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Bispecific antibody DNA constructs for intramuscular administration

A technology of antibodies and vectors, applied in the direction of recombinant DNA technology, antibodies, DNA / RNA vaccination, etc., can solve the problems of low transfer efficiency, weeks or months, etc.

Inactive Publication Date: 2005-05-18
伊诺维奥埃斯
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, transfer is very inefficient, and transient expression is generally observed for weeks or months at most

Method used

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  • Bispecific antibody DNA constructs for intramuscular administration
  • Bispecific antibody DNA constructs for intramuscular administration
  • Bispecific antibody DNA constructs for intramuscular administration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Serum expression specific for I-E following electroporation of expression vectors in muscle cells d or IgD a chimeric human / mouse antibody

[0065] This example shows that injection of expression vectors, in this case encoding antibody heavy and light chains, into skeletal muscle results in the production of intact complete antibodies and release of the antibodies into the individual's circulatory system. Intramuscular injection of a mixture of vectors, each encoding either the heavy or light chain of the antibody, or one vector encoding both chains, followed by electroporation at the injection site, followed by an assay designed to assess the full-length Antibody expression. In this regard, three expression vectors were prepared, a heavy chain vector constructed to contain DNA encoding a chimeric heavy chain IgG3 (murine V gene and human C gene), and a light chain vector constructed to contain DNA encoding a chimeric human IgG3 Kappa light chain (mouse V ...

Embodiment 2

[0098] Example 2: Removing heterogeneous sequences can enable long-term expression of antibodies in serum in vivo

[0099] Removal of heterologous sequences from expressed mAbs determines whether reduced immunogenicity increases the amount or duration of recombinant antibody expression in serum. In the first experiment, chimeric IgD a Antibody (in this antibody, V H Derived from Ig(5a)7.2, while C H A heavy chain vector derived from the human γ3 chain) was modified to remove the human γ3 constant region and replace it with the mouse γ2b constant region. The resulting vector pLNOH2γ2bV H T (Lunde et al., supra, 1999) with the variable region of Ig(5a)7.2, and the corresponding human / mouse chimeric light chain vector (pLNO κ V L T; Lunde et al., supra, 1999) co-injected into muscle. The expressed antibody is thus partially chimeric, with a safe mouse heavy chain, and a chimeric human / mouse light chain.

[0100] The serum analysis method is as follows: the NIP 2.6 BSA was...

Embodiment 3

[0105] Example 3: Lysis of cells by antibodies expressed in serum mediated by complement

[0106] The integrity of expressed antibodies is assessed by determining whether serum-expressed antibodies and their antigen binding activate complement. Complement-mediated cell lysis (CML) was performed as described. Michaelsen et al., Scand. J. Immunol. 32, 517-528 (1990); Aase et al., J. Immunol. Methods 136, 185-191. Briefly, by co-cultivating cells with rabbit anti-SRBC NIP-15-Fab' fragments, it was found that 51 CR-labeled sheep red blood cells (SRBC) are sensitive to NIP. According to the present invention, serial dilutions of NIP murine antibodies expressed in serum are added to NIP-sensitive 51 CR SRBC. Human serum was used as a source of complement. The same NIP antibody expressed from recombinant cells as well as purified NIP antibody were used as controls. The cytotoxicity index (CI) was calculated according to the formula: %CI=[(detected cpm-spontaneous cpm) / (maximum ...

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Abstract

Multi-chain proteins can be produced in a subject by intramuscular injection of one or more vectors encoding the protein chains, optionally by applying one or more electrical pulses at the injection site. Preferred multi-chain proteins are immunoglobulins. Methods of generating antibodies have diverse applications, including in vivo expression of multi-chain proteins for the treatment of disease, and can also be used to elicit an immune response against one or more foreign epitopes of the expressed protein.

Description

Background of the invention [0001] The present invention relates to the in vivo expression of multichain proteins from muscle. [0002] Many genetic changes have been identified to produce diseases (such as cancer, muscular dystrophy, and cystic fibrosis), and introduction of functional foreign genes into cells (ie, gene delivery) has emerged as a therapeutic approach. In gene delivery, different approaches have been considered with limited success. See Rosenberg et al., New Eng. J. Med. 323, 570 (1990). [0003] Viral vectors have been widely used for gene delivery due to their relatively high transfection efficiency and the long-term expression capability of vectors due to the integration of vector DNA into the host genome. However, the use of viruses, such as the activation of proto-oncogenes, the reversion of viruses that cannot replicate themselves to wild-type viruses, the immunogenicity of viral proteins, and the auxiliary effects of viral proteins on the immunogenici...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K35/76A61K39/00A61K39/395A61K48/00A61P21/04A61P35/00C07K16/28C07K16/42
CPCA61K39/00A61K2039/505A61K2039/53A61K2039/54C07K16/28C07K16/4283C07K2317/24C07K2317/31A61P21/04A61P35/00A61K48/00A61K39/395C12N15/63
Inventor 比亚尼·博根雅各布·马蒂森托热恩·伊丽莎白·蒂耶里
Owner 伊诺维奥埃斯
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