Challenges and the Evolving Landscape of Assessing Blood-Based PD-L1 Expression as a Biomarker for Anti-PD-(L)1 Immunotherapy.

While promising, PD-L1 expression on tumor tissues as assessed by immunohistochemistry has been shown to be an imperfect biomarker that only applies to a limited number of cancers, whereas many patients with PD-L1-negative tumors still respond to anti-PD-(L)1 immunotherapy. Recent studies using patient blood samples to assess immunotherapeutic responsiveness suggests a promising approach to the identification of novel and/or improved biomarkers for anti-PD-(L)1 immunotherapy. In this review, we discuss the advances in our evolving understanding of the regulation and function of PD-L1 expression, which is the foundation for developing blood-based PD-L1 as a biomarker for anti-PD-(L)1 immunotherapy. We further discuss current knowledge and clinical study results for biomarker identification using PD-L1 expression on tumor and immune cells, exosomes, and soluble forms of PD-L1 in the peripheral blood. Finally, we discuss key challenges for the successful development of the potential use of blood-based PD-L1 as a biomarker for anti-PD-(L)1 immunotherapy.

IL-10 Signaling Elicited by Nivolumab-Induced Activation of the MAP Kinase Pathway Does Not Fully Contribute to Nivolumab-Modulated Heterogeneous T Cell Responses.

Immune checkpoint inhibitor (ICI) therapy has revolutionized anti-cancer treatment for many late-stage cancer patients. However, ICI therapy has thus far demonstrated limited efficacy for most patients, and it remains unclear why this is so. Interleukin 10 (IL-10) is a cytokine that has been recognized as a central player in cancer biology with its ability to inhibit anti-tumor T cell responses. Recent studies suggest that IL-10 might also exert some intrinsic anti-tumor T cell responses, and clinical studies using recombinant IL-10 alone or in combination with ICI are underway. This paradoxical effect of IL-10 and its underlying mechanisms impacting ICI-modulated T cell responses remain poorly understood. In this study, using an in vitro mixed lymphocyte reaction assay, we found that treatment with ICIs such as the anti-programmed cell death receptor-1 (PD-1) mAb nivolumab elicits a strong expression of IL-10. While neutralization of IL-10 signaling with an anti-IL-10 specific mAb significantly decreases the production of IFN-γ by T cells in a cohort of donor cells, the opposite effect was observed in other donor cells. Similarly, neutralization of IL-10 signaling significantly decreases the expression of T cell activation markers Ki67 and CD25, as well as the production of Granzyme B in a cohort of donor cells, whereas the opposite effect was observed in others. Furthermore, we found that nivolumab and IL-10 differentially modulate the signal transducer and activator of transcription 3 (STAT3) and AKT serine-threonine kinase pathways. Finally, we found that nivolumab activates the mitogen-activated protein kinase (MAPK) pathway, which in turn is responsible for the observed induction of IL-10 production by nivolumab. These findings provide new insights into the mechanisms underlying anti-PD-1-modulated T cell responses by IL-10, which could lead to the discovery of novel combination treatments that target IL-10 and immune checkpoint molecules.

Exploring the Potential Use of a PBMC-Based Functional Assay to Identify Predictive Biomarkers for Anti-PD-1 Immunotherapy.

The absence of reliable, robust, and non-invasive biomarkers for anti- Programmed cell death protein 1 (PD-1) immunotherapy is an urgent unmet medical need for the treatment of cancer patients. No predictive biomarkers have been established based on the direct assessment of T cell functions, the primary mechanism of action of anti-PD-1 therapy. In this study, we established a model system to test T cell functions modulated by Nivolumab using anti-CD3 monoclonal antibody (mAb)-stimulated peripheral blood mononuclear cells (PBMCs), and characterized T cell functions primarily based on the knowledge gained from retrospective observations of patients treated with anti-PD-1 immunotherapy. During a comprehensive cytokine profile assessment to identify potential biomarkers, we found that Nivolumab increases expression of T helper type 1 (Th1) associated cytokines such as interferon-γ (IFN-γ) and interleukin-2 (IL-2) in a subset of donors. Furthermore, Nivolumab increases production of Th2, Th9, and Th17 associated cytokines, as well as many proinflammatory cytokines such as IL-6 in a subset of donors. Conversely, Nivolumab treatment has no impact on T cell proliferation, expression of CD25, CD69, or Granzyme B, and only modestly increases in the expansion of regulatory T cells. Our results suggest that assessment of cytokine production using a simple PBMC-based T cell functional assay could be used as a potential predictive marker for anti-PD-1 immunotherapy.

Defining the right diluent for intravenous infusion of therapeutic antibodies.

