Membrane Ubiquitin ligases to target protein degradation

Abstract

The invention pertains to a heterobifunctional molecule comprising a first and a second binding domain, wherein i) the first binding domain is capable of specific binding to a transmembrane E3 ubiquitin ligase; and ii) the second binding domain is capable of specific binding to a transmembrane protein, wherein simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the transmembrane protein results in ubiquitination and internalisation of the transmembrane protein. The invention further pertains to the heterobifunctional molecule for use in the treatment of a disease, wherein preferably the disease is at least one of cancer, an auto-immune disease, an inflammatory disease, an infectious disease and a hereditary disease.

Claims

1. A heterobifunctional molecule comprising a first and a second binding domain, wherein i) the first binding domain is capable of specific binding to a transmembrane E3 ubiquitin ligase; and ii) the second binding domain is capable of specific binding to a transmembrane protein, wherein simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the transmembrane protein preferably results in ubiquitination and internalisation of the transmembrane protein.

2. A heterobifunctional molecule according to claim 1, wherein the molecule binds an extracellular portion of the transmembrane E3 ubiquitin ligase and an extracellular portion of the transmembrane protein.

3. A heterobifunctional molecule according to claim 1, wherein simultaneous binding of the molecule to the transmembrane E3 ubiquitin ligase and the transmembrane protein results in degradation, preferably lysosomal degradation, of the transmembrane protein.

4. A heterobifunctional molecule according to claim 1, wherein the transmembrane E3 ubiquitin ligase ubiquitinates the transmembrane protein with monoubiquitin, multiubiquitin, Lys48-linked or Lys63-linked polyubiquitin chains.

5. A heterobifunctional molecule according to claim 1, wherein the transmembrane protein is a receptor, preferably a receptor involved in cancer.

6. A heterobifunctional molecule according to claim 1, wherein the transmembrane E3 ubiquitin ligase is selected from the group consisting of RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130 and RNF128 and/or wherein the transmembrane protein is selected from the group consisting of TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, IGF1R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CMTM6, CMTM4 and WLS.

7. A heterobifunctional molecule according to claim 6, wherein the transmembrane E3 ubiquitin ligase is RNF43, and the transmembrane protein is selected from the group consisting of PD-L1, FZD7, FLT3, TGFβR2 and EGFR.

8. A heterobifunctional molecule according to claim 6, wherein the transmembrane E3 ubiquitin ligase is RNF167, and the transmembrane protein is selected from the group consisting of PD-1, CTLA4, FLT3, TGFβR2 and EGFR.

9. A heterobifunctional molecule according to claim 6, wherein the transmembrane E3 ubiquitin ligase is RNF128, and the transmembrane protein is at least one of PD-1, PD-L1 and FLT3.

10. A heterobifunctional molecule according to claim 6, wherein transmembrane E3 ubiquitin ligase is RNF130, and the transmembrane protein is at least one of PD-1 and PD-L1.

11. A heterobifunctional molecule according to claim 1, wherein the molecule comprises a linker between the first binding domain and the second binding domain.

12. A heterobifunctional molecule according to claim 1, wherein at least one the first domain and the second domain is a small organic molecule or a proteinaceous molecule, wherein preferably the heterobifunctional molecule is a bicyclic peptide.

13. A heterobifunctional molecule according to claim 1, wherein at least one of the first domain and the second domain is an antibody or a functional fragment thereof, wherein preferably the functional fragment is a nanobody.

14. A heterobifunctional molecule according to claim 13, wherein the heterobifunctional molecule is a bi-specific antibody, preferably a bi-specific nanobody.

15. A heterobifunctional molecule according to claim 1, wherein at least one of the first domain and the second domain is an aptamer.

