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EP-4740964-A1 - NANO DELIVERY SYSTEM, PREPARATION METHOD THEREFOR AND USE THEREOF

EP4740964A1EP 4740964 A1EP4740964 A1EP 4740964A1EP-4740964-A1

Abstract

Provided are a nano-delivery system, comprising ferritin, a targeting protein and an active molecule, a preparation method thereof, and a use thereof. This can provide targeted delivery system and a targeted therapeutic method that are safe, stable, low toxic, highly targeted, multispecific, and convenient to prepare.

Inventors

  • CHEN, HAIYAN

Assignees

  • Chen, Haiyan
  • Guangzhou Nanopac Biotechnology Ltd

Dates

Publication Date
20260513
Application Date
20240619

Claims (14)

  1. A nano-delivery system, comprising a ferritin nanoparticle, a targeting protein, and an active molecule, wherein the targeting protein is conjugated onto the surface of the ferritin nanoparticle via the amino acid sequence set forth in SEQ ID NO. 19, and the active molecule is encapsulated within the ferritin nanoparticle.
  2. The nano-delivery system according to claim 1, wherein the amino acid sequence of a ferritin in the ferritin nanoparticle is set forth in SEQ ID NO. 11.
  3. The nano-delivery system according to claim 1 or 2, wherein the targeting protein is an antibody or an antigen-binding fragment thereof.
  4. The nano-delivery system according to claim 3, wherein the antibody is an IgG antibody.
  5. The nano-delivery system according to any one of claims 1 to 4, wherein the conjugation between the ferritin nanoparticle and the targeting protein is formed using Sortase A.
  6. A method for preparing a nano-delivery system, comprising: 1) encapsulating an active molecule within a ferritin nanoparticle; and 2) conjugating the ferritin nanoparticle to a targeting protein using Sortase A.
  7. The method according to claim 6, wherein steps 1) and 2) are performed in one reaction or in two separate reactions.
  8. The method according to claim 6 or 7, further comprising a step of providing a ferritin having the amino acid sequence set forth in SEQ ID NO. 20 (GGGG) linked to its N-terminus and a targeting protein having the amino acid sequence set forth in SEQ ID NO. 21 (LPXTGG) linked to its C-terminus.
  9. The method according to any one of claims 6 to 8, wherein the amino acid sequence of the ferritin is set forth in SEQ ID NO. 11.
  10. The method according to any one of claims 6 to 9, wherein the targeting protein is an antibody or an antigen-binding fragment thereof, and the ferritin nanoparticle is connected to the C-terminus of a heavy chain of the antibody or antigen-binding fragment thereof using Sortase A.
  11. A modified ferritin, having a sequence set forth in SEQ ID NO. 11.
  12. An isolated polynucleotide, encoding a ferritin having a sequence set forth in SEQ ID NO. 11
  13. Use of the modified ferritin according to claim 11 or the polynucleotide according to claim 12 in the preparation of a nano-delivery system.
  14. Use of the nano-delivery system according to any one of claims 1 to 5 in the preparation of a medicament for treating a tumor.

Description

FIELD OF THE INVENTION This invention belongs to the field of drug delivery systems for disease diagnosis and treatment. BACKGROUND OF THE INVENTION Small molecule drugs, such as tumor chemotherapeutic agents, are widely used in disease treatment. However, the clinical application of such drugs is limited by their aqueous solubility, distribution in tissues, specificity for cells and toxicity (Z.G. Chen, Small-molecule delivery by nanoparticles for anticancer therapy. Trends Mol Med 16, 594-602 (2010); L. M. Ickenstein and P. Garidel, Lipid-based nanoparticle formulations for small molecules and RNA drugs. Expert Opin Drug Deliv 16, 1205-1226 (2019)). The high expression of specific molecules on the surface of certain cells, e.g. tumor cells, enables antibody-based therapies, which achieve precise cell-targeting by binding to these specific molecules. Such antibody-mediated targeting therapies include CAR T-cell therapies, antibody-drug conjugate (ADC) therapies, bispecific antibody therapies, etc. However, currently developed CAR-T cell therapies only target hematological tumors, exhibit significant side effects, and are expensive, time-consuming and only applicable to specific subjects. The ADC therapies have cytotoxic drugs exposed to normal cells or tissues, resulting in insufficient stability and significant side effects. The bispecific antibody therapies may cause adverse reactions and have issues such as low stability. Therefore, there is still a need for safe, convenient, stable, low toxic, high targeting specificity, and commonly used targeted delivery systems and targeting therapies. SUMMARY OF THE INVENTION This disclosure addresses the need for the targeted delivery systems and targeting therapies that are safe, stable, low toxic, high targeting specificity, multispecific, and convenient to prepare. The disclosure is based on a surprising finding by the inventors that conjugating antibodies to ferritin nanoparticles using Sortase A and encapsulating active molecules, such as small molecule therapeutics, within the nanoparticles may conveniently generate a safe, highly stable, low-toxic, precisely targeting, and commonly used targeted delivery system. Since a ferritin nanoparticle is composed of 24 ferritin monomers (or referred to as subunits), the nanoparticle may theoretically conjugate 24 antibody molecules using Sortase A, enabling the enhanced antibody affinity. Alternatively, a plurality of different antibodies may be conjugated, thereby enabling targeting of multiple targets and achieving multispecificity. Earlier studies have utilized human ferritin to target human transferrin receptor 1 (TfR1). Human TfR1 is highly expressed in tumor cells, enabling targeted delivery of drugs. However, human TfR1 is also widely expressed in healthy tissues in human, such as bone marrow, lung, colon, and liver, to transport irons into cells. Therefore, the use of human ferritin may lead to undesirable risks of accumulating drugs in healthy tissues and of autoimmunity. The inventors have further surprisingly found that modifying the ferritin-encoding gene derived from Helicobacter pylori and expressing the modified gene in mammalian cells (e.g., CHO cells) can address these problems. Furthermore, the ferritin obtained in this way exhibits a conformation more closely resembling its native status, and simultaneously has a high yield ( more than 80 mg/L cells ). In one aspect, herein provided is a nano-delivery system, comprising a ferritin nanoparticle, a targeting protein, and an active molecule, wherein the targeting protein molecule is conjugated onto the surface of the ferritin nanoparticle through the linker set forth in SEQ ID NO. 19 (LPXTGGGG, where X can be any amino acid), and the active molecule is encapsulated within the ferritin nanoparticle. In some embodiments, the amino acid sequence of the ferritin in the nano-delivery system is set forth in SEQ ID NO. 11. In the nano-delivery system described herein, the targeting protein may be any protein with the ability to target, e.g., with the ability to target specific cells, such as tumor cells. For example, the targeting protein is an antibody molecule e.g., IgG antibody, or an antigen-binding fragment thereof. In the nano-delivery system described herein, the active molecule is any molecule desired for targeted delivery that can be encapsulated in the ferritin nanoparticle. For example, the active molecule is any suitable therapeutic agent, such as a small molecule drug, e.g., an anti-tumor drug, as long as it can be encapsulated in the ferritin nanoparticle as used herein. In the nano-delivery system as described herein, the ferritin nanoparticle and the targeting protein are conjugated together using Sortase A, thereby forming a linker set forth in SEQ ID NO. 19 (LPXTGGGG, wherein X is any amino acid) between them ( the conjugate is in the form of N-terminus - targeting molecule - linker - ferritin - C-terminus). In another aspect, herein provid