Safety and Immunogenicity of Tyrosinase DNA Vaccines in Patients with Melanoma

original article

© The American Society of Gene Therapy

Safety and Immunogenicity of Tyrosinase DNA Vaccines in Patients with Melanoma Jedd D Wolchok1, Jianda Yuan1, Alan N Houghton1, Humilidad F Gallardo1, Teresa S Rasalan1, Jian Wang1, Yan Zhang1, Rajaram Ranganathan1, Paul B Chapman1, Susan E Krown1, Philip O Livingston1, Melanie Heywood1, Isabelle Riviere1, Katherine S Panageas1, Stephanie L Terzulli1 and Miguel A Perales1 Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

1

Immunity to self antigens on cancer is constrained by tolerance/ignorance. DNA vaccines encoding xenogeneic differentiation antigens, such as tyrosinase (TYR), mediate tumor protection and regression in implantable mouse models, and dogs with spontaneous melanoma. We conducted a trial of mouse and human TYR DNA vaccines in stage III/IV melanoma patients. Eighteen human leukocyte antigen (HLA)-A*0201+ melanoma patients were randomized as follows: one group received three mouse TYR DNA injections followed by three human TYR DNA injections; the other group received the same vaccines in opposite sequence. The study was conducted at three dose levels: 100, 500, and 1,500 µg DNA/injection, administered intramuscularly (IM) every 3 weeks. Most toxicities were grade 1 injection site reactions. Seven patients developed CD8+ T-cell responses, defined by a >3 SD increase in baseline reactivity to TYR peptide in tetramer or intracellular cytokine staining (ICS) assays. There was found to be no relationship between dose, assigned schedule, and T-cell response. At a median of 42 months follow-up, median survival has not been reached. Mouse and human TYR DNA vaccines were found safe and induced CD8+ T-cell responses in 7 of 18 patients. T cells recognizing a native TYR peptide had a phenotype consistent with that of effector memory cells. Received 4 May 2007; accepted 23 July 2007; published online 28 August 2007. doi:10.1038/sj.mt.6300290

Introduction Tyrosinase (TYR), a prototypical differentiation antigen expressed by cells of melanocytic origin, is recognized by CD8+ T cells from melanoma patients.1–3 Compared with other melanosomal membrane glycoproteins, TYR is expressed homogeneously by most melanoma specimens,4–6 and is a logical protein to target in melanoma vaccination strategies. Various methods for vaccination have been used to immunize against TYR and, in particular, measurable CD8+ T-cell responses have been induced against individual TYR epitopes using synthetic peptides.7,8 The full-length antigen is more advantageous than just

using individual peptides because it has potential to present multiple epitopes. DNA immunization was selected based on the ease of engineering a non-infectious vector, its relative efficiency, and low cost of manufacture as well as the presence of unmethylated CpG motifs in the vector backbone which stimulate the innate immune system through TLR9 ligation.9 We have shown in pre-clinical mouse models that immunization with DNA, (by encoding the xenogeneic orthologues of self antigens, including TYR family antigens), is an effective strategy for inducing cancer immunity, and for overcoming immunologic ignorance/tolerance which frequently constrains responses to self proteins. Efficacy is due at least in part to the presence of class I and II epitopes that have improved binding capacity to human major histocompatibility complex molecules and T-cell receptors as a consequence of divergence of polypeptide sequences in orthologues of different mammalian species.10 Injection of mice with plasmid DNA, encoding xenogeneic (human) TYR, induces antibody and CD8+ T-cell responses to TYR. This immune response is capable of protecting mice from a poorly immunogenic syngeneic tumor.11 In addition to TYR, our group has demonstrated the efficacy of this approach using other melanocytic antigens as well as in mouse models of breast cancer, prostate cancer, and lymphoma.10,12–16 We had previously conducted a series of clinical trials of xenogeneic TYR DNA vaccines in companion animals (pet dogs) with spontaneous melanoma at the Animal Medical Center of New York.17,18 These trials showed that injection of xenogeneic DNA resulted in antibody production that recognized recombinant human TYR and canine melanoma cells.19 These studies were performed in dogs with clinically evident metastases and/or with a very high risk of recurrence after surgery and radiotherapy. It is therefore important to note that we observed meaningful clinical responses in dogs with such metastases and a marked prolongation of survival in dogs vaccinated in the adjuvant setting compared with historical controls.18 Consequently, we conducted a trial in human melanoma patients.

