Tagged chimeric effector molecules and receptors thereof

• US 10,494,434 B2
• Filed: 12/22/2014
• Issued: 12/03/2019
• Est. Priority Date: 12/20/2013
• Status: Active Grant

• Current Assignee: Fred Hutchinson Cancer Research Center (Fred Hutchinson Cancer Research Center|Argus Genetics|Mars)
• Original Assignee: Fred Hutchinson Cancer Research Center (Fred Hutchinson Cancer Research Center|Argus Genetics|Mars)
• Inventors: Riddell, Stanley R., Liu, Lingfeng
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US15/106,657
• Publication Number:
  US 20170267756A1
• Time in Patent Office: 1,807 Days
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/507 Comprising a combination of two or more separate antibodies
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61K 38/1774 Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
• CPC Class Codes: A61K 38/2013 IL-2
• CPC Class Codes: A61K 38/2086 IL-13 to IL-16
• 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: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/7051 : T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70521 : CD28, CD152
• 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 16/2803 : against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2818 : against CD28 or CD152
• CPC Class Codes: C07K 16/2878 : against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/64 : comprising a combination of variable region and constant region components
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/20 : containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C07K 2319/22 : containing a Strep-tag
• CPC Class Codes: C07K 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C07K 2319/95 : containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
• CPC Class Codes: C12N 2501/2302 : Interleukin-2 (IL-2)
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0636 : T lymphocytes
• CPC Class Codes: C12N 5/0637 : Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
• CPC Class Codes: C12N 5/0638 : Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
• CPC Class Codes: G01N 33/56972 : White blood cells

Abstract

The present disclosure relates to tagged chimeric effector molecules and receptor molecules thereof for genetically engineering a host cell, wherein the recombinant host cell can be identified, isolated, sorted, induced to proliferate, tracked or eliminated using the tag. An exemplary receptor molecule is a chimeric antigen receptor (CAR) having an extracellular domain comprising a binding domain for a target, a hinge region, and a tag cassette, a hydrophobic portion as a transmembrane domain and an intracellular part with an effector domain. An exemplary target is CD19. An exemplary tag is a Strep Tag®. T cells recombinantly modified for expression of such molecules may be used in adoptive immunotherapy.

Claims

1. A single chain fusion protein, comprising an extracellular component and an intracellular component connected by a hydrophobic portion,

wherein the extracellular component comprises a plurality of tag cassettes and a connector region comprising a hinge,

wherein the intracellular component comprises an effector domain, and

wherein at least one of the plurality of tag cassettes comprises a peptide having a length of up to 20 amino acids that includes the amino acid sequence of SEQ ID NO.:

1 or 2.

2. The single chain fusion protein according to claim 1, wherein the connector region further comprises a linker module.

3. The single chain fusion protein according to claim 2, wherein the linker module is a (Gly_xSer_y)_n, wherein n is an integer from 1 to 10, and x and y are independently an integer from 0 to 10 provided that x and y are not both 0;

or wherein the linker module comprises a CH2CH3 or a CH3.

4. The single chain fusion protein according to claim 1, wherein:

one or more of the plurality of tag cassettes are located within the connector region;

or

one or more of the plurality of tag cassettes are located within the connector region, wherein each tag cassette is connected to one or two linker modules comprising a (Gly_xSer_y)_n, wherein n is an integer from 1 to 10, and x and y are independently an integer from 0 to 10 provided that x and y are not both 0;

or

one or more of the plurality of tag cassettes are located within the connector region, wherein each tag cassette is connected to one or two linker modules having the amino acid sequence of Gly-Gly-Gly-Gly-Ser (SEQ ID NO;

10), (Gly-Gly-Gly-Gly-Ser)₂ (SEQ ID NO;

11), (Gly-Gly-Gly-Ser)₂-Gly-Gly-Ser (SEQ ID NO;

