The bacterium Bacillus subtilis (B. subtilis) has long been an important subject for basic studies. However, this organism has also had industrial applications due to its easy genetic manipulation, favorable culturing characteristics for large-scale fermentation, superior capacity for protein secretion, and its generally recognized as safe (GRAS) status. In addition, as the metabolically dormant form of B. subtilis, its spores have attracted great interest due to their extreme resistance to many environmental stresses, which makes spores a novel platform for a variety of applications. In this review, we summarize both conventional and emerging applications of B. subtilis spores, with a focus on how their unique characteristics have led to innovative applications in many areas of technology, including generation of stable and recyclable enzymes, synthetic biology, drug delivery, and material sciences. Ultimately, this review hopes to inspire the scientific community to leverage interdisciplinary approaches using spores to address global concerns about food shortage, environmental protection, and healthcare.

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA-suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual-ncAA incorporation. Using these platforms, we site-specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site-specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.

Site‐specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl‐tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA‐suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual‐ncAA incorporation. Using these platforms, we site‐specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site‐specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.

Antibody drug conjugates are entering a renaissance period as a promising treatment option for a variety of cancers. Despite some success in the past few decades, developing effective ADCs remains a challenge due to extensive inefficiencies of industry standard conjugation technologies. Industry available methods can be limited by lack of site-specificity, inflexibility on the site of conjugation, and poor overall biophysical characteristics which can alter the efficacy, safety, and bioavailability of these therapeutics. BrickBio’s unique bioconjugation methodology enables precise (site-specific), flexible (offering numerous options for conjugation site and chemistry), efficient, and scalable generation of antibody-drug conjugates which overcome these limitations and achieve the full potential of this highly promising class of therapeutics. In this work, we incorporate an azide-containing UAAs (unnatural amino acid) into full-length antibodies targeting receptors overexpressed in breast and gastric cancer. Specifically, we have engineered variants of IgG1 antibodies to contain the aforementioned UAA, enabling site-specific attachment of cytotoxic payloads, such as auristatins, via click chemistry to generate ADCs with homogeneous DARs and at different conjugation sites. The resulting BrickADCs exhibit high therapeutic efficacy against receptor-positive cell lines, particularly lines expressing low levels of HER2, in vitro as well as in mouse xenograft models. Not only are the BrickADCs 100x more potent in demonstrated cell lines, but current research indicate that they have an improved safety profile over T-DM1. In other efforts, the BrickADC platform is leveraging a proprietary Modular Multisite Platform (MMP) to address the unmet need of eventual ADC resistance by conjugating multiple distinct payloads in a homogenous fashion. Continued preclinical work is underway for these pipeline candidates as well as investigation into other target areas. Citation Format: James Sebastian Italia, Nikos Biris, Zhi Li, Myer Hussain, John Boyce, Audrey Warner. A next generation site-specific ADC targeting breast and gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1767.

e15024 Background: Antibody drug conjugates are entering a renaissance period as a promising treatment option for a variety of cancers. Despite some success in the past few decades, developing effective ADCs remains a challenge due to extensive inefficiencies of industry standard conjugation technologies. Industry available methods can be limited by lack of site-specificity, inflexibility on the site of conjugation, and poor overall biophysical characteristics which can alter the efficacy, safety, and bioavailability of these therapeutics. BrickBio’s unique bioconjugation methodology enables precise (site-specific), flexible (offering numerous options for conjugation site and chemistry), efficient, and scalable generation of antibody-drug conjugates which overcome these limitations and achieve the full potential of this highly promising class of therapeutics. Methods: In this work, we incorporate an azide-containing UAA (unnatural amino acid) into full-length antibodies targeting receptors overexpressed in breast and gastric cancer. Specifically, we have engineered variants of IgG1 antibodies to contain the aforementioned UAA, enabling site-specific attachment of cytotoxic payloads, such as auristatins, via click chemistry to generate ADCs with homogeneous DARs. Results: The resulting BrickADCs exhibit high therapeutic efficacy against receptor-positive cell lines, particularly lines expressing low levels of HER2, in vitro as well as in mouse xenograft models. Not only are the BrickADCs 100x more potent in demonstrated cell lines, but current research indicate that they have an improved safety profile over T-DM1 and extended half-life. Conclusions: It is conclusive that the BrickADC platform enables an increased therapeutic window. In other efforts, the BrickADC platform is leveraging a new Modular Multisite Platform to address the unmet need of eventual ADC resistance by conjugating multiple distinct payloads in a homogenous fashion. Continued preclinical work is underway for these pipeline candidates as well as investigation into other target areas.