Simlukafusp alfa (FAP-IL2v) immunocytokine is a versatile combination partner for cancer immunotherapy

Abstract

Simlukafusp alfa (FAP-IL2v, RO6874281/RG7461) is an immunocytokine comprising an antibody against fibroblast activation protein α (FAP) and an IL-2 variant with a retained affinity for IL-2Rβγ > IL-2 Rβγ and abolished binding to IL-2 Rα. Here, we investigated the immunostimulatory properties of FAP-IL2v and its combination with programmed cell death protein 1 (PD-1) checkpoint inhibition, CD40 agonism, T cell bispecific and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The binding and immunostimulatory properties of FAP-IL2v were investigated in vitro and compared with FAP-IL2wt. Tumor targeting was investigated in tumor-bearing mice and in a rhesus monkey. The ability of FAP-IL2v to potentiate the efficacy of different immunotherapies was investigated in different xenograft and syngeneic murine tumor models. FAP-IL2v bound IL-2 Rβγ and FAP with high affinity in vitro, inducing dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells while being significantly less potent than FAP-IL2wt in activating immunosuppressive regulatory T cells (Tregs). T cells activated by FAP-IL2v were less sensitive to Fas-mediated apoptosis than those activated by FAP-IL2wt. Imaging studies demonstrated improved tumor targeting of FAP-IL2v compared to FAP-IL2wt. Furthermore, FAP-IL2v significantly enhanced the in vitro and in vivo activity of therapeutic antibodies that mediate antibody-dependent or T cell-dependent cellular cytotoxicity (TDCC) and of programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with an anti-PD-L1 antibody and an agonistic CD40 antibody was most efficacious. These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies.

Keywords: FAP-il2v; fibroblast activation protein; immunocytokine; interleukin-2; rg7461.

Figure 1.

Structure of FAP-IL2v

Figure 2.

Activation of CD4+/CD8+ T cells and NK cells and cell killing in vitro. (a) Phosphorylation of STAT5 on CD4 T cells (CD3+ CD4+ CD25-), NK cells (CD3-CD56+), CD8 T cells (CD3+ CD8+ CD25-), and Tregs (CD4+ CD25+ FOXP3+) 20 min after treatment with different IL2/IL2v-containing constructs. (b) Tumor cell lysis and upregulation of NK cell activation markers following co-culture of LS174T colon cancer cells, GM05389 FAP-expressing fibroblasts, and human PBMCs with FAP-IL2v alone, cetuximab (1000 ng/mL fixed concentration) alone or the combination of FAP-IL2v and cetuximab, for 48 hours. (c) Tumor cell lysis, IL-2Rα (CD25) upregulation on CD8+ T cells, and cytokine release upon treatment of MKN45 cells with FAP-IL2v alone or in combination with CEA-TCB (0.2 nM fixed concentration) in the presence of PBMCs. Data are mean and standard deviation of triplicate experiments

Figure 3.

Biodistribution in animal models. (a) SPECT/CT imaging of 111In-labeled FAP-IgG, FAP-IL2v, FAP-IL2wt and DP47-IL2v in immunocompetent BALB/c mice (n = 3 per treatment) bearing renal carcinoma in the right kidney. Imaging was performed 3 days after IV injection of 0.3 μg 111In-labeled antibodies co-injected with 25 μg of corresponding unlabeled antibodies. (b) FDG-PET/CT axial view imaging of 89Zr-FAP-IL2v localization in lesions of a single rhesus monkey with breast carcinoma. The monkey was treated with 0.5 mg/kg of FAP-IL2v IV mixed with tracer amounts of 89Zr-labeled FAP-IL2v. PET scans were performed on the day of administration and 3 days (67 hours) and 6 days (154 hours) after IV injection. On-treatment biopsy was performed on Day 3 following the PET scan

Figure 4.

Efficacy in combination with an ADCC-competent antibody. (a) Targeted muFAP-muIL2v (2 mg/kg), untargeted muDP47-muIL2v (0.7 mg/kg), and anti-ratHER2 muIgG2a (20 mg/kg) were administered as single agents and in combination in the BALB-neuT genetically engineered mouse mammary tumor model (n = 6 per group). Treatments were given IV once weekly for 5 weeks starting at Week 19 or Week 21. (b-c) Mammary fat pad tumors from these mice were harvested 5 days after the last treatment and analyzed by immunohistochemistry for CD3 T cells, NK cells, and CD68-expressing cells. Representative immunohistochemistry staining images are shown in Supplementary Figure 12 and 13. * p < .05; ** p < .01; *** p < .001 vs vehicle control (unpaired t-test)

Figure 5.

Efficacy in combination with a TDCC-competent antibody. muFAP-muIL2v (2 mg/kg IV once weekly) was administered in combination with the TDCC-competent antibody muCEA-TCB (2.5 mg/kg IV twice weekly) in the inflamed orthotopic Panc02-CEA model in CEA transgenic C57BL/6 mice (n = 8 mice per group). Treatments (four in total) started 7 days after tumor cell injection

Figure 6.

Efficacy in combination with PD-L1 checkpoint inhibition. (a) Efficacy of muFAP-muIL2v (2 mg/kg IP) and anti-muPD-L1 (10 mg/kg IP) as single agents and in combination given once weekly for 5 weeks starting on Day 7 in the Panc02 pancreatic orthotopic syngeneic model in C57BL/6 mice (n = 10 mice per group). (b) Efficacy in the same mouse model with the addition of muCD40 (10 mg/kg IP given once on Day 7) as single agent and in combination with muFAP-muIL2v (2 mg/kg IP) and anti-muPD-L1 (10 mg/kg IP) given once weekly for 3 weeks (n = 6–8 mice per group). (c) Tumor growth in surviving mice from experiments shown in part (b) that were re-challenged subcutaneously with Panc02 cells (5 × 105) on Day 121 (Day 0 in Figure 6c). No additional antibody therapy was given in these experiments. A new group of vehicle-only treated mice was used for comparison in this experiment