Andrew W. Pawluczkowycz

Binding of Submaximal C1q Promotes Complement-Dependent Cytotoxicity (CDC) of B Cells Opsonized with Anti-CD20 mAbs Ofatumumab (OFA) or Rituximab (RTX): Considerably Higher Levels of CDC Are Induced by OFA than by RTX

The CD20 mAb ofatumumab (OFA) is more effective than rituximab (RTX) in promoting complement-dependent cytotoxicity (CDC) of B cells via the classical pathway (CP) of complement. CP activation is initiated by C1q binding to cell-bound IgG. Therefore, we examined the role of C1q in the dynamics of complement activation and CDC of B cell lines and primary cells from patients with chronic lymphocytic leukemia, reacted with OFA or RTX. C1q binding, complement activation, and colocalization of C1q with cell-bound mAbs were determined by flow cytometry and high-resolution digital imaging. C1q binds avidly to OFA-opsonized Raji and Daudi cells (KD = 12–16 nM) and colocalizes substantially with cell-bound OFA. Cells opsonized with OFA undergo high levels of complement activation and CDC in C1q-depleted serum supplemented with low concentrations of C1q. Under comparable conditions, RTX-opsonized cells bind less C1q; in addition, even when higher concentrations of C1q are used to achieve comparable C1q binding to RTX-opsonized cells, less complement activation and CDC are observed. Greater CDC induced by OFA may occur because C1q is bound in close proximity and with high avidity to OFA, resulting in effective CP activation. Moreover, OFA binds to the small, extracellular CD20 loop, placing the mAb considerably closer to the cell membrane than does RTX. This may facilitate effective capture and concentration of activated complement components closer to the cell membrane, potentially shielding them from inactivation by fluid phase agents and promoting efficient generation of the membrane attack complex.

Complement activation on B lymphocytes opsonized with rituximab or ofatumumab produces substantial changes in membrane structure preceding cell lysis.

Binding of the CD20 mAb rituximab (RTX) to B lymphocytes in normal human serum (NHS) activates complement (C) and promotes C3b deposition on or in close proximity to cell-bound RTX. Based on spinning disk confocal microscopy analyses, we report the first real-time visualization of C3b deposition and C-mediated killing of RTX-opsonized B cells. C activation by RTX-opsonized Daudi B cells induces rapid membrane blebbing and generation of long, thin structures protruding from cell surfaces, which we call streamers. Ofatumumab, a unique mAb that targets a distinct binding site (the small loop epitope) of the CD20 Ag, induces more rapid killing and streaming on Daudi cells than RTX. In contrast to RTX, ofatumumab promotes streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Generation of streamers requires C activation; no streaming occurs in media, NHS-EDTA, or in sera depleted of C5 or C9. Streamers can be visualized in bright field by phase imaging, and fluorescence-staining patterns indicate they contain membrane lipids and polymerized actin. Streaming also occurs if cells are reacted in medium with bee venom melittin, which penetrates cells and forms membrane pores in a manner similar to the membrane-attack complex of C. Structures similar to streamers are demonstrable when Ab-opsonized sheep erythrocytes (non-nucleated cells) are reacted with NHS. Taken together, our findings indicate that the membrane-attack complex is a key mediator of streaming. Streamer formation may, thus, represent a membrane structural change that can occur shortly before complement-induced cell death.

Binding of Rituximab, Trastuzumab, Cetuximab, or mAb T101 to Cancer Cells Promotes Trogocytosis Mediated by THP-1 Cells and Monocytes

More than 20 years ago clinical investigations in the immunotherapy of cancer revealed that infusion of certain immunotherapeutic mAbs directed to tumor cells induced loss of targeted epitopes. This phenomenon, called antigenic modulation, can compromise mAb-based therapies. Recently we reported that rituximab (RTX) treatment of chronic lymphocytic leukemia patients induced substantial loss of targeted CD20 on B cells found in the circulation after RTX infusion; this “shaving” of RTX-CD20 complexes from B cells is also promoted in vitro by THP-1 monocytes and by PBMC in a reaction mediated by Fcγ receptors. The mechanism responsible for shaving appears to be trogocytosis, a process in which receptors on effector cells remove and internalize cognate ligands and cell membrane fragments from target cells. We now report that three therapeutic mAbs approved by the U.S. Food and Drug Administration for the treatment of cancer, RTX, cetuximab, and trastuzumab, as well as mAb T101, which has been shown to induce antigenic modulation in the clinic, promote trogocytosis in vitro upon binding to their respective target cells. Trogocytosis of the mAb-opsonized cells is mediated by THP-1 monocytes and by primary monocytes isolated from PBMC. In view of these results, it is likely that these mAbs and possibly other anticancer mAbs now used in the clinic may promote trogocytic removal of the therapeutic mAbs and their cognate Ags from tumor cells in vivo. Our findings may have important implications with respect to the use of mAbs in cancer immunotherapy.

