Constitutively active GSK3 beta as a means to bolster dendritic cell functionality in the face of tumor-mediated immune suppression

In patients with cancer, the functionality of Dendritic Cells (DC) is hampered by high levels of tumor-derived suppressive cytokines, which interfere with DC development and maturation. Poor DC development can limit the efficacy of immune checkpoint blockade and in vivo vaccination approaches. Interference in intracellular signaling cascades downstream from the receptors of major tumor-associated suppressive cytokines like IL-10 and IL-6, might improve DC development and activation, and thus enhance immunotherapy efficacy. We performed exploratory functional screens on arrays consisting of >1000 human kinase peptide substrates to identify pathways involved in DC development and its inhibition by IL-10 or IL-6. The resulting alterations in phosphorylation of the kinome substrate profile pointed to glycogen-synthase kinase-3 beta (GSK3 beta) as a pivotal kinase in both DC development and suppression. GSK3 beta inhibition blocked human DC differentiation in vitro, which was accompanied by decreased levels of IL-12p70 secretion, and a reduced capacity for T cell priming. More importantly, adenoviral transduction of monocytes with a constitutively active form of GSK3 beta induced resistance to the suppressive effects of IL-10 and melanoma-derived supernatants alike, resulting in improved DC development, accompanied by up-regulation of co-stimulatory markers, an increase in CD83 expression levels in mature DC, and diminished release of IL-10. Moreover, adenovirus-mediated intratumoral manipulation of this pathway in an in vivo melanoma model resulted in DC activation and recruitment, and in improved immune surveillance and tumor control. We propose the induction of constitutive GSK3 beta activity as a novel therapeutic means to bolster DC functionality in the tumor microenvironment.Peer reviewe

Genetic Diversity of the Hemagglutinin Genes of Influenza a Virus in Asian Swine Populations

Swine influenza (SI) is a major respiratory disease of swine; SI is due to the influenza A virus of swine (IAV-S), a highly contagious virus with zoonotic potential. The intensity of IAV-S surveillance varies among countries because it is not a reportable disease and causes limited mortality in swine. Although Asia accounts for half of all pig production worldwide, SI is not well managed in those countries. Rigorously managing SI on pig farms could markedly reduce the economic losses, the likelihood of novel reassortants among IAV-S, and the zoonotic IAV-S infections in humans. Vaccination of pigs is a key control measure for SI, but its efficacy relies on the optimal antigenic matching of vaccine strains with the viral strains circulating in the field. Here, we phylogenetically reviewed the genetic diversity of the hemagglutinin gene among IAVs-S that have circulated in Asia during the last decade. This analysis revealed the existence of country-specific clades in both the H1 and H3 subtypes and cross-border transmission of IAVs-S. Our findings underscore the importance of choosing vaccine antigens for each geographic region according to both genetic and antigenic analyses of the circulating IAV-S to effectively manage SI in Asia

Genetic Diversity of the Hemagglutinin Genes of Influenza a Virus in Asian Swine Populations

Swine influenza (SI) is a major respiratory disease of swine; SI is due to the influenza A virus of swine (IAV-S), a highly contagious virus with zoonotic potential. The intensity of IAV-S surveillance varies among countries because it is not a reportable disease and causes limited mortality in swine. Although Asia accounts for half of all pig production worldwide, SI is not well managed in those countries. Rigorously managing SI on pig farms could markedly reduce the economic losses, the likelihood of novel reassortants among IAV-S, and the zoonotic IAV-S infections in humans. Vaccination of pigs is a key control measure for SI, but its efficacy relies on the optimal antigenic matching of vaccine strains with the viral strains circulating in the field. Here, we phylogenetically reviewed the genetic diversity of the hemagglutinin gene among IAVs-S that have circulated in Asia during the last decade. This analysis revealed the existence of country-specific clades in both the H1 and H3 subtypes and cross-border transmission of IAVs-S. Our findings underscore the importance of choosing vaccine antigens for each geographic region according to both genetic and antigenic analyses of the circulating IAV-S to effectively manage SI in Asia

MCLA-117, a CLEC12AxCD3 bispecific antibody targeting a leukaemic stem cell antigen, induces T cell-mediated AML blast lysis

Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23–98%) at low effector-to-target ratios (1:3–1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML

Potent antitumor immunity generated by a CD40-targeted adenoviral vaccine.

