ALBERT

Description: Key Points This study shows the effective anticancer activity of a T-cell receptor mimic antibody targeting WT1 in resistant human Ph+ ALL. In combination with tyrosine kinase inhibitors, ESKM can result in cure of Ph+ ALL in murine models.

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: HuM195, a humanized anti-CD33 monoclonal antibody, targets myeloid leukemia cells and has activity against minimal residual disease. To enhance the potency of native HuM195 and avoid nonspecific cytotoxicity seen with β-emitting radioimmunoconjugates, the α-emitting radiometal 213Bi was conjugated to HuM195. The feasibility, safety, and antileukemic activity of therapy with 213Bi-HuM195 were shown in a phase I trial (Jurcic et al. Blood 2002). Because of the short-range (50–80 μm) and high linear energy transfer (8400 keV) of α particles, radioimmunotherapy with 213Bi is ideally suited for the treatment of residual disease. To determine the effects of 213Bi-HuM195 after partial cytoreduction with chemotherapy, we treated 25 patients (median age, 67 years; range, 49–80) with cytarabine 200 mg/m2/day for 5 days followed by 213Bi-HuM195 in a phase I/II trial. Fourteen patients had relapsed or primary refractory AML; 5 patients had previously untreated de novo AML, and 6 patients had untreated secondary AML. Sixteen patients had intermediate-risk cytogenetics, and 9 had poor-risk cytogenetics. During the phase I portion of the study, cohorts of 3–6 patients were treated with 0.5, 0.75, 1, and 1.25 mCi/kg. At the 1.25 mCi/kg dose level, 2 of the 4 patients had dose-limiting myelosuppression (grade 4 leukopenia lasting ≥ 35 days) and 1 patient died of progressive pneumonia. Therefore, the maximum tolerated dose was determined to be 1 mCi/kg. No responses were seen at the first two dose levels. Seven of the 19 patients (37%) who received 1 mCi/kg (n=15) or 1.25 mCi/kg (n=4) responded. There were 2 CRs lasting 9 and 12 months; 3 CRp (CR with incomplete platelet recovery) lasting 1, 2, and 6 months and 2 PRs lasting 3 and 8 months. The median time from initiation of chemotherapy to recovery of leukocyte counts was 34 days (range, 21–59 days). Delayed count recovery was attributed to persistent leukemia in 6 patients. Neutropenic fever occurred in all patients, and 19 patients had documented infections. At the phase II dose level, two of the 15 patients died of progressive infections, and one had grade 4 hyperbilirubinemia. The most common extramedullary toxicities were transient, low-grade elevations in liver function tests (n=19) and serum creatinine (n=11). Thirteen patients had infusion-related reactions following the first injection of 213Bi-HuM195, typically characterized by reversible grade 1 or 2 fever and/or chills. One patient had orthostatic hypotension and syncope after the first antibody infusion associated with concomitant bacteremia. Sequential administration of cytarabine and 213Bi-HuM195 is tolerable and can produce complete remissions in patients with AML.

Description: HuM195, a humanized anti-CD33 antibody, targets myeloid leukemia cells and has modest activity alone against AML. To increase the antibody’s potency and allow single cell killing, but avoid the nonspecific cytotoxicity associated with β-emitting isotopes, the α-emitter bismuth-213 (213Bi) was initially conjugated to HuM195. In phase I and II trials, 213Bi-HuM195 was capable of inducing remissions in AML after partial cytoreduction with cytarabine. Therapeutic applications of 213Bi, however, are limited by its 46-minute half-life. The isotope generator, 225Ac, a radiometal which yields 4 α-emitting isotopes and has a 10-day half life, can be conjugated to a variety of antibodies using the bifunctional chelate DOTA-SCN. 225Ac-containing immunoconjugates can kill in vitro at radioactivity doses 1000 times lower than 213Bi analogs and prolong survival of animals in several xenograft models (McDevitt et al. Science 2001). We are conducting a first-in-man phase I dose escalation trial to determine the safety, pharmacology, and biological activity of such an in vivo isotope generator using 225Ac-HuM195. Seven patients (median age, 61 years; range, 46–77) with relapsed (n=3) or refractory (n=4) AML were treated to date. Three had poor-risk cytogenetics. Patients received a single infusion of 225Ac-HuM195 at doses of 0.5 (n=3), 1 (n=3), or 2 μCi/kg (n=1). Total administered activities of 225Ac ranged from 23–170 μCi, and HuM195 doses ranged from 1–1.9 mg. No acute toxicities were seen. One of 2 patients evaluable for neutropenia developed an ANC 33% of BM blasts in 4 patients at 4 weeks following treatment. One patient had 3% bone marrow blasts after therapy. This is the first study to show that targeted therapy with an in vivo α-particle generator is feasible in humans. 225Ac-HuM195 appears safe and has antileukemic activity. Accrual to this trial continues.

