Lipid-Encapsulated mRNAs Encoding Complex Fusion Proteins Potentiate Antitumor Immune Responses.

Lipid nanoparticle (LNP)-encapsulated mRNA has been used for in vivo production of several secreted protein classes, such as IgG, and has enabled the development of personalized vaccines in oncology. Establishing the feasibility of delivering complex multispecific modalities that require higher-order structures important for their function could help expand the use of mRNA/LNP biologic formulations. Here, we evaluated whether in vivo administration of mRNA/LNP formulations of SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT could achieve oligomerization and extend exposure, on-target activity, and antitumor responses comparable with that of the corresponding recombinant fusion proteins. Intravenous infusion of the formulated LNP-encapsulated mRNAs led to rapid and sustained production of functional hexameric proteins in vivo, which increased the overall exposure relative to the recombinant protein controls by ∼28 to 140 fold over 96 hours. High concentrations of the mRNA-encoded proteins were also observed in secondary lymphoid organs and within implanted tumors, with protein concentrations in tumors up to 134-fold greater than with the recombinant protein controls 24 hours after treatment. In addition, SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT mRNAs induced a greater increase in antigen-specific CD8+ T cells in the tumors. These mRNA/LNP formulations were well tolerated and led to a rapid increase in serum and intratumoral IL2, delayed tumor growth, extended survival, and outperformed the activities of benchmark mAb controls. Furthermore, the mRNA/LNPs demonstrated improved efficacy in combination with anti-PD-L1 relative to the recombinant fusion proteins. These data support the delivery of complex oligomeric biologics as mRNA/LNP formulations, where high therapeutic expression and exposure could translate into improved patient outcomes. SIGNIFICANCE: Lipid nanoparticle-encapsulated mRNA can efficiently encode complex fusion proteins encompassing immune checkpoint blockers and costimulators that functionally oligomerize in vivo with extended pharmacokinetics and durable exposure to induce potent antitumor immunity.

Analysis of Butyrophilin-Mediated Activation of γδ T Cells from Human Spleen.

There is considerable interest in therapeutically engaging human γδ T cells. However, due to the unique TCRs of human γδ T cells, studies from animal models have provided limited directly applicable insights, and human γδ T cells from key immunological tissues remain poorly characterized. In this study, we investigated γδ T cells from human spleen tissue. Compared to blood, where Vδ2+Vγ9+ T cells are the dominant subset, splenic γδ T cells included a variety of TCR types, with Vδ1+ T cells typically being the most frequent. Intracellular cytokine staining revealed that IFN-γ was produced by a substantial fraction of splenic γδ T cells, IL-17A by a small fraction, and IL-4 was minimal. Primary splenic γδ T cells frequently expressed NKG2D (NK group 2 member D) and CD16, whereas expression of DNAM-1 (DNAX accessory molecule 1), CD28, PD-1, TIGIT, and CD94 varied according to subset, and there was generally little expression of natural cytotoxicity receptors, TIM-3, LAG-3, or killer Ig-like receptors. In vitro expansion was associated with marked changes in expression of these activating and inhibitory receptors. Analysis of functional responses of spleen-derived Vδ2+Vγ9+, Vδ1+Vγ9+, and Vδ1+Vγ9- T cell lines to recombinant butyrophilin BTN2A1 and BTN3A1 demonstrated that both Vδ2+Vγ9+ and Vδ1+Vγ9+ T cells were capable of responding to the extracellular domain of BTN2A1, whereas the addition of BTN3A1 only markedly enhanced the responses of Vδ2+Vγ9+ T cells. Conversely, Vδ1+Vγ9+ T cells appeared more responsive than Vδ2+Vγ9+ T cells to TCR-independent NKG2D stimulation. Thus, despite shared recognition of BTN2A1, differential effects of BTN3A1 and coreceptors may segregate target cell responses of Vδ2+Vγ9+ and Vδ1+Vγ9+ T cells.

Reconciling intrinsic properties of activating TNF receptors by native ligands versus synthetic agonists.

