Supplementary Figure S1: Study design for the evaluation of FS118 in subjects with advanced solid tumors whoprogressed on or after prior PD-(L)1 therapy.Supplementary Figure S2: Time on FS118 treatment in patients with primary or acquired resistance to prioranti-PD-(L)1 therapies.Supplementary Figure S3: Total time on prior ICB therapies compared to the timeon FS118 treatment in patients with primary or acquired resistance to prior antiPD-(L)1 therapies.Supplementary Figure S4: Levels of soluble LAG-3 (sLAG-3) detected inthe serum of patients in the FS118 phase 1 trial.Supplementary Figure S5: Changes in circulating immune cells in FS118-treated patients.

Supplementary Table S1: Representativeness of Study Participants.Supplementary Table S2: Immunogenicity in patients treated with FS118.Supplementary Table S3: PK of FS118 by Dose Level.

AbstractPurpose:This phase 1 study (NCT03440437) evaluated the safety, tolerability, pharmacokinetics (PK), 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.Patients and Methods: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 to 20 mg/kg). Primary objectives were safety, tolerability, and PK. 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 (DLT) were observed, and an 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.Conclusions: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.See related commentary by Karapetyan and Luke, p. 835

Kraman et al Supplementary figures

Abstract Background: CD137 (4-1BB, TNFRSF9) is expressed on activated lymphocytes, and its clustering leads to agonism of the receptor resulting in lymphocyte proliferation and pro-inflammatory cytokine release. First-generation CD137 antibodies for cancer therapy were high affinity and enabled for FcγR engagement with either severe toxicity or weak activity limiting their therapeutic benefit. However, CD137 remains a promising target for bispecific antibodies designed to re-direct T cell activity to the tumor whilst limiting unwanted toxicities. We have rationally designed and developed a unique tetravalent bispecific antibody targeting CD137 and PD-L1 with reduced FcγR binding for safe and efficacious cancer therapy (Lakins MA et al. 2020). Methods: A CD137/PD-L1 bispecific mAb2 antibody (FS222) was generated by introducing a bivalent affinity-optimized CD137-binding Fcab into a human IgG1 bivalent PD-L1 mAb. LALA mutations were introduced to abrogate FcγR activity. To elucidate its mechanism of action, FS222's binding valency was assessed by chemically-crosslinked mass spectrometry mapping (XL/MS). Immune pharmacology models were also used to evaluate PD-L1 dependent FS222 agonism against variants with differing valency for each target. Results: FS222 was specifically designed to bind CD137 with moderate affinity, and without PD-L1-mediated crosslinking it does not cluster or activate the receptor on immune cells. When PD-L1 was bound by FS222, this lead to crosslinking and subsequent clustering and activation of CD137. This clustering was enabled by avid bivalent binding as demonstrated by tetravalent FS222 being biologically more active than variants with reduced valency. Moreover, XL/MS demonstrated all four binding sites were able to concurrently bind target antigens. FS222 was well tolerated in a GLP toxicity study up to the maximal dose (30mg/kg QW). It was pharmacologically active in peripheral blood and lymphoid tissues and had a predicted t½ of 286 hours in humans. A surrogate anti-mouse CD137/PD-L1 mAb2 significantly reduced tumor growth in a murine syngeneic model and this correlated with a significant survival benefit and T cell activation. Conclusions: FS222 was designed to be a potent anti-human CD137/PD-L1 tetravalent conditional agonist with a unique combination of high affinity PD-L1 binding and moderate monovalent affinity, and highly avid binding, to CD137 on activated T cells. A favorable safety profile and immunopharmacology was observed with FS222 in a primate GLP toxicity study. Tetravalent binding by FS222 was required for optimal activity in pharmacology studies. Preclinically, FS222 was well-tolerated with an effective mechanism of action. Citation Format: Matthew A. Lakins, Jose Munoz-Olaya, Christel Veyssier, Daniel Jones, Emma Goodman, Quincy Kaka, Jennifer Ofoedu, Robert Hughes, Daniel Gliddon, Michelle Morrow, Neil Brewis. FS222, a tetravalent bispecific antibody targeting CD137 and PD-L1, is designed for optimal CD137 interactions resulting in potent T cell activation without toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1864.

