Non-activated t cells expressing exogenous virus-specific T cell receptor (TCR)

Abstract

The present invention relates to T cells, in particular a non-activated T cell, comprising an exogenous nucleic acid encoding a T cell Receptor (TCR) specific for a virus. An embodiment of the invention is directed to a non-activated (resting) T cell expressing Hepatitis B virus (HBV) envelope s183-191 TCR capable of inhibiting viral replication and which shows reduced expression of perforins and/or granzymes in response to stimulation as compared to an activated T cell expressing the said TCR. Also encompassed are methods for producing such cells, compositions, pharmaceutical compositions and kits comprising such cells and medical uses thereof.

Claims

1. An isolated T cell comprising an exogenous nucleic acid encoding a T Cell Receptor (TCR) specific for a hepatitis virus, wherein the T cell is a non-activated T cell and wherein the non-activated T cell has a CD45RA.sup.high phenotype, which is capable of inhibiting replication of the virus in a cell infected with the virus, wherein the T cell exhibits a reduced level of expression or activity of one or more cytotoxic factors as compared to the level of expression or activity of an activated T cell which is not modified for reduced expression or activity of a cytotoxic factor, and wherein the T-cell displays reduced cytotoxicity against the cells infected with, or comprising a peptide of, the virus as compared to an activated T cell comprising the TCR specific for the virus which is not modified for reduced expression or activity of a cytotoxic factor.

2. The T cell according to claim 1, wherein the non-activated T cell does not display increased expression of perforin or granzyme in response to stimulation with peptide for which the TCR is specific.

3. The T cell according to claim 1, wherein the hepatitis virus is hepatitis B virus.

4. An in vitro method for producing a modified T cell specific for a hepatitis virus, the method comprising modifying a T cell to express or comprise a T Cell Receptor (TCR) specific for the virus, wherein modifying the T cell to express or comprise the TCR specific for the virus comprises introducing a nucleic acid encoding the TCR specific for the virus into the T cell, and wherein the modified T cell is a non-activated T cell having a CD45RA.sup.high phenotype, which is capable of inhibiting replication of the virus in a cell infected with the virus, and wherein the method comprises modifying a T cell to reduce expression or activity of one or more cytotoxic factors, and wherein the T cell displays reduced cytotoxicity against the cells infected with, or comprising a peptide of the virus as compared to an activated T cell comprising a TCR specific for the virus which is not modified for reduced expression or activity of a cytotoxic factor.

5. The method according to claim 4, wherein the nucleic acid is introduced into the T cell by a transduction, a transfection a transposon-based system, a retroviral transduction, or a mRNA electroporation.

6. The method according to claim 4, wherein the modified, non-activated T cell does not display increased expression of perforin or granzyme in response to stimulation with peptide for which the TCR is specific.

7. The method according to claim 4, wherein the modified T cell displays reduced cytotoxicity against cells infected with, or comprising a peptide of, the virus as compared to an activated T cell comprising the TCR specific for the virus which is not modified for reduced expression or activity of the cytotoxic factor.

8. The T cell according to claim 4, wherein the hepatitis virus is hepatitis B virus.

9. An isolated T cell, wherein the T cell is obtained or obtainable by the method according to claim 4.

10. A pharmaceutical composition comprising a T cell according to claim 1 and a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

11. The T cell according to claim 1 for use in a method of treating or preventing a disease or disorder which is caused or exacerbated by hepatitis virus infection.

12. A method of treating or preventing a disease or disorder which is caused or exacerbated by hepatitis virus infection, comprising administering to a subject a therapeutically or prophylactically effective amount of the T cell according to claim 1.

