Compositions and methods for making antibodies based on use of expression-enhancing loci

A technology of gene loci and marker genes, applied in the field of improving eukaryotic cells, site-specific integration and expression, and expression of antigen-binding proteins, can solve problems such as instability and changes

Active Publication Date: 2019-01-11
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Integration of additional genes may cause large changes in expression and instability due to local genetic environment (i.e., position effects)

Method used

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  • Compositions and methods for making antibodies based on use of expression-enhancing loci
  • Compositions and methods for making antibodies based on use of expression-enhancing loci
  • Compositions and methods for making antibodies based on use of expression-enhancing loci

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0194] Example 1: Expression of monospecific antibodies (Abs) in two specific expression enhancing loci (via site-specific integration)

[0195] Cloning the Ab chains (AbC1, AbC2) into vectors where the RSS sites are flanked by Ab expression cassettes and expression cassettes for selectable markers, e.g. figure 1 depicted. Both Ab chains can be cloned into separate vectors or combined into one vector with 2 expression cassettes arranged in tandem in any possible order: AbC1, AbC2 and selectable markers, e.g. AbC1 is equivalent to conventional LC and AbC2 is equivalent to than conventional heavy chains.

[0196] Briefly, DNA encoding VH and VL domains can be isolated directly by PCR from single antigen-positive B cells. The heavy and light chain PCR products were cloned into Sap I linearized antibody vectors containing IgG heavy chain constant region and kappa light chain constant region, respectively. The heavy chain plasmid (AbC2) has RRS3 and RRS2 sites flanking the heavy...

Embodiment 2

[0197] Example 2: Expression of bispecific antibodies (BsAbs) in two specific expression enhancing loci (via site-specific integration)

[0198] For bispecific antibody expression, the three antibody chains and two selectable markers were cloned into a plasmid similar to Example 1 such that AbC1, AbC2, and selectable marker 1 flank the first locus Compatible RRS sites, or integration sites ( SEQ ID NO:1; locus 1), and AbC1, AbC3, selectable marker 2 are compatible with the second locus or integration site (SEQ ID NO:2). In our observation, AbC1 as a conventional LC does not require two gene copies for full expression. For each site, 1 or 2 plasmids were prepared, wherein the 3 expression cassettes were arranged in tandem or into 2 plasmids, wherein the 2 expression cassettes were cloned into one vector and the remaining expression cassettes were Cloned into the second vector. see image 3 .

[0199] When recombinant plasmids expressing heavy and light chain genes are co-...

Embodiment 3

[0200] Example 3: Large scale production of bispecific and monospecific antibodies following specific integration

[0201] Host cells (CHO-K1) were generated as described above analogously to Example 1 (see also image 3 bispecific antibodies and figure 1 monospecific antibodies). will be able to transfer the gene cassette RMCE to The host cell in the locus (locus 1) and SEQ ID NO:2 (locus 2) is able to RMCE the gene cassette to only one integration site (locus 1 / locus 2). ) host cells for comparison. will carry antibody light and heavy chains (AbC1, AbC2, AbC3) and the requisite RRS and selectable marker nucleic acids (see image 3 ) vector recombined into the production cell line (RSX 2BP ) to generate host cells expressing Ab E, Ab F, Ab G, and Ab H. Thus, each bispecific antibody host cell expresses a common light chain, and two heavy chains that bind different antigens, wherein the one heavy chain is engineered in its CH3 domain to differentially bind protein A (e...

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Abstract

This invention relates to site-specific integration and expression of recombinant proteins in eukaryotic cells. In particular, the invention includes compositions and methods for improved expression of antigen-binding proteins including monospecific and bispecifc antibodies in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, by employing multiple expression-enhancinglocus.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 325,400, filed April 20, 2016, which is hereby incorporated by reference in its entirety. field of invention [0003] The present disclosure relates to the site-specific integration and expression of recombinant proteins in eukaryotic cells. In particular, the present disclosure relates to the improvement of antigen-binding proteins, including monospecific and bispecific antibodies, in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, by employing expression-enhancing loci Compositions and methods of expression. Background technique [0004] Cellular expression systems are intended to provide a reliable and efficient source for the production of a given protein, whether for research or therapeutic use. Recombinant protein expression in mammalian cells is a preferred method for the production of therape...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K16/46C12N15/90
CPCC12N5/0682C07K16/00C12N15/85C12N15/907C12N2510/02C07K2317/31C12N2800/107C07K16/468C07K2317/526
Inventor R·巴布D·布拉科夫陈刚J·P·梵迪Y·赵
Owner REGENERON PHARM INC
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