Abstract Introduction: Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand aberrantly expressed on SCLC cells and largely absent in healthy tissues, making it a promising therapeutic target. However, low DLL3 surface density may limit tumor accumulation of a targeted radiopharmaceutical. MP0712, a half-life extended DLL3-targeting DARPin (Designed Ankyrin Repeat Protein) molecule combined with the short-lived alpha-emitting therapeutic isotope 212Pb, showed favorable safety, biodistribution, and antitumor efficacy in mice (Croset et al. AACR 2025). Under South Africa’s Section 21 compassionate care framework, MP0712 was administered for imaging with 203Pb to patients with SCLC and other DLL3-positive neuroendocrine cancers (NECs). Initial human imaging data indicate specific tumor uptake, supporting MP0712’s intended mode of action (Steiner et al. TRP EU 2025). Here, we describe MP0712’s molecular features, focusing on binding properties, internalization, and circulatory half-life. Methods: DLL3-expressing SCLC cell lines and xenograft models were used to evaluate MP0712 biodistribution in vivo. Internalization was assessed using flow cytometry and confocal microscopy. PK/PD properties were determined in mice. The affinity to DLL3 protein was determined by Surface Plasmon Resonance. Results: We identified sub-nanomolar affinity as a critical parameter for achieving efficient tumor uptake. MP0712 comprises a high-affinity, DLL3-specific DARPin (KD of 0.2 nM) that binds efficiently and selectively to DLL3+ SCLC cells. Given the low surface density of DLL3, we reasoned that to maximize accumulation in tumor, rapid and repeated internalization of a Radio-DARPin would be beneficial. Consistent with this hypothesis, we could demonstrate that up to 80% of surface-bound DLL3-DARPin internalizes within 30 min into SCLC cells in vitro. Importantly, we observed progressive intracellular accumulation over time upon continuous supply of DLL3-targeting DARPins, suggesting that repeated cycles of internalization occur. Finally, we hypothesized that extending systemic exposure of the DLL3-targeting DARPin would further enhance tumor uptake by leveraging these internalization dynamics. Indeed, we observed that intermediate half-life extension with an albumin-binding moiety improved tumor accumulation in xenograft models compared to non-half-life extended molecules. Conclusions: MP0712 demonstrates a compelling preclinical profile that suggests it may benefit from rapid receptor turnover-mediated intracellular accumulation, offsetting the effects of low DLL3 surface density. Together with emerging 203Pb-imaging data from compassionate care, these findings support further clinical evaluation of MP0712 therapeutic application with 212Pb-payload in patients with SCLC and other DLL3-positive NECs. Citation Format: Stefanie Riesenberg, Amal Saidi, Francesca Malvezzi, Aaron Schatzmann, Christian Reichen, Tania Stallons, Nicole Pina, Aline Eggenschwiler, Amy Wong, Madlaina Mettier, Jitka Rantanen, Marcela Guzman-Ayala, Julien Torgue, Daniel Steiner. Molecular characteristics of MP0712, a clinical stage 212Pb-based Radio-DARPin candidate for targeted anti-DLL3 radiotherapy of small cell lung cancer (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7197.

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.

Abstract The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell–mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell–mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen–targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).

Supplementary Fig. S17. TAA expression analysis on AML PDX cells.

