Overcoming lung cancer immunotherapy resistance by combining nontoxic variants of IL-12 and IL-2

Abstract

Engineered cytokine-based approaches for immunotherapy of cancer are poised to enter the clinic, with IL-12 being at the forefront. However, little is known about potential mechanisms of resistance to cytokine therapies. We found that orthotopic murine lung tumors were resistant to systemically delivered IL-12 fused to murine serum albumin (MSA, IL12-MSA) because of low IL-12 receptor (IL-12R) expression on tumor-reactive CD8+ T cells. IL2-MSA increased binding of IL12-MSA by tumor-reactive CD8+ T cells, and combined administration of IL12-MSA and IL2-MSA led to enhanced tumor-reactive CD8+ T cell effector differentiation, decreased numbers of tumor-infiltrating CD4+ regulatory T cells, and increased survival of lung tumor-bearing mice. Predictably, the combination of IL-2 and IL-12 at therapeutic doses led to significant dose-limiting toxicity. Administering IL-12 and IL-2 analogs with preferential binding to cells expressing Il12rb1 and CD25, respectively, led to a significant extension of survival in mice with lung tumors while abrogating dose-limiting toxicity. These findings suggest that IL-12 and IL-2 represent a rational approach to combination cytokine therapy whose dose-limiting toxicity can be overcome with engineered cytokine variants.

Keywords: Cytokines; Immunology; Immunotherapy; T cells.

Figure 1. KP lung tumors are resistant to IL12-MSA monotherapy.

(A and B) Mice were inoculated with KP lung or flank tumors and treated intravenously with IL12-MSA on days 7 and 14 of tumor growth. (A) Survival of mice with KP lung tumors, control n = 7, IL12-MSA n = 9, pooled data from 2 independent experiments. Log-rank test. (B) Survival of mice with KP flank tumors, control n = 6, IL12-MSA n = 6, pooled data from 2 independent experiments. Log-rank test. (C–F) Mice were inoculated with KP.SIY lung or flank tumors, treated intravenously with IL12-MSA on day 7, and analyzed on day 10 of tumor growth. (C) Absolute number of SIY-reactive CD8⁺ T cells from TdLN, spleen, or tumor. (D) CD25 expression by SIY-reactive CD8⁺ T cells from TdLN, spleen, or tumor. (E) GzmB expression by SIY-reactive CD8⁺ T cells from TdLN, spleen, or tumor. (C–E) n = 12, pooled data from 4 independent experiments, 1-way ANOVA. (F) TCF-1 and TIM-3 expression by SIY-reactive CD8⁺ T cells from TdLN, spleen, or tumor. (G) The percentage of SIY-reactive CD8⁺ T cells that are PD-1⁺TCF-1⁺. (F and G) n = 6, pooled data from 2 independent experiments, 2-way ANOVA. Comparisons in F are between control and IL12-MSA–treated samples. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. TdLN, tumor-draining lymph node.

Figure 2. IL-2 increases CD8⁺ T cell binding of IL12-MSA.

(A) Example histogram plot of IL12-MSA–Alexa Fluor 647 (IL12-MSA-AF647) binding by SIY-reactive CD8⁺ T cells from KP.SIY lung or flank TdLN assessed by flow cytometry. (B and C) Quantification of IL12-MSA-AF647 binding by (B) SIY-reactive CD8⁺ T cells and (C) FoxP3⁺CD4⁺ Tregs from KP.SIY lung or flank TdLN. n = 6, pooled data from 2 independent experiments, 1-way ANOVA. (D) Experimental design. KP.SIY lung or flank tumor–bearing mice were treated on day 6 of tumor growth with IL2-MSA, and T cells were analyzed ex vivo on day 7 of tumor growth (E and F). Binding of IL12-MSA-AF647 by (E) SIY-reactive CD8⁺ T cells and (F) FoxP3⁺CD4⁺ Tregs. KP.SIY lung tumors n = 5, all conditions. KP.SIY flank tumors TdLN control = 5, TdLN IL2-MSA n = 6, spleen control n = 4, spleen IL2-MSA n = 6, tumor control n = 5, tumor IL2-MSA n = 5. Pooled data from 2 independent experiments. One-way ANOVA. (G) Naive CD8⁺ T cells were CTV labeled and primed ex vivo with agonist anti-CD3 and anti-CD28 antibodies in the presence or absence of IL-2 neutralizing antibodies. (H) Example (left) and quantification (right) of primed CD8⁺ T cell proliferation, n = 8, pooled data from 2 independent experiments, Mann-Whitney U test. (I) Example (left) and quantification (right) of IL12-MSA binding, n = 4, Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3. IL12-MSA and IL2-MSA induce synergistic changes to the phospho-proteome.

