Human epidermal growth factor polypeptide fusion protein for tumor targeted therapy
By designing tumor-targeted fusion proteins containing hEGF polypeptide and Fc domains, the problem of short half-life of EGF-related proteins in tumor treatment was solved, and effective inhibition of tumor cells with high expression of EGFR was achieved.
Patent Information
- Application Number
- CN202510143486.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-10
- Publication Date
- 2025-07-08
AI Technical Summary
In the prior art, EGF-related proteins are not used for tumor targeted therapy, and the hEGF polypeptide has a short half-life, making it difficult to effectively inhibit the growth of tumor cells with high EGFR expression.
A tumor-targeted fusion protein is designed, including hEGF polypeptide, linker and Fc domain, which blocks the EGFR signaling pathway by specifically acting on tumor cells with high EGFR expression, and uses the Fc domain to prolong the circulating half-life of hEGF polypeptide.
Targeted treatment of tumor cells with high expression of EGFR was achieved, significantly inhibiting tumor cell growth, and enhancing the action time and effect of hEGF polypeptide.
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Figure CN120271719A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the application of human epidermal growth factor (hEGF) polypeptide in tumor targeted therapy, and specifically relates to the anti-tumor effect of hEGF polypeptide fusion protein. Background Art
[0002] The EGFR family is mainly involved in the regulation of cell proliferation genes, the regulation of apoptosis genes, and the regulation of genes related to tumor growth, metastasis, and invasion. Epidermal growth factor (EGF) is an important ligand of EGFR. Currently, there is no EGF-related protein for tumor targeted therapy at the EGFR target. Summary of the Invention
[0003] The present invention provides a tumor-targeted fusion protein, which has the targeted anti-tumor effect of hEGF polypeptide on the one hand and overcomes the problem of short half-life of hEGF polypeptide on the other hand.
[0004] The tumor-targeted fusion protein provided by the present invention at least comprises (i) hEGF polypeptide, hEGF polypeptide variant, or its functional fragment, and (ii) Fc domain, Fc variant, or its functional fragment. Among them, the hEGF polypeptide consists of the third loop structure and subsequent amino acids.
[0005] In one embodiment of the present invention, the arrangement order of the components of the fusion protein is hEGF polypeptide - linker - Fc.
[0006] In one embodiment of the present invention, the hEGF polypeptide consists of the third loop structure and subsequent amino acids, which has the sequence shown in SEQ ID NO.1, or a derivative sequence obtained by substituting, deleting, or adding one or more amino acids to the sequence shown in SEQ ID NO.1 and having the same function as the hEGF polypeptide domain.
[0007] In one embodiment of the present invention, the Fc domain is composed of murine IgG1, which has the sequence shown in SEQ ID NO.2, or a derivative sequence obtained by substituting, deleting, or adding one or more amino acids to the sequence shown in SEQ ID NO.2 and having the same function as the Fc domain, such as human IgG1, CH2+CH3 of human or murine Fc, etc., including but not limited to the above derivative sequences.
[0008] In one embodiment, the linker has the sequence shown in SEQ ID NO.3, or a derivative sequence obtained by substituting, deleting, or adding one or more amino acids to the sequence shown in SEQ ID NO.3 and having the same function as the linker.
[0009] In one embodiment, the amino acid sequence of the tumor-targeting fusion protein is selected from: (a) the sequence shown in SEQ ID NO. 4; (b) the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO. 5; (c) the amino acid sequence encoded by the degenerate sequence of the sequence shown in SEQ ID NO. 5; and (d) a derivative sequence of the sequence shown in SEQ ID NO. 4 with one or more amino acids substituted, deleted or added and having the same function as the fusion protein.
[0010] On the other hand, the present invention provides a pharmaceutical composition comprising the tumor-targeting fusion protein of the present invention and a pharmaceutically acceptable additive, and the additive includes a carrier, a stabilizer and / or an excipient.
[0011] Another aspect of the present invention provides a pharmaceutical composition comprising the tumor-targeting fusion protein of the present invention and another anti-cancer agent.
[0012] In one embodiment of the present invention, the tumor is a tumor with high EGFR expression. In one embodiment, the tumor cells are human gastric cancer cells N87.
[0013] On the other hand, the present invention provides a nucleic acid sequence encoding the tumor-targeting fusion protein of the present invention, an expression vector containing the nucleic acid sequence, or a host transformed or transfected with the expression vector.
[0014] On the other hand, the present invention further provides a kit comprising the tumor-targeting fusion protein of the present invention, a nucleic acid sequence encoding the tumor-targeting fusion protein of the present invention, an expression vector containing the nucleic acid sequence, or a host transformed or transfected with the expression vector. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 For Figure 1 is a schematic diagram of the protein structure of one embodiment of the present invention.
[0016] Figure 2 is the inhibitory effect of the fusion protein of the embodiment herein on the growth of gastric cancer tumor cells. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0017] In the present invention, the "fusion protein" at least includes (i) an hEGF polypeptide, an hEGF polypeptide variant, or a functional fragment thereof, and (ii) an Fc domain, an Fc variant, or a functional fragment thereof. Component (ii) is used to extend the circulating half-life of the hEGF polypeptide. Component (i) is a targeting molecule that acts on the surface of tumor cells with high EGFR expression with high affinity and specificity, thereby blocking the EGFR signaling pathway and further inhibiting the growth of tumor cells.
[0018] In the present invention, the components in the fusion protein are arranged in a reasonable order so that the overall fusion protein achieves the intended purpose of the present invention. In one embodiment, the arrangement order of the components in the fusion protein is hEGF polypeptide - linker - Fc domain. The fusion proteins composed of different orders are also within the scope protected by this patent.
