ALBERT

Description: Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder of early childhood. Although allogeneic stem cell transplantation can induce long-term remissions, relapse rates remain high, and innovative approaches are needed. Since donor lymphocyte infusion in JMML is efficacious, T cell mediated immunotherapy may be effective, and appropriate antigenic targets must be identified. One candidate tumor-associated antigen for the immunotherapy of JMML is γ-globin, which is expressed at high levels in most JMML patients. Most clonogenic JMML cells constitutively express this onco-fetal protein, which is not necessary for the normal erythropoesis of children and adults. To determine whether γ-globin can serve as a target for immunotherapy in JMML, we sought to determine whether γ-globin is naturally processed and presented by the HLA complex. Using conventional bioinformatic techniques and the T2 binding assay to predict candidate epitopes, we identified 4 γ-globin derived peptides (g031, g071, g105, and g106) that were predicted to bind to the HLA-A2 molecule in vitro. Since this strategy provides no evidence for which predicted epitopes are processed and presented by tumor cells in vivo, we employed a biochemical strategy to determine which peptides are naturally processed and presented. This step is critical in certifying that a candidate peptide epitope is an appropriate target for immunotherapy treatments. Using our K562-derived artificial APC (aAPC), an APC that expresses A2 and no other HLA allele, we introduced the EGFP-γ-globin fusion gene. We then acid stripped peptides directly from the surface of one billion aAPC/EGFP-γ-globin cells without subjecting the cells to detergent mediated lysis. Peptides less than 5 kDa in size were fractionated by reverse phased HPLC analysis and analyzed by mass spectrometry. We identified two mass spectrometry peaks which corresponded to γ-globin derived peptides, g031 and g105. Of these, the identity of one peak, g105, was successfully confirmed by peptide sequencing, providing strong evidence that g105 is naturally processed and presented by aAPC/EGFP-γ-globin cells. Next, to confirm that g105 is processed and presented by primary JMML cells, we generated γ-globin specific CD8+ cytotoxic T cells (CTL) from A2 positive healthy donors using synthetic g105 peptide. γ-Globin specific CTL were able to specifically cytolyze A2+ γ-globin+ JMML cells but not A2+ γ-globin- JMML cells. Specific cytotoxicity was blocked by anti-A2 mAb but not isotype control. These results show for the first time that the γ-globin derived peptide, g105, can serve as a target epitope for the CTL directed immunotherapy of JMML. Furthermore, these results illustrate an innovative aAPC based strategy that can identify the antigenic peptide epitopes of putative tumor associated antigens that are naturally processed by tumor cells, presented via HLA class I, and can serve as targets for effective anti-cancer immunotherapy.

Description: In normal T-cell development interleukin-7 (IL-7) functions as an antiapoptotic factor by regulating bcl-2 expression in immature thymocytes and mature T cells. Similar to what occurs in normal immature thymocytes, prevention of spontaneous apoptosis by IL-7 in precursor T-cell acute lymphoblastic leukemia (T-ALL) cells correlates with up-regulation of bcl-2. IL-7 is also implicated in leukemogenesis because IL-7 transgenic mice develop lymphoid malignancies, suggesting that IL-7 may regulate the generation and expansion of malignant cells. This study shows that in the presence of IL-7, T-ALL cells not only up-regulated bcl-2 expression and escaped apoptosis but also progressed in the cell cycle, resulting in sequential induction of cyclin D2 and cyclin A. Down-regulation of p27kip1 was mandatory for IL-7–mediated cell cycle progression and temporally coincided with activation of cyclin-dependent kinase (cdk)4 and cdk2 and hyperphosphorylation of Rb. Strikingly, forced expression of p27kip1 in T-ALL cells not only prevented cell cycle progression but also reversed IL-7–mediated up-regulation of bcl-2 and promotion of viability. These results show for the first time that a causative link between IL-7–mediated proliferation and p27kip1 down-regulation exists in malignant T cells. Moreover, these results suggest that p27kip1 may function as a tumor suppressor gene not only because it is a negative regulator of cell cycle progression but also because it is associated with induction of apoptosis of primary malignant cells.

