[Show abstract][Hide abstract] ABSTRACT: Cancer cells display novel phosphopeptides in association with MHC class I and II molecules. In this study, we evaluated two HLA-A2-restricted phosphopeptides derived from the insulin receptor substrate (IRS)-2 and the cell cycle regulator CDC25b. These proteins are both broadly expressed in multiple malignancies and linked to cancer cell survival. Two phosphopeptides, termed pIRS-21097-1105 and pCDC25b38-46, served as targets of strong and specific CD8 T-cell memory responses in normal human donors. We cloned T-cell receptor (TCR) cDNAs from murine CD8 T-cell lines specific for either pIRS-21097-1105 or pCDC25b38-46. Expression of these TCR in human CD8 T-cells imparted high-avidity phosphopeptide-specific recognition and cytotoxic and cytokine-secreting effector activities. Using these cells, we found that endogenously processed pIRS-21097-1105 was presented on HLA-A2+ melanomas and breast, ovarian, and colorectal carcinomas. Presentation was correlated with the level of the Ser1100-phosphorylated IRS-2 protein in metastatic melanoma tissues. The highest expression of this protein was evident on dividing malignant cells. Presentation of endogenously processed pCDC25b38-46 was narrower, but still evident on HLA-A2+ melanoma, breast carcinoma and lymphoblastoid cells. Notably, pIRS-21097-1105-specific and pCDC25b38-46-specific TCR-expressing human CD8 T-cells markedly slowed tumor outgrowth in vivo. Our results define two new antigens that may be developed as immunotherapeutic agents for a broad range of HLA-A2+ cancers.
[Show abstract][Hide abstract] ABSTRACT: Cancers produce soluble and cell-associated molecules that can suppress or alter antitumor immunity. Preclinical studies suggest the disease burden may alter the cytokine profile of helper T cell responses to cancer antigens. We studied cytokine production by helper T cells responding to vaccination with 6 melanoma helper peptides (6MHP) in blood and lymph nodes.
Journal for immunotherapy of cancer. 01/2014; 2:23.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: Survival after amputation for melanoma is short; however, rare long-term survivors are reported. The mechanism for durable systemic tumor control in patients with regional failure is not known. To explore whether systemic tumor immunity may be implicated, tumor and circulating immune responses were examined in a patient who survived disease-free 14 years after hip disarticulation. METHODS: A 71-year-old female with extensive regional metastases of melanoma in the left lower extremity underwent amputation for palliative reasons. Tumor was collected at surgery, and blood was collected during follow-up. Tumor sections were evaluated for lymphocytic infiltration and NY-ESO-1 expression by immunohistochemistry. Cellular immune responses to defined tumor antigens were evaluated by ELISPOT assay, and antibody responses to a panel of tumor antigens were assayed by ELISA. RESULTS: The patient's tumor had minimal lymphocytic infiltrate (immunotype A). NY-ESO-1 was strongly expressed by the melanoma cells. Circulating T-cell responses to NY-ESO-1 peptides were observed 6 and 12 years postoperatively, and antibodies to NY-ESO-1 were detected 2-6 years after surgery. CONCLUSION: The patient described in this report experienced relentless regional tumor progression, with intravascular metastases, and then 14-year systemic disease-free survival after palliative resection, without evidence of melanoma recurrence before death from other causes. Her immune response to NY-ESO-1 likely failed to control established regional metastases because T cells were unable to infiltrate them. It is possible, however, that among other factors, the host immune response may have contributed to systemic protection.
