Targeted/TGF-ßRII fusion proteins and methods for making same
The present invention relates generally to the field of generating fusion proteins to be used in cancer therapy, and more specifically, to nucleotide sequences encoding the fusion proteins, wherein the chimeric fusion proteins comprises at least one targeting moiety and at least one immunomodulatory moiety that counteracts the immune tolerance of cancer cells.
1. A method of preparing a therapeutically active antibody-peptide fusion protein, the method comprising,
preparing a codon optimized nucleotide sequence of the antibody-peptide fusion protein, wherein the codon optimized nucleotide sequence comprises an increase of CG sequences, wherein the antibody-protein fusion protein comprises a targeting moiety and immunomodulating moiety, wherein the targeting moiety and the immunomodulating moiety are linked by an amino acid spacer selected from SEQ ID NO: 3 or SEQ ID NO: 11, wherein the immunomodulating moiety is TGF-βRII comprising an amino acid sequence of SEQ ID NO: 4; wherein the targeting moiety is selected from the group consisting of an Anti-EGFR1 antibody, consisting of heavy chain SEQ ID NO: 5 and light chain SEQ ID NO: 6, an Anti-HER2/Neu antibody consisting of heavy chain SEQ ID NO: 1 and light chain SEQ ID NO: 2; and anti-CTLA4 antibody consisting of heavy chain of SEQ ID NO: 7 and a light chain of SEQ ID NO: 8, wherein SEQ ID NO: 4 is attached via the amino acid spacer to the C-terminus of SEQ ID NO: 1 or SEQ ID NO: 2 of Anti-HER2/Neu; C-terminus of SEQ ID NO: 5 or SEQ ID NO: 6 of Anti-EGFR1; or C-terminus of SEQ ID NO: 7 or SEQ ID NO: 8 of Anti-CTLA-4;
cloning the optimized sequence of said antibody-peptide fusion protein in a Chinese Hamster Ovary (CHO) host cell capable of transient or continued expression;
growing the CHO host cell in a fermentation medium under suitable conditions for growing and allowing the CHO host cell to express a cloned protein, wherein the fermentation medium comprises a divalent transitional metallic salt; and
purifying the expressed antibody-peptide fusion protein and optionally checking the bi-specific binding capabilities of the antibody-peptide fusion protein to its targets.
2. The method of claim 1 , wherein the divalent transitional metallic salt is introduced into the cell culture either initially or in fed-batch mode.
3. The method of claim 1 , wherein the divalent transitional metallic salt includes a zinc ion.
4. The method of claim 3 , wherein the divalent transitional metallic salt is zinc sulphate hepta hydrate salt.
5. The method of claim 1 , wherein the divalent transitional metallic salt is in an amount sufficient to reduce accumulation of lactate during culturing.
6. The method of claim 5 , wherein the lactate reduces by about 10-40% throughout the run.
7. The method of claim 1 , wherein the divalent transitional metallic salt is added in the medium at a concentration of between about 0.3 mM to about 0.4 mM.
8. The method of claim 1 , wherein the fermentation medium is maintain at a temperature of 37±1° C., the first 3-4 days and reduced to 31±1° C. until the 7th day.
9. The method of claim 1 , wherein fermentation medium comprises a initial cell count of about 0.3-0.45×10 6 .
10. The method of claim 1 , wherein the expressed antibody-peptide fusion protein is subjected to affinity chromatography using a Mab Select Xtra protein A column having a specific pH.
11. The method of claim 1 , wherein the supernatant binding and passing through the Mab Select Xtra protein A column is pH adjusted to the specific pH of the protein A column.
12. The method of claim 1 , wherein the antibody-proteins fusion protein binds through the Fc region of the antibody to the column while impurities are eliminated as flow through.
13. The method of claim 1 , wherein the antibody-protein fusion protein bound to the column is eluted using glycine at pH 3.0 and adjusted to neutral pH for storage.
14. The method of claim 1 , wherein the purified protein is stored at −20° C. or at 2-8° C.