Open access Original research Systemic cellular viroimmunotherapy for canine high-­grade gliomas Ana Cloquell ‍ ‍ ,1 Isidro Mateo ‍ ‍ ,1,2 Stefano Gambera ‍ ‍ ,3,4 Martí Pumarola,5 Ramon Alemany,6 Javier García-­Castro ‍ ‍ ,3 Ana Judith Perisé-Barrios ‍ ‍ 7 To cite: Cloquell A, Mateo I, ABSTRACT Gambera S, et al. Systemic Background Oncolytic viruses constitute a growing field WHAT IS ALREADY KNOWN ON THIS TOPIC cellular viroimmunotherapy for of interest, both in human and veterinary oncology, given ⇒ High-­grade gliomas have a poor short-­term prog- canine high-­grade gliomas. nosis, despite the use of the most advanced or ag- that they are particularly helpful for treating non-­surgical Journal for ImmunoTherapy gressive therapies when possible, and the efficacy of Cancer 2022;10:e005669. tumors and disseminated cancer, such as high-­grade gliomas. Companion dogs present malignant gliomas of oncolytic viruses is limited by difficulties in tar- doi:10.1136/jitc-2022-005669 with biological, genetic, phenotypic, immunological, and geting brain tumors. Therapies, therefore, need to clinical similarities to human gliomas. These features be improved to reach the brain. ► Additional supplemental material is published online only. favor comparative approaches, leading to the treatment WHAT THIS STUDY ADDS To view, please visit the journal of canine oncological patients to achieve translational ⇒ The systemic endovenous treatment is safe, en- online (http://​dx.​doi.​org/​10.​ applications to the human clinic. The systemic sures that the virus is delivered to the brain tumor, 1136/​jitc-​2022-​005669). administration of oncolytic viruses presents a challenge and response evaluation suggest clinical efficacy. due to their limitations in effectively targeting tumors and AC and IM contributed equally. JG-­C and AJP-­B contributed metastases. Therefore, the aim of this study is to evaluate HOW THIS STUDY MIGHT AFFECT RESEARCH, equally. the safety and antitumor activity of a virotherapy used in PRACTICE OR POLICY spontaneous canine tumors. ⇒ These data are relevant to the ongoing phase Ib reg- Accepted 19 October 2022 Methods Ten dogs with high-­grade rostrotentorial ulated human clinical trial that is already adminis- gliomas underwent weekly systemic endovenous cellular tering this virotherapy to children, adolescents, and virotherapy with dCelyvir (canine mesenchymal stem young adults with diffuse pontine glioma. cells infected with the canine oncolytic adenovirus ICOCAV17) for 8 weeks. Efficacy was determined in seven dogs according to the Response Assessment in BACKGROUND Veterinary Neuro-­Oncology criteria considering clinical status and MRI measurements. Medical history, physical Oncolytic viruses (OVs) as therapeutic agents and neurological examinations, and vaccination status constitute a growing field of interest, both were evaluated prior to and during follow-­up. Safety was in human and veterinary oncology with evaluated by physical examinations and hematological several ongoing clinical trials and prom- and biochemical changes in peripheral blood. Immune ising results.1 OVs can reverse the microen- populations were analyzed by flow cytometry in peripheral vironment’s immunosuppressive status2 and blood and by gene expression and immunohistochemistry induce intratumoral immunostimulatory in the tumor microenvironment. signals increasing immune cell infiltration.3 Results The treatment was well tolerated and major The virus’ lytic activity induces the release of adverse effects were not observed. Two dogs had partial tumor neoantigens, leading to an antitumor responses (76% and 86% reduction in tumor size), and response.4 5 Due to its nature and mechanism 3/7 showed stable disease. ICOCAV17 was detected in of action, adenovirus is effective in promoting peripheral blood in nine dogs, and a correlation between immune response, controlling tumor growth, the ICOCAV17 particles and anti-­canine adenovirus (CAV) antibodies was observed. ICOCAV17 was detected in and improving survival.6 7 Conditionally repli- 3/9 tumor tissues after necropsies. Regarding tumor-­ cating adenoviruses (CRAds) are an option infiltrating lymphocytes, the dogs with disease stabilization for patients with cancer because CRAds © Author(s) (or their and partial response tended to have reduced memory have a selective potential for replication, employer(s)) 2022. Re-­use B-­cell infiltration and increased monocyte/macrophage achieving a successful viral cycle in tumor permitted under CC BY-­NC. No commercial re-­use. See rights lineage cells. cells, resulting in cell destruction. Most and permissions. Published by Conclusions These findings indicate that dCelyvir is safe CRAds rely on tumor-­specific promoters to BMJ. and presents efficacy in canine rostrotentorial high-­grade control the master key gene E1a and in dele- For numbered affiliations see gliomas. These data are relevant to the ongoing phase Ib tions in domains of E1 genes that are needed end of article. regulated human clinical trial that is administering this to block cell cycle regulators such as pRB or virotherapy to children, adolescents, and young adults with Correspondence to p53. Due to the lack of function of such regu- diffuse pontine glioma. Celyvir should be further explored Dr Javier García-­Castro; latory proteins in tumors, such deletion only as a treatment in veterinary and human neuro-­oncology. ​jgcastro@​isciii.​es has a detrimental effect on virus replication Cloquell A, et al. J Immunother Cancer 2022;10:e005669. doi:10.1136/jitc-2022-005669 1 Open access in normal cells but not in tumor cells. CRAds can also be significantly different from those obtained in previous genetically modified to enhance their infectivity and/or studies in dogs with extracranial tumors, given that the intratumoral spread.8 brain environment and the blood-­brain barrier can play a The use of CRAds can be particularly helpful for treating role that could affect the clinical outcome. Therefore, it non-­surgical tumors, such as high-­grade gliomas.9 10 Glio- would be useful to describe the dCelyvir therapy effects in blastoma multiforme (GBM) is the most frequent malig- a brain tumor population. nant primary brain tumor in dogs and humans, and the Domestic dogs develop spontaneous neoplasms in the standard patient care includes incomplete surgical resec- central nervous system (CNS),7 19–21 present intratumor tion combined with radiation