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.

Peritoneal carcinomatosis in mouse models.

Peritoneal carcinomatosis (PCa) represents a metastatic stage of a disease with unmet therapeutic options. Malignant cells from primary tumors (gastrointestinal or gynecologic malignancies) invade the peritoneal cavity and eventually seed onto peritoneal surfaces, with the omentum being the most common nest area. With a median survival of less than 6 months, PCa has a dismal prognosis that can be improved with treatments only available to a select few individuals with low tumor burden. Thus, the discovery of novel and effective therapies for this disease depends on reliable animal models. Here, we describe a method to generate syngeneic PCa mouse models based on intraperitoneal (i.p.) administration of tumor cells. This model allows to follow-up cancer progression in PCa models from ovarian and colorectal origins monitoring mice bodyweight changes, ascites development and overall survival. Moreover, luciferase-expressing tumor cells can also be used to assess tumor growth after i.p. injection through in vivo bioluminescence quantification. The establishment of reliable, easy-to-monitor and reproducible intraperitoneal syngeneic tumors models, as described here, is the first step to develop cutting-edge therapies against PCa.

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.

Statins act as transient type I interferon inhibitors to enable the antitumor activity of modified vaccinia Ankara viral vectors.

BACKGROUND: Modified vaccinia virus Ankara (MVA) are genetically engineered non-replicating viral vectors. Intratumoral administration of MVA induces a cyclic GMP-AMP synthase-mediated type I interferon (IFN) response and the production of high levels of the transgenes engineered into the viral genome such as tumor antigens to construct cancer vaccines. Although type I IFNs are essential for establishing CD8-mediated antitumor responses, this cytokine family may also give rise to immunosuppressive mechanisms. METHODS: In vitro assays were performed to evaluate the activity of simvastatin and atorvastatin on type I IFN signaling and on antigen presentation. Surface levels of IFN α/ß receptor 1, endocytosis of bovine serum albumin-fluorescein 5 (6)-isothiocyanate, signal transducer and activator of transcription (STAT) phosphorylation, and real-time PCR of IFN-stimulated genes were assessed in the murine fibroblast cell line L929. In vivo experiments were performed to characterize the effect of simvastatin on the MVA-induced innate immune response and on the antitumor effect of MVA-based antitumor vaccines in B16 melanoma expressing ovalbumin (OVA) and Lewis lung carcinoma (LLC)-OVA tumor models. RNAseq analysis, depleting monoclonal antibodies, and flow cytometry were used to evaluate the MVA-mediated immune response. RESULTS: In this work, we identified commonly prescribed statins as potent IFNα pharmacological inhibitors due to their ability to reduce surface expression levels of IFN-α/ß receptor 1 and to reduce clathrin-mediated endocytosis. Simvastatin and atorvastatin efficiently abrogated for 8 hours the transcriptomic response to IFNα and enhanced the number of dendritic cells presenting an OVA-derived peptide bound to major histocompatibility complex (MHC) class I. In vivo, intraperitoneal or intramuscular administration of simvastatin reduced the inflammatory response mediated by peritumoral administration of MVA and enhanced the antitumor activity of MVA encoding tumor-associated antigens. The synergistic antitumor effects critically depend on CD8+ cells, whereas they were markedly improved by depletion of CD4+ lymphocytes, T regulatory cells, or NK cells. Either MVA-OVA alone or combined with simvastatin augmented B cells, CD4+ lymphocytes, CD8+ lymphocytes, and tumor-specific CD8+ in the tumor-draining lymph nodes. However, only the treatment combination increased the numbers of these lymphocyte populations in the tumor microenvironment and in the spleen. CONCLUSION: In conclusion, blockade of IFNα functions by simvastatin markedly enhances lymphocyte infiltration and the antitumor activity of MVA, prompting a feasible drug repurposing.

Scavenger Receptor Class B Type I is Required for 25-Hydroxycholecalciferol Cellular Uptake and Signaling in Myeloid Cells.

