Radioligand therapies have emerged as promising treatment modality for certain cancers. This therapeutic approach relies on a suitable tumor-targeting moiety labelled with a radioactive payload to allow the delivery of the radionuclide to cancer cells with high specificity. Due to its expression profile in tumor vs healthy tissues, MSLN is an attractive protein for targeted therapies. MSLN is a GPI-anchored membrane protein which is overexpressed in many tumor types, including ovarian cancer and pancreatic adenocarcinoma. Therapeutic approaches targeting MSLN have encountered obstacles due to the high levels of its proteolytically cleaved soluble form (sMSLN), which can sequester anti-MSLN therapeutics and potentially diminish their efficacy. To circumvent this caveat, we sought to generate a Designed Ankyrin Repeat Protein (DARPin) that binds to a membrane proximal epitope of MSLN and therefore avoids binding to sMSLN. Thanks to their small size and high affinity, DARPins are ideal vectors for targeted radiotherapies, dubbed as Radio-DARPin therapies (RDT). As radioactive payload we used 212Pb, an alpha particle-emitting radionuclide with a short half-life and a favorable decay profile which allows high energy deposition on tumor in a short time frame. Herein we report for the first time the development and initial preclinical characterization of our anti-MSLN 212Pb-RDT approach. In vitro assays revealed that the membrane proximal MSLN targeting DARPin specifically interacts with MSLN expressed on cancer cells with high affinity, and cell binding was not affected by the presence of sMSLN. The DARPin was then labelled with 212Pb and its preclinical pharmacokinetic and biodistribution profile was evaluated in MSLN-expressing xenograft tumor models. In these tumor bearing mice, the anti-MSLN 212Pb Radio-DARPin demonstrated substantial uptake into MSLN-positive tumors, while other organs only showed limited accumulation. We designed an approach to specifically target MSLN expressed on cancer cells employing DARPin technology combined with 212Pb. Our early preclinical results indicative of a favorable biodistribution profile of the 212Pb labelled MSLN DARPin motivate further development of this RDT for the treatment of patients with MSLN-positive solid tumors. Stephan Wullschleger,Amal Saidi,Delphine Buffet,Tania Stallons,Stefanie Riesenberg,Amy Wong,Maria Paladino,Federico Rojas,Mischa Müller,Christel Herzog,Justin Walter,Denis Villemagne,Yvonne Kaufmann,Nicole Pina,Henri Fernandez,Tamara Lekishvili,Eleni Tselempi,Jacqueline Blunschi,Liridon Abduli, Chloe Iss,Amelie Croset,Clara Domke,Aaron Schatzmann,Anne Goubier,Julien Torgue,Daniel Steiner. Development of 212Pb-based Radio-DARPin therapy (RDT) for the treatment of mesothelin (MSLN)-positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 339.
Radioligand therapy has shown strong clinical potential for the treatment of certain neuroendocrine and prostate tumors. Nevertheless, effective strategies in various cancer types are currently restricted by the lack of appropriate targeting agents for suitable tumor-associated antigens. Not expressed in healthy tissue, DLL3 is one of these promising targets highly upregulated in SCLC and other high-grade neuroendocrine tumors. Herein we present preclinical results of our DLL3-targeting 212Pb-based Radio-DARPin Therapeutic (RDT) combining the advantages of a small protein-based delivery vector and the short-lived alpha particle-emitting radioisotope 212Pb. Designed Ankyrin Repeat Proteins (DARPins) are a class of binding proteins with high specificity and affinity that can be generated against a broad range of tumor targets. Leveraging the learnings from our DARPin platform optimization allowed us to achieve efficient tumor uptake and penetration, while limiting exposure of healthy tissues. Our Radio-DARPin platform was optimized to effectively reduce DARPin uptake to kidneys, a major drawback of all small-size polypeptide vectors. This can be addressed by engineering the DARPin scaffold surface in conjunction with half-life extension (HLE) through serum albumin binding. 