Phase III trial of 5-fluorouracil and leucovorin plus either 3H1 anti-idiotype monoclonal antibody or placebo in patients with advanced colorectal cancer.

BACKGROUND: The monoclonal antibody 3H1 mimics the external structure of the carcinoembryonic antigen (CEA). It therefore has the potential, via the anti-idiotypic network, to stimulate immune responses to CEA that may benefit colorectal cancer patients. PATIENTS AND METHODS: A total of 630 patients with previously untreated metastatic colorectal cancer were randomised in a 2:1 fashion to receive bolus 5-fluorouracil (5-FU) and leucovorin (LV) plus either 3H1 (n = 422) or placebo (n = 208). RESULTS: The addition of 3H1 to 5-FU and LV did not result in increased toxicity. Survival for the full intent-to-treat population was 14.7 months for the 3H1 arm and 15.2 months for the placebo arm (P = 0.80). Anti-CEA antibody responses were observed in 70% of patients treated with 3H1. Patients with a negative CEA response had a median survival of 8.3 months (95% CI 7.5-11.0) compared with patients with a strong response: median survival not reached (P <0.001). CONCLUSION: 3H1 is safe and effectively induces immune responses to CEA. Addition of 3H1 to 5-FU and LV was not shown to improve overall patient outcomes. However, improved survival in patients developing anti-CEA responses to 3H1 are provocative and should be studied in further clinical trials.

Engineering receptors activated solely by synthetic ligands (RASSLs).

The functional and molecular diversity of G-protein-coupled receptors presents a significant challenge to understanding the connection between a single receptor signaling pathway and a specific physiological or pathological response. To gain control over the timing and specificity of a G-protein signal, receptors activated solely by synthetic ligands (RASSLs) have been developed. These engineered receptors no longer respond to endogenous peptides, but can still be activated by a specific small-molecule drug. Further control over the location of the signal can be achieved by using RASSLs in conjunction with tissue-specific expression systems in vivo. Existing RASSLs have clarified the role of G(i) signaling in cardiac physiology and are currently being used to study cardiomyopathy, muscle remodeling, sensory transduction and complex neurobehavioral responses.

Abnormal contraction caused by expression of G(i)-coupled receptor in transgenic model of dilated cardiomyopathy.

Although increased G(i) signaling has been associated with dilated cardiomyopathy in humans, its role is not clear. Our goal was to determine the effects of chronically increased G(i) signaling on myocardial function. We studied transgenic mice that expressed a G(i)-coupled receptor (Ro1) that was targeted to the heart and regulated by a tetracycline-controlled expression system. Ro1 expression for 8 wk resulted in abnormal contractions of right ventricular muscle strips in vitro. Ro1 expression reduced myocardial force by >60% (from 35 +/- 3 to 13 +/- 2 mN/mm(2), P < 0.001). Nevertheless, sensitivity to extracellular Ca(2+) was enhanced. The extracellular [Ca(2+)] resulting in half-maximal force was lower with Ro1 expression compared with control (0.41 +/- 0.05 vs. 0.88 +/- 0.05 mM, P < 0.001). Ro1 expression slowed both contraction and relaxation kinetics, increasing the twitch time to peak (143 +/- 6 vs. 100 +/- 4 ms in control, P < 0.001) and the time to half relaxation (124 +/- 6 vs. 75 +/- 6 ms in control, P < 0.001). Increased pacing frequency increased contractile force threefold in control myocardium (P < 0.001) but caused no increase of force in Ro1-expressing myocardium. When stimulation was interrupted with rests, postrest force increased in control myocardium, but there was postrest decay of force in Ro1-expressing myocardium. These results suggest that defects in contractility mediated by G(i) signaling may contribute to the development of dilated cardiomyopathy.

Conditional expression of a Gi-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy.

Cardiomyopathy is a major cause of morbidity and mortality. Ventricular conduction delay, as shown by prolonged deflections in the electrocardiogram caused by delayed ventricular contraction (wide QRS complex), is a common feature of cardiomyopathy and is associated with a poor prognosis. Although the G(i)-signaling pathway is up-regulated in certain cardiomyopathies, previous studies suggested this up-regulation was compensatory rather than a potential cause of the disease. Using the tetracycline transactivator system and a modified G(i)-coupled receptor (Ro1), we provide evidence that increased G(i) signaling in mice can result in a lethal cardiomyopathy associated with a wide QRS complex arrhythmia. Induced expression of Ro1 in adult mice resulted in a >90% mortality rate at 16 wk, whereas suppression of Ro1 expression after 8 wk protected mice from further mortality and allowed partial improvement in systolic function. Results of DNA-array analysis of over 6,000 genes from hearts expressing Ro1 are consistent with hyperactive G(i) signaling. DNA-array analysis also identified known markers of cardiomyopathy and hundreds of previously unknown potential diagnostic markers and therapeutic targets for this syndrome. Our system allows cardiomyopathy to be induced and reversed in adult mice, providing an unprecedented opportunity to dissect the role of G(i) signaling in causing cardiac pathology.

Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice.

To control G protein signaling in vivo, we have modified G protein-coupled receptors to respond exclusively to synthetic small molecule agonists and not to their natural agonist(s). These engineered receptors are designated RASSLs (receptor activated solely by a synthetic ligand). A prototype RASSL (Ro1) based on the Gi-coupled K opioid receptor was expressed in transgenic mice under the control of the tetracycline transactivator (tet) system. Activation of Ro1 expressed in the heart decreased heart rate by up to 80%, an expected effect of increased Gi signaling. Maximal heart rate changes occurred in less than 1 min, demonstrating the speed of this inducible signaling system. This Ro1-mediated slowing of heart rate was also subject to desensitization, which lasted more than 24 h. Both the initial effect on heart rate and the desensitization occurred, even though Ro1 is derived from a human opioid receptor not normally involved in heart rate control. In addition, the tet system was used to induce Ro1 expression in hepatocytes and salivary gland, where Gi signaling is known to control physiologic events such as proliferation and secretion. These studies demonstrate that a RASSL can be inducibly expressed in several mouse tissues and used in vivo to activate G protein signaling in a controllable fashion.

In vivo-like drug responses of human tumors growing in three-dimensional gel-supported primary culture.

An in vitro test of cell sensitivity to drugs that indicates in vivo response is an important need in cancer therapy and cancer drug development. Toward this end, we previously developed a collagen gel-supported culture system for growth of human tumors. This three-dimensional culture system is general and grows tumors at high frequency directly from surgery or biopsy that maintain important in vivo properties in vitro, including tissue architecture. We report here that with autoradiographic techniques measuring cellular DNA synthesis the drug responses of individual cells within the tissue structure of in vitro-grown tumors can be determined. Twenty tumor classes, including all the major ones, have been measured in toto at greater than 50% frequency. Quantitative and qualitative results show increasing cell kill with rising cytotoxic drug concentration, differential drug sensitivities of multiple cell types within individual cultured tumors, differential sensitivities of a series of tumors of the same histopathological classification to a single drug, differential sensitivities of individual tumors to a series of drugs, and sensitivity patterns of various tumor types similar to the sensitivities found in vivo. Therefore, the results indicate that potentially important therapeutic data can be obtained from tumor specimens growing in vitro for the individual cancer patient as well as for rational and relevant screening for new agents active against human solid tumors.