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`
`Plenary paper
`
`B-cell depletion and remissions of malignancy along with cytokine-associated
`toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor–transduced
`T cells
`James N. Kochenderfer,1 Mark E. Dudley,2 Steven A. Feldman,2 Wyndham H. Wilson,3 David E. Spaner,4 Irina Maric,5
`Maryalice Stetler-Stevenson,6 Giao Q. Phan,2 Marybeth S. Hughes,2 Richard M. Sherry,2 James C. Yang,2
`Udai S. Kammula,2 Laura Devillier,2 Robert Carpenter,1 Debbie-Ann N. Nathan,2 Richard A. Morgan,2 Carolyn Laurencot,2
`and Steven A. Rosenberg2
`
`1Experimental Transplantation and Immunology Branch, 2Surgery Branch, and 3Metabolism Branch, National Cancer Institute (NCI), Bethesda, MD;
`4Sunnybrook Health Sciences Center, Toronto, ON; 5Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, MD; and
`6Laboratory of Pathology, NCI, Bethesda, MD
`
`We conducted a clinical trial to assess
`adoptive transfer of T cells genetically
`modified to express an anti-CD19 chime-
`ric Ag receptor (CAR). Our clinical proto-
`col consisted of chemotherapy followed
`by an infusion of anti–CD19-CAR–
`transduced T cells and a course of IL-2.
`Six of the 8 patients treated on our proto-
`col obtained remissions of
`their ad-
`vanced, progressive B-cell malignancies.
`Four of the 8 patients treated on the
`protocol had long-term depletion of nor-
`mal polyclonal CD19ⴙ B-lineage cells.
`Introduction
`
`Cells containing the anti-CD19 CAR gene
`were detected in the blood of all patients.
`Four of the 8 treated patients had promi-
`nent elevations in serum levels of the
`inflammatory cytokines IFN␥ and TNF.
`The severity of acute toxicities experi-
`enced by the patients correlated with
`serum IFN␥ and TNF levels. The infused
`anti–CD19-CAR–transduced T cells were
`a possible source of these inflammatory
`cytokines because we demonstrated pe-
`ripheral blood T cells that produced TNF
`and IFN␥ ex vivo in a CD19-specific man-
`
`ner after anti–CD19-CAR–transduced
`T-cell
`infusions. Anti–CD19-CAR–trans-
`duced T cells have great promise to im-
`prove the treatment of B-cell malignan-
`cies because of a potent ability to
`eradicate CD19ⴙ cells in vivo; however,
`reversible cytokine-associated toxicities
`occurred after CAR–transduced T-cell in-
`fusions. This trial was registered with
`ClinicalTrials.gov
`as NCT00924326.
`(Blood. 2012;119(12):2709-2720)
`
`Chimeric Ag receptors (CARs) are fusion proteins that incorporate Ag
`recognition moieties and T-cell activation domains.1-3 The Ag recogni-
`tion moieties of CARs are usually variable regions of mAbs.1-3 T cells
`genetically modified to express CARs acquire the ability to
`specifically recognize targeted Ags.2-8 CD19 is a protein that is
`expressed on almost all B-lineage cells.9 Because expression of
`CD19 is limited to normal and malignant B-lineage cells, CD19
`is an attractive target for immunotherapies aimed at B-cell
`malignancies.9 Many groups have conducted preclinical experi-
`ments with T cells expressing anti-CD19 CARs, and these experi-
`ments have shown that anti–CD19-CAR–expressing T cells can
`recognize and destroy target cells in a CD19-specific manner.10-18
`The CARs used in these experiments have contained T-cell
`activation domains from molecules such as CD3␨ and a variety of
`costimulatory domains such as those from CD28 and 4-1BB.12-17
`Murine studies have shown that syngeneic T cells genetically
`modified to express anti-CD19 CARs can cure lymphoma and
`cause long-term eradication of normal B cells.19,20 Based on these
`preclinical experiments, clinical trials of anti-CD19 CARs have
`been initiated, and some early results from these trials have been
`reported.21-27 Similar to the murine studies, these early clinical
`reports have suggested an anti-malignancy effect of T cells express-
`ing anti-CD19 CARs, and Ag-specific eradication of normal B cells
`has been demonstrated.21,23,24,27
`
`Significant toxicities including hypotension, fevers, and renal
`insufficiency have occurred after infusions of anti–CD19-CAR–
`expressing T cells.22-24,27 Three patients with elevations in serum
`levels of inflammatory cytokines such as IFN␥ after anti–CD19-
`CAR–transduced T-cell infusions have been reported22-24; however,
`in one of these cases,
`the elevation in serum inflammatory
`cytokines was present before CAR-transduced T cells were in-
`fused.22 Determining the causes of elevated cytokine levels after
`anti–CD19-CAR–transduced T-cell infusions is not straightforward
`because only a small number of patients with elevated serum
`cytokine levels have been reported, and there are other possible
`causes of elevated serum cytokines such as sepsis.28 Inflammatory
`cytokines such as IFN␥ and TNF (formerly known as TNF␣) are
`produced by anti–CD19-CAR–transduced T cells in vitro.10,12,15
`IFN␥ and TNF can cause significant
`toxicity in humans29-32;
`however, an association between inflammatory cytokine produc-
`tion by anti–CD19-CAR–transduced T cells and clinical toxicity
`has not been demonstrated. A better understanding of the relation-
`ship between cytokine production by CAR-transduced T cells and
`clinical toxicity is necessary to rationally plan future research
`aimed at
`increasing the safety of anti–CD19-CAR–transduced
`T cells.
