Continuous infusion of the anti-CD22 immunotoxin IgG-RFB4-SMPT-dgA in patients with B-cell lymphoma: a phase I study

IgG-RFB4-SMPT-dgA consists of deglycosylated ricin A chain (dgA) coupled to the monoclonal antihuman CD22 antibody, RFB4. This study determined the maximally tolerated dose (MTD) of this immunotoxin (IT) administered as a continuous 8-day infusion to 18 patients with B-cell lymphoma (30% CD22+ tumor cells) over 8 days. The MTD was 19.2 mg/m2/192 h (maximum toxicity grade 1), with vascular leak syndrome (VLS) as dose-limiting toxicity (DLT) at 28.8 mg/m2/192 h (grades 3 through 5 in 7 of 11 patients). Predictors of severe VLS included serum IT concentrations greater than 1,000 ng/mL and the absence of circulating tumor cells. Decreased urine sodium excreted in 24 hours provided evidence for mild VLS without notable changes in serum albumin. Four partial responses, 3 minor responses, 6 stable disease, and 3 progression of disease were observed. The mean maximal serum concentration (Cmax) in initial courses at the MTD (19.2 mg/m2) was 443 +/- 144 ng/mL (n = 3; range, 326 to 604). At 28.8 mg/m2/192 h, the Cmax was highly variable (n = 11; mean, 1,102 +/- 702; range, 9.6 to 2,032 ng/mL). Human antimouse or antiricin antibodies developed in 6 of 16 (37.5%) patients after one course of IT. However, 10 eligible patients received multiple courses of IT. Changes in serum cytokines and cytokine receptors did not correlate with toxicity but decreased soluble interleukin-2 receptor concentrations correlated with clinical response. Comparison to a prior study with the same IT administered by intermittent bolus infusions (Amlot et al, Blood 82:2624, 1993) suggests similar clinical response, toxicity, and immunogenicity.     

A phase I study of bolus versus continuous infusion of the anti-CD19 immunotoxin, IgG-HD37-dgA, in patients with B-cell lymphoma

IgG-HD37-SMPT-dgA is a deglycosylated ricin A chain (dgA)-containing immunotoxin (IT) prepared by conjugating the monoclonal murine (MoAb) anti-CD19 antibody, HD37, to dgA using the heterobifunctional hindered disulfide linker, N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2- pyridyldithio) toluene (SMPT). In this report, we have used two regimens for the administration of IgG-HD37-SMPT-dgA to patients with non-Hodgkin's lymphoma (NHL) in two concomitant phase I trials. One trial examined four intermittent bolus infusions administered at 48- hour intervals. The other studied a continuous infusion (CI) administered over the same 8-day period. In the intermittent bolus regimen, the maximum tolerated dose (MTD) was 16 mg/m2/8 d and the dose- limiting toxicity (DLT) consisted of vascular leak syndrome (VLS), aphasia, and evidence of rhabdomyolysis encountered at 24 mg/m2/8 d. Using the CI regimen, the MTD was defined by VLS at 19.2 mg/m2/8 d. At the MTD of both regimens, a novel toxicity, consisting of acrocyanosis with reversible superficial distal digital skin necrosis in the absence of overt evidence of systemic vasculitis, occurred in 3 patients. Of 23 evaluable patients on the bolus schedule, there was 1 persisting complete response (CR; > 40 months) and 1 partial response (PR). Of 9 evaluable patients on the continuous infusion regimen, there was 1 PR. Pharmacokinetic parameters for the bolus regimen at the MTD showed a mean maximum serum concentration (Cmax) of 1,209 +/- 430 ng/mL, with a median T1/2 beta for all courses of 18.2 hours (range, 10.0 to 80.0 hours), a volume of distribution (Vd) of 10.9 L (range, 3.1 to 34.5 L), and a clearance (CL) of 0.45 L/h (range, 0.13 to 2.3 L/h). For the CI regimen at MTD, the mean Cmax was 963 +/- 473 ng/mL, with a median T1/2 beta for all courses of 22.8 hours (range, 24.1 to 30.6 hours), a Vd of 9.4 L (range, 4.4 to 19.5 L), and a CL of 0.32 L/h (range, 0.12 to 0.55 L/h). Twenty-five percent of the patients on the bolus infusion regimen and 30% on the CI regimen made antibody against mouse Ig (HAMA) and/or ricin A chain antibody (HARA). We conclude that this IT can be administered safely and that both regimens achieve comparable peak serum concentrations at the MTD; these concentrations are similar to those achieved previously using other regimens with IgG-dgA ITs at their respective MTDs. Thus, toxicity is related to the serum level of the IT and does not differ with different targeting MoAbs.     

