BR-122026002520-A2 - CELL, VECTOR SET FOR EXPRESSING A BIOSPECIFIC ANTIGEN-BINDING PROTEIN IN A CELL, VECTOR SET, SYSTEM, METHOD, AND METHOD FOR PRODUCING AN ANTIGEN-BINDING PROTEIN

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 enhanced expression of antigen-binding proteins, including monospecific and bispecific antibodies in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, through the use of multiple expression enhancement loci.

Inventors

• Robert Babb
• Darya Burakov
• Gang Chen
• JAMES P. FANDL
• Yu Zhao

Assignees

• REGENERON PHARMACEUTICALS, INC.

Dates

Publication Date

20260310

Application Date

20170420

Priority Date

20160420

Claims (12)

1. 1. A set of vectors for expressing a bispecific antigen-binding protein in a mammalian cell, characterized in that it comprises a first vector comprising, from 5' to 3': a first recombinase recognition site (RRS), a first nucleic acid, a 5' portion of a first selectable marker gene, comprising a 5' portion of a first intron, and a second RRS, wherein the second RRS is located at the 3' end of the 5' portion of the first intron, which is also the 3' end of the 5' portion of the first selectable marker gene; a second vector comprising, from 5' to 3': a third RRS, a second nucleic acid, a 5' portion of a second selectable marker gene comprising a 5' portion of a second intron, and a fourth RRS, wherein the fourth RRS is placed at the 3' end of the 5' portion of the second intron, which is also the 3' end of the 5' portion of the second selectable marker gene; a third vector comprising 5' to 3': a 5' RRS, the remaining 3' portion of the first selectable marker gene comprising the remaining 3' portion of the first intron, a third nucleic acid, and a 3' RRS, wherein the 5' RRS is placed at the 5' end of the remaining 3' portion of the first intron, which is also the 5' end of the 3' portion of the first selectable marker gene, and wherein the 5' RRS is the same as the second RRS, and wherein the 3' RRS is different from the first and second RRSs; and a fourth vector comprising, from 5’ to 3’: a 5’ RRS, the remaining 3’ portion of the second selectable marker gene, comprising the remaining 3’ portion of the second intron, a fourth nucleic acid and a 3’ RRS, wherein the 5’ RRS is located at the 5’ end of the remaining 3’ portion of the second intron, which is also the 5’ end of the 3’ portion of the second selectable marker gene, and wherein the 5’ RRS is the same as the fourth RRS, and wherein the 3’ RRS of the fourth vector is different from the third and fourth RRSs; wherein the first, second, third and fourth RRSs are different from each other; wherein one of the first and third nucleic acids comprises a nucleotide sequence encoding a first HCF, and the other comprises a nucleotide sequence encoding a first LCF; wherein one of the nucleic acids, the second and the fourth, comprises a nucleotide sequence encoding a second HCF, and the other comprises a nucleotide sequence encoding a second LCF; wherein the bispecific antigen-binding protein comprises the first HCF, the second HCF, the first LCF and the second LCF, and wherein the first and second HCFs differ from each other in sequence.
2. 2. A set of vectors according to claim 1, characterized in that the first and second LCFs are sequentially equal.
3. 3. Vector set according to claim 1 or 2, characterized in that the bispecific antigen-binding protein is a full-length bispecific antibody.
4. 4. A set of vectors according to any one of claims 1 to 3, characterized in that the selectable marker genes confer antibiotic resistance.
5. 5. Vector set according to claim 4, characterized in that the antibiotic is hygromycin, neomycin or kanamycin.
6. 6. System for expressing a bispecific antigen-binding protein in a mammalian cell, characterized in that it comprises the set of vectors as defined in claim 1; and the mammalian cell, wherein the mammalian cell comprises: integrated within a first enhanced expression locus: from 5' to 3', a first recombinase recognition site (RRS), a first exogenous nucleic acid and an RRS that is identical to the 3' RRS in the third vector; and; integrated within a second enhanced expression locus: from 5' to 3', a third RRS, a second exogenous nucleic acid and a fourth RRS.
7. 7. System according to claim 6, characterized in that the first and second LCFs are the same in sequence.
8. 8. System according to claim 6 or 7, characterized in that the bispecific antigen-binding protein is a full-length bispecific antibody.
9. 9. A system according to any one of claims 6 to 8, characterized in that selectable marker genes confer antibiotic resistance.
10. 10. System according to claim 9, characterized in that the antibiotic is hygromycin, neomycin or kanamycin.
11. 11. System according to any one of claims 6 to 10, characterized in that the cell is a CHO cell.
12. 12. A method for producing a bispecific antigen-binding protein, characterized in that it comprises: providing a system as defined in any one of claims 6 to 11; introducing vector sets of the system into mammalian cells of the system by transfection; and selecting a transfectant wherein the first and third nucleic acids have integrated into the first locus of increased expression, and the second and fourth nucleic acids have integrated into the second locus of increased expression; and culturing the selected transfectant to express the bispecific antigen-binding protein.

