RESUMO
Antibody responses during infection and vaccination typically undergo affinity maturation to achieve high-affinity binding for efficient neutralization of pathogens1,2. Similarly, high affinity is routinely the goal for therapeutic antibody generation. However, in contrast to naturally occurring or direct-targeting therapeutic antibodies, immunomodulatory antibodies, which are designed to modulate receptor signalling, have not been widely examined for their affinity-function relationship. Here we examine three separate immunologically important receptors spanning two receptor superfamilies: CD40, 4-1BB and PD-1. We show that low rather than high affinity delivers greater activity through increased clustering. This approach delivered higher immune cell activation, in vivo T cell expansion and antitumour activity in the case of CD40. Moreover, an inert anti-4-1BB monoclonal antibody was transformed into an agonist. Low-affinity variants of the clinically important antagonistic anti-PD-1 monoclonal antibody nivolumab also mediated more potent signalling and affected T cell activation. These findings reveal a new paradigm for augmenting agonism across diverse receptor families and shed light on the mechanism of antibody-mediated receptor signalling. Such affinity engineering offers a rational, efficient and highly tuneable solution to deliver antibody-mediated receptor activity across a range of potencies suitable for translation to the treatment of human disease.
Assuntos
Anticorpos Monoclonais , Afinidade de Anticorpos , Imunomodulação , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD40/efeitos dos fármacos , Antígenos CD40/imunologia , Imunomodulação/efeitos dos fármacos , Imunomodulação/imunologia , Nivolumabe/imunologia , Nivolumabe/farmacologiaRESUMO
The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated T cells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated T cells in vitro and promote CD8 T cell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs in vivo. Anti-4-1BB mAbs also promoted effector T cell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by either agonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic.
Assuntos
Anticorpos Monoclonais/farmacologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Imunomodulação/efeitos dos fármacos , Neoplasias/imunologia , Neoplasias/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Expressão Gênica , Humanos , Imunoglobulina G/farmacologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Knockout , Neoplasias/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
The anti-CD28 superagonist antibody TGN1412 caused life-threatening cytokine release syndrome (CRS) in healthy volunteers, which had not been predicted by preclinical testing. T cells in fresh peripheral blood mononuclear cells (PBMCs) do not respond to soluble TGN1412 but do respond following high-density (HD) preculture. We show for the first time that this response is dependent on crystallizable fragment gamma receptor IIb (FcγRIIb) expression on monocytes. This was unexpected because, unlike B cells, circulating monocytes express little or no FcγRIIb. However, FcγRIIb expression is logarithmically increased on monocytes during HD preculture, and this upregulation is necessary and sufficient to explain TGN1412 potency after HD preculture. B-cell FcγRIIb expression is unchanged by HD preculture, but B cells can support TGN1412-mediated T-cell proliferation when added at a frequency higher than that in PBMCs. Although low-density (LD) precultured PBMCs do not respond to TGN1412, T cells from LD preculture are fully responsive when cocultured with FcγRIIb-expressing monocytes from HD preculture, which shows that they are fully able to respond to TGN1412-mediated activation. Our novel findings demonstrate that cross-linking by FcγRIIb is critical for the superagonist activity of TGN1412 after HD preculture, and this may contribute to CRS in humans because of the close association of FcγRIIb-bearing cells with T cells in lymphoid tissues.
Assuntos
Anticorpos Monoclonais Humanizados/química , Monócitos/citologia , Receptores de IgG/metabolismo , Regulação para Cima , Animais , Linfócitos B/citologia , Antígenos CD28/metabolismo , Células CHO , Proliferação de Células , Técnicas de Cocultura , Cricetinae , Cricetulus , Citocinas/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Linfócitos T/citologia , Linfócitos T/imunologia , TransfecçãoRESUMO
Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo.
