Expansion of cancer germline antigen-specific cytotoxic T lymphocytes for immunotherapy.

The cancer germline antigens MAGE-A1, MAGE-A3, and NY-ESO-1 can be used to target relapsed or therapy-resistant malignant solid tumors, and previous studies have demonstrated that these antigens can be epigenetically upregulated on the surface of tumor cells following exposure to low-dose demethylating chemotherapy agents, such as decitabine. The extent to which cancer germline antigen cytotoxic T lymphocytes can be reliably expanded from healthy donors has not been well characterized, specifically in terms of whether these T cells consistently kill antigen-bearing targets or simply produce interferon-γ in the presence of the antigen. Cancer germline antigen cytotoxic T lymphocytes were generated using conventional method and high-density lymphocyte culture method. We demonstrate that there is no difference in the extent of antigen-specific killing with or without CD25 depletion when interleukin-21 is added to the cultures. Cancer germline antigen-specific killer cells could be expanded from 8/12 healthy donors using overlapping peptide mixes derived from MAGE-A1, MAGE-A3, and NY-ESO-1 and from 7/9 healthy donors using HLA-restricted epitopes. Furthermore, cytotoxic T lymphocyte derived from 4/5 patients displayed specific cytotoxicity of target cells expressing respective cancer germline antigen and HLA partially matched tumor lines. High-density lymphocyte culture prior to stimulation with cancer germline antigen peptides resulted in antigen-specific cytotoxic T lymphocyte from healthy donors and patients from whom cancer germline antigen cytotoxic T lymphocyte culture with conventional methods was not feasible. These data demonstrate that MAGE-A1-, MAGE-A3-, and NY-ESO-1-specific T cells with antigen-specific cytotoxicity can be cultured from healthy donors and patient-derived cells making adoptive immunotherapy with these cytotoxic T lymphocyte feasible.

Cytomegalovirus Treatment in Pediatric Hematopoietic Stem Cell Transplant Patients.

Cytomegalovirus (CMV) is a frequent complication of hematopoietic stem cell transplant in pediatric patients, with significant morbidity and mortality. Antiviral drugs are used as prophylactic, preemptive or therapeutic medicines; however, no uniform guidelines exist for the best strategy to prevent CMV disease. Resistance to standard antiviral therapies can lead to further difficulty in managing CMV disease. Studies for investigational therapies are underway and could provide options for treatment of resistant CMV, while limiting toxicities associated with currently used antiviral therapies.

Dendritic cell vaccines: A review of recent developments and their potential pediatric application.

For many cancers the use of conventional chemotherapy has been maximized, and further intensification of chemotherapy generally results in excess toxicity with little long-term benefit for cure. Many tumors become resistant to chemotherapy, making the investigation of novel approaches such as immunotherapy of interest. Because the tumor microenvironment is known to promote immune tolerance and down regulate the body's natural defense mechanisms, modulating the immune system with the use of dendritic cell (DC) therapy is an attractive approach. Thousands of patients with diverse tumor types have been treated with DC vaccines. While antigen specific immune responses have been reported, the duration and magnitude of these responses are typically weak, and objective clinical responses have been limited. DC vaccine generation and administration is a multi-step process with opportunities for improvement in source of DC for vaccine, selection of target antigen, and boosting effector cell response via administration of vaccine adjuvant or concomitant pharmacologic immunomodulation. In this review we will discuss recent developments in each of these areas and highlight elements that could be moved into pediatric clinical trials.

Cellular and Antibody Based Approaches for Pediatric Cancer Immunotherapy.

Progress in the use of traditional chemotherapy and radiation-based strategies for the treatment of pediatric malignancies has plateaued in the past decade, particularly for patients with relapsing or therapy refractory disease. As a result, cellular and humoral immunotherapy approaches have been investigated for several childhood cancers. Several monoclonal antibodies are now FDA approved and commercially available, some of which are currently considered standard of practice. There are also several new cellular immunotherapy approaches under investigation, including chimeric antigen receptor (CAR) modified T cells, cancer vaccines and adjuvants, and natural killer (NK) cell therapies. In this review, we will discuss previous studies on pediatric cancer immunotherapy and new approaches that are currently being investigated in clinical trials.

