Therapeutic Immune Modulation Against Solid Cancers with Intratumoral Poly-ICLC: a Pilot Trial

Author Manuscript Published OnlineFirst on June 27, 2018; DOI: 10.1158/1078-0432.CCR-17-1866 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Title: Therapeutic Immune Modulation Against Solid Cancers with Intratumoral Poly-ICLC: A Pilot Trial

Running Title: Intratumoral Poly-ICLC in Solid Cancer Treatment

Authors: Chrisann Kyi1*, Vladimir Roudko1*, Rachel Sabado1, Yvonne Saenger2, William Loging1, John Mandeli1, Tin Htwe Thin1, Deborah Lehrer1, Michael Donovan1, Marshall Posner1, Krzysztof Misiukiewicz1, Benjamin Greenbaum1, Andres Salazar3, Philip Friedlander1, Nina Bhardwaj1 Tisch Cancer Center, Icahn School of Medicine at Mount Sinai, New York, NY1; Columbia University Medical Center, New York, NY2; Oncovir, Inc, Washington, DC 3

Grant Support: This research was supported by grants from the Cancer Research Institute, the Melanoma Research Alliance and NIH. N.B. is a member of the Parker Institute for Cancer Immunotherapy, which supported the Mount Sinai Hospital Cancer Immunotherapy Program.

Disclosures of Potential Conflicts of Interest: N B. is on the scientific advisory board of Neon, CPS companion diagnostics, Genentech and Curevac, Inc. R.S. works as clinical scientist at Genentech, Inc. A.S. is the scientific director of Oncovir, Inc. No potential conflicts of interest were disclosed by other authors.

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 27, 2018; DOI: 10.1158/1078-0432.CCR-17-1866 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

TRANSLATIONAL RELEVANCE We present a novel vaccine approach using intratumoral poly-ICLC, a viral mimic of the double-stranded RNA in viral replication, as a strategy of “autovaccination,” i.e. the use of the tumor itself as the antigen source in-situ. In this first evaluation of poly-ICLC in melanoma and head and neck cancer patients, we investigate the safety and tolerability of poly-ICLC administered intratumorally to induce tumor immune infiltration and intramuscularly to induce systemic inflammation. Treatment was well-tolerated with minimal toxicities noted. In the one patient with clinical benefit (stable disease), there was evidence of upregulation of genes associated with chemokine activity, T cell activation and antigen presentation (RNA sequencing), and increased CD4, CD8, PD1 and PDL1 levels (quantitative immunohistochemistry) compared to patients with progressive disease. Although only one patient demonstrated clinical benefit, these findings prompt further investigation into optimal dosing and delivery of intratumoral poly-ICLC, and combinations with immune checkpoint blockade and/or other immunomodulators.

ABSTRACT Purpose: Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (Poly-ICLC), a synthetic double-stranded RNA complex, is a ligand for toll-like receptor-3 (TLR3) and MDA-5 that can activate immune cells such as dendritic cells and trigger NK cells to kill tumor cells. Methods: In this pilot study, eligible patients included those with recurrent metastatic disease who failed prior systemic therapy (head and neck squamous cell cancer (HNSCC), melanoma). Patients received 2 treatment cycles, each cycle consisting of 1mg Poly-ICLC 3x weekly intratumorally (IT) for 2 weeks followed by intramuscular (IM) boosters biweekly for 7 weeks with a 1-week rest period. Immune response was evaluated by immunohistochemistry (IHC) and RNA Sequencing (RNASeq) in tumor and blood. Results: Two patients completed 2 cycles of IT treatments and one achieved clinical benefit (stable disease, PFS 6 months), while the remainder had progressive disease. Poly-ICLC was well tolerated with principal side effects of fatigue and inflammation at injection site (< grade 2). In the patient with clinical benefit, IHC analysis of tumor showed increased CD4, CD8, PD1 and PDL1 levels compared to patients with progressive disease. RNASeq analysis of the same patient’s tumor and PBMC showed dramatic changes in response to Poly-ICLC treatment including upregulation of genes associated with chemokine activity, T cell activation and antigen presentation. Conclusions: Poly-ICLC was well tolerated in solid cancer patients, and generated local and systemic immune responses as evident in the patient achieving clinical benefit. These results warrant further investigation, and are currently being explored in a multicenter phase II clinical trial (NCT02423863).

INTRODUCTION The last decade has ushered in an exciting new age of immunotherapy with the FDA approvals of the first cancer vaccine, Provenge or sipuleucel-T, and checkpoint blockade, e.g. ipilimumab (Yervoy; anti- CTLA-4), pembrolizumab (Keytruda; anti-programmed death (PD1)), and nivolumab (Opdivo; anti-PD1). However, even with advances in immune checkpoint blockade and other systemic chemotherapies, there remain a significant fraction of patients who either fail to respond or become resistant to treatment. Proposed interventions to broaden the fraction of patients benefiting from immunotherapies and increase response rates rely on reversing T cell exhaustion, reducing immune suppression in the tumor microenvironment (TME), and transforming a non-inflamed TME to a “responsive” TME (e.g. immune cell infiltration, upregulation of PDL-1 etc). Oncolytic viruses in the treatment of cancer work presumably through their effects not only upon tumor cells but by activating innate immunity and inducing tumor specific immunity 1-4. An intratumoral approach that mimics viral infection, without associated significant side effects or the complications of inducing dominant antiviral immunity, is one proposed strategy5.

Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (Poly-ICLC, Hiltonol, Oncovir, Inc) is a synthetic double-stranded RNA viral mimic for a pathogen associated molecular pattern (PAMP) or ‘danger signal’ that binds to toll-like receptor 3 (TLR3), MDA5 and other pathogen receptors to activate dendritic cells (DCs) and subsequently to also trigger NK cells to kill tumor cells. While initially developed as an IFN inducer, Poly-ICLC has been found to have much broader biological effects, including specific anti-tumor and anti-viral actions6. It activates multiple elements of innate and adaptive immunity, including induction of a ‘natural mix’ of IFNs, other cytokines and chemokines, NK cells, T cells, myeloid DCs, the P68 protein kinase (PKR), and other dsRNA-dependent host defense systems7,8. Thus, when properly combined with antigen, Poly-ICLC has the potential to generate a 'live virus vaccine equivalent' with a comprehensive immune response that includes activation of myeloid DCs, other antigen-presenting cells, and NK cells, and generation of a polyfunctional Th1-polarized and CTL response with increase in CD8 to CD4 / regulatory T cell ratio, which via the induction of specific chemokines can home to tumor or pathogen 9-14.

While most cancer vaccines are generally designed to utilize known or presumptive tumor antigens, an alternative strategy is ‘autovaccination,’ i.e. the use of the tumor itself as the antigen source, in-situ. Poly- ICLC can be given intramuscularly (IM) to induce systemic inflammation and/or intratumorally (IT) to induce immune infiltration of tumors. We observed a dramatic response in the first sarcoma case being treated with repeated IT and IM Poly-ICLC, an 18-year-old patient with a malignant embryonal rhabdomyosarcoma, who failed eight different regimens of chemotherapy as well as radiation and proton- beam therapy, and was in hospice. Treatment with Poly-ICLC resulted in necrosis and regression of facial, oral, retro-orbital and a large intracerebral tumor. Although the patient eventually succumbed, his life was extended well beyond expectations 15. A phase II trial of single-dose treatment of ultrasound guided IT Poly-ICLC followed by IM Poly-ICLC was found to be safe in patients with advanced primary or metastatic liver cancers, with evidence of regression of non-injected metastatic lesions as well as the targeted lesions16,17.

Based upon these early indications of clinical response, we conducted a pilot study using this autovaccination strategy with intratumoral and intramuscular Poly-ICLC at our institution in advanced treatment refractory head and neck cancers and melanoma. We hypothesize that the therapeutic in-situ autovaccination strategy using intratumoral (IT) and intramuscular (IM) Poly-ICLC administration of TLR3 ligand Poly-ICLC can reverse DC inhibition in the treated tumor micro-environment, increase the efficiency of antigen presentation to CTLs, prevent tolerization to tumor antigens, and elicit systemic antitumor immunity. Here, we present the first-ever published results of our phase I trial of intratumoral poly- ICLC in treatment of solid cancer patients.

MATERIALS AND METHODS

Patients Between 2013 and 2015, 8 patients (7 head and neck squamous cell cancers (HNSCC) and 1 melanoma) were enrolled in pilot phase of clinical trial. This trial is now accruing as a phase II multicenter clinical trial (NCT02423863). Eligible patients had unresectable recurrent or metastatic disease that had failed prior systemic therapy and was radiologically or visually measurable disease at least 10mm in longest dimension. At least one accessible primary or metastatic tumor site was necessary for intratumoral injection with poly-ICLC with or without ultrasound guidance. All patients had ECOG performance status of ≤ 2 and acceptable hematologic, renal and liver function per laboratory parameters. Exclusion criteria included bulky intracranial metastatic disease, history of active immunotherapy in the previous month, AIDS defined as CD4 count <200, and life expectancy of less than 6 months in the judgement of the study physician. Written consent was obtained from patients, and the study was conducted in accordance with the provisions of the Declaration of Helsinki. The study protocol and all amendments were approved by the institutional review board at Mount Sinai Hospital.

Treatment plan Patients were to receive two cycles of poly-ICLC treatment, each cycle including a priming and boosting treatment course (Figure 1), with dosing and frequency based on prior preclinical and phase I trials 18,19. For cycle 1, patients were treated with 1 mg of Poly-ICLC thrice weekly during week 1-2 (priming treatment course, a total of 6 IT injections) into the same lesion. During weeks 3-9, patients were treated with IM maintenance boosters biweekly (1mg per dose, boosting course, total 14 IM injections), followed by a rest week (week 10) without treatment. This 10-week cycle was repeated in cycle 2. Weeks 20 – 26 were a “no treatment rest period” to allow for evaluation of response in the absence of inflammation at week 26. At week 26, patients were assessed and response determined, and those patients with complete response (CR), partial response (PR), or stable disease (SD) were offered option of maintenance therapy from week 27-36 with administration of 1 mg IM poly-ICLC twice weekly.

Disease assessment Tumor response was evaluated using immune-related response criteria (irRC) in Solid Tumors 20: complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). All the patients with measurable disease at the time of enrollment on the study were eligible for response assessment.

