Multimeric protein engineering
The invention described herein encompasses (1) artificial preproproteins and the polynucleotides encoding them, (2) methods for producing these biomolecules, and (3) methods for their use. The artificial preproproteins of this invention comprise a protein assembly capable of producing a multimeric protein from a single protein. FIG. 4 illustrates generally the process by which a polynucleotide encoding the artificial preproprotein is introduced into a cell and a biomolecule of interest is produced.
1 . A targeted immunotherapy comprising administering to a subject a therapeutic amount of an immunoglobulin molecule capable of binding to a target antigen wherein the immunoglobulin molecule is produced from an artificial proprotein and is essentially free of a propeptide of the proprotein.
2 . The targeted immunotherapy of claim 1 wherein the artificial proprotein comprises a signal peptide sequence attached to the N-terminus of the proprotein.
3 . The targeted immunotherapy of claim 2 wherein the artificial proprotein comprises
(a) a first peptide sequence;
(b) a propeptide sequence attached to the C-terminus of the first peptide sequence; and
(c) a second peptide attached to the C-terminus of the propeptide sequence,
wherein the first peptide sequence and the second peptide sequence comprise an antibody light chain peptide and an antibody heavy chain peptide.
4 . The targeted immunotherapy of claim 3 wherein the first peptide sequence comprises the antibody light chain peptide and the second peptide sequence comprises the antibody heavy chain peptide.
5 . The targeted immunotherapy of claim 3 wherein the first peptide sequence comprises the antibody heavy chain peptide and the second peptide sequence comprises the antibody light chain peptide.
6 . The targeted immunotherapy of claim 2 wherein the artificial proprotein comprises
(a) a first peptide sequence;
(b) a propeptide sequence attached to the C-terminus of the first peptide sequence; and
(c) a second peptide attached to the C-terminus of the propeptide sequence,
wherein the first peptide sequence and the second peptide sequence comprise an antibody light chain peptide and an antibody heavy chain fragment selected from the group consisting of a Fd and an Fd′.
7 . The targeted immunotherapy of claim 6 wherein the first peptide sequence comprises the antibody light chain peptide and the second peptide sequence comprises the antibody heavy chain fragment selected from the group consisting of a Fd and an Fd′.
8 . The targeted immunotherapy of claim 6 wherein the first peptide sequence comprises the antibody heavy chain fragment selected from the group consisting of a Fd and an Fd′ and the second peptide sequence comprises the antibody light chain peptide.
9 . The targeted immunotherapy of claim 2 wherein the artificial proprotein comprises
(a) a first peptide sequence;
(b) a propeptide sequence attached to the C-terminus of the first peptide sequence; and
(c) a second peptide attached to the C-terminus of the propeptide sequence,
wherein the first peptide sequence and the second peptide sequence comprise a light chain peptide of an antibody fragment and a heavy chain peptide of an antibody fragment.
10 . The targeted immunotherapy of claim 9 wherein the antibody fragment is an Fab fragment or an Fv fragment.
11 . The targeted immunotherapy of claim 9 wherein the first peptide sequence comprises a light chain peptide of an antibody fragment and the second peptide sequence comprises a heavy chain peptide of an antibody fragment.
12 . The targeted immunotherapy of claim 9 wherein the first peptide sequence comprises a heavy chain peptide of an antibody fragment and the second peptide sequence comprises a light chain peptide of an antibody fragment.
13 . The targeted immunotherapy of claim 2 wherein the immunoglobulin molecule is a full-size antibody or an antibody fragment.
14 . The targeted immunotherapy of claim 13 wherein the antibody fragment is selected from the group consisting of an Fab fragment, an Fab′ fragment, an F(ab′) 2 fragment and an Fv fragment.
15 . The targeted immunotherapy of claim 2 wherein the target antigen is from a malignant cell.
16 . The targeted immunotherapy of claim 2 wherein the target antigen is a marker on a clonal tumor.
17 . The targeted immunotherapy of claim 2 wherein the target antigen is from a pathogen.
18 . The targeted immunotherapy of claim 2 wherein the immunoglobulin molecule is linked to a toxin.
19 . The targeted immunotherapy of claim 2 wherein the immunoglobulin molecule is linked to a radioisotope.
20 . The targeted immunotherapy of claim 2 wherein the immunoglobulin is linked to an enzyme.
21 . The targeted immunotherapy of claim 2 wherein the immunoglobulin molecule is PEGylated.
22 . The targeted immunotherapy of claim 2 wherein the immunoglobulin molecule is produced in a plant.
23 . The targeted immunotherapy of claim 2 wherein the immunoglobulin molecule is produced in a prokaryotic or eukaryotic microbial host cell culture.