Cancer Immunology and Immunotherapy 05/2013; · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immune cells infiltrating the microenvironment of melanoma metastases may either limit or promote tumor progression, but the characteristics that distinguish these effects are obscure. In this study, we systematically evaluated the composition and organization of immune cells that infiltrated melanoma metastases in human patients. Three histologic patterns of immune cell infiltration were identified, designated immunotypes A, B, and C. Immunotype A was characterized by no immune cell infiltrate. Immunotype B was characterized by infiltration of immune cells limited only to regions proximal to intratumoral blood vessels. Immunotype C was characterized by a diffuse immune cell infiltrate throughout a metastatic tumor. These immunotypes represented 29%, 63%, and 8% of metastases with estimated median survival periods of 15, 23, and 130 months, respectively. Notably, from immunotypes A to C, there were increasing proportions of B cells and decreasing proportions of macrophages. Overall, the predominant immune cells were T cells (53%), B cell lineage cells (33%), and macrophages (13%), with natural killer and mature dendritic cells only rarely present. Whereas higher densities of CD8(+) T cells correlated best with survival, a higher density of CD45(+) leukocytes, T cells, and B cells also correlated with increased survival. Together, our findings reveal striking differences in the immune infiltrate in melanoma metastases in patients, suggesting microenvironmental differences in immune homing receptors and ligands that affect immune cell recruitment. These findings are important, not only by revealing how the immune microenvironment can affect outcomes but also because they reveal characteristics that may help improve individualized therapy for patients with metastatic melanoma.
Cancer Research 03/2012; 72(5):1070-80. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell (DC) activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in 2 clinical trials of melanoma patients. In 1 study (Mel38), patients received 1 injection with an adjuvant mixture alone, composed of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature DCs, and vessels resembling high endothelial venules (HEVs). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DCs, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4and CD8 T lymphocytes, as well as FoxP3CD4 lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination.
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) is an angiogenic factor that also functions as an autocrine growth factor for VEGF receptor (VEGFR)-2(+) melanomas. In multiple studies, VEGFR-2 was detected by immunostaining in 78-89% of human melanoma cells, suggesting that most patients with melanoma would benefit from anti-VEGF therapy. Here, we evaluated 167 human melanoma specimens in a tissue microarray to verify the presence of VEGFR-2, but found disparities in staining with commercial antibodies A-3 and 55B11. Antibody A-3 stained melanoma cells in 79% of specimens, consistent with published results; however, we noted extensive nonspecific staining of other cells such as smooth muscle and histiocytes. In contrast, antibody 55B11 stained melanoma cells in only 7% (95% confidence interval: 3.3-11.5) of specimens. As an internal positive control for VEGFR-2 detection, vascular endothelial cells were stained with antibody 55B11 in all specimens. We compared VEGFR-2(+) and VEGFR-2(-) melanoma cell lines by immunoblotting and immunohistochemistry after small interfering RNA (siRNA) knockdown and transient overexpression of VEGFR-2 to validate antibody specificity. Immunoblotting revealed that A-3 primarily cross-reacted with several proteins in both cell lines and these were unaffected by siRNA knockdown of VEGFR-2. In contrast, 55B11 staining of VEGFR-2(+) cells was mostly eliminated by siRNA knockdown of VEGFR-2 and increased in VEGFR-2(-) melanoma cell lines following transfection to express ectopic VEGFR-2. Our results show that relatively few melanoma cells (<10%) express detectable levels of VEGFR-2, and therefore, the majority of patients with melanoma are unlikely to benefit from antiproliferative effects of anti-VEGF therapy.
International Journal of Cancer 02/2011; 129(12):2807-15. · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Melanoma vaccines have not been optimized. Adjuvants are added to activate dendritic cells (DCs) and to induce a favourable immunologic milieu, however, little is known about their cellular and molecular effects in human skin. We hypothesized that a vaccine in incomplete Freund's adjuvant (IFA) would increase dermal Th1 and Tc1-lymphocytes and mature DCs, but that repeated vaccination may increase regulatory cells.
During and after 6 weekly immunizations with a multipeptide vaccine, immunization sites were biopsied at weeks 0, 1, 3, 7, or 12. In 36 participants, we enumerated DCs and lymphocyte subsets by immunohistochemistry and characterized their location within skin compartments.
