G-CSF analog compositions and methods
AssigneeAmgen Inc.
InventorTimothy David Osslund
Life of this patent
Estimating the term…
Abstract
The present invention relates to granulocyte colony-stimulating factor (“G-CSF”) hybrid molecules which retain the internal core helices of G-CSF. Also provided are pharmaceutical compositions containing hybrid molecules.
Claims59
A method for preparing a G-CSF analog comprising the steps of:
- (a) viewing information conveying the three dimensional structure of a G-CSF molecule;
- (b) selecting from said viewed information at least one site on said G-CSF molecule for alteration;
- (c) preparing a G-CSF molecule having such alteration;
- and
- (d) optionally, testing such G-CSF molecule for a desired characteristic.
A computer based method for preparing a G-CSF analog comprising the steps of:
- (a) providing computer expression of the three dimensional structure of a G-CSF molecule;
- (b) selecting from said computer expression at least one site on said G-CSF molecule for alteration;
- (c) preparing a G-CSF molecule having such alteration;
- and,
- (d) optionally, testing such G-CSF molecule for a desired characteristic.
A method for preparing a G-CSF analog with the aid of a computer comprising:
- (a) providing said computer with the means for displaying the three dimensional structure of a G-CSF molecule including displaying the composition of moieties of said G-CSF molecule, preferably displaying the three dimensional location of each amino acid, and more preferably displaying the three dimensional location of each atom of a G-CSF molecule;
- (b) viewing said display;
- (c) selecting a site on said display for alteration in the composition of said molecule or the location of a moiety;
- and
- (d) preparing a G-CSF analog with such alteration.
A computer-based method for preparing a G-CSF analog comprising the steps of:
- (a) viewing the three dimensional structure of a G-CSF molecule via a computer, said computer having been previously programmed (i) to express the coordinates of a G-CSF molecule in three dimensional space, and (ii) to allow for entry of information for alteration of said G-CSF expression and viewing thereof;
- (b) selecting a site on said visual image of said G-CSF molecule for alteration;
- (c) entering information for said alteration on said computer;
- (d) viewing a three dimensional structure of said altered G-CSF molecule via said computer;
- (e) optionally repeating steps (a)-(e) above;
- (f) preparing a G-CSF analog with said alteration;
- and
- (g) optionally testing said G-CSF analog for a desired characteristic.
In a computer-based apparatus for displaying the three dimensional structure of a molecule, the improvement comprising means for correlating said three dimensional structure of a G-CSF molecule with the composition of said G-CSF molecule.
A method for crystallization of a protein comprising the steps of:
- (a) combining, optionally by automated means, aqueous aliquots of said protein with either (i) aliquots of a salt solution, each aliquot having a different concentration of salt;
- or (ii) aliquots of a precipitant solution, each aliquot having a different concentration of precipitant;
- (b) selecting at least one of said combined aliquots, said selection based on the formation of precrystalline forms, or, if no precrystalline forms are so produced, increasing the protein starting concentration of said aqueous aliquots of protein and repeating step (a);
- (c) after said salt or said precipitant concentration is selected, repeating step (a) with said previously unselected solution in the presence of said selected concentration;
- and,
- (d) repeating step (b) and step (a) until a crystal of desired quality is obtained.
A G-CSF analog having an amino acid sequence different from that of FIG. 1 in that:
- (a) the N-terminal methionine is optional;
- and
- (b) one or more of amino acids 58-72 (i) is substituted with one or more different amino acids or (ii) deleted;
- or (iii) chemically modified.
A G-CSF analog having an amino acid sequence different from that of FIG. 1 in that:
- (a) the N-terminal methionine is optional;
- and
- (b) one or more of amino acids 119-125 (i) is substituted with one or more different amino acids or (ii) deleted;
- or (iii) chemically modified.
14 A G-CSF analog of claim 12 wherein at least one of said amino acids is chemically modified by the addition of a polyethylene glycol molecule.
A G-CSF molecule having the AB loop stabilized by connecting such loop to one or more of helices A, B, C, or D.
A G-CSF molecule having the CD loop stabilized by connecting such loop to one or more of helices A, B, C, or D.
A G-CSF analog, optionally in a pharmaceutically effective carrier, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Lys17->Arg17 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Lys35->Arg35 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Lys41->Arg41 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Lys17,24,35->Arg17,24,35 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Lys17,35,41->Arg17,35,41 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Lys24,35,41->Arg24,35,41 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Lys17,24,35,41->Arg17,24,35,41 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Lys17,24,41->Arg17,24,41 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Gln68->Glu68 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Cys37,43->Ser37,43 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Gln26->Ala26 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Gln174->Ala174 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Arg170->Ala170 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Arg167->Ala167 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that there is a deletion at position 167 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Lys41->Ala41 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that His44->Lys44 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Glu47->Ala47 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Arg23->Ala23 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Lys24->Ala24 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Glu20->Ala20 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Asp28->Ala28 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Met127->Glu127 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from tha of FIG. 1 in that Met138->Glu138 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Met127->Leu127 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Met138->Leu138 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Cys18->Ala18 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Gln12,21->Glu12,21 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Gln12,21,68->Glu12,21,68 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Glu20->Ala20;
- Ser13->Gly13 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Met127,138->Leu127,138 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Ser13->Ala13 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Lys17->Ala17 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Gln121->Ala121 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Gln21->Ala21 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that His44->Ala44 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein said amino acid sequenc differs from that of FIG. 1 in that His53->Ala53 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Asp110->Ala110 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Asp113->Ala113 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Thr117->Ala117 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Asp28->Ala28;
- Asp110->Ala110 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Glu124->Ala124 and the N-terminal methionine is optional.
A G-CSF analog, optionally in a pharmaceutically effective carrier, wherein the amino acid sequence differs from that of FIG. 1 in that Phe114->Val114, Thr117->A117 and the N-terminal methionine is optional.
Dependencies are inferred from the claim text.