ZA200306930B - Modified erythropoietin (EPO) with reduced immunogenecity. - Google Patents
Modified erythropoietin (EPO) with reduced immunogenecity. Download PDFInfo
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Description
v MODIFIED ERYTHROPOIETIN (EPO) WITH REDUCED
IMMUNOGENICITY
The present invention relates to polypeptides to be administered especially to humans and ‘ in particular for therapeutic use. The polypeptides are modified polypeptides whereby the modification results in a reduced propensity for the polypeptide to elicit an immune response upon administration to the human subject. The invention in particular relates to the modification of human erythropoietin (EPO) to result in erythropoietin protein variants that are substantially non-immunogenic or less immunogenic than any non- modified counterpart when used in vivo. The invention relates furthermore to T-cell epitope peptides derived from said non-modified protein by means of which it is possible to create modified erythropoietin variants with reduced immunogenicity.
There are many instances whereby the efficacy of a therapeutic protein is limited by an unwanted immune reaction to the therapeutic protein. Several mouse monoclonal antibodies have shown promise as therapies in a number of human disease settings but in certain cases have failed due to the induction of significant degrees of a human anti- murine antibody (HAMA) response [Schroff, R. W. et al (1985) Cancer Res. 45: 879-885;
Shawler, D.L. et al (1985) J. Immunol. 135: 1530-1535]. For monoclonal antibodies, a number of techniques have been developed in attempt to reduce the HAMA response [WO 89/09622; EP 0239400; EP 0438310; WO 91/06667]. These recombinant DNA approaches have generally reduced the mouse genetic information in the final antibody construct whilst increasing the human genetic information in the final construct.
Notwithstanding, the resultant “humanized” antibodies have, in several cases, still elicited an immune response in patients [Issacs J.D. (1990) Sem. Immunol. 2: 449, 456; Rebello,
P.R. et al (1999) Transplantation 63: 1417-1420].
Antibodies are not the only class of polypeptide molecule administered as a therapeutic agent against which an immune response may be mounted. Even proteins of human origin and with the same amino acid sequences as occur within humans can still induce an immune response in humans. Notable examples include the therapeutic use of granulocyte-macrophage colony stimulating factor [Wadhwa, M. et al (1999) Clin.
CONFIRMATION COPY
Cancer Res. 5: 1353-1361] and interferon alpha 2 [Russo, D. et al (1996) Bri. J. Haem. 94: 300-305; Stein, R. et al (1988) New Engl. J. Med. 318: 1409-1413] amongst others.
A principal factor in the induction of an immune response is the presence within the . protein of peptides that can stimulate the activity of T-cell via presentation on MHC class
II molecules, so-called "T-cell epitopes. Such potential T-cell epitopes are commonly ' defined as any amino acid residue sequence with the ability to bind to MHC Class II molecules. Such T-cell epitopes can be measured to establish MHC binding. Implicitly, a "T-cell epitope” means an epitope which when bound to MHC molecules can be recognized by a T-cell receptor (TCR), and which can, at least in principle, cause the activation of these T-cells by engaging a TCR to promote a T-cell response. It is, however, usually understood that certain peptides which are found to bind to MHC Class
II molecules may be retained in a protein sequence because such peptides are recognized as "self" within the organism into which the final protein is administered.
It is known, that certain of these T-cell epitope peptides can be released during the degradation of peptides, polypeptides or proteins within cells and subsequently be presented by molecules of the major histocompatability complex (MHC) in order to trigger the activation of T-cells. For peptides presented by MHC Class II, such activation of T-cells can then give rise, for example, to an antibody response by direct stimulation of
B-cells to produce such antibodies.
MHC Class II molecules are a group of highly polymorphic proteins which play a central role in helper T-cell selection and activation. The human leukocyte antigen group DR (HLA-DR) are the predominant isotype of this group of proteins and are the major focus of the present invention. However, isotypes HLA-DQ and HLA-DP perform similar functions, hence the resent invention is equally applicable to these. The MHC class II DR molecule is made of an alpha and a beta chain which insert at their C-termini through the cell membrane. Each hetero-dimer possesses a ligand binding domain which binds to : peptides varying between 9 and 20 amino acids in length, although the binding groove can accommodate a maximum of 11 amino acids. The ligand binding domain is FT comprised of amino acids 1 to 85 of the alpha chain, and amino acids 1 to 94 of the beta chain. DQ molecules have recently been shown to have an homologous structure and the
DP family proteins are also expected to be very similar. In humans approximately 70
: different allotypes of the DR isotype are known, for DQ there are 30 different allotypes and for DP 47 different allotypes are known. Each individual bears two to four DR alleles, two DQ and two DP alleles. The structure of a number of DR molecules has been . solved and such structures point to an open-ended peptide binding groove with a number ofhydrophobic pockets which engage hydrophobic residues (pocket residues) of the ‘ peptide [Brown et al Nature (1993) 364: 33; Stern et al (1994) Nature 368: 215].
