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`Cancer Res.2011 May I; 71(9): 3175-3181. doi: I 0.1158/0008-5472.CAN-10-4035 .
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`
`Considerations for the Clinical Application of Chimeric Antigen
`Receptor (CAR) T Cells: Observations from a Recombinant DNA
`Advisory Committee (RAC) Symposium June 15, 2010
`
`Hildegund C.J. Ert1 1, John Zaia2, Steven A. Rosenberg3, Carl H. June4, Gianpietro Dotti5,
`Jeffrey Kahn6, Laurence J.N. Cooper7, Jacqueline Corrigan-Curay8 , and Scott E. Strome9
`1Wistar Institute, Philadelphia, PA
`
`2Beckman Research Institute, City of Hope, Duarte, CA
`
`3National Cancer Institute, NIH
`
`4University of Pennsylvania, Philadelphia PA
`
`5Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX
`
`6Center for Bioethics, University of Minnesota, MN
`
`7Division of Pediatrics, MD Anderson Cancer Center, Houston TX
`
`80ffice of Biotechnology Activities, Office of the Director, NIH
`
`9Department of Otorhinolaryngology-Head and Neck Surgery, University of Maryland, MD
`
`Abstract
`
`T cells which are genetically modified to express single-chain chimeric antigen receptors (CARs)
`have shown promise in early cancer immunotherapy clinical trials. Unfortunately, two recent
`deaths in cancer patients treated with CAR-T cells have created some uncertainty on how to best
`mitigate patient risk, while continuing to advance this very promising therapeutic avenue. In order
`to address these concerns, the Recombinant DNA Advisory Committee (RAC) held a symposium,
`the objectives of which were to first review the reported treatment-associated toxicities and
`second, to discuss methods for improving safety and efficacy. This report highlights the issues
`raised as part of this discussion with a specific focus on protocols infusing CAR-T cells. Because
`this was not a consensus conference, the opinions described should not be construed to represent
`those of any individual RAC member, the RAC as a body, conference participants, NIH or the
`FDA.
`
`Keywords
`
`Chimeric antigen receptor; T-cell therapy; gene therapy; CD 19; adverse events
`
`Corresponding author: Scott E. Strome, MD, Department ofOtorhinolaryngology-Head and Neck Surgery, Cniversity of Maryland,
`16 South Eutaw Street: Suite 500. Baltimore, MD, 21201-168, CSA, sstrome(':llsmail.umaryland.edu.
`Conflict of Interest: Dr. Strome is the cofounder and a major stockholder in Gliknik, Inc., a biotechnolob'Y company. He also receives
`royalties through the Mayo Clinic College of Medicine for the Ii censure of IP relating to 4-1 BB (CD 13 7) and B7-H I (PD-LI).
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`Evolution of CARs
`
`Page 2
`
`Following appropriate preconditioning regimens that deplete circulating lymphocytes, the
`adoptive transfer of tumor infiltrating lymphocytes (TILs) into patients with metastatic
`melanoma leads to objective clinical responses in select individuals (Supplemental
`references (SR) 1,2). Building on this approach, recent studies suggest that autologous T
`cells, genetically engineered to express tumor associated antigen (T AA)-specific TC Rs, can
`replace TILs in certain clinical settings. (I) Importantly, tumor regressions are observed
`even under conditions of widely metastatic and bulky disease, and in patients who have
`failed both prior surgical extirpation and medical therapy. Unfortunately, the potential for
`widespread use of adoptive transfer strategies using Tl Ls or T cells with recombinant TC Rs
`is limited (SR#3 ).
