>> GOOD AFTERNOON, EVERYBODY AND

WELCOME TO TODAY'S GRAND ROUNDS.

BOTH OF OUR SPEAKERS TODAY ARE

NIH BENCH TO BEDSIDE AWARD

RECIPIENTS.

SPEAKING FIRST IS DR. PETER D.

BURBELO, WHO IS A STAFF

SCIENTIST IN NEUROBIOLOGY AND

PAIN THERAPEUTICS BRANCH OF

LABORATORY OF SENSORY BIOLOGY AT

NIDCR.

HE HAS SERVED AS INVESTIGATOR ON

TWO AWARDED PROJECTS AND HE WILL

ADDRESS ILLUMINATING HUMAN

DISEASE AND INFECTION.

HE DR. FOWLER--FOWLER IS A

SENIOR INVESTIGATOR IN

EXPERIMENTAL TRANSPLANTATION AND

IMMUNOLOGY BRANCH AT NCI, HE

WORKED IN DEVELOPMENTAL BIOLOGY

AND ALSO A RESEARCH FELLOW IN

THE LABORATORY OF MOLECULAR CELL

BIOLOGY AT UNIVERSITY COLLEGE IN

LONDON.

HE SERVED AS AN ASSISTANT

PROFESSOR OF ONCOLOGY AT THE

LOMBARDI CANCER CENTER AT GEORGE

DOWN UNIVERSITY, PRIOR TO COMING

TO THE NIH AND IN 2006, HE'S ON

THE EDITORIAL BOARD OF JOURNAL

OF CLINICAL AND CELLULAR

IMMUNOLOGY AND THE AMERICAN

JOURNAL OF TRANSLATIONAL

RESEARCH.

HIS RESEARCH CENTERS ON B-CELL

MEDIATED HUMORAL RESPONSES AND

TODAY HE WILL DESCRIBE THE USE

OF LUCIFERASE

IMMUNOPRECIPITATION SYSTEMS

WHICH IS A HIGH DEFINITION

ANTIBODY PROFILING TECHNOLOGY

THAT CAN BE USED AS A DIAGNOSTIC

TOOL AND A TOOL IN UNDERSTANDING

THE PATHOGENESIS OF DISEASE AND

INFECTION AND I WELCOME

DR. BURBELO.

>> GOOD AFTERNOON AND THANK YOU

VERY MUCH.

SO TODAY I'M GOING TO TELL YOU

ABOUT OUR WORK ON HIGH

DEFINITION ANTIBODY PROFILING

WITH A NOVEL TECHNOLOGY WE

DEVELOPED TO REALLY INTERROGATE

VARIOUS HUMAN DISEASES IN

INFECTION AND WHAT I'M SHOWING

YOU RIGHT HERE IS A

BRENO C-PANSY THAT CONTAINS A

LUCIFERASE, A LOT LIKE THE FIRE

FLY YOU MIGHT SEE ON A SUMMER

NIGHT EXCEPT IT'S ABOUT HALF THE

SIZE AND WHAT I WILL TELL YOU

ABOUT IS HOW WE MADE RECOMBINANT

LIGHT EMITTING PROTEINS WITH

THIS LUCIFERASE USE INDEED A

SIMPLE ASSAY, TO MONITOR DISEASE

AND INFECTION, AND WHAT I

HOPE,--I'LL ACTUALLY COVER QUITE

A FEW DISEASES BUT WHAT I HOPE

TO DO IS REALLY ILLUSTRATE,

COMPARED TO AMINO ACIDS AND

REALLY THE NEW TYPES OF

INFORMATION THAT WE CAN GATHER

FROM THIS.

SO I DON'T HAVE ANY DISCLOSURES

AND THE OBJECTIVES OF MY TALK

ARE TO REALLY TALK ABOUT

QUANTITATIVE SIRROLOGY OF

DIAGNOSIS OF HUMAN DISEASE ASK

INFECTION AND OPPORTUNITIES FOR

ANTIBODY PROFILING FOR

PERSONALIZED MEDICINE AND LASTLY

TALK ABOUT HOW PATHOGENIS

ANTIBODIES MAY CAUSE DISEASE.

OKAY, SO TO START OFF WITH,

HUMAN DISEASE IS REALLY

OBVIOUSLY QUITE COMPLEX AND IT

INVOLVES INTERACTIONS BETWEEN

GENE AND THE ENVIRONMENT, AND

SO, SOME EXAMPLES ARE OBVIOUSLY

WHEN CELLS ARE DYING THEY

RELEASE A LOT OF DIFFERENT

PROTEINS, SOME OF THEM ARE

MISFOLDED OR MODIFIED IN SOME

WAY, OBVIOUSLY WE CAN ALSO HAVE

INFECTIOUS AGENTS THAT ARE

PRESENT IN THE DISEASE AS WELL,

AND MEDIATE BY STANDARDS.

BUT A MAJOR ROLE ACTUALLY IS OF

COURSE THE IMMUNE SYSTEM CAN

RECOGNIZE MANY OF THESE PROTEINS

THAT ARE BEING RELEASED AND EVEN

OF COURSE THE INFECTIOUS AGENTS

AND WHAT I'M SHOWING YOU YOU

HERE OF COURSE IS I'M PRIMARILY

GOING TO BE TALKING ABOUT

ANTIBODY RESPONSES AND HOW KEY

CAN USE THOSE, NOT JUST TO

DETECT THESE ANTIBODY RESPONSES

BUT HOPEFULLY AS BIOMARKERS OF

DISEASE OF INFECTION.

AND AGAIN, ANTIBODIES ARE

WELL-KNOWN TO BE IMPORTANT

BIOMARKERS FOR VARIOUS

INFECTIOUS DISEASE AND

AUTOIMMUNE DISEASES, PROBABLY

ANTIBODIES WERE ALSO PRESENT IN

ALL TYPES OF DISEASES BUT WE'VE

REALLY NOT CATALOGED THEM AND

FOR THE MAIN REASONS THE

TECHNOLOGIES ARE NOT THAT GOOD

AND I'M GOING TO AGAIN,

HIGHLIGHT THE ADVANCES THAT

WE'VE MADE.

SO, AGAIN, THE TECHNOLOGY, I'M

GOING TO BE TELLING YOU ABOUT IS

CALLED LUCIFERASE AND

IMMUNOPRECIPITATION SYSTEMS AND

LIPS FOR SHORT, AND AGAIN IT'S

BASED ON TAKING THIS GENE THAT'S

FROM THE RENILA, AND PUTTING IT

IN A SIMPLE PLASMID AND WHAT WE

DO IS--WHATEVER ANTIGEN TARGET

WHETHER IT'S INFLUENZA OR HIV OR

AUTOANTIGENS WE SUBCLONE THEM

DOWN STREAM AND BECAUSE THESE

AGAIN, THIS IS A PLASMA BASED

VECTOR WHAT WE CAN DO IS

TRANSVECT THESE INTO CELLS AND

COLLECT THE ANTIGEN TAG, LIGHT

EMITTING PROTEINS THAT PRORE

DUCEDDED AND WE DON'T HAVE TO

PURIFY THEM, SO AS FAR AS WE CAN

CLONE SOMETHING WE CAN PRODUCE

AN ANTIGENIC TARGET SO WHAT WE

DO IN A VERY RELATIVELY HIGH

THROUGH PUT APPROACH AND WE

BUILD MICRO FLUIDIC DEVICES AND

OTHER THINGS, MOST OF THESE

ASSAYS CAN OCCUR VERY, VERY,

RAPIDLY BECAUSE THEY'RE SOLUTION

BASED ASSAYS AND WHAT HAPPEN

WHEN IS YOU INCUBATE ANTIBODIES

WITH THESE TARGET ANTIGENS, THE

BODY BINDS.

WE CAN FURTHER THIS PROCESS

PLATE BY TRANSFERRING IT TO A

MICROFILTER PLATE AND CALCULATE

ALL THOSE AND WASH AWAY ANY

UNBOUND LIGHT EMITTING ANTIGEN

AND ONLY

BOUND BY ANTIBODIES REMAIN AND

OF COURSE THEY PRODUCE LIGHT IN

WHAT'S EXTRAORDINARY OF COURSE

ABOUT THE LUCIFERASE IS THAT

IT'S LINEAR OVER SIX LOGS SO YOU

DON'T NEED TO DO ANY CUMBERSOME

DILUTIONS OR ANYTHING LIKE THAT

AND IT'S ROBUST AND HAS A LO

BACKGROUND.

SO I'M GOING TO BREAK MY TALK

INTO TWO PARTS.

I WILL TALK ABOUT INFECTIOUS

DISEASE PROFILES AND ALSO

AUTOIMMUNE DISEASE ASK COMPLEX

PROFILING AND REALLY FOCUS ON

THREE AREAS, HOW THEY CAN USE

DIAGNOSTICALLY FOR DISEASE

MONITORING STRATIFICATION AND

FOR DISCOVERY.