The antitumor activity of an anti-CD22 immunotoxin in SCID mice with disseminated Daudi lymphoma is enhanced by either an anti-CD19 antibody or an anti-CD19 immunotoxin.

The antitumor activities of immunotoxins (ITs) constructed with deglycosylated ricin A chain (dgA) and either anti-CD19 (HD37) or anti-CD22 (RFB4) monoclonal antibodies were compared in SCID mice with disseminated human Daudi lymphoma (SCID/Daudi). As reported previously, after intravenous injection with Daudi cells, SCID mice develop disseminated lymphoma, which infiltrates the vertebral column and causes paralysis of the hind legs before death. The mean paralysis time (MPT) has been taken as an end point in this tumor model. We have previously reported that early treatment of SCID/Daudi mice with RFB4 coupled to dgA prolongs the MPT in a manner consistent with the killing of 4 logs of tumor cells. In the present study, we show that HD37-dgA kills 2 logs of tumor cells. The lower potency of the HD37-dgA is consistent with its lower IC50 on Daudi cells in vitro. We further show that the antitumor activity of a mixture of HD37-dgA and RFB4-dgA is significantly enhanced in SCID/Daudi mice and is consistent with the killing in excess of 5 logs of tumor cells. However, identical enhancement was observed when a mixture of the RFB4-dgA and the HD37 antibody was administered. In contrast, enhancement was not observed when mice were injected with a mixture of the RFB4 antibody and the HD37-dgA. The results indicate that a "cocktail" of HD37 antibody and RFB4-dgA immunotoxin can have significant antitumor activity in this mouse model of lymphoma and suggest that combinations of particular antibodies and ITs may have cooperative antitumor activity.     

Use of an anti-pan T-lymphocyte ricin a chain immunotoxin in steroid-resistant acute graft-versus-host disease

Acute steroid-resistant graft-versus-host disease (AGVHD) after allogeneic bone marrow transplantation is frequently fatal. A new treatment for this T-lymphocyte-mediated condition uses an immunotoxin, H65-RTA, comprised of a monoclonal antibody that recognizes the CD5 lymphocyte differentiation antigen coupled to ricin A chain, a cytotoxic enzyme that inhibits protein synthesis. The safety and efficacy of this lymphocyte-targeted immunotoxin was evaluated in patients with severe AGVHD in a phase I-II dose escalation study with group expansion at the two middle doses. Thirty-four patients received up to 14 daily intravenous infusions of the immunotoxin. The principal side effects were constitutional symptoms such as fatigue and myalgias, and hypoalbuminemia with weight gain was seen at all doses. Thirty-two patients were evaluated for improvement or resolution of disease. Durable complete or partial responses were not dose-related and were seen in 16 patients. Skin GVHD had the highest incidence of response (73%), although improvement or resolution in gastrointestinal tract (45%) and liver (28%) GVHD was also noted. Survival in responding patients was significantly prolonged at all times as compared with those with no response (P = .03). Treatment was associated with a rapid decrease in peripheral blood T lymphocytes, which persisted for greater than 1 month after therapy. Anti-immunotoxin antibodies were seen in 6 of the 23 patients tested; these were of low titer and did not block immunotoxin binding to T cells. Results of this study indicate that anti-T-lymphocyte immunotoxins may form a new class of immunosuppressive agents useful in T-lymphocyte-mediated diseases.     

