Construction and characterization of multimeric IL-15-based molecules with CD3 binding domains
The invention features soluble fusion protein complexes comprising at least two soluble fusion proteins. For example, the first fusion protein is an anti-CD3 antibody covalently linked to an interleukin-15 (IL-15) polypeptide or functional fragment thereof. The second fusion protein comprises a binding domain that recognizes disease antigens, wherein this domain is covalently linked to a soluble interleukin-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof. One or both of the first and second fusion proteins further includes an immunoglobulin Fc domain or a functional fragment thereof, and the IL-15 domain of first fusion protein binds to the soluble IL-15Rα domain of the second fusion protein to form a soluble fusion protein complex. The invention further provides methods for making and using the complexes of the invention.
1. An isolated soluble fusion protein complex comprising at least two soluble fusion proteins, wherein
the first fusion protein comprises (a) a first binding domain covalently linked to (b) a interleukin-15 (IL-15) polypeptide domain; and the second fusion protein comprises (c) a second binding domain covalently linked to (d) a soluble IL-15 receptor alpha sushi-binding domain (IL-15RαSu) fused to an immunoglobulin Fc domain,
wherein the first binding domain comprises an anti-CD3 antibody and the second binding domain comprises a means for binding an antigen on a tumor cell, and
wherein the IL-15 domain of the first fusion protein binds to the soluble IL-15RαSu domain of the second fusion protein to form a soluble fusion protein complex, and wherein the first fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 2.
2. The soluble fusion protein complex of claim 1 , wherein the IL-15 polypeptide is an IL-15 variant comprising an N72D mutation (IL-15N72D).
3. The soluble fusion protein complex of claim 1 , wherein the anti-CD3 antibody comprises an immunoglobulin light chain variable domain covalently linked to an immunoglobulin heavy chain variable domain by a polypeptide linker sequence.
4. The soluble fusion protein of claim 1 , wherein the means for binding an antigen on a tumor cell binds an antigen on a tumor cell and comprises an anti-CD20 antibody.
5. The soluble fusion protein complex of claim 4 , wherein the anti-CD20 antibody comprises an immunoglobulin light chain variable domain covalently linked to an immunoglobulin heavy chain variable domain by a polypeptide linker sequence.
6. The soluble fusion protein complex of claim 4 , wherein the second fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 4.
7. A soluble fusion protein complex comprising a first soluble fusion protein complex of claim 1 covalently linked to a second soluble fusion protein complex of claim 1 .
8. The soluble fusion protein complex of claim 7 , wherein the first soluble fusion protein complex is covalently linked to the second soluble fusion protein complex by a disulfide bond linking the Fc domain of the first soluble fusion protein complex to the Fc domain of the second soluble fusion protein complex.
9. A nucleic acid sequence encoding the first fusion protein of claim 1 , wherein said nucleic acid sequence comprises the sequence set forth in SEQ ID NO: 1.
10. The nucleic acid sequence of claim 9 , wherein the nucleic acid sequence further comprises a promoter, translation initiation signal, and leader sequence operably linked to the sequence encoding the fusion protein.
11. The soluble fusion protein complex of claim 1 , wherein a nucleic acid sequence encoding the second fusion protein comprises the sequence set forth in SEQ ID NO: 3.
12. The soluble fusion protein complex claim 11 , wherein the nucleic acid sequence further comprises a promoter, translation initiation signal, and leader sequence operably linked to the sequence encoding the fusion protein.
13. The soluble fusion protein complex of claim 1 , wherein the antigen-specific binding domain recognizes disease antigens.
14. The soluble fusion protein complex of claim 13 , wherein the disease antigens are associated with neoplasia, infectious disease or autoimmune disease.
15. The soluble fusion protein complex of claim 1 , wherein the second binding domain comprises a tumor cell antigen-specific binding domain.