Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia.

PURPOSE: Gemtuzumab ozogamicin (Mylotarg; Wyeth Laboratories, Philadelphia, PA) consists of a semisynthetic derivative of calicheamicin, a cytotoxic antibiotic linked to a recombinant monoclonal antibody directed against the CD33 antigen present on leukemic myeloblasts in most patients with acute myeloid leukemia (AML). In this study, we review the preclinical and clinical profiles of this immunoconjugate and the regulatory review that led to marketing approval by the United States Food and Drug Administration. EXPERIMENTAL DESIGN: From the literature and manufacturer's data, we review the activity, tolerability, and pharmacokinetics of gemtuzumab ozogamicin in preclinical and Phase I studies and its activity, efficacy, and side effects in three Phase 2 trials of 142 patients with relapsed AML. RESULTS: In Phase I studies, the major toxicity was myelosuppression, especially neutropenia and thrombocytopenia, resulting from the expression of CD33 on myeloid progenitor cells. The Phase 2 dose was 9 mg/m(2) infused i.v. over 4 h, repeated on day 14. A minority of patients experienced acute infusion-related symptoms, usually transient and occasionally requiring hospitalization. The complete response (CR) rate with full recovery of hematopoiesis was 16%. A subset of patients [CRs with incomplete platelet recovery (CRps)] was identified with blast clearance and neutrophil recovery but incomplete platelet recovery. The duration of responses of CRps appeared to be similar to those of the CRs, although the numbers were small. The question of the equivalence of these response groups was a central issue in the review of this new drug application (NDA). After considerable discussion, the Oncology Drugs Advisory Committee recommended allowing inclusion of CRps resulting in an overall response rate in the Phase 2 studies of 30%. In the subgroup of patients over 60 years of age, the overall response rate was 26%. Response duration was difficult to establish because of the high prevalence of postremission therapies. Tolerability and ease of administration may be improved compared with conventional chemotherapy, except for hepatotoxicity, with 31% of patients exhibiting abnormal liver enzymes. One patient died of liver failure in the Phase 2 trials. CONCLUSIONS: Marketing approval of gemtuzumab ozogamicin was granted on May 17, 2000 by the United States Food and Drug Administration under the Accelerated Approval regulations. Gemtuzumab ozogamicin is indicated for the treatment of patients with CD33 positive AML in first relapse who are 60 years of age or older and who are not considered candidates for cytotoxic chemotherapy. The approved dose was 9 mg/m(2) i.v. over 4 h and repeated in 14 days. Completion of the ongoing studies of gemtuzumab ozogamicin in relapsed AML and initiation of randomized clinical trials comparing the effects of gemtuzumab ozogamicin in combination with conventional induction chemotherapy to conventional chemotherapy alone on survival are mandated to confirm clinical benefit under the accelerated approval Subpart H regulations. Postmarketing reports of fatal anaphylaxis, adult respiratory distress syndrome (ARDS), and hepatotoxicity, especially venoocclusive disease (VOD) in patients treated with gemtuzumab ozogamicin, with and without associated hematopoietic stem cell transplantation (HSCT), have required labeling revisions and the initiation of a registration surveillance program. Tumor lysis and ARDS have been reported in patients with leukocytes above 30,000/ml treated with gemtuzumab ozogamicin; therefore, the reduction of leukocyte counts to below 30,000/ml is recommended prior to treatment. Patients should be carefully monitored for acute hypersensitivity, hypoxia, and delayed hepatotoxicity following treatment with gemtuzumab ozogamicin.

Human CD8 beta, but not mouse CD8 beta, can be expressed in the absence of CD8 alpha as a beta beta homodimer.

The T cell coreceptor CD8 exists on mature T cells as disulfide-linked homodimers of CD8 alpha polypeptide chains and heterodimers of CD8 alpha- and CD8 beta-chains. The function of the CD8 alpha-chain for binding to MHC class I and associating with the tyrosine kinase p56lck was demonstrated with CD8 alpha alpha homodimers. CD8 alpha beta functions as a better coreceptor, but the actual function of CD8 beta is less clear. Addressing this issue has been hampered by the apparent inability of CD8 beta to be expressed without CD8 alpha. This study demonstrates that human, but not mouse, CD8 beta can be expressed on the cell surface without CD8 alpha in both transfected COS-7 cells and murine lymphocytes. By creating chimeric proteins, we show that the murine Ig domain of CD8 beta is responsible for the lack of expression of murine CD8 beta beta dimers. In contrast to CD8 alpha alpha, CD8 beta beta is unable to bind MHC class I in a cell-cell adhesion assay. Detection of this form of CD8 should facilitate studies on the function of the CD8 beta-chain and indicates that caution should be used when interpreting studies on CD8 function using chimeric protein with the murine CD8 beta beta Ig domain. In addition, we demonstrate that the Ig domains of CD8 alpha are also involved in controlling the ability of CD8 to be expressed. Mutation of B- and F-strand cysteine residues in CD8 alpha reduced the ability of the protein to fold properly and, therefore, to be expressed.

