Riverbend Rd

ENGINEERED PAN-SPECIFIC REAGENTS FOR N-GLYCAN DETECTION AND ENRICHMENT - PROJECT SUMMARY GLYCANS PLAY CRUCIAL ROLES IN NEARLY EVERY ASPECT OF BIOLOGICAL PROCESSES, AND THEIR DISTINCT PROPERTIES MAKE THEM APPEALING AS DISEASE BIOMARKER TARGETS. HOWEVER, DUE TO THEIR HIGHLY BRANCHED AND VARIABLY LINKED NATURE, GLYCANS POSE A CHALLENGE FOR DETECTION, PURIFICATION, AND STRUCTURAL ANALYSIS. ALTHOUGH ADVANCED ANALYTICAL TECHNIQUES AND INSTRUMENTATION HAVE BEEN DEVELOPED THAT TARGET GLYCANS, THERE IS STILL A GREAT NEED FOR REAGENTS WITH WELL-DEFINED CARBOHYDRATE SPECIFICITY AND HIGH AFFINITY THAT CAN BE USED TO INDEPENDENTLY INTERROGATE OR ENRICH CARBOHYDRATE SAMPLES PRIOR TO TRADITIONAL ANALYSES. LECTENZ BIO HAS BEEN ENGINEERING GLYCAN-PROCESSING ENZYMES AND GLYCAN-BINDING PROTEINS INTO HIGH-AFFINITY GLYCAN-SPECIFIC REAGENTS WITH TUNABLE PROPERTIES. THE FBS1 COMPONENT OF THE E3 UBIQUITIN LIGASE COMPLEX RECOGNIZES GLYCANS AS PART OF ITS BIOLOGICAL ROLE IN UBIQUITYLATION. IT IS THEREFORE A PROMISING PROTEIN FOR CONVERSION INTO A BINDING REAGENT FOR ASPARAGINE-LINKED GLYCANS (N-GLYCANS), WHICH WE WILL ACHIEVE BY EMPLOYING COMPUTATIONAL MODELING AND DIRECTED EVOLUTION. INITIAL STUDIES DEMONSTRATE THAT FBS1 CAN BE READILY EXPRESSED IN E. COLI, AND BINDS SPECIFICALLY TO N-GLYCOSYLATED PEPTIDES AND PROTEINS, AND NOT TO THE CORRESPONDING NON-GLYCOSYLATED PEPTIDES AND PROTEINS. HOWEVER, IT DOES DEMONSTRATE A PREFERENCE FOR CERTAIN N-GLYCAN STRUCTURES. A PAN-SPECIFIC N-GLYCAN BINDING REAGENT WOULD DIRECTLY ADDRESS THE URGENT NEED TO TOOLS TO HELP DETECT GLYCO- BIOMARKERS AND COULD IMMEDIATELY BENEFIT THE FIELD OF GLYCOMICS/GLYCOPROTEOMICS BY ENABLING SAMPLE PRE- ENRICHMENT. AFTER ENGINEERING AND OPTIMIZING THE PRODUCTION OF RECOMBINANT FBS1 AND ITS ENGINEERED VARIANTS, LECTENZ BIO WILL DEVELOP A SUITE OF KITS FOR ITS APPLICATION IN WESTERN BLOTTING, IMMUNOHISTOCHEMISTRY, ELISA, FLOW CYTOMETRY, AND AFFINITY CHROMATOGRAPHY.

ENGINEERED PAN-SPECIFIC REAGENTS FOR N-GLYCAN DETECTION AND ENRICHMENT - PROJECT SUMMARY GLYCANS PLAY CRUCIAL ROLES IN NEARLY EVERY ASPECT OF BIOLOGICAL PROCESSES, AND THEIR DISTINCT PROPERTIES MAKE THEM APPEALING AS DISEASE BIOMARKER TARGETS. HOWEVER, DUE TO THEIR HIGHLY BRANCHED AND VARIABLY LINKED NATURE, GLYCANS POSE A CHALLENGE FOR DETECTION, PURIFICATION, AND STRUCTURAL ANALYSIS. ALTHOUGH ADVANCED ANALYTICAL TECHNIQUES AND INSTRUMENTATION HAVE BEEN DEVELOPED THAT TARGET GLYCANS, THERE IS STILL A GREAT NEED FOR REAGENTS WITH WELL-DEFINED CARBOHYDRATE SPECIFICITY AND HIGH AFFINITY THAT CAN BE USED TO INDEPENDENTLY INTERROGATE OR ENRICH CARBOHYDRATE SAMPLES PRIOR TO TRADITIONAL ANALYSES. LECTENZ BIO HAS BEEN ENGINEERING GLYCAN-PROCESSING ENZYMES AND GLYCAN-BINDING PROTEINS INTO HIGH-AFFINITY GLYCAN-SPECIFIC REAGENTS WITH TUNABLE PROPERTIES. THE FBS1 COMPONENT OF THE E3 UBIQUITIN LIGASE COMPLEX RECOGNIZES GLYCANS AS PART OF ITS BIOLOGICAL ROLE IN UBIQUITYLATION. IT IS THEREFORE A PROMISING PROTEIN FOR CONVERSION INTO A BINDING REAGENT FOR ASPARAGINE-LINKED GLYCANS (N-GLYCANS), WHICH WE WILL ACHIEVE BY EMPLOYING COMPUTATIONAL MODELING AND DIRECTED EVOLUTION. INITIAL STUDIES DEMONSTRATE THAT FBS1 CAN BE READILY EXPRESSED IN E. COLI, AND BINDS SPECIFICALLY TO N-GLYCOSYLATED PEPTIDES AND PROTEINS, AND NOT TO THE CORRESPONDING NON-GLYCOSYLATED PEPTIDES AND PROTEINS. HOWEVER, IT DOES DEMONSTRATE A PREFERENCE FOR CERTAIN N-GLYCAN STRUCTURES. A PAN-SPECIFIC N-GLYCAN BINDING REAGENT WOULD DIRECTLY ADDRESS THE URGENT NEED TO TOOLS TO HELP DETECT GLYCO- BIOMARKERS AND COULD IMMEDIATELY BENEFIT THE FIELD OF GLYCOMICS/GLYCOPROTEOMICS BY ENABLING SAMPLE PRE- ENRICHMENT. AFTER ENGINEERING AND OPTIMIZING THE PRODUCTION OF RECOMBINANT FBS1 AND ITS ENGINEERED VARIANTS, LECTENZ BIO WILL DEVELOP A SUITE OF KITS FOR ITS APPLICATION IN WESTERN BLOTTING, IMMUNOHISTOCHEMISTRY, ELISA, FLOW CYTOMETRY, AND AFFINITY CHROMATOGRAPHY.

