Phase 2 Trial of Oncolytic H-1 Parvovirus Therapy Shows Safety and Signs of Immune System Activation in Patients With Metastatic Pancreatic Ductal Adenocarcinoma

According to epidemiologic estimations for 40 European countries, the overall incidence of pancreatic ductal adenocarcinoma (PDAC) in the year 2012 amounted to approximately 10.5 cases per 100,000 inhabitants (1). The figures for mortality were basically equal with 10.1 cases per 100,000, indicating the limited treatment options for this disease (1, 2). Unlike in other neoplasms, the apparent mortality from PDAC has increased gradually in the past decades and was approximately 20% to 30% higher in 2014 than in 1970, possibly due to an improvement of diagnostic procedures (3). The disease is typically diagnosed at advanced stages, where no curative resection can be performed and thus, the majority of patients are treated with palliative chemotherapy (4–6). Modest advances have been made in the treatment of PDAC over the few last decades. Even with the most effective chemotherapy protocol FOLFIRINOX, median survival in stage IV patients is only 11.1 months (5). A combination of gemcitabine and nab-paclitaxel results in a prolongation of median survival to 8.5 months as compared with 6.7 months under monotherapy with gemcitabine (6). Immunotherapy is now an important treatment option in many types of cancer but has been largely unsuccessful in PDAC (7).

Oncolytic viruses (OVs) are considered a promising cancer treatment modality due to their dual capacity to both killing tumor cells and inducing anticancer immunity (8, 9). OVs are indeed able to cause an immunogenic type of tumor cell death and “warm up” an immunologically “cold” tumor microenvironment (TME) through the induction of local and systemic anticancer immune responses. Numerous clinical trials with different OVs have been carried out in various oncological indications. In 2015, the first oncolytic virus immunotherapy using talimogene laherparepvec (T-VEC) was approved by the FDA and EMA for late-stage melanoma. OVs have raised substantial interest as therapeutic agents in particular against PDAC. The first OVs tested against PDAC belonged to the Adenoviridae and Herpesviridae virus families. The E1B 55-kDa gene-deleted, restricted replication-competent adenoviruses ONYX-015 and AxE1Adb, have demonstrated antitumor effects in both animal (10) and clinical studies (11). Another early preclinical work described the efficiency of hrR3, a replication-competent ICP6 gene-deleted herpesvirus, in suppressing peritoneally disseminated PDAC in mice (12). On the basis of these pioneering observations, the consideration of oncolytic virotherapy as a novel strategy against pancreatic cancer aroused a further surge of interest in recent years when it was realized that oncolytic virotherapy represents a novel type of immunotherapy. This is especially promising in light of the physical and immunologic barriers posed by the PDAC TME to other systemic therapeutic agents. By now, various OVs, including oncolytic adeno-, herpes-, reo-, vaccinia-, measles-, and parvo-viruses have been investigated in PDAC preclinical and clinical studies (13).

A promising candidate for oncolytic immunotherapy is the rodent protoparvovirus H-1 (H-1PV). H-1PV is a small nonenveloped single-stranded DNA virus (14) whose natural host is the rat (15). Humans are not naturally infected with the virus and therefore lack neutralizing antibodies (16). Parvovirus oncoselectivity is a complex phenomenon based on multiple cancer cell–specific molecular determinants, which are underrepresented in nonmalignant cells. These include the availability of cellular replication/transcription factors and the activation of metabolic pathways in neoplastic cells, which provide an intracellular milieu that is particularly permissive for the parvovirus life cycle (17, 18).

The oncosuppressive activity of H-1PV was demonstrated in several preclinical investigations in PDAC and several other preclinical tumor models (19, 20). In animal models, cellular immune responses have been found to potentiate the oncosuppressive effect of H-1PV (15). Furthermore, synergistic therapeutic effects could be demonstrated in combination with gemcitabine in an immunocompetent orthotopic rat PDAC model (21).