A modified cell surface marker gene for transgenic animal studies.

We developed a marker gene encoding a human cell surface molecule called CD8 for use in transgenic animal studies. The CD8 cDNA contains three mutations: one in the extracellular domain which prevents interaction with its ligand MHC class I and the other two in the cytoplasmic domain which inhibit its signalling function. The cDNA was linked to a fragment of the human growth hormone gene and in transgenic animal studies, expression was observed in the appropriate cell types using a CD2 enhancer. The advantage of the CD8 marker gene is that it is incapable of signalling via its only known signalling pathway and its expression can be monitored using monoclonal antibodies and microscopy or flow cytometry.

Appropriate developmental expression of human CD8 beta in transgenic mice.

The human CD8 glycoprotein is expressed either as an alpha beta heterodimer or as an alpha alpha homodimer on thymocytes, mature T cells, and subpopulations of intestinal intraepithelial lymphocytes (IELs). The homodimeric form of CD8 is exclusively expressed on TCR gamma delta IELs and on subsets of NK cells and TCR alpha beta IELs. To understand the molecular mechanisms by which these genes are regulated, we created transgenic mice with a 95-kb human genomic DNA fragment containing the entire CD8 beta gene as well as a cluster of tissue-specific DNase I-hypersensitive sites 7 to 10 kb upstream of the gene. These sites were present in CD8 alpha beta+- but not CD8 alpha beta- T cell lines nor in a B cell line. We found that transgenic mice had correct developmental expression of human CD8 beta on thymocytes and mature CD8+ cells and no expression on mature CD4+ T cells or B cells. Interestingly, the percentage of mouse CD8 alpha+ cells that were human CD8 beta+ varied, depending on the founder line, from 4 to 88%, whereas the percentage among siblings was similar, indicative of a variegated phenotype resulting from site of integration effects. Expression was also observed on intestinal IELs, but only on those expressing the TCR alpha beta receptor and not the TCR gamma delta cells, which exclusively express CD8 alpha alpha. Of the TCR alpha beta+ cells, the transgene was expressed in both the CD8 alpha alpha and alpha beta subpopulations. These results indicate that this 95-kb fragment affords developmentally correct expression of the human CD8 beta gene on thymus-derived T cells in transgenic animals. Therefore, CD8 lineage-specific regulatory sequences must be located within the fragment.

Paclitaxel by 96-hour continuous infusion in combination with cisplatin: a phase I trial in patients with advanced lung cancer.

PURPOSE: To determine the maximum-tolerated dose (MTD) of paclitaxel administered by 96-hour continuous infusion in combination with cisplatin, to determine if the addition of granulocyte colony-stimulating factor (G-CSF) permits significant paclitaxel dose escalation, and to assess the toxicity and preliminary activity of this combination in patients with advanced lung cancer. PATIENTS AND METHODS: Fifty patients with untreated lung cancer were enrolled: 42 had advanced non-small-cell lung cancer (NSCLC) and eight had extensive-stage small-cell lung cancer (SCLC). Patients received paclitaxel doses of 100 to 180 mg/m2/96 hours and cisplatin doses of 60 to 80 mg/m2 as a single 30-minute bolus injection at the end of the paclitaxel infusion. RESULTS: Two of six patients experienced dose-limiting neutropenia at a dose of paclitaxel 140 mg/m2/96 hours and cisplatin 80 mg/m2. With G-CSF support, one of three patients experienced both dose-limiting mucositis and fatal neutropenic sepsis at a dose of paclitaxel 180 mg/m2/96 hours and cisplatin 80 mg/m2. Significant peripheral neuropathy developed in five patients and occurred after six or more cycles of therapy. Thirty-three of 42 patients with NSCLC had measurable disease; the objective response rate was 55%, with two complete responses and 16 partial responses. For all 42 patients with NSCLC, the median time to progression and median survival duration were 5 months and 10 months, respectively. The actuarial 1-year survival rate was 41%. Of eight SCLC patients, four responded to therapy, and the median survival duration for all SCLC patients was 11 months. CONCLUSION: The MTD without G-CSF is paclitaxel 120 mg/m2/96 hours and cisplatin 80 mg/m2, and the MTD with G-CSF is paclitaxel 160 mg/m2/96 hours and cisplatin 80 mg/m2. Infusional paclitaxel with cisplatin is well tolerated and active in patients with advanced NSCLC.

