Mark R. Stroud

Selectin GMP-140 (CD62; PADGEM) binds to sialosyl-Lea and sialosyl-Lex, and sulfated glycans modulate this binding

GMP-140 (CD62; PADGEM) is a member of the selectin family expressed highly at the surface of platelets and endothelial cells by agonists such as thrombin or phorbol esters. Previous studies indicate that the lectin domain of GMP-140 recognizes sialosyl-Le(x) (SLex) and to a lesser extent Le(x) (Polley MJ, et al., Proc Natl Acad Sci USA 88:6224, 1991). We now report that GMP-140 binds to sialosyl Lea (SLea) and to SLex, and that degree of binding to SLea is greater than that to SLex under our experimental conditions. Binding of activated platelets to SLea or SLex was inhibited to various degrees in the presence of sulfated glycans, suggesting that sulfated glycans induce conformational change in the lectin domain of GMP-140 and modulates its binding affinity to SLea and SLex.

Further studies on cell adhesion based on Le(x)-Le(x) interaction, with new approaches: embryoglycan aggregation of F9 teratocarcinoma cells, and adhesion of various tumour cells based on Le(x) expression.

We previously proposed specific interaction of Lex (Galβ1 → 4[Fucα1 → 3]-GlcNAcβ1 → 3Gal) with Lex as a basis of cell adhesion in pre-implantation embryos and in aggregation of F9 teratocarcinoma cells, based on several lines of evidence (Eggenset al., J Biol Chem (1989)264:9476–9484). We now present additional evidence for this concept, based on autoaggregation studies of plastic beads coated with glycosphingolipids (GSLs) bearing Lex or other epitopes, and affinity chromatography on Lex-columns of multivalent lactofucopentaose III (Lex oligosaccharide) conjugated with lysyllysine. Comparative adhesion studies of Lex-expressing tumour cellsvs their Lex-non-expressing variants showed that only Lex-expressing cells adhere to Lex-coated plates and are involved in tumour cell aggregation, in analogy to F9 cell aggregation. The major carrier of Lex determinant in F9 cells is not GSL but rather polylactosaminoglycan (‘embryoglycan’), and we demonstrated autoaggregation of purified embryoglycan in the presence of Ca2+, and reversible dissociation in the absence of Ca2+ (addition of EDTA). Defucosylated embryoglycan did not show autoaggregation under the same conditions. Thus, Lex-Lex interaction has been demonstrated on a lactosaminoglycan basis as well as a GSL basis. A molecular model of Lex-Lex interaction based on minimum energy conformation with involvement of Ca2+ is presented.

Monoclonal antibodies directed to the blood group a associated structure, galactosyl-A: Specificity and relation to the thomsen-friedenreich antigen

Two monoclonal antibodies, HH8 and HH9, have been established after immunization of mice with galactosyl-A glycolipid antigen having the terminal structure, Gal beta 1----3GalNAc alpha 1----3[Fuc alpha 1----2]Gal beta 1----R, which is the precursor for type 3 chain A (repetitive A) and type 3 chain H (A-associated H). Both antibodies react strongly and specifically with galactosyl-A, but HH8 (IgM) showed strong hemagglutination of blood group A1, A2, O and B erythrocytes after sialidase treatment, while HH9 (IgG1) did not react with human erythrocytes even after sialidase treatment. HH8 and anti-T antibody, but not HH9, reacted with glycophorin A after sialidase treatment. The reactivity of HH8 with glycophorin A was abolished by beta-galactosidase and was inhibited by liposomes containing galactosyl-A, but not other glycolipids. In addition, anti-T antibody and peanut lectin reacted specifically with galactosyl-A glycolipids. These findings indicate that HH8 recognizes the terminal disaccharide Gal beta 1----3GalNAc alpha 1----R, which is the same sequence as the classically known Thomsen-Friedenreich antigen (T-antigen), whereas HH9 does not cross-react with T-antigen but recognizes the entire galactosyl-A structure. The T-antigen was also demonstrated by immunohistology with HH8 after neuraminidase treatment in a subset of cells in stratified epithelium.

