Mantle Biotech

The coronavirus pandemic has exposed significant gaps in the global diagnostic infrastructure. National control efforts and epidemiological studies have been hampered by the poorly scalable nature of existing diagnostic techniques - a scarcity of raw materials (e.g. nasal swabs and RNA extraction kits), long testing backlogs, and limited trained personnel have all contributed to the unchecked global transmission of this disease. In order to address the poor scalability of existing diagnostic techniques, we are producing the world's most mass-manufacturable diagnostic format, by marrying two high-throughput production platforms: rapid bacterial production of biological reagents and roll-to-roll manufacturing of paper-based assays. This approach enables the production of low-cost, sensitive assays which can be administered and interpreted without training, and produced on a scale of tens of millions of tests per week. This approach extends medical coverage to the poorly served patient groups and communities which need them the most.

Traditional point-of-care diagnostics detect disease biomarkers using a class of target-specific binding proteins called antibodies. These antibodies can exhibit lock-and-key specificity to their binding target, enabling the selective detection of a disease marker in the context of a complex patient sample (e.g. blood, saliva, etc). However, antibodies also suffer from a number of limitations that make them ill-suited for addressing urgent medical needs on a global scale - new antibodies require 3-6 months to develop, they can only be produced via slow-growing mammalian cells at high cost, they have poor intrinsic thermal stability, they offer limited flexibility, and they are often poorly characterized and understood. These drawbacks have become evident in the context of the coronavirus pandemic - although some COVID-19 specific antibodies do exist thanks to a coincidental genetic similarity between SARS-CoV-2 biomarkers and markers from the 2003 SARS outbreak), we would otherwise still be struggling to produce antibodies that could detect SARS-CoV-2. Regardless, development of the reagents is only the first challenge - the highest-quality antibodies must be identified via an exhaustive search process, and these antibodies must be produced at scale to meet global demand. These challenges have significantly delayed the production of rapid coronavirus diagnostics. 

Over the last seven years, our work in the Sikes Lab at MIT has focused on the development of a novel class of molecules specifically engineered to yield low-cost, scalable, heat-stable, and sensitive medical diagnostics. To achieve this end, we have re-purposed a suite of proteins which originate in thermophilic microbes found in hot springs, selectively mutating these proteins in order to imbue them with novel function. Over the course of two weeks in March of this year, we developed a suite of over thirty heat-stable binding proteins which bind to the SARS-CoV-2 nucleoprotein with exquisite sensitivity and specificity. By using targeted development techniques, we identified optimal molecules without engaging in an extensive search process, and we are now integrating these reagents into low-cost paper-based immunoassays which can be produced via roll-to-roll manufacturing, and which yield a result that can be read with the naked eye within five minutes. Because we are using small, engineered binding proteins which we can tailor at the genetic level for our specific application, we are able to design diagnostic reagents which self-immobilize to a paper surface in under 10 seconds, enabling mass manufacture of user-friendly, non-instrumented tests.

We see this platform as a unique means of addressing the pressing diagnostic challenges of this current crisis, and providing the case-finding apparatus with much greater immediacy and community coverage. This low-cost, simple solution directly serves individuals around the world who would otherwise not have access to intensive centralized diagnostic services, providing them with life-saving insight into their own health, and helping to protect their family members and communities from further disease transmission. This solution can be readily adapted to the at-home format, enabling patients to diagnose themselves in private without incurring further risk. Looking beyond COVID-19, we see this approach enabling the next generation of point-of-care diagnostics for a broad range of other medical conditions (e.g. emergent disease outbreaks, pre-diabetes, malaria, sepsis). We have solutions in development for tuberculosis, and have spent much of the past three years engaging with patients and stakeholders to understand the needs of this community. Unfortunately the same socioeconmic determinants of poor medical access which impact TB patients will lead to highly disparate outcomes in the context of coronavirus; we see this approach as a modular solution which can broadly improve medical equity.

