RESUMO
The classical complement pathway contributes to the natural immune defense against pathogens and tumors. IgG antibodies can assemble at the cell surface into hexamers via Fc:Fc interactions, which recruit complement component C1q and induce complement activation. Biophysical characterization of the C1:IgG complex has remained elusive primarily due to the low affinity of IgG-C1q binding. Using IgG variants that dynamically form hexamers efficient in C1q binding and complement activation, we could assess C1q binding in solution by native mass spectrometry and size-exclusion chromatography. Fc-domain deglycosylation, described to abrogate complement activation, affected IgG hexamerization and C1q binding. Strikingly, antigen binding by IgG hexamers or deletion of the Fab arms substantially potentiated complement initiation, suggesting that Fab-mediated effects impact downstream Fc-mediated events. Finally, we characterized a reconstituted 2,045.3 ± 0.4-kDa complex of intact C1 bound to antigen-saturated IgG hexamer by native mass spectrometry, providing a clear visualization of a complete complement initiation complex.
Assuntos
Antígenos/metabolismo , Ativação do Complemento , Complemento C1q/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Reações Antígeno-Anticorpo , Antígenos/química , Antígenos/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular Tumoral , Cromatografia em Gel , Complemento C1q/química , Complemento C1q/imunologia , Glicosilação , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Mutação , Ligação Proteica , Estabilidade Proteica , Espectrometria de Massas em TandemRESUMO
Immunoglobulin (Ig) G molecules are essential players in the human immune response against bacterial infections. An important effector of IgG-dependent immunity is the induction of complement activation, a reaction that triggers a variety of responses that help kill bacteria. Antibody-dependent complement activation is promoted by the organization of target-bound IgGs into hexamers that are held together via noncovalent Fc-Fc interactions. Here we show that staphylococcal protein A (SpA), an important virulence factor and vaccine candidate of Staphylococcus aureus, effectively blocks IgG hexamerization and subsequent complement activation. Using native mass spectrometry and high-speed atomic force microscopy, we demonstrate that SpA blocks IgG hexamerization through competitive binding to the Fc-Fc interaction interface on IgG monomers. In concordance, we show that SpA interferes with the formation of (IgG)6:C1q complexes and prevents downstream complement activation on the surface of S. aureus. Finally, we demonstrate that IgG3 antibodies against S. aureus can potently induce complement activation and opsonophagocytic killing even in the presence of SpA. Together, our findings identify SpA as an immune evasion protein that specifically blocks IgG hexamerization.
Assuntos
Ativação do Complemento , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Multimerização Proteica , Proteína Estafilocócica A/metabolismo , Sítios de Ligação , Células Cultivadas , Humanos , Fagócitos/imunologia , Fagocitose , Ligação Proteica , Staphylococcus aureus/imunologiaRESUMO
Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody's constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes to C1q-IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q-IgG stability, both in the absence and presence of C1r2s2 In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.
Assuntos
Membrana Celular/metabolismo , Complemento C1q/metabolismo , Complemento C1r/metabolismo , Complemento C1s/metabolismo , Imunoglobulina G/metabolismo , Ativação do Complemento , Humanos , Microscopia de Força Atômica , Mutação/genética , Fagocitose , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Staphylococcus aureus/imunologiaRESUMO
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.
