WE'RE ONLINE? OK
SO THEN I CAN GREET EVERYONE WHO
IS ONLINE AS WELL AS HERE FOR
TODAY'S GRAND ROUNDS.
I'M REALLY DELIGHTED TO BE HERE
TO INTRODUCE OUR SPEAKER,
MITCHELL HO.
SO I'M SUSAN GOTTESMAN, CHIEF OF
THE LABORATORY OF MOLECULAR
BIOLOGY.
IRA PASTAN WHO FORMED THE LAB
AND HAS BEEN THE GUIDING FORCE
BEHIND IT IS HERE WITH US AS
WELL, AND MITCHELL FIRST CAME TO
US TO WORK WITH -- SO A LITTLE
ABOUT MITCHELL.
HE DID HIS UNDERGRADUATE
EDUCATION IN CHINA.
HE CAME TO THE U.S. AS A
POSTBAC, WHICH I DIDN'T KNOW
UNTIL I READ YOUR CV, AT UCSF,
SPENT SOME TIME IN CALIFORNIA AT
SOME COMPANIES, AND THEN GOT HIS
PH.D. IN IMMUNOLOGY AT ILLINOIS
URBANA-CHAMPAIGN AND STARTED A
POSTBAC WITH IRA IN THE
LABORATORY OF MOLECULAR BIOLOGY,
DID SOME BEAUTIFUL WORK, AND WE
WERE ABLE TO DO A TENURE TRACK
SEARCH AND FOUND MITCHELL IN
2008, AND HE STARTED HIS OWN
ANTIBODY THERAPY GROUP WITH LMB,
HE'S SINCE A LONG TIME AGO NOW
BEEN TENURED AND NOW IS DEPUTY
CHIEF OF THE LABORATORY OF
MOLECULAR BIOLOGY.
IN ADDITION, A FEW YEARS AGO, HE
STARTED THE ANTIBODY ENGINEERING
PROGRAM, WHICH SOME OF YOU MAY
HAVE TAKEN ADVANTAGE OF OVER THE
YEARS, AND IS DIRECTOR OF THAT
ALSO IN LMB.
SO MITCHELL DOES A LOT OF THINGS
AND HE DOES A LOT OF THEM WELL.
HE'LL BE TELLING YOU ABOUT
ASPECTS OF HIS RESEARCH IN A FEW
MINUTES, BUT JUST A FEW OTHER
THINGS, HE WAS THE LEAD
INSTRUCTOR FOR THE FAES
BIOCHEMISTRY COURSE FOR 14
YEARS, AND NOW IS ON THE FAES
BOARD OF DIRECTORS.
I THINK THIS IS A PATTERN.
HE GETS INVOLVED WITH THINGS,
DOES THEM WELL AND THEN GETS
DRAGGED INTO LEADERSHIP.
SO HE'S ALSO DOING -- HE'S A
LEADERSHIP IN THE ANTIBODY
SOCIETY AND THE CHINESE ANTIBODY
SOCIETY, HAS A NUMBER OF HONORS
AND LOTS OF INVITATIONS AND WAS
ELECTED AS A FELLOW IN THE
AMERICAN INSTITUTE FOR MEDICAL
AND BA BIOLOGICAL ENGINEERING IN
2003?
2023.
I KNEW I TYPED IT WRONG.
LAST YEAR.
SO TODAY, HE'S GOING TO TELL YOU
SOME ASPECT OF HIS VERY ACTIVE
RESEARCH PROGRAM ABOUT GIEP GLIP
CANS AS CANCER TARGETS FOR CAR-T
IMMUNOTHERAPY, SO LOOKING
FORWARD TO IT.
[APPLAUSE]
>> THANK YOU, SUSAN, FOR A VERY
KIND INTRODUCTION.
IT'S WONDERFUL TO SEE COLLEAGUES
HERE.
AS SUSAN INTRODUCED, I STARTED
MY POSTDOC MORE THAN 20 YEARS
AGO IN DR. IRA HAS TA PASTAN'S ,
GLAD THAT DR. PASTAN IS HERE
TODAY, AND ACTUALLY JUST TO MAKE
SURE I'M STILL DOING GOOD.
OKAY.
SO IN THE NEXT 40, 45 MINUTES,
I'M GOING TO TALK ABOUT MY WORK
ON THE GLYPICAN AS A CANCER
TARGET.
MANY OF YOU PROBABLY KNOW I'M
WORKING ON GLYPICANS FOR A
WHILE, WE STARTED CHEMISTRY, WE
MADE ANTIBODIES, WE ACTUALLY
TRIED TO DEVELOP A CELL THAT
EVENTUALLY CAN BENEFIT PATIENTS
AND IN RECENT YEARS, WE GET INTO
THE FIELD OF CAR-T CELLS, TRY TO
USE OUR ANTIBODY WE DEVELOPED TO
MAKE CAR-T CELLS, HOPEFULLY WE
CAN TREAT PATIENTS.
IN FACT, WE HAVE ONE CLINICAL
TRIAL ONGOING IN THE CLINICAL
CENTER AND THREE UPCOMING NEXT
YEAR, AND WE ARE VERY EXCITED.
I THINK IT'S A GOOD TIME FOR ME
TO SHARE SOME OF OUR WORK.
THE WORK IS ABOUT 15 OR 16
YEARS' WORK HERE WITH MY
COLLEAGUES, AND WE'RE ALWAYS
LOOKING FOR ADVICE AND
COLLABORATIONS.
SO THIS IS MY DISCLOSURE, JUST
SIMPLY SAY I'M AN INVENTOR FOR
MANY PATENTS THAT BELONG TO THE
NIH ABOUT ANTIBODY THERAPIES.
MY LAB FORTUNATELY GETS RESEARCH
FUNDS OVER THE YEARS FROM -- AND
PHARMACEUTICAL COMPANIES.
THEY'RE NOT JUST PROVIDING
USEFUL RESOURCE BUT ALSO EDUCATE
US HOW TO MOVE FROM THE LAB TO
THE CLINIC, ULTIMATELY BENEFIT
THE PATIENTS.
SO I WANT TO START MY TALK WITH
AN OVERVIEW WHAT ANTIBODY CAN DO
FOR CANCER THERAPY.
SO ANTIBODY AS I SHOW HERE IN
MOST RECENT REVIEW WE RAO
WRITING, AND THAT IS IT'S A Y
SHAPE OF PROTEINS AS SHOWN HERE.
I DON'T HAVE POINTER BUT I TRY
TO MAKE A DIGITAL POINTER.
OKAY.
SO THIS IS A Y SHAPE ANTIBODY,
AND THE WAY ANTIBODY ATTACKS THE
CANCER CELLS, THEY NORMALLY --
ADCC, ANTIBODY DEPENDENT
CELLULAR TOXICITY, AND ENGAGE
THE NK CELL MIKE PHAG MICROPHAGE
ANTIBODY.
CAN ALSO BLOCK THE CHECKPOINT,
PD-1 -- INTERACTIONS.
AND ANTIBODY COULD ACTUALLY
ENGAGE THE T CELLS THAT'S
RELATED TO WHAT I'M GOING TO
TALK ABOUT, THEY ACTUALLY BRING
THE T CELLS USING SO-CALLED --
ANTIBODY THE T-CELL ENGAGE TO
HAVE ANOTHER ARM -- ON T CELLS,
BRING THE T CELLS TO THE TUMORS,
AND KILL IT.
AND THE ANTIBODY COULD ALSO
BRING OTHER IMMUNE CELLS LIKE NK
CELLS, ATTACK TUMORS, BUT IN
THIS CASE LIKE LUNG CANCER
CELLS, THEY EXPRESS BOTH EGFR
AND MET, SO ANTIBODY COULD HAVE
BISPECIFIC TARGETED -- EGFR,
BLOCK THE HGF BINDING AND EGF
BINDING AND THE INHIBITOR CANCER
SIGNALING ON THE OTHER HAND
BRING THE NK CELLS TO THE TUMOR
CELLS.
AND THE OTHER THING THAT'S VERY
INTERESTING AND STILL VERY
ACTIVE FIELD IS THE ANTIBODY
COULD BRING THE CYTOTOXIC DRUGS
LIKE SMALL MOLECULES, ADC, AND
IT CAN ALSO -- RADIO ISOTOPE, MY
COLLABORATORS HERE, WE ARE
WORKING ACTIVELY ON THIS, I
DON'T HAVE TIME TO SHARE THE
VERY BEAUTIFUL DATA WE HAVE NOW
USING OUR ANTIBODY TO PUT THE
RADIO ISOTOPE ON IT.
AND THE OTHER THINGS,
IMMUNOTOXIN BRING TO THE HOME,
AND THAT IS DR. IRA PASTAN AND
HIS TEAM IN LABORATORY OF
MOLECULAR BIOLOGY OVER THE YEARS
DEVELOPED AN IMMUNOTOXIN, AND
THAT'S THE FIRST AND THE ONLY
IMMUNOTOXIN APPROVED BY FDA
TREATING -- LEUKEMIA, QUITE A
MAZE HERE LEADING THE TRIALS.
THEN WHEN WE LOOK AT THE CANCER
FIELD, THESE ARE THE CANCER MOST
FREQUENTLY KILLED PATIENTS IN
THE UNITED STATES.
NUMBER ONE IS LUNG CANCER
FOLLOWED BY COLORECTAL CANCER,
PANCREATIC CANCER, BREAST
CANCER, PROSTATE, LIVER AND
BILE -- THERE ARE REALLY NOT
MANY TARGETS FOR THOSE TOP
CANCERS.
