letters to the editor
`
`© The American Society of Gene & Cell Therapy
`
`See pages 661 and 843
`Treatment of Chronic
`Lymphocytic Leukemia
`With Genetically Targeted
`Autologous T Cells: Case
`Report of an Unforeseen
`Adverse Event in a Phase I
`Clinical Trial
`
`To the editor:
`Most patients with B-cell malignan-
`cies will die from their disease or are
`incurable. For this reason, innovative
`therapeutic approaches are direly needed.
`Patient T cells may be genetically modi-
`fied to target antigens expressed on tumor
`cells through the expression of chimeric
`antigen receptors (CARs), which are
`antigen receptors designed to recognize
`cell surface antigens in a human leu-
`kocyte antigen–independent manner.1
`CD19, which is expressed on most B-cell
`malignancies—including most non-
`Hodgkin’s lymphomas, acute lymphoblas-
`tic leukemias, and chronic lymphocytic
`leukemias (CLLs)—is an attractive antigen
`for this approach.2 It is present on normal
`B-lineage cells from the early pre–B-cell
`stage until plasma cell differentiation.
`In a model of CD19+ acute lymphoblas-
`tic leukemia, we found that that a CAR
`termed 19-28z, which comprises the
`CD28 cytoplasmic domain in addition to
`that of the CD3 ζ-chain,3 induced better
`responses than a ζ-chain-based receptor.4
`In preclinical in vitro studies, we demon-
`strated that human T cells that express
`CD19-specific CARs efficiently lyse
`human CD19+ tumor cell lines and that
`CLL patient–derived T cells effectively lyse
`autologous tumor cells.2,4 These results,
`and others,5 supported a phase I clinical
`trial treating refractory CLL patients with
`autologous T cells modified by retroviral
`gene transfer of the 19-28z CAR.
`We have thus far enrolled six patients
`in this clinical trial. The cohort of subjects
`treated with modified T cells alone at the
`first dose level of T cells tolerated therapy
`well without dose-limiting toxicities. How-
`ever, the first subject (subject 4) enrolled
`in the second cohort of patients, in whom
`cyclophosphamide lymphodepleting
`
`chemotherapy was administered before
`infusion of the same T-cell dose, devel-
`oped a syndrome of hypotension, dyspnea,
`and renal failure following T-cell infusion.
`Subject 4 died 4 days after administra-
`tion of cyclophosphamide and modified
`T cells. Herein we describe the chronology
`of his treatment and report the findings of
`an extensive postmortem analysis.
`
`Clinical trial design
`Subject 4 was treated in a phase I clinical
`trial (IRB no. 06-138, NIH-RAC no.
`0507-721, NCT00466531) designed to
`assess the safety of infusing autologous
`T cells modified to express the CD19-
`targeted CAR 19-28z in subjects with
`relapsed or purine analog–refractory
`CLL. For 2–3 days following T-cell infu-
`sion, the subjects are closely monitored
`for tumor lysis and unforeseen adverse
`events. If stable, subjects are discharged
`and subsequently closely followed in the
`outpatient clinic setting.
`This phase I clinical trial has a three-
`step design (Table 1). In the first step,
`subjects are treated with dose level 1 of
`modified T cells (1.2–3.0 × 107 CAR+
`T cells/kg) without prior lymphodepleting
`chemotherapy. The subject of the current
`report was enrolled in cohort 1 of step 2
`and treated with 1.5 g/m2 of cyclophos-
`phamide followed 2 days later by infusion
`of modified T cells at dose level 1. The
`enrollment thus far is summarized in
`Table 1.
`
`Case report
`Subject 4 was a 69-year-old man with
`refractory CLL who was enrolled in clini-
`cal trial IRB no. 06-138. At the time of
`enrollment, three previous subjects had
`been treated on this protocol without sig-
`nificant adverse events in the first planned
`cohort, receiving the lowest planned dose
`
`of modified T cells alone. Subject 4 was
`the first to receive lymphodepleting che-
`motherapy with cyclophosphamide (1.5
`g/m2) followed 2 days later by infusion of
`modified T cells at the same dose tolerated
`earlier by the first three subjects enrolled
`in cohort 1 of this trial.
