Therapeutic Immune Modulation Against Solid Cancers with Intratumoral Poly-ICLC

Published OnlineFirst June 27, 2018; DOI: 10.1158/1078-0432.CCR-17-1866

Clinical Trial Brief Report Clinical Cancer Research Therapeutic Immune Modulation against Solid Cancers with Intratumoral Poly-ICLC: A Pilot Trial 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, and Nina Bhardwaj1

Abstract

Purpose: Polyinosinic-polycytidylic acid-poly-l-lysine survival 6 months), whereas the remainder had progressive carboxymethylcellulose (poly-ICLC), a synthetic double- disease. Poly-ICLC was well tolerated, with principal side stranded RNA complex, is a ligand for toll-like receptor-3 effects of fatigue and inflammation at injection site and MDA-5 that can activate immune cells, such as (chemokine activity, T-cell activation, and antigen each cycle consisting of 1 mg poly-ICLC 3 weekly intratu- presentation. morally (IT) for 2 weeks followed by intramuscular (IM) Conclusions: Poly-ICLC was well tolerated in patients boosters biweekly for 7 weeks, with a 1-week rest period. with solid cancer and generated local and systemic immune Immune response was evaluated by immunohistochemistry responses, as evident in the patient achieving clinical ben- (IHC) and RNA sequencing (RNA-seq) in tumor and blood. efit. These results warrant further investigation and are Results: Two patients completed 2 cycles of IT treatments, currently being explored in a multicenter phase II clinical and 1 achieved clinical benefit (stable disease, progression-free trial (NCT02423863). Clin Cancer Res; 1–12. 2018 AACR.

Introduction T-cell exhaustion, reducing immune suppression in the tumor microenvironment (TME), and transforming a noninflamed The last decade has ushered in an exciting new age of TMEtoa"responsive"TME(e.g.,immunecellinfiltration, immunotherapy with the FDA approvals of the first cancer upregulation of PD-L1, etc.). Oncolytic viruses in the treatment vaccine, Provenge or sipuleucel-T, and checkpoint blockade, of cancer work presumably through their effects not only upon e.g., ipilimumab (Yervoy; anti–CTLA-4), pembrolizumab tumor cells but by activating innate immunity and inducing [Keytruda; anti-programmed death (PD1)], and nivolumab tumor-specific immunity (1–4). An intratumoral (IT) approach (Opdivo; anti-PD1). However, even with advances in immune that mimics viral infection, without associated significant side checkpoint blockade and other systemic chemotherapies, there effects or the complications of inducing dominant antiviral remains a significant fraction of patients who either fail to immunity, is one proposed strategy (5). respond or become resistant to treatment. Proposed interven- Polyinosinic-polycytidylic acid-poly-l-lysine carboxymeth- tions to broaden the fraction of patients benefiting from ylcellulose (poly-ICLC, Hiltonol, Oncovir, Inc.) is a synthetic immunotherapies and increase response rates rely on reversing 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 recep- 1 Tisch Cancer Center, Icahn School of Medicine at Mount Sinai, New York, New tors to activate dendritic cells (DC) and subsequently to also York. 2Columbia University Medical Center, New York, New York. 3Oncovir, Inc., trigger natural killer (NK) cells to kill tumor cells. Although Washington, DC. initially developed as an IFN inducer, poly-ICLC has been Note: Supplementary data for this article are available at Clinical Cancer found to have much broader biological effects, including Research Online (http://clincancerres.aacrjournals.org/). specific antitumor and antiviral actions (6). It activates mul- Current address for R. Sabado: Genentech, Inc., South San Francisco, California. tiple elements of innate and adaptive immunity, including Corresponding Author: Nina Bhardwaj, Tisch Cancer Institute, Icahn School of induction of a "natural mix" of IFNs, other cytokines and Medicine at Mount Sinai, Hess Center for Science and Medicine, 1470 Madison chemokines, NK cells, T cells, myeloid DCs, the P68 protein Avenue, Room 116, New York, NY 10029. Phone: 212-824-8427; Fax: 646-537- kinase (PKR), and other dsRNA-dependent host defense sys- 9571; E-mail: [email protected] tems (7, 8). Thus, when properly combined with antigen, doi: 10.1158/1078-0432.CCR-17-1866 poly-ICLC has the potential to generate a "live virus vaccine 2018 American Association for Cancer Research. equivalent" with a comprehensive immune response that