Rituximab-CD20 Complexes Are Shaved from Z138 Mantle Cell Lymphoma Cells in Intravenous and Subcutaneous SCID Mouse Models

Infusion of standard-dose rituximab (RTX) in chronic lymphocytic leukemia (CLL) patients promotes rapid complement activation and deposition of C3 fragments on CLL B cells. However, immediately after RTX infusions, there is substantial loss (shaving) of CD20 from circulating malignant cells. Because shaving can compromise efficacies of anticancer immunotherapeutic mAbs, we investigated whether shaving occurs in SCID mouse models. Z138 cells, a B cell line derived from human mantle cell lymphoma, were infused i.v. or s.c. The i.v. model recapitulates findings we previously reported for therapeutic RTX in CLL: i.v. infused RTX rapidly binds to Z138 cells in lungs, and binding is accompanied by deposition of C3 fragments. However, within 1 h targeted cells lose bound RTX and CD20, and these shaved cells are still demonstrable 40 h after RTX infusion. Z138 cells grow in tumors at s.c. injection sites, and infusion of large amounts of RTX (0.50 mg on each of 4 days) leads to considerable loss of CD20 from these cells. Human i.v. Ig blocked shaving, suggesting that FcγRI on cells of the mononuclear phagocytic system promote shaving. Examination of frozen tumor sections from treated mice by immunofluorescence revealed large areas of B cells devoid of CD20, with CD20 intact in adjacent areas; it is likely that RTX had opsonized Z138 cells closest to capillaries, and these cells were shaved by monocyte/macrophages. The shaving reaction occurs in neoplastic B cells in tissue and in peripheral blood, and strategies to enhance therapeutic targeting and block shaving are under development.

A novel approach to preventing the hemolysis of paroxysmal nocturnal hemoglobinuria: both complement-mediated cytolysis and C3 deposition are blocked by a monoclonal antibody specific for the alternative pathway of complement

The clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) is chronic intravascular hemolysis that is a consequence of unregulated activation of the alternative pathway of complement (APC). Intravascular hemolysis can be inhibited in patients by treatment with eculizumab, a monoclonal antibody that binds complement C5 thereby preventing formation of the cytolytic membrane attack complex of complement. However, in essentially all patients treated with eculizumab, persistent anemia, reticulocytosis, and biochemical evidence of hemolysis are observed; and in a significant proportion, their PNH erythrocytes become opsonized with complement C3. These observations suggest that PNH patients treated with eculizumab are left with clinically significant immune-mediated hemolytic anemia because the antibody does not block APC activation. With a goal of improving PNH therapy, we characterized the activity of anti-C3b/iC3b monoclonal antibody 3E7 in an in vitro model of APC-mediated hemolysis. We show that 3E7 and its chimeric-deimmunized derivative H17 block both hemolysis and C3 deposition on PNH erythrocytes. The antibody is specific for the APC C3/C5 convertase because classical pathway-mediated hemolysis is unaffected by 3E7/H17. These findings suggest an approach to PNH treatment in which both intravascular and extravascular hemolysis can be inhibited while preserving important immune functions of the classical pathway of complement.

Hematin Promotes Complement Alternative Pathway-Mediated Deposition of C3 Activation Fragments on Human Erythrocytes: Potential Implications for the Pathogenesis of Anemia in Malaria

Childhood malaria caused by Plasmodium falciparum is often characterized by severe anemia at low parasite burdens; the mechanism(s) responsible for this pathology remain to be defined. We have reported, based on clinical observations and in vitro models, that complement control proteins on erythrocytes such as CR1, the immune adherence receptor specific for C3b, may be reduced in childhood malaria, suggesting a possible role for complement in erythrocyte destruction. Intravascular lysis of iE by P. falciparum leads to release of erythrocyte breakdown products such as hemoglobin and hematin, which have inflammatory properties. In the present article, we demonstrate that in serum and in anticoagulated whole blood, moderate concentrations of hematin activate the alternative pathway of complement and promote deposition of C3 activation and breakdown products on erythrocytes. The degree of C3 fragment deposition is directly correlated with erythrocyte CR1 levels, and erythrocytes opsonized with large amounts of C3dg form rosettes with Raji cells, which express CR2, the C3dg receptor which is expressed on several types of B cells in the spleen. Thus, the reaction mediated by hematin promotes opsonization and possible clearance of the youngest (highest CR1) erythrocytes. A mAb specific for C3b, previously demonstrated to inhibit the alternative pathway of complement, completely blocks the C3 fragment deposition reaction. Use of this mAb in nonhuman primate models of malaria may provide insight into mechanisms of erythrocyte destruction and thus aid in the development of targeted therapies based on inhibiting the alternative pathway of complement.

Fractionated subcutaneous rituximab is well-tolerated and preserves CD20 expression on tumor cells in patients with chronic lymphocytic leukemia

A pilot study previously demonstrated that thrice-weekly, fractionated-dose intravenous rituximab (RTX) limits CD20 loss from chronic lymphocytic leukemia (CLL) B cells, thereby enhancing immunotherapeutic targeting. Here, we investigated the feasibility of giving 20 mg rituximab subcutaneously thrice weekly for up to 12 weeks in 4 previously treated CLL patients. Subcutaneous rituximab was well-tolerated with minimal injection site reactions; a variable degree of efficacy was observed, likely influenced by the size of the patients’ B cell/CD20 burden. Subcutaneous RTX largely preserved CD20 expression on leukemic cells but the most effective therapeutic dosing regimen needs to be established (ClinicalTrials.gov Identifier: NCT00366418).