The anti-CD20 antibody rituximab augments the immunospecific therapeutic effectiveness of an anti-CD19 immunotoxin directed against human B-cell lymphoma

The chimaeric anti-CD20 antibody rituximab (Rituxan) sensitises lymphoma cells to small molecule cytotoxic drugs and to protein toxins. We have explored the augmentive effect of rituximab on the anti-CD19 immunotoxin BU12-SAPORIN in a model of human lymphoma. Intact rituximab and its F(ab)2 derivative both augmented the immunospecific protein synthesis inhibitory effects of BU12-SAPORIN in a complement-independent manner. A combination of rituximab + BU12-SAPORIN completely abolished the proliferation of Ramos cells in vitro and also induced a significantly greater degree of apoptosis in these cells. Treatment with rituximab, BU12-SAPORIN or a combination of both induced poly(ADPribose) polymerase and caspase 3 cleavage, although this was always consistently greater in combination-treated cells. zVAD almost completely inhibited apoptosis in rituximab- or BU12-SAPORIN-treated cells but only partially in combination-treated cells. In severe combined immunodeficient (SCID)-Ramos mice the combination of rituximab + BU12-SAPORIN was significantly better therapeutically than either single agent. The immunological fidelity of the therapeutic effect because of combination treatment was demonstrated through the failure of rituximab to augment an irrelevant anti-CD7 immunotoxin. The therapeutic efficacy of rituximab and combination treatment was reduced in SCID-Ramos mice depleted of serum complement while natural killer cell depletion failed to show any convincing role for antibody-dependent cellular cytotoxicity. This study shows a clear therapeutic advantage from using rituximab to immunospecifically augment immunotoxin cytotoxicity warranting further investigation.     

A phase I study of a combination of anti-CD19 and anti-CD22 immunotoxins (Combotox) in adult patients with refractory B-lineage acute lymphoblastic leukaemia

Novel agents are needed for patients with refractory and relapsed acute lymphoblastic leukaemia (ALL). Combotox is a 1:1 mixture of two immunotoxins (ITs), prepared by coupling deglycosylated ricin A chain (dgRTA) to monoclonal antibodies directed against CD22 (RFB4‐dgRTA) and CD19 (HD37‐dgRTA). Pre‐clinical data demonstrated that Combotox was effective in killing both pre‐B‐ALL cell lines and cells from patients with pre‐B ALL. A clinical study of paediatric patients in which 3 of 17 patients with ALL experienced complete remission, supported the preclinical work and motivated this study. This study was a Phase I, dose‐escalation trial using Combotox in adults with refractory or relapsed B‐lineage‐ALL. A cycle consisted of three doses, with one dose given every other day. Dose levels were 3, 5, 6, 7 and 8 mg/m² per dose. Seventeen patients, aged 19–72 years, were enrolled in this multi‐institution study. The maximum tolerated dose was 7 mg/m²/dose (21 mg/m²/cycle) and vascular leak syndrome was the dose‐limiting toxicity. Two patients developed reversible grade 3 elevations in liver function tests. One patient achieved partial remission and proceeded to allogeneic stem cell transplantation. All patients with peripheral blasts experienced decreased blast counts following the administration of Combotox. Thus, Combotox can be safely administered to adults with refractory leukaemia.     

