CHIMERIC ANTIGEN RECEPTOR

Abstract

Chimeric Antigen Receptors (CARs) comprising a costimulatory sequence which is, or which is derived from, the intracellular domain of CD226, or a fragment thereof, are disclosed. Also disclosed are compositions comprising such CARs, and uses and methods using the same.

Claims

1. A chimeric antigen receptor (CAR), comprising a costimulatory sequence which is, or which is derived from, the intracellular domain of CD226, or a fragment thereof.

2. The CAR according to claim 1, wherein the costimulatory sequence which is, or which is derived from, the intracellular domain of CD226, or a fragment thereof comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:16, 58 or 59.

3. The CAR according to claim 1 or claim 2, wherein the CAR additionally comprises a costimulatory sequence which comprises or consists of an amino acid sequence which is, or which is derived from, the intracellular domain of CD28.

4. The CAR according to any one of claims 1 to 3, wherein the CAR additionally comprises a costimulatory sequence which comprises or consists of an amino acid sequence which is, or which is derived from, the intracellular domain of 4-1BB.

5. The CAR according to any one of claims claim 1 to 4, wherein the CAR comprises a costimulatory sequence which comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:17.

6. The CAR according to any one of claims 1 to 5, wherein the CAR comprises a costimulatory sequence which comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:18.

7. The CAR according to any one of claims 1 to 6, wherein the CAR additionally comprises a dimerization domain.

8. The CAR according to claim 7, wherein the dimerization domain is an inducible dimerization domain.

9. The CAR according to claim 7 or claim 8, wherein the dimerization domain comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:20.

10. The CAR according to any one of claims 1 to 9, wherein the CAR comprises a transmembrane domain which comprises or consists of an amino acid sequence which is, or which is derived from, the transmembrane domain of CD28, CD8a or CD226.

11. The CAR according to any one of claims 1 to 10, wherein the CAR comprises a transmembrane domain which comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:11, 10 or 57.

12. The CAR according to any one of claims 1 to 11, wherein the CAR additionally comprises a hinge region which is, or which is derived from, the human IgG1 hinge region.

13. The CAR according to claim 12, wherein the hinge region comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:19.

14. The CAR according to any one of claims 1 to 13, wherein the CAR comprises an antigen-binding domain which comprises: a heavy chain variable region sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1, and a light chain variable region sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:5.

15. The CAR according to any one of claims 1 to 13, wherein the CAR comprises an antigen-binding domain which comprises: a heavy chain variable region sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:48, and a light chain variable region sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:52.

16. A chimeric antigen receptor (CAR) according to any one of A, B, C, D, E, F, G or H, I, J, K, L or M as shown in Table 1, or V, W, X, Z, AA, BB, CC, DD, EE, FF, GG, HH, II, JJ, KK, LL or MM as shown in Table 3.

17. A chimeric antigen receptor (CAR) comprising, or consisting of, an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:22, 23, 24, 25, 26, 27, 28, 29, 38, 39, 40, 41, 42, 81, 83, 84, 85, 86, 88, 89, 90, 92, 93, 94, 95, 96, 97 or 98.

18. A chimeric antigen receptor (CAR) comprising, or consisting of, an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:30, 31, 32, 33, 34, 35, 36, 37, 43, 44, 45, 46, 47, 62, 64, 65, 66, 67, 69, 70, 71, 73, 74, 75, 76, 77, 78 or 79.

19. A nucleic acid encoding the chimeric antigen receptor (CAR) according to any one of claims 1 to 18.

20. A vector comprising the nucleic acid of claim 19.

21. A cell comprising the chimeric antigen receptor (CAR) according to any one of claims 1 to 18, the nucleic acid according to claim 19, or the vector according to claim 20.

22. A method for producing a cell expressing a chimeric antigen receptor (CAR), comprising introducing into a cell a nucleic acid according to claim 19, or a vector according to claim 20, and culturing the cell under conditions suitable for expression of the nucleic acid or vector by the cell.

23. A cell which is obtained or obtainable by the method according to claim 22.

24. A pharmaceutical composition comprising a chimeric antigen receptor (CAR) according to any one of claims 1 to 18, a nucleic acid according to claim 19, a vector according to claim 20, or a cell according to claim 21 or claim 23, and a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

25. A chimeric antigen receptor (CAR) according to any one of claims 1 to 18, a nucleic acid according to claim 19, a vector according to claim 20, a cell according to claim 21 or claim 23, or a pharmaceutical composition according to claim 24, for use in a method of treating or preventing a disease or disorder.

26. Use of a chimeric antigen receptor (CAR) according to any one of claims 1 to 18, a nucleic acid according to claim 19, a vector according to claim 20, a cell according to claim 21 or claim 23, or a pharmaceutical composition according to claim 24, in the manufacture of a medicament for treating or preventing a disease or disorder.

