CLINICAL TRIALS AND OBSERVATIONS
`
`Safety and persistence of adoptively transferred autologous CD19-targeted T cells
`in patients with relapsed or chemotherapy refractory B-cell leukemias
`*Renier J. Brentjens,1-3 *Isabelle Rivie` re,1-4 Jae H. Park,1,2 Marco L. Davila,1,2 Xiuyan Wang,2-4 Jolanta Stefanski,2-4
`Clare Taylor,2-4 Raymond Yeh,1,2 Shirley Bartido,2,3 Oriana Borquez-Ojeda,2-4 Malgorzata Olszewska,2-4 Yvette Bernal,1
`Hollie Pegram,1,2 Mark Przybylowski,2-4 Daniel Hollyman,2-4 Yelena Usachenko,1,2 Domenick Pirraglia,2-4 James Hosey,2-4
`Elmer Santos,3,5 Elizabeth Halton,1 Peter Maslak,1 David Scheinberg,1-3 Joseph Jurcic,1 Mark Heaney,1 Glenn Heller,6
`Mark Frattini,1 and Michel Sadelain1-3
`
`1Department of Medicine, 2Center for Cell Engineering, 3Molecular Pharmacology and Chemistry Program, 4Cell Therapy and Cell Engineering Facility,
`5Department of Radiology, and 6Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY
`
`We report the findings from the first 10
`patients with chemotherapy-refractory
`chronic lymphocytic leukemia (CLL) or
`relapsed B-cell acute lymphoblastic leuke-
`mia (ALL) we have enrolled for treatment
`with autologous T cells modified to ex-
`press 19-28z, a second-generation chime-
`ric antigen (Ag) receptor specific to the
`B-cell lineage Ag CD19. Eight of the 9
`treated patients tolerated 19-28zⴙ T-cell
`infusions well. Three of 4 evaluable pa-
`tients with bulky CLL who received prior
`conditioning with cyclophosphamide ex-
`Introduction
`
`hibited either a significant reduction or a
`mixed response in lymphadenopathy
`without concomitant development of B-
`cell aplasia. In contrast, one patient with
`relapsed ALL who was treated in remis-
`sion with a similar T-cell dose developed
`a predicted B-cell aplasia. The short-term
`persistence of infused T cells was enhanced
`by prior cyclophosphamide administration
`and inversely proportional to the peripheral
`blood tumor burden. Further analyses
`showed rapid trafficking of modified T cells
`to tumor and retained ex vivo cytotoxic
`
`potential of CD19-targeted T cells retrieved
`8 days after infusion. We conclude that this
`adoptive T-cell approach is promising and
`more likely to show clinical benefit in
`the setting of prior conditioning chemo-
`therapy and low tumor burden or mini-
`mal residual disease. These studies are
`registered at www.clinicaltrials.org as
`#NCT00466531 (CLL protocol) and
`#NCT01044069 (B-ALL protocol). (Blood.
`2011;118(18):4817-4828)
`
`Despite currently available therapies, most patients with B-cell
`leukemias, including chronic lymphocytic leukemia (CLL) and
`B-cell acute lymphoblastic leukemia (B-ALL), are incurable.1,2 For
`this reason, novel therapeutic strategies are needed. The adoptive
`transfer of genetically engineered immune effector cells aims to
`rapidly establish T cell–mediated tumor immunity.3,4 In this ap-
`proach, the patient’s own T cells are targeted to tumor cells through
`a transgene-encoded Ag receptor consisting of either TCR chains or
`a chimeric Ag receptor (CAR). CARs are composed of an
`extracellular Ag recognition domain, most commonly a single
`chain fragment variable derived from a mAb, fused to a transmem-
`brane domain, and a cytoplasmic signaling domain, most com-
`monly including that of the CD3␨ chain.3-10 When expressed in
`T cells, CARs efficiently redirect T-cell specificity and cytotoxicity
`to cells expressing the targeted Ag in HLA-independent manner.11-18
`We have previously generated a series of CARs specific for the
`CD19 Ag,11,12 a member of the Ig superfamily and component of a
`B-cell surface signal transduction complex.19 Expression of CD19
`is restricted to B-lineage cells and possibly follicular dendritic cells
`and is found in most B-cell malignancies, including CLL and
`B-ALL.19-23 Significantly, CD19 is not expressed in hematopoietic
`stem cells. The immunologic targeting of CD19 therefore carries a
`
`minimal risk of autoimmunity or BM toxicity other than the
`potential induction of B-cell aplasias.
`In preclinical studies, human T cells expressing CD19-specific
`CARs efficiently lysed a wide panel of human CD19⫹ tumor cell
`lines as well as freshly isolated patient B-cell tumors.11 Signifi-
`cantly, intravenously administered CD19-targeted human periph-
`eral blood T cells eradicated systemic CD19⫹ tumors established in
`SCID-Beige mice.11,12 Our in vivo studies further showed enhanced
`antitumor efficacy by providing costimulatory signals to adoptively
`transferred T cells. Because most B-cell leukemias fail to express
`ligands for activating costimulatory receptors,24,25 we overcame
`this limitation by replacing the inert CD8 transmembrane domain
`with the transmembrane and cytoplasmic signaling domains of the
`T-cell costimulatory CD28 receptor,26 resulting in the 19-28z CAR,
`which enhances antitumor efficacy in SCID-Beige mice bearing
`CD19⫹ leukemias.12 On the basis of these preclinical data we chose
`to translate this approach to the clinical setting with the use of the
`19-28z CAR.
