Rituximab Pretreatment Research Articles

Abstract 4179: High valency of IGM-2323, a CD20xCD3 IgM bispecific T cell engager, displaces rituximab binding and induces potent B lymphoma cell killing

Abstract Rituximab-containing treatment regimens are the standard of care for patients with non-Hodgkin’s lymphoma (NHL). However, the majority of patients ultimately experience disease relapse or progression indicating resistance to rituximab therapy. IGM-2323 is an engineered high-affinity, high-avidity anti-CD20 pentameric IgM antibody with an anti-CD3 scFv fused to the joining chain. IGM-2323 offers a novel treatment strategy in NHL through two mechanisms: 1) the recruitment of T cells to kill CD20-expressing tumor cells through T cell dependent cellular cytotoxicity (TDCC) and, 2) complement-dependent cytotoxicity (CDC). We evaluated the activity of IGM-2323 in the presence of rituximab since rituximab can persist in patients after treatment discontinuation, and it can bind to an overlapping epitope on CD20 as IGM-2323. We hypothesized that the high valency of IGM-2323 could displace rituximab, thus enabling potent B cell killing by IGM-2323 even in the presence of high concentrations of rituximab. The affinity of IGM-2323 and its corresponding bivalent anti-CD20 IgG antibody to recombinant human CD20 protein were measured by surface plasmon resonance. IGM-2323 bound to human CD20 with an apparent 300-fold higher binding affinity (KD) and ~100-fold slower off-rate (kdis) than the bivalent anti-CD20 IgG. Human B cell lines with a range of CD20 expression levels, including a CD20-low rituximab-resistant Ramos cell variant, were pre-treated with escalating concentrations of rituximab, and subsequently evaluated in vitro for cell binding, TDCC, and CDC by IGM-2323. At high concentrations of rituximab, which correspond to reported peak serum concentrations (Cmax) found in rituximab-treated patients, IGM-2323 displaced the binding of rituximab on human B cell lines. In contrast, binding of a bispecific CD20xCD3 IgG was severely inhibited by the Cmax of rituximab. In TDCC assays with healthy donor effector T cells, pre-treatment with high concentrations of rituximab only modestly inhibited IGM-2323 activity. Furthermore, only a minor impact to the maximum killing activity (Emax) of IGM-2323 was observed at the Cmax of rituximab. In contrast, rituximab pre-treatment resulted in a distinctly lower Emax of a bispecific CD20xCD3 IgG. Live cell imaging of CDC kinetics was utilized to quantify the extent of CDC by IGM-2323 with or without rituximab. Pre-treatment with rituximab enhanced CDC of IGM-2323 compared to single agent activity. Our preclinical data indicate that IGM-2323 maintains activity in the presence of rituximab. IGM-2323 is currently being studied in a phase 1 clinical trial in relapsed/refractory NHL, where it has been generally well tolerated, with both complete and partial responses observed (NCT04082936). Clinical studies will continue to evaluate these findings, including the treatment of patients with circulating serum levels of rituximab. Citation Format: Kevin C. Hart, Kathryn Logronio, Mandy Li, Poonam Yakkundi, Marigold Manlusoc, Keyu Li, Paul R. Hinton, Dean Ng, Maya K. Leabman, Genevive Hernandez, Thomas Manley, Angus M. Sinclair, Stephen F. Carroll, Bruce A. Keyt, Maya F. Kotturi. High valency of IGM-2323, a CD20xCD3 IgM bispecific T cell engager, displaces rituximab binding and induces potent B lymphoma cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4179.

The Role of SMPDL3b in Regulating Radiation Nephropathy

Radiation nephropathy (RN) is functionally characterized by an initial acute phase (proteinuria) followed by a chronic decline of glomerular filtration rate leading to renal failure. RN is irreversible and affects the quality of life of both short- and long-term cancer survivors. The molecular mechanisms responsible for RN remain largely unknown. The current study investigates the role of sphingolipids in radiation-induced podocytopathy and RN. Sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) expression was assessed in cultured podocytes after X-irradiation (1-8 Gy) by real-time PCR and Western blotting. Changes in podocyte morphology and radiation-induced DNA damage repair were assessed in wild-type, SMPDL3b-KO and SMPDL3b-over-expressing podocytes. Wild-type C57BL/6 male and female mice (10–14 weeks) were X-irradiated using CT image-guidance (SD 14 Gy, fractionated 24x2 Gy; and 4 Gy SD + 6 mg/kg cisplatin). 50 mg/kg Rituximab (or IgG) was administered IP 30 min before irradiation to protect SMPDL3b. Functional kidney parameters (proteinuria and albumin/creatinine urine ratio, along with serum BUN [blood urea nitrogen], creatinine and hematocrit) were analyzed 10–40 weeks post treatment. Kidney histology, podocyte number and SMPDL3b levels were assessed by immunohistochemistry and Western blotting. Following irradiation, SMPDL3b expression was significantly reduced in vitro and in vivo . In irradiated kidneys, podocyte number decreased significantly and H&E kidney sections demonstrated a multifocal increase in the number of pericytes, tubular atrophy and glomerular damage. Transmission electron microscopy indicated radiation-mediated changes to glomerular base membrane thickness and podocyte foot process effacement. Periodic Acid-Schiff (PAS) staining demonstrated increases in glomerular mesangial matrix accumulation post-irradiation, along with diffuse intertubular fibrosis as determined by Sirius red staining, especially in the renal cortex. Rituximab improved kidney functional parameters, vascular structure, normalization of pericyte coverage and suppressed the development of fibrosis and tubular damage post irradiation. SMPDL3b expression was identified as a molecular determinant of podocyte injury after single dose and fractionated X-irradiation. Rituximab pretreatment protected mice against radiation-induced nephrotoxicity, which may have therapeutic implications for radiation-induced injuries in cancer patients.