Assuntos
Anticorpos Monoclonais/química , Modulação Antigênica , Antígenos CD20/química , Antígenos/química , Animais , Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais Murinos/química , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linfócitos B/imunologia , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina G/química , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Fagocitose , Receptores de IgG/metabolismo , RituximabRESUMO
FcγRs are key regulators of the immune response, capable of binding to the Fc portion of IgG Abs and manipulating the behavior of numerous cell types. Through a variety of receptors, isoforms, and cellular expression patterns, they are able to fine-tune and direct appropriate responses. Furthermore, they are key determinants of mAb immunotherapy, with mAb isotype and FcγR interaction governing therapeutic efficacy. Critical to understanding the biology of this complex family of receptors are reagents that are robust and highly specific for each receptor. In this study, we describe the development and characterization of mAb panels specific for both mouse and human FcγR for use in flow cytometry, immunofluorescence, and immunocytochemistry. We highlight key differences in expression between the two species and also patterns of expression that will likely impact on immunotherapeutic efficacy and translation of therapeutic agents from mouse to clinic.
Assuntos
Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Receptores de IgG/biossíntese , Receptores de IgG/imunologia , Animais , Medula Óssea/imunologia , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Citometria de Fluxo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tonsila Palatina/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Ratos , Ratos Wistar , Baço/imunologiaRESUMO
Immunomodulatory mAbs, led by the anti-CTLA4 mAb ipilimumab, are an exciting new class of drugs capable of promoting anticancer immunity and providing durable control of some tumors. Close analysis of a number of agents has revealed a critical yet variable role for Fcγ receptors in their efficacy. In this article, we reveal that agonistic anti-CD40 mAbs have an absolute requirement for cross-linking by inhibitory FcγRIIB when used systemically to treat established BCL1 syngeneic lymphoma, and therapy is lost when using a mouse IgG2a mAb not cross-linked by FcγRIIB. Furthermore, in FcγRIIB-deficient mice the lymphoma itself can provide FcγRIIB to cross-link anti-CD40 on neighboring cells, and only when this is blocked does therapy fail. The dependence on FcγRIIB for immunostimulatory activity was not absolute, however, because when anti-CD40 mAbs were administered systemically with the TLR3 agonist polyinosinic:polycytidylic acid or were given subcutaneously, activatory FcγR could also provide cross-linking. Using this mechanistic insight, we designed multimeric forms of anti-CD40 mAb with intrinsic FcγR-independent activity that were highly effective in the treatment of lymphoma-bearing mice. In conclusion, FcγR-independent anti-CD40 activation is a viable strategy in vivo. These findings have important translational implications, as humans, unlike mice, do not have IgG that binds strongly to FcγRIIB; therefore FcγR-independent derivatives represent an attractive therapeutic option.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD40/imunologia , Linfoma/terapia , Multimerização Proteica/imunologia , Receptores de IgG/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Células Cultivadas , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Imunoterapia , Linfoma/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli I-C/uso terapêutico , Receptores de IgG/genética , Ressonância de Plasmônio de Superfície , Receptor 3 Toll-Like/agonistasRESUMO
Although long considered as a disease of failed apoptosis, it is now clear that chronic lymphocytic leukemia (CLL) cells undergo extensive cell division in vivo, especially in progressive disease. Signaling via the B-cell receptor is thought to activate proliferation and survival pathways in CLL cells and also has been linked to poor outcome. Here, we have analyzed the expression of the proto-oncoprotein MYC, an essential positive regulator of the cell cycle, after stimulation of surface IgM (sIgM). MYC expression was rapidly increased after sIgM stimulation in a subset of CLL samples. The ability of sIgM stimulation to increase MYC expression was correlated with sIgM-induced intracellular calcium fluxes. MYC induction was partially dependent on the MEK/ERK signaling pathway, and MYC and phosphorylated ERK1/2 were both expressed within proliferation centers in vivo. Although stimulation of sIgD also resulted in ERK1/2 phosphorylation, responses were relatively short lived compared with sIgM and were associated with significantly reduced MYC induction, suggesting that the kinetics of ERK1/2 activation is a critical determinant of MYC induction. Our results suggest that ERK1/2-dependent induction of MYC is likely to play an important role in antigen-induced CLL cell proliferation.