The first-in-man trial with H-1PV (ParvOryx01) investigated safety, tolerability, and antitumor activity of escalating virus doses in patients with recurrent glioblastoma (22). It showed safety and good tolerability of H-1PV with no detection of dose-limiting toxicities (DLTs). Moreover, favorable pharmacokinetics, dose-dependent induction of virus-specific antibodies, and virus-specific T-cell responses, as well as evidence of virus replication and some hints for improvement of clinical outcome were observed (23).

The current ParvOryx02 trial is a noncontrolled, single-arm, open-label, phase II study of intravenous and intratumoral administration of ParvOryx in patients with metastatic, inoperable PDAC (24). The primary objectives of this approach were to test safety and feasibility, assessment of humoral immune response to ParvOryx, and investigation of virus tumor homing and shedding of the viral agent, but also its ability to boost inflammatory status of these rather immunologically cold tumors. Specifically, we evaluated changes in the effector cell repertoire and cytokine profile in patients with objective tumor responses versus nonresponders. ParvOryx02 met all primary objectives. Thus, the study paves the way for future clinical investigation.

Eligible were adult patients with stage IV, histologically confirmed PDAC, and at least one hepatic metastasis. Patients had previously progressed on first-line therapy and were eligible for second-line treatment with gemcitabine. They had radiologically measurable tumor according to RECIST 1.1, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, an adequate bone marrow, liver, and renal function, and a normal thyroid function. Patients were excluded if they were eligible for surgical treatment, had symptomatic cerebral, pulmonary, or osseous metastases; had relevant peritoneal carcinomatosis, liver cirrhosis, respiratory impairment, or any signs of active, systemic infection. Recent chemotherapy, radiotherapy, or hospitalization due to a reason other than PDAC also led to patient exclusion.

Prior to enrolment, all patients provided written informed consent. ParvOryx02 was registered with EudraCT (2015–001119–11) and ClinicalTrials.gov (NCT02653313). It was conducted according to the principles of the Declaration of Helsinki. Approvals by the German competent authority (PEI) and the Ethics Committee of the Medical Faculty Heidelberg were obtained.

ParvOryx02 was a noncontrolled, single-arm, open-label, dose-escalating, single-center trial with a GMP-grade pharmaceutical formulation of H-1PV (ParvOryx). Its design, as shown in Fig. 1, has already been reported (24). The primary objective was to investigate safety, tolerability, immunogenicity, PK, and shedding of the escalating doses of H-1PV. The secondary objectives included a range of accompanying evaluations of tumor tissue and immunological patterns in blood to explore tumor suppressing properties of the virus. The clinical response was assessed by means of progression-free survival (PFS) and overall survival (OS).

Each patient was assigned to one of three sequentially escalating total dose levels, that is, 1E09 pfu (N = 1), 5E09 pfu (N = 3), and 1E10 pfu (N = 3; see Fig. 1). The sequential patient inclusion had to stop in case of any unacceptable treatment toxicity (DLT). DLT was defined as any Common Terminology Criteria for Adverse Events (CTCAE) grade 4, or a CTCAE grade 3 hepatic, neurological, or cardiovascular treatment-related toxicity, or a relevant increase/decrease of blood neutrophil count. Because no organ-specific toxicities had been observed in previous preclinical and clinical studies, no formal definition of the MTD was implemented.

Eligible patients received H-1PV on an in-patient basis. The initial administrations of the virus were performed by 2-hour intravenous infusions, given in four equal fractions of 10% of the total dose on 4 consecutive days. Between 6 and 13 days later, a fraction of 60% of the total dose was administered as a single, ultrasound-guided injection into a hepatic metastasis of PDAC. The exact timing of intrametastatic injection was defined for each patient prior to the start of the trial and thus it depended on the timing of the individual study inclusion. Patients were monitored closely, partly in-patient, until 28 days after the first administration of H-1PV. In case of a DLT or a substantial delay in administration sequence of the virus, the individual treatment with H-1PV was to be discontinued. The principal investigator could also decide to discontinue individual patient treatment if he considered it medically imperative. Patients who withdrew from the trial for a reason other than treatment toxicity were to be replaced. If they consented, they were followed up until the end of the regular trial. On day 28, a treatment with gemcitabine was commenced. If in the course of the study a disease progression occurred, an additional treatment with nab-paclitaxel had to be introduced. After day 28, the follow-up examinations were performed at months 2, 4, and 6. If applicable, patients were asked to consent in a long-term follow-up for survival after completion of their regular trial participation. A Data Safety Monitoring Board (DSMB) regularly reviewed safety data and gave recommendations on trial continuation.

