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 isotypes. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy.

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.

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.

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 CD20 mAb ofatumumab (OFA) induces complement-mediated lysis of B cells. In an investigator-initiated phase II trial of OFA plus chemotherapy for chronic lymphocytic leukemia (CLL), OFA treatment promoted partial CLL B cell depletion that coincided with reduced complement titers. Remaining CLL B cells circulated with bound OFA and covalently bound complement breakdown product C3d, indicative of ongoing complement activation. Presumably, neither complement- nor effector cell-based mechanisms were sufficiently robust to clear these remaining B cells. Instead, almost all of the bound OFA and CD20 was removed from the cells, in accordance with previous clinical studies that demonstrated comparable loss of CD20 from B cells after treatment of CLL patients with rituximab. In vitro experiments with OFA and rituximab addressing these observations suggest that host effector mechanisms that support mAb-mediated lysis and tumor cell clearance are finite, and they can be saturated or exhausted at high B cell burdens, particularly at high mAb concentrations. Interestingly, only a fraction of available complement was required to kill cells with CD20 mAbs, and killing could be tuned by titrating the mAb concentration. Consequently, maximal B cell killing of an initial and secondary B cell challenge was achieved with intermediate mAb concentrations, whereas high concentrations promoted lower overall killing. Therefore, mAb therapies that rely substantially on effector mechanisms subject to exhaustion, including complement, may benefit from lower, more frequent dosing schemes optimized to sustain and maximize killing by cytotoxic immune effector systems.

The diversity in the heterotrimeric G protein ␣, ␤, and ␥ subunits may allow selective protein-protein interactions and provide specificity for signaling pathways. We examined the ability of five ␣ subunits (␣ i1 , ␣ i2 , ␣ o , ␣ s , and ␣ q ) to associate with three ␤ subunits (␤ 1 , ␤ 2 , and ␤ 5 ) dimerized to a ␥ 2 subunit containing an amino-terminal hexahistidine-FLAG affinity tag (␥ 2HF ). Sf9 insect cells were used to overexpress the recombinant proteins. The hexahistidine-FLAG sequence does not hinder the function of the ␤ 1 ␥ 2HF dimer as it can be specifically eluted from an ␣ i1 -agarose column with GDP and AlF 4 ؊ , and purified ␤ 1 ␥ 2HF dimer stimulates type II adenylyl cyclase. The ␤ 1 ␥ 2HF and ␤ 2 ␥ 2HF dimers immobilized on an anti-FLAG affinity column bound all five ␣ subunits tested, whereas the ␤ 5 ␥ 2HF dimer bound only ␣ q . The ability of other ␣ subunits to compete with the ␣ q subunit for binding to the ␤ 5 ␥ 2HF dimer was tested. Addition of increasing amounts of purified, recombinant ␣ i1 to the ␣ q in a Sf9 cell extract did not decrease the amount of ␣ q bound to the ␤ 5 ␥ 2HF column. When G proteins in an extract of brain membranes were activated with GDP and AlF 4 ؊ and deactivated in the presence of equal amounts of the ␤ 1 ␥ 2HF or ␤ 5 ␥ 2HF dimers, only ␣ q bound to the ␤ 5 ␥ 2HF dimer. The ␣ q -␤ 5 ␥ 2HF interaction on the column was functional as GDP, and AlF 4 ؊ specifically eluted ␣ q from the column. These results indicate that although the ␤ 1 and ␤ 2 subunits interact with ␣ subunits from the ␣ i , ␣ s , and ␣ q families, the structurally divergent ␤ 5 subunit only interacts with ␣ q .All cells possess multiple signaling pathways that transmit signals from the hormones, autacoids, neurotransmitters, and growth factors in their environment. Complex biochemical mechanisms exist to discriminate, integrate, and modulate a cell's response to this constantly changing set of stimuli. One of the best characterized signal transduction systems is the pathway used by receptors coupled to heterotrimeric G proteins 1 (1, 2). Our current understanding of this signaling pathway shows it to be surprisingly complex with large families of proteins comprising the receptors, G proteins, and effectors (1, 3, 4) and important roles for both the ␣ and ␤␥ subunits of the heterotrimer in activating effectors (2, 4, 5). Moreover, some ligands activate multiple G proteins (1, 2, 6), and certain receptors activate the MAP kinase pathway (6, 7) and/or other tyrosine kinase signaling pathways (8). Thus, an important unsolved question in cell signaling is how a cell selects a response from the multiple possibilities available. Current evidence holds that specificity is determined at many levels. In addition to the tissue-specific expression of receptors, G proteins, or effectors (3), there are important protein-protein interactions involving the ␣ and ␤␥ subunits of the G protein heterotrimer that determine specificity. For example, the ␣ t subunit couples selectively to rhodopsin and the ␣ s subunit ...

Treatment of chronic lymphocytic leukemia (CLL) patients with standard dose infusion of rituximab (RTX), 375 mg/m2, induces clearance of malignant cells from peripheral blood after infusion of 30 mg of RTX. After completion of the full RTX infusion, substantial recrudescence of CLL cells occurs, and these cells have lost >90% of CD20. To gain insight into mechanism(s) of CD20 loss, we investigated the hypothesis that thrice-weekly low-dose RTX (20 or 60 mg/m2) treatment for CLL over 4 wk would preserve CD20 and enhance leukemic cell clearance. During initial infusions in all 12 patients, the first 30 mg of RTX promoted clearance of >75% leukemic cells. Four of six patients receiving 20 mg/m2 RTX retained ≥50% CD20, and additional RTX infusions promoted further cell clearance. However, four of six patients receiving 60 mg/m2 had CD20 levels <20% baseline 2 days after initial infusions, and additional RTX infusions were less effective, presumably due to epitope loss. Our results suggest that when a threshold RTX dose is exceeded, recrudesced RTX-opsonized cells are not cleared, due to saturation of the mononuclear phagocytic system, but instead are shaved of RTX-CD20 complexes by acceptor cells. Thrice-weekly low-dose RTX may promote enhanced clearance of circulating CLL cells by preserving CD20.

Protein splicing is the protein analogue of RNA splicing in which the central portion (spacer) of a protein precursor is excised and the amino‐ and carboxy‐terminal portions of the precursor reconnected. The yeast Tfp1 protein undergoes a rapid protein splicing reaction to yield a spliced 69 kDa polypeptide and an excised 50 kDa spacer protein. We have demonstrated that the 69 kDa species arises by reformation of a bona fide peptide bond. Deletion analyses indicate that only sequences in the central spacer protein of the Tfp1 precursor are critical for the protein splicing reaction. A fusion protein in which only the Tfp1 spacer domain was inserted into an unrelated protein also underwent efficient splicing, demonstrating that all of the information required for protein splicing resides within the spacer domain. Alteration of Tfp1p splice junction residues blocked or kinetically impaired protein splicing. A protein splicing model is presented in which asparagine rearrangement initiates the self‐excision of the spacer protein from the Tfp1 precursor. The Tfp1 spacer protein belongs to a new class of intervening sequences that are excised at the protein rather than the RNA level.