Margaret A. Lindorfer

Rituximab Infusion Promotes Rapid Complement Depletion and Acute CD20 Loss in Chronic Lymphocytic Leukemia

Complement plays an important role in the immunotherapeutic action of the anti-CD20 mAb rituximab, and therefore we investigated whether complement might be the limiting factor in rituximab therapy. Our in vitro studies indicate that at high cell densities, binding of rituximab to human CD20+ cells leads to loss of complement activity and consumption of component C2. Infusion of rituximab in chronic lymphocytic leukemia patients also depletes complement; sera of treated patients have reduced capacity to C3b opsonize and kill CD20+ cells unless supplemented with normal serum or component C2. Initiation of rituximab infusion in chronic lymphocytic leukemia patients leads to rapid clearance of CD20+ cells. However, substantial numbers of B cells, with significantly reduced levels of CD20, return to the bloodstream immediately after rituximab infusion. In addition, a mAb specific for the Fc region of rituximab does not bind to these recirculating cells, suggesting that the rituximab-opsonized cells were temporarily sequestered by the mononuclear phagocytic system, and then released back into the circulation after the rituximab-CD20 complexes were removed by phagocytic cells. Western blots provide additional evidence for this escape mechanism that appears to occur as a consequence of CD20 loss. Treatment paradigms to prevent this escape, such as use of engineered or alternative anti-CD20 mAbs, may allow for more effective immunotherapy of chronic lymphocytic leukemia.

Complement Is Activated by IgG Hexamers Assembled at the Cell Surface

Hexing Complement Complement activation is an immediate and potent immune defense mechanism, but how immunoglobulin G (IgG) antibodies activate complement at the molecular level is poorly understood. Using high-resolution crystallography, Diebolder et al. (p. 1260) show that human IgGs form hexameric structures by interacting with neighboring IgG molecules, and the complex then activates complement. Thus, IgG molecules and the complement system can coexist in the blood because complement activation will only be triggered after IgG senses a surface antigen and starts to aggregate. Hexameric platforms of antibodies on the cell surface trigger the complement cascade. Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune defense, a number of disease processes, and immunotherapies. How antibodies activate the complement cascade, however, is poorly understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and killing of target cells, using all four human IgG subclasses. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy.

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.

The Shaving Reaction: Rituximab/CD20 Complexes Are Removed from Mantle Cell Lymphoma and Chronic Lymphocytic Leukemia Cells by THP-1 Monocytes

Clinical investigations have revealed that infusion of immunotherapeutic mAbs directed to normal or tumor cells can lead to loss of targeted epitopes, a phenomenon called antigenic modulation. Recently, we reported that rituximab treatment of chronic lymphocytic leukemia patients induced substantial loss of CD20 on B cells found in the circulation after rituximab infusion, when rituximab plasma concentrations were high. Such antigenic modulation can severely compromise therapeutic efficacy, and we postulated that B cells had been stripped (shaved) of the rituximab/CD20 complex by monocytes or macrophages in a reaction mediated by FcγR. We developed an in vitro model to replicate this in vivo shaving process, based on reacting rituximab-opsonized CD20+ cells with acceptor THP-1 monocytes. After 45 min at 37°C, rituximab and CD20 are removed from opsonized cells, and both are demonstrable on acceptor THP-1 cells. The reaction occurs equally well in the presence and absence of normal human serum, and monocytes isolated from peripheral blood also promote shaving of CD20 from rituximab-opsonized cells. Tests with inhibitors and use of F(ab′)2 of rituximab indicate transfer of rituximab/CD20 complexes to THP-1 cells is mediated by FcγR. Antigenic modulation described in previous reports may have been mediated by such shaving, and our findings may have profound implications for the use of mAbs in the immunotherapy of cancer.

Immunotherapeutic Mechanisms of Anti-CD20 Monoclonal Antibodies

The anti-CD20, B-cell-specific mAb rituximab (RTX) has been approved for treatment of non-Hodgkins B cell lymphoma and rheumatoid arthritis. Under conditions of high B cell burden, exhaustion of the bodys effector mechanisms, for example, NK-cell-mediated killing, may lead to substantial decreases in the immunotherapeutic efficacy of this mAb. Moreover, RTX treatment of patients with chronic lymphocytic leukemia and high levels of circulating B cells can lead to removal of CD20 from the cells, thus allowing them to persist and resist clearance. RTX therapy for several autoimmune diseases has proven to be effective, but in numerous instances there has been little correlation between reductions in disease activity and changes in titers of pathogenic autoantibodies. This paradox may be explained by a separate mechanism: Binding of RTX to B cells generates immune complexes that act as decoys to attract monoycte/macrophages and thus reduce their inflammatory activity in certain autoantibody-mediated diseases. Several second-generation anti-CD20 mAbs with enhanced cytotoxic action have been developed and are being tested in the clinic for treatment of cancer and autoimmune diseases. The application of these mAbs, potentially in combination with immune effector modifying drugs, may successfully address the shortcomings of current anti-CD20 immunotherapy.

