Jacqueline E. Testa

Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen

We have previously used a subtractive immunization (SI) approach to generate monoclonal antibodies (mAbs) against proteins preferentially expressed by the highly metastatic human epidermoid carcinoma cell line, M+HEp3. Here we report the immunopurification, identification and characterization of SIMA135/CDCP1 (subtractive immunization M+HEp3 associated 135 kDa protein/CUB domain containing protein 1) using one of these mAbs designated 41-2. Protein expression levels of SIMA135/CDCP1 correlated with the metastatic ability of variant HEp3 cell lines. Protein sequence analysis predicted a cell surface location and type I orientation of SIMA135/CDCP1, which was confirmed directly by immunocytochemistry. Analysis of deglycosylated cell lysates indicated that up to 40 kDa of the apparent molecular weight of SIMA135/CDCP1 is because of N-glycosylation. Western blot analysis using a antiphosphotyrosine antibody demonstrated that SIMA135/CDCP1 from HEp3 cells is tyrosine phosphorylated. Selective inhibitor studies indicated that an Src kinase family member is involved in the tyrosine phosphorylation of the protein. In addition to high expression in M+HEp3 cells, the SIMA135/CDCP1 protein is expressed to varying levels in 13 other human tumor cell lines, manifesting only a weak correlation with the reported metastatic ability of these tumor cell lines. The protein is not detected in normal human fibroblasts and endothelial cells. Northern blot analysis indicated that SIMA135/CDCP1 mRNA has a restricted expression pattern in normal human tissues with highest levels of expression in skeletal muscle and colon. Immunohistochemical analysis indicated apical and basal plasma membrane expression of SIMA135/CDCP1 in epithelial cells in normal colon. In colon tumor, SIMA135/CDCP1 expression appeared dysregulated showing extensive cell surface as well as cytoplasmic expression. Consistent with in vitro shedding experiments on HEp3 cells, SIMA135/CDCP1 was also detected within the lumen of normal and cancerous colon crypts, suggesting that protein shedding may occur in vivo. Thus, specific immunodetection followed by proteomic analysis allows for the identification and partial characterization of a heretofore uncharacterized human cell surface antigen.

Targeting the proteome/epitome, implementation of subtractive immunization

Monoclonal antibody technology has generated invaluable tools for both the analytical and clinical sciences. However, standard immunization approaches frequently fail to provide monoclonal antibodies with the desired specificity. Subtractive immunization provides a powerful alternative to standard immunization and allows for the production of truly unique antibodies. With the intent of targeting specific epitopes within the proteome, subtractive immunization has been broadly and successfully implemented for the production of monoclonal antibodies otherwise unobtainable by standard immunization. Subtractive immunization utilizes a distinct immune tolerization approach that can substantially enhance the generation of monoclonal antibodies to desired antigens. The approach is based on tolerizing the host animal to immunodominant or otherwise undesired antigen(s) (tolerogen) that may be structurally or functionally related to the antigen of interest. Tolerization of the host animal can be achieved through one of three methods: High Zone, Neonatal, or Drug-induced tolerization. The tolerized animal is then inoculated with the desired antigen (immunogen) and antibodies generated by the subsequent immune response are screened for the desired antigenic reactivity. Over the past 15 years a large number of investigators have used the subtractive approach with cleverly chosen tolerogen-immunogen combinations and successfully generated uniquely reactive antibodies which are often neutralizing or function-blocking. This review will focus on the implementation of subtractive immunization for the production of antibodies otherwise unobtainable by standard immunization.

In vivo proteomic imaging analysis of caveolae reveals pumping system to penetrate solid tumors

Technologies are needed to map and image biological barriers in vivo that limit solid tumor delivery and, ultimately, the effectiveness of imaging and therapeutic agents. Here we integrate proteomic and imaging analyses of caveolae at the blood-tumor interface to discover an active transendothelial portal to infiltrate tumors. A post-translationally modified form of annexin A1 (AnnA1) is selectively concentrated in human and rodent tumor caveolae. To follow trafficking, we generated a specific AnnA1 antibody that targets caveolae in the tumor endothelium. Intravital microscopy of caveolae-immunotargeted fluorophores even at low intravenous doses showed rapid and robust pumping across the endothelium to enter mammary, prostate and lung tumors. Within 1 h, the fluorescence signal concentrated throughout tumors to exceed the peak levels in blood. This transvascular pumping required the expression of caveolin 1 and annexin A1. Tumor uptake with other antibodies were >100-fold less. This proteomic imaging strategy reveals a unique target, antibody and caveolae pumping system for solid tumor penetration.