Description: Neo-vascularization has been implicated in a number of inflammatory diseases as well as tumor growth. Both angiogenesis, the sprouting of resident tissue endothelial cells (ECs), and vasculogenesis, the recruitment of bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs), are thought to participate in neo-vascularization. EPCs have been implicated in tumor growth, however, the biologic significance of EPCs during inflammation is unclear. We studied neo-vascularization and the role of EPCs during inflammation in well-characterized murine models of graft-versus-host disease (GVHD). We found a significantly increased number of donor BM-derived EPCs in peripheral blood and BM in allogeneic bone marrow transplantation (allo-BMT) recipients with GVHD at different time points after BMT. We next quantified neo-vascularization during inflammation in GVHD target organs by immunofluorescence microscopy and by flow cytometry. We found significantly increased numbers of donor-derived ECs in the liver as well as a significantly higher vessel density in the liver, illeum and colon. We adoptively transferred selected GFP+ EPCs and observed incorporation into the neo-vasculature of the inflamed intestines (Fig. 1A) and liver during GVHD. Taken together, these data suggest that neovascularization during GVHD is due to vasculogenesis from donor EPCs. Next we used an antibody (E4G10) against the vascular endothelial adhesion molecule VE-cadherin, which recognizes a terminal epitope that is exposed on circulating EPCs, but is masked in the established vasculature, and found a significant reduction of EPCs in the peripheral blood and BM. We observed that depletion of EPCs was associated with a significant inhibition of donor BM-derived neo-vascularization in the liver, illeum and colon during GVHD. E4G10 treated recipients had significantly better survival and lower clinical GVHD scores in all tested models (B6BALB/c [1×106 T], B6B6D2F1 [1×106 T], B6B6D2F1 [2×106 T], B6B6D2F1 [3×106 T]). We found significantly reduced numbers of allo-reactive donor T cells in secondary lymphoid organs during GVHD, but no changes in the expression of activation markers and homing molecules, as a consequence of E4G10 administration. In blinded histopathological analyses we found significantly less GVHD and reduced numbers of tissue-infiltrating CD3+ T cells in the liver, illeum and colon in E4G10-treated allo-BMT recipients. To better emulate the clinical setting, we first assessed the role of EPCs in tumor growth in allo-BMT recipients. We transferred sorted GFP+ EPCs as well as renal carcinoma (RENCA) cells to BALB/c recipients and found that GFP+ EPCs were recruited to the neo-vasculature of lung metastases. We detected a significant inhibitory effect of E4G10 administration on tumor growth, as determined by in vivo bioluminescence imaging, in both tumors tested (RENCA, A20 lymphoma) as well as a significant survival prolongation in tumor-bearing mice that were treated with E4G10 in the RENCA and C1498 (AML) model. Finally, we performed experiments in which tumor-bearing allo-BMT recipients received allogeneic T cells, which mediate the favorable graft-versus-tumor (GVT) effect but also cause inflammation in GVHD target organs. We found that administration of E4G10 led to a significantly higher rate of tumor-free survival in all models (B6'BALB/c [1×105 B6 T and 2×105 RENCA], B10BR'B6 [1×105 B10BR T and 2×105 C1498], B6'BALB/c [2×105 B6 T and 5×105 A20]), which was due to both attenuation of GVHD as well as inhibition of tumor growth (Fig. 1B). We conclude that depletion of EPCs is a strategy to simultaneously ameliorate inflammatory disease and tumors, providing a new approach to improve therapeutic outcome of allogeneic hematopoietic stem cell transplantation. This study demonstrates the biological significance of EPCs for neo-vascularization during inflammation and identifies the specific targeting of EPCs to disrupt neo-vascularization as a novel therapeutic concept to decrease inflammation. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group.

