Oncolytic Adenovirus Coding for a Variant Interleukin 2 (vIL-2) Cytokine Re-Programs the Tumor Microenvironment and Confers Enhanced Tumor Control

The notion of developing variants of the classic interleukin 2 (IL-2) cytokine has emerged from the limitations observed with the systemic use of human IL-2 in the clinic: severe adverse events accompanied by low therapeutic response rate in treated patients. Modifications made in the IL-2 receptor-binding structure leads to preferential binding of IL-2 variant cytokine to receptors on effector anti-tumor lymphocytes over T regulatory (TReg) cells. Because of their inherent immunogenicity, oncolytic adenoviruses are useful for expression of immunomodulatory molecules in tumors, for induction of a pro-inflammatory state in the tumor microenvironment. In the present study, we constructed an adenovirus coding for an IL-2 variant (vIL-2) protein, Ad5/3-E2F-d24-vIL2. Functionality of the new virus was tested in vitro, and anti-tumor efficacy and mechanism of action studies were performed in immunocompetent hamsters bearing pancreatic tumors. Ad5/3-E2F-d24-vIL2 treatment elicited efficient anti-tumor response, with 62.5% monotherapy complete response. Moreover, it promoted substantial repression of genes associated with myeloid cells mediated immunosuppression (CD11b, ARG1, CD206). This was seen in conjunction with upregulation of genes associated with tumor-infiltrating lymphocyte (TIL) cytotoxicity (CD3G, SAP, PRF1, GZMM and GZMK). In summary, Ad5/3-E2F-d24-vIL2 demonstrates therapeutic potential by counteracting immunosuppression and in efficiently coordinating lymphocytes mediated anti-tumor response in immunosuppressive tumors. Thus, Ad5/3-E2F-d24-vIL2 is a promising candidate for translation into clinical trials in human immunosuppressive solid tumors.

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Duration of response after DEB-TACE compared to lipiodol-TACE in HCC-naïve patients: a propensity score matching analysis

European Radiology ◽

10.1007/s00330-021-07905-x ◽

2021 ◽

Author(s):

Irene Bargellini ◽

Valentina Lorenzoni ◽

Giulia Lorenzoni ◽

Paola Scalise ◽

Gianni Andreozzi ◽

...

Keyword(s):

Propensity Score ◽

Tumor Progression ◽

Tumor Response ◽

Complete Response ◽

Tumor Burden ◽

Local Tumor Control ◽

Lower Number ◽

Tumor Control ◽

Duration Of Response

Abstract Objectives To retrospectively compare long-term outcomes of first-line drug-eluting particle (DEB)- transarterial chemoembolization (TACE) and lipiodol-TACE, in patients with unresectable hepatocellular (HCC). Methods We retrospectively reviewed our database to identify adult patients with treatment-naïve unresectable HCC, who underwent TACE from 2006 to 2013. Patients were excluded in the absence of complete medical records relative to first TACE, 1-month follow-up, and/or sufficient follow-up data. Periprocedural complications, duration of hospitalization, 1-month tumor response by mRECIST, time to tumor progression (TTP) and target tumor progression (TTTP), and overall survival (OS) were evaluated. Results Out of an initial series of 656 patients, 329 patients were excluded for unavailability of sufficient baseline and/or follow-up data. The remaining 327 patients underwent either lipiodol-TACE (n = 160) or DEB-TACE (n = 167). Patients treated with lipiodol-TACE had a significantly higher tumor burden. By propensity score, patients were matched according to baseline differences (BCLC stage, uninodular or multinodular HCC, and unilobar or bilobar HCC), resulting in 101 patients in each treatment group. Lipiodol-TACE was associated with a significantly higher incidence of adverse events (p = 0.03), and longer hospitalization (mean, 2.5 days vs 1.9 days; p = 0.03), while tumor response, TTP, and OS were comparable. In patients achieving 1-month complete response (CR) of target tumor, TTTP was significantly (p = 0.009) longer after DEB-TACE compared to lipiodol-TACE (median, 835 vs 353 days), resulting in a lower number of re-treatments during the entire follow-up (0.75 vs 1.6, p = 0.01). Conclusion Compared to lipiodol-TACE, DEB-TACE offers higher tolerability, reduced hospitalization, and more durable target tumor response after CR. Key Points • Compared to lipiodol-TACE, DEB-TACE is better tolerated and has reduced side effects, which translates into shorter hospitalization. • When complete radiological response according to the mRECIST is obtained 1 month after the procedure, DEB-TACE offers a more durable local tumor control compared to lipiodol-TACE. • In these patients, the longer duration of response after DEB-TACE translates into a lower number of re-interventions.