Study-specific hygienic measures included in-patient stay until the first detection of H-1PV specific antibodies or until viral genomes (Vg) were no longer detectable in feces, urine, and saliva. Patients should entirely avoid contact with pregnant women and infants for 2 months after the first administration of the virus. On the basis of the findings from the ParvOryx01 trial, no patient isolation was considered necessary in this study.

Investigation of safety and tolerability, along with determination of antidrug antibodies (ADAs), pharmacokinetics, and shedding of Vg, was the primary objective of the trial. Safety and tolerability were assessed on the basis of adverse events (AEs), including serious adverse events (SAEs), vital signs, 12-lead electrocardiograms (ECGs), and blood evaluations. The main characteristics of AEs/SAEs, that is, severity, causality, countermeasures, and outcome, were documented from the first administration of H-1PV throughout the trial. The severity of each AE was graded according to the CTCAE version 4.0. Vital signs (pulse rate, systolic and diastolic blood pressure, and body temperature), pathologic findings in ECG, and blood laboratory parameters were determined at pre-specified study visit and at any time if required for clarification of changes in a patient's clinical condition. Blood evaluations included clinical chemistry, hematology, and coagulation parameters.

Blood, urine, saliva, and feces were collected at each study visit. In addition, thorough PK investigations (PK profiles) were carried out once in each patient at varying study times, that is, either after the last intravenous or after the intrametastatic administration. Blood, urine, and feces DNA were extracted with commercial kits (Qiagen or Epicentre) and saliva DNA was extracted with Quick Extract DNA extraction solution 1.0 (Lucigen). DNA extracted from each matrix was analyzed by qPCR. The assay was validated and performed by Eurofins GmbH in a LightCycler 480 (Roche). The methodology was previously described (23). The lower limits of quantification (LLOQ) were 40 Vg/μL for blood, 20 ± 2 Vg/mg for feces, 8.57 Vg/μL for urine, and 9E04 Vg/swab for saliva.

In addition, measurements of infectious H-1PV particles were performed by plaque assay in all shedding samples. Samples were diluted in MEM media and virus release was performed with three thaw cycles followed by a 48 W sonication for 1 minute. Feces, urine, and saliva samples were filtered through a 0.2-μm cellulose acetate filter. The plaque assay method was described previously (25).

Recovery of quality control (QC) had to be ≥20% for blood, ≥10% for urine and feces, and ≥5% for saliva. To detect as low as 1 pfu/200 μL H-1PV in blood, 50 pfu/g in feces, 5 pfu/mL in urine, and 1 pfu/swab in saliva, samples were propagated on rat glioma cells (RG2). Infectious H-1PV as well as positive control (PC), QC spiked with a specific amount of H-1PV, and QC-NC were detected after three rounds of H-1PV propagation (each round for 5 days) on RG2 cells via DNA hybridization assay and qPCR as described previously (26).

Blood for determination of ADA was sampled at each study visit. To obtain results on ADA titers on short notice, they were measured by inhibition of virus-mediated hemagglutination (HI assay) in the course of the trial. Antibody titers were determined as the highest serum dilution causing complete inhibition of hemagglutination.

After the end of the trial, a neutralizing antibody assay was additionally performed. Virus-specific neutralizing antibodies were detected by measuring the ability of serum to inhibit lytic infection of permissive NB-324K cells by H-1PV. Cell viability was determined by crystal violet staining and photometric quantification of residual cells 4 days after infectionem (0.16 pfu/cell) in the presence and absence of serially diluted patient serum. Dose–response curves were calculated. The neutralizing antibody titer was defined as the serum dilution causing 50% inhibition of virus-induced toxicity. This was performed according to a previously described methodology (23).

