Purpose: Transforming growth factor β (TGFβ) is a pleiotropic cytokine that affects tumor growth, metastasis, stroma, and immune response. We investigated the therapeutic efficacy of anti-TGFβ receptor II (TGFβ RII) antibody in controlling metastasis and tumor growth as well as enhancing antitumor immunity in preclinical tumor models.Experimental Design: We generated neutralizing antibodies to TGFβ RII and assessed the antibody effects on cancer, stroma, and immune cells in vitro. The efficacy and mechanism of action of the antibody as monotherapy and in combination with chemotherapy in suppression of primary tumor growth and metastasis were evaluated in several tumor models.Results: Anti-TGFβ RII antibody blocked TGFβ RII binding to TGFβ 1, 2, and 3, and attenuated the TGFβ-mediated activation of downstream Smad2 kinase, invasion of cancer cells, motility of endothelial and fibroblast cells, and induction of immunosuppressive cells. Treatment with the antibody significantly suppressed primary tumor growth and metastasis and enhanced natural killer and CTL activity in tumorbearing mice. Immunohistochemistry analysis showed cancer cell apoptosis and massive necrosis, and increased tumor-infiltrating T effector cells and decreased tumor-infiltrating Gr-1+ myeloid cells in the antibody-treated tumors. Fluorescence-activated cell sorting analysis indicated the significant reduction of peripheral Gr-1+/CD11b+ myeloid cells in treated animals. Concomitant treatment with the cytotoxic agent cyclophosphamide resulted in a significantly increased antitumor efficacy against primary tumor growth and metastasis.Conclusions: These preclinical data provide a foundation to support using anti-TGFβ RII antibody as a therapeutic agent for TGFβ RII-dependent cancer with metastatic capacity.

Recent advances in mammalian cell culture processes have significantly increased product titers, but have also resulted in substantial increases in cell density and cellular debris as well as process and product related impurities. As such, with improvements in titer, corresponding improvements in downstream processing are essential. In this study we have developed an alternative antibody harvest process that incorporates flocculation using a novel stimulus responsive polymer, benzylated poly(allylamine), followed by depth filtration. As tested on multiple antibodies, this process demonstrates high process yield, improved clearance of cells and cell debris, and efficient reduction of aggregates, host cell proteins (HCP) and DNA. A wide operating window was established for this novel flocculation process through design of experiments condition screening and optimization. Residual levels of impurities in the Protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. In addition, efficient clearance of residual polymer was demonstrated using a fluorescence tagged polymer in the presence of a stimulus reagent. The mechanism of HCP and aggregates removal during flocculation was also explored. This novel and efficient process can be easily integrated into current mAb purification platforms, and may overcome downstream processing challenges. Biotechnol. Bioeng. 2013;110: 2928–2937. © 2013 Wiley Periodicals, Inc.

Differentially processed human cytomegalovirus (HCMV) UL37 RNAs encode biologically significant proteins. Due to the recent discovery of alternative UL37 exon 3 (UL37x3) splice donors, permissively infected cells were thoroughly examined for additional alternatively spliced UL37 RNAs. Newly described donors within UL37 exon 1 (nt 52520) and intron 1 (nt 52209) as well as UL37x3 di (nt 50770) and dii (nt 50782) were differentially spliced to known downstream UL37 acceptors. The alternatively spliced UL37 S , UL37 L , UL37 di and UL37d ii RNAs predictably encode proteins of 83, 163, 217 and 213 residues, respectively, which share UL37x1 N-terminal sequences but differ downstream in their C termini. Moreover, temporal expression of the alternatively spliced UL37 RNAs differs during HCMV infection. The complexity of UL37 pre-mRNA processing is evidenced by the detection of 11 UL37 spliced and unspliced UL37x1 RNAs in HCMV-infected cells. Based upon these data, a revised HCMV UL37 gene map is presented, which incorporates all RNA species detected during permissive infection.The human cytomegalovirus (HCMV) UL37 immediateearly (IE) locus encodes proteins that play important roles for anti-apoptosis, viral DNA replication and growth (Colberg-Poley, 1996;Colberg-Poley et al., 1992, 1998Goldmacher et al., 1999; Hayajneh et al., 2001a, b;Smith & Pari, 1995). The UL37 exon 1 (UL37x1) protein (pUL37x1 or vMIA) and UL37 glycoproteins (gpUL37 and gpUL37 M ) contain two domains, at aa 5-34 and 118-147, which are required and sufficient for anti-apoptotic activities (Goldmacher, 2002;Goldmacher et al., 1999; Hayajneh et al., 2001a). HCMV UL37 proteins unconventionally traffic into the endoplasmic reticulum and mitochondria, where they inhibit apoptosis by blocking cytochrome C release (AlBarazi & Colberg-Poley, 1996;Colberg-Poley et al., 2000;Goldmacher, 2002;Goldmacher et al., 1999; Hayajneh et al., 2001a;McCormick et al., 2003).The UL37 gene is known to encode three alternatively processed transcripts (Chee et al., 1990;Goldmacher et al., 1999;Kouzarides et al., 1988; Tenney & Colberg-Poley, 1991a, b). Because of our recent discovery of unreported UL37 exon 3 (UL37x3) splice donors (Su et al., 2003), we thoroughly examined HCMV-infected cells for more alternatively processed UL37 RNA species. To that end, we generated a UL37 cDNA library, screened 100 clones by PCR (as described below) and sequenced any previously unreported UL37 spliced cDNAs. The UL37 spliced junctions and cDNAs found in HCMV-infected cells are shown in Table 1 and Fig. 1(A). pUL37x1, gpUL37 and gpUL37 M , as well as the newly described UL37 ORFs, are represented in Fig. 1(B).To generate the UL37 cDNA library, 2610 8 human foreskin fibroblasts (HFFs) were infected with HCMV strain AD169 in the absence of any inhibitor treatment. To insure that all cells were synchronously infected, an m.o.i. of 3 p.f.u. per cell was used. Cells were harvested at 8 h postinfection (p.i.) and poly(A) + RNA was isolated using the Poly(A) Pure Isolation kit (Ambion). Reverse...