13. 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 T Cell Receptor (TCR) specific for a virus wherein modifying the T cell to express or comprise the TCR specific for the virus comprises includes introducing a nucleic acid encoding the TCR specific for the virus into the at least one T cell; and (c) administering the modified at least one T cell to the subject; wherein the modified at least one T cell is a non-activated T cell having a CD45RA.sup.high phenotype, which is capable of inhibiting replication of the virus in a cell infected with the virus, wherein the T cell exhibits a reduced level of expression or activity of one or more cytotoxic factors as compared to the level of expression or activity of an activated T cell which is not modified for reduced expression or activity of a cytotoxic factor, and wherein the T cell displays reduced cytotoxicity against the cells infected with, or comprising a peptide of, the virus as compared to an activated T cell comprising the TCR specific for the virus which is not modified for reduced expression or activity of a cytotoxic factor.

14. The method according to claim 13, wherein the nucleic acid is introduced into the at least one T cell by transduction, transfection or transposon-based system.

15. The method according to claim 13, wherein the hepatitis virus is hepatitis B virus.

16. The method according to claim 11, wherein the disease or disorder which is caused or exacerbated by hepatitis virus infection is selected from acute hepatitis, fulminant hepatic failure, chronic hepatitis, cirrhosis, liver cancer, hepatocellular carcinoma (HCC) and pancreatic cancer.

17. The method according to claim 11, wherein the disease or disorder which is caused or exacerbated by hepatitis B virus infection.

18. A kit of parts comprising a predetermined quantity of the T cell according to claim 1.

19. The T cell according to claim 1, wherein the cytotoxic factor is selected from a group consisting of perforin, granyme B, granzyme A, granulysin, FASL, and any combination thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

(2) FIG. 1. Graphs showing HBV TCR expression on T cells. (A) scatterplots showing expression of HBV TCR on resting and activated T cells electroporated with mRNA encoding HBV TCR, as measured by pentamer staining. (B) Graphs showing the level of expression of the TCR over time following electroporation. MFI=mean fluorescence intensity.

(3) FIG. 2. Graphs showing phenotypes of T cells electroporated with HBV TCR mRNA. (A) scatterplots showing CD45RA, CD62L, CD27 and CD28 expression by electroporated resting T cells, and chart showing % of CD8+ dextramer+ cells amongst resting T cell subsets. (B) scatterplots showing CD45RA, CD62L, CD27 and CD28 expression by electroporated activated T cells, and chart showing % of CD8+ dextramer+ cells amongst activated T cell subsets. CM=central memory, EM=effector memory, TEM=Terminally effector memory.

(4) FIG. 3. Graphs showing expression of cytotoxic factors by T cells electroporated with HBV TCR mRNA. (A) Histograms showing expression of perforin and granzyme by electroporated, activated T cells stimulated with peptide with as compared to unstimulated activated, electroporated T cells. (B) Histograms showing expression of perforin and granzyme by electroporated, resting T cells stimulated with peptide as compared to unstimulated, resting electroporated T cells. (C) Chart showing the percentage of granzyme B+ CD8 cells of the total number of lymphocytes, (D) Chart showing the percentage of perforin+ CD8 cells of the total number of lymphocytes and (E) Chart showing the percentage of IFN-γ+ CD8 cells of the total number of lymphocytes, for activated, electroporated and resting, electroporated T cells, after 5 hours or 24 hours of peptide-specific stimulation, or in the absence of stimulation (unstimulated).

(5) FIG. 4. Graphs and bar chart showing cytotoxicity of T cells electroporated with HBV TCR mRNA to cells expressing HBV antigens. (A) Graph showing % cell killing of cells expressing HBV antigen by resting, electroporated T cells at different effector:target cell ratios. (B) Graph showing % cell killing of cells expressing HBV antigen by activated, electroporated T cells at different effector:target cell ratios. (C) Bar chart showing % cell killing of cells expressing HBV Env antigen by activated, electroporated T cells, resting, electroporated T cells, activated, non-electroporated T cells and resting, non-electroporated T cells.

(6) FIG. 5. Image and bar chart showing expression of various soluble factors by activated, electroporated T cells, resting, electroporated T cells, activated, non-electroporated T cells and resting, non-electroporated T cells.

