Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to...

doi:10.1182/blood-2008-01-135319Prepublished online June 2, 2008;

Timothy M ClayMichael A Morse, Amy C Hobeika, Takuya Osada, Delila Serra, Donna Niedzwiecki, H. Kim Lyerly and specific immune responses to cancer vaccinesDepletion of human regulatory T cells specifically enhances antigen

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Depletion of Human Regulatory T Cells Specifically Enhances Antigen Specific Immune

Responses to Cancer Vaccines

Michael A. Morse1, Amy C. Hobeika2, Takuya Osada2, Delila Serra2, Donna

Niedzwiecki5, H. Kim Lyerly2-4, 6, and Timothy M. Clay2

Departments of 1Medicine, 2Surgery, 3Immunology, 4Pathology, 5Biostatistics and

Bioinformatics, and 5Duke Comprehensive Cancer Center, Duke University Medical

Center, Durham, NC

Corresponding Author: Michael A. Morse

Duke University Medical Center

Box 3233 MSRB 1 Rm 403

Durham, NC 27710

Tel: (919) 681-3480

Fax: (919) 681-7970

Email: [email protected]

Running Title: Depletion of Treg enhances immune responses

1

Blood First Edition Paper, prepublished online June 2, 2008; DOI 10.1182/blood-2008-01-135319

Copyright © 2008 American Society of Hematology


mailto:[email protected]



Abstract

CD4+CD25highFoxP3+ regulatory T cells (Treg) limit antigen-specific immune responses

and are a cause of suppressed anti-cancer immunity. In pre-clinical and clinical studies,

we sought to assess the immune consequences of FoxP3+ Treg depletion in patients with

advanced malignancies. First, we demonstrated that a CD25high targeting immunotoxin

(denileukin diftitox (ONTAKTM)) depleted FoxP3+ Treg, decreased Treg function, and

enhanced antigen-specific T cell responses in vitro. We then attempted to enhance anti-

tumor immune responses in patients with carcinoembryonic antigen (CEA)-expressing

malignancies by pre-vaccination Treg depletion. In a pilot study (n=15), denileukin

diftitox, given as single dose or repeated dosing, was followed by immunizations with

dendritic cells modified with the fowlpox vector rF-CEA(6D)-TRICOM. By

multiparameter flow cytometric analysis, we report the first direct evidence that

circulating CD4+CD25highFoxP3+ Treg are depleted following multiple doses of

denileukin diftitox. Earlier induction of, and overall greater exposure to, the T cell

response to CEA was observed in the multiple dose group, but not the single dose group.

These results indicate the potential for combining Treg depletion with anti-cancer

vaccines to enhance tumor antigen-specific immune responses and the need to explore

dose and schedule of Treg depletion strategies in optimizing vaccine efforts. This trial

was registered at www.clinicaltrials.gov #NCT00128622.

2




Introduction

Vaccines to treat or prevent recurrence of cancer have been of considerable interest, 1, 2

but challenges to their efficacy has been an immune suppressive effect of the tumor

microenvironment and modulation of T cell expansion by inhibitory cells such as

regulatory T cells. Regulatory T cells (Treg), defined by their expression of CD4,

persistently high expression of the IL-2 receptor component CD25, and intracellular

expression of the transcription factor FoxP3, 3 prevent uncontrolled proliferation of

antigen-specific T cells. 4-7 Increasing evidence implicates a contribution of Treg to the

impaired host immune response against cancer. 8, 9 Elevated Treg levels in the peripheral

blood, regional lymph nodes, and the tumor microenvironment of cancer patients are

associated with reduced survival.10-13 Depletion of Treg in animal models leads to

enhanced anti-tumor immune responses. 14-17 A recent human study reported that Treg

depletion before immunization enhanced tumor antigen-specific T cell responses.18

Potential strategies for depleting regulatory T cells, such as anti-CD25 antibodies

and cyclophosphamide, are limited by the persistence of the antibodies which would also

deplete or inhibit activated T cells that express CD25 and the non-specific toxicity of

chemotherapeutics. We therefore studied denileukin diftitox (ONTAK), a fusion between

the active domain of diphtheria toxin and IL-2. Denileukin diftitox binds to cells

expressing high levels of CD25 whereupon it is internalized, leading to blockade of

protein synthesis and cell death.19 Denileukin diftitox has direct antitumor activity against

CD25-expressing T cell malignancies.20 Cells expressing the high affinity IL-2 receptor

are most susceptible to the effects of denileukin diftitox, but the short half-life of

denileukin diftitox (70-80 minutes) should limit its impact on subsequently activated

3




effector T cells. In murine models and in humans with melanoma and renal cell

carcinoma, denileukin diftitox reduced CD4+CD25high levels.18, 21-23 With the availability

of techniques to directly identify FoxP3 by permeabilization and intracellular staining, we

can now directly assess the circulating levels of the more precisely defined

CD4+CD25highFoxP3+ Tregs over time.

In order to address the hypothesis that Treg depletion would enhance immune

responses against a cancer vaccine, we performed a pre-clinical study that demonstrated

depletion of Treg prior to application of antigen in vitro enhanced antigen-specific T cell

responses. Subsequently, patients with advanced CEA-expressing malignancies were

administered denileukin diftitox before immunization with a vaccine consisting of

dendritic cells (DC) modified with the viral vector rF-CEA(6D)-TRICOM vaccine. We

observed depletion of CD4+CD25highFoxP3+ Tregs and enhanced CEA specific T cell

immunity.

