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ABSTRACT: Numerous antibody-drug conjugates (ADCs) are being developed for cancer immunotherapy. Although several of these agents have demonstrated considerable clinical efficacy and have won Food and Drug Administration (FDA) approval, in many instances, they have been characterized by adverse side effects (ASEs), which can be quite severe in a fraction of treated patients. The key hypothesis in this perspective is that many of the most serious ASEs associated with the use of ADCs in the treatment of cancer can be most readily explained and understood due to the inappropriate processing of these ADCs via pathways normally followed for immune complex clearance, which include phagocytosis and trogocytosis. We review the key published basic science experiments and clinical observations that support this idea. We propose that it is the interaction of the ADC with Fcγ receptors expressed on off-target cells and tissues that can most readily explain ADC-mediated pathologies, which therefore provides a rationale for the design of protocols to minimize ASEs. We describe measurements that should help identify those patients most likely to experience ASE due to ADC, and we propose readily available treatments as well as therapies under development for other indications that should substantially reduce ASE associated with ADC. Our focus will be on the following FDA-approved ADC for which there are substantial literatures: gemtuzumab ozogamicin and inotuzumab ozogamicin; and trastuzumab emtansine and trastuzumab deruxtecan.
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Imunoconjugados , Humanos , Imunoconjugados/uso terapêutico , Imunoconjugados/efeitos adversos , Complexo Antígeno-Anticorpo/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Animais , Receptores de IgG/metabolismo , Fagocitose/efeitos dos fármacos , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/uso terapêuticoRESUMO
Trogocytosis is a process in which receptors on acceptor cells remove and internalize cognate ligands from donor cells. Trogocytosis has a profound and negative impact on mAb-based cancer immunotherapy, as seen in the treatment of chronic lymphocytic leukemia (CLL) with CD20 mAbs, such as rituximab (RTX) and ofatumumab (OFA). Our clinical observations of RTX/OFA-mediated loss of the CD20 target from circulating CLL cells have been replicated in our in vitro studies. Here we describe flow cytometry and fluorescence microscopy experiments, which demonstrate that acceptor cells, such as monocytes/macrophages that express FcγR, remove and internalize both antigen and donor cell-bound cognate IgG mAbs for several different mAb-donor cell pairs. Fluorescent mAbs and portions of the plasma cell membrane are transferred from donor cells to acceptor cells, which include the THP-1 monocytic cell line as well as freshly isolated monocytes. We describe rigorous controls to validate the reactions and eliminate dissociation or internalization as alternative mechanisms. Trogocytosis is likely to contribute to neutropenia, thrombocytopenia, and liver damage associated with use of antibody-drug conjugates. The methods we have described should allow for examination of strategies focused on blocking trogocytosis and its adverse effects. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trogocytosis of mAb-opsonized donor cells mediated by adherent THP-1 cells Alternate Protocol: Application of fluorescence microscopy to examine THP-1 cell-mediated trogocytosis Support Protocol 1: Alexa labeling of mAbs and determination of F/P ratios Support Protocol 2: Standard washing procedure Support Protocol 3: Labeling and opsonization of cells Basic Protocol 2: Trogocytosis mediated by human monocytes as acceptor cells Support Protocol 4: Isolation of human monocytes Basic Protocol 3: Trogocytosis mediated by THP-1 cells in solution Support Protocol 5: Retinoic acid treatment of THP-1 cells Support Protocol 6: Culturing of SCC-25, BT-474, MOLT-4 and THP-1 cell lines.
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Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Anticorpos Monoclonais Murinos/uso terapêutico , Trogocitose , Antígenos CD20/uso terapêutico , Rituximab/farmacologia , Rituximab/uso terapêuticoRESUMO
Vaso-occlusive pain episodes (VOE) cause severe pain in patients with sickle cell disease (SCD). Vaso-occlusive events promote ischemia/reperfusion pathobiology that activates complement. We hypothesized that complement activation is linked to VOE. We used cold to induce VOE in the Townes sickle homozygous for hemoglobin S (HbSS) mouse model and complement inhibitors to determine whether anaphylatoxin C5a mediates VOE. We used a dorsal skinfold chamber to measure microvascular stasis (vaso-occlusion) and von Frey filaments applied to the plantar surface of the hind paw to assess mechanical hyperalgesia in HbSS and control Townes mice homozygous for hemoglobin A (HbAA) mice after cold exposure at 10°C/50°F for 1 hour. Cold exposure induced more vaso-occlusion in nonhyperalgesic HbSS mice (33%) than in HbAA mice (11%) or HbSS mice left at room temperature (1%). Cold exposure also produced mechanical hyperalgesia as measured by paw withdrawal threshold in HbSS mice compared with that in HbAA mice or HbSS mice left at room temperature. Vaso-occlusion and hyperalgesia were associated with an increase in complement activation fragments Bb and C5a in plasma of HbSS mice after cold exposure. This was accompanied by an increase in proinflammatory NF-κB activation and VCAM-1 and ICAM-1 expression in the liver. Pretreatment of nonhyperalgesic HbSS mice before cold exposure with anti-C5 or anti-C5aR monoclonal antibodies (mAbs) decreased vaso-occlusion, mechanical hyperalgesia, complement activation, and liver inflammatory markers compared with pretreatment with control mAb. Anti-C5 or -C5aR mAb infusion also abrogated mechanical hyperalgesia in HbSS mice with ongoing hyperalgesia at baseline. These findings suggest that C5a promotes vaso-occlusion, pain, and inflammation during VOE and may play a role in chronic pain.