AND EVEN FOR LATE STAGE CLINICAL
TRIALS, THERE ARE NOT MANY --
AVAILABLE FOR TREATING THOSE
CANCERS.
AND WHEN I STARTED MY CAREER
HERE AND HAVE MY OWN LAB, I GET
ADVICE SAYING WE SHOULD WORK ON
LIVER CANCER BECAUSE THAT'S THE
CANCER VERY POORLY UNDERSTOOD
AND WE NEED THERAPY.
SO THAT'S WHY I STARTED MY
JOURNEY HERE, THE LAB WORKING ON
THE GPC ON LIVER CANCER, THEN
LATER ON IN MOST RECENT YEARS WE
EXPRESS -- EXPAND OUR PROGRAM TO
GAAP 2 IN NEED TRICK C PEDIATRID
ALSO PANCREATIC CANCER SO I'M
GOING TO TALK ABOUT THAT.
SO CAR-T CELLS BASICALLY MEANS
YOU HAVE TO REPROGRAM THE T
CELLS, LEAD THE T CELLS TO THE
TUMORS AND IT HAS TO RECOGNIZE
THE TUMOR ENERGY.
AS I SAID, TO BE ABLE TO CURE
TUMOR CELLS, YOU REALLY NEED TO
HAVE THE RIGHT ANTIGENS.
AND WHEN CAR-T CELLS BE VERY
SUCCESSFUL SO FAR TO KILL TUMOR
CELLS, OUR NCI COLLEAGUE STEVE
ROSENBERG, THEY ACTUALLY SHOWED
THE CD19 CAR-T CELL COULD KILL
THE LEUKEMIA CANCER CELLS, BUT
WHEN YOU USE THE SAME APPROACH
TO THE SOLID TUMOR, THEN WE ARE
FACING MANY BARRIERS OF
CHALLENGES HERE.
SO THE CAR-T CELLS NEED TO GET
INTO THE TUMOR MICROENVIRONMENT
AND OVERCOME THE EXTRACELLULAR
MATRIX, AND THEN HAS TO BE
ENCOUNTERED WITH
IMMUNOSUPPRESSIVE CELLS AND
IMMUNOSUPPRESSIVE MOLECULES.
AND IN THE END, WE REALLY NEED
THE TUMOR ANTIGENS, AND EVEN
FOR -- THOSE ARE HETEROGENEOUS
MEANING THEIR EXPRESSION MAY BE
VERY DIFFERENT FROM PATIENT TO
PATIENT AND EVEN IN THE SAME
PATIENT, FROM TUMOR CELLS TO THE
OTHER TUMOR CELLS, THEY ARE VERY
DIFFERENT.
SO MY LAB HAS BEEN FOCUSED ON
STUDIES OF GLYPICAN AS NEW
TARGETS OVER THE YEARS.
SO THERE ARE SIX MEMBERS IN THE
GLYPICAN FAMILY.
THEY ALL HAVE A CORE PROTEIN
WITH THE HEPARAN -- THEY DISPLAY
ON THE CELL SURFACE AS AT
GLYCOLIPID, ALL OF THEM HAS
THESE TWO -- THEY'RE CANONICAL,
DOESN'T MATTER IN DROSOPHILA OR
C. ELEGANS OR -- WHICH
INDICATING THE EVOLUTIONARY
SELECTION PRESSURE, THESE ARE
IMPORTANT FOR THEIR FUNCTION.
AND WE STARTED WITH AS I SAID
THE GPC3 IN LIVER CANCER AND
THEN WE GRADUALLY EXPAND OUR
PROGRAM STARTING GPC -- WHERE WE
STARTED GPC1 -- TO INDUCE WPT WT
SIGNALING IN THE CANCER CELLS
WHILE GPC 4, 5 AND 6 INHIBIT WNT
SIGNALING SO OPPOSITE.
SO THIS IS GPC3.
AND WE STUDIED THE GPC3 WHEN --
SO WE STUDIED GPC3, WE FOCUS ON
THE WNT FRIZZLED/GPC AS
SIGNALING ACTIVATION COMPLEX ON
THE CANCER CELL SURFACE.
THERE'S QUITE CONFLICTING DATA
FROM MY LAB AND OTHER LAB TO TRY
TO UNDERSTAND HOW GPC3 -- WNT.
WE NEED TO DO BIOCHEMISTRY.
SO HERE IS THE MODEL WE HAVE.
THE WNT ACTUALLY ATTRACTS THE
GPC3 AS CON CONCENTRATEWITH FRL
AND ULTIMATELY INDUCE THE BETA
CAR-T SIGNALING AND UPREGULATE
WNT TARGET GENES AND LEAD TO THE
HCC LIVER CANCER CELL GROWTH.
WHEN ONE OF MY FIRST POSTDOC
CAME TO THE LAB, SHE WAS VERY
INTERESTED IN CANCER SIGNALING.
SO I SAID WELL, WHY DON'T YOU
JUST TRY TO DO SOME BIOCHEMISTRY
OF THE WNT AND THE GPC3 TO
FIGURE OUT THE INTERACTION, AS I
SAID FOR A WHILE, THIS KIND OF
INTERACTION IS NOT CLEAR.
SO THE BIOCHEMISTRY OF GPC3 IS
NOT KNOWN.
AND SO WE START TO MAP THE WNT
BINDING DOMAIN ON THE GPC3 --
CHAIN AND LUCKILY WE HAVE A --
JIAN LIU -- WITH DEFINE THE
RESIDUES.
USING THOSE KIND OF TOOLS, WE
SHOULD BE ABLE TO MAP THE --
EXACTLY THE BINDING MOTIF ON THE
HEPARAN SULFATE GLYCATE CHAINS
FOR THE WNT.
THIS IS IMPORTANT FINDING
BECAUSE IT DOES NOT SEEM LINE
THE WNT BINDING TOTALLY RELIES
ON THE NEGATIVE CHARGE BECAUSE
WE HAVE MORE NEGATIVE CHARGES --
RESIDUES OR -- WOULD NOT DO THE
WNT BINDING AND WOULD NOT
FUNCTIONALLY INDUCE THE WNT
SIGNALING.
SO ULTIMATELY, YOU NEED A
STRUCTURE OF MINIMUM SIX
SACCHARIDE RESIDUES, YOU NEED A
2-O AND 6-O SULFATIONS AND
CONFIRM NATION -- NOT JUST
NEGATIVE CHARGE AND KNEE 3-O
SULFATION TO ENHANCE THE WNT
SIGNALING.
FURTHERMORE, WHEN WE LOOK AT THE
WNT BINDING SIDE ON THE PROTEIN
CORE AND ACTUALLY IT WAS WEI
GAO'S IDEA, SHE CAME TO ME AND
SAID HEY -- ON THE ENVELOPE OF
GLYCAN 3 MIGHT BE INTERESTING TO
LOOK BECAUSE IT'S SIMILAR LIKE A
FRIZZ EL.
SO I CONSULTED MY COLLEAGUE B.K.
LEE AND B.K. ACTUALLY DESIGNED
SUBMUTATION IN THIS REGION AND
WEIGHTED ALL THE MUTATIONS.
WE ALSO TESTED THE CELL IN A
MOUSE MODEL, INDICATING, YES,
THAT'S THE WNT BINDING SITE ON
THE GPC3.
AND MORE RECENTLY, BEFORE AND
DURING THE PANDEMIC, I
COLLABORATED WITH STANFORD TO
TRY TO FIGURE OUT MORE ABOUT THE
GLYPICAN, HOW GLYPICAN ACTIVATE
WNT SIGNALING.
SO THIS IS THE WNT WHERE IT'S
NOT ACTIVATED.
SO THERE'S NO GPC3 HERE.
THERE'S A PROTEIN CALLED ZNRF3,
RNF43, I USUALLY CALL IT ZNR, IT
ACTUALLY DEGRADE FRIZZLE, IN LOW
LEVELS.
SO WHEN GLYPICAN COMES, THE
GLYPICAN COULD BE ELEVATED IN
THE CANCER LIKE IN LIVER CANCER,
PROTEOGLYCAN IS A CORE RECEPTOR
FOR HEPATITIS AND HEPATITIS B
AND HEPATITIS C, EVEN IN THE
COVID, THEY ARE ALSO CO-RECEPTOR
AND ATTACHMENT FOR THE SARS COV.
SO INCLUDING POTENTIALLY CHRONIC
INFLAMMATION, AND THOSE
RECEPTORS MAY BE ELEVATED IN THE
LIVER CANCER, AND THEN ACTIVATE
WNT SIGNALING BY INTACT WITH WNT
AND FRIZZLE AS I JUST DESCRIBED,
THEY HAVE VERY DEFINED
BIOCHEMICAL INTERACTIONS.
AND WHAT THE GLYPICAN COULD DO,
SO I CALLED THE SECOND
MECHANISM, ACTUALLY ANOTHER
PROTEIN RELATIVELY NEW FOR THE
WNT FIELD, AND IT SEEMS LIKE A
BROAD CANONICAL RECEPTOR CALLED
LGR456 TO THE CELL SURFACE AND
THEY COULD ACTUALLY -- WITH THE
ZNR PROTEIN AND CAUSE MAYBE ENDO
CYTOSIS, SO THE FRIZZLE WILL NOT
BE UBIQUITINNIZED ANYMORE.
SO THAT WAY YOU CAN -- THE WNT
SIGNALING.
