AU2005224597A1 - Cytotoxicity assay - Google Patents

Description

WO 2005/090990 PCT/US2005/001993 CYTOTOXICITY ASSAY Field of the Invention The present invention relates to methods for detecting CTL activity using flow cytometry to detect cleavage of cytotoxicity-related enzymes. 5 Background of the Invention The 51 Cr-release assay has been the gold standard to measure CTL activity during an immune response for over 3 decades. However, the assay has a number of technical limitations and recent data has brought into question the physiological 10 relevance of the 5 sCr-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 distinct mechanisms: 1) A membranolysis process mediated when perforin is simply inserted into the target cell membrane; and, 2) DNA fragmentation (apoptosis) induced by the action of 15 Granzyme B and Granzyme A entering a target cell via perforin 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 20 relevant. Based on these observations, the investigator must remain cognizant of the fact that effector cells determined to be cytotoxic based on 5 sCr-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 immunotherapy literature in which effector cell responses monitored in vitro may not correlate with vaccine efficacy in 25 the patient. For example, peptide vaccines selected by virtue of positive effector cell cytotoxicity date generated using 5 Cr-release assays may not accurately reflect the actual epitopes active in inducing CTL lysis in vivo via DNA fragmentation. In addition, there are a number of other issues plaguing the use of 5 Cr-based assays, including the need to use hazardous radioactivity, and problems related to the use of 30 scintillation counters. 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

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thymidine or 12 5 -deoxyuridine. This method invariably causes DNA damage and 1 WO 2005/090990 PCT/US2005/001993 changes the physiology of the target cell before the CTL assay. In addition, the 3

