Philip G. Kasprzyk

Insulin-like growth factor-I can mediate autocrine proliferation of human small cell lung cancer cell lines in vitro.

The effect of insulin-like growth factor I (IGF-I) on growth of small cell lung cancer (SCLC) cell lines was studied. Western blot analysis of whole cell lysates of cell lines NCI-H345 and NCI-N417 demonstrated the presence of a 16-kD band consistent with an IGF-I precursor molecule. Scatchard plot analysis of cell line NCI-H345 using 125I-labeled IGF-I demonstrated two high affinity specific binding sites (Kd 1.3 and 4.0 nM with maximal rate (Bmax) 200 and 500 fmol/mg protein, respectively). The exogenous addition of IGF-I, IGF-II, or insulin resulted in marked proliferation of human SCLC cells as evaluated using an in vitro growth assay. These peptides stimulated the growth of SCLC cell lines NCI-H82, NCI-H209, NCI-H345, and NCI-N417. The concentration of IGF-I producing maximal SCLC cell growth was 10-100-fold less than that of insulin or IGF-II, whereas the maximal growth stimulated by the optimal concentration of these peptides were similar. An MAb that specifically binds to the IGF-I receptor (but not to the insulin receptor) mediates a dose-dependent inhibition of cell growth in basal media as well as IGF-I, IGF-II, or insulin-supplemented media. The IGF-I receptor thus appears to be the common pathway for the mitogenic activity by IGF-I, IGF-II, and insulin for human SCLC cell lines. The demonstration of an IGF-I precursor molecule, specific IGF-I receptor binding, IGF-I-mediated growth stimulation, and inhibition of basal cell growth by an MAb to the IGF-I receptor suggests that an IGF-I-like molecule can function in vitro as an autocrine growth factor for human SCLC cell lines.

Anticancer activity of BIM-46174, a new inhibitor of the heterotrimeric Gα/Gβγ protein complex

A large number of hormones and local agonists activating guanine-binding protein-coupled receptors (GPCR) play a major role in cancer progression. Here, we characterize the new imidazo-pyrazine derivative BIM-46174, which acts as a selective inhibitor of heterotrimeric G-protein complex. BIM-46174 prevents the heterotrimeric G-protein signaling linked to several GPCRs mediating ( a ) cyclic AMP generation (Gαs), ( b ) calcium release (Gαq), and ( c ) cancer cell invasion by Wnt-2 frizzled receptors and high-affinity neurotensin receptors (Gαo/i and Gαq). BIM-46174 inhibits the growth of a large panel of human cancer cell lines, including anticancer drug-resistant cells. Exposure of cancer cells to BIM-46174 leads to caspase-3-dependent apoptosis and poly(ADP-ribose) polymerase cleavage. National Cancer Institute COMPARE analysis for BIM-46174 supports its novel pharmacologic profile compared with 12,000 anticancer agents. The growth rate of human tumor xenografts in athymic mice is significantly reduced after administration of BIM-46174 combined with either cisplatin, farnesyltransferase inhibitor, or topoisomerase inhibitors. Our data validate the feasibility of targeting heterotrimeric G-protein functions downstream the GPCRs to improve anticancer chemotherapy. (Cancer Res 2006; 66(18): 9227-34)

Clinical use of a monoclonal antibody to bombesin-like peptide in patients with lung cancer.

Small cell lung cancer (SCLC) is one of four major types of lung cancer and constitutes roughly 25% of all the new cases. Unfortunately, despite major improvements in overall patient survival as a result of combination chemotherapy, over 90% of all SCLC patients die of their tumor.’,’ More effective treatments are clearly needed. Drug development programs based on empiric screening of various compounds have been successful for treatment of certain tumors, but not of lung cancer, despite massive investments of time and money.’*‘ Many cancer investigators feel that therapeutic progress for lung cancer will be made by exploiting the increased understanding of tumor cell biology in order to use rationally based therapies. The observation that a monoclonal antibody which binds to gastrin-releasing

Homocamptothecins: E‐Ring Modified CPT Analogues

Abstract: Homocamptothecins (hCPT) are modified camptothecins (CPT) with a seven‐membered β‐hydroxylactone instead of the naturally occurring six‐membered α‐hydroxylactone. This E‐ring modification fully conserves the ability to stabilize topo I‐DNA single‐strand breaks and stimulates high levels of DNA cleavage. A key feature is the irreversibility of E‐ring opening, which should give reduced toxicity. Substituted hCPTs have been selected for their high antiproliferative activity on a panel of tumor cell lines, including those with cross resistance, and were found to be active at very low doses in a variety of human tumor xenografts when administered orally. BN 80915, a difluoro‐hCPT, has entered clinical trials.

Growth Factor Effects on Small Cell Lung Cancer Cells Using a Colorimetric Assay: Can a Transferrin-Like Factor Mediate Autocrine Growth?

