A drug design method and the obtained drug and its application
A technology of drugs and biologically active drugs, which is applied in the field of drug design and development and pharmaceutical preparations, and can solve problems such as unsatisfactory effects, congestive heart failure, and liver toxicity
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Embodiment 1
[0147] Embodiment 1: detect the ED-B protein in the blood
[0148] (A) Expression and purification of ED-B protein antibody
[0149]The DNA sequence of the single-chain antibody (scFv) structure of B5 and L19 was synthesized respectively (see patent application number CN201480001324.5, Pini, Viti et al. (1998). J Biol Chem.273:21769-21776 and Borsi, Balza et al . (2002). Int J Cancer. 102:75-85). Add a DNA sequence encoding a signal peptide at the 5' end of the antibody DNA sequence and a DNA sequence encoding an IgG1 Fc tag at the 3' end (Cao, Cao et al. (2009). Appl Biochem Biotechnol.157:562-574) . The N-terminal and C-terminal of the sequence were respectively digested with Nhe I and Not I restriction endonucleases and cloned into pCI-neo vector to form pCI-neo-B5-Fc vector and pCI-neo-L19-Fc vector. The vector construction and expression method of antibody protein can be found in (Borsi, Balza et al. (2002). Int J Cancer. 102:75-85.).
[0150] The vector containing th...
Embodiment 2
[0158] Example 2: Design and expression of biologically inert and biologically active drugs
[0159] 1) Design and expression of biologically inert drug B5-Fc fusion protein (SEQ ID NO: 5)
[0160] The B5 antibody heavy chain (VH) (SEQ ID NO: 1) and light chain (VL) (SEQ ID NO: 2) are connected with a long linker fragment (SEQ ID NO: 7) to form a single-chain antibody B5-ScFv ( SEQ ID NO: 3), the Fc fragment of antibody IgG4 (SEQ ID NO: 4) is fused to the back end, and the hinge region of the Fc fragment is retained, that is, cysteine is retained to form a dimer, and the flexibility of the protein is improved (B5 For antibodies, see patent publication WO2014 / 194784). The carboxy-terminal of the B5 antibody is connected to the amino-terminal hinge region sequence of Fc with three alanine residues. The fusion forms protein B5-Fc (SEQ ID NO: 5).
[0161] 2) Design and expression of bioactive drug B5-IL2 fusion protein (SEQ ID NO: 6)
[0162] The B5 antibody heavy chain (VH)...
Embodiment 3
[0167] Embodiment 3: animal modeling, grouping and administration method
[0168] Solution preparation: in PBS (NaCl 8g / L, KCl 0.2g / L, NaCl 2 HPO 4 1.42g / L,KH 2 PO 4 0.27g / L, pH=7.4) the B5-Fc and B5-IL2 fusion proteins prepared in Example 1 were prepared into 0.4mg / ml solutions respectively.
[0169] Establishment of mouse xenograft tumor model: Healthy Balb / c nu nude mice were raised in SPF level barrier system until 8 weeks old, and mouse teratoma cells (F9) were subcutaneously inoculated on the back of the mice (Shanghai Fuxiang Biotechnology Co., Ltd.) , 2.5×10 per injection point 6 cells. On the 6th day of inoculation, the tumor volume was selected to be about 60mm 3 mice for group experiments. According to 8 groups per group, set up groups A, B, C, D, and E, and administer them through tail vein injection on the 6th day, 7th day, and 8th day after inoculation, wherein:
[0170] Group A was the negative control group, and they were given normal saline twice a d...
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