Chimeric antigen receptor specific for interleukin-23 receptor

Abstract

The present invention relates to a chimeric antigen receptor (CAR) specific for an IL-23 receptor, and to a nucleic acid encoding the same. The present invention further relates to a T cell expressing said CAR, and to the use thereof for treating an autoimmune and/or inflammatory disease or disorder.

Claims

1. A chimeric antigen receptor (CAR) specific for at least one IL-23 receptor (IL-23R), wherein said CAR comprises: i) an extracellular binding domain, wherein said binding domain binds to said IL-23R, ii) optionally an extracellular hinge domain, iii) a transmembrane domain, iv) an intracellular signaling domain, and, v) optionally a tag and/or a leader sequence.

2. The CAR according to claim 1, wherein the extracellular binding domain comprises an scFv fragment directed against said IL-23R.

3. The CAR according to claim 1, wherein the extracellular binding domain comprises an scFv fragment directed against said IL-23R, wherein said scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) and optionally a linker between the VH and the VL, wherein the VH has the sequence of SEQ ID NO: 37, and the VL has a sequence selected from the group consisting of SEQ ID NOs: 38, 46 and 56.

4. The CAR according to claim 1, wherein the extracellular binding domain comprises an scFv fragment directed against said IL-23R, wherein said scFv has the sequence of SEQ ID NO: 55.

5. The CAR according to claim 1, wherein the hinge domain is a hinge region of human CD8.

6. The CAR according to claim 1, wherein the hinge domain is a hinge region of human CD8 having the sequence of SEQ ID NO: 13.

7. The CAR according to claim 1, wherein the transmembrane domain is a transmembrane domain derived from the human CD8a.

8. The CAR according to claim 1, wherein the transmembrane domain is a transmembrane domain derived from human CD8a having the sequence of SEQ ID NO: 21.

9. The CAR according to claim 1, wherein the intracellular signaling domain comprises a costimulatory signaling domain of a molecule selected from the group consisting of 4-1BB, ICOS, CD27, OX40, CD28, CTLA4 and PD-1 and a T cell primary signaling human CD3 zeta.

10. The CAR according to claim 1, wherein the intracellular signaling domain comprises a costimulatory signaling domain of human 4-1BB having the sequence of SEQ ID NO: 29 and a T cell primary signaling human CD3 zeta having the sequence of SEQ ID NO: 26.

11. The CAR according to claim 1, wherein the intracellular signaling domain comprises a costimulatory signaling domain of human CD28 having the sequence of SEQ ID NO: 31 and a T cell primary signaling human CD3 zeta having the sequence of SEQ ID NO: 26.

12. The CAR according to claim 1, comprising i) an anti-IL-23R scFv comprising a VH that has the sequence of SEQ ID NO: 37 and a VL that has the sequence of SEQ ID NO: 38, linked by a (G45).sub.3 linker (SEQ ID NO: 3), ii) a hinge domain derived from CD8a, iii) a human CD8a transmembrane domain, iv) an intracellular signaling domain comprising a costimulatory signaling domain of a molecule selected from the group consisting of 4-1BB, ICOS, CD27, OX40, CD28, CTLA4 and PD-1, and a human CD3 zeta domain, and v) optionally a tag and/or a leader sequence.

13. A chimeric antigen receptor (CAR) specific for at least one IL-23 receptor (IL-23R), comprising i) an anti-IL-23R scFv comprising a VH that has the sequence of SEQ ID NO: 37 and a VL that has the sequence of SEQ ID NO: 38, linked by a (G-45).sub.3 linker (SEQ ID NO: 3), ii) a hinge domain derived from CD8a having the sequence of SEQ ID NO: 13, iii) a human CD8a transmembrane domain having the sequence of SEQ ID NO: 21, iv) an intracellular signaling domain comprising a human 4-1BB signaling domain having the sequence of SEQ ID NO: 29 and a human CD3 zeta domain having the sequence of SEQ ID NO: 26, and v) optionally a tag and/or a leader sequence.

14. A chimeric antigen receptor (CAR) specific for at least one IL-23 receptor (IL-23R), comprising i) an anti-IL-23R scFv comprising a VH that has the sequence of SEQ ID NO: 37 and a VL that has the sequence of SEQ ID NO: 38, linked by a (G-45).sub.3 linker (SEQ ID NO: 3), ii) a hinge domain derived from CD8a having the sequence of SEQ ID NO: 13, iii) a human CD8a transmembrane domain having the sequence of SEQ ID NO: 21, iv) an intracellular signaling domain comprising a CD28 signaling domain having the sequence of SEQ ID NO: 31 and a human CD3 zeta domain having the sequence of SEQ ID NO: 26, and v) optionally a tag and/or a leader sequence.

