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Combined immunotherapy encompassing intratumoral poly-ICLC, dendritic-cell vaccination and radiotherapy in advanced cancer patients
Annals of Oncology 29: 1312–1319, 2018 doi:10.1093/annonc/mdy089 Published online 14 March 2018
ORIGINAL ARTICLE
Combined immunotherapy encompassing intratumoral poly-ICLC, dendritic-cell vaccination and radiotherapy in advanced cancer patients ~ate2,3,5, G. Pe´rez2,3,5, M. E. Rodrı´guez-Ruiz1,2,3,4†, J. L. Perez-Gracia1,3,4†, I. Rodrı´guez2,3,5, C. Alfaro2,3,5, C. On I. Gil-Bazo1,2,3,4, A. Benito6, S. Inoge´s4,5, A. Lo´pez-Diaz de Cerio4,5, M. Ponz-Sarvise1,2,4, L. Resano1, P. Berraondo2,3,4, B. Barbe´s4,7, S. Martin-Algarra1,4, A. Gu´rpide1,3,4, M. F. Sanmamed1,8, C. de Andrea3,4,9, A. M. Salazar10‡ & I. Melero1,2,3,4,5*‡ 1
Department of Oncology, Clı´nica Universidad de Navarra, Pamplona; 2Centro de Investigacio´n Me´dica Aplicada (CIMA), Universidad de Navarra, Pamplona; CIBERONC, Centro de Investigacio´n Biome´dica en Red de Ca´ncer, Madrid; 4Navarra Health Research Insititute (IDISNA), Pamplona; 5Departments of Immunology; 6 Radiology; 7Physics, Clı´nica Universidad de Navarra, Pamplona, Spain; 8Immunobiology Department, Yale University School of Medicine, New Haven, USA; 9 Department of Pathology, Clı´nica Universidad de Navarra, Pamplona, Spain; 10Oncovir, Washington, USA 3
*Correspondence to: Dr Ignacio Melero, Department of Immunology, University Clinic, Avenida Pio XII, 55, 31008 Pamplona, Navarra, Spain. Tel: þ34-948255400; E-mail: [email protected] †
Both authors equally share credit.
‡
Both authors will share credit for senior authorship.
Background: Combination immunotherapy has the potential to achieve additive or synergistic effects. Combined local injections of dsRNA analogues (mimicking viral RNA) and repeated vaccinations with tumor-lysate loaded dendritic cells shows efficacy against colon cancer mouse models. In the context of immunotherapy, radiotherapy can exert beneficial abscopal effects. Patients and methods: In this two-cohort pilot phase I study, 15 advanced cancer patients received two 4-week cycles of four intradermal daily doses of monocyte-derived dendritic cells preloaded with autologous tumor lysate and matured for 24 h with poly-ICLC (Hiltonol), TNF-a and IFN-a. On days þ8 and þ10 of each cycle, patients received intratumoral image-guided 0.25 mg injections of the dsRNA-analogue Hiltonol. Cyclophosphamide 600 mg/m2 was administered 1 week before. Six patients received stereotactic ablative radiotherapy (SABR) on selected tumor lesions, including those injected with Hiltonol. Expression of 25 immune-relevant genes was sequentially monitored by RT-PCR on circulating peripheral blood mononuclear cell (PBMCs) and serum concentrations of a cytokine panel were sequentially determined before and during treatment. Pre- and posttreatment PBMC from patients achieving durable stable disease (SD) were studied by IFNc ELISPOT-assays responding to tumor-lysate loaded DC and by TCRb sequencing. Results: Combined treatment was, safe and well tolerated. One heavily pretreated castration-resistant prostate cancer patient experienced a remarkable mixed abscopal response to SABRþ immunotherapy. No objective responses were observed, while nine patients presented SD (five of them in the six-patient radiotherapy cohort). Intratumoral Hiltonol increased IFN-b and IFN-a mRNA in circulating PBMC. DC vaccination increased serum IL-12 and IL-1b concentrations, especially in patients presenting SD. IFNc-ELISPOT reactivity to tumor lysates was observed in two patients experiencing durable SD. Conclusions: This radio-immunotherapy combination strategy, aimed at resembling viral infection in tumor tissue in combination with a dendritic-cell vaccine and SABR, is safe and shows immune-associated activity and signs of preliminary clinical efficacy. Key words: intratumoral injections, poly-ICLC (Hiltonol), dendritic cell vaccination, radiotherapy and abscopal effects
C The Author(s) 2018. Published by Oxford University Press on behalf of the European Society for Medical Oncology. V
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Annals of Oncology Introduction Dendritic cells (DCs) mediate key events in priming, shaping and sustaining tumor immunity [1, 2]. Consequently, many investigators have tested ex vivo differentiated DC to formulate vaccines against tumor antigens [3]. The best results to date have been reported with DC loaded with tumor neoantigens [4], DC transfected with clinical grade mRNAs to transfer antigens and enhance their performance [5], antigen-loaded DCs with autologous tumor lysates [6, 7] and with myeloid DC directly isolated from peripheral blood [3]. Mimicking an acute viral infection inside malignant tissue is becoming a paradigm to maximize tumor immunity [8–11]. In this regard, poly-I: C is well known to mimic double stranded RNA (dsRNA) as a TLR-3 agonist in endosomes and as a RIG-I or MDA-5 agonist if reaching the cytosol [12]. These functions confer poly I: C a strong adjuvant activity when formulated in vaccines to elicit robust cytotoxic T lymphocytes [13]. Hiltonol (Poly-ICLC) is a GMP-grade form of poly I: C stabilized by poly L-lysine and formulated for injection [14]. Safety is excellent and it has been catalogued as an endogenous type I interferon inducer. It shows antitumor activity in mice [11, 15, 16] and has been tested in clinical trials [17–19]. Subcutaneous injection in a cohort of healthy volunteers induced transient local dermatitis and a rapid and prominent upregulation of a type I IFN-related transcriptional signature in peripheral blood mononuclear cells (PBMCs) [17]. Moreover, Hiltonol has been delivered intratumorally in advanced cancer patients [18, 19] and intramuscularly in advanced glioblastoma patients with non-surgically amenable disease in combination with peptide-pulsed DC vaccines [20]. Evidence of clinical activity was reported in some of the cases, along with an excellent safety profile.
Patients and methods Patient characteristics, radiotherapy, DC-vaccine manufacture, flow cytometry, determination of serum cytokines, quantitative RT-PCR, immunohistochemistry, IFNc ELISPOT, TCRb sequencing and statistics are in supplementary Patients and methods section, available at Annals of Oncology online.
Results Clinical trial rationale, description and results Hiltonol (poly-ICLC) intratumoral injection showed signs of therapeutic activity in BALB/c mice against 10-day established CT26-derived subcutaneous tumors, in particular when combined with a vaccine approach based on bone marrow-derived GM-CSF DCs loaded with tumor lysate and matured in the presence of Hiltonol, IFN-a and TNF-a (supplementary Figure S1, available at Annals of Oncology online). To implement a similar strategy in humans, DC vaccines were prepared from autologous immunomagnetically isolated CD14þ monocytes in 7-day differentiation cultures with GM-CSF and IL-4, loaded with freeze/thaw tumor lysates from a needle tumor biopsy, and matured for 24 h with Hiltonol, IFN-a and TNF-a. Identity, purity and maturation of the DC products were as
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Original article shown in supplementary Figure S2, available at Annals of Oncology online and consistent with our previously reported trials of similar cell therapy products [21, 22]. There were no consistent differences in the phenotype and maturation status of DC in patients who achieved stable disease (SD) as compared with those progressing to treatment. In matured DC cultures, a tendency to produce larger amounts of IL-6 and IL-8 was noted in those patients who eventually progressed (supplementary Figure S2, available at Annals of Oncology online). Study treatment is presented in Figure 1A and consisted of cyclophosphamide (600 mg/m2) on day –7 of each cycle to reduce Treg cells and myeloid suppressor cells [21]; four daily intradermal DC vaccinations in alternating upper thigh regions from day 1 to 4 of each cycle; and image-guided intratumoral injection of 0.25 mg of Hiltonol in 0.5 ml of saline per dose into an accessible lesion on days 8 and 10 of each cycle. Two patient cohorts were included (cohort-1 and cohort-2) and their clinical features are summarized in Table 1. Ten patients were recruited in cohort-1 and nine completed treatment (Table 1). Patient 8 signed consent, but was a screening failure due to laboratory values that did not meet inclusion criteria. Treatment was well tolerated and only grades 1–2 side effects attributable to treatment were observed. Four patients experienced SD by RECIST1.1 in the first tumor evaluation carried out 6–7 weeks after treatment onset and five patients progressed. Supplementary Table S1, available at Annals of Oncology online differentially shows CTSCAN evaluation of directly treated and distant tumor lesions without evidence for distinct outcomes. Subsequent lines of treatment and duration of treatment until a new line of therapy are included in supplementary Table 2, available at Annals of Oncology online. One of the patients (patient 6) with metastatic head and neck cancer continues to show SD at month 51, although this patient was treated subsequently in month 34 with a recombinant IL-2 agent in a different clinical trial (NCT02627274) despite not having ever progressed. After treatment of the first 10 planned patients (cohort-1), the trial protocol was amended to recruit six additional patients who received fractionated stereotactic ablative radiotherapy (SABR) to the lesion injected with Hiltonol and other local or regional lesions (cohort-2). Radiotherapy consisted of 24 Gy in three fractions given every other day from day 7 to 11 (Figure 1A). Among patients treated with SABR only grades 1–2 adverse events were observed (Table 1). In spite of being heavily pretreated patients, five experienced disease stabilization by RECIST1.1 criteria as their best response. All patients were examined per-protocol by 18F-FDG PET-CT before treatment and 6–7 weeks after immunotherapy onset. Table 1 shows the summatory change in standard up-take value (SUV) in target lesions that excluded those which received irradiation in cohort-2. In five cases there was reduction of SUV (Table 1). Patient 11, one of the patients presenting SD, was a 68-year-old black male diagnosed with advanced castration-resistant prostate carcinoma presenting a prostatic mass, lung, mediastinal and inguinal lymph-node metastases and bone marrow infiltration. Hiltonol was injected into metastatic inguinal lymph nodes and SABR was administered to the prostatic tumor and the inguinal lymph nodes (Figure 1B). PET imaging carried out 3 months following treatment showed evidence of response in the thoracic
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Figure 1. Clinical trial treatment scheme and mixed clinical response in patient 11. (A) Sequence of combined immunotherapy encompassing DC vaccination and intratumoral injections of Hiltonol. (B) Dosimetry of patient 11. (C and D) PET and PET-SCAN of patient 11 three months after therapy onset. (E) CT-SCAN of patient 11 six months following treatment onset.
