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 https://clinicalcenter.nih.gov OUR SPEAKER IS DR. IRENE CORTESE, A STAFF CLINICIAN IN THE NEUROIMMUNOLOGY BRANCH AND DIRECTOR OF THE NEUROIMMUNOLOGY CLINIC AT THE NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE. DR. CORTESE EARNED HER MEDICAL DEGREE FROM AND COMPLETED RESIDENCY TRAINING AT THE UNIVERSITY IN VERGATA, ROME. SHE WENT ON TO PURSUE A RESEARCH FELLOWSHIP IN THE LABORATORY OF DR. MARTIN AT NINDS. THEREAFTER, SHE RELOCATED TO BALTIMORE, TO COMPLETE AN INTERNSHIP IN INTERNAL MEDICINE AT JOHNS HOPKINS, BAY VIEW MEDICAL CENTER, FOLLOWED BY NEUROLOGY RESIDENCY TRAINING AND A CLINICAL FELLOWSHIP AT JOHNS HOPKINS HOSPITAL. SHE THEN RETURNED TO THE NIH TO COMPLETE A CLINICAL NEUROIMMUNOLOGY FELLOWSHIP UNDER DR. McFARLAND AT NINDS. DR. CORTESE JOINED THE FACULTY AS A STAFF CLINICIAN IN 2008, AND BECAME HEAD OF THE CLINICAL GROUP IN THE NEUROIMMUNOLOGY BRANCH IN 2011. SHE WAS APPOINTED DIRECTOR OF THE NEWLY ESTABLISHED NEUROIMMUNOLOGY CLINIC IN 2013, AND AS DIRECTOR OF THIS CLINIC, DR. CORTESE COLLABORATES WITH TRANSLATIONAL RESEARCH LABS AT NINDS AND THE NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES, DEVELOPING AND IMPLEMENTING OBSERVATIONAL AND INTERVENTIONAL STUDIES OF MULTIPLE SCLEROSIS AND HTLV1 ASSOCIATED MYELOPATHY. MORE RECENTLY, SHE HAS LED EFFORTS EXPLORING STRATEGIES FOR PROMOTING IMMUNE RECONSTITUTION AS A TREATMENT FOR PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY, WHICH YOU'LL HEAR A LITTLE BIT MORE ABOUT TODAY. DR. CORTESE HAS BEEN RECOGNIZED WITH AN INDIVIDUAL MERIT AWARD, A PEER RECOGNITION AWARD AND A SPECIAL ACT AWARDS BY NINDS, AND SHE IS PUBLISHED WIDELY ON THE TOPIC OF NEUROIMMUNOLOGICAL DISEASES IN THE BIOMEDICAL LITERATURE. DR. CORTESE WILL SPEAK ON IMMUNOTHERAPIES FOR PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY. SO LET'S BEGIN. [APPLAUSE] >> GOOD AFTERNOON AND THANK YOU FOR THE OPPORTUNITY TO TALK WITH YOU A BIT ABOUT SOME OF THE WORK THAT WE'VE BEEN DOING IN PML. SO DURING THIS TALK, I'LL BE GIVING YOU AN OVERVIEW OF PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY AS WELL AS THE BIOLOGY OF JC VIRUS, MUCH OF WHICH HAS BEEN WORKED OUT HERE AT NINDS OVER SEVERAL DECADES OF WORK IN JEAN MAJOR'S LAB AND NINDS. IN THE SECOND PART OF MY TALK I'LL TELL YOU A LITTLE ABOUT THE IMMUNOTHERAPEUTIC STRATEGIES THAT WE HAVE USED FOR THE TREATMENT OF PML, NAMELY WITH CHECKPOINT INHIBITORS AS WELL AS VIRAL-SPECIFIC T-CELLS. SO I WANTED TO START WITH A BRIEF CLINICAL DESCRIPTION OF A PATIENT THAT WE SAW HERE. THIS WAS A 43-YEAR-OLD WOMAN WITH PAST MEDICAL HISTORY RELEVANT FOR DISCOID LUPUS DIAGNOSED AT AGE 38 AND SYSTEMIC LUPUS ERYTHEMATOSUS WITH LUPUS DIAGNOSED AT AGE 42 FOR WHICH SHE WAS TREATED FOR EIGHT MONTHS PRIOR TO HER NEUROLOGICAL PRESENTATION, WHICH WAS JUNE 10TH OF 2018, WHEN SHE STARTED NOTICING THAT HERBAL ANS WAS A BIT HER -- HER& BALANCE WAS A BIT OFF. VERY QUICKLY THESE NEUROLOGICAL SYMPTOMS PROGRESSED WITH DEVELOPMENT OF WEAKNESS IN THE RIGHT LEG AS WELL AS SOME COGNITIVE CHANGES, AND THEN WITHIN A WEEK, CLUMSINESS IN HER RIGHT HAND AS WELL AS VISUAL CHANGES. FOR THIS REASON, SHE SOUGHT ATTENTION IN THE EMERGENCY ROOM, WHERE AN MRI WAS DONE, AND WE SEE THE IMAGES RIGHT HERE WHICH SHOWED THE PRESENCE OF WHITE MATTER LESIONS, MULTIFOCAL, SCATTERED IN THE SUBCORTICAL WHITE MATTER WHICH WERE NOT ENHANCING, AND FINAL FLUID ANALYSIS SHOWED THE PRESENCE OF POSITIVE JC VIRUS PCR. SO WITH THIS, THEY WERE ABLE TO ESTABLISH A DIAGNOSIS OF PROGRESSIVE -- HERE TREATMENTS WERE DISCONTINUED AND THE PATIENT WAS SENT HOME AND OVER THE SUBSEQUENT WEEKS, SHE HAD PROGRESSIVE WORSENING OF HER NEUROLOGICAL SYMPTOMS INITIALLY REQUIRING A WALKER AND THEN WITHIN JUST A FEW WEEKS, REQUIRING A WHEELCHAIR. BY JULY 2ND, NOT EVEN A MONTH SINCE THE ONSET OF PRESENTATION, SHE WAS UNABLE TO BATHE OR TOILET INDEPENDENTLY. JULY 15TH, SHE CAME FOR HER FIRST VISIT AT NIH, WHERE WE SEE HER MRIs ACROSS HERE ON THE BOTTOM. THERE HAD BEEN SOME PROGRESSION OF THE EXTENSION OF INVOLVEMENT BY THE PML WITH MULTIFOCAL WHITE MATTER CHANGES THAT YOU CAN SEE, AND WE WERE ABLE TO DETERMINE ALSO THAT SHE WAS RELATIVELY IMMUNE-SUPPRESSED WITH CD4 COUNT OF 136, CD8 COUNT OF 296, AND JUST 5 CD19 CELLS. JCV PCR WAS POSITIVE AGAIN, AND WE WERE ABLE TO QUANTITATE THIS AT 899 COPIES PER ML, WHICH IS A REASONABLY LOW VALUE ACROSS THE SPECTRUM OF PML AS WE'LL SEE LATER IN MY TALK. SO THE PATIENT WENT HOME WITH A PLAN TO RETURN HOPEFULLY FOR TREATMENT WITH US IN ONE OF OUR EXPERIMENTAL STUDIES BUT OVER THE SUBSEQUENT WEEKS, SHE BECAME PROGRESSIVELY MORE CONFUSED AND LESS RESPONSIVE, AND THEN AUGUST 8TH, JUST TWO MONTHS AFTER THE ONSET OF SYMPTOMS, SHE DIED. THIS CASE REALLY DOES ILLUSTRATE HOW AGGRESSIVE PML CAN BE. SO PROGRESSIVE MULTIFOCAL LEUKOENSELF LOP KEY IS A RARE DEVASTATING BRAIN INFECTION SEEN ALMOST EXCLUSIVELY IN PATIENTS WITH IMPAIRED CELLULAR IMMUNITY CAUSED BY THE