ALBERT

Description: A pilot study was undertaken to assess the safety, activity, and immunogenicity of a polyvalent Wilms tumor gene 1 (WT1) peptide vaccine in patients with acute myeloid leukemia in complete remission but with molecular evidence of WT1 transcript. Patients received 6 vaccinations with 4 WT1 peptides (200 μg each) plus immune adjuvants over 12 weeks. Immune responses were evaluated by delayed-type hypersensitivity, CD4+ T-cell proliferation, CD3+ T-cell interferon-γ release, and WT1 peptide tetramer staining. Of the 9 evaluable patients, 7 completed 6 vaccinations and WT1-specific T-cell responses were noted in 7 of 8 patients. Three patients who were HLA-A0201-positive showed significant increase in interferon-γ–secreting cells and frequency of WT1 tetramer-positive CD8+ T cells. Three patients developed a delayed hypersensitivity reaction after vaccination. Definite related toxicities were minimal. With a mean follow-up of 30 plus or minus 8 months after diagnosis, median disease-free survival has not been reached. These preliminary data suggest that this polyvalent WT1 peptide vaccine can be administered safely to patients with a resulting immune response. Further studies are needed to establish the role of vaccination as viable postremission therapy for acute myeloid leukemia. This study was registered at www.clinicaltrials.gov as #NCT00398138.

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: Despite profound T-cell immunodeficiency, most patients treated with chemotherapy do not succumb to infection. The basis for residual protective immunity in lymphopenic patients is not known. We prospectively measured T-cell numbers, thymopoiesis, and T-cell memory in 73 children undergoing a 2-year chemotherapy regimen for acute lymphoblastic leukemia (ALL) and compared them to an age-matched cohort of 805 healthy children. Most patients had profound defects in CD4 and CD8 T-cell numbers at diagnosis that did not recover during the 2 years of therapy. Thymic output and the fraction of naive T cells were significantly lower than in healthy controls. However, the remaining T-cell compartment was enriched for antigen-experienced, memory T cells defined both by phenotype and by function. This relative sparing of T-cell memory may, in part, account for the maintenance of protective immunity in lymphopenic patients treated for ALL. Moreover, because the memory T-cell compartment is least affected by ALL and its treatment, strategies to induce immunity to pathogens or tumor antigens in cancer patients may be most successful if they seek to expand pre-existing memory T cells. (Blood. 2005; 106:1749-1754)

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: Although highly responsive, advanced stage follicular lymphoma (FL) is not curable with conventional treatment. This relative resistance is thought to be due to the t(14;18) that results in the constitutive overexpression of the death-inhibiting protein bcl-2. However, the observation that FL cells are sensitive to treatment in vivo and prone to apoptosis on in vitro culture questions whether bcl-2 alone is responsible for the pathogenesis and clinical behavior of this disease. Therefore, multiple genes are likely to be involved in both the lymphomagenesis and the clinical course of FL. We examined whether expression of other bcl-2 family genes might also be operative. Here, we show that FL cells display a different pattern of expression of bcl-2 family proteins from normal germinal center (GC) B cells that are thought to be their normal counterpart. FL cells express the death-suppressor proteins bcl-2, bcl-xL, and mcl-1; whereas GC B cells express bcl-xL and mcl-1 but also the proapoptotic proteins bax-α and bad. Although maintaining constitutive levels of bcl-2 and mcl-1, FL cells are not protected from apoptosis when cultured in vitro. Their propensity to undergo apoptosis is temporally associated with downregulation of bcl-xL. More importantly, activation of FL cells via CD40 not only prevents downregulation but increases the level of bcl-xL expression and results in promotion of survival. These results support the hypothesis that the overexpression of bcl-2 is not the only antiapoptotic mechanism responsible for the pathogenesis of FL. Survival of FL cells is determined by a number of death-inhibiting proteins, among which bcl-xL appears to have the most critical role. Moreover, these findings are consistent with the hypothesis that, although FL cells are malignant, they respond to microenvironmental signals such as CD40L that appear to contribute to their survival through the upregulation of death-inhibiting proteins.

Description: Follicular lymphomas (FLs) rarely induce clinically significant T-cell–mediated responses. We showed that freshly isolated tumor infiltrating T cells (T-TILs) lack tumor-specific cytotoxicity. Stimulation of these T cells with FL cells in the presence of interleukin-2 (IL-2) and/or costimulation via CD28 does not lead to T-cell activation and expansion. In contrast, when stimulated with FL cells preactivated via CD40, autologous T-TILs can be expanded by the addition of exogenous IL-2. These T cells can be further expanded in vitro by the addition of exogenous IL-4, IL-7, or interferon-γ, but not IL-12. Once activated, these T cells showed FL-directed cytotoxicity in four of five patients tested. We concluded that autologous cytotoxic anti-FL–specific T cells exist, but can only be detected in vitro under optimized conditions for T-cell stimulation and expansion. This suggests that their frequency in vivo is either very low or that the microenvironment does not provide the necessary signals to activate these T cells. This model system allows dissection of the requisite conditions for activation and expansion of lymphoma-directed cytotoxicity and may permit expansion of previously activated cytotoxic T cells for adoptive transfer.

