Polysialic acid derivatives

Abstract

A polysialic acid compound is reacted with a hetero-bifunctional reagent to introduce a pendant functional group for site-specific conjugation to sulfhydryl groups, for instance side chains of cysteine units in drugs, drug delivery systems, proteins or peptides. The functional group is, for instance, an N-maleimide group.

Claims

1. A compound comprising a polysaccharide having at least two sialic acid units linked 2.8 and/or 2.9 to one another, and having reducing and non-reducing terminal units, said polysaccharide having a pendant moiety XRY, wherein the pendant moiety is covalently linked to the reducing terminal sialic acid unit through Y; in which: X is a N-maleimido, N-iodoacetamido, S-vinylsulphonyl or S-orthopyridyldisulphide group, R is alkene-diyl, arylene or aralkylene alkarylene, alkylene-oxaalkylene, or alkylene-oiigooxa-alkylene or alkyl-oligopeptidyl-alkyl group; and Y is a hydrazide, amine or N-hydroxysuccinimide group.

2. The polysaccharide of claim 1, wherein R is C.sub.1-6 alkanediyl, C.sub.2-3-alkyl-oxa-C.sub.2-3-alkylene, C.sub.2-3 alkyl-oligo(oxa-C.sub.2-3 alkylene), or C.sub.2-6 alkylene phenyl.

3. The polysaccharide of claim 1, wherein X is N-maleimido or orthopyridyldisulphide.

4. The polysaccharide of claim 1, wherein Y is a hydrazide or a hydroxyl succinimide.

5. The compound of claim 1, wherein the polysaccharide is colominic acid.

6. The compound of claim 1, wherein the polysaccharide comprises glycosyl units other than sialic acid units.

7. The compound of claim 1, wherein the polysaccharide is a B polysaccharide.

8. The compound of claim 1, wherein the polysaccharide comprises at least 2, at least 5, at least 10, or at least 50 sialic acid units.

9. A polysaccharide protein conjugate comprising the polysaccharide of claim 1 linked through a thioether bond to the side chain of a cysteine unit of the protein.

10. The polysaccharide protein conjugate of claim 9, wherein the polysaccharide is linked through a moiety joined at one or both terminal units of the polysaccharide.

11. The polysaccharide protein conjugate of claim 9, wherein the protein contains a single cysteine.

12. The polysaccharide protein conjugate of claim 9, wherein the polysaccharide is a B polysaccharide.

13. The polysaccharide protein conjugate of claim 9, wherein the polysaccharide is linked through an N-succinimidyl group with the thioester linked at the -carbon atom.

14. The polysaccharide protein conjugate of claim 9, wherein the moiety comprises a secondary amine, a hydrazine, or an amide bond.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 shows an SOS-PAGE gel of triplicate samples of the conjugate of colominic acid maleimide (CAM) with Fab and relevant controls.

(2) FIG. 2 shows an SOS-PAGE gel of the conjugate of colominic acid iodoacetate (CAI) with P-galactosidase and of CAM with p-galactosidase.

(3) FIG. 3 shows SOS-PAGE gels of conjugates of P-galactosidase with alternate colominic acid derivatives.

DETAILED DESCRIPTION

(4) In the invention, the moiety which includes the functional group, may be linked directly to the polysialic acid unit or, more conveniently, may be linked via a difunctional organic group, such as an alkane diyl group, an arylene group or an oligo(alkyleneoxy) alkane group, or alternatively an oligo peptidyl group. The linkage to the polysialic acid (from the linker or the moiety including the functional group may be a secondary amine, a hydrazone, an alkyl hydrazine an ester or a peptidyl group. The moiety may be generated by reaction of a polysialic acid substrate with a heterobifunctional reagent. The process form a further aspect of the invention.

