Search

Translation on this Article from EBV-Related Lymphoproliferative Disease Complicating Therapy with the Anti-CD2 Monoclonal Antibody, Siplizumab, in Patients with T-Cell Malignancies

Purpose: We report an increased incidence of EBV-induced B-cell lymphoproliferative disease (LPD) in patients treated with siplizumab, an anti-CD2 antibody. The development of EBV-LPD has been associated with the use of immunosuppressive agents used in solid organ, bone marrow, and stem cell transplantation and in certain congenital immunodeficiencies.Experimental Design: We conducted a single-institution phase I dose-escalation trial of siplizumab, a humanized monoclonal antibody to CD2, in 29 patients with T-cell malignancies.Results: Although initial responses were encouraging, 4 (13.7%) patients developed EBV-LPD and the trial was stopped. Reductions in CD4+ and CD8+ cell count numbers in response to therapy were seen in all patients, but in those patients developing EBV-LPD a significantly greater reduction in natural killer (NK) cell number and CD2 expression on T cells was seen. These findings highlight the importance of NK-cell depletion and CD2 expression in addition to T-cell depletion in the etiology of EBV-LPD.Conclusions: The emergence of EBV-LPD may be associated with the ability of siplizumab to deplete both T and NK cells without affecting B cells. Agents that deplete T- and NK-cell populations without affecting B cell number should be screened for this potentially serious adverse event.

Supplementary Tables S1-S3 from EBV-Related Lymphoproliferative Disease Complicating Therapy with the Anti-CD2 Monoclonal Antibody, Siplizumab, in Patients with T-Cell Malignancies

Neuroblastoma is the most common extracranial solid tumor in children and tumor ganglioside composition has been linked to its biological and clinical behavior. We recently found that high expression of complex gangliosides that are products of the enzyme GM1a/GD1b synthase predicts a more favorable outcome in human neuroblastoma, and others have shown that complex gangliosides such as GD1a inhibit metastasis of murine tumors. To determine how a switch from structurally simple to structurally complex ganglioside expression affects neuroblastoma cell behavior, we engineered IMR32 human neuroblastoma cells, which contain almost exclusively (89%) the simple gangliosides (SG) GM2, GD2, GM3, and GD3, to overexpress the complex gangliosides (CG) GM1, GD1a, GD1b and GT1b, by stable retroviral-mediated transduction of the cDNA encoding GM1a/GD1b synthase. This strikingly altered cellular ganglioside composition without affecting total ganglioside content: There was a 23-fold increase in the ratio of complex to simple gangliosides in GM1a/GD1b synthase-transduced cells (IMR32-CG) vs. wild type (IMR32) or vector-transfected (IMR32-V) cells with essentially no expression of the clinical neuroblastoma marker, GD2, confirming effectiveness of this molecular switch from simple to complex ganglioside synthesis. Probing for consequences of the switch, we found that among functional properties of IMR32-CG cells, cell migration was inhibited and Rho/Rac1 activities were altered, while proliferation kinetics and cell differentiation were unaffected. These findings further implicate cellular ganglioside composition in determining cell migration characteristics of tumor cells. This IMR32 model system should be useful in delineating the impact of ganglioside composition on tumor cell function.

Purpose: We report an increased incidence of EBV-induced B-cell lymphoproliferative disease (LPD) in patients treated with siplizumab, an anti-CD2 antibody. The development of EBV-LPD has been associated with the use of immunosuppressive agents used in solid organ, bone marrow, and stem cell transplantation and in certain congenital immunodeficiencies.Experimental Design: We conducted a single-institution phase I dose-escalation trial of siplizumab, a humanized monoclonal antibody to CD2, in 29 patients with T-cell malignancies.Results: Although initial responses were encouraging, 4 (13.7%) patients developed EBV-LPD and the trial was stopped. Reductions in CD4+ and CD8+ cell count numbers in response to therapy were seen in all patients, but in those patients developing EBV-LPD a significantly greater reduction in natural killer (NK) cell number and CD2 expression on T cells was seen. These findings highlight the importance of NK-cell depletion and CD2 expression in addition to T-cell depletion in the etiology of EBV-LPD.Conclusions: The emergence of EBV-LPD may be associated with the ability of siplizumab to deplete both T and NK cells without affecting B cells. Agents that deplete T- and NK-cell populations without affecting B cell number should be screened for this potentially serious adverse event.

