Abstract

The ability of immune-competent donor T cells to mediate a beneficial graft-versus-leukemia (GVL) effect was first identified in the setting of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematologic malignancies. Unfortunately, with the exception of chronic myelogenous leukemia and EBV-induced lymphoproliferative disease, allo-HSCT GVL lacks the potency to significantly affect disease progression or recurrence in most other hematologic malignancies. The inadequacy of a GVL effect using past approaches is particularly evident in patients with lymphoid malignancies. However, with the advent of improved gene transfer technology, genetically modified tumor-specific immune effectors have extended cellular immunotherapy to lymphoid malignancies. One promising strategy entails the introduction of genes encoding artificial receptors called chimeric antigen receptors (CARs), which redirect the specificity and function of immune effectors. CAR-modified T cells targeted to the B cell–specific CD19 antigen have demonstrated promising results in multiple early clinical trials, supporting further investigation in patients with B-cell cancers. However, disparities in clinical trial design and CAR structure have complicated the discovery of the optimal application of this technology. Recent preclinical studies support additional genetic modifications of CAR-modified T cells to achieve optimal clinical efficacy using this novel adoptive cellular therapy.

Introduction

Leukemia is the most common pediatric malignancy, accounting for 31% of all pediatric cancers diagnosed in the United States for children less than 15 years of age and 25% in patients less than 20 years of age.1  Acute lymphoblastic leukemia (ALL) accounts for the majority of cases (23% of all pediatric cancer) and is predominantly of B-cell origin (approximately 85% of ALL cases). In 2012, it is estimated that more than 6000 cases of ALL will be diagnosed in adults and children, with approximately two-thirds occurring in children.2  Acute myelogenous leukemia (AML) occurs less commonly in children, at 4% of all pediatric cancers diagnosed, but still represents approximately 20% of new cases of pediatric leukemia.1  The majority of the estimated 14 000 cases of AML diagnosed each year in the United States will be in adults.2 

Most pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) are cured with standard chemotherapy regimens, with overall survival exceeding 80% in many reported series.3–6  For some patients with very-high-risk features, the use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission is recommended.7  In contrast, most adult patients diagnosed with ALL have a poor prognosis, with survival rates < 40% in most series.8  For pediatric patients with AML, the chance of cure with chemotherapy alone is significantly less, with only 50%-65% of patients achieving long-term survival.9–12  Despite optimal therapy, long-term survival is less likely in adults, with older patients having a worse prognosis.13,14  Recommendations have been made for the use of allo-HSCT as a consolidative treatment for both adult and pediatric patients with high-risk features or with a suitable matched related donor.15,16  Unfortunately, the survival of both adult and pediatric patients with relapsed or refractory ALL or AML remains dismal.17–23  In fact, even with advances in chemotherapy or the use of allo-HSCT, the leading cause of mortality for children diagnosed with cancer is relapsed leukemia.1  To address the problem of limited therapeutic success with current options for treatment of refractory or relapsed leukemia, novel adoptive cellular therapies have been developed.

allo-HSCT

To date, allo-HSCT is the most common and successful example of cellular therapy for leukemia. Conditioning chemotherapy and/or radiation provides sufficient immunosuppression of the recipient to prevent donor HSC rejection and facilitates the destruction of residual malignant cells. Subsequent infusion of HSCs from healthy donors provides a hematologic and immunologic recovery for recipients. Optimizing the balance between antileukemic efficacy and patient toxicity has been a long-term objective of clinical BM transplantation research. Significantly, the antileukemic effect of allo-HSCT is not limited to the cytoreductive regimen. Immunocompetent donor T cells can mediate a beneficial GVL effect facilitated through the recognition of allo-Ags presented on residual tumor cells by donor T cells. Evidence to support the GVL of allo-HSCT was first demonstrated by Weiden et al in patients with acute leukemia.24  In that landmark paper illustrating the GVL effect of allo-HSCT, higher relapse rates were seen in patients with syngeneic donors compared with patients with allogeneic donors who experienced acute and/or chronic GVHD.24  Evidence supporting the presence of a GVL of allo-HSCT includes higher relapse rates in patients with chronic myelogenous leukemia after T cell–depleted allo-HSCT and anecdotal reports of remissions achieved in patients with relapsed/refractory leukemia after allo-HSCT following withdrawal of immune suppression or a GVHD flare.25–28  Unfortunately, attempts to enhance this GVL benefit (eg, using HLA disparate donors) has been met with the untoward consequences of increasing GVHD with its associated morbidity and mortality, thus counteracting the associated potential GVL benefit derived from allo-HSCT.

Recommendations for the use of allo-HSCT in both adult and pediatric patients with ALL and AML have been described previously.7,15,16,29  HLA matched related donors are the favored source of HSCs for allo-HSCT. Unfortunately, the majority of patients requiring allo-HSCT do not have an appropriately HLA matched relative. Alternative HSC sources include: unrelated donors, umbilical cord blood (UCB), or haploidentical related donors. The use of alternative HSC sources for allo-BMT has expanded the pool of eligible patients markedly. Recent reports have shown equivalent leukemia-free survival after UCB transplantations compared with BM transplantations in children and UCB transplantations vs peripheral blood stem cell or BM transplantations in adults.30,31  Haploidentical related donor transplantation has gained renewed interest after reports of improved engraftment and reduced GVHD after posttransplantation cyclophosphamide (in the setting of reduced intensity conditioning) or improved methods of T-cell depletion through CD34+ selection (in the setting of myeloablative conditioning).32–34 

Donor leukocyte infusion

One approach that attempts to use the GVL effect of donor T cells is donor leukocyte infusion (DLI) after allo-HSCT. In the setting of relapsed chronic myelogenous leukemia after allo-HSCT, additional therapy with DLI has demonstrated the most compelling direct evidence of a clinically meaningful GVL effect.35–37  In addition, in the setting of EBV-induced lymphoproliferative disease (EBV-LPD), DLI from EBV-seropositive donors or the infusion of EBV-specific cytotoxic T cells (EBV-CTLs) generated ex vivo can induce durable disease remissions by restoring cell-mediated viral immunity.38–40  However, in the setting of relapsed adult and pediatric acute leukemia after allo-HSCT, the use of DLI has only rarely induced durable remissions, is associated with a high incidence of acute and chronic GVHD, and does not provide a meaningful survival benefit for the majority of patients.36,37,41,42 

NK-cell infusion

The identification of a GVL effect mediated by alloreactive natural killer (NK) cells was first described in the studies by Ruggeri et al.43  This phenomenon is most evident in the setting of haploidentical transplantations for AML in which donor NK cells improve transplantation outcomes by reducing leukemia relapse in the setting of killer cell Ig–like receptor ligand mismatches, thus triggering donor NK cell versus recipient leukemia alloreactivity.43  Several centers have attempted to use this apparent GVL effect of alloreactive NK cells in clinical trials through the infusion of haploidentical NK cells, primarily in patients with either poor-risk or relapsed AML.44,45  To date, reported beneficial clinical outcomes have been reported in a minority of treated patients.44,45 

CARs: genetically engineered adoptive cell therapy

Conventional approaches to adoptive cellular therapy of leukemia, including allo-HSCT and DLI, in most cases mediate at best a modest GVL effect. Further, this modest antitumor effect is offset by the significant risk of donor T cell–mediated GVHD, which has diminished enthusiasm for these cellular therapeutic approaches. To address the problem of limited success with associated significant toxicity, novel cellular therapies using genetically modified, tumor-specific T cells have gained significant interest among investigators.

