The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.

Although Akt is known as a survival kinase, inhibitors of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway do not always induce substantial apoptosis. We show that silencing Akt1 alone, or any combination of Akt isoforms, can suppress the growth of tumors established from phosphatase and tensin homologue–null human cancer cells. Although these findings indicate that Akt is essential for tumor maintenance, most tumors eventually rebound. Akt knockdown or inactivation with small molecule inhibitors did not induce significant apoptosis but rather markedly increased autophagy. Further treatment with the lysosomotropic agent chloroquine caused accumulation of abnormal autophagolysosomes and reactive oxygen species, leading to accelerated cell death in vitro and complete tumor remission in vivo. Cell death was also promoted when Akt inhibition was combined with the vacuolar H+–adenosine triphosphatase inhibitor bafilomycin A1 or with cathepsin inhibition. These results suggest that blocking lysosomal degradation can be detrimental to cancer cell survival when autophagy is activated, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PI3K–Akt pathway inhibition.

Overexpression of FGF receptor 3 (FGFR3) is implicated in the development of t(4;14

The usual paradigm for developing kinase inhibitors in oncology is to use a high-affinity proof-of-concept inhibitor with acceptable metabolic properties for key target validation experiments. This approach requires substantial medicinal chemistry and can be confounded by drug toxicity and offtarget activities of the test molecule. As a better alternative, we have developed inducible short-hairpin RNA xenograft models to examine the in vivo efficacy of inhibiting oncogenic BRAF. Our results show that tumor regression resulting from BRAF suppression is inducible, reversible, and tightly regulated in these models. Analysis of regressing tumors showed the primary mechanism of action for BRAF to be increased tumor cell proliferation and survival. In a metastatic melanoma model, conditional BRAF suppression slowed systemic tumor growth as determined by in vivo bioluminescence imaging. Taken together, gain-of-function BRAF signaling is strongly associated with in vivo tumorigenicity, confirming BRAF as an important target for small-molecule and RNA interferencebased therapeutics. (Cancer Res 2006; 66(2): 999-1006)

In the past decade, there has been an explosion of interest in hyaluronan, an often misunderstood, biochemically simple, yet functionally complex carbohydrate polymer that is a resident of many extracellular matrices. Previously thought of as a passive, space-filling component of the extracellular matrix, the so-called "goo" concept, hyaluronan has risen to a much higher regard in recent years, even being called "magic glue" in a recent perspective. Hyaluronan is likely to be the common thread in many morphogenetic processes, including condensation events and epithelial-to-mesenchymal transformation. Hyaluronan is comparatively unique as a component of the extracellular matrix as it is solely composed of carbohydrate. In order to truly understand this biopolymer, one must first understand its biosynthesis, then understand its uptake and turnover, then identify its binding proteins and receptors. Major advances have been made in all of these arenas within the past decade. Hyaluronan synthases, hyaluronidases, and the hyaladherins have been molecularly identified and cloned. Furthermore, many have now been inactivated, employing gene targeting strategies, to create mice deficient in the respective gene product function. Collectively, huge strides have been made in our understanding of the diverse biological functions for this fascinating molecule. Hyaluronan appeared in metazoans immediately prior to the arrival of the vertebrates, and may be required for the differentiation, development, and/or function of most cell lineages, structures, and tissues that we associate with vertebrates, such as the neural crest, the skeleton, including the teeth, skin, and hair, and the chambered heart. In this review, we will update the reader on the advances of the past decade and provide insight into those morphogenetic processes through which hyaluronan regulates vertebrate development.

Oncogenic activation of the BRAF serine/threonine kinase has been associated with initiation and maintenance of melanoma tumors. As such, development of pharmacologic agents to target RAF proteins or their effector kinases is an area of intense investigation. Here we report the biological properties of GDC-0879, a highly selective, potent, and orally bioavailable RAF small-molecule inhibitor. We used extracellular signal-regulated kinase (ERK)-1/2 and mitogen-activated protein kinase/ERK kinase (MEK)-1/2 phosphorylation as biomarkers to explore the relationship between tumor outcome and pharmacodynamic inhibition of the RAF-MEK-ERK pathway. In GDC-0879-treated mice, both cell line-and patient-derived BRAF V600E tumors exhibited stronger and more sustained pharmacodynamic inhibition (>90% for 8 hours) and improved survival compared with mutant KRAS-expressing tumors. Despite the involvement of activated RAF signaling in RAS-induced tumorigenesis, decreased time to progression was observed for some KRAS-mutant tumors following GDC-0879 administration. Moreover, striking differences were noted for RAF and MEK inhibition across a panel of 130 tumor cell lines. Whereas GDC-0879-mediated efficacy was associated strictly with BRAF V600E status, MEK inhibition also attenuated proliferation and tumor growth of cell lines expressing wild-type BRAF (81% KRAS mutant, 38% KRAS wild type). The responsiveness of BRAF V600E melanoma cells to GDC-0879 could be dramatically altered by pharmacologic and genetic modulation of phosphatidylinositol 3-kinase pathway activity. These data suggest that GDC-0879-induced signaling changes are dependent on the point of oncogenic activation within the RAS network. Taken together, these studies increase our understanding of the molecular determinants for antitumor efficacy resulting from RAF pathway inhibition and have implications for therapeutic intervention in the clinic.

Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated. Here, we report that through chemical modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chemical synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clinically approved ADC, trastuzumab-MCC-DM1.

We have used in situ hybridization to study the expression of the vertebrate hyaluronan synthase (Has) gene family members, designated Has1, Has2, and Has3, during mouse development. At embryonic day (E) 7.5, Has1 and Has2 are expressed throughout the gastrulating embryo. After E8.5, Has1 expression disappears, but Has2 continues to be strongly, albeit transiently, expressed in numerous tissues, including the branchial arches and craniofacial structures such as the palatal shelves and lens pit. Has2 is also expressed during cardiac, skeletal, and tail development. Has3 transcripts are first detected at E10.5 in the maxillary and mandibular components of the first branchial arch. Notably, Has3 expression in the developing teeth, vibrissae hair follicles, nasal cavity, and inner ear complements the expression pattern of Has2. Our results indicate that, whereas Has2 is exclusively expressed in some tissues, its expression pattern overlaps and/or complements that of Has1 and Has3 in others. Developmental Dynamics 233: 130 -141, 2005.