(5) Reagents useful for introducing the selected functional groups are commercially available. A compound which will introduce a maleimide group onto an amine moiety without introducing any additional linker moiety is N-methoxy-carbonyl-maleimide. Generally the reagents include a second functional group for reaction with a group on the polysialic acid which may be the aldehyde group described above, or an amine group. Suitable second functional groups include N-hydroxy succinimide esters and their sulfosuccimide analogues and hydrazides. Preferably the compound is a N-maleimido-alkanoic acid hydrazide or an N-maleimidoarylalkanoic acid hydrazide i.e. a compound having the general formula
XRY in which: X is a N-maleimido, N-iodoacetamido, S-vinylsulphonyl or S-orthopyridyldisulphide group, R is alkane-diyl, arylene or aralkylene alkarylene, alkylene-oxaalkylene, or alkylene-oligooxa-alkylene or alkyl-oligopeptidyl-alkyl group; and Y is a hydrazide, amine or N-hydroxysuccinimide group. Preferably R is C.sub.1-6 alkanediyl, C.sub.2-3-alkyl-oxa-C.sub.2-3-alkylene, C.sub.2-3 alkyloligo(oxa-C.sub.2-3 alkylene), or C.sub.2-6 alkylene phenyl.

(6) Preferably X is N-maleimido or orthopyridyldisulphide. Preferably Y is a hydrazide or a hydroxyl succinimide. Compounds which may be reacted with an aldehyde group, and which include a linker moiety and introduce a maleimide group include N-[-maleimidopropionic acid]hydrazide and 4-(4-N-maleimidophenyl)butyric acid hydrazide. The hydrazide group reacts with the aldehyde to form a stable hydrazone group. A suitable heterobifunctional compound which includes an oligo(ethyleneoxy) ethylene group is a compound comprising a polyethylene glycol (poly(ethyelenoxy)) group with, at one end, N-hydroxy succinimide (NHS) group and at the other end the functional group. The NHS group reacts with amine groups to form stable amide linkages. Heterobifunctional polyethyleneglycols with NHS at one end and either vinylsulphone or maleimide at the other end are available. Other examples of heterobifunctional reagents include, 3-(2-pyridyldithio)propionyl hydrazide, N-succinimidyl-3-[2-pyridyldithio]propionate, succinimidyl-H[N-maleimidomethyl]cyclohexane-1-carboxylate), m-maleimidobenzoyl-N-hydroxysuccinimide ester, N-succinimidyl-[4-iodoacetyl]amino benzoate, N-[gamma-maleimidobutyryloxy]succinimide ester, N-[epsilon-maleimidocaproyloxy]succinimide ester and N-succinimidyl iodoacetate. Other reagents are available from Pierce Biotechnology and Shearwater Corporation (polyethylene glycol-based).

(7) Sialic acids (also known as nonulosonic acids) are members of a family of amino containing sugars containing 9 or more carbon atoms. The most important of the sialic acids is N-acetylneuraminic acid (also known as 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-nonulosonic, lactaminic acid and O-sialic acid) which has the formula:

(8) ##STR00001##

(9) Polysialic acids may be linked 2.fwdarw.8 and/or 2.fwdarw.9, usually in the -configuration. In some polysialic acids the linkages are alternating 2.fwdarw.8 and 2.fwdarw.9. The invention is also of utility for heteropolymeric polysaccharides comprising glycosyl units other than sialic acid units.

(10) Polysialic acids are generally found to be non-toxic and substantially non-immunogenic. Furthermore the biodegration units, sialic acid, is not known to be toxic and, indeed sialic acids are widely found in animal proteins and cells, including blood cells and circulating proteins.

(11) Polysaccharide compounds containing many sialic acid units are polysaccharides produced by Escherichia coli, Moraxella nonliquifaciens, Pasteurella aeroginosis and Neisseria meningitidis or derivatives thereof. For instance colominic acid derived (by hydrolysis to shorten the chain lengths) from E. coli K1 comprises 2.fwdarw.8 linked sialic acid units. Polysaccharide from E. coli K92 strain comprises alternating 2.fwdarw.8 and 2.fwdarw.9 linked sialic acid units. Polysaccharide C of N. meningitidis group C has 2.fwdarw.9 linked sialic acid units.

(12) One group of polysaccharide compounds which has been found to be of particular utility in the invention is group B polysaccharides. These compounds are produced by N. meningitidis, M. nonliquifaciens, P. aeroginosis A2 and E. coli K1. These compounds comprise a polysaccharide component comprising sialic acid residues and a phospholipid component. The sialic acid residues are linked a 2.fwdarw.8 the naturally occurring polymer consisting of about 200 residues. Some of the glycolipid molecules, especially the high molecular weight compounds appear to have a covalently attached phospholipid at the reducing end of the polysaccharide component.

