Molecular targets and cancer therapeutics: discovery, development and clinical validation

PERSPECTIVES Molecular targets and cancer therapeutics: discovery, development and clinical validation Beverly A.Teicher

Table 1 Kinase targets Malignant cells: Intracellular:

Plasma Membrane:

Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, USA

Abstract The AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics held in Washington, DC on 16–19 November 1999 provided a forum for cancer research clearly showing evolution of a target and mechanism-driven science.The notion of the tumor as a tissue composed of heterogeneous populations of normal and abnormal cells as viable targets is coming to the fore with the advent of agents directed toward non-malignant cell targets. Growth control rather than eradication as a treatment strategy for malignant disease is being tested preclinically and clinically.Among targets, kinases are in the lead with nuclear, cytoplasmic and membrane kinases being selectively inhibited by small molecules and macromolecules. First generation tumor vasculature-directed agents are progressing through early clinical studies.The interest in tumor vasculature as a target has renewed interest in imaging technology to discern biological effect and in tumor hypoxia.This has resulted in elucidation of molecular responses triggered by a low oxygen environment. Challenges remain in the areas of cellular and immune therapies. Dendritic cell-based vaccines are being tested preclinically in many systems. Interleukin-12 is proceeding through clinical trials.Apoptosis-protective molecules such as bcl-2, and apoptosis-stimulating molecules such as bax, are being pursued as targets for inhibition and activation, respectively. Finally, methods and technology to aid in the identification of new targets were highlighted.This perspective, while it is by no means an exhaustive review of the presentations, brings forward some of the main topics and interests that are current in cancer research.Targets were the topic but methods of target identification and the need for increased chemical diversity to selectively focus agents to targets with small differences were also major topics of discussion. 2000 Harcourt Publishers Ltd

KINASES, KINASES, KINASES rowth factor receptor kinases, intermediary signaling pathway kinases, nuclear cyclin-dependent kinases, and kinases approached directly or indirectly through chaperones formed a major cohort of molecular targets under investigation.1 Apparently, there was no doubt that selectivity for the desired kinase target could be achieved in vivo. Several molecules emerged as high interest target:agent pairs (Table 1). Epidermal growth factor receptor: ZD1839 (Iressa™), a quinazoline-derivative selectively inhibits the epidermal

G

Endothelial cells: Intracellular:

Plasma Membrane:

Flavopiridol UCN-01 NU2058, NU6027 3-ATA Benzothiopyrans, quinolinedisulfides Indirubin ZD 1839 PK1166 Lefluomide WHI-P97 C225 CEP-751, CEP-701 Endostatin TNP-470 Squalamine Combretastatin A4 SU5416, SU6668

growth factor receptor (EGFR) tyrosine kinase, is effective in a wide variety of human tumor xenografts administered orally at doses of 100–200 mg/kg alone and especially in combination with cytotoxic anticancer agents. ZD1839 has been assessed in phase I clinical trial. In one trial, ZD1839 was given twice on day 1 and once daily for 28 consecutive days. This trial is continuing enrollment at 300 and 400 mg dose levels (planned dose escalation 150 mg/day to 1000 mg/day); however, this trial confirms the acceptable tolerability of ZD1839 orally administered as a continuous once-daily schedule.Antitumor activity was seen in non-small cell lung cancer and head and neck cancers with ZD1839 monotherapy. In another Phase I study, ZD1839 was given orally (50–700 mg) once daily for 14 consecutive days followed by 14 days without treatment in repeating cycles. Responses with rapid symptom improvement and significant anti-tumor response occurred at doses of 300–525 mg. Mechanistically, the cellular growth inhibition induced by ZD1839 is associated with upregulation of p21 and p27 cyclin-dependent kinase inhibitors. ZD1839 is clearly and exciting new anticancer agent. Other small molecule EGFR inhibitors include PK1166, lefluomide (LFM-A12) and WHI-P97. The humanized monoclonal antibody, C225 directed toward the epidermal growth factor receptor continues to be studied both preclinically and clinically. C225 treatment increased the efficacy of paclitaxel in human transitional cell carcinoma 253JUB-V implanted orthotopically in nude mice. Inhibition of angiogenesis and induction of apoptosis mediated this effect. In orthotopically implanted human L3.6p1 pancreatic carcinoma, treatment with C225 increased the antitumor activity of gemcitabine. Herceptin and HER family receptor tyrosine kinase targets were another source of excitement. Geldanamycin is a natural product that binds to a specific pocket of the protein chaperone hsp90.2–4 Geldanamycin binding to hsp90 results in proteasomal degradation of several key-signaling proteins  2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 67–73 DOI: 10.1054/drup.2000.0123, available online at http://www.idealibrary.com on

