The~NEW ENGLAND
`JOU NAL of MEDICINE
`
`VOL. 365 NO. 8
`
`BSTABLISKED IN l s12
`
`AUGUST 25, 2011
`
`NEJM.ORG
`
`688
`
`THIS WEEK AT NEJM,ORG
`
`681
`
`PERSPECTIVE
`Impossible? Outlawing State Safety Laws
`for Generic Drugs L.H. Glantz and G.J. Annas
`683 The Effects of Medicaid Coverage - Learning from e16
`the Oregon Experiment K. Baicker and A. Finkelstein
`685 HIV Surveillance, Public Health, and Clinical Medicine
`- Will the Walls Come Tumbling Down?
`A.L. Fairchild and R. Bayer
`
`742
`
`743
`
`689
`
`699
`
`709
`
`ORICINAL ARTICLES
`Azithromycin for Prevention of Exacerbations of COPD
`R.K. Albert and Others
`) .
`Apixaban with Antiplatelet Therapy after Acute
`Coronary Syndrome
`J. H. Alexander and Others
`Origins of the E.coli Strain Causing an Outbreak
`of Hemolytic-Uremic Syndrome in Germany
`O.A. Rasko and Others
`718 Brief Report: Open-Source Genomic Analysis
`of Shiga-Toxin-Producing E.coli O104:H4
`H. Rohde and Others
`725 Brief Report: Chimeric Antigen Receptor-Modified 758
`T Cells in Chronic Lymphoid Leukemia
`O.L. Porter and Others
`CLINICAL THERAPEUT ICS
`734 Thrombopoietin-Receptor Agonists for Primary
`Immune Thrombocytopenia
`P. Imbach and M. Crowther
`Ow,!~ &:pirblifflnf &,_v t.ht MASSACHUSllJTS Mf:l)JCAl. SOCtnYC, 2011.
`AIJ rig.flu ,esnwd. lS.1N 002S•• 1U,
`
`753
`
`754
`
`e17
`
`761
`
`University of Southern California
`
`August 26, 2011
`
`Norris Medical Library
`
`73
`73
`75
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`_..._ " ' :11 •vvv •
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`• a~afll IN O.:tN U.l~I fJ tll IUA
`
`IMAGES IN CLINICAL MEDICINE
`Bal6's Concentric Sclerosis
`0. Wengert and E. Siebert
`Pneumatosis Cystoides Intestinalis
`S.-S. Wu and H.-H. Yen
`
`CASE RECORDS OF THE MASSACHU S ETTS
`GENERAL HOSPITAL
`A Boy with a Complex Cyst in the Kidney
`E. Paul and Others
`
`EDITORIALS
`Preventing Exacerbations of COPD - Advice
`from Hippocrates
`N.M. Siafakas
`
`Redirecting T Cells
`W.J. Urba and D. L. Longo
`
`CLI N ICAL DECISIO NS
`Treatment of a 6-Year-Old Girl with Vesicoureteral
`Reflux - Polling Results
`O.J. Lamas,J.R. lngelflnger, and L.S. Rosen baum
`
`CLINICAL IMPLICATIONS OF BASIC RESEARC H
`Systemic Lupus Erythematosus and the Neutrophil
`X. Bosch
`
`CORRES~ON DEN CE
`Transmission of Resistant Bacteria in Intensive Care
`Abiraterone and Increased Survival in Metastatic
`Prostate Cancer
`FOLFIRINOX versus Gemcitabine for Metastatic
`Pancreatic Cancer
`Selenium and the Course of Mild Graves' Orbitopathy
`gpl00 Peptide Vaccine in Melanoma
`Childhood Diarrhea Deaths after Rotavirus
`Vaccination in Mexico
`CORRECTIO.N S
`
`NOTICES
`
`CONTINUING M EDICAL EDUCATION
`
`Miltenyi Ex. 1012 Page 1
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`

