Sentinel Immunized Node Research Articles

A pilot trial of vaccination with Carcinoembryonic antigen and Her2/neu peptides in advanced colorectal cancer.

Checkpoint-blockade therapy (CBT) is approved for select colorectal cancer (CRC) patents, but additional immunotherapeutic options are needed. We hypothesized that vaccination with carcinoembryonic antigen (CEA) and Her2/neu (Her2) peptides would be immunogenic and well tolerated by participants with advanced CRC. A pilot clinical trial (NCT00091286) was conducted in HLA-A2+ or -A3+ Stage IIIC-IV CRC patients. Participants were vaccinated weekly with CEA and Her2 peptides plus tetanus peptide and GM-CSF emulsified in Montanide ISA-51 adjuvant for 3 weeks. Adverse events (AEs) were recorded per NIH Common Terminology Criteria for Adverse Events version 3. Immunogenicity was evaluated by interferon-gamma ELISpot assay of in vitro sensitized peripheral blood mononuclear cells and lymphocytes from the sentinel immunized node. Eleven participants were enrolled and treated; one was retrospectively found to be ineligible due to HLA type. All 11 participants were included in AEs and survival analyses, and the 10 eligible participants were evaluated for immunogenicity. All participants reported AEs: 82% were Grade 1-2, most commonly fatigue or injection site reactions. Two participants (18%) experienced treatment-related dose-limiting Grade 3 AEs; both were self-limiting. Immune responses to Her2 or CEA peptides were detected in 70% of participants. Median overall survival (OS) was 16 months; among those enrolled with no evidence of disease (n=3), median OS was not reached after 10 years of follow-up. These data demonstrate that vaccination with CEA or Her2 peptides is well tolerated and immunogenic. Further study is warranted to assess potential clinical benefits of vaccination in advanced CRC either alone or in combination with CBT.

A randomized pilot trial testing the safety and immunologic effects of a MAGE-A3 protein plus AS15 immunostimulant administered into muscle or into dermal/subcutaneous sites.

Methods to induce T cell responses to protein vaccines have not been optimized. The immunostimulant AS15 has been administered with the recombinant MAGE-A3 protein (recMAGE-A3) i.m. but not i.d. or s.c. This study tests hypotheses that the i.d./s.c. route is safe and will increase CD4(+) and CD8(+) T cell responses to MAGE-A3. Twenty-five patients with resected stage IIB-IV MAGE-A3(+) melanoma were randomized to immunization with recMAGE-A3 combined with AS15 immunostimulant (MAGE-A3 immunotherapeutic) either i.m. (group A, n=13) or i.d./s.c. (group B, n=12). Adverse events were recorded. Ab responses to MAGE-A3 were measured by ELISA. T cell responses to overlapping MAGE-A3 peptides were assessed in PBMC and a sentinel immunized node (SIN) after 1 in vitro stimulation with recMAGE-A3, by IFN-γ ELISPOT assay and by flow cytometry for multifunctional (TNF-α/IFN-γ) responses. Both routes of immunization were well tolerated without treatment-related grade 3 adverse events. All patients had durable Ab responses. For all 25 patients, the T cell response rate by ELISPOT assay was 30% in SIN (7/23) but only 4% (1/25) in PBMC. By flow cytometry, multifunctional CD8(+) T cell responses were identified in one patient in each group; multifunctional CD4(+) T cell response rates for groups A and B, respectively, were 31 and 64% in SIN and 31 and 50% in PBMC. The MAGE-A3 immunotherapeutic was well tolerated after i.d./s.c. administration, with trends to higher CD4(+) T cell response rates than with i.m. administration. This study supports further study of AS15 by i.d./s.c. administration.

Immunologic hierarchy, class II MHC promiscuity, and epitope spreading of a melanoma helper peptide vaccine.

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4(+) T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8(+) T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund's adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4(+) and CD8(+) T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4(+) T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3281-295 (49 %) and tyrosinase386-406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8(+) T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4(+) and CD8(+) T cell responses against melanoma antigens. CD4(+) T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8(+) T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.

A melanoma helper peptide vaccine increases Th1 cytokine production by leukocytes in peripheral blood and immunized lymph nodes.

