THIS IS LARRY TABAK. AS THE NIHERS KNOW, WHEN THEY SEE ME, IT USUALLY MEANS THAT FRANCIS HAD SOMETHING ELSE TO DO, BUT ACTUALLY THE TRUTH IS, I FOUGHT HIM FOR THE ABILITY TO INTRODUCE OUR SPEAKER TODAY, CAROLYN BERTOZZI, BECAUSE OF MY OWN PERSONAL INTEREST IN THE GLYCOBIOLOGY FIELD, SO IT'S REALLY WONDERFUL TO BE ABLE TO WELCOME CAROLYN VIRTUALLY. AS YOU KNOW, WE'VE BEEN DOING THE NIH DIRECTOR'S WEDNESDAY AFTERNOON LECTURE SERIES, THE WALS LECTURE SERIES, AS AN AT-HOME EDITION, AS SOME ARE CALLING IT. WE'RE GOING TO BE DOING THIS REMOTE-ONLY THROUGH DECEMBER, AND OBVIOUSLY WE'LL SEE WHAT NEXT YEAR BRINGS. NEVERTHELESS, WE'VE REALLY BEEN THRILLED TO SEE THAT THE WALS HAS REMAINED SUCH A ROBUST VENUE FOR SHARING GREAT IDEAS. OF COURSE NOTHING BEATS IN-PERSON LECTURES, BUT THE FACT THAT SO MANY OF YOU ARE TUNING IN EACH WEEK TO HEAR THESE GREAT TALKS IS BOTH A REASSURING AND POSITIVE SIGN THAT THE NIH COMMUNITY REMAINS AS FOCUSED AS EVER ON OUR MISSION. IT'S MY GREAT PLEASURE TO INTRODUCE DR. CAROLYN BERTOZZI, THE ANNE AND ROBERT BASS PROFESSOR OF CHEMISTRY AT STANFORD UNIVERSITY, AND HOWARD HUGHES MEDICAL INSTITUTE INVESTIGATORS. DR. BERTOZZI'S ACCOMPLISHMENTS ARE MANY. IN PARTICULAR, SHE HAS REALLY ESTABLISHED AN ENTIRE NEW FIELD OF CHEMISTRY TERMED BIOORTHOGONAL CHEMISTRY, ALMOST TWO DECADES AGO NOW, AND THIS AS MANY OF YOU KNOW REFERS TO CHEMICAL REACTIONS THAT CAN BE TRIGGERED INSIDE A LIVING CELL WITHOUT INTERFERING OR PERTURBING THE NATIVE BIOCHEMICAL PROCESSES. IT'S A NEAT TRICK, AND OFFERS UP EXTRAORDINARY OPPORTUNITIES FROM GLYCOENGINEERING TO PROTEIN AND LIPID MODIFICATION, TO CELL SURFACE IMAGING. AND WHILE LABS NOW AROUND THE GLOBE ARE USING THESE APPROACHES, DR. BERTOZZI'S OWN LAB HAS USED THIS PLATFORM FOR IN SITU DRUG ASSEMBLY, GLYCOPROTEOMICS-BASED DISCOVERY OF CANCER, AND STEM CELL BIOMARKERS, AND FUNDAMENTAL REACTION ETHODOLOGY. SO THE ACCOMPLISHMENTS HAVE BEEN MANY. HER MOST RECENT WORK HAS FOCUSED ON PATHOGENIC GLYCANS IN TUMOR MICRO ENVIRONMENTS, AND NEW THERAPEUTIC MODALITIES BASED ON THE CONCEPT OF TARGETED DEGRADATION. A VERY BRIEF OUTLINE OF HER BACKGROUND AND MANY, MANY HONORS. SHE RECEIVED HER AB FROM HARVARD, A PH.D. IN CHEMISTRY FROM BERKLEY, AND I GUESS DECIDED TO STAY ON THE WEST COAST, WHERE SHE HAS BUILT A REMARKABLE CAREER. AGAIN I MENTIONED THE HONORS WHICH ARE NUMEROUS. SHE IS A MEMBER OF THE NATIONAL ACADEMY OF SCIENCES, THE NATIONAL ACADEMY OF MEDICINE, THE NATIONAL ACADEMY OF INVENTORS, THE AMERICAN ACADEMY OF ARTS AND SCIENCES, AND THE ROYAL SOCIETY AS A FOREIGN MEMBER. THE LIST OF AWARDS THAT WERE HANDED TO ME WAS PRINTED IN 8-POINT FONT SO THERE'S JUST NO WAY I CAN READ THESE THINGS, BUT THERE ARE A COUPLE THAT I'LL JUST MENTION THAT STAND OUT. THE PRESIDENT'S INNOVATOR AWARD FROM THE SOCIETY FOR GLYCOBIOLOGY, CHEMISTRY FOR FUTURE -- PRIZE, AND THE NATIONAL ACADEMY OF SCIENCES JOHN J. CARDY AWARD FOR THE ADVANCEMENT OF SCIENCE. WE ARE ABSOLUTELY DELIGHTED FOR CAROLYN TO BE WITH US TODAY. THE TITLE OF HER TALK: THERAPEUTIC OPPORTUNITIES IN GLYCOSCIENCE. CAROLYN, THANK YOU FOR BEING WITH US, AND WELCOME. >> THANK YOU, LARRY, AND LET ME JUST START BY THANKING ALL OF THE WONDERFUL SCIENTISTS AT NIH. THANK YOU FOR YOUR SERVICE AND THANK YOU FOR JOINING US TODAY, AND HOPEFULLY YOU'LL ENJOY HEARING ABOUT SOME OF OUR LATEST WORK. IN THIS BROAD AREA OF FOCUS OF MY LAB, WHICH IS TO BASICALLY MINE THE FIELD OF GLYCOSCIENCE FOR THERAPEUTIC OPPORTUNITIES. I THOUGHT I WOULD JUST START WITH AN OVERVIEW OF THE RESEARCH PROJECTS IN MY LAB. JUST TO KIND OF GIVE YOU A HIGH LEVEL OF HOW MUCH ENJOYMENT THAT WE HAVE HAD FROM WORKING IN THE FIELD OF GLYCOSCIENCE. IT'S AN AREA THAT I FIRST LEARNED ABOUT AS A GRADUATE STUDENT STARTING WAY BACK IN 1988 AT UC BERKLEY WITH A YOUNG ASSISTANT PROFESSOR WHO INTRODUCED ME TO CARBOHYDRATE CHEMISTRY, AND I JUST GOT HOOKED, I THOUGHT THE MOLECULES WERE BEAUTIFUL AND COMPLICATED, AND THE BIOLOGY WAS AT THAT TIME EVEN MORE SO THAN TODAY QUITE MYSTERIOUS, BUT CLEARLY QUITE IMPORTANT AS WELL. SO WHEN I STARTED MY OWN LAB 25ISH YEARS AGO, IT WAS WITH THE MISSION OF BASICALLY BRINGING CHEMISTRY AND BIOLOGY TOGETHER UNDER ONE ROOF TO TRY AND ADVANCE THIS FIELD OF GLYCOSCIENCE, AND I FOUND TO BE INCREDIBLY FERTILE WITH ALL KINDS OF INTERESTING OPPORTUNITIES. SO IF YOU TOOK A SNAPSHOT OF MY LAB TODAY, YOU WOULD SEE THAT WE'RE VERY INTERESTED IN WHAT WE CALL DECODING THE CANCER GLYCOCALYX, WHICH HAS LONG BEEN KNOWN TO BE SORT OF AN ALTERED MOLECULAR SIGNATURE OF MALIGNANCY WHOSE FUNCTIONAL SIGNIFICANCE HAS NOT BEEN VERY WELL UNDERSTOOD, AND WE WORK ON THAT. AND ALONG THE LINES, WE'VE DEVELOPED SOME TOOLS IN WHAT WE CALL CHEMICAL GLYCOPROTEOMICS. THIS HAS ALLOWED US TO DEPLOY SOME CUTTING EDGE MASS SPECTROMETRY TECHNOLOGIES TO HELP DEFINE THE GLYCO SIGNATURES IN CANCER, AND WE'VE ALSO, FROM OUR LEARNINGS IN THE BASIC SCIENCE, COME UP WITH SOME IDEAS FOR THERAPEUTIC INTERVENTIONS, AND LOOKING BACK THROUGH MY FILES, THE LAST TIME I GAVE A TALK AT NIH, WHICH WAS NOW FOUR OR FIVE YEARS AGO, I TALKED ABOUT THE DEVELOPMENT OF ANTIBODY ENZYME CONJUGATES THAT WE USE BASICALLY FOR TARGETED REMODELING OF THE CANCER GLYCOCALYX FOR AN APPLICATION FOR CANCER IMMUNE THERAPY, SO I THOUGHT TODAY WHAT I WOULD DO IS INSTEAD TALK ABOUT A DIFFERENT AND NEWER THERAPEUTIC MODALITY THAT WE HAVE BEEN WORKING ON, WHICH WE CALL THE LYTAC TECHNOLOGY. THAT'S AN ACRONYM THAT STANDS FOR LYSOSOME TARGETING CHIMERAS. AS YOU'LL SEE, OUR DEVELOPMENT OF LYTACs WAS VERY MUCH ROOTED IN SOME FUNDAMENTAL CELLULAR GLYCOSCIENCE. THEN FINALLY FOR THE SAKE OF COMPLETION, WE HAVE A PROGRAM IN MY LAB THAT FOCUSES ON STUDIES OF MICRO BACTERIUM TUBERCULOSIS AND UNDERSTANDING THE GLYCOSCIENCE OF THE CELL WALL AND THE CELL MEMBRANE, AGAIN WITH AN EYE FOR DEVELOPING DIAGNOSTICS AND THERAPEUTICS. NOFN OF THIS WOULD HAVE BEEN POSSIBLE WITHOUT CONTINUOUS AND VERY GENEROUS SUPPORT FROM VARIOUS INSTITUTES AT THE NIH AND IT ALL BEGAN WITH NIGMS, MY VERY FIRST NIH GRANT TO STUDY CANCER GLYCOSYLATION CAME FROM THAT INSTITUTE AND SINCE THEN I'VE BEEN LUCKY TO HAVE SUPPORT FROM THE NATIONAL CANCER INSTITUTE ON SOME OF THESE PROJECTS AS WELL AS FROM THE NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASE FOR OUR TUBERCULOSIS PROJECT. IN THAT CONTEXT, I'VE GOTTEN TO KNOW THE INCREDIBLE SCIENTISTS AND PROGRAM OFFICERS AT NIH WHO TURNED OUT TO BE STAUNCH SUPPORTERS OF GLYCOSCIENCE, AND I THINK HISTORY WILL LOOK BACK ON NIH'S DECADE OF INVESTMENT IN GLYOSCIENCE THROUGH THE