The use of lipid-formulated RNA vaccines for cancer or COVID-19 is associated with dose-limiting systemic inflammatory responses in humans that were not predicted from preclinical studies. Here, we ...show that the 'interleukin 1 (IL-1)-interleukin 1 receptor antagonist (IL-1ra)' axis regulates vaccine-mediated systemic inflammation in a host-specific manner. In human immune cells, RNA vaccines induce production of IL-1 cytokines, predominantly IL-1β, which is dependent on both the RNA and lipid formulation. IL-1 in turn triggers the induction of the broad spectrum of pro-inflammatory cytokines (including IL-6). Unlike humans, murine leukocytes respond to RNA vaccines by upregulating anti-inflammatory IL-1ra relative to IL-1 (predominantly IL-1α), protecting mice from cytokine-mediated toxicities at >1,000-fold higher vaccine doses. Thus, the IL-1 pathway plays a key role in triggering RNA vaccine-associated innate signaling, an effect that was unexpectedly amplified by certain lipids used in vaccine formulations incorporating N1-methyl-pseudouridine-modified RNA to reduce activation of Toll-like receptor signaling.
Glucocorticosteroids (GCS) have an established role in oncology and are administered to cancer patients in routine clinical care and in drug development trials as co-medication. Given their strong ...immune-suppressive activity, GCS may interfere with immune-oncology drugs. We are developing a therapeutic cancer vaccine, which is based on a liposomal formulation of tumor-antigen encoding RNA (RNA-LPX) and induces a strong T-cell response both in mice as well as in humans. In this study, we investigated in vivo in mice and in human PBMCs the effect of the commonly used long-acting GCS Dexamethasone (Dexa) on the efficacy of this vaccine format, with a particular focus on antigen-specific T-cell immune responses.
We show that Dexa, when used as premedication, substantially blunts RNA-LPX vaccine-mediated immune effects. Premedication with Dexa inhibits vaccine-dependent induction of serum cytokines and chemokines and reduces both the number and activation of splenic conventional dendritic cells (cDC) expressing vaccine-encoded antigens. Consequently, priming of functional effector T cells and therapeutic activity is significantly impaired. Interestingly, responses are less impacted when Dexa is administered post-vaccination. Consistent with this observation, although many inflammatory cytokines are reduced, IFNα, a key cytokine in T-cell priming, is less impacted and antigen expression by cDCs is intact. These findings warrant special caution when combining GCS with immune therapies relying on priming and activation of antigen-specific T cells and suggest that careful sequencing of these treatments may preserve T-cell induction.
Virus-based cancer vaccines are nowadays considered an interesting approach in the field of cancer immunotherapy, despite the observation that the majority of the immune responses they elicit are ...against the virus and not against the tumor. In contrast, targeting tumor associated antigens is effective, however the identification of these antigens remains challenging. Here, we describe ExtraCRAd, a multi-vaccination strategy focused on an oncolytic virus artificially wrapped with tumor cancer membranes carrying tumor antigens. We demonstrate that ExtraCRAd displays increased infectivity and oncolytic effect in vitro and in vivo. We show that this nanoparticle platform controls the growth of aggressive melanoma and lung tumors in vivo both in preventive and therapeutic setting, creating a highly specific anti-cancer immune response. In conclusion, ExtraCRAd might serve as the next generation of personalized cancer vaccines with enhanced features over standard vaccination regimens, representing an alternative way to target cancer.