12), or any combination thereof;

or

one to five of the plurality of tag cassettes are located within the connector region;

or

one to five of the plurality of tag cassettes are located within the connector region, wherein each tag cassette is connected to one or two linker modules comprising a (Gly_nSer_y)_n, wherein n is an integer from 1 to 10, and x and y are independently an integer from 0 to 10 provided that x and y are not both 0;

or

the connector region comprises from one to five tag cassettes, wherein each tag cassette is connected to one or two linker modules having the amino acid sequence of Gly-Gly-Gly-Gly-Ser (SEQ ID NO;

10), (Gly-Gly-Gly-Gly-Ser)₂ (SEQ ID NO;

11), (Gly-Gly-Gly-Ser)₂-Gly-Gly-Ser (SEQ ID NO;

12), or any combination thereof.

5. The single chain fusion protein according to claim 1, wherein the single chain fusion protein further comprises a His tag, a tag of SEQ ID NO.:

3, an Xpress tag, a tag of SEQ ID NO.;

5, a Calmodulin tag, a Polyglutamate tag, an HA tag, a Myc tag, a Nus tag, an S tag, an SBP tag, a tag of SEQ ID NO.;

9 or 32, a V5 tag, a CBP, a GST, an MBP, a GFP, a Thioredoxin tag, or any combination thereof.

6. The single chain fusion protein according to claim 5, wherein the at least one of the tag cassettes consists of the amino acid sequence of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO.:

1) or Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO.;

2).

7. The single chain fusion protein according to claim 1, wherein the hydrophobic portion is a transmembrane domain and the transmembrane domain comprises a CD4, CD8, CD28 or CD27 transmembrane domain.

8. The single chain fusion protein according to claim 1, wherein the effector domain or an effector portion thereof is, or is an effector domain of, one or more of the group consisting of a 4-1BB (CD137), a CD3ε

, a CD3δ

, a CD3ζ

, a CD25, a CD27, a CD28, a CD79A, a CD79B, a CARD11, a DAP10, a FcRα

, a FcRβ

, a FcRγ

, a Fyn, a HVEM, an ICOS, a Lck, a LAG3, a LAT, a LRP, a NKG2D, a NOTCH1, a NOTCH2, a NOTCH3, a NOTCH4, an OX40 (CD134), a ROR2, a Ryk, a SLAMF1, a Slp76, a pTα

, a TCRα

, a TCRβ

, a TRIM, a Zap70, and a PTCH2.

9. The single chain fusion protein according to claim 1, wherein the effector domain or an effector portion thereof comprises, or comprises an effector domain of, (a) a CD3ζ

and (b) one or more of 4-1BB (CD137), CD27, CD28, and OX40 (CD134).

10. The single chain fusion protein according to claim 1, wherein the fusion protein comprises, from amino-terminus to carboxy-terminus:

(a) an extracellular binding domain, a tag cassette, a connector region comprising a hinge, a hydrophobic portion, and an intracellular component comprising an effector domain;

or

(b) an extracellular binding domain, a first connector region, a tag cassette, a second connector region comprising a hinge, a hydrophobic portion, and an intracellular component comprising an effector domain;

or

(c) an extracellular binding domain, a first tag cassette, a first connector region, a second tag cassette, a second connector region comprising a hinge, a hydrophobic portion, and an intracellular component comprising an effector domain;

or

(d) an extracellular binding domain, a first tag cassette, a first connector region, a second tag cassette, a second connector region, a third tag cassette, a third connector region comprising a hinge, a hydrophobic portion, and an intracellular component comprising an effector domain;

or

(e) a tag cassette, an extracellular binding domain, a connector region comprising a hinge, a hydrophobic portion, and an intracellular component comprising an effector domain;

or

(f) an extracellular binding domain, two to five tag cassettes, a connector region comprising a hinge, a hydrophobic portion, and an intracellular component comprising an effector domain;

or

(g) an extracellular scFv or scTCR binding domain comprising a variable region linker disposed between the variable regions and containing a tag cassette, a connector region comprising a hinge, a hydrophobic portion, and an intracellular component comprising an effector domain;

or

(h) an extracellular scFv or scTCR binding domain, a tag cassette, a connector region comprising an IgG hinge, a transmembrane domain, and an intracellular component comprising an effector domain, wherein the effector domain (A) comprises, or comprises an effector domain of, each of 4 1BB and CD3ζ