With the advent of enhanced gene-transfer technology, especially in the arena of gamma-retroviral and lentiviral vector design, it is now possible to efficiently introduce tumor-specific cloned TCR genes46,47  or genes encoding chimeric Ag receptors (CARs)48  into immune effector cells. Through this genetic reprogramming, these immune effector cells are redirected to target Ags expressed by leukemic cells. In most clinical applications to date, a patient's own T cells may be reprogrammed to express these tumor-specific receptors, largely minimizing the potential risk of adverse GVHD-related toxicities as seen in the setting of allo-HSCT and DLI.

The use of tumor-targeted TCR gene transfer has to date had limited clinical application in the treatment of hematological malignancies. This approach is further limited by complications including: a lack of efficient cloned TCR expression49 ; theoretical mismatched pairing of cloned alpha- and beta-chains with endogenous alpha- and beta-chains, resulting in highly unpredictable and potentially autoimmune TCR pairings50 ; and the HLA-restricted nature of TCR recognition that limits the application of this technology to specific HLA repertoires.

In contrast, genetic modification of immune effectors via a CAR is not restrained by these limitations and has several advantages. Because tumor targeting is HLA independent, the use of CARs is applicable to a broad range of patients irrespective of HLA phenotype. Furthermore, CAR modification of immune effectors overcomes the tumor's ability to escape immunodetection by down-regulation of HLA molecules on the cell surface. Targeting of tumor Ags by CAR-modified T cells is applicable to any cell-surface Ag, including proteins, carbohydrates, and glycolipids, for which a mAb can be generated.51  This enables CAR-modified T cells to respond to a broader range of targets compared with TCR-modified immune effector cells. Finally, the ability to generate a large quantity of tumor-specific T cells in a relatively short period of time makes this strategy feasible for use in the clinical setting.52 

CARs are composed of an Ag-specific binding domain (most commonly a single-chain–variable fragment derived from the fused variable heavy- and light-chain domains of a tumor-targeted mAb) fused to a transmembrane domain followed by one or more cytoplasmic signaling domains (Figure 1). In initial designs, CARs were designed to contain a single cytoplasmic signaling domain derived most commonly from the TCR-derived CD3ζ chain. These “first-generation” CARs, when expressed in T cells, mediate a primary activation signal upon encounter with the targeted Ag (termed signal 1), but in the absence of additional costimulation (ie, signal 2) undergo activation-induced apoptosis or senescence, termed anergy.53  To address this limitation, we and others further modified CAR structure to add additional cytoplasmic signaling domains, including T cell–costimulatory signaling domains (eg, CD28, 4-1BB, or OX-40), resulting in “second-generation” CARs. These second-generation CARs containing costimulatory signaling domains are capable of delivering both signal 1 and signal 2 upon encounter with the targeted tumor Ag.54–58  Given the promising preclinical results with T cells modified to express second-generation CARs, investigators have favored this CAR design in initial clinical trials. Further modification of CAR design includes adding more than one costimulatory signaling domain in addition to the CD3ζ chain, resulting in “third-generation” CARs; this hasalso been translated to the clinical setting (Figure 1).59–62 

Figure 1.

CAR technology evolution through the generation of more potent CARs. First-generation CARs classically contain only one signaling domain, typically the cytoplasmic signaling domain of the CD3 TCRζ chain. Second-generation CARs containing 2 signaling domains typically include the addition of the cytoplasmic signaling domains of the costimulatory receptors CD28, 4-1BB, or OX-40, among others. Third-generation CARs attempt to harness the signaling potential of 2 costimulatory domains: classically, the CD28 domain followed by either the 4-1BB or OX-40 signaling domains. CAR-modified T-cell potency may be further enhanced through the introduction of additional genes, including those encoding proproliferative cytokines (ie, IL-12) or costimulatory ligands (ie, 4-1BBL), thus producing “armored” fourth-generation CAR-modified T cells.

Figure 1.

CAR technology evolution through the generation of more potent CARs. First-generation CARs classically contain only one signaling domain, typically the cytoplasmic signaling domain of the CD3 TCRζ chain. Second-generation CARs containing 2 signaling domains typically include the addition of the cytoplasmic signaling domains of the costimulatory receptors CD28, 4-1BB, or OX-40, among others. Third-generation CARs attempt to harness the signaling potential of 2 costimulatory domains: classically, the CD28 domain followed by either the 4-1BB or OX-40 signaling domains. CAR-modified T-cell potency may be further enhanced through the introduction of additional genes, including those encoding proproliferative cytokines (ie, IL-12) or costimulatory ligands (ie, 4-1BBL), thus producing “armored” fourth-generation CAR-modified T cells.

Adoptive therapy with CD19-targeted, CAR-modified T cells: rationale and preclinical studies

Genetically engineered tumor-targeted cell therapy first and foremost requires the identification of a suitable target Ag to avoid the toxicity associated with targeting Ags expressed on normal tissues. To this end, most clinical trials in the field of cellular therapy for leukemia have targeted the CD19 Ag. Although CD19 is expressed on normal B cells and most B-cell malignancies, including B-ALL, chronic lymphocytic leukemias (CLL), and most non-Hodgkin lymphomas, the Ag is not expressed on normal HSCs. Whereas optimal immune targeting of the CD19 Ag will result in the eradication of B-cell tumors and may induce subsequent B-cell aplasia, successful targeting of this Ag will otherwise spare other normal hematopoietic precursors and BM function.

Early preclinical studies using adoptive transfer of CD19-targeted, CAR-modified human T cells into CD19+ human tumor–bearing immunocompromised mice demonstrated marked antitumor responses mediated by these genetically modified T cells.58,63  In addition, preclinical studies demonstrated superior proliferative and antitumor activity, both in vitro and in vivo, of T cells modified to express second-generation CD19-targeted CARs containing either the costimulatory CD28 or 4-1BB cytoplasmic signaling domains.58,64  Given these promising preclinical findings, several centers, including our own, have translated this technology to the clinical setting as phase 1 clinical trials treating patients with CD19+ malignancies (Table 1).

Table 1.