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47/M 63/M 46/M 60/M 72/F 67/F
40/F
65/F 70/M 68/M 58/M 57/F 51/F 43/M 42/F
01 02 03 04 05 06
07
09 10 11 12 14 15 16 17
Years-old/ Gender
Head&Neck Prostate Prostate Mesothelioma Ovarian Cervix Head&Neck Ovarian
Thyroid
Kidney Mesothelioma Kidney Head&Neck Kidney Head&Neck
Primary Tumor
L, LU, R, PE L LN, L, B PE, LU L, LU LN, PE L, LU LN, SC
L, LU, B
LN, PE LN, L, LU, B LN, L, SR LN, LU PE, SR, P L, LU
Metastases
QT, RT, Surg H, QT, TT Surg, RT,>5QT, TT QTRT QT Surg, QT QT, RT QT
Surg, RT, TT, QT
TT, IT QT Surg, IT, QT, RT, TT Surg, RT,>5QT Surg, TT, IT Surg, RT
Previous lines
Liver Liver Lymph nodes Peritoneum node Peritoneum node Lymph node Lung SC node
Esternal
SC node Paraesternal node Kidney cervical Breast Liver
Hiltonol Injection Site Asthenia G1 DC Local ReactionG1, HyporexiaG1 DC Local ReactionG1 DC Local ReactionG1 DC Local reactionG1, vomitG1 Hiltonol Infusion ReactionG1, DC Local ReactionG1 AstheniaG1, DC Local ReactionG1tumoral painG1 VomitG1, Anemia G2 DC Local ReactionG1, AST G2 DC Local ReactionG1 AstheniaG1 AstheniaG1, VomitG1, AstheniaG1, ChillsG1 No AE DC Local ReactionG1
Treatment-Related AE (grade)b
PD(þ48, 5%) Not evaluated MIXED (-34, 1%) SD (þ11%) PD (þ55%) SD (-24%) SD (-8, 8%) SD (-4%)
MIXED (-11, 4%)
SD (þ15%) PD (þ26%) SD (þ8, 8%) PD (þ44, 5%) SD (-1, 4%) SD (-7, 6%)
Best Response (% change)
2 Not evaluated 30, 6 3, 5 8, 1 32, 4 9, 7 0, 3
2, 29
1, 49 2, 61 8, 44 1, 23 0, 44 5, 41
SUVc Change
12 3 14 12 4 15 4 12þ
7
6 3 18 5 15 51þ
Overall Survivald
b
Color code used through the figures: Blue (no radiotherapy) versus red (SABR, stereoatactic ablative radiotherapy). AE, adverse events (AE refer to degree of toxicity according to Common Toxicity Criteria). c Absolute value. d Months (þongoing survival). B, Bone; IT, immunotherapy; L, liver; LN, lymph node; Lu, lung; Pe, peritoneum; P, pancreas; QT, chemotherapy; R, renal; RT, radiotherapy; SC, subcutaneous; SR, suprarrenal; Surg, surgery; TT, target therapy. Mixed: Mixed response patients with clear evidence of tumor shrinkage not meeting RECIST1.1 criteria for response.
a
COHORT 2(SABR)
COHORT 1
Patient Numbera
Table 1. Patient characteristics (n 5 17)
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Figure 2. CD3/CD8 infiltration in a pre and post-treatment biopsy of patient 11. IHC assessment of CD3 and CD8 T cells in cylinder biopsies from an inguinal adenopathy locally treated with Hiltonol and radiotherapy. Serial sections were also stained for PD-L1 expression. Evaluation was carried out in tumor containing areas (identified by H&E). Quantitative data are presented in each microphotograph as the percentage of positive cells versus total nuclei in all the tumor containing areas.
and retroperitoneal metastatic lesions, which became cystic (Figure 1C and D). Moreover, a drastic reduction in the size of mediastinal and retroperitoneal lesions was observed in a CTSCAN carried out 6 months following treatment onset (Figure 1E). A PSA serum concentration reduction followed the radiological pattern (from 120.7 to 97.5 ng/ml). Despite this evidence of activity, resulting in a mixed response, the patient did not achieve a partial response according to RECIST1.1 criteria and radiologically progressed 12 months later with corresponding increases in serum PSA. This patient consented to a post-treatment biopsy being carried out immediately before the second intratumoral injection of Hiltonol of the second treatment cycle (day þ30). Figure 2 shows pre and post core needle biopsies of the Hiltonol plus SABR treated lesion from patient 11 at baseline and on day 30. At day 30 posttreatment morphological changes are seen with a slight increase in CD3þ and CD8þ T cells, with no detectable increase in PD-L1 immunostaining on tumor cells.
Exploratory studies in peripheral blood on immune parameters related to outcome To assess signs of increased immune activity, we studied expression in PBMC of 25 relevant immune genes, based on previous experience with Hiltonol administration [17] and DC vaccination [21, 23]. Supplementary Figure S3, available at Annals of Oncology online represents the relative changes in transcript expression comparing each patient’s baseline PBMC with that obtained on day 14 (color code red indicates patients receiving SABR). As can be seen, transcripts for IFNb and IFNa increased in the majority of the patients. Of note, mRNA encoding the chemokines CXCL9 and CXCL10 increased in a fraction of patients experiencing SD. Importantly, no increases in T-cell activation markers, including PD1 and CD137 were found at least at this time point in peripheral blood, while quite surprisingly IFNc transcripts decreased in all patients. However, a multicolor flow cytometry panel on these PBMC samples assessing proportions of T, Treg, NK, B and myeloid cells did not detect noticeable
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changes or any evidence for variation in T-cell activation markers (data not shown). The serum concentrations of a panel of 16 cytokines were sequentially studied at baseline, on day 4 and on day 24. In addition, to monitor for vascular inflammation and T-cell activation, serum concentrations of soluble forms of ICAM-1, VCAM and CD25 were monitored. Results in Figure 3 are presented as percentage of change over each patient’s baseline value. Interestingly, IL-12 and IL-1b increased in most patients, but were indistinguishable between SD and PD patients on day 4. However, on day 24 the concentration elevations of IL-12 and IL1b were significantly higher in those patients experiencing SD as compared with those progressing at this late time-point. Of note, IFNa was not detectable in serum by ELISA in any of these samples. Interestingly, IL-8 decreased or remained unchanged in SD patients on day 24 while it increased in patients presenting progressive disease. A similar tendency was found for sICAM-1. Furthermore, patient 6 who remains a long-term non-progressor behaved as an outlier in terms of marked increases in IL-12, IL-1b and IL-10 in serum as well as in PBMC increases of CXCL9, CXCL10, PD-1 and PD-L1 transcripts (supplementary Figure S3, available at Annals of Oncology online and Figure 3 where this patient is marked with a blue star). Moreover, we carried out IFNcELISPOT assays co-culturing PBMC with mature DC loaded with autologous tumor lysate, as those used for treatment. Increased T-cell reactivity was found on day þ42 samples and TCRb sequencing showed a discrete increase of some clones (Figure 4A). Patient 11, who experienced the mixed response, was not an outlier for any of the parameters assessed with the exception of IL-6 serum concentration elevations that were markedly high in this case. These patients’ values are specified with stars in supplementary Figure S3, available at Annals of Oncology online and Figure 3. In terms of tumor-lysate specific IFNc-ELISPOTs, PBMC showed clear pretreatment reactivity to autologous tumor lysate that was stable or marginally decreased on day þ42, while TCRb sequencing indicated enrichment of some clones (Figure 4B).
