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AssigneeDyax Corp.
InventorsJames P. Beltzer, M. Daniel Potter, Marilou Potter, Tony J. Fleming, Robert Charles Ladner
Estimating the term…
Binding polypeptides comprising specific amino acid sequences are disclosed that bind B Lymphocyte Stimulator (BLyS) protein or BLyS-like polypeptides. The binding polypeptides can be used in methods of the invention for detecting or isolating BLyS protein or BLyS-like polypeptides in solutions or mixtures, such as blood, tissue samples, or conditioned media.
A B Lymphocyte Stimulator (BLyS) binding polypeptide comprising the amino acid sequence:
A BLyS binding polypeptide comprising an amino acid sequence according to one of the following formulae:
A BLyS binding polypeptide comprising an amino acid sequence of the following formula:
A recombinant bacteriophage expressing exogenous DNA encoding a BLyS binding polypeptide comprising an amino acid sequence:
A recombinant bacteriophage expressing exogenous DNA encoding a BLyS binding polypeptide comprising an amino acid sequence of the formula:
A polynucleotide encoding a BLyS binding polypeptide comprising the amino acid sequence:
A polynucleotide encoding a BLyS binding polypeptide of the formula:
A BLyS affinity maturation library, comprising a population of at least 103 polypeptides, wherein the polypeptides of said population comprise the amino acid sequence:
A DNA template encoding a multiplicity of BLyS binding polypeptides, comprising the sequence:
Dependencies are inferred from the claim text.
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From animals; from humans: receptors; Cell surface antigens; Cell surface determinants
On carriers: simultaneous synthesis of different peptide species; Peptide libraries
Tetrapeptides: with the first amino acid being acidic
Linear peptides containing only normal peptide links: having 5 to 11 amino acids
Term end not estimated
The present invention relates to polypeptides that bind to B lymphocyte stimulator protein (BLyS). Such polypeptides have uses for example, in the detection, isolation, and/or purification of BLyS. The invention also relates to nucleic acid molecules encoding these BLyS binding polypeptides, vectors and host cells containing these nucleic acids, and methods for producing the same.
B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (“TNF”) superfamily that induces both in vivo and in vitro B cell proliferation and differentiation (Moore et al., Science, 285: 260-263 (1999)). BLyS is distinguishable from other B cell growth and differentiation factors such as IL-2, IL-4, IL-5, IL-6, IL-7, IL-13, IL-15, CD40L, or CD27L (CD70) by its monocyte-specific gene and protein expression pattern and its specific receptor distribution and biological activity on B lymphocytes. BLyS expression is not detected on natural killer (“NK”) cells, T cells or B cells, but is restricted to cells of myeloid origin. BLyS expression on resting monocytes is upregulated by interferon-gamma (IFN-gamma). The gene encoding BLyS has been mapped to chromosome 13q34.
BLyS is expressed as a 285 amino acid type II membrane-bound polypeptide and a soluble 152 amino acid polypeptide (Moore et al., 1999, supra). The membrane-bound form of BLyS has a predicted transmembrane spanning domain between amino acid residues 47 and 73. The NH2-terminus of the soluble form of BLyS begins at Ala134 of the membrane-bound form of BLyS. Both the soluble and membrane-bound forms of the protein form homotrimers. Soluble recombinant BLyS has been shown to induce in vitro proliferation of murine splenic B cells and to bind to a cell-surface receptor on these cells (Moore et al., 1999, supra). Soluble BLyS administration to mice has been shown to result in an increase in the proportion of CD45Rdull, Ly6Dbright (also known as ThB) B cells and an increase in serum IgM and IgA levels (Moore et al., 1999, supra). Thus, BLyS displays a B cell tropism in both its receptor distribution and biological activity.
Screening of Phage Display Libraries
Streptavidin-coated magnetic beads (Dynal M-280) were chosen for presentation of the target during screening because of their superior binding capacity compared to that of a 96 well plate. The binding capacity of the beads for biotinylated antibodies was 5-10 μg/mg of beads as stated by the manufacturer. For this study and the screening to follow, 5 μg of biotinylated recombinant BLyS (obtained from Human Genome Sciences, Inc.) was allowed for each mg of beads. This amount of biotinylated BLyS represents a 10-fold excess of target, for saturation of the beads. Unbound BLyS was washed away. Bound biotinylated BLyS was confirmed with detection using Mab 16C9 (murine anti-BLyS, Human Genome Sciences) primary antibody and a goat anti-mouse HRP conjugate as the secondary antibody. An irrelevant monoclonal antibody (anti-TNFα) was used to probe a second set of beads to control for nonspecific binding. The color reagent TMB was used and the assay read at OD 630 nm.
Nine libraries, TN6/6, TN7/4, TN8/9, TN9/4, TN10/9, TN12/1, Substrate Phage #2, PhD7, and PhD12, were screened for BLyS binders. The makeup of these libraries was as follows: