TREM2 CHIMERIC RECEPTOR

Abstract

The present invention relates to chimeric receptors (e.g. CARs including both single chain and multichain CARs) that bind to TREM2 ligands and their use in therapy. In particular, the invention provides a chimeric receptor comprising: (a) an exodomain comprising the ligand binding domain of TREM2 or a functional variant thereof, optionally wherein said exodomain is resistant to cleavage by a sheddase; (b) a transmembrane domain; and (c) an endodomain comprising an intracellular signalling domain.

Claims

1. A chimeric receptor comprising: (a) an exodomain comprising the ligand binding domain of TREM2 or a functional variant thereof; (b) a transmembrane domain; and (c) an endodomain comprising an intracellular signalling domain.

2. The chimeric receptor of claim 1, wherein said exodomain is resistant to cleavage by a sheddase.

3. The chimeric receptor of claim 1 or claim 2, wherein the chimeric receptor comprises (a)-(c) in a single polypeptide chain.

4. The chimeric receptor of claim 1, 2 or 3, wherein the chimeric receptor comprises two or more polypeptide chains, wherein at least one of the polypeptide chains comprises linked domains from two or more proteins, optionally wherein the exodomain and endodomain are in different polypeptide chains.

5. The chimeric receptor of any one of claims 2 to 4, wherein the sheddase is a member of the ADAM (a disintegrin and metalloproteinase) protein family or is a member of the metalloproteinases, such as meprin β.

6. The chimeric receptor of any one of claims 2 to 5, wherein the sheddase is ADAM10 and/or ADAM 17.

7. The chimeric receptor of any one of claims 1 to 6, wherein the exodomain comprises: (i) a functional variant of an amino acid sequence as set forth in SEQ ID NO: 3; or (ii) a functional variant of an amino acid sequence as set forth in SEQ ID NO: 4, wherein the amino acid at the position equivalent to position 78 of SEQ ID NO: 4 is a basic amino acid, preferably lysine or arginine.

8. The chimeric receptor of any one of claims 1 to 7, wherein the exodomain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 5 or 6 or a functional variant thereof, wherein the amino acid at the position equivalent to position 78 of SEQ ID NO: 6 is a basic amino acid, preferably lysine or arginine.

9. The chimeric receptor of any one of claims 1 to 7, wherein the exodomain comprises or consists of: (i) an amino acid sequence as set forth in SEQ ID NO: 7 or 8 or a functional variant thereof, wherein (a) the amino acid at the position equivalent to position 78 of SEQ ID NO: 8 is a basic amino acid, preferably lysine or arginine; (b) the amino acids at positions equivalent to positions 139-140 of SEQ ID NO: 7 or 8 are: (1) not histidine and/or serine, respectively; and/or (2) modified to make the exodomain resistant to cleavage by the sheddase; and optionally (c) the amino acids at the position equivalent to positions 118-119 of SEQ ID NO 7 or 8 are: (1) not arginine and/or aspartic acid, respectively; and/or (2) modified to make the exodomain resistant to cleavage by a sheddase.

10. The chimeric receptor of any one of claims 1 to 9, wherein the chimeric receptor comprises a hinge domain between the ligand binding domain of TREM2 and the transmembrane domain.

11. The chimeric receptor of claim 10, wherein the hinge domain is, or is derived from, the hinge region or stalk domain of human CD8α, CD4, CD28, CD7 or TREM2.

12. The chimeric receptor of any one of claims 1 to 11, wherein the chimeric receptor comprises a signal sequence upstream of the ligand binding domain of TREM2.

13. The chimeric receptor of claim 12, wherein the signal sequence is a CD8a signal sequence.

14. The chimeric receptor of any one of claims 1 to 13, wherein the chimeric receptor comprises one or more co-stimulatory signalling domains.

15. The chimeric receptor of claim 14, wherein the one or more co-stimulatory signalling domains is from a protein selected from CD27, CD28, 4-IBB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2 and a ligand that specifically binds CD83.

