Results of a phase I-II study of fenretinide and rituximab for patients with indolent B-cell lymphoma and mantle cell lymphoma.

Fenretinide, a synthetic retinoid, induces apoptotic cell death in B-cell non-Hodgkin lymphoma (B-NHL) and acts synergistically with rituximab in preclinical models. We report results from a phase I-II study of fenretinide with rituximab for B-NHLs. Eligible diagnoses included indolent B-NHL or mantle cell lymphoma. The phase I design de-escalated from fenretinide at 900 mg/

Anti-CD45 Pretargeted Radioimmunotherapy Prior to Bone Marrow Transplantation without Total Body Irradiation Facilitates Engraftment From Haploidentical Donors and Prolongs Survival in a Disseminated Murine Leukemia Model

s / Biol Blood Marrow Transplant 19 (2013) S211eS232 S228 chemotherapy was HIDAC (1-3 grams/m2 for 6-8 doses)/ Etoposide(15-40mg/kg) in 16 patients and growth factor alone in one patient. Median time from diagnosis to ASCT was 4.2 (range 3.6-7) months. Preparative regimen for ASCT was Busulfan (3.2mg/kg x 4)/Etoposide (60 mg/kg) in 12 patients and high dose melphalan in 5 patients. The median CD34 cells infused was 4.9 x 10e6/kg (range 2.8 to 15.9).All patients engrafted with a median time to neutrophil engraftment of 11 (range10-12) days. The median time to platelet engraftment was 20 (range15-40) days. The median length of inpatient stay during the ASCT admission was 14 (range 10-25) days. One patient died of progressive disease 14 months post ASCT. Two patients died in remission on day 53 (sepsis) and day 836 (unknown cause) post ASCT. Fourteen patients (82%) are currently alive in complete remission. at a median follow-up of 20 (range 140) months post ASCT. Conclusion: Consolidation of good risk AML patients with ASCT following induction of complete remission is safe and effective in preventing relapse in good risk AML patients

Pretargeted Radioimmunotherapy Using Genetically Engineered Antibody-Streptavidin Fusion Proteins for Treatment of Non-Hodgkin Lymphoma

Pretargeted radioimmunotherapy (PRIT) using streptavidin (SAv)-biotin technology can deliver higher therapeutic doses of radioactivity to tumors than conventional RIT. However, “endogenous” biotin can interfere with the effectiveness of this approach by blocking binding of radiolabeled biotin to SAv. We engineered a series of SAv FPs that down-modulate the affinity of SAv for biotin, while retaining high avidity for divalent DOTA-bis-biotin to circumvent this problem

Bendamustine (Treanda®), Etoposide and Dexamethasone (BED) Followed by GCSF Effectively Mobilizes Autologous Peripheral Blood Hematopoietic Stem Cells