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Translational Relevance Patients and Methods Patients We present a novel vaccine approach using intratumoral Between 2013 and 2015, 8 patients [7 head and neck squamous polyinosinic-polycytidylic acid-poly-l-lysine carboxymethyl- cell cancers (HNSCC) and 1 melanoma] were enrolled in pilot cellulose (poly-ICLC), a viral mimic of the double-stranded phase of clinical trial. This trial is now accruing as a phase II RNA in viral replication, as a strategy of "autovaccination," that multicenter clinical trial (NCT02423863). Eligible patients had is, the use of the tumor itself as the antigen source in situ. In this unresectable recurrent or metastatic disease that had failed prior first evaluation of poly-ICLC in melanoma and head and neck systemic therapy and was radiologically or visually measurable cancer patients, we investigate the safety and tolerability of disease at least 10 mm in longest dimension. At least one poly-ICLC administered intratumorally to induce tumor accessible primary or metastatic tumor site was necessary for IT immune infiltration and intramuscularly to induce systemic injection with poly-ICLC with or without ultrasound guidance. inflammation. Treatment was well-tolerated, with minimal All patients had Eastern Cooperative Oncology Group perfor- toxicities noted. In the 1 patient with clinical benefit (stable mance status of 2 and acceptable hematologic, renal, and liver disease), there was evidence of upregulation of genes associ- function per laboratory parameters. Exclusion criteria included ated with chemokine activity, T-cell activation, and antigen bulky intracranial metastatic disease, history of active immuno- presentation (RNA sequencing), and increased CD4, CD8, therapy in the previous month, AIDS defined as CD4 count <200, PD1, and PD-L1 levels (quantitative immunohistochemistry) and life expectancy of less than 6 months in the judgment of the compared with patients with progressive disease. Although study physician. Written consent was obtained from patients, only 1 patient demonstrated clinical benefit, these findings and the study was conducted in accordance with the provisions prompt further investigation into optimal dosing and delivery of the Declaration of Helsinki. The study protocol and all of intratumoral poly-ICLC, and combinations with immune amendments were approved by the Institutional Review Board checkpoint blockade and/or other immunomodulators. at Mount Sinai Hospital.

Treatment plan includes activation of myeloid DCs, other antigen-presenting Patients were to receive two cycles of poly-ICLC treatment, cells, and NK cells, and generation of a polyfunctional Th1- each cycle including a priming and boosting treatment course polarized and cytotoxic T lymphocyte (CTL) response with (Fig. 1), with dosing and frequency based on prior preclinical increase in CD8 to CD4/regulatory T-cell ratio, which via the and phase I trials (18, 19). For cycle 1, patients were treated induction of specific chemokines can home to tumor or with 1 mg of poly-ICLC thrice weekly during weeks 1 and 2 pathogen (9–14). (priming treatment course, a total of 6 IT injections) into the Although most cancer vaccines are generally designed to utilize same lesion. During weeks 3to 9, patients were treated with IM known or presumptive tumor antigens, an alternative strategy is maintenance boosters biweekly(1mgperdose,boosting "autovaccination," i.e., the use of the tumor itself as the antigen course, total 14 IM injections), followed by a rest week (week source, in situ. Poly-ICLC can be given intramuscularly (IM) to 10) without treatment. This 10-week cycle was repeated in cycle induce systemic inflammation and/or IT to induce immune 2. Weeks 20 to 26 were a "no treatment rest period" to allow for infiltration of tumors. We observed a dramatic response in the evaluation of response in the absence of inflammation at week first sarcoma case being treated with repeated IT and IM poly- 26. At week 26, patients were assessed and response deter- ICLC, an 18-year-old patient with a malignant embryonal rhab- mined, and those patients with complete response (CR), partial domyosarcoma, who failed eight different regimens of chemo- response (PR), or stable disease (SD) were offered option of therapy as well as radiotherapy and proton-beam therapy, and maintenance therapy from weeks 27 to 36 with administration was in hospice. Treatment with poly-ICLC resulted in necrosis and of 1 mg IM poly-ICLC twice weekly. regression of facial, oral, retro-orbital, and a large intracerebral tumor. Although the patient eventually succumbed, his life was Disease assessment extended well beyond expectations (15). A phase II trial of single- Tumor response was evaluated using immune-related response dose treatment of ultrasound-guided IT poly-ICLC followed by IM criteria in solid tumors (20): CR, PR, SD, and progressive disease poly-ICLC was found to be safe in patients with advanced primary (PD). All the patients with measurable disease at the time of or metastatic liver cancers, with evidence of regression of non- enrollment on the study were eligible for response assessment. injected metastatic lesions as well as the targeted lesions (16, 17). Based upon these early indications of clinical response, we Correlative biology studies conducted a pilot study using this autovaccination strategy with Serial blood samples collected at baseline and at selected time IT and IM poly-ICLC at our institution in advanced treatment points during and after treatment (Fig. 1) were processed to collect refractory head and neck cancers and melanoma. We hypoth- plasma/serum and peripheral blood mononuclear cells (PBMC), esize that the therapeutic in situ autovaccination strategy using and used to evaluate humoral and cellular immunity induced by IT and IM poly-ICLC administration of TLR3 ligand poly-ICLC IT and IM poly-ICLC injections. Tumor biopsies were performed can reverse DC inhibition in the treated tumor microenviron- at baseline, week 3, and week 26 (if possible). Pre- and postvac- ment, increase the efficiency of antigen presentation to CTLs, cination tumor biopsies of both IT-treated tumors, and prevent tolerization to tumor antigens, and elicit systemic nontreated distant tumors taken when possible, were analyzed antitumor immunity. Here, we present the first-ever published by quantitative multiplex immunohistochemistry (IHC) for results of our phase I trial of IT poly-ICLC in treatment of lymphocyte infiltration [e.g., CD4 and CD8 T-cell subsets, þ patients with solid cancer. T regulatory cells (FoxP3 ), PD-1 or CTLA-4 expressing T cells,

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CYCLE 1

WEEK 1, Intratumor WEEK 2, Intratumor WEEK 3−9, IM 2x/wk WEEK 10, rest

Biopsy Biopsy CYCLE 2

WEEK 11, Intratumor WEEK 12, Intratumor WEEK 13−19, IM 2x/wk WEEK 20−26, rest

Biopsy

MAINTENANCE (12 weeks)

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

Blood draws for immune response evaluaon

Figure 1. Protocol schema. Patients received two cycles of poly-ICLC treatment, each cycle including a priming and boosting treatment course (Fig. 1). In cycle1, patients were treated with 1 mg of poly-ICLC 3 times weekly during weeks 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 was determined, and patients with CR, PR, or SD were offered the option of maintenance therapy (from weeks 27 to 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 postvaccination tumors were evaluated by quantitative multiplex IHC and RNA-seq. Blood samples were collected at baseline and throughout treatment cycles (as indicated) for immune response evaluations.

