CONJUGATES OF PROTEIN DRUGS AND P/A PEPTIDES

Abstract

The present invention relates to conjugates of a protein drug and two or more P/A peptides, and pharmaceutical compositions comprising them. The conjugates of the invention exhibit an advantageously reduced immunogenicity as compared to the respective unmasked protein drugs as well as a favorable safety and tolerability profile, which render them particularly suitable for therapeutic use. The conjugates further show an enhanced plasma half-life and, thus, a prolonged duration of action as compared to the respective unmasked protein drugs, which allows for a reduction in the dosing frequency and, thus, side-effect burden. The invention also provides processes of preparing such conjugates as well as activated P/A peptides that are useful as synthetic intermediates in the preparation of the conjugates.

Claims

1. A conjugate of a protein drug and two or more P/A peptides, wherein each P/A peptide is independently a peptide R.sup.N-(P/A)-R.sup.C, wherein (P/A) is an amino acid sequence consisting of about 7 to about 1200 amino acid residues, wherein at least 80% of the number of amino acid residues in (P/A) are independently selected from proline and alanine, wherein (P/A) includes at least one proline residue and at least one alanine residue, wherein R.sup.N is a protecting group which is attached to the N-terminal amino group of (P/A) or R.sup.N is absent, and wherein R.sup.C is an amino acid residue which is bound via its amino group to the C-terminal carboxy group of (P/A) and which comprises at least two carbon atoms between its amino group and its carboxy group, wherein each P/A peptide is conjugated to the protein drug via an amide linkage formed from the carboxy group of the C-terminal amino acid residue R.sup.C of the P/A peptide and a free amino group of the protein drug, and wherein at least one of the free amino groups, which the P/A peptides are conjugated to, is not an N-terminal -amino group of the protein drug.

2. The conjugate of claim 1, wherein (P/A) is an amino acid sequence consisting of about 8 to about 400 amino acid residues, wherein at least 85% of the number of amino acid residues in (P/A) are independently selected from proline and alanine, wherein at least 95% of the number of amino acid residues in (P/A) are independently selected from proline, alanine, glycine and serine, and wherein (P/A) includes at least one proline residue and at least one alanine residue.

3. The conjugate of claim 1, wherein (P/A) is an amino acid sequence consisting of 10 to 60 amino acid residues independently selected from proline, alanine, glycine and serine, wherein at least 95% of the number of amino acid residues in (P/A) are independently selected from proline and alanine, and wherein (P/A) includes at least one proline residue and at least one alanine residue.

4. The conjugate of claim 1, wherein (P/A) is an amino acid sequence consisting of 15 to 45 amino acid residues independently selected from proline and alanine, wherein (P/A) includes at least one proline residue and at least one alanine residue.

5. The conjugate of claim 1, wherein the proportion of the number of proline residues comprised in (P/A) to the total number of amino acid residues comprised in (P/A) is 10% and 70%, preferably 20% and 50%, more preferably 25% and 40%.

6. The conjugate of claim 1, wherein (P/A) consists of (i) two or more partial sequences independently selected from AAPA and APAP, and (ii) optionally one, two or three further amino acid residues independently selected from proline and alanine.

7. The conjugate of claim 1, wherein (P/A) consists of (i) one or more partial sequences AAPAAPAP, (ii) optionally one or two partial sequences AAPA, and (iii) optionally one, two or three further amino acid residues independently selected from proline and alanine.

8. The conjugate of claim 1, wherein (P/A) consists of (i) the sequence ASPAAPAPASPAAPAPSAPA, (ii) the sequence APASPAPAAPSAPAPAAPSA, (iii) the sequence AASPAAPSAPPAAASPAAPSAPPA, (iv) a fragment of any of the aforementioned sequences, or (v) a combination of two or more of the aforementioned sequences.

9. The conjugate of claim 1, wherein R.sup.N is selected from formyl, CO(C.sub.1-4 alkyl), pyroglutamoyl and homopyroglutamoyl, wherein the alkyl moiety comprised in said CO(C.sub.1-4 alkyl) is optionally substituted with one or two groups independently selected from OH, O(C.sub.1-4 alkyl), NH(C.sub.1-4 alkyl), N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl) and COOH, or R.sup.N is absent.

10. The conjugate of claim 1, wherein R.sup.N is selected from formyl, acetyl, hydroxyacetyl, methoxyacetyl, ethoxyacetyl, propoxyacetyl, malonyl, propionyl, 2-hydroxypropionyl, 3-hydroxypropionyl, 2-methoxypropionyl, 3-methoxypropionyl, 2-ethoxypropionyl, 3-ethoxypropionyl, succinyl, butyryl, 2-hydroxybutyryl, 3-hydroxybutyryl, 4-hydroxybutyryl, 2-methoxybutyryl, 3-methoxybutyryl, 4-methoxybutyryl, glycine betainyl, glutaryl, pyroglutamoyl, and homopyroglutamoyl.

11. The conjugate of claim 1, wherein R.sup.N is absent.

12. The conjugate of claim 1, wherein R.sup.C is H.sub.2N(C.sub.2-12 hydrocarbyl)-COOH, wherein it is preferred that R.sup.C is selected from H.sub.2N(CH.sub.2).sub.3-10COOH, H.sub.2N-phenyl-COOH, and H.sub.2N-cyclohexyl-COOH, and wherein it is more preferred that R.sup.C is selected from H.sub.2N(CH.sub.2).sub.4COOH, H.sub.2N(CH.sub.2).sub.5COOH, H.sub.2N(CH.sub.2).sub.6COOH, H.sub.2N(CH.sub.2).sub.7COOH, H.sub.2N(CH.sub.2).sub.8COOH, ##STR00009##

13. The conjugate of claim 1, wherein R.sup.C is alanine or proline.

14. The conjugate of claim 1, wherein the P/A peptides comprised in said conjugate adopt a random coil conformation.

15. The conjugate of claim 1, wherein all of the P/A peptides comprised in said conjugate are the same.

16. The conjugate of claim 1, wherein at least one of the free amino groups, which the P/A peptides are conjugated to, is an -amino group of a lysine residue of the protein drug.

17. The conjugate of claim 1, wherein the free amino groups, which the P/A peptides are conjugated to, are selected from the -amino group(s) of any lysine residue(s) of the protein drug, the N-terminal -amino group(s) of the protein drug or of any subunit(s) of the protein drug, and any combination thereof.

18. The conjugate of claim 1, wherein said conjugate is composed of the protein drug and the P/A peptides at a ratio m.sub.(P/A peptides)/m.sub.(protein drug) which assumes a value from 0.1 to 50, wherein m.sub.(P/A peptides) is the combined total number of amino acid residues in the moieties (P/A) of all P/A peptides comprised in the conjugate and wherein m.sub.(protein drug) is the total number of amino acid residues in the protein drug comprised in the conjugate.

