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DARPin

Designed Ankyrin Repeat Proteins (DARPins) are small, engineered single-domain binding proteins, typically 14–21 kDa in size, derived from the natural ankyrin repeat scaffold found in eukaryotic proteins.^[1] These proteins consist of 2–3 modular internal repeats of approximately 33 amino acids each, flanked by N- and C-terminal capping repeats that ensure structural stability, forming a rigid, non-immunoglobulin-based α-helical bundle with a concave binding surface for target recognition.^[2] DARPins are generated through combinatorial library design, where surface-exposed residues in the repeats are randomized to select variants with picomolar affinities for specific targets via ribosomal display or phage display, making them versatile tools for protein engineering.^[3] Developed in the early 2000s at the University of Zurich, DARPins represent a class of antibody mimetics inspired by the modular nature of repeat proteins, with foundational work published in 2003 demonstrating their high-affinity binding capabilities.^[2] Unlike traditional antibodies, which rely on disulfide bonds and complex folding, DARPins lack cysteines, exhibit exceptional thermodynamic stability (melting temperatures often exceeding 80°C), and resist aggregation even under harsh conditions, enabling straightforward high-yield production in Escherichia coli at levels up to 30% of total cellular protein.^[3] Mutations such as Asp17Leu in the N-cap further enhance thermostability by up to 16°C, reducing flexibility and improving inter-repeat interactions, which is particularly valuable for therapeutic formulations.^[3] DARPins offer significant advantages over antibodies, including superior solubility, tissue penetration due to their compact size, and the ability to format into multispecific constructs (e.g., ensovibep with five DARPin domains) without compromising function, facilitating applications in diagnostics, imaging, and intracellular targeting.^[1] In research, they serve as crystallization chaperones, biosensors, and tools for studying protein interactions, while in medicine, they have advanced to clinical trials for conditions like age-related macular degeneration (abicipar pegol) and cancer, with ensovibep demonstrating potent neutralization of SARS-CoV-2 by blocking viral entry.^[3] Their half-life can be extended via fusion to albumin-binding modules, reaching weeks in vivo, underscoring their potential as next-generation therapeutics.^[1]

Origins and Engineering

Historical Development

The development of Designed Ankyrin Repeat Proteins (DARPins) originated in the late 1990s at the University of Zurich, led by Andreas Plückthun and his team, as an extension of their pioneering work on recombinant antibody fragments and in vitro protein evolution techniques. Motivated by the challenges of producing stable, soluble antibody-based binders—such as aggregation and poor expression in non-eukaryotic systems—the researchers sought alternative protein scaffolds that could offer modular architectures for high-affinity interactions without the liabilities of disulfide bonds or glycosylation requirements. They identified ankyrin repeat proteins, a ubiquitous class of natural mediators of protein-protein interactions found across all domains of life, as ideal candidates due to their inherent stability and repetitive beta-hairpin-helix motifs that facilitate target recognition.^[4]^[5] Building on sequence alignments from databases like SMART and PFAM, which encompassed thousands of natural ankyrin repeats, the team derived consensus sequences to engineer robust scaffolds. These consensus designs minimized variability while preserving structural integrity, allowing the creation of artificial repeat modules with randomized paratopes for binding diversification. The first DARPin prototypes, consisting of N- and C-terminal capping repeats flanking two or three internal consensus modules, were expressed in E. coli around 2000–2002, demonstrating exceptional solubility (up to 200 mg/L yield), monomeric behavior, and thermal stability (melting temperatures exceeding 66°C). This foundational work was detailed in a 2003 publication, marking the initial validation of these scaffolds as viable alternatives to antibodies.00896-9)^[6] Key milestones followed rapidly in the early 2000s, including the adaptation of Plückthun's earlier invention of ribosome display—a cell-free selection method developed in 1997 for evolving antibody fragments—to DARPin libraries, enabling the isolation of binders with sub-nanomolar affinities. The core DARPin framework, showcasing high-affinity selections against diverse targets like maltose-binding protein, was published in 2004, establishing the technology's potential for research, diagnostics, and therapeutics. That same year, Plückthun and colleagues founded Molecular Partners AG in Zurich to commercialize DARPins, securing an exclusive license from the University of Zurich and accelerating their translation from academic prototypes to clinical candidates.^[5]^[7]