16.-17. (canceled)

18. A heterobifunctional molecule according to claim 1, wherein the transmembrane E3 ubiquitin ligase and the membrane-bound protein are selected using a selection method comprising the steps of: a) providing a cell expressing a transmembrane E3 ubiquitin ligase and a membrane-bound protein at its cell surface, and wherein the transmembrane E3 ubiquitin ligase comprises a first non-native epitope tag in the extracellular portion; and the membrane-bound protein comprises a second non-native epitope tag in the extracellular portion; b) exposing the cell to a heterobifunctional molecule, wherein the heterobifunctional molecule comprises: a first binding domain capable of specific binding to the first non-native epitope tag; and a second binding domain capable of binding to the second non-native epitope tag; c) determining the surface levels of the membrane-bound protein of the cell; and d) selecting the transmembrane E3 ubiquitin ligase and the membrane-bound protein when the surface levels of the membrane-bound protein are decreased at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 100%, and wherein the decrease is a decrease as compared to the surface levels of the membrane-bound protein of the cell prior to step b).

19.-20. (canceled)

21. A method for decreasing the surface level of a membrane-bound protein of a cell, wherein the method comprises the steps of a) Providing a cell expressing a transmembrane E3 ubiquitin ligase and the membrane-bound protein at its cell surface; b) Exposing the cell to a heterobifunctional molecule, wherein the heterobifunctional molecule comprises: i) a first binding domain capable of specific binding to an extracellular portion of the transmembrane E3 ubiquitin ligase; and ii) a second binding domain capable of specific binding to an extracellular portion of the membrane-bound protein; and c) optionally determining the surface levels of the membrane-bound protein of the cell, wherein the decrease is a decrease as compared to the surface levels of the membrane-bound protein of the cell prior to step b).

22. A method according to claim 21, wherein at least one of: the transmembrane E3 ubiquitin ligase comprises a first non-native epitope tag in the extracellular portion, and wherein the first binding domain of the heterobifunctional molecule binds to the first non-native epitope tag; and the membrane-bound protein comprises a second non-native epitope tag in the extracellular portion, and wherein the second binding domain of the heterobifunctional molecule binds to the second non-native epitope tag.

23. A method for the treatment of cancer, the method comprising, administering the heterobifunctional molecule according to claim 1 to a subject in need thereof.

24. The method according to claim 24, wherein the subject suffers from a cancer selected from the group consisting of colorectal cancer, ovarian cancer, breast cancer, oesophagal cancer, gastric cancer, prostate cancer, lung cancer, melanoma, leukemia, pancreatic cancer and bladder cancer

Description

FIGURE LEGENDS

[0461] FIG. 1. Schematic representation of an exemplary embodiment of the invention. A heterobifunctional molecule of the invention simultaneously binds to a transmembrane E3 ubiquitin ligase and a transmembrane protein. As a consequence, the transmembrane protein will become ubiquitinated, internalized and degraded.

[0462] FIG. 2. Functional assessment of the A/C dimerizer. HEK293T cells were transfected with RNF43-FKBP and TβRII-Flag-FRB and treated with the A/C dimerizer or a similar volume of 100% ethanol overnight. Flag-M2 beads were used to immunoprecipitate the TβRII construct from the cell lysates. IP samples and whole cell lysates were separated by SDS-page, blotted and stained for Flag and RNF43 to detect binding between the two constructs.

[0463] FIG. 3. Forced dimerization of RNF43 and TβRII induces the relocalization of both proteins to perinuclear lysosomes. (a) Confocal images of HEK293T cells transfected with TβRII-Flag-FRB. (B) Confocal images of HEK293T cells transfected with RNF43-FKBP and TβRII-Flag-FRB. Cells were treated with A/C dimerizer or a similar volume of 100% ethanol overnight. TβRII and RNF43 were visualized by Flag and RNF43 staining, respectively. (C) Confocal images of HEK293T cells transfected with CD63-GFP, RNF43-FKBP and TβRII-Flag-FRB. Cells were treated with A/C dimerizer overnight and TβRII-Flag-FRB was visualized by Flag staining. Arrows indicate perinuclear lysosomes.

