Epitope specificity of the anti-(B cell lymphoma) monoclonal antibody, LL2

Abstract

LL2 is a murine monoclonal antibody IgG2a reactive with B cells and non-Hodgkin's B-cell lymphoma, which, in a radioiodinated form, induces responses in lymphoma patients [Goldenberg et al. (1991) J Clin Oncol 9:548-564]. In this report we identify LL2 as a member of the CD22 cluster. The molecular size of the antigen, its expression profile, and competitive blocking studies were used to establish this identification. By Western blot analysis and immunoprecipitation studies using the Raji Burkitt's lymphoma cell line metabolically labelled with [3H]leucine, the LL2 antigen was determined to correspond to a molecular mass of 140 kDa. The molecular mass of the LL2 antigen, and the B-cell-restricted reactivity of the LL2 antibody, were consistent with both the CD21 and CD22 clusters. To assess additional similarities and differences between LL2 and anti-CD22 and anti-CD21, the binding of these mAb to cultured cell lines, Nalm-6 and Molt-4, was compared by flow cytometry. The binding profile of LL2 on these cell lines was consistent with anti-CD22, but not anti-CD21. Sequential immunoprecipitation and cross-blocking studies with anti-CD22 monoclonal antibodies recognizing established CD22 epitopes were performed to confirm that LL2 reacts with CD22 and to determine which epitope LL2 recognizes. Binding of 131I-LL2 to Raji cells is inhibited over 90% by prior incubation of the target cells with unlabelled RFB4, indicating that LL2 belongs to the same epitope group as RFB4, i.e., epitope B.

Potent and specific antitumor effects of an anti-CD22–targeted cytotoxic ribonuclease: potential for the treatment of non-Hodgkin lymphoma

“…In vitro cytotoxicity of LL2-onconase: duration of exposure LL2 is known to be rapidly internalized 4 and our experiments have shown that more than 95% of the antibody is specifically bound to the cell surface by 2 hours (data not shown). To determine whether a short-term exposure to the conjugate would be sufficient to cause cytotoxicity, inhibition of protein synthesis was examined in an experiment that compared the effects of LL2-onconase on Daudi cells after a 1-hour treatment compared with continuous exposure of Daudi cells to the conjugate ( Figure 5).…”

“…LL2 has a highly restricted specificity; it does not cross-react with peripheral blood cells, including the blood's normal B cells, yet is reactive with virtually all cases of NHL. 4 Recently, preliminary results of a clinical trial in which the humanized version of LL2, Epratuzumab, was used have been reported. 5 No dose-limiting toxicity nor human antiEpratuzumab antibody response was observed at the highest dose administered (1000 mg/m 2 per week), whereas objective responses were noted.…”

“…It is rapidly internalized, and then the CD22 antigen is re-expressed on the cell surface. 4 In fact, LL2 was previously linked to a derivative of Pseudomonas exotoxin by Kreitman et al 16 and shown to very effectively induce regression of subcutaneous human B-cell lymphomas in mice.…”

Identification and characterization of fully human anti-CD22 monoclonal antibodies

“…This is in agreement with the published data showing that RFB4, the parental IgG of HA22, competed partially with another mAb that binds to domain 3. 17 The two new antibodies reacted only to the region including domains 5 to 7 (Fig. 2).…”

Preclinical and clinical evaluation of epratuzumab (anti-CD22 IgG) in B-cell malignancies

“…Several anti-CD22 monoclonal antibodies have been developed, with immunoprecipitation studies demonstrating that they react to different Ig domains. Epratuzumab, a humanized anti-CD22 antibody, which is the subject of this review, and the RFB4 antibody compete for binding to the third Ig domain (Stein et al, 1993).…”