`
`In order to overcome the historic problems associated with the cellular targeting of HLA
`restricted T AA, Eshhar et al. developed a strategy to redirect T-cell specificity using
`Chimeric Antigen Receptors (CARs) or T-bodies (Figure I). First generation vectors for the
`production of CAR-T cells contain the heavy and light chain immunoglobulin (lg) variable
`regions, fused as a single chain to the epsilon, gamma or zeta signaling sequences of the T
`cell receptor (TCR) or the signaling region of the Fey domain. (2-8) T cells expressing such
`first generation CARs recognize surface T AAs, independent of HLA restriction, but cannot
`recognize intracellular T AAs (Table I). Early clinical experiences with I st generation
`CAR-T cells demonstrated that (a) the survival of adoptively transferred CAR-T cells was
`limited in cancer patients and (b) few objective anti-tumor responses were observed. (8-10)
`
`To enhance survival and/or increase proliferation of transferred CAR-T cells, investigators
`have incorporated signaling moieties from costimulatory molecules including CD28 (87-1 ),
`CD 134 (OX40), and CD 13 7 ( 4-1 88), alone (2nd generation) or in sequence (3rd
`generation). ( I 1-15) A recent study comparing simultaneously infused CD 19-specific CAR(cid:173)
`T cells with or without the CD28 signaling sequence indeed demonstrates improved survival
`of CD28-modified cells .. (16) Additionally, combining CD28 with 4-188 signaling
`sequences further promotes engraftment of CAR-T cells. ( 17, 18) As an alternative to
`incorporating costimulatory moieties into the vector, some investigators express first
`generation CARs in T cells specific to endogenous viral antigens e.g. Epstein Barr virus
`(EBY). In this setting, antigen recognition by the CAR provides TCR ~ signaling, while
`recognition of processed EBY peptides in the context of appropriate HLA molecules by the
`physiologic urncR, allows for intrinsic costimulation.( 19)
`
`Focus Points for Future Consideration
`
`Reported serious adverse events (SAE) following CAR-T cell transfer
`
`At least 40 trials using CAR-T cells for treatment of cancer have been registered with the
`NIH Office of Biotechnology Activities (OBA) and undergone NIH RAC review and at least
`120 subjects have been dosed across all trials (Supplemental Table I). (20) Overall, most
`SA Es that were viewed as possibly related to transfer of CAR-T cells have been mild, self(cid:173)
`limited and occurred shortly after infusion (Supplemental Table 2). Unfortunately, 2 patients
`died shortly after adoptive transfer of CAR-T cells. (21, 22)
`
`Cancer Res. Author manuscript; available in PMC 2014 August 11
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`Following RAC review of the death in the HER2-CAR-T cell trial at its December 2009
`meeting [http /, oba.od.nih.gov/rdna _ rac 'rac past meetings _ 2000.htrnl] the Committee
`recommended holding a safety symposium to review clinical trials employing CAR-T cells
`and to then formulate a report to both facilitate subsequent RAC and Institutional Biosafety
`Committee (IBC) reviews of these trials and to assist investigators in designing future
`studies. A planning committee, consisting of RAC members, leading investigators in this
`field, and OBA staff, identified key components of these trials based on their potential
`impact on patient safety and their role in therapy. These issues were discussed in a panel
`format and are summarized under the heading "Points to Consider in the Design and
`Implementation of Clinical Trials using CAR-T cells." A paradigm for considering how to
`incorporate these considerations into clinical trial design is provided in Table 2 and
`Supplemental Tables 3-5.
`
`Points to Consider in the Design and Implementation of Clinical Trials
`using CAR-T cells
`
`1. Unwanted On-Target Effects ofCAR-T Cells
`
`Recognition of antigen expressed on non-tumor cells by CAR-T cells is emerging as the
`major risk factor of CAR-T-cell transfer. Such recognition may become manifest as: (a)
`immediate toxicity and (b) late or sustained toxicity resulting from long term depletion of
`cells with important homeostatic functions.
`
`A. Mitigating the potential for early toxicity-A feared complication after infusion of
`CAR-T cells is their massive activation leading to the research participant's death. This is
`especially of concern with CARs directed against untested and/or endogenously prevalent
`T AAs, as T cells with high avidity receptors can respond to cells that express their targets at
`levels that are currently too low for detection by conventional means. One approach to limit
`such toxicity is an inter-patient (and sometimes intra-patient) dose-escalation scheme.
`Alternatively one could conduct the initial dose escalation with I st generation CAR-T cells
`without conditioning or cytokine supplementation under the expectation that stimulation of
`these adoptively transferred T cells may be suboptimal, although this is not yet proven. Once
`the I st generation CAR-T cells are shown to be safe, 2nd (or perhaps 3rd) generation CAR-T
`cells, with a potential for more pronounced responses and prolonged persistence, could be
`explored in combination with supplementary treatments. (23) An important caveat lo this
`statement is that it is currently unclear whether lack of toxicity using I st generation CAR-T
`cells will translate into a similar safety profile for 2nd or 3rd generation CAR-T cells
`combined with lymphodepletion and/or cytokine support. As an alternative approach, if 2nd
`or 3rd generation CARs targeting a new antigen are evaluated without previous experience
`with a I st generation CAR, a very conservative dose escalation strategy should be adopted
`(see: Starting dose ofCAR-T cells in phase I trials).