Correlative biology studies Serial blood samples collected at baseline, and at selected time points during and post treatment (Figure 1) were processed to collect plasma/serum and peripheral blood mononuclear cells (PBMCs), and used to evaluate humoral and cellular immunity induced by IT and IM poly-ICLC injections. Tumor biopsies were performed at baseline, week 3, and week 26 (if possible). Pre- and post-vaccination tumor biopsies of both intratumorally treated tumors, and non-treated distant tumors taken when possible, were analyzed by quantitative multiplex immunohistochemistry (IHC) for lymphocyte infiltration (e.g. CD4 and CD8 T cell subsets, T regulatory cells (FoxP3+), PD-1 or CTLA-4 expressing T cells, DC subsets, as well as other immune markers (CD86 antigen-presenting cells, CD68 macrophages / monocytes, CD16 natural killer cells, and HLA encoding the major histocompatibility complex (MHC) proteins using standard immunohistochemistry techniques 21. Individual antigens were quantified using the CRI Inform software (Perkin Elmer) which applies user-directed antigen thresholds to generate percentages normalized to the total tumor area.

RNA sequencing analysis RNA sequencing analysis (RNA-Seq) and TCR sequencing (Adaptive, Inc) were done on PBMC and tumor tissue. PBMCs were isolated from patients at multiple time points prior to and post treatment with

Poly-ICLC. Paired end sequencing of cDNA created from isolated mRNA was performed using the Illumina HiSeq 2500 platform at a depth of 30M-35M reads. The read quality was assessed using FastQC 22. Corresponding sequence files were processed using publicly available RNA-seq data analysis pipelines 23. Briefly, aligned reads are assigned to genes using the FeatureCounts function of Rsubread. Gene expression in terms of log2-CPM (counts per million reads) was computed and normalized across samples. Genes with low expression (ones not having at least 10 reads per million reads in at least two samples) were filtered out. Differential expression analysis was performed using the limma software package (R, Bioconductor). Gene set enrichment analysis (GSEA) was computed following published recommendations 24,25. Analysis of blood transcriptome modules (BTM) was done accordingly 26 Comparisons between time points as well as pre and post treatment were conducted addressing both gene and pathway specific changes 24.

For TCR analysis, genomic DNA was purified from total PBMCs and tumor samples using the Qiagen DNeasy Blood extraction kit. The TCRβ CDR3 regions were amplified and sequenced using immunoSEQ® (Adaptive Biotechnologies, Seattle, WA), as previously described 27.

Survival analysis Kaplan-Meyer survival curve analyses were performed using publicly available on-line server Tumor Immune Estimation Resource (TIMER, https://cistrome.shinyapps.io/timer/).

RESULTS The primary objective of this phase I pilot study was to evaluate the safety of intratumoral (IT) plus intramuscular (IM) poly-ICLC for treatment of patients with advanced accessible solid tumors. Secondary objectives were to investigate the changes in humoral and adaptive cellular immunity induced by autovaccination with poly-ICLC.

Patient characteristics and disease response Eight patients with treatment refractory solid tumors (1 melanoma, 7 head and neck cancer) were enrolled in this study between January 2014 and July 2014. Two subjects completed 2 cycles of IT treatment though one of these patient did not complete a second IM boosting cycle; the remaining 6 subjects completed 1 cycle or less of IT poly-ICLC due to progression of disease (Table 1).

One of two patients who completed 2 cycles of IT poly-ICLC achieved clinical benefit. Patient 002, was a 54-year-old man with metastatic EBV-positive nasopharynx HNSCC, previously treated and refractory to chemotherapy. He tolerated treatment to completion of 2 cycles (12 IT and 28 IM injections in total) over a 30-week period, and was the one patient who achieved clinical benefit with stable disease (progression-free survival of 6 months) (Figure 2). At 6 months he developed diplopia due to new brain metastasis; patient treatment was switched to chemotherapy with radiation with control of disease. Patient was still alive on subsequent line of chemotherapy 18 months out from start of poly-ICLC treatment.

The other 7 patients had progressive disease while on poly-ICLC treatment (Table 1). Patient 001 was the only other patient (HNSCC) in our pilot trial to complete 2 cycles of IT poly-ICLC treatments (12 IT injections) though only had 20 of 28 IM boosting treatments. Patient was initially thought to have clinical improvement on exam. CT scans at 10 weeks suggested pseudoprogression and as the patient was clinically stable, he remained on study for cycle 2. Unfortunately, repeat imaging at week 15 showed clear progression of disease with innumerable pulmonary nodules and patient was taken off study in the middle of second IM boosting cycle (after 12 IT and 20 IM poly-ICLC injections total) and switched to salvage chemotherapy.

Patients 003, 004, 005, and 008 were patients with metastatic HNSCC (range 70-80 years old) who also stopped treatment shortly after initiation of poly-ICLC treatment due to rapidly progressive disease (Table 1). Patient 003 received only 3 IT injections, while patients 004, 005 and 008 received the first course of 6 IT injections and subsequently 3-6 IM injections before progressing. As an example, patient 008 was a 70 male patient with metastatic HNSCC whose treatment was halted particularly early, in cycle 1 at week 3-4 due to progressive disease (6 IT and 3 IM injections in total). CT scans at 10 weeks showed progressive disease with new lung nodules and enlarging liver and right abdominal wall lymph nodes. Tissue biopsies were obtained at week 3 and end-of treatment (week 9) per protocol.

Patient 006 was a patient with BRAF-wildtype subungal melanoma of the right fourth digit, who developed multiple recurrences through chemotherapy, radiation, targeted therapy and checkpoint blockade. The patient completed 6 IT and 5 IM injections but treatment was stopped prior to cycle 2 due to rapidly progressive disease. At 9 week scans, he had progressive disease of pulmonary nodules and lower extremity lesions. The patient was initiated on anti-PD1 blockade (2014), but subsequently progressed through treatment and died.

