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Advances in Brief |
Swim Across America Laboratory of Tumor Immunology [S. G. S., W-J. H., P. O. L., J. J. L., A. N. H., P. B. C.], and Departments of Medicine [V. M. K., C. M. M, L. B., W-J. H., P. O. L., L. W., J. J. L., A. N. H., P. B. C.], Surgery [J. J. L.], and Biostatistics and Epidemiology [K. S. P.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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 Top ABSTRACT IntroductionPatients and MethodsResultsDiscussionREFERENCES |
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release by CD8+ T cells against tyrosinase and gp100 peptides measured by enzyme-linked immunospot assays without in vitro prestimulation measured pretreatment, 2 and 8 weeks after the third vaccination. Four of 9 and 4 of 8 patients immunized using QS-21 and GM-CSF, respectively, developed increased frequencies of CD8+ T cells against tyrosinase 370D peptide compared with 0 of 9 patients immunized using IFA (P = 0.045). T-cell responses against a gp100-related peptide showed similar results, but their relevance to T-cell reactivity against native gp100 209–217 is uncertain. These results show that: (a) QS-21 and GM-CSF are superior to IFA as immunological adjuvants for vaccination against tyrosinase 370D peptide; and (b) with appropriate adjuvants, increased frequencies of peptide-specific T cells after vaccination can be detected by enzyme-linked immunospot without prolonged prestimulation in vitro. |
Introduction |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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40% of patients. Both tyrosinase 368–376(370D) peptide and gp100 209–217 peptide are HLA-A2.1-restricted peptides that can be processed, presented, and recognized by T cells in the immune repertoire. Alteration of the gp100 209–217 peptide at the second amino acid (changing threonine to a methionine) was found to increase markedly the affinity for HLA-A2.1 resulting in enhanced induction of T cells reactive to native gp100 (6) . Immunization with this heteroclytic peptide, gp100 209–217(210M), with IFA3 induced T cells against native gp100 209–217 peptide in 10 of 11 patients vaccinated (7) . Immunogenicity of tyrosinase peptide appears to be lower. In the absence of adjuvant, 0 of 6 patients immunized with tyrosinase 368–376(370D) peptide developed a cytotoxic lymphocyte reaction to the peptide (1) . However, immunization with tyrosinase peptide plus QS-21 can induce T-cell responses (5) .
Because peptides derived from self proteins are minimally immunogenic alone (1 , 8) , they must be administered with a potent immunological adjuvant to induce detectable T-cell responses. Most trials have used IFA as an adjuvant (7 , 9 , 10) . Another potent adjuvant is QS-21, a purified saponin (11) , which can enhance both B-cell (12, 13, 14) and T-cell responses (15 , 16) . Multiple clinical studies have shown QS-21 to be well tolerated by patients (5 , 13 , 17) . We have also been interested in GM-CSF as a vaccine adjuvant because of its role as one of the primary growth and maturation factors for DCs. We and others have shown that daily intradermal injections of GM-CSF for 10 days recruit DCs to the site of injection (18 , 19) and, in 6 patients, administration of GM-CSF seemed to enhance the immunogenicity of peptides derived from melanosomal differentiation proteins (20) . We reasoned that introduction of peptides into the area prepared by the daily GM-CSF injections could be an efficient method of loading DCs in vivo and immunizing the patient.
Despite the potentially critical role of adjuvants in vaccine design, we were aware of no data directly comparing adjuvants in patients receiving cancer vaccines. Therefore, we conducted a randomized trial in which patients were vaccinated with both the tyrosinase 368–376(370D) and gp100 209–217(210M) peptides using one of three different adjuvants: IFA, QS-21, or GM-CSF. The primary end point of the study was the induction of peptide-specific CD8+ T cells.
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Patients and Methods |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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18 years, Karnofsky performance status 60%, WBC 3,500 cells/mm3, platelets 75,000/mm3, lactate dehydrogenase < 2 x the upper limit of normal, albumin 3.5 mg/dl, and ability to give written informed consent. Patients were excluded if they had received chemotherapy, immunotherapy, or radiation therapy within the previous 4 weeks, or if they had a known immunodeficiency, previous splenectomy, or radiation therapy to the spleen. Patients could not have received prior vaccines containing IFA, tyrosinase, or gp100. Patients were also excluded if they had a serious underlying medical disease, active infection requiring antimicrobial drugs, or active bleeding. While on study, patients could not receive anti-inflammatory or antihistamine drugs. Women who were pregnant, <3 months postpartum, or lactating were not eligible. Patients with preexisting retinal or choroidal eye disease were also excluded.
Peptides.
