A CHIMERIC ANTIGEN RECEPTOR CONSTRUCT ENCODING A CHECKPOINT INHIBITORY MOLECULE AND AN IMMUNE STIMULATORY CYTOKINE AND CAR-EXPRESSING CELLS RECOGNIZING CD44v6

Abstract

A recombinant nucleic acid expression construct including a first nucleic acid sequence region encoding a chimeric antigen receptor (CAR), a second nucleic acid sequence region encoding a checkpoint inhibitory molecule, and a third nucleic acid sequence region encoding an immune stimulatory cytokine. A recombinant nucleic acid expression construct encoding the CAR specifically recognizes CD44v6, and includes a PD1 checkpoint inhibitory molecule, and an immune stimulating cytokine. Further aspects relate to genetically modified cells, including a recombinant nucleic acid expression construct encoding the CAR, wherein the cells are preferably immune cells, more preferably NK cells or cytotoxic T lymphocytes or T helper cells. Medical use of the cells may be in the treatment of a medical disorder associated with the presence of pathogenic cells expressing CD44v6, preferably cancer cells, more preferably cancer stem cells of solid or liquid malignancies.

Claims

1. A recombinant nucleic acid expression construct, comprising: (a.) a first nucleic acid sequence region encoding a chimeric antigen receptor (CAR), (b.) a second nucleic acid sequence region encoding a checkpoint inhibitory molecule, and (c.) a third nucleic acid sequence region encoding an immune stimulatory cytokine.

2. The recombinant nucleic acid expression construct according to claim 1, wherein the first nucleic acid sequence region encoding the CAR comprises: (d.) a nucleic acid sequence encoding an extracellular antigen-binding domain, said antigen-binding domain preferably comprising an antibody or antibody fragment, (e.) a nucleic acid sequence encoding a transmembrane domain, and (f.) a nucleic acid sequence encoding an intracellular co-stimulatory domain.

3. The recombinant nucleic acid expression construct according to claim 1, wherein at least the first nucleic acid sequence region encoding the CAR is constitutively expressed by a promoter or promoter/enhancer combination.

4. (canceled)

5. The recombinant nucleic acid expression construct according to claim 1, wherein at least the first nucleic acid sequence region encoding the CAR and the second nucleic acid sequence region encoding the checkpoint inhibitory molecule, are configured to encode a polycistronic mRNA comprising coding regions for the polypeptide sequences of the CAR and the checkpoint inhibitory molecule, and wherein an amino acid sequence comprising a polypeptide cleavage site is disposed between the CAR polypeptide and the checkpoint inhibitory molecule polypeptide.

6. The recombinant nucleic acid expression construct according to claim 5, wherein the polypeptide cleavage site is selected from the group consisting of P2A, T2A, E2A and F2A.

7. The recombinant nucleic acid expression construct according to claim 1, wherein the checkpoint inhibitory molecule encoded by the second nucleic acid sequence region is a dominant negative polypeptide and/or an antibody inhibiting and/or blocking an immune checkpoint protein.

8. The recombinant nucleic acid expression construct according to claim 7, wherein the checkpoint inhibitory polypeptide is a dominant negative truncated PD1 polypeptide or a PD1 antibody.

9. The recombinant nucleic acid expression construct according to claim 1, wherein the third nucleic acid sequence region encoding an immune stimulatory cytokine comprises a nucleic acid sequence encoding one or more immune stimulatory cytokines operably linked to one or more promoters, wherein at least one of said cytokines is selected from the group consisting of IL-15, IL-15RA, IL-2, IL-7, IL-12, IL-21, IFN gamma, and IFN beta.

10. The recombinant nucleic acid expression construct according to claim 9, wherein the third nucleic acid sequence region encoding the immune stimulatory cytokine is operably linked to one or more constitutive promoters, and wherein the immune stimulatory cytokine maintains or enhances the activity, survival and/or number of immune cells within and/or in proximity to tumor tissue.