BackgroundToll-like receptors (TLRs) are an integral part of innate immune cell activation and play an essential role in mounting effective antigen-specific adaptive immune responses. TLR7 and TLR8 ...are highly homologous TLR family members that recognize ssRNA derived from viruses and induce distinct cytokine profiles due to differential expression in immune cells. Recent studies demonstrate that TLR7 and TLR8 recognize degraded ssRNA products generated by endosomal RNases. We hypothesized that site-specific modifications to foundational ssRNA sequences containing both TLR7 and TLR8 binding motifs could slow or inhibit RNase-mediated degradation of TLR8 binding site motifs. Here we designed oligoribonucleotides (ORNs) that reduce TLR8 activity while maintaining TLR7 agonism and developed a high throughput automated in vitro assay to identify TLR7 selective ORNs.MethodsWe engineered 20–25 base pair ssRNA foundation sequences containing both TLR7 and TLR8 binding motifs with 2’ modifications placed on select nucleotides to slow or stop RNase-mediated processing of ORNs into ligands activating TLR8. This allowed for the abrogation of TLR8 and preservation of TLR7 activity. We established an automated screening platform to assemble and deliver lipid complexes containing a TLR-selective ORN and mRNA encoding GFP, and assessed their immunostimulatory activity in vitro. Cytokine responses from human PBMCs were evaluated to identify TLR7 selective agonists by Luminex, and monocyte maturation and antigen expression were assessed by flow cytometry. Top performing TLR7 specific ORNs were administered systemically to mice. DC maturation and serum cytokines were measured ex vivo by flow cytometry and Luminex.ResultsIntroduction of 2’ sugar-modified bases into ORNs limited or blocked RNase-mediated cleavage 3’ to the modified nucleotide at specific phosphodiester bonds. The nucleotide modifications of ssRNAs modulate endosomal RNase processing of these sequences, preventing degradation into monomeric uridine required for TLR8 activation, while preserving TLR7 activation. To mimic the effects of a co-delivered mRNA-based therapeutic, ssRNA ORN and mRNA were assembled into lipid complexes with DOTAP and cultured with human PBMCs. We identified top performing TLR7 specific oligos, classified as S2-PS-F.F and S5-F, that induced a robust IFNα response and weak TNFα response, accompanied by monocyte maturation and mRNA encoded GFP expression. These modified ORNs induced splenic DC maturation and cytokine release in vivo.ConclusionsUsing a rationale design we created ORNs that have diminished TLR8 and preserved TLR7 activity. This design strategy and screening platform can be applied and expanded to identify additional novel TLR7 selective agonists for potential use with mRNA-based therapeutics.Ethics ApprovalObtained informed consent from healthy adult volunteer apheresis donors who have passed the standard blood donor health history questionnaire. All animal experiments were performed under protocols approved by the Genentech Institutional Animal Care and Use Committee.
Pancreatic ductal adenocarcinoma (PDA) is characterized by its highly immunosuppressive tumor microenvironment (TME) that limits T cell infiltration and induces T cell hypofunction. ...Mesothelin-redirected chimeric antigen receptor T cell (meso-CAR T cell) therapy has shown some efficacy in clinical trials but antitumor efficacy remains modest. We hypothesized that combined meso-CAR T cells with an oncolytic adenovirus expressing TNF-α and IL-2 (Ad5/3-E2F-D24-TNFa-IRES-IL2, or OAd-TNFa-IL2) would improve efficacy. OAd-TNFa-IL2 enhanced the antitumor efficacy of meso-CAR T cells in human-PDA-xenograft immunodeficient mice and efficacy was associated with robustly increased tumor-infiltrating lymphocytes (TILs), enhanced and prolonged T cell function. Mice treated with parental OAd combined with meso-CAR T developed tumor metastasis to the lungs even if primary tumors were controlled. However, no mice treated with combined OAd-TNFa-IL2 and meso-CAR T died of tumor metastasis. We also evaluated this approach in a syngeneic mouse tumor model by combining adenovirus expressing murine TNF-α and IL-2 (Ad-mTNFa-mIL2) and mouse CAR T cells. This approach induced significant tumor regression in mice engrafted with highly aggressive and immunosuppressive PDA tumors. Ad-mTNFa-mIL2 increased both CAR T cell and host T cell infiltration to the tumor and altered host tumor immune status with M1 polarization of macrophages and increased dendritic cell maturation. These findings indicate that combining cytokine-armed oncolytic adenovirus to enhance the efficacy of CAR T cell therapy is a promising approach to overcome the immunosuppressive TME for the treatment of PDA.
Multiple injections of oncolytic adenovirus could enhance immunologic response. In the first part of this article, the focus was on immunologic aspects. Sixty patients previously naïve to oncolytic ...virus and who had white blood cells available were treated. Thirty-nine of 60 were assessed after a single virus administration, whereas 21 of 60 received a "serial treatment" consisting of three injections within 10 weeks. In the second part, we focused on 115 patients treated with a granulocyte macrophage colony-stimulating factor (GM-CSF)-coding capsid chimeric adenovirus, CGTG-102.
