Definition of an Immunologic Response Using the Major Histocompatibility Complex Tetramer and Enzyme-Linked Immunospot Assays

Imaging, Diagnosis, Prognosis

DefinitionofanImmunologicResponseUsingtheMajor Histocompatibility Complex Tetramer and Enzyme-Linked Immunospot Assays Begon‹ aComin-Anduix,1 Antonio Gualberto,4 John A. Glaspy,2,3 Elisabeth Seja,2 Maribel Ontiveros,2 Deborah L. Reardon,4 Roberto Renteria,5 Brigitte Englahner,4 James S. Economou,1, 3 Jesus Gomez-Navarro,4 and Antoni Ribas1,2,3

Abstract Purpose: Define an immunologic response using the tetramer and enzyme-linked immunospot (ELISPOT) assays. Experimental Design: Ten healthy subjects and 21patients with melanoma (all HLA-A*0201) donated a total of 121blood samples to determine the lower limit of detection (LLD), analytic coefficient of variation (aCV), and physiologic CV (pCV) of the tetramer and ELISPOTassays. The mean, SD, and reference change value (RCV) were calculated to define changes beyond the assay imprecision, andits application was testedin the monitoring ofT-cell expansion after CTLA4 blockade with ticilimumab (CP-675,206). Results: The LLD for the tetramer assay was 0.038% CD8+ cells and seven spots per 105 peripheral blood mononuclear cells for the ELISPOTassay. The aCV of the tetramer assay was <10% and was higher for the ELISPOT (24.69-36.32%). There was marked between-subject variability on baseline homeostatic values, which was correlated to prior antigen exposure. An immunologic response was defined as an increase beyond the mean + 3 SD in antigen-specific cells for subjects with baseline levels below the LLD, or beyond the assay RCV for baseline levels above the LLD. Infour patients receiving ticilimumab, expansions of antigen-specificTcells beyond the assay variability were noted for EBVand MART1antigens. Conclusions: A combined approach of change from negative (below the LLD) to positive (above the LLD) and a percentage change beyond the assay variability using the RCV score can be computed to define which change in circulating antigen-specificTcells represents a response to immunotherapy.

Modern cellular immunologic assays, like the enzyme-linked rapidly replaced older assays requiring extensive ex vivo immunospot (ELISPOT; refs. 1–4), the MHC tetramer binding manipulations, like the limiting dilution microcytotoxicity assay (5, 6), and the intracellular cytokine flow cytometry assay and the lymphoproliferative assay (10). However, few assay (7, 8), allow for the enumeration of antigen-specific studies had been conducted to define key methodologic T lymphocytes at a single cell level (9–11). These assays have variables, like accuracy, precision, and reproducibility (10). These variables are critical to determine which level of T-cell expansion is beyond the baseline variability of the assay and therefore would represent a positive immune response. Authors’ Affiliations: 1Department of Surgery, Division of Surgical; 2Department of Medicine, Division of Hematology/Oncology; 3Jonsson Comprehensive Cancer The most commonly used approach to define a positive Center, Oncology, University of California at Los Angeles, Los Angeles, California; immune response is the detection of circulating antigen-specific 4Pfizer Global Research and Development, Groton-New London, Connecticut; and T cells after immunotherapy beyond the mean and 2 or 3 SD 5Beckman Coulter, Inc., San Diego, California from negative values, using results from a negative control Received 1/18/05; revised 9/11/05; accepted 10/10/05. antigen or a positive antigen in a negative population (10, 12). Grant support: Flow cytometry and enzyme-linked immunospot assays were done in the University of California at Los Angeles Jonsson Comprehensive Cancer This approach is logical for values that are below a certain Center and Center for AIDS Research Flow Cytometry Core Facility that is threshold of negativity at baseline, which would reflect the assay supported by NIH grants CA-16042 and AI-28697, Jonsson Comprehensive background. However, the baseline values of circulating antigen- Cancer Center, University of California at Los Angeles AIDS Institute, and David specific T cells for many infectious disease and tumor antigens Geffen School of Medicine at University of California at Los Angeles. can be positive at baseline for a significant subset of patients The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance (12, 13). In this case, the definition based on a fixed value with 18 U.S.C. Section 1734 solely to indicate this fact. (an increase beyond the mean + SD from negatives) would not Requests for reprints: Antoni Ribas, University of California at Los Angeles be easily applicable. This approach could only be used if several Medical Center, 11-954 Factor Building, 10833 Le Conte Avenue, Los Angeles, CA baseline values were collected for each patient, allowing defining 90095. Phone: 310-206-3928; Fax: 310-206-0914; E-mail: aribas@mednet. ucla.edu. a mean and SD for their individual baseline values of circulating F 2006 American Association for Cancer Research. antigen-specific T cells, and then comparing it to its change after doi:10.1158/1078-0432.CCR-05-0136 immunotherapy. This is not feasible in routine practice.

