Sacituzumab govitecan, a Trop-2-directed antibody-drug conjugate, for patients with epithelial cancer: final safety and efficacy results from the phase I/II IMMU-132-01 basket trial.

BACKGROUND: Sacituzumab govitecan (SG), a trophoblast cell surface antigen-2 (Trop-2)-directed antibody-drug conjugate, has demonstrated antitumor efficacy and acceptable tolerability in a phase I/II multicenter trial (NCT01631552) in patients with advanced epithelial cancers. This report summarizes the safety data from the overall safety population (OSP) and efficacy data, including additional disease cohorts not published previously. PATIENTS AND METHODS: Patients with refractory metastatic epithelial cancers received intravenous SG (8, 10, 12, or 18 mg/kg) on days 1 and 8 of 21-day cycles until disease progression or unacceptable toxicity. Endpoints for the OSP included safety and pharmacokinetic parameters with investigator-evaluated objective response rate (ORR per RECIST 1.1), duration of response, clinical benefit rate, progression-free survival, and overall survival evaluated for cohorts (n > 10 patients) of small-cell lung, colorectal, esophageal, endometrial, pancreatic ductal adenocarcinoma, and castrate-resistant prostate cancer. RESULTS: In the OSP (n = 495, median age 61 years, 68% female; UGT1A1∗28 homozygous, n = 46; 9.3%), 41 (8.3%) permanently discontinued treatment due to adverse events (AEs). Most common treatment-related AEs were nausea (62.6%), diarrhea (56.2%), fatigue (48.3%), alopecia (40.4%), and neutropenia (57.8%). Most common treatment-related serious AEs (n = 75; 15.2%) were febrile neutropenia (4.0%) and diarrhea (2.8%). Grade ≥3 neutropenia and febrile neutropenia occurred in 42.4% and 5.3% of patients, respectively. Neutropenia (all grades) was numerically more frequent in UGT1A1∗28 homozygotes (28/46; 60.9%) than heterozygotes (69/180; 38.3%) or UGT1A1∗1 wild type (59/177; 33.3%). There was one treatment-related death due to an AE of aspiration pneumonia. Partial responses were seen in endometrial cancer (4/18, 22.2% ORR) and small-cell lung cancer (11/62, 17.7% ORR), and one castrate-resistant prostate cancer patient had a complete response (n = 1/11; 9.1% ORR). CONCLUSIONS: SG demonstrated a toxicity profile consistent with previous published reports. Efficacy was seen in several cancer cohorts, which validates Trop-2 as a broad target in solid tumors.

Biomarker analyses in the phase III ASCENT study of sacituzumab govitecan versus chemotherapy in patients with metastatic triple-negative breast cancer.

BACKGROUND: The pivotal phase III ASCENT trial demonstrated improved survival outcomes associated with sacituzumab govitecan (SG), an anti-trophoblast cell-surface antigen 2 (anti-Trop-2) antibody-drug conjugate linked with the topoisomerase-inhibitor SN-38, over single-agent chemotherapy treatment of physician's choice (TPC) in previously treated metastatic triple-negative breast cancer (mTNBC). This prespecified, exploratory biomarker analysis from the ASCENT trial evaluates the association between tumor Trop-2 expression and germline BRCA1/2 mutation status with clinical outcomes. PATIENTS AND METHODS: Patients with mTNBC refractory to or progressing after two or more prior chemotherapies, with one or more in the metastatic setting, were randomized to receive SG (10 mg/kg intravenously days 1 and 8, every 21 days) or TPC (capecitabine, eribulin, vinorelbine, or gemcitabine) until disease progression/unacceptable toxicity. Biopsy or surgical specimens were collected at study entry to determine Trop-2 expression level using a validated immunohistochemistry assay and histochemical scoring. Germline BRCA1/2 mutation status was collected at baseline. RESULTS: Of 468 assessable patients, 290 had Trop-2 expression data [64% (n = 151 SG) versus 60% (n = 139 TPC)] and 292 had known BRCA1/2 mutation status [63% (n = 149 SG) versus 61% (n = 143 TPC)]. Median progression-free survival in SG- versus TPC-treated patients was 6.9, 5.6, and 2.7 months versus 2.5, 2.2, and 1.6 months for high, medium, and low Trop-2 expression, respectively. Median overall survival (14.2, 14.9, and 9.3 months versus 6.9, 6.9, and 7.6 months) and objective response rates (44%, 38%, and 22% versus 1%, 11%, and 6%) were numerically higher with SG versus TPC in patients with high, medium, and low Trop-2 expression, respectively. Efficacy outcomes were numerically higher with SG versus TPC in patients with and without germline BRCA1/2 mutations. CONCLUSIONS: SG benefits patients with previously treated mTNBC expressing high/medium Trop-2 compared with standard-of-care chemotherapy and regardless of germline BRCA1/2 mutation status. The small number of patients with low Trop-2 expression precludes definitive conclusions on the benefit of SG in this subgroup.

