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Purpose: Small molecule drug conjugates (SMDC) are modular anticancer prodrugs that include a tumor-targeting small organic ligand, a cleavable linker, and a potent cytotoxic agent. Most of the SMDC products that have been developed for clinical applications target internalizing tumor-associated antigens on the surface of tumor cells. We have recently described a novel non-internalizing small organic ligand (named OncoFAP) of fibroblast activation protein (FAP), a tumor-associated antigen highly expressed in the stroma of most solid human malignancies. Experimental Design: In this article, we describe a new series of OncoFAP-Drug derivatives based on monomethyl auristatin E (MMAE; a potent cytotoxic tubulin poison) and dipeptide linkers that are selectively cleaved by FAP in the tumor microenvironment. Results: The tumor-targeting potential of OncoFAP was confirmed in patients with cancer using nuclear medicine procedures. We used mass spectrometry methodologies to quantify the amount of prodrug delivered to tumors and normal organs, as well as the efficiency of the drug release process. Linkers previously exploited for anticancer drug conjugates were used as benchmark. We identified OncoFAP-Gly-Pro-MMAE as the best performing SMDC, which has now been prioritized for further clinical development. OncoFAP-Gly-Pro-MMAE selectively delivered more than 10% injected dose per gram of MMAE to FAP-positive tumors, with a tumor-to-kidney ratio of 16:1 at 24 hours post-injection. Conclusions: The FAP-specific drug conjugates described in this article promise to be efficacious for the targeting of human malignancies. The extracellular release of potent anticancer payloads mediates durable complete remission in difficult-to-treat animal models of cancer.

Purpose:Small molecule drug conjugates (SMDC) are modular anticancer prodrugs that include a tumor-targeting small organic ligand, a cleavable linker, and a potent cytotoxic agent. Most of the SMDC products that have been developed for clinical applications target internalizing tumor-associated antigens on the surface of tumor cells. We have recently described a novel non-internalizing small organic ligand (named OncoFAP) of fibroblast activation protein (FAP), a tumor-associated antigen highly expressed in the stroma of most solid human malignancies.Experimental Design:In this article, we describe a new series of OncoFAP-Drug derivatives based on monomethyl auristatin E (MMAE; a potent cytotoxic tubulin poison) and dipeptide linkers that are selectively cleaved by FAP in the tumor microenvironment.Results:The tumor-targeting potential of OncoFAP was confirmed in patients with cancer using nuclear medicine procedures. We used mass spectrometry methodologies to quantify the amount of prodrug delivered to tumors and normal organs, as well as the efficiency of the drug release process. Linkers previously exploited for anticancer drug conjugates were used as benchmark. We identified OncoFAP-Gly-Pro-MMAE as the best performing SMDC, which has now been prioritized for further clinical development. OncoFAP-Gly-Pro-MMAE selectively delivered more than 10% injected dose per gram of MMAE to FAP-positive tumors, with a tumor-to-kidney ratio of 16:1 at 24 hours post-injection.Conclusions:The FAP-specific drug conjugates described in this article promise to be efficacious for the targeting of human malignancies. The extracellular release of potent anticancer payloads mediates durable complete remission in difficult-to-treat animal models of cancer.

