- Document Number:
20230157972
- Appl. No:
17/311480
- Application Filed:
October 14, 2019
- نبذة مختصرة :
The invention relates to a method for providing a virus-like particle (VLP) derived from John Cunningham virus (JCV), relates to a VLP associated with a cargo, in particular a protein, and a drug delivery system (DDS) obtainable by said method, in particular for crossing the blood brain barrier (BBB), and a VLP containing composition. The method comprises steps of disassembly of VLP into pentamers, inducing the pentamers to aggregate and reassembly into VLP.
- Assignees:
NEUWAY Pharma GmbH (Bonn, DE)
- Claim:
1. Method for providing a virus-like particle (VLP) derived from John Cunningham virus (JCV) comprising the following steps: a) providing a composition comprising VP1 proteins, b) exposing the VP1 proteins of the composition of a) to conditions inducing the VP1 to assemble into VLP, c) exposing the VLP of the composition of b) to conditions disassembling the VLP into pentamers, d) exposing the pentamers of the composition of c) to conditions inducing the pentamers to reassemble into VLP c) exposing the VLP of the composition of d) to conditions disassembling the VLP into pentamers, f) exposing the pentamers of the composition of e) to conditions inducing the aggregation of the pentamers, g) exposing the aggregated pentamers of the composition of f) to a cargo, in particular a protein, under conditions inducing the aggregated pentamers to assemble into a VLP associated with the cargo, in particular the protein.
- Claim:
2. The method according to claim 1, whereas the aggregated pentamers in step g) are induced to assemble into a VLP incorporating the cargo, in particular the protein.
- Claim:
3. The method according to claim 1, whereas before step d) the pentamers of the composition of c) are exposed to conditions inducing the aggregation of the pentamers.
- Claim:
4. The method according to claim 1, whereas the aggregation is induced by a precipitation agent, preferably selected from the group consisting of polyethylene glycol (PEG), alcohol, a salt or a combination thereof, most preferably by a salt.
- Claim:
5. The method according to claim 4, whereas the salt comprises an anion and a cation selected from the group consisting of citrate (C6H5O73−), phosphate (PO43−), sulfate (SO42−), hydrogen phosphate (HPO42−), dihydrogen phosphate (H2PO4−), iodate (IO3−), hydroxide (OH−), fluoride (F−), bromate (BrO3−) or acetate (CH3COO−), quaternary ammonium compounds (NR4+) with R being an alkyl or an aryl group, preferably tetramethylammonium ((CH3)4N+) or dimethylammonium ((CH3)2N2+), ammonium (NH4+), potassium (K+), caesium (Cs+), rubidium (Rb+) or lithium (Li+), preferably comprising SO42− and/or NH4+.
- Claim:
6. The method according to claim 4, whereas the salt is selected from the group consisting of (NH4)2SO4, K2SO4, Na2SO4, (NH4)2HPO4, K2HPO4 and Na2HPO4, preferably (NH4)2SO4.
- Claim:
7. The method according to claim 4, whereas the pentamers are brought into contact with the precipitation agent, preferably by dialysis or cross-flow filtration.
- Claim:
8. The method according to claim 1, whereas after inducing the aggregation, the pentamers of composition f) and/or d) are separated from the conditions inducing the aggregation of the pentamers.
- Claim:
9. The method according to claim 8, whereas the separation is achieved by a dialysis against a composition comprising a salt and a buffer and a pH of 6 to 8.5, preferably of 6.5 to 8.5, more preferably of 7 to 8, most preferably of 7.2 to 7.5, in particular of 7.5.
- Claim:
10. The method according to claim 1, whereas in step c) and/or in step e) the VLP are exposed to a reducing agent, preferably selected from the group consisting of 2-mercaptoethanol, Tris-2-carboxyethylphosphine hydrochloride (TCEP), hydrogen peroxide (H2O2), Dithiothreitol (DTT), thiosulfate and formic acid, more preferably selected from the group consisting of 2-mercaptoethanol, TCEP and DTT, most preferably DTT.
- Claim:
11. The method according to claim 1, whereas the cargo, in particular the protein, has a molecular weight of about 10 kDa to about 4000 kDa.
