Welcome to the Clinical Center Grand Rounds, a weekly series of educational lectures for physicians and health care professionals broadcast from the Clinical Center at the National Institutes of Health in Bethesda, MD. The NIH Clinical Center is the world's largest hospital totally dedicated to investigational research and leads the global effort in training today's investigators and discovering tomorrow's cures. Learn more by visiting us online at http://clinicalcenter.nih.gov >> GOOD AFTERNOON. WELCOME TO THE CLINICAL CENTER GRAND ROUNDS. THE HAVE THE DISTINCT OF PRIVILEGE OF INTRODUCING TWO OF THE ALL-STARS OR HALL OF FAME INVESTIGATORS HERE AT THE CLINICAL CENTER WHO ARE GOING TO TELL YOU VERY EXCITING STORIES ABOUT USE OF GENES AND CELLS IN THE -- WHAT WE MIGHT CALL PRECISION MEDICINE. THE FUTURE OF THERAPY. SO OUR FIRST SPEAKER IS HARRY MALECH, WHO HAPPENS TO HAVE BEEN A COLLEAGUE OF MINE SINCE I'VE BEEN HERE AT THE NIH. I WON'T SAY WHEN. BUT HARRY IS NOW CHIEF OF THE -- HIS INTERNSHIP AND RESIDENCY AT THE UNIVERSITY OF PENNSYLVANIA. 1974 AS A RESEARCH FELLOW WITH THE CELL BIOLOGY -- IN 1978, AN ASSOCIATE PROFESSOR OF MEDICINE AT YALE IN 1983. HE CAME BACK TO THE NIH IN 1986, JOINING THE LAB I WAS IN, AND THEN THE OCI AND NIAID AND A FEW YEARS LATER, A LABORATORY OF HOST DEFENSES WAS CREATED AND LARRY -- HARRY BECAME THE DEPUTY CHIEF OF THAT LABORATORY AS WELL AS CHIEF OF THE GENETIC IMMUNOTHERAPY SECTION, AND HE BECAME CHIEF OF THE OF THE LABORATORY IN 2003. DR. MALECH CARES FOR PATIENTS WITH SEVERE IMMUNODEFICIENCIES, AND HE'S SPENT A LOT OF TIME FOCUSING ON CHILDREN WHO HAVE SERIOUS IT DISORDERS SUCH AS CHRONIC GRANULOMATOUS DISEASE. HE CURRENTLY FOCUSES MUCH OF HIS WORK ON ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANT AND EX VIVO GENE THERAPY FOR CGD, AS WELL AS THE SEVERE COMBINED IMMUNODEFICIENCY AND OTHER IMMUNODEFICIENCIES. HIS RESEARCH IS FOCUSED ON ACHIEVING EFFICIENT GENETIC CORRECTION OF THE PATIENT'S HEMATOPOIETIC STEM CELLS. HE'S SERVED ON NUMEROUS EDITORIAL BOARDS INCLUDING BLOOD AND THE EUROPEAN JOURNAL OF HEMATOLOGY, AND HE'S WORKED WITH THE JCI AND ALONG THE WAY, HE'S RECEIVED MANY AWARDS, INCLUDING THE NIH DIRECTOR'S AWARD. HE'S BEEN ELECTED TO THE ASCI AND THE AAP, AND HE SERVED AS PRESIDENT OF THE AMERICAN SOCIETY OF GENE AND CELL THERAPY, WHICH HE COMPLETED JUST THIS PAST MAY. TODAY HE'S GOING TO TELL US ABOUT HIS WORK TI TITLED LENTIVECTOR GENE THERAPY FOR X-LINKED SEVERE COMBINED IMMUNODEFICIENCY IN OLDER CHILDREN AND YOUNG ADULTS WITH IMPAIRED IMMUNITY AND RECURRENT INFECTIONS DESPITE PRIOR HAPLO-TRANSPLANTS. HARRY, WELCOME. [APPLAUSE] >> THANK YOU FOR THAT KIND INTRODUCTION, AND THANK YOU FOR INVITING ME TO SPEAK. I HAVE NO DISCLOSURES, AND I WILL BE TALKING ABOUT AN UNAPPROVED GENE THERAPY PRODUCT. I WON'T READ ALL THESE EDUCATIONAL OBJECTIVES BUT HOPEFULLY WE'LL ACHIEVE ALL OF THEM DURING THIS BRIEF TALK. X-LINKED SEVERE COMBINED IMMUNE DEFICIENCY IS A PROFOUND LACK OF T, B AND NK CELL IMMUNITY. T CELLS AND NK CELLS ARE ABSENT, B CELLS ARE PRESENT, THOUGH OFTEN SMALL NUMBERS, BUT LACK FUNCTION. THEY GET RECURRENT INFECTIONS, SEVERE DEFICIENCY IS FATAL IN INFANCY WITHOUT SOME TREATMENT PROVIDING IMMUNE RECONSTITUTION, AND XSCID IS THE MOST COMMON TYPE OF SCID. THE GENE DEFECT IS DUE TO MUTATIONS IN THE INTERLEUKIN RECEPTOR G GENE THAT ENCODES THE COMMON GAMMA CHAIN REQUIRED FOR INTRACELLULAR SIGNALING BY THIS WHOLE GROUP OF RECEPTORS. NEWLY DIAGNOSED SKID IN INFANCY REQUIRES URGENT MEASURES TO CONTROL INFECTION AND PROVIDE HEMATOPOIETIC STEM CELL TRANSPLANT TO RESTORE IMMUNITY, TRANSPLANT MARROW CONDITIONING IS NOT REQUIRED AND GENERALLY HAS NOT BEEN USED. AN HLA MATCHED SIBLING TRANSPLANT IS THE CURRENT TREATMENT OF CHOICE BUT MOST LACK A MATCHED SIBLING. HAPLO-IDENTICAL T CELL DEPLETED BONE MARROW TRANSPLANT FIRST BEGAN IN THE EARLY 1980s, FROM A PATIENT WITHOUT CONDITIONING CAN BE LIFE SAVING, AND IF THIS IS DONE IN THE FIRST THREE MONTHS OF LIFE BEFORE OPPORTUNISTIC INFECTIONS, SURVIVAL CAN BE 90%. HOWEVER, THERE ARE LIMITATIONS TO T-DEPLETED PARENT-DERIVED HAPLOIDENTICAL BONE MARROW TRANSPLANT WITHOUT CONDITIONING IN THAT PLEAS LITTLE OR NO INGRAFTMENT LONG TERM OF DONOR HEMATOPOE IHEMATOPOIETIC STEM CELLS, AND YOU CAN SEE MYELOID CELLS ARE ONLY FROM THE PATIENT, DONOR GRAFT IS LI.ED T LIMITED TO T CELLS, DERIVED FROM THE MOTHER, X CHROMOSOME ONLY. THERE ARE FEW OR NO DONOR B CELLS AND FEW OR NO DONOR NK CELLS, YOU CAN SEE A TINY BIT OF DONOR B CELLS BUT IN MOST PATIENTS IT LOOKS LIKE THIS, SO THERE'S IN B CELL DONOR INGRAFTMENT AS WELL. UNFORTUNATELY, ALTHOUGH THIS IS LIFE SAVING IN INFANCY, AS CHILDREN GET OLDER, THERE'S PROGRESSIVE DECREASE IN DONOR T CELL NUMBER AND FUNCTION. THE LATE CONSEQUENCES OF THIS INCOMPLETE IMMUNE RECONSTITUTION IS THAT PATIENTS REQUIRE