A phase I/II trial of iodine-131-tositumomab (anti-CD20), etoposide, cyclophosphamide, and autologous stem cell transplantation for relapsed B-cell lymphomas

Relapsed B-cell lymphomas are incurable with conventional chemotherapy and radiation therapy, although a fraction of patients can be cured with high-dose chemoradiotherapy and autologous stem-cell transplantation (ASCT). We conducted a phase I/II trial to estimate the maximum tolerated dose (MTD) of iodine 131 ((131)I)-tositumomab (anti-CD20 antibody) that could be combined with etoposide and cyclophosphamide followed by ASCT in patients with relapsed B-cell lymphomas. Fifty-two patients received a trace-labeled infusion of 1.7 mg/kg (131)I-tositumomab (185-370 MBq) followed by serial quantitative gamma-camera imaging and estimation of absorbed doses of radiation to tumor sites and normal organs. Ten days later, patients received a therapeutic infusion of 1.7 mg/kg tositumomab labeled with an amount of (131)I calculated to deliver the target dose of radiation (20-27 Gy) to critical normal organs (liver, kidneys, and lungs). Patients were maintained in radiation isolation until their total-body radioactivity was less than 0.07 mSv/h at 1 m. They were then given etoposide and cyclophosphamide followed by ASCT. The MTD of (131)I-tositumomab that could be safely combined with 60 mg/kg etoposide and 100 mg/kg cyclophosphamide delivered 25 Gy to critical normal organs. The estimated overall survival (OS) and progression-free survival (PFS) of all treated patients at 2 years was 83% and 68%, respectively. These findings compare favorably with those in a nonrandomized control group of patients who underwent transplantation, external-beam total-body irradiation, and etoposide and cyclophosphamide therapy during the same period (OS of 53% and PFS of 36% at 2 years), even after adjustment for confounding variables in a multivariable analysis.     

Anti-CD38-blocked ricin: an immunotoxin for the treatment of multiple myeloma [see comments]

We report the development of a potent anti-CD38 immunotoxin capable of killing human myeloma and lymphoma cell lines. The immunotoxin is composed of an anti-CD38 antibody HB7 conjugated to a chemically modified ricin molecule wherein the binding sites of the B chain have been blocked by covalent attachment of affinity ligands (blocked ricin). Conjugation of blocked ricin to the HB7 antibody has minimal effect on the apparent affinity of the antibody and no effect on the ribosome-inactivating activity of the ricin A-chain moiety. Four to six logs of CD38+ tumor cell line kill was achieved at concentrations of HB7-blocked ricin in the range of 0.1 to 3 nmol/L. Low level of toxicity for normal bone marrow (BM) granulocyte-macrophage colony- forming units (CFU-GM), burst-forming units-erythroid (BFU-E), colony- forming units-granulocyte/erythroid/monocyte/macrophage (CFU-GEMM) cells was observed. Greater than two logs of CD38+ multiple myeloma cells were depleted from a 10-fold excess of normal BM mononuclear cells (BMMCs) after an exposure to HB7-blocked ricin under conditions (0.3 nmol/L) that were not very toxic for the normal BM precursors. HB7- blocked ricin was tested for its ability to inhibit protein synthesis in fresh patients' multiple myeloma cells and in normal BMMCs isolated from two healthy volunteers; tumor cells from four of five patients were 100-fold to 500-fold more sensitive to the inhibitory effect of HB7-blocked ricin than the normal BM cells. HB7 antibody does not activate normal resting peripheral blood lymphocytes, and HB7-blocked ricin is not cytotoxic toward these cells at concentrations of up to 1 nmol/L. The potent killing of antigen-bearing tumor cells coupled with a lack of effects on peripheral blood T cells or on hematopoietic progenitor cells suggests that HB7-blocked ricin may have clinical utility for the in vivo or in vitro purging of human multiple myeloma cells.     

