RESUMEN
Antibodies protect from infection, underpin successful vaccines and elicit therapeutic responses in otherwise untreatable cancers and autoimmune conditions. The human IgG2 isotype displays a unique capacity to undergo disulfide shuffling in the hinge region, leading to modulation of its ability to drive target receptor signaling (agonism) in a variety of important immune receptors, through hitherto unexplained molecular mechanisms. To address the underlying process and reveal how hinge disulfide orientation affects agonistic activity, we generated a series of cysteine to serine exchange variants in the hinge region of the clinically relevant monoclonal antibody ChiLob7/4, directed against the key immune receptor CD40. We report how agonistic activity varies with disulfide pattern and is afforded by the presence of a disulfide crossover between F(ab) arms in the agonistic forms, independently of epitope, as observed in the determined crystallographic structures. This structural "switch" affects directly on antibody conformation and flexibility. Small-angle x-ray scattering and ensemble modeling demonstrated that the least flexible variants adopt the fewest conformations and evoke the highest levels of receptor agonism. This covalent change may be amenable for broad implementation to modulate receptor signaling in an epitope-independent manner in future therapeutics.
Asunto(s)
Disulfuros , Inmunoglobulina G , Anticuerpos Monoclonales , Disulfuros/química , Epítopos , Humanos , Conformación ProteicaRESUMEN
Since the approval of Rituximab in the late 1990s, the first chimeric monoclonal antibody for the treatment of non-Hodgkin lymphoma, antibody engineering for cancer immunotherapy has become a rapidly growing field, with almost 50 antibody therapeutics approved in the USA and EU and hundreds undergoing testing in clinical trials [...].
RESUMEN
Anti-CD40 monoclonal antibodies (mAbs) comprise agonists and antagonists, which display promising therapeutic activities in cancer and autoimmunity, respectively. We previously showed that epitope and isotype interact to deliver optimal agonistic anti-CD40 mAbs. The impact of Fc engineering on antagonists, however, remains largely unexplored. Here, we show that clinically relevant antagonists used for treating autoimmune conditions can be converted into potent FcγR-independent agonists with remarkable antitumor activity by isotype switching to hIgG2. One antagonist is converted to a super-agonist with greater potency than previously reported highly agonistic anti-CD40 mAbs. Such conversion is dependent on the unique disulfide bonding properties of the hIgG2 hinge. This investigation highlights the transformative capacity of the hIgG2 isotype for converting antagonists to agonists to treat cancer.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos CD40/inmunología , Ligando de CD40/inmunología , Células Dendríticas/inmunología , Cambio de Clase de Inmunoglobulina/inmunología , Inmunoglobulina G/inmunología , Neoplasias/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/inmunología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células Dendríticas/efectos de los fármacos , Cambio de Clase de Inmunoglobulina/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Receptores de IgE/fisiología , Receptores de IgG/fisiología , Neoplasias del Timo/tratamiento farmacológico , Neoplasias del Timo/inmunología , Neoplasias del Timo/metabolismo , Neoplasias del Timo/patologíaRESUMEN
Anti-CD40 monoclonal antibodies (mAbs) that promote or inhibit receptor function hold promise as therapeutics for cancer and autoimmunity. Rules governing their diverse range of functions, however, are lacking. Here we determined characteristics of nine hCD40 mAbs engaging epitopes throughout the CD40 extracellular region expressed as varying isotypes. All mAb formats were strong agonists when hyper-crosslinked; however, only those binding the membrane-distal cysteine-rich domain 1 (CRD1) retained agonistic activity with physiological Fc gamma receptor crosslinking or as human immunoglobulin G2 isotype; agonistic activity decreased as epitopes drew closer to the membrane. In addition, all CRD2-4 binding mAbs blocked CD40 ligand interaction and were potent antagonists. Thus, the membrane distal CRD1 provides a region of choice for selecting CD40 agonists while CRD2-4 provides antagonistic epitopes.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos CD40/química , Antígenos CD40/metabolismo , Epítopos/química , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Antígenos CD40/agonistas , Ligando de CD40/metabolismo , Reactivos de Enlaces Cruzados , Humanos , Modelos Moleculares , Unión Proteica/efectos de los fármacosRESUMEN
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
Monoclonal antibody (mAb) drugs that stimulate antitumor immunity are transforming cancer treatment but require optimization for maximum clinical impact. Here, we show that, unlike other immunoglobulin isotypes, human IgG2 (h2) imparts FcγR-independent agonistic activity to immune-stimulatory mAbs such as anti-CD40, -4-1BB, and -CD28. Activity is provided by a subfraction of h2, h2B, that is structurally constrained due its unique arrangement of hinge region disulfide bonds. Agonistic activity can be transferred from h2 to h1 by swapping their hinge and CH1 domains, and substitution of key hinge and CH1 cysteines generates homogenous h2 variants with distinct agonistic properties. This provides the exciting opportunity to engineer clinical reagents with defined therapeutic activity regardless of FcγR expression levels in the local microenvironment.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/uso terapéutico , Inmunoglobulina G/química , Inmunoglobulina G/uso terapéutico , Receptores de IgG/inmunología , Timoma/prevención & control , Neoplasias del Timo/prevención & control , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD28/inmunología , Antígenos CD40/inmunología , Células Cultivadas , Humanos , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos C57BL , Timoma/tratamiento farmacológico , Timoma/inmunología , Neoplasias del Timo/tratamiento farmacológico , Neoplasias del Timo/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Vacunación/métodosRESUMEN