HUMAN POLYOMA VIRUS JC VIRUS. I SAY DEVASTATING BECAUSE AS WE SAW IN OUR PATIENT CASE UNTREATED, THE DISEASE CAN BE FATAL WITHIN JUST A FEW MONTHS AND OF THE FEW SURVIVORS, 80% REALLY HAVE NO MEANINGFUL RECOVERY FROM DEFICITS. OVER HERE ON THE RIGHT, WE SEE AN MRI IMAGE SHOWING TYPICAL WHITE MATTER CHANGES THAT ARE HYPERINTENSE ON T2 WEIGHTED SEQUENCES. THEY'RE MULTIFOCAL AND SCATTERED IN THE SUBCORTICAL WHITE MATTER. A CHARACTERISTIC OF CLASSIC PML IS THAT THESE LESIONS DO NOT CONTRAST-ENHANCE AND TYPICALLY DON'T HAVE MUCH EDEMA SURROUNDING THEM, AND THIS IS BASICALLY REFLECTING THE LACK OF ANY SIGNIFICANT INFLAMMATORY RESPONSE AGAINST THE INFECTION. SO PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY WAS REALLY A FAIRLY OBSCURE DISEASE, AND FOLLOWING ITS FIRST DESCRIPTION, IN 1958, THERE WERE JUST 200 CASES DESCRIBED BETWEEN 1958 AND 1982, WHEN THE FIRST CASES OF PML WERE DESCRIBED IN AIDS. THIS SORT OF SIGNALED THE BEGINNING OF A SURGE IN INCIDENCE OF PML ASSOCIATED WITH HIV, WHERE THERE WAS A 50 FOLD INCREASE BASICALLY IN THE INCIDENCE WHICH AT ITS PEAK, PRIOR TO THE DEVELOPMENT OF ANTIRETROVIRAL THERAPY, WAS ESTIMATED TO BE AT ABOUT 7,000 NEW CASES PER YEAR. FOLLOWING THE INTRODUCTION OF ANTI-RETRO RETROVIRAL THERAPY, THE SIGNIFICANCE WENT DOWN SIGNIFICANTLY TO ABOUT ONE-THIRD OFITY PEAK. IN 2005, THE VERY FIRST WERE FOUND IN PATIENTS WITH MULTIPLE SCLEROSIS AND CROHN'S DISEASE TREATED WITH AN IMMUNE MODULATOR, NATALIZUMAB. PML ASSOCIATED WITH VARIOUS MEDICATIONS USED TO TREAT CHRONIC INFLAMMATORY DISEASES AS WELL AS CANCERS WITH IMMUNOSUPPRESSIVE EFFECTS. OVERALL, THE TOTAL NUMBER OF CASES ASSOCIATED WITH NAT LIZ MAN WAS 756 SO CERTAINLY A MUCH LESS DRAMATIC SURGE THAN WHAT WAS SEEN DURING THE AIDS EPIDEMIC. SO THERE AREN'T VERY GOOD STATISTICS, HOWEVER, THE BEST ESTIMATES ARE THAT PML DEVELOPS IN ABOUT ONE IN 200,000 OF THE GENERAL POPULATION WITH ABOUT 4,000 NEW CASES PER YEAR IN THE UNITED STATES AND EUROPE COMBINED. THE MAJORITY OF CASES ARE STILL REPRESENTED IN HIV PATIENTS AND AMONG THE NON-HIV CASES, WE HAVE A VARIETY OF DIFFERENT CONDITIONS. THE MAJORITY ARE STILL REPRESENTED BY HEMOTOE LOGICAL MALIGNANCY AND DIFFERENT CONDITIONS THAT CAN CAUSE PROLONGED AND PROFOUND CELLULAR IMMUNE IMPAIRMENT SUCH AS I HAD YOEPATHIC IMMUNE DEFICIENCIES, TRANSPLANT PATIENTS AS WELL, AS WELL AS TREATED AND UNTREATED INFLAMMATORY DISEASE. IMPORTANTLY, ABOUT 14 FDA-APPROVED MEDICATIONS NOW CARRY A BLACK BOX WARNING FOR PML, WHICH HAS GREATLY INCREASED THE ATTENTION FOR THIS DISEASE. SO JC VIRUS IS A COMMON VIRUS. IT IS -- THE SERO PREVALENCE IS ESTIMATED AT 50 TO 80% IN THE GENERAL POPULATION DEPENDING ON THE ASSAY USED. IT INCREASES WITH EACH DECADE OF LIFE AND THIS TYPE OF DISTRIBUTION IS OBSERVED THROUGHOUT THE WORLD. FOLLOWING PRIMARY INFECTION, THE JC VIRUS ESTABLISHES A PERSISTENT INFECTION IN THE UROEPITHELIUM AND ABOUT 30 TO 50% OF INFECTED INDIVIDUALS, THE VIRUS IS ACTUALLY EXCRETED IN THE URINE WHICH IS IMPORTANT FOR ITS TRANSMISSION. THAT SAID, PML ITSELF IS ACTUALLY QUITE RARE, AND IN THIS CARTOON, YOU SEE A DEPICTION OF THE -- WITH CONCENTRIC CIRCLES REPRESENTING THE CATEGORIES OF PATIENTS AT RISK FOR DEVELOPING PML, AND THE YELLOW CIRCLES REPRESENTING THE PROPORTION OF PML CASES WITHIN THOSE CATEGORIES. SO EVEN AMONG PATIENTS AT HIGH RISK FOR DEVELOPING PML, THE NUMBER OF CASES ARE ACTUALLY RATHER INFREQUENT EVENTS. AND TO SOME EXTENT, WE UNDERSTAND THE REASON FOR THIS NOW, AND TO LOOK AT THAT, WE NEED TO LOOK AT THE VIRUS A LITTLE BIT MORE CLOSELY. SO YOU SEE IN THIS CARTOON, THE VIRAL GENOME, IT'S A RATHER -- IT'S A SMALL VIRUS, VERY SIMPLE WITH TWO CODING REGIONS, CODING FOR THE EARLY GENES THAT ARE LARGELY PROTEINS INVOLVED IN REGULATORY PROCESS OF VIRAL REPLICATION, AND THE LATE GENES WHICH ARE REPRESENTED MAINLY BY STRUCTURAL PROTEINS AND IN PARTICULAR, VP1, WHICH IS THE MAJOR STRUCTURAL PROTEIN. THESE REGIONS ARE RELATIVELY WELL CONSERVED ACROSS PATIENT POPULATIONS, AND IN FACT, THERE'S JUST ONE SEROTYPE OF THIS VIRUS. HOWEVER, THE NON-CODING CONTROL REGION OR NCCR RIGHT HERE IS HYPERVARIABLE, AND IT IS THIS REGION THAT ACTUALLY -- PATHOGENICITY OF THE VIRUS. SO IF WE LOOK AT THIS REGION MORE CLOSELY, AND WE TAKE ISOLATES FROM THE URINE OF HEALTHY INDIVIDUALS, WE CAN SEE THAT THE ARCHITECTURE OF THIS AREA IS VERY STABLE AND RELATIVELY WELL CONSERVED IN THE INDIVIDUAL PATIENT AND ALSO ACROSS PATIENT POPULATIONS, AND THIS GENETIC REARRANGEMENT OF THE NCCR, OF THE REGULATORY REGION, HAS BEEN TERMED ARCHETYPE, AND IT IS THE COMMON SORT OF ARRANGEMENT OF THE NCCR FOUND IN VIRUS IN URBAN SEWAGE, FOR EXAMPLE, AND SO THE FORM THAT IS EXCRETED IN THE URINE IN THE GENERAL POPULATION. ON THE OTHER HAND, ISOLATES OBTAINED FROM LESIONS OF PATIENTS WITH PML HAVE A GROSSLY REARRANGED CONTROL REGION WITH AREAS OF DUPLICATION AS WELL AS DELETIONS THAT HAVE BEEN SHOWN TO DERIVE FROM THE ARCHETYPE SEQUENCE WITHIN AN INDIVIDUAL PATIENT. IN THE