Mature DCs aggregated with lymphocytes around superficial vessels, however, immature DCs were randomly distributed. Over time, there was no change in mature DCs. Increases in T and B-cells were noted. Th2 cells outnumbered Th1 lymphocytes after 1 vaccine 6.6:1. Eosinophils and FoxP3+ cells accumulated, especially after 3 vaccinations, the former cell population most abundantly in deeper layers.
A multipeptide/IFA vaccine may induce a Th2-dominant microenvironment, which is reversed with repeat vaccination. However, repeat vaccination may increase FoxP3+T-cells and eosinophils. These data suggest multiple opportunities to optimize vaccine regimens and potential endpoints for monitoring the effects of new adjuvants.
ClinicalTrials.gov Identifier: NCT00705640.
Journal of Translational Medicine 01/2010; 8:79. · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human cancer vaccines incorporating autologous tumor cells carry a risk of implantation and subsequent metastasis of viable tumor cells into the patient who is being treated. Despite the fact that the melanoma cell preparations used in a recent vaccine trial (Mel37) were gamma-irradiated (200 Gy), approximately 25% of the preparations failed quality control release criteria which required that the irradiated cells incorporate 3H-thymidine at no more than 5% the level seen in the non-irradiated cells. We have, therefore, investigated ultraviolet (UV)-irradiation as a possible adjunct to, or replacement for gamma-irradiation.
Melanoma cells were gamma- and/or UV-irradiated. 3H-thymidine uptake was used to assess proliferation of the treated and untreated cells. Caspase-3 activity and DNA fragmentation were measured as indicators of apoptosis. Immunohistochemistry and Western blot analysis was used to assess antigen expression.
UV-irradiation, either alone or in combination with gamma-irradiation, proved to be extremely effective in controlling the proliferation of melanoma cells. In contrast to gamma-irradiation, UV-irradiation was also capable of inducing significant levels of apoptosis. UV-irradiation, but not gamma-irradiation, was associated with the loss of tyrosinase expression. Neither form of radiation affected the expression of gp100, MART-1/MelanA, or S100.
These results indicate that UV-irradiation may increase the safety of autologous melanoma vaccines, although it may do so at the expense of altering the antigenic profile of the irradiated tumor cells.
[Show abstract][Hide abstract] ABSTRACT: A phase I/II trial was performed to evaluate the safety and immunogenicity of a novel melanoma vaccine comprising six melanoma-associated peptides defined as antigenic targets for melanoma-reactive helper T cells. Source proteins for these peptides include MAGE proteins, MART-1/MelanA, gp100, and tyrosinase.
Thirty-nine patients with stage IIIB to IV melanoma were vaccinated with this six-peptide mixture weekly at three dose levels, with a preceding phase I dose escalation and subsequent random assignment among the dose levels. Helper T-lymphocyte responses were assessed by in vitro proliferation assay and delayed-type hypersensitivity skin testing. Patients with measurable disease were evaluated for objective clinical response by Response Evaluation Criteria in Solid Tumors.
Vaccination with the helper peptide vaccine was well tolerated. Proliferation assays revealed induction of T-cell responses to the melanoma helper peptides in 81% of patients. Among 17 patients with measurable disease, objective clinical responses were observed in two patients (12%), with response durations of 1 and 3.9+ years. Durable stable disease was observed in two additional patients for periods of 1.8 and 4.6+ years.
Results of this study support the safety and immunogenicity of a vaccine comprised of six melanoma helper peptides. There is also early evidence of clinical activity.
Journal of Clinical Oncology 10/2008; 26(30):4973-80. · 17.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8(+) T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient's spontaneous T cell response adapted, gaining the ability to recognize and to lyse "edited" tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.
The Journal of Immunology 07/2005; 174(11):6863-71. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A phase II trial was performed to test whether systemic low-dose interleukin-2 (IL-2) augments T-cell immune responses to a multipeptide melanoma vaccine. Forty patients with resected stage IIB-IV melanoma were randomly assigned to vaccination with four gp100- and tyrosinase-derived peptides restricted by human leukocyte antigen (HLA) -A1, HLA-A2, and HLA-A3, and a tetanus helper peptide plus IL-2 administered daily either beginning day 7 (group 1), or beginning day 28 (group 2).
