Ellen S. Vitetta

Amplification and overexpression of the HER-2 oncogene in breast cancer is felt to be stable over the course of disease and concordant between primary tumor and metastases. Therefore, patients with HER-2-negative primary tumors rarely will receive anti-Her-2 antibody (trastuzumab, Herceptin) therapy. A very sensitive blood test was used to capture circulating tumor cells (CTCs) and evaluate their HER-2 gene status by fluorescence in situ hybridization. The HER-2 status of the primary tumor and corresponding CTCs in 31 patients showed 97% agreement, with no false positives. In 10 patients with HER-2-positive tumors, the HER-2/chromosome enumerator probe 17 ratio in each tumor was about twice that of the corresponding CTCs (mean 6.64 +/- 2.72 vs. 2.8 +/- 0.6). Hence, the ratio of the CTCs is a reliable surrogate marker for the expected high ratio in the primary tumor. Her-2 protein expression of 10 CTCs was sufficient to make a definitive diagnosis of the HER-2 gene status of the whole population of CTCs in 19 patients with recurrent breast cancer. Nine of 24 breast cancer patients whose primary tumor was HER-2-negative each acquired HER-2 gene amplification in their CTCs during cancer progression, i.e., 37.5% (95% confidence interval of 18.8-59.4%). Four of the 9 patients were treated with Herceptin-containing therapy. One had a complete response and 2 had a partial response.

Single-walled carbon nanotubes (CNTs) emit heat when they absorb energy from near-infrared (NIR) light. Tissue is relatively transparent to NIR, which suggests that targeting CNTs to tumor cells, followed by noninvasive exposure to NIR light, will ablate tumors within the range of NIR. In this study, we demonstrate the specific binding of antibody-coupled CNTs to tumor cells in vitro, followed by their highly specific ablation with NIR light. Biotinylated polar lipids were used to prepare stable, biocompatible, noncytotoxic CNT dispersions that were then attached to one of two different neutralite avidin-derivatized mAbs directed against either human CD22 or CD25. CD22(+)CD25(-) Daudi cells bound only CNTs coupled to the anti-CD22 mAb; CD22(-)CD25(+) activated peripheral blood mononuclear cells bound only to the CNTs coupled to the anti-CD25 mAb. Most importantly, only the specifically targeted cells were killed after exposure to NIR light.

The proteins on surfaces of living splenic lymphocytes from normal BALB/c mice were iodinated enzymatically. Such cells were fractionated into two sub-populations: one composed almost exclusively of small lymphocytes and the other mainly of large lymphocytes and plasma cells. Specific immunoprecipitation of radiolabeled surface Ig obtained from lysates of these cell populations indicated that approximately 2-3% of the acid-precipitable radioactivity from the cell surface is Ig. Moreover, 95% of the H chain radioactivity from the Ig of the small lymphocyte fraction and 90% from the large lymphocyte-plasma cell fraction was characterized as micro by precipitation with anti-micro sera as well as by molecular weight determination on polyacrylamide gels in sodium dodecyl sulfate. The Ig was recovered from the cell surface in the form of an IgM monomer. Control experiments suggested that the monomer did not result from depolymerization of 19S IgM by the methods used to radiolabel and isolate the molecule. (3)H-tyrosine labeling of IgM produced by meyloma cells and radio-iodination of IgM in solution gave the same ratios of microL radioactivity as radiolabeling of IgM on cells, indicating that the tyrosine residues of L and micro-chains of cell surface IgM are available to the lactoperoxidase during the iodination. This is consistent with the hypothesis that cell surface IgM is entirely on the outside of the plasma membrane presumably attached to it by its Fc fragment. These results, together with previous reports by others, suggest that IgM, in its monomeric form, is the main antigen-specific receptor on lymphocytes of normal mice.

We have characterized a murine IgM monoclonal antibody, TEC-11, that recognizes endoglin and may be suitable for targeting cytotoxic agents to human tumor vasculature. TEC-11 strongly stains endothelial cells in a broad range of solid human tumors while staining endothelial cells in the majority of normal, healthy adult tissues relatively weakly. Human umbilical vein endothelial cells (HUVECs) in sections of the umbilical vein react weakly with TEC-11, whereas proliferating HUVECs in tissue culture react strongly and uniformly. HUVEC cultures grown to confluence and then rested contain two subpopulations having high and low levels of endoglin expression. Flow cytometry revealed that a significant proportion of cells with high endoglin expression are cycling, having markedly increased levels of cellular protein, RNA, and DNA by comparison to low endoglin-expressing cells, which appear to be noncycling. Taken together, the increased binding of TEC-11 to tumor vasculature and to dividing as opposed to noncycling HUVECs in vitro suggests that endoglin is an endothelial cell proliferation-associated marker. An immunotoxin [TEC-11.deglycosylated ricin A chain (dgA)] composed of TEC-11 and dgA was 3000-fold more potent at inhibiting protein synthesis in proliferating HUVEC cultures than in confluent cultures. The confluent cells were no more sensitive to TEC-11.dgA than they were to an isotype-matched immunotoxin of irrelevant specificity. These findings suggest that TEC-11.dgA might have therapeutic value in the treatment of solid tumors in humans by selectively killing dividing endothelial cells which are prevalent in such tumors.