An IL-7 fusion protein targeting EDA fibronectin upregulates TCF1 on CD8+ T-cells, preferentially accumulates to neoplastic lesions, and boosts PD-1 blockade

Abstract

Background: Anti-PD-1 antibodies have revolutionized cancer immunotherapy due to their ability to induce long-lasting complete remissions in a proportion of patients. Current research efforts are attempting to identify biomarkers and suitable combination partners to predict or further improve the activity of immune checkpoint inhibitors. Antibody-cytokine fusions are a class of pharmaceuticals that showed the potential to boost the anticancer properties of other immunotherapies. Extradomain A-fibronectin (EDA-FN), which is expressed in most solid and hematological tumors but is virtually undetectable in healthy adult tissues, is an attractive target for the delivery of cytokine at the site of the disease.

Methods: In this work, we describe the generation and characterization of a novel interleukin-7-based fusion protein targeting EDA-FN termed F8(scDb)-IL7. The product consists of the F8 antibody specific to the alternatively spliced EDA of FN in the single-chain diabody (scDb) format fused to human IL-7.

Results: F8(scDb)-IL7 efficiently stimulates human peripheral blood mononuclear cells in vitro. Moreover, the product significantly increases the expression of T Cell Factor 1 (TCF-1) on CD8+T cells compared with an IL2-fusion protein. TCF-1 has emerged as a pivotal transcription factor that influences the durability and potency of immune responses against tumors. In preclinical cancer models, F8(scDb)-IL7 demonstrates potent single-agent activity and eradicates sarcoma lesions when combined with anti-PD-1.

Conclusions: Our results provide the rationale to explore the combination of F8(scDb)-IL7 with anti-PD-1 antibodies for the treatment of patients with cancer.

Keywords: Antibodies, Bispecific; CD8-Positive T-Lymphocytes; Combined Modality Therapy; Cytokines; Immune Checkpoint Inhibitors.

Figure 1. Tumor homing properties of IL-7-based immunocytokines. Quantitative biodistribution analysis of ¹²⁵I radio labeled L19(scDb)-IL7 (A), L19 IgG4 KIH-IL7 (B), L19 IgG4-IL7 (C), F8(scDb)-IL7 (D) and KSF(scDb)-IL7 (E) in immunocompetent mice bearing F9 teratocarcinoma tumors. About 10 µg of radio-labeled fusion protein was injected into the lateral tail vein and mice were sacrificed 24 hours after injection. Organs and tumor were excised, weighed and the radioactivity of each sample was measured. Results are corrected on tumor growth and expressed as a percentage of injected dose per gram of tissue (% ID/g±SEM), (n=5 for F8(scDb)-IL7; n=4 for L19 IgG4 KIH-IL7 and KSF(scDb)-IL7; n=3 for L19(scDb)-IL7 and L19 IgG4-IL7). A schematic representation of each fusion protein is depicted. (F) Microscopic fluorescence analysis of EDA and EDB expression (green) in F9, WEHI-164, MC38 and GL261 tumors. An anti-CD31 antibody was used to stain blood vessels (red). 20×magnification, scale bar=100 µm. EDA, extra domain A.

Figure 2. Antigen expression analysis of human cancer sections. Microscopic fluorescence analysis of EDA and EDB expression (green) on various human cancer sections using F8(scDb)-IL7 (aEDA) and L19(scDb)-IL7 (aEDB) fusion proteins. An anti-CD31 antibody was used to stain blood vessels (red). 20×magnification, scale bar=100 µm. EDA, extra domain A.

Figure 3. Biochemical and functional characterization of F8(scDb)-IL7. (A) Schematic representation of F8(scDb)-IL7. (B) SDS-PAGE analysis on 12% gel in reducing (R) and non-reducing (NR) conditions of F8(scDb)-IL7. (C) Size exclusion chromatogram of F8(scDb)-IL7. (D) SPR of F8(scDb)-IL7 on EDA-coated CM5 sensor chip. (E) Differential scanning fluorimetry of F8(scDb)-IL7. (F) Activity assay based on hPBMCs proliferation by exposure to F8(scDb)-IL7 and rhIL-7. (G) IFNg release by hPBMCs exposed to titration of F8(scDb)-IL7 in coated EDA (+) and non-coated EDA (-) wells. (H) IFNγ release on hPBMCs exposed to rhIL-7. Results are expressed as a percentage proliferation (proliferation%±SEM) and as IFNγ measured in pg/mL±SEM (n=3). (**p<0.01, ***p<0.001). EDA, extra domain A; hPBMCs, human peripheral blood mononuclear cells; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Figure 4. Fluorimetry analysis on hPBMCs exposed to F8(scDb)-IL7 reveals increased expression of TCF1 compared with L19IL2. Expression levels of CD69 and CD44 on inactivated CD8+T cells exposed to F8(scDb)-IL7 (A) or L19IL2 (B). Expression levels of CD69 and CD44 on activated CD8+T cells exposed to F8(scDb)-IL7 (C) or L19IL2 (D). Percentage of CD3+ and CD4+ T cells exposed to F8(scDb)-IL7 (E) or L19IL2 (F). Contour plot displaying expression levels of Ki67 (x-axis) and TCF1 (y-axis) on CD8+CD44+ T cells exposed to F8(scDb)-IL7 at 62.5 nM (G) or L19IL2 at 50 nM (H). Expression of TCF1+Ki67+ on CD8+ CD44+ exposed to F8(scDb)-IL7 (blue) or L19IL2 (green) at three distinct concentrations (1000 nM, 250 nM and 62.5 nM for F8(scDb)-IL7 and 100 nM, 50 nM and 10 nM for L19IL2) (I). Results are expressed as marker positivity percentage (% ±SEM). (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). hPBMCs, human peripheral blood mononuclear cells.

