© The American Society of Gene & Cell Therapy
`
`See page 666
`
`original article
`
`Case Report of a Serious Adverse Event Following
`the Administration of T Cells Transduced With
`a Chimeric Antigen Receptor Recognizing Erbb2
`Richard A Morgan1, James C Yang1, Mio Kitano1, Mark E Dudley1, Carolyn M Laurencot1
`and Steven A Rosenberg1
`
`1Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
`
`In an attempt to treat cancer patients with ERBB2
` overexpressing tumors, we developed a chimeric anti-
`gen receptor (CAR) based on the widely used humanized
`monoclonal antibody (mAb) Trastuzumab (Herceptin).
`An optimized CAR vector containing CD28, 4-1BB, and
`CD3ζ signaling moieties was assembled in a γ-retroviral
`vector and used to transduce autologous peripheral
`blood lymphocytes (PBLs) from a patient with colon
`cancer metastatic to the lungs and liver, refractory to
` multiple standard treatments. The gene transfer effi-
`ciency into autologous T cells was 79% CAR+ in CD3+
`cells and these cells demonstrated high-specific reactiv-
`ity in in vitro coculture assays. Following completion of
`nonmyeloablative conditioning, the patient received
`1010 cells intravenously. Within 15 minutes after cell infu-
`sion the patient experienced respiratory distress, and
`displayed a dramatic pulmonary infiltrate on chest X-ray.
`She was intubated and despite intensive medical inter-
`vention the patient died 5 days after treatment. Serum
`samples after cell infusion showed marked increases in
`interferon-γ (IFN-γ), granulocyte macrophage-colony
`stimulating factor (GM-CSF), tumor necrosis factor-α
`(TNF-α), interleukin-6 (IL-6), and IL-10, consistent with
`a cytokine storm. We speculate that the large number
`of administered cells localized to the lung immedi-
`ately following infusion and were triggered to release
`cytokine by the recognition of low levels of ERBB2 on
`lung epithelial cells.
`Received 14 January 2010; accepted 22 January 2010; published online
`23 February 2010. doi:10.1038/mt.2010.24
`
`IntroductIon
`ERBB2 (HER-2/neu) is a member of the epidermal growth factor
`receptor family. Epidermal growth factor receptor–ligand interac-
`tion induces the heterodimerization of receptors, which in turn
`results in the activation of intracellular tyrosine kinase domain
`signaling cascades that mediate cell growth, differentiation, and
`survival.1–3 Overexpression of ERBB2 can induce dimerization of
`ERBB2 and initiates signal transduction activities without ligand
`
`binding. ERBB2 overexpression/amplification occurs in ~15–25%
`of human breast cancer patients, and is associated with more
`aggressive disease.4 A proportion of other human cancers are also
`associated with ERBB2 gene amplification and protein overexpres-
`sion; including cancers of the colon, ovary, stomach, kidney, mela-
`noma, and others.5–7 Investigation of agents that target the ERBB2
`protein led to the development of Trastuzumab (Herceptin), a
`humanized monoclonal antibody (mAb) that binds to the extra-
`cellular domain of the receptor.8 Trastuzumab has been shown
`to be of clinical benefit for metastatic breast cancer patients with
`ERBB2 overexpression/amplification, either alone or in combina-
`tion with chemotherapy regimens.9,10 ERBB2 has also been the
`target of several cancer vaccine trials,11–13 as well as, adoptive cell
`therapy using anti-ERBB2 cytotoxic T lymphocyte lines.14
`Adoptive cell therapy has emerged as the most effective treat-
`ment for patients with metastatic melanoma. Adoptive cell therapy
`using tumor-reactive autologous tumor infiltrating lymphocytes
`(TIL) in combination with nonmyeloablative but lymphodeplet-
`ing conditioning resulted in 50% objective clinical regression in
`melanoma patients.15 Intensifying the lymphodepletion by adding
`total-body irradiation to the chemotherapy conditioning regi-
`men improved the objective response rate to 72%.16 This potent
`therapy, however, has been limited by the requisite surgery to pro-
`cure tumor-reactive TIL, by ex vivo identification and expansion
`of these cells, and by the failure to reproducibly isolate similar cells
`from common epithelial tumors.
