Antibody Fusion Protein and Preparation Method and Use Thereof

Abstract

The present invention provides an antibody fusion protein, comprising an antitumor antigen-specific antibody or a Fab fragment thereof, a single domain antibody or single chain antibody, and further comprising a human NKG2D ligand or ligand fragment. The human NKG2D ligand or ligand fragment and the antitumor antigen-specific antibody or Fab fragment thereof and the single domain antibody or single chain antibody are mutually connected by a linker peptide.

Claims

1. An antibody fusion protein, characterized in that, comprising an antitumor antigen-specific antibody or a Fab fragment thereof, a single domain antibody or single chain antibody, and further comprising a human NKG2D ligand or ligand fragment; Said human NKG2D ligand or ligand fragment and the antitumor antigen-specific antibody or Fab fragment thereof and the single domain antibody or single chain antibody are mutually connected by a linker peptide.

2. The antibody fusion protein according to claim 1, characterized in that, the amino acid sequence of said human NKG2D ligand are shown in from SEQ ID NO: 1 to SEQ ID NO: 6.

3. The antibody fusion protein according to claim 1, characterized in that, the coding gene sequence of said human NKG2D ligand are shown in from SEQ ID NO: 7 to SEQ ID NO: 12.

4. The antibody fusion protein according to claim 1, characterized in that, said antitumor antigen-specific antibody is a specific antibody targeting human tumor.

5. A recombinant vector, a recombinant cell, a recombinant bacterium, or an expression cassette, which contains the coding gene of any of the antibody fusion protein according to claim 1.

6. A preparation method of any of the antibody fusion protein according to claim 1, characterized in that, comprising the following steps: (a) Insert the heavy chain coding gene, NKG2D ligand coding gene and linker peptide coding gene of said antibody fusion protein into pBluescript II SK (+) vector to construct the pBS-SK-H plasmid; (b) Insert the light chain coding gene of said antibody fusion protein into pBluescript II SK (+) vector to construct the pBS-SK-L plasmid; (c) Cleave the pBS-SK-H plasmid by enzyme to obtain vector fragment, which contains the heavy chain coding gene, NKG2D ligand coding gene and linker peptide coding gene of said antibody fusion protein; (d) Cleave the pBS-SK-L plasmid by enzyme to obtain vector fragments, which contains the light chain coding gene of said antibody fusion protein; (e) Insert the vector fragments obtained in the step (c) and the step (d) into expression vector to obtain the recombinant expression vector; (f) Transform the recombinant expression vector obtained in the step (e) into recipient cell to express said antibody fusion protein.

7. The preparation method according to claim 6, characterized in that, the expression vector in the step (e) is pcDNA3.0-FLAG.

8. The preparation method according to claim 6, characterized in that, the recipient cell in the step (f) is 293T cell.

9. A use of any of the fusion protein antibody according to claim 1 in preparation of anti-tumor drugs.

10. A drug containing any of the fusion protein antibody according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows the structure diagram of antibody fusion protein in the embodiment of the invention.

[0033] FIG. 2 shows the structure diagram of pBluescript II SK (+) vector used in the embodiment of the invention.

[0034] FIG. 3 shows the structure diagram of heavy chain and light chain of antibody fusion protein in the embodiment of the invention.

[0035] FIG. 4 shows the structure diagram of expression vector pcDNA3.1-FLAG used in the embodiment of the invention.

[0036] FIG. 5 shows the identification figure of the successfully constructing pBS-SK-L of light chain in the embodiment of the invention.

[0037] FIG. 6 shows the identification figure of the successfully constructing pBS-SK-Linker of heavy chain in the embodiment of the invention.

[0038] FIG. 7 shows the identification figure of the successfully constructing pBS-SK-VH-CH1-linker of heavy chain in the embodiment of the invention.

[0039] FIG. 8 shows the identification figure of the successfully constructing pBS-SK-H of heavy chain in the embodiment of the invention (Take MICA as an example).

[0040] FIG. 9 shows the identification figure of the successfully constructing pcDNA3.1-FLAG-L of light chain in the embodiment of the invention.

[0041] FIG. 10 shows the identification figure of the successfully constructing pcDNA3.1-FLAG-H of heavy chain in the embodiment of the invention (Take MICA as an example).