BRAINS OF PEOPLE WITH PML AND ALSO IN THE SPINAL FLUID AND EVEN IN THE BLOOD, THESE PROTOTYPE SEQUENCES AS THEY'VE BEEN TERMED ARE QUITE VARIED, SO YOU CAN SEE QUITE A BIT OF DIVERSITY OF THESE VARIANTS. TYPICALLY WITH A DOMINANT VARIANT THAT CAN BE IDENTIFIED. BUT THE COMMON FEATURE OF THESE VARIANTS IS THAT COMPARED TO THE ARCHETYPE, WHICH IS REALLY NOT ABLE TO INFECT GLIAL CELLS IN THE BLAINE, THESE PROTOTYPE VARIANTS ALL HAVE GAINED NEUROTROPISM AS WELL AS GREATLY IMPROVED REPLICATION EFFICIENCY. SO HOW DOES THIS REARRANGEMENT REALLY HAPPEN? WE DON'T KNOW EXACTLY HOWEVER THE IMMUNE RESPONSE SEEMS TO BE EXTREMELY IMPORTANT AND IN SUSTAINED SUPPRESSION, IT SEEMS THAT THE VIRUS IS ABLE TO REACTIVATE, AND ONCE IT REACTIVATES, IT CAN -- THIS CAN LEAD TO REPLICATION-DRIVEN RECOMBINATION EVENTS THAT ARE SEQUENTIAL AND ULTIMATELY LEAD TO THAT GROSSLY REARRANGED PROTO TYPIC VARIANT WHICH IS PROGRESSIVELY SELECTED OVER TIME AND CAN LEAD TO PML. IT'S IMPORTANT TO NOTE THAT PROBABLY SOME OF THESE EVENTS MAY OCCUR EVEN IN HEALTHY POPULATIONS OR IMMUNE-SUPPRESSED INDIVIDUALS, BUT THE RARITY OF PML IS REALLY RELATED TO THE MULTIPLICATIVE PROBABILITY OF THESE SERIAL REARRANGEMENTS OCCURRING AND LEADING TO THAT VIRAL QUASISPECIES THAT'S ACTUALLY ABLE TO CAUSE PML. AND SO TO SORT OF SUMMARIZE WHAT WE KNOW ABOUT THE JC LIFE CYCLE, THE ARCHETYPE VIRUS, WHICH IS THAT STABLE FORM, IS THOUGHT TO ENTER VIA THE OROPHARYNX, AND FROM THERE, TO SPREAD BY HEMATOGENOUS SPREAD TO OTHER SITES, MOST IMPORTANTLY THE KIDNEY, THE PRIMARY SITE OF LATENT INFECTION. AND FROM THERE, THERE IS SHEDDING OF THE VIRUS INTO THE URINE, INTO THE ENVIRONMENT, AND THIS IS OBVIOUSLY THE MODE OF TRANSMISSION. THERE ARE OTHER SITES OF LATENT INFECTION THAT HAVE BEEN IDENTIFIED. IMPORTANTLY, THE BONE MARROW AS WELL AS B CELLS, AND THEN THERE IS ALSO THE SUGGESTION THAT LATENT INFECTION MAY ALSO OCCUR IN THE BRAIN. IN THE SETTING OF SUSTAINED IMMUNE SUPPRESSION, THIS VIRUS CAN MUTATE AS WE SAW, AND THEN FROM THERE, CAN EITHER REACH THE BRAIN OR IF IT'S ALREADY THERE, IT CAN CAUSE THE CLINICAL SYNDROME OF PML. SO AT PRESENT, THERE ARE NO ANTIVIRAL TREATMENTS THAT HAVE PROVEN TO BE EFFECTIVE. THERE HAVE BEEN VERY FEW INTERVENTIONAL STUDIES, AND EVEN MOLECULES THAT HAVE PROVEN OR WERE THOUGHT TO HAVE PRE-CLINICAL EFFECT WERE NOT PROVEN IN PATIENT POPULATIONS. AND SO AT PRESENT REALLY THE ONLY TREATMENT AVAILABLE IS IMMUNE RECONSTITUTION. WHEN IMMUNE RECONSTITUTION CAN BE ACHIEVED RAPIDLY AND EFFECTIVELY, THEN FOR EXAMPLE, IN MULTIPLE SCLEROSIS PATIENTS TREATED WITH THE DRUG NAT LEWIS MAN WHICH IS NOT IMMUNE DEPLETED, DEPLETING, THE SURVIVAL RATE COULD BE AS HIGH AS 80%. IN PATIENTS WITH HIV/AIDS TREATED WITH ANTIRETROVIRAL THERAPY, THE SURVIVAL IS OF APPROXIMATELY 50 TO 70%. BUT IN PATIENTS IN WHOM IMMUNE RECONSTITUTION CANNOT BE ACHIEVED, RAPIDLY OR EFFECTIVELY, MORTALITY REMAINS AT ABOUT 90%. IT'S IMPORTANT TO NOTE THAT EVEN WITH IMMUNE RECONSTITUTION, THE VIRUS IS OFTEN NOT CLEARED. YOU SEE HERE BELOW A GRAPH SHOWING 65 PATIENTS THAT WERE FOLLOWED LONGITUDINALLY WITH CSF PCR OF JC VIRUS, AND DESPITE IMMUNE RECONSTITUTION RELATED TO DISCONTINUATION OF TREATMENT WITH NAT NATILUZIMAB, VIRUS COULD STILL BE DETECTED. SO IMMUNE RECONSTITUTION CAN REALLY ONLY ACHIEVE TO REGAIN CONTROL OF VIRAL REPLICATION AND TO HALT DISEASE PROGRESSION, EVEN IF NOT FULLY CLEARING THE VIRUS. SO IN OUR CLINIC, OUR ATTENTION HAS REALLY BEEN FOCUSED ON PATIENTS IN WHOM WE WERE UNABLE TO RAPIDLY ACHIEVE IMMUNE RECONSTITUTION, AND OUR APPROACH WAS REALLY THAT OF TRYING TO FACILITATE IMMUNE RECONSTITUTION AND THIS WAS USING TWO STRATEGIES, THE FIRST USING CHECKPOINT INHIBITORS. THIS WORK WAS PUB PUBLISHED THIS PAST YEAR AND I'LL TELL YOU A BIT ABOUT THE RATIONALE BEHIND THIS APPROACH AS WELL AS SOME OF THE KEY FINDINGS. OUR SECOND APPROACH WAS OF ADOPTIVE IMMUNOTHERAPY USING VIRAL-SPECIFIC T-CELLS. SO CHECKPOINT INHIBITORS OBVIOUSLY HAVE FIRMLY ESTABLISHED THEMSELVES IN THE TREATMENT OF SEVERAL CANCERS, BUT THE PATHWAYS OF IMMUNE EXHAUSTION WERE REALLY FIRST DESCRIBED IN MODELS OF VIRAL INFECTION. AND SO AS WE'VE LEARNED FROM THE LCMV ANIMAL MODEL, WHEN THERE IS AN ACUTE INFECTION, NAIVE T-CELLS EXPAND RAPIDLY, GENERATING A ROBUST AND BROAD ANTIVIRAL RESPONSE THAT IS ABLE TO EFFECTIVELY CLEAR VIRUS AND SURVIVE THE INFECTION. ON THE OTHER HAND, WHEN T-CELLS ARE UNABLE TO EFFECTIVELY CLEAR VIRUS, EITHER BECAUSE THERE IS VERY HIGH ANTIGEN BURDEN OR BECAUSE PERHAPS THERE IS A DEFECTIVE IMMUNE RESPONSE, WHAT CAN HAPPEN IS THE ESTABLISHMENT OF A PERSISTENT INFECTION. AND IN THIS SCENARIO, PROGRESSIVELY OVER TIME, THE IMMUNE RESPONSE WILL PROGRESSIVELY LOSE EFFECTOR FUNCTIONS AND THIS HAS BEEN TERMED IMMUNE EXHAUSTION. THIS WAS FIRST DESCRIBED IN ANIMAL MODELS BUT HAS ALSO BEEN CLEARLY DESCRIBED IN HUMAN DISEASE SUCH AS CHRONIC INFECTIONS AS WELL AS IN CANCER. IMMUNE EXHAUSTION IS CHARACTERIZED AND ALSO MEDIATED BY A SERIES OF