T h e new england journal o f medicine

Brief Report

Chimeric Antigen Receptor–Modified T Cells in Chronic Lymphoid 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 [4-1BB]) and CD3-zeta (a signal-transduction component of the T-cell antigen receptor) signaling domains. A low dose (approximately 1.5×105 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 from 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 mar- 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.

ith the use of gene-transfer techniques, T cells can be genet- From the Abramson Cancer Center (D.L.P., ically modified to stably express on their surface, conferring B.L.L., M.K., A.B., C.H.J.), the Department of Medicine (D.L.P.), the Department of new antigen specificity. Chimeric antigen receptors combine an antigen- Pathology and Laboratory Medicine (B.L.L., W M.K., A.B., C.H.J.), and the Abramson recognition domain of a specific with an intracellular domain of the CD3- zeta chain or FcγRI protein into a single chimeric protein.1,2 Although chimeric Family Cancer Research Institute (B.L.L., M.K., C.H.J.), Perelman School of Medi- antigen receptors can trigger T-cell activation in a manner similar to that of endogenous cine, University of Pennsylvania, Philadel- T-cell receptors, a major impediment to the clinical application of this technique to phia. Address reprint requests to Dr. Porter date has been limited in vivo expansion of chimeric antigen receptor T cells and dis- at the Division of and On- 3,4 cology, University of Pennsylvania Medical appointing clinical activity. Chimeric antigen receptor–mediated T-cell responses Center, 3400 Civic Center Blvd., PCAM 2 can be further enhanced with the addition of a costimulatory domain. In preclinical West Pavilion, Philadelphia, PA 19104, or models, we found that inclusion of the CD137 (4-1BB) signaling domain significantly at [email protected]. increases antitumor activity and in vivo persistence of chimeric antigen receptors as This article (10.1056/NEJMoa1103849) was compared with inclusion of the CD3-zeta chain alone.5,6 published on August 10, 2011, at NEJM.org. In most cancers, tumor-specific antigens for targeting are not well defined, but in N Engl J Med 2011;365:725-33. B-cell neoplasms, CD19 is an attractive target. Expression of CD19 is restricted to Copyright © 2011 Massachusetts Medical Society. 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 (CART19); three patients have been treated. Here we report on the immuno- logic and clinical effects of in vivo T-cell treatment with chimeric antigen receptors in one of the patients, who had advanced, p53-deficient CLL.

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Study Procedures Case Report We designed a self-inactivating lentiviral vector The patient received a diagnosis of stage I CLL in (GeMCRIS 0607-793), which was subjected to pre- 1996. He first required treatment after 6 years of clinical safety testing, as reported previously.5 observation for progressive and ade- Methods of T-cell preparation have also been de- nopathy. In 2002, he was treated with two cycles of scribed previously.8 Quantitative polymerase-chain- 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- 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- counts and partial regression of adenopathy. This able but not quantifiable) are considered approxi- 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 copies 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 row was extensively infiltrated with CLL. Cyto- that was separated into aliquots for single use and genetic analysis showed that 3 of 15 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 of 200 cells had a deletion involving TP53 on chro- Technologies. mosome 17p. He received rituximab with benda- mustine for one cycle and three additional cycles Results of bendamustine without rituximab (because of a severe allergic reaction). This treatment resulted in Cell Infusions only transient improvement in . Pro- Autologous T cells from the patient were thawed gressive adenopathy was documented by means of and transduced with lentivirus to express the CD19- computed tomography (CT) after therapy. specific chimeric antigen receptor (Fig. 1A). Four In December 2009, autologous T cells were days before cell infusion, the patient received che- collected by means of leukapheresis and cryopre- motherapy designed for depletion of served. The patient then received alemtuzumab (an (pentostatin at a dose of 4 mg per square meter of anti-CD52, mature-, cell-surface anti- body-surface area and cyclophosphamide at a dose gen) for 11 weeks, with improved hematopoiesis of 600 mg per square meter) without rituximab.9 and a partial resolution of adenopathy. Over the Three days after chemotherapy but before cell in- next 6 months, he had stable disease with persis- fusion, the bone marrow was hypercellular with tent, extensive marrow involvement and diffuse approximately 40% involvement by CLL. Leukemia adenopathy with multiple 1- to 3-cm lymph nodes. cells expressed kappa light chain and CD5, CD19, In July 2010, the patient was enrolled in a phase 1 CD20, and CD23. Cytogenetic analysis showed two clinical trial of chimeric antigen receptor–modified separate clones, both resulting in loss of chromo- T cells. some 17p and the TP53 locus (46,XY,del(17)(p12) [5]/46,XY,der(17)t(17;21)(q10;q10)[5]/46,XY[14]). Methods Four days after chemotherapy, the patient received a total of 3×108 T cells, of which 5% were trans- Study Design duced, for a total of 1.42×107 transduced cells The trial (ClinicalTrials.gov number, NCT01029366) (1.46×105 cells per kilogram) split into three con- was designed to assess the safety and feasibility of secutive daily intravenous infusions (10% on day infusing autologous CART19 T cells in patients 1, 30% on day 2, and 60% on day 3). No postinfu- with relapsed or refractory B-cell neoplasms. The sion cytokines were administered. No toxic effects trial was approved by the institutional review board of infusions were noted. at the University of Pennsylvania. The study was conducted in accordance with the protocol (avail- Clinical Response and Evaluations able with the full text of this article at NEJM.org). Fourteen days after the first infusion, the patient No commercial sponsor was involved in the study. began having chills and low-grade fevers associ-