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Teicher including Erb2/HER2. A geldanamycin derivative, 17-allylaminogeldanamycin, is currently undergoing Phase I clinical trial. In cell culture, human breast cancer cells that overexpress Erb2/HER2 are much more sensitive to 17-allylaminogeldanamycin than low espressors whether they are wild type or mutated in Rb. The neurotrophins and their high-affinity tyrosine kinase (trk) receptors have been linked to prostate cancer and other malignancy progression.1,5 CEP-751 and CEP-701, Trk inhibitors, impede the growth of human prostate carcinoma xenografts as well as human neuroblastoma and human medulloblastoma xenografts. The cyclin-dependent kinases remain a major focus as molecular targets with agents such as flavopiridol which is in Phase II clinical trial and UCN-01 which is in Phase I clinical trial leading the way. Flavopiridol is a non-specific inhibitor of cyclin-dependent kinases.6–8 In early clinical trials, flavopiridol showed anti-cancer activity in renal cancer, prostate cancer, colon cancer and non-Hodgkin’s lymphoma. In addition to inhibiting cyclin-dependent kinases, flavopiridol affects glycogen and glucose metabolism. Flavopiridol acted antagonistically with the differentiating agent PMA and potently induced apoptosis in combination with PMA in human U937 leukemia cells in culture. In cell culture, flavopiridol also enhanced the cytotoxicity of radiation therapy in human HCT-116 colon carcinoma cells by a mechanism involving the loss of p21 protein. In a Phase I clinical trial of 24 h (and later 3 h) paclitaxel on day 1, followed on day 2 both by escalating doses of 24 h flavopiridol and later by 20 min cisplatin, repeated every 21 days, clinical responses were observed in patients with taxane refractory esophagus, lung and prostate cancers. UCN-01, 7-hydroxy-staurosporine, is a non-specific protein kinase inhibitor currently in Phase I clinical trials. Preclinically, cell culture studies are examining the role of UCN-01 in combination regimens and combinations of UCN01 with mitomycin C or fludarabine appear promising.7,8 The cyclin-dependent kinases especially cdk1, cdk2 and cdk4, are the targets for several compounds. Under study are the cyclin B1/cdk1 and cyclin A/cdk2 inhibitors 6-o-cyclohexylmethyloxyguamine (NU2058) and 6-amino-4-cyclohexylmethyloxy-5-nitrosopyrimidine (NU6027). The diaminothiazoles appear especially promising inhibitors. Using combinatorial chemistry and structure-based drug design, diaminothiazoles equipotent for cdk4, cdk2 and cdk1 inhibition and diaminothiazoles with over 100-fold selectivity for cdk4 have been identified. Several of the compounds are active against human tumor xenografts. Benzothiopyrans and quinolinedisulfides have been identified in a highthroughput screen as cyclin/cdk inhibitors but these molecules lack molecular specificity.Through analysis of the p16 status of the NCI 60 cell line panel, one compound, 3-aminothioacridone (3-ATA; NSC 680434) was identified whose growth inhibition pattern in the cell lines correlated with p16 status.9,10 3-ATA was a non-competitive inhibitor of cyclin D1/cdk4. Cyclin D1 and cdk4 alterations were found in 13 and 3 of the 60 cell lines, respectively. The bisindole, indirubin, and its analogs are potent selective inhibitors of cyclin/cdks, most specifically cyclin B/cdk1. Mechanistically, cyclin/cdk inhibitors may also act by inducing the 68