The extracellular domain of tumor necrosis factor receptors (TNFR) generally require assembly into a homotrimeric quaternary structure as a prerequisite for initiation of signaling via the cytoplasmic domains. TNF receptor homotrimers are natively activated by similarly homo-trimerized TNF ligands, but can also be activated by synthetic agonists including engineered antibodies and Fc-ligand fusion proteins. A large body of literature from pre-clinical models supports the hypothesis that synthetic agonists targeting a diverse range of TNF receptors (including 4-1BB, CD40, OX40, GITR, DR5, TNFRSF25, HVEM, LTßR, CD27, and CD30) could amplify immune responses to provide clinical benefit in patients with infectious diseases or cancer. Unfortunately, however, the pre-clinical attributes of synthetic TNF receptor agonists have not translated well in human clinical studies, and have instead raised fundamental questions regarding the intrinsic biology of TNF receptors. Clinical observations of bell-shaped dose response curves have led some to hypothesize that TNF receptor overstimulation is possible and can lead to anergy and/or activation induced cell death of target cells. Safety issues including liver toxicity and cytokine release syndrome have also been observed in humans, raising questions as to whether those toxicities are driven by overstimulation of the targeted TNF receptor, a non-TNF receptor related attribute of the synthetic agonist, or both. Together, these clinical findings have limited the development of many TNF receptor agonists, and may have prevented generation of clinical data which reflects the full potential of TNF receptor agonism. A number of recent studies have provided structural insights into how different TNF receptor agonists bind and cluster TNF receptors, and these insights aid in deconvoluting the intrinsic biology of TNF receptors with the mechanistic underpinnings of synthetic TNF receptor agonist therapeutics.

Cutting Edge: Bispecific γδ T Cell Engager Containing Heterodimeric BTN2A1 and BTN3A1 Promotes Targeted Activation of Vγ9Vδ2+ T Cells in the Presence of Costimulation by CD28 or NKG2D.

Vγ9Vδ2+ T cell-targeted immunotherapy is of interest to harness its MHC-independent cytotoxic potential against a variety of cancers. Recent studies have identified heterodimeric butyrophilin (BTN) 2A1 and BTN3A1 as the molecular entity providing "signal 1" to the Vγ9Vδ2 TCR, but "signal 2" costimulatory requirements remain unclear. Using a tumor cell-free assay, we demonstrated that a BTN2A1/3A1 heterodimeric fusion protein activated human Vγ9Vδ2+ T cells, but only in the presence of costimulatory signal via CD28 or NK group 2 member D. Nonetheless, addition of a bispecific γδ T cell engager BTN2A1/3A1-Fc-CD19scFv alone enhanced granzyme B-mediated killing of human CD19+ lymphoma cells when cocultured with Vγ9Vδ2+ T cells, suggesting expression of costimulatory ligand(s) on tumor cells is sufficient to satisfy the "signal 2" requirement. These results highlight the parallels of signal 1 and signal 2 requirements in αß and γδ T cell activation and demonstrate the utility of heterodimeric BTNs to promote targeted activation of γδ T cells.

Differentiation of renal masses with multi-parametric MRI: the de Silva St George classification scheme.

PURPOSE: To develop a system for multi-parametric MRI to differentiate benign from malignant solid renal masses and assess its accuracy compared to the gold standard of histopathological diagnosis. METHODS: This is a retrospective analysis of patients who underwent 3 Tesla mpMRI for further assessment of small renal tumours with specific scanning and reporting protocol incorporating T2 HASTE signal intensity, contrast enhancement ratios, apparent diffusion coefficient and presence of microscopic/macroscopic fat. All MRIs were reported prior to comparison with histopathologic diagnosis and a reporting scheme was developed. 2 × 2 contingency table analysis (sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV)), Fisher Exact test were used to assess the association between suspicion of malignancy on mpMRI and histopathology, and descriptive statistics were performed. RESULTS: 67 patients were included over a 5-year period with a total of 75 renal masses. 70 masses were confirmed on histopathology (five had pathognomonic findings for angiomyolipomas; biopsy was therefore considered unethical, so these were included without histopathology). Three patients were excluded due to a non-diagnostic result, non-standardised imaging and one found to be an organising haematoma rather than a mass. Therefore 72 cases were included in analysis (in 64 patients, with seven patients having multiple tumours). Unless otherwise specified, all further statistics refer to individual tumours rather than patients. 52 (72.2%) were deemed 'suspicious or malignant' and 20 (27.8%) were deemed 'benign' on mpMRI. 51 cases (70.8%) had renal cell carcinoma confirmed. The sensitivity, NPV, specificity and PPV for MRI for detecting malignancy were 96.1%, 90%, 85.7% and 94.2% respectively, Fisher's exact test demonstrated p < 0.0001 for the association between suspicion of malignancy on MRI and histopathology. CONCLUSION: The de Silva St George classification scheme performed well in differentiating benign from malignant solid renal masses, and may be useful in predicting the likelihood of malignancy to determine the need for biopsy/excision. Further validation is required before this reporting system can  be recommended for clinical use.