19. The conjugate of claim 18, wherein the ratio m.sub.(P/A peptides)/m.sub.(protein drug) assumes a value from 0.5 to 5.

20. The conjugate of claim 1, wherein the protein drug is an enzyme.

21. The conjugate of claim 1, wherein the protein drug is selected from urate oxidase, adenosine deaminase, purine nucleoside phosphorylase, an L-phenylalanine degrading enzyme, phenylalanine hydroxylase, phenylalanine ammonia lyase, an antioxidant enzyme, superoxide dismutase, catalase, rhodanese, an organophosphate degrading enzyme, phosphotriesterase, organophosphorus anhydrolase, an alcohol oxidizing enzyme, alcohol dehydrogenase, alcohol oxidase, an acetaldehyde degrading enzyme, aldehyde dehydrogenase, an L-glutamine degrading enzyme, glutaminase, an L-arginine degrading enzyme, arginase, arginine deiminase, a plasminogen activating enzyme, tissue plasminogen activator, reteplase, streptokinase, urokinase, a fibrinogenolytic enzyme, ancrod, batroxobin, cystathionine--synthase, a homocysteine thiolactone degrading enzyme, paraoxonase 1, bleomycin hydrolase, human serum HTase, human biphenyl hydrolase-like protein, a methionine degrading enzyme, methioninase, cystathionine--lyase engineered for methionine specificity, a homocysteine degrading enzyme, a cysteine degrading enzyme, a cystine degrading enzyme, hyaluronidase, -glucosidase, -glucuronidase, -galactosidase, -galactosidase A, glucocerebrosidase, imiglucerase, a broad-spectrum protease without activity for P/A peptides, ananain, comosain, ocriplasmin, an acetylcholine degrading enzyme, butyrylcholinesterase, acetylcholinesterase, a cocaine degrading enzyme, cocaine esterase, chondroitinase, collagenase, N-acetylgalactosamine-4-sulfatase, iduronate-2-sulfatase, -L-iduronidase, porphobilinogen, a DNase, dornase , an oxalate degrading enzyme, oxalate decarboxylase, N-sulphoglucosamine sulphohydrolase, acetyl CoA -glucosaminide acetyltransferase, N-acetylglucosamine-6-sulfatase, N--acetylglucosaminidase, N-acetylgalactosamine-6-sulfate sulfatase, tripeptidyl peptidase 1, phosphoglycerate kinase, coagulation factor IX, coagulation factor VIII, coagulation factor VIIa, coagulation factor Xa, coagulation factor IV, coagulation factor XIII, a protease with specificity for a protein of the complement pathway, a version of membrane type serine protease 1 engineered for factor C3 specificity, a protease with specificity for VEGF or VEGF receptor, an engineered version of membrane type serine protease 1, human angiotensin converting enzyme 2, an RNase, onconase, ranpirnase, bovine seminal RNase, RNase T1, -sarcin, RNase P, actibind, RNase T2, alkaline phosphatase, human tissue-nonspecific alkaline phosphatase, asfotase alfa, aspartylglucosaminidase, aspartoacylase, -mannosidase, galactosylceramidase, glutamate oxaloacetate transaminase 1, granzyme B, a bacteriolysin, an endolysin, an ectolysin, an N-acetylmuramidase, an N-acetyl-P3-D-glucosaminidase, an N-acetylmuramoyl-L-alanine amidase, an L-alanoyl-D-glutamate endopeptidase, a cysteine/histidine-dependent amidohydrolase/peptidase, lysostaphin, a phage tail-associated muralytic enzyme, a fusion protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme catalytic domain and the cell-wall-binding SH3b domain of lysostaphin, ectonucleotide pyrophosphatase/phosphodiesterase-1, an endo-P3-N-acetylglucosaminidase, EndoS or EndoS2 from Streptococcus pyogenes, an immunoglobulin degrading enzyme, IdeS of Streptococcus pyogenes, IgA protease of Neisseria gonorrhoeae, lecithin cholesterol acyl transferase, thymidine phosphorylase, arylsulfatase A, cyclin-dependent kinase-like 5 protein, gliadin peptidase, a kynurenine-degrading enzyme, kynureninase, myotubularin, and a catalytic antibody or a functional fragment thereof.

22. A pharmaceutical composition comprising a conjugate as defined in claim 1 and a pharmaceutically acceptable excipient.

23. (canceled)

24. A process of preparing a conjugate as defined in claim 1, the process comprising: (a) coupling an activated P/A peptide of the formula R.sup.N-(P/A)-R.sup.C-act, wherein R.sup.C-act is a carboxy-activated form of R.sup.C, wherein R.sup.C and (P/A) are as defined in the conjugate to be prepared, and wherein R.sup.N is a protecting group which is attached to the N-terminal amino group of (P/A), with a protein drug to obtain a conjugate of the protein drug and the P/A peptides in which R.sup.N is a protecting group; and (b) optionally removing the protecting groups R.sup.N from the P/A peptides contained in the conjugate obtained in step (a) to obtain a conjugate of the protein drug and the P/A peptides in which R.sup.N is absent.

25. The process of claim 24, wherein the activated carboxy group of the amino acid residue R.sup.C-act in the activated P/A peptide is an active ester group; wherein said active ester group is preferably selected from any one of the following groups: ##STR00010## ##STR00011## ##STR00012## ##STR00013## and wherein said active ester group is more preferably a 1-hydroxybenzotriazole active ester group of the following formula: ##STR00014##

26. The process of claim 24, wherein the activated carboxy group of the amino acid residue R.sup.C-act in the activated P/A peptide is an anhydride group; wherein said anhydride group is preferably (i) a propylphosphonic anhydride (T3P) group of the following formula: ##STR00015## or (ii) a mixed carbonic acid anhydride group, such as a group of the following formula: ##STR00016##

27. The process of claim 24, wherein the activated carboxy group of the amino acid residue R.sup.C-act in the activated P/A peptide is an acyl halide group, wherein said acyl halide group is preferably COCl or COF.

28. The process of claim 24, wherein the process comprises, before step (a), a further step of converting a P/A peptide of the formula R.sup.N-(P/A)-R.sup.C, wherein R.sup.C and (P/A) are as defined in the conjugate to be prepared, and wherein R.sup.N is a protecting group which is attached to the N-terminal amino group of (P/A), into the activated P/A peptide.