SO ONE OF THE SINGLE ANTIGEN

TESTS I'M GOING TO START OFF

SIMPLE, ONE IS ONE FOR LIME

DISEASE THAT WE DEVELOPED AND

DID THIS WORK WITH ADRIANA

MçRQUEZ AND JO COHEN, LIME

DISEASE IS IN THE UNITED STATES

AND NORMALLY YOU GET THIS MIRROR

IMAGE GRANS BULLS EYE AND IT CAN

BE MISSED AND THERE'S A REAL

NEED TO HAVE A HIGH THROUGH PUT

ROBUST LINE TEST AND WHAT WE DID

AND I PUBLISH, I'M NOT GOING

INTO ALL THE DETAILS BUT WE SEE

IMMUNO DOMINANT EPITOPES OF

THE--OF SOME OF THE ANTIGENIC

PROTEINS OF THAT TO RENAL

CANCERILLA AND EXPRESS THESE

FROM A SINGLE DISH CELL, WE'RE

ABLE TO MAKE ABOUT--WE CAN DO

ABOUT 20,000 CLINICAL TESTS, SO

IT'S REALLY INEXPENSIVE AND TO

PRODUCE THE ANTIGEN AND WHAT I'M

GOING TO SHOW YOU HERE AND THIS

IS THE TYPEICAL TYPE OF PLOT TO

START OFF WITH AND I'LL SHOW YOU

THE ANTIBODY TIGHTERS ARE ON THE

Y AXIS AND HERE ARE CLINICAL

SAMPLES AND AGAIN, THE FIRST

THING TO POINT OUT IS THAT THIS

IS A LOG 10 SCALE AND YOU CAN

SEE HA IN OUR CONTROL

GROUP--THAT IN OUR CONTROL GROUP

THESE WERE BLINDED SAMPLES, THE

VALUE OF THE LIGHT UNIT IS ABOUT

200, BUT IN EARLY INFECTION WITH

THE ARRHYTHMIA GRAMS ARE

MULTIPLE, CAN YOU SEE LITERALLY

A HUNDRED FOLD TIGHTERS AND

OBVIOUSLY IN MORE CHRONIC MARKED

ARTHRITIS OR OTHER PROBLEMS,

YOU'RE SEEING MUCH, MUCH, HIGHER

TIGHTERS, AGAIN, USING VERY

SIMPLE STATISTICS OR ROCK CEIVER

OPERATED CHARACTERISTICS WE CAN

COME UP WITH VARIOUS CUT OFFS

AND USING THIS TEST, WE FOUND IT

WAS ESSENTIALLY 99% SENSITIVE

WITH A HUNDRED% SPECIFICITY AND

AGAIN, REALLY ROBUST TEST TO

DETECT AGAIN, AND ORGANISM THAT

UP TO NOW PEOPLE HAVE USED,

PEOPLE USE WESTERN BLOTS THAT

ARE NOT SO USEFUL.

ANOTHER THING WE'RE INTERESTED

IN IS OF COURSE AS YOU TREAT THE

INFECTION WITH ANTIBIOTICS, WHAT

HAPPENS OF COURSE IS THE LOAD

CAN GO DOWN OVER TIME AND

CONVERSELY OR ANALOGOUSLY,

AMOUNT OF ANTIGEN CAN DECREASE

AND IT'S USEFUL FOR MONITORING

POTENTIAL TREATMENT AND THESE

STUDIES ARE ONGOING, BUT I JUST

WANT TO POINT OUT THAT WHEN WE

DID THIS MONITORING, OF EACH,

YOU KNOW LATE TERM INFECTION,

LID WAS STILL ABLE TO DETECT LOW

LEVELS OF TIGHTER TO THE LINE,

BUT EALIZA WAS ONLY 76%, SO

AGAIN THE TAKE HOME MESSAGE IS

IT'S THIS ROBUST TEST AND DO

HIGH THROUGH PUT AND THERE'S NO

SUBJECTIVE READING OF THE

WESTERN BLOT.

SO, AGAIN, I'M GOING TO PUT A

MOVE TO OTHER INFECTIOUS AGENTS

BUT AGAIN, HIV, IS A VIRUS WHERE

WE'RE ALL INTERESTED IN AND HAS

OBVIOUSLY MANY DIFFERENT

PROTEINS INCLUDING OF COURSE, GP

120.

SOME OF THE ENVELOPE PROTEINS IN

GAG PROTEINS, AND BACK IN 2005

WHEN I WAS STILL IN GEORGE TOWN,

I WORK WIDE JOE CO VAC AND WE

WERE THE FIRST ACTUALLY TO

PROFILE THE ENTIRE PROTEIN PROTEOME OF

HIV WITH USING INTACT PROTEINS

AND I'M JUST SORT OF SUMMARIZING

THE GREEN BEING CONTROLLED AND

VALUES OVER THE CONTROL IN

SIMPLE HEAT MAP.

AGAIN EACH PATIENT GOING ACROSS

AND WHAT YOU CAN SEE, REMARKABLY

IS THAT ALL OF THE HIV PATIENTS

AND WE'VE NOW DONE PROBABLY FIVE

OR 600 DIFFERENT PATIENTS HAVE

EXTREMELY HIGH TIGHTERS OF

REVERSE TRANSCRIPT ACE AND THESE

ARE TYPICAL PROGRESSOR PATIENTS

AND THEY HAVE ANTIBODY GB41 AND

EVEN WHAT'S USE INDEED MOST

COMMERCIAL OR RAPID TESTS ARE

LIKE THE P24 GAG PROTEIN IS LESS

INFORMATIVE THAN THESE OTHER

ANTIGENS, OTHER LINE OF PROTEINS

WE ALSO HAD SAW RESPONSES SO

AGAIN, YOU KNOW WE CAN OBTAIN

ALL THIS INFORMATION AND IS IT

USEFUL AND RECENTLY WE WORKED

WITH STEPHEN AND DANIEL AND

PUBLISHED A PAPER IN BLOOD

LOOKING AT A VERY UNIQUE GROUP

OF PATIENTS THESE ARE ELITE HIV

NONPROGRESSORS AND THESE

PATIENTS ARE--HAVE TYPICAL--HAVE

STREAMLY LOW LEVELS OF HIV VIRAL

LOAD AND THEY ALSO TONED HAVE

WEAK IMMUNE RESPONSES AND I

THINK YOU CAN APPRECIATE THAT

AGAIN, HERE ARE THE CONTROLS AND

HERE ARE SOME OF THE TYPICAL

PROGRESSORS, YOU SEE THIS QUITE

DRAMATIC DIFFERENCE IN THE BLUE

LINE AND ALL THESE HAVE

GEOMETRIC MEAN TIGHTER AND YOU

CAN SEE THAT THREE OF THE FOUR

ELITE PATIENTS, REALLY DO HAVE

THESE BLUNTED GP 41 AND REALLY

NOT SEEN THAT WE'VE DONE MUCH

LARGER STUDIES, SIMILARLY, ALL

FOUR OR THREE OF THE FOUR AGAIN

HAD DEPRESSED P24 ANTIBODY

LEVELS AND LASTLY, THE REVERSE

TRANSCRIPT ACE WHICH IS REALLY,

REALLY USEFUL FOR DETECTING HIV,

WAS COMPLETELY ABSENT, REALLY

AGAIN.

HERE'S THE CUT OFF WITH THE

THREE OF THE FOUR LEAD

PROGRESSORS, NONPROGRESSORS AND

SO I THINK THIS AGAIN, JUST IT'S

JUST POINTING OUT THAT THESE

QUANTITATIVE PROFILE K'S GIVE

YOU MORE THAN A YES NO BUT

POTENTIALLY GIVE YOU INSIGHT

INTO WHAT'S GOING ON AND WE'RE

CURRENTLY INTERESTED IN LOOKING

AT THINGS LIKE THE REVERSE

TRANSCRIPT ACE ANTIBODY PATIENTS

WHO HAVE BEEN TREATED FOR A LONG

TIME WITH ANTIRETROVIRALS OR

RIGHT AFTER INFECTION TO SEE IF

WE CAN HAVE THEM WITH A

BIOMARKER.

AND JUST TO FINISH THIS VIRUS,

ANOTHER VIRUS IS WE'VE LOOKED AT

FOR TRYING TO UNDERSTAND AGAIN,

IMPORTANT SUBSET OF THE DISEASE

OF THAT CAUSED BY THE FIRST

RETROVIRUS IS HTLV ONE AND MOST

PEOPLE REMAINS ASYMPTOMATIC FOR

THE LIFETIME BUT CAN ALSO CAUSE

A T-CELL LEUKEMIA LYMPHOMA OR IN

A DIFFERENT SUBSET CAN RESULT IN

AN MS DISEASE OF MILE OPERATING

GLOBALLYATHY, CALLED GSP AND

WORKING WITH STEVE JACOB SEN

ILLEGALSEN, WE'RE INTERESTED IN

THE UTILITY OF LITS FOR

DISTINGUISHING THESE THREE

GROUPS AND WHAT WE FOUND, AGAIN

WAS THAT WHEN WE PROFILED THE

GAG, WHICH AGAIN IS PART OF THE

CAPSID AND WE TEST THE FULL GAG,

THE ANTIBODIES WERE EXTREMELY

INFORMATIVE, AGAIN HERE ARE ARE

THE CONTROLS AND WE HAD MUCH

HIGHER TIGHTERS AGAINST IN THE

ASYMPTOMATIC PATIENTS OR THE

PATIENTS WITH THE LYMPHOMA

LEUKEMIA AND WITH THE TSP, BUT

YOU NOTICE THEY'RE NOT DIFFERENT

AMONGST THE THREE EFFECTIVE

GROUPS.

IN THIS CONTRAST WHAT WE FOUND

WHEN WE LOOK AT THE ENVELOPE AND

AGAIN WE FOUND MUCH, MUCH,

HIGHER TIGHTERS IN THE HAND PSP

PATIENTS COMPARED TO OTHER

SUBGROUPS.

SO AGAIN, STEVE JACOB SEN

ILLEGALSEN HAS DONE THIS

INDEPENDENTLY AND LOOKED AT

JAMAICAN COHORT AND USED THESE

AND REPLICATED WHAT WE FOUND

HERE THAT AGAIN THERE ARE THESE

TIGHTERS THAT POTENTIALLY THEy

TAX THAT I'M NOT SHOWING THAT

YOU CAN BE INFORMATIVE FOR

STRATIFYING AND RISK ASSESSMENT.

ALL RIGHT.

SO, ONE, I'M STILL WORKING ON

THIS PROJECT BUT I'M TRYING TO

DEVELOP A SIROLOGICAL RAPID TEST

FOR TD.

AND AGAIN AS YOU ALL KNOW,

MICROBACTERIA AND TUBERCULOSIS

INFECTS A THIRD OF THE WORLD,

CIRCULATING ANTIBODIES FIRST

READ THIS LITERATURE AND WERE

DETECT INDEED 1894 USING A

SIMPLE GLUTENATION ASSAY, BUT,

THERE WAS STILL REALLY A GREAT

DEAL OF DIFFICULTY IN DIAGNOSING

TB AND WE DO THE TBB TESTING AND

THERE'S MORE EXPENSIVE NOW,

EALIZA SPOT TEST AND SO ONE

THING I'VE BEEN TRYING, THERE'S

3000 PROTEINS IN THIS GENOME AND

OBVIOUSLY BECAUSE WE CAN CLONE

THEM VERY RAPIDLY, I'VE BEEN

TRYING TO DEVELOP A TEST FOR TB

AND I'M AGAIN SHOWING YOU SHALL

TIDBIT.