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Figure 3. Changes in serum concentrations of cytokines and soluble surface markers. Relative changes of pre and post treatment serum concentrations assessed by ELISA on days þ4 and þ24. (A) Presents 10 cytokines of 16 sequentially tested and (B) soluble forms of surface markers denoting inflammation. Data are normalized as percentage of increase or decrease and shown for patients experiencing SD versus PD. Patients receiving radiotherapy are color-coded in red and those without radiotherapy in blue. Stars mark values of the indicated patients.
Observations in peripheral blood do not accurately estimate more relevant changes in the tumor microenvironment but at least are indicative of overall immune-associated reactivity in this series of patients.
Discussion Synergistic cancer Immunotherapy combinations [24] may include local interventions to transform a tumor lesion into an immunogenic vaccine [9]. In this pilot clinical trial we have tested two local interventions, namely Hiltonol intratumoral injections and radiotherapy, in combination with a reportedly safe and bioactive DC vaccine in an intensive regimen [21, 22]. Efficacious local interventions in combination immunotherapy have been clinically pioneered by intratumoral recombinant herpes virus (T-VEC) þ pembrolizumab, resulting in excellent efficacy in a phase I clinical trial for melanoma patients [25]. Intratumor injections of other microbial-denoting agents are also under active clinical research in combination regimens, including LPS analogues (NCT02501473), other dsRNA analogues [25], TLR-7 agonists (NCT03276832), TLR-8 agonists [26, 27], TLR-9 agonists [28] and STING agonists (NCT03172936). Arguably the most promising of such combinations are those that include PD-1/PD-L1 blockade. We observed five disease stabilizations among six previously progressing and heavily pretreated patients who were treated in the radiotherapy cohort, suggesting that this radioimmunotherapy
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combination is beneficial to some extent. Some degree of benefit had been previously reported in lymphoma patients with a scheme based on intratumoral injection of TLR-9 agonists and low dose radiotherapy [28]. The proimmune effects of radiotherapy have been extensively reported in mouse models [29, 30] and observed in clinical trials [31, 32]. The mechanisms behind of these phenomena involve immunogenic cell death and proinflammation. We selected our SABR doses in accordance with previously reported experience [33]. We were able to show increases in IFNa/b mRNA related to intratumor Hiltonol administration [17] and increases in circulating IL-12 and IL1b related to DC administration and perhaps produced at least in part by the injected DC themselves. Associations with outcome, albeit promising, remain speculative. ELISPOT reactivity in durable SD patients is indicative of T-cell priming against tumor antigens in durable SD patients. Interestingly IL-8 levels decreased in SD in comparison to PD patients possibly because IL-8 is a surrogate biomarker of tumor burden [21].
Conclusion In conclusion, local and systemic immunotherapy interventions including cancer vaccines could be combined. Refinements in the formulation of the vaccines to focus on tumor neoantigens and the combined instigation of radiotherapy þ intratumoral viralmimicking agents are to be considered in our quest to improve immunotherapeutic results.
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Figure 4. ELISPOT tumor reactivity and TCRb sequencing of peripheral blood lymphocytes in durable SD patients. Thawed PBMC samples from patient 6 (A) and 11 (B), that had been taken pre-treatment and on day þ42, were co-incubated 36h with mature autologous DC with or without loading autologous tumor lysates. Mean ELISPOT numbers per 106 cells are shown 6SD of triplicate wells. Positive controls were SAB superantigen (A) and PMAþionomycin (B). Genomic DNA from the same thawed PBMC samples were quantitatively TCRb-sequenced by ADAPTIVE to assess T-cell clonality comparing pretreatment samples and samples on day þ42. Results for patient 6 (A right) and patient 11 (B right) show the evolution of the different clones. Clones showing increase in frequency are marked with color and followed with the corresponding CDR3 sequence provided.
Acknowledgements We are grateful to the Cell Therapy Unit and Radiotherapy Department staff at the Clinica Universidad de Navarra and especially to their heads Dr. Felipe Prosper and Dr. Rafael Martinez Monge. Excellent work by personnel at the Unidad Central de Ensayos Clinicos of CUN (UCEC) is acknowledged. Scientific discussions with Drs Lasarte, Sarobe, Hervas-Stubbs and Bendandi are also acknowledged.
Commission VII Framework and Horizon 2020 programs (IACT and PROCROP) (no grant number), Cancer Research Institute (CRI) CLIP Grant 2017, Fundacio´n de la Asociacio´n Espa~ nola Contra el Ca´ncer (AECC) (no grant number) and Fundacio´n BBVA (no grant number). MER-R has received a Rio Hortega contract (ISCIII) (no grant number).
Disclosure Funding ICI Ministerio de Sanidad (EC10-113 and TRA-007), MINECO (SAF2014-52361-R and SAF2017-83267-C2-1R), European
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IM is a consultant for Bristol Myers-Squibb, Roche-Genentech, Medimmune, Merck Serono, Bioncotech, F-STAR, Genemab and Tusk. He receives grants from Bristol Myers Squibb, RocheGenentech. JLP-G is an advisor to Bristol Myers, Roche-Genentech
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Annals of Oncology and Servier. AMS is employed by Oncovyr. All remaining authors have declared no conflicts of interest.