`

`Th, NEW ENGLAND J OURNAL of MEDI C IN E
`
`l~l _______ s_R_r_EF_R_E_P_o_RT _ _ _ _ _ _ _ ~II
`
`Chimeric Antigen Receptor-Modified
`T Cells in Chronic Lymphoid Leukemia
`David L. Porter, M.D., Bruce L. Levine, Ph.D., Michael Kalos, Ph.D.,
`Adam Bagg, M.D., and Carl H.June, M.D.
`
`SUMMARY
`
`We designed a lentiviral vector expressing a chimeric antigen receptor with speci•
`ficity for the B-cell antigen CD19, coupled with CD137 (a costimulatory receptor in
`T cells [4-lBB]) ~nd CD3-zeta (a signal-transduction component of the T-cell antigen
`receptor) signaling domains. A low dose (approximately 1.SxlO' cells per kilogram
`of body weight) of autologous chimeric antigen receptor-modified T cells reinfused
`into a patient with refractory chronic lymphocytic leukemia (CLL) expanded to a
`level that was more than 1000 times as high as the initial engraftment level in vivo,
`with delayed development of the tumor lysis syndrome and with complete remission.
`Apart ftom the tumor lysis syndrome, the only other grade 3/4 toxic effect related
`to chimeric antigen receptor T cells was lymphopenia. Engineered cells persisted at
`high levels for 6 months in the blood and bone marrow and continued to express the
`chimeric antigen receptor. A specific immune response was detected in the bone ma.r(cid:173)
`row, accompanied by loss of normal B cells and leukemia cells that express CD19.
`Remission was ongoing 10 months after treatment. Hypogammaglobulinemia was
`an expected chronic toxic effect
`
`W ITH THE USE OF GENE-TRANSFER TECHNIQUES, TCELLS CAN BE GENET·
`
`ically modified to stably express antibodies on their surface, conferring
`new antigen specificity. Chimeric antigen receptors combine an antigen(cid:173)
`recognition domain of a specific antibody with an intracellular domain of the CD3-
`zeta chain or Fc-yRl protein into a single chimeric protein.'•' Although chimeric
`antigen receptors can trigger T-cell activation in a manner similar to that of endogenous
`T·cell receptors, a major impediment to the clinical application of this technique to
`date has been limited in vivo expansion of chimeric antigen receptor T cells and dis(cid:173)
`appointing clinical activity.••• Chimeric antigen receptor-mediated T-cell responses
`can be further enhanced with the addition of a costimulatory domain. In preclinical
`models, we found that inclusion of the CD137 (4·1BB) signaling domain significantly
`increases antitumor activity and in vivo persistence of chimeric antigen receptors as
`compared with inclusion of the CD3.zeta chain alone.•••
`In most cancers, tumor-specific antigens for targeting are not well defined, but in
`B-cell neoplasms, CD19 is an attractive target. Expression of CD19 is restricted to
`normal and malignant B cells and B-cell precursors.7 We have initiated a pilot clinical
`trial of treatment with autologous T cells expressing an anti-CD19 chimeric antigen
`receptor (CAR'.f19); three patients have been treated. Here we report on the immuno(cid:173)
`logic and clinical effects of in vivo T•cell treatment with chimeric antigen receptors
`in one of the patients, who bad advanced, p53-deficient CLL.
`
`From the Abramson Cancer Center (D.L.P.,
`8.L.L., M.K., A.8., C.H.J.), the Oep• rtment
`of Medicine (O.l.P.), the Department of
`Pathology and Laboratory Medicine (B.l.L,
`M.K., A.8., C.HJ.), and the Abramson
`Ft1milyCancer Researc.h Institute (B.l.L.,
`M.K., C.H.J.), Pe,elman School of Med;.
`cine, University of Pennsytvanla, Philadel•
`phia. Address "print requests to Or. Porter
`at the Division of Hematology and On(cid:173)
`cology, University of Pennsylvania Medical
`Center, 3400 Civic Center Blvd., PCAM 2
`West Pavilion, Philadelphia, PA 19104, or
`at david.porter@uphs.upenn.edu.
`
`Th;, artkle (10.1056/N EJMo,11038-49) was
`publlthed onAU&lJSt 10, 2011, it NEJM.o,g.
`
`N EnglJ Med 2011;365:725-33.
`C.,,,,.,.0201JM_M _ _ _
`
`N IENC.:t.J Mt:D 365;8 H!JM. 011:G AUGUST 25, 2.011
`
`725
`
`Miltenyi Ex. 1012 Page 2
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`