726 n engl j med 365;8 nejm.org august 25, 2011 The New England Journal of Medicine Downloaded from nejm.org by J ROBINSON on May 13, 2014. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. brief report ated with grade 2 fatigue. Over the next 5 days, the grade 3 tumor lysis syndrome described above. the chills intensified, and his temperature esca- The patient had grade 1 lymphopenia at baseline lated to 39.2°C (102.5°F), associated with rigors, and grade 2 or 3 lymphopenia beginning on day diaphoresis, anorexia, nausea, and diarrhea. He 1 and continuing through the most recent follow- had no respiratory or cardiac symptoms. Because up visit, 10 months after therapy. Grade 4 lym- of the fevers, chest radiography and blood, urine, phopenia, with an absolute lymphocyte count of and stool cultures were performed, and were all 140 cells per cubic millimeter, was recorded on day negative or normal. The tumor lysis syndrome was 19, but from day 22 through the most recent follow- diagnosed on day 22 after infusion (Fig. 1B). The up visit, the absolute lymphocyte count ranged uric acid level was 10.6 mg per deciliter (630.5 μmol between 390 and 780 cells per cubic millimeter per liter), the phosphorus level was 4.7 mg per (grade 2 or 3 lymphopenia). The patient had tran- deciliter (1.5 mmol per liter) (normal range, 2.4 to sient grade 1 thrombocytopenia (platelet count, 4.7 mg per deciliter [0.8 to 1.5 mmol per liter]), 98,000 to 131,000 per cubic millimeter) from day and the lactate dehydrogenase level was 1130 U per 19 through day 26 and grade 1 or 2 neutropenia liter (normal range, 98 to 192). There was evidence (absolute neutrophil count, 1090 to 1630 per cubic of acute kidney injury, with a creatinine level of millimeter) from day 17 through day 33. Other 2.60 mg per deciliter (229.8 μmol per liter) (base- signs and symptoms that were probably related to line level, <1.0 mg per deciliter [<88.4 μmol per li- the study treatment included grade 1 and 2 eleva- ter]). The patient was hospitalized and treated with tions in aminotransferase and alkaline phospha- fluid resuscitation and rasburicase. The uric acid tase levels, which developed 17 days after the first level returned to the normal range within 24 hours, infusion and resolved by day 33. Grade 1 and 2 and the creatinine level within 3 days; he was dis- constitutional symptoms consisted of fevers, chills, charged on hospital day 4. The lactate dehydroge- diaphoresis, myalgias, headache, and fatigue. nase level decreased gradually, becoming normal Grade 2 hypogammaglobulinemia was corrected over the following month. with infusions of intravenous immune globulin. By day 28 after CART19-cell infusion, adenop- athy was no longer palpable, and on day 23, there Analysis of Serum and Bone Marrow Cytokines was no evidence of CLL in the bone marrow (Fig. The patient’s clinical response was accompanied 1C). The karyotype was now normal in 15 of 15 by a delayed increase in levels of inflammatory cells (46,XY), and FISH testing was negative for cytokines (Fig. 2A through 2D), with levels of deletion TP53 in 198 of 200 cells examined; this is interferon-γ, the interferon-γ–responsive chemo- considered to be within normal limits in negative kines CXCL9 and CXCL10, and interleukin-6 that controls. Flow-cytometric analysis showed no re- were 160 times as high as baseline levels. The tem- sidual CLL, and B cells were not detectable (<1% poral rise in cytokine levels paralleled the clinical of cells within the CD5 + CD10 – CD19 + CD23 + symptoms, peaking 17 to 23 days after the first lymphocyte gate). CT scanning performed on day CART19-cell infusion. 31 after infusion showed resolution of adenopathy The supernatants from serial bone marrow as- (Fig. 1D). pirates were measured for cytokines and showed Three and 6 months after CART19-cell in­ evidence of immune activation (Fig. 2E). Signifi- fusion, the physical examination remained unre- cant increases in interferon-γ, CXCL9, interleu- markable, with no palpable adenopathy, and CT kin-6, and soluble interleukin-2 receptor were not- scanning performed 3 months after CART19-cell ed, as compared with the baseline levels on the day infusion showed sustained remission (Fig. 1D). before T-cell infusion; the values peaked on day 23 Bone marrow studies at 3 and 6 months also after the first CART19-cell infusion. The increase showed no evidence of CLL by means of morpho- in bone marrow cytokines coincided with the logic analysis, karyotype analysis (46,XY), or flow- elimination of leukemia cells from the marrow. cytometric analysis, with a continued lack of nor- Serum and marrow tumor necrosis factor α re- mal B cells as well. Remission had been sustained mained unchanged. for 10 months as of this writing. Expansion and Persistence of Chimeric Toxicity of CART19 Cells Antigen Receptor T Cells The cell infusions had no acute toxic effects. The Real-time PCR detected DNA encoding anti-CD19 only serious (grade 3 or 4) adverse event noted was chimeric antigen receptor (CAR19) beginning on