(13) It is preferable for the bacteria from which the polysaccharide compound is derived to be non-pathogenic for convenience during production. It is particularly suitable therefore for the polysaccharide to be derived from a non-pathogenic strain of E. coli such as E. coli K92 or, preferably, K1 which is non-immunogenic. E. coli K92 and K1 isolates are well-known and any such type of any such strains can be used as sources of suitable polysaccharide. Preferably the polysialic acid should have at least 2, preferably at least 5, more preferably at least 10, for instance more than 50 sialic acid units.

(14) According to the invention, there is also provided a conjugate of a protein and the novel activated polysaccharide. The novel compound comprises a protein with at least one cysteine unit and, linked through a thioether bond to the side chain of a cysteine unit, a polysialic acid through a moiety joined at one or both terminal unit of the polysialic acid.

(15) Where the polysialic acid derivative was a N-maleimido group the moiety will include a N-succinimidyl group, with the thioester linked at the -carbon atom. Preferably the moiety also comprises a secondary amine, a hydrazone or an amide bond.

(16) There is also provided in the invention a new process in which a polysialic acid is reacted with a heterobifunctional reagent having a first functional group for reaction with sulfhydryl groups and a second functional group different to the first group whereby the said second functional group reacts with a terminal unit of the polysialic acid to form a covalent bond therewith and form a capable of reaction with a sulfhydryl group functional polysialic acid.

(17) In one embodiment the second functional group is a nucleophilic group, preferably hydrazine. This is of particular utility where the polysialic acid comprises an aldehyde group in the terminal unit whose carbonyl group is attacked by the nucleophilic group.

(18) In another embodiment of the process the second functional group is electrophilic such as an N-alkoxyl carbonyl-imide such as N-hydroxysuccinimide ester or sulphosuccinimide ester, or carbodiimide. The terminal group in such cases is preferably nucleophilic such as amine.

(19) In the process it is preferred that the reagent comprises a bifunctional organic group linking the first and second functional groups. Preferably the bifunctional organic group is selected from a C.sub.2-18-alkanediyl group, an arylene group, an oligo peptide and an oligo(alkoxy)alkyl group.

(20) Examples of suitable reagents are given above.

(21) Most usefully the process involves a subsequent step in which the functional polysialic acid is reacted with a polypeptide or a protein having at least one free and unprotected Cys unit whereby the functional group forms a thioether linkage with the thiol group of a Cys unit to form a polysialyated polypeptide or protein. The process is particularly suitable where the protein contains a single Cys unit, whereby site-controlled derivatisation is achieved.

(22) The invention is illustrated in the accompanying examples.

(23) ##STR00002##

Example 1

1.1 Synthesis

(24) Three separate preparations were carried out as follows:

(25) Colominic acid aldehyde (CAO) produced according to WO-A-9222331 (100 mg, 4.410.sup.6 mol) was dissolved into 500 l 0.1 M sodium acetate, to this 5 molar equivalents of N-[-Maleimidopropionic acid]hydrazide (6.5 mg, 2.210.sup.5 mol) was added. This mixture was then vortex mixed and wrapped in foil and allowed to incubate at 37 C. for 2 h on a rotary mixer. The polymer was then precipitated by the addition of 2 volumes (1.0 ml) of ethanol. The precipitate was collected by centrifugation (13,000 rpm 2 min) in a bench top microcentrifuge. The supernatant was discarded and the pellet dissolved in 500 l 0.1 M acetate. This process was repeated a further 2 times and the final pellet dissolved in deionised water and freeze dried overnight.

1.2 Assay for Maleimide Content

(26) In this assay cysteine is reacted with the maleimide on the polymer preventing further reaction with Ellman's Reagent (5,5-dithiobis(2-nitrobenzoic acid)) which contains a disulphide which forms an intense yellow colour when it is substituted for a thiol not adjacent to an aromatic ring. Thus maleimide content can be calculated by measuring the inhibition of reaction between cysteine and Ellman's reagent assay.