`

`Th, NEW ENGLAND J01JR.NAL ,fMEOICINE
`
`CASE REPORT
`
`STUDY PIIOCEDURES
`We designed a self-inactivating lentiviral vector
`The patient received a diagnosis of stage I CLL in (GeMCRJS 0607-793), which was subjected to pre-
`1996. He first required treatment after 6 years of clinical safety testing, as reported previously.'
`observation for progressive leukocytosis and ade- Methods ofT-cell preparation have also been de(cid:173)
`nopathy. ln 2002, he was treated with two cycles of scribed previously.• Quantitative polymerase-chain(cid:173)
`rituximab plus fludarabine; this treatment result• . reaction (PCR) analysis was performed to detect
`ed in normalization of blood counts and partial chimeric antigen receptor T cells in blood and bone
`resolution of adenopathy. In 2006, he received four marrow. The lower limit of quantification was de(cid:173)
`cycles of rituximab and fludarabine for disease
`termined from the standard curve; average values
`progression, again with normalization of blood below the lower limit of quantification (i.e., report(cid:173)
`counts and partial regression of adenopathy. This able but not quantifiable) are considered approxi(cid:173)
`response was followed by a 20-month progression- mate. The lower limit of quantification of the assay
`free interval and a 2-year treatment-free interval. was 25 oopies per microgram of genomic DNA.
`In February 2009, he had rapidly progressive leuko-
`Soluble-factor analysis was performed with the
`cytosis and recurrent adenopathy. His bone mar- use of serum from whole blood and bone marrow
`that was separated into aliquots for single use and
`row was extensively infiltrated with CLL. Cyto-
`genetic analysis showed that 3 oflS cells contained stored at -80"C. Quantification of soluble cytokine
`a deletion of chromosome 17p, and fluorescence in factors was performed with the use of Luminex
`situ hybridization (FISH) testing showed that 170 bead-array technology and reagents from Life
`of200 cells had a deletion involving TP5J on chro- Technologies.
`mosome 17p. He received rituximab with benda-
`mustine for one cycle and three additional cycles
`of bendamustine without rituximab (because of a
`severe allergic reaction). This treatment resulted in
`only transient improvement in lymphocytosis. Pro(cid:173)
`gressive adenopathy was documented by means of
`computed tomography (CT) after therapy.
`In December 2009, autologous T cells were
`collected by means ofleukapheresis and cryopre(cid:173)
`served. The patient then received alemtuzumab (an
`anti·CD52, mature-lymphocyte, cell-surface anti(cid:173)
`gen) for 11 weeks, with improved bematopoiesis
`and a partial resolution of adenopathy. Over the
`next 6 months, he had stable disease with persis(cid:173)
`tent, extensive marrow involvement and diffuse
`adenopathy with multiple 1· to 3,-cm lymph nodes.
`In July 2010, the patient was enrolled in a phase 1
`clinical trial of chimeric antigen receptor-modified
`T cells.
`
`RESULTS
`
`CELL INFUSIONS
`Autologous T cells from the patient were thawed
`and transduced with lentivirus to express the CD19-
`specific chimeric antigen receptor (Fig. lA). Four
`days before cell infusion, the patient received che(cid:173)
`motherapy designed for depletion oflympbocytes
`(pentostatin at a dose of 4 mg per square meter of
`body-surface area and cyclopbosphamide at a dose
`of 600 mg per square meter) without rituximab.9
`Three days after chemotherapy but before cell in(cid:173)
`fusion, the bone marrow was hypercellular with
`approximately 400/o involvement by CLL. Leukemia
`cells expressed kappa light chain and CDS, CD19,
`CD20, and CD23. Cytogenetic analysis showed two
`separate clones, both resulting in loss of chromo(cid:173)
`some 17p and the TP53 locus (46,l{Y,del(17)(p12J
`[5]/46,XY,der(17)t(17;21)(q10;q10)[5)/46,XY[14]).
`Four days after chemotherapy, the patient received
`a total of 3x10• T cells, of which 5"/o were trans•
`duced, for a total of 1.42xl07 transduced cells
`(l.46xl0' cells per kilogram) split into three con(cid:173)
`secutive daily intravenous infusions (10% on day
`1, 30% on day 2, and 60% on day 3). No postinfu(cid:173)
`sion cytokines were administered. No toxic effects
`of infusions were noted.
`
`CLINICAL RESPONSE ANO EVALUATIONS
`Fourteen days after the first infusion, the patient
`began having chills and low-grade fevers associ-
`
`MET HODS
`
`STUDY DESICN
`The trial (ClinicalTrials.gov number, NCT01029366)
`was designed to assess the safety and feasibility of
`infusing autologous CARI19 T cells in patients
`with relapsed or refractory B-cell neoplasms. The
`trial was approved by the institutional review board
`at the University of Pennsylvania. The study was
`conducted in accordance with the protocol (avail(cid:173)
`able with the full text of this article atNEJM.org).
`No commercial sponsor was involved in the study.
`
`726
`
`N liNC:.1.J MEO 365;8 NEJM.ORG AUGUST 25, 2011
`
`Miltenyi Ex. 1012 Page 3
`
`