`Subject 4’s CLL treatment history.
`Subject 4 was initially diagnosed with CLL
`8 years before treatment on this protocol,
`when he was noted to have an elevated
`lymphocyte count on a routine complete
`blood count in the context of lymphade-
`nopathy. The subject had a significant past
`medical history of myocardial infarction,
`coronary artery disease, hypertension, and
`chronic renal insufficiency. Two years after
`diagnosis, because of progressive symp-
`tomatic abdominal lymphadenopathy
`and a rapidly doubling peripheral blood
`lymphocyte count, he was treated per
`Memorial Sloan–Kettering Cancer Center
`(MSKCC) IRB protocol no. 98-080 with
`sequential fludarabine (25 mg/m2) daily
`for 5 days every 4 weeks for six cycles,
`followed by high-dose cyclophos phamide
`(3 g/m2) given once every 3 weeks for
`three cycles, followed by rituximab (375
`mg/m2) given weekly for 8 weeks. He
`achieved a durable partial response. Five
`years later, he developed evidence of
`progressive disease as shown by increasing
`lymphadenopathy, increasing peripheral
`blood lymphocyte counts, and cytopenias.
`The subject was enrolled in MSKCC IRB
`protocol no. 05-077 and treated with six
`monthly cycles of combination therapy
`with pentostatin (4 mg/m2), cyclophos-
`phamide (600 mg/m2), rituximab (375
`mg/m2 given only on cycles 2–6), and
`mitoxantrone (10 mg/m2). The subject
`once more achieved a partial response.
`Two years later, the subject presented with
`a rapidly increasing peripheral blood lym-
`phocyte count, worsening cytopenias, and
`
`Table 1 Cyclophosphamide and T-cell doses in IRB protocol no. 06-138
`Step
`Cyclophosphamide
`CAR + T-cell dose
`No. of enrolled subjects
`1
`0
`3
`1.2–3.0 × 107/kg
`2
`1.5 g/m2
`1
`1.2–3.0 × 107/kg
`3.0 g/m2
`0
`1.2–3.0 × 107/kg
`MTD
`3
`0
`0.4–1.0 × 108/kg
`1.5 g/m2
`–1
`2
`4.0–10 × 106/kg
`CAR, chimeric antigen receptor; MTD, maximum-tolerated dose.
`
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`© The American Society of Gene Therapy
`
`Figure 1 Clinical assessment of subject 4 on IRB no. 06-138, NIH-RAC no. 0507-721,
`NCT00466531. Assessment of patient from the time of admission to the hospital before cyclo-
`phosphamide chemotherapy (–48 hours), through time of modified T-cell infusion (0 hours), to
`time of death (44 hours). Clinical status was assessed by routine vital sign parameters, including
`(a) temperature and systolic blood pressure, as well as by laboratory chemistry measurements,
`including (b) renal function as measured by creatinine, potassium, phosphorus, and uric acid con-
`centrations. Vital signs over time are consistent with a sepsis syndrome (fever with hypotension),
`whereas laboratory chemistry studies demonstrate an initial rise in creatinine coinciding with the
`patient’s anuric state, followed by rising potassium, phosphorus, and uric acid at concentrations
`that are consistent with tumor lysis but confounded by the antecedent acute renal failure. The
`vertical arrow indicates the time of T-cell infusion.
`
`increasing lymphadenopathy and he was
`enrolled in IRB protocol no. 06-138 (NIH-
`RAC no. 0507-721, NCT00466531). The
`subject was assessed and met all criteria
`for enrollment in this trial.
