Collagen-Anchored Interleukin-2 and Interleukin-12 Safely Reprogram the Tumor Microenvironment in Canine Soft-Tissue Sarcomas

Abstract

Purpose: Cytokine therapies such as IL2 and IL12 suffer from impractically small therapeutic windows driven by their on-target, off-tumor activity, limiting their clinical potential despite potent antitumor effects. We previously engineered cytokines that bind and anchor to tumor collagen following intratumoral injection, and sought to test their safety and biomarker activity in spontaneous canine soft-tissue sarcomas (STS).

Experimental design: Collagen-binding cytokines were canine-ized to minimize immunogenicity and were used in a rapid dose-escalation study in healthy beagles to identify a maximum tolerated dose. Ten client-owned pet dogs with STS were then enrolled into trial, receiving cytokines at different intervals prior to surgical tumor excision. Tumor tissue was analyzed through IHC and NanoString RNA profiling for dynamic changes within treated tumors. Archived, untreated STS samples were analyzed in parallel as controls.

Results: Intratumorally administered collagen-binding IL2 and IL12 were well tolerated by STS-bearing dogs, with only Grade 1/2 adverse events observed (mild fever, thrombocytopenia, neutropenia). IHC revealed enhanced T-cell infiltrates, corroborated by an enhancement in gene expression associated with cytotoxic immune function. We found concordant increases in expression of counter-regulatory genes that we hypothesize would contribute to a transient antitumor effect, and confirmed in mouse models that combination therapy to inhibit this counter-regulation can improve responses to cytokine therapy.

Conclusions: These results support the safety and activity of intratumorally delivered, collagen-anchoring cytokines for inflammatory polarization of the canine STS tumor microenvironment. We are further evaluating the efficacy of this approach in additional canine cancers, including oral malignant melanoma.

Figure 1.. Characterization and activity of canine collagen-binding cytokines

(A) Schematic of IL-2 and IL-12 (single-chain) fusion proteins. (B) Purified canine cytokines on a non-reducing SDS-PAGE gel with Coomassie blue (Simple Blue) stain. (C-D) Size exclusion chromatograms of 100 μg purified canine cytokines using a Superdex 200 Increase 10/300 GL column. Black bars above the trace signify the fractions isolated, filtered, frozen and used for therapeutic injections. (E) IL-12 bioactivity as measured by the absorbance readout for the HEK-blue IL12 reporter cells. (F) IL-2 bioactivity measured via CTLL-2 proliferation. CTLL-2 cells were incubated with indicated protein for 2 days in incomplete media, then cell viability was measured using CellTiter-Glo. (G) LAIR fusion protein binding to rat collagen type I was measured by enzyme-linked immunosorbent assay. MSA (E, G) or mLAIR-MSA (F) serve as a nonspecific protein controls.

Figure 2.. Dose-escalation study in healthy beagles determines maximum tolerated dose (MTD).

(A) Dogs were injected intradermally (i.d.) with escalating doses of cytokines on days 0 and 14. A single dog was treated at each increasing dose level until toxicity was observed (MTD), then 2 additional dogs (one de-escalated, one naïve) were treated at the previous dose (n=4 total dogs). In total, 9 doses of cytokine were administered. (B) Heatmaps of white blood cell count (WBC), neutrophil count, platelet count, and alanine transaminase (ALT) levels measured via bloodwork at indicated timepoints (NR: normal range). (C) Serum from the indicated time points after dosing was collected and analyzed for cytokines and chemokines. Heatmap columns show average data for sera from each dose level (n per group as indicated), reported as log10 fold change in concentration compared to pre-treatment (t=0hr). Raw data are in Supplementary Data File.

Figure 3.. Collagen-anchored cytokines show safe toxicity profile in soft tissue sarcoma dogs.

(A) Dogs were injected intratumorally (i.t.) with cytokines (yellow arrows), then surgically resected (black arrow) on the schedules shown (patient information in SI-T3, T4). All dogs dosed at 17.4 ug/kg cLAIR-CSA-cIL-2 and 0.041 mg/m² cIL-12-CSA-cLAIR. Dogs were monitored for 48 hours post-treatment. (B) Heatmaps of white blood cell (WBC), neutrophil, and platelet count, and alanine transaminase (ALT) levels measured via bloodwork at indicated timepoints (NR: normal range). (C) Body temperature was measured at indicated time points. (B-C) Heatmap rows represent individual patients. (D) Serum from the indicated time points after dosing was collected and analyzed for cytokines and chemokines. Heatmap columns show average data for sera from each dose level (n per group as indicated), reported as log10 fold change in concentration compared to pre-treatment (t=0hr). Bloodwork values and serum analyte concentrations are available in the Supplementary Data File.

Figure 4.. Cytokine treatment increases abundance of tumor-infiltrating lymphocytes and monocytes.

Immunohistochemical staining for CD3 and Iba-1 was performed on a single section from each patient within each cohort. Representative images for a single patient in each cohort are shown. Inflammatory cells corresponding to (A) CD3+ (T lymphocytes), (B) Iba-1+ immunohistochemical staining were quantified. The average count for each stained slide (1 per patient) with each cohort was calculated by taking the mean of the positive cell count from seven fields per sample. Each field of quantification is 0.34 mm². Scale bar = 100 μm.

Figure 5.. Nanostring RNA profiling of canine soft tissue sarcomas

(add more description) (A) Volcano plots for cohorts 1 (C1), 2 (C2), and 3 (C3). Fold-change is determined relative to untreated control data. Genes associated with significant p-adj values (< 0.05) are highlighted in red and quantified for each cohort. (B) Venn diagram of all significant differentially expressed (DE) genes highlighted in (A), grouped by cohort in which they are found. C2 displays no significant DE genes and is therefore not represented. The top 10 to 20 DE genes per category are listed by absolute magnitude of fold-change in expression. (C) Pathway scoring for Nanostring annotated pathways involved in canine immune response. Pathway scores are calculated as the first principal component of the pathway genes’ normalized expression. Heatmap columns represent individual patient soft tissue sarcomas. (D) Z-scored expression data for top DE genes (15 max) in select pathways associated with IL-2/IL-12 activity. Heatmap rows represent individual patients. (E) Normalized expression (log 2) of counter-regulatory genes (one-way ANOVA with Dunnett’s multiple comparisons test; *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

Figure 6.. Nanostring RNA profiling of murine melanoma reveals conserved and distinct responses to cytokine therapy

(A) Mice were inoculated with 10⁶ B16F10 cells, injected intratumorally (i.t.) with cytokines (yellow arrows), then euthanized for tumor analysis (black arrow) on the schedules shown. mLAIR-MSA-mIL-2 dose was 0.11 nmol (10.8 ug), mIL-12-MSA-mLAIR dose was 14 pmol (2 ug). (B) Volcano plots for murine cohorts 1 (C1), 2 (C2), and 3 (C3). Fold-change is determined relative to untreated control data. Genes associated with significant p-adj values (< 0.05) are highlighted in red and quantified for each cohort. (C) Pathway scoring for Nanostring annotated pathways shared by murine and canine panels (pathway gene sets are not identical, but represent functionally similar sets). (D) Z-scored gene expression for homologous genes to those contained in the human 18-gene Tumor Inflammation Signature(60), which is known to correlated with human clinical response to ICB(59). (E) Comparison of differential expression of shared genes in canine soft tissue sarcoma and murine melanoma tumors, cohort 1 (C1) and cohort 3 (C3). Genes that are significantly differentially expressed (p <0.05) in canine and/or murine cohorts are shown. (F) Venn diagram of all significant differentially expressed (DE) genes shown in (D), grouped by cohort and species. The top 20 DE genes shared by both species and both cohorts (left) and genes that are only significantly differentially expressed in either canine cohorts (right, top) or murine cohorts (right, bottom) are shown. (G) Normalized expression (log 2) of counter-regulatory genes (one-way ANOVA with Dunnett’s multiple comparisons test). (H) Tumor survival for mice inoculated with B16F10 tumors, treated with cytokines ± aPD-1, aCTLA-4, or IDOi. Survival P values were determined by log-rank (Mantel-Cox) test (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).