`
`The question of whether the co-expression of conditional suicide genes might safeguard
`against some of the potential side effects of non-tumor cell recognition by CAR-T cells was
`discussed. (24) As exemplified by the fatal SAE using HER2-CAR-T cells, the CAR-T cells
`may act within minutes after engagement of their target antigen and symptoms are not
`
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`expected until significant damage has occurred. Therefore, inclusion of a suicide gene,
`whose benefit will necessarily take time, was deemed to have limited potential for
`preventing acute toxicity although it may have utility in modulating late toxicities.
`
`A third approach to reduce risks of immediate toxicity is splitting of the T-ccll dose over
`two or more days as is being tested in recipients of CD 19-specific T cells. (21) Using this
`scheme it would be desirable to monitor cytokines or chemokines in the serum as potential
`indicators for toxicity after the first of the split doses. A fourth approach using a very
`conservative dose escalation strategy is discussed below under the heading: Starting dose of
`CAR-T cells in Phase I trials. Importantly, in all trials, pre-clinical studies should carefully
`test for expression of the TCR's target antigen in healthy tissues. Furthermore, the use of
`CART cells against antigens that are widely expressed on non-tumor cells essential for
`important physiological functions should, to the extent feasible, not be chosen as targets
`unless there is convincing preclinical data that their expression are at such low levels that
`on-target toxicity is unlikely to occur.
`
`B. Mitigating the potential for late toxicity-Another risk ofCAR-T cells is the long
`term depletion of cells, which are important for normal human function. For example, CAR(cid:173)
`T cells for treatment of 8-cell malignancies have targeted CD 19 and CD20, markers
`expressed on normal 8 cells. (25) Treatment with CAR-T cells directed against either of
`these antigens has the potential to deplete the patients' 8 cells.(26) Furthermore, unlike the
`u-CD20 monoclonal antibody (mAb), RituxanTM, which has a defined half-life, these CAR(cid:173)
`T cells may potentially survive and function for the life of the patient. The lack of CD 19 or
`CD20 expression on most plasma cells allows the maintenance of physiologic antibody
`levels in the majority of patients following depletion of CD 19+ or CD20- cells ( SR#4 ). In
`addition, it is expected that 8 cells would rapidly be replaced from lymphoid progenitors
`once the CAR-T cells die or become functionally impaired. Finally, even under conditions
`that would allow for long-term persistence of functioning CDI 9- or CD20-specific CAR-T
`cells, resulting in continuous depiction of 8 cells, this could be managed by lg transfer and
`would thus be preferable to a fatal cancer.
`
`A related concern is the potential for CAR-T cells to negatively impact organ function
`secondary to low levels of target antigen expression in a non-tumor site e.g. vascular
`endothelial growth factor receptor 2 (VEGFR2)-specific CARs targeting the tumor
`vasculature (SR#5,6 ). VEGFR2 is expressed on endothelial cells during physiological
`angiogenesis, vasculogenesis, arteriogencsis, and lymphangiogencsis and is needed for
`processes such as wound healing and embryogencsis (SR#7). As such, VEGFR2-spccific
`persisting C AR-T cells could interfere with formation of indispensable vasculature. While
`there is no universal strategy for mitigating these late CAR-T-cell associated toxicities, the
`resultant conditions could potentially be managed medically. Alternatively, risks of long(cid:173)
`term toxicity could be mitigated by the insertion of suicide genes into the CAR-T cells.
`
`2. Effects of Costimulatory Signaling
`Results to date demonstrate that while target cell lysis by I st generation CAR-T cells is
`independent of costimulatory signaling, their proliferation, production of cytokines and up-
`
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`regulation of other effector cell molecules depends on and/or is improved by co-stimulation.
`(27) Furthermore, based on what is understood regarding natural functions of CD28 and
`4-188 in vivo, it is anticipated that CAR-T cells bearing these domains might be more
`resistant to activation-induced cell death (SR#8). Therefore, the majority of participants
`concluded that the incorporation of CD28 and/or 4-188 into CAR constructs offers an
`important potential for therapeutic benefit. Evaluations of other cosignaling moieties, which
`may enhance the proliferation and/or survival of CAR-T cells are appropriate. In addition to
`modifying the CAR to improve T cell engraftment it may also be possible to a priori identify
`T-cell sub-populations with the capacity for sustained proliferation. In fact, there is evidence
`that the starting population ofT cells used for CAR modification may affect their ability to
`survive and expand (SR#9). For example, in non-human primates, effector cos+ T cells
`generated ex vivo from central rather than effector memory populations, enjoy enhanced
`survival following adoptive transfer (SR# I 0). Because the ratios of different T-cell subsets
`varies between patients, selective use of T-cell subsets prior to genetic manipulation may
`provide a more discriminating path to predict the long-term outcome of CAR-T cell transfer.