Description: Generating antigen specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy requires the use of antigen presenting cells (APCs) such as dendritic cells, activated B cells, and/or artificial APC engineered to express immunoaccessory molecules. Although clearly useful, each approach presents certain drawbacks preventing their widespread application to the treatment of minimal residual disease in cancer and the treatment and prophylaxis of infectious disease. For instance, generating autologous APC for each patient is costly and is dependent on a limited supply of patient material. Most artificial APC approaches, while bypassing the individualized preparation of APC, are limited by the requirement to produce clinical grade HLA/peptide tetramers, monoclonal antibodies, polystyrene beads, and/or non-human cell lines. To circumvent all these issues, we have developed an immortalized APC line that can uniquely support the priming and prolonged expansion of antigen specific T cells through the engagement of TCR, CD28, and CD83 ligand. Co-engagement of CD28 and CD83 ligand preferentially enriches and significantly amplifies the number of antigen specific T cells, obviating the necessity to perform any tetramer sorting procedures. Furthermore, we are consistently successful in generating immunity to a wide array of peptide epitopes derived from antigens such as MART1, NY-ESO-1, Her-2/neu, influenza virus, telomerase, and HIV. Since this strategy can provide an unlimited APC source that can be used to prime, expand and select for antigen specific CTLs, we have generated a GMP grade APC for the treatment of patients. Our CASE (clinical grade antigen specific expansion) line was produced by simultaneously transfecting K562 with linearized plasmids encoding the HLA-A*0201 (A2), CD80, CD83, and puromycin resistance genes using a lipofection method. Following drug selection and cloning by limiting dilution, 5 CASE clones were then studied for their ability to induce expansion of antigenic peptide specific CTL, using MART1 as a model antigen. HLA-A2+ CD8 T cells were stimulated with irradiated, peptide pulsed CASE clones, and the percentage of tetramer staining T cells was determined by flow cytometry. The stable CASE clone, #33, was found to consistently generate the highest number of antigen specific T cells. Using the CASE33 clone, we successfully generated several CTL lines, succeeding in 8/8 cases. The generated CTL lines achieved several logs of antigen specific expansion, with tetramer staining up to 57%, and function has been demonstrated by high antigen specific killing and gamma-interferon secretion. Both master and working cell banks for CASE33 have been established and have successfully passed extensive testing including in vitro cultures, testing for bovine adventitious agents, in vivo testing and PCR for viral pathogens, karyotype analysis, and analysis of cellular morphology and growth. The highly effective APC, CASE33, was generated in a relatively short timeframe, and the production of additional clinical grade APCs expressing HLA molecules such as A11, A24, and A30, would be technically straightforward. With such an APC bank, most patients encountered in the clinic could be treated using one or more of these APCs and the widespread testing of adoptive immunotherapy in cancer and infectious diseases will commence.

Description: High dose therapy and autologous stem cell transplantation (ASCT) has been employed as salvage therapy for patients (pts) with relapsed follicular non-Hodgkin’s lymphoma (FL). Recently, the randomized European CUP trial demonstrated that following 3 cycles of CHOP, ASCT significantly improved both disease free and overall survival when compared to continued CHOP in pts with relapsed FL. In an attempt to improve the results of high dose therapy in FL, we undertook 2 sequential studies for pts with FL in first remission. In the first study, 77 pts with previously untreated advanced stage FL (median age 43), received 6–8 cycles of standard dose CHOP (SD-CHOP) + involved field radiotherapy followed by high dose chemoradiotherapy and anti-B cell purged autologous bone marrow transplantation (ABMT). Following SD-CHOP induction, 27 of 77 pts (36%) were in CR and 50 in a minimal disease state (〈 2 cm masses, 〈 20% BM involvement). At BM harvest, 36 pts had histologic evidence of BM involvement. In the second trial, 19 pts with advanced stage disease (median age 44) were treated with 4 cycles of dose intensified CHOP (HD-CHOP) (cyclophosphamide 1.5 g/m2 day 1 and 2) with G-CSF support followed by high dose chemoradiotherapy and anti-B cell purged autologous ABMT. Following HD-CHOP, 13 of 19 pts were in CR. At BM harvest, 7 of the 19 HD-CHOP pts had histologic BM involvement. Following ABMT, there were 2 acute in-hospital deaths in the pts receiving SD-CHOP induction and none in the pts who received HD-CHOP induction. Nine late deaths in remission were observed in the SD-CHOP pts including 6 from MDS/AML, and 2 late deaths in the HD-CHOP pts. Thirty-three pts who received SD-CHOP remain alive without relapse, with a median follow up of 12 years. Ten pts who received HD-CHOP induction remain alive without relapse, with a median follow up of 9.1 years. For pts receiving SD-CHOP induction, the DFS and overall survival at 10 years are 42% (90% CI: 33%–52%) and 64% (90% CI: 55%–73%), respectively. For pts who received HD-CHOP induction, the DFS and overall survival at 10 years are 59% (90% CI: 38%–79%) and 75% (90% CI: 57%–93%), respectively. The impact of BM treatment and DFS was examined. Pts with known bcl-2 translocations for whom post-BM purging samples were available for pcr examination were analyzed. Following ABMT, pts who were reinfused with pcr negative BM had a significantly better DFS than the pts who were reinfused with pcr positive BM. This study suggests that a subset of pts with FL experience long term remission following ABMT in first remission.

