CN115873774A - A bacterium that degrades intracellular tumor-proliferating proteins - Google Patents

Abstract

The invention relates to the preparation and application of a drug for treating tumors based on bacterial carrier-specific degradation of intracellular proteins, belonging to the field of biomedicine. The invention uses bacteria as a carrier to express a fusion protein capable of specifically targeting and degrading intracellular proteins, which can cause cell immunogenic death while blocking tumor cell proliferation, so as to achieve the purpose of tumor treatment.

Description

A bacterium that degrades intracellular tumor-proliferating proteins

1. Technical field

The invention belongs to the field of biomedicine, and in particular relates to the preparation and application of a drug for treating tumors based on bacterial carrier-specific degradation of intracellular proliferation proteins.

2. Background technology

Cancer is still a major disease that is difficult to solve for human beings. At present, more and more researchers are focusing on immunotherapy, such as immune checkpoint inhibitors such as PD-1, small molecule inhibitors targeting different targets, and tumor vaccines targeting tumor antigens.

Targeted protein degradation (Targeted Protein Degradation, TPD) provides unprecedented opportunities for drug discovery. Proteolysis-targeting chimeras (PROTACs), which utilize the ubiquitin-proteasome system to selectively induce targeted protein degradation, are an emerging therapeutic technology with the potential to modulate traditionally undruggable targets. One end of PROTAC can target and bind to the target protein, and the other end can recruit E3 ubiquitin ligase (E3 ligase), which ubiquitinates the target protein and degrades the target protein through the ubiquitin-proteasome pathway. This technology has entered into clinical trials and has changed the prospects of small molecule drugs, but it also faces new challenges, such as the difficulty in screening molecules targeting target proteins with high specificity, and there are fewer types of E3 ligase to choose from. In addition, the molecular weight of the protein is large, and the delivery efficiency to the cell is low, which is an urgent problem to be solved. At present, some studies have used mRNA to encode PROTAC fusion protein and express it directly in cells. However, mRNA itself is easily degraded by ribozymes in the body, and the delivery efficiency is low. Therefore, cationic liposomes are commonly used as delivery systems, and cationic liposomes are enriched in the liver after administration, making it difficult to target tumors for precise delivery. , and the cationic liposome itself has toxic side effects.

The PROTAC target used in this study is proliferating cell nuclear antigen (PCNA), which is an auxiliary protein required for DNA replication in the process of cell proliferation. After it is degraded, DNA replication cannot proceed normally, so the cell cycle arrests, and tumor cells Amplification is stopped. At the same time, because PCNA is the key protein of DNA replication, the degradation of PCNA can lead to abnormal DNA replication, and the damaged DNA fragments flow into the cytoplasm, activating the cGAS-STING signaling pathway, thereby causing immunogenic cell death, which can induce tumor cell death. It causes a cascade reaction internally and promotes the body's immune killing of tumors.

In this study, attenuated Salmonella VNP20009 with knockout flagellin was used as a vector to introduce and express a fusion protein targeting PCNA, and the bacterial vector was used as a delivery system for PROTAC. Due to its anaerobic characteristics, attenuated Salmonella will be enriched in the hypoxic tumor microenvironment after entering the body, and a small number of bacteria free in the body will be phagocytized and cleared by macrophages without affecting normal cells. After the attenuated Salmonella is enriched in the tumor, because its flagellin is knocked out, it cannot escape from the cell, so it can colonize in the cytoplasm and continuously express the fusion protein targeting PCNA, becoming a "protein factory". PCNA protein degradation. The bacterial carrier used in this study to deliver PROTAC can specifically target the PCNA protein in the tumor, degrade it, block the tumor cell cycle, and inhibit tumor growth.

3. Contents of the invention

1. Purpose of the invention

The invention constructs a drug for treating tumors based on a bacterial carrier that specifically degrades intracellular proteins, uses attenuated Salmonella to express a fusion protein, one end is a domain that recruits E3 ubiquitin ligase (E3 ligase), and the other end is a specificity Targeting the proliferating cell nuclear antigen (PCNA) binding domain, the linker linker is used in the middle to form a fusion protein. The E3 ligase recruitment domain can enrich the E3 ubiquitin ligase, ubiquitinate the PCNA bound by the PCNA binding domain, and degrade it through the ubiquitin-proteasome pathway. As a result, the tumor cell cycle stagnates and no longer proliferates. At the same time, because PCNA is a key protein for DNA replication, the degradation of PCNA can lead to abnormal DNA replication, and the damaged DNA fragments flow into the cytoplasm, activating the cGAS-STING signaling pathway, thereby causing cellular immunogenicity. Immunogenic cell death can cause a cascade reaction inside the tumor and promote the body's immune killing of the tumor.