SCOPE: Vitamin D3 is a critical molecule for the properly controlled activity of the immune system. In myeloid-derived cells, vitamin D3 induces the production of the antimicrobial and antitumor peptide cathelicidin. In this study, the mechanism of the entry of 25-hydroxycholecalciferol (25(OH)D) in myeloid-derived cells is explored. METHODS AND RESULTS: Here, a novel regulatory pathway of vitamin D3 biology is described. Using a polyclonal antibody, two different chemical inhibitors, and a high-density lipoprotein as a competing ligand, it is demonstrated here that the 25(OH)D signaling pathway in myeloid cells depends on scavenger receptor class B type I (SR-B1). This effect is observed in the THP-1 monocytic cell line and in human primary monocytes. SR-B1 blockade abrogates the cellular uptake of 25(OH)D leading to a general shut down of the gene transcription program modulated by 25(OH)D. The results obtained at the transcriptional level are confirmed at the protein and functional level for CD14 in the THP-1 cell line. CONCLUSION: In conclusion, SR-B1 plays a critical role in vitamin D3 biology, paving the way for novel therapeutic interventions.

Insulin Fused to Apolipoprotein A-I Reduces Body Weight and Steatosis in DB/DB Mice.

Background: Targeting long-lasting insulins to the liver may improve metabolic alterations that are not corrected with current insulin replacement therapies. However, insulin is only able to promote lipogenesis but not to block gluconeogenesis in the insulin-resistant liver, exacerbating liver steatosis associated with diabetes. Methods: In order to overcome this limitation, we fused a single-chain insulin to apolipoprotein A-I, and we evaluated the pharmacokinetics and pharmacodynamics of this novel fusion protein in wild type mice and in db/db mice using both recombinant proteins and recombinant adenoassociated virus (AAV). Results: Here, we report that the fusion protein between single-chain insulin and apolipoprotein A-I prolonged the insulin half-life in circulation, and accumulated in the liver. We analyzed the long-term effect of these insulin fused to apolipoprotein A-I or insulin fused to albumin using AAVs in the db/db mouse model of diabetes, obesity, and liver steatosis. While AAV encoding insulin fused to albumin exacerbated liver steatosis in several mice, AAV encoding insulin fused to apolipoprotein A-I reduced liver steatosis. These results were confirmed upon daily subcutaneous administration of the recombinant insulin-apolipoprotein A-I fusion protein for six weeks. The reduced liver steatosis was associated with reduced body weight in mice treated with insulin fused to apolipoprotein A-I. Recombinant apolipoprotein A-I alone significantly reduces body weight and liver weight, indicating that the apolipoprotein A-I moiety is the main driver of these effects. Conclusion: The fusion protein of insulin and apolipoprotein A-I could be a promising insulin derivative for the treatment of diabetic patients with associated fatty liver disease.

Long-Term Liver Expression of an Apolipoprotein A-I Mimetic Peptide Attenuates Interferon-Alpha-Induced Inflammation and Promotes Antiviral Activity.

Apolipoprotein A-I mimetic peptides are amphipathic alpha-helix peptides that display similar functions to apolipoprotein A-I. Preclinical and clinical studies have demonstrated the safety and efficacy of apolipoprotein A-I mimetic peptides in multiple indications associated with inflammatory processes. In this study, we evaluated the effect of the long-term expression of L37pA in the liver by an adeno-associated virus (AAV-L37pA) on the expression of an adeno-associated virus encoding interferon-alpha (AAV-IFNα). Long-term IFNα expression in the liver leads to lethal hematological toxicity one month after AAV administration. Concomitant administration of AAV-L37pA prevented the lethal toxicity since the IFNα expression was reduced one month after AAV administration. To identify the mechanism of action of L37pA, a genomic and proteomic analysis was performed 15 days after AAV administration when a similar level of IFNα and interferon-stimulated genes were observed in mice treated with AAV-IFNα alone and in mice treated with AAV-IFNα and AAV-L37pA. The coexpression of the apolipoprotein A-I mimetic peptide L37pA with IFNα modulated the gene expression program of IFNα, inducing a significant reduction in inflammatory pathways affecting pathogen-associated molecular patterns receptor, dendritic cells, NK cells and Th1 immune response. The proteomic analysis confirmed the impact of the L37pA activity on several inflammatory pathways and indicated an activation of LXR/RXR and PPPARα/γ nuclear receptors. Thus, long-term expression of L37pA induces an anti-inflammatory effect in the liver that allows silencing of IFNα expression mediated by an adeno-associated virus.

