MALT1 inhibition suppresses antigen-specific T cell responses.

The aim of this study was to assess the potential use of a selective small molecule MALT1 inhibitor in solid tumor treatment as an immunotherapy targeting regulatory T-cells (Tregs). In vitro, MALT1 inhibition suppressed the proteolytic cleavage of the MALT1-substrate HOIL1 and blocked IL-2 secretion in Jurkat cells. It selectively suppressed the proliferation of PBMC-derived Tregs, with no effect on conventional CD4+T-cells. In vivo, however, no evident anti-tumor effect was achieved by MALT1 inhibition monotherapy or in combination with anti-CTLA4 in the MB49 cancer model. Despite decreased Treg-frequencies in lymph nodes of tumor-bearing animals, intratumoral Treg depletion was not observed. We also showed that MALT1-inhibition caused a reduction of antigen-specific CD8+T-cells in an adoptive T-cell transfer model. Thus, selective targeting of Tregs would be required to improve the immunotherapeutic effect of MALT1-inhibition. Also, various dosing schedules and combination therapy strategies should be carefully designed and evaluated further.

Formation of Immune Complexes with a Tetanus-Derived B Cell Epitope Boosts Human T Cell Responses to Covalently Linked Peptides in an Ex Vivo Blood Loop System.

Enhancing T cell responses against both viral and tumor Ags requires efficient costimulation and directed delivery of peptide Ags into APCs. Long peptide vaccines are considered favorable vaccine moieties from a clinical perspective, as they can harbor more than one immunogenic epitope enabling treatment of a broader target population. In addition, longer peptides are not extracellularly loaded on MHC class I; rather, they require intracellular processing and will thereby be presented to T cells mainly by professional APCs, thereby avoiding the risk of tolerance induction. The drawback of peptide vaccines regardless of peptide length is that naked peptides are not actively targeted to and taken up by APCs, and the standard nonconjugated adjuvant-peptide mixtures do not ensure cotargeting of the two to the same APC. We have identified a tetanus toxin-derived B cell epitope that can mediate the formation of immune complexes in the presence of circulating Abs. In this study, we show that these immune complexes improve both Ag uptake by APCs (blood monocytes and CD1c+ dendritic cells) and consequently improve CD8+ T cell recall responses in a human ex vivo blood loop system. The uptake of the peptide conjugate by blood monocytes is dependent on Abs and the complement component C1q. We envision that this strategy can be used to facilitate active uptake of Ags into APCs to improve T cell responses against pathogens or cancer.

Glucocorticoid response to both predictable and unpredictable challenges detected as corticosterone metabolites in collared flycatcher droppings.

In most vertebrate animals, glucocorticoid hormones are the chief mediators of homeostasis in response to ecological conditions and as they progress through their lifecycle. In addition, glucocorticoids are a major part of the stress response and stress induced elevations of the hormone can make it difficult to assess glucocorticoid secretion in response to changes in life-stage and current environmental conditions in wild animals. Particularly when quantifying circulating levels of glucocorticoids in the blood which fluctuate rapidly in response to stress. An alternative method of quantifying glucocorticoids is as hormone metabolites in faeces or urine giving a historical sample related to the gut passage time and urinary tract that is less sensitive to stressful events which cause spikes in the circulating hormone level. Although the concentration of glucocorticoid metabolites are influenced by faecal mass thereby potentially affecting any differences in hormone metabolites detected amongst samples. In the present study, we aimed to detect changes in levels of corticosterone, the primary bird glucocorticoid, in relation to the phase of reproduction, in a breeding population of collared flycatchers by sampling corticosterone metabolites in droppings. We also tested how corticosterone metabolite concentrations were affected by ambient temperature and related to body condition in adult birds. Our results indicate that the upregulation of corticosterone between incubation and nestling feeding in female birds is crucial for successful reproduction in this species. Also, females appear to downregulate corticosterone during incubation in response to lower ambient temperature and poorer body condition. Our results did not indicate a relationship between dropping mass and corticosterone metabolite concentrations, which suggests that our findings were linked to the regulation of corticosterone in response to predictable and unpredictable challenges.

Extracorporeal human whole blood in motion, as a tool to predict first-infusion reactions and mechanism-of-action of immunotherapeutics.

First infusion reactions along with severe anaphylactic responses can occur as a result of systemic administration of therapeutic antibodies. The underlying mechanisms by which monoclonal antibodies induce cytokine release syndrome (CRS) can involve direct agonistic effects via the drug target, or a combination of target-engagement along with innate receptor interactions. Despite the wide variety of pathways and cells that can play a role in CRS, many currently used assays are devoid of one or more components that must be present for these responses to occur. One assay that has not been assessed for its capacity to predict CRS is the modified Chandler loop model. Herein we evaluate a plethora of commercially available monoclonal antibodies to evaluate the modified Chandler loop model's potential in CRS prediction. We demonstrate that in a 4-hour loop assay, both the superagonistic antibodies, anti-CD3 (OKT3) and anti-CD28 (ANC28.1), display a clear cytokine response with a mixed adaptive/innate cytokine source. OKT3 induce TNFα and IFN-γ release in 20 out of 23 donors tested, whereas ANC28.1 induce TNF-α, IL-2 and IFN-γ release in all donors tested (n=18-22). On the other hand, non-agonistic antibodies associated with no or low infusion reactions in the clinic, namely cetuximab and natalizumab, neither induce cytokine release nor cause false positive responses. A TGN1412-like antibody also display a clear cytokine release with an adaptive cytokine profile (IFN-γ and IL-2) and all donors (n=9) induce a distinct IL-2 response. Additionally, the value of an intact complement system in the assay is highlighted by the possibility to dissect out the mechanism-of-action of alemtuzumab and rituximab. The loop assay can either complement lymph node-like assays or stand-alone to investigate drug/blood interactions during preclinical development, or for individual safety screening prior to first-in-man clinical trial.