11. The recombinant nucleic acid expression construct according to claim 1, wherein said construct optionally comprises an additional nucleic acid sequence region encoding a chemokine receptor.

12. The recombinant nucleic acid expression construct according to claim 11, wherein the chemokine receptor is CC chemokine receptor type 4 (CCR4).

13. The recombinant nucleic acid expression construct according to claim 1, wherein said construct optionally comprises a further nucleic acid sequence region encoding a suicide gene.

14. The recombinant nucleic acid expression construct according to claim 1, wherein the extracellular antigen-binding domain of the CAR encoded by the first nucleic acid sequence region specifically recognizes human CD44v6.

15. The recombinant nucleic acid expression construct according to claim 14, wherein the said CAR comprises: a CAR signal sequence; an antigen-binding domain of a CAR that specifically recognizes CD44v6; an immunoglobulin heavy chain extracellular constant region of a CAR; a CD28 signalling domain; wherein the CD28 signaling domain comprises a transmembrane domain; and a CD3 zeta signaling domain.

16. The recombinant nucleic acid expression construct according to claim 15, wherein the CAR comprises: a CAR signal sequence according to SEQ ID NO 14 or a sequence with at least 80% sequence identity to SEQ ID NO 14; an antigen-binding domain of a CAR that specifically recognizes CD44v6 according to SEQ ID NO 15 and SEQ ID NO 19 or a sequence with at least 80% sequence identity to SEQ ID NO 15 and 19; an immunoglobulin heavy chain extracellular constant region of a CAR according to SEQ ID NO 23 or a sequence with at least 80% sequence identity to SEQ ID NO 23; a CD28 signalling domain according to SEQ ID NO 24 or a sequence with at least 80% sequence identity to SEQ ID NO 24; wherein the CD28 signaling domain comprises a transmembrane domain according to SEQ ID NO 25 or a sequence with at least 80% sequence identity to SEQ ID NO 25; and a CD3 zeta signaling domain according to SEQ ID NO 26 or a sequence with at least 80% sequence identity to SEQ ID NO 26.

17. (canceled)

18. The recombinant nucleic acid expression construct according to claim 1, wherein the checkpoint inhibitory molecule comprises: a dominant negative truncated form of a checkpoint protein, wherein said checkpoint protein is positioned adjacently to a polypeptide cleavage site for cleaving the checkpoint inhibitory molecule from the CAR polypeptide.

19. The recombinant nucleic acid expression construct according to claim 18, wherein the dominant negative truncated form of a checkpoint protein is dominant negative truncated PD1 according to SEQ ID NO 28 or a sequence with at least 80% sequence identity to SEQ ID NO 28 and wherein the cleavage site is selected from the group consisting of P2A, T2A, E2A and F2A.

20. The recombinant nucleic acid expression construct according to claim 1, wherein the immune stimulatory cytokine comprises: A signal sequence; A N-terminal IL15RA polypeptide; A linking loop sequence; and An IL-15 polypeptide.

21. The recombinant nucleic acid expression construct according to claim 20, wherein the immune stimulatory cytokine comprises: A signal sequence according to SEQ ID NO 29 or a sequence with at least 80% sequence identity to SEQ ID NO 29; A N-terminal IL15RA polypeptide according to SEQ ID NO 30 or a sequence with at least 80% sequence identity to SEQ ID NO 30; A linking loop sequence according to SEQ ID NO 31 or a sequence with at least 80% sequence identity to SEQ ID NO 31; and An IL-15 polypeptide according to SEQ ID NO 32 or a sequence with at least 80% sequence identity to SEQ ID NO 32.

22. The recombinant nucleic acid expression construct according to claim 1, wherein said construct comprises nucleic acid sequence regions encoding: a CAR that specifically recognizes human CD44v6, a checkpoint inhibitory molecule dominant negative truncated PD1 polypeptide, and an immune stimulatory cytokine, comprising a signal sequence, a N-terminal IL15RA polypeptide, a linking loop sequence, and an IL-15 polypeptide.