Alternative processing of human cytomegalovirus (HCMV) UL37 pre-mRNA predominantly produces the unspliced UL37 exon 1 (UL37x1) RNA and multiple, lower abundance, alternatively spliced UL37 RNAs. The relative abundance of UL37x1 unspliced RNA is surprising because it requires the favoured use of a polyadenylation signal within UL37 intron 1, just upstream of the UL37 exon 2 (UL37x2) acceptor. Here, it was shown that a downstream element (DSE) in UL37x2 strongly enhanced processing at the UL37x1 polyadenylation site, but did not influence UL37x1-x2 splicing. There was a potential binding site (UCUU) for polypyrimidine tract-binding protein (PTB) at the UL37x1 polyadenylation/cleavage site and its mutation to UGGG reduced both polyadenylation and splicing of UL37x1-x2 minigene pre-mRNA, suggesting a role in both RNA processing events. To determine whether lytic HCMV infection altered the balance of RNA processing factors, which bind to UL37 pre-mRNA cis elements, these were investigated in permissively infected primary and immortalized human diploid fibroblasts (HFFs) and epithelial cells. Induction of polyadenylation factors in HCMV-infected, serum-starved (G 0 ) HFFs was also investigated. Permissive HCMV infection consistently increased, albeit with different kinetics, the abundance of cleavage stimulation factor 64 (CstF-64) and PTB, and altered hypo-phosphorylated SF2 in different cell types. Moreover, the preponderance of UL37x1 RNA increased during infection and correlated with CstF-64 induction, whereas the complexity of the lower abundance UL37 spliced RNAs transiently increased following reduction of hypo-phosphorylated SF2. Collectively, multiple UL37 RNA polyadenylation cis elements and induced cellular factors in HCMV-infected cells strongly favoured the production of UL37x1 unspliced RNA. INTRODUCTIONThe human cytomegalovirus (HCMV) UL37 immediateearly (IE) gene locus encodes several protein isoforms, of which at least one, the UL37 exon 1 protein (pUL37x1), is essential for HCMV growth in humans (Hayajneh et al., 2001a) and in cultured human foreskin fibroblasts (HFFs) (Dunn et al., 2003;Yu et al., 2003). pUL37x1 shares aminoterminal sequences, including its anti-apoptotic domains, with the UL37 glycoprotein, gpUL37, and the UL37 medium protein, pUL37 M (Kouzarides et al., 1988;Goldmacher et al., 1999). While the carboxy terminus of gpUL37 is nonessential for HCMV growth in HFFs (Borst et al., 1999), it appears to play a role in HCMV growth in humans, as it does for mouse CMV pathogenesis in vivo (Lee et al., 2000;Hayajneh et al., 2001b). pUL37x1 is the product of the UL37x1 unspliced RNA and is the predominant UL37 protein produced throughout permissive HCMV infection of HFFs (Mavinakere & Colberg-Poley, 2004a). Nonetheless, gpUL37, the product of the UL37 3?4 kb spliced RNA is also produced, at lower abundance, and is N-glycosylated (Al- Barazi & ColbergPoley, 1996). Both HCMV UL37 proteins traffic dually into the endoplasmic reticulum (ER) and into mitochondria, where they block the release of cyt...