HOOK FUSION PROTEIN FOR REGULATING THE CELLULAR TRAFFICKING OF A TARGET PROTEIN

Abstract

The present invention relates to a hook fusion protein comprising a hook domain and at least one cytoplasmic carboxy terminal endoplasmic reticulum (ER) retention signal and/or at least one cytoplasmic amino terminal endoplasmic reticulum (ER) retention signal; wherein the hook fusion protein is a soluble protein that localizes in the cytoplasm. The present invention also relates to a nucleic acid system for intracellular targeting control comprising a nucleic acid encoding a hook fusion protein as herein disclosed, and a nucleic acid encoding a target fusion protein comprising a hook-binding domain; wherein said target fusion protein in a membrane protein; and wherein the hook fusion protein localizes in the ER when bound to the target fusion protein. The invention also encompasses a vector system, viral particle system, host cell and kit comprising said nucleic acids. The invention also includes the vector system, viral particle system, host cell or kit for use as a medicament, in particular for immunotherapy.

Claims

1. A hook fusion protein comprising a hook core at least one cytoplasmic carboxy terminal endoplasmic reticulum (ER) retention signal and/or at least one cytoplasmic amino terminal endoplasmic reticulum (ER) retention signal; wherein the hook fusion protein is a soluble protein that localizes in the cytoplasm.

2. A hook fusion protein according to claim 1, wherein the hook core is a streptavidin sequence.

3. A hook fusion protein according to claim 1 wherein the carboxy terminal endoplasmic reticulum (ER) retention signal is K(X)KXX, with X being any amino acids and/or the amino terminal endoplasmic reticulum (ER) retention signal is a fragment of the isoform of the human invariant chain of the major histocompatibility complex protein Ii, optionally wherein the fragment of the isoform of the human invariant chain of the major histocompatibility complex protein Ii has an amino acid sequence selected from SEQ ID NO: 5 or 14.

4. A hook fusion protein according to claim 1 further comprising an endocytosis signal, preferably consisting of YXXI with X being any amino acids.

5. A nucleic acid comprising a nucleic acid sequence encoding the hook fusion protein according to claim 1.

6. The nucleic acid according to claim 5 further comprising a nucleic acid sequence encoding a target fusion protein comprising a hook-binding domain, wherein said target fusion protein is a chimeric antigen receptor comprising: a binding domain; a hook-binding domain, and at least one activation domain; or alternatively comprising: the full NKG2D or a functional variant thereof, at least one activation domain, and a hook-binding domain.

7. A nucleic acid system for intracellular targeting control comprising (a) a nucleic acid encoding a hook fusion protein according to claim 1, and (b) a nucleic acid encoding a target fusion protein comprising a hook-binding domain; wherein said target fusion protein is a membrane protein; and wherein the hook fusion protein localizes in the ER when bound to the target fusion protein; optionally wherein the hook fusion protein comprises a streptavidin domain and the target fusion protein comprises a streptavidin-binding domain, optionally wherein the target fusion protein is a chimeric antigen receptor as defined in claim 6 comprising: a binding domain; a hook-binding domain, and at least one activation domain; or alternatively comprising: the full NKG2D or a functional variant thereof, at least one activation domain, and a hook-binding domain.

8. A vector system comprising one or more vectors comprising (a) the nucleic acid sequence of claim 6, and optionally (b) a nucleic acid encoding a target fusion protein comprising a hook-binding domain; wherein the nucleic acids (a) and (b) are located on the same or on different vectors; optionally wherein the hook fusion protein comprises a streptavidin domain and the target fusion protein comprises a streptavidin-binding domain.

9. A vector system according to claim 8, wherein the nucleic acids (a) and (b) are located on the same vector and wherein the nucleic acid (a) is inserted upstream of an IRES sequence and the nucleic acid (b) is inserted downstream of said IRES sequence.

10. A vector system according to claim 8, wherein the nucleic acids (a) and (b) are located on the same vector and wherein: i) the nucleic acid (a) comprises an Ii retention signal in its N terminal sequence and is inserted upstream of a 2A peptide sequence, or ii) the nucleic acid (a) comprises a K(X)KXX retention signal in its C terminal sequence and is inserted downstream of a 2A peptide sequence.

11. A vector system according to claim 10 further comprising the nucleic acid sequence (b) wherein said nucleic acid sequence (b) comprises a streptavidin-binding domain, and wherein said nucleic acid sequence (b) is inserted downstream of the 2A peptide in the i) configuration or upstream of the 2A peptide in the ii) configuration.

