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Keywords:

  • vaccine;
  • immunotherapy;
  • cytotoxic T lymphocyte (CTL);
  • tumor antigen;
  • poxvirus

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Recombinant plasmid DNA and attenuated poxviruses are under development as cancer and infectious disease vaccines. We present the results of a phase I clinical trial of recombinant plasmid DNA and modified vaccinia Ankara (MVA), both encoding 7 melanoma tumor antigen cytotoxic T lymphocyte (CTL) epitopes. HLA-A*0201-positive patients with surgically treated melanoma received either a “prime-boost” DNA/MVA or a homologous MVA-only regimen. Ex vivo tetramer analysis, performed at multiple time points, provided detailed kinetics of vaccine-driven CTL responses specific for the high-affinity melan-A26, 27, 28, 29, 30, 31, 32, 33, 34, 35 analogue epitope. Melan-A26-35-specific CTL were generated in 2/6 patients who received DNA/MVA (detectable only after the first MVA injection) and 4/7 patients who received MVA only. Ex vivo ELISPOT analysis and in vitro proliferation assays confirmed the effector function of these CTL. Responses were seen in smallpox-vaccinated as well as vaccinia-naïve patients, as defined by anti-vaccinia antibody responses demonstrated by ELISA assay. The observations that 1) CTL responses were generated to only 1 of the recombinant epitopes and 2) that the magnitude of these responses (0.029–0.19% CD8+ T cells) was below the levels usually seen in acute viral infections suggest that to ensure high numbers of CTL specific for multiple recombinant epitopes, a deeper understanding of the interplay between CTL responses specific for the viral vector and recombinant epitopes is required.

The characterization of tumor antigens recognized by cytotoxic T lymphocytes (CTL) has led to large numbers of human trials of vaccines designed to stimulate anti-tumor CTL responses. A variety of approaches have been employed, including peptides,1, 2, 3, 4, 5 whole proteins and dendritic cells,6, 7, 8 many of which have shown promising clinical and immunological responses. However, vaccine-driven CTL responses have not always correlated with clinical response. The expression of tumor antigen on tumor cells within an individual is known to be heterogeneous,9 and it has been proposed that this is a result of selection pressure by tumor antigen-specific CTL.10, 11 Vaccination strategies against multiple tumor antigens are therefore aimed at counteracting this tumor escape mechanism. Recently, recombinant gene technology has led to the development of viral and plasmid DNA vaccine delivery systems, and a recombinant “polyepitope” approach, involving a “string” of known tumor antigen epitopes inserted into a vector delivery system, allows multiple epitopes to be delivered simultaneously.

The Poxviridae family of viruses has proven to be particularly suited to the development of recombinant vaccines, and large numbers of animal studies have shown them to be efficient inducers of CTL responses specific for recombinant gene products.12 MVA, a highly attenuated vaccinia virus, incapable of replication in human and most other mammalian cells, has been shown to be capable of inducing strong CTL and antibody responses against recombinant antigens, leading to protective immunity in preclinical animal models.13, 14, 15, 16 Preclinical studies have also demonstrated that some of the most successful vaccination protocols are heterologous “prime-boost” regimens, involving sequential injections of different vectors encoding the same recombinant antigen.17 These regimens are designed to focus the CTL response on the recombinant antigen, which contains the only CTL epitopes shared by the different delivery vectors.18 A “prime-boost” combination of plasmid DNA and MVA has been shown to be particularly effective in animal models.15, 19, 20, 21 Although McConkey et al.5 have recently published results from human trials demonstrating effective generation of anti-malaria T cell responses using DNA/MVA “prime-boost” protocols, evidence of the immunogenicity of recombinant plasmid DNA in humans remains controversial.22, 23, 24

We have engineered 2 recombinant vaccine constructs: plasmid DNA (DNA.Mel3) and MVA (MVA.Mel3), both encoding the same polyepitope string (Mel3) of 7 HLA-A*0201- and HLA-A*01-restricted CTL epitopes from 5 well-defined melanoma tumor antigens (melan-A, NY-ESO-1, MAGE-1, MAGE-3 and tyrosinase),15 (Fig. 1). We have previously shown that each of the epitopes is properly processed and presented to epitope-specific CTL clones.15 We have also demonstrated in HLA-A2-transgenic mice that a heterologous “prime-boost” protocol of DNA.Mel3 and MVA.Mel3 generated up to 100-fold higher CTL expansions than sequential injections of the same vaccine vector.15

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Figure 1. The Mel3 polyepitope string.

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To assess the safety and immunogenicity of DNA.Mel3 and MVA.Mel3, we performed a phase I clinical trial in HLA-A*0201-positive patients with resected melanoma and a high risk of disease recurrence. We examined 2 vaccination regimens: a heterologous “prime-boost” DNA/MVA regimen and a homologous MVA-only regimen. We performed detailed immunomonitoring, including ex vivo tetramer and ELISPOT analyses and in vitro proliferation assays of CTL responses specific for the recombinant tumor antigen epitopes in the Mel3 polyepitope string. We also measured antibody responses generated against the MVA viral vector. Our results confirmed the safety of both vaccine constructs and demonstrated that recombinant MVA is capable of eliciting a CTL response specific for the high affinity melan-A26, 27, 28, 29, 30, 31, 32, 33, 34, 35 analogue peptide in 50% of the patients, even in the context of previous vaccination against smallpox. Importantly however, no vaccine-driven CTL responses specific for any of the other epitopes in the Mel3 polyepitope string were demonstrated. These results have important implications for future recombinant polyepitope vaccine design and demonstrate that alternative strategies need to be undertaken to generate broad CTL responses.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Investigational agents

