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EP1706744A2 - Essai de cytotoxicite - Google Patents

Essai de cytotoxicite

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Publication number
EP1706744A2
EP1706744A2 EP05736477A EP05736477A EP1706744A2 EP 1706744 A2 EP1706744 A2 EP 1706744A2 EP 05736477 A EP05736477 A EP 05736477A EP 05736477 A EP05736477 A EP 05736477A EP 1706744 A2 EP1706744 A2 EP 1706744A2
Authority
EP
European Patent Office
Prior art keywords
cells
caspase
assay
cell
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05736477A
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German (de)
English (en)
Inventor
Pamela Dunn
Liwei He
Laszlo Radvanyi
Danielle Salha
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Sanofi Pasteur Inc
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Sanofi Pasteur Inc
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Publication date
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Publication of EP1706744A2 publication Critical patent/EP1706744A2/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to methods for detecting CTL activity using flow cytometry to detect cleavage of cytotoxicity-related enzymes.
  • the 51 Cr-release assay has been the gold standard to measure CTL activity during an immune response for over 3 decades.
  • the assay has a number of technical limitations and recent data has brought into question the physiological relevance of the 51 Cr-release assay and whether it is a true measure of CTL activity measured against target cells in vivo, especially in solid tumors.
  • the lysis of target cells in in vitro CTL assays can be induced via two cHsctinct mechanisms: 1) A. membranolysis process mediated when perform is simply inserted into the target cell membrane; and, 2) DNA fragmentation (apoptosis) induced by the action of Granzyme B and Granzyme A entering a target cell via perform and/or vesicle fusion events at the target cell surface.
  • the 51 Cr-release assay measures the first of these two read-outs.
  • the latter mechanism has more physiologic relevance and is required in vivo for target cell killing.
  • Recent observations indicate that measurement of effector cell cytotoxicity by the 51 Cr-release assay may not be accurate or in some cases, even relevant. Based on these observations, the investigator must remain cognizant of the fact that effector cells determined to be cytotoxic based on 51 Cr-release assays may indeed not be CTL capable of tumor cell killing via DNA fragmentation in vivo. This may account for many of the results in the cancer irnmunotherapy literature in which effector cell responses monitored in vitro may not correlate with vaccine efficacy in the patient.
  • peptide vaccines selected by virtue of positive effector cell cytotoxicity date generated using 51 Cr-release assays may not accurately reflect the actual epitopes active in inducing CTL lysis in vivo via DNA fragmentation.
  • the JAM assay is a newer method that solves some of the issues with 51 Cr- release. Although it measures DNA fragmentation effectively, it has serious limitations due to the need to label the DNA of target cells with agents such as 3 H- thymidine or 125 I-deoxyuridine.
  • This method invariably causes DNA damage and changes the physiology of the target cell before the CTL assay.
  • the 3 H- thymidine labelling method does not afford the same degree of sensitivity as 51 Cr and 125 I is a dangerous agent requiring considerable lead shielding of both the lab worker as well as the lab area.
  • a non-radioactive method to measure apoptosis induction and DNA fragmentation in target cells during CTL attack would be a more ideal assay, especially when contemplating its use in clinical trial monitoring. Detection of capsase cleavage using fluorogenic caspase substrates has also been used to demonstrate cytotoxic T cell activity (Liu, et al.
  • the present invention provides a cytotoxicity assay that detects DNA fragmentation- and/or apoptosis-related changes in target cells following contact with effector cells.
  • the assay is conducted using one or more reagents that detect cleavage of a caspase or phosphorylation of histones.
  • cleavage of caspase is detected using a monoclonal antibody.
  • phosphorylation of histones is detected using a monoclonal antibody.
  • cleaved caspase and/or phosphorylated histones are detected using at least one monoclonal antibody coupled with flow cytometric analysis.
  • Fig. 1 Stability of target cell labeling dye and gating strategy developed for monitoring CTL activity using the caspase 3-cleavage in target cells.
  • DDAO-SE-labeled P815 cells with C57BL/6 anti-Balb/c MLR cells An example of DDAO-SE-labeled P815 cells with C57BL/6 anti-Balb/c MLR cells is shown.
  • the first plot shows FSC versus SSC with gating of the entire population (Rl).
  • Gate Rl cells were then analyzed using SSC and FSC versus FL4 fluorescence (DDAO-SE positive).
  • DDAO-SE positive cells were gated as shown (R2 and R3) excluding low FSC (dead) cells.
  • R2*R3 cells were then analyzed using FL2 (PE-labeled cleaved caspase 3) versus FL4 (DDAO-SE) to determine the percentage of apoptotic targets (R4).