Results Clinical trial design The goals of this study were to assess the safety and immuno­ genicity of two different immunization schedules with TYR DNA,

Correspondence: Jedd D. Wolchok, Assistant Attending Physician, Melanoma-Sarcoma Service, Associate Director, Ludwig Center for Cancer Immunotherapy, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Room Z-1462, New York, New York 10021, USA. E-mail: [email protected]

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DNA Vaccine Safety and Immunogenicity in Melanoma

in three different dose cohorts. Patients were randomized so as to receive either three mouse TYR DNA injections followed by three human TYR DNA injections, or the same vaccines in the opposite sequence. This crossover design was chosen based on pre-clinical observations in which the initial injection of the xenogeneic gene for priming, followed by the syngeneic gene as a boost, yielded ­better immune responses compared to the xenogeneic gene alone for all injections.16 Groups of six patients were randomized between the two schedules for each of three different dose levels: 100, 500, and 1,500 µg. DNA was administered intramuscularly (IM) with the Biojector2000 needle-free delivery system. This method was preferred since results obtained from DNA vaccine trials conducted for malaria, had shown that the IM jet injection produced superior CD8+ T-cell responses compared to the standard intradermal and IM needle methods.20,21

Patient demographics The trial enrolled mainly stage III melanoma patients who were free of disease after surgery but were still at high risk for recurrence (Table 1). Two patients had active stage IV disease at the time of enrollment (slowly progressive M1a and M1b) and one patient was classified under stage IV but with no evidence of disease. There was an equal gender distribution and the age range was 39–77 (median 59). All patients had Karnofsky performance status >80 with minimal or no laboratory abnormalities at the time of entry into the study. All eligible patients with stage III disease refused interferon (IFN) therapy, after a complete discussion of the data from the relevant Eastern Cooperative Oncology Group trials with a physician investigator. Stage III patients were enrolled within 1 year of definitive surgery for resection of primary tumor

and/or regional lymph nodes. Two of the stage III patients had previously received adjuvant therapy with temozolomide and thalidomide.

Toxicity No patient enrolled in this study developed a dose-limiting toxicity, defined as any event specified in the National Cancer Institute Common Toxicity Criteria (CTC v2) at grade 3 or 2 allergic/ immunologic toxicity (Table 2). Since patients were injected with double-stranded DNA, we measured anti-DNA antibodies during and after treatment. Similar to prior DNA vaccination trials,22 we did not detect any persistent elevation of anti-DNA antibodies. In accordance with the Food and Drug Administration guidelines for conducting gene transfer studies, all patients will be under surveillance for 15 years in case they present with any second malignancies, neurologic, or autoimmune disease. One patient in this study was diagnosed with non-small cell lung cancer within 1 year of vaccination; however, this was considered unlikely to be related to vaccination because the patient had a substantial history of tobacco use. Evaluation of CD8+ T-cell responses Peripheral blood mononuclear cells (PBMCs) were collected and stored at −120 °C at two time points before the first immunization (A, B), at the time of cross-over immunization (C), at 3 weeks (D) and at 8 weeks (E) following the last immunization. None of Table 2  Incidence and adverse events in the study Grade 1 Toxicity

Table 1  Patient demographics and T-cell responses Patient TYR 01

Stage Prior therapy III

Temozolomide + Thalidomide

Vaccine arm

T-cell response

Mouse

No

No. of Patients

Grade 2 %

No. of Patients

Hematologic and laboratory abnormalities occurring in ≥5% of patients

PFS OS (months) (months) 22

48

  Hypoalbuminemia

14

78

—

  Hyponatremia

2

11

—

  Hyperkalemia

2

11

—

67

—

TYR 03

III

None

Mouse

No

>60

>60

  Hyperglycemia

12

TYR 04

III

None

Human

Yes

>55

>55

  Bilirubin

4

22

—

TYR 05

III

None

Human

No

>50

>50

  Alkaline phosphatase

2

11

—

TYR 06

III

None

Mouse

Yes

>51

>51

  Hemoglobin (Hgb)

3

17

—

TYR 07

III

None

Human

No

>49

>49

6

1

III

None

Human

Yes

>48

>48

  SGOT (AST)

1

TYR 08 TYR 09

IV

Biochemotherapy Mouse

No

4

37

  SGPT (ALT)

