The HB22.7 Anti-CD22 monoclonal antibody enhances bortezomib-mediated lymphomacidal activity in a sequence dependent manner.

Most non-Hodgkin's lymphomas (NHL) initially respond to chemotherapy, but relapse is common and treatment is often limited by chemotherapy-related toxicity. Bortezomib, is a highly selective proteasome inhibitor with anti-NHL activity; it is currently FDA approved for second-line treatment of mantle cell lymphoma (MCL). Bortezomib exerts its activity in part through the generation of reactive oxygen species (ROS) and also by the induction of apoptosis.We previously validated CD22 as a potential target in treating NHL and have shown that the anti-CD22 ligand blocking antibody, HB22.7, has significant independent lymphomacidal properties in NHL xenograft models. We sought to determine whether or not these agents would work synergistically to enhance cytotoxicity. Our results indicate that treatment of NHL cell lines with HB22.7 six hours prior to bortezomib significantly diminished cell viability. These effects were not seen when the agents were administered alone or when bortezomib was administered prior to HB22.7. Additionally, HB22.7 treatment prior to bortezomib increased apoptosis in part through enhanced ROS generation. Finally, in a mouse xenograft model, administration of HB22.7 followed 24 hours later by bortezomib resulted in 23% smaller tumor volumes and 20% enhanced survival compared to treatment with the reverse sequence. Despite the increased efficacy of HB22.7 treatment followed by bortezomib, there was no corresponding decrease in peripheral blood cell counts, indicating no increase in toxicity. Our results suggest that pre-treatment with HB22.7 increases bortezomib cytotoxicity, in part through increased reactive oxygen species and apoptosis, and that this sequential treatment combination has robust efficacy in vivo.

The Bs20x22 anti-CD20-CD22 bispecific antibody has more lymphomacidal activity than do the parent antibodies alone.

Previous studies have shown that bispecific antibodies that target both CD20 and CD22 have in vivo lymphomacidal properties. We developed a CD20-CD22 bispecific antibody (Bs20x22) from anti-CD20 and the anti-CD22 monoclonal antibodies (mAb), rituximab and HB22.7, respectively. Bs20x22 was constructed using standard methods and was shown to specifically bind CD20 and CD22. In vitro cytotoxicity assays showed that Bs20x22 was three times more effective than either parent mAb alone and twice as effective as a combination of both parent mAb used at equimolar concentrations. Bs20x22 was also nearly four times more effective at inducing apoptosis than either mAb alone. Examination of the MAPK and SAPK signaling cascades revealed that Bs20x22 induced significantly more p38 phosphorylation than either mAb alone. In an in vivo human NHL xenograft model, treatment with Bs20x22 resulted in significantly greater tumor shrinkage and improved overall survival when compared to either mAb alone or treatment with a combination of HB22.7 and rituximab. The effect of the initial tumor volume was assessed by comparing the efficacy of Bs20x22 administered before xenografts grew versus treatment of established tumors; significantly, greater efficacy was found when treatment was initiated before tumors could become established.

Efficacy, biodistribution, and pharmacokinetics of CD22-targeted pegylated liposomal doxorubicin in a B-cell non-Hodgkin's lymphoma xenograft mouse model.