4




Materials and methods

Blood collection and cell preparation

Blood and leukapheresis products were obtained following signed informed consent from

cancer patients and healthy donors according to institutional review board-approved

protocols. Peripheral blood mononuclear cells (PBMC) were separated over a Ficoll

gradient. Relevant Duke University IRB or Ethics Committee approval was obtained for

each trial.

Treg depletion in vitro with denileukin diftitox detected using flow cytometry

PBMC were cultured for 18-20 hours (day 1) in complete media (RPMI 1640, 10% huAB

serum, 25mM Hepes, 100U/ml penicillin, 100μg/ml streptomycin, 2mM glutamine) with

denileukin diftitox (0-8nM) (ONTAK, Eisai, Inc., Woodcliff Lake, NJ), washed, and

replated in complete media with 10 U/ml IL-2 for 3 days to allow recovery. On day 4,

Treg were identified using an anti-human FoxP3 staining set (eBioscience, San Diego,

CA). Briefly, cell surface staining with anti-CD25-FITC, anti-CD3-PerCP, and anti-CD4-

APC (BD Biosciences) was followed by fixation and permeabilization prior to

intracellular staining with anti-Foxp3-PE. Events collected using a FACS Calibur (BD

Biosciences) were analyzed using CellQuest (BD Biosciences). CD4+CD25+FoxP3+ T

cells were identified by gating on the CD4+CD25high cells expressing > 90% FoxP3+.

Functional analysis of Treg depletion using proliferation assay

5




PBMC treated with various concentrations of denileukin diftitox were analyzed for their

proliferative capacity (on day 4) in response to stimulation with anti-CD3 (OKT3).

PBMC (105 cells/well in a total volume of 200 μl) were stimulated with media alone or

0.5 μg/ml soluble OKT3 for 96 hours. Proliferation was determined by [3H]Thymidine

(1 μCi/well) incorporation using a Wallac scintillation counter and was reported as mean

counts per minute +/- standard deviation.

In vitro analysis of antigen-specific T cell expansion following Treg depletion

PBMC from normal healthy HLA-A*0201 positive volunteers, treated as above with 4

nM denileukin diftitox and recovered in 10 U/ml IL-2 for 3 days, were stimulated with 1

μg/ml CMV pp65(495-503) peptide or 1 μg/ml MART-1(27-35) peptide at 2 x 106

cells/well in 24 well plates. 300 U/ml IL-2 was added every 3 days. For CMVpp65

expansions, stimulated cells were analyzed by peptide-MHC tetramer binding at days 7

and 10. For MART-1 expansion, responding cells were restimulated (at an 8:1 S:R ratio)

on day 8 using irradiated, autologous PBMC pre-treated with 4 nM denileukin diftitox

and pulsed with 10 μg/ml MART-1(27-35) peptide. On day 8 of the re-culture, MART-1

expanded cells were analyzed by peptide-MHC tetramer (Beckman Coulter, San Diego,

CA) staining. Cells were stained with αCD3-FITC, αCD8-PerCP, αCD4, αCD14, and

αCD19-APC, and tetramer-PE. Tetramer positive cells were identified by gating on the

population of cells that were forward scatterlo side scatterlo CD4-CD14-CD19-CD3+.

Phase I clinical trial of denileukin diftitox plus DC-rF-CEA(6D)-TRICOM

Participants were recruited from the medical and surgical oncology clinics of Duke

University Medical Center and provided signed informed consent approved by the Duke

6




University Medical Center Institutional Review Board before enrollment. The study was

performed under an FDA approved Investigational New Drug Exemption (IND).

Participants were required to have a metastatic cancer expressing CEA defined by

immunohistochemical analysis or elevated CEA in peripheral blood, adequate

hematologic (WBC >4.0), renal (Cr <2.0), and hepatic function (bilirubin <1.5). They

must have received and had progressive disease on prior therapy. They were excluded if

they had had chemotherapy, radiation therapy, or immunotherapy within the prior 4

weeks, history of auto-immune disease including inflammatory bowel disease, presence

of an active acute or chronic infection, HIV, or viral hepatitis, and if they had used

immunosuppressives in the preceding 4 weeks.

Generation of vaccines

Participants underwent a 4 hour peripheral blood leukapheresis and the product was

separated by density gradient centrifugation over Ficoll in a Cell Separator (Cobe BCT,

Inc., Lakewood, CO) to obtain. The DC vaccine was generated using patient DC and rF-

CEA(6D)-TRICOM (manufactured by Therion Biologics Corporation, Cambridge, MA

and supplied by Cancer Treatment Evaluation Program, NCI) as previously reported.2

For vaccines that were to be administered fresh, the DC cell preparations were

washed and resuspended at 5 x 106 cells in 0.5 ml of saline. They were then mixed with

the rF-CEA(6D)-TRICOM (25 x 106 particles) in a total volume of 1 ml saline for at least

30 minutes before administration. All cellular products were required to pass lot release

criteria consisting of expression of HLA-DR and CD86 in at least 50% of the (large,

dendritic) cells, viability >70%, and no evidence of bacterial or fungal contamination. For

7




vaccines administered following cryopreservation of the DC, one vial of DC product was

thawed and the required number of DC (5 x 106 cells) was mixed with the rF-CEA(6D)-

TRICOM (25 x 106 particles) in a total volume of 1 ml saline for at least 30 minutes

before administration. We found no significant differences in the mean expression of

CEA, CD80, CD54 or CD58 between cohorts or between DC that were fresh and those

previously cryopreserved (data not shown).

