Canine parvovirus (CPV) is the number one viral cause of enteritis, morbidity, and mortality in 8-week-old young puppies. We have developed twin assays (slide agglutination test [SAT] for CPV antigen and slide inhibition test [SIT] for CPV antibody) that are sensitive, specific, cost-effective, generic for all genotypes of CPV, and provide instant results for CPV antigen detection in feces and antibody quantification in serum. We found these assays to be useful for routine applications in kennels with large numbers of puppies at risk. The results of these assays are available in 1 min and do not require any special instrumentation. SAT-SIT technology will find applications in rapid screening of samples for other hemagglutinating emerging viruses of animals and humans (influenza virus and severe acute respiratory syndrome coronavirus).Canine parvovirus (CPV) is the number one viral cause of puppy enteritis and mortality (8). Unique properties of CPV make it an emerging and reemerging pathogen of dogs worldwide (2,5,16,17). Parvoviruses have a single-stranded DNA genome of 5,000 bases with a hairpin structure (4). Parvoviruses have exceptional evolutionary ability (10). Parvoviruses are extremely stable in the environment and relatively resistant to disinfectants because they are nonenveloped viruses (19). CPV multiplies in the rapidly dividing cells in the crypts of the intestine, leading to diarrhea and dehydration (4).In the kennel environment, the availability of a large number of susceptible puppies, environmental stress, and unique properties of CPV combine to form an ideal scenario for the rapid spread of CPV. Effective commercial modified live virus vaccines that vary in the genotype (CPV type 2 [CPV-2] and CPV-2b) of CPV in the vaccine are available. There is currently no commercial vaccine with CPV-2c in the vaccine. However, induction of active immunity in puppies is blocked by maternal immunity in the puppies (18). The stability of CPV in the kennel environment and excretion of large amounts of CPV by sick puppies can expose susceptible puppies to massive infectious doses of CPV. This CPV susceptibility window coincides with weaning in puppies in the age group of about 6 to 8 weeks old. Eight weeks is the age when the largest number of puppies succumb to CPV. Moreover, there are variations in the decay of antibodies and induction of active immunity after vaccination directed by the genetic makeup (canine major histocompatibility antigens) of the puppies.It would be clinically useful if there were diagnostic tests that could detect the amount of CPV in a sample, genotype the virus, and quantify the antibodies against different CPV subtypes quickly in the kennel environment. With this goal, we have developed and validated instant CPV antigen and antibody tests (slide agglutination test [SAT] for CPV antigen and slide inhibition test [SIT] for CPV antibody in serum). These tests are instant (real time), sensitive, quantitative, and generic for all the CPV types. We are confident that these safe, econo...

8081 Background: Ocaratzumab, previously known as AME-133v, is a humanized next-generation anti-CD20 monoclonal antibody. It has been optimized with a 13 to 20-fold increase in binding affinity to CD20 and improved binding to the low-affinity (F/F and F/V) polymorphisms of FcγRIIIa (CD16), which are thought to predict lower response rates and shorter duration of responses to rituximab. Methods: In a phase I dose escalation study in relapsed follicular lymphoma (FL) patients, ocaratuzumab was well-tolerated at doses up to 375 mg/m2 (Forero-Torres et al. CCR 2012). In a follow-on phase II trial, 44 patients with relapsed FL following prior rituximab and the low-affinity FcγRIIIa polymorphism (F-carriers) received 375 mg/m2 of ocaratuzumab weekly for 4 doses. In this study, overall response rate (ORR) was 36% and median progression free survival (PFS) was 91 weeks (Ganjoo et al. Haematologica 2011). Results: Amongst the 56 patients receiving 100 and 375 mg/m2 of ocaratuzumab, 8 patients had a previous time to progression of ≤ 180 days following their last rituximab treatment. These patients had a median of 2 prior rituximab treatments, (range 1-6 treatments), and median PFS following last treatment of 159 days. Five of the 8 patients showed a longer PFS after ocaratuzumab administration, compared with last rituximab treatment. All 5 patients expressed the homozygous low-affinity genotype of FcγRIIIa (F/F). At the time of study closure, 3 of the patients were still in remission (indicated by * in the table). Conclusions: This retrospective analysis suggests that ocaratuzumab may be non-cross-resistant to rituximab in patients with the low-affinity FcγRIIIa polymorphism. Prolonged PFS in selected patients following ocaratuzumab suggests that the increased binding affinity to CD16 and improved antibody-dependent cell-mediated cytotoxicity (ADCC) of this antibody is clinically relevant. As a single agent, ocaratuzumab may provide prolonged clinical benefit in relapsed FL patients and a clinical trial comparing ocaratuzumab to rituximab is in preparation. [Table: see text]