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thymidine labelling method does not afford the same degree of sensitivity as 51 Cr and 1251 is a dangerous agent requiring considerable lead shielding of both the lab worker as well as the lab area. Overall, a non-radioactive method to measure apoptosis 5 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. Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic 10 caspase substrates. Nature Medicine, Jan. 2003, 9(1):4-5.) In addition, other highly sensitive methods to enumerate antigen-specific CD8 + T-cells have been introduced, such as intracellular cytokine staining, the ELISPOT assay, aind staining for T-cells using class I MHC tetramers. However, these assays do not measure ultimate cytolytic function, which is especially important in analyzing anti-tumor immune 15 responses. Assays offering a suitable levels of sensitivity, specificity, safety and ease of-use are lacking. A new and sensitive assay system for detecting CTL activity is provided herein. Summary of the Invention 20 The present invention provides a cytotoxicity assay that detects DNA fragmentation- and/or apoptosis-related changes in target cells following contact with effector cells. In one embodiment, the assay is conducted using one or more reagents that detect cleavage of a caspase or phosphorylation of histones. In a preferred embodiment, cleavage of caspase is detected using a monoclonal antibody. In another 25 preferred embodiment, phosphorylation of histones is detected using a monoclonal antibody. In other embodiments, cleaved caspase and/or phosphorylated histones are detected using at least one monoclonal antibody coupled with flow cytometric analysis. 30 Brief Description of the Drawings Fig. 1. Stability of target cell labeling dye and gating strategy developed for monitoring CTL activity using the caspase 3-cleavage in target cells. A. DDAO SE cell tracker dye brightly and stably labels CTL target cell lines. Shown is staining of a number of commonly used suspension-cultured and adherent cell lines: P815, 2 WO 2005/090990 PCT/US2005/001993 EL4, T2 lymphoma, NIH 3T3, 4T1 breast cancer, and B16F10 melanoma. Cells were stained with DDAO-SE and incubated for the indicated time intervals (0, 1, 3, or 15 h) and analyzed by FACS. The mean fluorescent intensity and the coefficient of variation (CV) are shown for each cell line. The result of one out of two similar 5 experiments is shown. B. Gating strategy used to enumerate caspase 3-cleavage in DDAO-SE-labeled targets. 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 (R1). Gate R1 cells were then analyzed using SSC and FSC versus FL4 fluorescence (DDAO-SE positive). DDAO-SE positive cells were 10 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 15 cleavage assay and dependence of caspase 3 cleavage in the target on perforin 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. B. P815 or 20 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. C. Concanamycin A, an 25 inhibitor of cytolytic granule secretion by CTL prevents caspase 3 cleavage in P815 targets incubated with C57BL/6 anti-Balb/c MLR effector cells. Five-day MLR cultures were isolated and diluted with naive syngeneic C57BL/6 spleen lymnphocytes 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 naive C57BL/6 spleen cells with no MLR 30 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 5 sCr-release for monitoring CTL activity using murine MLR effectors and CTL isolated after 3 WO 2005/090990 PCT/US2005/001993 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 s 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 5 and 0.1:1 E:T ratio. The results of 1 out of 2 similar experiments are shown. B. Caspase 3 cleavage assay is equally reliable and specific as the 5 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. 10 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-y ELISPOT analysis. HLA-A2.1/Kb transgenic mice were immunized with ALVAC-gpl 100 and spleen cells re-stimulated for 5 days with a pool of gpl00 peptides. Viable cells were isolated and tested for CTL activity using 15 caspase 3 cleavage with at a 20:1 E:T ratio (left panel), or for IFN-y production in ELISPOT assays (right panel) at 100,000 cells per well. Targets were P815-A2/Kb transfectants pulsed with gpl00 peptides or a control A2-binding CMV pp65 peptide. The results of two independent experiments are shown. Points represent results from individual vaccinated mice. 20 Fig. 5. Monitoring of antigen-specific human CD8 T cell responses using the caspase 3-cleavage assay. Primary peptide-specific 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 25 stimulation before being tested for CTL activity. A. Application of the caspase 3 cleavage assay to foreign antigen-specific (HIV rev peptide) T cell lines. T cells were harvested and incubated with DDAO-SE-labeled T2 lymphoma targets pulsed with the HIV rev or CMV pp65 peptides, or with no peptide. The T cells were diluted with naive autologous PBMC at a 1:1 or 1:5 ratio before mixing with 100,000 pulsed target 30 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 4 WO 2005/090990 PCT/US2005/001993 Annexin-V plus 7-AAD staining assays. Effector cells were diluted at the indicated ratios with naive autologous PBMC before mixing with peptide-pulsed T2 cells pulsed with the CAP1 specific peptide or non-specific CMV pp 6 5 peptide. Target cells alone ("T alone") and naive PBMC from the donor were used as negative 5 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. Fig. 6. The caspase 3-cleavage is a highly sensitive assay for monitoring murine 10 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 naive syngeneic spleen cells at the ratios indicated before mixing with the P815 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 15 cleavage after FACS analysis at different dilutions of the MLR effector cells. Naive B6 spleen cells mixed with target cells are shown as a negative control. B. Results of two independent experiments showing caspase 3 cleavage at the indicated b-4d MLR effector cell dilutions. The effectors and P815 target cells were incubated for 2 or 4 h before staining for cleaved caspase 3. 