Assuntos
Ativação do Complemento/imunologia , Gonorreia/imunologia , Neisseria gonorrhoeae/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos de Bactérias , Proteína de Ligação ao Complemento C4b/imunologia , Fator H do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Epitopos/imunologia , Feminino , Voluntários Saudáveis , Humanos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neisseria gonorrhoeae/patogenicidadeRESUMO
The sialylatable lacto-N-neotetraose (LNnT; Gal-GlcNAc-Gal-Glc) moiety from heptose I (HepI) of the lipooligosaccharide (LOS) of Neisseria gonorrhoeae undergoes positive selection during human infection. Lactose (Gal-Glc) from HepII, although phase variable, is commonly expressed in humans; loss of HepII lactose compromises gonococcal fitness in mice. Anti-LOS monoclonal antibody (MAb) 2C7, a promising antigonococcal immunotherapeutic that elicits complement-dependent bactericidal activity and attenuates gonococcal colonization in mice, recognizes an epitope comprised of lactoses expressed simultaneously from HepI and HepII. Glycan extensions beyond lactose on HepI modulate binding and function of MAb 2C7 in vitro Here, four gonococcal LOS mutants, each with lactose from HepII but fixed (unable to phase-vary) LOS HepI glycans extended beyond the lactose substitution of HepI (lactose alone, Gal-lactose, LNnT, or GalNAc-LNnT), were used to define how HepI glycan extensions affect (i) mouse vaginal colonization and (ii) efficacy in vitro and in vivo of a human IgG1 chimeric derivative of MAb 2C7 (2C7-Ximab) with a complement-enhancing E-to-G Fc mutation at position 430 (2C7-Ximab-E430G). About 10-fold lower 2C7-Ximab-E430G concentrations achieved similar complement-dependent killing of three gonococcal mutants with glycan extensions beyond lactose-substituted HepI (lactose alone, LNnT, or GalNAc-LNnT) as 2C7-Ximab (unmodified Fc). The fourth mutant (Gal-lactose) resisted direct complement-dependent killing but was killed approximately 70% by 2C7-Ximab-E430G in the presence of polymorphonuclear leukocytes and complement. Only mutants with (sialylatable) LNnT from HepI colonized mice for >3 days, reiterating the importance of LNnT sialylation for infection. 2C7-Ximab-E430G significantly attenuated colonization caused by the virulent mutants.
Assuntos
Anticorpos Antibacterianos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Gonorreia/terapia , Lipopolissacarídeos/imunologia , Neisseria gonorrhoeae/imunologia , Animais , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos BALB C , Resultado do Tratamento , Vagina/microbiologiaRESUMO
IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design.
Assuntos
Ativação do Complemento , Complemento C1/química , Imunoglobulina G/química , Multimerização Proteica , Complemento C1/imunologia , Humanos , Imunoglobulina G/imunologiaRESUMO
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.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD20/imunologia , Antígenos de Neoplasias/imunologia , Proteínas do Sistema Complemento/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Leucemia Linfocítica Crônica de Células B/genética , Tetraspaninas/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Linhagem Celular Tumoral , Complemento C1q/imunologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Imunoglobulina G/imunologia , Leucemia Linfocítica Crônica de Células B/sangue , Mutação , Ligação Proteica , Rituximab/farmacologiaRESUMO
IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Imunoglobulina G/metabolismo , Imunoterapia/métodos , Animais , Linhagem Celular Tumoral , Ativação do Complemento , Feminino , Humanos , Imunoglobulina G/genética , Camundongos SCID , Mutação , Transplante de Neoplasias , PolimerizaçãoRESUMO
