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The molecular changes induced by alemtuzumab following binding of CD52 on B tumor cells were investigated. Alemtuzumab alone had no detectable impact on cell signaling but cross-linking of alemtuzumab on the surface of B tumor lines with anti-human Fc antibodies induced a transient Ca(2+) flux followed by phosphorylation of several kinases involved in stress and survival pathways, and expression of associated proteins including TNF-α. Cross-linking of alemtuzumab also induced capping and caspase-dependent apoptosis of the tumor lines. When using primary cells from B-CLL patients, alemtuzumab alone was capable of inducing protein phosphorylation and apoptosis through the cross-linking of alemtuzumab by FcγRIIb receptors on B-CLL cells. Apoptosis was prevented by blocking of FcγRIIb receptors with anti-CD32 antibody. Overall, our results indicate that cross-linking of alemtuzumab on B tumor cells can occur naturally through Fc receptor interaction and leads to the activation of specific cellular pathways and induction of apoptosis.

sFLT01 is a novel fusion protein that consists of the VEGF/PlGF (placental growth factor) binding domain of human VEGFR1/Flt-1 (hVEGFR1) fused to the Fc portion of human IgG1 through a polyglycine linker. It binds to both human VEGF (hVEGF) and human PlGF (hPlGF) and to mouse VEGF (mVEGF) and mouse PlGF (mPlGF). In vitro, sFLT01 inhibited the proliferation of human umbilical vein endothelial cells and pericytes stimulated by either hVEGF or hPlGF. In vivo, sFLT01 had robust and significant antitumor activity in numerous preclinical subcutaneous tumor models including H460 non–small cell lung carcinoma, HT29 colon carcinoma, Karpas 299 lymphoma, MOLM-13 AML (acute myeloid leukemia), 786-O, and RENCA renal cell carcinoma (RCC). sFLT01 also increased median survival in the orthotopic RENCA RCC model. sFLT01 had strong antiangiogenic activity and altered intratumoral microvessel density, blood vessel lumen size and perimeter, and vascular and vessel areas in RCC models. sFLT01 treatment resulted in fewer endothelial cells and pericytes within the tumor microenvironment. sFLT01 in combination with cyclophosphamide resulted in greater inhibition of tumor growth than either agent used alone as a monotherapy in the A673 Ewing's sarcoma model. Gene expression profiling indicated that the molecular changes in the A673 sarcoma tumors are similar to changes observed under hypoxic conditions. sFLT01 is an innovative fusion protein that possessed robust antitumor and antiangiogenic activities in preclinical cancer models. It is a dual targeting agent that neutralizes both VEGF and PlGF and, therefore, has potential as a next generation antiangiogenic therapeutic for oncology. Mol Cancer Ther; 10(3); 404–15. ©2011 AACR.

Lipids can perform many useful functions that modify or otherwise enhance drug absorption or disposition. They may influence release/absorption-controlling processes such as gastro retention and muco-adhesion. They can facilitate lymphatic transport of lipophilic drugs, and make them less susceptible to “first pass effects” by avoiding or reducing the hepatic portal route. Understanding lipid digestion and structure formation in vivo is key to exploiting possibilities for their use in controlling absorption or disposition. In particular, liquid crystalline structures are being increasingly investigated for their roles in the aforementioned processes. Such phenomena and possibilities are discussed in this chapter.

This study is the first to demonstrate the ability of nanostructured liquid crystal particles to sustain the absorption of a poorly water soluble drug after oral administration. Cubic (V(2)) liquid crystalline nanostructured particles (cubosomes) formed from phytantriol (PHY) were shown to sustain the absorption of cinnarizine (CZ) beyond 48h after oral administration to rats. Plasma concentrations were sustained within the range of 21.5±1.5ng/mL from 12 to 48h. In stark contrast, cubosomes prepared using glyceryl monooleate (GMO) did not sustain the absorption of CZ and drug concentrations fell below quantifiable levels after 24h. Sustained absorption of CZ from PHY cubosomes lead to a significant enhancement (p0.05) in oral bioavailability (F%=21%) compared to a CZ suspension (9%) and oleic acid emulsion (12%). Analysis of the nanostructured particles in simulated gastric and intestinal fluids using small angle x-ray scattering (SAXS) revealed that the V(2)Pn3m nanostructure of PHY cubosomes was maintained for extended periods of time, in contrast to GMO cubosomes where the V(2)Im3m nanostructure was lost within 18h after exposure, suggesting that degradation of the LC nanostructure may limit sustained drug release. In addition, PHY cubosomes were shown to be extensively retained in the stomach (24h) leading to the conclusion that in the case of non-digestible PHY cubosomes, the stomach may act as a non-sink reservoir that facilitates the slow release of poorly water soluble drugs, highlighting the potential use of non-digestible LC nanostructured particles as novel sustained oral drug delivery systems.