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ORIGINAL ARTICLE
Combined immunotherapy encompassing intratumoral poly-ICLC, dendritic-cell vaccination and radiotherapy in advanced cancer patients ~ate2,3,5, G. Pe´rez2,3,5, M. E. Rodrı´guez-Ruiz1,2,3,4†, J. L. Perez-Gracia1,3,4†, I. Rodrı´guez2,3,5, C. Alfaro2,3,5, C. On I. Gil-Bazo1,2,3,4, A. Benito6, S. Inoge´s4,5, A. Lo´pez-Diaz de Cerio4,5, M. Ponz-Sarvise1,2,4, L. Resano1, P. Berraondo2,3,4, B. Barbe´s4,7, S. Martin-Algarra1,4, A. Gu´rpide1,3,4, M. F. Sanmamed1,8, C. de Andrea3,4,9, A. M. Salazar10‡ & I. Melero1,2,3,4,5*‡ 1
Department of Oncology, Clı´nica Universidad de Navarra, Pamplona; 2Centro de Investigacio´n Me´dica Aplicada (CIMA), Universidad de Navarra, Pamplona; CIBERONC, Centro de Investigacio´n Biome´dica en Red de Ca´ncer, Madrid; 4Navarra Health Research Insititute (IDISNA), Pamplona; 5Departments of Immunology; 6 Radiology; 7Physics, Clı´nica Universidad de Navarra, Pamplona, Spain; 8Immunobiology Department, Yale University School of Medicine, New Haven, USA; 9 Department of Pathology, Clı´nica Universidad de Navarra, Pamplona, Spain; 10Oncovir, Washington, USA 3
*Correspondence to: Dr Ignacio Melero, Department of Immunology, University Clinic, Avenida Pio XII, 55, 31008 Pamplona, Navarra, Spain. Tel: þ34-948255400; E-mail: [email protected] †
Both authors equally share credit.
‡
Both authors will share credit for senior authorship.
Background: Combination immunotherapy has the potential to achieve additive or synergistic effects. Combined local injections of dsRNA analogues (mimicking viral RNA) and repeated vaccinations with tumor-lysate loaded dendritic cells shows efficacy against colon cancer mouse models. In the context of immunotherapy, radiotherapy can exert beneficial abscopal effects. Patients and methods: In this two-cohort pilot phase I study, 15 advanced cancer patients received two 4-week cycles of four intradermal daily doses of monocyte-derived dendritic cells preloaded with autologous tumor lysate and matured for 24 h with poly-ICLC (Hiltonol), TNF-a and IFN-a. On days þ8 and þ10 of each cycle, patients received intratumoral image-guided 0.25 mg injections of the dsRNA-analogue Hiltonol. Cyclophosphamide 600 mg/m2 was administered 1 week before. Six patients received stereotactic ablative radiotherapy (SABR) on selected tumor lesions, including those injected with Hiltonol. Expression of 25 immune-relevant genes was sequentially monitored by RT-PCR on circulating peripheral blood mononuclear cell (PBMCs) and serum concentrations of a cytokine panel were sequentially determined before and during treatment. Pre- and posttreatment PBMC from patients achieving durable stable disease (SD) were studied by IFNc ELISPOT-assays responding to tumor-lysate loaded DC and by TCRb sequencing. Results: Combined treatment was, safe and well tolerated. One heavily pretreated castration-resistant prostate cancer patient experienced a remarkable mixed abscopal response to SABRþ immunotherapy. No objective responses were observed, while nine patients presented SD (five of them in the six-patient radiotherapy cohort). Intratumoral Hiltonol increased IFN-b and IFN-a mRNA in circulating PBMC. DC vaccination increased serum IL-12 and IL-1b concentrations, especially in patients presenting SD. IFNc-ELISPOT reactivity to tumor lysates was observed in two patients experiencing durable SD. Conclusions: This radio-immunotherapy combination strategy, aimed at resembling viral infection in tumor tissue in combination with a dendritic-cell vaccine and SABR, is safe and shows immune-associated activity and signs of preliminary clinical efficacy. Key words: intratumoral injections, poly-ICLC (Hiltonol), dendritic cell vaccination, radiotherapy and abscopal effects
C The Author(s) 2018. Published by Oxford University Press on behalf of the European Society for Medical Oncology. V
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Annals of Oncology Introduction Dendritic cells (DCs) mediate key events in priming, shaping and sustaining tumor immunity [1, 2]. Consequently, many investigators have tested ex vivo differentiated DC to formulate vaccines against tumor antigens [3]. The best results to date have been reported with DC loaded with tumor neoantigens [4], DC transfected with clinical grade mRNAs to transfer antigens and enhance their performance [5], antigen-loaded DCs with autologous tumor lysates [6, 7] and with myeloid DC directly isolated from peripheral blood [3]. Mimicking an acute viral infection inside malignant tissue is becoming a paradigm to maximize tumor immunity [8–11]. In this regard, poly-I: C is well known to mimic double stranded RNA (dsRNA) as a TLR-3 agonist in endosomes and as a RIG-I or MDA-5 agonist if reaching the cytosol [12]. These functions confer poly I: C a strong adjuvant activity when formulated in vaccines to elicit robust cytotoxic T lymphocytes [13]. Hiltonol (Poly-ICLC) is a GMP-grade form of poly I: C stabilized by poly L-lysine and formulated for injection [14]. Safety is excellent and it has been catalogued as an endogenous type I interferon inducer. It shows antitumor activity in mice [11, 15, 16] and has been tested in clinical trials [17–19]. Subcutaneous injection in a cohort of healthy volunteers induced transient local dermatitis and a rapid and prominent upregulation of a type I IFN-related transcriptional signature in peripheral blood mononuclear cells (PBMCs) [17]. Moreover, Hiltonol has been delivered intratumorally in advanced cancer patients [18, 19] and intramuscularly in advanced glioblastoma patients with non-surgically amenable disease in combination with peptide-pulsed DC vaccines [20]. Evidence of clinical activity was reported in some of the cases, along with an excellent safety profile.
