Structure for enhancing antibody drug stability

Abstract

The invention belongs to the field of a gene engineering drug. In the invention, an amino terminal and a carboxyl terminal of a recombinant protein are respectively connected to a heavy chain variable zone and a light chain variable zone of the antibody through a peptide bond, and a structure of antibody variable zone-recombination protein-antibody variable zone is formed, the recombination protein is connected with the antibody by two peptide bonds; thus the stability and targeting ability of the recombination protein are improved.

Description

technical field

[0001] The invention belongs to the field of genetic engineering. Using DNA recombination technology to connect the light chain and heavy chain of the antibody into a recombinant protein to form a new antibody fusion protein, which can be used as a drug. Background technique

[0002] Antibodies have good specificity and targeting, and the fusion or coupling of antibodies and drugs can make the drugs have corresponding specificity and targeting, so that the drugs can be enriched in the target tissue at a higher concentration. The antibody-recombinant protein drug fusion protein composed of antibody and recombinant protein drug molecule is one of the directions of drug design and development, but the structure of this type of drug is characterized by the fusion of recombinant protein drug upstream or downstream of the single-chain antibody (Muller (2014). BioDrugs. 28: 123-131.), the remarkable feature of the antibody drug structure is that there is only one p...

Examples

Embodiment 1

[0020] Example 1 Structural Design of Antibody Fusion Protein

[0021] The antibody fusion protein structure of the present invention is shown in figure 1 . VH and VL are the heavy chain variable region and light chain variable region of the antibody, respectively, and FP represents the recombinant protein. The three parts are connected by two peptide chains. Antibody fusion protein can be expressed by host cells through DNA recombinant technology.

Embodiment 2

[0022] Example 2 Antibody fusion protein drug

[0023] Fusion of the B5 antibody to human interleukin 2 (hIL2) to form the B5H-hIL2-B5L protein. The B5H-hIL2-B5L structure is that the heavy chain variable region of the protein B5 antibody is in the front, interleukin 2 is in the middle, and the light chain variable region of the B5 antibody is behind, and the three domains are connected by glycine-rich peptide chains . The amino acid sequence is shown in SEQ ID No.1.

[0024] The B5H-hIL2-B5L protein was obtained by secreting and expressing the protein from CHO (Chinese hamster ovary cells) cells and purifying it by cationic chromatography.

[0025] The purified B5H-hIL2-B5L protein was analyzed by SDS-PAGE denaturing electrophoresis and non-denaturing electrophoresis, and the molecular weight of the electrophoresis bands under both conditions was about 50kD. It suggested that B5H-hIL2-B5L was a monomer.

Embodiment 3

[0026] Example 3 Analysis of biological activity of antibody fusion protein

[0027] (1) IL2 biological activity of B5H-hIL2-B5L

[0028] B5H-IL2-B5L was added at 100 ng / ml to RPM1640 medium containing 10% fetal bovine serum to cultivate CTLL2 cells (mouse cytotoxic T lymphocytes), which can maintain the normal growth and continuous passage of CTLL2 cells. The hIL2 fused in the B5H-hIL2-B5L fusion protein has the biological activity of recombinant human IL2.

[0029] (2) Stability test of B5H-hIL2-B5L in plasma

technical field

[0001] The invention belongs to the field of genetic engineering. Using DNA recombination technology to connect the light chain and heavy chain of the antibody into a recombinant protein to form a new antibody fusion protein, which can be used as a drug. Background technique

[0002] Antibodies have good specificity and targeting, and the fusion or coupling of antibodies and drugs can make the drugs have corresponding specificity and targeting, so that the drugs can be enriched in the target tissue at a higher concentration. The antibody-recombinant protein drug fusion protein composed of antibody and recombinant protein drug molecule is one of the directions of drug design and development, but the structure of this type of drug is characterized by the fusion of recombinant protein drug upstream or downstream of the single-chain antibody (Muller (2014). BioDrugs. 28: 123-131.), the remarkable feature of the antibody drug structure is that there is only one p...

Examples

Embodiment 1

[0020] Example 1 Structural Design of Antibody Fusion Protein

[0021] The antibody fusion protein structure of the present invention is shown in figure 1 . VH and VL are the heavy chain variable region and light chain variable region of the antibody, respectively, and FP represents the recombinant protein. The three parts are connected by two peptide chains. Antibody fusion protein can be expressed by host cells through DNA recombinant technology.

Embodiment 2

[0022] Example 2 Antibody fusion protein drug

[0023] Fusion of the B5 antibody to human interleukin 2 (hIL2) to form the B5H-hIL2-B5L protein. The B5H-hIL2-B5L structure is that the heavy chain variable region of the protein B5 antibody is in the front, interleukin 2 is in the middle, and the light chain variable region of the B5 antibody is behind, and the three domains are connected by glycine-rich peptide chains . The amino acid sequence is shown in SEQ ID No.1.

[0024] The B5H-hIL2-B5L protein was obtained by secreting and expressing the protein from CHO (Chinese hamster ovary cells) cells and purifying it by cationic chromatography.

[0025] The purified B5H-hIL2-B5L protein was analyzed by SDS-PAGE denaturing electrophoresis and non-denaturing electrophoresis, and the molecular weight of the electrophoresis bands under both conditions was about 50kD. It suggested that B5H-hIL2-B5L was a monomer.

Embodiment 3

[0026] Example 3 Analysis of biological activity of antibody fusion protein

[0027] (1) IL2 biological activity of B5H-hIL2-B5L

[0028] B5H-IL2-B5L was added at 100 ng / ml to RPM1640 medium containing 10% fetal bovine serum to cultivate CTLL2 cells (mouse cytotoxic T lymphocytes), which can maintain the normal growth and continuous passage of CTLL2 cells. The hIL2 fused in the B5H-hIL2-B5L fusion protein has the biological activity of recombinant human IL2.

[0029] (2) Stability test of B5H-hIL2-B5L in plasma

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