Fusion immunomodulatory proteins and methods for making same
The present invention relates generally to the field of generating recombinant chimeric fusion proteins to be used in the cancer therapy, and more specifically, to fusion molecules of Anti-EGFR1-TGFβRII, Anti-EGFR1-PD1 and Anti-CTLA4-PD1 and methods of generating same, wherein the methods reduce production costs and increase homogeneity of the recombinant chimeric fusion proteins.
1. A method for preparing therapeutically active fusion protein, wherein the fusion protein comprises a tumor targeting moiety and an immunomodulatory molecule wherein the immunomodulatory molecule counteracts immune tolerance of a cancer cell, wherein the tumor targeting moiety is an antibody that binds to EGFR1 and wherein the fusion protein is prepared by the following steps:
preparing a codon optimized nucleotide sequence encoding the fusion protein, wherein the codon optimized nucleotide sequence has been modified to increase CG sequences, wherein the codon optimized nucleotide sequence for the antibody is lacking nucleotides for expression of a lysine at the C-terminal end of the heavy chain of the antibody, wherein the immunomodulatory moiety binds to transforming growth factor beta (TGF-β) and wherein the codon optimized nucleotide sequences consist of SEQ ID NOs: 1, 2, 4 and 3;
cloning the optimized sequence of said fusion protein in a CHO host cell capable of transient or stable expression;
growing the host cell in a media under suitable conditions for growing and allowing the host cell to express the fusion protein; and
collecting secreted fusion proteins and optionally for further purification.
2. The method of claim 1 , wherein the immunomodulatory molecule is linked to the antibody by an amino acid sequence of sufficient length to allow bi-specific binding of the fusion protein.
3. The method of claim 1 , wherein the immunomodulatory molecule is linked directly or through a linker to the heavy chain of the antibody, light chain of the antibody or both chains.
4. The method of claim 1 , wherein the immunomodulatory molecule is linked directly or through a linker to the N or C terminus of the heavy chain of the antibody, N or C terminus of the light chain of the antibody or both N and C terminus of both chains.
5. A method of treating cancer in a subject, the method comprising:
a) preparing a therapeutically active fusion protein having anti-proliferative activity, wherein the fusion protein comprises a tumor targeting moiety and at least one immunomodulatory molecule, wherein the tumor targeting moiety is an antibody that binds to EGFR1 and wherein the fusion protein is prepared by the following steps:
i) preparing a codon optimized nucleotide sequence encoding the fusion protein, wherein the codon optimized nucleotide sequence has been modified to increase CG sequences, wherein the codon optimized nucleotide sequence for the antibody is lacking nucleotides for expression of a lysine at the C-terminal end of the heavy chain of the antibody, wherein the immunomodulatory moiety binds to transforming growth factor beta (TGF-β) and wherein the codon optimized nucleotide sequences consisting of SEQ ID NOs: 1, 2, 4 and 3;
ii) cloning the optimized sequence of said fusion protein in a CHO host cell capable of transient or stable expression;
iii) growing the host cell in a media under suitable conditions for growing and allowing the host cell to express the fusion protein; and
iv) collecting secreted fusion proteins for optional purification;
b) administering a therapeutically active amount of the fusion proteins to the subject.