Lipid-based liquid crystals formed from phytantriol (PHY) and glyceryl monooleate (GMO) retain their cubic-phase structure on dilution in physiologically relevant simulated gastrointestinal media, suggesting their potential application as sustained-release drug-delivery systems for poorly water-soluble drugs. In this study the potential of PHY and GMO to serve as sustained-release lipid vehicles for a model poorly-water-soluble drug, cinnarizine, was assessed and compared to that of an aqueous suspension formulation.Small-angle X-ray scattering was used to confirm the nanostructure of the liquid-crystalline matrix in the presence of the selected model drug, cinnarizine. Oral bioavailability studies were conducted in rats, and disposition of lipid and drug in segments of the gastrointestinal tract was determined over time. Differences in the digestibility and stability of formulations under digestion conditions were investigated using an in-vitro lipolysis model.The oral bioavailability of cinnarizine using the PHY formulation was 41%, compared to 19% for the GMO formulation and 6% for an aqueous suspension. The PHY formulation provided a T(max) for cinnarizine of 33 h, with absorption apparent up to 55 h after administration. In contrast, the T(max) for the GMO formulation was only 5 h. The PHY formulation was retained in the stomach for extended periods of time, with 56% of lipid remaining in the stomach after 24 h, in contrast to less than 1% of the GMO formulation after 8 h, suggesting that gastric retention was a key aspect of the prolonged period of absorption, which correlated with the formulations' relative susceptibility to in-vitro lipolysis and degradation.PHY provides a dramatic sustained-release effect for cinnarizine on oral administration, which is linked to gastric retention of the formulation and its ability to resist digestive processing. Poorly digested liquid crystal lipid formulations therefore offer a novel class of sustained-release matrices for oral administration.

The potential utility of liquid crystalline lipid-based formulations in oral drug delivery is expected to depend critically on their structure formation and stability in gastrointestinal fluids. The phase behaviour of lipid-based liquid crystals formed by phytantriol and glyceryl monooleate, known to form a bicontinuous cubic phase in excess water, was therefore assessed in physiologically-relevant simulated gastrointestinal media.Fixed composition phase studies, crossed polarised light microscopy (CPLM) and small angle X-ray scattering (SAXS) were used to determine the phase structures formed in phosphate-buffered saline, simulated gastric and intestinal fluids in the presence of model poorly water soluble drugs cinnarizine, diazepam and vitamin E acetate.The phase behaviour of phytantriol in phosphate-buffered saline was very similar to that in water. Increasing concentrations of bile components (bile salts and phospholipids) caused an increase in the lattice parameter of the cubic phase structure for both lipids. Incorporation of cinnarizine and diazepam did not influence the phase behaviour of the phytantriol- or glyceryl monooleate-based systems at physiological temperatures; however, an inverse hexagonal phase formed on incorporation of vitamin E acetate.Phytantriol and glyceryl monooleate have the potential to form stable cubic phase liquid crystalline delivery systems in the gastrointestinal tract. In-vivo studies to assess their sustained-release behaviour are warranted.

The granulocyte colony-stimulating factor receptor (G-CSFR) regulates the proliferation, differentiation and survival of neutrophilic progenitor cells. In these studies, we introduced mutant G-CSFRs with cytoplasmic domains truncated approximately every 30 amino acids from the C-terminus into interleukin-3 (IL-3)-dependent myeloid LGM-1 cells. The G-CSFR membrane proximal region containing the Box 2 homology sequence was determined to be critical for proliferative signaling, as well as for activation of Janus kinase (JAK2) and p44/42 mitogen-activated protein kinase (MAPK) following G-CSF stimulation. In the presence of increasing concentrations of JAK2 or p44/42 MAPK inhibitors, LGM-1 cells expressing the full-length G-CSFR exhibited a decreased capacity to proliferate in response to G-CSF. These results demonstrate that JAK2 and p44/42 MAPK activation is involved in proliferative signaling through the G-CSFR membrane proximal region containing the Box 2 homology sequence.