LIFE-LONG IGG SUPPLEMENTATION, JUST TO REMIND YOU, THAT THIS COSTS MAYBE NOT THEM BUT CERTAINLY SOCIETY ANYWHERE FROM 30 TO $50,000 A YEAR. AND THIS DOESN'T ACCOUNT FOR HOSPITALIZATIONS AND OTHER PROBLEMS THAT THEY MAY HAVE. THEY GET RECURRENT BACTERIAL AND VIRAL INFECTIONS, SKIN WARDS AND MOLLUSCUM, AND PROGRESSIVE BRONCHIECTASIS AND PULMONARY DYSFUNCTION. THEY HAVE AUTO AND ALLO-IMMUNE RASHES, SOME ELEMENT OF CHRONIC INFLAMMATORY BOWEL DISEASE WITH MALL ABSORPTION, DIARRHEA AND IMPAIRED NUTRITION. SOME MAY HAVE PROTEIN LOSING ENTEROPATHY THAT CAN BE SO SEVERE THAT IT GREATLY INCREASES THE FREQUENCY AND DOSING REQUIREMENTS FOR IGG. SOME HAVE VERY SIGNIFICANT FAILURE IT TO GROW. SO IN THE LAST 10 TO 15 YEARS, THERE'S BEEN DEVELOPED GENE THERAPY USING MURINE RETROVIRUS VECTOR WITHOUT CONDITIONING, TRANSFERRING THIS VECTOR IN AUTOLOGOUS MARROWCD34 HEMATOPOIETIC STEM CELLS. TREATMENT IN PARIS AND LONDON, THE PROBLEM IS THAT VECTOR INSERTIONAL MUTAGENESIS DUE TO THE ACTIVATOR REGIONS CAUSED FIVE TO DEVELOP T CELL LYMPHOPROLIFERATIVE DISORDER. HOWEVER YOU LOOK AT THE LONG PICTURE, 13 ACHIEVED EVENT-FREE T CELL CORRECTIONTION THAT WAS CLINICALLY BENEFICIAL, THE FOUR WHO DEVELOPED LEUKEMIA ACHIEVED REMISSION WITH CONTINUED GENE MEDIATED T CELL CORRECTION. ONE DID DIE OF THE T CELL LEUKEMIA AND TWO DID NOT ACHIEVE SIGNIFICANT BENEFIT REQUIRING TRANSPLANT. VERY RECENTLY, ANOTHER STUDY USING A MURINE GAMMA RETROVIRUS BUT REMOVING SOME OF THE ACTIVATOR AND ENHANCERS FROM THE LTR SO THE VIRUS IS WHAT'S CALLED SELF INACTIVATING -- WITH SIMILAR OUT COME LIMITED TO T CELL IMMUNITY. SO IF YOU ADD IT ALL UP, 26 OF 29 INFANTS TREATED WITH RETROVIRUS GENE THERAPY WITHOUT CONDITIONING IT DID BENEFIT FROM THE GENE TRANSFER. BUT THERE WAS NO B CELL MARKING, POOR OR NO CORRECTION OF HUMORAL IMMUNITY AND NO CORRECTION OF NK CELL. DURING THIS ERA, WE AND ADRIAN THRASHER FROM LONDON SAID TO OURSELVES, WELL, THIS WORKED IN INFANTS, LET'S TRY THESE OLDER PATIENTS SO WE'RE SORT OF -- THAT HAVE IMMUNE DYSFUNCTION DESPITE THE HAPLO-TRANSPLANT. ONLY ONE UNFORTUNATELY ACHIEVED ANY SUBSTANTIAL GENE MARKING AND DESPITE THAT GENE MARKING, CONTINUED TO HAVE INFECTIONS, FAILURE TO GROW AND NUTRITIONAL DEFICIENCIES, AND HE SUBSEQUENTLY HAD A SUCCESSFUL MATCHED UNRELATED DONOR TRANSPLANT, FULLY CONDITIONING TRANSPLANT BY US AT THE NIH. WE CON INCLUDE CLEUDED IN OLDER CHILDREN IT REQUIRES IMPROVED ME CAN TORE AND MARROW CONDITIONING. SO WE WORKED WITH OUR COLLEAGUES AT ST. JUDE TO DEVELOP A SELF ACTIVATED LENTIVECTOR, AS WITH ALL, A DEFECT IS MADE IN THE 3 PRIME REGION SUCHS THAT WHEN THE VIRUS INSERTS IN THE PATIENT'S CELLS, THIS IS BROUGHT OVER TO THE FRONT SO THAT YOU LACK A PROMOTER OR THE ACTIVATOR ACTIVITY OF THIS LTR. FOR THAT THEN, WE NEED AN INTERNAL PROMOTER, WHICH IN THIS CASE IS THE HUMAN ELONGATION FACTOR 1 SHORT PROMOTER. WE ALSO ADDED SOME INSULATORS, AND CODON OPTIMIZED THE HUMAN IL2 RECEPTOR GAMMA CHAIN CDNA. STUDIES WERE DONE TO SHOW THAT THIS VECTOR NO LONGER ACTIVATES ONCOGENES WHEN IT LANDS NEXT TO THESE ONCOGENES. AND ONE OTHER NEW FEATURE OF THIS IS THAT THE GROUP AT ST. JUDE HAVE DEVELOPED A STABLE PRODUCER LINE FROM WHICH LENTIVECTOR CAN BE PRODUCED. AND SO THIS IS THE FIRST IN HUMAN TRIAL OF LENTIVECTOR TO TREAT XSCID, LENTIVECTOR FROM A STABLE PRODUCER LINE, AND THE USE OF BUSULFANE CONDITIONING FOR X-LINKED SCID GENE THERAPY. JUST TO GO BACK TO THE SLIDE AND JUST POINT OUT THAT DR. DERAVEN IN OUR GROUP IS THE P.I. ON THE MEDICALLY RESPONSIBLE INVESTIGATOR, BRIAN SORENTINO FROM ST. JUDE IS THE OVERALL PROJECT LEADER AND THEY HAVE A PROTOCOL TO TREAT INFANTS WITH THIS SAME VECTOR, OPEN NOW AT ST. JUDE, THOUGH THEY HAVEN'T YET TREATED ANY INFANTS IN THEIR PROGRAM. I MENTIONED THE BUSULFAN, WE USE SOTHAT THE CELLS CAN SETTLE IN TO THE MARROW HOPEFULLY AND PROVIDE MORE BENEFIT THAN WE'VE SEEN WITHOUT CONDITIONING. SO THE GENE TRANSFER STANDARD OPERATING PROCEDURE IS THAT WE TAKE PERIPHERAL BLOOD MOBILIZE 34 CELLS, WE PUT THEM IN CULTURE, WE EXPOSE THEM TO THE LENTIVECTOR TWICE ON CULTURE DAY, ONE AND TWO, AND THE MORNING OF DAY THREE, THESE CELLS ARE HARVESTED, WASHED, AND THEN FUSED IN THE PATIENT WHO HAS BEEN CONDITIONED WITH BUSULFANE. JUST TO RETURN THEN TO THE TRIAL, I WON'T READ ALL OF THESE, BUT BOTH THE FDA AND THE RAC AND OTHER REGULATORY GROUPS SAID, WELL, YOU KNOW, THIS IS NEW, WE'VE HAD BAD PROBLEMS WITH INFANTS, WE NEED TO HAVE YOUR SICKEST PATIENTS GO INTO THIS TRIAL. SO KEEP IN MIND THAT THESE PATIENTS WERE ONES WHO HAD MANY, MANY PROBLEMS, EVEN IF THEY HAD SURVIVED INTO THEIR YOUNG ADULTHOOD, THEY WERE QUITE ILL. SUBJECTS ONE AND TWO, WHO WERE TREATED A BIT MORE THAN TWO YEARS AGO, WERE BOTH YOUNG ADULTS, THEY WERE STATUS-POST T CELL DEPLETED BONE MARROW TRANSPLANT, HAPLO-TRANSPLANT WITHOUT CONDITIONING IN