Abstract 4126 Background: High dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard of care for patients with advanced or treatment refractory multiple myeloma (MM) and non-Hodgkin lymphoma (NHL). Stem cell proliferation and mobilization can be enhanced though the addition of myelosuppressive chemotherapy to GCSF administration. Chemotherapeutic agents without cross resistance to prior therapies may support peripheral blood stem cell (PBSC) collection and improve patient outcomes by exacting a more potent direct anti-tumor effect prior to ASCT. Bendamustine (Treanda®) is a synthetic chemotherapeutic agent that shares structural similarities to both purine analog and alkylating agents without significant cross resistance to other compounds in either drug class. Bendamustine appears to have low stem cell toxicity in vitro, is well tolerated, and has activity in MM and NHL. We hypothesized that bendamustine9s activity in patients with disease resistant to first line therapies makes it a logical candidate for chemotherapy based PBSC mobilization. Methods: Patients were eligible if they had relapsed or refractory MM, B-cell NHL or T-cell NHL and were candidates for ASCT. Other criteria included: age >18 years, ANC >1,500/mm 3 , platelets >100,000/mm 3 , adequate renal and hepatic function, 2 IV d 1, 2 - provided along with financial support for this study by Teva Pharmaceuticals), etoposide (200 mg/m 2 IV d 1– 3), dexamethasone (40 mg PO d 1– 4), delivered as an outpatient, followed by filgrastim (10 mcg/kg/day; starting on d 5 through end of collection)]. Apheresis was initiated when peripheral blood CD34 cell counts were >5/μL. The primary endpoint was successful mobilization, defined as collection of >2.0 × 10 6 CD34 cells/kg. Adverse events (AEs) were graded using the CTCAE v4.0. Results: Twenty patients (16 MM, 3 B-cell NHL, 1 NK/T-cell NHL) were treated. The median age was 59 years (range 43–70), and the median number of prior therapies was 1 (range 1–3) for MM and 2 (range 2–3) for NHL patients. All patients (20/20) were successfully mobilized. The median number of CD34 + cells collected was 19.11 × 10 6 /kg (Mean 22.49; range 4.35 to 55.51 × 10 6 ). All MM patients collected >10 × 10 6 CD34 + cells/kg. The median time from BED mobilization therapy to the first day of CD34 stem cell collection was 12 days (mean 12.05; range 10 to 20 days). The median number of days of apheresis was 1 (mean 1.45; range 1 to 4). A predictable pattern of leucocyte nadir and recovery was demonstrated (88% of patients started apheresis between days 10–12). One patient (5%) was given plerixafor and for 2 patients (10%) the dose of GCSF was increased to 16 mcg/kg twice daily. Among the 20 patients mobilized and collected, 12 have thus far undergone ASCT and 100% (12/12) have achieved an unsupported neutrophil count >500/μL at an average of 14.3 days after PBSC infusion and a platelet count >20K/μL at an average of 10 days. Serious AEs (SAEs) were observed in 5 patients and 1 patient died due to disease progression. No unexpected grade 3 or greater treatment related SAEs were seen. Disease response assessments are ongoing. The original protocol design involved 3 agents (bendamustine, dexamethasone and GCSF [BDG]). After the first 3 patients enrolled, the mobilization regimen was modified to include etoposide because BDG did not yield a predictable pattern of leucocyte nadir and recovery, thus complicating timing for apheresis (median time to collection 22 days). The first 3 patients were censored from the analysis, however all 3 patients were successfully mobilized and collected. Conclusions: The initial experience with PBSC mobilization after BED in this phase II study suggests the regimen is safe and effective, while the use of BDG does not yield predictable CD34 kinetics. Time to neutrophil and platelet engraftment after ASCT appears unimpaired when compared with other chemotherapy based mobilization regimens. Large numbers of stem cells were rapidly mobilized and resulted in short durations of apheresis. No patient with MM collected 6 CD34 + cells/kg (sufficient for 2 ASCTs). The regimen was very well tolerated and these findings suggest that the role of bendamustine in PBSC mobilization should be further explored. Disclosures: Green: Teva Pharmaceuticals: Research Funding. Holmberg: Millenium: Research Funding; Otsuka: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Sanofi: Research Funding. Budde: Teva Pharmaceuticals: Research Funding. Gopal: Teva Pharmaceuticals: Research Funding

Combining a CD20 chimeric antigen receptor and an inducible caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma.

Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a "suicide gene" relying on inducible activation of caspase 9 (iC9), and a truncated CD19 selectable marker. Rapid expansion (2000 fold) of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20(+) malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned

Pretargeted radioimmunotherapy using genetically engineered antibody-streptavidin fusion proteins for treatment of non-hodgkin lymphoma.

Purpose: Pretargeted radioimmunotherapy (PRIT) using streptavidin (SAv)-biotin technology can deliver higher therapeutic doses of radioactivity to tumors than conventional RIT. However, "endogenous" biotin can interfere with the effectiveness of this approach by blocking binding of radiolabeled biotin to SAv. We engineered a series of SAv FPs that downmodulate the affinity of SAv for biotin, while retaining high avidity for divalent DOTA-bis-biotin to circumvent this problem.Experimental Design: The single-chain variable region gene of the murine 1F5 anti-CD20 antibody was fused to the wild-type (WT) SAv gene and to mutant SAv genes, Y43A-SAv and S45A-SAv. FPs were expressed, purified, and compared in studies using athymic mice bearing Ramos lymphoma xenografts.Results: Biodistribution studies showed delivery of more radioactivity to tumors of mice pretargeted with mutant SAv FPs followed by (111)In-DOTA-bis-biotin [6.2 +/- 1.7% of the injected dose per gram (%ID/gm) of tumor 24 hours after Y43A-SAv FP and 5.6 +/- 2.2%ID/g with S45A-SAv FP] than in mice on normal diets pretargeted with WT-SAv FP (2.5 +/- 1.6%ID/g; P = 0.01). These superior biodistributions translated into superior antitumor efficacy in mice treated with mutant FPs and (90)Y-DOTA-bis-biotin [tumor volumes after 11 days: 237 +/- 66 mm(3) with Y43A-SAv, 543 +/- 320 mm(3) with S45A-SAv, 1129 +/- 322 mm(3) with WT-SAv, and 1435 +/- 212 mm(3) with control FP (P < 0.0001)].Conclusions: Genetically engineered mutant-SAv FPs and bis-biotin reagents provide an attractive alternative to current SAv-biotin PRIT methods in settings where endogenous biotin levels are high. Clin Cancer Res; 17(23); 7373-82. (C)2011 AACR