`

`BRIEF R EPORT
`
`ated with grade 2 fatigue. Over the next 5 days,
`the chills intensified, and his temperature esca(cid:173)
`l~ted to 3~.2•c (102.S-F), associated with rigors,
`diaphores1s, anorexia, nausea, and diarrhea. He
`had no respiratory or cardiac symptoms. Because
`of the fevers, chest radiography and blood, urine,
`and s~ol cultures were performed, and were all
`negative or normal. The tumor lysis syndrome was
`diagnosed on day 22 after infusion (Fig. 18). The
`uric acid level was 10.6 mg per deciliter (630.5 µ.mol
`per liter), the phosphorus level was 4.7 mg per
`deciliter (1.5 mmol per liter) (normal range, 2.4 to
`4.7 mg per deciliter [0.8 to 1.5 mmol per liter)),
`and the lactate dehydrogenase level was 1130 U per
`liter (normal range, 98 to 192). There was evidence
`of acute kidney injury, with a creatinine level of
`2.60 mg per deciliter (U9.8 µ,mol per liter) (base(cid:173)
`line level, <1.0 mg per deciliter [<88.4 µ.mol per li(cid:173)
`ter)]. The patient was hospitalized and treated with
`fluid resuscitation and rasburicase. The uric acid
`level returned to the normal range within 24 hours,
`and the creatinine level within 3 days; he was dis(cid:173)
`charged on hospital day 4. The lactate dehydroge(cid:173)
`nase level decreased gradually, becoming normal
`over the following month.
`By day 28 after CARJ'19-cell infusion, adenop(cid:173)
`athy was no longer palpable, and on day 23, there
`was no evidence of CLL in the bone marrow (Fig.
`lC). The karyotype was now normal in 15 of 15
`cells (46,XY), and FISH testing was negative for
`deletion TP53 in 198 of 200 cells examined; this is
`considered to be within normal limits in negative
`controls. Flow-cytometric analysis showed no re(cid:173)
`sidual CLL, and B cells were not detectable (<1%
`of cells within the CDS+CD10-CD19+CD23 +
`lymphocyte gate). CT scanning performed on day
`31 after infusion showed resolution of adenopathy
`(Fig. 10).
`Three and 6 months after CART19-cell in(cid:173)
`fusion, rhe physical examination remained unre(cid:173)
`markable, with no palpable adenopathy, and CT
`scanning performed 3 months after CART19-cell
`infusion showed sustained remission (Fig. 1D).
`Bone marrow studies at 3 and 6 months a.lso
`showed no evidence of CLL by means of morpho(cid:173)
`logic analysis, karyotype analysis (46,XY), or flow(cid:173)
`cytometric analysis, with a continued lack of nor(cid:173)
`mal B cells as well. Remission had been sustained
`for 10 months as of rhis writing.
`
`the grade 3 tumor lysis syndrome described above.
`The patient had grade 1 lymphopenia at baseline
`and grade 2 or 3 lymphopenia beginning on day
`l and continuing throu_gh the most recent follow(cid:173)
`up visit, 10 months after therapy. Grade 4 lym(cid:173)
`phopenia, with an absolute lymphocyte count of
`140 cells per cubic milliimeter, was recorded on day
`19, but from day U through the most recent follow(cid:173)
`up visit, the absolute .lymphocyte count ranged
`between 390 and 780 ,cells per cubic millimeter
`(grade 2 or 3 lymphopenia). The patient had tran(cid:173)
`sient grade 1 thrombo,cytopenia (platelet count,
`98,000 to 131,000 per cubic millimeter) from day
`19 through day 26 and grade 1 or 2 neutropenia
`(absolute neutrophil count, 1090 to 1630 per cubic
`millimeter) from day :17 through day 33. Other
`signs and symptoms that were probably related to
`the study treatment included grade 1 and 2 eleva(cid:173)
`t ions in aminotransfetase and alkaline phospha(cid:173)
`tase levels, which devel,,ped 17 days after the first
`infusion and resolved by day 33. Grade 1 and 2
`constitutional symptoms consisted of fevers, chills,
`diaphoresis, myalgias, headache, and fatigue.
`Grade 2 hypogammagl.obulinemia was corrected
`with infusions of intra~enous immune globulin.
`
`ANALYSIS OF SERU M A.NC• ION E MAIROW CYTOltlN ES
`The patient's clinical r,esponse was accompanied
`by a delayed increase in levels of inflammatory
`cytokines (Fig. 2A through 2D), with levels of
`interferon--y, the interforon--y-responsive chemo(cid:173)
`kines CXCL9 and CXCLl0, and intedeukin-6 that
`were 160 times as high as baseline levels. The tem(cid:173)
`poral rise in cytokine levels paralleled the clinical
`symptoms, peaking li' to 23 days after rhe first
`CARl'19·cell infusion.
`The supematants from serial bone marrow as(cid:173)
`pirates were measured for cytokines and showed
`evidence of immune a,:tivation (Fig. 2E). Signifi(cid:173)
`cant increases in interferon--y, CXCL9, interleu(cid:173)
`kin-6, and soluble intedeukin-2 receptor were not(cid:173)
`ed, as compared with the baseline levels on the day
`before T-cell infusion; the va.lues peaked on day 23
`after rhe first CARJ'19 .. cell infusion. The increase
`in bone marrow cyto,kines coincided with rhe
`elimination ofleukemia cells from rhe marrow.
`Serum and marrow tumor necrosis factor a re(cid:173)
`mained unchanged.
`
`IDPAN SION AND PERSISTENCE O F CHIM ERIC
`ANTIGEN RECEPTOR T CIELLS
`TOXICITY O F CAltT19 CELLS
`The cell infusions had no acute toxic effects. The Real-time PCR detecte,d DNA encoding anti-CD19
`on.ly serious (grade 3 or 4) adverse event noted was chimeric antigen receptor (CAR19) beginning on
`
`N INClJ MlO }6S;8 NIJM.OltG AUGUST 25, :1011
`
`727
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`Miltenyi Ex. 1012 Page 4
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`D Contrast-Enhanced CT
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`
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`728
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`NENGL) MED 365;8 NEJM.ORG AUGUST 25, 2011
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`Miltenyi Ex. 1012 Page 5
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`Miltenyi Ex. 1012 Page 5
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`