INFANCY, THEY HAD 100% DONOR T CELLS, NO 34 CELLS, NO MYELOID CELLS, NO B CELLS FROM THE DONOR, VERY FEW NK CELLS. SUBJECT ONE HAD SEVERE PROTEIN LOSING ENTEROPATHY, SUBJECT TWO HAD SUCH SEVERE BRONG YEK TA CYST THAT THEY HAD ONLY 20 TO 30% OF NORMAL LUNG FUNCTION, BOTH SUBJECTS HAD ABNORMALLY LOW BODY MASS AND LONG-STANDING CHRONIC NOROVIRUS INFECTION. NOW MOST RECENTLY, THIS YEAR, WE'VE TREATED THREE MORE PATIENTS. NOW THESE ARE YOUNGER CHILDREN, THEY'RE VERY SIMILAR IN THEIR CHARACTERISTICS TO THE OLDER PATIENTS. PATIENT FOUR HAD SEVERE PROTEIN LOSING ENTEROPATHY LIKE PATIENT ONE, SUBJECT THREE HAD ABNORMALLY LOW BODY MASS, AND SUBJECTS THREE, FOUR AND FIVE HAD LONG-STANDING CHRONIC GI NOROVIRUS INFECTION. NOROVIRUS INFECTION COMES AND IT STAYS, NEVER GOES AWAY IN THESE PATIENTS. SO SO THIS IS THE TRANSDUCKTION PRODUCT AT HARVEST SO EACH OF THESE PATIENTS RECEIVED A FAIR NUMBER OF AUTOLOGOUS STEM CELLS THAT WERE QUITE REASONABLY TRANSDUCED IN TERMS OF PERCENT TRANSDUCTION. THESE ARE THE DATES THAT THEY OCCURRED, YOU CAN SEE THAT THIS IS ALMOST THREE YEARS AGO, A LITTLE MORE THAN TWO YEARS AGO. EARLIER THIS YEAR, THEN VERY RECENTLY. I'M JUST GOING TO SHOW GRAPHIC DATA ON THE FIRST THREE PATIENTS BECAUSE THE LAST TWO PATIENTS ARE A LITTLE EARLY FOR THAT, BUT I DO HAVE SOME DATA TO SHOW ABOUT THOSE PATIENTS AS WELL. NOW, YOU CAN SEE THAT B CELLS ARE COMING UP AND K CELLS ARE COMING UP IN PATIENT ONE AND TWO. T CELLS ARE COMING UP ONLY MODESTLY. AND IF YOU LOOK AT GENE MARKING, THESE ARE A LITTLE BUSY SO I'M GOING TO POINT YOU DOWN HERE AND BASICALLY SAY THAT MYELOID CELLS, WHICH ARE A SURROGATE FOR LOOKING AT 34 CELLS, IN PATIENT ONE LONG TERM, AT 30 MONTHS, 24 MONTHS, 8 TO 12%. PATIENT THREE AT THREE MONTHS HAD 7%. WE HAD A NEW BATCH OF VECTOR TO TREAT PATIENTS FOUR AND FIVE THAT I THINK MADE A DIFFERENCE AND WE HAVE HIGHER TRANSDUCTION RATES IN PATIENTS FOUR AND FIVE, BUT YOU CAN SEE THAT IN EVERY SINGLE ONE OF THE PATIENTS, THERE'S A SELECTIVE OUTGROWTH OF MARKED B CELLS. THIS DID NOT OCCUR IN ANY OF THE INFANTS TREATED WITHOUT CONDITIONING. AS WITH THE INFANTS, THERE'S A DELAY IN T CELL MARKING. YOU CAN SEE THE EARLY PATIENTS STILL HAVE LOW T CELL MARKING, BUT SIMILAR TO THE FIRST TWO PATIENTS AT EARLY TIMES, AND THEN THESE NUMBERS COME UP AFTER ABOUT FOUR OR FIVE MONTHS. IN FACT, IN THESE PATIENTS, WAS PASSED SIX MONTHS THAT THESE CAME UP. NK CELLS IN FOUR OF THE FIVE PATIENTS ARE QUITE HIGHLY MARKED, AND I HAVEN'T LOOKED AT 34 CELLS YET IN THESE PATIENTS, BUT THE 34 CELLS TRACK THE MYELOID CELLS. SO WHAT HAPPENS TO THE DONOR T CELLS? REMEMBER, THESE PATIENTS ARE -- ANY HEALTH THEY HAVE IS BECAUSE THEY HAD THEIR DONOR T CELLS. WE HAD A LOT OF OPINIONS ABOUT WHETHER WE SHOULD HAVE KILLED OFF THOSE DONOR T CELLS, BUT WE FELT THAT THAT WAS NOT SAFE, SO WE IT DIDN'T USE ANY T CELL CONDITIONING AND HOPED THAT WHAT WOULD HAPPEN IS WHAT YOU SEE HERE, WHICH IS THAT DONOR T CELLS -- AS PATIENT T CELLS ARE COMING IN, THEY'RE DISPLACING THE DONOR MYELOID T CELL KIEMERISM -- THE DONOR KIEMERISM IS GOING DOWN. EQUALLY IMPORTANT IS THAT THOSE DONOR T CELLS AREN'T FUNCTIONING VERY WELL. THEY DON'T RESPOND TO TEC TETANUS, BUT AS THEY ACQUIRED AUTOLOGOUS T CELLS, THEY ACQUIRED TETANUS RESPONSES IN RESPONSE TO TETANUS IMMUNIZATION. THIS IS A SLIDE SHOWING AS YOU MIGHT EXPECT THESE OLD DONOR T CELLS HAVE VERY LOW TREKS AS T CELL EXCISION CIRCLE -- RESTRICTION EXCISION CIRCLES >> Dr. Anderson: THAT'S A MEASURE OF THYMIC FUNCTION AND ALSO COMING UP IN THESE PATIENTS INTO THE NORMAL RANGE IN PATIENT ONE AND TWO. THIS IS A BUSY SLIDE, AND I'M NOT GOING TO GO THROUGH THE WHOLE THING EXCEPT TO POINT OUT THAT AT THE START, EVEN AT SIX MONTHS IN THIS PATIENT ONE, THERE ARE NO IGG-PRODUCING B CELLS, THERE ARE FEW OR NO MEMORY B CELLS, BUT BY TWO YEARS OUT, THIS PATIENT HAS MEMORY B CELLS AND THEI THEY'RE IGG-POSITIVE. THIS IS TRUE IN PATIENT TWO AS WELL. PATIENT THREE, FOUR AND FIVE WERE BEGINNING TO SEE THE BEGINNINGS OF -- I DON'T HAVE TIME TO GO INTO IT BUT BEGINNING TO SEE SIMILAR KINDS OF CHANGES IN THESE PATIENTS. WHAT'S MOST MIRACULOUS TO US, BECAUSE WE TRULY DID NOT EXPECT THIS FROM THIS TRIAL, IS THAT THESE PATIENTS INITIALLY BEGAN MAKING IGM, PATIENT ONE, PATIENT TWO, EARLY ON, AND PATIENT THREE. AND THEN BEGAN TO MAKE SO MUCH IGG THAT THEY WERE OFF THEIR IGG SUPPLEMENTS, SUBJECTS ONE AND TWO RESPONDED NORMALLY TO TETANUS, DIPHTHERIA. ME NIN JOE COCCUS AND INFLUENZA IMMUNIZATION, SUBJECT ONE RESPONDED NORMALLY TO A RABIES VACCINE GIVEN UNDER ANOTHER IRB-APPROVED PROTOCOL, AND SUBJECTS ONE AND TWO MOST IMPORTANT OF ALL, AFTER MANY, MANY YEARS, CLEARED THEIR CHRONIC NOROVIRUS. SO WHAT ABOUT INTEGRATION? THIS IS JUST A COLORFUL SLIDE DEMONSTRATING THAT MARKING IS POLL OWE CLONAL WITHOUT EVIDENCE FOR ANY PERSISTENCE OF ANY CLONAL