Methods of Generation

DARPin libraries are constructed by diversifying the internal repeat modules of the ankyrin repeat scaffold through randomized amino acid substitutions at key target-binding residues, typically positions in the convex binding surface such as 2, 3, 5, 13, and 14.^[8] This randomization employs degenerate oligonucleotides or trinucleotide phosphoramidites to introduce controlled variability, avoiding stop codons, cysteines, and prolines while favoring polar and charged residues to enhance solubility and reduce hydrophobicity.^[9] The resulting libraries incorporate three internal repeats flanked by N- and C-terminal capping modules, assembled via overlap extension PCR and type IIS restriction enzymes, yielding diversities exceeding 10¹² unique variants.^[10] Selection of target-specific DARPins primarily relies on ribosome display, an in vitro evolution technique that physically links the protein (phenotype) to its mRNA (genotype) in ribosomal complexes, enabling iterative rounds of binding enrichment against immobilized or soluble targets.^[11] This process involves 3-5 rounds of selection, including pre-panning to remove non-specific binders and counter-selection for specificity, often supplemented by phage display for in vivo validation and further diversification using signal recognition particle targeting.^[12] Ribosome display's key advantage lies in its capacity to handle vast library sizes without transformation limits, facilitating the isolation of binders with nanomolar affinities from initial pools.^[8] Sequence optimization integrates computational design to preserve the scaffold's structural integrity during randomization, ensuring the helical repeats maintain their modular, non-covalent assembly.^[8] This involves deriving consensus sequences from alignments of natural ankyrin repeats across diverse proteins, identifying invariant framework residues (e.g., leucines at inter-repeat interfaces) while permitting variation only on binding surfaces.^[13] Computational tools model these changes to minimize entropy on non-binding regions and stabilize capping modules, such as optimizing the C-cap to prevent aggregation.^[14] For scale-up and refinement, high-throughput screening follows initial selections, employing yeast or bacterial expression for parallel affinity maturation via error-prone PCR or site-directed mutagenesis on promising candidates.^[12] Iterative ribosome display rounds on these variants typically achieve picomolar binding affinities (e.g., 30-90 pM) within 2-3 cycles, as demonstrated for targets like HER2 and BCL-2, by enriching aromatic residues like tyrosine and tryptophan at paratope hotspots.^[15]^[16]

Structural Features

Protein Architecture

DARPins are engineered proteins derived from the ankyrin repeat scaffold, consisting of multiple consensus ankyrin repeat modules that assemble into a right-handed solenoid structure with a continuous hydrophobic core and a large concave surface.^[14] Each internal repeat module comprises approximately 33 amino acids, folding into a β-hairpin motif followed by two short antiparallel α-helices connected by a flexible loop, which stacks with adjacent repeats to form an elongated helical solenoid approximately 2 nm in diameter.^[8] The typical DARPin incorporates 3 to 5 such internal repeats, providing a rigid, non-covalent framework that maintains structural integrity without relying on disulfide bonds.^[1] To protect the hydrophobic core at the termini and prevent aggregation or proteolytic degradation, DARPins are flanked by specialized capping domains. The N-terminal cap (N-cap) consists of about 40 residues forming a short α-helix that interacts with the first repeat, presenting a hydrophilic surface to solvent.^[6] Similarly, the C-terminal cap (C-cap) comprises roughly 50 residues in an extended helical conformation, shielding the C-terminal end of the solenoid and further stabilizing the overall fold through hydrophobic interactions.^[14] These caps, derived from natural ankyrin proteins and optimized via consensus design, ensure the scaffold's solubility and resistance to unfolding in diverse environments.^[8] Standard DARPins adopt an N3C configuration with three internal repeats, yielding a total length of approximately 180 residues and a molecular weight of 18–20 kDa, suitable for a wide range of applications due to its balanced size and stability.^[1] A compact variant, N2C, features only two internal repeats for a total of about 130–140 residues and 14 kDa, facilitating rapid renal clearance in vivo while retaining the core architectural principles.^[6] The DARPin scaffold inherently lacks cysteine residues for disulfides and asparagine-based glycosylation sites, enabling straightforward high-yield expression in prokaryotic systems without the need for eukaryotic modifications.^[1]