Patients and methods Patient characteristics, radiotherapy, DC-vaccine manufacture, flow cytometry, determination of serum cytokines, quantitative RT-PCR, immunohistochemistry, IFNc ELISPOT, TCRb sequencing and statistics are in supplementary Patients and methods section, available at Annals of Oncology online.
Results Clinical trial rationale, description and results Hiltonol (poly-ICLC) intratumoral injection showed signs of therapeutic activity in BALB/c mice against 10-day established CT26-derived subcutaneous tumors, in particular when combined with a vaccine approach based on bone marrow-derived GM-CSF DCs loaded with tumor lysate and matured in the presence of Hiltonol, IFN-a and TNF-a (supplementary Figure S1, available at Annals of Oncology online). To implement a similar strategy in humans, DC vaccines were prepared from autologous immunomagnetically isolated CD14þ monocytes in 7-day differentiation cultures with GM-CSF and IL-4, loaded with freeze/thaw tumor lysates from a needle tumor biopsy, and matured for 24 h with Hiltonol, IFN-a and TNF-a. Identity, purity and maturation of the DC products were as
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Original article shown in supplementary Figure S2, available at Annals of Oncology online and consistent with our previously reported trials of similar cell therapy products [21, 22]. There were no consistent differences in the phenotype and maturation status of DC in patients who achieved stable disease (SD) as compared with those progressing to treatment. In matured DC cultures, a tendency to produce larger amounts of IL-6 and IL-8 was noted in those patients who eventually progressed (supplementary Figure S2, available at Annals of Oncology online). Study treatment is presented in Figure 1A and consisted of cyclophosphamide (600 mg/m2) on day –7 of each cycle to reduce Treg cells and myeloid suppressor cells [21]; four daily intradermal DC vaccinations in alternating upper thigh regions from day 1 to 4 of each cycle; and image-guided intratumoral injection of 0.25 mg of Hiltonol in 0.5 ml of saline per dose into an accessible lesion on days 8 and 10 of each cycle. Two patient cohorts were included (cohort-1 and cohort-2) and their clinical features are summarized in Table 1. Ten patients were recruited in cohort-1 and nine completed treatment (Table 1). Patient 8 signed consent, but was a screening failure due to laboratory values that did not meet inclusion criteria. Treatment was well tolerated and only grades 1–2 side effects attributable to treatment were observed. Four patients experienced SD by RECIST1.1 in the first tumor evaluation carried out 6–7 weeks after treatment onset and five patients progressed. Supplementary Table S1, available at Annals of Oncology online differentially shows CTSCAN evaluation of directly treated and distant tumor lesions without evidence for distinct outcomes. Subsequent lines of treatment and duration of treatment until a new line of therapy are included in supplementary Table 2, available at Annals of Oncology online. One of the patients (patient 6) with metastatic head and neck cancer continues to show SD at month 51, although this patient was treated subsequently in month 34 with a recombinant IL-2 agent in a different clinical trial (NCT02627274) despite not having ever progressed. After treatment of the first 10 planned patients (cohort-1), the trial protocol was amended to recruit six additional patients who received fractionated stereotactic ablative radiotherapy (SABR) to the lesion injected with Hiltonol and other local or regional lesions (cohort-2). Radiotherapy consisted of 24 Gy in three fractions given every other day from day 7 to 11 (Figure 1A). Among patients treated with SABR only grades 1–2 adverse events were observed (Table 1). In spite of being heavily pretreated patients, five experienced disease stabilization by RECIST1.1 criteria as their best response. All patients were examined per-protocol by 18F-FDG PET-CT before treatment and 6–7 weeks after immunotherapy onset. Table 1 shows the summatory change in standard up-take value (SUV) in target lesions that excluded those which received irradiation in cohort-2. In five cases there was reduction of SUV (Table 1). Patient 11, one of the patients presenting SD, was a 68-year-old black male diagnosed with advanced castration-resistant prostate carcinoma presenting a prostatic mass, lung, mediastinal and inguinal lymph-node metastases and bone marrow infiltration. Hiltonol was injected into metastatic inguinal lymph nodes and SABR was administered to the prostatic tumor and the inguinal lymph nodes (Figure 1B). PET imaging carried out 3 months following treatment showed evidence of response in the thoracic
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Figure 1. Clinical trial treatment scheme and mixed clinical response in patient 11. (A) Sequence of combined immunotherapy encompassing DC vaccination and intratumoral injections of Hiltonol. (B) Dosimetry of patient 11. (C and D) PET and PET-SCAN of patient 11 three months after therapy onset. (E) CT-SCAN of patient 11 six months following treatment onset.