Abstract 4161: Protecting Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3b) enhances kidney function and reduces concurrent chemoradiotherapy-induced nephrotoxicity

Abstract Background: Cisplatin is a widely used radiosensitizer. Dose-related and cumulative renal insufficiency, including acute renal failure, are the primary dose-limiting toxicities of cisplatin. The lipid-modulating enzyme sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) and Sphingosine 1-Phosphate Receptor-1 (S1P1) are critical determinants of renal podocyte injury. In the current study, we investigated the functions of SMPDL3b and S1P in cisplatin and radiation-induced renal injury, and the use of rituximab (RTX) to attenuate nephrotoxicity. Material/Methods: Podocytes were incubated for 2 h with S1P (100 nM) or vehicle (2% dodecane/98% ethanol) and irradiated with 0-8 Gy, or podocytes were irradiated (8 Gy) in the presence of RTX (100 mg/ml) or IgG (100 mg/ml). In other experiments, 10-14-week old C57BL/6 mice were given bilateral kidney X-irradiation (4 Gy) using an image-guided small animal arc radiation treatment system (iSMAART), 6mg/kg cisplatin or combined cisplatin plus radiation. Some animals received a single intravenous (IV) injection of rituximab mAb or IgG (50 mg/kg) prior to treatment. Functional kidney parameters and immuno-histopathological assessments of renal damage (e.g. oil red staining, H & E, Periodic Acid-Schiff (PAS) and SMPDL3B expression) were measured post-RT. iSMAART dynamic contrast-enhanced (DCE) imaging was used for estimating glomerular filtration rate 20 weeks post radiation. Results: Exogenous S1P rescued podocytes from concurrent chemoradiotherapy-induced cellular cytoskeletal remodeling, and RTX pretreatment mitigated podocytopathy in vitro. iSMAART dynamic contrast-enhanced (DCE) imaging analysis showed that rituximab pretreatment improved the GFR and kidney function parameters post concurrent chemoradiotherapy. SMPDL3b expression at the protein level and podocyte counts dropped significantly in irradiated kidney post-RT in cisplatin-treated mice. Rituximab pretreatment in cisplatin-treated mice, improved GFR, kidney functional parameters, vascular structure, normalization of pericyte coverage, suppress the development of fibrosis and tubular damage post-irradiation. Conclusion: This study shows that rituximab pretreatment protects mice against concurrent chemoradiation induced kidney injury. SMPDL3b and Sphingosine 1-Phosphate Receptor-1 may be important therapeutic targets for radiation-induced kidney injuries in cancer patients. Citation Format: Anis Ahmad, Alla Mitrofanova, Saba Ansari, Junwei Shi, Yidong Yang, Sandra Merscher, Alessia Fornoni, Youssef Zeidan, Brian Marples. Protecting Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3b) enhances kidney function and reduces concurrent chemoradiotherapy-induced nephrotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4161.

Beneficial effect of ABO incompatibility on de novo DSA production after kidney transplantation

Introduction De novo donor specific HLA antibodies (DSA)-induced chronic antibody-mediated rejection (ABMR) is one of major obstacles to long term graft survival. Recently, ABO incompatible kidney transplantation (ABO-I) has showed favorable graft survival. We have reported that anti-A/B antibody binding could cause graft accommodation, which demonstrated protective effects against graft injury due to upregulation of complement regulatory proteins (CD55/CD59) and downregulation of HLA class II antigens. The purpose of this study was to elucidate the effect of ABO incompatibility on de novo DSA production after kidney transplantation. Methods HLA-DRB1/3/4/5 and DQB1 epitope mismatch (MM) levels and de novo DSA production were examined in 681 living donor kidney transplantations [449 ABO-identical/compatible (ABO-Id/C), 232 ABO-I] performed between 2008 and 2015. Desensitization in ABO-I included rituximab (RIT, n=164), splenectomy (SPX, n=10) and neither (NONE, n=58) due to low anti-A/B antibody titers. Results De novo DSA were detected in 80 recipients (11.7%) including DSA against class I (n=9), class I+DQ (n=2), DR (n=14), DQ (n=50) and DR+DQ (n=5). Biopsy-proven chronic ABMR was significantly associated with DR DSA (HR 14.7, P=0.0003) and DQ DSA (HR 33.1, P<0.0001), but not class I DSA. Therefore, only class II (DR and/or DQ) DSA were considered in this study. Epitope MM levels of DRB1/3/4/5 and DQB1 were significantly correlated with production of DR and DQ DSA, respectively (Fig. 1). De novo DSA free graft survivals with moderate MM levels (6-15 DRB1/3/4/5 MM and 1-5 DQB1 MM) were significantly higher in ABO-I (DR 100%, DQ 100%) than in ABO-Id/C (DR 96.3% P=0.0378, DQ 90.7% P=0.0143) (Fig. 2). ABO-I without rituximab pretreatment also exhibited the favorable results for de novo DSA production (Fig. 3)..Conclusions DRB1/3/4/5 and DQB1 epitope MM levels could predict the risk of de novo DSA production. Protective effect against de novo DSA production was observed in ABO-I with moderate levels of DRB and DQB epitope MM. Beneficial effect of ABO incompatibility may be elicited by anti-A/B antibody binding to the graft, rather than rituximab pretreatment.

Favorable results in ABO-incompatible renal transplantation without B cell-targeted therapy: Advantages and disadvantages of rituximab pretreatment.

The effectiveness of desensitization with rituximab in ABO-incompatible renal transplantation (ABO-I) has been widely reported. However, ABO-I outcomes are still worse than those of ABO-identical or ABO-compatible renal transplantation (ABO-Id/C). We retrospectively examined the outcomes in consecutive living donor ABO-Id/C (n=412) and ABO-I (n=205) cases to elucidate the causes of inferiority in ABO-I. ABO-I cases included recipients treated with rituximab (RIT, n=131), splenectomy (SPX, n=21), or neither because of low anti-A/B antibody titers (NoR/S, n=53). Graft survival, infection, and denovo HLA antibody production were compared for ABO-I and ABO-Id/C, followed by stratification into RIT and NoR/S groups. Propensity score-based methods were employed to limit selection bias and potential confounders. Overall graft survival for ABO-I was significantly lower than that for ABO-Id/C (92.8% vs 97.2% after 5years, P=.0037). Graft loss due to infection with ABO-I was significantly more frequent than that with ABO-Id/C, whereas acute antibody-mediated rejection (AMR) caused no graft failure in ABO-I recipients. Stratified analysis demonstrated significantly higher infection risk with RIT than with NoR/S. Safe reduction or avoidance of rituximab in desensitization protocols might contribute to further improvement of ABO-I outcome.

