Xerna™ TME Panel is a machine learning-based transcriptomic biomarker designed to predict therapeutic response in multiple cancers.

Introduction: Most predictive biomarkers approved for clinical use measure single analytes such as genetic alteration or protein overexpression. We developed and validated a novel biomarker with the aim of achieving broad clinical utility. The Xerna™ TME Panel is a pan-tumor, RNA expression-based classifier, designed to predict response to multiple tumor microenvironment (TME)-targeted therapies, including immunotherapies and anti-angiogenic agents. Methods: The Panel algorithm is an artificial neural network (ANN) trained with an input signature of 124 genes that was optimized across various solid tumors. From the 298-patient training data, the model learned to discriminate four TME subtypes: Angiogenic (A), Immune Active (IA), Immune Desert (ID), and Immune Suppressed (IS). The final classifier was evaluated in four independent clinical cohorts to test whether TME subtype could predict response to anti-angiogenic agents and immunotherapies across gastric, ovarian, and melanoma datasets. Results: The TME subtypes represent stromal phenotypes defined by angiogenesis and immune biological axes. The model yields clear boundaries between biomarker-positive and -negative and showed 1.6-to-7-fold enrichment of clinical benefit for multiple therapeutic hypotheses. The Panel performed better across all criteria compared to a null model for gastric and ovarian anti-angiogenic datasets. It also outperformed PD-L1 combined positive score (>1) in accuracy, specificity, and positive predictive value (PPV), and microsatellite-instability high (MSI-H) in sensitivity and negative predictive value (NPV) for the gastric immunotherapy cohort. Discussion: The TME Panel's strong performance on diverse datasets suggests it may be amenable for use as a clinical diagnostic for varied cancer types and therapeutic modalities.

Vidutolimod in Combination With Atezolizumab With and Without Radiation Therapy in Patients With Programmed Cell Death Protein 1 or Programmed Death-Ligand 1 Blockade-Resistant Advanced NSCLC.

Introduction: Vidutolimod, a CpG-A TLR9 agonist, was investigated in a phase 1b study (CMP-001-003; ClinicalTrials.gov, NCT03438318) in combination with atezolizumab with and without radiation therapy (RT) in patients with advanced NSCLC. Methods: Patients with progressive disease after anti-programmed cell death protein 1 or programmed death-ligand 1 therapy received either vidutolimod and atezolizumab (part A) or vidutolimod, atezolizumab, and RT (part B). The primary objective was to evaluate the safety of vidutolimod and atezolizumab with and without RT. Key secondary end point was best objective response rate per Response Evaluation Criteria in Solid Tumors, version 1.1. Results: Between March 28, 2018, and July 25, 2019, a total of 29 patients were enrolled and received at least one dose of vidutolimod (part A, n = 13; part B, n = 16). Intratumoral injections of vidutolimod were administered successfully, including injection of visceral lesions. The most common treatment-related adverse events (≥30%) were flu-like symptoms and hypotension. No objective responses were observed; 23.1% and 50.0% of the patients in parts A and B, respectively, had stable disease as best response. In parts A and B, 15.4% and 25.0% of the patients, respectively, had tumor shrinkage (<30% decrease in tumor size, nonirradiated). Enrollment was stopped owing to lack of objective responses. In the two patients with initial tumor shrinkage in part A, a strong serum induction of C-X-C motif chemokine ligand 10 was observed. Conclusions: Vidutolimod and atezolizumab with and without RT had a manageable safety profile, with minimal clinical activity in heavily pretreated patients with programmed cell death protein 1 or programmed death-ligand 1 blockade-resistant NSCLC.

Considerations in the Preclinical Assessment of the Safety of Antisense Oligonucleotides.