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Antigen-Specific Regulatory T Cell Therapy in Autoimmune Diseases and Transplantation

Frontiers in Immunology ◽

10.3389/fimmu.2021.661875 ◽

2021 ◽

Vol 12 ◽

Author(s):

Claudia Selck ◽

Margarita Dominguez-Villar

Keyword(s):

Autoimmune Diseases ◽

Treg Cell ◽

Therapeutic Potential ◽

Treg Cells ◽

T Cell Therapy ◽

Cell Therapies ◽

Heterogenous Population ◽

Clinical Trial Results

Regulatory T (Treg) cells are a heterogenous population of immunosuppressive T cells whose therapeutic potential for the treatment of autoimmune diseases and graft rejection is currently being explored. While clinical trial results thus far support the safety and efficacy of adoptive therapies using polyclonal Treg cells, some studies suggest that antigen-specific Treg cells are more potent in regulating and improving immune tolerance in a disease-specific manner. Hence, several approaches to generate and/or expand antigen-specific Treg cells in vitro or in vivo are currently under investigation. However, antigen-specific Treg cell therapies face additional challenges that require further consideration, including the identification of disease-relevant antigens as well as the in vivo stability and migratory behavior of Treg cells following transfer. In this review, we discuss these approaches and the potential limitations and describe prospective strategies to enhance the efficacy of antigen-specific Treg cell treatments in autoimmunity and transplantation.

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Single-Cell Sequencing Reveals the Transcriptome and TCR Characteristics of pTregs and in vitro Expanded iTregs

Frontiers in Immunology ◽

10.3389/fimmu.2021.619932 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zhenzhen Hui ◽

Jiali Zhang ◽

Yu Zheng ◽

Lili Yang ◽

Wenwen Yu ◽

...

Keyword(s):

Colorectal Cancer ◽

Single Cell ◽

Cancer Patients ◽

Peripheral Blood ◽

Critical Role ◽

Interleukin 2 ◽

Treg Cells ◽

Single Cell Sequencing ◽

Colorectal Cancer Patients

Regulatory T cells (Tregs) play a critical role in the maintenance of immune tolerance and tumor evasion. However, the relative low proportion of these cells in peripheral blood and tissues has hindered many studies. We sought to establish a rapamycin-based in vitro Treg expansion procedure in patients diagnosed with colorectal cancer and perform single-cell sequencing to explore the characteristics of Treg cells. CD25+ cells enriched from peripheral blood mononuclear cells (PBMC) of colorectal tumor patients were cultured in X-VIVO15 medium, supplemented with 5% human AB serum, L-glutamine, rapamycin, interleukin-2 (IL-2), and Dynabeads human Treg expander for 21 days to expand Tregs. Treg cells with satisfactory phenotype and function were successfully expanded from CD4+CD25+ cells in patients with colorectal cancer. The median expansion fold was 75 (range, 20–105-fold), and >90.0% of the harvest cells were CD4+CD25+CD127dim/− cells. The ratio of CD4+CD25+Foxp3+ cells exceeded 60%. Functional assays showed that iTregs significantly inhibited CD8+T cell proliferation in vitro. Single-cell sequencing showed that the transcriptome of pTreg (CD4+CD25+CD127dim/− cells isolated from PBMC of colorectal cancer patients) and iTreg (CD4+CD25+CD127dim/− cells expanded in vitro according to the above regimen) cells were interlaced. pTregs exhibited enhanced suppressive function, whereas iTregs exhibited increased proliferative capacity. TCR repertoire analysis indicated minimal overlap between pTregs and iTregs. Pseudo-time trajectory analysis of Tregs revealed that pTregs were a continuum composed of three main branches: activated/effector, resting and proliferative Tregs. In contrast, in vitro expanded iTregs were a mixture of proliferating and activated/effector cells. The expression of trafficking receptors was also different in pTregs and iTregs. Various chemokine receptors were upregulated in pTregs. Activated effector pTregs overexpressed the chemokine receptor CCR10, which was not expressed in iTregs. The chemokine CCL28 was overexpressed in colorectal cancer and associated with poor prognosis. CCR10 interacted with CCL28 to mediate the recruitment of Treg into tumors and accelerated tumor progression. Depletion of CCR10+Treg cells from tumor microenvironment (TME) could be used as an effective treatment strategy for colorectal cancer patients. Our data distinguished the transcriptomic characteristics of different subsets of Treg cells and revealed the context-dependent functions of different populations of Treg cells, which was crucial to the development of alternative therapeutic strategies for Treg cells in autoimmune disease and cancer.

