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Background
OH2 is an oncolytic virus derived from herpes simplex virus type 2. A phase Ia/Ib clinical trial in China was conducted in patients with unresected stage III–IV melanoma, the majority of whom had the acral type, to assess the safety and preliminary efficacy of OH2.
Methods
The trial enrolled patients with histologically confirmed unresectable stage III or advanced stage IV melanoma. In phase Ia, nine patients received OH2 single-dose treatment across three dose levels (106, 107, and 108 CCID50/mL, where CCID50 represents cell culture infectious dose 50%) while six patients underwent multidose therapy. Phase Ib expanded the proposed dose. Antitumor efficacy was evaluated using the Response Evaluation Criteria in Solid Tumors and immune-RECIST guidelines.
Results
All 44 patients were enrolled. OH2 was well tolerated without serious adverse events (AEs) or deaths reported. No Grade 3 or higher treatment-related AEs occurred. In phase Ia, the 1-year survival rate was 92.9% (95% CI, 59.1% to 99.0%), with a median overall survival of 28.9 months (95% CI, 12.7 to not reached). In phase Ib, 10 patients achieved immune-partial response (iPR)/partial response (PR), yielding an objective response rate (ORR) of 37.0% (95% CI, 19.4% to 57.6%), with 6 patients still responding. The rate of the durable response (PR or complete response lasting at least 6 months) was at least 29.6% (8/27). Notably, 7 of 12 III–IVM1a patients who previously received programmed cell death protein-1 (PD-1) therapy achieved iPR/PR, with an ORR of 58.3% (95% CI, 27.7% to 84.8%) and a disease control rate of 75.0% (95% CI, 42.8% to 94.5%). Biomarker analysis indicated that elevated baseline neutrophil activation state correlated with poorer clinical outcomes. A phase III clinical trial is ongoing in China (
Conclusions
OH2 oncolytic virotherapy exhibited a favorable safety profile without dose-limiting toxicities (DLTs) and demonstrated durable antitumor efficacy in patients with melanoma, especially in those who had progressed on anti-PD-1 treatment.
Trial registration number
ClinicalTrials.gov identifier
Full text
Correspondence to Dr Chuanliang Cui; [email protected] ; Dr Yan Kong; [email protected]
WHAT IS ALREADY KNOWN ON THIS TOPIC
Intratumoral administration of OH2, a genetically engineered oncolytic herpes simplex virus type 2, has previously shown durable antitumor activity in patients with metastatic esophageal and rectal cancer.
WHAT THIS STUDY ADDS
We report results from an open-label, single-arm, phase Ia/Ib study evaluating intratumoral OH2 in patients with advanced melanoma.
OH2 showed promising antitumor efficacy, with a 1-year survival rate of 94.3% (95% CI, 79.2% to 98.6%) in phase Ia+Ib and an objective response rate (ORR) of 37.0% in phase Ib.
Seven of 12 III–IVM1a patients who had previously received programmed cell death protein-1 (PD-1) therapy achieved immune-partial response/partial response, yielding an ORR of 58.3%.
Biomarker analysis revealed a correlation between elevated baseline neutrophil levels and poorer clinical outcomes.
OH2 demonstrated a favorable safety profile in patients with unresectable stage III–IV melanoma.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
In this phase Ia/Ib study, OH2 administered intratumorally had manageable toxicity, and responses were robust and durable in patients with advanced melanoma, including patients who progressed after anti-PD-1 therapy. Baseline neutrophil was found to be correlated with the OH2 treatment.
Background
The incidence of malignant melanoma is increasing globally. Although the incidence rate has increased, survival rates have improved over the past decade. The improvement in outcomes is mainly due to the use of immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1) monoclonal antibodies.1 2 However, many of the anti-PD-1 treated patients eventually develop resistance to these antibodies. Treatment of PD-1 antibody resistance has therefore developed into an unmet medical need for melanoma. Especially for acral lentiginous and mucosal subtypes, which are more common in Asian nations,3 but usually associated with more aggressive and mediate lower response rates to anti-PD-1 therapy.4–9
Oncolytic viruses (OVs) are genetically modified or naturally occurring viruses that selectively replicate in tumor cells, leading to their destruction. Multiple preclinical and clinical studies have highlighted the ability of OVs to remodel the tumor microenvironment (TME), transforming the non-immunogenic “cold” tumor into an immunogenic “hot” tumor, thereby enhancing the susceptibility of the tumor to ICI treatment.10–12 In a phase Ib trial that treated patients with unresectable stage IIIB–IV cutaneous melanoma (CM) with intralesional T-VEC followed by systemic pembrolizumab,13 an increase in circulating T cells was observed in most patients following administration of T-VEC. Furthermore, increases in programmed cell death ligand 1 (PD-L1) protein levels, interferon-γ (IFN-γ) gene expression, and T cells were observed within the tumors of responder patients following treatment with pembrolizumab and T-VEC. We therefore questioned whether OVs could serve as a catalyst in overcoming resistance to PD-1 monoclonal antibodies particularly among Chinese melanoma populations, and especially in acral melanoma, which exhibits a significant prevalence of inherent immune-resistance.