A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host disease

This study evaluated the anti-graft versus host disease (GVHD) potential of a combination of immunotoxins (IT), consisting of a murine CD3 (SPV-T3a) and CD7 (WT1) monoclonal antibody both conjugated to deglycosylated ricin A. In vitro efficacy data demonstrated that these IT act synergistically, resulting in an approximately 99% elimination of activated T cells at 10(-8 )mol/L (about 1.8 microg/mL). Because most natural killer (NK) cells are CD7(+), NK activity was inhibited as well. Apart from the killing mediated by ricin A, binding of SPV-T3a by itself impaired in vitro cytotoxic T-cell cytotoxicity. Flow cytometric analysis revealed that this was due to both modulation of the CD3/T-cell receptor complex and activation-induced cell death. These results warranted evaluation of the IT combination in patients with refractory acute GVHD in an ongoing pilot study. So far, 4 patients have been treated with 3 to 4 infusions of 2 or 4 mg/m(2) IT combination, administered intravenously at 48-hour intervals. The T(1/2) was 6.7 hours, and peak serum levels ranged from 258 to 3210 ng/mL. Drug-associated side effects were restricted to limited edema, fever, and a modest rise of creatine kinase levels. One patient developed low-titer antibodies against ricin A. Infusions were associated with an immediate drop of circulating T cells, followed by a more gradual but continuing elimination of T/NK cells. One patient mounted an extensive CD8 T-cell response directly after treatment, not accompanied with aggravating GVHD. Two patients showed nearly complete remission of GVHD, despite unresponsiveness to the extensive pretreatment. These findings justify further investigation of the IT combination for treatment of diseases mediated by T cells. (Blood. 2000;95:3693-3701)     

Successful treatment and re-treatment of resistant B-cell chronic lymphocytic leukemia with the monoclonal anti-CD 20 antibody rituximab

We report on two patients with chemoresistant B-cell chronic lymphocytic leukemia who were treated successfully with the monoclonal anti-CD 20 antibody rituximab. Both patients suffered from severe thrombocytopenia requiring platelet transfusions over a period of several months. Neither chemotherapy nor immunosuppressive agents (corticoids, immunoglobulins) were effective. After four doses of rituximab (375 mg/m² weekly), both patients recovered within a few weeks to hematological partial remission. One patient was re-treated successfully three times after relapses. Both patients were premedicated with prednisone (100 mg) 30 min prior to the infusion to prevent cytokine release and the antibody infusions were well tolerated. Received: 5 May 1999 / Accepted: 18 October 1999     

Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20

A controlled study has been carried out to assess the efficacy of rituximab, a chimeric antibody that binds to the B-cell surface antigen CD20, in 20 patients with mixed cryoglobulinemia (MC) and hepatitis C virus (HCV)-positive chronic active liver disease, resistant to interferon alpha (IFN-alpha) therapy. They received an intravenous infusion of 375 mg/m(2) rituximab once a week for 4 consecutive weeks. Infusion of rituximab had a good safety profile and no severe side effects were reported. Sixteen patients (80%) showed a complete response (CR), characterized by rapid improvement of clinical signs (disappearance of purpura and weakness arthralgia and improvement of peripheral neuropathy), and decline of cryocrit. CR was associated with a significant reduction of rheumatoid factor (RF) activity and anti-HCV antibody titers. Decline of IgG anti-HCV titers in the cryoprecipitates was usually associated with a favorable response (r = 0.81; P <.005). No differences in the dynamics of B-cell depletion and recovery were found between responders and nonresponders. Molecular monitoring of the B-cell response revealed disappearance/deletion of peripheral clones in the responders and great stability in the nonresponders. Rituximab had a deep impact on hepatitis C viremia; HCV RNA increased approximately twice the baseline levels in the responders, whereas it remained much the same in the nonresponders. Twelve (75%) of 16 responders remained in remission throughout the follow-up. The results indicate that rituximab has clinical and biologic activity in patients with HCV(+) MC. However, in view of the increased viremia in the responders, additional modes of application and combination of rituximab with other agents need to be investigated.     