Supplementary Information

The use of cytokine-based therapeutics has traditionally been limited by systemic toxicity, but it is now becoming evident that it may be possible to circumvent this problem in a number of ways. Tumor-targeting antibody-cytokine fusion proteins (also called “immunocytokines”) typically allow dose escalation to therapeutically active regimens as a result of their selective localization at the site of disease (Neri 2019; Helguera, Morrison, and Penichet 2002). Despite the enhancement of the therapeutic index, when the concentration of the payload in blood exceeds a certain threshold, patients may experience dose-limiting immune-related adverse events (irAEs). In most cases, irAEs disappear when the immunocytokine is cleared from circulation (Ko et al. 2004; Spitaleri et al. 2013; Rudman et al. 2011). Here, we describe an innovative approach to mask off-target activity of immunocytokines by transient inhibition of their intracellular signaling cascade. Small molecules are particularly attractive for this application since their serum half-life matches the clearance rate of the immunocytokine from circulation. L19-TNF and L19-IL12 are two clinical-stage antibody-cytokine fusion proteins that display potent activity by triggering the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and Janus Kinase 2 (JAK2) signaling pathways, respectively (Amin et al. 2018; Hu et al. 2021). In a first study, GSK’963, a potent small molecule inhibitor of RIPK1, was tested in tumor-bearing mice with the aim to reduce acute systemic toxicity associated with TNF signaling. Transient inhibition of RIPK1 allowed the administration of L19-murTNF doses which would otherwise be lethal. The combination with GSK’963 did not affect the selective localization of the immunocytokine to tumors. Moreover, L19-murTNF was still able to induce tumor necrosis and to exert potent anti-cancer activity. In a second study, tumor-bearing mice receiving L19-murIL12 were pretreated with Ruxolitinib, a commercially available JAK2 inhibitor. The addition of Ruxolitinib could significantly improve the tolerability profile of L19-murIL12 without affecting the anti-cancer properties of the immunocytokine. In clinical trials, it has been observed that IL12 treatment can induce cytokine release syndrome and liver damage (Atkins et al. 1997). Our data demonstrate that the pre-treatment with Ruxolitinib restricted the pro-inflammatory effects of L19-murIL12 to the tumor site, protecting the mice from body weight loss as well as reducing blood cytokine levels and hepatotoxicity. Overall, this preclinical work is of clinical relevance, as patients treated with targeted cytokines could potentially benefit from judicious combinations treatments using small molecule inhibitors. Citation Format: Giulia Rotta, Ettore Gilardoni, Domenico Ravazza, Jacqueline Mock, Samuele Cazzamalli, Roberto De Luca, Emanuele Puca, Dario Neri, Sheila Dakhel Plaza. Decreasing toxicity of immunocytokines by transient and selective inhibition of their intracellular signaling activation. [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 5744.

Conventional cancer chemotherapy relies on the use of cytotoxic drugs which are not designed and capable of selective accumulation to neoplastic lesions. The conjugation to tumour-targeting delivery vehicles has been proposed as a strategy to enhance the therapeutic index of potent cytotoxic drugs. To date, fifteen Antibody-Drug Conjugate products have received marketing approval for the treatment of solid and liquid human malignancies. We present an in vivo comparative evaluation of ADCs against products based on small organic delivery vehicles (Small Molecule-Drug Conjugates, or SMDCs) or on tumour-targeting peptides (Peptide-Drug Conjugates, or PDCs). All products are designed to target and to be activated by Fibroblast Activation Protein in the tumour microenvironment. Our results reveal that both targeted ADC and SMDC products mediate potent anti-cancer activity and complete tumour remission in a FAP-positive preclinical murine model of cancer, when administered at the same molar dose. Non-targeted albumin and antibody conjugates included in our comparative studies delayed tumour growth but presented suboptimal anti-cancer efficacy in the same cancer model. Our results indicate that both FAP-targeted ADCs and SMDCs are promising product candidates for the treatment of multiple human malignancies. Citation Format: Aureliano Zana, Claudia Puig Moreno, Ettore Gilardoni, Dario Neri, Samuele Cazzamalli. FAP-targeted non-internalising drug conjugates: A comparative evaluation between small molecules, antibodies and peptides [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 1988.