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

Abstract Background Activating T cells via clustering tumour necrosis factor receptor superfamily (TNFRSF) has shown promise in cancer therapy. This has typically been achieved by crosslinking of targeting antibodies via Fc-gamma receptor (FcγR). TNFRSF member CD137 is expressed by activated lymphocytes, and its clustering results in lymphocyte proliferation and agonism. First generation CD137 antibodies induced liver immune pathology, causing clinical toxicity and death. It remains a promising target for bispecific antibodies designed to limit unwanted toxicities. Methods An anti-human CD137/PD-L1 mAb2 (FS222) was generated by introducing a CD137-binding specificity into a human IgG1 targeting PD-L1 mAb with reduced FcγR binding. Cell binding and in vitro activity was used to evaluate FS222 agonism conditional on PD-L1 crosslinking. An anti-mouse CD137/PD-L1 mAb2 was generated to assess anti-tumour activity, liver pharmacology and PK/PD in murine models. To predict the clinical toxicity and PK/PD profiles of FS222, studies were performed in non-human primates. Results FS222 is designed to bind human PD-L1 with sub-nanomolar affinity and crosslink to enable CD137 clustering and conditional agonism (low nM EC50). The toxicity, PK and PD of FS222 was tested in cynomolgus monkey. FS222 was well tolerated in a GLP toxicity study up to the maximal dose (30mg/kg QW) and showed no evidence of liver toxicity. The molecule has a t½ of 211h in monkeys, with treatment resulting in pharmacology in peripheral blood which included lymphocyte proliferation and increases in soluble receptors. The anti-mouse CD137/PD-L1 mAb2 significantly reduced tumor growth in mouse tumor models. This correlated with a significant survival benefit and increases in tumor and peripheral activated lymphocytes. An anti-mouse CD137 antibody (clone 3H3) showed crosslink-independent agonism and sustained increases in activated T cells in liver whereas the liver effects of the anti-mouse CD137/PD-L1 mAb2 were of similar magnitude but peaked earlier and resolved. Conclusions FS222 was observed to be a potent anti-human CD137/PD-L1 tetravalent conditional agonist. A favourable safety profile and immunopharmacology was observed with FS222 in a primate GLP toxicity study. FS222 did not show toxicity and the anti-mouse CD137/PD-L1 mAb2 had self-limiting immunopharmacology outside the tumor microenvironment, suggesting a well-tolerated and effective mechanism of action with a broad therapeutic window. Citation Format: Matthew A. Lakins, Alexander Koers, Raffaella Giambalvo, Robert Hughes, Sylwia Marshall, Mateusz Wydro, Cristian Gradinaru, Sarah Batey, Daniel Gliddon, Michelle Morrow, Neil Brewis. Clustering CD137 via cell-expressed PD-L1 crosslinking avoided Fc-mediated agonism and resulted in safe and potent conditional lymphocyte activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4547.

AbstractPurpose:Although programmed death-ligand 1 (PD-L1) antibody–based therapy has improved the outcome of patients with cancer, acquired resistance to these treatments limits their clinical efficacy. FS118 is a novel bispecific, tetravalent antibody (mAb2) against human lymphocyte activation gene-3 (LAG-3) and PD-L1 with the potential to reinvigorate exhausted immune cells and overcome resistance mechanisms to PD-L1 blockade. Here, using FS118 and a murine surrogate, we characterized the activity and report a novel mechanism of action of this bispecific antibody.Experimental Design:This study characterizes the binding activity and immune function of FS118 in cell lines and human peripheral blood mononuclear cells and further investigates its antitumor activity and mechanism of action using a surrogate murine bispecific antibody (mLAG-3/PD-L1 mAb2).Results:FS118 demonstrated simultaneous binding to LAG-3 and PD-L1 with high affinity and comparable or better activity than the combination of the single component parts of the mAb2 in blocking LAG-3- and PD-L1–mediated immune suppression and enhancing T-cell activity. In syngeneic tumor mouse models, mLAG-3/PD-L1 mAb2 significantly suppressed tumor growth. Mechanistic studies revealed decreased LAG-3 expression on T cells following treatment with the mouse surrogate mLAG-3/PD-L1 mAb2, whereas LAG-3 expression increased upon treatment with the combination of mAbs targeting LAG-3 and PD-L1. Moreover, following binding of mLAG-3/PD-L1 mAb2 to target-expressing cells, mouse LAG-3 is rapidly shed into the blood.Conclusions:This study demonstrates a novel benefit of the bispecific approach over a combination of mAbs and supports the further development of FS118 for the treatment of patients with cancer.

