RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales , Afinidad de Anticuerpos , Inmunomodulación , Humanos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD40/efectos de los fármacos , Antígenos CD40/inmunología , Inmunomodulación/efectos de los fármacos , Inmunomodulación/inmunología , Nivolumab/inmunología , Nivolumab/farmacologíaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Inmunomodulación/efectos de los fármacos , Neoplasias/inmunología , Neoplasias/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/antagonistas & inhibidores , Animales , Expresión Génica , Humanos , Inmunoglobulina G/farmacología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Ratones Noqueados , Neoplasias/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Immunotherapy using mAbs, such as rituximab, is an established means of treating hematological malignancies. Abs can elicit a number of mechanisms to delete target cells, including complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity, and Ab-dependent cellular phagocytosis. The inherent properties of the target molecule help to define which of these mechanisms are more important for efficacy. However, it is often unclear why mAb binding to different epitopes within the same target elicits different levels of therapeutic activity. To specifically address whether distance from the target cell membrane influences the aforementioned effector mechanisms, a panel of fusion proteins consisting of a CD20 or CD52 epitope attached to various CD137 scaffold molecules was generated. The CD137 scaffold was modified through the removal or addition of cysteine-rich extracellular domains to produce a panel of chimeric molecules that held the target epitope at different distances along the protein. It was shown that complement-dependent cytotoxicity and Ab-dependent cellular cytotoxicity favored a membrane-proximal epitope, whereas Ab-dependent cellular phagocytosis favored an epitope positioned further away. These findings were confirmed using reagents targeting the membrane-proximal or -distal domains of CD137 itself before investigating these properties in vivo, where a clear difference in the splenic clearance of transfected tumor cells was observed. Together, this work demonstrates how altering the position of the Ab epitope is able to change the effector mechanisms engaged and facilitates the selection of mAbs designed to delete target cells through specific effector mechanisms and provide more effective therapeutic agents.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Membrana Celular/inmunología , Epítopos/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales de Origen Murino/inmunología , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD20/genética , Antígenos CD20/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígeno CD52 , Línea Celular Tumoral , Glicoproteínas/genética , Glicoproteínas/inmunología , Humanos , Inmunoterapia , Ratones , Fagocitosis , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
Monoclonal antibody (mAb) therapeutics are revolutionizing cancer treatment; however, not all tumors respond, and agent optimization is essential to improve outcome. It has become clear over recent years that isotype choice is vital to therapeutic success with agents that work through different mechanisms, direct tumor targeting, agonistic receptor engagement, or receptor-ligand blockade, having contrasting requirements. Here we summarize how isotype dictates mAb activity and discuss ways in which this information can be used for the development of enhanced therapeutics.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Isotipos de Inmunoglobulinas/inmunología , Humanos , Factores Inmunológicos/inmunología , Modelos Biológicos , Receptores de IgG/metabolismo , Vacunas/inmunologíaRESUMEN