Summary of actively recruiting clinical trials with CD19-targeted CARs

Summary of actively recruiting clinical trials with CD19-targeted CARs
Summary of actively recruiting clinical trials with CD19-targeted CARs

MDACC indicates MD Anderson Cancer Center; NIH, National Institutes of Health; FHCRC, Fred Hutchinson Cancer Research Center; NHL, non-Hodgkin lymphoma; and scFv, single-chain–variable fragment.

*Donor-derived EBV-CTLs.

†Donor-derived Tri-virus CTLs.

‡Donor-derived CD8+ central memory viral specific (EBV or CMV) T cells.

Published results of adoptive therapy with autologous CD19-targeted, CAR-modified T cells

Early reported clinical outcomes from the City of Hope and Baylor College of Medicine (BCM) in patients with low-grade B-cell malignancies treated with CD19-targeted, CAR-modified T cells were disappointing, with patients achieving at best stable disease.65,66  It is notable that the clinical trial designs of these early studies entailed infusion of CD19-targeted, CAR-modified T cells in the absence of prior lymphodepleting or conditioning chemotherapy, a likely clinically relevant variable. The first truly promising clinical outcome with CD19-targeted, CAR-modified autologous T cells was reported by researchers at the National Cancer Institute (NCI). In that case report, a heavily pretreated patient with follicular non-Hodgkin lymphoma received conditioning cyclophosphamide and fludarabine chemotherapy, followed by infusion of T cells modified to express a second-generation CD28-containing CD19-targeted CAR, followed by additional exogenous IL-2 cytokine support.67  The patient achieved a partial response and developed a long-standing B-cell aplasia, a surrogate marker for a persistent anti–CD19-targeted effect mediated by the infused CAR-modified T cells. Soon thereafter, investigators from the University of Pennsylvania (UPenn) published 2 reports detailing a partial response in one patient and long-term molecular complete responses in an additional 2 patients with CLL after conditioning chemotherapy and infusion of T cells modified to express a CD19-targeted, 4-1BB–containing, second-generation CAR.68,69  In addition to impressive antitumor responses, all patients in this cohort exhibited the predicted long-term B-cell aplasia. In similar trials conducted at our institution, treatment with autologous T cells modified to express a CD28-containing second-generation CD19-targeted CAR resulted in a significant radiographic response to disease in 1 of 4 evaluable CLL patients who received prior cyclophosphamide conditioning chemotherapy, with an additional 2 patients achieving stable disease despite rapid tumor progression before therapy.70  In addition, in one patient with relapsed B-ALL, we reported an expected long-term B-cell aplasia after reinduction chemotherapy and genetically modified T-cell infusion before subsequent allo-HSCT. Most recently, investigators at the NCI published the results of a subsequent cohort of 8 patients with low-grade B-cell malignancies.71  That study, which included an analysis of the first published patient after reinfusion of genetically modified T cells, used a CD28-containing second-generation CAR targeted to the CD19 Ag. In these studies, the investigators reported one complete response, one death on study unrelated to T-cell infusion, and partial responses observed in the remaining treated patients. Four of 8 treated patients in this report developed the anticipated B-cell aplasia.

Relevant variables in published CD19-targeted, CAR-modified T-cell clinical trials

Given the increasing number of reported clinical trial results from multiple academic centers, we are now able to speculate on factors that may be relevant to the optimal clinical approach of CAR-modified T-cell therapy. Multiple differences in clinical trial design using genetically modified T cells to treat B-cell malignancies exist, and the optimal clinical trial design using this technology is still to be determined (Figure 2). Specifically, potentially relevant variables between published trials include the design of the CD19-targeted CAR, in vitro T-cell production, the role of prior conditioning chemotherapy, and the roles of tumor burden as well as tumor sensitivity to conditioning therapy at the time of modified T-cell infusion (Table 2).

Figure 2.

Variables in clinical trial design. Multiple, potentially clinically relevant variables exist between various published clinical trial outcomes treating patients with CD19-targeted, CAR-modified T cells. There are variables in the methodology of CAR gene transfer (1), the design of the CAR (2), the inclusion or exclusion of prior conditioning chemotherapy (3), whether conditioning chemotherapy may reduce tumor burden (4), and whether additional cytokine support with IL-2 is provided exogenously after modified T-cell infusion (5). Whether one or more of these variables are indeed relevant to ultimate clinical outcomes awaits additional multicenter trials resolving these variables by direct comparison to establish the optimal conditions in which these CAR-modified T cells may induce an optimal clinical response.

Figure 2.

Variables in clinical trial design. Multiple, potentially clinically relevant variables exist between various published clinical trial outcomes treating patients with CD19-targeted, CAR-modified T cells. There are variables in the methodology of CAR gene transfer (1), the design of the CAR (2), the inclusion or exclusion of prior conditioning chemotherapy (3), whether conditioning chemotherapy may reduce tumor burden (4), and whether additional cytokine support with IL-2 is provided exogenously after modified T-cell infusion (5). Whether one or more of these variables are indeed relevant to ultimate clinical outcomes awaits additional multicenter trials resolving these variables by direct comparison to establish the optimal conditions in which these CAR-modified T cells may induce an optimal clinical response.

Table 2.

Comparison of recently published clinical trials using CD19-targeted CAR-modified T cells

Comparison of recently published clinical trials using CD19-targeted CAR-modified T cells
Comparison of recently published clinical trials using CD19-targeted CAR-modified T cells

*Data were available for only 1 of 8 patients.

†In the setting of B-ALL patients in complete remission.

CAR design

Preclinical studies have shown superior in vivo antitumor responses mediated by second- and third-generation CARs (Figure 1) containing T cell–costimulatory signaling domains compared with first-generation CARs lacking costimulatory signaling.58,62,64,72  Consistent with these preclinical studies, elegant studies in patients with low-grade B-cell malignancies infused with a combination of first- and second-generation CAR-modified CD19-targeted T cells demonstrated superior persistence of CD28-containing second-generation CAR-modified T cells compared with first-generation CAR-modified T cells.66  Although it is reasonable, in light of these data and subsequent promising clinical data in patients treated with second-generation CARs,66  to predict the clinical superiority of second-generation over first-generation CAR-modified T cells, direct in vivo comparisons between various second-generation CD19-targeted constructs containing either the CD28 or the 4-1BB signaling domains have not been conducted in patients to date. Both complete tumor responses and B-cell aplasia have been reported in patients treated with T cells modified to express either CD28- or 4-1BB–containing second-generation CARs.67–71  Whether either second-generation costimulatory construct has clinical superiority over the other remains to be evaluated directly in the clinical setting. Also untested in the clinical setting is whether variation of the hybridoma-derived single-chain–variable fragment used in these variable CARs, with the FMC63 MAb being incorporated in the NCI and UPenn CARs and the SJ25C1 MAb incorporated in the MSKCC CAR, plays a relevant role in antitumor efficacy and subsequent clinical outcomes (Table 2).