`After the validation of a robust process for large-scale human
`T-cell transduction and expansion,27 we enrolled 10 patients with
`either chemotherapy refractory CLL or relapsed B-ALL on 2 phase
`1 dose escalation clinical trials. The primary objective of these
`
`Submitted April 13, 2011; accepted August 5, 2011. Prepublished online as
`Blood First Edition paper, August 17, 2011; DOI 10.1182/blood-2011-04-348540.
`
`*R.J.B. and I.R. contributed equally to this study.
`
`The online version of this article contains a data supplement.
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
`
`An Inside Blood analysis of this article appears at the front of this issue.
`
`© 2011 by The American Society of Hematology
`
`BLOOD, 3 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 18
`
`4817
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`4818
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`BRENTJENS et al
`
`BLOOD, 3 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 18
`
`trials is to assess the safety of infusing 19-28z⫹ T cells with or
`without prior cyclophosphamide-conditioning chemotherapy. Sec-
`ondary objectives include assessment of clinical responses to
`therapy and the effect of cyclophosphamide conditioning on
`disease response, T-cell persistence, and T-cell function. Herein, we
`report our findings on the first 10 patients, 9 of which were infused
`with the manufactured T cells. We were able to generate sufficient
`CAR-transduced T cells from leukapheresis products derived from
`all enrolled patients. Either a marked reduction in tumor burden
`stable disease or B-cell aplasia was observed in 4 of the 5 evaluable
`patients given cyclophosphamide before T-cell infusion. We dem-
`onstrate rapid trafficking of 19-28z⫹ T cells to sites of tumor
`involvement and report on the short-term persistence and function
`of the adoptively transferred T cells. Collectively, these data show
`the promise of CD19-targeted T cells for the treatment of B-cell
`malignancies and provide insights into how to optimally apply this
`strategy in the clinical setting.
`
`rus by PCR and marker rescue cell culture assay; residual Dynabeads
`ClinExVivo CD3/CD28; negative bacterial, fungal, and Mycoplasma
`cultures; endotoxin level no ⬎ 5 EU/kg; Gram stain–negative on day of
`infusion; ⬎ 80% cell viability; and CD3⫹, CD8⫹, CD5⫹ (patients with
`CLL), CD10⫹ (patients with ALL), and CD19⫹ phenotype by flow
`cytometry and CD19-specific cytotoxicity. The assays were performed as
`described in Hollyman et al27 and Taylor et al.29
`
`Restimulation of T cells from postinfusion PBMCs with
`Dynabeads ClinExVivo CD3/CD28
`
`PBMCs collected in cell preparation tubes (BD Biosciences) were purified
`from whole blood according to the manufacturer’s recommendations.
`Patient CD3⫹ T cells were subsequently selected and activated with
`Dynabeads ClinExVivo CD3/CD28 (Invitrogen) at ratio of 3 beads to
`1 CD3⫹ T cell. CD3⫹ T cells were cultured in X-VIVO 15 medium (Lonza)
`supplemented with 100 U/mL IL-2. Cell samples were taken at different
`time points to determine the average vector copy number by quantitative
`RT-PCR and the expression of 19-28z CAR by flow cytometry.
`
`Methods
`
`Clinical protocols
`
`CLL protocol (NCT00466531). Patients with relapsed purine analog-
`refractory CLL are eligible for enrollment. This is a phase 1 dose escalation
`trial. Patients initially undergo leukapheresis for T-cell collection. In the
`first step, one cohort of 3-6 patients is treated with the lowest initial dose of
`T cells, level 1 (1.2-3.0 ⫻ 107 19-28z⫹ T cells/kg), without prior cyclophos-
`phamide administration. In the second step with 2 cohorts, patients are
`treated with dose-escalating cyclophosphamide chemotherapy (1.5 and
`3.0 g/m2) followed 2 days later by infusion of modified T cells at dose level
`1. After the death on study of the first patient treated in the second cohort,28
`the ⫺1 T-cell dose level
`the subsequent 3 patients were treated at
`(0.4-1.0 ⫻ 107 19-28z⫹ T cells/kg) with T-cell infusions split over 2 days to
`enhance safety. Enrollment on this second cohort has been completed.
`B-ALL protocol (NCT01044069). Adult patients with CD19⫹ B-ALL
`are eligible for enrollment. Patients can be either enrolled in first complete
`remission (CR1) after ⱖ 1 round of consolidation therapy or on presenta-
`tion with relapsed or refractory disease, defined as no CR after ⱖ 2 induc-
`tion regimens. Patients enrolled in CR1 underwent leukapheresis but are
`treated with T cells only after relapse. Patients enrolled with relapsed or
`refractory B-ALL leukapheresed, treated with a salvage reinduction regi-
`men, and regardless of remission status, receive cyclophosphamide (3.0 g/
`m2) followed 2 days later by split dose infusion of autologous 19-28z⫹
`T cells. This is a standard phase 1 dose-escalation trial with 3 planned T-cell
`doses, 3 ⫻ 106, 1 ⫻ 107, and 3 ⫻ 107 19-28z⫹ T cells/kg. Patients enrolled
`on this protocol are not precluded, if eligible, to undergo additional therapy
`with allogeneic stem cell transplantation after the modified T-cell infusion.