Linking T cell epitopes to a common linear B cell epitope: A targeting and adjuvant strategy to improve T cell responses.

Immune complexes are potent mediators of cellular immunity and have been extensively studied for their disease mediating properties in humans and for their role in anti-cancer immunity. However, a viable approach to use antibody-complexed antigen as vehicle for specific immunotherapy has not yet reached clinical use. Since virtually all people have endogenous antibodies against tetanus toxoid (TTd), such commonly occurring antibodies are promising candidates to utilize for immune modulation. As an initial proof-of-concept we investigated if anti-tetanus IgG could induce potent cross-presentation of a conjugate with SIINFEKL, a MHC class I presented epitope of ovalbumin (OVA), to TTd. This protein conjugate enhanced OVA-specific CD8+ T cell responses when administrated to seropositive mice. Since TTd is poorly defined, we next investigated whether a synthetic peptide-peptide conjugate, with a chemically defined linear B cell epitope of tetanus toxin (TTx) origin, could improve cellular immune responses. Herein we identify one linear B cell epitope, here after named MTTE thru a screening of overlapping peptides from the alpha and beta region of TTx, and by assessment of the binding of pooled IgG, or individual human IgG from high-titer TTd vaccinated donors, to these peptides. Subsequently, we developed a chemical protocol to synthesize defined conjugates containing multiple copies of MTTE covalently attached to one or more T cell epitopes of choice. To demonstrate the potential of the above approach we showed that immune complexes of anti-MTTE antibodies with MTTE-containing conjugates are able to induce DC and T cell activation using model antigens.

The human agonistic CD40 antibody ADC-1013 eradicates bladder tumors and generates T-cell-dependent tumor immunity.

PURPOSE: Local administration of immune-activating antibodies may increase the efficacy and reduce the immune-related adverse events associated with systemic immunotherapy of cancer. Here, we report the development and affinity maturation of a fully human agonistic CD40 antibody (IgG1), ADC-1013. EXPERIMENTAL DESIGN: We have used molecular engineering to generate an agonistic antibody with high affinity for CD40. The functional activity of ADC-1013 was investigated in human and murine in vitro models. The in vivo effect was investigated in two separate bladder cancer models, both using human xenograft tumors in immune deficient NSG mice and using a syngeneic bladder cancer model in a novel human CD40 transgenic mouse. RESULTS: Activation of dendritic cells (DC) by ADC-1013 results in upregulation of the costimulatory molecules CD80 and CD86, and secretion of IL12. ADC-1013 also activates DCs from human CD40 transgenic mice, and peptide-pulsed and ADC-1013-stimulated DCs induce antigen-specific T-cell proliferation in vitro. In vivo, treatment with ADC-1013 in a syngeneic bladder cancer model, negative for hCD40, induces significant antitumor effects and long-term tumor-specific immunity. Furthermore, ADC-1013 demonstrates significant antitumor effects in a human bladder cancer transplanted into immunodeficient NSG mice. CONCLUSIONS: Our data demonstrate that ADC-1013 induces long-lasting antitumor responses and immunologic memory mediated by CD40 stimulation. To the best of our knowledge, ADC-1013 represents the first immunomodulatory antibody developed for local immunotherapy of cancer.

Antibody induced CD4 down-modulation of T cells is site-specifically mediated by CD64(+) cells.

Treatment of PBMC with the CD4-specific mAb BT-061 induces CD4 down-modulation of T cells. Here we report that addition of BT-061 to purified T cells did not confer this effect, whereas incubation of T cells in BT-061 coated wells restored CD4 down-modulation. These results implied that Fcγ receptor mediated cell-cell interactions played a role. In consistence with this hypothesis PBMC depleted of CD64(+) monocytes did not confer CD4 down-modulation of BT-061 decorated T cells. Strikingly, CD4 down-modulation was observed in BT-061 treated synovial fluid punctuated from patients' inflamed joints that comprised enhanced numbers of CD64(+) cells. In contrast, in a circulating whole blood system injection of BT-061 did not induce CD4 down-modulation, due to CD64 saturation by serum IgG. Similarly, tonsil derived mononuclear cells devoid of CD64(+) cells did not show CD4 down-modulation, whereas addition of blood derived monocytes restored the effect. Thus, the interaction of BT-061 decorated T cells with CD64(+) cells is needed for CD4 down-modulation, implying that in patients BT-061 would primarily induce CD4 down-modulation at inflammatory sites. These results highlight the need not only to examine the interaction of a given mAb with single FcγR, but also the immunological environment that is appropriate to support such interactions.