T-cell production

Gene-transfer methodology also varies between centers: either gamma-retroviral vectors or lentiviral vectors are used by most centers, but some use electroporation or transposon technology.74–76  Given the time in culture required for T-cell production using electroporation, this approach has largely been abandoned.76  Published studies lack direct comparisons between alternative gene-transfer approaches, leaving open the question of whether there are clinically meaningful differences between gamma-retroviral-, lentiviral-, and transposon-mediated gene transfer. However, given the published data, it is worthwhile to note that clinical CRs and resultant B-cell aplasia were achieved in the setting of both gamma-retroviral67,70,71  and lentiviral68,69  gene-modified, CD19-targeted, CAR-modified T-cell therapies. In addition, the concern regarding the risk of insertional oncogenesis after gene transfer in the T cell is unknown, but recent studies have demonstrated a decade-long safety using gamma-retroviral vectors.40,76,77  Furthermore, the relative risk of insertional oncogenesis between the various gene-transfer methodologies has yet to be determined.

Prior conditioning chemotherapy

Conditioning chemotherapy, also termed lymphodepleting therapy, is a well-recognized and requisite adjunct to enhancing antitumor efficacy in the setting of autologous tumor-infiltrating lymphocyte therapy for patients with melanoma.79  Based on the currently published clinical outcomes from patients treated with CD19-targeted CAR+ T cells, this paradigm appears to be relevant to the field of CAR-modified T-cell adoptive therapy. Specifically, in 2 initial published clinical trials using CD19-targeted, CAR-modified T cells patients treated in the absence of prior conditioning chemotherapy achieved at best modest clinical responses.65,66  In contrast, unique trials at our institution allowed for the comparison between an initial cohort of patients treated with CAR-modified T cells in the absence of prior conditioning chemotherapy followed by a similar cohort treated with an even lower dose of T cells after prior cyclophosphamide chemotherapy. In these studies, we demonstrated both improved persistence of modified T cells and improved clinical outcomes in the cohort receiving prior conditioning chemotherapy.70  Similarly, studies from both the NCI and UPenn included prior chemotherapy conditioning of patients, which resulted in a marked clinical benefits.67–69,71  Comparisons of the clinical outcomes from these studies support the use of prior conditioning therapy to achieve the optimal clinical outcomes after CAR-modified T-cell therapy.

Tumor burden and tumor sensitivity to conditioning chemotherapy

Analysis of CD19-targeted CAR+ T-cell persistence in treated patients with either CLL or B-ALL at Memorial Sloan-Kettering Cancer Center (MSKCC) demonstrated an inverse correlation between tumor burden and detectable infused modified T cells over time.70  This finding, in combination with our reported clinical outcomes, is consistent with an enhanced anti-CD19 efficacy mediated by CAR-modified T cells in the setting of lower tumor burden. In light of this finding, there remains the additional question of what role potential tumor sensitivity to conditioning chemotherapy plays in the ultimate clinical outcomes of patients treated with CD19-targeted, CAR-modified T cells. Specifically, in our studies, patients were conditioned with cyclophosphamide before genetically modified T-cell infusion. In our cohort of treated CLL patients, patients had received one or more prior chemotherapy regimens containing cyclophosphamide. Patients with tumors already refractory to cyclophosphamide treated on our protocols achieved no additional reduction in tumor burden mediated by this conditioning regimen before infusion with modified T cells.70  In contrast, CLL patients treated on the UPenn protocol were naive to conditioning chemotherapies with standard second-line CLL agents, including bendamustine and pentostatin.68,69  Similarly, the initial reported patient at the NCI was treated with a combination conditioning regimen of cyclophosphamide and fludarabine, the latter of which the patient had not received previously.67  These results, at the very least, suggest a potential correlation between clinical outcomes and potential tumor sensitivity to prior conditioning chemotherapies. Unfortunately, the more recently published clinical data from the NCI failed to report prior patient chemotherapy regimens to allow for further correlation between genetically modified T-cell antitumor efficacy and tumor sensitivity with respect to the prior conditioning chemotherapy.71 

Additional currently ongoing clinical trials of B-cell malignancies using CD19-targeted, CAR-modified T cells

Given the promising published clinical outcomes of patients with B-cell malignancies treated with CD19-targeted, CAR-modified T cells to date, there has been a rapid expansion of additional trials at multiple centers using this CD19-targeted CAR technology (Table 1). Specifically, at our institution, we have recently opened an additional clinical trial treating CLL patients in the upfront setting with CD19-targeted, CAR-modified T cells as a consolidation after initial therapy with pentostatin, cyclophosphamide, and rituximab–based chemotherapy. Additional CD19-targeted CAR clinical trials are currently open using transposon gene-transfer technology73  and CAR-modified T cells generated in vitro from previously selected T cells with a central memory phenotype, which may enhance subsequent in vivo persistence79  (Table 1).

Several centers, including MSKCC, have transferred this technology from the autologous to the allogeneic setting. Specifically, at our institution, we have initiated enrollment and treatment of pediatric patients with B-ALL who have relapsed after allo-HSCT with donor-derived EBV-specific T cells modified to express our CD28-containing second-generation CAR targeted to the CD19 Ag. The rationale of this approach is based on the reported safety and minimal GVHD-mediated toxicity that these enriched EBV-specific allogeneic T cells have in patients with EBV-LPD. Our institution has recently reported on the safety of infused donor EBV-CTLs for patient with EBV-LPD with the development of GVHD.39  Our aim is to provide a GVL effect through the anti-CD19 CAR in the absence of a GVHD despite the allogeneic nature of these T cells. Similar studies using CD19-specific, CAR-modified viral specific T cells with specificity against 3 viruses (EBV, CMV, and adenovirus)80  are currently being conducted by investigators at BCM (Table 1).

Genetically engineered adoptive therapy of B-cell malignancies is not solely restricted to T cells. In fact, based on the clinical potential of NK cells as antileukemic immune effectors in the haploidentical setting, investigators have reported promising preclinical studies using CD19-targeted, CAR-modified NK cells.81  Based on these promising preclinical data, investigators at St Jude Research Hospital have opened a clinical trial treating patients with haploidentical NK cells also modified to express a second-generation 4-1BB–containing, CD19-targeted CAR (Table 1).