`The transfer of genes into primary human lymphocytes per-
`mits the introduction of tumor antigen receptor molecules that
`can endow the engineered cell with antitumor specificity.17–19 We
`reported the first clinical trials using autologous peripheral blood
`lymphocytes (PBLs) modified to express a tumor antigen- reactive
`T-cell receptor in the treatment of patients with metastatic mela-
`noma that resulted in objective tumor regressions.20,21 These strat-
`egies, however, have a lower response rate than TIL, and only
`a minority of patients are eligible for current protocols, as they
`must express human leukocyte antigen-A*0201 in order to be
` recognized by the T-cell receptor-engineered cells.
`An alternative to T-cell receptor gene therapy is the use of
`a chimeric antigen receptor (CAR) that is capable of relaying
`excitatory signals to T cells in a non-Major histocompatibility
`
`Correspondence: Richard A Morgan, Surgery Branch, National Cancer Institute, National Institutes of Health, 10 Center Drive, CRC-3W5940, Bethesda,
`Maryland 20892, USA. E-mail: rmorgan@mail.nih.gov
`
`Molecular Therapy vol. 18 no. 4, 843–851 apr. 2010
`
`843
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`Serious Adverse Event With ERBB2-based CAR
`
`© The American Society of Gene & Cell Therapy
`
`complex-restricted manner. These hybrid proteins, composed of
`an extracellular antigen recognition domain fused to an intra-
`cellular T-cell activation domain,22,23 may therefore be used in
`patients regardless of their human leukocyte antigen genotype.
`The absence of human leukocyte antigen-restricted antigen rec-
`ognition is achieved by harnessing the antigen-binding properties
`of mAb; this recognition is also independent of antigen process-
`ing, thus bypassing a potential mechanism by which tumor cells
`can evade the immune system in vivo. Several clinical trials using
`CAR-transduced T cells have been reported.24–27 ERBB2-based
`CARs reported thus far are composed of single-chain Fv fragment
`from murine mAb, which have been shown to induce anti-CAR
`immune responses in humans.25,26 The anti-ERBB2 CAR used in
`this case report was a next generation CAR containing both the
`humanized Herceptin single-chain Fv fragment and optimized
`costimulatory signaling domains designed for increased cytokine
`secretion, lytic activity, and shown to display robust in vivo anti-
`tumor activity in a human breast cancer xenograft model.28
`
`results
`In vitro characteristics of the erbB2-cAr
`transduced t cells for patient treatment
`Leukophoresis was performed to obtain patient peripheral blood
`mononuclear cells (PBMCs), which were stimulated with an anti-
`CD3 mAb and interleukin-2 (IL-2) to initiate T-cell expansion
`followed by transduction with the 4D5-CD8-28BBZ ERBB2-
`
`CAR vector as described in Materials and Methods section. At
`4 days before infusion, cells were analyzed for the expression of
`the ERBB2 CAR using an ERBB2-Fc fusion protein as previously
`described.28 As shown in Figure 1, 79% of CD3+ T cells expressed
`the CAR with gene transfer into both CD4+ (17%) and CD8+
`(63%) T-cell subsets. To determine functional activity, transduced
`T cells were cocultured with ERBB2+ melanoma, breast cancer,
`and ovarian cancer cell lines, or ERBB2− breast cancer and T
`lymphoblastoid cell lines. ERBB2-specific reactivity was demon-
`strated by production of effector cytokine interferon-γ (IFN-γ)
`only in cell lines expressing ERBB2 (Table 1). Background
`cytokine production of ERBB2-CAR transduced cells, when
`cocultured with ERBB2-targets, was similar to untransduced
`control cells. To complete the certificate of analysis for patient
`treatment, transduced cells were also tested to be negative for the
`presence of replication competent retrovirus by PCR and passed
`sterility testing (data not shown). Retrospective testing using an
`amplification-based S+/L− assay was also negative for replication
`competent retrovirus.
