CAN-3110
(2026)Objective
Characterize spatial T cell programs, clonal dynamics, and resistance mechanisms in recurrent glioblastoma after a single intratumoral oncolytic herpes simplex virus (rQNestin34.5v.2 / CAN-3110) injection.
Study Summary
• Expansion of pre-existing tumor-infiltrating T cell clones associated with longer overall survival: median OS 445 vs 235 days (log-rank p=0.033)
• No new adverse event data reported; residual HSV confined to necrotic regions (nucleic acids 4/8 samples by Xenium; protein 32.1% [9/28] regions by IHC)
Intervention
Single intratumoral injection of rQNestin34.5v.2 (CAN-3110, linoserpaturev) — a genetically engineered oncolytic HSV-1 in which expression of viral ICP34.5 is restricted by the nestin promoter, enabling selective replication in nestin-high GBM cells.
Inclusion Criteria
Analyzed cohort (paired samples from parent phase 1 trial NCT03152318, arm A): adults with recurrent glioblastoma at first to fourth recurrence; age 27–65 years; baseline KPS 70–100; 8 female / 8 male; IDH-mutant in 5/16; HSV-1 seropositive pre-treatment in 8/16; all of European ancestry; paired pre-treatment biopsy and post-treatment resection with adequate FFPE tissue for CODEX and/or Xenium. Formal trial-level eligibility criteria are not re-stated in this publication.
Study Design
Arms: Single-arm, within-patient paired pre- vs post-oHSV analysis of tissue from parent phase 1 trial (arm A of NCT03152318); no separate control arm.
Patients per Arm: 16 patients contributed paired pre/post tumor tissue (CODEX cohort); 8 of 16 had matched Xenium spatial transcriptomics; bulk TCRβ sequencing from 33 patients (parent trial cohort). Single treated cohort with within-patient paired samples.
Outcome
• Post-oHSV, substantial increase in CD8, CD4, and B cell infiltration; CD8/Treg ratio 6.8 → 21.1 (p=0.0042); T cell density 30.4 → 138 cells/mm² by Xenium (p=0.0078)
• Survival correlate: shorter average distance from tumor cells to GZMBhi-med T cells (CODEX, Fig 2I) associated with above-median PFS (175 vs 262 µm, p=0.038) and lower tumor growth rate (p=0.028)
• Clonal expansion: patients with expanded shared TCR repertoire had median OS 445 vs 235 days (log-rank p=0.033); productive clonality increased in tumor (p=0.032) but stable in PBMC (p=0.62)
• Tissue-resident CD8 T cells showed largest post-treatment increase (log2FC 3.03, p=0.0078); plasma cells log2FC 3.47 (p=0.0156)
• HSV nucleic acids detected in 4/8 post-treatment samples by Xenium (possibly residual viral DNA per authors) and HSV protein detected by IHC in 32.1% (9/28) of post-treatment regions; both restricted to necrotic regions; T cells depleted from HSV+ zones (log2FC −3.47 CD8, −2.01 CD4)
• Hypoxic MES-like 2 tumor cells expanded post-treatment (log2FC 1.38, p=0.0078) and excluded T cells (constituting up to 78% of tumor cells in most distant zones)
Clinical Question
Does a single intratumoral injection of the oncolytic HSV-1 rQNestin34.5v.2 (CAN-3110) induce durable, tumor-directed T cell-mediated immunity in recurrent glioblastoma, and what spatial and clonal features correlate with patient outcomes?
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 average distances from tumor cells to GZMBhi-med T cells (CODEX, Fig 2I: 175 vs 262 µm, p=0.038) 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); T cell density 30.4 → 138 cells/mm² by Xenium (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 (p=0.62); up to 1,024-fold local intratumoral expansion of individual clones.
- Shorter average distance from tumor cells to GZMBhi-med T cells (CODEX, Fig 2I) correlated with above-median PFS (175 vs 262 µm, p=0.038) and lower tumor growth rate (p=0.028); GZMB+ CD8 T cells were captured in situ in close proximity to cleaved caspase-3+ apoptotic tumor cells, consistent with cytotoxicity.
- 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 to tumor cells) interacted directly with tumor cells with tissue-resident (ITGAE/CD103, ZNF683/HOBIT) and early activation (NR4A1/Nur77, CD69, IFNG, TNF) programs.
- Residual HSV was restricted to necrotic regions: HSV nucleic acids (Xenium; possibly residual viral DNA rather than RNA per authors) were detected in 4 of 8 post-treatment samples, and HSV protein by IHC was detected in 32.1% (9/28) of examined post-treatment regions; 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 (Pearson 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.
Study Design
- Study Type
- Translational spatial immunology analysis of phase 1 single-arm clinical trial samples (arm A of NCT03152318)
- Randomization
- No
- Blinding
- Open-label phase 1 trial; spatial/molecular analyses not blinded
- Sample Size
- 16
- Follow-up
- Post-treatment tissue collected across months 6 to 25 following single oHSV injection (>2 years in one patient, P32); additional early samples 24–331 days (Figure 2B); the earliest and latest post-treatment resections shown in Figure 2D correspond to P46 (15 days) and P32 (801 days)
- Centers
- 1
- Countries
- USA
Primary Outcome
Definition: No formally pre-specified primary endpoint (translational spatial analysis); paper's stated goal was to investigate spatial interactions of T cells with tumor and other immune cells. Key primary observation: post-oHSV increase in intratumoral T cell density from a median of 30.4 to 138 cells/mm² (paired Xenium)
| Control | Intervention | HR/OR | P-value |
|---|---|---|---|
| 30.4 cells/mm² (pre-treatment median, Xenium; within-patient baseline) | 138 cells/mm² (post-treatment median, Xenium) | - (Not reported) | 0.0078 (paired Wilcoxon) |
Limitations & Criticisms
- Single treated cohort, n=16 with paired pre/post within-patient comparison — no separate 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.
- No adverse event / toxicity data are re-reported in this publication; safety must be inferred from the parent trial (Ling et al., Nature 2023).
Citation
Cell 2026;189(5):1287-1304.e7