Carbamylation reduces the capacity of IgG for hexamerization and complement activation.

Carbamylation is a post-translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca-IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement-dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca-IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca-IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca-IgG to activate complement was analyzed in enzyme-linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca-IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca-IgG lost its capacity to activate complement in both solid-phase and CDC assays. Mixing ca-IgG with non-modified IgG did not result in effective inhibition of complement activation by ca-IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca-IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers.

The novel tribody [(CD20)(2)xCD16] efficiently triggers effector cell-mediated lysis of malignant B cells.

Bispecific antibodies (bsab) offer a promising approach for optimizing antibody-based therapies. In the present study, [(CD20)(2)xCD16], a recombinant CD20- and CD16-directed bsab in the tribody format, was designed to optimize recruitment of FcγRIII (CD16)-positive effector cells. [(CD20)(2)xCD16] retained the antigen specificities of the parental monoclonal antibodies and binding to FcγRIIIa was not compromised by the F/V polymorphism at amino-acid position 158. [(CD20)(2)xCD16] mediated potent lysis of lymphoma cell lines and freshly isolated tumor cells from patients, even at low picomolar concentrations (∼10 pM). Irrespective of the CD16a allotype, potency as well as efficacy of lysis obtained with the tribody was significantly higher than lysis triggered by rituximab. Tumor cell killing also occurred when autologous NK cells were used as effector cells. Compared with rituximab, the tribody demonstrated depletion of autologous B cells in ex vivo whole blood assays at 100-fold lower antibody concentration. In mice with a reconstituted humanized hematopoietic system, established by transplantation of human CD34-positive cord blood cells, this novel tribody significantly depleted autologous human B cells. Thus, tribodies such as [(CD20)(2)xCD16], recruiting CD16-positive effector cells, may represent promising candidates for clinical development.

Antibody CR1-2B11 recognizes a non-polymorphic epitope of human CR1 (CD35).

Measurement of erythrocyte [red blood cells (RBC)] complement receptor type 1 (CR1, CD35) has the potential to serve as a sensitive assessment of complement activation and immune complex clearance. All previously reported monoclonal antibodies (MoAb) to the extracellular region of CR1 recognize epitopes within the long homologous repeats (LHR) of CR1 and the epitopes for the most frequently used MoAbs are repeated at least twice per CR1 molecule. Furthermore, CR1 exhibits structural polymorphism characterized by a variable number of LHR per molecule. Thus, accurate enumeration of cell surface CR1 using currently available MoAb would require that the results be corrected for the number of antibody epitopes per CR1 molecule encoded by each individual's alleles. To obtain a MoAb to a non-polymorphic epitope on human CR1, hybridomas were generated from mice immunized with recombinant soluble CR1 (sCR1) and MoAb were screened for those that recognized the full-length extracellular domain but failed to bind to all four recombinant LHR fragments. A single antibody, CR1-2B11, was identified and was found to recognize an epitope located wholly within SCR29-30 of CR1, NH2-terminal to an elastase cleavage site. Like other CR1 MoAb, the CR1-2B11 epitope expression decreased on old erythrocytes compared to younger cells and CR1-2B11 did not identify a CR1 'stump' on RBC. Importantly, CR1-2B11 immunofluorescence did not change with storage or handling of RBC, unlike the apparent decrease in immunofluorescence observed with other MoAb. CR1-2B11 should be useful for the accurate enumeration of RBC CR1.

Mouse complement components C4 and Slp act synergistically in a homologous hemolytic C4 assay.