LIGHT (TNFSF14) Costimulation Enhances Myeloid Cell Activation and Antitumor Immunity in the Setting of PD-1/PD-L1 and TIGIT Checkpoint Blockade.

Coinhibition of TIGIT (T cell immunoreceptor with Ig and ITIM domains) and PD-1/PD-L1 (PD-1/L1) may improve response rates compared with monotherapy PD-1/L1 blockade in checkpoint naive non-small cell lung cancer with PD-L1 expression >50%. TIGIT mAbs with an effector-competent Fc can induce myeloid cell activation, and some have demonstrated effector T cell depletion, which carries a clinical liability of unknown significance. TIGIT Ab blockade translates to antitumor activity by enabling PVR signaling through CD226 (DNAM-1), which can be directly inhibited by PD-1. Furthermore, DNAM-1 is downregulated on tumor-infiltrating lymphocytes (TILs) in advanced and checkpoint inhibition-resistant cancers. Therefore, broadening clinical responses from TIGIT blockade into PD-L1low or checkpoint inhibition-resistant tumors, may be induced by immune costimulation that operates independently from PD-1/L1 inhibition. TNFSF14 (LIGHT) was identified through genomic screens, in vitro functional analysis, and immune profiling of TILs as a TNF ligand that could provide broad immune activation. Accordingly, murine and human bifunctional fusion proteins were engineered linking the extracellular domain of TIGIT to the extracellular domain of LIGHT, yielding TIGIT-Fc-LIGHT. TIGIT competitively inhibited binding to all PVR ligands. LIGHT directly activated myeloid cells through interactions with LTßR (lymphotoxin ß receptor), without the requirement for a competent Fc domain to engage Fcγ receptors. LIGHT costimulated CD8+ T and NK cells through HVEM (herpes virus entry mediator A). Importantly, HVEM was more widely expressed than DNAM-1 on T memory stem cells and TILs across a range of tumor types. Taken together, the mechanisms of TIGIT-Fc-LIGHT promoted strong antitumor activity in preclinical tumor models of primary and acquired resistance to PD-1 blockade, suggesting that immune costimulation mediated by LIGHT may broaden the clinical utility of TIGIT blockade.

RESIRT: A Phase 1 Study of Selective Internal Radiation Therapy Using Yttrium-90 Resin Microspheres in Patients With Primary Renal Cell Carcinoma.

