Most patients with lung cancer still die from their disease, necessitating additional options to improve treatment. Here, we provide evidence for targeting CD22, a cell adhesion protein known to influence B-cell survival that we found is also widely expressed in lung cancer cells. In characterizing the antitumor activity of an established anti-CD22 monoclonal antibody (mAb), HB22.7, we showed CD22 expression by multiple approaches in various lung cancer subtypes, including 7 of 8 cell lines and a panel of primary patient specimens. HB22.7 displayed in vitro and in vivo cytotoxicity against CD22-positive human lung cancer cells and tumor xenografts. In a model of metastatic lung cancer, HB22.7 inhibited the development of pulmonary metastasis and extended overall survival. The finding that CD22 is expressed on lung cancer cells is significant in revealing a heretofore unknown mechanism of tumorigenesis and metastasis. Our work suggests that anti-CD22 mAbs may be useful for targeted therapy of lung cancer, a malignancy that has few tumor-specific targets.

Most patients with lung cancer still die from their disease, necessitating additional options to improve treatment. Here, we provide evidence for targeting CD22, a cell adhesion protein known to influence B-cell survival that we found is also widely expressed in lung cancer cells. In characterizing the antitumor activity of an established anti-CD22 monoclonal antibody (mAb), HB22.7, we showed CD22 expression by multiple approaches in various lung cancer subtypes, including 7 of 8 cell lines and a panel of primary patient specimens. HB22.7 displayed in vitro and in vivo cytotoxicity against CD22-positive human lung cancer cells and tumor xenografts. In a model of metastatic lung cancer, HB22.7 inhibited the development of pulmonary metastasis and extended overall survival. The finding that CD22 is expressed on lung cancer cells is significant in revealing a heretofore unknown mechanism of tumorigenesis and metastasis. Our work suggests that antiCD22 mAbs may be useful for targeted therapy of lung cancer, a malignancy that has few tumor-specific targets. Cancer Res; 72(21); 5556–65. 2012 AACR.

Purpose To examine the role of phosphatase inhibition on anti-CD22, HB22.7-mediated lymphomacidal effects. Experimental design CD22 is a cell-surface molecule expressed on most B cell lymphomas (NHL). HB22.7 is an anti-CD22 monoclonal antibody that binds a unique CD22-epitope, blocks ligand binding, initiates signaling, and has demonstrated lymphomacidal activity. The SHP-1 tyrosine phosphatase is associated with the cytoplasmic domain of CD22. Sodium orthovanadate (NaV) is a phosphatase inhibitor. The SHP-1-CD22 interaction presents an opportunity to manipulate CD22-mediated signaling effects. In vitro cell culture assays and in vivo human NHL xenograft studies were used to assess the effects of phosphatase inhibition. Results NaV caused dose dependent killing of NHL cells in vitro; when HB22.7 was given with NaV, antibody-mediated cell death was augmented. Flow cytometry showed that NaV-pretreatment resulted in less CD22 internalization after ligation with HB22.7 than did control cells. Studies in mice bearing Raji NHL xenografts showed that the combination of NaV and HB22.7 shrank NHL tumors more rapidly, had a higher complete response rate (80%), and produced the best survival compared to controls; no toxicity was detected. Studies using Raji cells stably transfected with SHP-1DN confirmed that these observations were due to SHP-1 inhibition. Conclusion The relatively specific association of SHP-1 with CD22 suggests that CD22-specific signal augmentation by phosphatase inhibitors can improve the clinical outcome of anti-CD22 based immunotherapy.

CD22 is a cell-surface molecule found on most B-cell lymphomas (NHL). HB22.7 is an anti-CD22 antibody that blocks CD22 ligand binding, initiates signaling, and kills NHL cells. The SHP-1 tyrosine phosphatase is disproportionately associated with the cytoplasmic domain of CD22. Sodium orthovanadate (NaV) and dephostatin (DP) are phosphatase inhibitors. The interaction of SHP-1 with CD22 presents an opportunity to manipulate CD22-mediated signaling effects. NaV caused dose dependent killing of NHL cells in vitro; when HB22.7 was given with NaV, antibody-mediated cell death increased. NaV caused a substantial increase in CD22-mediated SAPK and ERK-1/2 activation when CD22 was crosslinked by HB22.7; NaV did not significantly affect IgM-mediated signals. Studies using Raji NHL cells stably transfected with a SHP-1 dominant negative (DN) confirmed that these observations were due to SHP-1 inhibition. The relatively specific association of SHP-1 with CD22 suggests that CD22-specific signaling may be altered by phosphatase inhibition in ways that could prove useful for anti-CD22-based immunotherapy.