The development of effective radioligand therapeutics (RLTs) is frequently hampered by the lack of high-quality targeting agents that selectively deliver radioactive payloads to the site of disease while sparing healthy tissues. Antibodies can have high affinity and specificity to tumor targets, but their large size results in limited tumor penetration and long systemic half-life is frequently causing haematological toxicities. Alternatively, targeting agents with low molecular weight such as small molecules and peptides often suffer from limited affinity and specificity to the tumor target, resulting in off-target effects and limited tumor retention. DARPins (Designed Ankyrin Repeat Proteins) developed by Molecular Partners combine small size (15 kDa) and ideal binding properties. Due to their rigid-body target binding mode DARPins combine very high affinity and specificity and unless engineered accordingly, DARPins have very short systemic half-lives. Thanks to a simple and robust architecture, DARPins can be efficiently coupled with radioactive payloads, even at elevated temperatures; and they can tolerate sequence-engineering approaches, which are not compatible with other protein scaffolds. To establish the DARPin platform for RLT, we have used DARPin candidates against different tumor targets. We have previously shown that increasing affinity to the tumor target correlates with elevated tumor uptake and long tumor residence in preclinical mouse models. We now also show that DARPins exhibit a homogeneous and deep tumor penetration in vivo that is highly superior to antibody benchmarks. Globular proteins below 60 kDa in size are typically cleared from the bloodstream via the renal pathway. This generally results in a strong kidney accumulation of small sized, protein-based targeting agents and their coupled residualizing radionuclides, leading to dose-limiting kidney toxicities. To overcome this limitation, we have undertaken an extensive engineering approach of the DARPin scaffold. Our results show that sequence engineering strongly reduces kidney uptake of DARPins without affecting their tumor uptake. This effect was confirmed with independent DARPin candidates suggesting a general applicability of the approach. Combined with other orthogonal strategies, we are able obtain favourable tumor to kidney ratios in preclinical mouse models. These results show that our proprietary optimized DARPin platform offers an attractive solution to the limitations of protein-based targeting agents for RLT applications. Together with the fact that high-affinity DARPins can be generated against a large variety of tumor targets, we conclude that our platform provides a powerful basis for the development of next-generation RLTs. Several DARPin-RLT programs in indications with high unmet medical need are currently in development. Citation Format: Andreas Bosshart, Stephan Wullschleger, Martin Behe, Alain Blanc, Stefan Imobersteg, Alexandra Neculcea, Jacqueline Blunschi, Liridon Abduli, Sarah Schütz, Julia Wolter, Christian Reichen, Amelie Croset, Alessandra Villa, Christian Lizak, Anne Goubier, Roger Schibli, Daniel Steiner. DARPins as powerful targeting agents for 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 5037.

Introduction: Acute myeloid leukemia (AML) is driven by leukemia stem cells (LSCs) that resist conventional chemotherapy and are the major cause of relapse. Newer therapies, including chimeric antigen receptor (CAR) T cells and T cell engagers (TCE) that target specific tumor associated antigens (TAAs) overexpressed on stem cells and leukemic blasts, are promising options for AML. We postulated that a TCE able to simultaneously target multiple TAAs could allow highly efficient and specific T cell-mediated killing of AML LSCs and circulating blast cells while minimally affecting healthy cells. Moreover, the simultaneous targeting of different TAAs has the potential to address tumor heterogeneity, allowing targeting of AML cells with different co-expression patterns and/or expression levels of each TAA. Methods: Our DARPin (Designed Ankyrin Repeat Protein) libraries contain trillions of molecules allowing the generation of highly diverse binders against target proteins that can be easily combined into multi-specific DARPins to elicit desired biological effects. We leveraged this proprietary platform to screen multi-specific CD3 engaging DARPin molecules, including serum albumin binding DARPins for systemic half-life extension, to generate MP0533, the first half-life extended avidity-engineered CD3 engaging DARPin capable of targeting CD33, CD123, and CD70 simultaneously. Efficacy and safety of MP0533 was extensively tested in vitro and ex vivo, with cell lines and primary cells in both allogenic and autologous settings, and in vivo in different cell line-derived xenograft mouse models. Results: We demonstrated the avidity effect of the multi-targeting MP0533 in co-culture assays with genetically engineered Molm-13 cells and allogenic T cells, where MP0533 induced up to 10-fold higher T cell mediated killing of cells expressing any combination of at least 2 of the 3 targeted TAAs vs. cells expressing a single TAA. Overall, an EC50 potency ranging from 5 to 25 pM was measured on multiple cell lines showing different levels of expression and co-expression of all TAAs (Molm-13, THP-1, RPMI8226). Efficacy was additionally assessed on primary unsorted AML bone marrow samples: MP0533 demonstrated the ability to induce autologous T cell activation and killing of AML cells in samples from newly diagnosed and previously treated patients with different TAA expression/co-expression levels and E:T ratios ranging from 10:1 to 1:28. MP0533-induced killing of LSC and selectivity over healthy hematopoietic stem cells (HSCs) was analyzed in clonogenic assays in co-cultures of sorted CD34+ cells with allogenic T cells. After 4 days initial killing assay, and 2 weeks colony culture in semi-solid media, MP0533 was able to show depletion of LSC at doses where HSC where only minimally affected. While T-cell activation, proliferation, and cytotoxicity were not affected, the multi-specific TCE format of MP0533 resulted in a significant decrease in cytokine release in both tumor cell killing assays and whole blood test systems when compared to other mono-targeting TCEs, indicating potential for improved safety. Moreover, the avidity approach and the safer profile on single-TAA expressing cells was confirmed by the absence of toxicity measured on CD123-expressing endothelial cells, indicating a low risk of capillary leak syndrome induced by MP0533. Also, potential T cell fratricide induced by CD70 expression on activated T cells was not observed. Finally, efficacy and safety profiles were also confirmed in in vivo xenograft models in human PBMC- or CD34-reconstituted mice. In such models MP0533 induced T cell infiltration in s.c. implanted tumors, leading to anti-tumor activity. Importantly, activation of T cells and release of cytokines were only localized in the tumors, indicating absence of potential systemic on-target off-tumor toxicities induced by MP0533. Conclusion: We generated a multi-specific avidity-driven CD3 engaging DARPin molecule with tailored affinities towards different TAAs showing high efficacy and with the potential for better LSC selectivity and lower cytokine release compared with mono-specific TCE approaches. Further, our approach to achieve systemic half-life extension could ensure a more convenient Q1W IV therapy. A Phase I clinical trial to evaluate safety and dose of MP0533 in humans is imminent.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).