The Drosophila Gene brainiac Encodes a Glycosyltransferase Putatively Involved in Glycosphingolipid Synthesis

The Drosophila genesfringe and brainiac exhibit sequence similarities to glycosyltransferases. Drosophila and mammalian fringe homologs encode UDP-N-acetylglucosamine:fucose-O-Ser β1,3-N-acetylglucosaminyltransferases that modulate the function of Notch family receptors. The biological function of brainiac is less well understood. brainiac is a member of a large homologous mammalian β3-glycosyltransferase family with diverse functions. Eleven distinct mammalian homologs have been demonstrated to encode functional enzymes forming β1–3 glycosidic linkages with different UDP donor sugars and acceptor sugars. The putative mammalian homologs with highest sequence similarity tobrainiac encode UDP-N-acetylglucosamine:β1,3-N-acetylglucosaminyltransferases (β3GlcNAc-transferases), and in the present study we show thatbrainiac also encodes a β3GlcNAc-transferase that uses β-linked mannose as well as β-linked galactose as acceptor sugars. The inner disaccharide core structures of glycosphingolipids in mammals (Galβ1–4Glcβ1-Cer) and insects (Manβ1–4Glcβ1-Cer) are different. Both disaccharide glycolipids served as substrates for brainiac, but glycolipids of insect cells have so far only been found to be based on the GlcNAcβ1–3Manβ1–4Glcβ1-Cer core structure. Infection of High FiveTM cells with baculovirus containing full coding brainiac cDNA markedly increased the ratio of GlcNAcβ1–3Manβ1–4Glcβ1-Cer glycolipids compared with Galβ1–4Manβ1–4Glcβ1-Cer found in wild type cells. We suggest that brainiac exerts its biological functions by regulating biosynthesis of glycosphingolipids.

Preclinical Validation of the Utility of BLZ-100 in Providing Fluorescence Contrast for Imaging Spontaneous Solid Tumors

There is a need in surgical oncology for contrast agents that can enable real-time intraoperative visualization of solid tumors that can enable complete resections while sparing normal surrounding tissues. The Tumor Paint agent BLZ-100 is a peptide-fluorophore conjugate that can specifically bind solid tumors and fluoresce in the near-infrared range, minimizing light scatter and signal attenuation. In this study, we provide a preclinical proof of concept for use of this imaging contrast agent as administered before surgery to dogs with a variety of naturally occurring spontaneous tumors. Imaging was performed on excised tissues as well as intraoperatively in a subset of cases. Actionable contrast was achieved between tumor tissue and surrounding normal tissues in adenocarcinomas, squamous cell carcinomas, mast cell tumors, and soft tissue sarcomas. Subcutaneous soft tissue sarcomas were labeled with the highest fluorescence intensity and greatest tumor-to-background signal ratio. Our results establish a foundation that rationalizes clinical studies in humans with soft tissue sarcoma, an indication with a notably high unmet need.

Solid-phase synthesis and immunoreactivity of penta-O-(N-acetyl-α-D-galactosaminyl)-MUC1 eicosapeptide, a glycosylated counter part of the highly immunogenic tandem repeat sequence of carcinoma-associated mucin

Abstract Penta- O -glycosylated MUC1 peptide HVal-Thr * -Ser * -Ala-Pro-Asp-Thr * -Arg-Pro-Ala-Pro-Gly-Ser * -Thr * -Ala-Pro-Pro-Ala-His-Gly-OH ( * α- D -GalNA cp ) ( 2 ) has been synthesized by a solid-phase method on Sasrin resin using Fmoc-glycoaminoacids 3 and 4 as building blocks. The monoclonal antibodies SM3 and HMPV, specific to the MUC1 peptide 1 , also react strongly with 2 .

Abstract 4245: BLZ-100 provides optical contrast in animal models of breast cancer

