First-in-human phase 1 study of margetuximab (MGAH22), an Fc-modified chimeric monoclonal antibody, in patients with HER2-positive advanced solid tumors.

Background: Margetuximab is an anti-HER2 antibody that binds with elevated affinity to both the lower and higher affinity forms of CD16A, an Fc-receptor important for antibody dependent cell-mediated cytotoxicity (ADCC) against tumor cells. A Phase 1 study was initiated to evaluate the toxicity profile, maximum tolerated dose (MTD), pharmacokinetics, and antitumor activity of margetuximab in patients with HER2-overexpressing carcinomas. Patients and methods: Patients with HER2-positive breast or gastric cancer, or other carcinomas that overexpress HER2, for whom no standard therapy was available, were treated with margetuximab by intravenous infusion at doses of 0.1-6.0 mg/kg for 3 of every 4 weeks (Regimen A) or once every 3 weeks (10-18 mg/kg) (Regimen B). Results: Sixty-six patients received margetuximab (34 patients for Regimen A and 32 patients for Regimen B). The MTD was not reached for either regimen. Treatment was well-tolerated, with mostly Grade 1 and 2 toxicities consisting of constitutional symptoms such as pyrexia, nausea, anemia, diarrhea, and fatigue. Among 60 response-evaluable patients, confirmed partial responses and stable disease were observed in 7 (12%) and 30 (50%) patients, respectively; 26 (70%) of these patients had received prior HER2-targeted therapy. Tumor reductions were observed in over half (18/23, 78%) of response-evaluable patients with breast cancer including durable (>30 weeks) responders. Ex vivo analyses of patient peripheral blood mononuclear cell samples confirmed the ability of margetuximab to support enhanced ADCC compared with trastuzumab. Conclusions: Margetuximab was well-tolerated and has promising single-agent activity. Further development efforts of margetuximab as single agent and in combination with other therapeutic agents are ongoing. Trial Registration ID: NCT01148849.

Variability of total and pathogenic Vibrio parahaemolyticus densities in northern Gulf of Mexico water and oysters.

Vibrio parahaemolyticus is indigenous to coastal environments and a frequent cause of seafood-borne gastroenteritis in the United States, primarily due to raw-oyster consumption. Previous seasonal-cycle studies of V. parahaemolyticus have identified water temperature as the strongest environmental predictor. Salinity has also been identified, although it is evident that its effect on annual variation is not as pronounced. The effects of other environmental factors, both with respect to the seasonal cycle and intraseasonal variation, are uncertain. This study investigated intraseasonal variations of densities of total and pathogenic V. parahaemolyticus organisms in oysters and overlying waters during the summer of 2004 at two sites in the northern Gulf of Mexico. Regression analyses indicated significant associations (P < 0.001) between total V. parahaemolyticus densities and salinity, as well as turbidity in water and in oysters at the Mississippi site but not at the Alabama site. Pathogenic V. parahaemolyticus organisms in Mississippi oyster and water samples were detected in 56% (9 out of 16) and 78% (43 out of 55) of samples, respectively. In contrast, 44% (7 out of 16) of oyster samples and 30% (14 out of 47) of water samples from Alabama were positive. At both sites, there was greater sample-to-sample variability in pathogenic V. parahaemolyticus densities than in total V. parahaemolyticus densities. These data suggest that, although total V. parahaemolyticus densities may be very informative, there is greater uncertainty when total V. parahaemolyticus densities are used to predict the risk of infection by pathogenic V. parahaemolyticus than previously recognized.

Splicing and polyadenylylation at cryptic sites in RNA transcribed from pSV2-neo.

Mapping the structures of RNAs transcribed from the chimeric plasmid pSV2-neo in transfected COS cells revealed discontinuities within the neo portion of the transcripts. Two cryptic 5' splice sites and three cryptic 3' splice sites were identified. The cryptic 5' splice sites matched 5 or 7 bases of the 5' consensus sequence. Each cryptic 3' splice site consisted of an AG that was preceded by a 15 nucleotide region which was 73% pyrimidine. They differed from the 3' consensus sequence mainly by the presence of a purine, rather than a pyrimidine, immediately adjacent to the AG at the splice site. Greater than 99% of the transcripts were spliced at the SV40 intron. Approx. 50% of these transcripts were spliced, in a complex pattern, at the additional sites within the neo region. Deletion of the SV40 early polyadenylylation signal from pSV2-neo revealed the presence of a cryptic polyadenylylation signal in a region of pBR322 sequence. It is located in the 5' portion of the beta-lactamase gene and occurs 6 +/- 2 nucleotides downstream from the sequence AATAATAATGAA, which contains three overlapping variant hexanucleotides. The cryptic signal appears to be approx. 10-fold less efficient than the SV40 polyadenylylation signal.