Blood was drawn in a whole-blood syringe at screening, on study day 28 and at all follow-up visits to assess the cellular immune response to H-1PV by FACS. Blood was immediately aliquoted and stored at −80°C until further processing. Relative and absolute cell counts of selected cell subsets were determined by using four-color-fluorescence flow cytometry and commercially available antibodies. The following cell subpopulations were differentiated: CD45⁺, CD45⁺CD3⁺CD16⁻CD56⁻CD19⁻, CD45⁺CD3⁻CD16⁺CD56⁺CD19⁻, CD45⁺CD3⁻CD16⁻CD56⁻CD19⁺, CD45⁺CD3⁺CD4⁺CD8⁻, CD45⁺CD3⁺CD4⁻CD8⁺, CD45⁺CD3⁺CD4⁺DR⁺, CD45⁺CD3⁺CD8⁺DR⁺, CD45⁺CD3⁺DR⁺, CD45⁺CD3⁺CD25⁺, CD4⁺CD25⁺CD127⁻, and CD4⁺CD25⁺CD127⁻Foxp3⁺.

Blood was collected and peripheral blood mononuclear cells (PBMCs) were freshly prepared at screening, on study day 28, and at all follow-up visits to detect the immune response against H-1PV (23). The assay was carried out by Lophius Biosciences GmbH on the basis of a previously described method (27).

Three timely subsequent tumor biopsies of a hepatic metastasis were obtained from each patient at (i) screening, (ii) between study day 7 and 14, and (iii) either on study day 28 or at month 2 visit. Dependent on the tumor content, the following sequence of investigations was carried out: (i) general pathological evaluations, (ii) FISH for virus mRNA, (iii) qPCR for H-1PV DNA and mRNA detection, (iv) assessment of tumor infiltration with immune cells and determination of the content of cytokines and chemokines, and (v) detection of infectious H-1PV.

Each liver biopsy was evaluated by frozen section to determine tumor cell content (tumor cellularity). Subsequently, biopsies were fixed with formalin and embedded in paraffin (FFPE). In addition, cryomaterial was stored and provided for nucleic acid extraction, if possible. FFPE biopsies were processed for haematoxylin–eosin staining to assess the amount of tumor necrosis. In addition, the TUNEL assay for evaluation of apoptotic cells (late stage) and Ki67 staining for assessment of the proliferative tumor compartment were performed.

Viral RNA transcripts in the paraffin-embedded tumor were detected via FISH analysis. The applied methodology was already reported in detail (23).

Viral RNA and DNA in paraffin-embedded tumor were detected via qPCR. The applied methodology was already reported in detail (23).

Frozen tumor tissue was homogenized and potential virus was propagated for three rounds on RG2 cells. Virus was detected afterwards via DNA hybridization assay and qPCR (23, 26).

Biopsy samples were stained by IHC and analyzed by automated whole-slide microscopy to dissect patterns of tumor-infiltrating immune cells. Using laser capture microdissection, cytokine, and chemokine profiling of central tumor tissue and the invasive margin were performed to gain insight into immune signatures associated with response. Tumor-infiltrating T cells from biopsies as well as T cells from the PBMC fraction were enriched by magnetic cell sorting.

Tumor response, assessed by the revised RECIST 1.1. criteria (28), and CA19–9 serum levels were determined at screening, on study day 28, and at all follow-up visits. The survival status was documented at each study visit up to the end of the regular trial participation. Where applicable, after obtaining patient's consent, survival status was followed up beyond the regular end of the trial.

Because of the exploratory approach of the current trial, no statistical sample size calculation was performed. Demography, adverse events, findings in physical examinations, and ECG were listed and tabulated, including breakdowns by system organ class (SOC) and preferred term (PT) of the Medical Dictionary for Regulatory Activities (MedDRA). Continuous variables were tabulated by basic descriptive statistics, where appropriate. PFS and OS were listed and displayed as Kaplan–Meier plots. Other study variables were listed and/or tabulated as appropriate. No hypothesis testing was performed.