INTRODUCTION: Selective internal radiation therapy (SIRT) is a potential treatment of primary renal cell carcinoma (RCC) deemed unsuitable for conventional therapy. RESIRT is the first-in-human study to evaluate safety and feasibility of SIRT for primary RCC. PATIENTS AND METHODS: Patients with RCC, unsuitable for, or who declined conventional therapy, were eligible. A single transfemoral micro-catheter administration of yttrium-90 (Y-90) resin microspheres (SIR-Spheres) was delivered super selectively via the renal artery to the tumour at intended radiation doses of 75, 100, 150, 200, 300 Gy and a final cohort with a procedural endpoint of "imminent stasis," in a dose-escalation design. Post-SIRT follow-up was 12 months. Study endpoints included safety and toxicity 30-days and 12-months post-SIRT and tumour response (RECIST v1.1). RESULTS: In total, 21 patients were enrolled, mean (SD) age was 75 (9.3) years, WHO performance status was 0 in 81%, 12 (57%) had stage 3 chronic kidney disease, and 7 (33%) had prior contralateral nephrectomy. Overall, 71% of patients completed 12 months of follow-up. Intended doses were delivered without any dose-limiting toxicity. Seventeen out of 21 (81%) patients experienced an adverse event (AE) from any cause within 30 days post-SIRT; all SIRT-related AEs were grade 1 to 2. Best overall tumour responses were partial response 1/21 (4.8%), stable disease 19/21 (90.5%) and progressive disease 1/21 (4.8%). CONCLUSION: This study demonstrated good tolerability of SIRT at all dose levels including "imminent stasis" in treating primary tumours in RCC patients otherwise unsuitable for conventional therapy. SIRT with Y-90 resin microspheres may be a feasible treatment option for RCC.

The evolving role of multi-parametric MRI in the evaluation of bladder cancer: Revealing what lies beneath the surface.

Conventional staging for bladder cancer involves CT and TURBT, and is thought to understage up to fifty-percent of T1 tumours. This report details the case of a 64-year-old male who whilst on cystoscopic surveillance for high grade bladder cancer, underwent a multi-parametric MRI Bladder due to clinical suspicion of occult muscle invasive disease. Despite minimal urothelial changes at cystoscopy, MRI demonstrated a well-defined T3 lesion. The patient proceeded to radical cystectomy and final pathology verified the MRI findings. The role of MRI in bladder cancer is yet to be defined but should be considered if clinical suspicion for understaging exists.

The diagnostic utility of diffusion weighted MRI imaging and ADC ratio to distinguish benign from malignant renal masses: sorting the kittens from the tigers.

BACKGROUND: MRI is playing an increasing role in risk stratification and non-invasive diagnosis of the undifferentiated small renal mass. This study was designed to assess the reliability of MRI in diagnostic evaluation of renal masses, specifically characterising lesions with diffusion weighted imaging (DWI) and apparent diffusion coefficient (ADC) values. METHODS: This is a retrospective analysis of patients undergoing MRI as part of their clinical workup for a renal mass suspicious for renal cell carcinoma (RCC) on CT or ultrasound followed by biopsy and/or surgical excision. All cases were conducted on 3 Tesla MRI, with conventional breath-held sequences, DWI and dynamic contrast enhanced phases. Tumour regions of interest were evaluated on ADC maps and compared with T2 weighted and post-contrast images. RESULTS: Of the 66 renal tumours included, 33 (50.0%) were Clear Cell RCC, 11 (16.7%) were Oncocytoma, nine (13.6%) were Angiomyolipoma (AML), nine (13.6%) were Papillary RCC and four (6.1%) were Chromophobe RCC. Oncocytoma had the largest ADC values, significantly larger than AMLs and all RCC subtypes (p < 0.001). The average ADC value was also significantly larger in Clear Cell RCCs compared to AMLs, and other RCC subtypes (p < 0.001). CONCLUSIONS: MRI with DWI/ADC imaging may aid the differentiation of oncocytomas from RCCs and stratify RCC subtypes, Further studies are required to validate these findings. TRIAL REGISTRATION: Not applicable/retrospective study.

Chemical shift imaging in the identification of those renal tumours that contain microscopic fat and the utility of multiparametric MRI in their differentiation.