Abstract 904: Podocyte-specific SMPDl3b modulates radiation-induced renal dysfunction

Abstract Background: The underlying mechanisms responsible for the renal failure and proteinuria in radiation nephropathy remain largely unknown. Radiotherapy with or without chemotherapy may result in radiation-induced kidney injury in pelvic malignancies such as gynecologic cancers, lymphomas, gastrointestinal cancers, sarcomas of the upper abdomen and during total body irradiation. The current study investigates the role of sphingolipids in radiation-induced podocytopathy using a murine model. The molecular and functional effects of kidney irradiation were evaluated after single-dose exposures. Material/Methods: In cell culture, SMPDL3b expression post radiation (8Gy) was determined by real-time PCR (RT-PCR) and Western blotting. Morphological changes and DNA damage were detected post radiation using immunofluorescence microscopy. Wild-type C57BL/6 male and female mice (age 10-14 weeks) were irradiated with a single dose of X-ray (14 Gy) using an image guided small animal arc radiation treatment system (iSMAART). Rituximab/IgG was administered (50 mg/kg, intraperitoneal injection) 30 min before the single dose of irradiation. Functional kidney parameters, kidney histology, and gene expression were analyzed at 20, 30 and 40 weeks after irradiation. Results: Following irradiation, SMPDL3b expression at protein level was significantly reduced in vitro and in vivo. However, no significant changes were observed at the transcriptional level. Podocyte number also decreased significantly post radiation in vivo. iSMAART dynamic contrast enhanced (DCE) imaging data analysis showed reduced glomerular filtration rate post radiation. In agreement with the functional data, hematoxylin and eosin staining of kidney sections showed a multifocal increase in the number of pericytes, tubular atrophy, and glomerular damage. Periodic Acid-Schiff (PAS) staining showed an increase in glomerular mesangial matrix accumulation post radiation. Sirius red staining showed diffuse intertubular fibrosis, especially in the renal cortex post radiation. These histological changes were paralleled by the change in serum Creatinine, urine albumin. Rituximab pretreatment to mice, improved kidney functional parameters, vascular structure, normalization of pericyte coverage, suppress the development of fibrosis and tubular damage post irradiation. Conclusion: This study shows that rituximab pretreatment protects mice against radiation-induced nephrotoxicity, which may have therapeutic implications for radiation-induced injuries in cancer patients. Citation Format: Anis Ahmad, Alla Mitrofanova, Saba Ansari, Thirupandiyur Udayakumar, Jacek Bielawski, Alan Pollack, Alesia Fornoni, Brian Marples, Youssef Zeidan. Podocyte-specific SMPDl3b modulates radiation-induced renal dysfunction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 904. doi:10.1158/1538-7445.AM2017-904

Remission Induction in a Phase 1 Study of a Bispecific Single Chain Immunotoxin Targeting CD22 and CD19 (DT2219) for Refractory B-Cell Malignancies

DT2219 is a recombinant fusion protein composed of catalytic and translocation domains of diphtheria toxin (DT) fused with bispecific single chain variable fragments (sFv) of antibodies targeting human CD19 and CD22 cell surface receptors. Preclinical testing determined that the combination of two different sFvs and a toxin on the same single-chain molecule resulted in efficient toxin delivery and greater anticancer activity compared to monomeric immunotoxins sFv alone or anti-CD19 and anti-CD22 parental monoclonal antibodies. Murine studies demonstrated clinical efficacy and enhanced therapeutic effect with repetitive dosing (Vallera D, Clin Cancer Res 2009).We conducted a phase 1 study to assess safety, to determine the maximum tolerated dose (MTD) and to evaluate preliminary efficacy in patients with chemotherapy refractory B cell leukemia or lymphoma expressing CD19 and/or CD22. DT2219 was administered in a single course intravenously over 4 hours (1-2 hours for subsequent doses) every other day for 4 total doses. The starting dose was 0.5 µg/kg/dose (2.0 ug/kg/course) (1/1000th of the MTD in rabbits). The dose was escalated by accelerated design until dose limited toxicity (DLT) was observed. Twenty-two patients were enrolled, with a median age of 55 years (range, 34-78). Five patients had pre-B acute lymphoblastic leukemia (4 refractory, 1 with extra-medullary disease, 1 had chronic lymphocytic leukemia (CLL) and 7 had non-Hodgkin lymphoma). All were chemo-refractory with a median of 4 prior therapies. All patients had biopsy-confirmed tumor expression of CD19 and/or CD22. The first 9 patients were treated at doses ranging from 0.5 ug/kg/dose to 20 ug/kg/dose. No drug was detectable in those patients and they experienced no DLT or clinical responses. DT2219 was detectable in the serum at doses ≥40 µg/kg/dose (N=13). Dose levels were 40 µg/kg/day N=5; 60 µg/kg/day N=5; 80 µg/kg/day N=3. The area under the curve (AUC ) range was 1,104 -1,346 and the half-life ranged from 54-84 minutes. After a single course of DT2219 we observed partial remission in a 77 year old patient with rituximab/chemotherapy-refractory CLL (dose level 40 µg/kg). The 40% reduction in the abdominal tumor mass is shown in Figure 1. This patient did not experience any DLT. A second partial remission was observed in 53 year old patient with relapsed marginal zone lymphoma (dose level 60 µg/kg) who experienced a DLT of capillary leak syndrome. After regulatory approval, that patient received a second treatment course 8 weeks later at a reduced dose of 40 µg/kg which resulted in a complete remission (Figure 2). Both patients are alive and in remission at 6 and 4 months, respectively, after therapy. Adverse events were observed in all patients who received ≥40 µg/kg. The most common was grade 1-2 capillary leak syndrome, grade 1-2 hematologic toxicity, elevated liver function tests and fatigue. Two patients experienced DLTs: at the 40 µg/kg dose (grade 3 lower extremity weakness) and 60 µg/kg dose ( grade 4 neutropenia and grade 3 capillary leak). All adverse reactions resolved completely within one week. Immunogenicity and formation of neutralizing antibodies is a major barrier in development of DT-based immunotoxins. Remarkably, none of the 8 patients with B-cell lymphoma/CLL who had received rituximab within weeks preceding therapy with DT2219 had detectable neutralizing antibody. These patients had no detectable B cells at the time of study drug administration. This suggests that rituximab pre-treatment may prevent the development of neutralizing antibodies and allow for repetitive dosing. In conclusion, we have demonstrated promising clinical efficacy of the novel immunotoxin DT2219 in refractory B cell lymphoid malignancies. We determined that the biologically active dose of DT2219 is between 40-80 µg/kg and that repetitive dosing is feasible in patients pre-treated with rituximab. Phase 2 trial is in development. [Display omitted] [Display omitted] DisclosuresVallera:The Lion Fund, William Lawrence and Blanche Hughes Foundation : Other, Research Funding.