15. A nucleic acid sequence encoding a CAR according to claim 1.

16. A T cell population, engineered to express on the cell surface a CAR according to claim 1.

17. The T cell population according to claim 16, wherein said T cell population is a regulatory T cell (Treg) population.

18. The T cell population according to claim 17, wherein said Treg population is selected from the group consisting of CD4.sup.+CD25.sup.+Foxp3.sup.+ Treg, Tr1 cells, TGF-? secreting Th3 cells, regulatory NKT cells, regulatory ?? T cells, regulatory CD8.sup.+ T cells, and double negative regulatory T cells.

19. A method for treating an IL-23R-expressing cell-mediated disease or disorder in a subject in need thereof, comprising administering to the subject a T cell population according to claim 16, wherein said IL-23R-expressing cell-mediated disease or disorder is an autoimmune and/or inflammatory disease and/or disorder selected from the group consisting of inflammatory bowel diseases, systemic lupus erythematosus, multiple sclerosis, psoriasis, psoriatic arthritis and uveitis.

20. The method according to claim 19, wherein said IL-23R expressing cell-mediated disease or disorder is Crohn's disease or ulcerative colitis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 represents a schematic view of an anti-IL-23R Chimeric Antigen Receptor (CAR) construct of the invention (such as, for example, the CAR having a sequence SEQ ID NO: 54). The anti-IL23R CAR comprises a human CD8 leader sequence (CD8), a scFv directed against the human IL23R (?IL23R), a CD8 hinge (CD8 linker), a transmembrane domain derived from the human CD8 alpha (CD8 TM), an activation domain of human 4-1BB (4-IBB) and CD3 zeta (CD3Z). The CAR construct is in frame with a P2A-GFP coding sequence.

(2) FIG. 2 is a combination of dot plots of flow cytometry showing Treg transduction efficacy and membrane expression of the CAR. Results obtained with two Treg clones are shown (representative results). Transduction efficacy was assessed by GFP expression in flow cytometry. Membrane expression of the CAR was assessed by protein-L staining in flow cytometry. Results obtained with two Treg clones (clone 1: A-B; clone 2: C-D) are shown (representative results). (A, C) GFP-transduced Treg (MOCK), (B, D): Treg transduced with IL-23R CAR of the invention.

(3) FIG. 3 is a histogram showing the IL-23R CAR mediated Treg activation. Treg cell activation was assessed by CD69 expression by flow cytometry 24 h after activation of untransduced Treg (MOCK) or IL-23R-CAR transduced Treg (IL-23R-CAR) with coated recombinant human IL-23R (rhuIL-23R) (5 ?g/ml) or with CD3/CD28 coated beads (at a 1:1 bead-to-cell ratio). Results were expressed as mean of 2 different donors?SEM.

(4) FIG. 4 is a schematic view of an anti-murine IL-23R Chimeric Antigen Receptor (CAR) construct of the invention. The anti-IL23R CAR comprises a human CD8 leader sequence (CD8), a cross reactive scFv directed against the human/murine IL23R (?IL23R), a hinge and transmembrane domain derived from the murine CD28 (mCD28 linker & mCD28 TM), an activation domain of murine CD28 (mCD28) and murine CD3 zeta (mCD3Z).

(5) FIG. 5 is a combination of dot plots of flow cytometry showing murine Treg transduction efficacy. Transduction efficacy was assessed by NGFR staining in flow cytometry.

(6) FIG. 6 is a histogram showing the IL-23R CAR mediated murine Treg (mTreg) activation. mTreg cell activation was assessed by CD69 expression by flow cytometry 24 h after activation of untransduced mTreg (Poly Treg) or IL-23R-CAR transduced mTreg (IL-23R-CAR mTreg) with CD3/CD28 coated beads, or plate-coated recombinant murine IL-23R (mIL23R plate-coated 1 ?g/ml) or beads coated with human or murine IL-23R.

(7) FIG. 7 is a graph showing that IL-23R CARs mTregs exhibit efficient CAR-mediated suppressive activity in vitro. Contact dependent suppression mediated by untransduced Treg (A) or IL-23R-CAR Treg (B) stimulated through their CAR with mIL-23R coated beads (ratios 1:1 or 2:1), with CD3/CD28 coated beads or unstimulated (NS) was evaluated by measuring the proliferation of conventional T cells (Tconv) in flow cytometry.