(7) FIG. 6. Graphs showing antiviral activity and cytotoxicity of T cells to HCV replicon-infected liver cells at different effector:target cell ratios. Bars showing inhibition of viral replication as determined by % reduction of luminescence, and lines showing cytolysis as determined by aspartate aminotransferase (AST) levels. (A) resting, electroporated T cells and resting, non-electroporated T cells. (B) activated, electroporated T cells and activated, non-electroporated T cells.

(8) FIG. 7. Graph showing antiviral activity and cytotoxicity of T cells to HBV virus-infected liver cells at an effector:target ratio of 1:3. Bars showing inhibition of viral replication as determined by qPCR for HBV DNA, and lines showing cytolysis as determined by aspartate aminotransferase (AST) levels.

(9) FIG. 8. Scatterplots showing expression of lymphotoxin B or LIGHT on resting CD8+ T cells. (A) Expression on HBV TCR-positive, unstimulated CD8+ T cells. (B) Expression on HBV TCR-positive, CD8+ T cells following 24 h and 48 h of stimulation with cells pulsed with TCR specific peptide. (C) Expression on HBV TCR-positive, CD8+ T cells following 24 h and 48 h of activation through CD3 and CD28. (D) Expression on HBV TCR-positive, CD8+ T cells following 24 h of treatment with IFN-γ.

EXAMPLES

(10) The inventors describe in the following Examples the generation and characterisation of virus-specific T cells.

Example 1: Cell Lines

(11) HepG2-Env and HepG2-Core expressing luciferase cell lines were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 20 mM HEPES. 0.5 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, MeM amino acids, Glutamax, MeM nonessential amino acids, (Invitrogen, Carlsbad, Calif.), and 2 μg/ml puromycin (BD Biosciences, San Jose, Calif.) was added to maintain selection. T2 cells were cultured in RPMI 1640 supplemented as described above. HepG2 cells that stably expressed the entire HBV genome and produced infectious virus (HepG2.2.15) were cultured in DMEM supplemented with 10% heat-inactivated FBS. 20 mM HEPES. 0.5 mM sodium pyruvate, and 150 μg/ml G418 (Sigma-Aldrich, St. Louis, Mo.). HepG2 cells that stably expressed human sodium taurocholate co-transporting polypeptide (hNTCP) were cultured in DMEM supplemented with 10% heat-inactivated FBS. Glutamax, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5 μg/ml puromycin was added to maintain selection. Huh7 cells expressing HLA-A2 and harboring HCV replicon encoding luciferase were cultured in DMEM supplemented with 10% heat-inactivated FBS, Glutamax, MeM nonessential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mg/ml G418 and 3 μg/ml blasticidin S hydrochloride were added to maintain selection.

Example 2: T Cells

(12) Peripheral blood mononuclear cells (PBMC) were collected under informed consent from healthy donors. To produce activated T cells, PBMC were stimulated with 600 IU/ml interleukin-2 (rlL-2; R&D Systems) and 50 ng/ml anti-CD3 (OKT-3; eBioscience, San Diego, Calif.) in AIM-V+2% human AB serum for 8 days, and rlL-2 was increased to 1000 IU/ml one day before electroporation. Non-activated (resting) T cells were isolated using the pan T cell isolation kit (Miltenyi Biotec, GmbH, Germany) and cultured overnight in 100 IU/ml rlL-2 before electroporation.

Example 3: Flow Cytometry

(13) Antibodies for cell surface staining were obtained from BD Biosciences (anti-human CD8-PE-Cy7, CD8-V500, CD45RA-APC, CD62L-PECy7), eBioscience (anti-human CD45RO-eFluor650), Immudex and Proimmune (HLA-A201-HBs183-91-PE dextramer or pentamer), and R&D Systems (human LTβ receptor-Fc chimera). Antibodies for intracellular cytokine staining were obtained from BD Biosciences (anti-human IFN-γ-APC, TNF-α-Alexa488, IL-2-PE, Granzyme-APC) and Diaclone (anti-human perforin-FITC). Intracellular cytokine staining was performed by fixing and permeabilizing cells with cytofix/cytoperm (BD Biosciences). Flow cytometry was performed using a FACS Canto flow cytometer or LSRII (BD Biosciences) and data was analyzed with FACS Diva program (BD Biosciences).