A NEW APPROACH TO MONITORING GLYCOSYLATION DURING GLYCOPROTEIN PRODUCTION - PROJECT SUMMARY DURING THE PRODUCTION OF THERAPEUTIC BIOLOGICALS, GLYCOSYLATION HAS BEEN IDENTIFIED AS A CRITICAL QUALITY ATTRIBUTE (CQA) THAT MUST BE CAREFULLY MONITORED TO ENSURE THAT THE DESIRED PROTEIN STABILITY, IMMUNOGENICITY, ANTIBODY EFFECTOR FUNCTION, PHARMACOLOGICAL SAFETY AND POTENCY, AND SERUM HALF-LIFE ARE CONSISTENTLY OBTAINED. MANY FACTORS CAN AFFECT THE COMPOSITION OF THE GLYCAN CHAINS DURING PRODUCTION, SUCH AS MEDIA, TEMPERATURE, DISSOLVED GASES, INDUCTION TIMES, ETC., AND CONSEQUENTLY VARIATIONS IN GLYCOSYLATION ARE AMONG THE LEADING CAUSES OF PHARMACEUTICAL BATCH REJECTION. CURRENTLY, THE CHARACTERIZATION OF PROTEIN GLYCOSYLATION RELIES HEAVILY ON METHODS THAT EMPLOY CHROMATOGRAPHY AND/OR MASS SPECTROMETRY, WHICH REQUIRE A HIGH LEVEL OF EXPERTISE, ARE TIME-CONSUMING AND COSTLY, AND, BECAUSE THEY ARE CHALLENGING TO IMPLEMENT DURING BIOLOGICS PRODUCTION AT-LINE, ARE GENERALLY PERFORMED ONLY POST-PRODUCTION. IN RECOGNITION OF THE CONTINUING NEED FOR NOVEL ANALYTICAL TECHNOLOGIES THAT CAN BE USED DURING THERAPEUTIC PRODUCTION, THE US FOOD AND DRUG ADMINISTRATION (FDA) ENCOURAGES THE ADOPTION OF NEW PROCESS ANALYTICAL METHODS (PAT) THAT OFFER MECHANISMS TO DESIGN, ANALYZE, OR FACILITATE THE MEASUREMENT OF PROCESS PARAMETERS THAT AFFECT CQAS. HERE WE PROPOSE TO OPTIMIZE AND COMMERCIALIZE A SIMPLIFIED APPROACH FOR MONITORING GLYCOSYLATION FEATURES DURING GLYCOPROTEIN PRODUCTION, WHICH WE CALL GLYCOSENSETM, THAT IS FULLY ALIGNED WITH THE FDA PAT INITIATIVE. THE GLYCOSENSE TECHNOLOGY USES FLOW CYTOMETRY WITH GLYCAN-SPECIFIC REAGENTS CONJUGATED TO MULTIPLEX BEADS TO PROVIDE A RAPID, INEXPENSIVE, AND EASY-TO-USE METHOD FOR MONITORING PROTEIN GLYCOSYLATION PATTERNS. FLOW CYTOMETRY WAS CHOSEN AS THE PREFERRED PLATFORM BECAUSE THE DATA ARE STATISTICALLY ROBUST, THE EQUIPMENT IS RELATIVELY COMMONPLACE, THE ANALYSES CONSUME VERY LOW QUANTITIES OF SAMPLE, AND IT WILL ENABLE LECTENZ BIO TO LEVERAGE ITS PROPRIETARY EXPERTISE IN PRODUCING CARBOHYDRATE-SENSING REAGENTS. IN PRELIMINARY STUDIES REPORTED HERE, GLYCOSENSE PERFORMANCE METRICS WERE ESTABLISHED AND PUBLISHED USING GLYCANS AND GLYCOPROTEIN STANDARDS. IN PHASE II, THE TECHNOLOGY WILL BE OPTIMIZED FOR MONITORING GLYCOSYLATION CHANGES DURING CELL CULTURE. THIS WILL RESULT IN GLYCOSENSE KITS THAT CAN BE USED FOR A VARIETY OF APPLICATIONS IN BASIC RESEARCH AND IN BIOPHARMA.

INNOVATIVE GLYCAN-SPECIFIC REAGENTS TO ACCELERATE THE DETECTION OF DISEASE BIOMARKERS - PROJECT SUMMARY GLYCANS HAVE SEVERAL DISTINCT PROPERTIES THAT MAKE THEM EXCELLENT TARGETS FOR DISEASE BIOMARKERS. FIRSTLY, BECAUSE THE STRUCTURES OF CELL SURFACE GLYCANS ARE DETERMINED BY A COMPLEX ENZYMATIC PATHWAY WITHIN THE CELL, ANY ALTERATION TO THIS HOMEOSTASIS, SUCH AS FROM DISEASE, MAY RESULT IN ABERRANT PROTEIN GLYCOSYLATION. THUS, SPECIFIC GLYCAN STRUCTURES THAT ARE NOT PRESENT, OR ARE IN LOW AMOUNTS, IN NORMAL STATES PROLIFERATE IN DISEASE STATES, SUCH AS CANCER. SECONDLY, THEIR LOCATION ON CELL SURFACES MAKES THEM READILY ACCESSIBLE TO DETECTION REAGENTS. THIRDLY, ANY CHANGE IN CELLULAR GLYCOSYLATION MACHINERY MAY IMPACT A LARGE NUMBER OF GLYCOPROTEINS, OFFERING MANY POTENTIAL GLYCAN-RELATED BIOMARKERS PER DISEASED CELL. TO EFFECTIVELY EMPLOY AND DISCOVER GLYCAN DISEASE MARKERS A WIDER RANGE OF HIGHLY-SPECIFIC REAGENTS ARE URGENTLY NEEDED. THE MONOSACCHARIDE MANNOSE HAS BEEN IDENTIFIED IN MANY DISEASE MARKERS, BUT IS DIFFICULT TO DETECT SPECIFICALLY WITHIN THE CONTEXT OF OTHER GLYCANS WITH EXISTING REAGENTS. USING STRUCTURALLY-GUIDED MUTAGENESIS, WE WILL CONVERT AN Α-MANNOSIDASE ENZYME INTO A HIGH AFFINITY REAGENT FOR THE DETECTION OF HIGH MANNOSE GLYCANS THAT ARE KNOWN BIOMARKERS FOR A NUMBER OF CANCERS. SUCH ENGINEERED LECTIN-LIKE REAGENTS DERIVED FROM ENZYMES ARE CALLED “LECTENZ®”, AND HAVE SEVERAL ADVANTAGES OVER LECTINS AND ANTIBODIES. THE PRINCIPAL ADVANTAGES OF AN ENGINEERED LECTENZ® OVER AN ANTIBODY ARE THAT THE LECTENZ® IS SPECIFIC TO THE CARBOHYDRATE SEQUENCE, BUT, IN CONTRAST TO ANTIBODIES, WILL RECOGNIZE THAT SEQUENCE IN A BROAD RANGE OF GLYCANS. FURTHER, IN CONTRAST TO CARBOHYDRATE REAGENTS BASED ON PLANT LECTINS, ENGINEERED LECTENZ® ARE DERIVED FROM ENZYMES THAT HAVE EXQUISITE SUBSTRATE SPECIFICITIES AND LOW TOXICITIES. ADDITIONAL ADVANTAGES OF LECTENZ® INCLUDE PRECISE DEFINITION OF SPECIFICITY, TUNABLE BINDING PROPERTIES, AND EASE OF RECOMBINANT EXPRESSION, ENABLING THEIR POTENTIAL USE IN AFFINITY PURIFICATION, WESTERN BLOTTING, IN SITU HISTOLOGICAL STAINING, AND IN VIVO IMAGING. WE WILL GENERATE MANNOSE-BINDING LECTENZ® THAT CAN BIND HIGH MANNOSE GLYCANS WITH HIGH SPECIFICITY. GLYCOSYLATION DETECTION IS ESSENTIAL IN FULLY CHARACTERIZING AND EXPLOITING GLYCANS AS MARKERS OF SPECIFIC DISEASE STATES, AND YET CURRENT REAGENTS HAVE BROAD SPECIFICITY.