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production of inhibitory proteins such as p21 in cells. The ubiquitin-proteasome pathway is responsible for proteolytic destruction of key inhibitors of cell cycle progression. The 26S-proteasome complex has emerged as a potential target for cancer therapies.11 PS-341, a boronate dipeptidyl inhibitor of the chymotryptic site of the 26 S proteasome, is being evaluated in clinical studies. Preclinical studies in nude mice bearing human pancreatic carcinoma xenograft tumors treated with PS-341 for up to 4 weeks resulted in decreased tumor cell proliferation as well as increased tumor cell apoptosis via blockade of NF-κB activation. In a Phase I clinical trial, PS-341 is being administered intravenously weekly for 4 weeks in a 6-week cycle at doses up to 0.75 mg/m2. Trichostatin A, an antifungal antibiotic, is a potent and specific inhibitor of histone deacetylase activity. Trichostatin A activates immediate c-jun expression via the p42/44 MAP kinase pathway and increased AP-1 activity, a process separate from histone acetylation. Trichostatin A had antitumor activity when administered subcutaneously to mice bearing human breast carcinoma xenografts at doses between 0.5 and 5 mg/kg for 4 weeks. TRIGGERING APOPTOSIS: BCL-2 AND BAX The bcl-2 family of proteins represents the interesting complexity of cellular biology.12–13 These proteins are expressed very heterogeneously in human tumors. Bcl-2 is known to be a negative regulator of apoptosis and thus to protect cells from cytotoxic agents; however, the exact biochemical functions of bcl-2 remain unclear. Bax is an apoptosis-promoting member of the bcl-2 family and may be a key factor influencing the chemosensitivity of tumor cells. On cell culture, transfecting the bax gene into head and neck squamous carcinoma cells increased their sensitivity to cisplatin. Delivery of the bax gene to tumors in mice using particlemediated (gene gun) delivery resulting in overexpression of bax improved the response of the tumors to cisplatin. On the other hand, transfecting bcl-2 into U937 cells, protected the cells from the cytotoxicity of flavopiridol but only if the cells expressed very high levels of bcl-2. Intracellularly, bcl-2 is located endoplasmic reticulum and nuclear membrane. In normal liver (rat), bcl-2 is located predominantly in the inner membrane and crista of the mitochondria. In a cell line selected for resistance to buthionine sulfoximine, bcl-2 was expressed to a much higher level than in the parent CC531 cell line.Transfection of CC531 cells with the gene for bcl-2 resulting in a 9-fold increase in expression also resulted in buthionine sulfoximine resistance. Bcl-2 overexpression prevented reactive oxygen species-related necrosis and decreased apoptosis induced by doxorubicin but did not alter survival as determined by colony formation. Overexpression of bcl-2 did not alter the response of bcl-2 transfected MX-1 xenograft tumor to taxanes, platinum complexes or anthracyclines. Bcl-2 phosphorylation, which has been associated with the apoptotic response to these agents, was a common event in response to these drugs. Neuroblastoma is resistant to even the most aggressive treatment strategies suggesting that there maybe a defect in the apoptotic mechanism in these cells. Neuroblastoma cells