Objective: A proportion of RA and SLE patients treated with standard doses of rituximab (RTX) display inefficient B cell deletion and poor clinical responses that can be augmented by delivering higher doses, indicating that standard-dose RTX is a sub-optimal therapy in these patients. This study aimed to investigate whether better responses could be achieved with mechanistically different anti-CD20 mAbs. Methods: We compared RTX with obinutuzumab (OBZ), a new-generation, glycoengineered type II anti-CD20 mAb, in a series of in vitro assays measuring B cell cytotoxicity in RA and SLE patient samples. Results: We found that OBZ was at least 2-fold more efficient than RTX at inducing B-cell cytotoxicity in in vitro whole blood assays. Dissecting this difference, we found that RTX elicited more potent complement-dependent cellular cytotoxicity than OBZ. In contrast, OBZ was more effective at evoking Fc gamma receptor-mediated effector mechanisms, including activation of NK cells and neutrophils, probably due to stronger interaction with Fc gamma receptors and the ability of OBZ to remain at the cell surface following CD20 engagement, whereas RTX became internalized. OBZ was also more efficient at inducing direct cell death. This was true for all CD19 + B cells as a whole and in naïve (IgD + CD27 - ) and switched (IgD - CD27 + ) memory B cells specifically, a higher frequency of which is associated with poor clinical response after RTX. Conclusion: Taken together, these data provide a mechanistic basis for resistance to rituximab-induced B-cell depletion, and for considering obinutuzumab as an alternative B-cell depleting agent in RA and SLE.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Apoptosis/efectos de los fármacos , Artritis Reumatoide/tratamiento farmacológico , Linfocitos B/efectos de los fármacos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Rituximab/farmacología , Adulto , Antirreumáticos/farmacología , Artritis Reumatoide/sangre , Células Cultivadas , Femenino , Humanos , Técnicas In Vitro , Lupus Eritematoso Sistémico/sangre , Masculino , Persona de Mediana Edad , Receptores de IgG/efectos de los fármacos , Receptores de IgG/metabolismo , Muestreo , Estadísticas no Paramétricas , Adulto JovenRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Monocitos/citología , Receptores de IgG/metabolismo , Regulación hacia Arriba , Animales , Linfocitos B/citología , Antígenos CD28/metabolismo , Células CHO , Proliferación Celular , Técnicas de Cocultivo , Cricetinae , Cricetulus , Citocinas/metabolismo , Humanos , Leucocitos Mononucleares/citología , Linfocitos T/citología , Linfocitos T/inmunología , TransfecciónRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Inmunoglobulina G/inmunología , Receptores de IgG/biosíntesis , Receptores de IgG/inmunología , Animales , Médula Ósea/inmunología , Células CHO , Línea Celular , Cricetinae , Cricetulus , Citometría de Flujo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Tonsila Palatina/inmunología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Ratas , Ratas Wistar , Bazo/inmunologíaRESUMEN
Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20·mAb·FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20·mAb internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/metabolismo , Antígenos CD20/metabolismo , Endocitosis , Receptores de IgG/metabolismo , Biotinilación , Western Blotting , Línea Celular Tumoral , Humanos , Microscopía Confocal , Mutación , Receptores de IgG/genética , Rituximab , Transducción de SeñalRESUMEN
CD70-mediated stimulation of CD27 is an important cofactor of CD4(+) T-cell licensed dendritic cells (DCs). However, it is unclear how CD70-mediated stimulation of T cells is integrated with signals that emanate from signal 3 pathways, such as type-1 interferon (IFN-1) and IL-12. We find that while stimulation of CD27 in isolation drives weak Eomesodermin(hi) T-bet(lo) CD8(+) T-cell responses to OVA immunization, profound synergistic expansion is achieved by cotargeting TLR. This cooperativity can substantially boost antiviral CD8(+) T-cell responses during acute infection. Concomitant stimulation of TLR significantly increases per cell IFN-γ production and the proportion of the population with characteristics of short-lived effector cells, yet also promotes the ability to form long-lived memory. Notably, while IFN-1 contributes to the expression of CD70 on DCs, the synergy between CD27 and TLR stimulation is dependent upon IFN-1's effect directly on CD8(+) T cells, and is associated with the increased expression of T-bet in T cells. Surprisingly, we find that IL-12 fails to synergize with CD27 stimulation to promote CD8(+) T-cell expansion, despite its capacity to drive effector CD8(+) T-cell differentiation. Together, these data identify complex interactions between signal 3 and costimulatory pathways, and identify opportunities to influence the differentiation of CD8(+) T-cell responses.
