Specific inhibition of the classical complement pathway with an engineered single-chain Fv to C1q globular heads decreases complement activation by apoptotic cells.

Apoptotic cells are potent complement activators; and proposed mechanisms include IgM-mediated classical pathway activation, C-reactive protein (CRP)-mediated classical pathway activation, and IgM-mediated lectin pathway activation. While complement activation is beneficial in clearing apoptotic cells, the resulting complement-mediated inflammation may extend damage to the surrounding cells and tissues, as observed in ischemia/reperfusion injury. We previously engineered and characterized a single-chain Fv against C1q globular heads (scFv(QuVHVL)) that blocked C1q binding to immobilized IgG and to IgG-sensitized cells, and thereby inhibited IgG-mediated classical pathway activation [Hwang H.Y., Duvall M.R., Tomlinson S., Boackle R.J., 2008. Highly specific inhibition of C1q globular-head binding to human IgG: a novel approach to control and regulate the classical complement pathway using an engineered single-chain antibody variable fragment. Molecular Immunology 45, 2570-2580]. In the present study, this scFv(QuVHVL) was examined for its ability to restrict complement deposition on apoptotic cells in the presence of fresh normal human serum (NHS). Interestingly, the addition of scFv(QuVHVL) to NHS decreased C1-mediated C4b deposition on apoptotic cells by 60% as compared to appropriate buffer-treated control serum. By inhibiting initiation of the early complement components, the subsequent C3b and membrane attack complex depositions were inhibited by 70%. Apoptotic cells may acquire serum CRP, a known classical complement pathway activator. It was observed that scFv(QuVHVL) blocked C1 binding to CRP and blocked CRP-mediated classical pathway activation using an ELISA format. However, under the experimental conditions used, the addition of exogenous CRP to apoptotic cells did not further increase the levels of C4b, C3b, or MAC deposition significantly, suggesting predominance by other activation mechanisms, such as antibody-C1-mediated complement activation. In summary, the results indicated that C1-mediated classical pathway activation was a highly significant mechanism for complement activation by apoptotic cells. In the future, specific inhibition of classical complement pathway activation by a humanized form of scFv(QuVHVL) may be useful in reducing inadvertent damage to healthy bystander tissue in a variety of acute, complement-mediated inflammatory conditions, including ischemia/reperfusion injury.

Complement C1q activates tumor suppressor WWOX to induce apoptosis in prostate cancer cells.

BACKGROUND: Tissue exudates contain low levels of serum complement proteins, and their regulatory effects on prostate cancer progression are largely unknown. We examined specific serum complement components in coordinating the activation of tumor suppressors p53 and WWOX (also named FOR or WOX1) and kinases ERK, JNK1 and STAT3 in human prostate DU145 cells. METHODOLOGY/PRINCIPAL FINDINGS: DU145 cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated, and hyaluronan (HA) reversed the effect. Under C6-free conditions, HA induced activation of STAT3, an enhancer of metastasis. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) microscopy, it was determined that C1q destabilized adherence of WOX1-expressing DU145 cells by partial detaching and inducing formation of clustered microvilli for focal adhesion particularly in between cells. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues. CONCLUSIONS/SIGNIFICANCE: We conclude that complement C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation.

Highly specific inhibition of C1q globular-head binding to human IgG: a novel approach to control and regulate the classical complement pathway using an engineered single chain antibody variable fragment.

We sought to specifically regulate the binding of human C1q, and thus the activation of the first complement component, via the construction of a single chain antibody variable binding region fragment (scFv) targeting the C1q globular heads. Here we describe details of the construction, expression and evaluation of this scFv, which was derived from a high-affinity hybridoma (Qu) specific for the C1q globular heads. The scFv was comprised of the Qu variable heavy chain domain (VH) linked to the Qu variable light chain domain (VL) and was termed scFv-QuVHVL. When mixed with either purified C1q or with human serum as a source of C1, scFv-QuVHVL bound to C1q and competitively restricted the interaction of C1q or C1 with immobilized IgG or with IgG1 antibody-coated cells, and prevented the activation of native C1 in human serum as determined by analyses of C1-mediated C4 deposition and fluid-phase C4 conversion. However scFv-QuVHVL could be manipulated to become a C1 activator when it was irreversibly immobilized onto microtiter ELISA plates, prior to contact with human serum complement. This functional dichotomy can be a useful tool in selectively elucidating, differentiating, inducing or inhibiting specific roles of human C1q and the classical complement pathway in complement-mediated physiological processes. We project that once fully humanized, fluid-phase scFv-QuVHVL could become a useful therapeutic in limiting inadvertent host tissue damage elicited by the classical complement pathway.