and/or chemotherapy; heterogeneity, and have clinical and pathophysiological unfortunately, these treatments achieve a poor short-­ features and genetic changes equivalent to human CNS term prognosis in humans and dogs.11 Treatment with tumors.22 23 The conserved glioma drivers and aneuploidy CRAds has shown promising results in veterinary trials, have been identified as a hallmark of high-­grade disease, and further in human clinical trials including a phase I showing high similarity especially between human pedi- using intratumoral injections of DNX-­2401, showing clin- atric and canine gliomas.19 In addition, dogs have an ical benefit in 12% of cases, with long-­term survival in intact immune system and present similarities with patients with recurrent high-­grade gliomas, and a link to humans in tumor immune infiltration, making them a an immune-­mediated response.12 13 Further, the infusion valuable immunocompetent model.19 23–25 of OV DNX-­2401 followed by radiotherapy in pediatric Moreover, the absence of effective gold standards for patients with diffuse intrinsic pontine glioma resulted in managing high-­grade gliomas in dogs11 26 27 makes these a reduction or stabilization of tumor size.14 tumors excellent candidates for developing novel ther- Although CRAds have also been systemically admin- apeutics, with the aim of obtaining responses allowing istered, they have shown limited efficacy due to various comparative approaches that favor a clinical translation barriers that viral particles encounter in targeting of the findings from dogs to humans.19 20 28 Gliomas are neoplastic lesions, such as neutralization in blood and one of the most common tumors in dogs, that represent liver uptake.15 Intracranial solid tumors are not always 36%–70% of primary brain tumors in those animals, surgically accessible, and the intratumoral delivery of which have a poor prognosis due to the lack of an acces- CRAds is not a feasible option for these patients in the sible and effective treatment for this disease. Radio- regular clinic. To improve the systemic administration therapy is an expensive treatment and not recommended of CRAds, the use of mesenchymal stem cells (MSCs) as for some diffuse tumors (unless novel irradiation equip- cellular carriers for CRAds in humans and dogs has been ment is available, which unfortunately is not common), previously demonstrated.6 16 MSCs were infected in vitro and benefit from resection surgery appears to be limited. with CRAds and employed as a ‘Trojan horse’, avoiding Fortunately, the availability of advanced imaging for the systemic immunity against the virus and even further diagnosis and follow-­ up, makes dogs excellent animal reaching the tumor due to their tumor-­homing capacity. models to test novel therapeutic strategies, as immuno- This treatment, named Celyvir, has been demonstrated therapies.21 26 Therefore, veterinary trials with companion to be effective in humans and canine patients without dogs are useful in translational research, and should take secondary effects or toxicity.6 17 a position between basic research and human clinical The most complete study developed in the veteri- trials.25 29 30 nary field have been performed using a canine onco- The aim of this study was to evaluate the safety and lytic adenovirus named ICOCAV17.6 This CRAd has the efficacy of systemic infusions of dCelyvir and its immune-­ same characteristics as the human oncolytic adenovirus related response in canine patients with high-­ grade ICOVIR17 (used in clinical trials as VCN-­01): In partic- gliomas. The findings will allow us to address improve- ular, ICOCAV17 has four palindromic E2F to repress E1a ments in current therapies in human gliomas, leading us promoter in normal cells though the E2F-­pRB complex to propose the use of the therapy described here as a treat- and to activate E1a expression in tumor cells through free ment for people with brain tumors, even as a first line of E2F. In addition, the pRB binding domain of E1a has been treatment. The findings reported here are indeed novel deleted in ICOCAV17. To increase its antitumor potency and the first to describe the therapy effects in patients this virus express hyaluronidase linked to the major late with brain tumors. promoter and has an RGD motif inserted in the HI-­loop of the fiber.18 In this regard, we previously published a veterinary clinical trial, in which 27 dogs with extracranial MATERIAL AND METHODS solid tumors were treated with canine Celyvir (dCelyvir) Patient selection by using allogenic canine MSCs (dMSCs) infected with The study followed the ARRIVE guidelines 2.0, and the ICOCAV17, showing a response rate of 74%, including information can be found in the manuscript. The study’s 14.8% of complete remission, with an excellent toxicity inclusion criteria were as follows: (1) client-­owned dogs profile.6 The safety and efficacy of this treatment in canine with a single intra-­axial rostrotentorial mass suspected intraparenchymal primary brain tumors have never been to be a high-­grade glioma based on MRI findings; (2) explored and remain unknown. The results could be no previous treatments except for palliative care with 2 Cloquell A, et al. J Immunother Cancer 2022;10:e005669. doi:10.1136/jitc-2022-005669 Open access Figure 1 (A) Schematic representation of the dCelyvir treatment. (B) MRI and macroscopic tumor images from the dCelyvir-­ treated patients. MRI prior to dCelyvir treatment (first MRI), and 2 months after starting weekly treatment (second MRI) showing tumors (white arrows). Clinical benefit is indicated according to RAVNO criteria. Bottom figures show macroscopic tumor tissue (white arrows) in necropsies. PD, progressive disease; PR, partial response; RAVNO, response assessment in veterinary neuro-­ oncology; SD, stable disease. anticonvulsants and/or prednisone; (3) good physical neuro-­oncology (RAVNO) criteria with a modification31 : condition with no hematological or biochemical evidence lesions were measured even if their longest diameter was of organ dysfunction; (4) a mild increase in liver enzymes smaller than 10 mm. Treatment was discontinued in those with a normal bile acid stimulation test was considered dogs with progressive disease (PD) or severe biochemical acceptable in cases under corticosteroid or anticonvul- abnormalities (such as elevation of serum liver enzyme’s sant therapy; and (5) dogs whose owners declined other activity