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47/M 63/M 46/M 60/M 72/F 67/F
40/F
65/F 70/M 68/M 58/M 57/F 51/F 43/M 42/F
01 02 03 04 05 06
07
09 10 11 12 14 15 16 17
Years-old/ Gender
Head&Neck Prostate Prostate Mesothelioma Ovarian Cervix Head&Neck Ovarian
Thyroid
Kidney Mesothelioma Kidney Head&Neck Kidney Head&Neck
Primary Tumor
L, LU, R, PE L LN, L, B PE, LU L, LU LN, PE L, LU LN, SC
L, LU, B
LN, PE LN, L, LU, B LN, L, SR LN, LU PE, SR, P L, LU
Metastases
QT, RT, Surg H, QT, TT Surg, RT,>5QT, TT QTRT QT Surg, QT QT, RT QT
Surg, RT, TT, QT
TT, IT QT Surg, IT, QT, RT, TT Surg, RT,>5QT Surg, TT, IT Surg, RT
Previous lines
Liver Liver Lymph nodes Peritoneum node Peritoneum node Lymph node Lung SC node
Esternal
SC node Paraesternal node Kidney cervical Breast Liver
Hiltonol Injection Site Asthenia G1 DC Local ReactionG1, HyporexiaG1 DC Local ReactionG1 DC Local ReactionG1 DC Local reactionG1, vomitG1 Hiltonol Infusion ReactionG1, DC Local ReactionG1 AstheniaG1, DC Local ReactionG1tumoral painG1 VomitG1, Anemia G2 DC Local ReactionG1, AST G2 DC Local ReactionG1 AstheniaG1 AstheniaG1, VomitG1, AstheniaG1, ChillsG1 No AE DC Local ReactionG1
Treatment-Related AE (grade)b
PD(þ48, 5%) Not evaluated MIXED (-34, 1%) SD (þ11%) PD (þ55%) SD (-24%) SD (-8, 8%) SD (-4%)
MIXED (-11, 4%)
SD (þ15%) PD (þ26%) SD (þ8, 8%) PD (þ44, 5%) SD (-1, 4%) SD (-7, 6%)
Best Response (% change)
2 Not evaluated 30, 6 3, 5 8, 1 32, 4 9, 7 0, 3
2, 29
1, 49 2, 61 8, 44 1, 23 0, 44 5, 41
SUVc Change
12 3 14 12 4 15 4 12þ
7
6 3 18 5 15 51þ
Overall Survivald
b
Color code used through the figures: Blue (no radiotherapy) versus red (SABR, stereoatactic ablative radiotherapy). AE, adverse events (AE refer to degree of toxicity according to Common Toxicity Criteria). c Absolute value. d Months (þongoing survival). B, Bone; IT, immunotherapy; L, liver; LN, lymph node; Lu, lung; Pe, peritoneum; P, pancreas; QT, chemotherapy; R, renal; RT, radiotherapy; SC, subcutaneous; SR, suprarrenal; Surg, surgery; TT, target therapy. Mixed: Mixed response patients with clear evidence of tumor shrinkage not meeting RECIST1.1 criteria for response.
a
COHORT 2(SABR)
COHORT 1
Patient Numbera
Table 1. Patient characteristics (n 5 17)
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Figure 2. CD3/CD8 infiltration in a pre and post-treatment biopsy of patient 11. IHC assessment of CD3 and CD8 T cells in cylinder biopsies from an inguinal adenopathy locally treated with Hiltonol and radiotherapy. Serial sections were also stained for PD-L1 expression. Evaluation was carried out in tumor containing areas (identified by H&E). Quantitative data are presented in each microphotograph as the percentage of positive cells versus total nuclei in all the tumor containing areas.
and retroperitoneal metastatic lesions, which became cystic (Figure 1C and D). Moreover, a drastic reduction in the size of mediastinal and retroperitoneal lesions was observed in a CTSCAN carried out 6 months following treatment onset (Figure 1E). A PSA serum concentration reduction followed the radiological pattern (from 120.7 to 97.5 ng/ml). Despite this evidence of activity, resulting in a mixed response, the patient did not achieve a partial response according to RECIST1.1 criteria and radiologically progressed 12 months later with corresponding increases in serum PSA. This patient consented to a post-treatment biopsy being carried out immediately before the second intratumoral injection of Hiltonol of the second treatment cycle (day þ30). Figure 2 shows pre and post core needle biopsies of the Hiltonol plus SABR treated lesion from patient 11 at baseline and on day 30. At day 30 posttreatment morphological changes are seen with a slight increase in CD3þ and CD8þ T cells, with no detectable increase in PD-L1 immunostaining on tumor cells.
Exploratory studies in peripheral blood on immune parameters related to outcome To assess signs of increased immune activity, we studied expression in PBMC of 25 relevant immune genes, based on previous experience with Hiltonol administration [17] and DC vaccination [21, 23]. Supplementary Figure S3, available at Annals of Oncology online represents the relative changes in transcript expression comparing each patient’s baseline PBMC with that obtained on day 14 (color code red indicates patients receiving SABR). As can be seen, transcripts for IFNb and IFNa increased in the majority of the patients. Of note, mRNA encoding the chemokines CXCL9 and CXCL10 increased in a fraction of patients experiencing SD. Importantly, no increases in T-cell activation markers, including PD1 and CD137 were found at least at this time point in peripheral blood, while quite surprisingly IFNc transcripts decreased in all patients. However, a multicolor flow cytometry panel on these PBMC samples assessing proportions of T, Treg, NK, B and myeloid cells did not detect noticeable
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changes or any evidence for variation in T-cell activation markers (data not shown). The serum concentrations of a panel of 16 cytokines were sequentially studied at baseline, on day 4 and on day 24. In addition, to monitor for vascular inflammation and T-cell activation, serum concentrations of soluble forms of ICAM-1, VCAM and CD25 were monitored. Results in Figure 3 are presented as percentage of change over each patient’s baseline value. Interestingly, IL-12 and IL-1b increased in most patients, but were indistinguishable between SD and PD patients on day 4. However, on day 24 the concentration elevations of IL-12 and IL1b were significantly higher in those patients experiencing SD as compared with those progressing at this late time-point. Of note, IFNa was not detectable in serum by ELISA in any of these samples. Interestingly, IL-8 decreased or remained unchanged in SD patients on day 24 while it increased in patients presenting progressive disease. A similar tendency was found for sICAM-1. Furthermore, patient 6 who remains a long-term non-progressor behaved as an outlier in terms of marked increases in IL-12, IL-1b and IL-10 in serum as well as in PBMC increases of CXCL9, CXCL10, PD-1 and PD-L1 transcripts (supplementary Figure S3, available at Annals of Oncology online and Figure 3 where this patient is marked with a blue star). Moreover, we carried out IFNcELISPOT assays co-culturing PBMC with mature DC loaded with autologous tumor lysate, as those used for treatment. Increased T-cell reactivity was found on day þ42 samples and TCRb sequencing showed a discrete increase of some clones (Figure 4A). Patient 11, who experienced the mixed response, was not an outlier for any of the parameters assessed with the exception of IL-6 serum concentration elevations that were markedly high in this case. These patients’ values are specified with stars in supplementary Figure S3, available at Annals of Oncology online and Figure 3. In terms of tumor-lysate specific IFNc-ELISPOTs, PBMC showed clear pretreatment reactivity to autologous tumor lysate that was stable or marginally decreased on day þ42, while TCRb sequencing indicated enrichment of some clones (Figure 4B).
