Adoptive transfer of membrane-restricted IL-12-TCR T cells promotes antigen spreading and elimination of antigen-negative tumor variants

Abstract

Background: Adoptive T-cell therapy has demonstrated clinical activity in B-cell malignancies, offering hope for its application to a broad spectrum of cancers. However, a significant portion of patients with solid tumors experience primary or secondary resistance to this treatment modality. Target antigen loss resulting either from non-uniform antigen expression or defects in antigen processing and presentation machinery is one well-characterized resistance mechanism. Constitutively expressed membrane-anchored interleukin-12 (caIL-12) has demonstrated enhanced antitumor activity and low systemic exposure in multiple preclinical adoptive T-cell treatment models with homogeneous tumor antigen expression. In this study, we assess the therapeutic impact of caIL-12 on target antigen-negative variants in syngeneic mouse models.

Methods: Target antigen-positive tumors were generated by transducing B16F10 melanoma cells (B16) or Lewis Lung Carcinoma cells (LLC) with a construct expressing the OVA antigen, SIINFEKL, tagged to ubiquitin (B16-U-OVA, LLC-U-OVA), while B16 or LLC tumors served as antigen-negative variants. C57BL/6J mice were subcutaneously injected with heterogeneous tumors composed of 80% B16-U-OVA and 20% B16. Bilateral tumors were established by injecting the left flank with B16-U-OVA or LLC-U-OVA tumors and the right flank injected with B16 or LLC tumors. The tumor-bearing mice then underwent 5.5 Gy total body irradiation, followed by adoptive transfer of OT-I TCR-T cells engineered with or without caIL-12.

Results: TCR-T cells (OT-I) delivered caIL-12 to the B16-U-OVA tumor sites and induced robust tumor control and survival benefits in mice bearing a heterogeneous tumor with OVA-negative variants. caIL-12 exerted its effect on OVA-negative B16 variants primarily by priming and activating endogenous antitumor CD8 T cells via antigen spreading. In addition, antigen spreading induced by OT-I-caIL-12 resulted in controlling OVA-negative tumors implanted at distant sites. This therapeutic effect required antigen-specific TCR-T cells and caIL-12 to colocalize at the tumor site, along with endogenous CD8 T cells capable of recognizing shared tumor antigens.

Conclusion: Expression of caIL-12 by tumor-targeting T cells demonstrated therapeutic effect against target-antigen-negative tumor variants, primarily through the induction of antigen spreading. These findings highlight the potential of caIL-12 to address challenges of antigen escape and tumor heterogeneity that may limit the efficacy of T-cell therapy against solid tumors.

Keywords: Adoptive cell therapy - ACT; Cytokine; Solid tumor; T cell Receptor - TCR; Tumor infiltrating lymphocyte - TIL.

Figure 1. OT-I T cells engineered with membrane-anchored IL-12 demonstrated enhanced tumor regression in tumors with pre-existing OVA-negative variants. (A) Experimental schema of treatment. (B) Tumor growth curve of all treatment groups. All mice received 5.5 Gy of whole-body radiation before cell infusion. P values represent the comparison between OT-I-control and OT-I-caIL-12 using a matched-pairs, repeated-measures, two-way ANOVA test. *p<0.05, **p<0.01, ***p<0.001. (C) 100% B16-U-OVA-positive tumor or (D) 80% B16-U-OVA/20% B16 mixed tumor-bearing mice underwent OT-I-control or OT-I-caIL-12 T cell transfer. Tumors were measured twice a week. N=5 mice per group. (E) The survival curve of tumor-bearing mice that underwent the same T-cell transfer. The significance is tested by the log-rank (Mantel-Cox) test. **p<0.01. ANOVA, analysis of variance. TBI, totoal body irradiation.

Figure 2. Antitumor activity of OT-I-caIL-12 is associated with an increase of activated tumor-infiltrating endogenous CD8 T cells. (A) Schedule of experimental design. (B) Tumor growth curve of mice with a heterogeneous OVA antigen tumor who received OT-I or OT-I-caIL-12 T-cell transfer. P values represent the comparison between OT-I-control and OT-I-caIL-12 using a matched-pairs, repeated-measures, two-way ANOVA test. *p<0.05, **p<0.01, ***p<0.001. (C) Percentage of caIL-12 on transferred T cells in the TME following ACT. D0, transferring T cells. (D) Representative flow cytometric analysis of the total number of T cells, (E) CD8 T cells, (F) CD4 T cells, (G) transferred OT-I CD8 T cells, and (H) endogenous CD8 T cells infiltrated within the TME at day 5, day 13, and day 19 post-T-cell transfer. *p<0.05 (I) Summary of the frequency of CD4 T cells, transferred OT-I CD8 T cells, and endogenous CD8 T cells in the TME of tumor-bearing mice that underwent OT-I or OT-I-caIL-12 treatment. (J) Tumor-bearing mice received antibody-targeting CD4 and NK cells via IP injection on the same day as T-cell transfer; this was repeated every 3–4 days until the endpoint. Tumor growth curve (K) and mice survival curve (L) of tumor-bearing mice that underwent indicated treatments. **P<0.01. ANOVA, analysis of variance. TBI, total body irradiation.

