Patent Application: US-57705304-A

Abstract:
the invention discloses the human chemokine hcc - 1 , n - terminally truncated hcc - 1 molecules and glycosylated hcc - 1 which improve the homing of stem cells into the - bone marrow during stem cell transplantation . it is also provided a procedure for producing the polypeptides by recombinant techniques or chemical synthesis and for producing antibodies against such polypeptide . furthermore , it is disclosed the modification of the polypeptide by coupling of amino acid residues and / or chemical groups or deleting amino - acids generating potent derivatives of the polypeptide . another aspect of the invention provides a combination of the polypeptide of the present invention and a suitable pharmaceutical carrier for providing a therapeutically effective amount of the polypeptide for the treatment of various associated diseases . the invention concerns also the use of the hcc - 1 molecules to increase engraftment of stem cells in the course of the stem cell transplantation performed in stem cell transplantation related diseases .

Description:
the present invention concerns a poly peptide having at least 90 % homology with the amino acid sequence whereby r is an oligosaccharide composed out of n - acetylgalactosamine galactose or an oligosacharide composed out of n - acetylgalactosamine galactose and n - acetylneuraminic acids its biologically active fragments , analogs and derivatives , in particular amidated , acylated , and / or phosphorylized derivatives wherein the two cystein residues in positions 16 and 40 linked together by a disulfide bond and wherein the two cystein residues in positions 17 and 56 are linked together by a disulfide bond . in the context of the description of the invention the term “ homology ” means identical amino acids in an amino acid sequence , as well as amino acids which are modified without altering the function of the molecule . also amino acids may be substituted in the polypeptide chain which amino acids are conservatively exchanged amino acids . such amino acids are e . g . neutral amino acids , aromatic amino acids charged amino acids and the like . for example an exchange of serine against valine or lysine against asparagine may not alter the function of the polypeptide of the invention . in particular , a polypeptide of the invention has at least 90 % identity to the polypeptide sequence of the invention . the polypeptide of the invention is in particular the glycosylated chemokine hcc - 1 . the processed chemokine of the invention comprises a polypeptide wherein ( a ) the n - terminus is modified by coupling a chemical group generating a chemokine having the structure of [ glyoxyloyl1 ] phc 1 - pentane oxime , nonanyl - phc , [ glyoxyloyl1 ] phc 1 - heptane oxime , [ glyoxyloyl1 ] phc 1 - hexane oxime , [ glyoxyloyl1 ] phc 1 - pentene oxime or nonaoyl - phc and wherein the modification is influencing the biological activity of phc or ( b ) wherein amino acid residues of the n - terminus or of the c - terminus are deleted . the polypeptides , of the invention comprise modifications which are increasing the plasma half - life time of hcc - 1 which by way of example may be achieved by introducing one or more lysine , histidine , glutamate , aspartate , or cysteine residues which are e . g . modified by coupling a chemical group having the structure of poly ethylene glycol . subject - matter of the invention is also an antibody against an amino acid sequence of the invention . the skilled person knows very well how to obtain antibodies against a polypeptide by immunizing e . g . animal with the respective polypeptide . from polyclonal antibodies monoclonals may be derived by established methods based on clonal selection techniques . polyclonal or monoclonal antibodies against the polypeptide such as chemokine hcc - 1 of the invention may serve as starting material for diagnostic agents or may be used directly for detecting the level of the polypeptide of the invention . from the polypeptide and / or the antibody of the invention a medicament can be manufactured . the skilled person knows very well how to provide an appropriate galenic preparation . a process for producing a polypeptide is also subject matter of the invention . the polypeptide of the invention can be manufactured using recombinant techniques or chemical synthesis . the polypeptides of the invention may also be manufactured by utilizing the cellular expression system . a process for producing cells capable of expressing a polypeptide of the invention is also subject - matter of the invention . according to the invention the polypeptide of the invention e . g . hcc - 1 , hcc - 1 molecules without glycosylation and n - terminally