INTRODUCTION: The aim of this study was to assess the qualitative and MRI findings of renal tumours, to determine which lesions contain microscopic fat, one of the potential differentiating factors between tumour types. METHODS: 73 patients who underwent 3 Tesla MRI including chemical shift imaging, with subsequent biopsy or excision for histopathological diagnosis, were included in the study. The images were reviewed for a decrease in signal intensity (SI) on the opposed phase compared with the in-phase gradient echo T1 images, indicating the presence of microscopic fat. The chemical shift index was then calculated as a percentage of SI change and compared with the pathological diagnosis. RESULTS: In total, 38 (52%) of lesions demonstrated a decrease in SI, consistent with microscopic fat. Microscopic fat was found in 28 (80%) clear cell renal cell carcinomas (RCCs), 6 (66.7%) angiomyolipomas, 2 (20%) papillary RCCs, 1 (20%) chromophobe RCC and 1 (9.1%) oncocytoma. Pairwise comparison of means indicated that the amount of microscopic fat was significantly larger only for angiomyolipomas compared with clear cell RCCs (P < 0.001) and other renal lesions (P < 0.001). CONCLUSIONS: A decrease in SI on opposed phase compared with in-phase chemical shift imaging favours the diagnosis of either clear cell RCC or an angiomyolipoma. When combined with other parameters in mpMRI, this may aid differentiation of benign from malignant tumours and differentiation of aggressive from indolent RCC subtypes. This may be of value where biopsy is non-diagnostic, not feasible due to location or in high-risk patients.

CD40 Enhances Type I Interferon Responses Downstream of CD47 Blockade, Bridging Innate and Adaptive Immunity.

Disrupting the binding of CD47 to SIRPα has emerged as a promising immunotherapeutic strategy for advanced cancers by potentiating antibody-dependent cellular phagocytosis (ADCP) of targeted antibodies. Preclinically, CD47/SIRPα blockade induces antitumor activity by increasing the phagocytosis of tumor cells by macrophages and enhancing the cross-presentation of tumor antigens to CD8+ T cells by dendritic cells; both of these processes are potentiated by CD40 signaling. Here we generated a novel, two-sided fusion protein incorporating the extracellular domains of SIRPα and CD40L, adjoined by a central Fc domain, termed SIRPα-Fc-CD40L. SIRPα-Fc-CD40L bound CD47 and CD40 with high affinity and activated CD40 signaling in the absence of Fc receptor cross-linking. No evidence of hemolysis, hemagglutination, or thrombocytopenia was observed in vitro or in cynomolgus macaques. Murine SIRPα-Fc-CD40L outperformed CD47 blocking and CD40 agonist antibodies in murine CT26 tumor models and synergized with immune checkpoint blockade of PD-1 and CTLA4. SIRPα-Fc-CD40L activated a type I interferon response in macrophages and potentiated the activity of ADCP-competent targeted antibodies both in vitro and in vivo These data illustrated that whereas CD47/SIRPα inhibition could potentiate tumor cell phagocytosis, CD40-mediated activation of a type I interferon response provided a bridge between macrophage- and T-cell-mediated immunity that significantly enhanced durable tumor control and rejection.

Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy.

Simultaneous blockade of immune checkpoint molecules and co-stimulation of the TNF receptor superfamily (TNFRSF) is predicted to improve overall survival in human cancer. TNFRSF co-stimulation depends upon coordinated antigen recognition through the T cell receptor followed by homotrimerization of the TNFRSF, and is most effective when these functions occur simultaneously. To address this mechanism, we developed a two-sided human fusion protein incorporating the extracellular domains (ECD) of PD-1 and OX40L, adjoined by a central Fc domain, termed PD1-Fc-OX40L. The PD-1 end of the fusion protein binds PD-L1 and PD-L2 with affinities of 2.08 and 1.76 nM, respectively, and the OX40L end binds OX40 with an affinity of 246 pM. High binding affinity on both sides of the construct translated to potent stimulation of OX40 signaling and PD1:PD-L1/L2 blockade, in multiple in vitro assays, including improved potency as compared to pembrolizumab, nivolumab, tavolixizumab and combinations of those antibodies. Furthermore, when activated human T cells were co-cultured with PD-L1 positive human tumor cells, PD1-Fc-OX40L was observed to concentrate to the immune synapse, which enhanced proliferation of T cells and production of IL-2, IFNγ and TNFα, and led to efficient killing of tumor cells. The therapeutic activity of PD1-Fc-OX40L in established murine tumors was significantly superior to either PD1 blocking, OX40 agonist, or combination antibody therapy; and required CD4+ T cells for maximum response. Importantly, all agonist functions of PD1-Fc-OX40L are independent of Fc receptor cross-linking. Collectively, these data demonstrate a highly potent fusion protein that is part of a platform, capable of providing checkpoint blockade and TNFRSF costimulation in a single molecule, which uniquely localizes TNFRSF costimulation to checkpoint ligand positive tumor cells.