27. A method of treating or preventing a disease or disorder, comprising administering to a subject a therapeutically or prophylactically effective amount of a chimeric antigen receptor (CAR) according to any one of claims 1 to 18, a nucleic acid according to claim 19, a vector according to claim 20, a cell according to claim 21 or claim 23, or a pharmaceutical composition according to claim 24.

28. A method of treating or preventing a disease or disorder in a subject, comprising: (a) isolating at least one T cell from a subject; (b) modifying the at least one T cell to express or comprise a chimeric antigen receptor (CAR) according to any one of claims 1 to 18, a nucleic acid according to claim 19, or a vector according to claim 20, and; (c) administering the modified at least one T cell to a subject.

29. A method of treating or preventing a disease or disorder in a subject, comprising: (a) isolating at least one T cell from a subject; (b) introducing into the at least one T cell a nucleic acid according to claim 19, or a vector according to claim 20, thereby modifying the at least one T cell and; (c) administering the modified at least one T cell to a subject.

30. The CAR, nucleic acid, vector, cell, or pharmaceutical composition for use according to claim 25, the use according to claim 26, or the method according to any one of claims 27 to 29, wherein the disease or disorder is a cancer.

31. The CAR, nucleic acid, vector, cell, or pharmaceutical composition for use, the use, or the method according to according to claim 30, wherein the cancer is a GPC3-expressing cancer or an EpCAM-expressing cancer.

32. A kit of parts comprising a predetermined quantity of a chimeric antigen receptor (CAR) according to any one of claims 1 to 18, a nucleic acid according to claim 19, a vector according to claim 20, a cell according to claim 21 or claim 23, or a pharmaceutical composition according to claim 24.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0797] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0798] FIGS. 1A and 1B. Schematic representations of the GPC3 targeted CAR constructs of the present invention in the pELNS lentiviral vector.

[0799] FIGS. 2A and 2B. Scatterplots showing expression of anti-GPC3 at the cell surface of T cells transduced with anti-GPC3 CAR constructs, as determined by flow cytometry. FIG. 2A show the results of analysis of non-transduced T cells (negative control), T cells transduced with a construct encoding GFP (transduction control), or T cells transduced with (FIG. 2A) T, KK, LL, W or X (FIG. 2B) S, CC, FF, U, Z, BB, or EE GPC3-CAR constructs.

[0800] FIGS. 3A to 3C. Bar charts showing cell killing of GPC3-expressing cells by T cells transduced with anti-GPC3 CAR constructs having different domains, as determined by Delfia cytotoxicity assay. FIGS. 3A and 3B show specific cytolysis of HepG2 cells by non-transduced T cells (negative control), or T cells transduced with T, KK, LL, W, or X GPC3-CAR constructs, at target cell:CAR-T cell ratios of (FIG. 3A) 10:1 and (FIG. 3B) 20:1.

[0801] FIG. 3C shows specific cytolysis of HepG2 cells by T cells transduced with construct encoding GFP (negative control), or transduced with Z, S, BB, CC, U, EE, FF GPC3-CAR constructs, at target cell:CAR-T cell ratios of 10:1 and 20:1.

[0802] FIG. 4. Bar chart showing cell killing of GPC3-expressing cells by T cells transduced with anti-GPC3 CAR constructs having different domains. Percent cytolysis of HepG2 cells in the absence of T cells, in the presence of Triton X-100 (positive control), by T cells transduced with construct encoding GFP (negative control), or transduced with T or X GPC3-CAR constructs is shown, as determined by xCELLigence assay.

[0803] FIGS. 5A and 5B. Graph and Bar chart showing cell killing of GPC3-expressing cells by T cells transduced with anti-GPC3 CAR constructs having different domains. FIG. 5A shows cytolysis over time. FIG. 5B shows percent cytolysis of HepG2 cells in the absence of T cells, by non-transduced T cells (negative control), or transduced with T, KK, LL, W or X GPC3-CAR constructs, as determined by xCELLigence assay.

[0804] FIGS. 6A and 6B. Graph and Bar chart showing cell killing of GPC3-expressing cells by T cells transduced with anti-GPC3 CAR constructs having different domains. FIG. 6A shows cytolysis over time. FIG. 6B shows percent cytolysis of HepG2 cells in the absence of T cells, by T cells transduced with construct encoding GFP (negative control), or transduced with T, KK, LL, W, X, GG or MM GPC3-CAR constructs, as determined by xCELLigence assay.

