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Aim: Despite the huge advancements in cancer therapies and treatments over the past decade, most patients with metastasized melanoma still die from the disease. This poor prognosis largely results from resistance to conventional chemotherapies and other cytotoxic drugs. We have previously identified 6 antigenic peptides derived from melanomas that have proven efficacious for activating CD4+ T cells in clinical trials for melanoma. Our aim was to improve pharmacodynamics, pharmacokinetic and toxicological parameters by individually encapsulating each of the 6 melanoma helper peptides within their own immunogenic nanoliposomes. Methods: We modified these liposomes as necessary to account for differences in the peptides’ chemical properties, resulting in 3 distinct formulations. To further enhance immunogenicity, we also incorporated KDO2, a TLR4 agonist, into the lipid bilayer of all nanoliposome formulations. We then conducted in vivo imaging studies in mice and ex vivo cell studies from 2 patient samples who both strongly expressed one of the identified peptides. Results: We demonstrate that these liposomes, loaded with the different melanoma helper peptides, can be readily mixed together and simultaneously delivered without toxicity in vivo. These liposomes are capable of being diffused to the secondary lymphoid organs very quickly and for at least 6 days. In addition, we show that these immunogenic liposomes enhance immune responses to specific peptides ex vivo. Conclusion: Lipid-based delivery systems, including nanoliposomes and lipid nanoparticles, have now been validated for pharmacological (small molecules, bioactive lipids) and molecular (mRNA, siRNA) therapeutic approaches. However, the utility of these formulations as cancer vaccines, delivering antigenic peptides, has not yet achieved the same degree of commercial success. Here, we describe the novel and successful development of a nanoliposome-based cancer vaccine for melanoma. These vaccines help to circumvent drug resistance by increasing a patient’s T cell response, making them more susceptible to checkpoint blockade therapy.

Supplemental text for Methods and Results.

Supplementary Data from Effect of Granulocyte/Macrophage Colony-Stimulating Factor on Circulating CD8+ and CD4+ T-Cell Responses to a Multipeptide Melanoma Vaccine: Outcome of a Multicenter Randomized Trial

Blood cell counts for all patients on study, except lymphocytes.

Purpose: Granulocyte/macrophage colony-stimulating factor (GM-CSF) administered locally together with vaccines can augment T-cell responses in animal models. Human experience has been limited to small and uncontrolled trials. Thus, a multicenter randomized phase II trial was done to determine whether local administration of GM-CSF augments immunogenicity of a multipeptide vaccine. It also assessed immunogenicity of administration in one versus two vaccine sites.Experimental Design: One hundred twenty-one eligible patients with resected stage IIB to IV melanoma were vaccinated with 12 MHC class Irestricted melanoma peptides to stimulate CD8+ T cells plus a HLA-DRrestricted tetanus helper peptide to stimulate CD4+ T cells, emulsified in incomplete Freund's adjuvant, with or without 110 g GM-CSF. Among 119 evaluable patients, T-cell responses were assessed by IFN- ELIspot assay and tetramer analysis. Clinical outcomes were recorded.Results: CD8+ T-cell response rates to the 12 MHC class Irestricted melanoma peptides (by day 50) with or without GM-CSF were 34 and 73, respectively (P < 0.001), by direct ELIspot assay. Tetramer analyses corroborated the functional data. CD4+ T-cell responses to tetanus helper peptide were higher without GM-CSF (95 versus 77; P = 0.005). There was no significant difference by number of vaccine sites. Three-year overall and disease-free survival estimates (95 confidence interval) were 76 (67-83) and 52 (43-61), respectively, with too few events to assess differences by study group.Conclusions: High immune response rates for this multipeptide vaccine were achieved, but CD8+ and CD4+ T-cell responses were lower when administered with GM-CSF. These data challenge the value of local GM-CSF as a vaccine adjuvant in humans. (Clin Cancer Res 2009;15(22):703644)

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Supplementary Figure 2. Increased Ab responses over time to 3 of the 4 peptides assessed.

Purpose: This multicenter randomized trial was designed to evaluate whether melanoma helper peptides augment cytotoxic T lymphocyte (CTL) responses to a melanoma vaccine and improve clinical outcome in patients with advanced melanoma.Experimental Design: One hundred seventy-five patients with measurable stage IV melanoma were enrolled into 4 treatment groups, vaccinated with 12 MHC class I-restricted melanoma peptides to stimulate CTL (12MP, group A), plus a tetanus peptide (group B), or a mixture of 6 melanoma helper peptides (6MHP, group C) to stimulate helper T lymphocytes (HTL), or with 6 melanoma helper peptide (6MHP) alone (group D), in incomplete Freund's adjuvant plus granulocyte macrophage colony-stimulating factor. CTL responses were assessed using an in vitro-stimulated IFN-γ ELIspot assay, and HTL responses were assessed using a proliferation assay.Results: In groups A to D, respectively, CTL response rates to 12 melanoma peptides were 43%, 47%, 28%, and 5%, and HTL response rates to 6MHP were in 3%, 0%, 40%, and 41%. Best clinical response was partial response in 7 of 148 evaluable patients (4.7%) without significant difference among study arms. Median overall survival (OS) was 11.8 months. Immune response to 6 MHP was significantly associated with both clinical response (P = 0.036) and OS (P = 0.004).Conclusion: Each vaccine regimen was immunogenic, but MHPs did not augment CTL responses to 12 melanoma peptides. The association of survival and immune response to 6MHP supports further investigation of helper peptide vaccines. For patients with advanced melanoma, multipeptide vaccines should be studied in combination with other potentially synergistic active therapies. Clin Cancer Res; 19(15); 4228–38. ©2013 AACR.