BackgroundCancers 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.MethodsTwenty-three patients with stage IIIB-IV melanoma received a 6MHP vaccine. Antigen-reactive T cells from blood and draining lymph nodes were cultured, exposed to antigen, and then supernatants (days 2 and 5) were assayed for Th1 and Th2 cytokines. Results from 4 time points were compared to pre-vaccine levels.ResultsCytokine responses to vaccinating peptides were observed in 83% of patients. Th1 favoring responses were most common (17 of 19 responders). The most abundant cytokines produced were IFN-γ and IL-5 in the PBMC’s. IL-2 responses predominated in cells obtained from draining lymph nodes in 2-day culture but not in 5-day cultures. Patients with clinically measurable disease produced similar levels of total cytokine and similar degree of Th1 polarization as patients with no evidence of disease (NED).ConclusionsThe MHC class II-associated peptides used in this study induced helper T cells with a Th1-biased cytokine response in both PBMC and sentinel immunized nodes. Most patients can mount a Th1 dominant response to these peptides. Future studies are needed to test newer vaccine adjuvants in combination with these peptides.Trial registrationCDR0000378171, Clinicaltrials: NCT00089219.

A multi-epitope melanoma helper peptide vaccine durably increases Th1 cytokine production by responding lymphocytes

Background 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. We describe here the cytokine responses as change from baseline. Experimental design Thirty-nine patients with stage IIIB-IV melanoma received a 6MHP vaccine. Antigen-reactive T cells from blood and draining lymph nodes were exposed to antigen, and supernatants (days 2 and 5) were assayed by cytokine bead arrays for Th1 and Th2 cytokines. Results The greatest absolute cytokine responses detected were the levels of IFN-g and IL-5, though high levels of IL-2 were also produced early by lymph node-derived lymphocytes. Lymphocytes from patients with clinically measurable disease produced similar levels of total cytokine as patients with no evidence of disease (NED). For both disease statuses, the cytokine profile was Th1dominant in most patients. Total cytokine production and individual cytokine levels varied significantly among patients and varied over time but persisted up to nine months post-vaccine. Conclusions The MHC class II-associated peptides used in this study induced helper T cells with a Th1-biased cytokine response in both PBMC and sentinel immunized nodes in most patients. Patients with or without measurable disease can mount a Th1 dominant response to these peptides, which may persist long after the vaccination sequence. Future studies are needed to test newer vaccine adjuvants in combination with these peptides.

A multipeptide vaccine plus toll-like receptor agonists in melanoma patients, with evaluation of the vaccine site microenvironment and sentinel immunized node (Mel58; NCT01585350).

TPS3125 Background: Recent data show clinical activity of cancer vaccines containing a defined cancer antigen, and a peptide vaccine for melanoma. However, immune responses to peptide vaccines are often transient and of low magnitude. The most common adjuvant for peptide vaccines for melanoma has been an incomplete Freund’s adjuvant (IFA), which may have suboptimal adjuvant properties. Toll-like receptor (TLR) agonists offer the potential to improve the magnitude and persistence of antitumor T cell responses, either in combination with IFA or alone. CD40 ligation at the vaccine site microenvironment (VSME) may also improve adjuvant activity of TLR agonists and may be provided by CD4 T cell activation. We report a clinical trial of a multipeptide vaccine using TLR agonists and IFA, with correlative studies in 3 immunologic compartments. Methods: This trial is enrolling patients with resected stage IIB-IV melanoma (n=48) and is designed to evaluate the safety and immunogenicity of vaccination with peptides and either of 2 toll-like receptor agonists (TLR3 agonist polyICLC; TLR4 agonist endotoxin), with or without IFA. Patients are vaccinated 6 times over 12 weeks with 12 Class I MHC-restricted nonamer peptides. An immunogenic tetanus helper peptide is included to activate CD4 T cells in the VSME and secondarily to ligate CD40. Goals include safety assessment, measures of CD8 T cell responses, and characterization of cellular and molecular events induced in the blood, VSME and vaccine-draining node (sentinel immunized node, SIN), as well as a preliminary assessment of whether vaccination with TLR agonists improves the persistence of CD8 and CD4 T cell responses to melanoma antigens compared to prior studies with IFA. This includes a first-in-humans evaluation of the safety and immunogenicity of LPS, a classic TLR4 agonist, as a vaccine adjuvant. Thus, there is a novel dose-escalation phase with this adjuvant, which has been safely administered in other settings by intravenous and inhalation routes. An aim of this study is to identify an improved adjuvant for use in future trials combining peptides with other immune therapies. Clinical trial information: NCT01585350.