[0805] FIGS. 7A and 7B. Graph and Bar chart showing cell killing of GPC3-expressing cells by T cells transduced with anti-GPC3 CAR constructs having different domains. FIG. 7A shows cytolysis over time. FIG. 7B shows percent cytolysis of HepG2 cells by T cells transduced with construct encoding GFP (negative control), or transduced with T, W or X GPC3-CAR constructs, as determined by xCELLigence assay.

[0806] FIGS. 8A to 8D. Graph and Bar charts showing cell killing of GPC3-expressing cells by T cells transduced with anti-GPC3 CAR constructs having different domains. FIG. 8A shows cytolysis over time. FIGS. 8B to 8D show specific cytolysis of HepG2 cells by T cells transduced with construct encoding GFP (negative control), or transduced with Z, S, BB, CC, T, EE or FF GPC3-CAR constructs, at (FIG. 8B) 4 hours, (FIG. 8C) 12 hours, and (FIG. 8D) 36 hours, as determined by xCELLigence assay.

[0807] FIGS. 9A to 9D. Graph and Bar charts showing cell killing of GPC3-expressing cells by T cells transduced with anti-GPC3 CAR constructs having different domains. FIG. 9A shows cytolysis over time. FIGS. 9B to 9D show specific cytolysis of HepG2 cells by T cells transduced with construct encoding GFP (negative control), or transduced with Z, S, BB, CC, T, EE or FF GPC3-CAR constructs, at (FIG. 9B) 4 hours, (FIG. 9C) 12 hours, and (FIG. 9D) 24 hours, as determined by xCELLigence assay.

[0808] FIGS. 10A to 10D. Graph and Bar charts showing cell killing of GPC3-expressing cells by T cells transduced with anti-GPC3 CAR constructs having different domains. FIG. 10A shows cytolysis over time. FIGS. 10B to 10D show specific cytolysis of HepG2 cells by T cells transduced with construct encoding GFP (negative control), or transduced with Z, S, BB, CC, T, EE or FF GPC3-CAR constructs, at (FIG. 10B) 4 hours, (FIG. 100) 8 hours, and (FIG. 10D) 16 hours, as determined by xCELLigence assay.

[0809] FIGS. 11A and 7B. Graph and Bar chart showing cell killing of GPC3-expressing cells by T cells transduced with anti-GPC3 CAR constructs having different domains. FIG. 11A shows cytolysis over time. FIG. 11B shows percent cytolysis of HepG2 cells by T cells transduced with construct encoding GFP (negative control), or transduced with S or BB GPC3-CAR constructs, as determined by xCELLigence assay.

[0810] FIGS. 12A and 12B. Graph and Bar chart showing cell killing of GPC3-expressing cells by T cells transduced with anti-GPC3 CAR constructs having different domains. FIG. 12A shows cytolysis over time. FIG. 12B shows percent cytolysis of HepG2 cells by T cells transduced with construct encoding GFP (negative control), or transduced with S or BB GPC3-CAR constructs, as determined by xCELLigence assay.

[0811] FIGS. 13A to 13H. Bar charts showing levels of cytokines in co-cultures of GPC3-expressing cells and T cells transduced with anti-GPC3 CAR constructs. Bar charts shown the level of (FIG. 13A) IL-2, (FIG. 13B) IFNg, (FIG. 13C) TNFa, (FIG. 13D) GM-CSF, (FIG. 13E) MIP-1a, (FIG. 13F) MIP-1b, (FIG. 13G) RANTES, and (FIG. 13H) TNFb in cell culture supernatants of co-cultures of HepG2 cells with T cells transduced construct encoding GFP (negative control), or transduced with T, W or X GPC3-CAR constructs after 16 hours of co-culture.

[0812] FIGS. 14A to 14H. Graphs showing levels of cytokines in co-cultures of GPC3-expressing cells and T cells transduced with anti-GPC3 CAR constructs. Bar charts shown the level of (FIG. 14A) IL-2, (FIG. 14B) IFNg, (FIG. 14C) TNFa, (FIG. 14D) GM-CSF, (FIG. 14E) MIP-1a, (FIG. 14F) MIP-1b, (FIG. 14G) RANTES, and (FIG. 14H) TNFb in cell culture supernatants of co-cultures of HepG2 cells with T cells transduced construct encoding GFP (negative control), or transduced with T, W or X GPC3-CAR constructs after 16 hours of co-culture.

[0813] FIGS. 15A and 15B. Bar charts showing proliferation by T cells transduced with anti-GPC3 CAR constructs following co-culture with GPC3-expressing cells. Bar charts show proliferation of (FIG. 15A) CD4+ and (FIG. 15B) CD8+ T cells transduced with construct encoding GFP (negative control), or transduced with T, W or X GPC3-CAR constructs following 5 days of co-culture with HepG2 cells.