Polymorphism identifying the different allotypes of class II molecule contributes to a wide diversity of different binding surfaces for peptides within the peptide binding grove and at the population level ensures maximal flexibility with regard to the ability to recognize foreign proteins and mount an immune response to pathogenic organisms.
There is a considerable amount of polymorphism within the ligand binding domain with distinct "families" within different geographical populations and ethnic groups. This polymorphism affects the binding characteristics of the peptide binding domain, thus different "families" of DR molecules will have specificities for peptides with different sequence properties, although there may be some overlap. This specificity determines 2 recognition of Th-cell epitopes (Class II T-cell response) which are ultimately responsible 7 for driving the antibody response to B-cell epitopes present on the same protein from g which the Th-cell epitope is derived. Thus, the immune response to a protein in an i individual is heavily influenced by T-cell epitope recognition which is a function of the ‘ 20 peptide binding specificity of that individual’s HLA-DR allotype. Therefore, in order to identify T-cell epitopes within a protein or peptide in the context of a global population, it is desirable to consider the binding properties of as diverse a set of HLA-DR allotypes as possible, thus covering as high a percentage of the world population as possible.
An immune response to a therapeutic protein such as the protein which is object of this invention, proceeds via the MHC class II peptide presentation pathway. Here exogenous proteins are engulfed and processed for presentation in association with MHC class II molecules of the DR, DQ or DP type. MHC Class II molecules are expressed by ¢ professional antigen presenting cells (APCs), such as macrophages and dendritic cells amongst others. Engagement of a MHC class II peptide complex by a cognate T-cell ) receptor on the surface of the T-cell, together with the cross-binding of certain other co- receptors such as the CD4 molecule, can induce an activated state within the T-cell.
Activation leads to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
The ability of a peptide to bind a given MHC class II molecule for presentation on the surface of an APC is dependent on a number of factors most notably its primary sequence. This will influence both its propensity for proteolytic cleavage and also its affinity for binding within the peptide binding cleft of the MHC class II molecule. The ,
MHC class II / peptide complex on the APC surface presents a binding face to a particular
T-cell receptor (TCR) able to recognize determinants provided both by exposed residues ' of the peptide and the MHC class II molecule.
In the art there are procedures for identifying synthetic peptides able to bind MHC class IT molecules (e.g. WO98/52976 and W0O00/34317). Such peptides may not function as T- cell epitopes in all situations, particularly, in vivo due to the processing pathways or other phenomena. T-cell epitope identification is the first step to epitope elimination. The identification and removal of potential T-cell epitopes from proteins has been previously disclosed. In the art methods have been provided to enable the detection of T-cell epitopes usually by computational means scanning for recognized sequence motifs in experimentally determined T-cell epitopes or alternatively using computational techniques to predict MHC class II-binding peptides and in particular DR-binding : peptides.
WO98/52976 and WO00/34317 teach computational threading approaches to identifying polypeptide sequences with the potential to bind a sub-set of human MHC class II DR allotypes. In these teachings, predicted T-cell epitopes are removed by the use of judicious amino acid substitution within the primary sequence of the therapeutic antibody or non-antibody protein of both non-human and human derivation.
Other techniques exploiting soluble complexes of recombinant MHC molecules in combination with synthetic peptides and able to bind to T-cell clones from peripheral blood samples from human or experimental animal subjects have been used in the art [Kern, F. et al (1998) Nature Medicine 4:975-978; Kwok, W.W. et al (2001) TRENDS in
Immunology 22: 583-588] and may also be exploited in an epitope identification strategy. )
As depicted above and as consequence thereof, it would be desirable to identify and to remove or at least to reduce T-cell epitopes from a given in principal therapeutically valuable but originally immunogenic peptide, polypeptide or protein.