A phase I trial combining decitabine/dendritic cell vaccine targeting MAGE-A1, MAGE-A3 and NY-ESO-1 for children with relapsed or therapy-refractory neuroblastoma and sarcoma.

Antigen-specific immunotherapy was studied in a multi-institutional phase 1/2 study by combining decitabine (DAC) followed by an autologous dendritic cell (DC)/MAGE-A1, MAGE-A3 and NY-ESO-1 peptide vaccine in children with relapsed/refractory solid tumors. Patients aged 2.5-15 years with relapsed neuroblastoma, Ewing's sarcoma, osteosarcoma and rhabdomyosarcoma were eligible to receive DAC followed by DC pulsed with overlapping peptides derived from full-length MAGE-A1, MAGE-A3 and NY-ESO-1. The primary endpoints were to assess the feasibility and tolerability of this regimen. Each of four cycles consisted of week 1: DAC 10 mg/m(2)/day for 5 days and weeks 2 and 3: DC vaccine once weekly. Fifteen patients were enrolled in the study, of which 10 were evaluable. Generation of DC was highly feasible for all enrolled patients. The treatment regimen was generally well tolerated, with the major toxicity being DAC-related myelosuppression in 5/10 patients. Six of nine patients developed a response to MAGE-A1, MAGE-A3 or NY-ESO-1 peptides post-vaccine. Due to limitations in number of cells available for analysis, controls infected with a virus encoding relevant genes have not been performed. Objective responses were documented in 1/10 patients who had a complete response. Of the two patients who had no evidence of disease at the time of treatment, one remains disease-free 2 years post-therapy, while the other experienced a relapse 10 months post-therapy. The chemoimmunotherapy approach using DAC/DC-CT vaccine is feasible, well tolerated and results in antitumor activity in some patients. Future trials to maximize the likelihood of T cell responses post-vaccine are warranted.

Cancer testis antigen and immunotherapy.

The identification of cancer testis (CT) antigens has been an important advance in determining potential targets for cancer immunotherapy. Multiple previous studies have shown that CT antigen vaccines, using both peptides and dendritic cell vaccines, can elicit clinical and immunologic responses in several different tumors. This review details the expression of melanoma antigen family A, 1 (MAGE-A1), melanoma antigen family A, 3 (MAGE-A3), and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) in various malignancies, and presents our current understanding of CT antigen based immunotherapy.

Adoptive immunotherapy with CMV-specific cytotoxic T lymphocytes for stem cell transplant patients with refractory CMV infections.

Adoptive immunotherapy with cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) is an effective strategy for preventing and treating viral reactivation after allogeneic stem cell transplantation (SCT). We have shown previously that CMV CTL can be generated in 1 to 2 weeks by stimulating donor lymphocytes with peptide mixes derived from full-length pp65 and IE1. We conducted a multi-institutional study of CMV-specific CTL for patients with persistent or anti-viral-resistant CMV infections after allogeneic SCT, to determine the safety, feasibility, and immunologic effects of this approach. We were successful in stimulating CTL from 10/10 donors with pooled CMV overlapping peptide mixes. Five of the 7 subjects who met infusion criteria had new onset CMV-specific CTL activity detected within 4 to 6 weeks after infusion. Of the 2 subjects who did not have immunologic responses after infusion, 1 received CTL with a low viability after thawing, and the other patient received cyclosporine A and systemic corticosteroids at the time of the infusion, achieving only a low, transient increase (10%) in pp65-specific activity. There was no graft-versus-host disease attributable to these infusions. These findings indicate that the infusion of CTL stimulated over 1 to 2 weeks with overlapping CMV peptides can result in virus-specific immune reconstitution in SCT recipients, without exacerbations of graft-versus-host disease.

Wilms' tumor 1-specific cytotoxic T lymphocytes can be expanded from adult donors and cord blood.