PORTAL OF THE COMMON FUND AND OTHER INITIATIVES AS BEING INCREDIBLY TRANSFORMATIVE OF THE FIELD. I'VE BEEN A DIRECT BENEFICIARY OF THOSE INVESTMENTS. SO THIS MY OPPORTUNITY TO SAY THANK YOU TO ALL OF YOU. OKAY. SO BEFORE I TALK ABOUT THE SCIENCE, LET ME JUST DISCLOSE MY CONFLICTS OF INTEREST. I AM COFOUNDER AND SCIENTIFIC ADVISORY BOARD CHAIR OF TWO RELATIVELY NEW BIOTECH COMPANIES THAT BOTH HAVE AN INTEREST IN TARGETED DEGRADATION STRATEGIES FOR CANCER THERAPY AND OTHER THERAPEUTIC AREAS. SO THE WORK THAT I'M TALKING ABOUT TODAY HAS BEEN LICENSED EXCLUSIVELY BY THIS COMPANY, LYCIA THERAPEUTICS, AND THERE ARE SOME RELATED TECHNOLOGIES THAT ARE BEING PURSUED BY PALLEON PHARMACEUTICAL, AND FINALLY I'M A MEMBER OF THE BOARD OF DIRECTORS OF ELI LILLY. WITH THAT, LET ME INTRODUCE THE TWO CO-WORKERS WHOSE RESEARCH I WILL TALK ABOUT TODAY AROUND THIS LYSOSOME TARGET CHIMERA TECHNOLOGY. THE FIRST IS POSTDOC STEVEN BANIK, WHO CAME TO MY LAB WITH A TRAINING IN WHAT WE CALL HARD CORE CHEMISTRY, NO GLYCOSCIENCE, NO BIOLOGY. AND IN THE THREE YEARS THAT HE'S BEEN A POSTDOC IN MY LAB, HE HAS LAUNCHED THIS AMAZING PROJECT WITH SO MUCH POTENTIAL FOR NEW THERAPEUTIC MODALITIES, SO I OWE HIM A DEEP DEBT OF GRATITUDE FOR HIS CREATIVITY AND CONTRIBUTIONS, AND I'M ALSO VERY LUCKY THAT WE WERE ABLE TO LAND HIM AS A NEW PROFESSOR IN STANFORD'S CHEMISTRY DEPARTMENT, WHERE HE WILL BEGIN HIS OWN INDEPENDENT POSITION IN JANUARY. AND MEANWHILE, HE HAS HANDED THE BATON TO FOURTH YEAR GRADUATE STUDENT GREEN AHN, WHO HAS TAKEN THE PROJECT TO A WHOLE NEW LEVEL, AND I'LL BE TALKING ABOUT HER WORK IN THE SECOND PART OF THE TALK. SO LET ME START BY TALKING ABOUT THE CONCEPT OF TARGETED DEGRADATION AS A NEW THERAPEUTIC MODALITY. THIS IS A FIELD THAT WAS CONCEPTUALLY LAUNCHED BY A GOOD FRIEND OF MINE, CRAIG C REWS. CRAIG STARTED HIS OWN INDEPENDENT LAB BACK IN 1995, THE SAME YEAR THAT I DID. HE WAS AND STILL IS AT YALE UNIVERSITY, AND HE STUDIED BASICALLY THE UBIQUITIN PROTEASOME PATHWAY FOR INTERCELLULAR PROTEIN DEGRADATION. AND HE DEVELOPED A TECHNOLOGY IN WHICH A SMALL MOLECULE CAN TARGET A PROTEIN NOT JUST FOR INHIBITION BUT TO BE DEGRADED BY THIS PROTEASOME SYSTEM. AND HE WROTE THIS VERY NICE REVIEW ARTICLE A FEW YEARS AGO, ONE OF MANY THAT HE'S WRITTEN, DESCRIBING HOW THIS KIND OF DEGRADATION IS A REALLY DIFFERENT AND POTENTALLY SUPERIOR PHARMACOLOGY FOR DRUGGING A TARGET AS COMPARED TO THE MORE CONVENTIONAL DRUG MOA, WHICH IS TO JUST INHIBIT THE TARGET. AND SO AS SHOWN IN THIS CARTOON, PICTURE A SCENARIO WHERE THERE'S A PROTEIN IN A DISEASE SETTING THAT'S PATHOGENIC AND YOU WANT TO INHIBIT ITS FUNCTION, SO IT'S AN ENZYME AND YOU WANT TO MAKE AN INHIBITOR OF ITS ACTIVE SITE, FOR EXAMPLE. YOU MAKE A DRUG AND WHEN THE DRUG IS PRESENT, IT BINDS IN AN EQUILIBRIUM WITH THAT PROTEIN, SOME FRACTION OF THE PROTEIN IS PLUGGED UP WITH YOUR INHIBITOR SO YOU'RE BASICALLY TAKING THE BIOLOGY FROM A DISEASE SETTING TO A NORMAL SETTING THROUGH WHAT WOULD BE CALLED OCCUPANCY DRIVEN PHARMACOLOGY. SO THE TARGET IS OCCUPIED, AT LEAST IN PART BY THIS DRUG. AND THAT WORKS IN A LOT OF SETTINGS AND THERE ARE OF COURSE VERY IMPORTANT MEDICINES THAT FUNCTION THROUGH THIS OCCUPANCY-DRIVEN PHARMACOLOGY, BUT WHAT WE ALSO NOW KNOW IS THAT THERE ARE MANY TARGETS THAT ARE NOT SO AMENABLE TO OCCUPANCY DRIVEN PHARMACOLOGY, THAT IS, THEY ARE THE SO CALLED UNDRUGGABLE PROTEINS. MAYBE THEY DON'T HAVE AN ACTIVE SITE THAT'S THE RIGHT SHAPE FOR A SMALL MOLECULE DRUG TO BIND WITH HIGH AFFINITY, FOR EXAMPLE. PROTEINS THAT FALL INTO THIS HISTORICALLY UNDRUGGABLE CATEGORY INCLUDE TRANSCRIPTION FACTORS, SCAFFOLDING PROTEINS, SOMETIMES PROTEINS WITH LOTS OF TRANS MEMBRANE DOMAINS BUT NOT A LOT OUTSIDE OF THE MEMBRANE. MANY PROTEINS THAT WE KNOW ARE PATHOGENIC DRIVERS IN DISEASE, BUT HAVE NOT BEEN ABLE TO MAKE DRUGS AGAINST. AND IT WAS THE FRUSTRATION AROUND THESE KINDS OF TARGETS THAT MOTIVATED CRAIG AND NOW MANY OTHER RESEARCHERS TO DEVELOP THIS ALTERNATIVE APPROACH THROUGH DEGRADATION. SO HERE THE IDEA IS THAT WHAT COULD TAKE THE STATE FROM BEING A DISEASE STATE TO THE HEALTHY STATE NOT BY JUST BLOCKING THE PROTEIN BUT LITERALLY REMOVING IT ALL TOGETHER, TARGETING FOR DEGRADATION, AND THESE UNDRUGGABLE PROTEINS, ACTUALLY SOME OF THEM ARE REASONABLE CANDIDATE FOR A DEGRADER TECHNOLOGY. NOW OVER THE LAST 20 YEARS, THIS CONCEPT OF DEGRADATION HAS GONE FROM THESE EARLY ACADEMIC PROOF OF CONCEPT EXPERIMENTS OUT OF CRAIG CREWS' LAB ALL THE WAY TO A HUGE INDUSTRY, MANY COMPANIES FOCUSED ENTIRELY ON THIS, EVERY MAJOR BIOPHARMA COMPANY HAS A PLAY IN TARGETED DEGRADATION. THERE ARE MANY OF THESE SO-CALLED TPD OR TARGETED PROTEIN DEGRADATION PLATFORMS THAT ARE BEING TRANSLATED INTO HUMAN MEDICINES. SO THE EARLY WAVE OF THESE TECHNOLOGIES UTILIZE THIS UBIQUITIN PROTEASOME SYSTEM, SO THIS IS A PRIMARY PATHWAY TO DEGRADE CYTOSOLIC PROTEINS. THERE ARE TWO TECHNOLOGIES NOW, BOTH OF WHICH ARE IN HUMAN CLINICAL TESTING, THAT USE THIS SYSTEM TO DEGRADE A TARGET OF INTEREST. THERE'S CRAIG'S PROTACs, THAT'S AN ACRONYM FOR PROTEOLYSIS TARGETING CHIMERA, AND THE PROTAC IS A BIFUNCTIONAL MOLECULE WHERE ONE PART BINDS THE TARGET OF INTEREST AND THE OTHER PART BINDS AN E3 LIE GAS ENZYME. BASICALLY THE MOLECULE BRIDGES THE DIVIDE BETWEEN A TARGET AND THE E3 LIGASE, WHICH IS AN ENZYME THAT'S CRITICAL FOR PUTTING UBIQUITIN ON TO THAT TARGET AND DIRECTING IT FOR DEGRADATION. THEN THERE'S THE IMID CLASS OF MOLECULES VERY MUCH LIKE PROTACs BUT THEY TEND TO BE SMALLER MOLECULES INSPIRED BY THE DRUG THALIDOMIDE, A NOTORIOUS DRUG FROM THE PAST BUT HAS TURNED INTO A VERY IMPORTANT CANCER MECHANISM WHOSE MECHANISM OF ACTION WAS DISCOVERED A DECADE AFTER IT WAS BEING USED TO TREAT PATIENTS, BUT WE NOW KNOW IT'S BASICALLY A TARGETING FOR DEGRADATION. THEN MORE RECENTLY, PEOPLE HAVE COME UP WITH REALLY CLEVER WAYS OF TARGETING INTRACELLULAR PATHWAYS FOR AUTOPHAGY, PLATFORMS CALLED THE ATTECS AND AUTACS, THAT BIND ON ONE HAND AND MACHINERY THAT TARGETS IT FOR THE OTHER. SO MY LAB HAS BEEN FOLLOWING THIS STORY BECAUSE IT'S SUCH A GREAT EXAMPLE OF TRANSLATIONAL CHEMICAL BIOLOGY. IT SUCH A GREAT STORY FOR TAKING A COOL CHEMICAL BUY BIOLOGY CONCEPT FROM ACADEMIA TO HUMAN -- BUT WE WERE ALSO KIND OF FRUSTRATED WITH WHAT WAS GOING ON BECAUSE ALL OF THESE BEAUTIFUL TECHNOLOGIES WERE LIMITED TO PROTEINS THAT WERE IN THE CYTOSOL, INSIDE THE CELL, BECAUSE THAT'S WHERE THE PROTEASOME MACHINERY IS, AND THAT'S WHERE THE AUTOPHAGY MACHINERY IS, AND MEANWHILE, THERE'S LOTS OF PROTEINS THAT ARE