A comparison of an anti-CD25 immunotoxin, Ontak and anti-CD25 microbeads for their ability to deplete alloreactive T cells in vitro

Ex vivo depletion of alloreactive CD25(+) T cells from a stem cell transplant (SCT) can reduce the incidence of graft-versus-host disease (GVHD) while preserving antimicrobial and perhaps antileukemia activity. However, the most effective methods for allodepleting T cells prior to transplant have not been determined. In this study, we have compared three agents that deplete CD25(+) activated, alloreactive T cells. These included Ontak (Denileukin Diftitox), an IL-2 fusion toxin, anti-CD25 microbeads (MACS), an anti-CD25 immunotoxin (IT) and a combination of the IT and MACS. Peripheral blood mononuclear cells (PBMCs) activated in a primary mixed lymphocyte reaction (MLR) were allodepleted using optimal amounts of each agent, and the cells were then analyzed by flow cytometry. The treated cells were examined both for remaining alloreactivity and for the preservation of third party reactivity by testing them in a secondary MLR. Our data demonstrate that both the anti-CD25 IT and the anti-CD25 MACS were equally effective in depleting CD4(+)CD25(+) cells and in sparing T cells that were reactive with third party cells. The anti-CD25 IT was, however, superior in depleting alloreactive CD8(+)CD25(+) cells. In contrast, Ontak did not eliminate alloreactive cells and the Ontak-treated cells retained significant reactivity against the original stimulator cells.     

Monoclonal antibody therapy for B-cell lymphoma: clinical trials of an anti-CD20 monoclonal antibody for B-cell lymphoma in Japan

Twelve patients with relapsed CD20+ B-cell non-Hodgkin's lymphoma (B-NHL) were enrolled in a phase I study of rituximab; 4 received rituximab 250 mg/m2 and 8 received rituximab 375 mg/m2 once weekly for 4 weeks. Grade 1 or 2 infusion-related toxicity was observed. Of the 11 eligible patients, 2 achieved complete responses and 5 achieved partial responses. The elimination half-life (T1/2) of rituximab was 445 +/- 361 hours, and serum rituximab levels were detectable at 3 months. Thereafter, 90 relapse patients with indolent B-NHL or mantle cell lymphoma (MCL) were enrolled in a phase II study and treated with rituximab at 375 mg/m2 per infusion in 4 weekly infusions. Sixteen patients were ineligible in protocol compatible analyses. The overall response rates (ORR) in indolent B-NHL and MCL were 61% (37 of 61 patients) and 46% (6 of 13 patients), respectively. Factors affecting response and progression-free survival (PFS) were analyzed for 77 patients whose histopathology was centrally confirmed as indolent B-NHL or MCL. The ORR in patients receiving 1 prior chemotherapy regimen was higher than the ORR in those receiving > or = 2 regimens (P < .05). The median PFS was shorter in MCL patients, in those with extranodal disease, and in those receiving > or = 2 prior chemotherapy regimens (P < .01). The PFS of patients with higher serum rituximab levels (> or = 70 microg/mL) immediately before the third infusion was longer than that of other patients (P < .01). Several pretreatment factors and serum rituximab levels are useful for predicting the efficacy of rituximab monotherapy. Rituximab re-treatment was well tolerated in 13 patients with no grade 3 or 4 nonhematological toxicities. A partial response was observed in 5 patients (38%), and the median PFS after re-treatment was 5.1 months. In conclusion, rituximab is a highly effective agent in relapsed indolent and aggressive B-NHL and MCL and has acceptable toxicities.     

Prognostic significance of receptors for the third component of complement an heavy chain phenotype in diffuse B-cell lymphomas

As part of a larger study to determine the prognostic significance of cell marker phenotype in diffuse lymphomas, 51 patients with monoclonal B-cell lymphoma were further characterized by receptors for C3 (EAC rosettes) as well as heavy and light chain phenotypes. Patients with greater than 10% EAC rosette-forming cells were found to have a statistically significant longer survival than those with less than 10% EAC rosettes (p = 0.005). A similar trend in survival duration was found for patients whose cells expressed mu heavy chain on their surfaces when compared to those with gamma heavy chain on their cell surfaces (p = 0.05). No difference was observed for light chain phenotype. No correlation was observed between these prognostic groups and any of the three most frequently used histologic classifications (i.e., Rappaport, Lukes, Kiel).     