The key to a successful pandemic response is the ability to quickly identify cases, isolate known patients, and limit community spread. In order to do so, medical practitioners need sensitive diagnostic tests which yield real-time results and which can be produced at a scale that permits presumptive community testing. Paper-based tests can be printed in these massive quantities, and the binding proteins we produce are uniquely enabling for this approach. We have also demonstrated our rapid response capability, generating the reagents required for coronavirus diagnosis in two weeks - six times faster than can be achieved using standard antibodies.

The idea of paper-based diagnostics emerged in 2007 with the creation of Diagnostics for All. While this organization was able to produce chemical assays for testing medical conditions like liver function, they were stymied in their attempts at producing a important class of diagnostic assays called "immunoassays," which capture disease biomarkers from a patient sample. Cellulose is a relatively inert material, and so immobilization of the antibodies required to capture a disease marker was found to be difficult and inconsistent. Competitive technologies within this space are lateral flow assays (e.g. pregnancy tests), which use a different material (nitrocellulose) which enables more straightforward protein immobilization - companies operating within this space include names like Abbott, Quidel, and Access Bio. However, these lateral flow assays are much more complex to manufacture, and thus suffer from limited scalability.

We solve the problem of immobilizing target-specific binding proteins on cellulose by stitching our engineered binding proteins to a cellulose-anchoring domain that facilitates rapid "self-assembly" of paper-based immunoassays. Furthermore, because we are developing diagnostic reagents that can be readily produced in bacteria, we can produce these reagents in much higher yields that match the potential scale of mass-manufacturable paper-based assays. Throughout our development process, our platform development has also been informed by considerations of modularity - every format innovation can be easily adapted to address new disease targets, such that in the future we can readily accessible diagnostic testing to additional indications beyond coronavirus. 

We have produced a new class of diagnostic reagents which are based on proteins sourced from thermophilic microbes found living in hot springs. This original protein scaffold is stable up to 98 degrees Celsius, and can be readily produced in bacteria at low cost. We are able to develop new versions of this protein which recognize target disease markers by leveraging protein engineering techniques developed in the pharmaceutical industry. In short, we create billions of random variations based on this common protein scaffold. We then rapidly screen through this "library" to identify protein variants which bind specifically and strongly to disease biomarkers, while retaining the thermal stability of the original scaffold. Because the entire process happens in a test tube, we can exert intentional, concerted selective pressures throughout this process, ensuring that our reagents exhibit limited cross-reactivity and function well in complex patient samples. We are able to produce sequenced, individual reagents 6-12x faster than the broader diagnostic industry, and have unparalleled flexibility to improve and tailor these reagents as needed to improve their target-binding performance. 

Furthermore, we can use recombinant DNA technology to fuse these binding proteins to other functional proteins in a plug-and-play manner - in particular, we have found that fusing our target-binding proteins to a cellulose anchoring domain enable the straightforward manufacturing of paper-based diagnostic assays. This enables us to manufacture assays within 30 seconds (compared to the previous standard of 32 hours), making roll-to-roll mass-manufacturing of low-cost paper-based diagnostics feasible. 

Our work is documented in 8 peer-reviewed articles: 

•Sung, K.-J., Jabbour Al Maalouf, Y., Johns, Q. R., Miller, E. A. & Sikes, H. D. Functional comparison of paper-based immunoassays based on antibodies and engineered binding proteins. Analyst 145, 2515–2519 (2020). https://doi.org/10.1039/D0AN00299B

•Miller, E.A.*, Sung, K-J.*, Kongsuphol, P., Baniya, S., Aw-Yong, H.Q., Tay, V., Tan, Y., Kabir, F.M., Pang-Yeo, K., Kaspriskie, I.G., Sikes, H.D., 2019. Beyond epitope binning: directed in vitro selection of complementary pairs of binding proteins. ACS Comb.Sci. https://doi.org/10.1021/acscombsci.9b00176

