Abstract 4356: Cytotoxicity of the anti-CD22 immunotoxin HA22 against pediatric acute lymphoblastic leukemia (ALL) (2010)

Abstract Cure rates for children with acute lymphoblastic leukemia (ALL) have steadily improved, but a proportion of patients still relapse despite intensive and prolonged chemotherapy. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. CD22 is a B-lineage differentiation antigen expressed on most B-lineage ALL blasts. We have developed recombinant anti-CD22 immunotoxins composed of the Fv portion of the anti-CD22 mAb RFB4 linked to a 38kDa truncated protein derived from Pseudomonas exotoxin A (PE38). These produce cell death through a unique and highly specific mechanism of action, entailing binding to cell surface CD22, rapid internalization, intracellular processing, and inhibition of protein synthesis through ADP-ribosylation of elongation factor-2. In phase I and II trials of patients with hairy cell leukemia the first generation agent, BL22, produced complete responses (CR) of 61% and 47% respectively. However, activity was modest in children with ALL (Blood 2007;110:262a). A modified agent, HA22, was derived by complementarity-determining region mutagenesis resulting in a significantly higher binding affinity for the CD22 antigen. We cultured pre-B ALL cells from newly diagnosed (n = 13) and relapsed children (n= 22), as well as human ALL cells derived from xenografts in immunodeficient mice (n = 4) and the cell line KOPN-8 on telomerase-immortalized human bone marrow mesenchymal cells. On day 2 of culture, HA22 was added at concentrations ranging from 0 to 500ng/ml and the cells were incubated for an additional 72 hours. Dexamethasone 10umol/L was used as a positive control. The number of viable CD19+ cells at the end of the cultures was determined by flow cytometry. Viability was assessed using gates set for light-scatter properties and Annexin-PE/7-AAD staining on Days 2 and 5. The percentage of CD19+ viable cells relative to the control was determined and the 50% inhibitory concentration (IC50) calculated. Mesenchymal-supported cell cultures maintained ALL cell viability (mean 77%, median 52%, range 6% - 280% viable cells at 96 hours). ALL samples varied in their sensitivity to HA22. IC50s ranged from 1-80 ng/mL in 29 of 40 samples. In 11 samples 50% killing was not achieved. Resistance to cytotoxicity from dexamethasone was also observed, which correlated with resistance to HA22 for samples from newly diagnosed patients (r = 0.55, p = 0.053). Peripheral blood blasts appeared to be more sensitive to HA22 in comparison to bone marrow derived blasts (p = 0.008). Blasts with higher CD22 site densities showed increased cytotoxicity to HA22 (r = −0.34, p = 0.16). These results indicate that most cases of childhood B-lineage ALL are sensitive to HA22 at concentrations well below clinically achievable blood levels. Clinical testing of HA22 in children with drug-resistant ALL has been initiated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4356.