An engineered fc fusion protein inducing dual-targeted protein degradation across the blood-brain barrier and application thereof

Abstract

The present application relates to a kind of engineering Fc fusion protein and application of double target protein degradation induced, the fusion protein includes cell penetration peptide, linker, EAG2 binding peptide, the Fc segment of engineered IgG and brain glioma targeting peptide in turn, the sequence of the Fc segment of engineered IgG is as shown in SEQ ID NO:1.The present application solves the delivery in brain of fusion protein degradation agent across BBB by means of engineering Fc, while using the characteristics that E3 ubiquitin ligase TRIM21 with Fc receptor function and self-ubiquitination of glioblastoma high expression, can mediate the synchronous ubiquitination degradation of self and target protein, to form the double target effect of glioblastoma that TRIM21 and EAG2 are both high expression, enhanced anti-tumor effect, provide new ideas for developing new anti-tumor drugs and delivery strategy.

Description

Technical Field

[0001] This invention belongs to the field of macromolecular drugs, specifically relating to an engineered Fc fusion protein that induces the degradation of dual-targeting proteins, and the application of this fusion protein in the treatment of glioblastoma. Background Technology

[0002] Glioblastoma (GBM) is one of the most malignant and difficult-to-cure primary brain tumors. Due to its high invasiveness and genetic heterogeneity, GBM is difficult to surgically remove, and radiotherapy, chemotherapy, and immunotherapy have limited effectiveness. It also carries a high risk of complications, resulting in extremely poor patient prognosis and making treatment very challenging. Therefore, developing new targets and strategies for GBM treatment has significant scientific and clinical value.

[0003] In recent years, an increasing number of studies have shown that the nervous system plays a crucial role in the development, progression, invasion, and metastasis of tumors. Recent research indicates that the interaction between glioma and neurons is fundamental to glioma progression, and this interaction represents a potential therapeutic target for gliomas. In 2023, Nature Cancer reported a novel therapeutic target for GBM: a potassium channel formed by the EAG2–Kvβ2 complex. This study demonstrated that both EAG2 and Kvβ2 proteins are highly expressed in glioblastoma and form potassium channels at the junction of tumor cells and normal tissue, regulating glioma-neuron interactions and playing a vital role in glioma growth, invasion, and chemotherapy resistance. Based on this, the research team designed a membrane-permeable peptide (K90-114). TAT By disrupting the interaction between two proteins, this peptide drug showed a significant antitumor effect against GBM, even inhibiting GBM resistant to temozolomide, without obvious toxic side effects. However, because the peptide has difficulty crossing the blood-brain barrier (BBB), the drug can only be administered intraventricularly via an implanted osmotic pump and cannula, greatly limiting its in vivo application. Therefore, there is an urgent need to develop effective delivery strategies to facilitate its clinical application.

[0004] Targeted protein degradation (TPD) is an emerging technology in drug development that utilizes functional molecules (protein degraders) to degrade disease-related proteins using naturally occurring intracellular protein degradation systems, thereby achieving therapeutic goals. This technology directly and efficiently induces the degradation of target proteins, employing a different mechanism of action than traditional small-molecule drugs that inhibit protein function. This makes many currently "drug-resistant" targets "drug-like," enabling the targeting of points that traditional small-molecule inhibitors struggle to reach, such as protein-protein interactions. Designing protein degraders that simultaneously bind to E3 ubiquitin ligases and target proteins, utilizing the intracellular ubiquitin-proteasome system (UPS) to degrade intracellular disease-related proteins, is the fastest-growing and most promising type of targeted protein degradation technology. Summary of the Invention

[0005] The purpose of this invention is to construct a fusion protein of an EAG2-Kvβ2 complex targeting peptide and an engineered Fc segment. The engineered Fc segment is used to solve the problem of intrabrain delivery of the EAG2-Kvβ2 complex targeting peptide. At the same time, a targeted protein degradation technology is introduced to develop the fusion protein of the EAG2-Kvβ2 complex targeting peptide and the engineered Fc segment into a degrading agent. E3 ubiquitin ligase, which can recognize the antibody Fc segment, mediates proteasome degradation to degrade EAG2, thereby completely destroying the function of the EAG2-Kvβ2 ion channel and enhancing the anti-tumor effect.

