Efficacy of LCMV-based cancer immunotherapies is unleashed by intratumoral injections of polyI:C.

BACKGROUND: Lymphocytic choriomeningitis virus (LCMV) belongs to the Arenavirus family known for inducing strong cytotoxic T-cell responses in both mice and humans. LCMV has been engineered for the development of cancer immunotherapies, currently undergoing evaluation in phase I/II clinical trials. Initial findings have demonstrated safety and an exceptional ability to activate and expand tumor-specific T lymphocytes. Combination strategies to maximize the antitumor effectiveness of LCMV-based immunotherapies are being explored. METHODS: We assessed the antitumor therapeutic effects of intratumoral administration of polyinosinic:polycytidylic acid (poly(I:C)) and systemic vaccination using an LCMV-vector expressing non-oncogenic versions of the E6 and E7 antigens of human papillomavirus 16 (artLCMV-E7E6) in a bilateral model engrafting TC-1/A9 cells. This cell line, derived from the parental TC-1, exhibits low MHC class I expression and is highly immune-resistant. The mechanisms underlying the combination's efficacy were investigated through bulk RNA-seq, flow cytometry analyses of the tumor microenvironment, selective depletions using antibodies and clodronate liposomes, Batf3 deficient mice, and in vivo bioluminescence experiments. Finally, we assessed the antitumor effectiveness of the combination of artLCMV-E7E6 with BO-112, a GMP-grade poly(I:C) formulated in polyethyleneimine, currently under evaluation in clinical trials. RESULTS: Intratumoral injection of poly(I:C) enhanced the antitumor efficacy of artLCMV-E7E6 in both injected and non-injected tumor lesions. The combined treatment resulted in a significant delay in tumor growth and often complete eradication of several tumor lesions, leading to significantly improved survival compared with monotherapies. While intratumoral administration of poly(I:C) did not impact LCMV vector biodistribution or transgene expression, it significantly modified leucocyte infiltrates within the tumor microenvironment and amplified systemic efficacy through proinflammatory cytokines/chemokines such as CCL3, CCL5, CXCL10, TNF, IFNα, and IL12p70. Upregulation of MHC on tumor cells and a reconfiguration of the gene expression programs related to tumor vasculature, leucocyte migration, and the activation profile of tumor-infiltrating CD8+ T lymphocytes were observed. Indeed, the antitumor effect relied on the functions of CD8+ T lymphocytes and macrophages. The synergistic efficacy of the combination was further confirmed when BO-112 was included. CONCLUSION: Intratumoral injection of poly(I:C) sensitizes MHClow tumors to the antitumor effects of artLCMV-E7E6, resulting in a potent therapeutic synergy.

Intraperitoneal administration of a modified vaccinia virus Ankara confers single-chain interleukin-12 expression to the omentum and achieves immune-mediated efficacy against peritoneal carcinomatosis.

BACKGROUND: Peritoneal carcinomatosis is an advanced stage of cancer in which the disease has spread to the peritoneal cavity. In order to restore antitumor immunity subverted by tumor cells in this location, we evaluated intraperitoneal administrations of modified vaccinia virus Ankara (MVA) engineered to express single-chain interleukin 12 (scIL-12) to increase antitumor immune responses. METHODS: MVA encoding scIL-12 (MVA.scIL-12) was evaluated against peritoneal carcinomatosis models based on intraperitoneal engraftment of tumor cells. CD8-mediated immune responses, elucidated antitumor efficacy, and safety were evaluated following intravenous, intratumoral, or intraperitoneal administration of the viral vector. The immune response was measured by ELISpot (enzyme-linked immunosorbent spot), RNA sequencing, flow cytometry, intravital microscopy, and depletion of lymphocyte subsets with monoclonal antibodies. Safety was assessed by body-weight follow-up and blood testing. Tissue tropism on intravenous or intraperitoneal administration was assessed by bioluminescence analysis using a reporter MVA encoding luciferase. RESULTS: Intraperitoneal or locoregional administration, but not other routes of administration, resulted in a potent immune response characterized by increased levels of tumor-specific CD8+ T lymphocytes with the ability to produce both interferon-γ and tumor necrosis factor-α. The antitumor immune response was detectable not only in the peritoneal cavity but also systemically. As a result of intraperitoneal treatment, a single administration of MVA.scIL-12 encoding scIL-12 completely eradicated MC38 tumors implanted in the peritoneal cavity and also protected cured mice from subsequent subcutaneous rechallenges. Bioluminescence imaging using an MVA encoding luciferase revealed that intraperitoneal administration targets transgene to the omentum. The omentum is considered a key tissue in immune protection of the peritoneal cavity. The safety profile of intraperitoneal administration was also better than that following intravenous administration since no weight loss or hematological toxicity was observed when the vector was locally delivered into the peritoneal cavity. CONCLUSION: Intraperitoneal administration of MVA vectors encoding scIL-12 targets the omentum, which is the tissue where peritoneal carcinomatosis usually begins. MVA.scIL-12 induces a potent tumor-specific immune response that often leads to the eradication of experimental tumors disseminated to the peritoneal cavity.