29. The process of claim 28, wherein the activated carboxy group of the amino acid residue R.sup.C-act in the activated P/A peptide is a 1-hydroxybenzotriazole active ester group having the formula ##STR00017## and wherein the step of converting the P/A peptide into the activated P/A peptide is conducted by reacting the P/A peptide with a salt of a phosphonium, uronium or immonium ester of 1-hydroxybenzotriazole in the presence of a base; wherein the salt of a phosphonium, uronium or immonium derivative of 1-hydroxybenzotriazole is preferably selected from BOP, PyBOP, BDP, HBTU, TBTU, BCC, TDBTU, BOMI and BDMP, and is more preferably TBTU.

30. An activated P/A peptide of the formula R.sup.N-(P/A)-R.sup.C-act, wherein R.sup.N is a protecting group which is attached to the N-terminal amino group of (P/A), wherein (P/A) is an amino acid sequence consisting of about 7 to about 1200 amino acid residues, wherein at least 80% of the number of amino acid residues in (P/A) are independently selected from proline and alanine, wherein (P/A) includes at least one proline residue and at least one alanine residue, and wherein R.sup.C-act is an amino acid residue which has an activated carboxy group, which is bound via its amino group to the C-terminal carboxy group of (P/A), and which comprises at least two carbon atoms between its amino group and its activated carboxy group.

31. The activated P/A peptide of claim 30, wherein the activated carboxy group of the amino acid residue R.sup.C-act is an active ester group; wherein said active ester group is preferably selected from any one of the following groups: ##STR00018## ##STR00019## ##STR00020## ##STR00021## and wherein said active ester group is more preferably a 1-hydroxybenzotriazole active ester group of the following formula: ##STR00022##

32. The activated P/A peptide of claim 30, wherein the activated carboxy group of the amino acid residue R.sup.C-act is an anhydride group; wherein said anhydride group is preferably (i) a propylphosphonic anhydride (T3P) group of the following formula: ##STR00023## or (ii) a mixed carbonic acid anhydride group, such as a group of the following formula: ##STR00024##

33. The activated P/A peptide of claim 30, wherein the activated carboxy group of the amino acid residue R.sup.C-act is an acyl halide group, wherein said acyl halide group is preferably COCl or COF.

34. The activated P/A peptide of claim 30, wherein (P/A) is an amino acid sequence consisting of about 8 to about 400 amino acid residues, wherein at least 85% of the number of amino acid residues in (P/A) are independently selected from proline and alanine, wherein at least 95% of the number of amino acid residues in (P/A) are independently selected from proline, alanine, glycine and serine, and wherein (P/A) includes at least one proline residue and at least one alanine residue.

35. The activated P/A peptide of claim 30, wherein (P/A) is an amino acid sequence consisting of 10 to 60 amino acid residues independently selected from proline, alanine, glycine and serine, wherein at least 95% of the number of amino acid residues in (P/A) are independently selected from proline and alanine, and wherein (P/A) includes at least one proline residue and at least one alanine residue.

36. The activated P/A peptide of claim 30, wherein (P/A) is an amino acid sequence consisting of 15 to 45 amino acid residues independently selected from proline and alanine, wherein (P/A) includes at least one proline residue and at least one alanine residue.

37. The activated P/A peptide of claim 30, wherein the proportion of the number of proline residues comprised in (P/A) to the total number of amino acid residues comprised in (P/A) is 10% and 70%, preferably 20% and 50%, more preferably 25% and 40%.

38. The activated P/A peptide of claim 30, wherein (P/A) consists of (i) two or more partial sequences independently selected from AAPA and APAP, and (ii) optionally one, two or three further amino acid residues independently selected from proline and alanine.

39. The activated P/A peptide of claim 30, wherein (P/A) consists of (i) one or more partial sequences AAPAAPAP, (ii) optionally one or two partial sequences AAPA, and (iii) optionally one, two or three further amino acid residues independently selected from proline and alanine.

40. The activated P/A peptide of claim 30, wherein (P/A) consists of (i) the sequence ASPAAPAPASPAAPAPSAPA, (ii) the sequence APASPAPAAPSAPAPAAPSA, (iii) the sequence AASPAAPSAPPAAASPAAPSAPPA, (iv) a fragment of any of the aforementioned sequences, or (v) a combination of two or more of the aforementioned sequences.

41. The activated P/A peptide of claim 30, wherein R.sup.N is selected from formyl, CO(C.sub.1-4 alkyl), pyroglutamoyl and homopyroglutamoyl, wherein the alkyl moiety comprised in said CO(C.sub.1-4 alkyl) is optionally substituted with one or two groups independently selected from OH, O(C.sub.1-4 alkyl), NH(C.sub.1-4 alkyl), N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl) and COOH.

42. The activated P/A peptide of claim 30, wherein R.sup.N is selected from formyl, acetyl, hydroxyacetyl, methoxyacetyl, ethoxyacetyl, propoxyacetyl, malonyl, propionyl, 2-hydroxypropionyl, 3-hydroxypropionyl, 2-methoxypropionyl, 3-methoxypropionyl, 2-ethoxypropionyl, 3-ethoxypropionyl, succinyl, butyryl, 2-hydroxybutyryl, 3-hydroxybutyryl, 4-hydroxybutyryl, 2-methoxybutyryl, 3-methoxybutyryl, 4-methoxybutyryl, glycine betainyl, glutaryl, pyroglutamoyl, and homopyroglutamoyl.

43. The activated P/A peptide of claim 30, wherein R.sup.C-act is H.sub.2N(C.sub.2-12 hydrocarbyl)-COOH and wherein the COOH group of said H.sub.2N(C.sub.2-12 hydrocarbyl)-COOH is in the form of an activated carboxy group.

44. The activated P/A peptide of claim 30, wherein R.sup.C-act is selected from H.sub.2N(CH.sub.2).sub.3-10COOH, H.sub.2N-phenyl-COOH, and H.sub.2N-cyclohexyl-COOH, and wherein the COOH group of each one of the aforementioned groups R.sup.C-act is in the form of an activated carboxy group.

45. The activated P/A peptide of claim 30, wherein R.sup.C-act is selected from H.sub.2N(CH.sub.2).sub.4COOH, H.sub.2N(CH.sub.2).sub.5COOH, H.sub.2N(CH.sub.2).sub.6COOH, H.sub.2N(CH.sub.2).sub.7COOH, H.sub.2N(CH.sub.2).sub.8COOH, ##STR00025## and wherein the COOH group of each one of the aforementioned groups R.sup.C-act is in the form of an activated carboxy group.

46. The activated P/A peptide of claim 30, wherein R.sup.C-act is alanine having an activated carboxy group, or R.sup.C-act is proline having an activated carboxy group.