WE HAVE MUCH MORE DATA THAN

THIS.

I THINK WE'RE UP TO TESTING

ABOUT 2000, 900--OKAY, THE POINT

OF THIS IS HERE'S SAMPLE OF ONE

SUCH ANTIGEN THAT WE CAN DETECT

SIGNIFICANT ANTIBODY RESPONSES

TO ONE OF THESE ANTIGENS AND

USING A DIFFERENT ANTIGEN, YOU

MIGHT FIND A FEW MORE AND A

THIRD ANTIGEN, A FEW MORE AND

THE POINT I WANT TO MAKE HERE IS

AN ACTUAL FACT IS REAL

HETEROGENEITY, THEY'RE NOT

ALWAYS THE SAME PATIENTS

REACTING AND SO ONE OF THE OTHER

FEATURES OF LIT SYSTEM THAT WE

CAN COMBINE ALL THREE ANTIGENS

IN A MIXTURE AND WE THINK WE CAN

PROBABLY EVEN ADD POTENTIALLY

EVEN 20 OR MORE, DEPENDS ON HOW

CONCENTRATED THE EXTRACT IS, BUT

YOU CAN THEN SEE HOW SENSITIVITY

CAN DRAMATICALLY IMPROVE AND BE

USED IN A RAP RAPID TEST FOR

DIAGNOSIS.

MOST OF THESE TESTS WE DO FOR

TWO HOURS BUT NUMEROUS STUDY K'S

BE DONE IN FIVE MINUTES.

OKAY SO I WILL ALMOST FINISH THE

INFECTION ROUND UP SO SOME OF

OUR STUDIES DOING VIRUS HUNTING

AND WORKING WITH OTHER GROUPS

WHO ARE DISCOVERING LOTS OF

VIRUSES BY JUST DOING--TAKING

SOME TISSUE OR POOP OR THINGS

LIKE THAT AND SEQUENCING IT,

RUNNING IT THROUGH

BIOINFORMATICS, THEY'RE FINDING

NEW VIRUSS AND THE BIG QUESTION

IS HOW MANY ARE REAL PATHOGENS

OR NOT.

SO HERE'S AN EXAMPLE OF ONE

STUDY THAT WE DID WITH

COLLEAGUES AND IT FOLLOWS ON

THEIR FIRST OBSERVATION THAT

THEY FOUND A HEPATITIS C LIKE

VIRUS IN DOGS AND THEY PUBLISHED

THIS LAST YEAR IN PNAS, AND WHY

THIS IS INTERESTING IS UP TO

POINT THERE WERE NO CLOSE

RELATIVES AND THEY'RE ALL JUST

OF COURSE CHIMPANZEES OR HUMANS

INFECTION WITH THE HCV, THERE

WERE NO NONHUMAN PRIMATES AND SO

THIS CAINAIN STORY WAS QUITES

EXCITING, IT'S 40% SIMILAR AND

WORKING WITH THEM, WE WANT TO

VALIDATE AFTER THEY PUBLISH THIS

PAPER WERE DOINGS POTENTIALLY

EQUAL OF THE HUMAN HEPATITIS C,

SO WE BUILT A LIPS TEST BUILDING

THE HELIX ON THE CANINE AND WE

STARTED SCREENING DOGS DOGS AND

BASICALLY DIDN'T FIND ANYTHING

AND WE TRIED THE CORD AND DIDN'T

FIND ANY ANTIBODIES.

SO I DID THE NONAPOPTOTIC'S ARK

THEORY, I TESTED CAT, RABBITS,

COWS, FOR THE MOST PART THEY'RE

ALL NEGATIVE, WE HAD ONE COW

THAT HIT.

BUT THE REAL SEQUENCE IS WHEN WE

PROFILE HORSE SERUM, 40% OF THE

HORSES ARE INFECTED WITH THE

HEPATITIS C AND WHAT'S

INTERESTING ABOUT THIS IS I

IMMEDIATELY SENT BACK THE SIR O

POSITIVES THAT WE WERE TESTING

BACK TO COLUMBIA ANDAD MITT WAS

ABLE TO--ADMIT WAS ABLE TO CLONE

GENOTYPES AND BUILD A PC R BASED

TEST AND LIKE HUMAN HCV

INFECTION, YOU CAN SUH SEE ONLY

THE ONES I'M COLORS HERE IN RED

ARE DNA POSITIVE SO IN OTHER

WORDS IT LOOKS LIKE MOST OF

THESE HORSES ARE CLEARING THE

INFECTION, THIS IS OBVIOUSLY

VERY DIFFERENT THAN THE HUMAN

SITUATION.

BUT I THINK THIS IS KIND OF A

NEAT, NEAT THING IN THE SENSE WE

NOW SORT OF UNDERSTAND THE

NATURAL RESERVOIR OR POTENTIAL

RESERVOIR OF HEPATITIS C O

RELATED VIRUSS AND OBVIOUSLY

THIS IS STILL A LOT TO BE DONE

CAN I'M EAG TORE TEST THE ZEBRA

NEXT.

OKAY, SO I'M JUST GOING TO VERY

BRIEFLY MENTION THAT THERE ARE A

LOT OF VIRUSES OUT THERE, YOU

CAN OPEN ANY OF THE TOP JOURNALS

OR VIROLOGY JOURNALS AND PEOPLE

ARE DESCRIBING NEW HUMAN

INFECTIOUS AGENT, THERE NEEDS TO

BE CAUTION THERE BECAUSE MOST OF

THAT IS ALL JUST BASED ON

NUCLEIC ACID, AMPLIFICATION AND

WE WANT TO KNOW ARE THESE INFECT

YOWS AGENTS INFECTING HUMANS SO

WE BUILT LIPS TEST FOR NEW

VIRUSES THAT EMITg#rd> THESE AND

THEY'VE BEEN DISCOVERING IT AND

I'M SHOWING A FEW PUBLIC

EXAMPLES, WE PUBLISHED THIS ONE

ALREADY AND NOVELS, AND VIRUSES

LOOK LIKE THE ANIMAL VERSION ASK

THERE'S A STUDY OUT THERE THAT'S

FOUND ACTUALLY IN THE BRAIN OF A

CHILD.

AND WE'RE SEEING ABOUT 60% OF A

CHILD WITH ENCEPHALITIS AND IT

APPEARS TO BE QUITE A COMMENT.

INFECT YOWS AGENT, THIS VIRUS

CALLEDICOSA VIRUS, IT'S A PC

ORNA VIRUS AND WE'RE DETECTING

ABOUT 50%, IN THE STUDIES DONE

SO FAR, IT LOOKS LIKE ONE OF THE

MOST COMMON IN YOU HUMAN STOOL AND

UP TO THIS POINT NO ONE'S PROVEN

IT'S AN INFECT YOWS AGENT.

AND LASTLY I'M NOT GOING TO TELL

YOU TOO MUCH ABOUT THIS BUT IT'S

A NOVEL PLANT LIKE VIRUS THAT'S

ASSOCIATE WIDE THE DISEASE AND

WHAT'S REALLY QUITE INTERESTING

IS I TESTED TWO DIFFERENT PIECES

FROM THIS VIRUS AND YOU CAN SEE

THE SAME PATIENTS TONED HIT.

THIS IS SOLIDIFYING THE IDEA

THAT IT MIGHT BE THE REAL

INFECTION.

ALL RIGHT.

HOPEFULLY I'M STILL ON TARGET

HERE, I THINK I'M RUNNING

BEHIND.

SO ONE THING WE'RE ALSO VERY

INTERESTED IN DOING IS TO

PROFILE AUTOANTIGENS AND THESE

ARE ANTIBODIES AGAINST CELL

PROTEINS AND MANY OF THESE ARE

CONFIRM ASSESSAL AND THEY'RE

IMPORTANT WITH AUTOIMMUNE

DISEASE, AND I'M GOING TO GIVE

YOU AS A STARTING EXAMPLE, ONE

AUTOIMMUNE DISEASE WE'RE

PARTICULARLY INTERESTED IN TYPE

ONE DIABETES.

AND OF COURSE THESE ARE MEDIATED

DISEASE, BUT A LOT OF THE

ANTIGENS ARE RECOGNIZED BY THE

SAME REPPER 24S AS TARGETS AND

THINGS LIKE INSULIN AND IATWO

GAD, AND BETA AND THESE RECENTLY

DISCOVERED ZINC TRANSPORTER AND

WHAT I'M JUST SHOWING YOU HERE

IS THAT WE'VE DEVELOPED SOME

REALLY QUITE ROBUST TESTS FOR

MANY OF THESE AUTOANTIGENS AND

AGAIN UP TO NOW, ALL THIS TEST

SUGGEST DONE RADIOACTIVELY WITH

ANOTHER APPROACH REALLY CAN'T

DETECT MANY OF THESE

CONFIRMATIONAL ANTIBODIES, ONE

OF THE IMPEDIMENTS FOR USING

THESE PROFILES CLINICALLY.

AND ALL I'M GOING TO BRIEFLY GO

OVER AT THIS POINT IS TO SAY

THAT THESE ANTIBODY RESPONSES

AGAINST MANY OF THESE BETA CELL

TARGETS ARE EXTREMELY

HETEROGEANIOUS, I WANT TO POINT

OUT HOW IMPORTANT CONFIRMATIONAL

EPITOPES ARE.

THIS TRANSPORT SER A POLYMORPHIC

VERSION.