CCR Translation for the Article from Effect of Granulocyte/Macrophage Colony-Stimulating Factor on Circulating CD8+ and CD4+ T-Cell Responses to a Multipeptide Melanoma Vaccine: Outcome of a Multicenter Randomized Trial

Supplementary Figure Legends. Legends for supplementary figures.

Supplementary Figure 1. Positive control response to 6MHP peptide components.

Detail of treatment-related toxicities.

CCR Translation for This Article from A Randomized Phase II Trial of Multiepitope Vaccination with Melanoma Peptides for Cytotoxic T Cells and Helper T Cells for Patients with Metastatic Melanoma (E1602)

Supplementary Table 1: Patient demographics. Supplementary Table 2: T cell response rates by arm. Supplementary Table 3: Peptide-specific immune response by HLA type. Supplementary Table 4: Number of patients in each group defined by sex, visceral disease and ECOG PS. Supplementary Table 5: Predicted 1-year OS rate (using Korn method) and observed 1-year OS rate for E1602 patients. Supplementary Table 6: treatment related toxicity.

PDF file - 126K, Detailed data are shown for selected proteins assayed by RPPA.

PDF file - 39K, Lymphocyte counts for all patients on study - detail.

PDF - 433K, Supplementary Figure 1: Study Schema. Supplementary Figure 2: Association of overall survival and immune response to 6MHP, detail by study arm (Arm C and Arm D.)

Purpose: A melanoma vaccine incorporating six peptides designed to induce helper T-cell responses to melanoma antigens has induced Th1-dominant CD4+ T-cell responses in most patients, and induced durable clinical responses or stable disease in 24% of evaluable patients. The present study tested whether this vaccine also induced antibody (Ab) responses to each peptide, and whether Ab responses were associated with T-cell responses and with clinical outcome.Experimental Design: Serum samples were studied from 35 patients with stage III-IV melanomas vaccinated with 6 melanoma helper peptides (6MHP). IgG Ab responses were measured by ELISA. Associations with immune response and overall survival were assessed by log-rank test and χ2 analysis of Kaplan–Meier data.Results: Ab responses to 6MHP were detected by week 7 in 77% of patients, and increased to peak 6 weeks after the last vaccine and persisted to 6 months. Ab responses were induced most frequently to longer peptides. Of those with T-cell responses, 82% had early Ab responses. Survival was improved for patients with early Ab response (P = 0.0011) or with early T-cell response (P < 0.006), and was best for those with both Ab and T-cell responses (P = 0.0002).Conclusions: Vaccination with helper peptides induced both Ab responses and T-cell responses, associated with favorable clinical outcome. Such immune responses may predict favorable clinical outcome to guide combination immunotherapy. Further studies are warranted to understand mechanisms of interaction of these Abs, T-cell responses, and tumor control. Clin Cancer Res; 21(17); 3879–87. ©2015 AACR.

Purpose: A CTEP-sponsored phase II trial was conducted to evaluate safety and clinical activity of combination therapy with CCI-779 (temsirolimus) and bevacizumab in patients with advanced melanoma.Experimental Design: Patients with unresectable stage III to IV melanoma were treated intravenously with temsirolimus 25 mg weekly and bevacizumab 10 mg every 2 weeks. Adverse events were recorded using CTCAE v3.0. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors and overall survival was recorded. Correlative studies measured protein kinases and histology of tumor biopsies and immune function in peripheral blood.Results: Seventeen patients were treated. Most patients tolerated treatment well, but 2 had grade 4 lymphopenia and 1 developed reversible grade 2 leukoencephalopathy. Best clinical response was partial response (PR) in 3 patients [17.7%, 90% confidence interval (CI) 5, 0–39.6], stable disease at 8 weeks (SD) in 9 patients, progressive disease (PD) in 4 patients, and not evaluable in 1 patient. Maximal response duration for PR was 35 months. Ten evaluable patients had BRAFWT tumors, among whom 3 had PRs, 5 had SD, and 2 had PD. Correlative studies of tumor biopsies revealed decreased phospho-S6K (d2 and d23 vs. d1, P < 0.001), and decreased mitotic rate (Ki67+) among melanoma cells by d23 (P = 0.007). Effects on immune functions were mixed, with decreased alloreactive T-cell responses and decreased circulating CD4+FoxP3+ cells.Conclusion: These data provide preliminary evidence for clinical activity of combination therapy with temsirolimus and bevacizumab, which may be greater in patients with BRAFwt melanoma. Mixed effects on immunologic function also support combination with immune therapies. Clin Cancer Res; 19(13); 3611–20. ©2013 AACR.

PDF file - 208K, Blood cell counts for all patients on study, except lymphocytes.

Detailed data are shown for selected proteins assayed by RPPA.

