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`
`CLINICAL TRIALS AND OBSERVATIONS
`
`Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell
`lymphoma using genetically modified autologous CD20-specific T cells
`Brian G. Till,1,2 Michael C. Jensen3, Jinjuan Wang,1 Eric Y. Chen,1 Brent L. Wood,4 Harvey A. Greisman,4 Xiaojun Qian,1
`Scott E. James,1 Andrew Raubitschek,5 Stephen J. Forman,6 Ajay K. Gopal,1,2 John M. Pagel,1,2 Catherine G. Lindgren,2
`Philip D. Greenberg,1,2 Stanley R. Riddell,1,2 and Oliver W. Press1,2
`
`1Clinical Research Division of the Fred Hutchinson Cancer Research Center, Seattle, WA; 2Department of Medicine, University of Washington, Seattle;
`3Department of Pediatric Hematology-Oncology, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA; 4Department of Laboratory
`Medicine, University of Washington, Seattle; and 5Department of Radioimmunotherapy and 6Division of Hematology and HCT, City of Hope National Medical
`Center and Beckman Research Institute, Duarte, CA
`
`Adoptive immunotherapy with T cells ex-
`pressing a tumor-specific chimeric T-cell
`receptor is a promising approach to can-
`cer therapy that has not previously been
`explored for the treatment of lymphoma
`in human subjects. We report the results
`of a proof-of-concept clinical trial in which
`patients with relapsed or refractory indo-
`lent B-cell lymphoma or mantle cell lym-
`phoma were treated with autologous
`T cells genetically modified by electropo-
`ration with a vector plasmid encoding a
`Introduction
`
`CD20-specific chimeric T-cell receptor and
`neomycin resistance gene. Transfected
`cells were immunophenotypically similar
`to CD8ⴙ effector cells and showed CD20-
`specific cytotoxicity in vitro. Seven pa-
`tients received a total of 20 T-cell infu-
`sions, with minimal toxicities. Modified
`T cells persisted in vivo 1 to 3 weeks in
`the first 3 patients, who received T cells
`produced by limiting dilution methods,
`but persisted 5 to 9 weeks in the next
`4 patients who received T cells produced
`
`in bulk cultures followed by 14 days of
`low-dose subcutaneous interleukin-2
`(IL-2) injections. Of the 7 treated patients,
`2 maintained a previous complete re-
`sponse, 1 achieved a partial response,
`and 4 had stable disease. These results
`show the safety, feasibility, and potential
`antitumor activity of adoptive T-cell
`therapy using this approach. This trial
`was registered at www.clinicaltrials.gov
`as #NCT00012207.
`(Blood. 2008;112:
`2261-2271)
`
`Several lymphoma subtypes are incurable with standard chemo-
`therapy and radiation, but immune-based therapies have emerged
`as effective treatment and offer a potential for cure. Monoclonal
`antibodies (Abs) against the B-cell lymphoma marker CD20 have
`activity alone,1,2 in combination with chemotherapy,3-5 or conju-
`gated with radiation-emitting nuclides.6-8 Adoptive cellular therapy
`with nonmyeloablative allogeneic stem cell transplantation (SCT)
`or donor lymphocyte infusion (DLI) can eradicate tumors, resulting
`in long-term survival, even in highly chemotherapy-refractory
`lymphomas.9-11 Both of these immunotherapy approaches have
`limitations, however, because antibodies fail to cure many types of
`lymphoma, and SCT and DLI, although potentially curative, cannot
`be used in many patients because of significant
`toxicity and
`transplantation-related mortality.