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The calculation of the reference change value (RCV) may immunologic studies at redosing at 3 or 6 mg/kg under a separate allow a more reliable definition of an immune response if redosing study (IRB 03-01-014). Two were redosed after being enrolled baseline values are above the assay lower limit of detection at the initial low dose cohorts, and two were enrolled after having (LLD). The RCV allows determining the change that must clinical benefit to the initial dose. Peptides and tetramers. The following peptides were used: AFP occur in an individual’s serial test results before the change is 325-332 (GLSPNLNRFL), CMVpp65 (NLVPMVATV), EBV BMLF1 considered significant by taking into account the main 495-503 259-267 (GLCTLVAML), MART-126-35 (ELAGIGILTV), MART-127-35 (AAGIGILTV), components of assay variability and a desired statistical and a HLA-A*0201-binding nonrelevant peptide (ref. 21; referred to as significance. Its calculation has the goal to define the the negative peptide from here on). All tetramers were purchased from magnitude of change in results that would be beyond the Beckman Coulter, Inc. (San Diego, CA), except a PE-labeled HLA-A*0201 assay imprecision. Since its original description by Harris and MART127-35 tetramer, which was obtained from the National Institute of Brown in 1979 (14), different versions of the formula have Allergy and Infectious Disease (Emory University, Atlanta, GA). been proposed (15–19). We reasoned that when applied to Tetramer-binding assay. Cryopreserved PBMC aliquots were thawed immunologic monitoring, it would allow us to define positive and diluted with RPMI supplemented with 10% human AB serum, 1% and negative changes in circulating antigen-specific T cells after penicillin, streptomycin, amphotericine (PSA, OmegaSci), and DNase j an immunotherapy intervention for subjects with baseline (0.002%, Sigma) for an hour at 37 C. PBMCs were then washed and resuspended at 1 to 3 106/100 AL in 100% adult bovine serum measurable values (above the assay LLD), thereby representing (OmegaSci). Only samples with viability of >90% by trypan blue a true positive or negative immune response and not the assay exclusion were used. PBMCs were stained for 30 minutes at room noise. temperature in the dark with 8 AL MHC tetramer, 8 AL FITC-conjugate anti-CD8 (SFI21Thy2D3), and 8 AL iMASC antibodies (a pre-prepared Materials and Methods mixture with PC5-conjugated CD4 13B8.2, CD13 IMMU103.44, and CD19 J4.119, all from Beckman Coulter), which were used to Study eligibility and screening procedures for the methodology study. gate out CD8 lymphocytes. Cells were washed in 3 mL of PBS and, HLA-A*0201-positive healthy subjects and HLA-A*0201-positive immediately before flow cytometric analysis, 10 AL of 7-amino- patients with melanoma were eligible, provided that they were not actinomycin D was added to gate out dead cells. PBMCs were analyzed receiving active therapy or having an intercurrent event (like an on a FACSCalibur (BD Biosciences, San Jose, CA) within 1 hour of influenza infection or symptoms of common cold) for a minimum staining, without adding any fixative. Stained cells were kept constantly period of 30 days before the blood draws and during the blood draws. at 4jC. A minimum of 30,000 CD8+ T cells were acquired, and up to Eligible patients with melanoma included stage II to IV melanoma, a 200,000 were preferred. Analysis was done both with CellQuest Karnofsky performance status of >70%, and adequate hematopoietic, (Beckman Coulter) and FCS Express (DeNovo Software, Thornhill, hepatic, and renal function. To be eligible, tumors were required to Ontario, Canada) software. In addition to the routine daily FACSCa- express Melan-A/MART-1 (MART-1) by immunohistochemistry or libur calibration, FLOW-SET Fluorospheres (Beckman Coulter) were reverse transcription-PCR. Exclusion criteria included prior therapy < used to set peak channels before every tetramer flow cytometry 4 weeks before study entry, untreated central nervous system metastasis, experiment. pregnancy, or concurrent immunosuppressive conditions or therapy. For tetramer spiking experiments, CD8+ T cells were isolated by All subjects signed a written informed consent approved by the magnetic column separation (Miltenyi Biotec, Auburn, CA). CD8+ cells University of California at Los Angeles Institutional Review Board were stained with the CMVpp65 tetramer and sorted into tetramer- (UCLA IRB 01-12-029). Screening procedures included complete blood positive or tetramer-negative cells using a FACSAria (BD Biosciences). counts; serum electrolyte determination; liver function tests; coagula- Immediately after sorting, tetramer-negative but CD8+ cells were mixed tion tests; serology determination of antibodies to HBV, HCV, with the CD8+/CMVpp65 tetramer-positive cells at different rations cytomegalovirus (CMV), EBV, and HIV; and an autoimmunity panel and analyzed on a FACSCalibur. At least 80,000 CD8+ T cells were testing, including anti-nuclear antibodies, rheumatoid factor, anti- acquired. neutrophilic cytoplasmic, anti-tyroglobin, anti-LK microsome, anti-Ro, IFN-g ELISPOT assay. Multiscreen HA plates (Millipore, Bedford, anti-La, and anti-phospholipid. MA) were coated overnight at 4jC with 4 Ag/mL of purified antihuman Blood collection and cryopreservation. Subjects underwent three to IFN-g monoclonal antibody (BD PharMingen, San Diego, CA) in a four peripheral blood collections of 40 mL of whole blood: an initial 0.1 mol/L sodium bicarbonate buffer (pH 8.2). Unbound antibody was one on study day 1, a second one between study days 2 and 7, a third removed by five washings with PBS, and nonspecific binding to plates one at weeks 2 to 4, and an optional fourth one between weeks 5 was blocked with PBS/10% (v/v) heat-inactivated human AB serum and 7. Additionally, two patients (study subjects 103 and 109) with a (1 hour, 37jC). Different concentrations of thawed PBMCs and clearly defined population of antigen-positive cells underwent leuka- antigen-presenting cells (APC) were seeded in triplicate at a final pheresis under a different protocol (UCLA IRB 93-07-289). Peripheral volume of 200 AL/well in X-Vivo 10 media (Bio Whittaker, Walkersville, blood mononuclear cells (PBMC) were obtained by Ficoll-Hypaque MD) supplemented with 10% (v/v) heat-inactivated human AB serum. (Amersham Pharmacia, Piscataway, NJ) centrifugation of whole blood The following APC were tested: T2, purchased from American Type or the leukapheresis product. PBMCs were cryopreserved in liquid Culture Collection (Rockville, MD); JY, provided by Dr. Martin Kast nitrogen in RPMI (Life Technologies Bethesda Research Laboratories, (Loyola University, Mayfield, IL); and HLA-A*0201-transfected K562 Gaithersburg, MD) supplemented with 20% (all as v/v) heat-inactivated (K562/A*0201), provided by Prof. Wolfgang Herr and Dr. Cedrik M. AB human serum (OmegaSci., Inc., Tarzana, CA) and 10% DMSO Britten (Johannes Gutenberg-University of Mainz, Mainz, Germany; (Sigma, St. Louis, MO). PBMCs were cryopreserved at a concentration ref. 22). Plates were incubated for 24 hours at 37jCin5%CO2 in a of 5 106/mL. water-saturated atmosphere. After washings with ice-cold PBS/0.05% Subject enrollment and blood collection in the validation study. Four Tween 20 (Fisher Scientific, Chino, CA), plates were incubated HLA-A*0201-positive patients with metastatic melanoma receiving the overnight with 3 Ag/mL of biotinylated mouse anti-human IFN-g anti-CTLA4 monoclonal antibody ticilimumab (Pfizer, Inc., New antibody (BD PharMingen) at 4jC. Avidin-peroxidase (1:2,000 London, CT) were included in the methodology validation part of the dilution; Vector, Burlingame, CA) was added for 1 hour in the dark study. These four patients had been previously enrolled in a phase I at room temperature, and spots were developed using 0.4 mg/mL of single-dose escalation trial at the University of California at Los Angeles 3-amino-9-ethylcarbazole (Sigma) in N,NV-dimethylformamide, freshly (IRB 01-09-054; ref. 20). The patients consented to donate blood for diluted 1:25 in 0.05 mol/L sodium acetate (pH 5), and analyzed in