SOME OF US HAVE RGUIN AND TRIP O

FEIGN ONE OF TWOAN 20 AMINO

ACIDS I'M USING AND WHAT'S

REMARKABLE IS TAKEN--THEY YOU

AGAIN GOING

PATIENTS WHO HAVE ANTIBODIES AND

MOST LIKELY DIDN'T GENOTYPE BUT

MOST LIKELY THE RGENERATEDDINE

VARIANT HIGH LEVELS OF

ANTIBODIES TO THIS FORM BUT NOT

AGAINST THE TRIP TO FAN FORM AND

VICE VERSA THIS AGAIN, SORT OF

AGAIN, KNOCKS THE IDEA THAT THE

MOLECULAR MIMICKRY, IS QUITE

SPECIFIC TO THE GENOTYPE AND

LESS LIKELY THAT THERE'S SOME

INFECT YOWS AGENT OUT THERE

THAT'S CAUSING IT.

ONE OF THE REAL INTERESTING

THINGS THAT WE CAN DO, AND I'M

ONLY JUST FOR TIME CONSTRAINTS

GIVING YOU JUST A SNAPSHOT OF

THIS, START LOOKING AT COMORBID

CONDITIONS ASSOCIATE WIDE A LOT

OF THESE AUTOIMMUNE DISEASES.

AND SO, IN TYPE ONE DIABETES,

THERE ARE A LOT OF SILLIAC AND

THYROID, HARBY MOTOS,

THYROIDITEIS, AUTOIMMUNE

GASTRITIS AND SO WE BUILT A

LARGE PANEL SCREENING AND THIS

IS ALL WORK DONE BLINDLY.

THE EYELET PROFILE ALONG WITH

THE ANTIBODIES AGAIN, THESE

EXTRA PANCREATIC TARGETS AND

AGAIN, THE TAKE HOME MESSAGE IS

THAT APPROXIMATELY 50% OF THESE

TYPE ONE DIABETIC CHILDREN ARE

SHOWING ANTIBODIES AGAINST SOME

OF THESE OTHER TARGETS AND THIS

ISvXC

HIGHER AND MAYBE IN MANY CASES

THESE ARE SELF-CLINICAL

CONDITIONS BUT IT DOES HIGHLIGHT

THE IDEA THAT THESE

AUTOANTIBODIES, AUTOIMMUNITY CAN

BE DIRECTED NOT JUST AGAINST THE

BETA TARGETS BUT THE BETA CELL

TARGETS.

SO JUST VERY QUICKLY, ALSO,

AUTOANTIBODIES CAN BE USED TO

NOT ONLY DIAGNOSE DISEASE, BUT

ALSO TO PREDICT DISEASE AND THIS

IS JUST A CARTOON OF SUMMARIZING

THE WORK THAT AGAIN, NONDIABETIC

CHILDREN DON'T HAVE THESE

ANTIBODIES BUT CHILDREN WHO GO

ON TO DEVELOP DIABETES, WE CAN

ALREADY DETECT IN THIS EARLY

WINDOW, AUTOANTIBODIES AGAINST

SOME OF THESE EYELET TARGETS,

YEARS BEFORE THEY ACTUALLY

REQUIRE IT OR DEVELOP THE TYPE

ONE DIABETES, SO THEY HAVE

ROLEs AS PREDICTORS AND

THERE'S A LARGE INTEREST IN

TRYING TO PRESERVE THE BETA CELL

MASK WITH CLINICAL PROTOCOL SO

PEOPLE ARE INTERESTED IN TRYING

TO IMPROVE THESE TYPES OF

PROFILES SO, I GOT--WE RECEIVED

SAMPLES FROM MARY ANN ROYERS AT

UNIVERSITY OF COLORADO WHO

COLLECTS THESE TYPES OF

PROTECTIVE STUDIES AND I'M

SHOWING THREE DIFFERENT PATES

WHERE WE PROFILES AGAIN,

ANTIBODIES AGAINST THIS GHASTIC

ATTP, ON THIS COHORT ABOUT 25

CHILDREN WITH TYPE ONE DIABETES

AND THREE OF THEM AGAIN HAD

ANTIBODIES AND YOU CAN SEE SOME

OF THESE CHILDREN ARE ALREADY

DEVELOPING ANTIBODIES AGAINST

THE STOMACH AT AGE TWO, WAY

BEFORE AGE 14 WHEN THE

AUTOANTIBODIES ARE WITH THE

EYELET OR THEY DEVELOP DIABETES.

SO AGAIN THESE TYPES OF PROFILES

SUGGEST THAT THERE IS THIS

B-CELL DYSFUNCTION IN MANY OF

THESE PATIENTS YEARS BEFORE EVEN

THEY START ATTACKING OR YOU CAN

RECOGNIZABLY SEE THE ANTIBODIES

AGAINST THE BETA CELL TARGETS.

WE'RE ALSO INTERESTED IN RHEUM

TO LOGICAL DISEASES AND I WAS

GOING TO SORT OF SUMMARIZE THE

KEY POINTS HERE.

SLE AND CHILDREN'S AGAIN ARE TWO

RHEUM TO LOGICAL DISEASES,

THEY'RE THOUGHT TO BE ON THE

SAME SPECTRUM AND SOMETIMES IT'S

DIFFICULT TO DIAGNOSE THEM AND

USING A SIX ANTIGENERATED PANEL

BEEN ABLE TO PRETTY MUCH DETECT

CLOSE TO 95% OF THE LUPUS

PATIENTS AS BEING AUTOANTIBODY

POSITIVE AND WE'VE IDENTIFIED

TWO DIFFERENT SUBSETS AND ONE

SUBSET AGAIN AS VERY HIGH

TIGHTERS OF SMITH, SMITH R&  P

ANTIGEN VERSES THE OTHER SUBSET

THAT HAS A VERY HIGH LEVELS OF

ROW, LAW AND ANTIBODIES AND WE

THINK THAT AGAIN IT HONES IN ON

THE PATHOGENESIS POTENTIALLY OF

LUPUS, THESE ARE RNA BINDING

PROTEINS, IT'S JUST A

POSSIBILITY THAT EVEN THOUGH

LUPUS HAS MANY DIFFERENT GENETIC

VARIANTS THAT ARE BEING

DISCOVERED, IT ALL SEEMS TO

FUNNEL IN ON THESE FEW

AUTOANTIGEN TARGETS THAT LEADS

TO THE DIAGNOSIS POTENTIALLY

SUGGESTING THEY'RE IMPORTANT.

WE'VE DONE THE SAME THING OF

COURSE IN CHILDREN'S SYNDROME

AND IT'S WELL KNOWN THAT THE

ANTIBODIES AGAINST SSA AND SSB,

BUT WE PROFILED AND FOUND A

LARGE CLUSTER IN THE LUPUS--IN

IMPROVING THE TESTS THAT ARE OUT

THERE, FOR EXAMPLE, THE LAW TEST

WITH THE EALIZA MIGHT PICK UP

45%, WE'RE ALMOST ABLE TO TAKE

80% OF THOSE AS BEING POSITIVE

AND WE'VE ALSO IDENTIFIED

SIMILAR LIKE LUPUS CLUSTERS IN A

SUBSET AND PROBABLY NOT BEEN

RECOGNIZED AND WE'RE ALSO SEEING

AUTOANTIBODIES AGAIN.

MANY OTHER EXTRA GLANDULAR

TARGETS INCLUDING AQUA PORIN

FOUR AND WHEN WE BROKE THE SATTA

ON A SUBSET, WE'RE REPEATING

MANY OF THESE PATIENTS HAVE

NEUROLOGICAL DISEASE SUCH AS

NEUROPATHY.

I'M GOING TO SORT OF END HERE

AND I KNOW I'VE GONE VERY

QUICKLY OVER MANY DIFFERENT

DISEASES.

BUT WHAT I LIKE TO POINT OUT

HERE IS THAT, THERE ALSO CAN BE

ANTIBODIES AGAINST A LOT OF

EXTRA CELLULAR TARGETS AND YOU

PROBABLY ALL KNOW THAT

MYOSINNAIA GRAFFIS IS THE

CLASSIC IMMUNE DISEASE AGAINST

THE END CLAVE OF THE RECEPTOR

AND IT CAN CAUSE RECEPTOR TO

DOWN REGULATE AND OBVIOUSLY

CAUSE WEAKNESS IN CLINICAL

SYMPTOMS, THERE ARE OTHER

DISEASE LIKE GOOD PASTOR'S

DISEASE AGAINST COLLAGEN FOUR OR

PROGNOSEIS AGAINST EVEN

CYTOKINES AND WORKING WITH SARAH

BROWN AND STEVE HOLLAND WE'VE

BEEN INTERESTED IN THE

POSSIBILITY THAT CYTOKINES AND

ANTIBODIES TO CYTOKINES MIGHT

CAUSE DIFFERENT IMMUNO

DEFICIENCY, SO THE IDEA AGAIN IS

THAT NORMALLY THE CYTOKINE BINDS

TO RECEPTOR AND CAN ACTIVATE,

THE STAT PATHWAY AND TURN ON

GENE ACTIVATION AND HOST DEFENSE

AND IMMUNE ACTIVATION.

OBVIOUSLY IF THERE ARE

ANTIBODIES THAT BLOCK THIS, THIS

WOULD BE A REAL PROBLEM.

SO SARAH HAS COLLECTED A LARGE

GROUP OF COHORT OF PATIENTS FROM

THAILAND AND IN ASIA AND WHAT

SHE'S FOUND IS THAT THEY HAVE

SOME SYMPTOMS THAT RESEMBLE

ALMOST LIKE AN HIV DISEASE BUT

THEY DON'T HAVE HIV.

THEY HAVE A LOT OF DESIMINATED

MICROBACTERIAL INFECTION, THAT'S

GROUP ONE.

THERE'S ALSO A SECOND GROUP THAT

HAVE TOX O AND HISTOAND FOR

EXAMPLE, SIGNIFY SEMESTERINATED

VCV WITH AND WITHOUT

MICROBACTERIAL INFECTIONS AND

WHAT WE DID WAS WE PROFILED

THOSE TWO GROUPS AS WELL AS

THREE OTHER GROUPS INCLUDING

HEALTHY CONTROL, PATIENTS WITH

PULL MONITORAR SCHEDISSEMINATED

TD AND WHAT WE FOUND AGAIN WAS

THAT THESE PATIENTS WITH THESE

MAINLY MICROBACTERIAL BUT OTHER

OPPORTUNISTIC INFECTION HAD HIGH

TIGHTERS OF ANTIBODIES AGAINST

GAMMA INTERFERON AND HAD

NEUTRALIZING ACTIVITY IN VARIOUS

FUNCTIONAL ASSAYS.