Lymphocyte counts for all patients on study - detail

BackgroundImmune-mediated melanoma regression relies on melanoma-reactive T cells infiltrating tumor. Cancer vaccines increase circulating melanoma-reactive T cells, but little is known about vaccine-induced circulating lymphocytes (viCLs) homing to tumor or whether interventions are needed to enhance infiltration. We hypothesized that viCLs infiltrate melanoma metastases, and intratumoral interferon (IFN)-γ or Toll-like receptor 7 (TLR7) agonism enhances infiltration.MethodsPatients on two clinical trials (Mel51 (NCT00977145), Mel53 (NCT01264731)) received vaccines containing 12 class I major histocompatibility complex-restricted melanoma peptides (12MP). In Mel51, tumor was injected with IFN-γ on day 22, and biopsied on days 1, 22, and 24. In Mel53, dermal metastases were treated with topical imiquimod, a TLR7 agonist, for 12 weeks, and biopsied on days 1, 22, and 43. For patients with circulating T-cell responses to 12MP by IFN-γ ELISpot assays, DNA was extracted from peripheral blood mononuclear cells (PBMCs) pre-vaccination and at peak T-cell response, and from tumor biopsies, which underwent T-cell receptor sequencing. This enabled identification of clonotypes induced in PBMCs post-vaccination (viCLs) and present in tumor post-vaccination, but not pre-vaccination.ResultsSix patients with T-cell responses post-vaccination (Mel51 n = 4, Mel53 n = 2) were evaluated for viCLs and vaccine-induced tumor infiltrating lymphocytes (viTILs). All six patients had viCLs, five of whom were evaluable for viTILs in tumor post-vaccination alone. Mel51 patients had viTILs identified in day 22 tumors, post-vaccination and before IFN-γ (median = 2, range = 0–24). This increased in day 24 tumors after IFN-γ (median = 30, range = 4–74). Mel53 patients had viTILs identified in day 22 tumors, post-vaccination plus imiquimod (median = 33, range = 2–64). Three of five evaluable patients across both trials had viTILs with vaccination alone. All five had enhancement of viTILs with tumor-directed therapy. viTILs represented 0.0–2.9% of total T cells after vaccination alone, which increased to 0.6–8.7% after tumor-directed therapy.ConclusionCancer vaccines induce expansion of new viCLs, which infiltrate melanoma metastases in some patients. Our findings identify opportunities to combine vaccines with tumor-directed therapies to enhance T-cell infiltration and T cell-mediated tumor control. These combinations hold promise in improving the therapeutic efficacy of antigen-specific therapies for solid malignancies.

There is a need for better classification and understanding of tumor infiltrating lymphocytes (TILs). Here we applied advanced functional genomics to interrogate 9000 human tumors, and multiple single-cell sequencing sets using benchmarked T cell states, comprehensive T cell differentiation trajectories, human and mouse vaccine responses, and other human TILs. Compared to other T cell states, enrichment of T memory/resident memory programs was observed across solid tumors. Trajectory analysis of single-cell melanoma CD8(+) TILs also identified a high fraction of memory/resident memory-scoring TILs in anti-PD-1 responders, which expanded post therapy. In contrast, TILs scoring highly for early T cell activation, but not exhaustion, associated with non-response. Late/persistent, but not early activation signatures, prognosticate melanoma survival, and co-express with dendritic cell and IFN-γ response programs. These data identify an activation-like state associated to poor response and suggest successful memory conversion, above resuscitation of exhaustion, is an under-appreciated aspect of successful anti-tumoral immunity.

BackgroundWe performed a clinical trial to evaluate safety and immunogenicity of a novel long peptide vaccine administered in combinations of incomplete Freund’s adjuvant (IFA) and agonists for TLR3 (polyICLC) and TLR7/8 (resiquimod). We hypothesized that T cell responses to minimal epitope peptides (MEPs) within the long peptides would be enhanced compared with prior vaccines with MEP themselves and that T cell responses would be enhanced with TLR agonists, compared with IFA alone.MethodsParticipants with resected stage IIB-IV melanoma were vaccinated with seven long melanoma peptides (LPV7) from tyrosinase, gp100, MAGE-A1, MAGE-A10, and NY-ESO-1, each containing a known MEP for CD8+ T cells, plus a tetanus helper peptide (Tet) restricted by Class II MHC. Enrollment was guided by an adaptive design to one of seven adjuvant combinations. Vaccines were administered at weeks 1, 2, 3, 6, 9, 12 at rotating injection sites. T cell and IgG antibody (Ab) responses were measured with IFN-gamma ELIspot assay ex vivo and ELISA, respectively.ResultsFifty eligible participants were assigned to seven study groups, with highest enrollment on arm E (LPV7+Tet+IFA+polyICLC). There was one dose-limiting toxicity (DLT) in Group E (grade 3 injection site reaction, 6% DLT rate). All other treatment-related adverse events were grades 1–2. The CD8+ T cell immune response rate (IRR) to MEPs was 18%, less than in prior studies using MEP vaccines in IFA. The CD8+ T cell IRR trended higher for IFA-containing adjuvants (24%) than adjuvants containing only TLR agonists (6%). Overall T cell IRR to full-length LPV7 was 30%; CD4+ T cell IRR to Tet was 40%, and serum Ab IRR to LPV7 was 84%. These IRRs also trended higher for IFA-containing adjuvants (36% vs 18%, 48% vs 24%, and 97% vs 60%, respectively).ConclusionsThe LPV7 vaccine is safe with each of seven adjuvant strategies and induced T cell responses to CD8 MEPs ex vivo in a subset of patients but did not enhance IRRs compared with prior vaccines using short peptides. Immunogenicity was supported more by IFA than by TLR agonists alone and may be enhanced by polyICLC plus IFA.Trial registration numberNCT02126579.