  • Fig. 2 Measurement of CTL activity in murine MLR using the caspase 3 cleavage assay and dependence of caspase 3 cleavage in the target on perform and granzyme secretion by CTL.
  • C57BL/6 anti-Balb/c or Balb/c anti-C57BL/6 MLR effector cells were incubated with labeled P815 targets (A) for different time intervals (0.5, 1, 2, and 3 h) followed by staining for caspase 3 cleavage as shown.
  • Caspase 3 cleavage was found only with the anti-Balb/c MLR effectors.
  • P815 or EL4 targets labeled with DDAO-SE were incubated with C57BL/6 anti-Balb/c or Balb/c anti-C57BL/6 MLR effectors for different time intervals as shown.
  • the percentage caspase 3 -cleaved target cells is shown.
  • the results show the specificity of the assay with MLR effectors only killing the H-2 unmatched targets.
  • the results are representative of 3 experiments with similar results.
  • MLR cultures Five-day MLR cultures were isolated and diluted with na ⁇ ve syngeneic C57BL/6 spleen lymphocytes at a 1 : 1 , 1:3, 1 :9 or with no dilution, as shown. The diluted effectors were incubated with labeled P815 cells. Controls were na ⁇ ve C57BL/6 spleen cells with no MLR cells or labeled P815 cells alone. The assays were stained for caspase 3-cleavage after 3 h.
  • FIG. 3 Comparison of the caspase 3 cleavage assay and 51 Cr-release for monitoring CTL activity using murine MLR effectors and CTL isolated after peptide-based vaccine responses in mice.
  • A Effector cells from a murine MLR (b->d) were incubated with 100,000 P815 or EL4 target cells (control) in a 4 h assay at the indicated E:T ratios.
  • For the 51 Cr-release assay we started at a 16:1 dilution and diluted down to 0.2:1, while the caspase 3 cleavage assay was tested between 1:1 and 0.1:1 E:T ratio. The results of 1 out of 2 similar experiments are shown.
  • B Effector cells from a murine MLR (b->d) were incubated with 100,000 P815 or EL4 target cells (control) in a 4 h assay at the indicated E:T ratios.
  • For the 51 Cr-release assay we started at a 16:1 dilution and diluted down to 0.2:
  • Caspase 3 cleavage assay is equally reliable and specific as the 51 Cr-release assay for monitoring recall responses from peptide-immunized mice. Mice were immunized with the melanoma TRP2 peptide and tested at the indicated E:T ratios.
  • Fig. 4 Monitoring CD8 T-cell responses following viral vector-based vaccination in mice using the caspase 3-cleavage CTL assay yields comparable results as IFN- ⁇ ELISPOT analysis.
  • HLA-A2.1/K b transgenic mice were immunized with ALVAC-gplOO and spleen cells re-stimulated for 5 days with a pool of gplOO peptides.
  • Viable cells were isolated and tested for CTL activity using caspase 3 cleavage with at a 20:1 E:T ratio (left panel), or for IFN- ⁇ production in ELISPOT assays (right panel) at 100,000 cells per well.
  • Targets were P815-A2/K b transfectants pulsed with gplOO peptides or a control A2 -binding CMV pp65 peptide. The results of two independent experiments are shown. Points represent results from individual vaccinated mice.
  • Fig. 5 Monitoring of antigen-specific human CD8 + T cell responses using the caspase 3-cleavage assay.
  • human T-cell lines were generated from PBMC of HLA-A*0201 donors using peptide-pulsed autologous DC and activated B cells as described. T-cell underwent four rounds of peptide-specific stimulation before being tested for CTL activity.
  • T cells were diluted with na ⁇ ve autologous PBMC at a 1:1 or 1:5 ratio before mixing with 100,000 pulsed target at a 1:1 ratio.
  • the cells were incubated for 1 or 3 h, as indicated, and stained for cleaved caspase 3.
  • Target cells alone ("T alone") were also used as controls. Results of one out of 3 similar experiments are shown.
  • B CTL activity measured in self- antigen-specific (CEA CAP1 peptide) human T-cell lines using caspase 3 cleavage or Annexin-V plus 7-AAD staining assays.
  • Effector cells were diluted at the indicated ratios with na ⁇ ve autologous PBMC before mixing with peptide-pulsed T2 cells pulsed with the CAP1 specific peptide or non-specific CMV pp65 peptide.
  • Target cells alone (“T alone") and na ⁇ ve PBMC from the donor were used as negative controls. The results of 3 h incubation are shown. Similar results were obtained after 1 or 4 h incubation (not shown). Results of one of out two similar experiments are shown.
  • the caspase 3-cleavage is a highly sensitive assay for monitoring murine CTL activity.