DC subsets], as well as other immune markers (CD86 antigen- (23). Briefly,alignedreadsareassignedtogenesusingthe presenting cells, CD68 macrophages/monocytes, CD16 NK cells, FeatureCounts function of Rsubread. Gene expression in terms and HLA encoding the MHC proteins) using standard IHC tech- of log2-CPM (counts per million reads) was computed and niques (21). Individual antigens were quantified using the CRI normalized across samples. Genes with low expression (ones Inform software (Perkin Elmer) which applies user-directed anti- nothavingatleast10readspermillionreadsinatleasttwo gen thresholds to generate percentages normalized to the total samples) were filtered out. Differential expression analysis was tumor area. performed using the limma software package (R, Bioconduc- tor). Gene set enrichment analysis (GSEA) was computed RNA sequencing analysis following published recommendations (24, 25). Analysis of RNA sequencing (RNA-seq) analysis and T-cell receptor blood transcriptome modules (BTM) was done accordingly (TCR) sequencing (Adaptive, Inc.) were done on PBMC (26) Comparisons between time points as well as before and and tumor tissue. PBMCs were isolated from patients at after treatment were conducted addressing both gene- and multiple time points prior to and after treatment with poly- pathway-specific changes (24). ICLC. Paired-end sequencing of cDNA created from isolated For TCR analysis, genomic DNA was purified from total PBMCs mRNA was performed using the Illumina HiSeq 2500 platform and tumor samples using the Qiagen DNeasy Blood extraction at a depth of 30 M–35 M reads. The read quality was assessed Kit. The TCRb CDR3 regions were amplified and sequenced using FastQC (22). Corresponding sequence files were pro- using immunoSEQ (Adaptive Biotechnologies), as previously cessed using publicly available RNA-seq data analysis pipelines described (27).

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Survival analysis new brain metastasis; patient treatment was switched to chemo- Kaplan–Meyer survival curve analyses were performed using therapy with radiotherapy with control of disease. Patient was still publicly available on-line server Tumor Immune Estimation alive on subsequent line of chemotherapy 18 months out from Resource (TIMER, https://cistrome.shinyapps.io/timer/). start of poly-ICLC treatment. The other 7 patients had PD while on poly-ICLC treatment (Table 1). Patient 001 was the only other patient (HNSCC) in our Results pilot trial to complete 2 cycles of IT poly-ICLC treatments (12 IT The primary objective of this phase I pilot study was to evaluate injections) though only had 20 of 28 IM boosting treatments. the safety of IT plus IM poly-ICLC for treatment of patients with Patient was initially thought to have clinical improvement on advanced accessible solid tumors. Secondary objectives were to exam. CT scans at 10 weeks suggested pseudoprogression, and as investigate the changes in humoral and adaptive cellular immu- the patient was clinically stable, he remained on study for cycle 2. nity induced by autovaccination with poly-ICLC. Unfortunately, repeat imaging at week 15 showed clear progression of disease with innumerable pulmonary nodules, and patient Patient characteristics and disease response was taken off study in the middle of second IM boosting cycle Eight patients with treatment refractory solid tumors (1 mel- (after 12 IT and 20 IM poly-ICLC injections total) and switched to anoma and 7 head and neck cancer) were enrolled in this study salvage chemotherapy. between January 2014 and July 2014. Two subjects completed 2 Patients 003, 004, 005, and 008 were patients with metastatic cycles of IT treatment, though one of these patients did not HNSCC (range, 70–80 years old) who also stopped treatment complete a second IM boosting cycle; the remaining 6 subjects shortly after initiation of poly-ICLC treatment due to rapidly PD completed 1 cycle or less of IT poly-ICLC due to progression of (Table 1). Patient 003 received only 3 IT injections, whereas disease (Table 1). patients 004, 005, and 008 received the first course of 6 IT One of 2 patients who completed 2 cycles of IT poly-ICLC injections and subsequently 3 to 6 IM injections before progres- achieved clinical benefit. Patient 002 was a 54-year-old man sing. As an example, patient 008 was a 70-year-old male patient with metastatic Epstein–Barr virus (EBV)-positive nasopharynx with metastatic HNSCC whose treatment was halted particularly HNSCC, previously treated and refractory to chemotherapy. He early, in cycle 1 at week 3 to 4 due to PD (6 IT and 3 IM injections in tolerated treatment to completion of 2 cycles (12 IT and 28 IM total). CT scans at 10 weeks showed PD with new lung nodules injections in total) over a 30-week period and was the one patient and enlarging liver and right abdominal wall lymph nodes. Tissue who achieved clinical benefit with SD (progression-free survival biopsies were obtained at week 3 and end of treatment (week 9) of 6 months; Fig. 2). At 6 months, he developed diplopia due to per protocol.

