Novel recombinant protein RB1-PDL1 and application thereof

Abstract

The invention discloses a novel recombinant protein RB1-PDL1 and application thereof, and belongs to the technical field of biological medicine. The invention has the advantages that: proper hydrophilic modification design is performed on the existing non-functional regions of the retinoblastoma related protein and PD-L1 protein by means of synthetic biology, the anti-tumor activity function of the protein is reserved, the water solubility of the protein is improved, and the defect of poor water solubility of the traditional anti-tumor drug is effectively overcome. The target protein with the effect of killing breast cancer cells and lung cancer cells is obtained on a large scale by means of synthetic biology, and the production cost of protein anti-tumor drugs can also be reduced.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to a novel recombinant protein RB1-PDL1 and its application. Background Art

[0002] Programmed death ligand 1 (PD-L1) is encoded by the PDCDL1 gene, and its most important function is to bind to programmed death receptor-1 (PD-1; CD279). The binding of PD-L1 and PD-1 to cancer cells can activate the phosphatase containing the Src homology 2 domain, resulting in the inhibition of the T cell receptor (TCR) pathway. The inhibition of the TCR pathway makes it difficult for T cells to exert their anti-cancer activity. Based on this, if the binding of PD-1 and PD-L1 is blocked, the occurrence and development of tumors can be effectively inhibited, and the apoptosis of tumor cells can be promoted. In existing studies, a variety of molecular techniques targeting PD-L1 have been proposed, and their principle is usually to competitively bind to PD-1, thereby blocking the binding of PD-L1 and PD-1. For example, the patent with publication number CN116063401B and invention name "Ultra-high affinity small protein blocking type targeting PD-L1 and uses thereof" discloses a fusion protein targeting PD-L1. However, existing clinical studies show that many patients cannot produce sufficient responses to monospecific therapies. For example, among patients treated with PD-L1 / PD-1 targeting antibodies, only a part shows durable responses or survival. To address the above limitations, some scholars have also proposed to develop bispecific or multispecific recombinant proteins targeting two or more different antigens or different epitopes of the same antigen.

[0003] The protein Retinoblastoma-associated protein encoded by the RB1 gene is a negative regulator of the cell cycle. Under normal conditions, RB1 plays a role in maintaining the stability of chromatin structure. The dephosphorylated form of RB1 can bind to the transcription factor E2F1, and then act as a negative regulator of the cell cycle to prevent cells from passing through the G1-S checkpoint smoothly, resulting in cell cycle arrest. Therefore, RB1 plays a key role in inhibiting the occurrence of various tumors.

[0004] However, currently, bispecific or multispecific proteins face huge challenges in design. Because a large number of variable factors need to be considered, including the compatibility of molecules, whether they can be linked together, whether the resulting recombinant protein can be successfully expressed, affinity, drug stability, etc. It has been recognized that simply linking two or more antibodies or proteins together usually does not cause a synergistic effect, and even produces worse effects. In summary, it is necessary to propose new improvements for the deficiencies of the existing technology. Summary of the Invention

[0005] The object of the present invention is to provide a novel recombinant protein RB1-PDL1 for treating breast cancer or lung cancer. By means of synthetic biology, the existing Retinoblastoma-associated protein and PD-L1 are redesigned. The original functional regions of Retinoblastoma-associated protein and PD-L1 are retained to exert their biological functions. The partial non-coding regions at their starting ends are appropriately mutated and modified. The mutated Retinoblastoma-associated protein and PD-L1 are linked by a flexible protein linker (GGGGSGGGGSGGGGS) to obtain a brand-new recombinant protein RB1-PDL1 with the killing effect on breast cancer cells and lung cancer cells, which can effectively improve the water solubility of the protein and the killing effect on breast cancer and lung cancer cells. The present invention scales up to obtain the target protein with the killing effect on breast cancer cells and lung cancer cells by means of synthetic biology, and can also reduce the production cost of natural protein-based anti-tumor cell drug preparations.

[0006] The above technical object of the present invention is achieved by the following technical solutions:

[0007] A novel recombinant protein RB1-PDL1, characterized in that it includes the entire amino acid sequence of the Retinoblastoma-associated protein and the amino acid sequence of PD-L1, and the 42nd leucine (L), 44th leucine (L), 45th valine (V), 47th leucine (L), 49th phenylalanine (F), 57th phenylalanine (F), 60th leucine (L), and 64th leucine (L) of the Retinoblastoma-associated protein, which are all hydrophobic, are successively mutated into hydrophilic glutamine (Q), glutamine (Q), threonine (T), glutamine (Q), tyrosine (Y), tyrosine (Y), glutamine (Q), and glutamine (Q), respectively, with a total of 8 hydrophobic amino acids mutated; the 1st phenylalanine (F), 3rd isoleucine (I), 5th valine (V), 9th leucine (L), 11th valine (V), and 12th valine (V) of PD-L1, which are all hydrophobic, are successively mutated into hydrophilic tyrosine (Y), threonine (T), threonine (T), glutamine (Q), threonine (T), and threonine (T), respectively, with a total of 6 hydrophobic amino acids mutated. The amino acid sequence of the novel recombinant protein is shown in SEQ ID NO.1.