Additional target Ags for genetically modified immune effectors in the adoptive immune cell therapy of leukemias

The restricted nature of the CD19 Ag on normal B cells and most B-cell malignancies has resulted in this target receiving the majority of attention in both preclinical and clinical investigations for adoptive T-cell therapy in leukemia. However, additional target Ags with the potential to broaden the application of this approach to other hematologic malignancies are under investigation. Specifically, clinical trials using CAR-modified T cells targeted to the CD20 Ag have demonstrated safety and some promising initial clinical outcomes.65,75  Additional targets for CAR-modified T cells include the receptor tyrosine kinase-like orphan receptor 1 (ROR1) or kappa-light chain of human Ig, which expands the application of this therapy for hematologic malignancies.82,83  Furthermore, this adoptive T-cell approach may be extrapolated to myeloid malignancies based on promising preclinical data using CAR-modified immune effector cells targeted to the CD33 Ag and the Lewis Y Ag,84–86  as well as TCR-modified T cells targeted to the WT-1 Ag and the hyaluronan-mediated motility receptor (HMMR/Rhamm).87,88  With the exception of anti-kappa chain CAR-modified T cells, it is unclear if these latter Ags will be targeted through adoptive T-cell therapies in future clinical trials.

Further modification of tumor targeted T cells: The next generation

To date, CAR-modified T cells have demonstrated promising initial clinical responses; however, most patients who have been treated with CD19-targeted T cells ultimately develop progressive disease and succumb to their disease. We and others have developed additional genetic approaches to enhance the in vivo antitumor efficacy of CAR-modified T cells. Specifically, we have demonstrated the additional genetic modification of CAR-modified T cells to express proproliferative T cell–costimulatory ligands (4-1BBL)89  or proinflammatory cytokines (IL-12),90  resulting in “armored” fourth-generation CAR-modified T cells (Figure 1). These cells have shown enhanced in vivo antitumor efficacy in preclinical tumor models compared with T cells modified to express the tumor-targeted CAR alone. Based on the very promising preclinical outcomes of these studies, we anticipate translating these more potent tumor-targeted T cells to the clinical setting as the next generation of CAR-modified T-cell trials.

Conclusions

Under ideal conditions, immune effectors may mediate a GVL effect, which in turn may result in an enhanced clinical response in the allogeneic setting for patients with hematologic malignancies. Unfortunately, this GVL effect in the setting of most hematologic malignancies is at best modest, with many patients experiencing relapsed disease despite allo-HSCT and DLI therapy. Gene therapy of immune effectors to target these cells to Ags expressed on tumor cells have demonstrated promising clinical responses in the autologous setting. However, despite promising but anecdotal early clinical trial results in the setting of T cells targeted to the B-cell CD19 Ag, significant variables in clinical trial design make it difficult to determine the optimal clinical setting in which to apply this novel technology. Variables in CAR design, gene-transfer technology, and prior conditioning therapies need to be addressed directly in the clinical setting to answer these questions. Given the increasing number of clinical trials using CD19-targeted, CAR-modified T cells currently open at multiple centers, these questions are doomed to remain largely unanswered through direct clinical comparisons in the absence of collaborative trials between centers. Our institution, in collaboration with investigators at UPenn and the Children's Hospital of Philadelphia through a National Institutes of Health Special Translational Research Acceleration Project (STRAP) award, are currently addressing several of these variables directly by harmonizing our respective clinical protocols. Through this collaboration, patients with B-cell malignancies will be treated with equal numbers of autologous T cells modified with the gamma-retroviral gene transfer of the MSKCC CD28 containing second-generation, CD19-targeted CAR and the UPenn lentiviral gene transfer of the 4-1BB–containing second-generation CD19-targeted CAR. These studies are designed to address the questions of both optimal second-generation CAR design (CD28 versus 4-1BB containing CARs) and gene-transfer technology (gamma-retroviral versus lentiviral vectors) directly. However, these studies do not directly address additional questions regarding optimal conditioning therapy and the relationship between CAR-modified T-cell antitumor efficacy and tumor sensitivity to conditioning chemotherapy. We hope that this initial collaborative effort will serve as a template to other investigators in this field and spur additional collaborative trials to further compare additional variables, not only with respect to targeting tumors expressing the CD19 Ag, but also with respect to clinical trials with CAR-modified T cells targeting additional promising Ags expressed on a wider array of hematologic and solid tumor malignancies.

Disclosures

Conflict-of-interest disclosure: The authors declare no competing financial interests. Off-label drug use: None disclosed.

Acknowledgments

The authors thank Hollie Pegram for designing the figures; Michel Sadelain and Joe Olechnowicz for reviewing the manuscript; and Isabelle Riviere, Nancy Kernan, Jae Park, Marco Davila, Xiuyan Wang, and the entire CTCEF team for their efforts on behalf of the patients.

Correspondence

Renier Brentjens, MD, PhD, Associate Member, Leukemia Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10065; Phone: 212-639-7053; Fax: 212-717-3569; e-mail: brentjer@mskcc.org.

References

References
1
Ries
 
LAG
Smith
 
MA
Gurney
 
JG
, et al. 
Cancer Incidence and Survival among Children and Adolescents: United States SEER Program 1975-1995
1999
Bethesda, MD
National Cancer Institute, National Institutes of Health
 
publication no. 99-4649
2
American Cancer Society
Cancer Facts and Figures 2012
2012
Atlanta, GA
American Cancer Society
3
Möricke
 
A
Reiter
 
A
Zimmermann
 
M
, et al. 
Risk-adjusted therapy of acute lymphoblastic leukemia can decrease treatment burden and improve survival: treatment results of 2169 unselected pediatric and adolescent patients enrolled in the trial ALL-BFM 95
Blood
2008
, vol. 
111
 
9
(pg. 
4477
-
4489
)
4
Mitchell
 
CD
Richards
 
SM
Kinsey
 
SE
Lilleyman
 
J
Vora
 
A
Eden
 
TO
Benefit of dexamethasone compared with prednisolone for childhood acute lymphoblastic leukaemia: results of the UK Medical Research Council ALL97 randomized trial
Br J Haematol
2005
, vol. 
129
 
6
(pg. 
734
-
745
)
5
Moghrabi
 
A
Levy
 
DE
Asselin
 
B
, et al. 
Results of the Dana-Farber Cancer Institute ALL Consortium Protocol 95-01 for children with acute lymphoblastic leukemia
Blood
2007
, vol. 
109
 
3
(pg. 
896
-
904
)
6
Pui
 
CH
Campana
 
D
Pei
 
D
, et al. 
Treating childhood acute lymphoblastic leukemia without cranial irradiation
N Engl J Med
2009
, vol. 
360
 
26
(pg. 
2730
-
2741
)
7
Oliansky
 
DM
Camitta
 
B
Gaynon
 
P
, et al. 
Role of cytotoxic therapy with hematopoietic stem cell transplantation in the treatment of pediatric acute lymphoblastic leukemia: update of the 2005 evidence-based review
Biol Blood Marrow Transplant
2012
, vol. 
18
 
4
(pg. 
505
-
522
)
8
Pui
 
CH
Evans
 
WE
Treatment of acute lymphoblastic leukemia
N Engl J Med
2006
, vol. 
354
 
2
(pg. 
166
-
178
)
9
Gibson
 
BE
Wheatley
 
K
Hann
 
IM
, et al. 
Treatment strategy and long-term results in paediatric patients treated in consecutive UK AML trials
Leukemia
2005
, vol. 
19
 