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Inhibitory Effect of Dihydroartemisinin on the Proliferation and Migration of Melanoma Cells and Experimental Lung Metastasis From Melanoma in Mice

Frontiers in Pharmacology ◽

10.3389/fphar.2021.727275 ◽

2021 ◽

Vol 12 ◽

Author(s):

Qi Zhang ◽

Linbo Jin ◽

Quanxin Jin ◽

Qiang Wei ◽

Mingyuan Sun ◽

...

Keyword(s):

Tumor Microenvironment ◽

Early Stage ◽

Metastatic Tumor ◽

Treg Cells ◽

Inhibitory Effect ◽

B16f10 Cells ◽

And Migration ◽

Migration Capacity

Melanoma is aggressive and can metastasize in the early stage of tumor. It has been proved that dihydroartemisinin (DHA) positively affects the treatment of tumors and has no apparent toxic and side effects. Our previous research has shown that DHA can suppress the formation of melanoma. However, it remains poorly established how DHA impacts the invasion and metastasis of melanoma. In this study, B16F10 and A375 cell lines and metastatic tumor models will be used to investigate the effects of DHA. The present results demonstrated that DHA inhibited the proliferative capacity in A375 and B16F10 cells. As expected, the migration capacity of A375 and B16F10 cells was also reduced after DHA administration. DHA alleviated the severity and histopathological changes of melanoma in mice. DHA induced expansion of CD8+CTL in the tumor microenvironment. By contrast, DHA inhibited Treg cells infiltration into the tumor microenvironment. DHA enhanced apoptosis of melanoma by regulating FasL expression and Granzyme B secretion in CD8+CTLs. Moreover, DHA impacts STAT3-induced EMT and MMPS in tumor tissue. Furthermore, Metabolomics analysis indicated that PGD2 and EPA significantly increased after DHA administration. In conclusion, DHA inhibited the proliferation, migration and metastasis of melanoma in vitro and in vivo. These results have important implications for the potential use of DHA in the treatment of melanoma in humans.

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Long-lasting analgesia via targeted in situ repression of NaV1.7 in mice

Science Translational Medicine ◽

10.1126/scitranslmed.aay9056 ◽

2021 ◽

Vol 13 (584) ◽

pp. eaay9056 ◽

Cited By ~ 1

Author(s):

Ana M. Moreno ◽

Fernando Alemán ◽

Glaucilene F. Catroli ◽

Matthew Hunt ◽

Michael Hu ◽

...

Keyword(s):

Chronic Pain ◽

Therapeutic Potential ◽

Thermal Hyperalgesia ◽

Structural Similarity ◽

Loss Of Function ◽

Sensory Afferents ◽

Inflammatory State ◽

Epigenetic Repression

Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)–dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of NaV1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.

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Echinochrome A Reduces Colitis in Mice and Induces In Vitro Generation of Regulatory Immune Cells

Marine Drugs ◽

10.3390/md17110622 ◽

2019 ◽

Vol 17 (11) ◽

pp. 622 ◽

Cited By ~ 1

Author(s):

Su-Jeong Oh ◽

Yoojin Seo ◽

Ji-Su Ahn ◽

Ye Young Shin ◽

Ji Won Yang ◽

...