OUTSIDE THE CELL THAT ARE DISEASE-RELEVANT PROTEINS THAT ONE MIGHT WANT TO DEGRADE IN A THERAPEUTIC STRATEGY. AND THE ESTIMATES FROM INFORMATICS ANALYSES SUGGEST THAT THIS COULD BE ABOUT 40% OF THE HUMAN PROTEOME. SO THESE ARE GOING TO BE PROTEINS THAT ARE SECRETED, THEY'RE JUST RELEASED FROM THE CELL OUT INTO CIRCULATION, INTO TISSUES, BODY FLUIDS, THEY DON'T SPEND TIME IN THE CYTOSOL AT ALL, THEREFORE ARE NOT ACCESSIBLE TO THE PROTEASOME. AND ALSO MANY MEMBRANE-ASSOCIATED PROTEINS HAVE A MAJOR DOMAIN OUTSIDE THE CELL BUT NOT REALLY ENOUGH INSIDE ON THE CYTOPLASMIC SIDE OF THE MEMBRANE IN ORDER TO BE LIKE BOUND THROUGH A SMALL MOLECULE LIGAND. AND AMONG THIS UNIVERSE OF EXTRACELLULAR PROTEINS, THERE ARE SOME VERY IMPORTANT POTENTIAL THERAPEUTIC TARGETS. FOR EXAMPLE, CYTOKINES, CHEMOKINES, THESE PRO INFLAMMATORY MOLECULES INVOLVED IN CYTOKINE STORMS AS WELL AS IN CHRONIC INFLAMMATORY CONDITIONS, IMMUNE COMPLEXES THAT CAN CAUSE KIDNEY DAMAGE, A VARIETY OF AMYLOID-TYPE DISEASES, YOU KNOW, AGGREGATE-BASED DISEASES, BOTH IN THE CNS AND ALSO IN THE PERIPHERY. AND THEN THERE ARE MEMBRANE PROTEINS THAT HAVE PROVEN TO BE DIFFICULT TO DRUG, USING THE CONVENTIONAL OCCUPANCY-DRIVEN PHARMACOLOGY TECHNIQUES. SO SOME RECEPTOR TYROSINE KINASES FALL INTO THIS CATEGORY, ADHESION MOLECULES LIKE INTERGRINS, IMMUNOMODULATORY RECEPTORS LIKE MANY OF THE GLYCOPROTEINS WE WERE STUDYING IN THE CONTEXT OF CANCER IMMUNE THERAPY ARE THINGS YOU MIGHT JUST WANT TO DEGRADE. SO HOW COULD YOU TAKE ON THESE KIND OF TARGETS WITH A DEGRADATION STRATEGY? AND THAT WAS THE QUESTION THAT STEVE BANIK ASKED BH WHEN HE JOINED MY LAB, SO HOW DOES NATURE NORMALLY DEGRADE THESE EXTRACELLULAR GLYCOPROTEINS AND MEMBRANE PROTEINS? SHE DOES IT THROUGH THE ENDOSOME LYSOSOME SYSTEM. SO THIS IS THE PATHWAY THROUGH WHICH MEMBRANE BOUND OR SOLUBLE MOLECULES ARE GRABBED INTO ENDOSOMAL VESICLES AND AN EARLY ENDOSOME THEN MATURES THROUGH THIS PATHWAY EVENTUALLY TO FORM A LATE ENDOSOME, AND THE LATE ENDOSOME FUSES WITH THE LYSOSOME, AND THE LYSOSOME IS THE ORGANELLE THAT CONTAINS ALL THE DEGRADATION MACHINERIES THAT WE NEED TO TAKE GLYCOPROTEINS AND GLYCOLIPIDS AND CHOP THEM DOWN INTO THEIR INDIVIDUAL BUILDING BLOCKS. SO THE LYSOSOME HAS PROTEASES, GLIE COST DAYS IS, LIPASES, PHOSPHATASES, SULFA TASTES. IT'S A GREAT DEGRADATION MACHINERY IF YOU CAN GET YOUR TARGET INTO THAT ORGANELLE. SO THAT BECAME THE QUESTION, IS THERE A MECHANISM BY WHICH WE COULD TAKE A PROTEIN OF INTEREST AND TARGET IT IN SOME WAY SO THAT IT GETS BASICALLY GRABBED AND DRAGGED THROUGH THE ENDOSOME LYSOSOME SYSTEM AND DEPOSITED IN THE LYSOSOME FOR DEGRADATION. SO HERE, WE WERE ABLE TO TAKE A PAGE RIGHT OUT OF THE BOOK OF GLYCOSCIENCE. BECAUSE ANYONE WHO STUDIES GLYCOSCIENCE, IF YOU'VE GOT THE TEXTBOOK, FOR EXAMPLE, ON YOUR SHELF, YOU CAN READ ABOUT HOW GLYCOSYLATION CAN AFFECT SUBCELLULAR TRAFFICKING, AND THERE'S A VERY FAMOUS LYSOSOMAL TRAFFICKING RECEPTOR WHOSE JOB IT IS TO GRAB GLYCOPROTEINS THAT HAVE GLYCANS THAT TERMINATE WITH THIS MANNOSE-6-PHOSPHATE MARK. IT'S A VERY INTERESTING TRAFFICKING SYSTEM, IT'S RATHER COMPLEX, BECAUSE THIS MANNOSE-6-PHOSPHATE RECEPTOR HAS THE PRIMARY FUNCTION OF BASICALLY GRABBING LYSOSOMAL ENZYMES FROM THE GOLGI DEPARTMENT AND THEN TRAFFICKING THEM WITHIN THE CELL THROUGH A RETROMER COMPLEX AND A LATE ENDOSOME INTO THE LYSOSOME. THEY GOT THERE BECAUSE THEY HAVE MANNOSE-6-PHOSPHATE AND THEY WERE TRAFFICKED FROM THE GOLGI TO THE LYSOSOME. BUT AT ANY GIVEN TIME, ABOUT 10% OF THE MANNOSE-6-PHOSPHATE RECEPTOR CYCLES OUT TO THE PLASMA MEMBRANE, AND FROM THE MEMBRANE, IT CAN ACTUALLY GRAB HOLD OF GLYCOPROTEINS WITH MANNOSE 6 PHOSPHATE THAT ARE OUTSIDE THE CELL IN CIRCULATION AND JUST PULL THEM IN THROUGH THE ENDOSOME LYSOSOME SYSTEM AND DEPOSIT THEM IN THE LYSOSOME. THE WAY THAT THESE MOLECULES ARE RELEASED FROM THE RECEPTOR IS THROUGH A PH SWITCH. SO MANNOSE 6 PHOSPHATE RECEPTOR BINDS ITS LIE GAFNED WITH LIGAND WITH HIG H AFFINITY BUT THE PH DROPS AND THESE MORE ACIDIC PHs, THE AFFINITY DROPS AND THE CARGO IS RELEASED SO IT CAN GO TO THE LYSOSOME WHEREAS THE RECEPTOR CAN THEN CONTINUE TO RECYCLE AROUND IN THE CELL. ABSOLUTELY BEAUTIFUL SYSTEM. AND IN FACT, WELL-KNOWN IN THE BIOPHARMACEUTICAL INDUSTRY AS THE PRIMARY TRAFFICKING MECHANISM FOR LYSOSOMAL STORAGE DISEASE ENZYME REPLACEMENT THERAPIES. INTO SO WE ALREADY KNEW FROM, YOU KNOW, COMPANIES LIKE BIOMARIN AND SANOFI GENZYME THAT RECOMBINANT PROTEINS, BH INJECTED WHEN INJECTED INTO THE HUMAN BODY CAN CORRECT AN ENZYME DEFICIENCY. WE BASICALLY TOOK A PAGE FROM THAT LITERATURE AND BASICALLY CREATED A TARGETED DEGRADATION TECHNOLOGY BENEFITING FROM ALL OF THIS PRIOR WORK. SO WE COINED THE TERM LYTAC BECAUSE IT'S LIKE THE COUSIN OF THE PROTACK AND THE LYTAC IS AN ACRONYM FOR LYSOSOME TARGETING CHIMERA, AND WE ENVISIONED THAT THESE CHIMERAS WOULD HAVE TWO PARTS. ON THE ONE HAND, THERE WOULD BE A PART THAT BINDS THE TARGET OF INTEREST AND I'M SHOWING A MONOCLONAL ANTIBODY CARTOON HERE BECAUSE WE HAVE MADE LYTACs FROM MANY DIFFERENT ANTIBODIES, BUT ONE SHOULD THINK AGNOSTICALLY ABOUT THE BINDER. THE BINDER COULD BE A SMALL MOLECULE LIGAND THAT BINDS WITH HIGH AFFINITY TO A TARGET OF INTEREST, FOR EXAMPLE. AND THEN THE OTHER PART IS THE PART THAT ENGAGES THE MANNOSE 6 PHOSPHATE RECEPTOR, AND AS I'LL SHOW YOU, WE TYPICALLY WOULD USE A LIGAMERS OF SYNTHETIC ANALOG. THE IDEA IS THAT WITH THIS KIND OF A LYTAC, WE COULD TARGET EITHER SOLUBLE PROTEINS OR MEMBRANE PROTEINS FOR DEGRADATION, AND THE ANTIBODY OR THE BINDER WOULD ATTACH TO THE TARGET, AND THEN THOSE MANNOSE 6 PHOSPHATE GROUPS WOULD BIND THE RECEPTOR AND THE WHOLE ASSEMBLY WOULD BE TAKEN UP INTO THE CELL, DEPOSITED IN THE LYSOSOME. WE WERE PRETTY CONFIDENT THAT A SOLUBLE TARGET COULD BE TARGETED TO THE LYSOSOME THIS WAY. WHAT WAS LESS CLEAR WAS WHETHER A MEMBRANE ASSOCIATED TARGET COULD BE TRAFFICKED TO THE LYSOSOME BECAUSE THERE WASN'T REALLY A KNOWN NATURAL BIOLOGY WHEREIN A MOLECULE ON THE MEMBRANE WOULD BE ABLE TO INTERACT WITH THE MANNOSE 6 PHOSPHATE RECEPTOR ON THE SAME MEMBRANE AND TRAFFICKED TO THE LYSOSOME. SO THAT WAS KIND OF LIKE THE BIG QUESTION MARK AT THE OUTSET. OKAY. SO WE DID SOME CHEMISTRY TO MAKE THESE MOLECULES, AND FOR HISTORIC REASONS, WE DECIDED TO MAKE GLYCOPOLYPEPTIDES AS THE MANNOSE 6 PHOSPHATE -- SIMPLY BECAUSE WE WERE DOING THIS CHEMISTRY IN MY LAB FOR OTHER PURPOSES, NOT WITH MANNOSE 6 PHOSPHATE, BUT WE KNEW THAT IT WAS A PRETTY EASY WAY TO JUST MAKE A LIGAMER AS QUICKLY OF KNOWN SIZE AND