Frequency of Angiomyolipomas Among Echogenic Nonshadowing Renal Masses (> 4 mm) Found at Ultrasound and the Utility of MRI for Diagnosis.

OBJECTIVE: The purpose of this study is to evaluate what percentage of echogenic nonshadowing renal lesions larger than 4 mm found at ultrasound are angiomyolipomas (AMLs) and to review how to diagnose AMLs, with particular emphasis on the increasing role played by MRI. MATERIALS AND METHODS: The study data were obtained at a single institution over a period of 45 months. Although some patients were being reviewed for specific symptoms, such as hematuria, pain, or recurrent urinary tract infections, most of the findings were incidental. Follow-up data on 158 lesions in 132 patients were available. Confirmation of diagnosis was made with follow-up imaging or with histopathologic examination. RESULTS: Ninety-eight (62.0%) of the lesions were AMLs, eight (5.1%) were renal cell carcinomas, three (1.9%) were oncocytomas, 17 (10.8%) were artifacts, seven (4.4%) were fat, five (3.2%) were calculi, another eight (5.1%) were scars, and 12 (7.6%) were complicated cysts. The mean age of patients with AML was significantly lower than that of patients without AML (61.71 [SD, 13.25] years vs 68.80 [SD, 17.85] years; p = 0.005). There was a female association with AMLs (p < 0.001). CONCLUSION: Echogenic nonshadowing renal lesions larger than 4 mm seen at ultrasound should not be assumed to represent an AML without follow-up because a percentage of renal cell carcinomas will be missed. Although certain ultrasound features can be useful in differentiating an AML from a renal cell carcinoma and CT is frequently diagnostic, an understanding of MRI is important because it can potentially detect lipid-poor AMLs.

Histological Comparison of Kidney Tissue Following Radioembolization with Yttrium-90 Resin Microspheres and Embolization with Bland Microspheres.

BACKGROUND: Intra-arterial brachytherapy with yttrium-90 (90Y) resin microspheres (radioembolization) is a procedure to selectively deliver high-dose radiation to tumors. The purpose of this research was to compare the radioembolic effect of 90Y-radioembolization versus the embolic effect of bland microspheres in the porcine kidney model. METHODS: In each of six pigs, ~25-33 % of the kidney volume was embolized with 90Y resin microspheres and an equivalent number of bland microspheres in the contralateral kidney. Kidney volume was estimated visually from contrast-enhanced fluoroscopy imaging. Morphologic and histologic analysis was performed 8-9 weeks after the procedure to assess the locations of the microspheres and extent of tissue necrosis from 90Y-radioembolization and bland embolization. A semi-quantified evaluation of the non-acute peri-particle and perivascular tissue reaction was conducted. All guidelines for the care and use of animals were followed. RESULTS: Kidneys embolized with 90Y-radioembolization decreased in mass by 30-70 % versus the contralateral kidney embolized with bland microspheres. These kidneys showed significant necrosis/fibrosis, avascularization, and glomerular atrophy in the immediate vicinity of the 90Y resin microspheres. By contrast, glomerular changes were not observed, even with clusters of bland microspheres in afferent arterioles. Evidence of a foreign body reaction was recorded in some kidneys with bland microspheres, and subcapsular scarring/infarction only with the highest load (4.96 × 106) of bland microspheres. CONCLUSION: This study showed that radioembolization with 90Y resin microspheres produces localized necrosis/fibrosis and loss of kidney mass in a porcine kidney model. This result supports the study of 90Y resin microspheres for the localized treatment of kidney tumors.

Gp96-Ig/Costimulator (OX40L, ICOSL, or 4-1BBL) Combination Vaccine Improves T-cell Priming and Enhances Immunity, Memory, and Tumor Elimination.