`

`BRIEF REPORT
`
`Figure 1 (facing page). Clinical Response in the Patient .
`Panel A shows the lentiviral vector used to infect
`T cells from the patient . A pseudotyped, clinical-grade
`lentiviral vector of vesicular stomatitis virus protein G
`(pELPs 19-BB-z) d irecting e,pression of anti-CDl9 scFv
`derived from FMC63 murine monoclonal antibody, hu•
`man CO!a- hinge and transmembrane domain, and
`human ◄•lBB and C03' signaling domains was pro(cid:173)
`duced. Details of the CAR19 transgene, at the bottom
`o f the panel, show the major functional elements. The
`figure is not to scale. 3"LTR denotes 3' long terminal
`repeat; 5'LTR, s· long terminal repeat; Amp R, ampicil•
`lin resistance gene; Bovine GH Poly A, bovine growth
`hormone with polyadenylation tail; cPPT/CTS, central
`polypurine tract with central termination sequence;
`EF•la, elongation factor 1-alpha; env, envelope; gag,
`group-specific antigen; pol, HIV gene encoding poly•
`merase and reverse transcriptase; R, repeat; RRE, rev
`response e lement; scfv, single-chain variable frag•
`me nt; TM, transmembrane; and WPRE, woodchuck
`hepatitis virus posMranscriptional regulatory element.
`Panel B shows serum creatinine, uric acid, and lactate
`dehydrogenase (LDH) levels from day I to day 28 after
`the first CART19-cell infusion. The peak levels coincid(cid:173)
`ed with hosp italization for the tumor lysis syndrome.
`Panel C shows bone marrow-biopsy specimens ob(cid:173)
`tained 3 days after chemotherapy (day -1, before
`CART19-cell infusion) and 23 days and 6 months after
`CART19-cell infusion (hematoxylin and eosin). The
`baseline specimen shows hypercellular bone marrow
`(60%) with trilineage hematopoiesis, infiltrated by pre•
`dominantly interstitial aggregates of small, mature
`lymphocytes that account for 40% of tot al cellularity.
`The specimen obtained on day 23 shows residual lym(cid:173)
`phoid aggregates (10%) that were negative for chronic
`lymphoid leukemia (CLL), with a mixture ofT cells and
`COS-negative 8 cells. The specimen obtained 6 months
`after infusion shows trilineage hematopoiesis, without
`lymphoid aggregates and continued absence of CLL.
`Panel D shows contrast-enhanced CT scans obtained
`before the patient was enrolled in the study and 31 days
`and 104 days after the first infusion. The pre.infusion
`CT scan reveals l-to-3-cm bilateral masses. Regression
`of axillary lymphadenopathy occurred within 1 month
`after infusion and was sustained. Arrows highlight vari(cid:173)
`ous enlarged lymph nodes before therapy and lymph(cid:173)
`node responses on comparable CT scans after therapy.
`
`day 1 after the first infusion (Fig. 3A). More than
`a 3-log expansion of the cells in vivo was noted by
`day 21 after infusion. At peak levels, CARJ1.9 cells
`in blood accounted for more than 20% of circulat(cid:173)
`ing lymphocytes; these peak levels coincided with
`the occurrence of constitutional symptoms, the tu·
`mor Jysis syndrome (Fig. lB), and elevations in se(cid:173)
`rum cytokine levels (Fig, 2A through 2D). CART19
`cells remained detectable at high levels 6 months
`after the infusions, though the values decreased by
`a factor of 10 from peak levels. The doubling time
`
`of chimeric antigen receptor T cells in blood was
`approximately 1.2 days, with an elimination half..
`life of 31 days.
`
`C H I MERIC ANTIG EN RECEPTOR T CELLS t N I O N E
`MARROW
`CARJ1.9 cells were identified in bone marrow spec(cid:173)
`imens beginning 23 days after the first infusion
`(Fig. 3B) and persisted for at least 6 months, with
`a decay half.life of 34 days. The highest levels of
`CART19 cells in the bone marrow were identified
`at the first assessment 23 days after the first infu•
`sion and coincided with induction of an immune
`response, as indicated by cytokine-secretion pro·
`files (Fig. 2E). Flow-cytometric analysis of bone
`marrow aspirates indicated a clonal expansion
`of CD5+CD19+ cells at baseline that was absent
`1 month after infusion and in a sample obtained
`3 months after infusion (data not shown). Normal
`B cells were not detected after treatment (Fig. 3C).
`
`DISCUSS I ON
`
`We report the delayed development of the tumor
`lysis syndrome and a complete response 3 weeks
`after treatment with autologous T cells genetically
`modified to target CD19 through transduction with
`a lentivirus vector expressing a.nti-CD19 linked to
`CD3-zeta and CD137 (4·1BB) signaling domains.
`Genetically modified cells were present at high lev•
`els in bone marrow for at least 6 months after infu(cid:173)
`sion. The generation of a CD19-specific immune
`response in bone marrow was demonstrated by
`temporal release of cytokines and ablation ofleu(cid:173)
`kemia cells that coincided with peak infiltration of
`chimeric antigen receptor T cells. Development of
`the tumor Jysis syndrome after cellular immuno(cid:173)
`therapy has not been reported previously. 10 The
`finding that chimeric antigen receptor T cells are
`capable of extensive proliferation and cytotoxicity
`in vivo indicates the need for caution in the design
`of clinical trials when chimeric antigen receptors
`with new specificities are tested.11
`Genetic manipulation of autologous T cells to
`target specific tumor antigens is an attractive strat·
`egy for cancer therapy.>,• An important feature of
`this approach is that chimeric antigen receptor
`T cells can recognize tumor targets in an HLA(cid:173)
`unrestricted manner, so that "off-the-shelf' chime(cid:173)
`ric antigen receptors can be constructed for tumors
`with a wide variety of histologic features. We used
`HIV-derived lentiviral vectors for cancer therapy,
`
`N (NGLJ M(O 365;8 NIEJM.OltC AUGUST 25, 2011
`
`729
`
`Miltenyi Ex. 1012 Page 6
`
`