Bendamustine (Treanda®)-Based Regimens Are Effective In Mobilizing Peripheral Blood Hematopoietic Stem Cells For Autologous Transplantation

Background High dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard of care for patients with advanced or treatment refractory multiple myeloma (MM) and non-Hodgkin lymphoma (NHL). Stem cell proliferation and mobilization can be enhanced though the addition of myelosuppressive chemotherapy prior to GCSF administration. Chemotherapeutic agents without cross resistance to prior therapies may support peripheral blood stem cell (PBSC) collection and improve patient outcomes by exacting a more potent direct anti-tumor effect prior to ASCT. Bendamustine (Treanda®) is a synthetic chemotherapeutic agent that shares structural similarities to both purine analog and alkylating agents without significant cross resistance to other compounds in either drug class. Bendamustine appears to have low stem cell toxicity in vitro, is well tolerated, and has activity in MM and NHL, but the potential for the purine moiety to adversely impact stem cell reserve is unknown. We hypothesized that bendamustine’s activity in patients with disease resistant to first line therapies makes it a logical candidate for chemotherapy based PBSC mobilization and tested its impact on stem cell yield. Methods Patients were eligible if they had relapsed or refractory MM, B-cell NHL or T-cell NHL and were candidates for ASCT. Other criteria included: age >18 years, ANC >1,500/mm 3 , platelets >100,000/mm 3 , adequate renal and hepatic function, 2 IV d 1, 2 - provided along with financial support for this study by Teva Pharmaceuticals), etoposide (200 mg/m 2 IV d 1- 3), dexamethasone (40 mg PO d 1- 4), delivered as an outpatient, followed by filgrastim (initially 10 mcg/kg/d sc; starting on d 5 through end of collection)]. Apheresis was initiated when peripheral blood CD34 cell counts were >5/µL. The primary endpoint was successful mobilization, defined as collection of >2.0 x 10 6 CD34 cells/kg. AEs were graded using the CTCAE v4.0. Results Thirty-seven patients (32 MM, 4B-cell NHL, 1 NK/T-cell NHL) were treated. The median age was 60 years (range 43-70). The median number of prior therapies was 1 (range 1-3) for MM and 2 (range 1-3) for NHL patients. All patients (37/37) were successfully mobilized. The median number of CD34 + cells collected was 19.43 x 10 6 /kg (range 4.35 to 55.51 x 10 6 ). All MM patients collected >10 x 10 6 CD34 + cells/kg. The median time from the start of BED mobilization therapy to the first day of CD34 stem cell collection was 12 days (range 9 to 20 days). The median number of apheresis days was 1 (range 1 to 4). A predictable pattern of leucocyte nadir and recovery was demonstrated (95% of patients started apheresis between days 9-13). Two patients (5%) were given plerixafor and for 2 patients (5%) GCSF was increased to 16 mcg/kg twice daily. Among the 37 patients mobilized and collected, 31 have thus far undergone ASCT and 100% (31/31) achieved an unsupported neutrophil count >500/µL at a median of 15 days (range 7-19) after PBSC infusion and a platelet count >20K/µL at a median of 11 days (range 8-14). Ten SAEs were observed in 8 patients and 1 patient died due to disease progression prior to ASCT. SAEs include: neutropenic fever (1, grade [GR] 3), bone pain (2, GR 3), renal insufficiency (1, GR 1), atrial fibrillation (1, GR 2), hypotension (1, GR 3), stroke (1, GR 2), and one patient accounted for 3 SAEs including GR 3 tumor lysis syndrome and sepsis and GR 5 disease progression. Among twenty-nine evaluable patients to date, responses include: CR= 4 PR=2, SD=19 and PD=4. The ORR to this single cycle of therapy was 21%. Conclusions PBSC mobilization with BED is safe and effective. BED is not an acute stem cell toxin. Large numbers of stem cells were rapidly mobilized and resulted in short durations of apheresis. No patient with MM collected 6 CD34 + cells/kg (sufficient for 2 ASCTs). Twenty-one percent of patients demonstrated a measurable response to a single cycle of BED therapy and an additional 65% of patients had stable disease. In patients who were transplanted, the time to neutrophil and platelet engraftment was comparable to other chemotherapy based mobilization regimens. The BED regimen was well tolerated and these findings suggest that the role of BED in PBSC mobilization should be further explored. Disclosures: No relevant conflicts of interest to declare