BUT WE AGAIN USE SORT OF LISTS

TO IDENTIFY THAT AND WE ALSO

SCREENED AGAIN 40 OTHER

CYTOKINES AND AS YOU CAN SEE,

WE'RE LOOKING FOR REALLY THE RED

GROUP BEING HIGH, THERE'S REALLY

THIS DISEASE IS REALLY

EXCLUSIVELY HIGH TIGHTER

ANTIBODIES WITH GAMMA INTERFERON

AND THERE'S ONE EXAMPLE OF ONE

PATIENT WHO HAD SCRIPT O CAUCUS

AND MENINGITIS THAT HAD A HIGH

LEVEL OF J&  C.

SO THE TAKE HOME MESSAGE IS

THERE'S PROBABLY BASED ON THIS

STUDY AGAIN FUNCTIONALLY HOW

IT'S WORK SUGGEST BLOCKING

RECEPTOR, BINDING, THE LIGAND

BLOCKING SIGNALING AND WE THINK

THERE ARE MANY, MANY, OTHER--NOT

MANY, MANY, OTHERS BUT THERE'S

SOME OUT THERE THAT ARE OTHER

DISEASES THAT COULD BE CAUSED BY

ANTIBODY BINDING TO THE EXTRA

CELLULAR LIGAND.

SO IN SUMMARY, VERY QUICKLY, I

TRIED TO GIVE YOU A PERSPECTIVE

ON HOW WE USED THIS VERY

POWERFUL ANTIBODY PROFILING

TECHNOLOGY TO DEVELOP THESE

PROFILES IN INFECT YOWS AGENTS

AND AUTOIMMUNE TARGETS AND THESE

PREDICTED MEDICINE SINCE I DON'T

HAVE MUCH TIME, I WILL THANK MY

MANY, MANY, COLLABORATORS, OUR

LABORATORY AND OBVIOUSLY ADRIANA

MARK QUESTIONS AND COLLEAGUES

AND THE DIABETES WORK WITH DAVID

WHO WAS FORMERLY HERE AND OTHER

COLLABORATORS.

THANK YOU.

[ APPLAUSE ]

>> I THINK GIVEN THE TIME WE'LL

CONTINUE WITH THE SECOND SPEAKER

AND TAKE QUESTIONS LATER.

>> SENIOR INVESTIGATOR AT NCI IN

THE EXPERIMENTAL TRANSPLANTATION

BRANCH AND SERVED AS PRINCIPLE

INVESTIGATOR AT BENCH TO BEDSIDE

PROG EXPECT WILL PRESENT

RAPAMYCIN RESISTANT ALOE GENIC

T-CELLS.

DR. FOWLERIS A SENIOR

INVESTIGATOR AT EXPERIMENTAL

TRANSPLANTATION AND IMMUNOLOGY

BRANCH.

HE'S ON THE EDITORIAL BOARDS OF

THE JOURNAL OF CLINICAL

IMMUNOLOGY AND BIOLOGY OF BLOODS

AND BONE MARROW TRANSPLANTATION,

MEMBERSSHIPS INCLUDE THE

AMERICAN SOCIETY FOR CLINICAL

INVESTIGATION AND LEUKEMIA

SOCIETY OF AMERICA AND THE

INTERNATIONAL SOCIETY FOR

CELLULAR THERAPY.

HIS RESEARCH FOCUSES ON ADOPTIVE

T-CELL THERAPY THERAPY AND ALOE

GENERATED AIC STEM CELL

TRANSPLANTATION, AND THEY USE

MOUSE MODELS TO IDENTIFY THAT

RAPAMYCIN T-CELLS MODULATE

IMMUNE RESPONSES INCLUDING GRAPH

VERSES HOST DISEASE AND GRAFT

REJECTION.

ONGOING CLINICAL TRIALS ARE

EVALUATING RAPAMYCIN T-CELLS FOR

REFRACTORY OF LYMPHOMA ANOTHER

HEMOTO LOGIC MALIGNANCYS AND

WITH THAT I TURN THE PODIUM OVER

TO DR. FOWLER.

>> THANK YOU, FRIENDS, IT'S AN

HONOR TO BE HERE TODAY TO TALK

ABOUT OUR RESEARCH.

I UNDERSTAND IT'S GETTING LATE

IN THE AFTERNOON, EVERYONE'S

READY FOR LUNCH BUT I REMIND YOU

THAT RAPAMYCIN INDUCES A

STARVATION IN STATE SO IF

EVERYONE COULD HOLD ON FOR

ANOTHER HALF OUR.

OUR DISCLOSURE VS TO DO WITH

U.S. PATENT APPLICATIONS WE HAVE

SUBMITTED BASED ON THIS WORK

WITH RAPAMYCIN AND WITH GENETIC

MODIFICATION OF THESE T-CELLS.

THE OBJECTIVES TODAY WOULD BE TO

DISCUSS THE DIFFERENTIAL

MECHANISM OF ACTION, OF

RAPAMYCIN TO THE MORE COMMONLY

UTILIZED DRUGS INHIBITORS SUCH

AS CYCLOSPORIN AND TACK RALAMIS

AND NUMBER TWO TO EXPLAIN THE

EFFECT OF RAPAMYCIN ON T-CELLS

AND THREE TO DESCRIBE THE ROLE

FOR GRAFT ENGINEER NOTHING THE

SETTING OF ALOE GENETIC

HEMATOPOIETIC STEM CELL

TRANSPLANT.

THIS IS A SCHEMA I USED FOR MANY

YEARS TO HELP DESCRIBE THE

CELLULAR PLAYERS THAT ARE

IMPORTANT WHEN WE CONSIDER A

STEM CELL TRANSPLANT FOR THE

TREATMENT OF CANCER.

ON THE LEFT ARE THE DONOR

POPULATIONS FROM A GENETICALLY

DESPERATE INDIVIDUAL IN OUR

STUDIES, A MATCHED SIBLING

DONOR, RELATIVE TO THE PATIENT

HERE, I DESIGNATED LEUKEMIC

HOST, SO YOU HAVE THE CD34 STEM

CELLS THAT ARE INFUSED AND THESE

SEMESTER CELLS FROM THE DONORROR

SUSEPTIBLE OF GRAFT INJECTION

AFTER THEY REMAIN AFTER ANY

CONDITIONING REGIMEN THEY MAY

ADMINISTER, WE USE SO CALLED LOW

INTENSITY TRANSPLANT IN OUR

PROTOCOLS, SO THERE'S MORE OF

THESE T-CELLS REMAINING SO THE

RISK OF GRAFT REJECTION IS

HIGHER IN OUR STUDIES.

YOU ALSO HAVE CELLULAR PLAYERS

FROM THE DONEAR INCLUDING CD4

AND CDEIGHT POSITIVE T-CELLS

THAT ARE HARVESTED ALONG WITH

THE STEM CELL GRAFT AND THIS

COMBINATION IS THE TRANSPLANT,

THESE T-CELLS GOING INTO THE

BODY, TIME OF TRANSPLANT CAN

CAUSE A BENEFICIAL GRAFT VERSES

LYMPHOMA OR GRAFT VERSES

LEUKEMIA EFFECT AGAINST ANY

TUMOR CELLS.

DESIGNATED AS CML HERE BECAUSE

THAT'S THE CLASSICAL CASE OF A

GDL EFFECT.

AND THESE T-CELLS CAN ALSO GO

INTO THE BODY AND RECOGNIZE THE

NORMAL TISSUE, LIVER INTESTINE

AND SKIN PREDOMINANTLY WHICH ARE

THE TARGET ORGAN SIGHTS FOR

GRAFT VERSES HOST DISEASE, SO

OUR SUCCESS IN ANY ONE OR

INDIVIDUAL TRANSPLANT IS A

BALANCING ACT BETWEEN NOT

GETTING GRAFT REJECTION, GETTING

ENOUGH TO THE ANTITUMOR EFFECT

AND AVOIDING TOO MUCH TOXICITY

TO THE NORMAL TARGETS.

SO ONE STRATEGY THAT WE'VE TAKEN

OVER MANY YEARS NOW, SINCE I

CAME HERE AT NIH, WAS TO

UNDERSTAND WHETHER WE COULD TAKE

THE DONOR T-CELL COMPARTMENT AND

BREAK IT INTO DIFFERENT

FUNCTIONAL SUBSET SO INSTEAD OF

LOOKING AT CD4 AND CDEIGHT, LOOK

AT WHETHER THE CD4 CELLS WERE A

THONE PHENOTYPE THAT IS CELLS

THAT WHEN THEY RESPOND TO

ANTIGEN RELEASE INTERLEUKIN TO

AN INTERFERON-GAMMA OR IF THEY

WERE A THTWO PHENOTYPE THEY MAY

SECRETE THESE IL410, AND THEN,

CYTOKINES.

DO THESE HAVE A PRESET

DISPOSITION TO CAUSE GBHD AND

ANTITUMOR EFFECTS.

THAT WAS OUR INITIAL FOCUS THONE

AND TWO BECAUSE THESE WERE THE

SUB SETS IDENTIFIED, MANY MORE

HAS BEEN IDENTIFIED RECENTLY

WITH THE FoxpTHREE SUBSET

WHICH IS AN IMMUNE SUPPRESSOR

TH17 SUBSET.

I'LL FOCUS MOSTLY ON TH1 AND TH2

SUBSETS.

WE HAVE STUDIED IN THE ROLE OF

THIS MOLECULE PROGRAM DEATH

LIGAND 1 AS A MOLECULE IS THAT

CAN ENCODE A REGULATORY T-CELL

EFFECT AND THIS MOLECULE CAN

BIND TO PD1 ON A TH1 CELL AND

TURN THIS INTO A T-REG TYPE

SUBSET.