2

11

—

TYR 10

III

None

Human

No

11

31

TYR 11

III

None

Human

Yes

17

22

TYR 12

III

None

Mouse

Yes

>44

>44

TYR 14

IV

Temozolomide + Thalidomide

Mouse

Yes

>57

>57

TYR 15

III

None

Human

No

8

20

TYR 16

III

None

Mouse

No

6

35

TYR 17

III

Temozolomide

Mouse

No

>39

  Injection site reaction   Nausea   Vomiting

14

78

—

1

6

—

—

1

  Diarrhea (patients with   colitis)

1

6

—

  Fatigue

1

6

—

>39

  Dizziness

1

6

—

1

6

—

TYR 18

IV

None

Human

Yes

8

>21

TYR 19

III

None

Human

No

>36

>36

  Edema

1

6

—

TYR 21

III

None

Mouse

No

>40

>40

  Rigors, chills

1

6

—

Abbreviations: OS, overall survival; PFS, progression free survival.

6

Non-hematologic toxicities occurring in ≥5% of patients

  Pruritus

Molecular Therapy vol. 15 no. 11 nov. 2007

%

6

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase.

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DNA Vaccine Safety and Immunogenicity in Melanoma

a

C

Pre-vac 0.03

0.1

0.09

103

103

103

103

2

2

10

10

2

102

101

101

101

101

0

0

0

0

10

TYR tetramer APC

E

D

0.13

101

0

102

103

101

0

0.04

102

103

101

0

0.15

102

103

0.33 103

103

102

102

102

102

101

101

101

101

0

0

0

0

102

103

101

0

102

103

101

0

102

103

0.1

103

101

101

0

103

0

TYR-04

102

103

TYR-18

101

0

102

103

CD8-ECD

b

Pre-vac

4

10

10

1.08

104

1.29

D

104

3.38

103

103

103

103

102

102

102

102

1

1

1

1

10 IFN-�-FITC

C

4

10

0

10

10

0

100

101

102

103

0.15

101

102

10 104 100

103

0.19

101

102

103

100 104 100

0.32

103

103

103

102

102

102

102

101

101

101

101

0 0

101

102 103

0 0

101

102 103

101

102

103

104

0.27

103

0

TYR-06

10

0

10 104 100

E 1.77

TYR-08

0 0

101

102 103

0

101

102 103

CD8-PE-Cy7

Figure 1 DNA vaccination induced tyrosinase (TYR)-specific CD8+ T cells assessed by tetramer binding and interferon-γ (IFN-γ) production. Peripheral blood mononuclear cells were collected pre-­vaccination, and at several time points after vaccination (C = week 10, D = week 21, and E = week 26) and analyzed by tetramer and intracellular cytokine staining (ICS) IFN-γ assays. (a) Two tetramer positive and (b) two ICS IFN-γ posi-­ tive patients are shown. APC, antigen presenting cell; FITC, fluorescein isothiocyanate; Pre-vac, pre-vaccination.

the samples from any of the patients demonstrated responses to the human leukocyte antigen (HLA)-A*0201-restricted TYR369–377 YMDGTMSQV peptide in an IFN-γ enzyme-linked immunosorbent spot assay on freshly thawed PBMCs without any prior in vitro stimulation. We then used a more sensitive assay, in which PBMCs are incubated with peptide-pulsed K562 cells expressing HLAA*0201 for 10 days to expand previously activated CD8+

T cells,23 prior to tetramer and intracellular cytokine staining (ICS) analysis. We defined a positive response as one in which: (i) the population of responding cells was >0.1% of total CD3+CD8+ cells, and (ii) the post-vaccination specimen was ≥3 SDs above the pre-vaccination specimens. Seven of eighteen patients demonstrated a positive response to TYR369–377 at one or more post-vaccination time points by either assay (Figure 1, Table 3). In the tetramer assay, the peak response was found to be 2.5-fold to 8.6-fold greater than the respective pre-treatment values, while the range in the ICS assay was a 2.1-fold to 4.5-fold increase. Positive responses were generally detected 3 weeks and up to 3 months after completion of DNA injections; however, 2046

