Inactive iC3b Research Articles

Abstract 2915: Targeting complement factor H renders STK11-mutant lung adenocarcinoma sensitive to anti-PD-1 therapy in mice

Abstract STK11 mutations occurs in 15-20% of lung adenocarcinoma (LUAD) and is associated with KRAS mutations, poor survival and resistance to immune checkpoint inhibitors (ICI). We observed tumor cell lines and primary human STK11-mutant LUAD upregulates of expression of C3 and complement factor H (FH). Deletion of C3 in STK11 knock-out tumor cells resulted in reduced FH expression. FH is a co-factor of Factor I, which converts C3b to proteolytically inactive iC3b thereby limiting activation of the alternative pathway (AP) C3 convertase. FH binds to proteoaminoglycans on the cell membrane and protects cells from AP complement-dependent cytotoxicity. With increased C3 generation by STK11-mutant tumor cells, we reasoned that systemic or tumor-derived FH protects tumor cells from complement attack. We hypothesized that tumor-derived C3 would promote STK11-mutant LUAD growth in immune intact mice by promoting immune evasion and would drive ICI resistance. Based on the expected tumor-protective effects of FH, we also hypothesized that anti-FH antibodies would control tumor growth and render tumor more sensitive to ICI. We evaluated growth of subcutaneously administered CMT167-STK11KO with and without C3 deletion (CMT167-STK11KO is murine KRASG12V LUAD with CRISPR/Cas9 deletion of STK11). Deletion of C3 in CMT167-STK11KO (CMT167-STK11KO/C3KO) resulted in dramatic inhibition of tumor growth and in sensitivity to anti-PD-1 therapy in vivo. In contrast, deletion of C3 in STK11-KO tumors had no effect on tumor growth in nude mice or following CD8 depletion in WT mice. These findings support a role of tumor-derived C3 in evasion of T cell anti-tumor immunity and as an obstacle to anti-PD-1 therapy. GT103 is a therapeutic antibody that binds tumor cell-attached CFH, causes complement activation, kills tumor cells, initiates a shift from a pro- to an anti-tumor immune microenvironment, and is currently being evaluated in a Phase 1b-2 clinical trial in combination with pembrolizumab (NCT05617313). We observed that single agent GT103 or anti-PD-1 has limited efficacy in CMT167-STK11KO tumor in WT mice. However, the combination of GT103 and anti-PD-1 was very effective in limiting tumor growth. Our results support a role of tumor-derived C3 in suppressing CD8 T-cell immunity and in driving ICI resistance and that the addition of GT103 renders STK11-KO tumors sensitive to anti-PD-1 in mice. These studies establish rationale for evaluating this combination in patients with STK11-mutant LUAD. Citation Format: Sora Suzuki, Anm Nazmul Khan, Thejaswini Giridharan, Catrina Ting, Ryan Bushey, Elizabeth Gottlin, Michael Campa, Edward F. Patz, Edwin H. Yau, Brahm Segal. Targeting complement factor H renders STK11-mutant lung adenocarcinoma sensitive to anti-PD-1 therapy in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2915.

Thrombomodulin enhances complement regulation through strong affinity interactions with factor H and C3b-Factor H complex

IntroductionCoagulation and complement systems are simultaneously activated at sites of tissue injury, leading to thrombin generation and opsonisation with C3b. Thrombomodulin (TM) is a cell-bound regulator of thrombin activation, but can also enhance the regulatory activity of complement factor H (FH), thus accelerating the degradation of C3b into inactive iC3b. ObjectivesThis study sought to determine the biophysical interaction affinities of two recombinant TM analogs with thrombin, FH and C3b in order to analyze their ability to regulate serum complement activity. MethodsSurface plasmon resonance (SPR) analysis was used to determine binding affinities of TM analogs with FH and C3b, and compared to thrombin as positive control. The capacity of the two recombinant TM analogs to regulate complement in serum was tested in standard complement hemolytic activity assays. ResultsSPR analysis showed that both TM analogs bind FH and C3b-Factor H with nanomolar and C3b with micromolar affinity; binding affinity for its natural ligand thrombin was several fold higher than for FH. At a physiological relevant concentration, TM inhibits complement hemolytic activity in serum via FH dependent and independent mechanisms. ConclusionsTM exhibits significant binding affinity for complement protein FH and C3b-FH complex and its soluble form is capable at physiologically relevant concentrations of inhibiting complement activation in serum.