DNA.Mel3 and MVA.Mel3 were manufactured by Qiagen GmbH/DSM Biologics and Impfstoffwerk Dessau-Tornau GmbH (IDT) (Dessau, Germany), respectively, according to cGMP guidelines. DNA.Mel3 was administered at a dose of 1,000 μg intramuscularly, and MVA.Mel3 was administered at 5 × 107 plaque forming units intradermally. The Mel3-encoded epitopes are: tyrosinase1-9, melan-A26–35 analogue (substitution Ala to Leu at residue 2725), tyrosinase369–377,26 MAGE-3168–176, MAGE-3271–279, MAGE-1161–169, NY-ESO-1155–167 and a murine H2-Db restricted influenza virus (flu) nucleoprotein epitope,27 which was included in the construct for preclinical analyses (see Palmowski et al.15 for full insert sequence) (Fig. 1). The tumor antigen epitopes are restricted by HLA-A*0201, except MAGE-3168–176 and MAGE-1161–169, which are HLA-A*01-restricted (Fig. 1).

Protocol

Treatment was carried out at the Cancer Research UK Medical Oncology Unit, Oxford, U.K. and The Christie Hospital, Manchester, U.K. The protocol was approved by the relevant Local Research Ethics Committees and was carried out according to the declaration of Helsinki. The protocol was also approved by the U.K. Gene Therapy Advisory Committee. All patients gave written, informed consent prior to study entry.

Patients were randomized to receive 2 injections of DNA.Mel3 followed by 2 injections of MVA.Mel3 (Arm A), or 4 injections of MVA.Mel3 (Arm B). Injections were administered every 2 weeks. Randomization was performed to avoid treatment group allocation bias, but the study was not designed to perform statistical comparisons between the 2 treatment groups.

Patients

Fourteen patients were enrolled (Table I). Inclusion criteria were the following: HLA-A*0201-positivity, adequate surgery for cutaneous melanoma, a 50% or greater chance of disease recurrence (primary tumor ≥ 4.0 mm thick or ≥ 2.5 mm with ulceration, stage III, or excised skin metastases) and no evidence of metastatic disease on computerized tomography of brain, chest, abdomen and pelvis performed within 4 weeks prior to study entry. Exclusion criteria were the following: surgery, radiotherapy or vaccination within 4 weeks; chemotherapy or immunotherapy within 1 year; immunodeficiency; autoimmune disease and egg allergy. Whilst on study, 3 patients (08, 10 and 11) developed skin nodules that were confirmed to be melanoma on excision after study completion. Patient 01 withdrew from the study early for reasons unrelated to the clinical trial and was not included for immunomonitoring.

Table I. Patient Characteristics and Randomization
Patient numberAgeSexTumor stage1Previous treatmentRandomization2Control peptide3
  • 1

    Tumor stage according to AJCC staging system.52

  • 2

    Randomization, Arm A:DNA/MVA, Arm B: MVA only.

  • 3

    Viral control peptide to which the patient had the greatest response on ex vivo tetramer staining, and used as a positive control in ELISPOT and recall assays: Influenza matrix58–66, EBV BMLF1 lytic protein280–288 or CMV pp65 lower matrix protein495–503.

  • 4

    Not vaccinated against smallpox.

  • 5

    HLA-A *01-positive.

  • 6

    Did not complete study and not included for immunomonitoring.

01657MIIISurgery, chemotherapyAN/A
0258FIIISurgeryACMV
0367MIIISurgery, chemotherapyBEBV
0441MIIISurgery, chemotherapyBInfluenza
0556FIIbSurgeryBEBV
0672MIIbSurgeryAEBV
0776MIIbSurgeryBCMV
0850FIIISurgery, radiotherapyACMV
09476FIIISurgeryBInfluenza
1063FIV (skin metastases)SurgeryBCMV
114556MIV (skin metastases)SurgeryACMV
12567FIIISurgeryACMV
1376MIIbSurgeryAEBV
1471FIIbSurgeryBCMV

Safety assessments

Safety was assessed by monitoring systemic and local toxicities, which were graded according to the National Cancer Institute Common Toxicity Criteria (NCI CTC) scale. Patients were observed closely for 3 hr after each injection. Two days after each injection an assessment of the local skin reaction was performed. Clinical haematology and biochemistry tests were performed on days 0, 2, 7, 14, 28, 42, 56 and 70.

Immunoassays

Sample Collection.

Blood samples were taken at 16 time points during the 70-day monitoring period. Peripheral Blood Mononuclear Cells (PBMC) were separated, cryopreserved in 10% DMSO and 40% FCS in RPMI and stored in liquid nitrogen. Serum samples were stored at −20°C. For each assay, PBMC or serum from the time points studied were thawed and analyzed concurrently to ensure consistent comparison across time points.

Reagents.

Peptides were synthesized by FMOC chemistry in house, or purchased (Sigma-Genosys, The Woodlands, TX, or Invitrogen, Carlsbad, CA). Tetramers were synthesized as previously described,28 and doses determined by titration against CTL clones and PBMC, including HLA-A*0201-negative PBMC as negative controls.29 The melan-A26–35 tetramer was synthesized using the analogue peptide. In every experiment, relevant CTL clones and HLA-A*0201-negative PBMC were included to validate tetramer staining. Anti-CD8α (PerCP) and anti-CD45R0 (APC) were from BD Biosciences (Mountain View, CA); anti-CD45RA (FITC), anti-CD27 (FITC) and anti-CD28 (APC) were from BD PharMingen, (San Diego, CA).