(27) First an initial stock of cysteine at 1210.sup.3 M (0.145 mg/ml) was prepared in PBS. In a clean microtitre plate 100 l volume doubling dilutions from 1210.sup.3 M to 0.37510.sup.3 M were made from row B to row H. In row A 100 l PBS was used as a zero standard. Samples of CA and CA-maleimide (CAM) were prepared at 5 or 10 mg/ml and 100 l of each sample added to duplicate columns of the cysteine dilutions. In one set 100 l PBS without any CA was added as a control. The plate was covered and allowed to incubate at 37 C. for 1 h. After this time 20 l of Ellman's reagent (4 mg/ml) was added to each well and the plate incubated in the dark at room temperature for 15 min. Absorbance was the measured in wells at 405 nm. Standard curves were then plotted for the samples and the amount of maleimide present calculated from inhibition of the signal.

1.3 (Intentionally Omitted)

1.4 Thiolation of FAb and Conjugation to CAM

(28) In the first step a thiol group is introduced into a model protein by thiolation of an amine of lysine.

(29) Ovine FAb (anti Desipramine/Norityrptaline, 4 mg, 7.210.sup.8 mol) was dissolved in 0.25 ml PBS+10 mM EDTA to this was added 0.498 mg 2-iminothiolane (2-IT or Traut's reagent 50 mol equiv 3.610.sup.6 mol) in 0.25 ml of the same buffer. The tube was wrapped in foil and left to incubate stirring end over end for 1 h at 37 C.

(30) ##STR00003##

(31) Thiolated Fab was purified from free 2-IT (Traut's reagent) by gel filtration (PD-10) and 0.5 ml fractions assayed for presence of protein (BCA assay) or thiol (Ellman's assay). The first eluting peak containing both was pooled and protein and thiols quantified.

1.5 Conjugation of Fab-Thiol to CAM

(32) To thiolated FAb (3.6 mg, 6.610.sup.8 mol) in 2 ml PBS/EDTA 22.5 mg CAM was added (910.sup.7 mol, 15 molar equiv). The tube was sealed and allowed to incubate at 37 C. for 1 h whilst gently mixing. The resulting conjugate was then purified according to accepted protocols to remove free CAM. Both CA and protein content were assayed on the conjugate to calculate conjugation ratio.

(33) Control reactions were carried out with CA as a negative control.

(34) Several batches of CAM-Fab were prepared with various degrees of thiolation but maintaining the 15:1 ratio of CAM: Fab. Results are shown in table 1 below:

(35) TABLE-US-00001 TABLE 1 Thiol per FAb Conjugate ratio (CA:Fab) 1 0.53:1 2 0.9:1 5 (triplicate reaction) 1.925:1 +/ 0.19:1 Fab 10 3.51:1

(36) FIG. 1 shows an SDS-PAGE gel of triplicate samples and relevant controls

1.6 Conclusion

(37) The results show that in all control wells (samples of thiolated FAb) the migration of the sample is similar to that for fresh FAb (below that of the 50 kDa marker) indicating no cross linking of FAb molecules during the process of conjugation. In the replicate lanes there is considerable band broadening with maximum intensity between the 98 and 250 kDa markers which typically indicates an increase in mass which is indicative of a of polysialylated product.

1.7 Conjugation of CAM to Beta Galactosidase

(38) To E. coli -galactosidase (-gal: 5.0 mg, 4.310.sup.8 mol) in 1 ml PBS 15 mg CAM was added (6.5910.sup.7 mol, 15 molar equiv). The tube was sealed wrapped in foil and allowed to incubate at room temperature for 1 h whilst gently mixing. The resulting conjugate was analysed by SDS page and then purified according to accepted protocols to remove free CAM. Samples were assayed for polymer and protein content as outlined elsewhere.

(39) Control reactions were carried out with CA as a negative control.

1.8 Assay for Enzyme Activity

(40) Standards from 60 g/ml to 3.75 g/ml of fresh -galactosidase were prepared in PBS. Sample of CAM--gal were diluted to 60 g/ml in the same buffer. Enzyme activity of the conjugates was measured as follows: In a microtitre plate, to 100 l of sample or standard was added 100 l of All-in-One -gal substrate (Pierce). The plate was incubated at 37 C. for 30 min and absorbance read at 405 nm. A calibration curve was prepared from the standards and the activity of the samples calculated from the equation for the linear regression of the curve.