12. A vector system according to claim 8, wherein the target fusion protein encoded by the nucleic acid (b) is a chimeric antigen receptor comprising: a binding domain; a hook-binding domain, and at least one activation domain; or alternatively comprising: the full NKG2D or a functional variant thereof, at least one activation domain, and a hook-binding domain.

13. A viral particle system comprising a vector system according to claim 8; optionally wherein the viral particle system is a lentiviral particle.

14. An isolated cell comprising a vector system as defined in claim 8.

15. An in vitro method for regulating the intracellular trafficking in a host cell of a target protein; wherein said target protein is a fusion protein comprising a hook binding domain; and wherein the method comprises expressing in said host cell a vector system according to claim 8; wherein the hook fusion protein and the target fusion protein are capable of conditional interaction in the absence of a ligand for the hook core domain, optionally wherein the hook core domain is streptavidin, the hook-binding domain is a streptavidin-binding domain and the ligand is biotin.

16. (canceled)

17. (canceled)

18. (canceled)

Description

FIGURE LEGENDS

[0212] FIG. 1: Schematic representation of the bicistronic plasmid coding for the known (Cs-Ii), newly Hooks and reporter gene. The Hooks are (A) cytoplasmic hook (Str-Ii, streptavidin (str)) fused to the isoform of the human invariant chain of the major histocompatibility complex (Ii; a type II protein) containing an ER retention arginine-based motif at the N-terminal); (B) soluble Cytoplasmic Streptavidin with the endocytosis signal (YXXI, X any aa) and ER retention signal (KKXX, X any aa); (C) cytoplasmic mini Hook with a Ii retention signal in the N-terminal and the endocytosis signal in C-terminal. These gene are expressed under the control of the CMV promoter and separated by a synthetic intron, i.e. intervening sequence (IVS) followed by an internal ribosome entry site (IRES)(Boncompain, Divoux et al. 2012).

[0213] FIG. 2: Traffic of RUSH based constructs using the streptavidin containing an endocytosis signal and the ER retention signal KKXX hook. HeLa cells expressing A) scFv (CD19)-myc-DAP10-sSBP reporter (anti-myc stained) or B) CD3-SBP-NKG2D co-transfected with myc-DAP10 (DAP10 is required for NKG2D traffic) (anti-NKG2D stained). The cells were non-treated (NT) and treated with biotin and different time points were recorded. Overnight treatment (ON) was performed by adding biotin immediately after adding transduction solution and it is representative of the protein steady state. Nucleus was stained using DAPI.

[0214] FIG. 3: Traffic of RUSH based constructs using the soluble mini hook containing an endocytosis signal. HeLa cells expressing A) scFv (CD19)-GFP-DAP10-sSBP reporter or B) scFv (CD19)-GFP-DAP10CD3-sSBP. Streptavidin in the mini hook was stained with anti-Str. The cells were non-treated (NT) and treated with biotin and different time points were recorded. Overnight treatment (ON) was performed by adding biotin immediately after adding transduction solution and it is representative of the protein steady state. Nucleus was stained using DAPI.

[0215] FIG. 4: The soluble Str-endoKKXX Hook allows retention at the level of the ER and retrieval from the cell surface. HeLa cells were transfected by BACE1-GFP-SBP (BACE1-RUSH) together with an Invariant chain-based Hook (Ii Hook, a-c) or a cytoplasmic Hook bearing both an ER transport signal and an endocytosis signal (Str-endoKKXX, d-f). Upon transfection in the absence of releasing molecule (without biotin, a,d), BACE1-RUSH is retained in the ER. Addition of the biotin-mimetic molecule ALiS-1 overnight (b, e) allows efficient release of BACE1-RUSH and its transport to the cell surface. Washing-out ALiS-1 for 1 hour (c,f) does not allow to capture cell surface localized BALI-RUSH if transfected with the Ii-based Hook (c) while it is efficiently transported back to the ER when expressed with Str-endoKKXX highlighting the capacity of the new Hook to mediate transport form the plasma membrane to the ER in addition to its ability to retain proteins in the ER.

[0216] FIG. 5: The soluble miniIi Hook allows retention at the level of the ER and retrieval from the cell surface. HeLa cells were transfected by scFv (CD19)-myc-DAP10-sSBP together with a cytoplasmic Hook bearing both an ER transport signal and an endocytosis signal (Str-endoKKXX, a-c) or soluble mini hook containing an endocytosis signal (mini hook, d-f). Upon transfection in the absence of releasing molecule (without ALIS, a,d), scFv (CD19)-myc-DAP10-sSBP is retained in the ER. Addition of the biotin-mimetic molecule ALiS-1 45 min (b, e) allows efficient release of scFv (CD19)-myc-DAP10-sSBP and its transport to the cell surface. Washing-out ALiS-1 for 2 hours (c,f) efficiently transported back scFv (CD19)-myc-DAP10-sSBP to the ER when expressed with Str-endoKKXX or mini hook highlighting the capacity of the new Hooks to mediate transport form the plasma membrane to the ER in addition to its ability to retain proteins in the ER.

