Targeted/immunomodulatory fusion proteins and methods for making same

Patent metadata

Field	Value
Publication number	US-2016237155-A1
Application number	US-201615145135-A
Country	US
Kind code	A1
Filing date	May 3, 2016
Priority date	Apr 30, 2012
Publication date	Aug 18, 2016
Grant date	—

Abstract

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.

First claim

That which is claimed is:

1 . A chimeric fusion protein comprising a targeting moiety to target a cancer cell and an immunomodulating moiety that counteracts immune tolerance, 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 SEQ ID NO: 4, wherein the targeting moiety is anti-CTLA4 having heavy chain SEQ ID NO: 7 and light chain SEQ ID NO: 8; wherein SEQ ID NO: 4 is attached via the amino acid spacer to the C-terminus of SEQ ID NO 7 or SEQ ID NO: 8.

2 . A method of treating a neoplastic disease, the method comprising the administration to a subject in need thereof a chimeric fusion protein according to claim 1 .

3 . A process of preparing the chimeric fusion protein according to claim 1 , comprising:
transfecting a host cell with polynucleotide sequences that encode the chimeric fusion protein and maintaining the transformed host cell under biological conditions sufficient for expression of the chimeric fusion protein.

4 . A method of preparing the chimeric fusion protein according to claim 1 , the method comprising:
a) preparing a codon optimized sequence of the said chimeric fusion protein; b) cloning the optimized sequence of said chimeric fusion protein a host cell capable of transient or continued expression; c) growing the host cell in a media sufficiently and allowing it to express the cloned chimeric fusion protein; and d) subjecting the expressed chimeric fusion protein to purification and optionally checking the bi-specific binding capabilities of the chimeric fusion protein to its targets.

5 . The method of claim 4 , wherein the immunomodulating moiety is either bound to the C-terminus of the heavy or light chain of the antibody.

6 . The method of claim 4 , wherein the host cell is a transient cell line or a stable cell line

7 . The method of claim 6 , wherein transient expression is done by transfecting or transforming the host with vectors carrying the chimeric fusion proteins into mammalian host cells

8 . The method of claim 7 , wherein the transiently expressed chimeric fusion peptides are subjected to purification and in-vitro tests to check its bi-specificity

9 . The method of claim 8 , wherein the in-vitro test are ELISA or NK/T-cell binding assays to validate bi-functional target binding or immune cell stimulation.

10 . The method of claim 9 , wherein the peptides demonstrating desired bi-specificity are selected for sub-cloning into a stable cell line for larger scale expression and purification.

11 . The method of claim 10 , wherein the expression levels in stable cell line are comparable to the earlier generations.

Assignees

• Biocon Ltd

Inventors

• Govindappa Nagaraj
• Sastry Kedarnath
• Soares Maria Melina

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61P37/04

  Immunostimulants · CPC title

• C07K14/65

  Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2 · CPC title

• C07K2317/31

  multispecific · CPC title

• A61K38/10

  Peptides having 12 to 20 amino acids {(A61K38/043 - A61K38/046 take precedence)} · CPC title