Triggering of the complement cascade induces tumor cell lysis via complement-dependent cytotoxicity (CDC) and attracts and activates cytotoxic cells. It therefore represents an attractive mechanism for mAb in cancer immunotherapy development. The classical complement pathway is initiated by IgG molecules that have assembled into ordered hexamers after binding their Ag on the tumor cell surface. The requirements for CDC are further impacted by factors such as Ab epitope, valency, and affinity. Thus, mAb against well-validated solid tumor targets, such as the epidermal growth factor receptor (EGFR) that effectively induces complement activation and CDC, are highly sought after. The potency of complement activation by IgG Abs can be increased via several strategies. We identified single-point mutations in the Fc domain (e.g., E345K or E430G) enhancing Fc:Fc interactions, hexamer formation, and CDC after Ab binds cell-surface Ag. We show that EGFR Abs directed against clinically relevant epitopes can be converted into mAb with unprecedented CDC activity. Alternative strategies rely on increasing the affinity of monomeric IgG for C1q by introduction of a quadruple mutation at the C1q binding site or via generation of an IgG1/IgG3 chimera. In this study we show that selective enhancement of C1q binding via avidity modulation is superior to the unattended increase in C1q binding via affinity approaches, particularly for target cells with reduced EGFR expression levels. Improving Fc:Fc interactions of Ag-bound IgG therefore represents a highly promising and novel approach for potentiating the anti-tumor activity of therapeutic mAb against EGFR and potentially other tumor targets.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Ativação do Complemento , Receptores ErbB/imunologia , Imunoglobulina G/imunologia , Anticorpos Monoclonais/genética , Sítios de Ligação , Linhagem Celular Tumoral , Complemento C1q/imunologia , Complemento C1q/metabolismo , Receptores ErbB/genética , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoterapia/métodos , Mutação , Mutação PuntualRESUMO
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.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD20/metabolismo , Antígenos/imunologia , Sítios de Ligação de Anticorpos , Complemento C9/metabolismo , Glicoproteínas de Membrana/metabolismo , ADP-Ribosil Ciclase 1/imunologia , Alemtuzumab , Anticorpos Monoclonais Humanizados/imunologia , Antígenos CD20/imunologia , Linfócitos B/imunologia , Linhagem Celular Tumoral , Ativação do Complemento , Complemento C3b/metabolismo , Complemento C9/imunologia , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Humanos , Glicoproteínas de Membrana/imunologiaRESUMO
Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20+ B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab')2 fragments, as well as C1q-binding-deficient IgG mutants, retained an ability to induce CDC, albeit with lower efficiency than for whole or unmodified IgG. Experiments using human serum depleted of specific complement components demonstrated that the observed lytic activity, which we termed "accessory CDC," remained to be dependent on C1 and the classical pathway. We hypothesized that CD20 Ab-induced clustering of the IgM or IgG BCR was involved in accessory CDC. Indeed, accessory CDC was consistently observed in B cell lines expressing an IgM BCR and in some cell lines expressing an IgG BCR, but it was absent in BCR- B cell lines. A direct relationship between BCR expression and accessory CDC was established by transfecting the BCR into CD20+ cells: OFA-F(ab')2 fragments were able to induce CDC in the CD20+BCR+ cell population, but not in the CD20+BCR- population. Importantly, OFA-F(ab')2 fragments were able to induce CDC ex vivo in malignant B cells isolated from patients with mantle cell lymphoma and Waldenström macroglobulinemia. In summary, accessory CDC represents a novel effector mechanism that is dependent on type I CD20 Ab-induced BCR clustering. Accessory CDC may contribute to the excellent capacity of type I CD20 Abs to induce CDC, and thereby to the antitumor activity of such Abs in the clinic.
Assuntos
Anticorpos Monoclonais/metabolismo , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD20/metabolismo , Linfócitos B/efeitos dos fármacos , Via Clássica do Complemento , Imunoterapia Adotiva/métodos , Linfoma de Células B/terapia , Rituximab/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antígenos CD20/imunologia , Linfócitos B/imunologia , Linhagem Celular Tumoral , Complemento C1/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Linfoma de Células B/imunologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Rituximab/genética , Rituximab/uso terapêuticoRESUMO
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.