Figure 5. Therapeutic performance of F8(scDb)-IL7 in subcutaneous tumor models. (A) WEHI-164 tumor-bearing BALB/c mice received either Saline, KSF(scDb)-IL7 130 µg, F8(scDb)-IL7 130 µg and L19IL2 100 µg three times every 48 hours (black arrow; n=4 per group). (B) WEHI-164 tumor-bearing BALB/c mice received either Saline, αPD-1 200 µg, F8(scDb)-IL7 130 µg three times every 48 hours (black arrow) or αPD-1 200 µg (red arrow) in combination with F8(scDb)-IL7 130 µg (black arrow) 24 hours later. (C) MC38 tumor-bearing C57BL/6 mice received either Saline, αPD-1 200 µg, F8(scDb)-IL7 130 µg three times every 48 hours (black arrow) or αPD-1 200 µg (red arrow) in combination with F8(scDb)-IL7 130 µg (black arrow) 24 hours later.(D) F9 tumor-bearing 129/SvEv mice received either Saline, αPD-1 200 µg, F8(scDb)-IL7 130 µg three times every 48 hours (black arrow) or αPD-1 200 µg (red arrow) in combination with F8(scDb)-IL7 130 µg (black arrow) 24 hours later. Curves were halted on day 13 because animals were sacrificed earlier in the study due to small ulcer formation, in compliance with our animal license. Data represent mean tumor volume and body weight change % (±SEM). (*p<0.05, ****p<0.0001). CR, complete response.

Figure 6. Mechanism of action of F8(scDb)-IL7 and aPD-1 combination in BALB/c immunocompetent mice. (A) Photograph of spleens harvested from BALB/c tumor-bearing mice treated either with either saline, αPD-1 200 µg, F8(scDb)-IL7 130 µg three times every 48 hours or with αPD-1 200 µg in combination with F8(scDb)-IL7 130 µg. Scale bar=1 cm. (B) Weight comparison of spleens from BALB/c treated mice. (C) IFNγ levels in plasma of BALB/c treated mice. (D) Ex vivo immunofluorescence analysis on WEHI-164 sarcoma 24 hours after the third injection of saline, αPD-1, and F8(scDb)-IL7 alone or in combination with 200 µg of αPD-1. Markers specific for Tregs (Foxp3), NK cells (NCR1), CD4+T cells (CD4), and CD8+T cells (CD8) were used (green). Blood vessels were stained with an anti-CD31 antibody (red). Magnification: ×20; scale bars=100 µm. (E) Principal component analysis (PCA) of spleens proteomics data. (F) Pathway enrichment analysis was used to identify significantly impacted biological pathways after αPD-1 + F8(scDb)-IL7 combination treatment. (*p<0.05, **p<0.01, ****p<0.0001).

Figure 7. Therapeutic performance of F8(scDb)-IL7 in GL-261 orthotopic glioma model. (A) Treatment schedule and schematic overview of the therapeutic agents investigated in C57BL/6 orthotopic GL-261 glioma-bearing mice. (B) Survival studies on GL-261 orthotopically implanted C57BL/6 female mice. Treatments started on day 5 post-tumor implantation as shown in the scheme. (C) Body weight change (%) of treated mice. (D) FMT of mice treated either with saline, αPD-1 200 μg, F8(scDb)-IL7 130 µg, or combination. (E) Tumor fluorescence signal at day 18 post-tumor implantation (n=3). Survival data are presented as Kaplan-Meier plots. P values were calculated with the log-rank test (treatment vs other treatments: **p<0.01).