Example 4: Production of HBV Envelope s183-191 TCR mRNA

(14) The HBV envelope s183-191 TCR construct (HBV s183-TCR) was derived from a pUC57-s183cys b2Aa vector, and sub-cloned it into the pVAX1 vector. Plasmids were propagated in and purified from E. coli using the One Shot Top10 E. coli kit (Invitrogen), purified using QIAGEN Endo Free Plasmid Maxi Kit (QIAGEN, Valencia, Calif.), and linearized using the XbaI restriction enzyme. The linearized DNA was used to produce the TCR mRNA using the mMESSAGE mMACHINE T7 Ultra Kit (Ambion, Austin, Tex.) or T7 mScript Standard mRNA Production System (Cellscript, Madison, Wis.); T7 RNA polymerase was added to start transcription; RNA was capped with Anti-Reverse Cap Analog (ARCA). Then, poly(A)-tail was added by E. coli Poly(A) Polymerase and ATP. The resulting product was concentrated by lithium chloride precipitation and re-dissolved in buffer.

Example 5: mRNA Electroporation of T Cells

(15) For electroporation with the 4D-nucleofector system (Lonza, Cologne, Germany), 10×10.sup.6 activated T cells or non-activated (resting) T cells as described above were suspended in 100 μl of supplemented 40-Nucleofector Solution at room temperature and HBV s183-TCR mRNA was added at 200 μg/ml. The mixture was placed in a nucleocuvette and electroporated using program for stimulated or unstimulated T cells. After electroporation, cells were resuspended in AIM-V 10% human AB serum+100 IU/ml rlL-2, and cultured at 37° C. and 5% CO.sub.2 until analysis.

Example 6: Function of mRNA Electroporated T Cells

(16) HLA-A2+ T2 cells were pulsed with 1 μg/ml of s183-191 peptide for 1 h at 10.sup.6 cells/ml and then washed twice. HBV s183-TCR mRNA electroporated activated (activated EP) or non-activated (resting EP) T cells were co-cultured with peptide-loaded T2 cells for 5 h or overnight in the presence of 10 μg/ml or 2 μg/ml brefeldin A respectively, and stained for CD8, IFN-γ, granzyme and perforin.

Example 7: Cytotoxicity Assays

(17) HepG2-Core or HepG2-Env expressing luciferase cell lines were plated overnight in 96-well Rat bottom plate to permit adherence. Target cells were washed and co-cultured with HBV s183-TCR mRNA electroporated activated or non-activated (resting) T cells at various effector:target (E:T) ratios (effectors calculated based on frequency of CD8+pentamer+ cells) in triplicates in AIM-V+2% human AB serum for 24 h. Cytotoxicity was measured by quantifying luciferase expression in remaining target cells. Briefly, culture medium was discarded and 100 μl of Steady-Glo reagent (Promega, Madison, Wis.) was added to each well and incubated for 5 min to allow cell lysis. Luminescence was measured with a microplate reader (Tecan, Mannedorf, Switzerland). Target cells without effectors were used as a reference for maximum luminescence. Results were expressed as % lysis=100%−(luminescence remaining after lysis/maximum luminescence) % and calculated as mean of triplicate measurements+/−standard deviation.

