The kinetics of growth and antibody formation of an anti-interleukin-2 producing hybridoma line were studied in suspension culture at temperatures ranging from 34 degrees C to 39 degrees C. Flow cytometry was used to determine the effect of temperature on the cell cycle. Maximum cell density and monoclonal antibody yield were observed at 37 degrees C. The specific monoclonal antibody production rate was approximately constant throughout each batch experiment. Lower temperatures caused cells to stay longer in the G(1)-phase of the cell cycle, but temperature had only a marginal effect on the specific antibody production rate. Arresting of cells in the G(1)-phase by means of temperature was, therefore, not suited for enhanced monoclonal antibody production. Rather, antibody production for this hybridoma was directly linked to viable cell concentration.

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BackgroundRecombinant human interleukin-2 (rhIL-2, aldesleukin) is Food and Drug Administration approved for the treatment of metastatic melanoma and renal cell carcinoma and has achieved durable response in a subset of patients. However, its utility as an immunotherapeutic drug is limited by undesirable activation of immune suppressive regulatory T cells (Tregs) and a short half-life requiring frequent high dose administration, leading to unacceptable toxicities. We have engineered MDNA11, a long-acting IL-2 superkine, to overcome these limitations by (1) modifying receptor selectivity in favor of anti-cancer immune cells to increase therapeutic efficacy and (2) fusion to human albumin to extend the pharmacokinetic (PK) profile, circumventing the need for frequent dosing.MethodsMDNA11 was evaluated using in vitro and in vivo studies including: binding analyses to measure receptor affinity, IL-2 pathway signaling, PK studies in mice, and efficacy studies in syngeneic tumor models as single agent and in combination with immune checkpoint inhibitors. Finally, the safety and pharmacodynamic profile of MDNA11 was assessed in non-human primate (NHP).ResultsBinding studies with MDNA11 demonstrated increased affinity for IL-2Rβ (CD122) and no binding to IL-2Rα (CD25). As a result, MDNA11 exhibits reduced/limited Treg stimulation while triggering an enhanced activation of natural killer and naïve CD8 T cells compared with rhIL-2. When administered to animals with pre-established tumors, MDNA11 controlled tumor growth in a monotherapy setting and in combination with anti-PD1 or anti-CTLA4 to induce durable tumor clearance with a once weekly dosing regimen. In a NHP model, MDNA11 was well tolerated while triggering durable and potent immune responses including expansion of lymphocytes without significant effect on Tregs and eosinophils, the latter been linked to an increased risk of vascular leak syndrome.ConclusionMDNA11 is a next generation long-acting IL-2 immunotherapeutic with a highly favorable pharmacodynamic profile that translates to a strong therapeutic efficacy in preclinical tumor models and a strong and durable immune response in NHP.

The current study compared the standard response assessment in neuro-oncology (RANO), immunotherapy RANO (iRANO), and modified RANO (mRANO) criteria as well as quantified the association between progression-free (PFS) and overall survival (OS) in an immunotherapy trial in recurrent glioblastoma (rGBM).Patients and Methods: A total of 47 patients with rGBM were enrolled in a prospective phase II convection-enhanced delivery of an IL4R-targeted immunotoxin (MDNA55-05, NCT02858895). Bidirectional tumor measurements were created by local sites and centrally by an independent radiologic faculty, then standard RANO, iRANO, and mRANO criteria were applied.Results: A total of 41 of 47 patients (mean age 56 AE 11.7) were evaluable for response. PFS was significantly shorter using standard RANO compared with iRANO (log-rank, P < 0.0001; HR ¼ 0.3) and mRANO (P < 0.0001; HR ¼ 0.3). In patients who died and had confirmed progression on standard RANO, no correlation was observed between PFS and OS (local, P ¼ 0.47; central, P ¼ 0.34). Using iRANO, a weak association was observed between confirmed PFS and OS via local site measurements (P ¼ 0.017), but not central measurements (P ¼ 0.18). A total of 24 of 41 patients (59%) were censored using iRANO and because they lacked confirmation of progression 3 months after initial progression. A strong correlation was observed between mRANO PFS and OS for both local (R 2 ¼ 0.66, P < 0.0001) and centrally determined reads (R 2 ¼ 0.57, P ¼ 0.0007).Conclusions: No correlation between radiographic PFS and OS was observed for standard RANO or iRANO, but a correlation was observed between PFS and OS using the mRANO criteria. Also, the iRANO criteria was difficult to implement due to need to confirm progression 3 months after initial progression, censoring more than half the patients.

Background MDNA55 is an IL4R-targeting toxin in development for recurrent GBM, a universally fatal disease. IL4R is overexpressed in GBM as well as cells of the tumor microenvironment. High expression of IL4R is associated with poor clinical outcome. Method MDNA55-05 is an open-label, single-arm Phase 2b study of MDNA55 in recurrent GBM (rGBM) patients with an aggressive form of GBM (de novo GBM, IDH wild-type, and non-resectable at recurrence) on their 1 st or 2 nd recurrence. MDNA55 was administered intratumorally as a single dose treatment (dose range of 18 to 240 ug) using convection enhanced delivery (CED) with up to 4 stereo-tactically placed catheters. It was co-infused with a contrast agent (Gd-DTPA, Magnevist®) to assess distribution in and around the tumor margins. The flow rate of each catheter did not exceed 10μL/min to ensure that the infusion duration did not exceed 48 hours. Primary endpoint was mOS, with secondary endpoints determining the effects of IL4R status on mOS and PFS. Results MDNA55 showed an acceptable safety profile at doses up to 240 μg. In all evaluable patients (n=44) mOS was 11.64 months (80% one-sided CI 8.62, 15.02) and OS-12 was 46%. A sub-group (n=32) consisting of IL4R High and IL4R Low patients treated with high dose MDNA55 (&gt;180 ug) showed best benefit with mOS of 15 months, OS-12 of 55%. Based on mRANO criteria, tumor control was observed in 81% (26/32), including those patients who exhibited pseudo-progression (15/26). Conclusions MDNA55 demonstrated tumor control and promising survival and may benefit rGBM patients when treated at high dose irrespective of IL4R expression level.