20 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 25 with naive 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 naive PBMC with no effector cells were used as negative controls. A. 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. 30 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 naive PBMC as indicated. In each case, 150,000 diluted effectors were incubated with 150,000 DDAO-SE-labeled, 5 WO 2005/090990 PCT/US2005/001993 peptide-pulsed targets. As a comparison, the same T cells were stained with an HLA gag peptide pentamer (ProImmune, Oxford, UK) in parallel (C). A CMV pp65 peptide-specific T-cell line was used as the specificity control. In 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). Detailed Description The present invention relates to reagents and assays for performing 10 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 15 apoptotic process is detected. In another, a change in the characteristic of a DNA associated protein during the apoptotic process is detected. Such changes are suitable to detection using devices that measure cell fluorescence, such as a Fluorescence Activated Cell Sorter (FACS) machine or flow cytometer. In one embodiment, the present invention relates to an assay for measuring the 20 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. In one embodiment, caspase 3 is detected using the assay described herein. Another enzyme known to be cleaved during the apoptotic process is poly-ADP 25 ribose polymerase (PARP). Cleavage of caspases, in particular caspase 3, and PARP are early events during apoptosis and are triggered in CTL targets. In addition to the cleavage of caspases and PARP, a number of intracellular substrates are phosphorylated during the induction of apoptosis. For instance, histone H2A.X is phosphorylated in wide variety of cell lineages. Antibodies with specific binding 30 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. In certain embodiments, dyes are utilized to stain the target cells prior to contact with effector cells. Suitable dues include Vial T, CFSE, and DDAO-SE, each 6 WO 2005/090990 PCT/US2005/001993 of which are known in the art. A preferred dye is DDAO-SE (Molecular Probes). Other suitable dyes are also known in the art and may be useful in practicing the present invention. In one embodiment, the assay of the present invention provides is carried out 5 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) 10 fixing and permeabilizing the mixed cells; 6) staining the cells with a detectable reagent having the ability to bind a protein or fri-agment thereof that undergoes a change during or results from the apoptotic process; and, 7) detecting the detectable reagent. In certain embodiments, the detectable reagent is detectable by virtue of a fluorescent tag or moiety contained within the reagent or by detecting a secondary 15 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. In preferred embodiments, 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. In one 20 preferred embodiment, the detectable reagent is an anti-Caspase 3 antibody (such as the PE Conjugated Monoclonal Rabbit Anti-Active Caspase 3 Antibody, BD Pharrningen Catalog No. 550914). Other suitable reagents are known in the art. In certain embodiments, it may be beneficial or desirable to contact or "pulse" a target cell with a peptide to which the effector cells may respond prior to staining 25 the target cells and / or mixing the target and effector cells. For instance, 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, gpl00 (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) 30 (Wang et al., J. Exp. Med., 186:1131-1140 (1996)), tyrosinase (Wolfel et al., Eur. J. Inmnzunol., 24:759-764 (1994)), NY-ESO-1 (WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom et al., . Immunol., 130:1467-1472 (1983)), 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 7 WO 2005/090990 PCT/US2005/001993 (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. 6,013,765), 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., 5 J. Exp. Med., 183:1173-1183 (1996)), p15 (Robbins et al., J. Immunol. 