Keyword(s):

Therapeutic Potential ◽

Sea Urchins ◽

Biological Activities ◽

Cardiac Injury ◽

Body Weight Loss ◽

Experimental Colitis ◽

Treg Cells ◽

Echinochrome A ◽

Intestinal Immune System

Echinochrome A (Ech A), a natural pigment extracted from sea urchins, is the active ingredient of a marine-derived pharmaceutical called ‘histochrome’. Since it exhibits several biological activities including anti-oxidative and anti-inflammatory effects, it has been applied to the management of cardiac injury and ocular degenerative disorders in Russia and its protective role has been studied for other pathologic conditions. In the present study, we sought to investigate the therapeutic potential of Ech A for inflammatory bowel disease (IBD) using a murine model of experimental colitis. We found that intravenous injection of Ech A significantly prevented body weight loss and subsequent lethality in colitis-induced mice. Interestingly, T cell proliferation was significantly inhibited upon Ech A treatment in vitro. During the helper T (Th) cell differentiation process, Ech A stimulated the generation regulatory T (Treg) cells that modulate the inflammatory response and immune homeostasis. Moreover, Ech A treatment suppressed the in vitro activation of pro-inflammatory M1 type macrophages, while inducing the production of M2 type macrophages that promote the resolution of inflammation and initiate tissue repair. Based on these results, we suggest that Ech A could provide a beneficial impact on IBD by correcting the imbalance in the intestinal immune system.

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Prodigiosin Modulates the Immune Response and Could Promote a Stable Atherosclerotic Lession in C57bl/6 Ldlr-/- Mice

International Journal of Molecular Sciences ◽

10.3390/ijms21176417 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6417 ◽

Cited By ~ 1

Author(s):

Alejandro Cuevas ◽

Nicolás Saavedra ◽

Luis A. Salazar ◽

Marcela F. Cavalcante ◽

Jacqueline C. Silva ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

Atherosclerotic Plaque ◽

Therapeutic Potential ◽

Inflammatory Marker ◽

Interleukin 2 ◽

Atheromatous Plaque ◽

In Vitro Tests ◽

Significant Difference

Atherosclerosis is a chronic inflammatory disease, whose progression and stability are modulated, among other factors, by an innate and adaptive immune response. Prodiginines are bacterial secondary metabolites with antiproliferative and immunomodulatory activities; however, their effect on the progression or vulnerability of atheromatous plaque has not been evaluated. This study assessed the therapeutic potential of prodigiosin and undecylprodigiosin on inflammatory marker expression and atherosclerosis. An in vitro and in vivo study was carried out. Migration, low-density lipoprotein (LDL) uptake and angiogenesis assays were performed on cell types involved in the pathophysiology of atherosclerosis. In addition, male LDL receptor null (Ldlr-/-) C57BL/6J mice were treated with prodigiosin or undecylprodigiosin for 28 days. Morphometric analysis of atherosclerotic plaques, gene expression of atherogenic factors in the aortic sinus and serum cytokine quantification were performed. The treatments applied had slight effects on the in vitro tests performed, highlighting the inhibitory effect on the migration of SMCs (smooth muscle cells). On the other hand, although no significant difference in atherosclerotic plaque progression was observed, gene expression of IL-4 and chemokine (C-C motif) ligand 2 (Ccl2) was downregulated. In addition, 50 µg/Kg/day of both treatments was sufficient to inhibit circulating tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in serum. These results suggested that prodigiosin and undecylprodigiosin modulated inflammatory markers and could have an impact in reducing atherosclerotic plaque vulnerability.

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3373 Modulation of Hedgehog Signaling Alters Immune Infiltration in Pancreatic Cancer

Journal of Clinical and Translational Science ◽

10.1017/cts.2019.40 ◽

2019 ◽

Vol 3 (s1) ◽

pp. 16-16

Author(s):

Nina Steele ◽

Valerie Irizarry-Negron ◽

Veerin Sirihorachai ◽

Samantha Kemp ◽

Eileen Carpenter ◽

...