Therapeutic monoclonal antibodies (mAbs) are commonly administered to patients through intravenous (IV) infusion, which involves diluting the medication into an infusion solution (e.g., saline and 5% dextrose). Using the wrong diluent can cause product aggregation, which may compromise patient safety. We and others have shown that Herceptin® (trastuzumab) and Avastin® (bevacizumab) undergo rapid aggregation upon mixing with dextrose and human plasma in vitro. In this study, we evaluated the compatibility of a panel of 11 therapeutic mAbs with dextrose or saline and human serum. These mAbs were randomly selected for their distinct formulations and IgG isotypes (IgG1, IgG2, IgG4, and Fc-fusion protein). All the mAbs appeared to be compatible with saline and human serum. However, mAbs that were formulated at acidic pH (≤ 6.5) exclusively formed insoluble aggregates upon mixing with dextrose and serum. Such aggregation was not detected for the mAbs that are at neutral pH (7.2-7.5) or in buffers containing sodium chloride. Mass spectrometric analysis revealed that the insoluble aggregates were composed of mAb molecules and several serum proteins (e.g., complement proteins, apolipoprotein, fibronectin) that are characterized by an isoelectric point of pH 5.4-6.7. At proximate pH to the isoelectric point values, those abundant serum proteins appeared to undergo isoelectric precipitation with mAb molecules. Our observations highlight a potential risk of protein aggregation at the blood-IV interface if a diluent is incompatible with a specific mAb formulation. This information has implications in guiding the design of product formulations and the selection of the right diluent for intravenous infusion of therapeutic mAbs.Abbreviations: ADC: antibody-drug conjugate; D5W: 5% dextrose in water; IM: intramuscular; IV: intravenous; LC-MS/MS: liquid chromatography-tandem mass spectrometry; mAb: monoclonal antibody; SC: subcutaneous; pI: isoelectric point.

BRAF and MEK inhibitors differentially affect nivolumab-induced T cell activation by modulating the TCR and AKT signaling pathways.

Immune checkpoint inhibitors (ICIs) such as the anti-PD-1 antibody Nivolumab, achieve remarkable clinical efficacy in patients with late stage cancers. However, only a small subset of patients benefit from this therapy. Numerous clinical trials are underway testing whether combining ICIs with other anti-cancer therapies can increase this response rate. For example, anti-PD-1/PD-L1 therapy combined with MAP kinase inhibition using BRAF inhibitors (BRAFi) and/or MEK inhibitors (MEKi) are in development for treatment of late stage melanomas. However, the benefits and underlying mechanisms of these combinatorial therapies remain unclear. In the current study, we assess the effects of MAPK inhibition on Nivolumab-induced T cell responses. Using an in vitro mixed lymphocyte reaction assay, we demonstrate that Nivolumab-induced T cell activation is highly heterogeneous. While BRAFi inhibits Nivolumab-induced cytokine production, T cell proliferation, activation markers (CD69, CD25), and Granzyme B in a substantial proportion of donor pairs, a small subset of donor pairs shows an additive effect. MEKi alone significantly inhibits Nivolumab-induced T cell activation; the addition of BRAFi significantly enhances this inhibitory effect. Mechanistically, the effects of BRAFi and/or MEKi on Nivolumab-induced T cell activation may be due to alteration of the activation of the AKT and T cell receptor (TCR) signaling pathways. Our results suggest that MAPK inhibition may not provide a clinical benefit for most melanoma patients being treated with anti-PD-1 therapy.

Pathogenic prion protein fragment (PrP106-126) promotes human immunodeficiency virus type-1 infection in peripheral blood monocyte-derived macrophages.

Transfusion of blood and blood products contaminated with the pathogenic form of prion protein Prp(sc), thought to be the causative agent of variant a Creutzfeldt-Jakob disease (vCJD), may result in serious consequences in recipients with a compromised immune system, for example, as seen in HIV-1 infection. In the present study, we demonstrate that treatment of peripheral blood monocyte-derived macrophages (MDM) with PrP106-126, a synthetic domain of PrP(sc) that has intrinsic functional activities related to the full-length protein, markedly increased their susceptibility to HIV-1 infection, induced cytokine secretion, and enhanced their migratory behavior in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Live-cell imaging of MDM cultured in the presence of PrP106-126 showed large cell clusters indicative of cellular activation. Tyrosine kinase inhibitor STI-571, protein kinase C inhibitor K252B, and cyclin-dependent kinase inhibitor olomoucine attenuated PrP106-126-induced altered MDM functions. These findings delineate a previously undefined functional role of PrP106-126-mediated host cell response in promoting HIV-1 pathogenesis.

Annexin A2 tetramer activates human and murine macrophages through TLR4.