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A Lentiviral Vector B Serum Creatinine, Uric Acid, and LDH R region 3.5 12 1400 Truncated gag/pol 5'LTR cPPT/CTS 3.0 Creatinine 1200 Amp R 10 RRE Uric acid 2.5 LDH 1000 Truncated 8 Bacterial pELPS 19-BB-ζ env 2.0 800 replication 11556 bp origin 6 EF-1α 1.5 600

3'LTR (truncated) 4 LDH (IU/liter) Uric Acid (mg/dl) Bovine GH Poly A Creatinine (mg/dl) 1.0 400 R region (truncated) CD19 BBζ 0.5 2 200 WPRE U3 (truncated) 0.0 0 0 FMC63 Human CD8α 0 5 10 15 20 25 30 scFv Hinge and TM 4-1BB CD3ζ Days after Infusion VH VL

C Bone Marrow–Biopsy Specimens Day –1 (baseline) Day 23 6 Mo

D Contrast-Enhanced CT Axial Coronal

Before Therapy

1 Mo of Treatment

3 Mo of Treatment

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of chimeric antigen receptor T cells in blood was Figure 1 (facing page). Clinical Response in the Patient. approximately 1.2 days, with an elimination half- Panel A shows the lentiviral vector used to infect T cells from the patient. A pseudotyped, clinical-grade life of 31 days. lentiviral vector of vesicular stomatitis virus protein G (pELPs 19-BB-z) directing expression of anti-CD19 scFv Chimeric Antigen Receptor T Cells in Bone derived from FMC63 murine , hu- Marrow man CD8α hinge and transmembrane domain, and CART19 cells were identified in bone marrow spec- human 4-1BB and CD3ζ signaling domains was pro- duced. Details of the CAR19 transgene, at the bottom imens beginning 23 days after the first infusion of the panel, show the major functional elements. The (Fig. 3B) and persisted for at least 6 months, with figure is not to scale. 3′LTR denotes 3′ long terminal a decay half-life of 34 days. The highest levels of repeat; 5′LTR, 5′ long terminal repeat; Amp R, ampicil- CART19 cells in the bone marrow were identified lin resistance gene; Bovine GH Poly A, bovine growth at the first assessment 23 days after the first infu- hormone with polyadenylation tail; cPPT/CTS, central polypurine tract with central termination sequence; sion and coincided with induction of an immune EF-1α, elongation factor 1-alpha; env, envelope; gag, response, as indicated by cytokine-secretion pro- group-specific antigen; pol, HIV gene encoding poly- files (Fig. 2E). Flow-cytometric analysis of bone merase and reverse transcriptase; R, repeat; RRE, rev marrow aspirates indicated a clonal expansion response element; scFv, single-chain variable frag- of CD5+CD19+ cells at baseline that was absent ment; TM, transmembrane; and WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. 1 month after infusion and in a sample obtained Panel B shows serum creatinine, uric acid, and lactate 3 months after infusion (data not shown). Normal dehydrogenase (LDH) levels from day 1 to day 28 after B cells were not detected after treatment (Fig. 3C). the first CART19-cell infusion. The peak levels coincid- ed with hospitalization for the tumor lysis syndrome. Panel C shows bone marrow–biopsy specimens ob- Discussion tained 3 days after chemotherapy (day –1, before CART19-cell