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Antibody responses during infection and vaccination typically undergo affinity maturation to achieve high-affinity binding for efficient neutralization of pathogens 1 , 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. In contrast to direct-targeting monoclonal antibodies, low affinity confers agonistic monoclonal antibodies with more potency.

A promising strategy in cancer immunotherapy is activation of immune signalling pathways through antibodies that target co-stimulatory receptors. h IgG2, one of four human antibody isotypes, is known to deliver strong agonistic activity, and modification of h IgG2 hinge disulfides can influence immune-stimulating activity. This was shown for antibodies directed against the h CD40 receptor, where cysteine-to-serine exchange mutations caused changes in antibody conformational flexibility. Here we demonstrate that the principles of increasing agonism by restricting antibody conformation through disulfide modification can be translated to the co-stimulatory receptor h 4-1BB, another member of the tumour necrosis factor receptor superfamily. Furthermore, we explore structure-guided design of the anti- h CD40 antibody ChiLob7/4 and show that engineering additional disulfides between opposing F(ab’) arms can elicit conformational restriction, concomitant with enhanced agonism. These results support a mode where subtle increases in rigidity can deliver significant improvements in immunostimulatory activity, thus providing a strategy for the rational design of more powerful antibody therapeutics. Immunostimulatory antibodies have strong potential as anti-cancer therapeutics. Here Elliott et al. show that conformational rigidity determines agonistic activity of antibodies targeting CD40 and 4-1BB and demonstrate further enhancement of agonism through disulfide engineering.

BackgroundDespite 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.MethodsMouse 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.ResultsEngagement 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.ConclusionsOur 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.

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.Yu et al examined a panel of both experimental and clinically-relevant TNF agonists in order to advance our understanding of the mechanisms underlying their varying activities and anti-tumor responses. They demonstrated that agonists with greater activity induced higher density receptor clustering and specific super-structures as opposed to simply larger receptor clusters.

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.

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. 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). Whereas tCD70 failed to costimulate CD8 T cells, both dtCD70-Fc and an agonist anti-CD27 antibody were capable of enhancing T cell proliferation . Initial studies demonstrated that dtCD70-Fc was less efficacious than anti-CD27 in boosting a CD8 T cell vaccine response , 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 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 . 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. 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.

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. Agonistic CD27 monoclonal antibodies can be used to enhance the efficacy of depleting antibodies such as anti-CTLA-4 in a colon adenocarcinoma tumour model. CD27 antibody agonism is dependent on isotype and epitope specificity.

We searched for cell-surface-associated proteins overexpressed on B cell chronic lymphocytic leukemia (CLL) to use as therapeutic antibody targets. Antibodies binding the immunosuppressive molecule CD200 were identified by cell panning of an antibody phage display library derived from rabbits immunized with primary CLL cells. B cells from 87 CLL patients exhibited 1.6- to 5.4-fold cellsurface up-regulation of CD200 relative to normal B cells. An effect of increased CD200 expression by CLL cells on the immune system was evaluated in mixed lymphocyte reactions. Addition of primary CLL but not normal B cells to macrophages and T cells down-regulated the Th1 response, as seen by a 50-95% reduction in secreted IL-2 and IFN-γ. Antibodies to CD200 prevented downregulation of the Th1 response in most B cell CLL samples evaluated, indicating abrogation of the CD200/CD200R interaction can be sufficient to restore the Th1 response. A disease-progression-associated shift of the immune response from Th1 to Th2 has been observed in numerous cancers. Because this cytokine shift is also believed to promote the induction of regulatory T cells, reverting the immune response to Th1 through direct targeting of the cancer cells may provide therapeutic benefits in CLL by encouraging a cytotoxic T cell response.

BackgroundPrevious data suggests that anti-OX40 mAb can elicit anti-tumor effects in mice through deletion of Tregs. However, OX40 also has powerful costimulatory effects on T cells which could evoke therapeutic responses. Human trials with anti-OX40 antibodies have shown that these entities are well tolerated but to date have delivered disappointing clinical responses, indicating that the rules for the optimal use of anti-human OX40 (hOX40) antibodies is not yet fully understood. Changes to timing and dosages may lead to improved outcomes; however, here we focus on addressing the role of agonism versus depleting activity in determining therapeutic outcomes. We investigated a novel panel of anti-hOX40 mAb to understand how these reagents and mechanisms may be optimized for therapeutic benefit.MethodsThis study examines the binding activity and in vitro activity of a panel of anti-hOX40 antibodies. They were further evaluated in several in vivo models to address how isotype and epitope determine mechanism of action and efficacy of anti-hOX40 mAb.ResultsBinding analysis revealed the antibodies to be high affinity, with epitopes spanning all four cysteine-rich domains of the OX40 extracellular domain. In vivo analysis showed that their activities relate directly to two key properties: (1) isotype—with mIgG1 mAb evoking receptor agonism and CD8+ T-cell expansion and mIgG2a mAb evoking deletion of Treg and (2) epitope—with membrane-proximal mAb delivering more powerful agonism. Intriguingly, both isotypes acted therapeutically in tumor models by engaging these different mechanisms.ConclusionThese findings highlight the significant impact of isotype and epitope on the modulation of anti-hOX40 mAb therapy, and indicate that CD8+ T-cell expansion or Treg depletion might be preferred according to the composition of different tumors. As many of the current clinical trials using OX40 antibodies are now using combination therapies, this understanding of how to manipulate therapeutic activity will be vital in directing new combinations that are more likely to improve efficacy and clinical outcomes.

BackgroundImmune compromised mice are increasingly used for the preclinical development of monoclonal antibodies (mAb). Most common are non-obese diabetic (NOD) severe combined immunodeficient (SCID) and their derivatives such as NOD SCID interleukin-2 γ-/- (NSG), which are attractive hosts for patient-derived xenografts. Despite their widespread use, the relative biological performance of mAb in these strains has not been extensively studied.MethodsClinically relevant mAb of various isotypes were administered to tumor and non-tumor-bearing SCID and NOD SCID mice and the mAb clearance monitored by ELISA. Expression analysis of surface proteins in both strains was carried out by flow cytometry and immunofluorescence microscopy. Further analysis was performed in vitro by surface plasmon resonance to assess mAb affinity for Fcγ receptors (FcγR) at pH 6 and pH 7.4. NOD SCID mice genetically deficient in different FcγR were used to delineate their involvement.ResultsHere, we show that strains on the NOD SCID background have significantly faster antibody clearance than other strains leading to reduced antitumor efficacy of clinically relevant mAb. This rapid clearance is dependent on antibody isotype, the presence of Fc glycosylation (at N297) and expression of FcγRII. Comparable effects were not seen in the parental NOD or SCID strains, demonstrating the presence of a compound defect requiring both genotypes. The absence of endogenous IgG was the key parameter transferred from the SCID as reconstituting NOD SCID or NSG mice with exogenous IgG overcame the rapid clearance and recovered antitumor efficacy. In contrast, the NOD strain was associated with reduced expression of the neonatal Fc Receptor (FcRn). We propose a novel mechanism for the rapid clearance of certain mAb isotypes in NOD SCID mouse strains, based on their interaction with FcγRII in the context of reduced FcRn.ConclusionsThis study highlights the importance of understanding the limitation of the mouse strain being used for preclinical evaluation, and demonstrates that NOD SCID strains of mice should be reconstituted with IgG prior to studies of mAb efficacy.