OH2 is a recombinant herpes simplex virus type 2 (HSV-2) that selectively replicates in tumor cells.14–16 The ICP34.5 and ICP47 genes were removed to attenuate virulence and improve oncolytic activity. In addition to these deletions, a GM-CSF expression cassette was inserted to enhance the immune response.17 18 OH2 is similar to T-VEC (a modified HSV-1) except that in OH2 the viral backbone originates from HSV-2. Preclinical studies have shown that modified HSV-2 has a better tumor-suppressive effect than modified HSV-1 in several tumor-bearing models like breast cancer, colon cancer, and metastatic ovarian cancer models in vivo.19–21 A multicenter phase I/II study (NCT03866525) of OH2 is being conducted, which showed that OH2 was well tolerated and demonstrated long-term antitumor activity in patients with metastatic colorectal and esophageal cancer.16
However, in advanced melanoma, there are still many problems to be solved. For example, it is unknown if OVs and ICI must be given individually or in combination. Furthermore, biomarkers for these treatments have not been identified that could help tailor drugs to the melanoma population that is likely to benefit most from treatment.
To begin addressing these questions, we initiated a phase Ia/Ib study to evaluate the safety, biodistribution, and antitumor activity of OH2 in patients with unresectable stage III–IV melanoma. A related phase III clinical trial is ongoing in China (NCT05868707). OH2 received breakthrough designation from the National Medical Products Administration in February 2023 and fast-track designation from the US Food and Drug Administration in June 2023.
Methods
Patients
During this phase Ia/Ib study, we recruited patients with histologically confirmed melanoma who had progressed after previous therapy, had not received (or had declined) therapy, or were intolerant to therapy. All patients were 18–75 years old. Inclusion criteria included: tumors suitable for virus injection; at least one assessable lesion as determined by the guidelines of Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1; the Eastern Cooperative Oncology Group (ECOG) score was 1 or 0; and serum chemistry, complete blood count, and coagulation tests were used to assess organ function. Patients were excluded if they had a history of or evidence of primary brain tumors, or had clinically active cerebral metastases, or had been treated with antiviral agents within 4 weeks before the start of therapy.
Study design and treatment
The study consisted of a dose escalation (phase Ia) and a dose expansion (phase Ib) part (online supplemental figure 1). The phase Ia part involved three single ascending dose cohorts (ie, 106, 107, and 108 CCID50/mL, where CCID50 represents cell culture infectious dose 50%) and two multiple-dose ascending (MAD) cohorts (ie, 107 and 108 CCID50/mL) (online supplemental figure 1). Patients in the MAD cohorts received 3 doses of OH2 2 weeks apart. If dose-limiting toxicity (DLT) was not observed within 21 days of injection, patients received extended therapy every 2 weeks. If DLTs were not observed in the 3 weeks of DLT assessment for the first three patients, dose escalation proceeded; if not, an additional cohort of three patients was enrolled. The previous lower dose was used to determine the maximum-tolerated dose (MTD) if two or more DLTs were found at the same dose level. The proposed dose for OH2 injection was chosen after dose escalation considering the safety, MTD, biodistribution, and efficacy. Patients were assigned to OH2 injection with the proposed dose in the phase Ib study.
Intratumoral injection was performed directly for subcutaneous or cutaneous lesions, or under ultrasound guidance for organ metastases or deep-located nodes. The volume administered into each tumor was based on the lesion’s longest diameter (>5.0 cm, up to 8 mL; >2.5 to ≤5.0 cm, up to 4 mL; >1.5 to ≤2.5 cm, up to 2 mL; ≤1.5 cm, up to 1 mL). Multiple tumor injections were permitted.
Safety and efficacy assessment
Adverse event (AEs) and DLTs were graded according to the guidelines of the National Institute Common Terminology Criteria for Adverse Events (V.5.0). DLTs must have occurred in the initial 3 weeks after treatment and be considered treatment-related by the investigators. Information regarding AEs was recorded throughout the trial and within 60 days of the last dose of OH2.
Tumor assessment was done at screening and then every 8 weeks after therapy initiation. CT or MRI were used for radiographic evaluation. The size of subcutaneous or cutaneous lesions was measured with calipers. Efficacy was evaluated using guidelines of both the RECIST V.1.1 and the immune-RECIST (iRECIST) criteria.
In order to identify potential biomarkers associated with clinical efficacy, 44 serum samples from 22 patients at treatment baseline and after treatment (at the time of maximal efficacy) were collected. Cytokines and chemokines in the samples were profiled using the proximity extension assays developed by Olink Proteomics (Sweden) and performed by the Shanghai Biotechnology Corporation. Standard protocols for quality control and data normalization by referencing internal and external controls were carried out in the Olink normalized protein expression (NPX) Manager software (V.3.3.2.434).