Assuntos
Complemento C9/deficiência , Proteínas do Sistema Complemento/imunologia , Síndromes de Imunodeficiência/imunologia , Leucemia Linfocítica Crônica de Células B , Rituximab/farmacologia , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Complemento C9/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Doenças da Deficiência Hereditária de Complemento , Humanos , Imunoterapia , PolimerizaçãoRESUMO
Complement-dependent cytotoxicity (CDC) has been suggested to be an important mechanism of action of tumor-targeting Abs. However, single unmodified epidermal growth factor receptor (EGFR)-targeting IgG1 Abs fail to trigger efficient CDC. For the current study, we generated a CDC-optimized variant of the EGFR Ab matuzumab (H425 wt) by introducing amino acid substitutions K326A/E333A (H425 mt). This Ab was then used to elucidate the impact of complement activation on the capacity of effector cells such as mononuclear cells (MNC) and polymorphonuclear cells (PMN) to exert Ab-dependent cell-mediated cytotoxicity (ADCC). H425 mt, but not H425 wt, significantly induced complement deposition, release of anaphylatoxins, and CDC against distinct tumor cell lines, whereas no differences in ADCC by MNC or PMN were detected. Notably, stronger cytotoxicity was induced by H425 mt than by H425 wt in whole blood assays and in experiments in which MNC or PMN were combined with serum. Although MNC-ADCC was not affected by C5 cleavage, the cytotoxic activity of PMN in the presence of serum strongly depended on C5 cleavage, pointing to a direct interaction between complement and PMN. Strong cell surface expression of C5a receptors was detected on PMN, whereas NK cells completely lacked expression. Stimulation of PMN with C5a led to upregulation of activated complement receptor 3, resulting in enhanced complement receptor 3-dependent PMN-ADCC against tumor cells. In conclusion, complement-optimized EGFR Abs may constitute a promising strategy to improve tumor cell killing by enhancing the interaction between humoral and cellular effector functions in Ab-based tumor therapy.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Antineoplásicos/farmacologia , Complemento C5a/imunologia , Receptores ErbB/antagonistas & inibidores , Leucócitos/imunologia , Neoplasias/tratamento farmacológico , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/imunologia , Humanos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Receptor da Anafilatoxina C5a/imunologiaRESUMO
The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3-3 µM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 µM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD20/imunologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Linfoma de Células B/tratamento farmacológico , Purinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinazolinonas/farmacologia , Adenina/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Western Blotting , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ativação do Complemento/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , PiperidinasRESUMO
Monoclonal antibodies constitute a major treatment option for many tumor patients. Due to their specific recognition sites in their constant Fc regions, antibodies are able to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). While the contribution of ADCC to clinical efficacy has been strengthened by observations that patients with favorable Fcγ receptor polymorphisms display better response rates to therapeutic antibodies, the contribution of CDC to their clinical efficacy remains controversial. In the background of high expression of complement-regulatory proteins on tumor cells as well as of the fact that some therapeutic antibodies lack the capacity to trigger efficient CDC, strategies have been implemented to improve either the capacity of antibodies to initiate the complement cascade or to interfere with tumor cells' resistance mechanisms. Although both strategies have demonstrated therapeutic benefit in vitro and in murine models, CDC-enhanced antibodies-to the best of our knowledge-have not been clinically tested, and evidence for the potential of CDC-optimizing approaches has yet to be generated in humans. Hence, the potency of complement activation and its impact on the clinical efficacy of therapeutic antibodies still remains to be elucidated in clinical trials encompassing novel complement-enhancing molecules.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Proteínas do Sistema Complemento/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Terapia de Alvo MolecularRESUMO