Description: Imatinib mesylate is now standard treatment for chronic myelogenous leukemia. In spite of its high efficacy in controlling the disease, residual bcr-abl cells measured by RT-PCR are still detectable in more than 95% of the patients treated with this drug. In addition, recent reports describe the emergence of resistance to Imatinib mesylate. A major mechanism for the appearance of resistance, are point mutations changing amino acids in the bcr-abl tyrosine kinase that make imatinib ineffective. We hypothesized that some bcr-abl kinase mutations selected by imatinib would generate new immunogenic peptide-epitopes that could be recognized by the human immune system. For this purpose, we selected with predictive computer algorithms potentially immunogenic mutated amino acid sequences and synthetic analogues of mutated bcr-abl kinase sequences to study for immunogenicity and anti-leukemia activity in an in vitro human system. Initially we designed a number of synthetic peptides derived from the most frequent bcr-abl kinase mutations induced by imatinib in which single amino acid substitution was introduced at different MHC-binding positions. We investigated the most common mutations: E255V, E255K, Y253H, Y253F, F311L, T315I, M351T and H396P. Specific single and combined mutations improved the predicted binding of peptides to HLA A0201. Further additional single amino-acid substitutions on mutated peptides generated high predicted binding. Selected sequences were evaluated for eliciting MHC-restricted, peptide-specific CTL responses in an in vitro model using T-cells from different HLA A0201 healthy donors after stimulation with monocyte-derived DC. CTL lines were assessed by either IFN gamma ELISPOT or by a chromium51 release assay. Mutated derived peptides from E255K, E255V, Y253F and T315I were shown to be immunogenic in vitro by the specific release of IFN gamma. Furthermore, single amino-acid substitutions in key residues improved the immunogenicity of these peptides. Finally, T cell lines generated with optimized peptides derived from E255K and T315I (the most common imatinib-induced mutations) were able to kill peptide pulsed targets; more important, these T cell lines cross-reacted with the mutated peptides (a heteroclitic response). In conclusion, Imatinib-selected mutations of peptides from bcr-abl kinase have a better predicted binding than native kinase sequences. Selected peptides from the most common mutations are immunogenic in vitro in a HLA A0201 restricted manner. T cells generated with optimized peptides from these mutations are able to elicit heteroclitic peptide-specific cytotoxicity.

Description: The t(15;17) translocation in acute promyelocytic leukemia (APL) yields a PML/RAR-α fusion messenger RNA species that can be detected by reverse transcription–polymerase chain reaction (RT-PCR) amplification. Breakpoints within intron 3 of PML produce a short PML/RAR-α isoform, whereas breakpoints within intron 6 result in a longer form. Using RT-PCR, serial evaluations were performed on the bone marrow of 82 patients with APL (median follow-up, 〉 63 months) who received retinoic acid (RA) induction followed by postremission treatment with chemotherapy, RA, and biologic agents. Sixty-four patients attained a clinical complete remission and had at least 2 RT-PCR assays performed after completing therapy. Forty of 47 patients (85%) with newly diagnosed APL who were induced using RA had residual disease detectable by RT-PCR before additional therapy. After 3 cycles of consolidation therapy, residual disease was found in only 4 of 40 evaluable patients (10%). Among newly diagnosed patients who had 2 or more negative RT-PCR assays, only 3 of 41 (7%) had a relapse, whereas all 4 patients (100%) who had 2 or more positive results had a relapse. Among 63 newly diagnosed patients, those who expressed the short isoform appeared to have shorter disease-free and overall survival durations than patients who expressed the long isoform. These data indicate that 2 or more negative RT-PCR assays on bone marrow, performed at least 1 month apart after completing therapy, are strongly associated with long-term remissions. Conversely, a confirmed positive test is highly predictive of relapse.

Description: Abstract 2599 The Wilms' tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in leukemias and a wide range of cancers. WT1 may be expressed in leukemia stem cells. RMFPNAPYL, “RMF”, a WT1-derived CD8 T cell epitope presented by HLA-A0201, is a validated target for T cell-based immunotherapy, used in multiple clinical trials of vaccines and cellular therapies. Therefore, we hypothesized that a “TCR-like antibody” specific for the WT1 peptide/HLA-A0201 complex might be an effective therapeutic agent. Using phage display technology, we generated 2 lead, high avidity (Kd 〈 0.2nM), fully human monoclonal antibodies (mAb) specific for the WT1 RMF peptide/HLA-A0201 complex. One version, ESK1, is a native human IgG1. A second version, ESKM, with enhanced antibody dependent human effector cell cytotoxicity (ADCC) function due to altered Fc glycolsylation was also prepared. ESK mAb bind to leukemia lines and other cancer cell lines, as well as primary leukemia cells that are both WT1+ and HLA-A0201+. In vitro, both ESK mAb mediated ADCC against CML cells, at concentrations below 3 ug/ml, but ESKM was about 8–10 fold more potent and could kill targets with far fewer peptide/MHC complexes expressed on the cell surface. At therapeutic doses of ESKM, there was no difference in biodistribution between the 2 mAb in C57BL6 mice or in mice that were transgenic for HLA-A0201. Low doses of ESK (25–100ug twice) effectively treated an established disseminated, ALL or human bcr/abl + lymphoid leukemias in a NSG mouse model (T, B and NK deficient) and prolonged survival. In mice, ESKM was slightly more effective than ESK1. An F(Ab')2 version of the antibody had no anti-tumor effect, indicating that an Fc-mediated mechanism plays the major role in therapy. When combined with a single infusion of human CD34-, CD3-, human NK and monocyte effectors in the NSG mice, therapeutic effects of the mAb were more pronounced and more durable. There was no therapeutic effect of either mAb on WT1 low/A0201 negative disseminated Daudi ALL in mice. There was no observed toxicity in HLA-A0201 transgenic mice at the therapeutic mAb dose and schedule. ESK mAb are potential therapeutic agents for ALL, CML, other leukemias and cancers over-expressing the WT1 oncoprotein. Its expression in early leukemia cells may allow for elimination of the progenitors. The data also provide proof of concept for developing therapeutic mAb targeting important intracellular oncogenic proteins. Disclosures: Yan: Eureka: Employment, Equity Ownership. Liu:Eureka: Employment, Equity Ownership.