SARS-CoV-2 has infected millions of people globally and continues to undergo evolution. Emerging variants can be partially resistant to vaccine induced and therapeutic antibodies, emphasizing the urgent need for accessible, broad-spectrum therapeutics. Here, we report a comprehensive study of ensovibep, the first trispecific clinical DARPin candidate, that can simultaneously engage all three units of the spike protein trimer to potently inhibit ACE2 interaction, as revealed by structural analyses. The cooperative binding of the individual modules enables ensovibep to retain inhibitory potency against all frequent SARS-CoV-2 variants, including Omicron, as of December 2021. Moreover, viral passaging experiments show that ensovibep, when used as a single agent, can prevent development of escape mutations comparably to a cocktail of monoclonal antibodies (mAb). Finally, we demonstrate that the very high in vitro antiviral potency also translates into significant therapeutic protection and reduction of pathogenesis in Roborovski dwarf hamsters infected with either the SARS-CoV-2 wild-type or the Alpha variant. In this model, ensovibep prevents fatality and provides substantial protection equivalent to the standard of care mAb cocktail. These results support further clinical evaluation and indicate that ensovibep could be a valuable alternative to mAb cocktails and other treatments for COVID-19.

Globally accessible therapeutics against SARS-CoV-2 are urgently needed. Here, we report the generation of the first anti-SARS-CoV-2 DARPin molecules with therapeutic potential as well as rapid large-scale production capabilities. Highly potent multivalent DARPin molecules with low picomolar virus neutralization efficacies were generated by molecular linkage of three different monovalent DARPin molecules. These multivalent DARPin molecules target various domains of the SARS-CoV-2 spike protein, thereby limiting possible viral escape. Cryo-EM analysis of individual monovalent DARPin molecules provided structural explanations for the mode of action. Analysis of the protective efficacy of one multivalent DARPin molecule in a hamster SARS-CoV-2 infection model demonstrated a significant reduction of pathogenesis. Taken together, the multivalent DARPin molecules reported here, one of which has entered clinical studies, constitute promising therapeutics against the COVID-19 pandemic. ### Competing Interest Statement Molecular Partners authors own performance share units and/or stock of the company. HKB owns stock of the company.

AML is driven by leukemic stem cells (LSC) that resist conventional chemotherapies and remain unaffected in their niche, continually replenishing circulating blast cells. We postulated that an avidity-engineered CD3 engaging DARPin ® (Designed Ankyrin Repeat Protein) able to simultaneously target LSC-specific CD70 as well as CD123 and CD33 could allow highly efficient and specific T cell-mediated killing of AML LSCs and circulating blast cells while preserving a therapeutic window towards healthy cells. Moreover, this simultaneous targeting of three different tumor associated antigens (TAAs) has the potential to address tumor heterogeneity, allowing targeting of AML cells with different co-expression patterns and/or expression levels of each single TAA. To achieve this ambitious goal we used our DARPin ® platform to build a novel class of triple targeting CD3 engaging molecules.

