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original article
Adoptive Transfer of Chimeric Antigen Receptor Re-directed Cytolytic T Lymphocyte Clones in Patients with Neuroblastoma Julie R Park1, David L DiGiusto2, Marilyn Slovak3, Christine Wright2, Araceli Naranjo4, Jamie Wagner4, Hunsar B Meechoovet4, Cherrilyn Bautista4, Wen-Chung Chang4, Julie R Ostberg4 and Michael C Jensen5 Department of Pediatric Hematology–Oncology, Children’s Hospital and Medical Center, Seattle, Washington, USA; 2Division of Hematology and Hematopoietic Cell Transplant, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA; 3Department of Cytogenetics, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA; 4Division of Molecular Medicine, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA; 5Division of Cancer Immunotherapeutics and Tumor Immunology, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA 1
Metastatic neuroblastoma is a poor-prognosis malignancy arising during childhood that overexpresses the L1-cell adhesion molecule (CD171). We have previously described a tumor L1-cell adhesion molecule-specific, single chain antibody-derived, chimeric antigen receptor designated CE7R for re-directing the antigen-specific effector functioning of cytolytic T lymphocytes. Here, we report on the feasibility of isolating, and the safety of infusing, autologous CD8+ cytolytic T lymphocyte clones co-expressing CE7R and the selection-suicide expression enzyme HyTK in children with recurrent/ refractory neuroblastoma. The cytolytic T lymphocyte products were derived from peripheral blood mononuclear cells that were subjected to polyclonal activation, plasmid vector electrotransfer, limiting dilution hygromycin selection, and expansion to numbers sufficient for adoptive transfer. In total, 12 infusions (nine at 108 cells/m2, three at 109 cells/m2) were administered to six patients. No overt toxicities to tissues known to express L1-cell adhesion molecule (e.g., central nervous system, adrenal medulla, and sympathetic ganglia) were observed. The persistence of cytolytic T lymphocyte clones in the circulation, measured by vector-specific quantitative polymerase chain reaction, was short (1–7 days) in patients with bulky disease, but significantly longer (42 days) in a patient with a limited disease burden. This first-in-humans pilot study sets the stage for clinical trials employing adoptive transfer in the context of minimal residual disease. Received 29 September 2006; accepted 14 December 2006; advance online publication 13 February 2007. doi:10.1038/mt. sj.6300104
INTRODUCTION The ex vivo derivation of tumor-reactive T lymphocytes by their genetic modification to express chimeric antigen receptors
(CARs) is becoming a clinically feasible method for generating cell products for adoptive therapy of cancer. Antibody-derived scFv-targeting chimeras are HLA unrestricted and thus can be used for patients with target epitope–positive tumors and, unlike tumor recognition based on T cell receptors (TCRs), their use is not subject to tumor escape as a consequence of tumor down regulation of restricting HLA molecules or pertubations in the cellular antigen-processing machinery. We have constructed a CAR using a single-chain antibody extracellular domain (scFv) derived from the L1-cell adhesion molecule (L1-CAM)-specific murine CE7 hybridoma.1 This chimeric immunoreceptor, CE7R, is specific for an epitope in the extracellular domain of L1-CAM present on neuroblastoma cells and, to a limited extent, on adrenal medulla and sympathetic ganglia.1 More recently, L1-CAM expression has been not only associated with recurrent neuroblastoma2 but also found to correlate with tumor progression and metastasis in several types of cancer, including colon carcinoma,3 malignant glioma,4 cutaneous malignant melanoma,5 ovarian carcinoma,6–8 prostate cancer,9 renal cell carcinoma,10 and uterine carcinoma.6 Furthermore, L1-CAM has been found to participate in the regulation of tumor cell differentiation, proliferation, migration, and invasion.3,11–14 Overall, these findings provide a rationale for developing L1-CAM targeted immunotherapy of cancer. We have previously reported on CE7R-expressing cytolytic T cells that execute re-directed specificity for L1-CAM+ human neuroblastoma cell lines and are activated for tumor cell cytolysis and TC1 cytokine production.15 Although a variety of CARs have been described for potential application to human tumor immunotherapy, relatively few have been tested in human trials. To take the CE7R construct into human trials, we have developed a T lymphocyte plasmid vector gene transfer platform using electroporation. This platform relies on drug selection of T cell transfectants and results in the chromosomal integration of vector DNA, typically in a single site and copy number.16 We have also constructed a plasmid vector that directs the
Correspondence: Michael C Jensen, Division of Cancer Immunotherapeutics and Tumor Immunology, Beckman Research Institute, Department of Pediatric Hematology–Oncology, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010-3000, USA. E-mail:
[email protected] Molecular Therapy vol. 15 no. 4, 825–833 april 2007
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Administration of Re-Directed CTLs in Neuroblastoma
t ranscription of CE7R and the selection-suicide fusion protein HyTK, from the human EF-1 and cytomegalovirus promoters, respectively (see Figure 1). The electroporated T cells are amenable to cloning and selection in limiting dilution, as well as to expanding to large numbers using T-cell culture systems devised by Riddell et al.17 Here we report our initial experience in manufacturing and infusing autologous CE7R/HyTK+ CD8+ cytolytic T-lymphocyte (CTL) clones in heavily treated children with advanced recurrent metastatic neuroblastoma. This is the first human trial targeting L1-CAM via genetically engineered CTLs, and, combined with the association of L1-CAM expression with tumor metastasis and poor prognosis, the initial safety profile described here strongly supports the further development of this adoptive T-cell therapy approach for malignancies that express L1-CAM.