infusion) and 23 days and 6 months after We report the delayed development of the tumor CART19-cell infusion (hematoxylin and eosin). The lysis syndrome and a complete response 3 weeks baseline specimen shows hypercellular bone marrow after treatment with autologous T cells genetically (60%) with trilineage hematopoiesis, infiltrated by pre- modified to target CD19 through transduction with dominantly interstitial aggregates of small, mature lymphocytes that account for 40% of total cellularity. a lentivirus vector expressing anti-CD19 linked to The specimen obtained on day 23 shows residual lym- CD3-zeta and CD137 (4-1BB) signaling domains. phoid aggregates (10%) that were negative for chronic Genetically modified cells were present at high lev- lymphoid leukemia (CLL), with a mixture of T cells and els in bone marrow for at least 6 months after infu- CD5-negative B cells. The specimen obtained 6 months sion. The generation of a CD19-specific immune after infusion shows trilineage hematopoiesis, without lymphoid aggregates and continued absence of CLL. response in bone marrow was demonstrated by Panel D shows contrast-enhanced CT scans obtained temporal release of cytokines and ablation of leu- before the patient was enrolled in the study and 31 days kemia cells that coincided with peak infiltration of and 104 days after the first infusion. The preinfusion chimeric antigen receptor T cells. Development of CT scan reveals 1-to-3-cm bilateral masses. Regression the tumor lysis syndrome after cellular immuno- of axillary lymphadenopathy occurred within 1 month 10 after infusion and was sustained. Arrows highlight vari- therapy has not been reported previously. The ous enlarged lymph nodes before therapy and lymph- finding that chimeric antigen receptor T cells are node responses on comparable CT scans after therapy. capable of extensive proliferation and cytotoxicity in vivo indicates the need for caution in the design day 1 after the first infusion (Fig. 3A). More than of clinical trials when chimeric antigen receptors a 3-log expansion of the cells in vivo was noted by with new specificities are tested.11 day 21 after infusion. At peak levels, CART19 cells Genetic manipulation of autologous T cells to in blood accounted for more than 20% of circulat- target specific tumor antigens is an attractive strat- ing lymphocytes; these peak levels coincided with egy for cancer therapy.3,4 An important feature of the occurrence of constitutional symptoms, the tu- this approach is that chimeric antigen receptor mor lysis syndrome (Fig. 1B), and elevations in se- T cells can recognize tumor targets in an HLA- rum cytokine levels (Fig. 2A through 2D). CART19 unrestricted manner, so that “off-the-shelf” chime- cells remained detectable at high levels 6 months ric antigen receptors can be constructed for tumors after the infusions, though the values decreased by with a wide variety of histologic features. We used a factor of 10 from peak levels. The doubling time HIV-derived lentiviral vectors for cancer therapy,