WHAT WE FIND HERE IS GLYPICAN
COULD DO THE SAME THING, IT
COULD BRING THE -- 40 TO THE
CELL SURFACE.
WE KNOCK OUT THE 456, COULD
STILL BRING TO THE CELL SURFACE
AND ACTIVATE WNT.
MEANING THE GLYPICAN COULD DO
BOTH THINGS.
IT COULD ACTUALLY -- WNT,
BROUGHT WNT TO THE CELL SURFACE
WITH FRIZZLE TO ACTIVATE THE
BETA CATENIN, IT COULD ALSO
BRING THE R-SPONDIN TO THE CELL
SURFACE AND UBIQUINATION BY R
PROTEINS.
SO BASED ON ALL THIS RESEARCH,
WE THINK TARGETING WNT IS A
REALLY GOOD IDEA BUT NOT
SPECIFIC BECAUSE WNT AND FRIZZLE
ALSO EXPRESS NORMAL TISSUES.
AND INDEED ON A LOT OF LAB
INCLUDING COMPANIES WORKING ON
WNT AND FRIZZLE TRIED TO DEVELOP
DRUGS BUT WE BELIEVE THE BETTER
STRATEGY MAY BE JUST TARGET
TARGETING -- GLYPICAN BECAUSE
THEY ARE MORE TUMOR SPECIFIC
ANTIGENS AND THE SAME COMPANY IN
THE LAB ALSO TARGETED -- USING
ANTIBODY BUT AGAIN IT ALSO CAN
BE ON THE NORMAL TISSUES AND WE
THINK GLYPICAN MIGHT BE A BETTER
ANTIGEN.
MANY YEARS AGO, WHEN I START TO
MAKE GPC3 ANTIBODY, IT WAS VERY
CHALLENGING.
WE FAILED AT THREE -- NONE OF
THEM WORKED, AND I JUST CAME
BACK LAST WEEK, STILL SCIENTISTS
CAME TO THE MEETING AND TOLD ME
THEY TRIED TO MAKE ANTIGPC3
ANTIBODY LIKE YP7 WE MADE, AND
THEY EITHER FAILED OR THEY ONLY
GET ANTIBODY WITH LOW AFFINITY.
THE REASON IS THE GLYPICAN --
IT'S VERY HARD TO MAKE
ANTIBODIES.
THEY'RE EXPRESSED IN THE FETAL
LIVER AND PLACENTA AND HIGH
EXPRESSION OF -- THE WAY YOU
MINIMIZE HCC, USING PEPTIDE WE
SCREENED A LOT OF CELLS AND
TISSUES AND MANY, MANY CLONES.
TO DO THAT, REMEMBER WE HAVE A
TH96 WELL FLOW CYTOMETRY IN THE
LAB AND EACH PLATE, YOU HAVE TO
RUN ONE HOUR.
SO THAT MEANS MANY, MANY HOURS,
WE STAYED OVERNIGHT TO SCREEN
ALL THE CLONES AND TRY TO FIND A
WP7.
SO YEN PHUN GWAS A POSTBAC AT
THAT TIME, DID ALL THIS WORK,
SHE IS NOW AT HARVARD AS A
RESIDENT AND DOING -- BEING A
DOCTOR THERE IN THE SURGERY
DEPARTMENT.
SO NOW WE FIND Y 7 HAS NO BINDO
BINDING ON ANY HUMAN O ORGAN AND
THAT IS GREAT -- IN THE CART
CELLS AT THE MOMENT.
SO THEN IT COMES TO THE CAR-T.
SO WE TRY TO MAKE CAR-T CELLS
USING THE ANTIBODY WE DEVELOPED
FOR THE GPC3.
THEN A LOT OF PEOPLE CONFUSED
WITH THEM BUT TO BE HONEST, BOTH
LIs ARE REALLY IMPORTANT, THEY
BOTH REALLY CONTRIBUTED EQUALLY
TO MANY OF OUR PROJECTS.
SO ACTUALLY WHEN DAN CAME TO THE
LAB, SHE WAS A PH.D. STUDENT
FROM PROGRAM FROM CHINA AND
DECIDED TO STAY AS A POSTDOC,
SHE JUST GOT OUTSTANDING AWARD
YESTERDAY AND GAVE KEYNOTE IN
THE FELLOW RETREAT.
AND NAN CAME AS A POSTDOC IN THE
LAB, AND EVENTUALLY PROMOTED AS
A STAFF SCIENTIST BY DOING CAR-T
CELLS AND LAST YEAR, SHE LEFT
AND NOW LEADING CAR-T --
ALLOGENIC CAR-T ENGINEERING
PROGRAM IN ASTRAZENECA.
SO BOTH OF THEM ARE DOING
IMPORTANT WORK HERE.
SO WE ACTUALLY PUT THE ANTIBODY
SINGLE CHAIN -- FUSED WITH CD8H
AND THE H-1BB CD3 AS A CAR AND
WE PUT HEGFR TRUNK BECAUSE WE
WANT TO REMOVE IT, WE PUT OFF
SWITCH ON IT SO IT CAN BE REM
REMOVED.
WE DON'T WANT TO GO VERY DETAIL
BUT WE ACTUALLY COMPARE THE TWO
ANTIBODY, ONE BIND -- TERMINAL,
ONE THE C TERMINAL AND WE FIND
THE ONE CAR-T DERIVED FROM THE
HUMANIZED YP7 CLOSE TO THE
MEMBRANE IS THE MOST POTENT AND
ON DOSE DEPENDENT YOU CAN SEE
IT'S ACTUALLY VERY EFFECTIVE
WHEN IT'S A HIGH DOSE, ALL THE
MICE RECOVER, WERE TUMOR-FREE.
BUT WE ACTUALLY LIKE -- IT'S A
SINGLE DOMAIN BODY.
I'M GOING TO TALK ABOUT IT WHEN
I MATCH THE GPC1 PROJECT.
AND WE REALLY WANT TO --
EVENTUALLY WE THINK MAYBE WE CAN
OPTIMIZE CAR-T CELL IN
PARTICULAR FOR NANOBODY, BECAUSE
THE CURRENT FORMAT CAR-T DOES
NOT SEEM COMPATIBLE WITH
ANTIBODY SO WE NEED TO DO MORE
ENGINEERING TO MAKE IT WORK, TO
FORM -- USING NANOBODY WITH
CAR-T CELLS.
BUT BECAUSE OF THIS WORK, WE ARE
NOW RUNNING A CLINICAL TRIAL AT
THE NIH.
I THINK HE'S NOW IN LIVER CANCER
MEETING AT SALT LAKE CITY, AND
HE HAPPILY SHARED ME SOME OF THE
MOST UPDATED CLINICAL DATA AND
SO THE TRIAL IS STILL GOING ON.
WE ARE ON THE SECOND DOSAGE, AND
WE ALREADY PASS THE FIRST DOS
DOSAGE, SECOND DOSAGE OF THE
TRIAL SO WE'RE RECRUITING
PATIENTS.
AND AS A MOLECULAR BIOLOGIST, I
ALWAYS WANT TO KNOW WHY, THE
MECHANISMS, AND WHY CAR-T CELLS
COULD WORK, SO WE ACTUALLY
ISOLATED SINGLE CAR-T CELLS FROM
MICE AND TRY TO STUDY THEM.
SO ONE WAY WE DO IT, WE ACTUALLY
ISOLATE CAR-T CELL FROM SPLEEN
OR FROM THE MOUSE BLOOD AND
EVENTUALLY IN THE PATIENTS, WE
CAN COLLECT THE BLOOD FROM THE
PATIENTS IN THE CLINICAL TRIAL.
SO WE CO-CULTURE WITH TUMOR
CELLS AND ANALYZE THEM.
WE ANALYZE -- IT'S LIKE A
NANOFNANOFLUIDIC CHAMBER, WE HA5
CHEMOKINE CYTOKINE WHICH LISTED
VERY SMALL HERE, SORRY FOR THAT,
ALL THE CYTOKINE, CHEMOKINE AND
WE CAN TRACK THEM AND WE CAN
ANALYZE FROM EACH SINGLE CAR-T
CELLS, WHAT CYTOKINE, CHEMOKINE,
CYTOTOXIC EFFECT RELEASED
SIMULTANEOUSLY.
AND IN THIS MICROFLUIDIC
CHAMBERS.
TOTAL 500 -- CAR-T CELLS SO BY
DOING THAT, WE ACTUALLY FIND --
LITTLE SURPRISING AT THAT TIME
IS, WHEN WE CO-CULTURED WITH
TUMOR CELLS OR KNOCKOUT CELLS
OBVIOUSLY, THE CAR-T CELLS WILL
RECOVER THE -- MICE.
THOSE MUSE TREATED WITH CAR-T TE
TREATED WITH CAR-T CELLS FOR
MANY, MANY WEEKS, THEY ARE STILL
SPECIFIC FOR THE ANTIGEN.
SO IF IN CO-CULTURE WITH THE
ANTIGEN POSITIVE TUMOR CELLS,
YOU CAN SEE THEY RELEASE A LOT
OF CYTOKINE OR CYTOTOXIC
FACTORS, BUT IF YOU CO-CULTURE
WITH KNOCKOUT TUMOR CELLS, THE
SAME TUMOR CELLS, JUST KNOCK OUT
THE GPC3, YOU HAVE MUCH LESS.
SO THEY'RE PERSISTENT, THEY ARE
IN MICE FOR MANY WEEKS, AND THEY
ARE -- BUT VERY FEW CAR-T CELLS.