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Anemia Falciforme , Traço Falciforme , Camundongos , Humanos , Animais , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Camundongos Transgênicos , Dor , Anemia Falciforme/complicações , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Traço Falciforme/complicações , Ativação do ComplementoRESUMO
Monoclonal antibody induced infusion reactions (IRs) can be serious and even fatal. We used clinical data and blood samples from 37 treatment naïve patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) initiating therapy for progressive disease with a single 50 mg dose of intravenous (IV) rituximab at 25 mg/h. Twenty-four (65 %) patients had IRs at a median of 78 min (range 35-128) and rituximab dose of 32 mg (range 15-50). IR risk did not correlate with patient or CLL characteristics, CLL counts or CD20 levels, or serum rituximab or complement concentrations. Thirty-five (95 %) patients had cytokine release response with a ≥ 4-fold increase in serum concentration of ≥ 1 inflammatory cytokine. IRs were associated with significantly higher post-infusion serum concentrations of gamma interferon induced cytokines IP-10, IL-6 and IL-8. IP-10 concentrations increased ≥ 4-fold in all patients with an IR and were above the upper limit of detection (40,000 pg/ml) in 17 (71 %). In contrast, to only three (23 %) patients without an IR had an ≥ 4-fold increase in serum concentrations of IP-10 (highest 22,013 pg/ml). Our data suggest that cytokine release could be initiated by activation of effector cells responsible for clearance of circulating CLL cells with IRs occurring in those with higher levels of gamma interferon induced cytokines. These novel insights could inform future research to better understand and manage IRs and understand the role of cytokines in the control of cytotoxic immune responses to mAb.
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Antineoplásicos , Leucemia Linfocítica Crônica de Células B , Humanos , Rituximab , Citocinas , Quimiocina CXCL10/uso terapêutico , Leucemia Linfocítica Crônica de Células B/patologia , Interferon gama/uso terapêutico , Anticorpos Monoclonais Murinos , Antineoplásicos/uso terapêuticoRESUMO
CR3 (CD11b/CD18; αmß2 integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and microbial ligands, leading to actin-dependent phagocytosis. There are conflicting reports about how CR3 engagement affects the fate of phagocytosed substrates. Using imaging flow cytometry, we confirmed that binding and internalization of iC3b-opsonized polystyrene beads by primary human neutrophils was CR3-dependent. iC3b-opsonized beads did not stimulate neutrophil reactive oxygen species, and most beads were found in primary granule-negative phagosomes. Similarly, Neisseria gonorrhoeae that does not express phase-variable Opa proteins suppresses neutrophil reactive oxygen species and delays phagolysosome formation. Here, binding and internalization of Opa-deleted (Δopa) N. gonorrhoeae by adherent human neutrophils was inhibited using blocking antibodies against CR3 and by adding neutrophil inhibitory factor, which targets the CD11b I-domain. No detectable C3 was deposited on N. gonorrhoeae in the presence of neutrophils alone. Conversely, overexpressing CD11b in HL-60 promyelocytes enhanced Δopa N. gonorrhoeae phagocytosis, which required the CD11b I-domain. Phagocytosis of N. gonorrhoeae was also inhibited in mouse neutrophils that were CD11b-deficient or treated with anti-CD11b. Phorbol ester treatment upregulated surface CR3 on neutrophils in suspension, enabling CR3-dependent phagocytosis of Δopa N. gonorrhoeae. Neutrophils exposed to Δopa N. gonorrhoeae had limited phosphorylation of Erk1/2, p38, and JNK. Neutrophil phagocytosis of unopsonized Mycobacterium smegmatis, which also resides in immature phagosomes, was CR3-dependent and did not elicit reactive oxygen species. We suggest that CR3-mediated phagocytosis is a silent mode of entry into neutrophils, which is appropriated by diverse pathogens to subvert phagocytic killing.
