In this issue

Director's Corner by David G. Poplack, M.D.

Gastrointestinal stromal tumors (GIST)
by Alberto Pappo, M.D.

Turning the immune system against neuroblastoma
by Doug Myers, M.D. and Malcolm Brenner, M.D., Ph.D.

Pediatric Hematology Oncology Subspecialty
Training in the United States
by Lindsay B. Kilburn, M.D. and C. Philip Steuber, M.D.

Regulatory and Ethical Issues in the Treatment of Children
by Bambi Grilley, R.Ph., C.C.R.P., C.C.R.C., C.I.P., Stacey Berg, M.D.
and Cynthia Boudreaux, M.Ed., C.C.R.P.

Turning the Immune System Against Neuroblastoma by Doug Myers, M.D. and Malcolm Brenner, M.D., Ph.D.
	Dr. Doug Myers		Dr. Malcolm Brenner

Neuroblastoma is the most common extra-cranial solid tumor in children. When the tumor occurs in infants, it is frequently localized and responds well to therapy.¹ Even disseminated disease can be eradicated in about 75 percent of infants and ,indeed, may undergo spontaneous remission.^2,3

In older children, the prognosis is far worse. Although patients with localized disease still may be cured by conventional therapy, 80 percent or more of those with disseminated tumors can be expected to relapse within three years, and virtually none of this subgroup will become long-term survivors.⁴

Over the past decade, attempts to improve the outcome of advanced neuroblastoma have focused on greater intensification
of the induction and consolidation phases of chemo-radiotherapy, with or without stem cell rescue.^5-8 Despite improvements in remission rates, long-term survival remains poor. This failure has lead to a resurgence of interest in alternative methods of disease eradication. Immune modulation is one particular option.^9-10

Immunotherapeutic approaches have taken many forms. The most widely used immunotherapy for neuroblastoma consists of murine or human-murine chimeric monoclonal antibodies directed against GD2, a disialoganglioside expressed in tumors of neuroectodermal origin. There have been phase I and phase II studies of these antibodies, and the reported phase I data revealed an apparently significant response rate. Other studies have coupled these antibodies to toxins or to radionucleotides with broadly comparable results.^11-14 These therapies are not without their toxicities. Most patients experience significant pain during therapy.

A second approach has been vaccination with autologous or allogeneic tumor cell lines. In murine models, tumor cell lines modified to express cytokine genes have been shown to increase tumor immunogenicity to such an extent that tumors were rejected.¹⁵

Our group has clinical experience with autologous and allogeneic neuroblastoma cells transduced to express IL-2 +/- lymphotactin, which were then injected into children with advanced neuroblastoma.^16-18 In patients receiving IL-2 alone, significant tumor responses were seen, including three complete remissions among 28 treated patients. These studies as well as others have demonstrated the ability to generate a lymphocyte-mediated cytotoxic response to the tumor, but results have not been optimal.

We plan to extend the study of allogeneic tumor cell vaccines in a phase II clinical trial. With the safety of retrovirally transduced, IL-2 and lymphotactin-secreting allogeneic tumor cell vaccines demonstrated, we will introduce the vaccine to patients in a minimal residual disease state. This will involve immunization soon after completion of standard therapy, after high-dose chemotherapy and hematopoietic stem cell rescue. Immediately following high-dose chemotherapy with hematopoietic stem cell rescue, the opportunity for expansion of tumor specific lymphocytes stimulated with vaccine is optimal.

In yet a third immunotherapeutic approach to targeting neuroblastoma, we have combined these successful clinical cellular and antibody-mediated immunotherapies by generating cytotoxic T-cells expressing chimeric single chain antibodies (chimeric antigen receptors or CAR) directed against GD2. Variable regions of a monoclonal antibody are coupled to the cytoplasmic portion of the CD3 zeta chain via a linker to form a CAR (Figure 1).

Figure 1

Engagement of a single CAR suffices to induce cellular activation and proliferation.^19-22

CAR-transduced T-cells have numerous advantages over immunotherapies based on monoclonal antibodies or T lymphocytes alone. Since there is no need to select and expand tumor-specific lymphocytes from scanty precursors, large populations of antigen-redirected T lymphocytes can be obtained in a matter of weeks. Moreover, chimeric T-cell receptors are MHC-unrestricted, so that tumor escape by downregulation of HLA class I molecules or defects in antigen processing is bypassed. Finally, since both CD4+ and CD8+ T cells can express the same chimeric receptor, the full network of T-cell function is directed against tumor cells.

However, there is little doubt from clinical studies that activated chimeric T-cells rapidly lose their function and may disappear from the circulation. The principal problem appears to be that CAR- expressing T-cells rapidly return to a resting state. We are overcoming this deficit by expressing the tumor-specific CAR in EBV-specific cytotoxic T lymphocytes (CTL). These cells recognize EBV-expressing cells (which are present lifelong in patients infected with the virus) through their native receptor and receive all relevant co-stimulatory signals. Simultaneously, they express the chimeric TCR targeting GD-2 expressed on neuroblastomas and, having been activated through their native receptors can kill neuroblastoma cells recognized through their chimeric receptors. (Figure 2).

Figure 2

We treated eight patients in a phase I study of this approach and have seen greatly improved levels and persistence of chimeric EBV—CTL compared to chimeric primary T-cells. As we increase cell doses, we hope the clinical responses will be more complete and longer sustained.^23,24

About the authors
Doug Myers, M.D., is an assistant professor of pediatrics and a pediatric hematologist/oncologist in the Texas Children’s Cancer Center and a member of the Center for Cell and Gene Therapy at Baylor College of Medicine. His interests center on translational and clinical research in the fields of solid tumor immunotherapy and immune reconstitution following hematopoietic stem cell transplantation. He is currently the primary investigator on a study of gene-modified T-cells redirected through retroviral gene transduction to target the tumor antigen GD2 on neuroblastoma.

Malcolm Brenner, M.D., Ph.D., is a professor in the Departments of Molecular and Human Genetics, Medicine and Pediatrics and director, Center for Cell and Gene Therapy at Baylor College of Medicine. Brenner's primary research interest is the use of gene transfer to augment the immune response to human tumors. In collaboration with Dr. Rooney's laboratory, he directs studies using gene-modified cytotoxic T lymphocytes to prevent and treat the Epstein-Barr virus-associated malignancies, immunoblastic lymphoma, Hodgkin disease and nasopharyngeal cancer (NPC). He also directs studies to redirect T-cells toward solid tumors such as meduloblastoma, bone tumors and neuroblastoma via transduction with retroviral vectors carrying genes for chimeric antigen receptors targeting tumor-associated antigens.

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