BackgroundA vaccine containing 6 melanoma-associated peptides to stimulate helper T cells (6MHP) is safe, immunogenic, and clinically active. A phase I/II trial was designed to evaluate safety and immunogenicity of 6MHP vaccines plus programmed death 1 (PD-1) blockade.Participants and methodsParticipants with advanced melanoma received 6MHP vaccines in an incomplete Freund’s adjuvant (6 vaccines over 12 weeks). Pembrolizumab was administered intravenously every 3 weeks. Tumor biopsies at baseline and day 22 were analyzed by multiplex immunohistochemistry. Primary end points were safety (Common Terminology Criteria for Adverse Events V.4.03) and immunogenicity (ex vivo interferon-γ ELISpot assay). Additional end points included changes in the tumor microenvironment (TME) and clinical outcomes.ResultsTwenty-two eligible participants were treated: 6 naïve to PD-1 antibody (Ab) and 16 PD-1 Ab-experienced. Median follow-up was 24.4 months. Most common treatment-related adverse events (any grade) included injection site reactions, fatigue, anemia, lymphopenia, fever, elevated aspartate aminotransferase, pruritus, and rash. Treatment-related dose-limiting toxicities were observed in 3 (14%) participants, which did not cross the study safety bound. A high durable T cell response (Rsp) to 6MHP was detected in only one participant, but twofold T cell Rsps to 6MHP were detected in 7/22 (32%; 90% CI (16% to 52%)) by week 13. Objective clinical responses were observed in 23% (1 complete response, 4 partial responses), including 4/6 PD-1 Ab-naïve (67%) and 1/16 PD-1 Ab-experienced (6%). Overall survival (OS) was longer for PD-1 Ab-naïve than Ab-experienced participants (HR 6.3 (90% CI (2.1 to 28.7)). In landmark analyses at 13 weeks, OS was also longer for those with T cell Rsps (HR 6.5 (90% CI (2.1 to 29.2)) and for those with objective clinical responses. TME evaluation revealed increased densities of CD8+T cells, CD20+B cells, and Tbet+cells by day 22.ConclusionsTreatment with the 6MHP vaccine plus pembrolizumab was safe, increased intratumoral lymphocytes, and induced T cell Rsps associated with prolonged OS. The low T cell Rsp rate in PD-1 Ab-experienced participants corroborates prior murine studies that caution against delaying cancer vaccines until after PD-1 blockade. The promising objective response rate and OS in PD-1 Ab-naïve participants support consideration of a larger study in that setting.

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 (AE) 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 (SIN). Eleven participants were enrolled and treated; one was retrospectively found to be ineligible due to HLA type. All eleven participants were included in AE and survival analyses, and the ten eligible participants were evaluated for immunogenicity. All participants reported AE: 82% were Grade 1-2, most commonly fatigue or injection site reactions. Two participants (18%) experienced treatment-related dose-limiting Grade 3 AE; both were self-limiting. Immune responses to Her2 or CEA peptides were detected in 70% of participants. Median overall survival was 16 months; among those enrolled with no evidence of disease (n=3), median overall survival 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.

BackgroundPeptide vaccines designed to stimulate melanoma-reactive CD4+ T cells can induce T cell and antibody (Ab) responses, associated with enhanced overall survival. We hypothesized that adding toll-like receptor 3 agonist polyICLC to an incomplete Freund’s adjuvant (IFA) would be safe and would support strong, durable CD4+ T cell and Ab responses. We also hypothesized that oral low-dose metronomic cyclophosphamide (mCy) would be safe, would reduce circulating regulatory T cells (T-regs) and would further enhance immunogenicity.Participants and methodsAn adaptive design based on toxicity and durable CD4+ T cell immune response (dRsp) was used to assign participants with resected stage IIA-IV melanoma to one of four study regimens. The regimens included a vaccine comprising six melanoma peptides restricted by Class II MHC (6MHP) in an emulsion with IFA alone (Arm A), with IFA plus systemic mCy (Arm B), with IFA+ local polyICLC (Arm C), or with IFA+ polyICLC+ mCy (Arm D). Toxicities were recorded (CTCAE V.4.03). T cell responses were measured by interferon γ ELIspot assay ex vivo. Serum Ab responses to 6MHP were measured by ELISA. Circulating T-regs were assessed by flow cytometry.ResultsForty-eight eligible participants were enrolled and treated. Early data on safety and dRsp favored enrollment on arm D. Total enrollment on Arms A-D were 3, 7, 6, and 32, respectively. Treatment-related dose-limiting toxicities (DLTs) were observed in 1/7 (14%) participants on arm B and 2/32 (6%) on arm D. None exceeded the 25% DLT threshold for early closure to enrollment for any arm. Strong durable T cell responses to 6MHP were detected ex vivo in 0%, 29%, 67%, and 47% of participants on arms A-D, respectively. IgG Ab responses were greatest for arms C and D. Circulating T-regs frequencies were not altered by mCy.Conclusions6MHP vaccines administered with IFA, polyICLC, and mCy were well tolerated. The dRsp rate for arm D of 47% (90% CI 32 to 63) exceeded the 18% (90% CI 11 to 26) rate previously observed with 6MHP in IFA alone. Vaccination with IFA+ polyICLC (arm C) also showed promise for enhancing T cell and Ab responses.

Ipilimumab (IPI) can enhance immunity to the cancer-testis antigen NY-ESO-1. A clinical trial was designed to assess safety, immunogenicity, and clinical responses with IPI + NY-ESO-1 vaccines and effects on the tumor microenvironment (TME). Patients with measurable NY-ESO-1+ tumors were enrolled among three arms: A) IPI + NY-ESO-1 protein + poly-ICLC (pICLC) + incomplete Freund’s adjuvant (IFA); B) IPI + NY-ESO-1 overlapping long peptides (OLP) + pICLC + IFA; and C) IPI + NY-ESO-1 OLP + pICLC. Clinical responses were assessed by irRC. T cell and Ab responses were assessed by ex vivo IFN-gamma ELIspot and ELISA. Tumor biopsies pre- and post-treatment were evaluated for immune infiltrates. Eight patients were enrolled: 5, 2, and 1 in Arms A-C, respectively. There were no DLTs. Best clinical responses were SD (4) and PD (4). T-cell and antibody (Ab) responses to NY-ESO-1 were detected in 6 (75%) and 7 (88%) patients, respectively, and were associated with SD. The breadth of Ab responses was greater for patients with SD than PD (p = .036). For five patients evaluable in the TME, treatment was associated with increases in proliferating (Ki67+) CD8+ T cells and decreases in RORγt+ CD4+ T cells. T cell densities increased for those with SD. Detection of T cell responses to NY-ESO-1 ex vivo in most patients suggests that IPI may have enhanced those responses. Proliferating intratumoral CD8+ T cells increased after vaccination plus IPI suggesting favorable impact of IPI plus NY-ESO-1 vaccines on the TME. List of Abbreviations: Ab = antibody; CTCAE = NCI Common Terminology Criteria for Adverse Events; DHFR/DHRP = dihydrofolate reductase; DLT = Dose-limiting toxicity; ELISA = enzyme-linked immunosorbent assay; IFA = incomplete Freund’s adjuvant (Montanide ISA-51); IFNγ = Interferon gamma; IPI = Ipilimumab; irRC = immune-related response criteria; mIFH = multispectral immunofluorescence histology; OLP = NY-ESO-1 overlapping long peptides; PBMC = peripheral blood mononuclear cells; PD = Progressive disease; pICLC = poly-ICLC (Hiltonol), a TLR3/MDA-5 agonist; RLT = Regimen-limiting Toxicity; ROI = regions of interest; RT = room temperature; SAE = serious adverse event; SD = stable disease; TEAE = treatment-emergent adverse events; TLR = toll-like receptor; TME = tumor microenvironment; TRAE = treatment-related adverse events.