Olink standard differential analysis uses Student’s t-test to assess the expression differences of proteins in standardized NPX data across distinct experimental conditions. Proteins exhibiting a p value<0.05 (Student’s t-test) are designated as differentially expressed. Functional enrichment analysis of differentially expressed proteins involved comparing all differentially expressed proteins with all proteins identified in the experiment based on Gene Ontology (GO) or Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotations. The statistical significance of disparities between the two sets was determined through Fisher’s exact test, thereby identifying functional categories enriched in all differentially expressed proteins (p value<0.05).
Biodistribution and biological activity
PCR detection of DNA copies in blood, saliva, and urine was used to assess OH2 distribution during phase Ia. The injection site was swabbed for OH2 shedding the day after every treatment. Anti-HSV-2 antibody was detected in serum samples using a chemiluminescence immunoassay until HSV seropositivity was confirmed.
A validated OH2-specific quantitative PCR assay was used to determine OH2 DNA levels in samples (non-target nucleic acids, including wild-type HSV-2 and HSV-1, were not detected). The assay’s lower limit of quantification for blood, urine, and saliva samples was 5 copies/μL.
Statistical analysis and endpoints
During phase Ia, the primary endpoints were safety and tolerability as defined by MTD and DLT, and evaluation of the biodistribution and effects of intratumoral injection of OH2. The secondary endpoints were preliminary evaluations of the efficacy of OH2 injection. For phase Ib, the primary endpoints were further evaluation of the safety and tolerability of OH2 in patients with advanced solid tumor (mainly patients with melanoma). The secondary endpoints were further evaluation of the efficacy of OH2, evaluation of the biodistribution of OH2 in patients with advanced tumor, and the immune response of patients with advanced tumor to OH2. To estimate time-to-event variables, the Kaplan-Meier method was applied. R software (V.4.1.1; https://www.r-project.org/) was used for statistical analysis.
Results
Patients
Between November 2018 and July 2023, we enrolled 44 patients with melanoma. Baseline characteristics are listed in table 1 and online supplemental table 1. 28 patients (63.6%) had clinical stage III–IV M1a disease. Acral melanoma was the predominant subtype 75%. More than half (59.1%) of the patients had been previously treated with and progressed on anti-PD-1 antibodies, and were therefore considered resistant to these agents. 42 patients were included in the efficacy analysis and 43 patients were included in the safety analysis.
Table 1Baseline demographic and clinical characteristics
| Characteristic | Phase Ia (n=15) | Phase Ib (n=29) | Overall (n=44) |
| Age, years | |||
| Median | 56.0 | 60 | 59.5 |
| Range | 18–73 | 47–75 | 18–75 |
| Sex, No. (%) | |||
| Male | 6 (40) | 13 (44.8) | 19 (43.2) |
| Female | 9 (60) | 16 (55.2) | 25 (56.8) |
| HSV-2 serostatus, No. (%) | |||
| Negative | 11 (73.3) | 27 (93.1) | 38 (86.3) |
| Positive | 4 (26.7) | 2 (6.9) | 6 (13.6) |
| AJCC stage, No. (%) | |||
| III–IVM1a | 11 (73.3) | 17 (58.6) | 28 (63.6) |
| IVM1b | 3 (20.0) | 8 (27.6) | 11 (25.0) |
| IVM1c | 1 (6.7) | 3 (10.3) | 4 (9.1) |
| Unknown* | 0 (0) | 1 (3.4) | 1 (2.3) |
| ECOG PS, No. (%) | |||
| 0 | 7 (46.7) | 14 (48.3) | 21 (47.7) |
| 1 | 8 (53.3) | 15 (51.7) | 23 (52.3) |
| Melanoma subtype, No. (%) | |||
| Acral | 10 (66.7) | 23 (79.3) | 33 (75.0) |
| Non-acral cutaneous | 4 (26.7) | 3 (10.3) | 7 (15.9) |
| Mucosal | 0 (0) | 1 (3.4) | 1 (2.3) |
| Unknown primary | 1 (6.7) | 2 (6.9) | 3 (6.8) |
| Prior adjuvant therapy, No. (%) | |||
| Yes | 7 (46.7) | 15 (51.7) | 22 (50.0) |
| No | 8 (53.3) | 14 (48.3) | 22 (50.0) |
| Line of treatment†‡, No. (%) | |||
| First | 3 (20.0) | 10 (34.5) | 13 (29.5) |
| ≥ Second | 12 (80.0) | 14 (48.3) | 26 (59.1) |
| Previously immunotherapy | |||
| PD-1-treated | 7 (46.7) | 19 (65.5) | 26 (59.1) |
| PD-1-naïve | 8 (53.3) | 10 (34.5) | 18 (40.9) |
| BRAF status, No. (%) | |||
| Mutation | 2 (13.3) | 1 (3.4) | 3 (6.8) |
| Wild type | 8 (53.3) | 16 (55.2) | 24 (54.5) |
| Unknown/missing | 5 (33.3) | 12 (41.4) | 17 (38.6) |
*At phase Ib, one patient with left supraclavicular lymph node metastasis was pathologically diagnosed with melanoma, but the primary lesion was unknown.