`
`clinical course
`The patient was a 39-year-old female who 3 years earlier had
`undergone a sigmoid resection for colon cancer that on patho-
`logic ana lysis exhibited
`lymphatic
`invasion and vascular
`involvement, with spread to 6 of 21 lymph nodes and the pres-
`ence of synchronous liver metastases. She was treated with a
`
`5′ LTR
`
`sd
`
`ψ
`
`sa
`
`4D5 scFv
`
`CD8 Hinge &TM
`
`CD28
`
`4-1BB
`
`CD3ζ
`
`3’ LTR
`
`0.2
`
`0.5
`
`0.4
`
`0.3
`
`UnTd
`
`21
`
`78
`
`102
`101
`100
`103
`APC-Cy7-A:: CD8 APC-Cy7-A
`
`103
`
`102
`
`101
`
`100
`
`PE-A:ErbB2 + GAMPE PE-A
`
`0.6
`
`0.1
`
`75
`
`24
`
`100
`
`102
`101
`103
`PE-Cy7-A:: CD4 PE-Cy7-A
`
`CD4
`
`CD8
`
`16
`
`6
`
`CAR
`
`63
`
`14
`
`102
`101
`100
`103
`APC-Cy7-A:: CD8 APC-Cy7-A
`
`CD8
`
`103
`
`102
`
`101
`
`100
`
`PE-A:ErbB2 + GAMPE PE-A
`
`62
`
`15
`
`17
`
`5.0
`
`100
`
`102
`101
`103
`PE-Cy7-A:: CD4 PE-Cy7-A
`
`CD4
`
`103
`
`102
`
`101
`
`100
`
`PE-A:ErbB2 + GAMPE PE-A
`
`103
`
`102
`
`101
`
`100
`
`PE-A:ErbB2 + GAMPE PE-A
`
`98
`
`79
`
`20
`
`103
`
`102
`
`101
`
`100
`
`PE-A:ErbB2 + GAMPE PE-A
`
`0.5
`
`100
`
`101
`103
`102
`FITC-A:: CD3 FITC-A
`
`CD3
`
`102
`101
`103
`FITC-A:: CD3 FITC-A
`
`0.4
`
`0.3
`
`100
`
`103
`
`102
`
`101
`
`100
`
`PE-A:ErbB2 + GAMPE PE-A
`
`CD3
`
`a
`
`b
`
`ErbB2
`CAR
`
`ErbB2
`CAR
`
`Figure 1 expression of the erBB2 cAr. Diagram of the ERBB2-CAR vector (MSGV1-4D5-CD8-28BBZ) used in this trial is as shown on the top of
`the figure. As described in Materials and Methods section, patient PBMC were stimulated to induced T-cell division and then transduced with the
`CAR vector. Four days before cell infusion samples were removed for analysis of ERBB2-CAR gene expression by FACS (along with the CD3, CD4, or
`CD8 T-cell markers). CAR, chimeric antigen receptor; LTR, long terminal repeats; PBMC, peripheral blood mononuclear cell; scFv, single-chain Fv
`fragment.
`
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`
`Serious Adverse Event With ERBB2-based CAR
`
`table 1 erbB2-cAr certificate of analysis
`
`erbB2+ target
`MdA361
`Mel526
`Mel624
`Mel938
`Mel888
`Media
`effector
`57
`44
`66
`68
`49
`24
`None
`37
`3,700
`4,900
`35
`37
`26
`MART-1 TCR
`>26,815
`11,430
`>21,065
`13,210
`12,745
`46
`Patient CAR
`82
`93
`127
`81
`69
`46
`Mock Td PBL
`Abbreviations: CAR, chimeric antigen receptor; IFN-γ, interferon-γ; PBL, peripheral blood lymphocyte.
`Cell reactivity following coculture of 1e5 effectors with 1e5 targets. Data are IFN-γ (pg/ml) following overnight incubation. Mock Td PBL, untransduced PBL maintained
`under identical culture conditions; MART-1 TCR, MART-1 TCR-transduced PBL; patient CAR, ErbB2 CAR-transduced patient PBL. ErbB2 antigen expression was as
`indicated. Only melanoma lines 526 and 624 expressed both HLA-A2 and MART-1, required for TCR recognition.
`
`erbB2− target
`ceM
`MdA468
`37
`99
`29
`55
`114
`134
`117
`103
`
`sK-Br3
`143
`68
`>33,385
`144
`
`sK-oV3
`82
`47
`>58,480
`146
`
`chemotherapy regimen consisting of 5-fluorourocil, leucovorin
`and oxaliplatin plus the antivascular endothelial growth factor
`mAb, bevacizumab. The tumor progressed and the patient was
`then treated with an alternate chemotherapy regimen in which
` irinotecan was substituted for oxaliplatin (FOLFIRI). The patient
`again progressed and after desensitization to oxaliplatin for an
`allergic reaction, she received a third chemotherapy regimen con-
`sisting of capecitabine, oxaliplatin, and bevacizumab. The tumors
`in the lung and liver continued to progress and the patient was
`referred to the Surgery Branch, National Cancer Institute (NCI;
`Bethesda, MD) and signed an informed consent for our protocol.
`The protocol was reviewed and approved by the National Institutes
`of Health Institutional Biosafety Committee, the NCI Institutional
`Review Board, the National Institutes of Health Office of
`Biotechnology Activities, and the Food and Drug Administration
`(all Bethesda, MD). Patient inclusion criteria included metastatic
`cancer that expressed ERBB2 (Her-2/neu) at ≥2+ as assessed by
`immunohistochemistry.
