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CN-122003429-A - Phage particle complexes, methods of making and uses thereof

CN122003429ACN 122003429 ACN122003429 ACN 122003429ACN-122003429-A

Abstract

Phage particle complexes comprising a polypeptide displayed on the surface of the phage, a nucleic acid sequence encoding the displayed polypeptide within the phage, and a linking compound, wherein the linking compound forms a bicyclic polypeptide containing at least 3 discrete ionic bonds with the polypeptide, methods of making the same, and uses thereof are disclosed.

Inventors

  • LI ZIGANG
  • YIN FENG
  • LIU NA

Assignees

  • 深圳湾实验室坪山生物医药研发转化中心
  • 深圳湾实验室
  • 北京大学深圳研究生院

Dates

Publication Date
20260508
Application Date
20240926
Priority Date
20230928

Claims (20)

  1. A phage particle complex comprising a polypeptide displayed on the phage surface, a nucleic acid sequence encoding the displayed polypeptide inside the phage, and a linking compound, wherein the linking compound forms a bicyclic polypeptide containing at least 3 discrete ionic bonds with the polypeptide.
  2. The phage particle complex of claim 1, wherein said linking compound has molecular symmetry corresponding to the number of said ionic bonds.
  3. The phage particle complex of claim 1 or 2, wherein the linking compound has triple molecular symmetry and the linking compound is linked to the polypeptide by three discrete ionic bonds.
  4. A phage particle complex according to any one of claims 1 to 3, wherein the linking compound comprises a chemical group of rigid structure, preferably comprising a benzene ring structure or a cyclic olefin structure, more preferably 1,3, 5-tribromotoluene, 1,3, 5-tris (bromomethyl) -2,4, 6-triethylbenzene, 1,3, 5-tris (bromomethyl) cyclohexene.
  5. Phage particle complex according to any one of claims 1 to 4, wherein the polypeptide comprises at least 3 methionine residues, preferably discrete ionic bonds are formed by linking a linking compound to methionine in the polypeptide.
  6. The phage particle complex of any one of claims 1-5, wherein said polypeptide sequence comprises MXmMXnM, wherein X represents a natural amino acid residue, M represents methionine, M is an integer selected from 2 to 20, and n is an integer selected from 2 to 20.
  7. The phage particle complex of any one of claims 1-6, wherein the bicyclic polypeptide has the structure: wherein R is selected from side chains of 20 natural amino acids, m is an integer selected from 2 to 20, and n is an integer selected from 2 to 20.
  8. The phage particle complex of any one of claims 1 to 6, wherein said bicyclic polypeptide is selected from the group consisting of SEQ ID NO 9 to SEQ ID NO 23.
  9. A library of polypeptides comprising the phage particle complex of any one of claims 1 to 8.
  10. A method of preparing a phage particle complex comprising forming a bicyclic polypeptide comprising at least 3 discrete ionic bonds from a polypeptide displayed on the phage surface and a linker compound.
  11. The method of claim 10, wherein the bicyclic polypeptide is formed in a polar solvent-containing, preferably polar solvent selected from an aqueous acetonitrile solution, an aqueous methanol solution, or any mixture thereof.
  12. The method of claim 10 or 11, wherein the bicyclic polypeptide is formed at 4 to 40 ℃.
  13. The method of any one of claims 10 to 12, wherein the bicyclic polypeptide is formed at a pH of 3 to 10.
  14. The method of any one of claims 10 to 13, wherein the reaction time of the polypeptide with the linking compound to form the bicyclic polypeptide is 1 to 36 hours.
  15. The method of any one of claims 10 to 14, wherein the linking compound comprises a chemical group of rigid structure, preferably comprising a benzene ring structure or an olefin structure, more preferably 1,3, 5-tribromotoluene, 1,3, 5-tris (bromomethyl) -2,4, 6-triethylbenzene, 1,3, 5-tris (bromomethyl) cyclohexene.
  16. The method of any one of claims 10 to 15, wherein the structure of the bicyclic polypeptide is: wherein R is selected from residues of natural amino acids, m is an integer selected from 2 to 20, and n is an integer selected from 2 to 20.
  17. The method of any one of claims 10 to 16, wherein the bicyclic polypeptide is selected from the group consisting of SEQ ID No. 9 to SEQ ID No. 23.
  18. A phage particle complex prepared by the method of any one of claims 10 to 17.
  19. The phage particle complex of claim 18, further comprising a nucleic acid sequence encoding said polypeptide.
  20. Phage particle complex according to claim 18 or 19, wherein the polypeptide comprises at least 3 methionine residues, preferably discrete ionic bonds are formed by linking a linking compound to methionine in the polypeptide.