CD22 is a cell-surface adhesion molecule on most B-cell NHL, so it is a promising target for immunotherapy. HB22.7 is an anti-CD22 mAb that binds the two NH 2 -terminal immunoglobulin domains and specifically blocks the interaction of CD22 with its ligand. CD22-blocking mAbs induce apoptosis in neoplastic B-cells and are functionally distinguishable from other anti-CD22 mAbs. This study assessed the optimal dose, route, schedule, and the targeted CD22 epitope. Raji NHL-bearing nude mice were studied. A non-blocking anti-CD22 mAb (HB22.27) was used as a control. HB22.27 had minimal effect, whereas HB22.7 improved survival and shrank tumors substantially. HB22.7 doses greater than 1.4 mg/week did not further increase efficacy (or toxicity). Tumors less than 200 mm 3 had a higher response rate than did larger tumors. Various schedules of HB22.7 administration were tested; one dose every other week was more effective than more or less frequent dosing. Pharmacokinetic studies revealed that the half-life of HB22.7 was 28 days; this correlated with the time needed to re-populate cell-surface CD22 after treatment with HB22.7. Immuno-PET showed that NHL was rapidly and specifically targeted by copper-64-labeled-HB22.7. This study provided data as to an optimal dose, route, schedule and interval between doses of HB22.7.

CD22 is a B-cell specific membrane glycoprotein that mediates homotypic and heterotypic cell adhesion; it also regulates B-cell receptor (BCR)-mediated signals. Monoclonal antibodies (mAb) directed at the ligand binding domain of CD22 initiate CD22-mediated signal transduction and apoptosis in B-cell lymphomas (NHL). Amino acid analysis of the complimentary determining regions (CDRs) of six different anti-CD22 ligand blocking mAb revealed a high level of sequence conservation. The heavy chain CDRs 1, 2, and 3 are 85, 40, and 38% conserved, respectively; light chain CDRs 1, 2, and 3, are 95, 90 and 90% conserved, respectively. Based on these conserved sequences, five peptides were designed and synthesized. Only the sequence derived from heavy chain CDR2 (Peptide 5) demonstrated significant B-cell binding. Peptide 5 bound to both malignant and primary B-cells with very little T-cell binding. The affinity had a Km of 5 × 10−6 M. Peptide 5 mediated killing of several NHL cell lines to a degree similar to that of the parent mAb (HB22.7). Peptide 5’s loop structure was shown to be crucial for B-cell binding and ligand blocking. Mutational analysis revealed that most Peptide 5 amino acids were critical for B cell binding. Using a CD22 transfected COS cell line, we demonstrated CD22-specific binding and CD22 ligand blocking to a degree similar to HB22.7. Finally Peptide 5 was used as a vehicle to deliver a pro-apoptotic peptide into NHL cells. Peptide 5 was fused to a BH3 death domain-containing peptide which demonstrated more effective NHL cell killing than the parent peptide.