A phase I study of bendamustine,lenalidomide and rituximab in relapsed and refractory lymphomas

Many patients with non‐Hodgkin (NHL) or Hodgkin lymphoma (HL) relapse or are refractory to initial therapy and require additional options. Bendamustine (B), lenalidomide (L) and rituximab (R) each have activity in this setting. This study was performed to determine the safety of BLR and its optimal phase II dose. Patients with NHL or HL failing standard therapies received B (90 mg/m² days 1, 2 every 28 days), and L (escalating from 5 mg 21/28 days) for six cycles, followed by 6 months of L. At the highest dose R 375 mg/m² on day one of each cycle was added for patients with B‐NHL. Histologies included diffuse large B‐cell lymphoma (DLBCL, 11), marginal zone lymphoma (3), HL (2), and one each of transformed follicular lymphoma, Sézary syndrome, Waldenström macroglobulinaemia and mantle cell lymphoma. Neutropenia was the most common grade 3 and 4 toxicity, but no maximum tolerated dose was identified. Of 20 patients, seven responded (35%), including four complete remissions, with five unmaintained responses from 28+ to 37+ months, including 2 DLBCL. BR with 20 mg l at, 21/28 days achieved durable responses; however, in light of its modest activity, and the availability of newer targeted therapies, the future of BLR is uncertain.     

Combined therapy in the treatment of primary mediastinal B-cell lymphoma: conventional versus escalated chemotherapy

Treatment of patients with primary mediastinal B-cell lymphoma (PMBCL) remains controversial. We started a controlled clinical trial to evaluate the efficacy and toxicity of a conventional versus more intensive regimen of combined chemotherapy followed by radiotherapy to the mediastinum with the mantle technique. From 1989 to 1997, 68 patients diagnosed with previously untreated PMBCL, aged 18-65 years and negative for immunodeficiency virus test, were considered candidates to receive either conventional chemotherapy with CEOP-Bleo (cyclophosphamide 750 mg/m(2), vincristine 1.4 mg/m(2), prednisone 40 mg/m(2), epirubicin 70 mg/m(2), and bleomycin 10 mg/m(2)) or mega CEOP-Bleo (cyclophosphamide 1000 mg/m(2), epirubicin 120 mg/m(2), vincristine, prednisone, and bleomycin at the same doses) every 21 days for six cycles, followed by radiotherapy to the mediastinum with the mantle technique (35-45 Gy, mean 38 Gy). Complete response (CR) rates were not statistically different: 64% [95 percent confidence interval (CI): 58 percent to 70 percent] for conventional arm vs 81 percent (95 CI: 77-86 percent) in the intensive group (p=0.2). However, failure-free survival (FFS) and overall survival (OS) had statistical differences. At 5 years, actuarial FFS for patients treated with conventional chemotherapy was 51 percent (95 percent CI: 44-59 percent) compared to 70 percent (95 percent CI: 65-76 percent) in the intensive arm (p>0.01). OS rates were also different: 54 percent (95 percent CI: 48-57 percent) vs 70 percent (95 percent CI: 65-76 percent), respectively (p<0.01). Toxicity was mild and no therapy-related deaths were observed. At a median follow-up of 7.3 years, no second neoplasia or acute leukemia has been observed. The international prognostic index was not useful to define clinical risk in this selected group of patients. Multivariate analysis identified pleural and pericardial effusion and chemotherapy regimen as prognostic factors influencing FFS and OS. We feel that patients with PMBCL should be treated with more intensive, but not myeloablative chemotherapy, followed by adjuvant radiotherapy to achieve an improvement in outcome in this setting of patients. Patients with pleural or pericardial effusion are considered at high risk for failure with the actual programs of treatment and probably will be considered for experimental therapeutic approaches.     

Effects of anti-CD33 blocked ricin immunotoxin on the capacity of CD34+ human marrow cells to establish in vitro hematopoiesis in long-term marrow cultures.

