Faculty Bibliography

PURPOSE: This phase I trial was undertaken to determine the maximum-tolerated dose (MTD) and dose-limiting toxicities (DLTs) of the B-cell-restricted immunotoxin anti-B4-blocked ricin (anti-B4-bR) when it is administered by 7-day continuous infusion. PATIENTS AND METHODS: Thirty-four patients with relapsed and refractory B-cell neoplasms (26 non-Hodgkin's lymphoma [NHL], four chronic lymphocytic leukemia [CLL], four acute lymphoblastic leukemia [ALL]) received 7-day continuous infusion anti-B4-bR. Successive cohorts of at least three patients were treated at doses of 10 to 70 micrograms/kg/d for 7 days with the dose increased by 10 micrograms/kg/d for each cohort. The initial three cohorts of patients (10, 20, and 30 micrograms/kg/d x 7 days) also received a bolus infusion of 20 micrograms/kg before beginning the continuous infusion. RESULTS: The MTD was reached at 50 micrograms/kg/d x 7 days. The DLTs were National Cancer Institute Common Toxicity Criteria (NCI CTC) grade IV reversible increases in AST and ALT, and grade IV decreases in platelet counts. Adverse reactions included fevers, nausea, headaches, myalgias, hypoalbuminemia, dyspnea, edema, and capillary leak syndrome. Potentially therapeutic serum levels of anti-B4-bR could be sustained for 4 days in patients treated at the MTD. Two complete responses (CRs), three partial responses (PRs), and 11 transient responses (TRs) were observed. CONCLUSION: Anti-B4-bR can be administered safely by 7-day continuous infusion with tolerable, reversible toxicities to patients with relapsed B-cell neoplasms. Although occasional responses were seen, future trials will use anti-B4-bR in patients with lower tumor burdens to circumvent the obstacle of immunotoxin delivery to bulk disease.

Anti-B4-blocked ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody and the protein toxin, "blocked ricin." In blocked ricin, the galactose-binding sites of the ricin B-chain which mediate nonspecific binding to cells are blocked by covalently linked affinity ligands prepared from N-linked oligosaccharides of fetuin. Blocked ricin consists of two species, one with two covalently attached ligands and one with three covalently attached ligands. In a Phase I dose escalation clinical trial, Anti-B4-bR was administered to patients with relapsed and refractory B-cell neoplasms by 7-day continuous infusion. Although several different lots of Anti-B4-bR had similar IC37 values as determined by in vitro cytotoxicity testing on cultured human cell lines, these lots differed in their in vivo toxicity when administered to patients. Thus, IC37 values alone were not sufficient to predict in vivo toxicity. We report that the degree of cell kill at concentrations of drug that saturate the B4 antigen and murine 50% lethal dose values provide additional parameters that may be predictive of in vivo cytotoxicity. Furthermore, we performed detailed cytotoxicity studies of the ricin species containing two and three covalently attached ligands, respectively. In vitro cytotoxicity testing using these samples revealed that Anti-B4-bR made with blocked ricin containing two covalently attached ligands is capable of depleting five logs of target cells in an in vitro cytotoxicity assay, while Anti-B4-bR comprised of blocked ricin with three ligands can deplete only one log of cells. Log cell kill at antigen saturating concentration, murine 50% lethal dose and biochemical analysis of the composition of blocked ricin are therefore important considerations for establishing the potential efficacy and safety of Anti-B4-bR.

Anti-B4-blocked Ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody (MoAb) and the protein toxin "blocked ricin." The anti-B4 MoAb is directed against the B-lineage-restricted CD19 antigen expressed on more than 95% of normal and neoplastic B cells. Blocked ricin is an altered ricin derivative that has its nonspecific binding eliminated by chemically blocking the galactose binding domains of the B chain. In vitro cytotoxicity studies demonstrate that the IC37 of Anti-B4-bR is 2 x 10(-11) mol/L compared with 4 x 10(-12) mol/L for native ricin. A phase I dose escalation clinical trial was conducted in 25 patients with refractory B-cell malignancies. Anti-B4-bR was administered by daily 1-hour bolus infusion for 5 consecutive days at doses ranging from 1 microgram/kg/d to 60 micrograms/kg/d. Serum levels above 1 nmol/L were achieved transiently in the majority of patients treated at the maximum tolerated dose of 50 micrograms/kg/d for 5 days for a total dose of 250 micrograms/kg. The dose-limiting toxicity was defined by transient, reversible grade 3 elevations in hepatic transaminases, without impaired hepatic synthetic function. Minor toxicities included transient hypoalbuminemia, thrombocytopenia, and fevers. Human antimouse antibody and human anti-ricin antibody were detected in nine patients. One complete response, two partial responses, and eight mixed or transient responses were observed. These results show the in vitro and in vivo cytotoxicity of Anti-B4-bR and indicate that this immunotoxin can be administered as a daily bolus infusion for 5 days with tolerable, reversible toxicity.