`

`TIit NEW ENGLAND JOU RNAL, of M EO JC INE
`
`A lntene<on-y
`300
`
`250
`
`200
`
`lSO
`
`100
`
`50
`
`0, . . ................... .--.---.--..,.....:. , . , - _ . . . , . _
`- 1 0
`I
`2
`3 15 17 21 23 31 76 lOS 1'3 176
`
`Days after Infusion
`
`C CXCL9
`8000
`
`6000
`
`I 4000
`
`!)
`~
`V
`
`2000
`
`8 cxcuo
`10,000
`
`a,ooo
`'W J 6,000
`~ 4,000
`
`2,000
`
`D lnterleukin-6
`100
`
`80
`
`20
`
`Days aft.et Infusion
`
`o_.,,,'f!=~ '!""'f-,-----,-----,-----,-----,::,~~Hl--
`- 1 0
`l
`2
`3
`IS 17 21 23 31 76 lOS 10 176
`Days aftet Infusion
`
`0
`
`I
`
`2 3 15 17 21 23 31 76 105 143 176
`Days after Infusion
`
`E
`
`Immune Response in Bone Marrow
`II l OitY l>d°Oft
`lnfus.On
`
`so
`
`O 23 Oays afttr ■ 31 Days after
`rnf,,,sion
`inf,,,sion
`
`■ IOS Oays afttr ■ 176 Days aftu
`lnfusk>n
`infusion
`12,000
`
`E 40
`I 10
`-8
`
`20
`
`1 10
`
`10.000
`
`i
`1 ,.ooo
`
`6,000
`
`4,000
`
`-8 s !
`
`2,000
`
`CXCL9
`
`lntet1eukln-2 ,eceptor
`
`an approach that may have some advantages over
`the use of retroviral vectors."
`In previous trials of chimeric antigen receptor
`T cells, objective tumor responses have been mod·
`est, and in vivo proliferation of modified cells has
`not been sustained.,. . ., We developed a second(cid:173)
`generation chimeric antigen receptor designed to
`
`address this limitation by incorporation of the
`CD137 (4-188) signaling domain, on the basis of
`our preclinical observation that this molecule
`promotc!d the persistence of antigen-specific and
`antigen••nonspecific chimeric antigen receptor
`T-cells. • ·• Brentjens and colleagues reponed pre(cid:173)
`hmmaiy results of a clinical trial ofCD19-targeted
`
`730
`
`N lN<.LJ Mt:O ;65;& NlJM,OIIC AUGUST 25, 2011
`
`Miltenyi Ex. 1012 Page 7
`
`

