tumor digests or malignant effusions were co-cultured with exogenously added fluorescently labeled Skov3 cells at an E:T ratio of 1:1 for 24h in the presence or absence of a PD-1 blocking antibody and FolR1-TCB. The increase in specific killing by the α-PD-1 antibody was compared in PD-1hi scarce and abundant tumors. _P_-values were calculated using the unpaired Mann–Whitney test.

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