AB0526 Sustained efficacy responses and a consistent safety profile with rituximab repeat treatment over 5 years in patients with rheumatoid arthritis and an inadequate response to tumour necrosis factor inhibitors in the reflex study

BackgroundIn the REFLEX study conducted in anti-TNF inadequate responder (TNF-IR) patients (pts) with RA, a single course of rituximab (RTX) in combination with methotrexate (MTX) significantly improved disease activity at...

Multiple Courses of Rituximab Produce Sustained Clinical and Radiographic Efficacy and Safety in Patients with Rheumatoid Arthritis and an Inadequate Response to 1 or More Tumor Necrosis Factor Inhibitors: 5-Year Data from the REFLEX Study

This 5-year observational posthoc analysis of the REFLEX study and its open-label extension assessed clinical efficacy, radiographic response, and safety of rituximab (RTX) in patients with rheumatoid arthritis (RA) who had an inadequate response to tumor necrosis factor (TNF) inhibitors. Patients in REFLEX were originally randomized to placebo (PBO) + methotrexate (MTX; PBO-randomized) or RTX + MTX (RTX-randomized). PBO-randomized patients were rescued with RTX as appropriate. Patients responding to initial RTX treatment could receive further RTX courses. For clinical efficacy and safety analyses, PBO-randomized patients were re-baselined prior to first RTX treatment and the data were pooled with RTX-randomized patient data. Efficacy outcomes 24 weeks after each course were calculated relative to first RTX pretreatment baseline. Radiographic outcomes were assessed relative to randomization baseline for both PBO-randomized and RTX-randomized groups. A total of 480 patients received ≥ 1 RTX course. At 24 weeks, American College of Rheumatology 20/50/70 responses were 62.0%, 30.8%, and 13.0%, respectively at course 1 (n = 400) and 70.3%, 41.8%, and 22.0% at course 5 (n = 91). European League Against Rheumatism good/moderate responses were 77.2% and 84.4% at courses 1 (n = 390) and 5 (n = 90). Rates of adverse events (AE), serious AE, and infections generally remained stable. Rate of progressive joint damage (PJD; change in mean Total Sharp Score) decreased over time in both PBO-randomized (n = 79) and RTX-randomized (n = 105) groups. Mean change from baseline in PJD over 5 years was greater in PBO-randomized versus RTX-randomized patients (5.51 vs 3.21). RTX re-treatment over 5 years is associated with maintained or improved efficacy, continued inhibition of PJD, and a safety profile consistent with that previously reported. A delay in initiating RTX treatment may result in increased PJD.

High Dose Rituximab Pretreatment in a New Human cd20-GAA−/− Mouse Model Causes Complete B-Cell Depletion and Reduction in Effector and Memory T-Cell Activation

High Dose Rituximab Pretreatment in a New Human cd20-GAA−/− Mouse Model Causes Complete B-Cell Depletion and Reduction in Effector and Memory T-Cell Activation

Results of the Interim Analysis of the “HUSOM” Trial: Hungarian Study of Maintenance After Rituximab Pretreatment, A Multicentre, Phase III, Open-Label Study Evaluating the Benefit of a Long-Term MabThera (rituximab) Maintenance Therapy in Patients with Advance Follicular Lymphoma After Induction of Response (CR(u) or PR) with a MabThera (rituximab)-Containing First Line Regimen

