Enhancement of the Tumor Penetration of Monoclonal Antibody by Fusion of a Neuropilin-Targeting Peptide Improves the Antitumor Efficacy

Abstract

The limited localization and penetration of monoclonal antibodies (mAb) into solid tumors restricts their antitumor efficacy. Here, we describe a solid tumor-targeting antibody with enhanced tumor penetration activity. We designed a 22-residue peptide (A22p), which was extracted from the C-terminal basic region of semaphorin 3A (Sema3A) but modified to have higher affinity with neuropilin receptors (NRP), and genetically fused it to the C-terminus of Fc of human immunoglobulin G1 via a 15-residue (G 4 S) 3 linker, generating FcA22p, for the bivalent binding to NRPs. In contrast to Fc or the monovalent A22p peptide alone, Fc-A22p homed to tumor vessels and induced vascular permeability through VE-cadherin downregulation and penetrated tumor tissues by interacting with NRPs in mice bearing human tumor xenografts. We extended the Fc-A22p platform by generating mAb-A22p antibodies of two clinically approved solid tumor-targeting mAbs, the anti-EGF receptor mAb cetuximab (erbitux), and the anti-Her2 mAb trastuzumab (herceptin). The mAb-A22p antibodies retained the intrinsic antigen binding, natural Fc-like biophysical properties, and productivity in mammalian cell cultures, comparable with those of the parent mAbs. In mouse xenograft tumor models, the mAb-A22p antibodies more efficiently homed to tumor vessels and spread into the extravascular tumor parenchyma, which significantly enhanced antitumor efficacy compared with the parent mAbs. Our results suggest that mAb-A22p is a superior format for solid tumor-targeting antibodies due to its enhanced tumor tissue penetration and greater antitumor efficacy compared with conventional mAbs.

Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency

“…Tumor size, calculated as length £ width 2 £ 0.5, and body weights were recorded at regular intervals. 44 Mice were euthanized with CO 2 asphyxiation. All experiments were conducted in accordance with institutional guidelines and under an Institutional Animal Care and Use Committee (IACUC) protocol.…”

Humanization of a phosphothreonine peptide-specific chicken antibody by combinatorial library optimization of the phosphoepitope-binding motif

“…The two isolated Fab clones (huPT3 and huPT9) were reformatted into IgG1 through sub-cloning the HC (VHeCH1) gene into the modified pcDNA 3.4 vector (Invitrogen) carrying the human IgG1 Fc (hinge-CH 2 -CH 3 ) gene and LC (VL-CL) gene into the pcDNA 3.4 vector, as described previously [16,20]. Antibodies were produced by transient transfection of the plasmids into HEK293F cells using the Freestyle 293-F expression and media system (Invitrogen), as described in previous studies [16,20].…”

“…Antibodies were produced by transient transfection of the plasmids into HEK293F cells using the Freestyle 293-F expression and media system (Invitrogen), as described in previous studies [16,20]. Antibodies were purified from the culture supernatants using a CaptivA™ PriMAB protein A column (Repligen, USA) following the manufacturer's protocol.…”

“…2B) [17,20]. After two rounds of sorting, 30 clones were randomly analyzed to yield two unique high-affinity binders, designated as huPT3 and huPT9 (Fig.…”

“…The plasmids encoding HC and LC, respectively, were transiently co-transfected into HEK293F cells to express the IgG antibody [16,20]. Both huPT3 and huPT9 IgG antibodies were well-expressed in the correctly assembled form (Fig.…”