Immunotargeting and cloning of two CD34 variants exhibiting restricted expression in adult rat endothelia in vivo

Mapping protein expression of endothelial cells (EC) in vivo is fundamental to understanding cellular function and may yield new tissue-selective targets. We have developed a monoclonal antibody, MAb J120, to a protein expressed primarily in rat lung and heart endothelium. The antigen was identified as CD34, a marker of hematopoietic stem cells and global marker of endothelial cells in human and mouse tissues. PCR-based cloning identified two CD34 variant proteins, full length and truncated, both of which are expressed on luminal endothelial cell plasma membranes (P) isolated from lung. Truncated CD34 predominated in heart P, and neither variant was detected in P from kidney or liver. CD34 in lung was readily accessible to (125)I-J120 inoculated intravenously, and immunohistochemistry showed strong CD34 expression in lung EC. Few microvessels stained in heart and kidney, and no CD34 was detected in vessels of other organs or in lymphatics. We present herein the first complete sequence of a rat CD34 variant and show for the first time that the encoded truncated variant is endogenously expressed on EC in vivo. We also demonstrate that CD34 expression in rat EC, unlike mouse and human, is restricted in its distribution enabling quite specific lung targeting in vivo.

Ubiquitous yet distinct expression of podocalyxin on vascular surfaces in normal and tumor tissues in the rat

Background/Aims: The vasculature has become an important target in the development of therapies for increasing numbers of human diseases, yet there are few reliable markers available to identify the endothelium in rodent models. We have characterized the expression, subcellular localization and accessibility of the rat pan-endothelial marker podocalyxin (podxl) using a newly developed monoclonal antibody (mAb), G278. Methods: podxl expression and accessibility to binding by G278 were determined in the rat by a variety of experimental approaches. Results: mAb G278 reliably immunostained blood vessels of all types and of every size in fresh-frozen, fixed-frozen and paraffin-embedded sections of all tissues, but did not stain lymphatic vessels. Western blotting, in vivo imaging and biodistribution analyses demonstrated that the highest levels of endothelial podxl were found in the lung and heart. We also determined that podxl is not enriched in caveolae and that its expression can be modulated in the tumor microenvironment. Conclusion: Our study shows that podxl is a better identifier of rat endothelia than are some of the more commonly used markers and that mAb G278 is a robust antibody for use not only in identifying rat blood vessels but also as a tool to elucidate podxl function.

Avian urokinase-type plasminogen activator (u-PA) lacks the putative binding site for plasminogen activator inhibitor (PAI) and is resistant to inhibition by human PAI-1 and PAI-2

Summary In mammalian cultures, the activation and catalytic activity of both urokinase-type plasminogen activator (u-PA) and tissue-type PA (t-PA) is regulated in part by naturally occurring plasminogen activator inhibitors PAI-1 and PAI-2, members of the serpin family of serine protease inhibitors. Interaction of human PAIs with human u-PA (h-u-PA) and t-PA appears to be mediated by a putative PAI binding sequence in the enzymes. Comparison of the amino acid sequences of human, murine, porcine, bovine, simian and avian u-PA revealed specific sequences in the mammalian enzymes that are homologous to the recently discovered PAI-binding site in human u-PA and t-PA which is characterized by conserved basic amino acids. However, no homology was seen in the same region in chicken u-PA (c-u-PA), which is devoid of the consensus arginine and/or lysine residues. The chicken enzyme is a close structural and catalytic homolog of mammalian u-PA. The lack of the putative PAI binding sequence would predict that c-u-PA cannot interact with and be inhibited by human PAIs. To test this hypothesis, the effects of human PAI-1 and PAI-2 on avian u-PA were examined, using purified enzyme and inhibitors. Under conditions where h-u-PA activity is inhibited by human recombinant PAI-1 (rPAI-1), c-u-PA retains 95% activity. c-u-PA was also resistant to inhibition by human PAI-2. High molecular weight, SDS-stable complexes do not form readily when c-u-PA is incubated with PAI-1 or PAI-2, while human as well as murine u-PA formed complexes with both PAIs under the identical conditions. The c-u-PA molecule, thus, is a naturally occurring u-PA variant which lacks a PAI-binding region. The resistance of this enzyme to inhibition by human PAI-1 and PAI-2 supports the functional relevance of this sequence in mammalian u-PA and t-PA.

Co-inoculation of human and murine carcinoma cells induces reciprocal suppression of metastasis by both cell lines.

The interactions of two cell lines having different metastatic properties, and the subsequent effects on dissemination were investigated using the chicken embryo metastasis assay. The highly aggressive human epidermoid cell line HEp-3 was tested alone or mixed with the mouse colon carcinoma cell line CL26 in this assay. When inoculated individually, each cell line forms experimental metastases in the chicken embryo, but only the HEp-3 cells give rise to spontaneous metastases. In embryos co-inoculated with both cell lines there was an overall reduction in metastatic burden in both the spontaneous and experimental metastasis assays. Furthers studies revealed that CL26 cells, when co-inoculated with HEp-3 cells did not acquire the ability to spontaneously metastasize. However, in the presence of CL26 cells, spontaneous HEp-3 metastasis was reduced. Intravenous co-inoculation of HEp-3 and CL26 cells also resulted in a reciprocal suppression of experimental metastasis by both cell lines. These studies demonstrate that the interactions of adjacent, phenotypically different tumor cells can have a suppressive effect on dissemination of one or both cell types.