`

`BRIEF REPORT
`
`Figure 2 (facing page). Serum and Bone Marrow
`Cytokines before and after Chimeric Antigen Receptor
`T-Cell Infusion.
`Serial measurements of the cytokine interferon--y
`(Panel A), the interferon-y-stimulated chemokines C-X-C
`motif chemokine 10 (CXCLIO) (Panel 8) and C·X·C motif
`ligand 9 (CXCL9) (Panel C), and interleukin-6 (Panel 0)
`were measured at the indicated time points. The in(cid:173)
`creases in these innammatory cytokines and chemo•
`kines coincided with the onset of the tumor lysis syn•
`drome. low levels of interleukin-6 were detected at
`baseline, whereas interferon-y, CXCL9, and CXCUO
`were below the limits of detection at biillseline. Standard(cid:173)
`curve ranges for the analytes and baseline values in
`the patient, given in parentheses, were as follows:
`interferon--,,, 11.2 to 23,972 pg per m illiliter (1.4 pg per
`m'.ml~er); CXCLIO, 2.1 to 5319 pg per milliliter (274 pg per
`molhl,ter); CXCL9, 48.2 to 3700 pg per milliliter (177 pg
`per milliliter); interleukin-6, 2.7 to 4572 pg per milliliter
`(8.3 pg per milliliter); tumor necrosis factor a (TNF-a),
`1.9 to 4005 pg per milliliter (not detectable); and soluble
`interleukin-2 receptor, 13.4 to 34,210 pg per milliliter
`(644 pg per milliliter). Panel E shows the induction of
`the immune response in bone marrow. The cytokines
`TNF-a, interleukin-6, intcrferon--y, chemokine CXCL9,
`and soluble intcrleukin-2 receptor were measu,ed in
`supernatant fluids obtained from bone marrow aspirates
`on the indicated days before and after CART19-cell
`infusion. The increases in levels of interleukin-6,
`interferon--y, CXCL9, and soluble interleukin-2 receptor
`coincided with the tumor lysis syndrome, peak chimeric
`antigen receptor T~ccll infiltration, and eradication of
`the leukemic infiltrate.
`
`chimeric antigen receptors linked to a CD28 sig•
`naling domain and found transient tumor respons•
`es in two of three patients with advanced CLL16;
`however, the chimeric antigen receptors rapidly
`disappeared from the circulation.
`It was unexpected that the very low dose of
`chimeric antigen receptor T cells that we infused
`would result in a clinically evident antitumor re(cid:173)
`sponse. Indeed, the infused dose of 1.5x10' chi·
`meric antigen receptor T cells per kilogram was
`several orders of magnitude below doses used in
`previous studies of T cells modified to express
`chimeric antigen receptors or transgenic T-cell
`receptors.u,16-10 We speculate that the chemother•
`apy may potentiate the effects of chimeric antigen
`receptor T cells in several ways, including increas·
`ing engraftment and migration to tumor cells,19
`as well as potentiating the ability of chimeric anti·
`gen receptor T cells to kill stressed tumor cells that
`would otherwise survive the chemotherapy. 20
`21
`,
`Whether the inclusion of exogenous cytokines
`would further increase the activity of chimeric an(cid:173)
`tigen receptor T cells is not known.
`
`The prolonged persistence of CAR:r19 cells in
`the blood and bone marrow of our patient may
`result ftom inclusion of the 4-lBB signaling do(cid:173)
`main. It is likely that the CART19·cell-mediated
`elimination of normal B cells facilitated the induc·
`tion of immunologic tolerance to the chimeric
`antigen receptor, since the CART19 cells that ex(cid:173)
`press the single-chain Fv antibody fragment con•
`raining murine sequences were not rejected. Given
`the absence of detectable CO19-positive leukemia
`cells in this patient, it is possible that homeostasis
`of the chimeric antigen receptor T cells was
`achieved at least in part from stimulation deliv(cid:173)
`ered by early B·cell progenitors as they began to
`emerge in the bone nurrow. We speculate that this
`would be a new mechanism to maintain "memo(cid:173)
`ry" chimeric antigen receptor T cells.
`Although C019 is an attractive tumor target,
`with expression limited to normal and malignant
`B cells, there is concern that persistence of the
`chimeric antigen receptor T cells will mediate
`long·tenn B•cell deficiency. In fact, in our patient,
`B cells were absent from the blood and bone mar(cid:173)
`row for at least 6 months after infusion. This pa·
`tient did not have recurrent infections. Targeting
`B cells through CD20 with rituximab is an effec(cid:173)
`tive and relatively safe strategy for patients with
`B-cell neoplasms, and long-term B-cell lymphope·
`nia is manageable. 22 Patients treated with ritux(cid:173)
`imab have been reported to have a return ofB cells
`within months after discontinuation of therapy. It
`is not yet clear whether such recovery will occur in
`patients whose anti-B-cell T cells persist in vivo.
`Patients who have CLL with TP53 deletions have
`short remissions after standard therapies. 23 Alloge(cid:173)
`neic bone marrow transplantation has been the
`only approach that has induced long-term remis(cid:173)
`sions in patients with advanced CLL.24 However,
`the resulting potent graft-versus-tumor effect is as•
`sociated with considerable morbidity because of the
`high frequency of chronic graft-versus-host disease,
`which is often especially severe in older patients
`those who are typically affected by CLL. ••·"
`-
`Our study suggests that genetically modified au(cid:173)
`tologous T cells may circumvent this limitation.
`The delayed onset of the tumor lysis syndrome
`and cytokine secretion, combined with vigorous in
`vivo chimeric antigen receptor T-cell expansion
`and prominent antileukemia activity, points to
`substantial and sustained effector functions of the
`CART19 cells. Our early research highlights the
`potency of this therapy and provides support for
`the detailed study of autologous T cells geneti-
`
`N EMGI.J Ml:0 )65;8 Nl!JM.O.-G AUC.UST 25, 2011
`
`73l
`
`Miltenyi Ex. 1012 Page 8
`
`