Background Upregulation of immune checkpoints, such as LAG-3, plays an important role in promoting resistance to anti-PD-(L)1 therapy. Targeting PD-L1 and LAG-3 using a bispecific antibody may overcome resistance to PD-(L)1 blockade.1 We report initial data from a first-in-human study evaluating FS118 in patients with advanced cancer and resistance to PD-(L)1 therapy. Methods The ongoing Phase I FIH study (NCT03440437) is being conducted to evaluate safety, tolerability, immunogenicity, PK/PD and clinical activity of FS118 administered IV weekly to heavily pre-treated patients who had previously received anti-PD-(L)1 therapy for a minimum of 12 weeks. Adverse events were assessed using CTCAEv4.03 and tumor responses assessed using RECISTv1.1 and iRECIST. Single subject dose escalation cohorts were followed by a 3+3 ascending dose design. Three cohorts (3, 10, 20 mg/kg) were expanded to evaluate PK, PD and clinical activity. Pharmacodynamic studies examined soluble LAG-3 production and peripheral T-cell expansion. Results Forty-three patients (median 6 lines of prior therapy, including ICB) with solid tumors received FS118 at doses from 0.8 mg up to 20 mg/kg across 8 dose levels. Weekly administration of FS118 was well tolerated and did not result in dose- or treatment-limiting toxicities. An MTD was not reached. No safety signals unexpected for the drug class of immune-checkpoint inhibitors were identified in the early study population. The majority (95%) of treatment-emergent adverse events (TEAE) considered by the Safety Review Committee (SRC) to be treatment-related were Grade 1 and 2. Grade 3 TEAEs toxicities (elevated liver enzymes) were observed in 2 patients (5%). No SAEs or deaths were attributed to FS118 treatment. Anti-drug antibodies, observed in half of patients, were typically transient in nature. The pharmacokinetic profile confirmed preclinical predictions and PD parameters included a dose-dependent increase in serum soluble LAG-3 and expansion of peripheral T cells. Long-lasting disease stabilisation (>6 months) was observed in a subset of patients with acquired resistance (defined as a CR, PR or SD ≥3 months on previous PD-(L)1 treatment), but not in patients with primary resistance. Two patients remain on FS118 treatment as of 2 Jul 2020 (duration 10 and 16 months). Retrospective IHC analysis of PD-L1 and LAG-3 co-expression in the tumor was assessed as a potential biomarker associated with clinical outcome. Conclusions Weekly treatment with FS118 was well tolerated up to 20 mg/kg and was associated with pharmacodynamic markers of FS118 activity. Encouraging signs of clinical activity were observed in highly pre-treated patients who had acquired resistance to prior PD-(L)1 therapy. Trial Registration Registered at www.clinicaltrials.gov, NCT03440437 Reference Kraman 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.