`We are conducting a clinical trial to assess the anti-malignancy
`efficacy, toxicity, and in vivo persistence of T cells transduced with
`
`Submitted October 6, 2011; accepted December 5, 2011. Prepublished online
`as Blood First Edition paper, December 8, 2011; DOI 10.1182/blood-2011-10-
`384388.
`
`There is an Inside Blood commentary on this article in this issue.
`
`The online version of this article contains a data supplement.
`
`BLOOD, 22 MARCH 2012 䡠 VOLUME 119, NUMBER 12
`
`2709
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`2710
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`KOCHENDERFER et al
`
`BLOOD, 22 MARCH 2012 䡠 VOLUME 119, NUMBER 12
`
`Figure 1. Anti–CD19-CAR–transduced T-cell produc-
`tion and clinical treatment protocols. (A) PBMCs were
`stimulated with the anti-CD3 mAb OKT3 on day 0. The
`cells were transduced with gammaretroviruses encoding
`the anti-CD19 CAR on days 2 and 3. On day 10, a rapid
`expansion protocol was started, and the cells were ready
`for infusion on day 24. (B) Patients received 60 mg/kg
`cyclophosphamide chemotherapy daily for 2 days. Next,
`patients received 25 mg/m2 fludarabine chemotherapy
`daily for 5 days. One day later, the patients received a
`single infusion of anti–CD19-CAR–transduced T cells.
`Starting on the same day as the T-cell
`infusion, the
`patients received IV IL-2 every 8 hours.
`
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`
`an anti-CD19 CAR. All of the patients on our clinical trial had
`advanced, progressive B-cell malignancies that were incurable by
`any standard treatment except allogeneic stem cell transplantation.
`Six of the 8 patients treated on our trial obtained objective
`remissions of their malignancies, and 4 of 8 patients had long-term
`elimination of CD19⫹ B-lineage cells. Significant toxicities that
`correlated with elevations in serum IFN␥ and TNF occurred after
`infusions of anti–CD19-CAR–transduced T cells. In addition, we
`demonstrated CD19-specific IFN␥ and TNF production by T cells
`from the blood of patients who had received infusions of anti–CD19-
`CAR–transduced T cells.
`
`Methods
`
`Clinical trial design
`
`The trial was reviewed by the US Food and Drug Administration and the
`Institutional Review Board of the National Cancer Institute and allowed to
`proceed. Patients provided written informed consent before participation in this
`study in accordance with the Declaration of Helsinki. Patients underwent an
`apheresis to obtain PBMCs for producing anti–CD19-CAR–transduced T cells.
`Cyclophosphamide was administered daily on days ⫺7 and ⫺6 at a dose of
`60 mg/kg. On days ⫺5 through ⫺1, patients received 25 mg/m2 fludarabine
`daily. On day 0, patients received a single infusion of CAR-transduced T cells.
`Three hours after CAR-transduced T cells were administered, IL-2 was initiated.
`IL-2 was administered intravenously at a dose of 720 000 international units/kg
`every 8 hours until toxicity precluded additional doses. Remissions of chronic
`lymphocytic leukemia (CLL) or lymphoma were defined according to standard
`international criteria.33,34
`
`In vitro and ex vivo assays
`
`Anti-Fab Ab staining and staining with labeled CD19 protein were used in
`flow cytometry to detect surface expression of the anti-CD19 CAR as
`described in the supplemental Methods. ELISAs, intracellular cytokine
`staining assays (ICCS), and CD107a degranulation assays were performed
`as described in supplemental Methods and as previously described.12,21
`Serum ELISAs were carried out as described in supplemental Methods.