Promising clinical performance of pretargeted immuno-PET with anti-CEA bispecific antibody and gallium-68-labelled IMP-288 peptide for imaging colorectal cancer metastases: a pilot study.

INTRODUCTION: This pilot study evaluated the imaging performance of pretargeted immunological positron emission tomography (immuno-PET) using an anti-carcinoembryonic antigen (CEA) recombinant bispecific monoclonal antibody (BsMAb), TF2 and the [68Ga]Ga-labelled HSG peptide, IMP288, in patients with metastatic colorectal carcinoma (CRC). PATIENTS AND METHODS: Patients requiring diagnostic workup of CRC metastases or in case of elevated CEA for surveillance were prospectively studied. They had to present with elevated CEA serum titre or positive CEA tumour staining by immunohistochemistry of a previous biopsy or surgical specimen. All patients underwent endoscopic ultrasound (EUS), chest-abdominal-pelvic computed tomography (CT), abdominal magnetic resonance imaging (MRI) and positron emission tomography using [18F]fluorodeoxyglucose (FDG-PET). For immuno-PET, patients received intravenously 120 nmol of TF2 followed 30 h later by 150 MBq of [68Ga]Ga-labelled IMP288, both I.V. The gold standard was histology and imaging after 6-month follow-up. RESULTS: Eleven patients were included. No adverse effects were reported after BsMAb and peptide injections. In a per-patient analysis, immuno-PET was positive in 9/11 patients. On a per-lesion analysis, 12 of 14 lesions were positive with immuno-PET. Median SUVmax, MTV and TLG were 7.65 [3.98-13.94, SD 3.37], 8.63 cm3 [1.98-46.64; SD 14.83] and 37.90 cm3 [8.07-127.5; SD 43.47] respectively for immuno-PET lesions. Based on a per-lesion analysis, the sensitivity, specificity, positive-predictive value and negative-predictive value were, respectively, 82%, 25%, 82% and 25% for the combination of EUS/CT/MRI; 76%, 67%, 87% and 33% for FDG-PET; and 88%, 100%, 100% and 67% for immuno-PET. Immuno-PET had an impact on management in 2 patients. CONCLUSION: This pilot study showed that pretargeted immuno-PET using anti-CEA/anti-IMP288 BsMAb and a [68Ga]Ga-labelled hapten was safe and feasible, with promising diagnostic performance. TRIAL REGISTRATION: ClinicalTrials.gov NCT02587247 Registered 27 October 2015.

Sacituzumab govitecan in previously treated hormone receptor-positive/HER2-negative metastatic breast cancer: final results from a phase I/II, single-arm, basket trial.

