Mammalian cell lines containing SIRT-1 gene knockout

Abstract

To provide a method for generating a recombinant mammalian cell expressing a heterologous polypeptide, and a method for producing a heterologous polypeptide using the recombinant mammalian cell, wherein in the recombinant cell, the expression of the endogenous SIRT-1 gene has been reduced.SOLUTION: The reduction in the expression of the endogenous SIRT-1 gene in a recombinant cell, for example, the knockout of the sirtuin-1 gene (SIRT-1) in mammalian cells, such as CHO cells, improves recombinant productivity and reduces lactate production by the cells.SELECTED DRAWING: None

Claims

1. Mammalian cells containing deoxyribonucleic acid encoding a heterologous polypeptide, and exhibiting reduced expression of the endogenous SIRT-1 gene, The reduction in SIRT-1 gene expression is caused by SIRT-1 gene knockout, and the SIRT-1 gene knockout is heterozygous or homozygous, and The productivity of the heterologous polypeptide in the mammalian cells is increased by at least 10% compared to cells cultured under the same conditions that have the same genotype but endogenous SIRT-1 gene expression. The aforementioned mammalian cells.

2. The mammalian cell according to claim 1, wherein the SIRT-1 gene knockout is mediated by a nuclease-assisted gene targeting system.

3. The mammalian cell according to claim 2, wherein the nuclease-supported gene targeting system is selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nuclease, and TALEN.

4. The mammalian cell according to any one of claims 1 to 3, wherein the SIRT-1 gene knockout is performed before or after the introduction of an exogenous nucleic acid encoding a heterologous polypeptide.

5. The mammalian cell according to any one of claims 1 to 4, wherein the heterologous polypeptide is an antibody.

6. The mammalian cell according to claim 5, wherein the antibody is a multispecific antibody.

7. The multispecific antibody described above i) A full-length antibody with domain exchange comprising a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment, a) The light chain of the first Fab fragment comprises the VL and CH1 domains, and the heavy chain of the first Fab fragment comprises the VH and CL domains, b) The light chain of the first Fab fragment contains VH and CL domains, and the heavy chain of the first Fab fragment contains VL and CH1 domains, or c) The light chain of the first Fab fragment comprises VH and CH1 domains, and the heavy chain of the first Fab fragment comprises VL and CL domains, The second Fab fragment comprises a light chain containing VL and CL domains and a heavy chain containing VH and CH1 domains. Full length antibody; ii) A full-length antibody having domain exchange and an additional heavy chain C-terminal binding site, - A full-length antibody comprising two pairs each of full-length antibody light chains and full-length antibody heavy chains, wherein the binding sites formed by each pair of full-length heavy chains and full-length light chains specifically bind to a first antigen, and - One additional Fab fragment, the additional Fab fragment fused to the C-terminus of one heavy chain of the full-length antibody, the binding site of the additional Fab fragment specifically binds to the second antigen. Includes, An additional Fab fragment that specifically binds to a second antigen includes: i) a) a domain crossover in which the light chain variable domain (VL) and heavy chain variable domain (VH) are substituted for each other, or b) a domain crossover in which the light chain constant domain (CL) and heavy chain constant domain (CH1) are substituted for each other, or ii) a single-stranded Fab fragment. Full length antibody; iii) A one-arm, single-chain antibody comprising a first binding site that specifically binds to a first epitope or antigen, and a second binding site that specifically binds to a second epitope or antigen, - Light chain containing a variable light chain domain and a light chain kappa or lambda constant domain, - A light / heavy chain combination including a variable light chain domain, a constant light chain domain, a peptide linker, a variable heavy chain domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 with a knob mutation. - Heavy chain containing a variable heavy chain domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain with a hole mutation. A single-arm, single-chain antibody containing; iv) A two-arm single-chain antibody comprising a first binding site that specifically binds to a first epitope or antigen, and a second binding site that specifically binds to a second epitope or antigen, - A first light chain / heavy chain combination comprising a variable light chain domain, a constant light chain domain, a peptide linker, a variable heavy chain domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 with a hole mutation, - A second light / heavy chain combination including a variable light chain domain, a constant light chain domain, a peptide linker, a variable heavy chain domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain with a knob mutation. A two-armed single-chain antibody containing; v) A common light chain bispecific antibody comprising a first binding site that specifically binds to a first epitope or antigen, and a second binding site that specifically binds to a second epitope or antigen, - Light chain including variable light chain domain and constant light chain domain, - The first heavy chain comprises a variable heavy chain domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain with a hole mutation. - A second heavy chain comprising a variable heavy chain domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain with a knob mutation. Common light chain bispecific antibodies, including; vi) A full-length antibody having an additional heavy chain N-terminal binding site accompanied by domain exchange, - A first Fab fragment and a second Fab fragment, wherein each binding site of the first Fab fragment and the second Fab fragment specifically binds to the first antigen, - A third Fab fragment wherein the binding site of the third Fab fragment specifically binds to the second antigen, and the third Fab fragment includes a domain crossover in which a variable light chain domain (VL) and a variable heavy chain domain (VH) are substituted for each other, and - Fc region comprising a first Fc region polypeptide and a second Fc region polypeptide Includes, The first Fab fragment and the second Fab fragment each contain a heavy chain fragment and a complete long light chain, The C-terminus of the heavy chain fragment of the first Fab fragment is fused to the N-terminus of the first Fc region polypeptide. The C-terminus of the heavy chain fragment of the second Fab fragment is fused to the N-terminus of the variable light chain domain of the third Fab fragment, and the C-terminus of the CH1 domain of the third Fab fragment is fused to the N-terminus of the second Fc region polypeptide. Full length antibody; Furthermore vii) An immunoconjugate comprising a full-length antibody and, optionally, a non-immunoglobulin moiety linked to it via a peptide linker. A mammalian cell according to claim 6, selected from the group consisting of the following.

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