Table 1. Patient demographics, treatment, and toxicities of phase I/pilot trial Biopsy obtained Patient Age/ Recent prior therapy Best number sex Diagnosis (<6 months) Treatment course Wk 1 Wk 3 Wk 26 Toxicities response 001 68F HNSCC Cetuximab plus Cycle 1: 6 IT and 14 IM injections X X Gr 1 injection site pain and PD radiotherapy Cycle 2: 6 IT and 6 IM injections inflammation (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 SD Cycle 2: 6 IT and 14 IM injections and necrosis; Gr 1 injection site pain, flu-like symptoms, fever, fatigue, myalgia, arthralgia 003 80M HNSCC Radiotherapy Cycle 1: 3 IT injections, stopped X Gr 1 fever, fatigue PD due to rapidly POD and infection (bibasilar pneumonia) 004 70M HNSCC Docetaxel!cetuximab Cycle 1: 6 IT and 4 IM injections, X X Gr 2 injection pain (left neck), PD stopped due to POD malaise; Gr 1 fatigue, myalgia 005 70M HNSCC Carboplatin plus Cycle 1: 6 IT and 6 IM injections, X X Gr 1 injection site pain and PD radiation stopped due to POD drainage, fatigue, myalgia 006 88M Melanoma Ipilimumab with Cycle 1: 6 IT and 5 IM injections, X Gr 2 fatigue, Gr 1 injection site PD immune-related stopped due to POD pain and swelling, fever adverse events requiring steroids 007 66M HNSCC None Cycle 1: 1 IT injection, stopped due X Gr 3 pneumonitis (likely PD to complication of aspiration unrelated); Gr 2 fever and pneumonitis (likely not study hypoxia, Gr 1 tachycardia related) 008 70M HNSCC PD-1 inhibitor Cycle 1: 6 IT and 3 IM injections X X Wk 9 (EOT) Gr 1 injection site discomfort; PD Gr 1 fatigue, flu-like symptoms, fever Abbreviations: EOT, end of treatment; Gr, grade; I, IHC; N, inadequate tissue; R, RNA sequencing.

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Figure 2. Clinical data. A, Postinjection site demonstrating necrosis after IT poly-ICLC treatment. B, Patient 002 was an HNSCC patient who showed clinical beneﬁt (SD), with CT scans over the treatment course demonstrating SD (PFS over 6 months).

Patient 006 was a patient with BRAF-wild-type subungal patients 003 and 007 due to complication of bibasilar pneumonia melanoma of the right fourth digit, who developed multiple requiring hospitalization and grade 3 aspiration pneumonitis, recurrences through chemotherapy, radiotherapy, targeted ther- respectively. Week-26 biopsy was not obtained for patients 001, apy, and checkpoint blockade. The patient completed 6 IT and 004, 005, and 006 due to PD requiring study termination and 5 IM injections, but treatment was stopped prior to cycle 2 due to change in treatment plan. Except for patients 002, 004, and 008, rapidly PD. At 9-week scans, he had PD of pulmonary nodules and although the week-3 biopsy was sufficient for pathology diagnosis lower extremity lesions. The patient was initiated on anti-PD1 and IHC analysis, tissue was insufficient for RNA-seq due to blockade (2014), but subsequently progressed through treatment inadequate sample. A summary of treatment courses and biopsies and died. (including if tissue adequate for IHC and RNA-seq analysis) for Patient 007 was a patient with advanced HPV-negative, EBV- each subject is outlined in Table 1. positive, progressive tumor after induction chemotherapy and radiotherapy. He was consented and enrolled in the poly-ICLC IHC data trial, but after one IT poly-ICLC injection, had aspiration pneu- Tumor biopsies were obtained of injected site for IT Poly-ICLC monia/pneumonitis, which was temporally related to aspiration at baseline and at week 3 (after 6 IT injections) and week 26 if and not considered directly related to treatment. Subsequently, possible. Quantitative IHC from tumor biopsies of patients with patient was taken off study. PD (001, 004, 005, and 008) showed unchanged or decreased levels of CD4, CD8, PD-1, and PD-L1 over the course of the Toxicities treatment period (Fig. 3; Supplementary Table S1). In contrast, Poly-ICLC was generally well tolerated with the principal side IHC analysis of tumor biopsies of patient 002, the one patient effects of treatment, fatigue, and inflammation at primary injec- who had clinical benefit (SD), showed increased levels of CD4 tion sites, less than grade 2. One case of overt necrosis of tumor (60-fold), CD8 (10-fold), PD-1 (20-fold), and PD-L1 (3-fold). In (grade 2) was observed. There was a case of grade 3 pneumonitis addition, tumor biopsies obtained in patient 002 showed marked þ in 1 patient (007), but this was temporally related to aspiration increases in immune cells (CD86 antigen-presenting cells, þ þ pneumonia and not considered directly related to poly-ICLC CD68 macrophages/monocytes, CD16 NK cells, HLA encoding treatment. Full range of toxicities is listed in detail in Table 1. the MHC expression) after treatment (Fig. 3).

Tumor biopsies and samples RNA-seq analysis Baseline, week-3 (post 6 IT injections), and week-26 (post IT RNA-seq was used to characterize immune response in the and IM injection) biopsies were obtained if possible. Unfortu- blood compartment (PBMC) versus the tumor site. Analysis nately, week-3 (and subsequent) biopsy was not obtained in of over 18,000 transcripts revealed that poly-ICLC treatment

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Patient 008 (PD) Patient 002 (SD)

CD4: Yellow CD4: Yellow CD86, green CD8: Red CD8: Red CD16, yellow PD-L1: Green PD-L1: Green HLA ABC, PD-1: Magenta PD-1: Magenta red CD68, magenta Baseline

CD4: Yellow CD4: Yellow CD86, green CD8: Red CD8: Red CD16, yellow PD-L1: Green PD-L1: Green HLA ABC, red PD-1: Magenta PD-1: Magenta CD68, magenta After 6 IT Injections 6 IT After

Figure 3. IHC of tumor biopsy samples taken at baseline (top row) and after 6 IT injections (bottom row). In patient 008 (PD, left column), quantitative IHC showed unchanged or decreased levels of CD4, CD8, PD-1, and PD-L1 over the treatment period. In patient 002 with clinical beneﬁt (SD, right two columns), IHC analysis of tumor showed increased CD4 (60x), CD8 (10x), PD1 (20x), and PD-L1 (3x; also see Supplementary Table S1), as well as marked increases in immune cells (CD86 antigen-presenting cells, CD68 macrophages/monocytes, CD16 NK cells, HLA encoding the MHC proteins) after treatment.

resulted in changes in gene expression in IFN-related genes both at beta, CXCL8, antigen processing-related marker CD83, and T-cell the tumor and PBMC level indicative of local and systemic activation–related protein IFN gamma (see Table 2A). Fewer immune activation. genes were specifically upregulated in patients 004 and 008 Nonsupervised hierarchical clustering of patients' PBMC (66 genes, lower-right quadrant, Fig. 4B), with some related to revealed three major groups of clustering (Fig. 4A). First, similar an IFN response: ISGs OAS and DDX60 (see Table 2B). blood RNA expression profiles were observed between patient To better understand these trends in PBMC of the patient 002, 002 pretreatment and off-treatment samples, and screening sam- we performed GSEA conditioned upon two independent sources ples of patients 004 and 008 who developed PD along with one of relevant published gene sets: BTM (23) and the Broad Institute sample during IM poly-ICLC injection. We assigned these samples immune gene collection (ref. 21; C7). GSEA using BTM demon- as baseline cluster, "minimal or absence of immune activation." strated upregulated presence of activated DCs in the blood of Second, similar expression clustering was observed in patient 002 patient 002 with IT poly-ICLC treatment, as well as upregulation who had SD, indicative of systemic inflammation resulting in a of stimulatory cytokine expression. Interestingly, enrichment of clinical benefit response (inflamed cluster). A clustering of "inter- these gene sets was specific for the patient 002, whereas NK/T-cell mediate" signaling or activation of immune system was observed gene expression was similar between patients with SD and PD in blood samples derived from patients 004 and 008 undergoing responses (Fig. 5). Notably, immune gene expression changed IT poly-ICLC injections and patient 002 undergoing IM poly-ICLC minimally in PBMC upon IM poly-ICLC treatment in both SD and injections (intermediate cluster). PD patients, but strongly upon IT poly-ICLC treatments, suggest- In order to gain insight on expression alterations with poly- ing the importance of IT poly-ICLC injection to achieve systemic ICLC treatment, we calculated differential gene expression con- immune cell activation. Analysis of GSEA of immune gene sets sidering samples within clusters as biological parallels. Resulting from the Broad Institute yielded similar trends (Supplementary scatter plot of expression fold changes was inferred from two Fig. S1). Taken together, our observations suggest systemic acti- comparisons: patient 004 and 008 samples from intermediate vation of the immune system including T-cell activation, elevated cluster versus baseline (PD, Fig. 4B) and patient 004 samples from antigen-presentation cells, and inflammatory cytokine expression inflamed cluster versus baseline (SD, Fig. 4B) revealed several in patient 002 blood upon IT injections of poly-ICLC as defined trends. The majority of gene expression changes upon poly-ICLC by BTM and Broad gene sets. IT injections were similar between patients 004, 008, and 002. To gain insight into IT immune cell infiltration profiles, we However, specific gene sets were upregulated in patient 002, but performed similar gene set enrichment analyses on tumor RNA- repressed or unchanged in patients 004 and 008 (624 genes, seq samples. Both immune gene set collections detected gene þ upper-left quadrant, Fig. 4B). These included genes regulating signatures of activated DCs, B cells, CD8 T cells, Th17-polarized þ antitumor activity: stimulatory cytokines and chemokines: IL1 CD4 T cells, NK cells, monocytes, and neutrophils. Expression of