BACKGROUND: Trophoblast cell-surface antigen-2 (Trop-2) is expressed in epithelial cancers, including hormone receptor-positive (HR+) metastatic breast cancer (mBC). Sacituzumab govitecan (SG; Trodelvy®) is an antibody-drug conjugate composed of a humanized anti-Trop-2 monoclonal antibody coupled to SN-38 at a high drug-to-antibody ratio via a unique hydrolyzable linker that delivers SN-38 intracellularly and in the tumor microenvironment. SG was granted accelerated FDA approval for metastatic triple-negative BC treatment in April 2020. PATIENTS AND METHODS: We analyzed a prespecified subpopulation of patients with HR+/human epidermal growth factor receptor 2-negative (HER2-) HR+/HER2- mBC from the phase I/II, single-arm trial (NCT01631552), who received intravenous SG (10 mg/kg) and whose disease progressed on endocrine-based therapy and at least one prior chemotherapy for mBC. End points included objective response rate (ORR; RECIST version 1.1) assessed locally, duration of response (DOR), clinical benefit rate, progression-free survival (PFS), overall survival (OS), and safety. RESULTS: Fifty-four women were enrolled between 13 February 2015 and 1 June 2017. Median (range) age was 54 (33-79) years and all received at least two prior lines of therapy for mBC. At data cut-off (1 March 2019), 12 patients were still alive. Key grade ≥3 treatment-related toxicities included neutropenia (50.0%), anemia (11.1%), and diarrhea (7.4%). Two patients discontinued treatment due to treatment-related adverse events. No treatment-related deaths occurred. At a median follow-up of 11.5 months, the ORR was 31.5% [95% confidence interval (CI), 19.5%-45.6%; 17 partial responses]; median DOR was 8.7 months (95% CI 3.7-12.7), median PFS was 5.5 months (95% CI 3.6-7.6), and median OS was 12 months (95% CI 9.0-18.2). CONCLUSIONS: SG shows encouraging activity in patients with pretreated HR+/HER2- mBC and a predictable, manageable safety profile. Further evaluation in a randomized phase III trial (TROPiCS-02) is ongoing (NCT03901339). TRIAL REGISTRATION: ClinicalTrials.gov NCT01631552; https://clinicaltrials.gov/ct2/show/NCT01631552.

Anti-CEA antibody fragments labeled with [(18)F]AlF for PET imaging of CEA-expressing tumors.

A facile and rapid method to label peptides with (18)F based on chelation of [(18)F]AlF has been developed recently. Since this method requires heating to 100 °C, it cannot be used to label heat-sensitive proteins. Here, we used a two-step procedure to prepare (18)F-labeled heat-labile proteins using the [(18)F]AlF method based on hot maleimide conjugation. 1,4,7-Triazacyclononae-1,4-diacetate (NODA) containing a methyl phenylacetic acid group (MPA) functionalized with N-(2-aminoethyl)maleimide (EM) was used as a ligand which was labeled with [(18)F]AlF and then conjugated to the humanized anti-CEA antibody derivatives hMN-14-Fab', hMN-14-(scFv)2 (diabody), and a Dock-and-Lock engineered dimeric fragment hMN-14 Fab-AD2 at room temperature. The in vivo tumor targeting characteristics of the (18)F-labeled antibody derivatives were determined by PET imaging of mice with s.c. xenografts. NODA-MPAEM was radiolabeled with [(18)F]AlF at a specific activity of 29-39 MBq/nmol and a labeling efficiency of 94 ± 2%. The labeling efficiencies of the maleimide conjugation ranged from 70% to 77%, resulting in [(18)F]AlF-labeled hMN14-Fab', hMN14-Fab-AD2, or hMN14-diabody with a specific activity of 15-17 MBq/nmol. The radiolabeled conjugates were purified by gel filtration. For biodistribution and microPET imaging, antibody fragments were injected intravenously into BALB/c nude mice with s.c. CEA-expressing LS174T xenografts (right flank) and CEA-negative SK-RC-52 xenografts (left flank). All [(18)F]AlF-labeled conjugates showed specific uptake in the LS174T xenografts with a maximal tumor uptake of 4.73% ID/g at 4 h after injection. Uptake in CEA-negative SK-RC-52 xenografts was significantly lower. Tumors were clearly visualized on microPET images. Using a [(18)F]AlF-labeled maleimide functionalized chelator, antibody fragments could be radiofluorinated within 4 h at high specific activity. Here, we translated this method to preclinical PET imaging studies and showed feasibility of [(18)F]AlF-fluorinated hMN-14-Fab', [(18)F]AlF-hMN-14-Fab-AD2, and [(18)F]AlF-hMN-14-diabody for microPET imaging of CEA-expressing colonic cancer.