Patient 007 was a patient with advanced HPV-negative, EBV-positive, progressive tumor after induction chemotherapy and radiation. He was consented and enrolled in the poly-ICLC trial but after one IT poly- ICLC injection, had aspiration pneumonia / pneumonitis, that was temporally related to aspiration and not considered directly related to treatment. Subsequently, patient was taken off study.

Toxicities Poly-ICLC was generally well tolerated with the principal side effects of treatment, fatigue and inflammation at primary injection sites, less than grade 2. One case of overt necrosis of tumor (grade 2) was observed. There was a case of grade 3 pneumonitis in 1 patient (007) but this was temporally related to aspiration pneumonia and not considered directly related to poly-ICLC treatment. Full range of toxicities are listed in detail in Table 1.

Tumor biopsies and samples Baseline, week 3 (post-6 IT injections) and week 26 (post IT and IM injection) biopsies were obtained if possible. Unfortunately, week 3 (and subsequent) biopsy was not obtained in patients 003 and 007 due to complication of bibasilar pneumonia requiring hospitalization and grade 3 aspiration pneumonitis, respectively. Week 26 biopsy was not obtained for patients 001, 004, 005, and 006 due to progressive disease requiring study termination and change in treatment plan. Except for patients 002, 004, 008, while the week 3 biopsy was sufficient for pathology diagnosis and IHC analysis, tissue was insufficient for RNA sequencing due to inadequate sample. A summary of treatment courses, and biopsies (including if tissue adequate for IHC and RNA sequencing analysis) for each subject is outlined in Table 1.

Immunohistochemistry data Tumor biopsies were obtained of injected site for intratumoral Poly-ICLC at baseline and at week 3 (after 6 IT injections) and week 26 if possible. Quantitative IHC from tumor biopsies of patients with progressive disease (001, 004, 005, and 008) showed unchanged or decreased levels of CD4, CD8, PD-1, and PDL-1 over the course of the treatment period (Figure 3, supplemental table 1). In contrast, IHC analysis of tumor biopsies of patient 002, the one patient who had clinical benefit (stable disease) showed increased levels of CD4 (60-fold), CD8 (10-fold), PD-1 (20-fold) and PDL-1 (3-fold). Additionally, tumor biopsies obtained in patient 002 showed marked increases in immune cells (CD86+ antigen- presenting cells, CD68+ macrophages / monocytes, CD16+ natural killer cells, HLA encoding the major histocompatibility complex (MHC) expression) post treatment (Figure 3).

RNA sequencing analysis

RNA sequencing was used to characterize immune response in the blood compartment (PBMC) versus the tumor site. Analysis of over 18,000 transcripts revealed that poly-ICLC treatment resulted in changes in gene expression in interferon-related genes both at the tumor and PBMC level indicative of local and systemic immune activation.

Non-supervised hierarchical clustering of patients’ PBMC revealed three major groups of clustering (Figure 4A). First, similar blood RNA expression profiles were observed between patient 002 pre- treatment and off-treatment samples, and screening samples of patients 004 and 008 who developed progressive disease (PD) along with one sample during intra-muscular poly-ICLC injection. We assigned these samples as baseline cluster, “minimal or absence of immune activation”. Second, similar expression clustering was observed in patient 002 who had stable disease (SD), indicative of systemic inflammation resulting in a clinical benefit response (inflamed cluster). A clustering of “intermediate” signaling or activation of immune system was observed in blood samples derived from patients 004 and 008 undergoing IT poly-ICLC injections and patient 002 undergoing IM poly-ICLC injections (intermediate cluster).

In order to gain insight on expression alterations with poly-ICLC treatment, we calculated differential gene expression considering samples within clusters as biological parallels. Resulting scatter plot of expression fold changes, were inferred from two comparisons: patient 004 and 008 samples from intermediate cluster versus baseline (PD, Figure 4B) and patient 004 samples from inflamed cluster versus baseline (SD, Figure 4B), revealed several trends. The majority of gene expression changes upon poly-ICLC intratumoral injections were similar between patients 004, 008 and 002. However, specific gene sets were up-regulated in patient 002, but repressed or unchanged in patients 004 and 008 (624 genes, upper-left quadrant Figure 4B). These included genes regulating anti-tumor activity: stimulatory cytokines and chemokines: IL-1 beta, CXCL8; antigen processing-related marker CD83; T cell activation-related protein IFN gamma (see Table 2A). Fewer genes were specifically up-regulated in patients 004 and 008 (66 genes, lower-right quadrant, Figure 4B), with some related to an interferon response: ISGs OAS, DDX60 (see Table 2B).

To better understand these trends in PBMC of the patient 002, we performed gene set enrichment analyses (GSEA) conditioned upon two independent sources of relevant published gene sets: blood transcriptome modules23 (BTM) and the Broad Institute immune gene collection21 (C7). GSEA using BTM demonstrated upregulated presence of activated DCs in the blood of patient 002 with IT poly-ICLC treatment, as well as upregulation of stimulatory cytokine expression. Interestingly, enrichment of these gene sets was specific for the patient 002, while NK/T cell gene expression was similar between patients with SD and PD responses (Figure 5). Notably, immune gene expression changed minimally in PBMC upon IM poly-ICLC treatment in both SD and PD patients, but strongly upon IT poly-ICLC treatments - suggesting the importance of IT poly-ICLC injection to achieve systemic immune cell activation. Analysis of GSEA of immune genesets from the Broad Institute yielded similar trends (Supplemental Figure 1). Taken together, our observations suggest systemic activation of the immune system including T cell activation, elevated antigen presentation cells and inflammatory cytokine expression in patient 002 blood upon intratumoral injections of poly-ICLC] as defined by BTM and Broad genesets.