ONLY .5 TO 1%.
WE CALL IT POLYFUNCTIONAL
BECAUSE THEY CAN PRODUCE --
GAMMA INTERFERON -- SO THAT'S A
LITTLE SURPRISING TO US BUT IN
THE BEGINNING WE THOUGHT MAYBE
EVEN MORE THAN THAT, MAYBE 5%,
10% BUT ACTUALLY IT'S ONLY LESS
THAN 1%.
WHEN WE LOOK AT THE CAR-T CELLS,
WE ACTUALLY LOOKED IN THE
CLINICAL, WE FIND THIS NUMBER IS
QUITE RIGHT FOR MOST OF THE
CAR-T CELLS.
SO WHEN WE LOOK AT THE GENOME,
THE PHENOTYPE WE WANT TO LOOK AT
THE GENOME TYPE.
WE WANT TO SEE WHAT ARE THESE
CAR-T CELLS, WHERE THE TRANS
GENE GOES -- NORMALLY LAB USE TO
SEQUENCE THE HIV VIRUS IN THE
T-CELL GENOME.
SO WE ACTUALLY PARTICULARLY
INTEGRATION SITE IN THE T-CELL
GENOME.
WHEN WE LOOK AT THAT, WE FIND IN
MULTIPLE MICE AFTER TREATMENT,
LIKE THREE WEEKS, FIVE WEEKS OR
SIX WEEKS IN DIFFERENT MICE, WE
CAN SEE CLEARLY THEY CAN'T GO TO
SO-CALLED HOT SPOT IN THE T-CELL
GENOME.
SO BEFORE THE TREATMENT, CAR
COULD GO TO ANYWHERE IN THE
GENOME.
SO WE ACTUALLY SEE IT FROM THE
CHROMOSOME 1 TO 22 AND THEN YOU
SEE THE X CHROMOSOME.
JUST NOT MANY IN THE Y
CHROMOSOME BECAUSE Y CHROMOSOME
IS VERY, VERY SMALL, AND
ESPECIALLY -- IT'S -- YOU WILL
LESS FREQUENTLY SEE THE CAR IN A
Y CHROMOSOME BUT OTHER THAN
THAT, YOU SEE IT EVERYWHERE.
BUT THEN YOU'RE SELECTING THE
MICE UNDER THE SELECTION
PRESSURE FOR MANY WEEKS AND YOU
RECOVER THOSE CAR-T CELLS, YOU
RUN THE SINGLE CELL AND THEN
SEND YOU TO THE GENOME SEQUENCE,
YOU COULD FIND THAT THOSE
GENES -- NUPL1 IS PROBABLY
RELATED TO T-CELL SURVIVAL AND
THEY ACTUALLY FREQUENTLY HAVE
CAR INTEGRATED INTO THAT GENE
AND SURVIVE AND BE VERY ACTIVE
FOR MANY WEEKS.
AND NOT ONLY FOR ONE SIDE, IT
COULD BE MORE THAN ONE SIDE, IN
THE SAME DIRECTION, MEANING THE
SELECTION PRESSURE IS PRETTY
HIGH.
SO THAT'S KIND OF INTERESTING.
WE KIND OF HYPOTHESIZED WE
PROBABLY JUST NEED A FEW
FUNCTIONAL CAR-T CELLS TO EXPAND
AND KILL TUMOR CELLS IN MICE OR
EVEN IN HUMANS.
TO GO TO THE EXTREME WE PROBABLY
NEED ONE CAR-T CELL TO BE ABLE
TO WORK.
SO THAT'S THE GOAL.
SO MAYBE WE CAN ENGINEER JUST
ONE CAR-T CELL TO MAKE IT
POLYFUNCTIONAL AND PERSISTENT
INSTEAD OF A POOL OF CAR-T
CELLS.
SO BESIDES THIS, WE ALSO DO
SINGLE CELL RNA SEQ, WE SEPARATE
THE HIGH AND LOW POLYFUNCTIONAL
CAR-T CELLS, SO WE SEQUENCE A
SUBSET OF HIGH POLYFUNCTIONAL
CAR-T CELL AND LOW BASED ON THE
PHENOTYPE I JUST DESCRIBED, THE
CYTOKINE AND CHEMOKINE RELEASE.
AND THEN -- SO THOSE ARE THE
CYTOKINE, CHEMOKINE FACTOR WE
USE TO SEPARATE THEM, THE HIGH
OR LOW CAR-T CELLS, AND THEN WE
DO THE SINGLE CELL RNA SEQ SO
ALL OF THEM, AND THEN WE LIST
ALL OF THE GENES THAT MAY BE
RELATED TO THE HIGH AND THE LOW
POLYFUNCTIONAL CAR-T CELLS.
SO I THINK WHEN WE'RE DOING
THAT, WE'RE KEEPING IN MIND
MAYBE ONE DAY WE CAN BUILD A
POLYFUNCTIONAL CAR-T CELLS, THEY
ARE ALLOGENIC, AND THEY ARE --
SO WE CAN DO GENOME EDITING TO
BUILD THE CAR-T CELLS.
SO WHEN WE DO THE GPC3, WE START
TO THINK ABOUT MAYBE WE SHOULD
WORK ON OTHER GLYPICANS.
I SAID IN THE BEGINNING
PARTICULARLY GPC2 AND 1 BECAUSE
THEY ALSO INDUCE THE WNT
SIGNALING.
AND WE LOOKED AT MICRO ARRAY
DATABASE AND OTHERS, WE FIND
GPC2 IS ACTUALLY ONE OF THE
TRANSCRIPT, YOU CAN FIND IT
PARTICULARLY IN PEDIATRIC
CANCERS AND IN GLIOMA DATABASE.
I THINK ONE OF THE DATABASE DONE
HERE IN POB BY RIMA AND CHRIS'
GROUP AND THEY ACTUALLY DID ALL
THE MICRO ARRAY IN MANY
PEDIATRIC CANCER TISSUES.
SO GPC2, WE ACTUALLY VALIDATE
THAT.
IT'S THE ONLY ONE, THE PROTEIN
EXPRESSION CAN BE ELEVATED IN
THE NEUROBLASTOMA.
SO WE FOCUS ON NEUROBLASTOMA,
AND THEN WE ALSO LOOK AT THE
DATABASE AND WE FIND THAT THE
HIGH GPC2 IS ACTUALLY RELEVANT
TO THE POOR SURVIVAL OF THESE
NEUROBLASTOMA PATIENTS.
JUST LIKE THE GPC3, WE STUDIED
GPC2 TO ALSO LOOK AT THE WNT
SIGNALING.
WHAT WE'RE FINDING IS IF WE
KNOCK DOWN OR KNOCK OUT THE GPC3
IN THIS CASE, WE ACTUALLY COULD
SEE ACTIVE BETA CATENIN ALSO
DECREASE.
AND THE OTHER PROTEIN WE ARE
INTERESTED IN IS CALLED N MIBG.,
SO WHEN WE KNOCK DOWN GPC2, YOU
CAN SEE THE THE N-MYC SIGNIFICY
GOING DOWN IN THE NEUROBLASTOMA
CELL LINES.
WHEN WE REPEAT IT WITH KNOCKOUT,
YOU SEE THE SAME THING, IN THE
SAME CELL LINE, KNOCKDOWN AND
THE KNOCKOUT, YOU WILL SEE
DECREASE OF THE N-MYC IN THESE
CELLS.
SO INDICATING THE GPC2 KNOCKDOWN
AND KNOCKOUT WERE ACTUALLY
REDUCED EXPRESSION OF N-MYC.
TO MAKE AN ANTIBODY AGAINST
GPC2, WE DID THE SAME THING WE
DID FOR GPC3, SINCE WE ALREADY
KNOW HOW TO MAKE THIS ANTIBODY,
AND WE LEARN A LOT FROM OUR
PROJECT TO TRY TO MAKE A GPC3
ANTIBODY.
SO HERE WE KNOW GLYPICANS ARE
NOT IMMUNOGENIC SO WE HAVE TO
DESIGN A PEPTIDE TO BREAK THE
IMMUNE TOLERANCE.
EXON 10 SEQUENCE, THEN WE ALSO
USE MEMBRANE-PROXIMAL EPITOPE
BECAUSE WE THINK BASED ON OUR
DATA AND OTHER PEOPLE'S DATA, IN
SINGLE CHAIN BASED CAR-T CELLS
SEEMS LIKE -- MEMBRANE WILL BE
MORE POTENT MEMBRANE EP TOAD FOR
THE CAR-Ts.
SO WE ACTUALLY INJECT INTO THESE
MICE AND SCREEN AS WE DID FOR
GPC3, AND THEN WE SCREEN
NEUROBLASTOMA CELLS, WE DO A LOT
OF SINGLE CELL FACT TO ACTUALLY
IDENTIFY THE ANTIBODY.
SO NAN LI DID THIS WORK,
SCREENED ALL THESE ANTIBODIES
AND 3 IS THE MOST PROMISING ONE
BECAUSE IT BINDS THE TISSUE
LEAST.
EVEN THOUGH WE USE THE SAME
PEPTIDE TO IMMUNIZE MICE, WE
WILL GET ANTIBODY EVEN WE SCREEN
ON THE CELLS ALREADY, WE STILL
GET ANTIBODY ON THE TISH EURL,
SOME IMPACT ON THE LIVER, MAYBE
ON THE STOMACH OR MAYBE ON
PANCREAS, SO CT3 IS THE ONLY ONE
THAT HAS THE MOST CLEAN
BACKGROUND.