154:5944-5950 (1995)), 1-catenin (Robbins et al., J. Exp. Med., 183:1185-1192 (1996)), MUM-1 (Coulie et al., Proc. Natl. Acad. Sci. USA, 92:7976-7980 (1995)), cyclin dependent kinase-4 (CDK4) (Wolfel et al., Science, 269:1281-1284 (1995)), p21-ras (Fossum et at., Int. J. Cancer, 56:40-45 (1994)), BCR-abl (Bocchia et al., Blood, 85:2680-2684 10 (1995)), p53 (Theobald et al., Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995)), p 185 HER2/neu (erb-B1; Fisk et al., J. Exp. Med., 181:2109-2117 (1995)), epidermal growth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat, 29:1-2 (1994)), carcinoembryonic antigens (CEA) (Kwong et al., J. Natl. Cancer Inst., 85:982-990 (1995) U.S. Pat. Nos. 5,756,103; 5,274,087; 5,571,710; 6,071,716; 15 5,698,530; 6,045,802; EP 263933; EP 346710; and, EP 784483); carcinoma associated mutated mucins (i.e., MUC-1 gene products; Jerome et al., J. Immunol., 151:1654-1662 (1993)); EBNA gene products of EBV (i.e., EBNA-1; Rickinson et al., Cancer Surveys, 13:53-80 (1992)); E7, E6 proteins of human papillomavirus (Ressing et al., J. Immunol, 154:5934-5943 (1995)); prostate specific antigen (PSA; 20 Xue et al., The Prostate, 30:73-78 (1997)); prostate specific membrane antigen (PSMA; Israeli, et al., Cancer Res., 54:1807-1811 (1994)); idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes (Chen et al., J. Immunol., 153:4775-4787 (1994)); KSA (U.S. Patent No. 5,348,887), kinesin 2 (Dietz, et al. Biochem Biophys Res Commun 2000 Sep 7;275(3):731-8), HIP-55, 25 TGF3-1 anti-apoptotic factor (Toomey, et al. Br J Biomed Sci 2001;58(3):177-83), tumor protein D52 (Bryne J.A., et al., Genomics, 35:523-532 (1996)), H1FT, NY-BR 1 (WO 01/47959), NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87 and NY-BR-96 (Scanlan, M. Serologic and Bioinformatic Approaches to the Identification of Human Tumor Antigens, in Cancer Vaccines 2000, Cancer Research Institute, New York, 30 NY), among others, including wild-type, modified, mutated versions thereof. In certain embodiments, 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. 8 WO 2005/090990 PCT/US2005/001993 Other aspects of the present invention 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 5 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 10 will be had from the following examples, given by way of illustration. EXAMPLES Example 1 MA TERIALS AND METHODS 15 A. Reagents and cell culture media Anti-cleaved caspase 3 (reactive against both human and mouse proteins) was phycoerythrin (PE)-labeled and purchased from BD Biosciences (Mississauga, ON). Anti-phospho-histone H2A.X-FITC was purchased from Upstate Cell Signaling Solutions (Lake Placid, NY). The cell tracker dye, DDAO-SE was obtained from 20 Molecular Probes (Eugene, OR). All cell culture medium was from Invitrogen (Mississauga, ON). Cytokines (IL-2, IL-2, IL-12, and IL-6) were from R&D Systems (Minneapolis, MN). Anti-IFN- 7 y antibody pairs for human and mouse IFN-y ELISPOT analysis was from MabTech (Amsterdam, NL) and BD Biosciences (Mississauga, ON), respectively. slCr ( 51 Sodium Chromate) was from Amersham 25 (Toronto, Ontario). The following HLA-A2.1-binding and murine H-2Kb-binding peptides were used in the experiments: ILKEPVHGV (HIV rev), SLYNTVATL (HIV gag), YLSGANLNL (CEA tumor antigen CAP1 epitope), IMDQVPVSV, YLEPGPVTV, KTWGQYWQV (gp 100 melanoma epitopes), NLVPMVATV (CMV pp65 epitope), DAPIYTNV (1-galactosidase H-2Kb epitope), TPHPARIGL ((P3 30 galactosidase H-2Ld), and TLDSQVMSL (TRP-2 melanoma epitope). Peptides were synthesized in a 396 Multiple Biomolecular Synthesizer (Advanced Chemtech, Louisville, KY) and purified by HPLC. ALVAC-gal and ALVAC-gpl00 were propagated in chick embryonic fibroblasts (CEF) and purified on a sucrose cushion by 9 WO 2005/090990 PCT/US2005/001993 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). 5 B. Mice Balb/c and C57BL/6 mice were purchased from Charles River (Montreal, QC). HLA-A2.1/Kb 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 10 Campus, Toronto, ON) adhering to Canadian Council on Animal Care (CACC) guidelines. C. 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 15 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. 20 D. Murine MLR and preparation of effector and target cells Spleens from C57BL/6 or Balb/c mice were harvested in mouse cell culture medium (ca-MEM, 10% FCS, 50 gM 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 gm 25 pore size strainer (BD Biosciences). The cells were washed and re-suspended in a culture medium. 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 106 cells/ml. Responder cells (20 x 106 cells) and irradiated stimulator cells (20 x 106 cells) were placed into upright T-25 flasks in a total of 20 30 ml. The cultures were incubated for 5 days and viable cells harvested using centrifugation over Lympholyte-M (Cedarlane Labs, Canada) at 100xg for 20 min. The cells were washed in culture medium at 1.5 xl 06/ml. 10 WO 2005/090990 PCT/US2005/001993 E. Mouse immunization studies and in vitro T-cell re-stimulation Peptide immunization was performed using a melanoma H-2b-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 5 anti-peptide CTL response. C57BL/6 mice were immunized with 50 pg of the TRP-2 peptide subcutaneously and boosted after 3 weeks. Three weeks after boosting spleens were harvested and spleen cells re-stimulated with TRP-2 peptide for 5 days and then subjected to CTL assays using either 51 Cr-release or using the caspase 3 cleavage assay. Immunization with a canarypox viral vector (ALVAC-gpl00) was 10 performed subcutaneously in HLA-A2.1/K b transgenic mice (Borenstein et al., 2000). The mice were immunized with 2 x 107 pfu of ALVAC-gpl00 followed by a booster at the same dose 2 weeks later. Splenocytes were harvested 5 weeks and 7 weeks after boosting and re-stimulated (5 x 106 cells/well in 24-well plates) with a pool of dominant HLA-A2.1-binding gpl00 peptides (IMDQVPVSV, YLEPGPVTV, and 15 KTWGQYWQV), at 0.2 pg/ml/peptide, for 5 days. The cells were harvested and subjected to analysis using IFN-y ELISPOT or the caspase 3-cleavage assay. The ELISPOT analysis was performed with 100,000 re-activated spleen cells mixed with 10,000 gpl00 peptide-pulsed (2 gg/ml) P815 stably transfected with an HLA-A2/Kb expression plasmid (P815-A2/K> cell line). 