Fusion protein, and preparation method and use thereof

Patent metadata

Field	Value
Publication number	US-12508311-B2
Application number	US-202117766450-A
Country	US
Kind code	B2
Filing date	May 18, 2021
Priority date	Apr 14, 2021
Publication date	Dec 30, 2025
Grant date	Dec 30, 2025

Abstract

Disclosed are a fusion protein, and a preparation method and use thereof, which belong to the field of biomedicine technologies. The fusion protein comprises a polypeptide specifically bound to a KRas protein, a specific tumor-cell-penetrating peptide and a lysosome recognition peptide. The fusion protein with tumor targeting, penetrability and specific protein degradation is designed and constructed direct to an undruggable protein for the first time, thus providing a new idea for development of an anti-cancer targeted drug. Different from the prior art in which one molecule can only target one target protein, the fusion protein can simultaneously induce degradation of wild-type and mutant-type KRas proteins. Meanwhile, the fusion protein can improve the sensitivity of the KRas mutant-type tumor to the tumor-targeted drug by inducing degradation of KRas, thus expanding an application range of existing anti-cancer targeted drugs and having important significance in tumor clinic treatment.

First claim

The invention claimed is:

1 . A fusion protein, comprising an anti-KRas protein nanobody, a ganglioside binding peptide and a lysosome recognition peptide; and
wherein the amino acid sequence of the anti-KRas protein nanobody comprises:

(SEQ ID NO: 1)

DVQLQESGGGLVQAGGSLRLSCVASGRTFSTYPTGWFRQA

PGKEREFVARINLSGGITNYADSVKGRFTISRDNAKNTVY

LQMNSLKPEDTAVYYCGGGSTTWAGGIPTNFDYWGQGTQV

TVSSGR;

wherein the amino acid sequence of the ganglioside binding peptide comprises:

(SEQ ID NO: 2)

RAGLQFPVGRLLRRLLR;

and
wherein the amino acid sequence of the lysosome recognition peptide comprises:

(SEQ ID NO: 3)

KFERQKILDQRFFE.

2 . A nucleic acid molecule encoding the fusion protein according to claim 1 .

3 . A vector, comprising the nucleic acid molecule according to claim 2 .

4 . A host cell, comprising the vector according to claim 3 .

5 . A preparation method of the fusion protein according to claim 1 , comprising the step of: culturing the host cell according to claim 4 to obtain the fusion protein.

6 . A drug, comprising the fusion protein according to claim 1 and a pharmaceutically acceptable excipient.

7 . A combination drug, comprising a fusion protein and an epidermal growth factor receptor (EGFR)-targeted drug, wherein the fusion protein comprises an anti-KRas protein nanobody, a ganglioside binding peptide and a lysosome recognition peptide; and
wherein the amino acid sequence of the anti-KRas protein nanobody comprises:

(SEQ ID NO: 1)

DVQLQESGGGLVQAGGSLRLSCVASGRTFSTYPTGWFRQA

PGKEREFVARINLSGGITNYADSVKGRFTISRDNAKNTVY

LQMNSLKPEDTAVYYCGGGSTTWAGGIPTNFDYWGQGTQV

TVSSGR;

wherein the amino acid sequence of the ganglioside binding peptide comprises:
RAGLQFPVGRLLRRLLR (SEQ ID NO: 2); and
wherein the amino acid sequence of the lysosome recognition peptide comprises:
KFERQKILDORFFE (SEQ ID NO: 3).

8 . The host cell according to claim 4 , wherein the host cell is selected from the group consisting of prokaryotic cell and eukaryotic cell.

9 . The drug according to claim 6 , wherein the drug is any one selected from the group consisting of a preparation for degrading a KRas protein, an anti-tumor drug, and a drug for improving a sensitivity of a KRas mutant-type colorectal cancer to a tumor-targeted drug.

10 . The drug according to claim 9 , the KRas protein is selected from wild-type KRas protein or mutant-type KRas protein.

11 . The drug according to claim 10 , the mutant-type KRas protein is the KRas protein mutated at one or more amino acid positions of G12, G13, S17, P34, A59, Q61 and A146.

12 . The drug according to claim 9 , wherein the KRas mutant-type tumor is a tumor related to a mutant-type KRas protein.

13 . The drug according to claim 9 , wherein the tumor-targeted drug is a human epidermal growth factor receptor (EGFR)-targeted drug.

14 . The drug according to claim 13 , wherein the tumor-targeted drug is gefitinib.

15 . A method for treating or ameliorating colorectal cancer, comprising administering to a subject in need thereof an effective amount of the fusion protein according to claim 1 and gefitinib.

Assignees

• Univ Sun Yat Sen
• Sun Yat Sen Univ Cancer Center Sysucc

Inventors

• Zeng Musheng
• Yang Jie
• Feng Guokai
• Wang Qiaoli

Classifications

• C12Y304/21

  Serine endopeptidases (3.4.21) · CPC title

• C12N9/6424

  Serine endopeptidases (3.4.21) · CPC title

• C07K2319/33

  fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies · CPC title

• C07K2317/569

  Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® · CPC title

• C07K16/40 Primary

  against enzymes · CPC title