FcγRIIB controls antibody-mediated target cell depletion by ITIM-independent mechanisms.

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.

Antibody Afucosylation Augments CD16-Mediated Serial Killing and IFNγ Secretion by Human Natural Killer Cells.

One mechanism by which monoclonal antibodies (mAb) help treat cancer or autoimmune disease is through triggering antibody-dependent cellular cytotoxicity (ADCC) via CD16 on Natural Killer (NK) cells. Afucosylation is known to increase the affinity of mAbs for CD16 on NK cells and here, we set out to assess how mAb afucosylation affects the dynamics of NK cell interactions, receptor expression and effector functions. An IgG1 version of a clinically important anti-CD20 mAb was compared to its afucosylated counterpart (anti-CD20-AF). Opsonization of CD20-expressing target cells, 721.221 or Daudi, with anti-CD20-AF increased NK cell cytotoxicity and IFNγ secretion, compared to anti-CD20. The afucosylated mAb also caused a more rapid and greater loss of CD16 from NK cell surfaces. Loss of CD16 has recently been shown to be important for NK cell detachment and sequential engagement of multiple target cells. Here, live-cell time-lapse microscopy of individual cell-cell interactions in an aqueous environment and a three-dimensional matrix, revealed that anti-CD20-AF induced more rapid killing of opsonized target cells. In addition, NK cells detached more quickly from target cells opsonized with anti-CD20-AF compared to anti-CD20, which increased engagement of multiple targets and enabled a greater proportion of NK cells to perform serial killing. Inhibition of CD16 shedding with TAPI-0 led to reduced detachment and serial killing. Thus, disassembly of the immune synapse caused by loss of cell surface CD16 is a factor determining the efficiency of ADCC and antibody afucosylation alters the dynamics of intercellular interactions to boost serial killing.

Novel Bispecific Domain Antibody to LRP6 Inhibits Wnt and R-spondin Ligand-Induced Wnt Signaling and Tumor Growth.

UNLABELLED: Aberrant WNT signaling is associated with the formation and growth of numerous human cancer types. The low-density lipoprotein receptor-related protein 6 (LRP6) is the least redundant component of the WNT receptor complex with two independent WNT ligand-binding sites. Using domain antibody (dAb) technology, a bispecific antibody (GSK3178022) to LRP6 was identified that is capable of blocking stimulation in the presence of a range of WNT and R-spondin (RSPO) ligands in vitro GSK3178022 was also efficacious in reducing WNT target gene expression in vivo, in both cancer cell line and patient-derived xenograft models, and delays tumor growth in a patient-derived RSPO fusion model of colorectal cancer. IMPLICATIONS: This article demonstrates the inhibition of a key oncogenic receptor, intractable to mAb inhibition due to multiple independent ligand interaction sites, using an innovative dAb approach. Mol Cancer Res; 14(9); 859-68. ©2016 AACR.

Pharmacokinetic and Pharmacodynamic Characterisation of an Anti-Mouse TNF Receptor 1 Domain Antibody Formatted for In Vivo Half-Life Extension.

Tumour Necrosis Factor-α (TNF-α) inhibition has been transformational in the treatment of patients with inflammatory disease, e.g. rheumatoid arthritis. Intriguingly, TNF-α signals through two receptors, TNFR1 and TNFR2, which have been associated with detrimental inflammatory and beneficial immune-regulatory processes, respectively. To investigate if selective TNFR1 inhibition might provide benefits over pan TNF-α inhibition, tools to investigate the potential impact of pharmacological intervention are needed. Receptor-deficient mice have been very insightful, but are not reversible and could distort receptor cross-talk, while inhibitory anti-TNFR1 monoclonal antibodies have a propensity to induce receptor agonism. Therefore, we set out to characterise a monovalent anti-TNFR1 domain antibody (dAb) formatted for in vivo use. The mouse TNFR1 antagonist (DMS5540) is a genetic fusion product of an anti-TNFR1 dAb with an albumin-binding dAb (AlbudAb). It bound mouse TNFR1, but not human TNFR1, and was an antagonist of TNF-α-mediated cytotoxicity in a L929 cell assay. Surprisingly, the dAb did not compete with TNF-α for TNFR1-binding. This was supported by additional data showing the anti-TNFR1 epitope mapped to a single residue in the first domain of TNFR1. Pharmacokinetic studies of DMS5540 in mice over three doses (0.1, 1.0 and 10 mg/kg) confirmed extended in vivo half-life, mediated by the AlbudAb, and demonstrated non-linear clearance of DMS5540. Target engagement was further confirmed by dose-dependent increases in total soluble TNFR1 levels. Functional in vivo activity was demonstrated in a mouse challenge study, where DMS5540 provided dose-dependent inhibition of serum IL-6 increases in response to bolus mouse TNF-α injections. Hence, DMS5540 is a potent mouse TNFR1 antagonist with in vivo pharmacokinetic and pharmacodynamic properties compatible with use in pre-clinical disease models and could provide a useful tool to dissect the individual contributions of TNFR1 and TNFR2 in homeostasis and disease.

Pharmacokinetic characteristics, pharmacodynamic effect and in vivo antiviral efficacy of liver-targeted interferon alpha.

Interferon alpha (IFNα) is used for the treatment of hepatitis B virus infection, and whilst efficacious, it is associated with multiple adverse events caused by systemic exposure to interferon. We therefore hypothesise that targeting IFN directly to the intended site of action in the liver would reduce exposure in blood and peripheral tissue and hence improve the safety and tolerability of IFNα therapy. Furthermore we investigated whether directing IFN to the reservoir of infection in the liver may improve antiviral efficacy by increasing local concentration in target organs and tissues. Our previous results show that the mIFNα2 fused to an ASGPR specific liver targeting antibody, DOM26h-196-61, results in a fusion protein which retains the activity of both fusion partners when measured in vitro. In vivo targeting of the liver by mIFNα2-DOM26h-196-61, hereafter referred to as targeted mIFNα2, was observed in microSPECT imaging studies in mice. In this study we show by pharmacokinetic analysis that antibody mediated liver-targeting results in increased uptake and exposure of targeted mIFNα2 in target tissues, and correspondingly reduced uptake and exposure in systemic circulation, clearance organs and non-target tissues. We also show that cytokine activity and antiviral activity of liver-targeted IFN is observed in vivo, but that, contrary to expectations, liver-targeting of mIFNα2 using ASGPR specific dAbs actually leads to a reduced pharmacodynamic effect in target organs and lower antiviral activity in vivo when compared to non-targeted mIFNα2-dAb fusions.

Methods for determining the PK parameters of AlbudAbs™ and of long serum half-life drugs made using the AlbudAb™ technology.

Increasing serum residence time of drugs by means of fusing them to albumin-binding domain antibodies (AlbudAbs™) has previously been documented. AlbudAbs™ provide a valuable method for increasing the efficacy of drugs by extending the time for which therapeutic levels of drug are present in the body and also for increasing the convenience to the patient by reducing the need for frequent dosing. Here, we describe methods that could be used preclinically to determine the suitability of drug-AlbudAbs™ for development. Particular focus is given to suggested in vivo study design which could enable the fitting of accurate PK parameters, assay methods for concentration determination of AlbudAbs™ in blood samples, and to the protocols used to fit PK parameters to AlbudAb™ concentration data. Whilst the examples cited here are focussed on the AlbudAb™ technology, similar methods could be used for assessing the success of other half-life extension technologies (drug Fc fusions, PEGylated drugs).

Pharmacodynamics of DT-IgG, a dual-targeting antibody against VEGF-EGFR, in tumor xenografted mice.

PURPOSE: DT-IgG is a fully humanized dual-target therapeutic antibody being developed to simultaneously target epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), important signaling molecules for tumor growth. The antitumor pharmacodynamics (PD) of DT-IgG was studied in nude mice bearing human tumor xenografts with different EGFR and VEGF expressions and K-ras oncogene status and compared with bevacizumab, cetuximab and bevacizumab + cetuximab. METHODS: Mice bearing human oral squamous cell carcinoma (Tu212), lung adenocarcinoma (A549), or colon cancer (GEO) subcutaneous xenografts were administered with the antibodies intraperitoneally (i.p.), and tumor volumes were measured versus time. Nonlinear mixed effects modeling (NONMEM) was used to study drug potencies (IC(50)) and variations in tumor growth. RESULTS: The PD models adequately described tumor responses for the antibody dose regimens. In vivo IC(50) values varied with EGFR and K-ras status. DT-IgG had a similar serum t (1/2) as cetuximab (~1.7 vs. 1.5 day), was more rapid than bevacizumab (~6 day), and had the largest apparent distribution volume (DT-IgG > cetuximab > bevacizumab). The efficacy of DT-IgG was comparable to bevacizumab despite lower serum concentrations, but was less than bevacizumab + cetuximab. CONCLUSIONS: A lower IC(50) of DT-IgG partially compensated for lower serum concentrations than bevacizumab and cetuximab, but may require higher doses for comparable efficacy as the combination. The model adequately predicted variations of tumor response at the DT-IgG doses tested and could be used for targeting specific tumor efficacies for future testing.

A dual-targeting antibody against EGFR-VEGF for lung and head and neck cancer treatment.

An antibody simultaneously targeting epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), two major tumor growth-driving machineries, may provide a novel effective strategy for optimizing tumor targeting and maximizing potential clinical benefits. Human domain antibodies selected against VEGF and EGFR were formatted into a fully human dual-targeting IgG (DT-IgG) to directly target both antigens in a single molecule. We evaluated the efficacy of DT-IgG in comparison with bevacizumab and cetuximab alone and in combination in the lung cancer cell line A549 (low EGFR expression and KRAS mutant) and the head and neck squamous cell carcinoma (HNSCC) cell line Tu212 (high EGFR expression and KRAS wild type) in vitro and in vivo. DT-IgG suppressed Tu212 and A549 cell growth, inhibited EGFR activation and induced apoptosis as effectively as cetuximab, and neutralized VEGF as effectively as bevacizumab. DT-IgG induced EGFR-dependent VEGF internalization, constituting a novel antiangiogenesis mechanism. In xenograft models with lung and head and neck cancer cell lines, DT-IgG displayed efficacy equivalent to bevacizumab in diminishing tumor growth despite its short serum half-life (36 hr in rats) and both agents may constitute preferable alternatives to cetuximab in KRAS-mutant tumors. Immunofluorescence staining revealed that localization of DT-IgG was similar to that of cetuximab, largely associated with EGFR+tumor cells. Our proof of principle study suggests a DT-IgG against EGFR and VEGF as an alternative therapeutic strategy with potentially enhanced clinical benefit.

Anti-serum albumin domain antibodies in the development of highly potent, efficacious and long-acting interferon.

Serum albumin-binding domain antibodies (AlbudAbs) have previously been shown to greatly extend the serum half-life of the interleukin-1 receptor antagonist IL-1ra. We have subsequently extended this approach to look at the in vitro activity, in vivo efficacy and pharmacokinetics of an agonist molecule, interferon (IFN)-alpha2b, fused to an AlbudAb. Here we describe this molecule and show that in this format AlbudAb half-life extension technology displays significant advantages in comparison with other methods of half-life extension, in particular genetic fusion to serum albumin. When compared directly IFN-alpha2b fused to an Albudab shows higher potency, increased serum half-life and greater efficacy than human serum albumin fused to IFN-alpha2b. AlbudAbs are therefore an ideal platform technology for creation of therapeutics with agonist activity and long serum half-lives.