Description: The presence of breast cancer cells in the patient’s bone marrow (BM) at diagnosis is associated with resistance to treatment, disease relapse and poor prognosis. Identification of the factors implicated in the homing, survival and latency of breast cancer cells in the BM should contribute to the design of more efficient therapeutic strategies for breast cancer. There is evidence that breast cancer can recruit endothelial progenitors from the BM. Also, other epithelial tumors seem to preferentially adhere to BM endothelial cells. Therefore, we hypothesized that BM endothelium may play a significant role in the biology of breast cancer cells residing in the BM. Co-cultures in Matrigel showed that breast cancer cells interact with BM endothelium to form heterotypic multicellular networks. Moreover, breast cancer cells migrate towards BM endothelium assembled as capillary-like structures, but not to structures of BM mesenchymal stem cells or BM stroma. This migration was abrogated by pertussis toxin-mediated blockade of chemokine receptor signaling, suggesting the involvement of endothelium-secreted chemokine(s). We then evaluated the impact of breast cancer cells in the survival and proliferation of BM endothelium. All breast cancer lines tested (n=4) promoted the proliferation of BM-derived endothelial cells. This effect is mediated through the engagement of the PI3K/Akt pathway (phosphorylation of Akt at Ser437 and Thr308, and activation of its downstream substrates GSK3β, PRAS-40 and FKHRL1) since its specific blockade abrogated the stimulatory effects of breast cancer on BM endothelium. We next determined whether, reciprocally, BM endothelium impacts on breast cancer cell survival. These experiments were performed in serum-free media to enhance dependency of breast cancer cells from microenvironmental stimuli. In all cases tested, BM endothelium promoted survival/proliferation of breast cancer cells. This stimulation was accompanied by the engagement of the PI3K/Akt pathway in breast cancer cells and, in three of the four lines, the phosphorylation of Erk1/2. These effects were also observed for breast cancer cells that showed constitutive activation of Akt (MCF-7 and ZR-75-1 cells). Specific blockade of PI3K/Akt abrogated the BM endothelium-promoted survival of breast cancer cells, thus demonstrating the critical role of this pathway. These studies show that crosstalk between BM endothelial cells and breast cancer cells may impact on the survival of both cell types. These findings provide new light on the mechanisms that may facilitate the development of a tumor-permissive BM microenvironment in breast cancer, and the creation of breast cancer-supporting BM niches. Importantly, this study implicates BM endothelium as a therapeutic target in breast cancer and suggest that blockade of PI3K/Akt may impact the outcome of patients with metastatic breast cancer.