Figure 1 Schemas illustrating the experiment reported in this article. (a) Generation of re-directed T-cell clones; (b) treatment of patients.
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RESULTS Patient characteristics Patients in this study had either primary refractory or relapsed metastatic neuroblastoma (see Supplementary Table S1). All 10 research participants enrolled had received intensive alkylatorbased induction chemotherapy, and 7 of the 10 had received additional radiation therapy, surgery, myeloablative autologous stem cell transplantation, and a variety of salvage therapies before enrollment and leukapheresis. Two patients (UPN018 and UPN021) were withdrawn from the study as a result of interim changes in their eligibility status. The average time from apheresis to first infusion was 172 days and was affected by the time required to manufacture the T cell product and/or the time taken for the patient to recover from salvage therapy.
Successful generation of genetically modified T cells Cell products meeting all quality control release tests (Table 1) were available for six of the eight patients eligible to undergo adoptive therapy (see Supplementary Table S1). All leukapheresis products were obtained during breaks in ongoing salvage chemotherapy, typically 14–21 days after the end of a cycle. The hygromycin-resistant CAR+ CD8+ CTL clones were isolated from each of the eight patients in the trial. The failure to release clones for patients UPN020 and UPN023 was due to more than one band on the Southern blot and low-level expression of endogenous TCR-αβ, respectively. The Southern blots indicating singlesite insertions of the CE7R transgene within the released clones are depicted in Figure 2. The western blot and cell-surface density profiles are also depicted, confirming expression of the CAR protein by these T cell clones. Phenotype and function of autologous CE7R+/HYTK+ CD8+ cytolytic T lymphocyte clones Clones were subjected to flow cytometric analysis for the T cell subtype markers TCR-αβ, CD3, CD8 (Figure 3a), and CD4 (data not shown). These clones were also analyzed for additional differentiation markers. This evaluation revealed that the CTL clones were differentiated, the effectors being CD45RO+, CD62L—, CD27—, and CD28—. All the clones also expressed low to negative amounts of PD-1, a marker for T cell exhaustion, and relatively high levels of the co-stimulatory molecule NKG2D, which recognizes stress-induced ligands on tumor cells such as MICA/B. The expression levels of CD56, a natural killer– associated marker, varied among CTL clones, with those from patients UPN014, UPN016, UPN019, and UPN022 being significantly positive. Examination of receptors specific for chemokines that have been reported to be expressed on neuroblastomas revealed varied positive expression of CCR2, CCR5, and CXCR4 (receptors for chemokines MCP-1, RANTES, and SDF-1, respectively) on these T cell clones (Figure 3b). All of the clones used in the therapy also expressed cytolytic effector molecules granzyme A and perforin, and exhibited re-directed killing of allogeneic L1-CAM+ human neuroblastoma Be-2 targets in 4-hour chromium release assays (Figure 3c). Interestingly, however, there was no direct correlation between the measured level of surface CAR expression (Figure 2) and the relative amount of cytotoxic activity (for example, compare “low” expressor clone 1H1 of UPN015 www.moleculartherapy.org vol. 15 no. 4, april 2007
Administration of Re-Directed CTLs in Neuroblastoma
© The American Society of Gene Therapy
Table 1 T cell product release criteria Release test
Test methodology
Criteria for passing
Plasmid vector copy number
Southern w/HyTK-specific probe
Single band
CE7R expression
Western w/CD3ζ specific antibody
Unique 66-kd band
Cell-surface phenotype
Flow cytometry
Uniform expression of TCR-αβ and CD8
Anti-neuroblastoma cytolytic activity
4-hour 51Cr release assay
>50% specific lysis at an effector-to-targetratio of 25:1
Sensitivity to Ganciclovir
14-day Ganciclovir culture/trypan blue
90% viability
Antigen/IL-2-independent growth
3H-TdR uptake