truncated hcc - 1 molecules , especially hcc - 1 ( 2 - 74 ), hcc - 1 ( 3 - 74 ), hcc - 1 ( 4 - 74 ), hcc - 1 ( 5 - 74 ), hcc - 1 ( 6 - 74 ), hcc - 1 ( 7 - 74 ), hcc - 1 ( 8 - 74 ), hcc - 1 ( 9 - 74 ), hcc - 1 ( 10 - 74 ), hcc - 1 ( 11 - 74 ) and hcc - 1 ( 12 - 74 ) can be used to increase engraftment of stem cells , for transplantation of progenitor and stem cells , for treatment of progenitor - and stem cells prior to transplantation , for in vivo application of such a molecule into patients which are receiving stem cell transplantation prior to and / or in the course of stem cell transplantation . this is in particular useful , if the host patient is not conditioned or conditioned e . g . under sublethal , lethal , or supralethal conditions . sublethal , lethal , or supralethal conditions include treatment with total body irradiation , optionally followed by treatment with myeloablative or immunosuppressive agents ; myeloablative or immunosuppressive treatment without total body irradiation . furthermore , the polypeptide of the invention can be used for the transplantation of hematopoietic progenitor and stem cells , umbilical cord blood and placental stem and progenitor cells , liver stem and progenitor cells ( oval cells ), mesenchymal stem and progenitor cells , endothelial progenitor cells , skeletal muscle stem and progenitor cells ( satellite cells ), smooth muscle stem and progenitor cells , intestinal stem and progenitor cells , embryonic stem cells , and genetically modified embryonic stem cells , adult islet / beta stem - and progenitor cell , epidermal progenitor and stem cells , keratinocyte stem cells of cornea , skin and hair follicles , olfactory ( bulb ) stem and progenitor cells and side population cells from diverse adult tissues . the polypeptide of the invention may be used as well for the treatment of leukemias , lymphoproliferative disorders , aplastic anemia , congenital disorders of the bone marrow , solid tumors , autoimmune disorders , inflammatory diseases , primary immunodeficiencies , primary systemic amyloidosis , systemic sclerosis , heart diseases , liver diseases , neurodegenerative diseases , multiple sclerosis , m . parkinson , stroke , spinal cord injury diabetes mellitus , bone diseases , skin diseases , replacement therapy of the skin , retina or cornea , other congenital disorders , vessel diseases like atherosclerosis or cardiovascular disease . 900 l of human hemofiltrate ( hf ) for large scale recovery of plasma peptides were obtained from chemotherapy - treated patients with renal failure . ultrafilters used for hemofiltration had a specified molecular mass cut - off of 20 kd . the sterile filtrate was immediately cooled to 4 ° c . and acidified to ph 3 to prevent bacterial growth and proteolysis . for peptide extraction the hf was ultrafiltrated a second time . the filtrate was conditioned to ph 2 . 7 and applied onto the strong cation exchanger , fractogel tsk sp 650 ( m ), 100 × 250 mm ( merck , darmstadt , germany ) using an autopilot chromatography system ( perseptive biosystems , wiesbaden , germany ). bound peptides were eluted using seven buffers with increasing ph resulting in seven ph - pools . the seven buffers were composed as follows : i : 0 . 1 m citric acid monohydrate , ph 3 . 6 ; ii : 0 . 1 m acetic acid + 0 . 1 m sodium acetate , ph 4 . 5 ; iii : 0 . 1 m malic acid , ph 5 . 0 ; iv : 0 . 1 m succinic acid , ph 5 . 6 ; v : 0 . 1 m sodium dihydrogen phosphate , ph 6 . 6 ; vi : : 0 . 1 m disodiumhydrogen phosphate , ph 7 . 4 ; vii : 0 . 1 m ammonium carbonate , ph 9 . 0 . the seven pools ( ph pools ) were collected and each of them was loaded onto a rp column , 125 mm × 100 mm i . d ., source rpc , 15 μm ( pharmacia ) and eluted in a gradient from 100 % a ( 0 . 01 m hcl in water ) to 60 % b ( 0 . 01 m hcl in 80 % acetonitrile ). fractions of 200 ml were collected . in the screening for chemotactic activities using the fdcp - mix stem cell line the predominant activity was identified in ph pool vi . this chemotactic activity was purified in four further chromatographic steps a to d . ( a ) reverse phase chromatography , using a bakerbond cartrige ( 47 mm i . d .× 300 mm ) with an acetonitril gradient . ( b ) size exclusion chromatography using sec superdex 16 / 60 high load column with the eluent pbs , ph 7 . 4 . ( c ) reverse phase chromatography , using a ymc c18 ( 10 mm i . d .× 250 mm ) with an acetonitril gradient . ( d ) reverse phase chromatography , using a ymc c18 ( 4 mm i . d .× 250 mm ) with an acetonitril gradient . amino acid sequencing by edman degradation of the purified material revealed the sequence of hcc - 1 . mass spectrometric analysis of the isolated material revealed a glycosylated molecule . the isolated molecules revealed molecular weights ( mw ) of 9038 . 