Clin Cancer Res 2006;12(1) January 1, 2006 108 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Tetramer and ELISPOTAnalytic Performance a series 1 Immunospot Image Analyzer (Cellular Technology Ltd., cryopreserved samples were analyzed for reactivity to two Columbus, OH). The murine anti-HLA-A*02 antibody BB7.2 candidate negative control epitopes, the AFP325-332 peptide (24) (BD PharMingen) was used to confirm surface expression of HLA- and an irrelevant HLA-A*0201-binding peptide (ref. 21; termed A*0201 on K562/A*0201 cells. K562/A*0201 cell cultures used in this negative peptide). The negative tetramer resulted in low and study were free of Mycoplasma infection, tested using a Mycoplasma PCR tight tetramer binding (Table 1), with all 31 subjects having ELISA kit (Roche Diagnostic, Indianapolis, IN). Statistical analysis. The mean and SDs were calculated using similar mean values (0.000-0.04%). For AFP325-332, the mean Microsoft Excel. The data set was inspected for outliers, which were values had a wider range (0.00-0.22%). Four subjects (two evaluated using Reed’s criterion (18). We defined the LLD as the mean + healthy donors and two patients with melanoma) had a clearly 2 SDs for an HLA-A*0201-matched negative peptide not eliciting a evident AFP325-332-positive population in peripheral blood and detectable response by both assays in the majority of the study were excluded from the computation of the LLD for this population. The coefficient of variation (CV) was calculated as antigen. The LLD was defined as 38 CD8+ cells nonspecifically CV = (SD / mean) 100. Two CVs were calculated for each epitope binding to the negative tetramer within a population of 105 and assay, the analytic CV (aCV) and the physiologic CV (pCV). The CD8+ cells (0.038%, Table 1), which provides a stringent level aCV defines the intra-assay variability, whereas the pCV defines the to define the assay background noise. within-subject biological variability. We defined an acceptable aCV as We then compared this result with the biological limit an assay with V20% CV. The RCV to define a significant negative or positive change in baseline values above the LLD was calculated as of detection, which can be derived from a spiked sample RCV = 21/2 Z (aCV2 + pCV2)1/2, where a bidirectional Z score with whose concentration approximates the detection limit (23). a 95% or 99% probability of significance was used, and the median aCV CMVpp65495-503 tetramer-positive cells were sorted by flow and pCV derived from subjects with mean values above the assay LLD cytometry and spiked into the CMVpp65495-503 tetramer- (17, 18, 23). The Z score was adjusted to the degrees of freedom negative population. In these studies, five CMVpp65495-503 corresponding to the mean minus one from the mean number of tetramer-positive cells could be detected in a background of 106 samples analyzed for each assay and condition. + CD8 but CMVpp65495-503 tetramer-negative cells (0.0005%; Fig. 1B). This apparent improvement in assay sensitivity is Results achieved by measuring high-intensity tetramer staining spiked events, as opposed to the determination of the LLD, which Subject characteristics more adequately reflects the problem of nonspecific low- Thirty-one HLA-A*0201-positive subjects were enrolled, intensity tetramer staining representing the assay background. including 10 healthy subjects and 21 patients with a diagnosis Analytic variability. The analytic variability of the assay was of melanoma. Self-referred healthy subjects were younger determined using replicate cryopreserved aliquots derived from (median age of 34 versus 59 for patients with melanoma) the same blood draw (either a peripheral blood draw with and were predominantly female (90% versus 35% for patients multiple aliquots or a leukapheresis) that were rerun at different with melanoma). Nine patients with melanoma had no time points (Fig. 1D). Only samples with CMVpp65495-503,EBV evidence of disease, and 12 had measurable melanoma during BMLF1259-267,andMART-126-35 tetramer values above the LLD blood collections. Eligibility criteria required no active onco- were used (values below the LLD should not be considered logic treatment or intercurrent illness for at least 30 days before ‘‘measurable’’). aCV ranged from 6.55% to 8.30%, depending on enrollment and during the entire blood collection time. the epitope tested (Table 1), indicating that the tetramer assay had an acceptable precision (below 20%). Tetramer assay Physiologic variability. The availability of three (in three Assay optimization. Standardizing the conditions of blood subjects) or four (in 28 subjects) PBMC samples obtained over procurement, processing, and assay methodology allows for a period of time (3-9 weeks) without a concurrent treatment or minimizing preanalytic variation (18). Therefore, initial trial new illness, for a total of 121 blood draws, allowed us to runs were done to establish the optimal conditions and determine the physiologic (within person or biological) reagents for the tetramer assay, with the goal of generating variability in baseline reactivity of multiple tetramer assays standard operating procedures. Using PBMCs from subjects (Fig. 1D). Figure 2A, B, and C shows large differences among with known populations of tetramer-positive cells, different the set points of different individuals. In addition, results for tetramers sources (National Institute of Allergy and Infectious subjects vary around their own homeostatic set point (depicted Disease or Beckman Coulter) and algorithms for gating as the SE for the different assay results for each subject). To tetramer/CD8 double-positive cells were tested. We noted minimize the effect of this between-subject variability, we marked variations in the absolute number of CD8+ cells divided the population between subjects that may have been within subjects (data not shown). To maintain consistency, previously exposed to the antigen (and therefore should be eligible samples were required to allow the collection of a more likely to have developed a T-cell response), using sur- + minimum of 30,000 CD8 events, and up to 200,000 were rogate clinical and laboratory data. Circulating CMVpp65495-503 collected if feasible. Results are presented as the percentage of and EBV BMLF1259-267 tetramer-binding cells were significantly tetramer-positive cells among total CD8+ cells throughout this higher in seropositive than in seronegative subjects (Fig. 2A study. Additional details on the preferred reagents and and B). For the MART-126-35 tetramer, healthy subjects had approach are described in Materials and Methods and in significantly lower numbers of tetramer-positive/CD8+ cells Fig. 1A. than patients with prior diagnosis of melanoma but currently LLD. The LLD is usually estimated as the mean of a blank no evidence of disease, whereas they were not significantly sample + 2 SD, which is sometimes described as the limit of different from patients with active melanoma (Fig. 2C). absence (23). It describes the range of numbers that are Table 2A presents data on the physiologic variability for possible when the correct result is 0. PBMC aliquots from subjects with mean values above the LLD. Values below the