`Treatment course. For IRB proto-
`col no. 06-138, the subject underwent a
`leukapheresis procedure, and the product
`was processed and frozen. Subsequently,
`T cells were activated and retrovirally
`transduced with the 19-28z retroviral
`vector as described.6 The subject was
`then admitted and, per protocol, received
`tumor lysis prophylaxis with hydration
`and allopurinol followed by cyclophos-
`phamide (1.5 g/m2) infusion. He toler-
`ated therapy well, and twice-daily serum
`electrolyte studies revealed no evidence of
`tumor lysis. On the day of T-cell infusion,
`the subject’s tumor lysis laboratory results
`were unremarkable, with the exception
`of a mildly elevated phosphorus level (4.8
`mg/dl). His creatinine was 1.3 mg/dl.
`The T-cell infusion was completed over 3
`hours without complication. Twenty hours
`after modified T-cell infusion, the subject
`developed a fever, a transient finding
`observed in all three subjects treated on
`step 1 of this protocol; however, in con-
`trast to previously treated subjects, subject
`4’s fever persisted and was associated with
`concomitant hypotension (Figure 1a).
`At the same time, the subject developed
`
`respiratory distress despite a negative chest
`X-ray. After bacterial blood cultures had
`been obtained, the patient was started on
`broad-spectrum antibiotics (piperacil-
`lin/tazobactam and ciprofloxacin) with
`pressor support. He was subsequently
`transferred to the intensive care unit.
`Laboratory studies obtained at 24 hours
`after T-cell infusion demonstrated an
`elevated creatinine concentration (2.2 mg/
`dl) and rising phosphorus (7.4 mg/dl),
`potassium (5.0 mEq/L), and uric acid (8.3
`mg/dl) concentrations.
`The subject became anuric, consistent
`with acute renal failure. In the intensive
`care unit, his blood pressure responded
`to inotropic support, from which he was
`successfully and fully weaned over the
`course of the day. However, he remained
`anuric with increasing serum potas-
`sium and phosphorus concentrations
`(Figure 1b). By the early evening, he once
`more became hypotensive, and inotropic
`support was restarted. A worsening
`respiratory status led to intubation and
`mechanical ventilation. Supportive care
`was withdrawn shortly thereafter at the
`request of the subject’s health-care proxy.
`The subject expired 44 hours after infusion
`of modified T cells. Laboratory studies
`just before his death demonstrated an
`increasing serum creatinine concentration
`at 3.7 mg/dl, as well as markedly elevated
`
`letters to the editor
`
`potassium and phosphorus concentrations
`at 9.1 mEq/L and 14.2 mg/dl, respectively
`(Figure 1b). The subject’s peripheral blood
`lymphocytosis remained generally stable
`over time, beginning with initial chemo-
`therapy and following modified T-cell
`infusion (data not shown).
`Postmortem pathology report. Both
`gross and histologic postmortem analyses
`of tissues from subject 4 revealed extensive
`CLL with diffuse bulky adenopathy, in-
`cluding a large abdominal tumor (2.5 kg)
`and associated increased mesentery
`lymphadenopathy. Microscopic evalua-
`tion revealed diffuse CLL involvement in
`multiple organs, including the liver, pan-
`creas, adrenal glands, and bone marrow,
`as well as the lymph nodes. Renal tissues
`were generally normal, other than scat-
`tered calcium crystals. Overall, these data
`fail to support a diagnosis of tumor lysis
`syndrome as the primary source of renal
`failure. Histology of the lung and cardiac
`tissues showed no significant pathology.
`Furthermore, initial blood cultures, as well
`as all subsequent cultures obtained after
`antibiotic administration, were negative.
`The infused cell product was sterile at the
`time of infusion, a result we reconfirmed
`after the occurrence of the serious adverse
`event (SAE).