Asunto(s)
Ligando CD27/fisiología , Linfocitos T CD8-positivos/inmunología , Interferón Tipo I/farmacología , Proteínas de Dominio T Box/fisiología , Animales , Memoria Inmunológica , Interleucina-12/farmacología , Ratones , Ratones Endogámicos C57BL , Receptores Toll-Like/fisiologíaRESUMEN
A major feature that distinguishes type I from type II anti-CD20 monoclonal antibodies (mAbs) and reduces their therapeutic efficacy is the tendency to internalize from the cell surface. We have shown previously that the extent of internalization correlates with the capacity of type I mAb to simultaneously engage both CD20 and the inhibitory Fcγ receptor, FcγRIIb, in a bipolar configuration. Here, we investigated whether mAbs directed at other B-cell surface receptors also engaged FcγRIIb and whether this interaction promoted internalization. Most mAbs engaged and activated FcγRIIb, with the strength of activation related to the level of mAb bound to the cell surface. However, engagement did not affect internalization of most mAb-ligated receptors, either in cell lines or primary chronic lymphocytic leukemia cells with the exception of CD19 and CD38. Furthermore, at high cell concentrations/density both cis and trans interactions between cell-surface bound mAb and FcγRIIb were evident, but trans interactions did not inhibit type I anti-CD20 mAb-mediated internalization. These data identify that FcγRIIb is engaged by many mAbs in both cis and trans configurations, triggering its activation, but that internalization via FcγRIIb occurs for only a select subset. These findings have implications when designing new antibody-based therapeutics.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptores de IgG/inmunología , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Antígenos CD20/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Humanos , Isoformas de Proteínas/inmunología , Transporte de ProteínasRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD40/inmunología , Linfoma/terapia , Multimerización de Proteína/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Células Cultivadas , Inmunoglobulina G/inmunología , Inmunoglobulina G/uso terapéutico , Inmunoterapia , Linfoma/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Poli I-C/uso terapéutico , Receptores de IgG/genética , Resonancia por Plasmón de Superficie , Receptor Toll-Like 3/agonistasRESUMEN
Immunotherapy is a promising strategy for the treatment of Alzheimer's disease (AD). Antibodies directed against Amyloid Beta (Aß) are able to successfully clear plaques and reverse cognitive deficits in mouse models. Excitement towards this approach has been tempered by high profile failures in the clinic, one key issue has been the development of inflammatory side effects in the brain (ARIAs). New antibodies are entering the clinic for Alzheimer's disease; therefore, it is important to learn all we can from the current generation. In this study, we directly compared 3 clinical candidates in the same pre-clinical model, with the same effector function, for their ability to clear plaques and induce inflammation in the brain. We produced murine versions of the antibodies: Bapineuzumab (3D6), Crenezumab (mC2) and Gantenerumab (chGantenerumab) with an IgG2a constant region. 18-month transgenic APP mice (Tg2576) were injected bilaterally into the hippocampus with 2 µg of each antibody or control. After 7 days, the mice tissue was analysed for clearance of plaques and neuroinflammation by histology and biochemical analysis. 3D6 was the best binder to plaques and in vitro, whilst mC2 bound the least strongly. This translated into 3D6 effectively clearing plaques and reducing the levels of insoluble Aß, whilst chGantenerumab and mC2 did not. 3D6 caused a significant increase in the levels of pro-inflammatory cytokines IL-1ß and TNFα, and an associated increase in microglial expression of CD11B and CD68. chGantenerumab increased pro-inflammatory cytokines and microglial activation, but minimal changes in CD68, as an indicator of phagocytosis. Injection of mC2 did not cause any significant inflammatory changes. Our results demonstrate that the ability of an antibody to clear plaques and induce inflammation is dependent on the epitope and affinity of the antibody.