BackgroundA multipeptide vaccine containing 6 melanoma-associated peptides to stimulate helper T cells (6MHP) is safe and immunogenic and has clinical activity. A phase I/II trial was designed to evaluate safety and immunogenicity of 6MHP vaccines plus PD1 blockade.MethodsParticipants with measurable advanced melanoma, age ≥ 18 years, and ECOG performance status 0–1 were administered 6MHP vaccine intradermally and subcutaneously in an incomplete Freund’s adjuvant on days (D) 1, 8, 15, 43, 64, and 85. Pembrolizumab 200 mg was administered intravenously every 3 weeks for up to two years. Biopsies of accessible tumors at baseline and D22 were analyzed by multiparameter immunofluorescence histology. Primary endpoints were safety (CTCAE 4.03) and immunogenicity (ex vivo IFNγ ELIspot assay). Secondary and exploratory endpoints included changes in the tumor microenvironment (TME), and clinical outcomes.ResultsTwenty-two eligible participants were enrolled and treated, including 6 naïve to PD-1 Ab and 16 anti-PD-1 Ab-experienced. Median follow-up was 20 months. Treatment-related adverse events (any grade) experienced by >20% were injection site reactions, fatigue, anemia, nausea, fever, bruising, and rash. Treatment-related dose limiting toxicities (grade 3 elevated AST, skin ulcer, or uveitis) were observed in 3 (14%), which did not cross the study safety bound. Objective clinical responses were observed in 23% (1 CR, 4 PR), including 4/6 anti-PD-1 Ab-naive (67%) and one 1/16 anti-PD1 Ab-experienced (6%). Four participants (18%) had SD as best radiographic response (18%), all in the Ab-experienced cohort. T cell responses to 6MHP were detected in seven participants (32%) by week 13 and were associated with clinical response (CR/PR 80% vs. SD/PD 18%; p = 0.01). Overall survival was prolonged for anti-PD-1 Ab naïve vs experienced (p = 0.0048), for those with T cell response (p = 0.045, landmark analysis after week 13), and for those with objective response (p = 0.0148). TME evaluation in 12 participants revealed significant increases by D22 in the densities (per mm2) of CD8+ T cells (p = 0.0186), CD20+ B cells (P = 0.002), and Tbet+ cells (p = 0.034).ConclusionsIn patients with advanced melanoma, combined treatment with the 6MHP vaccine plus pembrolizumab was safe, increased intratumoral T and B cells, as well as Th1 (Tbet+) cells, and induced T cell responses that were associated with objective response and with overall survival. The promising objective response rate and overall survival in patients naive to PD1 blockade supports consideration of a larger study to assess definitive benefit in that clinical setting.AcknowledgementsWe acknowledge the support of Merck for providing pembrolizumab at no charge, and the University of Virginia Cancer Center Support grant P30 CA044579 for support of shared resource facilities.Trial RegistrationThe clinical trial Mel64 (PATHVACS) is registered with Clinicaltrials.gov (NCT02515227).Ethics ApprovalThe clinical trial Mel64 (PATHVACS) was performed with IRB (#18174) and FDA approval (IND #10825) and is registered with Clinicaltrials.gov (NCT02515227). Written informed consent was obtained from each participant prior to participation in the study.

Background Breast cancer remains a leading cause of cancer death worldwide. There is evidence that immunotherapy may play a role in the eradication of residual disease. Peptide vaccines for immunotherapy are capable of durable immune memory, but vaccines alone have shown sparse clinical activity against breast cancer to date. Toll-like receptor (TLR) agonists and helper peptides are excellent adjuvants for vaccine immunotherapy and they are examined in this human clinical trial. Methods A vaccine consisting of 9 MHC class I-restricted breast cancer-associated peptides (from MAGE-A1, −A3, and -A10, CEA, NY-ESO-1, and HER2 proteins) was combined with a TLR3 agonist, poly-ICLC, along with a helper peptide derived from tetanus toxoid. The vaccine was administered on days 1, 8, 15, 36, 57, 78. CD8+ T cell responses to the vaccine were assessed by both direct and stimulated interferon gamma ELIspot assays. Results Twelve patients with breast cancer were treated: five had estrogen receptor positive disease and five were HER2 amplified. There were no dose-limiting toxicities. Toxicities were limited to Grade 1 and Grade 2 and included mild injection site reactions and flu-like symptoms, which occurred in most patients. The most common toxicities were injection site reaction/induration and fatigue, which were experienced by 100% and 92% of participants, respectively. In the stimulated ELIspot assays, peptide-specific CD8+ T cell responses were detected in 4 of 11 evaluable patients. Two patients had borderline immune responses to the vaccine. The two peptides derived from CEA were immunogenic. No difference in immune response was evident between patients receiving endocrine therapy and those not receiving endocrine therapy during the vaccine series. Conclusions Peptide vaccine administered in the adjuvant breast cancer setting was safe and feasible. The TLR3 adjuvant, poly-ICLC, plus helper peptide mixture provided modest immune stimulation. Further optimization is required for this multi-peptide vaccine/adjuvant combination. Trial registration ClinicalTrials.gov (posted 2/15/2012): NCT01532960. Registered 2/8/2012. https://clinicaltrials.gov/show/NCT01532960 Electronic supplementary material The online version of this article (10.1186/s40425-017-0295-5) contains supplementary material, which is available to authorized users.