Human CD8 alpha expression in NK cells but not cytotoxic T cells of transgenic mice.

In our previous work, DNase hypersensitivity mapping was used to identify an enhancer within the human CD8 alpha (hCD8 alpha) gene which allowed T cell-specific expression of a reporter construct in transiently transfected cell lines. To study the role of this intronic enhancer in vivo, transgenic mice were made using human CD8 genomic constructs. We found that while a 14 kb wild-type human CD8 alpha (WThCD8) genomic construct did not lead to expression in mature peripheral CD8+ T cells, this transgene was consistently expressed in small populations of T cells and B cells, and in a subset of mouse NK cells. While murine CD8 is not normally expressed on resting NK cells, expression of the human CD8 transgene on mouse NK cells is appropriate since CD8 is expressed on a subset of human NK cells. Deletion of the intronic enhancer resulted in a complete loss of transgene expression in most lines and a loss of expression only in NK cells in one line. Our results indicate, firstly, that cis-acting sequences within the 14 kb genomic fragment are sufficient for NK cell-specific expression. In addition, our results suggest that the enhancer may have dual roles in regulation of transgene expression. It may enhance general expression of the transgene and may also be required for NK cell-specific expression.

Cyclophilin-40, a protein with homology to the P59 component of the steroid receptor complex. Cloning of the cDNA and further characterization.

We have reported previously the isolation and preliminary characterization of a 40-kDa cyclosporin A (CsA)-binding protein, cyclophilin-40 (CyP-40). To determine the sequence of this protein, degenerate oligonucleotide primers based on bovine brain CyP-40 tryptic peptides were used to generate a polymerase chain reaction fragment of CyP-40 cDNA. This was used to isolate the complete cDNA from a human pancreatic islet cell library. Northern analysis indicated ubiquitous distribution of CyP-40 mRNA throughout human tissues. The CyP-18 domain of CyP-40 is most similar to maize CyP (64.3% identity), whereas 150 amino acids of the non-CyP-18 domain of CyP-40 share 30.7% identity with P59, a member of the steroid receptor complex. Failure to detect glycosylation and mass spectroscopy with isolated CyP-40 indicate minimal, if any, posttranslational modification. Employing a new assay for calcineurin protein phosphatase activity to compare the effects of CyP-40.CsA and CyP-18.CsA complexes, IC50 values of 320 nM +/- 20 and 195 nM +/- 15, respectively, were obtained. A chemical cross-linking study revealed that CyP-40 competes for 125I-CyP-18 binding to calcineurin in the presence of CsA. The homology of CyP-40 to P59 suggests that CyP-40 might be involved in modulating the activity of biologically important receptors.

Isolation and partial characterization of membrane-associated cyclophilin and a related 22-kDa glycoprotein.

The presence of membrane-associated proteins which stereospecifically bind cyclosporin A and react with anti-cyclophilin antibodies has been documented in rat tissues. Extraction of membranes with 6 M urea or 0.5% Chaps releases cyclosporin-binding activity that is 5-12% of that found in cytosol. Cyclosporin-A-binding proteins are present in most subcellular organelles of liver, but microsomes contain the greatest activity. These proteins can be purified by adsorption onto a cyclosporin-A affinity column and elution with cyclosporin A. Two major fractions are resolved on SDS/PAGE: an 18-kDa fraction is comprised of two isoforms that are similar if not identical to the two major cytosolic isoforms of cyclophilin. In addition, in microsomes an approximately equal quantity of a 22-kDa glycoprotein was detected. Based on partial sequencing (five peptides, 89 amino acids) this protein is similar but not identical to human cyclophilin B. This 22-kDa isoform is poorly recognized by affinity-purified anti-cyclophilin antibodies and comprises several predominant isoforms (pI approximately 9.3-9.6). Selective binding of membrane 22-kDa cyclophilin to peanut lectin suggests the oligosaccharides contain a terminal galactosyl-N-galactosamine residue.

Isolation and characterization of a 40-kDa cyclophilin-related protein.

