Cancer Immunology Research (AACR)2020ResearchNon-viral Gene Delivery

Optimization of T-cell Receptor–Modified T Cells for Cancer Therapy

Dylan J. Drakes, Sarwish Rafiq, Terence J. Purdon, Andrea V. Lopez, Smita S. Chandran, Christopher A. Klebanoff, Renier J. Brentjens29 September 2026DOI 10.1158/2326-6066.CIR-19-0910

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Summary

T-cell receptor (TCR)-modified T-cell gene therapy can target a variety of extracellular and intracellular tumour-associated antigens, yet has had little clinical success. A potential explanation for limited antitumor efficacy is a lack of T-cell activation in vivo, particularly when tumour cells downregulate costimulatory molecules. A method to provide a durable proinflammatory signal to TCR-modified T cells could enhance persistence, effector. ### In Vitro T Cell Activation & Cytotoxicity | Parameter | Control | mIL12 pmel-1 | mIL18 pmel-1 | |---------------|-------------|------------------|------------------| | IFNγ secretion | Baseline | Significantly.

Keywords

T cells TCR signalling Tumour microenvironment Macrophages Immune cells Nanocarriers Gene delivery

Purpose: T-cell receptor (TCR)-modified T-cell gene therapy can target a variety of extracellular and intracellular tumour-associated antigens, yet has had little clinical success. A potential explanation for limited antitumor efficacy is a lack of T-cell activation in vivo, particularly when tumour cells downregulate costimulatory molecules. A method to provide a durable proinflammatory signal to TCR-modified T cells could enhance persistence, effector function, and antitumor efficacy while avoiding toxicity.

Hypothesis: Expression of proinflammatory cytokines (IL18 or IL12) in tumour-directed TCR-modified T cells will activate T cells and enhance antitumor efficacy. IL18 will provide a superior and more durable proinflammatory signal than IL12, promoting persistent and functional effector T cells, a proinflammatory tumour microenvironment, and improved survival without the toxicity associated with IL12 secretion, particularly when combined with lymphodepleting preconditioning.

Aims: 1. Compare IL18 vs. IL12 as proinflammatory signals for enhancing the effector function of TCR-modified T cells in vitro 2. Evaluate in vivo antitumor efficacy of IL18- and IL12-secreting pmel-1 TCR T cells in syngeneic B16F10 melanoma model 3. Characterise the tumour microenvironment and T cell persistence after adoptive transfer of cytokine-armored T cells 4. Test the combination of IL18-secreting T cells with sublethal irradiation for tumour eradication 5. Determine whether IL18 acts in cis (on adoptively transferred T cells) or in trans (on host immune cells) 6. Demonstrate translational relevance using human T cells transduced with NY-ESO-1-specific TCR (1G4) secreting human IL18

5. Biological System:

Component: TCR System (Murine); Description: pmel-1 TCR transgenic T cells — recognise gp100 melanoma antigen in H-2Dᵇ context

Component: TCR System (Human); Description: 1G4 TCR — recognises NY-ESO-1 (SLLMWITQC) in HLA-A02:01 context

Component: Cytokine Constructs (Murine); Description: SFG retroviral vector with mCD19t (truncated CD19, transduction marker) + either: • mIL12 (IRES; α and β subunit fusion) • mIL18 (P2A self-cleaving peptide; mature form)

Component: Cytokine Constructs (Human); Description: SFG retroviral vector with 1G4 TCR α/β chains + equine 2A + signal peptide + mature hIL18

Component: Tumour Models; Description: • Syngeneic: C57BL/6 mice; B16F10 melanoma (gp100⁺, CD80/86⁻) • Xenograft: NSG mice; A375 human melanoma (HLA-A2⁺, NY-ESO-1⁺)

Component: Host Models; Description: • Wild-type C57BL/6 • CD80/86⁻/⁻ (costimulation-deficient) • IL18R⁻/⁻ (for cis vs. trans experiments) • pmel-1 × IL18R⁻/⁻ donor T cells