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales/farmacología , Factores Inmunológicos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/genética , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígeno CD11b/metabolismo , Línea Celular , Evaluación Preclínica de Medicamentos , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/inmunología , Hipocampo/patología , Humanos , Interleucina-1beta/metabolismo , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/inmunología , Microglía/patología , Neuroinmunomodulación/efectos de los fármacos , Neuroinmunomodulación/fisiología , Placa Amiloide/tratamiento farmacológico , Placa Amiloide/inmunología , Placa Amiloide/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The CD70/CD27 pathway plays a significant role in the control of immunity and tolerance, and previous studies demonstrated that targeting murine CD27 (mCD27) with agonist mAbs can mediate antitumor efficacy. We sought to exploit the potential of this pathway for immunotherapy by developing 1F5, a fully human IgG1 mAb to human CD27 (hCD27) with agonist activity. We developed transgenic mice expressing hCD27 under control of its native promoter for in vivo testing of the Ab. The expression and regulation of hCD27 in hCD27-transgenic (hCD27-Tg) mice were consistent with the understood biology of CD27 in humans. In vitro, 1F5 effectively induced proliferation and cytokine production from hCD27-Tg-derived T cells when combined with TCR stimulation. Administration of 1F5 to hCD27-Tg mice enhanced Ag-specific CD8(+) T cell responses to protein vaccination comparably to an agonist anti-mCD27 mAb. In syngeneic mouse tumor models, 1F5 showed potent antitumor efficacy and induction of protective immunity, which was dependent on CD4(+) and CD8(+) T cells. The requirement of FcR engagement for the agonistic and antitumor activities of 1F5 was demonstrated using an aglycosylated version of the 1F5 mAb. These data with regard to the targeting of hCD27 are consistent with previous reports on targeting mCD27 and provide a rationale for the clinical development of the 1F5 mAb, for which studies in advanced cancer patients have been initiated under the name CDX-1127.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias/terapia , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Proliferación Celular , Humanos , Inmunoglobulina G/inmunología , Inmunoterapia , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistas , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genéticaRESUMEN
Genetic deficiency of the inhibitory Fc receptor, FcγRIIB (CD32b), has been shown to augment the activity of activatory FcγR and promote mAb immunotherapy. To investigate whether mAbs capable of blocking FcγRIIB have similar capacity, we recently generated a panel of specific anti-mouse FcγRIIB mAbs that do not cross-react with other FcRs, allowing us to study the potential of FcγRIIB as a therapeutic target. Previous work revealed a number of these mAbs capable of eliciting programmed cell death of targets, and in the present study we demonstrated their ability to promote target cell phagocytosis. However, in a variety of murine tumor models, anti-FcγRIIB mAbs demonstrated limited therapeutic activity despite optimized treatment regimens. Unexpectedly, we observed that the anti-FcγRIIB mAbs are rapidly and extensively consumed in vivo, both by the tumor and host cells, including B cells, leading to a precipitous loss from the circulation. Closer analysis revealed that the anti-FcγRIIB mAbs become extensively internalized from the cell surface within 24 h in vivo, likely explaining their suboptimal efficacy. Subsequent studies revealed that anti-FcγRIIB mAb immunotherapy was effective when used against FcγRIIB(+) tumors in FcγRIIB(-/-) recipients, indicating that consumption of the mAb by nontumor cells is the primary limitation of these reagents. Importantly, similar rates of internalization were not seen on human target cells, at least in vitro. These studies further highlight the need to determine the propensity of mAb therapeutics to internalize target receptors and also identify potential key differences between human and mouse cells in this respect.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfoma de Células B/inmunología , Macrófagos/inmunología , Mieloma Múltiple/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Apoptosis/inmunología , Células Cultivadas , Femenino , Humanos , Inmunoterapia , Linfoma de Células B/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Mieloma Múltiple/terapia , Receptores de IgG/genéticaRESUMEN
Neuroblastoma, a childhood tumour of neuroectodermal origin, accounts for 15 % of paediatric cancer deaths, which is often metastatic at diagnosis and despite aggressive therapies, it has poor long-term prognosis with high risk of recurrence. Monoclonal antibody (mAb) therapy targeting GD2, a disialoganglioside expressed on neuroblastoma, has shown promise in recent trials with natural killer cell (NK)-mediated antibody-dependent cellular cytotoxicity (ADCC) thought to be central to efficacy, although other immune effectors may be important. To further enhance therapy, immunomonitoring of patients is essential to elucidate the in vivo mechanisms of action and provides surrogate end points of efficacy for future clinical trials. Our aim was to establish