`To facilitate homeostatic expansion of the transduced cells, the
`patient received a lymphodepleting regimen (60 mg/kg cyclophos-
`phamide daily for 2 days followed by flurodarabine 25 mg/m2 for
`the next 5 days). On the day following the last chemotherapy dose
`the patient received an intravenous infusion of 1010 cells trans-
`duced with the ERBB2 CAR in 125 ml over 30 minutes. This was
`the largest number of cells permitted in the first dose- escalation
`cohort. Within 15 minutes after completing the infusion, the
`patient developed respiratory distress with decreased blood oxy-
`gen saturation that worsened over the next hour. Chest X-ray
`obtained 40 minutes after completion of the infusion showed pul-
`monary edema, which appeared worse on chest X-rays repeated
`at 2 and 4 hours after the infusion. Because of decreasing respi-
`ratory function the patient was transferred to the intensive care
`unit and was intubated about 1 hour after the cell infusion. The
`patient then developed severe hypotension requiring vasopres-
`sors. Dexamethasone, 8 mg every 6 hours, was administered start-
`ing at about 5 hours after the cell infusion (it was continued for 2
`days, after which the dose was tapered). The patient experienced
`two cardiac arrests in the next 12 hours after cell infusion both
`requiring cardiopulmonary resuscitation. She was maximally sup-
`ported with vasopressors and ventilatory support. She remained
`severely ill with maximum intensive care unit support for the next
`5 days at which time progressive hypotension and bradycardia as
`well as gastrointestinal bleeding resulted in cardiac arrest from
`which she could not be resuscitated.
`
`Postmortem analysis
`At autopsy, multiple organs exhibited signs of systemic ischemia
`and hemorrhagic microangiopathic injury. The lungs also showed
`diffuse alveolar damage consistent with the clinical findings of
`acute respiratory distress syndrome. Autopsy also revealed a
` generalized rhabdomyolysis. Copious blood in the small intestine
`indicated that the patient succumbed to hemorrhage in the set-
`ting of multiple organ failure secondary to systematic microangio-
`pathic injury. The autopsy findings appeared to be a combination
`of the initial pulmonary injury followed by the sequela of several
`days of hypotension and organ ischemia.
`Beginning at about 4 hours after cell infusion serum samples
`were obtained and stored for analysis. Compared to pretreat-
`ment samples, the patient’s serum displayed a rapid and marked
`increase in the levels of five cytokines; IFN-γ, granulocyte mac-
`rophage-colony stimulating factor (GM-CSF), tumor necrosis
`factor-α (TNF-α), IL-6, and IL-10 (Figure 2). At 4 hours after
`infusion, four of the five cytokines displayed peak serum levels:
`IFN-γ,11, 456 pg/ml; TNF-α, 380 pg/ml; GM-CSF, 10,191 pg/ml;
`and IL-6, 34,467 pg/ml. Preinfusion levels for IFN-γ, GM-CSF,
`and IL-6 were undetectable, whereas TNF-α values varied from
`0 to 51 pg/ ml. The levels of these cytokines decreased over the
`next 3 days but remained above baseline values. The levels of
`seven additional cytokines (IL-1β, IL-2, IL-4, IL-7, IL-10, IL-12,
`and TRAIL) were determined by cytokine array (SearchLight
`assay) with only IL-10 showing increased levels after infusion,
`which unlike the other cytokines, was sustained through the study
`period (8 pg/ml preinfusion, 219 pg/ml at about 100 hours after
`infusion, Figure 2). An increased amount of IL-2 was observed
`at 4 hours only and was likely associated with the administra-
`tion of the cell product, which was given in saline with 300 IU/ ml
`IL-2 (the patient received no other IL-2). The possible involve-
`ment of an anaphylactic response to the infused cells was deemed
`unlikely because we measured only a modest twofold increase in
`serum tryptase levels at the 4 hours time point (from 7–9 pg/ml
` preinfusion to 15 pg/ml at 4 hours).