Intratumoral injection of IL-12-encoding mRNA targeted to CSFR1 and PD-L1 exerts potent anti-tumor effects without substantial systemic exposure.

IL-12 is a potent cytokine for cancer immunotherapy. However, its systemic delivery as a recombinant protein has shown unacceptable toxicity in the clinic. Currently, the intratumoral injection of IL-12-encoding mRNA or DNA to avoid such side effects is being evaluated in clinical trials. In this study, we aimed to improve this strategy by further favoring IL-12 tethering to the tumor. We generated in vitro transcribed mRNAs encoding murine single-chain IL-12 fused to diabodies binding to CSF1R and/or PD-L1. These targeted molecules are expressed in the tumor microenvironment, especially on myeloid cells. The binding capacity of chimeric constructs and the bioactivity of IL-12 were demonstrated in vitro and in vivo. Doses as low as 0.5 µg IL-12-encoding mRNA achieved potent antitumor effects in subcutaneously injected B16-OVA and MC38 tumors. Treatment delivery was associated with increases in IL-12p70 and IFN-γ levels in circulation. Fusion of IL-12 to the diabodies exerted comparable efficacy against bilateral tumor models. However, it achieved tethering to myeloid cells infiltrating the tumor, resulting in nearly undetectable systemic levels of IL-12 and IFN-γ. Overall, tethering IL-12 to intratumoral myeloid cells in the mRNA-transferred tumors achieves similar efficacy while reducing the dangerous systemic bioavailability of IL-12.

Intratumoral neoadjuvant immunotherapy based on the BO-112 viral RNA mimetic.

BO-112 is a poly I:C-based viral mimetic that exerts anti-tumor efficacy when intratumorally delivered in mouse models. Intratumoral BO-112 synergizes in mice with systemic anti-PD-1 mAbs and this combination has attained efficacy in PD1-refractory melanoma patients. We sought to evaluate the anti-tumor efficacy of BO-112 pre-surgically applied in neoadjuvant settings to mouse models. We have observed that repeated intratumoral injections of BO-112 prior to surgical excision of the primary tumor significantly reduced tumor metastasis from orthotopically implanted 4T1-derived tumors and subcutaneous MC38-derived tumors in mice. Such effects were enhanced when combined with systemic anti-PD-1 mAb. The anti-tumor efficacy of this neoadjuvant immunotherapy approach depended on the presence of antigen-specific effector CD8 T cells and cDC1 antigen-presenting cells. Since BO-112 has been successful in phase-two clinical trials for metastatic melanoma, these results provide a strong rationale for translating this pre-surgical strategy into clinical settings, especially in combination with standard-of-care checkpoint inhibitors.

mRNAs encoding IL-12 and a decoy-resistant variant of IL-18 synergize to engineer T cells for efficacious intratumoral adoptive immunotherapy.