Abstract Purpose: With the increased prevalence in checkpoint therapy resistance, there remains a significant unmet need for additional therapies for patients with relapsing or refractory cancer. We have developed FS222, a bispecific tetravalent antibody targeting CD137 and PD-L1, to induce T-cell activation to eradicate tumors without the current toxicity and efficacy limitations seen in the clinic. Experimental Design: A bispecific antibody (FS222) was developed by engineering CD137 antigen–binding sites into the Fc region of a PD-L1 IgG1 mAb. T-cell activation by FS222 was investigated using multiple in vitro assays. The antitumor efficacy, survival benefit, pharmacodynamics, and liver pharmacology of a murine surrogate molecule were assessed in syngeneic mouse tumor models. Toxicology and the pharmacokinetic/pharmacodynamic profile of FS222 were investigated in a non-human primate dose-range finding study. Results: We demonstrated simultaneous binding of CD137 and PD-L1 and showed potent T-cell activation across CD8+ T-cell activation assays in a PD-L1–dependent manner with a CD137/PD-L1 bispecific antibody, FS222. FS222 also activated T cells in a human primary mixed lymphocyte reaction assay, with greater potency than the monospecific mAb combination. FS222 showed no signs of liver toxicity up to 30 mg/kg in a non-human primate dose-range finding study. A surrogate molecule caused significant tumor growth inhibition and survival benefit, concomitant with CD8+ T-cell activation, in CT26 and MC38 syngeneic mouse tumor models. Conclusions: By targeting CD137 agonism to areas of PD-L1 expression, predominantly found in the tumor microenvironment, FS222 has the potential to leverage a focused, potent, and safe immune response augmenting the PD-(L)1 axis blockade.

FS118, currently tested in Phase I clinical trial in patients with advanced malignancies (NCT03440437), is a first-in-class bispecific antagonistic antibody (known as mAb2) targeting LAG-3 (Lymphocyte-Activation Gene 3) and PD-L1 (Programmed Death-Ligand 1), two immune checkpoint molecules that promote tumour escape from immune surveillance. Resistance to anti-PD-1 treatment is associated with upregulation of other checkpoint inhibitor receptors such as LAG-3. Dual targeting in immune checkpoint blockade (ICB) using bispecific monoclonal antibodies could potentially overcome this resistance, further increase the clinical benefit of ICB therapy and prevent relapse or resistance after immunotherapy. In a syngeneic model we used a surrogate mAb2 of FS118 (blocking both mPD-L1 and mLAG-3 binding to their receptors) to determine the effect of dual checkpoint blockade on tumour growth and to elucidate the modulation of the underlying immune mechanisms following treatment with the mAb2. Dual blockade of LAG-3 and PD-L1 with the surrogate mAb2 in the MC38 tumour-bearing mice model resulted in an increased anti-tumour activity comparable to a combination of the single agents targeting LAG-3 and PD-L1. Moreover, the mAb2 and single agent combination resulted in distinct modulations of LAG-3 and PD-L1 cell surface expression within the spleen and tumour microenvironment (TME). While both the mAb2 and combination therapy significantly reduced the number of free PD-L1 binding sites on CD4+ and CD8+ T cells in spleen and TME, total LAG-3 cell surface expression increased following treatment with the combination, whereas it reduced with mAb2 treatment. In addition, analysis of serum samples collected at various timepoints confirmed evidence of drug target engagement with increasing levels of both soluble LAG-3 (sLAG-3) and soluble PD-L1 (sPD-L1) in the mAb2 treated animals. Modulation of sPD-L1 levels in the serum of treated animals was also observed in cynomolgus monkeys treated with FS118. These data provide a strong rationale for investigating both cell surface and soluble LAG-3 and PD-L1 levels as potential pharmacodynamic biomarkers in further clinical development. Citation Format: Mustapha Faroudi, Matthew Kraman, Natalie Fosh, Claire Reader, Daniel Gliddon, Claire Seal, Christa Lucas, Alexander Koers, Mateusz Wydro, Michelle Morrow, Neil Brewis. LAG-3/PD-L1 mAb2 can overcome PD-L1-mediated compensatory upregulation of LAG-3 induced by single-agent checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2399.