`To determine the relative tissue distribution of vector-containing
`cells, DNA was isolated from samples obtained at autopsy and
`subjected to quantitative-PCR using vector-specific primers and
`probe. As a reference for this analysis, DNA was extracted from
`the infusion sample (79% ERBB2-CAR+) and arbitrarily assigned
`a value of 100 for comparison to tissue samples. There was a wide
`variation in the presence of vector-containing cells found in mul-
`tiple tissues, though the highest levels were seen in the lung and
`
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`Serious Adverse Event With ERBB2-based CAR
`
`© The American Society of Gene & Cell Therapy
`
`Pre
`
`~4 hours
`
`~18 hours
`
`
`
`~26 hours~48 hours
`
`~72 hours
`
`400
`
`350
`
`300
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`
`TNF−α (pg/ml)
`
`35,000
`
`30,000
`
`25,000
`
`20,000
`
`15,000
`
`10,000
`
`5,000
`
`IL-6 (pg/ml)
`
`12,000
`
`10,000
`
`8,000
`
`6,000
`
`4,000
`
`2,000
`
`IFN-γ (pg/ml)
`
`0
`
`Pre
`
`~4 hours
`
`~18 hours
`
`
`
`~26 hours~48 hours
`
`~72 hours
`
`12,000
`
`10,000
`
`8,000
`
`6,000
`
`4,000
`
`2,000
`
`GM-CSF (pg/ml)
`
`0
`
`Pre
`
`~4 hours
`
`~18 hours
`
`
`
`~26 hours~48 hours
`
`~72 hours
`
`0
`
`Pre
`
`~4 hours
`
`~18 hours
`
`
`
`~26 hours~48 hours
`
`~72 hours
`
`Pre
`
`~4 hours
`
`~18 hours
`
`~26 hours
`
`~100 hours
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`
`IL-10 (pg/ml)
`
`Figure 2 serum cytokine levels. Serum samples obtained at the approximate times indicated after cell infusion were assayed for cytokine expression
`using commercial ELISA kits for cytokines IL-6, TNF-α, GM-CSF, and IFN-γ. The levels of cytokine IL-10 were independently determined by cytokine
`array (SearchLight) assay. All samples were diluted as necessary as to be in the linear range of the assay. ELISA, enzyme-linked immunosorbent assay;
`GM-CSF, granulocyte macrophage-colony stimulating factor; IFN-γ, interferon-γ; IL, interleukin; TNF-α, tumor necrosis factor-α.
`
`abdominal/mediastinal lymph nodes (Table 2). There did not
`appear to be a preferential accumulation of vector-containing
`cells in metastatic deposits in the liver or lungs. DNA from the
`pretreatment PBMC was also subjected to analysis of single-
` nucleotide polymorphisms associated with the activity/function
`of five cytokine genes (IL-6, IL-10, IFN-γ, TGF-β1, and TNF-α).
`PCR using sequence-specific primers (Figure 3) indicated that
`the patients’ genotype was: IL-6, heterozygous −741G/C; IL-10,
`homozygous −1082G, −819C, −592C; IFN-γ, homozygous 874A;
`transforming growth factor-β1, homozygous 10T, 25G; and TNF-α
`homozygous −308G. This genotype is consistent with a phenotype
`of increased synthesis of transforming growth factor-β, IL-10, and
`IL-6, but lower production of TNF-α and IFN-γ.
`Analysis of the ERBB2-CAR transduced T cells before infu-
`sion demonstrated specific recognition of ERBB2-expressing
`tumor cells (Table 1). To confirm and extend this analysis, an
`aliquot of the cryopreserved infusion sample was thawed and
`
`retested. In addition to cell lines used for the certificate of analy-
`sis, the patient’s PBMC were used to derive autologous dendritic
`cell and macrophage cultures and several cultures of allogeneic
`primary cells adapted for growth in culture by a commercial sup-
`plier were obtained. Coculture results presented in Figure 4 con-
`firm the data obtained in the certificate of analysis. There was no
`reactivity seen to the dendritic cell and macrophage autologous
`cell cultures. Cytokine release was observed in several cocultures
`using allogeneic primary cells adapted for growth in culture.