47. The activated P/A peptide of any claim 30, wherein the activated P/A peptide adopts a random coil conformation.

48. (canceled)

Description

[0113] The invention is also described by the following illustrative figures. The appended figures show:

[0114] FIG. 1: Reaction scheme for the coupling of P/A peptides to proteins via lysine residues. In the presence of the non-nucleophilic base N,N-diisopropylethylamine (DIPEA, Hnig's base) and with DMSO as solvent the N-terminally protected P/A peptide (e.g. acetyl-P/A #1(40)) is activated via its C-terminus with O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium tetrafluoroborate (TBTU). The resulting hydroxybenzotriazol (HOBt) active ester of the peptide is subsequently used to selectively derivatize the amino groups (-amino groups of lysine residues or -amino group of N-terminus) of a protein with the P/A peptide through formation of a peptide or isopeptide bond while free HOBt is released. This coupling step is performed in aqueous solution (e.g. PBS buffer) with a content of organic solvent 30%. The P/A-protein conjugate may be purified from residual P/A peptide/coupling reagent by dialysis and/or chromatography (e.g. ion exchange chromatography).

[0115] FIG. 2: SDS-PAGE analysis of RNase A conjugated with Ac-P/A #1(40) peptide. RNase A from bovine pancreas was conjugated with Ac-P/A #1(40) (SEQ ID NO: 1) as described in Example 1 (10 mg P/A peptide per 1 mg RNase A), After quenching residual TBTU with glycine in molar excess, the SDS-polyacrylamide gel was loaded both with unmodified RNase A (2 g or 8 g in lanes 1 and 2, respectively) and with the Ac-P/A #1(40)-RNase A conjugate (2 g or 8 g in lanes 3 and 4, respectively). The conjugate appeared as three distinct bands with high apparent molecular weight. The individual bands correspond to protein conjugates varying by one coupled P/A peptide each. After the coupling reaction, unmodified RNase A was not detectable. Lane M: Pierce Unstained Protein MW Marker (Thermo Fisher Scientific).

[0116] FIG. 3: Chemical structures of P/A #1(20) peptides. P/A(20) peptides differing in their C-terminal linker amino acid, all obtained by solid-phase peptide synthesis: A, glycine (reference example); B, none (corresponding to L-alanine of the P/A(20) peptide sequence); C, D-alanine; D, -alanine; E, L-proline; F, -aminobutyric acid (GABA); G, 5-aminovaleric acid (Ava); H, 6-aminohexanoic acid (Ahx); I, 8-aminooctanoic acid (Aoa); J, 4-aminocyclohexanecarboxylic acid (ACHA); K, 4-aminobenzoic acid (Abz). In order to avoid polymerization of the peptides upon chemical activation of the C-terminus, the N-terminus was protected with a pyroglutamoyl (Pga) residue in these examples.

[0117] FIG. 4: SDS-PAGE analysis of RNase A conjugated with Pga-P/A #1(20)-Ahx peptide. RNase A from bovine pancreas was conjugated with Pga-P/A #1(20)-Ahx peptide (SEQ ID NO: 9) as described in Example 2. The P/A peptide-to-protein ratio during the coupling reaction was varied between 0.5 mg and 15 mg P/A peptide per 1 mg RNase A. The gel was loaded with 7 g of conjugated RNase A from each coupling reaction. Additionally, unconjugated RNase A was loaded onto the SDS-polyacrylamide gel (lane 0). The number of coupled P/A peptides as determined by counting the bands in the successive ladders starting from the unconjugated RNase A are marked on the right. Lane M: Pierce Unstained Protein MW Marker (Thermo Fisher Scientific).

[0118] FIG. 5: SDS-PAGE analysis of B. fastidiosus uricase conjugated with Pga-P/A #1(20) peptides differing in their C-terminal amino acid. Recombinant B. fastidiosus uricase was conjugated with different Pga-P/A #1(20) peptides (SEQ ID NOs: 2 to 12), differing in their C-terminal amino acid (see FIG. 3) which acts as a linker between P/A moiety and protein. The coupling was performed as described in Example 2. The P/A peptide-to-protein ratio during the coupling reaction was varied between 0.5 mg and 10 mg P/A peptide per 1 mg uricase. The gel was loaded with 7 g of conjugated uricase from each coupling reaction. Additionally, unconjugated uricase was loaded onto the SDS-polyacrylamide gel (arrows). PageRuler Plus Prestained (Thermo Fisher Scientific) was applied to lane M.

[0119] FIG. 6: Coupling efficiency of B. fastidiosus uricase depending on the C-terminal (linker) amino acid of the conjugated P/A(20) peptide. SDS-PAGE (see FIG. 5) of uricase conjugated to Pga-P/A(20) peptides differing in their C-terminal linker amino acid (see FIG. 3) was evaluated densitometrically, and the arithmetic mean of the number of coupled peptides, weighted for the corresponding band intensities (P), was plotted against the mass ratio between peptide and protein (R) applied during the coupling reaction. Data were fitted using a saturation function and maximal coupling ratio (P.sub.max) as well as half-maximal mass ratio (R.sub.1/2) were extrapolated from the corresponding curves (listed in Table 2, see Example 3).

[0120] FIG. 7: SDS-PAGE analysis of uricase/Pga-PA(20)-Ahx conjugate. Recombinant B. fastidiosus uricase was purified by size exclusion chromatography and conjugated with a 2-fold mass ratio of Pga-P/A(20)-Ahx (lane 3). Application of unmodified uricase (lane 1) and uricase conjugated with a 0.5-fold mass ratio of Pga-P/A(20)-Ahx (lane 2) allowed counting of the bands in successive ladders starting from the unconjugated protein. Thus, the number of coupled P/A peptides could be precisely determined (indicated on the right). Lane M: PageRuler Plus Prestained (Thermo Fisher Scientific).

[0121] FIG. 8: Size exclusion chromatography of uricase/Pga-PA(20)-Ahx conjugate. (A) Overlay of elution profiles for recombinant B. fastidiosus uricase conjugated to Pga-P/A(20)-Ahx (described in Example 4, see FIG. 7) and unmodified uricase (dotted line). 150 L of the purified protein at a concentration of 1 mg/ml was applied to a Superdex S200 10/300 GL column equilibrated with PBS buffer. Absorption at 280 nm was monitored and the peak of each chromatography run was normalized to 100%. (B) Calibration curve for the chromatograms from (A) using a Superdex S200 10/300 GL column. The logarithm of the molecular weight of marker proteins (ovalbumin, 43.0 kDa; bovine serum albumin, 66.3 kDa; alcohol dehydrogenase, 150 kDa, -amylase, 200 kDa, apo-ferritin, 440 kDa) was plotted vs. their elution volumes (black circles) and fitted by a straight line. From the observed elution volumes of the tetrameric uricase and its Pga-P/A(20)-Ahx peptide conjugate (black squares) the apparent molecular sizes were determined as follows: uricase, 132 kDa (true mass 142 kDa); uricase/Pga-P/A(20)-Ahx conjugate, 408 kDa (true mass: 197 kDa). These data show that the chemically conjugated P/A peptides confer a much enlarged hydrodynamic volume.

[0122] FIG. 9: ESI-MS analysis of P/A #1(20) active esters. Active esters of the Pga-P/A #1(20)-Ahx peptide with 1-hydroxybenzotriazol (A), 4-nitrophenyl (B) or pentafluorophenyl (C) were prepared as described in Example 5 and m/z spectra were measured by ESI-MS using the positive ion mode. Mass peaks that correspond either to the unmodified or the activated P/A peptide as well as detectable adducts of a single water molecule to those peptides are labelled with their predicted and measured masses.

[0123] FIG. 10: SDS-PAGE analysis of uricase conjugated with different P/A(20) active esters. 1-Hydroxybenzotriazol (HOBt), 4-nitrophenyl (pNP) and pentafluorophenyl (PFP) active esters of the Pga-P/A #1(20)-Ahx peptide were prepared as described in Example 5 and coupled to Bacillus fastidiosus uricase. For the coupling of the HOBt active ester, the P/A peptide-to-protein ratio during the coupling reaction was varied between 1 mg and 10 mg P/A peptide per 1 mg uricase. For the coupling of the pNP and the PFP active esters, the applied P/A peptide to uricase mass ratio was 6:1. Lane 0: unconjugated uricase. Lane M: PAGE Ruler Prestaind Protein MW Marker (Thermo Fisher Scientific). Samples were analysed by 10% SDS-PAGE followed by Coomassie staining.

[0124] FIG. 11: SDS-PAGE analysis of alcohol dehydrogenase conjugated with different P/A(40) peptides. Alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae was conjugated either (A) with Pga-P/A #1(40)-Ahx (SEQ ID NO: 18) or (B) with Pga-P/A #3(40)-Ahx (SEQ ID NO: 19) as described in Example 6. The P/A peptide-to-protein ratio during the coupling reaction was varied between 1 mg and 10 mg P/A peptide per 1 mg. After dialysis against PBS, 5 g of each coupling mixture was analyzed by SDS-PAGE. The conjugates appear as ladders of distinct bands with increasing molecular weight differing by one coupled P/A peptide each. Lane 0: unconjugated ADH. Lane M: PAGE ruler prestained MW Marker (Thermo Fisher Scientific).

[0125] FIG. 12: SDS-PAGE analysis of adenosine deaminase conjugated with different P/A(40) peptides. Adenosine deaminase (ADA) from Bos taurus was conjugated either with Pga-P/A #1(40)-Ahx (SEQ ID NO: 18) or with Pga-P/A #3(40)-Ahx (SEQ ID NO: 19) as described in Example 7. The P/A peptide-to-protein ratio during the coupling reaction was varied between 1 mg and 10 mg P/A peptide per 1 mg protein. After dialysis against PBS, 5 g of each coupling mixture was analyzed by SDS-PAGE. The conjugates appear as ladders of distinct bands with increasing molecular weight differing by one coupled P/A peptide each. Lane 0: unconjugated ADA. Lane M: PAGE ruler prestained MW Marker (Thermo Fisher Scientific).

[0126] FIG. 13: SDS-PAGE analysis of RNase A conjugated with Pga-PAS #1(40)-Ahx peptide. RNase A from bovine pancreas was conjugated with Pga-PAS #1(40) (SEQ ID NO: 22) as described in Example 8 (4 mg PAS peptide per 1 mg RNase A). After quenching residual TBTU with glycine in molar excess, the Pga-PAS #1(40)-Ahx-RNase A conjugate was loaded in different amounts on an SDS-polyacrylamide gel (0.5 g, 1 g, 2 g or 10 g in lanes 1, 2, 3 and 4, respectively). The conjugate appeared as four distinct bands with high apparent molecular weight. The individual bands correspond to protein conjugates varying by one coupled PAS peptide each. After the coupling reaction, remaining unmodified RNase A was no longer detectable. Lane M: PAGE ruler prestained MW Marker (Thermo Fisher Scientific).

[0127] The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

EXAMPLES

Example 1: Preparation of Acetyl-PA(40)-RNase A Conjugate

[0128] 35 mg of the Ac-P/A #1(40) peptide (SEQ ID NO: 1) (TFA salt, purity 98%; Peptide Specialties Laboratories, Heidelberg, Germany) was dissolved in 1268 L of anhydrous DMSO (99.9%; Sigma-Aldrich, Taufkirchen, Germany). To achieve chemical activation of the P/A peptide via its terminal carboxylate group, 214 L of a solution of 500 mM TBTU (CAS #125700-67-6; Iris Biotech, Marktredwitz, Germany) in DMSO and, after mixing, 18 L DIPEA (99.5%, biotech. Grade, Sigma-Aldrich) was added. The whole mixture was vortexed briefly and incubated for 20 min at 25 C. (see FIG. 1). In this setup, the peptide concentration was 7.14 mM and the molar ratio between DIPEA, TBTU and Ac-P/A #1(40) was 10:10:1.

[0129] Ribonuclease A from bovine pancreas (RNase A; Sigma-Aldrich, catalogue No. 83831; SEQ ID NO: 16) was dissolved in phosphate-buffered saline (PBS: 115 mM NaCl, 4 mM KH.sub.2PO.sub.4 and 16 mM Na.sub.2HPO.sub.4, pH 7.4) to obtain a protein concentration of 2 mg/mL and cooled on ice. 3.5 mL of the RNase A solution was mixed with the activated peptide solution (1.5 mL), resulting in a mass ratio between Ac-P/A #1(40) and protein of 5:1, and incubated at room temperature for 30 min to allow coupling. To quench residual TBTU, glycine (pH 8, adjusted with Tris base) was added to the protein sample (final glycine concentration: 250 mM) prior to heating the sample for SDS-PAGE (as shown in FIG. 2). The resulting conjugate revealed three distinct bands with high apparent molecular weight. The individual bands correspond to a distribution of protein conjugates differing by the number of coupled P/A peptides. Unmodified RNase A was not detectable.