This study assessed the safety, immunogenicity, and pharmacokinetics of etaracizumab, a monoclonal antibody directed against the alphavbeta3 integrin, in patients with advanced malignancies. Four cohorts of four patients received escalating dose of etaracizumab as a 30-min intravenous infusion, first as a single test dose, followed-up 2-5 weeks later by weekly doses. Sixteen patients with advanced solid tumors received a total of 309 cycles of etaracizumab at doses ranging 1-6 mg/kg. The mean number of weekly infusions was 19 (ranging 5-53). Frequently reported adverse events were grades 1-2 asthenia (15 patients) and infusion reactions (9 patients). At 1 mg/kg, one patient experienced grade 3 chills with the first infusion. Other grade 3 toxicities included reversible hyponatremia, hypophosphatemia and hyponatremia in one patient each at 1, 4 and 6 mg/kg, respectively. No patient experienced treatment delay/discontinuation due to an adverse event. The half-life of etaracizumab ranged 49-180 h with a nonlinear increase in terminal half-life with increasing doses. There was no objective response but five patients experienced a stable disease of6-month duration. Etaracizumab was well-tolerated at doses up to 6 mg/kg with no evidence of immunogenicity. The safety profile of etaracizumab warrants further exploration in ongoing phase I/II trials.

Low (or = 35%) or absent expression of the complex 'b' pathway gangliosides GD1b, GT1b and GQ1b (CbG) correlates with an aggressive biological phenotype in human neuroblastoma tumors. To develop an in vitro model to probe mechanisms by which CbG may contribute to neuroblastoma behavior, we have comprehensively evaluated ganglioside expression in nine well-established human neuroblastoma cell lines, all derived from poor prognosis tumors. Total cellular ganglioside content ranged from 8 to 69 nmol/10(8) cells. High performance thin layer chromatography revealed that the simple disialoganglioside GD2 was prominent in eight of the cell lines (up to 60% of total gangliosides), whereas CbG were low (1-21%) in all nine cell lines. The structurally most complex 'b' pathway species, GQ1b, was not detected in any of the cell lines. The prominence of GD2 in neuroblastoma cell lines mirrors the high expression of GD2 that characterizes human neuroblastoma tumors, and the low CbG expression in the cell lines is analogous to that found in clinically and biologically unfavorable neuroblastoma tumors, thus establishing these neuroblastoma cell lines as valuable model systems for study of the role of CbG in the pathobiology of human neuroblastoma.

Recent findings link increased expression of the structurally complex 'b' pathway gangliosides GD1b, GT1b, GQ1b (CbG) to a favourable clinical and biological behaviour in human neuroblastoma (NB). Seeking a model to probe these observations, we evaluated four human NB cell lines. Very low CbG content (4-10%) in three of the four cell lines (LAN-5, LAN-1, SMS-KCNR) reflected the ganglioside pattern observed in the most aggressive NB tumours. Pharmacological alterations of complex ganglioside synthesis in vitro by a 5-7 day exposure to 5-10 microM retinoic acid, which is employed in maintenance therapy of disseminated NB, included markedly increased (i) relative expression of CbG (6.6+/-2.0-fold increase, P=0.037), (ii) relative expression of the analogous 'a' pathway gangliosides, termed CaG (6.4+/-1.4-fold increase in GM1a and GD1a; P=0.010), and (iii) total cellular ganglioside content (2.0-6.3-fold), which in turn amplified the accumulation of structurally complex gangliosides. Substantial increases (2.7-2.9-fold) in the activity of GD1b/GM1a synthase (beta-1,3-galactosyltransferase), which initiates the synthesis of CbG and CaG, accompanied the all-trans retinoic acid (ATRA)-induced ganglioside changes. Thus, increased CbG synthesis in NB cell lines is attributable to a specific effect of ATRA, namely induction of GD1b/GM1a synthase activity. Since the shift towards higher expression of CbG and CaG during retinoic acid-induced cellular differentiation reflects a ganglioside pattern found in clinically less-aggressive tumours, our studies suggest that complex gangliosides may play a role in the biological and clinical behaviour of NB.

