MITHRIDATISM TECHNOLOGIES
ADVANCED BIOENGINEERING
MEETS NATURAL HYPERIMMUNITY
NEXTGEN ANTIBODY REPERTOIRE SEQUENCING
From Tim's blood, we have extracted RNA, converted it into cDNA, and then PCR amplified the antibody Fv DNA instructions from all of his B-cells using specially engineered DNA oligo primer sets that contain flapped adaptors suitable for both multiplex DNA barcoded high-throughput sequencing as well as restriction-digest cloning into phage display plasmids. These DNA amplicons have then been sequenced using Illumina MiSeq high throughput sequencing technology to generate approximately 15 million 2x300bp DNA sequence reads. The resulting data is being analyzed on the Amazon Cloud using Distributed Bio's AbGenesis platform and algorithms. By analyzing B-cell diversity generated across different immunizations by different snakes, it will be possible to computationally identify the candidate sequences most likely associated with broad neutralization, narrow down their likely toxin target, and characterize those individual sequences.
ANTIBODY PHAGE DISPLAY
In addition to high-throughput sequencing, the DNA amplicons contain restriction digest positions that enable us to search through the hundreds of million antibodies for neutralizing binders using a technology called phage display. The diversity has been cloned into an m13, pIII-displayed scFv and Fab formatted phage display vector. Distributed Bio's vector has been engineered to both package displayed phage as well as secrete scFvs or Fabs for soluble characterization in ELISA, Surface Plasmon Resonance (SPR) kinetics measurements as well as in-vitro and in-vivo neutralization assays. The resulting libraries are being panned using a 24-plex Kingfisher automated magnetic bead instrument, where venoms from 12 different snake species are biotin-conjugated and then displayed on streptavidin-conjugated magnetic beads. The resulting scfv and Fab molecules can be validated for binding and specificity by ELISA and SPR, then confirmed in in-vivo challenge on B6 mouse models. The goal will be to identify the minimum set of broadly neutralizing antibodies to produce a stable, universal, fully human antivenom.
THERAPEUTIC OPTIMIZATION
Antibody therapeutics are vulnerable to degradation during heating and drying if not carefully engineered. Existing antivenoms are drawn directly from animals and have received no engineering optimization. Distributed Bio specializes in therapeutic developability optimization of monoclonal antibodies. This includes analysis and out-engineering of biochemical liabilities including N-linked glycosylation sites, deamination sites, acid hydrolysis sites, free Cys, solvent accessible Met, framework mutations, inferior framework deselection, and enrichment for heat stability mutations. The resulting molecules will be suitable for a therapeutic formulation of the last antivenom that the world will ever need.
TECHNOLOGY RESEARCH
THAT ENABLES
MITHROS BIO PIPELINE
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Looney, Timothy J., et al. "Human B-cell isotype switching origins of IgE." Journal of Allergy and Clinical Immunology (2015).
Leighton, Philip A., et al. "A diverse repertoire of human immunoglobulin variable genes in a chicken B cell line is generated by both gene conversion and somatic hypermutation." Frontiers in immunology 6 (2015).
Wei, Yu-Ling, et al. "A highly focused antigen receptor repertoire characterizes γδ T cells that are poised to make IL-17 rapidly in naive animals." Frontiers in immunology 6 (2015).
Avnir, Yuval, et al. "IGHV1-69 polymorphism modulates anti-influenza antibody repertoires, correlates with IGHV utilization shifts and varies by ethnicity." Scientific reports 6 (2016).
Yeung, Yik Andy, et al. "Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire." Nature Communications 7 (2016): 13376.
Levin, Mattias, et al. "Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy." Journal of Allergy and Clinical Immunology 137.5 (2016): 1535-1544.
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Watson, Corey T., et al. "Comment on “A Database of Human Immune Receptor Alleles Recovered from Population Sequencing Data”." The Journal of Immunology 198.9 (2017): 3371-3373.
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