`Finally, clinical experience with the expression of CARs in non-T cell populations e.g. NK
`cells, is limited. (28) Therefore, special care must be taken with CAR use in T-cell subsets
`and non-T cell lymphocyte populations.
`
`3. Systemic Conditioning
`
`Adoptive transfer of CAR-T cells can be further modified by auxiliary therapies such as
`partial or complete myeloablation to improve preservation of the transferred T-cell
`populations. The rationale behind lymphodepletion prior to T-cell infusion is multifactorial
`and includes, but is not limited to, the elimination of"cytokine sinks," creation of space for
`the expansion of adoptively transferred cells, and removal of suppressor cell populations. In
`fact, depletion of lymphocytes in melanoma patients infused with ex vivo expanded TILs is
`requisite for treatment efficacy and recent studies suggest a correlation between the degree
`of lymphocyte depletion and objective response rates (SR# 11 ). Despite the recognized risks
`of myelosuppression, the majority of the RAC meeting participants felt that administration
`of CAR-T cells that do not carry an endogenous TCR to a persisting virus is unlikely to
`result in significant clinical benefit without prior conditioning.
`
`4. Cytokine supplements
`
`Recombinant soluble cytokines such as IL-2 are administered upon CAR-T cell transfer with
`the expectation that they will promote the expansion and survival of transferred cells. If and
`to what degree cytokines may exacerbate the potential toxicity of 2nd or 3rd generation
`CAR-T cells is uncertain. It is also unclear if 2nd and 3rd generation CAR-T cells need to be
`supplemented with IL-2 either at all or at the high and potentially toxic doses used for TIL
`transfer (SR# 12). For the initial evaluation of CAR-T cells to novel targets, dosing without
`addition of cytokines or with injection of low to moderate amounts of cytokines may be
`prudent and would facilitate differentiation of SA Es induced by CAR-T cells alone from
`those caused by the potential synergy between these drugs. Furthennore, the design of later
`Phase studies employing 2nd and 3rd generation CAR-T cells should consider incorporating
`treatment arms with and without cytokine support. Ongoing studies exploring the use of
`CAR-T cells genetically modified to secrete cytokines such as IL-15 or IL-12 may eliminate
`
`Cancer Res . Author manuscript ; available in PMC 2014 August 11
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`the need for systemic cytokine administration. (29) If and to what degree this approach will
`improve the potential benefit of CAR-T cells or instead pose additional risks, is currently
`unknown.
`
`5. CARs expressed in T cells targeting persisting viruses
`
`Some groups have combined the advantages ofCAR-T cells with those of traditional
`antigen-specific T cells (SR#l3). Specifically, T cells to persisting viruses such as EBY
`have been enriched in vitro and then genetically modified by insertion ofa transgene
`encoding a T AA-specific CAR. In a neuroblastoma trial, 11 individuals were treated
`simultaneously with EBV-CTLs expressing a GD2-specific first generation CAR and
`activated T lymphocytes expressing the same CAR. Only CAR EBV-CTLs showed
`significant in vivo persistence, and four of eight patients with evaluable tumors had evidence
`of tumor necrosis or regressions, including a sustained complete remission."
`Lymphodcpletion did not improve CAR-T cell persistence or clinical outcome. ( 19)
`Potential deleterious effects due to virus-specific CAR-T cells without a costimulatory
`endodomain arc anticipated to be similar to those of other T cells expressing 2nd or 3rd
`generation CARs.
`
`6. Starting dose of CAR-T cells in Phase I trials
`
`Phase I trials are primarily designed to assess safety and feasibility rather than efficacy;
`biological activity and proof of concept are usually secondary aims. Phase I trials for CAR-T
`cells typically start with reduced cell doses which are then gradually escalated. The starting
`dose should be adjusted depending on the type of CAR, i.e .. T cells with a 2nd or yd
`generation CARs should start at a lower dose than those with a I st generation CAR.