4977 Sustained Depletion of B-Cells by a Humanized, Fc-Engineered Anti-CD20 Antibody, AME-133v, in Patients with Relapsed Follicular Lymphoma J Wayne,1 K Ganjoo,2 A Forero,3 B Pohlman,4 S de Vos,5 S Carpenter,6 J Wooldridge,6 S Marulappa,1 V Jain11Mentrik Biotech, LLC, Dallas, TX, 2Standford University Medical Center, Stanford, CA, 3Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL,4Cleveland Clinic Taussig Cancer Institute, Cleveland, OH, 5David Geffen School of Medicine at University of California, Los Angeles, CA, 6Eli Lilly and Company, Indianapolis, Indiana Introduction AME-133v is a humanized anti-CD20 monoclonal antibody that has a 13 to 20-fold increase in binding affinity and approximately 6-fold more potent effector function in antibody-dependent cell-mediated cytotoxicity (ADCC) compared to rituximab. Phase I/II clinical trials of AME-133v in patients with relapsed follicular lymphoma have demonstrated an overall response rate of greater than 30% with a complete response rate of 16%. The extent and duration of depletion of CD19+ B-cells in peripheral blood was used as a surrogate of therapeutic levels of AME-133v. Analysis from the Phase I/II clinical trials is presented in this report. Methods CD-19 positive B-cells in peripheral blood were measured in 77 patients with relapsed follicular lymphoma enrolled in two phase I/II clinical trials of AME-133v. These studies assessed five different doses of AME-133v (from 2 mg/m2 to 375 mg/m2). AME-133v was administered intravenously four times at weekly intervals in both trials. Blood samples were taken at multiple time points throughout the trial and a central lab measured levels of circulating CD19+ B-cells using fluorescence-activated cell sorting (FACS). Results Excluding the four patients enrolled in the 2 mg/m2 dose cohort, depletion of peripheral B-cells occurred in all patients and was sustained over time (Table 1). Baseline levels of B-cell counts ranged from 4 × 103 to 1,187 × 103 cells/μL, with an average of 102 × 103 cells/μL and a median of 60 × 103 cells/μL. Within 24 hours of the first infusion, all patients had depletion of circulating B-cells; ninety-six percent of patients had less than 10 × 103 cells/μL and two patients had less than 20 × 103 cells/μL. Interestingly, AME-133v was effective at depleting B-cells even at doses as low as 7.5 mg/m2. To assess sustainability of B-cell depletion after four doses of AME-133v, CD19+ cell counts were evaluated at nine weeks after the fourth infusion and every three months thereafter. Complete depletion of CD19+ lymphocytes was sustained for nine weeks. At five months after the last infusion of AME-133v, nearly two-thirds of patients had no detectable circulating B-cells. Sustained B-cell depletion lasted for at least eight months following the last infusion in 63% of patients. Table 1. B-cell counts for all patients in 7.5, 30, 100 and 375 mg/m2 cohorts. Percentages are cumulative Time Point Cell Count (x 103 cells/μL) 0 < 1 2 to 10 11 to 30 31 to 50 < 100 Day 1 (24 hours after last infusion) 62 % 66 % 96 % 100 % 100% 100% Day 7 (day of infusion 2) 75% 80% 95% 97% 97% 98% Day 28 (1 week after last infusion) 78 % 87% 95% 98% 98% 100% Day 84 (9 weeks after last infusion) 78% 87% 91% 96% 96% 98% Day 174 (5 months after last infusion) 60% 60% 70% 86% 93% 100% Day 264 (8 months after last infusion) 26% 26% 41% 63% 81% 89% Day 354 (11 months after last infusion) 0% 0% 15% 40% 55% 80% DEMOGRAPHIC CHARACTERISTICS (EVALUABLE POPULATION) “\f C \l 1 Demographic and Disease Characteristics on evaluable population (N=30) Conclusion The rapid and sustained effect of AME-133v on B-cell depletion, even in low-affinity FcγRIIIa patients, indicates a potentially relevant biological activity of the antibody in treating B-cell non-Hodgkin lymphoma. Notably, this depletion occurred even at very low doses of drug administration and persisted over time. This may be related to its higher affinity for CD20, increased ADCC, or both. The sustained B-cell depletion may result in prolonged clinical response and might mitigate the need for maintenance therapy. A randomized trial is being planned to compare efficacy of AME-133v vs. rituximab. Disclosures: No relevant conflicts of interest to declare.