(A) Experimental schematic. Naive splenocytes were activated and expanded in vitro for 7 days. CD8⁺ T cells were enriched by magnetic separation, rested, and stimulated with IL12-MSA, IL2-MSA, or the combination. Cells were lysed, and peptides were analyzed by mass spectrometry to quantify phospho-tyrosine residues. (B–D) Volcano plots quantifying changes in phospho-tyrosine sites induced by cytokine treatments. Dotted lines: x axis, no fold-change; y axis, threshold of significance. Data in B–D were generated from 3 independent biological replicates for each condition and were analyzed for significance using multiple paired t tests. (E–G) Plots displaying the relative abundance of specific phospho-tyrosine sites across treatment conditions.

Figure 4. Combined IL12-MSA and IL2-MSA coordinates CD8⁺ T cell and Treg responses.

(A–E) Mice were inoculated with KP.SIY lung tumors, then treated with IL12-MSA, IL2-MSA, or the combination on day 7 of tumor growth. TdLNs, spleens, and lungs were analyzed on day 10 of tumor growth. (A) The fraction of CD4⁺ T cells that express FoxP3. (B) Ratio of SIY-reactive CD8⁺ T cells to Tregs. (C) Percentage of SIY-reactive CD8⁺ T cells that express CD25. (D) Percentage of SIY-reactive CD8⁺ T cells that express GzmB. (E) Percentage of SIY-reactive CD8⁺ T cells that express PD-1 and TCF-1. (A–E) TdLNs n = 5 per condition, spleens n = 6 per condition, lungs n = 6 per condition, combined data from 2 independent experiments. (A and B) One-way ANOVA. (C–E) Two-way ANOVA. (F and G) Mice were inoculated with KP lung tumors, then treated with IL12-MSA, IL2-MSA, or the combination on days 7 and 14 of tumor growth. (F) Survival of KP lung tumor–bearing mice, n = 5 per condition, log-rank test (Mantel-Cox). Representative data from 2 experiments. (G) Weight loss of KP lung tumor–bearing mice treated with IL12-MSA, IL2-MSA, or the combination on day 7. Weight loss was measured from day 7 to day 14 of tumor growth. Shown statistical comparisons are with the control group, n = 5 per condition, 2-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5. Combined administration of IL-12 and IL-2 variants extends survival of KP lung tumor–bearing mice without toxicity.

(A–D) Mice were inoculated with KP.SIY lung tumors and treated with IL2-MSA or IL2-REH-MSA in vivo on day 6, and then T cells from TdLNs, spleen, and lungs were analyzed on day 7. (A) Binding of IL12-MSA-AF647 by SIY-reactive CD8⁺ T cells. (B) Binding of IL12-MSA-AF647 by bystander, SIY-nonreactive CD8⁺ T cells. (C) The percentage of CD4⁺ T cells that are FoxP3⁺ Tregs. (D) Binding of IL12-MSA-AF647 by CD4⁺FoxP3⁺ Tregs. (A–D) n = 6. Combined data from 2 independent experiments. One-way ANOVA. (E and F) Mice were inoculated with KP lung tumors and treated with either IL12-MSA and IL2-MSA or IL2-REH-MSA and IL12-3XA-MSA on days 7 and 14 of tumor growth. (E) Weight loss of KP lung tumor–bearing mice treated with either IL12-MSA and IL2-MSA or IL12-3XA-MSA and IL2-REH-MSA on day 7. Weight loss was measured from day 7 to day 14 of tumor growth. (F) Survival of KP lung tumor–bearing mice treated with either IL12-MSA and IL2-MSA or IL2-REH-MSA and IL12-3XA-MSA. (E and F) Control n = 15, IL12-MSA + IL2-MSA n = 15, IL2-REH-MSA + IL12-3XA-MSA 54 n = 5, IL2-REH-MSA + IL12-3XA-MSA 30 n = 5, IL2-REH-MSA + IL12-3XA-MSA 15 n = 10, combined data from 3 independent experiments. (E) Two-way ANOVA. (F) Log-rank test (Mantel-Cox). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.