T-cell costimulation typically occurs in a defined microenvironment that is not recapitulated by agonistic antibody therapy. To deliver such stimulation under more favorable conditions, we investigated whether an allogeneic cell-based vaccine that secreted Fc-OX40L, Fc-ICOSL, or Fc-4-1BBL would activate and expand T cells comparably with systemically administered agonist antibodies. Among these costimulators, locally secreted Fc-OX40L provided superior priming of antigen-specific CD8(+) T cells, compared with combinations with OX40 antibodies or vaccine alone. Vaccine-expressed Fc-OX40L also stimulated IFNγ, TNFα, granzyme B, and IL2 by antigen-specific CD8(+) T cells similarly to OX40 antibodies, without off-target consequences such as proinflammatory cytokine induction. Vaccine-secreted Fc-OX40L increased CD127(+)KLRG-1(-) memory precursor cells during the contraction phase, resulting in improved proliferation upon secondary antigen challenge, as compared with OX40 antibody. A cell-based vaccine cosecreting gp96-Ig and Fc-OX40L led to even more pronounced tumor control, complete tumor rejection, and increased tumor antigen-specific T-cell proliferation, including in tumor-infiltrating lymphocytes, as compared with combinations of gp96-Ig vaccine and OX40 antibodies, in mice with established melanoma or colorectal carcinoma. These data suggest that local modulation of the vaccine microenvironment has unexpected advantages over systemic costimulation with agonistic antibodies, which may simplify the clinical translation of such combination immunotherapies into humans. Cancer Immunol Res; 4(9); 766-78. ©2016 AACR.

Phosphorylation of mouse SAMHD1 regulates its restriction of human immunodeficiency virus type 1 infection, but not murine leukemia virus infection.

Human SAMHD1 (hSAMHD1) restricts HIV-1 infection in non-dividing cells by depleting intracellular dNTPs to limit viral reverse transcription. Phosphorylation of hSAMHD1 at threonine (T) 592 by cyclin-dependent kinase (CDK) 1 and CDK2 negatively regulates HIV-1 restriction. Mouse SAMHD1 (mSAMHD1) restricts HIV-1 infection in non-dividing cells, but whether its phosphorylation regulates retroviral restriction is unknown. Here we identified six phospho-sites of mSAMHD1, including T634 that is homologous to T592 of hSAMHD1 and phosphorylated by CDK1 and CDK2. We found that wild-type (WT) mSAMHD1 and a phospho-ablative mutant, but not a phospho-mimetic mutant, restricted HIV-1 infection in differentiated U937 cells. Murine leukemia virus (MLV) infection of dividing NIH3T3 cells was modestly restricted by mSAMHD1 WT and phospho-mutants, but not by a dNTPase-defective mutant. Our results suggest that phosphorylation of mSAMHD1 at T634 by CDK1/2 negatively regulates its HIV-1 restriction in differentiated cells, but does not affect its MLV restriction in dividing cells.

A Novel Histone Deacetylase Inhibitor, AR-42, Reactivates HIV-1 from Chronically and Latently Infected CD4+ T-cells.

Human immunodeficiency virus type 1 (HIV-1) latency is a major barrier to a cure of AIDS. Latently infected cells harbor an integrated HIV-1 genome but are not actively producing HIV-1. Histone deacetylase (HDAC) inhibitors, such as vorinostat (SAHA), have been shown to reactivate latent HIV-1. AR-42, a modified HDAC inhibitor, has demonstrated efficacy against malignant melanoma, meningioma, and acute myeloid leukemia and is currently used in clinical trials for non-Hodgkin's lymphoma and multiple myeloma. In this study, we evaluated the ability of AR-42 to reactivate HIV-1 in the two established CD4+ T-cell line models of HIV-1 latency. In HIV-1 chronically infected ACH-2 cells, AR-42-induced histone acetylation was more potent and robust than that of vorinostat. Although AR-42 and vorinostat were equipotent in their ability to reactivate HIV-1, AR-42-induced maximal HIV-1 reactivation was twofold greater than vorinostat in ACH-2 and J-Lat (clone 9.2) cells. These data provide rationale for assessing the efficacy of AR-42-mediated HIV-1 reactivation within primary CD4+ T-cells.