Design and Optimization

DARPins are engineered through modular assembly, where multiple binding domains are fused in tandem using short, flexible linkers to create bispecific or multispecific constructs typically comprising 2-4 domains. This approach leverages the compact, non-immunoglobulin scaffold to enable rigid or semi-rigid linkages that maintain structural integrity and binding functionality without steric hindrance. For instance, bispecific DARPins targeting HER2 and VEGF have been constructed by fusing distinct modules, allowing simultaneous engagement of multiple targets for enhanced therapeutic efficacy.^[17]^[18] Surface engineering of the DARPin scaffold involves the strategic introduction of conjugation tags or extension modules to tailor pharmacokinetics and enable payload attachment. Sortase-mediated sites, such as the LPXTG motif, are incorporated at the C-terminus to facilitate site-specific ligation of cytotoxic drugs or imaging agents, ensuring homogeneous conjugates with preserved binding affinity. Additionally, fusion with albumin-binding modules, often derived from streptococcal protein G, extends serum half-life by non-covalent association with serum albumin, as demonstrated in HER2-targeting DARPins where such fusions reduced renal clearance and improved tumor accumulation.^[19]^[20]^[21] Optimization of DARPin constructs relies on directed evolution techniques, such as ribosome display, to refine specificity and affinity in multi-domain formats by iteratively selecting variants with minimal cross-reactivity. Computational modeling further aids design by predicting inter-domain interactions and overall stability in tandem assemblies, using standardized protocols to simulate target complexes and guide linker length selection. These strategies ensure that the core ankyrin repeat modules—consisting of consensus repeats flanked by cap domains—retain their helical bundle architecture during modifications.^[22]^[23]^[24] A notable example is the design of biparatopic DARPins, which incorporate two identical modules targeting the same epitope to boost avidity through cooperative binding, as seen in constructs against tumor antigens where this format increased apparent affinity by orders of magnitude compared to monovalent versions. Such optimizations have been pivotal in developing high-potency multispecific agents while avoiding aggregation in multi-domain configurations.^[25]

Biophysical Characteristics

Binding Affinity and Specificity

DARPins recognize target proteins through their characteristic concave binding surface, formed by the β-turns and α-helices of the ankyrin repeats, which complements convex epitopes on the target. This interface primarily involves non-covalent interactions such as hydrogen bonds and van der Waals forces, enabling precise molecular recognition without the need for disulfide bridges.^[26]^[27] The paratope typically engages 15-25 residues from the DARPin, contributing to a compact interaction area that supports high specificity in diverse protein targets.^[28] The binding affinity of DARPins is engineered to reach picomolar dissociation constants (K_d) in the range of 1-100 pM for many targets, achieved through selection methods like ribosome display that optimize both association and dissociation kinetics.^[29] High specificity is further enhanced by slow off-rates, often below 10^{-5} s^{-1}, which minimize dissociation and reduce non-specific interactions in complex biological environments.^[30] Specificity is refined during engineering via orthogonal selection strategies, such as negative selection in ribosome display to eliminate cross-reactive binders, ensuring targeted recognition without off-target effects. For instance, DARPins selected against green fluorescent protein (GFP) demonstrate over 1,000-fold selectivity compared to related fluorescent proteins like mCherry or unrelated cellular proteins.^[31]^[32] In multispecific DARPin constructs, avidity effects arise from cooperative binding of multiple domains to the same or adjacent epitopes, amplifying the effective affinity by 10- to 100-fold compared to monovalent binders. This is particularly evident in constructs targeting multimeric targets like viral spike proteins, where linked DARPins achieve functional affinities in the femtomolar range through enhanced local concentration and reduced off-rates.^[33]^[34]

