Tumours have developed strategies to interfere with most steps required for anti-tumour immune responses. Although many populations contribute to anti-tumour responses, tumour-infiltrating cytotoxic T cells dominate, hence, many suppressive strategies act to inhibit these. Tumour-associated T cells are frequently restricted to stromal zones rather than tumour islands, raising the possibility that the tumour microenvironment, where crosstalk between malignant and “normal” stromal cells exists, may be critical for T cell suppression. We provide evidence of direct interactions between stroma and T cells driving suppression, showing that cancer-associated fibroblasts (CAFs) sample, process and cross-present antigen, killing CD8+ T cells in an antigen-specific, antigen-dependent manner via PD-L2 and FASL. Inhibitory ligand expression is observed in CAFs from human tumours, and neutralisation of PD-L2 or FASL reactivates T cell cytotoxic capacity in vitro and in vivo. Thus, CAFs support T cell suppression within the tumour microenvironment by a mechanism dependent on immune checkpoint activation.

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 þ 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 þ 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.

Summary Biofilms are an important element of the natural ecosystems but can be detrimental in health care and industrial settings. To improve our ability to combat biofilms, we need to understand the processes that facilitate their formation and dispersal. One approach that has proven to be invaluable is to image biofilms as they grow. Here we describe tools and protocols to visualize biofilms with multiphoton laser scanning microscopy, compare this with single photon laser scanning confocal microscopy and highlight best working procedures. Furthermore, we describe how with multiphoton laser scanning microscopy the laser can be used to manipulate the biofilm, specifically to achieve localized bleaching, killing or ablation within the biofilm biomass. These applications open novel ways to study the dynamics of biofilm formation, regeneration and dispersal.

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 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.

<p>All supplementary figures and tables within one powerpoint document</p>

<p>All supplementary figures and tables within one powerpoint document</p>

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.