Goals of the present study were to compare the hemolytic activities of mouse C4 and Slp in a homologous system and to study a possible interaction between these proteins during complement activation. As reagents for mouse C4 and Slp, we used serum of C4(- / -) knockout C57BL / 6 (C4(-) / Slp(-)) mice and sensitized rabbit erythrocytes as target cells. Sera to be tested contained none, either of the two or both proteins. We found that C4(-) / Slp(+) serum has some hemolytic C4 activity, but less than C4(+) / Slp(-) serum. Comparing C4 activities of C4(+) / Slp(-) and C4(+) / Slp(+) sera, we found a threefold enhanced activity in double-positive serum. Hemolytic C4 levels of mixtures of solely C4- and Slp-sufficient sera did not overlap with expected C4 levels, but rather these sera showed synergy. This explains the enhanced activity of double-positive serum. Similar results were observed for total complement activation. In conclusion, Slp has measurable, but poor C4 activity as compared with mouse C4. Using our homologous system, we showed that the enhanced classical pathway activity of double-positive sera is most probably based on synergy between C4 and Slp. Our results answer an old question as to why C4(+) / Slp(+) mice have higher complement levels than C4(+) / Slp(-) mice.

Sex-limited protein: in vitro and in vivo functions.

Mouse complement component C4 exists in two isoforms, C4 and a protein with expression restricted to male animals called sex-limited protein (Slp). Although Slp is about 95% homologous to C4, it is generally believed to be non-functional, at least in conventional haemolytic complement assays. In a previous study, however, we showed that Slp is haemolytically active in a C1-inhibitor (C1INH)-regulated, EDTA-resistant mouse complement activation pathway. To study other possible implications of this finding, we generated constitutively expressing Slp-transgenic mice. The transgene was crossed into otherwise Slp-deficient C57Bl/6J and NZB mice. Members of the third backcross generation of C57Bl/6J mice were tested for functional Slp and classical and alternative complement pathway activities (CH50 and AP50 levels, respectively). Slp-transgenic C57Bl/6J mice showed enhanced CH50, but normal AP50 levels when compared with non-transgenic littermates. To discover a possible protective role for Slp in spontaneous systemic lupus erythematosus (SLE) in NZBxNZW (NZBxW) mice, the third backcross generation of Slp-transgenic NZB mice was mated with NZW mice and the development of SLE in the female offspring was followed. In these introductory experiments, Slp-transgenic NZBxW animals presented with a significantly extended life span. Our results imply that Slp is a mouse complement component with functions which partially resemble some of those of human C4A.

Antibody responses and opsonic activity in sera of preterm neonates with coagulase-negative staphylococcal septicemia and the effect of the administration of fresh frozen plasma.

Coagulase-negative staphylococcal septicemia is the most prominent nosocomial infection in neonatal intensive care units. Immaturity of host defenses in premature neonates is assumed to constitute an important risk factor. Opsonophagocytosis is considered to be the key host defense system against staphylococci with IgG antibodies as a major opsonin. For this reason we have studied serum IgG antibody titers and opsonic activity to coagulase-negative staphylococci in 20 infants with septicemia and 40 matched control subjects. In addition, we assessed the effect of administration of fresh frozen plasma (FFP) on IgG antibody titer and serum opsonic activity in 12 patients with septicemia. IgG antibodies, quantified by ELISA and opsonic activity, determined by flow cytometry, were expressed as a percentage of the value of pooled normal human reference serum. Both patients and control subjects showed low IgG titers (median, 21%; range, 1-192%) and a low opsonic activity (median, 33%; range, 8-484%) at birth. During the first 2 postnatal wk IgG titers decreased significantly in septicemia patients (from a median of 30 to 17%, p = 0.025), but not in control subjects, whereas opsonic activity remained unchanged. The titer of IgG antibodies increased significantly in septicemia patients after FFP administration (from a median of 17 to 41%, p = 0.002), whereas the effect on opsonic activity was unpredictable, showing a moderate increase in 10 out of 12 infants, and in 2 patients even a substantial decrease (>50%), despite adequate opsonic activity in the corresponding FFP batches. Immunoblotting of sepsis isolates with the corresponding patient sera demonstrated that septicemic infants may generate IgG antibodies against their blood isolate. Neonates who acquire coagulase-negative staphylococcal septicemia cannot be distinguished from control subjects on the basis of IgG antibodies and opsonic activity to staphylococci either at birth or during the first 2 postnatal wk. The administration of FFP to septicemia neonates has an unpredictable effect on opsonic activity and therefore does not seem to be a useful addition to antibiotic therapy.