SO THAT'S WHY WE PICKED THE CT3,
JUST BECAUSE OF THE TISSUE
STAINING.
SO THEN WE HAVE A FLEX AWARD, I
HAVE A NEW POSTDOC, RISHI CAME
TO THE LAB AND WE ANALYZED THE
ISOFORM OF THE GPC2 BECAUSE WE
THINK ISOFORM PLAYS IMPORTANT
ROLE IN CANCER, PARTICULARLY
CANCER-SPECIFIC ISOFORM IS VERY
INTERESTING TO US.
SO WE LOOKED AT GPC2.
THEY HAVE TWO ISOFORM, 201 AND
203.
201 IS IN CANCER, 203 IN NORMAL
TISSUE.
BUT IN THE CANCER, 201 HAS EXTRA
EXON 3 AND EXON 8, 9, 10 EXONS,
INDICATING IF WE DEVELOP AN
ANTIBODY AGAINST THE 3, 8, 9 #,
10, 910,THAT WOULD BE MORE
TUMOR-SPECIFIC THAN IF WE
TARGETED OTHER EXONS.
LUCKILY WE ALREADY HAVE ANTIBODY
BIND ON EXON 10, THAT'S THE CT3,
SO WE THINK WE SHOULD GET
ANTIBODY AGAINST CT3.
I MEAN EXON 3.
THE REASON IS YOU CAN SEE HERE,
EXON 3 IS VERY CLEAN, EXCEPT THE
TESTES, DID NOT HAVE ANY IN ANY
NORMAL TISSUE.
SO WE THINK WE SHOULD HAVE
ANTIBODY AGAINST EXON 3 AND I I
ASSIGNED RUIXUE TO DESIGN
PROPOSAL IN THE FLEX PROJECT.
BUT THEN RUIXUE DILIGENTLY MADE
THE PROTEIN AND ONE DAY CAME TO
MY OFFICE AND SAID HEY, I THINK
CT3 ALSO BINDS TO EXON 3 EVEN
THOUGH WE MADE CT3 AGAINST EXON
10 BUT ALSO BIND EXON 3.
I DON'T BELIEVE IT.
SO I SAID YOU HAVE TO REPEAT IT.
SO WE REPEAT IT, NAN ALSO REPEAT
IT.
IT SHOWS LIKE, YES, EVEN THOUGH
WE IMMUNIZED THE MICE WITH EXON
10, THE ANTIBODY STILL BINDS TO
EXON 3.
AND WE PICKED THIS ONE BECAUSE
THE LOWEST BACKGROUND IN THE
TISSUE ARRAY, AS I TOLD YOU.
SO THAT START TO MAKE SENSE, AND
THEN WE SENT TO FREDERICK, WE
HAVE A CCR CENTER FOR MOLECULAR
MICROSCOPY DOING EM, AND WE
ACTUALLY SEE THE CT3 -- ACTUALLY
BIND EXON 3 AND EXON 10.
OKAY, SO WE COULD VISUALIZE IT.
SO THAT'S GREAT.
AND THEN, WE LOOK AT THE
CT3 ANTIBODY ON A NORMAL TISSUE,
WE FIND THAT, YES, INDEED, THEY
DON'T BIND TO PRETTY MUCH
ANYTHING EXCEPT THE TESTIS, VERY
HIGHLY TUMOR SPECIFIC ANTIBODY
DEVELOPED.
SO IN CONCLUSION, I THINK IT'S
FEASIBLE TO GENERATE TUMOR
SPECIFIC ANTIBODY BY TARGETING
TUMOR ASSOCIATED ISOFORM OR
EXONS AND THE TUMOR ASSOCIATED
ISOFORM/EXON, I THINK THEY'RE
MORE IN THE CANCER, AND WE NEED
TO FIND THEM.
AND THAT THE CURRENT, WE HAVE
EXPERT HERE, AND DOING THE
ANALYSIS DEEP SEQUENCING, I
THINK THE CURRENT METHODOLOGY
MIGHT NEED TO BE OPTIMIZED OR
IMPROVED TO BE ABLE TO FIND MORE
ISOFORM AND EXONS IN THE TUMORS.
WE ALSO TEST THE CT3 ANTIBODY ON
OTHER PEDIATRIC CANCER, THEY ARE
ALL VERY POSITIVE IN THOSE
PEDIATRIC CANCER, BUT NEGATIVE
IN THE NORMAL TISSUES.
SO POTENTIALLY WE CAN TREAT
OTHER PEDIATRIC CANCER, NOT ONLY
JUST NEUROBLASTOMA.
SO LIKE WE DO FOR GPC3 WE
ACTUALLY MADE CAR-T CELLS THAT'S
THE SAME CONSTRUCT, I'M NOT
GOING TO GO INTO DETAIL.
AND THEN WE TEST THE GPC2 IN
NEUROBLASTOMA.
THIS IS A VERY COMPLICATED
MODEL.
WE EITHER INJECTED THE
NEUROBLASTOMA FOR ABOUT A MONTH,
28 TO 35 DAYS, DEPENDS ON WHAT
THE EXPERIMENT WE ACTUALLY TAKE.
SO USUALLY ABOUT A MONTH.
YOU -- EVERYWHERE IN THE MICE,
FROM THE SKULL TO ADRENAL GLAND
TO THE LEG TO EVERYWHERE, AND
THEN YOU START TO TREAT IT WITH
CAR-T CELLS, AND THEN YOU CAN
SEE WITH THE TREATMENT, IT JUST
TOTALLY ELIMINATED AND ALL THE
MICE SURVIVED.
SO BECAUSE OF THIS DATA AND
OTHER DATA, WE ALSO DO THE
MODEL, BASICALLY MICRO INJECT IN
THE SAME CELL LINE INTO THE
ADRENAL GLAND AND WE SHOW
SIMILAR DATA.
SO BECAUSE OF THIS DATA, WE ARE
NOW COLLABORATING WITH PEDIATRIC
ONCOLOGY BRANCH HERE TO START A
CLINICAL TRIAL IN 2025 ERK 2025T
A CANCER MOONSHOT GRANT,
DR. ROSA NGUYEN IS GOING TO LEAD
THIS TRIAL.
AT THE SIMILAR TIME AS I SAID WE
WORK ON GPC2, WE ALSO WORK ON
GPC1.
AND IT'S THE SAME KIND OF
REPORTER EVEN THOUGH IT'S QUITE
CONTROVERSIAL, IT'S BEEN
REPORTED THAT GPC1 IS SECRETED
FROM PANCREATIC CANCER CELLS AS
EXOSOME, EVEN THOUGH THAT THAT D
OF ARGUMENT IS DEBATED IN MANY
MEETINGS WHETHER IT'S TRUE OR
NOT, BUT WE ACTUALLY START TO
LOOK AT GPC1 TO SEE WHETHER IT'S
TRULY EXPRESSED IN THE
PANCREATIC CANCER, WE DO RNA AND
THE PROTEIN LEVELS AND USED
NORMAL PANCREATIC CELL LINES AS
A CONTROL.
YOU CAN SEE GPC1 INDEED IS
HIGHLY ELEVATED IN PANCREATIC
CANCER.
BUT UNLIKE GPC2 AND GPC3, GPC1
IS MORE LIKE A PANCANCER TARGET,
EXPRESSED NOT ONLY IN PANCREATIC
CANCER BUT IN OTHER CANCERS --
SOLID TUMORS.
SO LIKE A GPC2, WE ALSO LOOK AT
THE WNT IN THE PANCREATIC
CANCER.
WE ALSO DO THE REPORTER -- USE
GPC3 AS A CONTROL.
LIKE A GPC3, IF WE OVEREXPRESS
GPC1, IT WILL ELEVATE THE BETA
CATENIN SIGNALING AND THAT'S
SIMILAR LIKE WHAT THE GPC3 DOES
IN LIVER CANCER.
JIAJIA PAN IS A PH.D. STUDENT IN
THE LAB.
SHE MADE A GP GPC1 KNOCKOUT, EVN
THOUGH WE DO CRISPR, MEANING
THEY ARE TOTALLY INDEPENDENT
KNOCKOUT CLONE, AND WE SEE ALL
THIS SINGLE INDEPENDENT KNOCKOUT
GPC1 CLONE IN PANCREATIC CANCER
CELLS, AND -- SIGNIFICANT
DECREASE.
IT'S AMAZING, THE DECREASE AND I
THINK IT'S JUST BECAUSE THE GPC1
IS TOTALLY -- THOSE PANCREATIC
CANCERS ARE VERY ADDICTED TO THE
GPC1 WHEN YOU KNOCK OUT THE BETA
CATENIN LEVEL IT REALLY GOES TO
VERY, VERY LOW.
TO MAKE THE GPC1 ANTIBODY THIS
TIME, IN ADDITION TO WHAT WE DID
FOR GPC3 AND GPC3
2 IMMUNIZATION, NOW WE HAVE --
LIBRARY.
SO WE MADE -- FROM THE CAM
1 SHOCK.
THOSE ARE VERY SPECIAL ANIMALS.
THEY ACTUALLY PRODUCE THE SO
CALLED SINGLE-DOMAIN ANTIBODY
WHICH IS HEAVY CHAIN WITHOUT THE
LIGHT CHAIN AND THOSE ANTIBODIES
ARE VERY SMALL, SO THEY ACTUALLY
ONLY HAVE 12 OR 15 KILODALTON
INSTEAD OF 150 KILODALTON
ANTIBODY IGG I SHOW IN THE
BEGINNING.