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Fig. 1. Assay optimization studies. A, gating algorithm for the MHC tetramer assay. PBMCs cryopreserved from a subject with a known defined population of CMVpp65495-503 tetramer-positive cells were thawed and stained with CMVpp65495-503 tetramers. i, cells positive for iMASC (non-CD8 WBC) or 7-amino-actinomycin D (7-AAD, dead cells) were excluded and gating was focused on the R1population. ii, from the R1population, only cells with high surface expression of CD8+ were gated on. iii, from the R1 + R2 population, cells with a size consistent with lymphocytes were gated on in a forward and side scatter. iv, from the R1 + R2 + R3 population, we selected the CD8/CMVpp65495-503 tetramer double-positive population excluding double-positive cells with low CD8 expression. B, biologic limit of detection derived from a spike + experiment. CD8 cells from a subject with a clearly defined population of CMVpp65495-503-specificTcells were stained with the CMVpp65495-503 tetramer and sorted by flow cytometry.The CMVpp65495-503 tetramer-negative population was then spiked with different concentrations of cells from the CMVpp65495-503 tetramer-positive population.The resulting mixtures were restained with the CMVpp65495-503 tetramer and analyzed by flow cytometry. C, choice of antigen-presenting cells for the ELISPOT assay. PBMCs from a subject with known CMVpp65495-503 reactivity were restimulated with JYorT2 cells pulsed with the CMVpp65495-503 peptide and analyzed from IFN-g-producing cells. In a second experiment, PBMCs from a subject with known reactivity to EBVBMLF1259-267 were restimulated withT2 or K562/A*0201cells pulsed with the CMVpp65495-503 peptide and analyzed for IFN-g-producing cells. D, calculations and significance of the RCV.Calculation of the aCV.The aCV was calculated by analyzing samples derived from a single blood draw that had been cryopreserved in multiple aliquots.The mean value and SD from the different aliquots were used tocalculatethe aCV for each blood draw, and the overall aCV was calculated using the median of the individual aCV. Calculation of the pCV.The pCV was calculated by analyzing samples derived from three or four blood draws taken from the same subject without intercurrent treatments.The mean value and SD from the different blood draws were used to calculate the pCV for each subject, and the overall pCV was calculated using the median of the individual pCV. Calculation of the RCV.The formula for the RCV incorporates a constant, a Z score that reflects the desired level of significance (95% or 99%), and the computed median aCVand pCV for each assay and antigen. E, significance of the RCV. The mean and SD for the MART-126-35 tetramer results in two extreme cases are used to illustrate the problem of using a single definition for a positive immune response. The tetramer results for patient 102, with a baseline number of circulating MART-126-35-specificTcells below the LLD (red), and for patient 109, with a baseline number of circulating MART-126-35-specificTcells above the LLD, are presented in a semilogarithmic plot. A fixed change (defined by the mean + 3 SD based on results of a negative antigen) would allow to define a response in patient 102, who has homeostatic set value below the assay LLD. However, only the RCVcould define a responseinpatient109, because adding the mean + 3 SD of results based on a negative antigen would not be considered a biologically significant change.

LLD would be expected to vary widely due to the assay T2, and K562/A*0201 cells (Fig. 1B). Higher background was inaccuracy at that level. Above the LLD, the pCV of the tetramer noted with the use of JY and T2 cells. Therefore, K562/A*0201 assay ranged from 16.71% to 35.95%, which shows that the cells were chosen for further testing. The standardized levels of circulating antigen-specific T cells can vary over time conditions of 1:10 APC to PBMC ratio, with APC pulsed with without any immunotherapy treatment (Table 2A). 10 Ag of peptide, were defined in peptide and APC/PBMC ratio titration studies. Additional optimization studies concluded IFN-; ELISPOT assay that 1 105 PBMCs per well and performing the restimulation Assay optimization. We based our assay methodology on our in the plate were optimal. K562/A*0201 cells were periodically previously published experience (25, 26) and then tested phenotyped, with an average of 96 F 1% expression of HLA- several key conditions to attempt to decrease preanalytic A*0201, and were free of Mycoplasma contamination (data not variation. We compared three widely used surrogate APC: JY, shown).