[0008] A recombinant expression vector comprising the novel recombinant protein RB1-PDL1 described in claim 1.

[0009] Preferably, the type of the vector is a plasmid vector, a phage vector, or an animal and plant virus vector.

[0010] A recombinant bacterium that expresses the novel recombinant protein RB1-PDL1.

[0011] Preferably, the recombinant bacterium is selected from one or more of Escherichia coli, Bacillus subtilis, Pichia pastoris, and / or Saccharomyces cerevisiae.

[0012] Use of the above novel recombinant protein RB1-PDL1 in the preparation of a pharmaceutical preparation for treating breast cancer or lung cancer.

[0013] Preferably, the pharmaceutical preparation further comprises a pharmaceutically acceptable carrier and / or adjuvant.

[0014] Preferably, the drug is for internal use.

[0015] Preferably, the dosage form of the drug for internal use is selected from one of injection, tablet, or pill.

[0016] Use of the above novel recombinant protein RB1-PDL1 in the preparation of a reagent for killing breast cancer cells or lung cancer cells in vitro, including biochemical reagents for experimental use.

[0017] In summary, the present invention has the following beneficial effects:

[0018] 1. In the present invention, the amino acid sequences of partial non-functional regions of Retinoblastoma-associated protein and PD-L1 are mutated and fused through a flexible linker to obtain a novel recombinant protein named RB1-PDL1. The specific amino acid sequence of the novel recombinant protein is shown in SEQ ID NO.1. This novel recombinant protein has significant killing effects on breast cancer cells and lung cancer cells and better hydrophilicity.

[0019] 2. A series of tumor cell killing performance tests were carried out on the designed novel recombinant protein RB1-PDL1, including tumor cell killing performance experiments on 4T1 and A549 cells. The experimental results show that the novel recombinant protein RB1-PDL1 has high killing ability for breast cancer cells and non-small cell lung cancer cells, and has good basic research and clinical application value. Description of the Drawings

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual scale. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to these drawings.

[0021] Figure 1 Electrophoresis strip diagram of the novel recombinant protein RB1-PDL1 prepared by the present invention;

[0022] Figure 2 Advanced structure prediction result of the novel recombinant protein RB1-PDL1 designed by the present invention;

[0023] Figure 3 Hydrophilicity and hydrophobicity prediction result of the novel recombinant protein RB1-PDL1 designed by the present invention;

[0024] Figure 4 Cytotoxicity experiment of the novel recombinant protein RB1-PDL1 designed by the present invention on L929 and HEK293T cells;

[0025] Figure 5 Cytotoxicity experiment result of the novel recombinant protein RB1-PDL1 designed by the present invention on 4T1 cells (mouse breast cancer cells);

[0026] Figure 6 Cytotoxicity experiment result of the novel recombinant protein RB1-PDL1 designed by the present invention on A549 cells (human non-small cell lung cancer cells). Detailed implementation manners

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0028] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0029] As used herein, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0030] It should be noted that, in this article, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not preclude the presence of additional identical elements in the process, method, article or device comprising that element.

[0031] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0032] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and the individual numerical values within that range. For example, the description of the range 1 - 6 should be regarded as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0033] Materials and Reagents:

[0034] HEK293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences' Committee for Type Culture Collection, catalog number: SCSP-502, and the Chinese cell name: human embryonic kidney cells. L929 cells were purchased from the Cell Bank of the Chinese Academy of Sciences' Committee for Type Culture Collection, catalog number: SCSP-5039, and the Chinese cell name: mouse fibroblasts. 4T1 and A549 cells were also both purchased from the Cell Bank of the Chinese Academy of Sciences' Committee for Type Culture Collection, with catalog numbers: SCSP-5056 and SCSP-503 respectively, and the Chinese cell names: mouse breast cancer cells and human non-small cell lung cancer cells

[0035] Escherichia coli BL21(DE3) competent cells were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0036] The pET-22b(+) plasmid is a commercially available E. coli expression vector, purchased from Beijing Tsingke Biotechnology Co., Ltd. The vector tags are N-pelB and C-His, and the vector resistance is Ampicillin.