T-cell responses were assessed by an interferon gamma ELIspot assay in peripheral blood lymphocytes (PBL) and in a lymph node draining a vaccination site (sentinel immunized node [SIN]). Patients were followed for disease-free and overall survival.
T-cell responses to the melanoma peptides were observed in 37% of PBL and 38% of SINs in group 1, and in 53% of PBL and 83% of SINs in group 2. The magnitude of T-cell response was higher in group 2. The tyrosinase peptides DAEKSDICTDEY and YMDGTMSQV were more immunogenic than the gp100 peptides YLEPGPVTA and ALLAVGATK. T-cell responses were detected in the SINs more frequently, and with higher magnitude, than responses in the PBL. Disease-free survival estimates at 2 years were 39% (95% CI, 18% to 61%) for group 1, and 50% (95% CI, 28% to 72%) for group 2 (P = .32).
The results of this study support the safety and immunogenicity of a vaccine composed of four peptides derived from gp100 and tyrosinase. The low-dose IL-2 regimen used for group 1 paradoxically diminishes the magnitude and frequency of cytotoxic T lymphocyte responses to these peptides.
Journal of Clinical Oncology 12/2004; 22(22):4474-85. · 17.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effectiveness of peptide-based cancer vaccines depends on the ability of peptides to bind to MHC molecules on the surface of antigen-presenting cells, where they reconstitute epitopes for cytotoxic T lymphocytes (CTLs). Multivalent vaccines have advantages over single-peptide vaccines; however, peptides may compete for binding to the same MHC molecules. In particular, it is possible that peptides with high affinity for MHC molecules prevent the binding of lower-affinity peptides. However, only small numbers of peptide/MHC complexes per cell are required for CTL recognition. Thus, the authors hypothesized that competition of peptides for MHC binding would not significantly reduce CTL recognition of individual peptides within a multiple-peptide mixture, and this hypothesis was tested by a series of experiments performed in vitro. In multiple experiments, two peptides with different affinities for HLA-A*0201 molecules were mixed at various concentrations and pulsed onto HLA-A2 cells, which were then evaluated for susceptibility to lysis by HLA-A*0201-restricted CTLs. CTL recognition of the melanoma peptides gp100(154-162) (KTWGQYWQV), gp100(280-288) (YLEPGPVTA), and tyrosinase(369-377D) (YMDGTMSQV) was maintained even when target cells were co-pulsed with equimolar concentrations of peptides with comparable or higher affinity for HLA-A2. In some cases, CTL recognition was maintained even when the higher-affinity peptide was present at concentrations several orders of magnitude higher than the target peptide. In addition, CTLs generated by in vitro stimulation with a peptide mixture developed reactivity to three different peptides, at a level comparable to that obtained by stimulation with each individual peptide separately. These data suggest that CTLs can respond to multiple peptides presented on the same antigen-presenting cells and justify further investigation, in clinical trials, of multiple-peptide cancer vaccines.
Journla of Immunotherapy 01/2004; 27(6):425-31. · 3.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The p68-derived peptide, QIVDVCHDV, was identified by a reverse immunology approach as capable of reconstituting an epitope recognized by the melanoma-reactive cytotoxic T lymphocyte (CTL) line VMM5. The peptide has not been demonstrated definitively on the cell surface by mass spectrometry; thus, it is not yet considered appropriate for use in human melanoma vaccines. Interestingly, however, the antigenicity of this peptide was affected by spontaneous modifications at two distinct residues. Spontaneous modification of the QIVDVCHDV peptide can occur at the cysteine residue at position 6 or at the N-terminal glutamine residue, and both modifications dramatically affect CTL recognition. Avoidance of an acidic environment prevents the conversion of the N-terminal glutamine residue to pyroglutamic acid, a conversion that inhibits binding of the peptide to HLA-A2 and diminishes recognition by CTLs. Substitution of asparagine for the N-terminal glutamine and substitution of serine for the cysteine were shown to enhance the binding of the peptide to HLA-A2 and to enhance the recognition of the peptide by CTLs. These findings suggest general strategies for enhancing the antigenicity of other peptides containing similar amino acids in their sequence.