Stability and Production

DARPins exhibit exceptional thermal stability, with unfolding free energies exceeding 9.5 kcal/mol and melting temperatures ranging from 70°C to 90°C, enabling them to maintain structural integrity under harsh conditions.^[27] This robustness extends to resistance against proteolytic degradation and stability across a wide pH range, attributes that stem from their engineered ankyrin repeat scaffold lacking cysteines, which prevents unwanted disulfide bond formation.^[27]^[33] Their high solubility, often surpassing 100 mg/mL in aqueous buffers, further underscores their practical utility, as the capped core design minimizes aggregation propensity even at elevated concentrations.^[27] This intrinsic solubility facilitates downstream processing without the need for complex stabilization strategies. Production of DARPins is highly efficient, primarily through cytoplasmic expression in Escherichia coli, achieving yields greater than 10 g/L via optimized fermentation processes.^[27] If expressed as inclusion bodies, refolding protocols can recover functional protein effectively, followed by straightforward purification using affinity tags such as His-tags.^[27] The scalability of DARPin manufacturing benefits from bacterial fermentation systems, which circumvent the complexities of mammalian cell culture, including the absence of required post-translational modifications.^[27] This approach supports large-scale production suitable for therapeutic and diagnostic applications while maintaining cost-effectiveness and consistency.

Therapeutic Advantages

Comparisons to Antibodies

DARPins exhibit significant advantages over conventional antibodies in terms of molecular size and tissue accessibility. With a molecular weight of 14-18 kDa for typical three-repeat modules, DARPins are substantially smaller than the 150 kDa immunoglobulin G (IgG) antibodies, facilitating superior penetration into dense tissues such as solid tumors and the eye. This reduced size allows DARPins to diffuse more effectively through extracellular matrices and vascular barriers that hinder larger antibody molecules.^[35]^[7] The pharmacokinetic profile of DARPins further distinguishes them from antibodies, primarily due to their compact structure. Unmodified DARPins undergo rapid renal clearance, resulting in a plasma half-life of approximately 30 minutes, compared to the extended 21-day half-life of IgG antibodies, which is sustained by FcRn-mediated recycling. This swift clearance can be beneficial for applications requiring minimal systemic exposure, such as diagnostic imaging, though half-life extension strategies are often employed for therapeutic uses.^[7]^[36] In terms of immunogenicity, DARPins present a lower risk profile than many antibody formats, stemming from their engineering based on consensus sequences derived from naturally occurring human ankyrin repeat proteins, which are abundant in human cells and less likely to provoke immune responses. Preclinical models have demonstrated reduced formation of anti-drug antibodies with DARPins relative to non-humanized antibodies, enhancing their tolerability in repeated dosing scenarios.^[37]^[38] DARPins also surpass antibodies in development efficiency and manufacturing economics. High-affinity DARPins can be selected and optimized in 6-12 months via in vitro techniques like ribosome display, a process far quicker than the 2-3 years typically required for monoclonal antibody discovery, humanization, and validation. Production occurs in Escherichia coli systems, yielding gram-per-liter quantities at substantially lower costs than those for mammalian-cell-derived antibodies, owing to simpler fermentation and purification without glycosylation needs.^[35]^[39] The inherent modularity of DARPins enables straightforward construction of bispecific or multispecific agents by fusing independent binding domains, bypassing the intricate Fc-domain modifications and potential mispairing challenges encountered in bispecific antibody engineering. Additionally, bacterial expression eliminates glycan heterogeneity, a common source of variability and immunogenicity in antibody production that can compromise batch consistency and efficacy.^[7]^[40]