[0042] FIG. 11 shows the expression identification figure of the pcDNA3.1-FLAG-L protein of light chain in the embodiment of the invention.

[0043] FIG. 12 shows the expression identification figure of the pcDNA3.1-FLAG-H protein of heavy chain in the embodiment of the invention (Take MICA as an example).

[0044] FIG. 13 shows the expression identification figure of EGFR on the surface of Hep3B cell strains in the embodiment of the invention.

[0045] FIG. 14 shows the expression identification figure of MICA on the surface of Hep3B cell strains in the embodiment of the invention.

[0046] FIG. 15 shows the killing efficiency of NK cells on tumor cell Hep3B when antibody fusion protein exists (Take anti EGFR antibody-MICA fusion protein as an example).

DETAILED DESCRIPTION

[0047] In order to describe the present invention in more detail, the following examples and figures are provided in order to better understand the solution of the present invention and its advantages of various aspects.

[0048] The experiment methods used in the following examples are all conventional methods unless special version.

[0049] The materials and reagents used in the following examples are all commercial available unless special version.

Example 1, Construction of pBS-SK-L Plasmid

[0050] Nucleotide sequence KpnI-EcoRV-VL-XhoI-HINDIII-CL-PacI-SpeI (nucleotide sequence is shown as SEQ ID NO:19) is synthesized and 747 bp in full length, in which KpnI cleavage site, EcoRV cleavage site, variable region sequence VL of antibody light chain (amino acid sequence is shown as SEQ ID NO:15), XhoI cleavage site, HindIII cleavage site, constant region CL of antibody light chain (amino acid sequence is shown as SEQ ID NO:16), Pad cleavage site and SpeI cleavage site are included.

[0051] Inserting the above nucleotide sequence which has been cleaved by KpnI and SpeI into the vector pBluescript II SK (+) which has been cleaved by the same enzyme, pBS-SK-L is constructed and then verified by PCR (FIG. 5).

Example 2, Construction of pBS-SK-H Plasmid

[0052] a. The nucleotide sequence fragment of linker peptide is inserted into the cleavage site of the vector pBluescript II SK (+) (purchased from Stratagen Co.) between HindIII and KpnI (nucleotide sequence of linker is shown as SEQ ID NO: 17), the vector pBS-SK-Linker is constructed and then verified by PCR (FIG. 6).

[0053] b. Nucleotide sequence KpnI-AvrII-VH-BglI-CH1-XhoI (nucleotide sequence is shown as SEQ ID NO: 18) is synthesized and 745 bp in full length, in which KpnI cleavage site, AvrII cleavage site, variable region sequence VH of antibody heavy chain (amino acid sequence is shown as SEQ ID NO: 13), BglI cleavage site, constant region CH1 of antibody heavy chain (amino acid sequence is shown as SEQ ID NO: 14) and XhoI cleavage site are included. Inserting the above nucleotide sequence which has been cleaved by KpnI and XhoI into the vector pBS-SK-Linker which has been cleaved by the same enzyme, pBS-SK-VH-CH1-linker is constructed and then verified by PCR (FIG. 7).

[0054] c. Six of the ligand coding gene sequence of NKG2D (the six ligand coding gene sequence of NKG2D are shown as from SEQ ID NO:7 to SEQ ID NO:12) are respectively cloned into the vector BS-SK-VH-CH1-linker, between two cleavage sites of XbaI and SacII, six different kinds of pBS-SK-H are constructed and then verified by PCR (FIG. 8).

Example 3, Construction of the Expression Vector of Antibody Fusion Protein

[0055] a. KpnI-EcoRV-VL-XhoI-HINDIII-CL-PacI-SpeI (nucleotide sequence is shown as SEQ ID NO:19), which is obtained from pBS-SK-L plasmid in Example 1, is cloned into the vector pcDNA3.1-FLAG (purchased from Thermofisher Co.) by PCR cloning method. Wherein, the cleavage sites are KpnI and NotI, the length of the sequence is 747 bp. Light chain expression plasmid pcDNA3.1-FLAG-L is constructed and then verified by PCR (FIG. 9).