Major and minor isoforms of cyclophilin (CyP-18), a 17.8-kDa protein with peptidyl-prolyl cis-trans isomerase activity, comprise the primary intracellular binding proteins for cyclosporin A. Additional CyP-like proteins with approximate molecular masses of 22 (CyP-22) and 40 kDa (CyP-40) have been recovered from the soluble fraction of calf brain along with CyP-18 by adsorption onto a cyclosporin A affinity column and elution with cyclosporin A. Based on a limited number of peptide sequences from CyP-22, it appears that we have isolated from tissue CyPB, a protein whose sequence was deduced previously from cloned cDNA. The 40-kDa protein was separated from CyP-18 and CyP-22 on a molecular sieving column. Isoelectric focusing of CyP-40 yielded two bands at pI 5.3 and 5.5, in contrast to the basic pI values of CyP-18. Some tryptic peptides from CyP-40 were found to be highly homologous but not identical to bovine CyP-18; others were not significantly homologous to CyP-18 or any other protein in the data base. Unlike the major and minor isoforms of Cyp-18, monospecific polyclonal anti-CyP-18 antibodies did not cross-react with CyP-22 and CyP-40. Likewise, anti-CyP-40 serum minimally cross-reacts with CyP-18 and CyP-22. Cyp-40 possesses peptidyl-prolyl cis-trans isomerase activity which is less sensitive to inhibition by cyclosporin A (IC50 = 300 nM) than is CyP-18 (IC50 = 20 nM).

Estradiol decreases retention of rhodamine 123 fluorescence in GH4C1 pituitary tumor cells.

Rhodamine 123 is a lipophilic cationic fluorescent dye that localizes in mitochondria. We found that 17 beta-estradiol changes the ability of GH4C1 cells, clonal rat pituitary tumor cells, to retain rhodamine 123. Cells incubated with 10 micrograms/ml rhodamine 123 for 30 min at 37 C took up about equal amounts of rhodamine 123, as determined by fluorescence microscopy, regardless of whether they had been treated with estradiol. After three 5-min washes at 37 C, cells treated with 1 nM estradiol for 7 days before incubation with rhodamine 123 had lost more fluorescence than untreated cells. We further characterized the effect by flow cytometry. The difference in fluorescence between control and treated cells ranged from 50- to 500-fold. The effect of estradiol was maximal at 10(-10) M and took a week to develop fully. The effect is specific for estradiol, because estradiol and diethylstilbestrol reduced retention of rhodamine 123 fluorescence at 10(-10) M, but the same concentrations of dihydrotestosterone, progesterone, dexamethasone, and cholesterol did not. To test if the effect on rhodamine 123 fluorescence was caused by activation of the multidrug resistance transport system, we examined the effect of estradiol on the retention of daunomycin, a known substrate of the transport system. Estradiol treatment caused a 3-fold decrease in daunomycin fluorescence. We isolated clones resistant to estradiol-induced loss of rhodamine 123 fluorescence by flow cytometry and found that two clones still showed an estradiol-induced decrease in daunomycin fluorescence equivalent to that of the parent line.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational requirements of substrates for activity with phenylethanolamine N-methyltransferase.

beta-Phenylethanolamines have long been known to be substrates for the enzyme that converts norepinephrine to epinephrine (phenylethanolamine N-methyltransferase, PNMT, EC 2.1.1.28). In an effort to determine which, if any, particular conformation of the aminoethyl side chain of phenylethanolamines is required for PNMT active site binding and catalysis, we have prepared and evaluated conformationally restricted phenylethanolamine analogues 8-10. The folded phenylethanolamine derivative 4-hydroxy-1,2,3,4-tetrahydroisoquinoline (8) is not a substrate and does not interact with the enzyme active site as an inhibitor as well as 1,2,3,4-tetrahydroisoquinoline (6). In the cyclic 2-aminotetralol systems, only cis-phenylethanolamine derivative 9 demonstrates activity as a PNMT substrate. The corresponding trans isomer 10 is not a substrate, in spite of enhanced active site interactions with respect to the parent analogue (2-aminotetralin, 4). Comparison of the inhibition constants for the folded (8,Ki = 175 microM) and extended (10,Ki = 9 microM) phenylethanolamine analogues strongly suggests that simultaneous binding of both the amino and hydroxyl functionalities to the PNMT active site requires an extended aminoethyl side chain conformation.

Effects of concurrent negative feedback on performance of two motor tasks.

The effects of stress on state anxiety and on HR of male high school Ss were investigated using two psychomotor tasks. In the Stress Condition, Ss received negative feedback about performance; Ss in the Nonstress Condition were given rest intervals. Ss in the two conditions showed similar pretask, p> .05, A-State and HR measures. However, during the tasks the groups showed differing regression lines. The Nonstress Group maintained the same A-State level across the tasks with increased HR occurring as a result of the motor task. The Stress Group increased in A-State and HR measures. The two groups were significantly different, p<.01, in all within-task measures. The Stress Group performed better on the two motor skill tasks.