James D. Marks, Bin Liu, Zhou Yu, Marie A. Poul, Tara Heitner, Ulrik B. Nielsen, Baltazar Becerill, Dave O’Connell, Mike Huie
University of California, San Francisco, CA

Selection of Internalizing Antibodies from Phage Libraries for Targeted Drug Delivery

Many approaches to treating cancer require the delivery of a therapeutic drug, protein or gene into the cytosol of the cancer cell. Since many of these therapies are toxic, it is essential that they be delivered only to cancer cells. Thus discovery of molecules capable of binding to cancer cells but not normal cells is an important first step in developing targeted drugs for cancer therapy. We hypothesized that we could develop a method to select single chain Fv (scFv) antibody fragments capable of triggering receptor-mediated endocytosis from libraries of antibodies displayed on filamentous bacteriophage (phage display).

Using a model system, we showed using immunofluorescence that phage displaying a scFv that binds ErbB2 could be endocytosed into human cancer cells that expressed ErbB2. Moreover, infectious phage could be recovered from the cytosol. Dimeric scFv (diabodies) and display of scFv multivalently in a phage vector led to more efficient endocytosis.

The selection methodology developed using the model system was applied to the selection of a human phage antibody library on the breast tumor cell line SKBR3. After three rounds of selection, more than 50% of the phage antibodies bound SKBR3 cells, but not a normal breast cell line. Further antibody characterization identified two of the antibodies as binding ErbB2 and the transferrin receptor. Both antibodies were endocytosed into SKBR3 cells, both as phage and as native scFv. The anti-transferrin receptor antibody also caused dose dependent growth inhibition of SKBR3 cells. The anti-ErbB2 scFv (F5) was used to target doxorubicin containing stealth liposomes (ILs). In a mouse model, ILs were more effective than untargeted liposomes in causing tumor regression and cures. Based on the success of preclinical studies, expression of the F5 scFv has been scaled up and transferred to the GMP fermenters at the NCI MARP facility to manufacture antibody for a phase 1 clinical trial.

This method can be used to both: 1) identify new receptors on the surface of cancer cells which can be utilized for specific therapeutic targeting; and at the same time 2) generates human antibodies capable of targeting the receptor.