Example 2: Optimization of Coupling Ratio for the Preparation of Pyroglutamoyl-P/A-(20)-aminohexanoyl-RNase A

[0130] 3 mg Pga-P/A #1(20)-Ahx peptide (TFA salt, purity 98%; Almac Group, Craigavon, UK) (SEQ ID NO: 9) was dissolved in 37.3 l of a 435 mM TBTU solution in DMSO. The chemical activation of the P/A peptide via its terminal carboxylate group was started by addition of 2.7 L DIPEA to the peptide solution and vortexing. In this setup, the concentration of the peptide was 40.6 mM and the molar ratio between DIPEA, TBTU and Pga-P/A #1(20)-Ahx was 10:10:1. After 10 min incubation at 25 C. the mixture was diluted with DMSO in Eppendorf tubes according to Table 1. Each Eppendorf tube finally contained a volume of 15 L of the diluted peptide solution.

[0131] A solution of Ribonuclease A from bovine pancreas (RNase A; Sigma-Aldrich, catalogue No. 83831) with a concentration of 2 mg/mL was prepared in PBS. 35 L of this protein solution were pipetted into each Eppendorf tube and mixed by repeated pipetting and vortexing. The coupling reaction was allowed to take place at 25 C. for 30 min. The reaction was quenched by addition of glycine (pH 8.0, adjusted with Tris base) to a final concentration of 250 mM. SDS-PAGE analysis of the conjugates is shown in FIG. 4. The individual bands correspond to protein conjugates varying by one coupled P/A peptide each. The application of coupling reactions with lower ratios between peptide and protein allowed counting of the bands in successive ladders starting from the unconjugated protein, thus allowing precise determination of the number of coupled P/A peptides. A coupling ratio of 3 mg Pga-P/A #1(20)-Ahx peptide per 1 mg RNase A was sufficient to achieve coupling of all amino groups (10 lysine residues and N-terminus). In this case, the ratio between amino acid residues in the coupled P/A #1(20) peptides and in the enzyme (RNase A) was 1.77.

TABLE-US-00002 TABLE 1 Typical dilution series of activated P/A peptide for coupling with the test protein Peptide stock Mass ratio solution [L] DMSO [L] 15x 15 0 10x 10 5 6x 6 9 3x 3 12 2x 2 13 1x 1 14 0.5x.sup. 0.5 14.5

Example 3: Preparation of Pga-P/A #1(20)-Uricase Conjugates with Different Linkers

[0132] Freeze-dried recombinant Bacillus fastidiosus Uricase (Sigma-Aldrich, catalogue No. 94310; SEQ ID NO: 17) was dissolved in PBS and dialyzed against PBS over night at 4 C. using a Slide-A-Lyzer dialysis cassette (MWCO 10.000; Thermo Fisher Scientific, Waltham, Mass.) to remove small molecular weight contaminants.

[0133] 3 mg of each of the Pga-P/A #1(20) peptides with either glycine, L-alanine, D-alanine, -alanine, L-proline, 4-aminobutanoic acid (GABA), 5-aminopentanoic acid (Ava), 6-aminohexanoic acid (Ahx), 8-aminooctanoic acid (Aoa), 4-aminobenzoic acid (Abz) or 4-aminocyclohexanecarboxylic acid (ACHA) as C-terminal amino acid, R.sup.c (see FIG. 3; TFA salts, purity 98%; Peptide Specialties Laboratories) were conjugated to the Uricase (2 mg/mL in PBS) in the same manner as described for RNase A in Example 2. SDS-PAGE analysis of the conjugates is shown in FIG. 5. In order to quantify the average number of coupled P/A peptides for each peptide-to-protein ratio applied during the coupling reaction, the SDS-polyacrylamide gels were scanned after staining with Coomassie Brilliant Blue R-250 on a Perfection V700 Photo scanner (Epson, Meerbusch, Germany) and densitometrically evaluated using the Quant v12.2 software (TotalLab, Newcastle upon Tyne, UK). The number of coupled P/A peptides for each band (i.e., the molar ratio or peptide-to-protein stoichiometry) was assigned by counting the bands starting from the unconjugated Uricase. Then, the average number of coupled peptides per enzyme, calculated as the arithmetic mean of the number of coupled peptides weighted for the corresponding band intensities (P) as seen in SDS-Page, was plotted against the mass ratio (R) applied during the coupling reaction (see FIG. 6). Using Kaleidagraph v4.1 software (Synergy Software, Reading, Pa.), the data was fitted to the following saturation function:

P(R)=P.sub.maxR/(R.sub.1/2+R)

P.sub.max corresponds to the maximal (asymptotic) average number of coupled peptides, while R.sub.1/2 corresponds to the coupling ratio with half-maximal number of coupled peptides.

[0134] The P.sub.max and R.sub.1/2 values determined for each of the tested peptides are listed in Table 2. While the C-terminal amino acid, R.sup.c, of the tested P/A peptides has only insignificant influence on R.sub.1/2, this linker group showed a pronounced effect on the maximum number of coupled peptides (P.sub.max). Saturation of all Uricase amino groups (16 lysine residues and the N-terminus of each subunit) was achieved with Ahx or with Ava as linker amino acid, as indicated by P.sub.max values 17. The P/A peptide with C-terminal glycine had the lowest P.sub.max value of 3.5. Intermediate coupling efficacy, with P.sub.max values in the range of 6.9 to 9.9, was achieved with C-terminal alanine and proline. Increasing the length of the aliphatic linker amino acid resulted in an increased coupling efficacy (as indicated by P.sub.max), reaching a maximum with the C5 amino acid Ava.

[0135] Both the aliphatic and aromatic C6 cyclic linkers showed high P.sub.max values, similar to the linear 6-aminohexanoic acid linker.

TABLE-US-00003 TABLE 2 P/A peptide coupling efficacy R.sub.1/2 P.sub.max Glycine 3.5 0.6 3.5 0.3 L-Alanine 1.6 0.5 6.9 0.6 D-Alanine 5.8 1.1 8.7 0.9 -Alanine 1.9 0.2 13.9 0.5 L-Proline 1.8 0.1 9.9 0.1 4-Aminobutanoic acid (GABA) 2.2 0.2 15.6 0.4 5-Aminopentanoic acid (Ava) 1.5 0.2 18.6 0.7 6-Aminohexanoic acid (Ahx) 1.3 0.2 17.9 0.9 8-Aminooctanoic acid (Aoa) 1.1 0.1 18.3 0.5 4-Aminocyclohexanecarboxylic 1.9 0.2 18.9 0.6 acid (ACHA) 4-Aminobenzoic acid (Abz) 1.0 0.1 18.4 0.6

Example 4: Characterisation of P/A 20-Uricase Conjugates

[0136] Freeze-dried recombinant Bacillus fastidiosus Uricase (Sigma-Aldrich, catalogue No. 94310; SEQ ID NO: 17) was dissolved in PBS and purified as a tetramer by size exclusion chromatography on a Superdex 200 increase 10/300 column (GE Healthcare) equilibrated with PBS.