[0814] FIGS. 16A and 16B. Bar charts showing proliferation by T cells transduced with anti-GPC3 CAR constructs following co-culture with GPC3-expressing cells. Bar charts show proliferation of (FIG. 16A) CD4+ and (FIG. 16B) CD8+ T cells transduced with construct encoding GFP (negative control), or transduced with S, AA or BB GPC3-CAR constructs following 5 days of co-culture with HepG2 cells.

[0815] FIGS. 17A and 17B. Graph and Bar chart showing cell killing of GPC3-expressing cells by T cells transduced with anti-GPC3 CAR constructs having different domains, in the presence or absence of TGF. FIG. 17A shows cytolysis over time. FIG. 17B shows percent cytolysis of HepG2 cells by T cells transduced with construct encoding GFP (negative control), or transduced with S or BB GPC3-CAR constructs, as determined by xCELLigence assay.

EXAMPLES

[0816] The inventors describe in the following Examples construction of GPC3-targeted CARs, transduction into human T lymphocytes to generate GPC3-targeted CAR-T cells, antigen-specific killing of GPC3-expressing cells by the GPC3-targeted CAR-T cells, and anti-cancer activity of GPC3-targeted CAR-T cells in vivo, and reduced sensitivity to immunosuppressive factors, improved selectivity for tumour targets, improved priming of CTL to eradicate tumour cells, improved trafficking, tumour migration and penetration, and increased expression of growth factors for CAR-T cells expressing CARs comprising a CD226 costimulatory region as compared to CAR-T cells expressing CARs lacking a CD226 intracellular domain.

Example 1: Generation of CARs Comprising CD226 Intracellular Domain and Lentivirally-Transduced Human T Lymphocytes

[0817] The cDNA of GC33 scFv and CD226 intracellular domain is amplified by PCR and inserted into the lentiviral vector pELNs using BamHI and NheI restriction sites to generate lentiviral vector pELNs/GC33 CARs having a CD226 intracellular domain.

[0818] For lentiviral transduction, 510.sup.6 HEK 293T cells are plated on 10 cm.sup.2 dish pre-coated with 0.002% Poly-L-lysine (Sigma, St. Louis Mo.). The lentiviral vector pELNS-CARs are then co-transfected with the plasmid pMD.G, pMDLg/pRRE, and pRSV-Rev. The virus-containing supernatant is collected and passed through a 0.45 m filter. The supernatant is then concentrated by ultracentrifugation at 25,000 rpm, titered, and then stored at 80 C. until use.

[0819] Primary human T lymphocytes isolated from healthy donors are acquired. T cells are cultured in complete medium (RPMI 1640 supplemented with 10% inactivated FBS, penicillin and streptomycin sulfate), and activated by stimulation with anti-CD3 and anti-CD28mAb-coated beads (Invitrogen). 12 hours after activation, the T cells are transduced with lentiviral vectors in presence of polybrene. Human T lymphocytes are expanded and maintained by addition of IL-2 every other day.

Example 2: GPC3-Specific CAR Construction and T Lymphocyte Transduction

[0820] GC33 scFv is selected to construct GPC3-specific CARs with high antigen-binding affinity. A lentiviral CAR vector is used to make CAR constructs including different domains by sub-cloning of cDNA sequences encoding the domains into the vector. The following constructs are generated:

TABLE-US-00021 TABLE 1 Trans- Antigen-binding membrane Dimerization domain domain domain Signaling domain A GPC3-binding scFV CD8 CD226, CD3 B GPC3-binding scFV CD8 F36V-FKBP CD226, CD3 C GPC3-binding scFV CD8 CD226, CD28, CD3 D GPC3-binding scFV CD8 F36V-FKBP CD226, CD28, CD3 E GPC3-binding scFV CD8 CD226, 4-1BB, CD3 F GPC3-binding scFV CD8 F36V-FKBP CD226, 4-1BB, CD3 G GPC3-binding scFV CD8 CD226, CD28, 4-1BB, CD3 H GPC3-binding scFV CD8 F36V-FKBP CD226, CD28, 4-1BB, CD3 I GPC3-binding scFV CD28 F36V-FKBP 4-1BB, CD3 J GPC3-binding scFV CD8 4-1BB, CD3 K GPC3-binding scFV CD8 F36V-FKBP 4-1BB, CD3 L GPC3-binding scFV CD8 CD28, CD3 M GPC3-binding scFV CD8 F36V-FKBP CD28, CD3

[0821] A signalling deficient construct containing a truncated CD3 intracellular domain is prepared as a negative control for evaluating initiation of signal transduction by the constructs.

[0822] The vectors are transformed into 293T cells, and lysates are analysed by western blot to confirm successful expression of the vectors.