Molecular targets and cancer therapeutics express high levels of the somatostatin receptors and internalize somatostatin. Fusion proteins produced with somatostatin and pro-apoptotic proteins, bax, bcl-xs or HRK, retained pro-apoptotic activity and may be useful therapeutics for neuroblastoma. Antimycin A, an inhibitor of mitochondrial electron transfer complex III, binds to recombinant bcl-2 protein and demonstrates competitive binding with a 16-amino acid BH3 peptide, a hydrophobic groove for bcl-xL. Non-peptidyl compounds related to antimycin A may be useful in cells overexpressing bcl-xL. In a clinical study of Merkel cell carcinoma, 85% of the patients’ tumors were positive for bcl-2 by immunohistochemical staining. BLOCKING BLOOD VESSEL GROWTH Among the widely varied targets, agents that constitute antiangiogenic and antimetastatic agents, SU5416 is progressing well through clinical trial. SU5416 is a small molecule inhibitor of Flk-1 kinase activity. Flk-1 is the murine form of the vascular endothelial growth factor (VEGF) receptor KDR. SU5416 must be administered intravenously. In one trial, patients with AIDS-associated Kaposi’s sarcoma were treated with SU5416 twice weekly at doses of 65–145 mg/m2 for up to four cycles (29 days per cycle).Among the first 18 patients, 11 had evidence of biological activity. SU5416 is also under study in metastatic colorectal cancer in combination with 5-fluorouracil/leucovorin administered at standard doses. SU5416 (85–145 mg/m2) twice weekly intravenously did not add significantly to the toxicity of the cytotoxic chemotherapy. SU5416 is currently undergoing Phase II/II clinical trial. While SU5416 is administered intravenously in the clinic, in mice bearing human tumor xenografts, it can be administered orally, subcutaneously and intraperitoneally as well as intravenously and be an effective tumor growth inhibitor.The new compound SU6668, directed towards the same target, is currently in Phase I clinical trial. Unlike SU5416, SU6668 is administered orally to patients. SU6668 is a competitive inhibitor at the ATP-binding site of Flk-1/KDR, PDGFR and FGFR. In mice, SU6668 is 100% orally bioavailable. In human tumor xenograft models, SU6668 caused regression of large established tumors. Endostatin, a 20-kDa C-terminal fragment of collagen XVIII, was discovered to be an antiangiogenic agent by Dr Michael O’Reilly working in the laboratory of Dr Judah Folkman.14–15 Human endostatin has been expressed in several recombinant systems. When prepared from E. coli, human endostatin was a potent inhibitor of endothelial cell proliferation and vascularization of the rabbit cornea with the potency varying with the angiogenic stimulus. Soluble, recombinant endostatin expressed in Pichia pastoris was assessed for safety following intravenous administration to Cynomolgus monkeys for 28 days at doses up to 300 mg/m2 per day. Serum trough level determinations of circulating endostatin protein revealed drug accumulation over the 28 days and some degree of antigenicity as determined by IgM and IgG antibody titers. Circulating levels of endostatin reached 2.1 µg/ml at 20 min post injection of the highest dose tested. A longer study of 90 consecutive days is underway. Spontaneous canine tumor patients were used to assess

the safety of endostatin gene therapy with the gene administered intravenously in cationic liposome-DNA complexes. The side-effects observed in 19 canine patients included moderate thrombocytopenia and fever that resolved in 48 h. There was one complete response (malignant melanoma) and one stabilization of disease in this group. TNP-470, a fumagillin derivative, is a potent inhibitor of endothelial cell proliferation and is currently in Phase II clinical trial. Preclinical studies were carried out with TNP-470 in nude mice bearing human neuroblastoma xenograft tumors.16,17 TNP-470 administered as a single agent or sequentially with cyclophosphamide showed that TNP-470 is a potent inhibitor of human neuroblastoma and may be useful as adjuvant therapy in high-risk neuroblastoma patients. TNP-470 was also proven to be safe in a preclinical model of stem cell transplantation and did not adversely impact stem cell engraftment. The aminosterol, squalamine, represents an interesting non-traditional antiangiogenic agent. The mechanism of action of squalamine appears to involve the redistribution of calmodulin and increases in calcium in endothelial cells. Squalamine was an antiangiogenic agent in nude mice bearing human 2008 ovarian carcinoma xenografts and was especially effective when administered in combination with cisplatin. Squalamine is currently in Phase II clinical trial along with paclitaxel and carboplatin in-patients with stage IIIb or IV non-small cell lung cancer. In the Phase I clinical trial, toxicities included nausea/vomiting, fatigue and weight loss. Squalamine is well tolerated at doses up to 255 mg/m2 and plasma levels predicted to be sufficient for antiangiogenic activity were reached. The tubulin-binding agent, combretastatin A4 phosphate, that binds to the colchicine-binding site of tubulin and inhibits microtubule assembly, has a tumor vascular targeting action and thus represents another type of non-traditional antiangiogenic agent. Exposure to combretastatin A4 induces a rapid change in endothelial cell shape and decrease in cell area.The time course of this effect on endothelial cells correlates with the increase in vascular resistance observed in an ex vivo model. In tumor-bearing rats and in patients dynamic MRI measurements demonstrated a reduction in tumor blood flow of about 70% at 6 h post combretastatin A4 administration that returned to near pre-treatment levels by 24 h. Combretastatin A4 phosphate is a pro-drug that is dephosphorylated to the active combretastatin. In a Phase I clinical trial combretastatin A4 phosphate was administered intravenously in a 10 min. infusion at 3 week intervals at doses up to 90 mg/m2. The dose-limiting toxicities were shortness of breath and a reversible episode of acute coronary ischemia. The extracellular matrix remains an interesting target for antiangiogenic strategies.Vitaxin is a humanized monoclonal antibody, which targets the αvβ3 integrin.Vitaxin was administered in a pilot clinical study at doses up to 200 mg. Serum levels fit a two-compartment model with a β half-life of about one week.The data from this pilot study suggest that there is an αvβ3 pool which causes rapid clearance of lower doses of Vitaxin that can be exceeded at higher doses (200 mg) of the humanized antibody and thus circulating levels in excess of 5 µg/ml can be maintained on an every 3 week schedule.  2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 67–73