A PARAINFLUENZA VIRUS 5 (PIV5)-BASED BIVALENT VACCINE FOR RESPIRATORY SYNCYTIAL VIRUS (RSV) AND HUMAN METAPNEUMOVIRUS (HMPV) - ABSTRACT IN THIS R21 APPLICATION, WE PROPOSE TO DEVELOP A UNIVERSAL, INTRANASAL, PARAINFLUENZA VIRUS 5 (PIV5)-BASED RESPIRATORY SYNCYTIAL VIRUS (RSV) AND HUMAN METAPNEUMOVIRUS (HMPV) BIVALENT VACCINE. RSV AND HMPV ARE TWO OF THE LEADING CAUSES OF ACUTE RESPIRATORY INFECTIONS (ARIS) IN CHILDREN, IMMUNOCOMPROMISED INDIVIDUALS, AND THE ELDERLY. ILLNESS RANGES FROM ASYMPTOMATIC INFECTION TO SEVERE BRONCHIOLITIS AND PNEUMONIA, WITH 90-100% OF CHILDREN INFECTED WITH RSV BY 2 YEARS OF AGE, AND HMPV BETWEEN THE AGES OF 5-10 YEARS OLD. NO LICENSED RSV OR HMPV VACCINE IS AVAILABLE AND THERE IS AN UNMET MEDICAL NEED TO DEVELOP SAFE AND EFFECTIVE VACCINES FOR BOTH DISEASES. A RECENT PROOF-OF-PRINCIPLE STUDY HAS SHOWN THAT IT IS POSSIBLE TO CREATE A CHIMERIC F PROTEIN (RHMS-1) BY COMBINING IMMUNODOMINANT EPITOPES FROM RSV F AND HMPV F THAT RETAINS ANTIGENICITY FOR BOTH VIRUSES, THE PURIFIED PROTEIN IS IMMUNOGENIC AND PROTECTIVE AGAINST RSV AND HMPV CHALLENGE IN MICE. HOWEVER, THIS PURIFIED PROTEIN REQUIRES AN ADJUVANT AND A MULTI-DOSE APPROACH, THE CELL-MEDIATED IMMUNE RESPONSE WAS NOT STUDIED, THE DURATION OF THE ANTIBODY RESPONSE HAS NOT BEEN DETERMINED, AND THERE IS A POTENTIAL SAFETY CONCERN FOR CHILDREN DUE TO THE HIGH DOSE OF PROTEIN REQUIRED FOR THE VACCINE TO BE IMMUNOGENIC. PIV5 IS A SAFE DELIVERY VECTOR FOR INTRANASAL IMMUNIZATION. A PIV5-VECTORED RSV CANDIDATE VACCINE (BLB-201) HAS ALREADY BEEN CLEARED BY THE FDA FOR A PHASE I CLINICAL TRIAL THIS YEAR (NCT05281263). DUE TO THE SIMILARITIES BETWEEN RSV AND HMPV F PROTEINS, DISEASE MANIFESTATION, AND TARGET POPULATIONS, A BIVALENT VACCINE IS DESIRABLE TO PROTECT AGAINST ARI DISEASES CAUSED BY BOTH VIRUSES. HERE, WE PROPOSE TO INTRODUCE THE RHMS-1 NOVEL SEQUENCE INTO THE PIV5- VECTORED VACCINE PLATFORM. WE WILL ALSO MAKE A MODIFICATION TO THE RHMS-1 SEQUENCE TO IMPROVE F PROTEIN EXPRESSION AND ITS IMMUNOGENICITY. THE CONSTRUCTS TO BE EVALUATED INCLUDE: 1) RHMS-1 PRE-FUSION; AND 2) RHMS-1 PRE-FUSION FORM WITH THE TRIMERIZATION DOMAIN REPLACED WITH THE TRANSMEMBRANE DOMAIN AND CYTOPLASMIC TAIL FROM THE PIV5 F PROTEIN. THE CANDIDATE VACCINE VIRUSES WILL BE COMPARED FOR THEIR REPLICATION AND ANTIGEN EXPRESSION IN VITRO, AND IMMUNOGENICITY AND PROTECTIVE EFFICACY AGAINST RSV AND HMPV CHALLENGE INFECTION IN VIVO. THE NOVELTY OF THE VACCINE PROPOSED IN THIS R21 APPLICATION RELATES TO: 1) THE USE OF A CHIMERIC RSV+HMPV F PROTEIN AGAINST TWO PATHOGENS (BIVALENT VACCINE); 2) A NEEDLE-FREE INTRANASAL DELIVERY METHOD IN A SAFE, HIGHLY IMMUNOGENIC VIRAL VECTOR; 3) EASE OF ADMINISTRATION; AND 4) THE ABILITY TO INDUCE CELLULAR AND ANTIBODY RESPONSES INCLUDING MUCOSAL IMMUNITY, WHICH IS NECESSARY FOR PROTECTING AGAINST RESPIRATORY PATHOGENS. THE PROJECT IS VERY PROMISING IN GENERATING AN EFFECTIVE BIVALENT VACCINE THAT COULD PROVIDE PROTECTION AGAINST THE TWO LEADING CAUSES OF ACUTE LOWER RESPIRATORY TRACT INFECTIONS IN CHILDREN AND OLDER ADULTS.

A NEW APPROACH TO MONITORING GLYCOSYLATION DURING GLYCOPROTEIN PRODUCTION - PROJECT SUMMARY DURING THE PRODUCTION OF THERAPEUTIC BIOLOGICALS, GLYCOSYLATION HAS BEEN IDENTIFIED AS A CRITICAL QUALITY ATTRIBUTE (CQA) THAT MUST BE CAREFULLY MONITORED TO ENSURE THAT THE DESIRED PROTEIN STABILITY, IMMUNOGENICITY, ANTIBODY EFFECTOR FUNCTION, PHARMACOLOGICAL SAFETY AND POTENCY, AND SERUM HALF-LIFE ARE CONSISTENTLY OBTAINED. MANY FACTORS CAN AFFECT THE COMPOSITION OF THE GLYCAN CHAINS DURING PRODUCTION, SUCH AS MEDIA, TEMPERATURE, DISSOLVED GASES, INDUCTION TIMES, ETC., AND CONSEQUENTLY VARIATIONS IN GLYCOSYLATION ARE AMONG THE LEADING CAUSES OF PHARMACEUTICAL BATCH REJECTION. CURRENTLY, THE CHARACTERIZATION OF PROTEIN GLYCOSYLATION RELIES HEAVILY ON METHODS THAT EMPLOY CHROMATOGRAPHY AND/OR MASS SPECTROMETRY, WHICH REQUIRE A HIGH LEVEL OF EXPERTISE, ARE TIME-CONSUMING AND COSTLY, AND, BECAUSE THEY ARE CHALLENGING TO IMPLEMENT DURING BIOLOGICS PRODUCTION AT-LINE, ARE GENERALLY PERFORMED ONLY POST-PRODUCTION. IN RECOGNITION OF THE CONTINUING NEED FOR NOVEL ANALYTICAL TECHNOLOGIES THAT CAN BE USED DURING THERAPEUTIC PRODUCTION, THE US FOOD AND DRUG ADMINISTRATION (FDA) ENCOURAGES THE ADOPTION OF NEW PROCESS ANALYTICAL METHODS (PAT) THAT OFFER MECHANISMS TO DESIGN, ANALYZE, OR FACILITATE THE MEASUREMENT OF PROCESS PARAMETERS THAT AFFECT CQAS. HERE WE PROPOSE TO OPTIMIZE AND COMMERCIALIZE A SIMPLIFIED APPROACH FOR MONITORING GLYCOSYLATION FEATURES DURING GLYCOPROTEIN PRODUCTION, WHICH WE CALL GLYCOSENSETM, THAT IS FULLY ALIGNED WITH THE FDA PAT INITIATIVE. THE GLYCOSENSE TECHNOLOGY USES FLOW CYTOMETRY WITH GLYCAN-SPECIFIC REAGENTS CONJUGATED TO MULTIPLEX BEADS TO PROVIDE A RAPID, INEXPENSIVE, AND EASY-TO-USE METHOD FOR MONITORING PROTEIN GLYCOSYLATION PATTERNS. FLOW CYTOMETRY WAS CHOSEN AS THE PREFERRED PLATFORM BECAUSE THE DATA ARE STATISTICALLY ROBUST, THE EQUIPMENT IS RELATIVELY COMMONPLACE, THE ANALYSES CONSUME VERY LOW QUANTITIES OF SAMPLE, AND IT WILL ENABLE LECTENZ BIO TO LEVERAGE ITS PROPRIETARY EXPERTISE IN PRODUCING CARBOHYDRATE-SENSING REAGENTS. IN PRELIMINARY STUDIES REPORTED HERE, GLYCOSENSE PERFORMANCE METRICS WERE ESTABLISHED AND PUBLISHED USING GLYCANS AND GLYCOPROTEIN STANDARDS. IN PHASE II, THE TECHNOLOGY WILL BE OPTIMIZED FOR MONITORING GLYCOSYLATION CHANGES DURING CELL CULTURE. THIS WILL RESULT IN GLYCOSENSE KITS THAT CAN BE USED FOR A VARIETY OF APPLICATIONS IN BASIC RESEARCH AND IN BIOPHARMA.