To gain insight on intratumoral immune cell infiltration profiles, we performed similar geneset enrichment analyses on tumor RNAseq samples. Both immune geneset collections detected gene signatures of activated DCs, B cells, CD8+ T cells, Th17 polarized CD4+ T cells, NK cells, monocytes and neutrophils. Expression of gene signatures related to IFN, IFN, IFN, IL17 stimulation were similarly elevated at the tumor sites of patients with SD and PD responses: at approximately similar levels, before and after poly-ICLC treatment (Supplemental Figures 2-4). The latter suggests the immune system is activated at the tumor site, but fails to execute its function due to perhaps different levels and/or distribution of immune cells in tumor and/or exhaustion states as observed in the IHC data.

Combined together, PBMC and tumor RNAseq gene expression analyses revealed differences in immune profiles between patient 002 and patients 004 and 008. Notably, we detected differential cytokine expression and immune cell activation related to DC-NK cell crosstalk (Figure 6, Supplemental Table 2). DC infiltration in blood correlated with specific up-regulation of XCL1/2 chemokines by NK or T cells in the patient 002 (Figure 6A). This observation is in agreement with specific up-regulation of IL15 and IL23A at the end of the treatment cycle by the same patient, as well as elevated expression of Clec9A. We speculate that XCL1/2 expression may attract the migratory DC subset XCR1-DC, known for its ability to produce IL15, type I IFN, as well as cross-present tumor derived antigens through the Clec9A receptor. Interestingly, the chemokines CXCL1 and CXCL5 were both elevated at the tumor site of patients 004 and 008 (Figure 6B) prior to and after poly-ICLC treatment. Both cytokines are ligands of the CXCR2 receptor, activation of which in tumors has been shown to interfere with PD-1/PDL-1 immunotherapies in pre-clinical studies 28,29. The trend of increased CXCL1 and CXCL5 expression may identify a pro-tumorigenic inflammatory profile, possibly interfering with the poly-ICLC regimen, while expression of IL15, IL23A, Clec9A, XCR1, XCL1, XCL2 may reflect a better response to immunotherapy.

Our data is limited to the analysis of just a few patients, therefore we sought verification of these signatures in larger data sets from TCGA as a potential approach to define prognostic biomarkers of response to poly-ICLC. Survival analyses of available cancer patient data derived from the TCGA database supports our hypothesis (Figure 6). The high expression of gene markers associated with clinical benefit of poly-ICLC, correlates with better prognosis in HNSCC (HNSC) and melanoma (SKMC) patients (Figure 6C; e.g. DC associated factors IL-15, XCR1). In contrast, elevated expression of pro-tumorigenic receptor CXCR2 and its ligand CXCL1 correlates with poor prognosis (Figure 6D). These observations, though clinical trends, suggest that presence of antigen-presenting cells, IL15 and IL23A at tumor sites have a beneficial effect on patient survival and perhaps, on response to immunotherapy, while expression of CXCR2/CXCL1/CXCL5 axis may interfere with the treatment.

Seromics and humoral response No significant findings were noted in analysis of pre- and post- IT poly-ICLC treatment in patients.

T-Cell Receptor (TCR) Analysis TCR sequencing revealed that the patient 002 with clinical benefit (SD) showed increases in TCR clonality (~20% increase) and density (~30% increase) after poly-ICLC treatment. Unfortunately, similar analyses of other patients were not possible due to inadequate tissue sampling size or no available tissue after other tests performed (IHC and RNA sequencing).

DISCUSSION AND CONCLUSIONS Here we present the results of a pilot trial testing a strategy of therapeutic in situ autovaccination with intratumoral injections of the dsRNA viral mimic and TLR agonist, poly-ICLC. Poly-ICLC was well tolerated and generated local immune response in tumor microenvironment and systemic immune response as evident in the patient achieving clinical benefit.

A major limitation of the trial was the study population of patients with refractory disease who had failed several other lines of treatment (often with recent chemotherapy) (Table 1), and thus already highly immunosuppressed. Certain chemotherapies can suppress T cells proliferating in response to antigen or poly-ICLC, and this effect is seen for as long as 6 months after cessation of chemotherapy30. In this pilot trial, there were 3 rapid progressers who did not complete even 1 cycle, and may not have received enough treatment with single agent poly-ICLC to generate an antitumor immune response. Therefore, single agent poly-ICLC may not be adequate treatment for patients with advanced, rapidly progressing HNSCC; however, information learned from our study can inform combination treatments in the future.

In the patient achieving stable disease, our pilot results suggest how sequential IT injections of poly-ICLC can potentially induce an effective personalized systemic therapeutic “autovaccination” against tumor antigens in patients. We postulate that a therapeutic in-situ autovaccination strategy using Poly-ICLC works via three immunomodulatory steps. First, IT Poly-ICLC potentially activates NK cells, triggers TLR3 and MDA-5 receptors present at many APCs, and induces other pro-apoptotic mechanisms, resulting in local tumor killing and release of tumor antigens 31-33. In prior studies, it has been shown that poly-ICLC can upregulate several hundreds of genes closely representing some 10 canonical innate immune pathways 6,34,35. Our RNA sequencing analysis detected expression of over 18,000 transcripts and revealed poly-ICLC treatment changed gene expression of interferon-related genes both at the tumor and PBMC level indicative of local and systemic immune activation. Upregulation of genes in response to poly-ICLC treatment included chemokines, interferon stimulated genes, genes associated with antigen processing, T cell activation, and apoptosis.