  • Spleen cells from b- d MLR were harvested and incubated with DDAO-SE-labeled P815 target cells.
  • the MLR effector cells were diluted with na ⁇ ve syngeneic spleen cells at the ratios indicated before mixing with the P8 5 targets at a 1:1 ratio.
  • One hundred thousand diluted effector cells and 100,000 target cells were in the assay.
  • the upper panel (A) shows dot plots for DDAO-SE versus caspase 3- cleavage after FACS analysis at different dilutions of the MLR effector cells. Na ⁇ ve B6 spleen cells mixed with target cells are shown as a negative control.
  • Fig. 7 The caspase 3-cleavage is a highly sensitive assay for monitoring human CD8 + T-cell activity with a similar level of sensitivity as TCR staining with HLA- peptide pentamers.
  • HLA-A*0201 -restricted HIV gag peptide-specific human T-cell lines were generated as before and tested for CTL activity.
  • the T cells were diluted with na ⁇ ve autologous PBMC at the ratio indicated before adding at a 1:1 ratio with HIV or CMV (specificity control) peptide-pulsed T2 targets. Targets alone and na ⁇ ve PBMC with no effector cells were used as negative controls.
  • Dot plots show representative results when highly diluted HIV gag peptide-specific T cells are incubated T2 cells pulsed with HIV gag peptide or non-specific CMV pp65 peptide. Significant differences in percent caspase cleavage were routinely detected even at the 1 :199 dilution.
  • B Bar graph showing the average and standard deviation of three separate experiments testing the caspase 3 cleavage assay to measure CTL activity in HIV gag peptide-specific T cells lines diluted with na ⁇ ve PBMC as indicated. In each case, 150,000 diluted effectors were incubated with 150,000 DDAO-SE-labeled, peptide-pulsed targets.
  • T cells were stained with an HLA- gag peptide pentamer (Prolmmune, Oxford, UK) in parallel (C).
  • HLA- gag peptide pentamer Prolmmune, Oxford, UK
  • CMV pp65 peptide-specific T-cell line was used as the specificity control, hi order to compare the pentamer assay to the caspase 3-cleavage assay, 150,000 gated T cells were 5 collected for each data point (the same amount of effector cells added to the CTL assay).
  • the present invention relates to reagents and assays for performingO cytotoxicity assays.
  • the assays are suitable to detecting cytotoxicity mediated by immune effector cells including but not limited to T cells, natural killer (NK) cells, granulocytes, monocytes, macrophages, and the like,
  • the assays are generally based on the detection of DNA fragmentation- and/or apoptosis-related changes in a target cell. For instance, in certain embodiments, cleavage of an enzyme involved in the 5 apoptotic process is detected. In another, a change in the characteristic of a DNA- associated protein during the apoptotic process is detected.
  • the present invention relates to an assay for measuring theO cleavage of one or more caspases during the apoptotic process.
  • Caspases shown to play a role in apoptosis that may be suitable for detection in an assay as shown herein many include but are not limited to caspase 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.
  • caspase 3 is detected using the assay described herein.
  • Another enzyme known to be cleaved during the apoptotic process is poly-ADP5 ribose polymerase (PARP).
  • Cleavage of caspases, in particular caspase 3, and PARP are early events during apoptosis and are triggered in CTL targets.
  • a number of intracellular substrates are phosphorylated during the induction of apoptosis.
  • histone H2A.X is phosphorylated in wide variety of cell lineages.
  • Antibodies with specific bindingO capacity for cleaved caspases, as well as antibodies specific for phosphorylated histone H2.AX are commercially available and suitable for use in practicing the present invention.
  • dyes are utilized to stain the target cells prior to contact with effector cells.
  • the assay of the present invention provides is carried out by: 1) preparing effector cells (E); 2) preparing target cells (T), including tagging the cells with a detectable marker such as a fluorescent dye; 3) mixing the effector cells and the target cells at a sufficient number of E:T ratios; 4) incubating the mixture for a sufficient period of time and under conditions suitable for at least some of the effector cells to become cytotoxic toward at least some of the target cells; 5) fixing and permeabilizing the mixed cells; 6) staining the cells with a detectable reagent having the ability to bind a protein or fragment thereof that undergoes a change during or results from the apoptotic process; and, 7) detecting the detectable reagent.
  • the detectable reagent is detectable by virtue of a fluorescent tag or moiety contained within the reagent or by detecting a secondary reagent that binds to the detectable reagent, the secondary reagent being detectable by virtue of a fluorescent tag or moiety contained within the secondary reagent.
  • the detectable reagent and / or the secondary reagent is an antibody, which may be fluorescently labeled.
