CAN-3110
Persistent T cell activation and cytotoxicity against glioblastoma following single oncolytic virus treatment in a clinical trial
Bottom Line
A single intratumoral injection of CAN-3110 produced deep and persistent T cell infiltration in recurrent GBM by locally expanding pre-existing tumor-infiltrating T cell clones; shorter GZMB+ CD8 T cell–apoptotic tumor cell distances correlated with longer PFS, and patients with expanded shared TCR repertoires had longer overall survival (445 vs 235 days, p=0.033). Hypoxic mesenchymal tumor regions excluded T cells, identifying a key resistance mechanism.
Major Points
- Single intratumoral oncolytic HSV-1 (CAN-3110) drives sustained T cell infiltration into rGBM at late time points (months 6–25, including >2 years in one patient).
- CD8/Treg ratio increased from 6.8 pre to 21.1 post (p=0.0042); CD8 T cell density 30.4 → 138 cells/mm² (p=0.0078); tissue-resident CD8 T cells showed largest expansion (log2FC 3.03).
- Productive TCR clonality increased in tumor (p=0.032) but not in PBMC; up to 1,024-fold local intratumoral expansion of individual clones.
- Proximity of GZMB+ CD8 T cells to cleaved caspase-3+ apoptotic tumor cells correlated with better PFS (175 vs 262 µm, p=0.038) and lower tumor growth rate (p=0.028).
- Pre-existing tumor-infiltrating T cell clones expanded after oHSV; expansion of shared TCR repertoire was associated with median OS 445 vs 235 days (log-rank p=0.033).
- Xenium spatial TCR mapping showed clonally expanded T cells (median 32.3 vs 51.8 µm) interacted directly with tumor cells with tissue-resident (ITGAE/CD103, ZNF683/HOBIT) and early activation (NR4A1/Nur77, CD69, IFNG, TNF) programs.
- Residual HSV viral protein and RNA were restricted to necrotic regions in only 4/8 post-treatment samples; T cells were depleted near HSV+ areas (log2FC −3.47 CD8, −2.01 CD4), supporting that late T cell infiltration is driven by tumor antigen rather than viral antigen recognition.
- Hypoxic mesenchymal (MES-like 2) tumor regions expanded post-treatment (log2FC 1.38, p=0.0078) and excluded T cells (constituting up to 78% of tumor cells in the most distant zones), identifying a spatial resistance mechanism.
- Dexamethasone use >100 days correlated with reduced T cell clonality (r=−0.414, p=0.04), implicating chronic steroid exposure in impaired immunity.
- Screening of expanded TCRs against VDJdb, McPAS-TCR, and HSV-specific databases found only 15 of 1,246 clonotypes with known specificities and none matched HSV, consistent with tumor (not viral) antigen reactivity.
Design
Study Type: Translational spatial immunology analysis of phase 1 single-arm clinical trial samples (NCT03152318)
Randomization:
Blinding: Open-label phase 1 trial; spatial/molecular analyses not blinded
Enrollment Period: Parent phase 1 trial enrolled 2018–2022 (Ling et al., 2023)
Follow-up Duration: Tissue collected up to 331 days post-injection (one patient >2 years)
Centers: 1
Countries: USA
Sample Size: 16
Analysis: Paired pre- vs post-treatment within-patient comparisons; Wilcoxon signed-rank, Mann-Whitney, Spearman correlation, Kaplan-Meier with log-rank tests
Inclusion Criteria
- Adults with histologically confirmed recurrent glioblastoma (rGBM)
- Disease at first to fourth recurrence
- Surgically accessible tumor amenable to stereotactic intratumoral injection of oHSV
- Subsequent surgical resection or biopsy yielding tissue for paired pre/post analysis
- Baseline Karnofsky Performance Status (KPS) 70–100
- Adequate FFPE tissue quality for CODEX and/or Xenium spatial profiling
Exclusion Criteria
- Inadequate tumor tissue volume or quality on paired specimens
- Lack of matched pre-treatment biopsy or post-treatment resection material
- Insufficient morphological quality for spatial multiplex imaging
- Inability to undergo intratumoral injection of investigational oHSV
- (Phase 1 trial standard exclusions for active systemic infection, uncontrolled comorbidities, etc., as per NCT03152318)
Arms
| Field | Control | Post-oHSV resection |
|---|---|---|