†Patients with disease progression within 6 months after receiving adjuvant therapy were considered as having failed first-line therapy.
‡Four patients were enrolled directly after surgery because there was no available standard antitumor therapy, one patient had disease progression less than 6 months after receiving adjuvant therapy and was enrolled directly because there was no available standard antitumor therapy.
AJCC, American Joint Committee on Cancer; ECOG PS, Eastern Cooperative Oncology Group performance status; HSV-2, herpes simplex virus type 2; PD-1, programmed cell death protein-1.
Response rate and survival
For the 15 patients in phase Ia, no clinical response was observed, but the disease control rate (DCR) was 60.0%. Online supplemental table 2 presents details of clinical responses observed in the phase Ib dose expansion cohort (n=27 of 42). 10 patients achieved immune-PR (immune-partial response/partial response), with an objective response rate (ORR) of 37.0% (95% CI, 19.4% to 57.6%). Stable disease was observed in five patients. DCR was 55.6% (95% CI, 35.3% to 74.5)%. The durable response rate (partial or complete response lasting at least 6 months) was at least 29.6% (8/27). Figure 1A,B show the alterations in target lesion size from baseline. Shrinkage could be observed in both injected and non-injected lesions (figure 1C,D). Within the 51 assessable lesions that received OH2 injection, 26 (51.0%) regressed. The maximum reduction was 82% from baseline (figure 1C). Five (9.8%) lesions remained stable. Of the 20 non-injected lesions, 8 (40.0%) regressed, with the maximum reduction being 100% (figure 1D). Two (10.0%) non-injected lesions remained stable. Figures 1E and 1F present two representative cases, for injection lesion and no-injection lesion. We observed heterogeneity in treatment responses across tumor lesions in some patients, with 4 out of 27 showing a mixed response. Additionally, the development of new lesions while the target lesion shrinks was noted in 2 out of 27 cases. However, some newly developed lesions may still respond positively to subsequent OH2 rescue injections.
Figure 1. Response to OH2 treatment. Chronological change of target lesions from baseline in phase Ia (A) and in phase Ib (B). Maximal changes in tumor burden from baseline in injected lesions (C) and uninjected lesions (D) from phase Ib. Each bar represents an individual lesion, with colors corresponding to patients in different response categories. (E) A biopsy of the scalp nodule (injection lesion) of patient 027 was performed with a histological finding of malignant melanoma. The patient achieved a PR duration of 13.1 months after OH2 treatment. (F) Patient 026 was diagnosed in June 2019 with melanoma in the left foot heel with excision surgery and lymph node dissection. Observation lesion (non-injection lesion): after 12 months treatment, iliac artery lymph nodes (short diameter) decreased from 21 to 9 mm. iPR, immune-partial response; PR, partial response; PD, progressive disease; SD, stable disease; TL, target lesion.
With respect to the 27 phase Ib patients, ORR was 50.0% (95% CI, 24.7% to 75.3%) in stage III–IVM1a patients but only 20.0% (95% CI, 2.5% to 55.6%) in stage IVM1b–IVM1c patients (online supplemental table 2). Among 22 patients with normal lactate dehydrogenase (LDH) levels, the ORR was 45.5% (95% CI, 24.4% to 67.8%). The ORR was 0% in the above upper limit of the normal cohort. The ORR of the major subtype, acral melanoma, was 33.3% (95% CI, 14.6% to 57.0%), which is similar to the total ORR in phase Ib. Patients with smaller tumor sizes, fewer than three metastasis-involved organs, or no visceral involvement exhibited a higher objective response rate (ORR), though these findings did not reach statistical significance (online supplemental table 2).
The total progression-free survival (PFS) for phase Ia/Ib (42 patients) was 2.7 months (95% CI, 2.0 to 3.7). Subgroup analyses showed that patients with normal LDH show better median PFS (3.6 months (CI, 2.1 to 11.7) than patients with high LDH (2.0 months (CI, 1.8 to 3.7) (p=0.04).
Based on Kaplan-Meier estimation, the 1 year and 2-year survival rates in the phase Ia+Ib study were 94.3% (95% CI, 79.2% to 98.6%) and 73.3% (95% CI, 54.9% to 85.1%. The median overall survival (mOS) for the 42 patients in phase Ia+Ib was not reached, with a median follow-up of 27.5 months (95% CI, 25.3 to 35.4) (figure 2A). For phase Ia, the median OS was 28.9 months, with a median follow-up of 49 months (95% CI, 39.3 to 50.5). OS for phase Ib was not reached, with limited follow-up time (25.3 months 95% CI, 14.4 to 30.4). Based on the treatment clinical benefit, patients were divided into the clinical benefit cohort (receiving OH2 treatment for≥12 months, n=30) and a clinical non-benefit cohort (receiving OH2 treatment for<12 months, n=12). The mOS were not reached in the clinical benefit cohort and 39.33 months in the clinical non-benefit cohort (figure 2A). On univariate analysis, tumor stage, gender, subtype, age, LDH, ECOG performance status and other factors were not significantly associated with OS (online supplemental figure 3).