[0037] The restriction enzymes NdeI and XhoI were purchased from NEB (Beijing) Co., Ltd. The His-tag protein purification resin (nickel column) was purchased from Shanghai Lianmai Biotechnology Co., Ltd., with the product number LM-616. IPTG, Ampicillin, and DMSO (dimethyl sulfoxide) were all purchased from Beijing Solarbio Science & Technology Co., Ltd. The DMEM medium was purchased from GIBCO. The CCK8 reagent was purchased from Chongqing Baoguang Technology Co., Ltd. and was used according to the reagent instruction manual.

[0038] Medium:

[0039] Each liter of LB medium contains: 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride, and the pH is adjusted to 7.0.

[0040] Preparation method: Dissolve 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride in 950 mL of double-distilled water, then adjust the pH to 7.0 with sodium hydroxide solution, and make up the volume to 1 L with double-distilled water. If preparing solid medium, add agar at 1.5 g / 100 mL. Autoclave at 121 °C for 30 min.

[0041] The experimental reagents not specifically described in the present invention are all conventional reagents in the art, and can be prepared by conventional methods in the art or obtained from relevant reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and relevant experimental manuals can be referred to, such as the Molecular Cloning Experimental Manual or the instruction manuals of relevant reagent manufacturers.

[0042] Example 1

[0043] 1. RB1-PDL1 gene design and protein expression

[0044] Design a gene with the nucleotide sequence shown in SEQ ID NO.2, with a full length of 3513 bp. This gene encodes a protein with the amino acid sequence shown in SEQ ID NO.1, with a full length of 1171 amino acids, named recombinant protein RB1-PDL1. When synthesizing this gene, NdeI restriction enzyme site (CATATG) and XhoI restriction enzyme site (CTCGAG) were added to the 5' end and 3' end of the gene respectively. The gene sequence with restriction enzyme sites is shown in SEQ ID NO.3. The synthesized gene was sequenced and verified, and the gene with correct sequence was used for subsequent vector construction. The specific sequences involved are shown in Table 1.

[0045] Table 1

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] 2. Vector construction

[0053] In this example, the pET-22b(+) plasmid is preferably used as the expression vector. The pET-22b(+) plasmid and the target gene (SEQ ID NO.1) are respectively double digested with the restriction endonucleases NdeI and XhoI, and then the target gene is ligated into the pET-22b(+) vector through a ligation reaction. It can be understood that this example can also be achieved by using a phage vector or an animal and plant virus vector, etc.

[0054] 3. Recombinant bacteria transformation

[0055] (1) Take out the Escherichia coli BL21(DE3) competent cells (Beijing Solarbio) from the -80°C refrigerator and place them on ice for 5 min.

[0056] (2) After the glycerol for storing the BL21(DE3) competent cells melts, add the competent cells to the ligation product, pipette up and down 3 times with a pipette tip to mix evenly, and let it stand on ice for 30 min.

[0057] (3) Quickly dry the moisture on the tube wall with absorbent paper, then perform heat shock at 42°C for 90 s, and immediately place it on ice for 2 min.

[0058] (4) Under sterile conditions, add 800 μL of LB liquid medium, culture at 37°C and 150 rpm for 45 min.

[0059] (5) Centrifuge at 8000 rpm for 5 min to collect the bacterial cells, discard part of the supernatant, resuspend the Escherichia coli with the remaining about 100 μL of supernatant, and then evenly spread it on the LB solid medium containing 100 μg / mL ampicillin, and place it in a 37°C incubator for inverted culture for about 12 h.

[0060] (6) Pick a single colony and inoculate it into the liquid LB medium containing 100 μg / mL ampicillin. After culturing at 37°C for about 13 h, perform identification of positive clones.

[0061] This embodiment preferably uses Escherichia coli as the recombinant bacterium. It can be understood that this embodiment can also be implemented using Bacillus subtilis, Pichia pastoris or Saccharomyces cerevisiae strains, etc.

[0062] 4. Protein expression and purification

[0063] (1) Inoculation: Prepare liquid LB medium and sterilize it. After sterilization, place the liquid LB medium in a laminar flow hood and cool it to room temperature. Add ampicillin to the LB medium in the laminar flow hood and mix well to make the final concentration of ampicillin 100 μg / mL. Then inoculate Escherichia coli (positive clone) containing the plasmid with the target gene into the LB medium at a rate of 200 μL / L. Then place this LB medium in a shaker and culture it for 8 h under the conditions of a rotation speed of 170 rpm and a temperature of 37 °C.