[0464] FIG. 4. TβRII is degraded upon forced dimerization of RNF43 and TβRII. HEK293T cells were transfected with RNF43-FKBP and TβRII-Flag-FRB and treated with the A/C dimerizer or a similar volume of 100% ethanol overnight. Cell lysates were separated by SDS-page, blotted and stained for Flag and RNF43 to visualize protein levels.

[0465] FIG. 5. VHH-mediated dimerization of RNF43 or RNF167 with the receptors TβRII or EGFR induces receptor internalization and co-clustering in the perinuclear area. Cells were treated for 5 h with 100 nM of the bi-VHH before fixation. E3 ligases were visualized by Myc staining and the receptors by Flag staining. Confocal images of HEK293T cells transfected with (A) E6-Flag-TβRII and Alpha-Myc-RNF43, (B) E6-Flag-TβRII and Alpha-Myc-RNF167, (C) E6-Flag-EGFR and Alpha-Myc-RNF43 and (D) E6-Flag-EGFR and Alpha-Myc-RNF167. Arrows indicate co-clustering of the E3 ligases and receptors in the perinuclear area.

[0466] FIG. 6. Bi-functional VHH treatment promotes membrane-bound E3 ligase-mediated internalization of transmembrane receptors from the cell surface. HEK293T cells were transfected with one of the E3 ligases RNF43, RNF128, RNF130 or RNF167 and with the receptors CTLA-4, FLT-3, PD-1 and PD-L1. Cells were left untreated or were treated overnight with 50 nM of the bi-VHH before fixation. The receptors present at the cell surface were visualized by Flag staining in unpermeabilized cells. Confocal images are shown of HEK293T cells transfected with (A) E6-Flag-CTLA-4 and Alpha-Myc-RNF43 or Alpha-Myc-RNF167, (B) E6-Flag-FLT-3 and Myc-RNF43, Alpha-Myc-RNF128 or Alpha-Myc-RNF167, (C) E6-Flag-PD-1 and Alpha-Myc-RNF43, Alpha-Myc-RNF128, Alpha-Myc-RNF130 or Alpha-Myc-RNF167 and (D) E6-Flag-PD-L1 and Alpha-Myc-RNF43, Alpha-Myc-RNF128, Alpha-Myc-RNF130 or Alpha-Myc-RNF167.

[0467] FIG. 7. Validation effect bi-VHH on E3 ligase-target combinations at the endogenous level. (A) Strategy to generate endogenously tagged proteins. (B) Schematic of the approach for cell surface removal of targets by bi-VHH using endogenously tagged versions of E3 ligase-target combinations.

EXAMPLES

Example 1

[0468] Materials and Methods

[0469] Cell Culture and Transfection

[0470] Human Embryonic Kidney (HEK) 293 T cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (GE Healthcare), 2 mM UltraGlutamine (Lonza), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen). Cells were cultured at 37° C. in 5% CO.sub.2. Transfections were performed using FuGENE 6 (Promega) according to the manufacturer's protocol for microscopy, or using PEI for biochemistry. The A/C Heterodimerizer (Takara Bio, #635056) was used at 1 uM overnight at 37° C., control conditions were treated with an equal volume of 100% ethanol. TGFβ was used at 1.5 ng/mL for 45 minutes.

[0471] Constructs and Antibodies

[0472] TGF-β type II serine/threonine kinase receptor (TβRII)-Flag-FKBP and -Flag-FRB were provided by Peter ten Dijke (LUMC, Leiden). RNF43-FKBP and -FRB were obtained by inserting the coding sequence for FKBP.sup.36V or FRB, respectively, in the C-tail of human RNF43 using Q5 High-Fidelity 2× Master Mix (NEB). All constructs were sequence verified. CD63-GFP was a gift of J. Klumperman (UMCU, Utrecht). The following primary antibodies were used for immunoblotting (IB), immunofluorescence (IF) or immunoprecipitation (IP): rabbit anti-FLAG (Sigma-Aldrich), rat anti-HA (Roche), mouse anti-FLAG (M2; Sigma-Aldrich), mouse anti-Actin (MP Biomedicals) and rabbit anti-RNF43 (Sigma-Aldrich). Primary antibodies were diluted conform manufacturer's instructions. Secondary antibodies used for IB or IF were used 1:8000 or 1:300 respectively and obtained from either Rockland or Invitrogen.

