The balance between affinity and specificity in T cell receptor (TCR)-dependent targeting of HLA-restricted tumor-associated antigens presents a significant challenge for immunotherapy development. T cell engagers that circumvent these limitations are therefore of particular interest. We established a process to generate bispecific designed ankyrin repeat proteins (DARPins) that simultaneously target HLA-I/peptide complexes and CD3e. These high-affinity T cell engagers elicited CD8+ T cell activation against tumor targets with strong peptide specificity, as confirmed by X-scanning mutagenesis and functional killing assays. A cryo-EM structure of the ternary DARPin/HLA-A∗0201/NY-ESO1157-165 complex revealed a rigid, concave DARPin surface spanning the full length of the peptide-binding cleft, contacting both α-helices and the peptide. The present findings reveal promising immuno-oncotherapeutic approaches and demonstrate the feasibility of rapidly developing DARPins with high affinity and specificity for HLA/peptide targets, which can be readily combined with a new generation of anti-CD3e-specific DARPins.

Clinical studies have proven antiviral effectiveness of treatment with a Designed Ankyrin Repeat Protein (DARPin) specific against the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). More information on transport mechanisms and efficiency to the site of action is desirable. Transepithelial migration through air–liquid interface (ALI) cultures of reconstituted human bronchial epithelia (HBE) was assessed by Enzyme-Linked Immunosorbent Assays and Confocal Laser Scanning Microscopy for different DARPin designs in comparison to a monoclonal antibody. Antiviral efficacy against authentic SARS-CoV-2, applied apically on HBE, was investigated based on viral titers and genome equivalents, after administration of therapeutic candidates on the basal side. Transepithelial translocation of all DARPin candidates and the monoclonal antibody was efficient and dose dependent. Small DARPins and the antibody migrated more efficiently than larger molecules, indicating different transport mechanisms involved. Microscopic analyses support this, demonstrating passive paracellular transport of smaller DARPins and transcellular migration of the larger molecules. All therapeutic candidates applied to the basal side of HBE conferred effective protection against SARS-CoV-2 infection. In summary, we have shown that DARPins specific against SARS-CoV-2 translocate across intact airway epithelia and confer effective protection against infection and viral replication.

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.

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.

Abstract Development of biologics or cell therapeutics which target MHC class I:peptide (pMHC) complexes for recognition and elimination of tumor cells is hindered by low affinity, cross-reactivity or challenging biochemical properties of antibody- and T-cell receptor-based binders. We hypothesized that DARPin® proteins may be particularly effective in solving this problem due to structural characteristics of their antigen binding surface and excellent biophysical properties. Panels of binders highly specific for a given pMHC complex were isolated from DARPin® libraries through several rounds of selection and counter selection on the relevant or irrelevant but structurally similar pMHC complexes using ribosome display. DARPin® binders were successfully isolated against pMHC complexes composed of different MHC class I alleles with various peptides derived from either tumor associated antigens or non-self viral proteins. A selected panel of DARPin® binders specific to HLA-A2 molecule in association with SLLMWITQC (SLL peptide), a peptide derived from NY-ESO-1, was used to create bi-specific T-cell engagers containing another moiety binding to the epsilon component of the CD3 complex, thus allowing highly sensitive analysis of pMHC specificity and potential cross-reactivity. Using a number of cellular assays, including peptide pulsing of TAP-deficient T2 cells, we confirmed high specificity of selected DARPin® proteins to the HLA-A2:SLL complex. This was manifested as effective activation of T-cells in the presence of relevant DARPin® constructs tested at a range of concentrations and HLA A2 positive cells pulsed with the SLL or irrelevant HLA A2 binding peptides. Furthermore, HLA-A2+/NY-ESO-1+ cells but not HLA-A2+/NY-ESO-1- cells were effectively killed in the presence of HLA-A2:SLL-specific T-cell engagers. Alanine scanning mutagenesis demonstrated that, in many cases, interactions with several peptide residues located across the entire peptide sequence are critical for DARPin® protein binding to the pMHC complex. These data suggest that peptide residues exposed outside of the MHC peptide binding grove create the focal point of MHC:peptide:DARPin interactions. Further molecular and cellular analysis of DARPin® protein specificity using additional molecular and cellular approaches will be presented in order to de-risk for potential clinically relevant toxicity which may result from off-target interactions with other pMHC complexes structurally resembling the tumor-associated pMHC complex. In conclusion, we show that the DARPin® technology platform may be highly instrumental in developing a new class of anti-cancer therapeutics based on specific targeting of pMHC complexes presented selectively by cancer cells. Citation Format: Marcel Walcer, Natalia Venetz, Tim Schulte, Stefanie Fischer, Nicole Bassler, Maria Paladino, Nicole Pina, Denis Villemagne, Sandra Bruckmaier, Andreas Cornelius, Tanja Hospodarsch, Loic Duffet, Tatjana Sandalova, Adnane Achour, Victor Levitsky. Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes [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 690.