Keyword(s):

Pancreatic Cancer ◽

Bone Marrow ◽

Tumor Microenvironment ◽

Mouse Model ◽

Tumor Cells ◽

Pancreatic Tumors ◽

Hh Signaling ◽

Immune Profiling

OBJECTIVES/SPECIFIC AIMS: Pancreatic ductal adenocarcinoma (PDA) has a dismal 5-year survival rate of 9%, making this disease one of the deadliest human malignancies (https://seer.cancer.gov/). Primary barriers to the treatment of pancreatic cancer include extensive stromal interactions and sustained immune suppression. Aberrant Hedgehog (HH) pathway activity is a hallmark of pancreatic tumorigenesis. Tumor-derived HH ligands signal in a paracrine fashion to the surrounding stroma to influence tumor growth. Expression of HH ligands increases during PDA progression, and previous work has shown that genetic deletion of Sonic HH (Shh) from the epithelium of mice with pancreatic tumors results in increased Indian HH (Ihh) expression. This research aims to investigate the translational impact of changes in immune infiltration following deletion of IHH in a preclinical mouse model of pancreatic cancer. METHODS/STUDY POPULATION: Ihh was deleted in tumor cells lines (IhhKO) derived from a genetically engineered mouse model of pancreatic cancer (LSL-KrasG12D/+;LSL-TrpR270H;P48-Cre), using CRISPR/Cas-9 gene editing to assess the role of Ihh in the tumor microenvironment. The level of HH signaling was determined using tumor cell co-cultures with Gli1lacZ fibroblasts (derived from mice with a lacZ reporter allele knocked into the Gli1 locus), in which Beta Galactosidase activity serves as a readout for HH signaling. WT and IhhKO tumor cells were orthotopically transplanted into the pancreas of syngeneic C57BL/6 mice. Human pancreas samples were obtained from surgical resection of pancreatic adenocarcinoma, or fine needle biopsy procedure (FNB). Immune profiling of mouse and human pancreatic tumors was performed using Cytometry Time-of-Flight analysis (CyTOF), and tumor composition was analyzed by single-cell RNA sequencing (scRNA seq). In vitro cultures with pancreatic fibroblasts treated with either WT or IhhKO tumor cell conditioned media (CM) were cultured with bone-marrow derived macrophages to assess tumor crosstalk. RESULTS/ANTICIPATED RESULTS: Tumor cells lacking Ihh were generated through CRISPR/Cas-9 deletion, and this was confirmed by qRT-PCR. Co-culture of IhhKO tumor cells with Gli1lacZ fibroblasts results in decreased Gli1 expression both in vitro and in vivo. Immune profiling revealed that tumors lacking Ihh have significantly fewer tumor associated macrophages (CD11b+/F4/80+/CD206+), resulting in decreased presence of immunosuppressive factors such as arginase 1 and PDL1. Immune phenotyping of human pancreatic tissues revealed similar populations of immunosuppressive myeloid cells present in tumors. In vitro co-cultures demonstrated that, in the presence of bone-marrow derived macrophages, immunosuppressive IL-6 production was reduced in pancreatic fibroblasts cultured with IhhKO-CM, as compared to fibroblasts cultured with WT-CM, providing mechanistic insight into the in vivo phenotype observed. Further, scRNA seq analysis suggests that modulation of HH signaling in the tumor microenvironment alters chemokine and immunomodulatory signaling pathways driven by fibroblasts in the pancreatic tumor microenvironment. DISCUSSION/SIGNIFICANCE OF IMPACT: HH signaling in pancreatic fibroblasts contributes to the establishment of an immune suppressive environment in pancreatic cancer. Combining methods to target HH signaling and immune checkpoint therapy has translational potential in treating pancreatic cancer patients.

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Long-lasting Analgesia via Targetedin vivoEpigenetic Repression of Nav1.7

10.1101/711812 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ana M. Moreno ◽

Glaucilene F. Catroli ◽

Fernando Alemán ◽

Andrew Pla ◽

Sarah A. Woller ◽

...