Assuntos
Membrana Celular/metabolismo , Imunoglobulina M/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Western Blotting , Ciclo Celular , Proliferação de Células , Humanos , Técnicas Imunoenzimáticas , Leucemia Linfocítica Crônica de Células B/genética , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais CultivadasRESUMO
B-cell receptor and microenvironment-derived signals promote accumulation of chronic lymphocytic leukemia (CLL) cells through increased proliferation and/or decreased apoptosis. In this study, we investigated the regulation of BIM, a proapoptotic BCL2-related protein, which is tightly regulated by phosphorylation. Surface IgM stimulation increased phosphorylation of 2 BIM isoforms, BIM(EL) and BIM(L), in a subset of CLL samples. In contrast, in normal B cells, anti-IgM triggered selective phosphorylation of BIM(EL) only. In CLL, anti-IgM-induced BIM phosphorylation correlated with unmutated IGHV gene status and with progressive disease. Strikingly, it was also associated with progressive disease within the mutated IGHV gene subset. BIM phosphorylation was dependent on MEK1/2 kinase activity, and we identified BIM(EL) serine 69, previously linked to pro-survival responses, as the major site of phosphorylation in CLL and in Ramos cells. BIM(EL)/BIM(L) phosphorylation was associated with release of the pro-survival protein MCL1. Coculture of CLL cells with HK cells, a model of the CLL microenvironment, promoted CLL cell survival and was associated with MEK1/2 activation and BIM(EL) phosphorylation. Hence, BIM phosphorylation appears to play a key role in apoptosis regulation in CLL cells, potentially coordinating antigen and microenvironment-derived survival signals. Antigen-mediated effects on BIM may be an important determinant of clinical behavior.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Anticorpos Anti-Idiotípicos/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2 , Biomarcadores Tumorais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: OX40 has been widely studied as a target for immunotherapy with agonist antibodies taken forward into clinical trials for cancer where they are yet to show substantial efficacy. Here, we investigated potential mechanisms of action of anti-mouse (m) OX40 and anti-human (h) OX40 antibodies, including a clinically relevant monoclonal antibody (mAb) (GSK3174998) and evaluated how isotype can alter those mechanisms with the aim to develop improved antibodies for use in rational combination treatments for cancer. METHODS: Anti-mOX40 and anti-hOX40 mAbs were evaluated in a number of in vivo models, including an OT-I adoptive transfer immunization model in hOX40 knock-in (KI) mice and syngeneic tumor models. The impact of FcγR engagement was evaluated in hOX40 KI mice deficient for Fc gamma receptors (FcγR). Additionally, combination studies using anti-mouse programmed cell death protein-1 (mPD-1) were assessed. In vitro experiments using peripheral blood mononuclear cells (PBMCs) examining possible anti-hOX40 mAb mechanisms of action were also performed. RESULTS: Isotype variants of the clinically relevant mAb GSK3174998 showed immunomodulatory effects that differed in mechanism; mIgG1 mediated direct T-cell agonism while mIgG2a acted indirectly, likely through depletion of regulatory T cells (Tregs) via activating FcγRs. In both the OT-I and EG.7-OVA models, hIgG1 was the most effective human isotype, capable of acting both directly and through Treg depletion. The anti-hOX40 hIgG1 synergized with anti-mPD-1 to improve therapeutic outcomes in the EG.7-OVA model. Finally, in vitro assays with human peripheral blood mononuclear cells (hPBMCs), anti-hOX40 hIgG1 also showed the potential for T-cell stimulation and Treg depletion. CONCLUSIONS: These findings underline the importance of understanding the role of isotype in the mechanism of action of therapeutic mAbs. As an hIgG1, the anti-hOX40 mAb can elicit multiple mechanisms of action that could aid or hinder therapeutic outcomes, dependent on the microenvironment. This should be considered when designing potential combinatorial partners and their FcγR requirements to achieve maximal benefit and improvement of patient outcomes.
