CHIMERIC ANTIGEN RECEPTOR (CAR)-EXPRESSING CELLS RECOGNIZING CEA

Abstract

Genetically modified cells, including a recombinant nucleic acid expression construct including a first nucleic acid sequence region encoding a chimeric antigen receptor (CAR) that includes an extracellular antigen-binding domain recognizing a carcinoembryonic antigen (CEA) protein, a second nucleic acid sequence region encoding a checkpoint inhibitory molecule, and a third nucleic acid sequence region encoding an immune stimulatory cytokine. In some aspects, the genetically modified cells are T cells or NK cells, preferably cytotoxic T lymphocytes. Anti-CEA CAR-T cells or anti-CEA CAR-NK cells preferentially recognize a membrane-bound CEA protein and express a checkpoint inhibitory molecule and/or an immune stimulatory interleukin in proximity to tumor tissue. Medical use of the cells may relate to treatment of a medical disorder associated with the presence of pathogenic cells expressing CEA, preferably cancer cells, more preferably cancer cells of solid malignancies.

Claims

1. Genetically modified cells, comprising a recombinant nucleic acid expression construct encoding a CAR, said construct comprising: (a.) a first nucleic acid sequence region encoding a chimeric antigen receptor (CAR), said CAR comprising an extracellular antigen-binding domain that recognizes a carcinoembryonic antigen (CEA) protein, (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 genetically modified cells according to claim 1, wherein the extracellular antigen-binding domain recognizes a non-soluble form of the carcinoembryonic antigen (CEA) protein.

3. The genetically modified cells 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 that recognizes a CEA protein, said antigen-binding domain 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.

4. The genetically modified cells 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.

5. (canceled)

6. The genetically modified cells 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.

7. The genetically modified cells construct according to claim 6, wherein the polypeptide cleavage site is selected from the group consisting of P2A, T2A, E2A and F2A.

8. The genetically modified cells 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.

9. The genetically modified cells construct according to claim 8, wherein the checkpoint inhibitory polypeptide is a dominant negative truncated PD1 polypeptide or a PD1 antibody.

10. The genetically modified cells 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.

11. The genetically modified cells construct according to claim 10, 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.

12. The genetically modified cells according to claim 1, wherein the recombinant nucleic acid expression construct optionally comprises an additional nucleic acid sequence region encoding a chemokine receptor.

13. The genetically modified cells construct according to claim 12, wherein the chemokine receptor is C—C chemokine receptor type 4 (CCR4).

14. The genetically modified cells according to claim 1, wherein said construct optionally comprises a further nucleic acid sequence region encoding a suicide gene.

15. The genetically modified cells according to claim 1, comprising a recombinant nucleic acid expression construct that encodes a CAR, said CAR comprising: a CAR signal sequence; an antigen-binding domain of a CAR that specifically recognizes CEA; an immunoglobulin heavy chain extracellular constant region of a CAR; a CD28 signaling domain, wherein the CD28 signaling domain comprises a transmembrane domain; and a CD3 zeta signaling domain.

16. The genetically modified cells according to claim 15, comprising a recombinant nucleic acid expression construct that encodes a CAR, said CAR comprising: 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 CEA, 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 signaling 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. The genetically modified cells according to claim 1, wherein the checkpoint inhibitory molecule comprises: (a.) a dominant negative truncated form of a checkpoint protein, (b.) wherein said checkpoint protein is positioned adjacently to a polypeptide cleavage site for cleaving the checkpoint inhibitory molecule from the CAR polypeptide.

18. The genetically modified cells according to claim 17, 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.

19. The genetically modified cells according to claim 1, wherein the immune stimulatory cytokine comprises: (c.) A signal sequence; (d.) A N-terminal IL15RA polypeptide; (e.) A linking loop sequence; and (f.) An IL-15 polypeptide.

20. The genetically modified cells according to claim 19, wherein the immune stimulatory cytokine comprises: (g.) A signal sequence according to SEQ ID NO 29, or a sequence with at least 80% sequence identity to SEQ ID NO 29; (h.) 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; (i.) 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 (j.) An IL-15 polypeptide according to SEQ ID NO 32, or a sequence with at least 80% sequence identity to SEQ ID NO 32.

21. (canceled)

22. The genetically modified cells according to claim 1, wherein the recombinant nucleic acid expression construct comprises nucleic acid sequence regions encoding: A CAR comprising an extracellular antigen-binding domain that specifically recognizes a carcinoembryonic antigen (CEA) protein, 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 genetically modified cells according to claim 1, wherein the cells are selected from immune cells, induced pluripotent stem cells (iPSC), immortalized immune cells, Natural Killer (NK) cells, NK T cells, cytokine-induced killer cell (CIK), T lymphocytes.

24. (canceled)

25. (canceled)

26. The genetically modified cells according to claim 1, wherein the cells are induced pluripotent stem cell (iPSC) line ND50039.

27. (canceled)

28. (canceled)

29. (canceled)

30. A method for the treatment of a medical disorder associated with the presence of pathogenic cells expressing CEA, comprising administering the genetically modified stem cells according to claim 1 to a subject.

31. The method according to claim 30, wherein the medical disorder comprises cancer cells expressing CEA.

32. The method according to claim 31, wherein the cancer is a solid malignancy expressing CEA, or a cancer expressing CEA selected from the group consisting of breast cancer, pancreatic cancer colon cancer, rectal cancer, lung cancer, breast cancer, liver cancer, stomach cancer and ovarian cancer.

33. (canceled)

34. (canceled)

35. A recombinant nucleic acid expression construct encoding a chimeric antigen receptor (CAR), said construct comprising: (a.) a first nucleic acid sequence region encoding a chimeric antigen receptor (CAR), said CAR comprising an extracellular antigen-binding domain that recognizes a carcinoembryonic antigen (CEA) protein, (b.) a second nucleic acid sequence region encoding checkpoint inhibitory molecule, and (c.) a third nucleic acid sequence region encoding an immune stimulatory cytokine.