Ex vivo tetramer analysis.

Thawed PBMC were stained with PE-labeled tetramer and antibodies as described previously.29 Cells were kept on ice without fixation and analyzed on a Becton Dickinson FACScalibur® using CellQuest® software. Small lymphocytes were gated according to forward/side scatter, and dead cells were excluded on the basis of atypical fluorescence. Tetramer+ CD8+ cells were defined as CD8high with a PE-fluorescence equivalent to that of a tetramer-stained clone. A detection threshold for a tetramer+ cell population in PBMC was defined as 100 tetramer+ cells/106 CD8+ cells (0.01% CD8+).4 We defined a positive response to the vaccine as an expansion in the number of melan-A26–35 tetramer+ cells either to > 0.02% CD8+ on 2 consecutive time points if the baseline value was <0.01% CD8+, or to > 3× the baseline value on 2 consecutive time points if it was above 0.01% CD8+ cells. Where replicate analyses were performed, results are given as the arithmetic mean, and the error bars signify ± 1 SD. For phenotypic analysis, profiles of tetramer+ CD8+ cells were compared to whole PBMC for that patient.

Recall assay.

The recall assay induces proliferation of peptide-specific CD8+ cells in PBMC samples to increase the frequency of cells detectable with tetramers and validate ex vivo results. PBMC were incubated at 1 × 105 cells/well of a 96-well round-bottomed plate, in 200 μl Iscove's Modified Dulbecco's Medium (IMDM) with 20 μM peptide, for 1 hr. For analysis of melan-A26, 27, 28, 29, 30, 31, 32, 33, 34, 35-specific CTL responses the analogue peptide was used. After washing, 4 × 105 unpulsed PBMC were added to each well in 250 μl IMDM with 5% human serum (I5) and 100 U/ml IL-2 (Chiron, Emeryville, CA). After 7-day culture, cells were stained with appropriate tetramer and PerCP anti-CD8α. Every assay testing recall responses to tumor epitopes also included a viral control peptide recognized by each patient (Table I), which was selected from Influenza (flu) matrix protein58–66, Epstein Barr virus (EBV) BMLF1 lytic protein280–288, and cytomegalovirus (CMV) pp65 lower matrix protein495–503, according to the largest peptide-specific CTL population observed on tetramer staining of a PBMC sample for each patient.

Enzyme-linked immunospot (ELISPOT) assay.

ELISPOT analysis of Interferon-gamma (IFN-γ) secretion (Mabtech, Stockholm, Sweden) was performed as previously described.29 Thawed PBMC were rested overnight, plated in duplicates of 5 × 105 cells/well in I5 with or without 10 μM peptide, and incubated for 36 hr. For analysis of melan-A26–35-specific CTL responses the analogue peptide was used. Each assay included a viral control peptide as in the recall assay. Spots were counted using an automated AID ELISPOT reader (Autoimmun-Diagnostika, Strassberg, Germany). Results were calculated by subtracting the mean background spot count from the mean spot count for each peptide and adjusted to % of total CD8+ cells (determined by flow cytometry). Assays were excluded if counts were > 25 spots per well in the absence of peptide, and a detection threshold was defined as 10 spots per well above no-peptide controls (equivalent to 0.01% CD8+ cells).30

Anti-MVA IgG ELISA.

Serum ELISA for anti-viral IgG antibodies was performed as previously described,31 using 96-well Maxisorp ELISA plates (Nunc, Denmark) coated with 50 μl per well of MVA.Mel3 at 10 μg protein/ml buffer.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Immune responses to the recombinant Mel3 epitopes

Expansion of melan-A26–35-specific CTL.

We and others have shown that HLA-A*0201-positive individuals have a higher frequency of melan-A26–35-specific CTL precursors than for any of the other Mel3 epitopes.29, 30, 32 Therefore, we first analyzed the frequency of melan-A26–35-specific CTL by ex vivo tetramer analysis of PBMC from all 16 study time points for each patient (Fig. 2). This analysis was performed in duplicate (Fig. 2). According to the stringent criteria we set for a positive vaccine-driven CTL response determined by ex vivo tetramer analysis (outlined in the Material and Methods), 6/13 patients developed a melan- A26–35-specific CTL response over the course of the study. Two patients (02 and 08) had received DNA/MVA, and 4 (04, 07, 09 and 10) patients had received MVA only (Fig. 2). The range of peak responses was 0.029–0.19% CD8+ cells.

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Figure 2. Kinetics of vaccine-driven melan-A26–35-specific CTL responses. For each patient, a time course of melan-A26–35-specific CTL response determined by ex vivo tetramer analysis is presented. Melan-A26-35-specific response is shown as tetramer+ cells/106 CD8+ cells. Values are the arithmetic mean of replicate analyses, and error bars signify ± 1 SD. *Responding patient, ▵Patient 02: different scale.

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The large number of immunomonitoring time points in this clinical trial enabled the kinetics of vaccine-driven epitope-specific CTL responses to be visualized in unprecedented detail for a clinical trial (Fig. 2). Expansions in the number of melan-A26–35-specific CTL were seen after 1 or 2 MVA.Mel3 injections. All of the responding patients who received 4 injections of MVA.Mel3 (04, 07, 09 and 10) demonstrated a plateau in the expansion of melan-A26–35-specific CTL after 2 or 3 MVA.Mel3 injections. Responses in both of the responding patients who received the DNA/MVA “prime-boost” regimen (02 and 08), melan-A26–35-specific CTL were detectable ex vivo only after the first MVA.Mel3 injection.