Figure 2. (A) Kaplan-Meier survival curves. (B) Response of patients in subgroups of different disease stages and PD1 treatment states to OH2. Note: there was one case of unknown primary lesion and unknown tumor stage in subjects with PD-1- naïve status. DCR, disease control rate; iPR, immune-partial response; ORR, objective response rate; OS, overall survival; PD-1, programmed cell death protein-1; PR, partial response; PD, progressive disease; SD, stable disease.
It should be noted that 15 patients evaluated as PD based on RECIST 1.1 continued to receive OH2 (at least five doses) due to clinical benefit. These patients appeared to gain a survival benefit, as the median OS for these patients was not reached with a median follow-up of 27.3 months (ranging from 11.3 to 50.7).
OH2 achieved an elevated ORR rate in patients progressing on PD-1 immunotherapy
In phase Ib, the ORR was elevated in patients progressing on anti-PD-1 therapy (ORR=47.1% (95% CI, 23.0% to 72.2%) (online supplemental table 2). Post hoc subset analysis identified a cohort of 12 III–IVM1a patients progressing on anti-PD-1 therapy with ORR 58.3% (95% CI, 27.7% to 84.8%) and DCR of 75.0% (95% CI, 42.8% to 94.5%) (figure 2B). The median OS for the 24 patients progressing on anti-PD-1 therapy in phase Ia/Ib was not reached, with a median follow-up of 27.5 months (95% CI, 23.2 to 39.3) (figure 2A). These results demonstrate that OH2 may have greater efficacy in patients progressing on anti-PD-1 therapy. Further analysis revealed that patients with secondary resistance as well as those receiving adjuvant therapy, demonstrated relatively better outcomes with OH2 (online supplemental table 3). The definitions of primary and secondary resistance were outlined by the Society for Immunotherapy of Cancer.22
Safety results
At the cut-off date of July 2023, all patients had received an average of 20.4 (range, 2–94) doses of OH2 injection. 38 of 43 (88.4%) patients experienced at least one treatment-related AE (TRAE). Most TRAEs were grade 1 or 2. No patient had grade≥3 TRAEs. The TRAEs are listed in table 2. The most frequent TRAE was fever (n=23, 53.5%). Other common TRAEs were skin depigmentation (n=6, 14.0%) and nausea (n=3, 7.0%). No DLTs were observed, suggesting that OH2 has a favorable safety profile.
Table 2Treatment-related adverse events (n=43)
| AEs, all grades n (%) | |||
| Adverse event (PT) | Grade 1 | Grade 2 | ≥Grade 3 |
| Fever | 22 (51.2) | 3 (7.0) | 0 |
| Skin depigmentation | 6 (14.0) | 0 | 0 |
| Creatine phosphokinase serum increased | 3 (7.0) | 0 | 0 |
| Rash | 3 (7.0) | 0 | 0 |
| Nausea | 3 (7.0) | 0 | 0 |
| Gamma GT increased | 2 (4.7) | 0 | 0 |
| Decreased white cell count | 2 (4.7) | 0 | 0 |
| Aching in limb | 2 (4.7) | 1 (2.3) | 0 |
| Peripheral swelling | 2 (4.7) | 0 | 0 |
| Decreased appetite | 1 (2.3) | 0 | 0 |
| Hyperglycemia | 1 (2.3) | 0 | 0 |
| Skin infection | 0 | 1 (2.3) | 0 |
| Bilirubin conjugated increased | 1 (2.3) | 0 | 0 |
| Aspartate aminotransferase increase | 1 (2.3) | 0 | 0 |
| Blood bilirubin increased | 1 (2.3) | 0 | 0 |
| Hemoglobin decreased | 1 (2.3) | 0 | 0 |
| Blood creatine phosphokinase MB increased | 1 (2.3) | 0 | 0 |
| Blood glucose increased | 1 (2.3) | 0 | 0 |
| Platelet count increased | 1 (2.3) | 0 | 0 |
| Neutrophil count decreased | 1 (2.3) | 0 | 0 |
| Congenital lymphoedema | 1 (2.3) | 0 | 0 |
| Dry throat | 1 (2.3) | 0 | 0 |
| Skin exfoliation | 1 (2.3) | 0 | |
| Vitiligo | 1 (2.3) | 0 | |
| Alopecia | 1 (2.3) | 0 | |
| Asthenia | 1 (2.3) | 0 | |
AEs, adverse events; GT, glutamyl transferase; PT, preferred term.