The nucleic acid therapeutics field has made tremendous progress in the past decades. Continuous advances in chemistry and design have led to many successful clinical applications, eliciting even more interest from researchers including both academic groups and drug development companies. Many preclinical studies in the field focus on improving the delivery of antisense oligonucleotide drugs (ONDs) and/or assessing their efficacy in target tissues, often neglecting the evaluation of toxicity, at least in early phases of development. A series of consensus recommendations regarding regulatory considerations and expectations have been generated by the Oligonucleotide Safety Working Group and the Japanese Research Working Group for the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S6 and Related Issues (WGS6) in several white papers. However, safety aspects should also be kept in sight in earlier phases while screening and designing OND to avoid subsequent failure in the development phase. Experts and members of the network "DARTER," a COST Action funded by the Cooperation in Science and Technology of the EU, have utilized their collective experience working with OND, as well as their insights into OND-mediated toxicities, to generate a series of consensus recommendations to assess OND toxicity in early stages of preclinical research. In the past few years, several publications have described predictive assays, which can be used to assess OND-mediated toxicity in vitro or ex vivo to filter out potential toxic candidates before moving to in vivo phases of preclinical development, that is, animal toxicity studies. These assays also have the potential to provide translational insight since they allow a safety evaluation in human in vitro systems. Yet, small preliminary in vivo studies should also be considered to complement this early assessment. In this study, we summarize the state of the art and provide guidelines and recommendations on the different tests available for these early stage preclinical assessments.

Overcoming PD-1 Blockade Resistance with CpG-A Toll-Like Receptor 9 Agonist Vidutolimod in Patients with Metastatic Melanoma.

Patients with advanced melanoma that is resistant to PD-1 blockade therapy have limited treatment options. Vidutolimod (formerly CMP-001), a virus-like particle containing a CpG-A Toll-like receptor 9 (TLR9) agonist, may reverse PD-1 blockade resistance by triggering a strong IFN response to induce and attract antitumor T cells. In the dose-escalation part of this phase Ib study, vidutolimod was administered intratumorally at escalating doses with intravenous pembrolizumab to 44 patients with advanced melanoma who had progressive disease or stable disease on prior anti-PD-1 therapy. The combination of vidutolimod and pembrolizumab had a manageable safety profile, and durable responses were observed in 25% of patients, with tumor regression in both injected and noninjected lesions, including visceral lesions. Patients who responded to vidutolimod and pembrolizumab had noninflamed tumors at baseline and induction of an IFNγ gene signature following treatment, as well as increased systemic expression of the IFN-inducible chemokine CXCL10. SIGNIFICANCE: In this phase Ib study in patients with advanced melanoma, intratumoral TLR9 agonist vidutolimod in combination with pembrolizumab had a manageable safety profile and showed promising clinical activity, supporting the further clinical development of vidutolimod to overcome PD-1 blockade resistance through induction of an IFN response. See related commentary by Sullivan, p. 2960. This article is highlighted in the In This Issue feature, p. 2945.

Antibody Opsonization of a TLR9 Agonist-Containing Virus-like Particle Enhances In Situ Immunization.

The immunologic and therapeutic effects of intratumoral (IT) delivery of a novel virus-like particle as a lymphoma immunotherapy were evaluated in preclinical studies with human cells and a murine model. CMP-001 is a virus-like particle composed of the Qß bacteriophage capsid protein encapsulating an immunostimulatory CpG-A oligodeoxynucleotide TLR9 agonist. In vitro, CMP-001 induced cytokine production, including IFN-α from plasmacytoid dendritic cells, but only in the presence of anti-Qß Ab. In vivo, IT CMP-001 treatment of murine A20 lymphoma enhanced survival and reduced growth of both injected and contralateral noninjected tumors in a manner dependent on both the ability of mice to generate anti-Qß Ab and the presence of T cells. The combination of IT CMP-001 with systemic anti-PD-1 enhanced antitumor responses in both injected and noninjected tumors. IT CMP-001 alone or combined with anti-PD-1 augmented T cell infiltration in tumor-draining lymph nodes. We conclude IT CMP-001 induces a robust antitumor T cell response in an anti-Qß Ab-dependent manner and results in systemic antitumor T cell effects that are enhanced by anti-PD-1 in a mouse model of B cell lymphoma. Early-phase clinical evaluation of CMP-001 and anti-PD-1 combination therapy in lymphoma will begin shortly, based in part on these results.