Description

CROSS-REFERENCE ON RELATED REQUEST [001] This application claims the benefit of priority under U.S. Provisional Application No. 62/325,400, filed April 20, 2016, the full contents of which are incorporated herein by reference. FIELD OF THE INVENTION [002] This disclosure relates to the integration and site-specific expression of recombinant proteins in eukaryotic cells. In particular, the disclosure relates to compositions and methods for the enhanced expression of antigen-binding proteins (including monospecific and bispecific antibodies) in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, using expression-enhancing loci. FUNDAMENTALS OF THE TECHNIQUE [003] Cellular expression systems aim to provide a reliable and efficient source for the manufacture of a given protein, whether for research or therapeutic use. The expression of recombinant proteins in mammalian cells is a preferred method for the manufacture of therapeutic proteins due, for example, to the ability of mammalian expression systems to appropriately modify recombinant proteins post-traditionally. [004] Despite the availability of various expression systems, the challenge of efficient gene transfer and stability of the integrated gene for the expression of a recombinant protein still exists. For long-term expression of a target transgene, one consideration is the minimal disruption of cellular genes to avoid alterations in the cell line phenotype. [005] Manipulating stable cell lines to accommodate multiple genes for expression, such as multiple antibody chains as in multispecific antibodies, is particularly challenging. Large variations in the expression levels of integrated genes can occur. Integrating additional genes can lead to greater variation in expression and instability due to the local genetic environment (i.e., position effects). Expression systems for the production of multispecific antigen-binding proteins often require the expression of two or more different immunoglobulin chains intended to pair as a specific multimeric format and may often tip in favor of producing homodimers rather than the desired combination of heterodimers or multimers. In this sense, there is a need in the technique for improved mammalian expression systems. SUMMARY OF THE INVENTION [006] In one aspect, this disclosure provides a cell containing multiple exogenous nucleic acids integrated in a site-specific manner at two expression-enhancing loci, where several exogenous nucleic acids together encode an antigen-binding protein. The antigen-binding protein can be a bispecific antigen-binding protein, or a conventional monospecific antigen-binding protein. [007] In some embodiments, a supplied cell contains a first exogenous nucleic acid integrated within a first locus of enhanced expression, and a second exogenous nucleic acid integrated within a second locus of enhanced expression; wherein the first and second exogenous nucleic acids together encode an antigen-binding protein. [008] In some embodiments, the first exogenous nucleic acid contains a nucleotide sequence encoding a first heavy chain fragment (HCF), and the second exogenous nucleic acid contains a nucleotide sequence encoding a first light chain fragment (LCF). [009] In some embodiments, the second exogenous nucleic acid also includes a nucleotide sequence encoding a second HCF (also HCF*). The first and second HCFs may be the same, or different, as in a bispecific antigen-binding protein. Each nucleotide sequence encoding HCF or LCF may encode amino acids from a constant region. In some embodiments, the nucleotide sequence encoding the first HCF encodes a first CH3 domain and the nucleotide sequence encoding the second HCF (HCF*) encodes a second CH3 domain. In some embodiments, the first and second CH3 domains may differ at least at one amino acid position, such as a position that results in different Protein A binding characteristics. In other embodiments, the nucleotide sequences encoding the first and second CH3 domains differ from each other, one of the nucleotide sequences having been modified at the codon level. [0010] In some embodiments, the first exogenous nucleic acid (which contains the first nucleotide sequence encoding HCF) further comprises a nucleotide sequence encoding a second LCF. The second LCF may be the same as or different from the first LCF in the second exogenous nucleic acid. [0011] In many embodiments of a cell provided in this document, each of the nucleotide sequences encoding an HCF or LCF is operationally linked to a promoter independently so that the transcription of each HCF or LCF coding sequence is regulated separately. [0012] In some embodiments, a first RRS and a second RRS are positioned at 5' and 3', respectively, relative to the first exogenous nucleic acid, and a third RRS and a fourth RRS are positioned at 5' and 3', respectively, relative to the second exogenous nucleic acid, wherein the first and second RRSs are differen