[0473] Immunofluorescence and Confocal microscopy. HEK293T cells were grown on glass coverslips coated with laminin (Sigma) in 24-well plates. After overnight transfection cells were fixed in 4% formaldehyde in phosphate buffered saline (PBS). Cells were blocked in buffer containing 2% BSA and 0.1% saponin in PBS for 30 min at room temperature (RT). Subsequently, cells were incubated with primary and secondary antibodies for 1 h at RT in blocking buffer. Cells were mounted in Prolong Diamond (Life technologies) and images were acquired with a LSM700 confocal microscopes. Images were analysed and processed with ImageJ.

[0474] Immunoprecipitation and western blotting. After transfection, cells were grown to 80% confluency in 10 cm dishes. After washing cells with PBS, cells were scraped and lysed in cell lysis buffer containing 100 mM NaCL, 50 mM Tris pH 7.5, 0.25% Triton X-100, 10% Glycerol, 50 mM NaF, 10 mM Na.sub.3VO.sub.4, 10 μM leupeptin, 10 μM aprotinin and 1 mM PMSF. Lysates were cleared by centrifugation at 16.000×g for 15 min at 4° C. Lysates were taken up in SDS sample buffer and heated for 1 hour at 37° C. For immunoprecipitation, lysates were incubated with 25 μl pre-coupled Flag-M2 beads (Sigma) and incubated overnight at 4° C. After washing, beads were eluted with sample buffer and heated for 1 hour at 37° C. After SDS-PAGE, proteins were transferred via Western blotting onto Immobilon-FL PVDF membranes (Milipore). After blocking with Odyssey blocking buffer (LI-COR), proteins were labelled with the indicated primary antibodies that were detected with goat anti-mouse/rabbit Alexa 680 (Invitrogen), donkey anti-rat Alexa 680 (Invitrogen) or goat anti-mouse/rabbit IRDye 800 (Rockland) using a Amersham Typhoon Biomolecular Imager (GE Health Care).

[0475] Results and Discussion

[0476] Forced Dimerization of RNF43 and TGF-β Type II Serine/Threonine Kinase Receptor (TβRII) Induces Lysosomal Localization and Degradation of TβRII

[0477] To show proof of concept for redirection of a transmembrane E3 ligase to target a selected cell surface protein for internalization and lysosomal degradation, we used the FKBP/FRB dimerization system. We fused either the FKBP domain or the FRB domain to the C-terminal tails of both TβRII and RNF43. When co-expressed in HEK293T cells, these proteins do not interact (FIG. 2). Upon addition of the A/C dimerizer, however, co-immunoprecipitation of RNF43 and TβRII is induced (FIG. 2). The dimerizer by itself does not interfere with TβRII or RNF43 stability (FIG. 2, whole cell lysate). We next asked whether the forced interaction of RNF43 with TβRII changes the subcellular localization of TβRII. In the absence of dimerizer, TβRII predominantly localizes to the plasma membrane, both in the absence of RNF43 (FIG. 3A) and upon co-expression of RNF43 (FIG. 3B). The addition of the dimerizer, however, strongly induced relocalization of both TβRII and RNF43 to a perinuclear vesicle cluster that is positive for the lysosomal marker CD63 (FIG. 3C). These findings indicate that forced dimerization of RNF43 and TβRII directs increased levels of TβRII to the lysosome.