BACKGROUND: The use of autologous bone marrow transplantation is increasing in the management of advanced cancers. Many investigators have attempted to "purge" autologous marrow of residual tumor cells because of concern that reinfused tumor cells might contribute to relapse. The efficacy of purging remains unproved. METHODS: We performed clonogenic assays in a tumor cell line in culture to determine the efficiency of immunologic purging. Amplification by the polymerase chain reaction (PCR) was used to detect residual lymphoma cells before and after purging of bone marrow from 114 patients with B-cell non-Hodgkin's lymphoma in whom a translocation (t(14;18] that could be amplified by PCR was detected at the time of their initial evaluation. RESULTS: Immunologic purging in vitro resulted in a 3-to-6-log destruction of cells in the tumor cell line. Residual lymphoma cells were detected by PCR in the bone marrow of all patients before purging. No lymphoma cells could be detected in the marrow of 57 patients after purging. Disease-free survival was increased in these 57 patients as compared with those whose marrow contained detectable residual lymphoma (P less than 0.00001). The ability to purge residual lymphoma cells was not associated with the degree of bone marrow involvement (P = 0.4494) or the previous response to therapy (P = 0.1298). CONCLUSIONS: The inability to purge residual lymphoma cells was the most important prognostic indicator in predicting relapse. These results provide evidence of the clinical usefulness of ex vivo purging of autologous bone marrow in the treatment of patients with lymphoma and suggest that the reinfusion of malignant cells in autologous marrow contributes to relapse

Similar to Streptococcus mutans, buffer suspensions of Lactobacillus casei, Lactobacillus plantarum, and Fusobacterium nucleatum all undergo cell lysis when treated with the lysozyme-protease-inorganic monovalent anion antibacterial system. For Lactobacillus species treated with lysozyme and proteases at pHs of 4 and 5.3, lysis resulted when a lytic activating concentration of bicarbonate anion followed enzyme treatment. Furthermore, synergistic lysis of these bacteria was noted when lysozyme-protease treatment was followed by bicarbonate anion used in combination with chloride or fluoride anions. Noteworthy, the halides were not active in promoting lysis when used by themselves in the absence of bicarbonate. For F. nucleatum suspended at pH 6.9, lysis was dependent upon the ionic strength of the buffer and resulted when lysozyme-protease treatment of the organism was followed by 100 mmol/L bicarbonate activation. When lysozyme and proteases were omitted from the incubation mixtures and replaced by stimulated whole saliva, pH 5.3, lysis was observed only with L. plantarum and S. mutans, but not with L. casei. The latter could be lysed, however, if suspended in saliva which was diluted several-fold with distilled water. In experiments where lysozyme was selectively depleted from whole saliva by immunoadsorption affinity chromatography, the great majority of the lysis capability of the saliva for L. plantarum was lost, although a significant degree of lysis appeared to be due to salivary factors other than lysozyme. F. nucleatum was also found to lyse in saliva at neutral pH, suggesting that both Gram-positive and Gram-negative oral bacteria may be susceptible to this antibacterial system in vivo.

Bile pigment composition (biliverdin, bilirubin and their conjugates) was analyzed in stored gallbladder bile and newly synthesized hepatic bile from the small skate (Raja erinacea). During a five day period of captivity, gallbladder volume remained relatively constant while bilirubin and biliverdin content increased two to three fold. Biliverdin which accounted for 50% of the pigments did not increase as a percentage of tetrapyrroles during this period. The relative proportion of bilirubin and its conjugates also remained constant, averaging 65% for bilirubin monoglucuronide, 30% for bilirubin diglucuronide and 5% for unconjugated bilirubin as measured by HPLC methods. Intravenous administration of biliverdin resulted in significant increases in the biliary excretion of both biliverdin and all bilirubin tetrapyrroles. Insignificant quantities of 3H-biliverdin were detected in hepatic bile following the intravenous administration of 3H-bilirubin. These studies indicate that the small skate excreted both biliverdin and bilirubin conjugates in bile and that the biliverdin was not produced by in vitro oxidation of bilirubin or its metabolites.

The ability of lysozyme to aggregate and lyse the gram-negative capnophilic periodontal microorganism Capnocytophaga gingivalis 2010 was monitored optically at 540 nm. Both hen egg white and chromatographically purified human lysozymes had significant but similar aggregation potentials for both logarithmic- and stationary-phase bacteria. In general, an increase in enzyme concentration resulted in a graded increase in both the initial and maximum changes in turbidity which occurred during the reaction period. The greatest change in turbidity occurred within the initial minutes of interaction of lysozyme and the cells, and the extent of aggregation paralleled a rapid depletion of lysozyme by the suspensions during the first minute of its incubation with the bacteria. Interestingly, the muramidase inhibitors N-acetyl-D-glucosamine and histamine did not block aggregation, whereas maleylation of lysozyme completely inhibited its aggregating ability. Demaleylation, however, restored aggregation activity comparable to the native enzyme, indicating that maleylated lysozyme retained its integrity and that aggregation was primarily dependent on charge. The addition of up to physiological concentrations of NaHCO3 and NaCl to cell aggregates resulted in varying degrees of deaggregation and lysis. Surprisingly, ultrastructural analysis of lysozyme-treated cells revealed morphological changes with or without the addition of salt. Damage appeared to occur at the blunted polar end of the cells where there was a large spherical outpouching bordered by a damaged cell envelope. Damaged cells uniformly contained dense granular cytoplasmic debris. In effect, the cationic enzyme lysed C. gingivalis 2010, which was not apparent in the spectrophotometric assay. The paradoxical finding that during bacterial aggregation there was lysis may be of significance to the further elucidation of lysozyme's antibacterial role in the gingival sulcus.