Assuntos
Receptores OX40 , Animais , Humanos , Camundongos , Receptores OX40/agonistas , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Linhagem Celular Tumoral , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Modelos Animais de DoençasRESUMO
Fc receptors (FcRs) play a key role in regulating and coordinating responses from both innate and adaptive arms of the immune system. The inhibitory Fc gamma receptor II (FcγRIIB; CD32) is central to this regulation with FcγRIIB(-/-) mice demonstrating augmented responses to mAb immunotherapy, elevated incidence and severity of auto-immunity, and increased response to mAb-mediated cancer therapy. To date, these observations have remained unexploited therapeutically, partly through a lack of specific mAb reagents capable of exclusively binding mouse FcγRIIB. Thus almost all of the FcγRIIB-binding mAb currently available, such as 2.4G2, also bind FcγRIII (CD16), and polyclonal reagents have limited availability and are of unproven specificity and avidity, making in vivo manipulation of FcγRIIB impossible. Following an extensive immunisation protocol using FcγRIIB(-/-) mice, we recently produced three unique mAb that are suitable for this purpose. Here we characterise these novel reagents and demonstrate that they fall into two distinct categories; those which cause phosphorylation and subsequent activation of FcγRIIB (agonistic) and those that block receptor phosphorylation (antagonistic). These two types of mAb exhibit different characteristics in a range of biochemical, cellular, and functional assays relevant to FcγRIIB activity and mAb therapy.
Assuntos
Anticorpos Monoclonais/imunologia , Receptores de IgG/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Morte Celular , Células Cultivadas , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosforilação , Receptores de IgG/genéticaRESUMO
The anti-CD20 mAb rituximab is central to the treatment of B-cell malignancies, but resistance remains a significant problem. We recently reported that resistance could be explained, in part, by internalization of rituximab (type I anti-CD20) from the surface of certain B-cell malignancies, thus limiting engagement of natural effectors and increasing mAb consumption. Internalization of rituximab was most evident in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but the extent of internalization was heterogeneous within each disease. Here, we show that the inhibitory FcγRIIb on target B cells promotes this process and is largely responsible for the observed heterogeneity across a range of B-cell malignancies. Internalization correlated strongly with FcγRIIb expression on normal and malignant B cells, and resulted in reduced macrophage phagocytosis of mAb-coated targets. Furthermore, transfection of FcγRIIb into FcγRIIb negative Ramos cells increased internalization of rituximab in a dose-dependent manner. Target-cell FcγRIIb promoted rituximab internalization in a cis fashion and was independent of FcγRIIb on neighboring cells. It became phosphorylated and internalized along with CD20:anti-CD20 complexes before lysosomal degradation. In MCL patients, high FcγRIIb expression predicted less durable responses after rituximab-containing regimens. Therefore, target-cell FcγRIIb provides a potential biomarker of response to type I anti-CD20 mAb.
Assuntos
Anticorpos Monoclonais Murinos/metabolismo , Anticorpos Antineoplásicos/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , Antígenos CD20/metabolismo , Antígenos de Neoplasias/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos B/imunologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Endocitose/fisiologia , Linfoma de Célula do Manto/tratamento farmacológico , Receptores de IgG/metabolismo , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Antineoplásicos/administração & dosagem , Anticorpos Antineoplásicos/imunologia , Antígenos CD20/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos B/metabolismo , Biomarcadores , Linhagem Celular Tumoral/imunologia , Linhagem Celular Tumoral/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Linfoma de Célula do Manto/patologia , Lisossomos/metabolismo , Macrófagos/fisiologia , Fagocitose , Fosforilação , Processamento de Proteína Pós-Traducional , Receptores de IgG/genética , Proteínas Recombinantes de Fusão/metabolismo , Rituximab , Transfecção , Resultado do TratamentoRESUMO
A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10-100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.