| Intervention | Diagnostic/baseline rGBM specimen prior to oHSV injection | Single intratumoral injection of rQNestin34.5v.2 (CAN-3110, linoserpaturev) at escalating doses; tissue collected at subsequent resection for recurrence |
| Duration | Baseline (within-patient control) | Single injection; tissue 24–331+ days later |
Outcomes
| Outcome | Type | Control | Intervention | HR / OR / RR | P-value |
|---|---|---|---|---|---|
| Change in intratumoral T cell infiltration density (cells/mm²) from pre- to post-oHSV treatment (paired analysis) | Primary | 30.4 cells/mm² (pre-treatment median, Xenium) | 138 cells/mm² (post-treatment median, Xenium) | 0.0078 (paired Wilcoxon) | |
| CD8/Treg ratio change pre vs post | Secondary | 6.8 (pre) | 21.1 (post) | 0.0042 | |
| Productive TCR clonality change in tumor | Secondary | Pre-treatment baseline | Increased post-oHSV | 0.032 | |
| Productive TCR clonality in PBMC | Secondary | Pre-treatment baseline | Unchanged | 0.62 (NS) | |
| GZMB+ T cell to apoptotic tumor cell distance — above-median PFS vs below | Secondary | 262 µm (below median PFS) | 175 µm (above median PFS) | 0.038 | |
| Overall survival — expanded vs contracted shared TCR repertoire | Secondary | Median 235 days (contracted) | Median 445 days (expanded) | 0.033 (log-rank) | |
| Tissue-resident CD8 T cell expansion (post vs pre, log2FC) | Secondary | Pre baseline | log2FC = 3.03 | 0.0078 | |
| Plasma cell expansion (post vs pre, log2FC) | Secondary | Pre baseline | log2FC = 3.47 | 0.0156 | |
| MES-like 2 (hypoxic) tumor cell expansion post-treatment | Secondary | Pre baseline | log2FC = 1.38 | 0.0078 | |
| Tumor growth rate vs GZMB+ T cell–tumor distance | Secondary | Larger distance | Shorter distance → lower growth | 0.028 | |
| Dexamethasone >100 days vs post-treatment clonality | Secondary | n/a | r = −0.414 (Spearman) | 0.04 | |
| Note | Adverse | This spatial/translational publication did not re-report toxicity. Safety/adverse events for the rQNestin34.5v.2 (CAN-3110) phase 1 dose escalation were reported in the parent trial publication (Ling et al., Nature 2023), where single intratumoral injection was generally well tolerated with no dose-limiting toxicities at the doses studied; common events were headache, seizure, and transient neurologic deficits typical of intracranial procedures. | |||
Subgroup Analysis
Patients with expanded shared TCR repertoire (vs contracted) had significantly longer OS (445 vs 235 days, p=0.033). Patients with above-median PFS had shorter GZMB+ T cell–tumor cell distances (175 vs 262 µm, p=0.038). Dexamethasone exposure >100 days associated with reduced T cell clonality (r=−0.414, p=0.04). Hypoxia/MES-like 2 enrichment correlated with T cell exclusion (r=0.457, p<10e−4).
Criticisms
- Single-arm, n=16 with paired pre/post comparison — no randomized control arm.
- Cohort exclusively of European ancestry; demographic associations across ancestral backgrounds not assessable.
- Translational/correlative analysis of a phase 1 trial, not powered for clinical efficacy endpoints.
- Tissue available only at recurrence rather than at defined on-treatment time points (intracranial location limits serial biopsy).
- Only TCRβ chain sequences available for most patients; paired TCRα and antigen specificity not directly established.
- No matched tumor cell lines available to functionally validate T cell tumor-reactivity.
- Bulk TCR (not single-cell) for most patients; in situ TCR mapping limited to 2 patients (P28, P34) with the strongest responses, with potential selection bias.
- Causal direction between immune infiltration and outcomes cannot be established from observational spatial data alone.
Funding
NCI grants P01 CA236749 (KWW & EAC); P01 CA163222, R01 CA238039, R01 CA251599 (KWW); P01 CA163205, R01 NS110942 (EAC); Parker Institute for Cancer Immunotherapy (PICI); Cancer Research Institute Immuno-Informatics Fellowship (MM, CRI #CRI5000); Institut Servier and Philippe Foundation mobility grants.
Based on: CAN-3110 (Cell, 2026)
Authors: Meylan M, Tian Y, Wu L, ..., Wucherpfennig KW
Citation: Cell 2026;189(5):1287-1304.e18
Content summarized and formatted by NeuroTrials.ai.