ACHIEVING HOMEOSTASIS IN TRAUMATIC INJURIES W ITH A NITRIC OXIDE-DRIVEN ANTIMICROBIAL DR ESSING COMBINED W ITH A RAPID BLOOD CLOTTING AGENT EXTEND THE PERIOD OF PERFORMANCE

ACHIEVING HOMEOSTASIS IN TRAUMATIC INJURIES WITH A NITRIC OXIDE-DRIVEN ANTIMICROBIAL DRESSING COMBINED WITH A RAPID BLOOD CLOTTING AGENT INCORPORATE REVISED STATEMENT OF WORK AND ADD NEW SUBAWARD AND REVISED BUDGET

A NEW APPROACH TO MONITORING GLYCOSYLATION DURING GLYCOPROTEIN PRODUCTION - PROJECT SUMMARY DURING THE PRODUCTION OF THERAPEUTIC BIOLOGICALS, GLYCOSYLATION HAS BEEN IDENTIFIED AS A CRITICAL QUALITY ATTRIBUTE (CQA) THAT MUST BE CAREFULLY MONITORED TO ENSURE THAT THE DESIRED PROTEIN STABILITY, IMMUNOGENICITY, ANTIBODY EFFECTOR FUNCTION, PHARMACOLOGICAL SAFETY AND POTENCY, AND SERUM HALF-LIFE ARE CONSISTENTLY OBTAINED. MANY FACTORS CAN AFFECT THE COMPOSITION OF THE GLYCAN CHAINS DURING PRODUCTION, SUCH AS MEDIA, TEMPERATURE, DISSOLVED GASES, INDUCTION TIMES, ETC., AND CONSEQUENTLY VARIATIONS IN GLYCOSYLATION ARE AMONG THE LEADING CAUSES OF PHARMACEUTICAL BATCH REJECTION. CURRENTLY, THE CHARACTERIZATION OF PROTEIN GLYCOSYLATION RELIES HEAVILY ON METHODS THAT EMPLOY CHROMATOGRAPHY AND/OR MASS SPECTROMETRY, WHICH REQUIRE A HIGH LEVEL OF EXPERTISE, ARE TIME-CONSUMING AND COSTLY, AND, BECAUSE THEY ARE CHALLENGING TO IMPLEMENT DURING BIOLOGICS PRODUCTION AT-LINE, ARE GENERALLY PERFORMED ONLY POST-PRODUCTION. IN RECOGNITION OF THE CONTINUING NEED FOR NOVEL ANALYTICAL TECHNOLOGIES THAT CAN BE USED DURING THERAPEUTIC PRODUCTION, THE US FOOD AND DRUG ADMINISTRATION (FDA) ENCOURAGES THE ADOPTION OF NEW PROCESS ANALYTICAL METHODS (PAT) THAT OFFER MECHANISMS TO DESIGN, ANALYZE, OR FACILITATE THE MEASUREMENT OF PROCESS PARAMETERS THAT AFFECT CQAS. HERE WE PROPOSE TO OPTIMIZE AND COMMERCIALIZE A SIMPLIFIED APPROACH FOR MONITORING GLYCOSYLATION FEATURES DURING GLYCOPROTEIN PRODUCTION, WHICH WE CALL GLYCOSENSETM, THAT IS FULLY ALIGNED WITH THE FDA PAT INITIATIVE. THE GLYCOSENSE TECHNOLOGY USES FLOW CYTOMETRY WITH GLYCAN-SPECIFIC REAGENTS CONJUGATED TO MULTIPLEX BEADS TO PROVIDE A RAPID, INEXPENSIVE, AND EASY-TO-USE METHOD FOR MONITORING PROTEIN GLYCOSYLATION PATTERNS. FLOW CYTOMETRY WAS CHOSEN AS THE PREFERRED PLATFORM BECAUSE THE DATA ARE STATISTICALLY ROBUST, THE EQUIPMENT IS RELATIVELY COMMONPLACE, THE ANALYSES CONSUME VERY LOW QUANTITIES OF SAMPLE, AND IT WILL ENABLE LECTENZ BIO TO LEVERAGE ITS PROPRIETARY EXPERTISE IN PRODUCING CARBOHYDRATE-SENSING REAGENTS. IN PRELIMINARY STUDIES REPORTED HERE, GLYCOSENSE PERFORMANCE METRICS WERE ESTABLISHED AND PUBLISHED USING GLYCANS AND GLYCOPROTEIN STANDARDS. IN PHASE II, THE TECHNOLOGY WILL BE OPTIMIZED FOR MONITORING GLYCOSYLATION CHANGES DURING CELL CULTURE. THIS WILL RESULT IN GLYCOSENSE KITS THAT CAN BE USED FOR A VARIETY OF APPLICATIONS IN BASIC RESEARCH AND IN BIOPHARMA.

ENGINEERED PAN-SPECIFIC REAGENTS FOR N-GLYCAN DETECTION AND ENRICHMENT - PROJECT SUMMARY GLYCANS PLAY CRUCIAL ROLES IN NEARLY EVERY ASPECT OF BIOLOGICAL PROCESSES, AND THEIR DISTINCT PROPERTIES MAKE THEM APPEALING AS DISEASE BIOMARKER TARGETS. HOWEVER, DUE TO THEIR HIGHLY BRANCHED AND VARIABLY LINKED NATURE, GLYCANS POSE A CHALLENGE FOR DETECTION, PURIFICATION, AND STRUCTURAL ANALYSIS. ALTHOUGH ADVANCED ANALYTICAL TECHNIQUES AND INSTRUMENTATION HAVE BEEN DEVELOPED THAT TARGET GLYCANS, THERE IS STILL A GREAT NEED FOR REAGENTS WITH WELL-DEFINED CARBOHYDRATE SPECIFICITY AND HIGH AFFINITY THAT CAN BE USED TO INDEPENDENTLY INTERROGATE OR ENRICH CARBOHYDRATE SAMPLES PRIOR TO TRADITIONAL ANALYSES. LECTENZ BIO HAS BEEN ENGINEERING GLYCAN-PROCESSING ENZYMES AND GLYCAN-BINDING PROTEINS INTO HIGH-AFFINITY GLYCAN-SPECIFIC REAGENTS WITH TUNABLE PROPERTIES. THE FBS1 COMPONENT OF THE E3 UBIQUITIN LIGASE COMPLEX RECOGNIZES GLYCANS AS PART OF ITS BIOLOGICAL ROLE IN UBIQUITYLATION. IT IS THEREFORE A PROMISING PROTEIN FOR CONVERSION INTO A BINDING REAGENT FOR ASPARAGINE-LINKED GLYCANS (N-GLYCANS), WHICH WE WILL ACHIEVE BY EMPLOYING COMPUTATIONAL MODELING AND DIRECTED EVOLUTION. INITIAL STUDIES DEMONSTRATE THAT FBS1 CAN BE READILY EXPRESSED IN E. COLI, AND BINDS SPECIFICALLY TO N-GLYCOSYLATED PEPTIDES AND PROTEINS, AND NOT TO THE CORRESPONDING NON-GLYCOSYLATED PEPTIDES AND PROTEINS. HOWEVER, IT DOES DEMONSTRATE A PREFERENCE FOR CERTAIN N-GLYCAN STRUCTURES. A PAN-SPECIFIC N-GLYCAN BINDING REAGENT WOULD DIRECTLY ADDRESS THE URGENT NEED TO TOOLS TO HELP DETECT GLYCO- BIOMARKERS AND COULD IMMEDIATELY BENEFIT THE FIELD OF GLYCOMICS/GLYCOPROTEOMICS BY ENABLING SAMPLE PRE- ENRICHMENT. AFTER ENGINEERING AND OPTIMIZING THE PRODUCTION OF RECOMBINANT FBS1 AND ITS ENGINEERED VARIANTS, LECTENZ BIO WILL DEVELOP A SUITE OF KITS FOR ITS APPLICATION IN WESTERN BLOTTING, IMMUNOHISTOCHEMISTRY, ELISA, FLOW CYTOMETRY, AND AFFINITY CHROMATOGRAPHY.