Journla of Immunotherapy 01/2004; 27(3):177-83. · 3.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine clinical and immunologic responses to a multipeptide melanoma vaccine regimen, a randomized phase II trial was performed.
Twenty-six patients with advanced melanoma were randomly assigned to vaccination with a mixture of four gp100 and tyrosinase peptides restricted by HLA-A1, HLA-A2, and HLA-A3, plus a tetanus helper peptide, either in an emulsion with granulocyte-macrophage colony-stimulating factor (GM-CSF) and Montanide ISA-51 adjuvant (Seppic Inc, Fairfield, NJ), or pulsed on monocyte-derived dendritic cells (DCs). Systemic low-dose interleukin-2 (Chiron, Emeryville, CA) was given to both groups. T-lymphocyte responses were assessed, by interferon gamma ELIspot assay (Chiron, Emeryville, CA), in peripheral-blood lymphocytes (PBLs) and in a lymph node draining a vaccine site (sentinel immunized node [SIN]).
In patients vaccinated with GM-CSF in adjuvant, T-cell responses to melanoma peptides were observed in 42% of PBLs and 80% of SINs, but in patients vaccinated with DCs, they were observed in only 11% and 13%, respectively. The overall immune response was greater in the GM-CSF arm (P <.02). Vitiligo developed in two of 13 patients in the GM-CSF arm but in no patients in the DC arm. Helper T-cell responses to the tetanus peptide were detected in PBLs after vaccination and correlated with T-cell reactivity to the melanoma peptides. Objective clinical responses were observed in two patients in the GM-CSF arm and one patient in the DC arm. Stable disease was observed in two patients in the GM-CSF arm and one patient in the DC arm.
The high frequency of cytotoxic T-lymphocyte responses and the occurrence of clinical tumor regressions support continued investigation of multipeptide vaccines administered with GM-CSF in adjuvant.
Journal of Clinical Oncology 12/2003; 21(21):4016-26. · 17.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A melanoma vaccine composed of HLA-A2-restricted peptide YLEPGPVTA (gp100(280)), with or without a modified T-helper epitope from tetanus toxoid AQYIKANSKFIGITEL, has been evaluated in a Phase I trial to assess safety and immunological response. The vaccines were administered s.c. in either of two adjuvants, Montanide ISA-51 or QS-21, to 22 patients with high-risk resected melanoma (stage IIB-IV). Local and systemic toxicities were mild and transient. We detected CTL responses to the gp100(280) peptide in peripheral blood in 14% of patients. Helper T-cell responses to the tetanus helper peptide were detected in 79% of patients and had a Th1 cytokine profile. One patient with a CTL response to gp100 had a recurrence in a lymph node 2 years later; her nodes contained CD8+ cells reactive to gp100(280) (0.24%), which proliferated in response to peptide. The overall survival of patients is 75% (95% confidence interval, 57-94%) at 4.7 years follow-up, which compares favorably with expected survival. Four of 14 patients who completed at least six vaccines subsequently developed metastases, all of which were solitary and surgically resectable. They remain alive and clinically free of disease at last follow-up. Data from this trial demonstrate immunogenicity of the gp100(280) peptide and suggest that immune responses may persist long-term in some patients. The frequency and magnitude of the CTL response may be improved with more aggressive vaccination regimens. Although this Phase I study was not intended to evaluate clinical benefit, the excellent survival of patients on this protocol suggests the possibility of a benefit that should be assessed in future studies.