`Immunohistochemistry and flow cytometry were carried out as detailed in
`the supplemental Methods and as previously described.21
`
`Real-time qPCR
`
`Real-time quantitative PCR (qPCR) was carried out to determine the
`percentage of peripheral blood mononuclear cells containing the CAR gene
`as described in supplemental Methods.
`
`Calculation of SOFA scores
`
`The sequential organ failure assessment (SOFA) score is an established
`method of quantifying the overall severity of illness.36 The SOFA score
`includes an assessment of hypotension, the platelet count, and measure-
`ments of respiratory, liver, renal, and central nervous system func-
`tion.36,37 We calculated daily SOFA scores for each patient by using
`clinical records from the day of CAR-tranduced T-cell infusion and each
`of the first 10 days after CAR-transduced T-cell infusion.36 For each
`patient, the sum of the SOFA scores from each day was calculated to
`give the total SOFA score.36
`
`Results
`
`Anti-CD19 CAR retroviral vector design
`
`Production of anti–CD19-CAR–transduced T cells
`
`We previously reported the design and construction of the murine stem cell
`virus–based splice-gag vector (MSGV)–FMC63-28Z that encoded the
`anti-CD19 CAR used in our clinical trial.12
`
`Anti–CD19-CAR–transduced T cell preparation
`
`Anti–CD19-CAR–transduced T cells were prepared as described in supple-
`mental Methods (available on the Blood Web site; see the Supplemental
`Materials link at the top of the online article) and as previously described.35
`
`Autologous PBMCs were stimulated with an anti-CD3 mAb and
`transduced with gammaretroviruses encoding an anti-CD19
`CAR (Figure 1A). The anti-CD19 CAR used in our clinical trial
`contained the variable regions of a murine anti–human-CD19
`Ab, a portion of the CD28 molecule, and the signaling domain of
`the CD3␨ molecule.12 The anti-CD19 CAR could be detected on
`the surface of transduced T cells by flow cytometry (Figure 2A).
`For the 8 treated patients,
`the mean percentage of T cells
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`BLOOD, 22 MARCH 2012 䡠 VOLUME 119, NUMBER 12
`
`ANTI-CD19 CAR CLINICAL TRIAL
`
`2711
`
`Figure 2. Anti–CD19-CAR–transduced T cells pro-
`duced cytokines in a CD19-specific manner and
`recognized autologous leukemia cells. (A) Staining
`with an anti-Fab Ab revealed expression of the anti-
`CD19 CAR on the surface of T cells that were adminis-
`tered to patient 7. Staining with an isotype control Ab is
`also shown. Both plots were gated on CD3⫹ lympho-
`cytes, which made up 99% of the cells in the culture.
`(B) On the day of
`infusion, T cells of patient 7 up-
`regulated CD107a expression after a 4-hour culture with
`the CD19⫹ target cell CD19-K562 but not the negative
`control cell NGFR-K562 that does not express CD19.
`(C) On the day of infusion, anti–CD19-CAR–transduced
`T cells of patient 7 produced IFN␥, TNF, and IL-2 when
`cultured for 6 hours with the CD19⫹ target cell CD19-
`K562 but not the negative control cell NGFR-K562 that
`does not express CD19. The results shown in panels A
`through C are representative of the results obtained for
`all of the patients on the protocol. (D) Anti–CD19-CAR–
`transduced T cells of patient 3 were cultured with either
`autologous pretreatment lymphocytes or autologous re-
`mission lymphocytes overnight, and an IFN␥ ELISA was
`performed on the supernatant. Anti–CD19-CAR–
`transduced T cells of patient 3 specifically recognized
`pretreatment
`lymphocytes but not remission lympho-
`cytes obtained 7 weeks after CAR-transduced T-cell
`infusion. Sixty-four percent of the pretreatment lympho-
`cytes were CD19⫹ leukemia cells. The remission lympho-
`cytes contained only 0.1% CD19⫹ cells. (E) Anti–CD19-
`CAR–transduced T cells of patient 6 were cultured with
`either pretreatment autologous lymphocytes or autolo-
`gous remission lymphocytes overnight and an IFN␥
`ELISA was performed on the supernatant. Anti–CD19-
`CAR–transduced T cells of patient 6 specifically recog-
`nized pretreatment lymphocytes but not remission lym-
`phocytes obtained 2 weeks after CAR–transduced T-cell
`infusion. Seventy-six percent of the pretreatment lympho-
`cytes were CD19⫹ leukemia cells. The remission lympho-
`cytes contained only 0.1% CD19⫹ cells. In both panels D
`and E, pretreatment lymphocytes cultured alone did not
`produce detectable quantities of IFN␥.