[0019] The Fc domain "Fc domain" or "Fc fragment" refers to the "crystallizable fragment" region of the immunoglobulin heavy chain, and its circulating half-life can be as long as 21 days. The Fc domain can bind to cell surface receptors (Fc receptors) and / or proteins of the complement system, or can be modified to weaken or enhance this binding activity. The Fc domain can be derived from IgG, IgA, IgD, IgM or IgE antibodies and produce immune functions, such as opsonization, cell lysis, mast cell degranulation and other Fc receptor-dependent processes.
[0020] In the present invention, in one embodiment, the Fc domain is composed of CH1, CH2 and CH3 of murine IgG1.
[0021] In certain embodiments, "Fc variant" refers to a molecule or sequence that is modified from natural Fc and can still bind to Fc receptors. "Fc variants" include molecules or sequences that are humanized from non-human natural Fc. In addition, due to certain structural features or biological activities that are not required for the fusion molecules of the present invention, certain sites of natural Fc can be removed. Therefore, in certain embodiments, "Fc variants" include molecules or sequences lacking one or more natural Fc sites or residues. The "Fc domain" includes molecules or sequences of natural Fc and Fc variants as described above, which include molecules in monomeric or polymeric forms.
[0022] One embodiment of the fusion protein of the present invention further includes a linker located between the components, usually a short peptide composed of 4 - 20 amino acids. These linkers enable the components to be reasonably positioned to achieve the functional activities of the components. In one embodiment of the fusion protein of the present invention, a linker is contained, and the sequence of the linker is Gly Ser Gly Gly Gly Gly Ser, and other sequences can also be used.
[0023] The structure of the hEGF polypeptide fusion protein is as Figure 1 shown, mainly composed of hEGF polypeptide, Fc domain, and linker ( Figure 1 ). The DNA sequence was subcloned into the pcDNA3.1(+) vector, and the vector was transfected into 293F cells by transient transfection and expressed.
[0024] In the cell viability assay experiment, N87 cells in the logarithmic growth phase were harvested and seeded into a 96-well cell culture plate. The hEGF fusion protein was diluted to a final concentration of 300 nM in the assay medium and serially diluted. Incubation was continued for 48 or 72 hours. Finally, the CCK-8 reagent was used to determine the cell growth inhibition curve, and the experimental results showed that the growth of gastric cancer cells could be significantly inhibited( Figure 2 ).
[0025] Although one embodiment of the present invention has been disclosed, those skilled in the art can make modifications and variations to the ideas disclosed herein, and such modifications and variations still fall within the scope of the present invention defined by the appended claims.
[0026]
[0027]
[0028]
[0029]
Claims
1. A tumor-targeting fusion protein comprises at least (i) an hEGF polypeptide, an hEGF polypeptide variant, or a functional fragment thereof, and (ii) an Fc domain, an Fc variant, or a functional fragment thereof; wherein, The hEGF polypeptide consists of the third loop structure and subsequent amino acids.
2. The tumor-targeting fusion protein according to claim 1, wherein the arrangement order of each component of the fusion protein is hEGF polypeptide - Fc domain or Fc domain - hEGF polypeptide.
3. The tumor-targeting fusion protein according to claim 1, wherein the amino acid sequence of the hEGF polypeptide is as shown in SEQ ID NO.1, or a derivative sequence with substitution, deletion or addition of one or more amino acids in the sequence shown in SEQ ID NO.1 and having the same function as the hEGF polypeptide.
4. The tumor-targeting fusion protein according to claim 1, wherein the amino acid sequence of the Fc domain is as shown in SEQ ID NO.2, or a derivative sequence with substitution, deletion or addition of one or more amino acids in the sequence shown in SEQ ID NO.2 and having the same function as the Fc domain.
5. The tumor-targeting fusion protein according to claim 1, which may or may not have a linker. The sequence of the linker is as shown in SEQ ID NO.3, or a derivative sequence with substitution, deletion or addition of one or more amino acids in the sequence shown in SEQ ID NO.3 and having the same function as the linker domain.
6. The tumor-targeting fusion protein according to claim 1, wherein the amino acid sequence of the tumor-targeting fusion protein is selected from: (a) the sequence as shown in SEQ ID NO.4; (c) the amino acid sequence encoded by the nucleic acid sequence as shown in SEQ ID NO.5; (d) the amino acid sequence encoded by the degenerate sequence of the sequence as shown in SEQ ID NO.5; (b) a derivative sequence with substitution, deletion or addition of one or more amino acids in the sequence shown in SEQ ID NO.4 and having the same function as the fusion protein.
7. The tumor-targeting fusion protein according to any one of claims 1 - 6, wherein the tumor is an EGFR-positive expressing tumor.
8. A pharmaceutical composition comprising the tumor-targeting fusion protein according to any one of claims 1 - 6 and a pharmaceutically acceptable excipient.
9. A pharmaceutical composition comprising the tumor-targeting fusion protein according to any one of claims 1 - 6 and another anti-cancer agent.
10. A nucleic acid encoding the tumor-targeting fusion protein according to any one of claims 1 - 6.
11. An expression vector comprising the nucleic acid according to claim 11.
12. A non-human host cell transformed or transfected with the expression vector according to claim 12.
13. A kit comprising the tumor-targeting fusion protein according to any one of claims 1 - 6, the nucleic acid according to claim 10, the expression vector according to claim 11, or the non-human host cell according to claim 12.