Although MTD was not achieved in the OH2 dose escalation, it was found that the frequency of fever appeared to be dose-related (2/3 of patients at 106 CCID50/mL, 4/6 of patients at 107 CCID50/mL, and 6/6 of patients at 108 CCID50/mL). This result suggested a higher risk of fever in patients injected with 108 CCID50/mL. Because patients from both the 106 CCID50/mL and 107 CCID50/mL groups had clinical responses, the proposed dose for OH2 in phase Ib was set at 107 CCID50/mL.
Biodistribution and shedding of OH2
Blood, saliva, and urine samples were collected at various time points in the phase Ia study to detect viral nucleic acids (online supplemental figure 2A). OH2 DNA was detected in the blood of 60.0% of patients (9/15) and in 6.3% of samples (13/207). The highest frequency of OH2 DNA detection occurred within 24 hours following OH2 administration, after which OH2 DNA was cleared (online supplemental figure 2B). The number of OH2 DNA copies did not correlate with the baseline HSV-2 serostatus (online supplemental table 4). OH2 DNA was detected in saliva in 60.0% of patients (9/15) and 11.7% of samples (24/206) (online supplemental figure 2C). OH2 DNA was not detected in any of the 180 urine samples. A total of 192 swabs from the injection sites of 15 patients between 5 min and 24 hours after the first injection were collected to detect viral shedding. All the swabs were negative. Among the 44 patients, seroconversion occurred in the 36 negative patients within 8 weeks (the serostatus of 2 patients were unknown), which was consistent with previous findings.16
Biomarker analysis
We leveraged the Olink technology (Olink Proteomics AB, Sweden) to quantify circulating inflammatory proteins (liquid biopsy). 44 serum samples were collected from 22 patients before (baseline) and after treatment (corresponding to the time of maximal efficacy) for analysis. Based on the treatment clinical benefit, patients were divided into the clinical benefit cohort (receiving OH2 treatment for≥12 months) and the clinical non-benefit cohort (receiving OH2 treatment for<12 months). At baseline, nine plasma proteins could be effectively distinguished between the two cohorts, including IFN-γ, IL18, VEGFR-2, CXCL9, ARG1, CSF-1, CXCL11, CXCL12, CD5 (t-test, adjusted p<0.05, and all inflammatory proteins were up-regulated in the clinical non-benefit cohort) (figure 3A). We performed GO pathway and KEGG enrichment analyses on all the nine treatment-responsive inflammatory proteins, and found they were mostly enriched in the cytokine activity, signaling pathways and inflammatory response (figure 3A).
Figure 3. (A) Representative box plot of the differentially expressed proteins between the clinical benefit and clinical non-benefit group before treatment (baseline) were measured with Olink and expressed as NPX units (left panel). GO enrichment analysis based on the nine differentially expressed proteins (middle panel). KEGG enrichment analysis based on the nine differentially expressed proteins (right panel). (B) Representative box plot of the differentially expressed proteins between the clinical benefit and clinical non-benefit group after treatment (maximal efficacy time) were measured with Olink and expressed as NPX units (left panel). GO enrichment analysis based on the 21 differentially expressed proteins (middle panel). KEGG enrichment analysis based on the 21 differentially expressed proteins (right panel). A t-test was performed to analyze the difference between the differentially expressed proteins. (C) immune cell counts of patients in the clinical benefit cohort and clinical non-benefit cohort at baseline. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
After treatment, 21 proteins could be effectively distinguished between the two cohorts, namely IFN-γ, GZMH, CXCL9, Gal-1, IL18, CD5, PD-L1, GZMA, Gal-9, GZMB, CD70, CXCL10, CSF-1, TIE2, PGF, CCL23, CD8A, TNFRSF9, CXCL11, CD4 and NCR1 (t-test, p<0.05) (figure 3B). We performed GO and KEGG enrichment analyses on all the 21 treatment-responsive inflammatory proteins, and found they were mostly enriched in the neutrophil chemotaxis, chemokine-mediated signaling pathway and chemokine activity (figure 3B).
Neutrophil activation was further verified through peripheral blood tests. For all 44 patients, during the baseline period before treatment, the peripheral blood neutrophil levels in the clinical non-benefit cohort were significantly higher than in the clinical benefit cohort (t-test; mean 3.93 vs 3.16×109/L, p=0.04687) (figure 3C). Other cells such as lymphocytes, natural killer cells, Treg were also analyzed, but no significant difference was observed (figure 3C). We further analyzed the peripheral blood neutrophil levels at baseline in all the 44 patients grouped according to different treatment durations, and found that the trend persisted (table 3).
