ALBERT

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: Combination therapy with purine analogs, alkylators, and monoclonal antibodies has transformed the treatment paradigm in patients with CLL by dramatically enhancing both the quality and frequency of responses that can be achieved in these patients. However, combinations utilizing fludarabine as the purine analog have augmented myelosuppression and immunosuppression requiring careful attention to dosing and schedule in order to minimize these complications. Even with these precautions many patients are unable to complete the entire treatment program at full dose and for the planned number of cycles. Comparative experience with pentostatin indicates that it is less myelosuppressive than either fludarabine or cladribine. We previously reported our experience with pentostatin and cyclophosphamide. Subsequently, we have added rituximab to this active combination (PCR regimen) and treated a second cohort of 46 patients with previously treated CLL (32 patients) and other low grade lymphoid neoplasms (14 patients). The PCR regimen consists of pentostatin 4mg/m2, cyclophosphamide 600mg/m2, and rituximab 375mg/m2 all given on a single day with anti-emetics, hydration, and careful monitoring of renal function. The treatment was administered every 3 weeks for a total of 6 treatments. Rituximab was not given during the first cycle to reduce the frequency and severity of infusion reactions. Filgrastim, sulfamethoxazole/trimethoprim, and acyclovir were administered prophylactically. The median age of the patients treated was 62 (range 44–80) and the median number of prior regimens was 2 (range 1–7). The overall frequency of response was 75% with 25% achieving a complete response, 3% a nodular response, and 47% a partial response. We have compared these results to the recently reported MD Anderson FCR regimen. In terms of pre-treatment characteristics the patient groups in both studies appear comparable with the exception of a higher proportion of high-risk patients treated with PCR (78%) compared to FCR (50%) (P=0.003). The response frequencies are virtually identical in both studies with responses seen 75% of PCR treated patients and 73% of FCR treated patients and CR achieved in 25% in both studies. In terms of toxicity, however, PCR compares favorably to FCR in the following categories: Grade 3/4 neutropenia PCR 53% vs FCR 81% (P=0.0007), thrombocytopenia PCR 16% vs FCR 34% (P=0.04), anemia PCR 9% vs FCR 24% (P=0.06), and grade 3/4 infections (including fever of unknown origin) PCR 28% vs FCR 47% (P=0.05). PCR also appeared to be better tolerated than FCR as indicated by the fraction of patients completing all planned cycles of chemotherapy at full dose 72% vs 38% (P=0.0004). An important caveat in these comparisons is that myeloid growth factor was routinely administered to patients on the PCR study but was not routinely administered to patients treated with FCR. In conclusion, PCR appears to be equivalent in activity to FCR but may be better tolerated and less toxic. These results indicate that a prospective randomized comparison is warranted.

Description: Peptides derived from BCR-ABL have been used to trigger immune responses in CML pts. The native peptides may have low immunogenicity because they bind with low affinity to the major histocompatibility complex (MHC) molecules. Synthetic peptides containing mutations in the sequences of these peptides can bind with higher affinity than parental peptides and may generate a cross-reactive T cell response to the native sequence (known as a heteroclitic response). We used a mixture of heteroclitic and native peptides derived from both b3a2 and b2a2 sequences in a pilot study to vaccinate pts with CML in complete cytogenetic remission on imatinib (IM) therapy with stable bcr-abl transcript levels. Montanide ISA51 and GM-CSF were used as immunological adjuvants. Pts were required to have received IM for ≥12 mo with no change in dose for at least 6 mo and were not allowed any dose increases after registration. Pts were vaccinated with the peptides corresponding to their BCR-ABL breakpoint as follows: every 2 wks x4, then once 3 wks later, followed by 10 monthly vaccinations for a total of 15 vaccinations in 12 mo. After registration, pts had 3 additional measures of bcr-abl transcripts to better define baseline values. 8 pts have been vaccinated. At enrollment, pts had a median age of 45 yrs (range 29–63) and had been receiving IM for a median of 63 mo (range 35–68 mo) on a median dose of 600mg (range 300–800mg). Pts have been followed for a median of 15 wks (range 2–20) and have received a median of 7 vaccinations (range 1 to 8). To determine the effect of vaccination on immune leukocytes, we measured peripheral blood T-cell subsets, B, NK, and dendritic cells by FACS at baseline and at 3-month intervals thereafter. The percentage of myeloid DC (DC1) and plasmacytoid DC (DC2) cells were identified by the mutually exclusive expression of CD11c and CD123; Treg cells by the surface co-expression of CD3, CD4 and CD25 together with cytoplasmic expression of CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and FoxP3 (forkhead/winged helix transcription factor). Vaccinations have been well tolerated. The only adverse event attributable to the vaccine is local site reactions grade 1 or 2 in 5 pts. Among the 5 pts who have had an evaluation at 3 mo from the 1st vaccination, 1 has achieved a major molecular response (bcr-abl/abl

