Multipartite signaling proteins and uses thereof

• US 10,196,444 B2
• Filed: 07/23/2014
• Issued: 02/05/2019
• 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
• Primary Examiner(s): Chen, Shin Lin
• Application Number:
  US14/908,734
• Publication Number:
  US 20160311901A1
• Time in Patent Office: 1,658 Days
• Field of Search: 424 9321, 530350, 536 234
• 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. An isolated non-natural cell, comprising:

(a) a first nucleic acid molecule encoding a first fusion protein comprising a first multimerization domain, a transmembrane 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 secretion signal, a binding domain that comprises a single chain antibody, a receptor ectodomain, or a ligand, and a second multimerization domain, wherein the second fusion protein localizes extracellularly when expressed;

wherein a 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 isolated non-natural cell of claim 1, wherein the first and second multimerization domains are the same or different.

3. The isolated non-natural cell of claim 1, wherein the multimerization domains of the first and second fusion proteins associate with a bridging factor selected from the group consisting of:

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, FK506/cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim (Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof.

4. The isolated non-natural cell of claim 1, wherein the first and second multimerization domains are a pair selected from the group consisting of:

FKBP and FKBP12-rapamycin binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin, and PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).

5. The isolated non-natural cell of claim 1, wherein 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.

6. The isolated non-natural cell of claim 1, wherein 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.

7. The isolated non-natural cell of claim 5 or claim 6, wherein the bridging factor is AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus.

8. The isolated non-natural cell of claim 1, wherein the transmembrane domain is a CD4, CD8 or CD28 transmembrane domain.

9. The isolated non-natural cell of claim 1, wherein the actuator domain comprises a lymphocyte receptor signaling domain.

10. The isolated non-natural cell of claim 1, wherein the actuator domain comprises one or a plurality of immunoreceptor tyrosine-based activation motifs (ITAMs).

11. The isolated non-natural cell of claim 1, wherein the actuator domain comprises a lymphocyte receptor signaling domain of a polypeptide selected from the group consisting of:

CD3ε

, CD36δ

, CD3ζ

, pre-T cell receptor α

(pTα

), T cell receptor 60 (TCRα

), T cell receptor β

(TCRβ

), Fc receptor α

(FcRα

), Fc receptor β

(FcRβ

), Fc receptor γ

(FcRγ

), natural-killer group 2, member D (NKG2D), CD79A, CD79B, and any combination thereof.

12. The isolated non-natural cell of 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.

13. The isolated non-natural cell of claim 12, wherein the costimulatory domain is from a polypeptide selected from the group consisting of:

CD27, CD28, CD30, CD40, linker for activation of T-cells (LAT), zeta-chain-associated protein kinase 70 (Zap70), inducible T-cell costimulator (ICOS), DNAX-activation protein 10 (DAP10), 4-1BB, caspase recruitment domain family member 11 (CARD11), herpesvirus entry mediator (HVEM), lymphocyte activating gene 3 (LAG3), signaling lymphocytic activation molecule family member 1 (SLAMF1), lymphocyte-specific protein tyrosine kinase (Lck), fyn oncogene related to src, fgr, yes (Fyn), SH2 domain-containing leukocyte protein of 76 KDa (Slp76), OX40, and any combination thereof.

14. The isolated non-natural cell of claim 1, wherein the actuator domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein.

15. The isolated non-natural cell of claim 14, wherein the cytoplasmic signaling protein 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.

16. The isolated non-natural cell of claim 15, wherein the lymphocyte receptor or signaling domain thereof is selected from the group consisting of:

CD3ε

, CD3δ

, CD3ζ

, pre-T cell receptor α

(pTα

), T cell receptor α

(TCRα

), T cell receptor β

(TCRβ

), Fc receptor α

(FcRα

), Fc receptor β

(FcRβ

), Fc receptor γ

(FcRγ

), natural-killer group 2, member D (NKG2D), CD22, CD79A, CD79B, and any combination thereof.

