Treatment Plan

In this phase 1 study, patients received DT2219 in a single course at doses ranging from 0.5 μg/kg/day (1/500th of the MTD in rabbits) to 80 μg/kg/day intravenously (IV) over 2 hours (4 hours for 1^st dose) every other day for 4 total doses (days 1,3,5 & 8). The dose was escalated in 9 cohorts until a dose limiting toxicity (DLT) was observed (Table 2). The first 15 patients were treated by rapid escalation design (dose cohorts 1-3) or by standard 3+3 dose escalation design (cohorts 4-6). We applied Continual Reassessment Method (9) to the last 10 patients (dose cohorts 8,9) with the goal to identify the dose level which corresponds to a desired toxicity rate of 33% or less using grade 3 or greater DT2219 related toxicity except blood pressure changes and fever as the targeted toxicity (based on NCI Common Terminology Criteria for Adverse Events version (CTCAE) 4). Administration of doses 2-4 was permitted if pre-dose creatinine was <1.5× ULN and absence of DLT. Supportive care included allopurinol (300 mg/day orally); intravenous fluids; and premedication with diphenylhydramine (25 mg IV), acetaminophen (325 mg orally), hydrocortisone (100 mg IV), and ranitidine (50 mg IV) 30 minutes prior to each DT2219 dose.

Disease Reassessment and Correlative Studies

Disease assessment included physical examination for lymph node and spleen weekly; blood and marrow evaluation including flow cytometry assessment for CD19 and CD22 expression and assessment for minimal residual disease, and computerized tomography (CT) scan 21-28 days after treatment using Chesson criteria for lymphoma and leukemia staging (10, 11). Adverse event collection focused on targeted and unexpected adverse events before and after each dose at the following time-points: 1-4 hours, 24 hours, and days 9, 15, 22, 29 of the cycle.

Correlative studies included assessment of pharmacokinetics, neutralizing antibody, and immunophenotype of peripheral blood and marrow for CD22, CD19, and CD20. Cell suspensions were stained with the following mAbs: PerCPCy5.5-anti-CD3 (OKT3, Tonbo biosciences65-0037); APC anti-CD45 (HI30. Tonbo biosciences 20-0459),; FITC-anti CD19 (BU-12), FITC-anti CD20 (clone 2H7, eBioscience, 11-0209-42); FITC-anti CD22 (Invitrogen MHCD2201). Phenotypic acquisition of cells was carried out on the BD Accuri C6 and analyzed with BD Accuri C6 software. The presence of DT2219 in serum was measured by the ability of diluted serum to inhibit proliferation of CD22⁺CD19⁺ Raji indicator cells and then extrapolating DT2219 concentration using standard curve comparison, as described previously (12). The presence of CD19 and CD22 on lymphoid tumor samples obtained from patients prior to the therapy has been evaluated using standard immunohistochemistry on formalin fixed, paraffin embedded tissues and, where possible, by flow cytometry. We also measured CD19, CD22, CD20 and CD3 expressing peripheral blood cells at weekly intervals. Finally, the presence of neutralizing antibodies was measured with an assay where patient serum was used to block the activity of DT2219 in vitro (5). Peripheral mononuclear cells (MNC) and serum samples were collected pre-treatment and post-treatment at days 1, 8, 15, 21, and 28 and stored at -80C.

Pharmacologic and immunologic studies

At the time of enrollment, most patients exhibited low peripheral blood (PB) B-cells counts (median B cell count 3.5% (<0.1 ×10⁶ cells/μL); range 0-52%; n=10) often associated with prior rituximab, corticosteroids and chemotherapy. The effect of DT2219 on B lymphocytes in a patient with an extramedullary ALL relapse shortly after allogeneic HCT was observed with gradual decline in number of PB CD19- and CD22-expressing cells after 4 doses of DT2219 (Figure 1A). The possibility that DT2219 may interfere with fluorochrome-labeled anti-CD19 and anti-CD22 was excluded by examining CD20-positive cells, which also declined over time. The B cell depletion was specific as CD3-positive T cell levels remained constant during the testing interval.