`Because the graft-versus-tumor effect of SCT and DLI appears
`to be mediated by alloreactive donor T lymphocytes,12,13 generating
`T cells specific for tumor antigens minimally expressed in normal
`tissues is an attractive strategy for harnessing this antitumor
`effector activity. One technique involves genetically modifying
`autologous T cells to express a chimeric T-cell receptor (cTCR) that
`targets a tumor antigen and induces antigen-specific T-cell activa-
`tion, proliferation, and killing. Because this antigen-induced activa-
`tion of the T cell occurs in an MHC-independent fashion, a single
`vector can be used universally to confer recognition of a selected
`target antigen. By introducing the cTCR into autologous T cells, the
`
`risk of graft-versus-host disease is eliminated. Such genetically
`modified T cells have been designed to target antigens associated
`with a variety of tumors, with success in animal models14-16 and
`some early evidence of clinical efficacy in human subjects.17
`Our group has developed a technique to manufacture CD20-
`specific T cells by transfecting peripheral blood mononuclear cells
`(PBMCs) with a linearized naked DNA plasmid encoding a cTCR
`derived from a murine anti–human CD20 Ab.18-20 The cell-surface
`antigen CD20 is an attractive target for immune-based therapies
`because it is present in more than 90% of B-cell lymphomas, is
`expressed at a high copy number, is stable on the cell surface, and
`does not internalize on binding Abs.21 These modified T cells
`secrete interleukin-2 (IL-2) in an antigen-dependent manner,19
`selectively kill CD20⫹ target cells in vitro,20 and eradicate human
`xenograft tumors in mice.22 Application of this approach to the
`treatment of lymphoma in human subjects has not yet been
`described. We report here the results of a proof-of-concept clinical
`trial in which ex vivo–expanded, genetically modified autologous
`CD20-specific T cells were used as adoptive cellular therapy for
`patients with relapsed or refractory indolent B-cell non-Hodgkin
`lymphoma (NHL) and mantle cell lymphoma (MCL). We show that
`these T cells can be reproducibly generated and expanded to
`therapeutic numbers, exhibit in vitro antitumor cytotoxicity, persist
`in vivo for up to 9 weeks, and appear to be safe, well tolerated, and
`potentially capable of mediating in vivo antitumor activity.
`
`Submitted December 14, 2007; accepted May 3, 2008. Prepublished online as
`Blood First Edition paper, May 28, 2008; DOI 10.1182/ blood-2007-12-128843.
`
`An Inside Blood analysis of this article appears at the front of this issue.
`
`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.
`
`The online version of this article contains a data supplement.
`
`© 2008 by The American Society of Hematology
`
`BLOOD, 15 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 6
`
`2261
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`
`2262
`
`TILL et al
`
`Methods
`
`Clinical protocol
`
`This clinical protocol was approved by the Fred Hutchinson Cancer
`Research Center Institutional Review Board, the University of Washington
`Institutional Biosafety Committee, the US Food and Drug Administration,
`and the Recombinant DNA Advisory Committee of the National Institutes
`of Health. Informed consent was obtained in accordance with the Declara-
`tion of Helsinki. Patients were eligible if they had a pathologically
`confirmed diagnosis of CD20⫹ MCL or indolent B-cell lymphoma, had
`relapsed or refractory disease after at least one prior chemotherapy, were
`deemed not to be candidates for (or refused) stem cell transplantation, and
`had serologic evidence of prior Epstein-Barr virus (EBV) exposure
`(because the TM-LCL cell line used in T-cell culture is EBV-transformed).
`Patients were excluded if they received fludarabine or cladribine within
`2 years before apheresis (but could receive these drugs as cytoreductive
`therapy after apheresis), anti-CD20 Ab within 4 months of T-cell
`infusions, or chemotherapy within 4 weeks of T-cell infusions; had lymph
`nodes more than 5 cm or more than 5000 circulating lymphoma cells in the
`peripheral blood at the time of T-cell infusions, a previous allogeneic stem
`cell transplantation, or human anti–mouse Ab (HAMA) seropositivity;
`required corticosteroids during the study period; had pulmonary or central
`nervous system involvement with lymphoma; were HIV-seropositive;
`or were pregnant.
`Patients underwent leukapheresis after signing informed consent, and
`then they were allowed to receive cytoreductive chemotherapy for disease
`control or debulking during the 2- to 4-month period of T-cell generation, at
`the discretion of their referring physician. For patients A to E, PBMCs were
`activated, transfected, and plated at limiting dilution with the intention of
`isolating and subsequently expanding T-cell clones. This approach proved
`to be laborious and inefficient, however, and the protocol was modified for
`patients F to I to allow expansion of modified cells in bulk culture. Patients
`subsequently received 3 infusions of autologous CD20-specific T cells
`2 to 5 days apart in escalating doses (108 cells/m2, 109 cells/m2, and
`3.3 ⫻ 109 cells/m2) followed by 14 days of subcutaneous low-dose
`(500 000 IU/m2) interleukin-2 (IL-2) injections twice daily (patients F-I
`only). Patients then underwent clinical follow-up to evaluate toxicities
`related to therapy, which were assessed according to National Institutes of
`Health Common Terminology Criteria for Adverse Events, version
`3.0 (http://ctep.cancer.gov/). A Data and Safety Monitoring Board was
`assembled that performed reviews of the safety data every 6 months.
