Suzanne Grothe

Link protein has greater affinity for versican than aggrecan.

The function of link protein in stabilizing the interaction between aggrecan and hyaluronan to form aggrecan aggregates, via the binding of link protein to the aggrecan G1 domain and hyaluronan, is well established. However, it is not known whether link protein can function with similar avidity with versican, another member of the large hyaluronan-binding proteoglycan family that also binds to hyaluronan via its G1 domain. To address this issue, we have compared the interaction of the versican and aggrecan G1 domains with link protein and hyaluronan using recombinant proteins expressed in insect cells and BIAcore analysis. The results showed that link protein could significantly improve the binding of both G1 domains to hyaluronan and that its interaction with VG1 is of a higher affinity than that with AG1. These observations suggest that link protein may function as a stabilizer of the interaction, not only between aggrecan and hyaluronan in cartilage, but also between versican and hyaluronan in many tissues.

Kinetic Analysis of the Interactions between Troponin C and the C-terminal Troponin I Regulatory Region and Validation of a New Peptide Delivery/Capture System used for Surface Plasmon Resonance

Using surface plasmon resonance (SPR)-based biosensor analysis and fluorescence spectroscopy, the apparent kinetic constants, k(on) and k(off), and equilibrium dissociation constant, K(d), have been determined for the binding interaction between rabbit skeletal troponin C (TnC) and rabbit skeletal troponin I (TnI) regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). To carry out SPR analysis, a new peptide delivery/capture system was utilized in which the TnI peptides were conjugated to the E-coil strand of a de novo designed heterodimeric coiled-coil domain. The TnI peptide conjugates were then captured via dimerization to the opposite strand (K-coil), which was immobilized on the biosensor surface. TnC was then injected over the biosensor surface for quantitative binding analysis. For fluorescence spectroscopy analysis, the environmentally sensitive fluoroprobe 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid (1,5-IAEDANS) was covalently linked to Cys98 of TnC and free TnI peptides were added. SPR analysis yielded equilibrium dissociation constants for TnC (plus Ca(2+)) binding to the C-terminal TnI regulatory peptides TnI(96-131) and TnI(96-139) of 89nM and 58nM, respectively. The apparent association and dissociation rate constants for each interaction were k(on)=2.3x10(5)M(-1)s(-1), 2.0x10(5)M(-1)s(-1) and k(off)=2.0x10(-2)s(-1), 1.2x10(-2)s(-1) for TnI(96-131) and TnI(96-139) peptides, respectively. These results were consistent with those obtained by fluorescence spectroscopy analysis: K(d) being equal to 130nM and 56nM for TnC-TnI(96-131) and TnC-TnI(96-139), respectively. Interestingly, although the inhibitory region peptide (TnI(96-115)) was observed to bind with an affinity similar to that of TnI(96-131) by fluorescence analysis (K(d)=380nM), its binding was not detected by SPR. Subsequent investigations examining salt effects suggested that the binding mechanism for the inhibitory region peptide is best characterized by an electrostatically driven fast on-rate ( approximately 1x10(8) to 1x10(9)M(-1)s(-1)) and a fast off-rate ( approximately 1x10(2)s(-1)). Taken together, the determination of these kinetic rate constants permits a clearer view of the interactions between the TnC and TnI proteins of the troponin complex.

Applications of pullulan in aqueous two-phase systems for enzyme production, purification and utilization

SummaryPullulan, a microbial polysaccharide, was employed with polyethylene glycol (PEG) to form an aqueous two-phase system. The phase diagram of the PEG-Pullulan systems as well as the partition of enzymes depended strongly on the molecular weight of PEG. This behavior provided a basis for the separation of cellulase from β-galactosidase. The extractive hydrolysis of lactose, using a commercial preparation of β-galactosidase, could also be performed in a PEG-Pullulan system (16% PEG 1400, 14% Pullulan). The enzyme was effectively contained in the bottom phase while a high percentage of the glucose produced could be removed in the top phase. The enzyme activity was preserved very well after several repeated hydrolyses.Furthermore, it was demonstrated that the PEG-Pullulan system was capable of retaining Trichoderma reesei in the bottom phase, allowing the production of cellulase which could be intermittently removed in the top phase. Such a system permitted a semicontinuous production scheme although the enzyme production was observed to decrease after five cycles.In view of the low cost and low viscosity of pullulan, this polysaccharide could be considered a suitable replacement for dextran, a component widely used in aqueous two-phase formulation.