NON-REDUNDANT INHIBITORY RECEPTORS, OF WHOM PD IS IS PD1 IS REALLY THE DOMINANT RECEPTOR, AND TARGETING OF PD1 HAS BEEN DEMONSTRATED TO REINVIGORATE THE IMMUNE RESPONSE AND IN THE VIRAL INFECTIOUS MODELS TO ALSO LEAD TO VIRAL CLEARANCE. SO THE FIRST SUGGESTION THAT USING CHECKPOINT OR RATHER THAT THIS PATHWAY MIGHT BE RELEVANT IN PML CAME FROM THIS REPORT IN WHICH THIS GROUP WAS ABLE TO SHOW THAT PD-1 WAS IN FACT ENRICHED ON THE SURFACE OF JC VIRUS SPECIFIC -- ON THIS BASIS, WE TREATED EIGHT PATIENTS WITH PML WITH THE CHECKPOINT INHIBITOR PEMBROLIZUMAB. YOU SEE THE PATIENTS LISTED HERE. NONE OF OUR PATIENTS HAD UNDERLYING AUTOIMMUNE DISEASE BUT HAD VARIOUS CAUSES FOR THEIR CELLULAR IMMUNE IMPAIRMENT. PATIENT NUMBER ONE HAD ACTUALLY ALREADY CLINICALLY STABILIZED BEFORE RECEIVING HIS FIRST DOZE OF PEM BROAL PEMBROLIZUMAB BUT HE REMAINE D LYMPHOPENIC, WE WISHED TO SEE IF WE COULD CLEAR THE VIRUS FULLY USING THE CHECKPOINT INHIBITOR. THE REMAINING PATIENTS WERE ALL ACTIVELY PROGRESSING AT THE TIME OF TREATMENT WITH PEMBROLIZUMAB. SO WE GAVE THE PATIENTS 2 MILLIGRAMS PER KILOGRAM, PATIENTS RECEIVED BETWEEN ONE AND THREE DOSES, AND THIS WAS REALLY TOLERATED QUITE WELL. WE HAD BEEN CONCERNED ABOUT THE POSSIBILITY OF PROVOKING AN EXUBERANT INFLAMMATORY RESPONSE IN THE BRAIN, BUT ACTUALLY WE DID NOT SEE THIS. THERE WAS NO SYMPTOMATIC IRIS AND REALLY THERE WAS NO TREATMENT LIMITING TOXICITY OBSERVED WITH JUST VERY MINOR MANIFESTATION OF RASH, MOSTLY RECURRENCE OF A PREVIOUS RASH IN TWO PATIENTS. WE BELIEVE THAT PERHAPS THE REASON THIS WAS TOLL TOLERATED SO WELL IS THAT THESE PATIENTS WERE REALLY SEVERELY LYMPHOPENIC, SO THIS MAY HAVE MITIGATED SOME OF THE INFLAMMATORY RESPONSE SEEN TYPICALLY WITH CHECKPOINT INHIBITORS. WE WERE ABLE TO SHOW EFFECTIVE PD-1 BLOCKADE IN THE BLOOD ON CD8 AND CD4 CELLS AS WELL AS IN THE SPINAL FLUID TO A LESSER EXTENT, IT WAS COMPLETE BLOCKADE IN BLOOD LYMPHOCYTES AND BETWEEN 50 AND 80% BLOCKADE IN THE SPINAL FLUID. THE DEGREE OF BLOCKADE IN THE SPINAL FLUID DID NOT RELATE TO CLINICAL RESPONSE. WE DEFINED CLINICAL RESPONSE BASED ON CLINICAL SYMPTOMS, RADIOLOGICAL AS WELL AS VIROLOGICAL PARAMETERS AND AS SUCH, THERE WERE FIVE RESPONDERS. FIVE OF THESE SUBJECTS HAD CLINICAL AND RADIOLOGICAL IMPROVEMENT OR STABILIZATION WITH DECREASE IN JC COPY NUMBER IN THE SPINAL FLUID. THREE OF THOSE PATIENTS SURVIVED GREATER THAN TWO YEARS FROM THEIR LAST INFUSION, WHICH IS A PRETTY REMARKABLE ACHIEVEMENT. TWO OF THEM DIED AT 16 AND 18 MONTHS AFTER THEIR LAST INFUSION DUE TO UNDERLYING DISEASE WHICH WAS NOT LONGER BEING TREATED WITH SUPPRESSIVE MEDICATION. AMONG THE NON-RESPONDERS, THREE PATIENTS, ONE PATIENT HAD NO INCREMENTAL BENEFIT FROM THIS VIRAL MEDICATION. THE REMAINING TWO PATIENTS DIED OF PML WITHIN SIX MONTHS OF THEIR FIRST VISIT TO NIH. YOU SEE HERE DEPICTED JC COPY NUMBERS, IN RED ARE THE NON-RESPONDERS, AND IN BLUE, THE RESPONDERS. AMONG THE RESPONDERS, YOU SEE IN EVERYBODY SOME DEGREE OF DECLINE IN JC COPY NUMBER IN THE CSF. THIS PATIENT RIGHT HERE IS PATIENT NUMBER ONE WHO JUST HAD A STABLE VIRAL COPY NUMBER, AND THESE ARE THE OTHER TWO PATIENTS WHO DIED OF PML. AND THESE OBVIOUSLY HAVE A MUCH HIGHER COPY NUMBER THAN THE OTHER PATIENTS AND PERHAPS THIS SIGNIFIES OVERWHELMING INFECTION, MORE SEVERE EXHAUSTION THAT COULD NOT BE OVERCOME WITH OUR TREATMENT INTERVENTION. AS PROOF OF PRINCIPLE, IMPORTANTLY WE WERE ABLE TO DEMONSTRATE WITH SAMPLES THAT WE HAD PRE AND POST TREATMENT IN SIX PATIENTS THAT AMONG THE NON-RESPONDERS, THERE FS WAS REALLY& NO BOOSTING OF ANTIVIRAL RESPONSES TO THE IMMUNODOMINANT PEPTIDES VP1 AND LT OF JC FOLLOWING ADMINISTRATION OF PEMBROLIZUMAB AT TIME ZERO SO THESE ARE COMPLETELY FLAT. IN THE RESPONDERS, ON THE OTHER HAND, THERE WAS A TEMPORALLY ASSOCIATED BOOST IN ANTIVIRAL RESPONSES TO THE IMMUNODOMINANT PEPTIDES. FOR ONE PATIENT, WE WERE LUCKY ENOUGH TO HAVE SAMPLES DATING BACK MORE THAN THREE YEARS BECAUSE THIS PATIENTS WAS ACTUALLY FOLLOWED FOR HIS CLL AT NIH, AND 40 MONTHS PRIOR TO TREATMENT WITH PEMBROLIZUMAB, THESE ARE HIS ANTIVIRAL ACTIVITIES. THIS IS THE PERCENT OF CD4 REACTIVE CELLS TO EITHER VP1 OR LT AND JC IMMUNE RESPONSES AGAINST THE RELATED POLYOWE MA VIRUS BK AND EBV. WITH TREATMENT FOR CLL, THAT ANTIVIRAL RESPONSE WENT DOWN SIGNIFICANTLY FOR ALL THREE VIRUSES THAT WE LOOKED AT. HE DEVELOPED PML AT MONTH MINUS ZERO, AND AT TIME .0, RECEIVED TREATMENT WITH PEMBROLIZUMAB WHICH WAS ASSOCIATED WITH A BOOSTING OF ANTIVIRAL RESPONSES THAT WAS ACROSS THE BOARD TO ALL THE VIRUSES TESTED. WHILE HE DID NOT REGAIN PRE-MORBID ANTIVIRAL ACTIVITY, THIS WAS SUFFICIENT FOR HIM TO CONTAIN INFECTION AND TO SURVIVE HIS PML. SO IN SUMMARY, OUR EXPERIENCE OF PD-1 INHIBITORS SHOWED THAT THIS WAS SAFE WITH NO SERIOUS ADVERSE REACTIONS AND NO IRIS. WE WERE ABLE TO SHOW THAT PEMBROLIZUMAB DID REDUCE THE DETECTION OF PD-1 IN THE BLOOD AND IN THE SPINAL FLUID OF THESE PATIENTS AND THAT THIS WAS ASSOCIATED WITH ENHANCED