Abstract 2714 Background:The addition of immunotherapy–rituximab–to the treatment regimen of follicular lymphomas has led to a significant improvement in therapeutic efficacy. In the mid-2000’s, immuno-chemotherapy was approved by European authorities as part of the induction treatment for untreated and relapsed follicular lymphomas followed by maintenance therapy. Data about efficacy and safety of rituximab maintenance following first-line induction with R-CVP or R-CHOP have not been available. Therefore the Hungarian National Working-Group initiated a trial: Hungarian Study of Maintenance after Rituximab Pretreatment. Methods: Between July 2005 and December 2008 126 newly diagnosed patients with follicular lymphoma who had achieved partial or complete remission after induction treatment with either R-CVP or R-CHOP were treated with two years of rituximab maintenance. In this open-label, single-arm study rituximab was administered in standard dose (375 mg/m2) every 8 weeks, 12 times in total. Results:126 patients have been included. Data of 99 patients are available for this interim analysis. Average age of the 30 male (30%) and 69 female (70%) patients were 53.2 (29–80) years. Histological subtypes were grade-1 in 32% of patients, grade-2 in 49%, and grade-3a in 19%. Twenty-eight percent of patients had tumors with size over 7 cm. Sixty-four percent of patients were in the moderate risk group (FLIPI: 1–2) and 36% in the high risk group. Fifty-six percent of patients received R-CVP and 44% R-CHOP induction treatment. After induction 61% achieved complete remission and 39% partial remission. With respect to induction treatment regimen, patients treated with R-CVP 52% showed CR and 48% PR. In patients treated with R-CHOP 75% had CR and 25% had PR. Fourteen patients (37%) in PR converted to CR during or after rituximab maintenance therapy. Three-year OS after initiation of maintenance was 94%, EFS was 84%, and PFS was 88%. With respect to FLIPI, OS rates were 97% and 89% for moderate and high risk groups respectively. Patients treated with R-CHOP achieved significantly better PFS (98% vs. 80%; p=0.0096) and EFS (93% vs. 76%; p=0.0332) than those treated with R-CVP. Patients achieving CR compared to PR showed significantly higher PFS (100% vs. 68 %; p<0.0001) and EFS (95 % vs. 65 %; p<0.0001) values respectively. Safety: Four patients experienced SAEs. Three patients died (due to causes other than lymphoma). Two hepatitis B reactivations occurred. Conclusion:These data confirm that rituximab maintenance is a safe and effective treatment for patients after induction with rituximab based immuno-chemotherapy. Interestingly those patients who were treated with more intense induction regimen R-CHOP and those who achieved a better remission status seemed to have a favorable outcome. Thirty-seven percent of patients receiving rituximab maintenance converted from PR to CR. Therefore these data support the use of rituximab maintenance in 1st line treatment. Disclosures:No relevant conflicts of interest to declare.

Fractionated 90y Ibritumomab Tiuxetan (Zevalin™) Radioimmunotherapy As An Initial Therapy of Follicular Lymphoma – First Results From a Phase II Study in Patients Requiring Treatment According to GELF/BNLI Criteria

Abstract 102 Background:Radioimmunotherapy (RIT) has proven to be highly active in relapsed follicular lymphoma (FL) and the best single agent efficacy results in frontline therapy of FL were obtained with Iodine-131 Tositumomab (Bexxar™) (Kaminski et al NEJM 2005); albeit half of the patients had low tumor burden. In patients with higher tumor burden, using more than one fraction of RIT increases the overall radiation dose over that of a single fraction of treatment, thereby potentially improving both the response rates and survival (Illidge et al, Blood 2009). Methods:We conducted an international, multicenter phase 2 trial to evaluate the efficacy and toxicity of Fractionated 90Y Ibritumomab tiuxetan (Zevalin™) RIT as an initial therapy of Follicular Lymphoma. Eligible patients had untreated follicular lymphoma (grade 1, 2, or 3a) and at least one criterion of high tumour burden - one lymphoma lesion greater than 7 cm or three separate nodes of 3 cm or more; symptomatic splenic enlargement; raised serum concentrations of either lactate dehydrogenase or β2-microglobulin; compressive syndrome; or the presence of B symptoms. Treatment consisted of two doses of 90Y-ibritumomab tiuxetan (11.1 MBq/kg) given 8–12 weeks apart. Patients with greater than 20% bone marrow involvement (BM) with lymphoma received 4 weekly infusions Rituximab (375 mg/m2) and proceeded to fractionated RIT only if a repeat BM biopsy demonstrated clearing of lymphoma with less than or equal to 20% involvement. The primary endpoint was end of treatment response (EOR) of the intent-to-treat (ITT) population according to IWC 1999, assessed 12 weeks after last 90Y Ibritumomab tiuxetan infusion (21 weeks after treatment start). Secondary objectives were safety and progression free survival (PFS). Results:74 patients with a median age of 61 years (28−80), including 58 (78%) with stage III–IV stage, 23 (31%) intermediate risk FLIPI 2 and 34 (46%) with high risk FLIPI 3–5; were included between June 2007 and June 2010 in 7 centres. Thirteen (18%) patients with >20% BM involvement required rituximab pre-treatment, 2/74 did not qualify for RIT, meaning 72 received the first 90Y Ibritumomab tiuxetan infusion and 55 (76%) completed the full treatment schedule. The 2nd infusion of RIT was withheld secondary to hematologic toxicity with 1st infusion (n=12, 17%) or human anti murine antibodies positive testing (n = 4; 5.6%) or other (n = 1, 1.4%). Two out of 72 patients did not have recorded response data and the EOR was 95.7% (67/70) with CR/CRu of 57.1% (40/71). Six patients subsequently improved response making an ORR of 97.1% (68/70) (95% CI 90.0% – 99.7%), and CR/CRu of 64.3%% (45/70) (95% CI 51.9% – 75.4%). For the subset of 17 patients who only received a single 90Y Ibritumomab tiuxetan infusion, ORR (CR/CRu) was 100% (76,5%). At a median follow-up of 1.52 years (range 0.13 – 3.69 years) the PFS is 67 %, 20 patients have progressed and 12 of these have required further treatment (8 chemotherapy, 2 radiotherapy, 2 other). Updated data with median follow-up of more than 2 years will be presented.Ten patients experienced at least one SAE during the treatment period, with 3 related to study treatment (one case of rigors associated with the first infusion of rituximab and 2 cases of neutropenic sepsis both associated with the second RIT dose. The most common toxicity was hematologic: after the first 90Y Ibritumomab tiuxetan dose, related G3-4 hematological AEs were transient neutropenia (20.8%, 18 days median duration) and thrombocytopenia (20.8%; 20 days median duration). After the second 90Y Ibritumomab tiuxetan dose, related G3-4 hematological AEs increased to 36.4% for neutropenia (31days median duration), 14% for anemia (8/55 required transfusion) and 56.4% for thrombocytopenia (40 days median duration). There has been one case of MDS diagnosed 26 months after treatment and one death due to metastatic breast cancer diagnosed 9 months post last dose of 90Y Ibritumomab tiuxetan Conclusion:Fractionated RIT using 90Y-ibritumomab tiuxetan is an effective frontline treatment of advanced-stage follicular lymphoma in patients with high tumor burden requiring treatment and delivers high response rates. The treatment was well tolerated by patients with few infectious episodes and AE's and manageable hematologic toxicity. Disclosures:Illidge:Bayer Schering Pharma: Honoraria, Research Funding, Speakers Bureau. Morschhauser:Roche: Consultancy, Honoraria.