`Clinical responses were assessed according to International Working
`Group criteria.23
`
`BLOOD, 15 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 6
`
`Generation and expansion of genetically modified T cells. Trans-
`fected cells from patients A through E were selected in G418, and attempts
`were made to generate T-cell clones by limiting dilution as previously
`described.24,25 Although the intention was to isolate clonal populations
`derived from a single progenitor cell, the plating density required to yield
`reliable growth of T cells resulted in the presence of 1 to 3 clones per well,
`as subsequently determined by V␤ TCR spectratyping. For patients
`F through I, G418-resistant transfected cells were grown in bulk cultures as
`previously described.25 As cell numbers increased, T cells were transferred
`to 1-L or 3-L tissue culture bags (Lifecell, Branchburg, NJ). During the
`expansion, 5 to 8 stimulation cycles were performed. Fresh T cells were
`infused in patients A and B. For logistic reasons, T cells from patients D, F,
`G, H, and I were cryopreserved between days 70 and 132 after apheresis in
`Plasmalyte-A containing 5% HSA and 10% DMSO and thawed 3 to 4 hours
`before infusion (48 hours before infusion for patient D). Release criteria
`included detectable cTCR expression by flow cytometry, negative bacterial,
`fungal, and Mycoplasma cultures, endotoxin level no more than 5 EU/kg
`per hour, Gram stain–negative on day of infusion, greater than 80% cell
`viability, TCR␣/␤⫹ and CD3⫹ phenotype by flow cytometry, IL-2 growth
`dependence, and CD20-specific cytotoxicity.
`
`T-cell clonality assays
`
`T-cell clonality was determined by polymerase chain reaction (PCR)
`amplification of rearrangements at the T-cell receptor gamma (TCR␥) locus
`as previously described,26 except that V␥I-J␥1/2, V␥II-J␥1/2, V␥I-J␥P1/2,
`and V␥II-J␥P1/2 rearrangements were amplified in a single multiplex PCR
`reaction and analyzed by capillary electrophoresis on an Applied Biosys-
`tems Model 3130 (Foster City, CA). See Document S1 (available on the
`Blood website; see the Supplemental Materials link at the top of the online
`article) for detailed methods.
`V␤ spectratyping was also performed by flow cytometry. Cells were
`labeled with monoclonal antibodies CD8 ECD and IOTest Beta Mark Kit
`(Beckman Coulter, Fullerton, CA). The expression of each of the 24 T-cell
`receptor isoforms present
`in the Beta Mark Kit (approximately 70%
`coverage of the normal human TCR V␤ repertoire) were determined
`independently on the CD8⫹ T-cell populations, and a threshold of 85%
`positivity for a single isoform or an absence of expression of all 24 isoforms
`outside the reference range was considered to represent a clonal expansion.
`Samples showing 2 or more isoforms outside the reference range were
`considered oligoclonal.
`
`Western blot assay
`
`lysates of modified T cells were probed with a mouse
`Whole cell
`anti–human CD3␨ monoclonal Ab (BD PharMingen, San Diego, CA) as
`previously described.20
`
`T-cell transfection, selection, and expansion
`
`Cytotoxicity assays
`
`All cell culture for therapeutic use was performed in the Cell and Gene
`Therapy Core Laboratory at the University of Washington General Clinical
`Research Center, under current good manufacturing practice standards.
`PBMCs collected by apheresis were diluted 1:2 with PBS containing
`200 mg/L EDTA, isolated by density gradient centrifugation over Ficoll-
`Paque (GE Healthcare, Little Chalfont, United Kingdom), washed, and
`resuspended in RPMI 1640 medium containing 2 mmol of L-glutamine,
`25 mmol HEPES, and 10% fetal calf serum. Cells were activated with
`30 ng/mL OKT3, and after overnight incubation recombinant human IL-2
`was added (50 U/mL).
`Electroporation and selection. On day 4 of culture, cells were
`harvested and resuspended in chilled hypo-osmolar electroporation buffer
`(Eppendorf North America, New York, NY) at 20 ⫻ 106 cells/mL. Cell
`suspensions were mixed with linearized plasmids (25 ␮g/mL) encoding a
`CD20-specific scFvFc:␨ cTCR,18-20,24 and divided into aliquots into chilled
`0.2-cm electroporation cuvettes. Cells were electroporated with an Eppen-
`dorf Multiporator at 250 V for 40 microseconds (␮sec) as previously
`described.20 Approximately 3 days after electroporation, G418 was added to
`flasks (at 0.8 mg/mL). Cells were selected by G418 for 8 days before
`generating cells by limiting dilution.