Table 3Neutrophil level and mOS of patients grouped according to different treatment time (phase Ia/Ib)
| Received OH2 treatment time (months) | Baseline neutrophil counts (109/L) | P value | mOS (months) | P value |
| <3 (n=12) | 4.7±0.4 | 0.01229 | 18.5 | 0.00141 |
| ≥3 (n=32) | 3.4±0.3 | NA | ||
| <6 (n=24) | 4.1±0.4 | 0.0404 | 26.6 | 0.00063 |
| ≥6 (n=20) | 3.2±0.2 | NA | ||
| <9 (n=29) | 4.0±0.3 | 0.04027 | 26.6 | 0.00406 |
| ≥9 (n=15) | 3.2±0.2 | NA | ||
| <12 (n=32) | 3.9±0.3 | 0.04687 | 39.3 | 0.01556 |
| ≥12 (n=12) | 3.2±0.2 | NA | ||
| <15 (n=33) | 3.9±0.3 | 0.01911 | 39.3 | 0.0209 |
| ≥15 (n=11) | 3.1±0.2 | NA |
mOS, median overall survival; OH2, genetically engineered oncolytic herpes simplex virus type 2.
Discussion
OVs represent an important immunotherapy for melanoma. Numerous clinical studies have focused on the use of OVs in melanoma therapy.23–27 While OVs have demonstrated a favorable safety profile, more compelling evidence of their efficacy is still required.23 HSV-2 has shown superior antitumor activity than HSV-1 in several preclinical studies.20 21 28 This phase Ia/Ib trial was designed to evaluate the safety, biodistribution, and preliminary efficacy of OH2 therapy.
The 1-year and 2-year survival rates in the phase Ia/Ib study were 94.3% (95% CI, 79.2% to 98.6%) and 73.3% (95% CI, 54.9% to 85.1%), respectively. The median OS was not reached still the last follow-up 27.5 months (95% CI, 25.2% to 35.4%). In a recent report, the efficacy of a modified HSV-1 was assessed in China in a phase Ib study, and all patients with unresectable IIIC–IV melanoma (n=26) had a median OS of 19.2 months.29 The estimated 1-year and 2-year survival rates in the randomized open-label phase III study assessing the efficacy of T-VEC in patients with unresectable stage IIIB–IV melanoma in USA were 74% and 50%, respectively.23
In the present trial, good efficacy was observed in patients who had progressed on anti-PD-1 treatment. In phase Ib, ORR was 47.1% (95% CI, 23.0% to 72.2%) for these patients. Furthermore, this advantage is particularly pronounced in cases of secondary resistance to PD-1, as compared with primary resistance (37.5% vs 26.7%). According to published data, patients with advanced melanoma respond poorly after progressing on PD-1 therapy. A retrospective study showed that chemotherapy combined with antiangiogenic drugs had limited efficacy, with ORR being merely 5.8%.30
Other trials also support our findings. For example, ONCOS-102 with pembrolizumab mediated an ORR of 35% in patients with anti-PD-1–refractory advanced melanoma, whereas the ORRs of single-agent T-VEC, Toll-like receptor 9 agonists (CMP-001) plus pembrolizumab, RP1 plus nivolumab are 26%, 24% and 33%, respectively.31–34 It is likely that OH2 is a strong driver of the efficacy observed in the current study, given that response to single-agent treatment of patients progressing on prior checkpoint inhibitor therapy is quite rare.
So far, combinations of OV and PD-1 monoclonal antibody therapy have been disappointing. For example, a combination of T-Vec with pembrolizumab in a phase III trial of advanced melanoma did not significantly improve PFS or OS compared with the placebo–pembrolizumab combination.35 Similarly, combination therapy is not superior to monotherapy in the neoadjuvant setting either as KEYMAKER-U02.36
In an interesting research published by Nguyen et al, a patient with melanoma who had progressed after anti-PD-1 therapy was found to have a loss of JAK1/JAK2 function.37 This genetic alterations in key immune signaling pathways, such as the JAK/STAT and IFN-γ pathways, are one of the driving mechanisms of secondary resistance.38 The anti-PD-1 resistant cancer cells lacking JAK1/JAK2 function are much more vulnerable to OVs killing. Another lab found that the loss of function of STING, a known biomarker for anti-PD-1 resistance, was related to the enhanced antitumor activity of T-VEC.39 Dynamic changes in the TME during immune checkpoint blockade therapy—such as the recruitment of immunosuppressive cells, the formation of an immunosuppressive extracellular matrix, and shifts in cytokine and chemokine expression—can alter immune cell infiltration and function, thereby contributing to secondary resistance.40 There is evidence that intratumoral administration of T-Vec causes a systemic increase in circulating CD4+ and CD8+ T cells, which leads to increased CD8+ T cell infiltration into tumors. Injection of T-Vec may change the TME by attracting T cells that may induce a systemic response in distant metastases after subsequent PD-1 blockade with pembrolizumab.13 These apparently higher efficacies of combination therapies against drug-resistant or immunosuppressed populations might explain why OH2 showed better efficacy in anti-PD-1-treated patients especially for secondary resistance, than in those who did not receive anti-PD-1 treatment.