Results of a Phase I-II Study of Fenretinide and Rituximab for Patients with B-Cell Lymphomas

Background: Despite improved treatment options, indolent non-Hodgkin lymphoma (NHL) and Mantle Cell Lymphoma (MCL) remain incurable for most patients. Fenretinide (4-hydroxy(phenyl)retinamide; 4-HPR), an orally bioavailable synthetic retinoid, has been shown to induce apoptotic cell death in a variety of tumor types, presumably via intratumoral induction of oxygen free radicals. Our group has shown single-agent fenretinide anti-B-NHL activity as well as synergy with anti-CD20 antibody therapy when tested in vitro (Shan et al Clin Cancer Res . 2001) and in human xenograft models (Gopal et al, Blood 2004). These preclinical data support the first trial to evaluate this strategy of fenretinide and rituximab in patients with B-cell malignancies. Here we report results from a phase I-II study evaluating the safety and efficacy of fenretinide with rituximab for indolent NHL and MCL. Methods: This was an open-label, phase I/II study conducted at the Fred Hutchinson Cancer Research Center (FHCRC) and Seattle Cancer Care Alliance, Seattle, WA (ClinicalTrials.gov identifier: [NCT00288067][1]). The study was approved by the institutional review board at FHCRC and written informed consent was obtained from all patients. Major eligibility criteria included: CD20 positive lymphoid malignancy, radiographically measurable disease, ECOG PS ²2, and adequate organ function. The phase I portion evaluated the safety of dosing single agent fenretinide at 900 mg/m2 PO BID for days 1-5 of a 7 day cycle and allowed de-escalation for excess toxicity. The phase II portion added 375 mg/m2 IV rituximab weekly on weeks 5-9 then every 3 months. All patients could remain on therapy until disease progression or unacceptable toxicity. Response assessment occurred approximately every 3 months. Correlative studies included fenretinide pharmacokinetics, serum retinol concentrations, and evaluation of tumor expression of BCL-2 family proteins. Response was scored by standard criteria (Cheson 1999). Results: Thirty-two patients enrolled in the study: 7 patients in phase I, and 25 patients in phase II. The median age was 64 years (range, 40 - 78), 81% were male, and the median number of prior therapies was 1 (range, 0 - 10) with 22 (69%) having received prior rituximab and 8 (25%) with rituximab-refractory disease. Histologies included 13 CLL/SLL, 10 follicular (FL), 7 mantle cell (MCL), 1 lymphoplasmacytic, and 1 marginal zone. No dose limiting toxicities were observed in the phase I portion, and 900 mg/m2 was the dose level delivered in combination with rituximab for the phase II component. The most common treatment-related adverse events (AE) of any grade were reversible night blindness (n=18, 56%), other eye disorders (n=17, 53%), rash (n=12, 38%), and photosensitivity (n=8, 25%). The most common treatment related AEs of grade 3 or higher included: maculopapular rash, night blindness, and decreased lymphocyte count. Three patients (9%) discontinued treatment early due to toxicity (rash-2, GI toxicity-1). One patient with MCL in the phase I portion experienced stable disease (SD) lasting 35 months. In the phase II portion of the study, 5 (20%) patientOs disease responded (Figure), with 2 (8%) achieving complete remission (both SLL/CLL), and 3 (12%) achieving partial remission (2 FL, 1 MCL). In addition 16 (64%) patients had SD. The median progression-free survival was 9 months (range, 6 - 31 months), and the median overall survival was not reached (range, 33 months to not reached). Median time to progression of responders was 14.5 months. The one-month median peak and trough concentrations of fenretinide were 11.45 µM and 2.5 µM, respectively. Correlative studies assessing Bcl-2, BAX, and apoptosis in the 13 patients with circulating tumor cells were not able to associate these data with response, or survival. Discussion: In this phase I/II study, the combination of fenretinide and rituximab was well tolerated with predictable, reversible toxicities with up to 4.5 years of continuous therapy. Though the ORR was modest (20%), the majority had of patients had disease control (ORR + SD=84%) which lasted for > 6 months. Further study of this novel combination should focus on identifying the subset of B-NHL that is most likely to respond or the rational addition of other agents to augment these anti-tumor effects. ![Figure 1.][2] Figure 1. Disclosures Pagel: Actinium Pharmacetuicals, Inc.: Equity Ownership. Gopal: Merck: Research Funding; Emergent/Abbott: Research Funding; Cephalon/Teva: Research Funding; BioMarin: Research Funding; Sanofi-Aventis: Honoraria; Millenium: Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Piramal: Research Funding; Biogen Idec, BMS: Research Funding. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00288067&atom=%2Fbloodjournal%2F126%2F23%2F2728.atom [2]: pending:ye