•Miller, E.A., Traxlmayr, M.W., Shen, J., Sikes, H.D., 2016. Activity-based assessment of an engineered hyperthermophilic protein as a capture agent in paper-based diagnostic tests. Mol.Syst.Des.Eng. 1, 377–381. https://doi.org/10.1039/C6ME00032K

•Miller, E.A., Baniya, S., Osorio, D., Al Maalouf, Y.J., Sikes, H.D., 2018. Paper-based diagnostics in the antigen-depletion regime: High-density immobilization of rcSso7d-cellulose-binding domain fusion proteins for efficient target capture. Biosens.Bioelectron. 102, 456–463. https://doi.org/10.1016/j.bios.2017.11.050

•Miller, E.A., Jabbour Al Maalouf, Y., Sikes, H.D., 2018. Design Principles for Enhancing Sensitivity in Paper-Based Diagnostics via Large-Volume Processing. Anal.Chem. 90, 9472–9479. https://doi.org/10.1021/acs.analchem.8b02113

•Sung, K.-J., Miller, E.A., Sikes, H.D., 2018. Engineering hyperthermostable rcSso7d as reporter molecule for in vitro diagnostic tests. Mol.Syst.Des.Eng. 3, 877–882. https://doi.org/10.1039/C8ME00049B

•Zhang, Q., Zeininger, L., Sung, K.-J., Miller, E.A., Yoshinaga, K., Sikes, H.D., Swager, T.M., 2019. Emulsion Agglutination Assay for the Detection of Protein–Protein Interactions: An Optical Sensor for Zika Virus. ACS Sensors 4, 180–184. https://doi.org/10.1021/acssensors.8b01202

•Paloni, J.M., Miller, E.A., Sikes, H.D., Olsen, B.D., 2018. Improved Ordering in Low Molecular Weight Protein-Polymer Conjugates Through Oligomerization of the Protein Block. Biomacromolecules 19, 3814–3824. https://doi.org/10.1021/acs.biomac.8b00928

Our intervention is designed to enable front-line clinicians to perform their roles more effectively, identifying patients earlier in the course of disease progression and preventing disease transmission within families and local communities. One of the key barriers to achieving this outcome is the development of robust and sensitive diagnostics which can be produced at scale. DNA-based assays have shown their limited scalability throughout the course of this crisis, serology assays fail to identify patients in time to avert disease transmission, and the only existing platform for point-of-care antigen detection has limited manufacturing scalability. We believe that easy-to-use, mass-manufacturable paper-based assays can address these shortfalls, and that our protein engineering platform is uniquely well-suited to enable this product profile. 

By producing paper-based assays enabling the early detection of coronavirus, we believe that we can provide health officials with the tools they need to control local outbreaks - rather than sending patients home without a clear indication of their infection status, results would be available within the same visit, informing treatment decisions and personal behavior. We envision producing tests at such a scale that they become ubiquitous, and can be used for presumptive community testing (rather than rationing testing resources only for suspected cases). In so doing, these tests would enable the detection of asymptomatic carriers and patients early in the course of disease, averting compounding cycles of disease transmission.

Because we've designed this platform for modularity and development speed, we are able to extend these capabilities to a broad range of additional diseases, as well as to enable rapid response in future disease outbreaks. Further, because these reagents are shelf-stable, assays can be supplied in bulk to local clinics without any requirement of intensive infrastructure. Our goal is for clinics of all sizes to have drawers full of color-coded, non-instrumented paper-based assays that provide rapid diagnostic results for a range of medical conditions. This ready access and the simplified distribution logistics can make this the world's most wide-ranging diagnostic technology, stopping disease outbreaks in their tracks and enabling clinicians to diagnose medical conditions early in the course of disease.