Assuntos
Anticorpos Monoclonais Murinos , Antineoplásicos , Antígenos CD40/imunologia , Capeamento Imunológico/efeitos dos fármacos , Receptores de IgG/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacocinética , Anticorpos Monoclonais Murinos/farmacologia , Antineoplásicos/imunologia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Capeamento Imunológico/imunologia , Camundongos , Camundongos Knockout , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
Introduction: T cell expressed CD27 provides costimulation upon binding to inducible membrane expressed trimeric CD70 and is required for protective CD8 T cell responses. CD27 agonists could therefore be used to bolster cellular vaccines and anti-tumour immune responses. To date, clinical development of CD27 agonists has focussed on anti-CD27 antibodies with little attention given to alternative approaches. Methods: Here, we describe the generation and activity of soluble variants of CD70 that form either trimeric (t) or dimer-of-trimer proteins and conduct side-by-side comparisons with an agonist anti-CD27 antibody. To generate a dimer-of-trimer protein (dt), we fused three extracellular domains of CD70 to the Fc domain of mouse IgG1 in a 'string of beads' configuration (dtCD70-Fc). Results: Whereas tCD70 failed to costimulate CD8 T cells, both dtCD70-Fc and an agonist anti-CD27 antibody were capable of enhancing T cell proliferation in vitro. Initial studies demonstrated that dtCD70-Fc was less efficacious than anti-CD27 in boosting a CD8 T cell vaccine response in vivo, concomitant with rapid clearance of dtCD70-Fc from the circulation. The accelerated plasma clearance of dtCD70-Fc was not due to the lack of neonatal Fc receptor binding but was dependent on the large population of oligomannose type glycosylation. Enzymatic treatment to reduce the oligomannose-type glycans in dtCD70-Fc improved its half-life and significantly enhanced its T cell stimulatory activity in vivo surpassing that of anti-CD27 antibody. We also show that whereas the ability of the anti-CD27 to boost a vaccine response was abolished in Fc gamma receptor (FcγR)-deficient mice, dtCD70-Fc remained active. By comparing the activity of dtCD70-Fc with a variant (dtCD70-Fc(D265A)) that lacks binding to FcγRs, we unexpectedly found that FcγR binding to dtCD70-Fc was required for maximal boosting of a CD8 T cell response in vivo. Interestingly, both dtCD70-Fc and dtCD70-Fc(D265A) were effective in prolonging the survival of mice harbouring BCL1 B cell lymphoma, demonstrating that a substantial part of the stimulatory activity of dtCD70-Fc in this setting is retained in the absence of FcγR interaction. Discussion: These data reveal that TNFRSF ligands can be generated with a tunable activity profile and suggest that this class of immune agonists could have broad applications in immunotherapy.
Assuntos
Receptores de IgG , Vacinas , Animais , Camundongos , Ligante CD27/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , ImunizaçãoAssuntos
Anticorpos Monoclonais/imunologia , Receptores de IgG/imunologia , Transferência Adotiva , Animais , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos CD20/imunologia , Reações Cruzadas , Humanos , Depleção Linfocítica , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Baço/citologiaRESUMO
Surface IgM (sIgM) has a key influence on the clinical behavior of chronic lymphocytic leukemia (CLL). We now report that it exists in 2 forms with different N-glycosylation patterns in the mu-constant region. One glycoform is similar to normal B cells in bearing mature complex glycans common to most cell-surface glycoproteins. The other is an immature mannosylated form more characteristic of mu chains in the endoplasmic reticulum. Unmutated CLL (U-CLL) expresses a higher proportion of mannosylated surface mu chains than mutated CLL. Normal B cells express only the mature glycoform but can express the immature form after persistent engagement of sIgM, suggesting that glycan modification is a consequence of antigen exposure. CLL cells express variable proportions of the mannosylated form and can revert to the mature form after incubation in vitro. Both glycoforms are able to signal after sIgM engagement in vitro, leading to enhanced tyrosine phosphorylation. These findings support the concept that CLL cells are continuously exposed to antigen in vivo, driving the N-glycosylation pattern of expressed sIgM toward a mannosylated form, especially in U-CLL. Strikingly, this is reminiscent of follicular lymphoma, where mannosylated Ig is expressed constitutively via N-glycosylation sites in the variable region, suggesting a functional asset for this glycoform.