ANTIVIRAL ACTIVITY IN VITRO. IN TERMS OF EFFICACY, FIVE OUT OF THE EIGHT PATIENTS DEMONSTRATED DECLINE IN CSF COPY NUMBER WHICH WAS ASSOCIATED WITH CLINICAL AND RADIOLOGICAL IMPROVEMENT OR STABILIZATION. SINCE OUR REPORT, THERE HAVE BEEN SEVERAL OTHER REPORTS OF USE OF CHECKPOINT INHIBITORS IN PML AND THIS STAGE WHAT WE'LL NEED TO FOLLOW IS AN ADEQUATELY POWERED STUDY TO UNDERSTAND THE ROLE OF THIS TYPE OF TREATMNT STRATEGY IN PML. SO OUR SECOND APPROACH HAS BEEN THAT OF ADOPTIVE IMMUNOTHERAPY USING VIRAL-SPECIFIC T-CELLS. SO FOR THOSE OF YOU NOT FAMILIAR WITH VIRAL-SPECIFIC T-CELLS, THESE ARE CELLS THAT ARE GENERATED BY COLLECTING PERIPHERAL BLOOD MONO NOW CLEAR CELLS MONO NUCLEAR CELLS FROM A DONOR. THESE ARE THEN EXPANDED IN VITRO, AND IN OUR CASE, IN THE CENTER FOR CELLULAR ENGINEERING HERE AT NIH, WE USED PEPTIDE LIBRARIES THAT WERE 15-MER PEPTIDES WITH 11 AMINO ACID OVERLAP PEPTIDE, AFTER ABOUT 14 DAYS OF CULTURE, YOU CAN HARVEST A VIRAL-SPECIFIC CELL PRODUCT WHICH CAN THEN EITHER BE CRYOPRESERVED OR ADMINISTERED TO THE PATIENT FOR TREATMENT OF THE INFECTION. SO THIS TYPE OF STRATEGY IS USED MOST COMMONLY IN THE SETTING HEMATOPOIETIC STEM CELL TRANSPLANTATION EITHER FOR THE PREVENTION OR FOR THE TREATMENT OF REFRACTORY INFECTIONS WHICH CAN GREATLY INCREASE MORBIDITY AND MORTALITY IN THIS SETTING, SO TYPICALLY AGAINST BK, CMV, ADENOVIRUS, AS WELL AS EBV. THE FIRST SUGGESTION THAT THIS TYPE OF STRATEGY MIGHT BE USED IN PML CAME WITH THIS REPORT OF A PATIENT WHO HAD A HISTORY OF BONE MARROW TRANSPLANTATION, SUBSEQUENTLY TREATED FOR DVHD FOR SEVERA YEARS WHO THEN DEVELOPED PML AND WAS TREATED WITH VIRAL-SPECIFIC T-CELLS GENERATED FROM THE SAME DONOR WHO HAD BEEN USED FOR THE TRANSPLANTATION AND THIS PATIENT DID SURVIVE. A SECOND REPORT IN THIS PAST YEAR WAS OF THREE PATIENTS WHO WERE ACTUALLY TREATED USING OFF THE SHELF THIRD PARTY ALLOGENIC BK VIRUS-SPECIFIC T-CELLS WHICH THE GROUP ALREADY HAD AVAILABLE FOR THEIR HIGH PRESSURE STEM CELL TRANSPLANTATION PROTOCOLS AND ALSO I THINK FOR KIDNEY TRANSPLANT PATIENTS. BECAUSE OF THE DEGREE OF CROSS REACTIVITY BETWEEN BK AND JC, THEY SPECULATED THEY COULD USE BK VIRAL CELLS FOR THE TREATMENT OF PML AND, IN FACT, IN THESE THREE PATIENTS, TWO OF THEM SURVIVED PML, SUGGESTING THAT THIS WAS, IN FACT, A GOOD STRATEGY. WE HAD SIMILAR OPPORTUNITY HERE AT NIH BECAUSE OF A EXISTING PROTOCOL USING MULTI-VIRUS-SPECIFIC T-CELLS IN COLLABORATION WITH THE CENTER FOR CELLULAR ENGINEERING, AND AS PART OF THEIR MULTI-VIRUS CELL PRODUCT, THEY WERE ALSO GENERATING CELLS AGAINST BK VIRUS. AND SO WE WERE ABLE TO SHOW THAT THE DEGREE OF CROSS REACTIVITY BETWEEN BK AND JC WAS SUCH THAT WE COULD REASONABLY EXPECT THAT THIS CELL PRODUCT MIGHT ALSO BE EFFECTIVE FOR TREATMENT OF PML, AND THIS IS WHAT WE SET OUT TO EXPLORE. SO OUR GOAL WAS TO TREAT 10 PATIENTS WITH PML AS PART OF A PILOT STUDY. OUR PATIENTS HAD TO HAVE CLINICALLY DEFINITE PML WITH A POSITIVE JC VIRUS PCR IN THE CSF, HAD TO HAVE ACTIVE PML PROGRESSION, AND WE WERE, OF COURSE, FOE FOCUSING OUR EFFORTS ON PATIENTS WHO DID NOT HAVE READILY REVERSIBLE IMMUNE SUPPRESSED STATE. FOR DONORS, WE USED FIRST DEGREE RELATIVES WHO HAD AT LEAST PARTIAL HLA COMPATIBILITY AND WHEN MULTIPLE DONORS WERE AVAILABLE, WE GAVE PREFERENCE TO THOSE WITH THE BEST HLA MATCH AND WHO WERE JC AND BODY POSITIVE. SO OUR STUDY OUTCOMES, IN TERMS OF OUR PRIMARY OUT COME, SAFETY, FIRST OF ALL, WE WERE CONCERNED ABOUT THE POSSIBILITY OF INDUCING AN INFLAMMATORY RESPONSE IN THE BRAIN AGAINST THE PML, AND WE WERE ALSO CONCERNED ALTHOUGH LESS SO OF THE POSSIBILITY OF DEVELOPING JVHD. THIS IS NOT REALLY DESCRIBED SO MUCH USING VIRAL-SPECIFIC T-CELLS BUT IT'S ALWAYS A CONCERN. OUR OTHER PRIMARY QUESTION WAS IF THIS WAS EVEN FEASIBLE AS A THERAPEUTIC APPROACH, WOULD WE BE ABLE TO GENERATE A VIRAL T-CELL-SPECIFIC -- WITHIN A TIME FRAME TO ACTUALLY CHANGE THE CLINICAL OUTCOME OF THE PATIENT. AND OF COURSE SECONDARY OUTCOMES WERE LOOKING AT EFFICACY USING CLINICAL RADIOLOGICAL AS WELL AS IMMUNOLOGICAL OUTCOME MEASURES. THIS IS A SCHEMA OF OUR TRIAL. IN ORDER TO TRY TO STREAMLINE OUR TIMING, WE WERE TRYING TO SCREEN PATIENTS AS WELL AS POTENTIAL DONORS AT THE SAME TIME. IDEALLY, THE DONOR SCREENING AND CELL MANUFACTURING PROCESS FROM START TO FINISH WOULD BE COMPLETED WITHIN 15 TO 20 DAYS. THIS CLEARLY PROVED TO BE QUITE AMBITIOUS, AND I WOULD SAY THAT MOST OFTEN IT TOOK AT LEAST ONE TO TWO MONTHS AND AT TIMES LONGER THAN THAT AS WELL. ONCE PATIENTS RECEIVED THEIR FIRST INFLEUTION, THEY WERE INFUSION, THEY WERE FOLLOWED FOR A 28-DAY SAFETY MONITORING PERIOD WITH CLINICAL, RADIOLOGICAL AS WELL AS LABORATORY OUTCOMES INCLUDING SPINAL FLUID, AND THEN FOLLOWING THAT 28-DAY PERIOD, PATIENTS WERE ELIGIBLE FOR UP TO TWO ADDITIONAL INFUSIONS FOR THE SECOND AND THIRD INFUSION, WE WERE ALLOWED TO DOUBLE THE DOSE OF THE CELLS ADMINISTERED SO THE FIRST INFUSION WAS 1 MILLION CELLS PER KILOGRAM, FOR THE SECOND AND THIRD, WE