15 and 9331 . 9 . whereas hcc - 1 ( 1 - 74 ) carries mw of 8673 . 09 the increase of the mw in the isolated molecules was identified as an o - glycosylation of the amino acid serine in position 7 with n - acetylgalactosamine galactose and with oligosacharide composed of n - acetylgalactosamine galactose and n - acetylneuraminic acid . fig1 shows the purification step a : reverse phase chromatography , using a bakerbond cartrige ( 47 mm i . d .× 300 mm ) with an acetonitril gradient . fig2 shows the purification step b : size exclusion chromatography using sec superdex 16 / 60 high load column with the eluent pbs , ph 7 . 4 . fig3 shows the purification step c : reverse phase chromatography , using a ymc c18 ( 10 mm i . d .× 250 mm ) with an acetonitril gradient . fig4 shows the purification step d : reverse phase chromatography , using a ymc c18 ( 4 mm i . d .× 250 mm ) with an acetonitril gradient . chemotactic activity of hcc - 1 molecules to the murine fdcp - mix stem cell line fig5 and 6 are showing fdcp - mix cells which were subjected to in vitro chemotactic assays . chemotaxis was assessed in 96 - transwell chambers ( neuroprobe , cabin john , md ) by using polyvinylpyrrolidone - free polycarbonate membranes ( nucleopore , neuroprobe ) with 5 - μm pores . four hundred microliters of imdm medium was added to the bottom of the well , and was supplemented with varying concentrations of hcc - 1 molecules . 200 μl of imdm medium containing 100 . 000 fdcp - mix cells were added to the upper wells of the chemotaxis chamber . all assays were carried out in triplicate , and the migrated cells were counted in 4 randomly selected fields at 63 - fold magnification after migration for 14 h . enriched mononulcear cells , cd34 + progenitor cells from human cord blood , mobilized peripheral blood , or bone marrow are incubated with hcc - 1 in concentrations between 100 pm and 10 μm for a time period which is between 5 minutes and 12 hours . fig7 describes the concept of the modulation of homing mechanisms by preincubation with hcc - 1 . after preincubation stem cells are transplanted into the blood flow . in a competitive repopulation model using ly 5 . 1 and ly 5 . 2 mice it was shown that preincubation of the cells gives an advantage in the engraftment of the bone marrow over cells which were not treated with hcc - 1 . the potential of hcc - 1 ( 1 - 74 ) to increase the adhesion of fdcp - mix progenitor cells to huvec endothelial cells under a shear stress of 2 dynes / cm 2 was validated . with and without preincubation of progenitor cells with hcc - 1 ( 1 - 74 ). the chemokine hcc - 1 ( 1 - 74 ) was shown to improve the adhesion of hematopoietic progenitor cells to the endothelium . cells from the hematopoietic progenitor cell line ( fdcp - mix ) were primed with 1000 ng / ml hcc - 1 ( 1 - 74 ) and subsequently injected into a flow chamber . adhesion of the progenitor cells to endothelial cells was detected under a shear stress of 2 dynes / cm 2 . in the competitive ly 5 . 1 / ly 5 . 2 repopulation model ly5 . 1 bone marrow cells were preincubated with 1000 ng / ml hcc - 1 ( 1 - 74 ). subsequently the cells were washed , mixed in a ratio of 50 : 50 with ly5 . 2 bone marrow cells , and injected i . v . into sublethally irradiated ly5 . 2 mice . after 5 weeks animals were killed and the amount of ly5 . 1 cells in the bone marrow was detected . the results show that preincubation with hcc - 1 induces a significant increase of the engraftment of the ly5 . 1 cells ( p = 0 . 03 ) ( fig9 ). it was investigated , if the priming of human progenitor cells with hcc - 1 for 30 min increases the engraftment of the cells in non - obese diabetic / severe combined immunodeficiency ( nod / scid ) mice ( fig1 ). in these experiments hcc - 1 ( 1 - 74 ) as well as hcc - 1 ( 9 - 74 ) were tested . by priming of the cells with hcc - 1 ( 1 - 74 ) an increase of the engraftment could be achieved . the priming with hcc - 1 ( 9 - 74 ) showed a dose dependent increase of the engraftment . the highest concentrations of 100 ng / ml and 1000 ng / ml resulted in a significant increase of the engraftment 1 . lapidot t , p . f ., doedens m , murdoch b , williams d e , dick j e , cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in scid mice . science , 1992 . 255 : p . 255 . 2 . larochelle a , v . j ., hanenberg h , wang j c , bhatia m , lapidot t , moritz t , murdoch b , xiao x l , kato i , williams d a , dick j e , identification of primitive human hematopoietic cells capable of repopulating nod / scid mouse bone marrow : implications for gene therapy . nat med , 1996 . 2 : p . 1329 - 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