Description: Targeted disruption of signaling pathways essential for tumor cell survival and proliferation offer great promise for the design of more efficient and specific therapies. mTOR plays a central role in sensing mitogenic, nutrient and energy signals, and in the integration of different signaling pathways. Moreover, increasing evidence implicates mTOR in tumorigenesis and in the maintenance of transformed phenotypes. Therefore, we hypothesized that the mTOR pathway may play a significant role in T-cell acute lymphoblastic leukemia (ALL). BM and peripheral blood specimens from 15 pediatric patients were analyzed for the phosphorylation status of two downstream substrates of mTOR, S6 Ribosomal Protein and eIF4G. In all cases, primary leukemia T cells showed marked phosphorylation of S6 Ribosomal Protein (Ser235/236) and phosphorylation of the eukaryotic initiation factor eIF4G (Ser1108), showing that the mTOR pathway is constitutively active in T-ALL cells. Since leukemia T cells did not show significant basal phosphorylation of Akt/PKB, this observation suggests that the mTOR activation observed in T-ALL is Akt-independent. Specific blockade of mTOR by Rapamycin (100nM) reduced or abrogated phosphorylation of S6 ribosomal protein. Moreover, the mTOR blockers Rapamycin (10–100nM) and CCI-779 (10–100nM) significantly inhibited cell survival, and IL-7-promoted proliferation (40% to 85% inhibition) and cell cycle progression of T-ALL cells. Since we have shown previously that PI3K/Akt is critically involved in T-ALL responses to exogenous stimuli, we next evaluated whether inhibition of PI3K/Akt signals would synergize or potentiate the effects of mTOR blockade. We observed that the PI3K-inhibitor LY294002 (10μM) synergized with Rapamycin to abrogate IL-7-promoted leukemia T-cell proliferation. Finally, we explored the possibility that mTOR blockade can enhance the cytotoxic effects on T-ALL of the conventional drugs Dexamethasone (100nM) and Doxorubicin (100nM). Rapamycin potentiated the cytotoxic effects of both Dexamethasone (1.3 to 10 fold) and Doxorubicin (1.7 to 7.3 fold), suggesting that the combination of mTOR blockade with chemotherapy can improve the anti-leukemia efficacy of conventional regimens. In conclusion, we observed that the mTOR pathway is constitutively activated in primary leukemia T cells, and that the mTOR blockers Rapamycin and CCI-779 significantly inhibit T-ALL survival and proliferation. In addition, Rapamycin potentiates the inhibitory activity of other signaling inhibitors and the cytotoxic effects of the conventional drugs Dexamethasone and Doxorubicin. This study suggests that the mTOR pathway is a valid target for the treatment of T-ALL and lay the groundwork for the inclusion of mTOR blockade in the current therapeutic regimens.

Description: Appropriate processing and presentation of tumor associated antigens (TAA) by antigen presenting cells (APC) is absolutely required for the development of clinically relevant anti-tumor T cell responses. One common approach which utilizes the exogenous pulsing of synthetic peptides onto APC can sometimes generate ineffective immune responses. This failure may, in part, be the consequence of erroneous conformations of HLA/synthetic pulsed peptides which can differ from conformations formed with HLA/endogenous peptides that are derived from intracellular proteins. Also, endogenously processed peptides sometimes undergo post-translational modifications during transport to the cell surface, a process that does not occur with exogenously loaded peptides. Since our goal is to induce immunity that can recognize TAA that are endogenously presented by tumors, it is logical that the ideal APC would not only express the required immunoaccessory molecules, but would also endogenously process and appropriately present target antigenic peptides. In this report, we employed our artificial APC (aAPC) that expresses HLA-A2, CD80, and CD83 and is capable of priming and supporting the prolonged expansion of peptide specific CD8+ cytotoxic T cells (CTL). We hypothesized that aAPC can endogenously process and present HLA class I peptides and can induce functional T cell immunity. To test this, aAPC was transduced with an EGFP-mini MP1 (aa 55–66) fusion gene containing the sequence for the influenza derived peptide MP58 (aa 58–66). We observed that this HLA-A2 restricted peptide is processed and presented by aAPC by demonstrating that MP58 specific CTL are able to recognize aAPC/mini MP1 target cells. This was completely abrogated by treating aAPC/mini MP1 with proteasome inhibitors, suggesting that MP58 is endogenously processed by the proteasome. This was confirmed by the elevation of EGFP-mini MP1 fusion protein and the accumulation of ubiquitinated forms as detected by flow cytometry and Western blot analysis, respectively. At the protein level, aAPC was shown to express all proteasome subunits examined and to upregulate immunoproeosome subunits with exposure to IFN-γ. We biochemically confirmed the presence of MP58 in the A2 groove on the surface of aAPC/mini MP1, by performing reverse phased HPLC, mass spectrometry and peptide sequencing of peptides directly acid stripped from the cell surface. Since aAPC expresses only one HLA allele, A2, this finding provides strong support that MP58 is processed and presented in the groove of the A2 molecule on aAPC/mini MP1. We next evaluated the density of MP58 presented by HLA-A2 on aAPC/mini MP1. A2 positive CD8+ T cells were stimulated at weekly intervals by either aAPC/mini MP1 or parental aAPC exogenously pulsed with graded concentrations of synthetic MP58. After three stimulations, peptide specificity of generated CTL was examined by tetramer analysis. The comparison of tetramer staining revealed that the density of endogenously processed and presented MP58 corresponded to pulsing aAPC with 100 μg/ml. In order to extend this strategy to a TAA, we transduced aAPC with EGFP-mini MART1 (aa 27–35) mini gene. We have demonstrated that MART1 peptide is processed and presented on the cell surface and have induced the expansion of MART1 peptide specific T cells. These results suggest that our APC can naturally process and present class I restricted peptides, resulting in the efficient priming and expansion of clinically relevant antigen specific CTL.