Complement susceptibility in glutamine deprived breast cancer cells.

BACKGROUND: Membrane complement regulatory proteins (mCRPs) inhibit complement-mediated killing of human cells by human complement, a property that confers protection from complement to malignant breast cancer cells and that thwarts some immunotherapies. Metabolic mechanisms may come into play in protecting cancer cells from the complement system subsequent to relatively low levels of complement deposition. RESULTS: In differentiating these mechanisms, two types of human breast cancer cell lines, MCF7 (adenocarcinoma) and Bcap37 (medullary carcinoma) were cell-cycle synchronized using glutamine-deprivation followed by restoration. These cells were examined for the expression of two mCRPs (CD59 and CD55), and for subsequent susceptibility to antibody-mediated complement-induced membrane damage. After glutamine restoration, MCF7 and Bcap37 cells were synchronized into the G2/M phase and an average increased expression of CD59 and CD55 occurred with a corresponding resistance to complement-mediated damage. Blocking CD59 inhibitory function with monoclonal antibody revealed that CD59 played a key role in protecting unsynchronized Bcap37 and MCF7 cancer cells from the complement membrane attack complex. Interestingly, glutamine-deprivation did not significantly affect the expression of proteins e.g., the surface level of CD59 or CD55, but did increase the susceptibility to complement-mediated killing. One possible explanation is that glutamine-deprivation may have slowed the turnover rate of mCRPs, preventing the cells from replacing pre-existing mCRPs, as they became neutralized by covalent C4b and C3b depositions. CONCLUSION: Taken together the findings are consistent with the conclusion that future immunotherapies should aim to achieve a highly specific and profound activation and deposition of complement as well as to disrupt the synthesis and expression of CD59 and CD55 by the cancer cells.

Conformationally altered hyaluronan restricts complement classical pathway activation by binding to C1q, C1r, C1s, C2, C5 and C9, and suppresses WOX1 expression in prostate DU145 cells.

Linear non-sulfated hyaluronan (HA) does not bind complement proteins yet inhibits their hemolytic function. We have previously induced the complement inhibitory function of HA by heat treatment. However, heated HA readily loses its anti-complementary activity probably due to instantaneous interchain re-association. Here, HA solutions were heated and then freeze-dried. Compared to native HA, heated/freeze-dried HA stably restricted serum complement-mediated hemolysis via the classical pathway, in which serum C1 hemolytic function and C3 activation were blocked. Also, treated HA had a significantly increased binding of component C1q, C1r, C1s, C2, C5, C9, P, D and H. Further, when HA was gel-fractionated by electrophoresis and then freeze-dried, its anti-complementary activity was stably induced. Both native and heated/freeze-dried HA stimulated ERK phosphorylation in prostate DU145 cells. However, treated HA suppressed the expression of tumor suppressors WOX1 and WOX2. Together, HA with an altered conformation stabilizes its inhibition and binding of complement proteins. It may recognize cell surface receptors differently from native HA, thereby differentially regulating the expression of cellular proteins.

OxLDL immune complexes activate complement and induce cytokine production by MonoMac 6 cells and human macrophages.

