A method of preparing a modified granulocyte colony stimulating factor (G-CSF) protein having reduced immunogenicity relative to human G-CSF comprises the steps of (i) identifying one or more potential T-cell epitopes within the amino acid sequence of human G-CSF (SEQ ID NO: 1); (ii) designing at least one sequence variant of at least one potential T-cell epitope identified in step (i), wherein the sequence variant eliminates or substantially reduces the MHC class II binding activity of the potential T-cell epitope; (iii) preparing, by recombinant DNA techniques, at least one modified G-CSF protein including a sequence variant designed in step (ii); (iv) evaluating at least one modified G-CSF protein prepared in step (iii) for G-CSF activity and immunogenicity; and (v) selecting a modified G-CSF protein evaluated in step (iv) that has substantially the same therapeutic G-CSF biological activity as, but substantially less immunogenicity than, human G-CSF.

Description of the Invention

SUMMARY AND DESCRIPTION OF THE INVENTION

The present invention provides for modified forms of "granulocyte colony stimulating factor (G-CSF)", in which the immune characteristic is modified by means of reduced or removed numbers of potential T-cell epitopes. The present invention provides for modified forms of human G-CSF with one or more T-cell epitopes removed. The invention discloses sequences identified within the G-CSF primary sequence that are potential T-cell epitopes by virtue of MHC class II binding potential. This disclosure specifically pertains to both recognized forms of the human G-CSF protein being the 177 amino acid species and the 174 amino acid species.

The invention may be applied to any G-CSF species of molecule with substantially the same primary amino acid sequences as those disclosed herein and would include therefore G-CSF molecules derived by genetic engineering means or other processes and may not contain either 177 or 174 amino acid residues.

G-CSF proteins such as identified from murine, bovine, canine and other mammalian sources have in common many of the peptide sequences of the present disclosure and have in common many peptide sequences with substantially the same sequence as those of the disclosed listing. Such protein sequences equally therefore fall under the scope of the present invention.

The invention discloses also specific positions within the primary sequence of the molecule according to the invention which has to be altered by specific amino acid substitution, addition or deletion without affecting the biological activity in principal. In cases in which the loss of immunogenicity can be achieved only by a simultaneous loss of biological activity it is possible to restore said activity by further alterations within the amino acid sequence of the protein.

The invention discloses furthermore methods to produce such modified molecules, above all methods to identify said T-cell epitopes which have to be altered in order to reduce or remove immunogenetic sites.

The protein according to this invention would expect to display an increased circulation time within the human subject and would be of particular benefit in chronic or recurring disease settings such as is the case for a number of indications for granulocyte colony stimulating factor (G-CSF). The present invention provides for modified forms of G-CSF proteins that are expected to display enhanced properties in vivo. These modified G-CSF molecules can be used in pharmaceutical compositions.

