This Small Business Innovation Research (SBIR) Phase II project will develop methods for the control of larval settlement, metamorphosis and postlarval growth of Megathura crenulata (keyhole limpet) to support the production of commercial quantities of Keyhole Limpet Hemocyanin (KLH), a unique and medically valuable marine natural product. Unlike many other prospective medical products from marine organisms, KLH is already in extensive use in over 20 KLH-based therapeutic vaccine trials. Phase I research successfully identified a critical "cue" for settlement of M. crenulata larvae and demonstrated the feasibility of achieving the long-term commercial objectives of this research. Phase II studies will translate the results from Phase I studies into prototype designs for testing and optimization of systems, diets and aquaculture methods for cultivation of the age-specific developmental phases, from metamorphosis to fully developed adults for KLH production.
The broader impacts of this research are; 1) The elucidation of the underlying biochemical factors that promote settlement, metamorphosis and early postlarval survival of this carnivorous gastropod thus adding significantly to the body of scientific knowledge in this field and improving the potential for cultivation of other commercially important species with biomedical potential; 2) Providing sustainable commercial supplies of KLH for new, life-saving therapeutic vaccines for cancer, arthritis, hypertension, and other debilitating diseases, without continued dependence on the limited and threatened fishery, and; 3) Providing regulators and resource managers the opportunity to formulate management policies to protect the wild population without imposing limitations on KLH or the important KLH-based vaccines under development.
This Small Business Innovation Research was proposed to develop methods for the reliable control of settlement and metamorphosis of Megathura crenulata larvae (giant keyhole limpet) in quantities sufficient to support the production of commercial quantities of Keyhole Limpet Hemocyanin (KLH), a unique, medically important marine natural product. This key developmental step was identified in previous research as the remaining constraint limiting aquaculture production of M. crenulata to eliminate the biomedical industry’s dependence on wild-harvested animals for commercial KLH supplies. Megathura crenulata comprises a limited population of wild animals found from southern central California, USA, to northern Baja California, Mexico. While the anatomy and general ecology of this organism was described more than a century ago, larval development was uncharacterized until NSF funded Phase-I/II studies. This species is the sole source of KLH, a most important pharmaceutical protein that finds important uses as an adjuvant and carrier protein for the presentation of antigens in active immune therapies for cancers and autoimmune diseases. Due to its high immunogenicity and established safety profile, KLH also is used as an injectable diagnostic antigen for testing patient immune status, as well as the possible toxicology of new pharmaceutical agents, both in the clinic and in animal models. The long-term survival of commercially harvested marine gastropods is quickly becoming a global issue with observed declines and reproductive alterations in many species, and the complete collapse of other fisheries such as the California abalone (Haliotis sp.). Unable now to rely on natural resources, maintaining a biopharmaceutical source of KLH depends on developing reliable culture methods to produce M. crenulata at a scale sufficient to support commercial needs. To effectively bring M. crenulata under controlled production and to maintain uniform high quality of the resultant KLH products, reliable methods were needed to breed limpets, then to support development, metamorphosis, settlement and rearing of larvae (Figure 1.), and an understanding of the biochemical parameters that affect the immunogenicity of the KLH protein with a functional assay for KLH potency were still needed. This has been the focus of our NSF funded SBIR research efforts. NSF SBIR funded research successfully addressed and resolved each of these constraints. Completion of the technical objectives of this research demonstrated the feasibility of culturing M. crenulata at commercial scale in a fully controlled land-based aquaculture system (figure 2.), and producing medical grade KLH from animals that have been domesticated and grown specifically for KLH production (Figure 3.) These successes have led to the deployment of the resulting technology at a scale suitable to support production and marketing of medical-grade KLH with product sales to commercial vaccine developers and pharmaceutical customers. Commercialization goals achieved during this Phase I through Phase IIB SBIR funded research included successes: (i) demonstrating feasibility for production of Megathura crenulata, (the gastropod mollusk from which keyhole limpet hemocyanin, KLH is harvested), at commercial scale and under conditions suitable for compliance with FDA cGMP; (ii) establishing reliable culture methods; (iii) attracting customers and strategic pharmaceutical partners (Bayer Innovation GmbH, a subsidiary of Bayer Healthcare AG, SAFC Division of Sigma Aldrich, Neovacs S.A., and others); (iv) securing $10M in equity investments through private placements; (v) achieving a public listing on the US OTC QB (SBOTF) (vi) building a professional management team, Board of Directors, and Scientific Advisory Board; and, (vii) advancing products in development toward sales and marketing of vaccine excipients, diagnostic, and immunotoxicology products. The results of this research established the physiological, developmental and nutritional requirements needed for M. crenulata aquaculture. Uniform methods applied in the hatchery and nursery have led to multiple generation of Megathura crenulata to be cultured on site thus completing the life cycle in captivity and providing a truly sustainable and environmentally sound source of medical-grade KLH. Assays for the functional potency of KLH were developed and used to characterize the parameters affecting immunogenicity and to optimize the immunogenic quality of KLH produced from this sustainable aquaculture system. Scientific Advances from this NSF SBIR Research: Identified the mechanisms that control larval metamorphosis and support post-larval development of M. crenulata. Identified the production parameters for hatchery and nursery phase cultivation of M. crenulata. Developed and optimized systems and culture methods to support controlled, commercial-scale production of juvenile M. crenulata. Successfully produced multiple generations of M. crenulata (F-1, F-2) demonstrating feasibility of producing KLH from a domesticated strain. Developed methods for detection of KLH isoforms in larval tissues and identified the larval development phase in which the KLH isoforms (KLH1 and KLH2) are initially synthesized. Developed a cell-based assay for the immunogenicity (functional potency) of KLH. Characterized bio-chemical parameters of KLH affecting functional potency. Optimized KLH purification methods for functional potency of GMP-grade KLH
