M. Jules Mattes

CD74: A New Candidate Target for the Immunotherapy of B-Cell Neoplasms

CD74 is an integral membrane protein that functions as a MHC class II chaperone. Moreover, it has recently been shown to have a role as an accessory-signaling molecule and has been implicated in malignant B-cell proliferation and survival. These biological functions combined with expression of CD74 on malignant B cells and limited expression on normal tissues implicate CD74 as a potential therapeutic target. The anti-CD74 monoclonal antibody LL1 has been humanized (hLL1 milatuzumab or IMMU-115) and can provide the basis for novel therapeutic approaches to B-cell malignancies, particularly because this antibody shows rapid internalization into CD74+ malignant cells. This article reviews the preclinical evaluations of LL1, its humanized form, and isotope, drug, and toxin conjugates. These studies show that unconjugated hLL1 and conjugates of hLL1 constructs with radioisotopes, doxorubicin, and frog RNase have high antitumor activity in non–Hodgkins lymphoma and multiple myeloma in vitro and in tumor xenograft models. Single-dose studies of hLL1 in monkeys showed no adverse effects but did decrease circulating B and T lymphocytes and natural killer cells. When evaluated in combination with rituximab, either equivalent or improved efficacy, compared with either antibody alone, was observed. CD74 is a new candidate target for the immunotherapy of neoplasms expressing this antigen, which can be exploited using either a naked antibody or conjugated to isotopes, drugs, or toxins.

Mouse strains with typical mammalian levels of complement activity

Common laboratory mouse strains have very low complement levels relative to humans, rats, guinea pigs, rabbits and other mammals, which limits the value of the mouse as an experimental model. We therefore tested serum complement levels of 43 mouse strains and 11 rat strains, for the purpose of selecting a convenient laboratory animal having high complement levels. Total complement activity was determined with both erythrocytes and human tumor cells as targets. Eight mouse strains were identified that have complement levels comparable to those of other mammals. These mouse sera lyse tumor cell targets as well as sera from humans, rats or guinea pigs, although they are somewhat less active than rabbit sera. They are relatively inefficient in lysing erythrocyte targets, yet are as active as rabbit serum in this assay. Target cell lysis was demonstrated to be via the classical pathway of complement activation. Of the eight high complement mouse strains, four were recently derived from wild mice, and one, SF/CamEi, was derived from wild mice in 1951. The three other strains, BUB/BnJ, DA/HuSn and RIIIS/J, were developed more than 40 years ago, but apparently were not tested previously for complement activity. Using the BUB mouse as a representative of the high complement mice, we assayed levels of the nine complement components, in an attempt to identify the cause of high complement activity. No difference in levels of C1, C2, C4, C8 or C9 was detected between BUB and BDF1 mice. C2 activity was very low in both strains. C3, C5, C6 and C7 activities were higher in BUB mice than in BDF1 mice, indicating that variation in these complement components is responsible for the difference in total complement activity. The genes determining the high complement phenotype appeared to be semi-dominant in F1 hybrids. The high-complement mouse strains, and recombinant strains derived from them, will be useful in a wide range of biomedical research.

Therapy of Advanced B-Lymphoma Xenografts with a Combination of 90Y-anti-CD22 IgG (Epratuzumab) and Unlabeled Anti-CD20 IgG (Veltuzumab)

Purpose: Antibodies are effective therapeutic agents in cancer, but cures are rarely if ever obtained. Combination therapies are likely to be more effective than a single agent. In this study, the combination of a new unconjugated humanized anti-CD20 IgG, veltuzumab, with a 90Y-conjugated humanized antibody to CD22 (epratuzumab) was evaluated for the treatment of B-cell lymphoma in a nude mouse model system. Experimental Design: Nude mice were grafted with the Ramos human B-lymphoma and treatment initiated when tumors were >0.1 cm3. In most experiments, mice were injected first with unconjugated anti-CD20, then with 90Y-anti-CD22 1 day later. Additional weekly injections of the unconjugated veltuzumab were administered for 3 weeks. Controls included a single agent only and a nonreactive control radiolabeled antibody. Results: Unconjugated anti-CD20 veltuzumab alone did not have a significant therapeutic effect, even at a total dose of 2.5 mg per mouse. The 90Y-anti-CD22 epratuzumab alone induced marked regressions of all tumors, but they regrew in a few weeks. The combination of these agents cured ∼80% of the mice. A nonreactive control antibody labeled with 90Y, used without veltuzumab, had no therapeutic effect. The therapeutic effect of 90Y-epratuzumab required the maximum tolerated dose of radioactivity, which was 160 μCi per mouse. Conclusions: These studies illustrate how combinations of unconjugated and radioconjugated antibodies against different B-cell markers can improve therapeutic outcome, and offer a new therapeutic paradigm for the treatment of B-cell lymphomas.

Characterization of antibody-containing vesicles shed from B-lymphoma cell lines: exposure of annexin V binding sites

Antibodies (Abs) to CD20 or HLA-DR, after binding to the B-lymphoma cell line RL following an overnight incubation at 37°C, accumulate in the form of shed vesicles, which develop in the center of the cell clusters that are spontaneously formed by this cell line. These vesicles coalesce into fairly stable large structures, which we refer to as conglomerates of shed vesicles (CSVs). In the present study, we have extended our previous investigations into the nature of this material. Electron microscopy revealed a conglomerate of heterogeneous vesicles, which looked like pinched-off cytoplasmic projections. CSVs developed similarly either with or without Ab, demonstrating that CSV production is a spontaneous process that incorporates bound Abs if they are present. Before delivery to CSVs, the Abs capped on the cell surface. CSVs had high expression of annexin V binding sites, which are phagocytic signals that are exposed on damaged cells. For CSVs that were cell bound, which are frequently observed, the annexin V binding sites were only in the CSVs, and not on the surface of the intact cell. Although all CSVs contained both Abs and annexin V binding sites, the precise distribution of these two ligands was generally different. Annexin V binding sites were present on caps as well as on CSVs, and appear as soon as caps are formed. In cells incubated with anti-HLA-DR, CD20 was delivered to the CSVs together with HLA-DR, suggesting an association between these two molecules. CSVs prepared with anti-HLA-DR, but not CSVs prepared with anti-CD20, contained considerable numbers of nuclear fragments, identified by propidium iodide staining.