Evaluating combinations of costimulatory antibody-ligand fusion proteins for targeted cancer immunotherapy

Abstract

Combinatory strategies are becoming of increasing interest in cancer immunotherapy. Costimulation by individual members of the immunoglobulin-like (Ig)- and TNF superfamily have already shown promising antitumor potential, thus prompting the exploration of their synergistic abilities in combinatorial approaches. Here, we pursued a targeted strategy with antibody-fusion proteins composed of a tumor-directed antibody and the extracellular domain of the costimulatory ligand B7.1, 4-1BBL, OX40L, GITRL or LIGHT, respectively. Costimulatory activity was assessed in an experimental setting where initial T cell activation was induced by a bispecific antibody (tumor-related antigen × CD3). Advantage of combined targeted costimulation was shown for either B7.1 or 4-1BBL with OX40L, GITRL, LIGHT and 4-1BBL in terms of T cell proliferation and IFN-γ release. Since encouraging results were obtained by the combination of B7.1 and 4-1BBL, we adapted the model system for a time-shift setting. Here, enhanced proliferation and granzyme B expression as well as reduced PD-1 expression on the T cell population demonstrated the benefit of costimulation-assisted restimulation. Finally, the antitumor potential of this combinatorial setting was confirmed in vivo in a lung metastasis mouse model. Thus, combinatorial approaches with costimulatory antibody-ligand fusion proteins seem a promising strategy to be further investigated for cancer immunotherapy.

Fig. 1

a Modular schema of the bispecific antibody and antibody–ligand fusion proteins. V _H/L variable region of the heavy/light antibody chain, FAP fibroblast activation protein, EDG endoglin, scDb single-chain diabody, Db diabody, ECD extracellular domain, H Histidine tag, TNF tenascin, L _n linker of n amino acids. Schema of the combinatorial settings: b targeting scDb/B7-Db to FAP and the scFv-ligand to endoglin; c Targeting scDb to FAP and scFv-4-1BBL plus a second scFv-ligand to EDG. CR costimulatory receptor. d SDS-PAGE analysis of the scFv–ligand fusion proteins (3 μg/lane) under reducing (R) and non-reducing (NR) conditions on a 12 % SDS-PAGE and 4–12 % Bis–Tris Gel, respectively; Coomassie staining. e HPLC analysis on a TSK-GEL G3000SW_XL column (Tosoh Bioscience) of scFv-OX40L (2 μg), scFv-LIGHT (15 μg) and scFv-TNC-GITRL (15 μg). Mobile phase was PBS at a flow rate of 0.5 ml/min

Fig. 2

Functional properties of scFv-OX40L, scFv-LIGHT and scFv-TNC-GITRL. a Antibody binding by flow cytometry. HT1080-FAP (EDG⁺) and HEK293 (EDG⁻) cells were incubated with 300 nM scFv-ligand. Bound construct were detected by an anti-hexahistidyl-tag-PE conjugated antibody. Gray filled: cells only; gray line: detection antibody only; black line: scFv-ligand followed by detection antibody. b Ligand activity of the fusion proteins in coated and soluble form. scDbFAPCD3 was coated and incubated with CFSE-labled PBMC together with scFv-ligand (50nM) presented either coated or in solution. Proliferation was determined after 4 days by flow cytometry. [n = 3. Mean ± SD, One-way ANOVA, Tukey post test, *p < 0.05, **p < 0.01, ***p < 0.0001]. c Costimulatory properties of the fusion proteins in a cellular assay. Target cells (HT1080-FAP) were incubated with scDb in combination with the scFv-ligands for 1 h before the addition of CFSE-labeled PBMC. Proliferation of PBMC was measured after 4 days by flow cytometry

Fig. 3

Costimulatory effects mediated by the combined application of the antibody-fusion protein panel. Target cells (HT1080-FAP) were cocultured with PBMCs in presence of scDb (16 pM) in combination with one or two costimulatory antibody-fusion proteins (10 nM). a IFN-γ-release was measured after 4 days by Sandwich-ELISA. b Proliferation of T cells was determined (CFSE/anti-CD3-PerCP) after 7 days by flow cytometry. c Cytotoxic potential of T cells was measured after 7 days via granzyme B staining. Therefore, PBMC were fixed, permeabilized and granzyme B expressing T cells identified (anti-CD3-FITC/anti-granzyme B-PE) by flow cytometry. [n = 3. Mean ± SD, One-way ANOVA, Tukey post test, *p < 0.05, **p < 0.01, ***p < 0.0001]

Fig. 4

Costimulatory effects of the combination of B7.1-Db and scFv-4-1BBL in a time-shift setting. HT1080-FAP cells were incubated with scDbFAPCD3 (33 pM) for 1 h, followed by washing and the addition of PBMC. After 3 days, PBMC were transferred to a well with freshly seeded HT1080-FAP and cocultured for another 7 days. For restimulation purpose, HT108-FAP had been previously incubated with the scDb (33 pM), followed by washing. Costimulation was applied by the addition of B7.1-Db (10 nM) and scFv-4-1BBL (10 nM) either simultaneously during initial stimulation or during restimulation with the scDb. Alternatively, B7.1-Db was applied together with initial scDb-mediated stimulation followed by scDb-mediated restimulation in presence of scFv-4-1BBL. T cell proliferation (a) and expression of granzyme B (b), PD-1 (c) and CTLA-4 (d) was measured after 7 days via flow cytometry. [n = 3. Mean ± SD, One-way ANOVA, Tukey post test, *p < 0.05, **p < 0.01, ***p < 0.0001]

Fig. 5

Antitumor effect of the combined treatment with scDb(m), B7.1-Db and scFv-4-1BBL(m) in a metastatic tumor mouse model. C57BL/6 mice were injected i.v. with B16-FAP cells on day 0. Treatment with the scDb(m) (4 pmol) and the antibody–ligand fusion proteins (0.2 nmol) in the indicated combinations were performed once a day on day 1, 2, 3, 10, 11 and 12. Lungs were removed on day 21 and metastasis counted. [n = 5–6 mice/group, One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001]