2. Technical solutions

2.1. Flagellar gene flhD knockout vector preparation

The strain used as a bacterial carrier in this study was attenuated Salmonella typhimurium VNP20009, which has been proven safe in the literature. The flagellar gene flhD was knocked out by the Red homologous recombination method to obtain a bacterial vector.

2.2 Preparation of fusion protein particles expressing targeted degradation of PCNA

The protein sequence was synthesized in GenScript. After receiving it, primers were designed to amplify the target fragment E3LR-PCNAtag by PCR, and it was integrated into the linearized plasmid vector pUC57 (with Amp resistance) through homologous recombination, and then introduced into E. coli for amplification. The extracted plasmid is the target plasmid pUC57-E3LR-PCNAtag.

2.3 Preparation of target plasmid into bacterial vector

The target plasmid pUC57-E3LR-PCNAtag described in 2.2 was introduced into the bacterial vector described in 2.1 by electroporation, the expression of CD47 antagonistic protein was verified by Western Blot, and the strains capable of normal protein expression were amplified and counted, and stored in 25% in glycerol and stored in a -80°C refrigerator.

2.4 Validation of Drug Efficacy in Mouse Tumor Models

Dilute the attenuated bacterial strains prepared in 2.3 to three concentrations of 1×10^ ⁷ , 1×10^ ⁶ , and 1×10^ ⁵ respectively, and design a normal saline (solvent) control group, carry out dose climbing verification, and screen out the drug Optimal dose and maximum tolerated dose.

Select the optimal drug efficacy dose within the safe range, compare the drug effect of blocking the cell cycle with the cell cycle small molecule inhibitor CDK4/6 inhibitor, and verify the activation and infiltration of immune cells by flow cytometry.

3. Technical effects

In this study, attenuated Salmonella was used to express a fusion protein, which can recruit E3 ubiquitin ligase (E3ligase) and specifically target proliferating cell nuclear antigen (PCNA), and degrade PCNA through the ubiquitin-proteasome pathway. As a result, the tumor cell cycle stagnates, no longer proliferates, and at the same time causes DNA damage, activates the cGAS-STING signaling pathway, causes cell immunogenic death, causes a cascade reaction inside the tumor, and promotes the body's immune killing of the tumor.

4. Specific implementation

Example 1. Preparation of a plasmid vector expressing a fusion protein targeting degradation of PCNA

1.1 Target gene amplification

The target fragment was synthesized in GenScript, and the upstream and downstream primers p1 and p2 were designed. Put the bacterial solution containing the target plasmid in 10ml LB medium, shake it overnight at 37°C, and use the plasmid mini-extraction kit to lyse the bacterial cells to extract the plasmid. After measuring the concentration of the plasmid, perform high-fidelity PCR amplification. The system is as follows: high-fidelity enzyme 25 μl; deionized water 17 μl; primers p1 and p2 2 μl each; template (target plasmid) 4 μl; total volume 50 μl. PCR cycle parameters: pre-denaturation at 98°C for 5 minutes; 30 cycles at 98°C for 10s, 55°C for 30s, and 72°C for 1 min; extension at 72°C for 10 minutes, and storage at 4°C. 10 μl of the PCR product was taken for agarose gel electrophoresis to detect the amplification effect, and the remaining products were recovered and purified with an agarose gel recovery kit or a rapid PCR product recovery kit, and finally dissolved in an appropriate amount of deionized water to determine the concentration of recovered DNA.

1.2 Linearization of pUC57 plasmid vector

The pUC57 plasmid vector was digested and linearized with BamHI endonuclease. The reaction system: 10 μg of vector plasmid; 10 μl of 10× digestion buffer; 2 μl of BamHI endonuclease; made up to 100 μl of deionized water. Enzyme digestion at 37°C for 2 hours, adding 1 μl of rapid CIP reagent for dephosphorylation, and bathing at 37°C for 30 minutes to prevent circularization of the cut vector. Use an agarose gel recovery kit or a rapid PCR product recovery kit to recover and purify, and finally dissolve in an appropriate amount of deionized water and measure the recovered DNA concentration.

1.3 Homologous recombination ligation of pUC57 plasmid vector and target fragment

Using a homologous recombination kit, the concentration of the target fragment and the carrier ratio are 3:1 for homologous recombination connection, the reaction system: pUC57 restriction enzyme digestion vector 50ng; target fragment 150ng; buffer 4μl; homologous recombination reaction enzyme 2μl; deionized water Make up to 20μl. 37°C water bath for 30 minutes, immediately ice bath for 5 minutes, and transform Escherichia coli DH5α.

