Ankyrons: There Might Be a “Gold Mine” for Students, Scientists, and Clinicians

Graduation requirements for students often mandate publications, and the promotion of scientists and clinicians in academic radiology hinges significantly on their publication achievements.¹ In light of these challenges, we introduce Ankyrons, a potentially transformative entity poised to become a “gold mine” for publications. If successful, this proposal has the potential to characterize thousands of protein targets, opening avenues for new discoveries in target identification, disease diagnosis, and therapy, particularly in the realm of translational science in molecular imaging. Our analysis positions Ankyrons as an innovative solution addressing the challenges associated with publishing and discovery in translational science within molecular imaging. Ankyrons stand out as a promising avenue for impactful publications, emphasizing strong and selective ligand binding coupled with rapid circulation clearance, facilitating high-contrast in vivo imaging. The introduction of Ankyrons signifies a noteworthy development, poised to make a substantial contribution to scientific literature. It has the potential to advance our understanding and application of cutting-edge technologies in the fields of molecular imaging and therapeutic interventions for various diseases.

What are Ankyrons?

The term “Ankyron” might not be widely recognized, and it only came to our attention a few months ago, sparking immediate interest. Specifically, it piqued our interest as 15 kDa proteins with the remarkable ability to bind to a variety of targets. This discovery resonates with our ongoing work on nanobodies (Nbs) spanning several years, making Ankyrons a particularly enticing prospect for further exploration. Initial speculation arose about the potential overlap between Ankyrons and Nbs. Upon engaging in discussions with sales representatives and delving deeper into the information available on their website, we discovered a crucial distinction—Ankyrons feature repeating ribbon-like structures, setting them apart from the structural characteristics of Nbs. No research papers related to ’Ankyron’ have been located, and the Ankyron company asserts that there have been no publications on the subject. As a result, the exact structure of Ankyrons remains inadequately explored. Nonetheless, our hypothesis posits that Ankyrons derive from Ankyrins, a well-established family of proteins with a history spanning more than 30 years, characterized by ribbon-like structures. The ankyrin repeat is a 33-residue sequence motif, initially identified in 1987,² and characterized by 24 copies of this sequence.³ Hence, we venture to propose that Ankyrons are simplified domains of ankyrin repeats, with a similarity, albeit with only 3– 5 repeating units, as indicated by the molecular weight calculations. As shown in Figure 1, a representative proposed Ankyron ribbon-like structure was created by Chimera, based on the sequence of 1K1A. We’d like to explain why Ankyrons could be a valuable resource for scientists and students, particularly those in the field of molecular imaging.

Figure 1.

Representative proposed Ankyron structure, modeled on the Ankyrin repeats from the 1K1A crystal structure (PDB ID: 1K1A): A repetitive segment (291–323) was extracted from the crystal structure (PDB ID: 1K1A), replicated four times, and presented in Chimera software using the ribbon representation with appropriate coloration.

Why Ankyrons for Molecular Imaging?

The company’s claim that Ankyrons signify the next generation of target-binding reagents, surpassing limitations linked to research antibodies, is grounded in their distinctive features. A pivotal factor driving their effectiveness is their ability to bind to target proteins robustly and selectively. There is no doubt that Ankyrons have the potential to bind to a target, given the success observed with peptides, Nbs, and affibodies—all of which share the commonality of being a polypeptide chain. This shared characteristic underscores the potential of Ankyrons to follow suit and become a powerful force in the domain of target-specific binding reagents. Notably, a characterized PDL1 Ankyron clone has demonstrated a remarkable binding affinity as low as 0.7 pM.⁴ Given their robust binding capabilities, Ankyrons emerge as a promising and potent tool in the field of molecular imaging. With their proven ability to precisely target proteins of interest, Ankyrons hold the potential to revolutionize molecular imaging techniques and further enhance our capabilities in biomedical research.

Another crucial characteristic is that Ankyrons may exhibit favorable in vivo pharmacokinetics, aligning with the prerequisites for molecular imaging. The success of in vivo molecular imaging techniques relies on two crucial factors: the rapid and specific accumulation of tracers at their intended targets, and efficient clearance of the unbound tracer from the circulation.⁵ Given their molecular weight of 15 kDa, similar to small proteins such as Nbs, we speculate that Ankyrons may exhibit comparable in vivo properties. This conjecture is further supported by evidence from PET imaging techniques illustrating the behavior of ankyrin repeats in vivo (Figure 2).⁶ Drawing parallels with ankyrin repeats and Nbs, we anticipate that Ankyrons’ pharmacokinetics should align closely, considering their shared components and size (15 kDa). Consequently, we expect Ankyrons to undergo efficient clearance from the bloodstream, akin to affibodies and Nbs. If Ankyrons meet the criteria for molecular in vivo imaging, we anticipate their potential as ideal imaging contrasts for disease diagnosis and possibly radiation therapy.