THE POINT HERE IS THESE SUBSETS

AREINER CONVERTIBLE AND NOT

STATIC IN THEIR FUNCTIONAL

PHENOTYPE.

AND COMPARED TO THE INHIBITORS

THESE ARE THE COMMON USED ORGANS

ISSUES THESE DRUGS HAVE A

RELATIVELY SIMPLE ACTION AND

THAT THEY INHIBIT CALCIUM FLUX

THROUGH INHIBITION AND THIS

LEADS TO T-CELLS LARGELY AT THE

MESSENGER RNA LEVEL,

TRANSCRIPTIONAL INHIBITION AND

IN CONTRAST RAPAMYCIN DISCOVERED

ALMOST 50 YEARS AGO, RECENTLY

THE MECHANISM OF ACTION ONLY

ELUCIDATED THROUGH

IDENTIFICATION OF THIS PROTEIN

HERE, THE MAMMALIAN TARGET OF

RAP MICEIN OR mTOR, SO WHAT

RAPAMYCIN DOES IS IT BINDS TO AN

FBKBB12, AND THIS INHIBITS

MTOR WHICH IS A GATE KEEPER FER

CELL SURFACE RECEPTOR SIGNALING.

INITIALLY IT WAS THOUGHT TO BE

MAINLY THE IL2 RECEPTOR AND NOW

IT'S MANY DIFFERENT CYTOKINES,

NUCLEOTIDES TREENT SIGNALING

SUCH AS IGF PATHWAYS AND CO

STIMLATTORY PATHWAYS, ET CETERA.

SO IF THE RAP O MICEIN INHIBITS

mTOR, HAVE YOU MANY DOWN STREAM

EFFECTS BLOCKING MANY DIFFERENT

CELL SURFACE SIGNALS, AND WHAT

THIS RESULTS IN, IS A REDUCTION

IN PROTEIN TRANSLATION THROUGH 4

EDP 1 INHIBITION.

REDUCED PHOSPHORYLATION THROUGH

THE P-70 S6 KINASE PATHWAY,

REDUCE CELL CYCLE THROUGH

INHIBITION OF SICK LYNN PATHWAYS

SO VERY MUCH MORE PLEOMORPHIC

EFFECTS ON NOT ONLY T-CELLS BUT

EVERY OTHER CELL IN THE BODY,

LEADING TO CELL CYCLE ARREST AND

INHIBITION OF PROTEIN

TRANSLATION AND I'LL ALSO

MENTION IF YOU INHIBIT mTOR A

CELL WILL UNDERGO AUTOPHAGY OR

THE DOWN SIZING OF ORGANELLE AS

AN ATTEMPT TO SURVIVE IN A STATE

OF STAFFATION.

SO WE WERE INTERESTED IN

RAPAMYCIN BECAUSE THERE WAS

EVIDENCE THAT IT MAY HELP

PROMOTE A TH2 EFFECT.

SO WE WENT TO THE MOUSE MODEL

WHERE WE TRY TO GENERATE TH2

CELLS IN OUR EXPERIMENTAL

SYSTEMS TO ASK THAT QUESTION, DO

TH2 CELLS HEPATITIS E REGULATE

GRAFT VERSES HOST DISEASE AND

THIS IS OUR SYSTEM FOR TRYING TO

MANUFACTURE A TH2 CELL.

WE PURIFY CD4 CELLS AND CO

STIMULATE THEM IN A ANTIGEN FREE

SYSTEM, THAT IS WE HAVE MAGNETIC

BEADS THAT HAVE CO STIMLATTORY

ANTIBODIES TO THE KORESEPTORSOR,

AND TO THE CD28 CO STIMLATTORY

PATHWAY AND THEN WE HAVE

CYTOKINE SUCH AS IL4 AND 2 TO

DIFFERENTIATE TOWARD TH2 AND IN

THIS TYPE OF SYSTEM, WE ADDED

RAPAMYCIN TO ASK COULD WE GROW A

SO CALLED TH2 CELLS UNDER THE

INFLUENCE OF RAPAMYCIN, TH2

RAPPA CELL.

WE'RE SOMEWHAT SURPRISED BY OUR

INITIAL RESULTS BECAUSE THEY RAN

SOMEWHAT AGAINST OUR HYPOTHESIS,

THAT IS IF WE MANUFACTURED CELLS

UNDER A TYPE 1 POLARIZING

CONDITION, THIS WAS THE CONTROL,

TH1 CELLS OR CD8 TC-1 CELLS, YOU

CAN SEE SLEEP APNEA AND OBESITY

HERE'S THE EXPANSION WITHOUT

RAPAMYCIN, MANY LOG GROWTH

BECAUSE WE COULD GROW CELLS THAT

WERE RAPAMYCIN RESISTANT IN A

TH1 TC-1 PHENOTYPE AND THESE

CELLS ACTUALLY CAUSE A LOT OF

GRAFT VERSES HOST DISEASE.

SO IN A VERY MARKED CONTRAST TO

STUDIES THAT EARLIER HAD SHOWN

RAPAMYCIN TO TOLL-LIKE RECEPTOR

RISE T-CELLS IN OUR EXPERIMENT

IF ENOUGH CO STIMULATION WAS

PROVIDED AND ENOUGH POLARIZING

CYTOKINES WAS PROVIDED CELLS

COULD BECOME RAPAMYCIN RESISTANT

AND THEY COULD BECOME POLARIZED

TO BOTH THE TH1 PHENOTYPE AND ON

THE RIGHT TO THE TH2 PHENOTYPE.

SO WE'RE GREEN CELLING RAPAMYCIN

RESISTANT CELLS IN A TH2

POLARIZING CONDITION.

WHAT WE FOUND RECENTLY STUDIES

DONE IN OUR LAB BY A COLLEAGUE,

HERE'S AN IMAGE OF A HUMAN

T-CELL BEING POLARIZED TO THE

TH2 PHENOTYPE IN RAPAMYCIN

UNDERGOING AUTOPHAGY.

SO THESE ARE CELLULAR ORGANELLES

SUCH AS MITOCHONDRIA AND

ENDORETICUE LIMP, AS A

NUTRITIONAL SOURCE FOR THAT CELL

WHILE IT'S STARVED BY RAP O

MICEIN AND THIS REMINDS 1 OF THE

QUOTE BY CHARLES DARWIN ABOUT

THE STRONGEST SPECIES SURVIVES

IS THE 1 WHO IS ADAPTABLE TO

CHANGE AND THAT'S HOW WE THINK

THESE T

RAP O MICEIN AND THEY UNDERGO

INTENSIVE CHANGES THAT ALLOW

THEM TO SURVIVE.

AND THIS IS ILLUSTRATED HERE,

THESE ARE HUMAN T-CELLS GROWN IN

RAPAMYCIN AND AT THE END OF THAT

EXPOSURE AS THE CELLS ARE

GROWING AUTOPHAGY, WE REDUCE THE

MASS, THERE'S A FLOW CYTOMETRY

ASSAY--IN CONTRAST IF YOU LOOK

AT MITOCHONDRIAL STABILITY OR

ANTIAPOPTOTIC PHENOTYPE, THEY

HAVE AN INCREASE IN THEIR

MITOCHONDRIAL STABILITY SO WE

HAVE A PREFERENTIAL ELIMINATION

OF OLDER OR INSUFFICIENT

MITOCHONDRIA IN FAVOR OF A

T-CELL PHENOTYPE, A METABOLIC

ANTIA POP TO THETIC PHENOTYPE

WHICH SHOWN GIVE THESE IN RAP O

MICEIN AND INCREASED IN VIVO

FUNCTION.

ABILITY TO PROLIFERATE AND

SURVIVE IN VIVO.

SO WE ASK HOW ARE THESE GROWN IN

RAP O MICEIN, HOW DO THEY WORK

TO TREAT GRAFT VERSES HOST

DISEASE AND IT WAS A COMPLICATED

MECHANISM OF ACTION, USING

KNOCKOUT MICE, VERY BRIEFLY IF

APPROXIMATE YOU TAKE TH2 CELLS

GROWN IN RAPAMYCIN IF YOU

GENERATE THEM FROM AN IL10 KNOCK

OUT MOUSE, THEY DON'T WORK WELL

ANYMORE BECAUSE IL10 IS

IMPORTANT IN MODULATING THE HOST

APC, ASPECT GEN PRESENTING CELLS

THAT PREVENT ALOE ANTIGEN AND

SECRETE IL12, THESE ARE WHAT

DRIVES GRAFT VERSES HOST DISEASE

SO IF YOU BLOCK THESE THROUGH

IL10, SECRETION FROM T-CELL YOU

GET LESS GBHD AND THESE CELLS’I[

MAKE IL4, IF YOU USE AN IL4

KNOCK OUT, TH2 CELL, THESE IL4

WILL NOT BE PRODUCED AND YOU

WILL BE UNABLE TO MODULATE THE

EFFECT OF TH1, TC-1 CELLS THAT

WILL CAUSE GRAFT VERSES HOST

DISEASE AND THESE CELLS HAVE

HIGH LEVELS OF CD25.

THE IL2 RECEPTOR.

THEY'RE AVID SYNCH FOR IL2,

ESSENTIALLY STARVING THESE CELLS

FROM THE IL2 THAT'S REQUIRED FOR

THEM TO PROPAGATE GBHD.

SO A MULTIFACETED MECHANISM

WHEREBY THESE CELLS MAY BE

IMMUNO REG LATTER SCHEIN

ADDITION, THESE CELLS WE HAVE

SHOWN BY MAKING IL4 CAN BE HOST

T-CELLS, THE PRECURIOUSOR CTL,

SO THE IL4 BINDS TO THE IL4

RECEPTOR AND ACTIVATES STAT 6

AND THESE HOST T-CELLS ADOPT A

TH2 PHENOTYPE AND THEREFORE THIS

TH2 PHENOTYPE IS---WE HAVE SHOWN

LESS ABLE TO MEDIATE A REJECTION

RESPONSE AGAINST THE STEM CELLS.