Drug Insight: the mechanism of action of rituximab in autoimmune disease—the immune complex decoy hypothesis

Inflammatory responses to cell-associated or tissue-associated immune complexes are key elements in the pathogenesis of several autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus and immune thrombocytopenic purpura. Effector cells, such as monocytes, macrophages and neutrophils, bind immune complexes in a process mediated by Fcγ receptors, and these cells then initiate inflammatory reactions that lead to tissue destruction. Rituximab is an anti-CD20 monoclonal antibody that suppresses inflammation effectively in autoimmune diseases. It was initially approved by the FDA for the treatment of B-cell lymphomas and later for rheumatoid arthritis refractory to anti-tumor necrosis factor therapies. Rituximab is hypothesized to suppress disease injury in autoimmune diseases by promoting rapid and long-term elimination of circulating and possibly lymphoid-tissue-associated B cells. We suggest, however, that a different mechanism may underlie much of the therapeutic action of rituximab in autoimmune diseases: binding of tens of thousands of rituximab−IgG molecules to B cells generates decoy sacrificial cellular immune complexes that efficiently attract and bind Fcγ receptor-expressing effector cells, which diminishes recruitment of these effector cells at sites of immune complex deposition and, therefore, reduces inflammation and tissue damage.

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.

Role of the prenyl group on the G protein gamma subunit in coupling trimeric G proteins to A1 adenosine receptors.

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein α subunits and by the composition of the βγ dimers. To determine the role of the γ subunit prenyl modification in this interaction, the CaaX motifs in the γ1 and γ2 subunits were altered to direct modification with different prenyl groups, recombinant βγ dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the βγ dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the α subunit. The β1γ1 dimer (modified with farnesyl) was significantly less effective than β1γ2 (modified with geranylgeranyl) in either assay. The β1γ1-S74L dimer (modified with geranylgeranyl) was nearly as effective as β1γ2 in either assay. The β1γ2-L71S dimer (modified with farnesyl) was significantly less active than β1γ2. Using 125I-labeled βγ subunits, it was determined that native and altered βγ dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the γ subunit is an important determinant of the interaction between receptors and G protein γ subunits.

The complement receptor 2/factor H fusion protein TT30 protects paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and C3 fragment.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis because of the lack from erythrocyte surface of the complement regulators CD55 and CD59, with subsequent uncontrolled continuous spontaneous activation of the complement alternative pathway (CAP), and at times of the complement classic pathway. Here we investigate in an in vitro model the effect on PNH erythrocytes of a novel therapeutic strategy for membrane-targeted delivery of a CAP inhibitor. TT30 is a 65 kDa recombinant human fusion protein consisting of the iC3b/C3d-binding region of complement receptor 2 (CR2) and the inhibitory domain of the CAP regulator factor H (fH). TT30 completely inhibits in a dose-dependent manner hemolysis of PNH erythrocytes in a modified extended acidified serum assay, and also prevents C3 fragment deposition on surviving PNH erythrocytes. The efficacy of TT30 derives from its direct binding to PNH erythrocytes; if binding to the erythrocytes is disrupted, only partial inhibition of hemolysis is mediated by TT30 in solution, which is similar to that produced by the fH moiety of TT30 alone, or by intact human fH. TT30 is a membrane-targeted selective CAP inhibitor that may prevent both intravascular and C3-mediated extravascular hemolysis of PNH erythrocytes and warrants consideration for the treatment of PNH patients.

The G Protein β5 Subunit Interacts Selectively with the Gq α Subunit

The diversity in the heterotrimeric G protein α, β, and γ subunits may allow selective protein-protein interactions and provide specificity for signaling pathways. We examined the ability of five α subunits (αi1, αi2, αo, αs, and αq) to associate with three β subunits (β1, β2, and β5) dimerized to a γ2 subunit containing an amino-terminal hexahistidine-FLAG affinity tag (γ2HF). Sf9 insect cells were used to overexpress the recombinant proteins. The hexahistidine-FLAG sequence does not hinder the function of the β1γ2HF dimer as it can be specifically eluted from an αi1-agarose column with GDP and AlF4 −, and purified β1γ2HF dimer stimulates type II adenylyl cyclase. The β1γ2HF and β2γ2HF dimers immobilized on an anti-FLAG affinity column bound all five α subunits tested, whereas the β5γ2HF dimer bound only αq. The ability of other α subunits to compete with the αqsubunit for binding to the β5γ2HF dimer was tested. Addition of increasing amounts of purified, recombinant αi1 to the αq in a Sf9 cell extract did not decrease the amount of αq bound to the β5γ2HF column. When G proteins in an extract of brain membranes were activated with GDP and AlF4 − and deactivated in the presence of equal amounts of the β1γ2HF or β5γ2HF dimers, only αq bound to the β5γ2HF dimer. The αq-β5γ2HF interaction on the column was functional as GDP, and AlF4 −specifically eluted αq from the column. These results indicate that although the β1 and β2 subunits interact with α subunits from the αi, αs, and αq families, the structurally divergent β5 subunit only interacts with αq.