The vaccine consisted of tyrosinase 368–376(370D) peptide (YMDGTMSQV) and gp100 209–217(210M) peptide (IMDQVPFSV), which were prepared and administered separately. Both peptides are HLA-A2.1 restricted. The gp100 209–217(210 M) peptide was modified at the second position to increase the affinity of binding to HLA-A2.1 (6)
and to enhance induction of T cells against the native peptide expressed by melanoma. Both peptides were provided by Cancer Treatment Evaluation Program/NCI under an Investigational New Drug application held by the NCI.
Adjuvants.
IFA formulated as Montanide ISA-51 was manufactured by Seppic, Inc. and supplied by the NCI as single-use vials containing 3 ml of sterile adjuvant solution without preservative. QS-21 was prepared by Antigenics, Inc. (Woburn, MA) from Quillaja saponaria Molina tree bark by silica and reverse-phase chromatography (11
, 16)
. The QS-21 was supplied in 0.5 mg/ml in PBS vials [(pH 6.8), volume 0.6 ml]. Human GM-CSF, made by Immunex Corporation (Seattle, WA), was supplied through the NCI.
Vaccine Preparation and Administration.
The vaccine peptides were sterile and endotoxin-free. The tyrosinase 368–376(370D) peptide was supplied in vials containing 1.2 ml of a sterile 1 mg/ml solution for injection with 0.1 N HCl added to adjust the pH. The gp 100 209–217(210M) peptide was supplied in a 1 mg/ml solution in single use-vials containing 1 ml volume. Intact vials were stored frozen at -10°C to -20°C. Once thawed, the peptides were used within 3 h.
Treatment Plan.
Patients were assigned by block randomization to receive IFA, GM-CSF, or QS-21 to ensure 9 patients in each adjuvant arm. Patients were stratified for age ([65 versus >65) and previous chemotherapy. All of the patients received 3 monthly vaccinations with both tyrosinase 368–376(370D) peptide and gp100 209–217(210M) peptide at a dose of 500 µg/peptide administered at separate sites. If patients failed to receive all three of the planned vaccinations, they were considered inevaluable for immunological response and were replaced. For patients randomized to the IFA arm, 500 µg of each peptide in aqueous solution at room temperature was mixed with Montanide in a ratio of 1:1 and injected s.c. For patients receiving QS-21, 500 µg of each peptide in aqueous solution was mixed with 100 µg of QS-21 and administered s.c. Patients randomized to GM-CSF received 40 µg of GM-CSF injected intradermally at a single skin site for 10 consecutive days for each vaccination. On day 7 of the GM-CSF treatment, 500 µg of each peptide was administered intradermally at separate sites within the erythematous reaction induced by the GM-CSF.
Physical examination, blood cell counts, and standard blood tests were performed before each immunization, and 4 and 8 weeks after the last immunization. Blood for T-cell studies was drawn pretreatment, and 2 weeks and 8 weeks after the last immunization.
The protocol was reviewed and approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center. All of the patients signed informed consent before participating in the clinical trial.
PBMCs.
PBMCs were collected pretreatment 2 weeks and 8 weeks after the last vaccine. For each time point, 80 ml of heparinized blood were drawn, and PBMCs were isolated by centrifugation on a Ficoll-Paque gradient (Pharmacia, Piscataway, NJ) and stored in FCS/10% DMSO in aliquots of 5 x 106 cells at -180°C until needed.
Immunological Monitoring by ELISPOT.
T-cell reactivity against tyrosinase 368–376(370D) peptide was measured by IFN- ELISPOT without previous in vitro stimulation. ELISPOT assays against gp100 (209–217) were carried out using a gp100-related peptide containing tyrosine at position 217 based on an early publication that contained an error in the gp100 peptide sequence (21)
. This results in a peptide with low avidity for HLA-A2 molecule compared with the native sequence, which contains valine at position 217. As a consequence, the significance of the gp100 ELISPOT results was difficult to interpret and these results [although very similar to the ELISPOT results with tyrosinase 368–376(370D) peptide] are not reported here. PBMCs were thawed, and CD8+ T cells were purified by magnetic microbeads (Miltenyi Biotec Inc., Auburn, CA) following the manufacturer’s instructions, which yielded populations of >98% pure CD8+ T cells.
HA-Multiscreen plates (Millipore, Burlington, MA) were coated with 100 µl of mouse antihuman IFN- antibody (10 µg/ml; clone 1-D1K; Mabtech, Nacka, Sweden) overnight at 4°C, washed, and blocked with human serum for 1 h at 37°C. CD8+ T cells were plated in duplicate or triplicate (depending on the number of cells available) at 5 x 104/well with 1 x 104 irradiated T2 cells/well pulsed with 10 µg/ml ß2-microglobulin (Sigma Chemical Co., St. Louis, MO) and 10 µg/ml tyrosinase 368–376(370D) peptide. In some assays, peptide was added at lower concentrations (1 µg/ml) to detect high affinity T cells. Background activity was measured as CD8+ T-cell reactivity against unpulsed T2 cells. Negative control wells were included in which T2 cells were pulsed with the irrelevant peptide KTWGQYWQV, a HLA-A2.1-restricted peptide derived from the gp100 protein but completely distinct from the gp100 209–217 peptide, and not generally recognized by T cells from melanoma patients. Negative control wells were also included that contained only CD8+ T cells (no T2 cells), only pulsed T2 cells (no T cells), and medium alone.