(8) FIG. 8 is a combination of graphs showing (A) a schematic view of the in vivo experimental design and (B) the PASI score of imiquimod-induced skin inflammation model of different group of mice: untreated (saline), treated i.p. with anti-IL-23, treated i.v. with poly-mTreg (8?10.sup.6) or IL-23R-CAR mTreg (8?10.sup.6).

EXAMPLES

(9) The present invention is further illustrated by the following examples.

Example 1: Anti-IL23R CAR humanTreg

(10) Material and Methods

(11) Cells and Reagents

(12) HEK293T cells (LentiX, Ozyme, France) were cultured in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS).

(13) FoxP3 Treg isolation

(14) CD4.sup.+CD25.sup.+CD127.sup.low Treg cells were freshly isolated from buffy coats using the EasySep? Human CD4.sup.+CD127.sup.lowCD25.sup.+ Regulatory T Cell Isolation Kit from StemCell. After isolation purity was assessed by FoxP3 staining. Isolated Treg cells were plated at 5?10.sup.5 cells per well in a 24-well plate (Costar) into X-VIVO 15 media (Lonza) and activated with anti-CD3/anti-CD28 coated microbeads (Invitrogen, Carlsbad, CA) at a 1:1 bead-to-cell ratio. Tregs cells received rapamycin (100 ng/ml) at the same time that the activation. At day 2, 5, 7, and 9 IL-2 was added (1000 units/ml, Miltenyi).

(15) CAR Construct

(16) The anti-IL23R CAR construct comprises a human CD8 leader sequence (aa1-22) (e.g., having the sequence SEQ ID NO: 39), a scFv directed against the human IL23R (e.g., having the sequence SEQ ID NO: 55), a CD8 hinge (e.g., having the sequence SEQ ID NO: 13) and transmembrane domain derived from the human CD8 alpha (aa138-206) (e.g., having the sequence SEQ ID NO: 21), an activation domain of human 4-1BB (aa214-255) (e.g., having the sequence SEQ ID NO: 29) and CD3 zeta (aa52-164) (e.g., having the sequence SEQ ID NO: 26). The CAR construct is in frame with a P2A-GFP coding sequence.

(17) Vector and Titration

(18) The CAR constructs were produced using a classical 4-plasmid lentiviral system. Briefly, HEK293T cells were transfected with a third-generation lentiviral transfer vector (pTX266), the plasmid expressing HIV-1 gagpol (pMDLgpRRE), the plasmid expressing HIV-1 rev (pRSV.Rev) and the plasmid expressing VSV-G, the envelope glycoprotein of the vesicular stomatitis virus (pMD2.G). One-day post-transfection, viral supernatants were harvested, concentrated by centrifugation, aliquoted and frozen at ?80? C. for long term storage. The infectious titers, expressed as the number of transducing units per milliliter (TU/ml), were obtained after transduction of Jurkat T cells with a serial dilution of viral supernatants and transduction efficiency evaluated after 3 days by monitoring the green fluorescent protein (GFP) expression using flow cytometry.

(19) Lentiviral Transduction

(20) Tregs were transduced 2 days after their activation with a chimeric receptor composed of the sequence leader of the CD8 followed by an scFv anti-IL-23R. This extracellular domain is linked to the signaling sequence via the hinge and the transmembrane region of the human CD8. The signaling sequence is composed to the intracellular domain of 4-1BB followed by intracellular human CD3? chain. Briefly transduction was carried out by loading 0.5?10.sup.7 Transduction unit (TU) per ml to each well. After 6 hours at 37? C., viral particles were removed by washout. The plates were then incubated at 37? C. with 5% CO2. The efficiency of transduction (assessed by GFP expression) and the level of chimeric receptors at cell surface (assessed by protein-L staining) were checked 5 days after transduction.

(21) Protein-L Staining

(22) The quantification of cell surface CAR expression was performed by labelling the CAR with APC-conjugated protein L and analyzed using flow cytometry. Briefly, after wash, cells were resuspended in 0.2 ml of the ice-cold wash buffer (PBS 4% BSA) and incubated with 5 ?g of protein L at 4? C. for 20 minutes. Cells were washed with 0.2 ml of the ice-cold wash buffer three times, and then incubated (in the dark) with 1 ?l of APC-conjugated streptavidin in 0.2 ml of the wash buffer. Immunofluorescence staining was analyzed on a MACQUANT (Miltenyi) using MacsQuantify software (Miltenyi).

