RESUMEN
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.
Asunto(s)
Inmunoconjugados , Humanos , Inmunoconjugados/uso terapéutico , Inmunoconjugados/efectos adversos , Complejo Antígeno-Anticuerpo/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Animales , Receptores de IgG/metabolismo , Fagocitosis/efectos de los fármacos , Antineoplásicos Inmunológicos/efectos adversos , Antineoplásicos Inmunológicos/uso terapéuticoRESUMEN
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.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/metabolismo , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Trogocitosis , Antígenos CD20/uso terapéutico , Rituximab/farmacología , Rituximab/uso terapéuticoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Anticuerpos Monoclonales Humanizados/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Humanos , Inmunoterapia , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Resultado del TratamientoRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Complemento C3/inmunología , Factor H de Complemento/inmunología , Vía Alternativa del Complemento , Enfermedades por Deficiencia de Complemento Hereditario/inmunología , Enfermedades Renales/inmunología , Animales , Complemento C5/inmunología , Factor D del Complemento/inmunología , Humanos , Riñón/inmunología , Riñón/patología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
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.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antígenos de Neoplasias/inmunología , Leucemia Linfocítica Crónica de Células B/terapia , Linfoma de Células B/terapia , Receptores Fc/inmunología , Tetraspaninas/inmunología , Animales , Anticuerpos Biespecíficos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/patología , Línea Celular Tumoral , Desarrollo de Medicamentos , Células HEK293 , Xenoinjertos , Humanos , Inmunoglobulina G/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Linfoma de Células B/inmunología , Ratones , Ratones SCID , Terapia Molecular Dirigida , Receptores Fc/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antígenos CD20/inmunología , Antígenos de Neoplasias/inmunología , Proteínas del Sistema Complemento/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Leucemia Linfocítica Crónica de Células B/genética , Tetraspaninas/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Línea Celular Tumoral , Complemento C1q/inmunología , Transferencia Resonante de Energía de Fluorescencia , Humanos , Inmunoglobulina G/inmunología , Leucemia Linfocítica Crónica de Células B/sangre , Mutación , Unión Proteica , Rituximab/farmacologíaRESUMEN
Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD.
Asunto(s)
Anemia de Células Falciformes/inmunología , Anticuerpos Neutralizantes/farmacología , Trastornos Cerebrovasculares/inmunología , Complemento C3/inmunología , Complemento C5a/inmunología , Receptor de Anafilatoxina C5a/inmunología , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/patología , Animales , Trastornos Cerebrovasculares/tratamiento farmacológico , Trastornos Cerebrovasculares/genética , Trastornos Cerebrovasculares/patología , Complemento C3/genética , Complemento C5a/antagonistas & inhibidores , Complemento C5a/genética , Complejo de Ataque a Membrana del Sistema Complemento/genética , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Modelos Animales de Enfermedad , Selectina E/genética , Selectina E/inmunología , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Riñón/inmunología , Riñón/patología , Hígado/irrigación sanguínea , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/patología , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/inmunología , Selectina-P/antagonistas & inhibidores , Selectina-P/genética , Selectina-P/inmunología , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/genética , Transducción de Señal , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
The complement cascade is an ancient defense system that destroys and eliminates threats to normal homeostasis in the bloodstream and tissues. Although multiple controls keep complement in check to minimize innocent bystander injury to normal cells and tissues, defects in complement regulation due to mutations in, or autoantibodies to, complement control proteins underlie the pathogenesis of several hemolytic diseases including paroxysmal nocturnal hemoglobinuria, and atypical hemolytic uremic syndrome. In autoimmune hemolytic anemias complement plays an important role in erythrocyte destruction mediated by antierythrocyte antibodies. The pathogenic mechanisms of these hemolytic diseases are discussed, with an emphasis on pivotal steps in complement activation.
Asunto(s)
Proteínas del Sistema Complemento/fisiología , Enfermedades Hematológicas/fisiopatología , HumanosRESUMEN
BACKGROUND: Endothelial microparticles are associated with chronic kidney disease (CKD) and complement activation. We hypothesized that the complement pathway is activated in patients with CKD via endothelial microparticles and that complement activation correlates with endothelial dysfunction in CKD. METHODS AND RESULTS: We analyzed complement data of 30 healthy subjects, 30 patients with stage III/IV CKD, and 30 renal transplant recipients with stage III/IV CKD, evaluating the potential correlation of complement fragments with brachial artery flow-mediated dilation, Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and urinary albumin/creatinine ratio. Endothelial microparticles were characterized via proteomic analysis and compared between study groups. Complement fragment Ba was significantly increased in CKD and post-kidney transplant CKD. Plasma Ba levels correlated significantly with lower brachial artery flow-mediated dilation, lower Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and higher urinary albumin/creatinine ratio. Factor D levels were significantly higher in the plasma microparticles of patients with CKD versus healthy controls. Plasma microparticles isolated from patients with CKD and containing factor D activated the alternative pathway in vitro. CONCLUSION: The alternative complement pathway is activated in CKD and correlates with endothelial dysfunction and markers of CKD. Future studies are needed to evaluate whether endothelial microparticles with increased factor D play a pathologic role in CKD-associated vascular disease. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02230202.
Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Factor B del Complemento/metabolismo , Factor D del Complemento/metabolismo , Vía Alternativa del Complemento , Endotelio Vascular/fisiopatología , Insuficiencia Renal Crónica/metabolismo , Adulto , Anciano , Arteria Braquial/diagnóstico por imagen , Arteria Braquial/fisiopatología , Estudios de Casos y Controles , Activación de Complemento , Complemento C4a/metabolismo , Complemento C5a/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/metabolismo , Células Endoteliales , Femenino , Tasa de Filtración Glomerular , Humanos , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Proyectos Piloto , Proteómica , Insuficiencia Renal Crónica/fisiopatología , Insuficiencia Renal Crónica/cirugía , Índice de Severidad de la Enfermedad , VasodilataciónRESUMEN
We examined complement-dependent cytotoxicity (CDC) by hexamer formation-enhanced CD20 mAb Hx-7D8 of patient-derived chronic lymphocytic leukemia (CLL) cells that are relatively resistant to CDC. CDC was analyzed in normal human serum (NHS) and serum from an individual genetically deficient for C9. Hx-7D8 was able to kill up to 80% of CLL cells in complete absence of C9. We conclude that the narrow C5b-8 pores formed without C9 are sufficient for CDC due to efficient antibody-mediated hexamer formation. In the absence of C9, we observed transient intracellular increases of Ca2+ during CDC (as assessed with FLUO-4) that were extended in time. This suggests that small C5b-8 pores allow Ca2+ to enter the cell, while dissipation of the fluorescent signal accompanying cell disintegration is delayed. The Ca2+ signal is retained concomitantly with TOPRO-3 (viability dye) staining, thereby confirming that Ca2+ influx represents the most proximate mediator of cell death by CDC.
Asunto(s)
Complemento C9/deficiencia , Proteínas del Sistema Complemento/inmunología , Síndromes de Inmunodeficiencia/inmunología , Leucemia Linfocítica Crónica de Células B , Rituximab/farmacología , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Complemento C9/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/metabolismo , Enfermedades por Deficiencia de Complemento Hereditario , Humanos , Inmunoterapia , PolimerizacionRESUMEN
Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.
Asunto(s)
Complemento C1q/inmunología , Citotoxicidad Inmunológica/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Inmunoterapia , Neoplasias/tratamiento farmacológico , Fagocitosis/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/inmunología , Línea Celular Tumoral , Cromatografía en Gel , Cromatografía Liquida , Complemento C1q/metabolismo , Cristalización , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Humanos , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Técnicas In Vitro , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/inmunología , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/inmunología , Espectrometría de Masas , Ratones , Neoplasias/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Receptores de IgG/metabolismo , Resonancia por Plasmón de Superficie , Espectrometría de Masas en TándemRESUMEN
During malarial anemia, 20 uninfected red blood cells (RBCs) are destroyed for every RBC infected by Plasmodium falciparum (Pf). Increasing evidence indicates an important role for complement in destruction of uninfected RBCs. Products of RBC lysis induced by Pf, including the digestive vacuole and hematin, activate complement and promote C3 fragment deposition on uninfected RBCs. C3-opsonized cells are then subject to extravascular destruction mediated by fixed tissue macrophages which express receptors for C3 fragments. The Compstatin family of cyclic peptides blocks complement activation at the C3 cleavage step, and is under investigation for treatment of complement-mediated diseases. We demonstrate, that under a variety of stringent conditions, second-generation Compstatin analogue Cp40 completely blocks hematin-mediated deposition of C3 fragments on naïve RBCs. Our findings indicate that prophylactic provision of Compstatin for malaria-infected individuals at increased risk for anemia may provide a safe and inexpensive treatment to prevent or substantially reduce malarial anemia.