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Fig. 1

Several matrix metalloproteinase inhibitors were updated at the meeting including: marimastat, Bay 12-9566,AG3340 and MM1270B (CGS 27023A). Marimastat is currently in Phase III clinical trials and is undergoing Phase II study in Japan. Bay 12-9566 has been withdrawn from clinical trial. This compound was unique amongst the matrix metalloproteinase inhibitors because it is non-peptidic and had much lower occurrence of the dose-limiting musculoskeletal effects seen with other matrix metalloproteinase inhibitors. Disease stabilization was observed in some patients treated with Bay 129566.AG 3340 (prinomastat), a potent inhibitor of MMP-2, -9, -13 and -14, delays the growth of many human tumor xenografts. Nude mice-bearing human colon carcinoma xenografts were treated orally for up to one year with continuing evidence of activity. MM1270B is a matrix metalloproteinase inhibitor with activity against several matrix metalloproteinases and preclinical activity in cells and human tumor xenografts. The effect of food intake was assessed in a study of 17 patients with oral doses of MM1270B up to 600 mg. Food intake did not result in a significant change in the AUC of MM1270B or in the peak plasma levels of the compound. AE-941, a standardized complex of shark cartilage-derived angiogenesis inhibitor, has completed Phase I and Phase II clinical trials. AE-941 given orally at a dose higher than 3 ml/kg/day significantly improved survival in-patients with

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unresectable stage IIIA, IIIB and IV non-small cell lung cancer. Phase III clinical studies of AE-941 will evaluate the agent given in combination with standard therapy. HYPOXIA RETURNS Through the study of tumor angiogenesis, there has been renewed interest in understanding the molecular biology of tumor hypoxia since hypoxia has been identified as a major trigger for secretion of angiogenic factors.18–20 In a workshop, the transcription factors, hypoxia inducible factor-1α (hif-1α) and hypoxia inducible factor-2α (hif-2α) and their significance in tumor growth were addressed. Under hypoxic conditions, hypoxia inducible factor-1α is stabilized and induces transcription of VEGF, enzymes in the glycolytic pathway and endothelin-1. Hypoxia inducible factor-2α has been found to be especially upregulated in renal cell cancer. In colon cancer cells exposure in culture to insulin-like growth factor-1 and insulin-like growth factor-2 lead to induction of VEGF production. In preclinical tumor models, administration of anti-VEGF therapies such as small molecule VEGFR inhibitors decreased tumor growth. The incorporation of antiangiogenic agents into therapeutic treatment schemas assigned to cure the major solid tumors remains an issue. In this workshop, data were presented that indicated that some of the antiangiogenic agents under clinical