, (B) comprises, or comprises an effector domain of, each of CD27 and CD3ζ

, (C) comprises, or comprises an effector domain of, each of CD28 and CD3ζ

, (D) comprises or comprises an effector domain of each of OX40 and CD3ζ

, (E) comprises, or comprises an effector domain of, each of CD28, 4-1BB and CD3ζ

, (F) comprises, or comprises an effector domain of, each of OX40, 4-1BB and CD3ζ

, or (G) comprises, or comprises an effector domain of, each of CD28, OX40 and CD3ζ

;

or

(i) an extracellular binding domain comprising a receptor ectodomain, a tag cassette, a connector region comprising a hinge, a hydrophobic portion, and an intracellular component comprising an effector domain, wherein the effector domain comprises, or comprises an effector domain of, a 4-1BB, CD27, CD28, or OX40.

11. The single chain fusion protein according to claim 1, wherein:

the extracellular component comprises from two to five tag cassettes; and

/or

the fusion protein further comprises;

(i) one or more linker modules comprising a (Gly_zSer_y)_m, wherein m is an integer from 1 to 10, and z and y are independently an integer from 0 to 10 provided that z and y are not both 0;

(ii) a (Gly_xSer)_n linker module disposed between an extracellular binding domain and one or more tag cassettes, wherein x is an integer from 2 to 4 and n is an integer from 1 to 3;

(iii) an extracellular (Gly_xSer)_n linker module disposed between one or more tag cassettes and the intracellular component comprising an effector domain, wherein x is an integer from 2 to 4 and n is an integer from 1 to 3; and

/or (iv) two extracellular (Gly_xSer)_n linker modules, wherein x is an integer from 2 to 4 and n is an integer from 1 to 3, and wherein the first linker module is amino-terminal to at least one of the plurality of tag cassettes and the second linker module is carboxy-terminal to at least one of the plurality of tag cassettes; and

/or

the extracellular component comprises two tag cassettes and the fusion protein further comprises two extracellular (Gly_xSer)_n linker modules, wherein x is an integer from 2 to 4 and n is an integer from 1 to 3, and wherein a first of the two tag cassettes is disposed between an extracellular binding domain and the first linker module, a second of the two tag cassettes is disposed between the first and second linker modules, and the second linker module is disposed between the second tag cassette and the effector domain, optionally wherein the fusion protein further comprises a third tag cassette and a third extracellular (Gly_xSer)_n linker module, wherein x is an integer from 2 to 4 and n is an integer from 1 to 3, and wherein the third tag cassette is disposed between the second linker module and the third linker module, and the third linker module is disposed between the third tag cassette and the effector domain.

12. The single chain fusion protein of claim 1, wherein the extracellular component further comprises a binding domain that specifically binds a target.

13. The single chain fusion protein of claim 1, wherein the fusion protein is non-covalently associated with a binding domain that specifically binds a target.

14. The single chain fusion protein of claim 13, wherein:

the non-covalently associated binding domain is associated with one or more of the plurality of tag cassettes of the fusion protein; and

/or

the non-covalently associated binding domain is multi-specific, optionally bi-specific, with at least a first binding end and a second binding end, wherein (i) the first binding end is specific for one or more of the plurality of tag cassettes of the fusion protein and the second binding end is specific for the target, which is a target other than the plurality of tag cassettes; and

/or (ii) the first and the second binding ends are specific for one or more of the plurality of tag cassettes; and

/or

the non-covalently associated binding domain is multispecific, with a first end that binds to one or more of the plurality of tag cassettes and one or more additional ends that are specific for one or more targets other than the plurality of tag cassettes.