36. (canceled)

37. (canceled)

38. A method for producing a genetically modified cell comprising delivering or transferring a nucleic acid construct according to claim 35 into a cell in vitro.

39. (canceled)

40. A chimeric antigen receptor (CAR) polypeptide encoded by the recombinant nucleic acid expression construct according to claim 35.

41. A pharmaceutical composition comprising the genetically modified cells according to claim 1 and a pharmaceutically acceptable carrier.

42. (canceled)

43. (canceled)

44. (canceled)

45. The method according to claim 38, wherein the nucleic acid construct is transferred or delivered into the cell in vitro using electroporation.

Description

FIGURES

[0333] 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.

[0334] Short description of the figures:

[0335] FIG. 1: Cytotoxicity of CEA CAR-transduced YT cells to MCF-7 cells.

[0336] FIG. 2: Checkpoint inhibition by a dominant negative PD1 (dnPD1opt) leads to an improvement in NFAT promoter activity.

[0337] FIG. 3: The activity IL-15 superagonists (15R15) compared to IL-2 or IL-15.

[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).

[0339] FIG. 5: The cytotoxicity of CEA CAR induced YT cells to MC32A cells.

[0340] FIG. 6: Specific release of IL-15 superagonist upon stimulation with CEA expressing tumor cell line MCF-7.

DETAILED DESCRIPTION OF THE FIGURES

[0341] FIG. 1: Cytotoxicity of CEA CAR-transduced YT cells to MCF-7 cells: YT cells were transduced with CEA CAR construct encoding lentivirus with dnPD1opt and 15R15 (YT CEA CAR 15R15) or empty vector (YT control construct). YT CEA 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.

[0342] 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.

[0343] FIG. 3: The activity IL-15 superagonists (15R15) compared to IL-2 or IL-15: 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).

[0344] 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.

[0345] FIG. 5: The cytotoxicity of CEA CAR induced YT cells to MC32A cells: The cytotoxicity of CEA CAR transduced scBW431/26-hFcz YT cells to CEA positive MC32A cells was analyzed in a 6 hr chromium release assay in the absence or presence of soluble CEA protein in the medium. The cell lysis of target cells was given with the standard deviation.

[0346] FIG. 6: Specific release of IL-15 superagonist upon stimulation with CEA expressing tumor cell line MCF-7: CEA-expressing cells were mixed with different ratios of responder cells, namely (A) CEA CAR YT on MCF-7 cells and (B) CEA CAR on MCF 7 cells or the same number of responder cells expressing a control vector (Empty CAR). After 18 hours supernatant was collected and the amount of IL15 activity was determined by IL-2/IL-15 reporter cell line.

EXAMPLES

[0347] 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.

[0348] Lentiviral vectors encoding the anti-CEA 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 CEA-positive cancer cell line MCF-7, while the immune cell line YT Is not modified with the anti-CEA CAR and cannot sufficiently lyse the cancer cell line MCF-7 (Example 1, FIG. 1). Further examples show [0349] the successful PD-1 checkpoint inhibition (Example 2, FIG. 2), [0350] the high activity of the IL-15 superagonist (Example 3, FIG. 3), [0351] the synergistic effect of the combination of anti-CEA CAR, PD-1 checkpoint inhibition and IL-15 superagonist (Example 4, FIG. 4), [0352] binding of anti-CEA CAR to cell membrane bound CEA proteins independent of the presence of soluble CEA proteins (Example 5, FIG. 5).

[0353] 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 cell transfection was performed using polyethyleneimine according to the manufacturers instructions (Polyplus).

[0354] 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-CEA CAR Construct

[0355] 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 CEA, a checkpoint inhibitor dnPD1opt, and/or an immune stimulatory cytokine 15R15 (YT CEA CAR 15R15). (FIG. 1) YT CEA 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 CEA-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).

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

[0356] 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. (FIG. 2) Jurkat cells express GFP under the control of the NFAT promoter. 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

[0357] The example relates to experiments that proves superagonist activity of the IL-15 transgene 15R15. (FIG. 4) 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)

[0358] The example relates 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. (FIG. 4) 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. 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.

Example 5: The Cytotoxicity of Anti-CEA CAR Induced YT Cells to MC32A Cells

[0359] The example relates to experiments showing that the anti-CEA CAR recognition of cell membrane bound CEA proteins is independent from the presence of soluble CEA proteins. (FIG. 5) The cytotoxicity of anti-CEA CAR transduced scBW431/26-hFcz YT cells to CEA positive MC32A cells was analyzed in a 6 hr chromium release assay in the absence or presence of soluble CEA protein in the medium. The cell lysis of target cells was given with the standard deviation. The cytotoxic activity against MC32A cells of scBW431/26-hFcz YT cells transduced with a lentiviral vector encoding an anti-CEA CAR remains at comparable levels irrespective whether or not soluble CEA protein was added to medium.

Example 6: Specific Release of IL-15 Superagonist Upon Stimulation With CEA Expressing Tumor Cell Line MCF-7

[0360] CEA-expressing cells were mixed with different ratios of responder cells, namely (A) CEA CAR YT on MCF-7 cells and (B) CEA CAR on MCF 7 cells or the same number of responder cells expressing a control vector (Empty CAR). After 18 hours supernatant was collected and the amount of IL15 activity was determined by IL-2/IL-15 reporter cell line. As can be determined from the figure, CEA-CAR expressing cells induce a dose-dependent response after incubation with CEA-expressing MCF 7 cells.

REFERENCES

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