1.9 Results

(41) From the protein and polymer assays the conjugation ratio was determined to be 1.23 CAM:1 -Gal. There was also a corresponding increase in apparent molecular mass from the SDS page of the samples (FIG. 2). Enzyme activity in the purified sample was calculated to be 100.4% compared to the free enzyme.

Example 2

Synthesis Route 2

(42) Step 1 Amination of CA-Aldehyde (CHO)

(43) ##STR00004##

(44) Step 2 Introduction of Maleimide Ring

(45) ##STR00005##

Synthesis

2.1 Step 1 Amination of Oxidised CA

(46) Oxidised colominic acid at (CAO) 10-100 mg/ml was dissolved in 2 ml of deionised water with a 300-fold molar excess of NH.sub.4Cl, in a 50 ml tube and then NaCNBH.sub.3 (5 M stock in 1 N NaOH(aq)) was added at a final concentration of 5 mg/ml. The mixture was incubated at room temperature for 5 days. A control reaction was also set up with colominic acid (A) instead of CAO. Product colominic acid amine derivative was precipitated by the addition of 5 ml ice-cold ethanol. The precipitate was recovered by centrifugation at 4000 rpm, 30 minutes, room temperature in a benchtop centrifuge. The pellet was retained and resuspended in 2 ml of deionised water, then precipitated again with 5 ml of ice-cold ethanol in a 10 ml ultracentrifuge tube. The precipitate was collected by centrifugation at 30,000 rpm for 30 minutes at room temperature. The pellet was again resuspended in 2 ml of deionised water and freeze-dried.

2.2. Assay for Amine Content

(47) The TNBS (picrylsulphonic acid or 2, 4, 6-tri-nitro-benzene sulphonic Acid) assay was used to determine the amount of amino groups present in the product.

(48) In the well of a microtitre plate TNBS (0.5 l of 15 mM TNBS) was added to 90 l of 0.1 M borate buffer pH 9.5. To this was added 10 l of a 50 mg/ml solution of CA-amide the plate was allowed to stand for 20 minutes at room temperature, before reading the absorbance at 405 nm. Glycine was used as a standard, at a concentration range of 0.1 to 1 mM. TNBS trinitrophenylates primary amine groups. The TNP adduct of the amine is detected.

(49) Testing the product purified with a double cold-ethanol precipitation using the TNBS assay showed close to 90% conversion.

2.3 Maleimidation of CA-Amine

(50) CA-Amine (17 mg) was dissolved in 1 ml deionised water, to this was added 6 mg methoxy-carbonyl-maleimide (MCM). The mixture was left to react at room temperature for 30 min. To the sample 100 l water and 200 l acetonitrile was added and then incubated at room temperature for 4 h, after which 300 l CHCl.sub.3 was added, the tube shaken and the aqueous fraction collected. Then the fraction was purified over a PD-10 column to remove small molecules. The eluate was freeze dried and assayed for maleimide content. The molar concentration of maleimido was 44 mol %.

Example 3

Preparation of Iodoacetate Derivative of Colominic Acid (CAI)

(51) ##STR00006##

3.1 Synthesis

(52) To 40 mg colominic acid amine (85 mol % amine) as (described in Example 2.1) dissolved in 1 ml of PBS pH 7.4 was added 5 mg of N-succinimidyl iodoacetate (SIA). The mixture was left to react for 1 h at 37 C., after which excess SIA was removed by gel filtration over a 5 ml HIGHTRAP Desalting column (AP Bioscience) eluted with PBS. 0.5 ml fractions were collected from the column and samples from each fraction tested for colominic acid content (resorcinol assay) and reactivity with cysteine indicating Iodide (Ellman's Assay). Fractions positive for both iodide and CA were pooled.

3.2 Conjugation of CAI to -Galactosidase

(53) To E. coli -galactosidase (5.0 mg, 4.310.sup.8 mol) in 1 ml PBS 15 mg CAI was added (6.59107 mol, 15 molar equiv). The tube was sealed wrapped in foil and allowed to incubate at room temperature for 1 h whilst gently mixing. The resulting conjugate was analysed by SDS page and then purified according to accepted protocols to remove free CAI. Samples were assayed for polymer and protein content as outlined elsewhere.

(54) Control reactions were carried out with CA as a negative control. All samples were analysed for -gal activity as example 1.8 above.