one patient (TYR 04) had a positive response by tetramer assay during vaccination at the cross-over immunization. Positive responses were found for all three dose levels and showed no relationship to the use of xenogeneic or syngeneic DNA for initial priming injections. To confirm that these results were a consequence of vaccination and not due to assay variability, data from all pre-treatment specimens were analyzed to determine if they would qualify as a response when compared with the post-treatment values. In only one instance was a pre-treatment specimen scored as positive, supporting the validity of positive response to vaccination. In this patient, the difference between the post- and pre-treatment tetramer specimens was small (0.10 versus 0.15%) compared to those patients defined to be positive responders to vaccination (see Figure 1). Thus, the statistical significance of difference in this patient is likely to be due to the small SD of 0.01%. Interestingly, this patient had a very high risk for recurrence after resection of both nodal and soft tissue metastases and had been treated with adjuvant temozolomide and thalidomide for 6 months immediately prior to entry into this study. We cannot exclude the www.moleculartherapy.org vol. 15 no. 11 nov. 2007

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DNA Vaccine Safety and Immunogenicity in Melanoma

Phenotype of responding CD8+ T cells We assessed the phenotype of responding CD8+ T cells (Figures 2 and 3, Table 3). The specimens from two of three patients found to be positive by tetramer assay were CD45R0high CD62Lhigh, indicating a central memory phenotype.24 One patient who had Table 3  Immune responsesa Patient No.

Positive assay

Time

Phenotype

Clinical status

TYR 04

Tetramer

C, D, E

CD62Lhigh

48 months NED

TYR 06

Tetramer

E

CD62L

44 months NED

ICS

D

CD107aint

ICS

D

CD62Llow; CD45R0high; Granhigh; CD107aint; CD127low

41 months NED

TYR 08

low

TYR 11

ICS

D, E

CD62Llow; CD107aint; CD127low

DOD

TYR 12

ICS

D

CD62Llow; CD45R0high; Granlow; CD107aint; CD127int

42 months NED

TYR 14

ICS

D

CD62Llow; CD45R0high; Granhigh; CD107aint; CD127low

50 months NED

TYR 18

Tetramer

D

CD62Lhigh; CD45R0high; 26 months NED CD122low; CD127low

Abbreviations: DOD, died of disease; ICS, intracellular cytokine staining; NED, no evidence of disease. a Markers not indicated were not examined. Low = 0–30%, intermediate = 30–60%, and high = 60–100% of cells that are positive for above-mentioned markers.

86.4

0.45

CD8-ECD 0

0

13.1

0.05

Antibody responses No immunoglobulin G antibodies against TYR were detected in pre- or post-vaccination sera, analyzed using an enzymatic immunoprecipitation assay.11 To confirm this result, we transfected COS7 cells with human TYR containing a C-terminal truncation that eliminates the di-leucine lysosomal sorting signal, permitting cell surface expression.28 These cells, after staining with pre- and postvaccination sera and analyzed by flow cytometry, confirmed the absence of detectable immunoglobulin G anti-TYR antibodies. Clinical observations We have continued to follow patients for a median period of 42 months since enrollment (Figure 4). The Kaplan–Meier overall survival curve is typical of what might be expected from this patient population and we have not reached a median overall survival rate as of yet. It is interesting to note that six of seven patients

44.2

0.43

55.3

0.07

3.17

0.06

96.3

0.42

CD127-PE

0.48 CD45RO-PC7

99.5

CD62L-APC-Cy7

a

a positive response by tetramer staining with a predominantly CD62Llow population, consistent with an effector memory phenotype (TYR 06), was also the only patient who responded by both ICS and tetramer assays. The responses scored positive by ICS comprised T cells that were CD45R0high CD62Llow, indicative of an effector memory population. In three of these responders, we also detected expression of the effector molecule granzyme B in responding T cells (Figure 3). This was supported by moderate cell surface expression of CD107a in these specimens, consistent with degranulation and lytic function.25 Both IFN-γ-secreting and non-secreting cells expressed intracellular granzyme B, whereas only the IFN-γ-secreting cells expressed CD107a, consistent with a degranulation following in vitro stimulation with the cognate peptide. Expression of CD127 was generally low on tetramerpositive or IFN-γ-secreting cells, consistent with an effector phenotype.26,27

13.9

0.11

85.6

0.37

CD122-FITC

­ ossibility that the chemotherapy and thalidomide suppressed the p patient’s reactivity to the vaccine.