BackgroundExperimental cancer vaccines are traditionally administered by injection in subcutaneous tissue or muscle, commonly with adjuvants that create chronic inflammatory depots. Injection of melanoma-derived peptides induces T cell responses; however, the depots that form following injection may inhibit optimization of the immune response. In skin, epidermal Langerhans cells (LC) are a dominant source of professional antigen presenting cells. We hypothesized that: (1) applying melanoma-derived peptides topically, in proximity to LC, could be immunogenic and safe, with low vaccine-site toxicity and (2) topical toll-like receptor 7 (TLR7) agonist would increase immunogenicity of the peptide vaccine.MethodsTwelve melanoma peptides plus a tetanus helper peptide were combined with granulocyte macrophage colony stimulating factor (GM-CSF) and were administered topically on days 1, 8, and 15, to 28 patients randomized to one of four adjuvant preparations: (1) incomplete Freund’s adjuvant (IFA); (2) IFA plus a TLR7 agonist (imiquimod) administered on days 0, 7, 14; (3) dimethyl sulfoxide (DMSO) or (4) DMSO+ imiquimod administered on day 0, 7, 14. Every 3 weeks thereafter (x 6), the peptides were combined with GM-CSF and were injected into the dermis and subcutis in an emulsion with IFA. Toxicities were recorded and immune responses assayed by ELIspot.ResultsCD8+ T cell responses to transdermal vaccination in DMSO occurred in 83% of participants in group 3 and 86% in group 4, and responses to vaccination in IFA were observed in 29% of participants in group 1 and 14% in group 2. Overall, 61% of participants had CD4+ T cell immune responses to the tetanus peptide, with large, durable responses in groups 3 and 4. Five of seven participants in group 4 had a severe rash, one that was dose limiting. Ten-year overall survival was 67% and disease-free survival was 44%.ConclusionsThese data provide proof of principle for immunogenicity in humans of transdermal immunization using peptides in DMSO. Further study is warranted into the pharmacokinetics and immunobiology of TLR agonists as vaccine adjuvants during transcutaneous application. Overall survival is high, supporting further investigation of this immunization approach.

Optimal approaches to induce T cell infiltration of tumors are not known. Chemokines CXCL9, CXCL10, and CXCL11 support effector T cell recruitment and may be induced by IFN. This study tests the hypothesis that intratumoral administration of IFNγ will induce CXCL9-11 and will induce T cell recruitment and anti-tumor immune signatures in melanoma metastases.Nine eligible patients were immunized with a vaccine comprised of 12 class I MHC-restricted melanoma peptides and received IFNγ intratumorally. Effects on the tumor microenvironment were evaluated in sequential tumor biopsies. Adverse events (AEs) were recorded. T cell responses to vaccination were assessed in PBMC by IFNγ ELISPOT assay. Tumor biopsies were evaluated for immune cell infiltration, chemokine protein expression, and gene expression.Vaccination and intratumoral administration of IFNγ were well tolerated. Circulating T cell responses to vaccine were detected in six of nine patients. IFNγ increased production of chemokines CXCL10, CXCL11, and CCL5 in patient tumors. Neither vaccination alone, nor the addition of IFNγ promoted immune cell infiltration or induced anti-tumor immune gene signatures.The melanoma vaccine induced circulating T cell responses, but it failed to infiltrate metastases, thus highlighting the need for combination strategies to support T cell infiltration. A single intratumoral injection of IFNγ induced T cell-attracting chemokines; however, it also induced secondary immune regulation that may paradoxically limit immune infiltration and effector functions. Alternate dosing strategies or additional combinatorial treatments may be needed to promote trafficking and retention of tumor-reactive T cells in melanoma metastases.

Integrins α1β1 (CD49a), α2β1 (CD49b) and αEβ7 (CD103) mediate retention of lymphocytes in peripheral tissues, and their expression is upregulated on tumor infiltrating lymphocytes (TIL) compared to circulating lymphocytes. Little is known about what induces expression of these retention integrins (RI) nor whether RI define subsets in the tumor microenvironment (TME) with a specific phenotype. Human metastatic melanoma-derived CD8 TIL could be grouped into five subpopulations based on RI expression patterns: RI(neg), CD49a(+) only, CD49a(+)CD49b(+), CD49a(+)CD103(+), or positive for all three RI. A significantly larger fraction of the CD49a(+) only subpopulation expressed multiple effector cytokines, whereas CD49a(+)CD103(+) and CD49a(+)CD49b(+) cells expressed IFNγ only. RI(neg) and CD49a(+)CD49b(+)CD103(+) CD8 TIL subsets expressed significantly less effector cytokines overall. Interestingly, however, CD49a(+)CD49b(+)CD103(+) CD8 expressed lowest CD127, and highest levels of perforin and exhaustion markers PD-1 and Tim3, suggesting selective exhaustion rather than conversion to memory. To gain insight into RI expression induction, normal donor PBMC were cultured with T cell receptor (TCR) stimulation and/or cytokines. TCR stimulation alone induced two RI(+) cell populations: CD49a single positive and CD49a(+)CD49b(+) cells. TNFα and IL-2 each were capable of inducing these populations. Addition of TGFβ to TCR stimulation generated two additional populations; CD49a(+)CD49b(neg)CD103(+) and CD49a(+)CD49b(+)CD103(+.) Taken together, our findings identify opportunities to modulate RI expression in the TME by cytokine therapies and to generate subsets with a specific RI repertoire in the interest of augmenting immune therapies for cancer or for modulating other immune-related diseases such as autoimmune diseases.