Ligand-dependent regulation of plasmid-based transgene expression in vivo.

As gene therapy advances, the ability to regulate transgene expression will become paramount for safety and efficacy. In this study, we investigate the ability of the mifepristone-dependent GeneSwitch system to regulate the expression of trangenes delivered to mice by nonviral methods. Two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene for secreted human placental alkaline phosphatase (SEAP), were delivered to the hind-limb muscles of adult mice. Modulation of the level of secretion of the transgene product into serum was achieved by intraperitoneal administration of low doses of the drug mifepristone (MFP). The EC50 for induction of transgene expression by MFP was 0.03 +/- 0.005 mg/kg. The maximal level of transgene expression after induction was equal to or higher than that displayed by a plasmid driven by the CMV enhancer/promoter. The average magnitude of induction was 14- to 19-fold. Multiple rounds of drug-dependent regulation of transgene expression in vivo were demonstrated. In BALB/c mice, the ability to regulate transgene expression persisted for approximately 3 weeks, until the appearance of neutralizing antibodies to the secreted transgene product. In immune-deficient mice, the ability to repetitively regulate transgene expression persisted for at least 5 weeks. Although the dynamic range of regulation needs improvement, the plasmid-based GeneSwitch system has features that are attractive for gene therapy applications.

Combination of interleukin 12 and interferon alpha gene therapy induces a synergistic antitumor response against colon and renal cell carcinoma.

The antitumor effect and mechanism of action of IL-12 gene therapy combined with IFN-alpha gene therapy were investigated in tumor-bearing mice using renal and colon carcinoma models, Renca and CT26, respectively. Tumors were treated with murine IL-12 plasmid alone or in combination with IFN-alpha plasmid formulated with a polymeric interactive noncondensing (PINC) gene delivery system. Intratumoral injection of IL-12 DNA/polyvinyl pyrrolidone (PVP) alone induced rejection of 58 and 17% of Renca and CT26 tumors, respectively, whereas 25% (Renca) and 0% (CT26) rejection was observed in mice treated with IFN-alpha plasmid/PVP. Combination gene therapy of formulated plasmids, IL-12 with IFN-alpha, synergistically increased the antitumor response against Renca (100% tumor rejection) and CT26 (50%). In vivo depletion of leukocyte subsets indicated that CD8(+) T and NK cells were the primary effectors of the antitumor response induced by the combined cytokine gene therapy. Moreover, mice that rejected the primary tumors after combined treatment with IL-12 and IFN-alpha plasmid formulation developed protective immunity against a subsequent tumor challenge. Analysis of tumor-infiltrating leukocytes from mice treated with the combined IL-12 and IFN-alpha gene therapy showed upregulation of CD40 molecules on antigen-presenting cells (Mac-1(hi) cells). Finally, levels of mRNA for the chemokines IP-10 and TCA-3 were higher in tumors treated with the combination of cytokine plasmids than in tumors treated with either cytokine gene alone. These data provide evidence that IL12 gene therapy combined with IFN-alpha gene therapy synergistically induces regression of established tumors and may represent a novel therapeutic strategy for cancer treatment.

Intratracheal administration of interleukin 12 plasmid-cationic lipid complexes inhibits murine lung metastases.

Administration of plasmid/lipid complexes to the lung airways for the treatment of metastatic pulmonary diseases represents a new strategy of gene therapy. In this study we present evidence that intratracheal administration of a plasmid encoding murine IL-12 complexed with N-[1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride:cholesterol inhibits the growth of lung metastases, using a renal cell carcinoma model. Instillation of pIL-12/lipid complexes resulted in expression of biologically active IL-12 (170-240 pg/ml) and IFN-gamma (100-190 pg/ml) in the bronchoalveolar lavage fluid. A significantly reduced number of lung metastases (26+/-24) was observed in mice instilled with IL-12/lipid complexes 24 hr after tumor challenge, whereas more than 250 metastatic foci were counted in lungs of untreated mice. Moreover, IL-12/lipid inhibited the growth of 3-day-old established metastases when compared with empty plasmid/lipid or IL-12 plasmid in saline. Mice receiving IL-12 gene therapy survived significantly longer (median survival of 43 days) than untreated mice (median survival of 31 days) or mice treated with control plasmid/lipid complexes (median survival of 35 days). These data demonstrate that a nonviral IL-12 gene therapy employing synthetic cationic lipids as a delivery system can be used to inhibit the development of lung metastases. Thus, this method provides support for the use of IL-12/lipid complexes to control the growth of pulmonary metastases and represents a potentially safer alternative to IL-12 protein immunotherapy.

Advances in plasmid gene delivery and expression in skeletal muscle.