23. The recombinant nucleic acid expression construct according to claim 22, wherein the checkpoint inhibitory molecule is positioned adjacent to a polypeptide cleavage site P2A for cleaving the checkpoint inhibitory molecule from the CAR polypeptide.

24. A chimeric antigen receptor (CAR) polypeptide encoded by the recombinant nucleic acid expression construct according to claim 1.

25. Genetically modified cells, comprising a recombinant nucleic acid expression construct according to claim 1.

26. The genetically modified cells according to claim 25, wherein the cells are selected from the group consisting of induced pluripotent stem cells (iPSC), immortalized immune cells, primary immune cells, Natural Killer (NK) cells, NK T cells, cytokine-induced killer cells (CIK), and T lymphocytes.

27. (canceled)

28. (canceled)

29. The genetically modified cells according to claim 25, wherein the cells are induced pluripotent stem cells (iPSC) line ND50039.

30. (canceled)

31. A method for the treatment of a medical disorder associated with the presence of pathogenic cells expressing CD44v6, comprising administering the genetically modified stem cells according to claim 25 to a subject, wherein the extracellular antigen-binding domain of the CAR specifically recognizes human CD44v6.

32. The method according to claim 31, wherein the medical disorder comprises cancer cells expressing CD44v6.

33. (canceled)

34. (canceled)

35. (canceled)

36. (canceled)

37. A method for producing genetically modified cells , comprising delivering or transferring a nucleic acid construct according to claim 1 into cells in vitro.

38. (canceled)

39. (canceled)

40. A pharmaceutical composition comprising genetically modified cells according to claim 25 and a pharmaceutically acceptable carrier.

41. (canceled)

42. (canceled)

43. (canceled)

44. The method according to claim 37, wherein the nucleic acid construct is transferred or delivered into the cell in vitro using electroporation.

Description

FIGURES

[0327] The invention is demonstrated by way of the example through the figures disclosed herein. The figures provided represent particular, non-limiting embodiments and are not intended to limit the scope of the invention.

SHORT DESCRIPTION OF THE FIGURES

[0328] FIG. 1: Cytotoxicity of CD44v6 CAR-transduced YT cells to MCF-7 cells.

[0329] FIG. 2: Checkpoint inhibition by a dominant negative PD1 (dnPD1opt) leads to an improvement in NFAT promoter activity.

[0330] FIG. 3: The activity IL-15 superagonists (15R15) compared to IL-2 or IL-I5.

[0331] FIG. 4: The NF-kB promoter activity of anti-CEA CAR expressing Jurkat cells stimulates with target cells (MCF-7) with or without checkpoint inhibition (dnPD1opt) or IL-IS superagonists (15R15).

[0332] FIG. 5: Specific kill of MCF-7 cells with primary NK cells transduced with CD44v6 CAR construct.

[0333] FIG. 6: PD1 transfected reporter Jurkat cells stimulated with MCF-7.

[0334] FIG. 7: Evaluation of disease indication by CD44v6 expression analysis.

DETAILED DESCRIPTION OF THE FIGURES

[0335] FIG. 1: Cytotoxicity of CD44v6 CAR-transduced YT cells to MCF-7 cells: YT cells were transduced with CD44v6 CAR construct encoding lentivirus with dnPD1opt and 15R15 (YT CD44v6 CAR 15R15) or empty vector (YT control construct). YT CD44v6 CAR 15R15 cells or YT control construct were then added to duplicate wells of a 96-well plate containing MCF-7 cells, and a non-specific cytotoxic signal (etoposide 10 ug/ml) was added to further wells. Cytotoxicity was determined after 18 hours.