Treatment of Experimental Autoimmune Encephalomyelitis by Sustained Delivery of Low-Dose IFN-α.

Multiple sclerosis (MS) is a chronic autoimmune disease with no curative treatment. The immune regulatory properties of type I IFNs have led to the approval of IFN-ß for the treatment of relapsing-remitting MS. However, there is still an unmet need to improve the tolerability and efficacy of this therapy. In this work, we evaluated the sustained delivery of IFN-α1, either alone or fused to apolipoprotein A-1 by means of an adeno-associated viral (AAV) system in the mouse model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. These in vivo experiments demonstrated the prophylactic and therapeutic efficacy of the AAV-IFN-α or AAV-IFN-α fused to apolipoprotein A-1 vectors in experimental autoimmune encephalomyelitis, even at low doses devoid of hematological or neurologic toxicity. The sustained delivery of such low-dose IFN-α resulted in immunomodulatory effects, consisting of proinflammatory monocyte and T regulatory cell expansion. Moreover, encephalitogenic T lymphocytes from IFN-α-treated mice re-exposed to the myelin oligodendrocyte glycoprotein peptide in vitro showed a reduced proliferative response and cytokine (IL-17A and IFN-γ) production, in addition to upregulation of immunosuppressive molecules, such as IL-10, IDO, or PD-1. In conclusion, the results of the present work support the potential of sustained delivery of low-dose IFN-α for the treatment of MS and likely other T cell-dependent chronic autoimmune disorders.

Antitumor effect of an adeno-associated virus expressing apolipoprotein A-1 fused to interferon alpha in an interferon alpha-resistant murine tumor model.

Interferon alpha (IFNα) is a cytokine approved for the treatment of several types of cancer. However, the modest effect on overall survival and the high toxicity associated with the treatment has reduced the clinical use of this cytokine. In this study, we have developed a tumor model that reproduces this clinical setting. A high dose of an adeno-associated virus encoding IFNα (AAV-IFNα) was able to eradicate a liver metastases model of colon cancer but induced lethal pancytopenia. On the other hand, a safe dose of AAV-IFNα was not able to eliminate the liver metastases of colon cancer. In this IFNα-resistant tumor model, administration of an adeno-associated vector encoding apolipoprotein A-1 fused to IFNα was able to fully eradicate the tumor in 43% of mice without toxicity. This antitumor effect was limited by suboptimal long-term CD8+ T cell activation and the expansion of T regulatory cells. In contrast, IFNα upregulated suppressor molecules such as PD-1 and interleukin 10 on CD8+ T lymphocytes. In conclusion, we show that apolipoprotein A-1 fused to IFNα is a novel antitumor drug that differs from IFNα in the modulation of suppressor mechanisms of the immune response. These differential properties pave the way for rational combinations with other immunomodulatory drugs.

Interferon alpha bioactivity critically depends on Scavenger receptor class B type I function.