SO MOVING TO CLINICAL

TRANSLATION, I WANT TO POINT

THIS OUT WHY WE'RE INTERESTED IN

THE LOW INTENSITY TRANSPLANT SO

AS WE'RE DEVELOP THANKSGIVING

NOVEL T-CELL THERAPY, TO USE IN

THE SETTING OF ALOE GENERATED

AIC TRANSPLANT IF YOU LOOK AT

THE NONHODGE KIN LYMPHOMA WHICH

IS THE PRIMARY DATIONIS WE

TREAT, IF YOU LOOK AT DATABASE A

COUPLE THINGS COME TO BEAR.

IN THIS TIME IRPT VAL YOU HAVE A

CERTAIN INCIDENCE OF LYMPHOMA

AND IN THIS RECENT INTERVAL YOU

SEE A HER INCIDENCE THIS, IS 1

OF THE CANCERS THAT IS

INCREASING IN INCIDENCE OVER THE

LAST 30 OR 40 YEARS.

THE OTHER THING IS IF YOU LOOK

AT AGE OF DIAGNOSIS, MOST OF

THESE PATIENTS, 80 OR 90% ARE

OVER THE AGE OF 50.

NOW THAT'S IMPORTANT BECAUSE

WHEN YOU LOOK AT ALOE

GENERATEDAIC TRANSPLANT, THE

TRADITIONAL WAY IS TO DO IT

THROUGH A MILE O ABLATIVE

PROCEDURE THAT IS GIVING A

PATIENT HIGH DOSES OF CHEMO

THERAPY AND TOTAL BODY RADIATION

AT THE AGE OF 50 IS A CUT OFF IN

THAT THE MORTALITY AND MORBIDITY

FROM A PLAYED PANZ PLANT

INCREASES DRAMATICALLY SO IF

WE'RE GOING TO ADDRESS

REFRACTORY LYMPHOMA FOR THE

MAJORITY OF PATIENTS WE FEEL WE

HAVE TO GIVE SAFER TRANSPLANT

PROCEDURES AND WE THOUGHT THAT

THE LOW INTENSITY TRANSPLANT

WOULD BE A GOOD SETTING TO START

THERE.

SO WE DEVELOP THIS REGIMEN HERE

SO WE NOW COMPLETED A PHASE 2

CLINICAL TRIAL, USING THIS

REGIMEN, IT'S A LOW INTENSITY

TRANSPLANT THAT, IS WE TAKE

PATIENTS WITH HEMOTO LOGIC

DISEASE, MEETLY IMFOAMA, MOST

PATIENTS COME IN WITH A FAIRLY

LOW IMMUNE SIZE, SO THE RISK OF

GRAFT REJECTION IS SOMEWHAT

MITIGATED, AND BEFORE THE

TRANSPLANT WE GIVE THESE 2 DRUGS

FLU DERRA BINE AND CYCLOPHOS

FOCUSED ON MID, THE DOSE IS

SHOWN HERE, THE 1000-MILLIGRAM

FOR 2-METER SQUARED THAT IS A

75% REDUCTION COMPARED TO THE

EXAMPLE WE USED 12 OR 14 YEARS

AGO.

SO IT'S A VERY LOW DOSE OF CHEMO

THERAPY AND ACTIVITIES AND

PROJECTS THAT'S CAN BE

ADMINISTERED IN THE OUTPATIENT

SETTING.

SO LOW INTENSITY TRANSPLANT.

WE GIVE CYCLOSPORIN TO THE

PATIENTS BEFORE THE TRANSPLANT.

GIVE A SHORT COURSE OF RAP O

MICEIN.

ALSO KNOWN AS SEROLIMEUS THROUGH

DAY 14 OF THE TRANSPLANT.

THE IDEA IS TO GIVE 2 AGENTS TO

PREVENT GBHD MEDIATED BY THE

T-CELLS THAT ARE CONTAINED IN

THE PERIPHERAL BLOOD SEMESTER

CELL PRODUCTS SO WE GIVE THE

DONORS GCSF TO MOBILIZE STEM

CELL AND WITHIN THAT PRODUCT

THERE ARE VERY LARGE AMOUNTS OF

T-CELLS SO WE USE 2 AGENTS TO

PRY TO PREVENT GBHD FROM THIS

PROD AND YOU CAN THEN AT DAY 14

POST TRANSPLANT WE ADMINNISTER

THE T-RAPID CELLS OR OTHER

T-CELLS THAT WE MANUFACTURE FROM

THE DONOR OF THE CD4 CELLS AND

WE GIVE THEM AT THIS DOSE SHOWN

THERE.

SO THAT'S OUR CLINICAL DESIGN SO

HOW DO WE MANUFACTURE THESE

CELLS, WE MIMIC STUDIES, THIS IS

ALL DONE IN THE DEPARTMENT OF

TRANSFUSION MEDICINE THROUGH OUR

COLLABORATORS THERE.

WE PURIFY CDCELLS THROUGH A

COLUMN WE USE CLINICAL GRADE AND

BEADS TO ACTIVATE THESE T-CELLS,

WE ADD CLINICAL GRADE CYTOKINES

TO POLARIZE AND ADD A DOSE OF

RAPAMYCIN, AND GROW THESE IN A

CULTURE BAG AND THESE WERE GROWN

FOR 12 DAYS, WE CRYOPRESERVE THE

PRODUCT AND MAKE SURE THEY'RE

GOOD FOR INJECTION AND THEN WE

CAN USE THEM HERE AND INTO OR A

MULTICENTER STUDY WHERE WE CAN

EXPORT TECHNOLOGY AND BROUGHT

COLTO MULTICENTER SITUATION TO

TRY TO GET OUR TRANSPLANT

PERFORM TED OUT THERE IN THE

COMMUNITY.

SOPHISTICATED WHAT DO THESE LOOK

LIKE.

THAT'S BASED ON CLINICAL

PRODUCTS AND CAN YOU LOOK AT

TRANSCRIPTION FACTORS THAT

DICTATE THE TH1 AND T

HPHENOTYPES SO EACH 1 OF THESE

REPRESENTS A CLINICAL PRODUCT

AND THE GATTA 3 IS THE TH2

TRANSCRIPTION FACTOR.

YOU SEE A LOT OF CELLS POSITIVE

ON AVERAGE, T-BET IS A TH1

TRANSCRIPTION FACTOR.

STILL QUIT A BIT OF POSITIVITY,

IT'S A MIX OF TH1 AND 2 CELLS.

THEY'RE NOT HIGHLY PURIFIED FOR

TH2 BUT A SUITABLE MIXTURE AND

Foxp3 IS A REGULATORY T-CELL

MARKER AND NOT A LOT OF TREG

TYPE PHENOTYPE IN THESE

PRODUCTS.

TAKE A MORE INDEPTH LOOK AT

THESE PRODUCTS INFUSED IN THE

PATIENTS AND WHAT YOU CAN SEE,

IS A BASIC NIGHT AND DAY

DIFFERENCE BETWEEN THE CELLS AT

THE TIME OF THEIR INPUT INTO

CULT AND YOU ARE AFTER THEY 12

DAY CULT NUR RAP O MICEIN ABOUT

20% OF THE MRNASPECIES ARE

DRAMATICALLY DIFFERENT AFTER YOU

CULTURE THE CELLS IN RAP O

MICEIN SO VERY BROAD CHANGES TO

THE CELLULAR POPULATION, YOU CAN

SEE ABOUT AN EQUAL NUMBER OF

GENEP GENES UPREGULATE INDEED

RAP O MICEIN AND EQUAL NUMBER

DOWN REGULATED AND WHEN YOU LOOK

AT THE GENES THAT ARE

UPREGULATED, IT'S INHIBITION OF

THE CELL CYCLE, INDUCTION OF A

METABOLISM, AND MAY BE IMPORTANT

IN THESE CELLS SURVIVING IN VIVO

AND TUMOR ENVIRONMENT.

AND INCREASED STABILITY TO

HANDLE A STRESS RESPONSE.

GENES THAT ARE DOWN REGULATED

ARE THE T-CELL ACTIVATION GENES,

APOPTOSIS GENES AND INFLAMMATORY

GENES.

WE DID 40 PATIENTS USING THIS

PLATFORM AND FOUND IT TO BE SAFE

RELATIVE TO OUR PRIOR TRANSPLANT

METHODS.

THE MAIN PROBLEMS IN TRANSPLANT

ARE SHOWN HERE, OCCLUSIVE

DISEASE INGRAFTMENT SYNDROME,

TRANSPLANT ASSOCIATED

MICROANGIOPATHY, TRANSPLANTED

RELATED MORTALITY, VERY LITTLE

IF ANY OF THESE EVENTS ON THIS

PHASE 2 STUDY.

IN TERMS OF GRAFT VERSES HOST

DISEASE, WE ALSO SAW WHAT WE

THOUGHT WAS A MUTED GBHD EFFECT

USING THIS PROD COL.

WE SAW 1 CLASS OUT OF 40 ACUTE

GBHD AND WE SAW 3 CASES TO

FURTHER DONOR LYMPHOCYTE

INFUSIONS AND WE SAW HERE

CHRONIC GHHD TO A THOIRD A HALF

WITH THE CHRONIC GBHD SO

RELATIVE TO HISTORICAL DATA WE

THOUGHT THIS WAS A VERY SAFE

TRANSPLANT PLATFORM AS WE

THOUGHT WE WERE DESIGNING.

AND WE FOUND TO BE VERY

EFFECTIVE IN MANY OF OUR

PATIENTS IN PARTICULAR, PATIENTS

WITH LOW GRADE LYMPHOMA NOT SO

EFFECTIVE FOR HIGH GRADE

LYMPHOMA, PATIENTS WITH CHEMO

THERAPY REFRACTORY DISEASE.

HAD ONLY ABOUT A 20% CHANCE OF

ENTERING INTO A COMPLETE

REMISSION, SO WE KNEW WE HAD

WORK TO DO.

IN TERMS OF ADDRESSING CERTAIN

PATIENT POPULATIONS, SO WE

MODIFIED OUR CULTURE SYSTEM HERE

INSTEAD OF A 12 DAY CULTURE

SYSTEM, WE FOUND THAT A 6 DAY

CULTURE SYSTEM IN RAP O MICEIN

WAS POTENTIALLY MORE ADVAPT

AGEIOUS.