Expanded melan-A26–35-specific CTL have effector phenotype and function.

Phenotypic analysis of melan-A26–35-specific CTL in the prevaccination samples confirmed our previous results that showed that early stage melanoma patients, as well as normal healthy individuals,29, 30 have melan-A26–35-specific CTL with functional and phenotypic markers of a naïve population. In prevaccination samples, tetramer+ melan-A26–35-specific CTL were predominantly CD45RA+/CD45R0, consistent with a naïve phenotype (Fig. 3a). In contrast, vaccine-driven expanded populations of tetramer+ melan-A26–35-specific CTL also included cells with antigen-experienced CD45RA/CD45R0+ and CD45RA+/CD45R0+ phenotypes (Fig. 3a).

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Figure 3. Vaccine-driven melan-A26–35-specific CTL have effector phenotype and function. Melan-A26–35-specific responses are presented for 4 immunomonitoring time points in patients 02 and 04. (a) Ex vivo melan-A26–35 tetramer analysis. Numbers refer to melan-A26–35 tetramer+/CD8high cells (gated as shown) as a percentage of total CD8+ cells and are representative of replicate analysis. For each time point, CD45RA/CD45R0 profiles of melan-A26–35-specific cells, gated in above plots, are also shown. (b) ELISPOT analysis of PBMC from the same time points as above. Representative wells of PBMC incubated with no peptide and melan-A26–35 peptide are shown. Numbers indicate melan-A26–35-specific cells as percentage of total CD8+ cells, when above the detection threshold of 0.01%, and are representative of replicate analyses with background subtracted.

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Ex vivo ELISPOT analysis confirmed that a proportion of vaccine-expanded melan-A26–35-specific CTL were capable of secreting IFN-γ. No responses were detected in prevaccination samples of any of the patients. Responses were demonstrated in postvaccination samples of 3 patients. In Arm A (DNA/MVA), patient 02 showed a response on ELISPOT of 0.08% CD8+ at day 56 and 0.061% CD8+ at day 70 (Fig. 3b). Patient 08 was excluded from analysis because of persistently high background levels. In Arm B (MVA only), the 2 patients with the highest melan-A26–35-specific responses on tetramer analysis also demonstrated a positive response to melan-A26–35 on ELISPOT: patient 04 with 0.021% CD8+ at day 42 (Fig. 3b), and patient 10 with 0.027% CD8+ at day 42 (data not shown). The differences in the magnitude of epitope-specific CTL responses detected on ex vivo ELISPOT and tetramer analyses (approximately 3 times higher with tetramers) are consistent with the previous experience of ourselves and others.4, 29, 30, 33, 34

Recall in vitro proliferation assays confirmed that, for every patient in whom a vaccine-driven expansion of melan-A26–35-specific CTL was detectable on ex vivo tetramer analysis, the expanded CTL were capable of rapidly proliferating in response to melan-A26–35 peptide (Fig. 4 and data not shown). To further address the question of priming by DNA.Mel3, we performed recall assays for the melan-A26–35 peptide at day 28 (after 2 DNA.Mel3 injections, prior to MVA.Mel3) for the 2 patients (02 and 08) in the DNA/MVA Arm who both demonstrated an ex vivo melan-A26–35-specific CTL responses on tetramer analysis after receiving MVA.Mel3. Although ex vivo tetramer analysis of PBMC from time points after DNA.Mel3 and prior to MVA.Mel3 showed no evidence of melan-A26–35-specific CTL, recall assays were performed to exclude the presence of melan-A26–35-specific cells below the limit of detection. For patient 02, there was no difference between prevaccination and day 28 recall responses (Fig. 4), providing no evidence of DNA.Mel3 priming. For patient 08, we observed a prevaccination recall response of 0.04% CD8+ cells after averaging triplicate assays, and the results of triplicate assays performed at day 28 were 0.03%, 0.04% and 1.4% CD8+ cells (data not shown). While these results raise the possibility of priming by DNA.Mel3, they also illustrate the inherent variability associated with in vitro proliferation assays and provide reason for caution when using these assays as the sole readout for immunotherapy trials. This is particularly relevant for epitopes such as melan-A26–35 for which even healthy donors have an atypically elevated baseline level, and for which in vitro priming cannot be excluded.32

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Figure 4. Melan-A26–35-specific recall response. Melan-A26–35-specific responses are presented for 4 immunomonitoring time points for patients 02 (DNA/MVA). Tetramer analysis is shown both ex vivo, and after a 1-week in vitro peptide restimulation (recall response). Numbers indicate melan-A26–35-specific cells as percentage of total CD8+ cells. No recall response is seen pre-vaccination (day 0) or after 2 injections of DNA.Mel3 prior to the first MVA.Mel3 injection (day 28). In contrast, strong recall responses are seen at day 56 and 70. The fold expansion after 1-week in vitro restimulation is shown.

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Indirect evidence of effector function in vaccine-driven CTL was provided by clinical findings in patient 02 (DNA/MVA), who demonstrated the highest peak of melan-A26–35-specific CTL (0.19% CD8+ T cells). At the same time point that the expansion of melan- A26–35-specific CTL first became detectable by ex vivo tetramer analysis (day 35, 7 days after the first MVA.Mel3 injection), this patient developed redness, itching and crusting of 3 melanocytic naevi, as well as a diffuse macular rash, most prominent around the neck and shoulders. Excision biopsies of 2 of the naevi were performed on day 42. In both cases, histology revealed a lymphocytic infiltrate, consisting of both CD8+ and CD4+ lymphocytes. The rash resolved spontaneously over the following 6 weeks. No vitiligo developed.