CXCR4 is a chemokine receptor frequently over-expressed on hematological cancers and solid tumors. CXCR4 and its ligand SDF1α (stromal derived factor-1α or CXCL12) play an important role in cancer metastasis, regulation of stem cell trafficking and neovascularization. We have observed previously that treatment with the small molecule CXCR4 antagonists Plerixafor (AMD3100, Mozobil) and AMD3465 in combination with Campath or Rituxan had a synergistic effect resulting in a prolongation of survival greater than that obtained with either agent alone in disseminated human B-lymphoma tumor models. We next explored the mechanism of action of CXCR4 antagonists in vitro using the human B-lymphoma cell lines Ramos, Raji, B104, BL31, IM9 or Wien133 which display similarly strong levels of cell surface CXCR4 expression. All cell lines responded to SDF1α but, in spite of comparable levels of CXCR4 expression, showed varying degrees of calcium influx and cell migration when stimulated. There was a good correlation between calcium influx and migratory responses for a given cell line. Addition of the CXCR4 antagonists Plerixafor or AMD3465 inhibited both responses. The compounds did not show any direct cytotoxicity and failed to inhibit spontaneous proliferation of Ramos, Raji and B104 human B-lymphoma cell lines. However, they did inhibit SDF1α-induced proliferation of these cell lines in agreement with their ability to block CXCR4. Moreover, CXCR4 antagonists inhibited SDF1α-induced phosphorylation of multiple proteins involved in cell migration, proliferation and survival signal pathways as well as some transcription factors, including Akt, ERK1/2, JNK, p70S6, p90RSK, GSK-3a/b, Tyk2, p38, MEK1, c-Jun, STAT3 and CREB. These results suggest that the CXCR4/SDF1α axis plays an important role in the migration, proliferation and survival of human B-lymphoma cells and that disruption of these functions with CXCR4 antagonists may contribute to their observed ability to enhance the anti-tumor activity of Campath and Rituxan in human B-lymphoma models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4460.

Introduction: Anti-angiogenic agents have shown clinical value in combination with chemotherapy by targeting the VEGF pathways. The development of tumor vasculature in human renal cell carcinomas (RCC) is stimulated not only by VEGF but by other angiogenic growth factors such as placental growth factor (PlGF) [1,2]. In the current study, we used a novel fusion protein, sFLT01, that includes human VEGFR1 domain 2 and an IgG1 Fc, which binds to both human VEGF and PlGF. The anti-angiogenic activity of sFLT01 was evaluated in several preclinical models of RCC. Methods: The binding affinity of sFLT01 for PlGF was evaluated in vitro in binding assays quantified by ELISA and Octet and in proliferation assays where endothelial cells are stimulated by PlGF. The ability of sFLT01 to slow tumor growth was evaluated in the 786-O human RCC xenograft model as well as in the mouse RENCA RCC orthotopic and subcutaneous tumor models. sFLT01 was delivered by intraperitoneal injection twice per week at doses ranging from 5–25 mg/kg. Microvessel density (MVD) and other vascular parameters were analyzed in the syngeneic and xenograft tumors by immunohistochemical methods with an antibody against mouse CD31 to identify endothelial cells. Results: sFLT01 bound to human PlGF with great affinity and inhibited HUVEC proliferation with an IC90 of approximately 2.3 nM. Subcutaneous 786-O RCC tumors were inhibited at a dose of 25 mg/kg and median survival time was increased by 33% in the orthotopic RENCA model. sFLT01 reduced MVD in subcutaneous RENCA tumors by approximately 50% and lumen area by approximately 66%. In the 786-O xenograft model, 5 or 25 mg/kg sFLT01 reduced the blood vessel area, lumen size, and vessel perimeter compared to control. Conclusions: sFLT01 is effective at inhibiting blood vessel growth in tumors by binding VEGF and PlGF thereby preventing the development of new vasculature and decreasing existing vasculature. sFLT01 reduces intratumoral blood vessel count, prolongs survival, and delays tumor progression in the RENCA and 786-O renal cell carcinoma models. As an anti-angiogenic agent, sFLT01 may be useful in treating human renal cell carcinomas. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A14.

TMPRSS4, a cell surface transmembrane serine protease, is over expressed at the transcriptional level in pancreatic, colorectal and thyroid cancers compared with normal tissues. Recent studies have indicated a role for TMPRSS4 in tumor cell migration and tumor metastasis. We examined TMPRSS4 expression in dissected non-small cell lung cancer (NSCLC) tissues by quantitative RT-PCR and immunohistochemisty. At the transcriptional level, TMPRSS4 expression was elevated in adeno and squamous cell (SC) carcinomas when compared to the matching normal tissues (p=0.0035). At the protein level, over 100 tumor and normal specimens were examined with rabbit polyclonal anti-TMPRSS4 antibodies via immunohistochemistry. Adeno and SC carcinomas were positive for TMPRSS4 (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C166.

Background Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization. Methods In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep. Results The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation. Conclusion Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (