Impact of isotype on the mechanism of action of agonist anti-OX40 antibodies in cancer: implications for therapeutic combinations.

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.

Inhibition of TNF Receptor p55 By a Domain Antibody Attenuates the Initial Phase of Acid-Induced Lung Injury in Mice.

BACKGROUND: Tumor necrosis factor-α (TNF) is strongly implicated in the development of acute respiratory distress syndrome (ARDS), but its potential as a therapeutic target has been hampered by its complex biology. TNF signals through two receptors, p55 and p75, which play differential roles in pulmonary edema formation during ARDS. We have recently shown that inhibition of p55 by a novel domain antibody (dAb™) attenuated ventilator-induced lung injury. In the current study, we explored the efficacy of this antibody in mouse models of acid-induced lung injury to investigate the longer consequences of treatment. METHODS: We employed two acid-induced injury models, an acute ventilated model and a resolving spontaneously breathing model. C57BL/6 mice were pretreated intratracheally or intranasally with p55-targeting dAb or non-targeting "dummy" dAb, 1 or 4 h before acid instillation. RESULTS: Acid instillation in the dummy dAb group caused hypoxemia, increased respiratory system elastance, pulmonary inflammation, and edema in both the ventilated and resolving models. Pretreatment with p55-targeting dAb significantly attenuated physiological markers of ARDS in both models. p55-targeting dAb also attenuated pulmonary inflammation in the ventilated model, with signs that altered cytokine production and leukocyte recruitment persisted beyond the very acute phase. CONCLUSION: These results demonstrate that the p55-targeting dAb attenuates lung injury and edema formation in models of ARDS induced by acid aspiration, with protection from a single dose lasting up to 24 h. Together with our previous data, the current study lends support toward the clinical targeting of p55 for patients with, or at risk of ARDS.

siRNA high-throughput kinase library screen identifies protein kinase, DNA-activated catalytic polypeptide to play a role in MyD88-induced IFNA2 activation and IL-8 secretion.

The discovery of RNA interference has led to the development of short interfering RNA (siRNA) screening, which has been widely used to study biological pathways. Here, we describe the development and validation of a system suitable to identify cellular genes involved in interferon A2 (IFNA2) promoter activation and interleukin (IL)-8 secretion downstream of MyD88. Forty genes were identified. Five genes were selected for further study. One gene, protein kinase, DNA-activated catalytic polypeptide (PRKDC), was confirmed to play a role in MyD88-induced IFNA2 activation and IL-8 secretion.

Selective inhibition of intra-alveolar p55 TNF receptor attenuates ventilator-induced lung injury.

BACKGROUND: Tumour necrosis factor (TNF) is upregulated in the alveolar space early in the course of ventilator-induced lung injury (VILI). Studies in genetically modified mice indicate that the two TNF receptors play opposing roles during injurious high-stretch mechanical ventilation, with p55 promoting but p75 preventing pulmonary oedema. AIM: To investigate the effects of selective inhibition of intra-alveolar p55 TNF receptor on pulmonary oedema and inflammation during ventilator-induced lung injury using a newly developed domain antibody. METHODS: Anaesthetised mice were ventilated with high tidal volume and given an intratracheal bolus of p55-specific domain antibody or anti-TNF monoclonal antibody ('pure' VILI model). As a model of enhanced inflammation, a subclinical dose of lipopolysaccharide (LPS) was included in the intratracheal antibody bolus (LPS+VILI model). Development of lung injury was assessed by respiratory mechanics and blood gases and protein levels in lavage fluid. Flow cytometry was used to determine leucocyte recruitment and alveolar macrophage activation, while lavage fluid cytokines were assessed by ELISA. RESULTS: The ventilation protocol produced deteriorations in respiratory mechanics and gas exchange with increased lavage fluid protein levels in the two models. The p55-specific domain antibody substantially attenuated all of these changes in the 'pure' VILI model, while anti-TNF antibody was ineffective. In the LPS+VILI model, p55 blockade prevented deteriorations in respiratory mechanics and oxygenation and significantly decreased neutrophil recruitment, expression of intercellular adhesion molecule 1 on alveolar macrophages, and interleukin 6 and monocyte chemotactic protein 1 levels in lavage fluid. CONCLUSIONS: Selective inhibition of intra-alveolar p55 TNF receptor signalling by domain antibodies may open new therapeutic approaches for ventilated patients with acute lung injury.