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Figure 4. A, Hierarchical clustering of PBMC RNA expression from patients with PD and SD. I, Baseline cluster, including samples, taken at "screen," "rest," or during IM injection time points, samples are 1, 4, 9, 11, 12, 14, and 17. II, Intermediate cluster, including samples from PD patients, taken during IT injections: 13 and 15; and samples from SD patient, taken during IM injections: 3 and 8. III,Inﬂamed cluster, including samples from SD patient, taken after IT 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 week 1. 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 (FDR < 0.05) are plotted. gene signatures related to IFNa, IFNb, IFNg, and IL17 stimulation upregulation of XCL1/2 chemokines by NK or T cells in the was similarly elevated at the tumor sites of patients with SD and patient 002 (Fig. 6A). This observation is in agreement with PD responses: at approximately similar levels, before and after specific upregulation of IL15 and IL23A at the end of the poly-ICLC treatment (Supplementary Figs. S2–S4). The latter treatment cycle by the same patient, as well as elevated expres- suggests the immune system is activated at the tumor site, but sion of Clec9A. We speculate that XCL1/2 expression may fails to execute its function due to perhaps different levels and/or attract the migratory DC subset XCR1-DC, known for its ability distribution of immune cells in tumor and/or exhaustion states as to produce IL15, type I IFN, as well as cross-present tumor- observed in the IHC data. derived antigens through the Clec9A receptor. Interestingly, the Combined together, PBMC and tumor RNA-seq gene expres- chemokines CXCL1 and CXCL5 were both elevated at the sion analyses revealed differences in immune profiles between tumor site of patients 004 and 008 (Fig. 6B) prior to and after patient 002 and patients 004 and 008. Notably, we detected poly-ICLC treatment. Both cytokines are ligands of the CXCR2 differential cytokine expression and immune cell activation receptor, activation of which in tumors has been shown to relatedtoDC–NK cell cross-talk (Fig. 6; Supplementary interfere with PD-1/PD-L1 immunotherapies in preclinical Table S2). DC infiltration in blood correlated with specific studies (28, 29). The trend of increased CXCL1 and CXCL5

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Table 2A. Selected genes (FDR < 0.05), upregulated in PBMC with poly-ICLC treatment in patient 002 with clinical beneﬁtSD

Gene name Group Fold change (log2) Average expression (log2) CXCL8 Chemokine 6.0 1.9 CD83 Antigen presentation 4.3 0.6 GPR183 GPCR receptor 3.0 0.2 ICAM1 Adhesion molecule 2.6 0.2 IL1B Cytokine 2.3 0.1 FOXO3 Transcription factor 1.9 0.3 IFNG Cytokine 1.9 2.6 XCL2 Chemokine 1.7 2.6 FCAR IgA receptor 1.7 0.1 MIR22HG miRNA, inﬂammation 1.5 0.7 TIGIT Immune receptor 1.0 0.2 NOD2 Immune receptor 1.0 0.2

Table 2B. Selected genes (FDR < 0.05), upregulated in PBMC with poly-ICLC treatment in patients 004 and 008 with PD

Gene name Group Fold change (log2) Average expression (log2) CCR2 Chemokine receptor 3.0 2.4 TLR8 Toll-like receptor 2.1 1.9 GIMAP6 Immune-associated gene 1.5 1.8 SAMD9L Endosome fusion 1.9 1.7 MT-CO1 Cytochrome oxidase 1.9 3.0 GIMAP5 Immune-associated gene 1.8 0.8 CX3CR1 Chemokine receptor 1.5 1.8 DDX60 IFN-inducible gene 1.3 0.9 IFI44 IFN-inducible gene 1.3 0.5 BTK Tyrosine kinase 1.3 0.8 OAS2 IFN-inducible gene 1.2 1.3 IL16 Cytokine 1.1 0.8

expression may identify a protumorigenic inflammatory pro- to inadequate tissue sampling size or no available tissue after file, possibly interfering with the poly-ICLC regimen, whereas other tests performed (IHC and RNA-seq). expression of IL15, IL23A, Clec9A, XCR1, XCL1, and XCL2 may fl re ect a better response to immunotherapy. Discussion Our data are limited to the analysis of just a few patients; therefore, we sought verification of these signatures in larger Here, we present the results of a pilot trial testing a strategy of data sets from The Cancer Genome Atlas (TCGA) as a potential therapeutic in situ autovaccination with IT injections of the dsRNA approach to define prognostic biomarkers of response to poly- viral mimic and TLR agonist, poly-ICLC. Poly-ICLC was well ICLC. Survival analyses of available cancer patient data derived tolerated and generated local immune response in tumor micro- from the TCGA database support our hypothesis (Fig. 6). The environment and systemic immune response as evident in the high expression of gene markers associated with clinical patient achieving clinical benefit. benefit of poly-ICLC correlates with better prognosis in A major limitation of the trial was the study population of HNSCC (HNSC) and melanoma (SKMC) patients (Fig. 6C; patients with refractory disease who had failed several other lines e.g., DC-associated factors IL15 and XCR1). In contrast, ele- of treatment (often with recent chemotherapy; Table 1) and thus vated expression of protumorigenic receptor CXCR2 and its already highly immunosuppressed. Certain chemotherapies can ligand CXCL1 correlates with poor prognosis (Fig. 6D). These suppress T cells proliferating in response to antigen or poly-ICLC, observations, though clinical trends, suggest that the presence and this effect is seen for as long as 6 months after cessation of of antigen-presenting cells, IL15 and IL23A, at tumor sites has chemotherapy (30). In this pilot trial, there were three rapid abeneficial effect on patient survival and perhaps on response progressors who did not complete even 1 cycle and may not have to immunotherapy, whereas expression of CXCR2/CXCL1/ received enough treatment with single-agent poly-ICLC to gen- CXCL5 axis may interfere with the treatment. erate an antitumor immune response. Therefore, single-agent poly-ICLC may not be adequate treatment for patients with Seromics and humoral response advanced, rapidly progressing HNSCC; however, information No significant findings were noted in analysis of pre- and post- learned from our study can inform combination treatments in IT poly-ICLC treatment in patients. the future. In the patient achieving SD, our pilot results suggest how TCR analysis sequential IT injections of poly-ICLC can potentially induce an TCR sequencing revealed that the patient 002 with clinical effective personalized systemic therapeutic "autovaccination" benefit (SD) showed increases in TCR clonality (20% increase) against tumor antigens in patients. We postulate that a therapeutic and density (30% increase) after poly-ICLC treatment. Unfor- in situ autovaccination strategy using poly-ICLC works via three tunately, similar analyses of other patients were not possible due immunomodulatory steps. First, IT Poly-ICLC potentially