[0137] 1 mg Pga-P/A(20)#1-Ahx peptide dissolved in DMSO was activated with TBTU and DIPEA as described in Example 3 and mixed with 0.5 mg of the purified uricase (2 mg/mL in PBS). The reaction mixture was incubated at 25 C. for 30 min and subsequently dialyzed against 5 L AEX buffer (25 mM Na-borate pH 8.8, 1 mM EDTA) over night at 4 C. using a regenerated cellulose membrane dialysis tube (MWCO 50 kDa; Spectrum Laboratories, Los Angeles, Calif.). In order to remove unreacted coupling reagents, the dialyzed enzyme conjugate was subjected to anion exchange chromatography on a 1 mL Resource Q column (GE Healthcare). The column was equilibrated with AEX buffer and the protein conjugate was eluted using a linear NaCl concentration gradient from 0 to 300 mM over 30 column volumes.

[0138] Applying eluate samples to SDS-PAGE, alongside a coupling reaction carried out with a lower ratio of 0.5 mg peptide per mg uricase, allowed determination of the coupling ratio observed for the preparative setup described in the preceding paragraph, thus yielding 6-9 PA peptides per uricase monomer (see FIG. 7).

[0139] Size exclusion chromatography (SEC) was carried out on a Superdex S200 increase 10/300 GL column (GE Healthcare Europe, Freiburg, Germany) at a flow rate of 0.5 mL/min using an kta Purifier 10 system (GE Healthcare) with PBS as running buffer. 150 L samples of the uricase-P/A(20) conjugate and of unmodified uricase were individually applied to the column and the chromatography profiles were superimposed (see FIG. 8A). Both proteins eluted in a single homogenous peak.

[0140] For column calibration (see FIG. 8B), 150 L of an appropriate mixture of the following globular proteins (Sigma, Deisenhofen, Germany) were applied in PBS at protein concentrations between 0.5 mg/ml and 1.0 mg/ml: cytochrome c, 12.4 kDa; ovalbumin, 43.0 kDa; bovine serum albumin, 66.3 kDa; alcohol dehydrogenase, 150 kDa; 1-amylase, 200 kDa; apo-ferritin, 440 kDa; thyroglobulin, 660 kDa.

[0141] As result, the chemically conjugated uricase preparation exhibited a significantly larger size during SEC than corresponding globular proteins with the same molecular weight. The apparent size increase for uricase-P/A(20)n was 3.1-fold compared with the unmodified uricase, whereas the true mass of the conjugate was only larger by 1.3 to 1.5-fold. This observation clearly indicates a much increased hydrodynamic volume conferred to the biologically active uricase enzyme by conjugation with Pro/Ala peptides according to this invention.

[0142] Urate oxidase activity of both the uricase-P/A(20) conjugate and unmodified uricase was determined by the decrease in absorbance at 293 nm resulting from the oxidation of uric acid to allantoin. Briefly, 10 L of enzyme solution was mixed with 200 L of a 300 M uric acid solution (sodium salt; Sigma-Aldrich), in 100 mM Na-borate buffer pH 9.2 containing 1 mM EDTA and incubated for 5 min at 30 C. Absorbance of this solution at 293 nm was measured using a SpectraMax 250 microwell plate reader (Molecular Devices, Sunnyvale, Calif.). The activity was calculated from the decrease in absorbance using a calibration curve that was obtained from a dilution series of uric acid. The results are summarized in Table 3.

TABLE-US-00004 TABLE 3 Enzymatic activity of uricase-P/A(20) conjugate mol PA Specific Rel. peptide/mol activity activity monomer [U/mg]* [%] unmodified uricase 0 7.0 0.7 100 Uricase-P/A(20).sub.n 6-9 4.5 0.6 64 *The specific activity relates to the mass of the enzyme component only, i.e. neglecting the additional mass of the conjugate contributed by the coupled P/A#1(20) peptides.

Example 5: Synthesis, Isolation and Conjugation of Various Pga-P/A(20)-Ahx Active Esters

[0143] For the preparation of Pga-P/A(20)-Ahx peptides activated as esters with either 1-hydroxybenzotriazol (HOBt), 4-nitrophenyl (pNP) or pentafluorophenyl (PFP), 10 mg Pga-P/A #1(20)-Ahx peptide (TFA salt, purity 98%; Almac Group, Craigavon, UK) (SEQ ID NO: 9) was dissolved in 360 l of a 150 mM DIPEA solution in DMF for each activation. The chemical activation of the P/A peptide via its terminal carboxylate group was then started by addition of 360 l of a 150 mM solution of either TBTU, 4-nitrophenyl trifluoroacetate (Sigma-Aldrich) or pentafluorophenyl diphenylphosphinate (Sigma-Aldrich), respectively, in DMF to the peptide/DIPEA solution and vortexing. In this setup, the concentration of the peptide was 7.5 mM and the molar ratio between DIPEA, coupling reagent and Pga-P/A #1(20)-Ahx was 10:10:1. The formation of the pNP active ester was facilitated by addition of 22 l of a 50 mM 4-(dimethylamino)pyridine (Sigma-Aldrich) solution in DMF. After 20 min incubation at 25 C. aliquots of 72 l of each mixture were withdrawn. The activated peptides were precipitated by addition of 500 l diethyl ether. After centrifugation (13.500g, 4 C.) the supernatant was removed and the sediments were washed with 500 l diethyl ether, dried using a vacuum evaporator (SpeedyDry RVC 2-18 CDplus, Martin Crist Freeze Dryers, Germany) and stored at 20 C., e.g. for 14 days.

[0144] For ESI-MS analysis, a dried aliquot of each of the different P/A(20) active esters was dissolved in 10 mL acetonitrile/water (1:1) and injected into a maXis instrument (Bruker Daltonik, Bremen, Germany) using the positive ion mode. The raw m/z spectra of the Pga-P/A #1(20)-Ahx-HOBt active ester, the Pga-P/A #1(20)-Ahx-pNP active ester and the Pga-P/A #1(20)-Ahx-PFP active ester are shown in FIGS. 9A, 9B and 9C, respectively. For all prepared active esters, the detected main mass species corresponded to a single water adduct of the calculated/predicted mass of the respective Pga-P/A #1(20)-Ahx active ester.