From February 2016 to January 2018, nine patients were screened and seven entered the trial (see Fig. 1). One patient discontinued the trial prematurely at her own wish after having received all doses of H-1PV. She was not eligible for assessment of treatment response (see Supplementary Table S1) but she consented to a follow-up for survival. The mean age at screening was 55.1 ± 10.6 (35–69) years [±SD (range)]. All patients received at least two cycles of FOLFIRINOX chemotherapy prior to the study inclusion and had progressive disease. Two patients had a history of surgery for PDAC (see Supplementary Table S2). The mean CA19–9 serum level was 3,354 ± 5,144 (17–12,182) U/mL. As required per protocol, at least one liver metastasis amenable to percutaneous puncture and injection of H-1PV was identified in each patient. All enrolled patients received all administrations of H-1PV according to the study protocol (see Fig. 1). There were no major protocol deviations that might have influenced the study results.

No DLTs were seen at any investigated dose level. Overall, 91 AEs were documented in the trial, with no dose dependency. Only two AEs, slightly and moderately increased C-reactive protein, were rated as possibly related to the H-1PV treatment. All six SAEs observed in the trial were attributable to PDAC and thus assessed as not related to the study treatment (see Supplementary Table S3). H-1PV had no impact on safety laboratory parameters, vital signs, including body temperature or ECG.

Median PFS was 72 days and median OS was 175 days (see Supplementary Fig. S1). Two patients showed partial response (PR) according to RECIST criteria; patient No. 4 at month 2 with PR continuing up to the last evaluation at month 6 (confirmed PR) and patient No. 6, who had stable disease (SD) at months 2 and 4 but developed PR at the last evaluation at month 6 (nonconfirmed PR, because no further evaluations were performed), see Supplementary Table S1. Because no response evaluations according to RECIST were performed beyond month 6, the proven PFS was 177 and 176 days for patient Nos. 4 and 6, respectively. Both patients gave their consent for a survival follow-up after their regular end of the trial. They survived 326 days (patient No. 4) and 555 days (patient No. 6). CA19–9 levels rose in most patients in the course of the trial and only in part correlated with radiologic responses (see Supplementary Fig. S2).

Concentrations of Vg and infectious particles were measured to evaluate systemic virus availability and the extent of virus excretion in body fluids.

Blood concentrations of Vg were discernible for the three investigated escalating dose levels (see Fig. 2). The duration of Vg detection in blood was slightly dose-dependent. The maximum Vg concentrations were elevated by approximately one order of magnitude after rapid intrametastatic injection as compared with the foregoing 2-hour intravenous infusions. However, within 30 minutes after intrametastatic injection, blood concentrations dropped to the range detected 30 minutes after the end of intravenous infusions. Several hours after administration by either route, blood concentrations of Vg decreased by approximately two orders of magnitude and remained at this level for days. In general, blood concentrations of infectious virus were dose-dependent after intravenous administrations (see Fig. 2). The duration of their detectability strongly correlated with the appearance of ADA (proven to have neutralizing properties) in blood. Only in one patient, relevant levels of infectious virus were measured in blood after intrametastatic administration. This patient was the only one in whom no ADA occurred prior to the intrametastatic administration (patient No. 6; see Fig. 2).

Vg were found in feces of each patient (see Supplementary Fig. S3), with dose-dependent concentrations after intravenous administrations. The concentrations of Vg in feces after intrametastatic injections apparently depended on the amount of virus particles, which directly entered the biliary tract during the administration procedure. Infectious virus particles were detected in five patients. In four patients, the concentrations were just above the border level of LLOQ (after H-1PV propagation); in one patient, they reached a maximum of 3,100 pfu/g feces on day 12, again probably due to a direct passage of virus to the biliary system during administration. Only in one patient substantial concentrations of Vg in urine were found (see Supplementary Fig. S4). This was clearly due to a blood contamination of the concerned urine samples. No infectious virus particles were found in any of the other urine samples. Only two saliva samples showed traces of Vg, with no infectious virus detected in any sample (see Supplementary Fig. S4).

All patients developed (neutralizing) ADA shortly after beginning of the administration of H-1PV with increasing levels in the course of the trial (see Fig. 2).