[0064] (2) Induction: After culturing in the shaker for 8 h, take out 2 mL of the bacterial solution and measure its OD 600 value. When the OD 600 value of the bacterial solution reaches 0.6 - 0.8, add IPTG with a final concentration of 200 μL / mL to this bacterial solution, and then culture it in a shaker for 8 h under the conditions of 37 °C and 170 rpm.

[0065] (3) Purification: After adding IPTG and culturing in the shaker for 8 h, take out the bacterial solution, centrifuge it at 8000 rpm for 5 min at 4 °C. After centrifugation, discard the supernatant and retain the precipitate (the precipitate is Escherichia coli).

[0066] (4) Ultrasonic disruption: Ultrasonically disrupt Escherichia coli and then centrifuge it. The precipitate obtained by centrifugation contains the target protein. First, wash the precipitate obtained by centrifugation with washing solution I (50 mmol / L Tris-HCl, 1 mol / L urea, 10 mL / L Triton X-100), then wash it with washing solution II (50 mmol / L Tris-HCl, 2 mol / L urea, 5 mL / L Triton X-100), and then dissolve it with inclusion body dissolution solution (50 mmol / L Tris-HCl, 8 mol / L urea, 100 mmol / L NaCl).

[0067] (5) Finally, the inclusion body solution was subjected to gradient renaturation as follows: the inclusion body solution was placed in a dialysis bag (Beijing Solebow Technology Co., Ltd., catalog number: YA1071), and the dialysis bag was placed in 6M, 4M, 2M, 1M, and 0.5M urea solutions in sequence for gradient renaturation. The renaturation was performed for 3 hours at each urea concentration, and the renaturation was performed at a low temperature of 4°C. The renaturation solution was purified by His-tag protein purification resin (nickel column, Shanghai Lianmai) to obtain the recombinant protein RB1-PDL1 (the gene sequence of the target protein was designed with a histidine tag (HHHHHHHHHH)). After purification, polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify whether the purified target protein was successfully obtained. The results are as follows. Figure 1 As shown, a target protein of about 133.33 kDa was obtained.

[0068] The detailed information of the new recombinant protein RB1-PDL1 is as follows:

[0069]

[0070] The higher-order structure of the recombinant protein RB1-PDL1 prepared in this example was predicted, and the results are shown in Figure 2 The higher-order structure of the novel recombinant protein RB1-PDL1 contains a large number of "α-helical" secondary structures.

[0071] Furthermore, the hydrophilicity and hydrophobicity of the novel recombinant protein RB1-PDL1 prepared in this example were predicted. Figure 3 . Figure 3 The horizontal axis represents the amino acid sequence of the protein; the vertical axis represents hydrophilicity, and the larger the value, the more hydrophobic it is. If it is a negative value, it represents hydrophilicity. The total hydrophilicity value of the new recombinant protein RB1-PDL1 (-0.481) is smaller than the total hydrophilicity value of the natural Retinoblastoma-associated protein (-0.406), indicating that the new recombinant protein with breast cancer cell and lung cancer cell killing function prepared in the present invention is more hydrophilic.

[0072] Example 2

[0073] Advanced structural prediction of the novel recombinant protein RB1-PDL1

[0074] Enter the entire amino acid sequence of the new recombinant protein RB1-PDL1 into the Alphafold3 online prediction website (https: / / alphafoldserver.com / ), submit it, and start prediction.

[0075] Open the prediction result file using Pymol software, such as Figure 2As shown, the obtained advanced structure prediction results are analyzed.

[0076] Example 3

[0077] Analysis of the total hydrophilicity-hydrophobicity value of the novel recombinant protein RB1-PDL1

[0078] Input the entire amino acid sequence of the novel recombinant protein RB1-PDL1 into the online website https: / / web.expasy.org / protscale / , click submit and predict, and the hydrophilicity-hydrophobicity analysis results of different amino acid sites of the novel recombinant protein can be obtained.

[0079] As Figure 3 shown, in the obtained hydrophilicity-hydrophobicity result picture, the horizontal axis represents the amino acid sites, the vertical axis represents the hydrophilicity-hydrophobicity, a positive value on the vertical axis indicates hydrophobicity, and a negative value on the vertical axis indicates hydrophilicity. The larger the value on the vertical axis, the more hydrophobic, and the smaller the value on the vertical axis, the more hydrophilic.

[0080] Example 4

[0081] Cytotoxicity evaluation of the novel recombinant protein RB1-PDL1

[0082] Verify the toxicity of the recombinant protein RB1-PDL1 prepared in Example 1 to normal cells HEK293T, L929 and tumor cells 4T1, A549 by the CCK8 method to evaluate its biosafety and the killing effect on mouse breast cancer cells 4T1 and human non-small cell lung cancer cells A549.