[0823] For effective transduction, human T lymphocytes isolated from peripheral blood samples are activated by stimulation with CD3/CD28 beads. To evaluate transduction efficiency, T cells are transduced with GFP-expressing lentiviral vector, and stable consistent GFP expression is observed 10 days after transduction.

[0824] To analyse CAR expression at the T cell membrane, a FLAG-tag is artificially inserted at the N-terminus of the CAR, and expression is detected by flow cytometry following staining of the cells with an anti-FLAG mAb. The results suggest that around 50% T cells are transduced and express CAR receptor at the cell surface.

Example 3: Comparison of T Cells Expressing a GPC3 CAR Including a CD226 Costimulatory Region to T Cells Expressing a GPC3 CAR Lacking a CD226 Costimulatory Region

[0825] GPC3 CART T cells with CD226 costimulatory regions display reduced sensitivity to immunosuppressive factors as compared to a CAR not comprising a costimulatory sequence of CD226. Expression of GC33/CD226 CARs in T cells is sufficient to protect CAR T cells from the potent inhibitory effect of treatment with TGF-.

[0826] Selectivity of T cells expressing GC33/CD226 CAR is compared to T cells expressing GC33 CAR lacking a CD226 intracellular domain in vitro using targets that recapitulate normal vs. tumor tissue. CAR T cells expressing GC33/CD226 CAR selectively eliminate only tumor targets and not normal surrogate targets. The selectively of these CAR-T cells is confirmed in vivo.

Example 4: Efficacy of T Cells Expressing GPC3/CD226 CAR in CTL Priming to Eradicate Tumor Cells

[0827] T cells expressing a GPC3 CAR having a CD266 intracellular domain are tested in in vitro priming systems and compared to T cells expressing GC33 CAR lacking a CD226 intracellular domain. Human CAR-expressing T cells are co-cultured with irradiated tumor cells, in the presence of a pool of non-engineered T cells and optionally DCs.

[0828] T cells expressing a GPC3/CD226 CAR display improved priming of CTL to eradicate tumour cells as compared to CARs lacking a CD226 intracellular domain.

Example 5: Migration Assays Determining Cellular Localization after Infusion

[0829] A transwell migration assay indicates that GPC3/CD226 CAR-T cells are able to migrate towards tumor cell line supernatant more efficiently than GPC3 CAR-T cells lacking a CD226 intracellular domain.

[0830] GPC3 CAR-T cells are labeled with GFP and placed in the upper chamber of the 24-well transwell chamber. Media alone or LCL tumor supernatant is placed in the bottom chamber. Plates are then incubated for 3 h at 37 C. Cells in the bottom chamber are then harvested and analysed to determine migration of T cells from the upper chamber to the lower chamber. Specific migration is calculated using the following equation:

Specific Migration (%)=(Experimental[LCL supernatant]Spontaneous[media alone])/(Maximum[1.510.sup.5 cells]Spontaneous[media alone])100.

[0831] CAR-T cells expressing the CAR construct including a CD226 intracellular domain exhibit trafficking to the lower chamber, and display better tumor migration and penetration as compared to GPC3 CAR-T cells lacking a CD226 intracellular domain.

Example 6: Cytokine Assays Using Multiplex Cytokine Analysis

[0832] Levels of interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, interferon-gamma (IFN-gamma), granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor-alpha (TNF-alpha) in the cell culture supernatant of the CAR T cells co-expressing CD226 is analysed by multiplex technique. Intracellular cytokine staining (ICS) is employed to detect IFN-gamma, IL-2, IL-4 and IL-13 in CD3.sup.+ cells.

[0833] The multiplex analysis detects representative cytokine profiles for the majority of the cytokines on day 7 by identifying peak levels or good correlation with peak levels.

[0834] CAR-T cells expressing CAR including a CD226 intracellular domain have increased expression of growth factors as compared to GPC3 CAR-T cells lacking a CD226 intracellular domain.

Example 7: Generation of GP3C-Specific CAR and Lentivirally-Transduced Human T Lymphocytes

[0835] The cDNA of GC33 scFv is amplified by PCR and inserted into the lentiviral vector pELNs using BamHI and NheI restriction sites, to generate lentiviral vector pELNs/GC33 CARs.

[0836] For lentiviral transduction, 510.sup.6 HEK 293T cells are plated on 10 cm.sup.2 dish pre-coated with 0.002% poly-L-lysine (Sigma, St. Louis Mo.). The lentiviral vector pELNS-CARs are then co-transfected with the plasmid pMD.G, pMDLg/pRRE, and pRSV-Rev. The virus-containing supernatant is collected and passed through a 0.45 m filter. The supernatant is then concentrated by ultracentrifugation at 25,000 rpm, titered, and then stored at 80 C. until use.