DOMINANCE. I MIGHT POINT OUT IN SUBJECT TWO WHO HAD TERRIBLE, TERRIBLE WARTS, HE ACTUALLY BEGAN TO CLEAR SOME OF THOSE WARTS, AND HIS MOLLUSCUM ALSO CLEARED. WE DO HAVE ONE UNFORTUNATE AND VERY SAD THING, WHICH IS THAT PATIENT TWO, WHO HAD VERY SEVERE BRONCHIECTASIS, SUDDENLY ABOUT 10 DAYS AGO, HAD A BLEED AND BLED OUT AND DIED. OBVIOUSLY THIS IS A TRAGIC THING FOR US, FOR FAMILY, FOR EVERYBODY. ONE OF THE THINGS HIS MOTHER SAID TO ME WAS WELL, HE HAD A YEAR AND A HALF WITHOUT HIS IGG AND IT MADE HIM REALLY HAPPY, BUT I WISH I'D GIVEN HIM A LOT MORE THAN THAT. SO THE OUTCOME OF SUMMARY OF ENT VECTOR TRIAL, EX VIVO GENE THERAPY, SIGNIFICANTLY IMPROVED BOTH CELLULAR AND HUMORAL IMMUNITY IN THE FORM OF IMPROVED T CELL FUNCTION, INCREASED NUMBERS OF NK CELLS AND INCREASED SERUM IGG IN ASSOCIATION WITH PROGRESSIVELY INCREASING B CELL MARKING. THERE'S EVIDENCE FOR B CELL CLASS SWITCHING, AND RESPONSES TO IT IMMUNIZATIONS, SUBJECTS THREE I MENTIONED IGM IS BEGINNING TO COME UP. THIS UNEQUIVOCALLY PROVEN LEVEL OF REEF CONSTITUTION OF HUMORAL IMMUNITY IS UNIQUE TO THE STUDY AND HAS NOT BEEN OBSERVED IN ANY PREVIOUS STUDY OF GENE THERAPY FOR XSCID. AND IN FACT, I MIGHT ADD, THE OTHER PEOPLE DOING STUDIES IN INFANTS HAVE CHANGED THEIR PROTOCOLS AND GOTTEN FDA PERMISSION TO GET SOME CONDITIONING SO AS TO TRY TO MATCH THIS IN THE INFANTS. BOTH SUBJECTS HAD WEIGHT GAIN. SUBJECT ONE HAD CORRECTION OF HIS LONG TERM CHRONIC PROTEIN-LOSING ENTEROPATHY. BOTH PATIENTS AS I MENTIONED CLEARED THEIR CHRONIC NOROVIRUS INFECTION. I MENTIONED THE WARTS AND THE MOLLUSCUM. SO IN SUMMARY, LENTIVECTOR GENE THERAPY WITH CONDITIONING FOR OLDER XSCID SUBJECTS SEEMS TO BE SAFE AND WELL TOLERATED, RESULTS IN PERSISTENT MULTI-LINEAGE GENE MARKING, IMPROVED CELLULAR AND HUMORAL IMMUNITY, SUSTAINED IMPROVEMENT OF CLINICAL STATUS. HOWEVER, GENE THERAPY CANNOT REVERSE SEVERE DAMAGE TO LUNGS AND IS A RATIONALE FOR RECOMMENDING EARLY INTERVENTION TO CORRECT HUMERAL AND NK IMMUNITY AND XSCID BY USE OF BUSULFAN CONDITIONING WITH GENE THERAPY BEFORE IRREVERSIBLIBLE DAMAGE OCCURRED. I'M HOPE THE GROUPS THAT ARE TREATING INFANTS WILL TREAT ALL INFANTS SO I WILL HAVE NO MORE OLDER PATIENTS TO TREAT WHO ARE SICK. I'LL THANK THE PATIENTS AND THEIR CURRENT PRIMARY PHYSICIAN, AND I'LL SHOW MY CREDITS. THANK YOU VERY MUCH. [APPLAUSE] >> THANK YOU, HAIR EE. WE HAVE TIME FOR A FEW QUESTIONS. IF YOU HAVE ANY, PLEASE USE THE MICROPHONES. I'LL START OFF WITH A QUESTION. YOU COMMENTED ABOUT BONE MARROW TRANSPLANT FOR THE -- SOME OF THE PATIENTS WHO DIDN'T BENEFIT. DO YOU THINK WE NEED TO DO A HEAD TO HEAD COMPARISON OF GENE THERAPY IN BONE MARROW TRANSPLANT AS YOU LOOK INTO THE FUTURE FUTURE? >> THE QUESTION ANSWER IS YES, I THINK THEY SHOULD BE COMPARED AS COMPARISONS. OUR GROUP IS PART OF A NORTH AMERICAN PRIMARY IMMUNE DEFICIENCY TREATMENT CONSORTIUM, WHICH IS VERY INTERESTED IN LOOKING AT AND COMPARING HOW PATIENTS DO WITH DIFFERENT KINDS OF TRANSPLANTS, HOW THEY DO WITH GENE THERAPY AND TRANSPLANT, AND WITHOUT ACTUALLY DOING A STUDY, THEY'RE GATHERING ALL THE DATA FROM ALL THE PATIENTS WHO GET GENE THERAPY OR TRANSPLANT, AND HOPE TO MAKE THESE KINDS OF COMPARISONS. ONE OF THE BIG QUESTIONS TO THEM IS THE SAME QUESTION WE'RE ASKING WITH THE GENE THERAPY, WHICH IS, I ENTRANTS WHO RECEIVE HAPLOTRANSPLANTS, WOULD CONDITIONING HELP? THE ANSWER FROM LOOKING BACKWARDS IS THAT UNFORTUNATELY, MANY OF THOSE INFANTS HAVE INFECTIONS, AND WHEN THEY GET CONDITIONING, THEY HAVE HIGHER MORTALITY RATES, BUT IF THEY SURVIVE, THEY GET BETTER IMMUNE RECONSTITUTION. SO THE QUESTION IS QUITE COMPLEX AND I THINK THAT THE TOTAL ANSWER TO YOUR QUESTION ISN'T A SIMPLE ONE. >> I'LL JUST REMIND PEOPLE THAT I THINK IT WAS LAST WEEK OR THE WEEK BEFORE, WAS THE 25TH ANNIVERSARY OF THE FIRST USE OF GENES IN PATIENTS WITH, THINK, ADA DEFICIENCY HERE AT THE CLIN WILL CAL CENTER, SO WE HAVE LOTS TO CELEBRATE AND IT'S GREAT TO SEE THIS PROGRESS. SO THANK YOU VERY MUCH. >> THANK YOU. [APPLAUSE] >> SO NOW I HAVE THE GREAT OPPORTUNITY TO INTRODUCE OUR SECOND SPEAKER, STEVE ROSENBERG, WHO'S CHIEF OF THE SURGERY BRANCH IN THE NCI. HE'S ALSO PROFESSOR OF SURGERY AT THE UNIFORMED SERVICES UNIVERSITY OF OF THE HEALTH SIGH INSCIENCES AT GEORGE WASHINGTON UNIVERSITY SCOOFL MEDICINE. DR. ROSENBERG RECEIVED HIS MEDICAL DEGREE AT JOHNS HOPKINS IN BALTIMORE AND THEN WENT TO HARVARD, WHERE HE RECEIVED A PH.D. IN BIOPHYSICS. HE THEN DID RESIDENCY TRAINING IN SURGERY IN 1974, WHERE IT WAS COMPLETED AT THE BRIGHAM HOSPITAL IN BOSTON, AND HE BECAME CHIEF OF SURGERY AT THE NCI IMMEDIATELY FOLLOWING HIS RESIDENCY. DR. ROSENBERG HAS PIONEERED THE DEVELOPMENT OF IMMUNOTHERAPY AND I THINK MOST PEOPLE THINK HE'S THE FATHER OF IMMUNE THERAPY. THAT HAS RESULTED IN THE FIRST EFFECTIVE TREATMENT FOR SELECTED PATIENTS WITH ADVANCED