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LLD. Replicate aliquots from cryopreserved samples were high for all conditions and peptides, between 45% and analyzed for reactivity to the two candidate negative control 100.13% (Table 2B). epitopes (negative and AFP325-332) to determine the LLD Definition of significant changes in circulating antigen-specific (Table 1). Four of 31 subjects had clear baseline reactivity T cells after immunotherapy. Given the marked between-subject (mean values over the mean + 2 SDs from the rest of variability on homeostatic levels of circulating antigen-specific subjects) to the negative peptide by ELISPOT assay (the same T cells (Fig. 2), we used our definition of the LLD as a threshold subjects had no negative tetramer-binding cells). Two subjects to separate subpopulations of subjects with baseline negative had clear reactivity to AFP325-332. Results from these subjects and positive values for each assay. We reasoned that patients were considered outliers compared with the overall popula- with levels of circulating antigen-specific T cells below the LLD tion and were not used to calculate the assay LLD (18). In could be defined as responders to immunotherapy if their this assay, both the negative and AFP325-332 peptides provided antigen-specific cells expanded beyond the mean + 2 SD (95% a similar LLD, which was set at seven IFN-g spots per 1 significance) or 3 SD (99% significance) from baseline values. 105 PBMCs. For patients with baseline values above the LLD, we reasoned Analytic variability. The within-run replication experiment, that the RCV would allow quantifying changes in response to using samples with values above the LLD, showed a slightly immunotherapy beyond the assay variability. This algorithm higher aCV for the native self-melanosomal antigen MART-127- is based on a preliminary analysis of the use of these two 35 compared with the xenoantigens CMVpp65495-503 and EBV approaches in two extreme cases within our series (Fig. 1E). BMLF1259-267 (Table 1). Additionally, it showed that the Table 3A provides the values for EBV, CMV, and MART-1 to ELISPOT assay has much higher analytic variation than the be used as cutoff points to define a positive immune response tetramer assay, even beyond the 20% CV that is routinely to immunotherapy in subjects with baseline levels of circulating considered as an acceptable assay variability for analytic antigen-specific T cells below the LLD for both assays. methods (23). Calculations for the EBV BMLF1259-267 tetramer could not be Physiologic variability. Figure 2D, E, and F depicts the results done, because all samples were above the assay LLD. For each of testing all the available samples for IFN-g-producing cells epitope and assay, the use of mean + 2 SD gave results very detected in the ELISPOT assay in response to K562/A*0201 close to the LLD. Therefore, we choose to consider a significant cells pulsed with the CMVpp65495-503, EBV BMLF1259-267, and change from below the LLD to equal or greater than the mean + MART-127-35 peptides. As with the tetramer assay, subjects 3 SD. Despite the high analytic variability of the ELISPOT assay without prior exposure shown by a negative humoral response above the LLD, there was little variability in background levels, to CMV or EBV had low levels of T-cell responses in the with a tight SD. This is due to the low nonspecific reactivity ELISPOT assay, whereas subjects with prior exposure had with K562-A*0201 as APC (Fig. 1C), with many samples higher mean values of IFN-g-producing cells (Fig. 2D and E). having values of 0 spot in the four time points. Using the For MART-127-35, again a higher reactivity was noted for mean + 3 SD approach for the ELISPOT assay, a change from patients who were with no evidence of disease at the time of below seven to nine spots would be considered a positive blood donations compared with healthy subjects and patients immune response. However, given the assay variability (pCV up with active melanoma (Fig. 2F). to 100%), an increase from seven to nine spots cannot be The pCV above the LLD is described in Table 2B. It could not considered biologically relevant. Therefore, the mean + 3 SD be calculated for the MART-127-35 peptide in the healthy donor approach is valid for the tetramer assay, where there is a wide population, because all subject had values below the LLD. range of values below the assay LLD but not for the ELISPOT Consistent with the high assay analytic variability, the pCV was assay.

Ta b l e 1. LLD and aCVof the tetramer and ELISPOTassays

Negative AFP325-332 EBV BMLF1259-267* CMV pp65495-503*MART-126-35* MART-127-35* Te t r a m e r No. samples 129 109 29 30 76 ö No. subjects 31 27 3 4 3 No. time points 121 109 4 5 17 Mean (SD) 0.017(0.01) 0.040 (0.02) 3.25 (0.34) 0.37 (0.05) 3.88 (0.41) ö LLD 0.038 0.084 ö ö ö ö aCV (%) ö ö 8.24 6.55 8.30 ö ELISPOT No. samples 310 328 121 79 81 61 No. subjects 27 29 5 6 2 2 No. time points 108 112 15 12 4 3 Mean(SD) 2(3) 2(2) 113(41) 99(25) 137(37) 68(20) LLD 7 7 ö ö ö ö aCV ö ö 27.96 24.69 36.32 34.18

*Calculated by only taking into account the samples with values above the LLD defined using the negative peptide.

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Fig. 2. Physiological variability of circulating antigen-specific cells. Cryopreserved PBMC samples collected from different time points were thawed and analyzed with tetramers after exclusion with iMASC antibodies or were restimulated in ELISPOT plates with K562/A*0201pulsed with each peptide. Plates were incubated for 24 hours at 37jCin5%CO2 and the number of IFN-g-producing cells in the ELISPOTassay were quantitated using a series 1Immunospot Image Analyzer. A, CMVpp65495-503 tetramer. B, EBV BMLF1259-267 tetramer. C, MART-126-35 tetramer. D, CMVpp65495-503 ELISPOT. E, EBV BMLF1259-267 ELISPOT. F, MART-127-35 ELISPOT.

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Ta b l e 2 . Physiologic variability of the tetramer and ELISPOTassays

Epitope Clinical group No. subjects No. samples Mean (SD) pCV A. Physiologic variability of the tetramer assay above the LLD

EBV BMLF1259-267 Seronegative 11 42 0.53 (0.21) 25.91 Seropositive 13 50 1.27 (0.34) 21.02

CMVpp65495-503 Seronegative 14 53 0.10 (0.05) 35.95 Seropositive 10 39 0.97 (0.06) 16.71

MART-126-35 Healthy Donors 7 27 0.29 (0.08) 29.46 Pts with Melanoma 15 55 0.76 (0.21) 27.40

B. Physiologic variability of the ELISPOTassay above the LLD

EBV BMLF1259-267 Seronegative 3 33 32 (26) 100.13 Seropositive 7 82 48 (34) 60.47

CMVpp65495-503 Seronegative 5 31 16 (16) 75.17 Seropositive 8 90 47 (22) 45.00

MART-127-35 Healthy Donors 0 0 ö ö Pts with Melanoma 4 54 43 (25) 62.47

Abbreviation: Pts, patients.

