Potentiation of PD-L1 blockade with a potency-matched dual cytokine–antibody fusion protein leads to cancer eradication in BALB/c-derived tumors but not in other mouse strains

Abstract

We have recently described a novel therapeutic antibody product (IL2-F8-TNF), featuring the simultaneous fusion of murine IL2 and of a TNF mutant with scFv(F8), an antibody specific to the alternatively-spliced extra domain A of fibronectin (EDA). Here, we report on the in vivo characterization of the anti-cancer activity of IL2-F8-TNF in four immunocompetent murine models of cancer, CT26, WEHI-164, F9 teratocarcinoma and Lewis lung carcinoma (LLC), using the product alone or in combination with a monoclonal antibody specific to murine PD-L1. All four models exhibited a strong expression of EDA-fibronectin, which was confined to vascular structures for F9 tumors, while the other three malignancies exhibited a more stromal pattern of staining. A complete and long-lasting tumor eradication of CT26 and WEHI-164 tumors was observed in BALB/c mice when IL2-F8-TNF was used in combination with PD-L1 blockade. The combination treatment led to improved tumor growth inhibition in 129/SvEv mice bearing murine teratocarcinoma or in C57BL/6 mice bearing murine LLC, but those cancer cures were difficult to achieve in those models. A microscopic analysis of tumor sections, obtained 24 h after pharmacological treatment, revealed that the PD-L1 antibody had homogenously reached tumor cells in vivo and that the combination of PD-L1 blockade with IL2-F8-TNF stimulated an influx of NK cells and of T cells into the neoplastic mass. These data indicate that potency-matched dual-cytokine fusion proteins may be ideally suited to potentiate the therapeutic activity of immune check-point inhibitors.

A novel fully-human potency-matched dual cytokine-antibody fusion protein targets carbonic anhydrase IX in renal cell carcinomas

“…IL2-and TNF-based products have shown to be synergistically active against various type of malignancies (4,6,8,(22)(23)(24)(25)27,42) by two distinct complementary mechanism of actions. On one hand, TNF is capable of inducing hemorrhagic necrosis and apoptosis of the tumor endothelial cells and also of cancer cells (4,6,25,43,44).…”

“…depicts a schematic representation of a fully-human fusion protein (termed IL2-XE114-TNF mut ), featuring a sequential arrangement of IL2, a scFv fragment specific to CAIX (named XE114 (31)) and TNF [Supplementary Figure 1]. The protein arrangement is reminiscent of the one previously described for the murine fusion protein IL2-F8-TNF mut (6,27) [ Supplementary Figure 2], but here we used human payloads in order to facilitate clinical translational activities. The TNF moiety was de-potentiated by a single amino acid substitution (R108A), in order to achieve a similar cytokine activity for both IL2 and TNF moieties.…”

“…We tested the therapeutic activity of IL2-XE114-TNF mut and IL2-F8-TNF mut in nude mice bearing CAIX-transfected CT26 tumors [Figure 4]. Since the human TNF moiety is only partly active in mice, we also studied the therapeutic activity of the mIL2-XE114-mTNF mut and mIL2-F8-mTNF mut analogues, bearing an attenuated version of murine TNF (6,27) [ Supplementary Figure 2, 5]. A tumor-growth retardation was observed in this model, which lacked a functional set of T lymphocytes, but still retained natural killer (NK) cells [Figure 4].…”

An antibody-tumor necrosis factor fusion protein that synergizes with oxaliplatin for treatment of colorectal cancer

“…However, although we detected AH1-specific T cells in the CEA-transfected tumors using tetramer reagents [ Supplementary Figure 6], there was no apparent link to a treatment regime. We have previously shown that the targeted delivery of TNF to colorectal tumors and to other cancer types in immunocompetent mouse models potently synergizes with other therapeutic modalities, including other cytokines (e.g., IL2(43) and IL12 (44)), cancer vaccines(37) and immune checkpoint inhibitors (45). In the current manuscript, we studied the combination Sm3E-TNF with 5-FU and with oxaliplatin, as these drugs are commonly used in chemotherapeutic strategies for the treatment of patients with metastatic colorectal cancer (46).…”

The immunocytokine L19-TNF eradicates Sarcomas in combination with Chemotherapy agents or with immune check-point inhibitors

“…TNF is a homo-trimeric pro-inflammatory cytokine, mainly produced by macrophages, NK and T cells [10,11]. TNF is involved in inflammatory processes and as indicated by its name, it may display a potent anticancer activity against some malignancies, by promoting hemorrhagic necrosis and by inducing direct apoptosis of tumor cells [12][13][14][15][16][17]. Moreover, TNF may activate the immune system as a result of the increased inflammation [18][19][20].…”