The PD-L1/4-1BB Bispecific Antibody-Anticalin Fusion Protein PRS-344/S095012 Elicits Strong T-Cell Stimulation in a Tumor-Localized Manner.

PURPOSE: While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis and localizes 4-1BB costimulation to a PD-L1-positive (PD-L1+) tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti-4-1BB mAbs. EXPERIMENTAL DESIGN: We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumoral activity. RESULTS: PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB costimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4-positive (CD4+) and CD8-positive (CD8+) T-cell effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti-PD-L1-resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity. CONCLUSIONS: The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB-mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 toward clinical development. See related commentary by Shu et al., p. 3182.

Antibody targeting of Cathepsin S induces antibody-dependent cellular cytotoxicity.

BACKGROUND: Proteolytic enzymes have been implicated in driving tumor progression by means of their cancer cell microenvironment activity where they promote proliferation, differentiation, apoptosis, migration, and invasion. Therapeutic strategies have focused on attenuating their activity using small molecule inhibitors, but the association of proteases with the cell surface during cancer progression opens up the possibility of targeting these using antibody dependent cellular cytotoxicity (ADCC). Cathepsin S is a lysosomal cysteine protease that promotes the growth and invasion of tumour and endothelial cells during cancer progression. Our analysis of colorectal cancer patient biopsies shows that cathepsin S associates with the cell membrane indicating a potential for ADCC targeting. RESULTS: Here we report the cell surface characterization of cathepsin S and the development of a humanized antibody (Fsn0503h) with immune effector function and a stable in vivo half-life of 274 hours. Cathepsin S is expressed on the surface of tumor cells representative of colorectal and pancreatic cancer (23%-79% positive expression). Furthermore the binding of Fsn0503h to surface associated cathepsin S results in natural killer (NK) cell targeted tumor killing. In a colorectal cancer model Fsn0503h elicits a 22% cytotoxic effect. CONCLUSIONS: This data highlights the potential to target cell surface associated enzymes, such as cathepsin S, as therapeutic targets using antibodies capable of elicitingADCC in tumor cells.

Tumor-Localized Costimulatory T-Cell Engagement by the 4-1BB/HER2 Bispecific Antibody-Anticalin Fusion PRS-343.

PURPOSE: 4-1BB (CD137) is a key costimulatory immunoreceptor and promising therapeutic target in cancer. To overcome limitations of current 4-1BB-targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific molecule. PRS-343 is designed to facilitate T-cell costimulation by tumor-localized, HER2-dependent 4-1BB clustering and activation. EXPERIMENTAL DESIGN: PRS-343 was generated by the genetic fusion of 4-1BB-specific Anticalin proteins to a variant of trastuzumab with an engineered IgG4 isotype. Its activity was characterized using a panel of in vitro assays and humanized mouse models. The safety was assessed using ex vivo human cell assays and a toxicity study in cynomolgus monkeys. RESULTS: PRS-343 targets 4-1BB and HER2 with high affinity and binds both targets simultaneously. 4-1BB-expressing T cells are efficiently costimulated when incubated with PRS-343 in the presence of cancer cells expressing HER2, as evidenced by increased production of proinflammatory cytokines (IL2, GM-CSF, TNFα, and IFNγ). In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human peripheral blood mononuclear cells, PRS-343 leads to tumor growth inhibition and a dose-dependent increase of tumor-infiltrating lymphocytes. In IND-enabling studies, PRS-343 was found to be well tolerated, with no overt toxicity and no relevant drug-related toxicologic findings. CONCLUSIONS: PRS-343 facilitates tumor-localized targeting of T cells by bispecific engagement of HER2 and 4-1BB. This approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity compared with current monospecific approaches. The reported data led to initiation of a phase I clinical trial with this first-in-class molecule.See related commentary by Su et al., p. 5732.

Inhibition of Cathepsin S by Fsn0503 enhances the efficacy of chemotherapy in colorectal carcinomas.