Asunto(s)
Complemento C3b/metabolismo , Eritrocitos/efectos de los fármacos , Hemina/metabolismo , Péptidos Cíclicos/farmacología , Anemia/tratamiento farmacológico , Eritrocitos/metabolismo , Humanos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparumRESUMEN
Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation-enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than their wild-type counterparts. To further investigate the CDC potential of these mAbs, we used flow cytometry, high-resolution digital imaging, and four-color confocal microscopy to examine their activity against B cell lines and primary chronic lymphocytic leukemia cells in sera depleted of single complement components. We also examined the CDC activity of alemtuzumab (anti-CD52) and mAb W6/32 (anti-HLA), which bind at high density to cells and promote substantial complement activation. Although we observed little CDC for mAb-opsonized cells reacted with sera depleted of early complement components, we were surprised to discover that the Hexabody mAbs, as well as ALM and W6/32, were all quite effective at promoting CDC in sera depleted of individual complement components C6 to C9. However, neutralization studies conducted with an anti-C9 mAb verified that C9 is required for CDC activity against cell lines. These highly effective complement-activating mAbs efficiently focus activated complement components on the cell, including C3b and C9, and promote CDC with a very low threshold of MAC binding, thus providing additional insight into their enhanced efficacy in promoting CDC.
Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD20/metabolismo , Antígenos/inmunología , Sitios de Unión de Anticuerpos , Complemento C9/metabolismo , Glicoproteínas de Membrana/metabolismo , ADP-Ribosil Ciclasa 1/inmunología , Alemtuzumab , Anticuerpos Monoclonales Humanizados/inmunología , Antígenos CD20/inmunología , Linfocitos B/inmunología , Línea Celular Tumoral , Activación de Complemento , Complemento C3b/metabolismo , Complemento C9/inmunología , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Humanos , Glicoproteínas de Membrana/inmunologíaRESUMEN
Several mAbs that have been approved for the treatment of cancer make use of complement-dependent cytotoxicity (CDC) to eliminate tumor cells. Comprehensive investigations, based on in vitro studies, mouse models and analyses of patient blood samples after mAb treatment have provided key insights into the details of individual steps in the CDC reaction. Based on the lessons learned from these studies, new and innovative approaches are now being developed to increase the clinical efficacy of next generation mAbs with respect to CDC. These improvements include engineering changes in the mAbs to enhance their ability to activate complement. In addition, mAb dosing paradigms are being developed that take into account the capacity as well as the limitations of the complement system to eliminate a substantial burden of mAb-opsonized cells. Over the next few years it is likely these approaches will lead to mAbs that are far more effective in the treatment of cancer.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Proteínas del Sistema Complemento/metabolismo , Citotoxicidad Inmunológica , Inmunoterapia/métodos , Neoplasias/terapia , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos de Neoplasias/inmunología , Activación de Complemento , Ingeniería Genética , Humanos , Neoplasias/inmunologíaRESUMEN
Complement-dependent cytotoxicity is an important mechanism of action of certain mAbs used in cancer immunotherapy, including ofatumumab and rituximab. However, the detailed sequence of cellular changes that occur in nucleated cells attacked by mAb and complement has not been delineated. Recently developed CD20 mAbs, engineered to form hexamers on binding to cells, react with B-cells in serum, chelate C1q, and then activate complement and promote cell killing considerably more effectively than their wild-type precursors. We used these engineered mAbs as a model to investigate the sequence of events that occur when mAbs bind to B-cell lines and to primary cells from patients with chronic lymphocytic leukemia and then activate complement. Based on four-color confocal microscopy real-time movies and high resolution digital imaging, we find that after CD20 mAb binding and C1q uptake, C3b deposits on cells, followed by Ca(2+) influx, revealed by bright green signals generated on cells labeled with FLUO-4, a Ca(2+) indicator. The bright FLUO-4/Ca(2+) signal fades, replaced by punctate green signals in mitochondria, indicating Ca(2+) localization. This step leads to mitochondrial poisoning followed by cell death. The entire sequence is completed in <2 min for hexamerization-enhanced CD20 mAb-mediated killing. To our knowledge this is the first time the entire process has been characterized in detail in real time. By identifying multiple discrete steps in the cytotoxic pathway for nucleated cells our findings may inform future development and more effective application of complement-fixing mAbs to cancer treatment.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/inmunología , Linfocitos B/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos/farmacología , Línea Celular , Línea Celular Tumoral , Activación de Complemento/inmunología , Humanos , Microscopía Confocal , Rituximab/inmunología , Rituximab/farmacologíaRESUMEN
Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.