`Both trials were approved by the Memorial Sloan-Kettering Cancer
`Center Institutional Review Board,
`the Recombinant DNA Advisory
`Committee of the National Institutes of Health, and are supported by
`BB-IND 13266 approved by the US Food and Drug Administration. In both
`trials, informed consent is obtained from eligible patients in accordance
`with the Declaration of Helsinki. Patients enrolled on these trials do not
`receive IL-2. Adverse events during and after therapy were assessed
`according to the National Institutes of Health Common Terminology
`Criteria for Adverse Events Version 3.0 (http://ctep.cancer.gov/). The
`Memorial Sloan-Kettering Cancer Center Data and Safety Monitoring
`Board review all safety data every 6 months.
`
`Generation and expansion of genetically modified T cells
`
`19-28z–transduced T cells were generated as described.27 Briefly, T cells
`were isolated from leukapheresis product and activated with Dynabeads
`ClinExVivo CD3/CD28. Release criteria include 19-28z CAR expression
`by flow cytometry; average vector copy number by quantitative PCR;
`vector identity by Southern blot; absence of replication competent retrovi-
`
`Restimulation of T cells from postinfusion PBMCs with
`CD19ⴙCD80ⴙ artificial APCs
`
`Human CD19 and CD80 expressing fibroblasts (3T3-CD19-CD80)11 were
`irradiated at 30 Gy. Postinfusion PBMCs were thawed and plated on the
`3T3-CD19-CD80 cells in X-VIVO 15 media (Lonza) supplemented with
`5% human AB serum (GEMINI) and 100 U/mL IL-2. Lysis of 3T3 cells
`was monitored, and PBMCs were fed on day 3. Cells were counted 7 days
`after restimulation and stained with anti–human CD3-FITC, CD8-eFluor
`450, CD4-PE-Cy7, biotinylated goat anti–mouse IgG F(ab)2 followed by
`APC-labeled streptavidin, and 7-amino-actinomycin D (7-AAD) with the
`use of standard staining procedures. Data acquisition was performed on a
`LSRII flow cytometer, and data analysis was performed with FlowJo
`software (TreeStar Inc).
`
`Characterization of cytokine profiles secreted by EOP
`19-28z–transduced T cells
`
`Samples of patient T cells transduced with 19-28z CAR were taken at the
`end of production (EOP). T cells (1.0 ⫻ 106) were plated on 3T3 artificial
`APCs (AAPCs) expressing CD19 or untransduced 3T3 fibroblasts as
`controls, in complete X-Vivo culture media in 5% human AB serum and in
`the absence of ILs. After 48 hours supernatants were collected and assayed
`for human cytokines on a Luminex IS100 instrument as per manufacturer’s
`instructions (Millipore Corp). The cytokine levels were normalized to
`transduction efficiency. In addition, background levels measured on untrans-
`duced 3T3 fibroblasts were subtracted.
`
`Other methods
`
`including manufacture of clinical grade PG13-19-28z
`Other methods,
`vector stocks, serum cytokine analyses, cytotoxic T-lymphocyte assays,
`quantitative real-time PCR, Abs and reagents, flow cytometry, statistics,
`and IHC can be found in supplemental Methods (available on the Blood
`Web site; see the Supplemental Materials link at the top of the online
`article).
`
`Results
`
`Patient characteristics
`
`Eight patients with relapsed purine analog-refractory CLL have
`been enrolled and treated (Table 1). The median age of patients was
`68 years with 7 of 8 being men. All patients were previously treated
`with 1-4 different chemotherapy regimens, with 7 of 8 patients
`having received ⱖ 2 different regimens before enrollment. Chromo-
`somal and genetic analyses of CLL tumor cells showed adverse
`prognostic features, including del17p with concomitant p53 gene
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`BLOOD, 3 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 18
`
`AUTOLOGOUS CD19-TARGETED T CELLS IN CLL/ALL
`
`4819
`
`Table 1. Patient characteristics