A semiautomated colorimetric assay (MTT assay), based on the ability of live cells to reduce a tetrazolium-based compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), to a purplish colored formazan product that can be measured spectrophotometrically, has recently been adapted for use in drug sensitivity analysis of cultured human tumor cell lines. We report the application of this assay for the evaluation of the growth factor requirements of human small cell lung cancer (SCLC) cell lines. Specifically, the growth stimulation of each constituent of a previously reported serum-free defined medium system for SCLC including various concentrations of hydrocortisone, insulin, transferrin, 17 beta-estradiol, and selenium (HITES) was evaluated. The optimal concentrations for insulin, transferrin, and selenium derived in the previously reported experiments with direct counting of viable cells were similar to optimal concentrations determined for the growth of three SCLC cell lines (NCI-H82, NCI-N417, NCI-H526) using the MTT assay. In contrast to the previous report, the growth-stimulating effects of hydrocortisone and 17 beta-estradiol were negligible. Using the MTT we have shown that a SCLC cell line, NCI-H345 (which has been previously reported to produce a transferrin-like molecule), was growth-inhibited by an anti-transferrin receptor antibody, when grown in transferrin-free media. The conditioned media from this cell line is stimulatory to other transferrin-sensitive cell lines, suggesting the possibility of an autocrine role for this transferrin-like molecule at least in that cell line. With carefully defined conditions for a given cell line in which cell density and other parameters are within a range of constant MTT metabolism, the assay is well suited for precise analysis of growth factor effects.

The dual topoisomerase inhibitor, BN 80927, is highly potent against cell proliferation and tumor growth.

BN 80927 belongs to a novel family of camptothecin (CPT) analogues, the homocamptothecins (hCPTs),1 developed on the concept of topoisomerase (topo) I inhibition and characterized by the unique feature of a seven-membered βhydroxylactone ring. The lower reactivity of the lactone ring results in enhanced plasma stability compared to that of conventional camptothecin analogues such as topotecan and irinotecan. BN 80927, in addition to its topo I poisoning activity, has been shown to be a topoisomerase II catalytic inhibitor.2 We report here the results of studies evaluating the antiproliferative activity of BN 80927 in a panel of human tumor cell lines, including cells overexpressing P-glycoprotein (PgP). We also evaluated the cytotoxicity of BN 80927 on resting cells, because human tumors are a heterogeneous population of cells including cells that are not actively proliferating. Finally, we report the antitumor activity in xenograft models after oral administration of BN 80927.

Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation☆

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones.

Autocrine Growth Factors and Lung Cancer

Lung cancer is rapidly fatal in all but a small minority of patients regardless of therapy [1]. This situation reflects our inability to detect this disease prior to systemic dissemination, and the inadequacy of our systemic therapies for this disease [2]. The enormous investment of the National Cancer Institute and others in empiric new drug development has not had the same impact in lung cancer and other solid tumors as was seen in the hematologic malignancies. Both in response to the unfulfilling drug screening experience and to enhance understanding of tumor biology, many investigators are looking to more rationally developed anticancer therapies to lead to breakthroughs in the control of solid tumors. In lung cancer biology, there are many different directions that could be explored [3]. Recent developments in growth factor biology and the availability of specific antagonists that can be administered to patients, as is the case with monoclonal antibody [4], make this area especially promising. In this chapter we will review the development of growth factor research, attempting to summarize the current state of understanding of the role of growth factors especially as they relate to lung cancer, and discuss how that knowledge can be exploited for the successful therapy of lung cancer.

In vivo diagnosis and therapy of human tumors with monoclonal antibodies: Selection of antibodies and preliminary clinical studies in small cell carcinoma of the lung

Initial monoclonal antibody therapy trials include an attempt to control malignant proliferation of small cell lung cancer by blocking the autocrine stimulation of gastrin releasing peptide. A critical issue is the adequacy of antibody penetration into the tumor bed to effect immunologic blockade of the mitogenic peptide. The use of an indium-111 antibody chelate which is coadministered with the first therapeutic antibody administration facilitates analysis of the pharmacokinetic dynamics for this trial. If this approach is successful with gastrin releasing peptide, other peptide hormones with autocrine effects could also be targeted. A combination of anti growth factor therapies could lead to successful therapeutic control of this lethal disease.

Chapter 19 Some considerations for the therapeutic use of monoclonal antibodies in lung cancer

Abstract Ten years after the revolutionary work of Kohler and Milstein1 clinical trials in lung cancer employing monoclonal antibodies are just beginning2. Clinical investigators are familiar with the critical issues in the design of clinical trials of chemotherapy. The design of monoclonal antibody trials will borrow heavily from that experience and yet will break new ground. In this review we will consider the issues governing the development of clinical trials strategies.