- Claim:
12. The method according to claim 1, whereas the protein is an antibody, antibody fragment, preferably a nanobody, derivative of the antibody or the antibody fragment, or an antibody-drug-conjugate (ADC).
- Claim:
13. The method according to claim 12, whereas the antibody is an IgG antibody, an IgM antibody, an IgA antibody, an IgD antibody or an IgE antibody, preferably an IgG antibody or an IgM antibody.
- Claim:
14. The method according to claim 1, whereas the composition of step d) is characterized by a) a polydispersity index (PDI) of less than 0.3, preferably less than 0.2, preferably less than 0.1, more preferably is in a range between 0.01 and 0.09 and/or b) at least 70% of the VLP having an average diameter of 20 nm to 70 nm, preferably of 30 nm to 70 nm, more preferably of 35 nm to 65 nm, more preferably of 40 to 60 nm.
- Claim:
15. The method according to claim 1, comprising a step of storing the VLP from the composition of step d) for at least 10 h, 15 h, 20 h, preferably for at least 24 h at a temperature of about −80° C. to about 4° C., more preferably at a temperature of about −80° C. for at least 24 h.
- Claim:
16. The method according to claim 15, whereas storing takes place in a composition comprising a cryoadditive, preferably selected from the group comprising polyols, sugars, inorganic salts, organic salts, amino acids, polymers, extremolytes or derivatives or combinations thereof.
- Claim:
17. The method according to claim 16, whereas the inorganic salt comprises a sulfate anion and preferably is ammonium sulfate and/or the amino acid is glycine, glutamine, proline, alanine and/or the amino acid derivative is betain.
- Claim:
18. The method according to claim 1, whereas the VLP of step b), the pentamers of step c), the VLP of step d) and/or the VLP of step g) are subject to purification.
- Claim:
19. The method according to claim 1, whereas the VP1 comprises an amino acid sequence which is at least 80%, more preferably at least 90% identical to the amino acid sequence according to SEQ ID NO: 1 over its entire length, preferably has the amino acid sequence of SEQ ID NO: 1.
- Claim:
20. The method according to claim 1, whereas a nucleotide sequence of the VP1 protein is at least 70%, more preferably at least 80%, more preferably at least 90% identical to the nucleotide sequence of SEQ ID NO: 2 over its entire length, preferably is the nucleotide sequence of SEQ ID NO: 2.
- Claim:
21. A VLP associated with a cargo, in particular a protein, obtainable by the method according to claim 1.
- Claim:
22. The VLP according to claim 21, whereas the VLP incorporates the cargo, in particular the protein.
- Claim:
23. Composition comprising virus-like particles (VLP) derived from John Cunningham virus (JCV) characterized by one or more of the following parameters: a) a polydispersity index (PDI) of less than 0.3, preferably less than 0.2, preferably less than 0.1, more preferably in a range between 0.01 and 0.09, b) at least 70% of VLP with an average diameter from 20 nm to 70 nm, preferably of 30 nm to 70 nm, more preferably of 35 nm to 65 nm, more preferably of 40 to 60 nm, c) a VLP content within the composition of at least 80% (v/v), preferably at least 85% (v/v), preferably at least 90% (v/v), preferably at least 95% (v/v).
- Claim:
24. Drug Delivery System obtainable by the method according to claim 1.
- Claim:
25. The Drug Delivery System according to claim 24 for use in a method of therapy and/or diagnosis, preferably for the treatment of neurological disorders, in particular of CNS diseases, in a subject in the need thereof.
- Claim:
26. The Drug Delivery System according to claim 24, whereas the VLP cross the blood brain barrier (BBB).
- Claim:
27. The Drug Delivery System according to claim 26, whereas VLP are detectable in the CNS within 10 days or less after administration of the drug delivery system, preferably after i.v. administration.
- Claim:
28. The Drug Delivery System according to claim 26, whereas the VLP cross the physiologically intact BBB.
- Claim:
29. The Drug Delivery System according to claim 26, whereas the crossing does not require a loss of integrity or increased permeability of the BBB.
- Claim:
30. The Drug Delivery System according to claim 26, whereas the subject has not received a treatment of increasing the permeability of his BBB or inducing disruption thereof.
- Current International Class:
61; 07
- الرقم المعرف:
edspap.20230157972
No Comments.