Description: Allogeneic hematopoietic progenitor cell transplantation (HPCT) remains an established curative approach for selected patients with chronic myelogenous leukemia (CML). Patients for whom an allogeneic donor cannot be found or who are unable to tolerate allotransplantation require post-Gleevec alternatives. Autologous HPCT causes less morbidity/mortality than allotransplantation but CML cells in the autograft pose a potential risk for post-transplant relapse. An effective purging method for eliminating leukemic cells without compromising normal reconstituting activity would make autotransplantation a more attractive strategy for selected CML patients. In previous studies, we and others have shown that primitive CML cells are selectively killed after brief treatments in vitro with Mafosfamide (Maf) or Gleevec. Additionally, cultured primitive CML cells differentiate much more rapidly than their normal counterparts resulting in a selective depletion of CML stem cells after 1–2 weeks. Here we report the development of a novel autograft purging strategy that combines the use of a brief exposure of CML cells to Gleevec plus Maf followed by extended culture without cytotoxic agents but with added recombinant cytokines that favor the maintenance of normal stem cells. Methods: CD34+ cells were isolated from the peripheral blood progenitor cell (PBPC) products of CML patients and normal donors, treated for 72 hrs with graded concentrations of Gleevec (0.5–1.0 uM) followed by 0, 30, 60 or 90 ug/ml of Maf for 30 min. Cells were then washed and cultured for 14 days in medium containing 10% FBS and 100 ng/ml each of stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF) and thrombopoietin (TPO). Following treatment, cells from each group were evaluated for total viable cells, BCR-ABL message by real time polymerase chain reaction (RT-PCR) assays and colony-forming unit-granulocyte macrophage (CFU-GM) content in methylcellulose assays. Results: BCR-ABL+ cells were eliminated in the CML PBPC products from patients in chronic phase (n=3), but not blast crisis (n=1) when treated with Gleevec (0.75 uM) and Maf (60 ug/ml) followed by 2 weeks in vitro with SCF+G-CSF+TPO. Residual Ph positive cells by RT PCR Patient/Disease stage Culture alone Drugs alone (Gleevec 0.75 or 1uM + Maf 60ug/ml) Culture + Drugs 1-chronic phase/Gleevec refractory Positive Positive Negative 2-chronic phase/Gleevec refractory Positive Positive Negative 3-chronic phase/Gleevec naïve Positive Negative Negative 4-blastic phase/Gleevec naïve Positive Positive Positive Treatment of CD34+ PBPCs from normal donors with 0.75 uM Gleevec + 60 ug/ml Maf reduced CFU-GMs to 15% of input controls immediately post drug-exposure but these recovered to 320% of input after the additional 2 weeks in culture. Conclusion: Triple purging of CML cells with Gleevec, Maf and 2 weeks of culture appears to eradicate chronic phase BCR-ABL+ cells while retaining most of the normal progenitor activity as assessed by in vitro clonogenic assays. This approach may be useful as a strategy for purging CML autografts for Gleevec-refractory patients and, pending confirmatory results from in vivo xenograft assays, will be tested in a clinical autotransplant trial.