BACKGROUND Gangliosides are membrane-bound glycolipid molecules particularly prominent in neural tissue. Changes in ganglioside expression during embryologic development result from a shift in biosynthesis from the fetal b pathway to the adult a pathway. Tumor gangliosides may play a role in the clinical behavior of certain subtypes of neuroblastoma. Because neuroblastoma, which presents in infancy, has a different biologic and clinical phenotype than that which presents in older children, the authors determined whether differences in ganglioside biosynthesis exist between these two neuroblastoma subgroups. METHODS Sixty-eight tumor specimens (25 diagnosed by screening and 43 diagnosed clinically) were obtained from the Quebec Neuroblastoma Screening Project. Gangliosides were isolated and purified by solvent partitioning, separated by high performance thin-layer chromatography, and quantitated by scanning densitometry. The sum of a and b pathway gangliosides were determined for each tumor. RESULTS Gangliosides of the b (fetal) pathway predominated in both screened and clinically diagnosed tumors of patients younger than 1 year of age. Twenty-three of 25 screened patients (92%) and 21 of 23 patients with clinically diagnosed tumors at younger than 1 year of age (91%) had tumor b pathway ganglioside content greater than 60%. In contrast, tumors of only 8 of 20 patients 1 year or older (40%) had b pathway ganglioside predominance. Predominance of b pathway tumor gangliosides correlated with improved outcome. Event free survival was significantly higher among patients with b pathway ganglioside tumor content greater than 60% versus those with b pathway ganglioside tumor content less than 60% (118.1 ± 3.9 months vs. 69.2 ± 8.6 months, P < 0.01). CONCLUSIONS Fetal patterns of ganglioside biosynthesis predominate in neuroblastoma tumors from patients younger than 1 year of age and adult patterns of ganglioside biosynthesis predominate in tumors from older children, supporting the view that neuroblastoma consists of distinct but overlapping disorders, and that gangliosides may play a biologic role in the clinical differences among these patients. Cancer 2001;91:785–93. © 2001 American Cancer Society.

Ganglioside metabolism has been linked to the clinical and biological behavior of human neuroblastoma. This study investigated the importance of differences in complex "b" ganglioside (GD1b, GT1b, and GQ1b; designated CbG) expression in this tumor. Gangliosides of 74 neuroblastomas were analyzed by high-performance TLC. Associations of CbG expression with known prognostic markers and with event-free survival (EFS) were evaluated. Higher CbG expression characterized nonprogressive versus progressive tumors (median 41% versus 18% of total gangliosides; P = 0.001) and completely accounted for the observed higher overall "b" pathway ganglioside expression (median 81% versus 68%; P = 0.003). In contrast, expression of the structurally simpler "b" pathway gangliosides (GD2 and GD3) did not differ (median 31% versus 35%; P = 0.4). Absolute CbG content differed even more (median 93 versus 29 nmol/g among nonprogressive versus progressive tumors; P = 0.02) and was most striking in the case of GQ1b content (8-fold higher in nonprogressive tumors). High CbG (or =35% of total gangliosides) expression was strongly predictive of a favorable outcome in: (a) the entire study population (90% versus 60% EFS at 25 months; P = 0.001); and (b) among patients assigned a low-risk status by a either single genetic or biochemical tumor marker (MYCN, DNA, NSE, or ferritin), or by both unamplified MYCN and aneuploid DNA (22-28% difference in EFS at 25 months). These data suggest that high tumor CbG content may substratify "good prognosis" neuroblastoma patients, identifying patients at very low risk of relapse or death, and that the biological roles of CbG in neuroblastoma will be of importance to define.