`Analysis of serum cytokines. As
`stipulated in the protocol, serial serum
`samples were routinely obtained from
`all subjects before and after each stage
`of the treatment regimen. Analyses of
`these serum samples revealed a sig-
`nificant increase in the concentrations of
`interleukin (IL)-2, IL-7, IL-15, and IL-12
`cytokines following cyclophosphamide
`chemotherapy as compared with the
`pretreatment serum sample obtained 30
`days earlier (Figure 2). An interpretation
`implicating the cyclophosphamide chemo-
`therapy in this rise in serum cytokines is
`hampered by the 30-day time lag between
`obtaining the pre- and post-chemotherapy
`serum samples. Alternatively, the eleva-
`tion of these cytokines may have been
`secondary to a prior subacute infectious
`process that was subsequently exacerbated
`by cyclophos phamide-mediated immune
`suppression, resulting in the sepsis
`syndrome seen in this subject. Signifi-
`cantly, serum tumor necrosis factor-α and
`interferon-γ were unchanged immediately
`before and after T-cell infusion (Figure 2).
`
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`letters to the editor
`
`Conclusion
`Etiology of SAE. Subject 4, a 69-year-old
`patient with bulky CLL, was the first to
`receive T cells following prior lympho-
`depleting chemotherapy (IRB protocol no.
`06-138, step 2, cohort 1). In the first co-
`hort (step 1), in which three subjects were
`treated with the lowest planned modified
`T-cell dose alone, all experienced tran-
`sient fevers following T-cell infusion but
`otherwise tolerated therapy well. These
`subjects had no evidence of hypotension,
`tumor lysis, or acute renal failure. In con-
`trast to subjects treated in the first cohort,
`subject 4 developed persistent fevers fol-
`lowing T-cell infusion, became hypoten-
`sive, and developed acute renal failure, all
`consistent with a clinical picture of sepsis.
`Significantly, acute renal failure developed
`in the absence of any clinical evidence
`suggestive of tumor lysis syndrome. A
`later rise in serum potassium, phospho-
`rus, and uric acid concentrations may
`be indicative of a subsequent incipient
`tumor lysis. This sequence of events thus
`suggests that the patient developed renal
`failure due primarily to hypotension as a
`consequence of sepsis-like syndrome, a
`conclusion supported by the postmortem
`examination of renal tissues.
`Although consistent with an infec-
`tious etiology, the subject’s blood cultures
`as well as postmortem cultures failed to
`detect any bacterial growth, noting that
`the latter may have been compromised by
`broad-spectrum antibiotic therapy.
`Serum cytokine analysis revealed
`markedly elevated levels of the proinflam-
`matory and homeostatic cytokines IL-2,
`IL-7, IL-15, and IL-12 following cyclo-
`
`phosphamide chemotherapy and preced-
`ing the T-cell infusion. The etiology of this
`elevated cytokine profile remains unclear,
`because the pretreatment serum sample
`was obtained 30 days before chemothera-
`py. Nevertheless, regardless of the etiology,
`this cytokine milieu, highly favorable
`to T-cell persistence, activation, and
`proliferation, may account for the possible
`incipient tumor lysis seen in subject 4 but
`not in subjects 1–3.
`Combining this biological evidence
`with the unremarkable lung, heart, and
`kidney pathology, this SAE, despite
`negative blood cultures, is consistent
`with sepsis due to infection, leading to
`hypotension, leading to acute renal failure
`and, ultimately, death. This scenario is also
`consistent with infection as a prevalent
`and leading cause of morbidity and mor-
`tality in patients with advanced CLL.