A specific T-cell response against H-1PV virion proteins was shown in all six and anti-NS response in three of six patients in whom respective investigations were performed (see Supplementary Table S4).

CD3 staining showed a pronounced increase of T-cell density in the tumor specimen of one of the treatment responders (patient No. 4) after local administration of the virus (see Fig. 3).

The results of analyses for H-1PV nucleic acids (DNA, RNA) are shown in Table 1. H-1PV DNA and RNA transcripts were detected by qPCR in three patients treated with different dose levels after intravenous and in part after intratumoral administrations. FISH analysis showed a weak signal for RNA transcripts in one additional patient. Infectious virus was detected only after intravenous administrations, in three patients treated with the intermediate and highest dose level, respectively.

A diagnosis of PDAC could be confirmed histologically in each patient. Other pathologic and immunologic investigations were limited by the small amount of tumor material.

An increased area of necrosis was seen in five patients following the treatment with H-1PV. However, no changes in the proliferative activity of the tumor cells (staining for Ki-67) or in the percentage of apoptotic cells could be systematically detected.

All investigated patients showed a similar course of the proinflammatory and promigratory molecules IL1ra and CXCL9, respectively (see Fig. 4). An interesting immunological pattern was apparent, mainly in one of the two patients who showed PR after the virus treatment (patient No. 4). The proinflammatory interleukins IL8, IL9, and IL12 and the interferons INFα2 and INFγ showed a substantial increase after the treatment with H-1PV (see Fig. 4). In another patient with PR (patient No. 6), the available data indicated a similar tendency; however, because no biopsy material was available at screening, the course of the immunologic parameters cannot be entirely evaluated. The immunologic pattern described above was not observed in any of the other patients.

The immunologic findings observed in the tumor biopsies of both patients with PR were accompanied by some interesting immunologic observations in blood. Neutrophil-to-lymphocyte (NLR), platelet-to-lymphocyte (PLR), and CD4⁺/CD8⁺ ratio, all postulated as immunologic prognostic factors for PDAC and other malignancies, showed a unique course in both concerned patients (see Fig. 5A). Prior to treatment initiation, NLR and PLR assumed the lowest values in both patients. In contrast, CD4⁺/CD8⁺ ratios were at the highest level as compared with other patients. All three parameters remained at approximately constant levels during the course of the trial in both patients with PR.

In addition to the three parameters described above, another immunologic feature observed at screening in both patients with the favorable clinical outcome deserves some attention. Both patients had relatively low counts of activated T lymphocytes, as evaluated by HLA-DR expression. The ratios of HLA-DR expressing CD3⁺, CD4⁺, and CD8⁺ to the entire population of the respective cell lineages are plotted in Fig. 5B. Furthermore, the blood count of CD4⁺CD25⁺CD127⁻Foxp3⁺ regulatory T lymphocytes was low at the beginning of the trial in both patients (see Fig. 5C). After administration of H-1PV, the fraction of activated CD8⁺HLA-DR⁺ cytotoxic T lymphocytes increased toward day 28 but again decreased thereafter (see Fig. 5B). The levels of regulatory T lymphocytes in patient No. 4 and in patient No. 6 developed in an opposite direction, that is, they decreased or remained constant at day 28 but showed a tendency to increase thereafter (see Fig. 5C). The contrasting course of cytotoxic and regulatory T lymphocytes in both patients with PR may be best illustrated by plotting the CD8⁺HLA⁻DR⁺/CD4⁺CD25⁺CD127⁻Foxp3⁺ ratios over time (see Fig. 5C).

Metastatic PDAC has a dismal prognosis with a median overall survival of less than 1 year. Approved chemotherapy regimens including the gold standard FOLFIRINOX do not deliver long-term responses in the majority of patients. The poster child of cancer immunotherapy, immune checkpoint inhibitors, are not effective in PDAC with response rates around 0% (7). Targeted therapy is only rarely possible, for example, in patients with MSI-high tumors, germline BRCA mutations, or rare oncogenic fusions like NTRK. However, for the majority of patients, there are currently very limited treatment options (4).