Example 8: Co-Culture Experiments of mRNA Electroporated T Cells with Targets

(18) HBV s183-TCR mRNA electroporated activated or non-activated (resting) T cells were co-cultured with either HepG2.2.15 or HBV-infected HepG2-hNTCP at 1:3 and 1:1 E:T ratios (effectors calculated based on frequency of CD8+pentamer+ cells) for 24 h. HCV NS3 TCR mRNA electroporated non-activated (resting) T cells were co-cultured with Huh7 HCV replicon cells for 18 h, followed by quantification of luminescence as described above. To assess for target cell lysis after co-culture with T cells, supernatants from co-culture experiments were collected for measurement of alanine aminotransferase (ALT) after 24 h, or viability assays were performed using the Cell Proliferation Kit II (XTT) (Roche Applied Science, Mannheim, Germany). For blocking IFN-γ, 20 μg/ml purified anti-human IFN-γ or isotype control mouse IgG1 (BioLegend) was added. To block lymphotoxin (LT)α1β2, LTα2β1 and LIGHT, 1 μg/ml recombinant human LTβ receptor-Fc chimera (R&D Systems, Minneapolis, USA) was used.

Example 9: HBV Infection

(19) Approximately 80 to 100-fold concentrated supernatant of HepAD38 cells was used as HBV inoculum. HepG2-hNTCP cells seeded overnight in 24-well plates were inoculated for 24 h with approximately multiplicities of genome equivalents of 3000/well HBV in medium containing 4% polyethylene glycol (Sigma-Aldrich). After infection, cells were washed with PBS 3 times and culture medium with DMSO was added and changed every 2 days. Viral infection was analysed by measuring hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) expression on infected cells by flow cytometry, and HBV DNA was quantified by real-time PCR.

Example 10: Quantification of HBV Genome Equivalent Copies

(20) RLT buffer with β-mercaptoethanol or lysis buffer containing 50 mM Tris, 140 mM NaCl, 1.5 mM MgCl, 0.5% NP-40 at pH 8 was added to lyse the cells for isolation of intracellular viral nucleic acids using QIAamp MinElute Virus Spin kit (Qiagen, Valencia, Calif.), and HBV DNA was quantified by real-time PCR. Real-time PCRs were performed using the artus HBV RG PCR kit following the manufacturer's instructions in a Rotor-Gene Q 2-plex instrument Qiagen).

Example 11: Three-Dimensional Microdevice-Based Assay

(21) To prepare 200 μl of a 2.5 mg/ml type-I collagen gel solution containing homogenously dissociated HepG2 targets, 20 μl of 1 OX PBS was mixed with 4 μl of NaOH (0.5 M), 129.2 μl of collagen type I (Rat Tail, Dow Corning), 20 μl of freshly trypsinized and dissociated HepG2 targets at 5×10.sup.6 cells/ml and 22.9 μl of cell culture water. The final pH of the gel solution was approximately 7 as determined using a pH indicator strip. The collagen gel solution containing the HepG2 targets was then injected into the device dedicated gel region of the device and polymerized for 40 min in the cell culture incubator (37° C., 5% CO.sub.2). Immediately after gel polymerization, the media channels were filled with R10 media in order to hydrate the gel and keep the HepG2 targets vital. The cell impermeable nuclear dye DRAQ7 (BioLegend, San Diego, Calif.) was also added in the R10 media at a concentration of 3 μM to discriminate between live and dead cells. The devices were then incubated for 24 hr to permit the interaction of the HepG2 targets with the collagen matrix.

(22) Devices with empty gel regions (control) were prepared similarly by adding collagen gel solution containing 20 μl of 10× PBS, 4 μl of NaOH (0.5 M), 129.2 μl of collagen I and 42.8 μl of cell culture water. Prior to injection of the T cells in the device R10 media in the device was replaced with DRAQ7 containing AIM-V 2% human AB serum+100 IU/ml rlL-2.

(23) In order to visualize the spatial position of the engineered T cells, the cells were stained with 3 μM of CellTracker Violet BMQC (Life Technologies Co., Carlsbad, Calif.) in RPMI 1640 for 30 min at 37° C. T cell suspensions were then washed with AIM-V 2% human AB serum, followed by another 30 min incubation at 37° C. The stained T cells were then washed, and resuspended in the corresponding media at 3×10.sup.6 cells/ml. 30 μl of the T cell suspension was then added into one of two culture media channels flanking the central gel region of each device. Finally, the devices were incubated overnight in the indicated conditions.