[0083] Method steps:

[0084] (1) In a 96-well plate, inoculate 1×10 4 human embryonic kidney cells HEK293T into DMEM medium, set 5 experimental groups and 1 control group, and set 3 replicates for each group.

[0085] (2) After culturing the cells at 37°C for 24 h, add the novel recombinant protein RB1-PDL1 to the wells of the experimental groups (experimental wells) so that the final concentrations of the novel recombinant protein RB1-PDL1 in the 5 experimental groups are 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL respectively. Do not add the novel recombinant protein RB1-PDL1 to the cells in the wells of the control group (control wells).

[0086] (3) Continue to culture at 37°C for 24 h, and use the CCK8 method to detect the number of live cells. The steps are as follows: Add 10 μL of CCK8 reagent to each well and incubate at 37°C for 3 h. Then use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at 450 nm.

[0087] The results are shown inFigure 4 , Figure 4 When the concentration of the novel recombinant protein RB1-PDL1 reaches 400 μg / mL, the survival rate of HEK293T cells still remains at about 85%, indicating that the novel recombinant protein RB1-PDL1 has no obvious cytotoxicity and has no obvious inhibitory or killing effect on normal cells, and can be further applied to in vitro and in vivo research, diagnosis and treatment.

[0088] The cytotoxicity test procedures for L929 cells, mouse breast cancer cells 4T1, and human non-small cell lung cancer cells A549 are the same as those for HEK293T cells.

[0089] As Figure 4 , the cytotoxicity test results of L929 cells also show that the novel recombinant protein RB1-PDL1 has no obvious toxicity to normal cells L929 and has good biocompatibility.

[0090] The experimental results of mouse breast cancer cells 4T1 and human non-small cell lung cancer cells A549 are respectively as Figure 5 , Figure 6 shown. When the concentration of the novel recombinant protein RB1-PDL1 reaches 200 μg / ml, the survival rates of 4T1 cells and A549 cells are both less than 50%. The results indicate that the novel recombinant protein RB1-PDL1 can significantly kill mouse breast cancer cells 4T1 and human non-small cell lung cancer cells A549 and has excellent anti-breast cancer and non-small cell lung cancer capabilities.

[0091] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the present invention and the claims. All of these are within the protection scope of the present invention.

Claims

1. A novel recombinant protein RB1-PDL1, characterized in that: The novel recombinant protein comprises the entire amino acid sequence of a retinoblastoma-associated protein and the amino acid sequence of PD-L1, and the 42nd leucine, 44th leucine, 45th valine, 47th leucine, 49th phenylalanine, 57th phenylalanine, 60th leucine, and 64th leucine of the retinoblastoma-associated protein, which are all hydrophobic, are mutated into hydrophilic glutamine, glutamine, threonine, glutamine, tyrosine, tyrosine, glutamine, and glutamine, respectively, for a total of 8 hydrophobic amino acids; the 1st phenylalanine, 3rd isoleucine, 5th valine, 9th leucine, 11th valine, and 12th valine of PD-L1, which are all hydrophobic, are mutated into hydrophilic tyrosine, threonine, threonine, glutamine, threonine, and threonine, for a total of 6 hydrophobic amino acids. The amino acid sequence of the novel recombinant protein is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that: Contains the novel recombinant protein RB1-PDL1 as described in claim 1.

3. The recombinant expression vector according to claim 2, characterized in that The type of the vector is a plasmid vector, a phage vector or an animal or plant virus vector.

4. A recombinant bacterium, characterized in that Expressing the novel recombinant protein RB1-PDL1 described in claim 1.

5. The recombinant bacterium according to claim 4, characterized in that The recombinant bacteria are selected from one or more of Escherichia coli, Bacillus subtilis, Pichia pastoris and / or Saccharomyces cerevisiae.

6. Use of the novel recombinant protein RB1-PDL1 according to claim 1 in the preparation of a pharmaceutical preparation for treating breast cancer or lung cancer.

7. The use according to claim 6, characterized in that The pharmaceutical preparation further comprises a pharmaceutically acceptable carrier and / or adjuvant.

8. The use according to claim 6, characterized in that The medicine is for internal use.

9. The use according to claim 8, characterized in that The dosage form of the internal medicine is selected from one of injection, tablet or pill.

10. Use of the novel recombinant protein RB1-PDL1 according to claim 1 in the preparation of an in vitro breast cancer cell or lung cancer cell killing reagent, including experimental biochemical reagents.

Images