[0336] FIG. 2: Checkpoint inhibition by a dominant negative PD1 (dnPD1opt) leads to an improvement in NFAT promoter activity: Jurkat cells expressing GFP under the control of the NFAT promoter were transduced with a dentPD1opt expressing lentiviral vector or a control vector. The cells were then exposed 1 day earlier to a cell line expressing high levels of PD-L1 (U251-PD-L1) in duplicate wells of a 96-well plate with increasing levels of a TCR-linked proliferative signal (phytohemagglutinin: PHA). The number of GFP expressing cells was determined by flow cytometry.

[0337] FIG. 3: The activity IL-I5 superagonists (15R15) compared to IL-2 or IL-I5: The IL-15 transgene 15R15 or an empty plasmid was expressed in HEK293 cells after transient transfection and collected 2 days after transfection. The supernatant was double tested for IL-2/IL-15 specific activity in a bioassay using the Hek-Blue IL-2 reporter cell line as described by the manufacturer (Invivogen).

[0338] FIG. 4: The NF-kB promoter activity of anti-CEA CAR expressing Jurkat cells stimulates with target cells (MCF-7) with or without checkpoint inhibition (dnPD1opt) or IL-15 superagonists (15R15): Jurkat cells expressing GFP under the control of an NF-kB promoter were transduced with a lentiviral vector encoding (1) a CEA-CAR construct combined with dnPD1opt and 15R15 (Jurkat-CEA-CAR-dnPD1opt-IL15R15), (2) a CEA-CAR construct combined with dnPD1opt (Jurkat-CEA-CAR-dnPD1opt), (3) a CEA-CAR construct (Jurkat CEA-CAR) or (4) an empty lentiviral vector (empty vector). The transduced Jurkat cells were transferred to MCF-7 target cells. After 1 day, positive cells were analysed by flow cytometry.

[0339] FIG. 5: Specific kill of MCF-7 cells with primary NK cells transduced with CD44v6 CAR construct: Primary NK cells were transduced with CD44v6 CAR construct or empty vector and compared with non-transduced primary NK cells. Cells were added to duplicate wells of a 96-well plate containing MCF-7 cells, and a non-specific cytotoxic signal (etoposide 10 ug/ml) was added to further wells. Cytotoxicity was determined after 18 hours.

[0340] FIG. 6: PD1 transfected reporter Jurkat cells stimulated with MCF-7: PD1 transfected reported Jurkat cells were transduced with a lentiviral vector under the control of an SSFV promoter encoding (1) an empty lentiviral vector (empty vector, left bar), (2) a CD44v6-CAR (CD44v6 CAR, middle bar) or (3) a CD44v6-CAR construct combined with dnPD1opt (CD44v6 checkpoint inhibition CAR, right bar). The transduced Jurkat cells were transferred to MCF-7 target cells. Signal intensity of positive cells was analysed by flow cytometry.

[0341] FIG. 7: Evaluation of disease indication by CD44v6 expression analysis: CD44v6 is highly expressed in different (A) Multiple Myeloma cell lines and (B) AML cell lines.

EXAMPLES

[0342] The invention is demonstrated through the examples disclosed herein. The examples provided represent particular embodiments and are not intended to limit the scope of the invention. The examples are to be considered as providing a non-limiting illustration and technical support for carrying out the invention.

[0343] Lentiviral vectors encoding the anti-CD44v6 CAR, a checkpoint inhibitor, the dominant negative truncated PD-1 protein, an immunostimulatory cytokine, the IL-15 transgenic 15R15, or a combination of these, are introduced into immune cell lines using the well-known method of lentiviral gene transfer. The cell line modified in this way lyses the CD44v6-positive cancer cell line MCF-7, while the immune cell line YT Is not modified with the anti-CD44v6 CAR and cannot sufficiently lyse the cancer cell line MCF-7 (Example 1, FIG. 1).

