The engineered CD80 variant fusion therapeutic davoceticept combines checkpoint antagonism with conditional CD28 costimulation for anti-tumor immunity.

Despite the recent clinical success of T cell checkpoint inhibition targeting the CTLA-4 and PD-1 pathways, many patients either fail to achieve objective responses or they develop resistance to therapy. In some cases, poor responses to checkpoint blockade have been linked to suboptimal CD28 costimulation and the inability to generate and maintain a productive adaptive anti-tumor immune response. To address this, here we utilize directed evolution to engineer a CD80 IgV domain with increased PD-L1 affinity and fuse this to an immunoglobulin Fc domain, creating a therapeutic (ALPN-202, davoceticept) capable of providing CD28 costimulation in a PD-L1-dependent fashion while also antagonizing PD-1 - PD-L1 and CTLA-4-CD80/CD86 interactions. We demonstrate that by combining CD28 costimulation and dual checkpoint inhibition, ALPN-202 enhances T cell activation and anti-tumor efficacy in cell-based assays and mouse tumor models more potently than checkpoint blockade alone and thus has the potential to generate potent, clinically meaningful anti-tumor immunity in humans.

A dual-targeting PDGFRbeta/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo.

Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VEGF and PDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting PDGFRbeta and VEGF-A were selected for superior stability. The scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. The resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. The assembly of a bsAb using stable monomeric units allowed development of an anti-PDGFRB/VEGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.

Engineering of stable bispecific antibodies targeting IL-17A and IL-23.

Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.