a 'real-time' ex vivo wholeblood (WB) immunomonitoring strategy to perform within the logistical constraints such as limited sample volumes, anticoagulant effects, sample stability and shipping time. A fluorescent dye release assay measuring target cell lysis was coupled with flow cytometry to monitor specific effector response. Significant target cell lysis with anti-GD2 antibody (p < 0.05) was abrogated following NK depletion. NK up-regulation of CD107a and CD69 positively correlated with target cell lysis (r > 0.6). The ADCC activity of WB correlated with peripheral blood mononuclear cells (r > 0.95), although WB showed overall greater target cell lysis attributed to the combination of NK-mediated ADCC, CD16+ granulocyte degranulation and complement- dependent cytotoxicity. Response was maintained in heparinised samples stored for 24 h at room temperature, but not 4 °C. Critically, the assay showed good reproducibility (mean % CV < 6.4) and was successfully applied to primary neuroblastoma samples. As such, WB provides a resourceful analysis of multiple mechanisms for efficient end point monitoring to correlate immune modulation with clinical outcome.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Gangliósidos/metabolismo , Leucocitos Mononucleares/inmunología , Neuroblastoma/inmunología , Adulto , Citometría de Flujo , Humanos , Leucocitos Mononucleares/metabolismo , Neuroblastoma/sangre , Neuroblastoma/patología , Células Tumorales CultivadasRESUMEN
Fully functional CD8(+) T cell memory is highly dependent upon CD4(+) T cell support. CD4(+) T cells play a critical role in inducing the expression of CD70, the ligand for CD27, on dendritic cells. In this study, we demonstrate that CD27 stimulation during primary CD8(+) T cell responses regulates the ability to mount secondary CD8(+) T cell responses. CD27 stimulation during vaccinia and dendritic cell immunization controls the expression of the IL-7R (CD127), which has been shown to be necessary for memory CD8(+) T cell survival. Furthermore, CD27 stimulation during primary CD8(+) T cell responses to vaccinia virus restrained the late expression on memory precursor cells of cytokine receptors that support terminal differentiation. The formation of CD8(+) T cell memory precursors and secondary CD8(+) T cell responses was restored in the absence of CD27 costimulation when endogenous IL-12 was not available. Similarly, the lesion in CD8(+) T cell memory that occurs in the absence of CD4(+) T cells did not occur in mice lacking IL-12. These data indicate that CD4(+) T cell help and, by extension, CD27 stimulation support CD8(+) T cell memory by modulating the expression of cytokine receptors that influence the differentiation and survival of memory CD8(+) T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Receptores de Interleucina-7/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Traslado Adoptivo , Animales , Ligando CD27/genética , Ligando CD27/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Diferenciación Celular , Células Dendríticas/citología , Células Dendríticas/inmunología , Regulación de la Expresión Génica , Inmunización , Interleucina-12/genética , Interleucina-12/inmunología , Activación de Linfocitos/inmunología , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interleucina-7/genética , Transducción de Señal , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Virus Vaccinia/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Muerte Celular , Células Cultivadas , Inmunoterapia , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fosforilación , Receptores de IgG/genéticaRESUMEN
Neutrophils potently kill tumour cells in the presence of anti-tumour antibodies in vitro. However, for in vivo targeting, the neutrophils need to extravasate from the circulation by passing through endothelial barriers. To study neutrophil migration in the presence of endothelial cells in vitro, we established a three-dimensional collagen culture in which SK-BR-3 tumour colonies were grown in the presence or absence of an endothelial barrier. We demonstrated that - in contrast to targeting FcγR on neutrophils with mAbs - targeting the immunoglobulin A Fc receptor (FcαRI) instead triggered neutrophil migration and degranulation leading to tumour destruction, which coincided with release of the pro-inflammatory cytokines interleukin (IL)-1ß and tumour necrosis factor (TNF)-α. Interestingly, neutrophil migration was enhanced in the presence of endothelial cells, which coincided with production of significant levels of the neutrophil chemokine IL-8. This supports the idea that stimulation of neutrophil FcαRI, but not FcγR, initiates cross-talk between neutrophils and endothelial cells, leading to enhanced neutrophil migration towards tumour colonies and subsequent tumour killing.