TYR tetramer APC

9.3

14

4.7

83.7

5.1

39.2

0.2

2.7

6.2

49.5

10.4

86.7

0

0

25.6

7

67.4

0.3

11.5

CD122-FITC

2.3 CD127-PE

79.1

CD122-FITC

99.5 CD62L-APC-Cy7

CD8-ECD

0.48

6.9

CD127-PE

b

CD62L-APC-Cy7

TYR tetramer APC

12.2

76

CD45RO-PC7

Figure 2 Tyrosinase (TYR) tetramer-reactive CD8+ cells in the responder population have an effector memory phenotype. Peripheral blood mononuclear cells were analyzed by tetramer assay after in vitro culture using TYR369–377 peptide YMDGTMSQV. (a) Dot plots from TYR-04 at time point D is shown. (b) Contour plots show CD3+CD8+ T cells analyzed for tetramer reactivity. Upper plots gated CD3+CD8+tetramer+ T cells; lower row plots gated on CD3+CD8+tetramer-T cells. APC, ­antigen presenting cells; FITC, fluorescein isothiocyanate.

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0

0.43

18.1

0.07

3

0.02

96.6

0.42

0.73

0.01

98.8

0.43

CD107a-PECy5

0

81.4

CD127-PE

0.46

CD62L-APC-Cy7

99.54

CD45RO-ECD

CD8-PE-Cy7

a

0.8

0.16

98.8

0.28

Granzyme B-AF647

© The American Society of Gene Therapy

DNA Vaccine Safety and Immunogenicity in Melanoma

90.4

0.45

9.08

0.05

2.3

4.16

93.1

94.2

21.7

0

78.3

0.04

0.92

4.55

94.5

0.46

IFN-�-FITC

0.1

1.6

4.5

93.8

Granzyme B-AF647

0.4

0

0

Granzyme B-AF647

95.6

5.8

CD107a-PECy5

0

0

CD107a-PECy5

4.4 CD127-PE

0

CD127-PE

99.54

CD62L-APC-Cy7

CD8-PE-Cy7

b

CD62L-APC-Cy7

IFN-�-FITC 0

95.3

0

4.4

2.73

87.1

1.87

8.28

CD45RO-ECD

Figure 3  Phenotypic characterization of cells secreting interferon-γ (IFN-γ) in intracellular cytokine staining (ICS) assays. ICS assays were performed with CD45RO, CD62L, CD127, CD107a, and granzyme B. (a) Representative dot plots from patient TYR-08 at time point D are shown. (b) Contour plots show the gated CD3+CD8+IFN-γ+ T cells. Upper plots gated on CD3+CD8+IFN-γ+ T cells; lower row plots gated on CD3+CD8+ IFN-γ -T cells. APC, antigen presenting cell; FITC, fluorescein isothiocyanate; TYR, tyrosinase.

Overall survival 1.0 0.9

Proportion surviving

0.8 0.7 0.6 0.5 0.4 N = 18 (6 DOD) Median survival: not reached Median follow–up for survivors: 42 months

0.3 0.2 0.1

0.0 Months 0 Number at Risk: 18

6

12 18

18

24 14

30

36

42

48

54

60

10

Figure 4 The Kaplan–Meier overall survival curve. Median survival time has not been reached with >42 months of median follow-up. DOD, died of disease.

with detectable T-cell responses remain alive, whereas only six of eleven of the immunologic non-responders are alive. It remains uncertain whether, non-responsiveness is merely indicative of a patient population with a poor prognosis, or the induced T-cell responses provided benefit.

Discussion We treated stage III and IV melanoma patients with two different sequences of xenogeneic mouse and human TYR DNA vaccines. We report induction of CD8+ T-cell responses in 7 of 18 patients by tetramer or ICS with 10 day in vitro stimulated cells, which is sufficient to expand previously activated T cells but should not substantially activate and expand naïve T cells. No T-cell responses were detected with overnight in vitro stimulation. Our results are 2048