IT BINDS TO THE -- FUNCTION SITE
AND IT HAS A BETTER TRANSDUCTION
EFFICIENCY BECAUSE THEY ARE
SMALL, AND MAY BE IDEAL FOR
ALLOGENIC CAR-T BECAUSE YOU CAN
DO A A LOT OF ENGINEERING TO THE
GENOME AND EASY TO EXPRESS AND
ENGINEER AS BISPECIFIC OR
MULTI-SPECIFIC.
IT'S LIKE A KEY IN THE HOLE, AS
LONG AS THE HOLE IS THE SAME
SIZE, SAME SHAPE, THEY ALWAYS
CAN GET IN.
SO THE NANOBODY REALLY CROSSES A
LOT OF SPECIES.
AND WE ALSO DO LIKE WE GO IN
GPC2, WE DO THE EM TO TRY TO
FIGURE OUT THE EPITOPE.
THIS IS THE EM COMPLEX -- SO WE
CAN SEE THE D4, THE NANOBODY
ACTUALLY BINDS TO THE END OF THE
GPC1, AND HM2 FAB ACTUALLY BINDS
TO C TERMINAL OF THE -- CLOSE TO
THE CELL SURFACE.
SO AS I JUST DESCRIBED, THIS
WILL SHOW YOU ONE EXAMPLE.
THE WHOLE IGG WHICH IS HEAVY IN
THE LIGHTER CHAIN BUT INTERFACE
WITH THE ANTIGEN, PROTEIN
ANTIGEN, ARE RELATIVELY FLAT,
BUT IF WE USE THE NANOBODY,
BECAUSE THE LOOP IS VERY LONG
SOMETIMES THEY HAVE -- BOND TO
STABILIZE IT, THEY COULD GO VERY
DEEP TO THE VA CAVITY OF THE
PROTEIN, THAT WE THEY ACTUALLY
PROTRUDE INTO THE BACK POCKET
BECAUSE THIS IS ENZYME POCKET.
AND SO THIS IS SHARK BUT THE
CAMEL NANOBODY COULD DO THE SAME
THING.
I JUST WANT TO SHOW HERE, WE
HAVE A CAMEL NANOBODY -- THAT
CAN BE USED FOR -- DISCOVERY FOR
NOT ONLY CANCER BUT ALSO FOR
VIRUS, AND SCIENT SCIENTIST IN ,
HUA AND JESSICA BOTH SPEND ONE
OR TWO YEARS FULL TIME WORKING
ON THIS AND GENERATED THOSE
CAMEL LIBRARY.
THIS LIBRARY, WE MADE EACH CAMEL
A LIBRARY SO TOTAL WE HAVE 20
LIBRARY, VERY BIG LIBRARY, AND
USING THOSE LIBRARY, JESSICA
HONG, BIOLOGIST IN MY LAB
ACTUALLY ISOLATED THE SARS CROW
V ANTIBODY DURING THE PANDEMIC
AND SHOW THEY ARE VERY BROADLY
NEUTRALIZING THE COV VARIANTS
AND THAT'S PRETTY EXCITING WORK
DURING THE PANDEMIC.
IT SHOWS THE NANOBODY COULD WORK
BOTH FOR CANCER AND VIRUS
ANTIGENS.
AND BACK TO GPC1.
SO NAN ACTUALLY FOUND AN
ANTIBODY CALLED D4.
IT ACTUALLY HAS A VERY HIGH
AFFINITY FOR MOUSE AND HUMAN
GPC1.
THAT MEANS YOU CAN START IN
MOUSE PANCREATIC CANCER MODELS
USING THIS NANOBODY.
AND ACTUALLY, AFFINITY IS A
SUBNANOMOLAR AFFINITY, EVEN
THOUGH IT'S SMALL, .7, IT'S NOT
LIKE A PEPTIDE, VERY HIGH
AFFINITY, SO -- MATURATION OR
DIRECTED EVOLUTION TO IMPROVE
THE AFFINITY BECAUSE IT'S -- THE
AFFINITY IS ALREADY WITHIN THE
THERAPEUTIC ANTIBODY WINDOWS.
SO WE ACTUALLY MAKE THE CAR-T
CELLS USING THIS NANOBODY.
WE FIND IF WE USE DIFFERENT --
IT WILL HAVE DIFFERENT IMPACT.
I WILL NOT GO INTO DETAIL
BECAUSE WE MADE A LOT OF
DIFFERENT BUT I ONLY WANT TO
SHOW TWO HERE.
THE CD8 IN THE GPC3 AND
2 PROJECT.
IGG 4 HINGE IS SHORT, CD8 HINGE
IS 45 ACID SO WHEN WE SWITCH TO
THE SHORT HINGE, IGG 4 HINGE,
THEY'RE CLOSE TO EACH OTHER AND
THEY FORM PROBABLY BETTER DIMERS
AND EVEN INTRACELLULAR PART OF
THE MOLECULE CD3 DATA IS MORE
STRUCTURED THAN THE CD8 HINGE SO
THAT MEANS POTENTIALLY MAKES IT
MORE ACTUALLY ACTIVE.
SO INDEED, ACTUALLY SHOW USING
NANOBODY T CELLS WITH IGG HINGE
AND IT SHOWS MUCH BETTER KILLING
EFFECT THAN THE ORIGINAL
CD8 HINGE.
ANOTHER PH.D. STUDENT CAME AT
PH.D. PROGRAM FROM MAYO AND SHE
DID A SIMILAR EXPERIMENT USING
ANTIGPC3 NANOBODY.
AS YOU CAN SEE HERE, WHEN SHE
SWITCHED TO IGG 4 HINGE, THE
ANTIGPC NANOBODY CAR-T-CELL IS
VERY POTENT, EVEN MORE POTENT
THAN THE ONE I TALK ABOUT,
SINGLE CHAIN TARGETED IN GPC3 SO
WE'RE VERY EXCITED ABOUT THIS,
INDICATING IGG 4 IS PROBABLY THE
RIGHT HINGE FOR THE NANOBODY
CAR-T CELLS.
SO NOW IN THE LAST FEW MINUTE,
I'M GOING TO TALK ABOUT HOW CAN
WE MAKE EVEN MORE ACTIVE CAR-T
CELLS.
SO TUMOR ANTIGEN AS I SAID IN
THE BEGINNING IS HETEROJEAN US,
HETEROGENEOUSMOST ARE VERY LOW.
FOR HIGH TUMOR -- LIKE GPC2 AND
GPC3 SINCE WE HAVE A GOOD
ANTIBODY BUT ONLY TUMOR, NOT ANY
NORMAL TISSUES, WE COULD
PROBABLY AFFORD TO IMPROVE THE
ACTIVE -- OF THE CAR-T CELLS
WITH RESULT ON TARGET OF OFF
TUMOR TOXICITY BECAUSE THEY ARE
VERY TUMOR SPECIFIC.
SO KEEPING THAT IN MIND, WE
ACTUALLY COLLABORATEED WITH A
COMPANY SINCE 2018, SO WE
ACTUALLY COULD DEVELOP KIND OF
THE CONSTRUCT WHICH HAS A
GAMMA-DELTA CONSTRUCT WITH VLB
FAB, WITH TCR.
THE DIFFERENCE FROM THIS TO THE
CAR IS YOU CAN SEE THE CD3 PART
OF THE ACTIVATION PART C D3,
ANOTHER COVALENT BOND WITH THIS
CONSTRUCT, AND CD3 WITH PEPTIDE
BOND.
HERE WE DON'T USE ANY -- THEY
ACTUALLY RECRUIT CT3 JUST LIKE A
NATIVE TCR TO GET INTO THE
ACTIVATION COMPLEX.
SO WE ALSO SPLIT THE -- FACTOR
INTO A DIFFERENT MOLECULES AND
NOT COVALENT BOND WITH OUR CAR.
SO IN THAT WAY, WE ACTUALLY KEEP
EVERYTHING MORE LIKE A NATIVE
TCR EXCEPT THE VARIABLE REGION
OF THE TCR WE CHANGE IT WITH THE
FACT.
OBVIOUSLY WE WANT TO ASK WHETHER
THIS F TCR COULD REQUEST LOW
ANTIGEN DENSITY BECAUSE AS I
SHOW HERE, THIS IS VERY
IMPORTANT BECAUSE THIS IS THE
ANTIGEN SIZE PER CELL.
FOR MANY ANTIBODY THERAPY, WE'RE
TALKING ABOUT 100 SIZE PER CELL.
FOR CAR-T THERAPY IN GENERAL
WE'RE TALKING ABOUT MAYBE 1,000,
2,000 SIZE PER CELL AT THE
LOWEST CUTOFF YOU CAN KILL THE
TUMOR CELLS ACTUALLY
EFFICIENTLY.
TO GO DOWN TO SOMETHING LIKE
THIS, ONLY 500, EVEN LESS
BECAUSE WITH CONTROL IT'S HERE,
THE BACKGROUND, EVEN LESS, A
COUPLE OF HUNDRED SIZE PER CELL
IS ALMOST IMPOSSIBLE.
IF YOU USE ANY ANTIBODY
THERAPEUTIC DRUGS, OR EVEN
THE -- CAR-T CELLS AND USUALLY
THAT'S THE THRESHOLD, PROBABLY
1,000, 2,000 SIZE PER CELL.