[0006] To achieve the above objectives, the present invention provides an engineered Fc fusion protein that induces dual-target protein degradation, comprising, in sequence, a cell-penetrating peptide, a linker, an EAG2-binding peptide, an engineered IgG Fc segment, and a glioma-targeting peptide, wherein the sequence of the engineered IgG Fc segment is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0007] The engineered Fc segment in the Fc fusion protein is an engineered Fc segment of IgG. It specifically binds to the human transferrin receptor, which is highly expressed on brain endothelial cells, and delivers the macromolecules it carries across the brain via receptor-mediated transport. Furthermore, the mutated amino acid sites in the engineered Fc segment neither overlap with the key binding site of TRIM21 in sequence nor are spatially distant from it, thus not affecting the binding of the Fc segment to TRIM21.

[0008] The glioma-targeting peptide in the Fc fusion protein is a tumor-homing peptide used to target glioma cells. According to a preferred embodiment of the present invention, the sequence of the glioma-targeting peptide is shown in SEQ ID NO: 3, and it can target and bind to the GRP78 protein. GRP78 has been reported to be highly expressed in glioma cells, vascular endothelial cells, and glioma stem cells, and almost not expressed in normal cells, making it an excellent target for glioma drug delivery.

[0009] According to a preferred embodiment of the present invention, the sequence of the EAG2-binding peptide is shown in SEQ ID NO: 4, and the EAG2-binding peptide is the EAG2–Kvβ2 complex targeting polypeptide described above---K90-114. TAT In addition to the membrane-penetrating peptide portion, it can bind to EAG2.

[0010] According to a preferred embodiment of the present invention, the sequence of the cell-penetrating peptide is shown in SEQ ID NO: 5, and the cell-penetrating peptide is derived from human immunodeficiency virus-1 transcription activator (HIV-1 TAT).

[0011] The linker serves to effectively separate the cell-penetrating peptide and the EAG2-binding peptide to ensure their proper functioning. Through exploration and optimization, the inventors of this invention have determined a linker with the sequence shown in SEQ ID NO: 6.

[0012] In this invention, since the Fc segment of engineered IgG comprises two chains, the resulting fusion protein also comprises two peptide chains. The positions of each functional unit are such that they satisfy their respective functions. According to a preferred embodiment of this invention, one of the two peptide chains consists of a cell-penetrating peptide, a linker, an EAG2-binding peptide, and the Fc segment of engineered IgG, sequentially from the N-terminus to the C-terminus. The other chain consists of a glioma-targeting peptide and the Fc segment of engineered IgG, sequentially from the N-terminus to the C-terminus.

[0013] The engineered Fc fusion protein of the present invention can be obtained through fusion expression. Given that the sequence is known, those skilled in the art can obtain the target protein using conventional methods in the field.

[0014] The principle behind the multifunctional engineered Fc fusion protein degrader designed in this invention inducing tumor cell death is as follows: Figure 1As shown, when the Fc fusion protein enters the circulatory system, it first binds to the human transferrin receptor hTfR via its engineered Fc segment. Through hTfR-mediated transport, it crosses the body border (BBB) ​​and then accumulates at the tumor site by targeting glioma cells via its targeting peptide. Subsequently, it effectively crosses the cell membrane using its cell-penetrating peptide. In the cytoplasm, the Fc fusion protein binds to TRIM21, which is highly expressed on glioma cells, via its Fc segment, and simultaneously binds to the target protein EAG2 via its EAG2-binding peptide, forming a "TRIM21-Fc fusion protein-EAG2" ternary complex. At this point, the binding of the Fc segment to TRIM21 activates TRIM21, inducing its autoubiquitination, which leads to the recognition and degradation of the ternary complex by the proteasome. This dual degradation of TRIM21 and EAG2 creates a dual-target effect on glioblastoma, ultimately inducing tumor cell death.

[0015] Therefore, the engineered Fc fusion protein of the present invention, which induces dual-target protein degradation, can be used to prepare drugs for the treatment of glioblastoma.

[0016] The technical effects of this invention are as follows: (1) By cleverly exploring the multiple functions of the antibody Fc segment, the application dilemma caused by the difficulty of peptide / protein drugs to cross the BBB was solved by engineering the Fc segment. Furthermore, the Fc segment is the core of the interaction between TRIM21 and antibody degrading agents. A dual-targeting protein degrading agent with cross-BBB function and cell targeting and permeabilization was developed - the multi-kinetic engineered Fc fusion protein. This makes the degrading agent present a diversity of functions and types, thereby broadening the application scope of the original targeted protein degradation technology and developing a new strategy for treating GBM.

[0017] (2) By utilizing the specific correlation between TRIM21 and EAG2 and glioblastoma, the autoubiquitination of TRIM21 and its mediated dual degradation of itself and target proteins are regulated by a single molecule (multifunctional engineered Fc fusion protein degrader) to form a dual-target effect on glioblastoma cells that highly express both TRIM21 and EAG2, thereby enhancing the anti-tumor effect.