Oncolytic viruses (OVs) represent a unique class of anticancer agents, which preferentially infect and lyse cancer cells. Thereby, the virus drug can induce an antitumor immune response involving co-presentation of both viral and tumor antigens in an OV-induced inflammatory environment (29). These changes can lead to an increased number of activated effector cells in peripheral blood and at the tumor site, ultimately causing significant response and disease control rates (30).

The current ParvOryx02 trial met the primary endpoints safety and feasibility. A combination of sequential intravenous and intralesional virus administration offered a possibility to explore viral tropism and tumor homing after intravenous application. Moreover, tissue effects could be separately observed after systemic and local administration. In addition, it allowed to maximize the local availability of the booster treatment by preventing virus neutralization in the systemic circulation. Administration of the viral agent was tolerated very well without any associated clinical findings. The only adverse reaction was a transiently elevated serum CRP level in four of seven patients.

Safety was further validated by extensive viral shedding analyses. As expected, detection of viral genomes in blood, urine, saliva, and feces after intravenous administration was dose-dependent (e.g., >50 days in blood after intravenous injection). However, infectious viral particles could be detected over a period of 3 to 8 days in blood and ended with the appearance of neutralizing antibodies (ADAs) regardless of the administered dose. After injections into liver metastases, infectious particles were detected in feces in five of seven patients. In samples from one patient, higher amounts of up to 3,100 pfu/g were determined at days 11 and 12, which was most likely due to the direct passage of viral particles to the biliary tract during intralesional injections on day 10. No infectious viral particles were detectable in urine or saliva samples at any time. Altogether, these clinical investigations and shedding data attest an excellent safety profile of ParvOryx.

In addition, viral tumor homing after systemic administration was confirmed in four of seven patients by detection of H-1PV nucleic acids. In general, these assays were constrained by a small amount of biopsy material. However, viral detection was successfully achieved in patients of all dose levels (intravenous infusions in total in a range of 4 × 10E8 to 4 × 10E9 PFU).

In this trial, two of seven patients had a partial remission (one confirmed, one unconfirmed according to RECIST criteria), which was associated with a specific antitumor immune signature. The two patients with PR showed an initial increase in the number of activated CD4⁺ and CD8⁺ T cells in blood. Accordingly, changes toward a proinflammatory cytokine profile within the tumor microenvironment could be observed. This included increased levels of IL8, IL9, IL12, and IFNγ. Also, promigratory cytokines like CXCL9 were increased significantly after viral treatment. Interestingly, the Teff/Treg ratio in blood significantly increased in the early treatment phase (up to day 28) in patients with PR only. Moreover, T-cell density in the tumor increased more than twofold in one of the clinical responders. However, due to the limited number of subjects in this study, it remains unclear whether there is in fact a significant correlation between higher previrotherapy T-cell density or increase in T cells after virotherapy and response to the treatment. Further studies with a tailored translational research program aiming to identify immunologic and molecular signatures associated with clinical responses are highly warranted to confirm these findings.

In other studies, it could be demonstrated that OVs can synergize with, for example, anti–PD-1 immunotherapy due to beneficial remodeling of the tumor microenvironment. These changes ultimately resulted in increased antitumor activity and high response rates in early clinical trials (31).

Thus, future trials with ParvOryx will aim to combine the viral agent with clinically approved immunomodulators including checkpoint inhibitors to potentially amplify therapeutic outcome.