`
`T-cell cytotoxicity was analyzed 2 to 7 weeks before planned T-cell
`infusions to permit selection of optimal “clones” of T cells for expansion.
`CD20-specific cytotoxicity was assessed with the use of standard chromium-
`release assays with the following target cell lines: EL4-CD20 (a murine
`T-cell lymphoma line transfected to express the human CD20 molecule),
`the parental CD20–nontransfected EL4 cell line, or the Daudi Burkitt
`lymphoma cell line, as previously described.25 Cytotoxicity assays were
`repeated in some patients just before T-cell infusions and showed levels of
`cytotoxicity comparable to assays performed 2 to 7 weeks before infusion.
`
`Flow cytometry for immunophenotypic characterization
`of T cells and lymphocyte subset analysis
`
`Flow cytometry was performed with the use of standard methods. Briefly,
`cells cryopreserved within 1 day of the first T-cell infusion were thawed,
`washed, and labeled with the indicated monoclonal Ab for 15 minutes at
`room temperature in the dark. The samples were then washed once,
`resuspended in a dilute DNA binding dye (DAPI), incubated for 10 minutes,
`and approximately 20 000 events acquired on an LSRII flow cytometer
`(Becton Dickinson, Franklin Lakes, NJ). Data were analyzed using software
`
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`BLOOD, 15 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 6
`
`ANTI-CD20 T-CELL THERAPY FOR NHL
`
`2263
`
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`
`Figure 1. Schema of clinical protocol.
`
`T cells bearing a CD20-specific cTCR to treat indolent and mantle
`cell lymphomas. Autologous PBMCs were collected by apheresis,
`genetically modified, and expanded ex vivo, a process that
`typically required 2 to 4 months. During this interval patients
`underwent cytoreductive chemotherapy if necessary for tumor
`debulking or to maintain disease control. Subjects were then treated
`with 3 infusions of modified CD20-specific T cells, 2 to 5 days
`incremental doses (108 cells/m2, 109 cells/m2, and
`apart, at
`3.3 ⫻ 109 cells/m2) similar to those used in previous adoptive
`T-cell therapy trials,27 but with a shorter interval between infusions
`to limit the potential for development of an immune response
`against the transfected cells. The last 4 patients received low-dose
`subcutaneous injections of IL-2 twice daily for 14 days after the
`final T-cell
`infusion to enhance in vivo T-cell survival and
`proliferation. Patients then underwent follow-up for clinical and
`research end points, and long-term monitoring for adverse events
`for 2 years. The study design is outlined in Figure 1.
`Nine patients with relapsed or refractory indolent B-cell NHL or
`MCL were enrolled: 8 men and 1 woman between the ages of
`43 and 77 years; 8 had relapsed follicular lymphoma, and 1 had
`relapsed MCL. Patients had been treated with a median of 2 prior
`therapies (range, 1-7 therapies; Table 1).
`
`Generation and expansion of autologous CD20-specific T cells
`
`PBMCs collected by apheresis were stimulated with anti-CD3
`Ab (OKT3) and IL-2 and transfected by electroporation with a
`naked DNA plasmid encoding a cTCR consisting of a murine
`kappa leader sequence, CD20-specific scFv derived from the
`Leu16 murine Ab, human IgG1 CH2CH3 hinge, human CD4
`transmembrane, and human CD3␨ intracellular signaling do-
`main, as well as a neomycin resistance gene (neoR) under a
`separate promoter (Figure 2A).20,25 Anti-CD20 cTCR surface
`expression was confirmed by Western blot (Figure 2B) and flow
`cytometry (Figure S3).