more than three times the upper normal limit). therapeutic options such as surgery, radiotherapy, and Those dogs with partial response (PR) or stable disease chemotherapy. Dogs weighing less than 4 kg and dogs (SD) were selected for a second round of dCelyvir (four with concurrent neoplastic or inflammatory diseases were doses, every 2 weeks). Adverse effects were defined by excluded. the veterinary cooperative oncology group for common terminology criteria of adverse events in dogs and cats. Clinical study design The dogs were followed-­up until death, at which point The trial followed a schedule of the weekly intravenous administration of dCelyvir at 0.5×106 dMSC/kg for 8 they underwent a necropsy within 24 hours. Samples weeks, following the previously described protocol.6 from the tumor, the unaffected contralateral hemisphere, To avoid anaphylactic reactions, dogs were pretreated and the liver were used to perform histopathology and with metamizol (30 mg/kg/IV) and difenhidramine immunohistochemistry, or snap frozen to analyze the (0.5 mg/kg/IV). Tumor reevaluation by MRI at 2 months RNA expression. (figure 1A). At diagnosis, all dogs received a tapering course of prednisone for 4 weeks, and phenobarbital Cell culture and dCelyvir preparation was initiated and continued throughout the treatment in Isolation and characterization of dMSC from the adipose the dogs with seizures. The clinical outcome was defined tissue of healthy donor dogs, were performed as previ- according to the response assessment in veterinary ously described.6 The presence of mycoplasma (MycoAlert Cloquell A, et al. J Immunother Cancer 2022;10:e005669. doi:10.1136/jitc-2022-005669 3 Open access Mycoplasma Detection Kit, Lonza) and the preparation SSTII-­Advance), and serum was obtained and frozen at of dCelyvir treatment were as previously described.6 −20°C. Quantification of interleukin (IL)−2, IL-­6, IL-­7, IL-­8, IL-­10, IL-­15, IL-­18, interferon (IFN)-γ, CXC chemo- MRI and RAVNO criteria kine ligand (CXCL)10, granulocyte-­macrophage colony-­ Imaging studies were performed at pre-­treatment and 2 stimulating factor (GM-­ CSF), CC chemokine ligand months after the first dCelyvir treatment using an MRI (CCL)2 and keratinocyte-­ derived chemokine(KC)-­ like unit (Hitachi Airis Mate 0.2T model; Tokio, Japan). were performed with an Immunology Multiplex Assay The tumor size was calculated considering the contrast-­ (Milliplex MAP Canine Cytokine/Chemokine Magnetic enhancing lesion (online supplemental figure S1A); Bead Panel, Millipore; Darmstadt, Germany) following when no enhancement was detected, the size was calcu- the manufacturer’s protocol and were analyzed using lated by comparing the parenchymal intensity patterns Luminex technology. with the normal anatomy (online supplemental figure 1B-­C). Peritumoral edema (online supplemental figure Antiviral-specific antibodies 1D-­E) and the midline sift were measured (online supple- Blood samples were collected in BD vacutainer plasma mental figure 1F). Clinical outcomes were defined by separator tubes (BD PSTII); then plasma was obtained RAVNO criteria as follows: complete response, stable and frozen at −20°C until analysis. To determine specific or improved clinical status not receiving steroids, elimi- antibodies (IgG) against canine adenovirus (CAV), a nation of all enhancing tumors and stable or decreased solid-­phase ELISA was performed (ImmunoComb Canine T2W/fluid-­attenuated inversion recovery (FLAIR) lesion VacciCheck, Biogal Galed Laboratories Acs.; Kibbutz burden; PR: ≥50% decrease in the contrast enhancing Galed, Israel) following a previously described protocol.6 lesion burden in patients with stable or improved clinical status; SD <50% decrease or <25% increase in enhancing DNA extraction from blood and tissues and qPCR analysis tumor size in a patient with stable or improved clinical Blood samples were collected in PAXgene Blood DNA status requiring stable or decreased steroid doses; and tubes and processed using the QIAamp DNA Blood Mini PD ≥25% increase in enhancing tumor in T2W/FLAIR Kit (Qiagen; Madrid, Spain) following the manufac- images, new lesions and/or clinical deterioration. For turer’s instructions. Tumors from necropsies were snap further details regarding the MRI, see online supple- frozen at −80°C as dried pieces. Tissue samples were mental information. introduced into a metal cylinder submerged in liquid nitrogen and ground thoroughly with a pestle until they Histopathology, diagnosis, and immunohistochemistry were completely pulverized. DNA was isolated using the The morphological evaluation was performed on QIAamp DNA Mini Qit (Qiagen). DNA quantification formalin-­fixed, paraffin-­embedded sections stained with and purity (A260/280 and A260/230) were analyzed with hematoxylin and eosin in eight necropsies. Tumors were a Nanodrop 2000 spectrophotometer (Thermo Scien- classified according to the 2007 criteria of the WHO for tific). Dilutions of ICOCAV17 from 108 to 103 vp/mL in human CNS tumors32 and the subsequently revised diag- blood from healthy donors were used for the standard nostic scheme for canine gliomas.19 28 33 Samples were eval- curve. Samples were analyzed in triplicate by the quantita- uated for the presence of necrosis, extracellular matrix tive real-­time PCR (qRT-­PCR) 7500 Fast (Applied Biosys- degradation, vascular proliferation, mucinous secretion, tems; Madrid, Spain) using the Premix Ex Taq (Clontech; and hemorrhages. Saint-­Germain-­ en-­ Laye, France), with forward primer Immunohistochemistry was performed using primary (0.5 µmol/L) 5’​TGTGGGCCTGTGTGATTCCT-­3’; antibodies to detect glial fibrillar acidic protein (Dako reverse primer (0.5 µmol/L) 5’-​CCAG​AATC​AGCC​TGAG​ Z0334; Glostrup, Denmark), oligodendrocyte transcrip- TGCTC-­3’; and 10 pmol of Taqman probe FAM-­​CTCG​ tion factor 2 (Olig2, Merck Millipore AB9610; Billerica, AATC​AGTG​TCAG​GCTCCGCA-­TAMRA, which iden- Massachusetts, USA), CD3 (Dako A0452; Glostrup, tified the E1A region. The qRT-­PCR conditions were as Denmark), CD20 (Thermo Fisher PA5-­32313; Waltham, follows: holding stage 10 min at 95°C, cycling stage 15 s Massachusetts, USA), FOXP3 (forkhead box P3, eBio- at 95°C and 1 min at 60°C, repeated 40 times. Analysis science 14–7979; Waltham, Massachusetts, USA) and was performed using the 7500 Software V.2.0.6 (Applied ICOCAV17 (anti-­ Ad5 Abcam ab6982). Stained tumor Biosystems). slides were assessed using