Weekly Infusion of Bortezomib In Combination with Rituximab In Relapsed/Refractory Indolent Non-Follicular and Mantle Cell Lymphoma Is Safe and Effective: Two-Years Analysis of Phase II Trial BRIL06 of Intergruppo Italiano Linfomi (IIL)

AbstractAbstract 3965Introduction. Gene-profiling studies demonstrated a constitutive activation of the NFκB signalling pathway in Mantle Cell Lymphoma (MCL) and Marginal Zone Lymphoma (MZL). Bortezomib, a potent inhibitor of the 26S proteasome, is a good candidate to block this pathway and was tested in relapsed or refractory MCL with encouraging results (objective response up to 45%, with a median PFS of 5–7 months). In vitro, the combination of Bortezomib and Rituximab has been shown synergistic apoptosis and enhanced NFkB depletion in MCL and MZL cells. Aim. On these bases, the IIL conducted a phase II multicenter study to evaluate safety and efficacy of Rituximab and Bortezomib combination in relapsed/refractory indolent non-follicular lymphoma (Linfocytic Lymphoma, LL, or MZL) and MCL not eligible for high-dose chemotherapy. Patients and methods. Inclusion criteria were: age 18–75 years, histological proven relapsed or refractory LL, MZL and MCL after 1–4 lines of therapies. Treatment plan was: one course of four weekly intravenous bolus of 1.6 mg/sqm Bortezomib in combination with four infusion of 375 mg/sqm Rituximab followed by two courses of four weekly bolus of 1.6 mg/sqm Bortezomib. Patients with complete (CR), partial remission (PR) and stable disease at the intermediate evaluation were planned to be given three further courses with the same schedule. Results. From September 2006 to March 2008, 55 patients entered into the study. Central histology revision was performed. Forty-nine patients fulfilled inclusion criteria and were evaluable. Clinical characteristics were: median age 68 (50-74) years; 16 LL, eight MZL, 25 MCL; 42 stage III/IV; 33 bone marrow involvement; 20 at intermediate-high/high International Prognostic Index (IPI) risk. Thirty-eigh patients performed more than two prior lines of chemotherapy; 34 were Rituximab-pretreated; 21 refractory and 28 relapsed disease. Overall Response Rate (ORR) was 53% (CR 26.5%, PR 26.5%); no response 43% and 4% off therapy for other causes. ORR by histology was: 37% in LL, 50% in MZL and 64% in MCL. ORR was not adversely affected by Rituximab pretreatment: Rituximab-pretreated 62% and Rituximab-naïve 33%. ORR was higher in relapsed patients compared with refractory ones: 64% and 38% (p .06). (Table 1). With a median follow-up of 25 months, 2-year Overall Survival (OS) was 80% (95%CI: 66–89) and 2-year PFS was 25% (95%CI: 14–38) (Figure 1A, 1B). Two-year PFS by histology was shown in Figure 1C. A total of 233 courses were delivered with a median of 4.7 courses/patient. Thirty patients completed the treatment plan; 19 did not due to progression disease in 13, adverse events in five (concomitant gastric neoplasia, neurotoxicity grade II, sepsis, pleural effusion and toxic death due to interstitial pneumonia). Grade 3–4 CTC haematological toxicity was rare: neutropenia in 5% of the courses and thrombocytopenia in <2%. Grade 3–4 CTC cumulative non-hematological toxicity was observed in 4.7% of all courses. The most frequent non-hematological toxicities were: neurotoxicity grade III in four patients, with complete recover or return to grade I in all of them. Infections were observed in eight patient with ten events: viral reactivation in four, pneumonia in three, sepsis in one and micosis in two. Conclusions. The combination of Rituximab and Bortezomib in weekly schedule was effective and safe in treatment of relapsed/refractory indolent and MCL. PFS was promising also in Rituximab-pretreated patients and mainly in MZL and MCL.Table 1ORR (%)MCL/MZL/LL64/50/37Rituximab-pretreated/Rituximab-naive62/33N of prior therapies <2/≥275/46Relapsed/Refractory64/38 [Display omitted] Disclosures:Off Label Use: The use of Bortezomib is off-label in Relapsed/Refractory Indolent Non-Follicular and Mantle Cell Lymphoma. Vitolo:Roche-Italy: Advisory committee; Celgene-Italy: Advisory committee; Janssen-Cilag: Lecture Fee.

Accelerated Allogeneic Haemopoietic Engraftment after Preceding Rituximab-Therapy