  • the detectable reagent may detected by any of several well-known methods including but not limited to FACS.
  • the detectable reagent is an anti-Caspase 3 antibody (such as the PE Conjugated Monoclonal Rabbit Anti-Active Caspase 3 Antibody, BD Pharmingen Catalog No. 550914).
  • Other suitable reagents are known in the art.
  • a target cell may be pulsed with a peptide corresponding to the amino acid sequence of an infectious agent such as HIV or a tumor antigen.
  • Suitable tumor antigens include include, for example, gplOO (Cox et al., Science, 264:716-719 (1994)), MART- 1/Melan A (Kawakami et al., J. Exp. Med., 180:347-352 (1994)), gp75 (TRP-1) (Wang et al, J. Exp. Med., 186:1131-1140 (1996)), tyrosinase (Wolfel et al., Eur. J. Immunol, 24:759-764 (1994)), NY-ESO-1 (WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom et al., J.
  • MAGE family antigens i.e., MAGE-1, 2,3,4,6, and 12; Van der Bruggen et al., Science, 254:1643-1647 (1991); U.S. Pat. Nos. 6,235,525), BAGE family antigens (Boel et al., Immunity, 2:167-175 (1995)), GAGE family antigens (i.e., GAGE-1,2; Van den Eynde et al., J. Exp. Med., 182:689-698 (1995); U.S. Pat. No.
  • RAGE family antigens i.e., RAGE-1; Gaugler et at., Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526), N-acetylglucosaminyltransferase-V (Guilloux et at., J. Exp. Med., 183 : 1173-1183 (1996)), pi 5 (Robbins et al., J. Immunol. 154:5944-5950 (1995)), ⁇ -catenin (Robbins et al., J. Exp. Med., 183:1185-1192 (1996)), MUM-1 (Coulie et al., Proc. Natl.
  • EGFR epidermal growth factor receptor
  • CEA carcinoembryonic antigens
  • the time during which the target and effector cells are incubated may be modified. For instance, it may be desirable to incubate the mixture for one, two, three, four, five, six or more hours prior to further analysis.
  • kits for carrying out the assays described herein include kits for carrying out the assays described herein.
  • a kit may include materials useful in preparing effector cells, a suitable number of effector cells, a fluorescent dye with which to stain the target cells, a device such as a 96-well plate in which the target and effector cells may be mixed and incubated, materials required for fixing and permeabilizing the cells, a detectable reagent, and / or a secondary reagent. Different combinations of such materials may be organized as a kit in order to aid the skilled artisan in carrying out the assay of the present invention. A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.
  • Cytokines (IL-2, IL-2, IL-12, and IL-6) were from R&D Systems (Minneapolis, MN). Anti-IFN- ⁇ antibody pairs for human and mouse IFN- ⁇ ELISPOT analysis was from MabTech (Amsterdam, NL) and BD Biosciences (Mississauga, ON), respectively. 51 Cr ( 51 Sodium Chromate) was from Amersham (Toronto, Ontario).
  • HLA-A2.1 -binding and murine H-2K b -binding peptides were used in the experiments: ILKEPVHGV (HIV rev), SLYNTVATL (HIV gag), YLSGANLNL (CEA tumor antigen CAP1 epitope), IMDQVPVSV, YLEPGPVTV, KTWGQYWQV (g lOO melanoma epitopes), NLVPMVATV (CMV pp65 epitope), DAPIYTNV ( ⁇ -galactosidase H-2K b epitope), TPHPARIGL (( ⁇ - galactosidase H-2L d ), and TLDSQVMSL (TRP-2 melanoma epitope).
  • ALVAC-gal and ALVAC-gplOO were propagated in chick embryonic fibroblasts (CEF) and purified on a sucrose cushion by centrifugation from lysed cells, as previously described (Ferrari et al., 1997).
  • Viral titers in plaque-forming units (pfu) were determined in CEF, as previously described (Santra et al., 2002).
  • mice Balb/c and C57BL/6 mice were purchased from Charles River (Montreal, QC). HLA-A2.1/K b transgenic mice were generated as described (Borenstein et al., 2000). All mice were house under specific-pathogen fee (SPF) conditions in micro- isolator cages in the Animal Resources Department of Aventis Pasteur (Sunnybrook Campus, Toronto, ON) adhering to Canadian Council on Animal Care (CACC) guidelines.
  • SPF specific-pathogen fee
  • Human PBMC preparations Human PBMC were obtained from leukopherisis donors using Ficoll-Hypaque gradient centrifugation (Sigma, St. Louis, MO). All blood products were collected at the Sunnybrook and Women's Health Sciences Center (Toronto, ON) according to institutional (IRB guidelines with complete donor consent. Donors were pre-screened for HLA-A*0201 using DNA sequencing and only positive individuals subjected to leukopherisis collections.