After incubation for 20 h at 37°C, plates were washed with PBS/0.5% Tween. Bound IFN- was detected by adding 100 µl of biotinylated antihuman IFN- (2 µg/ml; clone 7-B6-1; Mabtech). Spot development was performed by using avidin-biotin complexes as described (22)
, and spots were counted using an automated ELISPOT reader system (Carl Zeiss Vision, Göttinger, Germany) with KS ELISPOT 4.2 software. The number of specific spots was the number of spots counted in the wells containing CD8+ T cells with T2 cells pulsed with the specific peptide of interest minus the number of the background activity spots (CD8+ T cells with unpulsed T2 cells). In most experiments, a similar number of spots was observed in wells containing CD8+ T cells and either unpulsed T2 cells or T2 cells pulsed with the irrelevant peptide. Negative control wells containing either CD8+ T cells alone or pulsed T2 cells alone (no CD8+ T cells) yielded no spots.
On the basis of intra-assay variability in healthy donors, a positive response to vaccination was defined as an increase in 12 spots compared with the prevaccination value (5)
.
Statistical Analysis.
A Fisher’s exact test was used to test for an overall association between adjuvant and immunological response to peptide. When an overall difference was observed, the QS-21 and GM-CSF arms were collapsed, and a Fisher’s exact test was performed adjusted for these post-hoc comparisons.
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Results |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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. Eighteen patients were free of disease at the time of entry on the trial; 2 patients had unresectable stage III in-transit disease. Sites of metastatic disease among the 7 patients enrolled with active stage IV melanoma were: liver (5)
, lymph nodes (4)
, lung (3)
, heart (1)
, spleen (1)
, adrenal (1)
, pancreas (1)
, and abdominal wall (1)
. Each of the 7 patients had at least two known sites of metastases. Nineteen of the patients had received some form of systemic therapy before entering the study.
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12 spots/well over pretreatment values (5)
. However, for 3 patients there were sufficient numbers of lymphocytes for only one round of assays. One patient, in the GM-CSF group, failed to return to clinic for PBMC collection and could not be assessed for T-cell response.
Overall, 8 of 26 patients (30%) developed T-cell responses to tyrosinase 368–370 (370D) peptide (Fig. 1)
. The response proportion in the QS-21 and GM-CSF groups (4 of 9 and 4 of 8, respectively) was significantly higher than the response proportion in the IFA group (0 of 9). An overall association between adjuvant and response was observed (P = 0.045). When the response rates of the QS-21 and GM-CSF groups were combined, the response rate of the IFA group was significantly lower (P = 0.04). At 8 weeks after immunization, the median number of T cells reactive to tyrosinase 368–370 (370D) peptide in the IFA, QS-21, and GM-CSF groups was 7, 32, and 22 per 105 CD8+ T cells (1 of 14,000, 1 of 3,125, and 1 of 4,545 T cells), respectively (Fig. 2)
. Although 2 weeks after completing immunization the median number of T cells reactive to tyrosinase 368–370 (370D) peptide was not significantly increased in any of the adjuvant groups, we noted that at this time point 3 of 9 patients in the QS-21 group demonstrated >60 tyrosinase-reactive T cells/105 CD8+ T cells (<1 of 1,700 T cells reactive with tyrosinase). Three of the 8 patients in whom antityrosinase T-cell responses were enhanced had received previous chemotherapy indicating that prior chemotherapy did not preclude an immunological response.
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. These results indicate that at least some of the patients developed responses consisting of T cells expressing T-cell receptors with relatively high avidity for the peptide.
Toxicity.
All 31 of the patients entered were evaluable for toxicity. In general, vaccines were well-tolerated, and serious toxicity (grade 3) was not observed in any patient. One patient developed vitiligo.