`
`expressing the anti-CD19 CAR at the time of infusion was 55%
`(Table 1). Most of the infused CAR-transduced cells were
`CCR7-negative, CD45RA-negative effector memory cells, but
`variable numbers of CCR7⫹, CD45RA-negative central memory
`
`cells were also present (Table 2). The CAR-expressing T cells
`specifically up-regulated CD107a when cultured with CD19-
`expressing target cells but not when cultured with negative
`control target cells that lacked CD19 expression (Figure 2B).
`
`Table 1. Patient data
`
`Patient Age, y
`
`Malignancy
`
`Total no. of
`infused
`cells/kg,
`ⴛ107
`
`No. of prior
`therapies
`
`Percentage of
`infused cells
`CARⴙ
`
`No. of infused
`CARⴙ cells/kg
`ⴛ107
`
`Infused cells
`CD4/CD8
`ratio
`
`Doses of IL-2
`administered
`
`Response and time
`since treatment, mo*
`
`1a†
`1b†
`2
`3
`4
`
`5
`6
`7
`8
`
`47
`48
`48
`61
`55
`
`54
`57
`61
`63
`
`Follicular lymphoma
`Follicular lymphoma
`Follicular lymphoma
`CLL
`Splenic marginal zone
`lymphoma
`CLL
`CLL
`CLL
`Follicular lymphoma
`
`4
`5
`5
`3
`3
`
`4
`7
`4
`7
`
`0.5
`2.1
`0.5
`2.5
`2.0
`
`0.6
`5.5
`5.4
`4.2
`
`64
`63
`65
`45
`53
`
`50
`30
`51
`71
`
`0.3
`1.3
`0.3
`1.1
`1.1
`
`0.3
`1.7
`2.8
`3.0
`
`29/63
`19/71
`23/73
`35/53
`72/24
`
`87/12
`37/57
`58/41
`54/43
`
`8
`10
`9
`2
`4
`
`2
`1
`2
`5
`
`PR (7)
`PR (18⫹)
`NE (died with influenza)
`CR (15⫹)
`PR (12)
`
`SD (6)
`PR (7)
`PR (7⫹)
`PR (8⫹)
`
`PR indicates partial remission; NE, not evaluable; CR, complete remission; SD, stable disease; and CLL, chronic lymphocytic leukemia.
`*Time since treatment is in months; ⫹ indicates an ongoing response as of the time of writing. The body surface area of each patient is given in supplemental Table 2.
`†Patient 1 was treated twice. His first treatment has been previously reported (21).
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`2712
`
`KOCHENDERFER et al
`
`BLOOD, 22 MARCH 2012 䡠 VOLUME 119, NUMBER 12
`
`Table 2. Memory phenotype of infused anti-CD19 CAR-expressing T cells
`CCR7ⴙCD45RAⴙ
`CCR7ⴙCD45RAⴚ
`Patient
`CD62L
`
`CCR7ⴚCD45RAⴚ
`
`CCR7ⴚCD45RAⴙ
`
`1a*
`1b*
`2
`3
`4
`5
`6
`7
`8
`
`21
`20
`25
`27
`35
`26
`10
`4
`8
`
`7
`8
`5
`1
`0
`3
`6
`4
`1
`
`30
`20
`21
`8
`5
`13
`27
`27
`9
`
`53
`55
`61
`87
`90
`71
`57
`60
`81
`
`10
`18
`13
`5
`5
`13
`10
`9
`8
`
`The phenotype of the infused CD19 CAR-expressing T cells was determined by flow cytometry. Values are the percentage of the CAR⫹ CD3⫹ cells that expressed the
`indicated markers.
`*Patient 1 was treated twice.
`
`Up-regulation of CD107a indicated degranulation, which is a
`prerequisite for perforin-mediated cytotoxicity.38 The CAR-
`transduced T cells produced IFN␥, TNF, and IL-2 in a CD19-
`specific manner (Figure 2C, Table 3).