The chimeric monoclonal anti-CD20 antibody rituximab and the graft-versus-leukaemia/lymphoma effect after allogeneic stem cell transplantation have improved therapy of lymphatic malignancies during the last decade. In addition, successful therapy of chronic graft-versus-host disease after stem cell transplantation with rituximab has been published. Other investigators correlated prolonged neutropenia after autologous stem cell transplantation with prior rituximab- therapy. To address the question, if rituximab pre-treatment prior to transplantation diminishes the graft versus lymphoma effect afterwards and can prolong engraftment of allogeneic haemopoiesis, we retrospectively analysed data from 34 patients al- lografted for lymphoid malignancies. 19 patients had received at least one course of rituximab prior to allogeneic stem cell transplantation and 15 did not. Both groups matched very good. Patients with rituximab pre-treatment engrafted with 1 leukocyte/nl significantly faster than patients without prior rituxi- mab therapy (12 days (median, range 1-32) vs. 18 days (median, range 12-31), p<0,005). Engraftment of platelets (PLT) occurred as well faster after rituximab pre-treatment. The differences were significant for all three steps (20PLT/nl: 11,5 days (median, range 1-28) vs. 27 days (median, range 11-54) p<0,001; 50PLT/nl: 16,5 days (median, range 11-37) vs. 42 days (median, range 22-70) p<0,001; 100PLT/nl: 22,5 days (median, range 13-52) vs. 60 days (median, range 25-117) p<0,005). The mechanism leading to faster engraftment remains unclear so far. Preceding therapy with rituximab had no influence on event-free survival, overall survival, or manifestation freedom from graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. Patients with GVHD had a significantly better overall survival (p=0,0001) comparing to patients without. In conclusion, this investigation gives no hint for a suppression of GvH and GvL effects by rituximab administration prior to allogeneic stem cell transplantation. In addition, in contrast to results published for autologous stem cell transplantation, no detrimental effects on establishment of graft haemopoiesis after allogeneic stem cell transplantation by preceding ri- tuximab therapy were found.

Presence of heterozygous ATM deletion may not be critical in the primary response of chronic lymphocytic leukemia cells to fludarabine

Abnormalities of the TP53 or ATM, cooperating tumor-suppressor genes, significantly worsen the treatment options for chronic lymphocytic leukemia (CLL) patients. Although the aberrations seem to be mutually exclusive in this leukemia, inactivation of the former gene leads to worse prognosis. We tested the in vitro sensitivity of the CLL samples with heterozygous ATM deletion to fludarabine and combination of fludarabine and rituximab; the responses were compared with the TP53-abnormal and wild-type (wt) cells to delimitate relative significance of ATM deletion. In vitro analysis was performed on fifty-nine characterized CLL samples using viability assay WST-1. Western blot and real-time RT-PCR were used to monitor the activation of the ATM/p53 pathway. At the clinically relevant concentration of fludarabine, TP53-abnormal samples exhibited markedly higher resistance to fludarabine than the remaining CLL samples (P = 0.012); cohort with ATM deletion was not more resistant than wt cells. A similar induction of the p53 protein and its downstream target genes PUMA and BAX in ATM-deleted and wt cells confirmed that the former subgroup has preserved a critical pro-apoptotic response. Proportions of the samples, which had been sensitized to fludarabine by rituximab pretreatment, were insignificantly lower (P = 0.22) in the TP53-abnormal and ATM-deleted subgroups compared to the wt cases (30%; 29%; 50%, respectively). The presence of ATM (11q22-23) deletion in the CLL cells should not be considered an indication of resistance to fludarabine or its combination with rituximab.

Impaired B-cell reconstitution in lymphoma patients undergoing allogeneic HSCT: an effect of pretreatment with rituximab?

Allogeneic hematopoietic SCT (HSCT) is increasingly considered an option in refractory or relapsing lymphoma. Today, most patients with B-cell lymphoma are treated with the monoclonal anti-CD20 antibody rituximab before HSCT. We hypothesized that prior therapy with rituximab might alter immune reconstitution after allogeneic transplantation due to in vivo depletion of B cells at the time of graft infusion. We studied B-cell immune reconstitution in 12 patients with lymphoma receiving rituximab 1-12 months before HSCT. Compared to an age- and sex-matched population of patients transplanted for myeloid malignancies, lymphoma patients with rituximab pretreatment showed significantly reduced B-cell counts at time of HSCT at +3, +6 and +12 months; B-cell counts reached values comparable to controls only 24 months after HSCT. In parallel, levels of immunoglobulins were markedly reduced for up to 2 years post transplant in patients with prior rituximab treatment. Two patients suffered from severe late bacterial infections to which the impaired humoral immunity may have contributed. In contrast, T- and NK-cell reconstitution was not different compared to control patients.In conclusion, B-cell reconstitution can be significantly delayed in allogeneic HSCT recipients with prior rituximab treatment. Rituximab appears to have clinical consequences beyond the immediate early post-transplant period.

Rituximab blocks binding of radiolabeled anti-CD20 antibodies (Ab) but not radiolabeled anti-CD45 Ab

AbstractRituximab therapy is associated with a long in vivo persistence, yet little is known about the effect of circulating rituximab on B-cell non-Hodgkin lymphoma (B-NHL) targeting by the other available anti-CD20 monoclonal antibodies (MoAbs) 131iodine-tositumomab and 90yttrium-ibritumomab tiuxetan. Therefore we assessed the impact of preexisting rituximab on the binding and efficacy of second anti-CD20 MoAbs to B-NHL and determined whether targeting an alternative lymphoma-associated antigen, CD45, could circumvent this effect. We demonstrated that rituximab concentrations as low as 5 μg/mL nearly completely blocked the binding of a second anti-CD20 MoAbs (P < .001), but had no impact on CD45 targeting (P = .89). Serum from patients with distant exposures to rituximab also blocked binding of anti-CD20 MoAbs to patient-derived rituximab-naive B-NHL at concentrations at low as 7 μg/mL, but did not affect CD45 ligation. A mouse xenograft model (Granta, FL-18, Ramos cell lines) showed that rituximab pretreatment significantly reduced B-NHL targeting and tumor control by CD20-directed radioimmunotherapy (RIT), but had no impact on targeting CD45. These findings suggest that circulating rituximab impairs the clinical efficacy of CD20-directed RIT, imply that novel anti-CD20 MoAbs could also face this same limitation, and indicate that CD45 may represent an alternative target for RIT in B-NHL.