`Age at
`Age at
`diagnosis,
`treatment,
`y
`y
`
`Patient
`ID
`
`Sex
`
`Indication
`for treatment
`
`CLL-1
`CLL-2
`CLL-3
`CLL-4
`CLL-5
`CLL-6
`CLL-7
`CLL-8
`
`ALL-1
`
`ALL-2
`
`44
`66
`62
`63
`65
`56
`52
`58
`
`66
`
`45
`
`51
`72
`73
`69
`68
`68
`62
`61
`
`67
`
`48
`
`M
`M
`F
`M
`M
`M
`M
`M
`
`M
`
`F
`
`Bulky LAD
`Bulky LAD
`Bulky LAD
`Bulky LAD
`Bulky LAD
`Bulky LAD
`Bulky LAD
`Bulky LAD
`
`Prior therapies
`
`FCR, PCRM
`FR, RCVP, PCRM
`Chlorambucil, PCR, PCRM
`R, PCRM
`PCR
`RCVP, PCR, Bendamustine
`CVP, RC, PCR, PCRM
`RCVP, Alemtuzumab
`
`Genetic
`abnormalities/IgVH
`mutation status
`
`WBC
`count,
`ⴛ 103/␮L
`
`ALC,
`ⴛ 103/␮L
`
`Hgb
`level,
`g/dL
`
`PLT
`count,
`ⴛ103/␮L
`
`del11q
`Unmutated IgVH
`Normal karyotype
`del11q
`del11q, trisomy 12
`del11q, inv1, unmutated IgVH
`del17p, unmutated IgVH
`del17p, monosomy 14,
`monosomy 15
`Normal karyotype
`
`Normal karyotype
`
`200.6
`4.2
`136.4
`187.1
`76.3
`97.1
`1.9
`5.4
`
`2.9
`
`ⴱ
`
`196.6
`3.4
`132.3
`174
`66.4
`92.2
`1
`3.3
`
`7.1
`9.9
`8.9
`9.9
`10
`8.9
`10
`11.6
`
`26
`60
`100
`189
`162
`174
`61
`41
`
`0.7
`
`8.6
`
`126
`
`ⴱ
`
`ⴱ
`
`ⴱ
`
`Relapsed disease C, Mitoxantrone, vincristine,
`etoposide
`Relapsed disease HyperCVAD, mitoxantrone,
`cytarabine, vincristine
`
`IgVH indicates immunoglobulin heavy chain; WBC, white blood cell; ALC, absolute lymphocyte count; Hgb, hemoglobin; PLT, platelet; LAD, lymphadenopathy;
`FCR, fludarabine, cyclophosphamide, rituximab; PCRM, pentostatin, cyclophosphamide, rituximab, mitoxantrone; FR, fludarabine, rituximab; RCVP, rituximab, cyclophosph-
`amide, vincristine, prednisone; PCR, pentostatin, cyclophosphamide, rituximab; R, rituximab; CVP, cyclophosphamide, vincristine, prednisone; RC, rituximab, cyclophosph-
`amide; C, cyclophosphamide; and HyperCVAD, cyclophosphamide, vincristine, doxorubicin, dexamethasone.
`*This patient is yet to be treated with modified T cells.
`
`deletion, del11q, and/or an unmutated Ig variable heavy chain
`domain. All patients exhibited advanced disease and tumor burden
`as evidenced by marked bulky lymphadenopathy.
`Two patients with relapsed B-ALL have been enrolled and 1
`patient has been treated (Table 1). The latter achieved an initial
`remission after induction therapy but subsequently relapsed after
`the third cycle of consolidation therapy at which point the patient
`was enrolled and T cells were obtained by leukapheresis. The patient
`achieved a second remission after reinduction chemotherapy and
`was treated on protocol. The second patient had a relapse of disease
`after initial induction therapy but became ineligible for treatment
`with modified T cells because of intervening complications.
`
`Generation of patient-derived 19-28z–transduced T cells
`
`Despite all patients having been heavily pretreated, we were able to
`obtain adequate numbers of T cells from the leukapheresis products
`in every case (Table 2). The mean time in culture to achieve the
`19-28z⫹ T-cell dose was 16 days (range, 11-19 days) with a mean
`120-fold expansion of T cells (range, 24- to 385-fold). The
`transduction efficiency in CD3⫹ T cells, as assessed by flow
`cytometry, ranged from 23% to 70% in CLL patient cells and from
`4% to 8.6% in ALL patient cells. The average vector copy number
`
`per cell in these cell products ranged from 0.06 to 1.5 with a mean
`of 0.67.
`
`Characterization of final 19-28z–transduced T cells
`
`The final T-cell products exhibited a predominant CD4⫹ pheno-
`type, particularly pronounced in the CLL EOP cells (mean CD4⫹
`T-cell fraction of 88%, CD4/CD8 ratio of 10.5; Tables 3 and 4). The
`patients with B-ALL showed a mean CD4⫹ T-cell fraction of 63%
`(Table 3). Despite a marked prevalence of CD4⫹ T cells, all EOP
`T cells displayed robust in vitro cytotoxic activity against both