Von Willebrand Factor Is a Cofactor in Complement Regulation

Complement, besides its involvement in eliminating microbes, participates in such diverse processes as synapse maturation, clearance of immune complexes, angiogenesis and tissue regeneration. Delicate balance between complement activation and regulation contributes to complement’s role in physiology. Any trigger that tips this balance can induce self-attack. Atypical hemolytic uremic syndrome (aHUS) is a systemic disease characterized by non-immune hemolytic anemia, thrombocytopenia, and renal impairment. In over 50% of cases, aHUS is known to be caused by uncontrolled activation of the complements. Von Willebrand factor (VWF) is a large multimeric glycoprotein that plays an important role in stopping the escape of blood from vessels following vascular injury. VWF and VWF cleavage enzyme ADAMTS13 gene defects have been identified in patients with aHUS, raising the possibility that VWF could have contributed to complement regulation. To examine the role of VWF in complement activation, we investigated whether VWF functions as a cofactor for FI-mediated C3b cleavage through in vitro assay. We found that C3b binds to VWF. In the presence of plasma-purified VWF (pVWF), FI cleaves C3b to 68kD and 43kD degradation products (iC3b) (Figure (A)). VWF alone, or FI alone, did not have any effect on C3b cleavage. C3b, not C3 or iC3b, was the mainly substrate for FI/VWF proteolysis. Increasing VWF concentration or prolonging the incubation time with VWF enhanced FI-mediated C3b cleavage. To remove the possibility that another plasma protein co-purified with pVWF that affects our results, we used recombinant VWF dimers purified from human embryonic kidney (HEK) 293 cell expressing VWF-Dpro cDNA, and detected a similar cofactor activity (Figure (B)).To investigate whether the size of VWF multimers have any effect on C3b cleavage, we compared the cofactor activity of pVWF, recombinant VWF dimers, and ULVWF multimers. While pVWF and dimer VWF enhanced C3b cleavage by FI, ULVWF did not have any effect on C3b cleavage (Figure (B)). In plasma, complement proteins Factor B and Factor D coexist with the inhibitory protein FI. To investigate the effect of VWF on complement activity in the presence of both pro-activation and inhibitory complement proteins, we incubated C3, FB, FD, and FI with VWF. In the presence of FB and FD, C3 be activated and resulted in the generation of C3a and C3b. Addition of recombinant VWF put a brake on complement activation and shifted C3 toward the generation of iC3b (Figure (C)). We conclude that normal plasma VWF, function as a cofactor, prevents complement activation through steers the complement pathway toward the generation of inactive iC3b. ULVWF multimers, as are present in patients with thrombotic microangiopathy, lack an inhibitory effect on complement and permit complement activation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Loa loaMicrofilariae Evade Complement Attack In Vivo by Acquiring Regulatory Proteins from Host Plasma

Loa loa is a filarial nematode that infects humans. The adults live in subcutaneous tissues and produce microfilariae that live for several weeks in the blood circulation in order to be transmitted to another person via blood meals of a dipterian vector. As microfilariae live in continuous contact with plasma, it is obvious that they evade the complement system. We studied markers of complement activation and signs of complement regulation on Loa loa microfilariae in vivo. The microfilariae were isolated from anticoagulated blood samples of a Loa loa-infected Caucasian patient. C1q and some mannose-binding lectin but only a limited amount of C3b or C4b fragments and practically no C5 or C5b-9 were present on the microfilariae. The covalently microfilaria-bound C3 and C4 depositions were mainly inactive iC3b, C3c, and iC4b fragments indicating that microfilariae had regulated complement activation in vivo. Also, in vitro deposition of C3b onto the microfilariae upon serum exposure was limited. The patient-isolated microfilariae were found to carry the host complement regulators factor H and C4b-binding protein on the outermost layer, so called sheath. The microfilaria-bound factor H was functionally active. Binding of the complement regulators to the microfilariae was confirmed in vitro using (125)I-labeled factor H and C4b-binding protein. In conclusion, our study shows that Loa loa microfilariae block complement activation and acquire the host complement regulators factor H and C4b-binding protein in blood circulation. This is the first time that binding of complement regulators onto nonviral pathogens has been demonstrated to occur in humans in vivo.

