Fine-tuned long-acting interleukin-2 superkine potentiates durable immune responses in mice and non-human primate.

BACKGROUND: Recombinant human interleukin-2 (rhIL-2, aldesleukin) is Food and Drug Administration approved for the treatment of metastatic melanoma and renal cell carcinoma and has achieved durable response in a subset of patients. However, its utility as an immunotherapeutic drug is limited by undesirable activation of immune suppressive regulatory T cells (Tregs) and a short half-life requiring frequent high dose administration, leading to unacceptable toxicities. We have engineered MDNA11, a long-acting IL-2 superkine, to overcome these limitations by (1) modifying receptor selectivity in favor of anti-cancer immune cells to increase therapeutic efficacy and (2) fusion to human albumin to extend the pharmacokinetic (PK) profile, circumventing the need for frequent dosing. METHODS: MDNA11 was evaluated using in vitro and in vivo studies including: binding analyses to measure receptor affinity, IL-2 pathway signaling, PK studies in mice, and efficacy studies in syngeneic tumor models as single agent and in combination with immune checkpoint inhibitors. Finally, the safety and pharmacodynamic profile of MDNA11 was assessed in non-human primate (NHP). RESULTS: Binding studies with MDNA11 demonstrated increased affinity for IL-2Rß (CD122) and no binding to IL-2Rα (CD25). As a result, MDNA11 exhibits reduced/limited Treg stimulation while triggering an enhanced activation of natural killer and naïve CD8 T cells compared with rhIL-2. When administered to animals with pre-established tumors, MDNA11 controlled tumor growth in a monotherapy setting and in combination with anti-PD1 or anti-CTLA4 to induce durable tumor clearance with a once weekly dosing regimen. In a NHP model, MDNA11 was well tolerated while triggering durable and potent immune responses including expansion of lymphocytes without significant effect on Tregs and eosinophils, the latter been linked to an increased risk of vascular leak syndrome. CONCLUSION: MDNA11 is a next generation long-acting IL-2 immunotherapeutic with a highly favorable pharmacodynamic profile that translates to a strong therapeutic efficacy in preclinical tumor models and a strong and durable immune response in NHP.

Ru(II) CONTAINING PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY: A CRITIQUE ON REPORTING AND AN ATTEMPT TO COMPARE EFFICACY.

Ruthenium(II)-based coordination complexes have emerged as photosensitizers (PSs) for photodynamic therapy (PDT) in oncology as well as antimicrobial indications and have great potential. Their modular architectures that integrate multiple ligands can be exploited to tune cellular uptake and subcellular targeting, solubility, light absorption, and other photophysical properties. A wide range of Ru(II) containing compounds have been reported as PSs for PDT or as photochemotherapy (PCT) agents. Many studies employ a common scaffold that is subject to systematic variation in one or two ligands to elucidate the impact of these modifications on the photophysical and photobiological performance. Studies that probe the excited state energies and dynamics within these molecules are of fundamental interest and are used to design next-generation systems. However, a comparison of the PDT efficacy between Ru(II) containing PSs and 1st or 2nd generation PSs, already in clinical use or preclinical/clinical studies, is rare. Even comparisons between Ru(II) containing molecular structures are difficult, given the wide range of excitation wavelengths, power densities, and cell lines utilized. Despite this gap, PDT dose metrics quantifying a PS's efficacy are available to perform qualitative comparisons. Such models are independent of excitation wavelength and are based on common outcome parameters, such as the photon density absorbed by the Ru(II) compound to cause 50% cell kill (LD50) based on the previously established threshold model. In this focused photophysical review, we identified all published studies on Ru(II) containing PSs since 2005 that reported the required photophysical, light treatment, and in vitro outcome data to permit the application of the Photodynamic Threshold Model to quantify their potential efficacy. The resulting LD50 values range from less than 1013 to above 1020 [hν cm-3], indicating a wide range in PDT efficacy and required optical energy density for ultimate clinical translation.

Lung gene therapy-How to capture illumination from the light already present in the tunnel.

Gene therapy has been considered as the most ideal medical intervention for genetic diseases because it is intended to target the cause of diseases instead of disease symptoms. Availability of techniques for identification of genetic mutations and for in vitro manipulation of genes makes it practical and attractive. After the initial hype in 1990s and later disappointments in clinical trials for more than a decade, light has finally come into the tunnel in recent years, especially in the field of eye gene therapy where it has taken big strides. Clinical trials in gene therapy for retinal degenerative diseases such as Leber's congenital amaurosis (LCA) and choroideremia demonstrated clear therapeutic efficacies without apparent side effects. Although these successful examples are still rare and sporadic in the field, they provide the proof of concept for harnessing the power of gene therapy to treat genetic diseases and to modernize our medication. In addition, those success stories illuminate the path for the development of gene therapy treating other genetic diseases. Because of the differences in target organs and cells, distinct barriers to gene delivery exist in gene therapy for each genetic disease. It is not feasible for authors to review the current development in the entire field. Thus, in this article, we will focus on what we can learn from the current success in gene therapy for retinal degenerative diseases to speed up the gene therapy development for lung diseases, such as cystic fibrosis.