Abstract 839 Although most children with ALL are cured, treatment is associated with multiple toxicities and outcome after relapse is poor. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. CD22 is a B-lineage differentiation antigen expressed on most B-lineage ALL blasts. The anti-CD22 immunotoxin RFB4(dsFv)-PE38 CAT-3888 (BL22) was recently shown to have clinical activity with an acceptable safety profile in children with ALL (Blood 2007;110:262a). We undertook a Phase I trial of a modified agent with higher CD22 binding affinity (CAT-8015 or HA22). Methods: Patients 6 months to 24 years of age with relapsed or refractory CD22 + B-lineage ALL or non-Hodgkin lymphoma were eligible for enrollment into this Phase I trial. CAT-8015 was administered at doses of 5, 10, 20, or 30 mcg/kg every-other-day for 6 doses every 21 days for up to 6 cycles. One patient was enrolled at each of the first 3 dose levels (5, 10, 20 mcg/kg) with standard 3+3 dose escalation commencing at 30 mcg/kg. All patients received acetaminophen, ranitidine and diphenhydramine to mitigate infusion-related symptoms, and prophylaxis for central-nervous-system leukemia with intrathecal hydrocortisone, cytarabine and methotrexate. Patients at high risk for tumor lysis syndrome received standard prophylaxis. Results: Seven patients with ALL (6 precursor-B, 1 mature B-cell) 5 to 17 years of age (median, 10) were treated on the clinical trial. All patients had been heavily pre-treated and had baseline cytopenias due to active malignancy and thus were not evaluable for hematologic toxicities. The most common adverse events observed to date have been hyperbilirubinemia, transaminase elevations, hypoalbuminemia, elevated creatinine, febrile neutropenia, abdominal pain, pyrexia, hypertension, microscopic proteinuria, hemoglobinuria, hypoxia and pleural effusion. Two of 4 patients treated at 30 mcg/kg experienced Grade 3 or greater toxicity consistent with capillary leak: 1 with Grade 3 pleural effusion and hypoxia and 1 with Grade 4 vascular leak syndrome. All toxicities attributed to CAT-8015 were reversible. Clinical activity was demonstrated in 4 of 7 subjects. One patient treated at 10 mcg/kg had a complete remission by morphology and flow cytometry. Three patients met the protocol definition for hematologic activity (blood count improvement). One of these patients developed high-titer neutralizing antibodies. Two patients met the protocol definition for stable disease. The patient treated at the lowest dose level had progressive disease. Conclusions: CAT-8015 appears to be active against chemotherapy-refractory ALL. Strategies to predict and/or prevent vascular leak syndrome are currently being developed. Disclosures: No relevant conflicts of interest to declare.

Abstract 888 Novel therapies are needed for patients with relapsed or refractory hairy cell leukemia (HCL), particularly those who have failed purine analogs. CD22 is expressed in the majority of B-cell malignancies and universally in HCL, making it an ideal therapeutic target. CAT-8015 is a targeted immunotoxin composed of an anti-CD22 antibody fused to a modified form of Pseudomonas exotoxin A. It has a novel mechanism of action compared with other CD22 targeted antibodies, as CAT-8015 is internalized upon binding to CD22, inhibiting protein translation and prompting apoptosis. CAT-8015 has shown significant antitumor activity in B-cell tumor cell lines and malignant cells isolated from patients with HCL. CAT-8015 (HA22) is a high-affinity derivative of CAT-3888 (BL22) that displays higher CD22-binding and inhibitory activity due to mutation of 3 amino acids. A multicenter, dose-escalation, phase I study is being conducted to estimate the maximum tolerated dose (MTD) of CAT-8015, and evaluate its safety, efficacy and immunogenicity profiles in HCL. Adult HCL patients who previously received at least 2 systemic therapies (including purine analogs) and had cytopenias or symptomatic splenomegaly requiring treatment are eligible to participate. A standard 3+3 dose-escalation design is being employed at doses of 5, 10, 20, 30, 40, and 50 ug/kg. CAT-8015 is administered as a 30-min IV infusion on days 1, 3, and 5 (QODx3) of each 28-day cycle for up to a total of 10 cycles until disease progression, intervening toxicity [e.g. dose-limiting toxicity (DLT)], completion of two cycles of treatment beyond documentation of complete response (CR), or other reason for which the patient might become ineligible. Patients receive premedication with hydroxyzine, ranitidine, and acetaminophen to prevent infusion reactions; and low-dose aspirin and IV hydration to prevent hemolytic uremic syndrome (HUS), which has been observed in association with CAT-3888. Data are electronically archived by each investigator and were pooled for analysis. The study is ongoing with 2 more patients expected to be treated at 50 ug/kg. A total of 26 patients have received CAT-8015 to date. Three patients enrolled at each of the 5, 10, 20, and 30 ug/kg dose levels; 4 patients at the 40 ug/kg dose level; and 10 patients at 50 ug/kg dose level. The median age is 59 years (range 40-77), and the majority (84.6%) are male. Patients were heavily pretreated (median number of prior therapies: 3, range 2-7). Among the 26 patients in total, 14 received prior rituximab (53.8%); among 10 patients in cohort 50 ug/kg, 7 received prior rituximab (70.0%). Patients have received a median of 3 treatment cycles (range 1-8). No DLTs have been observed and an MTD has not been reached. Expanded enrollment at 50 ug/kg has been undertaken to better characterize the safety profile and antitumor activity. The most common drug-related toxicities have been of grade 2 or lower severity: hypoalbuminemia (57.7%), peripheral edema (42.3%), pyrexia (38.5%), elevated ALT (34.6%) and AST (30.8%), headaches (26.9%), and nausea (26.9%). Four patients (15.4%) developed grade 2 vascular leak syndrome (VLS). One treatment-related serious adverse event occurred, a reversible grade 2 HUS that was reported in the 30 ug/kg dose cohort. Anti-drug antibodies developed in 10 patients (38.5%). CAT-8015 was highly active in HCL. Among the 26 patients treated, the objective response (OR) rate was 73.1% (19/26), with a CR rate of 34.6% (9 patients) and a partial response (PR) rate of 38.5% (10 patients). Responses were observed at all dose levels. Specific OR rates at the 5,10, 20, 30, 40, and 50 ug/kg/dose cohorts were 100%, 100%, 33%, 33%, 75% and 80%, respectively. At the time of this report, none of the patients achieving a CR has relapsed. Four of 9 (44.4%) patients with CR have a duration of response of >12 months. CAT-8015 exhibited an acceptable safety profile when administered up to 50 ug/kg QOD × 3, and demonstrated substantial antitumor activity in patients with relapsed/refractory HCL. These data demonstrate that CAT-8015 is a promising new product candidate for patients with advanced HCL. These data support further investigation in newly diagnosed patients with HCL and suggest that CAT-8015 may have clinical activity in other B-cell malignancies Disclosures: Kreitman: NIH: Patents & Royalties. Off Label Use: Recombinant immunotoxin HA22 for targeting CD22+ cells. Robak:MedImmune, LLC: Research Funding. FitzGerald:NIH: Patents & Royalties. Pastan:NIH: Patents & Royalties.