DENSITY. WE MADE THESE BY POLYMERIZING GLYCOSYLATED AMINO ACIDS THAT WERE FUNCTIONALIZED IN AN N CARBOXY AN AN HYDRIDE FORM AND THE SUGARS WERE PROTECTED WITH PROTECTING GROUPS. WE USED CHEMISTRY DEVELOPED BY TIM DEMMING'S GROUP AT UCLA. HE HAD SHOWN THAT ONE CAN DO A LIVING PORE MERRIZATION WITH THESE NCAs AND A NICKEL CATALYST TO MAKE LIGAMERS OF DEFINED SIZE BASED ON THE RATIO OF THE MONOMER TO THE CATALYST. IT'S A HALLMARK OF A LIVING POLYMERIZATION. AND WE COULD ALSO BLEND IN OTHER AMINO ACIDS TO THE POLYMERIZATION REACTION SUCH AS ALANINE SO THAT WE COULD SPACE THE MANNOSE 6 PHOSPHATE GROUPS OUT AT WILL. AND THEN AT THE END OF THE DAY, WE HAD A POLYPEPTIDE WHICH HAS A C-TERMINUS AND AN N-TERMINUS THAT WE COULD FUNCTIONALIZE AS NECESSARY TO ATTACH TO TARGET BINDERS. SO TO DETERMINE THAT THESE GLYCOPOLYPEPTIDES WERE ABLE TO INTERACT WITH MANNOSE 6 PHOSPHATE RECEPTOR AND GET TAKEN UP INTO THE CELL, STEVE MADE A SIMPLE LYTAC WHERE THE BIOTIN LYTAC WOULD BE ABLE TO BIND A FLUORESCENT LABELED PROTEIN IN THE CELL TUL CULTURE MEDIA, AND THE IDEA WAS TO SEE WHETHER THE SIGNAL WAS TAKEN INTO THE CELL AND CONCENTRATED IN THE LYSOSOME. THAT WORKED QUITE NICELY, AND SO WHAT YOU'RE LOOKING AT HERE ARE THE FLUORESCENCE INTENSITY OF CELLS AS MEASURED BY FLOW CYTOMETRY, AND THESE CELLS WERE TREATED WITH BIOTIN LYTACs HAVING DIFFERENT SUGAR STRUCTURES ON THEM, SO IN THIS COLLECTION OF FOUR DIFFERENT LYTACs, STEVE HAD CONTROL SUGARS THAT DON'T BIND MANNOSE 6 PHOSPHATE RECEPTOR, AND THEN TWO STRUCTURES THAT SHOULD BIND THE MANNOSE 6 PHOSPHATE RECEPTOR, WHICH WERE MANNOSE 6 PHOSPHATE AND AN ANALOG IN WHICH THERE'S A CARBON ATOM IN PLACE OF THIS OXYGEN ATOM AT C6, THIS IS A PHOSPHONATE, AND WE KNEW FROM THE LITERATURE THAT IT SHOULD STILL BIND WITH SIMILAR AFFINITY TO THE RECEPTOR, BUT IT'S NON-HYDROLYZABLE, SO IT WOULD BE MORE STABLE IN SERUM, IN CIRCULATION, BECAUSE THERE'S PHOSPHATASES THERE THAT ARE KNOWN TO BE ABLE TO CLIP THE NATIVE MANNOSE 6 PHOSPHATE STRUCTURE. THEN LOOKING AT THE UPTAKE OF THESE DIFFERENT CONSTRUCTS, WHAT STEVE OBSERVED IS THAT THE CONTROL LYTACs, WHICH ARE HERE, THESE SHORT BARS, WERE BASICALLY IGNORED BY THE CELLS, BUT ANY OF THESE CONSTRUCTS WITH EITHER MANNOSE 6 PHOSPHATE OR MANNOSE 6 FOS FA NATE, WHETHER THE POLYMERS WERE SHORT, OR LONG AS WE CALL THEM, MORE LIKE 60, ALL OF THESE WERE TAKEN UP QUITE EFFICIENTLY. AND THE UPTAKE WAS INHIBITABLE WITH HIGH CONCENTRATIONS OF MONOMERIC MANNOSE 6 PHOSPHATE IN SOLUTION BECAUSE IT'S BLOCKING THE RECEPTOR. SO THAT'S HOW WE KNOW IT REALLY WAS A RECEPTOR-MEDIATED PROCESS SO WITH THAT RESULT IN HAND, STEVE THEN WENT AHEAD TO GENERATE ANTIBODY-BASED LYTACs AGAINST WHAT WE THOUGHT TO BE SOME INTERESTING TARGETS, MOSTLY FOR CANCER. SOL WELL-KNOWN CANCER TARGETS. TOWARD THIS END, HE BUILT THE LYTAC MOLECULES USING THE PHOSPHONATE ANALOG, THE HYDROLITICALLY STABLE ANALOG, DID THE POLYMERIZATION CHEMISTRY TO MAKE OLIGOMERS, SPACED THEM OUT A LITTLE BIT THEN USED THE FREE N-TERMINUS IN ORDER TO FUNCTIONAL IEDZ THEM TO CONGRESS GATE THEM TO AN ANTIBODY. THIS IS WHERE WE WERE ABLE TO USE SOME BIOORTHOGONAL CHEMISTRY REAGENTS THAT OUR LAB HAD DEVELOPED MANY YEARS EARLIER, SO WHAT HE DID IN THIS FIRST ROUND OF LYTACs IS HE TOOK AN ANTIBODY, AND IT WAS ACTUALLY AN ANTIBODY CALLED CETUXIMAB, WE'LL GET TO THAT IN A BIT, BUT THIS WORKS WITH ANY ANTIBODY, AND HE BASICALLY NON-SPECIFICALLY CONJUGATED SOME OF THE LYSINE RESIDUES ON THE ANTIBODY WITH A LINGER, POLYETHYLENE GLYCOL LINGER TERMINATED WITH AN ASIDE. SO THEN IN PARALLEL, HE COULD TAKE THE GLYCOPOLYPEPTIDES, FUNCTIONALIZED THEM WITH A BUY CYCLOOCC TYNE, AND THEY UNDERGO A CLICK CHEMISTRY REACTION TO ATTACH THE GLYCOPOLYPEPTIDES TO THE ANTIBODY. IT'S KIND OF FUN TO FOLLOW THESE REACTIONS BY NATIVE GEL ELECTROPHORESIS, WHICH IS WHAT YOU'RE LOOKING AT HERE, AND YOU CAN SEE A NATIVE ANTIBODY RUNS AS A COLLECTION OF BANDS ON THE NATIVE GEL. THERE'S DIFFERENT GLYCOFORMS OF THE ANTIBODY, WHICH IS WHY IT LOOKS HETEROGENEOUS. AFTER WE DO THE CHEMISTRY TO MAKE THIS AZIDE LINGER, WE'VE BASICALLY AIZ LATED SOME OF THE LYSINE RESIDUES SO THEY GO TO NEUTRAL AND IT MIGRATES FASTER IN THIS NATIVE GEL SO THIS IS NOW THE LINGER FUNCTIONALIZED ANTIBODY AND FINALLY WHEN WE ATTACH THE MANNOSE 6 PHOSPHONATE OLIGOMERS, IT'S EVEN FASTER SO WE'VE FURTHER ENHANCED THE NEGATIVE CHARGE OF THE ANTIBODY. SO ONCE WE HAD MADE THESE LYTACs OUT OF ANTIBODIES, THE NEXT QUESTION WAS, CAN THEY DRIVE THE UPTAKE OF A TARGET PROTEIN BOTH IN CELL CULTURE BUT EVEN MORE IMPORTANTLY IN THE CONTEXT OF A MODEL ANIMAL? AND TOWARD THAT END, STEVE DID AN EXPERIMENT WHERE HE WAS MODELING THE CLEARANCE OF IMMUNE COMPLEKS FROM CIRCULATION IN A MOUSE, WHICH IS, OF COURSE, ALSO A THERAPEUTICALLY RELEVANT KIND OF SITUATION GIVEN THAT THERE ARE MANY HUMAN DISEASES WHICH ARE CHARACTERIZED BY IMMUNE COMPLEXES THAT MIGHT WANT TO CLEAR AND DEGRADE. SO HE TOOK THE ANIMALS AND DID IP INJECTIONS OF AN IMMUNE COMPLEX EITHER MADE FROM A LYTAC WHERE THERE'S LIE OR FROM A NAKED ANTIBODY AND FROM TIME POINTS HE TOOK BLOOD DRAWS AND EXTRACTED TISSUES TO SEE HOW MUCH OF THE CARGO HAD BEEN CLEARED. THE CARGO WAS AN ANTIBODY THAT HAD A FLUORESCENT DYE ON IT, JUST BY DOING LIKE A FLUORESCENCE GEL ANALYSIS. SO THAT'S WHAT YOU'RE LOOKING AT HERE. THE CARGO WAS A RABBIT IGG. IF YOU LOOK AT MICE TREATED JUST WITH THE NAKED ANTIBODY IMMUNE COMPLEX, THAT'S THIS COLUMN MINUS AND THIS COLUMN MINUS, YOU CAN SEE THERE'S LOTS OF THAT RABBIT ANTIBODY IN THE SERUM IN THE LIVER AND THE SPLEEN AFTER 24 HOURS, AND AFTER 48 HOURS, IT REALLY HASN'T CHANGED MUCH. SO THIS IMMUNE COMPLEX HAS A LONG CIRCULATION TIME IN THE MOUSE AS ONE WOULD EXPECT FOR ANTIBODY IMMUNE COMPLEXES. BUT BY CONTRAST, WHEN WE LOOKED AT THE LYTAC IMMUNE COMPLEX, YOU CAN SEE THERE'S A REDUCTION IN THE ABUNDANCE AFTER 24 HOURS AND ALMOST COMPLETE CLEARANCE OUT OF THE ANIMAL AFTER 48 HOURS. THAT WAS AN INDICATOR THAT THESE LYTACs ARE RECOGNIZED BY RECEPTORS IN VIVO WHICH CAN LEAD TO THE CLEARANCE OF CARGO FROM THE CIRCULATION OF AN ANIMAL. AS I MENTIONED BEFORE, THE BIG QUESTION MARK HANGING OVER THIS TECHNOLOGY FROM THE GET-GO WAS, COULD YOU TARGET A CELL SURFACE, A MEMBRANE ASSOCIATED PROTEIN FOR DEGRADATION BY VIRTUE OF MANNOSE 6 PHOSPHATE RECEPTOR ENGAGEMENT ON THE SAME MEMBRANE. AND THE REASON THIS WAS A BLACK BOX WAS BECAUSE MEMBRANE PROTEINS, UNLIKE CIRCULATING PROTEINS, MEMBRANE PROTEINS HAVE MORE OF A PERSONALITY. THEY HAVE THEIR OWN RESEEKING BEHAVIOR INHERENTLY SO THEY HAVE THEIR