In summary the invention relates to the following issues: a modified molecule having the biological activity of human granulocyte colony stimulating factor (G-CSF) and being substantially non-immunogenic or less immunogenic than any non-modified molecule having the same biological activity when used in vivo; an accordingly specified molecule, wherein said loss of immunogenicity is achieved by removing one or more T-cell epitopes derived from the originally non-modified molecule; an accordingly specified molecule, wherein said loss of immunogenicity is achieved by reduction in numbers of MHC allotypes able to bind peptides derived from said molecule; an accordingly specified molecule, wherein one T-cell epitope is removed; an accordingly specified molecule, wherein said originally present T-cell epitopes are MHC class II ligands or peptide sequences which show the ability to stimulate or bind T-cells via presentation on class II; an accordingly specified molecule, wherein said peptide sequences are selected from the group as depicted in Table 1 (see Original Patent); an accordingly specified molecule, wherein 1-9 amino acid residues, preferably one amino acid residue in any of the originally present T-cell epitopes are altered; an accordingly specified molecule, wherein the alteration of the amino acid residues is substitution, addition or deletion of originally present amino acid(s) residue(s) by other amino acid residue(s) at specific position(s); an accordingly specified molecule, wherein one or more of the amino acid residue substitutions are carried out as indicated in Table 2 (see Original Patent); an accordingly specified molecule, wherein (additionally) one or more of the amino acid residue substitutions are carried out as indicated in Table 3 (see Original Patent) for the reduction in the number of MHC allotypes able to bind peptides derived from said molecule; an accordingly specified molecule, wherein, if necessary, additionally further alteration usually by substitution, addition or deletion of specific amino acid(s) is conducted to restore biological activity of said molecule; A DNA sequence or molecule which codes for any of the modified molecules as specified above and below; a pharmaceutical composition comprising a modified molecule having the biological activity of granulocyte colony stimulating factor (G-CSF) as defined above and/or in the claims, optionally together with a pharmaceutically acceptable carrier, diluent or excipient; a method for manufacturing a modified molecule having the biological activity of granulocyte colony stimulating factor (G-CSF) as defined in any of the claims of the above-cited claims comprising the following steps: (i) determining the amino acid sequence of the polypeptide or part thereof; (ii) identifying one or more potential T-cell epitopes within the amino acid sequence of the protein by any method including determination of the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays; (iii) designing new sequence variants with one or more amino acids within the identified potential T-cell epitopes modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope as determined by the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays; (iv) constructing such sequence variants by recombinant DNA techniques and testing said variants in order to identify one or more variants with desirable properties; and (v) optionally repeating steps (ii)-(iv); an accordingly specified method, wherein step (iii) is carried out by substitution, addition or deletion of 1-9 amino acid residues in any of the originally present T-cell epitopes; an accordingly specified method, wherein the alteration is made with reference to a homologues protein sequence and/or in silico modeling techniques; an accordingly specified method, wherein step (ii) of above is carried out by the following steps: (a) selecting a region of the peptide having a known amino acid residue sequence; (b) sequentially sampling overlapping amino acid residue segments of predetermined uniform size and constituted by at least three amino acid residues from the selected region; (c) calculating MHC Class II molecule binding score for each said sampled segment by summing assigned values for each hydrophobic amino acid residue side chain present in said sampled amino acid residue segment; and (d) identifying at least one of said segments suitable for modification, based on the calculated MHC Class II molecule binding score for that segment, to change overall MHC Class II binding score for the peptide without substantially reducing therapeutic utility of the peptide; step (c) is preferably carried out by using a Bohm scoring function modified to include 12-6 van der Waal's ligand-protein energy repulsive term and ligand conformational energy term by (1) providing a first data base of MHC Class II molecule models; (2) providing a second data base of allowed peptide backbones for said MHC Class II molecule models; (3) selecting a model from said first data base; (4) selecting an allowed peptide backbone from said second data base; (5) identifying amino acid residue side chains present in each sampled segment; (6) determining the binding affinity value for all side chains present in each sampled segment; and repeating steps (1) through (5) for each said model and each said backbone; a 13 mer T-cell epitope peptide having a potential MHC class II binding activity and created from immunogenetically non-modified granulocyte colony stimulating factor (G-CSF), selected from the group as depicted in Table 1 and its use for the manufacture of G-CSF having substantially no or less immunogenicity than any non-modified molecule with the same biological activity when used in vivo; a peptide sequence consisting of at least 9 consecutive amino acid residues of a 13 mer T-cell epitope peptide as specified above and its use for the manufacture of G-CSF having substantially no or less immunogenicity than any non-modified molecule with the same biological activity when used in vivo;

The term "T-cell epitope" means according to the understanding of this invention an amino acid sequence which is able to bind MCH II, able to stimulate T-cells and/or also to bind (without necessarily measurably activating) T-cells in complex with MHC II. The term "peptide" as used herein and in the appended claims, is a compound that includes two or more amino acids. The amino acids are linked together by a peptide bond (defined herein below). There are 20 different naturally occurring amino acids involved int eh biological production of peptides, and any number of them may be linked in any order to form a peptide chain or ring. The naturally occurring amino acids employed in the biological production of peptides all have the L-configuration. Synthetic peptides can be prepared employing conventional synthetic methods, utilizing L-amino acids, D-amino acids, or various combinations of amino acids of the two different configurations. Some peptides contain only a few amino acid units. Short peptides, e.g., having less than ten amino acid units, are sometimes referred to as "oligopeptides". Other peptides contain a large number of amino acid residues, e.g. up to 100 ore more, and are referred to as "polypeptides". By convention, a "polypeptide" may be considered as any peptide chain containing three or more amino acids, whereas a "oligopeptide" is usually considered as a particular type of "short" polypeptide. Thus, as used herein, it is understood that any reference to a "polypeptide" also includes an oligopeptide. Further, any reference to a "peptide" includes polypeptides, oligopeptides, and proteins. Each different arrangement of amino acids forms different polypeptides or proteins. The number of polypeptides--and hence the number of different proteins--that can be formed is practically unlimited. "Alpha carbon (C.alpha.)" is the carbon atom of the carbon-hydrogen (CH) component that is in the peptide chain. A "side chain" is a pendant group to C.alpha. that can comprise a simple or complex group or moiety, having physical dimensions that can vary significantly compared to the dimensions of the peptide.