Description: Abstract 3054 Poster Board II-1030 Cancer therapies targeting tumor endothelium have shown promising results when combined with chemotherapeutics. Vascular endothelial (VE) cadherin is an endothelial cell specific molecule that is expressed constitutively throughout the vasculature and participates in the formation of adherens junctions between adjacent endothelial cells. The monoclonal antibody E4G10 specifically binds to a cryptic epitope exposed only on the monomeric unengaged form of VE-Cadherin found in neovasculature. We chemically conjugated E4G10 with the cytotoxic, short-range αa-particle emitter, 225Ac, and show that the agent dampens tumor growth as a single agent and this effect is enhanced when given in combination with chemotherapy in a xenograft mouse model. The therapeutic efficacy of the combination therapy is sequence dependent and most pronounced when 225Ac-E4G10 is administered prior to the chemotherapy. Immunohistochemical and immunofluorescence studies for endothelial cells (Meca32), vascular basement membrane (Collagen IV), and pericytes (alpha-smooth muscle actin (alpha-SMA), nerve/glial antigen 2 (NG2) ) showed that the vasculature of 225Ac-E4G10 treated tumors was depleted and the remaining vessels appeared normalized as evidenced by morphological changes, increased pericyte density and coverage. Fluorescence studies with Hoechst 33342 dye i.v. injected showed more homogenous distribution of the dye within 225Ac-E4G10 treated tumors, suggesting improved perfusion, and biodistribution studies with i.v. injected 111In-Diethylene triamine pentaacetic acid (DTPA), a freely diffusible small molecule, showed significantly (p=0.03) increased passive tumor accumulation of 111In-DTPA in 225Ac-E4G10-treated tumors when compared to controls. Quantitative autoradiographic studies on tumor sections also showed both stronger and more homogenous signal distribution in 225Ac-E4G10-treated tumors. These results show that 225Ac-E4G10 treatment leads to ablation and remodeling of the tumor vasculature allowing improved delivery of subsequent small molecules, such as chemotherapeutics, and thereby improves therapeutic outcome. Disclosures McDevitt: Actinium Pharma Inc.: Patents & Royalties. Scheinberg:Actinium Pharma Inc.: Patents & Royalties.

Description: Wilms tumor protein (WT1) is a transcription factor selectively over expressed in several types of leukemia and solid tumors, making it a promising potential target antigen for immunotherapy. Several open clinical trials use native or altered peptide sequences derived from the WT1 protein in order to overcome the weak immunogenicity of the self-antigen. Here we report a new strategy to circumvent tolerance by designing peptides that incorporate non-natural amino acids into the native sequence of WT1 peptides. Starting from the nonamer sequences WT1 187–195 and WT1 235–243, eight peptides containing natural amino acids and nine peptides in which different chemical modifications (fluorination, photo-reactive azido groups or benzophenone groups) were introduced at major histocompatibility complex (MHC) and T cell receptor binding positions, were synthesized. The new non-natural peptides could stabilize MHC class I molecules better than the native sequences and were also able to elicit strong specific T-cell responses. Photo-reactive peptides were additionally modified with biotin handles to allow streptavidin-biotin pull down and western blot analysis of kinetics of binding and catabolism. Upon UV irradiation, these peptides covalently bound to MHC molecules on the live cells; clearance of the peptide-MHC covalent complex occurred over 24 hours, consistent with the T2 thermo-stabilization data for the same peptide. Further catabolic studies may elucidate the important or novel cellular proteins involved in antigenic peptide processing and cross presentation and should aid in vaccine development. We are investigating whether covalent interaction with the MHC may lead to alterations in immune responses as well. T cells stimulated with one of the synthetic peptides (WT1J-W4WF) cross-reacted with the native WT1J sequence and were able to kill WT1 positive HLA-A0201 matched acute lymphoblastic leukemia cell lines. In conclusion, this study shows that peptides with non-natural amino acids can be successfully incorporated into T cell epitopes to provide increased immunogenicity and novel biological information.

Description: HuM195, a humanized anti-CD33 monoclonal antibody, targets myeloid leukemia cells and has single-agent activity against acute myeloid leukemia (AML). 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 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 against cytoreduced disease, we treated 31 patients (median age, 67 years; range, 37–80) with cytarabine 200 mg/m2/day for 5 days followed by 213Bi-HuM195 in a phase I/II trial. Thirteen patients had untreated AML (5 with de novo AML; 8 with secondary AML). Eight patients had AML in untreated first relapse, and ten patients had heavily pretreated relapsed AML (n=7) or primary refractory AML (n=3). Nine patients had poor-risk cytogenetics. During the phase I portion of the study, cohorts of 3–6 patients were treated with 18.5, 27.75, 37, and 46.25 MBq/kg. Prolonged myelosuppression with grade 4 leukopenia 〉 35 days was the most common dose-limiting toxicity. The maximum tolerated dose (MTD) was 37 MBq/kg. Extramedullary toxicity was primarily limited to ≤ grade 2 events, including infusion-related reactions; however, grade 3/4 liver function abnormalities were seen in four patients (13%). Treatment-related deaths occurred in two of 21 patients (10%) who received the MTD. Significant reductions in marrow blasts were seen at all dose levels, and clinical responses were observed in 6 of the 25 patients (24%) who received doses of at least 37 MBq/kg (2 CR, 2 CRp, 2 PR). The median response duration was 7.7 months (range, 2–12). Pharmacokinetic and biodistribution studies suggested that saturation of all available CD33 sites by 213Bi-HuM195 was possible after cytoreduction with cytarabine. Sequential administration of cytarabine and 213Bi-HuM195 is tolerable and can produce complete remissions in patients with AML.