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A Interferon-γ B CXCL10 300 10,000

250 8,000

200 6,000 150 4,000

100 CXCL10 (pg/ml) Interferon-γ (pg/ml) 2,000 50

0 0 −1 0 1 2 3 15 17 21 23 31 76 105 143 176 −1 0 1 2 3 15 17 21 23 31 76 105 143 176 Days after Infusion Days after Infusion

C CXCL9 D Interleukin-6 8000 100

80 6000

60 4000 40 CXCL9 (pg/ml)

2000 Interleukin-6 (pg/ml) 20

0 0 −1 0 1 2 3 15 17 21 23 31 76 105 143 176 −1 0 1 2 3 15 17 21 23 31 76 105 143 176 Days after Infusion Days after Infusion

E Immune Response in Bone Marrow 1 Day before 23 Days after 31 Days after 105 Days after 176 Days after infusion infusion infusion infusion infusion 50 12,000

10,000 40

8,000 30 6,000 20 4,000

Concentration (pg/ml) 10 Concentration (pg/ml) 2,000

0 0 TNF-α Interleukin-6 Interferon-γ CXCL9 Interleukin-2 receptor

AUTHOR: June (Porter) RETAKE: 1st an approach that may have some advantages over address this2nd limitation by incorporation of the FIGURE: 2 of12 3 3rd the use of retroviral vectors. CD137Revised (4-1BB) signaling domain, on the basis of ARTIST: ts In previous trials of chimeric antigen receptor our preclinicalSIZE observation that this molecule 7 col T cells, objective tumorTYPE: responsesLine haveCombo been4-C modH/T- promoted36p6 the persistence of antigen-specific and est, and in vivo proliferation of modifiedAUTHOR, PLEASE cells hasNOTE: antigen-nonspecific chimeric antigen receptor not been sustained.13-15Figure We developedhas been redrawn a second- and type has T-cells.been reset.5,6 Brentjens and colleagues reported pre- generation chimeric antigen receptorPlease designed check carefully. to liminary results of a clinical trial of CD19-targeted JOB: 36508 ISSUE: 08-25-11 730 n engl j med 365;8 nejm.org august 25, 2011 The New England Journal of Medicine Downloaded from nejm.org by J ROBINSON on May 13, 2014. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. brief report