(24) Live imaging (time-lapse) experiments were performed using either the LSM7800 confocal microscope (Zeiss, Germany) or FV1200 confocal microscope (Olympus, Japan) equipped with an environmental chamber set at 37° C. and 5% CO.sub.2. The microscope was programmed in order to acquire Z stacks of the selected regions at the stated time intervals. For static imaging experiments, confocal images of the same region of interest were acquired before T cell addition and after overnight incubation.

Example 12: Statistical Analysis

(25) Statistical analysis was performed in GraphPad Prism (Graph-Pad Software Inc). For comparisons involving more than two groups, statistical significance was determined using the Kruksal Wallis test with Dunn's post-test for multiple comparisons with p<0.05 taken as evidence of a significant difference.

Example 13: Expression of HBV TCR on Human Non-Activated T Cells Using TCR mRNA Electroporation

(26) It is known that mRNA electroporation can introduce transgenes in human primary lymphocytes without any pre-activation (Zhao et al. Mol Ther (2006), 13 (1): 151-159). This could have significant advantages with respect to clinical applications.

(27) The present inventors investigated whether HBV TCR could be expressed on unstimulated non-activated (resting) T cells.

(28) mRNA encoding the alpha and beta chains of a HLA-A2-restricted HBV envelope s183-TCR was electroporated into unstimulated, non-activated (resting) T cells, or T cells which had been activated for 8 days with anti-CD3 and IL-2.

(29) At 24 hours post-electroporation, 25% of non-activated (resting) T cells and 64% of activated T cells expressed the introduced HBV s183-TCR (FIG. 1A).

(30) The Kinetics of TCR expression following electroporation was also determined. Similar to activated T cells, HBV s183-TCR expressed on non-activated (resting) T cells could be detected at 6 hours post-electroporation; TCR expression peaked at 24 hours post-electroporation and disappeared after 72 hours (FIG. 1B).

Example 14: Differentiation Phenotype of HBV TCR mRNA Electroporated T Cells

(31) Preclinical animal models and retrospective analyses of human adoptive T cell therapy clinical trials have shown that infusion of less differentiated non-activated, stem cell memory or central memory T cell subsets can increase the therapeutic efficacy of adoptive T cell therapy (Klebanoff et al. J Immunother. (2012), 35(9): 651-660).

(32) The present inventors therefore analyzed the differentiation phenotypes of both non-activated and activated T cells expressing the introduced HBV s183-TCR.

(33) Whilst more than 80% of non-activated HBV s183-TCR T cells have a CD45RA+CD62L+ non-activated phenotype, activated HBV s183-TCR T cells comprise of a mixture of 40% CD45RA+CD62L+ non-activated, 45% CD45RA-CD62L+ central memory and 15% effector memory phenotypes (FIGS. 2A and 2B). More than 90% of both non-activated and activated HBV s183-TCR T cells expressed both costimulatory receptors CD27 and CD28 (FIGS. 2A and 2B), likely because electroporation can stimulate proliferation of cells (Qiabius et al. New Biotech (2015), 32(1): 229-235).

(34) No difference was observed in the differentiation phenotypes of non-TCR expressing compared to TCR expressing cells within non-activated or activated T cells, indicating that mRNA electroporation does not preferentially transfect cells of particular differentiation status (data not shown).

Example 15: Analysis of Cytolytic Enzyme Production by HBV TCR mRNA Electroporated T Cells

(35) It is known that the expression of cytolytic enzymes (granzyme and perforin) and cytokines is related to T cell maturity and differentiation; non-activated (resting) T cells express little or no cytolytic enzymes and less cytokines compared with more differentiated cell types (Chattopadhyay et al., J Leukoc Biol (2009) 85(1): 88-97, incorporated by reference hereinabove). The inventors therefore analyzed the expression of granzyme B, perforin and IFN-γ in non-activated and activated HBV TCR mRNA-electroporated T cells after 5 hours and 24 hours of peptide-specific stimulation.

