Despite the significant progress in the curative treatment of childhood hematologic malignancies, relapse remains one of the greatest challenges in pediatric oncology (3
). Further, survivors have life-long risks of treatment-associated morbidity and mortality (6
). New therapeutic approaches are needed to overcome chemotherapy resistance and to reduce side effects (23
CD22 is rapidly internalized upon antibody or immunotoxin binding (24
). As demonstrated, this antigen is expressed in high frequency in childhood ALL. Unconjugated MoAbs can induce cytotoxicity by direct and indirect (e.g., immune-mediated) mechanisms, the latter of which are expected to be defective in individuals with ALL (25
). Unconjugated MoAb against CD22 (epratuzumab) has recently been studied in childhood ALL and its activity as a single agent in the setting of relapsed ALL appears to be limited (26
). Notably, anti-RFB4 control showed no activity against ALL xenografts or in cytotoxicity assays with primary samples from children with ALL ().
The activity of MoAbs can be dramatically increased by linkage to toxic moieties. Plant and bacterial toxins cause cellular cytotoxicity via inhibition of protein synthesis after internalization. These are highly potent and active in minute quantities, such that even a single molecule in the cytoplasm is sufficient to kill a cell (27
). There have been limited studies of immunotoxins in childhood hematologic malignancies and previously evaluated agents have been associated with severe adverse events and a high incidence of immunogenicity (28
). Clinical development of immunotoxins in general has been hampered by non-specific toxicities, immunogenicity, and production complexities. Serial modifications in the Pseudomonas
-based immunotoxin constructs utilized at the NCI have reduced non-specific toxicities, increased stability, enhanced tissue penetration, and improved targeted cellular killing (30
BL22 is a potent immunotoxin that targets CD22, which as shown, is expressed in relatively high density on the surface of 100% of the blasts in the vast majority (96%) of cases of childhood B-lineage ALL. BL22 was demonstrated to have clinical activity with acceptable toxicity in adults with relapsed and refractory hairy cell leukemia, where a maximum tolerated dose of 40 µg/kg every other day × 3 every 28 days was defined (15
). This pediatric Phase I trial extends those observations and establishes that activity can be achieved in highly resistant childhood ALL with acceptable toxicity. Notably, BL22 was tolerated at greater dose intensity (i.e., every other day for 6 doses every 21 days) in comparison to adults, and hemolytic uremic syndrome, which was the DLT in adults, was not observed. Importantly, anti-leukemia activity was seen at all dose levels, however, clinical benefits in this highly refractory population were modest and transient at the doses tested. There are a number of possible explanations for the limited observed activity. Higher doses are likely required to achieve maximal benefit. Further, while peak levels at the upper doses exceeded concentrations required for in vitro
cytotoxicity, drug exposure was limited in most subjects due to rapid clearance associated with large disease burden. CD22 expression has been demonstrated to be a determinant of response to BL22 in vitro
), although there was no obvious influence of antigen density on clinical activity in this trial (). However, small numbers preclude definitive conclusions in this regard, and notably, all subjects without progressive disease had site densities that exceeded 3,000 sites/cell, whereas 6 of 16 with progressive disease had lower levels of expression.
This trial shows that BL22 can be administered at doses up to 40 µg/kg/dose every other day × 6 in children with ALL. No maximum tolerated dose was defined. Although two subjects treated at the highest dose level developed brief Grade 4 ALT elevations, this was not dose limiting given the short duration (1 to 2 days). We subsequently chose to close the trial and apply the schedule developed in this study (every other day × 6 every 21 days) to Phase I testing of a modified BL22 construct with higher affinity for the CD22 antigen. This second-generation agent, HA22 or CAT-8015, was engineered to replace three amino acid residues in the heavy chain complementary determining region 3 of the BL22 binding domain. This modification increased the binding affinity for CD22 by 14-fold, which resulted in approximately a 1-log10
improvement in cytotoxicity against a variety of CD22+ malignancies (31
In summary, CD22 represents an excellent target for pediatric B-lineage hematologic malignancies and these studies offer proof-of-principle that anti-CD22 Pseudomonas-based immunotoxins can be administered to children and have the potential to overcome chemotherapy resistance and induce cytotoxicity of CD22+ blasts refractory to standard therapy. Anti-CD22 immunotoxins hold therapeutic promise in this common subtype of pediatric cancer.
Statement of Translational Relevance
B-lineage hematologic malignancies remain a leading cause of cancer-related mortality in pediatrics and current therapies are associated with a wide array of toxicities. New agents are needed to overcome drug resistance and reduce non-specific adverse effects. We report results of the first pre-clinical studies and Phase I clinical trial of a novel anti-CD22 immunotoxin, RFB4(dsFv)-PE38, in the setting of childhood hematologic malignancies. An acceptable toxicity profile and transient clinical activity were observed. This Phase I clinical trial serves as proof-of-principle that this immunotoxin construct is cytotoxic to chemotherapy-resistant CD22+ blasts and that it can be administered to children in doses that achieve serum levels that exceed the expected in vitro IC50. The trial established a dose and schedule for subsequent testing of RFB4(dsFv)-PE38 with a modified Fv sequence that confers higher binding affinity for CD22. Future trials are planned in combination with standard chemotherapy agents.