WERE ALLOWED TO GIVE 2 MILLION CELLS PER KILOGRAM. FOLLOWING THE THIRD OR FINAL INFUSION, PATIENTS WOULD THEN ENTER INTO A FOLLOW-UP PERIOD WHICH WAS DESIGNED TO GO OUT FOR ONE YEAR. SO THIS IS A SCHEMA OF THE PATIENTS. WE SCREENED 26 PATIENTS. OF THESE, 14 WERE EXCLUDED, SIX BECAUSE THEY DIED BEFORE EVER RETURNING TO NIH, ONE OF THEM WAS THE PATIENT THAT I SHOWED YOU AT THE VERY BEGINNING OF THIS TALK. SEVEN OF THE PATIENTS ACTUALLY STABILIZED ON THEIR OWN, AND SO WERE NO LONGER ELIGIBLE FOR ENROLLMENT. OF INTEREST, FOUR OF THOSE PATIENTS ACTUALLY HAD UNDERLYING UNTREATED SARCOIDOSIS, WHICH IS A VERY INTERESTING CATEGORY OF PATIENTS THAT CAN DEVELOP PML. ONE PATIENT RESPONDED TO ALTERNATIVE TREATMENT, WHICH WAS ACTUALLY CHECKPOINT INHIBITION. 12 OF THE PATIENTS RECEIVED ONEINFUSION, 10 RECEIVED TWO INFUSIONS, AND SEVEN PATIENTS RECEIVED A TOTAL OF THREE INFUSIONS. THIS IS THE LIST OF OUR PATIENTS WITH VARIED UNDERLYING CAUSE FOR THEIR IMMUNE-SUPPRESSED STATE. IN THIS STUDY, WE DID INCLUDE SEVERAL PATIENTS WITH AUTOIMMUNE DISEASE WHO HAD BEEN TREATED WITH MEDICATIONS THAT COULD NOT BE READILY REVERSED. IMPORTANTLY WE SAW NO TREATMENT LIMITING TOXICITY, THE TREATMENT RELATED ADVERSE EVENTS WERE REALLY QUITE FEW. THE DONORS AT THE TOP WERE LARGELY RELATED TO LEUKOPHORESIS, AND FOR THE PATIENTS, MOST OF THE ADVERSE EVENTS THAT WE OBSERVED WERE RELATED TO EXPECTED EVENTS FOLLOWING LUMBAR PUNCTURE. IN TERMS OF SURVIVAL, SO RATHER THE WAY THAT WE GAUGED RESPONSE WAS REALLY BY SURVIVAL OF PML, AND SEVEN OF THE PATIENTS SURVIVED THEIR PML. TWO OF THE PATIENTS WITHDREW FROM STUDY PRIOR TO THEIR FOLLOW-UP, BUT WE KNOW THAT THEY WERE STABLE AT 16 AND 13 MONTHS FOLLOWING THEIR LAST INFUSION. OF THOSE THAT STAYED IN THE TRIAL AND COMPLETED FOLLOW-UP, ALL OF THEM HAD SOME DEGREE OF NEUROLOGICAL IMPROVEMENT AS ASSESSED BY NEUROLOGICAL EXAM. TWO DIED ONE YEAR AFTER THE LAST INFUSION RELATED TO THEIR UNDERLYING DISEASE. THE PATIENTS WHO DIED OF PML ON THE OTHER HAND ARE FIVE, AND THESE ALL DIED WITHIN THREE WEEKS TO THREE MONTHS AFTER THEIR LAST INFUSION OF VIRAL-SPECIFIC T-CELLS. THE PATIENTS WERE ASSESSED BY NEUROLOGICAL EXAM AND THEN ALSO BY THREE VALIDATED DISABILITY RATING SCALES, THE KARNOSKY PERFORMANCE SCALE, THE MODIFIED RANKIN SCALE AND ALSO -- I HAVE DEPICTED THE INITIAL BASELINE SCORE AS WELL AS THE FINAL SCORE OF THE MMSE, AS WELL AS A GRAPHIC TO SORT OF SHOW YOU QUICKLY THE DIRECTION OF THE CHANGE, WHICH AMONG THE SEVEN RESPONDERS FOR THE MOST PART REMAINED EITHER STABLE OR HAD SOME IMPROVEMENT IN THEIR SCORES, WHILE AMONG THE NON-RESPONDERS, THE MAJORITY SHOWED A CLEAR DECLINE IN SCORES AND THIS WAS TRUE ALSO FOR THE OTHER VALIDATED CLINICAL SCALES. HERE YOU SEE EXAMPLES OF MRIs OF TWO PATIENTS, ON THE TOP A NON-RESPONDER, ON THE BOTTOM, A RESPONDER. THE SCANS WERE TAKEN IN THE MONTHS LEADING UP TO THE FIRST INFUSION WHERE YOU SEE PROGRESSIVE INCREASE IN SIZE OF THE LESION EXTENSION. FOLLOWING THE FIRST INFUSION, AND THE SECOND AND THIRD INFUSION FOR THESE PATIENTS, THERE WAS IN THE NON-RESPONDER CONTINUED EXPANSION OF THE LESION SIZE, WHILE IN THE RESPONDER, THERE WAS STILL SOME EXTENSION OF THE LESION FOLLOWING THE FIRST INFUSION, BUT AFTER THAT, PROGRESSIVE DECREASE IN SIZE OF THE LESION AS WELL AS ASSOCIATED FOCAL ATROPHY. IN NEITHER OF THE CATEGORIES OF PATIENTS DID WE EVER SEE SIGNIFICANT CONTRAST ENHANCEMENT SUGGESTIVE OF EXUBERANT INFLAMMATORY RESPONSE. HERE YOU SEE DEPICTED JC COPY NUMBERS. IN RED ARE THE NON-RESPONDERS AND IN BLUE, THE RESPONDERS. IN THE RESPONDERS, WE SAW SOME DEGREE OF DECLINE IN THE COPY NUMBER OVER TIME WHILE THE NON-RESPONDERS REMAINED GROSSLY STABLE IN TERMS OF THEIR COPY NUMBER. AS BEFORE, THE NON-RESPONDERS TEND TO BE PATIENTS WITH HIGHER COPY NUMBER, AND THIS IS ALSO SUPPORTED IN THE LITERATURE THAT HIGH COPY NUMBER OF VIRUS IN THE SPINAL FLUID IS CERTAINLY PREDICTIVE OF A POOR PROGRESS PROGNOSIS AND PROBABLY SUGGESTIVE OF OVERWHELMING INFECTION AND NO ABILITY TO MOUNT ANY SORT OF ANTIVIRAL RESPONSE. LOW COPY NUMBER, ON THE OTHER HAND, IS PERHAPS A LITTLE LESS STRAIGHTFORWARD AND IF WE PLOT HERE THE COPY NUMBER OF THE PATIENTS WHO WERE UNABLE TO RETURN TO NIH BECAUSE THEY DIED IN THE INTERVENING TIME, WE CAN SEE THAT A LOW COPY NUMBER PROBABLY IS MORE SUGGESTIVE OF A WINDOW OF OPPORTUNITY FOR TREATMENT RATHER THAN NECESSARILY A GOOD PROGNOSIS. SO IN TRYING TO TEASE OUT WHAT FACTORS MAY BE USEFUL FOR UNDERSTANDING RESPONSE TO TREATMENT, WE NEED TO CONSIDER DONOR AND CELL PRODUCT FACTORS, OBVIOUSLY EACH CELL PRODUCT THAT IS MANUFACTURED IS A DIFFERENT AND INDEPENDENT PRODUCT, AND IT'S ALSO PATIENT FACTORS. SO IN TERMS OF THE CELL PRODUCTS, WHAT WE SAW AND HERE DEPICTED ARE THE CELL COMPOSITION BETWEEN THE GROUPS WITH NON-RESPONDERS DEPICTED IN THIS SORT OF PINK COLOR AND THE RESPONDERS IN BLUE. THE COMPOSITION OF CD3, CD8 AND CD4 CELLS BETWEEN PRODUCTS RECEIVED BY NON-RESPONDERS AND RESPONDERS DID NOT REALLY DIFFER