The CD20 mAb ofatumumab (OFA) induces complement-mediated lysis of B cells. In an investigator-initiated phase II trial of OFA plus chemotherapy for chronic lymphocytic leukemia (CLL), OFA treatment promoted partial CLL B cell depletion that coincided with reduced complement titers. Remaining CLL B cells circulated with bound OFA and covalently bound complement breakdown product C3d, indicative of ongoing complement activation. Presumably, neither complement- nor effector cell-based mechanisms were sufficiently robust to clear these remaining B cells. Instead, almost all of the bound OFA and CD20 was removed from the cells, in accordance with previous clinical studies that demonstrated comparable loss of CD20 from B cells after treatment of CLL patients with rituximab. In vitro experiments with OFA and rituximab addressing these observations suggest that host effector mechanisms that support mAb-mediated lysis and tumor cell clearance are finite, and they can be saturated or exhausted at high B cell burdens, particularly at high mAb concentrations. Interestingly, only a fraction of available complement was required to kill cells with CD20 mAbs, and killing could be tuned by titrating the mAb concentration. Consequently, maximal B cell killing of an initial and secondary B cell challenge was achieved with intermediate mAb concentrations, whereas high concentrations promoted lower overall killing. Therefore, mAb therapies that rely substantially on effector mechanisms subject to exhaustion, including complement, may benefit from lower, more frequent dosing schemes optimized to sustain and maximize killing by cytotoxic immune effector systems.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Murinos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Proteínas do Sistema Complemento/imunologia , Imunoterapia , Leucemia Linfocítica Crônica de Células B/terapia , Alemtuzumab , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Murinos/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD20/imunologia , Antígenos de Neoplasias/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Subpopulações de Linfócitos B/efeitos dos fármacos , Subpopulações de Linfócitos B/imunologia , Linhagem Celular Tumoral/efeitos dos fármacos , Terapia Combinada , Ativação do Complemento , Complemento C3b/análise , Complemento C4b/análise , Ciclofosfamida/administração & dosagem , Citotoxicidade Imunológica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Contagem de Linfócitos , Proteínas Opsonizantes/imunologia , Rituximab , Vidarabina/administração & dosagem , Vidarabina/análogos & derivadosRESUMO
We previously reported that 1 h after infusion of CD20 mAb rituximab in patients with chronic lymphocytic leukemia (CLL), >80% of CD20 was removed from circulating B cells, and we replicated this finding, based on in vitro models. This reaction occurs via an endocytic process called shaving/trogocytosis, mediated by FcγR on acceptor cells including monocytes/macrophages, which remove and internalize rituximab-CD20 immune complexes from B cells. Beers et al. reported that CD20 mAb-induced antigenic modulation occurs as a result of internalization of B cell-bound mAb-CD20 complexes by the B cells themselves, with internalization of â¼40% observed after 2 h at 37°C. These findings raise fundamental questions regarding the relative importance of shaving versus internalization in promoting CD20 loss and have substantial implications for the design of mAb-based cancer therapies. Therefore, we performed direct comparisons, based on flow cytometry, to determine the relative rates and extent of shaving versus internalization. B cells, from cell lines, from patients with CLL, and from normal donors, were opsonized with CD20 mAbs rituximab or ofatumumab and incubated for varying times and then reacted with acceptor THP-1 monocytes to promote shaving. We find that shaving induces considerably greater loss of CD20 and bound mAb from opsonized B cells in much shorter time periods (75-90% in <45 min) than is observed for internalization. Both shaving/trogocytosis and internalization could contribute to CD20 loss when CLL patients receive rituximab therapy, but shaving should occur more rapidly and is most likely to be the key mechanism of CD20 loss.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Antígenos CD20/metabolismo , Antineoplásicos/farmacologia , Linfócitos B/metabolismo , Endocitose/fisiologia , Monócitos/metabolismo , Animais , Linfócitos B/efeitos dos fármacos , Linhagem Celular , Separação Celular , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos , Receptores de IgG/metabolismo , Rituximab , TransfecçãoRESUMO
Most successful vaccines elicit neutralizing antibodies and this property is a high priority when developing an HIV vaccine. Indeed, passively administered neutralizing antibodies have been shown to protect against HIV challenge in some of the best available animal models. For example, antibodies given intravenously can protect macaques against intravenous or mucosal SHIV (an HIV/SIV chimaera) challenge and topically applied antibodies can protect macaques against vaginal SHIV challenge. However, the mechanism(s) by which neutralizing antibodies afford protection against HIV is not understood and, in particular, the role of antibody Fc-mediated effector functions is unclear. Here we report that there is a dramatic decrease in the ability of a broadly neutralizing antibody to protect macaques against SHIV challenge when Fc receptor and complement-binding activities are engineered out of the antibody. No loss of antibody protective activity is associated with the elimination of complement binding alone. Our in vivo results are consistent with in vitro assays indicating that interaction of Fc-receptor-bearing effector cells with antibody-complexed infected cells is important in reducing virus yield from infected cells. Overall, the data suggest the potential importance of activity against both infected cells and free virus for effective protection against HIV.