Development of an imaging-guided CEA-pretargeted radionuclide treatment of advanced colorectal cancer: first clinical results.

BACKGROUND: Radiolabelled antibody targeting of cancer is limited by slow blood clearance. Pretargeting with a non-radiolabelled bispecific monoclonal antibody (bsMAb) followed by a rapidly clearing radiolabelled hapten peptide improves tumour localisation. The primary goals of this first pretargeting study in patients with the anti-CEACAM5 × anti-hapten (HSG) bsMAb, TF2, and the radiolabelled hapten-peptide, IMP288, were to assess optimal pretargeting conditions and safety in patients with metastatic colorectal cancer (CRC). METHODS: Different dose schedules were studied in four cohorts of five patients: (1) shortening the interval between the bsMAb and peptide administration (5 days vs 1 day), (2) escalating the TF2 dose (from 75 to 150 mg), and (3) reducing the peptide dose (from 100 to 25 µg). After confirmation of tumour targeting by (111)In-IMP288, patients were treated with a bsMAb/(177)Lu-IMP288 cycle. RESULTS: Rapid and selective tumour targeting of the radiolabelled peptide was visualised within 1 h, with high tumour-to-tissue ratios (>20 at 24 h). Improved tumour targeting was achieved with a 1-day interval between the administration of the bsMAb and the peptide and with the 25-µg peptide dose. High (177)Lu-IMP288 doses (2.5-7.4 GBq) were well tolerated, with some manageable TF2 infusion reactions, and transient grades 3-4 thrombocytopaenia in 10% of the patients who received (177)Lu-IMP288. CONCLUSION: This phase I study demonstrates for the first time that pretargeting with bsMAb TF2 and radiolabelled IMP288 in patients with CEA-expressing CRC is feasible and safe. With this pretargeting method, tumours are specifically and rapidly targeted.

Epratuzumab for systemic lupus erythematosus.

Epratuzumab (EMab, UCB, Immunomedics) is a humanized monoclonal antibody targeting CD22 that is being studied in clinical trials for patients with a variety of rheumatic and hematologic conditions, including systemic lupus erythematosus (SLE). An overview of its mechanism of action is followed by a summary of completed lupus studies, and a preview of studies in progress. The agent clearly has anti-inflammatory activity and is a potentially useful agent in the management of autoimmune disorders.

Ethanol and isopropanol inactivation of human coronavirus on hard surfaces.

BACKGROUND: The coronavirus disease 2019 pandemic has greatly increased the frequency of disinfecting surfaces in public places, causing a strain on the ability to obtain disinfectant solutions. An alternative is to use plain alcohols (EtOH and IPA) or sodium hypochlorite (SH). AIM: To determine the efficacy of various concentrations of EtOH, IPA and SH on a human coronavirus (HCoV) dried on to surfaces using short contact times. METHODS: High concentrations of infectious HCoV were dried on to porcelain and ceramic tiles, then treated with various concentrations of the alcohols for contact times of 15 s, 30 s and 1 min. Three concentrations of SH were also tested. Reductions in titres were measured using the tissue culture infectious dose 50 assay. FINDINGS: Concentrations of EtOH and IPA from 62% to 80% were very efficient at inactivating high concentrations of HCoV dried on to tile surfaces, even with a 15-s contact time. Concentrations of 95% dehydrated the virus, allowing infectious virus to survive. The dilutions of SH recommended by the Centers for Disease Control and Prevention (1/10 and 1/50) were efficient at inactivating high concentrations of HCoV dried on to tile surfaces, whereas a 1/100 dilution had substantially lower activity. CONCLUSIONS: Multiple concentrations of EtOH, IPA and SH efficiently inactivated infectious HCoV on hard surfaces, typical of those found in public places. Often no remaining infectious HCoV could be detected.