Mutations in the temperature-sensitive murine cytomegalovirus (MCMV) mutants tsm5 and tsm30: a study of genes involved in immune evasion, DNA packaging and processing, and DNA replication.

A murine cytomegalovirus (MCMV) temperature-sensitive (ts) mutant, tsm5, of the K181 (Birmingham) strain, showed approximately 10-fold and approximately 10,000-fold reductions in yields at the permissive (33 degrees C) and non-permissive temperature (40 degrees C), respectively. It did not replicate to detectable levels in any tissue of 1-week-old Balb/c mice for up to 21 days following i.p. inoculation with 4 x 10(3) pfu although it did replicate, albeit with considerably delayed kinetics, in SCID mice. tsm5 expressed all kinetic classes of transcript (immediate-early, early and late) both in vitro at the non-permissive temperature and in vivo. To identify mutations contributing to this phenotype, chimaeric viruses produced from overlapping cosmids generated from tsm5 and the Smith strain of MCMV were examined. A virus, Smith/tsm5DGIK, comprising the central conserved region of the tsm5 genome, was not attenuated at 33 or 37 degrees C but was ts at 40 degrees C, although not to the same extent as tsm5. In contrast to tsm5, this chimaeric virus replicated to similar levels as parental viruses in adult BALB/c mice. These results suggested that genes contributing to reduced replication at 33 degrees C and lack of replication in vivo are located at the ends of the tsm5 genome while those contributing to the ts phenotype are located in the central conserved region of the genome. Sequencing of some immune evasion genes known to be located at the 3' or 5' ends of the MCMV genome showed that no mutations were present in ORFs m04, m06, M33, M37, m38.5, m144, m152, or m157 although mutations were found in M27 (A658S) and M36Ex1 (V54I). tsm5 made few capsids at 40 degrees C and these lacked DNA. DNA synthesis was significantly reduced in tsm5-infected cells at 40 degrees C although DNA cleavage occurred with close to wt efficiency. Sequencing of the herpesvirus conserved cis-acting elements, pac1 and pac2, and genes involved in DNA packaging and cleavage located in the central core region of the genome identified few point mutations. Two were identified that alter the encoded protein in tsm5 ORFs M98 (P324S) and M56 (G439R). Furthermore, a point mutation (C890Y) was identified in M70, the primase. Another mutant, tsm30, which is also defective in DNA packaging and processing, has a point mutation in M52 (D494N). Thus, a number of mutations have been identified in tsm5 that suggests that it is defective in genes involved in immune evasion, DNA replication and DNA encapsidation.

Conserved features in the extracellular domain of human toll-like receptor 8 are essential for pH-dependent signaling.

Toll-like receptor (TLR) 8 has an important role in initiating immune responses to viral single-stranded RNA and the antiviral compound resiquimod. Together with TLR3, -7, and -9, it forms a subgroup of the TLRs that are localized intracellularly and signal in response to pathogen-derived nucleic acids. In this work, we have used site-directed mutagenesis to identify regions of the TLR8 extracellular domain that are required for stimulus-induced signal transduction. We have shown that a cysteinerich sequence predicted to form a loop projecting from the solenoidal ectodomain structure at leucine-rich repeat 8 is essential for signaling in response to both single-stranded RNA and resiquimod. A second region, centered on an aspartic acid residue in leucine-rich repeat 17, is also required for TLR8 function. The corresponding residue in TLR9 is known to be important for pH-dependent binding and signaling in response to unmethylated CpG DNA, suggesting that the TLR7/8/9 subgroups share a common signaling mechanism. We have also shown that TLR8 is localized predominantly in the endoplasmic reticulum but that signaling is completely abolished by an inhibitor of vesicle-type H+ ATPases. This indicates that TLR8 is present at low levels in an acidified compartment and that a lowered pH is required for receptor function. We propose that pH-dependent changes in the ligand facilitate activation of the receptor. The protonated form of resiquimod, a cell-permeable weak base, is likely to concentrate significantly (approximately 100x) in acidified compartments, and this may potentiate low affinity interactions with either the receptor or a specific binding protein.

High-frequency interferon-gamma-secreting splenocytes specific for murine cytomegalovirus immediate-early-1 (IE-1) peptide 168YPHFMPTNL176 are insufficient to provide complete protection from viral challenge.

