Enrique Escandon

The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor

Neurotrophic factors are essential for neuronal survival and function. Recent data have demonstrated that the product of the tyrosine kinase trk proto-oncogene binds NGF and is a component of the high affinity NGF receptor. Analysis of the trkB gene product, gp145trkB, in NIH 3T3 cells indicates that this tyrosine kinase receptor is rapidly phosphorylated on tyrosine residues upon exposure to the NGF-related neurotrophic factors BDNF and NT-3. Furthermore, gp145trkB specifically binds BDNF and NT-3 in NIH 3T3 cells and in hippocampal cells, but does not bind NGF. Thus, the trk family of receptors are likely to be important signal transducers of NGF-related trophic signals in the formation and maintenance of neuronal circuits.

The rat trkC locus encodes multiple neurogenic receptors that exhibit differential response to neurotrophin-3 in PC12 cells

Members of the Trk tyrosine kinase family have recently been identified as functional receptors of the NGF family of neurotrophins. Here we show the rat trkC locus to be complex, encoding at least four distinct polypeptides. Three of the encoded polypeptides are full-length receptor tyrosine kinases that differ by novel amino acid insertions in the kinase domain. A fourth protein is a truncated receptor that lacks the catalytic domain. Tyrosine phosphorylation, cross-linking, and ligand binding assays indicate that TrkC receptors interact with NT-3 and not with the related neurotrophins NGF, BDNF, xNT-4, or hNT-5. Furthermore, high and low affinity NT-3-binding sites are associated with the TrkC receptors. Stable and transient expression of TrkC receptors in PC12 cells indicates that the neurite outgrowth response elicited by NT-3 is dramatic in receptors lacking the novel kinase insert (gp150trkC) but absent in receptors containing the 14 amino acid insert in the kinase domain (gp150trkC14). These data suggest that the trkC locus encodes receptors that may be capable of mediating different biological responses within the cell. This could have important implications in understanding the role of neurotrophins in the development of the vertebrate nervous system.

The binding epitopes of neurotrophin-3 to its receptors trkC and gp75 and the design of a multifunctional human neurotrophin.

Survival and maintenance of vertebrate neurons are influenced by neurotrophic factors which mediate their signal by binding to specific cell surface receptors. We determined the binding sites of human neurotrophin‐3 (NT‐3) to its receptors trkC and gp75 by mutational analysis and compared them to the analogous interactions of nerve growth factor (NGF) with trkA and gp75. The trkC binding site extends around the central beta‐strand bundle and in contrast to NGF does not make use of non‐conserved loops and the six N‐terminal residues. The gp75 epitope is dominated by loop residues and the C‐terminus of NT‐3. A novel rapid biological screening procedure allowed the identification of NT‐3 mutants that are able to signal efficiently through the non‐preferred receptors trkA and trkB, which are specific for NGF and BDNF respectively. Mutation of only seven residues in NT‐3 resulted in a human neurotrophin variant which bound to all receptors of the trk family with high affinity and efficiently supported the survival of NGF‐, BDNF‐ and NT‐3‐dependent neurons. Our results suggest that the specificity among neurotrophic factors is not solely encoded in sequence diversity, but rather in the way each neurotrophin interacts with its preferred receptor.

TISSUE DISTRIBUTION, STABILITY, AND PHARMACOKINETICS OF APO2 LIGAND/TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND IN HUMAN COLON CARCINOMA COLO205 TUMOR-BEARING NUDE MICE

Apo2L/TRAIL [Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand], a member of the TNF cytokine superfamily, induces cell death by apoptosis in a number of human cancer cells and is a potential agent for cancer therapy. We have characterized the in vitro stability of Apo2L/TRAIL in human serum and the tissue distribution and metabolism of Apo2L/TRAIL in a xenograft model of human colon carcinoma (COLO205). Apo2L/TRAIL was stable after incubation in human serum, with no significant high molecular weight complexes or degradation products observed. After i.v. administration of 125I-Apo2L/TRAIL to mice, a small percentage of the radiolabeled drug was seen as high molecular weight complex or as low molecular weight degradation products in plasma. However, the most abundant radioactive species corresponded to the intact Apo2L/TRAIL monomer, indicative of the relative stability of this recombinant protein in blood. Distribution of 125I-Apo2L/TRAIL to organs and solid xenograft tumors was limited. Intact 125I-Apo2L/TRAIL was detectable in the solid tumor at all time points and was the only tissue in which radioactivity transiently increased over time. Kidney contained the highest levels of radioactivity. Radioactive signal reached a tissue-to-blood ratio of 18 in the kidney cortex region when 125I-Apo2L/TRAIL was given in the presence of excess unlabeled ligand. In contrast to blood, extensive 125I-Apo2L/TRAIL degradation was observed in the kidney and, to a lesser degree, in the solid tumor and other organs, including liver, spleen, and lung. Our studies demonstrated that Apo2L/TRAIL is stable in the circulation, localizes to human solid xenograft tumors, and is primarily eliminated through the kidney.

