Design and Preclinical Translation Study of Mixed-Bispecific/Trispecific Antibody for Enhancing Therapeutic Effectiveness of Anticancer Drug-Loaded Pegylated Nanocarriers by Simultaneously Targeting to Tumor Cells and Cancer/Tumor Associated Fibrobla (I)

Antibody (Ab)-conjugated methoxy-PEGylated nanocarriers have been proven to improve the specific targeting and therapeutic efficacy toward tumors. However, covalent chemical modification of Ab/ligands with mPEG-nanocarriers often leads to heterogeneous orientation of Ab/ligand and difficulty in obtaining a reproducible product. In this project, we will develop multispecific antibodies by fusing a scFv against prostate surface membrane antigen (anti-PSMA), a scFv against breast cancer marker (anti-a9-nAChR or anti-HER2), or a scFv against tumor associated fibroblast antigen (anti-FAP) with a humanized anti-methoxy polyethylene glycol Fab (anti-mPEG Fab) to form a-tumor/a-mPEG BsAbs, a-FAP/a-mPEG BsAb, or a-tumor/a-FAP/a-mPEG TsAbs, respectively. The anti-mPEG end of TsAb (or BsAb) could noncovalently modify the terminal end of the mPEG on mPEG-nanocarriers, leading to the orientation of anti-tumor scFv and anti-FAP scFv to be homogeneous. The other anti-tumor and anti-FAP end offers non-targeted mPEG-nanocarriers to acquire the tumor/FAP-specificity for the targeting of prostate (or breast) cancer cells in vitro and in vivo. Based on this strategy, this project has the following specific aims: 1. Development and characterization of novel trispecific antibodies (TsAb) and bispecific antibodies (BsAb), 2. Optimize the formulation of TsAb (or BsAb)-micelle-DOC for anti-cancer efficacy. 3. Image the tumor targeting efficacy and endocytosis of TsAb (or BsAb)-micelle in vivo. 4. Analyze the pharmacokinetics (PK), biodistribution, and in vivo toxicity of TsAb (or BsAb)-micelle-DOC. 5. Determine the therapeutic index of TsAb (or BsAb)-micelle-DOC for primary and metastatic tumors. 6. Determine the therapeutic index of TsAb (or BsAb)-micelle-DOC to patient derived prostate / breast tumor xenografts. Successful development of this strategy is expected to provide valuable tools for non-covalent modification of any mPEG-nanocarriers, allowing increased specific targeting and therapeutic efficacy of mPEG-nanocarriers (Liposomes, Micelles…)for tumors in the clinic.