In the second step, Poly-ICLC danger signals activate macrophages and dendritic cells at the tumor site, in which they acquire tumor antigens that are cross-presented to CD4 helper cells and to CD8 T cells to generate antigen-specific cytotoxic T lymphocytes (CTLs). The repeated administration of the Poly-ICLC danger signal IT in the context of the patient's own tumor antigens may be critical for optimal priming of the system at this step. Gene set enrichment analysis of PBMC and tumor RNAseq also suggests systemic activation of immune system, increased infiltration of antigen presentation cells (activated DCs) in the blood, and upregulation of inflammatory cytokine expression upon intratumoral injections of poly- ICLC. Additionally, RNA sequencing analysis detected gene signatures of activated DCs, B cells, CD8 T cells, Th17 polarized CD4 T cells, NK cells, monocytes and neutrophils. Importantly, T-cell costimulatory cytokines IL15 and IL23A were highly up-regulated at the tumor site of SD patient upon completion of poly-ICLC treatment cycle, indicating the prolonged maintenance of immune activation.

The third step is attraction and maintenance of antigen-specific CTLs via various poly-ICLC induced chemokines, co-stimulatory factors, and other mechanisms 14. This was seen systemically in patient 002 with clinical benefit (SD) whose PBMC showed transcriptional upregulation of genes related to cytokines / chemokines, T cell activation and antigen processing in response to Poly-ICLC treatment. IHC analysis of tumor biopsies of patient 002 showed increased levels of CD4 (60-fold), CD8 (10-fold), PD-1 (20-fold) and PDL-1 (3-fold), consistent with T cell activation, migration and consequent upregulation of PDL-1. Other studies have demonstrated that poly-ICLC alone is sufficient to induce expression of the costimulatory molecules B7-H2, CD40, and OX40 36 37. Our findings as well as others suggests rationale for potential combination of poly-ICLC with OX40, anti–CTLA-4, anti–PD-L1, FLT3L, and other costimulatory factors for enhanced antitumor activity.

One interesting observation was the increased expression of pro-tumorigenic chemokines CXCL1/5 at the tumor site of patient 004 (progressive disease). CXCL1, expressed by tumor-associated macrophages, neutrophils and epithelial cells, has been suggested to have tumorigenic and mitogenic properties in cancer cells, and shown to be a major component required for serum-dependent melanoma cell growth. Importantly, CXCL1 and CXCL5 elicit its effects by signaling through the chemokine receptor CXCR2 found on MDSCs. Recent preclinical studies have shown that activated CXCR2 signaling modulates the tumor immune micoenvironment, reducing CD8+ T cell trafficking and activation, and dampening NK cell activity and promoting regular T cell expansiont24, 25. Therefore, in the patient with progressive disease, elevated expression of CXCL1/5 at the tumor site could indicate activated CXCR2 signaling, interfering with poly-ICLC treatment with ensuring tumor outgrowth. AZD5069 is an investigational selective CXCR2 antagonist that inhibits the migration of CXCR2+ MDSCs to tumor microenvironment and may enhance immune-mediated tumor attack. It is currently being evaluated for safety and efficacy in human clinical trials. Ongoing clinical trials attempt to explore the potentially synergistic combination of anti-PD-L1 inhibitors (durvalumab) in combination with anti CXCR2, AZD5069 (AstraZeneca, anti-

CXCR2) in HNSCC (NCT02499328) and advanced pancreatic ductal cancers (NCT02583477), respectively. Our results also suggest that a combination of intratumoral poly-ICLC with a CXCR2 inhibitor could be an effective strategy and warrants further investigation 28,29. Our ongoing adaptive phase II trial attempts to explore how such inflammatory responses can be harnessed to enhance therapeutic efficacy in such effective combinations.

While limited by a study population of patients with refractory disease who had failed other lines of treatment and were already highly immunosuppressed with significant tumor burden, results of our study should still be informative in the design of future combination studies of poly-ICLC with other systemic therapies including immune checkpoint blockade, or inhibitors of potential pro-tumorigenic signaling pathways, like CXCR2, which are being tested in the clinic. Additionally, other areas of ongoing exploration with poly-ICLC treatment is the optimal dose and schedule of treatment. Timed release or structured formulations of poly-ICLC could be adjusted to better mimic a viral infection, especially for deep tumors in which repeated IT administration is logistically more challenging. At the same time, the possibility of overstimulation with a PAMP must also be considered, and the optimal dose and schedule to mediate optimal anticancer effects remains a needed area of our investigation. Immune infiltrate at distal tumors should also be explored to evaluate whether in situ vaccination has systemic therapeutic effect at other metastatic tumor sites. These trials are on-going and under exploration in our current ongoing adaptive phase II trial that attempts to explore these combinations

In summary, we present a novel intratumoral approach using poly-ICLC, a viral mimic of the double- stranded RNA encountered in viral replication, demonstrating its capacity to induce immunogenic cell death and also an immune stimulation effects through activation of DCs, upregulation of cytokines and chemokines and T cell priming. Indeed, other recent clinical studies with engineered herpes simplex virus-1 expressing granulocyte-macrophage colony-stimulating factor (talimogene laherparepvec) also demonstrated response in distal tumors with evidence of enhanced TILs38. The encouraging results of these studies, intratumoral poly-ICLC, as well as other pattern recognition receptor (PRR agonists such as TLR and STING agonists, suggest how in situ autovaccination can generate a localized antitumor immune response ultimately driving systemic antitumor immunity, and provides a strong rationale for clinical exploration of combinations with immune checkpoint antibodies with poly-ICLC as well as other oncolytic viruses. These preliminary findings study warrant further investigation, and a larger multicenter phase II clinical trial (clinicaltrials.gov, NCT02423863) is now underway to confirm these findings in advanced solid cancers.