[0837] Primary human T lymphocytes isolated from healthy donors are acquired. T cells are cultured in complete medium (RPMI 1640 supplemented with 10% inactivated FBS, penicillin and streptomycin sulfate), and activated by stimulation with anti-CD3 and anti-CD28mAb-coated beads (Invitrogen). 12 hours after activation, the T cells are transduced with lentiviral vectors in presence of polybrene. Human T lymphocytes are expanded and maintained by addition of IL-2 every other day.

Example 8: Validation of the Ability of the CAR to Direct T-Cells Against G3PC Expressing Target Cells

[0838] The ability of the transduced T lymphocytes to lyse GPC3-positive tumor cells is confirmed by in vitro analysis by fluorescence-based killing assay, cytokine release assay, and high dimension flow cytometry.

Example 9: CAR+ T Cells Showed GPC3-Specific Cytotoxicity In Vitro

[0839] Engineered T cells are co-cultured with GPC3-positive or GPC3-negative tumor cells to determine whether the CAR-expressing T cells display antigen-specific cytotoxicity.

[0840] T cells are transduced by lentiviral vector and their transduction efficiency is assessed by FACS, and further equilibrated. T cells transduced with GFP lentiviral vector are included as a control. For target cells, several established tumor cell lines are selected and GPC3 protein expression levels are determined by FACS. Two tumor cell lines, hs578T (a GPC3-negative cell line) and HepG2.sh57 (a cell line which displays lower level of GPC3 expression), are also selected.

[0841] The results indicate that the GPC3-CAR transduced T cells display antigen-specific cytotoxicity to target cells expressing GPC3.

Example 10: Improved In Vivo Proliferation and Persistence and Enhanced Antitumor Efficacy of GPC3-CAR T Cells after Adoptive Transfer

[0842] GPC3-CAR T cells are injected subcutaneously into immune-compromised mice with GPC3-positive xenograft tumours.

[0843] The mice in the untreated control group start dying after 50 days. By contrast, mice treated with GPC3-CAR T cells continue to survive. After 130 days of treatment, most of the mice from the control group have died, but 80% of mice from CAR T group remain alive. These data indicate that GPC3-CAR T cells have improved in vivo persistence and proliferation, and enhanced long-term antitumor effects in vivo.

Example 11: Generation of Further CAR Constructs

[0844] Further CAR constructs were generated by PCR amplification and sub-cloning of cDNA encoding the different CAR domains into a lentiviral CAR vector. The following constructs were generated:

TABLE-US-00022 Trans- Antigen-binding Hinge membrane Signaling domain region domain domain R GPC3-binding scFV Human IgG1 CD28 CD3 S GPC3-binding scFV Human IgG1 CD28 CD28, CD3 T GPC3-binding scFV Human IgG1 CD28 4-1BB, CD3 U GPC3-binding scFV Human IgG1 CD28 CD28, 4-1BB, CD3 V GPC3-binding scFV Human IgG1 CD28 CD226 ICDv2 W GPC3-binding scFV Human IgG1 CD28 CD226 ICDv1, 4-1BB, CD3 X GPC3-binding scFV Human IgG1 CD28 CD226 ICDv2, 4-1BB, CD3 Y GPC3-binding scFV Human IgG1 CD28 CD226 ICDv1, CD3 Z GPC3-binding scFV Human IgG1 CD28 CD226 ICDv2, CD3 AA GPC3-binding scFV Human IgG1 CD28 CD226 ICDv1, CD28, CD3 BB GPC3-binding scFV Human IgG1 CD28 CD226 ICDv2, CD28, CD3 CC GPC3-binding scFV Human IgG1 CD28 CD28, CD226 ICDv1, CD3 DD GPC3-binding scFV Human IgG1 CD28 CD226 ICDv1, CD28, 4-1BB, CD3 EE GPC3-binding scFV Human IgG1 CD28 CD226 ICDv2, CD28, 4-1BB, CD3 FF GPC3-binding scFV Human IgG1 CD28 CD28, CD226 ICDv1, 4-1BB, CD3 GG GPC3-binding scFV Human IgG1 CD226 CD226 ICDv1, 4-1BB, CD3 HH EpCAM-binding scFV Human IgG1 CD226 CD226 ICDv1 II EpCAM-binding scFV Human IgG1 CD226 CD3 JJ EpCAM-binding scFV Human IgG1 CD226 CD226 ICDv1, CD3 KK GPC3-binding scFV Human IgG1 CD28 4-1BB, CD3, CD226 ICDv1 LL GPC3-binding scFV Human IgG1 CD28 4-1BB, CD226 ICDv1, CD3 MM GPC3-binding scFV Human IgG1 CD226 CD226 ICDv1

Example 12: Expression of Anti-GPC3 CARs on Transduced T Cells

[0845] CD3+ cells were obtained from peripheral blood samples, activated by stimulation with anti-CD3/anti-CD28 beads and then transduced with the following GPC3-CAR constructs described in Example 11: T, KK, LL, W or X (FIG. 2A), S, CC, FF, U, Z, BB, or EE (FIG. 2B) or lentivirus encoding GFP, as a transduction control.

