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Novel selection vectors and methods of selecting eukaryotic host cells

A technology of host cells and vectors, applied in the direction of nucleic acid vectors, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of weak transport affinity and inactivation of folic acid

Active Publication Date: 2016-05-18
NOVARTIS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The RFC selection system has several drawbacks: a) RFC-null recipient cells must be used in which the endogenous RFC locus has been knocked out or inactivated by targeted knockout or loss-of-function mutations
b) RFC has a very weak transport affinity for folate and therefore said oxidized folate cannot be used for selection

Method used

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  • Novel selection vectors and methods of selecting eukaryotic host cells
  • Novel selection vectors and methods of selecting eukaryotic host cells
  • Novel selection vectors and methods of selecting eukaryotic host cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0171] According to another embodiment, the expression cassette comprises at least

[0172] (i) a polynucleotide encoding the polypeptide of interest,

[0173] (ii) includes a 5' splice donor site and a 3' splice acceptor site, and an intron including an in-frame translation stop codon and polyadenylation signal, and

[0174] (iii) a polynucleotide encoding a membrane anchor and / or a membrane anchor signal downstream of said intron.

[0175] This expression cassette design has the effect that at least 2 different mature mRNAs (mRNA-POI) and (mRNA-POI-ANCHOR) are obtained from the expression cassette through transcription and transcript processing. mRNA-POI translation produces the target polypeptide. mRNA-POI-ANCHOR translation produces a fusion polypeptide containing the polypeptide of interest (POI) and the membrane anchor. Thus, the fusion polypeptide is again displayed on the cell surface, and cells displaying high levels of the membrane-anchored fusion polypeptide can ...

Embodiment 1

[0321] Example 1: Single transfection

[0322] For single transfection, wild-type human folate receptor alpha (vector: V-wtFRalpha) or mutant human folate receptor alpha (vector: V-mutFRalpha) was introduced into CHO cells as a selectable marker. This experiment was used to analyze the function of a folate receptor selection system which, in contrast to the DHFR / MTX-system, is not based on a toxic inhibition of cell growth, but on growth inhibition by loss of folic acid in the medium. Folic acid is the oxidized form of vitamin B9. The reduced form of folic acid, tetrahydrofolate (THF), is biologically active. Intracellular folate is reduced to its biologically active form tetrahydrofolate (THF) by dihydrofolate reductase (DHFR) via dihydrofolate (DHF) in a NADPH-dependent reaction. Folate uptake is essential for mammalian cells to maintain cell growth and cell proliferation.

[0323] Only cells that integrate the transfection vector into the genome and express wild-type fol...

Embodiment 2

[0339] Example 2: Determination of antibody production rate

[0340]To analyze the success of the transfection and selection based on the expression of the gene of interest (here the reference antibody), the cells obtained (i.e. selected according to Example 1) were batch cultured in shake flasks for 13 days to determine the cell yield after selection . Cells have greater than 90% viability. On day 13, the antibody concentration of the culture supernatant was determined by protein A affinity chromatography [mg / L]. The results are shown in Table 4.

[0341] Table 4: Antibody concentration (mAb) in batch culture supernatant (mg / L)

[0342]

[0343]

[0344] Antibodies expressed from the introduced expression vectors can be detected in all cell populations. Since small amounts of antibody were also detected in the pool of cells not transfected with DNA, only values ​​greater than 9 mg / l were determined to be significant. The reference population in 125nMMTX (standard D...

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Abstract

The disclosure pertains to an expression vector or a combination of at least two expression vectors comprising: a) a polynucleotide encoding a mutated folate receptor as selectable marker, wherein the mutated folate receptor has a decreased folate binding affinity compared to the wild type folate receptor; b) at least one polynucleotide encoding a polypeptide of interest, wherein when said expression vector or combination of at least two expression vectors is introduced into a host cell, the polypeptide of interest is secreted from said host cell. Also provided are suitable host cells, selection methods and methods for producing polypeptides with high yield.

Description

technical field [0001] The present invention relates to a novel selection system based on the use of mutated folate receptors as selectable markers to select host cells, especially mammalian host cells, expressing a polypeptide of interest. The present invention provides appropriate expression vectors, host cells and methods to select host cells expressing target recombinant polypeptides with high yield. In addition, the present invention relates to methods for efficiently producing recombinant polypeptides with high yields. Background technique [0002] The ability to clone and express products of interest such as recombinant peptides and proteins in large quantities is increasingly important. The ability to purify high levels of protein is important in the fields of human medicine and biotechnology, for example for the production of protein medicines, and in basic research, for example for crystallizing proteins so that their three-dimensional structures can be determined...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C12N15/65C12N15/69C12N15/85
CPCC07K14/705C12N15/69C12N15/85C12N15/65C12P21/02C07K16/00C12N2800/40
Inventor Y·G·阿萨拉夫T·约斯托克H-P·诺夫
Owner NOVARTIS AG
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