Description: The use of tumor cells as vaccines in cancer immunotherapy is critically dependent on their capacity to initiate and amplify tumor-specific immunity. Optimal responses may require the modification of the tumor cells not only to increase their immunogenicity but also to improve their ability to recruit effector cells to the tumor sites or sites of tumor antigen exposure. It has been reported that CD40 cross-linking of acute lymphoblastic leukemia (ALL) cells significantly increases their immunogenicity and allows the generation and expansion of autologous antileukemia cytotoxic T lymphocytes. This study demonstrates that the CD40 ligation of these tumor cells also induces the secretion of the CC-chemokines MDC and TARC. Supernatants from malignant cells cultured in the presence of sCD40L promote the migration of activated T cells that express CCR4, the common specific receptor for MDC and TARC. More importantly, the supernatants from CD40-stimulated tumor cells also support the transendothelial migration of autologous CCR4+ antileukemia T cells. Therefore, the results demonstrate that the delivery to leukemia cells of a single physiologic signal, that is, CD40 cross-linking, simultaneously improves tumor cell immunogenicity and induces potent chemoattraction for T cells.

Description: Following the engagement of the T cell receptor by HLA class I and antigenic peptide, naïve CD8+ T cells are primed to receive one or more costimulatory signals. Some of these signals, which are upregulated on and delivered by mature dendritic cells, include members of the immunoglobulin superfamily such as CD80 and CD83. Using a K562 derived artificial antigen presenting cell (aAPC) that expresses HLA-A2, CD80, and CD83, we have shown that the coengagement of CD83 ligand:CD83 and CD28:CD80 induces prolonged and preferential expansion of antigen specific CD8+ T cells. Furthermore, we have found that CD28:CD80 signaling is required for the induction of CD83 ligand expression on peripheral T cells. In order to identify additional immunoaccessory molecules that can augment this response, we have developed a system to efficiently transfer any chosen molecule into aAPC. This provides an excellent platform for studying a potentially immunogenic molecule given the relative lack of immunoaccessory molecules expressed by K562 (i.e. no expression of CD40, CD40 ligand, CD83, CD86, 4-1BB, 4-1BB ligand, OX40, OX40 ligand, HLA class I, or HLA class II). Following the transduction of a candidate molecule under study, the stimulatory capacity of a supertransduced aAPC can be compared to parental aAPC. Attractive candidates include members of the TNF superfamily since they have been shown to deliver important costimulatory signals to T cells. It has been suggested that 4-1BB signaling supports the survival of newly generated effector CD8+ T cells and that CD40 signaling confers “CD4+ T cell-like” help directly to CD8+ T cells. However, the impact of each of these molecules on the stimulation and expansion of antigen specific T cells has not been exhaustively studied. In this report, we transfected aAPC with either 4-1BB ligand or CD40 ligand, allowing us to compare the stimulatory capacity of aAPC/CD40 ligand, aAPC/4-1BB ligand and parental aAPC. We stimulated HLA-A2 positive CD8+ T cells from healthy donors three times at weekly intervals with A2-restricted MART1 peptide pulsed onto either irradiated aAPC/CD40 ligand, aAPC/4-1BB ligand or parental aAPC. Between the stimulations, cells were treated with IL2 and IL15 every three days. When MART1 peptide pulsed aAPC/CD40 ligand were used as stimulators, the total number of CD8+ T cells and number of MART1 specific CD8+ T cells was slightly smaller. IFN-γ ELISPOT analysis revealed that functional avidity of T cell receptors on MART1 specific CD8+ T cells was similar whether they were stimulated by aAPC/CD40 ligand or parental aAPC. These results indicate that CD40 ligand, at least in the human setting, does not directly provide “CD4+ T cell-like” help to antigen-specific CD8+ T cells. In contrast, stimulation with peptide pulsed aAPC/4-1BB ligand did generate a larger total number of CD8+ T cells. Surprisingly, however, most of these T cells were not antigen specific. In fact, significantly fewer MART1 specific CD8+ T cells were generated by aAPC/4-1BB ligand compared to aAPC alone. These results suggest that, unlike CD80 and CD83, 4-1BB ligand delivers a costimulatory signal resulting in the non-specific expansion of CD8+ T cells. This work demonstrates the versatility of our system to dissect the function of particular immunoaccessory molecules and determine the optimal conditions in the stimulation and expansion of antigen-specific human CD8+ T cells ex vivo.