Abstract 1778: Nimotuzumab, a humanized antiepidermal growth factor receptor antibody, interacts with EGFRvIII

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Glioblastomas frequently overexpress a variant form of EGFR, called variant III (EGFRvIII), which has an in-frame deletion of an 801-bp sequence in the extracellular domain. EGFRvIII does not bind EGF ligand but exhibits constitutive kinase activity that results in enhanced transformation, reduced apoptosis, and resistance to therapy. Nimotuzumab (Nmab) is an EGFR-targeting antibody that has demonstrated encouraging results in early clinical trials treating adult and pediatric brain tumors. Here we present data characterizing the interaction of Nmab with EGFRvIII. Methods: Binding of Nmab to the extracellular domain of wtEGFR and EGFRvIII was first characterized by SPR biosensor analysis: Nmab was captured via its Fc domain and varying concentrations of EGFRvIII and EGFRwt were flowed. Flow cytometry analysis was subsequently performed using a parental U87MG glioblastoma cell line and derivatives which were engineered to overexpress either wtEGFR or EGFRvIII. Results: Nmab bound EGFRvIII and EGFRwt with similar affinity, which was in the 10−8 M range. This KD is consistent with the previously reported affinity constant of Nmab for EGFRwt (Telavera et al, 2009). Binding was further analyzed by flow cytometry. Nmab bound both EGFRwt and EGFRvIII expressed on the surface of parental U87MG, U87MG EGFRvIII and U87MG wtEGFR cells. Additional data concerning effects of Nmab on EGFRvIII expressing cell lines will be presented. Conclusion: Nmab binds to wtEGFR and EGFRvIII with similar affinity, which supports development of the antibody as a therapeutic for glioblastoma. In U87MG glioblastoma cells, synergistic activity of Nmab in combination with radiotherapy has been previously reported (Diaz Miqueli et al, 2009). Nmab is currently being tested in an advanced randomized study in the first line setting for the treatment of adult glioma. Nmab is being tested in combination with radiation plus temozolomide, vs radiation plus temozolomide alone, with preliminary results expected in the second half of 2010. 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 1778.

Abstract 1467: AB-16B5, a therapeutic monoclonal antibody against human clusterin that blocks the epithelial-to-mesenchymal transition

Studies are increasingly implicating clusterin (CLU) as an important contributing factor in cancer promotion and invasion. Although there is evidence that secreted (s) CLU plays a pro-survival role in tumors, recently published results demonstrated that sCLU is also a potent stimulator of the epithelial-to-mesenchymal transition (EMT). A family of monoclonal antibodies (mAbs) specific for human sCLU was generated and a subset of these was found to inhibit the migration and invasion of several types of cancer cells. Importantly, this EMT-inhibiting subset of mAbs all bind to a specific amino acid sequence in sCLU and inhibit metastasis in vivo. The variable regions of the lead candidate mAb were modified using an in silico molecular modeling approach to generate a humanized IgG2, designated AB-16B5, which exhibited almost identical binding parameters compared to the original mouse antibody, with an apparent KD of 2 − 5 nM for recombinant human CLU. Treatment of 4T1 carcinoma cells with AB-16B5 inhibited TGFβ-induced EMT indicating that the biological activity in vitro was maintained in the humanized antibody. Moreover, AB-16B5 inhibited the motility of EMT6 cells in scratch assays and reduced the invasion of DU145 and PC-3 hormone-insensitive human prostate cancer cells when cultured in Matrigel. In animal studies, DU145 prostate cancer cells were implanted in SCID mice and treated with 5 mg/kg AB-16B5 twice per week as a monotherapy or in combination with the anti-mitotic drug, docetaxel (TxT). These experiments showed that the tumors in the AB-16B5 treated animals were 55% smaller than those in the control group. Furthermore, combining the AB-16B5 treatment with that of TxT caused a reduction of tumor size by 40% compared to the TxT-only group. PC-3-derived tumors in SCID mice treated with AB-16B5 showed a similar degree of tumor growth inhibition. Importantly, in both prostate cancer models, the mice exposed to AB-16B5 exhibited a marked increase in their overall survival. Furthermore, AB-16B5 treatment of Nude mice with intra-cardiac implantations of MDA-231 breast cancer cells resulted in a reduction of the number of metastatic bone lesions. The pharmacokinetic parameters of AB-16B5 are comparable to other IgG2 antibodies with its half-life being approximately 15 days following a bolus intravenous injection in mice. Finally, mice exposed to AB-16B5 at 10-times the therapeutic dose displayed no observable signs of toxicity and no major changes in serum biochemistry were detected. In conclusion, AB-16B5 is one of the rare therapeutic mAbs that directly targets EMT to reduce the invasion of tumors. Its therapeutic effects hold much promise to control metastasis from breast and prostate tumors, in addition to enhancing the response to chemotherapeutic drugs. 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 1467.