Figure 2.

Microsingle photon emission computed tomography/computed tomography imaging of HER2 expression in mice bearing SKOV3 xenografts at 1 h (A), 2 h (B), and 4 h (C). S, stomach; K, kidneys; T, tumor. Reproduced from ref (6). Copyright 2019 Spandidos Publications.

The Third, Yet Arguably the Most Compelling Reason for Now, Is the Affordability and Accessibility of Ankyrons

While Nbs have shown promise as excellent in vivo imaging agents, and some successful imaging studies indicate a prime time for research in this area, progress has not been as explosive as expected. Observing the landscape, it is apparent that not many imaging laboratories are extensively engaged in this domain. At the 2023 Society of Nuclear Medicine and Molecular Imaging meeting, for instance, only four Nb abstracts were presented out of 85 in the targeting molecule section. The primary obstacle appears to be the accessibility of Nbs and the high costs associated with contract research organization (CRO) services.⁵ Despite the availability of in vitro screening platforms, acquiring Nbs for most research laboratories remains challenging. Many imaging laboratories lack the necessary setup for biological screening, limiting the widespread application of Nbs in the field. In our laboratory, with a background in chemistry, we’ve faced challenges in Nb screening. While we possess several Nb libraries, the actual screening process has not commenced due to a shortage of personnel with the right biological background and insufficient funding to hire the necessary scientists. We believe this predicament is common in many imaging laboratories, hindering the broader adoption of Nbs in research. Ankyrons, with their affordability and accessibility, offer a promising solution to address these limitations.

Regarding Ankyrons, the company boasts a rapidly expanding library, having screened over 1500 protein targets with more than 7000 clones. Notably, they also offer custom screening services at no extra charge. While these clones are not fully characterized, they present a valuable opportunity for students to undertake this characterization for potential publication. Simultaneously, the collection offers novel binding entities for further exploration, particularly in vivo imaging applications. Ankyron clones, provided in protein form at a reasonable cost (recently $50 per clone), are ready for labeling and use as primary antibodies. This simplicity facilitates experimentation, requiring minimal biology and chemistry background. The updated affordable price makes it attractive for laboratories with budget constraints. However, it is imperative for the company to maintain an affordable price for Ankyrons and address potential delays in screening and production. Previous experiences with certain Ankyrons from their library have highlighted delays, and the free custom screening process has proven time-consuming.

Labeling Ankyrons Holds Potential Advantages

Given the repeating units, it is reasonable to assume that all units could bind to antigens, making them more robust to modification compared to Nbs. Ankyrons’ repeating units are polypeptides, suggesting that modifying one unit may not significantly affect their binding capability. Nbs, on the other hand, are sensitive to random labeling, particularly in the Complementarity Determining Regions (CDRs).⁷ Furthermore, it is worth noting that the pharmacokinetics (PK) of Ankyrons could potentially be finely tuned by controlling the number of repeating units. Regarding applications, it is crucial to highlight the immense value of both fluorescent and nucleotide-based imaging. Nucleotide-based imaging, highly sensitive and translational, is well-suited for in vivo molecular imaging. Simultaneously, fluorescent imaging plays a pivotal role by providing high-resolution data and establishing in vitro binding profiles, laying the groundwork for subsequent in vivo imaging techniques. One challenge worth noting is that our received Ankyrons were all stabilized by 1% BSA, posing difficulties in selective labeling of Ankyrons.

Potential Biosafety of the “Ankyrons” When Applying for Clinics

Addressing the potential biosafety of “Ankyrons” in clinical applications is paramount. The small size and polypeptide chain nature of Ankyrons suggest a favorable safety profile, aligning with the precedent set by numerous approved antibody and peptide drugs sharing similar characteristics. Furthermore, the practice of microdosing in nucleotide imaging serves to further diminish safety concerns, emphasizing the promising safety profile of Ankyrons in clinical contexts for imaging purposes.

Concluding Remarks and Perspective

In summary, for students grappling with the obligation of publishing papers for their graduation, this approach may offer a potentially expedited route to showcasing their work. The remarkable binding capabilities and possible swift clearance from the bloodstream of Ankyrons lead us to envision their application in expediting in vivo disease diagnosis. Additionally, with careful molecular tuning, it may prove valuable in the realm of radiation therapy. The most compelling aspect is the practicality of incorporating Ankyrons into actual research, offering an affordable option at $50 per clone and a broad spectrum of over 1500 targets. However, a significant caveat is the intellectual property belonging to the company, raising uncertainties about how any new discoveries with clinical potential will be handled. We are currently navigating the challenge of dealing with the company in such scenarios. Simultaneously, we express the hope that forward-thinking institutions or companies may consider launching similar services for Nbs or other types of small proteins with targeting capabilities. Should you have any inquiries or require guidance on implementing these experiments for your publication, please do not hesitate to reach out to us.

The authors declare no competing financial interest.