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Bispecific recombinant protein and application thereof

A technology for recombinant proteins and proteins, applied in immunoglobulins, peptide/protein components, hybrid immunoglobulins, etc.

Active Publication Date: 2018-11-23
SHANGHAI JMT BIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In addition, in view of the potential safety risks of monovalent or multivalent antibodies or recombinant proteins targeting CD47 (such as anemia, erythrocyte agglutination, CD47-positive non-tumor target cell killing, etc.), at the same time, due to the combination of human SIRPα and human CD47 However, the use of human blood is limited by ethics and genetic resources. Therefore, it is urgent to develop an early in vivo / in vitro immune safety evaluation method for evaluating the efficacy of CD47-targeting antibodies or recombinant proteins. immune safety

Method used

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  • Bispecific recombinant protein and application thereof
  • Bispecific recombinant protein and application thereof
  • Bispecific recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0287] The construction of embodiment 1 expression vector

[0288] According to the designed molecular structure, the amino acid sequences of each component are spliced ​​together, and the optimal DNA coding sequence is designed according to the codon preference of the Chinese hamster (Cricetulus griseus), excluding the need for gene cloning operations. site, then add a cloning site, a Kozak sequence, and a signal peptide coding sequence at the 5' end of the sequence, and add a stop codon and a cloning site at the 3' end of the sequence, such as figure 1 shown.

[0289] Carry out whole gene synthesis, use the 5' end and 3' end cloning sites to clone the whole gene into the expression vector pCHO-TE2 (purchased from Thermo Fisher) between the corresponding cloning sites, and after the sequence verification is correct, you can obtain expression plasmid. The cloning sites used at both the 5' end and the 3' end are EcoRV and PacI sites. figure 2 It is the plasmid map of the ex...

Embodiment 2

[0290] Example 2 Preparation of expression plasmid, cell transfection and expression and purification of target protein

[0291] Preparation of expression plasmids

[0292] Glycerol bacteria containing the expression plasmid (1 mL of Escherichia coli liquid containing the expression plasmid was added with 0.5 mL of 60% sterile glycerol solution and mixed well) was inoculated into the liquid LB medium at a ratio of 1:1000. After cultured on a shaker at 37° C. and 220 rpm for 16 hours, the bacterial cells were collected by centrifugation. Using the endotoxin-free plasmid large-scale extraction kit (DP117, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), the expression plasmid was extracted according to the standard procedure provided in the kit manual.

[0293] Cell transfection, protein expression

[0294] After filtering the obtained expression plasmid with a 0.22 μm filter membrane, absorb 3 mg of the plasmid (wherein, the product is a typical antibody mo...

Embodiment 3

[0306] Example 3 Detection of target affinity and target competition binding activity

[0307] 1. CD47, CD20, EGFR, Her2 target affinity detection method

[0308] Recombinant protein Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1 m -Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1 m -D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1 m -Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1 m - The binding affinities of D2-Fc2 and Obi-Fc1-D1-D2-D3-Fc2 to targets CD47 and CD20 are determined by ELISA and / or flow cytometry. The following method takes Ofa-Fc1-D1-Fc2 as an example, which is suitable for the detection of recombinant proteins whose left arm is CD20 target.

[0309] Recombinant protein Anti-Her2(T)-Fc1-D1-Fc2, Anti-Her2(T)-Fc1-D1 m -Fc2, Anti-Her2(T)-Fc1-D1-D2-Fc2, Anti-Her2(T)-Fc1-D1 m -D2-Fc2, Anti-Her2(T)-Fc1-D1-D2-D3-Fc2, Anti-Her2(P)-Fc1-D1-Fc2, Anti-Her2(P)-Fc1-D1 m -Fc2, Anti-Her2(P)-Fc1-D1-D2-Fc2, Anti-Her2(P)-Fc1-D1 m - The binding affinities of D2-Fc2 and Anti-Her2(P)-Fc1-D1-D2-D3-Fc2 to targets ...

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Abstract

The invention discloses bispecific recombinant protein. The bispecific recombinant protein comprises a high-affinity tumor-targeted arm and a low-affinity fusion protein for blocking interaction of CD47 and SIRPalpha, wherein an antibody corresponding to the high-affinity tumor-targeted arm is not combined with the CD47, and the bonding affinity to target antigens on tumor cells is at least 6 times of the bonding affinity of the monomeric fusion protein and dimer corresponding to the low-affinity fusion protein for blocking the interaction of the CD47 and the SIRPalpha to the CD47 on the tumorcells; the low-affinity fusion protein for blocking mutual action of the CD47 and SIRPalpha contains SIRPalpha extracellular truncation. The invention also discloses a nucleic-acid molecule for encoding the recombinant protein and application of the recombinant protein and the nucleic-acid molecule in preparation of medicines for treating tumors. The bispecific recombinant protein disclosed by the invention has the beneficial effects that the bonding abundance of tumor targeting saturation of the recombinant protein with the function of adjusting macrophage is obviously improved by the bispecific recombinant protein, the side effect of the non-tumor targeting is reduced and the application value is large clinically.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a recombinant protein and its application. Background technique [0002] With the deepening of research in the field of tumor treatment, the development and application of molecularly targeted therapeutic drugs for tumors has received more and more attention. Based on the advantages of strong targeting, few side effects, and significant curative effect, antibody drugs have quickly become hot drugs in the field of tumor targeted therapy. At present, dozens of tumor-targeting antibody drugs have been approved for clinical use, and have achieved remarkable curative effects. Such antibody drugs, such as MabThera for CD20 target molecules (Rituximab, the first monoclonal antibody approved in the United States for the treatment of cancer, originally for the treatment of non-Hodgkin's lymphoma, produced by Roche), (ibritumomab tiuxetan, which was originally approved by the US ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00
CPCC07K16/30C07K2319/30C07K16/2887C07K14/70596C07K2319/33C07K2317/24C07K2317/92A61K39/395A61P35/00C07K16/46C07K19/00C12N5/10G01N33/53C07K2317/31C07K2317/76C07K2319/70C07K2319/74C07K16/2896C07K16/2803C07K2317/55C07K2317/732A61K2039/505A01K67/0278A01K2207/15A01K2227/105A01K2267/0331A61K38/00C07K14/435C07K16/2827C07K16/2863C12N15/85
Inventor 宋利平崔笑添王健吴海祥贾佳娜范艺张甘良李涛徐红佘伊莎龙凯
Owner SHANGHAI JMT BIO INC
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