image analysis software (Nano- Zoomer Digital Pathology; Hamamatsu, Japan). A Flow cytometry number of CD3+, CD20+ and FOXP3+ lymphocytes were Blood samples were collected in a BD vacutainer determined. For further details, see online supplemental K3EDTA. Samples were incubated with human FcR information. Blocking (Miltenyi; Cologne, Germany) for 10 min at room temperature and incubated for 20 min at 4°C with Cytokine analysis directly conjugated antibodies diluted in phosphate-­ Blood samples were obtained at days 0, 1, 2, and 3 buffered saline with 1% fetal bovine serum. Samples and prior to every weekly dCelyvir infusion. Samples were incubated with Quicklisis buffer (Cytognos; Sala- were collected in BD vacutainer separator tubes (BD manca, Spain) to lyse erythrocytes. The acquisition and 4 Cloquell A, et al. J Immunother Cancer 2022;10:e005669. doi:10.1136/jitc-2022-005669 Open access analysis were conducted in a MacsQuant10 Flow Cytom- **p<0.01 and ***p<0.001. To test statistical association eter (Miltenyi). The antibodies employed and the flow between two continuous variables, a Pearson’s correla- cytometry gating strategy of the immune subpopulation tion coefficient test was performed. A two-­way analysis of analysis in dog-­derived samples have been previously variance and Bonferroni’s multiple comparison tests were described.6 used to analyze CIBERSORT results. RNA extraction, gene expression, and tumor-infiltrating immune cell profile Tumor tissues were snap frozen at −80°C, and RNA was RESULTS isolated using the RNeasy Mini Kit (Qiagen) and was Canine patient demographics and clinical status analyzed with a Nanodrop 2000 spectrophotometer Ten dogs were enrolled for the dCelyvir therapy. Brachy- (Thermo Scientific). The RNA Integrity Number was cephalic breeds were overrepresented (six French bull- evaluated using the Bioanalyzer RNA 6000 Nano assay, dogs, two Boxers, and one English bulldog). There were and samples with RIN values higher than 8.6 were used. seven males and three females with a median age of 8 years Tumor RNA expression was analyzed by GeneChip (6–10 years) and a mean weight of 17 kg (4.4–38.8 kg). Canine Gene V.1.0 ST Array (Affymetrix). Data were All the dogs had been vaccinated against CAV2 (table 1), analyzed using Transcriptome Analysis Console Soft- all had experienced seizures, and seven of them showed ware. Tumor-­ infiltrating leukocyte cell fractions of 6 other prosencephalic signs (abnormal mental status, tumors were estimated from bulk RNA expression data contralateral hemiparesis, menace response deficits and/ employing a CIBERSORT algorithm (https://cibersort.​ or abnormal facial sensation). The mean period from stanford.edu).34 The microarray expression matrix of first neurological signs to diagnosis was 40 days (online canine tumors was preprocessed to match human gene supplemental table S1). Prednisone treatment was main- identifiers with canine orthologues. Leucocyte infiltra- tained during the entire treatment in five dogs and the tion was estimated using a validated leukocyte gene signa- other five were treated intermittently (overall, the dogs ture matrix (LM22); canine orthologues matched 85% of received prednisone during 73% of the treatment time). the LM22 gene signature matrix. Analysis was performed All the dogs, except one, received phenobarbital during in relative mode, with 100 permutation runs and without the entire dCelyvir treatment. quantile normalization. The resulting cellular fractions were collapsed from 22 to 11 cell types based on their lineage, as previously described.34 Safety and toxicity of dCelyvir In most of the dogs, the dCelyvir treatment was well toler- Statistical analysis ated, except for one dog who showed a forelimb swelling The data were graphed and analyzed with Excel 2013 contralateral to the site of venous catheterization after (Microsoft Office 2013) and Prism (GraphPad) Soft- the first dCelyvir administration. The adverse event was ware. Comparisons between quantitative variables were resolved 48 hours after treatment with a single dose of performed with Wilcoxon’s rank-­sum test, and the Mann-­ meloxicam (0.2 mg/kg subcutaneous) and difenhidr- Whitney statistic for samples with non-­normal distribu- amine (0.5 mg/kg intramuscular). Two dogs showed mild tion. Differences were considered significant at *p<0.05; fatigue after treatment on two occasions each. Table 1 Patient characteristics, treatment, clinical outcome, and tumor characterization Patient Age Breed Gender CAV vacc. dCelyvir doses RAVNO Tumor location Diagnosis 1 8 French Bulldog M 8 8 PD Temp. Olig. III 2 6 French Bulldog M 3 11 PD Frontal Olig. III 3 7 Poodle M 1 8 PR Temp.+Occ. Olig. III 4 9 English Bulldog F 11 1 N/A Temp.+Th. N/A 5 9 Boxer F 35 12 SD Frontal Olig. III 6 9 Boxer M 5 14 SD Frontal GBM 7 10 French Bulldog M 4 12 SD Frontal+Th. GBM 8 6 French Bulldog M 5 5 N/A Frontal+parietal + Temp.+Th. Olig. III 9 8 French Bulldog F 10 17 PR Parietal+Th. N/A 10 6 French Bulldog M 3 4 N/A Frontal Olig. III Age in years; CAV vaccination in months. CAV, canine adenovirus; F, female; GBM, glioblastoma multiforme; M, male; N/A, not available; Occ., Occipital; Olig. III, oligodendroglioma grade III; PD, progressive disease; PR, partial response; RAVNO, response assessment in veterinary neuro-­oncology; SD, stable disease; Temp., temporal; Th., thalamus. Cloquell A, et al. J Immunother Cancer 2022;10:e005669. doi:10.1136/jitc-2022-005669 5 Open access There were no significant changes in the complete weeks before, which could not be attributed to the blood count during the treatment (online supplemental glioma. The images showed a lesion in the right temporal figure S2A). Total protein, albumin, globulin, glucose, lobe (online supplemental figure S3A, asterisk) and urea, creatinine, Na+, K+ and Ca++ levels also remained bilateral bone resorption (online supplemental figure within the reference ranges during the follow-­up (online 3B,C, arrows) affecting the orbital area of the frontal supplemental figure S2B). However, patient 3 had neutro- bones (online supplemental figure S3D, arrows). Also, philic and monocytic leucocytosis (online supplemental the wings of the presphenoid and basisphenoid bones figure S2A) and increased urea levels (online supple- and the ventral area of the temporal bone were affected mental figure S2B) before treatment, which were progres- (online supplemental figure S3E). The bone resorption