(36) The results are shown in FIG. 3. Electroporated, activated T cells were found to express higher levels of perforin, granzyme B and IFN-γ than electroporated non-activated (resting) T cells.

Example 16: Analysis of Ability of HBV TCR mRNA Electroporated T Cells to Lyse Target Cells

(37) Electroporated, activated or non-activated (resting) T cells were investigated for their ability to lyse target cells. The amount of target cell killing was quantified using both static and live imaging.

(38) The results are shown in FIG. 4. Overnight co-culture of activated, electroporated T cells normalized to the same antigen specific frequency as non-activated, electroporated T cells were able to lyse targets. However, non-activated, electroporated T cells did not lyse target cells.

Example 17: Analysis of Cytokine Production by HBV TCR mRNA Electroporated T Cells

(39) Electroporated, activated or non-activated T cells were analysed for expression of various cytokines, and the results are shown in FIG. 5.

(40) Electroporated, activated T cells produced more RANTES, IL-13, MIP-1α and MIP-1β than electroporated, non-activated T cells. Non-electroporated activated T cells produced more RANTES, IL-13, MIP-1α and MIP-1β than electroporated non-activated T cells.

Example 18: Analysis of Antiviral Activity of HBV TCR mRNA Electroporated T Cells

(41) A HCV replicon system was used to analyse whether electroporated T cells exhibited antiviral activity without causing lysis of target cells.

(42) Huh7 cells expressing HLA-A2 were transfected with a construct encoding the HCV JFH-1 strain and luciferase, described in Jo et al., Gastroenterology (2009) 136(4):1391-1401. Luciferase activity correlated with HCV RNA replication, and therefore viral replication could be analysed by measuring luminescence.

(43) The cells were pulsed with 1 μg/ml HBV env 183 peptide overnight, and then co-cultured for 24 h with HBV env TCR electroporated activated or non-activated T cells. Peptide-pulsed HCV replicon cells were co-cultured with T cells at various effector:target (E:T) ratios, and antiviral activity was determined by calculating the percentage reduction in luminescence. Supernatants of the co-cultures were collected and analysed for aspartate aminotransferase (AST) as a marker of target cell lysis.

(44) The results of the experiments are shown in FIGS. 6A and 6B. Both electroporated non-activated T cells and electroporated activated T cells were shown to have high antiviral activity, even at high E:T ratio. For example, even at E:T 1:500 a ˜80% reduction in luminescence was observed.

(45) Importantly, electroporated non-activated T cells were shown to possess this antiviral activity without extensive target cell lysis, as illustrated by detection of lower levels of AST in co-culture supernatant as compared to co-cultures with electroporated activated T cells.

(46) Further co-culture experiments were performed using electroporated non-activated T cells (either as a bulk population, or subsets sorted based on surface marker expression) and HBV producing HepG2 cells at 1:3 E:T for 24 h. Intracellular HBV DNA was quantified by real-time PCR, and AST levels were measured in the co-culture supernatant.

(47) The results are shown in FIG. 7. Both electroporated, activated and electroporated, non-activated (resting) T cells were able to inhibit HBV viral load. However, electroporated, activated T cells lysed the target cells as shown by higher AST levels, whilst electroporated, non-activated T cells did not lyse the target cells.

Example 19: Investigation of the Mechanism of Antiviral Activity of HBV TCR mRNA Electroporated T Cells

(48) Recent studies have suggested that lymphotoxin-β receptor (LTβR) activation on hepatocytes can lead to degradation of HBV nuclear covalently closed circular DNA (cccDNA) without hepatotoxicity (Lucifora et al., Science (2014) 343 (6176): 1221-1228; Haybaeck et al., Cancer Cell (2009) 16(4): 295-308).

(49) The ligands for LTβR are LTβ and LIGHT, and they are expressed on the non-activated (resting) electroporated T cells.