Component: Transduction; Description: Retroviral spinoculation (Phoenix-ECO for murine; 293 galv9 for human); RetroNectin plates

Component: Preconditioning; Description: 5 Gy total body irradiation (sublethal)

Component: Dosing; Description: • Murine: 5×10⁶ T cells i.v.; weekly ×3 or single infusion • Human: 2.5×10⁶ T cells i.v.; single infusion

Approach:

Parameter: In Vitro Studies; Details: • Cytokine secretion (Luminex) • Cytotoxicity: luciferase-based killing assay (24 h) • CD25 expression (flow cytometry) • IFNγ secretion (ELISA/Luminex)

Parameter: In Vivo Syngeneic Model; Details: B16F10 s.c. (day -10); T cell infusion (day 0, 7, 14); tumour volume measured; survival monitored; endpoint >1,000 mm³

Parameter: Irradiation Studies; Details: 5 Gy total body irradiation (day -1); single T cell infusion (day 0) or weekly ×3

Parameter: Tumour Microenvironment Analysis; Details: Tumour dissociation; flow cytometry: CD206⁺ M2 macrophages, Gr1⁺ MDSCs, CD11c⁺MHCII⁺ DCs, Thy1.1⁺ pmel-1 T cells; day 6 and 13 post-infusion

Parameter: Ex Vivo T Cell Function; Details: FACS-sorted Thy1.1⁺ T cells; coculture with B16F10 GFP/luc; IFNγ secretion (Luminex); cytotoxicity (luciferase loss)

Parameter: Cis vs. Trans Experiment; Details: pmel-1⁺/⁺ IL18R⁻/⁻ donor T cells; C57BL/6 or IL18R⁻/⁻ hosts; assess tumour growth and survival

Parameter: Human Xenograft Model; Details: NSG mice; A375 s.c. (day -10); 1G4 or 1G4hIL18 T cells (day 0); peripheral blood T cell counts (day 7); tumour volume and survival

Parameter: Controls; Details: mCD19t-only T cells (murine); mock-transduced T cells (human); no T cell control

Parameter: Sample Sizes; Details: n=4-17 per group (as indicated)

Parameter: Statistical Tests; Details: Unpaired two-tailed t-test; one-way/two-way ANOVA; Mann-Whitney (tumour volume); log-rank (survival); P<0.05 significant

Key methods:

Analysis Category: Cytokine Secretion; Methods: Luminex (Millipore; FlexMap3D); IFNγ, IL2, IL12, IL18, TNFα, IL6, IL10

Analysis Category: Cytotoxicity; Methods: Luciferase-based killing assay (Spark plate reader); % lysis = [1 - (sample/max)] × 100

Analysis Category: Flow Cytometry; Methods: Gallios (10-color) or Attune NxT (14-color); antibodies for mouse (CD3, CD4, CD8, CD44, CD62L, CD25, PD-1, TIM-3, LAG-3, CD80, CD86, CD206, Gr-1, CD11b, CD11c, MHCII, Thy1.1, FoxP3, IL18R) and human (CD3, CD4, CD8, CD80, CD86, HLA-A2, NY-ESO-1 tetramer); DAPI or LIVE/DEAD for viability; 123count eBeads for quantification

Analysis Category: T Cell Persistence; Methods: Flow cytometry: Thy1.1⁺ pmel-1 T cells in tumour, spleen, blood; human 1G4 T cells in peripheral blood

Analysis Category: Intracellular Staining; Methods: Foxp3/Transcription factor staining buffer set (eBioscience)

Analysis Category: Serum Analysis; Methods: Retro-orbital bleeds; serum cytokines by Luminex

Analysis Category: Ex Vivo T Cell Function; Methods: FACS sorting of Thy1.1⁺ T cells from tumours; coculture with B16F10; IFNγ secretion; cytotoxicity