Calculation of the RCV to define a positive or negative change Definition of an immune response based on the tetramer and in circulating antigen-specific T cells beyond the assay variability. ELISPOT assays. Based on the results of this methodology Table 3B provides the RCV for CMVpp65495-503,EBV study, we propose to use the following criteria to define an BMLF1259-267,MART-127-35,orMART-126-35 epitopes for immune response. (a) Tetramer assay: In subjects with baseline both assays. To minimize the effects of between-subject levels below the LLD, we would consider an increase from the variability (not incorporated into the RCV formula being tetramer assay LLD of 0.038% to z0.062% (the mean + 3 SD) applied here), all aCV and pCV were taken from antigen as being significant. In subjects with baseline values above the preexposed subjects with values above the LLD for each assay assay LLD, a significant change should at least reach the 95% (values from Tables 1 and 2). Thanks to its much lower analytic RCV for each peptide. (b) ELISPOT assay: Because an increase variability, the RCV was lower for the structural tetramer assay from below seven to at least nine spots per 105 PBMCs is not compared with the functional IFN-g ELISPOT assay for all considered biologically relevant given the assay imprecision, we epitopes and study populations. propose to use the 95% RCV for both subjects with baseline

Ta b l e 3 .

A. Calculation of significant antigen specific expansion for subjects with baseline levels of circulating antigen-specific T cells below the assay LLD Assay Epitope No. subjects No. samples Mean + 2 SD (95%) Mean + 3 SD (99%)

Tetramer EBV BMLF1259-267 öö ö ö

CMVpp65495-503 6 24 0.064 0.066

MART-126-35 9 36 0.050 0.062

ELISPOT EBV BMLF1259-267 16 188 7 9

CMVpp65495-503 19 224 7 9

MART-127-35 27 295 4 5

B. RCV to calculate significant antigen specific expansion for subjects with baseline levels of circulating antigen-specific T cells above the assay LLD Assay Epitope Degrees of freedom 95% Z score 99% Z score 95% RCV 99% RCV

Tetramer EBV BMLF1259-267 43 2.02 2.66 64.50 85.89

CMVpp65495-503 33 2.03 2.73 51.52 69.29

MART-126-35 66 1.99 2.65 80.57 107.29

ELISPOT EBV BMLF1259-267 99 1.98 2.62 186.55 246.85

CMVpp65495-503 71 1.99 2.65 144.45 192.36

MART-127-35 63 1.99 2.66 200.41 267.89

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Ta b l e 4 .

Patient Dose (mg/kg) Epitope Predose Within 1wk Within 2-4 wks Change 95% Change 99% A. Tetramer quantitation of antigen-specificT-cell expansion after administration of ticilimumab 113X 3 Negative 0.03 0.01 0.01

AFP325-332 0.03 0.05 0.02

EBV BMLF1259-267 4.25 4.0 3.76

CMVpp65495-503 <0.01 0.01 0.02

MART-126-35 0.07 0.05 0.07 119X 6 Negative 0.02 0.01 0.01

AFP325-332 0.03 0.01 0.02

EBV BMLF1259-267 0.81 1.15 (" 42%) 1.39(" 72%) 64.50% 85.89%

CMVpp65495-503 0.53 0.50 0.51

MART-126-35 0.02 0.03 0.04 120X 6 Negative 0.01 0.02 0.02

AFP325-332 3.35 4.22 3.25

EBV BMLF1259-267 0.46 0.38 0.45

CMVpp65495-503 0.01 0.04 0.02

MART-126-35 0.19 0.38 (" 100%) 0.22 80.57% 107.29% 12 5 X 10 Ne gati ve 0.0 2 <0.01 0.01

AFP325-332 öö ö

EBV BMLF1259-267 0.74 1.46 (" 97%) 0.87 64.50% 85.89%

CMVpp65495-503 öö ö

MART-126-35 0.05 0.04 0.02

B. IFN-; ELISPOT quantitation of antigen-specificT-cell expansion after administration of ticilimumab 113X 3 Negative 8 0 0

AFP325-332 00 0

EBV BMLF1259-267 12 9 24 6 ( " 91%) 247 (" 91%) 186.55% 246.85%

CMVpp65495-503 21 23 0 (# >100%) 144.45% 192.36%

MART-127-35 03 3

MART-126-35 11 3 0 ( " 173 % ) 11 NA NA 119X 6 Negative 36 30 33

AFP325-332 10 11 14

EBV BMLF1259-267 25 21 ö

CMVpp65495-503 24 21 24

MART-127-35 14 7 (# 50%) 9 (# 36%) 200.41% 267.89%

MART-126-35 21 5 (# 76%) 1 (# 95%) NA NA 12 5 X 10 Ne gati ve 6 3 0

AFP325-332 00 0

EBV BMLF1259-267 634(" 467%) 14 (" 133%) 186.55% 246.85%

CMVpp65495-503 32 1

MART-127-35 01 0

MART-126-35 32 0

Abbreviation: NA, not available. levels below and above the LLD. For patients starting below the negative regulatory pathway in T cells, which is expected to assay LLD, a significant positive change should be calculated then permit their expansion and activation leading to using the assay LLD plus the 95% RCV for each peptide. antitumor immune responses (27). These four patients Application of the definition of immunologic response in had received a prior dose of ticilimumab between 8 and patients receiving the anti-CTLA4 antibody ticilimumab. The 18 months before, and blood samples were collected during definitions of immune response based on the quantitation of redosing (see Materials and Methods). All patients had at circulating antigen-specific T cells using the tetramer and least one baseline PBMC sample before dosing, and at least ELISPOT assays were applied to the analysis of serial PBMC two follow-up PBMC samples. Table 4 provides the results of samples from four HLA-A*0201-positive patients with meta- the MHC tetramer and ELISPOT assays (not enough PBMCs static melanoma after dosing with ticilimumab (see Materials were available on patient 120X to perform an ELISPOT and Methods). This monoclonal antibody blocks a major assay).