One of the most striking recent advances for plasmid delivery in vivo has been that of electropermeabilization, commonly referred to as electroporation. This physical process exposes a muscle tissue to a brief, high intensity electric field that induces temporary and reversible breakdown of the plasma membrane. During the period of membrane destabilization, a variety of molecules, including plasmids, gain intracellular access. Electroporation has been shown to improve the efficiency of plasmid gene delivery to skeletal muscle of small animals by as much as two-orders of magnitude to levels comparable to that of adenoviral gene delivery. This technology will allow the muscle to be used as a bioreactor for the secretion of therapeutic proteins into the circulation. This method of gene delivery, which is simple, efficient and reproducible, has become valuable for basic research, with great potential for gene therapy and DNA vaccination. Moreover, significant progress has been made using a variety of molecular designs to achieve regulation of gene expression by low molecular weight drugs. The enhanced efficiency of plasmid delivery by electroporation and the resultant durability of transgene expression, combined with the effectiveness of drug-dependent transgene regulation systems, provide a powerful set of tools that will be broadly applicable to the development of plasmid-based gene therapies for the treatment of human disease.

Effect of intertidal exposure on Vibrio parahaemolyticus levels in Pacific Northwest oysters.

Interest in Vibrio parahaemolyticus (Vp) increased in the United States following Vp-associated gastroenteritis outbreaks in 1997 and 1998 involving the West Coast and other areas. The present study evaluated multiple aspects of Vp ecology in the Pacific Northwest with three objectives: (i) to determine the effect of low-tide exposure on Vp levels in oysters, (ii) to determine the relationship between total and pathogenic Vp, and (iii) to examine sediments and aquatic fauna as reservoirs for pathogenic Vp. Samples were collected from intertidal reefs along Hood Canal, Wash., in August 2001. Fecal matter from marine mammals and aquatic birds as well as intestinal contents from bottom-dwelling fish were tested. Total and pathogenic Vp levels in all the samples were enumerated with colony hybridization procedures using DNA probes that targeted the thermolabile direct hemolysin (tlh) and thermostable direct hemolysin (tdh) genes, respectively. The mean Vp densities in oysters were four to eight times greater at maximum exposure than at the corresponding first exposure. While tdh-positive Vp counts were generally < or = 10 CFU/g at first exposure, counts as high as 160 CFU/g were found at maximum exposure. Vp concentrations in sediments were not significantly different from those in oysters at maximum exposure. Pathogenic (tdh positive) Vp was detected in 9 of 42 (21%) oyster samples at maximum exposure, in 5 of 19 (26%) sediment samples, but in 0 of 9 excreta samples. These results demonstrate that summer conditions permit the multiplication of Vp in oysters exposed by a receding tide.

The ovalbumin gene: transcriptional regulation by estrogen.

De novo synthesis of RNA sequences corresponding to intervening as well as to structural sequences of the ovalbumin gene have been detected in isolated oviduct nuclei. Their presence in the nuclear transcripts and their time course of induction support the hypothesis that transcription of structural and intervening sequences of the natural ovalbumin gene are regulated by steroid hormones. These results are in agreement with out previous demonstration of high-molecular-weight species of ovalbumin RNA in nuclei that contain structural as well as intervening RNA sequences and are thus likely precursors to mature cytoplasmic mRNAov. Analysis of the size of in vivo nuclear RNA by gel electrophoresis under denaturing conditions, revealed that withdrawal of hormone depletes the level of high molecular weight ovalbumin RNA as well as that of nature mRNAov and that readministration of estrogen induces the accumulation of both species. These results are consistent also with transcriptional regulation of the ovalbumin gene. In addition, they rule out the possibility that the rapid accumulation of mature mRNAov after secondard stimulation results from processing of ovalbumin RNA precursors that might have been stored in the withdrawn oviduct. We conclude that steroid hormones exert a primary effect at the level of gene transcription. Following this event, a series of coordinated cellular responses may occur which involve RNA processing, mRNA transport to the cytoplasm, protein synthesis and mRNA degradation. The final consequence of this network of molecular reactions is the induced cellular function inherent to a specific steroid hormone.

Bipartite structure of the downstream element of the mouse beta globin (major) poly(A) signal.

The downstream region of the mouse beta (major) globin poly(A) signal was mutated and analyzed for function in transfected COS cells. From analysis of unidirectional Bal31 deletions, the 3' boundary of the downstream element was defined as +22 (22 nucleotides downstream from the cleavage site). Analysis of cluster mutations, in which 5 or 6 adjacent bases were replaced with a random CA-containing sequence in a manner that did not alter spacing, confirmed +22 as the 3' boundary of the downstream element. The analysis also revealed two short UG-rich sequences, located from +5 to +10 and from +17 to +22, as major functional components. In contrast, a more refined series of mutations, in which clusters of 3 bases were replaced, failed to cause loss of function. We conclude that the downstream element of the mouse beta globin poly(A) signal is bipartite in structure, and that portions of its sequence are functionally redundant.