The use of WT1-specific CTL is one potential strategy to treat leukemic relapse following allogeneic stem cell transplant (SCT). Previous studies have largely focused on generating WT1-CTL from adult donors by cloning. We demonstrate that WT1-CTL can be generated from healthy adult donors and from cord blood by stimulating with an overlapping pool of peptides derived from full length WT1 and selecting antigen-specific cells based on the expression of CD137. The rapid expansion with anti-CD3 and IL-2 resulted in a 100-200-fold expansion. These CTL lysed WT1 expressing targets, including leukemia lines, in a HLA restricted manner.

The detection of CMV pp65 and IE1 in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly lethal brain tumor affecting children and adults, with the majority of affected individuals dying from their disease by 2 years following diagnosis. Other groups have reported the association of cytomegalovirus (CMV) with GBM, and we sought to confirm these findings in a large series of patients with primary GBM from our institution. Immunohistochemical analysis of paraffin embedded tissue sections was performed on 49 newly diagnosed GBM tumors, the largest series reported to date. We confirmed the presence of CMV pp65 on 25/49 (51%) and of IE1 on 8/49 (16%) of these tumors. While pp65 and IE1 are generally found in the nucleus of cells that are permissibly infected by CMV, GBM in this series had mostly cytoplasmic staining, with only 16% having nuclear staining for one or both of these antigens. We infected GBM cell lines with a laboratory strain of CMV, and found that most of the staining was cytoplasmic, with some perinuclear localization of IE1. To test the potential for CMV infected GBM cells to be recognized by CMV pp65 and IE1 specific cytotoxic T lymphocytes (CTL), we used CMV infected GBM cell lines in cytotoxicity assays with human leukocyte antigen partially matched CMV CTL. Lysis of CMV infected GBM tumor cells was accentuated by pre-treating these cell lines with either the demethylating agent decitabine or interferon-γ, both of which were shown to increase MHC Class I and II expression on tumor cells in vitro. These studies confirm the presence of CMV pp65 or IE1 on approximately half of GBM, with the possibility that CMV positive tumor cells can be recognized by CMV pp65/IE1 specific T cells.

Efficacy and safety of ex vivo cultured adult human mesenchymal stem cells (Prochymal™) in pediatric patients with severe refractory acute graft-versus-host disease in a compassionate use study.

Preliminary studies using directed-donor ex vivo expanded human mesenchymal stem cells (hMSCs) have shown promise in the treatment of acute graft-versus-host disease (aGVHD). However, their production is cumbersome and standardization is difficult. We describe the first experience of using a premanufactured, universal donor, formulation of hMSCs (Prochymal) in children (n = 12; 10 boys; 9 Caucasian; age range: 0.4-15 years) with treatment-resistant grade III and IV aGVHD who received therapy on compassionate use basis between July 2005 and June 2007 at 5 transplant centers. All patients had stage III or IV gut (GI) symptoms and half had additional liver and/or skin involvement. Disease was refractory to steroids in all cases and additionally to a median of 3 other immunosuppressive therapies. The hMSCs (8 × 10(6)cells/kg/dose in 2 patients and 2 × 10(6)cells/kg/dose in the rest) were infused intravenously over 1 hour twice a week for 4 weeks. Partial and mixed responders received subsequent weekly therapy for 4 weeks. HLA or other matching was not needed. The hMSCs were started at a median of 98 days (range: 45-237) posttransplant. A total of 124 doses were administered, with a median of 8 doses (range: 2-21) per patient. Overall, 7 (58%) patients had complete response, 2 (17%) partial response, and 3 (25%) mixed response. Complete resolution of GI symptoms occurred in 9 (75%) patients. Two patients relapsed after initial response and showed partial response to retreatment. The cumulative incidence of survival at 100 days from the initiation of Prochymal therapy was 58%. Five of 12 patients (42%) were still alive after a median follow-up of 611 days (range: 427-1111) in surviving patients. No infusional or other identifiable acute toxicity was seen in any patient. Multiple infusions of hMSCs were well tolerated and appeared to be safe in children. Clinical responses, particularly in the GI system, were seen in the majority of children with severe refractory aGVHD. Given the favorable results observed in a patient population with an otherwise grave prognosis, we conclude that hMSCs hold potential for the treatment of aGVHD, and should be further studied in phase III trials in pediatric and adult patients.