CANCER, PARTICULARLY INITIALLY IN MELANOMA. HIS STUDIES OF CELL TRANSFER IMMUNOTHERAPY HAVE RESULTED IN DURABLE COMPLETE REMISSIONS IN PATIENTS WITH MELANOMA, AND YOU'RE GOING TO HEAR ABOUT SOME OTHERS IN A FEW MINUTES. HE'S PIONEERED, AS I MENTIONED, THE GENE THERAPY AS LONG AGO AS 25 YEARS. AND HIS STUDIES OF ADOPTIVE TRANSFER OF GENETICALLY MODIFIED LYMPHOCYTES RESULTED IN THE REGRESSION OF METASTATIC CANCER IN MELANOMAS, SARCOMAS AND LYMPHOMAS. HE'S BEEN RECOGNIZED AND RECEIVED MANY AWARDS, AND IN 2004, HE RECEIVED THE RICHARD V. SMILEY AWARD, THE HIGHEST GIVEN BY THE INTERNATIONAL SOCIETY FOR BIOLOGICAL THERAPY OF CANCER. IN 2006, HE WAS SELECTED TO GIVE OUR ASTUTE CLINICIAN LECTURE. HE RECEIVED THE MEMORIAL AWARD FROM THE AMERICAN ASSOCIATION OF BLOOD BANKS IN 2010. AND THE KO MEDICAL SCIENCE PRIZE IN 2012, AND THE MASSEY PRIZE IN 2014, AND RECENTLY RECEIVED THE MEDAL OF HONOR FROM THE AMERICAN CANCER SOCIETY OF. SOCIETY. HE'S A MEMBER OF THE INSTITUTE OF MEDICINE OF THE NATIONAL ACADEMY OF SCIENCES, AS WELL AS THE AMERICAN SOCIETY OF CLINICAL ONCOLOGY, IN WHICH HE SERVED ON THE BOARD OF DIRECTORS. HE'S ALSO A MEMBER OF THE SOCIETY OF UNIVERSITY SURGEONS, THE AMERICAN SURGICAL ASSOCIATION, AND THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, AS WELL AS THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS. HE'S AUTHORED OVER 1100 PAPERS, AND HE IS AN EDITOR OF THE PRINCIPLES AND PRACTICES OF ONCOLOGY, WHICH HE'S DONE IN PARTNERSHIP WITH VINCE DEVETER AND LAWRENCE, IN ITS TENTH EDITION. TODAY HE'S GOING TO PRESENT HIS WORK IN TALK TITLED A NEW APPROACH TO CANCER IMMUNOTHERAPY TARGETING UNIQUE CANCER MUTATIONS. WELCOME, STEVE. [APPLAUSE] I HAVE, SADLY, NO PERSONAL FINANCE TO REPORT NOW WE'RE GOING TO TALK ABOUT IMMUNOTHERAPY TODAY AS ONE OF THE FORMS OF CANCER TREATMENT. WE HAVE, OF COURSE, SURGERY, RADIATION THERAPY, CHEMOTHERAPY, THAT WILL CURE A LITTLE MORE THAN HALF OF PEOPLE WHO DEVELOPS CANCER -- WHO DEVELOP CANCER IN THIS COUNTRY. BUT THE NUMBER OF PEOPLE WHO CANNOT BE CURED WITH THOSE MODALITIES RESULT IN ABOUT 580,000 DEATHS IN THE UNITED STATES LAST YEAR ALONE. SO WE'VE SEEN THE EMERGENCE OF IMMUNOTHERAPY. NOW ALMOST INTO MAINSTREAM ONCOLOGY IN THE LAST SEVERAL YEARS. THERE ARE THREE MAIN APPROACHES TO CANCER IMMUNOTHERAPY AS SHOWN ON THIS SLIDE. ONE CAN NON-SPECIFICALLY STIMULATE IMMUNE REACTIONS, WE CAN DO THAT WITH INTERLEUKIN 2 OR ANALITY NAY WAY TO STIMULATE A REACTION IS TO INHIBIT AN INHIBIT TORE, AND THE INHIBITION OF REGULATORY FACTORS, ANTICTLA4 AND ANTIPD1 ARE ASSUMING INCREASING IMPORTANCE IN THE TREATMENT OF PATIENTS WITH MELANOMA AND SMOKING-INDUCED LUNG CANCER. A SECOND APPROACH IS AN APPROACH THAT INVOLVES CANCER VACCINES. THAT IS AN ATTEMPT TO ACTIVELY IMMUNIZE A PATIENT AGAINST THEIR CANCER. THERE HAVE BEEN WELL OVER A THOUSAND PUBLISHED PAPERS IN ANIMALS AND IN HUMANS THAT HAVE LOOKED AT THIS APPROACH. UNFORTUNATELY NO ONE HAS YET DETERMINED A METHOD TO ENABLE CANCER VACCINES TO WORK. THERE'S ONE APPROVED TO TREATMENT FOR PATIENTS WITH PROSTATE CANCER THAT CAN PROLONG SURVIVAL BY ABOUT FOUR MONTHS, BUT ALL PATIENTS PROGRESS AND ALL PATIENTS GO ON TO DIE OF THEIR DISEASE. SO THIS IS AN ACTIVE AREA OF INVESTIGATION, BUT ONE THAT HASN'T BEEN SOLVED AT ALL YET. FINALLY, THERE'S A SUBJECT OF TODAY'S DISCUSSION, AND THAT IS THE USE OF T CELLS AS A DRUG TO TREAT CANCER. IT'S CALLED ADOPTIVE IMMUNOTHERAPY. IT'S THE PASSIVE TRANSFER OF ACTIVATED IMMUNE CELLS THAT HAVE ANTI-TUMOR ACTIVITY. WE ACTUALLY USE A T CELL IN MUCH THE SAME WAY THAT WE MIGHT USE A DRUG IN A VIAL OFF THE SHELF, ONLY WE'RE USING THE PATIENT'S OWN T CELLS TO TREAT THAT PATIENT. NOW THE ADVANTAGES OF CELL TRANSFER IMMUNOTHERAPY ARE MANY. ONE CAN GROW EASILY 10 OF THE 11 LYMPHOCYTES FROM A PATIENT ON ONE SHELF OF A TABLETOP INCUBATOR. THAT'S ABOUT A TENTH THE TOTAL NUMBER OF LYMPHOCYTES THAT WE HAVE IN OUR BODIES. YOU CAN ADMINISTER THESE CELLS, ACTIVATE IT EX VIVO TO EXHIBIT ANTITUMOR EFFECTOR FUNCTION. THEY GO IN WITH GUNS BLAZING, THEY DO NOT -- ARE NOT TOLERIZED OR ENERGIZED BY THE IN VIVO FACTORS. BECAUSE WE HAVE THE CELLS IN A TEST TUBE, WE CAN POTENTIALLY IDENTIFY THE EXACT CELL SUBPOPULATIONS AND EFFECTOR FUNCTIONS THAT ARE REQUIRED TO SEE ANTITUMOR A ACTIVITY IN VIVO, AND PERHAPS MOST IMPORTANTLY, WE CAN MANIPULATE THE HOST PRIOR TO THE CELL TRANSFER TO PROVIDE AN ALTERED AND OPTIMAL MICRO ENVIRONMENT FOR THE TRANSFERRED CELLS. IN A WAY THAT WE CANNOT DO WITH ANY KIND OF TREATMENT THAT ACTUALLY INVOLVES LEAVING THE IMMUNE LYMPHOCYTES INSIDE THE BODY. WE DEVELOPED THIS ADOPTIVE T CELL THERAPY APPROACH USING A CELL CALLED A TUMOR INFILTRATING LYMPHOCYTE, AND WHAT BETTER PLACE INTUITIVELY TO FIND A CELL THAT'S DOING BATTLE AGAINST THE CANCER THAN WITHIN THE TUMOR IT SELF. WHAT WE DO IN THIS TREATMENT