Radioligand Therapeutics (RLTs) are products composed of a tumor-targeting small organic ligand and a biocidal radioactive payload. The therapeutic potential of RLTs strongly depends on their uptake and residence time in tumors and in the healthy structures after administration. Fibroblast Activation Protein (FAP) is a cell surface antigen abundantly expressed in the microenvironment of the majority of solid human cancers of epithelial origin. We have recently described OncoFAP, an ultra-high affinity ligand of FAP whose targeting performances have been validated by Nuclear Medicine in patients with a wide variety of solid malignancies. Multivalent OncoFAP radioligand therapeutics (OncoFAP-RLTs) show high and prolonged tumor uptake with favorable tumor-to-organ ratios. Here we describe the development of novel multivalent OncoFAP-RLTs and their combination with a clinical-stage tumor-targeted interleukin-2 (L19-IL2). We designed and synthesized a set of multivalent OncoFAP derivatives, aiming at maximizing their affinity for the target protein and their residence time in the catalytic pocket of FAP. The multivalent ligands were coupled to the DOTAGA chelator, suitable for radiolabeling with 177Lu for therapeutic application. The tumor targeting properties of the multivalent OncoFAP ligands were investigated in a comparative biodistribution study in SK-RC-52.hFAP tumor-bearing mice. 177Lu-OncoFAP-23 emerged for its superior uptake and residence time in tumors and for its favorable tumor-to-kidney ratio. Moreover, when administered at very low dose 177Lu-OncoFAP-23 exhibited an enhanced anticancer activity compared to a monovalent (177Lu-OncoFAP-DOTAGA) and bivalent (177Lu-BiOncoFAP-DOTAGA) version of OncoFAP. When OncoFAP-based RLTs were combined with L19-IL2, a strong synergistic anticancer activity was observed. This synergy is driven by NK-cell mediated immune response directed to the tumor, with the concomitant release of granzymes and other pro-apoptotic proteins in the tumor microenvironment. Our findings heighten OncoFAP-based RLTs as promising anti-cancer drugs for treatment of FAP-positive tumors, alone or in combination with targeted interleukin-2. Citation Format: Andrea Galbiati, Matilde Bocci, Ettore Gilardoni, Aureliano Zana, Dario Neri, Samuele Cazzamalli. Tumor-targeted interleukin-2 enhances the anti-cancer activity of multivalent OncoFAP radioligand therapeutics [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 2838.

Small Molecule-Drug Conjugates (SMDCs) are modular anti-cancer pro-drugs that include a tumor-targeting small ligand, a cleavable linker and a potent cytotoxic agent. SMDC products that have been developed for clinical applications are targeting internalizing tumor-associated antigens expressed on the surface of tumor cells. We have recently developed a novel non-internalizing small organic ligand (named OncoFAP) of Fibroblast Activation Protein (FAP), a tumor-associated antigen highly expressed in the stroma of most of solid human malignancies. The tumor targeting performance of OncoFAP has been validated by nuclear medicine studies in patients with various solid tumors. In a previous study, we showed that OncoFAP can be used to produce non-internalizing SMDCs that are effective and well tolerated. Here, we describe a new series of OncoFAP-Drug derivatives based on the MMAE tubulin poison and dipeptide linkers that are selectively cleaved by FAP in the tumor microenvironment. We benchmarked the new SMDCs against OncoFAP-MMAE conjugates displaying linker modules which are widely used in approved and clinical stage Antibody-Drug Conjugates, including structures cleaved by Cathepsin B and by reducing agents. We selected OncoFAP-GlyPro-MMAE as the most efficacious and safe SMDC for further clinical development after quantitatively analyzing the biodistribution of MMAE released by OncoFAP-MMAE conjugates. OncoFAP-GlyPro-MMAE selectively delivers high amounts of MMAE at the site of disease, with a tumor-to-kidney ratio of 7-to-1 and of 16-to-1 at 6- and 24-hours post-injection, respectively. Our molecules based on the OncoFAP tumor-targeting ligand and FAP-cleavable linkers promise to be safe and effective against most of human malignancies. Citation Format: Aureliano Zana, Andrea Galbiati, Ettore Gilardoni, Jacopo Millul, Theo Sturm, Riccardo Stucchi, Matilde Bocci, Abdullah Elsayed, Lisa Nadal, Martina Cirillo, Dario Neri, Samuele Cazzamalli. Fibroblast activation protein triggers the release of drug payload from non-internalizing small molecule-drug conjugates in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB522.