Lack of CTL responses to other Mel3 recombinant epitopes.

We screened for the presence of responses to all of the other epitopes in the Mel3 polyepitope string using recall assays and ex vivo ELISPOT analysis (including ELISPOT for HLA-A*01-restricted epitopes in patients 11 and 12). In simultaneous assays, we compared PBMC samples taken prevaccination, postvaccination and at time points at which a peak ex vivo melan-A26–35-specific response was seen on tetramer analysis. Whilst we always detected the presence of CD8+ T cells specific for viral control peptides, neither assay provided any evidence of the presence of CTL specific for the Mel3 epitopes other than melan-A26–35, in any of the samples tested, with only the following exception.

For patient 12 (DNA/MVA), in whom there was no vaccine-driven melan-A26–35-specific CTL response, we demonstrated NY-ESO-1155–167-specific CTL on ex vivo tetramer and ELISPOT analysis, and recall assay (data not shown). There was no difference in any of the assays between pre- and post-vaccination PBMC samples. Ex vivo tetramer analysis performed at each immunomonitoring time point revealed NY-ESO-1155–167-specific CD8+ cells, that were predominantly CD45RA/CD45R0+, at a frequency of 0.13–0.15% CD8+ across all time points (data not shown).

Anti-viral immune responses

Analysis of serum anti-MVA IgG antibodies demonstrated that all patients developed an increase in the serum titer over the course of the study. Two patterns of response were seen (Fig. 5). Eleven patients developed a rapid increase in the serum titer within 2 weeks of receiving the first dose of MVA.Mel3. The other 2 patients (09 and 11), who reported no previous history of vaccination against smallpox, had prevaccination titers no higher than the negative controls and did not develop an increase in the serum titer until 4 weeks after receiving the first dose of MVA.Mel3. These results are consistent with previously described kinetics of antibody responses, determined by ELISA, in vaccinia-naïve and -nonnaïve volunteers vaccinated with the New York City Board of Health strain of vaccinia.35 Recently, anti-poxviral antibodies determined by ELISA have been shown to correlate closely with the presence of neutralizing antibodies.36

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Figure 5. MVA.Mel3 induces anti-MVA IgG response. For 2 representative patients who received the MVA-only protocol, serum anti-MVA.Mel3 IgG responses are shown for serum samples taken at 2-weekly intervals during the study. Patient 04 gave a history of childhood smallpox vaccination, whereas patient 09 did not.

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Safety

During the clinical trial, a total of 14 DNA.Mel3 and 42 MVA.Mel3 injections were administered. No local reactions developed after any of the DNA.Mel3 injections. All patients developed a mild (NCI CTC Grade 1) local skin reaction 1–3 cm in diameter after each MVA.Mel 3 injection. These reactions resolved within 7 days. Several patients reported mild (NCI CTC Grade 1) flu-like symptoms, characteristically occurring within 24 hr after MVA.Mel3 injections and resolving within the following 24–48 hr. With the exception of the skin reaction in patient 02, described above, no adverse events considered to be likely due to the investigational agents occurred. None of the patients developed abnormalities in complete blood picture, electrolytes or liver function tests.

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Our study is the first tumor immunotherapy clinical trial to demonstrate conclusively that recombinant MVA is capable of priming a functional and sustained CTL response specific for a recombinant tumor antigen epitope. Vaccine-driven expansions of CTL specific for the melan-A26–35 epitope were demonstrated in approximately 50% of patients (Fig. 2), and functional analyses demonstrated that these cells were effector cells, as defined by their ability to secrete IFN-γ on ex vivo ELISPOT analysis (Fig. 3b) and to proliferate in vitro in recall assays in the absence of professional APC (Fig. 4).

Although our preclinical studies confirmed that each of the recombinant epitopes in the Mel3 polyepitope string is properly processed and presented to epitope-specific CTL clones,15 no expansion of CTL specific for any of the epitopes other than melan-A26–35 were seen. These results are consistent with our pre-clinical studies of the Mel3 polyepitope string in HLA-A2 transgenic mice, in which we also demonstrated dominance of the melan-A26–35 epitope.15 There are several factors which may have contributed to the dominance of the melan-A26–35 analogue epitope over the other Mel3 recombinant epitopes. Firstly, unlike the other Mel3-encoded epitopes, the melan-A26–35 analogue has a very high peptide binding affinity to HLA-A*0201 molecules.25 Second, healthy individuals as well as melanoma patients have atypically elevated frequencies of melan-A26–35-specific CTL in their peripheral blood.29, 30 We and others have shown that competition between CTL occurs at the surface of the APC and that the simultaneous presentation of different epitopes to a skewed repertoire of primed CTL leads to dominant expansion of a CTL of a limited range of specificities.15, 37, 38 These findings are relevant to the future design of recombinant polyepitope vaccines. To successfully generate CTL responses of multiple specificities, we suggest that epitopes should be separated in the boosting phase, thereby minimizing CTL competition.

Owing to the prevalence of childhood smallpox vaccination, concern has been raised about the possible detrimental effects of preexisting anti-poxviral immune responses on the generation of immune responses against recombinant gene products. Several studies have shown that preexisting immunity against the viral delivery vector impacts negatively on immune responses specific for recombinant gene products.15, 39, 40, 41 Our results showed that preexisting vaccinia-specific immunity, as defined by clinical history and pattern of antibody response, did not prevent a response specific for the recombinant melan-A26–35 epitope: 5 of the 6 melan-A26–35-responding patients, including patient 02 who demonstrated the greatest response, had been vaccinated against smallpox. Furthermore, the 2 vaccinia-naïve patients (patients 09 and 11) did not develop better responses than the smallpox-vaccinated patients.