The invention may be applied to any G-CSF species of molecule with substantially the same primary amino acid sequences as those disclosed herein and would include therefore G-CSF molecules derived by genetic engineering means or other processes and may not contain either 177 or 174 amino acid residues. granulocyte colony stimulating factor (G-CSF) proteins such as identified from other mammalian sources have in common many of the peptide sequences of the present disclosure and have in common many peptide sequences with substantially the same sequence as those of the disclosed listing. Such protein sequences equally therefore fall under the scope of the present invention.

The invention is conceived to overcome the practical reality that soluble proteins introduced into autologous organisms can trigger an immune response resulting in development of host antibodies that bind to the soluble protein. One example amongst others, is interferon alpha 2 to which a proportion of human patients make antibodies despite the fact that this protein is produced endogenously [Russo, D. et al (1996) ibid; Stein, R. et al (1988) ibid]. It is likely that the same situation pertains to the therapeutic use of granulocyte colony stimulating factor (G-CSF) and the present invention seeks to address this by providing granulocyte colony stimulating factor (G-CSF) proteins with altered propensity to elicit an immune response on administration to the human host.

The general method of the present invention leading to the modified granulocyte colony stimulating factor (G-CSF) comprises the following steps: (a) determining the amino acid sequence of the polypeptide or part thereof; (b) identifying one or more potential T-cell epitopes within the amino acid sequence of the protein by any method including determination of the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays; (c) designing new sequence variants with one or more amino acids within the identified potential T-cell epitopes modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope as determined by the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays. Such sequence variants are created in such a way to avoid creation of new potential T-cell epitopes by the sequence variations unless such new potential T-cell epitopes are, in turn, modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope; and (d) constructing such sequence variants by recombinant DNA techniques and testing said variants in order to identify one or more variants with desirable properties according to well known recombinant techniques.

The identification of potential T-cell epitopes according to step (b) can be carried out according to methods describes previously in the prior art. Suitable methods are disclosed in WO 98/59244; WO 98/52976; WO 00/34317 and may preferably be used to identify binding propensity of granulocyte colony stimulating factor (G-CSF)-derived peptides to an MHC class II molecule.

Another very efficacious method for identifying T-cell epitopes by calculation is described in the EXAMPLE which is a preferred embodiment according to this invention.

In practice a number of variant granulocyte colony stimulating factor (G-CSF) proteins will be produced and tested for the desired immune and functional characteristic. The variant proteins will most preferably be produced by recombinant DNA techniques although other procedures including chemical synthesis of granulocyte colony stimulating factor (G-CSF) fragments may be contemplated.

The results of an analysis according to step (b) of the above scheme and pertaining to the whole human G-CSF protein sequences of both the 174 and 177 forms is presented in Table 1 (see Original Patent).

The invention relates to granulocyte colony stimulating factor (G-CSF) analogues in which substitutions of at least one amino acid residue have been made at positions resulting in a substantial reduction in activity of or elimination of one or more potential T-cell epitopes from the protein. One or more amino acid substitutions at particular points within any of the potential MHC class II ligands identified in Table 1 may result in a granulocyte colony stimulating factor (G-CSF) molecule with a reduced immunogenic potential when administered as a therapeutic to the human host. Preferably, amino acid substitutions are made at appropriate points within the peptide sequence predicted to achieve substantial reduction or elimination of the activity of the T-cell epitope. In practice an appropriate point will preferably equate to an amino acid residue binding within one of the hydrophobic pockets provided within the MHC class II binding groove.