Abstract Purpose: The medical need due to high mortality in acute myeloid leukemia (AML) remains high, and the treatment of relapsed or refractory AML continues to be therapeutically challenging. MYLOTARG, the only approved anti-CD33 antibody drug conjugate (ADC), has provided proof-of-concept for targeted immunotherapies in AML. Currently, a plethora of ADCs and T-cell engager (TCE) therapies have entered clinical development in AML, but those therapies are often accompanied by dose limiting toxicities, preventing dose escalation to desired anti-tumor efficacy. The biggest challenges seem to be limited target specificity and hyperstimulation of the immune system leading to e.g. myelotoxicities and cytokine release syndrome, respectively. Therefore, more selective therapies are needed to allow for robust anti-tumor activity with a more acceptable safety profile. Experimental design: To address the selectivity challenge, we have generated multi-specific T-cell engaging DARPin® molecules, targeting two different tumor associated antigens (TAAs) with optimized affinity for their targets. In order to find the right target combination, the optimal affinity to increase tumor specificity via avidity, as well as the best molecular architecture, we took advantage of our unique modular DARPin® platform and screened 1000s of combinations of multi-specific DARPin® molecules, binding simultaneously to multiple TAAs in conjunction with our CD3-binding DARPin® molecule. Results: We constructed multi-specific TCEs targeting two different AML antigens with optimized affinity leading to a substantial avidity gain when both targets are co-expressed on tumor cells. The avidity gain resulted in strongly enhanced in vitro potency as shown by activation of both CD8+ and CD4+ T cells and subsequent killing of AML tumor cells, with bioactivities in the range of established TCE benchmark formats (e.g. BiTE® and DART®). In contrast, in an ex vivo whole blood assay the multi-specific DARPin® constructs induced profoundly less cytokine release as compared to benchmark molecules indicating an improved therapeutic window. Finally, we also demonstrated tumor regression in PMBC humanized mouse models bearing MOLM-13 tumors, using both half-life extended (HLE) and non-HLE lead constructs. In conclusion, we have generated TCEs based on multi-specific DARPin® constructs with high potency, selectivity and ultimately with the potential for an improved therapeutic window for the treatment of AML. Citation Format: Nina Reschke, Thamar Looser, Jennifer Krieg, Matteo Bianchi, Patricia Schildknecht, Nicole Bassler, Yvonne Gruebler, Sebastian Grimm, Laura Jeanbart, Tanja Hospodarsch, Alexandra Neculcea, Daniel Steiner, Bernd Schlereth, Christian Reichen. Novel DARPin multi-specific T-cell engager with an improved therapeutic window to overcome dose limiting toxicities in AML therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 525.

Globally accessible preventive and therapeutic molecules against SARS-CoV-2 are urgently needed. DARPin molecules are an emerging class of novel therapeutics based on naturally occurring repeat proteins (∼15 kDa in size) and can be rapidly produced in bacteria in large quantities. Here, we report the identification of 380 DARPin molecules specifically targeting the SARS-CoV-2 spike protein selected from a naïve library of 10 DARPin molecules. Using extensive biophysical and biochemical characterization, (pseudo)virus neutralization assays and cryo-EM analysis, 11 mono-DARPin molecules targeting either the receptor binding domain (RBD), the S1 N-terminal-domain (NTD) or the S2 domain of the SARS-CoV-2 spike protein were chosen. Based on these 11 mono-DARPin molecules, 31 anti-SARS-CoV-2 multi-DARPin molecules were constructed which can broadly be grouped into 2 types; multi-paratopic RBD-neutralizing DARPin molecules and multi-mode DARPin molecules targeting simultaneously RBD, NTD and the S2 domain. Each of these multi-DARPin molecules acts by binding with 3 DARPin modules to the SARS-CoV-2 spike protein, leading to potent inhibition of SARS-CoV-2 infection down to 1 ng/ml (12 pM) and potentially providing protection against viral escape mutations. Additionally, 2 DARPin modules binding serum albumin, conferring an expected half-life of about 3 weeks in humans, were included in the multi-DARPin molecules. The protective efficacy of one multi-DARPin molecule was studied in a Golden Syrian hamster SARS-CoV-2 infection model, resulting in a significant reduction in viral load and pathogenesis. In conclusion, the multi-DARPin molecules reported here display very high antiviral potency, high-production yield, and a long systemic half-life, and thereby have the potential for single-dose use for prevention and treatment of COVID-19.