The detailed kinetics of CTL responses generated in this clinical trial demonstrated that repeated vaccinations with MVA.Mel3 resulted in a plateau of melan-A26–35-specific CTL responses after 2 or 3 injections. This result is consistent with the findings of other groups who have used homologous recombinant viral vaccination strategies.16, 42 Neither this study nor others employing recombinant poxviral vectors have achieved epitope-specific CTL, of the levels that are associated with acute viral infections.43, 44, 45. The likely mechanism for these 2 observations is the generation of poxviral-specific antibody and/or cellular immune responses during the vaccination schedule, either by neutralization of infection or by the generation of immunodominant CTL specific for epitopes encoded within the viral vector itself.46 Therefore, poxviral delivery vectors should best utilized in “prime-boost” vaccination protocols, and consideration should be made to using “smaller” delivery vectors, such as lentivirus, to minimize the impact of vector-specific immune responses. Alternatively, the use of proteins or peptides in combination with adjuvants should be considered: a recent clinical study of repeated injections of peptide and adjuvant has demonstrated continued boosting of CTL responses, reaching levels as high as 8% of CD8+ cells.47, 48

Many preclinical studies have confirmed the ability of recombinant DNA vaccines to prime both humoral and cellular immune responses specific for recombinant gene products in mice and primates.49 Indeed, we confirmed that DNA.Mel3 primes responses specific for the melan-A26–35, tyrosinase369–377 and NY-ESO-1155–167 epitopes in HLA-A2 transgenic mice.15 In contrast, evidence of immunogenicity of plasmid DNA in humans is much more limited.5, 22, 23, 24 Here, we have generated no direct evidence that 2 injections of 1,000 μg DNA.Mel3 are capable of priming an immune response against the recombinant gene products, although the possibility that DNA.Mel3-primed CTL are below the detection limits of the ex vivo assays used in our study, or not recirculating and mainly localized in draining lymph nodes cannot be excluded. Possible contributing factors to the apparent interspecies differences in the immunogenicity of recombinant plasmid DNA are emerging in the literature. They include differences between immune systems, such as the differential expression of Toll-like receptor 9, which recognizes CpG repeat motifs in bacterial DNA.50, 51 Therefore, in addition to addressing such issues as dose, route and method of administration, future clinical trials of DNA vaccination strategies should take these factors into consideration.