WHY DO WE BELIEVE THAT?

WELL WHEN YOU COMPARE A 6 DAY

CULTURE PRODUCT TO THE 12 DAY,

THEY HAVE A MORE ANTIAPOPTOTIC

PHENOTYPE AND MORE OF AN

APOPTOTIC PHENOTYPE SHOWN HERE.

LESS CELL DETH--DEATH AND

THEREFORE BASED ON THESE STUDIES

SHOWING A POTENTIALLY DIFFERENT

CELL PRODUCT WE HAVE NOW

COMPLETED ANOTHER SEQUENTIAL

PHASE 2 STUDY WHERE WE KEPT ALL

THE VARIABLES THE SAME, THESE 40

PATIENTS I SHOWED YOU HAVE THE

12 DAY MANUFACTURE AND THEN

COMPARING OUR OTHER RAPAMYCIN

RESISTANT T-CELL PRODUCT.

THIS IS 1 EXAMPLE A PATIENT

RECEIVING A CELL, WITH

REFRACTORY LYMPHOMA NEVER

RESPONDED TO CHEMO THERAPY,

THESE ARE THE 4 DOSE HIGH

THROUGHOUT PUT WE ADMINISTERED,

NO SHRINKAGE OF TUMOR, WE TAKE

AROUND A PROTOCOL AND GIVE A LOW

DOSE OF CHEMO THERAPY AND THEN

GIVE RAP O--RAPAMYCIN TRANSPLANT

AND WE SAW DRAMATIC ABET TUMOR

RESPONSE.

ONE MONTH POST TRANSPLANT YOU

CAN SEE A LESION HERE IN THE

MEDIA SIGN UMPIRES ALREADY

HAVING REDUCTION BY PET SCAN AND

A LITTLE BIT BY CAT SCAN BY DAY

60 THIS TUMOR IS COMPLETELY PET

SCAN NEGATIVE AND THIS IS SCAR

PATIENT'S NOW A WE'RE AND HALF

POST TRANSPLANT.

SO WE BELIEVE IN THIS CASE AND

THEN IN THE OTHER PATIENTS WE'VE

CREATED, YOU CAN SEE HERE, THE

DIKES SAY RECIPIENTS WHEN WE

LOOK AT OVERALL SURVIVAL AND

PATIENTS SURVIVE NOTHING

COMPLETE RESPONSE, WE BELIEVE

WE'RE DOING BETTER THAN PATIENTS

THAT RECEIVED OUR 12 DAY

MANUFACTURED CELL.

SO BY MAKING THESE 7LE CHANGES

IN OW WE MANUFACTURE OURSELVES,

WE THINK WE CAN GET A BETTER

CLINICAL RESULT.

THESE ARE SEQUENTIAL STUDIES

ISSUES THESE ARE NOT RANDOMIZED,

THERE'S CAUTION THERE, BUT WE

CERTAINLY FEEL WE'RE MOVE

NOTHING THE RIGHT DIRECTION.

SO JUST TO FINISH UP, THEN WHAT

ELSE CAN WE DO WITH THESE

RAPAMYCIN RESIST ABT T-CELL, WE

CAN DO FURTHER CLINICAL TRIALS

AND MOVING TO MAKE THE

CONDITIONING REGIMEN LESS

INTENSIVE AND WE CAN DO

DEDICATED STUDIES AND SPECIFIC

DISEASE.

TRY TO DO RANDOMIZED STUDIES AND

SO ON AND SO FORTH BUT I WANT TO

TALK A LITTLE BIT ABOUT ANOTHER

DIRECTION THAT WE'RE GOING AND

THAT IS GENE MODIFICATION OF

THESE CELLS.

FOR EXAMPLE, CHRONIC GBHD MAY BE

MEDIATED BY THESELES CAUSING

AUTOIMMUNITY.

IF WE MOVE TO A HIGHER RISK

TRANSPLANT SETTING SUCH AS

UNRELATED DONOR OR MISMATCHED

SETTING WE CAN HAVE HIGHER

PROBLEMS WITH GBHD, SO IF WE'RE

ABLE TO PUT IN A CONTROL GENE,

SO CALLED SUICIDE GENE, WHICH

WOULD ALLOW US TO CONTROL THE

FATE OF THESE CELLS, WE WOULD BE

ABLE TO INCREASE FURTHER, THE

SAFETY OF THIS TRANSPLANT

METHODOLOGY AND WHAT WE

DEVELOPED IS AN ENZYME MUTATED

HUMAN TMPK WHICH I'LL TALK ABOUT

THAT'S A FUSION PROTEIN WITH

CD19 ON THE SURFACE SO WE CAN

SELECT THESE CELLS AND BY

PUTTING THIS ON A LENGTHY VIRAL

BACKBONE WE CAN GET THIS GENE

INTO OUR T-RAPID CELLS TO

CONTROL THE FATE OF THESE CELLS

IN VIVO SO WE TURN ON TRANSPLANT

RESPONSE AND THEN BY ENCODING

FOR CELL FATE GENE GET RID OF

THOSE CELLS IN VIVO.

AND THEN IN ANOTHER GENERATION

WE HAVE ANOTHER IN GETTING NOT

ONLY THE CELL FATE GENE BUT ALSO

THE PROTEIN DELIVERY GENE SUCH

AS PROGRAM 1 LIGAND GENE TO GIVE

THESE CELLS AN ADDITIONAL

FUNCTIONAL CHARACTERISTIC.

NOW WHAT IS THE TMPK, THIS IS

HUMAN KINASE GENE, MUTATED BY MY

COLLABORATOR, DR. METRICS DINE

AT TORONTO TO BE OPTIMIZED.

OPTIMIZED FOR PHOSPHORYLATION OF

AZT.

SO AZT IS A DRUG THAT'S FDA

APPROVED, DEVELOPED HERE IN THE

NCI, THIS ENZYME HERE AS LITTLE

IF ANY IMMUNO GENERATEDISSITY

SINCE IT'S HUMAN.

AND AZT, OF COURSE IS MINIMALLY

TOXIC TO CELLS THAT DO NOT

EXPRESS TMPK AND AZT WILL INDUCE

APOPTOSIS OF DIVIDING AND

NONDIVIDING CELLS SO THIS MAY BE

A GOOD CELL FATE OR SUICIDE GENE

AXIS AND WE LOOK AT THIS IN OUR

HUMAN T-RAPID CELL, THESE ARE

CELLS THAT ARE EXPRESSING A

CONTROLULENTY VIRAL GENE, GFP,

YOU CAN SEIZE THEE RAPID CELLS

IN AZT, THEY'RE GROWING JUST

FINE.

NAZT, AND WHEN THEY HAVE THE

TMPK GENE, THE TMPK GENE IS

ACTIVATING THE AZT AND

PHOSPHORYLATING AND AND THESE

ARE GOING THROUGH DEATH OVER

TIME AND YOU SEE THERE'S CELL

EFFECT AND YOU INCREASE THE

DOSE, THE CELLS ARE GROWING FINE

AND THESE CELLS ARE DYING WITHIN

2 DAYS OF AZT EXPOSURE.

SO WE THINK WE CAN CONTROL THE

FATE OF THESE T HAPPEN

EDUCATIONAL CELLS AND WE LOOK AT

THIS IN VIVO MODEL WHERE WE GROW

THE T-RAPID CELLS AND INSERT THE

CD19 AND TMPK TRANSGENE SO THESE

HAVE THE CELL FATE OR SUICIDE

GENE AND INJECT THEM INTO IMMUNO

DEFICIENT MICE SO THIS IS A

HUMAN IS A MOUSE EXTEN O

GENERATED AIC TRANSPLANT MODAND

HE WILL THEN IN THE FIRST WEEK

AFTER TRANSPLANT WE INJECT AZT

AT DOSES THAT ARE KNOWN TO BE

SAFE TO THE MICE AND ALSO

CLINICALLY ACHIEVABLE.

AND THEN WE CAN LOOK AT THE

SPLEEN FOR INGRAFTMENT OF ANY

CELLS THAT XREASES THE FUSION

PROTEIN.

--EXPRESS FUSION PROTEIN.

THIS IS THE RESULT WEB TAN.

WHAT YOU SEE IS YOU CAN LOOK AT

THE NUMBER OF HUMAN T-CELLS IN

THE SPLEEN OF THESE MICE IN

CONTROL INFECTION AND YOU SEE

ROBUST INGRAFTMENT OF THE

T-RAPID CELLS WHERE THIS IS THE

MICE IS CO ADMINISTER WIDE AZT,

WE HAVE ABOUT A 2 OR 3 LOG

REDUCTION IN THE HUMAN T-CELLS

SO WE THEN IS INITIAL DATA TO

TRY TO MOVE FORWARD TO FURTHER

CLINICAL TRIALS WOULD BE ABLE TO

TURN ON AND OFF THIS CELL

PRODUCT ONCE IT'S INFUSED INTO

PATIENTS.

I'D LIKE TO END HERE WITH A

CONCLUSION THAT BY STAFFING

T-CELL THREW'S RAPAMYCIN WE CAN

INDUCE AUTOPHAGY, WE CAN GET

TH1, TH2 DIFFERENTIATION AND GET

IN VIVO EFFICACY INCREASED AND

HAS ALLOWED US TO MOVE AWAY FROM

CHEMO THERAPY INTENSITY TO WHAT

WE TERM T-CELL INTENSITY

INFUSING MORE ADDRESSIVE T-CELLS

AND THIS CAN BE USED AS A

CELLULAR VEHICLE FOR GENE

THERAPY.

AND I WOULD LIKE TO THANK SO

MANY PEOPLE HERE AT THE NCI AND

THE CLINICAL CENTER AND BENCH TO

BODY SIDE PROGRAM AND MANY OF MY

COLLABORATORS AND OF COURSE

TRANSFUSION MEDICINE, DEPARTMENT

HERE AT NIH.

AND MOST IMPORTANTLY TO ALL THE

PATIENTS AND THEIR FAMILIES,

THANK YOU.

[ APPLAUSE ]