Pharmacokinetic and Formulation Enhancements

DARPins, typically ranging from 14 to 18 kDa in size, exhibit rapid renal clearance with plasma half-lives on the order of minutes to hours, necessitating enhancements to achieve therapeutic dosing intervals suitable for clinical use.^[41] One primary strategy involves fusion to albumin-binding DARPin domains, which leverage the long circulatory half-life of serum albumin (approximately 19 days in humans) through non-covalent association, thereby extending the terminal half-life of the fused construct.^[42] For instance, terminal half-lives of albumin-fused DARPins range from 27 to 80 hours in mice and 2.6 to 20 days in cynomolgus monkeys, with extrapolated human values of 5 to 50 days depending on the number and affinity of binding modules.^[42] Multispecific DARPins incorporating albumin-binding domains, such as those in ongoing oncology trials (e.g., MP0317 as of 2025), have demonstrated extended plasma half-lives enabling less frequent dosing.^[43] Alternatively, PEGylation with polymers of 20 to 40 kDa has been employed to prolong circulation; for example, PEGylated DARPins have shown extended half-lives in preclinical ocular models, supporting less frequent intravitreal administration.^[44] To enable targeted delivery of cytotoxic payloads, DARPins are conjugated to toxins or radionuclides using site-specific chemistries that preserve binding integrity and ensure homogeneity.^[45] Engineered cysteines introduced at non-interfering positions, such as the C-terminus, facilitate thiol-maleimide reactions for attaching monomethyl auristatin E (MMAE), achieving drug-to-protein ratios of 3.9 to 4.0 in mesothelin-targeting constructs while maintaining picomolar affinity and enabling efficient tumor cell internalization.^[46] Similarly, bivalent EGFR-targeting DARPin dimers conjugated to MMAE via enzymatic formylglycine generation and strain-promoted azide-alkyne cycloaddition yield homogeneous products with a drug ratio of 2.0, demonstrating potent cytotoxicity in vitro and reduced off-target effects compared to non-specific linkages.^[45] For imaging and therapy, DARPins are radiolabeled with isotopes like ⁹⁹ᵐTc or ⁶⁸Ga through chelator-mediated attachment, allowing rapid tumor visualization within hours due to favorable pharmacokinetics. Recent examples include ¹²³I-labeled DARPins for EpCAM imaging in lung and ovarian cancer trials as of 2025.^[41]^[47] Formulation strategies capitalize on the inherent stability of DARPins to support practical administration routes. Lyophilization enhances long-term storage stability, as demonstrated by HER2-targeting DARPin G3, which retains full binding activity and bioactivity for at least 9 months at room temperature in lyophilized chloroplasts, with yields up to 111 mg/g dry weight.^[48] Their small size and high solubility (enabling concentrations >100 mg/mL) further permit subcutaneous dosing, which benefits from reduced injection volumes and patient convenience, as seen in preclinical models of albumin-fused DARPins administered every three days without aggregation or loss of efficacy.^[44] These attributes allow modular fusion designs to incorporate pharmacokinetic modules without compromising manufacturability.^[7] The biodistribution profile of DARPins is characterized by rapid extravasation into tissues owing to their compact size (<18 kDa), facilitating quick tumor penetration and high contrast imaging as early as 1 to 4 hours post-injection.^[41] Clearance is predominantly renal, with monomeric formats exhibiting the fastest blood elimination (half-life <3 minutes in mice, ~90% cleared within 7.5 minutes), leading to efficient tumor accumulation (~8% injected dose per gram at 24 hours) but also notable kidney retention.^[49] Dimeric formats (around 30 kDa) offer tunable clearance, with slightly prolonged circulation and reduced tumor uptake (<2% ID/g at 24 hours) compared to monomers, allowing optimization of exposure based on therapeutic needs.^[49]