Figure 3. OT-I-caIL12 treatment enhances endogenous CD8 T cells function intrinsically. (A) TILs were isolated on day 8 post-T-cell treatment and processed to single-cell sequencing and α/β paired TCR sequencing. (B) Uniform manifold approximation and projection (UMAP) of total T cells harvested from a tumor on day 8 post-T-cell transfer and the projection of cells split by the experimental group. Clusters were curated based on signature gene expression profiles. (C) Transferred OT-I T cells were identified by α/β paired TCR sequencing and were separated from endogenous T cells in the UMAP. (D) Stacked bar charts showing the proportion of each T-cell phenotype that underwent indicated treatments.

Figure 4. Control of OVA-negative contralateral tumor by OT-I-caIL-12 is associated with increased tumor-infiltrating CD8 T cells. (A) Schema of bilateral tumor treatment model. (B) C57BL/6J mice were implanted with 1×10⁶ B16-U-OVA tumor at the left flank and 1×10⁵ B16 tumor at the right flank. On day 7, tumor-bearing mice were given 5.5 Gy total-body irradiation followed by T-cell transfer. Tumor growth curves of both B16-U-OVA and B16 were measured twice a week by caliper. n=5. P values represent the comparison between OT-I-control and OT-I-caIL-12 using a matched-pairs, repeated-measures two-way ANOVA test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (C) C57BL/6J mice were implanted with 2×10⁶ LLC-U-OVA tumor at the right flank and 2×10⁵ LLC tumor at the left flank. After day 7, tumor-bearing mice were given 5.5 Gy total-body irradiation followed by T-cell transfer. Tumor growth curves of both LLC-U-OVA and LLC were measured twice a week by caliper. N=5. P values represent the comparison between OT-I-control and OT-I-caIL-12 using a matched-pairs, repeated-measures, two-way ANOVA test. *p<0.05, **p<0.01, ***p<0.001. (D) On day 8 post-T-cell transfer, B16 tumors from either the OT-I-control or OT-I-caIL-12 treatment groups were reset and fixed. The tumor sections were then stained with CD45 (yellow), CD8 (blue), GzmB (green), CD98 (red), and DAPI (gray). (E) Densities of CD45+CD8+ cells and (F) CD45+CD8+GzmB cells were quantified at the core, the edge, and the total tumor section, *p<0.05, ***p<0.001. ANOVA, analysis of variance; LLC, Lewis lung carcinoma. TBI, totoal body irradiation.

Figure 5. caIL-12 induces contralateral OVA-negative tumor regression via antigen spreading. (A) Schema of treatment schedule. (B) C57BL/6 J mice were implanted with B16-U-OVA or B16 tumors on different flanks and then underwent T-cell transfer. The tumor growth curve of each tumor was measured twice a week. (C) On day 13 post-treatment, non-targeted B16 tumors were harvested and weighed. TILs were processed in vitro and analyzed by flow cytometry. (D) The number of endogenous cells, (E) transferred T cells, (F) gp100 tetramer+cells, and (G) Trp2 tetramer+cells were counted and calculated based on tumor weight. (H) Schema of treatment schedule. (I) Mice were implanted with 1×10⁶ B16-U-OVA on the left flank and 2×10⁵ LLC tumor on the right flank. On day 7, tumor-bearing mice were treated with OT-I-control T cells or OT-I-caIL-12 T cells. The tumor growth curve was measured twice per week. (J) Mice were implanted with 1×10⁶ B16-U-OVA tumor on the left flank and 1×10⁵ B16-β2MKO tumor on the right flank. On day 7, tumor-bearing mice were treated with OT-I-control T cells or OT-I-caIL-12 T cells. The tumor growth curve was measured twice per week. Tumor-bearing mice were euthanized if either side of the tumor reached the endpoint. N=5. P values represent the comparison between indicated treatment groups using a matched-pairs, repeated-measures two-way ANOVA test (multiple comparison). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; LLC, Lewis lung carcinoma. TBI, totoal body irradiation.

Figure 6. TCR-caIL-12 therapy triggers antigen spreading to eliminate TCR-target antigen-negative variants.