Clinical approaches to treat advanced melanoma include immune therapies, whose benefits depend on tumor-reactive T-cell infiltration of metastases. However, most tumors lack significant immune infiltration prior to therapy. Selected chemokines promote T-cell migration into tumors; thus, agents that induce these chemokines in the tumor microenvironment (TME) may improve responses to systemic immune therapy. CXCL10 has been implicated as a critical chemokine supporting T-cell infiltration into the TME. Here, we show that toll-like receptor (TLR) agonists can induce chemokine production directly from melanoma cells when combined with IFNγ treatment. We find that TLR2 and TLR6 are widely expressed on human melanoma cells, and that TLR2/6 agonists (MALP-2 or FSL-1) synergize with interferon-gamma (IFNγ) to induce production of CXCL10 from melanoma cells. Furthermore, melanoma cells and immune cells from surgical specimens also respond to TLR2/6 agonists and IFNγ by upregulating CXCL10 production, compared to treatment with either agent alone. Collectively, these data identify a novel mechanism for inducing CXCL10 production directly from melanoma cells, with TLR2/6 agonists +IFNγ and raise the possibility that intratumoral administration of these agents may improve immune signatures in melanoma and have value in combination with other immune therapies, by supporting T-cell migration into melanoma metastases.

Background Our research is focused on using the vaccine draining lymph node to better understand the immune response to cancer vaccines and as a possible source of anti-cancer reagents. We evaluated vaccine draining lymph nodes archived from a clinical study in melanoma patients and determined the reaction of B cells to the vaccine peptides. Methods Mononuclear cells (MNCs) were recovered from cryopreserved lymph nodes that were directly receiving drainage from multi-peptide melanoma vaccine. The patients were enrolled on a vaccine study (NCT00089219, FDA, BB-IND No. 10825). B cell responses in the vaccine-draining lymph nodes were studied under both stimulated and un-stimulated conditions. Cryopreserved cells were stimulated with CD40L, stained with multiple human cell-surface markers (CD19, CD27, IgM) to identify different categories of B cell sub populations with flow cytometry. Hybridomas were generated from the lymph node cells after CD40L-stimulation. Cells were fused to murine plasmacytoma P3X63.Ag8.653 using Helix electrofusion chamber. ELISA was used to evaluate hybridoma derived antibody binding to vaccine peptides. Results Viable MNCs were satisfactorily recovered from lymph nodes cryopreserved from six vaccine study patients 8–14 years previously. B cell ELISPOT demonstrated responses for each patient to multiple vaccine peptides. CD40L stimulation of lymph node cells increased the proportion of CD19+ CD27+ cells from 12 to 65% of the sample and increased the proportion of class-switched cells. Screening of IgG secreting clones demonstrated binding to melanoma vaccine peptides. Conclusions B cells were successfully recovered and expanded from human cryopreserved vaccine-draining lymph nodes. Individual B cells were identified that secreted antibodies that bound to cancer vaccine peptides. The ability to reliably generate in vitro the same antibodies observed in the blood of vaccinated patients will facilitate research to understand mechanisms of human antibody activity and possibly lead to therapeutic antibodies.

Successful therapeutic options remain elusive for pancreatic cancer. The exquisite sensitivity and specificity of humoral and cellular immunity may provide therapeutic approaches if antigens specific for pancreatic cancer cells can be identified. Here we characterize SAS1B (ovastacin, ASTL, astacin-like), a cancer-oocyte antigen, as an attractive immunotoxin target expressed at the surface of human pancreatic cancer cells, with limited expression among normal tissues. Immunohistochemistry shows that most pancreatic cancers are SAS1Bpos (68%), while normal pancreatic ductal epithelium is SAS1Bneg. Pancreatic cancer cell lines developed from patient-derived xenograft models display SAS1B cell surface localization, in addition to cytoplasmic expression, suggesting utility for SAS1B in multiple immunotherapeutic approaches. When pancreatic cancer cells were treated with an anti-SAS1B antibody-drug conjugate, significant cell death was observed at 0.01-0.1 μg/mL, while SAS1Bneg human keratinocytes were resistant. Cytotoxicity was correlated with SAS1B cell surface expression; substantial killing was observed for tumors with low steady state SAS1B expression, suggesting a substantial proportion of SAS1Bpos tumors can be targeted in this manner. These results demonstrate SAS1B is a surface target in pancreatic cancer cells capable of binding monoclonal antibodies, internalization, and delivering cytotoxic drug payloads, supporting further development of SAS1B as a novel target for pancreatic cancer.

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.

