Applied Biomedical Science Institute

MPER BROADLY NEUTRALIZING ANTIBODY KNOCKIN MICE TO STUDY ANTI-HIV BCELL RESPONSES

NOVEL VACCINE STRATEGIES TO INDUCE V2 APEX-DIRECTED BROAD NEUTRALIZING ANTIBODIES

BROADLY NEUTRALIZING SARS-COV-2 PEPTIDIC KNOBS - ABSTRACT TARGETED THERAPEUTIC AGENTS RANGE IN SIZE FROM VERY SMALL ORGANIC MOLECULES (100’S OF DA) TO PROTEIN-BASED MOLECULES LIKE MONOCLONAL ANTIBODIES (~150,000 DA). SMALL DISULFIDE BONDED PEPTIDES HAVE EVOLVED IN MANY SPECIES, INCLUDING PLANTS AND ANIMALS, TO HAVE IDEAL PHARMACOLOGICAL PROPERTIES INCLUDING HIGH AFFINITY TARGET BINDING, STABILITY TO PROTEASES, HEAT AND OTHER STRESSES. SUCH PEPTIDES INCLUDE “CYCLOTIDES” OR “KNOTTINS” WHICH CAN INHIBIT ENZYMES, ION CHANNELS, AND GPCRS WITH HIGH POTENCY AND ARE OFTEN THE MAJOR ACTIVE COMPONENT OF VENOMS OF MANY PREDATOR ORGANISMS. WE HAVE UNCOVERED CONVERGENT EVOLUTION BETWEEN ULTRALONG THIRD COMPLEMENTARY DETERMINING REGIONS (CDR H3S) IN THE HEAVY CHAIN OF AN UNUSUAL CLASS OF COW ANTIBODIES AND CYCLOTIDE/KNOTTIN PEPTIDES. WE CAN PRODUCE THESE “KNOB” PEPTIDES IN MICROBIAL SYSTEMS AND THEY RETAIN THE BINDING AND POTENCY PROPERTIES OF THE PARENT ANTIBODY. THESE TINY PEPTIDE-BASED MOLECULES ARE SMALL (~4-6 KDA), HIGHLY STABLE, AND CAN BIND TARGETS AT SUBNANOMOLAR KD. WE HAVE ALREADY DEVELOPED A PANEL OF VIRUS NEUTRALIZING KNOB PEPTIDES AGAINST SARS-COV-2 WHICH BIND UNIQUE EPITOPES, AND SOME OF WHICH MAINTAIN HIGH AFFINITY BINDING TO VARIOUS SARS-COV-2 VARIANTS, INCLUDING THE RECENT ‘DELTA’ AND ‘OMICRON’ STRAINS. THE HIGH STABILITY, POTENCY, AND STRAIGHTFORWARD MANUFACTURING PATH ENABLES MULTIPLE ROUTES OF ADMINISTRATION, POTENTIALLY INCLUDING INHALED OR INTRANASAL DELIVERY, WHICH COULD BE VERY IMPORTANT PROPHYLACTIC OR TREATMENT IN THE CURRENT OR FUTURE CORONAVIRUS PANDEMIC. OUR GOALS IN THIS PROJECT ARE TO FURTHER DEVELOP THE TECHNOLOGY TO IDENTIFY PEPTIDIC KNOB DOMAINS, EXPAND OUR PANEL OF KNOBS AGAINST SARS-COV-2 VARIANTS AND OTHER CORONAVIRUSES LIKE MERS-COV AND SARS-COV- 1, UNDERSTAND THE STRUCTURAL BASIS OF THEIR BINDING, AND VALIDATE THEIR ACTIVITY IN VITRO AND IN VIVO. THE KNOBS IDENTIFIED HERE CAN POTENTIALLY BE USED AS MONOTHERAPY OR COMBINATION THERAPY IN THE CURRENT OR A NEW CORONAVIRUS PANDEMIC, AND WILL BE A VALUABLE NEW THERAPEUTIC CLASS TO ADD TO THE ARSENAL AGAINST CORONAVIRUS DISEASE.