AND THEN AMAZINGLY, WE ACTUALLY
USE THIS LAN1, INOCULATE TO MICE
TO MAKE SUBCUTANEOUS TUMOR, WE
START TO SEE THE F TCR ACTUALLY
SHRINK THE TUMORS, WE STARTED
ABOUT 110 TO 140 SIZE, AND THEN
THEY ACTUALLY GO TO ZERO.
OR TUMOR REMISSIONS.
VERY INTERESTING, WE ARE STILL
TRYING TO UNDERSTAND THAT, IS
THE HUMANIZED IS MORE POTENT
THAN MOUSE CT3 AB TCR.
EVEN THOUGH THE MOUSE STILL IS
HIGH, MORE ACTIVE THAN CAR, BUT
THE HUMANIZED VERSION OF CT3
ABTCR IS THE MOST POTENT.
SO LAURA HUTCHINS, SHE'S A
POSTBAC IN THE LAB WORKING WITH
DAN ON THIS PROJECT, AND IT'S
VERY SURPRISING TO US IN THE
BEGINNING, THAT FACT.
REGARDLESS OF THAT, BECAUSE OF
THIS BEAUTIFUL DATA, AND THE
RESULT WE HAVE, THE COMPANY
DECIDE TO LICENSE OUR C
CT3 ANTIBODY AND ARE GOING TO
SPONSOR A CLINICAL TRIAL IN
2025, WE ALREADY SIGNED THE
CONTRACT AND GOING TO WORK IN
THE POB TO LEAD THE TRIAL HERE.
SO WE'RE GOING TO SEE MORE IN
THE CLINIC.
SO AGAIN, WE WANT TO UNDERSTAND
THE MECHANISM.
SO WHY THE F TCR IS MORE ACTIVE
THAN CAR, WE DON'T KNOW, THE
SHORT ANSWER IS WE DON'T KNOW.
MAYBE IT'S HUMANIZED CT3 FAB
SEQUENCE OF THE MICE FLOATING
WITH HUMAN SCAFFOLD BECAUSE THE
MOUSE WON'T WORK AS GOOD AS
HUMANIZED ONE, OR MAYBE NATIVE
TCR SIGNALING IS MORE REG
LATIVE, LESS EXHAUSTED, MORE
PERSISTENT, MORE TUMOR
INFILTRATION, SO WE CAN NAME ANY
OF THEM POTENTIALLY WHY IT'S
WORKING BETTER.
SO I JUST WANT TO SHOW ONE
PRELIMINARY DATA DONE BY ALEX
TRAN, ANOTHER POSTBAC IN THE
LAB, AND WE ACTUALLY TAKE THOSE
T CELLS, RECOVER THEM FROM THE
MICE, TREAT THEM WITH F TCR AND
COMPARE TO THE CAR.
FROM WEEK TWO TO WEEK FOUR.
WHICH IS VERY STRIKING.
IF YOU LOOK AT THE TSCM IS A
T-CELL LIKE MEMORY T CELLS.
IT SHOWS THE STEMNESS OF THE T
CELLS.
FROM WEEK TWO TO WEEK FOUR,
BASICALLY WE KEEP MORE OF THEM
40% STEMNESS, THE TSCM
POPULATION.
THAT MEANS IT'S VERY HIGH
STEMNESS OF THIS HUMANIZED AB
TCR.
MOST IS ONLY 20%, 30%, BUT MUCH
LESS IN THE CT3 CAR.
IT ONLY 8%.
SO THAT MEANS AB TCR IN GENERAL
HAS MORE MEMORY MARKERS, HAS
MORE STEMNESS THAN THE CAR.
AND THE HUMANIZED AB TCR EVEN
HAS MORE STEMNESS AND ENRICHED
MORE MEMORY MARKERS THAN THE
MOUSE CT3 AB TCR.
SO WE'RE STILL LEARNING IT AND
WHY THIS IS THE MECHANISM OF THE
EFFECT.
SO CONCLUSION, WE ACTUALLY SHOW
GPC123 TARGET FOR TREATING
PANCREATIC CANCER, PEDIATRIC
CANCER, LIVER CANCER.
UPCOMING CLINICAL TRIAL WILL --
AS A TUMOR TARGET IN HUMANS,
NANOBODY CAN BE ENGINEERED AS A
BUILDING BLOCK FOR CAR-T AND
ENGINEER -- MIGHT BE MORE ACTIVE
PARTICULARLY IN LOW ANTIGEN
DENSITY TUMORS.
AND FOR NANOBODY, WE NEED TO
ENGINEER THE HINGE SO WE USE THE
SHORT IGG 4 HINGE FOR
NANOBODIES.
AND FOR FUTURE OF CAR
ENGINEERING I THINK WE NEED A
MORE TARGETED CAR, BY TARGET
TARGETING -- LIKE B7 -- 3 PLUS
GPC2 WHICH IS -- AND WE ARE
WORKING WITH DR. -- IN
PANCREATIC CANCER AND WE STILL
NEED TO LOOK AT THE OFF THE
SHELF ALLOGENIC CAR, NOT ONLY T
CELLS, MAYBE MICROPHAGE,
NEUTROPHIL, NK CELLS, EVEN
GAMMA-DELTA T CELLS MAY BE THE
ONE WE WANT TO FOCUS ON TO
SELECT AND TO BE ABLE TO
ENGINEER THEM.
SO WITH THAT, I THI THANK MANY
MEMBERS AND COLLEAGUES THAT HAVE
HELPED ON THE PROJECT.
I THINK WITHOUT THOSE VERY
TALENTED POSTDOC PH.D. STUDENT
AND POSTBACS, MEDICAL STUDENTS,
NOTHING CAN BE DONE HERE IN THE
LAST 15, 16 YEARS.
I THINK WE OWE THEM BIG THANKS,
AND THE COLLABORATORS, WE HAVE
TIM GRETEN IS LEADING THE LIVER
CANCER TRIAL AND POB IS -- THE
CANCER TRIAL NEXT YEAR WITH
THAT, AND ALSO WE HAVE A LOT OF
FUNDING FROM MOONSHOT, AND
SUPPORTED BY LIVER CANCER
PROGRAM, MANY OTHER PROGRAMS,
PEOPLE ALWAYS HELP TO BRING THE
CRADA AND THE LICENSING
OPPORTUNITY TO US.
WITH THAT, THANK YOU VERY MUCH.
[APPLAUSE]
>> THAT WAS A GREAT TALK,
MITCHELL.
ONE QUESTION I WANT TO SORT OF
POSE, AND MAYBE SHIN WEI CAN
COMMENT IN ADDITION TO THIS.
YOU MENTIONED THE IMPORTANCE OF
TARGET IDENTIFICATION AND
FINDING SPECIFIC ISOFORMS THAT
ARE TUMOR-SPECIFIC VERSUS ONES
THAT MIGHT BE EXPRESSED IN
NORMAL TISSUES.
DO YOU HAVE ANY COMMENTS AS TO
HOW WE GO ABOUT DOING THAT,
USING NEXT GENERATION
SEQUENCING, TISSUE MICRO ARRAYS,
ET CETERA?
THANK YOU.
>> I THINK THIS IS A QUESTION
PROBABLY FOR SHIN WEI.
SO I THINK IN GENERAL, AS FAR AS
I KNOW, I HAVE VERY LIMITED
KNOWLEDGE.
THEY ARE THE EXPERTS.
SO I THINK LIMITED KNOWLEDGE,
CURRENT TCGA AND MANY OTHER
DATABASE, IT'S VERY DIFFICULT TO
DIG IN TO THE ISOFORM AND IT'S
NOT SET FOR THAT PURPOSE.
I THINK WITH MORE COMPUTATIONAL
SKILLS AND MORE DEEP SEQUENCING
AND IMPROVEMENT, WE'LL GET MORE
AND MORE DATA AND I THINK THE
ISOFORM OF THE ANTIGEN LIKE THIS
WILL BETHIS, WE'LL BE ABLE TO FE
AND MORE.
>> THANK YOU SO MUCH.
I ECHO WHAT FREDDIE SAID.
FANTASTIC TALK.
REALLY EXCITING STUDY.
SO I WAS ACTUALLY THINKING WHILE
YOU WERE PRESENTING THE STUDY,
ONE OF -- ACTUALLY THIS IS NOT
DIRECTED TO YOUR WORK, THE CAR-T
THERAPY OFTEN -- THE MAJOR
PROBLEM IS THAT THE ANTIGEN
OFTEN BECOMES DIMINISHED IN
TUMOR THAT AS A RESULT, YOU GET
CONSTANTLY RELAPSE, RIGHT?
BUT I ASSUME -- I LIKE TO SEE
YOU HAVE THE NEW APPROACH, AB
TCR.
DO YOU THINK YOU CAN DESIGN A
STUDY TO TITRATE DIFFERENT
ANTIGEN LEVEL IN CELLS WITH
DOING SORT OF MIXTURES OF CELL
AND THEN TESTING WHETHER THIS IS
POTENTIALLY WOULD BE ABLE TO
ACTUALLY ELIMINATE THOSE
PROBLEMS?
>> THE AB TCR WILL KILL THE
CELLS BECAUSE THEY'RE VERY LOW
DENSITY, SO WE DO N THEY DO NOTE
A CLANS TO ESCAPE, IS THAT WHAT
YOU'RE ASKING?
>> YEAH.
>> WE COULD CERTAINLY DO THAT,
AND WE ACTUALLY NEED TO DO MORE
SINGLE CELL-BASED STUDY TO SEE
WHETHER WE HAVE THIS ESCAPE.