similar to those of a study using prolonged intranodal infusion of similar doses of plasmid DNA encoding two major histocompatibility complex class I epitopes from TYR, in which immune responses were detected in 11 of 26 patients.29 It should be noted that we only examined responses to a single A0201 epitope, and may therefore be underestimating immune responses in patients recognizing other class I or class II epitopes. We have analyzed the sequences of mouse and human TYR for 9-mer peptides binding to HLA-A*0201 using a computerized algorithm.30 Interestingly, the peptide used for monitoring in this trial is highly conserved between species, as are most of the highest predicted binding peptides. One of the primary aims of this study was to investigate the safety and feasibility of injection of xenogeneic and syngeneic DNA vaccines, since this has not previously been established. We found that IM jet injection of all three doses of DNA was well tolerated, with transient grade 1 injection site reactions the only reproducibly reported side effect. No significant induction or exacerbation of anti-DNA antibodies was noted, even following 6 doses of DNA at 1.5 mg/dose. T-cell responses were detected in seven patients. The responding patients were divided between the two schedules of immunization: three responders received the human DNA followed by mouse and four received mouse DNA followed by human. One patient initially injected with human DNA had a positive response at the crossover, before receiving xenogeneic TYR DNA. The absence of T-cell responses at early time points was not unexpected, given the length of time that had been required to generate immune responses in other DNA vaccine trials in humans. In a human immunodeficiency virus DNA vaccine phase I clinical trial in healthy volunteers, approximately half of the immunologic responses were observed at approximately 1.5 months and the remaining responses were noted to require as long as 1 year to become apparent.31 The occurrence of a T-cell response after www.moleculartherapy.org vol. 15 no. 11 nov. 2007

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injection of only human TYR DNA could be a consequence of previous priming of T-cell precursors in the immune repertoire by the tumor, and subsequent expansion of this population by human TYR DNA injection. The absence of a response to syngeneic antigen in our mouse models would then be related to the immunization of animals without tumor. The lack of persistence in the T-cell responses detected has been noted in other melanoma vaccine trials using peptides and plasmid DNA. Reactivity to self antigens may be limited by several mechanisms: (i) inefficient antigen presentation, (ii) inhibition by molecules such as PD-1L and (iii) effects of regulatory T cells (Tregs). Five of seven patients responded by ICS assay, and three by tetramer assay (one of whom responded by both assays). Direct comparisons of these methods show that the tetramer assay is two to five times more sensitive than ICS, providing one explanation for T-cell responses by tetramer but not ICS assay.32 The oft-cited basis for this observation is that only a subset of intermediate or higher avidity CD8+ T cells with the cognate T-cell receptor for the target antigen is capable of responding by cytokine secretion. In addition, tetramer-positive T cells that recognize melanoma epitopes, which are functionally anergic, have been observed.33 This could provide another explanation for tetramer-positive, ICS-negative T-cell responses. The basis for ICS-positive, tetramer-negative T-cell responses are not as apparent. One possibility is that responding T cells downregulate T-cell receptors during the 10-day in vitro stimulation, leading to an inability to detect these T cells with tetramers. The ICS assay uses an additional overnight incubation with peptide-pulsed antigen presenting cells, which could induce IFN-γ responses in T cells, even those with low densities of T-cell receptors. The production of IFN-γ identifies T cells capable of effector functions. In this regard, T cells detected by ICS assays, but not tetramer staining, expressed granzyme B and CD107a which are involved with lytic function. A phenotypic characterization study of the responding cells was done. Patients responding to the ICS assay had reactive T cells with an effector memory phenotype (CD45R0high; CD62Llow), whereas, T cells of patients responding only to tetramer staining were typically CD62Lhigh. This probably indicates a central memory phenotype, because anergic T cells would not be expanded during the in vitro stimulation. Differences in phenotype that were observed may indicate that different subsets of CD8+ T cells are preferentially detected in each assay; however, larger studies are required to further delineate this. This trial was not designed or powered to demonstrate the clinical activity of TYR DNA vaccines and most patients had no evidence of disease at study entry. The Kaplan–Meier survival curve as a whole compares favorably with stage III and IV melanoma patients, including an analysis of 5,847 consecutive patients at Memorial Sloan-Kettering Cancer Center (MSKCC) between 1996 and 2004.34 Using the 2002 American Joint Committee on Cancer staging system and MSKCC Melanoma Database as benchmarks, stage III melanoma patients have a median survival time of 36 months; however in our trial, this median survival time has not been reached after >42 months of median followup. However, patient selection for clinical studies such as these can lead to marked skewing, and it is not possible to draw any conclusions. Molecular Therapy vol. 15 no. 11 nov. 2007

DNA Vaccine Safety and Immunogenicity in Melanoma

This is the first trial to compare immunization of melanoma patients with DNA vaccines encoding xenogeneic versus human antigens, and it is also the first study to demonstrate induction of T-cell responses to a self antigen using xenogeneic DNA. These results highlight the use of sensitive, standardized methods for detection of T-cell responses. We have shown that xenogeneic DNA vaccines can prime immune responses to self antigens. The next steps involve investigating optimized DNA vaccines, other methods of DNA delivery such as electroporation or gene gun,35 and novel adjuvants to enhance clinical activity.16,36,37