sively normalized after the second week of treatment. was more severe ipsilateral to the intracranial tumor. Liver enzymes were analyzed to assess whether the Three dogs subsequently added to the study underwent dCelyvir treatment affected liver function. Alkaline phos- CT study postmortem even if they did not have any clin- phatase (ALP) levels were increased before the treatment ical signs suggesting osteolysis of the cranial bones. Two in five dogs and remained elevated in three of them of them (patients 5 and 7) showed bone resorption. until death (online supplemental figure S2C). Patient Patient 5 showed involvement of the internal lamina of 5 was the only one who also showed increased ALP and the frontal bone adjacent to the tumor (online supple- aspartate transaminase levels during treatment (online mental figure S3F). Patient 7 showed bone resorption supplemental figure S2C). Serum alanine transaminase of the orbital area of the left frontal bone and ethmoid levels were over the baseline in eight dogs before treat- bone (online supplemental figures 3G,H) and bilaterally ment but were reduced or maintained during treatments, in the temporal bones, despite the tumor being in the left except for patient five who showed a slight increment frontal lobe. (online supplemental figure S2C). Most dogs developed neurological complications during treatment (abnormal Clinical outcome mental status, seizures, and changes in behavior), which Seven dogs survived more than 2 months after starting the are normally associated with the presence of the brain treatment, having a second MRI evaluation (figure 1B). tumor and could therefore not be attributed to the treat- Tumor size and midline shift reduction were observed ment itself. in 57% (4/7) and 43% (3/7) of dogs, respectively The patient 4 experienced severe elevation of liver (online supplemental table S1 and figure 2A). The clin- enzymes attributed to medical treatment with steroids ical benefit of these seven dogs was evaluated following and phenobarbital prior to the dCelyvir treatment. The the RAVNO criteria: two dogs presented PD, three SD, blood analysis performed 7 days after the treatment and two PR, showing a reduction of 76% and 86% in showed bilirubinemia, hypoalbuminemia, mild anemia, tumor size (online supplemental table S1, figures 1 and leukocytosis, and increased bile acids. Due to deterio- 2A). The main radiological features of the lesions (type rating clinical signs and liver dysfunction, the dCelyvir of enhancement, peritumoral edema, and mass effect) treatment was discontinued, and the dog was euthanized changed during the follow-­up in two dogs, one of which due to experiencing renal and hepatic failure. The final presented a moderate peritumoral edema prior to treat- histopathological classification was not available because ment that was undetectable after 2 months. The other necropsy was not allowed. peritumoral edema progressed from moderate to severe. Four dogs died naturally and six were euthanized due to Imaging findings clinical deterioration at the owner’s request. The median Before the dCelyvir treatment, all MRI studies showed survival time (MST) was 128 days from clinical onset, 123 the classic radiological features of high-­grade gliomas, days from diagnosis, and 90 days from the start of the including the presence of a single intra-­axial mass mainly dCelyvir treatment (online supplemental table S1 and hypointense in T1-­ weighted images and hyperintense figure 2B,C). The number of dCelyvir doses ranged from in T2-­weighted and FLAIR (online supplemental figure 1 to 17 (median of 8) (table 1). There was no correla- S1). The tumor location varied, affecting multiple lobes tion between survival time (from the start of treatment) (or one lobe and the thalamus) in five dogs (table 1). and tumor size evaluated at pretreatment (figure 2D) Contrast enhancement was severe in a ring-­like pattern or after 2 months of weekly treatment (figure 2E); and in nine dogs; however, the patient 2 did not have any the survival time lacked any correlation with the tumor enhancement (figure 1B). Before treatment, mass areas growth (figure 2F). However, there was a positive correla- ranged from 57 mm2 to 670 mm2 (mean 281 mm2), and tion (r=0.8455) between the number of dCelyvir doses most cases showed mass effect evidenced as ventricular and survival time (figure 2G). collapse (8/10) and/or midline shift (9/10), ranging The final diagnosis was performed by immunohisto- from 0 mm to 5.7 mm (mean 3 mm) (online supple- pathological study in eight dogs after necropsy (two dogs mental table S1). Perilesional edema was also observed in were not available for necropsy). All tumors were diag- most dogs (9/10). nosed as high-­grade gliomas: 6 of them were grade III Patient 3 was also imaged by CT after euthanasia to oligodendrogliomas and two were GBM (grade IV astro- investigate the cause of the jaw pain observed several cytomas) (table 1 and online supplemental figure S4). 6 Cloquell A, et al. J Immunother Cancer 2022;10:e005669. doi:10.1136/jitc-2022-005669 Open access Figure 2 Tumor growth and survival time from the dCelyvir-­treated patients. (A) Waterfall plot showing changes in tumor size. Tumor growth from baseline (first MRI) to 2 months after starting the dCelyvir treatment (second MRI) is shown for each patient. (B) Survival time. Bar plot showing the total survival time for each patient. The days from symptom onset to diagnosis, from diagnosis to starting dCelyvir treatment, and from treatment to death are indicated for each patient. (C) Survival analysis. Kaplan-­Meier curve showing the probability of survival for dCelyvir-­treated dogs (blue line) starting with day of treatment up to death. The dashed red line indicates the MST according to the literature and the historical cases treated with resective surgery. (D–G) Correlation analysis between survival time and tumor size at first MRI (D), at second MRI (E), tumor growth (F), and dCelyvir dose (G) calculated by Pearson’s correlation coefficient (r). Individual values (blue dots) and regression lines are shown. **p<0.005. MST, median survival time; PD, progressive disease; PR, partial response; SD, stable disease. Virus and antiviral antibody detection since the last vaccination) showed the highest virus titer The oncolytic adenovirus ICOCAV17 was detected in the 7 days after the second dose (d14) (figure 3A and table 1); blood of all of the