I THINK IN GENERAL, YOU WILL SEE
THE ANTIGEN ESCAPED IN THE
CLINICAL -- EVEN IN THE MICE,
AND SO I THINK THOSE KIND OF
THINGS WILL HAPPEN EVENTUALLY IN
CERTAIN PERCENTAGE WITH AB TCR
COULD ACTUALLY BE ABLE TO KILL
THEM BEFORE THEY ACTUALLY
ESCAPED.
SO I THINK THAT'S SOMETHING --
WE DEFINITELY SHOULD LOOK.
>> FIRST OF ALL I WOULD --
FANTASTIC TALK AND I HAVE
LEARNED A LOT.
MY QUESTION IS THAT BECAUSE GPC3
AND OTHER GPCs MAY BE
HETEROGENEOUS -- IN TUMOR CELLS,
IS THERE A CORRELATION BETWEEN
THE IMPRESSION OF THE CELLS AND
THE EFFICIENCY FOR THE
THERAPEUTIC AND WHETHER THERE
ARE CERTAIN MARKERS EXCEPT FOR
GPC3 THAT CAN BE PREDICTIVE
MARKERS FOR THE EITHER PATIENTS
OR THE MICE BENEFIT FROM THE
THERAPY?
THANK YOU.
>> VERY GOOD QUESTIONS.
I THINK YOUR FIRST QUESTION IS
KIND OF RELATED TO WHAT SHIN WEI
ASKING, BASICALLY IF YOU KILL
QUICKLY, THEY MAY NOT HAVE
CHANCE FOR SELECTION, AND THAT'S
WHAT AB TCR MIGHT BE DOING.
SO WHETHER YOU'RE ASKING WHETHER
THERE'S SOME PREDICTION, HIGH OR
LOW GPC, SO FIRST OF ALL HIGH OR
LOW GPC CORRELATE WITH
EFFICIENCY, I WOULD SAY WE DON'T
KNOW.
SO THAT'S WHY IN THE CLINICAL
TRIAL, WE SET THE -- VERY LOW,
25%, SO ANY PATIENT HAVE 25%
POSITIVITY FOR GPC3, WE WILL
RECRUIT THEM.
SO DOESN'T MATTER THEY HAVE
CHEMOTHERAPY, CHECKPOINT
THERAPY, WHATEVER, SO WE DON'T
CARE.
SO BECAUSE WE DON'T KNOW MUCH
ABOUT, YOU KNOW, WHAT ARE YOU
ASKING, WHAT IS THE BIOMARKER
FOR RESPONSE.
SO BASICALLY IN PHASE ONE
CLINICAL TRIAL WE INCLUDE
ANTIBODY.
AS LONG AS THEY HAVE 25%
POSITIVE, WE INCLUDE THEM.
WE DON'T CARE THEIR MEDICAL
HISTORY AND WHAT KIND OF
TREATMENT THEY HAVE.
SO WE LEARN THAT FROM THE PHASE
ONE CLINICAL TRIAL AND PROBABLY
YOU AND SHEN WEI EVENTUALLY
INVOLVED TO ANALYZE THOSE THINGS
TO BE ABLE TO HELP US TO
UNDERSTAND WHY SOME PATIENTS
RESPOND, SOME PATIENTS MAY NOT
RESPOND.
THESE ARE VERY COMPLICATED
THINGS I DON'T THINK WE KNOW
NOW.
>> ECHOING EVERYONE ELSE, THANK
YOU FOR THAT WONDERFUL TALK.
I APOLOGIZE BECAUSE I MIGHT HAVE
MISSED IT, BUT IN YOUR F TCR
CONSTRUCTS, IS THERE ANY
EVIDENCE OF WHETHER IT PAIRS
WITH ENDOGENOUS TCR CHAINS OR
NOT?
WITH YOUR F TCR CONSTRUCTS, IS
THERE ANY EVIDENCE OF WHETHER
ENDOGENOUS TCR LIKE ALPHA OR
BETA OR EVEN GAMMA-DELTA
COMPARE?
>> YES, YES.
THANK YOU FOR THE QUESTION.
YES, THE REASON WE DEVELOP
GAMMA-DELTA IS -- ALPHA BETA IS
TO TRY TO AVOID A MISMATCH WITH
ALPHA BETA BECAUSE GAMMA DELTA
PCR VERY LOW PERCENTAGE IN
SEPARATING T CELLS.
SO GAMMA DATA TCR IN GENERAL, IN
HUMANS IS TUCKED IN VIRUS, SO
THEY ARE PROTECTED FROM THE
VIRAL INFORECASTS.
SO THEY DON'T NEED TO HAVE A
LOT -- SO VERY SMALL NUMBER.
SO USING GAMMA BETA TCR WE WOULD
NOT HAVE A CHANCE TO MISMATCH
WITH ALPHA BETA BECAUSE MOST OF
THE T CELLS ARE ALPHA BETA.
AND IN TERMS OF WHETHER THEY
WERE MISMATCHED WITH THEIR OWN
NATIVE GAMMA-DELTA, SO WE DON'T
KNOW, AND SINCE THE NUMBER OF
THE GHANA DELTA TCR IN THE TOTAL
T-CELL POPULATION IS VERY, VERY
LOW.
MY FEELING IS NO, BECAUSE
SOMEBODY TOLD ME, I DID NOT DO
THIS IN MY LAB, BECAUSE
GAMMA-DELTA TCRs ARE DESIGNED
OR SELECTED DURING THE EVOLUTION
TO ATTACK THE VIRAL INFECTIONS
SO THEY DON'T LIKE VIRUS, AND WE
USE LENTIVIRUS TO INDUCE THE T
CELLS AND THEY USUALLY DON'T
LIKE IT.
SO IN FACT, YOU'RE VERY LESS
LIKELY TO INFECT OUR GAMMA-DELTA
TCR IN THE GAMMA-DELTA T CELLS
BECAUSE WE USE LENTIVIRUS.
THEY USUALLY KICK THEM OUT.
SO THEY DON'T LIKE ANY VIRUS.
IN FACT, MOST OF THE T CELLS WE
INFECT MIGHT BE ACTUALLY ALPHA
BETA, NOT GAMMA-DELTA, SO THERE
IS NO CHANCE TO MISMATCH WITH
OURS.
>> I SEE.
SO YOU JUST HAVE NEVER ASSESSED
IT, THOUGH --
>> SO WE DON'T KNOW -- BUT THE
COMPANY ACTUALLY WORKING ON
THAT, THEY ACTUALLY PRESENTED AT
ACR MEETING -- IN VITRO, TRY TO
SELECT AND GROW THEM, BUT IT'S
VERY HARD TO PUT THE -- BECAUSE
THEY DON'T LIKE THE CAR.
CAR USE LENTIVIRUS NEUTRALIZE
DOESN'T MATTER WHAT VIRUS YOU
USE UNLESS YOU USE --
JENNIFER -- TALK ABOUT THE --
SELECTING, THE VECTOR OR
VEHICLES, MAY BE SOMETHING ELSE,
BUT I DON'T KNOW THAT, BUT IF
YOU USE THE VIRUS TO DELIVER THE
CAR, MAYBE GAMMA-DELTA TCR IS
VERY DIFFICULT TO DO.
>> VERY INTERESTING BIOLOGY.
THANK YOU.
>> WE'LL DO A CAST QUESTION ANDD
THANK YOU ON BEHALF OF EVERYONE.
SO I WAS FASCINATED WHERE YOU
LOOKED AT INTEGRATION OF CAR-T.
SO DO YOU THINK THAT SOME OR ARE
MORE ACTIVE BECAUSE THEY'RE ALSO
KNOCKING OUT SOMETHING ELSE?
>> ABSOLUTELY.
YEAH.
WE THINK IT COULD BE KNOCKOUT,
COULD BE DOMINANT NEGATIVE.
>> I GUESS THE QUESTION IS --
>> SO WE DON'T KNOW MUCH ABOUT
THE BIOLOGY OF THAT.
WE HAVE TO DO THE FUNCTION ASSAY
TO --
>> SO SEPARATE THEM OUT, AND
THEN SO THAT WOULD PREDICT MAYBE
IF YOU KNOCKED THAT GENE FIRST,
ALL OF YOUR CAR-T WOULD BE
HYPERACTIVE.
>> RIGHT.
SO WE TRY TO DEVELOP THE SO-C
SO-CALLED NON-VIRAL -- METHOD
TO -- AND THEN TO STUDY THEM.
SO WE TRY TO AVOID THE VIRUS
BECAUSE VIRUS TEND TO BRING
EXTRA THINGS.
>> OTHER THINGS.
>> YEAH, SO WE TRY TO USE KNOWNO
VIRUSES IN THE LAB -- SEVERAL
MONTHS AGO, JUST FOCUS ON THAT,
AND JUST TRY TO MAKE A MESSAGE
WITH OTHER VIRUS AND BE ABLE TO
USE THE GENOME EDITING TO EDIT
THE T CELLS' GENOME SO WE CAN
PUT THE CAR IN THE SPECIFIC SITE
WE WANT.
>> GREAT.
SO WE'LL LOOK FORWARD TO THE
NEXT CHAPTER OR MANY CHAPTERS
AND THE RESULTS OF THE CLINICAL
TRIALS, AND I'LL THANK YOU FOR A
WONDERFUL TALK, AND BEAUTIFUL
WORK AND THANK THE AUDIENCE FOR
GREAT QUESTIONS.
[APPLAUSE]