[PMC free article] [PubMed] [Google Scholar] 21. results to any tumor model in which the antigen density and turnover rate are known, allowing facile quantitative prediction of the minimum antibody dose required for complete tumor saturation. Keywords: carcinoembryonic antigen, tumor penetration, microdistribution, transport barriers, mathematical models, protein delivery, cancer, drug transport, macromolecular drug delivery, preclinical pharmacokinetics INTRODUCTION Antibodies represent a significant and rapidly growing proportion of oncology therapeutics.1 Although many have found success in VER-49009 a range of cancers, particularly hematologic malignancies, there remain substantial barriers to the effective use of antibodies to treat solid tumors. Solid tumors present a number of barriers to tumor targeting and penetration, including blood clearance, extravasation, diffusion through the interstitial space, binding to antigen, endocytosis, and degradation.2,3 Many of these barriers are further exacerbated by the disordered physiology of solid tumors, which results in highly permeable and irregular vasculature and high interstitial fluid pressure.4C6 For decades researchers have noted that the penetration into solid tumor tissue is often limited for drugs ranging in size and mechanism of action from chemotherapeutics to antibodies and nanoparticles.3,4,7C11 Limited penetration has been linked to reduced therapeutic efficacy, even in cases in which bulk tumor uptake is high enough to exert an antitumor effect with a well-distributed therapeutic.12 Recently it was shown that the US Food and Drug Administration-approved monoclonal antibodies cetuximab and trastuzumab penetrate poorly into tumors in animal xenograft models.13,14 Quantitative studies of antibody delivery to and distribution within tumor spheroids have yielded insights into the roles that antibody affinity and antigen internalization play in this process.15,16 study of monoclonal antibody and antigen distribution around tumor blood vessels as a function of antibody dose covering two orders of magnitude. A computer-aided method of analyzing entire tumor cross sections in a quantitative and unbiased manner is utilized to generate data. These results are consistent with a Krogh cylinder model and scaling analysis, which predict the antibody dose necessary to saturate a tumor for a given antigen cell surface expression level and metabolic half-life. Although these modeling analyses are dramatic oversimplifications of the tumor microenvironment, they are nonetheless successful in quantitatively predicting the distribution of extravasated antibody averaged over the tumor cross section. MATERIALS AND METHODS Reagents A low-picomolar humanized antibody to carcinoembryonic antigen (CEA), designated sm3e, has previously been engineered and characterized.17 This antibody was secreted in transiently transfected human embryonic YWHAB kidney 293 cells (Invitrogen, Carlsbad, California), purified by protein A resin (Millipore, Billerica, Massachusetts) and buffer exchanged into phosphate-buffered saline (PBS). The antibody was fluorescently labeled using the Alexa Fluor 488 protein labeling kit from Invitrogen. Labeling was conducted in a single batch of approximately 3 mg protein to yield a homogenously labeled reagent source for all experiments presented. Anti-CEA monoclonal antibody M85151a was purchased from Fitzgerald (Acton, Massachusetts), and goat anti-rat 546 secondary antibody was from Invitrogen. Antibody M85151a was labeled with the Alexa Fluor 647 protein labeling kit (Invitrogen) and has been previously determined to be noncompetitive with sm3e.18 Animal Model Animal use and care was conducted in full compliance and under approval from the Committee on Animal Care of Massachusetts Institute of Technology. A CEA-positive human colorectal cancer cell line, LS174T, was used to induce xenograft formation in the flanks of 6C8 weeks old NCr nude mice (Taconic, Hudson, New York) by subcutaneous injection of 5 106 cancer cells. Tumors were allowed to establish and grow to a size of 5C10 mm, at which point antibody injections were conducted. Varying doses of fluorescently labeled sm3e, ranging from 5 to 500 g, were supplemented as needed with immunoglobulin (Ig) G from human serum (SigmaCAldrich, St. Louis, Missouri) to 500 g total IgG and then injected retroorbitally into tumor-bearing nude mice. Mice were sacrificed 24 h after antibody administration and tumors were immediately excised and snap frozen in optimal cutting temperature medium (Sakura Finetek USA, Torrance, California) VER-49009 via isopentane over liquid nitrogen. Frozen blocks were stored at ?80C until sectioned by the Histology Core Facility of Koch Institute. Frozen blocks were sectioned approximately 1C2 VER-49009 mm into the tumor tissue at a thickness of 8 m and.