[0344] Further examples show [0345] the successful PD-1 checkpoint inhibition (Example 2, FIG. 2), [0346] the high activity of the IL-15 superagonist (Example 3, FIG. 3), [0347] the killing activity in primary NK cells (Example 1, FIG. 5), [0348] the CD44v6 CAR expression in liquid cancer cell lines (Example 5, FIG. 7)

[0349] According to Dull et al. (1998), the packaging, production and cell transduction of lentiviruses was carried out using the described high-security 3rd generation plasmid system. The cells transfection was performed using polyethyleneimine according to the manufacturer's instructions (Polyplus).

[0350] Hek 293T and MC32A cells were cultivated in DMEM with 10% heat-inactivated FCS and penicillin/streptomycin. Jurkat cells were cultured in RPMI with 10% heat-inactivated FCS and penicillin/streptomycin. YT cells were cultivated in RPMI with 10% heat-inactivated FCS, penicillin/streptomycin and 10 IU/ml IL-2. GFP was measured by flow cytometry using a FACS-Calibur. Cytotoxicity was determined by a crystal violet assay or a chromium release assay according to standard procedures.

Example 1: Cytotoxicity of YT Cells to MCF-7 Cells Wherein YT Cells are Transduced with Anti-CD44v6 CAR Construct

[0351] This example refers to experimental results with YT cells transduced with empty a lenitviral vector (YT control construct) or a lentiviral vector comprising nucleic acid sequence regions encoding a chimeric antigen receptor (CAR) binding to CD44v6, a checkpoint inhibitor dnPD1opt, and/or an immune stimulatory cytokine 15R15 (YT CD44v6 CAR 15R15). YT CD44v6 CAR 15R15 cells or YT control construct cells were then added in duplicates to a 96-well plate containing MCF-7 cells, and a non-specific cytotoxic signal (etoposide 10 ug/ml) was added to further wells. Cytotoxicity was determined after 18 hours. The DNA sequence of the CAR was transferred to the immune cell line YT using the well-known method of lentiviral gene transfer. The cell line modified in this way lyses the CD44v6-positive cancer cell line MCF-7. The immune cell line YT, which is not modified with the CAR, does not sufficiently lyse the cancer cell line MCF-7 (FIG. 1).

[0352] Primary NK cells transduced with CD44v6 CAR construct efficiently lyses the CD44v6-positive cancer cell line MCF-7 compared to cells transduced with an empty vector or with non-transduced primary NK cells (FIG. 5).

Example 2: Checkpoint Inhibition by a Dominant Negative PD1 (dnPD1opt) Leads to an Improvement in NFAT Promoter Activity

[0353] The example relates to experiments that show successful inhibition of the checkpoint protein PD-1 through the dominant negative truncated form of PD-1, dntPD1opt. Jurkat cells expressing GFP under the control of the NFAT promoter (FIG. 2). The Jurkat cells were transduced with a control lentiviral vector or the checkpoint inhibitor (dntPD1opt) encoding lentiviral vector. One day before, these cells were exposed in duplicate in a 96 well plate to a cell line expressing high concentrations of PD-L1 (U251-PD-L1) and an increasing concentration of a TCR mediated T cell stimulus, phytohaemagglutinin (PHA). The number of GFP expressing cells, determined by flow cytometry, was higher for dntPD1opt expressing Jurkat cells compared to the negative control. This demonstrates a successful inhibition of the checkpoint protein PD-1 at biological relevant levels. Example 3: The activity IL-15 superagonists (15R15) compared to IL-2 or IL-15

[0354] The example relates to experiments that proves superagonist activity of the IL-15 transgene 15R15 (FIG. 3). The IL-15 transgene 15R15 or an empty plasmid was expressed in HEK293 cells after transient transfection and collected 2 days after transfection. The supernatant was tested twice for IL-2/IL-15 specific activity in a bioassay using the Hek-Blue IL-2 reporter cell line as described by the manufacturer (Invivogen). The OD260 was measured. This demonstrates successfully a superagonist activity of the IL-15 transgene 15R15 that is higher than the negative control, IL-2, and IL-15 at biological relevant levels.