Molecular targets and cancer therapeutics investigation may indeed result in increased malignant cell killing by causing tumor vasculature to become leaky and compromised compared with normal, well-formed vasculature.Thus, the strength of antiangiogenic therapies may be in their use in combination with traditional cytotoxic therapies. IMAGING Both preclinically and clinically, there was a major focus on the development of methods to detect a biological response to compounds directed toward targets that may not produce tumor growth delay or tumor shrinkage. Preclinically, the number of human and rodent cancer cells transfected with the gene for green fluorescent protein is increasing. If these tumor lines are stable and express high-levels of the green fluorescent protein then external high-resolution real-time optical imaging of internally growing tumors and metastases is possible.Tumors greater than 1 mm in diameter and up to 1.5 mm below the surface could be detected.The use of faxitron radiography with image analysis to identify bone lesions in nude mice bearing human breast and prostate tumors is increasing. By this technique, agents that may reduce the incidence and progression of bone metastases such as the bisphosphonate ester, SR-45023A can be studied. The role of magnetic resonance imaging (MRI) and spectroscopy (MRS) in imaging preclinical tumors (rats) and clinical disease was addressed in a plenary session.The goal is to establish the use of non-invasive imaging modalities for quantification of therapeutic outcome, gene delivery and monitoring of cellular events.The example used was treatment of intracranial rat 9L gliosarcoma with BCNU. Diffusion MRI that focuses on water translational mobility within tissue may be especially useful in quantifying the effects of therapy on the tumor. Magnetic resonance spectroscopy, on the other hand, detects agents that contain an appropriate atom such as fluorine and can detect conversion of 5-fluorocytosine to 5-fluorouracil in tumors transfected with the gene for enzyme cytosine deaminase. Antisense imaging using imagable radionuclides attached covalently to an antisense agent that localizes in tumor was discussed in the same plenary session. Finally, positron emission tomography (PET) was presented as a sensitive and specific technique for studying molecular interactions non-invasively in humans. PET requires molecules labeled with appropriate radionuclides such as [11C], [18F] or [15O]. Examples of pharmacokinetic applications of PET included 5-[18F]fluorouracil and [11C]temozolomide. Pharmacodynamic studies were described using [18F]fluorodeoxyglucose and 2-[11C]thymidine to detect changes in tumor metabolic status following treatment with chemotherapy. In addition, PET studies of tumor blood flow were described in connection with a Phase I trial of combretastatin 4A phosphate. CELLULAR THERAPIES AND THE IMMUNE SYSTEM The search continues for effective tumor vaccines.21–22 Methods to identify those antigens and peptides that can stimulate anti-tumor immune responses in vivo in patients now involve application of genomics technology. Many vaccine strategies are using dendritic cells. Dendritic cells are

