HER2 is a validated target in breast cancer therapy. Two drugs are currently approved for HER2-positive breast cancer: trastuzumab (Herceptin), introduced in 1998, and lapatinib (Tykerb), in 2007. Despite these advances, some patients progress through therapy and succumb to their disease. A variation on antibody-targeted therapy is utilization of antibodies to deliver cytotoxic agents specifically to antigenexpressing tumors. We determined in vitro and in vivo efficacy, pharmacokinetics, and toxicity of trastuzumab-maytansinoid (microtubule-depolymerizing agents) conjugates using disulfide and thioether linkers. Antiproliferative effects of trastuzumab-maytansinoid conjugates were evaluated on cultured normal and tumor cells. In vivo activity was determined in mouse breast cancer models, and toxicity was assessed in rats as measured by body weight loss. Surprisingly, trastuzumab linked to DM1 through a nonreducible thioether linkage (SMCC), displayed superior activity compared with unconjugated trastuzumab or trastuzumab linked to other maytansinoids through disulfide linkers. Serum concentrations of trastuzumab-MCC-DM1 remained elevated compared with other conjugates, and toxicity in rats was negligible compared with free DM1 or trastuzumab linked to DM1 through a reducible linker. Potent activity was observed on all HER2-overexpressing tumor cells, whereas nontransformed cells and tumor cell lines with normal HER2 expression were unaffected. In addition, trastuzumab-DM1 was active on HER2-overexpressing, trastuzumab-refractory tumors. In summary, trastuzumab-DM1 shows greater activity compared with nonconjugated trastuzumab while maintaining selectivity for HER2-overexpressing tumor cells. Because trastuzumab linked to DM1 through a nonreducible linker offers improved efficacy and pharmacokinetics and reduced toxicity over the reducible disulfide linkers evaluated, trastuzumab-MCC-DM1 was selected for clinical development. [Cancer Res 2008;68(22):9280-90]

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

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.

Physiological platelet synthesis is thought to require the humoral activities of meg-CSF and thrombopoietin, which respectively promote proliferation and maturation of megakaryocytic cells. A meg-CSF/thrombopoietin-like protein that is present in plasma of irradiated pigs has been purified and cloned. This protein binds to and activates the c-mpl protein, a member of the cytokine receptor superfamily. The isolated Mpl ligand shares homology with erythropoietin and stimulates both megakaryocytopoiesis and thrombopoiesis.

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We have investigated proteins which interact with the PEST-type protein tyrosine phosphatase, PTP hematopoietic stem cell fraction (HSCF), using the yeast two-hybrid system. This resulted in the identification of proline, serine, threonine phosphatase interacting protein (PSTPIP), a novel member of the actin- associated protein family that is homologous to Schizosaccharomyces pombe CDC15p, a phosphorylated protein involved with the assembly of the actin ring in the cytokinetic cleavage furrow. The binding of PTP HSCF to PSTPIP was induced by a novel interaction between the putative coiled-coil region of PSTPIP and the COOH-terminal, proline-rich region of the phosphatase. PSTPIP is tyrosine phosphorylated both endogenously and in v-Src transfected COS cells, and cotransfection of dominant-negative PTP HSCF results in hyperphosphorylation of PSTPIP. This dominant-negative effect is dependent upon the inclusion of the COOH-terminal, proline-rich PSTPIP-binding region of the phosphatase. Confocal microscopy analysis of endogenous PSTPIP revealed colocalization with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow. Overexpression of PSTPIP in 3T3 cells resulted in the formation of extended filopodia, consistent with a role for this protein in actin reorganization. Finally, overexpression of mammalian PSTPIP in exponentially growing S. pombe results in a dominant-negative inhibition of cytokinesis. PSTPIP is therefore a novel actin-associated protein, potentially involved with cytokinesis, whose tyrosine phosphorylation is regulated by PTP HSCF.

The orphan receptor CRF2-4 is a member of the class II cytokine receptor family (CRF2), which includes the interferon receptors, the interleukin (IL) 10 receptor, and tissue factor. CRFB4, the gene encoding CRF2-4, is located within a gene cluster on human chromosome 21 that comprises three interferon receptor subunits. To elucidate the role of CRF2-4, we disrupted the CRFB4 gene in mice by means of homologous recombination. Mice lacking CRF2-4 show no overt abnormalities, grow normally, and are fertile. CRF2-4 deficient cells are normally responsive to type I and type II interferons, but lack responsiveness to IL-10. By ∼12 wk of age, the majority of mutant mice raised in a conventional facility developed a chronic colitis and splenomegaly. Thus, CRFB4 mutant mice recapitulate the phenotype of IL-10–deficient mice. These findings suggest that CRF2-4 is essential for IL-10–mediated effects and is a subunit of the IL-10 receptor.

In this article, we show that, in transfected COS-1 cells, protein tyrosine phosphatase (PTP)-PEST translocates to the membrane periphery following stimulation by the extracellular matrix protein fibronectin. When plated on fibronectin, PTP-PEST (−/−) fibroblasts display a strong defect in motility. 3 h after plating on fibronectin, the number and size of vinculin containing focal adhesions were greatly increased in the homozygous PTP-PEST mutant cells as compared with heterozygous cells. This phenomenon appears to be due in part to a constitutive increase in tyrosine phosphorylation of p130CAS, a known PTP-PEST substrate, paxillin, which associates with PTP-PEST in vitro, and focal adhesion kinase (FAK). Another effect of this constitutive hyperphosphorylation, consistent with the focal adhesion regulation defect, is that (−/−) cells spread faster than the control cell line when plated on fibronectin. In the PTP-PEST (−/−) cells, an increase in affinity for the SH2 domains of Src and Crk towards p130CAS was also observed. In (−/−) cells, we found a significant increase in the level of tyrosine phosphorylation of PSTPIP, a cleavage furrow–associated protein that interacts physically with all PEST family members. An effect of PSTPIP hyperphosphorylation appears to be that some cells remain attached at the site of the cleavage furrow for an extended period of time. In conclusion, our data suggest PTP-PEST plays a dual role in cell cytoskeleton organization, by promoting the turnover of focal adhesions required for cell migration, and by directly or indirectly regulating the proline, serine, threonine phosphatase interacting protein (PSTPIP) tyrosine phosphorylation level which may be involved in regulating cleavage furrow formation or disassembly during normal cell division.