Example 4: The NF-kB Promoter Activity of Anti-CEA CAR Expressing Jurkat Cells Stimulates with Target Cells (MCF-7) with or without Checkpoint Inhibition (dnPD1opt) or IL-15 Superagonists (15R15)

[0355] In a further example, the inventors employed an antigen binding domain in the CAR that binds CEA. The carcinoembryonic antigen (CEA) is a transmembrane glycoprotein of the immunoglobulin superfamily. CEA is found both as a soluble form in blood and bound to cell membranes, mostly tumor cells. As a cell surface glycoprotein, the protein is involved in cell adhesion, intracellular signal transduction and tumor progression. CEA in its soluble form is used as a clinical biomarker for solid, especially malignant tumors, such as gastrointestinal cancers and may promote tumor development through its role as a cell adhesion molecule. It also plays a role as an oncogene by promoting tumor progression and inducing resistance of colorectal cancer cells to therapy. In addition, the encoded protein can regulate differentiation, apoptosis and cell polarity. Currently, no curative therapy exists for CEA high-positive tumor stem cells.

[0356] The remaining components of the CAR constructs were the same as those employed in the other examples. The antigen-binding domain of the CAR was adjusted to bind CEA. The employed CAR sequences are disclosed in the table above under SEQ ID NO 36-43, the coding sequence for the complete variable domain coding the antigen binding domain is disclosed in SEQ ID NO 44.

[0357] The example relates therefore to experiments showing a successful NF-kB promoter activity in Jurkat cells expressing anti-CEA CAR, checkpoint inhibitor dnPD1opt, and IL-15 transgene 15R15 and challenged with MCF-7 target cells. Refer FIG. 4.

[0358] GFP expression under the control of the NF-kB promoter was determined in Jurkat cells transduced with a lentiviral vector encoding (1) an anti-CEA CAR construct combined with checkpoint inhibitor dntPD1opt and IL-15 transgene 15R15 (Jurkat-CEA-CAR-dnPD1opt-IL15R15) or (2) an anti-CEA CAR construct combined with checkpoint inhibitor dntPD1opt (Jurkat-CEA-CAR-dnPD1opt), (3) an anti-CEA-CAR (Jurkat CEA-CAR) or (4) an empty lentiviral vector (empty vector). The transduced Jurkat cells were transferred to MCF-7 target cells. After 1 day, GFP positive cells were determined by flow cytometry.

[0359] The combination Jurkat-CEA-CAR-dnPD1opt-IL15R15 shows GFP expression at a very high level and higher than in Jurkat-CEA-CAR-dnPD1opt, Jurkat CEA-CAR, empty vector samples. The GFP expression level in Jurkat-CEA-CAR-dnPD1opt-IL15R15 is greater than an additive effect and thus proves a synergistic effect of the combination described in this invention.

[0360] The example also relates therefore to another experiment showing a successful SFFV promoter activity in Jurkat cells expressing CD44v6 CAR or CD44v6 checkpoint inhibition CAR and challenged with MCF-7 target cells. Refer FIG. 6.

Example 5: Evaluation of Disease Indication by CD44v6 Expression Analysis

[0361] The CAR target CD44v6 is strongly expressed on MM, Lymphomas, and Leukemias (FIG. 7), but is absent or weakly expressed in normal cells (non-cancerous cells). CD44v6 drives growth of cancer-initiating stem cells and tumor progression. Therefore, the CD44v6 CAR presents a high safety profile. For example, CD44v6-targeted T cells do not recognize hematopoietic stem cells and keratinocytes and can cause reversible monocytopenia.

REFERENCES

[0362] Kreye, J., et al Human cerebrospinal fluid monoclonal N-methyl-D-aspartate receptor autoantibodies are sufficient for encephalitis pathogenesis. Brain 139, 2641-2652 (2016).