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.
BackgroundUpregulation of immune checkpoints, such as LAG-3, plays an important role in promoting resistance to anti-PD-(L)1 therapy. FS118, currently being evaluated in a Phase 1 clinical trial in patients with advanced malignancies, 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.1 Here, we demonstrate a novel mechanism of action for FS118 in shedding of LAG-3 from the surface of T cells that is not observed with the combination of PD-L1 and LAG-3 antibodies.MethodsHuman ex vivo assays were performed by co-culturing activated CD4+ T cells with iDCs in the presence of Staphylococcal enterotoxin B and FS118, or control reagents. Soluble LAG-3 was measured by ELISA from day 4 to 13. A mouse tumor model used MC38 cells implanted subcutaneously into C57Bl/6 mice. Expression of surface markers was measured on tumor-infiltrating lymphocytes (TILs) from disaggregated tumors and soluble LAG-3 was measured in serum following dosing of mice intraperitoneally with FS118 surrogate or control reagents. Soluble LAG-3 in the serum of patients treated with FS118 was measured by ELISA (Phase 1 trial NCT03440437).ResultsIn an ex vivo T cell assay, FS118 resulted in an increase in the concentration of soluble LAG-3 in the cell culture medium, an effect that was greater than with the combination of the individual bispecific components. Addition of inhibitors of either ADAM10 or ADAM17 to the FS118-treated cells resulted in a decrease in the levels of soluble LAG-3 in the cell culture medium. In MC38 tumor-bearing mice, a mouse surrogate of FS118 decreased the levels of surface LAG-3 expressed by TILs, in contrast to the combination of the bispecific components where an increase in surface LAG-3 was observed. This corresponded with an increase in soluble LAG-3 in the serum following treatment with a mouse surrogate of FS118. Finally, in patients receiving treatment with FS118, a dose dependent increase in soluble LAG-3 was detected in the blood.ConclusionsFS118 mediates LAG-3 shedding from the surface of immune cells via a mechanism that is dependent upon simultaneous binding to both PD-L1 and LAG-3. This shedding was mediated by ADAM10 and ADAM17 metalloproteinases. Removing LAG-3 from the surface of TILs via shedding may be an important mechanism by which FS118 overcomes compensatory upregulation of LAG-3 induced by PD-L1 blockade. Soluble LAG-3 may be an important biomarker for monitoring the pharmacodynamic activity of FS118 in patients.Ethics ApprovalAll animal experiments were conducted under a UK Home Office Project Licence and approved by an Animal Welfare and Ethical Review Board (AWERB) in accordance with the UK Animal (Scientific Procedures) Act 1986 and with EU Directive EU 86/609ReferenceKraman M, Faroudi M, Allen N, Kmiecik K, Gliddon D, Seal C, Koers A, Wydro M, Winnewisser J, Young L, Tuna M, Doody J, Morrow M, Brewis N. FS118, a Bispecific Antibody Targeting LAG-3 and PD-L1, Enhances T-Cell Activation Resulting in Potent Antitumor Activity. Clin Cancer Res 2020;26:3333–3344
BackgroundImmune checkpoint inhibitors have demonstrated durable clinical responses and an increase in overall survival for some patients with cancer. Next generation cancer immunotherapies, such as tumor necrosis factor receptor superfamily (TNFRSF) agonists, have potential to further improve on this success. FS120 is a tetravalent bispecific antibody targeting OX40 and CD137 (4-1BB), currently being evaluated in a Phase I clinical trial (NCT04648202). FS120 activates CD4+ and CD8+ T cells by concurrent binding to both targets via an FcgR-independent mechanism [1]. In preclinical tumor models, FS120 induced T cell proliferation and cytokine production associated with significant tumor regression, better than that observed with a monoclonal antibody combination. Here, we demonstrate the ability of FS120 to improve anti-PD-1 induced T cell activity, increasing tumor growth inhibition and survival, in syngeneic mouse tumor models, compared to monotherapy.MethodsFS120 < i >in vitro</i > activity in combination with anti-PD-1 was assessed by utilizing staphylococcal enterotoxin A (SEA) superantigen assays and mixed leukocyte reaction (MLR) assays. An anti-mouse OX40/CD137 bispecific antibody (FS120 surrogate) was tested in CT26 syngeneic mouse tumor models in combination with an anti-mouse PD-1 antibody to assess efficacy and pharmacodynamic endpoints, including T cell proliferation by < i>ex vivo</i> flow cytometry and serum cytokine levels.ResultsFS120 in combination with anti-PD-1 enhanced primary human T cell activity, when compared to either monotherapy, in both SEA and MLR assays. FS120 surrogate significantly improved survival of CT26 tumor-bearing mice treated with anti-mPD-1 antibody. FS120 surrogate and anti-PD-1 combination significantly enhanced serum interferon-gamma levels and increased proliferating granzyme B+ CD8+ T cells in the blood of tumor-bearing mice, when compared to either monotherapy treatments.ConclusionsFS120 combination with anti-PD-1 enhances T cell activity in multiple human primary immune assays. In combination with anti-PD-1, FS120 surrogate increased the antitumor efficacy with pharmacodynamic changes related specifically to T cell activation, when compared to monotherapies. These data support the development of FS120 in combination with anti-PD-1 in patients with hard-to-treat cancers who may not benefit fully from either treatment as a monotherapy.ReferencesGaspar M, Pravin J, Rodrigues L, Uhlenbroich S, Everett K L, Wollerton F, Morrow M, Tuna M, Brewis N. CD137/OX40 Bispecific Antibody Induces Potent Antitumor Activity that Is Dependent on Target Coengagement. Cancer Immunol Res. 2020; (8) (6) 781–793Ethics ApprovalMurine studies were conducted under a U.K. Home Office License in accordance with the U.K. Animal (Scientific Procedures) Act 1986 and EU Directive EU 2010/63.
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