Over 230,000 new breast cancer cases occur in the US annually. Complete surgical removal of tumor tissue is critical to reduce the risk of local recurrence. About 24% of patients who undergo breast conservation surgery require a second surgery due to positive margins. Second surgeries can be psychologically and financially costly to patients. BLZ-100, a peptide-fluorophore conjugate, is a Tumor Paint product candidate being developed to provide real-time, high resolution, intraoperative optical contrast between tumor and normal tissue, potentially helping surgeons accomplish more complete and accurate tumor resections. Intraoperative imaging with BLZ-100 may potentially improve the percentage of breast cancer patients achieving a complete resection and negative margins at their first surgical procedure. To better define the relationship between tumor pathology and BLZ-100 fluorescence, data from mouse models and canine mammary cancer cases were evaluated. Human breast cancer was modeled in mice using cell lines and xenograft models using patient-derived tumors. Review of histopathology was also conducted for each dog case. Animals received a single IV dose of BLZ-100, and tumors were excised one day after dosing. Excised tumor and normal tissues were imaged using the Odyssey Clx (LI-COR) to determine the ratio of signal in tumor compared to normal (background) tissue (TBR). Detailed histopathology reports for the dog cases were also analyzed to determine factors potentially associated with fluorescence intensity and TBR. A significant BLZ-100 TBR was observed. Histopathologic features potentially associated with TBR in dogs included cellularity and the degree of infiltration and demarcation. The studies supported the concept that BLZ-100 might potentially serve as an intraoperative optical imaging agent for breast cancer surgery. Citation Format: Stacey Hansen, Janean Fidel, Katie Kennedy, William Dernell, Boel Fransson, Josh Ramsay, Valorie Wiss, Mark Stroud, Julia E. Parrish-Novak. BLZ-100 provides optical contrast in animal models of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4245.

Abstract LB-231: An optide (optimized knottin-peptide) that inhibits tumor cell growth In vitro and accumulates in sarcoma flank tumors in vivo

Antibody-drug conjugates (ADCs) have been FDA approved for the targeted delivery of chemotherapy to cancer. However, ADCs are limited for solid tumors and brain cancer by their poor penetration and inability to cross the blood-brain-barrier. A number of groups have shown that cystine-knot peptides (knottins) can be modified in ways that allow them to target cancer cells. Our lab demonstrated that an optide (optimized knottin-peptides) conjugate such as chlorotoxin-Cy5.5 are able to accumulate throughout tumors and cross an intact blood-brain barrier. However, chlorotoxin-Cy5.5 accumulates in mouse liver - a potential liability if used as a peptide-drug conjugate. We sought to identify a novel peptide that is capable of delivering chemotherapy selectively to tumor cells in vitro and in vivo. Our group has developed a mammalian protein expression system that is able to produce >20mg scale, endotoxin-free knottins and can rapidly generate variants of those peptides. We went in vivo to investigate if there was selective accumulation of our novel peptide-dye conjugates in sarcoma flank xenografts. Mice were injected intravenously with conjugate and accumulation was quantified in a number of tissues using IVIS imaging. We identified a novel peptide that accumulated in sarcoma flank tumors >10-fold relative to liver. We tested the ability of this peptide to deliver cytotoxic chemotherapy in vitro as a peptide-drug conjugate. We identified a novel conjugate capable of efficiently inhibiting the growth of a number of tumor cell lines in vitro. We tested the ability of various endocytosis inhibitors to prevent the uptake of this peptide and showed that inhibitors of GPI-anchored protein endocytosis prevented this accumulation. To verify tumor accumulation in preparation for efficacy studies we tracked the distribution of a radiolabeled form of the peptide in a whole body autoradiography model and observed sustained tumor uptake. Our novel peptide-conjugate is able to potently inhibit the growth of a number of tumor cell lines in vitro and the endocytosis of GPI-anchored proteins is involved in this accumulation. Additionally, this peptide accumulates in tumor tissue in vivo. Optide-drug conjugates may offer enhanced tumor penetration over antibody-drug conjugates and therefore increase the therapeutic index in delivering cytotoxic chemotherapy. Citation Format: Theo Sottero, Emily J. Girard, Colin Correnti, Mark R. Stroud, Brandon L. Kier, Andrew J. Mhyre, James Olson. An optide (optimized knottin-peptide) that inhibits tumor cell growth In vitro and accumulates in sarcoma flank tumors in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-231.

