Document text
Principal Investigator: Curtis Layton
Organization: PROTILLION BIOSCIENCES, INC.
Fiscal Year: 2022
Award: $855,260
Funding agency: National Institute of General Medical Sciences
Abstract
Monoclonal antibodies and antibody fragments are an important class of therapeutics comprising a $150B
industry. However, methods for discovering and optimizing antibodies to have desired affinity are generally
laborious laboratory procedures that require months of hands-on research performed by highly skilled
personnel (e.g. phage display, hybridoma, single cell). Additionally, the selection of leads to move forward in
the therapeutic development pipeline often must be made with limited information that does not necessarily
correspond to quantitative binding affinity. To address these challenges, Protillion has commercialized Prot-
MaP, a platform for measuring quantitative protein binding across large libraries of 105 to 109 variants on
automated instrumentation, with a time-to-result of approximately 2 days. We achieve this by generating
immobilized proteins directly on Illumina DNA sequencing flow cells through a process of in-situ transcription
and translation. This platform allows for direct, quantitative measurements of fluorescent antigen binding to
entire protein libraries at unprecedented scale—a scale that is finally a match for the sparseness of protein
function in amino acid mutation space. In our Phase I period, we adapted Prot-MaP to display VHHs
(nanobodies) capable of binding the SARS-CoV-2 spike (S1) receptor binding domain (RBD) protein. Our
multi-step optimization first comprehensively identified “beneficial” mutations, which were then combined into a
second combinatorial library. This strategy identified tens of thousands of protein variants with affinity superior
to wild type, with the best exhibiting the highest reported binding affinity for a VHH to this target, a 100-fold
improvement from the starting point. We also developed a strategy to humanize this nanobody, producing a
near-fully-human sequence that maintained high affinity. In Phase II, we will first improve automation and
commercial scalability of our instrumentation, and develop deep learning models for library design and
selection of therapeutic leads. We will next optimize other SARS-CoV-2 S1 RBD-binding nanobodies, as well
as nanobodies capable of binding PD-L1, a target relevant to cancer immunotherapy. We will develop a
universally applicable pipeline for identifying high-affinity, humanized, clinically-relevant VHH reagents. We will
also extend our display capabilities to larger, scFv domains, and carry out scFv affinity optimization against two
separate target ligands, including SARS-CoV-2 S1 RBD. Finally, we will adapt our methods to display up to 109
distinct protein variants on a NovaSeq sequencing chip, a scale sufficient to identify binders de novo from
naïve humanized VHH libraries. The activities outlined in this proposal will enable display multiple types of
antibody fragments, optimize affinity and humanize their sequences, and clearly define the landscape of
functional protein sequences. The capability of de novo discovery of new binders from untargeted libraries will
make the Protillion platform a vertically integrated “one stop shop” allowing both identification of “hits” from
untargeted libraries, as well as detailed mutational analysis and optimization of these variants.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Address><Affinity><Amino Acid Sequence><Amino Acids><Antibodies><Antibody Fragments><Antibody Repertoire><Assay><Automation><Automobile Driving><B7-H1><B7H1><Binding><Binding Proteins><Bioassay><Biochemical><Biologic Assays><Biological Assay><Biophysics><CD274><COVID-19><COVID-19 virus><COVID19><COVID19 virus><CV-19><CV19><Cancers><Cell Body><Cells><ChIP Sequencing><ChIP-seq><Clinical><Clinical Treatment Moab><CoV-2><CoV2><Color><DNA Replication><DNA Synthesis><DNA biosynthesis><DNA seq><DNA sequencing><DNAseq><Data><Data Set><Dataset><Detection><Development><Disease><Disorder><Drugs><Ecologic Systems><Ecological Systems><Ecosystem><Evaluation><Exhibits><Fluorescence><Gene Transcription><Generations><Genes><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Human><Human Resources><Hybridomas><Image><Immobilization><Immunoglobulin Fragments><In Situ><In Vitro><Individual><Industry><Infrastructure><Intellectual Property><Laboratory Procedures><Lead><Length><Libraries><Ligand Binding Protein><Ligand Binding Protein Gene><Ligands><Liquid substance><Malignant Neoplasms><Malignant Tumor><Manpower><Maps><Measurement><Measures><Medication><Methods><Modern Man><Molecular><Molecular Interaction><Monoclonal Antibodies><Mutation><Mutation Analysis><PD-L1><PDL-1><PDL1><Pb element><Peptides><Phage Display><Pharmaceutic Preparations><Pharmaceutical Preparations><Phase><Primary Protein Structure><Process><Programmed Cell Death 1 Ligand 1><Programmed Death Ligand 1><Property><Protein Array><Protein Binding><Protein Binding Domain><Protein Binding Motif><Protein Biochips><Protein Chips><Protein Engineering><Protein Microarray><Protein Microchips><Protein-Protein Interaction Domain><Proteins><Protocol><Protocols documentation><RNA Expression><Randomized><Reagent><Reporting><Runaway><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 inhibitor><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 inhibitor><Severe acute respiratory syndrome related corona virus 2><Specificity><Technology><Therapeutic><Therapeutic antibodies><Time><Transcription><Transgenic Animals><Translations><Variant><Variation><Viral><Work><Wuhan coronavirus><Yeasts><aminoacid><anti-cancer immunotherapy><anticancer immunotherapy><antigen binding><antigen bound><base><biophysical foundation><biophysical principles><biophysical sciences><block SARS-CoV-2><block severe acute respiratory syndrome coronavirus 2><bound protein><cancer immunotherapy><cell imaging><cellular imaging><chromatin immunoprecipitation-sequencing><clinical relevance><clinically relevant><combinatorial><corona virus disease 2019><coronavirus disease 2019><coronavirus disease 2019 virus><coronavirus disease-19><coronavirus disease-19 virus><coronavirus infectious disease-19><deep learning><deep learning based model><deep learning model><design><designing><developmental><driving><drug/agent><fighting><fluid><genetic protein engineering><genome mutation><hCoV19><hands on research><heavy metal Pb><heavy metal lead><high throughput analysis><imaging><immune-based cancer therapies><immunotherapy for cancer><immunotherapy of cancer><improved><inhibit SARS-CoV-2><inhibit severe acute respiratory syndrome coronavirus 2><instrument><instrumentation><liquid><mAbs><malignancy><nCoV2><nanobodies><nanobody><neoplasm/cancer><new drug treatments><new drugs><new therapeutics><new therapy><next generation therapeutics><novel><novel drug treatments><novel drugs><novel therapeutics><novel therapy><orthopedic freezing><personnel><programmed cell death ligand 1><programmed cell death protein ligand 1><prospective><protein design><protein expression><protein function><protein sequence><randomisation><randomization><randomly assigned><receptor binding><receptor bound><scaffold><scaffolding><sdAb><side effect><single cell analysis><single domain antibodies><success><therapeutic agent development><therapeutic development>