Following serial treatment, both increase and decrease in antitumor T cells in blood were seen more frequently, findings which are compatible with induction of T-cell immunity and trafficking of T cells to tumors, respectively. Safety was good in both groups. In 115 patients treated with CGTG-102 (Ad5/3-D24-GMCSF), median overall survival was 111 days following single and 277 days after serial treatment in nonrandomized comparison. Switching the virus capsid for avoiding neutralizing antibodies in a serial treatment featuring three different viruses did not impact safety or efficacy. A correlation between antiviral and antitumor T cells was seen (P = 0.001), suggesting that viral oncolysis can result in epitope spreading and breaking of tumor-associated immunologic tolerance. Alternatively, some patients may be more susceptible to induction of T-cell immunity and/or trafficking.
These results provide the first human data linking antiviral immunity with antitumor immunity, implying that oncolytic viruses could have an important role in cancer immunotherapy.
Unfavorable ratios between the number and activation status of effector and suppressor immune cells infiltrating the tumor contribute to resistance of solid tumors to T-cell based therapies. Here, we ...studied the capacity of FDA and EMA approved recombinant cytokines to manipulate this balance in favor of efficient anti-tumor responses in B16.OVA melanoma bearing C57BL/6 mice. Intratumoral administration of IFN-α2, IFN-γ, TNF-α, and IL-2 significantly enhanced the anti-tumor effect of ovalbumin-specific CD8+ T-cell (OT-I) therapy, whereas GM-CSF increased tumor growth in association with an increase in immunosuppressive cell populations. None of the cytokines augmented tumor trafficking of OT-I cells significantly, but injections of IFN-α2, IFN-γ and IL-2 increased intratumoral cytokine secretion and recruitment of endogenous immune cells capable of stimulating T-cells, such as natural killer and maturated CD11c+ antigen-presenting cells. Moreover, IFN-α2 and IL-2 increased the levels of activated tumor-infiltrating CD8+ T-cells concomitant with reduction in the CD8+ T-cell expression of anergy markers CTLA-4 and PD-1. In conclusion, intratumoral administration of IFN-α2, IFN-γ and IL-2 can lead to immune sensitization of the established tumor, whereas GM-CSF may contribute to tumor-associated immunosuppression. The results described here provide rationale for including local administration of immunostimulatory cytokines into T-cell therapy regimens. One appealing embodiment of this would be vectored delivery which could be advantageous over direct injection of recombinant molecules with regard to efficacy, cost, persistence and convenience.
Despite originating from several different tissues, soft‐tissue sarcomas (STS) are often grouped together as they share mesenchymal origin and treatment guidelines. Also, with some exceptions, a ...common denominator is that when the tumor cannot be cured with surgery, the efficacy of current therapies is poor and new treatment modalities are thus needed. We have studied the combination of a capsid‐modified oncolytic adenovirus CGTG‐102 (Ad5/3‐D24‐GMCSF) with doxorubicin, with or without ifosfamide, the preferred first‐line chemotherapeutic options for most types of STS. We show that CGTG‐102 and doxorubicin plus ifosfamide together are able to increase cell killing of Syrian hamster STS cells over single agents, as well as upregulate immunogenic cell death markers. When tested in vivo against established STS tumors in fully immunocompetent Syrian hamsters, the combination was highly effective. CGTG‐102 and doxorubicin (without ifosfamide) resulted in synergistic antitumor efficacy against human STS xenografts in comparison with single agent treatments. Doxorubicin increased adenoviral replication in human and hamster STS cells, potentially contributing to the observed therapeutic synergy. In conclusion, the preclinical data generated here support clinical translation of the combination of CGTG‐102 and doxorubicin, or doxorubicin plus ifosfamide, for the treatment of STS, and provide clues on the mechanisms of synergy.
What's new?
New treatment options are needed for patients with soft‐tissue sarcomas (STS) as management of metastatic disease is currently not very successful. Here the authors develop a new line of treatment, where they add an oncolytic adenovirus, CGTG‐102 (Ad5/3‐D24‐GMCSF) to doxorubicin and ifosfamide, the preferred chemotherapeutic regimen against most types of STS. They show in an immune‐competent Syrian hamster model that this combination is effective against established STS tumors. In an additional line of research, doxorubicin without ifosfamide synergized with CGTG‐102 to treat STS of human origin in a mouse xenograft tumor model. These results support a future clinical application of CGTG‐102 in combination with doxorubicin‐based chemotherapy for better treatment of STS.
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