PAN-SPECIFIC TOOLS AND REAGENTS FOR THE ENRICHMENT OF SIALO-GLYCANS - PROJECT SUMMARY GLYCANS HAVE SEVERAL DISTINCT PROPERTIES THAT MAKE THEIR DEVELOPMENT AS DISEASE BIOMARKERS APPEALING. FIRSTLY, THEIR LOCATION ON CELL SURFACES MAKES THEM THE FIRST POINT OF CONTACT FOR CELLULAR INTERACTIONS, AND THUS THEY ARE CRUCIAL IN THE CONTROL OF NORMAL METABOLIC PROCESSES, AND CONVERSELY, THEY FUNCTION AS PATHOGEN ADHESION RECEPTORS. SECONDLY, SPECIFIC GLYCAN STRUCTURES THAT ARE NOT PRESENT, OR ARE IN LOW AMOUNTS IN NORMAL STATE, PROLIFERATE OR ALTER THEIR SEQUENCE IN DISEASE STATES. AND, LASTLY, CHANGES IN GLYCOSYLATION MAY BE FOUND IN MANY PROTEINS, INCLUDING THOSE THAT ARE HIGHLY ABUNDANT. THUS CHANGES IN THE NORMAL LEVELS OF GLYCAN STRUCTURES, SUCH AS TERMINAL SIALIC ACID, MAY BE MARKERS OF DISEASE STATES. NEW HIGHLY-SPECIFIC REAGENTS ARE REQUIRED IN ORDER TO OVERCOME CURRENT LIMITATIONS IN THE DISCOVERY AND EXPLOITATION OF DISEASE-RELATED GLYCANS. USING STRUCTURALLY-GUIDED GENETIC MANIPULATIONS, WE ARE CONVERTING THE NANB SIALIDASE FROM S. PNEUMOCOCCUS INTO A HIGH-SPECIFICITY AFFINITY REAGENT FOR THE DETECTION OF ALL TYPES OF SIALIC ACID MODIFICATIONS OF GLYCOPEPTIDES AND GLYCOPROTEINS. BECAUSE SUCH A PROTEIN HAS LECTIN-LIKE PROPERTIES, BUT IS DERIVED FROM AN ENZYME, IT IS CALLED A "LECTENZ®". A NANB LECTENZ® ADDRESSES A KEY NEED IN DISEASE GLYCOMARKER DETECTION: NAMELY, A ROBUST AND EASY TO PRODUCE REAGENT SPECIFIC FOR DETECTING ALL TYPES OF SIALYLATED GLYCANS. THIS REAGENT COULD BE EMPLOYED IN AN AFFINITY MATRIX FOR SAMPLE ENRICHMENT, WHICH IN CONJUNCTION WITH EXISTING MS BASED METHODS COULD PROVIDE LINKAGE INFORMATION. GLYCOPEPTIDE SAMPLE ENRICHMENT AIDS GLYCOMIC ANALYSES BY ELIMINATING NON-GLYCOSYLATED PEPTIDES, WHICH WOULD OTHERWISE ATTENUATE THE SIGNALS FROM GLYCOPEPTIDES THAT HAVE LOW IONIZATION EFFICIENCY. GLYCOSYLATION SITE MAPPING IS ESSENTIAL IN FULLY CHARACTERIZING AND EXPLOITING GLYCANS AS MARKERS OF SPECIFIC DISEASE STATES, BUT AT PRESENT, NO REAGENT EXISTS THAT CAN DETECT SIALYLATED GLYCANS, INDEPENDENT OF THE TYPE OF LINKAGE ASSOCIATED WITH THE SIALIC ACID. THUS, AT PRESENT, A NUMBER OF REAGENTS WITH VARYING SPECIFICITIES AND AFFINITIES MUST BE EMPLOYED TO CAPTURE OR DETECT ALL FORMS OF SIALYLATED GLYCANS. LECTENZ® OFFER NUMEROUS ADVANTAGES OVER PLANT LECTINS: THEY ARE ENGINEERED TO BE HIGH AFFINITY AND YET RETAIN THE EXQUISITE SUBSTRATE SPECIFICITY OF THE ENDOGENOUS ENZYME, THEY MAY BE EFFICIENTLY PRODUCED, AND FOR HUMAN HOMOLOGUES HAVE THE POTENTIAL TO BE EMPLOYED IN VIVO WITH LOW TOXICITY. WHEREAS SOME ASPECTS OF LECTENZ® DEVELOPMENT PARALLEL THOSE OF ANTIBODY EVOLUTION, LECTENZ® HAVE THE TREMENDOUS BENEFIT OF EMPLOYING A PROTEIN NAÏVE TEMPLATE THAT HAS THE DESIRED SPECIFICITY.