Clinical Applications

Early and Discontinued Candidates

Abicipar pegol (MP0112), a monovalent DARPin targeting vascular endothelial growth factor A (VEGF-A), was developed for the treatment of neovascular age-related macular degeneration (wet AMD) through intravitreal administration.^[50] Initiated in collaboration between Molecular Partners and Allergan around 2011, it advanced to Phase 3 trials (CEDAR and SEQUOIA) starting in 2017, which demonstrated comparable efficacy to ranibizumab with a potential for extended dosing intervals of up to 12 weeks, reducing injection frequency by approximately 50%.^[51] However, the program faced challenges with higher rates of intraocular inflammation (15.4% versus 0.3% for ranibizumab), linked to immunogenicity and formulation issues in repeated dosing.^[52] Following a Complete Response Letter from the FDA in June 2020 citing unacceptable inflammation risks and subsequent withdrawal of the European marketing authorization application in July 2020, Allergan (acquired by AbbVie) terminated development in 2021 and returned global rights to Molecular Partners.^[53]^[54] Ensovibep (MP0420), a trispecific DARPin designed to neutralize SARS-CoV-2 by targeting three distinct epitopes on the spike protein, emerged as a rapid-response candidate during the COVID-19 pandemic.^[55] Developed by Molecular Partners in partnership with Novartis, it showed promising Phase 2 results in January 2022 from the EMPOWDER trial in non-hospitalized patients, achieving a significant reduction in viral load and symptom duration compared to placebo.^[55] Despite this, the Phase 3 EMPATHY trial in hospitalized patients, conducted under the ACTIV-3/TICO platform, failed to meet its primary endpoint of improved clinical status at day 5, with no significant benefit over standard care including remdesivir.^[56] The program was discontinued in 2022 after clinical work concluded without advancing to approval, with Novartis returning rights to Molecular Partners in January 2024.^[57] MP0250, a bispecific DARPin inhibiting both VEGF-A and hepatocyte growth factor (HGF) to disrupt tumor angiogenesis and growth signaling, targeted advanced solid tumors and hematologic malignancies.^[58] Molecular Partners initiated Phase 1 dose-escalation trials in 2014, followed by Phase 1b/2 combination studies with bortezomib and dexamethasone in relapsed/refractory multiple myeloma through 2018, which confirmed a favorable safety profile with manageable proteinuria as the primary dose-limiting toxicity and no maximum tolerated dose reached.^[58]^[59] While early data indicated stable disease in some patients and supported tolerability for multi-specific designs, limited efficacy signals—such as modest response rates and lack of robust tumor regression—led to pausing further development around 2018, with the program ultimately discontinued.^[60] These early DARPin candidates validated the platform's overall tolerability and manufacturing scalability in humans, with low rates of severe adverse events across oncology, ophthalmology, and infectious disease applications.^[7] A key challenge identified was the potential for anti-DARPin antibodies, particularly in repeated intravitreal dosing as seen with abicipar pegol, which triggered inflammation and underscored the need for immunogenicity mitigation strategies in chronic administration settings.^[61] In contrast, systemic candidates like MP0250 exhibited minimal immunogenicity, informing optimizations for half-life extension and multi-specificity without compromising safety.^[58]

Ongoing Trials and Recent Developments

One of the most advanced DARPin-based candidates in clinical development is MP0533, a tetravalent T-cell engager targeting CD33, CD123, CD70, and CD3 for the treatment of relapsed/refractory acute myeloid leukemia (R/R AML) and myelodysplastic syndrome (MDS).^[62] The Phase 1/2a trial, initiated in late 2022, reported encouraging interim results at the European Hematology Association (EHA) Congress in June 2025, with 3 out of 8 evaluable R/R AML patients achieving responses at doses of 100 µg/kg and above, demonstrating avidity-driven T-cell activation and leukemia cell killing.^[62] Updated data to be presented at the American Society of Hematology (ASH) Annual Meeting in December 2025 are expected to further support the candidate's tolerability, with cytokine release syndrome (CRS) events being mostly low-grade and manageable based on preliminary ongoing trial data as of November 2025.^[63] In solid tumor oncology, MP0317, a bispecific DARPin agonist targeting fibroblast activation protein (FAP) and CD40, remains in Phase 1 evaluation for advanced solid tumors.^[43] The ongoing trial, dosed up to 100 mg weekly or every three weeks across 46 patients as of mid-2024, has shown a favorable safety profile with no dose-limiting toxicities related to systemic CD40 activation, alongside evidence of tumor-localized immune activation via biomarkers like increased CD40-expressing cells in the tumor microenvironment.^[64] Preclinical-to-clinical transition programs include logic-gated CD3 Switch-DARPins, which demonstrated selective T-cell engagement against dual tumor antigen-expressing cells at the Society for Immunotherapy of Cancer (SITC) meeting in November 2025, with plans for Phase 1 initiation in 2026 targeting antigens such as HER2 in combination with CD3.^[65] Beyond oncology, DARPin development in virology builds on the ensovibep (MP0420) experience in COVID-19, where over 500 patients were treated across Phase 2/3 trials showing rapid viral clearance and low immunogenicity.^[66] While no new influenza-specific IND filings were reported in 2025, the platform's modular design supports rapid adaptation for emerging antivirals, with ongoing preclinical efforts leveraging multi-specific binding for respiratory pathogens.^[66] Key 2025 milestones include a systematic review published in May analyzing a decade of DARPin clinical data from over 750 systemically treated patients, confirming a well-tolerated profile with adverse events primarily mechanism- or target-specific (e.g., mild CRS in T-cell engagers) and no scaffold-related toxicities.^[66] In October, Molecular Partners filed an IND for MP0712, a DLL3-targeting radio-DARPin conjugate in partnership with Orano Med, with Phase 1 initiation anticipated by year-end for small cell lung cancer; on November 12, 2025, initial human images from the imaging and dosimetry step using 203Pb-labeled MP0712 were presented at the TRP Summit Europe, with full clinical data expected in 2026.^[67]^[68] Similarly, mesothelin-targeted radio-DARPin MP0726 advanced with preclinical data presented at the Society of Nuclear Medicine and Molecular Imaging (SNMMI) meeting in June, highlighting favorable tumor uptake and biodistribution.^[69] To date, six systemic DARPin therapeutics—MP0250, MP0274, MP0310, MP0317, MP0533, and ensovibep—have entered clinical testing, with recent formats emphasizing half-life extension to minimize immunogenicity risks.^[66] Anti-drug antibodies (ADAs) were detected in 20-75% of patients across programs, but rarely impacted efficacy or safety, underscoring the scaffold's low immunogenic potential derived from its ankyrin repeat structure mimicking natural proteins.^[66]