20 F. Generation of human peptide-specific T cells T-cell-enriched PBMC (plastic-adherent negative fraction of PBMC) were stimulated using peptide-pulsed autologous mature dendritic cell (DC) in human T cell culture medium (HTC-CM) consisting of Iscove's Modified Dulbecco's Medium, 25 1 mM Glutamax, 1 mM pyruvate (Invitrogen, Mississauga, ON), 5% human AB serum, 50 jtM 2-mercaptoethanol, 20 pg/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-a, and 100 U IFN-a. The DC were harvested, washed and pulsed with 10 jtg/ml 30 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). These peptides have been previously demonstrated to bind to HLA-A2.1 and elicit HLA-A2.1 11 WO 2005/090990 PCT/US2005/001993 restricted CD8 + T-cell responses (Stuber et al., 1992; Herr et al., 1996; Zaremba et al., 1997). The pulsed DC were washed and used to stimulate T-cell-enriched autologous PBMC (T cells) in 24-well plates at a ratio of 0.6 x 106 pulsed DC with 6 x 106 T cells. IL-2 (10 U/ml), IL-7 (5 ng/ml), IL-6 (1,000 U/ml), and IL-12 (10 ng/ml) were 5 added at culture initiation and the cells fed with IL-2 (additional 10 U/ml) after 4 days. Primary activations were performed for 10 to 12 days followed by 2 to 3 rounds of re-stimulation using peptide-pulsed CD40 ligand-activated autologous B lymphocytes, generated as previously described (Schultze et al., 1997). Effector cell assays (caspase 3-cleavage, IFN-y ELISPOT, CTL assays using Annexin-V/7-AAD, 10 and HLA-pentarner staining) were performed 5 days after the third or fourth stimulation. In some cases, the activated T cells were diluted with naive autologous PBMC before the assays, as indicated. G. Caspase 3-cleavage assay and staining methodology 15 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 pM DDAO-SE (Molecular Probes, Eugene, OR) in D-PBS and incubated at 37 0 C for 15 min. The cells were washed in culture medium and re-suspended in culture medium at 2 x 106 cell/ml and pulsed with the 20 indicated peptides at 1-3 pg/ml for 1 h. The pulsed targets were washed once in culture medium and re-suspended at 1 x 106 cells/ml. 2. CTL assay set-up and antibody staining. DDAO-SE-labeled target cells pulsed with peptide (0.1 ml) were mixed with 0.1 ml of effectors cells (1 x 106 cells/ml) in conical polypropylene Costar cluster tubes (Costar, Comrning, NY). The 25 cell mixture was centrifuged at low speed (200 rpm) for 1 minute. The mixtures were incubated at 37 0 C, 5% CO 2 in a humidified incubator for the indicated time periods (0.5 to 5 h). 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 30 at RT and then stored at 4 0 C for up to 24 h. Fixed and stored cells were centrifuged and re-suspended in Fix/Permnn 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. The cells were stained for 60 min on ice with 15 jil biotin-labeled 12 WO 2005/090990 PCT/US2005/001993 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. 5 3. Flow cytometric analysis. The stained cells were analyzed in a FACSCalibur flow cytometer (BD Biosciences, Mississauga, ON). Thirty to 50 thousand events were collected for each sample. Live cells and target cells were gated using the forward and side scatter parameters followed by gating of the DDAO-SE-labeled target cell population on the FL4 channel (see also Fig. 1B). Cleaved caspase 3 10 expression (FL2 channel) was then determined using the DDAO-SE gated target cells in an FL4 versus FL2 dot plot. IHI. IFN-y ELISPOT assay ELISPOT essay was using Millipore MultiScreen-HA 96-well filter plates 15 (Millipore Cat No. MAHAS4510). Plates were coated with 5 pg/ml anti-IFN-y mAb in pH 9.5 carbonate buffer (Sigma, St. Louis, MO) overnight at 4 0 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-y mAb for 1 h at RT. The plates were 20 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 lonomycin (Sigma) were used as positive controls in all experiments. 25 I. 5 sCr-release Assay Target cells (P815, P815-A2/Kb, EL4, and T2 cells) were labeled with 100 pCi Na 2 5 1 CrO 4 in cell culture medium containing 10% FCS for 45 min at 37 0 C. The cells were washed in culture medium and pulsed with the specific or non-specific control 30 peptides (1 to 3 tg/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. In some experiments, 13 WO 2005/090990 PCT/US2005/001993 the effector cells were diluted with naive lymphocytes before addition at various E:T ratios to the 5 lCr-release assay. After 4 h incubation, 25 itl 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). 