The prolonged persistence of CART19 cells in Figure 2 (facing page). Serum and Bone Marrow Cytokines before and after Chimeric Antigen Receptor the blood and bone marrow of our patient may T-Cell Infusion. result from inclusion of the 4-1BB signaling do- Serial measurements of the cytokine interferon-γ main. It is likely that the CART19-cell–mediated (Panel A), the interferon-γ–stimulated chemokines C-X-C elimination of normal B cells facilitated the induc- motif chemokine 10 (CXCL10) (Panel B) and C-X-C motif tion of immunologic tolerance to the chimeric ligand 9 (CXCL9) (Panel C), and interleukin-6 (Panel D) antigen receptor, since the CART19 cells that ex- were measured at the indicated time points. The in- creases in these inflammatory cytokines and chemo- press the single-chain Fv antibody fragment con- kines coincided with the onset of the tumor lysis syn- taining murine sequences were not rejected. Given drome. Low levels of interleukin-6 were detected at the absence of detectable CD19-positive leukemia baseline, whereas interferon-γ, CXCL9, and CXCL10 cells in this patient, it is possible that homeostasis were below the limits of detection at baseline. Standard- of the chimeric antigen receptor T cells was curve ranges for the analytes and baseline values in the patient, given in parentheses, were as follows: achieved at least in part from stimulation deliv- interferon-γ, 11.2 to 23,972 pg per milliliter (1.4 pg per ered by early B-cell progenitors as they began to milliliter); CXCL10, 2.1 to 5319 pg per milliliter (274 pg per emerge in the bone marrow. We speculate that this milliliter); CXCL9, 48.2 to 3700 pg per milliliter (177 pg would be a new mechanism to maintain “memo- per milliliter); interleukin-6, 2.7 to 4572 pg per milliliter ry” chimeric antigen receptor T cells. (8.3 pg per milliliter); tumor necrosis factor α (TNF-α), 1.9 to 4005 pg per milliliter (not detectable); and soluble Although CD19 is an attractive tumor target, interleukin-2 receptor, 13.4 to 34,210 pg per milliliter with expression limited to normal and malignant (644 pg per milliliter). Panel E shows the induction of B cells, there is concern that persistence of the the immune response in bone marrow. The cytokines chimeric antigen receptor T cells will mediate TNF-α, interleukin-6, interferon-γ, chemokine CXCL9, long-term B-cell deficiency. In fact, in our patient, and soluble interleukin-2 receptor were measured in supernatant fluids obtained from bone marrow aspirates B cells were absent from the blood and bone mar- on the indicated days before and after CART19-cell row for at least 6 months after infusion. This pa- infusion. The increases in levels of interleukin-6, tient did not have recurrent infections. Targeting interferon-γ, CXCL9, and soluble interleukin-2 receptor B cells through CD20 with rituximab is an effec- coincided with the tumor lysis syndrome, peak chimeric tive and relatively safe strategy for patients with antigen receptor T-cell infiltration, and eradication of the leukemic infiltrate. B-cell neoplasms, and long-term B-cell lymphope- nia is manageable.22 Patients treated with ritux- imab have been reported to have a return of B cells chimeric antigen receptors linked to a CD28 sig- within months after discontinuation of therapy. It naling domain and found transient tumor respons- is not yet clear whether such recovery will occur in es in two of three patients with advanced CLL16; patients whose anti–B-cell T cells persist in vivo. however, the chimeric antigen receptors rapidly Patients who have CLL with TP53 deletions have disappeared from the circulation. short remissions after standard therapies.23 Alloge- It was unexpected that the very low dose of neic bone marrow transplantation has been the chimeric antigen receptor T cells that we infused only approach that has induced long-term remis- would result in a clinically evident antitumor re- sions in patients with advanced CLL.24 However, sponse. Indeed, the infused dose of 1.5×105 chi- the resulting potent graft-versus-tumor effect is as- meric antigen receptor T cells per kilogram was sociated with considerable morbidity because of the several orders of magnitude below doses used in high frequency of chronic graft-versus-host disease, previous studies of T cells modified to express which is often especially severe in older patients chimeric antigen receptors or transgenic T-cell — those who are typically affected by CLL.24,25 receptors.13,16-18 We speculate that the chemother- Our study suggests that genetically modified au- apy may potentiate the effects of chimeric antigen tologous T cells may circumvent this limitation. receptor T cells in several ways, including increas- The delayed onset of the tumor lysis syndrome ing engraftment and migration to tumor cells,19 and cytokine secretion, combined with vigorous in as well as potentiating the ability of chimeric anti- vivo chimeric antigen receptor T-cell expansion gen receptor T cells to kill stressed tumor cells that and prominent antileukemia activity, points to would otherwise survive the chemotherapy.20,21 substantial and sustained effector functions of the Whether the inclusion of exogenous cytokines CART19 cells. Our early research highlights the would further increase the activity of chimeric an- potency of this therapy and provides support for tigen receptor T cells is not known. the detailed study of autologous T cells geneti-