`autologous CLL tumor cells and the CD19⫹ Raji Burkitt lymphoma
`cell line (Table 2). Further analyses of final T-cell products showed
`minimal numbers of CD4⫹ FoxP3⫹ T regulatory cells and a
`significant retention of cell surface markers,
`including CD27,
`CD28, and CD62L. Although the percentage of these markers
`showed some variability (Table 3), the absolute numbers of infused
`T cells were overall similar in all patients (supplemental Figure 1)
`as well as their distribution in CAR– versus CAR⫹ T cells (Table 3;
`supplemental Figure 1). To further assess the infused cell products,
`all EOP cells were activated in Ag-specific fashion in vitro under
`the same conditions with the use of CD19⫹ AAPCs.11,30 The
`cytokine signatures induced by exposure to CD19 were overall
`
`Table 2. Characteristics of the infused 19-28z–transduced T cells
`Test
`CLL-1
`CLL-2
`CLL-3
`
`CLL-4
`
`CLL-5
`
`CLL-6
`
`CLL-7
`
`CLL-8
`
`ALL-1
`
`ALL-2
`
`CD3ⴙ T cells, %
`Apheresis product
`EOP cells
`Tumor cells, %
`Apheresis product
`EOP cells
`CD3⫹/19-28z CAR⫹, %
`Cytotoxicity (25:1)
`Auto-B tumor cells
`Raji tumor cells
`Fold expansion after transduction
`(days in culture)
`Total 1928z⫹/CD3⫹ cells infused
`Total CD3⫹ cells infused
`
`5.4
`98
`
`94
`0.1
`23
`
`45
`100
`
`4
`0
`31
`
`7.2
`100
`
`90
`0
`53
`
`5.1
`99
`
`94
`0
`70
`
`15.9
`98
`
`50
`1
`32
`
`6.2
`99
`
`94
`0
`39
`
`25
`85
`
`28
`1.2
`25
`
`22.5
`100
`
`69
`0
`39.3
`
`45
`99
`
`3.1
`0
`8.6
`
`67
`100
`
`0
`0.2
`4
`
`58.8
`51.1
`53 (17)
`
`ND
`47.1
`184 (19)
`
`52.3
`52.3
`23.6 (18)
`
`48.7
`46.5
`112 (15)
`
`33.7
`43.3
`191 (18)
`
`59.5
`73.8
`51 (15)
`
`35.7
`72.9
`62.4 (15)
`
`28.3
`24.8
`385 (16)
`
`ND
`29.2
`49.4 (d11)
`
`ND
`8.6
`88.5 (d11)
`
`2.5 ⫻ 109
`11.1 ⫻ 109
`
`1.2 ⫻ 109
`3.7 ⫻ 109
`
`1.1 ⫻ 109
`2.1 ⫻ 109
`
`3.2 ⫻ 109
`4.6 ⫻ 109
`
`4 ⫻ 108
`1.2 ⫻ 109
`
`4 ⫻ 108
`1.0 ⫻ 109
`
`7.6 ⫻ 108
`3.0 ⫻ 109
`
`1.4 ⫻ 109
`3.5 ⫻ 109
`
`1.8 ⫻ 108
`2.1 ⫻ 109
`
`NA
`NA
`
`The average 19-28z vector copy number per cell from CLL-1 to ALL-2 patients was 0.34, 0.54, 0.66, 1.15, 0.81, 1.5, 0.86, 0.62, 0.14, and 0.06, respectively.
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`BRENTJENS et al
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`BLOOD, 3 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 18
`
`Table 3. Phenotype of the infused 19-28z–transduced T cells
`CD4ⴙ
`CD8ⴙ
`CD28ⴙCD27ⴚ
`CD28ⴙCD27ⴙ
`Patient ID
`
`CD62Lⴙ
`
`CCR7ⴙ
`
`CD25ⴙCD4ⴙFoxp3ⴙ
`
`Days in culture
`
`CLL-1
`CLL-2
`CLL-3
`CLL-4
`CLL-5
`CLL-5 (CAR⫹)
`CLL-6
`CLL-6 (CAR⫹)
`CLL-7
`CLL-7 (CAR⫹)
`CLL-8
`CLL-8 (CAR⫹)
`ALL-1
`ALL-1 (CAR⫹)
`ALL-2
`ALL-2 (CAR⫹)
`
`94
`96
`93
`99
`87
`87.4
`79
`78.6
`58
`66.6
`91.5
`90.3
`74
`62.8
`52
`47.9
`
`5
`5
`8
`0.7
`12
`11.9
`21
`20.8
`27
`21.5
`8.5
`9.7
`26
`25.6
`48
`51.8
`
`83.2
`86.1
`40.6
`86.9
`56.6
`62.9
`45.8
`57
`41.3
`49.1
`81.7
`82.4
`43.4
`52.3
`5.2
`28.5
`
`11.5
`6.1
`2
`5.9
`20.8
`16.6
`30.8
`19.3
`13.5
`13.3
`9.1
`7.8
`16.5
`15.9
`84.8
`64.1
`
`9.1
`15.5
`17.7
`34.4
`40
`40.4
`51.5
`52
`63.4
`54.3
`33.1
`28.1
`64.3
`78.1
`94.8
`86.1
`
`ND
`ND
`ND
`ND
`7.7
`10.1
`3.4
`3.7
`1.1
`1.4
`19.6
`16.6
`1.3
`1.3
`47.4
`36.5
`
`1.2
`0.61
`2.4
`0.9
`0.7
`ND
`1.3
`ND
`1.2
`ND
`ND
`ND
`1.7
`ND
`ND
`ND
`
`17
`19
`18
`15
`18
`ND
`15
`ND
`15
`ND
`16
`ND
`11
`ND
`13
`ND
`
`For patients CLL-1, CLL-2, CLL-3, and CLL-4, the CD4, CD8, and memory phenotypes were analyzed in 7-AAD⫺ populations. For patients CLL-5, CLL-6, CLL-7, ALL-1,
`and ALL-2, the CD4, CD8, and memory phenotypes were analyzed in both 7-AAD⫺ and CAR⫹ populations. All numbers are expressed in percentage of 7-AAD⫺ or CAR⫹
`populations as indicated.
`
`similar, showing in particular robust secretion of GM-CSF, IFN-␥,
`TNF-␣, MIP-1␣, MIP-1␤, and little or no IL-2, IL-4, and IL-10 in
`most patients (Figure 1).