`
`developed in our laboratory (WoodList). Positivity for DAPI was used to exclude
`nonviable cells, and thresholds for positivity were determined with unstained
`cells and isotype control Ab, as appropriate. Antibodies were used at the
`manufacturer’s recommended concentrations. A complete list of Abs used is
`included in Document S1. Flow cytometry to detect cTCR expression was
`performed using a FITC-labeled polyclonal goat anti–mouse IgG Fab-specific
`Ab (Sigma-Aldrich, St Louis, MO) as previously described.25
`
`Detection of modified T cells in vivo
`
`PBMCs collected serially after T-cell infusions were isolated by Ficoll density-
`gradient centrifugation, and genomic DNA was extracted using a QIAamp DNA
`Blood Mini Kit (Qiagen, Valencia, CA). The standard consisted of 10-fold serial
`dilutions of purified scFvFc:␨ plasmid DNA starting at 106 copies/␮L, with each
`sample containing 1 ␮g of preinfusion PBMC DNA to control for background
`signal. The negative control was preinfusion PBMC genomic DNA. A 72-bp
`(base pair) fragment containing portions of the CD3␨ chain and adjacent CD4
`transmembrane domain sequences was amplified using forward primer 5⬘-
`TCGCCGGCCTCCTGCTTT-3⬘ and reverse primer 5⬘-CGTCTGCGCTCCT-
`GCTGA-3⬘. The probe used was 5⬘-FAM-TGGGCTAGGCATCTTCTTCA-
`GAGTGAA-TAMRA-3⬘. Primers that amplify a fragment of the ␤-actin gene
`(TaqMan 〉-actin Detection Reagent Kit; Applied Biosystems) were used as an
`internal control and for normalization of DNA quantities. Quantitative real-time
`PCR was performed in triplicate with 1 ␮g DNA in each reaction, using TaqMan
`Universal PCR Master Mix in a 7900HT Sequence Detection System (all
`Applied Biosystems).
`
`Immune response assays
`
`Two assays were performed to test for humoral immune responses to the
`cTCR. In the first assay, 96-well enzyme-linked immunoabsorbent assay
`(ELISA) plates were coated with 0.5 ␮g Leu-16 murine anti–human CD20
`Ab (BD Biosciences, San Diego, CA) in pH 9.6 carbonate buffer and
`blocked with 5% milk before adding samples of goat anti–mouse IgG
`Fab-specific Ab (standard curve; Jackson ImmunoResearch Laboratories,
`West Grove, PA), serially diluted 2% BSA/PBS (negative control), baseline
`patient serum (negative control), HAMA⫹ patient serum (positive control),
`or study subject serum from serial postinfusion time points. Biotinylated
`Leu-16 murine anti–human CD20 Ab (BD Biosciences; 10 ␮g/mL) was
`added to each well as the primary Ab, followed by 1:1000 horseradish
`peroxidase-Avidin D (BD Biosciences). Samples were incubated for
`30 minutes at room temperature and washed 3 times with 0.01 M
`PBS/0.3% Tween between each step. Color reagent (2,2,-azino-bis[3-
`ethylbenzothiazoline-6-sulfonic acid] diammonium salt; Sigma-Aldrich) at
`0.42 mg/mL in citrate buffer (citrate 10.5 mg/mL, pH 4.0) plus hydrogen
`peroxide (100 ␮L/12 mL buffer) was added to each well; absorbency was
`read with a Bio-Tek XS ELISA reader (Bio-Tek Instruments, Winooski,
`VT). Optical density measurements were converted to concentration values
`as calculated from the standard curve. In the second assay, flow cytometry
`was used to assess the presence of anti-cTCR Ab in posttreatment patient
`serum samples (see Document S1 for detailed methods).
`Cellular immune response assays were performed by coincubating
`patient-derived PBMCs (106 cells/mL) serially collected after T-cell
`infusions with irradiated anti-CD20 cTCR-expressing T cells
`(106 cells/mL) from infused batches, at a 2:1 ratio. After 2 rounds of
`the PBMCs were tested in 51Cr release
`stimulation 1 week apart,
`cytotoxicity assays using either autologous T cells transfected with the
`cTCR-encoding plasmid or nontransfected autologous PBMC as target
`cells at a 25:1 E/T ratio. In the first 2 patients treated we also assessed
`the responsiveness of recovered T cells to histocompatibility locus
`antigen–disparate cells as a positive control.