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Figure 3. Changes in serum concentrations of cytokines and soluble surface markers. Relative changes of pre and post treatment serum concentrations assessed by ELISA on days þ4 and þ24. (A) Presents 10 cytokines of 16 sequentially tested and (B) soluble forms of surface markers denoting inflammation. Data are normalized as percentage of increase or decrease and shown for patients experiencing SD versus PD. Patients receiving radiotherapy are color-coded in red and those without radiotherapy in blue. Stars mark values of the indicated patients.
Observations in peripheral blood do not accurately estimate more relevant changes in the tumor microenvironment but at least are indicative of overall immune-associated reactivity in this series of patients.
Discussion Synergistic cancer Immunotherapy combinations [24] may include local interventions to transform a tumor lesion into an immunogenic vaccine [9]. In this pilot clinical trial we have tested two local interventions, namely Hiltonol intratumoral injections and radiotherapy, in combination with a reportedly safe and bioactive DC vaccine in an intensive regimen [21, 22]. Efficacious local interventions in combination immunotherapy have been clinically pioneered by intratumoral recombinant herpes virus (T-VEC) þ pembrolizumab, resulting in excellent efficacy in a phase I clinical trial for melanoma patients [25]. Intratumor injections of other microbial-denoting agents are also under active clinical research in combination regimens, including LPS analogues (NCT02501473), other dsRNA analogues [25], TLR-7 agonists (NCT03276832), TLR-8 agonists [26, 27], TLR-9 agonists [28] and STING agonists (NCT03172936). Arguably the most promising of such combinations are those that include PD-1/PD-L1 blockade. We observed five disease stabilizations among six previously progressing and heavily pretreated patients who were treated in the radiotherapy cohort, suggesting that this radioimmunotherapy
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combination is beneficial to some extent. Some degree of benefit had been previously reported in lymphoma patients with a scheme based on intratumoral injection of TLR-9 agonists and low dose radiotherapy [28]. The proimmune effects of radiotherapy have been extensively reported in mouse models [29, 30] and observed in clinical trials [31, 32]. The mechanisms behind of these phenomena involve immunogenic cell death and proinflammation. We selected our SABR doses in accordance with previously reported experience [33]. We were able to show increases in IFNa/b mRNA related to intratumor Hiltonol administration [17] and increases in circulating IL-12 and IL1b related to DC administration and perhaps produced at least in part by the injected DC themselves. Associations with outcome, albeit promising, remain speculative. ELISPOT reactivity in durable SD patients is indicative of T-cell priming against tumor antigens in durable SD patients. Interestingly IL-8 levels decreased in SD in comparison to PD patients possibly because IL-8 is a surrogate biomarker of tumor burden [21].
Conclusion In conclusion, local and systemic immunotherapy interventions including cancer vaccines could be combined. Refinements in the formulation of the vaccines to focus on tumor neoantigens and the combined instigation of radiotherapy þ intratumoral viralmimicking agents are to be considered in our quest to improve immunotherapeutic results.
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Figure 4. ELISPOT tumor reactivity and TCRb sequencing of peripheral blood lymphocytes in durable SD patients. Thawed PBMC samples from patient 6 (A) and 11 (B), that had been taken pre-treatment and on day þ42, were co-incubated 36h with mature autologous DC with or without loading autologous tumor lysates. Mean ELISPOT numbers per 106 cells are shown 6SD of triplicate wells. Positive controls were SAB superantigen (A) and PMAþionomycin (B). Genomic DNA from the same thawed PBMC samples were quantitatively TCRb-sequenced by ADAPTIVE to assess T-cell clonality comparing pretreatment samples and samples on day þ42. Results for patient 6 (A right) and patient 11 (B right) show the evolution of the different clones. Clones showing increase in frequency are marked with color and followed with the corresponding CDR3 sequence provided.
Acknowledgements We are grateful to the Cell Therapy Unit and Radiotherapy Department staff at the Clinica Universidad de Navarra and especially to their heads Dr. Felipe Prosper and Dr. Rafael Martinez Monge. Excellent work by personnel at the Unidad Central de Ensayos Clinicos of CUN (UCEC) is acknowledged. Scientific discussions with Drs Lasarte, Sarobe, Hervas-Stubbs and Bendandi are also acknowledged.
Commission VII Framework and Horizon 2020 programs (IACT and PROCROP) (no grant number), Cancer Research Institute (CRI) CLIP Grant 2017, Fundacio´n de la Asociacio´n Espa~ nola Contra el Ca´ncer (AECC) (no grant number) and Fundacio´n BBVA (no grant number). MER-R has received a Rio Hortega contract (ISCIII) (no grant number).
Disclosure Funding ICI Ministerio de Sanidad (EC10-113 and TRA-007), MINECO (SAF2014-52361-R and SAF2017-83267-C2-1R), European
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IM is a consultant for Bristol Myers-Squibb, Roche-Genentech, Medimmune, Merck Serono, Bioncotech, F-STAR, Genemab and Tusk. He receives grants from Bristol Myers Squibb, RocheGenentech. JLP-G is an advisor to Bristol Myers, Roche-Genentech
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Annals of Oncology and Servier. AMS is employed by Oncovyr. All remaining authors have declared no conflicts of interest.
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