STTR PHASE I: INTEGRATING VISION-GUIDED COLLABORATIVE ROBOTS FOR POSTHARVEST PROCESSING OF PRODUCE -THE BROADER IMPACT OF THIS SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PHASE I PROJECT IS TO EMPOWER THE PROCESSORS OF HARVESTED FRUITS AND VEGETABLES WITH THE FLEXIBILITY TO USE ROBOTIC AUTOMATION TO MEET THEIR LABOR NEEDS. THE AUTOMATION USES COLLABORATIVE ROBOTS (COBOTS) GUIDED BY COMPUTER VISION, WHICH ARE POTENTIALLY SAFE AROUND HUMANS. THE TECHNOLOGY WILL HELP ASSURE CONSISTENT PRODUCE QUALITY AND PROCESSING RATES. THROUGH A ROBUST COBOT-BASED SOLUTION, THE PROJECT WILL PROVIDE AN AFFORDABLE, SUSTAINABLE, AND SAFE MEANS FOR FARMS OF ALL SIZES TO KEEP UP WITH THEIR PRODUCTION GOALS, WHICH WILL SUSTAIN COMPETITION AND THE NATION?S FOOD SUPPLY. THIS PROJECT HAS THE ADDED BENEFIT OF UPSKILLING WORKERS IN FARMS BY CREATING OPENINGS FOR MORE TECHNICALLY ORIENTED POSITIONS, BOTH IN MONITORING AND MAINTAINING THE COBOTS. INSTEAD OF TEDIOUSLY PROGRAMMING THE COBOT FOR EACH USE, THE PROJECT IS INTRODUCING A NEW WAY OF TRANSLATING THE TASKS PERFORMED BY HUMANS TO THE COBOT BY LEARNING FROM CAMERA RECORDINGS. IT WILL ALSO IMPROVE UNDERSTANDING OF HOW COBOTS CAN SAFELY BE USED ALONGSIDE HUMANS IN A SHARED WORKING SPACE. THIS SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PHASE 1 PROJECT AIMS TO MAKE IT POSSIBLE TO USE COBOTS WITH HUMAN WORKERS ON TASKS THAT GO BEYOND THE TRADITIONAL PICK-AND-PLACE. THE PROPOSED TECHNOLOGY WILL AUTOMATE PROCESSING LINE TASKS THAT REQUIRE COMPUTER VISION, WHICH IS CHALLENGING BECAUSE ACCURATE AND RELIABLE PERCEPTION MUST GUIDE THE ROBOT?S MOTION. RESEARCH HAS COALESCED THE TECHNICAL CHALLENGES ON THE PATH TO A VIABLE COMMERCIAL PRODUCT AROUND FIVE STEPS. THESE START WITH A FORMAL DESCRIPTION OF THE TASK DOMAIN FOLLOWED BY USING ROBUST IMPLEMENTATIONS OF NOISE-TOLERANT MACHINE LEARNING ALGORITHMS FOR AUTOMATICALLY LEARNING THE TASK, AND END WITH A SOLUTION THAT INTEGRATES THE LEARNED TASK BEHAVIOR WITH A VISION-GUIDED COBOT SYSTEM. PHASE 1 WILL SUPPORT RESEARCH TOWARD ADDRESSING TWO PROBLEMS. THE FIRST IS TO DESIGN AN INTUITIVE WAY TO ELICIT A PRECISE SPECIFICATION OF THE CLIENT?S TASK DOMAIN. A DIGITAL CONVERSATIONAL ASSISTANT WILL UTILIZE MULTIPLE MODALITIES FOR THE ELICITATION. THE SECOND IS THE INABILITY OF AVAILABLE IMPLEMENTATIONS TO GENERATE COWORKER-AWARE AND EFFICIENT COBOT MOVEMENTS. THE RESEARCH WILL INVESTIGATE AND DEVELOP SIGNIFICANT IMPROVEMENTS TO THE COBOT MOTION TO IMPROVE COWORKER SAFETY WHILE REDUCING THE PROCESSING TIME BY AN EXPECTED 50%. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.

RECOGNITION OF O-GLCNAC MODIFIED PROTEINS USING SITE-SPECIFIC ANTIBODIES - PULMONARY ARTERIAL HYPERTENSION (PAH) IS A PROGRESSIVE DISORDER CHARACTERIZED BY HIGH BLOOD PRESSURE (HYPERTENSION) IN THE ARTERIES OF THE LUNGS (PULMONARY ARTERY) FOR NO APPARENT REASON. PAH IS A DISEASE OF PULMONARY VASCULAR ENDOTHELIAL DYSFUNCTION WITH NITRIC OXIDE (NO) DEFICIENCY AND DYSREGULATED GLUCOSE METABOLISM/UTILIZATION. IT IS A HETEROGENEOUS DISORDER LIKELY TO BE COMPRISED OF OVERLAPPING SYNDROMES WITH VARYING ORIGINS AND PATHOBIOLOGIES THAT PRESENTS WITH MANY PHENOTYPES. THE IDIOPATHIC FORM OF PULMONARY ARTERIAL HYPERTENSION (IPAH) IS PROGRESSIVE AND RESULTS IN THE DETERIORATION OF CARDIOPULMONARY FUNCTION AND PREMATURE DEATH. THE MEAN AGE AT DIAGNOSIS OF IPAH IS 50 YEARS WITH A HIGHER FEMALE TO MALE RATIO. PRESENTLY, PAH IS CONSIDERED A VASCULOPATHY, AND METABOLIC DYSREGULATION HAS EMERGED AS A MAJOR AREA OF RESEARCH IN THE PATHOBIOLOGY OF THE DISEASE. THESE METABOLIC CHANGES RESULTS IN STRUCTURAL AND MORPHOLOGICAL CHANGES WITHIN THE LUNG VASCULATURE AND CAUSE INCREASED PULMONARY ARTERY PRESSURE AND PULMONARY VASCULAR RESISTANCE, WHICH LEAD TO RIGHT VENTRICULAR FAILURE. OUR LAB AND OTHERS HAVE SHOWN THAT IPAH PATIENTS EXHIBIT ALTERED LEVELS OF NO, O-GLCNAC, GLUCOSE METABOLISM, AND INSULIN RESISTANCE. THE NATURE OF THE PRIMARY ABNORMALITIES THAT TRIGGER AND PERPETUATE THESE CHARACTERISTICS REMAINS UNCLEAR. CURRENTLY, ALL THERAPIES IN PAH TARGET VASODILATORS AND VASOCONSTRICTORS PATHWAYS (E.G. NO DEFICIENCY). THE FUTURE OF IPAH THERAPIES DEPEND ON OUR ABILITY TO IDENTIFY THE NEW MOLECULAR TARGETS WITHIN THESE PATHWAYS. HOWEVER, THE TOOLS AVAILABLE TO UNDERSTAND THE ROLE OF GLYCOSYLATION IN HUMAN HEALTH AND DISEASE ARE LACKING. IN PARTICULAR, HIGHLY SPECIFIC ANTIBODIES THAT CAN RECOGNIZE PEPTIDE SPECIFIC SEQUENCES MODIFIED BY O-GLCNAC IS NEEDED TO HELP US BETTER UNDERSTAND THE DISEASE PATHOBIOLOGY AND IMPROVE TREATMENT OF PATHOLOGIC PHENOTYPES IN IPAH. HERE WE PROPOSE TO CREATE THE FIRST ANTIBODIES TO ALLOW THE STUDY RESEARCHERS TO INVESTIGATE THE ROLE OF O-GLCNAC IN PAH. WE ANTICIPATE THAT THESE ANTIBODIES WILL HELP SHED LIGHT ON PAH DISEASE MECHANISM(S), AND POTENTIALLY OFFER NEW THERAPEUTIC APPROACHES.