Scavenger receptor class B type I (SR-B1) binds pathogen-associated molecular patterns participating in the regulation of the inflammatory reaction but there is no information regarding potential interactions between SR-B1 and the interferon system. Herein, we report that SR-B1 ligands strongly regulate the transcriptional response to interferon α (IFNα) and enhance its antiviral and antitumor activity. This effect was mediated by the activation of TLR2 and TLR4 as it was annulled by the addition of anti-TLR2 or anti-TLR4 blocking antibodies. In vivo, we maximized the antitumor activity of IFNα co-expressing in the liver a SR-B1 ligand and IFNα by adeno-associated viruses. This gene therapy strategy eradicated liver metastases from colon cancer with reduced toxicity. On the other hand, genetic and pharmacological inhibition of SR-B1 blocks the clathrin-dependent interferon receptor recycling pathway with a concomitant reduction in IFNα signaling and bioactivity. This effect can be applied to enhance cancer immunotherapy with oncolytic viruses. Indeed, SR-B1 antagonists facilitate replication of oncolytic viruses amplifying their tumoricidal potential. In conclusion, SR-B1 agonists behave as IFNα enhancers while SR-B1 inhibitors dampen IFNα activity. These results demonstrate that SR-B1 is a suitable pharmacology target to enhance cancer immunotherapy based on IFNα and oncolytic viruses.

Overexpression of apolipoprotein A-I fused to an anti-transforming growth factor beta peptide modulates the tumorigenicity and immunogenicity of mouse colon cancer cells.

Transforming growth factor beta (TGF-ß) promotes tumor growth, invasion and metastasis in established tumors. In this study, we analyzed the effect of overexpressing an anti-TGF-ß peptide fused to apolipoprotein A-I (ApoA-I) as a scaffold molecule. We generated and characterized stable MC38 colon carcinoma clones expressing ApoA-I fused to the anti-TGF-ß peptide P144 and ApoA-I as control cells. We evaluated in vitro the gene expression profile, cell cycle and anchorage-independent growth. The in vivo tumorigenic potential and immunogenicity were analyzed inoculating the MC38 clones into C57BL/6 mice, recombination-activating gene 1 knockout mice or mice deficient in NK cells either subcutaneously or intrasplenically to generate hepatic metastases. While overexpression of ApoA-I had no effect on the parameters analyzed, ApoA-I fused to P144 markedly diminished the tumorigenic capacity and metastatic potential of MC38 in vitro and in vivo, thus generating a highly immunogenic cell line. MC38 cells transfected with ApoA-I fused to P144 triggered memory T cell responses able to eliminate the parental cell line upon re-challenge. In summary, expression of ApoA-I fused to P144 is a novel strategy to modulate TGF-ß in tumor cells. These results highlight the potential of TGF-ß as a target in the development of new antitumor treatments.

Liver-directed gene therapy of chronic hepadnavirus infection using interferon alpha tethered to apolipoprotein A-I.

BACKGROUND & AIMS: Current hepatitis B virus (HBV) management is challenging as treatment with nucleos(t)ide analogues needs to be maintained indefinitely and because interferon (IFN)-α therapy is associated with considerable toxicity. Previously, we showed that linking IFNα to apolipoprotein A-I generates a molecule (IA) with distinct antiviral and immunostimulatory activities which lacks the hematological toxicity of IFNα. METHODS: Here, we analyse the antiviral potential of an adeno-associated vector encoding IFNα fused to apolipoprotein A-I (AAV-IA) in comparison to a vector encoding only IFNα (AAV-IFN) in two animal models of chronic hepadnavirus infection. RESULTS: In HBV transgenic mice, we found that both vectors induced marked reductions in serum and liver HBV DNA and in hepatic HBV RNA but AAV-IFN caused lethal pancytopenia. Woodchucks with chronic hepatitis virus (WHV) infection that were treated by intrahepatic injection of vectors encoding the woodchuck sequences (AAV-wIFN or AAV-wIA), experienced only a slight reduction of viremia which was associated with hematological toxicity and high mortality when using AAV-wIFN, while AAV-wIA was well tolerated. However, when we tested AAV-wIA or a control vector encoding woodchuck apolipoprotein A-I (AAV-wApo) in combination with entecavir, we found that AAV-wApo-treated animals exhibited an immediate rebound of viral load upon entecavir withdrawal while, in AAV-wIA-treated woodchucks, viremia and antigenemia remained at low levels for several weeks following entecavir interruption. CONCLUSIONS: Treatment with AAV-IA is safe and elicits antiviral effects in animal models with difficult to treat chronic hepadnavirus infection. AAV-IA in combination with nucleos(t)ide analogues represents a promising approach for the treatment of HBV infection in highly viremic patients.