Different mechanisms are responsible for the low accumulation of transcripts from intronless and 3' splice site deleted genes.

A large reduction in the accumulation of mouse beta-globin mRNA occurs when both introns are deleted from its gene. This reduction is ameliorated if a sequence from HSV-1 thymidine kinase (TK), a naturally intronless gene, is inserted into the intronless globin construct. A large reduction in globin mRNA accumulation also occurs when just the terminal 3' splice site region is deleted. This reduction is not ameliorated by the insertion of TK sequence. Different mechanisms therefore must be responsible for the reduced accumulation of mRNA from the intronless and 3' splice site deleted genes. The ability of TK sequence to enhance mRNA accumulation was dependent on its position within the intronless construct. Data are consistent with a model in which pre-mRNAs are co-transcriptionally channelled into intron-dependent or intron-independent metabolic pathways.

Polyadenylylation of sea urchin histone RNA sequences in transfected COS cells.

The region of pSV2neo that encompasses the simian virus 40 early polyadenylylation signal was replaced with a DNA fragment that spans the 3' end of a sea urchin (Psammechinus miliaris) histone H2A gene. This clone, pMK2.H2A(3'), was used to transfect COS cells. RNA analysis revealed that transcripts from pMK2.H2A(3') were polyadenylylated at a site 85 nucleotides downstream from the expected 3' end of mature H2A mRNA. Nucleotide sequencing showed that the site of poly(A) addition was located 10 nucleotides downstream from a cluster of four A-A-U-A-A-A sequences. The lower accumulation of MK2.H2A(3') mRNA, which was 5-10% that of SV2neo mRNA, suggests that the H2A polyadenylylation signal is relatively inefficient. The relationship of the above findings to the 3' end processing of other histone mRNAs is discussed.

Identification of potential ovomucoid mRNA precursors in chick oviduct nuclei.

Denaturing gel electrophoresis of chick oviduct nuclear RNA reveals multiple species of RNA that are 1.5-5 times larger than ovomucoid mRNA. By analogy with ovalbumin RNA processing, these data suggest that ovomucoid mRNA is derived from a primary transcript that contains intervening sequences.

Short oligonucleotides as external guide sequences for site-specific cleavage of RNA molecules with human RNase P.

Human RNase P recognizes a small model substrate consisting of only the 5' leader sequence, aminoacyl acceptor stem, and T stem and loop of a tRNA precursor. It was demonstrated here that a bimolecular construct in which the T loop is opened between G57 and A58 (tRNA numbering system) is still processed by RNase P. The strand that is cleaved can be considered the target RNA, whereas the other strand serves as an external guide sequence (EGS). The nucleotides corresponding to nt 58-60 in the T loop could be deleted without affecting cleavage of the substrate. Thus, the complete T loop can be replaced by the single-stranded sequence UUCG or UUCA (nt 55-57 in the T loop). The four nucleotides UUCR possibly form a structure that resembles the uridine turn in the T loop of tRNA. Because recognition by RNase P is independent of the helical sequence, this motif can be used for targeting RNA molecules for EGS-directed cleavage by human RNase P. Chemically modified EGSs with 2'-O-methyl groups also showed activity in inducing RNase P cleavage. Several 13-mer EGSs targeted to the 2.1-kb surface antigen mRNA of hepatitis B virus (HBV) were designed and tested using a co-transcriptional cleavage assay with a 2.1-kb HBV transcript. Some of the new EGSs were capable of inducing cleavage of the HBV RNA by RNase P.

Requirement of A-A-U-A-A-A and adjacent downstream sequences for SV40 early polyadenylation.

RNA mapping experiments and chloramphenicol acetyltransferase assays were used to analyze polyadenylation in COS cells of transcripts from derivatives of pSV2-neo and pSV2-cat, in which the SV40 early poly(A) signal has been modified. Neither the sequence A-A-U-A-A-A nor the sequences located immediately downstream from it in the SV40 early gene appear to function by themselves as a poly(A) signal. When combined, however, these two elements form a poly(A) signal whose efficiency and specificity closely resemble those of the wild type signal. The addition of six nucleotides between the A-A-U-A-A-A sequence and the poly(A) site has no detectable effect on the efficiency or site of polyadenylation. Deletion of the 60 nucleotides immediately upstream from the hexanucleotide also has no detectable effect on polyadenylation. Therefore, A-A-U-A-A-A and sequences downstream from it appear to be sufficient for SV40 early polyadenylation.