[0478] To determine if enhanced lysosomal localization of TβRII results in lower amounts of functional TβRII, we analyzed protein levels using Western Blot. Although RNF43 expression itself has no effect on the stability of TβRII, induced dimerization of RNF43 and TβRII clearly leads to reduced TβRII protein levels (FIG. 4). Together these results indicate that forced dimerization of the transmembrane E3 ligase RNF43 and a normally unrelated transmembrane receptor TβRII targets TβRII for lysosomal degradation.

Example 2

[0479] Materials and Methods

[0480] Cell Culture and Transfection

[0481] Human Embryonic Kidney (HEK) 293 T cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (GE Healthcare), 2 mM UltraGlutamine (Lonza), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen). Cells were cultured at 37° C. in 5% CO.sub.2. Transfections were performed using FuGENE 6 (Promega) according to the manufacturer's protocol.

[0482] Constructs and Antibodies

[0483] E6-Flag-TGF-β type II serine/threonine kinase receptor (TβRII) and -Epidermal Growth Factor Receptor (EGFR) and Alpha-myc-RNF43 and RNF167 were obtained by subcloning using Q5 High-Fidelity 2× Master Mix (NEB). All constructs were sequence verified. The following primary antibodies were used for immunofluorescence (IF): rabbit anti-Flag (Sigma-Aldrich) and mouse anti-Myc (hybridoma 9E10). Primary antibodies were diluted conform manufacturer's instructions. Secondary antibodies used for IF were used at 1:300 (Life technologies).

[0484] Immunofluorescence and Confocal microscopy. HEK293T cells were grown on glass coverslips coated with laminin (Sigma-Aldrich) in 24-well plates. After overnight transfection cells were incubated 1 h with 20 nM Bafilomycin A1 (Sigma-Aldrich) before and during a 5 h treatment with 100 nM of the bi-VHHs (VHH Alpha-(G4S)3-VHH E6). After treatment cells were washed two times with warm medium and fixed in 4% formaldehyde in 0.05 M Phosphate buffer pH 7.4. Cells were blocked in buffer containing 2% BSA and 0.1% saponin in PBS for 30 min at room temperature (RT). Subsequently, cells were incubated with primary antibodies against either Flag or Myc for 1 h at RT, followed by the secondary antibodies for 1 h at RT in blocking buffer. Cells were mounted in Prolong Diamond (Life technologies) and images were acquired with an LSM700 confocal microscope. Images were analysed and processed with ImageJ.

[0485] Results and Discussion

[0486] RNF43 and RNF167 Induce Cell Surface Removal of TβRII and EGFR Upon Forced Dimerization Using Bi-Specific VHHs.

[0487] To further confirm the functionality of the heterobifunctional molecules of the invention, selected receptors were targeted with E3 ligases by VHH-mediated dimerization of extracellular regions. To this end, we fused epitope tags to the extracellular domains of both the targets (E6 tag) and E3 ligases (Alpha tag). We selected these epitope tags for recognition by VHHs (Götzke et al., 2019, Nature Communications, 10(1), 1-12; Ling et al., 2019, Molecular Immunology, 114(July), 513-523) and we generated bi-specific VHHs (bi-VHHs) against these two epitopes to allow for VHH-mediated dimerization. To determine alterations in the localization of proteins, we also incorporated a Myc epitope tag for the E3 ligases and a Flag epitope tag for the receptors. When co-expressed in HEK293T cells, none of the receptors colocalized with any of the E3 ligases: E3 ligases mainly localized to intracellular compartments and the receptors mainly at the plasma membrane (data not shown). However, upon 5 h treatment with bi-VHH both RNF43 and RNF167 induced removal of TβRII as well as EGFR from the plasma membrane. Moreover, internalized proteins co-clustered in the perinuclear area in bafilomycin-treated cells, which inhibits lysosomal turnover, indicating accumulation of E3 ligases and their targets in late endosomal/lysosomal structures (FIG. 5A-D). These findings show that heterobifunctional molecules, such as bi-VHHs, can be used to deliberately dimerize a transmembrane E3 ligase with a selected transmembrane receptor, thereby inducing receptor removal from the cell surface.

