Recombinant antibody vector

A technology of recombinant antibodies and vectors, applied in the fields of biochemical equipment and methods, applications, and botanical equipment and methods, which can solve problems such as limited flexibility

Inactive Publication Date: 2012-09-19
ACYTE BIOTEC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing antibody reformatting vectors exhibit limited flexibility, and codons formed by restriction endonuclease recognition sequences often result in the addition of several "foreign" amino acids to the original sequence (Coloma et al., 1992; Persic et al. , 1997; Jostock et al., 2004)

Method used

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  • Recombinant antibody vector
  • Recombinant antibody vector
  • Recombinant antibody vector

Examples

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Embodiment 1

[0093] Design and Construction of Recombinant Antibody Vectors

[0094] Ig variable regions were identified to include glutamic acid (E) residues at or near the N-terminus and leucine (L) at or near the C-terminus. This feature appears to occur in about 10% of variable regions. By removing the spacer, a vector can be constructed that encodes the amino acid EL with the nucleotide sequence GAG ​​CTC, thereby forming a SacI site. Insertion of the variable region nucleotide sequence at the SacI site results in a "split" of the EL sequence such that the E residue is N-terminal to the variable region and L is C-terminal to the variable region. This avoids the addition of foreign amino acids, E and L residues are usually found at or near the N- and C-termini of variable regions. A schematic description of the "mAbXpress" recombinant antibody vector is in figure 1 available in .

[0095] method:

[0096] A single restriction site was included after the signal peptide to allow ins...

Embodiment 2

[0199] Production of anti-CD83 recombinant antibody

[0200] 1. Methods and materials:

[0201] 1.1 Design of expression vector.

[0202] mAbXpress vectors were assembled as described in Example 1 using publicly available human constant region heavy chain (IgG1 and IgG4 subtypes) and light chain (κ) sequences. The required DNA was synthesized and codon optimized for mammalian expression using Geneart AG (Germany). These cassettes are then placed into mammalian expression vectors containing sequences for expression, selection and amplification in mammalian cells ( figure 1 ). A single SacI site is included in the expression vector to facilitate linearization of variable regions and In Fusion TM Clone (see Section 3 for details).

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Abstract

A recombinant antibody vector for producing a single chain recombinant antibody comprises: (a) a contiguous nucleotide sequence: (i) that comprises a restriction endonuclease site that encodes an amino acid sequence of an immunoglobulin variable region; and (ii) that encodes an immunoglobulin constant region amino acid sequence in the same reading frame as (i), wherein another nucleotide sequence encoding (iii) an immunoglobulin variable region amino acid sequence, is insertable into the restriction endonuclease site in the same reading frame as (ii); and (b) one or more regulatory nucleotide sequences operably linked or connected to said nucleotide sequence, wherein the amino acid sequence in (i) comprises amino acids conserved in different immunoglobulin variable regions. The restriction endonuclease site may be a SacI site which encodes the conserved amino acids glutamate and leucine. In frame insertion of the nucleotide sequence of (iii) is facilitated by ligase independent cloning.

Description

technical field [0001] The present invention relates to nucleic acid vectors for producing recombinant antibodies, especially single-chain recombinant antibodies. Background technique [0002] Over the past 10-15 years, there has been tremendous interest in the use of recombinant monoclonal antibodies (mAbs) as therapeutic agents. In 2007, mAb sales in the US alone exceeded $14 billion, with a 22% annual growth rate (Aggarwal, 2008). With the number of approved mAbs approaching 30 and hundreds of new candidates in the pipeline, this trend shows no signs of slowing down. Most therapeutic recombinant mAbs are members of the IgG family, and due to their large size and complex glycosylation patterns, these molecules are currently produced in mammalian cells, the vast majority of which utilize Chinese hamster ovary (CHO) cells as production host (Wurm, 2004). [0003] The path from discovery to the clinic for therapeutic recombinant mAbs can be a long and tedious process, ofte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13
CPCC07K16/2803C07K2317/622C07K2317/21C07K2317/14C07K2317/732C07K16/00
Inventor T·P·芒罗M·L·琼斯M·G·斯梅德
Owner ACYTE BIOTEC
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