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FIGURE LEGENDS AND TABLES

Figure 1. Protocol schema. Patients received two cycles of poly-ICLC treatment, each cycle including a priming and boosting treatment course (Figure 1). In cycle 1, patients were treated with 1 mg of Poly- ICLC 3 times weekly during week 1-2 (“priming treatment”) into the same lesion. During weeks 3-9, patients were treated with IM maintenance boosters biweekly, followed by a rest week (week 10) without treatment. This 10-week cycle was repeated in cycle 2, followed by a “no treatment rest period” during weeks 20 – 26. At week 26, patients were assessed and response determined, and those patients with CR, PR, or SD were offered option of maintenance therapy (from week 27-36 with administration of 1 mg IM poly-ICLC twice weekly). Tumor biopsies were performed at baseline, week 3, and week 26 if possible. Pre- and post-vaccination tumors were evaluated by quantitative multiplex immunohistochemistry (IHC) and RNA sequencing. Blood samples were collected at baseline and throughout treatment cycles (as indicated) for immune response evaluations.

Figure 2 – Clinical data. (A) Post-injection site demonstrating necrosis after IT poly-ICLC treatment (B) Patient 002 was a HNSCC patient who showed clinical benefit (stable disease) with CT scans over the treatment course demonstrating stable disease (PFS over 6 months).

Figure 3. Immunohistochemistry (IHC) of tumor biopsies taken at baseline (top row) and after 6 IT injections (bottom row). In patient 008 (progressive disease, left column), quantitative IHC showed unchanged or decreased levels of CD4, CD8, PD-1, and PDL-1 over treatment period In the patient 002 with clinical benefit (stable disease, right two columns), IHC analysis of tumor showed increased CD4 (60x), CD8 (10x), PD1 (20x) and PDL1 (3x) (also see supplemental table 1), as well as marked increases in immune cells (CD86 antigen-presenting cells, CD68 macrophages / monocytes, CD16 natural killer cells, HLA encoding the major histocompatibility complex (MHC) proteins) post treatment.

Figure 4. A. Hierarchical clustering of PBMC RNA expression from patients with progressive disease PD and stable disease SD. I – baseline cluster, including samples, taken at “screen”, “rest” or during intra-muscular injection time points, samples are: 1, 4, 9, 11, 12, 14, 17. II – intermediate cluster, including samples from PD patients, taken during intratumoral injections: 13 and 15; and samples from SD patient, taken during intra-muscular injections: 3 and 8. III – inflamed cluster, including samples from SD patient, taken after intratumoral injections: 2, 5 and 6. Each column represents patient at specific treatment time point, as numbered below. For example, 002- C1W1 is patient 002 at time point Cycle 1 week1. Samples are: 1 – 002_Screen 10 – 002_C2W20 2 – 002_C1W1 11 – 002_C2W26 3 – 002_C1W3 12 – 004_Screen 4 – 002_C1W7 13 – 004_C1W2 5 – 002_C1W10 14 – 008_Screen 6 – 002_C2W11 15 – 008_C1W1 7 – 002_C2W12 16 – 008_C1W3 8 – 002_C2W13 17 – 008_C1W7 9 – 002_C2W17 18 – 008_C1W10 B. Scatter plot of log2-transformed fold changes, inferred from differential gene expression analyses of two comparisons: PBMC samples 15 and 13 versus cluster I, contrast called “PD”; PBMC samples 2 and 5 versus cluster I, contrast called “SD”. Statistically significant changes (false discovery rate < 0,05) are plotted.

Figure 5. BTM enrichments of selected genesets in PBMC of patients 002 with SD and patient 004 and 008 (PD) collected at different time points during poly-ICLC treatment cycle. (A) unsupervised hierarchical clustering of selected BTM genesets (false discovery rate FDR < 0,25). Patient samples are labeled according to cluster definitions (see Figure 4). Absolute (B) and relative (C) quantification of geneset enrichments in SD and PD patients.

Figure 6. Correlation of NK cell activation signatures and DC infiltration in blood with specific chemokine expression at tumor site and survival analysis of selected gene signatures. Survival analysis of head and neck squamous cell carcinoma (HNSC) and skin cutaneous melanoma (SKCM) patients from the cancer genome atlas (TCGA). Analysis is done by web-server TIMER, ranking patients by selected gene expression and using top 30% and bottom 30% of patients for survival estimation. (A) DC surface markers are highly up-regulated in blood of SD patient (RIGHT). DC presence correlates with XCL1/2 high expression in the blood of same patient (LEFT). (B) Up-regulation T/NK-cell co-stimulatory cytokines IL15 and IL23 at tumor site of patient 002 upon completion of poly-ICLC treatment (RIGHT). Patients 004 and 008 have up regulated pro-tumorigenic cytokines CXCL1 and CXCL5 at the beginning and the end of treatment (LEFT). Each column represents patient at specific treatment time point, as numbered on Figure 4. (C) Increased expression of IL15, IL23A, Clec9A, XCR1, XCL1, XCL2 correlates with better prognosis in HNSC and SKCM (p-value < 0,05; p = 0 indicates high level of significance, p-value substantially below 0.05). (D) Poor survival trend of elevated expression of CXCR2, CXCL1 and CXCL2. Note, increased level of CXCR2 is associated with poor prognosis in SKCM, while CXCL1 – in HNSC (p- value < 0.1)