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Discussion |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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Our results confirm that tyrosinase 368–370 (370D) peptide is immunogenic and can be used to induce a T-cell response in patients. The principal finding was that QS-21 and GM-CSF were more effective adjuvants than IFA for inducing T-cell responses against tyrosinase peptide; patients immunized with peptide and either QS-21 or GM-CSF were more likely to develop a T-cell response to tyrosinase compared with patients immunized using IFA. Among the 9 patients immunized with IFA, none developed a detectable T-cell response against the tyrosinase peptide. Although the route of administration may play a role in the efficiency of immunization, it is unlikely that the immunological differences observed were attributable simply to the route of immunization, because vaccines given with QS-21 and IFA were both administered via the s.c. route
Our inability to detect reactivity against tyrosinase peptide in T cells isolated directly from patients immunized with peptide and IFA is consistent with results reported by other groups immunizing patients with other peptides and IFA (7 , 9 , 29, 30, 31) . For example, among 7 patients immunized with gp100 209–217(210M) peptide and IFA at the Surgery Branch of the NCI, none had a detectable T-cell response to native gp100 209–217 peptide when T cells were tested immediately after being isolated from the patient (31) . To detect T cells against native gp100 209–217 peptide after immunization with gp100 209–217(210M) peptide and IFA, it has been necessary to preincubate PBMCs in vitro with target peptide for 10–12 days (with interleukin 2) before measuring T-cell response (7 , 31) . The requirement for in vitro expansion before detecting antipeptide T-cell responses has been observed in patients immunized with MART-1/MelanA and MAGE-3 peptides (9 , 29 , 30) presumably because of the low frequency of reactive T cells after immunization. This preincubation allows expansion of the specific T-cell population to a detectable level. In our study, CD8+ T cells were assayed by ELISPOT directly from the patient with only overnight incubation with peptide because we hypothesized that assaying cells directly out of the patient more accurately reflects the status of the cells in the patient. Indeed, data from the NCI suggest that the ability to expand peptide-specific T cells in vitro does not correlate with antitumor effects (10) .
This raises the important issue of how best to monitor T-cell responses in vaccine trials. A variety of techniques are used including limiting dilution assays, delayed-type hypersensitivity reactions, T-cell proliferation, ELISPOT, tetramers, cytokine secretion, or intracellular cytokine production. Each method has potential advantages and disadvantages. Some assays are quantitative but do not measure function (e.g., tetramer assays). Other assays measure function but do not provide information about T-cell frequencies (e.g., proliferation assays and delayed-type hypersensitivity reactivity). Limiting dilution assays measure function and are quantitative, but they are difficult to perform and can markedly underestimate T-cell frequencies.
In this study, we have used IFN- secretion by CD8+ cells as measured by ELISPOT assay. The advantage of this assay technique is that it measures T-cell function and can provide a quantitative estimate of T-cell frequency. It is common to use a stimulatory peptide concentration of 10 µg/ml when performing the ELISPOT assay. However, it is possible that this relatively high peptide concentration may detect T cells of such low avidity that they have little clinical relevance. In 2 patients from this trial who showed T-cell responses to peptide in our standard ELISPOT conditions, we were able to test CD8+ T cells using a 10-fold lower peptide concentration (1 µg/ml) and found we could still detect peptide-reactive T cells under these more stringent conditions. This suggests that at least some patients have T cells after immunization with relatively high avidity for peptide.
It has yet to be determined what magnitude of T-cell response is necessary for therapeutic effects and what is the best method to monitor T-cell responses in vaccine trials. We observed a maximum of 100 antityrosinase T cells/105 CD8+ T cells (Fig. 2)
, which corresponds to 0.1% of CD8+ T cells. Until a vaccine is developed with clear and reproducible beneficial clinical effects, it will be difficult to know how best to monitor T-cell responses and what magnitude of T-cell response correlates best with efficacy. The fact that clinical responses have been observed in the absence of detectable T-cell responses (1
, 7
, 8)
adds to the uncertainty. However, it seems likely that testing T cells directly from the patient is preferable to a technique using extensive in vitro prestimulation. On the basis of this assumption, our data suggest that QS-21 or GM-CSF is superior to IFA as an adjuvant for peptide vaccines.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the National Cancer Institute Grants CA33049 and CA81293, the Swim Across America Foundation, American Society of Clinical Oncology Young Investigator Award (to J. J. L.), and the Lacher Fellowship Fund (to V. M. K.). 
2 To whom requests for reprints should be addressed, at Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-5015; Fax: (212) 794-4352; E-mail: chapmap{at}mskcc.org
3 The abbreviations used are: IFA, incomplete Freund’s adjuvant; GM-CSF, granulocyte macrophage colony-stimulating factor; DC, dendritic cell; NCI, National Cancer Institute; PBMC, peripheral blood mononuclear cell; ELISPOT, enzyme-linked immunospot.
Received 7/16/01; revised 10/26/01; accepted 12/ 5/01.
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REFERENCES |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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in response to HLA-A2.1-binding melanoma and viral peptide antigens. J. Immunol. Methods, 191: 131-142, 1996.[CrossRef][Medline]
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), QS-21 (
), or GM-CSF (
) as adjuvants. Horizontal bars represent median values.