J. Hajda reports grants from ORYX GmbH & Co. KG during the conduct of the study. B. Leuchs reports other support from Oryx GmbH &Co. KG during the conduct of the study. A.L. Angelova reports grants from ORYX GmbH & Co. KG during the conduct of the study. V. Frehtman reports other support from Oryx GmbH & Co. KG during the conduct of the study. J. Rommelaere reports grants from ORYX GmbH & Co. KG. during the conduct of the study. M. Mertens reports other support from ORYX GmbH & Co. KG during the conduct of the study. M. Pilz reports other support from ORYX GmbH & Co. KG. during the conduct of the study. M. Kieser reports other support from ORYX GmbH & Co. KG during the conduct of the study. O. Krebs reports other support from Oryx GmbH & Co. KG during the conduct of the study. M.W. Dahm reports grants from ORYX GmbH & Co. KG. during the conduct of the study and personal fees from ORYX GmbH & Co. KG. outside the submitted work. B. Huber reports grants from ORYX GmbH & Co. KG during the conduct of the study and personal fees from ORYX GmbH & Co. KG outside the submitted work. N. Hohmann reports other support from Oryx GmbH & Co. KG during the conduct of the study. N. Halama reports grants from Bristol Myers Squibb, DH Foundation, and RR Pohl Foundation outside the submitted work. M.M. Gaida reports other support from Oryx GmbH & Co. KG during the conduct of the study. V. Daniel reports other support from ORYX GmbH & Co. KG. during the conduct of the study. C. Springfeld reports other support from ORYX during the conduct of the study and personal fees from Roche, MSD, Bayer, Servier, AstraZeneca and Eisai outside the submitted work. G. Ungerechts reports other support from ORYX GmbH & Co. KG. during the conduct of the study. No disclosures were reported by the other authors.

J. Hajda: Conceptualization, formal analysis, methodology, writing–original draft, project administration, writing–review and editing. B. Leuchs: Conceptualization, formal analysis, investigation, methodology, writing–review and editing. A.L. Angelova: Formal analysis, investigation, methodology, writing–review and editing. V. Frehtman: Formal analysis, investigation, methodology. J. Rommelaere: Formal analysis, investigation, methodology, writing–review and editing. M. Mertens: Resources, formal analysis, investigation, methodology, writing–review and editing. M. Pilz: Conceptualization, data curation, software, formal analysis, investigation, methodology, writing–original draft, writing–review and editing. M. Kieser: Conceptualization, data curation, software, formal analysis, investigation, methodology, writing–original draft, writing–review and editing. O. Krebs: Conceptualization, funding acquisition, methodology, project administration, writing–review and editing. M. Dahm: Conceptualization, funding acquisition, methodology, writing–review and editing. B. Huber: Conceptualization, funding acquisition, methodology, writing–review and editing. C.E. Engeland: Conceptualization, data curation, software, formal analysis, investigation, methodology, writing–review and editing. A. Mavratzas: Conceptualization, funding acquisition, investigation, methodology, project administration, writing–review and editing. N. Hohmann: Conceptualization, funding acquisition, investigation, methodology, writing–review and editing. J. Schreiber: Conceptualization, funding acquisition, investigation, methodology, writing–review and editing. D. Jäger: Conceptualization, investigation, methodology, writing–review and editing. N. Halama: Formal analysis, investigation, methodology. O. Sedlaczek: Formal analysis, investigation. M.M. Gaida: Formal analysis, investigation, methodology, writing–review and editing. V. Daniel: Formal analysis, investigation, methodology, writing–review and editing. C. Springfeld: Conceptualization, formal analysis, investigation, methodology, writing–original draft, writing–review and editing. G. Ungerechts: Conceptualization, formal analysis, investigation, methodology, writing–original draft, writing–review and editing.

We would like to thank the patients and their families for participation in this trial. We would also like to thank Eurofins BioPharma Product Testing Munich GmbH for viral genomes testing, Labor Prof. Dr. G. Enders MVZ GbR, Stuttgart for ADA and neutralizing ADA testing, as well as Lophius Biosciences GmbH, Regensburg for ELISPOT analysis. We acknowledge the expert support of Elke Jäger, Salah-Eddin Al-Batran, and Christoph Eisenbach as members of the data safety monitoring board. We are grateful to Birgit Beelte for her organizational support of the trial conduct, Alexandra Just for her assistance in conducting the FISH analysis, Jürg Nüesch for providing H-1PV NS1 protein, Marcus Müller, Barbara Liebetrau, and Jürgen Engel for assistance in pharamacokinetic and seroconversion analyses (DKFZ). The ParvOryx02 clinical trial was sponsored by the company ORYX GmbH & Co. KG.

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