A dual-targeted degradation agent of Fc fusion protein and its application

Abstract

The present invention belongs to the field of biomedicine, and relates to an Fc fusion protein dual-targeted degrader and its application. The Fc fusion protein dual-targeted degrader comprises: a tumor cell targeting peptide, an Fc segment of immunoglobulin G, a connector, a specific binding peptide of a tumor cell highly expressed protein, and a cell-penetrating peptide. The present invention utilizes the specific correlation between TRIM21 and CREPT and pancreatic cancer to improve the cell selectivity of the degrader; at the same time, the core of the interaction between TRIM21 and the antibody degrader, the Fc segment, is seized to develop an Fc-derived degrader with membrane permeability and targeting, and then the degrader regulates the self-ubiquitination of TRIM21 and its mediated dual degradation of itself and CREPT, forming a dual-target effect on pancreatic cancer cells with high expression of both TRIM21 and CREPT, improving the selectivity of the degradation effect, and enhancing the anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an Fc fusion protein dual-targeting degradation agent and its application. Background Art

[0002] Targeted Protein Degradation (TPD) is a new type of technology in the field of drug research and development in recent years. This technology uses functional molecules (protein degraders) to utilize the naturally existing protein degradation system (ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway) in cells to degrade disease-related proteins, thereby achieving the purpose of treating diseases. This type of technology directly and efficiently induces the degradation of target proteins, which is different from the mode of action of traditional small molecule drugs to inhibit protein function, thus making many currently "non-drugable" targets "drugable" and able to target targets that traditional small molecule inhibitors cannot target.

[0003] By designing protein degraders to bind E3 ubiquitin ligases and target proteins at the same time, using the intracellular ubiquitin-proteasome system UPS to degrade intracellular disease-related proteins is the fastest-growing type of targeted protein degradation technology with good application prospects. The Trim-Away system is a very noteworthy system in this type of technology. It uses antibodies as protein degraders to induce efficient and rapid degradation of intracellular proteins. The entire degradation process only takes about 10-20 minutes. The working principle of this system is based on the antibody-dependent intracellular antiviral neutralization mediated by TRIM21 protein (Tripartite Motif-Containing 21). Because TRIM21 has both Fc receptor and E3 ubiquitin ligase functions, when cells are invaded by antibody-coated viruses, TRIM21 in the cell can recognize the Fc segment of the antibody, initiate self-ubiquitination, recruit proteasomes, and mediate the degradation of the virus in the cell. Based on this, when antibodies (protein degraders) targeting intracellular proteins enter the cell, they can bind to TRIM21 and the target protein at the same time to form a "target protein-antibody-TRIM21" ternary complex, thereby initiating the autoubiquitination of TRIM21, recruiting proteasomes, and ultimately mediating the overall degradation of the target protein, TRIM21, and the antibody. Compared with another type of small molecule-based targeted protein degradation chimera (PROTAC) molecular degraders, antibody degraders can target proteins that lack small molecule binding cavities, showing their unique advantages. However, many tumor-related target proteins currently lack effective small molecule binding cavities, making it difficult for them to become targets for small molecules and thus lack drugability. Therefore, it is of great significance to develop antibody degraders for tumor-related target proteins.

[0004] Although antibody degraders based on the Trim-Away targeted protein degradation system have shown great potential in tumor treatment, the in vivo application of such degraders still faces difficulties such as cell selectivity and poor membrane permeability. Since the ubiquitin-proteasome protein degradation system UPS is ubiquitous in cells, when degraders targeting non-tumor-specific proteins enter the body, they can induce the action of UPS in normal cells and tumor cells at the same time, which can easily cause toxicity problems caused by protein degradation in normal cells. Therefore, improving the selectivity of degraders for tumor cells is a very important issue in the application of TPD technology in tumor targeted therapy. In addition, due to the poor membrane permeability of antibody degraders, the current application of Trim-Away technology requires the introduction of antibody molecules into cells by microinjection or electroporation, which also limits the application of this technology in a large number of cells and animals. Summary of the invention

[0005] In view of the problems of poor cell selectivity and membrane permeability of antibody degraders based on the Trim-Away targeted protein degradation system, the present invention provides a dual-targeted degrader based on the Trim-Away targeted protein degradation system. The dual-targeted degrader is a fusion protein, comprising two peptide segments that specifically bind to tumor cell-specific proteins, one is the Fc segment of immunoglobulin G (IgG) for identifying TRIM21, and the other is a specific binding peptide for tumor cell-specific highly expressed proteins. The dual-targeted degrader can bind to two target proteins at the same time, regulate the ubiquitin-mediated dual degradation of TRIM21 itself and another target protein, form a dual-target effect on tumor cells, thereby improving the selectivity of the antibody degrader and enhancing the anti-tumor effect.