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Figure 5. BTM enrichments of selected gene sets in PBMC of patient 002 with SD and patients 004 and 008 (PD) collected at different time points during poly-ICLC treatment cycle. A, Unsupervised hierarchical clustering of selected BTM gene sets (FDR < 0.25). Patient samples are labeled according to cluster deﬁnitions (see Fig. 4). Absolute (B) and relative (C) quantiﬁcation of gene set enrichments in SD and PD patients.

activates NK cells, triggers TLR3 and MDA-5 receptors present at genes both at the tumor and PBMC level indicative of local and many APCs, and induces other proapoptotic mechanisms, result- systemic immune activation. Upregulation of genes in response to ing in local tumor killing and release of tumor antigens (31–33). poly-ICLC treatment included chemokines, interferon-stimulated In prior studies, it has been shown that poly-ICLC can upregulate genes, genes associated with antigen processing, T-cell activation, several hundreds of genes closely representing some 10 canonical and apoptosis. innate immune pathways (6, 34, 35). Our RNA-seq analysis In the second step, poly-ICLC danger signals activate macro- detected expression of over 18,000 transcripts and revealed phages and DCs at the tumor site, in which they acquire tumor poly-ICLC treatment changed gene expression of IFN-related antigens that are cross-presented to CD4 helper cells and to CD8 T

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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 HNSC and SKCM patients from TCGA. Analysis is done by web-server TIMER, ranking patients by selected gene expression and using the top 30% and bottom 30% of patients for survival estimation. A, DC surface markers are highly upregulated in blood of SD patient (right). DC presence correlates with XCL1/2 high expression in the blood of the same patient (left). B, Upregulation T/NK-cell costimulatory cytokines IL15 and IL23 at tumor site of patient 002 upon completion of poly-ICLC treatment (right). Patients 004 and 008 have upregulated protumorigenic cytokines CXCL1 and CXCL5 at the beginning and the end of treatment (left). Each column represents the patient at a specific treatment time point, as numbered in Fig. 4. C, Increased expression of IL15, IL23A, Clec9A, XCR1, XCL1, and XCL2 correlates with better prognosis in HNSC and SKCM (P value < 0.05; P ¼ 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, as is CXCL1 in HNSC (P value < 0.1).

cells to generate antigen-specific CTLs. The repeated administra- anti–CTLA-4, anti–PD-L1, FLT3L, and other costimulatory factors tion of the poly-ICLC danger signal IT in the context of the for enhanced antitumor activity. patient's own tumor antigens may be critical for optimal priming One interesting observation was the increased expression of of the system at this step. GSEA of PBMC and tumor RNA-seq also protumorigenic chemokines CXCL1/5 at the tumor site of patient suggests systemic activation of immune system, increased infil- 004 (PD). CXCL1, expressed by tumor-associated macrophages, tration of antigen-presentation cells (activated DCs) in the blood, neutrophils, and epithelial cells, has been suggested to have and upregulation of inflammatory cytokine expression upon IT tumorigenic and mitogenic properties in cancer cells and shown injections of poly-ICLC. In addition, RNA-seq analysis detected to be a major component required for serum-dependent mela- gene signatures of activated DCs, B cells, CD8 T cells, Th17- noma cell growth. Importantly, CXCL1 and CXCL5 elicit its effects polarized CD4 T cells, NK cells, monocytes, and neutrophils. by signaling through the chemokine receptor CXCR2 found on Importantly, T-cell costimulatory cytokines IL15 and IL23A were MDSCs. Recent preclinical studies have shown that activated highly upregulated at the tumor site of SD patient upon comple- CXCR2 signaling modulates the tumor immune microenviron- þ tion of poly-ICLC treatment cycle, indicating the prolonged ment, reducing CD8 T-cell trafficking and activation, and damp- maintenance of immune activation. ening NK-cell activity and promoting regular T-cell expansion The third step is attraction and maintenance of antigen-specific (24, 25). Therefore, in the patient with PD, elevated expression of CTLs via various poly-ICLC–induced chemokines, costimulatory CXCL1/5 at the tumor site could indicate activated CXCR2 factors, and other mechanisms (14). This was seen systemically in signaling, interfering with poly-ICLC treatment with ensuring patient 002 with clinical benefit (SD) whose PBMC showed tumor outgrowth. AZD5069 is an investigational selective CXCR2 þ transcriptional upregulation of genes related to cytokines/ antagonist that inhibits the migration of CXCR2 MDSCs to chemokines, T-cell activation, and antigen processing in response tumor microenvironment and may enhance immune-mediated to poly-ICLC treatment. IHC analysis of tumor biopsies of patient tumor attack. It is currently being evaluated for safety and efficacy 002 showed increased levels of CD4 (60-fold), CD8 (10-fold), in human clinical trials. Ongoing clinical trials attempt to explore PD-1 (20-fold), and PD-L1 (3-fold), consistent with T-cell acti- the potentially synergistic combination of anti–PD-L1 inhibitors vation, migration, and consequent upregulation of PD-L1. Other (durvalumab) in combination with anti-CXCR2, AZD5069 studies have demonstrated that poly-ICLC alone is sufficient to (AstraZeneca, anti-CXCR2) in HNSCC (NCT02499328), and induce expression of the costimulatory molecules B7-H2, CD40, advanced pancreatic ductal cancers (NCT02583477), respective- and OX40 (36, 37). Our findings as well as others suggest ly. Our results also suggest that a combination of IT poly-ICLC rationale for potential combination of poly-ICLC with OX40, with a CXCR2 inhibitor could be an effective strategy and warrants

