Bispecific signaling agents and uses thereof
The present invention relates, in part, to bispecific chimeric proteins that find use in various immunotherapies based on various properties, including, for example, a dual immune cell recruitment and immune signal delivery function.
1. A chimeric protein comprising:
(a) a modified signaling agent, wherein the modified signaling agent consists of a single mutant human interferon alpha 2 (IFNα2), the single mutant human IFNα2 having at least 95% identity with SEQ ID NO: 336 and having a mutation at position R149;
(b) a first targeting moiety comprising a first recombinant heavy chain-only (VHH) antibody or single-chain variable fragment (scFv), wherein the first targeting moiety specifically binds to a first antigen or receptor of interest; and
(c) a second targeting moiety comprising a second recombinant heavy chain-only (VHH) antibody or single-chain variable fragment (scFv), wherein the second targeting moiety specifically binds to a second antigen or receptor of interest;
wherein the mutant human IFNα2 has a reduced affinity or activity for its receptor relative to the wild type human IFNα2; and
wherein the reduced affinity or activity of the mutant human IFNα2 for its receptor is restorable by one or more of the targeting moieties,
wherein the first targeting moiety is attached to the second targeting moiety and the second targeting moiety is attached to the modified signaling agent.
2. The chimeric protein of claim 1 , wherein the human IFNα2 further comprises one or more mutations at positions 144-154.
3. The chimeric protein of claim 2 , wherein the one or more mutations is at positions M148 or L153.
4. The chimeric protein of claim 3 , wherein the mutation is M148A.
5. The chimeric protein of claim 3 , wherein the mutation is L153A.
6. The chimeric protein of claim 1 , wherein the mutation is R149A.
7. The chimeric protein of claim 1 , wherein the first or the second antigen or receptor of interest is C-type lectin domain family 9 member A (Clec9A), Programmed Death-Ligand 1 (PD-L1), Cluster of Differentiation 3 (CD3), Cluster of Differentiation 8 (CD8), Cluster of Differentiation 20 (CD20), Signal Regulatory Protein Alpha (Sirp1α), or DNAX Accessory Molecule (DNAM).
8. The chimeric protein of claim 1 , wherein the first antigen or receptor of interest is PD-L1 and the second antigen or receptor of interest is CD20, Clec9A, CD8, Sirp1α, or DNAM.
9. The chimeric protein of claim 1 , wherein the first antigen or receptor of interest is Clec9A and the second antigen or receptor of interest is CD20.
10. The chimeric protein of claim 1 , wherein the first antigen or receptor of interest is PD-L1 and the second antigen or receptor of interest is Clec9A.
11. The chimeric protein of claim 1 , wherein:
(a) the first targeting moiety functionally modulates the activity of the first antigen or receptor of interest;
(b) the second targeting moiety functionally modulates the activity of the second antigen or receptor of interest;
(c) the first targeting moiety does not functionally modulate the activity of the first antigen or receptor of interest; or
(d) the second targeting moiety does not functionally modulate the activity of the second antigen or receptor of interest.
12. The chimeric protein of claim 1 , wherein the first targeting moiety, the second targeting moiety, or both comprise a VHH, or a humanized VHH.
13. The chimeric protein of claim 1 , wherein the first targeting moiety, the second targeting moiety, or both comprise a scFv.
14. A chimeric protein comprising:
(a) a modified signaling agent, wherein the modified signaling agent consists of a single mutant human interferon alpha 2 (IFNα2), the single mutant human IFNα2 having at least 95% identity with SEQ ID NO: 336 and having a mutation at position R149;
(b) a first targeting moiety comprising a first recombinant heavy chain-only (VHH) antibody or single-chain variable fragment (scFv), wherein the first targeting moiety specifically binds to a first antigen or receptor of interest; and
(c) a second targeting moiety comprising a second recombinant heavy chain-only (VHH) antibody or single-chain variable fragment (scFv), wherein the second targeting moiety specifically binds to a second antigen or receptor of interest;
wherein the mutant human IFNα2 has a reduced affinity or activity for its receptor relative to the wild type human IFNα2; and
wherein the reduced affinity or activity of the mutant human IFNα2 for its receptor is restorable by one or more of the targeting moieties,
wherein both the first and the second targeting moieties are attached to the modified signaling agent.
15. The chimeric protein of claim 14 , wherein the human IFNα2 further comprises one or more mutations at positions 144-154.
16. The chimeric protein of claim 15 , wherein the one or more mutations is at positions M148 or L153.
17. The chimeric protein of claim 16 , wherein the mutation is M148A.
18. The chimeric protein of claim 16 , wherein the mutation is L153A.
19. The chimeric protein of claim 14 , wherein the mutation is R149A.
20. The chimeric protein of claim 14 , wherein the first or the second antigen or receptor of interest is C-type lectin domain family 9 member A (Clec9A), Programmed Death-Ligand 1 (PD-L1), Cluster of Differentiation 3 (CD3), Cluster of Differentiation 8 (CD8), Cluster of Differentiation 20 (CD20), Signal Regulatory Protein Alpha (Sirp1α), or DNAX Accessory Molecule (DNAM).
21. The chimeric protein of claim 14 , wherein the first antigen or receptor of interest is PD-L1 and the second antigen or receptor of interest is CD20, Clec9A, CD8, Sirp1α, or DNAM.
22. The chimeric protein of claim 14 , wherein the first antigen or receptor of interest is Clec9A and the second antigen or receptor of interest is CD20.
23. The chimeric protein of claim 14 , wherein the first antigen or receptor of interest is PD-L1 and the second antigen or receptor of interest is Clec9A.
24. The chimeric protein of claim 14 , wherein the first antigen or receptor of interest is CD20 and the second antigen or receptor of interest is CD3.
25. The chimeric protein of claim 14 , wherein:
(a) the first targeting moiety functionally modulates the activity of the first antigen or receptor of interest;
(b) the second targeting moiety functionally modulates the activity of the second antigen or receptor of interest;
(c) the first targeting moiety does not functionally modulate the activity of the first antigen or receptor of interest; or
(d) the second targeting moiety does not functionally modulate the activity of the second antigen or receptor of interest.
26. The chimeric protein of claim 14 , wherein the first targeting moiety, the second targeting moiety, or both comprise a VHH, or a humanized VHH.
27. The chimeric protein of claim 14 , wherein the first targeting moiety, the second targeting moiety, or both comprise a scFv.
28. A chimeric protein comprising:
(a) a modified signaling agent, wherein the modified signaling agent consists of a single mutant human interferon alpha 2 (IFNα2), the single mutant human IFNα2 having at least 95% identity with SEQ ID NO: 336 and having a mutation at position R149;
(b) a first targeting moiety comprising a first recombinant heavy chain-only (VHH) antibody or single-chain variable fragment (scFv), wherein the first targeting moiety specifically binds to PD-L1; and
(c) a second targeting moiety comprising a second recombinant heavy chain-only (VHH) antibody or single-chain variable fragment (scFv), wherein the second targeting moiety specifically binds to Clec9A;
wherein the mutant human IFNα2 has a reduced affinity or activity for its receptor relative to the wild type human IFNα2; and
wherein the reduced affinity or activity of the mutant human IFNα2 for its receptor is restorable by one or more of the targeting moieties.
29. The chimeric protein of claim 28 , wherein the human IFNα2 further comprises one or more mutations at positions 144-154.
30. The chimeric protein of claim 29 , wherein the one or more mutations is at positions M148 or L153.
31. The chimeric protein of claim 30 , wherein the mutation is M148A.
32. The chimeric protein of claim 30 , wherein the mutation is L153A.
33. The chimeric protein of claim 28 , wherein the mutation is R149A.
34. The chimeric protein of claim 28 , wherein:
(a) the first targeting moiety functionally modulates the activity of PD-L1;
(b) the second targeting moiety functionally modulates the activity of Clec9A;
(c) the first targeting moiety does not functionally modulate the activity of PD-L1; or
(d) the second targeting moiety does not functionally modulate the activity of Clec9A.
35. The chimeric protein of claim 28 , wherein the first targeting moiety, the second targeting moiety, or both comprise a VHH or a humanized VHH.
36. The chimeric protein of claim 28 , wherein the first targeting moiety, the second targeting moiety, or both comprise a scFv.
37. A chimeric protein comprising:
(a) a modified signaling agent, wherein the modified signaling agent consists of a single mutant human interferon alpha 2 (IFNα2), the single mutant human IFNα2 having at least 95% identity with SEQ ID NO: 336 and having a mutation at position R149;
(b) a first targeting moiety comprising a first recombinant heavy chain-only (VHH) antibody or single-chain variable fragment (scFv), wherein the first targeting moiety specifically binds to CD20; and
(c) a second targeting moiety comprising a second recombinant heavy chain-only (VHH) antibody or single-chain variable fragment (scFv), wherein the second targeting moiety specifically binds to CD3;
wherein the mutant human IFNα2 has a reduced affinity or activity for its receptor relative to the wild type human IFNα2; and
wherein the reduced affinity or activity of the mutant human IFNα2 for its receptor is restorable by one or more of the targeting moieties.
38. The chimeric protein of claim 37 , wherein the human IFNa2 further comprises one or more mutations at positions 144-154.
39. The chimeric protein of claim 38 , wherein the one or more mutations is at positions M148 or L153.
40. The chimeric protein of claim 39 , wherein the mutation is M148A.
41. The chimeric protein of claim 39 , wherein the mutation is L153A.
42. The chimeric protein of claim 37 , wherein the mutation is R149A.
43. The chimeric protein of claim 37 , wherein:
(a) the first targeting moiety functionally modulates the activity of CD20;
(b) the second targeting moiety functionally modulates the activity of CD3;
(c) the first targeting moiety does not functionally modulate the activity of CD20; or
(d) the second targeting moiety does not functionally modulate the activity of CD3.
44. The chimeric protein of claim 37 , wherein the first targeting moiety, the second targeting moiety, or both comprise a VHH or a humanized VHH.
45. The chimeric protein of claim 37 , wherein the first targeting moiety, the second targeting moiety, or both comprise a scFv.