IS EXCISE THE TUMOR, USE A VARIETY OF TECHNIQUES TO IDENTIFY INDIVIDUAL T CELL CULTURES THAT WE CAN TEST IN VITRO TO SEE IF THEY HAVE ANTI-TUMOR ACTIVITY. WHEN WE FIND THOSE, WE CAN GROW THEM TO LARGE NUMBERS AND THEN RE-INFUSE THEM BACK INTO THE PATIENT AFTER FIRST LYMPHO-DEPLETING THE PATIENT WITH A NON-MYELOABLATIVE CHEMOTHERAPY. WE USE THEM AT DOSES THAT RESULT IN COMPLETE ELIMINATION OF CIRCULATING LYMPHOCYTES FOR ABOUT EIGHT TO NINE DAYS BEFORE THE BODY NATURALLY RECONSTITUTES THOSE T CELLS. SO OUR FIRST STUDY THAT WE BEGAN MANY YEARS AGO, YOU SEE THIS WITH A POTENTIAL MEDIAN FOLLOW-UP OF 8.8 YEARS WAS OF 93 PATIENTS WHO RECEIVED DIFFERENT LYMPHODEPLETING REGIMEN, ALL HAD METASTATIC MELANOMA WITH WIDESPREAD DISEASE, VIRTUALLY ALL HAD PREVIOUSLY HAD OTHER TREATMENTS AND HAD PROGRESSED. AS YOU CAN SEE IN THESE 93 PATIENTS, WE HAD A 56% OVERALL RESPONSE RATE AND, MOST INTERESTINGLY, 22% COMPLETE REGRESSION RATE IN THESE PATIENTS, AND I'LL SHOW SOME EXAMPLES AND AS YOU CAN SEE IN THE MONTHS NOW IN WHICH THOSE PATIENTS WERE IN COMPLETE REGRESSION, WE'VE ONLY HAD ONE PATIENT OUT OF THESE 20 THAT HAS EVER RECURRED WITH THE REMAINING PATIENTS IN ONGOING RESPONSE ANYWHERE FROM 80 MONTHS TO 124 MONTHS AND ONGOING COMPLETE REGRESSION. WE'VE DEVELOPED WHAT WE THINK IS A BLUEPRINT FOR THE GENERATION OF MUTATION REACTIVE T CELLS AGAINST COMMON CANCERS IN PATIENTS. AND THIS IS THE WORK OF MANY PEOPLE IN THE LABORATORY, PAUL ROBBINS, WILLIAM LU, ERIC TRAN AND OTHERS. HERE'S HOW IT WORKS. WE TAKE THE TUMOR, ISOLATE GENOMIC DNA AND RNA AND DO WHOLE EXOME AND TRANSCRIPTOME SEQUENCING TO IDENTIFY THE MUTATIONS. BY LACKING AT THE TRANSCRIP TOMORROW, LOOKED AT THOSE THAT ARE EXPRESSED AS WELL. AS YOU KNOW, THIS CAN BE DONE QUITE RAPIDLY. WE HAVE AN ALUMINUM MACHINE IN THE SURGERY BRANS, YOU CAN DO IT IN A MATTER OF ONE TO TWO WEEKS. WHAT WE THEN DO IS IDENTIFY EVERY INDIVIDUAL MUTATION BECAUSE WE'VE COMPARED THE TUMOR TO NORMAL IN THE SEQUENCING AND SINT SEISE IN VITRO SYNTHESIZE A GROUP OF MANY GENES THAT IN THE EXPRESSED PROTEINS OF THAT PATIENT. AND HERE'S HOW THAT WORKS. THE EXOMIC ANALYSIS IDENTIFIES AN INDIVIDUAL MUTATION IN A PROTEIN, BUT BECAUSE THE NORMAL SEQUENCE HAS BEEN DETERMINED, AND WE KNOW THE SEQUENCE OF THE HUMAN GENOME, WE KNOW THE 12 AMINO ACIDS ON EVERY SIDE OF THE MUTATION THAT GIVES RISE TO A NEW AMINO ACID AND A PROTEIN, IN THIS CASE, REPLACING THE NATURAL AMINO ACID. WELL, THIS 25MR PEPTIDE HAS TO CONTAIN EVERY POSSIBLE 9 TO 11 AMINO ACID PEPTIDE CONTAINING THIS MUTATION. RIGHT? THIS PEPTIDE THAT GOES TO THE SURFACE CAN HAVE THIS H AT THE FRONT OF THE PEPTIDE, TO BE AT THE END OF THE PEPTIDE OR IT CAN BE IN THE MIDDLE OF THE PEPTIDE, THIS BUT THIS CONTAINS EVERY POSSIBLE PEPTIDE THAT CONTAINS THE MUTATION. WE CALL THIS A MINI GENE, BECAUSE IT'S JUST THAT PART OF THE PROTEIN CONTAINING THE MUTATION, AND WE DO THAT FOR ALL OF THE MUTATIONS. AND WE STRING THEM TOGETHER INTO TANDEM MINI GENE, WE CAN PUT FROM 6 TO 16 OF THESE INDIVIDUAL MINI GENES IN A CONTINUOUS CONSTRUCT. WHAT WE THEN DO IS TAKE THESE TANDEM MINI GENES AND TRANSECT THEM INTO THE PATIENT'S OWN ANTIGEN-PRESENTING CELLS. PATIENTS' ANTIGEN PRESENTING CELLS OR DENDRITIC CELLS CAN EXPRESS ALL OF THE CLASS 1 AND CLASS 2 MOLECULES THAT ARE ON THE SURFACE OF A CELL THAT LEADS TO PRESENTATION OF THE PEPTIDES. THERE ARE SIX CLASS 1 AND 6 CLASS 2, AND BY LOOKING AT EVERY MUTATION, ON ALL OF THE ANTIGEN -- ON ALL OF THE MHC MOLECULES OF THAT PATIENT, WE HAVE A SUBSTANTIAL ADVANTAGE IN THAT WE HAVE NO NEED TO PREDICT PEPTIDE BINDING, EVERY CANDIDATE PEPTIDE AND MHC LOCUS IS INCLUDED, AND MOST IMPORTANTLY, WE DON'T NEED THE TUMOR CELL LINE. AND IT'S VIRTUALLY IMPOSSIBLE TO GROW TUMOR CELL LINES FROM MOST EPITHELIAL CANCERS GIVEN MODERN IT TECHNOLOGY. YOU JUST CAN'T GROW THEM FROM BREAST CANCERS, VERY DIFFICULT FROM ANY G.I. CANCER, PROSTATE CANCER IS DIFFICULT BUT IT CAN BE DONE. BECAUSE WE'VE NOW STRUNG TOGETHER ALL OF THESE TANDEM MINI GENES EXPRESSING EVERY MUTATION IN THAT PATIENT, WE BASICALLY PRODUCED AN IN VITRO SUBSTITUTE FOR THE TUMOR CELL FOR USE IN THIS TESTING. SO AGAIN THE BLUEPRINT IS TO IDENTIFY ALL OF THE MUTATIONS SH PUT THEM INTO TANDEM MINI GENE, IP CERTIFICATE THEM INTO A PATIENT'S OWN ANTIGEN PRESENTING CELL AND THEN TAKE THE TIL CELLS THAT CAUSE A COMPLETE REGRESSION IN PATIENTS WITH MELANOMA AND LOOK TO SEE IF ANY OF THESE TANDEM MINI GENES EXPRESSED IN THE TANDEM MINI GENE ARE RECOGNIZED. SO IF IT CAN RECOGNIZE THIS ANTIGEN-PRESENTING CELL, WE NOW HAVE A WAY IMMUNE LOGICALLY TO GET TO THE EXACT MUTATION THAT'S RECOGNIZED. AND SO WHAT WE DO IS WE HAVE THESE TIL CELLS, FOR EXAMPLE IN THIS ONE PATIENT WITH MELANOMA, HE HAD 71 NON-SUMMON NUSS MUTATIONS, WE MADE 12 TANDEM MINI GENES CONTAINING FIVE OR SIX OF THE INDIVIDUAL DPEENS, AND