[0846] Expression of the GPC3-CARs at the cell surface of the transduced cells was analysed by flow cytometry using biotinylated, anti-mouse-fab antibody and fluorescently-conjugated strepatavidin.

[0847] The results are shown in FIGS. 2A and 2B. GPC3-CAR expression was detected at the cell surface of the transduced cells.

Example 13: Analysis of Cell Killing by GPC3-Targeted CAR-T Cells

13.1 Analysis by Delfia Cytotoxicity Assay

[0848] Transduced T cells expressing GPC3-specific CAR constructs were analysed for their ability to lyse GPC3-expressing cells.

[0849] GPC3-expressing HepG2 hepatocarcinoma cells were loaded with Delfia fluorescence enhancer reagent. Lysis of target cells by the GPC3-targeted CAR-T cells releases the enhancer reagent into the culture media.

[0850] Culture media were collected after 2 hours co-incubation of HepG2 cells and GPC3-CAR-T cells; experiments were performed at target cell:CAR-T cell ratios of 10:1 and 20:1.

[0851] Fluorescence was measured with a fluorescence plate reader and compared to fluorescence released spontaneously, and fluorescence released by chemical lysis of Delfia-loaded HepG2 cells, to calculate percent specific cytolysis.

[0852] The results of experiments performed using T cells transduced with constructs T, KK, LL, W, or X constructs (see Example 11) are shown in FIGS. 3A and 3B.

[0853] The results of experiments performed using T cells transduced with constructs Z, S, BB, CC, U, EE or FF constructs (see Example 11) are shown in FIG. 3C.

[0854] The GPC3-CAR-T cells were shown to be capable of killing GPC3-expressing cells.

13.2 Analysis by xCELLigence Assay

[0855] Further analysis of lysis of HepG2 cells by GPC3-targeted CAR-T cells was performed using xCELLigence (ACEA Biosciences Inc) system, which measures changes in electrical resistance associated with changes in adherence of cells to gold microelectrodes. Interaction between the cells with the gold microelectrodes changes the flow of electric current between electrodes, and this impedance value is calculated as a Cell Index.

[0856] Briefly, HepG2 cells were seeded in xCELLigence plates and growth was monitored. When near-confluent or confluent, CAR-T cells were added to cultures at an effector:target cell ratio of 0.5:1. Lysis of HepG2 cells by CAR-T cells was monitored by xCELLigence machine and percent cytolysis was calculated using XIMT software.

[0857] The results performed using T cells transduced with GPC3-targeted constructs T or X are shown in FIG. 4. FIG. 4 shows percent cytolysis of the HepG2 cells at the end of the experiment. The T cells transduced with the X construct were found to display increased cytolytic activity against the GPC3-expressing cells as compared to T cells transduced with the T construct.

[0858] Further experiments were performed, using T cells isolated from blood samples obtained from different donors.

[0859] FIGS. 5A and 5B show the results obtained using T cells from donor ID1, 28 days after transduction with construct T, KK, LL, W or X. T cells transduced with the W and X constructs were found to display increased cytolytic activity against the GPC3-expressing cells as compared to T cells transduced with the T construct.

[0860] FIGS. 6A and 6B show the results obtained using T cells from donor ID2, 14 days after transduction with construct T, KK, LL, W, X, GG or MM. Once again, T cells transduced with the W and X constructs were found to display increased cytolytic activity against the GPC3-expressing cells as compared to T cells transduced with the T construct.

[0861] FIGS. 7A and 7B show the results obtained using T cells from donor ID4, 19 days after transduction with construct T, W or X. Once again, T cells transduced with the W and X constructs were found to display increased cytolytic activity against the GPC3-expressing cells as compared to T cells transduced with the T construct.

[0862] FIGS. 8A to 8D show the results obtained using T cells from donor ID3, at different time points in the experiment, from 10 days after transduction with construct Z, S, BB, CC, T, EE, FF.

[0863] FIGS. 9A to 9D show the results obtained using T cells from donor ID3, at different time points in the experiment, from 12 days after transduction with construct Z, S, BB, CC, T, EE or FF.

[0864] FIGS. 10A to 10D show the results obtained using T cells from donor ID3, at different time points in the experiment, from 20 days after transduction with construct Z, S, BB, CC, T, EE or FF.