12
(pg. 
2130
-
2138
)
10
Lange
 
BJ
Smith
 
FO
Feusner
 
J
, et al. 
Outcomes in CCG-2961, a children's oncology group phase 3 trial for untreated pediatric acute myeloid leukemia: a report from the children's oncology group
Blood
2008
, vol. 
111
 
3
(pg. 
1044
-
1053
)
11
Creutzig
 
U
Buchner
 
T
Sauerland
 
MC
, et al. 
Significance of age in acute myeloid leukemia patients younger than 30 years: a common analysis of the pediatric trials AML-BFM 93/98 and the adult trials AMLCG 92/99 and AMLSG HD93/98A
Cancer
2008
, vol. 
112
 
3
(pg. 
562
-
571
)
12
Kaspers
 
GJ
Creutzig
 
U
Pediatric acute myeloid leukemia: international progress and future directions
Leukemia
2005
, vol. 
19
 
12
(pg. 
2025
-
2029
)
13
Appelbaum
 
FR
Gundacker
 
H
Head
 
DR
, et al. 
Age and acute myeloid leukemia
Blood
2006
, vol. 
107
 
9
(pg. 
3481
-
3485
)
14
Rosenblat
 
TL
Jurcic
 
JG
Induction and postremission strategies in acute myeloid leukemia: state of the art and future directions
Hematol Oncol Clin North Am
2011
, vol. 
25
 
6
(pg. 
1189
-
1213
)
15
Oliansky
 
DM
Rizzo
 
JD
Aplan
 
PD
, et al. 
The role of cytotoxic therapy with hematopoietic stem cell transplantation in the therapy of acute myeloid leukemia in children: an evidence-based review
Biol Blood Marrow Transplant
2007
, vol. 
13
 
1
(pg. 
1
-
25
)
16
Oliansky
 
DM
Appelbaum
 
F
Cassileth
 
PA
, et al. 
The role of cytotoxic therapy with hematopoietic stem cell transplantation in the therapy of acute myelogenous leukemia in adults: an evidence-based review
Biol Blood Marrow Transplant
2008
, vol. 
14
 
2
(pg. 
137
-
180
)
17
Nguyen
 
K
Devidas
 
M
Cheng
 
SC
, et al. 
Factors influencing survival after relapse from acute lymphoblastic leukemia: a Children's Oncology Group study
Leukemia
2008
, vol. 
22
 
12
(pg. 
2142
-
2150
)
18
Einsiedel
 
HG
von Stackelberg
 
A
Hartmann
 
R
, et al. 
Long-term outcome in children with relapsed ALL by risk-stratified salvage therapy: results of trial acute lymphoblastic leukemia-relapse study of the Berlin-Frankfurt-Munster Group 87
J Clin Oncol
2005
, vol. 
23
 
31
(pg. 
7942
-
7950
)
19
Roy
 
A
Cargill
 
A
Love
 
S
, et al. 
Outcome after first relapse in childhood acute lymphoblastic leukaemia - lessons from the United Kingdom R2 trial
Br J Haematol
2005
, vol. 
130
 
1
(pg. 
67
-
75
)
20
Sander
 
A
Zimmermann
 
M
Dworzak
 
M
, et al. 
Consequent and intensified relapse therapy improved survival in pediatric AML: results of relapse treatment in 379 patients of three consecutive AML-BFM trials
Leukemia
2010
, vol. 
24
 
8
(pg. 
1422
-
1428
)
21
Wells
 
RJ
Adams
 
MT
Alonzo
 
TA
, et al. 
Mitoxantrone and cytarabine induction, high-dose cytarabine, and etoposide intensification for pediatric patients with relapsed or refractory acute myeloid leukemia: Children's Cancer Group Study 2951
J Clin Oncol
2003
, vol. 
21
 
15
(pg. 
2940
-
2947
)
22
Fielding
 
AK
Richards
 
SM
Chopra
 
R
, et al. 
Outcome of 609 adults after relapse of acute lymphoblastic leukemia (ALL); an MRC UKALL12/ECOG 2993 study
Blood
2007
, vol. 
109
 
3
(pg. 
944
-
950
)
23
Ferrara
 
F
Palmieri
 
S
Mele
 
G
Prognostic factors and therapeutic options for relapsed or refractory acute myeloid leukemia
Haematologica
2004
, vol. 
89
 
8
(pg. 
998
-
1008
)
24
Weiden
 
PL
Flournoy
 
N
Thomas
 
ED
, et al. 
Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts
N Engl J Med
1979
, vol. 
300
 
19
(pg. 
1068
-
1073
)
25
Porter
 
DL
Antin
 
JH
The graft-versus-leukemia effects of allogeneic cell therapy
Annu Rev Med
1999
, vol. 
50
 (pg. 
369
-
386
)
26
Higano
 
CS
Brixey
 
M
Bryant
 
EM
, et al. 
Durable complete remission of acute nonlymphocytic leukemia associated with discontinuation of immunosuppression following relapse after allogeneic bone marrow transplantation. A case report of a probable graft-versus-leukemia effect
Transplantation
1990
, vol. 
50
 
1
(pg. 
175
-
177
)
27
Horowitz
 
MM
Gale
 
RP
Sondel
 
PM
, et al. 
Graft-versus-leukemia reactions after bone marrow transplantation
Blood
1990
, vol. 
75
 
3
(pg. 
555
-
562
)
28
Wagner
 
JE
Thompson
 
JS
Carter
 
SL
Kernan
 
NA
Effect of graft-versus-host disease prophylaxis on 3-year disease-free survival in recipients of unrelated donor bone marrow (T-cell Depletion Trial): a multi-centre, randomised phase II-III trial
Lancet
2005
, vol. 
366
 
9487
(pg. 
733
-
741
)
29
Oliansky
 
DM
Larson
 
RA
Weisdorf
 
D
, et al. 
The role of cytotoxic therapy with hematopoietic stem cell transplantation in the treatment of adult acute lymphoblastic leukemia: update of the 2006 evidence-based review
Biol Blood Marrow Transplant
2012
, vol. 
18
 
1
(pg. 
18
-
36
)
30
Eapen
 
M
Rubinstein
 
P
Zhang
 
MJ
, et al. 
Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: a comparison study
Lancet
2007
, vol. 
369
 
9577
(pg. 
1947
-
1954
)
31
Eapen
 
M
Rocha
 
V
Sanz
 
G
, et al. 
Effect of graft source on unrelated donor haemopoietic stem-cell transplantation in adults with acute leukaemia: a retrospective analysis
Lancet Oncol
2010
, vol. 
11
 