Assuntos
Imunoglobulina M/química , Imunoglobulina M/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismo , Apresentação de Antígeno , Linfócitos B/imunologia , Estudos de Casos e Controles , Glicosilação , Humanos , Imunoglobulina M/genética , Cadeias mu de Imunoglobulina/química , Cadeias mu de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Modelos Imunológicos , Mutação , Polissacarídeos/química , Polissacarídeos/genética , Prognóstico , Receptores de Antígenos de Linfócitos B/genéticaRESUMO
The cell of origin of chronic lymphocytic leukemia (CLL) has long been sought, and immunoglobulin gene analysis provides new clues. In the unmutated subset (U-CLL), there is increased usage of the 51p1-related alleles of the immunoglobulin heavy chain variable 1-69 gene, often combined with selected genes and with immunoglobulin heavy chain diversity IGHJ6. Stereotypic characteristics of the HCDR3 result and suggest antigen selection of the leukemic clones. We have now analyzed 51p1/IGHJ6 combinations in normal blood B cells from 3 healthy persons for parallel sequence patterns. A high proportion (33.3% of sequences) revealed stereotypic patterns, with several (15.0%) being similar to those described in U-CLL. Previously unreported CLL-associated stereotypes were detected in 4.8%. Stereotypes (13.6%) not detected in CLL also were found. The HCDR2-IGHJ6 sequences were essentially unmutated. Junctional amino acids in normal B cells were heterogeneous, as in cases of stereotyped CLL. Phenotypically, normal B cells expressing 51p1-derived immunoglobulin M were naive. This snapshot of the naive B-cell repertoire reveals subsets of B cells closely related to those characteristic of CLL. Conserved patterns in the 51p1-encoded immunoglobulin M of normal B cells suggest a restricted sequence repertoire shaped by evolution to recognize common pathogens. Proliferative pressure on these cells is the likely route to U-CLL.
Assuntos
Linfócitos B/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Mutação/genética , Idoso , Alelos , Sequência de Aminoácidos , Sequência de Bases , Doadores de Sangue , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Feminino , Rearranjo Gênico de Cadeia Pesada de Linfócito B/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , FenótipoRESUMO
BACKGROUND: Despite extensive clinical use, the mechanisms that lead to therapeutic resistance to anti-programmed cell-death (PD)-1 monoclonal antibodies (mAbs) remain elusive. Here, we sought to determine how interactions between the Fc region of anti-PD-1 mAbs and Fcγ receptors (FcγRs) affect therapeutic activity and how these are impacted by the immune environment. METHODS: Mouse and human anti-PD-1 mAbs with different Fc binding profiles were generated and characterized in vitro. The ability of these mAbs to elicit T-cell responses in vivo was first assessed in a vaccination setting using the model antigen ovalbumin. The antitumor activity of anti-PD-1 mAbs was investigated in the context of immune 'hot' MC38 versus 'cold' neuroblastoma tumor models, and flow cytometry performed to assess immune infiltration. RESULTS: Engagement of activating FcγRs by anti-PD-1 mAbs led to depletion of activated CD8 T cells in vitro and in vivo, abrogating therapeutic activity. Importantly, the extent of this Fc-mediated modulation was determined by the surrounding immune environment. Low FcγR-engaging mouse anti-PD-1 isotypes, which are frequently used as surrogates for human mAbs, were unable to expand ovalbumin-reactive CD8 T cells, in contrast to Fc-null mAbs. These results were recapitulated in mice expressing human FcγRs, in which clinically relevant hIgG4 anti-PD-1 led to reduced endogenous expansion of CD8 T cells compared with its engineered Fc-null counterpart. In the context of an immunologically 'hot' tumor however, both low-engaging and Fc-null mAbs induced long-term antitumor immunity in MC38-bearing mice. Finally, a similar anti-PD-1 isotype hierarchy was demonstrated in the less responsive 'cold' 9464D neuroblastoma model, where the most effective mAbs were able to delay tumor growth but could not induce long-term protection. CONCLUSIONS: Our data collectively support a critical role for Fc:FcγR interactions in inhibiting immune responses to both mouse and human anti-PD-1 mAbs, and highlight the context-dependent effect that anti-PD-1 mAb isotypes can have on T-cell responses. We propose that engineering of Fc-null anti-PD-1 mAbs would prevent FcγR-mediated resistance in vivo and allow maximal T-cell stimulation independent of the immunological environment.