`
`Infusion of 19-28z–transduced T cells is well tolerated
`
`Nine patients have received intravenous infusion of 19-28z–
`transduced autologous T cells. Overall, patients tolerated therapy
`well with most patients experiencing rigors, chills, and transient
`fevers within 24 hours of modified T-cell infusions. In all cases,
`patient blood and urine cultures were obtained, and intravenous
`antibiotic therapy was initiated. However, with the exception of
`patient CLL-4, all patients readily recovered from these symptoms
`with negative blood and urine cultures and were released after an
`additional 48 hours of in-patient observation (Table 5). Patient
`CLL-4 experienced persistent fevers, developed a sepsis-like
`syndrome with hypotension and renal failure, and died within
`48 hours of T-cell infusion. Previously published extensive analy-
`ses of this case point to an infectious cause as evidenced by marked
`serum cytokine abnormalities that preceded T-cell infusion, argu-
`ing against the 19-28z⫹ T cells being the primary cause of this
`
`adverse outcome.28 The subsequent cohort of 4 patients with CLL
`was treated at a 3-fold lower T-cell dose than CLL-4 and
`administered in a split-dose manner over 2 consecutive days. In
`addition, serum cytokine levels were assessed before both cyclo-
`phosphamide and T-cell infusions. Cytokine elevations, as noted in
`CLL-4, were not observed in the subsequently treated patients
`before or after cyclophosphamide therapy or after T-cell infusion.
`Significantly, serial serum analyses in these patients did not show
`evidence of cyclophosphamide-induced alterations in cytokine
`profiles compared with patients treated with T cells without
`cyclophosphamide conditioning (Figure 2).
`
`Clinical responses to 19-28zⴙ T-cell infusion
`
`Eight patients with CLL have been treated. The first cohort of
`3 patients was treated without cyclophosphamide conditioning at
`the lowest planned T-cell dose of 1.2-3.0 ⫻ 107 19-28z⫹ T cells/kg
`with no objective disease responses (Table 6). Collectively, all
`3 patients on this cohort exhibited further progressive disease
`and soon required additional salvage chemotherapy. All patients
`on this cohort have died of their disease. Patient CLL-4 died soon after
`
`Table 4. CD4/CD8 ratio in apheresis, EOP 19-28z transduced T cells, and blood samples after infusion
`Apheresis
`EOP 19-28z T cells
`
`Patient ID
`
`CLL-1
`CLL-2
`CLL-3
`CLL-4
`CLL-5
`CLL-6
`CLL-7
`CLL-8
`Mean ⫾ SD
`ALL-1
`ALL-2
`
`CD4/CD8
`
`2.4
`0.3
`3.5
`0.5
`0.8
`1.5
`2.5
`4.5
`2.0 ⫾ 1.5
`1.0
`1.8
`
`CD4ⴙCD28ⴙ/CD8ⴙCD28ⴙ
`
`2.4
`0.3
`6.8
`4.0
`1.0
`2.3
`7.0
`6.0
`3.7 ⫾ 2.6
`2.1
`1.8
`
`CD4/CD8
`
`18.8
`19.2
`11.6
`141.4
`7.3
`3.8
`2.5
`10.1
`10.5 ⫾ 6.6ⴱ
`2.5
`0.9
`
`19-28z T cells after infusion
`
`CD4/CD8 (time after infusion)
`
`NA
`NA
`NA
`70.4 (1 h); 33.5 (24 h)
`NA
`NA
`NA
`19 (2 wk); 8.7 (4 wk); 8.8 (5 wk)
`NA
`1.1 (8 d)
`NA
`
`All phenotypes are analyzed by flow cytometry in 7-AAD⫺ and CD45⫹ populations. All numbers are expressed in ratio of percentages of CD4/CD8 or
`CD4⫹CD28⫹/CD8⫹CD28⫹.
`*The average CD4/CD8 ratio excludes the CD4/CD8 ratio for CLL-4. With CLL-4, the average CD4/CD8 ratio is 26.8 ⫾ 46.7. CLL-4 had the lowest CD8⫹CD28⫹ fraction
`(see supplemental Table 1).
`
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`
`AUTOLOGOUS CD19-TARGETED T CELLS IN CLL/ALL
`
`4821
`
`15000
`
`150
`
`15000
`
`1500
`
`2000
`
`25000
`
`20000
`
`10000
`
`100
`
`10000
`
`1000
`
`5000
`
`50
`
`5000
`
`500
`
`G-C SF
`
`0
`
`G M-C SF
`
`0
`
`IF N-alpha2
`
`m a
`
`0
`
`IF N-ga m
`
`0
`
`IL-3
`
`4000
`
`25000
`
`1500
`
`1000
`
`500
`
`0
`
`IL-17
`
`20000
`
`15000
`
`10000
`
`5000
`
`0
`
`MIP-1alpha
`
`300
`
`15000
`
`10000
`
`5000
`
`0
`
`MIP-1beta
`
`80
`
`60
`
`40
`
`20
`
`0
`
`pg/ml
`
`20000
`
`15000
`
`10000
`
`5000
`
`0
`
`3000
`
`2000
`
`1000
`
`0
`
`IL-10
`
`2000
`
`1500
`
`1000
`
`500
`
`200
`
`100
`
`CLL1
`CLL2
`CLL3
`CLL4
`CLL5
`CLL6
`CLL7
`CLL8
`ALL1
`ALL2
`3T3 CD19 ALONE
`
`IL-13
`
`0
`
`sC D40L
`
`0
`
`T N F-alpha
`
`2500
`
`2000
`
`1500
`
`1000
`
`500
`
`0
`
`150
`
`100
`
`50
`
`0
`
`pg/ml
`
`IL-2
`
`IL-4
`
`Figure 1. Cytokine analysis of EOP 19-28z–transduced T cells stimulated on CD19ⴙ AAPCs. EOP 19-28z–transduced T cells were plated on 3T3 fibroblast AAPCs
`expressing CD19. After 48 hours in culture, cell supernatants were assayed for cytokine levels. The cytokine levels were normalized to transduction efficiency. In addition,
`background cytokine levels measured on 3T3 fibroblasts that did not express CD19 were subtracted.