Currently we are not actively serving anyone with our solution - we are in the prototype stage, and are in the process of clinically validating our developed prototype. We are partnering with a large translational partner for the mass manufacture of these paper-based assays, and our aim is to have assays developed and in production by the end of August. Coronavirus is a difficult technical mark to hit by virtue of the low levels of the target biomarker - if we are able to do so, not only will this point-of-care assay be produced in the tens of millions, but a huge number of other medical conditions will be technically addressable. Our vision is to enable the production of billions of tests every year, ranging from coronavirus and malaria to tuberculosis and hypothyroidism. We believe that this may become the go-to platform for point-of-care diagnosis, extending the medical apparatus deep into communities that have previously gone neglected, and enabling the early diagnosis and treatment of many preventable, debilitating diseases.

Medical diagnostics are one of the most cost-effective health interventions ever developed, but the vast majority of the global populace doesn't have access to the sorts of tests taken for granted by those with high-quality medical care. Our five-year vision is to extend this access to the global population by developing sensitive assays that can be produced at low cost and on a massive scale. In the near term, we are leveraging this platform to address the growing coronavirus pandemic - we believe that these assays can be validated and produced at scale by the fall and we are actively pursuing that goal. If successful, this will be many patients' first point-of-contact with the medical apparatus, serving as a critical tool in case-finding efforts. In the long-term, we envision a broad operation, combining local community surveillance for emerging diseases with a highly efficient reagent development operation, and decentralized manufacturing of both the diagnostic reagents and paper-based assays. 

In order to scale to address the global burden of disease, we will need partners on all fronts - partners within the global health community (e.g. the WHO), clinical partners for sample collection and assay validation, translational partners for manufacturing scale-up, and funding partners to work with us to realize this vision. We 

We have established relationships with translational partners that would enable manufacturing of these paper-based assays at scale, and are building out the clinical network required for the procurement of patient samples required for assay validation and optimization. We have ongoing relationships with global health non-profits (e.g. Global Good and the Foundation for Innovative New Diagnostics), which can provide critical access to biobanked patient samples.

Currently our team is comprised of two official members (Dr. Eric Miller and Dr. Hadley Sikes, a chemical engineering professor at MIT). We are also supported by a small constellation of informal advisors with expertise ranging from diagnostic development and commercialization to regulatory affairs.

We are the inventors of this technology platform and have invented proprietary techniques for the rapid development of new binding proteins - we believe that we have developed the world's most robust and responsive binding protein production pipeline. Our expertise also extends into assay development - the marriage of protein engineering capabilities and the chemical engineering background required for assay optimization makes us particularly well-suited to deliver on this solution. 

We currently have translational partners that we are working with for manufacturing scale-up, but are not able to publicly disclose this collaboration at this time. We are also working with clinical lab organizations for the procurement of patient samples, including the Foundation for Innovative New Diagnostics and Brio Systems.

Because we can iterate on our reagent development process rapidly, we can quickly expand our portfolio of offerings to cover a wide range of different disease conditions. We would deploy these reagents via translational partners, and would adopt a royalty-based business model. The value that we create within the global community will ultimately be the provision of critical medical information that ensures patient safety and efficacious medical treatment. We want to play a role in providing this service, at a price point that is accessible for all patients - the low intrinsic cost-of-goods for these paper-based assays makes that sort of concessional pricing feasible, while maintaining sustainability for our organization.

We anticipate that we will raise money through governmental and philanthropic grants, as well as through traditional venture capital and strategic investments. As our portfolio of offerings grows, we hope to become revenue positive and self-sustaining, re-investing our profits back into R&D in order to yield an even more robust reagent development pipeline. 

We believe that this is a pivotal moment in human history, where the value of medical diagnostics has become imminently clear. The platform we have built over the course of the past seven years has been specifically designed to meet the needs of challenges on a global scale, and we feel a tremendous sense of moral urgency to deploy these solutions in service of humanity as the coronavirus pandemic unfolds.

The World Health Organization and Bill and Melinda Gates Foundation would be fantastic partners, as would Medecins Sans Frontieres and the Foundation for Innovative New Diagnostics.

We are producing solutions to respond to the emergent coronavirus crisis, and would deploy these funds to scale our platform and reagent development efforts. Furthermore, we would leverage these assets to expand our portfolio into other disease areas.