`
`dIscussIon
`The ERBB2 gene has been extensively studied as a target for
`both chemotherapy and immunotherapy. In breast cancer,
`overexpression of ERBB2 is correlated with a poor clinical out-
`come and at the same time, is a positive predictive factor for
`those women who are most likely to respond to therapy with
`the anti-ERBB2 antibody, trastuzumab.10 In randomized clinical
`
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`Serious Adverse Event With ERBB2-based CAR
`
`table 2 Vector tissue distribution
`
`signal
`normal tissue
`0.00
`Brain (left frontal lobe)
`0.00
`Pectoralis muscle (left)
`0.01
`Aorta
`0.04
`Atrium (right)
`0.05
`Neck LN (left)
`0.08
`Ventricle (left)
`0.09
`Small bowel
`0.11
`Liver (left lobe)
`0.13
`Liver (right lobe)
`0.14
`Abdominal para-aortic LN
`0.15
`Liver
`0.15
`Auxiliary LN
`0.16
`Kidney (left)
`0.17
`Adrenal gland (left)
`0.25
`Kidney (right)
`0.30
`Spleen
`0.44
`Lung left—A
`0.49
`Adrenal gland (right)
`0.56
`Lung left—B
`0.70
`Mediastinal
`0.78
`Lung right—A
`1.59
`Lung right—B
`1.99
`Para-aortic/mediastinal LN
`2.24
`Abdominal para-aortic LN
`2.58
`Abdominal LN
`2.64
`Mediastinal LN
`signal
`Metastasis
`0.03
`Liver metastasis
`0.08
`Lung metastasis (left)
`0.09
`Liver metastasis
`0.11
`Liver metastasis (left lateral)
`0.29
`Lung metastasis (left)
`0.34
`Liver metastasis (left anterior)
`Abbreviations: CAR, chimeric antigen receptor; FACS, fluorescence-activated cell
`sorting; LN, lymph node; PBL, peripheral blood lymphocyte; Q-PCR, quantitative-
`PCR.
`DNA extracted from tissues and metastasizes at autopsy were subjected to Q-PCR
`using ErbB2 CAR-vector specific primers/probe. Values were normalized to the
`infusion PBL (arbitrarily assigned a value of 100). Gene transfer efficiency of the
`infusion PBL was 79% as measured by FACS.
`
`trials, trastuzumab in combination with chemotherapy lead
`to increased disease-free and overall survival in breast cancer
`patients.9,29,30 The mechanism of action of trastuzumab appears
`to be multifactorial, but there are several studies that indicated
`the involvement of cell- mediated immunity (natural killer
`cell–based antibody-dependent cellular cytotoxicity) in patient
`responses.31–33 Furthermore, patient immune responses (anti-
`body-dependent cellular cytotoxicity) can be increased when
`trastuzumab is combined with concomitant cytokine (IL-2,
`
`IL-12) administration,34,35 or in vaccine trials where anti-ERBB2
`T cells responses have been reported.11,13
`Safety considerations that preceded our clinical trial included
`the use of trastuzumab in thousands of cancer patients, the lack
`of toxicity seen in multiple studies immunizing against epitopes
`of ERBB2, and the lack of toxicity seen in a report of the adop-
`tive transfer of autologous anti-ERBB2 cytotoxic T lymphocyte
`clones in the setting of breast cancer.14 In this report, three dif-
`ferent cytotoxic T lymphocyte clones were administered in five
`transfers given 2 weeks apart. A total of 2.65 × 109 total cells
`were administered along with low-dose IL-2. With the excep-
`tion of low-grade fever and chills following the third and fourth
`infusions, no side effects were noted. The therapy was associated
`with a decrease in tumor cells within the patient’s bone mar-
`row, but larger metastatic sites (such as liver) were not impacted.
`Radioimaging was performed using 111In-labeled cytotoxic T
`lymphocyte that demon strated an immediate accumulation of
`cells in the lung that decreased over 72 hours, whereas uptake
`to the liver and spleen increased over the first 24 hours then
`remained stable for the 72-hour study period. We have demon-
`strated similar uptake of 111In-labeled TIL36 and have observed
`that lung uptake of TIL happens at the first pass (at the 90%
`retention level) after which the TIL then leak out of the lungs
`to the liver and other organs (ref. 36 and J.C. Yang, unpublished
`results).
`The γ-retroviral vector construct used in this cancer gene
`therapy trial was designed for optimal ERBB2-CAR gene expres-
`sion and anti-ERBB2 reactivity. It was demonstrated to be highly
`specific, was able to recognize a wide range of tumor histologies,
`and was able to significantly prevent the growth of human breast
`cancer cells orthotopically implanted into the mammary fat pad of
`severe combined immunodeficiency mice.28 Part of the process of
`optimizing this anti-ERBB2-CAR vector was the inclusion of two
`T-cell costimulatory domains from CD28 and 4-1BB (CD137)
`that were linked to the CD3z signaling element. 4-1BB is essential
`for the optimal activity of CD8+ T cells37,38 and inclusion of 4-1BB
`signaling domains was shown to enhance the in vivo antitumor
`activity of CARs in tumor xenograft models.28,39–41 To date, the
`reported clinical application of CAR-engineered T cells has been
`limited to constructs containing CD3z alone.24–27,42 In one report
`targeting carbonic anhydrase IX, on-target toxicity was observed
`most likely due to recognition of antigen expression on biliary epi-
`thelium. The results with the carbonic anhydrase IX directed CAR
`suggest that on-target toxicity may be antigen dependant and does
`not require the presence of costimulatory signals (such as CD28
`and 4-1BB) in the CAR construct.