`
`Anti–CD19-CAR–transduced T cells can specifically recognize
`autologous leukemia cells
`
`The blood of some of the patients on our trial contained large
`numbers of CD19⫹ CLL cells. Access to pretreatment blood
`samples from these patients allowed us to determine whether
`anti–CD19-CAR–transduced T cells could specifically recognize
`unmanipulated autologous CLL cells. The blood lymphocytes of
`patient 3 contained 64% CD19⫹ CLL cells before treatment on our
`protocol, and the blood lymphocytes of patient 6 contained 76%
`CD19⫹ CLL cells before treatment on our protocol. After treatment
`when the patients were in remission, the blood lymphocytes of both
`patient 3 and patient 6 contained only 0.1% CD19⫹ cells. As shown
`in Figure 2D and E, the anti–CD19-CAR–transduced T cells from
`each of these patients produced large amounts of IFN␥ when cultured
`with the pretreatment lymphocytes that were mostly leukemia cells, but
`the CAR-transduced T cells produced only background levels of IFN␥
`when cultured with the lymphocytes that were obtained after treatment
`when the patients were in remission. IFN␥ was not produced by the
`leukemia cells of either patient (data not shown).
`
`Six of the 8 treated patients obtained objective remissions
`
`The 8 patients treated on our protocol had either B-cell lymphoma or
`CLL (Table 1). The patients all had progressive malignancy at the time
`of enrollment on our protocol despite a median of 4 prior therapies. All
`patients treated on our protocol received cyclophosphamide daily for
`
`2 days followed by fludarabine daily for 5 days. One day after the last
`dose of fludarabine, the patients received a single IV infusion of
`anti–CD19-CAR–transduced T cells (Figure 1B). Three hours after the
`CAR-transduced T-cell infusion, a course of IV IL-2 was initiated
`(Figure 1B). The patients received doses of CAR-expressing T cells that
`ranged from 0.3 ⫻ 107 to 3.0 ⫻ 107 CAR⫹ T cells/kg bodyweight.
`Patient 1 was treated twice. His first treatment course was previously
`reported.21 Patient 1 developed progressive CD19⫹ lymphoma 7 months
`after his first infusion of anti–CD19-CAR–transduced T cells. After his
`lymphoma progressed, patient 1 was treated a second time with the
`same regimen. He remains in a partial remission (PR) 18 months after
`the second treatment. Patient 2 died 18 days after CAR-transduced
`T-cell infusion with culture-proven influenza A pneumonia, nonbacterial
`thrombotic endocarditis, and cerebral infarction, so he is not evaluable
`for lymphoma response. Overall, 6 of the 7 evaluable patients treated on
`our trial obtained objective remissions (Table 1). Because the patients
`received chemotherapy with activity against B-cell malignancies imme-
`diately before the anti–CD19-CAR–transduced T-cell infusion, the
`contribution that the CAR-transduced T cells made to the remissions is
`unclear.
`
`Patient 3 had a prolonged complete remission and depletion of
`normal B cells
`
`At the time of enrollment on our protocol, patient 3 had progressive
`CLL after receiving 3 prior therapies. Just before treatment on our
`protocol, he had a blood B-cell count of 1544 B cells/␮L (Figure
`3A). Ninety-six percent of the blood B cells were CLL cells. In
`addition, 50%-60% of his cellular BM was CLL (Figure 3B-C). A
`prominent clonal leukemia population that expressed the character-
`istic CD19⫹ and CD5⫹ phenotype of CLL was detected by flow
`cytometry of BM cells before treatment on our protocol (Figure
`
`Table 3. Cytokine production by infused cells
`IFN␥, pg/mL
`
`TNF, pg/mL
`
`IL-2, pg/mL
`
`Patient
`
`CD19-positive target
`
`CD19-negative target
`
`CD19-positive target
`
`CD19-negative target
`
`CD19-positive target
`
`CD19-negative target
`
`1*
`2
`3
`4
`5
`6
`7
`8
`
`8190
`9850
`19 000
`27 900
`36 700
`14 800
`29 300
`9960
`
`411
`506
`916
`944
`734
`130
`341
`240
`
`448
`6250
`9312
`21 895
`21 515
`8288
`21 980
`9830
`
`⬍ 31
`⬍ 31
`⬍ 31
`⬍ 31
`⬍ 31
`⬍ 31
`⬍ 31
`73
`
`1156
`2002
`1683
`2768
`2421
`798
`1661
`1697
`
`48
`139
`51
`40
`32
`44
`36
`46
`
`Levels of the indicated cytokines were determined by standard ELISAs after an overnight culture of T cells from the time of infusion with either CD19-positive target cells or
`CD19-negative target cells. NGFR-K562 cells were used as CD19-negative target cells for all cultures. CD19-K562 cells were used as CD19-positive targets for the cultures
`preceding the TNF ELISAs. NALM6 cells were used as the CD19-positive target cells for the cultures preceding the IFN␥ and IL-2 ELISAs.