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Designed Ankyrin Repeat Protein (DARPin) to target chimeric ...
Sep 12, 2020 · There is some evidence that DARPins could be less immunogenic than other binding domains due to their presence in circulating red blood cells, ...
[38]
Thermostable designed ankyrin repeat proteins (DARPins) as ...
Nov 15, 2021 · Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development.
[39]
DARPins: a true alternative to antibodies - PubMed
DARPins are non-immunoglobulin proteins offering advantages over antibodies for target binding, with small size, high stability, and low-cost production.
[40]
Bispecific Designed Ankyrin Repeat Proteins (DARPins) Targeting ...
DARPins, alternative targeting molecules efficiently produced in bacteria, were shown to inhibit A431 cell proliferation and receptor recycling.
[41]
On the prevention of kidney uptake of radiolabeled DARPins - PMC
Feb 4, 2020 · The small size of DARPins enables good extravasation into tissues, fast accumulation in tumors, and rapid clearance from the blood, providing ...
[42]
Half-life extension using serum albumin-binding DARPin® domains
Aug 17, 2017 · Indeed, DARPin® domains binding to serum albumin exhibit clearly improved pharmacokinetic properties (Fig. 3), with domain #1 exhibiting a ...
[43]
Beyond Antibodies: The DARPin® Drug Platform | BioDrugs
Jun 24, 2020 · Pegylation of abicipar increases vitreal half-life, supporting a potential for up to 3 month duration of action in the clinic. In: The ...Beyond Antibodies: The... · 1.2 Darpin Platform · 2.2 Abicipar
[44]
Bivalent EGFR-Targeting DARPin-MMAE Conjugates - PMC
We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a ...
[45]
Mesothelin-directed protein-drug conjugates for mesothelin-low ...
Aug 23, 2025 · A gene fragment encoding four cysteines within a GCGGCGGCGGCG motif was inserted at the 3'-end of M7A or M7GA for site-specific drug conjugation ...
[46]
The lyophilized chloroplasts store synthetic DARPin G3 as bioactive ...
Oct 5, 2023 · In this study, we reported the prolonged stability and functionality of DARPin G3 in lyophilized transplastomic tobacco leaves and chloroplasts.
[47]
(PDF) Efficient Tumor Targeting with High-Affinity Designed Ankyrin Repeat Proteins: Effects of Affinity and Molecular Size
**Summary of Clearance of Monomeric vs Dimeric DARPin Formats**
[48]
Abicipar pegol: the non-monoclonal antibody anti-VEGF - PMC
Sep 30, 2019 · Allergan and Molecular Partners Announce Two Positive Phase 3 Clinical Trials for Abicipar pegol 8 and 12-week Regimens for the Treatment in ...
[49]
Allergan and Molecular Partners Announce Two Positive Phase 3 ...
Two pivotal head-to-head trials demonstrate the efficacy of Abicipar 12-week fixed dosing regimen with 50 percent fewer injections versus ranibizumab.Missing: MP0112 discontinuation
[50]
Allergan, an AbbVie Company, and Molecular Partners Receive ...
Jun 26, 2020 · Allergan, an AbbVie Company, and Molecular Partners Receive Complete Response Letter from FDA on Biologics License Application for Abicipar ...Missing: MP0112 discontinuation
[51]
Withdrawal of application for the marketing authorisation of Rayoqta ...
Jul 24, 2020 · The active substance in Rayoqta, abicipar pegol, attaches to VEGF-A in the eye and stops its activity. This was expected to stop the growth of ...Missing: MP0112 DARPin
[52]
PAbbVie Returns Eye Drug Abicipar Pegol Rights to Molecular ...
Aug 9, 2021 · AbbVie has terminated its license and collaboration deal with Molecular Partners for Abicipar pegol and returned all rights to the Swiss company ...