Fc gamma Receptor (FcγR) IIB (CD32B) is an immunoreceptor tyrosine inhibitory motif (ITIM)-bearing Fc receptor that is involved in abrogating the signalling and function delivered from other receptors; archetypally those arising from other, activatory, FcγR and from the B cell receptor (BCR) for antigen. In the context of immunotherapy, it has convincingly been shown to limit a variety of clinically important therapeutic monoclonal antibodies (mAb) such as rituximab and trastuzumab in preclinical models. However, recent exploration of so-called immunomodulatory mAb, for example agonist mAb directed against various members of the TNFR super-family, has cast new light on the ability of FcγRIIB to regulate immune responses and immunotherapy. These data, accumulated by several independent groups, have shown the seemingly paradoxical ability of FcγRIIB to augment or even be absolutely required for the activity of this class of mAb. In this review we highlight the key role of FcγRIIB in regulating agonistic mAb, detail the likely mechanism of action and propose new ways in which this information may be exploited therapeutically.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Receptores de IgG/fisiología , Animales , Humanos , InmunoterapiaRESUMEN
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
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
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.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino , Antineoplásicos , Antígenos CD40/inmunología , Recubrimiento Inmunológico/efectos de los fármacos , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/farmacocinética , Anticuerpos Monoclonales de Origen Murino/farmacología , Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Recubrimiento Inmunológico/inmunología , Ratones , Ratones Noqueados , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
In this study, we investigated the mouse dendritic cell (DC) receptor, complement receptor 4 (CR4; CD11c/CD18), as an immunotarget for triggering humoral immunity. Comparison of antibody titres generated against a panel of 13 anti-antigen-presenting cell receptor monoclonal antibodies, with or without conjugated ovalbumin (OVA), revealed uniquely rapid and robust responses following CR4 targeting, with antibody titres approaching 1 : 100 000 7 days after a single dose of antigen. Furthermore, using just 100 ng OVA conjugated to anti-CD11c Fab', we generated anti-OVA titres greater than those produced by a 100-fold higher dose of OVA in complete Freund's adjuvant at day 28. These anti-OVA antibody titres were sustained and could be boosted further with targeted OVA on day 21. Investigations to explain this vaccine potency showed that, in addition to targeting splenic DC, anti-CDl1c antibodies delivered a powerful adjuvant effect and could boost humoral immunity against OVA even when the OVA was targeted to other molecules on DC, such as major histocompatibility complex class II, CD11a and CD11b. However, interestingly, this adjuvant effect was lost if OVA was targeted to other cells such as B cells via CD21 or CD19. The adjuvant effect was mediated through a marked enhancement of both germinal centre and extrafollicular plasma cell formation in responding spleens. These results demonstrate that anti-CD11c monoclonal antibody can both target antigen and act as a powerful adjuvant for rapid and sustained antibody responses. They also point to an interesting role for CR4 on DC in triggering B cells during humoral immunity.
Asunto(s)
Formación de Anticuerpos/inmunología , Antígeno CD11c/inmunología , Antígeno CD11c/metabolismo , Centro Germinal/inmunología , Adyuvantes Inmunológicos , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Células Dendríticas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , VacunaciónRESUMEN
The magnitude and quality of T cell responses generated when Ag is targeted to receptors on DC is influenced by both the specific receptor targeted and its distribution among DC subsets. Here we examine the targeting of the model Ag OVA to potential DC targets, including CD11c, CD205, MHC class II, CD40, TLR2 and FcgammaRII/III, using a panel of (Fab' x OVA) conjugates. In vitro studies identified CD11c, CD205 and MHC class II as superior and comparably effective immunotargets for the delivery of OVA to APC for presentation to T cells. In vivo studies, however, showed a marked advantage of targeting Ag to CD11c for both CD4 (OT-II) and CD8 (OT-I) responses, with robust stimulation after a single, low dose (equivalent to 0.5 microg OVA); in contrast, (anti-CD205 x OVA) and (anti-MHC class II x OVA) resulted in markedly less proliferation of both OT-I and OT-II cells. Biodistribution and immunohistochemical studies suggest that the exceptional ability of CD11c to capture Ag in lymphoid tissues may, at least partially, explain its ability to promote T cell responses. These results suggest that targeting antigen via CD11c offers a previously unappreciated strategy for vaccine development which, unlike most targets, delivers robust responses of both CD4 and CD8 T cells.
Asunto(s)
Antígeno CD11c/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Animales , Presentación de Antígeno , Antígenos CD/fisiología , Inmunización , Lectinas Tipo C/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor , Ovalbúmina/inmunología , Receptores de Superficie Celular/fisiología , Bazo/inmunologíaRESUMEN
Human hepatic lipase (hHL) mainly exists cell surface bound, whereas mouse HL (mHL) circulates in the blood stream. Studies have suggested that the carboxyl terminus of HL mediates cell surface binding. We prepared recombinant hHL, mHL, and chimeric proteins (hHLmt and mHLht) in which the carboxyl terminal 70 amino acids of hHL were exchanged with the corresponding sequence from mHL. The hHL, mHL, and hHLmt proteins were catalytically active using triolein and tributyrin as substrates. In transfected cells, the majority of hHLs bound to the cell surface, with only 4% of total extracellular hHL released into heparin-free media, whereas under the same conditions, 61% of total extracellular mHLs were released. Like mHL, hHLmt showed decreased cell surface binding, with 68% of total extracellular hHLmt released. To determine the precise amino acid residues involved in cell surface binding, we prepared a truncated hHL mutant (hHL471) by deleting the carboxyl terminal five residues (KRKIR). The hHL471 also retained hydrolytic activity with triolein and tributyrin, and showed decreased cell surface binding, with 40% of total extracellular protein released into the heparin-free media. These data suggest that the determinants of cell surface binding exist within the carboxyl terminal 70 amino acids of hHL, of which the last five residues play an important role.