In Vitro Evaluation of Radioprotective and Radiosensitizing Effects of Rituximab

Clinical radioimmunotherapies with anti-CD20 monoclonal antibodies involve administering a predose of unlabeled anti-CD20 antibodies to favorably alter the biodistribution profile of the subsequently administered radiolabeled antibodies and mediate antitumor effects. Prior in vitro data suggested that unlabeled anti-CD20 monoclonal antibodies radiosensitize lymphoma cells as well. We assessed the antiproliferative and possible radiosensitizing capabilities of an anti-CD20 monoclonal antibody, rituximab. Luciferase-transfected (via a lentivirus vector) CD20+ human Raji lymphoma cells in log-phase growth were incubated with or without rituximab (20 microg/mL) for either 1 or 24 h before external-beam radiation exposure. Cell counts were measured with a luciferase assay at 24-h intervals. Subsets of these cells were also analyzed for cell cycle status by flow cytometry. Rituximab pretreatment and irradiation were found to significantly inhibit tumor cell growth compared with irradiation alone (by a factor of 0.40 at 1 Gy [P < 0.01]). One hour of rituximab pretreatment modestly radiosensitized tumor cells at a radiation dose of 1 Gy (by a factor of 1.03 compared with the results for nonirradiated cells). At higher radiation doses (2 and 12 Gy), 1 h of rituximab pretreatment paradoxically radioprotected tumor cells by factors of 0.25 (P < 0.01) and 0.54 (P < 0.05), respectively. Rituximab predosing for 24 h was found to be radiosensitizing at a radiation dose of 4 Gy (by a factor of 2.84 [P < 0.01]) but radioprotective at radiation doses of 1, 8, and 12 Gy (by factors of 0.10 [P < 0.01], 2.50 [P < 0.01], and 2.07 [P < 0.05], respectively). These results correlated with retardation of the cell cycle at 6 d after rituximab administration, as determined by flow cytometry. Rituximab demonstrated a direct tumor antiproliferative effect in the absence of radiation. At lower levels of radiation exposure, rituximab radiosensitized Raji lymphoma cells. At higher doses of radiation, rituximab paradoxically protected tumor cells against ionizing radiation, possibly through effects on the cell cycle. These radiobiologic effects of rituximab should be carefully considered in the design of radioimmunotherapeutic trials.

Rituximab Blocks Binding of Radiolabeled Anti-CD20 Antibodies (Ab) but Not Radiolabeled Anti-CD45-Ab.

The use of rituximab for treatment of B-cell non-Hodgkin lymphoma (B-NHL) is associated with a long in vivo persistence (months), which is enhanced by protracted maintenance regimens. At the time of progression, the radioimmunoconjates131I-Tositumomab and 90Y-Ibritumomab Tiuxetan are often employed to induce remissions in patients with relapsed B-NHL. Despite this common clinical scenario, little is known about the potential impact of high levels of circulating anti-CD20 antibody (Ab) on targeting of a second radiolabeled anti-CD20-Ab. Furthermore, alternate strategies to overcome potential blocking of the radiolabeled Ab have not been identified. In order to address this critical question we evaluated the impact of rituximab on both CD20 and CD45 targeting of B-NHL. First, we demonstrated that CD45 and CD20 were expressed in comparable densities on B-NHL lines (FL-18, Granta, Ramos). Next, blocking by rituximab was assessed by co-incubation of B-NHL lines with increasing concentrations of rituximab (0–2000μg/ml), followed by secondary incubation with either fluoresceinated tositumomab or fluoresceinated BC8 (anti-CD45). Evaluation by flow cytometry revealed that rituximab concentrations as low as 5μg/mL reduced tositumomab binding to CD20 by 86% (p=.00027), with complete blocking occurring at concentrations as low as 50μg/mL (p=0.00017). In contrast, rituximab had no effect on CD45 ligation (p=0.89). We then showed that serum derived from patients treated with rituximab (> 4 weeks prior to blood draw) had rituximab concentrations ranging from 14.6–82.2μg/ml and could fully block the binding of tositumomab to rituximab-naïve, patient-derived B-NHL cells at concentrations as low as 7.3μg/mL. Rituximab-containing serum exhibited no impact on binding of anti-CD45 Ab. Experiments assessing the biodistribution of radiolabeled Ab in human B-NHL xenografts in athymic mice demonstrated that the estimated radiation dose that would be delivered to tumor sites (% injected dose/g tumor) with 131I-labeled Ab was comparable with Tositumomab or BC8 in a rituximab-free system (p=0.54). In contrast, mice pre-treated with rituximab exhibited a 55% reduction in the radiation dose that would be delivered to tumor xenografts using 131I-labeled Tositumomab (p=0.00026), but no effect was observed on the estimated radiation dose that would be delivered by radioiodinated BC8 (p=.66). Therapy experiments confirmed the predictions of the biodistribution studies and demonstrated similar tumor control for 131I-Tositumomab and 131I-BC8 in the absence of rituximab (p=.21), but inferior tumor control for rituximab pre-treated mice receiving 131I-Tositumomab as compared to rituximab + 131I-BC8 (p=.00016). Likewise, survival was inferior for mice treated with rituximab followed by 131I-Tositumomab compared to mice treated with 131I-Tositumomab alone (p=.05), whereas the survival of mice treated with 131I-BC8 were not impacted by rituximab pre-treatment (p=.36). Our data indicate that even low concentrations of long half-lived Ab such as rituximab, can impair the binding of a secondary monoclonal Ab targeting the same antigen. These results identify major implications for the current clinical use of CD20-directed lymphoma therapy and uncover potential limitations for the development of novel therapeutic antibodies that compete for identical antigens.

Septic polyarthritis with Ureaplasma urealyticum in a patient with prolonged agammaglobulinemia and B-cell aplasia after allogeneic HSCT and rituximab pretreatment

A recent study in this journal1 raised the issue of prolonged hypogammaglobulinemia in patients receiving autologous hematopoietic stem cell transplantation (HSCT) for high-risk B-cell lymphoma and maintenance therapy with rituximab. Despite significant hypogammaglobulinemia, these patients did not experience significant infectious complications. With the following case report, we aim to increase the awareness of the potential for severe infectious complications associated with the hypogammaglobulinemia observed in lymphoma patients given lymphocytotoxic chemotherapy in combination with rituximab before allogeneic HSCT.2