Transforming growth factor-beta-responsive polypeptides and their methods for use
Aspects of the disclosure relate to polypeptides comprising a signal peptide, an antigen-binding domain that specifically binds TGF-β, a peptide spacer, a transmembrane domain, and an endodomain. When expressed in a cell, the polypeptides are capable of not only neutralizing the TGF-β but also specifically triggering T-cell activation in the presence of TGF-β. T-cell activation spurs the immune cell to produce immunostimulatory cytokines and proliferate, thus turning TGF-β from an immunosuppressive signal to an activating stimulus.
1. A method for stimulating an immune response comprising contacting an immune cell with TGF-β, wherein the immune cell comprises a polypeptide comprising a chimeric antigen receptor (CAR) and wherein the CAR comprises a signal peptide, an antigen-binding domain with a variable heavy (VH) and variable light (VL) region, a peptide spacer, a transmembrane domain, and an endodomain; wherein the antigen-binding domain specifically binds to TGF-β; and wherein the antigen-binding domain comprises: a VH region comprising SEQ ID NO:11 (HCDR1), SEQ ID NO:12 (HCDR2); and SEQ ID NO:13 (HCDR3) and a VL region comprising SEQ ID NO:14 (LCDR1), SEQ ID NO:15 (LCDR2); and SEQ ID NO:16 (LCDR3).
2. The method of claim 1 , wherein the immune cell is a T cell and wherein the cell or polypeptide further comprises a cancer specific chimeric antigen receptor (CAR).
3. The method of claim 1 , wherein the VH region comprises SEQ ID NO:3 and the VL region comprises SEQ ID NO:4.
4. The method of claim 1 , wherein the polypeptide further comprises a co-stimulatory region and wherein the co-stimulatory region is between the transmembrane domain and endodomain.
5. The method of claim 1 , wherein the transmembrane domain comprises a transmembrane domain of CD28.
6. The method of claim 1 , wherein the VH and VL regions are separated by a peptide linker.
7. The method of claim 1 , wherein the peptide spacer comprises less than 50 amino acids.
8. The method of claim 1 , wherein the peptide spacer comprises the hinge region of an IgG molecule.
9. The method of claim 1 , wherein the polypeptide further comprises a detection peptide.
10. The method of claim 1 , wherein the signal peptide comprises SEQ ID NO:18.
11. The method of claim 1 , wherein the CAR is a bi-specific CAR that further comprises a cancer molecule specific antigen-binding domain.
12. The method of claim 1 , wherein the antigen binding domain comprises a scFv.
13. The method of claim 2 , wherein the cancer specific chimeric antigen receptor is specific for GD2 or IL13Rα2.
14. The method of claim 11 , wherein the cancer molecule specific antigen-binding domain is specific for GD2 or IL13Rα2.
15. The method of claim 6 , wherein the endodomain comprises a CD28 or CD3 zeta signaling domain.
16. The method of claim 6 , wherein the peptide spacer comprises the hinge and CH 2 CH 3 region of an IgG molecule.
17. The method of claim 6 , wherein the VH is N-terminal to the VL.
18. The method of claim 6 , wherein the VL is N-terminal to the VH.
19. The method of claim 6 , wherein the peptide linker is a glycine-serine linker having at least 4 amino acids.
20. The method of claim 6 , wherein the immune cell is a CD4 + T cell, CD8 + T cell, T-regulatory cell, gamma-delta T cell, cytotoxic T cell, natural-killer (NK) cell, or a neutrophil.