[0018] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0019] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0020] Figure 1 This is a schematic diagram illustrating the mechanism of action of the engineered Fc fusion protein, which induces dual-target protein degradation, in the treatment of glioblastoma according to the present invention.

[0021] Figure 2 The Kaplan-Meier survival curves are based on a sample of 663 glioma patients. The vertical axis of Figure A represents overall survival, and the vertical axis of Figure B represents progression-free survival.

[0022] Figure 3 The results of TRIM21 and EAG2 expression level detection in human glioma cells U87 are shown.

[0023] Figure 4 Figures show the SDS-PAGE results of engineered Fc fusion protein (A) and its control protein (C), and the HPLC-SEC results of engineered Fc fusion protein (B) and its control protein (D). In Figure A, Lane 1 is the protein marker, Lane 2 is the engineered Fc fusion protein treated with reducing agent, and Lane 3 is the engineered Fc fusion protein. In Figure B, Lane 1 is the protein marker, Lane 2 is the control protein treated with reducing agent, and Lane 3 is the control protein.

[0024] Figure 5 The graph shows the effect of engineered Fc fusion protein degrading agent on the degradation of TRIM21 and EAG2 in glioma cells U87.

[0025] Figure 6 Figure A shows the distribution of engineered Fc-mCherry fusion protein in the mouse brain and major organs; Figure A shows the distribution of hTfR humanized C57BL / 6 mice (TfR). mu / hu Image B shows fluorescence imaging of brain tissue from KI mouse and normal mice 36 h after tail vein injection of engineered Fc-mCherry fusion protein; Image B shows hTfR humanized C57BL / 6 mice (TfR mu / hu Fluorescence imaging of the brain and major organs of KI mouse and normal mice 36 h after tail vein injection of engineered Fc-mCherry fusion protein. Detailed Implementation

[0026] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0027] Unless otherwise specified in the examples, all procedures were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0028] Example 1

[0029] Clinical data analysis of the association between TRIM21 and glioma

[0030] Based on the Cancer Genetic Information Database (TCGA), this embodiment analyzes the correlation between TRIM21 expression levels in tumor tissues of glioma patients and patient survival. The results show that lower TRIM21 expression levels are associated with prolonged overall survival and progression-free survival in glioma patients; conversely, patients with higher TRIM21 expression levels exhibit shorter overall survival and progression-free survival (see [link to study]). Figure 2 This indicates that the expression level of TRIM21 is closely related to the occurrence and development of glioma.

[0031] Example 2

[0032] Detection of the expression levels of TRIM21 and EAG2 proteins in glioma cells

[0033] This embodiment investigated the expression levels of TRIM21 and EAG2 in human glioma cells U87. Western blot results showed that TRIM21 and EAG2 were well expressed in glioma cells U87 (see...). Figure 3 ).

[0034] Example 3

[0035] Expression and purification of multifunctional engineered Fc fusion protein degraders.

[0036] The engineered Fc fusion protein degrader of the present invention consists of five parts: a cell-penetrating peptide, a linker, an EAG2-binding peptide, an engineered IgG Fc fragment, and a glioma-targeting peptide. It is obtained through fusion expression, and the sequences of each part are shown in Table 1.

[0037] Table 1

[0038] Expression and purification of engineered Fc fusion protein degrader: An expression plasmid encoding the Fc fusion protein degrader was constructed using the pCDNA3.4 vector. The "knob-into-hole" technique was employed to mutate relevant amino acid sites to prevent heavy chain mismatches that might occur during subsequent expression. The plasmid encoding the engineered Fc fusion protein degrader was then transfected into CHO cells and cultured at 37°C, 120 rpm, and 8% CO2 for 24 h. After 24 h, feed / sodium butyrate / antibiotics were added, and the cells were cultured for another 4 days. After expression, the cells were lysed, and then purified using Protein A affinity chromatography. Finally, the purified fusion protein was characterized for purity, molecular weight, etc., by SDS-PAGE, SEC-HPLC, and mass spectrometry. The purified protein was characterized as follows: Figure 4 As shown (the control protein is an engineered Fc fusion protein that does not contain the EAG2 binding peptide, i.e., it only includes cell-penetrating peptide, the Fc segment of engineered IgG, and glioma-targeting peptide), the results indicate that the present invention has successfully obtained the target protein.