5 The results are expressed as % specific lysis calculated as (Experimental release Spontaneous release / Total release - Spontaneous release) x 100. J. HLA-pentamner staining HIV gag peptide-specific and CEA CAP1 peptide-specific HLA-A*0201 human T-cell lines were stained with PE-conjugated recombinant HLA-peptide 10 pentamers (ProImmune, 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 CAPI peptide specific line. In both cases, an HLA-A*0201-restricted T-cell line consisting of a 15 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 106 cell/ml in pentamer staining buffer (PSB) consisting of D-PBS, 0.1% NaN 3 , 0.1% BSA. The cells were stained with 10 p1 Pro5T M for 20 min at RT, washed and then stained in PSB with 5 pl anti-human CD8-FITC (BD Biosciences) for 20 min at RT. The cells were washed in 20 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 + fluorescence on the y-axis. Results are shown as % pentamer + cells for 25 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 30 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. As a result, we searched for a non-toxic cell tracker dye that 14 WO 2005/090990 PCT/US2005/001993 would fluorescence in the far-red (FL4) channel and not require any compensation with the FL2 channel. We found that DDAO-SE (Molecular Probes) met these requirements. As shown in Fig. lA, 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, 5 Bl6F10 melanoma, 4TI breast cancer, human T2 thymoma, and COS cells, with no induction of non-specific caspase 3 cleavage. Using 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 10 were tested after 5 days of culture in comparison to 5 lCr-release. Effector cells were mixed with target cells (P815 or EL-4 cells) at different ratios and incubated for different time intervals and fixing and stained with anti-cleaved caspase 3. Fig. 1B shows the gating strategy employed top enumerate the percentage of caspase 3 cleaved target cells. This approach was used in all experiments. Forward scatter 15 (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 , caspase 3-cleaved (FL2 channel) targets. This gating strategy ensured that only live targets were taken into consideration. 20 Experiments with C57BL/6 anti-Balb/c MLR effector cells with P815 targets (1:1 E:T ratio) found that induction caspase 3- cleavage was specific for H-2d P815 targets and no marked cleavage was detected in H-2b EL4 targets (Fig. 2A). The time course shown in Fig. 2A and 2B also shows that the caspase 3 cleavage signal was maximal after 1 h incubation in this case. Similar results were found when Balb/c 25 anti-C57BL/6 MLR effector cells were tested (Fig. 2B). Overall, the caspase 3 cleavage assay exhibited the same specificity as using a 4-hour 51 Cr-release assay to measure allo-specific CTL activity in the MLR (data not shown). In order to confirm that the cleavage of caspase 3 in target cells was dependent on cytotoxic granules secreted by CTL, we incubated C57BL/6 anti-Balb/c MLR 30 effector cells with Concanamycin A, used as a CTL degranulation inhibitor in CTL assays, before mixing with the targets. Concanamycin A inhibited the appearance of caspase 3 cleavage in the P815 target cells (Fig. 2C). Similar results were obtained with an assay performed using human CTL line reactive against the CAP1 peptide from the CEA tumor antigen (data not shown). In addition, inhibition of caspase 3 15 WO 2005/090990 PCT/US2005/001993 activity 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). Together, these results indicate that caspase 3 cleavage in target cells is dependent on granzymes secreted by the CTL 5 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 5 lCr-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/Kb transgenic mice (Fig. 3). In the MLR model, 10 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/Kb target cells (Fig. 3B). The caspase 3-cleavage assay 15 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). 20 B. Use of caspase 3-cleavage assay to monitor CTL responses against viral vector vaccines The caspase 3 cleavage CTL assay was compared to the IFN-y ELISPOT assay to measure gp100 peptide-specific the CD8 recall response following immunization of HLA-A2.1/Kb transgenic mice with 2 x 107 pfu ALVAC-gpl00. 25 The mice were primed and boosted and spleen cells re-stimulated with 0.2 Pg/ml of a pool of HLA-A2.1-binding gpl00 peptides for 5 days and then subjected to effector cell assays using either caspase 3-cleavage or IFN-y ELISPOT. The caspase 3 cleavage assay used DDAO-SE-labeled gpl00 or control peptide-pulsed P815 cells stably transfected with HLA-A2.1/Kb plasmid. A similar approach was used in the 30 ELISPOT assays, except the P815-A2/Kb 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 16 WO 2005/090990 PCT/US2005/001993 3-cleavage read-out for gpl00 peptide-specific CD8 T-cell responses versus ELISPOT. Both assays yielded comparable results with no non-specific reactivity in mice immunized with an empty ALVAC control vector. The scatter of between individual animals was low indicating that monitoring CD8 T-cell responses using 5 caspase 3-cleavage in CTL targets can accurately measure antigen-specific CD8 T cell responses post-vaccination with high precision. These results indicate that measurement of caspase 3 cleavage in CTL target cells in murine systems is a viable alternative assay to 51Cr release and has the same degree of specificity but is markedly more sensitive that 51 Cr release. In addition, 10 measurement of CTL activity via caspase 3 cleavage can also be used as a supportive functional assay for IFN-y ELISPOT or as a substitute assay for the ELISPOT when target cells expressing the target antigen or peptide are available. C. Caspase 3-cleavage CTL assay for monitoring antigen-specific human T 15 cell responses In a first set of studies, human T cell lines were generated from HLA-A*0201 + donors against a 9-mer HIV rev peptide, an HIV gag peptide, or against the CAP1 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 20 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% TCRot3