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Modelos Animais de Doenças , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Camundongos , Microambiente TumoralRESUMO
Agonistic CD27 monoclonal antibodies (mAb) have demonstrated impressive anti-tumour efficacy in multiple preclinical models but modest clinical responses. This might reflect current reagents delivering suboptimal CD27 agonism. Here, using a novel panel of CD27 mAb including a clinical candidate, we investigate the determinants of CD27 mAb agonism. Epitope mapping and in silico docking analysis show that mAb binding to membrane-distal and external-facing residues are stronger agonists. However, poor epitope-dependent agonism could partially be overcome by Fc-engineering, using mAb isotypes that promote receptor clustering, such as human immunoglobulin G1 (hIgG1, h1) with enhanced affinity to Fc gamma receptor (FcγR) IIb, or hIgG2 (h2). This study provides the critical knowledge required for the development of agonistic CD27 mAb that are potentially more clinically efficacious.
Assuntos
Antineoplásicos Imunológicos , Neoplasias , Anticorpos Monoclonais/química , Antineoplásicos Imunológicos/uso terapêutico , Análise por Conglomerados , Epitopos , Humanos , Neoplasias/metabolismoRESUMO
Many therapeutic antibodies deplete target cells and elicit immunotherapy by engaging activating Fc gamma receptors (FcγRs) on host effector cells. These antibodies are negatively regulated by the inhibitory FcγRIIB (CD32B). Dogma suggests inhibition is mediated through the FcγRIIB immunoreceptor tyrosine-based inhibition motif (ITIM), negatively regulating immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling from activating FcγR. To assess this, we generated experimental models expressing human (h)FcγRIIB on targets or effectors, lacking or retaining ITIM signaling capacity. We demonstrate that signaling through the hFcγRIIB ITIM is dispensable for impairing monoclonal antibody (mAb)-mediated depletion of normal and malignant murine target cells through three therapeutically relevant surface receptors (CD20, CD25, and OX40) affecting immunotherapy. We demonstrate that hFcγRIIB competition with activating FcγRs for antibody Fc, rather than ITIM signaling, is sufficient to impair activating FcγR engagement, inhibiting effector function and immunotherapy.
Assuntos
Anticorpos Monoclonais , Receptores de IgG/imunologia , Animais , Humanos , Imunoterapia , Camundongos , Receptores de IgG/metabolismo , Transdução de SinaisRESUMO
Monoclonal antibodies (mAb) and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFR) exhibit a wide range of agonistic activities and antitumor responses. The mechanisms underlying these differential agonistic activities remain poorly understood. Here, we employ a panel of experimental and clinically-relevant molecules targeting human CD40, 4-1BB and OX40 to examine this issue. Confocal and STORM microscopy reveal that strongly agonistic reagents induce clusters characterized by small area and high receptor density. Using antibody pairs differing only in isotype we show that hIgG2 confers significantly more receptor clustering than hIgG1 across all three receptors, explaining its greater agonistic activity, with receptor clustering shielding the receptor-agonist complex from further molecular access. Nevertheless, discrete receptor clustering patterns are observed with different hIgG2 mAb, with a unique rod-shaped assembly observed with the most agonistic mAb. These findings dispel the notion that larger receptor clusters elicit greater agonism, and instead point to receptor density and subsequent super-structure as key determinants.