Variant immunoglobulins, particularly humanized antibody polypeptides are provided, along with methods for their preparation and use. Consensus immunoglobulin sequences and structural models are also provided.

Method for making humanized antibodies

The DNA encoding an antibody with an altered function, e.g. altered affinity for an effector ligand such as Fc receptor (FcR) on a cell or the Cl component of complement is produced by replacing the nucleic acid encoding at least one amino acid residue in the constant portion of the antibody with nucleic acid encoding a different residue.

DNA encoding antibodies with altered effector functions

Publisher Summary The family of fibroblast growth factors (FGF) is the largest family of growth factors involved in soft–tissue growth and regeneration. The FGF family includes seven members that share a varying degree of homology at the protein level and appear to have a similar broad mitogenic spectrum. They are angiogenic and promote the proliferation of a variety of cells of mesodermal and neuroectodermal origin . Three members of the family–namely, K–FGF/HST, FGF–5, INT–2 are identified originally as oncogenes, while two additions, FGF–6 and keratinocyte growth factor (KGF), are isolated by sequence homology or factor purification and cloning. FGF was identified as an activity in pituitary extracts that stimulated the proliferation of 3T3 cells in mice. The two prototypes of basic and acidic protein structure, the FGF genes, and the expressions of FGF are also discussed. FGF consist of three exons, separated by two introns of variable length and FGF genes map on several chromosomes. The molecular regulation of FGF expression, FGF receptors, and their interaction with extracellular matrix is described. The oncogenic potential of FGF members and their involvement in tumors is also discussed. All possible mechanisms operating on the expression of FGFs and their receptors: transcriptional controls, posttranscriptional regulation involving alternative splicing, alternative translation starts resulting in proteins with different properties, and control affecting the secretion of these proteins are discussed.

The FGF family of growth factors and oncogenes.

A unique method is disclosed for identifying and replacing immunoglobulin surface amino acid residues which converts the antigenicity of a first mammalian species to that of a second mammalian species. The method will simultaneously change immunogenicity and strictly preserve ligind binding properties. The judicious replacement of exterior amino acid residues has no effect on the ligind binding properties but greatly alters immunogenicity.

Method for reducing the immunogenicity of antibody variable domains

Humanized antibodies and methods for making them

The fibroblast or heparin-binding growth factors (HBGFs) are thought to be modulators of cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. A better understanding of the structural basis for the different activities of these proteins should facilitate the development of agonists and antagonists of specific HBGF activities and identification of the signal transduction pathways involved in the mechanisms of action of these growth factors. Chemical modification studies of Harper and Lobb (Harper, J. W., and R. R. Lobb. 1988. Biochemistry. 27:671-678) implicated lysine 132 in HBGF-1 (acidic fibroblast growth factor) as being important to the heparin-binding, receptor-binding, and mitogenic activities of the protein. We changed lysine 132 to a glutamic acid residue by site-directed mutagenesis of the human cDNA and expressed the mutant protein in Escherichia coli to obtain sufficient quantities for functional studies. Replacement of this lysine with glutamic acid reduces the apparent affinity of HBGF-1 for immobilized heparin (elutes at 0.45 M NaCl vs. 1.1 M NaCl for wild-type). Mitogenic assays established two points: (a) human recombinant HBGF-1 is highly dependent on the presence of heparin for optimal mitogenic activity, and (b) the change of lysine 132 to glutamic acid drastically reduces the specific mitogenic activity of HBGF-1. The poor mitogenic activity of the mutant protein does not appear to be due to a reduced affinity for the HBGF receptor. Similarly, the mutant HBGF-1 can stimulate tyrosine kinase activity and induce protooncogene expression. Differences in the biological properties of the wild-type and mutant proteins were observed in transfection studies. Mutant HBGF-1 expression in transfected NIH 3T3 cells did not induce the same transformed phenotype characteristic of cells expressing wild-type HBGF-1. Together these data indicate that different functional properties of HBGF-1 may be dissociated at the structural level.

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Possible dissociation of the heparin-binding and mitogenic activities of heparin-binding (acidic fibroblast) growth factor-1 from its receptor-binding activities by site-directed mutagenesis of a single lysine residue.

