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.

1Following the success of immune checkpoint blockade (ICB) therapy against cancer, agonistic 2 antibodies targeting T cell co-stimulatory pathways, are in clinical trials. The tumor necrosis factor 3 superfamily of receptors (TNFRSF) members CD137 and OX40 are co-stimulatory receptors that 4 stimulate T cell proliferation and activation upon interaction with their cognate ligands. Activating 5 CD137 and OX40 with agonistic monoclonal antibodies stimulates the immune system due to their 6 broad expression on CD4 + and CD8 + T cells and NK cells and has antitumor effects in pre-clinical 7 models. Most TNFRSF agonist antibodies require crosslinking via Fc receptors (FcRs), which can 8 limit their clinical activity. FS120 mAb 2 ™, a dual agonist bispecific antibody targeting CD137 and 9 OX40, activated both CD4 + and CD8 + T cells in a FcR-independent mechanism, dependent on 10 concurrent binding. A mouse surrogate version of the bispecific antibody displayed antitumor 11 activity in syngeneic tumor models, independent of T regulatory cell (Treg) depletion and of FcR-12interaction, but associated with peripheral T cell activation and proliferation. When compared to a 13 crosslink-independent CD137 agonist monoclonal antibody, the FS120 surrogate induced lower liver 14 T cell infiltration. These data support initiation of clinical development of FS120, a first-in-class dual 15 agonist bispecific antibody for the treatment of human cancer. 16 17

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Combining immunotherapeutic antibodies for treatment of cancer patients has shown benefits over single agent treatment. For example, the addition of anti-PD-1 antibodies to anti-CTLA-4 treatment in advanced melanoma patients has increased objective response rates from 19% to 57.6% (Larkin,J et al. 2015. NEJM 373:23-34). An alternative to combining two antibodies is the development of bispecific antibodies that not only bring two biologies together but may result in novel mechanisms of action that are impossible to attain with combinations. Lymphocyte Activation Gene-3 (LAG-3) is a member of the Ig superfamily expressed on activated T cells, NK cells, pDCs, B cells, γδ T cells and participates in immune suppression, particularly through persistent strong expression in a percentage of regulatory T cells (Tregs). Programmed Cell Death receptor (PD-1) binds to its ligand PD-L1, expressed not only on activated immune cells to inhibit cellular immune responses but also on tumour cells. Expression of LAG-3 and PD-1 leads to T cell exhaustion, allowing tumour escape from immune surveillance. Combining inhibitory antibodies to PD-1 and LAG-3 reactivates T cells leading to efficacy in murine models over single treatment (Woo,S-R et al. 2012. Cancer Res. 15: 917-27) which prompted interest in the development of a LAG-3 and PD-L1 bispecific antibody for inhibiting tumour growth. A murine-specific anti-LAG-3 and PD-L1 mAb²TM (bispecific antibody) was engineered which binds both antigens simultaneously and with nanomolar affinities. The anti-LAG-3/PD-L1 mAb² inhibits LAG-3 binding to MHCII and PD-L1 binding to PD-1 and CD80, resulting in T cell activation in an in vitro assay. This potency translates into in vivo efficacy, where the anti-LAG-3/PD-L1 mAb² decreased tumour burden in the MC38 colon carcinoma tumour model, both in early-established or well-established tumours. At the end of the study tumour-free animals were more numerous in the LAG-3/PD-L1 bispecific group than in the group given a combination of individual anti-LAG-3 and PD-L1 antibodies. The results were recapitulated in the CT26 murine colon cancer model, where the LAG-3/PD-L1 mAb² showed an increase of anti-tumour activity as compared to the combination of anti-LAG-3 and anti-PD-L1 antibodies. Thus, the preclinical data supports developing an anti-human LAG-3/PD-L1 mAb² for the treatment of cancer patients. This abstract is also being presented as Poster A27. Citation Format: Katy Everett, Matthew Kraman, Katarzyna Kmiecik, Natalie Allen, Mustapha Faroudi, Carlo Zimarino, Mateusz Wydro, Jacqueline Doody. A LAG-3/PD-L1 bispecific antibody inhibits tumour growth in two syngeneic colon carcinoma models. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR06.

Combining immunotherapeutic antibodies for treatment of cancer patients has shown benefits over single agent treatment. For example, the addition of anti-PD-1 antibodies to anti-CTLA-4 treatment in advanced melanoma patients has increased objective response rates from 19% to 57.6% (Larkin,J et al. 2015. NEJM 373:23-34). An alternative to combining two antibodies is the development of bispecific antibodies that not only bring two biologies together but may result in novel mechanisms of action that are impossible to attain with combinations. Lymphocyte Activation Gene-3 (LAG-3) is a member of the Ig superfamily expressed on activated T cells, NK cells, pDCs, B cells, γδ T cells and participates in immune suppression, particularly through persistent strong expression in a percentage of regulatory T cells (Tregs). Programmed Cell Death receptor (PD-1) binds to its ligand PD-L1, expressed not only on activated immune cells to inhibit cellular immune responses but also on tumor cells. Expression of LAG-3 and PD-1 leads to T cell exhaustion, allowing tumor escape from immune surveillance. Combining inhibitory antibodies to PD-1 and LAG-3 reactivates T cells leading to efficacy in murine models over single treatment (Woo,S-R et al. 2012. Cancer Res. 15: 917-27) which prompted interest in the development of a LAG-3 and PD-L1 bispecific antibody for inhibiting tumor growth. A murine-specific anti-LAG-3 and PD-L1 bispecific antibody was engineered which binds both antigens simultaneously and with nanomolar affinities. The anti-LAG-3/PD-L1 bispecific antibody inhibits LAG-3 binding to MHCII and PD-L1 binding to PD-1 and CD80, resulting in T cell activation in an in vitro assay. This potency translates into in vivo efficacy, where the anti-LAG-3/PD-L1 bispecific antibody decreased tumor burden in the MC38 colon carcinoma tumor model, both in early-established or well-established tumors. At the end of the study tumor-free animals were more numerous in the LAG-3/PD-L1 bispecific group than in the group given a combination of individual anti-LAG-3 and PD-L1 antibodies. The results were recapitulated in the CT26 murine colon cancer model, where the LAG-3/PD-L1 bispecific showed an increase of anti-tumor activity as compared to the combination of anti-LAG-3 and anti-PD-L1 antibodies. Thus, the preclinical data supports developing an anti-human LAG-3/PD-L1 bispecific for the treatment of cancer patients. Citation Format: Jacqueline Doody, Matthew Kraman, Katy Everett, Carlo Zimarino, Katarzyna Kmiecik, Mustapha Faroudi, Paul Thompson, Mateusz Wydro, Francisca Wollerton, Carlos Gaspar, Mihriban Tuna. A LAG-3/PD-L1 bispecific antibody inhibits tumor growth in two syngeneic colon carcinoma models [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B091.