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further investigation (28, 29). Our ongoing adaptive phase II trial other oncolytic viruses. These preliminary findings warrant fur- attempts to explore how such inflammatory responses can be ther investigation, and a larger multicenter phase II clinical trial harnessed to enhance therapeutic efficacy in such effective (clinicaltrials.gov, NCT02423863) is now underway to confirm combinations. these findings in advanced solid cancers. Although limited by a study population of patients with refractory disease who had failed other lines of treatment and Disclosure of Potential Conflicts of Interest were already highly immunosuppressed with significant tumor B. Greenbaum reports receiving speakers bureau honoraria from Merck and burden, results of our study should still be informative in the Bristol-Myers Squibb. A. Salazar reports receiving commercial research grants design of future combination studies of poly-ICLC with other from and holds ownership interest (including patents) in Oncovir, Inc. P. Friedlander is a consultant/advisory board member for Seattle Genetics and systemic therapies including immune checkpoint blockade, or Array. N. Bhardwaj is on the scientific advisory board of Neon, CPS Companion inhibitors of potential protumorigenic signaling pathways, like Diagnostics, Genentech, and CureVac. No potential conflicts of interest were CXCR2, which are being tested in the clinic. In addition, other disclosed by the other authors. areas of ongoing exploration with poly-ICLC treatment are the optimal dose and schedule of treatment. Timed release or struc- Authors' Contributions tured formulations of poly-ICLC could be adjusted to better Conception and design: C. Kyi, Y. Saenger, K. Misiukiewicz, A. Salazar, mimic a viral infection, especially for deep tumors in which N. Bhardwaj repeated IT administration is logistically more challenging. At Development of methodology: C. Kyi, A. Salazar, N. Bhardwaj Acquisition of data (provided animals, acquired and managed patients, the same time, the possibility of overstimulation with a PAMP provided facilities, etc.): C. Kyi, R. Sabado, Y. Saenger, T.H. Thin, M. Donovan, must also be considered, and the optimal dose and schedule to P. Friedlander, N. Bhardwaj mediate optimal anticancer effects remains a needed area of our Analysis and interpretation of data (e.g., statistical analysis, biostatistics, investigation. Immune infiltrate at distal tumors should also be computational analysis): C. Kyi, V. Roudko, R. Sabado, W. Loging, J. Mandeli, explored to evaluate whether in situ vaccination has systemic M. Donovan, M. Posner, B. Greenbaum, A. Salazar, P. Friedlander, N. Bhardwaj therapeutic effect at other metastatic tumor sites. These trials are Writing, review, and/or revision of the manuscript: C. Kyi, V. Roudko, M. Donovan, M. Posner, K. Misiukiewicz, A. Salazar, P. Friedlander, N. Bhardwaj on-going and under exploration in our current ongoing adaptive Administrative, technical, or material support (i.e., reporting or organizing phase II trial that attempts to explore these combinations. data, constructing databases): R. Sabado, Y. Saenger, N. Bhardwaj In summary, we present a novel IT approach using poly-ICLC, a Study supervision: Y. Saenger, K. Misiukiewicz, N. Bhardwaj viral mimic of the double-stranded RNA encountered in viral Other (coordination of care of research subjects and administration of replication, demonstrating its capacity to induce immunogenic investigational product): D. Lehrer cell death and also an immune stimulation effects through activation of DCs, upregulation of cytokines and chemokines, Acknowledgments and T-cell priming. Indeed, other recent clinical studies with This research was supported by grants from the Cancer Research Institute, the engineered herpes simplex virus-1 expressing granulocyte-mac- Melanoma Research Alliance, and NIH. N. Bhardwaj is a member of the Parker Institute for Cancer Immunotherapy, which supported the Mount Sinai Hos- rophage colony-stimulating factor (talimogene laherparepvec) pital Cancer Immunotherapy Program. also demonstrated response in distal tumors with evidence of enhanced TILs (38). The encouraging results of these studies, IT poly-ICLC, as well as other pattern recognition receptor (PRR The costs of publication of this article were defrayed in part by the in situ payment of page charges. This article must therefore be hereby marked agonists such as TLR and STING agonists) suggest how advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate autovaccination can generate a localized antitumor immune this fact. response ultimately driving systemic antitumor immunity and provide a strong rationale for clinical exploration of combinations Received July 31, 2017; revised December 12, 2017; accepted June 21, 2018; with immune checkpoint antibodies with poly-ICLC as well as published first July 20, 2018.

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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

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