Interleukin-12 (IL-12) gene transfer enhances the therapeutic potency of adoptive T cell therapies. We previously reported that transient engineering of tumor-specific CD8 T cells with IL-12 mRNA enhanced their systemic therapeutic efficacy when delivered intratumorally. Here, we mix T cells engineered with mRNAs to express either single-chain IL-12 (scIL-12) or an IL-18 decoy-resistant variant (DRIL18) that is not functionally hampered by IL-18 binding protein (IL-18BP). These mRNA-engineered T cell mixtures are repeatedly injected into mouse tumors. Pmel-1 T cell receptor (TCR)-transgenic T cells electroporated with scIL-12 or DRIL18 mRNAs exert powerful therapeutic effects in local and distant melanoma lesions. These effects are associated with T cell metabolic fitness, enhanced miR-155 control on immunosuppressive target genes, enhanced expression of various cytokines, and changes in the glycosylation profile of surface proteins, enabling adhesiveness to E-selectin. Efficacy of this intratumoral immunotherapeutic strategy is recapitulated in cultures of tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T cells on IL-12 and DRIL18 mRNA electroporation.

Intracavitary adoptive transfer of IL-12 mRNA-engineered tumor-specific CD8+ T cells eradicates peritoneal metastases in mouse models.

Previous studies have shown that local delivery of tumor antigen-specific CD8+ T lymphocytes engineered to transiently express single-chain IL-12 mRNA is highly efficacious. Peritoneal dissemination of cancer is a frequent and often fatal patient condition usually diagnosed when the tumor burden is too large and hence uncontrollable with current treatment options. In this study, we have modeled intracavitary adoptive T cell therapy with OVA-specific OT-I T cells electroporated with IL-12 mRNA to treat B16-OVA and PANC02-OVA tumor spread in the peritoneal cavity. Tumor localization in the omentum and the effects of local T-cell encounter with the tumor antigens were monitored, the gene expression profile evaluated, and the phenotypic reprogramming of several immune subsets was characterized. Intraperitoneal administration of T cells promoted homing to the omentum more effectively than intravenous administration. Transient IL-12 expression was responsible for a favorable reprogramming of the tumor immune microenvironment, longer persistence of transferred T lymphocytes in vivo, and the development of immunity to endogenous antigens following primary tumor eradication. The efficacy of the strategy was at least in part recapitulated with the adoptive transfer of lower affinity transgenic TCR-bearing PMEL-1 T lymphocytes to treat the aggressive intraperitoneally disseminated B16-F10 tumor. Locoregional adoptive transfer of transiently IL-12-armored T cells appears to offer promising therapeutic advantages in terms of anti-tumor efficacy to treat peritoneal carcinomatosis.

Intratumoral Gene Transfer of mRNAs Encoding IL12 in Combination with Decoy-Resistant IL18 Improves Local and Systemic Antitumor Immunity.

IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

Synergistic antitumor response with recombinant modified virus Ankara armed with CD40L and CD137L against peritoneal carcinomatosis.

Recombinant-modified vaccinia virus Ankara (rMVA) is known to elicit potent antitumor immune responses in preclinical models due to its inherent ability to activate the innate immune system and the activation of adaptive responses mediated by the expression of tumor antigens and costimulus-providing molecules, such as CD40L and CD137L. Here, we evaluated different rMVA vectors in preclinical peritoneal carcinomatosis models (ID8.OVA-Vegf/GFP and MC38). We compared rMVA vectors expressing a tumor antigen (OVA or gp70) either alone or co-expressed with CD40L or/and CD137L. In tumor-free mice, the vector coding for the triple combination was only slightly superior, whereas, in tumor-bearing animals, we observed a synergistic induction of T lymphocytes specific against vector-encoded and non-encoded tumor-associated antigens. The enhanced activation of the immune response was associated with improved survival in mice with peritoneal carcinomatosis treated with a rMVA vector encoding both CD40L and CD137L. Thus, the triple transgene combination in vaccinia viral vectors represents a promising strategy for the treatment of peritoneal carcinomatosis.

Intratumoral co-injection of the poly I:C-derivative BO-112 and a STING agonist synergize to achieve local and distant anti-tumor efficacy.