16. The chimeric receptor of any one of claims 1 to 15, wherein the transmembrane domain is from a protein selected from a receptor tyrosine kinase (RTK), an M-CSF receptor, CSF-1R, Kit, TIE3, an ITAM-containing protein, DAP12, DAP10, an Fc receptor, FcR-gamma, FcR-epsilon, FcR-beta, TCR-zeta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CD3-eta, CDS, CD22, CD79a, CD79b, CD66d, TNF-alpha, NF-kappaB, a TLR (toll-like receptor), TLRS, Myd88, lymphocyte receptor chain, IL-2 receptor, IgE, IgG, CD16α, FcγRIII, FcγRII, CD28, 4-1BB, CD4, CD8, e.g. CD8α, NKG2D (CD314) and TREM2.

17. The chimeric receptor of any one of claims 1 to 16, wherein the intracellular signalling domain is from a protein selected from a receptor tyrosine kinase (RTK), an M-CSF receptor, CSF-1R, Kit, TIE3, an ITAM-containing protein, DAP12, DAP10, an Fc receptor, FcR-gamma, FcR-epsilon, FcR-beta, TCR-zeta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CD3-eta, CDS, CD22, CD79a, CD79b, CD66d, TNF-alpha, NF-KappaB, a TLR (toll-like receptor), TLRS, Myd88, TOR/CD3 complex, lymphocyte receptor chain, IL-2 receptor, IgE, IgG, CD16α, FcγRIII, FcγCD28, 4-1BB, and any combination thereof.

18. The chimeric receptor of any one of claims 1 to 17, wherein the chimeric receptor comprises a signal peptide from CD8a; a hinge domain, transmembrane domain, co-stimulatory domain from CD28; and a CD3ζ intracellular signalling domain.

19. The chimeric receptor of any one of claims 1 to 18, wherein the chimeric receptor comprises an amino acid sequence of any one of SEQ ID NO. 32, 33 or 40 to 56, or an amino acid having at least 90% (e.g. at least 95%) sequence identity thereto.

20. One or a plurality of nucleic acid molecules encoding a chimeric receptor of any one of claims 1 to 19.

21. A vector comprising the one or plurality of nucleic acid molecules of claim 20, optionally wherein the vector encodes a chimeric receptor having an amino acid sequence of any one of SEQ ID Nos 32, 33 or 40 to 56, or a sequence having at least 90% identity thereto.

22. A cell, preferably an immune cell, comprising the one or more nucleic acid molecules of claim 20 or vector of claim 21 and/or expressing the chimeric receptor of any one of claims 1 to 19.

23. The cell of claim 22, wherein the cell is an NK cell, a dendritic cell, a NKT cell, a MDSC, a neutrophil, a macrophage or a T cell, such as a cytotoxic T lymphocyte (CTL), helper T cell or a Treg cell.

24. A cell population comprising the cell of claim 22 or 23.

25. A pharmaceutical composition comprising the cell of claim 22 or 23 or the cell population of claim 24.

26. A cell, cell population or pharmaceutical composition of any preceding claim for use in therapy.

27. A cell, cell population or pharmaceutical composition of any preceding claim for use in preventing, reducing risk of, or treating a neurological disease, disorder, or injury or liver disease in an individual in need thereof.

28. A cell, cell population or pharmaceutical composition for use of claim 27, wherein the neurological disease, disorder, or injury is selected from amyotrophic lateral sclerosis (ALS), dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Huntington's disease, Taupathy disease, Nasu-Hakola disease, central nervous system lupus, Parkinson's disease, dementia with Lewy bodies, Multiple System Atrophy (Shy-Drager syndrome), progressive supranuclear palsy, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, seizures, spinal cord injury, traumatic brain injury (e.g. ischemia and traumatic brain injury), depression, autism spectrum disorder and multiple sclerosis.

29. A cell, cell population or pharmaceutical composition for use of claim 27, wherein the neurological disease is amyotrophic lateral sclerosis (ALS) and the cell is a regulatory T cell (Treg) and the cell population is a regulatory T cell (Treg) population.

30. A cell, cell population or pharmaceutical composition for use of claim 27, wherein the liver disease is selected from fascioliasis, hepatitis (e.g. viral hepatitis, alcoholic hepatitis or autoimmune hepatitis), alcoholic liver disease, fatty liver disease (hepatic steatosis and/or steatohepatitis), hemochromatosis, Gilberts syndrome, cirrhosis, primary biliary cirrhosis and primary sclerosing cholangitis.

31. A cell, cell population or pharmaceutical composition of any preceding claim for use in preventing, reducing risk of, or treating fibrosis or atherosclerosis in an individual in need thereof.