Enhancing antisense efficacy with multimers and multi-targeting oligonucleotides (MTOs) using cleavable linkers.

The in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8-15 nucleotides and by the incorporation of high affinity RNA binders such as 2', 4'-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2',4'-constrained ethyl [cET]). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4-5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications.

Exon skipping therapy for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused mostly by internal deletions in the gene for dystrophin, a protein essential for maintaining muscle cell membrane integrity. These deletions abrogate the reading frame and the lack of dystrophin results in progressive muscle deterioration. DMD patients experience progressive loss of ambulation, followed by a need for assisted ventilation, and eventual death in mid-twenties. By the method of exon skipping in dystrophin pre-mRNA the reading frame is restored and the internally deleted but functional dystrophin is produced. Two oligonucleotide drugs that induce desired exon skipping are currently in advanced clinical trials.

The ability of CpG oligonucleotides to protect mice against Francisella tularensis live vaccine strain but not fully virulent F. tularensis subspecies holarctica is reflected in cell-based assays.

CpG DNA is a potent activator of the innate immune system. Here the protective effects of CpG DNA are assessed against the facultative intracellular pathogen Francisella tularensis. Dosing of mice with CpG DNA provided protection against disease caused by F. tularensis subsp. holarctica live vaccine strain (LVS) but did not protect against the fully virulent F. tularensis subsp holarctica strain HN63. Similarly, in vitro studies in J774A murine macrophage-like cells demonstrated that stimulation with CpG DNA enables control of intracellular replication of LVS but not HN63. These data confirm findings that CpG DNA may have limited efficacy in providing protection against fully virulent strains of F. tularensis and also suggest that in vitro assays may be useful for the evaluation of novel treatments for virulent F. tularensis.

Clinical evaluation of safety and immunogenicity of PADRE-cytomegalovirus (CMV) and tetanus-CMV fusion peptide vaccines with or without PF03512676 adjuvant.

BACKGROUND: It has been reported that cytomegalovirus (CMV) pp65-specific T cells can protect hematopoietic cell transplant (HCT) recipients from CMV complications. Two candidate CMV peptide vaccines composed of the HLA A*0201 pp65(495-503) cytotoxic CD8(+) T-cell epitope fused to 2 different universal T-helper epitopes (either the synthetic Pan DR epitope [PADRE] or a natural Tetanus sequence) were clinically evaluated for safety and ability to elicit pp65 T cells in HLA A*0201 healthy volunteers. METHODS: Escalating doses (0.5, 2.5, 10 mg) of PADRE or Tetanus pp65(495-503) vaccines with (30 adults) or without (28 adults) PF03512676 adjuvant were administered by subcutaneous injection every 3 weeks for a total of 4 injections. RESULTS: No serious adverse events were reported, although vaccines used in combination with PF03512676 had enhanced reactogenicity. Ex vivo responses were detected by flow cytometry exclusively in volunteers who received the vaccine coadministered with PF03512676. In addition, using a sensitive in vitro stimulation system, vaccine-elicited pp65(495-503) T cells were expanded in 30% of volunteers injected solely with the CMV peptides and in all tested subjects receiving the vaccines coinjected with PF03512676. CONCLUSIONS: Acceptable safety profiles and vaccine-driven expansion of pp65(495-503) T cells in healthy adults support further evaluation of CMV peptide vaccines combined with PF03512676 in the HCT setting. CLINICAL TRIALS REGISTRATION: NCT00722839.

Lipid-derived nanoparticles for immunostimulatory RNA adjuvant delivery.