15. The single chain fusion protein of claim 12, wherein the binding domain is a scFv, a scTCR, a receptor ectodomain, or a ligand of a receptor.

16. The single chain fusion protein of claim 12, wherein the target comprises CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis A, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, HLA, tumor or pathogen associated peptide bound to HLA, hTERT peptide bound to HLA, tyrosinase peptide bound to HLA, WT-1 peptide bound to HLA, LTβ

R, LIFRβ

, LRP5, MUC1, OSMRβ

, TCRα

, TCRβ

, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCH1, WT-1, Robo1, α

-fetoprotein (AFP), Frizzled, OX40, or CD79b.

17. The single chain fusion protein of claim 12, wherein the binding domain comprises:

(a) one or more of the tag cassettes;

or

(b) a scFv or scTCR comprising a variable region linker, wherein the variable region comprises one or more of the tag cassettes;

or

(c) one or more of the tag cassettes, wherein the one or more tag cassettes is located amino-terminal to the binding domain, carboxy-terminal to the binding domain, or both.

24. A method for activating a cell and/or promoting cell proliferation, comprising contacting a cell expressing the single Chain fusion protein of claim 1 with a cognate binding partner specific for one or more of the plurality of tag cassettes, thereby activating and/or promoting proliferation of the cell expressing the single chain fusion protein.

25. The method of claim 24 further comprising contacting the cell with a growth factor cytokine for a time sufficient to allow cell growth.

26. A method for identifying a cell or a population of cells, the method comprising:

contacting a sample, or a subject, comprising a cell or a population of cells expressing the single chain fusion protein of claim 1 with a binding domain specific for one or more of the plurality of tag cassettes, wherein the binding domain specific for one or more of the plurality of tag cassettes comprises a detectable moiety, and

detecting the presence of the cell or the population of cells expressing the single chain fusion protein in the sample or the subject.

27. A method for enriching for or isolating a cell or a population thereof, the method comprising

contacting a sample comprising a cell expressing the single chain fusion protein of claim 1 with a cognate binding partner specific for one or more of the plurality of tag cassettes, and

enriching for or isolating a cell bound by the cognate binding partner from other cells not bound by the cognate binding partner in the sample.

28. A method for depleting cells, comprising contacting a cell expressing the single chain fusion protein of claim 1 with a cognate binding partner specific for one or more of the plurality of tag cassettes, wherein binding between the cognate binding partner and the one or more of the plurality of tag cassettes leads to cell death and/or depletion of the cell expressing the single chain fusion protein.

29. A method of treating a disease in a subject, comprising administering to a subject a host cell expressing the single chain fusion protein according to claim 1.

30. The method of claim 29, wherein the extracellular component further comprises a binding domain that specifically binds a target, wherein the target is associated with the disease.

31. The method of claim 29, wherein the fusion protein is non-covalently associated with a binding domain that specifically binds a target, wherein the target is associated with the disease.

18. A nucleic acid molecule encoding a single chain fusion protein, the encoded single chain fusion protein comprising an extracellular component and an intracellular component connected by a hydrophobic portion,

wherein the extracellular component comprises a plurality of tag cassettes and a connector region comprising a hinge,

wherein the intracellular component comprises an effector domain, and

wherein at one of the plurality of tag cassettes comprises a peptide having a length of up to 20 amino acids that includes the amino acid sequence of SEQ ID NO.:

1 or 2.

19. A vector comprising a nucleic acid molecule according to claim 18.

20. The vector according to claim 19, wherein the vector is a viral vector.

21. The vector according to claim 20, wherein the viral vector is a retroviral vector or lentiviral vector.

22. A host cell, comprising the nucleic acid molecule according to claim 18.

23. The host cell according to claim 22, wherein the host cell is a T cell.