Matched unrelated umbilical cord blood transplantation for a patient with chemotherapy resistant Wilms tumor.

We report a patient with chemotherapy refractory Wilms tumor who underwent an unrelated donor cord blood transplant for chemotherapy refractory disease. The preparative regimen consisted of busulfan, melphalan, and anti-thymocyte globulin, and was well tolerated. This patient did not experience significant toxicity related to the chemotherapy regimen and did not develop any graft versus host disease from his HLA (A, B, DR) 6/6 matched cord blood transplant. Follow-up CT scans 2 years post-transplant have shown no evidence of disease progression, with only a few pulmonary nodules remaining, which are unchanged in size from his pre-transplant CT scan. It is possible that high-dose chemotherapy and stem cell transplantation can be curative in patients with tumors that are non-responsive to conventional chemotherapy.

Fusion of B lymphoblastoid and tumor cells expressing different antibiotic resistance genes facilitates selection of stable hybridomas.

Abstract The development of tumor vaccines or generation of tumor-specific cytotoxic T lymphocytes (CTL) is limited by the fact that many tumor cells downregulate the expression of major histocompatibility complex (MHC) Class I and II molecules, as well as key co-stimulatory molecules such as CD80 and CD86. An immune response to a vaccine or in vitro stimulation of tumor-specific CTL requires antigen-presenting cells conveying tumor antigens in the context of a host's MHC antigens. We have used a retroviral vector (murine stem cell virus) encoding neomycin resistance to transduce three pediatric tumor cell lines (two neuroblastoma, one neuroepithelial tumor). An EBV transformed B lymphoblastoid cell line (BLCL) was transduced with a separate vector encoding puromycin resistance and green fluorescent protein, individual tumor lines were fused with the BLCL, and the resulting hybridomas were selected using both antibiotics. The resulting hybridoma cells expressed the neural antigen GD2 as well as MHC Class I, Class II, CD 80, and CD86. A similar strategy could be used to produce stable hybridomas for either vaccination or for CTL expansion.

Expansion of cytomegalovirus pp65 and IE-1 specific cytotoxic T lymphocytes for cytomegalovirus-specific immunotherapy following allogeneic stem cell transplantation.

Adoptive immunotherapy with antigen-specific cytotoxic T lymphocytes (CTLs) has proven effective in restoring cellular immunity to cytomegalovirus (CMV) and preventing viral reactivation after allogeneic stem cell transplantation (SCT). In an effort to develop a cost-effective, relatively rapid method of CMV CTL expansion, we investigated the use of a pool of overlapping CMV peptides. Because the possibility exists of vaccinating CMV-seronegative donors, and these individuals may have T cell responses predominantly against IE-1, commercially available peptide mixes for pp65 as well as IE-1 were used to stimulate CTLs from 10 seropositive donors. Of these 10 donors, 4 responded to pp65 only, 1 did not respond to either pp65 or IE-1, 4 responded to both pp65 and IE-1, and 1 responded to IE-1 only. These CMV- specific T cells included a mixture of CD4(+) and CD8(+) effectors, and specific cytotoxicity correlated with interferon-gamma production. The costs associated with a 28-day maintenance course of intravenous ganciclovir, cidofovir, foscarnet, and valganciclovir, as well as the preparation and shipping a single dose of CTLs, were determined. The price of generating CMV CTLs using this method was comparable to or less expensive than a 28-day maintenance course for these agents, not including the costs associated with drug administration, supportive care, and the treatment of drug-related complications. Considering the relative ease, low cost, and the fact that CTL administration can result in CMV-specific immune reconstitution, this option should be considered for patients with CMV reactivation or for prophylaxis in patients at high risk for infection.

Allogeneic stem cell transplantation in a patient with relapsed Ewing sarcoma.