The optimal sequence of treatment is important for patients with advanced melanoma. By choosing the optimal treatment sequence, clinicians can maximize the potential for successful treatment outcomes for patients with advanced melanoma. For the treatment option of OV and anti-PD-1, sequencing or combination strategies still require further exploration. However, according to our data, treatment with OH2 after progression on anti-PD-1 therapy is a reasonable approach and thus merits further investigation in future clinical trials.
OH2 showed efficacy in injected and non-injected lesions as well. This phenomenon supports the immune activation mechanism of OH2 therapy, and is relevant for distant malignancies including micrometastatic disease. OH2 was broadly well tolerated with a good safety profile in line with that of other HSVs.16 41 42 A total of 878 OH2 doses were administered to 43 patients. No cumulative toxicity was observed. Viral nuclear acids were at low levels in both saliva and blood, while viral DNA was not detected in the urine samples. Therefore, the risk of OH2 spreading to close contacts, caregivers or the environment in general is very low.
Olink analysis demonstrated that in serum samples both at baseline and after treatment, immune-related cytokines such as ARG1, CXCL11 and CXCL12, were significantly lower in the clinical benefit cohort than in the non-clinical benefit cohort. GO enrichment analysis showed that the activated cytokines are enriched in the neutrophil chemotaxis axis. This suggests that the activation of the neutrophil pathway is negatively correlated with the efficacy of the OH2 OV. This finding has also been confirmed in the examination of neutrophils in the peripheral blood of patients collected at baseline. In fact, we have analyzed neutrophil data from different clinical trials of OH2 against other tumors and found that this is a common phenomenon (unpublished data). Consistent with our findings, a study of melanoma patients treated with pembrolizumab also reported that high neutrophil counts were negatively correlated with OS.43
Previous studies have provided evidence for both protumor and antitumor neutrophil activity.44 The basis of this apparent contradiction may lie in the nature of the cells studied. Earlier reports that focused on the role of neutrophils in cancer did not distinguish neutrophils from polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). Neutrophils are one of the major mechanisms of protection against invading pathogens, whereas PMN-MDSC have immune suppressive activity and restrict immune responses in cancer from T cell responses.45 Others have implicated a number of signaling pathways in the regulation of PMN-MDSC development and function.46 This has been confirmed with KEGG enrichment analysis in our study as well. Therefore, we hypothesize that PMN-MDSCs negatively regulate the antitumor efficacy of OVs by inhibiting T-cell function. Further investigation is needed to clarify the specific pathways and mechanisms involved in this.
Although the outcome of our trial was positive, it had certain limitations. First, it is important to note that the abovementioned data were obtained from a small sample size and should therefore be interpreted with caution. Second, this was an open-label study and there was no central review of investigator-assessed responses, which may lead to inadvertent bias.
Overall, OH2 oncolytic virotherapy exhibited a favorable safety profile without DLTs. It showed durable antitumor efficacy in patients with melanoma, especially in those who progressed on anti-PD-1 treatment. Sequential treatment with OH2 after progression on anti-PD-1 therapy merits further investigation in future clinical trials. A phase III clinical trial in China (NCT05868707) is ongoing.
We thank the patients and their caregivers, as well as the clinical trial investigators and their team members who participated in this study.
Data availability statement
Data are available upon reasonable request.
Ethics statements
Patient consent for publication
Not applicable.
Ethics approval
This study involves human participants and was approved by Ethics Committee of Peking University Cancer Hospital (2018YW91). Participants gave informed consent to participate in the study before taking part.
XW and HT contributed equally.
Presented at Presented at the ASCO Annual Meeting, 2022 (abstr e21375).
Contributors Conception and design: XW, JG, CC, and YK. Provision of study materials or patients: XW, ZC, LS, XS, SL, CL, BL, LZ, LM, BT, XY, XW, JL, JG, YK, and CC. Collection and assembly of data: XW, HT, ZC, LS, XS, HH, XG, SL, CL, BL, LZ, LM, BT, XY, XW, JL, BL, JG, YK, and CC. Data analysis and interpretation: XW, HT, XG, BL, JG, YK, and CC. Manuscript writing: XW, HH, BL, JG, CC. Guarantor: CC. Final approval of manuscript: All authors. Accountable for all aspects of the work: All authors.
Funding Funded by Clinical Medicine Plus X-Young Scholars Project of Peking University [PKU2022LCXQ030]; Beijing Municipal Administration of Hospitals Incubating Program [grant number PX2024040].
Competing interests JG serves consulting/advisory roles in Merck Sharp & Dohme, Roche, Bayer, Novartis, Simcere Pharmaceutical Group and Shanghai Junshi Biosciences. LS has received speakers’ honoraria from MSD, Roche, Novartis, and Shanghai Junshi Biosciences. BL is the founder of Wuhan Binhui Biopharmaceutical. XG is a salaried employee of Wuhan Binhui Biopharmaceutical. For the remaining authors, there are no conflicts of interest.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.
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