Materials and Methods Patients. Patients with American Joint Committee on Cancer stage IIb–IV

melanoma, HLA-A*0201+, and confirmed pathologic diagnosis were eligible to participate. Patients must have had any potentially curative surgery before being allowed entry. Patients with stages IIb–III disease must have either progressed on, been ineligible for or refused high dose IFN-α after a complete discussion of the results obtained from relevant Eastern Cooperative Oncology Group trials.38–40 Patients with stage IV disease had five or less anatomic sites of metastasis and no evidence of brain metastases. All patients signed an informed consent approved by the MSKCC Institutional Review Board. The protocol was approved by the National Institutes of Health Recombinant DNA Advisory Committee and the Food and Drug Administration. Vaccine design. Mouse and human TYR complementary DNAs were

cloned at MSKCC and inserted into the pING vector,11 a standard eukaryotic expression plasmid used extensively in pre-clinical models by our group,18 which conforms to criteria specified in the Food and Drug Administration points to consider for DNA vaccines. Vaccine manufacturing. Clinical grade material was manufactured in the

MSKCC Gene Transfer and Somatic Cell Engineering Facility using previously described methods.18

Immunization. Patients were injected IM every 3 weeks using the

Biojector2000 (Bioject, Tualatin, OR) in either the deltoid or gluteus muscles. Injection sites were rotated for each immunization and no injection was given to a site where draining lymph nodes had been removed.

T-cell stimulation in vitro. Thawed PBMC were stimulated with K562-

A*0201 cells pulsed with peptides (TYR369–377 YMDGTMSQV, EBVBMLF1280–288 GLCTLVAML)1,3 at 10 µg/ml as previously described.23 Immune assays. The following immune assays were performed as previ-

ously described with some modifications.7,8,23 For the enzyme-linked immunosorbent spot assay,7 freshly thawed PBMCs were added to the wells at a concentration of 5 × 105/50 μl culture medium. For tetramer and ICS,23 the following tetramers and fluorochrome-labeled antibodies were used: HLAA*0201-antigen presenting cell labeled tetramers loaded with Epstein-Barr virus and TYR peptides (Beckman Coulter, Fullerton, CA), CD45RO, CD3, and anti-IFN-γ (BD Biosciences, San Jose, CA), CD127, CD45RO, CD8, and Granzyme B (BD Pharmingen, San Jose, CA), CD3 (Caltag Laboratories, Burlingame, CA), CD122 (Sanquin, Amsterdam, NL), CD127 and CD8 (Beckman Coulter, Fullerton, CA), CD62L (eBioscience, San Diego, CA). Cells were analyzed by flow cytometry using a Cyan flow cytometer with Summit software (DakoCytomation California, Carpinteria, CA). The percentage of positive cells was determined by gating on the population of cells that were viable (forward scatterlow and side scatterlow), CD3high and CD8high. Serologic assays. Patients’ sera were analyzed using a previously described

enzymatic immunoprecipitation assay.11

Assay validation. For the enzyme-linked immunosorbent spot, tetramer and ICS assays, standard operating procedures were established in the

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DNA Vaccine Safety and Immunogenicity in Melanoma

Ludwig Center for Cancer Immunotherapy Immunologic Monitoring Facility for use in clinical trial monitoring. We have subjected these methods to validation criteria outlined by the Food and Drug Administration for cell based assays (http://www.fda.gov/CBER/summaries/120600bio10. htm). Statistical methods. In order to determine positive T-cell responses, we

calculated the SD of the pre-vaccination replicate values. Patients were considered to have positive T-cell responses if at any post-vaccination time point there was an increase of ≥3 SDs from baseline and the response was at least 0.1%. We also evaluated the number of patients that had a decrease of 3 SDs from baseline, as a means to determine the amount of extreme random fluctuation in T-cell measurements. Overall survival was estimated using the Kaplan–Meier method.

Acknowledgments This work was supported by Swim Across America, National Institutes of Health (NIH) grant CA33049, the Cancer Research Institute, the New York City Council Speakers Fund for Biomedical Research, and the Damon Runyon-Lilly Clinical Investigator Award (which has been awarded to J.D.W.). The NIH grant CA102606 was awarded to M.A.P. The NIH grants CA008748 and CA059350 were awarded to I.R.

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