(23) Results

(24) Human Treg cells were sorted using a kit from Stem Cell based on CD4.sup.+CD127.sup.lowCD25.sup.+ profile and pre-stimulated with anti-CD3/anti-CD28 coated beads as well as IL-2 for 2 days prior to viral transduction. Treg were transduced with a chimeric antigen receptor (IL-23R-CAR) composed of a single chain variable fragment (scFv) of an anti-IL-23R and is linked to a signaling sequence via the hinge and the transmembrane region of the human CD8, to the intracellular part of a human 4-1BB, which was in turn fused to an intracellular human CD3? chain (see FIG. 1).

(25) Viral supernatant was added to 5?10.sup.5 stimulated Tregs in 250 ?l Xvivo medium. Cells were cultured for 7 days with addition of IL-2 every 2 days. Transduction efficiency assessed by GFP expression in FoxP3 positive cells show around 80% of GFP.sup.+ and all of them expressed the CAR at cell surface (FIG. 2).

(26) We then examined the ability of transduced Tregs to be activated via the IL-23R CAR. Tregs were stimulated with plate-coated recombinant human IL-23R or beads-coated IL-23R. 24 h after stimulation the status of activation was analyzed through CD69 expression in flow cytometry.

(27) CD69 up-regulation in presence of plate or beads coated recombinant human IL-23R demonstrates that IL-23R CAR transduced Tregs can be activated through CAR triggering (FIG. 3).

Example 2: Anti-IL23R CAR Murine Treg

(28) Material and Methods

(29) FoxP3 Treg Isolation

(30) CD4.sup.+CD25.sup.+ murine Treg cells were freshly isolated from spleen of C57B16 mice using the Regulatory T Cell Isolation Kit from life technologies. After isolation purity was assessed by FoxP3 staining. Isolated Treg cells were plated at 5?10.sup.5 cells per well in a 24-well plate into RPMI 10% SVF and activated with anti-CD3/anti-CD28-coated microbeads (Invitrogen) at a 2:1 bead-to-cell ratio. Tregs cells received rapamycin (50 ng/ml) at the same time that the activation and at Day4. Finally, at day 0, 2, 4, IL-2 was added (1000 units/ml).

(31) CAR Construct

(32) The anti-murine IL23R CAR construct used in this experiment (e.g., having the nucleic acid sequence SEQ ID NO: 77 and the amino acid sequence SEQ ID NO: 78) comprises a human CD8 leader sequence (CD8), a cross reactive scFv directed against the human/murine IL23R (?IL23R), hinge and transmembrane domains derived from the murine CD28 (mCD28 linker & mCD28 TM), an activation domain of murine CD28 (mCD28) and murine CD3 zeta (mCD3Z).

(33) Vector and Titration

(34) The CAR constructs were produced using a classical 4-plasmid lentiviral system. Briefly, HEK293T cells were transfected with a third-generation lentiviral transfer vector, the plasmid expressing HIV-1 gagpol (pMDLgpRRE), the plasmid expressing HIV-1 rev (pRSV.Rev) and the plasmid expressing Eco-MLV, the envelope glycoprotein of the ecotropic murine leukemia virus (pCMV-Eco). One-day post-transfection, viral supernatants were harvested, concentrated by centrifugation, aliquoted and frozen at ?80? C. for long term storage. The infectious titers, expressed as the number of transducing units per milliliter (TU/ml), were obtained after transduction of Jurkat T cells with a serial dilution of viral supernatants and transduction efficiency evaluated after 3 days by monitoring the green fluorescent protein (GFP) expression using flow cytometry.

(35) Lentiviral Transduction

(36) Tregs were transduced 2 days after their activation with a chimeric receptor composed of the sequence leader of the CD8 followed by an scFv anti-IL-23R. This extracellular domain is linked to the signaling sequence via the hinge and the transmembrane region of the human CD8. The signaling sequence is composed to the intracellular domain of 4-1BB followed by intracellular human CD3? chain. Briefly transduction was carried out by loading 0.5?10.sup.7 TU/ml to each well. After 6 hours at 37? C., viral particles were removed by washout. The plates were then incubated at 37? C. with 500 CO.sub.2. The efficiency of transduction (assessed by GFP expression) and the level of chimeric receptors at cell surface (assessed by protein-L staining) were checked 5 days after transduction.

