Plasmid system for multigene expression
View Patent ↗The present invention provides a plasmid system which facilitates the construction of a single amplifiable plasmid that, having the potential to accommodate many independent expression cassettes, has the ability to express multi-subunit complex proteins such as antibodies and receptors.
1. A method for producing an antibody comprising culturing an isolated host cell comprising a recombined amplifiable vector under conditions favorable to expression of cassettes in said vector; which vector is the product of a method comprising:
(a) introducing one or more expression cassettes encoding an immunoglobulin light chain or immunoglobulin heavy chain of said antibody into the multiple cloning site of a first universal transfer vector comprising a first multiple cloning site which comprises nucleotides 620-766 of the nucleotide sequence set forth in SEQ ID NO: 2; wherein said multiple cloning site is in said orientation or in a reverse orientation;
(b) introducing one or more expression cassettes encoding an immunoglobulin light chain or heavy chain of said antibody, different from the cassettes introduced into said first universal transfer vector, into a second universal transfer vector comprising a second multiple cloning site which comprises nucleotides 620-772 of the nucleotide sequence set forth in SEQ ID NO: 1; wherein said multiple cloning site is in said orientation or in a reverse orientation; and
(c) introducing said cassettes from steps (a) and (b) into an amplifiable vector comprising a third multiple cloning site, comprising nucleotides 5037-5183 of the nucleotide sequence set forth in SEQ ID NO: 3; wherein said multiple cloning site is in said orientation or in a reverse orientation; and wherein the cassettes from steps (a) and (b) encode an immunoglobulin light chain and an immunoglobulin heavy chain, of said antibody.
2. The method of claim 1 wherein said first or second cassette comprises a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 20 and the other cassette comprises a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22.
3. The method of claim 2 wherein said polynucleotide encoding said polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 20 comprises the nucleotide sequence set forth in SEQ ID NO: 19 and wherein said polynucleotide encoding said polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22 comprises the nucleotide sequence set forth in SEQ ID NO: 21.
4. The method of claim 1 wherein said first universal transfer vector comprises the plasmid map set forth in FIG. 2 .
5. The method of claim 1 wherein said second universal transfer vector comprises the plasmid map set forth in FIG. 1 .
6. The method of claim 1 wherein said amplifiable vector comprises the plasmid map set forth in FIG. 3 .
7. The method of claim 1 wherein said first universal transfer vector comprises the nucleotide sequence set forth in SEQ ID NO: 2.
8. The method of claim 1 wherein said second universal transfer vector comprises the nucleotide sequence set forth in SEQ ID NO: 1.
9. The method of claim 1 wherein said amplifiable vector comprises the nucleotide sequence set forth in SEQ ID NO: 3.
10. The method of claim 1 wherein one set of expression cassettes comprises a polynucleotide, operably associated with a promoter, which polynucleotide encodes an anti-IGFR1 antibody immunoglobulin heavy chain and the other set of expression cassettes comprises a polynucleotide, operably associated with a promoter, which polynucleotide encodes an anti-IGFR1 antibody immunoglobulin light chain.