Keyword(s):

Chronic Pain ◽

Genome Engineering ◽

Therapeutic Potential ◽

Sequence Similarity ◽

Thermal Hyperalgesia ◽

Loss Of Function ◽

High Sequence Similarity ◽

Inflammatory State

ABSTRACTCurrent treatments for chronic pain rely largely on opioids despite their unwanted side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing, with the voltage-gated sodium channel, NaV1.7 (SCN9A), being perhaps the most promising candidate for analgesic drug development. Specifically, a hereditary loss-of-function mutation in NaV1.7 leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence similarity between NaVsubtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 via genome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins as a potential treatment for chronic pain. Towards this end, we first optimized the efficiency of NaV1.7 repressionin vitroin Neuro2A cells, and then by the lumbar intrathecal route delivered both genome-engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain and BzATP-induced pain. Our results demonstrate: one, effective repression of NaV1.7 in lumbar dorsal root ganglia; two, reduced thermal hyperalgesia in the inflammatory state; three, decreased tactile allodynia in the neuropathic state; and four, no changes in normal motor function. We anticipate this genomically scarless and non-addictivepainamelioration approach enablingLong-lastingAnalgesia viaTargetedin vivoEpigeneticRepression of Nav1.7, a methodology we dubpain LATER, will have significant therapeutic potential, such as for preemptive administration in anticipation of a pain stimulus (pre-operatively), or during an established chronic pain state.One sentence summaryIn situepigenome engineering approach for genomically scarless, durable, and non-addictive management of pain.

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CARG-2020, an oncolytic artificial virus co-delivering three immunomodulators, to regress and cure established tumors in mice.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e14560 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e14560-e14560

Author(s):

Bijan Almassian ◽

Bhaskara R Madina ◽

Ju Chen ◽

Marie M Krady ◽

Xianyong Ma ◽

...

Keyword(s):

Immune Modulation ◽

Semliki Forest Virus ◽

Therapeutic Potential ◽

Complete Response ◽

Therapy Resistance ◽

Dominant Negative ◽

Oncolytic Viruses ◽

Primary Tumors ◽

Artificial Virus

e14560 Background: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in humans. Treatment of late-stage CRC remains ineffective, even with the use of latest immunotherapies. Oncolytic viruses have shown limited use for the treatment of cancers, and further improvement of these agents with immune-modulating activities may prove crucial for patients with CRC and other malignancies. To this end, we developed CARG-2020 as an artificial virus for infectious diseases and immuno-oncology (AVIDIO) that employs virus-like vesicles (VLV). VLVs, which are membrane-encapsulated RNA replicons, are oncolytic and can deliver multiple genes resulting in the modulation of several independent immune pathways. Methods: The AVIDIO platform is comprised of in vitro evolved RNA-dependent RNA polymerase from an alphavirus, Semliki Forest virus, and envelope glycoproteins from vesicular stomatitis virus, which together form VLVs. Unarmed or empty vector (VLV), VLV armed with IL-12 (VLV-IL-12) and a VLV that simultaneously expresses IL-12, a dominant-negative form of IL-17 receptor A (dn-1L17RA) and shRNAs targeting PD-L1 (CARG-2020), were given intratumorally to test their therapeutic potential against established (500-600mm3) MC38 tumors in mice. We used tumor growth measurements and analyses of tumor-infiltrating cells after consecutive treatments with these agents to monitor their antitumor and immunomodulatory activities, respectively. Results: Both VLV-IL-12 and CARG-2020 regressed the tumors to undetectable levels in most mice harboring syngeneic MC38 tumors when given intratumorally. CARG-2020, carrying IL-12, dn-IL17RA and PD-L1 shRNA, exerts broader spectrum of immuno-responses and higher number of complete response rates compared with VLV-IL-12. Treatment of primary tumors with VLV-IL-12 or CARG-2020 also significantly repressed the growth of secondary tumors on the other flank of mouse’s body, suggesting an effective systemic immunity elicited by these two agents against the same type of tumors. In addition to the marked local and systemic activation of Th1 cells and CD8+ T cells by both vectors, CARG-2020 also downregulated PD-L1 expression in tumors, and suppressed expression of IL-17A-activated chemokines CXCL1 and CXCL2 that are known to promote cancer development and therapy resistance. Conclusions: As an oncolytic RNA replicon, AVIDIO platform-derived CARG-2020 encodes IL-12, dn-IL-17RA and PD-L1 shRNAs which are expressed concurrently within the same vector. CARG-2020 modulates IL-12, IL-17RA and PD-L1 signaling, and exerts broad immune modulation in tumor microenvironment. Treatment with CARG-2020 eliminates the majority of grafted large tumors in mice, and is effective against both primary and distal tumors. Based on this impressive efficacy results, further development of CARG-2020 in colorectal patients is warranted.

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