[0056] b. Six different kinds of VH-CH1-linker-NKG2DL, which are obtained from the six pBS-SK-H plasmids in Example 2, are respectively cloned into the vector pcDNA3.1-FLAG (purchased from Thermofisher Co.) by PCR cloning method. Wherein, the cleavage sites are NheI and NotI. Heavy chain expression plasmid pcDNA3.1-FLAG-H are constructed and then verified by PCR (FIG. 10).

Example 4, Antibody Fusion Protein Expressing in 293T Cell

[0057] a. Light chain expression plasmid pcDNA3.1-FLAG-L is transfected into 293T cell by lipofectamine 2000 (purchased from US ATCC cell bank). The total cellular protein is collected after transfecting for 24 hours, and the expression of the target protein is detected by immunoblotting method (FIG. 11);

[0058] b. Heavy chain expression plasmid pcDNA3.1-FLAG-H is transfected into 293T cell by lipofectamine 2000 (purchased from US ATCC cell bank). The total cellular protein is collected after transfecting for 24 hours, and the expression of the target protein is detected by immunoblotting method (FIG. 12);

Example 5, Activity Detection of Antibody Fusion Protein

[0059] In vitro identification of activity of fusion protein:

[0060] a. Antibody fusion protein obtained from Example 4 can target the tumor cell strains with high-expression EGFR (epidermal growth factor receptor), so the expression of EGFR (epidermal growth factor receptor) on the surface of Hep3B strains (human hepatocellular carcinoma cell, purchased from US ATCC cell bank) is detected by FACS (flow cytometry). The result has shown that the expression rate of EGFR on the surface of human hepatocellular carcinoma cell Hep3B is up to 98.7% (FIG. 13).

[0061] b. Antibody fusion protein obtained from Example 4 can express the ligand of NKG2D, and thus increase the ability of NK cells in recognizing and killing tumor cells, so the expression of MICA (the corresponding coding gene sequence is SEQ ID NO:7) on the surface of Hep3B strains is detected by FACS (flow cytometry). The result has shown that the expression rate of MICA on the surface of human hepatocellular carcinoma cell Hep3B is only 8.28% (FIG. 14).

[0062] c. Co-culture the NK-92 cells (human NK cell lines, purchased from US ATCC cell bank) and the Hep3B tumor cells in porous cell culture plate, and set control group and antibody fusion protein group (each group is repeated three times; in each hole, the cell number of target cell Hep3B is 1*10.sup.5 and the cell number of effector cell NK-92 is 5*10.sup.5). The target cell Hep3B is labeled with TFL-4 and the final concentration is 5 M, then the cells solution is counted and divided into groups after incubating at 37 C. for 20 min and washing with PBS for 3 times. The control group is prepared by adding effector cell (NK-92 cell), and the antibody fusion protein group is prepared by adding both the effector cell and antibody fusion protein obtained from Example 4 (the adding amount of antibody fusion protein is 12 ng per well), then the mixed solution is incubating at 37 C. for 2 h. After washing the above solution with Washing buffer for 2 times and resuspending the cells with 100 l buffer, 5 uL annexin FITC is used for staining at room temperature, then 200 l PBS is added 10 min later, and the positive rate of Annexin V is measured by flow cytometry. Annexin V is a reagent for the detection of apoptosis. Phosphatidyl serine is just distributed in internal area of cell membrane lipid bilayer in normal cells, while the phosphatidyl serine (PS) can move from the inside to the outside in the early stage of apoptosis. As a phospholipids-binding protein, Annexin V has high affinity with phosphatidyl serine, and it can combine with the membrane of early apoptotic cells by phosphatidyl serine exposed outside the cell. So, Annexin V can be seen as a sensitive index for detecting early stage apoptosis. The killing efficiency of NK-92 cells on tumor cells can be indicated by calculating the positive rate of Annexin V. As is shown in the figure, the killing rate of the antibody fusion protein group is almost double that of the control group (FIG. 15).

[0063] Finally, it should be stated that it is clear that the above examples are merely used for clearly illustrating the present invention, rather than a limitation of the implementation. For the skilled in the arts, different forms of changes can be made on the basis of the above explanation. There is no need or no way to give exhaustive implements. The obvious changes based on the above are still in the scope of the invention.