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Method for manufacturing recombinant polyclonal proteins

A protein and recombinant enzyme technology, applied in peptide/protein components, animal/human proteins, immunoglobulins, etc., can solve the problems of lack of original diversity, affecting long-term production of antibodies, mitotic instability of immunoglobulin loci, etc. The effect of avoiding ethical and clinical dilemmas

Inactive Publication Date: 2006-01-18
SYMPHOGEN AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, mitotic instability of the introduced immunoglobulin loci may affect long-term antibody production
Third, it is also technically challenging to delete an animal's own immunoglobulin loci so that, for example, the animal's antibody production does not exceed that of a human antibody
There is also nothing about how to maintain a specific V in the library H -V L Hints of raw diversity produced by combinations

Method used

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  • Method for manufacturing recombinant polyclonal proteins
  • Method for manufacturing recombinant polyclonal proteins
  • Method for manufacturing recombinant polyclonal proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0214] Site-specific integration versus random integration

[0215] For the following transfection experiments, CHO-Flp-In cells (Invitrogen, Carlsbad, CA) were used. The efficiency of the system was tested using human secreted alkaline phosphatase (SEAP) as a reporter gene. Prepare two plasmid constructs:

[0216] 1. Insert SEAP into pcDNA3.1 hygro+ (Invitrogen, Carlsbad, CA) (for random integration)

[0217] 2. Insertion of SEAP into pcDNA5 / FRT (Invitrogen, Carlsbad, CA) (for site-specific integration)

[0218] The two plasmid constructs were very similar in terms of regulatory elements (ie promoter, polyadenylation, etc.), which allowed the comparison of random versus site-specific integration using the plasmids.

[0219] CHO Flp-In cells were transfected with plasmid construct 1 alone or with plasmid construct 2 in combination with the recombinase-encoding plasmid pOG44 as described by Invitrogen. Transfectants were selected using hygromycin and the production of SEAP ...

Embodiment 2

[0222] Design and preparation of expression vectors for site-specific integration in host cells

[0223] Expression vectors suitable for site-specific integration into hotspot chromosomal regions of host cells can be assembled from the following DNA elements:

[0224] a) the FRT recombination site linked to the hygromycin resistance gene,

[0225] b) pUC replication initiation site,

[0226] c) Ampicillin resistance gene (bla),

[0227] d) the bla promoter allowing expression of the ampicillin (bla) resistance gene,

[0228] e) Genes encoding proteins of interest (GOIs),

[0229] f) a promoter allowing expression of said GOI, and

[0230] g) Optionally, additional transcriptional or translational regulatory elements, such as enhancers or UCOE's, to increase expression at the integration site or IRES.

[0231] In order to better understand the construction of the expression vector, a more detailed description of each element is given:

[0232] a) The FRT recombination sit...

Embodiment 3

[0248] Estimation of polyclonality preservation in developed production systems

[0249] In order to be able to assess the stability and reproducibility of the production system, cell lines expressing polyclonal compositions of different antibodies in known combinations were prepared. The polyclonal antibody composition is called a minisix composition. The nucleic acid sequence library encoding the mini six composition is called mini six library.

[0250] (a) Cloning origin

[0251] The following sequences encoding Fab fragments (genes of interest) reactive with antigens 1-6 were used in this example:

[0252] 1. Ovalbumin (OVA). The Fab coding fragment is selected from a mouse anti-OVA phage display library.

[0253] 2. Alkaline phosphatase (AP). The Fab coding fragment is selected from a mouse anti-AP phage display library.

[0254] 3. beta 2 - Microglobulin (β 2 m). The Fab coding fragment is cloned from hybridoma BBM.1 (gifted by Dr.L.Ф.Pedersen, Denmark), which t...

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Abstract

The invention relates to a method for manufacturing a recombinant polyclonal protein composition, in particular a recombinant polyclonal antibody composition. The method comprises obtaining a collection of cells transfected with a library of variant nucleic acid sequences, wherein each cell in the collection is transfected with and capable of expressing one member of the library, which encodes a distinct member of a polyclonal protein that binds a particular antigen and which is located at the same single site in the genome of individual cells in said collection, wherein said nucleic acid sequence is not naturally associated with said cell in the collection. The cells are cultured under suitable conditions for expression of the polyclonal protein, which is obtained from the cells or culture supernatant. The nucleic acid sequence id introduced into the cells by transfection with a library of vectors for site-specific integration. The present method is suitable for manufacturing recombinant polyclonal antibodies, thereby making available a superior replacement of plasma-derived therapeutic immunoglobulin products.

Description

field of invention [0001] The present invention forms the basis of a technological platform for the production of recombinant polyclonal proteins that can be used as novel therapeutic agents for the treatment, amelioration or prevention of various infections, inflammatory diseases, transplant rejection, cancer and allergic reactions , said polyclonal proteins are eg proteins derived from the immunoglobulin superfamily, eg soluble and membrane bound forms of B or T cell receptors. Background of the invention [0002] Some infectious diseases and cancers lack effective treatments. Monoclonal antibodies are often unsuccessful against these targets, in part because of the variability of the complex targets and adaptive mutations in the target proteins that lead to immune escape from recognition by the monoclonal antibodies. Polyclonal antibodies on the other hand can target most dynamic targets, such as viruses or cancer cells. Furthermore, polyclonal antibodies have the great...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N5/10C12N15/85C12N15/64C07K16/00C07K14/705A61K39/395A61K38/17
Inventor J・S・豪鲁姆F・C・维比尔格V・W・柯尔基J・莎伦杨秋英
Owner SYMPHOGEN AS
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