[0217] FIG. 6: Schematic representation of the NKG2D CAR. NKG2D (type II protein) is fused to CD3 zeta domain and to SBP in two distinct positions.

EXAMPLES

[0218] In the examples below, the term Hook refers to the hook fusion protein comprising the hook domain, and the term Reporter refers to the target membrane protein comprising the hook-binding domain.

[0219] Methods and Material:

[0220] Constructs

[0221] FIG. 1 shows a schematic representation of the Hook constructs by Boncompain et al (FIG. 1A), and of the new hooks according to the present invention (FIG. 1B, 1C). Those are inserted in the bicistronic vector using multicloning sites and the reporter using the typical cloning cassettes of the previously published RUSH vector. The soluble streptavidin containing an endocytosis signal and the ER retention signal KKXX were built by gene synthesis (gBlocks Gene FragmentsIntegrated DNA Technologies or GeneArt/Thermo-Fisher). The soluble mini hook containing a endocytosis signal was synthetized by gene syntheses (gBlocks Gene FragmentsIntegrated DNA Technologies) was generated by PCR amplification of the previously described luminal Ii-Str (Boncompain, Divoux et al. 2012), using the primers Fow-Nhe-IiMini-Str (5-CTAgctagccATGCACAGAAGAAGAAGCAGAAGCgaccctagcaaagactcaaaagc-3)(SEQ ID NO: 19) and Rev-mini-Ii-2nd-Xho (5-CTCGAGgcggctgcacttgctctc-3)(SEQ ID NO: 20) for amplification of the 46 aa of Ii and for streptavidin amplification the primer Fow-Xho-Str (5-CTCGAGGACCCTAGCAAAGACTCA-3)(SEQ ID NO: 21) and REV-Ires (5-GGATCAGTTATCTATGCG-3)(SEQ ID NO: 22). The fragments generated were digested with the respective enzymes and clone into the pCMV vector used previously in (Boncompain, Divoux et al. 2012). The sequences were evaluated and validate by sequencing. The several reporters were used tagged with a fluorescent protein (GFP). The sequence of some of the reporter were synthesized by gene syntheses (gBlocks Gene FragmentsIntegrated DNA Technologies), other were previously generated in (Boncompain, Divoux et al. 2012).

[0222] Cell Culture and Transfection:

[0223] HeLa cells were cultivated at 37 C. and 5% of CO.sub.2 in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS (Biowest), 1 mM sodium Pyruvate and 100 M of penicillin and streptomycin (Invitrogen). HeLa cells were transfected with the plasmid of interest using Calcium phosphate protocol in the presence of 25 mM of HEPES. Briefly, the plasmids coding the sequence of CAR based RUSH such as CD3-SBP-NKG2D (SEQ ID NO: 13), scFv(CD19)-GFP-DAP10CD3-sSBP (SEQ ID NO: 23), scFv(CD19)-GFP-DAP10-SBPdel (SEQ ID NO: 24), scFv(CD19)-mycDAP10-SBP (SEQ ID NO: 25) or BACE1-SBP-EGFP (SEQ ID NO: 26) (2.5 ug per 1 mL of final volume) were add to 1 mM tris-HCl pH 8.02 buffer followed by the addition of 10% of CaCl.sub.2 and incubated for 5 min (RT). Then this mix was add drop by drop into 2 concentrate HEBS buffer (160 mM NaCl, 1.5 mM Na.sub.2HPO.sub.4, 50 mM Hepes PH 7.04-7.05) while vortexing. The cells were incubated with this solution overnight at 37 C. and 5% of CO.sub.2.

[0224] Time Course Release Using Biotin:

[0225] The cells were seeded into a glass coverslips for fixed cell immunofluorescence and/or live imaging. In the next day the cells were transfected with the plasmids coding the construct of interest as previously described. For the steady state of the protein/construct, 40 M final concentration of biotin was added (4 mM stock solution) just after addition of the transfection solution. The presence of biotin prevented the interaction of the reporter (target membrane protein) with the hook, allowing the normal traffic of the reporter. In the next day, the cells in the coverslips were incubated at different time point with a final concentration of 40 M of biotin, allowing the traffic of the reporter and then prepared for immunofluorescence.

Biotin-mimetic molecule ALiS-1 was prepared in DMSO to 20 mM (stock solution) and the cells were treated with 40 M final concentration to prevent the interaction between the reporter (target membrane protein) and the hook.