Protocol schema and patient treatment

This phase I study enrolled patients into two sequential cohorts. In the first cohort (cohort

1), patients received one dose of denileukin diftitox 18 mcg/kg intravenously 4 days prior

to initiating DC injections. DC were given every 3 weeks for 4 immunizations.

Immunizations were given as a combination of intradermal (0.1 ml) and subcutaneous

(0.9 ml) into the same limb (predominantly the right thigh) and injection sites were

separated by 10 cm from each other.

The study protocol permitted escalation to the second cohort (cohort 2) if no more

than 1 of the first 6 patients experienced a dose limiting toxicity related to the vaccine. In

this cohort, based on data suggesting better Treg depletion with a lower dose of

denileukin diftitox,24 subjects received denileukin diftitox 9 mcg/kg intravenously 4 days

before every DC injection.

In order to compare the clinical and immunologic data from patients who received

denileukin diftitox with data from patients who received the same vaccine, but no

denileukin diftitox, we collected further clinical and immunologic data from patients who

8




had participated in our previous clinical trial of DC modified with rF-CEA(6D)-

TRICOM. 2 We refer to this group as cohort 0.

Analysis of clinical and immunologic activity

Clinical activity was assessed by applying the RECIST criteria to CT or MRI

scans obtained before and after all immunizations. For immunologic analyses, peripheral

blood was drawn before each immunization and following the final immunization. Fresh

PBMC were analyzed for antigen-specific reactivity. Blood was also drawn before and 4

days after each denileukin diftitox infusion for Treg analysis (performed as described

above). For cohort 0, PBMC from cryopreserved samples pre- and post-immunizations

were analyzed.

Enumeration of Treg

Whole blood from patients in cohort 2 was stained using anti-CD25, anti-CD4, anti-CD3

and anti-CD45 using BD Bioscience TruCount tubes. Using percent CD4+CD25+ that

were also FoxP3+ based on our intracellular staining described above, we determined the

number of CD4+CD25+FoxP3+ cells per μl blood.

Analysis of CEA-specific T cell responses from peripheral blood using

IFNγ ELISpot Assay

Multiscreen-HA 96-well plates (Millipore, Bedford, MA) were coated overnight with 10

μg/ml mouse anti-human IFNγ Mab (diaPharma Group, Inc., West Chester, OH) and

plated at 100,000 or 250,000 PBMC/well in the presence of rF-CEA(6D)-TRICOM

9




(multiplicity of infection (moi) 10), rF-wild type (moi 10), CAP-1 peptide (1μg/ml), CEA

peptide mix (overlapping peptides spanning entire CEA molecule, 1360μg/ml),

CMVpp65 (495-503) 1μg/ml, CMV peptide mix and HIV peptide mix

(1.75μg/ml/peptide) (Pharmingen, San Diego, CA), PMA 2 μg/ml (Sigma Chemicals, St.

Louis, MO), SEB 2 μg/ml (Sigma), or media. Following a 18-20 hour incubation, 100 μl

of mouse anti-human IFNγ biotinylated Mab (diaPharma Group, Inc., West Chester, OH)

was added to each well for 2 hours followed by Vectastain ABC Peroxidase (Vector

Labs, Inc., Burlingame, CA) 100 μl/well for 1 hour. Color was developed using 100

μl/well of 3-amino-9-ethyl-carbazole [AEC] (Sigma, St. Louis, MO).

Membranes were read using the KS ELISPOT Automated Reader System with

the KS ELISPOT 4.2 or 4.9 Software (Carl Zeiss, Inc., Thornwood, NY) to determine the

number of spots per well. The mean number of spots from the six replicate wells was

reported as the response to each antigen.

Analysis of CD4+ and CD8+ CEA-specific T cell responses using intracellular

cytokine staining

PBMCs (2-4 x 106/ml) were stimulated with the antigens (same concentrations as

described for ELISpot assay) 6 hours (rF-CEA(6D)-TRICOM (moi 1) and rF-wild type

(moi 1) incubated for 12 hours) in the presence of 10 μg/ml Brefelden A (Sigma) and 1

μg anti-CD28 (BD Biosciences, San Jose, CA) per 106 PBMC. Stimulated cells were

fixed with 1% paraformaldehyde and cryopreserved. Once all timepoints for a patient

were collected, cells were permeabilized (FACSTM Permeabilizing Solution (BD

Biosciences)) and stained with αIFNγ-FITC, αCD69-PE, αCD8-PerCP, and αCD4-APC.

10




The percentage of IFNγ/CD69 double positive CD8+ T cells was determined within

forward scatterlo side scatterlo CD8pos or CD4 pos cells.

Analysis of anti-CEA antibodies by ELISA

Patient serum was collected prior to and following immunization. Anti-CEA and anti-

fowlpox antibody (IgG) were quantified by ELISA. 96-well plates were coated with CEA

protein (100 ng/well), Fowlpox-wild type (1x107 pfu/well) or KLH (100 ng/well, Sigma).