DEVELOPING A PIV5-BASED HUMAN METAPNEUMOVIRUS (HMPV) VACCINE - ABSTRACT IN THIS PHASE I SBIR APPLICATION, WE PROPOSE TO DEVELOP AN INTRANASAL, PARAINFLUENZA VIRUS 5 (PIV5)-BASED HUMAN METAPNEUMOVIRUS (HMPV) VACCINE. HMPV IS ONE OF THE LEADING CAUSES OF ACUTE RESPIRATORY INFECTIONS (ARIS) IN CHILDREN, IMMUNOCOMPROMISED INDIVIDUALS, AND THE ELDERLY. ILLNESS RANGES FROM ASYMPTOMATIC INFECTION TO SEVERE BRONCHIOLITIS AND PNEUMONIA, WITH 90-100% OF CHILDREN INFECTED BETWEEN THE AGES OF 5-10 YEARS OLD. NO LICENSED HMPV VACCINE IS AVAILABLE AND THERE IS AN UNMET MEDICAL NEED TO DEVELOP A SAFE AND EFFECTIVE HMPV VACCINE. PIV5 IS A SAFE DELIVERY VECTOR FOR INTRANASAL IMMUNIZATION. THE PIV5-VECTORED COVID-19 VACCINE (CVXGA1) AND RESPIRATORY SYNCYTIAL VIRUS (RSV, A LEADING CAUSE OF LOWER RESPIRATORY TRACT INFECTION IN INFANTS AND ELDERLY) VACCINE ARE CURRENTLY IN PHASE 1 CLINICAL TESTING. PRECLINICAL DATA FOR PIV5-BASED RSV CANDIDATE VACCINES HAVE SHOWN EXCELLENT IMMUNOGENICITY, PROTECTION, AND SAFETY PROFILES IN VARIOUS ANIMAL MODELS, INCLUDING THE COTTON RAT MODEL, DEMONSTRATING LACK OF VACCINE-INDUCED ENHANCED DISEASE OBSERVED FOLLOWING FORMALIN-INACTIVATED RSV VACCINATION. IN THIS GRANT PROPOSAL, WE WILL PRODUCE TWO PIV5-BASED HMPV CANDIDATE VACCINE CONSTRUCTS, ONE IN THE W3A STRAIN WITH THE SH GENE DELETED (W3A!SH-HMPV-F) AND ANOTHER IN THE CANINE PARAINFLUENZA VIRUS (CPI) VACCINE STRAIN (CPI-HMPV-F) TO COMPARE THEIR REPLICATION IN VITRO, ANTIGEN EXPRESSION IN VITRO, IMMUNOGENICITY, AND PROTECTION AGAINST HMPV CHALLENGE INFECTION IN A MOUSE MODEL. THE NOVELTY OF THE VACCINE PROPOSED IN THIS PHASE I SBIR APPLICATION RELATES TO: 1) THE USE OF A CHIMERIC HMPV F PROTEIN CONTAINING THE PIV5 F CYTOPLASMIC TAIL TO POTENTIALLY INCREASE HMPV F ANTIGEN EXPOSURE ON THE VIRION SURFACE 2) A NEEDLE-FREE INTRANASAL DELIVERY METHOD IN A SAFE, HIGHLY IMMUNOGENIC VIRAL VECTOR AND 3) THE ABILITY TO INDUCE MUCOSAL IMMUNITY, WHICH IS NECESSARY FOR PROTECTING AGAINST RESPIRATORY PATHOGENS. ONCE THE PIV5-VECTORED HMPV VACCINE IS DEMONSTRATED TO BE IMMUNOGENIC IN THE MOUSE MODEL, THE PHASE II SBIR PROPOSAL WILL FOCUS ON PRECLINICAL STUDIES NEEDED FOR ENTERING PHASE 1 CLINICAL TRIALS WITH THE FINAL GOAL OF GENERATING A BIVALENT PIV5- VECTORED RSV AND HMPV VACCINE WHICH WOULD PROVIDE PROTECTION AGAINST THE TWO LEADING CAUSES OF LOWER RESPIRATORY TRACT INFECTIONS IN INFANTS AND ELDERLY.

DEVELOPING A LIQUID FORMULATION FOR CVXGA1 COVID-19 INTRANASAL VACCINE - DEVELOPING A LIQUID FORMULATION FOR CVXGA1 COVID-19 INTRANASAL VACCINE ABSTRACT IN THIS PHASE I SBIR APPLICATION, WE PROPOSE TO DEVELOP A LIQUID FORMULATION FOR CVXGA1 COVID-19 INTRANASAL VACCINE. THE ONGOING GLOBAL CORONAVIRUS DISEASE 2019 (COVID-19) PANDEMIC HAS RESULTED IN THE LOSS OF MANY LIVES AND SOCIAL ECONOMIC DISRUPTIONS. VARIANTS WITH HIGHER TRANSMISSION RATES AND ABILITY TO EVADE VACCINE OR NATURAL INFECTION ELICITED HOST IMMUNE RESPONSES HAVE CONTINUOUSLY EMERGED LEADING TO BREAKTHROUGH INFECTIONS DESPITE ADDITIONAL BOOSTER DOSES. THE NEXT GENERATION OF COVID-19 VACCINE, THAT IS ALSO ACCESSIBLE TO THE DEVELOPING COUNTRIES, IS NEEDED TO STOP SARS-COV-2 TRANSMISSION. PARAINFLUENZA VIRUS TYPE 5 (PIV5) IS AN EXCELLENT VIRAL VECTOR FOR EXPRESSING FOREIGN ANTIGENS TO FIGHT MANY VIRAL AND BACTERIAL PATHOGENS. A SINGLE INTRANASAL IMMUNIZATION WITH CVXGA1 (PIV5 EXPRESSING SARS-COV-2 S PROTEIN) INDUCES MUCOSAL, HUMORAL, AND CELLULAR IMMUNE RESPONSES, PROVIDING PROTECTION AGAINST SARS-COV-2 CHALLENGE IN MICE, HAMSTERS, FERRETS, CATS, AND AFRICA GREEN MONKEYS. CVXGA1 ALSO BLOCKED CONTACT TRANSMISSION. CVXGA1 VACCINE IS CURRENTLY IN PHASE 1 CLINICAL STUDIES (NCT04954287). THE CURRENT CVXGA1 VACCINE IS FORMULATED IN SUCROSE PHOSPHATE GLUTAMINE (SPG) BUFFER WHICH IS STABLE AT -80 OC AND -20 OC BUT NOT STABLE AT 4 OC. THE 4 OC STABLE LIQUID FORMULATION IS CRITICAL TO THE LAUNCH OF THIS INTRANASAL VACCINE FOR GLOBAL IMMUNIZATION. WE PROPOSE TO IDENTIFY A FORMULATION WHICH CAN PROVIDE CVXGA1 STABILITY AT 4 OC FOR A MINIMUM OF 4 MONTHS. WE PROPOSE TO FIRST OPTIMIZE THE PH FOR CVXGA1 STORAGE, THEN SCREEN MULTIPLE EXCIPIENTS TO SELECT AT LEAST TWO LEAD FORMULATIONS FOR FURTHER CHARACTERIZATION, INCLUDING IMMUNOGENICITY STUDIES. THE PHASE II SBIR PROPOSAL WILL FOCUS ON LONG-TERM STORAGE OF CVXGA1 IN THE CHOSEN DOSE DEVICE (SUCH AS PREFILLED SPRAYER) AND VACCINE EVALUATION AFTER STORAGE.