  • Murine MLR and preparation of effector and target cells Spleens from C57BL/6 or Balb/c mice were harvested in mouse cell culture medium (ot-MEM, 10% FCS, 50 ⁇ M mercaptoethanol, 1 mM Glutamax, Penicillin- Streptomycin) and cell suspensions were made using a stomacher machine (Stomacher 80 Biomaster, UK). The cell suspension was filtered through a 70 ⁇ m pore size strainer (BD Biosciences). The cells were washed and re-suspended in a culture medium.
  • mouse cell culture medium ot-MEM, 10% FCS, 50 ⁇ M mercaptoethanol, 1 mM Glutamax, Penicillin- Streptomycin
  • Stimulator cells were irradiated at 2000 Rads (Gammacell ® 1000 Elite, MDS Nordion) and washed once in culture medium and re-suspended in culture medium at 10 x 10 6 cells/ml.
  • Responder cells (20 x 10 6 cells) and irradiated stimulator cells (20 x 10 6 cells) were placed into upright T-25 flasks in a total of 20 ml.
  • the cultures were incubated for 5 days and viable cells harvested using centrifugation over Lympholyte-M (Cedarlane Labs, Canada) at lOOxg for 20 min. The cells were washed in culture medium at 1.5 xlO 6 /ml.
  • T-cell re-stimulation Peptide immunization was performed using a melanoma H-2 -binding TRP-2 tumour antigen peptide linked to a transcytosis peptide from the human Period 1 gene. This sequence induces the uptake of the TRP-2 sequence in APC and induces a strong anti-peptide CTL response.
  • C57BL/6 mice were immunized with 50 ⁇ g of the TRP-2 peptide subcutaneously and boosted after 3 weeks.
  • Splenocytes were harvested 5 weeks and 7 weeks after boosting and re-stimulated (5 x 10 6 cells/well in 24- well plates) with a pool of dominant HLA-A2.1 -binding gplOO peptides (IMDQVPVSV, YLEPGPVTV, and KTWGQYWQV), at 0.2 ⁇ g/ml/peptide, for 5 days.
  • the cells were harvested and subjected to analysis using IFN- ⁇ ELISPOT or the caspase 3-cleavage assay.
  • the ELISPOT analysis was performed with 100,000 re-activated spleen cells mixed with 10,000 gplOO peptide-pulsed (2 ⁇ g/ml) P815 stably transfected with an HLA-A2/K b expression plasmid (P815-A2/K b cell line).
  • T-cell-enriched PBMC plastic-adherent negative fraction of PBMC
  • DC peptide-pulsed autologous mature dendritic cell
  • HTC-CM human T- cell culture medium
  • Iscove's Modified Dulbecco's Medium 1 mM Glutamax, 1 mM pyruvate (Invitrogen, Mississauga, ON), 5% human AB serum, 50 ⁇ M 2-mercaptoethanol, 20 ⁇ g/ml gentamycin.
  • Mature DC were generated in a one-step protocol over 6 days from adherent monocytes using HTC-CM containing 2% human AB serum, 1,000 U/ml GM-CSF, 1,000 U/ml IL-4, 4 ng/ml TNF- ⁇ , and 100 U IFN- ⁇ .
  • the DC were harvested, washed and pulsed with 10 ⁇ g/ml of peptide.
  • the following HLA-A2.1 -binding nonamer peptides were used for T-cell generation: HIV rev peptide (ILKEPVHGV), HIV gag peptide (SLYNTVATL) and the CAP1 epitope from the CEA tumor antigen (YLSGANLNL).
  • G Caspase 3-cleavage assay and staining methodology 1.
  • Target cell labeling and peptide pulsing Harvested target cells (P815, EL4, T2 lymphoma cells) were washed once with D-PBS. The cells were re-suspended at 5 x 10 6 /ml in labeling buffer containing 0.6 ⁇ M DDAO-SE (Molecular Probes, Eugene, OR) in D-PBS and incubated at 37°C for 15 min. The cells were washed in culture medium and re-suspended in culture medium at 2 x 10 6 cell/ml and pulsed with the indicated peptides at 1-3 ⁇ g/ml for 1 h. The pulsed targets were washed once in culture medium and re-suspended at 1 x 10 6 cells/ml.
  • the cells were washed with D-PBS, 1% BSA at room temperature (RT) and either fixed and permeabilized with Fix/Perm solution (BD Biosciences, Mississauga, ON) 20 min at RT immediately or fixed in 1% paraformaldehyde for 20 at RT and then stored at 4°C for up to 24 h. Fixed and stored cells were centrifuged and re-suspended in Fix Perm buffer at RT for 20 min. Cells were then washed 2 times with staining buffer (D-PBS, 1% BSA, 0.1% saponin) and re-suspended in 0.1 ml staining buffer.