Placental growth factor (PlGF) enhances breast cancer cell motility by mobilising ERK1/2 phosphorylation and cytoskeletal rearrangement.

BACKGROUND: During metastasis, cancer cells migrate away from the primary tumour and invade the circulatory system and distal tissues. The stimulatory effect of growth factors has been implicated in the migration process. Placental growth factor (PlGF), expressed by 30-50% of primary breast cancers, stimulates measurable breast cancer cell motility in vitro within 3 h. This implies that PlGF activates intracellular signalling kinases and cytoskeletal remodelling necessary for cellular migration. The PlGF-mediated motility is prevented by an Flt-1-antagonising peptide, BP-1, and anti-PlGF antibody. The purpose of this study was to determine the intracellular effects of PlGF and the inhibiting peptide, BP-1. METHODS: Anti-PlGF receptor (anti-Flt-1) antibody and inhibitors of intracellular kinases were used for analysis of PlGF-delivered intracellular signals that result in motility. The effects of PlGF and BP-1 on kinase activation, intermediate filament (IF) protein stability, and the actin cytoskeleton were determined by immunohistochemistry, cellular migration assays, and immunoblots. RESULTS: Placental growth factor stimulated phosphorylation of extracellular-regulated kinase (ERK)1/2 (pERK) in breast cancer cell lines that also increased motility. In the presence of PlGF, BP-1 decreased cellular motility, reversed ERK1/2 phosphorylation, and decreased nuclear and peripheral pERK1/2. ERK1/2 kinases are associated with rearrangements of the actin and IF components of the cellular cytoskeleton. The PlGF caused rearrangements of the actin cytoskeleton, which were blocked by BP-1. The PlGF also stabilised cytokeratin 19 and vimentin expression in MDA-MB-231 human breast cancer cells in the absence of de novo transcription and translation. CONCLUSIONS: The PlGF activates ERK1/2 kinases, which are associated with cellular motility, in breast cancer cells. Several of these activating events are blocked by BP-1, which may explain its anti-tumour activity.

Malignant potential of murine stromal cells after transplantation of human tumors into nude mice.

Human malignant cancer tumors grafted into nude mice produce tumors containing both human cancer cells and the host's stromal cells. After short-term propagation of these tumors in vitro, the murine mesenchymal cells appear transformed and are tumorigenic in nude mice. However, established human cancer cell lines fail to similarly after adjacent murine stromal cells when used to produce tumors in nude mice. These experiments suggest that cancer cells may recruit normal cells to become malignant, qualifying the view of the clonal (unicellular) origin of cancer.

Carcinoembryonic antigen present in human colonic neoplasms serially propagated in hamsters.

Carcinoembryonic antigen, as measured by radioimmunoassay, is present in two different human colonic tumors that have been serially transplanted and maintained in the cheek pouches of unconditioned, adult golden hamsters. This finding shows that a human tumor-associated antigen can be produced in an animal host.

Electric enhancement of bone healing.

A human congenital pseudarthrosis of the tibia, unresponsive to conventional treatment, was stimulated to healing by direct electric current. The method was modeled after prior experimental work in vivo in rabbits. X-ray photographs, histological techniques, and electron microscopy confirmed the presence of newly formed bone in the defect region.

Clinical radioimmunodetection of cancer with radioactive antibodies to human chorionic gonadotropin.

Injection of iodine-131-labeled goat immunoglobulin G antibody to human chorionic gonadotropin (hCG) into patients with hCG-secreting trophoblastic and germinal tumors permitted tumor detection and location by external gamma-ray scintigraphy. Excision of one of the metastatic tumors located by this method indicated a tumor/nontumor ration of 39.29. The method appears to offer a new clinical tool for precisely locating hCG-producing tumors in the body, even when tumor identification by other clinical methods has failed.