3.3 Conclusions

(55) Fractions 3-6 were positive for both polymer and iodoacetate and were pooled. The SDS page (4-12% Bis/Tris gel; FIG. 2) showed an increase in apparent molecular mass for samples incubated with the iodoacetamide derivative but not with control polymer. From the protein and polymer assays the conjugation ratio was determined to be 1.63 CAI:1 -gal. -gal activity was calculated to be 100.9% for the conjugated sample, compared to the free enzyme.

Example 4

4.1 Synthesis: Non-Reducing End Activation with 4(4-N-maleimidophenyl)butyric acid hydrazide (CA-MBPH) and 3-(2-pyridyldithio)propionyl hydrazide (CA-PDPH)

(56) The preparations were made as follows:

(57) Colominic acid aldehyde (CAO; 22.7 kDa, Camida, Ireland) produced according to WO-A-9222331 (73 mg for MBPH, tube A; 99.3 mg for PDPH; tube B) was dissolved individually into 800 l 0.1 M sodium acetate (pH 5.5). To tube A 15 mg of MBPH (15:1 linker:CA ratio) dissolved in 200 l of DMSO was added. To tube B 15 mg (15:1 linker:CA ratio) of PDPH dissolved in 200 l of DMSO was added. The pH was adjusted to 5.5 in each case for each reaction vessel. These mixtures were then vortex mixed and wrapped in foil and allowed to incubate at 37 C. for 2 h on an orbital mixer. Each polymer solution was purified by gel filtration (PD 10 column) eluting with PBS pH 7.4 and the 1 ml fraction containing CA (by resorcinol assay) collected. These sample were freeze dried overnight and assayed for maleimide content as described in example 1.2.

4.2 Synthesis: Reducing End Activation with 4(4-N-maleimidophenyl)butyric acid hydrazide (MBPH-CA), (2-pyridyldithio)propionyl hydrazide (PDPH-CA) and N--Maleimidopropionic acid hydrazide)hydrazide (BMPH-CA)

(58) The preparations were carried out as follows all at molar ratios of 25:1 linker to CA:

(59) Colominic acid (CA; 22.7 kDa; 73 mg for MBPH, tube A; 99.3 mg for PDPH; tube B and 76.6 mg for BMPH; tube C) was dissolved individually into 800 l 0.1 M sodium acetate (pH 5.5) separately. To tube A, 25 mg of MBPH (dissolved in 200 l of DMSO), to tube B 25 mg of PDPH (dissolved in 200 l of DMSO) and to vial C 25 mg of BMPH (dissolved in 200 l of sodium acetate buffer) were added. The pH of the reaction mixture was adjusted to 5.5. Each mixture was then vortex mixed, wrapped in foil and allowed to incubate at 37 C. for 72 h on an orbital mixer. Each polymer solution was purified by gel filtration (PD 10 column) eluting with PBS pH 7.4 and the 1 ml fraction containing CA (by resorcinol assay) collected. These sample were freeze dried overnight and assayed for maleimide content as described in example 1.2.

4.3 Results

(60) The molar concentration of maleimido was 49.0 and 35.0 mol % for MBPH and PDPH respectively on the non-reducing (highly reactive) end. The molar concentration of maleimido was 41.5, 32.5 and 48.3 mol % for MBPH, PDPH and BMPH respectively on the reducing end (weakly reactive). The values on the reducing end are average of two values in each case.

4.4 Conjugation of -galactosidase (-gal) to Maleimide Activated Colominic Acids (Reducing End and Non-Reducing End)

(61) To -gal (1.0 mg; 1 ml in PBS) in separate tubes, a 15 molar excess of each maleimide activated CA (MBPH, PDPH and BMPH on non-reducing or reducing end, from above examples) was added separately. Each tube was sealed and allowed to incubate at 37 C. for 1 h whilst gently mixing. The resulting conjugate was then purified according to accepted protocols to remove free activated polymer. All samples were analysed by SDS PAGE and for -gal activity as in example 1.8

4.5 Results

(62) The results (FIG. 3) show that in all control well (with -gal) the migration of the sample is similar to that for fresh -gal. In the conjugate lanes there is considerable band broadening with maximum intensity between the 98 and 250 kDa markers which typically indicates an increase in mass which is indicative of a of polysialylated--gal. -gal activity was calculated to be 91.0-106% for the conjugated samples.