T-cell infiltration into the metastatic melanoma microenvironment (MME) correlates with improved patient survival. However, diffuse infiltration into tumor occurs in only 8% of melanoma metastases. Little is known about mechanisms governing T-cell infiltration into human melanoma metastases or about how those mechanisms may be altered therapeutically. We hypothesized that T cells in the MME would be enriched for chemokine receptors CCR4, CCR5, CXCR3 and homing receptors relevant to the tissue site. Viably cryopreserved single cell suspensions from nineteen melanoma metastases representing three metastatic sites (tumor-infiltrated lymph node, skin and small bowel) were evaluated by multiparameter flow cytometry and compared to benign lymph nodes and peripheral blood mononuclear cells from patients with Stage IIB-IV melanoma. T cells in the melanoma metastases contained large effector memory populations, high proportions of activated, moderately differentiated cells and few regulatory T cells. Site-specific homing was suggested in bowel, with high expression of CCR9. We neither encounter the anticipated enrichment of integrin α4β7 in bowel, cutaneous leukocyte antigen (CLA) in skin, nor integrin α4β1 or receptor CXCR3 in metastatic sites. Retention integrins αEβ7, α1β1 and α2β1 were significantly elevated in metastases. These data suggest limited tissue site-specific homing to human melanoma metastases, but a significant role for retention integrins in maintaining intratumoral T cells. Our findings also raise the possibility that T-cell homing, infiltration, and retention in melanoma metastases may be increased by increasing expression of ligands for CLA, α4β1 and CXCR3 on intratumoral endothelium.

Purpose: A CTEP-sponsored phase II trial was conducted to evaluate safety and clinical activity of combination therapy with CCI-779 (temsirolimus) and bevacizumab in patients with advanced melanoma. Experimental Design: Patients with unresectable stage III to IV melanoma were treated intravenously with temsirolimus 25 mg weekly and bevacizumab 10 mg every 2 weeks. Adverse events were recorded using CTCAE v3.0. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors and overall survival was recorded. Correlative studies measured protein kinases and histology of tumor biopsies and immune function in peripheral blood. Results: Seventeen patients were treated. Most patients tolerated treatment well, but 2 had grade 4 lymphopenia and 1 developed reversible grade 2 leukoencephalopathy. Best clinical response was partial response (PR) in 3 patients [17.7%, 90% confidence interval (CI) 5, 0–39.6], stable disease at 8 weeks (SD) in 9 patients, progressive disease (PD) in 4 patients, and not evaluable in 1 patient. Maximal response duration for PR was 35 months. Ten evaluable patients had BRAFWT tumors, among whom 3 had PRs, 5 had SD, and 2 had PD. Correlative studies of tumor biopsies revealed decreased phospho-S6K (d2 and d23 vs. d1, P < 0.001), and decreased mitotic rate (Ki67+) among melanoma cells by d23 (P = 0.007). Effects on immune functions were mixed, with decreased alloreactive T-cell responses and decreased circulating CD4+FoxP3+ cells. Conclusion: These data provide preliminary evidence for clinical activity of combination therapy with temsirolimus and bevacizumab, which may be greater in patients with BRAFwt melanoma. Mixed effects on immunologic function also support combination with immune therapies. Clin Cancer Res; 19(13); 3611–20. ©2013 AACR.

Infiltration of cancers by T cells is associated with improved patient survival and response to immune therapies; however, optimal approaches to induce T cell infiltration of tumors are not known. This study was designed to assess whether topical treatment of melanoma metastases with the TLR7 agonist imiquimod plus administration of a multipeptide cancer vaccine will improve immune cell infiltration of melanoma metastases.Eligible patients were immunized with a vaccine comprised of 12 melanoma peptides and a tetanus toxoid-derived helper peptide, and imiquimod was applied topically to metastatic tumors daily. Adverse events were recorded, and effects on the tumor microenvironment were evaluated from sequential tumor biopsies. T cell responses were assessed by IFNγ ELIspot assay and T cell tetramer staining. Patient tumors were evaluated for immune cell infiltration, cytokine and chemokine production, and gene expression.Four eligible patients were enrolled, and administration of imiquimod and vaccination were well tolerated. Circulating T cell responses to the vaccine was detected by ex vivo ELIspot assay in 3 of 4 patients. Treatment of metastases with imiquimod induced immune cell infiltration and favorable gene signatures in the patients with circulating T cell responses. This study supports further study of topical imiquimod combined with vaccines or other immune therapies for the treatment of melanoma.

Purpose: Granulocyte/macrophage colony-stimulating factor (GM-CSF) administered locally together with vaccines can augment T-cell responses in animal models. Human experience has been limited to small and uncontrolled trials. Thus, a multicenter randomized phase II trial was done to determine whether local administration of GM-CSF augments immunogenicity of a multipeptide vaccine. It also assessed immunogenicity of administration in one versus two vaccine sites.Experimental Design: One hundred twenty-one eligible patients with resected stage IIB to IV melanoma were vaccinated with 12 MHC class Irestricted melanoma peptides to stimulate CD8+ T cells plus a HLA-DRrestricted tetanus helper peptide to stimulate CD4+ T cells, emulsified in incomplete Freund's adjuvant, with or without 110 g GM-CSF. Among 119 evaluable patients, T-cell responses were assessed by IFN- ELIspot assay and tetramer analysis. Clinical outcomes were recorded.Results: CD8+ T-cell response rates to the 12 MHC class Irestricted melanoma peptides (by day 50) with or without GM-CSF were 34 and 73, respectively (P < 0.001), by direct ELIspot assay. Tetramer analyses corroborated the functional data. CD4+ T-cell responses to tetanus helper peptide were higher without GM-CSF (95 versus 77; P = 0.005). There was no significant difference by number of vaccine sites. Three-year overall and disease-free survival estimates (95 confidence interval) were 76 (67-83) and 52 (43-61), respectively, with too few events to assess differences by study group.Conclusions: High immune response rates for this multipeptide vaccine were achieved, but CD8+ and CD4+ T-cell responses were lower when administered with GM-CSF. These data challenge the value of local GM-CSF as a vaccine adjuvant in humans. (Clin Cancer Res 2009;15(22):703644)