`*Results are from the second treatment of patient 1.
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`BLOOD, 22 MARCH 2012 䡠 VOLUME 119, NUMBER 12
`
`ANTI-CD19 CAR CLINICAL TRIAL
`
`2713
`
`Figure 3. Normal and malignant B-lineage cells were eliminated from
`the blood and BM of patient 3. (A) Before treatment, the blood of patient
`3 contained an elevated number of B cells, 96% of which were leukemia
`cells. After treatment, the blood B-cell count has remained below normal
`and patient 3 has been in complete remission for 67 weeks. B cells were
`quantitated by flow cytometry staining of CD19⫹ cells. (B) CD19 IHC
`staining of the BM of patient 3 is shown before treatment and 13 weeks
`after treatment. The BM contained large numbers of CD19⫹ cells before
`treatment. Thirteen weeks after treatment, CD19⫹ cells were nearly
`absent. (C) CD20 IHC staining of the BM of patient 3 is shown before
`treatment and 13 weeks after treatment. The BM contained large numbers
`of CD20⫹ cells before treatment. Thirteen weeks after treatment, CD20⫹
`cells were nearly absent. (D) Flow cytometric results of a BM aspirate from
`patient 3 are shown. Plots are gated on lymphoid cells by forward and side
`scatter. A monoclonal population of B cells expressing the characteristic
`CD19⫹ and CD5⫹ phenotype of CLL (circled) was present before treat-
`ment but not 13 weeks after treatment.
`
`3D). After treatment, CLL was completely eradicated from the
`blood of patient 3, and the number of polyclonal blood B cells has
`levels of 17 to 40 B cells/␮L for
`stayed at below-normal
`⬎ 15 months (lower limit of the normal 61 B cells/␮L; Figure 3A).
`Patient 3’s blood B cells that have returned after treatment were
`determined to be polyclonal by flow cytometry staining for Ig ␬ and
`␭ proteins (data not shown). CLL was eliminated from the BM of
`patient 3 after treatment as shown by BM IHC staining (Figure
`3B-C) and by multicolor flow cytometry analysis of the BM cells
`(Figure 3D). B-lineage cells were nearly absent from the BM, but
`other hematopoietic cells had recovered (Figure 3B-C). Overall,
`the cellularity of the BM varied between 20% and 70%. A normal
`BM cellularity for a 61-year-old patient is ⬃ 40%. Patient 3
`continues in complete remission ⬎ 15 months after treatment on
`our protocol.
`
`adenopathy occurred during the time between a pretreatment
`computed tomography (CT) scan and a second CT scan that was
`performed 32 days after treatment (Figure 4A). Regression of the
`adenopathy continued between day 32 and day 132 after the
`CAR-transduced T-cell infusion. This continued regression, which
`occurred ⬎ 33 days after the last dose of chemotherapy suggested
`that the CAR-transduced T cells contributed to the regression of the
`adenopathy. In accordance with this possibility, anti–CD19-CAR–
`transduced T cells persisted in the blood of patient 7 until at least
`day 132 after infusion (Figure 5B). Because the patient received
`chemotherapy before the CAR-transduced T cells, an alternative
`explanation for the decreasing adenopathy between day 32 and
`132 after CAR-transduced T-cell infusion is continued resolution
`of adenopathy because of clearance of leukemia cells that were
`killed by the chemotherapy.
`
`Patient 7 had a substantial reduction in adenopathy after
`CAR-transduced T-cell infusion
`
`Before enrollment on our protocol, patient 7 had CLL that was
`progressive despite 4 prior therapies. Extensive regression of
`
`Patient 8 had a prolonged specific eradication of normal B cells
`
`Patient 8 had a normal level of polyclonal blood B cells before
`treatment on our protocol. After treatment, his blood B cells
`have been eliminated for 26 weeks as of his last follow-up
`
`UPenn Ex. 2020
`Miltenyi v. UPenn
`IPR2022-00853
`
`

`

`2714
`
`KOCHENDERFER et al
`
`BLOOD, 22 MARCH 2012 䡠 VOLUME 119, NUMBER 12
`
`Figure 4. Patients receiving infusions of anti–CD19-CAR–transduced
`T cells had reductions in adenopathy and elimination of normal
`B cells. CT scans of patient 7 showed extensive adenopathy before
`treatment. This adenopathy regressed by day 32 after CAR-transduced
`T-cell infusion (arrow). The enlarged lymph nodes continued to substan-
`tially regress between 32 and 132 days after CAR-transduced T-cell
`infusion. (B) Patient 8 had a normal number of polyclonal blood B cells
`before treatment. B cells were eliminated from the blood after treatment
`and had not recovered 26 weeks after anti–CD19-CAR–transduced T-cell
`infusion. B cell counts were determined by flow cytometry for CD19 and
`confirmed by flow cytometry for CD20. In contrast to the B cells, blood
`T-cell counts (C) and NK-cell counts (D) rapidly recovered after treatment.
