Methods of blocking cancer stem cell growth
Disclosed herein are antibodies against GPR49 and uses of such antibodies. The antibodies can be monoclonal, humanized, or fully human antibodies against GPR49, hybridomas or other cell lines expressing such antibodies, nucleic acids and vectors comprising nucleic acids encoding for such antibodies, and methods of blocking cancer stem cell growth with such antibodies.
1. A method to measure activity of a monoclonal antibody that binds a G-Protein Coupled Receptor 49 (GPR49) polypeptide to inhibit growth of cancer stem cells, comprising:
growing a tumorsphere of primary colon cancer stem cells, wherein the tumorsphere is derived from a single colon tumor cell and is grown in serum-free and low-anchorage conditions;
contacting a monoclonal antibody that binds to GPR49 polypeptide with a K d of less than 10×10 −9 M with the tumorsphere of cancer stem cells, wherein the GPR49 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, and wherein the monoclonal antibody has activity to block cancer stem cell growth in vitro and antitumor activity to human cells in vivo; and
measuring the activity of the monoclonal antibody to inhibit growth of the population of tumorspheres.
2. The method of claim 1 , wherein the antibody has activity to reduce growth-competent cancer stem cell abundance in a tumor formation assay in a mammalian subject.
3. The method of claim 1 , wherein the antibody has activity to inhibit growth of the tumorsphere of cancer stem cells by about 25%.
4. The method of claim 1 , wherein the monoclonal antibody has activity capable of causing internalization of GPR49 in cells contacted with the antibody.
5. The method of claim 1 , wherein the monoclonal antibody lacks specific binding to human leucine-rich repeat containing G-protein-coupled receptor 6 (LGR6).
6. The method of claim 1 , wherein the monoclonal antibody lacks specific binding to human leucine-rich repeat containing G-protein-coupled receptor (LGR4).
7. The method of claim 1 , wherein the monoclonal antibody specifically binds to murine GPR49.
8. A method to determine activity of a monoclonal antibody that binds specifically to a G-Protein Coupled Receptor 49 (GPR49) polypeptide to inhibit growth of cancer stem cells, comprising:
growing a tumorsphere of primary colon cancer stem cells, wherein the tumorsphere is derived from a single colon tumor cell and is grown in serum-free and low-anchorage conditions;
contacting a monoclonal antibody that binds to a GPR49 polypeptide with the tumorsphere of cancer stem cells; and
measuring the ability of the monoclonal antibody to inhibit growth of colon cancer stem cells in the tumorsphere, wherein the monoclonal antibody inhibits growth of the tumorsphere by greater than about 20% as compared to a tumorsphere not contacted with the antibody.
9. The method of claim 8 , further comprising measuring the activity of the monoclonal antibody to inhibit growth of the tumorsphere comprises comparing the growth of the tumorsphere contacted with the monoclonal antibody to growth of a control tumorsphere not contacted with the monoclonal antibody.
10. The method of claim 9 , wherein the antibody inhibits growth of the tumorsphere contacted with the antibody by greater than 40% compared to the control tumorsphere not contacted with the antibody.
11. The method of claim 8 , wherein the antibody is a human antibody.
12. The method of claim 8 , wherein the antibody is a mouse antibody.
13. The method of claim 8 , wherein the antibody is an IgG class antibody.
14. The method of claim 13 , wherein the antibody is an IgG1 class antibody.
15. The method of claim 8 , wherein the single colon tumor cell comprises a mutation in a human gene represented by a gene symbol selected from the group consisting of K-Ras, PI3K, PTEN, p53, H-Ras, APC, TP53, FGFR2, VANGL2, STK11, JAK2, and RB1.