In conclusion, our clinical trial demonstrates that recombinant MVA is capable of generating a functionally activated CTL response specific for recombinant gene products. Although recombinant viruses are powerful vaccine delivery vectors, our results highlight several issues relevant to recombinant vaccination protocols using polyepitope strategies and poxviral delivery vectors, which should be considered in future clinical trials.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank the Research Nurses and Registrars involved in this trial at the Cancer Research UK Medical Oncology Units in Oxford and Manchester, especially Dr. D. Eaton and D. Selman, and P. Bahl for her technical support. We are grateful to Dr. L.J. Old for his support and critical reading of the article.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL, Restifo NP, Dudley ME, Schwarz SL, Spiess PJ, Wunderlich JR, Parkhurst MR, et al. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med 1998; 4: 3217.
  • 2
    Marchand M, van Baren N, Weynants P, Brichard V, Dreno B, Tessier MH, Rankin E, Parmiani G, Arienti F, Humblet Y, Bourlond A, Vanwijck R, et al. Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int J Cancer 1999; 80: 21930.
  • 3
    Jager E, Gnjatic S, Nagata Y, Stockert E, Jager D, Karbach J, Neumann A, Rieckenberg J, Chen YT, Ritter G, Hoffman E, Arand M, et al. Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1+ cancers. Proc Natl Acad Sci U S A 2000; 97: 12198203.
  • 4
    Speiser DE, Lienard D, Pittet MJ, Batard P, Rimoldi D, Guillaume P, Cerottini JC, Romero P. In vivo activation of melanoma-specific CD8(+) T cells by endogenous tumor antigen and peptide vaccines: a comparison to virus-specific T cells. Eur J Immunol 2002; 32: 73141.
  • 5
    McConkey SJ, Reece WH, Moorthy VS, Webster D, Dunachie S, Butcher G, Vuola JM, Blanchard TJ, Gothard P, Watkins K, Hannan CM, Everaere S, et al. Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat Med 2003; 9: 72935.
  • 6
    Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, Burg G, Schadendorf D. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 1998; 4: 32832.
  • 7
    Schuler-Thurner B, Dieckmann D, Keikavoussi P, Bender A, Maczek C, Jonuleit H, Roder C, Haendle I, Leisgang W, Dunbar R, Cerundolo V, von Den Driesch P, et al. Mage-3 and influenza-matrix peptide-specific cytotoxic T cells are inducible in terminal stage HLA-A2.1+ melanoma patients by mature monocyte-derived dendritic cells. J Immunol 2000; 165: 34926.
  • 8
    Banchereau J, Palucka AK, Dhodapkar M, Burkeholder S, Taquet N, Rolland A, Taquet S, Coquery S, Wittkowski KM, Bhardwaj N, Pineiro L, Steinman R, et al. Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor-derived dendritic cell vaccine. Cancer Res 2001; 61: 64518.
  • 9
    Ohnmacht GA, Marincola FM. Heterogeneity in expression of human leukocyte antigens and melanoma-associated antigens in advanced melanoma. J Cell Physiol 2000; 182: 3328.
  • 10
    Lehmann F, Marchand M, Hainaut P, Pouillart P, Sastre X, Ikeda H, Boon T, Coulie PG. Differences in the antigens recognized by cytolytic T cells on two successive metastases of a melanoma patient are consistent with immune selection. Eur J Immunol 1995; 25: 3407.
  • 11
    Anichini A, Molla A, Mortarini R, Tragni G, Bersani I, Di Nicola M, Gianni AM, Pilotti S, Dunbar R, Cerundolo V, Parmiani G. An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions. J Exp Med 1999; 190: 65167.
  • 12
    Zavala F, Rodrigues M, Rodriguez D, Rodriguez JR, Nussenzweig RS, Esteban M. A striking property of recombinant poxviruses: efficient inducers of in vivo expansion of primed CD8(+) T cells. Virology 2001; 280: 1559.
  • 13
    Sutter G, Moss B. Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc Natl Acad Sci U S A 1992; 89: 1084751.
  • 14
    Hirsch VM, Fuerst TR, Sutter G, Carroll MW, Yang LC, Goldstein S, Piatak M Jr, Elkins WR, Alvord WG, Montefiori DC, Moss B, Lifson JD. Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara. J Virol 1996; 70: 374152.
  • 15
    Palmowski MJ, Choi EM, Hermans IF, Gilbert SC, Chen JL, Gileadi U, Salio M, Van Pel A, Man S, Bonin E, Liljestrom P, Dunbar PR, et al. Competition between CTL narrows the immune response induced by prime-boost vaccination protocols. J Immunol 2002; 168: 43918.
  • 16
    Amara RR, Villinger F, Staprans SI, Altman JD, Montefiori DC, Kozyr NL, Xu Y, Wyatt LS, Earl PL, Herndon JG, McClure HM, Moss B, et al. Different patterns of immune responses but similar control of a simian-human immunodeficiency virus 89.6P mucosal challenge by modified vaccinia virus Ankara (MVA) and DNA/MVA vaccines. J Virol 2002; 76: 762531.
  • 17
    Li S, Rodrigues M, Rodriguez D, Rodriguez JR, Esteban M, Palese P, Nussenzweig RS, Zavala F. Priming with recombinant influenza virus followed by administration of recombinant vaccinia virus induces CD8+ T-cell-mediated protective immunity against malaria. Proc Natl Acad Sci U S A 1993; 90: 52148.
  • 18
    Ramshaw IA, Ramsay AJ. The prime-boost strategy: exciting prospects for improved vaccination. Immunol Today 2000; 21: 1635.
  • 19
    Schneider J, Gilbert SC, Blanchard TJ, Hanke T, Robson KJ, Hannan CM, Becker M, Sinden R, Smith GL, Hill AV. Enhanced immunogenicity for CD8+ T cell induction and complete protective efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara. Nat Med 1998; 4: 397402.
  • 20
    Hanke T, Neumann VC, Blanchard TJ, Sweeney P, Hill AV, Smith GL, McMichael A. Effective induction of HIV-specific CTL by multi-epitope using gene gun in a combined vaccination regime. Vaccine 1999; 17: 589-96.
  • 21
    Amara RR, Villinger F, Altman JD, Lydy SL, O'Neil SP, Staprans SI, Montefiori DC, Xu Y, Herndon JG, Wyatt LS, Candido MA, Kozyr NL, et al. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 2001; 292: 6974.
  • 22
    Conry RM, Curiel DT, Strong TV, Moore SE, Allen KO, Barlow DL, Shaw DR, LoBuglio AF. Safety and immunogenicity of a DNA vaccine encoding carcinoembryonic antigen and hepatitis B surface antigen in colorectal carcinoma patients. Clin Cancer Res 2002; 8: 27827.
  • 23
    Yu Z, Restifo NP. Cancer vaccines: progress reveals new complexities. J Clin Invest 2002; 110: 28994.