  • D-PBS staining buffer
  • the cells were stained for 60 min on ice with 15 ⁇ l biotin-labeled anti-cleaved caspase 3 monoclonal antibody (BD Biosciences, Mississauga, ON). The cells were washed in staining buffer and counter-stained with Strepavidin-PE (Sigma, St. Louis, MO) for 30 min. on ice. The cells were washed in staining buffer 2 times and re-suspended in D-PBS, 1% BSA for analysis on a flow cytometer.
  • IFN- ⁇ ELISPOT assay ELISPOT essay was using Millipore MultiScreen-HA 96-well filter plates (Millipore Cat No. MAHAS4510). Plates were coated with 5 ⁇ g/ml anti-IFN- ⁇ mAb in pH 9.5 carbonate buffer (Sigma, St. Louis, MO) overnight at 4°C and blocked with D-PBS, 2% BSA. Human or murine T cells (100,000/well) were added, as indicated and incubated with relevant or irrelevant peptides overnight. The plates were washed and incubated with biotin-labelled anti-IFN- ⁇ mAb for 1 h at RT.
  • the plates were washed and then treated with 1 :5,000 dilution of Extravidin- Alkaline phosphatase (Sigma) for 1 h at RT.
  • the plates were developed with BCIP/NBT substrate (Sigma) until all spots were clearly visible.
  • Developed plates were dried and counted on an AID Version 3.1.1 ELISPOT reader (Cell Technologies, USA). Cells treated with PMA and Ionomycin (Sigma) were used as positive controls in all experiments.
  • I. sl Cr-release Assay Target cells (P815, P815-A2/K b , EL4, and T2 cells) were labeled with 100 ⁇ Ci Na 2 51 Cr0 4 in cell culture medium containing 10% FCS for 45 min at 37°C. The cells were washed in culture medium and pulsed with the specific or non-specific control peptides (1 to 3 ⁇ g/ml) for 1 h if the protocol required. The pulsed cells were washed once in culture medium and re-suspended at 30,000 cells/ml.
  • Effector cells (murine MLR, peptide re-stimulated spleen cells, or peptide-activated human T-cell lines) were added at different E:T ratios, as indicated in the results.
  • the effector cells were diluted with na ⁇ ve lymphocytes before addition at various E:T ratios to the 51 Cr-release assay. After 4 h incubation, 25 ⁇ l of the assay supernatant was placed into a 96-well Lumaplate (Packard). The plates were dried and radioactivity counted using a TopCount NXT v2.12 scintillation counter (Packard). The results are expressed as % specific lysis calculated as (Experimental release - Spontaneous release / Total release - Spontaneous release) x 100.
  • HLA-pentamer staining HIV gag peptide-specific and CEA CAPl peptide-specific HLA-A*0201 human T-cell lines were stained with PE-conjugated recombinant HLA-peptide pentamers (Prolmmune, Oxford, UK).
  • An HLA-A*0201 pentamer containing the SLYNTVATL sequence from HIV gag (Prolmmune code 010) was used to stain the HIV-specific lines, while an HLA-A*0201 pentamer containing the YLSGANLNL peptide from CEA (Prolmmune code 075) was used to stain the CAPl peptide- specific line.
  • an HLA-A* 0201 -restricted T-cell line consisting of a similar percentage of CD8 + T-cells was used as a non-specific pentamer staining control.
  • the T cells were washed and re-suspended at 2 x 10 6 cell/ml in pentamer staining buffer (PSB) consisting of D-PBS, 0.1% NaN 3 , 0.1% BSA.
  • PSB pentamer staining buffer
  • the cells were stained with 10 ⁇ l Pro 5TM for 20 min at RT, washed and then stained in PSB with 5 ⁇ l anti-human CD8-FITC (BD Biosciences) for 20 min at RT.
  • the cells were washed in PSB and fixed in D-PBS, 1% paraformaldehyde and run through a FACScalibur flow cytometry analyser.
  • the pentamer-positive cells were detected and quantitated by gating first on live cells using the forward scatter and side scatter discriminators and then analysing on a two-colour plot showing CD8 + fluorescence on the x-axis and pentamer 4" fluorescence on the y-axis. Results are shown as % pentamer 4" cells for each culture.
  • Example 2 A Assay procedure and choice of target cell tracking dye
  • One of the first issues we resolved is the problem of finding an effective target cell tracking dye that would not interfere with the caspase 3 signal during FACS analysis.