treated dogs that were analyzed (n=9) however, this dog presented a similar anti-­CAV2 antibody (figure 3A). Five of them showed the highest viral particles titer to that of the other dogs that had been vaccinated 2 days after the first treatment, showing a steady reduction more recently (figure 3B). Further, ICOCAV17-­positive until 14 days after the first treatment. Two dogs achieved cells were detected inside gliomas from necropsies in the highest values on the third day after the first doses; the 3 dogs (1, 6, and 10) (figure 3D). All the treated dogs presence of the virus in the blood samples subsequently showed anti-­CAV2 antibodies before the dCelyvir treat- decreased. Lastly, patient 5 (the one with the longest time ment, which increased gradually from the first dose; after Cloquell A, et al. J Immunother Cancer 2022;10:e005669. doi:10.1136/jitc-2022-005669 7 Open access Figure 3 Oncolytic virus and antibody response. (A) Adenovirus detection during follow-­up. Adenovirus presence analyzed by quantitative PCR (qPCR) in peripheral blood is shown. Quantification time refers to the time after the first day of treatment (day 0). Individual values from each patient are shown. (B) Specific antibodies. IgG α-CAV2 quantified by solid phase ELISA in the serum of canine patients during dCelyvir treatments. A Wilcoxon test was performed (***p<0.0001; **p<0.005). (C) Correlation between oncolytic adenovirus and specific antibodies was calculated by Pearson’s correlation coefficient (r), using data from all available dogs up to the second week of treatment (14th day after treatment). Individual values (blue dots) and a regression line are shown. *p<0.05. (D) The presence of adenovirus inside gliomas analyzed by immunohistochemistry. Representative images showing adenovirus-­positive cells (brown) on formalin-­fixed, paraffin-­embedded-­positive control tissue, and tumor necropsies from patients 6 and 10. Scale bars: 50 µm (negative and positive controls), 200 µm (top patients) and 50 µm (bottom patients). the second dose they tended to stabilize over the following Immune response in peripheral blood treatments (figure 3B). Although the dogs had similar Cytokines in peripheral blood were quantified during the antibody trend, they showed greater differences in viral first 4 weeks by analysis with a multiplex cytokine panel to particles detected in blood (figure 3A–B). There was a analyze whether the dCelyvir treatment could be modi- negative correlation (r=−0.4056) between the ICOCAV17 fying the dogs’ cytokine secretion. No alterations in IL-­2, particles in peripheral blood and the specific antibodies IL-­6, IL-­7, IL-­15, IL-­18, GM-­CSF, CCL2, or CXCL10 during against CAV2 (figure 3C). the treatment were detected (figure 4). However, patient 8 Cloquell A, et al. J Immunother Cancer 2022;10:e005669. doi:10.1136/jitc-2022-005669 Open access Figure 4 Cytokines in peripheral blood from treated patients quantified by a multiplex assay. Sera from treated dogs were analyzed prior to treatment (d0) and Weekly after the first four treatments (days 7, 14, 21, and 28) for IL-­2, IL-­6, IL-­7, IL-­15, IL-­ 18, GM-­CSF, CCL2, CXCL10, IL-­8, IL-­10, KC-­like, and IFN-ɣ. Individual values are shown. GM-­CSF, granulocyte-­macrophage colony-­stimulating factor. 7 showed an increase in IL-­ 2, IL6, IL-­ 7, IL-­ 15, IL-­18, tumor-­infiltrating lymphocytes (TILs), which could not be GM-­CSF, and CXCL10 levels after 4 weekly doses. Patient detected in two grade III oligodendrogliomas (table 2). 2 had the highest IL-­2 quantification before starting and In the rest of the dogs, lymphocytic infiltrates were found during the entire treatment. Immune cell populations around glomeruloid vessels forming perivascular cuffs or in peripheral blood were quantified by flow cytometry to as scattered cells within the tumor (online supplemental assess whether the dCelyvir treatment was affecting the figure S4). The most prominent lymphocytic population normal function of the immune system, but no significant was CD3+ TILs, in all tumors (mean of 36.05 cells/mm2) differences in neutrophil, T cell, macrophage, or natural and increased considerably in GBM (118.2 cells/mm2) killer cell counts were observed (data not shown). compared with grade III oligodendrogliomas (8.64 cells/ Tumor-infiltrating immune cells mm2) (figure 5A and table 2). FOXP3+ lymphocytes were Immunohistochemistry was performed to investigate detected in 25% (2/8) of the evaluated samples and were whether the dCelyvir treatment could induce changes in particularly severe in the GBM. A low number of CD20+ Cloquell A, et al. J Immunother Cancer 2022;10:e005669. doi:10.1136/jitc-2022-005669 9 Open access different organs, the use of dCelyvir did not demonstrate Table 2 Inflammatory pattern and quantification of TILs significant pathological changes or clinical signs in the TILs (mean) livers of dogs presenting extracranial solid tumors.6 36 Patient Inflammatory pattern FOXP3+ CD3+ CD20+ Considering these findings with the biochemical abnor- 1 None 0 0 0 malities that patient 4 presented before the dCelyvir 2 None 0 0 0 treatment, an undetected pre-­existing liver disease (toxic 3 Perivascular+intratumoral 0 28 2.4 hepatopathy secondary to phenobarbital and/or steroids, 5 Perivascular+intratumoral 0 4.8 1.6 infectious hepatitis, or diffuse neoplasia) could be the cause of its clinical deterioration. 6 Perivascular+intratumoral 17.6 226 27.2 Radiation therapy has become the gold standard for 7 Perivascular+intratumoral 0 10.4 1.6 treating canine high-­grade glioma, with MSTs from 255 to 8 Perivascular 0 12.8 0.8 282 days; however, it is expensive and there are few hospi- 10 Intratumoral 1.6 6.4 3.2 tals that offer this therapy.26 In dogs, lomustine has shown TILs, tumor-­infiltrating lymphocytes. a benefit in MST from diagnosis when compared with palliative care (138 vs 35 days).37 Interestingly, dCelyvir treatment shows a slight increase in MST from treatment cells was observed (mean of 4.6 cells/mm2). Patient 6 (90 days) when compared with palliatively treated dogs showed the highest number of CD20+ cells, as occurred (35 days)37 or surgically treated dogs.38 We have not with other evaluated lymphocytic populations (figure 5A found an association between MST and tumor volume or and table 2). its localization; however, it is important to note that most To determine whether the intratumor immune micro- of the patients presented large-­sized