212Pb is a radioisotope with a short half-life of 11h and a favorable decay chain, allowing high energy deposition on tumor in a short time frame. By combining 212Pb with a HLE anti-DLL3 DARPin candidate, we aimed to generate a targeted RDT with high efficacy and a favorable safety profile. After several screening rounds of anti-DLL3-binding DARPins combined with HLE in mice, MP0712 was selected as lead candidate for further development. This molecule showed specific binding to lung cancer cells in vitro (∼2nM on NCI-H82 ± HSA) and high affinity to human DLL3 (0.2nM by Surface Plasmon Resonance). MP0712 biodistributions were assessed in different mouse xenograft tumor models, matching clinically relevant DLL3 expression levels or overexpression of DLL3, and reached tumor:kidney ratios >2. Double xenografted mouse models showed selective uptake in DLL3-expressing tumors compared with tumors not expressing DLL3, confirming high target specificity of MP0712. The anti-tumor activity of MP0712 in different mouse xenograft tumor models showed promising efficacy. MP0712 led to tumor stabilization and significant effects on tumor versus necrotic tissue. In NCI-H82 tumors, median survival duration increased up to 3-fold with MP0317 compared with the buffer control. MP0712 also showed a favorable safety profile in vivo up to 30µCi. These preclinical results support our 212Pb-based RDT against DLL3 as a promising treatment option for SCLC, with encouraging in vivo antitumor activity and a good safety profile for our first DLL3-targeting 212Pb-RDT candidate for further development into the clinic. Amelie Croset, Amal Saidi, Francesca Malvezzi, Tania Stallons, Madlaina Mettier, Amy Wong, Jitka Rantanen, Federico Rojas, Stephan Wullschleger, Eleni Tselempi, Yvonne Kaufmann, Tamar Lekishvili, Stefanie Riesenberg, Nicole Pina, Jacqueline Blunschi, Liridon Abduli, Christian Reichen, Christian Lizak, Aaron Schatzmann, Anne Goubier, Julien Torgue, Daniel Steiner. MP0712, the first anti-DLL3 212Pb radio-DARPin (RDT) candidate for targeted radiotherapy of small cell lung cancer (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 346.
T-cell engagers (TCE) are powerful and clinically validated cancer therapeutics but are limited by high toxicity, lack of tumor-specific targets, and presence of exhausted/dysfunctional T cells particularly in solid tumors. Our CD3 Switch-DARPin platform provides a logic-gated “on/off” function (“Switch”) to increase tumor specificity and enable strong CD3 engagement and activation without compromising safety. Additionally, a DARPin with a co-stimulatory function can be added to enhance T-cell responses, especially in the context of an adverse tumor microenvironment. Using ovarian cancer (OC) as a proof-of-concept indication, we designed a multi-specific CD3 Switch-DARPin with CD2 co-engagement (CD2 x CD3 Switch-DARPin) which targets two tumor-associated antigens (TAAs); mesothelin and EpCAM. The logic-gated function of our Switch-DARPin ensures that the CD3 moiety is able to bind and activate T cells only when the TAAs are expressed on target cells. Herein we present a Switch-DARPin TCE that safely sustains anti-tumor efficacy, providing new TAA options and expanding TCE use in solid tumors. RNAseq phenotypic analyses of OC patient samples were validated on resected tumors and ascites using flow cytometry and immunohistochemistry. The specific activity and safety of our CD2 x CD3 Switch-DARPin were assessed in vitro and ex vivo using reporter cells, primary human T cells from healthy donors and OC patient ascites, whole blood cytokine release assays, and in vivo in PBMC-humanized NXG mice engrafted subcutaneously with mesothelin- and EpCAM-expressing cells. Our analysis confirmed that mesothelin and EpCAM are highly co-expressed in OC tumors. Furthermore, reduced expression of CD28 compared to CD2 on tumor-infiltrating lymphocytes, combined with CD58 downregulation on primary tumors and ascites, support the use of CD2 as a co-stimulatory receptor for T cell engagement in OC. Functionally, the CD2 x CD3 Switch-DARPin abrogated TAA-independent T-cell activation and reduced potential on-target off-tumor toxicity, while CD2 co-engagement sustained T-cell response and prevented T-cell dysfunction. Notably, in vivo, the CD2 x CD3 Switch-DARPin induced significant tumor regression without causing systemic release of cytokines in the blood. In contrast, a control molecule without the Switch led to systemic cytokine release and showed no antitumor efficacy, likely due to fratricide-mediated depletion of T cells. Our CD3 Switch-DARPin platform provides a novel approach for sustained tumor-specific T-cell engagement, by combining a logic-gated on/off Switch mechanism with CD2 co-stimulation. This allows immune activation to be directed to tumors, increasing efficacy and safety and paving the way for new opportunities for cancer treatment. Matteo Bianchi, Marco Franchini, Tamara Lekishvili, Eleni Tselempi, Joanna Robinson, Yvonne Kaufmann, Guillaume Ems, Chloé Friang, Clara Tissier, Mischa R. Müller, Alexander Link, Christophe Borg, Amelie Croset, Valérie Calabro, Anne Goubier, Marcela Guzman Ayala. Next-generation multi-specific and conditionally activated CD3 Switch-DARPins with CD2 co-stimulation to tackle the current limitations of T cell engagers in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3119.