Description: Recent studies have documented an increased risk of therapy-related myelodysplastic syndrome or acute myelogenous leukemia (t-MDS/AML) after autologous bone marrow transplant (ABMT) for non-Hodgkin's lymphoma (NHL). To develop methods to identify patients at risk for this complication, we have investigated the predictive value of clonal bone marrow (BM) hematopoiesis for the development of t-MDS/AML, as defined by an X-inactivation based clonality assay at the human androgen receptor locus (HUMARA), in a group of patients undergoing ABMT for NHL from a single institution (Dana-Farber Cancer Institute, Boston, MA). One hundred four female patients were analyzed. At the time of ABMT, the prevalence of polyclonal hematopoiesis was 77% (80/104), of skewed X-inactivation pattern (XIP) was 20% (21/104), and of clonal hematopoiesis was 3% (3/104). To determine the predictive value of clonality for the development of t-MDS/AML, a subgroup of 78 patients with at least 18 months follow-up was analyzed. As defined by the HUMARA assay, 53 of 78 patients had persistent polyclonal hematopoiesis, 15 of 78 had skewed XIP, and 10 of 78 (13.5%) either had clonal hematopoiesis at the time of ABMT or developed clonal hematopoiesis after ABMT. t-MDS/AML developed in 2 of 53 patients with polyclonal hematopoiesis and in 4 of 10 with clonal hematopoiesis. We conclude that a significant proportion of patients have clonal hematopoiesis at the time of ABMT and that clonal hematopoiesis, as detected by the HUMARA assay, is predictive of the development of t-MDS/AML (P = .004).

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: Poor and delayed immune reconstitution remains a major stumbling block to successful SCT especially when alternative donors are used. Strategies to selectively remove or inactivate alloreactive cells while leaving the other donor T cell repertoire intact might address this problem. A functional T cell response requires an antigen (Ag)-specific MHC-restricted signal (signal 1) to the T cell receptor (TCR) by an Ag presenting cell (APC) as well as a second, Ag independent costimulatory signal (signal 2) provided in large part by B7 family members on APC to CD28 on T cells. Without signal 2, T cells develop tolerance to the specific Ag. Costimulation can be blocked by either CTLA4-Ig, a fusion of Ig with human CTLA4 (the T cell high affinity B7 ligand) or a combination of humanized IgG2 isotype mutated monoclonal antibodies to the APC molecules B7-1 and B7-2. In 2 pilot studies of patients (pts) undergoing haploidentical SCT, donor T cell replete BM was incubated ex vivo with recipient irradiated peripheral blood mononuclear cells with CTLA4-Ig (pilot 1) or anti-B7-1+anti-B7-2 (pilot 2) to induce alloAg specific tolerance. 19 pts age 7 mos-50 yrs (median 15 yrs) were enrolled on pilot 1 and 5 aged 4–12 (median 6) on pilot 2. 3 pts had congenital BM failure. 21 pts with malignancy, ALL (11), AML(7), NHL(2), MDS(1), were 〉CR1and 14/21 had progressive disease (PD). Pts received TBI based ablative conditioning. Pts received a median of 3.3x106/kg CD34+ cells (0.5–12.3) containing a median of 2.8x 107/kg CD3+ (0.7–6.8), 1.6x 107/kg CD4+ (0.4–4.1), and 1x107/kg CD8+ (0.2–3.7) T cells. One pt got additional anergized cells for slow recovery and engrafted fully. One AML pt had autologous persistence and graft failure (GF). Evaluable pts engrafted at median 21 d (range, 13–29) with full donor chimerism. Of the 21 evaluable pts, 9 (43%) had findings consistent with acute GVHD graded B (n=4), C (n=4) and D (n=1) despite inconsistent pathology. GVHD symptoms were largely isolated to the GI tract and resolved with observation or moderate steroids. No death was attributable to GVHD. 11 pts died early of a combination of bacterial or fungal infection and/or regimen-related toxicity at a median of 35 d (8–159). Of the remaining 13 pts, the GF pt died after 2nd SCT elsewhere, 1 pt had sudden death d 176 at home and 2 pts with extramedullary AML died d 60 and 149 with PD. One T-ALL pt died of late PD d 1758. All BM failure and 3/14 transplanted with PD survive. All 8 survivors (8/19 〈 23 yrs) have 100% performance status at a median of 2423 d (1580–2875). None take medications or have chronic GVHD. 3 pts became CMV Ag + by d 100, (1 was transplanted with CMV), and responded to anti-viral therapy. Unlike many reported approaches to haploidentical SCT, aside from several CVL associated bacteremias, there have been no admissions for opportunistic infection and no late viral infections. All pts have good T cell counts, respond to vaccines and specific Ags and have good immunoglobulin levels. Costimulatory blockade, a method of limiting alloreactivity which leaves the remaining T cell repertoire intact, holds out promise as a method of overcoming alloreactivity while better preserving donor immune function and preserving anti-tumor activity. A new study combining costimulatory blockade and megadose stem cell SCT has been initiated.