PURPOSE: Non-Hodgkin's lymphoma (NHL) is the sixth most common cause of cancer death in the U.S. Pegylated liposomal doxorubicin (PLD) is a liposomal form of doxorubicin (DXR) that causes less toxicity than does free DXR. To further enhance efficacy and decrease toxicity, we conjugated HB22.7, an anti-CD22 monoclonal antibody to PLD, thus creating CD22-targeted immunoliposomal PLD (IL-PLD). EXPERIMENTAL DESIGN: In vitro cytotoxicity of IL-PLD and PLD was assessed in CD22-positive and CD22-negative cell lines. Biodistribution, myelotoxicity, and plasma pharmacokinetics were measured in NHL xenograft-bearing mice treated with IL-PLD or PLD. Survival, tumor volume, and toxicity (WBC counts, body weights) were assessed in mice receiving a single dose (8, 12, or 16 mg DXR/kg) or three doses (8 mg DXR/kg/dose) of IL-PLD; controls were PLD, free DXR, PLD plus unconjugated HB22.7, IL-null (HB22.7-conjugated empty liposome), and nontreated mice. RESULTS: IL-PLD improved cytotoxicity over PLD only in CD22-positive cells. IL-PLD displayed similar pharmacokinetics and toxicities as PLD. Tumor DXR accumulation was greater and tumor/normal tissue ratios were similar (spleen) or greater (kidney, lung, and liver) in mice treated with IL-PLD versus PLD. IL-PLD reduced tumor volume more effectively than PLD at all doses; the three-dose regimen was superior. The three-dose regimen was used in confirmatory studies, which showed that IL-PLD produced significantly greater tumor volume reduction and enhanced survival versus PLD. CONCLUSION: IL-PLD has increased efficacy without increased toxicity compared with PLD. This suggests that IL-PLD may be an improved form of DXR-based therapy of NHL.

Development and characterization of CD22-targeted pegylated-liposomal doxorubicin (IL-PLD).

Non-Hodgkin's lymphoma (NHL) is the sixth most common cause of cancer deaths in the U.S. Most NHLs initially respond well to chemotherapy, but relapse is common and treatment is often limited due to the toxicity of chemotherapeutic agents. Pegylated-liposomal doxorubicin (PLD, Ben Venue Laboratories, Inc), a produces less myelotoxicity than non-liposomal (NL) doxorubicin. To further enhance efficacy and NHL targeting and to decrease toxicity, we conjugated an anti-CD22 monoclonal antibody (HB22.7) to the surface of PLD, thereby creating CD22-targeted immunoliposomal PLD (IL-PLD). HB22.7 was successfully conjugated to PLD and the resulting IL-PLD exhibits specific binding to CD22-expressing cells as assessed by immunofluorescence staining. IL-PLD exhibits more cytotoxicity than PLD in CD22 positive cell lines but does not increase killing of CD22 negative cells. The IC(50) of IL-PLD is 3.1 to 5.4 times lower than that of PLD in CD22+ cell lines while the IC(50) of IL-PLD is equal to that of PLD in CD22- cells. Furthermore, IL-PLD remained bound to the CD22+ cells after washing and continued to exert cytotoxic effects, while PLD and NL- doxorubicin could easily be washed from these cells.

Imaging and pharmacokinetics of (64)Cu-DOTA-HB22.7 administered by intravenous, intraperitoneal, or subcutaneous injection to mice bearing non-Hodgkin's lymphoma xenografts.

PURPOSE: The aim of the study is to compare the tumor-specific targeting, pharmacokinetics, and biodistribution of (64)Cu-DOTA-HB22.7 when administered to xenograft-bearing mice intravenously (IV), intraperitoneally (IP), and subcutaneously (SQ). PROCEDURES: Mice bearing human non-Hodgkin's lymphoma (NHL) xenografts were injected IV, IP, or SQ with (64)Cu-DOTA-HB22.7. Xenograft targeting was evaluated by micro positron emission tomography (microPET) and confirmed by organ biodistribution studies. Blood measurements of (64)Cu were performed to determine the pharmacokinetics and clearance of (64)Cu-DOTA-HB22.7. RESULTS: (64)Cu-DOTA-HB22.7 demonstrated equivalent tumor targeting within 24-48 h, regardless of the route of administration. Organ biodistribution confirmed tumor-specific targeting. Blood pharmacokinetics demonstrated that (64)Cu-DOTA-HB22.7 accessed the bloodstream after IP and SQ administration to a similar degree as IV administration, albeit at a slower rate. CONCLUSIONS: These findings establish (64)Cu-DOTA-HB22.7 as a potential radioimmunotherapeutic and/or NHL-specific imaging agent. These findings provide evidence that IP and SQ administration can achieve results equivalent to IV administration and may lead to more efficient, reproducible treatment plans for antibody-based therapeutics.