Plates were incubated 4 hours with 100 µl of serum serially diluted 1:10 to 1:31,250, then

incubated with 100 µl of alkaline-phosphatase-conjugated goat anti-human IgG (working

dilution: x10,000, MP Biomedicals, Inc., OH) 3 hours, and developed with PNPP (p-

nitrophenyl phosphate, Sigma). Reaction was stopped with 3N NaOH and A405 nm was

measured using ELISA Plate Reader (BioRad). Titers were defined as the highest dilution

with A405 nm above background level (0.2 for CEA and KLH, 0.3 for fowlpox).

Statistical Methods

The percentage change in CD4+CD25+FoxP3+ Tregs from baseline (i.e., prior to

receiving denileukin diftitox (referred to as LPA)) for each cohort is determined by

dividing the difference between each patient value for each timepoint and baseline by the

baseline value (x 100%).

Immune response data for ELISpot, and cytokine flow cytometry assay were captured for

each patient at time 0, 3, 6, 9 and 10 weeks. Area under the curve of the immune response

(AUC) was estimated for each patient using the linear and log trapezoidal rules as

follows. The linear trapezoidal rule was used to estimate the area of a segment when the

11




curve was increasing; the log trapezoidal rule was used to estimate the area of a segment

when the curve was decreasing. A linear trapezoidal segment is estimated as

1/2 (ti+1-ti)(mi+1+mi) where t denotes timepoint, m denotes the immune response

measurement and i is in (1, 2, …. n-1). Similarly, a log trapezoidal segment is estimated

as (t i+1-ti)(m i+1-mi)/log(mi+1/mi). The total AUC for each patient is the sum of all

segments. The Wilcoxon Rank Sum test was used to compare the median AUC between

cohorts. The t-test was used to compare mean times to peak immune response between

cohorts.

A positive immune response by ELISpot was defined as described at the 2002 Society of

Biologic Therapy Workshop on “Immunologic Monitoring of Cancer Vaccine Therapy”:

a T cell response is considered positive if the mean number of spots in six wells with

antigen exceeds the mean number of spots in six control wells by 10 and the difference

between the mean of the six wells containing antigen and the six control wells is

statistically significant at a level of p ≤ 0.05 using the Student’s t test.25

12




Results

Denileukin diftitox depletes CD4+CD25highFoxP3+regulatory T cells leading to

enhanced T cell proliferation

PBMC from normal donors were treated with various concentrations of denileukin

diftitox for 18-20 hours and stained for CD4, CD25 and FoxP3 expression (day 1). These

cells were then washed and re-cultured in low dose IL-2 containing media for an

additional 3 days and again stained for CD4, CD25, and Foxp3 (day 4). The percentage

of CD4+CD25highFoxP3+ Treg decreased (approximately 75%) when treated with

increasing concentrations of denileukin diftitox (Figure 1A). This effect was observed on

day 1 and was still apparent after the rest period (day 4) although a greater percentage of

CD4+CD25highFoxP3+ Treg was observed at day 4. This suggests that Treg may

experience a rebound effect due to the influence of the IL-2 during the rest period.

PBMC depleted of CD4+CD25highFoxP3+ cells by denileukin diftitox

pretreatment exhibited greater proliferation in response to anti-CD3 stimulation (OKT3)

in a dose dependent manner (Figure 1B). Because the peak proliferative effect plateaued

at approximately 4nM denileukin diftitox, the concentration achieved in the peripheral

blood of patients treated with an 18mcg/kg dose, subsequent experiments used this

concentration. Importantly, these results required the 3 day rest period and administration

of the IL-2 to maintain viability of the PBMC and permit expansion of effector T cells.

Proliferation experiments attempted without the rest period led to no expansion of the

PBMC (data not shown). In summary, these data demonstrate that denileukin diftitox

depletes regulatory T cells in vitro and enhances the proliferation of effector T cells, but

13




that effector T cells may be affected by denileukin diftitox and require a rest period

before they can become activated.

Enhanced antigen specific T cell reactivity of PBMC after denileukin diftitox

Since denileukin diftitox increased non-specific T cell proliferation, we also determined

whether it could also enhance antigen-specific T cell expansion. PBMC from healthy

normal donors were depleted of Treg using denileukin diftitox as described above and

cultured in vitro short term with CMVpp65 peptide. On days 7 and 10 of the CMVpp65

peptide specific expansion, the percentage of CMVpp65 tetramer positive cells was

determined for untreated and denileukin diftitox treated cells (Figure 2A). PBMC treated

with denileukin diftitox showed a greater expansion of CMVpp65 specific T cells over

untreated as shown by CMV tetramer staining at day 7 (3.4% vs. 2.2% ) and day 10

(24.4% vs. 10.5%).

In order to demonstrate that Treg depletion would enhance tumor antigen-specific

T cell responses, we analyzed the effects of denileukin diftitox on antigen specific T cell

expansion with the melanoma antigen MART-1 peptide. PBMC exposed to denileukin

diftitox as described above were stimulated with MART-1 peptide. The expanded PBMC

were then restimulated with autologous PBMC that had again been treated with media

alone or denileukin diftitox, pulsed with MART-1 peptide, and irradiated. The percentage

of MART-1 specific T cells was determined by tetramer staining for both the initial and

repeated stimulation showing an increase in MART-1 specific T cells in the denileukin

diftitox treated PBMC (2.6% for denileukin diftitox treated PBMC compared with 0.55%

MART-1+ CD8+ T cells for the initial 8 day stimulation and 35.3% MART-1+ CD8+ T

14




cells compared with 22.5% for the second stimulation) (Figure 2B). These data

demonstrate that T cell responses against self and foreign recall antigens is enhanced

following Treg depletion with denileukin diftitox.