+» WO 02/062843 - 5 - PCT/EP02/01174 ’ One of these therapeutically valuable molecules is EPO. EPO is a glycoprotein hormone involved in the maturation of erythroid progenitor cells into erythrocytes. Naturally occurring EPO is produced by the liver during foetal life and by the kidney of adults and . circulates in the blood to stimulate production of red blood cells in bone marrow.
Anaemia is almost invariably a consequence of renal failure due to decreased production * of EPO from the kidney. Recombinant EPO is used as an effective treatment of anaemia resulting from chronic renal failure.
Recombinant EPO (expressed in mammalian cells) having the amino acid sequence 1-165 of human erythropoietin [Jacobs, K. et al (1985) Nature, 313: 806-810; Lin, F.-K. et al (1985) Proc. Natl. Acad. Sci. U.S.A. 82:7580-7585] contains three N-linked and one O- linked oligosaccharide chains each containing terminal sialic acid residues. The latter are significant in enabling EPO to evade rapid clearance from the circulation by the hepatic asialoglycoprotein binding protein.
The amino acid sequence of erythropoietin (EPO) (depicted as one-letter code) is as follows:
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQOQAVEVWQ
GLALLSEAVLRGOALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAIS PPDAASAAPL
RTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
Others have provides modified EPO molecules [e.g. US 5,856,298; US 5,955,422] but it is understood that these approaches have been directed towards improvements in the commercial production of EPO and approaches towards influencing the glycosylation status of the protein and particularly the recombinant form. None of these teachings recognize the importance of T-cell epitopes to the immunogenic properties of the protein nor have been conceived to directly influence said properties in a specific and controlled way according to the scheme of the present invention. io Despite the availability of commercial quantities of recombinant EPO, there is a continued need for erythropoietin (EPO) analogues with enhanced properties. Desired * enhancements include alternative schemes and modalities for the expression and purification of the said therapeutic, but also and especially, improvements in the biological properties of the protein. There is a particular need for enhancement of the in vivo characteristics when administered to the human subject. In this regard, it is highly desired to provide erythropoietin (EPO) with reduced or absent potential to induce an immune response in the human subject.
The present invention provides for modified forms of "erythropoietin", 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 erythropioetin with one or more T-cell epitopes removed.
The invention discloses sequences identified within the erythropoietin (EPO) primary sequence that are potential T-cell epitopes by virtue of MHC class II binding potential.
This disclosure specifically pertains the human erythropoietin (EPO) protein being the 165 amino acid residues.
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 erythropoietin (EPO).
The present invention provides for modified forms of EPO proteins that are expected to display enhanced properties in vivo. These modified erythropoietin molecules can be used in pharmaceutical compositions.
In summary the invention relates to the following issues: , e amodified molecule having the biological activity of erythropoietin (EPO) and being substantially non-immuno genic 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; e an accordingly specified molecule, wherein one T-cell epitope is removed; e 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; e an accordingly specified molecule, wherein said peptide sequences are selected from the group as depicted in Table 1; e 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; e an accordingly specified molecule, wherein (additionally) one or more of the amino acid residue substitutions are carried out as indicated in Table 3 for the reduction in the number of MHC allotypes able to bind peptides derived from said molecule; e 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 any of the modified molecules as defined; a pharmaceutical composition comprising a modified molecule having the biological . activity of erythropoietin (EPO) as defined above and / or in the claims, optionally together with a pharmaceutically acceptable carrier, diluent or excipient; v 30 eo amethod for manufacturing a modified molecule having the biological activity of erythropoietin (EPO) 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 ir 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 (Vv) optionally repeating steps (ii) — (iv); an accordingly specified method, wherein step (iil) 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; e 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; e a 13mer T-cell epitope peptide having a potential MHC class II binding activity and created from immunogenetically non-modified erythropoietin (EPO), selected from the
K WO 02/062843 - 9 - PCT/EP02/01174 } group as depicted in Table 1 and its use for the manufacture of erythropoietin (EPO) having substantially no or less immunogenicity than any non-modified molecule with the same biological activity when used in vivo;
R e a peptide sequence consisting of at least 9 consecutive amino acid residues of a 13mer
T-cell epitope peptide as specified above and its use for the manufacture of d erythropoietin (EPO) 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, €.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 (Ca)" is the carbon atom of the carbon-hydrogen (CH) component that is ’ in the peptide chain. A "side chain" is a pendant group to Co. 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 erythropoietin (EPO) species of molecule with substantially the same primary amino acid sequences as those disclosed herein and would include therefore erythropoietin (EPO) molecules derived by genetic engineering means or other processes and may not contain either 165 amino acid residues. . erythropoietin (EPO) 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 erythropoietin (EPO) and the present invention seeks to address this by providing erythropoietin 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 erythropoietin (EPO) comprises the following steps: (2) 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.