The Paramyxoviruses Simian Virus 5 and Mumps Virus Recruit Host Cell CD46 To Evade Complement-Mediated Neutralization

The complement system is a critical component of the innate immune response that all animal viruses must face during natural infections. Our previous results have shown that treatment of the paramyxovirus simian virus 5 (SV5) with human serum results in deposition of complement C3-derived polypeptides on virion particles. Here, we show that the virion-associated C3 component includes the inactive form iC3b, suggesting that SV5 may have mechanisms to evade the host complement system. Electron microscopy, gradient centrifugation, and Western blot analysis indicated that purified SV5 virions derived from human A549 cells contained CD46, a plasma membrane-expressed regulator of complement that acts as a cofactor for cleavage and inactivation of C3b into iC3b. In vitro cleavage assays with purified complement components showed that SV5 virions had C3b cofactor activity, resulting in specific factor I-mediated cleavage of C3b into inactive iC3b. SV5 particles generated in CHO cells, which do not express CD46, did not have cofactor activity. Conversely, virions derived from a CHO cell line that was engineered to overexpress human CD46 contained elevated levels of virion-associated CD46 and displayed enhanced C3b cofactor activity. In comparison with C3b, purified SV5 virions had very low cofactor activity against C4b, consistent with the known preference of CD46 for C3b versus C4b. Similar results were obtained for the closely related mumps virus (MuV), except that MuV particles derived from CHO-CD46 cells had higher C4b cofactor activity than SV5 virions. In neutralization assays with human serum, SV5 and MuV containing CD46 showed slower kinetics and more resistance to neutralization than SV5 and MuV that lacked CD46. Our results support a model in which the rubulaviruses SV5 and MuV incorporate cell surface complement inhibitors into progeny virions as a mechanism to limit complement-mediated neutralization.

Secretion of soluble complement inhibitors factor H and factor H-like protein (FHL-1) by ovarian tumour cells.

We observed that the soluble complement regulators factor H and factor H-like protein were abundantly present in ascites samples as well as in primary tumours of patients with ovarian cancer. RT–PCR and immunoblotting analyses showed that the two complement inhibitors were constitutively produced by the ovarian tumour cell lines SK-OV-3 and Caov-3, but not PA-1 or SW626 cells. The amounts of factor H-like protein secreted were equal to those of factor H. This is exceptional, because e.g. in normal human serum the concentration of factor H-like protein is below 1/10th of that of factor H. In ascites samples the mean level of factor H-like protein (130±55 μg ml−1) was 5.5-fold higher than in normal human serum (24±3 μg ml−1). Ovarian tumour cells thus preferentially synthesise factor H-like protein, the alternatively spliced short variant of factor H. The tumour cells were found to bind both 125I-labelled factor H and recombinant factor H-like protein to their surfaces. Surprisingly, the culture supernatants of all of the ovarian tumour cell lines studied, including those of PA-1 and SW626 that did not produce factor H/factor H-like protein, promoted factor I-mediated cleavage of C3b to inactive iC3b. Subsequently, the PA-1 and SW626 cell lines were found to secrete a soluble form of the membrane cofactor protein (CD46). Thus, our studies reveal two novel complement resistance mechanisms of ovarian tumour cells: (i) production of factor H-like protein and factor H and (ii) secretion of soluble membrane cofactor protein. Secretion of soluble complement inhibitors could protect ovarian tumour cells against humoral immune attack and pose an obstacle for therapy with monoclonal antibodies.British Journal of Cancer (2002) 87, 1119–1127. doi:10.1038/sj.bjc.6600614 www.bjcancer.com© 2002 Cancer Research UK

Role of sialic acid in the resistance of Trypanosoma cruzi trypomastigotes to complement.

Trypomastigotes of Trypanosoma cruzi, mammalian infective forms of the parasite, express an unusual cell surface trans-sialidase. This enzyme enables the parasite to rapidly sialylate its surface when supplied with alpha(2,3)-linked sialic acid from glycoconjugates in serum or on cell surfaces. Here we used a novel fluorescence-based, trypomastigote lysis assay to evaluate the role of sialic acid on the parasite's plasma membrane in providing protection against the complement cascade. Trypomastigotes were desialylated, and sialic acid removal was confirmed by a chemical assay and also by flow cytometry with the use of a mAb that recognizes a T. cruzi-sialylated epitope. Compared with sialylated trypomastigotes, which were completely refractory to lysis by human serum, only about 5% of the desialylated trypomastigotes were lysed by complement. However, further analysis revealed that the desialylated parasites had been resialylated during exposure to serum complement. Next we incubated desialylated trypomastigotes with samples of desialylated human serum. Although the sialidase-treated serum retained its full hemolytic activity, lysis of trypomastigotes increased only from 5 to 24%. This increase correlated with an enhanced deposition of complement protein C3 on the parasite surface. The ratio of C3b to lytically inactive iC3b was increased for desialylated, compared with sialylated, parasites. We conclude that although parasite sialic acid promotes C3b cleavage into iC3b, this mechanism alone does not account for the robust resistance of these parasites to complement lysis.