32. The cell, cell population or pharmaceutical composition for use of claim 31, wherein said fibrosis is fibrosis of the kidneys, lungs, liver, brain, intestines, heart or a combination thereof.

Description

[0307] The invention will now be further described by way of Figures and Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0308] FIG. 1 shows a schematic of a single chain chimeric receptor of the invention,comprising a ligand binding domain derived from TREM2 having a wild type sequence or a mutation of T96K, together with a stalk and a transmembrane domain ™ from CD28 or from CD8 alpha, a costimulatory domain from CD28 and an intracellular signalling domain from CD3zeta.

[0309] FIG. 2 shows a schematic of a multichain chimeric receptor of the invention, comprising a ligand binding domain derived from TREM2 having a wild type sequence or a mutation of T96K. The first example comprises a first polypeptide comprising a CD3zeta intracellular signalling domain and a second polypeptide comprising DAP10. The second example shows a first polypeptide comprising a CD3zeta intracellular signalling domain and a second polypeptide comprising DAP10 and a CD28 costimulatory domain. The third example show a first polypeptide with no intracellular signalling sequence or costimulatory domains, and a second polypeptide comprising DAP10 and an intracellular signalling domain of CD3zeta.

[0310] FIG. 3 shows a schematic of a multichain chimeric receptor of the invention, comprising a ligand binding domain derived from TREM2 having a wild type sequence or a mutation of T96K. The first example comprises a first polypeptide comprising a CD28 costimulatory domain and a second polypeptide comprising DAP12. The second example shows a first polypeptide comprising a CD28 costimulatory domain and a second polypeptide comprising DAP12 and a CD3zeta intracellular signalling domain. The third example show a first polypeptide with no intracellular signalling sequence or costimulatory domains, and a second polypeptide comprising DAP12 and a CD28 costimulatory domain.

[0311] FIG. 4 shows a schematic of a multichain chimeric receptor of the invention,comprising a ligand binding domain derived from TREM2 having a wild type sequence or a mutation of T96K. The first example comprises a first polypeptide comprising a CD28 costimulatory domain and a second polypeptide comprising DAP12. The second example shows a first polypeptide comprising a CD3zeta intracellular signalling domain and a second polypeptide comprising a truncated DAP12 (transmembrane only) and a CD28 costimulatory domain. The third example shows a first polypeptide with no intracellular signalling sequence or costimulatory domains, and a second polypeptide comprising a truncated DAP12 (transmembrane only), a CD28 costimulatory domain and a CD3zeta intracellular signalling domain. The fourth example shows a first polypeptide with no intracellular signalling sequence or costimulatory domains, and a second polypeptide comprising a truncated DAP12 (transmembrane only), a CD28 costimulatory domain and a CD3zeta intracellular signaling domain, wherein one ITAM sequence has been deleted.

[0312] FIG. 5 shows the expression of various proteases in Tregs. Whilst ADAM10 and 17 are expressed, Meprin beta (MEP1B) is not.

[0313] FIG. 6 shows the expression of various chimeric constructs of the invention (SEQ ID Nos 40 and 42-56) in Jurkat cells after transduction using two different concentrations of virus, using an anti-TREM2 antibody.

[0314] FIG. 7 shows the activation of Jurkat cells transduced with various chimeric constructs of the invention (SEQ ID Nos 44-54) using an anti-TREM2 antibody.

[0315] FIG. 8 shows the activation of Jurkat cells transduced with various chimeric constructs of the invention (SEQ ID Nos 44-54) using necrotic K562 cells.

[0316] FIG. 9 shows the activation of Jurkat cells transduced with various chimeric constructs of the invention (SEQ ID Nos 44-56) using HEK cell debris.

[0317] FIG. 10 shows the expression of various chimeric constructs of the invention (SEQ ID Nos 57-61) in Treg cells after transduction. SEQ ID Nos 57, 58, 59, 60 and 61 correspond to SEQ ID Nos 44, 45, 46, 48 and 49, except that they comprise an additional 2A cleavage sequence and eGFP sequence. These constructs are numbered as 5G, 6G, 7G, 9G and 10G in FIG. 10 to indicate the addition of eGFP.