Example 3

[0488] Materials and Methods

[0489] Cell Culture and Transfection

[0490] Human Embryonic Kidney (HEK) 293 T cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (GE Healthcare), 2 mM UltraGlutamine (Lonza), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen). Cells were cultured at 37° C. in 5% CO.sub.2. Transfections were performed using FuGENE 6 (Promega) or Effectene (Qiagen) according to the manufacturer's protocol.

[0491] Constructs and Antibodies

[0492] E6-Flag-Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), Receptor-type tyrosine-protein kinase FLT3 (FLT-3), Programmed cell death protein 1 (PD-1) and Programmed cell death 1 ligand 1 (PD-L1) and Alpha-myc-RNF43, RNF128, RNF130 and RNF167 were obtained by subcloning using Q5 High-Fidelity 2× Master Mix (NEB). All constructs were sequence verified. The following primary antibodies were used for immunofluorescence (IF): rabbit anti-Flag or mouse anti-Flag (Sigma-Aldrich). Primary antibodies were diluted conform manufacturer's instructions. Secondary antibodies used for IF were used at 1:300 (Life technologies).

[0493] Immunofluorescence and Confocal microscopy. HEK293T cells were grown on glass coverslips coated with laminin (Sigma-Aldrich) in 24-well plates. Six hours after transfection cells were incubated overnight with 50 nM of the bi-VHHs (VHH Alpha-(G4S)3-VHH E6). After treatment cells were washed two times with warm medium and fixed in 4% formaldehyde in 0.05 M Phosphate buffer pH 7.4. Cells were blocked in buffer containing 2% BSA in PBS for 30 min at room temperature (RT). Subsequently, cells were incubated with primary antibody against Flag for 1 h at RT, followed by the secondary antibody for 1 h at RT in blocking buffer. Cells were mounted in Prolong Diamond (Life technologies) and images were acquired with an LSM700 confocal microscope using a 5× objective lens or with an EVOS-M5000 microscope using a 20× objective lens. Images were analyzed and processed with ImageJ.

[0494] Results and Discussion

[0495] Specific E3 Ligase-Target Combinations Allow for Surface Removal of a Target Upon Forced Dimerization Using Bi-Specific VHHs.

[0496] To screen for additional candidate E3 ligase-receptor combinations, we generated the following constructs Alpha-Myc-RNF128, Alpha-Myc-RNF130, E6-Flag-CTLA-4, E6-Flag-FLT-3, E6-Flag-PD-1 and E6-Flag-PD-L1 in addition to the previously generated constructs. When co-expressed in HEK293T cells, CTLA-4, FLT-3, PD-1 and PD-L1 all localized to the cell surface. Upon overnight treatment with bi-VHHs, the following E3-target combinations lead to target removal of the surface: CTLA-4 and RNF167; FLT-3 and RNF43, RNF128 or RNF167, PD-1 and RNF128, RNF130 or RNF167 and PD-L1 and RNF43, RNF128 or RNF130 (FIG. 6A-D). These findings expand the range of use for heterobifunctional molecules, such as bi-VHHs, to deliberately dimerize various transmembrane E3 ligases with a selection of transmembrane receptors, thereby inducing removal of these receptors from the cell surface. These findings also underline that not all combinations are effective.

Example 4

[0497] To validate the promising combinations emerging from the above screening in a physiological setting, we will use CRISPR/Cas9 technology to generate cancer cell lines expressing endogenously tagged E3 ligases and targets. We will use guide RNAs in combination with donor DNA for the Alpha-Myc or E6-Flag tags that will facilitate the insertion of these tags between the signal peptide (SP) and the coding sequence for the first mature amino acid in the endogenous locus of the E3 ligase or the target (FIG. 7A). Using these cell lines, we will assess the removal of the endogenous target from the cell surface upon forced dimerization with the bi-VHH by either microscopy or Western blotting (FIG. 7B).