Assuntos
Vacinas contra a AIDS/imunologia , Proteínas do Sistema Complemento/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV/imunologia , Receptores Fc/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Complemento C1q/imunologia , Complemento C3/imunologia , Feminino , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Antígenos HIV/imunologia , Infecções por HIV/sangue , Infecções por HIV/virologia , Humanos , Imunidade nas Mucosas/imunologia , Macaca mulatta/imunologia , Macaca mulatta/virologia , Testes de Neutralização , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Fatores de Tempo , Carga ViralRESUMO
Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and an important cause of childhood mortality. Despite the introduction of successful vaccines, the global spread of both non-vaccine serotypes and antibiotic-resistant strains reinforces the development of alternative therapies against this pathogen. One possible route is the development of monoclonal antibodies (mAbs) that induce killing of bacteria via the immune system. Here, we investigate whether mAbs can be used to induce killing of pneumococcal serotypes for which the current vaccines show unsuccessful protection. Our study demonstrates that when human mAbs against pneumococcal capsule polysaccharides (CPS) have a poor capacity to induce complement activation, a critical process for immune protection against pneumococci, their activity can be strongly improved by hexamerization-enhancing mutations. Our data indicate that anti-capsular antibodies may have a low capacity to form higher-order oligomers (IgG hexamers) that are needed to recruit complement component C1. Indeed, specific point mutations in the IgG-Fc domain that strengthen hexamerization strongly enhance C1 recruitment and downstream complement activation on encapsulated pneumococci. Specifically, hexamerization-enhancing mutations E430G or E345K in CPS6-IgG strongly potentiate complement activation on S. pneumoniae strains that express capsular serotype 6 (CPS6), and the highly invasive serotype 19A strain. Furthermore, these mutations improve complement activation via mAbs recognizing CPS3 and CPS8 strains. Importantly, hexamer-enhancing mutations enable mAbs to induce strong opsonophagocytic killing by human neutrophils. Finally, passive immunization with CPS6-IgG1-E345K protected mice from developing severe pneumonia. Altogether, this work provides an important proof of concept for future optimization of antibody therapies against encapsulated bacteria.
Assuntos
Infecções Pneumocócicas , Streptococcus pneumoniae , Humanos , Animais , Camundongos , Infecções Pneumocócicas/prevenção & controle , Infecções Pneumocócicas/microbiologia , Neutrófilos , Sorogrupo , Imunoglobulina GRESUMO
We have reported that during complement-mediated cytolysis of B cells promoted by the CD20 mAbs rituximab or ofatumumab (OFA), long, thin structures that we call streamers (≥ 3 cell diameters) are rapidly generated and grow out from the cell surface. Streamers appear before cells are killed and contain opsonizing mAbs and membrane lipids. By exploiting the differential Ca(2+) requirements of discrete steps in the complement cascade, we determined that mAb-opsonized cells first tagged with C3b using C5-depleted serum are killed on addition of serum and EDTA, but the cells do not produce streamers. Also, cells first opsonized with OFA are lysed in serum containing Mg-EGTA by the alternative complement pathway but streamers are not produced. These findings indicate that Ca(2+) influx is necessary for streamer formation. Other mAbs that promote complement-mediated cytolysis also induce streamers on target cells. Streamer-like structures called nanotubes have been reported in several cellular systems, and are thought to promote intercellular communication/signaling. We tested whether this signaling could influence the susceptibility of neighboring cells contacted by streamers to complement attack and found that complement-mediated cytolysis of OFA-opsonized cells increases the resistance of unopsonized indicator cell populations to subsequent lysis when these cells are exposed to OFA and complement.