7
(pg. 
653
-
660
)
32
Munchel
 
A
Kesserwan
 
C
Symons
 
HJ
, et al. 
Nonmyeloablative, HLA-haploidentical bone marrow transplantation with high dose, post-transplantation cyclophosphamide
Pediatr Rep
2011
, vol. 
3
 
suppl 2
pg. 
e15
 
33
Aversa
 
F
Reisner
 
Y
Martelli
 
MF
The haploidentical option for high-risk haematological malignancies
Blood Cells Mol Dis
2008
, vol. 
40
 
1
(pg. 
8
-
12
)
34
Reisner
 
Y
Hagin
 
D
Martelli
 
MF
Haploidentical hematopoietic transplantation: current status and future perspectives
Blood
2011
, vol. 
118
 
23
(pg. 
6006
-
6017
)
35
Kolb
 
HJ
Mittermuller
 
J
Clemm
 
C
, et al. 
Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients
Blood
1990
, vol. 
76
 
12
(pg. 
2462
-
2465
)
36
Collins
 
RH
Shpilberg
 
O
Drobyski
 
WR
, et al. 
Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation
J Clin Oncol
1997
, vol. 
15
 
2
(pg. 
433
-
444
)
37
Kolb
 
HJ
Schattenberg
 
A
Goldman
 
JM
, et al. 
Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients
Blood
1995
, vol. 
86
 
5
(pg. 
2041
-
2050
)
38
Papadopoulos
 
EB
Ladanyi
 
M
Emanuel
 
D
, et al. 
Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation
N Engl J Med
1994
, vol. 
330
 
17
(pg. 
1185
-
1191
)
39
Doubrovina
 
E
Oflaz-Sozmen
 
B
Prockop
 
SE
, et al. 
Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation
Blood
2012
, vol. 
119
 
11
(pg. 
2644
-
2656
)
40
Heslop
 
HE
Slobod
 
KS
Pule
 
MA
, et al. 
Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients
Blood
2010
, vol. 
115
 
5
(pg. 
925
-
935
)
41
Collins
 
RH
Goldstein
 
S
Giralt
 
S
, et al. 
Donor leukocyte infusions in acute lymphocytic leukemia
Bone Marrow Transplant
2000
, vol. 
26
 
5
(pg. 
511
-
516
)
42
Levine
 
JE
Barrett
 
AJ
Zhang
 
MJ
, et al. 
Donor leukocyte infusions to treat hematologic malignancy relapse following allo-SCT in a pediatric population
Bone Marrow Transplant
2008
, vol. 
42
 
3
(pg. 
201
-
205
)
43
Ruggeri
 
L
Capanni
 
M
Urbani
 
E
, et al. 
Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants
Science
2002
, vol. 
295
 
5562
(pg. 
2097
-
2100
)
44
Miller
 
JS
Soignier
 
Y
Panoskaltsis-Mortari
 
A
, et al. 
Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer
Blood
2005
, vol. 
105
 
8
(pg. 
3051
-
3057
)
45
Rubnitz
 
JE
Inaba
 
H
Ribeiro
 
RC
, et al. 
NKAML: a pilot study to determine the safety and feasibility of haploidentical natural killer cell transplantation in childhood acute myeloid leukemia
J Clin Oncol
2010
, vol. 
28
 
6
(pg. 
955
-
959
)
46
Johnson
 
LA
Morgan
 
RA
Dudley
 
ME
, et al. 
Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen
Blood
2009
, vol. 
114
 
3
(pg. 
535
-
546
)
47
Parkhurst
 
MR
Yang
 
JC
Langan
 
RC
, et al. 
T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis
Mol Ther
2011
, vol. 
19
 
3
(pg. 
620
-
626
)
48
Sadelain
 
M
Brentjens
 
R
Riviere
 
I
The promise and potential pitfalls of chimeric antigen receptors
Curr Opin Immunol
2009
, vol. 
21
 
2
(pg. 
215
-
223
)
49
Jorritsma
 
A
Schotte
 
R
Coccoris
 
M
de Witte
 
MA
Schumacher
 
TN
Prospects and limitations of T cell receptor gene therapy
Curr Gene Ther
2011
, vol. 
11
 
4
(pg. 
276
-
287
)
50
Bendle
 
GM
Linnemann
 
C
Hooijkaas
 
AI
, et al. 
Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy
Nat Med
2010
, vol. 
16
 
5
(pg. 
565
-
570
)
51
Sadelain
 
M
Riviere
 
I
Brentjens
 
R
Targeting tumours with genetically enhanced T lymphocytes
Nat Rev Cancer
2003
, vol. 
3
 
1
(pg. 
35
-
45
)
52
Hollyman
 
D
Stefanski
 
J
Przybylowski
 
M
, et al. 
Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy
J Immunother
2009
, vol. 
32
 
2
(pg. 
169
-
180
)
53
Croft
 
M
Co-stimulatory members of the TNFR family: keys to effective T-cell immunity?
Nat Rev Immunol
2003
, vol. 
3
 
8
(pg. 
609
-
620
)
54
Imai
 
C
Mihara
 
K
Andreansky
 
M
, et al. 
Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia
Leukemia
2004
, vol. 
18
 
4
(pg. 
676
-
684
)
55
Finney
 
HM
Akbar
 
AN
Lawson
 
AD
Activation of resting human primary T cells with chimeric receptors: costimulation from CD28, inducible costimulator, CD134, and CD137 in series with signals from the TCR zeta chain
J Immunol
2004
, vol. 
172
 
1
(pg. 
104
-
113
)
56
Hombach
 
A
Wieczarkowiecz
 
A
Marquardt
 
T
, et al. 
Tumor-specific T cell activation by recombinant immunoreceptors: CD3 zeta signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3 zeta signaling receptor molecule
J Immunol
2001
, vol. 
167
 
11
(pg. 
6123
-
6131
)
57
Maher
 
J
Brentjens
 
RJ
Gunset
 
G
Riviere
 
I
Sadelain
 
M
Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor
Nat Biotechnol
2002
, vol. 
20
 
1
(pg. 
70
-
75
)
58
Brentjens
 
RJ
Santos
 
E
Nikhamin
 
Y
, et al. 
Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts
Clin Cancer Res
2007
, vol. 
13
 
18 pt 1
(pg. 
5426
-
5435
)
59
Pulè
 
MA
Straathof
 
KC
Dotti
 
G
Heslop
 
HE
Rooney
 
CM
Brenner
 
MK
A chimeric T cell antigen receptor that augments cytokine release and supports clonal expansion of primary human T cells
Mol Ther
2005
, vol. 
12
 
5
(pg. 
933
-
941
)
60
Morgan
 
RA
Yang
 
JC
Kitano
 
M
Dudley
 
ME
Laurencot
 
CM
Rosenberg
 
SA
Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2
Mol Ther
2010
, vol. 
18
 
4
(pg. 
843
-
851
)
61
Wilkie
 
S
Picco
 
G
Foster
 
J
, et al. 
Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor
J Immunol
2008
, vol. 
180
 