[0006] Specifically, the present invention provides an Fc fusion protein dual-targeting degrader, which includes: a tumor cell targeting peptide, an Fc segment of immunoglobulin G, a connector, a specific binding peptide for a protein highly expressed in tumor cells, and a cell-penetrating peptide CPP.

[0007] According to the design of the present invention, the dual-targeted degrader needs to specifically recognize the segment of TRIM21 and be easy to derivatize, so it is not appropriate to directly use the TRIM21 antibody. After optimization, the Fc segment of immunoglobulin G (IgG-Fc) of the TRIM21 antibody was selected, and the sequence is shown in SEQ ID NO: 1. The Fc segment of immunoglobulin G is used to bind to the TRIM21 molecule and mediate the overall degradation of the TRIM21, Fc-derivatized degrader and target protein ternary complex by the proteasome.

[0008] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1)

[0009] According to the present invention, the highly expressed protein in tumor cells can be determined according to the type of tumor cells. The system of the present invention is applicable to cancer types on which TRIM21 has a cancer-promoting effect, such as pancreatic cancer.

[0010] According to a preferred embodiment of the present invention, the tumor cells are pancreatic cancer cells, and the highly expressed protein in the tumor cells is a highly expressed cell cycle-related protein CREPT (Cell Cycle-Related and Expression-Elevated Protein in Tumor). CREPT is highly expressed in pancreatic cancer patients and is associated with a low disease-free survival rate. Overexpression of CREPT promotes the cloning and proliferation of pancreatic cancer cells, and the protein is a new target for pancreatic cancer treatment. However, this target currently lacks targeted small molecule inhibitors.

[0011] Preferably, the sequence of the specific binding peptide of the highly expressed protein in tumor cells is shown in SEQ ID NO: 2. The sequence is a short peptide taken from the amino acids 266-286 at the C-terminus of the CREPT protein. Since two molecules of CREPT can bind to form a homodimer through the C-terminus, the short peptide can simulate the binding of one molecule of CREPT to another molecule of CREPT.

[0012] KDVLSEKEKKLEEYKQKLARV(SEQ ID NO: 2)

[0013] The Fc fusion protein degrader designed by the present invention works through the following mechanism (eg Figure 1As shown in the figure): When the Fc fusion protein enters the circulatory system, it first targets pancreatic cancer cells through the pancreatic cancer targeting peptide, and then effectively enters the cell with the help of CPP. In the cell, it binds to TRIM21, which is highly expressed in pancreatic cancer cells, through the Fc segment, and at the same time binds to the target protein CREPT through the CREPT binding peptide part, thereby forming a "TRIM21-Fc fusion protein-CREPT" ternary complex; at the same time, the binding of the Fc segment to TRIM21 activates the activity of TRIM21, triggering its self-ubiquitination, prompting the ternary complex to be recognized and degraded by the proteasome. The dual degradation of TRIM21 and CREPT forms a dual-target effect on pancreatic cancer cells, and ultimately induces the death of pancreatic cancer cells.

[0014] The tumor cell targeting peptide is fused to enhance the in vivo targeting of the dual-targeted degrader. The targeting peptide can be selected from various targeting peptides for specific tumor cells known in the art, and the present invention is not particularly limited to this. According to a preferred embodiment of the present invention, for pancreatic cancer cells, the sequence of the tumor cell targeting peptide is shown in SEQ ID NO: 3.

[0015] KTLLPTP (SEQ ID NO: 3)

[0016] The inventors of the present invention have determined the linker of the sequence shown in SEQ ID NO: 4 through exploration and optimization.

[0017] GGGSGGGS (SEQ ID NO: 4)

[0018] The dual-targeted degrader fused with a cell-penetrating peptide (CPP) has membrane permeability, can simulate the binding of antibodies to TRIM21, mediate the degradation of target proteins, and solve the application dilemma caused by the poor membrane permeability of antibodies. The sequence of the cell-penetrating peptide can be selected from various known sequences in the art, as long as it has a cell-penetrating effect, and the present invention is not particularly limited to this. According to a preferred embodiment of the present invention, the sequence of the cell-penetrating peptide is shown in SEQ ID NO: 5.