Analysis of the effect of Treg depletion with denileukin diftitox in vivo: Phase I clinical

trial

Based on the preclinical data demonstrating that PBMC depleted of Treg with denileukin

diftitox exhibited enhanced immune responses, we performed a phase I clinical trial of a

dendritic cell vaccine modified to express CEA, administered to patients with advanced

CEA expressing malignancies following denileukin diftitox administration in 2 different

schedules (prior to the first dose of vaccine and prior to all 4 doses of the vaccine).

Patients (male 9, female 6; age 35-72) had colorectal cancer (n=14) or breast cancer

(n=1), all had extensive metastatic disease but a performance status of 90-100% and had

failed a median of 2 prior chemotherapeutic regimens. In cohort 1, 5 of 9 patients

completed all four immunizations. In the second cohort, 5 of 6 patients completed all

immunizations. Immunizations were well tolerated with only rare grade 3 (elevated ALT,

AST, and bilirubin attributed to disease progression and pleural effusion and dyspnea

attributed to disease progression) and one grade 4 event (elevated creatinine attributed to

initiation of an angiotensin converting enzyme inhibitor).

Clinical course

In cohort 1, 1/9 patients experienced a minor response and one had stable disease while

the remainder had progressive disease at the end of immunizations. One of these patients

15




with progressive disease, later underwent a surgical resection of an enlarged

retroperitoneal lymph node and only necrotic tissue was identified. A second patient who

initially had progressive disease, was later found to have stabilization of disease on a

subsequent CT scan. In the second cohort, all 6 patients had progression, but one

experienced stability on subsequent CT scans for > 6 months.

Effect of denileukin diftitox on peripheral blood CD4+CD25highFoxP3+ Treg

The circulating levels of CD4+CD25 highFoxp3+ Treg were determined for both cohorts

of this study as well as from our previous trial involving DC + rF-CEA(6D)-TRICOM

vaccine (n=5). This allowed a comparison of changes in Treg levels following

vaccinations alone (cohort 0), vaccinations following a single infusion of denileukin

diftitox (cohort 1), and denileukin diftitox given prior to each vaccination (cohort 2)

(Figure 3). No change occurred in percent CD4+CD25 highFoxp3+ cells between the pre

and post vaccination samples in patients receiving the DC vaccine without denileukin

diftitox (cohort 0). In cohort 1 (Figure 3B), although an initial increase is seen in the

mean Treg levels at week 1, the change in magnitude of CD4+CD25 highFoxp3+ cells dips

at weeks 6 and 9 before rebounding following the completion of the immunizations.

Overall, only 2/5 patients who completed all immunizations in cohort 1 had a decreased

percentage of Treg at the end of the immunizations. For the multidose cohort 2 (Figure

3C), the mean change in magnitude of CD4+CD25 highFoxP3+ cells continues to decrease

following the final vaccination. Overall, 4 of 4 patients in cohort 2 who completed all

immunizations had a lower percentage of Treg at the end of all immunizations. For the

second cohort, we also determined the total number of CD4+CD25 highFoxp3+ cells per

16




microliter of whole blood and found it to decrease beginning at week 5 and continue to

decline following the final vaccination (Figure 4). These data indicate that repeated

dosing of denileukin diftitox results in a consistent decrease in CD4+CD25 highFoxP3+

Treg.

Immunologic analysis of CEA-specific T cell response by ELISpot

ELISpot analysis was performed on freshly isolated PBMC prior to each immunization

and following the final immunization. Figure 5 demonstrates the mean number of IFNγ

producing cells per 100,000 PBMC for each cohort in response to rF-CEA(6D)-

TRICOM. Based on the definition of immune response proposed by the Immunotherapy

Working Group,25 we found 13 of 14 evaluable patients had a CEA-specific immune

response at some point during their immunizations for cohort 0, 4 of 5 for cohort 1, and 5

of 5 for cohort 2 (data not shown). Notably, denileukin diftitox was associated with an

earlier time to first immune response (6.7 weeks for cohort 0, 5.25 weeks for cohort 1,

and 3.6 weeks for cohort 2, P=.00374 for cohort 2 versus cohort 0) and greater area under

the curve (AUC) of the ELISPOT response for cohort 2 (Tables 1 and 2). The greater

AUC indicates that there was a greater period of time that the immune response occurred

in the multiple denileukin diftitox dose cohort.