The impact of a multi-epitope melanoma helper peptide vaccine on Th1 cytokine production by responding lymphocytes.

e19028 Background: Melanomas 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. To describe the vaccine-induced production of Th2 or Th1 responses to helper peptides in the setting of advanced melanoma, we measured cytokine production of helper T cells responding to vaccination by 6 melanoma helper peptides (6MHP). We then analyzed results by measurable disease status. Methods: Thirty-nine patients with stage IIIB-IV melanoma received a 6MHP vaccine as a part of the UVA Mel41 clinical trial. For analysis of this correlative endpoint, antigen-reactive T cells were exposed to antigen, and supernatants (days 2 and 5) were assayed by cytokine bead arrays for Th1 and Th2 cytokines. Results: The dominant Th1 and Th2 cytokines detected were IFN-g and IL-5, respectively, though high levels of IL-2 were also produced early by sentinel lymph node lymphocytes. Th1 cytokine responses predominated both pre and post vaccine. Peripheral blood lymphocytes from patients with clinically measurable disease produced similar levels of total cytokine as patients with no evidence of disease (111pg/ml versus 174pg/ml respectively, p=0.44). For both disease statuses, the cytokine profile was Th1-dominant in most patients. Total cytokine production varied significantly among patients and varied over time but persisted up to nine months post-vaccine. Conclusions: The MHC class II-associated peptides used in this study induced helper T cells with a Th1-biased cytokine response in both PBMC and sentinel immunized nodes. However, both groups of patients can mount a Th1 dominant response to these peptides, which may persist long after the vaccination sequence. Future studies are needed to test newer vaccine adjuvants in combination with these peptides.

Interferons Induce CXCR3-cognate Chemokine Production by Human Metastatic Melanoma

Immune-mediated cancer regression requires tumor infiltration by antigen-specific effector T cells, but lymphocytes are commonly sparse in melanoma metastases. Activated T cells express CXCR3, whose cognate chemokines are CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. Little is known about expression of these chemokines in lymph node (LN) metastases of melanoma. We evaluated whether metastatic melanoma induces these CXCR3-cognate chemokines in human LN-derived tissues. In addition, as these chemokines can be induced by interferon (IFN), we evaluated whether type I or II IFNs (IFN-α or IFN-γ, respectively) can modulate chemokine expression in an in vitro model of the human tumor microenvironment. Production of CXCL9-11 by melanoma-infiltrated nodes (MIN) was no different than tumor-free nodes; both produced less chemokine than activated LN (sentinel immunized nodes, SIN). These data suggest that melanoma infiltration into LN neither induces nor reduces CXCL9-11. Stimulation with IFN-α or IFN-γ increased production of CXCL10-11 from MIN, but not tumor-free node or SIN. IFN-γ also increased production of CXCL9 in MIN. In IFN-treated SIN, CD14+ cells were the primary source of CXCL9-11, whereas melanoma cells were the source of chemokine in MIN. Melanoma cells in MIN express IFN receptors. Consistent with these observations, multiple human melanoma lines expressed IFN receptors and produced CXCL9-11 in response to IFN treatment. Thus, melanoma infiltration of LN is insufficient to induce the production of CXCL9-11, but melanoma may be a significant source of IFN-induced chemokines. Collectively, these data suggest that IFN-α or IFN-γ may act in the tumor microenvironment to increase the chemotactic gradient for CXCR3+ T cells.

