Biodistribution and efficacy of [131I]A33scFv::CDy, a recombinant antibody-enzyme protein for colon cancer

Abstract

In antibody-directed enzyme-prodrug therapy (ADEPT), an antibody-bound enzyme localizes to tumor tissue, where it selectively converts a subsequently administered non-toxic prodrug into a cytotoxic drug. A33scFv::CDy is a bifunctional fusion construct comprising a single chain antibody against the gpA33 antigen and the prodrug-converting enzyme cytosine deaminase. gpA33 is highly and homogeneously expressed in >95% of all colorectal cancers. Here we describe the biodistribution and tumor-targeting capacity of 131 I labeled A33scFv::CDy. 131 I labeling of A33scFv::CDy was performed by the chloramine-T method, and the properties of the resulting [ 131 I]A33scFv::CDy conjugate were determined in vivo and in vitro, including biodistribution studies in nude mice bearing human LIM1215 colon carcinoma xenografts. The [ 131 I]A33scFv::CDy conjugate bound specifically to colorectal cancer cells in vitro with KD = 15.8 nM as determined by a saturation assay. in vivo, the tumor uptake of [ 131 I]A33scFv::CDy peaked at 87% injected dose/g 47 h post injection. Normal tissue uptake was low, and activity in blood was lower than in tumor at all time-points studied (6-92 h). The tumor-to-blood ratio increased over time with a maximum of 8.1 at 67 h post injection. [ 131 I]A33scFv::CDy thus shows a biodistribution that makes it attractive for both radioimmunotherapy (RIT) and ADEPT. Preliminary therapeutic experiments showed a significant reduction of tumor size in mice treated with the A33scFv::CDy-5fluorocytosine/5-fluorouracil ADEPT system. This work demonstrates the feasibility of ADEPT and RIT based on the A33scFv::CDy recombinant construct.

Targeted Tumor Therapy With a Fusion Protein of an Antiangiogenic Human Recombinant scFv and Yeast Cytosine Deaminase

“…A colon carcinoma nude mouse model based on human colon carcinoma cells is established in our laboratory. 34 Moreover, we established a mouse model with ED-B fibronectin-expressing murine colon carcinoma cells stably transfected with human glycoprotein A33. We expect a synergistic effect of the combination of A33CDy with L19CDy-His, resulting in inhibition of angiogenesis and eventually tumor cell death due to insufficient supply of oxygen and nutrients as well as cell killing by 5-FU either directly or indirectly due to the bystander effect of the membrane-permeable 5-FU.…”

Preparation of an engineered safer immunotoxin against colon carcinoma based on the ribotoxin hirsutellin A

“…Some issues have appeared regarding its low tumor penetration or secondary immune responses , leading to the use of different humanized antibody fragments as specific carriers of other molecules in immunoconjugate designs. In this regard, the use of the variable fragment of mAbA33 (scFvA33), which retains its binding specificity, could allow a better penetration into the tumor, as reported previously . However, the ability of a 131I‐huA33 antibody to penetrate into the necrotic centres of large tumors was not reduced .…”

Recent Innovations in Antibody-Mediated, Targeted Particulate Nanotechnology and Implications for Advanced Visualisation and Drug Delivery

“…Moreover, improvements in Ab production strategies and genetic engineering tools, such as DNA shuffling and mRNA or phage display, have enabled the production of specific binding monoclonal Ab against almost any antigen. Immunotargeting has been developed for a large spectrum of agents, such as fluorophores [76], cytokines [77] or enzymes [78,79] for AntibodyDirected Enzyme Prodrug Therapy approaches. In 2000, the first Ab-drug conjugate for human therapeutic use was approved by the FDA (Gemtuzumab, Mylotarg ® ), consisting of a chemotherapeutic agent ozogamicin conjugated to humanised CD33-specific monoclonal Ab [80].…”