Immunologic analysis of CEA-specific T cell response by intracellular cytokine

staining

In order to characterize both CD4+ and CD8+ T cell activation following immunization

and/or denileukin diftitox infusion, we performed cytokine flow cytometry of freshly

17




isolated PBMC from each time point. We identified T cells responding to CEA exposure

by the percentage of T cells that were CD69+ and producing IFNγ. Figure 6 depicts the

mean percent CD4+IFNγ+ and CD8+IFNγ+ cells in response to rF-CEA(6D)-TRICOM

stimulation pre and post each immunization. Based on our experience in assessing

positive and negative responses to various antigens,26 we chose a cut-off of 0.5% over

background as indication of a positive response. We found 14 of 14 patients had a CD4+

and CD8+ CEA-specific T cell response at some point during their immunizations for

cohort 0, 5 of 5 CD4+ response and 4 of 5 CD8+ response for cohort 1, and 5 of 5 CD4+

response and 4 of 5 CD8+ response for cohort 2 (data not shown). There were trends for

an earlier CD4 and CD8+ T cell response and for a greater AUC of the CD8+ immune

response in cohort 2 (Tables 1 and 2), but these did not achieve statistical significance.

Serum antibody titers following vaccination

Analysis of antibody titers to both fowlpox and CEA antigen was performed on serum

samples collected pre and post immunization (Figure 7). Fowlpox specific antibodies

were detected in all patients tested in all cohorts following vaccination with the exception

of one patient in the first cohort (Figure 7A). The titers ranged from 1:50 to 1:6250

among the responders, but were not greater in any of the cohorts. In contrast, CEA-

specific antibodies, although low titer, were more frequent in patients receiving a single

dose of denileukin diftitox (cohort 1) but were not detected in any patient after multiple

doses (cohort 2). These data suggest that tumor antigen-specific Treg may be more

sensitive to the effects of denileukin diftitox than foreign antigen-specific Treg.

18




Discussion

This translational study was performed by first determining the effect of denileukin

diftitox in vitro and then extending the observations into a phase I clinical trial. We

observed that denileukin diftitox depleted Treg in vitro and resulted in enhanced immune

reactivity. In the human clinical trial, we provide the first direct evidence that

CD4+CD25highFoxP3+ Treg are depleted following multiple administrations of

denileukin diftitox, but not after a single administration. CEA-specific T cell responses

were enhanced as measured by both ELISPOT and by cytokine flow cytometry, and these

responses occurred earlier in the multiple dose cohort. Denileukin diftitox was not

associated with an effect on primary/foreign antigen (fowlpox) antibody responses, but

was associated with differences in antibody titer against the self antigen, CEA.

Our study is unique because it directly assessed the level of circulating

CD4+CD25highFoxP3+ Tregs, evaluated 2 different dosing strategies and compared the

results with a group of patients not receiving denileukin diftitox. We also performed a

complete immune evaluation (ELISpot, cytokine flow cytometry, antibody responses, and

Treg phenotyping) at each immunization timepoint and thus have complete data

permitting a better understanding of the time-course of changes in immunity following

denileukin diftitox-mediated depletion of Treg.

Our results on in vitro depletion of Treg is consistent with the report of Litzinger

et al.22 who also observed that Treg decreased after denileukin diftitox treatment at 5nM

from 0.89 to 0.17% of CD4+ T cells. They observed little effect at 24 hours but a more

marked effect 48 and 72 hours after the application of denileukin diftitox, whereas we

saw an effect at 24 hours which became less pronounced after 72 hours. We suspect the

19




differences in our observations is due to the fact that we cultured the PBMC overnight

(18 hours) with denileukin diftitox whereas they applied it for only 2 hours. It is possible

that the IL-2 portion of the denileukin molecule persisting for a longer period of time in

our experiment caused an activation of T cells to an express a Treg phenotype on

subsequent days of culture. Dannull18 cultured sorted Treg with denileukin diftitox for 6

hours and observed a decrease in Treg at 48 hours which was greater at 72 hours. It is

possible that the lack of other PBMC in their purified Treg population did not permit the

observation that other PBMC could eventually develop a Treg phenotype. Mahnke23

similarly noted killing of isolated CD4+CD25+ T cells at concentrations of >8nM

denileukin diftitox after 24 hour in vitro incubation. In contrast to our work, Attia27

reported no evidence of reduced Foxp3 expression in purified CD4+ cells exposed to up

to 500nM denileukin diftitox in vitro. Furthermore, the denileukin diftitox failed to

remove cells capable of exerting a suppressive effect on effector cells. It is possible that

differences in our results may be related to how FoxP3 was measured (we used flow

cytometry and they used rt-PCR) and how the effect on effector T cells was measured.

Whether denileukin diftitox depletes Treg in vivo has been controversial.

Dannull18 reported a decrease in Treg percentage 4 days after a dose of 18mcg/kg/d of

denileukin diftitox when enumerating the cells by CD4+CD25high expression. They also

observed a decrease in FoxP3 copy number by rt-PCR. Mahnke23 reported that

CD4+CD25+ Treg decreased for approximately 13 days after 18mcg/kg/d for 3 days of

denileukin diftitox. The percentage of Treg decreased further after a second round of

denileukin diftitox. In one patient who received 5 doses of denileukin diftitox, there was a

slight decrease but never a complete depletion of Treg. Similar to our observation, they

20




found some patients with a transient increase in Treg in the first few days of beginning

the denileukin diftitox, but eventually all had a decrease in Treg. Barnett28 reported in a

small pilot study that CD4+CD25+ T cells decreased after 9-12mcg/kg dose of

denileukin diftitox. Schonfeld24 reported that when patients were injected with a dose of 5

mcg/kg denileukin diftitox, there was a reduction of CD4+CD25+ Tregs in peripheral

blood over a period of 2 weeks. However, when a dose of 18 mcg/Kg was used, no

significant reduction of Treg occurred. These data were one of the reasons we started the

second cohort at a lower dose of denileukin diftitox and may explain the better Treg

depletion observed in the multiple dose group (cohort 2). In contrast, Attia27 reported no

decrease in Treg. In their study, the denileukin diftitox was administered for 5

consecutive days every 21 days which could possibly have resulted in stimulation of Treg

by the IL-2 portion of the molecule. This report, using direct detection of

CD4+CD25highFoxP3+ Tregs provides compelling evidence about the effects of

denileukin diftitox in vivo.