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There is no clear trend of generalized antigen-specific T-cell doubling of the T-cell frequency observed after vaccine (32), expansion after ticilimumab administration evaluated using whereas others propose increases beyond the mean + 2 or 3 our definitions of immune response (Table 4). Analyzed with SD from negative values (12). These approaches interpret the tetramer assay, two of four patients had an increase in EBV change against fixed criteria (a population-based reference BMLF1259-267-specific T cells, whereas one of four had an value, a locally agreed protocol, a value proposed by expert increase in MART-126-35-specific T cells, at the 95% significance. committees, or multiples of the upper reference limit), which None had expansion of T cells specific for CMVpp65495-503, has been described as the ‘‘clinical fixed limits’’ or cutoff AFP325-332, or negative control peptides. Additional testing in point. Their advantage is that they are simple to implement patient 125X failed to reveal increases in the number of and use and are believed to be in some way related to the circulating gp100208-216 or tyrosinase368-376 antigen-specific T clinical outcome based on good evidence or in the views of cells (data not shown), two other immunodominant epitopes experts in the field (18). Definition of a positive immune in melanoma (28). response as an increase beyond the mean + 2 or 3 SD from Analyzed with the ELISPOT assay, one of three had an negative values can adequately address the situation of increase in EBV BMLF1259-267-reactive T cells at both the starting at a negative value (at or below the LLD for a 95% and 99% significance level, and an additional patient negative antigen). On the contrary, it is difficult to envision had a marked increase that did not reach the 95% signi- how this criterion can be used in the not infrequent situation ficance level. One patient had a marked decrease in IFN-g- that the prevaccination levels of antigen-specific T cells are producing cells specific for CMVpp65495-503.Nonehad above the LLD (Fig. 1E). We tested the approach of applying expansion of T cells reactive to the AFP325-332 or negative the RCV, which we reasoned would allow defining a positive control peptides. One of three had increase in the number or negative immune response based on a change in value of IFN-g-producing cells specific for the heteroclitic beyond the assay variability. The RCV calculation is MART-126-35 peptide but not the native MART-127-35 peptide unreliablebelowtheassayLLD,wheretheassayismost (significance could not be assessed, because our definition variable. In addition, a danger of RCV, compared with the was based on the MART-127-35 native peptide). One patient ‘‘fixed cutoff’’ point, is that RCV is changeable depending had a decrease in cells producing IFN-g in response to these on the experiment, sample size, and patient population. two MART-1-derived peptides, which did not reach signif- However, it allows defining if changes above the LLD are icance. true immune responses or just the variability that would be encountered when performing repeated measurements without any intercurrent treatments or illnesses. The LLD, Discussion analytic variability, and between-subject or within-subject biological variability in our study are in the same range as Much progress has been made in the past 10 years in the the ones defined at other laboratories (11, 12, 32–34). analysis of antigen-specific T-cell responses. The pioneering We tested the performance of our standardized assays and work of many groups (1–6, 9, 11, 12, 29, 30) has resulted in definitions of immune response in four HLA-A*0201 patients assays already being used as surrogate end points for immune receiving the CTLA4 blocking antibody ticilimumab. In this activation in experimental cancer immunotherapy trials (31). small group of patients, we were able to establish which The tetramer and ELISPOT assays quantitate antigen-specific changes from baseline levels of circulating antigen-specific T cells using two different approaches; therefore, it is not T cells were beyond the assay imprecision. Although some surprising that results are not directly concordant. Cells found values changed after dosing, there was no clear trend of to have a TCR specific for one antigen by the tetramer may antigen-specific T-cell expansion. Overall, these data do not or may not produce a particular cytokine (in our case IFN-g) specifically rule out an antitumor T-cell expansion by anti- detected in the ELISPOT assay. The available data suggest CTLA4 antibodies, because the relevant cells may be restricted that approximately one third of infectious disease or tumor to different epitopes or may only be notable in peripheral antigen-specific T cells detected using the tetramer assay blood transiently after the first antibody administration. produce IFN-g upon antigenic stimulation (12). The use of These possibilities are currently being tested in a larger set a combined tetramer and IFN-g intracellular cytokine flow of patients receiving ticilimumab under a separate protocol cytometry assay may allow to more directly define the (UCLA IRB 03-01-059), where immune response will be discrepancy between the two assay systems tested in the analyzed based on the definitions derived from this method- current study (12). ology study. A critical issue in the implementation of these assays for In conclusion, the MHC tetramer and ELISPOT assays can be the immune monitoring of vaccine and other immunother- used for the ex vivo analysis of T-cell responses after having apy approaches for human cancer is the definition of what is defined the assay LLD, between-subject variability in homeo- a positive immunologic response. Two consensus approaches static set points, and analytic and physiologic variability. for the ELISPOT assay were defined in an immune The ELISPOT assay has a much higher analytic imprecision monitoring workshop organized by the Society of Biological compared with the tetramer assay, even after optimizing its Therapy (10). These were the number of spots in six wells performance and using standardized operating procedures in exceeding by 10 the number of spots in six control wells, or replicate samples. An immunologic response should be the detection of a 2- or 3-fold increase over unstimulated considered positive only when the pre- and post-immunother- control wells. No consensus definition on an immunologi- apy determinations of circulating antigen-specific T cells cally positive response was proposed for the tetramer assay expand significantly above the LLD of the assay or are beyond (10); some groups have defined a positive response as a the RCV for the assay.