`Similarly, transfer of CAR-T cells into partially pre-conditioned patients should commence
`at a lower dose than transfer into non- myeloablated patients. Although one would expect
`that immediate toxicity due to transfer of CAR-T cells may directly correlate with numbers
`of injected cells, late adverse events may be independent of the injected dose due to
`construct-dependent dynamic changes in cell numbers.
`
`Providing reliable guidelines for starting doses ofCAR-T cells is currently not possible. To
`date, clinical trials have employed a fairly wide range of starting doses and some
`investigators dosed according to weight. others according to body surface area (BSA) and
`still others employed flat dosing schedules (Supplemental Table I). The use of unadjusted
`dosing should be avoided and a more uniform dosing scheme such as that based on cells/kg
`should be considered. An open question is whether cells/kg should be based on an ideal
`body weight or actual weight given that weight increases due to obesity may not justify an
`increase in dose (SR#l4).
`
`7. Ethical Considerations
`
`One of the ethical dilemmas facing investigators is the requirement in early phase research
`to design studies that arc safe while at the same time hoping to show biological activity or
`possibly even benefit to the individual subject. many of whom have few if any other
`therapeutic or even palliative options. In response to the unexpected death in the trial
`infusing HER2-specific CAR-T cells, initial starting doses in a number of trials
`
`Cancer Res. Author manuscript; available in PMC 2014 August 11
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`Summary
`
`administering HERZ-specific CAR-T cells, were lowered to 104 cells/m2. There was general
`agreement among the RAC members that a lower starting dose of 104 cells/m2 would likely
`be safe but would unlikely have potential benefit to the subject. Therefore, the risk-benefit
`calculus was such that any benefit realized from the research would most likely be a societal
`benefit, i.e. an increase in generalizable knowledge with minimized risk but little potential
`benefit for the patient-subject. It will be a challenge to design early phase studies which
`appropriately balance accurate disclosure of the risks and benefits posed by this research and
`appropriate informed consent among a seriously ill patient-subject population with few
`therapeutic options. While the expectation of clinical benefit must be discouraged in clinical
`trials, especially those at the earliest stages of research, selecting a dose that would be
`relatively safe but have potential biologic activity is an appropriate goal. Whether the
`acceptable level of risk should be adjusted in relation to the disease prognosis for a given
`patient cohort remains a subject worthy of future debate.
`
`CAR-T cells have shown some benefit in cancer patients.( 19, 26, 30) The major challenge
`for achieving therapeutic benefit by CAR-T cell transfer remains lack of sustained
`engraftment and loss of T cell functions. These hurdles may be overcome in part by
`incorporation of costimulatory domains into CAR constructs or by modifying EB V-speci tic
`T cells. Additional studies are needed to assess the performance of EBY-specific CAR-T
`cells in cancer patients of diverse ages. Results to date ( excluding those based on EBV(cid:173)
`specific T cells) suggest that partial myeloablation is required for survival ofCAR-T cells.
`Mechanisms that cause loss of transferred 2nd and 3rd generation CAR-T cells remain poorly
`understood and may relate to the differentiation status of the transduced T cell subsets. It is
`unclear if and at what doses cytokines are needed to improve the clinical outcome of 2nd and
`3rd generation CAR-T cells. Until this issue is clarified, the upfront use of high doses of
`systemic cytokines should be carefully justified.
`
`The major immediate risk factor for CAR-T cells remains their activity against non-tumor
`cells rather than genotoxicity from the gene transfer event. Long term risk factors, e.g.
`sustained depletion of normal cell populations or exacerbated expansion of CAR-T cells
`cannot yet be assessed. While the risk of insertional mutagenesis remains a concern, this has
`neither been observed clinically nor in recent preclinical studies that have attempted to
`recreate insertional mutagenesis (SR# 15). However, as the field moves towards the use of
`less differentiated CAR-T cells, the risk of insertional mutagenesis may increase.
`
`Supplementary Material
`
`Refer to Web version on PubMed Ce ntral for supplementary material.
`
`Acknowledgments
`
`The authors acknowledge the following staff from the Otlice of Biotechnology Activities, Dr. R. Jambou , Dr. E.
`Rosenthal, Dr. M. O'Reilly. Ms . M. Montgomery and Ms. L. Gargiulo, for thei r contributions in bringing together
`this symposium and in assembling the data on trials and adverse events. The authors also thank Dr. Richard
`Junghans for helpful discussions.
`
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