BACKGROUND: BO-112 is a nanoplexed form of polyinosinic:polycytidylic acid that acting on toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA5) and protein kinase RNA-activated (PKR) elicits rejection of directly injected transplanted tumors, but has only modest efficacy against distant untreated tumors. Its clinical activity has also been documented in early phase clinical trials. The 5,6-dimethylxanthenone-4-acetic acid (DMXAA) stimulator of interferon genes (STING) agonist shows a comparable pattern of efficacy when used via intratumoral injections. METHODS: Mice subcutaneously engrafted with bilateral MC38 and B16.OVA-derived tumors were treated with proinflammatory immunotherapy agents known to be active when intratumorally delivered. The combination of BO-112 and DMXAA was chosen given its excellent efficacy and the requirements for antitumor effects were studied on selective depletion of immune cell types and in gene-modified mouse strains lacking basic leucine zipper ATF-like transcription factor 3 (BATF3), interferon-α/ß receptor (IFNAR) or STING. Spatial requirements for the injections were studied in mice bearing three tumor lesions. RESULTS: BO-112 and DMXAA when co-injected in one of the lesions of mice bearing concomitant bilateral tumors frequently achieved complete local and distant antitumor efficacy. Synergistic effects were contingent on CD8 T cell lymphocytes and dependent on conventional type 1 dendritic cells, responsiveness to type I interferon (IFN) and STING function in the tumor-bearing host. Efficacy was preserved even if BO-112 and DMXAA were injected in separate lesions in a manner able to control another untreated third-party tumor. Efficacy could be further enhanced on concurrent PD-1 blockade. CONCLUSION: Clinically feasible co-injections of BO-112 and a STING agonist attain synergistic efficacy able to eradicate distant untreated tumor lesions.

UPEFinder: A Bioinformatic Tool for the Study of Uncharacterized Proteins Based on Gene Expression Correlation and the PageRank Algorithm.

The Human Proteome Project (HPP) is leading the international effort to characterize the human proteome. Although the main goal of this project was first focused on the detection of missing proteins, a new challenge arose from the need to assign biological functions to the uncharacterized human proteins and describe their implications in human diseases. Not only the proteins with experimental evidence (uPE1 proteins) but also the uncharacterized missing proteins (uMPs) were the objects of study in this challenge, neXt-CP50. In this work, we developed a new bioinformatic approach to infer biological annotations for the uPE1 proteins and uMPs based on a "guilt-by-association" analysis using public RNA-Seq data sets. We used the correlation of these proteins with the well-characterized PE1 proteins to construct a network. In this way, we applied the PageRank algorithm to this network to identify the most relevant nodes, which were the biological annotations of the uncharacterized proteins. All of the generated information was stored in a database. In addition, we implemented the web application UPEFinder (https://upefinder.proteored.org) to facilitate the access to this new resource. This information is especially relevant for the researchers of the HPP who are interested in the generation and validation of new hypotheses about the functions of these proteins. Both the database and the web application are publicly available (https://github.com/ubioinformat/UPEfinder).

Proteogenomics in the context of the Human Proteome Project (HPP).

INTRODUCTION: The technological and scientific progress performed in the Human Proteome Project (HPP) has provided to the scientific community a new set of experimental and bioinformatic methods in the challenging field of shotgun and SRM/MRM-based Proteomics. The requirements for a protein to be considered experimentally validated are now well-established, and the information about the human proteome is available in the neXtProt database, while targeted proteomic assays are stored in SRMAtlas. However, the study of the missing proteins continues being an outstanding issue. Areas covered: This review is focused on the implementation of proteogenomic methods designed to improve the detection and validation of the missing proteins. The evolution of the methodological strategies based on the combination of different omic technologies and the use of huge publicly available datasets is shown taking the Chromosome 16 Consortium as reference. Expert commentary: Proteogenomics and other strategies of data analysis implemented within the C-HPP initiative could be used as guidance to complete in a near future the catalog of the human proteins. Besides, in the next years, we will probably witness their use in the B/D-HPP initiative to go a step forward on the implications of the proteins in the human biology and disease.