Abstract 2248: Generation and formatting of a bispecific Affimer® biotherapeutic for the inhibition of the LAG-3 and PD-L1 pathway

Abstract

Programmed Cell Death-Ligand 1 (PD-L1) binds to its receptor, Programmed Cell Death protein (PD-1) and is expressed on both a range of activated immune cells and tumor cells. Lymphocyte Activation Gene-3 (LAG-3) is a member of the Ig superfamily and is expressed on activated T cells, NK cells, DCs and B cells. Both the PD-1/PD-L1 and the LAG-3 pathway are involved in immune suppression, leading to the down-regulation of T cell activity and the generation of exhausted T cells, allowing tumor escape and metastasis. Despite the impressive success of PD-L1 targeted immunotherapy, there remains a large proportion of patients with many tumor types that fail to benefit or acquire resistance during therapy. Consequently, it is necessary to consider future combinations of immunotherapies to enhance the clinical outcomes and reduce tumor escape. LAG-3 is upregulated on T-cells following PD-1/PD-L1 pathway blockade, indicating that targeting of both pathways as a combination may lead to more durable antitumor responses. To reach the full potential of LAG-3/PD-L1 dual blockade the development of a bispecific immunotherapy may further augment T-cell activation. Affimer biotherapeutics are a new protein scaffold with great potential for the generation of biotherapeutics. Based on the human protease inhibitor Stefin A, the scaffold is small (14kDa), lacks any post-translational modifications such as disulfide bonds and expresses at high levels. Affimers against a target are generated through the use of proprietary, highly diverse phage display libraries that have been created by engineering in two nine amino acid peptide loops into the scaffold backbone. We have identified a range of potent Affimer biotherapeutic antagonists to both human PD-L1 and LAG-3. When formatted as a bispecific Fc fusion to both the N and C-terminal of a human IgG1 Fc protein the Affimers demonstrate pM to nM binding affinities and binding to both target antigens simultaneously without compromising the affinities. The bispecific Fc fusion has been shown to block the interaction of LAG-3 with its ligand, MHC class II and PD-L1 with both PD-1 and CD80 by competitive ELISA. Functionally, the LAG-3/PD-L1 bispecific enhanced T-cell activation in vitro in both SEB-driven and mixed lymphocyte reaction assays. The Fc fusion format expresses over 500 mg/L from Expi293F mammalian transient transfections and can be purified to over 95% purity. The pharmacokinetic (PK) properties showed half-life extension of the Fc fusion format in mouse by using the IgG Fc-FcRn recycling pathway, dosing at 10 mg/kg resulted in a half-life extension of over 90 hours. We have demonstrated that the Affimer scaffold protein has all the necessary attributes to be developed as a bispecific LAG-3/PD-L1 immunotherapy. The Affimer scaffold was formatted as a bispecific Fc fusion molecule with ease, had good biophysical properties and shown to be well tolerated in vivo. Citation Format: Amrik Basran, Emma Jenkins, Estelle Adam, Michele Writer, Floriane Laurent, Assa Oumie, Jyrki Sivula, Jennifer Hillman, Lisa Strong, Maureen West, Emma Stanley. Generation and formatting of a bispecific Affimer® biotherapeutic for the inhibition of the LAG-3 and PD-L1 pathway [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 2248.