`
`infusion and therefore was not evaluable for clinical
`T-cell
`response.28
`
`Table 5. Adverse events
`Diagnosis-Patient
`
`Adverse events
`
`Grade
`
`CLL-1
`
`CLL-2
`
`CLL-3
`CLL-4
`
`CLL-5
`
`CLL-6
`
`CLL-7
`CLL-8
`ALL-1
`
`Febrile neutropenia
`Rigors, chills
`Fever, rigors, chills
`Chest pain
`Fever, rigors, chills
`Fever
`Rigors, chills, dyspnea
`Hypotension, renal failure
`Fever
`Rigors, chills
`Hyponatremia
`Fever
`Hypotension
`Febrile neutropenia
`Febrile neutropenia
`Fever
`Neutropenia
`Diarrhea
`Hypotension
`
`3
`2
`2
`2
`1
`2
`1
`5
`2
`1
`1
`1
`2
`3
`3
`2
`4
`2
`3
`
`Related
`
`Probable
`Probable
`Probable
`Probable
`Probable
`Probable
`Probable
`Possible
`Probable
`Probable
`Possible
`Probable
`Possible
`Possible
`Probable
`Probable
`Possible
`Possible
`Possible
`
`Patient ALL-2 is omitted because the patient became ineligible for modified T-cell
`infusion.
`
`The next 4 patients (CLL-5 to -8) were treated with
`cyclophosphamide-conditioning chemotherapy, followed by in-
`fusion of 0.4-1.0 ⫻ 107 19-28z⫹ T cells/kg. Patient CLL-5
`exhibited stable to progressive disease at 1 month after treatment as
`assessed by computed tomography scan but subsequently devel-
`oped marked objective reduction of peripheral lymphadenopathy in
`the absence of any further therapeutic interventions, as assessed by
`physical examination and computed tomography scans over the
`subsequent 2 months (Figure 3). This marked reduction of lymph-
`adenopathy remained stable over the subsequent 6 months, and
`thereafter the patient developed disease progression in the abdo-
`men with associated ascites and worsening cytopenias, which
`required further chemotherapy. The patient died 15 months after
`T-cell therapy with progressive chemotherapy refractory disease.
`Patient CLL-6 exhibited progressive disease ⬎ 1 month after
`therapy requiring salvage chemotherapy and died of infectious
`complications 2 months later. Patients CLL-7 and CLL-8, both
`treated in the setting of rapidly progressive disease with increasing
`lymphadenopathy and cytopenias, exhibited stable disease with
`respect to lymphadenopathy over a 4- and ⬎ 2-month period of
`expectant management, respectively, after cyclophosphamide and
`T-cell infusions (data not shown).
`Patient ALL-1 was a 67-year-old male who relapsed during
`consolidation therapy, achieved a second remission with salvage
`chemotherapy, and received a single consolidation with high-dose
`
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`4822
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`BRENTJENS et al
`
`BLOOD, 3 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 18
`
`CLL1
`CLL2
`CLL3
`CLL5
`CLL6
`CLL7
`ALL1
`
`No Cyclophosphamide
`
` Cyclophosphamide
`
`0
`
`10
`
`20
`Days
`
`30
`
`40
`
`0
`
`10
`
`20
`Days
`
`30
`
`40
`
`0
`
`10
`
`20
`Days
`
`30
`
`40
`
`10
`
`02468
`
`10
`
`02468
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`80
`
`60
`
`pg/ml
`
`pg/ml
`
`pg/ml
`
`0
`
`10
`
`20
`Days
`
`30
`
`40
`
`0
`
`10
`
`20
`Days
`
`30
`
`40
`
`0
`
`10
`
`20
`Days
`
`30
`
`40
`
`10
`
`02468
`
`10
`
`02468
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`80
`
`60
`
`40
`
`pg/ml
`
`pg/ml
`
`pg/ml
`
`IL-2
`
`IL-7
`
`IL-12 (p40)
`
`IL-15
`
`0
`
`10
`
`20
`Days
`
`30
`
`40
`
`0
`
`10
`
`20
`Days
`
`30
`
`40
`
`40
`
`20
`
`0
`
`150
`
`100
`
`50
`
`0
`250
`
`200
`
`150
`
`100
`
`50
`
`pg/ml
`
`pg/ml
`
`pg/ml
`
`0
`
`10
`
`20
`Days
`
`30
`
`40
`
`0
`
`10
`
`20
`Days
`
`30
`
`40
`
`pg/ml
`
`20
`
`0
`150
`
`100
`
`50
`
`0
`250
`
`200
`
`150
`
`100
`
`50
`
`pg/ml
`
`pg/ml
`
`INF-
`
`TNF-
`
`0
`
`0
`
`10
`
`30
`
`40
`
`20
`20
`Days
`Days
`Figure 2. Patient serum cytokine levels before and after cyclophosphamide and T-cell infusions. Serum cytokines were determined before and after 19-28z⫹ T-cell
`infusion. The first set of patients (CLL-1 to CLL-3) did not receive cyclophosphamide treatment before T-cell infusion, whereas the second set (CLL-5 to CLL-7 and ALL-1)
`received cyclophosphamide before T-cell infusion. The x-axis represents treatment time course with day 0 being the day of T-cell infusion and day ⫺2 being the day of
`cyclophosphamide treatment.