`
`Results
`
`Study design and patient characteristics
`
`The primary objective of this study was to assess the feasibility,
`safety, and toxicity of adoptive therapy using patient-derived
`
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`2264
`
`TILL et al
`
`Table 1. Patient characteristics
`
`BLOOD, 15 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 6
`
`Patient
`
`Age, y
`
`Sex
`
`Diagnosis
`
`Stage
`
`Prior therapies
`
`Cytoreductive therapy before
`T-cell infusions
`
`A
`B
`C
`D
`E
`F
`G
`H
`I
`
`44
`70
`47
`60
`63
`46
`43
`46
`77
`
`F
`M
`M
`M
`M
`M
`M
`M
`M
`
`FL
`FL
`FL
`FL
`MCL
`FL
`FL
`FL
`FL
`
`IV-B
`II-A
`IV-B
`IV-A
`IV-A
`IV-A
`IV-A
`IV-B
`III-A
`
`R-CHOP
`CHOP, rituximab, 131I-tositumomab
`ProMACE/MOPP, ASCT, fludarabine (10 cycles)
`Rituximab
`R-HyperCVAD, GCD-R
`R-CVP
`CHOP, IFN, CY ⫹ VP16, R-CY, CY ⫹ DEX, GCD-R, ASCT
`R-CHOP, fenretinide
`R-CVP, R-CHOP, GCD-R
`
`CVP
`CVP
`CVP
`CVP
`None
`FND
`None
`FND
`131I-tositumomab
`
`F indicates female; M, male; FL, follicular lymphoma; MCL, mantle cell lymphoma; CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisone; R, rituximab; CVP,
`cyclophosphamide, vincristine, and prednisone; ProMACE/MOPP, procarbazine, methotrexate with leucovorin, doxorubicin, cyclophosphamide, etoposide, mechlorethamine,
`vincristine, and prednisone; ASCT, high-dose therapy followed by autologous stem cell transplantation; HyperCVAD, cyclophosphamide, vincristine, doxorubicin, and
`dexamethasone alternating with cycles of high-dose cytarabine and methotrexate; GCD, gemcitabine, carboplatin, and dexamethasone; FND, fludarabine, mitoxantrone, and
`dexamethasone; IFN, interferon-␣; CY, cyclophosphamide; VP16, etoposide; and DEX, dexamethasone.
`
`Modified T cells were generated for the first 5 patients by
`limiting dilution and selected for CD20 cytotoxicity by chro-
`mium release assay and cTCR expression by flow cytometry. At
`the plating density required to reproducibly generate modified
`T cells,
`the resulting T-cell populations consisted of cells
`derived from 1 to 3 clones of T cells as assessed by V␤ TCR
`spectratyping and TCR␥ clonality testing by PCR (Figure 3A;
`Table S1). This expansion and selection process proved to be
`laborious and inefficient, requiring approximately 4 months to
`achieve the target cell dose. Moreover, T cells generated by
`limiting dilution could not be expanded adequately for infusions
`in 2 of these initial 5 patients, and in 2 of the other 3 patients the
`target cell doses could not be reached (Table 2).
`We subsequently elected to modify the protocol to include
`expansion of T-cell transfectants in bulk culture to circumvent the
`difficulties of expanding T cells after limiting dilution. Successful
`expansion of modified T cells was achieved for the subsequent
`4 patients using this approach, and the time required to reach the
`target cell dose was reduced by approximately 50% (Figure 4). V␤
`TCR spectratyping and TCR␥ clonality testing by PCR showed
`more heterogeneous T-cell populations in these bulk cultures,
`although several of the cultures contained prominent T-cell clones
`
`(Figure 3B; Table S1). Three of these 4 patients received all
`planned doses of T cells. The target cell number was reached for
`the fourth patient as well, but the third infusion consisted of only
`2 ⫻ 109 cells/m2 because of a loss of cells during a quality
`control assay.
`
`Immunophenotype of modified T cells
`
`The phenotype of ex vivo–expanded cTCR-bearing T cells has not
`been well described. We analyzed the immunophenotype of the
`infused T cells using multicolor flow cytometry and found it to be
`similar to that of activated effector T cells,28,29 expressing CD3,
`CD8, and CD45RO and lacking CD62L, CCR7, and CD127
`(Figure 5). As expected, patients treated with CD8⫹ T cells derived
`by limiting dilution received negligible numbers of CD4-
`expressing cells (0.67%-4.5%), whereas patients receiving infu-
`sions of T cells grown in bulk culture received 3.4% to 38.6%
`CD4⫹ cells. Infused T cells also expressed the activation marker
`CD95, but relatively few cells (1.3%-6.2%) expressed CD134
`(OX40; Table 3). We found negligible numbers of cells expressing
`central memory (CD62L⫹/CCR7⫹/CD45RA⫺/CD127⫹) and effec-
`tor memory (CD62L⫺/CCR7⫺/CD45RA⫺/CD127⫹) phenotypes.