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Neutrófilos , Fagocitose , Camundongos , Animais , Humanos , Espécies Reativas de Oxigênio/metabolismo , Antígeno de Macrófago 1/metabolismo , Complemento C3b/metabolismo , Receptores de Complemento/metabolismoRESUMO
There is increasing interest in the clinical implications and immunology of trogocytosis, a process in which the receptors on acceptor cells remove and internalize cognate ligands from donor cells. We have reported that this phenomenon occurs in cancer immunotherapy, in which cells that express FcγR remove and internalize CD20 and bound mAbs from malignant B cells. This process can be generalized to include other reactions including the immune adherence phenomenon and antibody-induced immunosuppression. We discuss in detail FcγR-mediated trogocytosis and the evidence supporting a proposed predominant role for liver sinusoidal endothelial cells via the action of the inhibitory receptor FcγRIIb2. We describe experiments to test the validity of this hypothesis. The elucidation of the details of FcγR-mediated trogocytosis has the potential to allow for the development of novel therapies that can potentially block or enhance this reaction, depending upon whether the process leads to unfavorable or positive biological effects.
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The complement system plays an important role in critical aspects of immune defense and in the maintenance of homeostasis in the bloodstream, as well as in essentially all tissues and organs [...].
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High-risk cytogenetics and minimal residual disease (MRD) after chemoimmunotherapy (CIT) predict unfavorable outcome in chronic lymphocytic leukemia (CLL). This phase 2 study investigated risk-adapted CIT in treatment-naïve CLL (NCT01145209). Patients with high-risk cytogenetics received induction with fludarabine, cyclophosphamide, and ofatumumab. Those without high-risk cytogenetics received fludarabine and ofatumumab. After induction, MRD positive (MRD+) patients received 4 doses of ofatumumab consolidation. MRD negative (MRD-) patients had no intervention. Of 28 evaluable for response, all responded to induction and 10 (36%) achieved MRD-. Two-year progression-free survival (PFS) was 71.4% (CI95, 56.5-90.3%). There was no significant difference in median PFS between the high-risk and the standard-risk groups. Ofatumumab consolidation didn't convert MRD + to MRD-. In the MRD + group, we saw selective loss of CD20 antigens during therapy. In conclusion, risk-adapted CIT is feasible in treatment-naïve CLL. Ofatumumab consolidation didn't improve depth of response in MRD + patients. Loss of targetable CD20 likely reduces efficacy of consolidation therapy.
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Leucemia Linfocítica Crônica de Células B , Anticorpos Monoclonais Humanizados/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Humanos , Imunoterapia , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Resultado do TratamentoRESUMO
Unconjugated anti-cancer IgG1 monoclonal antibodies (mAbs) activate antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells and antibody-dependent cellular phagocytosis (ADCP) by macrophages, and these activities are thought to be important mechanisms of action for many of these mAbs in vivo. Several mAbs also activate the classical complement pathway and promote complement-dependent cytotoxicity (CDC), although with very different levels of efficacy, depending on the mAb, the target antigen, and the tumor type. Recent studies have unraveled the various structural factors that define why some IgG1 mAbs are strong mediators of CDC, whereas others are not. The role of complement activation and membrane inhibitors expressed by tumor cells, most notably CD55 and CD59, has also been quite extensively studied, but how much these affect the resistance of tumors in vivo to IgG1 therapeutic mAbs still remains incompletely understood. Recent studies have demonstrated that complement activation has multiple effects beyond target cell lysis, affecting both innate and adaptive immunity mediated by soluble complement fragments, such as C3a and C5a, and by stimulating complement receptors expressed by immune cells, including NK cells, neutrophils, macrophages, T cells, and dendritic cells. Complement activation can enhance ADCC and ADCP and may contribute to the vaccine effect of mAbs. These different aspects of complement are also briefly reviewed in the specific context of FDA-approved therapeutic anti-cancer IgG1 mAbs.
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We propose use of bispecific monoclonal antibody (mAb) complexes bound to erythrocytes to redress the lack of efficacy of anti-amyloid beta mAbs in Alzheimer's disease treatment. Our paradigm leverages erythrocyte complement receptor 1 to promote rapid and quantitative removal of amyloid beta from the circulation, and its subsequent removal from the brain as well.