  • To maximize sensitivity we wanted to use a biotin-labeled primary anti- cleaved caspase 3 followed with a Strepavidin-PE counter-stain brightly fluorescing in the FL2 channel.
  • a non-toxic cell tracker dye that would fluorescence in the far-red (FL4) channel and not require any compensation with the FL2 channel.
  • DDAO-SE Molecular Probes
  • DDAO-SE was found to reproducibly and stably stain of a variety of murine and human cell lines for up to 15 h, including P815, EL-4, B16F10 melanoma, 4T1 breast cancer, human T2 thymoma, and COS cells, with no induction of non-specific caspase 3 cleavage.
  • DDAO-SE-stained target cells we first tested the caspase 3-cleavage assay to measure CTL activity in murine allogeneic T-cell responses. Mixed lymphocyte responses of C57BL/6 anti-Balb/c and Balb/c anti-C57BL/6 spleen cells were tested after 5 days of culture in comparison to 51 Cr-release.
  • Fig. IB shows the gating strategy employed top enumerate the percentage of caspase 3- cleaved target cells. This approach was used in all experiments. Forward scatter (FSC) and side scatter (SSC) plots were used to isolate the population of live DDAO- SE + (FL4 channel) target cells as shown (R2 and R3). The R2 and R3 gates were combined and used to determine the percentage DDAO-SE "1" , caspase 3 -cleaved (FL2 channel) targets. This gating strategy ensured that only live targets were taken into consideration. Experiments with C57BL/6 anti-Balb/c MLR effector cells with P815 targets
  • caspase 3 inhibition using the caspase inhibitor, Z-DEVD-FMK, or a general caspase inhibitor, Z- VAD-FMK did not prevent the appearance of cleaved caspase 3 in the P815 targets of the C57BL/6 anti-Balb/c MLR (data not shown).
  • caspase 3 cleavage in target cells is dependent on granzymes secreted by the CTL and is not triggered by endogenously activated caspase 3, or other caspases cleaving caspase 3 in the target during the killing process.
  • the caspase 3-cleavage assay was compared to the 51 Cr-release assay in two different assay systems, the murine C57B1/6 anti-Balb/c MLR system and in a peptide vaccination response in HLA-A2.1/K b transgenic mice (Fig. 3).
  • the caspase 3-cleavage assay showed the same specificity and reliability as the 51 Cr- release assay. However, it was markedly more sensitive being able to detect CTL activity at E:T ratios below 0.5:1 (Fig. 3A).
  • Spleen cells from mice vaccinated with a human TRP2 melanoma antigen peptide were also tested for CTL activity against peptide-pulsed P815-A2/K target cells (Fig.
  • the caspase 3-cleavage assay yielded similar results as 51 Cr-release when peptide re-stimulated spleen cells were tested at a 40:1 and 10:1 E:T. Again, as before, the assay showed a significantly higher degree of sensitivity with higher rates of specific CTL activity detected at the 10:1 E:T ratio (Fig. 3B).
  • caspase 3-cleavage assay to monitor CTL responses against viral vector vaccines
  • the caspase 3 cleavage CTL assay was compared to the IFN- ⁇ ELISPOT assay to measure gplOO peptide-specific the CD8 + recall response following immunization of HLA-A2.1/K transgenic mice with 2 x 10 pfu ALVAC-gplOO.
  • the mice were primed and boosted and spleen cells re-stimulated with 0.2 ⁇ g/ml of a pool of HLA-A2.1 -binding gplOO peptides for 5 days and then subjected to effector cell assays using either caspase 3-cleavage or IFN- ⁇ ELISPOT.
  • the caspase 3- cleavage assay used DDAO-SE-labeled gplOO or control peptide-pulsed P815 ceils stably transfected with HLA-A2.1/K b plasmid.
  • a similar approach was used in the ELISPOT assays, except the P815-A2/K b cells were not DDAO-SE labeled (see methods section).
  • Five replicate mice were used in each assay to test the precision of the results between individual animals when using the caspase 3-cleavage assay as a read-out.
  • Fig. 4 shows the results of two separate experiments comparing the caspase 3-cleavage read-out for gplOO peptide-specific CD8 + T-cell responses versus ELISPOT.
  • measurement of CTL activity via caspase 3 cleavage can also be used as a supportive functional assay for IFN- ⁇ ELISPOT or as a substitute assay for the ELISPOT when target cells expressing the target antigen or peptide are available.