`In our initial report we observed, in some transductions, T-cell
`recognition of the ERBB2−-tumor line MDA468 (this line is nega-
`tive for ERBB2 expression by fluorescence-activated cell sorting,
`but ERBB2 mRNA can be detected by PCR). Transduction of the
`current patient’s T cells with the identical ERBB2-CAR vector
`did not result in recognition of the MDA468 cell line (Table 1,
`Figure 4). The significance of the recognition of allogenic primary
`cells adapted for growth in culture (Figure 4) is not clear, as it is
`not known how the adaptation of these cells for growth in ex vivo
`culture influences the expression of ERBB2. For example, it was
`reported that while normal rat liver hepatocytes do not express
`
`Molecular Therapy vol. 18 no. 4 apr. 2010
`
`847
`
`UPenn Ex. 2033
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`Serious Adverse Event With ERBB2-based CAR
`
`© The American Society of Gene & Cell Therapy
`
`IL-10
`−1082A
`−819T
`
`TGFβ1
`25G
`
`TGFβ1
`25C
`
`TGFβ1
`10C
`
`TGFβ1
`10T
`
`TNFα
`−308G
`
`TNFα
`−308A
`
`Neg
`cntl
`
`IFNγ
`874A
`
`IFNγ
`874T
`
`IL-6
`−174G
`
`IL-6
`−174C
`
`IL-10
`−819C
`−592C
`
`IL-10
`−819T
`−592A
`
`IL-10
`−1082A
`−819C
`
`IL-10
`−1082G
`−819C
`
`β-Globin control
`
`Cytokine SNP
`
`Primers
`
`Figure 3 cytokine genotype. DNA extracted for patient PBMC was subject to PCR with sequence-specific primers (PCR-SSP) as described in
`Materials and Methods section. Primer pairs are designed to have perfect matches only with a single allele or group of alleles. Matched primer pairs
`result in the amplification of target sequences (i.e., a positive amplification band), whereas mismatched primer pairs do not result in amplification
`(i.e., a negative result). The specific genotypes detected by the SSP are shown above each lane (Neg cntl, reaction without cytokine primers). Shown
`is one of duplicate determinations. IFN-γ, interferon-γ; IL, interleukin; SNP, single-nucleotide polymorphism; TNF-α, tumor necrosis factor-α.
`
`with anti-CD28 mAb TGN1412.48–50 In that report,50 TNF-α lev-
`els peaked within 1 hour after infusion and IL-2, IL-6, IL-10, and
`IFN-γ reached maximum levels at the next time point, 4 hours
`after infusion (elevation in other cytokines included IL-4, IL-8,
`IL-12, and IL-1β). All six patients in that study required sup-
`portive care in an intensive care unit and two of the six required
`extensive intensive care unit stays of 11 and 21 days. Similar to
`patients suffering serious adverse events from other mAb infu-
`sions, these patients showed signs of cardiovascular instability
`and disseminated intravascular coagulation, but unlike other
`treatments, these patients manifested additional sequelae includ-
`ing early and acute lung injury. Of interest was the lack of toxic-
`ity seen with this agent in preclinical tests of the anti-CD28 mAb
`TGN1412 in nonhuman primate studies.48,49
`Perhaps the most relevant to our patient, were results observed
`in a phase I trial of a bispecific antibody that targeted both ERBB2
`and FcγRIII.51 In this study, attempting to target FcγRIII-expressing
`cells (e.g., natural killer cells) to ERBB2-expressing tumors, some
`patients experienced side effects including dyspnea with arterial
`oxygen desaturation and hypotension. Analysis of serum cytokine
`levels demonstrated an increase in TNF-α within 30 minutes that
`peaked at 2.3 hours before declining over the next 24 hours with
`slightly delayed increases in IL-6, IL-8, IL-2, IL-1β, GM-CSF, and
`IFN-γ. A major difference between therapeutic antibody admin-
`istration and CAR-engineered T cells, is that while antibodies are
`subject to clearance by the body (e.g., the bispecific mAb used in
`the cited trial had a t1/2 of 20 hours), T cells can continuously pro-
`duce effector cytokines and can expand in cell numbers following
`antigen stimulation. The severity of this patients’ response lead
`us to investigate her cytokine genotype (Figure 3), as poly-
`morphisms in cytokine genes are associated with both levels of
`cytokine production and immunological responses, such as organ
`graft rejection.52–54 Although her genotype for IFN-γ (−874A) and
`TNF-α (−308G) are associated with lower levels of cytokine pro-
`duction, her IL-6, IL-10, and transforming growth factor-β1 geno-
`types are associated with higher levels of cytokine production, and
`the specific IL-6 (−174G/C) and the IL-10 (−1082G) genotypes
`are associated with shock in patients with sepsis55 and increased
`mortality in severe sepsis,56 respectively.