+

, CD8 , CD16, CD56 proliferating T cells. These T cells were tested for effector cell activity using monitoring caspase 3-cleavage. IFN-y ELISPOT analysis confirmed the presence of peptide-specific T cells after the third and fourth round of 25 stimulation. These ranged from 1:100 to 1:250 for both the HIV peptide-specific and CEA peptide-specific lines (data not shown). Fig. 5A 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 HIV rev. The activated T cells were mixed with 30 pulsed T2 targets at a 5:1 or 1:1 E:T ratio (Fig. 5A). 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. In Fig. 5B, we compared the results of the caspase 3-cleavage assay to that of another FACS-based CTL assay 17 WO 2005/090990 PCT/US2005/001993 that detects apoptosis in targets using Annexin-V and 7-AAD staining (Fischer et al., 2002) with a kit (BD Bisciences) using T-cell lines specific for the CAP1 HLA-A2.1 epitope of CEA. The T-cell lines ( 4 th stimulation) were diluted with naive autologous PBMC from the donor at the ratios indicated and mixed with an equal number 5 (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-V + 7-AAD assay had a significantly higher non-specific background. In addition, we found that there was considerable variation in level of Annexin V fluorescence between samples (data not shown). Annexin V binding to 10 phosphatidyl serine is Ca 2 + sensitive and slight alterations in Ca 2+ concentration can alter the Annexin-V binding equilibrium. This variation in staining fluorescence was not found when staining for caspase 3-cleavage.. In addition to the lower background, the caspase 3-cleavage assay exhibited a significant degree of sensitivity in tracking CTL activity, as shown by the detection of 15 specific CTL activity at the 1:7 dilution of effector cells (Fig. SB; Caspase 3 cleavage). We also determined the frequency of CAP 1-specific T cells in the T line used in Fig. 5B with the IFN-y ELISPOT assay (not shown) and found a 1:237 frequency. Based on these results, we estimated that the frequency of CAP 1-specific T cells at the 1:3 and 1:7 dilutions in Fig. 5B was 1:711 (0.14%) and 1:1659 (0.06%), 20 respectively. This suggests that the assay is sensitive and capable of detecting rare antigen-specific CD8 + T cells, especially against TAA where CTL frequencies can be quite low. D. Sensitivity of the caspase 3-cleavage-based CTL assay 25 To further tests the sensitivity limits of the assay, a series of experiments were performed with mouse MLR effector cells and activated peptide-specific human T cell lines in which the effector cells were diluted up to 1:199 with naive syngeneic spleen cells or with naive autologous PBVIC, respectively. The first experiment was performed with C57BL/6 anti-Balb/c MLR effectors 30 cells diluted after harvesting the MLR at 1:0 (no dilution) to 1:199 with naive syngeneic C57BL/6 splenocytes (Fig. 6). The diluted effectors were incubated with DDAO-SE-labelled P815 cells at a 1:1 ratio. Target cells alone, and naive splenocytes served as negative controls for CTL activity and DDAO-SE-labeled EL-4 18 WO 2005/090990 PCT/US2005/001993 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 naive splenocytes (Fig. 6). An insignificant level of caspase 3 cleavage was seen at all dilutions in the non 5 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 naive 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 10 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 naive PBMC pool (Fig. 7). In the experiment shown in Fig. 9A, IFN-y ELISPOT analysis estimated that the frequency of HIV peptide-specific T cells in the cultures 15 was 1:156 (0.64%) after 3 stimulations. These T cell cultures were diluted with naive autologous PBMC at a 1:0 to 1:199 ratio. As before, DDAO-SE-labeled T2 lymphoma cell targets alone and naive 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 20 specificity. As shown in Fig. 7B and 9B, measurement of caspase 3-cleavage was remarkably sensitive detecting significant activity at effector cell dilutions as low as 1:99 and 1:199. A rate of caspase 3 cleavage of 5.7% and 6% was seen at 1:99 and 1:199, respectively, in the HIV peptide-pulsed T2 cells, while only 2.1% and 2.2% caspase 3 cleavage was seen in the non-specific pp65 CMV peptide-pulsed targets. 25 This low percentage of caspase 3-cleaved cells with the CMV peptide did not increase as the dilution of effector cells decreased. The percentage of caspase 3 cleavage in the targets minus the effectors and with naive PBMC plus targets was less than 2% (Fig. 7B). As a further test of the sensitivity of the assay, we also stained the HIV gag-specific T cell lines with an HLA-peptide pentamer (ProImmune, Oxford, UK) 30 that directly stains the TCR of the antigen-specific T cells (Fig. 7C). 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 19 WO 2005/090990 PCT/US2005/001993 gag peptide-specific T cells in the caspase 3-cleavage assay (Fig. 7B), based on the IFN-y 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. This represents a high level of sensitivity making the assay useful for quantitating CTL activity when antigen 5 specific T-cell frequencies are considerably low. Moreover, unlike pentamer- or tetramer- based methods, it is a measure of T cell function and not only T-cell frequency. 10 20 WO 2005/090990 PCT/US2005/001993 REFERENCES Andrade, F., Roy, S., Nicholson, D., Thomberry, N., Rosen, A. and Casciola-Rosen, L. (1998) Granzyme B directly and efficiently cleaves several downstream 5 caspase substrates: implications for CTL-induced apoptosis. Immunity 8, 451 60. Bashford, C.L., Menestrina, G., Henkart, P.A. and Pasternak, C.A. (1988) Cell damage by cytolysin. Spontaneous recovery and reversible inhibition by divalent cations. Journal of Immunology 141, 3965-74. 10 Betts, M.R., Brenchley, J.M., Price, D.A., De Rosa, S.C., Douek, D.C., Roederer, M. and Koup, R.A. (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. Journal of Immunological Methods 281, 65-78. Borenstein, S.H., Graham, J., Zhang, X.L. and Chamberlain, J.W. (2000) CD8+ T 15 cells are necessary for recognition of allelic, but not locus-mismatched or xeno-, HLA class I transplantation antigens. Journal of Immunology 165, 2341-53. Boyle, W. (1968) An extension of the 51Cr-release assay for the estimation of mouse cytotoxins. Transplantation 6, 761-4. 