T cells surface-loaded with immunostimulatory fusion molecules and uses thereof
Immunostimulatory fusion molecules that include an immune cell targeting moiety and a cytokine molecule, pharmaceutical and formulations thereof, and methods of using and making the same, are disclosed.
1. A composition comprising:
(i) an immunostimulatory fusion molecule comprising:
(a) an immunostimulatory cytokine molecule selected from the group consisting of IL-12, IL-15, IL-2, IL-6, IL-7, IL-18, IL-21, IL-23, and IL-27; and
(b) a targeting moiety comprising an antigen-binding fragment of an antibody having an affinity to a cell surface antigen expressed by or displayed on the surface of a T cell, wherein the immunostimulatory cytokine molecule is operably linked to the antigen-binding fragment; and
(ii) a population of T cells expressing or displaying the cell surface antigen,
wherein the immunostimulatory fusion molecule is bound to the surface of a T cell in the population of T cells through interaction with the cell surface antigen.
2. The composition of claim 1 , wherein the T cells are selected from the group consisting of effector T cells, CD4 + T cells, CD8 + T cells, and cytotoxic T lymphocytes (CTLs).
3. The composition of claim 2 , wherein the cell surface antigen is CD45.
4. The composition of claim 1 , wherein the targeting moiety comprises a Fab fragment, F(ab′)2, Fv, or a single chain Fv comprising:
(i) a heavy chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 94 and a light chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 95;
(ii) a heavy chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 59 and a light chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 60;
(iii) a heavy chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 23 and a light chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 24;
(iv) a heavy chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 96 and a light chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 97;
(v) a heavy chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 57 and a light chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 58; or
(vi) a heavy chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 61 and a light chain variable domain amino acid sequence at least 95% identical to SEQ ID NO: 62.
5. The composition of claim 1 , wherein the immunostimulatory cytokine molecule comprises an IL-12, a single chain IL-12, or a subunit of IL-12 selected from the group consisting of IL-12A and IL-12B.
6. The composition of claim 1 , further comprising a single-chain Fv having an affinity to an antigen on the surface of the T cell.
7. The composition of claim 6 , wherein the single-chain Fv has an affinity to the same antigen as the antigen-binding fragment of the targeting moiety.
8. The composition of claim 6 , wherein the single-chain Fv has an affinity to a different antigen than the antigen-binding fragment of the targeting moiety.
9. The composition of claim 1 , wherein the antigen-binding fragment is a Fab fragment.
10. The composition of claim 9 , wherein the Fab fragment comprises a light chain and a heavy chain fragment, and wherein the immunostimulatory cytokine molecule is operably linked to the Fab fragment at a C-terminus of the light chain.
11. The composition of claim 1 , wherein the immunostimulatory cytokine molecule is operably linked to the antigen-binding fragment by a linker.
12. The composition of claim 11 , wherein the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.
13. The composition of claim 1 , wherein the immunostimulatory fusion molecule has an affinity to a CD45 receptor and comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 82 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 79.
14. The composition of claim 5 , wherein the immunostimulatory cytokine molecule comprises a single-chain IL-12 molecule having an amino acid sequence at least 95% identical to SEQ ID NO: 50.
15. The composition of claim 14 , wherein the immunostimulatory cytokine molecule comprises a single-chain IL-12 molecule having an IL-12A subunit linked to an IL-12B subunit through a linker having an amino acid sequence at least 95% identical to SEQ ID NO: 70.
16. The composition of claim 12 , wherein the linker comprises a peptide linker having the amino acid sequence of SEQ ID NO: 36.
17. The composition of claim 1 , wherein the T cells are healthy and/or non-malignant.
18. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier, excipient, or stabilizer.
19. The composition of claim 1 , further comprising a nanoparticle.
20. The composition of claim 1 , further comprising a liposome.
21. The composition of claim 19 , wherein the nanoparticle comprises a protein nanogel, a nucleotide nanogel, a polymer nanoparticle, or a solid nanoparticle.
22. The composition of claim 21 , wherein the nanoparticle comprises a protein nanogel.
23. The composition of claim 22 , wherein the nanoparticle comprises at least one polymer, cationic polymer, or cationic block co-polymer on the nanoparticle surface.
24. The composition of claim 23 , wherein the nanoparticle comprises a nanogel that is cross linked by a reversible linker that is sensitive to redox state, pH, or one or more proteases.
25. The composition of claim 1 , further comprising a biodegradable polymer.
26. The composition of claim 1 , wherein the cell surface antigen is CD45.
27. The composition of claim 1 , wherein the population of T cells have specificity against multiple tumor-associated antigens.
28. The composition of claim 27 , wherein the tumor-associated antigens comprise one or more of MART-1, MAGE-A4, NY-ESO-1, SSX2, and Survivin.
29. The composition of claim 1 , wherein the cell surface antigen is selected from the group consisting of CD4, CD8, CD3, CD11a, CD11b, CD11c, CD18, CD25, CD127, CD19, CD20, CD22, HLA-DR, CD197, CD38, CD27, CD196, CXCR3, CXCR4, CXCR5, CD84, CD229, CCR1, CCR5, CCR4, CCR6, CCR8, CCR10, CD16, CD56, CD137, OX40, and GITR.
30. The composition of claim 1 , wherein the immunostimulatory fusion molecule lacks an Fc domain or comprises an Fc domain in which an antibody-dependent cellular cytotoxicity is reduced by at least 60% relative to a wild-type human IgG1 Fc domain having the amino acid sequence of SEQ ID NO: 67.