Clinical Cancer Research 11/2001; 7(10):3012-24. · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many peptide epitopes for cytotoxic T lymphocytes (CTLs) have been identified from melanocytic differentiation proteins. Vaccine trials with these peptides have been limited mostly to those associated with HLA-A2, and immune responses have been detected inconsistently. Cases of clinical regression have been observed after peptide vaccination in some trials, but melanoma regressions have not correlated well with T-cell responses measured in peripheral blood lymphocytes (PBLs). We vaccinated stage IV melanoma patients with a mixture of gp100 and tyrosinase peptides restricted by HLA-A1 (DAEKSDICTDEY), HLA-A2 (YLEPGPVTA and YMDGTMSQV) and HLA-A3 (ALLAVGATK) in an emulsion with GM-CSF and Montanide ISA-51 adjuvant. CTL responses were assessed in PBLs and in a lymph node draining a vaccine site (sentinel immunized node, SIN). We found CTL responses to vaccinating peptides in the SIN in 5/5 patients (100%). Equivalent assays detected peptide-reactive CTLs in PBLs of 2 of these 5 patients (40%). CTLs expanded from the SIN lysed melanoma cells naturally expressing tyrosinase or gp100. We demonstrated immunogenicity for peptides restricted by HLA-A1 and -A3 and for 1 HLA-A2 restricted peptide, YMDGTMSQV. Immune monitoring of clinical trials by evaluation of PBLs alone may under-estimate immunogenicity; evaluation of SIN provides a new and sensitive approach for defining responses to tumor vaccines and correlating these responses with clinical outcomes. This combination of an immunogenic vaccine strategy with a sensitive analysis of CTL responses demonstrates the potential for inducing and detecting anti-tumor immune responses in the majority of melanoma patients.
International Journal of Cancer 07/2001; 92(5):703-11. · 5.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The long-term survival of some patients with metastatic melanoma may be attributable in part to cellular immune responses to melanoma antigens. However, little is known about the level of CTL reactivity in vivo that is required for immunological control of tumor progression. In the present report, T-cell responses were evaluated with lymphocytes obtained from tumor-involved nodes and peripheral blood of a long-term melanoma survivor. Using an ELISPOT assay, naturally occurring functional T cells, which recognize the peptide ALLAVGATK (gp100(17-25)) plus two other HLA-A3 restricted peptides, were detected in a tumor-involved lymph node. The ALLAVGATK-reactive T cells were also evaluated by MHC-tetramers staining and were found to be CD8+ CD45RO+ L-selectin(-) CD11a+, suggesting that they are antigen experienced and have a memory phenotype. Unstimulated peripheral blood lymphocytes from the same patient demonstrated no detectable T-cell responses; however, a single stimulation with ALLAVGATK peptide in vitro resulted in a dramatic expansion of peptide-reactive CTLs. This patient, with evidence of tumor-reactive CTLs targeted to several tumor antigens in a tumor-involved lymph node and with evidence of a circulating memory T-cell response, has remained disease-free for 6 years, despite prior bulky nodal metastasis. In contrast, three HLA-A3+ patients with rapidly progressive metastatic melanoma had no detectable T-cell response in tumor-involved nodes or peripheral blood lymphocytes, even after peptide stimulation ex vivo. The presented data are consistent with a systemic polyvalent immune response against tumor in this long-term survivor. These data provide an estimate of the level of CTL response that may be associated with protection from tumor recurrence.
Clinical Cancer Research 04/2001; 7(3 Suppl):909s-916s. · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Melanoma-reactive HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched
allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pmel17/gp100. However,
a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A*0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in
expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pmel17/gp100, gp75/trp-1, and MART-1/Melan-A. This
concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a
subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines
expressed normal levels of HLA-A*0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A*0201-specific
CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived
antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was
restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and
HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1
or by HLA-A*0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique
or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest
that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique
antigens or other undefined antigens, especially in patients whose tumors do not express MDP.
Cancer Immunology and Immunotherapy 01/2000; 48(12):661-672. · 3.94 Impact Factor