A critical component of Treg depletion is the effect on immune homeostasis.

Mahnke23 reported vaccination with MART-1 and GP100 peptides following denileukin

diftitox and observed an increase in ELISpot and tetramer+ T cells, but they did not have

a control arm to determine if the immune responses were enhanced by the denileukin

diftitox. They argued that prior studies with peptides alone (without adjuvant) generally

yielded very low immune responses otherwise. Dannull18 immunized patients with

dendritic cells loaded with tumor RNA either following denileukin diftitox or without any

pre-treatment and observed an improved stimulation of tumor-specific T cell responses

following denileukin diftitox when compared with vaccination alone. Our study allows a

21




direct comparison of the immune response with data obtained in our prior study using an

identical vaccine in an identical patient population. In addition, the direct assessment of

CD4+CD25highFoxP3+ Tregs allowed us correlate Treg depletion with enhanced immune

response. Finally, assessment of the immune response to the neo-antigens found in the

vaccine vector backbone, allowed us to observe that there was specific enhancement of

tumor antigen specific immune responses, but not general enhancement of immune

responses.

The effect of the denileukin diftitox on antibody responses against CEA is

surprising. It would appear that multiple doses have no effect on the antibody response to

the foreign antigen (the fowlpox vector), but they cause a reduction in the self (CEA)

antibody response. Interestingly, Litzinger22 observed no effect of denileukin on the

antibody response to foreign antigens (the vaccinia vector and influenza A (NP34)) but

did not assess antibody responses to the tumor antigen CEA. One possibility for this

observation is that denileukin diftitox could have direct inhibitory or destructive effects

on tumor antigen-specific B cells. It is also possible that the apparent decrease in

antibody against CEA, observed in our study, is due to random variation because the anti-

CEA titers were low even in cohort 0 (the group not receiving denileukin).

In summary, denileukin diftitox eliminates regulatory T cells which is associated

with an enhanced specific immune response to tumor antigen vaccination, and an earlier

peak in T cell response after multiple doses. Tumor antigen-specific antibody responses

may be diminished after multiple doses. Future studies should better refine the number of

doses and timing required to maximize the immune response activated.

22




Acknowledgements

The authors thank Liz Anderson and Emily Privette for normal donor blood acquisition,

Manar Ghanayem, Wiguins Etienne, Amanda Summers, and Karrie Comatas for

generation of dendritic cells for the patient immunizations, Patti Frost for assistance with

the statistical analysis, and Jill Boy for help with figures. We thank Sharon Peplinski for

flow cytometry acquisition and analysis. We thank Eisai Inc., for supplying the

denileukin diftitox (ONTAK) for the second cohort of the study. We also thank the NCI

CTEP program for supplying the rF-CEA(6D)-TRICOM. This work was supported by a

grant from the NIH (1R21- CA117126-01A1) (M.A.M.). The authors declare no

competing financial interests.

Authorship

Contribution: D.S., T.O. and A.C.H. performed experiments; A.C.H., T.O. and M.A.M.

analyzed results and made the figures; H.K.L., M.A.M. and T.C. designed the research;

A.C.H., H.K.L, T.C., and M.A.M. wrote the paper; and D.N. performed statistical

analysis.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Michael A. Morse, Duke University Medical Center, Box 3233 MSRB

1 Rm 403, Durham, NC 27710; Email: [email protected]

23


mailto:[email protected]



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Table 1. Median (Range) of AUC for ELISPOT and CFC assays* Assay Cohort 0 (n=13) Cohort 1 (n=5) Cohort 2 (n=5) Elispot 177.98 (29.37-610.48) 317.43 (76.12-780.8) 532.05 (207.74-667.39) CFC % CD4 3.37 (1.28-8.17) 3.8 (1.8-8.19) 4.26 (3.02-13.16) CFC % CD8 6.03 (1.33-13.47) 7.04 (2.81-9.63) 13.25 (5.0, 15.16) *AUCs were computed based on ELISPOT measurements denoted as spots/100,000 PBMC x weeks and based on % cytokine-secreting cells x weeks for the CFC assay. Table 2. Two-sided Wilcoxon Rank Sum P values for the paired comparisons of AUCs among the 3 cohorts. Assay Cohort 0 vs 1 Cohort 0 vs 2 Cohort 1 vs 2 Elispot 0.49 0.044 0.425 CFC % CD4 0.84 0.44 0.56 CFC % CD8 0.49 0.09 0.32

29




Figure Legends

Figure 1: CD4+CD25 highFoxP3+ Treg levels and proliferation of PBMC following

in vitro treatment with denileukin diftitox. PBMC from a normal donor were treated

with up to 8 nM denileukin diftitox or media alone (0 nM) for 18-20 hours and then

cultured in media containing 10 U/ml IL-2 for 3 days. (A) Cells were stained for CD4,

CD25 and Foxp3 expression on days 1 (after 18 hours of denileukin diftitox) and 4.