OWN PREFERENCES AS TO WHERE THEY WANT TO BE AND HOW THEY'RE GOING TO GET THERE, AND THE QUESTION IN OUR MIND WAS, IF WE FORCE AN ENGAGEMENT WITH THE MANNOSE 6 PHOSPHATE RECEPTOR, IS IT GOING TO DOMINATE THE CYCLING AND INTERNALIZATION BEHAVIOR OR WILL THE PROTEIN FIGHT BACK WITH ITS OWN PREFERENCE. AND WE DIDN'T KNOW. SO TO ASK THAT QUESTION, WE STARTED WITH AN INTERESTING CANCER TARGET, WHICH IS THE EPIDERMAL GROWTH FACTOR RECEPTOR OR THE EGFR. EGFR EXPANSIONS AND MUTATIONS ARE QUITE PREVALENT IN HUMAN CANCERS. THERE ARE SEVERAL IMPORTANT CANCER MEDICINES THAT TARGET EGFR, EITHER THE WILD TYPE OR THE MUTANT FORMS. IT'S STILL AN OPEN NEED, HOWEVER, BECAUSE PATIENTS WHO TAKE THESE MEDICINES INEVITABLY BECOME RESISTANT AND THE RESISTANCE MECHANISMS ARE NOT FULLY UNDERSTOOD SO WE THOUGHT THERE WAS WOMB FOR ROOM FOR A DEGRADER IN THIS PARTICULAR SPACE. SO TO MAKE OUR FIRST LYTAC AGAINST EGFR, WE TOOK CLINICALLY APPROVED ANTIBODY DRUG CETUXIMAB, WHICH BLOCKS THE BINDING OF EGFR TO ITS LIGAND, THE EPIDERMAL GROWTH FACTOR. WE DID THE CHEMISTRY I SHOWED EARLIER, LINKED THEM ON TO CETUXIMAB NON-SPECIFICALLY THROUGH THESE LYSINE CONGREGATES, THEN ASKED THE QUESTION, WHAT DOES THE LYTAC DO TO THE ABUNDANCE OF EGFR IN CULTURED CELLS. SO WHAT YOU'RE LOOKING AT HERE ARE SOME OF THE DATA FROM HELA CELLS WHICH I EXPRESS ABUNDANTLY EGFR, AND THE PINK STAINING IS AN ANTIBODY AGAINST EGFR THAT DOES NOT OVERLAP WITH THE EPITOPE OF CETUXIMAB. YOU CAN SEE IN UNTREATED HELA CELLS, EGFR IS DENSELY POPULATED ON THE PLASMA MEMBRANE. WHEN YOU TREAT THESE CELLS WITH CETUXIMAB, IT BINDS EGFR BUT IT DOESN'T CHANGE EITHER THE LEVEL OF THAT PROTEIN OR ITS DISTRIBUTION, IT'S STILL ALL OVER THE PLASMA MEMBRANE. BUT WHEN WE TREAT THOSE CELLS WITH THE CETUXIMAB LYTAC, YOU CAN SEE THERE'S A DRAMATIC REDISTRIBUTION OF EGFR AND NOW IT BEEN DRIVEN INTO THE LYSOSOME QUITE DRAMATICALLY FROM THE CELL SURFACE. IMPORTANTLY IT'S DEGRADED THERE. SO HERE'S AN EXAMPLE OF A TIME COURSE FOR DEGRADATION OF EGFR INDUCED BY THE LYTAC. WHAT WE DID IS WE BASICALLY QUANTITATED THE PROTEIN LEVEL BY WESTERN BLOT NORMALIZED TO A HOUSEKEEPING PROTEIN, WHICH WAS VINCULIN. SO IN BLUE IS THE LEVEL OF EGFR, THE RELATIVE LEVEL UPON TREATMENT WITH THE NAKED ANTIBODY, CETUXIMAB, OVER THE COURSE OF FIVE DAYS, WHEREAS WHEN YOU TREAT WITH THE LYTAC OF CETUXIMAB, YOU CAN SEE THERE'S A LOSS OF THAT PROTEIN WHICH BASICALLY MAXES OUT AFTER MAYBE 24 HOURS AND THERE'S A NET REDUCTION IN PROTEIN LEVELS AT STEADY STATE TO ABOUT 20% OF WHAT THEY WOULD BE IN THE UNTREATED CELL. SO AT THIS POINT, YOU KNOW, STEVE WAS GETTING READY TO GO ON THE JOB MARKET AND WAS FLYING AROUND INTERVIEWING RIGHT BEFORE THE COVID CRISIS AND EVERYONE WAS TRYING TO RECRUIT HIM, STANFORD WAS SO HAPPY TO SUCCEED IN THAT COMPETITION, AND MEANWHILE GREEN HAD JOINED THE LAB AND WAS REALLY INTERESTED IN EXPANDING UPON THIS INITIAL RESULT THAT STEVE HAD HAD IN A NUMBER OF DIFFERENT DIMENSIONS. SO FIRST OF ALL, THESE QUESTIONS THAT SHE WANTED TO PURSUE WITH SECOND GENERATION LIE TAX HAD SORT OF THREE DIFFERENT BUCKETS. ONE WAS THAT WE'D HAD A LOT OF SUCCESS WITH THE MANNOSE OF PHOSPHATE RECEPTOR AS A LYSOSOME TRAFFICKING SYSTEM, BUT THERE'S MAYBE A DOZEN OTHER LYSOSOMAL TRAFFICKING RECEPTORS THAT HAVE BEEN IDENTIFIED THAT BIND TO OTHER TYPES OF MOLECULAR MOTIFS AND SHE WAS CURIOUS WHETHER OTHER LYSOSOMAL TRAFFICKING RECEPTORS MIGHT ALSO BE EXPLOITED FOR LYTAC DEVELOPMENT. INTERESTINGLY, SOME OF THOSE LYSOSOMAL TRAFFICKING SYSTEMS ARE TISSUE-SPECIFIC, WHEREAS MANNOSE 6 PHOSPHATE RECEPTOR IS EXPRESSED PRETTY MUCH IN EV CELL TYPE. IT'S REALLY A HOUSEKEEPING RECEPTOR. BUT THERE ARE OTHERS THAT ARE VERY TISSUE-SPECIFIC, AND WE THOUGHT THAT COULD BE INTERESTING, YOU KNOW, TO 345EUBG A MAKE A TISSUE-SPECIFIC LYTAC. WE WERE NOT HAPPY WITH THE CHEMISTRY IN OUR FIRST GENERATION LYTACs. WE THOUGHT IT WAS TOO HETEROGENEOUS. THE GLYCOPOLYPEPTIDES WE WERE MAKING HAD SOME HETEROGENEITY SINCE WE WERE MAKING THEM THROUGH POLYMERIZATION REACTIONS, AND WE WERE LINKING THEM TO LYSINE RESIDUS NON-SPECIFICALLY ON THE ANTIBODY. AND YOU KNOW, THOSE TWO LAYERS OF HETEROGENEITY MADE IT HARD FOR US TO DETERMINE WHETHER WE HAD REALLY MADE THE BEST LYTACs THAT WE COULD MAKE. WE COULDN'T REALLY OPTIMIZE THEIR STRUCTURES BECAUSE WE WEREN'T CONTROLLING THEM. MEANWHILE, WE HAD DEVELOPED SOME VERY NICE CHEMISTRIES IN MY LAB FOR SITE-SPECIFIC PROTEIN BIOCONJUGATION AND WE THOUGHT WE SHOULD BE USING THOSE TO MAKE HOMO GENIUS LYTACs TO BASICALLY OPTIMIZE THEIR PERFORMANCE, BOTH IFN VITRO AND IN VIVO, SO GREEN HAS NOW MADE SOME PROGRESS IN ALL THREE OF THESE AREAS. SO THE NEXT LYSOSOMAL TRAFFICKING RECEPTOR THAT WE TOOK ON WITH GREEN WAS ANOTHER ONE THAT BINDS A SUGAR. IT'S THE QUITE FAMOUS ASIALOGLYCOPROTEIN RECEPTOR OR ASGPR. AGAIN, WHEN YOU TAKE GLYCOSCIENCE 101, YOU LEARN ABOUT THE ROLE OF THIS RECEPTOR IN CLEARING ASIALOGLYCO -- MAKE ROOM FOR THE NEW ONES, AND IN FACT THIS RECEPTOR CAN ALSO CLEAR PARTICLES AND EVEN PLATELETS OUT OF CIRCULATION WHEN THEY HAVE LOST SIGH ALONG ACIDS AND EXPOSE IN UNDERLYING EITHER GA LACTOSE STRUCTURE. LIKE MANNOSE 6 PHOSPHATE RECEPTOR, THE ASGPR HAS A PH SWITCH THAT ALLOWS TO TAKE UP ITS CARGO, BRING IT INTO THE CELL, THE PH DROPS, IT LETS GO AND THEN IT RECYCLES BACK TO THE PLASMA MEMBRANE. WHAT'S REALLY COOL IS IT'S VERY ABUNDANT, THERE'S A MILLION OF THESE RECEPTORS PER HEPATOCYTE, IT WAS ALREADY KIND OF WELL ESTABLISHED AS A DELIVERY VEHICLE FOR MACRO MOLECULES TARGETED TO THE LIVER. SO QUITE FRAMESLY, BOTH THE TWO LEADING COMPANIES IN NUCLEIC ACID THERAPEUTICS ARE USING THE SO-CALLED TRIGALNAC MOTIF AS A MEANS TO INCREASE THE EFFICIENCY OF UPTAKE INTO THE LIVER OF THEIR VARIOUS NUCLEOTIDE THERAPEUTICS. IT'S VERY EFFICIENT, HAS VERY HIGH CAPACITY. SO WE THOUGHT THIS WAS REALLY CONVENIENT BECAUSE IT ALREADY TOLD US WHAT KIND OF STRUCTURE YOU WOULD NEED TO GET HIGH AFFINITY ENGAGEMENT OF ASGPR. WE'RE NOT ALONE IN OUR INTEREST IN THIS RECEPTOR AS A POTENTIAL VEHICLE FOR TARGETED DEGRADATION. AND IN FACT, JUST A FEW MONTHS AGO, GREEN POSTED HER FIRST PAPER AS A PRE-PRINT ON CHEM ARCHIVE AND WITHIN TWO WEEKS, THERE WERE TWO OTHER RELATED PAPERS THAT APPEARED THERE, ALL OF US USING THE RECEPTOR AS A MEANS TO TARGET THE DEGRADATION OF AN EXTRACELLULAR PROTEIN. SO LET ME MAKE MENTION OF THE BEAUTIFUL WORK OF DAVID SPIEGEL FROM YALE UNIVERSITY, WHO BASICALLY SHOWED THAT ONE COULD DO THIS WITH SMALL MOLECULE TRY GALNAC CONGRESS A -- THAT SHOWED THAT ANTIBODIES MODIFIED WITH THE