2475 Introduction: Anti-CD20 antibodies like rituximab are traditionally administered by the intravenous routes although clinical trials by subcutaneous routes have been initiated. AME-133 is a monoclonal anti-CD20 antibody, has been engineered to be more potent than rituximab. It has 10–20 fold higher binding affinity for the CD20 epitope and approximately 6-fold greater potency in ADCC assays relative to rituximab. The higher potency may provide benefit to patients with low affinity FcgIIIRa and enable subcutaneous administration (patient convenience). In addition, AME-133 has been engineered to be potentially safer than rituximab; AME-133 is humanized to reduce immunogenicity and the CDC activity has been diminished to potentially reduce side effects associated with tumor lysis syndrome. Phase I/II clinical studies of AME-133 in rituximab pretreated/relapsed, patients with low affinity FcγIIIRa, follicular non-Hodgkin's lymphoma (NHL) in the US and in Japan have shown excellent safety profile and response rate (RR) greater than 30%. As a prelude to investigating the clinical efficacy and safety of AME-133 in subcutaneous formulation, a safety/PK study was conducted in cynomolgus monkeys. Objective: The objective of this study was to evaluate the systemic toxicity and pharmacokinetics of AME-133 in cynomolgus monkeys at three different dose levels when administered subcutaneously once each week for 14 consecutive weeks. In addition, the effect of AME-133 on B-cell depletion was evaluated. Study Design: A total of 48 naive cynomolgus monkeys (24 male and 24 female) were randomly assigned to four dose groups of AME-133 administered subcutaneously once weekly (0.0 mg/kg (vehicle), 0.6 mg/kg, 1.9 mg/kg and 6.0 mg/kg). Within the dose groups, there were two (6 week and 14 week) dosing schedules. In the 6 week dosing schedule, two-third of animals were sacrificed immediately after 6 weeks, and one-third of animals were sacrificed after 4 to 8 weeks of recovery period. In the 14 week dosing schedule, two-third of animals were sacrificed immediately after 14 weeks, and one-third of animals were sacrificed after 4 to 8 weeks of recovery period. Results: There was no AME-133 related toxicity. All dose levels of AME-133 substantially depleted B-cells in peripheral blood. The extent and duration of B-cell depletion was dose-dependent (Figure 1). B-cell depletion was not affected by gender of the animal. All dose levels of AME-133 substantially depleted the B-cells in peripheral blood beginning with the first post-dose time point (4 hours after infusion). The mid and high doses resulted in delayed return to normal during recovery in cynomolgus monkeys in 14 week dosing schedule. Conclusion: This study in cynomolgus monkeys showed AME-133 caused no toxicity, well absorbed and rapidly depleted circulating B-cells when administered subcutaneously. The No Observed Adverse Effect Level (NOAEL) is 6.0 mg/kg, the highest administered dose. These preliminary results justify further investigation of AME-133v administered subcutaneously in follicular lymphoma and other B-cell malignancies. Disclosures: No relevant conflicts of interest to declare.