Percent CD4+CD25HighFoxp3+ cells are represented for each concentration. The

percentage of CD4+CD25HighFoxp3+ from freshly isolated PBMC prior to the addition of

denileukin diftitox is represented by a dashed line (1.82%). Data are representative of 3

repeated experiments. (B) PBMC similarly depleted of Treg with denileukin diftitox and

then rested in IL-2 for 3 days were stimulated in a proliferation assay with 0.5 mcg/ml

soluble OKT3 or media alone (unstimulated) for 4 days. Proliferation was determined by

3H-thymidine incorporation during a final 18 hours of culture and the data are represented

as mean cpm +/- SD. *(p< 0.01 compared with 0nM) Data are representative of 3

repeated experiments.

Figure 2: Expansion of PBMC pre-treated with denileukin diftitox by CMV and

MART-1 peptides. PBMC from a CMV+ donor were treated in vitro with denileukin

diftitox or media for 18h. (A) Cells were replated in 10 U/ml IL-2 for 3 days and then

cultured with CMV pp65 peptide and IL-2. Cells were analyzed by peptide-MHC

tetramer staining on days 7 and 10 of culture. Data is presented as the percent

CD8+CMV+ cells for untreated and denileukin diftitox pre-treated PBMC. (B) Cells

were replated in 10 U/ml IL-2 for 3 days and were then stimulated with MART-1 (26-35)

30




peptide and IL-2. Peptide-MHC tetramer staining was performed on day 8 of culture.

These cells were then re-stimulated with autologous PBMC pre-treated with denileukin

diftitox (or media) as described above, pulsed with MART-1 peptide and irradiated.

Cells were again analyzed by MART-1 specific tetramer 8 days after this second

stimulation. We present the percent CD8+MART-1+ cells for untreated and denileukin

diftitox pre-treated cells for both the first and second stimulation.

Figure 3: Changes in levels of Regulatory T cells following immunization and/or

administration of denileukin diftitox. PBMC pre and post vaccination for each cohort

were analyzed by flow cytometry for expression of CD4, CD25, and Foxp3. (A) Cohort

0: PBMC were collected pre and post TRICOM-CEA vaccination; (B) Cohort 1: PBMC

were collected pre TRICOM-CEA vaccination and denileukin diftitox administration

(LPA), pre vaccination and post denileukin diftitox (week 0), and weeks 1, 3, 6, 9, and

following the final vaccine dose; (C) Cohort 2: pre TRICOM-CEA vaccination and

denileukin diftitox (LPA), pre vaccination and post 1 dose of denileukin diftitox (week 0)

and weeks 1, 2, 3, 5, 6, 8, 9 and following the final vaccination. Arrows indicate time of

denileukin diftitox administration. The data is presented as the mean change in percent

of CD4+CD25+ cells that are > 90% Foxp3+ +/- SE for patients completing 4

vaccinations for each cohort (5 patients analyzed for each cohort). The data for each

cohort is presented as the mean percentage change in the percent CD4+CD25 highFoxp3+

cells from baseline (defined as the time of the leukapheresis (LPA), prior to any

denileukin diftitox) Arrows indicate time of administration of denileukin diftitox

31




Figure 4: Number of Regulatory T cells per μl blood following immunization and

administration of denileukin diftitox for Cohort 2. Whole blood was stained using BD

Bioscience TruCount tubes analyzed by flow cytometry to determine total number of

regulatory T cells per μl blood. The data is presented as the mean number of CD4+CD25

highFoxp3+ cells per ul blood +/- SE for 5 patients completing 4 vaccinations for cohort 2.

Analysis was performed prior to each vaccine injection and following the final

vaccination, and prior to each denileukin diftitox infusion.

Figure 5: Analysis of CEA-specific T cells by ELISpot assay for each Cohort.

PBMC collected prior to and following TRICOM-CEA vaccination were analyzed for

their response to rF-CEA(6D)-TRICOM by ELISpot. The data represents the mean

number of IFNγ producing cells per 100,000 PBMC +/- SE for patients completing 4

injections for each cohort. Arrows indicate time of denileukin diftitox administration.

Figure 6: Cytokine flow cytometry results at each time point. PBMC collected prior

to and following TRICOM-CEA vaccination were analyzed for intracellular production

of IFNγ in response to rF-CEA(6D)-TRICOM using flow cytometry. The data is

presented as the mean percentage of CD4+ or CD8+ CD69+ IFNγ + T cells for patients

completing 4 injections for each cohort. Arrows indicate time of denileukin diftitox

administration.

32




Figure 7: Fowlpox and CEA specific antibody induction for each cohort.

Patient sera for each cohort were tested by ELISA for antibodies to (A) fowlpox virus

and (B) CEA protein. The antibody titer for each patient tested is represented pre and

post vaccination by a star symbol.

33




0

20000

40000

60000

80000

100000

120000

140000

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unstimulatedOKT3 stimulated

Figure 1

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Concentration of Denileukin Diftitox

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Day 1Day 4

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A.

2.24% 3.37%

Day 7

Day 10

10.5% 24.45%

untreated Denileukin diftitox

CD8

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35




Figure 3

36




Figure 4

37




Figure 5

38




Figure 6

39




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40




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