THEN TESTED THEM TO SEE IF ANY OF THESE TANDEM MINI GENES PUT INTO THE PATIENT'S ANTIGEN-PRESENTING CELL WOULD BE RECOGNIZED BY THE TILL. SURE ENOUGH, TANDEM MINI GENE 1 WAS RECOGNIZED. IT ENCODED SIX 25MRs, EACH ONE OF THEM ENCODING A MUTATION, AND WE THEN NEEDED TO DECIDE WHICH OF THESE SIX PROTEINS REPRESENTED THE ACTUAL ANTIGEN. THAT COULD BE DONE IN THIS TECHNIQUE DEVELOPED BY ROBBINS BY, ONE BY ONE, TAKING THESE INDIVIDUAL MUTATIONS BACK TO WILD TYPE, WHEN ALL OF THE MUTATIONS ARE PRESENT, THE TANDEM MINI GENE IS RECOGNIZED, WHEN WE CHANGE THIS GENE BACK TO WILD TYPE, IT'S STILL RECOGNIZED, BUT WHEN WE CHANGE THIS SECOND GENE BACK TO WILD TYPE, ALL ACTIVITY IS LOST. THEREBY IDENTIFYING THIS PARTICULAR GENE AS THE GENE ENCODING THE PROTEIN RECOGNIZED BY THE TIL CELLS THAT MEDIATED COMPLETE REGRESSION IN THIS PATIENT, AND IT TURNS OUT TO BE QUITE AN IMPORTANT GENE A MEMBER OF THE KINESIN FAMILY MEMBER 2C INVOLVED IN THE STABILITY OF THE MITOTIC SPINDLE. WE'VE NOW DONE THIS ON 25 DIFFERENT PATIENTS WITH MELANOMA, AND THERE WAS QUITE AN ASTONISHING CONCLUSION THAT WAS REACHED, AND THAT IS OF THE 64 SOMATIC MUTATIONS THAT WERE IDENTIFIED USING TIL FROM 25 DIFFERENT PATIENTS, WE IDENTIFIED 64 COMPLETELY UNIQUE MUTATIONS. THAT IS, EVERY PATIENT THAT UNDERWENT A REGRESSION, WAS RECOGNIZING SOMETHING COMPLETELY DIFFERENT. AND AS YOU CAN SEE, COMPLETELY DIFFERENT NOT ONLY IN THE IDENTIFICATION OF THE MUTATED PROTEIN THAT COULD SERVE AS THE TARGET FOR THE IMMUNE SYSTEM, BUT ALSO A WHOLE VARIETY OF CLASS 2 AND CLASS 1 RESTRICTION ELEMENTS. WELL, OUR NEXT EFFORT IJ VOFLS ATTEMPTS TO USE THIS TANDEM MINI GENE APPROACH TO TREAT COMMON EPITHELIAL CANCERS BY TARGETING THE UNIQUE IMMUNOGENIC MUTATIONS THAT MIGHT BE EXPRESSED BY THOSE CANCERS BECAUSE THIS SAME BLUEPRINT COULD BE APPLIED TO VIRTUALLY ANY CANCER, ANY CANCER TYPE. AND THE FIRST PATIENT THAT WE TREATED THAT WAS REPORTED BY ONE OF THE IF HE LES IN TH FELLOWS IN THE LAB LAST YEAR WAS A PATIENT WHO WAS A 45-YEAR-OLD FEMALE. SHE HAD A BILE DUCT CANCER, A CLANK YOE CARCINOMA. SHE HAD UNDERGONE A RIGHT HELP TECH THOME, SHE HAD RECURRED WITH MULTIPLE LUNG AND LIVER METASTASES, RECEIVED CHEMOTHERAPY, HAD PROGRESSED. MORE CHEMOTHERAPY, PROGRESSED, RATHER TYPICAL STORY FOR PEOPLE WITH METASTATIC SOLID CANCERS. WE RESECTED A LUNG LESION TO GIVE HER CONVENTIONAL TIL. THEY WERE INFUSED. SHE HAD A TINY SHRINKAGE WHICH RAPIDLY START TODAY GROW, NEVER EVEN ACHIEVED A PARTIAL RESPONSE. WE THEN USED THE TANDEM MINI GENE APPROACH TO TARGET THE UNIQUE CANCER MUTATIONS THAT WERE PRESENT IN HER TUMOR THAT MIGHT BE RECOGNIZED BY HER TUMOR INFILTRATING LYMPHOCYTES. SHE HAD 26 DIFFERENT MUTATIONS PRESENT IN A LUNG METASTASIS AND PRESENT IN THREE RESECTED LUNG METASTATIC DEPOSITS. ERIC TRAN PRODUCED THREE TANDEM MI NEA GENE, EACH CONTAINING MULTIPLE INDIVIDUAL MINI GENE, TESTED THEM, AND YOU CAN SEE, TANDEM MINI GENE 1 BUT NOT 2 AND 3 ENCODED THE ANTIGEN RECOGNIZED BY THESE TIL CELLS, AND YOU CAN SEE THAT BY UPREGULATION OF OX40, AN ACTIVATION MOLECULE THAT SUGGESTED THAT THIS WAS, IN FACT, THE CD4 -- A CD4 CELL. WE COULD IDENTIFY THE EXACT PROTEIN BY AGAIN REVERTING THEM ALL BACK TO NORMAL, AND IT TURNED OUT REVERTING THIS ERBB2IP GENE BACK TO NORMAL ELIMINATED REACTIVITY, THERE BY IDENTIFYING ERBB2IP AS THE TARGET OF THE TIL IN THIS PATIENT. SO TUMOR SUPPRESSOR JEAN GENE THAT BINDS TO ERBB2 AT10 WAITS DOWNSTREAM RAS/ERK SIGNALING BY LAKELY IS A DRIVER BUT WE DON'T KNOW THAT. ERIC THEN IDENTIFIED A POPULATION THAT WAS ALMOST 90% PURELY REACTIVE AGAINST THIS MUTATED PROTEIN. AND WHEN WE TREATED THAT PATIENT WITH THESE CELLS, THE PATIENT HAS UNDERGONE AN ONGOING REGRESSION OF LUNG AND LIVER METASTASES, IN FACT, OUR PET SCAN IS NOW NORMAL, BUT SHE DOES HAVE SOME REMNANTS OF THE ORIGINAL DISEASE BUT AT HER LAST FOLLOW-UP IN JUNE, THEY WERE STILL SHRINKING AS WELL. SO HERE YOU CAN SEE THIS LESION, AGAIN, DOWN TO THIS STRUCK TEU MIGHT WELL BE SCAR. AGAIN, IT'S PET-NEGATIVE. THIS LEAGUES THE SAME. THIS LEAGUES HAS THIS RESIDUAL DEPOSIT STILL SHRINKING AS OF OUR LAST VISIT. THESE TWO LARGE METASTATIC DEPOSITS VIRTUALLY GONE, THIS ONE QUITE SMALLER AS WELL. AND THE PATIENT IS NOW, OF COURSE, LIVING NORMALLY WITHOUT DIFFICULTY FROM THIS. WELL, AGAIN, A STUNNING FINDING TO US, AND THAT IS FROM 22 PATIENTS THAT HAD EPITHELIAL CANCERS WHERE WE ACTUALLY IDENTIFIED THE EXACT ANTIGEN THAT WAS BEING RECOGNIZED, WE RECOGNIZED 57 SOMATIC MUTATIONS, RECOGNIZED BY PATIENTS' OWN T CELLS, OBTAINED FROM THEIR GROWING LESIONS, AND AGAIN, ALL WERE COMPLETELY UNIQUE. COMING FROM A WHOLE VARIETY OF CANCERS, CLANK YOE CARCINOMA, COLON CANCERS, RENAL CANCER, PANCREATIC, ESOPHAGEAL, LUNG CANCER, OVARIAN CERVICAL CANCER, THEY WERE ALL UNIQUE EXCEPT FOR ONE PROTEIN. TURNED OUT THAT TWO PATIENTS RECOGNIZED A K RAS