MOLECULAR AND STRUCTURAL STUDIES OF ANTIBODY DIVERSITY MECHANISMS

DEFINING CLINICALLY RELEVANT VIRAL EPITOPES WITH COW ANTIBODIES

UNIQUE ANTIBODY STRUCTURAL VARIEGATION MECHANISMS - ABSTRACT THE COW IMMUNE SYSTEM PRODUCES A SUBSET OF ANTIBODIES WITH EXTREMELY LONG CDR H3 REGIONS. THESE CDR H3S CAN BE UP TO 70 AMINO ACIDS IN LENGTH AND PROTRUDE FAR FROM THE TYPICAL ANTIBODY SURFACE, UTILIZING A -RIBBON “STALK” THAT SUPPORTS A DISULFIDE BONDED “KNOB” DOMAIN THAT IS SIMILAR IN SIZE AND SHAPE TO CYCLOTIDES AND KNOTTINS. THESE ANTIBODIES ARE ENCODED BY SPECIFIC VH, DH, AND JH GENE SEGMENTS, AND UNDERGO AN ATYPICAL VDJ RECOMBINATION EVENT WHICH INSERTS A SPECIFIC A-RICH SEQUENCE BETWEEN VH AND DH. THE MOLECULAR MECHANISM BEHIND THIS PHENOMENON IS CURRENTLY UNKNOWN. THE ULTRALONG CDR H3 ANTIBODIES BIND TO ANTIGEN THROUGH THEIR KNOB DOMAIN, WHICH WE HAVE SHOWN CAN BE PRODUCED INDEPENDENTLY OF THE ANTIBODY. THUS, THE COW CDR H3 KNOB IS THE SMALLEST KNOWN ANTIBODY FRAGMENT AT 1/3RD THE SIZE OF CAMELID VHH “NANOBODIES”. IN THIS PROPOSAL WE AIM TO UNDERSTAND THE MOLECULAR MECHANISM BEHIND THE UNUSUAL V(D)J RECOMBINATION EVENT THAT PRODUCES ULTRALONG CDR H3 GENES. THE RAG-1/RAG-2 RECOMBINASE IS KNOWN TO HAVE TRANSPOSASE ACTIVITY IN VITRO, HOWEVER THIS ACTIVITY HAS NOT BEEN OBSERVED IN NORMAL PHYSIOLOGY. WE WILL EVALUATE THE SOURCE OF THE UNUSUAL A-RICH INSERTIONAL EVENT BY BIOINFORMATIC, MOLECULAR, AND GENETIC ANALYSIS. UNDERSTANDING THIS MOLECULAR MECHANISM IN COWS MAY LEAD TO UNDERSTANDING HOW RARE BUT IMPORTANT BROADLY NEUTRALIZING ANTI-VIRAL ANTIBODIES WITH LONG CDR H3 FORM IN HUMANS, WHICH WOULD BE USEFUL INFORMATION FOR VACCINE APPROACHES ATTEMPTING TO INDUCE THESE ANTIBODIES. OUR SECOND MAJOR OBJECTIVE IS TO UNDERSTAND THE MOLECULAR FUNCTIONS OF CDR H3 KNOBS AT A STRUCTURAL AND PHYSIOLOGICAL LEVEL. WE WILL EMPLOY CRYSTALLOGRAPHY, ELECTRON MICROSCOPY, AND NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY TO UNDERSTAND KNOB STRUCTURE AND ANTIGEN BINDING, AND TO ASCERTAIN WHETHER THE FREE KNOB STRUCTURE IS IDENTICAL TO ITS PARENTAL ANTIBODY. FURTHER, WE WILL INVESTIGATE THE STABILITY, BIOAVAILABILITY AND PHARMACOKINETICS OF KNOBS IN VIVO IN MICE. KNOBS ARE SIMILAR IN STRUCTURE TO CYCLOTIDES, WHICH, REMARKABLY, ARE ORALLY BIOAVAILABLE. IF KNOBS ARE SIMILARLY BIOAVAILABLE, THEY COULD BECOME A NOVEL PROTEIN-BASED ORALLY AVAILABLE CLASS OF THERAPEUTIC. THESE STUDIES, THEREFORE, WILL LAY THE SCIENTIFIC FOUNDATIONS FOR KNOBS AS A NEW MODALITY OF RESEARCH TOOLS, AS WELL AS DIAGNOSTIC AND THERAPEUTIC AGENTS.

ULTRALONG CDR3 ANTIBODIES TARGETING EXHAUSTED T CELLS