`
`Downloaded from http://ashpublications.org/blood/article-pdf/119/12/2709/1350836/zh801212002709.pdf by guest on 19 July 2022
`
`(Figure 4B). The B-cell depletion in patient 8 was specific
`because blood T cells (Figure 4C) and NK cells (Figure 4D)
`recovered shortly after
`treatment. This specific,
`long-term
`elimination of normal CD19⫹ B cells indicates that anti–CD19-
`CAR–transduced T cells are destroying cells expressing the
`targeted CD19 Ag in vivo. Notably, this long-term elimination
`of B cells cannot be attributed to the chemotherapy that the
`patient received because we have previously shown that patients
`receiving the same chemotherapy regimen plus infusions of
`T cells targeting Ags that are not expressed on B cells recover
`normal blood B cell numbers in 8 to 19 weeks.21 Overall, we
`have noted B-cell depletion lasting at least 6 months in 4 of the
`8 patients treated on our protocol. As previously reported,
`patient 1 had a complete eradication of blood and BM B-lineage
`cells for 36 weeks after his first infusion of anti–CD19-CAR–
`transduced T cells.21 Nine weeks after his second treatment,
`patient 1 was found to have a normal number of polyclonal
`blood B cells. The reason for this recovery of normal B cells is
`unknown. We have noted prolonged B-cell depletion that lasted
`at least 6 months in 3 additional patients. Patient 7 had no
`detectable blood B cells at his last follow-up 7 months after
`infusion of anti–CD19-CAR–transduced T cells. The prolonged
`depressions of blood B cell numbers in patient 3 and patient 8
`are shown in Figures 3A and 4B, respectively. For all patients,
`the blood B-cells were quantitated 4 to 5 months after CAR-
`transduced T-cell infusion (supplemental Table 1).
`
`Varied levels of anti–CD19-CAR–transduced T cells could be
`detected in the blood of all patients
`
`To quantitate anti–CD19-CAR–transduced T cells in the blood of
`patients, we developed a qPCR assay that specifically detected the DNA
`sequence of the anti-CD19 CAR. Data from these quantitative PCR
`
`experiments were expressed as a percentage of total PBMCs that
`contained the anti-CD19 CAR gene. The persistence of cells containing
`the CAR gene varied widely, but in patient 1 through patient 6, cells
`containing the CAR gene could be detected at levels ⬎ 0.01% of total
`PBMCs for ⬍ 20 days (Figure 5A). In contrast, patient 7 and patient 8
`had high peak percentages and long-term persistence of PBMCs
`containing the CAR gene (Figure 5B). We confirmed the high levels of
`CAR-expressing cells in the blood of patient 7 and patient 8 by detecting
`CAR-expressing T cells with flow cytometry after staining with labeled
`CD19 protein (Figure 5C and D). We also detected cells containing the
`CAR gene in the BM of patient 7 and patient 8. As measured by qPCR,
`0.03% of the BM cells of patient 7 contained the CAR gene 14 weeks
`after CAR-transduced T-cell infusion, and 0.13% of the BM cells of
`patient 8 contained the CAR gene 8 weeks after CAR-transduced T-cell
`infusion.
`
`T cells that degranulated in a CD19-specific manner were
`detected in the blood of patient 7 ex vivo
`
`We demonstrated the presence of functional CD19-specific
`T cells in the blood of patient 7 by detecting T cells that
`up-regulated CD107a in a CD19-specific manner after anti–
`CD19-CAR–transduced T-cell infusion but not before CAR-
`transduced T-cell infusion (Figure 5E).