[0226] Immunofluorescence:

[0227] Cells coated in the coverslips were washed once in 1PBS buffer, fixed in 3% of paraformaldehyde (PFA) (10-15 min, RT), washed (2) and incubated with 50 mM of NH.sub.4Cl (5 min, RT) to quench free aldehydes. The cells were then permeabilized using a solution of PBS containing bovine serum Albumin (BSA, 0.5%, Sigma-Aldrich) and saponin (Sapo, 0.05% Sigma-Aldrich)(15 min, RT). When the protein was not fluorescent labelled, we used antibodies for their detection. These include the monoclonal anti human NKG2D (1/800, Biolegend), and anti-myc tag from mouse (1/2000, clone 9E10) or anti-myc from rabbit (1/500, Cell Signaling). The coverslip were mounted in Mowiol (Calbiochem) supplemented with DAPI (4,6-Diamidino-2-phenylindole) for DNA staining.

[0228] Results

[0229] Soluble Streptavidin Containing an Endocytosis Signal and the ER Retention Signal KKXX:

[0230] The soluble streptavidin containing an endocytosis signal and the ER retention signal KKXX was used to synchronized the traffic of the CAR, scFvCD19-Myc-DAP10-sSBP (sSBP; small streptavidin binding peptide, with 28 amino-acids (aa), instead of the typical 36 aa) (FIG. 2, A) and the NKG2D based CAR, with SBP into two different positions (FIG. 2, B). These include a SBP as CD3-SBP-NKG2D (FIG. 2, B). The NKG2D based CARs were always co-transfected with Myc-DAP10, since DAP10 is required for NKG2D traffic. The scFvCD19-Myc-DAP10-sSBP was well retain in the ER by this Hook and upon addition of biotin is released and 15 min later reached the Golgi apparatus (FIG. 2, A). 30 min later, part of the protein is localized in the cells surface although some remained in the Golgi and at 60 min the majority is at the cell surface (FIG. 2, A). Overnight with biotin allow the traffic of the protein to the cell surface, presumably as in its steady state (FIG. 2, A). The CD3-SBP-NKG2D (FIG. 2, B) were co-transfected with DAP10 for their traffic. Similar to the previous CAR, CD3-SBP-NKG2D (FIG. 2, B) is retained in the ER by this Hook. The traffic behavior of the CD3-SBP-NKG2D is very similar to the scFvCD19-Myc-DAP10-sSBP (FIG. 2, B). At 15 min the CD3-SBP-NKG2D is localized in the Golgi apparatus, 30 min after it started to reach the cell surface and 60 min, the majority of the NKG2D is at the cell surface, although some is still retain in the Golgi (FIG. 2, B).

[0231] Cytoplasmic Mini Hook:

[0232] To the cytoplasmic mini hook an endocytosis signal was added or not in the C-terminal. The endocytosis signal is similar to the one used for the soluble streptavidin containing an endocytosis signal and the ER retention signal KKXX (FIG. 3). This newly developed soluble mini hook is efficient to retain the CARs scFv (CD19)-GFP-DAP10-sSBP reporter (FIG. 3, A) or scFv (CD19)-GFP-DAP10CD3-sSBP (FIG. 3, B) in the endoplasmic reticulum. To the construct scFv (CD19)-GFP-DAP10-sSBP was fused a CD3 zeta domain (activation domain) after DAP10 that should increase the activation capacity of the CAR (scFv (CD19)-GFP-DAP10CD3-sSBP). The addition of the biotin leads to the release of the mentioned CARs and at 15 min they reached the Golgi apparatus and at 30 min at cell surface, although some still remain in the Golgi. At 60 min with biotin, the majority of the CARs are at the cell surface similar to the overnight treatment with biotin (FIG. 3). We also observed the scFv (CD19)-GFP-DAP10CD3-sSBP CAR at 60 min and ON in the presence of biotin, is still retained at the Golgi apparatus even when the majority reached the cells surface.

[0233] Cytoplasmic Mini Hook and Soluble Str-endoKKXX Hook Reversible Capacity:

[0234] We could observe that both cytoplasmic mini Hook and str-endoKKXX allow the retention and release using a biotin-mimetic molecule ALiS-1 (Terai et al, J. Am. Chem. Soc, 2015; 137(33):10464-7) (FIGS. 4 and 5). Washing-out ALiS-1 allows the retrieval from surface localized BACE1-RUSH and scFv (CD19)-myc-DAP10-sSBP back to the ER. These highlighting the capacity of the new Hooks to mediate a reversible transport from the plasma membrane to the ER while maintain their ability to retain and thus release proteins in the ER.