[0145] To achieve coupling of B. fastidiosus uricase with the isolated/preformed HOBt active ester of the P/A peptide, a dry aliquot (corresponding to 1 mg of the P/A peptide prior to activation) was dissolved either in 500 l, 250 l, 167 l, 83.3 l or 50 l of a solution of 2 mg/ml of the enzyme in 100 mM Na-borate pH 9 by vortexing, corresponding to P/A active ester-to-uricase mass ratios of 1:1, 2:1, 3:1, 6:1 or 10:1, respectively. The solution was incubated at room temperature for 1 h to allow coupling. In the same manner the pNP and PFP active esters of the Pga-P/A #1(20)-Ahx peptide were coupled to the B. fastidiosus uricase, applying a P/A active ester:uricase mass ratio of 1:6. After dialysing the coupled enzyme samples against PBS (4 C.) using Slide-A-Lyzer mini dialysis cassettes (MWCO 10.000, Thermo-Fisher), SDS-PAGE was performed under reducing conditions (see FIG. 10). It has thus been shown that conjugates of uricase and P/A peptides have been obtained with advantageously high coupling ratios. It has further been demonstrated that the activated P/A peptides according to the invention can be conveniently prepared and stored (even in a dried/solid state) over prolonged periods of time for later coupling to a protein drug, such as uricase.

Example 6: Preparation of Alcohol Dehydrogenase (ADH) Conjugates with Pga-P/A(40)-Ahx Peptides of Different Composition

[0146] 3.2 mg each of Pga-P/A #1(40)-Ahx peptide (Almac Group, Craigavon, UK) (SEQ ID NO: 18) or Pga-P/A #3(40)-Ahx peptide (Peptide Specialties Laboratories) (SEQ ID NO: 19) were dissolved in 3.5 l DMSO, and 18.5 l of a 500 mM TBTU solution in DMSO was added. The chemical activation of the P/A peptide via its terminal carboxylate group was started by addition of 1.6 L DIPEA to the peptide solution and vortexing. In this setup, the concentration of the peptide was 17.35 mM and the molar ratio between DIPEA, TBTU and Pga-P/A #1(40)-Ahx (or Pga-P/A #3(40)-Ahx) was 10:10:1. After 10 min incubation at 25 C. the mixture was diluted with DMSO in Eppendorf tubes similar to Example 2, to achieve enzyme-to-peptide mass ratios of 1:1, 1:3, 1:6 and 1:10. Each Eppendorf tube finally contained a volume of 25 L of the diluted and activated peptide solution.

[0147] Freeze-dried alcohol dehydrogenase (ADH, from Saccharomyces cerevisiae, Sigma-Aldrich) (SEQ ID NO: 20) was dissolved in PBS, and after additional dialysis against PBS, adjusted to a concentration of 2 mg/ml. 75 L of this protein solution was pipetted into each Eppendorf tube with the peptide from above and mixed by repeated pipetting and vortexing. The coupling reaction was allowed to proceed for 30 min at 25 C. After dialysing the coupled enzyme samples against PBS using Slide-A-Lyzer mini dialysis cassettes (MWCO 10.000, Thermo-Fisher) at 4 C. SDS-PAGE was performed (see FIG. 11). As also shown in FIG. 11, conjugates of alcohol dehydrogenase and P/A peptides have thus been obtained with high coupling ratios.

Example 7: Preparation of Adenosine Deaminase (ADA) Conjugates with Pga-P/A(40)-Ahx Peptides of Different Composition

[0148] 3.2 mg each of Pga-P/A #1(40)-Ahx peptide (Almac Group, Craigavon, UK) (SEQ ID NO: 18) or Pga-P/A #3(40)-Ahx peptide (Peptide Specialties Laboratories) (SEQ ID NO: 19) were dissolved in 3.5 l DMSO, and 18.5 l of a 500 mM TBTU solution in DMSO was added. The chemical activation of the P/A peptide via its terminal carboxylate group was started by addition of 1.6 L DIPEA to the peptide solution and vortexing. In this setup, the concentration of the peptide was 17.35 mM and the molar ratio between DIPEA, TBTU and Pga-P/A #1(40)-Ahx (or Pga-P/A #3(40)-Ahx) was 10:10:1. After 10 min incubation at 25 C. the mixture was diluted with DMSO in Eppendorf tubes similar to Example 2, to achieve enzyme-to-peptide mass ratios of 1:1, 1:3, 1:6 and 1:10. Each Eppendorf tube finally contained a volume of 25 L of the diluted and activated peptide solution.

[0149] Freeze-dried adenosine deaminase (ADA, from Bos taurus, Sigma-Aldrich) (SEQ ID NO: 21) was dissolved in PBS, and after additional dialysis against PBS, adjusted to a concentration of 2 mg/ml. 75 L of this protein solution was pipetted into each Eppendorf tube with the peptide from above and mixed by repeated pipetting and vortexing. The coupling reaction was allowed to proceed for 30 min at 25 C. After dialysing the coupled enzyme samples against PBS using Slide-A-Lyzer mini dialysis cassettes (MWCO 10.000, Thermo-Fisher) at 4 C. SDS-PAGE was performed (see FIG. 12). It has thus been shown that conjugates of adenosine deaminase and P/A peptides have been obtained with high coupling ratios.

Example 8: Preparation of RNase Conjugates with Pga-PAS #1 (40)-Ahx

[0150] 2 mg of Pga-PAS #1(40)-Ahx peptide (Peptide Specialties Laboratories) (SEQ ID NO: 22) were dissolved in 44 l of a 132 mM DIPEA solution in DMSO. The chemical activation of the PAS peptide via its terminal carboxylate group was started by addition of 11.6 L of a 500 mM TBTU solution in DMSO and vortexing. In this setup, the concentration of the peptide was 10.4 mM and the molar ratio between DIPEA, TBTU and Pga-PAS #1(40)-Ahx was 10:10:1. The whole mixture was vortexed briefly and incubated for 10 min at 25 C.

[0151] Ribonuclease A from bovine pancreas (RNase A; Sigma-Aldrich, catalogue No. 83831; SEQ ID NO: 16) was dissolved in PBS and, after dialysis against PBS, adjusted to a concentration of 2 mg/ml. 166.7 L of the RNase A solution was mixed with the activated peptide solution (55.6 L), resulting in a mass ratio between Pga-PAS #1(40)-Ahx and protein of 4:1, and incubated at room temperature for 30 min to allow coupling. After dialysing the coupled RNase sample against PBS using Slide-A-Lyzer mini dialysis cassette (MWCO 10.000, Thermo-Fisher) at 4 C., SDS-PAGE was performed (see FIG. 13). It has thus been shown that even with the serine-containing Pga-PAS #1(40)-Ahx peptide conjugates with RNase A have been obtained with high coupling ratios.

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