Targeting the EGF-Receptor and CD38 in Solid and Haematological Malignancies with Nanobody-Based Heavy Chain Antibodies and AAV Vectors

Abstract

Abstract Background Although monoclonal antibodies binding the EGF-receptor (cetuximab, panitumumab) or CD38 (daratumumab) show therapeutic efficacy, many cancer patients still develop resistance. Therefore, new therapies are necessary. Nanobodies are highly soluble immunoglobulin domains derived from heavy chain antibodies (hcAbs) that naturally occur in camelids. Replication defective AAV (adeno associated virus) vectors, when equipped with a tumor targeting ligand, can be used for the delivery of (suicide) genes to tumor cells. Methods We generated recombinant chimeric nanobody-based human IgG1 hcAbs, by fusing EGFR- or CD38-specific nanobodies derived from immunized llamas to the hinge, CH2 and CH3 domains of human IgG1 (1, 2). To promote cytotoxic effector functions we introduced CDC (complement dependent cytotoxicity)-enhancing and ADCC (antibody-dependent cell-mediated cytotoxicity)-enhancing mutations into the CH2 and CH3 domains of human IgG1. As a second strategy to target CD38-overexpressing cells, we inserted CD38-specific nanobodies into the VP1 capsid protein of AAV2 (3). We analyzed the capacity of these constructs to target EGFR and CD38 in tumor cell lines and multiple myeloma (MM) patient bone marrow samples using proliferation, CDC, ADCC and AAV-transduction assays. Results EGFR-specific hcAbs effectively blocked the proliferation of tumor cell lines (HNSCC, head and neck squamous cell carcinoma and mCRC, metastatic colorectal cancer) expressing WT EGFR and cetuximab/panitumumab-escape variants of EGFR. EGFR- and CD38-specific hcAbs mediated effective ADCC towards tumor cell lines (HNSCC, mCRC and MM respectively). HcAbs carrying the hexabody mutation were more potent at inducing CDC than parental hcAbs. Display of CD38-specific nanobodies on the AAV capsid resulted in a 20- to 100-fold enhanced transduction of myeloma cells in patient bone marrow samples. Some of these results have recently been published (1, 2) or accepted for publication (3). We will present updated data on tumor cell lines and patient samples. Conclusion Nanobody-based hcAbs and nanobody-displaying AAV hold promise as novel tools to target solid and hematological tumors. Legal entity responsible for the study Friedrich Koch-Nolte. Funding DFG (German Research Foundation) and SFB (Collaborative Research Centres). Disclosure The author has declared no conflicts of interest.