Direct in vivo evidence of CD4+ T cell requirement for CTL response and memory via pMHC-I targeting and CD40L signaling.

CD4(+) T cell help contributes critically to DC-induced CD8(+) CTL immunity. However, precisely how these three cell populations interact and how CD4(+) T cell signals are delivered to CD8(+) T cells in vivo have been unclear. In this study, we developed a novel, two-step approach, wherein CD4(+) T cells and antigen-presenting DCs productively engaged one another in vivo in the absence of cognate CD8(+) T cells, after which, we selectively depleted the previously engaged CD4(+) T cells or DCs before allowing interactions of either population alone with naïve CD8(+) T cells. This protocol thus allows us to clearly document the importance of CD4(+) T-licensed DCs and DC-primed CD4(+) T cells in CTL immunity. Here, we provide direct in vivo evidence that primed CD4(+) T cells or licensed DCs can stimulate CTL response and memory, independent of DC-CD4(+) T cell clusters. Our results suggest that primed CD4(+) T cells with acquired pMHC-I from DCs represent crucial "immune intermediates" for rapid induction of CTL responses and for functional memory via CD40L signaling. Importantly, intravital, two-photon microscopy elegantly provide unequivocal in vivo evidence for direct CD4-CD8(+) T cell interactions via pMHC-I engagement. This study corroborates the coexistence of direct and indirect mechanisms of T cell help for a CTL response in noninflammatory situations. These data suggest a new "dynamic model of three-cell interactions" for CTL immunity derived from stimulation by dissociated, licensed DCs, primed CD4(+) T cells, and DC-CD4(+) T cell clusters and may have significant implications for autoimmunity and vaccine design.

Dendritic cells recruit T cell exosomes via exosomal LFA-1 leading to inhibition of CD8+ CTL responses through downregulation of peptide/MHC class I and Fas ligand-mediated cytotoxicity.

Active T cells release bioactive exosomes (EXOs). However, its potential modulation in immune responses is elusive. In this study, we in vitro generated active OVA-specific CD8(+) T cells by cultivation of OVA-pulsed dendritic cells (DC(OVA)) with naive CD8(+) T cells derived from OVA-specific TCR transgenic OTI mice and purified EXOs from CD8(+) T cell culture supernatant by differential ultracentrifugation. We then investigated the suppressive effect of T cell EXOs on DC(OVA)-mediated CD8(+) CTL responses and antitumor immunity. We found that DC(OVA) uptake OTI T cell EXOs expressing OVA-specific TCRs and Fas ligand via peptide/MHC Ag I-TCR and CD54-LFA-1 interactions leading to downregulation of peptide/MHC Ag I expression and induction of apoptosis of DC(OVA) via Fas/Fas ligand pathway. We demonstrated that OVA-specific OTI T cell EXOs, but not lymphocytic choriomeningitis virus-specific TCR transgenic mouse CD8(+) T cell EXOs, can inhibit DC(OVA)-stimulated CD8(+) CTL responses and antitumor immunity against OVA-expressing B16 melanoma. In addition, these T cell EXOs can also inhibit DC(OVA)-mediated CD8(+) CTL-induced diabetes in transgenic rat insulin promoter-mOVA mice. Interestingly, the anti-LFA-1 Ab treatment significantly reduces T cell EXO-induced inhibition of CD8(+) CTL responses in both antitumor immunity and autoimmunity. EXOs released from T cell hybridoma RF3370 cells expressing OTI CD8(+) TCRs have a similar inhibitory effect as T cell EXOs in DC(OVA)-stimulated CTL responses and antitumor immunity. Therefore, our data indicate that Ag-specific CD8(+) T cells can modulate immune responses via T cell-released EXOs, and T cell EXOs may be useful for treatment of autoimmune diseases.

Optimal TLR9 signal converts tolerogenic CD4-8- DCs into immunogenic ones capable of stimulating antitumor immunity via activating CD4+ Th1/Th17 and NK cell responses.