`

`To, N EW ENG LAND JOURNAL of MEDI CINE
`
`A WholeBlood
`
`Tr:;in$gcn~ copie:s
`
`<
`z 10'
`,a,
`'S
`~ l()l
`f
`_; 10'
`...
`8
`l! 10'
`t
`~
`
`o
`
`20 ~ 60
`
`ao ~ rn ~ ~
`Oars after Infusion
`
`C Flow.Cytometric Analyses
`
`so
`
`40
`
`l
`JO t
`20 i. e s
`
`10
`
`B Bone Marrow Aspirates
`..
`< z 10'
`0
`'S
`~ 10'
`1
`l 10'
`I 101
`...
`~ JO"
`
`-1
`
`20
`
`40
`
`80 100 120
`60
`Days after Infusion
`
`140
`
`160 180
`
`""0'°"_4; -l_Oa~y'-~-,-l_nfu_ sion_,:-94:--:_g~- - - - - - --- - - - -_,-,o".2:-l:..Do=y_;before=f=lnfu_s,_·on_
`
`,oii:.Jil
`
`......... ,
`
`...................
`
`_ _
`t.oaio Units
`loC11t Units
`l Mc>nth aft.er lnNsion
`l Month after tnfustOn
`_,....,0,-,.7:----,r-----::0-::.4T - - - - - - - - - - - - -·=21"'_9:---, - - - - - ;46-;-_g.'l·
`
`',,,,.....,1.~o _
`
`.. ...
`1 .!l
`
`logio Unfu
`
`CD5•FITC
`
`2.7
`
`t.oci.Unit$
`
`lmmunoglobulin Kappa.APC
`
`Figure 3. Expansion and Persistence of Chimeric Antigen Receptor T Cells in Vivo.
`Genomic ONA (gDNA) was isolated from samples of the patient's whole blood (Panel A) and bone marrow aspirates (Panel B) collected at
`serial time points before and after chimeric antigen receptor T-cell infusion and used for quantitative real-time polymerase-chain-reaction
`(PCR) analysis. As assessed on the basis of transgenic DNA an~ the per~entag~ ~~lymphocytes expressing CAR19. the chimeric antigen
`receptor T cells expanded to levels that we,e mofe than 1000 t1mts as high as 1n1t1al cngraftment levels in the peripheral blood and bone
`marrow. Peak leve ls of chime,ic a ntigen ,cccpto, T cells were temporally cor,elatcd wlth the tumor lysis syndrome . A blood sample ob(cid:173)
`tained on day o and a bone mam:rw sample obtained on day - 1 had no PCR signal at base line. Flow.cytometric analysis of bone m arrow
`aspirates at baseline (Panel C) shows predominant infiltration with C019+CDS+ cells that were clonal, as assessed by means of immu(cid:173)
`nogfobulin kappa light-chain staining, with a paucity ofT cells. On day 31 afte, infusion, CDS+ T cells were present, and no normal or
`malignant B cells were detected. The numbers indicate the ,elative frequency of cells in each quadrant. Both the x axis and they axis
`show a log10 scale. The gating strategy invofved an lnltlal gati~g on COl9+ and CDS+ celfs in the boxes on the left, and the subsequent
`identification of immunoglobulin kappa and lambda expression on the CD19+CDS+ subset (boxes on the right).
`
`732
`
`N EN CLJ M ED 365;8 NEJM ,OJtC AUGUST 2 5, 201'
`
`Miltenyi Ex. 1012 Page 9
`
`

`

`BRIEF REPOR.T
`
`cally modified to target CD19 (and other targets)
`thr~gh transduction of a chimeric antigen recep(cid:173)
`tor _linked to po~nt signaling domains. Unlike
`annbody-media~ therapy, chimeric antigen re(cid:173)
`ceptor-modified T cells