Abstract 3827: Interferons induce CXCR3-cognate chemokine production by melanoma

Abstract Immune-mediated cancer regression requires tumor infiltration by Ag-specific effector T cells, but lymphocytes are commonly sparse in melanoma metastases. Activated T cells express CXCR3, whose cognate chemokines are CXCL9/MIG, CXCL10/IP-10 and CXCL11/I-TAC. Little is known about expression of these chemokines in lymph node (LN) metastases of melanoma. We evaluated whether metastatic melanoma induces these CXCR3-cognate chemokines in human LN-derived tissues. Also, because these chemokines can be induced by interferon (IFN), we evaluated whether type I or II IFNs (IFN-α or IFN-γ, respectively) can modulate chemokine expression in an in vitro model of the human tumor microenvironment. Production of CXCL9-11 by melanoma-infiltrated nodes (MIN) was no different than tumor-free nodes (TFN); both produced less than activated LN (sentinel immunized nodes, SIN). These data suggest melanoma infiltration into LN neither induces nor reduces CXCL9-11. Stimulation with IFN-α or IFN-γ increased production of CXCL10-11 from MIN, but not TFN or SIN. IFN-γ also increased production of CXCL9 in MIN. In IFN-treated MIN, the primary source of CXCL9-11 was melanoma cells themselves, whereas CD14+ cells were the source of these molecules in SIN. Melanoma cell lines expressed IFN receptors and produced CXCL9-11 in response to IFN treatment. Treatment of melanoma cell lines or primary melanomas in vitro with IFN-γ induced the migration of CXCR3+ T cells in transwell assays, whereas T cell did not significantly migrate in response to resting melanoma cells. Thus, melanoma infiltration of LN is insufficient to induce the production of CXCL9-11, but melanoma may be a significant source of IFN-induced chemokines. Collectively, these data suggest that IFN-α or IFN-γ may act in the tumor microenvironment to increase the chemotactic gradient for CXCR3+ T cells, thus enhancing the capacity of therapeutic vaccines to mediate clinical efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3827.

Evaluation of the Sentinel Immunized Node for Immune Monitoring of Cancer Vaccines

We hypothesized that lymph nodes draining sites of cutaneous vaccination could be identified by sentinel node biopsy techniques, and that measuring T-cell response with lymphocytes obtained from these lymph nodes would provide a more sensitive measure of immunogenicity than would the same measurement made with peripheral blood lymphocytes (PBL). ELISpot analysis was used to determine the magnitude of vaccine-specific T-cell response in the sentinel immunized nodes (SIN), random lymph nodes, and peripheral blood lymphocytes (PBL) obtained from patients enrolled in clinical trials of experimental melanoma vaccines. The SIN biopsy was successful in 97% of cases and morbidity was very low. The T-cell response to vaccination was detected with greater sensitivity in the SIN (57%) than in PBL (39%), and evaluation of T-cell responses in the SIN and the PBL together yielded T-cell responses in 63% of patients. When the T-cell responses from a SIN and a random lymph node were compared in four patients, immune responses were detected to one of the vaccine peptides in three of these four patients. In all of those cases, responses were present in the SIN but absent from the random lymph node. Measurements of T-cell responsiveness to cutaneous immunization are more frequently positive in the SIN than they are in the PBL, however evaluation of both the SIN and PBL permit a more sensitive measure of T-cell immunogenicity than use of either single source.

A Multipeptide Vaccine is Safe and Elicits T-cell Responses in Participants With Advanced Stage Ovarian Cancer

Nine participants with epithelial ovarian, fallopian tube, or primary peritoneal carcinoma, who were human leukocyte antigen (HLA)-A1, HLA-A2, or HLA-A3, were eligible to enroll in a phase 1 study designed to assess the safety and immunogenicity of a peptide-based vaccine. Participants received 5 class I major histocompatibility complex-restricted synthetic peptides derived from multiple ovarian cancer-associated proteins plus a class II major histocompatibility complex-restricted synthetic helper peptide derived from tetanus toxoid protein. The vaccines were administered with granulocyte macrophage-colony stimulating factor in Montanide ISA-51 adjuvant over a 7-week period. All vaccine-related toxicities were grade 1 to 2, the most common being injection site reaction (grade 2, 100%), fatigue (grade 1, 78%), and headache (grade 1, 67%). Lymphocytes from the peripheral blood and a node draining a secondary vaccine site (sentinel immunized node) were harvested during the course of vaccination and T-cell responses to the peptides were evaluated using an enzyme-linked immunosorbent spot assay. CD8 T-cell responses were detected in 1 participant ex vivo and in 8 of 9 participants (89%) after in vitro stimulation. All 4 HLA-A2 and HLA-A3-restricted peptides were immunogenic. This includes 2 peptides, folate binding protein (FBP191-199) and Her-2/neu754-762, which had not previously been evaluated in vaccines in humans. Responding T cells required over 200 nM for half-maximal reactivity. These data support continued investigation of these peptides as immunogens for patients with ovarian cancer but, owing to low potency, also suggest a need for additional immunomodulation in combination with vaccines to increase the magnitude and to improve the quality of the T-cell responses.