Key results: ### In Vitro T Cell Activation & Cytotoxicity

Parameter: IFNγ secretion; Control: Baseline; mIL12 pmel-1: Significantly increased; mIL18 pmel-1: Significantly increased

Parameter: CD25 expression; Control: Baseline; mIL12 pmel-1: ↑; mIL18 pmel-1: ↑

Parameter: Cytotoxicity (vs. B16F10); Control: Low; mIL12 pmel-1: Highest; mIL18 pmel-1: Intermediate (enhanced vs. control)

In Vivo Syngeneic Model (No Preconditioning):

Parameter: Tumour growth delay; Control: Minimal; mIL12 pmel-1: Significant; mIL18 pmel-1: Most significant

Parameter: Overall survival; Control: Baseline; mIL12 pmel-1: Enhanced; mIL18 pmel-1: Significantly enhanced vs. mIL12

Parameter: CD206⁺ M2 macrophages in tumour; Control: High; mIL12 pmel-1: Reduced; mIL18 pmel-1: Fewest

Parameter: Gr1⁺ MDSCs; Control: High; mIL12 pmel-1: Reduced; mIL18 pmel-1: Significantly reduced

Parameter: CD11c⁺MHCII⁺ DCs; Control: Low; mIL12 pmel-1: Moderate; mIL18 pmel-1: Significantly increased

Parameter: Thy1.1⁺ pmel-1 T cells in tumour; Control: Low; mIL12 pmel-1: Moderate; mIL18 pmel-1: Highest accumulation

T Cell Persistence & Function (Ex Vivo):

Parameter: Thy1.1⁺ T cell accumulation; Day 6: mIL18 > mIL12 = control; Day 13: mIL18 only maintained

Parameter: PD-1⁺ (activation); Day 6: All groups; Day 13: All groups

Parameter: PD-1⁺TIM-3⁺LAG-3⁺ (dysfunction); Day 6: mIL12 highest; Day 13: mIL18 lowest

Parameter: IFNγ secretion; Day 6: mIL12 & mIL18 enhanced; Day 13: mIL18 only maintained

Parameter: Cytotoxicity; Day 6: mIL12 & mIL18 enhanced; Day 13: mIL18 only maintained

CD80:

Parameter: Tumour growth; Control: Rapid; mIL12 pmel-1: Delayed; mIL18 pmel-1: Most delayed (comparable to WT)

Parameter: Survival; Control: Baseline; mIL12 pmel-1: Enhanced; mIL18 pmel-1: Significantly enhanced

Sublethal Irradiation (5 Gy) + T Cell Therapy:

Parameter: Tumour eradication; Control: None; mIL12 pmel-1: Toxicity (100% mortality); mIL18 pmel-1: 8/9 (88.8%) cured

Parameter: Serum IL12; Control: Low; mIL12 pmel-1: Extremely high; mIL18 pmel-1: Low

Parameter: Serum IFNγ, TNFα, IL6, IL10; Control: Low; mIL12 pmel-1: Extremely high (cytokine storm); mIL18 pmel-1: Elevated (tolerated)

Parameter: Tregs in tumour; Control: Present; mIL12 pmel-1: -; mIL18 pmel-1: Significantly reduced

Parameter: Granulocytic MDSCs; Control: Present; mIL12 pmel-1: -; mIL18 pmel-1: Significantly reduced

Parameter: Thy1.1⁺ pmel-1 T cells; Control: Low; mIL12 pmel-1: -; mIL18 pmel-1: Highest

IL18 Receptor Dependency (Cis vs. Trans):

Donor T Cells: pmel-1 (IL18R⁺); Host: WT; mIL18 Effect: Survival enhanced

Donor T Cells: pmel-1 (IL18R⁺); Host: IL18R⁻/⁻; mIL18 Effect: Survival enhanced

Donor T Cells: pmel-1 × IL18R⁻/⁻; Host: WT; mIL18 Effect: No survival benefit

Donor T Cells: pmel-1 × IL18R⁻/⁻; Host: IL18R⁻/⁻; mIL18 Effect: No survival benefit