SIGNIFICANTLY, BUT ONE DIFFERENCE WHICH WAS NOTABLE WAS THAT THE NON-RESPONDERS RECEIVED CELL PRODUCT THAT HAD SIGNIFICANTLY HIGHER NUMBER OF NON-T-CELL POPULATION WHICH WERE CD56-POSITIVE, MOST LIKELY REPRESENTING NK CELLS, WHICH ARE KNOWN TO INTERFERE WITH T-CELL FUNCTION IN VIRAL-SPECIFIC PRODUCTS. AND THIS MAY OFFER AN OPPORTUNITY OF HOW WE MIGHT BE ABLE TO OPTIMIZE THE CELL PRODUCT BY ACTUALLY SELECTING OUT THE NK CELLS. MORE INTERESTING PERHAPS IS THAT COMPARING RESPONDERS AND NON-RESPONDERS AND THE ROBUSTNESS OF THE ANTIVIRAL ACTIVITY IN THAT CELL PRODUCT, THE RESPONDERS ACROSS THE BOARD RECEIVED CELL PRODUCT THAT HAD MORE ROBUST ANTIVIRAL RESPONSES COMPARED TO THE NON-RESPONDERS AND THIS IS REALLY INTERESTING, BECAUSE IF WE CONSIDER THAT THE NON-RESPONDERS WERE THOSE PATIENTS WITH THE HIGHEST JC COPY NUMBERS WHO SEEM TO BE REALLY UNABLE TO MOUNT ANY SORT OF ANTIVIRAL RESPONSE AND THEIR FIRST DEGREE RELATIVES SIMILARLY WERE UNABLE TO GENERATE A SIGNIFICANT ANTIVIRAL RESPONSE AND THIS SUGGESTS THE POSSIBILITY THAT THERE MAY BE SOME GENETIC FACTORS UNDERLYING ANTIVIRAL ACTIVITY. SORT OF CONFOUNDING THIS, HOWEVER, IS THE FACT THAT THE PRODUCTS THAT WERE GIVEN TO RESPONDERS TENDED TO BE FROM JCV POSITIVE DONORS, WHETHER OR NOT THOSE DONORS -- OR RATHER THE JC ANTIBODY NEGATIVE DONORS ACTUALLY HAVE NEVER SEEN VIRUS OR WHETHER THERE'S SOMETHING DIFFERENT ABOUT THEIR IMMUNE RESPONSE AGAINST THE VIRUS, WE DON'T KNOW FOR SURE. BUT CERTAINLY IT LOOKS THAT TESTING FOR JC ANTIBODY POSITIVITY MAY BE A GOOD APPROACH TO OPTIMIZE CELL PRODUCT. ACTIVITY. SO AMONG THE PATIENTS, WE LOOKED AT VARIOUS FEATURES INCLUDING AGE, SEX, AS WELL AS DEGREE OF IMMUNE SUPPRESSION, AND ALSO TIME FROM SYMPTOM ONSET TO INITIAL INFUSION AND THERE REALLY WERE NO SIGNIFICANT DIFFERENCES BETWEEN RESPONDERS AND NON-RESPONDERS. THIS IS OBVIOUSLY A SMALL GROUP AND SO IT COULD BE THAT ENLARGING THESE NUMBERS, WE MAY BE ABLE TO SEE SOMETHING MORE RELEVANT. ONE OF THE BIG QUESTIONS THAT WE HAVE, OF COURSE, IS WHAT IS THE FATE OF THE TRANSFERRED CELLS, HOW LONG DO THE DONOR CELLS SUR VOIF AND SURVIVE AND DO THEY EVEN ENTER THE CENTRAL NERVOUS SYSTEM. ONE WAY THAT WE TRIED TO ADDRESS THIS IN THE ORIGINAL TRIAL DESIGN WAS LOOKING AT SORT TANDEM REPEAT KIE MAYORISM BUT IN THE FIRST PATIENTS TO RECEIVE CELL PRODUCT INFUSIONS WE WERE REALLY UNABLE TO DETECT ANY DONOR CELLS AT ALL SO WE STOPPED DOING THIS ASSAY, AND WHAT WE HAVE DONE IS TURN TO LOOKING AT HLA TYPING IN THE CSF OF LYMPHOCYTES POST TRANSFUSION TO LOOK FOR THAT MISMATCH BETWEEN DONORS AND PATIENTS. AND AT LEAST IN ONE PATIENT IN WHOM THIS ASSAY WAS COMPLETED, THERE SEEMS TO BE THE SUGGESTION THAT WE ARE, IN FACT, ABLE TO DETECT SOME NON-HOST HLA, BUT THIS IS STILL ONGOING. FOR ONE OF THE PATIENTS WHO DIED, A NON-RESPONDER, WE ACTUALLY WERE GIFTED HER BRAIN, AND WE HOPE TO LOOK FOR DONOR CELLS IN THE BRAIN TISSUE. WHAT WE KNOW IS THAT EVEN THAT VIRAL-SPECIFIC T-CELLS CAN ACTUALLY BE FOUND EVEN IN TISSUES OF PATIENTS WHO DON'T RESPOND AND DO SUCCUMB TO THE INFECTION, SO THE FACT THAT SHE WAS NOT A RESPONDER DOESN'T AUTOMATICALLY MEAN WE MAY NOT BE ABLE TO FIND DONOR CELLS. SO IN SUMMARY, IN TERMS OF SAFETY, WE REALLY DIDN'T SEE ANY INFUSION REACTIONS, WE SAW NO SEVERE IRIS, AND WE HAD NO SUGGESTION OF GBHD. IN TERMS OF FEASIBILITY, DEFINITELY THE TIMELY SCREENING AND MANUFACTURE OF CELL PRODUCTS WAS QUITE CHALLENGING AND WE WILL NEED TO TRY TO DO BETTER TO STREAMLINE THAT PROCESS CERTAINLY. WE ALSO RECOGNIZE NOW THE IMPORTANCE OF IDENTIFYING GOOD DONORS AND THINK THAT WE HAVE SOME THOUGHTS ABOUT HOW TO DO THAT AND ALSO TO GO ABOUT HOW TO OPTIMIZE THE CELL PRODUCT ITSELF. IN TERMS OF EFFICACY, CONSIDERED THE EXPECTED SUR VIEFERL OF THESE SURVIVAL AT THE OUTSET -- SO NEXT STEPS, WE WOULD LIKE TO IMPROVE OUR UNDERSTANDING OF THE DETERMINANTS OF IMMUNE RESPONSE TO JC VIRUS, WE'D LIKE TO OPTIMIZE OUR CELL PRODUCT POTENCY, WE MAY ALSO EXPLORE COMBINING OUR TWO TECHNIQUE, SO USING ADJUNCT IMMUNE MODULATORS SUCH AS CHECKPOINT INHIBITORS WITH VIRAL SPECIFIC T-CELL PRODUCTS, AND THEN, OF COURSE, WE'LL NEED TO DO LARGER TRIALS THAT ARE ACTUALLY POWERED TO DETERMINE EFFICACY. A LOT OF PEOPLE WERE INVOLVED IN THESE STUDIES AND I NEED TO THANK EVERYONE, AS WELL AS THE PATIENTS AND THEIR FAMILIES. SO WITH THAT, I AM HAPPY TO TAKE ANY QUESTIONS. [APPLAUSE] >> SO IF YOU HAVE QUESTIONS, CAN YOU PLEASE GO TO THE MICROPHONES AND WE'LL GET STARTED. OKAY. >> YOU MENTIONED THAT THERE'S -- YOU WERE LOOKING AT THE GENETIC PREDISPOSITION OF WHO COULD RESPOND OR NOT RESPOND TO YOUR PARTICULAR THERAPY. BUT DO THESE CASES, THE ONES THAT GO ON TO JC VIRUS, FOR EXAMPLE, THE ONES THAT WOULD BE SPORADIC AND DON'T HAVE A KNOWN ETIOLOGY, WOULD THEY HAVE SOME TYPE OF FAMILIAL BASIS THAT MIGHT BE DISCERNIBLE ON A GWAS OR A -- OR NEXT GENERATION SEQUENCING OR SOMETHING? IN OTHER WORDS, DO THEY CLUSTER IN SOME WAYS, THE ONES THAT WOULD BEING THOUGHT OF AS SPORADIC, BECAUSE