Harnessing high density lipoproteins to block transforming growth factor beta and to inhibit the growth of liver tumor metastases.

Transforming growth factor ß (TGF-ß) is a powerful promoter of cancer progression and a key target for antitumor therapy. As cancer cells exhibit active cholesterol metabolism, high density lipoproteins (HDLs) appear as an attractive delivery system for anticancer TGFß-inhibitory molecules. We constructed a plasmid encoding a potent TGF-ß-blocking peptide (P144) linked to apolipoprotein A-I (ApoA-I) through a flexible linker (pApoLinkerP144). The ApoLinkerP144 sequence was then incorporated into a hepatotropic adeno-associated vector (AAVApoLinkerP144). The aim was to induce hepatocytes to produce HDLs containing a modified ApoA-I capable of blocking TGF-ß. We observed that transduction of the murine liver with pApoLinkerP144 led to the appearance of a fraction of circulating HDL containing the fusion protein. These HDLs were able to attenuate TGF-ß signaling in the liver and to enhance IL-12 -mediated IFN-γ production. Treatment of liver metastasis of MC38 colorectal cancer with AAVApoLinkerP144 resulted in a significant reduction of tumor growth and enhanced expression of IFN-γ and GM-CSF in cancerous tissue. ApoLinkerP144 also delayed MC38 liver metastasis in Rag2-/-IL2rγ-/- immunodeficient mice. This effect was associated with downregulation of TGF-ß target genes essential for metastatic niche conditioning. Finally, in a subset of ret transgenic mice, a model of aggressive spontaneous metastatic melanoma, AAVApoLinkerP144 delayed tumor growth in association with increased CD8+ T cell numbers in regional lymph nodes. In conclusion, modification of HDLs to transport TGF-ß-blocking molecules is a novel and promising approach to inhibit the growth of liver metastases by immunological and non-immunological mechanisms.

Kinetic and dynamic computational model-based characterization of new proteins in mice: application to interferon alpha linked to apolipoprotein A-I.

Interferon alpha linked to apolipoprotein A-I has been recently proposed as an improved interferon-based therapy. In the present study, we aimed to develop a computational model to gain further insight into the in vivo behaviour of this new fusion protein. In order to facilitate in vivo evaluation of interferon and the fusion protein without altering their biological properties, green fluorescent protein was incorporated into their structures. Kinetic and dynamic behaviour of both compounds was successfully described after plasmid hydrodynamic administration and in situ synthesis of the studied proteins. Results from the modelling exercise showed that apolipoprotein A-I conferred a modified kinetic behaviour, varying molecule distribution and prolonging half-life without altering liver dynamic performance. However, differences in the gene expression activity were observed at brain level between both compounds. Those differences could be explained by modifications in the dynamic, but also in the biodistribution properties, which would be worth evaluating in future experiments. Therefore, the modelling approach provided a global comprehension of a complex system and allowed us to compare the in vivo behaviour of both compounds and to identify critical aspects that might be important to understand the system better and suggests a need for new model-based experiments.

The fusion protein of IFN-α and apolipoprotein A-I crosses the blood-brain barrier by a saturable transport mechanism.

IFN-α is widely used for the treatment of chronic viral hepatitis and malignancies. However, systemic IFN-α treatment causes severe neuropsychiatric complications in humans, including depression, anxiety, and cognitive impairments. We have previously reported that the fusion protein formed by IFN-α and apolipoprotein A-I (IA) circulates bound to high-density lipoproteins (HDLs) and exhibits liver targeting, increased half-life, enhanced immunostimulatory activity, and reduced cytotoxicity. As the transport of HDLs across the blood-brain barrier is a highly complex and regulated process, in this study, we examine the effects of IA on the brain. Determination of IFN-α in brain and serum after hydrodynamic administration of different doses of a plasmid encoding IFN-α or IA showed that IA penetrated into the brain by a saturable transport mechanism. Thus, at high serum levels of the transgenes, the induction of IFN-sensitive genes and the number of phospho-STAT1(+) cell nuclei in the brain were substantially higher with IFN-α than with IA. This was associated with attenuation of neurodepression in mice given IA, as manifested by shorter immobility time in the tail suspension test. However, when given low doses of rIFN-α or the same antiviral units of HDLs containing IA, the induction of IFN-stimulated genes in the brain was significantly greater with the latter. In conclusion, IA crosses the blood-brain barrier not by diffusion, as is the case of IFN-α, but by a facilitated saturable transport mechanism. Thus, linkage to apolipoprotein A-I may serve to modulate the effects of IFN-α on the CNS.