`
`B
`
`Patient B
`
`Negative Positive
`
`Chimeric Zeta Chain
`
`Ribosome
`Binding
`Sequence
`
`Leu 16
`scFv
`
`Transmembrane
`domain
`
`SV40
`promoter
`
`NeoR
`
`CD3ζ
`
`CH2-CH3
`Hinge
`
`CD4
`TM
`
`VH
`
`Linker
`
`VL
`
`Murine κ
`Signal
`Peptide
`
`Human
`IgG1 Fc
`
`Endogenous Zeta Chain
`
`A
`
`I/Epromoter
`
`CMV
`
`Figure 2. Expression of the CD20-specific cTCR. (A) Schematic diagram of the CD20-specific scFvFc:␨ chimeric T-cell receptor cDNA plasmid. (B) A representative Western
`blot analysis of cTCR expression performed using whole-cell lysates of preinfusion T cells from patient B, probed with mouse anti–human CD3␨ monoclonal Ab. Negative
`control was parental PBMCs, and positive control was transfected Jurkat cell line. A 21-kDa band corresponding to the endogenous CD3␨ chain and a 66-kDa band
`representing the expected cTCR protein were detected. The intermediate bands indicate degradation products or truncated forms of the cTCR.
`
`UPenn Ex. 2011
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

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`
`BLOOD, 15 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 6
`
`ANTI-CD20 T-CELL THERAPY FOR NHL
`
`2265
`
`VB2
`
`V B3
`
`V B5.1
`
`V B4
`
`V B7.1
`VB5.2V B5.3
`V B7.2
`
`V B8
`
`VB9
`
`V B11
`
`V B12
`VB13.6
`V B13.1
`VB13.2
`
`V B14
`
`V B16
`
`V B17
`
`VB18
`
`V B20
`V B21.3
`
`V B22
`
`VB23
`
`TCR VBETA FAMILY
`
`B
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`V B1
`
`% VBETA USAGE
`
`V B2
`
`V B3
`
`V B5.1
`
`V B4
`
`V B7.2
`V B7.1
`V B5.2V B5.3
`
`V B8
`
`V B9
`
`V B11
`
`V B12
`V B13.6
`V B13.2
`V B13.1
`
`V B14
`
`V B16
`
`V B17
`
`V B18
`
`V B20
`V B21.3
`
`V B22
`
`V B23
`
`TCR VBETA FAMILY
`
`A
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`V B1
`
`% VBETA USAGE
`
`Figure 3. Clonality of T cells produced by limiting dilution and in bulk culture. T-cell clonality was determined by flow cytometric T-cell receptor (TCR) V␤ spectratyping
`(top) and by PCR amplification of clonal V-J rearrangements at the TCR␥ locus (bottom). Representative results for T cells produced by limiting dilution (A) and in bulk culture
`(B) are shown. (A) T cells produced by limiting dilution (patient B), showing clonal expression of V␤17 in 98% of CD8⫹ T cells by V␤ spectratyping (top; f) and showing
`2 predominant TCR␥ rearrangements (bottom). Because each T-cell clone can rearrange one or both of its TCR␥ alleles, the 2 PCR products could represent either 1 T-cell
`clone with biallelic TCR␥ rearrangements or 2 singly rearranged clones, although the single predominant V␤17 clone identified by spectratyping would favor a single doubly
`rearranged clone. (B) T cells produced in bulk culture (patient G) showing oligoclonal V␤ expression in CD8⫹ T cells (16% V␤16; 9% V␤7.1; 3% each V␤3, V␤13.2 and V␤17;
`2% each V␤1 and V␤13.1; and 1% each V␤5.1, V␤13.6, V␤21.3, and V␤23) and 7 distinct TCR␥ rearrangements by PCR (bottom; f) that could correspond to between 4 and
`7 different T-cell clones, depending on the number of singly and doubly rearranged clones (see Table S1). The 䡺 in both top panels represent the average expression levels for
`each V␤ chain in normal polyclonal T-cell populations.
`
`T cells resulting from bulk culture and from limiting dilution were
`phenotypically similar, although the former generally contained higher
`proportions of CD4⫹ cells (3.4%-38.6% compared with 0.67%-4.5%)
`and cells with a regulatory T (Treg)–like phenotype (CD4⫹/CD25⫹/
`FoxP3⫹) (0.54%-1.9% compared with 0.61%-21.6%). Treg functional-
`ity studies were not performed, however. All T cells exhibited low
`expression of costimulatory markers CD28 (0.92%-5.4%) and CD137
`(0.47%-4.4%). High proportions of cells from all patients expressed
`adhesion molecules such as CD11a (98.7%-100%), CD44 (99.8%-
`100%), and CD49d (85.8%-99.6%).