[0318] FIG. 11 shows the amount of TREM2 cleavage in various chimeric constructs of the invention comprising a mutant TREM2 exodomain (SEQ ID Nos 57-60) compared to a chimeric construct of the invention comprising the wild-type TREM2 exodomain (SEQ ID NO: 69). FIG. 11a is a Western blot of samples from cells harvested at day 11. FIG. 11b is a Western blot of samples from cells harvested at day 14. FIG. 11c is an ELISA for TREM2 using media from cells harvested at day 24 and day 31.

EXAMPLES

Example 1a

Screening of TREM2 Constructs—Expression

[0319] Different constructs of the TREM2 CAR (encoding SEQ ID Nos 40 and 42-56) were cloned into a lentiviral backbone encoding a puromycin resistance gene, where the ligand binding domain of TREM2 (wildtype or mut (T96K)) was used to confer specificity to the CAR construct. Viral vectors were produced and used for the transduction of the Jurkat T cell line. Two days after transduction, Jurkat cells were selected with 4 μg/ml puromycin for one week. Cells were counted and 0.5*10.sup.6 cells were stained with an anti-TREM2 antibody (Human/mouse TREM2 APC-conjugated antibody from R&D systems (FAB17291A)) to determine the level of CAR expression. CAR expression was assessed by flow cytometry and can be seen in FIG. 6.

Example 1b

Screening of TREM2 Constructs—Activation with Antibody

[0320] Jurkat cells were transduced as described in Example 1a with constructs encoding SEQ ID Nos 44 to 54, except that the lentiviral backbone did not include a puromycin resistance gene and Jurkat cells were not selected for using puromycin. Transduced cells were activated with anti-TREM2 antibody. CAR-dependent activation levels were assessed by flow cytometry using CD69 staining and can be seen in FIG. 7,

Example 1c

Screening of TREM2 Constructs—Activation with Dead Cells or Cell Debris

[0321] Jurkat cells were transduced as described in Example 1a with constructs encoding either SEQ ID Nos 44-54 (FIG. 8) or SEQ ID Nos 44-56 (FIG. 9), except that the lentiviral backbone did not include a puromycin resistance gene and Jurkat cells were not selected for using puromycin. Transduced cells were either co-cultured with RPMI as control and with necrotic K562 cells to activate the cells (FIG. 8) or co-cultured with RPMI as control and with HEK cell debris for 24 or 48 hours to activate the cells. CAR-dependent activation levels were assessed by flow cytometry using CD69 staining and can be seen in FIGS. 8 and 9, respectively.

Example 2

NFAT/NfkB/STAT5 Signaling of CAR Constructs

[0322] Different constructs of the TREM2 CAR were cloned into a lentiviral backbone encoding a puromycin resistance gene, Viral vectors are produced and used for the transduction of a NFAT, NfkB and STAT5 Jurkat reporter cell line. Here,the NFAT, NfkB or STAT5 response element control the activity of a luc2 reporter gene. Two days after transduction, Jurkat cells are selected with 4 μg/ml puromycin for one week. Cells are then activated using ApoE2 or ApoE4 recombinant protiens coated on a cell culture plate and eight hours after activation luciferase is assessed in the different reporter cell lines using ONE-Glo™ Luciferase Assay System (Promega).

Example 3

Generation of Regulatory T Cells Expressing the TREM2 CAR

[0323] Regulatory T cells are purified and FACS sorted as CD4.sup.+ CD25.sup.+ CD127-cells from healthy donors. Cells are activated using Human T-Activator CD3/CD28 Dynabeads™ (ThermoFisher Scientific) in X-Vivo medium (Lonza) in the presence of Interleukin-2 (1000 IU/ml), After 48 hours of activation, cells are transduced with lentiviral particles,encoding TREM2 CAR constructs or encoding a control construct that comprises HLA-A2 instead of TREM2. All constructs also encode eGFP. Cells are further expanded, and expansion rate is compared between the different conditions. At day 14 cells are harvested and counted. 0.5*106 cells are stained with an anti-TREM2 antibody. The level of CAR expression and transduction efficiency was assessed by Flow Cytometry looking at the percentage of anti-TREM2 antibody and percentage of GFP expression, respectively. The Treg phenotype is assessed by surface staining with anti-CD4, anti-CD25, anti-CD127, anti-CD8, anti-GITR, anti-CD39, anti-CD45RA, anti-CD45RO, anti-ICOS and intracellular staining with anti-FOXP3 and anti-HELIOS, following fixation and permeabilization (Transcription Factor Staining Buffer Set, ThermoFisher Scientific)

[0324] FIG. 10 shows the expression of various TREM2 CAR constructs of the invention in regulatory T cells. Regulatory T cells were transduced with a control construct comprising HLA-A2 instead of TREM2 or with constructs comprising SEQ ID Nos 57, 58, 59, 60 and 61.