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References 1. Czerkinsky C, Andersson G, Ekre HP, et al. Reverse tracellular cytokine analysis. J Immunother 2005;28: 25. Butterfield LH, Ribas A, DissetteVB, et al. Determi- ELISPOTassay for clonal analysis of cytokine produc- 63^72. nant spreading associated with clinical response in tion. I. Enumeration of g-interferon-secreting cells. 13. Lee PP,Yee C, Savage PA, et al. Characterization of dendritic cell-based immunotherapy for malignant J Immunol Methods1988;110:29 ^ 36. circulatingT cells specific for tumor-associated anti- melanoma. Clin Cancer Res 2003;9:998 ^ 1008. 2. Tanguay S, Killion JJ. Direct comparison of ELISPOT gens in melanoma patients. Nat Med 1999;5:677^ 85. 26. Ribas A, Glaspy JA, Lee Y, et al. Role of dendrit- and ELISA-based assays for detection of individual 14. Harris EK, Brown SS.Temporal changes in the con- ic cell phenotype, determinant spreading, and neg- cytokine-secreting cells. Lymphokine Cytokine Res centrations of serum constituents in healthy men. Dis- ative costimulatory blockade in dendritic cell-based 1994;13:259^63. tributions of within-person variances and their melanoma immunotherapy. J Immunother 2004;27: 3. Herr W, Schneider J, Lohse AW, et al. Detection and relevance to the interpretation of differences between 354^67. quantification of blood-derived CD8+ T lymphocytes successive measurements. Ann Clin Biochem 1979; 27. Chambers CA, Kuhns MS, Egen JG, et al. CTLA-4- secreting tumor necrosis factor a in response to HLA- 16 :169 ^ 76. mediated inhibition in regulation of T cell responses: A2.1-binding melanoma and viral peptide antigens. 15. Harris EK,YasakaT.On the calculation of a‘‘reference mechanisms and manipulation in tumor immunother- J Immunol Methods1996;191:131 ^ 42. change’’ for comparing two consecutive measure- apy. Annu Rev Immunol 2001;19:565^94. 4. Schmittel A, Keilholz U, Scheibenbogen C.Evaluation ments. Clin Chem 1983;29:25 ^ 30. 28. Panelli MC, Riker A, Kammula U, et al. Expansion of of the interferon-g ELISPOT-assay for quantification of 16. Harris EK. Reference values for change: an adden- tumor-Tcell pairs from fine needle aspirates of melano- peptide specificT lymphocytes from peripheral blood. dum. Clin Chem 1983;29:997. ma metastases. J Immunol 2000;164:495 ^504. J Immunol Methods 1997;210:167 ^ 74. 17. Queralto JM, Boyd JC, Harris EK. On the calculation 29. Scheibenbogen C, Lee KH, Mayer S, et al. A sensi- 5. AltmanJD,MossPAH,GoulderPJR,etal.Phenotypic of reference change values, with examples from a tive ELISPOTassay for detection of CD8+ T lympho- analysis of antigen-specific T lymphocytes. Science long-term study. Clin Chem 1993;39:1398 ^ 403. cytes specific for HLA class I-binding peptide 1996;274:94 ^ 6. 18. Fraser CG. Biological variation: from principles to epitopes derived from influenza proteins in the blood 6. Murali-Krishna K, Altman JD, Suresh M, et al. Count- practice.Washington (DC): AACC Press; 2001. of healthy donors and melanoma patients. Clin Cancer ing antigen-specific CD8 Tcells: a reevaluation of by- 19. Trape J, Botargues JM, Porta F, et al. Reference Res 1997;3:221 ^ 6. stander activation during viral infection. Immunity change value for a-fetoprotein and its application in 30. Arlen P,Tsang KY,MarshallJL, et al.The use of a rap- 19 9 8;8 :177 ^ 87. early detection of hepatocellular carcinoma in patients id ELISPOTassay to analyze peptide-specific immune 7. Labalette-Houache M,Torpier G, Capron A, et al. Im- with hepatic disease. Clin Chem 2003;49:1209 ^ 11. responses in carcinoma patients to peptide vs. recom- proved permeabilization procedure for flow cytomet- 20. Ribas A, Camacho LH, Lopez-Berestein G, et al. binant poxvirus vaccines. Cancer Immunol Immun- ric detection of internal antigens. Analysis of Antitumor activity in melanoma and anti-self responses other 2000;49:517^ 29. interleukin-2 production. J Immunol Methods 1991; after CTLA4 blockade with the monoclonal antibody 31. Ribas A, Butterfield LH, Glaspy JA, et al. Current 138:143^53. CP-675.206. Journal of Clinical Oncology 2005;23: developments in cancer vaccines and cellularimmuno- 8. Chikanza IC, Corrigal V, Kingsley G, et al. Enumeration 10.1200/JCO.205.01.109. [epub ahead of print]. therapy. J Clin Oncol 2003;21:2415^ 32. of interleukin-1 aand h producing cells by flow cytom- 21. Maecker HT, MoonJ, Bhatia S, et al. Impact of cryo- 32. Speiser DE, Pittet MJ, Guillaume P, et al. Ex vivo etry.JImmunolMethods1992;154:173^8. preservation on tetramer, cytokine flow, and ELISPOT. analysis of human antigen-specific CD8+ T-cell 9. Clay TM, Hobeika AC, Mosca PJ, et al. Assays for BMC Immunol 2005;6:17. responses: quality assessment of fluorescent HLA- monitoring cellular immune responses to active immu- 22. Britten CM, Meyer RG, Kreer T, et al. The use of A2 multimer and interferon-g ELISPOTassays for pa- notherapy of cancer. Clin Cancer Res 2001;7:1127^35. HLA-A*0201-transfected K562 as standard anti- tient immune monitoring. J Immunother 2004;27: 10. Keilholz U, Weber J, Finke JH, et al. Immunologic gen-presenting cells for CD8(+) T lymphocytes in 298 ^308. monitoring of cancer vaccine therapy: results of a IFN-g ELISPOT assays. J Immunol Methods 2002; 33. HoffmannTK, DonnenbergVS, Friebe-Hoffmann U, workshop sponsored by the Society for Biological 259:95 ^ 110. et al. Competition of peptide-MHC class I tetrameric Therapy. J Immunother 2002;25:97 ^ 138. 23. Westgard JO. Westgard QC: tools, technology and complexes with anti-CD3 provides evidence for spec- 11. WhitesideTL. Monitoring of antigen-specific cytolyt- training for healthcare laboratories. Available from: ificity of peptide binding to theTCR complex. Cytome- icT lymphocytes in cancer patients receiving immuno- http://www.westgard.com 2005. try 2000;41:321 ^ 8. therapy. Clin Diagn Lab Immunol 2000;7:327 ^ 32. 24. Butterfield LH, Meng WS, Koh A, et al. T cell 34. Asai T, Storkus WJ,WhitesideTL. Evaluation of the 12. Hobeika AC, Morse MA, OsadaT, et al. Enumerating responses to HLA-A*0201-restricted peptides de- modified ELISPOTassay for g interferon production in antigen-specificT-cell responses in peripheral blood: a rived from human a fetoprotein. J Immunol 2001;166: cancer patients receiving antitumor vaccines. Clin comparison of peptide MHC Tetramer, ELISpot, and in- 5300^8. Diagn Lab Immunol 2000;7:145 ^ 54.

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Begoña Comin-Anduix, Antonio Gualberto, John A. Glaspy, et al.

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