Infection of Balb/c mice with murine cytomegalovirus (MCMV) has been used extensively as a model system with which to study host mechanisms of immunity to cytomegaloviruses. In this model, the cytotoxic T-lymphocyte (CTL) response crucial for clearing infected cells is dominated by CTLs specific for the MCMV nonapeptide 168YPHFMPTNL176 encoded by the immediate-early 1 (IE-1) gene. The intradermal injection of plasmid pcDNA89 encoding IE-1 has been shown to offer some protection from viral challenge. In the present studies, the protective efficacy of immunisation with pcDNA89 given by intradermal injection was compared with particle-mediated DNA delivery (PMDD) and contrasted with that induced by injection with the K181 MCMV strain and with temperature-sensitive mutants (tsm) derived from the K181 strain. Modest protection was afforded by pcDNA89 immunisation given by PMDD, but none was observed after intradermal injection. PMDD immunisation induced a frequency of 168YPHFMPTNL176-specific interferon-gamma (IFN-gamma)-secreting splenocytes, which was equivalent to that after K181 infection and significantly higher than tsm immunisation. Whereas tsm-immunised mice were completely protected from MCMV challenge, PMDD-immunised mice were only weakly protected. Tsm immunisation protected mice completely against challenge with natural isolates having sequence variation in the IE-1 nonapeptide, while PMDD-immunised mice were weakly protected from isolates encoding 168YPHFMPTNL176 and were not protected against isolates encoding 168YPHFMPPSL176 or 168YLDFMPPNL176. Thus, while IE-1-specific IFN-gamma-secreting splenocytes do contribute to immunity from MCMV challenge, their presence in isolation is insufficient to provide complete protection and they may not be involved in the protection observed against MCMV isolates having IE-1 sequence variation.

Characterization of 2 influenza A(H3N2) clinical isolates with reduced susceptibility to neuraminidase inhibitors due to mutations in the hemagglutinin gene.

Previous studies have shown that amino acid changes in the hemagglutinin (HA) gene of influenza viruses may result in decreased susceptibility to neuraminidase inhibitors (NAIs) in vitro. However, the emergence and characteristics of such HA variants in the clinical setting remain poorly studied. Herein, we report 2 influenza A(H3N2) isolates, from untreated patients, harboring an Arg229-->Ile substitution in the HA1 gene. The Ile229 variants were as sensitive as the Arg229 viruses to zanamivir and oseltamivir in neuroaminidase inhibition assays but were significantly less susceptible (by 60-140-fold) in cell-based assays. Although the Ile229 variants adsorbed less efficiently to Madin-Darby canine kidney (MDCK) cells in kinetic binding assays, they remained very sensitive to zanamivir in ferrets. Our study shows the importance of the HA1 229 residue in virus binding to MDCK cells and confirms the unreliability of cell-based assays in predicting the in vivo susceptibility of HA variants to NAIs.

Immunisation of Balb/c mice with severely attenuated murine cytomegalovirus mutants induces protective cellular and humoral immunity.

Previously, we showed that two temperature-sensitive mutants of murine cytomegalovirus (tsm5 and tsm30) expressed immediate-early (IE-1), early (E-1), and late (gB) phase genes in the tissues of immunocompetent Balb/c mice, yet failed to produce infectious progeny virus in any tissue at any time at 1-21 days post-infection. Mice inoculated intraperitoneally with tsm5 became latently infected, but this latent virus could not be reactivated as an infectious virus after immunosuppression, although all three transcripts were produced. Immunocompetent mice infected with tsm30 did not become latently infected. In the present study, immunodeficient SCID mice supported productive infection of both mutants, suggesting that low-level viral replication does occur in immunocompetent mice, but that it is limited by the host immune response. This is supported by the observation that immunocompetent mice were protected against virulent K181 challenge even after immunisation with as few as 40 pfu of mutant virus, whereas UV-inactivated mutant or K181 virus was not immunoprotective at doses of 40,000 pfu. Immunity induced by subcutaneous inoculation was also protective, whereas that induced by intragastric immunisation was not. Protection was lifelong (18 months). Although tsm5 induced high antibody titres, there was little evidence of an antibody response to tsm30. In contrast, a significant CD8(+) CTL response to the Balb/c immunodominant IE-1 nonapeptide (YPHFMPTNL) was elicited by both mutants, as determined by an interferon-gamma ELISPOT assay, although this response was lower than that induced by K181 infection. In addition, CTLs specific for m04 (YGPSLYRRF) and M84 (AYAGLFTPL) peptides could be detected at low frequency after K181, tsm5, and tsm30 immunisation. Such protective immunity did not prevent the challenge K181 virus from entering the latent state, but it appeared to reduce the frequency of reactivation.