Oxidized low density lipoprotein (OxLDL) is immunogenic and induces autoimmune responses in humans. OxLDL antibodies are predominantly of the proinflammatory IgG1 and IgG3 isotypes. We tested the capacity of immune complexes prepared with copper-oxidized human LDL and affinity chromatography-purified human OxLDL antibodies [OxLDL-immune complexes (ICs)] to activate complement and to induce cytokine release by MonoMac 6 (MM6) cells and by primary human macrophages. The levels of C4d and C3a were significantly higher in human serum incubated with OxLDL-ICs than after incubation with OxLDL or OxLDL antibody, indicating complement activation by the classical pathway. MM6 cells and primary human macrophages were incubated with OxLDL-ICs, with or without prior conditioning with interferon-gamma. After 18 h of incubation, both MM6 cells and primary human macrophages released significantly higher levels of proinflammatory cytokines after incubation with OxLDL-ICs than after incubation with OxLDL or with OxLDL antibody, both in primed and unprimed cells. OxLDL-ICs were more potent activators of MM6 cells than keyhole limpet hemocyanin-ICs. Blocking Fc gamma receptor I (FcgammaRI) with monomeric IgG1 significantly depressed the response of MM6 cells to OxLDL-ICs. In conclusion, human OxLDL-ICs have proinflammatory properties, as reflected by their capacity to activate the classical pathway of complement and to induce proinflammatory cytokine release from MM6 cells and primary human macrophages.

Complement-coated antibody-transfer (CCAT); serum IgA1 antibodies intercept and transport C4 and C3 fragments and preserve IgG1 deployment (PGD).

In periodontal disease, IgG1 and IgA1 antibodies produced in situ deposit on antigens in the affected tissues. Thus, there is an interest in the effect of co-deposited IgA1 antibodies on complement activation by IgG1-immune complexes. In the present study, we first analyzed the effect of IgA1-immune complexes on complement using human IgA1 antibodies to dansyl (with dansylated human serum albumin serving as the immobilized antigen). It was observed that these IgA1-immune complexes when incubated for prolonged times with 33% human serum as a source of complement received C4b and C3b deposition. As C4b and C3b deposited on the IgA1 antibodies and on the antigenic surface, the complement-coated IgA1 antibodies departed. These fluid-phase complement-coated IgA1 antibodies were transferred to antigen-coated microtiter-ELISA plates, where they became bound to the antigens. Thus, the complement-coated IgA1 antibodies retained their antigen-binding function, especially as a proportion of their covalently bound C3b progressively degraded to iC3b and C3d. Genetically engineered carbohydrate-deficient mutant human IgA1 antibodies were used to assess the role of carbohydrate in accepting the C4b and C3b depositions, and these studies indicated that the carbohydrate on the Fc-region of IgA1 played a positive role. Another interesting finding generated by this study was that when IgA1 was co-deposited with IgG1 antibodies, and serum complement was added, the IgG1 antibodies tended to remain on the antigenic surface. The co-deposited IgA1 antibodies not only controlled (reduced) the rate of the consumption of the first component of complement (C1) and of classical complement pathway activation by IgG1-immune complexes (and therein reduced the rate of complement-mediated dissolution of the IgG1-immune complexes), but also the co-deposited IgA1 antibodies simultaneously intercepted/accepted C4b and C3b, then departed, as complement began to cover the antigenic surfaces. The process in which complement-coated IgA1 antibodies transferred to non-complement-coated antigens is termed complement-coated antibody-transfer/transport (CCAT). In this way, IgA1 antibodies extended the efficiency of the complement system by insuring the specific IgA1 antibody-mediated transport of the captured biologically active complement fragments to those antigens stimulating the IgA1 antibody response but not yet neutralized (completely coated) with complement. Simultaneously by impeding the rate of C1 consumption and by intercepting C4b and C3b, IgA1 antibodies slowed C4b and C3b deposition on the antigenic surface and on the co-deposited IgG1 antibodies. Thus, in the presence of ongoing complement activation, the deposition of serum IgA1 antibodies enabled the co-deposited IgG1 antibodies to better maintain their ability to interact with antigens. We termed this latter phenomenon, preservation of IgG antibody deployment (PGD). In summary, co-deposited IgA1 antibodies maximized the efficiency of the complement system, transported their covalently bound complement fragments to specific antigens and sustained the effective deployment of IgG1 antibodies directed to those same antigens.