[0019] RRRRK (SEQ ID NO: 5)

[0020] According to a most preferred embodiment of the present invention, the sequence of the Fc fusion protein dual-targeting degrader is shown in SEQ ID NO:6.

[0021] KTLLPTPEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSKDVLSEKEKKLEEYKQKLARVRRRRK(SEQ ID NO:6)

[0022] The Fc fusion protein dual-targeted degradation agent of the present invention can be used to prepare a tumor therapeutic agent or a tumor cell proliferation inhibitor. The tumor is a cancer in which TRIM21 has a cancer-promoting effect, such as pancreatic cancer, and the tumor cell is, for example, a pancreatic cancer cell, specifically a PANC-1 cell or a SW1990 cell, preferably a PANC-1 cell.

[0023] The present invention utilizes the specific correlation between TRIM21 and CREPT and pancreatic cancer to improve the cell selectivity of the degradation agent; at the same time, the core of the interaction between TRIM21 and antibody degraders, namely the Fc segment, is seized to develop an Fc-derivatized degrader with membrane permeability and targeting, and then the autoubiquitination of TRIM21 and the dual degradation of itself and CREPT mediated by the degrader are regulated to form a dual-target effect on pancreatic cancer cells with high expression of both TRIM21 and CREPT, thereby improving the selectivity of the degradation effect and enhancing the anti-tumor effect.

[0024] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0026] Figure 1 The diagram is a working principle diagram of the dual targeted protein degradation mediated by ubiquitin ligase TRIM21 in the present invention for treating pancreatic cancer, wherein CPP is a cell penetrating peptide; CREPT is a cell cycle-related protein highly expressed in tumors; and Fc is the Fc segment of IgG.

[0027] Figure 2 The Kaplan–Meier survival curve is based on 178 pancreatic cancer patient samples.

[0028] Figure 3 Shown are the expression levels of TRIM21 and CREPT in pancreatic cancer cells.

[0029] Figure 4 The diagrams show the results of SDS-PAGE (left) and Western Blot (right) detection of Fc fusion protein. Western Blot was detected using anti-human IgG (Fc specific) antibody.

[0030] Figure 5 This figure shows the inhibitory effect of the degradation agent Fc fusion protein on the proliferation of pancreatic cancer cells PANC-1. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0032] Example 1

[0033] Clinical data analysis of the correlation between TRIM21 and pancreatic cancer.

[0034] Based on the cancer gene information database TCGA, the correlation between the expression level of TRIM21 in tumor tissues of pancreatic cancer patients and the survival of patients was analyzed.

[0035] The results of the analysis showed that lower TRIM21 expression levels were associated with longer disease-free survival in pancreatic cancer patients; conversely, patients with higher TRIM21 expression levels had shorter disease-free survival (see Figure 2 , the upper curve is low expression, and the lower curve is high expression). The expression level of TRIM21 is closely correlated with the occurrence and development of pancreatic cancer.

[0036] Example 2

[0037] Detection of TRIM21 and CREPT expression in pancreatic cancer cells.

[0038] The expression levels of two pancreatic cancer-related target proteins, CREPT and TRIM21, in pancreatic cancer cells PANC-1 and SW1990 were detected.

[0039] The expression levels of two pancreatic cancer-related target proteins, CREPT and TRIM21, in pancreatic cancer cells PANC-1 and SW1990 were detected by Western Blot using anti-TRIM 21 and anti-CREPT (also known as anti-RPRD1B) antibodies.

[0040] Western Blot results showed that TRIM21 and CREPT were well expressed in pancreatic cancer cells PANC-1 and SW1990 (see Figure 3 ). Based on the expression levels of the two proteins in two commonly used pancreatic cancer cells, PANC-1 and SW1990, pancreatic cancer cell PANC-1 was subsequently selected for cellular level research.

[0041] Example 3

[0042] The expression and purification conditions of Fc fusion protein were explored.