(37) Activation Assay of CAR-Tregs

(38) The activation assay was performed at day 7 of the culture. Briefly, 0.05?10.sup.6 Treg were seeded in 96 U bottom plate alone or in presence of anti CD28/antiCD3 coated beads (in a 2 to 1 Treg to beads ratio), or in presence of plate-coated murine IL-23R (1 ?g/ml) or in presence of dose escalation of beads coated human as well as murine IL-23R in a 200 ?l final volume. After 24 h at 37? C., 5% CO.sub.2, cells were stained for CD4 and CD69 and then analyzed using flow cytometry.

(39) Suppression Assay of T Cell Proliferation

(40) The suppression assay was performed at day 7 of the culture. Splenocytes from OTII mice (Tg for OVA specific TCR) were cocultured for 4 days in presence of ovalbumin (100 ?g/ml), untransduced Tregs (Poly Treg) or IL-23RmCAR Tregs with or without murine IL-23R coated beads or anti-CD3/CD28 beads. At day 4, cells were harvested, and proliferation of CD4 from splenocytes was assessed by flow cytometry through the determination of dye 450 dilution. The percentage of inhibition of Tconv proliferation was calculated as followed:

(41) $100-\frac{\%\mathrm{of}\mathrm{Tconv}\mathrm{proliferation}\mathrm{in}\mathrm{presence}\mathrm{of}\mathrm{CAR}-\mathrm{Treg}?100}{\%\mathrm{of}\mathrm{Tconv}\mathrm{proliferation}\mathrm{in}\mathrm{absence}\mathrm{of}\mathrm{CAR}-\mathrm{Treg}}$

(42) Imiquimod-Induced Psoriasis-Like Skin Inflammation

(43) Mice (C57BL/6) at 8 to 10 wk of age received a daily topical dose of 62.5 mg of commercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals) on the shaved back for 7 consecutive days, Control mice were treated similarly with a control vehicle cream (Vaseline Lanette cream; Fagron). AT day 2, Anti-IL-23 (100 ug) was injected intra peritoneally and 8?10.sup.6 Treg per mice (Poly or aIL-23R-CAR) were injected intravenously. All mice were sacrificed at Day 7. To score the severity of inflammation of the back skin, an objective scoring system was developed based on the clinical Psoriasis Area and Severity Index (PASI). Redness, scaling, and thickening were scored independently on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. The level of redness was scored using a scoring table with red taints. The cumulative score (redness plus scaling plus thickening) served as a measure of the severity of inflammation (scale 0-12). All experiments were approved by the animal ethics committee according to French legislation on animal experiments.

(44) Results

(45) After isolation, murine Treg (mTreg) cells were pre-stimulated with antiCD3/anti-CD28 coated beads as well as IL-2 for 2 days prior to viral transduction. mTreg were transduced with a chimeric antigen receptor (IL-23R-mCAR) composed of a single chain variable fragment (scFv) of the cross-reactive hu/ms anti-IL-23R and is linked to a signaling sequence via the hinge and the transmembrane region of the murin CD8 to the intracellular part of a murin CD28 which was in turn fused to an intracellular murin CD3? chain (FIG. 4).

(46) Viral supernatant was added to 5?10.sup.5 stimulated Tregs in 250 ?l Xvivo medium. Cells were cultured for 7 days with addition of IL-2 every 2 days. Transduction efficiency as well as CAR expression at cell surface, was assessed by NGFR staining and showed around 70% of transduced cells expressing CAR at cell surface. (FIG. 5).

(47) Then, the ability of transduced mTregs to be activated via the IL-23R CAR was examined. mTregs were stimulated with plate-coated recombinant murine IL-23R or beads-coated human as well as murine IL-23R. 24 h after stimulation the status of activation was analyzed through CD69 expression in flow cytometry. CD69 up-regulation in presence of plate or beads coated recombinant human/murine IL-23R demonstrates that IL-23R CAR transduced mTregs can be activated through CAR triggering (FIG. 6).

(48) Finally, the important feature is that FoxP3.sup.+ Treg cells transduced with IL-23R-CAR maintain their suppressive contact dependent function (FIGS. 7A and B) through the CAR triggering.

(49) Finally, as shown in FIGS. 8 A and B, the IL-23R CAR Treg was tested for its activity in vivo in the imiquimod-induced skin inflammation model, a model described to be driven by the IL-23/IL-23R axis. An anti-IL-23 antibody (white squares) confirmed that the blockade of IL-23 induce a reduction of clinical score. Interestingly, IL-23R-CAR mTreg (white circles) are also capable to induce a reduction, two days after their i.v. infusion, of clinical score whereas polyclonal Treg (white triangle) have no effect on the clinical course.