Donor T Cells: Conclusion; Host: IL18 acts in cis (on adoptively transferred T cells)

Human TCR System (1G4 NY-ESO-1 + hIL18):

Parameter: hIL18 secretion; Mock: None; 1G4: None; 1G4hIL18: Significant

Parameter: IFNγ secretion; Mock: Baseline; 1G4: Enhanced; 1G4hIL18: Enhanced

Parameter: IL2 secretion; Mock: Baseline; 1G4: Enhanced; 1G4hIL18: Enhanced

Parameter: Cytotoxicity (vs. A375); Mock: Low; 1G4: Enhanced; 1G4hIL18: Enhanced

Parameter: T cell persistence in blood (day 7); Mock: Low; 1G4: Moderate; 1G4hIL18: Highest

Parameter: Tumour growth (A375 xenograft); Mock: Rapid; 1G4: Delayed; 1G4hIL18: Most delayed

Parameter: Overall survival; Mock: Baseline; 1G4: Enhanced; 1G4hIL18: Significantly enhanced

Interpretation: The authors conclude that "IL18 provides a proinflammatory signal to T cells" and "IL18-engineered TCR-modified T cells promote an enhanced and persistent effector T-cell response." They state: "These results demonstrate a rationale for optimising the efficacy of TCR-modified T-cell cancer therapy through expression of IL18." The study establishes that "IL18 is a superior and safer proinflammatory signal to tumour-directed T cells" compared to IL12, particularly in the context of lymphodepleting preconditioning, where IL12 caused fatal toxicity. The authors highlight that "IL18 acts in cis" on adoptively transferred T cells, and that "1G4hIL18 TCR T cells were further shown to persist and enhance antitumor efficacy resulting in long-term survival of tumour-bearing mice in a xenograft model," providing "a rationale for the clinical application of IL18-armored TCR-modified T cells."

10. Limitations (Explicitly Stated or Evident):

1. Model antigen and tumour systems: The study uses model antigens (gp100, NY-ESO-1) and established cell lines; efficacy against patient-derived tumours or neoantigens was not tested.

2. Constitutive cytokine expression: IL18 was expressed constitutively from the LTR promoter, not under an inducible system. While no toxicity was observed in immune-competent mice, long-term safety of constitutive IL18 secretion remains to be established.

3. IL12 toxicity with preconditioning: IL12-secreting T cells were toxic only when combined with irradiation; the study did not explore lower doses of IL12 or more controlled expression systems.

4. Human xenograft model limitations: NSG mice lack a functional immune system; the human system studies did not assess effects on endogenous immune cells, and human T cells were not tested in immunocompetent models.

5. No direct comparison of IL18 vs. IL12 in human system: The human studies only tested IL18; IL12 was not tested in the 1G4 system, limiting direct translational comparison.

6. No assessment of tumour antigen escape or recurrence: Long-term follow-up beyond the study endpoint was not reported; potential for antigen loss variants was not evaluated.

7. Cytokine receptor expression not fully characterised: While IL18R expression was confirmed on pmel-1 T cells, the study did not comprehensively characterise IL18R expression on different T cell subsets or over time after adoptive transfer.

8. Single tumour model per system: Only B16F10 (melanoma) for murine and A375 (melanoma) for human; efficacy in other tumour types was not demonstrated.

9. No evaluation of IL18 on Tregs: IL18 can affect Treg function; this was not extensively characterised.

10. Ex vivo analyses limited to two time points: Day 6 and day 13 were the only time points examined; the kinetics of T cell persistence and dysfunction between these time points were not fully mapped.

11. Preconditioning regimen not optimised: Only one irradiation dose (5 Gy) was tested; dose-response and other lymphodepleting regimens were not explored.

12. No in vivo imaging: T cell trafficking and tumour localisation were inferred from tumour digestion, not directly visualised.

Report prepared based on the published Cancer Immunology Research article. For full experimental details, supplementary figures, and complete references, please refer to the original publication.

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