Ewing sarcoma (ES) can express tumor antigens which can be recognized by T cells, making allogeneic stem cell transplant (SCT) a potential option for those patients with refractory disease. A 6-year old with multifocal ES developed a recurrence of pulmonary metastases and underwent an allogeneic bone marrow transplant from her human leukocyte antigen (HLA) 10/10 matched mother. During a taper of her immunosuppression, she developed grade 1 skin and oral graft versus host disease (GVHD). CT scans performed 9 months post-transplant revealed a marked decrease in the size of her pulmonary lesions compared to scans 2 months post-transplant. This case highlights the possibility of treating patients with refractory metastatic ES with allogeneic SCT.

Epstein Barr virus induced lymphoma in a child with IPEX syndrome.

IPEX syndrome (immune deficiency, polyendocrinopathy, enteropathy, X-linked) is a disorder or regulatory T cell (Treg) function which can result in early death due to infection or complications related to autoimmunity. Therapeutic options for these patients can include allogeneic stem cell transplantation (SCT) or the use of immunosuppressive regimens to control the manifestations of autoimmunity. We report a patient with IPEX syndrome who was managed with rapamycin and subsequently developed EBV induced lymphoma.

Interferon-gamma expressing EBV LMP2A-specific T cells for cellular immunotherapy.

To generate therapeutic T cells for adoptive immunotherapy, T cells specific to Epstein-Barr virus LMP2A were enriched on the basis of antigen-specific production of interferon-gamma (IFNgamma). The enriched T cells, contained over 60% LMP2A-specific effectors, were polyclonal and targeted multiple LMP2A epitopes. A high proportion of the enriched T cells produced the Th1 cytokines interleukin (IL)-2 and granulocyte monocyte colony stimulating factor, while few cells expressed the Th2 cytokines IL4 and IL10. The enriched T cells specifically lysed LMP2A-expressing target cells, with concomitant production of IFNgamma and surface expression of CD107, suggesting the involvement of the granule exocytosis-mediated cytolytic pathway. In addition, the enriched T cells expressed CD45RO, CD28 and CD27, but not CD45RA, consistent with a differentiation stage capable of self-renewal for long-term persistence. LMP2A-specific T cells enriched based on IFNgamma-production may provide improved efficacy for the treatment of Epstein-Barr virus related malignancy.

Rapid generation of CMV pp65-specific T cells for immunotherapy.

Adoptive immunotherapy with cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) has been shown to be an effective means of restoring cellular immunity to this virus and preventing CMV infection after allogeneic stem cell transplantation. Problems with current strategies include requirements for generating dendritic cells or other antigen presenting cells for stimulating CTL and the time needed for cell culture. The adherent cell fraction of peripheral blood mononuclear cells from 6 CMV seropositive donors were pulsed with pooled CMV pp65 peptides and incubated with nonadherent peripheral blood lymphocytes. CTL lacking specific cytotoxicity to pp65 were restimulated at day 10 of culture using peptide pulsed adherent cells. Of the 6 CMV seropositive donors tested, 5 had specific cytotoxicity to CMV pp65 (range 31% to 75%), with no alloreactivity. The resulting pp65-specific CTL consisted of a mixture of CD4 and CD8 cells, with 1% to 29% of CD8 cells and 0.5% to 10% CD4 cells making interferon-gamma (IFN-gamma) in response to pp65. The donor from whom we could not detect CMV-specific cytotoxicity had detectable CD4 and CD8 CMV pp65 CTL by intracellular cytokine analysis for IFN-gamma. Using this simplified strategy for expanding CMV pp65 CTL, adoptive immunotherapy with pp65-specific CTL could be made available in a more timely manner for patients who have persistent or therapy refractory CMV infections.

Immunotherapy with virus-specific cytotoxic T lymphocytes after organ transplantation.

Adoptive immunotherapy with antigen specific cytotoxic T lymphocytes can favorably impact the outcome of serious herpesvirus infections in organ transplant recipients. Current challenges in this field include the determination of which patients are at highest risk, and the development of protocols that permit more rapid expansion of virus specific effector cells.