`Consequent modification of clini-
`cal trial protocol. Our findings fail to
`directly attribute the SAE in subject 4
`(IRB protocol no. 06-138) to the modified
`T cells. Nevertheless, because of the tem-
`poral relationship of the autologous T-cell
`infusion to this SAE, we conservatively
`attributed infusion of modified T cells as
`a “possible” source contributing to this
`SAE. As stipulated in the protocol, we
`reduced the CAR+ T-cell dose in the next
`cohort of patients to the –1 dose (0.4–1
`× 107 modified T cells/kg, Table 1). As
`a further precaution to enhance patient
`safety, we modified the protocol by
`administering T cells as a split infusion,
`infusing one-third of the dose on day 2
`following cyclophosphamide therapy and,
`in the absence of evidence of tumor lysis,
`
`Figure 2 Serum cytokine concentrations measured in subject 4. Serum samples were obtained
`30 days before cyclophosphamide (–30 d), 2 hours before T-cell infusion (–2 h), and 4 and 26 hours
`after T-cell infusion (+4 h, +26 h, respectively). The –2-h sample is therefore post-cyclophosphamide
`but pre-T-cell infusion. Pretreatment tumor necrosis factor-α (TNF-α) serum values were 200, 50,
`and 59 ng/ml in subjects 1, 2, and 3, respectively. IFN-γ, interferon-γ; IL, interleukin.
`
`© The American Society of Gene Therapy
`
`hypotension, or renal failure, adminis-
`tering the remaining two-thirds of the
`planned T-cell dose on day 3 following
`cyclophosphamide therapy. Significantly,
`the subsequent subject treated on this
`trial under these modified conditions did
`not exhibit evidence of a “cytokine storm”
`following cyclophosphamide chemo-
`therapy and further tolerated infusion
`of modified T cells without any notable
`toxicities. We will continue to focus on
`analyses of serum cytokine studies before
`and following both cyclo phosphamide
`chemotherapy and modified T-cell
`infusion, with special attention to the
`conditioning-induced cytokine response
`and its relationship to post-T-cell
`infusion cytokine concentrations.
`
`Note
`Since the submission of this letter, we
`have treated a sixth subject at the −1 treat-
`ment dose (see Table 1). This subject ex-
`hibited a transient hypotensive episode 24
`hours after T-cell infusion that responded
`to increased intravenous hydration, ac-
`companied by a mild, transient fever. This
`episode rapidly resolved with no evidence
`of infectious etiology or renal compro-
`mise. Cytokine serum analyses were
`unremarkable.
`
`doi:10.1038/mt.2010.31
`
`Renier Brentjens,1 Raymond Yeh,1
`Yvette Bernal,1 Isabelle Riviere1
`and Michel Sadelain1
`1Memorial Sloan–Kettering Cancer Center, New
`York, New York, USA
`Correspondence: Michel Sadelain (m-sadelain@
`ski.mskcc.org)
`
`ReFeReNCeS
`1
`Sadelain, M, Rivière, I and Brentjens, R (2003).
`Targeting tumours with genetically enhanced T
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`2. Brentjens, RJ, Latouche, JB, Santos, E, Marti, F, Gong,
`MC, Lyddane, C et al. (2003). Eradication of systemic
`B-cell tumors by genetically targeted human T lym-
`phocytes co-stimulated by CD80 and interleukin-15.
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`3. Maher, J, Brentjens, R, Gunset, G, Rivière, I and
`Sadelain, M (2002). Human T-lymphocyte cytotoxic-
`ity and proliferation directed by a single chimeric
`TCRzeta /CD28 receptor. Nat Biotechnol 20: 70–75.
`4. Brentjens, RJ, Santos, E, Nikhamin, Y, Yeh, R, Matsush-
`ita, M, La Perle, K et al. (2007). Genetically targeted T
`cells eradicate systemic acute lymphoblastic leukemia
`xenografts. Clin Cancer Res 13: 5426–5435.
`5. Sadelain, M, Brentjens, R and Rivière, I (2009). The
`promise and potential pitfalls of chimeric antigen
`receptors. Curr Opin Immunol 21: 215–223.
`6. Hollyman, D, Stefanski, J, Przybylowski, M, Bartido,
`S, Borquez-Ojeda, O, Taylor, C et al. (2009). Manu-
`facturing validation of biologically functional T cells
`targeted to CD19 antigen for autologous adoptive
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`
`668
`
`www.moleculartherapy.org vol. 18 no. 4 april 2010
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`Miltenyi v. UPenn
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