Cathepsin S is a lysosomal cysteine protease implicated in tumourigenesis with key roles in invasion and angiogenesis. We have previously shown that the specific inhibition of Cathepsin S using a monoclonal antibody (Fsn0503) blocks colorectal carcinoma tumour growth and angiogenesis in vivo. We investigated whether Cathepsin S expression levels were affected by chemotherapy in human cancer cell lines by RT-PCR. Using colorectal xenograft models, we examined the therapeutic benefit of Cathepsin S inhibition using Fsn0503 in combination with a metronomic dosing regimen of CPT-11. We analysed the effects of the combination therapy on tumour progression and on tumour vascularisation by immunohistochemical staining of tumours. Cathepsin S expression levels are upregulated in HCT116, LoVo, Colo205 cell lines and HUVECs after exposure to CPT-11 in vitro. The administration of Fsn0503 in combination with CPT-11 significantly attenuated tumour growth in comparison to CPT-11 alone in colorectal HCT116 xenograft models. Furthermore, analysis of tumour vascularisation revealed that this was also significantly disrupted by the combination treatment. These results show that the combination of Cathepsin S inhibition with CPT-11 enhances the therapeutic effect of the chemotherapy. This rationale may have clinical application in the treatment of colorectal cancer upon further evaluation.

Antibody targeting of cathepsin S inhibits angiogenesis and synergistically enhances anti-VEGF.

BACKGROUND: Angiogenesis is a key hallmark of tumourigenesis and its inhibition is a proven strategy for the development of novel anti-cancer therapeutics. An important aspect of early angiogenesis is the co-ordinated migration and invasion of endothelial cells through the hypoxic tumour tissue. Cathepsin S has been shown to play an important role in angiogenesis as has vascular endothelial growth factor (VEGF). We sought to assess the anti-angiogenic effect of Fsn0503, a novel cathepsin S inhibitory antibody, when combined with anti-VEGF on vascular development. METHODOLOGY/PRINCIPAL FINDINGS: Cathepsin S expression and secretion from endothelial cells was characterised using RT-PCR and western blotting. We further show that cathepsin S promotes pericellular hydrolysis of extracellular matrix components in the tumour microenvironment and facilitates endothelial invasion. The cathepsin S inhibitory antibody, Fsn0503, blocks extracellular proteolysis, inhibiting endothelial invasion and tube formation in cell-based assays. The anti-angiogenic effects of Fsn0503 were also shown in vivo where it significantly retarded the development of vasculature in human xenograft models. Furthermore, when Fsn0503 was combined with an anti-VEGF antibody, a synergistic inhibition of microvascular development was observed. CONCLUSIONS/SIGNIFICANCE: Taken together, this data demonstrates that the antibody-mediated targeting of cathepsin S represents a novel method of inhibiting angiogenesis. Furthermore, when used in combination with anti-VEGF therapies, Fsn0503 has the potential to significantly enhance current treatments of tumour neovascularisation and may also be of use in the treatment of other conditions associated with inappropriate angiogenesis.

Antibody-mediated inhibition of cathepsin S blocks colorectal tumor invasion and angiogenesis.

PURPOSE: Cathepsin S is a cysteine protease that promotes the invasion of tumor and endothelial cells during cancer progression. Here we investigated the potential to target cathepsin S using an antagonistic antibody, Fsn0503, to block these tumorigenic effects. EXPERIMENTAL DESIGN: A panel of monoclonal antibodies was raised to human cathepsin S. The effects of a selected antibody were subsequently determined using invasion and proteolysis assays. Endothelial cell tube formation and aorta sprouting assays were done to examine antiangiogenic effects. In vivo effects were also evaluated using HCT116 xenograft studies. RESULTS: A selected cathepsin S antibody, Fsn0503, significantly blocked invasion of a range of tumor cell lines, most significantly HCT116 colorectal carcinoma cells, through inhibition of extracellular cathepsin S-mediated proteolysis. We subsequently found enhanced expression of cathepsin S in colorectal adenocarcinoma biopsies when compared with normal colon tissue. Moreover, Fsn0503 blocked endothelial cell capillary tube formation and aortic microvascular sprouting. We further showed that administration of Fsn0503 resulted in inhibition of tumor growth and neovascularization of HCT116 xenograft tumors. CONCLUSIONS: These results show that blocking the invasive and proangiogenic effects of cathepsin S with antibody inhibitors may have therapeutic utility upon further preclinical and clinical evaluation.