`Since 2004, the Surgery Branch, NCI has conducted clini-
`cal trials involving the transfer of T-cell receptor genes into
`auto logous lymphocytes (either PBL or TIL) using the identical
`
`218,202
`
`UnTd
`CAR
`
`50,000
`
`40,000
`
`30,000
`
`20,000
`
`10,000
`
`0
`
`IFN-γ (pg/ml)
`
`SK-OV-3
`SK-BR-3
`MDA-361
`MDA-468
`CEM
`
`Auto-Mac
`Auto-DC
`
`HRE
`HMEC
`Keratinocyte
`HSMM
`PrEC
`NHBE
`EBV-B
`HUVEC
`HDF
`Media
`
`Figure 4 In vitro cytokine production. An aliquot of the ErbB2-CAR
`transduced (CAR) T lymphocytes infused into the patient or untrans-
`duced control T cells (UnTd) where assayed for IFN-γ cytokine produc-
`tion following overnight coculture with the indicated cell lines. ErbB2+
`target cells were SK-OV3, SK-BR3, and MDA361. ErbB2− tumor lines
`were MDA468 and CCRF-CEM (CEM). Primary cells adapted for growth
`in culture were HDF, human diploid fibroblast, HUVEC, human umbilical
`vein endothelial cells; EBV-B-EBV transformed B cell line, NHBE, normal
`human bronchial/tracheal epithelial cells; PrEC, human prostate epithe-
`lial cells, HSMM, human skeletal muscle myoblasts, keratinocytes-human
`keratinocytes, HMEC, human mammary epithelial cells, and HRE, human
`renal epithelial cells. Autologous patient cells were auto-DC-patient 6-day
`dendritic cell culture, and auto-mac-patient 6-day macrophage culture.
`All samples were diluted as necessary as to be in the linear range of the
`assay (sample SK-OV3 was off-scale in this assay and the value from a
`repeat determination was as indicated). CAR, chimeric antigen receptor;
`IFN-γ, interferon-γ.
`
`ERBB2, that upon ex vivo culture, ERBB2 expression was rapidly
`induced.43
`The most compelling finding in this case was the rapid rise
`in serum cytokine levels that has been associated with a multiple
`organ dysfunction syndrome.44–46 The administration of biolog-
`ics resulting in cytokine release syndrome was first reported to
`be associated with the administration of anti-CD3 mAb OTK3,
`which was administered as a systemic immunosuppressive agent
`during organ transplantation.47,48 Within 1–4 hours after OKT3
`injection, serum levels of proinflammatory cytokines such as
`TNF-α, IFN-γ, and IL-6 were markedly elevated. Most recently a
`cytokine storm was reported in six of six patients that were treated
`
`848
`
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`
`UPenn Ex. 2033
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`© The American Society of Gene & Cell Therapy
`
`Serious Adverse Event With ERBB2-based CAR
`
`nonmyeloablative conditioning regimen used in the present study
`(in some cases total body irradiation was added to further reduce
`endogenous lymphocytes). These treatments were undertaken in a
`variety of cancers ( melanoma, synovial cell sarcoma, and cancers of
`the breast, colon, and kidney) and there was no indication that this
`conditioning regime was associated with the tumor lysis syndrome
`most frequently observed in cancers of hematopoietic origin.57
`Similar numbers of cells were administered in these trials as well as
`the present trial, and review of the certificates of analysis of these
`products demonstrated similar levels of background cytokine pro-
`duction. A total of 143 patients were treated during this period
`without any treatment related mortality. These trials, included 11
`patients treated with PBL engineered to target the wild-type p53
`tumor suppressor protein,58,59 and no significant treatment related
`toxicity was seen (data not shown). In our recent report describing
`the use of high-avidity T-cell receptors targeting melanocyte dif-
`ferentiation antigens, on-target toxicity was