20 Ferrari, G., Berend, C., Ottinger, J., Dodge, R., Bartlett, J., Toso, J., Moody, D., Tartaglia, J., Cox, W.I., Paoletti, E. and Weinhold, K.J. (1997) Replication defective canarypox (ALVAC) vectors effectively activate anti-human immunodeficiency virus-1 cytotoxic T lymphocytes present in infected patients: implications for antigen-specific immunotherapy. Blood 90, 2406-16. 25 Fischer, K., Andreesen, R. and Mackensen, A. (2002) An improved flow cytometric assay for the determination of cytotoxic T lymphocyte activity. Journal of Immunological Methods 259, 159-69. Henkart, P.A., Williams, M.S., Zacharchuk, C.M. and Sarin, A. (1997) Do CTL kill target cells by inducing apoptosis? Seminars in Immunology 9, 135-44. 30 Hermans, I.F., Silk, J.D., Yang, J., Palmowski, M.J., Gileadi, U., McCarthy, C., Salio, M., Ronchese, F. and Cerundolo, V. (2004) The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo. Journal of Immunological Methods 285, 25-40. 35 Herr, W., Schneider, J., Lohse, A.W., Meyer zum Buschenfelde, K.H. and Wolfel, T. (1996) Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor alpha in response to HLA-A2.1-binding melanoma and viral peptide antigens. Journal of Immunological Methods 191, 131-42. 40 Huang, X., Traganos, F. and Darzynkiewicz, Z. (2003) DNA damrnage induced by DNA topoisomerase I- and topoisomerase II-inhibitors detected by histone H2AX phosphorylation in relation to the cell cycle phase and apoptosis. Cell Cycle 2, 614-9. Jerome, K.R., Sloan, D.D. and Aubert, M. (2003a) Measurement of CTL-induced 45 cytotoxicity: the caspase 3 assay. Apoptosis 8, 563-71. Jerome, K.R., Sloan, D.D. and Aubert, M. (2003b) Measuring T-cell-mediated cytotoxicity using antibody to activated caspase 3.[comment]. Nature Medicine 9, 4-5. 21 WO 2005/090990 PCT/US2005/001993 Kienzle, N., Olver, S., Buttigieg, K. and Kelso, A. (2002) The fluorolysis assay, a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers. Journal of Immunological Methods 267, 99-108. Lieberman, J. (2003) The ABCs of granule-mediated cytotoxicity: new weapons in 5 the arsenal. Nature Reviews. Immunology 3, 361-70. Liu, L., Chahroudi, A., Silvestri, G., Wernett, M.E., Kaiser, W.J., Safrit, J.T., Komoriya, A., Altman, J.D., Packard, B.Z. and Feinberg, M.B. (2002) Visualization and quantification ofT cell-mediated cytotoxicity using cell permeable fluorogenic caspase substrates.[see comment]. Nature Medicine 8, 10 185-9. Mattis, A.E., Bernhardt, G., Lipp, M. and Forster, R. (1997) Analyzing cytotoxic T lymphocyte activity: a simple and reliable flow cytometry-based assay. Journal of Immunological Methods 204, 135-42. Pham, C.T. and Ley, T.J. (1997) The role of granzyme B cluster proteases in cell 15 mediated cytotoxicity. Seminars in Immunology 9, 127-33. Roden, M.M., Lee, K.H., Panelli, M.C. and Marincola, F.M. (1999) A novel cytolysis assay using fluorescent labeling and quantitative fluorescent scanning technlmology. Journal of Immunological Methods 226, 29-41. Santra, S., Schmitz, J.E., Kuroda, M.J., Lifton, M.A., Nickerson, C.E., Lord, C.I., Pal, 20 R., Franchini, G. and Letvin, N.L. (2002) Recombinant canarypox vaccine elicited CTL specific for dominant and subdominant simian immunodeficiency virus epitopes in rhesus monkeys. Journal of Immunology 168, 1847-53. Schultze, J.L., Michalak, S., Seamon, M.J., Dranoff, G., Jung, K., Daley, J., Delgado, 25 J.C., Gribben, J.G. and Nadler, L.M. (1997) CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy. Journal of Clinical Investigation 100, 2757-65. Sheehy, M.E., McDermott, A.B., Furlan, S.N., Klenerman, P. and Nixon, D.F. (2001) 30 A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis.[erratum appears in J Immunol Methods 2001 Jun 1;252(1 2):219-20]. Journal of Immunological Methods 249, 99-110. Shiver, J.W. and Henkart, P.A. (1991) A noncytotoxic mast cell tumor line exhibits potent IgE-dependent cytotoxicity after transfection with the 35 cytolysin/perforin gene. Cell 64, 1175-81. Stuber, G., Modrow, S., Hoglund, P., Franksson, L., Elvin, J., Wolf, H., KICarre, K. and Klein, G. (1992) Assessment of major histocompatibility complex class I interaction with Epstein-Barr virus and human immunodeficiency virus peptides by elevation of membrane H-2 and HLA in peptide loading-deficient 40 cells. European Journal of Immunology 22, 2697-703. Sun, J., Whisstock, J.C., Harriott, P., Walker, B., Novak, A., Thompson, P.E., Smith, A.I. and Bird, P.I. (2001) Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop. Journal of Biological Chemistry 276, 15177 45 84. Thorn, R.M., Palmer, J.C. and Manson, L.A. (1974) A simplified 51Cr-release assay for killer cells. J Immunol Methods 4, 301-15. Trapani, J.A. and Sutton, V.R. (2003) Granzyme B: pro-apoptotic, antiviral and antitumor functions. Current Opinion in Immunology 15, 533-43. 22 WO 2005/090990 PCT/US2005/001993 Zaremba, S., Barzaga, E., Zhu, M., Soares, N., Tsang, K.Y. and Schlom, J. (1997) Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen. Cancer Research 57, 4570-7. 5 23

Claims (8)

1. A method for detecting cytotoxic activity of a population of immune effector cells against a population of target cells, the method comprising the steps of, in 5 combination: a) exposing a population of immune effector cells to a population of target cells; b) incubating the cells for a sufficient period of time and under conditions suitable for an immune effector cell to affect the target cell such that an apoptotic pathway in the target cell is activated; 10 c) fixing and permeabilizing the mixed population of cells; d) exposing the mixed population of cells to 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, e) detecting the detectable reagent. 15

2. The method of claim 1 wherein the immune effector cells are T cells.

3. The method of claim 1 wherein the target cells are virally-infected cells or tumor cells.

4. The method of claim 1 wherein the protein of step d) is a caspase.

5. The method of claim 4 wherein the easpase is caspase-3. 20

6. The method of claim 1 wherein the detectable reagent is an antibody.

7. The method of claim 1 wherein the detectable reagent is a fluorescently tagged antibody.

8.. The method of claim 1 wherein the detection of step e) is accomplished using a flow cytometer. 25 24