Example 4

Treg Suppressive Activity

[0325] For assessing the ability of Treg to suppress effector T cell activation, Teff cells are labeled with CFSE dye. Teff cells are co-cultured with different concentrations of Treg cells (ratios Treg:Teff of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:124) or no Treg cells. For activation, CD3/28 Beads (1:100) are added. For CAR-dependent activation, ApoE2 or ApoE4 recombinant proteins are added. 72 h after activation, cells are harvested and analyzed by flow cytometry. CFSE dilution is used as a surrogate marker for Teff cell proliferation.

Example 5

Treg Activation Assay

[0326] For analysis of CAR-dependent Treg activation, Treg are cultured in the presence of ApoE2 or ApoE4 recombinant proteins. Here, 0.1*10.sup.6 Treg are cultured. As a negative control, Treg are cultured in the absence of recombinant protein. As a positive control, Treg are cultured in the presence of CD3/CD28 activation beads. After 24 h cells are harvested and stained with anti-CD4, anti-CD25, anti-CD69, anti-CD137 and anti-GARP antibodies. Cells are acquired on a flow cytometer and percentage of CD69, CD137 and GARP up-regulation after stimulation are calculated.

Example 6

Generation of Effector CAR-T Cells

[0327] For the generation of Teff CAR-T cells, PBMCs were activated with anti-CD3 antibody (OKT3) for 48h. After activation, cells are washed and transduced with lentiviral vector encoding the TREM2 CAR. 48h after transduction, cells are washed and seeded for expansion until day 10. At day 10 cells are harvested and cryopreserved for further analysis. To assess CAR expression, cells are counted, and 0.5*106 cells are stained with TREM2 APC conjugated antibody to determine the level of CAR expression. CAR expression was assessed by flow cytometry. Cell phenotype was assessed by flow cytometry staining with anti-CD4, anti-CD8, anti-CD45RA, anti-CD45RO, anti-CD62L, anti-CCR7, anti-CD25 and anti-CD69 antibodies.

Example 7

TREM2 Cleavage

[0328] To confirm that the mutant TREM2 exodomain in the chimeric constructs of the invention is resistant to cleavage by the ADAM sheddases, Jurkat cells were transduced with four chimeric constructs of the invention comprising the mutant TREM2 exodomain, namely SEQ ID NOs: 57, 58, 59 and 60, and with a chimeric construct of the invention comprising the wild-type TREM2 exodomain, namely SEQ ID NO: 69.

[0329] The Jurkat cells were kept in culture and stained every 3 to 4 days with a TREM2 antibody, for up to 39 days, to confirm stable expression of the constructs in the cell membrane. Cell culture media was harvested the same day as staining. Harvested media was either frozen for later analysis by ELISA or protein was extracted for analysis by Western blot. A TREM2 antibody was used to detect the cleaved extracellular portion of TREM2, which is at around 28 kDa in the Western blot in FIGS. 11a and 11b. Cell lysates from Jurkat cells transduced with a chimeric construct of the invention (SEQ ID NO: 61) were used as a positive control for the Western blots to prove specificity of the TREM2 antibody and media from untransduced Jurkat cells (UTD—Jurkat media) was used as a negative control.

[0330] The Western blots in FIGS. 11a and 11b show that the constructs comprising the mutant TREM2 exodomain (SEQ ID NOs: 57-60) were resistant to cleavage by the ADAM sheddases, as less cleaved TREM2 was detected in media from cells transduced with these constructs compared to media from cells transduced with the construct comprising the wild-type TREM2 exodomain (SEQ ID NO: 69). This was the same at two different time points: day 11 (FIG. 11a) and day 14 (FIG. 11b). Similarly, it can be seen in the ELISA in FIG. 11c that there was less cleaved TREM2 in the media of cells transduced with constructs comprising the mutant TREM2 exodomain (SEQ ID NOs: 57-60) compared to cells comprising the wild-type TREM2 exodomain (SEQ ID NO: 69), at two different time points: day 24 and day 31.