  • 24
    Epstein JE, Gorak EJ, Charoenvit Y, Wang R, Freydberg N, Osinowo O, Richie TL, Stoltz EL, Trespalacios F, Nerges J, Ng J, Fallarme-Majam V, et al. Safety, tolerability, and lack of antibody responses after administration of a PfCSP DNA malaria vaccine via needle or needle-free jet injection, and comparison of intramuscular and combination intramuscular/intradermal routes. Hum Gene Ther 2002; 13: 155160.
  • 25
    Valmori D, Fonteneau JF, Lizana CM, Gervois N, Lienard D, Rimoldi D, Jongeneel V, Jotereau F, Cerottini JC, Romero P. Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues. J Immunol 1998; 160: 17508.
  • 26
    Skipper JC, Hendrickson RC, Gulden PH, Brichard V, Van Pel A, Chen Y, Shabanowitz J, Wolfel T, Slingluff CL Jr, Boon T, Hunt DF, Engelhard VH. An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J Exp Med 1996; 183: 52734.
  • 27
    Townsend AR, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 1986; 44: 95968.
  • 28
    Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996; 274: 946.
  • 29
    Dunbar PR, Smith CL, Chao D, Salio M, Shepherd D, Mirza F, Lipp M, Lanzavecchia A, Sallusto F, Evans A, Russell-Jones R, Harris AL, et al. A shift in the phenotype of melan-A-specific CTL identifies melanoma patients with an active tumor-specific immune response. J Immunol 2000; 165: 664452.
  • 30
    Pittet MJ, Valmori D, Dunbar PR, Speiser DE, Lienard D, Lejeune F, Fleischhauer K, Cerundolo V, Cerottini JC, Romero P. High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals. J Exp Med 1999; 190: 70515.
  • 31
    Ramirez JC, Gherardi MM, Rodriguez D, Esteban M. Attenuated modified vaccinia virus Ankara can be used as an immunizing agent under conditions of preexisting immunity to the vector. J Virol 2000; 74: 76515.
  • 32
    Salio M, Shepherd D, Dunbar PR, Palmowski M, Murphy K, Wu L, Cerundolo V. Mature dendritic cells prime functionally superior melan-A-specific CD8+ lymphocytes as compared with nonprofessional APC. J Immunol 2001; 167: 118897.
  • 33
    Tan LC, Gudgeon N, Annels NE, Hansasuta P, O'Callaghan CA, Rowland-Jones S, McMichael AJ, Rickinson AB, Callan MF. A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers. J Immunol 1999; 162: 182735.
  • 34
    Kuzushima K, Hoshino Y, Fujii K, Yokoyama N, Fujita M, Kiyono T, Kimura H, Morishima T, Morishima Y, Tsurumi T. Rapid determination of Epstein-Barr virus-specific CD8(+) T-cell frequencies by flow cytometry. Blood 1999; 94: 3094100.
  • 35
    Frey SE, Newman FK, Yan L, Lottenbach KR, Belshe RB. Response to smallpox vaccine in persons immunized in the distant past. Jama 2003; 289: 32959.
  • 36
    Hammarlund E, Lewis MW, Hansen SG, Strelow LI, Nelson JA, Sexton GJ, Hanifin JM, Slifka MK. Duration of antiviral immunity after smallpox vaccination. Nat Med 2003; 9: 11317.
  • 37
    Grufman P, Wolpert EZ, Sandberg JK, Karre K. T cell competition for the antigen-presenting cell as a model for immunodominance in the cytotoxic T lymphocyte response against minor histocompatibility antigens. Eur J Immunol 1999; 29: 2197204.
  • 38
    Kedl RM, Rees WA, Hildeman DA, Schaefer B, Mitchell T, Kappler J, Marrack P. T cells compete for access to antigen-bearing antigen-presenting cells. J Exp Med 2000; 192: 110513.
  • 39
    Cooney EL, Collier AC, Greenberg PD, Coombs RW, Zarling J, Arditti DE, Hoffman MC, Hu SL, Corey L. Safety of and immunological response to a recombinant vaccinia virus vaccine expressing HIV envelope glycoprotein. Lancet 1991; 337: 56772.
  • 40
    Kundig TM, Kalberer CP, Hengartner H, Zinkernagel RM. Vaccination with two different vaccinia recombinant viruses: long-term inhibition of secondary vaccination. Vaccine 1993; 11: 11548.
  • 41
    Rooney JF, Wohlenberg C, Cremer KJ, Moss B, Notkins AL. Immunization with a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: long-term protection and effect of revaccination. J Virol 1988; 62: 15304.
  • 42
    Eder JP, Kantoff PW, Roper K, Xu GX, Bubley GJ, Boyden J, Gritz L, Mazzara G, Oh WK, Arlen P, Tsang KY, Panicali D, et al. A phase I trial of a recombinant vaccinia virus expressing prostate-specific antigen in advanced prostate cancer. Clin Cancer Res 2000; 6: 16328.
  • 43
    Frey SE, Newman FK, Cruz J, Shelton WB, Tennant JM, Polach T, Rothman AL, Kennedy JS, Wolff M, Belshe RB, Ennis FA. Dose-related effects of smallpox vaccine. N Engl J Med 2002; 346: 127580.
  • 44
    Ennis FA, Cruz J, Demkowicz WE Jr, Rothman AL, McClain DJ. Primary induction of human CD8+ cytotoxic T lymphocytes and interferon-gamma-producing T cells after smallpox vaccination. J Infect Dis 2002; 185: 16579.
  • 45
    Klenerman P, Cerundolo V, Dunbar PR. Tracking T cells with tetramers: new tales from new tools. Nat Rev Immunol 2002; 2: 26372.
  • 46
    Terajima M, Cruz J, Raines G, Kilpatrick ED, Kennedy JS, Rothman AL, Ennis FA. Quantitation of CD8+ T cell responses to newly identified HLA-A*0201-restricted T cell epitopes conserved among vaccinia and variola (smallpox) viruses. J Exp Med 2003; 197: 92732.
  • 47
    Smith JW, 2nd, Walker EB, Fox BA, Haley D, Wisner KP, Doran T, Fisher B, Justice L, Wood W, Vetto J, Maecker H, Dols A, et al. Adjuvant immunization of HLA-A2-positive melanoma patients with a modified gp100 peptide induces peptide-specific CD8+ T-cell responses. J Clin Oncol 2003; 21: 156273.
  • 48
    Walker EB, Haley D, Miller W, Floyd K, Wisner KP, Sanjuan N, Maecker H, Romero P, Hu HM, Alvord WG, Smith JW II, Fox BA, et al. gp100(209-2M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells. Clin Cancer Res 2004; 10: 66880.
  • 49
    Donnelly JJ, Ulmer JB, Shiver JW, Liu MA. DNA vaccines. Annu Rev Immunol 1997; 15: 61748.
  • 50
    Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408: 7405.
  • 51
    Rehli M. Of mice and men: species variations of Toll-like receptor expression. Trends Immunol 2002; 23: 3758.
  • 52
    Balch CM, Buzaid AC, Atkins MB, Cascinelli N, Coit DG, Fleming ID, Houghton A Jr, Kirkwood JM, Mihm MF, Morton DL, Reintgen D, Ross MI, et al. A new American Joint Committee on Cancer staging system for cutaneous melanoma. Cancer 2000; 88: 148491.