PD-1/L1 immune checkpoint blockade shows durable responses and extends overall survival in a subset of cancer patients. Tumour Necrosis Factor Receptor (TNFR) activation is being tested clinically to improve patient responses. However, low affinity FcΓR-mediated crosslinking often limits monoclonal antibody (mAb) clinical efficacy, which is further restricted by adverse safety effects. The generation of a bispecific agonist of CD137, where potent agonist activity is conditional upon PD-L1 crosslinking, allows a greater therapeutic window. FS222, an anti-CD137/PD-L1 mAb2, was generated by introducing a CD137-binding specificity into the Fc-region of a human IgG1 mAb targeting PD-L1. A LALA mutation significantly reduces FcΓR binding. Binding characterisation was assessed by surface plasmon resonance (SPR) and cell binding, and in vitro activity measured in human primary T cell assays. A dose-range finding study in cynomolgus monkey was performed to investigate toxicity, pharmacokinetics (PK) and pharmacodynamics (PD). A murine surrogate molecule was generated and its anti-tumor activity and PK/PD was tested in multiple syngeneic mouse tumor models. FS222 binds to human PD-L1 with subnanomolar affinity. This PD-L1 binding is a prerequisite to subsequently enable highly potent CD137 agonism (low nM EC50 values in primary cell in vitro assays). We term this mechanism conditional agonism. In cynomolgus monkey FS222 has a half-life of ~150h and single and repeat dosing resulted in observable PD changes in lymphocytes and soluble receptor levels at low doses. Furthermore, no hepatotoxicity as defined by changes in clinical chemistry and histopathology was detected. A surrogate anti-mouse-mAb2 significantly reduced tumor growth in multiple syngeneic mouse tumor models. The observed dose-dependent tumor growth inhibition resulted in a significant survival benefit and was concomitant with increases in tumor and peripheral activated CD8+ T cells. FS222 is a conditional CD137/PD-L1 bispecific agonist antibody and has superior activity to control mAbs and relevant combinations in vitro assays. A favorable safety profile and immunopharmacology was observed in a cynomolgus dose-range finding study and with the surrogate molecule in multiple syngeneic mouse tumor models. Anti-CD137 agonistic mAbs in clinical development have so far shown limitations due to dose limiting toxicity or poor clinical activity. In preclinical tumor models anti-CD137/PD-L1 surrogate mAb2 treatment resulted in intra-tumoral and peripheral PD changes leading to an increase in CD8+ T cell proliferation. These changes were dose dependent and coincident with tumor growth inhibition. FS222 is a highly active bispecific molecule with a favorable safety profile with the potential to serve a significant unmet need in the immunotherapy of solid tumors.Citation Format: Matthew A. Lakins, Alexander Koers, Jose Munoz Olaya, Raffaella Giambalvo, Daniel Jones, Sarka Pechouckova, Emma Goodman, Sylwia Marshall, Mateusz Wydro, Cristian Gradinaru, Francisca Wollerton, Sarah Batey, Daniel Gliddon, Michael Davies, Michelle Morrow, Mihriban Tuna, Neil Brewis. FS222 mAb2, a bispecific conditional agonist antibody targeting CD137 and PD-L1, induces potent lymphocyte activation and has a favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1540.

