Pancreatic ductal adenocarcinoma (PDAC) has a 5‐year survival rate of less than 4% and desperately needs novel effective therapeutics. Integrin αvβ6 has been linked with poor prognosis in cancer but its potential as a target in PDAC remains unclear. We report that transcriptional expression analysis revealed that high levels of β6 mRNA correlated strongly with significantly poorer survival (n = 491 cases, p = 3.17 × 10−8). In two separate cohorts, we showed that over 80% of PDACs expressed αvβ6 protein and that paired metastases retained αvβ6 expression. In vitro, integrin αvβ6 promoted PDAC cell growth, survival, migration, and invasion. Treatment of both αvβ6‐positive human PDAC xenografts and transgenic mice bearing αvβ6‐positive PDAC with the αvβ6 blocking antibody 264RAD, combined with gemcitabine, significantly reduced tumour growth (p < 0.0001) and increased survival (log‐rank test, p < 0.05). Antibody therapy was associated with suppression of tumour cell activity (suppression of pErk growth signals, increased apoptosis seen as activated caspase‐3) and suppression of the pro‐tumourigenic microenvironment (suppression of TGFβ signalling, fewer αSMA‐positive myofibroblasts, decreased blood vessel density). These data show that αvβ6 promotes PDAC growth through both tumour cell and tumour microenvironment mechanisms and represents a valuable target for PDAC therapy. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Summary In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) can both promote and suppress tumor progression. Here, we show that the Rho effector protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 is associated with reduced PSC proliferation, contractility, and alpha-smooth muscle actin (α-SMA) stress fibers. In spheroid co-cultures with PDAC cells, loss of PKN2 prevents PSC invasion but, counter-intuitively, promotes invasive cancer cell outgrowth. PKN2 deletion induces a myofibroblast to inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo . Further, deletion of PKN2 in the pancreatic stroma induces more locally invasive, orthotopic pancreatic tumors. Finally, we demonstrate that a PKN2 KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast function can limit important stromal tumor-suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.
Purpose: This Phase 1 study (NCT03440437) evaluated the safety, tolerability, pharmacokinetics and activity of FS118, a bispecific antibody targeting LAG-3 and PD-L1, in patients with advanced cancer resistant to anti-PD-(L)1 therapy. Experimental Design: Patients with solid tumors, refractory to anti-PD-(L)1-based therapy, received intravenous FS118 weekly with an accelerated dose titration design (800 µg to 0.3 mg/kg) followed by 3+3 ascending dose expansion (1 mg/kg to 20 mg/kg). Primary objectives were safety, tolerability, and pharmacokinetics. Additional endpoints included antitumor activity, immunogenicity and pharmacodynamics. Results: Forty-three patients, with a median of three prior regimens in the locally advanced/metastatic setting including at least one anti-PD-(L)1 regimen, received FS118 monotherapy. FS118 was well tolerated with no serious adverse events relating to FS118 reported. No dose-limiting toxicities (DLTs) were observed, and a maximum tolerated dose (MTD) was not reached. The recommended Phase 2 dose of FS118 was established as 10 mg/kg weekly. The terminal half-life was 3.9 days. Immunogenicity was transient. Pharmacodynamic activity was prolonged throughout dosing as demonstrated by sustained elevation of soluble LAG-3 and increased peripheral effector cells. The overall disease control rate (DCR) was 46.5%; this disease control was observed as stable disease, except for one late partial response. Disease control of 54.8% was observed in patients receiving 1 mg/kg or greater who had acquired resistance to PD-(L)1-targeted therapy. Conclusion: FS118 was well tolerated with no DLTs observed up to and including 20 mg/kg QW. Further studies are warranted to determine clinical benefit in patients who have become refractory to anti-PD-(L)1 therapy.
FS118 is a tetravalent bispecific antibody targeting LAG-3 and PD-L1 that can overcome immune suppressive signals with greater preclinical activity than a combination of monoclonal antibodies. In vivo, FS118 downregulates LAG-3 on tumor-infiltrating lymphocytes (TILs) and increases soluble LAG-3 (sLAG-3) in the serum of mice. In a Phase 1 trial, FS118 demonstrated a dose-dependent increase in sLAG-3 in the serum of patients with advanced malignancies. Here, we demonstrate that the tetravalent structure of FS118 is required to mediate this novel LAG-3 shedding mechanism. Human ex vivo assays were performed by co-culturing expanded CD4+ T cells with iDCs in the presence of Staphylococcal enterotoxin B and FS118, or controls. sLAG-3 was measured by ECLIA. The binding valency of FS118 was determined using chemically-crosslinked mass spectrometry mapping (XL/MS). Variants of FS118 and FS118 surrogate molecules were generated with differing valency for LAG-3 and PD-L1. MC38 tumor-bearing C57BL/6 mice were dosed once interperitoneally with FS118 surrogate or valency variants. TILs were analyzed by flow cytometry, and sLAG-3 was measured in the serum by ECLIA. In patients receiving FS118 treatment, a dose-dependent increase in sLAG-3 was detected in the blood. In an ex vivo human T cell assay, the FS118-mediated increase in sLAG-3 was greater than with the combination of the individual bispecific components. sLAG-3 increase by FS118 was mediated by ADAM10 and ADAM17. FS118 demonstrated tetravalent binding to its target antigens using XL/MS. All four binding sites simultaneously bound to target antigens, with preferential binding to PD-L1 in the presence of equal amounts of both LAG-3 and PD-L1. Variants of FS118 with reduced binding valency for target antigens mediated lower levels of LAG-3 release than tetravalent FS118. Compared to FS118, monovalent PD-L1 binding reduced sLAG-3 release and importantly, a variant of FS118 with monovalent LAG-3 binding generated the lowest level of sLAG-3. Data will be presented showing the role of binding valency on LAG-3 downregulation by TILs and T cell activation in ex vivo murine samples. FS118 is tetravalent and can bind to two LAG-3 and two PD-L1 molecules simultaneously. Bivalent LAG-3 and PD-L1 binding is required for enhanced shedding of cell surface LAG-3 in vitro. Removing LAG-3 from the surface of exhausted TILs is a novel mechanism attributed to the tetravalent structure of FS118, which may be important to overcome compensatory upregulation of LAG-3 induced by blockade of PD-L1 in patients. Citation Format: Claire S. Reader, Wenjia Liao, Beatrice J. Potter-Landau, Christel Séguy Veyssier, Martyn C. Rhoades, Claire J. Seal, Michelle Morrow, Neil Brewis. The tetravalent structure of FS118, a bispecific antibody targeting LAG-3 and PD-L1, is required for its novel mechanism of LAG-3 shedding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2874.
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