Abstract TLR ligands have been reported to promote DC maturation and enhance CD8+ CTL responses. We have demonstrated previously that CD4-8- DCs secreting TGF-beta stimulate CD4+ Tr1 cell responses. Here, we have assessed whether TLR4 and TLR9 signaling through LPS and CpG stimulation can convert CD4-8- DC-induced tolerance. We demonstrate that immature OVA-pulsed CD4-8- DCs cultured in medium with LPS (2 microg/ml) and CpG (5 microg/ml) for 8 h became mature DCs (DCOVA) with no TGF-beta secretion. CpG-treated, CD4-8- DCOVA-secreting IL-6/IL-15 induced IFN-gamma/IL-17-secreting/T-bet- and ROR-gammat-expressing CD4+ Th1/Th17, whereas LPS-treated CD4-8- DCOVA stimulated IFN-gamma-secreting/T-bet-expressing CD4+ Th1 responses. The former also significantly stimulated more efficient OVA-specific CD8+ T cell responses and antitumor immunity against OVA-expressing BL6-10OVA tumor cells than the latter (P<0.05). CpG-treated, CD4-8- DCOVA-stimulated CD4+ Th1/Th17 cell responses and antitumor immunity were found to be reduced by using neutralizing anti-IL-6, IL-15, and NK1.1 antibodies in wild-type C57BL/6 mice, IL-15R-/- mice for immunization, or CD4-8- (IL-6-/-) DCOVA for immunization in C57BL/6 mice. Interestingly, in vitro-generated CD4+ Th17 cells significantly enhanced LPS-treated, CD4-8- DCOVA-induced in vivo antitumor immunity via increasing CD8+ CTL responses (P<0.05), although they did not show any direct killing activity against tumor cells in vitro. In addition, prolonged 48 h CpG-treated CD4-8- DCOVA dramatically diminished its cytokine secretion, stimulatory effect, and antitumor immunity. Taken together, our data demonstrate an effect of conversion of tolerogenic DCs into immunogenic ones capable of stimulating antitumor immunity via activating CD4+ Th1/Th17 and NK cell responses by optimal CpG signaling, which may advance current understanding of the importance of TLR9 signaling in a DC-based cancer vaccine.

Intercellular trogocytosis plays an important role in modulation of immune responses.

Intercellular communication is an important means of molecular information transfer through exchange of membrane proteins from cells to cells. Advent of the latest analytical and imaging tools has allowed us to enhance our understanding of the cellular communication through the intercellular exchange of intact membrane patches, also called trogocytosis, which is a ubiquitous phenomenon. Immune responses against pathogens or any foreign antigens require fine immune regulation, where cellular communications are mediated by either soluble or cell surface molecules. It has been demonstrated that the membrane molecule transfer between immune cells such as dendritic and T cells can be derived through internalization/recycling pathway, dissociation-associated pathway, uptake of exosomes and membrane nanotube formations. Recent evidence implicates the trogocytosis as an important mechanism of the immune system to modulate immune responses. Exchange of membrane molecules/antigens between immune cells has been observed for a long time, but the mechanisms and functional consequences of these transfers remain unclear. In this review, we discuss the possible mechanisms of trogocytosis and its physiological relevance to immune system, with special reference to T cells and the stimulatory or suppressive immune responses derived from T cells with acquired dendritic cell membrane molecules.

Antigen specificity acquisition of adoptive CD4+ regulatory T cells via acquired peptide-MHC class I complexes.

The Ag-specific CD4(+) regulatory T (Tr) cells play an important role in immune suppression in autoimmune diseases and antitumor immunity. However, the molecular mechanism for Ag-specificity acquisition of adoptive CD4(+) Tr cells is unclear. In this study, we generated IL-10- and IFN-gamma-expressing type 1 CD4(+) Tr (Tr1) cells by stimulation of transgenic OT II mouse-derived naive CD4(+) T cells with IL-10-expressing adenovirus (AdV(IL-10))-transfected and OVA-pulsed dendritic cells (DC(OVA/IL-10)). We demonstrated that both in vitro and in vivo DC(OVA/IL-10)-stimulated CD4(+) Tr1 cells acquired OVA peptide MHC class (pMHC) I which targets CD4(+) Tr1 cells suppressive effect via an IL-10-mediated mechanism onto CD8(+) T cells, leading to an enhanced suppression of DC(OVA)-induced CD8(+) T cell responses and antitumor immunity against OVA-expressing murine B16 melanoma cells by approximately 700% relative to analogous CD4(+) Tr1 cells without acquired pMHC I. Interestingly, the nonspecific CD4(+)25(+) Tr cells can also become OVA Ag specific and more immunosuppressive in inhibition of OVA-specific CD8(+) T cell responses and antitumor immunity after uptake of DC(OVA)-released exosomal pMHC I complexes. Taken together, the Ag-specificity acquisition of CD4(+) Tr cells via acquiring DC's pMHC I may be an important mean in augmenting CD4(+) Tr cell suppression.

T cell precursor frequency differentially affects CTL responses under different immune conditions.

Generation of effective CTL responses is the goal of many vaccination protocols. However, to what extant T cell precursor frequencies will generate a CD8(+) CTL response has not been elucidated properly. In this study, we employed a model system, in which naive CD4(+) and CD8(+) T cells derived from ovalbumin (OVA)-specific TCR transgenic OT II and OT I mice were used for adoptive transfer into wild-type, Ia(b-/-) gene knockout and transgenic RIP-mOVA mice, and assessed OVA-pulsed DC (DC(OVA))-stimulated CD8(+) CTL responses in these mice. We demonstrated that (i) a critical threshold exists above which T cells precursor frequency cannot enhance the CTL responses in wild-type C57BL/6 mice, (ii) increasing CD8(+) T cell precursors is required to generate CTL responses but with functional memory defect in absence of CD4(+) T cell help, and (iii) increasing CD4(+) and CD8(+) T cell precursors overcomes immune suppression to DC(OVA)-stimulated CD8(+) CTL responses in transgenic RIP-mOVA mice with OVA-specific self immune tolerance. Taken together, these findings may have important implications for optimizing immunotherapy against cancer.