EXPANDING THE GLYCOMICS TOOLBOX: CHEMO-ENZYMATIC SYNTHESIS OF WELL-DEFINED N-GLYCAN STANDARDS - PROJECT SUMMARY / ABSTRACT: SIGNIFICANCE: N-LINKED PROTEIN GLYCOSYLATION REPRESENTS AN IMPORTANT CLASS OF BIOMOLECULES RECEIVING SIGNIFICANT ATTENTION FOR THEIR APPLICATION IN DISEASE DIAGNOSIS AND THERAPEUTIC DEVELOPMENT. A MAJOR HURDLE WITH RAPID AND FACILE N-GLYCAN STRUCTURAL IDENTIFICATION STEMS FROM THE LACK OF WELL-DEFINED REAGENTS THAT CAN BE USED AS BIOANALYTICAL REFERENCE MATERIAL. THE INABILITY TO ROBUSTLY INTERPRET PROTEIN N-GLYCOSYLATION PREVENTS DEVELOPMENT OF NEW TECHNOLOGIES FOR UNCOVERING BIOMARKERS AND HINDERS THE ADVANCEMENT OF RECOMBINANT GLYCOPROTEIN ENGINEERING, SUCH AS ANTIBODIES. CURRENT COMMERCIALLY AVAILABLE REFERENCE MATERIAL EITHER PROVIDES A LIMITED NUMBER OF N-GLYCANS WHICH ARE NOT REPRESENTATIVE OF BIOLOGICAL COMPOSITION OR LACK DETAILS REGARDING EXACT STRUCTURAL ASSEMBLY. OUR LIBRARY WILL COMPRISE THE LARGEST COMMERCIALLY AVAILABLE COLLECTION OF WELL-DEFINED N- GLYCANS AND WILL REPRESENT A SIGNIFICANT ADVANCEMENT TO CURRENT AVAILABLE STANDARDS. STRATEGY: TAKING ADVANTAGE OF THE STRICT SUBSTRATE REQUIREMENTS OF GLYCOSYLTRANSFERASES AND HYDROLASES, AN ENZYMATIC APPROACH WILL BE UTILIZED TO PRODUCE A COLLECTION OF HIGH PURITY STRUCTURES. SPECIFIC AIM: THIS PROJECT DESCRIBES A ROBUST SEMI- SYNTHETIC, ENZYMATIC METHODOLOGY FOR PRODUCING THE 20 MOST ABUNDANT N-GLYCANS FOUND IN HUMAN SERUM ALL HAVING AN ABUNDANCE GREATER THAN 1%, AND TOGETHER, COMPRISE GREATER THAN 50% OF THE TOTAL SERUM N-GLYCOME. EACH COMPOUND WILL HAVE A PURITY OF >99% AND WILL CONTAIN A FREE-REDUCING ANOMERIC TERMINUS PROVIDING FLEXIBLITY FOR USE IN A MULTITUDE OF ANALYTICAL TECHNIQUES. TASK 1: EXTRACTION OF A SIALYLATED GLYCOPEPTIDE FROM COMMERCIALLY AVAILABLE EGG YOLK POWDER AND THE PREPARATION OF AN ADVANCED INTERMEDIATE WHICH IS COMMON TO ALL DESIRED TARGETS. TASK 2: SYNTHESIS OF BI-ANTENNARY TARGETS WITH SYMMETRIC AND ASYMMETRIC ARCHITECTURES. TASK 3: SYNTHESIZE A COLLECTION OF TRI-ANTENNARY, SIALYLATED REGIO-ISOMERS. TASK 4: DEVELOPMENT OF A SECOND PRODUCT LINE WHERE EACH N-GLYCAN CONTAINS ONE UNIFORMLY LABELED 13C6 GLCNAC WHICH CAN BE USED AS AN INTERNAL REFERENCE FOR QUANTIFICATION. PRELIMINARY DATA: A SCALABLE EXTRACTION STRATEGY HAS BEEN DEVISED FOR ACCESSING GRAM- QUANTITIES OF THE DESIRED SIALYLATED GLYCOPEPTIDE STARTING MATERIAL FROM EGG YOLK POWDER USING ROBUST EXTRACTION TECHNOLOGY. THE SYNTHETIC METHODOLOGIES TO BE EMPLOYED IN TASKS 1 – 4 HAVE BEEN VALIDATED REMOVING THE NEED FOR FURTHER OPTIMIZATION. DELIVERABLES: WHEN COMPLETE, THE DESCRIBED LIBRARY WILL REPRESENT THE MOST COMPREHENSIVE COLLECTION OF STRUCTURALLY DEFINED N-GLYCAN STANDARDS AND WILL DELIVER AN URGENTLY NEEDED TOOL TO THE SCIENTIFIC COMMUNITY.

CHEMOENZYMATIC SYNTHESIS OF A HUMAN MILK OLIGOSACCHARIDE ARRAY FOR DISCOVERY OF THERAPEUTIC LEADS - PROJECT SUMMARY / ABSTRACT: HUMAN MILK OLIGOSACCHARIDES (HMOS) ARE THE THIRD LARGEST COMPONENT OF BREAST MILK AND HAVE BEEN DESCRIBED TO CONVEY DEVELOPMENTAL BENEFITS TO THE NEWBORN. HMOS MAY COMPRISE A WEALTH OF UNTAPPED THERAPEUTIC OPPORTUNITIES AS THIS UNIQUE CLASS OF MOLECULES CAN INTERACT WITH, AND THEREBY POTENTIALLY MODULATE, MEDIATORS OF INFLAMMATION. UNFORTUNATELY, INVESTIGATION INTO THE THERAPEUTIC UTILITY OF HMOS HAS BEEN HINDERED BY LACK OF ACCESS TO INDIVIDUAL STRUCTURES IN APPROPRIATE QUANTITY AND PURITY FOR THOROUGH BIOLOGICAL EVALUATION AND PRECLINICAL DEVELOPMENT. CURRENT METHODS FOR EVALUATING THE BIOLOGICAL ROLE OF HMOS RELY ON: 1) PROBING A HETEROGENEOUS MIXTURE OF HMOS BEARING SIMILAR STRUCTURAL CHARACTERISTICS OR 2) ATTEMPTING TO ISOLATE INDIVIDUAL STRUCTURES FROM POOLED MILK USING EXHAUSTIVE CHROMATOGRAPHY TECHNIQUES. NEITHER STRATEGY PROVIDES A PLATFORM FOR READILY EXAMINING INDIVIDUAL HMOS AGAINST A PANEL OF BIOLOGICAL TARGETS, NOR AN EFFICIENT STRATEGY FOR TARGET SCALE-UP REQUIRED FOR MODE-OF-ACTION, STRUCTURE-ACTIVITY RELATIONSHIPS, AND PRE-CLINICAL DEVELOPMENT. THERE IS AN URGENT NEED TO DEVELOP A SYNTHETIC STRATEGY THAT CAN ACCESS WELL-DEFINED HMOS FOR HIGH-THROUGHPUT SCREENING AND IS SCALABLE TO PROVIDE IDENTIFIED LEADS IN APPROPRIATE QUANTITY FOR BIOLOGICAL EVALUATION AND LIGAND OPTIMIZATION. TO ADDRESS THE PROBLEM OF COMPOUND AVAILABILITY, A CHEMOENZYMATIC SYNTHETIC STRATEGY WILL BE EMPLOYED TO DEVELOP A LIBRARY COMPRISING 100 HMOS. THE STRATEGY AIMS AT CHEMICALLY SYNTHESIZING A LIMITED NUMBER OF ADVANCED INTERMEDIATES WHICH CAN BE FURTHER MODIFIED BY A PANEL OF GLYCOSYLTRANSFERASES TO PROVIDE A COLLECTION OF TARGETS WITH SIGNIFICANT STRUCTURAL DIVERSITY. THE LIBRARY WILL BE IMMOBILIZED ON MICROARRAY SLIDES FOR HIGH-THROUGHPUT SCREENING AND IN FIRST INSTANCE, WILL BE SCREENED WITH CARBOHYDRATE RECOGNIZING PROTEINS INVOLVED IN INTESTINAL INFLAMMATION. IDENTIFIED LEADS WILL BE EXAMINED FOR ANTI-INFLAMMATORY EFFICACY USING AN IN VITRO DENDRITIC CELL MODEL. FINALLY, A COMPLEMENTARY, CONVERGENT SYNTHETIC APPROACH WILL BE DEVELOPED TO PROVIDE LARGE-SCALE ACCESS TO EFFICACIOUS HITS REQUIRED FOR IN VIVO ANIMAL STUDIES AND PRECLINICAL DEVELOPMENT. THE SHORT-TERM GOAL OF THIS PROPOSAL IS THE SUCCESSFUL SYNTHESIS OF THE LARGEST COLLECTION OF SYNTHETIC HMOS TO DATE FOR ASSAY IMPLEMENTATION AND EARLY PHARMACOLOGICAL SCREENING. ADDITIONALLY, OUR CONVERGENT SYNTHETIC METHODOLOGY WILL PROVIDE A MEANS TO SCALE-UP ANY HMO LIBRARY MEMBER IN APPROPRIATE QUANTITY FOR PRECLINICAL DEVELOPMENT. THE LONG-TERM GOAL IS TO USE THIS METHODOLOGY TO FACILITATE THE DEVELOPMENT OF HMOS TO ADDRESS DEFICIENCIES IN NEONATAL AND INFANT FORMULAS AS WELL AS INVESTIGATE POTENTIAL AVENUES FOR DEVELOPMENT OF ANTI- INFLAMMATORY THERAPEUTICS.