Background The VEGF/VEGFR and the HGF/cMET pathways are key mediators of the interplay of tumor cells and their microenvironment. However, inhibition of VEGF has been shown to produce only limited clinical benefit and inhibition of the activation of cMET by HGF has not translated into clinical benefit in pivotal trials. MP0250, a DARPin® molecule that specifically inhibits both VEGF and HGF has been developed to explore the clinical potential of dual inhibition of these pathways. Results MP0250 binding to VEGF and HGF inhibited downstream signalling through VEGFR2 and cMET resulting in inhibition of proliferation of VEGF- and HGF-dependent cells. Antitumor activity was demonstrated in VEGF- and HGF-dependent xenograft and syngeneic models with activity superior to that of individual VEGF- and HGF-blocking DARPin® molecules. Combination therapy studies showed potentiation of the antitumor activity of chemotherapy and immunotherapy agents, including an anti-PD1 antibody. Materials and Methods Potency of MP0250 was assessed in cellular models and in a variety of xenograft models as monotherapy or in combination with standard-of-care drugs. Conclusions Dual inhibition of VEGF and HGF by MP0250 produced powerful single agent and combination antitumor activity. This, together with increasing understanding of the role of the HGF/cMET pathway in resistance to VEGF (and other agents), supports testing of MP0250 in the clinic.

MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin (R) domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin (R) drug candidate, little is known about DARPin (R) drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin (R) domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of similar to 30 hours in mouse and similar to 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin (R) proteins can be valuable next-generation drug candidates.

e19574 Background: In multiple myeloma, tumor cells and stroma exert a reciprocal effect on each other that leads to tumor expansion in the bone marrow and bone destruction. Hepatocyte growth factor (HGF) is a main player in osteolytic bone destruction and also stimulates myeloma cell proliferation, migration and adhesion in the bone marrow. Additionally, VEGF plays a key neovascularization role in multiple myeloma. Therefore, we investigated whether MP0250, a bispecific DARPin simultaneously inhibiting tumor stroma HGF and VEGF, could potentiate the anti-tumor effect of the standard of care proteasome inhibitor bortezomib. Methods: MP0250 was tested as monotherapy and in combination with 0.6 mg/kg of bortezomib in an orthotopic murine model, where human multiple myeloma cells are implanted in the murine bone marrow. Tumor growth and invasion into the muscle were monitored by X-ray, whereas bone destruction was analyzed by microCT. Results: MP0250 significantly inhibited bone lysis and tumor invasion both...

11039 Background: The interplay of tumor cells and their microenvironment is crucial in the growth of solid tumors. Key mediators of such interaction are the VEGF/VEGFR and the HGF/cMet pathways, which drive tumor survival, growth, angiogenesis, invasion and metastasis. It is anticipated that simultaneous inhibition of the VEGF/VEGFR and HGF/cMet pathways will interfere with crucial steps of tumor growth as well as with the onset of treatment resistance. MP0250 is a bispecific DARPin targeting both pathways simultaneously by specifically neutralizing VEGF and HGF. Its low toxicity offers the potential for combination with chemotherapy and other targeted therapies. Methods: Antitumor activity of MP0250 was tested in kidney, liver, lung and gastric patient-derived xenograft (PDX) models and compared to standard of care (SoC) therapies. Results: MP0250 showed consistent antitumor activity in 6 out of 7 investigated models. Its potency was superior to SoC in renal and gastric carcinoma models, and similar to SoC in liver and one lung cancer (NSCLC) model. Of note, neither MP0250 nor SoC (5-FU) showed potency in one SCLC lung cancer model. Tumor regression was induced by MP0250 in one liver and one renal model with optimal T/C values of 6.9% and 8.9%. Tumor growth inhibition was recorded in the other liver and one lung model (T/C values of 37% and 26%) whereas moderate efficacy was observed in the second renal cancer model (T/C value of 56%). Interestingly, MP0250 increased the potency of the SoC drug paclitaxel in the gastric cancer model (T/C values: MP0250 37%; paclitaxel 46%, MP0250/paclitaxel 21%). No toxicity of MP0250 was observed as monotherapy or in combination with SoC. Conclusions: MP0250, a bispecific DARPin blocking HGF and VEGF, is a potent inhibitor of tumor growth in PDX models, often surpassing the efficacy of SoC therapy. In a gastric cancer model, MP0250 increased potency of SoC. These results indicate that MP0250 has the potential to be used as monotherapy in a range of solid tumors and, as a result of its good safety profile, the possibility exists that it may be also combined with SoC and other tumor targeting molecules, i.e. TKIs. A phase I clinical trial is in preparation.

DARPinsTM (Designed Ankyrin Repeat Proteins) are a novel class of proteins that combine the affinity and specificity properties of antibodies with the solubility and tissue penetration properties of small molecules. The excellent biophysical properties of DARPin domains allows for simple engineering of multispecificity by linking domains that target different ligands. DARPin134 is a bispecific DARPin that inhibits binding of murine IL-13 and IL-4 to their receptors. The bispecific molecule (DARPin134) or either of the mono-specific molecules was delivered to the lungs of mice via intra-tracheal instillation during an acute ovalbumin sensitization and challenge model. Free IL-4 and IL-13 levels were reduced in the BAL following treatment with DARPin134. While the anti-IL-4 and anti-IL-13 DARPins each showed a trend towards reducing eosinophils in the lungs of mice, the bi-specific DARPin significantly reduced eosinophil infiltration. In addition, an improvement airway hyper-responsiveness in mice treated with DARPin134 was observed. A variety of studies have confirmed the unique and overlapping roles for IL-13 and IL-4 in the pathology of asthma. Molecules inhibiting IL-4 alone have failed in clinical trials of asthma and a variety of clinical studies with molecules targeting IL-13 are ongoing. In contrast, DARPin134 targets both IL-4 and IL-13 and in vitro data demonstrate the bispecific DARPin has high potency, affinity and excellent biophysical properties. Blockade of both IL-4 and IL-13 in the pulmonary compartment could provide a therapeutic option in pulmonary diseases without disruption of either cytokine systemically.