MUTATION THAT WAS RESTRICTED BY CWOO8. CAN YOU CAN SEE THIS PATIENT AND THIS PATIENT BOTH RECOGNIZED KRAS. NOW KRAS IS THE MOST COMMON SHARED MUTATION AMONG ALL CANCERS, AND IN THIS EXOA LAITION MADE BY JIM YANG IN THE SURGERY BRANCH, YOU CAN SEE 70% OF ALL PANCREATIC CANCERS CONTAIN KRAS MUTATIONS AND ATTEMPTS TO TARGET KRAS HAVE BEEN A VITAL PART OF MODERN ONCOLOGY, AND A LARGE PROGRAM OUT AT FREDERICK TRYING TO TARGET IT WITH SMALL MOLECULES. HERE FOR THE FIRST TIME NOW, WE MIGHT HAVE A T CELL RECEPTOR THAT CAN TARGET THIS KRAS G12D MUTATION IN PATIENTS, AND JIM YANG, BY IMMUNIZING HA11 TRANSGENIC MICE HAS IDENTIFIED TWO OTHER RECEPTORS THAT CAN RECOGNIZE G12 -- RESTRICTED BY HLA11, AGAIN, THAT ELEGANT WORK DONE BY JIM YANG. AND HIS LABORATORY GROUP. WE JUST RECENTLY TREATED ONE OF THOSE PATIENTS. WE'VE ONLY TREATED ABOUT SIX OR SEVEN PATIENTS THUS FAR WITH MOST OF THE EFFORT INVOLVED IN LABORATORY -- A LABORATORY EFFORTS. BUT THIS PATIENT WHO HAD A KRAS MUTATION, WE DID WHOLE EXOME SEQUENCING. SHE DID HAVE 61PUTATIVE MUTATIONS IDENTIFIED, WE MADE FIVE TANDEM MI NEA GENES AND MADE PEPTIDES, WE PUT THEM INTO THE PATIENT'S OWN ANTIGEN-PRESENTING CELL. WE COULD IDENTIFY THE NATURE OF THE BETA CHAIN OF THAT T CELL RECEPTOR, IDENTIFY THE 9 AND 10MR AMINO ACIDS THAT WERE MUTATED THAT WERE RECOGNIZED, COULD IDENTIFY THEN A CULTURE WITH 75% OF THE CELLS RECOGNIZING THIS KRAS MUTATION. AND THIS PATIENT WAS JUST TREATED 2 1/2 MONTHS AGO, AND IS HAVING WHAT APPEARS TO BE A SIGNIFICANT REGRESSION OF MULTIPLE LUNG METASTATIC DPO SITS FROM A COLORECTAL CANCER, AGAIN ATTACKING A KRAS MUTATION. YOU CAN SEE THAT TUMOR DEPOSIT HAS SHRUNK, AS HAVE THESE. YOU CAN SEE FROM THESE YELLOW ARROWS, EACH ONE OF THE DEPOSITS IS SHOWING SUBSTANTIAL SHRINKAGE AT 2 1/2 MONTHS. SO WE DON'T KNOW HOW LONG IT'S GOING TO LAST, BUT IT'S CERTAINLY A GOOD START. SO 33 PATIENTS IN WHICH WE'VE IDENTIFIED THE EXACT GENE, AT LEAST THE EXACT RESTRICTION ELEMENTS FOR THESE EPITHELIAL CANCERS, YOU CAN SEE FOR A WHOLE VARIETY OF DIFFERENT CANCER TYPES, 26 OF THE 33 PATIENTS WITH THIS VARIETY OF METASTATIC IT EPITHELIAL CANCERS EXPRESSED IMMUNOGENIC MUTATIONS. AGAIN, ALL UNIQUE WITH THE EXCEPTION OF KRAS. AND WE NOW HAVE AN ACTIVE EFFORT IN THE SURGERY BRANCH ATTEMPTING TO TREAT ADDITIONAL PATIENTS. SO CELL THERAPY USING AUTOLOGOUS T CELLS THAT TARGET RANDOM SOMATIC MUTATIONS OR, IN THE CASE OF KRAS, A SHARED MUTATION, BUT THERE ARE NOT VERY MANY SHARED MUTATIONS IN CANCERS THAT OCCUR AT A SINGLE -- IT'S A POSSIBLE TREATMENT THAT CAN BE STUDIED FOR MANY CANCERS THAT HAVE SUFFICIENT MUTATIONAL LOADS. AND IN FACT, IT APPEARS THAT THIS TARGETING OF RANDOM SOMATIC MUTATIONS IS THE FINAL COMMON PATHWAY OF ALL IMMUNOTHERAPIES. THAT'S THE HYPOTHESIS BUT INFORMATION IS GAINING THAT THAT MAY BE THE REASON THAT PATIENTS RESPOND TO INTERLEUKIN 2 TO CHECKPOINT MODULATORS LIKE ANTIPD1 AND CERTAINLY THESE T CELL TRANSFERS. I MIGHT JUST FINISH BY SHOWING TWO ADDITIONAL STU DID DIS AS STUDIES AS W E'RE LOOKING AT WAYS TO OPTIMALLY RECOGNIZE -- AND WAYS TO GENETICALLY ENGINEER THE T CELLS INTO AUTOLOGOUS NAIVE CELLS THAT WOULD HAVE AN EXPLOSIVE PROLIFERATIVE CAPACITY ONCE ADMINISTERED INTO THE PATIENTS. I'LL SHOW THIS ONE STUDY THAT WAS CONDUCTED BY ELENA GROSS, A POSTDOCTORAL FELLOW, WHO LOOKED AT A VARIETY OF DIFFERENT ACTIVATION AND INHIBITORY MARKERS TO SEE IF THERE WERE SUBPOPULATIONS THAT CONTAINED THE LYMPHOCYTES FROM TIL AND PERIPHERAL BLOOD THAT CONTAIN THEIR ANTITUMOR ACTIVITY. AS YOU CAN SEE, AS WE LOOK AT PD1, WHICH IS AN ACTIVATION MARKER THAT RESULTS FROM MULTIPLE STIMULATIONS OF A T CELL BY AN ANTIGEN, YOU CAN SEE PD1 IS ONLY A FEW PERCENT OF THE PERIPHERAL BLOOD CELLS, AND ABOUT 20 FOOF% MEDIAN IN TIL. INTERESTINGLY, ALENA FOUND THAT ALL OF THE ANTITUMOR ACTIVITY IN TIL OCCURRED IN THIS PD1-POSITIVE POPULATION, AND NOT IN THE PD1-NEGATIVE POP LAYING, AND THAT WAS ALSO TRUE FOR SEVERAL OTHER MARKERS AS WELL. OF WHAT I THINK IS SUBSTANTIAL IMPORTANCE IN THIS WORK JUST SUBMITTED BY ALENA GROS IS THIS IS ALSO TRUE IN PERIPHERAL BLOOD. EVEN THOUGH WE ONLY HAVE ROUGHLY 3 TO 4% OF PD1-POSITIVE CIRCULATING CELLS IN OUR PRIF LAL BLOOD AND THESE THREE CANCER PATIENTS, YOU COULD SEE VIRTUALLY ALL OF THE REACTIVITY BY A VARIETY OF ASSAYS IN CO-CULTURE ASSAYS -- UPREGULATION, ALL OF THE ANTITUMOR ACTIVITY IS IN THE PD1-POSITIVE POPULATION. AND THIS, THEREFORE, PROVIDES US WITH AN OPPORTUNITY TO OBTAIN HIGHLY ENRICHED POPULATIONS OF LYMPHOCYTES THAT CAN RECOGNIZE TUMOR ANTIGENS. I MIGHT FINISH WITH THIS ONGOING WORK DONE IN THE SURGERY BRANCH, WHERE THEY ACTUALLY TOOK THIS OBSERVATION THAT PD1 CELLS CONTAINED THE ANTITUMOR ACTIVITY AND LOOKED AT THE FREQUENCY OF THE INDIVIDUAL T CELL RECEPTORS WITHIN A FRESH TUMOR. SO YOU TAKE OUT A FRESH TUMOR, SEPARATE OUT THE PD1-POSITIVE