`
`0
`
`0
`
`10
`
`30
`
`40
`
`cyclophosphamide followed 2 days later with T-cell infusion. This
`patient was followed over the next 8 weeks before undergoing an
`allogeneic BM transplantation from a related sibling. Despite T-cell
`and neutrophil recovery, the patient exhibited a persistent B-cell
`
`aplasia in the BM and peripheral blood with only very low level
`CD19⫹ B-cell recovery just before transplantation (Figure 4).
`
`19-28zⴙ autologous T cells rapidly traffic to sites of CD19ⴙ tumor
`
`Table 6. Summary of patient responses
`Patient ID
`Response to T-cell infusions
`
`CLL-1
`CLL-2
`CLL-3
`CLL-4
`CLL-5
`
`CLL-6
`CLL-7
`CLL-8
`ALL-1
`
`No objective response
`No objective response
`No objective response
`Not evaluable
`Marked reduction in lymphadenopathy at 3 months, subsequently
`stable for 6 months
`Progressive disease
`Stable disease, lasting 4 months
`Stable disease, lasting ⬎ 8 weeks
`Persistent B-cell aplasia in BM and peripheral blood
`
`Cryopreserved autopsy tissue samples obtained from patient CLL-4
`were analyzed by IHC for 19-28z⫹ T cells. IHC studies showed
`infiltration of targeted 19-28z⫹ T cells into CLL tumor beds in
`lymph nodes, liver, and BM (Figure 5A), reflecting rapid CAR-
`mediated trafficking of 19-28z⫹ T cells to sites of CD19⫹ tumor.
`
`In vivo persistence of 19-28zⴙ autologous T cells
`
`19-28z⫹ T-cell persistence was measured by IHC of BM samples
`and by RT-PCR and flow cytometry of serial peripheral blood and
`BM aspirate samples. With the use of an anti-idiotypic monoclonal
`Ab specific for the 19-28z CAR, IHC analyses of BM samples from
`the first cohort showed either discrete (CLL-3) or no evidence
`(CLL-1 and CLL-2) of 19-28z⫹ T cells at 1 month (Figure 5C).
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`BLOOD, 3 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 18
`
`AUTOLOGOUS CD19-TARGETED T CELLS IN CLL/ALL
`
`4823
`
`Figure 3. Marked reduction of peripheral lymphadenopathy was observed after treatment with autologous 19-28zⴙ T cells. Representative computed tomography
`scan images of patient CLL-5 before treatment and from 4 weeks and 14 weeks after treatment show mild increase in axillary (top) and pelvic lymphadenopathy (bottom) at
`4 weeks but regression of lymphadenopathy at 14 weeks after therapy with modified T cells.
`
`However, in the second cohort, further treated with cyclophosph-
`amide conditioning, CLL-7 exhibited a significant number of
`retained 19-28z⫹ T cells within the BM ⱕ 6 weeks after T-cell
`infusion (Figure 5B-C). Between 1 and 0.02 vector copies per
`100 cells could also be detected by RT-PCR 2 and 3 weeks after
`infusion, respectively, in freshly obtained BM aspirates derived
`from the same patient. Similarly, patient ALL-1 exhibited 19-28z⫹
`T cells in the BM at 5 weeks after therapy consistent with the
`B-cell aplasia observed after T-cell infusion (Figure 5B-C).
`To overcome the high tumor burden background in most of the
`patients with CLL in postinfusion peripheral blood samples, T cells
`were selected and expanded with Dynabeads before analysis. RT-PCR
`(Figure 6A) and flow cytometry analyses (Figure 6B) of expanded
`peripheral blood samples showed an enhanced persistence of
`modified T cells in patients treated with prior cyclophosphamide
`(CLL-4 to CLL-8, ALL-1), despite a lower dose of infused 19-28z⫹
`T cells, compared with the first cohort of patients treated with
`19-28z⫹ T cells alone (CLL-1 to CLL-3).
`
`Persistent 19-28zⴙ T cells retrieved from patient peripheral
`blood samples proliferate in response to CD19 and are
`cytotoxic
`
`To assess the functional status of persisting 19-28z⫹ peripheral
`blood T cells, we investigated whether these cells could proliferate
`on coculture with CD19-expressing AAPCs (3T3-CD19-CD80).11,30
`Peripheral blood T cells collected 8 days after T-cell infusion from
`patient ALL-1 indeed expanded on 3T3-CD19-CD80 fibroblasts,
`with 19-28z⫹ T cells reaching 32.9% of the CD3⫹ T-cell fraction
`after 7 days in culture (Figure 6B). In contrast, the same blood
`sample expanded by nonspecific activation with the use of anti-CD3/
`CD28 beads showed that 4.2% of the CD3⫹ T-cell fraction
`expressed the 19-28z⫹ CAR. Confirming and extending these
`findings, quantitative measurements of vector sequences showed
`42.1