e14626 Background: Urelumab (BMS-663513) is a humanized monoclonal antibody binding to CD137 which, upon Fc-clustering, leads to activation of T-cells. Urelumab is currently in Phase 2 clinical development and has been reported to cause significant hepatotoxicities (around 15% Grade ≥2 ALT and AST elevation) when given as infusion every 3 weeks at doses ≥0.3 mg/kg. Currently ongoing clinical trials report decreased systemic toxicity but limited efficacy at lower doses of urelumab. We hypothesized that more effective triggering of CD137 without associated systemic toxicity may be achieved by targeting a CD137 agonistic engager without Fc to fibroblast activation protein (FAP) which is abundantly expressed in the stroma of many solid tumors. To achieve this, a targeted molecule belonging to the DARPin family of binding proteins was composed of one FAP- and two CD137-binding domains in a “beads on a string” format and tested in a mouse model with human PBMCs. Methods: Human PBMCs were used to reconstitute the immune system in NOG mice implanted subcutaneously with HT-29 human colon cancer cells. Mice were monitored for survival, body weight, and tumor size during the treatment phase of two weeks. Results: None of the mice in the control group died and no significant body weight loss was observed. Six of ten (60%) mice in the CD137 antibody group showed strong signs of graft vs. host disease and either died or reached the termination criterion of ≥20% body weight loss and were sacrificed. One of 30 (3%) mice died in the DARPin drug candidate groups but none of the animals showed body weight loss of ≥20% (p < 0.001, Log-rank test). Tumor growth inhibition was comparable for all treatment groups (around 20-30% at Day 18, p < 0.05 vs. control, Mann Whitney Test). Conclusions: This study confirms the hypothesis that systemic toxicities caused by the urelumab mode of action can be circumvented by FAP-targeting of a CD137 agonistic DARPin drug candidate while achieving comparable tumor growth inhibition. Consequently, higher clinical doses of tumor stroma-targeted agonistic DARPin drug candidates might be possible, and may result in stronger tumor growth inhibition.

A long systemic half-life is key for therapeutic proteins. To that end we have generated serum albumin-binding designed ankyrin repeat domains. These domains bind serum albumin of different species with nanomolar affinities, and have significantly improved pharmacokinetic properties both in mouse and cynomolgus monkey compared to non-serum albumin-binding DARPin® domains. In addition, they exhibit high thermal stability and long storage stability, which is an essential feature for their use in drug development. Covalently linking a serum albumin-binding DARPin® domain to domains with other target specificities results in improvements of multiple orders of magnitude in exposure and terminal half-life, both in mouse and cynomolgus monkey. Pharmacokinetic assessment of such constructs revealed terminal half-life values ranging from 27 h to 80 h in mouse, and from 2.6 days to 20 days in cynomolgus monkey. Extrapolation by allometric scaling on these findings suggests terminal half-life values of 5-50 days in human, indicating that pharmacokinetic properties in the range of monoclonal antibodies can be achieved with DARPin® drug candidates. Such serum albumin-binding DARPin® domains are thus valuable tools for the generation of multi-functional drugs with an extended in vivo half-life.

Introduction: Vascular inflammation is the underlying condition of several cardiovascular diseases and is mainly mediated by activated leukocytes. The leukocyte integrin αMβ2 with its activation specific epitope (I domain) is strongly involved in leukocyte adhesion to endothelial cells and thus represents an interesting therapeutic target. Designed Ankyrin Repeat Proteins (DARPins) are a novel class of linear, thermostable, highly specific recombinant binding proteins that overcome several limitations of immunoglobulins. Hypothesis: DARPins selected against the mouse I domain (mId) of αMβ2 bind specifically to activated leukocytes and can be used as a novel diagnostic tool as well as a therapeutic, anti-inflammatory agent. Methods: Using phage display, binding proteins were selected against recombinant I domain. Specific binding behavior to only activated leukocytes was assessed in FACS. Docking studies were used to define specific interaction sites of selected DARPins with the I domain. Therapeutic, anti-inflammatory effects of anti-mId DARPins was assessed in a sepsis mouse model. Results: DARPins selected against the I domain bind in FACS specifically to activated monocytes (activated vs. non-activated 61±4 % vs. 19±6 %, p<0.05). Docking studies revealed amino acid positions responsible for the specific binding behavior. Mutagenesis of these residues showed significantly reduced binding of the mutated DARPin using FACS analysis (anti-mId DARPin vs. mutated anti-mId DARPin 61±4 vs. 29±7, p<0.05) proving that binding of the wild type DARPin to its target is specific. Furthermore, anti- I domain DARPins showed anti-inflammatory effects in a mouse sepsis model (peritoneal cells: anti-mId DARPin vs control: 2,049±189 103/ml vs. 3,382±213 103/ml, p<0.01). Conclusions: DARPins selected against the I domain of αMβ2 bind specifically to activated leukocytes and inhibit leukocyte function as a new class of anti-inflammatory agents under in vivo conditions.