2584 Background: CD40 is a master switch for both innate and adaptive immune systems. The clinical development of CD40 agonists has been hampered by dose-limiting toxicity (DLT) due to systemic CD40 activation and peripheral target-mediated drug disposition (TMDD). MP0317 is a CD40-agonistic DARPin (designed ankyrin repeat protein), developed to reduce systemic toxicity. MP0317 is exclusively active in the presence of fibroblast activating protein (FAP) expressed by cancer associated fibroblasts in the tumor microenvironment. This allows reaching serum levels that overcome the TMDD and have the potential to deliver sustained and tumor-localized CD40 activation. Ongoing clinical testing aims at establishing its safety/tolerability profile, PK/PD characteristics, and a recommended dose for combination therapy. Methods: NCT05098405 is a Phase 1, multicenter, open label, dose escalation study followed by a safety expansion part in adult patients with advanced solid tumors. The dose escalation scheme uses an adaptive Bayesian logistic regression model with overdose control. Peripheral blood biomarkers are analyzed by immuno-assays and flow cytometry, and baseline and on-treatment tumor biopsies are characterized by RNA sequencing and immunofluorescence. Eligible patients are enrolled into 9 sequentially escalating dose cohorts of MP0317 (0.03-10 mg/kg), administered IV 3-weekly (Q3W) or 1-weekly (Q1W) until disease progression or unacceptable toxicity. Results: At submission, 23 patients across 6 cohorts completed the study, with no DLT observed. Patient enrolling in higher dose cohorts is ongoing. The most frequent AE was grade 2 infusion related reaction in 5 patients. The 23 patients received ≥2 (range 2-8) doses (range 0.03 – 3 mg/kg) of MP0317 across five Q3W and one Q1W cohorts and completed the 28-day DLT period. PK data confirmed all patients were exposed to MP0317 with broadly dose-dependent Cmax and no sign of accumulation. PD data was derived from patients dosed with 0.03 – 3 mg/kg. Soluble biomarkers (sFAP and sCD40) showed target engagement in periphery with signs of FAP saturation at ≥0.5 mg/kg. Colocalization of MP0317 with FAP and CD40 in the tumor was confirmed in 4 out of 8 evaluable paired tumor biopsies. Whole transcriptome and gene set enrichment analyses showed an upregulation of genes related to B-cell trafficking (CXCL3, CXCR5, CCR6, CCL20) upon treatment. Transient increase of IFNg-induced chemokines (CXCL9, CXCL10) was observed, whereas the pro-inflammatory cytokines (TNFa, IL-2, IL-6, IL-8) were not upregulated. Conclusions: Clinical data and the lack of pro-inflammatory circulating cytokines confirm MP0317 is safe and well-tolerated, while analysis of paired pre- and on-treatment tumor biopsies suggests early evidence of tumor-localized CD40 activation. The current data enables further evaluation in a combination setting. Clinical trial information: NCT05098405 .
The DARPin® drug platform was established with a vision to expand the medical use of biologics beyond what was possible with monoclonal antibodies. It is based on naturally occurring ankyrin repeat domains that are typically building blocks of multifunctional human proteins. The platform allows for the generation of diverse, well-behaved, multifunctional drug candidates. Recent clinical data illustrate the favorable safety profile of the first DARPin® molecules tested in patients. With the positive phase III results of the most advanced DARPin® drug candidate, abicipar, the DARPin® drug platform is potentially about to achieve its first marketing approval. This review highlights some of the key milestones and decisions encountered when transforming the DARPin® platform from an academic concept to a biotech drug pipeline engine.