[0043] This example explores the expression and purification conditions of the Fc fusion protein used. The Fc fusion protein of the present invention is composed of five parts: pancreatic cancer targeting peptide, IgG Fc segment, Linker, CREPT binding peptide and cell penetrating peptide CPP. The DNA sequence encoding the Fc fusion protein is linked to the p35 vector to construct an expression plasmid, and the target protein is expressed by transfecting HEK293 (see Figure 4 ).

[0044] Example 4

[0045] Inhibitory effect of Fc fusion protein on proliferation of pancreatic cancer cells

[0046] Using Fc fusion protein without CREPT binding peptide as control, the inhibitory effect of the degradation agent Fc fusion protein on the proliferation of pancreatic cancer cell PANC-1 was investigated by MTT experiment.

[0047] According to the MTT results ( Figure 5 ) It can be seen that compared with the control protein, the degrader Fc fusion protein showed a stronger inhibitory effect on the proliferation of pancreatic cancer cells PANC-1.

[0048] Compared with the target protein, the control protein lacks the CREPT binding peptide and has the following amino acid sequence:

[0049] KTLLPTPEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSRRRRK(SEQ ID NO:7)

[0050] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. Sequence Listing <110> Tianjin Medical University Nankai University <120> A dual-targeted degradation agent of Fc fusion protein and its application <130> 2200112 <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 232 <212> PRT <213> Artificial Sequence <400> 1 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 2 <211> 21 <212> PRT <213> Artificial Sequence <400> 2 Lys Asp Val Leu Ser Glu Lys Glu Lys Lys Leu Glu Glu Tyr Lys Gln 1 5 10 15 Lys Leu Ala Arg Val 20 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <400> 3 Lys Thr Leu Leu Pro Thr Pro 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <400> 4 Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <400> 5 Arg Arg Arg Arg Lys 1 5 <210> 6 <211> 273 <212> PRT <213> Artificial Sequence <400> 6 Lys Thr Leu Leu Pro Thr Pro Glu Pro Lys Ser Cys Asp Lys Thr His 1 5 10 15 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 20 25 30 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 35 40 45 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 50 55 60 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 65 70 75 80 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 85 90 95 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 100 105 110 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 115 120 125 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 130 135 140 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 145 150 155 160 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 165 170 175 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 180 185 190 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 195 200 205 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 210 215 220 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly 225 230 235 240 Gly Gly Ser Gly Gly Gly Ser Lys Asp Val Leu Ser Glu Lys Glu Lys 245 250 255 Lys Leu Glu Glu Tyr Lys Gln Lys Leu Ala Arg Val Arg Arg Arg Arg 260 265 270 Lys <210> 7 <211> 252 <212> PRT <213> Artificial Sequence <400> 7 Lys Thr Leu Leu Pro Thr Pro Glu Pro Lys Ser Cys Asp Lys Thr His 1 5 10 15 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 20 25 30 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 35 40 45 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 50 55 60 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 65 70 75 80 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 85 90 95 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 100 105 110 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 115 120 125 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 130 135 140 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 145 150 155 160 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 165 170 175 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 180 185 190 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 195 200 205 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 210 215 220 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly 225 230 235 240 Gly Gly Ser Gly Gly Gly Ser Arg Arg Arg Arg Lys 245 250

Claims

1. An Fc fusion protein dual-targeting degrader, the Fc fusion protein dual-targeting degrader consisting of: Tumor cell targeting peptides, Fc segment of immunoglobulin G, linkers, specific binding peptides for proteins highly expressed in tumor cells, and cell-penetrating peptides; The sequence of the Fc segment of immunoglobulin G is shown in SEQ ID NO: 1; The sequence of the specific binding peptide for the highly expressed protein in tumor cells is shown in SEQ ID NO: 2; The sequence of the tumor cell targeting peptide is shown in SEQ ID NO: 3; The sequence of the connector is shown in SEQ ID NO: 4; The sequence of the cell-penetrating peptide is shown in SEQ ID NO:

5.

2. The Fc fusion protein dual-targeting degradation agent according to claim 1, wherein: The sequence of the Fc fusion protein dual-targeting degrader is shown in SEQ ID NO:

6.

3. Use of the Fc fusion protein dual-targeting degradation agent according to any one of claims 1 to 2 in the preparation of a tumor therapeutic agent or a tumor cell proliferation inhibitor, wherein: The tumor is pancreatic cancer, and the tumor cells are pancreatic cancer cells.

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