Description: Abstract 2482 Despite the advent of prognostic factors such as cytogenetics and IGH mutational status, the natural history of chronic lymphocytic leukemia (CLL) remains heterogenous and difficult to predict in individual patients. We used the carbocyanine dye, JC-1, to investigate mitochondrial membrane potential in patients with untreated, predominantly early stage CLL. We found that JC-1 staining separated CD5+/CD19+ CLL cells into two discrete populations, Low Red (R)/Green (G) and High R/G, which mainly differed with regard to JC-1 green fluorescence (530 nm) in all patients (Fig. 1). In evaluating CLL patients, we found substantial variation in the proportion of cells in the two populations, with a range of 1.1% to 96.7% (median=79.9%) in the High R/G population. CD19+ B-cells from normal individuals typically had few cells with high JC-1 red fluorescence and had a JC-1 staining pattern that was distinct from CLL patients. Electron microscopy revealed disorganized mitochondrial christae, particularly in patients with elevated High R/G cell populations, compared with mononuclear cells from normal donors. Consistent with the morphologic disorganization, we found that the two populations of CLL cells had differential survival in culture with pyruvate and glucose as primary energy sources; cells in the High R/G population had inferior survival (p=0.016), while cells in the Low R/G population had equal survival in pyruvate compared with glucose (Fig. 2). This result indicates that the CLL clonal cell population can be subdivided into cells able to use oxidative phosphorylation and those dependent on aerobic glycolysis fro metabolic energy needs. Interestingly, patient samples with High R/G cell populations 〉90% had better cell survival in glucose-containing medium than samples derived from patients with 〈 75% High R/G cells (p=0.0085). We found that the mononuclear cells from patients with CLL expressed the M2 isoform of pyruvate kinase, which has been linked to the regulated utilization of aerobic glycolysis. We assessed the outcome of 100 patients with untreated CLL with more than 1 month of follow up (median 36 months) after measuring the proportion of cells in the mononuclear fraction of the peripheral blood with in the High R/G population. The clinical characteristics of the patients at the time of JC-1 staining are characteristic of patients with early stage, untreated CLL. The population was divided into approximate quartiles based on the percentage of mononuclear cells in the High R/G population, and progression-free survival (PFS) was estimated within each quartile. Patients with 50% or fewer cells in the High R/G gate appeared to have an indolent natural history; an estimated 88% (95% CI: 73%, 99%) of the patients had no evidence of significant clinical progression at two years. In contrast, patients with more than 90% of cells in the gate with High R/G cells had rapid progression with an estimated PFS of 50% (95% CI: 31%–82%) at two years (Fig. 3). Patients with 50–90% cells in the High R/G gate had an intermediate disease course. There was strong evidence of a difference in survival distributions across the four JC-1 categories (log-rank p-value 〈 0.001). The percentage of cells in the High R/G gate provided prognostic information that was independent of good risk or poor risk cytogenetic changes. In summary, we found that the clonal, neoplastic cells in patients with CLL comprise discrete populations separated on their metabolic dependence on aerobic glycolysis and aerobic glycolysis. We also show that the percentage of cells utilizing glycolysis has a significant adverse impact on the natural history of patients with CLL. Aerobic glycolysis, the so-called Warburg effect, is a common and incompletely understood aspect of cancer biology. These data tie the acquisition of aerobic glycolysis to more aggressive cancer biology, identify an important new prognostic measure for CLL and could lead to the development of therapies that target metabolic differences in cancer cells. Disclosures: No relevant conflicts of interest to declare.