i 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 erythropoietin (EPO)-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 erythropoietin (EPO) 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 erythropoietin (EPO) fragments may be contemplated. . 15 The results of an analysis according to step (b) of the above scheme and pertaining to the human erythropoietin (EPO) protein sequence of 165 amino acid residues is presented in
Table 1.
Table 1: Peptide sequences in human erythropoietin (EPO) with potential human MHC class II binding activity.
PRLICDSRVLERY, RLICDSRVLERYL, ICDSRVLERYLLE, CDSRVLERYLLEA,
SRVLERYLLEAKE, RVLERYLLEAKEA, LERYLLEAKEAEN, ERYLLEAKEAENT,
RYLLEAKEAENIT, YLLEAKEAENITT, LEAKEAENITTGC, KEAENITTGCAEH,
ENITTGCAEHCSL, CSLNENITVPDTK, NENITVPDTKVNF, ENITVPDTKVNEY,
NITVPDTKVNFYA, ITVPDTKVNFYAW, TKVNFYAWKRMEV, VNFYAWKRMEVGQ,
NFYAWKRMEVGQQ, YAWKRMEVGQQAV, KRMEVGQQAVEVW, RMEVGQQAVEVWQ,
MEVGQQAVEVWQG, QAVEVWQGLALLS, AVEVWQGLALLSE, VEVWQGLALLSEA,
EVWQGLALLSEAV, VWQGLALLSEAVL, WQGLALLSEAVLR, QGLALLSEAVLRG,
LALLSEAVLRGQA, ALLSEAVLRGQAL, LSEAVLRGQALLV, SEAVLRGQALLVN, ‘ 30 EAVLRGQALLVNS, AVLRGQALLVNSS, QALLVNSSQPWEP, ALLVNSSQPWEPL, . LLVNSSQPWEPLQ, QPWEPLQLHVDKA, EPLQLHVDKAVSG, LQLHVDKAVSGLR, ’ LHVDKAVSGLRSL, KAVSGLRSLTTLL, SGLRSLTTLLRAL, RSLTTLLRALGAQ,
SLTTLLRALGAQK, TTLLRALGAQKEA, TLLRALGAQKEAI, RALGAQKEAISPP,
AQKEAISPPDAAS, EAISPPDAASAAP, SPPDAASAAPLRT, ASAAPLRTITADT,
APLRTITADTFRK, RTITADTFRKLFR, TITADTFRKLFRV, DTFRKLFRVYSNF, ’
RKLFRVYSNFLRG, KLFRVYSNFLRGK, FRVYSNFLRGKLK, RVYSNFLRGKLKI,
YSNFLRGKLKLYT, SNFLRGKLKLYTG, NFLRGKLKLYTGE + RGKLKLYTGEACR, ’
GKLKLYTGEACRT, LKLYTGEACRTGD, KLYTGEACRTGDR
Peptides are 13mers, amino acids are identified using single letter code.
The results of a design and constructs according to step (c) and (d) of the above scheme and pertaining to the modified molecule of this invention is presented in Tables 2 and 3. .