tumors (>2 cm2). environment could be affected by the dCelyvir treatment, Tumor size was increased after 2 months of treatment a CIBERSORT algorithm that estimated TIL compo- in 3 dogs (3/7) in which revision MRI was performed, sition from bulk RNA expression data was employed which could be due to tumor cell proliferation and/or (figure 5B). The dogs with SD (2/5) and PR (1/5) tumor pseudoprogression, the latter of which has been presented a tendency for reduced memory B-­cell infiltra- observed in humans with recurrent high-­grade gliomas tion and increased B-­cell activation. Similarly, a tendency treated intratumorally with DNX-­2401, a CRAd homol- to increased monocyte/macrophage lineage infiltration ogous to ICOCAV17,12 and in dogs with brain tumors was observed in the dogs with SD and PR (figure 5C). treated with intrathecal Zika virus.39 This phenomenon Interestingly, dogs with osteolytic lesions (3/4) also appeared soon after the start of treatment, followed by a presented changes in TIL composition, with a significant progressive reduction in tumor size; however, the dogs’ reduction in memory B cells and a higher infiltration of short survival time did not allow us to confirm that possi- the monocyte/macrophage lineage (figure 5D). bility in this study. Unfortunately, given that there are no consensus criteria for the analysis of immunotherapies in veterinary medicine, RAVNO criteria have been used DISCUSSION even though they do not consider pseudoprogressions In this study, 9/10 dogs were brachycephalic according to as do the immune-­specific response criteria in human other reports that describe the predisposition of brachy- clinical practice, the Immune-­ related Response Evalu- cephalic dog breeds to gliomas.20 24 26 The median time ation Criteria in Solid Tumors and the Immune-­related from the onset of clinical symptoms to diagnosis (40 response criteria. days) agreed with previously reported values, as did the Severe skull bone resorption (calvarial erosion) was imaging characteristics.27 35 Therefore, our study popula- detected in 3 dogs (3/4) examined with CT soon before tion should be considered as a representative sample of or after death. This event has been previously reported in spontaneous high-­grade glioma in dogs. dogs, although is an uncommon finding in humans with The dCelyvir treatment was well tolerated, as we had brain tumors.40 41 The proposed mechanisms are direct previously reported with extracranial solid tumors.6 Only invasion of tumor cells into the skull, tumor pressure on minor adverse effects (limb swelling and fatigue) were the inner table of the skull, and the destruction caused sporadically observed and were self-­limiting or required by proteolytic enzymes secreted by the tumor cells.41 short-­term anti-­inflammatory treatment. There were no Calvarial erosion was found to be near the neoplasia but significant changes in the complete blood count, and also distally and bilaterally (patients 3 and 7). There is biochemical parameters were stable within the refer- the clinical impression that skull bone resorption in dogs enced ranges. The finding of adenovirus inside the with long-­standing gliomas is much more frequent than tumors is evidence that dMSCs are effective in delivering that reported to date in the veterinary literature. of ICOCAV17 into gliomas; but due to the strong hepatot- E1A-­mutant human Ads demonstrated potent cytolytic ropism of Ads, ICOCAV17 could have accumulated in the effects in human gliomas42 43 and canine Ads in extracra- liver, as previously reported with dCelyvir treatment.6 36 nial solid tumors, either administered intratumorally13 18 or Despite the prevalence of microscopic lesions, especially carried by dMSCs, with higher clinical benefit.6 Although previously reported chronic inflammatory responses in dCelyvir has been reported to reach an extracranial solid 10 Cloquell A, et al. J Immunother Cancer 2022;10:e005669. doi:10.1136/jitc-2022-005669 Open access Figure 5 Intratumor immune microenvironment characterization after dCelyvir treatment. (A) The presence of immune cells inside gliomas analyzed by immunohistochemistry. Representative images showing immune cells (brown) on formalin-­fixed, paraffin-­embedded tumors at necropsies. Scale bars: 50 µm. (B) Relative fraction of 11 types of TILs in 6 dogs estimated with CIBERSORT. (C) Immune cell relative fraction from patients depending on their clinical benefit, after dCelyvir treatment. (D) Immune cell relative fraction from patients, comparing those that present an osteolytic pattern at CT imaging (osteolytic) and those that do not (not osteolytic). Statistic test: Two-­way ANOVA and Bonferroni’s multiple comparison test; Alpha: 0.05; ns: not significant; *p<0.05. ANOVA, analysis of variance; DC, dendritic cells; Eos, Eosinophils; Mono/MP, Monocyte/Macrophages; PD, progressive disease; PMN, polymorphonuclear cells; PR, partial response; SD, stable disease; TILs, tumor-­infiltrating lymphocytes. tumor in a canine patient,44 this is the first time that system- (or other organs); or (4) by the large tumor size at the time ically administered viral treatment has been shown to effec- of inclusion in the study.45 46 tively reach the brain tumor and show biological activity, a All dogs presented an increase in IgG α-CAV-­2 until fact of vital importance for the efficacy of the therapy. Despite reaching a plateau after the second dCelyvir dose, in agree- that, the limited efficacy of dCelyvir in treating high-­grade ment with previous studies.6 13 The increase of the anti- gliomas could have been due to (1) the specific character- body levels did not preclude a clinical benefit in this study; istics of the immune system in the CNS; (2) the fact that the there was a correlation between antiviral antibodies and viral load reaching the tumor was reduced by the blood– ICOCAV17 particles, suggesting that a lower concentration brain barrier; (3) due to the MSC sequestration in the lungs of antibodies could reduce the number of neutralized viral Cloquell A, et al. J Immunother Cancer 2022;10:e005669. doi:10.1136/jitc-2022-005669 11 Open access 7 particles, thereby allowing a higher oncolytic viral load. The Unidad de Investigación Biomédica (UIB-­UAX), Universidad Alfonso X el Sabio, use of an OV, against which the dogs have not been previ- Villanueva de la Cañada, Spain ously vaccinated, should be considered in the future to favor Twitter Stefano