Human marrow cells that express the CD34 antigen but lack CD33 are able to initiate sustained, multilineage in vitro hematopoiesis in long-term Dexter cultures and are believed to include the primitive stem cells responsible for effecting long-term hematopoietic reconstitution in vivo following marrow transplantation. In studies described in this report we investigated the effects of a novel anti-CD33 immunotoxin on the clonogenic potential of normal human CD34+ marrow cells and on the ability of these cells to initiate hematopoiesis in two-stage Dexter cultures (long-term marrow cultures, LTMC). This immunotoxin (anti-CD33-bR), shown previously to kill both clonogenic myelogenous leukemia cells and normal mature myeloid progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM), consists of an anti-CD33 monoclonal antibody conjugated to purified ricin that has been modified by blocking the carbohydrate binding domains of the ricin B-chain to eliminate nonspecific binding. For our studies, normal CD34+ human marrow cells were isolated from the light-density (less than 1.070 g/ml) cells of aspirated marrow by positive selection with immunomagnetic beads linked to the monoclonal antibody K6.1. These cell isolates were highly enriched with both multipotential and lineage-restricted clonogenic, hematopoietic progenitors (mixed lineage colony-forming units, CFU-Mix; CFU-GM; and erythroid burst-forming units, BFU-E) which constituted greater than or equal to 20% of the cells. Recovery of clonogenic progenitors from these CD34+ cell preparations, following treatment with anti-CD33-bR (10 nM), was reduced by greater than or equal to 85% for CFU-GM and 20%-40% for CFU-Mix and BFU-E. However, the capacity of these cells to initiate hematopoietic LTMC was preserved. Indeed, the production of high proliferative potential (HPP) CFU-GM, BFU-E, and CFU-Mix in cultures seeded with 10(5) anti-CD33-bR-treated CD34+ marrow cells was substantially greater than that observed in LTMC seeded with equivalent numbers of untreated CD34+ cells. Moreover, concentrations of long-term culture initiating cells in CD34+ cell isolates, quantified by a limiting dilution technique, were found to be increased following anti-CD33-bR treatment. These findings support the potential usefulness of anti-CD33-bR for in vitro marrow purging or in vivo treatment to eliminate CD33+ leukemic clones, while sparing normal CD34+/CD33- stem cells that support normal hematopoiesis and hematopoietic reconstitution in vivo.     

Autologous transplantation of bone marrow purged in vitro with anti-CD7- (WT1-) ricin A immunotoxin in T-cell lymphoblastic leukemia and lymphoma

Seven patients with high-risk acute T-cell lymphoblastic leukemia (T- ALL) and six with T cell lymphoma (T-LL) were treated with autologous bone marrow transplantation (ABMT) after in vitro purging of their bone marrow with WT1 (CD7)-ricin A-chain immunotoxin. CD7 expression on the tumor cells showed large variations between the individual patients and was highly related to the specific cytotoxicity of WT1-ricin A. Incubation of bone marrow with up to 10(-8)mol/L WT1-ricin A in the presence of 6 mmol/L NH4Cl did not compromise the growth potential of the hematopoietic progenitors CFU-GM, CFU-GEMM, and BFU-E. Hematologic engraftment (greater than 10(9) leukocytes/L) occurred within a normal time period (median, 17 days). Seven patients are alive and in complete remission (CR) at 48+, 44+, 40+, 26+, 11+, 7+, and 6+ months after ABMT. Four patients relapsed within 6 months after ABMT. Two of them had the lowest CD7 expression on their tumor cells, the other two were transplanted in CR2 and CR3. Two patients died from transplantation related infections. The immunologic reconstitution was delayed, although the numbers of T cells reached normal levels within 1 month. The number of CD7+ cells remained low up to 1 year after transplantation. The T4/T8-ratio was decreased for at least 6 months. The T-cell response to mitogens recovered to normal levels after 1 year. This study shows that ABMT with WT1-ricin A purged bone marrow in high-risk T-cell malignancies results in a complete hematopoietic and a delayed immunologic reconstitution. The actuarial relapse free survival is 61% at 3 years.