[0865] FIGS. 11A and 11B show the results obtained using T cells from donor ID4, 19 days after transduction with construct S or BB. T cells transduced with the BB construct were found to display increased cytolytic activity against the GPC3-expressing cells as compared to T cells transduced with the S construct.

[0866] FIGS. 12A and 12B show the results obtained using T cells from donor ID5, 16 days after transduction with construct S or BB. T cells transduced with the BB construct were again found to display increased cytolytic activity against the GPC3-expressing cells as compared to T cells transduced with the S construct.

Example 14: Cytokine Production by GPC3-CAR Expressing CAR-T Cells

[0867] Cytokine production was analysed in 16 hour co-cultures of CAR-T cells transduced with GPC3-CAR lentivirus constructs and HepG2 cells. Cell-free supernatants were collected and analysed or frozen at 80 to be analysed later.

[0868] Multiplex analysis of the level of cytokines MIP-1a, MIP-1b, RANTES and TNFb produced by the cells in culture was performed using the Merck Immuno-monitoring reagent set, and the Luminex plate reader system.

[0869] The results obtained using T cells from three different donors are shown in FIGS. 13A to 13H and 14A to 14H. Overall, higher levels of the indicated cytokines were found in co-cultures comprising T cells transduced with the T construct, as compared to co-cultures comprising T cells transduced with the W and X constructs.

Example 15: Proliferation of GPC3-CAR Expressing CAR-T Cells

[0870] Proliferation of T cells transduced with different GPC3-CAR constructs was analysed following coculture with HepG2 cells for 5 days, or following culture for the same period in the absence of HepG2 cells.

[0871] Briefly, T cells were labelled with CFSE, a fluorescent label whose intensity is halved each time a labelled cell divide in 2. After labelling, T cells were analysed to ensure uniform labelling. HepG2 cells were irradiated to prevent further proliferation and co-incubated with labelled T cells. After 5 days, T cells were analysed by flow cytometry. Cells with fluorescence approximately equal to the original fluorescence were determined to be non-proliferating cells, and those cells with half or less than half of the original fluorescence intensity were determined to be proliferating cells.

[0872] FIGS. 15A and 15B shows the results of proliferation assays performed with T cells from donor ID4, performed 8 days after transduction with construct T, W or X constructs. T cells transduced with the W and X constructs were found to proliferate more following coculture with HepG2 cells as compared to T cells transduced with the T construct. T cells transduced with the W and X constructs were also found to proliferate more less than T cells transduced with the T construct in the absence of HepG2 cells.

[0873] FIGS. 16A and 16B shows the results of proliferation assays performed with T cells from donor ID4, performed 8 days after transduction with construct S, AA or BB. CD4+ T cells transduced with the BB construct were found to proliferate more following coculture with HepG2 cells as compared to CD4+ T cells transduced with the S construct. T cells transduced with the BB construct were also found to display substantial proliferation in the absence of HepG2 cells.

Example 16: Sensitivity to TGF

[0874] T cells transduced with different GPC3-CAR constructs were analysed for their sensitivity to immunosuppression by TGF.

[0875] Briefly, HepG2 cells were seeded on xCELLigence plates, and after 24 hours, T cells transduced with the GFP construct (negative control), or transduced with constructs S or BB, were added to wells in the presence or absence of 125 ng/ml TGF, and cytolysis was measured using the xCELLigence (ACEA Biosciences Inc) system.

[0876] The results are shown in FIGS. 17A and 17B. T cells transduced with the BB construct were found to be less sensitive to TGF-mediated suppression of cytolytic activity as compared to d T cells transduced with the S construct (compare FIG. 17B bars 3 and 5 with columns 4 and 6).

Example 17: Conclusions

[0877] Unexpectedly, T cells expressing CARs comprising CD226 intracellular domains were found to display enhanced cytotoxicity against target antigen-expressing cells as compared to T cells expressing equivalent CAR lacking a CD226 intracellular domain, whilst at the same time producing reduced levels of proinflammatory/effector cytokines in co-cultures with target antigen-expressing cells. Furthermore, T cells expressing CARs comprising CD226 intracellular domains were found to proliferate more following coculture with target-antigen expressing cells as compared to T cells expressing equivalent CAR lacking a CD226 intracellular domain.

[0878] For example, T cells transduced with constructs W and X displayed enhanced cytotoxicity against target antigen-expressing cells, and increased proliferation following coculture with target antigen expressing cells, as compared to T cells expressing construct T.

[0879] T cells transduced with construct BB displayed enhanced cytotoxicity against target antigen-expressing cells as compared to T cells expressing construct S, and were less susceptible to TGF-mediated suppression of effector function.