Breast cancer: Draining lymph node of vaccination site targeted by adjuvant GM-CSF in an autologous vaccine

13506 Background: We have reported the sensitization against Tumor Associated Antigens (TAA) and the tumor antiprogressive effect of Autologous Thermostable Hemoderivative-Cancer Vaccine (ATH-CV) in breast cancer patients (E. Garcia-Giralt et al ASCO, 2006). Systemic immune response elicited by TAA is started by a locoregional immune response at the microenvironment constituted by the antigens source and the draining lymph node. The antigens source can be a tumor or a vaccination site and respectively, the draining lymph node is the sentinel lymph node (SLN) or the Sentinel Immunized Node (SIN). Tolerogenic or protective locoregional immune response is decisional because it starts a corresponding systemic immune response, tolerogenic or protective. GM-CSF is a conditioner of SLN and SIN, switching the locoregional immune responses from tolerogenic to protective (ML Disis et al Blood, 1996; AJ Cochran et al Nat Rev Immunology, 2006). Therefore, to optimize the ATH-CV antitumoral effect, we have explored GM-CSF as a locoregional adjuvant. Methods: Thirty six M1 breast cancer patients, ER+,HER 2-, hormone and chemotherapy resistant, performance status = 2, and CA 15–3 rising level, were included in this prospective, IRB-approved phase I/II trial. Patients were 3-group (G) randomized (12 each), submitted to different treatments: G1, no additional oncology treatment; G2, ATH-CV; G3, ATH-CV plus GM-CSF at vaccination site 150 μg/day, 5 days. Statistic assessment (Student’s t-test) was performed at the 30-day end point: ATH sensitization by Delayed Type Hypersensibility test (DTH) and Lymphocyte Proliferative Assay (LPA); PSA serum level and immunophenotyping of lymph node cells in biopsies of SIN scintigraphy-localized. Results: Significant differences (p<0.05): DTH test > 5 mm: G1, 0/12; G2, 3/12 and G3, 6/12. LPA > 2.0: G1, 0/12; G2, 4/12 and G3, 7/12. Immunophenotyping: MHC-II+CD83+ (Mature Dendritic Cells):G3>G2>G1. 30 days CA 15–3 increase: G1>G2>G3. No relevant toxicities were evidenced. Conclusions: In advanced breast cancer, ATH-CV sensitization and tumor antiprogressive effect were potentiated by GM-CSF as local adjuvant. Increase of SIN mature dendritic cells is suggested as mechanism of action. No significant financial relationships to disclose.

MAGE-A1-, MAGE-A10-, and gp100-Derived Peptides Are Immunogenic When Combined with Granulocyte-Macrophage Colony-Stimulating Factor and Montanide ISA-51 Adjuvant and Administered as Part of a Multipeptide Vaccine for Melanoma

Twelve peptides derived from melanocyte differentiation proteins and cancer-testis Ags were combined and administered in a single mixture to patients with resected stage IIB, III, or IV melanoma. Five of the 12 peptides included in this mixture had not previously been evaluated for their immunogenicity in vivo following vaccination. We report in this study that at least three of these five peptides (MAGE-A1(96-104), MAGE-A10(254-262), and gp100(614-622)) are immunogenic when administered with GM-CSF in Montanide ISA-51 adjuvant. T cells secreting IFN-gamma in response to peptide-pulsed target cells were detected in peripheral blood and in the sentinel immunized node, the node draining a vaccine site, after three weekly injections. The magnitude of response typically reached a maximum after two vaccines, and though sometimes diminished thereafter, those responses typically were still detectable 6 wks after the last vaccines. Most importantly, tumor cell lines expressing the appropriate HLA-A restriction element and MAGE-A1, MAGE-A10, or gp100 proteins were lysed by corresponding CTL. This report supports the continued use of the MAGE-A1(96-104), MAGE-A10(254-262), and gp100(614-622) epitopes in peptide-based melanoma vaccines and thus expands the list of immunogenic peptide Ags available for human use. Cancer-testis Ags are expressed in multiple types of cancer; thus the MAGE-A1(96-104) and MAGE-A10(254-262) peptides may be considered for inclusion in vaccines against cancers of other histologic types, in addition to melanoma.

Immunologic and Clinical Outcomes of Vaccination With a Multiepitope Melanoma Peptide Vaccine Plus Low-Dose Interleukin-2 Administered Either Concurrently or on a Delayed Schedule

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.

