MULTIPARTITE SIGNALING PROTEINS AND USES THEREOF

• US 20150266973A1
• Filed: 01/28/2015
• Published: 09/24/2015
• Est. Priority Date: 07/29/2013
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: bluebird bio, Inc.
• Inventors: JARJOUR, Jordan, ASTRAKHAN, Alexander, CERTO, Michael
• Granted Patent: US 10,428,142 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 39/0005 Vertebrate antigens from snakes A61K39/38
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4631 Chimeric Antigen Receptors [CAR]
• CPC Class Codes: A61K 39/464412 CD19 or B4
• CPC Class Codes: A61K 39/464419 Receptors for interleukins [IL]
• CPC Class Codes: A61K 39/464424 CD20
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 14/70503 Immunoglobulin superfamily
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70514 CD4
• CPC Class Codes: C07K 14/70517 CD8
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 14/70535 Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
• CPC Class Codes: C07K 14/7056 : Lectin superfamily, e.g. CD23, CD72
• CPC Class Codes: C07K 14/70578 : NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 14/70596 : Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 16/2803 : against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2896 : against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/40 : against enzymes
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/70 : containing domain for protein-protein interaction
• CPC Class Codes: C12N 15/85 : for animal cells
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 9/003 : Dihydrofolate reductase [DHFR] (1.5.1.3)
• CPC Class Codes: C12N 9/12 : transferring phosphorus containing groups, e.g. kinases (2.7)
• CPC Class Codes: C12N 9/16 : acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/90 : Isomerases (5.)
• CPC Class Codes: C12Y 207/11001 : Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
• CPC Class Codes: C12Y 502/01008 : Peptidylprolyl isomerase (5.2.1.8), i.e. cyclophilin

Abstract

The present disclosure relates to compositions and methods for using cells having chemically-induced fusion protein complexes to spatially and temporally control immune cell signal initiation and downstream responses for treating disease.

Claims

1. A non-natural cell, comprising:

(a) a first nucleic acid molecule encoding a first fusion protein comprising a first multimerization domain, a first hydrophobic domain, and an actuator domain, wherein the first multimerization domain localizes extracellularly when the first fusion protein is expressed; and

(b) a second nucleic acid molecule encoding a second fusion protein comprising a binding domain and a second multimerization domain, and a second hydrophobic domain;

wherein a first bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second fusion proteins.

2. The non-natural cell according to claim 1, wherein:

a) the first and second multimerization domains are the same or different;

b) the first and second multimerization domains associate with a bridging factor selected from rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or any combination thereof;

c) the first and second multimerization domains are a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof;

d) the first multimerization domain comprises a first FKBP polypeptide or variant thereof, and the second multimerization domain comprises a first FRB polypeptide or variant thereof;

e) the first multimerization domain comprises a first FRB polypeptide or variant thereof, and the second multimerization domain comprises a first FKBP polypeptide or variant thereof;

or

f) the first fusion protein has at least one multimerization domain of FKBP, DHFR or GyrB.

7. The non-natural cell according to claim 1, wherein the bridging factor is sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus.

9. The non-natural cell according to claim 1, wherein the binding domain of the polypeptide complex specifically binds to a target located on a target cell surface.

10. The non-natural cell according to claim 1, wherein the first hydrophobic domain is a transmembrane domain selected from the group of a CD4, CD8, AMN, or CD28 transmembrane domain.

11. The non-natural cell according to claim 1, wherein the second hydrophobic domain comprises a CD154 transmembrane domain or a CD71 transmembrane domain.

13. The non-natural cell according to claim 1, wherein the first hydrophobic domain and the second hydrophobic domain do not increase cytotoxic activity of the non-natural cell in the absence of the bridging factor.

14. The non-natural cell according to claim 1, wherein the first hydrophobic domain and the second hydrophobic domain increase cytotoxic activity of the non-natural cell in the absence of the bridging factor, wherein the increase in cytotoxic activity is less than the cytotoxic activity in the presence of the bridging factor.

15. The non-natural cell according to claim 1, wherein:

a) the actuator domain comprises a lymphocyte receptor signaling domain;

b) the actuator domain comprises one or a plurality of immunoreceptor tyrosine-based activation motifs (ITAMs);

c) the actuator domain comprises CD3ε

, CD3δ

, CD3ζ

, pTα

, TCRα

, TCRβ

, FcRα

, FcRβ

, FcRγ

, NKG2D, CD22, CD79A, or CD79B, or any combination thereof;

or

d) the actuator domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein.

18. The non-natural cell according to claim 1, wherein the first nucleic acid molecule encodes the first fusion protein further comprising a different actuator domain, a costimulatory domain, an adhesion factor, or any combination thereof.

19. The non-natural cell according to claim 18, wherein the costimulatory domain is selected from CD27, CD28, CD30, CD40, LAT, Zap70, ICOS, DAP10, 4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM, OX40, or any combination thereof.