Asunto(s)
Antígenos CD/inmunología , Neoplasias de la Mama/inmunología , Quimiotaxis de Leucocito/inmunología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Neutrófilos/inmunología , Receptores Fc/inmunología , Neoplasias de la Mama/patología , Comunicación Celular/inmunología , Línea Celular Tumoral , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Inmunidad Innata/inmunología , Inmunohistoquímica , Interleucina-1beta/análisis , Interleucina-1beta/inmunología , Neutrófilos/citología , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Isotype plays a crucial role in therapeutic monoclonal antibody (mAb) function, mediated in large part through differences in Fcγ receptor (FcγR) interaction. Monoclonal Abs such as rituximab and alemtuzumab, which bind target cells directly, are designed for efficient recruitment of immune effector cells through their activatory FcγR engagement to mediate maximal target cell killing. In this setting, binding to inhibitory FcγRIIB is thought to inhibit function, making mAbs with high activatory/inhibitory (A/I) FcγR binding ratios, such as mouse IgG2a and human IgG1, the first choice for this role. In contrast, exciting new data show that agonistic mAbs directed against the tumour necrosis factor receptor superfamily member CD40 require interaction with FcγRIIB for in vivo function. Such ligation activates antigen-presenting cells, promotes myeloid and CTL responses and potentially stimulates effective anti-cancer immunity. It appears that the role of FcγRIIB is to mediate mAb hyper-crosslinking to allow CD40 downstream intracellular signalling. Previous work has shown that mAbs directed against other TNFR family members, Fas and death receptor 5 and probably death receptor 4, also require FcγRIIB hyper-crosslinking to promote target cell apoptosis, suggesting a common mechanism of action. In mouse models, IgG1 is optimal for these agents as it binds to FcγRIIB with tenfold higher affinity than IgG2a and hence has a relatively low A:I FcγR binding ratio. In contrast, human IgG isotypes have a universally low affinity for FcγRIIB, but in the case of human IgG1, engineering the Fc to increase its affinity for FcγRIIB can potentially overcome this problem. Thus, modifying the A/I binding ratio of human IgG Fc can be used to optimise different types of therapeutic activity by enhancing cytotoxic or hyper-crosslinking function.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Neoplasias/terapia , Receptores de IgG/inmunología , Receptores del Factor de Necrosis Tumoral/agonistas , Receptores del Factor de Necrosis Tumoral/inmunología , Animales , Apoptosis , Antígenos CD40/metabolismo , Humanos , Inmunoglobulina G/inmunología , Ratones , Neoplasias/inmunología , Unión Proteica , Transducción de SeñalRESUMEN
Attempts to generate robust anti-tumour cytotoxic T lymphocyte (CTL) responses using immunotherapy are frequently thwarted by exhaustion and anergy of CTL recruited to tumour. One strategy to overcome this is to retarget a population of virus-specific CTL to kill tumour cells. Here, we describe a proof-of-principle study using a bispecific conjugate designed to retarget ovalbumin (OVA)-specific CTL to kill tumour cells via CD20. A single-chain trimer (SCT) consisting of MHCI H-2K(b)/SIINFEKL peptide/beta 2 microglobulin/BirA was expressed in bacteria, refolded and chemically conjugated to one (1:1; F2) or two (2:1; F3) anti-hCD20 Fab' fragments. In vitro, the [SCT × Fab'] (F2 and F3) redirected SIINFEKL-specific OT-I CTL to kill CD20(+) target cells, and in the presence of CD20(+) target cells to provide crosslinking, they were also able to induce proliferation of OT-I cells. In vivo, activated OT-I CTL could be retargeted to kill [SCT × Fab']-coated B cells from hCD20 transgenic (hCD20 Tg) mice and also EL4 and B16 mouse tumour cells expressing human CD20 (hCD20). Importantly, in a hCD20 Tg mouse model, [SCT × Fab'] administered systemically were able to retarget activated OT-I cells to deplete normal B cells, and their performance matched that of a bispecific antibody (BsAb) comprising anti-CD3 and anti-CD20. [SCT × Fab'] were also active therapeutically in an EL4 tumour model. Furthermore, measurement of serum cytokine levels suggests that [SCT × Fab'] are associated with a lower level of inflammatory cytokine release than the BsAb and so may be advantageous clinically in terms of reduced toxicity.