The specific activation of Toll-like receptors (TLRs) has potential utility for a variety of therapeutic indications including antiviral immunotherapy and as vaccine adjuvants. TLR7 and TLR 8 may be activated by their native ligands, single-stranded RNA, or by small molecules of the imidazoquinoline family. However the use of TLR7/8 agonists for in vivo therapy is limited by instability, in the case of RNA, or systemic biodistribution and toxicity in the case of small molecule agonists. We hypothesized that unique lipid-like materials, termed "lipidoids," could be designed to efficiently deliver immunostimulatory RNA (isRNA) to TLR-expressing cells to drive innate and adaptive immune responses. A library of lipidoids was synthesized and screened for the ability to induce type I IFN activation in human peripheral blood mononuclear cells when combined with isRNA oligonucleotides. Effective lipidoid-isRNA nanoparticles, when tested in mice, stimulated strong IFN-α responses following subcutaneous injection, had robust antiviral activity that suppressed influenza virus replication, and enhanced antiovalbumin humoral and cell-mediated responses when used as a vaccine adjuvant. Further, we demonstrate that whereas all immunological activity was MyD88-dependent, certain materials were found to engage both TLR7-dependent and TLR7-independent activity in the mouse suggestive of cell-specific delivery. These lipidoid formulations, which are materials designed specifically for delivery of isRNA to Toll-like receptors, were superior to the commonly used N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate-RNA delivery system and may provide new tools for the manipulation of TLR responses in vitro and in vivo.

CpG still rocks! Update on an accidental drug.

The discovery of the CpG motif in 1995 led to a change in the perception of the immune stimulatory effects of oligodeoxynucleotides (ODN) from an unwanted nonspecific effect to a highly evolved immune defense that can be selectively triggered for a wide range of therapeutic applications. Over the last decade dozens of human clinical trials have been conducted with different CpG ODN in thousands of humans for applications ranging from vaccine adjuvant to immunotherapies for allergy, cancer, and infectious diseases. Along with many positive results have come some failures showing the limitations of several therapeutic approaches. This review summarizes these results to provide an overview of the clinical development of CpG ODN.

Combining vaccination and postexposure CpG therapy provides optimal protection against lethal sepsis in a biodefense model of human melioidosis.

The Gram-negative bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a major cause of lethal sepsis and morbidity in endemic areas of Southeast Asia and a potential bioterrorism threat. We have used susceptible BALB/c mice to evaluate the potential of targeting vaccination and generic immunotherapy to the lung for optimal protection against respiratory challenge. Intranasal vaccination with live attenuated B. pseudomallei increased survival and induced interferon-γ-secreting T cells in the lung. Intranasal delivery of CpG oligodeoxynucleotides also provided significant protection; however, combining preexposure vaccination with CpG treatment at the time of infection or up to 18 hours after infection, provided significantly greater protection than either treatment alone. This combination prolonged survival, decreased bacterial loads by >1000-fold, and delayed the onset of sepsis. This novel approach may be applicable to other potential biodefense agents for which existing countermeasures are not fully effective.

Immunostimulatory potential of silencing RNAs can be mediated by a non-uridine-rich toll-like receptor 7 motif.

Microbial infections trigger a multiplicity of responses in the host via innate immune sensors, including the Toll-like receptors (TLRs). TLR7 and TLR8, located in endosomes, detect pathogen-derived RNA, which can be mimicked by synthetic single-stranded oligoribonucleotides (ORNs). Detailed analysis of the immunostimulatory properties of numerous silencing RNAs (siRNAs) revealed that almost all tested siRNAs with a phosphodiester backbone actively stimulated cytokine production in human peripheral blood immune cells, but not all of them did contain previously described guanosine/uridine TLR7 or adenosine/uridine TLR8 motifs. By analysis of sequence variants of these siRNAs (as single- or double-strands), we were able to identify a new immunostimulatory, non-uridine-rich TLR7 motif that is present in many published siRNAs. Interestingly, the activity of this motif is dependent on the backbone chemistry. Phosphorothioate ORNs containing the motif did not stimulate immune activation, whereas phosphodiester ORNs of the same sequence induced a strong TLR7-biased immune response with high amounts of interferon-alpha. Using TLR7- and Myd88-deficient mice, we demonstrated that stimulation by ORNs containing this motif was TLR7 dependent. Our findings are of therapeutic relevance as this motif is present in many siRNA sequences and will to contribute to the immunostimulatory properties of unmodified siRNAs.