Anchoring interferon alpha to apolipoprotein A-I reduces hematological toxicity while enhancing immunostimulatory properties.

UNLABELLED: Interferon alpha (IFNα) is widely used for the treatment of viral hepatitis but substantial toxicity hampers its clinical use. In this work, we aimed at improving the efficacy of IFNα therapy by increasing the IFNα half-life and providing liver tropism. We selected apolipoprotein A-I (ApoA-I) as the stabilizing and targeting moiety. We generated plasmids encoding IFNα, albumin bound to IFNα (ALF), or IFNα linked to ApoA-I (IA) and mice were treated either by hydrodynamic administration of the plasmids or by injection of the corresponding recombinant proteins or high-density lipoproteins containing IA. The plasma half-life of IA was intermediate between IFNα and ALF. IA was targeted to the liver and induced higher hepatic expression of interferon-stimulated genes than IFNα or even ALF. IA exhibits stronger in vivo antiviral activity than IFNα and the hematologic cytopenic effects of IA are milder than those observed when using IFNα or ALF. In contrast to IFNα, IA does not cause activation-dependent cell death of lymphocytes in vitro. Accordingly, in vivo studies showed that IA boosts T-cell immune responses more efficiently than IFNα or ALF. The difference in immunostimulatory activity between IFNα and IA disappears in scavenger receptor class B type I (SR-BI) knockout mice, suggesting that crosstalk between SR-BI and IFNα receptor is essential for enhanced induction of cytotoxic T cells by IA. CONCLUSION: Anchoring IFNα to ApoA-I prolongs the half-life of IFNα and promotes targeting to the liver. Importantly, the fusion protein shows increased immunostimulatory properties and lower hematological toxicity.

Successful colon cancer eradication after chemoimmunotherapy is associated with profound phenotypic change of intratumoral myeloid cells.

IL-12 is a potent immunostimulatory cytokine, but its impact as an antitumor drug in clinical practice is limited. Upsurge of regulatory T cells (Treg) in the tumor milieu has been proposed to limit the efficacy of the treatment. In this paper, two drugs (cyclophosphamide [CPA] and anti-CD25 mAb) widely used to eliminate Treg were used in an attempt to enhance the antitumor effect of IL-12 gene therapy. Both anti-CD25 and CPA combined with IL-12 were able to deplete intratumoral Treg and myeloid-derived suppressor cells (MDSC), but only IL-12 plus CPA achieved significant antitumor activity in mice with large established s.c. colon carcinoma. This therapeutic effect was associated with the emergence of a heterogeneous population of myeloid cells within the tumor, termed inflammatory myeloid cells (IMC), composed of Ly6C(high)Ly6G(low) inflammatory monocytes and Ly6G(high)Ly6C(+) neutrophils. IMC showed a distinctive pattern of cytokine/chemokine production, and in contrast to MDSC, they did not induce conversion of naive CD4(+) T cells into Treg. The appearance of IMC coincided with intense tumor infiltration by effector T cells, which was abrogated by elimination of IMC by anti-Gr1 mAb, a maneuver that abolished the antitumor effect of the therapy. Therefore, the combination of IL-12 and CPA eliminates intratumoral Treg and MDSC, while it induces the appearance of IMC within the tumor microenvironment. The latter effect is essential to facilitate effector T cell infiltration and subsequent tumor elimination.