Background Despite advances with therapies targeting the PD-1/PD-L1 pathway, many patients are refractory or relapse following treatment. LAG-3 expression on exhausted T cells and T-regulatory cells (Tregs) in the tumor may be responsible for this resistance and provides a rationale for co-treatment with antibodies targeting LAG-3 and PD-L1. An alternative approach is the development of a bispecific antibody encompassing binding sites for two antigens. FS118 is a bispecific antibody targeting LAG-3 and PD-L1 that provides dual pathway blockade with the potential to drive unique biology via co-binding of PD-L1 and LAG-3. Methods FS118 was evaluated in vitro for antigen binding and de-repression of LAG-3 and PD-L1 function in both a D011.10 T-cell activation system and a super-antigen stimulated peripheral blood mononuclear cells (PBMC) assay. FS118 was also assessed in a human CD8 specific MHC I restricted antigen recall assay. Anti-tumor activity of a murine-specific molecule, mLAG-3/PD-L1 mAb2, was evaluated in vivo in the MC38 mouse tumor model and associated immunophenotypic changes were evaluated using flow cytometry. Results In murine in vitro assay systems, mLAG3/PD-L1 mAb2 recapitulates the function of FS118 in human systems. Furthermore, FS118 was shown to provide increased activation of human CD8+ T-cells compared to a PD-L1 mAb alone in response to stimulation with MHC Class I restricted peptides. In vivo, studies performed in MC38 tumor-bearing mice studies indicated that mLAG-3/PD-L1 could result in significant anti-tumor activity equivalent to a combination of antibodies targeting LAG-3 and PD-L1. Pharmacodynamic assessment demonstrated changes in the immunophenotype of tumor-infiltrating lymphocytes in the tumor of mLAG3/PD-L1 mAb2 treated mice. These changes were observed in cohorts which received the anti-mouse LAG-3/PD-L1 mAb2 and revealed a both a loss of LAG-3 surface expression on CD4+ and CD8+ T cells, as well as an increase in the CD8:Treg ratio. Conclusions Dual blockade of LAG-3 and PD-L1 with a bispecific antibody results in T-cell activation at least comparable to a combination of antibodies targeting LAG-3 and PD-L1 in primary T-cell assays and murine tumor models. Taken together, the human PBMC based assays and mouse tumor model data demonstrate that a LAG-3/PD-L1 mAb2 can not only potently suppress the checkpoint inhibitor LAG-3 at the tumor site, but does this in part through stimulating a CD8+ T cell mediated response. These data provide evidence to support the rationale for clinical development of FS118, a LAG-3/PD-L1 mAb2, for the treatment of human cancers. Citation Format: Matthew Kraman, Natalie Fosh, Katarzyna Kmiecik, Katy Everett, Carlo Zimarino, Mustapha Faroudi, Mateusz Wydro, Alexander Koers, Lesley Young, Daniel Gliddon, Michelle Morrow, Jacqueline Doody, Mihriban Tuna, Neil Brewis. Dual blockade of PD-L1 and LAG-3 with FS118, a unique bispecific antibody, induces CD8+ T-cell activation and modulates the tumor microenvironment to promote antitumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2719.

Nasal allergen challenge (NAC) is a human model of allergic rhinitis (AR) that delivers standardized allergens locally to the nasal mucosa allowing clinical symptoms and biospecimens such as peripheral blood to be collected. Although many studies have focused on local inflammatory sites, peripheral blood, an important mediator and a component of the systemic immune response, has not been well studied in the setting of AR. We sought to investigate immune gene signatures in peripheral blood collected after NAC under the setting of AR. Clinical symptoms and peripheral blood samples from AR subjects were collected during NAC. Fuzzy c-means clustering method was used to identify immune gene expression patterns in blood over time points (before NAC and 1, 2, and 6 h after NAC). We identified and validated seven clusters of differentially expressed immune genes after NAC onset. Clusters 2, 3, and 4 were associated with neutrophil and lymphocyte frequencies and neutrophil/lymphocyte ratio after the allergen challenge. The patterns of the clusters and immune cell frequencies were associated with the clinical symptoms of the AR subjects and were significantly different from healthy nonallergic subjects who had also undergone NAC. Our approach identified dynamic signatures of immune gene expression in blood as a systemic immune response associated with clinical symptoms after NAC. The immune gene signatures may allow cross-sectional investigation of the pathophysiology of AR and may also be useful as a potential objective measurement for diagnosis and treatment of AR combined with the NAC model.

The induction of cytokine release is a common consequence of the administration of therapeutic antibodies and in most cases is either tolerated by the patient or can be managed clinically by the administration of corticosteroids. However, in 2006, the administration of TGN1412 to six patients in a Phase I trial resulted in a unprecedentedly high level of cytokine release, systemic organ failure and the hospitalization of the subjects. Whilst the path to failure in this incident was multifactorial, at least one contributing factor was the lack of a robust in vitro model that would allow the prediction of the in vivo activity of a therapeutic antibody. In this article we review the current 'state of the art' of in vitro cytokine release assays and explore potential future developments.