Anti-CD45 Radioimmunotherapy Facilitates Donor Engraftment and Prolongs Survival in the Absence of TBI Prior to Haploidentical Bone Marrow Transplantation in a Disseminated Murine Leukemia Model

Abstract 4101 Background: Despite the curative promise of hematopoietic cell transplantation (HCT), many patients with hematologic malignancies relapse and others may not proceed to HCT due to the unavailability of a matched donor. Toxicities remain high with HCT, due in part to the administration of non-specific therapies such as total body irradiation (TBI) as part of preparative regimens. We aim to overcome these limitations by replacing TBI with anti-CD45 radioimmunotherapy (RIT) for haploidentical HCT to deliver radiation directly to leukemic cells while sparing normal organs and minimizing non-specific toxicities. Methods: We established an initial TBI HCT regimen in B6SJLF1/J mice (H-2D b haplotype) conditioned with fludarabine (FLU, days -6 to -2), followed by TBI (250, 500, 750 cGy; day -1). The mice then received 15 million donor (CB6F1/J, H-2D d ) BM cells (day 0), followed by cyclophosphamide (CY) for graft-versus-host disease (GvHD) prophylaxis (day +2). Subsequent RIT HCT studies involved B6SJLF1/J mice conditioned with and without fludarabine (FLU) and escalating doses (200–400 μCi) of 90 Y-anti-CD45 Ab (30F11) RIT without TBI, followed by infusion of haploidentical BM cells from CB6F1/J mice and a single dose of cyclophosphamide (CY) 2 days after HCT. Chimerism studies were performed using flow cytometric analysis to assay for engraftment of donor CD8 + cells. Therapeutic studies were performed in B6SJLF1/J mice given 10 5 syngeneic leukemia cells via tail vein (day -5), followed by 200 or 400 μCi 90 Y-30F11 (day -3), and 1.5 × 10 7 BM donor cells (day 0) and two doses of CY (days -2 and +2) without FLU. Results: Using this model we have demonstrated that mixed chimerism was established in mice transplanted with TBI or escalating doses (200–400 μCi) of 90 Y-30F11 RIT followed by injection of haploidentical BM donor rescue cells. TBI-based HCT showed that chimerism as determined by flow cytometric analysis for donor CD8 + cells was TBI dose-dependent; mice receiving ≥500 cGy were fully chimeric 4 weeks post-HCT, and persisted ≥12 months. RIT-based HCT also revealed mice with mixed chimerism, with up to 89% of donor CD8 + cells 1 month after HCT. Elimination of FLU from the conditioning regimen did not significantly decrease chimerism, as mice transplanted without FLU showed up to 70% donor CD8 + cells 1 month after HCT. Subsequent RIT experiments in B6SJLF1/J mice harboring AML were treated with escalating doses of 90 Y-30F11 prior to HCT without FLU. Mice treated with anti-CD45 RIT using 200 μCi and 400 μCi of 90 Y-30F11 had a median overall survival (OS) of 73 (p 90 Y-30F11 group were euthanized on day 3 for excessive weight loss, without gross histology abnormality in kidneys or liver. Conclusion: These studies suggest that anti-CD45 RIT in the absence of TBI and FLU prior to haploidentical HCT can lead to establishment of mixed chimerism. Moreover, this anti-CD45 RIT in combination with haploidentical HCT can lead to improvement in survival for mice with AML. These results suggest that clinical studies with anti-CD45 RIT in lieu of TBI and FLU in a haploidentical HCT regimen should be considered for further investigation. Disclosures: No relevant conflicts of interest to declare