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Figure 1

Immunologic and pharmacokinetic studies

A. Peripheral blood mononuclear cell (PBMC) analysis of a representative patient is shown. PBMCs were enriched from patient blood and collected at various times post-treatment. Flow cytometry was used to count cells expressing CD22, CD19, CD20, or CD3. B. A bioassay was used to determine the area under the curve (AUC) of serum DT2219 levels in serum by measuring the ability of diluted serum to inhibit proliferation of CD22+CD19+ Raji indicator cells. Drug serum levels at various times were analyzed using prism 5.0 software to calculate AUC. A concentration-time curve is shown for our second patient at 60ug/kg. T_1/2 was 59 minutes. C. DT2219 serum levels and neutralizing antibodies. Upper panels shows a patient treated at the 80 ug/kg dose level showing no evidence of DT2219 in serum (left) and high levels of neutralizing antibodies at day 8 through 22 (right). In contrast, lower panels shows that a patient treated at 60 ug/kg/day had a serum drug concentration (left) and no detectable neutralizing antibodies (right lower panel). DT2219 serum levels were calculated from assays in which various serum dilutions were tested for their ability to inhibit Raji cell proliferation. Serum collected prior to drug administration served as a negative control. Neutralization assays were performed based on the ability of undiluted patient serum samples to block the killing of a 99% inhibitory dose of DT2219. % neutralization was calculated

We also measured the circulating concentration of DT2219 in a functional pharmacokinetic bioassay. Patients treated at dose levels 0.5-20 μg/kg/day had no detectable drug in serum when sampled on day 1 and 8 at 15, 30, 45, 60, and 120 minutes post-infusion. All evaluable patients at the University of Minnesota treated with ≥40 μg/kg/dose (n=10) demonstrated detectable levels of DT2219 with the exception of one with preexisting antibodies to DT. The median area under the curve (AUC) after the 1^st dose (4 hours infusion) was lower at a median of 285 μg/mL × minutes; (range 0-2020; n=8) compared to drug levels after the 4^th dose (2 hours infusion; AUC median 1249 μg/ml × minutes; range 0-1692; n=7). A representative AUC is shown in Figure 1B. The drug half-life ranged from 59-110 minutes (n=4).

Because the recombinant immunotoxin contains a bacterial toxin, immunogenicity is expected and can be a major barrier to the potential activity of bacterial toxin-based drugs. We measured serum neutralizing antibodies (NAs) in all patients treated with ≥40 μg/kg/dose at days 1,8,15,29,35 and 42 (n=9). NAs developed in 3 evaluable patients (30%) at dose levels between 40-80 ug/kg at median of one week (range 1-2 weeks) after the 1^st dose of DT2219. One patient had pre-formed anti-diphtheria toxin antibody which we detected at screening and attributed to prior DT immunization. In some patients the presence of NA inversely correlated with the serum concentration of DT2219 (Figure 1C), however no consistent pattern was recognized.

Research reported in this publication was supported US Public Health Service grant R01CA36725 (DV), the Randy Shaver Foundation, the Lion's Children's Cancer Fund, the William Lawrence and Blanche Hughes Fund and by the National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1TR000114 (VB). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported in part by NIH P30 CA77598 utilizing the Biostatistics and informatics core, Masonic Cancer Center, University of Minnesota shared resource. We would like to thank Julie Curtsinger from University of Minnesota Cancer Center Translational Therapy Lab for assistance with blood samples retrieval and Michael Franklin for editorial assistance.

Funding: US Public Health Service grant R01CA36725 (DV), the Randy Shaver Foundation, the Lion's Children's Cancer Fund, the William Lawrence and Blanche Hughes Fund and by the National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1TR000114 (VB).