Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies.

Background: Based on its selective cell surface expression in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), receptor tyrosine kinase ROR1 has recently emerged as a promising target for therapeutic monoclonal antibodies (mAbs). To further assess the suitability of ROR1 for targeted therapy of CLL and MCL, a panel of mAbs was generated and its therapeutic utility was investigated.

Methodology And Principal Findings: A chimeric rabbit/human Fab library was generated from immunized rabbits and selected by phage display.

E. coli expression and purification of Fab antibody fragments.

The Fab molecule was the first generated antibody fragment and still dominates basic research and clinical applications. This unit describes the E. coli expression and purification of Fab antibody fragments with and without a His tag, and is designed to yield sufficient protein for the evaluation and characterization of a panel of Fab selected from a Fab library by phage display.

Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity.

In humans, NKG2D is an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet.

Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia.

Purpose: Gene expression profiling identified receptor tyrosine kinase ROR1, an embryonic protein involved in organogenesis, as a signature gene in B-cell chronic lymphocytic leukemia (B-CLL). To assess the suitability of ROR1 as a cell surface antigen for targeted therapy of B-CLL, we carried out a comprehensive analysis of ROR1 protein expression.

Experimental Design: Peripheral blood mononuclear cells, sera, and other adult tissues from B-CLL patients and healthy donors were analyzed qualitatively and quantitatively for ROR1 protein expression by flow cytometry, cell surface biotinylation, Western blotting, and ELISA.

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1.

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, by downregulating the HER-2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and development into tumors of HER-2/neu-overexpressing ovarian cancer cells. Here, we establish stable clones of the human breast cancer cell line MDA-MB-361DYT2 that express PEA3 under the control of a tetracycline-inducible promoter.

Nuclear translocation of the pro-apoptotic Bcl-2 family member Bok induces apoptosis.

The anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Bcl-XL, play a central role in preventing the induction of apoptosis via the intrinsic apoptotic pathway. It has been previously shown that induction of apoptosis by the pro-apoptotic Bcl-2 family member Bok is not antagonized by either Bcl-2 or Bcl-xL, suggesting that Bok might have a unique role in the apoptotic cascade. We showed here that human Bok is the only member of the Bcl-2 family to have a leucine-rich sequence indicative of a nuclear export signal within its BH3 domain.

Synchronous global assessment of gene and protein expression in colorectal cancer progression.

Well-established models of colorectal cancer progression are based on the idea that the disease evolves through a multistep process involving sequential genetic mutations, suggesting that progression through clinically defined stages should correlate with well-defined patterns of gene expression. The majority of studies to date, however, have assessed these processes one gene and one protein at a time. We report the first comprehensive assessment of both gene and protein expression performed in parallel across progressive stages of human colorectal neoplasia.

Molecular staging for survival prediction of colorectal cancer patients.

Purpose: The Dukes' staging system is the gold standard for predicting colorectal cancer prognosis; however, accurate classification of intermediate-stage cases is problematic. We hypothesized that molecular fingerprints could provide more accurate staging and potentially assist in directing adjuvant therapy.

Methods: A 32,000 cDNA microarray was used to evaluate 78 human colon cancer specimens, and these results were correlated with survival.

The limits of log-ratios.

Background: DNA microarray assays typically compare two biological samples and present the results of those comparisons gene-by-gene as the logarithm base two of the ratio of the measured expression levels for the two samples.

Results: Because of the fixed dynamic range of fluorescence and other detection systems, there is a limit to the range of comparisons that can be made using any array technology, and this must be taken into account when interpreting the results of any such analysis.

Conclusions: The dynamic range of microarray data collection systems results in limits in the comparative analyses that can be derived from such measurements and suggests that optimal results can be obtained by making measurements that avoid the boundaries of that dynamic range.

Multi-platform, multi-site, microarray-based human tumor classification.

The introduction of gene expression profiling has resulted in the production of rich human data sets with potential for deciphering tumor diagnosis, prognosis, and therapy. Here we demonstrate how artificial neural networks (ANNs) can be applied to two completely different microarray platforms (cDNA and oligonucleotide), or a combination of both, to build tumor classifiers capable of deciphering the identity of most human cancers. First, 78 tumors representing eight different types of histologically similar adenocarcinoma, were evaluated with a 32k cDNA microarray and correctly classified by a cDNA-based ANN, using independent training and test sets, with a mean accuracy of 83%.

The suppression of colon cancer cell growth in nude mice by targeting beta-catenin/TCF pathway.

The adenomatous polyposis coli (APC) or beta-catenin genes are frequently mutated in colorectal cancers, leading to activation of downstream genes with beta-catenin/T-cell factor (Tcf)-responsive promoters. We have developed a gene therapy approach selectively targeting colorectal cancer cells in which beta-catenin/Tcf4 pathway is activated by using a recombinant adenovirus AdTOP-CMV-TK, which carries a herpes simplex virus thymidine kinase gene (HSV TK) under the control of a beta-catenin/Tcf-response promoter linking to a minimum CMV promoter. AdTOP-CMV-TK and ganciclovir (GCV) treatment significantly suppressed the growth of human DLD-1 colon cancer cells in nude mice.

Proapoptotic and antitumor activities of adenovirus-mediated p202 gene transfer.

Purpose And Experimental Design: p202, a mouse IFN-inducible protein, is a member of the 200-amino acid repeat family. Enforced p202 expression in stable cancer cell lines resulted in growth inhibition in vitro and tumor suppression in vivo. However, to study the immediate effect of p202 and test the potential efficacy of p202 treatment, an efficient gene delivery system for p202 is required.