[0039] Example 4

[0040] Characterization of TRIM21 and EAG2 degradation induced by engineered Fc fusion protein degraders

[0041] Engineered Fc fusion protein degrader (EAG2-Fc) and a control protein without EAG2 binding peptide (EAG2-FcCtl) were incubated with U87 glioma cells for 48 h, followed by Western blotting. The results showed that the purified degrader successfully induced the dual degradation of TRIM21 and EAG2, which are highly expressed in U87 glioma cells (e.g.,...). Figure 5 As shown in the figure, this also illustrates that the engineering of the Fc segment does not affect its integration with TRIM21 or the functionality of TRIM21.

[0042] Example 5

[0043] In vivo validation of engineered Fc-mediated coupling molecules across the BBB

[0044] Given that engineered Fc exhibits high specificity for the human transferrin receptor hTfR and almost no binding to mouse TfR, to investigate whether engineered Fc can successfully carry conjugate molecules across the brain via the BBB, this invention constructs a fusion expression product of engineered Fc and red fluorescent protein mCherry. Using mCherry as a drug model molecule, the expression was conducted in hTfR-humanized C57BL / 6 mice (TfR... mu / hu In vivo imaging was used in KI mice to verify the feasibility of engineered Fc-carrying molecules crossing the BBB. During the humanization process in mice, only the apical domain of the mouse TfR was replaced with the apical domain of the human hTfR, rather than simply replacing it with the full-length human receptor hTfR. The resulting TfR... mu / hu KI mice retain the native mouse transferrin ligand-binding domain and most Tfrc introns, minimizing the potential risk of protein expression and dysfunction resulting from this.

[0045] The experiment used normal C57BL / 6 mice as controls. hTfR-humanized C57BL / 6 mice (6-8 weeks old) were randomly divided into groups. Engineered Fc-mCherry fusion protein (2.71 mg / kg) was injected intravenously into the tail vein of the hTfR-humanized C57BL / 6 mice. During this period, in vivo imaging was used to observe the entry of fluorescently labeled proteins into the brain at different time points. After 36 hours, the mice were sacrificed, and the brain and major organs (heart, liver, spleen, lung, and kidney) were harvested for imaging. The results showed that the red fluorescence in the brain of hTfR-humanized C57BL / 6 mice was significantly stronger than that of normal C57BL / 6 mice. This indicates that the engineered Fc can effectively carry the red fluorescent protein mCherry across the blood-brain barrier (BBB) ​​into the brain via transcytosis after binding to the human transferrin receptor hTfR. In contrast, because it cannot bind to mouse TfR, it cannot carry mCherry across the BBB into the brain of normal C57BL / 6 mice. Figure 6 (A). Furthermore, the fluorescence distribution of major organs (brain, heart, liver, spleen, lungs, and kidneys) showed that, except for the brain and the kidney (a major metabolic organ), no red fluorescence was detected in other tissues. This indicates that the engineered Fc possesses high specificity in targeting the brain. Figure 6 B).

[0046] The above experiments show that the fusion protein degrader of the present invention has cross-BBB function, and also has cell targeting and membrane permeability. It can regulate the dual degradation of GBM-related target proteins TRIM21 and EAG2, forming a dual-target effect on glioblastoma cells, and has an enhanced anti-tumor effect.

[0047] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. An engineered Fc fusion protein that induces dual-target protein degradation, comprising a cell-penetrating peptide, a linker, an EAG2-binding peptide, an Fc segment of engineered IgG, and a glioma-targeting peptide, wherein the sequence of the Fc segment of the engineered IgG is shown in SEQ ID NO: 1 and SEQ ID NO:

2.

2. The engineered Fc fusion protein for inducing dual-target protein degradation according to claim 1, wherein, The sequence of the glioma-targeting peptide is shown in SEQ ID NO:

3.

3. The engineered Fc fusion protein for inducing dual-target protein degradation according to claim 1, wherein, The sequence of the EAG2 binding peptide is shown in SEQ ID NO:

4.

4. The engineered Fc fusion protein for inducing dual-target protein degradation according to claim 1, wherein, The sequence of the cell-penetrating peptide is shown in SEQ ID NO:

5.

5. The engineered Fc fusion protein for inducing dual-target protein degradation according to claim 1, wherein, The sequence of the connector is shown in SEQ ID NO:

6.

6. The engineered Fc fusion protein for inducing dual-target protein degradation according to claim 1, wherein, The fusion protein comprises two peptide chains: one chain consisting of a cell-penetrating peptide, a linker, an EAG2-binding peptide, and the Fc segment of engineered IgG, arranged sequentially from the N-terminus to the C-terminus; and the other chain consisting of a glioma-targeting peptide and the Fc segment of engineered IgG, arranged sequentially from the N-terminus to the C-terminus.

7. The use of the engineered Fc fusion protein according to any one of claims 1-6, which induces dual-target protein degradation, in the preparation of a medicament for treating glioblastoma.

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