Description

Phage particle complexes, methods of making and uses thereof Citation of related application The present disclosure claims the full rights of the chinese patent application filed 28 at 9 months 2023 to the national intellectual property agency of the people's republic of China, application No. 202311282073.X, entitled "phage particle composite, method of preparation thereof, and uses thereof", and is incorporated by reference in its entirety into the present disclosure. FIELD The present disclosure relates generally to the field of bioengineering, and more specifically to phage particle complexes, methods of making the same, and uses thereof. Background The human body evolves a complex and precisely regulated protein-protein interactions (PPIs) network, which plays an important role in the vital activities of cells, such as signal transduction, cell cycle, immune system, gene regulation and the like, PPIs directly participate in influencing the occurrence and development of diseases and ultimately determining whether the diseases can be cured. Targeting intracellular protein-protein interactions is therefore considered a very promising therapeutic strategy. Polypeptide drug molecules are considered to be the most effective molecular forms for targeting intracellular protein-protein interactions. The polypeptide medicine has larger molecular action area, can form more complex conformation, can effectively expand the chemical space of the targeting molecule, and has smaller off-target effect compared with the small molecular medicine. Similar to biological macromolecules, polypeptide molecules have higher binding force and selectivity to targets, but compared with biological macromolecules, polypeptide drugs have lower cost and weaker immunogenicity, and simultaneously, due to smaller molecular size, the polypeptide drugs are easier to penetrate tissues. The traditional polypeptide medicine cannot effectively form a complex secondary structure due to the limited number of amino acid residues, has very high degree of freedom in physiological solution and is in a random linear state, so that the specificity of the polypeptide medicine is reduced, the polypeptide medicine is easily degraded by protease, and the cell membrane penetrating capacity of the polypeptide medicine is not very good. The polypeptide is modified by chemical means to stabilize the polypeptide into a conformation with a secondary structure, so that the stability of the polypeptide to protease can be improved, the cell membrane penetrating capacity of the polypeptide can be enhanced, and the binding capacity of the polypeptide and a target can be improved by reducing entropy change when the polypeptide is bound with the target. Polypeptides are used for the treatment of a variety of diseases, such as diabetes, allergies, anti-infections, obesity, diagnostics, tumors, arthritis and cardiovascular diseases, because of their broad active function. In 2019, the market of oral administration of cable Ma Lutai has also greatly promoted the enthusiasm for the development of polypeptide drugs. Polypeptide drug development is a complex process and is mainly divided into natural product mining, rational design, high-throughput screening and the like. Natural product mining involves a degree of contingency, and natural product ligands for most disease-related proteins are not yet known. The rational design is generally based on the existing protein-protein interaction crystal structure, a polypeptide sequence of the target protein is obtained, a series of identifications such as affinity, stability, cell membrane penetrating capacity and the like are carried out on the polypeptide sequence, the shortcomings of the polypeptide sequence are optimized and reformed, and finally, the polypeptide molecule with further improved affinity, strong polypeptide stability, proper polypeptide physicochemical property and good biological function is obtained. However, the polypeptide is limited by the existing crystal structure, and the physicochemical properties of the polypeptide sequence are different, so that the applicability of the polypeptide in drug development is limited. The polypeptide high-throughput screening technology mainly comprises phage display technology, mRNA display technology and the like. The high throughput screening technology does not rely on existing protein-protein interactions and can obtain more diverse polypeptide sequences through screening of numbers of library diversity of more than 10 hundred million, thus providing more possibilities in the development of polypeptide pharmaceuticals. In recent years, more polypeptide molecules obtained by high-throughput screening means have been introduced into clinical studies. Phage display technology is a widely used high-throughput screening means, and currently is mainly applied to screening of antibodies and polypeptides, and adalimumab is a typical case of the technology. Graagole, wentt do