`
`Cytotoxicity of modified T cells in vitro
`
`Our group showed in preclinical studies that T-cell clones
`bearing CD20-specific cTCRs exhibit antigen-specific cytotox-
`icity.20 We assessed the cytotoxicity of T cells used in this trial
`by coincubation with 51Cr-labeled CD20⫹ target cells (Daudi
`lymphoma cells and EL4 mouse lymphoma cells transfected to
`express human CD20), and the expanded T cells used for all
`7 patients killed CD20⫹ lymphoma cells in an antigen-specific
`manner (Figure 6).
`
`Table 2. T-cell infusions
`Infusion 1,
`cells/m2*
`
`Patient
`
`A
`B
`C
`D
`E
`F
`G
`H
`I
`
`108
`108
`
`108
`
`108
`108
`108
`108
`
`Infusion 2,
`cells/m2†
`
`109
`109
`
`4 ⫻ 108
`
`109
`109
`109
`109
`
`Infusion 3,
`cells/m2‡
`
`3.3 ⫻ 109
`2 ⫻ 109
`
`3.3 ⫻ 109
`3.3 ⫻ 109
`3.3 ⫻ 109
`2 ⫻ 109㛳
`
`Fresh versus
`thawed cells
`
`Time from apheresis
`to target cell number, d
`
`No. of stimulation
`cycles§
`
`Fresh
`Fresh
`
`Thawed
`
`Thawed
`Thawed
`Thawed
`Thawed
`
`130
`129⫹
`Expansion failed
`159⫹
`Expansion failed
`96
`90
`81
`104
`
`7
`7
`5
`7
`5
`6
`5
`5
`8
`
`For patients A through E, T cells were selected and expanded by limiting dilution. For patients F through I, T cells were expanded in bulk culture. Patients C and E did not
`receive T-cell infusions. Patients B and D received infusions but did not reach the target cell dose.
`*Target dose was 108 cells/m2.
`†Target dose was 109 cells/m2.
`‡Target dose was 3.3 ⫻ 109 cells/m2.
`§Defined as the number of times cells were stimulated with OKT3 in the presence of irradiated feeder PBMCs and LCL (repeated every 14 days, first cycle at day 14).
`㛳The target number of cells was generated for this patient, but nearly half of the last cell dose was lost during a quality control assay before infusion.
`
`UPenn Ex. 2011
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

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`
`2266
`
`TILL et al
`
`BLOOD, 15 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 6
`
`In vivo persistence of modified T cells
`
`We measured the in vivo persistence of modified T cells using
`quantitative real-time PCR of DNA from patient PBMCs collected
`at serial time points after T-cell infusion. Modified T cells were
`detectable by PCR for 5 to 21 days in the first 3 patients receiving
`T cells without adjuvant IL-2. In contrast, modified T cells were
`detectable for 5 to 9 weeks in the last 4 patients, who received
`T cells produced in bulk culture and 14 days of subcutaneous IL-2
`(Figure 7).
`Modified T cells were also detectable by PCR in the bone
`marrow 24 hours after the final T-cell infusion in patients A, D, F,
`and G (lymphoma cells were detectable in the marrow by flow
`cytometry only in patients G and H at this time point). Of the
`7 treated patients, only patients B and G had accessible lymph
`nodes for biopsy after T-cell infusions. The lymph node from
`patient B showed only fibroadipose tissue, and patient G’s lymph
`node showed tumor, but no modified T cells were detectable.
`
`Modified T cells were not immunogenic
`
`The introduction of foreign transgenes in therapeutic vectors has
`resulted in immune responses against modified T cells in previous
`gene therapy clinical trials.27,30 Given the significant immunosup-
`pression present in patients with lymphoma, we hypothesized that
`the transgenic cells in this trial might elicit lower immune response
`rates, and we used 3 assays to test this hypothesis. To detect cellular
`immune responses, 51Cr-release assays were performed with seri-
`ally collected patient PBMCs that had been coincubated with
`irradiated T cells expressing the scFvFc:␨ and neoR gene products
`(Figure S1A), using transfected and nontransfected T cells as target
`cells. In 2 patients the antigen responsiveness of recovered T cells
`was confirmed using allogeneic LCL as target cells; in both cases a
`cytotoxic response was elicited at all time points tested (Figure
`
`Figure 4. Growth curves of gen