Table 2: Substitutions leading to the elimination of potential T-cell epitopes of human erythropoietin (EPO) (WT = wild type). residue 5 L A C DE G H K N P Q R § T 6 I AC DE G H KN P Q R S§ T 11 V A C DE GH KN P Q R § T 12 L AC DE G H KN P Q R S§ T
Y A C DE G H K N P Q R § T 16 L A C DE G H KN P Q R S§ T 17 L A CDE 6G H KN P Q R § T
I A C DE GH KN P Q R § T
L A C DE G H KN P Q R S T
39 I1 A C DE G H KN P Q R S T 41 V AC DE G H KN P Q R SS T 46 Vv A C DE G H KN P Q R S T 48 F A C D E G H K N P Q R § T 49 Y A C DE G H K N P Q R S T 51 W A C DE G H KN P Q R S T 54 M A C DE G H KN P Q R S T 56 V A C DE G H K N P QQ R S T 61 Vv A C DE G H KN P Q R S T 63 V A ¢C DE 6G H KN P Q R S T 64 W A C DE G H K N P Q R S T 67 L A C DE G H KN P Q R S T 69 L A C DE G H KN P Q R S§ T 70 L A C DE G H KN P Q R S T 74 Vv A C bE G H KUN P Q R S T 75 L A C DE GH KN P Q R S T 80 L A C DE G H KN P 0 R S T 81 L A C DE G H KN P Q R S§ T 82 N A C DE G H KN P QQ R S T 88 W A C DE G H KN P QQ R S T 91 L A C DE G H KN P Q R § T 93 L A C DE G H KN P Q R S T 95 V A C DE G H K N P QQ R S§ T 99 V A C DE G H KN P Q R S T } 102 L A C DE G H KN P Q R S§ T 105 L A C DE G H KN P Q R S T 108 L A C DE G H KN P Q R S T 109 L AC DE G H KUN P Q R S T * 112 L A C DE G H KN P QQ R S T 119 I A C DE G H KN P QQ R S T 130 L A C DE GH KN P Q R S§ T 133 I A C DE G H KN P QQ R 8S T 138 F A C DE G H KN P gg R S T 141 L A C DE GH KN P Q R S T 142 F A C DE G H K N P OQ R S T
N Residue WT CL 144 VA CDE EGHK KN P Q R S§ T 145 Y A C bE G H K N P QQ R S T 148 F A C DE GH KN P Q R S T : 149 L AC DE G H KN P Q R S T ° 153 L AC DE G H KN P Q R S T 155 L AC DE G H KN P QQ R S T 156 Yy A C DE G H K N P Q R S T []
Table 3: Additional substitutions leading to the removal of a potential T-cell epitope for 1 or more MHC allotypes.
Residue WT _ 4 residue Substitution 4 R A C G P 6 I MW 8 D A C G P 9 Ss Pp T
R A C G P
11 V F I M W Y 12 L F I M V WwW Y 13 E A C G P 14 R A C G H P T 16 L W Y 17 L F I M V W Y 18 E A C G P T 19 A H P T
K H P T
1 21 E A C G P 22 A CC D E GG H K N P QQ R SS T 23 E P T 24 N A C G Pp 27 T A CC G P 28 G H T . 30 A D H P 33 C H T
L M WY
38 N T 40 T D H 41 Vv IY 43 D T 46 Vv M W Y 48 F M W 49 Y M W : 52 K A C G H Pp T 53 R A C G H P 54 M F I L V WwW Y 55 E A C G P T @ 56 v F I L WwW Y ' 57 G ¢C DE H K NP QO R S T 58 Q A C G P 0 59 Q A C G H Pp T 60 AC DEG H KN P Q R S§ T 6l Vv W : 62 E H P S T 63 v F I M W Y 65 Q A C G P 66 G DE H K N P Q R S T 67 L °F I M V WwW Y
Residue WT Lo ’ 4 residue Substitution 68 AC DE G H K N P Q R § T 69 L F I M W Y 70 LY 71 S A C G H P T 72 E H P T ’ 73 A E H P Q T 74 V F I W Y : 75 L F I V W Y . 76 R A C G P 77 G H P T 79 A H Pp 80 L F I Y 89 E A C G P 91 L F I M V W Y 92 Q A C G P 93 L M WY 94 H P T : 95 V F I M W Y 96 D AC G H Pp T 97 K A C G P 98 A C DE H K N P Q R § T 99 V WY 100 S D H N P OQ 101 G DEH KN P Q R S T 102 L F I W W Y 103 R DE H N P Q S§ T 104 S H Pp 105 L I Y 107 T H 108 IL WY 109 L F I M V W ¥Y 110 R A C G P 112 L F I M V W Y 113 G H T 114 A C DE G H K N P Q R SS T 115 Q P T 116 K A C G P 117 E H P T 118 A C DE G H K N P OQ R § T 119 I WY 120 S PT 124 A D H P 125 A PT 127 A H PT 130 L M WY 133 I WY 135 A H P 136 D P T 138 F M W Y . 139 R A C G P T 140 K A C G P 141 L F I M V W Y 142 F W 143 R A C G H P 146 S DH P T 147 N PT 148 F M 149 L F I M V WwW Y 150 R A ¢C D G H P T 151 G E H N P Q Ss T
Claims (29)
1. A modified molecule having the biological activity of human erythropoietin (EPO) : and being substantially non-immunogenic or less immunogenic than any non- modified molecule having the same biological activity when used in vivo.