TRIGALNAC UNIT COULD BE USED TO GRAB EXTRACELLULAR PROTEINS AND BRING THEM IN TO THE CELL FOR DEGRADATION. SO HERE'S WHAT GREEN DID. SHE SYNTHESIZED HER TRI-GALNAC -- BY MAKING THAT DENDROMER THAT'S ALMOST IDENTICAL TO THE ONE -- SIMPLY FUNCTIONALIZED IT WITH SIGH CROW OCCTINE CALLED DBCO. SO HERE'S THE CARTOON WE USE TO ABBREVIATE THAT. AND THEN JUST AS STEVE HAD DONE EARLIER, SHE TOOK ANTIBODIES OF INTEREST, MODIFIED THEIR LYSINE RESIDUES IN HER FIRST PASS WITH NHS ESTER PEG AZIDE LINGER AND CLICKED THAT UNIT ON TO THE AZIDE. THE FIRST WAVE OF GALNAC LYTACs THAT SHE MADE, IN RETROSPECT, WE NOW REALIZE THEY HAD PROBABLY WAY TOO MUCH GALNAC ON THEM SO SHE WAS MAKING CONSTRUCT THAT HAD ON AVERAGE 11 OF THOSE TRIMERS RANDOMLY ON LYSINES WHICH MEANS THEY'RE ON AVERAGE 33 GALNAC RESIDUES PER LYTAC BUT THAT'S WHAT WE MADE IN OUR FIRST PASS. AND WHAT SHE SHOWED IS THAT THOSE GALNAC LYTACs CAN VERY EFFICIENTLY TBRAB AND MEDIATE THE UPTAKE OF GAR GOA MOLECULES THAT ARE EXTRACELLULAR. SO THIS WAS A CELL CULTURE SMAIRMT EXPERIMENT GREEN PERFORMED USING A HEPATOCYTE CELL LINE THAT HAS ASGPR, AND SHE SIMPLY MADE A LYTAC FROM AN ANTIBODY THAT'S A SECONDARY ANTIBODY SPECIFIC FOR RABBIT IGG. AND WHAT SHE FOUND IS THAT A FLUORESCENTLY TAGGED RABBIT IGG COULD BE BOUND BY THIS LYTAC, TAKEN UP INTO CELLS THROUGH ASGPR, AND SHE DID A HEAD TO HEAD COMPARISON ACTUALLY BETWEEN GALNAC LYTAC AND A COMPARABLE MANNOSE 6 PHOSPHONATE -- WE COULD KIND OF COMPARE THEM SIDE BY SIDE ON THE SAME CELL TYPE. AS YOU CAN SEE HERE, THESE ARE BARS THAT REPRESENT THE FLUORESCENCE THAT ACCUMULATES IN THESE CELLS IN THE PRESENCE OF THE LYTAC. AND THIS MIDDLE BAR IS THE MANNOSE 6 PHOSPHATE LYTAC, SO IT MEDIATES THE UPTAKE BUT IT DOESN'T HOLD A CANDLE TO THE GALNAC HITAC. SO FOR UPTAKE OF A SOLUBLE PROTEIN, ASGPR IS MUCH MORE EFFICIENT AND MAYBE WE SHOULDN'T BE SURPRISED, THAT ISITY PRIMARY FUNCTION. WHAT WAS INTERESTING IS THAT WHEN SHE DID A HEAD TO HEAD COMPARISON OF GALNAC VER US MANNOSE 6 FOR DEGRADATION OF A MEMORY PROTEIN, IN THIS CASE, EGFR, ALL ELSE BEING EQUAL, THEY'RE ACTUALLY QUITE SIMILAR IN THEIR ABILITY TO DEGRADE A TARGET. CAN YOU SEE THAT IN THESE THREE DIFFERENT CELL LINES, ALL OF WHICH EXPRESS BOTH ASGPR, MANNOSE 6 PHOSPHATE RECEPTOR AND EGFR, THE TARGET WE WANTED TO DEGRADE. AND YOU CAN SEE THAT THE TWO DIFFERENT LYTACs FROM HARNESSING THE TWO DIFFERENT LIE SO SOMOL TRAFFICKING SYSTEMS ARE ACTUALLY QUITE COMPARABLE HERE, EVEN THOUGH THERE'S A BIG DIFFERENCE IN THEIR SOLUBLE PROTEIN UPTAKE CAPACITY. SO AT THIS POINT, WE THOUGHT IT WOULD BE IMPORTANT TO START LOOKING AT HOW THESE LYTACs BEHAVED IN VIVO, IN A LIVE ANIMAL MODEL. TOWARD THIS END, WHAT GREEN DID IS SHE MADE A LYTAC AGAINST THE KNOWN FAMOUS CANCER TARGET HER2, WHICH IS OVEREXPRESSED IN MANY TUMOR SETTINGS, BREAST CANCERS, LIVER CANCERS, OTHERS AS WELL. SHE DID THIS BY TAKING THE CLINICAL DRUG PER TEUZMAB AND CONGREGATING IT. AND IN THIS EXPERIMENT WHAT YOU'RE LOOKING AT IS THE DEGRADATION OF HER2 ON SUBCUTANEOUSLY INJECTED TUMOR GROWN FROM H E-PG 2 CELLS. AS SOON AS THE TUMORS WERE GREATER THAN 100 CUBIC MILLIMETERS, SHE DID THREE DAILY DOSES OF THE PERTUZUMAB AND LOOKED AT THE AMOUNT OF HER2 PROTEIN IN A WESTERN BLOT. I SHOULD POINT OUT IN THIS EXPERIMENT, BOTH OF THESE LYTACs WERE MADE THROUGH NON-SELF LYSINE CONJUGATION, AND THIS WAS THE GALNAC LYTAC THAT HAD A TON OF 33 ROUGHLY GALNACs PER PROTEIN. WHAT SH FOUND HERE IS UNLIKE THE CELL CULTURE SITUATION WHERE THE GALNAC LYTAC AND THE MANNOSE 6 PHOSPHATE LYTACs WERE COMPARABLE, IN THIS IN VIVO MODEL, THE MANNOSE 6 FOS PHOSPHONATE WAS VERY EFFICIENT BUT THE GALNAC LYTAC PRETTY MUCH FAILED. SO IT WAS NOT AN EFFECTIVE DEGRADER IN VIVO. SO THE QUESTION WAS WHAT HAPPENED, AND THEN WE REALIZED THAT WE HAD PUT SO MUCH GALNAC ON THIS LYTAC THAT IT JUST MAY HAVE BEEN CLEARED VERY RAPIDLY BEFORE IT HAD A CHANCE TO ENGAGE ITS TARGET HER2 IN THE TUMOR CELLS AND THEN MEDIATE DEGRADATION IN THE LOCAL ENVIRONMENT. SO THIS IS WHERE WE REALIZED WE REALLY HAD TO SIMPLIFY THE STRUCTURES AND SORT OF DOWNSCALE THE AMOUNT OF GALNAC ON THESE LYTACs TO TRY AND GET SOMETHING THAT HAD THE RIGHT PK PROPERTY TO PERFORM IN VIVO. WE HAD THIS BEAUTIFUL SITE-SPECIFIC TECHNOLOGY WE DEVELOPED YEARS EARLIER IN THE CONTEXT OF MAKING ANTIBODY DRUG CONJUGATES, AND THIS IS ALL PUBLISHED WORK, I'M NOT GOING TO GO INTO THE DETAILS HERE, BUT IT SUFFICES TO SAY WE CALLED THIS THE ALDEHYDE TAG TECHNOLOGY. WE HAD DEVELOPED A WAY TO GENETICALLY ENCODE A POST TRANSLATIONAL MODIFICATION AT A SITE OF INTEREST ON PROTEINS WHERE A CYSTINE RESIDUE GETS CONVERTED TO A RESIDUE THAT HAS AN ALD HEIGHT GROUP, AND AN ALDEHYDE IS UNIQUE ON THE SURFACE OF A PROTEIN. WE DEVELOPED A CHEMISTRY WHICH BASICALLY INVOLVES THIS FUNCTIONALIZED -- ALLOWING ONE TO ATTACH A LINGER OR A CARGO OF INTEREST AT A SITE THAT IS PRE-SPECIFIED BASED ON THE GENETIC ENGINEERING OF THE ALDEHYDE. SO WE DID THAT, AND INTRODUCED AZIDE GROUPS THAT WAY, CLICKED ON THE GAL KNACK DENDROMERS. SHE PUT THEM IN THREE DIFFERENT LOCATIONS, EITHER NEAR THE C-TERMINUS OF THE HEAVY CHAIN, IN THE HINGE REGION OR THE CH1 REGION OF THE HEAVY CHAIN. WE THOUGHT THIS WOULD BE INTERESTING BECAUSE MAYBE THERE WAS OPTIMAL GEOMETRY AND ONE COULD START TO TEASE THAT OUT. BUT MORE IMPORTANTLY THEY HAD A LOT LESS GALNAC ON THEM, RATHER THAN THE 11 DENDROMERS THAT WE HAD PREVIOUSLY MADE. SO NOW SHE COULD LINE UP THESE DIFFERENT LYTAC CONSTRUCTS AND ASK THE QUESTION, HOW DO THEY PERFORM BOTH IN VITRO AND IN VIVO. SO HERE'S SOME IN VITRO DEGRADATION REACTIONS USING THE HELP TB. THE H E-PG 2 CELL LINE. THIS IS THE LYTAC AGAINST EGFR, THIS IS THE LYTAC AGAINST HER2 AND LOOKING TERRELLTIVE AMOUNTS OF EGFR PROTEIN UNDER TREATMENT WITH ALL THESE DIFFERENT LYTACs, AND IT SUFFICES TO SAY THAT THESE MUCH LOWER SITE-SPECIFIC LYTACs -- PARTICULARLY IN THE CASE OF THE -- THIS IS THE NONSPEIFIC HEAVILY GALNAC MODIFIED LYTAC, TAKES YOU FROM 100 DOWN TO 25% OF PROTEIN AT STEADY STATE, WHEREAS THE SITE-SPECIFIC LYTACs WITH EITHER 1 OR 1 1/2 ON AVERAGE GALNAC DENDROMER, SO THREE RESIDUES HER LYTAC STILL ARE ABLE TO GET THE PROTEINS DOWN ALMOST I'DICALLY TO THIS HEAVILY MODIFIED. -- BY BRINGING THE GALNAC LEVELS WAY DOWN, AND THIS IS AN EXPERIMENT WHERE GREEN INJECTED HER LYTACs INTO MICE AND THEN AFTER TIME POINTS COLLECTED EITHER PLASMA OR DIFFERENT TISSUES, AND BY WESTERN BLOT, LOOKED FOR BASICALLY THE LYTAC ITSELF. SO YOU CAN SEE AFTER SIX HOURS, IF YOU INJECT NAKED CETUXIMAB INTO THESE ANIMALS, STILL THERE AFTER 24, AFTER 48, AFTER 72. IT HAS A LONG CIRCULATION TIME IN THE MOUSE, IN THE SERUM, AS YOU WOULD HOPE. WHEREAS THE LYTAC SHE HAD MADE WITH 11 OR 10 DENDROMERS, SO 30-SOMETHING GALNACS, IS COMPLETELY GONE AFTER SIX HOURS, TOTALLY CLEARED BY THE LIVE, SO NO WONDER IT DIDN'T FUNCTION WELL IN A TUMOR DEGRADATION MODEL. BUT IMPORTANTLY, THE LOWER VAI LAN SEE -- WERE STILL THERE AFTER SIX HOURS, AFTER 24, AFTER 48, AFTER # 72. SO HERE WE HAVE LYTAC THAT ARE EFFECTIVE DEGRADERS BUT HAVE THE SERUM RESIDENCE TIME OF A NAKED ANTIBODY. THEY'RE NOT JUST GETTING IMMEDIATELY TAKEN OUT OF SOLUTION. SO THAT'S REALLY THE PROPERTY THAT WE WANT, I THINK FOR THESE LYTACs TO PERFORM IN VIVO. IN A PROMISING PRELIMINARY EXPERIMENT, THE DATA FOR WHICH SHE JUST SENT ME LAST WEEK, WHAT SHE SHOWED IS THAT SHE CAN GET NOW GOOD DEGRADATION OF A TARGET IN A TUMOR MODEL WITH THESE SITE-SPECIFIC LOWER VALENCY LYTACs. SO ONCE AGAIN SHE FORMED H E-PG 2 TUMORS BY SUBQ INJECTION INTO THE MOUSE AND ON TWO SEQUENTIAL DAYS, SHE DOSED THEM WITH PERTUZEMAB -- BY WESTERN BLOT, AND NOW WHEREAS JUST AS BEFORE, THE HIGH VAI LEN SIEGAL KNACK LYTACs WERE NOT EFFECTIVE DEGRADERS. BY CONTRAST, SHE HAS A VERY LOW VALENCY LYTAC WHICH DOES SHOW DEGRADATION BEHAVIOR IN VIVO IN THIS TUMOR MODEL. SO THAT'S KIND OF WHERE THE PROJECT IS HEADED NOW AND THE HOPE IS THAT WE CAN MAKE LYTACs THAT CAN DEGRADE THEIR TARGET TO A LEVEL THAT IT ACTUALLY PROVIDES A THERAPEUTIC BENEFIT, AND SHE'S RIGHT IN THE MIDDLE RIGHT NOW OF A SERIES OF ANIMAL EXPERIMENTS TO SHOW THAT BY MEASURING TUMOR GROWTH. SO THAT KIND OF TAKES US TO THE PRESENT. AS I SAID FROM THE OUTSET, THIS IS ONE OF THE NEW PROJECTS IN THE LAB THAT WE'VE ONLY PUBLISHED ONE PAIB AND WE PAPER AND WE HA VE A PRE-PRINT UP BUT THERE'S A LOT LEFT ON THE DOCKET. SO FOR EXAMPLE, WHAT'S THE SPECTRUM OF MOLECULES ON THE CELL SURFACE THAT WE CAN DRUG WITH THE LYTAC FORMAT? AND WE'RE PARTICULARLY INTERESTED IN CELL SURFACE TARGETS FOR WHICH CONVENTIONAL ANTIBODIES OR BUY BIOLOGICS OR SMALL MOLECULES DON'T REALLY BLOCK THE FUNCTION OR DON'T GET A DEEP ENOUGH BLOCK TO HAVE A THERAPEUTIC BENEFIT, SO THESE WE THINK COULD BE TARGET FOR WHICH A DEGRADER COULD BE MORE EFFECTIVE THAN JUST A SIMPLE BLOCKER, AND WE'RE TRYING TO BUILD OUT THE SCOPE, WHERE IS THAT THE CASE. THEN FINALLY WE'RE VERY INTERESTED IN CIRCULATING PROTEINS, CIRCULATING PARTICLES LIKE PROTEIN AGGREGATES THAT ARE HARMFUL, DELETERIOUS, THEY CAN'T BE DRUGGED IN ANY CONVENTIONAL WAY AND NOW WE HAVE TECHNOLOGY TO GRAB THEM AND TAKE THUMB UP THEM UP THROUGH THIS LYSOSOME SYSTEM. EVERY TIME I GIVE THIS TALK SOMEBODY GIVES ME SOMETHING TO ADD TO MY GROWING LIST OF TARGETS AND HOPEFULLY THAT WILL HAPPEN TODAY. BUT MOST IMPORTANTLY, LET ME THANK ONCE AGAIN THE MANY PEOPLE WHO CONTRIBUTED TO THIS WORK, AND I CALLED OUT THE TWO PRIMARY PEOPLE AT THE FOREFRONT OF THIS PROJECT IN MY LAB STARTING WITH STEVEN BANIK, NOW CONTINUING IN THE CAPABLE HANDS OF GREEN AHN, BUT THEY ARE SURROUNDED BY A VERY TALENTED SUPPORTING CAST OF STUDENTS AND POSTDOCS AND STAFF, AND NONE OF THIS WOULD BE POSSIBLE AND THAT EXTENDS TO MY ENTIRE CAREER, I GUESS YOU COULD SAY, FAIRLY, WITHOUT THE GENEROUS SUPPORT FROM THE VARIOUS INSTITUTES AT THE NIH, AND ALSO FROM THE VISION AND THE VISIONARIES AMONG THE NIH PROGRAM OFFICERS AND LEADERS THAT HAVE FOUGHT SO HARD TO PROMOTE THIS VERY IMPORTANT FIELD OF GLYCOSCIENCE. SO ON BEHALF OF THE ENTIRE COMMUNITY OF GLYCOSCIENTISTS, I WOULD LIKE TO SAY THANK YOU VERY MUCH FOR YOUR SUPPORT, AND WITH THAT, I CAN TAKE QUESTIONS. >> GREAT. CAROLYN, THANK YOU SO VERY, VERY MUCH FOR A VERY CLEAR PRESENTATION. I GREATLY APPRECIATE THAT. THERE HAVE BEEN SOME QUESTIONS. WE'VE HAD ABOUT 400 PEOPLE SIGN IN TO THIS LECTURE, SO LET ME START WITH A COUPLE OF GENERAL QUESTIONS AND THEN I'LL TURN TO SOME MORE SPECIFIC ONES. ONE OF THE GENERAL QUESTIONS, CAN THIS TECHNOLOGY BE HARNESSED TO MODULATE THE HALF-LIFE OF BIOLOGICS FOR SMALL MOLECULE THERAPEUTICS? >> THAT'S A REALLY INTERESTING QUESTION. THE ANSWER IS, PERHAPS, AND IT WILL DEPEND OBVIOUSLY ON WHAT DIRECTION YOU WANT TO MODULATE. I CAN SAY THAT WE'VE ALREADY SEEN THAT WE CAN TUNE THE LYTAC STRUCTURES BOTH IN TERMS OF VALENCY AND INDIVIDUAL RECEPTOR BINDING AFFINITY IN ORDER TO ACCESS A RANGE OF DIFFERENT CLEARANCE KINETICS THAT GO ALL THE WAY FROM BEHAVING JUST LIKE ANY OLD ANTIBODY ALL THE WAY DOWN TO BEING LIKE RAPIDLY REMOVED WITHIN, LIKE, UNDER AN HOUR. AND EVERYTHING IN BETWEEN. SO I THINK, YEAH, I THINK IF YOU WANTED SOMETHING TO BE CLEARED FASTER THAN IT NORMALLY WOULD BE, THE LYTAC CAN HELP WITH THAT. IF YOU WANT SOMETHING TO HAVE AN EXTENDED RESIDENCE TIME BEYOND WHAT IT NORMALLY WOULD BE, I DON'T KNOW HOW WE COULD GET YOU IN THAT DIRECTION. >> OKAY, THANK YOU. NEXT A MORE GENERAL QUESTION, THE QUESTIONER IS EXPRESSING SOME CURIOSITY AS TO HOW MUCH OF A TARGET PROTEIN CAN YOU ACTUALLY REMOVE FROM THE CELL SURFACE? THEY POINT OUT THAT A WESTERN BLOT MAY NOT BE THE BEST MEASURE BECAUSE THEY REPRESENT A TOTAL AMOUNT OF PROTEIN IN THE CELL, THE AMOUNT REMOVED FROM THE SURFACE COULD BE MORE IMPORTANT WHEN CONSIDERING RECEPTOR TARGETS THAT MAY BE ABLE TO ENABLE SURVIVAL SIGNALING WITH WHAT FRACTION OF EXPRESS RECEPTORS ROMAINE. >> REMAIN. >> THAT'S AN EXCELLENT POINT. YOU'RE RIGHT. WHEN WE LICE THE WE LYSE -- THAT INCLUDES ALL THAT HASN'T YET MADE IT TO THE SURFACE. WE'VE DONE OTHER EXPERIMENTS FOCUSING, FOR EXAMPLE, ON QUANTITATIVE FLOW CYTOMETRY UNDER COLD TEMPERATURES AND SO ON, SHOWING THAT AT LEAST IN THE CASE OF EGFR AND HER2, THE MEMBRANE COMPONENT OF THAT PROTEIN IS DRIVEN DOWN TO, LIKE, BELOW 10%. THERE'S LIKE SINGLE DIGIT PERCENTAGES OF DETECTABLE PROTEIN WHEN YOU'RE IN THE PRESENCE OF A LYTAC AT MAYBE 10 NAN KNOW MOLAR CONCENTRATION SO YOU CAN REALLY GET RID OF THE CELL SURFACE PROTEIN, BUT OF COURSE THE MINUTE THE LYTAC IS WASHED OUT, THE PROTEIN THAT'S IN SORT OF TRANSIT STARTS TO APPEAR ON THE CELL SURFACE AGAIN AND YOU'RE DEALING WITH A SORT OF KINETICS OF DE NOVO PROTEIN BIOSYNTHESIS. BUT EVEN BEING ABLE TO DRIVE DOWN THE STEADY STATE LEVEL OF PROTEIN TO LIKE 20% OR LESS, THAT PUTS US SORT OF SQUARELY IN THE REGIME OF THE PROTAC SPACE. SO THAT'S OUR BENCHMARK, YOU KNOW, CAN WE ACHIEVE FOR THESE CELL SURFACE AND SECRETED PROTEINS THAT WHICH PEOPLE HAVE ACHIEVED FOR NUCLEAR AND CYTOSOLIC PROTEINS WITH SMALL MOLECULE PROTACs. >> NOW A COUPLE OF QUESTIONS ABOUT LYTACs THEMSELVES, VERSUS WHAT HAPPENS TO THE LYTACs AFTER THEY'RE TARGETED TO LYSOSOMES, ARE THEY ALSO DEGRADED OR ARE THEY RECYCLED, THEN A FOLLOW UP BY SOMEBODY ELSE, DO YOU HAVE ANY THOUGHTS ON HOW YOU MIGHT ENGINEER LYTACs WITH SPATIAL AND TEMPORAL CONTROL? >> THIS IS -- THOSE ARE EXCELLENT, EXCELLENT QUESTIONS.