Epstein Barr virus (EBV) has the capacity to transform B lymphocytes and in the setting of immunosuppression increases the risk of developing EBV induced lymphoproliferative disease (EBV LPD). This is a heterogeneous condition ranging from a benign polyclonal B cell proliferation to frank non-Hodgkin’s lymphoma. EBV LPD is associated with immunosuppressive agents used in solid organ, bone marrow and stem cell transplantation, and in certain congenital immunodeficiencies. We conducted a single institution phase I dose escalation study of siplizumab, a humanized monoclonal antibody to CD2, in patients with T-cell malignancies. A total of 29 patients (pts) were enrolled, of which 4 (13.7%) developed EBV LPD. In the original trial design (n=23) pts received escalating drug doses over 2 or 3 consecutive days per treatment week every 2 weeks (cohorts 1–7). In an attempt to increase the rate of drug delivery the trial was amended, for pts (n=6) to receive a single dose on day 0 and 14, and then once weekly thereafter (cohorts 8–10). While initial responses were exciting (2 complete responses, 7 partial responses and 10 stable disease) the development of EBV LPD was a concern. One of 23 (4.3%) pts in the original design and 3 of 6 (50%) in the revised schema developed EBV LPD within 6 months of starting therapy. The 29 pts included adult T-cell leukemia/lymphoma (n=15), large granular lymphocyte leukemia (n=7), cutaneous T-cell lymphoma (n=4) and peripheral T-cell lymphoma (n=3). A median of 2 (range 0–6) prior therapies and a median of 4 (range 1–26) courses of siplizumab with doses ranging from 0.4mg/kg to 4.8mg/kg were administered either biweekly or weekly depending on the cohort. Of the 4 EBV LPD cases, one responded to withdrawal of siplizumab, one to rituximab, another to combination chemotherapy and rituximab, and the remaining patient succumbed to a combination of her underlying disease and EBV-LPD. Review of T-cell trends in response to therapy demonstrated a reduction in the geometric mean of CD4 (84.9%, p