The heparin-binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. Relatively little is known regarding the precise mechanism of action of these growth factors or the structural basis for their action. A better understanding of the structural basis for the different activities of these proteins should lead to the development of agonists and antagonists of specific HBGF-1 can be dissociated from the receptor-binding activities of the growth factor by site-directed mutagenesis of a single lysine residue. Thus, the mutant HBGF-1 has normal receptor-binding activity and is capable of stimulating tyrosine kinase activity and proto-oncogene expression but is not able to elicit a mitogenic response. A similar dissociation of early events such as proto-oncogene expression from the mitogenic response is observed when the human wild-type HBGF-1 is used in the absence of added heparin. These results indicate that intracellular sites of action by the growth factor may be required to complete the mitogenic response. Further evidence for this idea is provided by transfection experiments where NIH 3T3 cells are engineered to produce large quantities of wild-type or mutant HBGF-1. Production of wild-type induces a transformed phenotype, whereas over-production of the mutant does not. The majority of both forms of the protein is found in the nuclear fraction of the transfected cells. Additional site-directed mutagenesis of putative nuclear translocation sequences in the wild-type protein do not affect mitogenic activity. Thus, the role of nuclear translocation in the mechanisms of action of HBGF-1 remains unclear.

Structure-function studies of heparin-binding (acidic fibroblast) growth factor-1 using site-directed mutagenesis.

Regulated expression of PDGF A-chain mRNA in human saphenous vein smooth muscle cells

Platelet-derived growth factor (PDGF) is mitogenic and chemotactic for vascular smooth muscle cells cultured in vitro, and, thus, may play a role in the smooth muscle cell proliferation and migration that occurs during atherosclerotic lesion development. Two related PDGF polypeptides, designated as the A and B chains, form functionally active PDGF-AA, AB, or BB dimers. The PDGF A- and B-chain genes are both transcribed in human umbilical vein endothelial (HUVE) cells and their expression is regulated by cytokines, growth factors, endotoxin, and phorbol ester. We reported previously that the angiogenic polypeptide heparin-binding growth factor (HBGF)-1 induces PDGF A-chain gene expression, but does not affect PDGF B-chain gene expression. In this study, we determined whether mRNA stabilization contributed to this induction by measuring the half-life of PDGF A-chain mRNA in quiescent, HBGF-1-stimulated, and proliferating HUVE cells. PDGF A-chain mRNA levels increase when quiescent HUVE cells are treated with the protein synthesis inhibitor cycloheximide; therefore, the effect of cycloheximide on PDGF A-chain mRNA decay was also investigated. The half-life of PDGF A-chain transcripts in quiescent cells was approximately 2.4 h and neither HBGF-1 nor cycloheximide significantly altered this decay rate. We also estimated the half-life of PDGF B-chain mRNA under the three different growth conditions and in the absence or presence of cycloheximide. The half-life in quiescent cells was approximately 1.8 h and was unaffected by HBGF-1 or protein synthesis inhibition. Therefore, the PDGF mRNAs have similar decay rates in HUVE cells, even though the 3' untranslated region of B-chain transcripts, but not A-chain transcripts, contains AU-rich sequence motifs postulated to confer rapid turnover in vivo.

The half-lives of platelet-derived growth factor A- and B-chain mRNAs are similar in endothelial cells and unaffected by heparin-binding growth factor-1 or cycloheximide.

The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet-derived growth factor (PDGF) A-chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson-Gilford (progeria) syndrome fibroblasts, AG3513, constitutively expresses PDGF A-chain mRNA and PDGF-AA homodimers. The PDGF A-chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have properties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AG3513 progeria fibroblasts to PDGF stimulation was examined in some detail. Studies using 125I-PDGF-BB, which binds with high affinity to both A- and B-type PDGF receptors, indicate that normal and AG3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF-stimulated AG3513 progeria fibroblasts, c-fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts is probably unrelated to their constitutive PDGF A-chain gene expression; further studies are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogen-inducible gene expression in normal cells.

Jeffrey A. Winkles

Papers

Possible dissociation of the heparin-binding and mitogenic activities of heparin-binding (acidic fibroblast) growth factor-1 from its receptor-binding activities by site-directed mutagenesis of a single lysine residue.

Structure-function studies of heparin-binding (acidic fibroblast) growth factor-1 using site-directed mutagenesis.

Regulated expression of PDGF A-chain mRNA in human saphenous vein smooth muscle cells

The half-lives of platelet-derived growth factor A- and B-chain mRNAs are similar in endothelial cells and unaffected by heparin-binding growth factor-1 or cycloheximide.

Altered regulation of platelet-derived growth factor A-chain and c-fos gene expression in senescent progeria fibroblasts.