Gambera @GamberaStefano and Javier García-­Castro @ dCelyvir effectiveness. JavierGC280 The absent systemic inflammatory response observed in Acknowledgements The authors would like to thank Miguel Ángel Rodríguez-­ the cytokine profile in most of the dogs could indicate that Milla, Isabel Cubillo and Álvaro Morales from Unidad de Biotecnología Celular, ICOCAV17 did not elicit an exaggerated systemic response, in Instituto de Salud Carlos III for their technical support in the study. The authors contrast to other intrathecally injected viruses that increased would also like to thank Dolors Pi, Ester Blasco and Tamara Rivero from Unitat IL-­8, keratinocyte chemoattractant (KC)-­ like, and CCL2 de Patologia Murina i Comparada (UPMiC) at Universitat Autònoma de Barcelona, and for their technical support with the diagnosis and immunohistochemistry. The in the serum of dogs with malignant intracranial tumors.39 authors would also like to thank Pablo Delgado-­Bonet from Universidad Alfonso X However, the intratumoral immune cells analysis of our treat- el Sabio for his help with the IHC images. The graphical abstract and figure 1 have ment suggested that changes in TILs could be relevant in been designed using images from ​Freepik.​com, which have free license. treatment response. The glioma composition of TILs in our Contributors Conception and design: JG-­C and AJP-­B; Development of tools treated dogs was according to that reported in non-­treated and methodology: AC, IM, RA, and AJP-­B; Acquisition of data (provided animals, managed patients, diagnosis and provided facilities): AC, IM, MP, JG-­C; Analysis dogs with gliomas, consisting in higher population of CD3+ and interpretation of data (imaging, statistical analysis and computational analysis): and FoxP3+, compared with CD20+ TILs. Also, the number IM, SG, MP, JG-­C and AJP-­B; Writing the paper: AC, IM, SG and AJP-­B; Review the of TILs were increased in GBM compared with lower grade paper: AC, IM, SG, MP, RA, JG-­C and AJP-­B. Guarantor: JG-­C gliomas.19 However, dogs showing a clinical benefit such as Funding This work was funded by Instituto de Salud Carlos III (PI14CIII/00005 SD or PR present a tendency for reduced memory B-­cell and PI17CIII/00013 grants to JG-­C), Consejeria de Educación, Juventud y Deporte infiltration and increased activated B cells and monocyte/ of Comunidad de Madrid (P2017/BMD-­3692 grant to JG-­C), the Madrid Regional Government (CellCAM; P2010/BMD-­2420 to JG-­C), the Asociación Pablo Ugarte, macrophage-­lineage cells infiltrated. It has now been proven and the Asociación AFANION. This work was also supported by Universidad Alfonso that the tumor-­infiltrating T lymphocytes play a critical role X el Sabio and Santander Universidades-­Fundación Universidad Alfonso X el Sabio in controlling or promoting tumor growth, but the function (1010909 grant to AJP-­B). of B lymphocytes is less clear, although recent data have indi- Competing interests None declared. cated a critical role for tumor-­infiltrating B-­cells in immu- Patient consent for publication Not applicable. notherapies.47 Changes in TIL populations have also been Provenance and peer review Not commissioned; externally peer reviewed. observed in dogs with osteolytic lesions. However, osteoclasts Data availability statement All data relevant to the study are included in the can have a monocytic-­macrophagic derivation,48–50 and dogs article or uploaded online as supplemental information. Not applicable. presenting an osteolytic pattern showed an increased rela- Supplemental material This content has been supplied by the author(s). It has tive fraction of this lineage. Thus, further studies should be not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been performed to clarify these relationships. peer-­reviewed. Any opinions or recommendations discussed are solely those In this study, dMSCs infected with a canine OV to treat of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and high-­grade gliomas in dogs were used without significant responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability toxicities or adverse reactions. The increased MST compared of the translations (including but not limited to local regulations, clinical guidelines, with previous reports describing the prognosis of palliative terminology, drug names and drug dosages), and is not responsible for any error treatment or surgery makes dCelyvir a candidate for combina- and/or omissions arising from translation and adaptation or otherwise. tion therapy with surgery or different administration routes. Open access This is an open access article distributed in accordance with the These findings could be relevant for clinical trials being Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-­commercially, developed in humans, which are similar to this study, such and license their derivative works on different terms, provided the original work is as the phase Ib clinical trial using this cellular virotherapy in properly cited, appropriate credit is given, any changes made indicated, and the use children, adolescents, and young adults with diffuse intrinsic is non-­commercial. See http://creativecommons.org/licenses/by-nc/4.0/. pontine glioma (EudraCT 2020-­004838-­37) and the trial with ORCID iDs the oncolytic adenovirus DNX-­2401 in patients with recur- Ana Cloquell http://orcid.org/0000-0002-1400-6134 rent high-­grade glioma (NCT03896568). The reported data Isidro Mateo http://orcid.org/0000-0002-2667-9459 using dogs as a preclinical model could serve as a transition Stefano Gambera http://orcid.org/0000-0003-2998-8502 in the therapeutic development of human medicine and can Javier García-­Castro http://orcid.org/0000-0001-7604-1640 Ana Judith Perisé-Barrios http://orcid.org/0000-0002-0136-3968 potentially improve the success of subsequent clinical trials. Author affiliations 1 Servicio de Neurología, Hospital Clínico Veterinario, Universidad Alfonso X el Sabio, Villanueva de la Cañada, Spain 2 Servicio de Neurología, Hospital Veterinario VETSIA, Leganés, Spain REFERENCES 3 1 Sánchez D, Cesarman-­Maus G, Amador-­Molina A, et al. Oncolytic Unidad de Biotecnología Celular, Instituto de Salud Carlos III, Madrid, Spain viruses for canine cancer treatment. Cancers 2018;10:404. 4 Molecular Genetics of Angiogenesis Group, Spanish National Centre for 2 Russell L, Peng KW, Russell SJ, et al. 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