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Artificial protein containing antigen-binding region of antibody and being fused with physiologically active peptide

A technology of physiological activity and binding activity, applied in the direction of fusion polypeptide, hybrid peptide, immunoglobulin, etc., can solve problems such as difficulties and loss of structural stability

Pending Publication Date: 2021-02-02
MIRABIOLOGICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, even if it is intended to randomize the amino acids of these loop regions to impart new physiological activities, since it usually causes a large loss in structural stability, it is more difficult to create a protein than the aforementioned Fcab and taFv.

Method used

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  • Artificial protein containing antigen-binding region of antibody and being fused with physiologically active peptide
  • Artificial protein containing antigen-binding region of antibody and being fused with physiologically active peptide
  • Artificial protein containing antigen-binding region of antibody and being fused with physiologically active peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0259] 1. Preparation of Peptide Fusion Proteins

[0260] The DNA encoding the Fab region in the H chain of the human IgG1 monoclonal antibody against neuropilin 1 (N1 antibody, clone name YW64.3) and the DNA encoding the Hisx6 tag were incorporated into the expression vector pcDNA3.1 (Thermo Fisher Scientific company) (antibody H chain Fab region expression vector).

[0261] The amino acid sequence (SEQ ID NO: 2) of the N1 antibody H chain Fab region produced by this vector is shown in Figure 48 . It should be noted that the amino acid sequence (SEQ ID NO: 1) of the N1 antibody H chain is as Figure 47 show.

[0262] The DNA encoding the full-length region of the Lκ chain of the N1 antibody was incorporated into the expression vector pcDNA3.1 (antibody Lκ chain expression vector). but assembled in an unlabeled way. The amino acid sequence (SEQ ID NO: 3) of the N1 antibody Lκ chain produced by this vector is shown in Figure 49 .

[0263] As the physiologically activ...

Embodiment 2

[0298] 1. Preparation of Peptide Fusion Proteins

[0299] The peptide-fused IgG protein comprising the peptide-fused antibody H chain or the peptide-fused antibody L chain was obtained by combining an antibody H chain-peptide fusion vector with an unfused antibody Lκ chain expression vector, Or antibody L chain-peptide fusion vector and unfused antibody H chain IgG region expression vector co-expressed and secreted into its culture supernatant.

[0300] 40 µL of Protein A Sepharose (manufactured by GE Healthcare) was added to 0.25 mL of the recovered culture supernatant, and vortexed for 1 hour. Precipitate the Sepharose by centrifugation, remove the supernatant, wash the Sepharose twice with 1 mL of Tris-buffered saline (TBS, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5), add 12.5 μL of TBS and 12.5 μL of SDS Add sample buffer for elution, and then heat at 95°C for 2 minutes to obtain a sample. 5 μL of the eluted sample was subjected to electrophoresis under reducing conditions an...

Embodiment 3

[0306] As the physiologically active peptide, aMD4 having the activity of binding to human Met receptor (Met) was used instead of mP6-9 having the activity of binding to human plexin B1, and the same procedure as in Example 1 was carried out.

[0307] aMD4 is a cyclic peptide having the amino acid sequence (SEQ ID NO: 34) of D-Tyr Arg Gln Phe Asn Arg Arg Thr His Glu Val Trp Asn LeuAsp Cys, and the chloroacetylated D-Tyr and Cys form a cyclic structure. The amino acid sequence (SEQ ID NO: 35) of aMD4 inserted into each site of the Fab region is Tyr Arg Gln Phe Asn Arg Arg ThrHis Glu Val Trp Asn Leu Asp.

[0308] It should be noted that, if Figure 24 and Figure 28 As shown in , Gly was used as a linker at the N-terminus and C-terminus of the inserted amino acid sequence (SEQ ID NO: 36-51).

[0309] The results are shown in Figure 25 ~ Figure 27 and Figure 29 ~ Figure 32 .

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Abstract

Provided is an artificial protein that is present in a folded part of the secondary structure in a structural region having the antigen-binding activity of a protein having at least an antigen-bindingregion of an antibody and that is fused with a physiologically active peptide at a site exposed on the protein surface.

Description

technical field [0001] The present invention relates to an artificial protein comprising an antigen-binding region of an antibody, a physiologically active peptide fused thereto, and the like. Background technique [0002] The function of an antibody is essentially to specifically bind to a single antigen molecule as a target molecule. Antibodies that recognize two or more different antigen molecules (multispecific antibodies: MsAbs) are known as modified antibodies obtained by protein engineering methods. [0003] Since one antibody binds to two or more target molecules at the same time, MsAb can be used as a drug that bridges different types of cells such as cancer cells and lymphocytes to exert an anticancer effect. In addition, by conferring a second binding specificity to an antibody having the first binding specificity inherent in the antibody, MsAbs can be accumulated in desired tissues and cells. Therefore, MsAbs are able to control diseases through a more complica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K16/00C07K19/00C12P21/00C12N15/62
CPCC07K2317/55C07K2318/10C07K16/2863C07K2317/31C07K2319/00C07K16/2809C07K16/283C07K7/08C07K7/64C07K2319/30
Inventor 高木淳一平井秀宪
Owner MIRABIOLOGICS INC
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