1.4 Transformation of Escherichia coli E.coli DH5α

Mix 10 μl of the ligation product, 10 μl of 5×KCM reagent, and 30 μl of deionized water in ice bath, add 50 μL of competent E.coli DH5α in ice bath for 30 min, heat shock at 42°C for 90 s, add 1 mL of LB and culture at 37°C for 1 h, centrifuge at 5000 rpm After 5 minutes, leave part of the liquid to resuspend the plate.

1.5 Colony PCR and sequencing verification

Pick the single colony after plating into LB liquid medium and shake it for 8 hours, design primers p3 and p4 for PCR amplification and agarose gel electrophoresis to detect the connection of the target fragments, take part of the bacterial liquid and send it to Nanjing Qingke Biotechnology Sequencing was carried out, and the sequence alignment was correct, stored in 25% glycerol, and stored in a -80°C refrigerator.

Example 2. Preparation of attenuated Salmonella VNP20009 vector with flagellar gene flhD knocked out

VNP20009 was shaken and cultured in liquid medium overnight (37°C, 150rpm), collected by centrifugation, and the flagellar gene flhD was knocked out by the Red homologous recombination method. It can be seen visually by electron microscope that VNP20009 does not express flagellin.

Example 3. Construction of attenuated Salmonella VNP20009 expressing the linked plasmid

The Escherichia coli containing the correctly connected plasmid described in Example 1 was amplified and cultured, and the concentration of the plasmid was extracted with a plasmid mini-extraction kit, and then introduced into the attenuated Salmonella VNP20009 with the flagellar gene knockout described in Example 2 by electroporation. The expression of secreted protein was detected by western blot. The strains with better expression were selected, counted after amplified culture, preserved in 25% glycerol, and stored in -80°C refrigerator.

Example 4. Attenuated Salmonella tumor vaccine animal efficacy verification

In this example, Balb/c mice were used to construct the CT26 colorectal cancer model, and C57 mice were used to construct the B16F10 melanoma model for animal drug efficacy verification. The experimental mice were purchased from Jicui Yaokang and were bred in a clean barrier in the Experimental Animal Center of Nanjing University. , The temperature of the feeding room is about 25°C, and the humidity is 40% to 70%. The newly introduced experimental animals are adapted for 1 to 3 days before the experiment.

CT26 and B16F10 cells were cultured and expanded in RPMI 1640 and DMEM medium respectively. Each mouse was inoculated with 5×10^ ⁵ cells, and the tumor size was measured 7 days later. The drug was started when it was about 40mm3, and the tumor size was measured every other day. , draw tumor growth curve and survival curve.

Dilute the attenuated bacterial strains prepared in Example 3 to three concentrations of 1×10^ ⁷ , 1×10^ ⁶ , and 1×10^ ⁵ respectively, and design a normal saline (solvent) control group for dose escalation verification and screening The most effective dose and the maximum tolerated dose.

Select the optimal drug efficacy dose within the safe range, compare the drug effect of blocking the cell cycle with the cell cycle small molecule inhibitor CDK4/6 inhibitor, and verify the activation and infiltration of immune cells by flow cytometry.

The results showed that the attenuated Salmonella VNP20009 could continuously express the fusion protein targeting the degradation of PCNA in the tumor, arrest the cell cycle, and stop the division of tumor cells. The safety evaluation shows that the safety of the bacterial carrier drug is good, indicating that the bacterial carrier prepared by the present invention expresses the fusion protein targeting degradation of PCNA to treat tumors and has good curative effect and high safety.

Claims (6)

1. A bacterium which degrades intracellular tumor proliferation protein, characterized by expressing an E3 ubiquitination fusion protein capable of specifically binding to intracellular proliferation protein, increasing ubiquitination degradation of cell proliferation protein, blocking proliferation of tumor cells.

2. The bacterial vector of claim 1, wherein the bacteria is attenuated salmonella typhimurium, including but not limited to attenuated listeria, escherichia coli, and the like.

3. The bacterial vector of claim 1, wherein the bacteria express a fusion protein targeted to degrade PCNA.

4. The composition of claim 1, wherein the delivery system dosage form is a solution, including but not limited to lyophilized powder, tablet, gel, and the like.

5. The composition of claim 1, wherein the administration is intravenous injection, including but not limited to intratumoral injection, subcutaneous injection, oral administration, intramuscular injection, etc.

6. The composition of claim 1, wherein the solvent used is physiological saline including, but not limited to, ringer's solution, phosphate buffered saline, water, and the like.