Monoclonal antibody therapeutics with up to five specificities: Functional enhancement through fusion of target-specific peptides

Abstract

The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvβ3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.

Tetraspecific ligand for tumor-targeted delivery of nanomaterials

“…The central barrier is to develop next generation targeting ligands with multispecificity that can be tuned and applied to different cancer types [10,11]. While it is a great achievement to engineer a monoclonal antibody to acquire multispecificity, the process is often limited [12], in addition to high manufacturing cost. In this study, we aimed to develop a targeting ligand with tetraspecificity capable of binding four cell surface biomarkers, namely, VEGFR2, α v β 3 integrin, EGFR, and HER2, which are of significant cancer targeting interest [13–16].…”

Novel multispecific heterodimeric antibody format allowing modular assembly of variable domain fragments

“…Moreover, all of these formats rely on the display of Fvs, which often exhibit poor stability and promote aggregation of the composite molecule 24,56 . Hence, other antibody formats that employ alternative binding moieties have been developed, such as Zybodies 57 and Fynomabs 7 . Variable-domain-only multispecifics with >2 specificities, such as the triplebody, have only been described in single-chain formats and only up to 3 specificities.…”

Antibody Conjugation Approach Enhances Breadth and Potency of Neutralization of Anti-HIV-1 Antibodies and CD4-IgG

“…To address this challenge, modified antibody formats (e.g., knobs into holes [40], IgG-scFv [41], and DVD-Ig [21]) have been created using protein engineering methods. Often these approaches suffer from protein instability and low expression yields, although the recently described peptide fusion approach overcomes many of these problems (42). The approach used here involves chemical conjugation of a peptide or small molecule to a monoclonal antibody that possesses a desired specificity.…”