7
(pg. 
4901
-
4909
)
62
Till
 
BG
Jensen
 
MC
Wang
 
J
, et al. 
CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results
Blood
2012
, vol. 
119
 
17
(pg. 
3940
-
3950
)
63
Brentjens
 
RJ
Latouche
 
JB
Santos
 
E
, et al. 
Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15
Nat Med
2003
, vol. 
9
 
3
(pg. 
279
-
286
)
64
Milone
 
MC
Fish
 
JD
Carpenito
 
C
, et al. 
Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo
Mol Ther
2009
, vol. 
17
 
8
(pg. 
1453
-
1464
)
65
Jensen
 
MC
Popplewell
 
L
Cooper
 
LJ
, et al. 
Antitransgene rejection responses contribute to attenuated persistence of adoptively transferred CD20/CD19-specific chimeric antigen receptor redirected T cells in humans
Biol Blood Marrow Transplant
2010
, vol. 
16
 
9
(pg. 
1245
-
1256
)
66
Savoldo
 
B
Ramos
 
CA
Liu
 
E
, et al. 
CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients
J Clin Invest
2011
, vol. 
121
 
5
(pg. 
1822
-
1826
)
67
Kochenderfer
 
JN
Wilson
 
WH
Janik
 
JE
, et al. 
Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19
Blood
2010
, vol. 
116
 
20
(pg. 
4099
-
4102
)
68
Kalos
 
M
Levine
 
BL
Porter
 
DL
, et al. 
T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced Leukemia
Sci Transl Med
2011
, vol. 
3
 
95
pg. 
95ra73
 
69
Porter
 
DL
Levine
 
BL
Kalos
 
M
Bagg
 
A
June
 
CH
Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia
N Engl J Med
2011
, vol. 
365
 
8
(pg. 
725
-
733
)
70
Brentjens
 
RJ
Riviere
 
I
Park
 
JH
, et al. 
Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias
Blood
2011
, vol. 
118
 
18
(pg. 
4817
-
4828
)
71
Kochenderfer
 
JN
Dudley
 
ME
Feldman
 
SA
, et al. 
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
Blood
2012
, vol. 
119
 
12
(pg. 
2709
-
2720
)
72
Teng
 
MW
Kershaw
 
MH
Moeller
 
M
Smyth
 
MJ
Darcy
 
PK
Immunotherapy of cancer using systemically delivered gene-modified human T lymphocytes
Hum Gene Ther
2004
, vol. 
15
 
7
(pg. 
699
-
708
)
73
Kowolik
 
CM
Topp
 
MS
Gonzalez
 
S
, et al. 
CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells
Cancer Res
2006
, vol. 
66
 
22
(pg. 
10995
-
11004
)
74
Jin
 
Z
Maiti
 
S
Huls
 
H
, et al. 
The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor
Gene Ther
2011
, vol. 
18
 
9
(pg. 
849
-
856
)
75
Till
 
BG
Jensen
 
MC
Wang
 
J
, et al. 
Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells
Blood
2008
, vol. 
112
 
6
(pg. 
2261
-
2271
)
76
Scholler
 
J
Brady
 
TL
Binder-Scholl
 
G
, et al. 
Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells
Sci Transl Med
2012
, vol. 
4
 
132
pg. 
132ra53
 
77
Biasco
 
L
Ambrosi
 
A
Pellin
 
D
, et al. 
Integration profile of retroviral vector in gene therapy treated patients is cell-specific according to gene expression and chromatin conformation of target cell
EMBO Mol Med
2011
, vol. 
3
 
2
(pg. 
89
-
101
)
78
Dudley
 
ME
Yang
 
JC
Sherry
 
R
, et al. 
Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens
J Clin Oncol
2008
, vol. 
26
 
32
(pg. 
5233
-
5239
)
79
Terakura
 
S
Yamamoto
 
TN
Gardner
 
RA
Turtle
 
CJ
Jensen
 
MC
Riddell
 
SR
Generation of CD19-chimeric antigen receptor modified CD8+ T cells derived from virus-specific central memory T cells
Blood
2012
, vol. 
119
 
1
(pg. 
72
-
82
)
80
Leen
 
AM
Myers
 
GD
Sili
 
U
, et al. 
Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals
Nat Med
2006
, vol. 
12
 
10
(pg. 
1160
-
1166
)
81
Marin
 
V
Kakuda
 
H
Dander
 
E
, et al. 
Enhancement of the anti-leukemic activity of cytokine induced killer cells with an anti-CD19 chimeric receptor delivering a 4-1BB-zeta activating signal
Exp Hematol
2007
, vol. 
35
 
9
(pg. 
1388
-
1397
)
82
Hudecek
 
M
Schmitt
 
TM
Baskar
 
S
, et al. 
The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor
Blood
2010
, vol. 
116
 
22
(pg. 
4532
-
4541
)
83
Vera
 
J
Savoldo
 
B
Vigouroux
 
S
, et al. 
T lymphocytes redirected against the kappa light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells
Blood
2006
, vol. 
108
 
12
(pg. 
3890
-
3897
)
84
Dutour
 
A
Marin
 
V
Pizzitola
 
I
, et al. 
In vitro and in vivo antitumor effect of anti-CD33 chimeric receptor-expressing EBV-CTL against CD33 Acute myeloid leukemia
Adv Hematol
2012
, vol. 
2012
 pg. 
683065
 
85
Peinert
 
S
Prince
 
HM
Guru
 
PM
, et al. 
Gene-modified T cells as immunotherapy for multiple myeloma and acute myeloid leukemia expressing the Lewis Y antigen
Gene Ther
2010
, vol. 
17
 
5
(pg. 
678
-
686
)
86
Schirrmann
 
T
Pecher
 
G
Specific targeting of CD33(+) leukemia cells by a natural killer cell line modified with a chimeric receptor
Leuk Res
2005
, vol. 
29
 
3
(pg. 
301
-
306
)
87
Spranger
 
S
Jeremias
 
I
Wilde
 
S
, et al. 
TCR-transgenic lymphocytes specific for HMMR/Rhamm limit tumor outgrowth in vivo
Blood
2012
, vol. 
119
 
15
(pg. 
3440
-
3449
)
88
Stauss
 
HJ
Thomas
 
S
Cesco-Gaspere
 
M
, et al. 
WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells
Blood Cells Mol Dis
2008
, vol. 
40
 
1
(pg. 
113
-
116
)
89
Stephan
 
MT
Ponomarev
 
V
Brentjens
 
RJ
, et al. 
T cell-encoded CD80 and 4-1BBL induce auto- and transcostimulation, resulting in potent tumor rejection
Nat Med
2007
, vol. 
13
 
12
(pg. 
1440
-
1449
)
90
Pegram
 
HJ
Lee
 
JC
Hayman
 
EG
, et al. 
Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning
Blood
2012
, vol. 
119
 
18
(pg. 
4133
-
4141
)