Virtually all patients with multiple myeloma become unresponsive to treatment with proteasome inhibitors over time. Relapsed/refractory multiple myeloma is accompanied by the clonal evolution of myeloma cells with heterogeneous genomic aberrations, diverse proteomic and metabolic alterations, and profound changes of the bone marrow microenvironment. However, the molecular mechanisms that drive resistance to proteasome inhibitors within the context of the bone marrow microenvironment remain elusive. In this review article, we summarize the latest knowledge about the complex interaction of malignant plasma cells with its surrounding microenvironment. We discuss the pivotal role of metabolic reprograming of malignant plasma cells within the tumor microenvironment with a subsequent focus on metabolic rewiring in plasma cells upon treatment with proteasome inhibitors, driving multiple ways of adaptation to the treatment. At the same time, mutual interaction of plasma cells with the surrounding tumor microenvironment drives multiple metabolic alterations in the bone marrow. This provides a tumor-promoting environment, but at the same time may offer novel therapeutic options for the treatment of relapsed/refractory myeloma patients.

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CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor receptor superfamily which can activate both innate and adaptive immune system, making it an interesting target for tumor immunotherapy. Systemic administration of agonistic CD40 antibodies (Ab) has shown signs of activity in cancer patients, but dose-limiting toxicity impaired the clinical efficacy. New approaches are therefore needed to increase the therapeutic index of CD40-targeting molecules and achieve better clinical outcomes. Here, we report an alternative approach designed to activate CD40 specifically in the tumor microenvironment (TME), and not systemically, in order to increase efficacy and reduce systemic toxicity. This novel approach is based on a bispecific DARPin® molecule, targeting CD40 and fibroblast activation protein (FAP) alpha, intended to induce immune activation only when clustered by binding to FAP-expressing cells in the TME. The bispecific FAP x CD40 DARPin®molecule was tested in a reporter assay and in additional cell assays using primary human 1) B cells, 2) macrophages and 3) dendritic cells. These studies demonstrated CD40 activation only in the presence of FAP-positive, but not with FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated cross-linking. A surrogate mouse-specific FAP x CD40 DARPin® molecule (mFAP x CD40) was generated and tested in similar in vitro assays and showed FAP-dependent activation of CD40 and comparable results as the human construct. In vivo experiments, performed in tumor-free mice, showed a comparable half-life between mFAP x CD40 and an anti-mouse CD40 Ab (clone FGK45). However, mFAP x CD40, in contrast to the FGK45 Ab, did not increase the serum level of IL-6, supporting a mode of action that is dependent on FAP-mediated crosslinking of CD40 receptor. In additional studies mFAP x CD40 was active and inhibited the growth of FAP+ tumors. There were no signs of toxicity with mFAP x CD40 in contrast to the FGK45 Ab which resulted in body weight loss. In conclusion, we have generated bispecific agonist FAP x CD40 DARPin® molecules able to activate the CD40 pathway with a targeting (FAP)-dependent mechanism of action. Citation Format: Nicolo Rigamonti, Anja Schlegel, Sophie Barsin, Jonas Schwestermann, Susanne Mangold, Yvonne Kaufmann, Christof Zitt, Niina Veitonmäki, Victor Levitsky, Clara Metz. Fibroblast activation protein (FAP)-selective delivery of CD40 agonistic DARPin®molecule for tumor-localized immune activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3251.

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CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily which can activate both innate and adaptive tumor immunity, making it an attractive target for cancer immunotherapy. Systemic administration of agonistic anti-CD40 antibodies has shown signs of activity in cancer patients, but dose-limiting toxicity has prevented the exploration of the full dose range and possibly has impaired clinical efficacy. To overcome this issue, we developed a multispecific DARPin® therapeutic candidate, MP0317, intended to activate CD40 receptor locally, rather than systemically, in the tumor microenvironment through cross-linking with fibroblast activation protein (FAP) that is abundantly present in the stroma of many solid tumors, but has limited expression in healthy adult tissues. Using primary human 1) B cells and 2) dendritic cells, MP0317 activated the CD40 pathway in vitro. Upregulation of CD80, CD86, CD69 and IL12 occurred exclusively in the presence of FAP-positive, but not with FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated cross-linking. A surrogate mouse-specific FAP x CD40 DARPin® molecule (mFAP x mCD40) was generated and tested in murine cell-based in vitro assays showing comparable results as MP0317 with a strict FAP-dependent activation of CD40. The mFAP x mCD40 was also active in vivo and inhibited substantially the progression of FAP-positive tumors. Moreover, in contrast to an anti-mouse CD40 antibody (clone FGK45), the antitumor activity of mFAP x mCD40 was neither associated with elevated blood cytokine levels nor with hepatotoxicity, both of which manifest as dose limiting toxicities of some of the clinical CD40 activating antibodies. These data support a mode of action, both in vitro and in vivo, that is dependent on FAP-mediated crosslinking of CD40 receptor resulting in a tumor-localized CD40 activation without peripheral or off-tumor organ toxicity. In conclusion, we have designed a tumor-targeted CD40 agonistic DARPin® therapeutic candidate that is able to activate the CD40 receptor locally in FAP-positive tumors producing impressive antitumor activity in the absence of systemic toxicity. Citation Format: Nicolo Rigamonti, Sarah Jetzer, Niels Van der Velden, Omar Abdelmotaleb, Sophie Barsin, Jonas Schwestermann, Susanne Mangold, Yvonne Kaufmann, Ralph Bessey, Andrea Valeri, Heidi Poulet, Christof Zitt, Pamela A. Trail, Victor Levitsky, Niina Veitonmäki, Clara Metz. A tumor-targeted CD40 agonistic DARPin® molecule leading to antitumor activity with limited systemic toxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1073.