Binding and release of C3 from Leishmania donovani promastigotes during incubation in normal human serum.

We have examined the nature and extent of C3 deposition on Leishmania donovani, strain 1S, clone 2D, promastigotes. Total molecules of C3 bound/parasite after 60 min was similar for parasites incubated in normal human serum, normal human serum adsorbed to remove natural antibody, or either serum source chelated with Mg-EGTA to limit activation to the alternative pathway. A comparison of parasites grown to early, mid, late-log or stationary phases revealed no difference in the extent and kinetics of C3 binding. C3 bound covalently to the parasite primarily through a hydroxylamine resistant (putatively amide) linkage. Of the bound C3, 75% was present as hemolytically inactive iC3b. Nearly 50% of the bound C3 was spontaneously released within 30 min at 37 degrees C. This spontaneous release was due to an unusual proteolytic cleavage event that released C3 from the C3 acceptor on the parasite surface. These results define and characterize the unusual features of C3 binding to L. donovani promastigotes during incubation in serum.

Lytic rabbit IgG for tissue culture trypomastigotes of Trypanosoma cruzi alters the extent and form of complement deposition

Infective and vertebrate stages of Trypanosoma cruzi are resistant to lysis by the alternative pathway of complement. To further elucidate the mechanism of complement evasion and to study how some immune sera render the infective stage sensitive to lysis, we compared the interaction of complement components C3 and C9 with the surface of complement susceptible, vector stage epimastigotes and vertebrate stage trypomastigotes of T. cruzi. Our studies showed that, upon incubation in human serum, complement resistant tissue culture trypomastigotes (TCT) bound five- to eightfold less C3 or C9 than complement sensitive epimastigotes (Epi). C3 bound to Epi is mainly in the hemolytically active C3b form, while TCT bear predominantly the hemolytically inactive iC3b fragment, which cannot participate in C5 convertase formation or lead to deposition of the lytic C5b-9 complex. Three- to sixfold more C3 and two- to threefold more C9 were deposited on TCT when lytic rabbit immune IgG with broad specificity was used to sensitize the parasites, and nearly one-half of bound C3 was present as C3b. In contrast, a comparison of three different sources of IgG from immune human serum showed a less clear correlation between the titer or specificity of anti- T. cruzi antibody, enhancement of C3 or C9 deposition, change in the form of bound C3, or killing. These results show that lytic rabbit IgG for T. cruzi changes the form and amount of bound complement components in anticipated fashion, but that human immune IgG does not give predictable changes in the extent or form of C3 or C9 deposition.

Pathogenesis of Campylobacter fetus infections. Failure of encapsulated Campylobacter fetus to bind C3b explains serum and phagocytosis resistance.

Campylobacter fetus ssp. fetus strains causing systemic infections in humans are highly resistant to normal and immune serum, which is due to the presence of high molecular weight (100,000, 127,000, or 149,000) surface (S-layer) proteins. Using serum-resistant parental strains (82-40 LP and 23D) containing the 100,000-mol wt protein and serum-sensitive mutants (82-40 HP and 23B) differing only in that they lack the 100,000-mol wt protein capsule, we examined complement binding and activation, and opsono-phagocytosis by polymorphonuclear leukocytes. C3 consumption was similar for all four strains but C3 was not efficiently bound to 82-40 LP or 23D even in the presence of immune serum, and the small amount of C3 bound was predominently the hemolytically inactive iC3b fragment. Consumption and binding of C5 and C9 was significantly greater for the unencapsulated than the encapsulated strains. Opsonization of 82-40 HP with heat-inactivated normal human serum caused greater than 99% killing by human PMN. Similar opsonization of 82-40 LP showed no kill, but use of immune serum restored killing. Findings in a PMN chemiluminescence assay showed parallel results. Association of 32P-labeled 82-40 HP with PMN in the presence of HINHS was 19-fold that for the 82-40 LP, and electron microscopy illustrated that the difference was in uptake rather than in binding. These results indicate that presence of the 100,000-mol wt protein capsule on the surface of C. fetus leads to impaired C3b binding, thus explaining serum resistance and defective opsonization in NHS, mechanisms that explain the capacity of this enteric organism to cause systemic infections.