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We examined the kinetics and mechanisms by which monoclonal antibodies (mAbs) utilize complement to rapidly kill targeted cancer cells. Based on results from flow cytometry, confocal microscopy and high-resolution digital imaging experiments, the general patterns which have emerged reveal cytotoxic activities mediated by substantial and lethal Ca2+ fluxes. The Ca2+ fluxes are common to the reported pathways that have been utilized by other toxins in killing nucleated cells. These reactions terminate in very high levels of cell killing, and based on these considerations, we suggest additional strategies to further enhance mAb-based targeting of cancer with complement.
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Atypical hemolytic uremic syndrome is an ultra-rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute kidney injury. Its pathogenesis is driven most frequently by dysregulated cell-surface control of the alternative pathway of complement secondary to inherited and/or acquired factors. Here we evaluated two unrelated patients with atypical hemolytic uremic syndrome. The first, a five-year-old Caucasian female, presented at 10 months with schistocytes, thrombocytopenia and kidney injury. The second, a 55-year-old Caucasian female, presented at age 31 following caesarean section for preeclampsia. Complement biomarker testing was remarkable for undetectable levels of C3 in both. Circulating levels of C5 and properdin were also low consistent with over-activity of the alternative and terminal pathways of complement. Genetic testing identified a heterozygous novel variant in CFB (c.1101 C>A, p.Ser367Arg) in both patients. Functional studies found strong fluid-phase C3 cleavage when normal and proband sera were mixed. Cell-surface C3b deposition was strongly positive when patient serum was supplemented with C3. In vitro control of C3 convertase activity could be restored with increased concentrations of factor H. Thus, CFB p.Ser367Arg is a gain-of-function pathogenic variant that leads to dysregulation of the alternative pathway in the fluid-phase and increased C3b deposition on cell surfaces. Our study highlights the complexities of complement-mediated diseases like atypical hemolytic uremic syndrome and illustrates the importance of functional studies at the variant level to gain insight into the disease phenotype.
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Síndrome Hemolítico-Urêmica Atípica , Adulto , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/genética , Cesárea , Pré-Escolar , Fator B do Complemento/genética , Fator H do Complemento/genética , Via Alternativa do Complemento/genética , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , GravidezRESUMO
Therapeutic complement inhibition is a major focus for novel drug development. Of upstream targets, factor D (FD) is appealing because it circulates in plasma at low concentrations and has a single function: to cleave factor B to generate C3 convertase of the alternative pathway (AP). Mice with a targeted deletion of factor H (FH; Cfh-/- mice) develop C3 glomerulopathy (C3G) due to uncontrolled AP activity. To assess the impact of FD inhibition, we studied Cfh-/- Cfd-/- mice. We show that C3G in Cfh-/- mice is not rescued by removing FD. We used serum from Cfh-/- Cfd-/- mice to demonstrate that residual AP function occurs even when both FD and FH are missing and that hemolytic activity is present due to the action of C3(H2O). We propose that uncontrolled tick-over leads to slow activation of the AP in Cfh-/- Cfd-/- mice and that a minimal threshold of FH is necessary if tissue deposition of C3 is to be prevented. The FD/FH ratio dictates serum C3 level and renal C3b deposition. In C3G patients with chronic renal disease, the FD/FH ratio correlates inversely with C3 and C5 serum levels, suggesting that continuous AP control may be difficult to achieve by targeting FD.
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Complemento C3/imunologia , Fator H do Complemento/imunologia , Via Alternativa do Complemento , Doenças da Deficiência Hereditária de Complemento/imunologia , Nefropatias/imunologia , Animais , Complemento C5/imunologia , Fator D do Complemento/imunologia , Humanos , Rim/imunologia , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Tetraspanin CD37 has recently received renewed interest as a therapeutic target for B-cell malignancies. Although complement-dependent cytotoxicity (CDC) is a powerful Fc-mediated effector function for killing hematological cancer cells, CD37-specific antibodies are generally poor inducers of CDC. To enhance CDC, the E430G mutation was introduced into humanized CD37 monoclonal IgG1 antibodies to drive more efficient IgG hexamer formation through intermolecular Fc-Fc interactions after cell surface antigen binding. DuoHexaBody-CD37, a bispecific CD37 antibody with the E430G hexamerization-enhancing mutation targeting two non-overlapping epitopes on CD37 (biparatopic), demonstrated potent and superior CDC activity compared to other CD37 antibody variants evaluated, in particular ex vivo in patient-derived chronic lymphocytic leukemia cells. The superior CDC potency was attributed to enhanced IgG hexamerization mediated by the E430G mutation in combination with dual epitope targeting. The mechanism of action of DuoHexaBody-CD37 was shown to be multifaceted, as it was additionally capable of inducing efficient antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vitro. Finally, potent anti-tumor activity in vivo was observed in cell line- and patient-derived xenograft models from different B-cell malignancy subtypes. These encouraging preclinical results suggest that DuoHexaBody-CD37 (GEN3009) may serve as a potential therapeutic antibody for the treatment of human B-cell malignancies.