Native and non-native intermediates in the BPTI folding pathway.

Recent studies of the disulfide-bonded intermediates in the refolding of bovine pancreatic trypsin inhibitor (BPTI) indicate that the most stable intermediates can take on much or all of the structure of the fully folded protein. Native-like structure in the intermediates probably causes steric inhibition of direct sequential formation of the three disulfides found in the native protein, thus accounting for the role of intramolecular rearrangements in the folding mechanism of this small protein.

Preclinical and clinical evaluation of epratuzumab (anti-CD22 IgG) in B-cell malignancies.

The vast majority of non-Hodgkin's lymphomas are of B-cell phenotype. Development of unlabeled or radiolabeled therapeutic monoclonal antibodies against the cell surface antigen, CD20, has revolutionized the treatment of these malignancies. It is clear that antibodies targeting other B-cell-specific molecules, such as CD22, also offer potential therapeutic benefit. Epratuzumab is a humanized anti-CD22 monoclonal, which has undergone preclinical and phase I/II clinical evaluation in patients with indolent or aggressive lymphoma. Data suggest that this agent is well tolerated, and can induce tumor regressions. Trials are currently evaluating its safety and activity in combination with rituximab (chimeric anti-CD20) and standard chemotherapy are ongoing. Initial results suggest that these regimens have acceptable toxicity, and that epratuzumab warrants further evaluation as an adjunct to standard lymphoma treatment regimens.

Novel radiolabeled antibody conjugates.

This article reviews the development of radioimmunoconjugates as a new class of cancer therapeutics. Numerous conjugates involving different antigen targets, antibody forms, radionuclides and methods of radiochemistry have been studied in the half-century since radioactive antibodies were first used in model systems to selectively target radiation to tumors. Whereas directly conjugated antibodies, fragments and subfragments have shown promise preclinically, the same approaches have not gained success in patients except in radiosensitive hematological neoplasms, or in settings involving minimal or locoregional disease. The separation of tumor targeting from the delivery of the therapeutic radionuclide in a multistep process called pretargeting has the potential to overcome many of the limitations of conventional, or one-step, radioimmunotherapy, with initial preclinical and clinical data showing increased sensitivity, specificity and higher radiation doses delivered. Our particular focus in pretargeting is the use of bispecific, trimeric (three Fab's) constructs made by a new antibody engineering method termed 'dock-and-lock.

Differential effects of epratuzumab on peripheral blood B cells of patients with systemic lupus erythematosus versus normal controls.

OBJECTIVE: B lymphocytes have been implicated in the pathogenesis of lupus and other autoimmune diseases, resulting in the introduction of B cell-directed therapies. Epratuzumab, a humanised anti-CD22 monoclonal antibody, is currently in clinical trials, although its effects on patients' B cells are not completely understood. METHODS: This study analysed the in vivo effect of epratuzumab on peripheral B cell subsets in 12 patients with systemic lupus erythematosus, and also addressed the in vitro effects of the drug by analysing anti-immunoglobulin-induced proliferation of isolated B cells obtained from the peripheral blood of 11 additional patients with lupus and seven normal subjects. RESULTS: Upon treatment, a pronounced reduction of CD27(-) B cells and CD22 surface expression on CD27(-) B cells was observed, suggesting that these cells, which mainly comprise naïve and transitional B cells, are preferentially targeted by epratuzumab in vivo. The results of in vitro studies indicate additional regulatory effects of the drug by reducing the enhanced activation and proliferation of anti-immunoglobulin-stimulated lupus B cells after co-incubation with CD40L or CpG. Epratuzumab inhibited the proliferation of B cells from patients with systemic lupus erythematosus but not normal B cells under all culture conditions. CONCLUSIONS: Epratuzumab preferentially modulates the exaggerated activation and proliferation of B cells from patients with lupus in contrast to normal subjects, thus suggesting that epratuzumab might offer a new therapeutic option for patients with systemic lupus erythematosus, as enhanced B cell activation is a hallmark of this disease.