% THE FIRST OF THE QIS QUESTIONS IS ONE EVERYONE ALWAYS ASKS ME AND ONE WE CONTINUE TO ASK OURSELVES. SO IN OUR FIRST PATH LYTACs, THE ONES WE'VE PUBLISHED ON SO FAR, WE BUILT THEM FROM ANTIBODIES AND THE GLYCOPOLYPEPTIDES ARE MADE FROM NATURAL SUGARS AND NATURAL AMINO ACIDS, AND SO ALL OF THOSE PARTS MAKE THEM SUSCEPTIBLE THEMSELVES TO DEGRADATION IN THE LYSOSOME. SO BARRING ANY INTERVENTION, ONE WOULD EXPECT THE LYTAC TO BIND ITS TARGET TO BE TAKEN UP BY THE RECEPTOR, THE REACCEPT RECEPTOR LETS GO AND RECYCLES BUT THEN THERE'S NO REASON THAT THE LYTAC WILL LET GO AND THEN YOU WOULD EXPECT THE LYTAC TO MAKE THE ULTIMATE SACRIFICE AND JOIN ITS TARGET IN THE DEGRADATION PROCESS IN THE LYSOSOME. THAT'S NOT IDEAL. WE WOULD PREFER THESE LYTACs COULD BE CATALYTIC, RIGHT, JUST AS PROTACs ARE CELEBRATED TO BE. IT WOULD BE BETTER IF THEY'D GO BACK OUT AND GRAB ANOTHER ONE AND BRING IT IN. THAT IS A FEASIBLE PROCESS AND WHAT WE NEED TO ACHIEVE THAT ARE TWO THINGS. FIRST OF ALL, WE WOULD WANT THE ANTIBODY TO LET GO THE TARGET IN A WAY THAT'S PH-SENSITIVE, JUST LIKE THE MANNOSE 6 PHOSPHATE RECEPTOR DOES. THERE ARE KNOWN STRATEGIES FOR ENG FEARING ENGINEERING A PH SWITCH IN THE COMBINING SITE. ONE COULD HAVE SELECTED AN ANTIBODY TO HAVE THAT FEATURE IF ONE MADE AN ANTIBODY FROM SCRATCH FOR THE PURPOSE OF A LYTAC. WE WERE BORROWING OTHER PEOPLE'S ANTIBO BODIES IN OUR FIRST GENERATION LYTACs SO THERE'S NO REASON CETUXIMAB WOULD HAVE BEEN ENGINEERED TO HAVE THAT PROPERTY BUT IF YOU START FROM SCRATCH, YOU COULD BUILD THAT INTO A CLONAL SELECTION PROCESS. NUMBER TWO, ANTIBODIES HAVE THE GREAT BENEFIT THAT THEY COMBINE FCRN, AND THE FCRN GIVES THEM THIS LONG PLASMA RESIDENCE TIME BECAUSE THEY CAN CONTINUOUSLY RECYCLE. AND SO LIKE BUILDING INTO THE ANTIBODY THE ABILITY TO HARNESS A RECYCLING RECEPTOR LIKE THE MANNOSE 6 PHOSPHATE RECEPTOR ALSO HAS WOULD BASICALLY GIVE YOU AN INJECTION METHOD. SO ALL OF THAT COULD BE ENGINEERED INTO AN ANTIBODY LYTAC. HAVING SAID THAT, ALL YOU HAVE TO DO TO SOLVE THIS PROBLEM IS SWITCH FROM USING ANTIBODIES TO USING A DIFFERENT KIND OF BINDING MOLECULE THAT IS NOT SUSCEPTIBLE TO LYSOSOMAL DEGRADATION. SO A SMALL ONE THAT'S NOT METABOLIZED ON THE TIME SCALE OF DEGRADATION COULD SOLVE THAT PROBLEM ENTIRELY. SO THAT'S KIND OF HOW WE SEE IT. BUT BOTH OF THESE ARE IMPORTANT DIRECTIONS FOR THE NEXT GENERATION OF SMARTER LYTACs TO BE SURE. >> WELL, AND IN THAT REGARD, ANOTHER QUESTION RELATED, COULD FAB FRAGMENTS BE USED IN STEAD OF THE WHOLE ANTIBODY, SPECULATION ABOUT STAIRK HINDRANCE POSSIBLY INTERFERING WITH THE BINDING. >> THAT'S A GREAT QUESTION. WE HAVE MADE LYTACs FROM FABs, THEY WORK GREAT. WE'VE MADE THEM FROM CETUXIMAB AND PERTUZIMAB, THEY'RE PRETTY INDISTINGUISHABLE IN CELL CULTURE MODELS. WE'VE MADE NANOBODIES AND MADE LYTACs OUT OF THEM. WE'VE MADE LYTACs OUT OF -- WE HAVE YET TO FIND A TYPE OF MOLECULE THAT'S KIND OF IMPOSSIBLE TO FORM A LYTAC, BUT WE HAVE CERTAINLY MADE CONSTRUCTS THAT DON'T FUNCTION WELL AND USUALLY IT'S BECAUSE WE HAVEN'T GOT ENOUGH SPACE BETWEEN THE TARGET BINDER AND THE MANNOSE 6 PHOSPHATE OR THE ESGPR BINDER. SO YOU HAVE TO HAVE ENOUGH SPACE THERE. AS LONG AS THERE'S ENOUGH SPACE, I THINK IT'S PRETTY MODALITY-AGNOSTIC. >> GREAT. ONE ADDITIONAL QUESTION ASKING IF LECTINS COULD POTENTIALLY BE USED AS THE TARGET BINDER TO SPECIFICALLY TARGET PATHOGENIC LOI CAN MOTIFS, FOR EXAMPLE, THOSE INVOLVED WITH PATHOGEN HOST INTERACTIONS? >> THAT WOULD AB REALLY COOL THING TO TRY. I DON'T SEE WHY NOT. WE HAVEN'T DONE ANYTHING LIKE THAT. BUT AGAIN THE LYSOSOME IS CHOCK FULL -- THERE'S RNASES IN THE LYSOSOMES. THERE ISN'T ANYTHING THAT CAN'T BE DEGRADED IF YOU CAN JUST GET IT THERE. SO I WOULD SAY YEAH, THAT WOULD BE AN INTERESTING THING TO TRY. >> GREAT. WELL, I KNOW WE'RE GETTING CLOSE TO THE HOUR HERE. I THINK -- I'M JUST SCANNING THROUGH BECAUSE ACTUALLY WE'VE ONLY BEEN ABLE TO TOUCH A FEW OF THE QUESTIONS, THERE ARE MANY. LET'S SEE IF THERE'S ANY FINAL ONE AND I'LL -- SO THIS IS A LITTLE BIT MORE TECHNICAL IN NATURE, BUT SO WHEN MAKING THE ORIGINAL LYTACs WITH THE ANTIBODIES AND LOOKING AT THEM BY -- GEL, ARE YOU ABLE TO DETERMINE THE EXTENT OF MODIFICATION OF THE ANTIBODY OR IS IT QUALITATIVE? IF THIS ISN'T A METHOD TO MEASURE THE AMOUNT OF CONJUGATION, ARE YOU USING ANOTHER METHOD TO QUANTITATE THAT? >> THAT'S A GREAT QUESTION. SO TO BE SURE, THESE NATIVE GEL -- IT JUST A FUN PICTURE TO LOOK AT AND IT'S VERY QUALITATIVE. SO WE ALWAYS DO DETAILED PEPTIDE MAPPING BY MASS SPECTROMETRY ON THESE LYTAC PRODUCT SO WE ACTUALLY DO KNOW EXACTLY WHICH LYSINES ARE MODIFIED TO WHAT EXTENT IN THIS HETEROGENEOUS POPULATION. BUT THOSE DATA ARE NOT AS FUN TO LOOK AT. >> GREAT. AND THEN I'LL JUST ASK ONE FINAL QUESTION, AGAIN APOLOGIES TO THE MANY LISTENERS BECAUSE THERE ARE MANY, MANY MORE QUESTIONS THAN WE HAVE TIME FOR. BUT THIS LAST ONE, REGARDING THE PH MECHANISM THAT GOVERNS THE MANNOSE 6 PHOSPHATE RECEPTOR BINDING, HAVE YOU CONSIDERED THE EFFECT OF AN ACIDIC MICROENVIRONMENT AND WHETHER THIS COULD DAMPEN THE THERAPEUTIC EFFECT OF THIS STRATEGY? >> THAT'S A REALLY, REALLY EXCELLENT QUESTION. AND YOU'RE RIGHT, ABSOLUTELY, YOU KNOW, WE DON'T REALLY HAVE A GOOD WAY TO TEST THAT EXPERIMENTALLY IN AN ANIMAL MODEL BECAUSE IT ISN'T AS STRAIGHTFORWARD TO KIND OF INTENTIONALLY PERTURB THE PH IN A TUMOR MICROENVIRONMENT AND SEE THE EFFECT ON EFFICACY, BUT I THINK THAT'S A VERY IMPORTANT CONSIDERATION. AND HOW WOULD YOU MITIGATE THAT IS THE NEXT QUESTION, IF THAT WAS A PROBLEM, AND I THINK THE WAY TO MITIGATE THAT WOULD BE TO CRANK UP THE VALENCY OF YOUR RECEPTOR LIGAND SO YOU'RE ABLE TO OVER COME A DEFICIT IN AFFINITY. I THINK THAT'S A GREAT QUESTION, I'VE BEEN ASKED THAT AND HAVE NO REALLY GOOD RESPONSE TO THAT OTHER THAN THANK YOU FOR KEEPING IT HIGH ON OUR RADAR SCREEN. >> AGAIN, APOLOGIES TO EVERYBODY BECAUSE THERE MANY MORE QUESTIONS THAN WE HAD TIME TO PROVIDE ANSWERS TO. CAROLYN, ON BEHALF OF THE SOME 400 PEOPLE WHO TUNED IN, WE WANT TO REALLY THANK YOU FOR THIS BEAUTIFUL SEMINAR I HOPE THOSE POSTBACKS WHO LISTENED IN, BEING ABLE TO DESCRIBE THINGS IN CLEAR, UNAMBIGUOUS WAY, IT'S SO IMPORTANT AND I THINK CAROLYN HAS GIVEN A BEAUTIFUL EXAMPLE OF THAT TODAY. SO CAROLYN, THANK YOU, BE WELL AND GREAT TO SEE YOU TODAY.