It is most preferred to alter binding within the first pocket of the cleft at the so-called P1 or P1 anchor position of the peptide. The quality of binding interaction between the P1 anchor residue of the peptide and the first pocket of the MHC class II binding groove is recognized as being a major determinant of overall binding affinity for the whole peptide. An appropriate substitution at this position of the peptide will be for a residue less readily accommodated within the pocket, for example, substitution to a more hydrophilic residue. Amino acid residues in the peptide at positions equating to binding within other pocket regions within the MHC binding cleft are also considered and fall under the scope of the present.

It is understood that single amino acid substitutions within a given potential T-cell epitope are the most preferred route by which the epitope may be eliminated. Combinations of substitution within a single epitope may be contemplated and for example can be particularly appropriate where individually defined epitopes are in overlap with each other. Moreover, amino acid substitutions either singly within a given epitope or in combination within a single epitope may be made at positions not equating to the "pocket residues" with respect to the MHC class II binding groove, but at any point within the peptide sequence. Substitutions may be made with reference to an homologues structure or structural method produced using in silico techniques known in the art and may be based on known structural features of the molecule according to this invention. All such substitutions fall within the scope of the present invention.

Amino acid substitutions other than within the peptides identified above may be contemplated particularly when made in combination with substitution(s) made within a listed peptide. For example a change may be contemplated to restore structure or biological activity of the variant molecule. Such compensatory changes and changes to include deletion or addition of particular amino acid residues from the granulocyte colony stimulating factor (G-CSF) polypeptide resulting in a variant with desired activity and in combination with changes in any of the disclosed peptides fall under the scope of the present.

In as far as this invention relates to modified granulocyte colony stimulating factor (G-CSF), compositions containing such modified G-CSF proteins or fragments of modified G-CSF proteins and related compositions should be considered within the scope of the invention. In another aspect, the present invention relates to nucleic acids encoding modified granulocyte colony stimulating factor (G-CSF) entities. In a further aspect the present invention relates to methods for therapeutic treatment of humans using the modified G-CSF proteins.

 

Claim 1 of 1 Claim

1. A method of preparing a modified granulocyte colony stimulating factor (G-CSF) protein comprising the steps of: (i) identifying one or more potential T-cell epitopes within the amino acid sequence of human G-CSF (SEQ ID NO: 1); (ii) designing at least one sequence variant of at least one potential T-cell epitope identified in step (i), wherein the sequence variant eliminates or substantially reduces the MHC class II binding activity of the potential T-cell epitope; (iii) preparing, by recombinant DNA techniques, at least one modified G-CSF protein including a sequence variant designed in step (ii), the amino acid sequence of the modified G-CSF consisting of SEQ ID NO: 1 with 1 to 9 amino acid substitutions or deletions therein or additions thereto, wherein substitutions are selected from the group of amino acid substitutions set forth in Table 2 and Table 3; (iv) evaluating at least one modified G-CSF protein prepared in step (iii) for therapeutic G-CSF biological activity and immunogenicity; and (v) selecting a modified G-CSF protein evaluated in step (iv) that has substantially the same therapeutic G-CSF biological activity as human G-CFS, but substantially less immunogenicity than human G-CSF; wherein step (i) is carried out by: (a) selecting a region of human G-CSF having a known amino acid sequence; (b) sequentially sampling overlapping amino acid residue segments of predetermined uniform size, and including at least three amino acid residues, from the selected region; and (c) calculating a MHC class II binding score for each sequentially sampled amino acid residue segment by summing assigned values for each hydrophobic amino acid residue side chain present in each sequentially sampled amino acid residue segment, and thereby obtaining a calculated MHC class II binding score therefor; step (ii) is carried out by: (d) identifying a desired segment from among the sequentially sampled amino acid residue segments that is suitable for modification, based on the calculated MHC class II binding score therefor; (e) calculating MHC class II binding scores for sequence variants of the desired segment; (f) selecting from said sequence variants a sequence variant that has a lower MHC class II binding score than the MHC class II binding score of the desired segment; and step (c) is carried out using a modified Bohm scoring function including 12-6 vander Waal's ligand-protien energy repulsive terms (1) selecting a model from a first database of MHC class II molecule models; (2) selecting an allowed peptide backbone from a second database of allowed peptide backbones for the MHC class II molecule models in step (1); (3) identifying amino acid residue side chains present in each sampled segment; (4) determining a binding affinity value for all side chains present in each sampled segment; and (5) repeating each of (1) through (4) for each model in the first database and for each backbone in the second database.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.