17. The isolated non-natural cell of claim 15, wherein the costimulatory domain is selected from the group consisting of:

CD27, CD28, CD30, CD40, linker for activation of T-cells (LAT), zeta-chain-associated protein kinase 70 (Zap70), inducible T-cell costimulator (ICOS), DNAX-activation protein 10 (DAP10), 4-1BB, caspase recruitment domain family member 11 (CARD11), herpesvirus entry mediator (HVEM), lymphocyte activating gene 3 (LAG3), signaling lymphocytic activation molecule family member 1 (SLAMF1), lymphocyte-specific protein tyrosine kinase (Lck), fyn oncogene related to src, fgr, yes (Fyn), SH2 domain-containing leukocyte protein of 76 KDa (Slp76), OX40, and any combination thereof.

18. The isolated non-natural cell of claim 1, further overexpressing a costimulatory factor, an immunomodulatory factor, an agonist for a costimulatory factor, an agonist for an immunomodulatory factor, or any combination thereof.

19. The isolated non-natural cell of claim 1, wherein the second nucleic acid molecule further encodes an anchor domain.

20. The isolated non-natural cell of claim 1, wherein the binding domain comprises an antibody, scFv, or Fab.

21. The isolated non-natural cell of claim 1, wherein the binding domain of the second fusion protein is amino terminal to the multimerization domain.

22. The isolated non-natural cell of claim 1, wherein the binding domain of the second fusion protein is carboxy terminal to the multimerization domain.

23. The isolated non-natural cell of 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.

24. The isolated non-natural cell of claim 1, wherein the cell further comprises a third nucleic acid molecule encoding a third fusion protein comprising a binding domain and a third multimerization domain, wherein the third fusion protein localizes extracellularly when expressed.

25. The isolated non-natural cell of claim 1, wherein the binding domain specifically binds to a target selected from the group consisting of:

α

-folate receptor, α

_vβ

₆ integrin, B cell maturation antigen (BCMA), Bb7 homolog 3 (B7-H3), B7 homolog 6 (B7-H6), Carbonic anhydrase IX (CAIX), CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, carcinoembryonic antigen (CEA), delta like canonical Notch ligand 4 (DLL4), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor receptor (EGFR), EGFR family including ErbB2 (HER2), EGFR variant III (EGFRvIII), epithelial cellular adhesion molecule (EPCAM), ephrin type-A receptor 2 (EphA2), fibroblast activation protein alpha (FAP), fetal acetylcholine receptor, frizzled class receptor 7 (Fzd7), ganglioside GD2 (GD2), ganglioside GD3 (GD3), Glypican-3 (GPC3), human oncofetal antigen 5T4 (h5T4), interleukin 11 receptor alpha (IL-11Rα

), interleukin 13 receptor alpha 2 (IL13R-α

2), κ

light chain, λ

light chain, Lewis Y antigen (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen gene family member A1 (MAGE-A1), mesothelin, major histocompatibility complex (MHC) presented peptides, mucin 1, cell surface associated (MUC1), mucin 16, cell surface associated (MUC16), neural cell adhesion molecule (NCAM), NKG2D ligands, Notch1, Notch2/3, New York esophageal squamous cell carcinoma 1 (NY-ESO-1), preferentially expressed antigen of melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), Survivin, tumor associate glycoprotein 72 (TAG-72), telomerase reverse transcriptase (TERT), vascular endothelial growth factor receptor 2 (VEGFR2), and receptor tyrosine kinase-like orphan receptor 1 (ROR1).

26. The isolated non-natural cell of 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 or BCMA and a second multimerization domain of FKBP12; and

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

27. The isolated non-natural cell of claim 26, wherein the first fusion protein has an amino acid sequence as set forth in SEQ ID NO.:

15 and the second fusion protein has an amino acid sequence as set forth in SEQ ID NO.;

1.