Table 1. Patient demographics, treatment and toxicities of phase I / pilot trial. Abbreviations: HNSCC: Head and neck squamous cell carcinoma; PD: Progressive disease; SD: Stable disease; EOT: End of treatment; Wk: week; Gr: Grade. I:IHC, R: RNA sequencing. N: Inadequate tissue

Table 2. Selected genes (false discovery rate FDR < 0,05), up-regulated in PBMC with poly-ICLC treatment in patient 002 with clinical benefit SD (A) and patients 004 and 008 with progressive disease PD (B)

Figure 1. CYCLE 1

WEEK 1, Intra-tumor WEEK 2, Intra- WEEK 3-9, IM 2x/wk WEEK 10, rest tumor

Biopsy Biopsy CYCLE 2

WEEK 11, Intra-tumor WEEK 12, Intra- WEEK 13-19, IM 2x/wk WEEK 20-26, rest tumor

Biopsy

MAINTENANCE (12 weeks)

WEEK 27-36 IM 2x/wk KEY Poly-ICLC Injection

Blood draws for immune response evaluation

A. Figure 2 A and B

Pre-Treatment Post-Cycle 1 Post-Cycle 2

Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Patient 008 (PD) 008 (PD) Patient Author ManuscriptPublishedOnlineFirstonJune27,2018;DOI:10.1158/1078-0432.CCR-17-1866 clincancerres.aacrjournals.org PD PD Red CD8: Yellow CD4: PD PD Red CD8: Yellow CD4: -1: Magenta Magenta -1: Green -L1: -1: Magenta Magenta -1: Green -L1: on September 27, 2021. © 2018American Association for Cancer Research. PD PD Red CD8: Yellow CD4: PD PD Red CD8: Yellow CD4: -1: Magenta Magenta -1: Green -L1: Magenta -1: Green -L1: Patient 002 (SD) 002 (SD) Patient CD68, magenta magenta CD68, red ABC, HLA yellow CD16, green CD86, magenta magenta CD68, red ABC, HLA yellow CD16, green CD86, Author Manuscript Published OnlineFirst on June 27, 2018; DOI: 10.1158/1078-0432.CCR-17-1866 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 4. A and B.

Figure 5 A-C..

Figure 6 A-D.

Table 1.

Patient # Age/Sex Diagnosis Recent Prior Treatment Course Biopsy Obtained Toxicities Best Therapy response (<6months) Wk1 Wk 3 Wk 26

001 68F HNSCC Cetuximab plus Cycle 1: 6IT and 14 IM injections X X Gr 1 injection site pain and inflammation PD radiation Cycle 2: 6IT and 6 IM injections (bilateral thighs), fever, fatigue, nausea, myalgia, malaise, fatigue, dizziness; gr 1 alkaline phosphatase increase 002 54M HNSCC Docetaxel Cycle 1: 6 IT and 14 IM injections X X X Gr 2 Periosteal inflammation and necrosis; SD Cycle 2: 6 IT and 14 IM injections Gr 1 injection site pain, flu-like symptoms, fever, fatigue, myalgia, arthralgia

003 80M HNSCC Radiation Cycle 1: 3 IT injections, stopped due X Gr 1 fever, fatigue PD to rapidly POD and infection (bibasilar pneumonia) 004 70M HNSCC Docetaxel Cycle 1: 6 IT and 4 IM injections, X X Gr 2 injection pain (left neck), malaise; Gr PD Cetuximab stopped due to POD 1 fatigue, myalgia 005 70M HNSCC Carboplatin plus Cycle 1: 6 IT and 6 IM injections, X X Gr 1 injection site pain and drainage, PD radiation stopped due to POD fatigue, myalgia, 006 88M Melanoma Ipilimumab with Cycle 1: 6 IT and 5 IM injections, X Gr 2 fatigue; Gr 1 injection site pain and PD immune related- stopper due to POD swelling, fever adverse events requiring steroids 007 66M HNSCC None Cycle 1: 1 IT injection, stopped due X Gr 3 pneumonitis (likely unrelated); Gr 2 PD to complication of aspiration fever and hypoxia; Gr 1 tachycardia, pneumonitis (likely not study related)

008 70M HNSCC PD-1 inhibitor Cycle 1: 6 IT and 3 IM injections X X Wk 9 Gr 1 injection site discomfort; Gr 1 PD (EOT) fatigue, flu like symptoms, fever

Table 2 A and B.

A Selected genes up-regulated in PBMC with polyIC-LC treatment in patient with clinical benefit Top genes, up regula(teSDd) ,i nFD PRB

Therapeutic Immune Modulation Against Solid Cancers with Intratumoral Poly-ICLC: A Pilot Trial

Chrisann Kyi, Vladimir Roudko, Rachel Sabado, et al.

Clin Cancer Res Published OnlineFirst June 27, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-1866

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2018/06/27/1078-0432.CCR-17-1866.DC1

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