Annexins are a large family of intracellular phospholipid-binding proteins, yet several extracellular roles have been identified. Specifically, annexin A2, found in a heterotetrameric complex with S100A10, not only serves as a key extracellular binding partner for pathogens and host proteins alike, but also can be shed or secreted. We reported previously that soluble annexin A2 tetramer (A2t) activates human monocyte-derived macrophages (MDM), resulting in secretion of inflammatory mediators and enhanced phagocytosis. Although a receptor for A2t has been cloned from bone marrow stromal cells, data contained in this study demonstrate that it is dispensable for A2t-dependent activation of MDM. Furthermore, A2t activates wild-type murine bone marrow-derived macrophages, whereas macrophages from myeloid differentiation factor 88-deficient mice display a blunted response, suggesting a role for Toll-like receptor (TLR) signaling. Small interfering RNA knockdown of TLR4 in human MDM reduced the response to A2t, blocking antibodies against TLR4 (but not TLR2) blocked activation altogether, and bone marrow-derived macrophages from TLR4(-/-) mice were refractory to A2t. These data demonstrate that the modulation of macrophage function by A2t is mediated through TLR4, suggesting a previously unknown, but important role for this stress-sensitive protein in the detection of danger to the host, whether from injury or invasion.

Activation by prion peptide PrP106-126 induces a NF-kappaB-driven proinflammatory response in human monocyte-derived dendritic cells.

Specific prion peptides have been shown to mimic the pathologic isoform of the prion protein (PrP) and to induce a neurotoxic effect in vitro and in vivo. As monocytic cells are thought to play a role in the transmission and pathogenesis of prion disease, the use of these peptides in regulating monocytic cell function is under intense investigation. In the current study, we characterize the ability of prion peptide PrP(106-126) to activate specific signaling pathways in human monocyte-derived dendritic cells (DCs). Electrophoretic mobility shift assays establish the activation of transcription factor nuclear factor-kappaB within 15 min of exposure, with as little as 25 micro M peptide. This signaling cascade results in the up-regulation of inflammatory cytokines interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) at the mRNA and protein levels. Phenotypic activation of DCs exposed to PrP(106-126) is partly a result of an autocrine TNF-alpha response and results in an increased ability of these cells to induce lymphocyte proliferation. The effects of PrP(106-126) on DCs were elicited through a receptor complex distinct from that used by human monocytes, demonstrating the ability of this peptide to interact with a multiplicity of receptors on various cell types. Together, these data suggest an involvement of DCs in prion disease pathogenesis.

Interaction of papillomavirus virus-like particles with human myeloid antigen-presenting cells.

Papillomavirus-like particles (VLPs) are potent inducers of humoral and cellular immune responses, making them attractive candidates for noninfectious viral subunit vaccines. To further our understanding of how VLPs activate the immune system, we have investigated their interaction with human myeloid antigen-presenting cells. We found that VLPs bound, with increasing density, to the cell surface of human monocytes, macrophages, and monocyte-derived dendritic cells (DCs). Interestingly, there was a negative correlation between binding intensity and CD83 expression in DCs, suggesting that the main receptor for binding of VLPs may be downregulated during maturation. Exposure to VLPs resulted in acute phenotypic activation of monocytes and DCs. Furthermore, VLPs rapidly induced production of inflammatory cytokines in monocytes, macrophages, and DCs, as assessed by intracellular cytokine staining. For each cell type, the patterns of interleukin-1beta, interleukin-12, tumor necrosis factor-alpha, and interleukin-6 production were distinct from the pattern induced by lipopolysaccharide (LPS), a bacterial activator of myeloid antigen-presenting cells. Our results indicate that VLPs target multiple cells of the immune system, which helps to account for VLPs being so effective in priming humoral and cellular immune responses even in the absence of adjuvant.

Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells.

Dendritic cells (DCs) are ideal accessory cells in the developing field of gene therapy. Although viral transfection of DCs has become widespread, non-viral transfection of DCs has shown disappointing results. Recently, a new technique for transfecting primary cells has become available -- the Amaxa Nucleofector. Here, we describe the use of this device in the successful non-viral transfection of human monocyte-derived DCs. Using enhanced green fluorescent protein as a reporter gene DCs were transfectable with efficiencies approaching 60%, remaining responsive to lipopolysaccharide-stimulated cytokine production in short-term experiments (though long-term functional assays were hampered by loss of viability). Although these data demonstrate the ease and efficiency with which human monocyte-derived DCs can now be non-virally transfected, they also suggest the limitations of this technology due to the gradual loss of cell viability. The potential use of this system in the development of DC-based cell and gene therapies will be hampered until cell viability can be maintained.