Enhanced tumor killing by Apo2L/TRAIL and CPT-11 co-treatment is associated with p21 cleavage and differential regulation of Apo2L/TRAIL ligand and its receptors

Apo2L/TRAIL exhibits enhanced apoptotic activity in tumor xenograft models when used in combination with the topoisomerase 1 inhibitor CPT-11. To investigate the cellular mechanisms involved in this increased tumor-killing activity, a series of in vitro experiments were conducted using the human colon carcinoma cell line (HCT116). Apo2L/TRAIL induced a transient upregulation of DR5 mRNA, while CPT-11 increased both death and decoy receptor expression. Upregulation of decoy receptors by CPT-11 was partially inhibited by co-administration of Apo2L/TRAIL. CPT-11 treatment resulted in accumulation of cells at G2M-phase and correlated with a substantial increase in the protein levels of the cyclin-dependent kinase inhibitor p21. However, cells co-treated with CPT-11 and Apo2L/TRAIL, or pretreated with CPT-11 for up to 24 h followed by 2 h Apo2L/TRAIL, resulted in a caspase-dependent degradation of p21, reversal of G2-M phase arrest with a concomitant increase in apoptosis. The sequential treatment produced the greatest induction of DR5 and DR4, caspase-3-like cleavage/activation and p21 degradation, as well as increased apoptosis. These data indicate that the up-regulation of Apo2L/TRAIL ligand and its death receptors as well as cleavage of p21 protein in the Apo2L/TRAIL plus CPT-11 treatment contributes to the positive cooperation between these agents in enhancing tumor cell apoptosis.

Effects of PEGylation and Immune Complex Formation on the Pharmacokinetics and Biodistribution of Recombinant Interleukin 10 in Mice

Interleukin 10 (IL-10) is a potent cytokine homodimer with multiple immunoregulatory functions. Here, we have characterized the effects of PEGylation and formation of human IL-10 (hIL-10)/humanized anti-human IL-10 (hαhIL-10) immune complexes in the pharmacokinetics, biodistribution, and biotransformation of IL-10 in mice. To assess the fate of native, PEGylated, and antibody-bound IL-10; we implemented an analytical set of fluorescence emission-linked assays. Plasma size exclusion chromatography analysis indicated that fluoro-labeled native and PEGylated murine IL-10 (PEG-mIL-10) are stable in the circulation. PEGylation of IL-10 resulted in a 21-fold increased exposure, 2.7-fold increase in half-life, and 20-fold reduction in clearance. Kidney is the major organ of disposition for both native and PEGylated mIL-10 with renal uptake directly related to systemic clearance. The fluorescence signal in the kidneys reached tissue/blood ratios up to 150 and 20 for native and PEG-mIL-10, respectively. hIL-10/hαhIL-10 immune complexes are detectable in the circulation without evidence of unbound or degraded hIL-10. The exposure of hIL-10 present in immune complexes versus that of hIL-10 alone increased from 0.53 to 11.28 μg · day/ml, with a half-life of 1.16 days and a 23-fold reduction in clearance. Unlike hIL-10 alone, antibody-bound hIL-10 was targeted mainly to the liver with minimal renal distribution. In addition, we found an 11-fold reduction (from 9.9 to 113 nM) in binding to the neonatal Fc receptor (FcRn) when the hαhIL10 antibody is conjugated to hIL-10. The potential changes in FcRn binding in vivo and increased liver uptake may explain the unique pharmacokinetic properties of hIL-10/hαhIL-10 immune complexes.