Item does not contain fulltextIn situ delivery of tumor-associated antigen (TAA) genes into dendritic cells (DC) has great potential as a generally applicable tumor vaccination approach. Although adenoviruses (Ad) are an attractive vaccine vehicle in this regard, Ad-mediated transduction of DCs is hampered by the lack of expression of the Ad receptor CAR on the DC surface. DC activation also requires interaction of CD40 with its ligand CD40L to generate protective T-cell-mediated tumor immunity. Therefore, to create a strategy to target Ads to DCs in vivo, we constructed a bispecific adaptor molecule with the CAR ectodomain linked to the CD40L extracellular domain via a trimerization motif (CFm40L). By targeting Ad to CD40 with the use of CFm40L, we enhanced both transduction and maturation of cultured bone marrow-derived DCs. Moreover, we improved transduction efficiency of DCs in lymph node and splenic cell suspensions in vitro and in skin and vaccination site-draining lymph nodes in vivo. Furthermore, CD40 targeting improved the induction of specific CD8(+) T cells along with therapeutic efficacy in a mouse model of melanoma. Taken together, our findings support the use of CD40-targeted Ad vectors encoding full-length TAA for in vivo targeting of DCs and high-efficacy induction of antitumor immunity

Low-dose IL-15 mediates efficient UCB-NK cell survival and expansion <i>in vivo</i>.

<p>Adult NSG mice were injected <i>i.v.</i> with 5×10⁶ UCB-NK cells with or without supportive IL-15. Recombinant human IL-15 was administered every 2–3 days for 2 weeks at the dose of 0.5 µg/mouse/injection, starting the day of UCB-NK cell infusion. (A) Percentages of circulating human CD45⁺ cells in all CD45⁺ cells were quantified by flow cytometry at the indicated time points. Each line corresponds to one mouse. (B) Percentages of human CD45⁺ cells in all CD45⁺ cells quantified in spleen and bone marrow (leg bones) 2 weeks after UCB-NK cell infusion. (C) Expression of CD16 and CD158a/h,b,e on UCB-NK cells before and 2 weeks after infusion into NSG mice. Percentages were determined on human CD45⁺CD56⁺ NK cells isolated from spleen and bone marrow. Graphs show the mean ± SD of 5 mice.</p

A single infusion of UCB-NK cells inhibits growth of BM-residing human leukemia cells.

<p>The potential of UCB-NK cells to attack human leukemia <i>in vivo</i> was evaluated in NSG mice bearing K562 intra-femoral (<i>i.f</i>.) tumors. (A) Experimental study design: adult NSG mice were injected in their right femur with 10⁵ K562.LucGFP cells. The day after, mice were treated with 20×10⁶ UCB-NK cells <i>i.v.</i> in combination with IL-15 administration (0.5 µg/mouse <i>i.p.</i> every 2–3 days for 14 days), or received PBS or IL-15 alone as control (n = 6 per group). Tumor load was monitored by BLI from day 8 after K562.LucGFP cell inoculation and next every 3–4 days for 2 weeks. At later time points, only mice with undetectable tumor load were imaged. (B) Two independent anti-leukemic studies were performed with similar outcomes. Tumor load per mouse measured at day 15 following K562.LucGFP cell IF injection from experiment 1 (black circles) and 2 (open circles). (C) BLI pictures acquired at day 15 after tumor cell <i>i.f.</i> injection, and (D) tumor load in time (mean ± SD, n = 6 per group). **<i>p</i><0.01 UCB-NK cells+IL-15 vs. PBS and IL-15. Data of experiment 2 are shown. (E) Time to first tumor detection by BLI and (F) mice survival analysed according to Mantel Cox test. One mouse from the IL-15 group (experiment 1) died at day 19 after luciferine injection and was excluded from the survival analysis.</p

Homing receptor expression profile of UCB-NK cells.

<p>(A–B) The expression level of homing receptors was analysed by flow cytometry on UCB-NK cells at the end of the culture process. (A) Dot plots gated on CD56⁺ cells from one representative donor; (B) Summary of 6 different donors analysed. (C) The capacity of UCB-NK cells to respond <i>in vitro</i> to gradients (10 to 250 ng/ml) of the chemokines CCL4 (CCR5 ligand), CCL20 (CCR6 ligand), CXCL10, CXCL11 (both CXCR3 ligand) and CXCL12 (CXCR4 ligand) was evaluated in transwell migration assays as described in materials and methods. Mean ± SEM of three independent experiments are shown, each performed with different UCB-NK cell donors. Migration towards specific chemokines was compared to non-specific migration (0 ng/ml) using a one way-ANOVA followed by Dunnett’s multiple comparison post-hoc test, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

<i>Ex vivo</i> flow cytometric analysis confirmed trafficking of UCB-NK cells through lymphoid tissues, liver and lungs following adoptive transfer in NSG mice.

<p>Two adult NSG mice were infused <i>i.v.</i> with 10×10⁶ UCB-NK cells. The day after, mice were sacrificed, organs collected and used to prepare cell suspension for <i>ex vivo</i> flow cytometric analysis following erythrocyte lysis. One additional non-injected mouse was used as control. Presence of human CD45⁺CD56⁺ NK cells was confirmed in all examined tissues except kidneys. Dot plots gated on total living cells are shown.</p