Immunological results of a phase II randomized trial of multipeptide vaccines for melanoma

7503 Background: To evaluate the immunogenicity of 12 peptides from cancer-testis and melanocyte differentiation proteins, and to assess the feasibility of complex peptide vaccines, a randomized phase II trial was performed in 51 patients with resected high-risk melanoma. Methods: Arm 1 received 4 peptides and arm 2 received 12 peptides. Each vaccine was an emulsion of 4 or 12 peptides (100ug each), a tetanus helper peptide (190ug), Montanide ISA-51 adjuvant (1 ml), and GM-CSF (110 ug). T-cell responses were evaluated in peripheral blood lymphocytes and in a sentinel immunized node by IFNγ ELIspot assay after one in vitro sensitization. Results: All patients have completed treatment; immunologic assessment is complete on 31 patients. Preliminary data from these 31 patients reveal immune responses in 11/14 patients (79%) on arm 1 and 17/17 patients (100%) on arm 2. Responses to 9 of the peptides are shown (Table). Patients on arm 2 responded to an average of 2.8 different peptides, those on arm 1 responded to an average of only 0.9 peptides. By comparing T cell responses to three index peptides in both peptide mixtures, it will be possible to assess whether co-administration of 4 peptides binding the same MHC molecule interferes with immunogenicity of the index peptides. Current results indicate that the median of the best immune responses for index peptides (fold-increase over background) was 9.7 in arm 1 and 11.1 in arm 2. The mean number of responses to index peptides per patient was 0.7 for Group 1, and 1.1 for Group 2. Conclusions: These findings support that (1) the most immunogenic peptides in this study were from tyrosinase, gp100, MAGE-A1, and MAGE-A10; (2) multiple peptides can be administered in combined aqueous mixture with safety and immunogenicity, and (3) immunogenicity is maintained despite peptide competition for binding to MHC. Further investigation of multiple-peptide vaccine mixtures is warranted. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration This was funded primarily by the NCI (R01 CA57653), but funding was provided by Immunex for purchase of GMCSF used in the vaccines

Immunological results of a phase II randomized trial of multipeptide vaccines for melanoma

7503 Background: To evaluate the immunogenicity of 12 peptides from cancer-testis and melanocyte differentiation proteins, and to assess the feasibility of complex peptide vaccines, a randomized phase II trial was performed in 51 patients with resected high-risk melanoma. Methods: Arm 1 received 4 peptides and arm 2 received 12 peptides. Each vaccine was an emulsion of 4 or 12 peptides (100ug each), a tetanus helper peptide (190ug), Montanide ISA-51 adjuvant (1 ml), and GM-CSF (110 ug). T-cell responses were evaluated in peripheral blood lymphocytes and in a sentinel immunized node by IFNγ ELIspot assay after one in vitro sensitization. Results: All patients have completed treatment; immunologic assessment is complete on 31 patients. Preliminary data from these 31 patients reveal immune responses in 11/14 patients (79%) on arm 1 and 17/17 patients (100%) on arm 2. Responses to 9 of the peptides are shown (Table). Patients on arm 2 responded to an average of 2.8 different peptides, those on arm 1 responded to an average of only 0.9 peptides. By comparing T cell responses to three index peptides in both peptide mixtures, it will be possible to assess whether co-administration of 4 peptides binding the same MHC molecule interferes with immunogenicity of the index peptides. Current results indicate that the median of the best immune responses for index peptides (fold-increase over background) was 9.7 in arm 1 and 11.1 in arm 2. The mean number of responses to index peptides per patient was 0.7 for Group 1, and 1.1 for Group 2. Conclusions: These findings support that (1) the most immunogenic peptides in this study were from tyrosinase, gp100, MAGE-A1, and MAGE-A10; (2) multiple peptides can be administered in combined aqueous mixture with safety and immunogenicity, and (3) immunogenicity is maintained despite peptide competition for binding to MHC. Further investigation of multiple-peptide vaccine mixtures is warranted. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration This was funded primarily by the NCI (R01 CA57653), but funding was provided by Immunex for purchase of GMCSF used in the vaccines

Evaluation of peptide vaccine immunogenicity in draining lymph nodes and peripheral blood of melanoma patients

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. © 2001 Wiley-Liss, Inc.

Evaluation of peptide vaccine immunogenicity in draining lymph nodes and peripheral blood of melanoma patients.

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.