professional antigen-presenting cells derived primarily from bone marrow. Traditionally, bone marrow-derived dendritic cells are differentiated in culture for 8–10 days using GMCSF/IL-4; however, combinations of GM-CSF/IL-4/IFNγ may be more effective. Dendritic cells may also be produced from peripheral blood adherent monocytes using the three cytokine-inducing regimen. Immunization of mice with dendritic cells generated in fetal bovine serum did not induce inhibition of tumor growth. Dendritic cells generated in normal mouse serum presented ‘self’ determinants. A vaccine based on the wild-type sequence mouse p53232–240 peptide was developed and used as a component of a peptide-pulsed dendritic cell vaccine in mice expressing H2-kd and -Db molecules to maximize vaccine efficacy. Carbohydrate structures in glycoproteins from carcinoma-associated glycoconjugates can be processed and presented by dendritic cells, which subsequently can induce naïve peripheral blood lymphocyte differentiation into functionally activated T lymphocytes. However, the physical state of the glycoconjugates may affect the efficacy of antigen presentation by the dendritic cells. Dendritic cells from ascites of ovarian cancer patients can be treated in vitro with cytokines or proteolytic enzymes to produce higher proportions of CD83+, CD40+ and CD80+ cells. The β-galactoside-specific recombinant mistletoe lectin is a type II ribosome-inactivating protein with cytotoxic and immunostimulating properties. Recombinant mistletoe lectin consists of a cytotoxic A chain linked by a disulfide bridge to a lectinic B chain. The A chain inactivates protein biosynthesis by enzymatic modification of 28 S ribosomyl RNA. Recombinant mistletoe lectin increases release of IFNγ and IL-1 from peripheral blood mononuclear cells as well as inducing other non-specific immunostimulatory events. Treatment of mice and rats with recombinant mistletoe lectin inhibited orthotopic bladder tumor formation and produced active antitumor activity against a variety of rodent tumors. As a class, interleukins administered exogenously tend to be better tolerated by rodents than by humans. Preclinical and clinical studies with two interleukins, IL-12 and IL-18, were highlighted at the meeting. Interleukin-12 was reported in studies administered to patients by intratumoral injection, subcutaneous injection and intraperitoneal injection.23 IL-12 acts through stimulation of cellular immunity by promoting T-helper 1 responses, induction of IFNγ production and activation and induction of proliferation of cytotoxic T-cells and natural killer cells. IL-12 (100 or 300 ng/kg) was administered once weekly for up to 24 weeks by intratumoral injection to patients with head and neck squamous carcinoma. The IL-12 treatments were well tolerated with some evidence of biological effect. The coagulative and fibrinolytic response after subcutaneous injection of IL-12 was studied in patients with advanced renal cell carcinoma at a dose of 500 ng/kg. Although routine coagulation tests were unaffected, IL-12 induction of a coagulation and fibrinolytic response could be demonstrated.A Phase I study of intraperitoneal IL12 was carried out in patients with peritoneal carcinomatosis from Mullerian and gastrointestinal malignancies at doses up to 300 ng/m2. Peritoneal exudates from patients demonstrated increased levels of the antiangiogenic α chemokine  2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 67–73

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Teicher IP10 and decreased levels of expression of the angiogenic factors VEGF and FGF2. In this study, there was no significant drug related toxicity, evidence of significant clinical benefit, enhancement of T-helper 1 type immune response and favorable modulation of angiogenesis related factors in the peritoneal tumor microenvironment. Interleukin-18 was identified as an IFNγ inducing factor that enhances natural killer cell activity. In tumor-bearing mice, IL-18 enhanced the antitumor activity of topotecan. GENE ARRAYS FOR IDENTIFYING TARGETS The dilemma of cancer is that malignant cells are very similar to normal cells.To this day, proliferation remains the target of cancer therapies. Several mRNA/cDNA differential gene expression systems were presented including the MICROMAX cDNA microarray system for high throughput and high sensitivity for differential gene expression; ValiGene’s Gene Identification (VGID) technology that isolates over-expressed and under-expressed transcripts from paired samples; GeneFilters GF200 micro-array which combines cDNA microarray analysis as a screening tool followed by competitive template reverse transcription polymerase chain reaction analysis for confirmation and quantification of differences; Clontech’s suppression subtractive hybridization followed by differential screening analysis of cDNA chips containing 20 000 cDNA clones and finally, mining the human EST database which currently has over 1.4 million entries. Each of these methods and others seek to identify targets that will cleanly and selectively allow malignant cells to be eradicated. CONCLUSIONS AND FUTURE The basic science and drive to effective therapies for cancer is a grand experiment on a global scale. The Molecular Targets meeting not only highlighted the global nature of cancer research but also the nearly seamless relationship between academia and industry in our field. Malignant cells are no longer envisioned as isolated entities. Malignant tumors are now being broadly approached as tissues composed of multiple relevant cell types, stromal components and host effectors. Molecular biology continues to increase our understanding of the interactive network of targets and effectors influencing tumor growth.The hope is that identification of therapeutic targets covering diverse aspects of malignant disease will result in less toxic, more effective therapies. There is no doubt that the general principles of cancer therapeutics established over the past 50 years remain valid. Combination treatment regimens covering multiple targets should result in improved treatment outcomes for cancer patients.

Received 7 January 2000; Revised 26 January 2000; Accepted 2 February 2000 Correspondence to: Beverly A.Teicher, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA,Tel: +1 317 276 2739; Fax: +1 317 277 6285; E-mail:TEICHER BEVERLY [email protected]

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