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BackgroundIn this study, we describe the generation of a fully human monoclonal antibody (named ‘7NP2’) targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types of solid neoplasms.Methods7NP2 was isolated from a synthetic antibody phage display library and was improved by one round of mutagenesis-based affinity maturation. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin (IL)-12 was generated and the anticancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. Furthermore, the safety of the fully human product (named IL12-7NP2) was evaluated in Cynomolgus monkeys.ResultsBiodistribution analysis in tumor-bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent antitumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates.ConclusionsThe results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2.

We present the first in vivo comparative evaluation of chemically defined antibody–drug conjugates (ADCs), small molecule–drug conjugates (SMDCs), and peptide–drug conjugates (PDCs) targeting and activated by fibroblast activation protein (FAP) in solid tumors. Both the SMDC (OncoFAP-Gly-Pro-MMAE) and the ADC (7NP2-Gly-Pro-MMAE) candidates delivered high amounts of active payload (i.e., MMAE) selectively at the tumor site, thus producing a potent antitumor activity in a preclinical cancer model.

Among the different pharmacological approaches for cancer treatment, antibody-cytokine fusion proteins (also called “immunocytokines”) represent an emerging class of biopharmaceutical products, capable of boosting the immune system to attack tumor cells as a result of a selective accumulation of pro-inflammatory molecules at the site of disease. When used as single agents, immunocytokines are often not able to cure mice and patients. However, it has been shown by our group that the combination of IL2- and TNF- based products was able to completely eradicate tumors in mouse models and to induce complete responses in cancer patients. We have previously described a novel class of immunocytokine products, termed “potency-matched dual cytokine antibody fusion proteins”, consisting of a tumor-targeting antibody (the F8 antibody, specific to the alternatively spliced EDA domain of fibronectin), simultaneously fused to both IL2 and TNF. To match biological activity of the two payloads a single-point mutation was inserted in the TNF sequence and the resulting product, named IL2-F8-TNFmut was able to selectively localize to tumors with excellent tumor-to-organ ratios and it was found to completely eradicate soft-tissue sarcomas in immunocompetent mice, which did not respond to standard chemotherapy. Moreover, the immunocytokine was able to induce complete responses in a variety of mouse models of cancer. The therapeutic activity was further improved when IL2-F8-TNFmut was used in combination with immune checkpoint inhibitors. At the mechanistic level we observed that the anticancer activity was completely abrogated when CD4+ and CD8+ T cells were depleted, while the contribution of NK cells was negligible. We now report the generation of a new potency-matched dual cytokine antibody fusion protein, named Tripokin based on the L19 antibody, specific to the alternatively spliced EDB domain of fibronectin. Compared to the previous product, Tripokin showed a favorable pharmacokinetic profile in monkey, an excellent localization to neoplastic lesions in mice and was easier to formulate for further clinical applications. When tested in tumor bearing mice, Tripokin revealed a superior anti-cancer activity compared to other IL2-based products.The results obtained in this work provided a rationale for future clinical translation activities using Tripokin as potential best-in-class tumor targeted IL2 product. Citation Format: Eleonora Prodi, Riccardo Corbellari, Mattia Matasci, Dario Neri, Roberto De Luca. Tripokin: potential best-in-class for tumor targeted interleukin-2 (IL2) potentiated by tumor necrosis factor (TNF) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1877.

The delivery of specific cytokine payloads to a neoplastic environment employing antibodies able to selectively accumulate at the tumor site represents an attractive strategy to stimulate an immune response to cancer. Whilst conventional antibody–cytokine fusions based on a single payload have shown potent anticancer activity, the concomitant delivery of two cytokine payloads may further improve the therapeutic outcome as the immune system typically adopts multiple signals to reinforce an antitumor strategy. We here describe a potency-matched dual-cytokine antibody fusion protein containing a tumor-targeting antibody fragment specific to human fibroblast activation protein (FAP), simultaneously linked to both interleukin-2 (IL2) and a tumor necrosis factor (TNF) mutant. The resulting fusion protein, termed IL2-7NP2-TNFmut, formed stable non-covalent trimers driven by the interaction of the tumor necrosis factor subunits. Both cytokine payloads retained their biological activity within the fusion protein, as shown by in vitro cellular assays. The tumor-targeting properties and the anticancer activity of IL2-7NP2-TNFmut were investigated in vivo in immunocompromised mice bearing SKRC52 cells transduced with human FAP. The fusion protein preferentially localized to the cancer site and induced partial tumor retardation.