`
`Patients experienced significant toxicity that correlated with
`elevated levels of IFN␥ and TNF
`
`As mentioned previously, 4 of the 8 patients on our clinical trial
`had long-term depletion of normal B cells. In accordance with
`an eradication of B-lineage cells, all of these 4 patients have
`developed hypogammaglobulinemia and have subsequently been treated
`
`UPenn Ex. 2020
`Miltenyi v. UPenn
`IPR2022-00853
`
`

`

`Downloaded from http://ashpublications.org/blood/article-pdf/119/12/2709/1350836/zh801212002709.pdf by guest on 19 July 2022
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`BLOOD, 22 MARCH 2012 䡠 VOLUME 119, NUMBER 12
`
`ANTI-CD19 CAR CLINICAL TRIAL
`
`2715
`
`Figure 5. Anti–CD19-CAR–transduced T cells can be
`detected in the blood of patients for up to 181 days
`after infusion. (A) The percentage of total PBMCs that
`contained the anti-CD19 CAR gene was determined by
`quantitative PCR. In patients 1 through 6, the peak levels
`and persistence of cells containing the CAR gene varied.
`(B) The percentage of total PBMCs from patient 7 and
`patient 8 that contained the anti-CD19 CAR gene was
`determined by quantitative PCR. Compared with the
`other patients, these patients had substantially greater
`persistence of cells that contained the CAR gene.
`(C) Anti-CD19 CAR expression was detected ex vivo on
`PBMCs of patient 7 by flow cytometry staining with
`labeled CD19 protein (CD19-DDK) 13 days after CAR-
`transduced T-cell
`infusion. Only background levels of
`cells expressing the anti-CD19 CAR were detected
`before treatment. (D) In patient 8, T cells expressing
`the anti-CD19 CAR were detected by flow cytometry
`staining with labeled CD19 protein 13 days after CAR-
`transduced T-cell infusion. Only background levels of
`cells expressing the anti-CD19 CAR were detected
`before treatment. (C-D) The plots are gated on CD3⫹
`lymphocytes. (E) PBMCs from patient 7 were cultured
`for 4 hours with either CD19⫹ CD19-K562 cells or
`NGFR-K562 control cells that do not express CD19.
`Before treatment, CD107a was not up-regulated on
`T cells after culture with either CD19-K562 cells or
`NGFR-K562 cells. In contrast, T cells from 8 days after
`infusion up-regulated CD107a after a 4-hour culture with
`CD19-K562 cells but not NGFR-K562 cells. All plots are
`gated on CD3⫹ lymphocytes.
`
`with infusions of IV Igs. B-cell depletion is a manageable form of
`autoimmunity, and it is not unexpected because autoimmunity has been
`observed after other immunotherapies.39 Except for B-cell depletion, the
`most prominent toxicities experienced by patients on our trial included
`hypotension, fevers, fatigue, renal failure, and obtundation (Table 4).
`These toxicities generally peaked during the first 8 days after CAR-
`transduced T-cell infusion and resolved completely over time. Many of
`the toxicities that were observed in our patients can be caused by
`cytokines such as IFN␥ and TNF. For example, TNF is a potent inducer
`of hypotension.30,31 IFN␥ can cause fever, fatigue, and myalgias.29,32 We
`have previously reported a patient who received an infusion of T cells
`transduced with an anti-ERBB2 (human epidermal growth factor
`receptor-2)–specific CAR without administration of exogenous IL-2.40
`This patient developed very high levels of serum inflammatory cyto-
`kines shortly after CAR-transduced T-cell infusion and later died.40 The
`course of this patient demonstrates that CAR-transduced T cells can
`cause large elevations in serum inflammatory cytokines.40 We have
`shown that the CAR-transduced T cells administered to our patients
`could produce IFN␥ and TNF in a CD19-specific manner in vitro
`(Figure 2, Table 3). To determine whether elevated levels of the
`inflammatory cytokines IFN␥ and TNF occurred in our patients after
`anti–CD19-CAR–transduced T-cell infusions, we performed ELISAs
`for IFN␥ and TNF on serum samples obtained before treatment and at
`multiple time-points after treatment (Figure 6). We found that the
`patients could be divided into 2 groups. One group, which included
`
`patients 1, 2, 4, and 5, did not have prominent elevations of serum IFN␥
`or TNF (Figure 6A and C). The other group, which included patients 3,
`6, 7, and 8, had prominent elevations in both IFN␥ and TNF during the
`first 10 days after anti–CD19-CAR–transduced T-cell infusion (Figure
`6B and D). We hypothesized that much of the toxicity that occurred in
`our patients was because of elevations in inflammatory cytokines such
`as IFN␥ and TNF. To quantify the toxicity experienced by our patients,
`we calculated SOFA scores for each patient.36,37 The SOFA score is an
`est