[53]
Novartis and Molecular Partners report positive topline data from ...
Jan 10, 2022 · Novartis and Molecular Partners report positive topline data from Phase 2 study for ensovibep (MP0420), a DARPin antiviral therapeutic for COVID ...Missing: discontinuation | Show results with:discontinuation
[54]
Efficacy and Safety of Ensovibep for Adults Hospitalized With COVID ...
Aug 9, 2022 · Compared with placebo, ensovibep did not improve clinical outcomes for hospitalized participants with COVID-19 receiving standard care, including remdesivir.
[55]
Partnering - Molecular Partners
Novartis returned the rights to the ensovibep program to Molecular Partners in January 2024. Clinical work on the ensovibep program ended in 2022. The ...Missing: discontinued | Show results with:discontinued
[56]
First-in-Human Phase I Study of MP0250, a First-in-Class DARPin ...
Dec 10, 2020 · DARPin molecules are a class of small, highly specific binding proteins that can be easily assembled to bind multiple molecular targets. MP0250 ...Pks And Adas · Discussion · Authors' Disclosures Of...<|separator|>
[57]
A phase 1b/2 study evaluating efficacy and safety of MP0250, a ...
Aug 1, 2024 · The results of this phase 1b/2 study show that treatment with the DARPin MP0250, a specific inhibitor of both VEGF-A and HGF, in combination ...2 Patients And Methods · 3 Results · 3.4 Antitumour Activity
[58]
MP-0250 - Drug Targets, Indications, Patents - Patsnap Synapse
MP-0250: a HGF inhibitors, VEGF-A inhibitors Drug, Initially developed by Molecular Partners AG, Now, its global highest R&D status is Discontinued, ...Missing: paused | Show results with:paused<|separator|>
[59]
Impact of Modifying Abicipar Manufacturing Process in Patients with ...
May 11, 2023 · Abicipar produced using a modified manufacturing process showed a moderately lower incidence and severity of IOI compared with Phase 3 abicipar studies.
[60]
Molecular Partners presents positive data from ongoing Phase 1/2a ...
Jun 11, 2025 · News Release Details. Molecular Partners presents positive data from ongoing Phase 1/2a trial of MP0533 in AML at EHA 2025.
[61]
NCT05098405 | First-in-human Safety and Tolerability of MP0317 in ...
This study is investigating a new experimental therapy, MP0317, a DARPin® drug candidate targeting fibroblast activation protein (FAP) and CD40.
[62]
Effect of MP0317, a FAP x CD40 DARPin, on safety profile and ...
May 29, 2024 · Effect of MP0317, a FAP x CD40 DARPin, on safety profile and tumor-localized CD40 activation in a phase 1 study in patients with advanced solid ...
[63]
News Release Details - Investors - Molecular Partners
Molecular Partners presents additional preclinical proof-of-concept data on logic-gated CD3 Switch-DARPin at SITC 2025. November 3, 2025.
[64]
[PDF] A Decade of Clinical Experience with DARPins in Oncology and ...
May 22, 2025 · The first DARPin drug candidate developed for systemic administration was MP0250, a multi- specific DARPin designed for dual-inhibition of ...
[65]
Molecular Partners Reports Q3 2025 Financial Results and Clinical ...
Oct 30, 2025 · The Phase 1 Investigational New Drug (IND) application for MP0712, a 212Pb-based Radio-DARPin therapy (RDT) candidate targeting the tumor- ...Missing: conjugate | Show results with:conjugate
[66]
Molecular Partners and Orano Med present preclinical data on ...
Jun 23, 2025 · Molecular Partners and Orano Med present preclinical data on mesothelin-targeting Radio-DARPin candidate MP0726 at SNMMI 2025. June 23, 2025 |.<|separator|>