Rituximab, a chimeric anti-CD20 monoclonal antibody (mAb) has become integral to non-Hodgkins lymphoma therapy. New, clinically effective, anti-NHL mAb may further improve outcomes. CD22 is a B-lymphocyte-specific adhesion molecule on the surface of most NHL, so it is a promising target for immunotherapy. A panel of anti-CD22 mAbs were developed and tested. Anti-CD22 mAbs that bind the two NH2-terminal immunoglobulin domains of CD22 and specifically block the interaction of CD22 with its ligand were identified. CD22-blocking mAbs induce apoptosis in neoplastic B-cells and are functionally distinguishable from other anti-B-cell, and even other anti-CD22 mAbs. MAbs that do not block ligand-binding have only modest functional effects. HB22.7 is the blocking anti-CD22 mAb chosen for study and characterization in a Raji NHL-bearing nude mouse model. The goal of this study was to characterize the dose, route and schedule of administration that can guide the translation of HB22.7 to a clinical trial. In addition to untreated Raji-bearing nude mice, a control, non-blocking mAb (HB22.27) was compared to HB22.7. The non-blocking mAb had minimal pre-clinical efficacy, whereas HB22.7 improved survival and caused substantial tumor shrinkage. Varying doses of HB22.7 were tested; doses greater than 1.4 mg did not further increase efficacy (or toxicity). An important consideration regarding translation of HB22.7 to clinical trials is the size of tumors to be treated. In this study, tumors less than 200 mm3 had a much higher response rate than did larger tumors. Varying schedules of HB22.7 administration (1.4 mg/dose) were tested; one dose every other week was more effective than more or less frequent dosing. Immuno-PET showed that NHL was effectively and specifically targeted when copper-64-labeled HB22.7 was administered either intravenously or subcutaneously. This study provided preclinical data as to a useful dose, route and schedule of administration of the anti-CD22 mAb, HB22.7

CD22 is a cell-surface molecule on most B-cells and B-cell non-Hodgkin's lymphoma (NHL). A panel of anti-CD22 monoclonal antibodies (mAb) were developed and characterized. Some anti-CD22 mAb bind CD22 but do not stimulate intracellular signaling. However, HB22.7 is an anti-CD22 mAb that binds a unique CD22-epitope, blocks ligand binding, and its binding initiates an intracellular signaling cascade. Sodium orthovanadate (NaV) is a non-specific phosphatase inhibitor whose effect on CD22-mediated signaling in NHL cells was tested with and without HB22.7. Compared to other B-cell receptors, the SHP-1 tyrosine phosphatase is disproportionately associated with the cytoplasmic domain of CD22; NaV inhibits SHP-1. The SHP-1/CD22 interaction presents an opportunity to manipulate CD22-mediated signaling and thus has implications for NHL therapy. Methods: Cultured human NHL cell lines were assessed by trypan blue exclusion for efficacy of treatment with varying concentrations of NaV and HB22.7. Flow cytometry was used to assess how pre-incubation of NHL-cells with NaV before treatment with HB22.7 effected the internalization of CD22 and its cell-surface concentration. Intracellular staining for phospho-p38 and activation of SAP kinase were assessed by flow cytometry. Finally, nude mice bearing Raji (human NHL) xenografts were treated with HB22.7 with and without repeated administrations of oral NaV. Tumor volume, response rate and survival were assessed over the 84-day study period. Results: NaV (1– 100 μM) caused a dose dependent killing of Ramos NHL cells in vitro. When HB22.7 (10 or 30 μg) was given along with NaV cell death was augmented. Flow cytometry of three NHL cell lines (DOHH-2, Karpas, and Ramos) demonstrated that pre-treatment with NaV resulted in less internalization, and more persistence of CD22 on the cell-surface after ligation with HB22.7 than did cells not pretreated with NaV. P38 is a known mediator of CD22 signals. When Ramos cells were pre-treated with NaV a substantial enhancement of phospho-p38 was detected when CD22 was ligated with HB22.7 as compared to either agent alone. Similarly, pretreatment of Ramos cells with NaV resulted in a substantial increase in HB22.7-mediated phosphorylation of SAP kinase as compared to the controls. Pretreatment with NaV did not affect IgM-mediated signals. Studies in nude mice bearing Raji NHL xenografts showed that the in vitro data had applicability to this murine model. Control (untreated) mice and those treated thrice weekly with NaV (150 μg) showed relentless tumor growth and no pattern of response. Mice treated with HB22.7 (2.1 mg, intravenously; 4 doses) had slowing tumor growth by day 21, then regression; there was a 50% complete response rate. Mice treated with the same doses of both NaV and HB22.7 had a more rapid anti-NHL response, less tumor growth, 80% complete responses and the best survival rate. No toxicity could be detected in mice due to NaV treatment. Conclusion: NaV can increase the expression and/or persistence of cell-surface CD22, effect signaling stimulated by HB22.7, and alter or enhance intracellular signaling pathways in ways beneficial to NHL cell killing without observed toxicity in the mouse model. Further experiments will refine the use of these agents in pre-clinical studies of NHL therapy.