,
2. A molecule according to claim 1, wherein said loss of immunogenicity is achieved by removing one or more T-cell epitopes derived from the originally non-modified molecule.
3. A molecule according to claim 1 or 2, wherein said loss of immunogenicity is achieved by reduction in numbers of MHC allotypes able to bind peptides derived from said molecule.
4. A molecule according to claim 2 or 3, wherein one T-cell epitope is removed.
5. A molecule according to any of the claims 2 — 4, 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.
6. A molecule according to claim 5, wherein said peptide sequences are selected from the group as depicted in Table 1.
7. A molecule according to any of the claims 2 — 6, wherein 1 — 9 amino acid residues in any of the originally present T-cell epitopes are altered.
8. A molecule according to claim 7, wherein one amino acid residue is altered.
9. A molecule according to claim 7 or 8, wherein the alteration of the amino acid residues is substitution of originally present amino acid(s) residue(s) by other amino acid residue(s) at specific position(s).
10. A molecule according to claim 9, wherein one or more of the amino acid residue substitutions are carried out as indicated in Table 2.
11. A molecule according to claim 10, wherein additionally one or more of the amino ‘ acid residue substitutions are carried out as indicated in Table 3 for the reduction in the number of MHC allotypes able to bind peptides derived from said molecule.
12. A molecule according to claim 9, wherein one or more amino acid substitutions are carried as indicated in Table 3. .
13. A molecule according to claim 7 or 8, wherein the alteration of the amino acid residues is deletion of originally present amino acid(s) residue(s) at specific : ~ position(s).
14. A molecule according to claim 7 or 8, wherein the alteration of the amino acid residues is addition of amino acid(s) at specific position(s) to those originally present.
15. A molecule according to any of the claims 7 to 14, wherein additionally further alteration is conducted to restore biological activity of said molecule.
16. A molecule according to claim 15, wherein the additional further alteration is substitution, addition or deletion of specific amino acid(s).
17. A modified molecule according to any of the claims 7 — 16, wherein the amino acid alteration is made with reference to an homologous protein sequence.
18. A modified molecule according to any of the claims 7 — 16, wherein the amino acid : alteration is made with reference to in silico modeling techniques.
19. A DNA sequence coding for a modified erythropoietin (EPO) of any of the claims 1 — 18. :
20. A pharmaceutical composition comprising a modified molecule having the biological activity of erythropoietin (EPO) as defined in any of the above-cited claims, optionally together with a pharmaceutically acceptable carrier, diluent or excipient.
) 21." A method for manufacturing a modified molecule having the biological activity of human erythropoietin (EPO) as defined in any of the claims of the above-cited claims comprising the following steps: . (1) determining the amino acid sequence of the polypeptide or part thereof. ' 5 (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; (iil) 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, or by binding of peptide-MHC complexes to T-cells; (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).
22. A method of claim 21, 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.
20 .
23. A method of claim 22, wherein the alteration is made with reference to a homologues protein sequence and / or in silico modeling techniques.
24. A method of any of the claims 21 — 23, wherein step (ii) 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 : 30 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.
25. A method of claim 24, wherein step (c) is carried out by using a Béhm 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.
26. A 13mer T-cell epitope peptide having a potential MHC class II binding activity and created from immunogenically non-modified erythropoietin (EPO), selected from the group as depicted in Table I.
27. A peptide sequence consisting of at least 9 consecutive amino acid residues of a 13mer T-cell epitope peptide according to claim 26.
28. Use of a 13mer T-cell epitope peptide according to claim 26 for the manufacture of erythropoietin (EPO) having substantially no or less immunogenicity than any non- modified molecule with the same biological activity when used in vivo.
29. Use of a peptide sequence according to claim 27 for the manufacture of erythropoietin (EPO) having substantially no or less immunogenicity than any non- modified molecule with the same biological activity when used in vivo.
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