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Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Linfoma de Células B/terapia , Receptores Fc/imunologia , Tetraspaninas/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Linhagem Celular Tumoral , Desenvolvimento de Medicamentos , Células HEK293 , Xenoenxertos , Humanos , Imunoglobulina G/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfoma de Células B/imunologia , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Receptores Fc/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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We have previously reported that complement activation precedes the development of kidney fibrosis; however, little is known about the cellular mechanisms involved in this transition. We hypothesized that increased expression of C1 complex protease C1r, the initiator of complement activation, contributes to tubulointerstitial fibrosis and tested this idea in mice with global deletion of C1r. Although expression of C1r in untreated wild-type (WT) mice was higher in the liver compared with kidney tissue, administration of folic acid (FA) led to upregulation of C1r mRNA and protein levels only in kidney tissue. Immunohistochemistry and in situ hybridization experiments localized increased expression of C1r and C1s proteases to renal tubular epithelial cells. C1r-null mice had reduced acute tubular injury and inflammation measured 2 days after FA administration compared with WT mice. C1r deletion reduced expression of C1s, C3 fragment formation, and organ fibrosis measured 14 days after FA administration. Differential gene expression performed in kidney tissue demonstrated that C1r-null mice had reduced expression of genes associated with the acute phase response, complement, proliferation of connective tissue cells (e.g., platelet-derived growth factor receptor-ß), and reduced expression of genes associated with inflammation compared with FA-treated WT mice. In vitro experiments in renal epithelial cells demonstrated that C1s expression is dependent on increased C1r expression and that interferon-γ induces the expression of these two proteases. We conclude that increased expression of C1 complex proteases is associated with increased tissue inflammation and complement C3 formation and represents an important pathogenic mechanism leading to FA-mediated tubulointerstitial fibrosis.
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Complemento C1r/metabolismo , Nefropatias/enzimologia , Animais , Linhagem Celular , Complemento C1r/genética , Complemento C1s/genética , Complemento C1s/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Ácido Fólico/farmacologia , Regulação Enzimológica da Expressão Gênica , Humanos , Inflamação , Rim/citologia , Nefropatias/genética , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Multidrug-resistant Neisseria gonorrhoeae is a global health problem. Monoclonal antibody (mAb) 2C7 recognizes a gonococcal lipooligosaccharide epitope that is expressed by >95% of clinical isolates and hastens gonococcal vaginal clearance in mice. Chimeric mAb 2C7 (human immunoglobulin G1 [IgG1]) with an E430G Fc modification that enhances Fc:Fc interactions and hexamerization following surface-target binding and increases complement activation (HexaBody technology) showed significantly greater C1q engagement and C4 and C3 deposition compared to mAb 2C7 with wild-type Fc. Greater complement activation by 2C7-E430G Fc translated to increased bactericidal activity in vitro and, consequently, enhanced efficacy in mice, compared with "Fc-unmodified" chimeric 2C7. Gonococci bind the complement inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human-specific manner, which dampens antibody (Ab)-mediated complement-dependent killing. The variant 2C7-E430G Fc overcame the barrier posed by these inhibitors in human FH/C4BP transgenic mice, for which a single 1 µg intravenous dose cleared established infection. Chlamydia frequently coexists with and exacerbates gonorrhea; 2C7-E430G Fc also proved effective against gonorrhea in gonorrhea/chlamydia-coinfected mice. Complement activation alone was necessary and sufficient for 2C7 function, evidenced by the fact that (1) "complement-inactive" Fc modifications that engaged Fc gamma receptor (FcγR) rendered 2C7 ineffective, nonetheless; (2) 2C7 was nonfunctional in C1q-/- mice, when C5 function was blocked, or in C9-/- mice; and (3) 2C7 remained effective in neutrophil-depleted mice and in mice treated with PMX205, a C5a receptor (C5aR1) inhibitor. We highlight the importance of complement activation for antigonococcal Ab function in the genital tract. Elucidating the correlates of protection against gonorrhea will inform the development of Ab-based gonococcal vaccines and immunotherapeutics.