YOU GAVE AN UNDERLYING CAUSE FOR THE INCREASE IN THE OTHER TYPES. >> RIGHT. SO WE DID NOT LOOK AT THIS SPECIFICALLY. WE JUST SPECULATE THAT THERE MAY BE SOME GENETIC FACTORS BASED ON THE POTENCY OF VIRAL PRODUCTS THAT WE DEVELOPED FROM FIRST DEGREE RELATIVES OF PEOPLE WITH PML, BUT THERE CERTAINLY ARE SOME HLA ASSOCIATIONS THAT HAVE BEEN IDENTIFIED WITH BETTER AND MORE POOR ANTIVIRAL RESPONSES. TO MY KNOWLEDGE, THE TYPE OF EFFORT THAT YOU'RE DESCRIBING HAS NOT BEEN DONE AND WE'RE CERTAINLY INTERESTED IN LOOKING AT THAT MORE CLOSELY. >> OKAY. THANK YOU VERY MUCH. >> SURE. >> SO I HAVE A FEW QUESTIONS. SO IT SOUNDS LIKE THE JC VIRUS DISSEMINATES PRETTY WIDELY ONCE IT INFECTS. BUT WHAT SPECIFIC CELL TYPES WITHIN THE CENTRAL NERVOUS SYSTEM DOES THE VIRUS ACTUALLY INFECT? IS THIS REALLY A DISORDER OF GLIAL CELLS, IS THIS A DISORDER REALLY OF NEURONS, COMBINATION OF BOTH? WHAT DO WE KNOW ABOUT THAT? >> RIGHT. SO, IT IS -- CLASSIC PML IS A DISORDER OF GLIAL CELLS, AND CLASSIC PML IS CAUSED BY ALLEY TICK INFECTION OF OLIGODENDROCYTES, ALTHOUGH ASTROCYTES CAN ALSO BE INFECTED. THERE ARE SOME VARIANTS. VP1 VARIANTS SPECIFICALLY THAT HAVE BEEN ASSOCIATED WITH THE ABILITY TO INFECT NEURONS AND SPECIFIC GRANULE CELLS IN THE CEREBELLUM AS WELL AS PYRAMIDAL CELLS THAT ARE ASSOCIATED WITH RELATIVELY UNIQUE LIN CLINICAL TEE FEWERS, IF HE TOURS BUT CLATIONICALLY THE INFECTION IS INVOLVING OLIGODENDROCYTES >> THAT'S INTERESTING BECAUSE AS I UNDERSTAND IT, ASTROCYTES REALLY ARE SORT OF DENDRITIC CELLS OR EQUIVALENT TO DENDRITIC CELLS IN THE CENTRAL NERVOUS SYSTEM, UNLESS I HAVE THAT MISTAKEN. IS THAT YOUR UNDERSTANDING? >> THAT'S NOT THE WAY THAT I THINK ABOUT IT, BUT -- YEAH, I DON'T -- I'M -- >> OKAY. WELL, SO I GUESS THAT WOULD BE SORT OF ANOTHER ASPECT FOR CLARIFICATION, BECAUSE I THINK ONE OF THE QUESTIONS THAT WAS RAISED AND I THINK YOU WERE ADDRESSING IT IS, WHAT IS THE SORT OF MECHANISM FOR RESPONSE FOR THESE TRANSFUSIONAL EVENT, THESE INFUSIONS THAT YOU'RE DOING, AND IS IT REALLY CYTOKINE-MEDIATED AND PERHAPS CYTOKINE-MEDIATED AT THE LEVEL OF EFFECTOR CELLS, LIKE ASTROCYTES, OR IS THIS REALLY SOMETHING RELATED TO THE ACTUAL CELLS THAT POTENTIALLY COULD GO ACROSS IN THE BLOOD BRAIN BARRIER AND HAVE A DIRECT EFFECT ON THESE CELLS. WHAT DO YOU THINK? >> SO WE DON'T REALLY KNOW. THE ASSUMPTION IS THEY ACTUALLY DO CROSS THE BLOOD BRAIN BARRIER AND HAVE A DIRECT EFFECT. THAT IS THE ASSUMPTION. WHETHER OR NOT THERE IS ALSO INDIRECT EFFECT, WE'RE NOT SURE. WHAT IS EXPECTED WITH THE INFUSION OF VIRAL-SPECIFIC T-CELLS IS THAT THESE EXPAND OVER THE WEEKS FOLLOWING INFUSION IN RESPONSE TO ANTIGEN ENCOUNTER, AND THAT THEN THEY DISTRIBUTE, YOU KNOW, TO -- THROUGHOUT THE BODY. >> OKAY. >> BUT I CAN'T EXCLUDE AND WE DON'T KNOW. >> OKAY. YES, SIR. >> QUICK TWO QUESTIONS. SOME OF THE NON-RESPONDERS HAD HIGHER COPY NUMBER OF THE VIRUS, AND I'M WONDERING IF YOU CAN GIVE THEM MORE ADAPTIVE CELL THERAPY, CAN YOU OVERCOME THOSE HIGHER BURDEN? AND MY SECOND QUESTION IS, SOME ADAPTIVE T-CELL THERAPY FOR CANCER OR CAR T THERAPY, YOU CAN PRECONDITION PATIENTS WITH SOME CHEMO OR SOMETHING LIKE FLEW DARE BEN TO PAVE THE WAY FOR THE T-CELL SURVIVAL, ADAPTIVE T-CELL SURVIVAL. HAVE YOU THOUGHT ABOUT THOSE KIND APPROACHES? >> SURE. SO WE OBVIOUSLY -- WE DON'T KNOW THE ANSWER TO THE FIRST QUESTION. WHAT WE CAN SAY IS THAT IN THE PREVIOUS STUDIES OF VIRAL-SPECIFIC T-CELLS, THE NUMBER OF CELLS THAT WERE ACTUALLY DELIVERED WERE EVEN LESS THAN WHAT WE DELIVERED. SO FOR EXAMPLE, I THINK IN THE VERY FIRST REPORT THAT I SHOWED YOU IN 2011, I THINK THAT 1 MILLION CELLS PER KILOGRAM WERE ADMINISTERED, AND IN THE 3BK THIRD PARTY PATIENTS, THEY WERE JUST HALF A MILLION CELLS PER KILOGRAM. SO I THINK THAT WHILE CERTAINLY WE'RE INTERESTED IN EXPLORING HIGHER DOSING AND OBSERVING WHETHER THERE'S ACTUALLY A DOSE EFFECT, I THINK THAT IT HAS MORE TO DO WITH THE POTENCY OF THE PRODUCT ITSELF, WHICH IS VERY VARIABLE ACROSS PATIENTS AND ALSO ON THE ABILITY OF THESE CELLS TO EXPAND ONCE THEY ACTUALLY ARE IN THE INDIVIDUAL INTO THE HOST AND SO IF THEY'RE ABLE TO EFFECTIVELY EXPAND IN THE HOST, THEN HOW MANY WE STARTED WITH POSSIBLY MATTERS LESS, BUT WE DON'T REALLY KNOW THE ANSWER TO THAT. IN TERMS OF PRECONDITIONING, THERE ARE TWO CONSIDERS SO FIRST OF ALL THESE PATIENTS OBVIOUSLY HAVE -- WE WANT TO PRESERVE ANY IMMUNE RESPONSE THAT THEY HAVE AND HITTING THEM WITH FURTHER CHEMOTHERAPY, THAT MIGHT WIPE OUT ANY OF THEIR OWN IMMUNE SYSTEM OR EVEN DELAY IMMUNE RECONSTITUTION FURTHER, I THINK THERE WOULD BE SOME CONCERN. BUT THAT SAID, THESE PATIENTS ARE REALLY PRETTY SEVERELY IMMUNE DEPLETED ALREADY AND SO THEY PROBABLY HAVE SPACE, I GUESS, FOR EXPANSION OF THESE DONOR CELLS TO HAPPEN AND SO WE HAVE NOT CONSIDERED THE NEED TO DO PRECONDITIONING FOR BOTH SAFETY CONCERNS AS WELL AS FOR THAT. THANK YOU. >> OKAY. SO IF THERE ARE NO MORE QUESTION, WE WANT TO THANK DR. CORTESE FOR A VERY STIMULATING PRESENTATION.