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A Whole Blood B Bone Marrow Aspirates 50 Transgene copies 104 104 40

103 103 30

102 102 20

1 1 10 Lymphocytes (%) 10 10 Lymphocytes 100 100 Transgene copies (no./µg of gDNA) 0 Transgene copies (no./µg of gDNA) 0 20 40 60 80 100 120 140 160 180 −1 20 40 60 80 100 120 140 160 180 Days after Infusion Days after Infusion

C Flow-Cytometric Analyses

1 Day before Infusion 1 Day before Infusion 0.4 94.8 0.2 0.3

3.5 1.3 1.0 98.5

Log10 Units Log10 Units 1 Month after Infusion 1 Month after Infusion 0.7 0.4 27.9 46.8 CD19-PerCP-Cy5.5 Immunoglobulin Lambda-PE

64.5 34.4 22.5 2.7

Log10 Units Log10 Units

CD5-FITC Immunoglobulin Kappa-APC

Figure 3. Expansion and Persistence of Chimeric Antigen Receptor T Cells In Vivo. Genomic DNA (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 and the percentage of lymphocytes expressing CAR19, the chimeric antigen receptor T cells expanded to levels that were more than 1000 times as high as initial engraftment levels in the peripheral blood and bone marrow. Peak levels of chimeric antigen receptor T cells were temporally correlated with the tumor lysis syndrome. A blood sample ob- tained on day 0 and a bone marrow sample obtained on day −1 had no PCR signal at baseline. Flow-cytometric analysis of bone marrow aspirates at baseline (Panel C) shows predominant infiltration with CD19+CD5+ cells that were clonal, as assessed by means of immu- noglobulin kappa light-chain staining, with a paucity of T cells. On day 31 after infusion, CD5+ T cells were present, and no normal or malignant B cells were detected. The numbers indicate the relative frequency of cells in each quadrant. Both the x axis and the y axis show a log10 scale. The gating strategy involved an initial gating on CD19+ and CD5+ cells in the boxes on the left, and the subsequent identification of immunoglobulin kappa and lambda expression on the CD19+CD5+ subset (boxes on the right).

732 n engl j med 365;8 nejm.org august 25, 2011 The New England Journal of Medicine Downloaded from nejm.org by J ROBINSON on May 13, 2014. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. brief report cally modified to target CD19 (and other targets) the Alliance for Cancer , and the Leukemia and Society (7000-02). through transduction of a chimeric antigen recep- Disclosure forms provided by the authors are available with tor linked to potent signaling domains. Unlike the full text of this article at NEJM.org. antibody-mediated therapy, chimeric antigen re- We thank Irina Kulikovskaya for the quantitative polymerase- chain-reaction (Q-PCR) assay; Erica Suppa and Casey Krebs for ceptor–modified T cells have the potential to rep- the Luminex assay; Jennifer Wright for Q-PCR assay development; licate in vivo, and long-term persistence could lead John Scholler for assay development; Tatiana Mikheeva for sample to sustained tumor control. Two other patients processing; Qun-Bin Xiong for flow-cytometric analysis; Zhaohui Zheng, Julio Cotte, Andrea Brennan, and members of the Clinical with advanced CLL have also received CART19 in- Cell and Vaccine Production facility for developing methods for fusions according to this protocol, and all three clinical-scale ex vivo lentiviral transduction and for cell manufactur- have had tumor responses.26 These findings war- ing; the Human Immunology Core for reagents; Boro Dropulic (Len- tigen) for clinical-grade vector production; Elizabeth Veloso, Lester rant continued study of CD19-redirected T cells for Lledo, Joan Gilmore, Gwendolyn Binder, and Anne Chew for assis- B-cell neoplasms. tance in clinical research support; Sharyn Katz for assistance with Supported in part by grants from the National Institutes of imaging; and Stephan Schuster, Elizabeth Hexner, Stephan Grupp, Health (K24 CA11787901, 1PN2-EY016586, and R01CA120409), Carmine Carpenito, Michael Milone, and Donald Siegel for advice.

References

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