21. The non-natural cell according to claim 15, wherein the cytoplasmic signaling protein of part d) is a lymphocyte receptor or signaling domain thereof, a protein comprising a plurality of immunoreceptor tyrosine-based activation motifs (ITAMs), a costimulatory domain, an adhesion factor, or any combination thereof.

22. The non-natural cell according to claim 21, wherein:

a) the lymphocyte receptor or signaling domain thereof is CD3ε

, CD3δ

, CD3ζ

, pTα

, TCRα

, TCRβ

, FcRα

, FcRβ

, FcRγ

, NKG2D, CD22, CD79A, or CD79B, or any combination thereof;

or

b) the costimulatory domain is selected from CD27, CD28, CD30, CD40, LAT, Zap70, ICOS, DAP10, 4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM, OX40, or any combination thereof.

25. The non-natural cell according to claim 1, wherein the second nucleic acid molecule further encodes a secretion signal such that the second fusion protein is secreted from the non-natural cell when expressed, and optionally further encodes an anchor domain.

26. The non-natural cell according to claim 1, wherein the binding domain of the second fusion protein is a single chain antibody variable region, a receptor ectodomain, or a ligand.

27. The non-natural cell according to claim 26, wherein the single chain antibody variable region is a domain antibody, sFv, scFv, F(ab′

)₂, or Fab.

28. The non-natural cell according to claim 1, wherein:

a) the binding domain of the second fusion protein is amino terminal to the multimerization domain;

or

b) the binding domain of the second fusion protein is carboxy terminal to the multimerization domain.

30. The non-natural cell according to claim 1, wherein the second nucleic acid molecule encoding the second fusion protein further comprises a sequence encoding a linker disposed between the binding domain and the second multimerization domain.

33. The non-natural cell according to claim 1, wherein the binding domain is specific for a target that is an antigen associated with a cancer, an inflammatory disease, an autoimmune disease, or a graft versus host disease.

34. The non-natural cell according to claim 33, wherein:

a) the cancer is a solid malignancy or a hematologic malignancy;

or

b) the cancer is a hematologic malignancy associated antigen target is CD19, CD20, CD22, CD33, or CD37.

36. The non-natural cell according to claim 1, wherein the binding domain specifically binds to a target selected from α

-folate receptor, α

_vβ

₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholine receptor, Fzd7, GD2, GD3, Glypican-3 (GPC3), h5T4, IL-11Rα

, IL13R-α

2, KDR, κ

light chain, λ

light chain, LeY, L1CAM, MAGE-A1, mesothelin, MHC presented peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notch1, Notch2/3, NY-ESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEMs, TERT, VEGFR2, and ROR1.

37. The non-natural cell according to claim 1, wherein the encoded first fusion protein comprises a first multimerization domain of FRB T2098L, a transmembrane domain, a costimulatory domain of 4-1BB, and actuator domain of CD3ζ

;

wherein the second encoded fusion protein comprises a binding domain of an scFv specific for CD19 and a second multimerization domain of FKBP12 and a CD154 or a CD71 transmembrane domain; and

wherein the first bridging factor that promotes the formation of a polypeptide complex on the non-natural cell surface is rapalog AP21967.

38. The non-natural cell according to claim 1, wherein the encoded first fusion protein comprises a first multimerization domain of FRB, a transmembrane domain, a costimulatory domain of 4-1BB, and actuator domain of CD3ζ

;

wherein the second encoded fusion protein comprises a binding domain of an scFv specific for CD19 and a second multimerization domain of FKBP12 and a CD154 or a CD71 transmembrane domain; and

wherein the first bridging factor that promotes the formation of a polypeptide complex on the non-natural cell surface is Rapamycin, temsirolimus or everolimus.

39. A method for treating a hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease, comprising:

(a) administering a non-natural cell according to claim 1; and

(b) administering a bridging factor, wherein the bridging factor promotes the formation of a polypeptide complex on the recombinant cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second fusion proteins;

wherein the binding domain of the polypeptide complex specifically binds a cell surface target on a hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease cell to promote an immunomodulatory response and thereby treats the hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease.

3-6. -6. (canceled)

8. (canceled)

12. (canceled)

16-17. -17. (canceled)

20. (canceled)

23-24. -24. (canceled)

29. (canceled)

31-32. -32. (canceled)

35. (canceled)

40-44. -44. (canceled)

45. A fusion polypeptide heterocomplex, comprising:

(a) a first fusion protein comprising a first multimerization domain, a first hydrophobic domain, and an actuator domain;

(b) a second fusion protein comprising an extracellular binding domain, a second multimerization domain, and a second hydrophobic domain; and

(c) a bridging factor;

wherein the first fusion protein, second fusion protein, and bridging factor associate to form a polypeptide heterocomplex with the bridging factor associated with and disposed between the multimerization domains of the first and second fusion proteins.

46-70. -70. (canceled)