  • T cell lines were generated from HLA-A*0201 + donors against a 9-mer HIV rev peptide, an HIV gag peptide, or against the CAPl peptide from the tumor-associated antigen CEA. All T-cell lines were generated from normal, non-HIV-infected and non-cancerous donors by repeated cycles of peptide stimulation and proliferation with IL-2 and IL-7 as outlined in the methods. After the third or fourth stimulation cycle, we routinely acquire T-cell lines consisting of >85% TCR ⁇ + , CD8 + , CD16 " , CD56 " proliferating T cells.
  • Fig. 5 A shows typical results of the caspase 3-cleavage assay for monitoring the activity of activated human T cells specific using an HLA-A2.1 -restricted T-cell line specific for a peptide from HTV rev.
  • the activated T cells were mixed with pulsed T2 targets at a 5:1 or 1:1 E:T ratio (Fig. 5 A).
  • the assay proved to be specific with only minimal level of caspase 3 cleavage in T2 targets pulsed with a non-specific HLA-A*0201 -binding peptide from the pp65 CMV antigen.
  • Fig. 5B we compared the results of the caspase 3-cleavage assay to that of another FACS-based CTL assay that detects apoptosis in targets using Annexin-N and 7-AAD staining (Fischer et al., 2002) with a kit (BD Bisciences) using T-cell lines specific for the CAPl HLA-A2.1 epitope of CEA.
  • the T-cell lines (4 th stimulation) were diluted with na ⁇ ve autologous PBMC from the donor at the ratios indicated and mixed with an equal number (100,000) peptide-pulsed T2 target cells. Both assays yielded comparable results with a similar level sensitivity (Fig. 5B). However, we noticed in a number of experiments that the Annexin-N + 7-AAD assay had a significantly higher non-specific background. In addition, we found that there was considerable variation in level of Annexin N fluorescence between samples (data not shown). Annexin N binding to phosphatidyl serine is Ca 2+ sensitive and slight alterations in Ca 2+ concentration can alter the Annexin-N binding equilibrium.
  • the diluted effectors were incubated with DDAO-SE-labelled P815 cells at a 1 : 1 ratio.
  • Target cells alone, and na ⁇ ve splenocytes served as negative controls for CTL activity and DDAO-SE-labeled EL-4 targets served as specificity controls.
  • the caspase 3-cleavage exhibited a high degree of sensitivity, as shown by the presence of significant caspase 3 cleavage even when the MLR effector cells were diluted at a ratio of 1 : 199 with na ⁇ ve splenocytes (Fig. 6). An insignificant level of caspase 3 cleavage was seen at all dilutions in the non- specific EL4 targets (Fig. 6).
  • the percentage of allo-reactive CD8 + splenocytes in a typical murine MLR has been found to be as high as 1:10. Thus, assuming that this frequency of anti-Balb/c effectors was present in the MLR, at the 1:199 dilution with na ⁇ ve splenocytes the assay was able to detect CTL activity at a 1:2,000 (0.05%) frequency of effector cells. This experiment was repeated at least 3 times with similar results. A similar dilution experiment was performed with HIV peptide-specific human HLA-A*0201 -restricted T cell lines after 3 to 4 rounds of stimulation from a na ⁇ ve PBMC pool (Fig. 7). In the experiment shown in Fig.
  • IFN- ⁇ ELISPOT analysis estimated that the frequency of HIV peptide-specific T cells in the cultures was 1:156 (0.64%) after 3 stimulations. These T cell cultures were diluted with na ⁇ ve autologous PBMC at a 1:0 to 1:199 ratio. As before, DDAO-SE-labeled T2 lymphoma cell targets alone and na ⁇ ve PBMC minus activated T cells incubated with the HIV peptide-pulsed T2 targets were used negative controls, while T2 cells pulsed with the HLA-A* 0201 -binding pp65 CMV peptide was used as a control for specificity. As shown in Fig.
  • Fig. 7B The percentage of caspase 3 cleavage in the targets minus the effectors and with na ⁇ ve PBMC plus targets was less than 2% (Fig. 7B).
  • Fig. 7C shows the results of the pentamer staining on the same T cells used in the caspase 3-cleavage assay shown in Fig. 7B. This analysis revealed that the caspase 3-cleavage assay had a comparable level of sensitivity as HLA pentamer staining.
  • the frequency of HIV gag peptide-specific T cells in the caspase 3-cleavage assay (Fig. 7B), based on the IFN- ⁇ ELISPOT frequency, was 1:15, 444 (0.007%) and 1:31,044 (0.003%) for the 1:99 and 1:199 dilutions of the effectors, respectively.
  • it is a measure of T cell function and not only T-cell frequency.
  • the VITAL assay a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo.

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Abstract

L'invention concerne des méthodes de détection de l'activité des lymphocytes T cytotoxiques (CTL) par cytométrie de flux pour la détection du clivage d'enzymes associés à la cytotoxicité.
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