Signaling by the p55 tumor necrosis factor (TNF) receptor and by the structurally related receptor Fas/APO1 is initiated by receptor clustering. Data presented here and in other recent studies (Wallach, D., Boldin, M., Varfolomeev, E. E., Bigda, Y., Camonis, H.J. and Mett, I. (1994) Cytokine 6, 556; Song, H.Y., Dunbar, J.D., and Bonner, D.B. (1994) J. Biol. Chem. 269, 22492-22495) indicate that part of that region within the intracellular domains of the two receptors that is involved in signaling for cell death, as well as for some other effects (the "death domain", specifically self-associates. We demonstrate also the expected functional consequence of this association; a mere increase in p55 TNF receptor expression, or the expression just of its intracellular domain, is shown to trigger signaling for cytotoxicity as well as for interleukin 8 gene induction, while expression of the intracellular domain of Fas/APO1 potentiates the cytotoxicity of co-expressed p55 TNF receptor. These findings indicate that the p55 TNF and Fas/APO1 receptors play active roles in their own clustering and suggest the existence of cellular mechanisms that restrict the self-association of these receptors, thus preventing constitutive signaling.

The human combinatorial antibody library Fab 1 (Hu-CAL®-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wö lle, J., Plü ckthun, A., and Virnekä s, B. (2000) J. Mol. Biol. 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 ؋ 10 10 different antibody fragments. The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes. Hu-CAL®-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far. A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence. Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry. Some of the selected Fab fragments completely inhibit targetmediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity. This study validates HuCAL®-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications.

Achondroplasia, the most common genetic form of human dwarfism, results from a point mutation (G380R) in the gene for fibroblast growth factor receptor 3 (FGFR-3). Heterozygotes for the mutation share disproportionate, proximal shortening of the limbs, mid-face hypoplasia and relative macrocephaly due to a failure in endochondral ossification. Here we have generated transgenic mice expressing the human mutant FGFR-3 under the transcriptional control of the mouse gene. Mice that are hemizygous for the mutant human gene display disproportionate dwarfism with skeletal phenotypes remarkably similar to those of human achondroplasia. Mice that are homozygous for the transgene suffer from a profound delay in skeletal development and die at birth, similar in that respect to humans homozygous for the achondroplasia mutant gene. Microscopic analysis of long bones demonstrates growth plate morphology compatible with that of human achondroplasia cases, sharing endochondral growth inhibition with restrained chondrocyte proliferation and maturation, penetration of ossification tufts and aberrant vascularization.

The association of fibroblast growth factor receptor 3 (FGFR3) expression with t(4;14) multiple myeloma (MM) and the demonstration of the transforming potential of this receptor tyrosine kinase (RTK) make it a particularly attractive target for drug development. We report here a novel and highly specific anti-FGFR3-neutralizing antibody (PRO-001). PRO IntroductionTreatment of multiple myeloma (MM) has advanced over the past decade, resulting in prolongation of median survival from 3 to 5 years. Despite the improved outcome with treatment regimens that include dose intensification, patients invariably relapse, and MM remains a universally fatal disease. [1][2][3] Because the limits of current chemotherapy have been reached, new approaches to therapy are urgently required. Various studies have delineated fundamental genetic lesions in MM that affect well-defined oncogenic pathways, growth, and survival signaling cascades. 4,5 These key cellular and genetic pathogenic processes provide a framework to identify novel therapeutic targets.The t(4;14)(p16.3;q32) translocation, which occurs in approximately 15% to 20% of MM tumors, 6,7 results in the dysregulated expression of 2 putative oncogenes, MMSET and fibroblast growth factor receptor 3 (FGFR3). 8 FGFR3 belongs to a family of 5 receptors, FGFR1-5, 4 of which harbor a functional tyrosine kinase. The FGFRs are characterized by 2 to 3 immunoglobulin (Ig)-like extracellular domains that bind ligand, a hydrophobic transmembrane domain, and a cytoplasmic region that contains a split tyrosine kinase domain. 9 Binding of fibroblast growth factor (FGF) ligand and heparin promotes receptor dimerization and activation of the kinase domain, resulting in autophosphorylation of specific tyrosines. Activation of FGFRs transduces signals through mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) pathways, among others that regulate multiple cellular processes, including cell growth, differentiation, migration, and survival depending on the cellular context. 9,10 Studies indicate that FGFR3 may play a significant, albeit not a singular, role in myeloma oncogenesis, thus making this receptor tyrosine kinase (RTK) an attractive target for therapeutic intervention. Activation of wild-type (WT) FGFR3 promotes proliferation of myeloma cells and is weakly transforming in a hematopoietic mouse model. 11,12 Subsequent acquisition of activating mutations of FGFR3 in some MMs is associated with disease progression and is strongly transforming in several experimental models. 12,13 In vitro studies suggest that FGFR3 can impart chemoresistance 14 consistent with clinical data that demonstrate poor responses to conventional chemotherapy 15,16 and shortened median survival of t(4;14) MM patients. 15,17,18 The data have spurred the development of selective FGFR3 tyrosine kinase inhibitors for the potential treatment of MM. To date, several small-molecule inhibitors have been reported to induce cytotoxic responses of FGFR3-expressing myeloma cells. [19][20][21][22...

Human embryonic stem cells (HESCs) are unique in their capacity to self-renew while remaining pluripotent. This undifferentiated state must be actively maintained by secreted factors. To identify autocrine factors that may support HESC growth, we have taken a global genetic approach. Microarray analysis identified fibroblast growth factor 4 (FGF4) as a prime candidate for autocrine signaling. Furthermore, the addition of recombinant FGF4 to HESCs supports their proliferation. We show that FGF4 is produced by multiple undifferentiated HESC lines, along with a novel fibroblast growth factor 4 splice isoform (FGF4si) that codes for the amino-terminal half of FGF4. Strikingly, although FGF4 supports the undifferentiated growth of HESCs, FGF4si effectively counters its effect.Furthermore, we show that FGF4si is an antagonist of FGF4, shutting down FGF4-induced Erk1/2 phosphorylation. Expression analysis shows that both isoforms are expressed in HESCs and early differentiated cells. However, whereas FGF4 ceases to be expressed in mature differentiated cells, FGF4si continues to be expressed after cell differentiation. Targeted knockdown of FGF4 using small interfering RNA increased differentiation of HESCs, demonstrating the importance of endogenous FGF4 signaling in maintaining their pluripotency. Taken together, these results suggest a growthpromoting role for FGF4 in HESCs and a putative feedback inhibition mechanism by a novel FGF4 splice isoform that may serve to promote differentiation at later stages of development.