High and long-term von Willebrand factor expression after Sleeping Beauty transposon-mediated gene therapy in a mouse model of severe von Willebrand disease.

Essentials von Willebrand disease (VWD) is the most common inherited bleeding disorder. Gene therapy for VWD offers long-term therapy for VWD patients. Transposons efficiently integrate the large von Willebrand factor (VWF) cDNA in mice. Liver-directed transposons support sustained VWF expression with suboptimal multimerization. SUMMARY: Background Type 3 von Willebrand disease (VWD) is characterized by complete absence of von Willebrand factor (VWF). Current therapy is limited to treatment with exogenous VWF/FVIII products, which only provide a short-term solution. Gene therapy offers the potential for a long-term treatment for VWD. Objectives To develop an integrative Sleeping Beauty (SB) transposon-mediated VWF gene transfer approach in a preclinical mouse model of severe VWD. Methods We established a robust platform for sustained transgene murine VWF (mVWF) expression in the liver of Vwf-/- mice by combining a liver-specific promoter with a sandwich transposon design and the SB100X transposase via hydrodynamic gene delivery. Results The sandwich SB transposon was suitable to deliver the full-length mVWF cDNA (8.4 kb) and supported supra-physiological expression that remained stable for up to 1.5 years after gene transfer. The sandwich vector stayed episomal (~60 weeks) or integrated in the host genome, respectively, in the absence or presence of the transposase. Transgene integration was confirmed using carbon tetrachloride-induced liver regeneration. Analysis of integration sites by high-throughput analysis revealed random integration of the sandwich vector. Although the SB vector supported long-term expression of supra-physiological VWF levels, the bleeding phenotype was not corrected in all mice. Long-term expression of VWF by hepatocytes resulted in relatively reduced amounts of high-molecular-weight multimers, potentially limiting its hemostatic efficacy. Conclusions Although this integrative platform for VWF gene transfer is an important milestone of VWD gene therapy, cell type-specific targeting is yet to be achieved.

Phage tailspike protein. A fishy tale of protein folding.

The crystal structure of bacteriophage P22 tailspike protein reveals a striking fold with a distinctive, fish-like appearance, and helps explain many of the properties of this unusual molecule and its folding pathway.

Transcription-coupled translation control of AML1/RUNX1 is mediated by cap- and internal ribosome entry site-dependent mechanisms.

AML1/RUNX1 belongs to the runt domain transcription factors that are important regulators of hematopoiesis and osteogenesis. Expression of AML1 is regulated at the level of transcription by two promoters, distal (D) and proximal (P), that give rise to mRNAs bearing two distinct 5' untranslated regions (5'UTRs) (D-UTR and P-UTR). Here we show that these 5'UTRs act as translation regulators in vivo. AML1 mRNAs bearing the uncommonly long (1,631-bp) P-UTR are poorly translated, whereas those with the shorter (452-bp) D-UTR are readily translated. The low translational efficiency of the P-UTR is attributed to its length and the cis-acting elements along it. Transfections and in vitro assays with bicistronic constructs demonstrate that the D-UTR mediates cap-dependent translation whereas the P-UTR mediates cap-independent translation and contains a functional internal ribosome entry site (IRES). The IRES-containing bicistronic constructs are more active in hematopoietic cell lines that normally express the P-UTR-containing mRNAs. Furthermore, we show that the IRES-dependent translation increases during megakaryocytic differentiation but not during erythroid differentiation, of K562 cells. These results strongly suggest that the function of the P-UTR IRES-dependent translation in vivo is to tightly regulate the translation of AML1 mRNAs. The data show that AML1 expression is regulated through usage of alternative promoters coupled with IRES-mediated translation control. This IRES-mediated translation regulation adds an important new dimension to the fine-tuned control of AML1 expression.