Abstract 4936: Tumor PaintTM technology detects naturally occurring solid tumors in dogs

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tumor PaintTM technology is designed to provide real-time visualization of tumors during surgery at improved resolution over existing methods. BLZ-100 is a Tumor Paint product consisting of a chlorotoxin (CTX) peptide conjugated to a near-infrared (NIR) fluorophore that is being advanced toward clinical studies. CTX-based Tumor Paint products have been shown to illuminate a broad range of cancers in mouse models. To facilitate clinical translation, a preclinical feasibility study was conducted in dogs with naturally occurring solid tumors. Many types of canine tumors resemble human disease, including sarcomas, mammary and lung cancers, mucosal squamous cell cancers, and gliomas. The diversity of tumor size and type, surrounding tissue, and patient body mass provides a model that is superior to the mouse in predicting the clinical characteristics of BLZ-100, including tumor penetration, background staining, and effective imaging dose. Twenty-seven dogs were given BLZ-100 intravenously 24 - 48 hours before surgery. Doses, normalized to body surface area (mg/m2), were 0.25 - 0.8 (7 dogs), 0.8 - 1.2 (15 dogs) and 1.2 - 1.6 (5 dogs). Tumor types were sarcoma, adenocarcinoma, mastocytoma, squamous cell carcinoma, and meningioma. Dogs received standard of care including tumor resection with intent to control or cure local disease. Excised tissues were imaged using the IVIS Spectrum (Caliper) and the Odyssey NIR scanner (Li-Cor) to determine overall signal in tumor and gross tumor to normal ratios. Tissues were then embedded in OCT, sectioned on a cryostat, and scanned on the Odyssey. Serial sections were stained with H&E, and comparison with the fluorescence scans was used to validate the specificity of BLZ-100 for tumor tissue. Ex vivo imaging data showed maximal tumor fluorescence at doses above 0.9 mg/m2. In dogs treated with 0.8 mg/m2 or higher (20 dogs in total), ratios of fluorescence in tumour to normal surrounding tissue ranged from 200, with good differentiation in several tumor types including meningioma, carcinomas (lung, thyroid, and mammary), and sarcomas. Highest signals and gross tumor to background ratios were seen in a subset of soft tissue sarcomas, suggesting preferential uptake of the conjugate in these tumor types. Histologic analysis of these cases showed 95% sensitivity and 85% specificity. Intraoperative imaging was conducted in several cases, using a prototype open NIR imaging device. These cases showed good contrast between gross tumors and surrounding structures, detection of tumor through 0.5 cm of normal fatty tissue, and low background in tissues such as fat and muscle. The information obtained in this study provides data that will be valuable in clinical translation of BLZ-100. This project was funded in whole with Federal funds from National Cancer Institute, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN2612012000054C. Citation Format: William S. Dernell, Janean Fidel, Katie Kennedy, Stacey Hansen, Valorie Wiss, Mark Stroud, Julia E. Parrish-Novak. Tumor PaintTM technology detects naturally occurring solid tumors in dogs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4936. doi:10.1158/1538-7445.AM2014-4936

The Globoseries Glycosphingolipid Sialosyl Galactosyl Globoside is Found in Urinary Tract Tissues and is a Preferred Binding Receptor In Vitro for Uropathogenic Escherichia Coli Expressing Pap-Encoded Adhesins

Women with a history of recurrent Escherichia coli urinary tract infections (UTIs) are significantly more likely to be nonsecretors of blood group antigens than are women without such a history, and vaginal epithelial cells (VEC) from women who are nonsecretors show enhanced adherence of uropathogenic E. coli isolates compared with cells from secretors. We previously extracted glycosphingolipids (GSLs) from native VEC and determined that nonsecretors (but not secretors) selectively express two extended globoseries GSLs, sialosyl galactosyl globoside (SGG) and disialosyl galactosyl globoside (DSGG), which specifically bound uropathogenic E. coli R45 expressing a P adhesin. In this study, we demonstrated, by purifying the compounds from this source, that SGG and DSGG are expressed in human kidney tissue. We also demonstrated that SGG and DSGG isolated from human kidneys bind uropathogenic E. coli isolates expressing each of the three classes of pap-encoded adhesins, including cloned isolates expressing PapG from J96, PrsG from J96, and PapG from IA2, and the wild-type isolates IA2 and R45. We metabolically 35S labeled these five E. coli isolates and measured their relative binding affinities to serial dilutions of SGG and DSGG as well as to globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4), two other globoseries GSLs present in urogenital tissues. Each of the five E. coli isolates bound to SGG with the highest apparent avidity compared with their binding to DSGG, Gb3, and Gb4, and each isolate had a unique pattern of GSL binding affinity. These studies further suggest that SGG likely plays an important role in the pathogenesis of UTI and that its presence may account for the increased binding of E. coli to uroepithelial cells from nonsecretors and for the increased susceptibility of nonsecretors to recurrent UTI.